Patent application title: TAILORED OILS
Inventors:
Scott Franklin (Woodside, CA, US)
Scott Franklin (Woodside, CA, US)
Aravind Somanchi (Redwood City, CA, US)
Aravind Somanchi (Redwood City, CA, US)
George Rudenko (Mountain View, CA, US)
George Rudenko (Mountain View, CA, US)
Riyaz Bhat (South San Francisco, CA, US)
Riyaz Bhat (South San Francisco, CA, US)
Xinhua Zhao (Foster City, CA, US)
Xinhua Zhao (Foster City, CA, US)
Jeffrey L. Moseley (Redwood City, CA, US)
Jeffrey L. Moseley (Redwood City, CA, US)
IPC8 Class: AC12P764FI
USPC Class:
1 1
Class name:
Publication date: 2019-01-03
Patent application number: 20190002934
Abstract:
Recombinant DNA techniques are used to produce oleaginous recombinant
cells that produce triglyceride oils having desired fatty acid profiles
and regiospecific or stereospecific profiles. Genes manipulated include
those encoding stearoyl-ACP desaturase, delta 12 fatty acid desaturase,
acyl-ACP thioesterase, ketoacyl-ACP synthase, and lysophosphatidic acid
acyltransferase. The oil produced can have enhanced oxidative or thermal
stability, or can be useful as a frying oil, shortening, roll-in
shortening, tempering fat, cocoa butter replacement, as a lubricant, or
as a feedstock for various chemical processes. The fatty acid profile can
be enriched in midchain profiles or the oil can be enriched in
triglycerides of the saturated-unsaturated-saturated type.Claims:
1.-84. (canceled)
85. A recombinant cell of the genus Prototheca or Chlorella, said recombinant cell comprising a knockout or knockdown of an endogenous Fatty acyl-ACP thioesterase gene, and further comprising exogenous nucleic acids that encode lysophosphatidic acid acyltransferase (LPAAT).
86. The recombinant cell of claim 85, wherein said exogenous nucleic acids that encode LPAAT encodes an LPPAT having at least 90% amino acid sequence identity to an LPAAT selected from the group consisting of SEQ ID NOs: 16, 77, 78, 79 and 157.
87. The recombinant cell of claim 86, wherein said exogenous nucleic acids that encode LPAAT encodes an LPPAT having at least 95% amino acid sequence identity to an LPAAT selected from the group consisting of SEQ ID NOs: 16, 77, 78, 79 and 157.
88. The recombinant cell of claim of claim 86, wherein said exogenous nucleic acids encodes an LPAAT comprising the amino acid sequences of SEQ ID NO: 157.
89. The recombinant cell of claim of claim 86, wherein said exogenous nucleic acids encodes an LPAAT comprising the amino acid sequences of SEQ ID NOs: 17, 77, 78 or 79.
90. The recombinant cell of claim 89, wherein said exogenous nucleic acids that encode LPAAT are nucleic acids having at least 90% sequence identity to the nucleic acids selected from the group consisting of SEQ ID NOs: 18, 80, 81, 82, 83, 84 and 85.
91. The recombinant cell of claim 85, wherein said exogenous nucleic acids that encode LPAAT are nucleic acids having at least 95% sequence identity to the nucleic acids selected from the group consisting of SEQ ID NOs: 18, 80, 81, 82, 83, 84 and 85.
92. The recombinant cell of claim 85, wherein triglyceride oil produced by the recombinant cell is enriched in stearate-oleate-stearate (SOS) triglycerides.
93. The recombinant cell of claim 92, wherein the SOS triglyceride content comprises at least 50% of the triglyceride oils.
94. The recombinant cell of claim 93, wherein the SOS triglyceride content comprises at least 60% of the triglyceride oils.
95. The recombinant cell of claim 85, wherein triglyceride oil produced by the recombinant cell is enriched in mid-chain fatty acid.
96. The recombinant cell of claim 95, wherein the triglyceride oil is enriched in C12:0.
97. The method of claim 85, wherein said recombinant cell is of the genus Prototheca.
98. The method of claim 97, wherein said recombinant cell is a Prototheca moriformis cell.
99. The method of claim 92, wherein said recombinant cell is of the genus Prototheca.
100. The method of claim 99, wherein said recombinant cell is a Prototheca moriformis cell.
101. A method of producing oil, the method comprising the steps of: a. cultivating a recombinant cell of the genus Prototheca or Chlorella, said recombinant cell comprising a knockout or knockdown of an endogenous Fatty acyl-ACP thioesterase gene, and further comprising exogenous nucleic acids that encode lysophosphatidic acid acyltransferase (LPAAT); and b. isolating the oil from said recombinant cell.
102. The method of claim 101, wherein said recombinant cell is of the genus Prototheca.
103. The method of claim 102, wherein said recombinant cell is a Prototheca moriformis cell.
104. An oil produced by the steps of: a. cultivating a recombinant cell of the genus Prototheca or Chlorella, said recombinant cell comprising a knockout or knockdown of an endogenous Fatty acyl-ACP thioesterase gene, and further comprising exogenous nucleic acids that encode lysophosphatidic acid acyltransferase (LPAAT); and b. isolating the oil from said recombinant cell.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent application Ser. No. 14/506,491, filed Oct. 3, 2014, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Nos. 61/887,268, filed Oct. 4, 2013; 61/892,399, filed Oct. 17, 2013; 61/895,355, filed Oct. 24, 2013; 61/923,327, filed Jan. 3, 2014; and 62/023,109, filed Jul. 10, 2014. Each of these applications is incorporated herein by reference in its entirety for all purposes. This application includes subject matter related to that disclosed in U.S. Provisional Patent Application No. 62/023,112, entitled "Novel Ketoacyl ACP Synthase Genes and Uses Thereof," filed Jul. 10, 2014, which is also hereby incorporated by reference in its entirety for all purposes. In particular, Tables 1, 7 and 8 of 62/023,112, and the corresponding sequences identified therein, are hereby incorporated by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2017, is named SOLAP059US-SL.txt and is 599,266 bytes in size.
[0003] This application includes an electronic sequence listing in a file named "452572-Sequence2.txt", created on Jan. 20, 2015, and containing 551,031 bytes, which is hereby incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0004] Embodiments of the present invention relate to oils/fats, fuels, foods, and oleochemicals and their production from cultures of genetically engineered cells. Specific embodiments relate to oils with a high content of triglycerides bearing fatty acyl groups upon the glycerol backbone in particular regiospecific patterns, highly stable oils, oils with high levels of oleic or mid-chain fatty acids, and products produced from such oils.
BACKGROUND OF THE INVENTION
[0005] PCT Publications WO2008/151149, WO2010/06032, WO2011/150410, WO2011/150411, WO2012/061647, and WO2012/106560 disclose oils and methods for producing those oils in microbes, including microalgae. These publications also describe the use of such oils to make oleochemicals and fuels.
[0006] Tempering is a process of converting a fat into a desired polymorphic form by manipulation of the temperature of the fat or fat-containing substance, commonly used in chocolate making.
[0007] Certain enzymes of the fatty acyl-CoA elongation pathway function to extend the length of fatty acyl-CoA molecules. Elongase-complex enzymes extend fatty acyl-CoA molecules in 2 carbon additions, for example myristoyl-CoA to palmitoyl-CoA, stearoyl-CoA to arachidyl-CoA, or oleoyl-CoA to eicosanoyl-CoA, eicosanoyl-CoA to erucyl-CoA. In addition, elongase enzymes also extend acyl chain length in 2 carbon increments. KCS enzymes condense acyl-CoA molecules with two carbons from malonyl-CoA to form beta-ketoacyl-CoA. KCS and elongases may show specificity for condensing acyl substrates of particular carbon length, modification (such as hydroxylation), or degree of saturation. For example, the jojoba (Simmondsia chinensis) beta-ketoacyl-CoA synthase has been demonstrated to prefer monounsaturated and saturated C18- and C20-CoA substrates to elevate production of erucic acid in transgenic plants (Lassner et al., Plant Cell, 1996, Vol 8(2), pp. 281-292), whereas specific elongase enzymes of Trypanosoma brucei show preference for elongating short and midchain saturated CoA substrates (Lee et al., Cell, 2006, Vol 126(4), pp. 691-9).
[0008] The type II fatty acid biosynthetic pathway employs a series of reactions catalyzed by soluble proteins with intermediates shuttled between enzymes as thioesters of acyl carrier protein (ACP). By contrast, the type I fatty acid biosynthetic pathway uses a single, large multifunctional polypeptide.
[0009] The oleaginous, non-photosynthetic alga, Prototheca moriformis, stores copious amounts of triacylglyceride oil under conditions when the nutritional carbon supply is in excess, but cell division is inhibited due to limitation of other essential nutrients. Bulk biosynthesis of fatty acids with carbon chain lengths up to C18 occurs in the plastids; fatty acids are then exported to the endoplasmic reticulum where (if it occurs) elongation past C18 and incorporation into triacylglycerides (TAGs) is believed to occur. Lipids are stored in large cytoplasmic organelles called lipid bodies until environmental conditions change to favor growth, whereupon they are mobilized to provide energy and carbon molecules for anabolic metabolism.
SUMMARY OF THE INVENTION
[0010] In accordance with an embodiment, a method includes cultivating a recombinant cell, the cell
[0011] (i) expressing an exogenous KASI or KASIV gene, optionally encoding a protein having at least 60, 65, 70, 75, 80, 85, 90, or 95% amino acid sequence identity to an enzyme encoded by any of SEQ ID NOs: 46-49, and at least one FATB acyl-ACP thioesterase gene optionally encoding a protein having at least 60, 65, 70, 75, 80, 85, 90, or 95% nucleic acid sequence identity to SEQ ID NOs: 11, 87, 89, 159, 162 or 163;
[0012] (ii) expressing a gene encoding a FATA, FATB, KASI, KASII, LPAAT, SAD, or FAD2 under the control of a nitrogen-sensitive promoter having at least 60, 65, 70, 75, 80, 85, 90, or 95% sequence identity to any of SEQ ID NOs: 129 to 147; or
[0013] (iii) having a knockout or knockdown of a SAD gene, a FAD2 gene, and a FATA gene, an overexpressing an exogenous C18-preferring FATA gene, an oleoyl-preferring LPAAT gene, and a KASII gene; and extracting oil from the cell.
[0014] In a related embodiment, the cell is of type (ii) and comprises at least a second acyl-ACP thioesterase, optionally encoding a protein having at least 60, 65, 70, 75, 80, 85, 90, or 95% nucleic acid sequence identity to any of SEQ ID NOS:: 11, 87, 89, 159, 162 or 163. The oil can have at least 30% C10:0 and at least 30% C12:0. The oil can have a viscosity of less than 30 cS and optionally of 25 cS.+-.20% at 40.degree. C. as measured by ASTM D445. The C10:0 and C12:0 fatty acids can be balanced to within 20%, 10% or 5%.
[0015] In a related embodiment, the cell is of type (iii) and the cell oil comprises at least 60% stearate-oleate-stearate (SOS). Optionally,the C18-preferring FATA gene encodes a protein with at least 60, 65, 70, 75, 80, 85, 90, or 95% amino acid identity to SEQ ID NO: 156, the LPAAT gene encodes a protein with at least 60, 65, 70, 75, 80, 85, 90, or 95% amino acid identity to SEQ ID NO: 157 and/or the KASII gene encodes a protein with at least 60, 65, 70, 75, 80, 85, 90, or 95% amino acid identity to SEQ ID NO 160 or 161.
[0016] Optionally, the cell is a microalga, optionally of Trebouxiophyceae, and optionally of the genus Prototheca.
[0017] In a related embodiment, there is an oil, soap, oleochemical, foodstuff, or other oil-derived product produced according to one of the aforementioned methods.
[0018] In accordance with an embodiment of the present invention, a method comprises cultivating an oleaginous recombinant cell. The cell comprises an exogenous gene encoding a palmitate ACP-desaturase enzyme active to produce an oil having a fatty acid profile characterized by a ratio of palmitoleic acid to palmitic acid of at least 0.1 and/or palmitoleic acid levels of 0.5% or more, as determined by FAME GC/FID analysis. Optionally, the cell is of an oleaginous recombinant eukaryotic microalga.
[0019] In related embodiments, the exogenous gene encodes a palmitoyl-ACP desaturase (PAD) having desaturating activity toward ACP-palmitate. Optionally, the exogenous PAD gene encodes a stearoyl-ACP desaturase variant having increased activity toward ACP-palmitate. The variant can be a L118W mutant. The gene can be in operable linkage with a promoter, plastid-targeting transit peptide, and 5'UTR active to express the gene product in a eukaryotic oleaginous microalga. The microalga can be of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca. Alternately, the microalga has 23 S rRNA with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide sequence identity to SEQ ID NO: 76.
[0020] Optionally, the fatty acid profile is further characterized by less than 3.5% saturated fatty acids. Optionally, the cell is cultivated to at least 40% oil by dry cell weight. Optionally, the microalga further comprises a knockout or knockdown of an endogenous acyl-ACP thioesterase and/or an exogenous KASII gene. This may reduce the levels of saturated fatty acids in the oil. For example, the exogenous KASII gene can be inserted into the coding region of the endogenous acyl-ACP thioesterase. Optionally, the inserted KASII gene is inverted in orientation relative to the endogenous acyl-ACP thioesterase.
[0021] In any of these embodiments, the oil can be produced by heterotrophically cultivating the microalga on sucrose and the microalga comprises an exogenous invertase gene that allows it to metabolize the sucrose.
[0022] The oil may be recovered. The recovered oil may be used for frying or as an ingredient in a prepared food. The oil may have a microalga sterol profile. In a specific embodiment, the microalga sterol profile is characterized by an excess of ergosterol over .beta.-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol.
[0023] In another embodiment, a method comprises cultivating an oleaginous cell, optionally a microalga, so that the cell produces an oil with less than 10% palmitic acid, greater than Optionally the cell is a microalga with FAD and FATA knockouts and expresses an exogenous KASII gene.
[0024] In a related embodiment, a method comprises cultivating an oleaginous cell, optionally a microalga, so that the cell produces an oil with a fatty acid profile in which: the sum of lauric and myristic acids is at least 50%; total saturated fatty acids are at least 50% and levels of capric and lauric fatty acids are balanced to within 20%; or capric acid is at least 45% and lauric acid is at least 45%. In specific related embodiments the sum of lauric and myristic acids is at least 60%, 70% or 7%%. Optionally, the cell comprises an exogenous plant FATB gene.
[0025] Optionally, the cell comprises an exogenous exogenous KASI or KASIV gene.
[0026] The oil may be recovered. The recovered oil may be used for frying or as an ingredient in a prepared food. The oil may have a microalgal sterol profile. In a specific embodiment, the microalgal sterol profile is characterized by an excess of ergosterol over .beta.-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol. The oil can be used to make a foodstuff or chemical.
[0027] In another embodiment, a method comprises cultivating an oleaginous cell, optionally a microalga, so that the cell produces an oil with a fatty acid profile characterized by 10% or less linolenic acid and 20% or more linoleic acid. The cell can comprise an overexpressed KASII gene and a FAD gene replacement. Optionally, the cell comprises an exogenous gene encoding an oleate-specific acyl-ACP thioesterase or a knockout of one or more FATA alleles, together with an exogenous gene encoding an oleate-specific acyl-ACP thioesterase. The overexpression of the FAD gene can be by environmental control of a regulatable promoter. The oil can be recovered and used to produce a foodstuff or chemicals. The oil may comprise a microalgal sterol profile.
[0028] In another aspect, the present invention provides a method for producing a triglyceride oil, in which the method comprises: (a) cultivating an oleaginous cell under nitrogen-replete conditions, thereby increasing the number of cells, then; (b) cultivating the cells under nitrogen-poor conditions thereby causing the cells to accumulate triglycerides to at least 20% by dry cell weight; comprising a FADc (FAD2) allele, optionally a sole allele, under control of a promoter that is active under the nitrogen replete conditions and inactive under the nitrogen-starved conditions, the promoter retaining at least half of its activity at pH 5.0 as compared to pH 7.0; and (c) obtaining the oil, wherein the oil comprises reduced linoleic acid due to the downregulation of the FADc gene under the nitrogen-starved conditions.
[0029] In some embodiments, the cell is cultivated at a pH of less than 6.5 using sucrose in the presence of invertase. In some cases, the invertase is produced by the cell. In some cases, the invertase is produced from an exogenous gene expressed by the cell.
[0030] In some embodiments, the oil obtained has a fatty acid profile with less than 3%, 2%, 1%, or 0.5% linoleic acid.
[0031] In some embodiments, the cell further comprises a FADc knockout so as to amplify the change in linoleic acid. In some cases, the transcript level of FADc decreases by a factor of 10 or more between the nitrogen-replete and nitrogen-starved conditions.
[0032] In another aspect, the present invention provides a method for producing a triglyceride cell oil comprising cultivating a recombinant cell comprising an exogenous FATB gene and an exogenous KASI gene, wherein the expression of the KASI gene causes the oil to have a shorter chain distribution relative to a control cell with the FATB gene but without the KASI gene.
[0033] In another aspect, the present invention provides a recombinant cell comprising a FATB acyl-ACP thioesterase gene having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 88% nucleotide identity to SEQ ID NOs: 90 or 91 or equivalent sequence due to the degeneracy of the genetic code, or encoding an enzyme having at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 88% amino acid identity to SEQ ID NOs: 90 or 91. In some embodiments, the cell produces triglycerides that are shifted in fatty acid profile due to expression of the FATB gene.
[0034] In an embodiment of the invention, there is a process for producing an oil. The process includes obtaining a cell oil from a genetically engineered microbe, optionally a microalga, and fractionating the cell oil to produce a stearin fraction. The stearin fraction can be characterized by a TAG profile having at least 70% SOS with no more than 4% trisaturates and an sn-2 profile characterized by least 90% oleate at the sn-2 position. Optionally, the microbe is a microalga comprising one or more of an overexpressed KASII gene, a SAD knockout or knockdown, or an exogenous C18-preferring FATA gene, an exogenous LPAAT, and a FAD2 knockout or knockdown. Optionally, the stearin fraction has a maximum heat-flow temperatures or DSC-derived SFC curve that is an essentially identical to the equivalent curve of Kokum butter. The fractionation can be a two step fractionation performed at a first temperature that removes 00S, optionally about 24.degree. C., and a second temperature that removes trisaturates, optionally about 29.degree. C.
[0035] In accordance with an embodiment of the invention a method produces a triglyceride oil characterized by a TAG profile. The method includes providing an oleaginous plastidic host cell overexpressing a KASII gene, an exogenous FATA gene and an exogenous LPAAT gene, cultivating the cell so as to produce the oil, and isolating the oil; the TAG profile has greater than 50% SOS an less than 10% trisaturates.
[0036] In related embodiments, the cell includes a knockdown or knockout of an endogenous SAD2 gene and/or knockdown or knockout of an endogenous FATA gene. The exogenous FATA gene can encode a functional FATA acyl-ACP thioesterase protein with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 92. The exogenous LPAAT gene can encode a functional Lysophosphatidic acid acyltransferase protein with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 93. Optionally, the host cell can be a microalga, optionally of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca, and optionally having 23S rRNA with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide sequence identity to SEQ ID NO: 76.
[0037] In an embodiment, a recombinant microlagal host cell optionally of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca, and optionally having 23S rRNA with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide sequence identity to SEQ ID NO: 76, expresses an exogenous FATA gene encodes a functional FATA acyl-ACP thioesterase protein with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 92.
[0038] In an embodiment, a recombinant microlagal host cell optionally of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca, and optionally having 23S rRNA with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide sequence identity to SEQ ID NO: 76, expresses an exogenous LPAAT gene encodes a functional Lysophosphatidic acid acyltransferase protein with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 93.
[0039] These and other aspects and embodiments of the invention are described and/or exemplified in the accompanying drawings, a brief description of which immediately follows, the detailed description of the invention, and in the examples. Any or all of the features discussed above and throughout the application can be combined in various embodiments of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1-14 show fatty acid profiles and melting curves of refined, bleached and deodorized oils from genetically engineered Prototheca moriformis strains, as discussed in Example 4;
[0041] FIG. 15 shows the stability of different oils as a function of antioxidant concentration, as discussed in Example 5;
[0042] FIG. 16 shows various properties of cell oils with very low levels of polyunsaturated fatty acids in accordance with an embodiment of the invention; and
[0043] FIG. 17 shows a plot of percent solid fat content for various oils as follows: (a) P. moriformis RBD oil without lipid pathway engineering; (b) Brazilian cocoa butter+25% milk fat; (c) three replicates of P. moriformis RBD oil from a strain expressing hairpin nucleic acids that reduce levels of a SAD allele thus reducing oleic acid and increasing stearic acid in the TAG profile; (d) P. moriformis RBD oil from a strain overexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, see Example 45); (e) Malaysian cocoa butter+25% milk fat; and (f) Malaysian cocoa butter. The cocoa butter and cocoa butter milk fat values are literature values (Bailey's Industrial Oils and Fat Products, 6.sup.th ed.).
[0044] FIG. 18 shows the results of thermal stability testing performed on methylated oil prepared from high-oleic (HO) and high-stability high-oleic (HSAO) triglyceride oils prepared from heterotrophically grown oleaginous microalgae, in comparison to a soya methyl ester control sample.
[0045] FIG. 19 shows various properties of high-oleic and high-stability high-oleic algal oils.
[0046] FIG. 20 shows TAG composition of Strain K-4, Strain AU and Strain AV oils from flask and fermenter biomass. La=laurate (C12:0), M=myristate (C14:0), P=palmitate (C16:0), Po=palmitoleate (C16:1), S=stearate (C18:0), O=oleate (C18:1), L=linoleate (C18:2), Ln=.alpha.-linolenate (C18:3), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0) S-S-S refers to the sum of TAGs in which all three fatty acids are saturated. In each block of bars, the strains are shown in the order illustrated at the bottom of the figure.
[0047] FIG. 21 shows TAG composition of Strain AW Strain AX and Strain AY oils from shake flask biomass. La=laurate (C12:0), M=myristate (C14:0), P=palmitate (C16:0), Po=palmitoleate (C16:1), S=stearate (C18:0), O=oleate (C18:1), L=linoleate (C18:2), Ln=.alpha.-linolenate (C18:3), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0). S-S-S refers to the sum of TAGs in which all three fatty acids are saturated. In each block of bars, the strains are shown in the order illustrated at the bottom of the figure.
[0048] FIG. 22 shows the fatty acid profile and solid fat content of a refined, bleached and deodorized myristate rich oil from a genetically engineered Prototheca moriformis strain as discussed in Example 52.
[0049] FIG. 23 shows the pairwise alignment of heterologous FAE proteins (SEQ ID NOS 165-171, respectively, in order of appearance) expressed in STRAIN Z.
[0050] FIG. 24 shows genetic modification of a microalgal strain to produced double knockouts of FAD2/FADc and FATA.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
[0051] An "allele" refers to a copy of a gene where an organism has multiple similar or identical gene copies, even if on the same chromosome. An allele may encode the same or similar protein.
[0052] In connection with two fatty acids in a fatty acid profile, "balanced" shall mean that the two fatty acids are within a specified percentage of their mean area percent. Thus, for fatty acid a in x % abundance and fatty acid b in y % abundance, the fatty acids are "balanced to within z %" if |x-((x+y)/2)| and |y-((x+y)/2)| are .ltoreq.100(z).
[0053] A "cell oil" or "cell fat" shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the cell oil or cell fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. For a cell oil produced by a cell, the sterol profile of oil is generally determined by the sterols produced by the cell, not by artificial reconstitution of the oil by adding sterols in order to mimic the cell oil. In connection with a cell oil or cell fat, and as used generally throughout the present disclosure, the terms oil and fat are used interchangeably, except where otherwise noted. Thus, an "oil" or a "fat" can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term "fractionation" means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms "cell oil" and "cell fat" encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching and/or degumming, which does not substantially change its triglyceride profile. A cell oil can also be a "noninteresterified cell oil", which means that the cell oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.
[0054] "Exogenous gene" shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a "transgene". A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
[0055] "FADc", also referred to as "FAD2" is a gene encoding a delta-12 fatty acid desaturase.
[0056] "Fatty acids" shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.
[0057] "Fixed carbon source" is a molecule(s) containing carbon, typically an organic molecule that is present at ambient temperature and pressure in solid or liquid form in a culture media that can be utilized by a microorganism cultured therein. Accordingly, carbon dioxide is not a fixed carbon source.
[0058] "In operable linkage" is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.
[0059] "Microalgae" are eukaryotic microbial organisms that contain a chloroplast or other plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca.
[0060] In connection with fatty acid length, "mid-chain" shall mean C8 to C16 fatty acids.
[0061] In connection with a recombinant cell, the term "knockdown" refers to a gene that has been partially suppressed (e.g., by about 1-95%) in terms of the production or activity of a protein encoded by the gene.
[0062] Also, in connection with a recombinant cell, the term "knockout" refers to a gene that has been completely or nearly completely (e.g., >95%) suppressed in terms of the production or activity of a protein encoded by the gene. Knockouts can be prepared by homologous recombination of a noncoding sequence into a coding sequence, gene deletion, mutation or other method.
[0063] An "oleaginous" cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An "oleaginous microbe" or "oleaginous microorganism" is a microbe, including a microalga that is oleaginous (especially eukaryotic microalgae that store lipid). An oleaginous cell also encompasses a cell that has had some or all of its lipid or other content removed, and both live and dead cells.
[0064] An "ordered oil" or "ordered fat" is one that forms crystals that are primarily of a given polymorphic structure. For example, an ordered oil or ordered fat can have crystals that are greater than 50%, 60%, 70%, 80%, or 90% of the .beta. or .beta.' polymorphic form.
[0065] In connection with a cell oil, a "profile" is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A "fatty acid profile" is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID), as in Example 1. The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids. A "sn-2 profile" is the distribution of fatty acids found at the sn-2 position of the triacylglycerides in the oil. A "regiospecific profile" is the distribution of triglycerides with reference to the positioning of acyl group attachment to the glycerol backbone without reference to stereospecificity. In other words, a regiospecific profile describes acyl group attachment at sn-1/3 vs. sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate) and SOP (stearate-oleate-palmitate) are treated identically. A "stereospecific profile" describes the attachment of acyl groups at sn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such as SOP and POS are to be considered equivalent. A "TAG profile" is the distribution of fatty acids found in the triglycerides with reference to connection to the glycerol backbone, but without reference to the regiospecific nature of the connections. Thus, in a TAG profile, the percent of SSO in the oil is the sum of SSO and SOS, while in a regiospecific profile, the percent of SSO is calculated without inclusion of SOS species in the oil. In contrast to the weight percentages of the FAME-GC-FID analysis, triglyceride percentages are typically given as mole percentages; that is the percent of a given TAG molecule in a TAG mixture.
[0066] The term "percent sequence identity," in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap.times.drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap.times.drop-off 50; Expect: 10; Word Size: 3; Filter: on.
[0067] "Recombinant" is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A "recombinant nucleic acid" is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
[0068] The terms "triglyceride", "triacylglyceride" and "TAG" are used interchangeably as is known in the art.
IL GENERAL
[0069] Illustrative embodiments of the present invention feature oleaginous cells that produce altered fatty acid profiles and/or altered regiospecific distribution of fatty acids in glycerolipids, and products produced from the cells. Examples of oleaginous cells include microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae and, where applicable, oil producing cells of higher plants including but not limited to commercial oilseed crops such as soy, corn, rapeseed/canola, cotton, flax, sunflower, safflower and peanut. Other specific examples of cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae and method of cultivation are also provided in Published PCT Patent Applications WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cell weight, .+-.5%. Optionally, the oils produced can be low in highly unsaturated fatty acids such as DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose) In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylose metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, "GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE", published Nov. 15, 2012, including disclosure of genetically engineered Prototheca strains that utilize xylose.
[0070] The oleaginous cells may, optionally, be cultivated in a bioreactor/fermenter. For example, heterotrophic oleaginous microalgal cells can be cultivated on a sugar-containing nutrient broth. Optionally, cultivation can proceed in two stages: a seed stage and a lipid-production stage. In the seed stage, the number of cells is increased from s starter culture. Thus, the seeds stage typically includes a nutrient rich, nitrogen replete, media designed to encourage rapid cell division. After the seeds stage, the cells may be fed sugar under nutrient-limiting (e.g. nitrogen sparse) conditions so that the sugar will be converted into triglycerides. For example, the rate of cell division in the lipid-production stage can be decreased by 50%, 80% or more relative to the seed stage. Additionally, variation in the media between the seed stage and the lipid-production stage can induce the recombinant cell to express different lipid-synthesis genes and thereby alter the triglycerides being produced. For example, as discussed below, nitrogen and/or pH sensitive promoters can be placed in front of endogenous or exogenous genes. This is especially useful when an oil is to be produced in the lipid-production phase that does not support optimal growth of the cells in the seed stage. In an example below, a cell has a fatty acid desaturase with a pH sensitive promoter so than an oil that is low in linoleic acid is produced in the lipid production stage while an oil that has adequate linoleic acid for cell division is produced during the seed stage. The resulting low linoleic oil has exceptional oxidative stability.
[0071] The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature cell oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.
[0072] The oleaginous cells (optionally microalgal cells) can be improved via classical strain improvement techniques such as UV and/or chemical mutagenesis followed by screening or selection under environmental conditions, including selection on a chemical or biochemical toxin. For example the cells can be selected on a fatty acid synthesis inhibitor, a sugar metabolism inhibitor, or an herbicide. As a result of the selection, strains can be obtained with increased yield on sugar, increased oil production (e.g., as a percent of cell volume, dry weight, or liter of cell culture), or improved fatty acid or TAG profile.
[0073] For example, the cells can be selected on one or more of 1,2-Cyclohexanedione; 19-Norethindone acetate; 2,2-dichloropropionic acid; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-trichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxyacetic acid; 2,4-dichlorophenoxyacetic acid, butyl ester; 2,4-dichlorophenoxyacetic acid, isooctyl ester; 2,4-dichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxybutyric acid; 2,4-dichlorophenoxybutyric acid, methyl ester; 2,6-dichlorobenzonitrile; 2-deoxyglucose; 5-Tetradecyloxy-w-furoic acid; A-922500; acetochlor; alachlor; ametryn; amphotericin; atrazine; benfluralin; bensulide; bentazon; bromacil; bromoxynil; Cafenstrole; carbonyl cyanide m-chlorophenyl hydrazone (CCCP); carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP); cerulenin; chlorpropham; chlorsulfuron; clofibric acid; clopyralid; colchicine; cycloate; cyclohexamide; C75; DACTHAL (dimethyl tetrachloroterephthalate); dicamba; dichloroprop ((R)-2-(2,4-dichlorophenoxy)propanoic acid); Diflufenican; dihyrojasmonic acid, methyl ester; diquat; diuron; dimethylsulfoxide; Epigallocatechin gallate (EGCG); endothall; ethalfluralin; ethanol; ethofumesate; Fenoxaprop-p-ethyl; Fluazifop-p-Butyl; fluometuron; fomasefen; foramsulfuron; gibberellic acid; glufosinate ammonium; glyphosate; haloxyfop; hexazinone; imazaquin; isoxaben; Lipase inhibitor THL ((-)-Tetrahydrolipstatin); malonic acid; MCPA (2-methyl-4-chlorophenoxyacetic acid); MCPB (4-(4-chloro-o-tolyloxy)butyric acid); mesotrione; methyl dihydrojasmonate; metolachlor; metribuzin; Mildronate; molinate; naptalam; norharman; orlistat; oxadiazon; oxyfluorfen; paraquat; pendimethalin; pentachlorophenol; PF-04620110; phenethyl alcohol; phenmedipham; picloram; Platencin; Platensimycin; prometon; prometryn; pronamide; propachlor; propanil; propazine; pyrazon; Quizalofop-p-ethyl; s-ethyl dipropylthiocarbamate (EPTC); s,s,s-tributylphosphorotrithioate; salicylhydroxamic acid; sesamol; siduron; sodium methane arsenate; simazine; T-863 (DGAT inhibitor); tebuthiuron; terbacil; thiobencarb; tralkoxydim; triallate; triclopyr; triclosan; trifluralin; and vulpinic acid.
[0074] The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. The raw oil may comprise sterols produced by the cells. WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/1504 disclose heterotrophic cultivation and oil isolation techniques for oleaginous microalgae. For example, oil may be obtained by providing or cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. Even after such processing, the oil may retain a sterol profile characteristic of the source. Microalgal sterol profiles are disclosed below. See especially Section XII of this patent application. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, drilling fluids, as animal feed, for human nutrition, or for fertilizer.
[0075] Where a fatty acid profile of a triglyceride (also referred to as a "triacylglyceride" or "TAG") cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell. Examples 1, 2, and 8 below give analytical methods for determining TAG fatty acid composition and regiospecific structure.
[0076] Broadly categorized, certain embodiments of the invention include (i) auxotrophs of particular fatty acids; (ii) cells that produce oils having low concentrations of polyunsaturated fatty acids, including cells that are auxotrophic for unsaturated fatty acids; (iii) cells producing oils having high concentrations of particular fatty acids due to expression of one or more exogenous genes encoding enzymes that transfer fatty acids to glycerol or a glycerol ester; (iv) cells producing regiospecific oils, (v) genetic constructs or cells encoding a newly discovered gene encoding an LPAAT enzyme from Cuphea PSR23 (see Example 43), (vi) cells producing low levels of saturated fatty acids and/or high levels of palmitoleic acid, (vii) cells producing erucic acid, and (viii) other inventions related to producing cell oils with altered profiles. The embodiments also encompass the oils made by such cells, the residual biomass from such cells after oil extraction, oleochemicals, fuels and food products made from the oils and methods of cultivating the cells.
[0077] In any of the embodiments below, the cells used are optionally cells having a type II fatty acid biosynthetic pathway such as microalgal cells including heterotrophic or obligate heterotrophic microalgal cells, including cells classified as Chlorophyta, Trebouxiophyceae , Chlorellales, Chlorellaceae, or Chlorophyceae, or cells engineered to have a type II fatty acid biosynthetic pathway using the tools of synthetic biology (i.e., transplanting the genetic machinery for a type II fatty acid biosynthesis into an organism lacking such a pathway). Use of a host cell with a type II pathway avoids the potential for non-interaction between an exogenous acyl-ACP thioesterase or other ACP-binding enzyme and the multienzyme complex of type I cellular machinery. In specific embodiments, the cell is of the species Prototheca moriformis, Prototheca krugani, Prototheca stagnora or Prototheca zopfii or has a 23S rRNA sequence with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide identity SEQ ID NO: 76. By cultivating in the dark or using an obligate heterotroph, the cell oil produced can be low in chlorophyll or other colorants. For example, the cell oil can have less than 100, 50, 10, 5, 1, 0.0.5 ppm of chlorophyll without substantial purification.
[0078] The stable carbon isotope value 613C is an expression of the ratio of .sup.13C/.sup.12C relative to a standard (e.g. PDB, carbonite of fossil skeleton of Belemnite americana from Peedee formation of South Carolina). The stable carbon isotope value .delta.13C (.Salinity.) of the oils can be related to the 613C value of the feedstock used. In some embodiments the oils are derived from oleaginous organisms heterotrophically grown on sugar derived from a C4 plant such as corn or sugarcane. In some embodiments the 613C (.Salinity.) of the oil is from -10 to -17.Salinity. or from -13 to -16.Salinity..
[0079] In specific embodiments and examples discussed below, one or more fatty acid synthesis genes (e.g., encoding an acyl-ACP thioesterase, a keto-acyl ACP synthase, an LPAAT, a stearoyl ACP desaturase, or others described herein) is incorporated into a microalga. It has been found that for certain microalga, a plant fatty acid synthesis gene product is functional in the absence of the corresponding plant acyl carrier protein (ACP), even when the gene product is an enzyme, such as an acyl-ACP thioesterase, that requires binding of ACP to function. Thus, optionally, the microalgal cells can utilize such genes to make a desired oil without co-expression of the plant ACP gene.
[0080] For the various embodiments of recombinant cells comprising exogenous genes or combinations of genes, it is contemplated that substitution of those genes with genes having 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity can give similar results, as can substitution of genes encoding proteins having 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% amino acid sequence identity. Likewise, for novel regulatory elements, it is contemplated that substitution of those nucleic acids with nucleic acids having 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid can be efficacious. In the various embodiments, it will be understood that sequences that are not necessary for function (e.g. FLAG.RTM. tags or inserted restriction sites) can often be omitted in use or ignored in comparing genes, proteins and variants.
[0081] Although discovered using or exemplified with microalgae, the novel genes and gene combinations reported here can be used in higher plants using techniques that are well known in the art. For example, the use of exogenous lipid metabolism genes in higher plants is described in U.S. Pat. Nos. 6,028,247, 5,850,022, 5,639,790, 5,455,167, 5,512,482,and 5,298,421 disclose higher plants with exogenous acyl-ACP thioesterases. WO2009129582 and WO1995027791 disclose cloning of LPAAT in plants. FAD2 suppression in higher plants is taught in WO 2013112578, and WO 2008006171.
[0082] As described in Example 63, transcript profiling was used to discover promoters that modulate expression in response to low nitrogen conditions. The promoters are useful to selectively express various genes and to alter the fatty acid composition of microbial oils. In accordance with an embodiment, there are non-natural constructs comprising a heterologous promoter and a gene, wherein the promoter comprises at least 60, 65, 70, 75, 80, 85, 90, or 95% sequence identity to any of the promoters of Example 63 (e.g., SEQ ID NOs: 130-147) and the gene is differentially expressed under low vs. high nitrogen conditions. Optionally, the expression is less pH sensitive than for the AMT03 promoter. For example, the promoters can be placed in front of a FAD2 gene in a linoleic acid auxotroph to produce an oil with less than 5, 4, 3, 2, or 1% linoleic acid after culturing under high, then low nitrogen conditions.
III. FATTY ACID AUXOTROPHS/REDUCING FATTY ACID LEVELS TO GROWTH INHIBITORY CONDITIONS DURING AN OIL PRODUCTION PHASE
[0083] In an embodiment, the cell is genetically engineered so that all alleles of a lipid pathway gene are knocked out. Alternately, the amount or activity of the gene products of the alleles is knocked down so as to require supplementation with fatty acids. A first transformation construct can be generated bearing donor sequences homologous to one or more of the alleles of the gene. This first transformation construct may be introduced and selection methods followed to obtain an isolated strain characterized by one or more allelic disruptions. Alternatively, a first strain may be created that is engineered to express a selectable marker from an insertion into a first allele, thereby inactivating the first allele. This strain may be used as the host for still further genetic engineering to knockout or knockdown the remaining allele(s) of the lipid pathway gene (e.g., using a second selectable marker to disrupt a second allele). Complementation of the endogenous gene can be achieved through engineered expression of an additional transformation construct bearing the endogenous gene whose activity was originally ablated, or through the expression of a suitable heterologous gene. The expression of the complementing gene can either be regulated constitutively or through regulatable control, thereby allowing for tuning of expression to the desired level so as to permit growth or create an auxotrophic condition at will. In an embodiment, a population of the fatty acid auxotroph cells are used to screen or select for complementing genes; e.g., by transformation with particular gene candidates for exogenous fatty acid synthesis enzymes, or a nucleic acid library believed to contain such candidates.
[0084] Knockout of all alleles of the desired gene and complementation of the knocked-out gene need not be carried out sequentially. The disruption of an endogenous gene of interest and its complementation either by constitutive or inducible expression of a suitable complementing gene can be carried out in several ways. In one method, this can be achieved by co-transformation of suitable constructs, one disrupting the gene of interest and the second providing complementation at a suitable, alternative locus. In another method, ablation of the target gene can be effected through the direct replacement of the target gene by a suitable gene under control of an inducible promoter ("promoter hijacking"). In this way, expression of the targeted gene is now put under the control of a regulatable promoter. An additional approach is to replace the endogenous regulatory elements of a gene with an exogenous, inducible gene expression system. Under such a regime, the gene of interest can now be turned on or off depending upon the particular needs. A still further method is to create a first strain to express an exogenous gene capable of complementing the gene of interest, then to knockout out or knockdown all alleles of the gene of interest in this first strain. The approach of multiple allelic knockdown or knockout and complementation with exogenous genes may be used to alter the fatty acid profile, regiospecific profile, sn-2 profile, or the TAG profile of the engineered cell.
[0085] Where a regulatable promoter is used, the promoter can be pH-sensitive (e.g., amt03), nitrogen and pH sensitive (e.g., amt03), or nitrogen sensitive but pH-insensitive (e.g., newly discovered promoters of Example 63) or variants thereof comprising at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity to any of the aforementioned promoters. In connection with a promoter, pH-insensitive means that the promoter is less sensitive than the amt03 promoter when environmental conditions are shifter from pH 6.8 to 5.0 (e.g., at least 5, 10, 15, or 20% less relative change in activity upon the pH-shift as compared to an equivalent cell with amt03 as the promoter).
[0086] In a specific embodiment, the recombinant cell comprises nucleic acids operable to reduce the activity of an endogenous acyl-ACP thioesterase; for example a FatA or FatB acyl-ACP thioesterase having a preference for hydrolyzing fatty acyl-ACP chains of length C18 (e.g., stearate (C18:0) or oleate (C18:1), or C8:0-C16:0 fatty acids. The activity of an endogenous acyl-ACP thioesterase may be reduced by knockout or knockdown approaches. Knockdown may be achieved, for example, through the use of one or more RNA hairpin constructs, by promoter hijacking (substitution of a lower activity or inducible promoter for the native promoter of an endogenous gene), or by a gene knockout combined with introduction of a similar or identical gene under the control of an inducible promoter. Example 34 describes the engineering of a Prototheca strain in which two alleles of the endogenous fatty acyl-ACP thioesterase (FATA1) have been knocked out. The activity of the Prototheca moriformis FATA1 was complemented by the expression of an exogenous FatA or FatB acyl-ACP thioesterase. Example 36 details the use of RNA hairpin constructs to reduce the expression of FATA in Prototheca, which resulted in an altered fatty acid profile having less palmitic acid and more oleic acid.
[0087] Accordingly, oleaginous cells, including those of organisms with a type II fatty acid biosynthetic pathway can have knockouts or knockdowns of acyl-ACP thioesterase-encoding alleles to such a degree as to eliminate or severely limit viability of the cells in the absence of fatty acid supplementation or genetic complementations. These strains can be used to select for transformants expressing acyl-ACP-thioesterase transgenes. Alternately, or in addition, the strains can be used to completely transplant exogenous acyl-ACP-thioesterases to give dramatically different fatty acid profiles of cell oils produced by such cells. For example, FATA expression can be completely or nearly completely eliminated and replaced with FATB genes that produce mid-chain fatty acids. Alternately, an organism with an endogenous FatA gene having specificity for palmitic acid (C16) relative to stearic or oleic acid (C18) can be replaced with an exogenous FatA gene having a greater relative specificity for stearic acid (C18:0) or replaced with an exogenous FatA gene having a greater relative specificity for oleic acid (C18:1). In certain specific embodiments, these transformants with double knockouts of an endogenous acyl-ACP thioesterase produce cell oils with more than 50, 60, 70, 80, or 90% caprylic, capric, lauric, myristic, or palmitic acid, or total fatty acids of chain length less than 18 carbons. Such cells may require supplementation with longer chain fatty acids such as stearic or oleic acid or switching of environmental conditions between growth permissive and restrictive states in the case of an inducible promoter regulating a FatA gene.
[0088] In an embodiment the oleaginous cells are cultured (e.g., in a bioreactor). The cells are fully auxotrophic or partially auxotrophic (i.e., lethality or synthetic sickness) with respect to one or more types of fatty acid. The cells are cultured with supplementation of the fatty acid(s) so as to increase the cell number, then allowing the cells to accumulate oil (e.g. to at least 40% by dry cell weight). Alternatively, the cells comprise a regulatable fatty acid synthesis gene that can be switched in activity based on environmental conditions and the environmental conditions during a first, cell division, phase favor production of the fatty acid and the environmental conditions during a second, oil accumulation, phase disfavor production of the fatty acid. In the case of an inducible gene, the regulation of the inducible gene can be mediated, without limitation, via environmental pH (for example, by using the AMT3 promoter as described in the Examples).
[0089] As a result of applying either of these supplementation or regulation methods, a cell oil may be obtained from the cell that has low amounts of one or more fatty acids essential for optimal cell propagation. Specific examples of oils that can be obtained include those low in stearic, linoleic and/or linolenic acids.
[0090] These cells and methods are illustrated in connection with low polyunsaturated oils in the section immediately below and in Example 6 (fatty acid desaturase auxotroph) in connection with oils low in polyunsaturated fatty acids and in Example 34 (acyl-ACP thioesterase auxotroph).
[0091] Likewise, fatty acid auxotrophs can be made in other fatty acid synthesis genes including those encoding a SAD, FAD, KASIII, KASI, KASII, KCS, elongase, GPAT, LPAAT, DGAT or AGPAT or PAP. These auxotrophs can also be used to select for complement genes or to eliminate native expression of these genes in favor of desired exogenous genes in order to alter the fatty acid profile, regiospecific profile, or TAG profile of cell oils produced by oleaginous cells.
[0092] Accordingly, in an embodiment of the invention, there is a method for producing an oil/fat. The method comprises cultivating a recombinant oleaginous cell in a growth phase under a first set of conditions that is permissive to cell division so as to increase the number of cells due to the presence of a fatty acid, cultivating the cell in an oil production phase under a second set of conditions that is restrictive to cell division but permissive to production of an oil that is depleted in the fatty acid, and extracting the oil from the cell, wherein the cell has a mutation or exogenous nucleic acids operable to suppress the activity of a fatty acid synthesis enzyme, the enzyme optionally being a stearoyl-ACP desaturase, delta 12 fatty acid desaturase, or a ketoacyl-ACP synthase. The oil produced by the cell can be depleted in the fatty acid by at least 50, 60, 70, 80, or 90%. The cell can be cultivated heterotrophically. The cell can be a microalgal cell cultivated heterotrophically or autotrophically and may produce at least 40, 50, 60, 70, 80, or 90% oil by dry cell weight.
IV. (A) LOW POLYUNSATURATED CELL OILS
[0093] In an embodiment of the present invention, the cell oil produced by the cell has very low levels of polyunsaturated fatty acids. As a result, the cell oil can have improved stability, including oxidative stability. The cell oil can be a liquid or solid at room temperature, or a blend of liquid and solid oils, including the regiospecific or stereospecific oils, high stearate oils, or high mid-chain oils described infra. Oxidative stability can be measured by the Rancimat method using the AOC S Cd 12b-92 standard test at a defined temperature. For example, the OSI (oxidative stability index) test may be run at temperatures between 110.degree. C. and 140.degree. C. The oil is produced by cultivating cells (e.g., any of the plastidic microbial cells mentioned above or elsewhere herein) that are genetically engineered to reduce the activity of one or more fatty acid desaturase. For example, the cells may be genetically engineered to reduce the activity of one or more fatty acyl .DELTA.12 desaturase(s) responsible for converting oleic acid (18:1) into linoleic acid (18:2) and/or one or more fatty acyl 415 desaturase(s) responsible for converting linoleic acid (18:2) into linolenic acid (18:3). Various methods may be used to inhibit the desaturase including knockout or mutation of one or more alleles of the gene encoding the desaturase in the coding or regulatory regions, inhibition of RNA transcription, or translation of the enzyme, including RNAi, siRNA, miRNA, dsRNA, antisense, and hairpin RNA techniques. Other techniques known in the art can also be used including introducing an exogenous gene that produces an inhibitory protein or other substance that is specific for the desaturase. In specific examples, a knockout of one fatty acyl .DELTA.12 desaturase allele is combined with RNA-level inhibition of a second allele.
[0094] In a specific embodiment, fatty acid desaturase (e.g., .DELTA.12 fatty acid desaturase) activity in the cell is reduced to such a degree that the cell is unable to be cultivated or is difficult to cultivate (e.g., the cell division rate is decreased more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97 or 99%). Achieving such conditions may involve knockout, or effective suppression of the activity of multiple gene copies (e.g. 2, 3, 4 or more) of the desaturase or their gene products. A specific embodiment includes the cultivation in cell culture of a full or partial fatty acid auxotroph with supplementation of the fatty acid or a mixture of fatty acids so as to increase the cell number, then allowing the cells to accumulate oil (e.g. to at least 40% by cell weight). Alternatively, the cells comprise a regulatable fatty acid synthesis gene that can be switched in activity. For example, the regulation can be based on environmental conditions and the environmental conditions during a first, cell division, phase favor production of the fatty acid and the environmental conditions during a second, oil accumulation, phase disfavor production of the oil. For example, culture media pH and/or nitrogen levels can be used as an environmental control to switch expression of a lipid pathway gene to produce a state of high or low synthetic enzyme activity. Examples of such cells are described in Example 7.
[0095] In a specific embodiment, a cell is cultivated using a modulation of linoleic acid levels within the cell. In particular, the cell oil is produced by cultivating the cells under a first condition that is permissive to an increase in cell number due to the presence of linoleic acid and then cultivating the cells under a second condition that is characterized by linoleic acid starvation and thus is inhibitory to cell division, yet permissive of oil accumulation. For example, a seed culture of the cells may be produced in the presence of linoleic acid added to the culture medium. For example, the addition of linoleic acid to 0.25 g/L in the seed culture of a Prototheca strain deficient in linoleic acid production due to ablation of two alleles of a fatty acyl .DELTA.12 desaturase (i.e., a linoleic auxotroph) was sufficient to support cell division to a level comparable to that of wild type cells. Optionally, the linoleic acid can then be consumed by the cells, or otherwise removed or diluted. The cells are then switched into an oil producing phase (e.g., supplying sugar under nitrogen limiting conditions such as described in WO2010/063032). Surprisingly, oil production has been found to occur even in the absence of linoleic acid production or supplementation, as demonstrated in the obligate heterotroph oleaginous microalgae Prototheca but generally applicable to other oleaginous microalgae, microorganisms, or even multicellular organisms (e.g., cultured plant cells). Under these conditions, the oil content of the cell can increase to about 10, 20, 30, 40, 50, 60, 70, 80, 90%, or more by dry cell weight, while the oil produced can have polyunsaturated fatty acid (e.g.; linoleic+linolenic) profile with 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05% or less, as a percent of total triacylglycerol fatty acids in the oil. For example, the oil content of the cell can be 50% or more by dry cell weight and the triglyceride of the oil produced less than 3% polyunsaturated fatty acids.
[0096] These oils can also be produced without the need (or reduced need) to supplement the culture with linoleic acid by using cell machinery to produce the linoleic acid during the cell division phase, but less or no linoleic acid in the lipid production phase. The linoleic-producing cell machinery may be regulatable so as to produce substantially less linoleic acid during the oil producing phase. The regulation may be via modulation of transcription of the desaturase gene(s) or modulation or modulation of production of an inhibitor substance (e.g., regulated production of hairpin RNA/RNAi). For example, the majority, and preferably all, of the fatty acid .DELTA.12 desaturase activity can be placed under a regulatable promoter regulated to express the desaturase in the cell division phase, but to be reduced or turned off during the oil accumulation phase. The regulation can be linked to a cell culture condition such as pH, and/or nitrogen level, as described in the examples herein, or other environmental condition. In practice, the condition may be manipulated by adding or removing a substance (e.g., protons via addition of acid or base) or by allowing the cells to consume a substance (e.g., nitrogen-supplying nutrients) to effect the desired switch in regulation of the desaturase activity.
[0097] Other genetic or non-genetic methods for regulating the desaturase activity can also be used. For example, an inhibitor of the desaturase can be added to the culture medium in a manner that is effective to inhibit polyunsaturated fatty acids from being produced during the oil production phase.
[0098] Accordingly, in a specific embodiment of the invention, there is a method comprising providing a recombinant cell having a regulatable delta 12 fatty acid desaturase gene, under control of a recombinant regulatory element via an environmental condition. The cell is cultivated under conditions that favor cell multiplication. Upon reaching a given cell density, the cell media is altered to switch the cells to lipid production mode by nutrient limitation (e.g. reduction of available nitrogen). During the lipid production phase, the environmental condition is such that the activity of the delta 12 fatty acid desaturase is downregulated. The cells are then harvested and, optionally, the oil extracted. Due to the low level of delta 12 fatty acid desaturase during the lipid production phase, the oil has less polyunsaturated fatty acids and has improved oxidative stability. Optionally the cells are cultivated heterotrophically and optionally microalgal cells.
[0099] Using one or more of these desaturase regulation methods, it is possible to obtain a cell oil that it is believed has been previously unobtainable, especially in large scale cultivation in a bioreactor (e.g., more than 1000 L). The oil can have polyunsaturated fatty acid levels that are 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or less, as an area percent of total triacylglycerol fatty acids in the oil.
[0100] One consequence of having such low levels of polyunsaturates is that oils are exceptionally stable to oxidation. Indeed, in some cases the oils may be more stable than any previously known cell cell oil. In specific embodiments, the oil is stable, without added antioxidants, at 110.degree. C. so that the inflection point in conductance is not yet reached by 10 hours, 15 hours, 20 hours, 30 hours, 40, hours, 50 hours, 60 hours, or 70 hours under conditions of the AOCS Cd 12b-92. Rancimat test, noting that for very stable oils, replenishment of water may be required in such a test due to evaporation that occurs with such long testing periods (see Example 5). For example the oil can have and OSI value of 40-50 hours or 41-46 hours at 110.degree. C. without added antioxidants. When antioxidants (suitable for foods or otherwise) are added, the OSI value measured may be further increased. For example, with added tocopherol (100ppm) and ascorbyl palmitate (500 ppm) or PANA and ascorbyl palmitate, such an oil can have an oxidative stability index (OSI value) at 110.degree. C. in excess 100 or 200 hours, as measured by the Rancimat test. In another example, 1050 ppm of mixed tocopherols and 500 pm of ascorbyl palmitate are added to an oil comprising less than 1% linoleic acid or less than 1% linoleic+linolenic acids; as a result, the oil is stable at 110.degree. C. for 1, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, 12, 13, 14, 15, or 16, 20, 30, 40 or 50 days, 5 to 15 days, 6 to 14 days, 7 to 13 days, 8 to 12 days, 9 to 11 days, about 10 days, or about 20 days. The oil can also be stable at 130.degree. C. for 1, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, 12, 13, 14, 15, or 16, 20, 30, 40 or 50 days, 5 to 15 days, 6 to 14 days, 7 to 13 days, 8 to 12 days, 9 to 11 days, about 10 days, or about 20 days. In a specific example, such an oil was found to be stable for greater than 100 hours (about 128 hours as observed). In a further embodiment, the OSI value of the cell oil without added antioxidants at 120.degree. C. is greater than 15 hours or 20 hours or is in the range of 10-15, 15-20, 20-25, or 25-50 hours, or 50-100 hours.
[0101] In an example, using these methods, the oil content of a microalgal cell is between 40 and about 85% by dry cell weight and the polyunsaturated fatty acids in the fatty acid profile of the oil is between 0.001% and 3% in the fatty acid profile of the oil and optionally yields a cell oil having an OSI induction time of at least 20 hours at 110.degree. C. without the addition of antioxidants. In yet another example, there is a cell oil produced by RBD treatment of a cell oil from an oleaginous cell, the oil comprises between 0.001% and 2% polyunsaturated fatty acids and has an OSI induction time exceeding 30 hours at 110C without the addition of antioxidants. In yet another example, there is a cell oil produced by RBD treatment of a cell oil from an oleaginous cell, the oil comprises between 0.001% and 1% polyunsaturated fatty acids and has an OSI induction time exceeding 30 hours at 110C without the addition of antioxidants.
[0102] In another specific embodiment there is an oil with reduced polyunsaturate levels produced by the above-described methods. The oil is combined with antioxidants such as PANA and ascorbyl palmitate. For example, it was found that when such an oil was combined with 0.5% PANA and 500ppm of ascorbyl palmitate the oil had an OSI value of about 5 days at 130.degree. C. or 21 days at 110.degree. C. These remarkable results suggest that not only is the oil exceptionally stable, but these two antioxidants are exceptionally potent stabilizers of triglyceride oils and the combination of these antioxidants may have general applicability including in producing stable biodegradable lubricants (e.g., jet engine lubricants). In specific embodiments, the genetic manipulation of fatty acyl .DELTA.12 desaturase results in a 2 to 30, or 5 to 25, or 10 to 20 fold increase in OSI (e.g., at 110.degree. C.) relative to a strain without the manipulation. The oil can be produced by suppressing desaturase activity in a cell, including as described above.
[0103] Antioxidants suitable for use with the oils of the present invention include alpha, delta, and gamma tocopherol (vitamin E), tocotrienol, ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, (3-carotene, lycopene, lutein, retinol (vitamin A), ubiquinol (coenzyme Q), melatonin, resveratrol, flavonoids, rosemary extract, propyl gallate (PG), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), N,N'-di-2-butyl-1,4-phenylenediamine,2,6-di-tert-butyl-4-methylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, 2,6-di-tert-butylphenol, and phenyl-alpha-naphthylamine (PANA).
[0104] In addition to the desaturase modifications, in a related embodiment other genetic modifications may be made to further tailor the properties of the oil, as described throughout, including introduction or substitution of acyl-ACP thioesterases having altered chain length specificity and/or overexpression of an endogenous or exogenous gene encoding a KAS, SAD, LPAAT, or DGAT gene. For example, a strain that produces elevated oleic levels may also produce low levels of polyunsaturates. Such genetic modifications can include increasing the activity of stearoyl-ACP desaturase (SAD) by introducing an exogenous SAD gene, increasing elongase activity by introducing an exogenous KASII gene, and/or knocking down or knocking out a FATA gene.
[0105] In a specific embodiment, a high oleic cell oil with low polyunsaturates may be produced. For example, the oil may have a fatty acid profile with greater than 60, 70, 80, 90, or 95% oleic acid and less than 5, 4, 3, 2, or 1% polyunsaturates. In related embodiments, a cell oil is produced by a cell having recombinant nucleic acids operable to decrease fatty acid .DELTA.12 desaturase activity and optionally fatty acid 415 desaturase so as to produce an oil having less than or equal to 3% polyunsaturated fatty acids with greater than 60% oleic acid, less than 2% polyunsaturated fatty acids and greater than 70% oleic acid, less than 1% polyunsaturated fatty acids and greater than 80% oleic acid, or less than 0.5% polyunsaturated fatty acids and greater than 90% oleic acid. It has been found that one way to increase oleic acid is to use recombinant nucleic acids operable to decrease expression of a FATA acyl-ACP thioesterase and optionally overexpress a KAS II gene; such a cell can produce an oil with greater than or equal to 75% oleic acid. Alternately, overexpression of KASII can be used without the FATA knockout or knockdown. Oleic acid levels can be further increased by reduction of delta 12 fatty acid desaturase activity using the methods above, thereby decreasing the amount of oleic acid the is converted into the unsaturates linoleic acid and linolenic acid. Thus, the oil produced can have a fatty acid profile with at least 75% oleic and at most 3%, 2%, 1%, or 0.5% linoleic acid. In a related example, the oil has between 80 to 95% oleic acid and about 0.001 to 2% linoleic acid, 0.01 to 2% linoleic acid, or 0.1 to 2% linoleic acid. In another related embodiment, an oil is produced by cultivating an oleaginous cell (e.g., a microalga) so that the microbe produces a cell oil with less than 10% palmitic acid, greater than 85% oleic acid, 1% or less polyunsaturated fatty acids, and less than 7% saturated fatty acids. See Example 58 in which such an oil is produced in a microalga with FAD and FATA knockouts plus expression of an exogenous KASII gene. Such oils will have a low freezing point, with excellent stability and are useful in foods, for frying, fuels, or in chemical applications. Further, these oils may exhibit a reduced propensity to change color over time. In an illustrative chemical application, the high oleic oil is used to produce a chemical. The oleic acid double bonds of the oleic acid groups of the triglycerides in the oil can be epoxidized or hydroxylated to make a polyol. The epoxidized or hydroxylated oil can be used in a variety of applications. One such application is the production of polyurethane (including polyurethane foam) via condensation of the hydroxylated triglyceride with an isocyanate, as has been practiced with hydroxylated soybean oil or castor oil. See, e.g. US2005/0239915, US2009/0176904, US2005/0176839, US2009/0270520, and U.S. Pat. No. 4,264,743 and Zlatanic, et al, Biomacromolecules 2002, 3, 1048-1056 (2002) for examples of hydroxylation and polyurethane condensation chemistries. Suitable hydroxyl forming reactions include epoxidation of one or more double bonds of a fatty acid followed by acid catalyzed epoxide ring opening with water (to form a diol), alcohol (to form a hydroxyl ether), or an acid (to form a hydroxyl ester). There are multiple advantages of using the high-oleic/low polyunsaturated oil in producing a bio-based polyurethane: (1) the shelf-life, color or odor, of polyurethane foams may be improved; (2) the reproducibility of the product may be improved due to lack of unwanted side reactions resulting from polyunsaturates; (3) a greater degree of hydroxylation reaction may occur due to lack of polyunsaturates and the structural characteristics of the polyurethane product can be improved accordingly.
[0106] The low-polyunsaturated or high-oleic/low-polyunsaturated oils described here may be advantageously used in chemical applications where yellowing is undesirable. For example, yellowing can be undesirable in paints or coatings made from the triglycerides fatty acids derived from the triglycerides. Yellowing may be caused by reactions involving polyunsaturated fatty acids and tocotrienols and/or tocopherols. Thus, producing the high-stability oil in an oleaginous microbe with low levels of tocotrienols can be advantageous in elevating high color stability a chemical composition made using the oil. In contrast to commonly used plant oils, through appropriate choice of oleaginous microbe, the cell oils of these embodiments can have tocopherols and tocotrienols levels of 1 g/L or less. In a specific embodiment, a cell oil has a fatty acid profile with less than 2% with polyunsaturated fatty acids and less than 1 g/L for tocopherols, tocotrienols or the sum of tocopherols and tocotrienols. In another specific embodiment, the cell oil has a fatty acid profile with less than 1% with polyunsaturated fatty acids and less than 0.5 g/L for tocopherols, tocotrienols or the sum of tocopherols and tocotrienols
[0107] Any of the high-stability (low-polyunsaturate) cell oils or derivatives thereof can be used to formulate foods, drugs, vitamins, nutraceuticals, personal care or other products, and are especially useful for oxidatively sensitive products. For example, the high-stability cell oil (e.g., less than or equal to 3%, 2% or 1% polyunsaturates) can be used to formulate a sunscreen (e.g. a composition having one or more of avobenzone, homosalate, octisalate, octocrylene or oxybenzone) or retinoid face cream with an increased shelf life due to the absence of free-radical reactions associated with polyunsaturated fatty acids. For example, the shelf-life can be increased in terms of color, odor, organoleptic properties or %active compound remaining after accelerated degradation for 4 weeks at 54.degree. C. The high stability oil can also be used as a lubricant with excellent high-temperature stability. In addition to stability, the oils can be biodegradable, which is a rare combination of properties.
[0108] In another related embodiment, the fatty acid profile of a cell oil is elevated in C8 to C16 fatty acids through additional genetic modification, e.g. through overexpression of a short-chain to mid chain preferring acyl-ACP thioesterase or other modifications described here. A low polyunsaturated oil in accordance with these embodiments can be used for various industrial, food, or consumer products, including those requiring improved oxidative stability. In food applications, the oils may be used for frying with extended life at high temperature, or extended shelf life.
[0109] Where the oil is used for frying, the high stability of the oil may allow for frying without the addition of antioxidant and/or defoamers (e.g. silicone). As a result of omitting defoamers, fried foods may absorb less oil. Where used in fuel applications, either as a triglyceride or processed into biodiesel or renewable diesel (see, e.g., WO2008/151149 WO2010/063032, and WO2011/150410), the high stability can promote storage for long periods, or allow use at elevated temperatures. For example, the fuel made from the high stability oil can be stored for use in a backup generator for more than a year or more than 5 years. The frying oil can have a smoke point of greater than 200.degree. C., and free fatty acids of less than 0.1% (either as a cell oil or after refining).
[0110] The low polyunsaturated oils may be blended with food oils, including structuring fats such as those that form beta or beta prime crystals, including those produced as described below. These oils can also be blended with liquid oils. If mixed with an oil having linoleic acid, such as corn oil, the linoleic acid level of the blend may approximate that of high oleic plant oils such as high oleic sunflower oils (e.g., about 80% oleic and 8% linoleic).
[0111] Blends of the low polyunsaturated cell oil can be interesterified with other oils. For example, the oil can be chemically or enzymatically interesterified. In a specific embodiment, a low polyunsaturated oil according to an embodiment of the invention has at least 10% oleic acid in its fatty acid profile and less than 5% polyunsaturates and is enzymatically interesterified with a high saturate oil (e.g. hydrogenated soybean oil or other oil with high stearate levels) using an enzyme that is specific for sn-1 and sn-2 triacylglycerol positions. The result is an oil that includes a stearate-oleate-stearate (SOS). Methods for interesterification are known in the art; see for example, "Enzymes in Lipid Modification," Uwe T. Bornschuer, ed., Wiley_VCH, 2000, ISBN 3-527-30176-3.
[0112] High stability oils can be used as spray oils. For example, dried fruits such as raisins can be sprayed with a high stability oil having less than 5, 4, 3, 2, or 1% polyunsaturates. As a result, the spray nozzle used will become clogged less frequently due to polymerization or oxidation product buildup in the nozzle that might otherwise result from the presence of polyunsaturates.
[0113] In a further embodiment, an oil that is high is SOS, such as those described below can be improved in stability by knockdown or regulation of delta 12 fatty acid desaturase.
[0114] Optionally, where the FADc promoter is regulated, it can be regulated with a promoter that is operable at low pH (e.g., one for which the level of transcription of FADc is reduced by less than half upon switching from cultivation at pH 7.0 to cultivation at pH 5.0). The promoter can be sensitive to cultivation under low nitrogen conditions such that the promoter is active under nitrogen replete conditions and inactive under nitrogen starved conditions. For example, the promoter may cause a reduction in FADc transcript levels of 5, 10, 15-fold or more upon nitrogen starvation. Because the promoter is operable at pH 5.0, more optimal invertase activity can be obtained. For example, the cell can be cultivated in the presence of invertase at a pH of less than 6.5, 6.0 or 5.5. The cell may have a FADc knockout to increase the relative gene-dosage of the regulated FADc. Optionally, the invertase is produced by the cell (natively or due to an exogenous invertase gene). Because the promoter is less active under nitrogen starved conditions, fatty acid production can proceed during the lipid production phase that would not allow for optimal cell proliferation in the cell proliferation stage. In particular, a low linoleic oil may be produced. The cell can be cultivated to an oil content of at least 20% lipid by dry cell weight. The oil may have a fatty acid profile having less than 5, 4, 3, 2, 1, or 0.5, 0.2, or 0.1% linoleic acid. Example 62 describes the discovery of such promoters.
IV. (B) HIGH 18:2/LOW 18:3 OILS OBTAINED USING FAD GENE REPLACEMENT
[0115] Surprisingly, while researching the production of low polyunsaturate oils as described above, an oil with high polyunsaturates but having a unique fatty acid profile was discovered. The discovery of this oil is described in Example 59. Thus, it is possible to use an oleaginous plastidic cell (e.g., microalgal) culture to produce an oil with a fatty acid profile characterized by 10% or less linolenic acid (C18:3) and 20% or more linoleic acid (C18:2). Such oils can be produced in an oleaginous microalga or other oleaginous plastidic cell by overexpression of a (endogenous or exogenous) KASII and gene replacement of FADc (also referred to as FAD2) and, if necessary based on the host cell, replacing native acyl-ACP thioesterase activity. In Example 58-59, an endogenous KASII was overexpressed and an endogenous FADc gene was placed under control of a pH-inducible promoter, although constitutive expression would also work. Interestingly, the oils were much higher in linoleic acid when the FADc was overexpressed in a linoleic acid auxotroph (e.g., a FADc double knockout). It is believed that this is due to the presence of a previously unrecognized gene-level regulatory system in microalgae that must be disabled in order to efficiently accumulate linoleic acid. In addition, two copies of the endogenous acyl-ACP thioesterase were knocked out and replaced with an oleate-specific plant acyl-ACP thioesterase. Under permissive pH conditions, an oil with 10% or less linolenic acid (C18:3) and 20% or more linoleic acid (C18:2). The oil can be extracted and used for various uses included in foodstuffs or chemicals. If the host cell is a microalga, the oil can comprise microalgal sterols. As with other embodiments, the host cell can be a microalga transformed to express an exogenous invertase, thus enable conversion of sucrose into the oil under conditions of heterotrophic cultivation.
[0116] In a specific embodiment, a host cell comprises a FADc knockdown, knockout, or FADc with a down-regulatable promoter combined with an exogenous KASII gene that expresses a protein having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to the protein encoded by the Prototheca moriformis KASII gene disclose in Example 58, and optionally expresses an acyl-ACP thioesterase gene producing an oleate-specific acyl-ACP thioesterase enzyme. Optionally, the cell can be an a plant cell, a microbial cell, or a microalgal cell.
V. CELLS WITH EXOGENOUS ACYLTRANSFERASES
[0117] In various embodiments of the present invention, one or more genes encoding an acyltransferase (an enzyme responsible for the condensation of a fatty acid with glycerol or a glycerol derivative to form an acylglyceride) can be introduced into an oleaginous cell (e.g., a plastidic microalgal cell) so as to alter the fatty acid composition of a cell oil produced by the cell. The genes may encode one or more of a glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), also known as 1-acylglycerol-3-phosphate acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP), or diacylglycerol acyltransferase (DGAT) that transfers an acyl group to the sn-3 position of DAG, thereby producing a TAG.
[0118] Recombinant nucleic acids may be integrated into a plasmid or chromosome of the cell. Alternately, the gene encodes an enzyme of a lipid pathway that generates TAG precursor molecules through fatty acyl-CoA-independent routes separate from that above. Acyl-ACPs may be substrates for plastidial GPAT and LPAAT enzymes and/or mitochondrial GPAT and LPAAT enzymes. Among further enzymes capable of incorporating acyl groups (e.g., from membrane phospholipids) to produce TAGs is phospholipid diacylglycerol acyltransferase (PDAT). Still further acyltransferases, including lysophosphosphatidylcholine acyltransferase (LPCAT), lysophosphosphatidylserine acyltransferase (LPSAT), lysophosphosphatidylethanolamine acyltransferase (LPEAT), and lysophosphosphatidylinositol acyltransferase (LPIAT), are involved in phospholipid synthesis and remodeling that may impact triglyceride composition.
[0119] The exogenous gene can encode an acyltransferase enzyme having preferential specificity for transferring an acyl substrate comprising a specific number of carbon atoms and/or a specific degree of saturation is introduced into a oleaginous cell so as to produce an oil enriched in a given regiospecific triglyceride. For example, the coconut (Cocos nucifera) lysophosphatidic acid acyltransferase has been demonstrated to prefer C12:0-CoA substrates over other acyl-CoA substrates (Knutzon et al., Plant Physiology, Vol. 120, 1999, pp. 739-746), whereas the 1-acyl-sn-3-glycerol-3-phosphate acyltransferase of maturing safflower seeds shows preference for linoleoyl-CoA and oleoyl-CoA substrates over other acyl-CoA substrates, including stearoyl-CoA (Ichihara et al., European Journal of Biochemistry, Vol. 167, 1989, pp. 339-347). Furthermore, acyltransferase proteins may demonstrate preferential specificity for one or more short-chain, medium-chain, or long-chain acyl-CoA or acyl-ACP substrates, but the preference may only be encountered where a particular, e.g. medium-chain, acyl group is present in the sn-1 or sn-3 position of the lysophosphatidic acid donor substrate. As a result of the exogenous gene, a TAG oil can be produced by the cell in which a particular fatty acid is found at the sn-2 position in greater than 20, 30, 40, 50, 60, 70, 90, or 90% of the TAG molecules.
[0120] In some embodiments of the invention, the cell makes an oil rich in saturated-unsaturated-saturated (sat-unsat-sat) TAGs. Sat-unsat-sat TAGS include 1,3-dihexadecanoyl-2-(9Z-octadecenoyl)-glycerol (referred to as 1-palmitoyl-2-oleyl-glycero-3-palmitoyl), 1,3-dioctadecanoyl-2-(9Z-octadecenoyl)-glycerol (referred to as 1-stearoyl-2-oleyl-glycero-3-stearoyl), and 1-hexadecanoyl-2-(9Z-octadecenoyl)-3-octadecanoy-glycerol (referred to as 1-palmitoyl-2-oleyl-glycero-3-stearoyl). These molecules are more commonly referred to as POP, SOS, and POS, respectively, where `P` represents palmitic acid, `S` represents stearic acid, and `O` represents oleic acid. Further examples of saturated-unsaturated-saturated TAGs include MOM, LOL, MOL, COC and COL, where `M` represents myristic acid, `L` represents lauric acid, and `C` represents capric acid (C8:0). Trisaturates, triglycerides with three saturated fatty acyl groups, are commonly sought for use in food applications for their greater rate of crystallization than other types of triglycerides. Examples of trisaturates include PPM, PPP, LLL, SSS, CCC, PPS, PPL, PPM, LLP, and LLS. In addition, the regiospecific distribution of fatty acids in a TAG is an important determinant of the metabolic fate of dietary fat during digestion and absorption.
[0121] According to certain embodiments of the present invention, oleaginous cells are transformed with recombinant nucleic acids so as to produce cell oils that comprise an elevated amount of a specified regiospecific triglyceride, for example 1-acyl-2-oleyl-glycero-3-acyl, or 1-acyl-2-lauric-glycero-3-acyl where oleic or lauric acid respectively is at the sn-2 position, as a result of introduced recombinant nucleic acids. Alternately, caprylic, capric, myristic, or palmitic acid may be at the sn-2 position. The amount of the specified regiospecific triglyceride present in the cell oil may be increased by greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 100-500%, or greater than 500% than in the cell oil produced by the microorganism without the recombinant nucleic acids. As a result, the sn-2 profile of the cell triglyceride may have greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the particular fatty acid.
[0122] The identity of the acyl chains located at the distinct stereospecific or regiospecific positions in a glycerolipid can be evaluated through one or more analytical methods known in the art (see Luddy et al., J. Am. Oil Chem. Soc., 41, 693-696 (1964), Brockerhoff, J. Lipid Res., 6, 10-15 (1965), Angers and Aryl, J. Am. Oil Chem. Soc.,Vol. 76:4, (1999), Buchgraber et al., Eur. J. Lipid Sci. Technol., 106, 621-648 (2004)), or in accordance with Examples 1, 2, and 8 given below.
[0123] The positional distribution of fatty acids in a triglyceride molecule can be influenced by the substrate specificity of acyltransferases and by the concentration and type of available acyl moieties substrate pool. Nonlimiting examples of enzymes suitable for altering the regiospecificity of a triglyceride produced in a recombinant microorganism are listed in Tables 1-4. One of skill in the art may identify additional suitable proteins.
TABLE-US-00001 TABLE 1 Glycerol-3-phosphate acyltransferases and GenBank accession numbers. glycerol-3-phosphate Arabidopsis BAA00575 acyltransferase thaliana glycerol-3-phosphate Chlamydomonas EDP02129 acyltransferase reinhardtii glycerol-3-phosphate Chlamydomonas Q886Q7 acyltransferase reinhardtii acyl-(acyl-carrier-protein): Cucurbita BAB39688 glycerol-3-phosphate moschata acyltransferase glycerol-3-phosphate Elaeis AAF64066 acyltransferase guineensis glycerol-3-phosphate Garcina ABS86942 acyltransferase mangostana glycerol-3-phosphate Gossypium ADK23938 acyltransferase hirsutum glycerol-3-phosphate Jatropha ADV77219 acyltransferase curcas plastid glycerol-3- Jatropha ACR61638 phosphate acyltransferase curcas plastidial glycerol- Ricinus EEF43526 phosphate acyltransferase communis glycerol-3-phosphate Vica faba AAD05164 acyltransferase glycerol-3-phosphate Zea mays ACG45812 acyltransferase
[0124] Lysophosphatidic acid acyltransferases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 2.
TABLE-US-00002 TABLE 2 Lysophosphatidic acid acyltransferases and GenBank accession numbers. 1-acyl-sn-glycerol-3- Arabidopsis AEE85783 phosphate acyltransferase thaliana 1-acyl-sn-glycerol-3- Brassica ABQ42862 phosphate acyltransferase juncea 1-acyl-sn-glycerol-3- Brassica ABM92334 phosphate acyltransferase juncea 1-acyl-sn-glycerol-3- Brassica CAB09138 phosphate acyltransferase napus lysophosphatidic acid Chlamydomonas EDP02300 acyltransferase reinhardtii lysophosphatidic acid Limnanthes AAC49185 acyltransferase alba 1-acyl-sn-glycerol-3- Limnanthes CAA88620 phosphate acyltransferase douglasii (putative) acyl-CoA:sn-1-acylglycerol- Limnanthes ABD62751 3-phosphate acyltransferase douglasii 1-acylglycerol-3-phosphate Limnanthes CAA58239 O-acyltransferase douglasii 1-acyl-sn-glycerol-3- Ricinus EEF39377 phosphate acyltransferase communis
[0125] Diacylglycerol acyltransferases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 3.
TABLE-US-00003 TABLE 3 Diacylglycerol acyltransferases and GenBank accession numbers. diacylglycerol Arabidopsis CAB45373 acyltransferase thaliana diacylglycerol Brassica AAY40784 acyltransferase juncea putative diacylglycerol Elaeis AEQ94187 acyltransferase guineensis putative diacylglycerol Elaeis AEQ94186 acyltransferase guineensis acyl CoA:diacylglycerol Glycine AAT73629 acyltransferase max diacylglycerol Helianthus ABX61081 acyltransferase annus acyl-CoA:diacylglycerol Olea AAS01606 acyltransferase 1 europaea diacylglycerol Ricinus AAR11479 acyltransferase communis
[0126] Phospholipid diacylglycerol acyltransferases suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 4.
TABLE-US-00004 TABLE 4 Phospholipid diacylglycerol acyltransferases and GenBank accession numbers. phospholipid:diacylglycerol Arabidopsis AED91921 acyltransferase thaliana Putative phospholipid: Elaeis AEQ94116 diacylglycerol guineensis acyltransferase phospholipid:diacylglycerol Glycine XP_003541296 acyltransferase 1-like max phospholipid:diacylglycerol Jatropha AEZ56255 acyltransferase curcas phospholipid:diacylglycerol Ricinus ADK92410 acyltransferase communis phospholipid:diacylglycerol Ricinus AEW99982 acyltransferase communis
[0127] In an embodiment of the invention, known or novel LPAAT genes are transformed into the oleaginous cells so as to alter the fatty acid profile of triglycerides produced by those cells, most notably by altering the sn-2 profile of the triglycerides. For example, by virtue of expressing an exogenous active LPAAT in an oleaginous cell, the percent of unsaturated fatty acid at the sn-2 position is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90% or more. For example, a cell may produce triglycerides with 30% unsaturates (which may be primarily 18:1 and 18:2 and 18:3 fatty acids) at the sn-2 position. In this example, introduction of the LPAAT activity increases the unsaturates at the sn-2 position by 20% so that 36% of the triglycerides comprise unsaturates at the sn-2 position. Alternately, an exogenous LPAAT can be used to increase mid-chain fatty acids including saturated mid-chains such as C8:0, C10:0, C12:0, C14:0 or C16:0 moieties at the sn-2 position. As a result, mid-chain levels in the overall fatty acid profile may be increased. Examples 43 and 44 describe altering the sn-2 and fatty acid profiles in an oleaginous microbe. As can be seen from those examples, the choice of LPAAT gene is important in that different LPAATs can cause a shift in the sn-2 and fatty acid profiles toward different acyl group chain-lengths or saturation levels. For example, the LPAAT of Example 43 increases C10-C14 fatty acids and the LPAAT of Example 44 causes an increase in C16 and C18 fatty acids. As in these examples, introduction of an exogenous LPAAT can be combined with introduction of exogenous acyl-ACP thioesterase. Combining a mid-chain preferring LPAAT and a mid-chain preferring FatB was found to give an additive effect; the fatty acid profile was shifted more toward the mid-chain fatty acids when both an exogenous LPAAT and FatB gene was present than when only an exogenous FatB gene was present. In a specific embodiment, the oil produced by a cell comprising an exogenous mid-chain specific LPAAT and (optionally) an exogenous FatB acyl-ACP thioesterase gene can have a fatty acid profile with 40, 50, 60, 70, 80% or more of C8:0, C10:0, C12:0, C14:0, or C16:0 fatty acids (separately or in sum).
[0128] Specific embodiments of the invention are a nucleic acid construct, a cell comprising the nucleic acid construct, a method of cultivating the cell to produce a triglyceride, and the triglyceride oil produced where the nucleic acid construct has a promoter operably linked to a novel LPAAT coding sequence. The coding sequence can have an initiation codon upstream and a termination codon downstream followed by a 3 UTR sequence. In a specific embodiment, the LPAAT gene has LPAAT activity and a coding sequence have at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any of the cDNAs of SEQ ID NOs: 80 to 85 or a functional fragment thereof including equivalent sequences by virtue of degeneracy of the genetic code. Introns can be inserted into the sequence as well. Alternately, the LPAAT gene codes for the amino acid sequence of SEQ ID NOs 77-79 or functional fragments thereof, or a protein having at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity . In addition to microalgae and other oleaginous cells, plants expressing the novel LPAAT as transgenes are expressly included in the embodiments and can be produced using known genetic engineering techniques.
VI. CELLS WITH EXOGENOUS ELONGASES OR ELONGASE COMPLEX ENZYMES
[0129] In various embodiments of the present invention, one or more genes encoding elongases or components of the fatty acyl-CoA elongation complex can be introduced into an oleaginous cell (e.g., a plastidic microalgal cell) so as to alter the fatty acid composition of the cell or of a cell oil produced by the cell. The genes may encode a beta-ketoacyl-CoA synthase (also referred to as 3-ketoacyl synthase, beta-ketoacyl synthase or KCS), a ketoacyl-CoA reductase, a hydroxyacyl-CoA dehydratase, enoyl-CoA reductase, or elongase. The enzymes encoded by these genes are active in the elongation of acyl-coA molecules liberated by acyl-ACP thioesterases. Recombinant nucleic acids may be integrated into a plasmid or chromosome of the cell. In a specific embodiment, the cell is of Chlorophyta, including heterotrophic cells such as those of the genus Prototheca.
[0130] Beta-Ketoacyl-CoA synthase and elongase enzymes suitable for use with the microbes and methods of the invention include, without limitation, those listed in Table 5.
TABLE-US-00005 TABLE 5 Beta-Ketoacyl-CoA synthases and elongases listed with GenBank accession numbers. Trypanosoma brucei elongase 3 (GenBank Accession No. AAX70673), Marchanita polymorpha (GenBank Accession No. AAP74370), Trypanosoma cruzi fatty acid elongase, putative (GenBank Accession No. EFZ33366), Nannochloropsis oculata fatty acid elongase (GenBank Accession No. ACV21066.1), Leishmania donovani fatty acid elongase, putative (GenBank Accession No. CBZ32733.1), Glycine max 3-ketoacyl-CoA synthase 11-like (GenBank Accession No. XP_003524525.1), Medicago truncatula beta-ketoacyl-CoA synthase (GenBank Accession No. XP_003609222), Zea mays fatty acid elongase (GenBank Accession No. ACG36525), Gossypium hirsutum beta- ketoacyl-CoA synthase (GenBank Accession No. ABV60087), Helianthus annuus beta-ketoacyl-CoA synthase (GenBank Accession No. ACC60973.1), Saccharomyces cerevisiae ELO1 (GenBank Accession No. P39540), Simmondsia chinensis beta- ketoacyl-CoA synthase (GenBank Accession No. AAC49186), Tropaeolum majus putative fatty acid elongase (GenBank Accession No. AAL99199, Brassica napus fatty acid elongase (GenBank Accession No. AAA96054)
[0131] In an embodiment of the invention, an exogenous gene encoding a beta-ketoacyl-CoA synthase or elongase enzyme having preferential specificity for elongating an acyl substrate comprising a specific number of carbon atoms and/or a specific degree of acyl chain saturation is introduced into a oleaginous cell so as to produce a cell or an oil enriched in fatty acids of specified chain length and/or saturation. Example 40 describes engineering of Prototheca strains in which exogenous fatty acid elongases with preferences for extending midchain fatty acyl-CoAs have been overexpressed to increase the concentration of stearate. Examples 42 and 54 describe engineering of Prototheca in which exogenous elongases or beta-ketoacyl-CoA synthases with preferences for extending monounsaturated and saturated C18- and C20-CoA substrates are overexpressed to increase the concentration of erucic acid.
[0132] In specific embodiments, the oleaginous cell produces an oil comprising greater than 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60 70, or 80% erucic and/or eicosenoic acid. Alternately, the cell produces an oil comprising 0.5-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99% erucic or eicosenoic acid. The cell may comprise recombinant acids described above in connection with high-oleic oils with a further introduction of an exogenous beta-ketoacyl-CoA synthase that is active in elongating oleoyl-CoA. As a result of the expression of the exogenous beta-ketoacyl-CoA synthase, the natural production of erucic or eicosenoic acid by the cell can be increased by more than 2, 3, 4, 5, 10, 20, 30, 40, 50, 70, 100, 130, 170 or 200 fold. The high erucic and/or eicosenoic oil can also be a high stability oil; e.g., one comprising less than 5, 4, 3, 2, or 1% polyunsaturates and/or having the OSI values described in Section IV or this application and accompanying Examples. In a specific embodiment, the cell is a microalgal cell, optionally cultivated heterotrophically. As in the other embodiments, the oil/fat can be produced by genetic engineering of a plastidic cell, including heterotrophic microalgae of the phylum Chlorophyta, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Preferably, the cell is oleaginous and capable of accumulating at least 40% oil by dry cell weight. The cell can be an obligate heterotroph, such as a species of Prototheca, including Prototheca moriformis or Prototheca zopfii.
[0133] In specific embodiments, an oleaginous microbial cell, optionally an oleaginous microalgal cell, optionally of the phylum Chlorophyta, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae expresses an enzyme having 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to an enzyme of Table 5.
VII. REGIOSPECIFIC AND STEREOSPECIFIC OILS/FATS
[0134] In an embodiment, a recombinant cell produces a cell fat or oil having a given regiospecific makeup. As a result, the cell can produce triglyceride fats having a tendency to form crystals of a given polymorphic form; e.g., when heated to above melting temperature and then cooled to below melting temperature of the fat. For example, the fat may tend to form crystal polymorphs of the .beta. or .beta.' form (e.g., as determined by X-ray diffraction analysis), either with or without tempering. The fats may be ordered fats. In specific embodiments, the fat may directly from either .beta. or .beta.' crystals upon cooling; alternatively, the fat can proceed through a .beta. form to a .beta.' form. Such fats can be used as structuring, laminating or coating fats for food applications. The cell fats can be incorporated into candy, dark or white chocolate, chocolate flavored confections, ice cream, margarines or other spreads, cream fillings, pastries, or other food products. Optionally, the fats can be semi-solid (at room temperature) yet free of artificially produced trans-fatty acids. Such fats can also be useful in skin care and other consumer or industrial products.
[0135] As in the other embodiments, the fat can be produced by genetic engineering of a plastidic cell, including heterotrophic eukaryotic microalgae of the phylum Chlorophyta, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Preferably, the cell is oleaginous and capable of accumulating at least 40% oil by dry cell weight. The cell can be an obligate heterotroph, such as a species of Prototheca, including Prototheca moriformis or Prototheca zopfii. The fats can also be produced in autotrophic algae or plants. Optionally, the cell is capable of using sucrose to produce oil and a recombinant invertase gene may be introduced to allow metabolism of sucrose, as described in PCT Publications WO2008/151149, WO2010/06032, WO2011/150410, WO2011/150411, and international patent application PCT/US12/23696. The invertase may be codon optimized and integrated into a chromosome of the cell, as may all of the genes mentioned here. It has been found that cultivated recombinant microalgae can produce hardstock fats at temperatures below the melting point of the hardstock fat. For example, Prototheca moriformis can be altered to heterotrophically produce triglyceride oil with greater than 50% stearic acid at temperatures in the range of 15 to 30.degree. C., wherein the oil freezes when held at 30.degree. C.
[0136] In an embodiment, the cell fat has at least 30, 40, 50, 60, 70, 80, or 90% fat of the general structure [saturated fatty acid (sn-1)-unsaturated fatty acid (sn-2)-saturated fatty acid (sn-3)]. This is denoted below as Sat-Unsat-Sat fat. In a specific embodiment, the saturated fatty acid in this structure is preferably stearate or palmitate and the unsaturated fatty acid is preferably oleate. As a result, the fat can form primarily .beta. or .beta.' polymorphic crystals, or a mixture of these, and have corresponding physical properties, including those desirable for use in foods or personal care products. For example, the fat can melt at mouth temperature for a food product or skin temperature for a cream, lotion or other personal care product (e.g., a melting temperature of 30 to 40, or 32 to 35.degree. C.). Optionally, the fats can have a 2 L or 3L lamellar structure (e.g., as determined by X-ray diffraction analysis). Optionally, the fat can form this polymorphic form without tempering.
[0137] In a specific related embodiment, a cell fat triglyceride has a high concentration of SOS (i.e. triglyceride with stearate at the terminal sn-1 and sn-3 positions, with oleate at the sn-2 position of the glycerol backbone). For example, the fat can have triglycerides comprising at least 50, 60, 70, 80 or 90% SOS. In an embodiment, the fat has triglyceride of at least 80% SOS. Optionally, at least 50, 60, 70, 80 or 90% of the sn-2 linked fatty acids are unsaturated fatty acids. In a specific embodiment, at least 95% of the sn-2 linked fatty acids are unsaturated fatty acids. In addition, the SSS (tri-stearate) level can be less than 20, 10 or 5% and/or the C20:0 fatty acid (arachidic acid) level may be less than 6%, and optionally greater than 1% (e.g., from 1 to 5%). For example, in a specific embodiment, a cell fat produced by a recombinant cell has at least 70% SOS triglyceride with at least 80% sn-2 unsaturated fatty acyl moieties. In another specific embodiment, a cell fat produced by a recombinant cell has TAGs with at least 80% SOS triglyceride and with at least 95% sn-2 unsaturated fatty acyl moieties. In yet another specific embodiment, a cell fat produced by a recombinant cell has TAGs with at least 80% SOS, with at least 95% sn-2 unsaturated fatty acyl moieties, and between 1 to 6% C20 fatty acids.
[0138] In yet another specific embodiment, the sum of the percent stearate and palmitate in the fatty acid profile of the cell fat is twice the percentage of oleate, .+-.10, 20, 30 or 40% [e.g., (% P+% S)/%O=2.0.+-.20%]. Optionally, the sn-2 profile of this fat is at least 40%, and preferably at least 50, 60, 70, or 80% oleate (at the sn-2 position). Also optionally, this fat may be at least 40, 50, 60, 70, 80, or 90% SOS. Optionally, the fat comprises between 1 to 6% C20 fatty acids.
[0139] In any of these embodiments, the high SatUnsatSat fat may tend to form .beta.' polymorphic crystals. Unlike previously available plant fats like cocoa butter, the SatUnsatSat fat produced by the cell may form .beta.' polymorphic crystals without tempering. In an embodiment, the polymorph forms upon heating to above melting temperature and cooling to less that the melting temperature for 3, 2, 1, or 0.5 hours. In a related embodiment, the polymorph forms upon heating to above 60.degree. C. and cooling to 10.degree. C. for 3, 2, 1, or 0.5 hours.
[0140] In various embodiments the fat forms polymorphs of the .beta. form, .beta.' form, or both, when heated above melting temperature and the cooled to below melting temperature, and optionally proceeding to at least 50% of polymorphic equilibrium within 5, 4, 3, 2, 1, 0.5 hours or less when heated to above melting temperature and then cooled at 10.degree. C. The fat may form .beta.' crystals at a rate faster than that of cocoa butter.
[0141] Optionally, any of these fats can have less than 2 mole % diacylglycerol, or less than 2 mole % mono and diacylglycerols, in sum.
[0142] In an embodiment, the fat may have a melting temperature of between 30-60.degree. C., 30-40.degree. C., 32 to 37.degree. C., 40 to 60.degree. C. or 45 to 55.degree. C. In another embodiment, the fat can have a solid fat content (SFC) of 40 to 50%, 15 to 25%, or less than 15% at 20.degree. C. and/or have an SFC of less than 15% at 35.degree. C.
[0143] The cell used to make the fat may include recombinant nucleic acids operable to modify the saturate to unsaturate ratio of the fatty acids in the cell triglyceride in order to favor the formation of SatUnsatSat fat. For example, a knock-out or knock-down of stearoyl-ACP desaturase (SAD) gene can be used to favor the formation of stearate over oleate or expression of an exogenous mid-chain-preferring acyl-ACP thioesterase gene can increase the levels mid-chain saturates. Alternately a gene encoding a SAD enzyme can be overexpressed to increase unsaturates.
[0144] In a specific embodiment, the cell has recombinant nucleic acids operable to elevate the level of stearate in the cell. As a result, the concentration of SOS may be increased. Example 9 demonstrates that the regiospecific profile of the recombinant microbe is enriched for the SatUnsatSat triglycerides POP, POS, and SOS as a result of overexpressing a Brassica napus C18:0-preferring thioesterase. An additional way to increase the stearate of a cell is to decrease oleate levels. For cells having high oleate levels (e.g., in excess of one half the stearate levels) one can also employ recombinant nucleic acids or classical genetic mutations operable to decrease oleate levels. For example, the cell can have a knockout, knockdown, or mutation in one or more FATA alleles, which encode an oleate liberating acyl-ACP thioesterase, and/or one or more alleles encoding a stearoyl ACP desaturase (SAD). Example 35 describes the inhibition of SAD2 gene product expression using hairpin RNA to produce a fatty acid profile of 37% stearate in Prototheca moriformis (UTEX 1435), whereas the wildtype strain produced less than 4% stearate, a more than 9-fold improvement. Moreover, while the strains of Example 35 are engineered to reduce SAD activity, sufficient SAD activity remains to produce enough oleate to make SOS, POP, and POS. See the TAG profiles of Example 47. In specific examples, one of multiple SAD encoding alleles may be knocked out and/or one or more alleles are downregulated using inhibition techniques such as antisense, RNAi, or siRNA, hairpin RNA or a combination thereof. In various embodiments, the cell can produce TAGs that have 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90 to about 100% stearate. In other embodiments, the cells can produce TAGs that are 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90 to about 100% SOS. Optionally, or in addition to genetic modification, stearoyl ACP desaturase can be inhibited chemically; e.g., by addition of sterculic acid to the cell culture during oil production.
[0145] Surprisingly, knockout of a single FATA allele has been found to increase the presence of C18 fatty acids produced in microalgae. By knocking out one allele, or otherwise suppressing the activity of the FATA gene product (e.g., using hairpin RNA), while also suppressing the activity of stearoyl-ACP desaturase (using techniques disclosed herein), stearate levels in the cell can be increased.
[0146] Another genetic modification to increase stearate levels includes increasing a ketoacyl ACP synthase (KAS) activity in the cell so as to increase the rate of stearate production. It has been found that in microalgae, increasing KASII activity is effective in increasing C18 synthesis and particularly effective in elevating stearate levels in cell triglyceride in combination with recombinant DNA effective in decreasing SAD activity. Recombinant nucleic acids operable to increase KASII (e.g., an exogenous KasII gene) can be also be combined with a knockout or knockdown of a FatA gene, or with knockouts or knockdowns of both a FatA gene and a SAD gene). Optionally, the KASII gene encodes a protein having at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid identity to the KASII Prototheca moriformis (mature protein given in SEQ ID NO: 161), or any plant KASII gene disclosed herein (e.g., in Example 60) or known in the art including a microalgal KASII.
[0147] Optionally, the cell can include an exogenous stearate-liberating acyl-ACP thioesterase, either as a sole modification or in combination with one or more other stearate-increasing genetic modifications. For example the cell may be engineered to overexpress an acyl-ACP thioesterase with preference for cleaving C18:0-ACPs. Example 9 describes the expression of exogenous C18:0-preferring acyl-ACP thioesterases to increase stearate in the fatty acid profile of the microalgae, Prototheca moriformis (UTEX 1435), from about 3.7% to about 30.4% (over 8-fold). Example 41 provides additional examples of C18:0-preferring acyl-ACP thioesterases function to elevate C18:0 levels in Prototheca. Optionally, the stearate-preferring acyl-ACP thioesterase gene encodes an enzyme having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 9% amino acid identity to the gene products of Example 9 or 41 (Seq ID NOS. 28, 65, 67, 69, 71, 73, or 75 omitting FLAG tags when present). Introduction of the acyl-ACP-thioesterase can be combined with a knockout or knockdown of one or more endogenous acyl-ACP thioesterase alleles. Introduction of the thioesterase can also be combined with overexpression of an elongase (KCS) or beta-ketoacyl-CoA synthase. In addition, one or more exogenous genes (e.g., encoding SAD or KASII) can be regulated via an environmental condition (e.g., by placement in operable linkage with a regulatable promoter). In a specific example, pH and/or nitrogen level is used to regulate an amt03 promoter. The environmental condition may then be modulated to tune the cell to produce the desired amount of stearate appearing in cell triglycerides (e.g., to twice the oleate concentration). As a result of these manipulations, the cell may exhibit an increase in stearate of at least 5, 10, 15, or 20 fold.
[0148] As a further modification, alone or in combination with the other stearate increasing modifications, the cell can comprise recombinant nucleic acids operable to express an elongase or a beta-ketoacyl-CoA synthase. For example, overexpression of a C18:0-preferring acyl-ACP thioesterases may be combined with overexpression of a midchain-extending elongase or KCS to increase the production of stearate in the recombinant cell. One or more of the exogenous genes (e.g., encoding a thioesterase, elongase, or KCS) can be regulated via an environmental condition (e.g., by placement in operable linkage with a regulatable promoter). In a specific example, pH and/or nitrogen level is used to regulate an amt03 promoter or any of the promoters of example 63 including those that are less pH-sensitive than amt03. The environmental condition may then be modulated to tune the cell to produce the desired amount of stearate appearing in cell triglycerides (e.g., to twice the oleate concentration). As a result of these manipulations, the cell may exhibit an increase in stearate of at least 5, 10, 15, or 20 fold. In addition to stearate, arachidic, behenic, lignoceric, and cerotic acids may also be produced.
[0149] In specific embodiments, due to the genetic manipulations of the cell to increase stearate levels, the ratio of stearate to oleate in the oil produced by the cell is 2:1.+-.30% (i.e., in the range of 1.4:1 to 2.6:1), 2:1.+-.20% or 2:1.+-.10%.
[0150] Alternately, the cell can be engineered to favor formation of SatUnsatSat where Sat is palmitate or a mixture of palmitate and stearate. In this case introduction of an exogenous palmitate liberating acyl-ACP thioesterase can promote palmitate formation. In this embodiment, the cell can produce triglycerides, that are at least 30, 40, 50, 60, 70, or 80% POP, or triglycerides in which the sum of POP, SOS, and POS is at least 30, 40, 50, 60, 70, 80, or 90% of cell triglycerides. In other related embodiments, the POS level is at least 30, 40, 50, 60, 70, 80, or 90% of the triglycerides produced by the cell.
[0151] In a specific embodiment, the melting temperature of the oil is similar to that of cocoa butter (about 30-32.degree. C.). The POP, POS and SOS levels can approximate cocoa butter at about 16, 38, and 23% respectively. For example, POP can be 16%.+-.20%, POS can be 38%.+-.20%, and SOS can be 23%.+-.20%. Or, POP can be 16%.+-.15%, POS can be 38%.+-.15%, an SOS can be 23%.+-.15%. Or, POP can be 16%.+-.10%, POS can be 38%.+-.10%, an SOS can be 23%.+-.10%.
[0152] As a result of the recombinant nucleic acids that increase stearate, a proportion of the fatty acid profile may be arachidic acid. For example, the fatty acid profile can be 0.01% to 5%, 0.1 to 4%, or 1 to 3% arachidic acid. Furthermore, the regiospecific profile may have 0.01% to 4%, 0.05% to 3%, or 0.07% to 2% AOS, or may have 0.01% to 4%, 0.05% to 3%, or 0.07% to 2% AOA. It is believed that AOS and AOA may reduce blooming and fat migration in confection comprising the fats of the present invention, among other potential benefits.
[0153] In addition to the manipulations designed to increase stearate and/or palmitate, and to modify the SatUnsatSat levels, the levels of polyunsaturates may be suppressed, including as described above by reducing delta 12 fatty acid desaturase activity (e.g., as encoded by a Fad gene) and optionally supplementing the growth medium or regulating FAD expression. It has been discovered that, in microalgae (as evidenced by work in Prototheca strains), polyunsaturates are preferentially added to the sn-2 position. Thus, to elevate the percent of triglycerides with oleate at the sn-2 position, production of linoleic acid by the cell may be suppressed. The techniques described herein, in connection with highly oxidatively stable oils, for inhibiting or ablating fatty acid desaturase (FAD) genes or gene products may be applied with good effect toward producing SatUnsatSat oils by reducing polyunsaturates at the sn-2 position. As an added benefit, such oils can have improved oxidatively stability. As also described herein, the fats may be produced in two stages with polyunsaturates supplied or produced by the cell in the first stage with a deficit of polyunsaturates during the fat producing stage. The fat produced may have a fatty acid profile having less than or equal to 15,10,7, 5, 4, 3, 2, 1, or 0.5% polyunsaturates. In a specific embodiment, the oil/fat produced by the cell has greater than 50% SatUnsatSat, and optionally greater than 50% SOS, yet has less than 3% polyunsaturates. Optionally, polyunsaturates can be approximated by the sum of linoleic and linolenic acid area% in the fatty acid profile.
[0154] In an embodiment, the cell fat is a Shea stearin substitute having 65% to 95% SOS and optionally 0.001 to 5% SSS. In a related embodiment, the fat has 65% to 95% SOS, 0.001 to 5% SSS, and optionally 0.1 to 8% arachidic acid containing triglycerides. In another related embodiment, the fat has 65% to 95% SOS and the sum of SSS and SSO is less than 10% or less than 5%.
[0155] The cell's regiospecific preference can be learned using the analytical method described below (Examples 1-2, 8). Despite balancing the saturates and unsaturates as describe above, it is possible that the cell enzymes do not place the unsaturated fatty acid at the sn-2 position. In this case, genetic manipulations can confer the desired regiospecificity by (i) reducing the activity of endogenous sn-2 specific acyl transferases (e.g., LPAAT) and/or (ii) introducing an exogenous LPAAT with the desired specificity (i.e., introduction of oleate at sn-2). Where an exogenous LPAAT is introduced, preferably the gene encoding the LPAAT is integrated into a host chromosome and is targeted to the endoplasmic reticulum. In some cases, the host cell may have both specific and non-specific LPAAT alleles and suppressing the activity of one of these alleles (e.g., with a gene knockout) will confer the desired specificity. For example, genes encoding the LPAATs of SEQ ID NO: 78 and SEQ ID NO: 79 or an LPAAT comprising at least 90, 95, 98, or 99% amino acid identity to either of these sequences, or a functional fragment thereof, can be used to add oleate to the sn-2 position in order to boost the levels of SatUnsatSat TAGs. The genes can have at least 80, 85, 90, 95, 96, 97, 98, or 99% nucleotide identity to any of SEQ ID NOs: 80 to 85 or equivalent sequences by virtue of the degeneracy of the genetic code. Alternatively, the proteins encoded by the genes can have at least 80, 85, 90, 95, 96, 97, 98, or 99% nucleotide identity to the gene products of any of SEQ ID NOs: 80 to 85. These genes can be manifest as recombinant nucleic acid constructs, vectors, chromosomes or host cells comprising these sequences or functional fragments thereof, which can be found by systematic deletion of nucleic acid from the sequences using known techniques. As a result of expression of the genes, the amount of sat-unsat-sat TAGs such as SOS, POS, POP, or triglycerides with C8 to C16 fatty acids at the sn-2 position can be increased in a host cell.
[0156] Among other discoveries, the above discussion and Examples below highlight certain pathways to obtain high Sat-Unsat-Sat oils in general and SOS oils in particular in microorganisms or in plants. Thus, it is possible that the use of genetic engineering techniques, optionally combined with classical mutagenesis and breeding, a microalga or higher plant can be produced with an increase in the amount of SatUnsatSat or SOS produced of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more relative to the starting strain. In another aspect, the SatUnsatSat or SOS concentration of a species for which the wild-type produces less than 20%, 30%, 40% or 50% SatUnsatSat or SOS can be increased so that the SatUnsatSat or SOS is increased to at least 30%, 40%, 50% or 60%, respectively. The key changes, relative to the starting or wild-type organism, are to increase the amount of stearate (e.g., by reducing the amount of oleate formed from stearate, e.g., by reducing SAD activity, and/or increasing the amount of palmitate that is converted to stearate by reducing the activity of FATA and/or increasing the activity of KASII) and by decreasing the amount of linoleate by reducing FAD2/FADc activity.
[0157] Optionally, the starting organism can have triacylglycerol (TAG) biosynthetic machineries which are predisposed toward the synthesis of TAG species in which oleate or unsaturated fatty acids, predominate at the sn-2 position. Many oilseed crops have this characteristic. It has been demonstrated that lysophosphatidic acyltransferases (LPAATs) play a critical role in determining the species of fatty acids which will ultimately be inserted at the sn-2 position. Indeed, manipulation, through heterologous gene expression, of LPAATs in higher plant seeds, can alter the species of fatty acid occupying the sn-2 position.
[0158] One approach to generating oils with significant levels of so-called structuring fats (typically comprised of the species SOS-stearate-oleate-stearate, POS-palmitate-oleate-stearate, or POP-palmitate-oleate-palmitate) in agriculturally important oilseeds and in algae, is through the manipulation of endogenous as well as heterologous LPAAT expression. Expression of LPAATs from seeds containing high levels of structuring fats, for example, would be one strategy to increase the level of structuring fats in an oil seed or oleaginous algae that normally contains only limited quantities of such fats.
[0159] An alternative or supplementary strategy, however, is to take advantage of the innate propensity of LPAATs in agriculturally important oilseeds (eg, safflower-Carthamus sp., sunflower-Helianthus sp., canola-Brassica sp., peanut-Arachis sp., soybean-Glycine sp., corn-Zea sp., olive-Olea sp., flax-Linum sp., palm-Elaeis sp. and cotton-Gossypium sp., see representative profiles in Table 5a below) and through either genetic engineering alone or a combination of genetic engineering and classical strain improvement (i.e. mutagenesis) selectively manipulate the species of fatty acids present in order to increase the levels of structuring fats. In the case of SOS, these manipulations are comprised of a series of discrete steps, which can be carried out independently. These include:
[0160] Increasing the level of stearate. This can be achieved, as we have demonstrated in microalgae here and others have shown in higher plants, through the expression of stearate specific FATA activities or down regulation of the endogenous SAD activity; e.g., through direct gene knockout, RNA silencing, or mutation, including classical strain improvement. Simply elevating stearate levels alone, by the above approaches,however, will not be optimal. For example, in the case of palm oil, the already high levels of palmitate, coupled with increased stearate levels, will likely overwhelm the existing LPAAT activity, leading to significant amounts of stearate and palmitate incorporation into tri-saturated fatty acids (SSS, PPP, SSP, PPS etc.). Hence, steps must be taken to control palmitate levels as well.
[0161] Palmitate levels must be minimized in order to create high SOS containing fats because palmitate, even with a high-functioning LPAAT, will occupy sn-1 or sn-3 positions that could be taken up by stearate, and, as outlined above, too many saturates will result in significant levels of tri-saturated TAG species. Palmitate levels can be lowered. for example, through down-regulation of endogenous FATA activity through mutation/classical strain improvement, gene knockouts or RNAi-mediated strategies, in instances wherein the endogenous FATA activity has significant palmitate activity. Alternatively, or in concert with the above, palmitate levels can be lowered through over expression of endogenous KASII activity or classical strain improvement efforts which manifest in the same effect, such that elongation from palmitate to stearate is enhanced. Simply lowering palmitate levels via the above methods may not be sufficient, however. Take again the example of palm oil. Reduction of palmitate and elevation of stearate via the previous methods would still leave significant levels of linoleic acid. The endogenous LPAAT activity in most higher plants species while they will preferentially insert oleate in the sn-2 position, will insert linoleic as the next most preferred species. As oleate levels decrease, linoleic will come to occupy the sn-2 position with increased frequency. TAG species with linoleic at the sn-2 position have poor structuring properties as the TAGs will tend to display much higher melting temperatures than what is desired in a structuring fat. Hence, increases in stearate and reductions in palmitate must in turn be balanced by reductions in levels of linoleic fatty acids.
[0162] In turn, levels of linoleic fatty acids must be minimized in order to create high SOS-containing fats because linoleate, even with a high functioning LPAAT will occupy sn-2 positions to the exclusion of oleate, creating liquid oils as opposed to the desired solid fat (at room temperature). Linoleate levels can be lowered, as we have demonstrated in microalgae and others have shown in plant oilseeds, through down regulation of endogenous FAD2 desaturases; e.g., through mutation/classical strain improvement, FAD2 knockouts or RNAi mediated down regulation of endogenous FAD2 activity. Accordingly, the linoleic acid level in the fatty acid profile can be reduced by at least 10, 20, 30, 40, 50, 100, 200, or 300%. For example, an RNAi construct with at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to those disclosed herein can be used to downregulate FAD2.
[0163] Although one can choose a starting strain with such an sn-2 preference one can also introduce an exogenous LPAAT gene having a greater oleate preference, to further boost oleate at the sn-2 position and to further boost Sat-Unsat-Sat in the TAG profile. Optionally, one can replace one or more endogenous LPAAT alleles with the exogenous, more specific LPAAT.
[0164] The cell oils resulting from the SatUnsatSat/SOS producing organisms can be distinguished from conventional sources of SOS/POP/POS in that the sterol profile will be indicative of the host organism as distinguishable from the conventional source. Conventional sources of SOS/POP/POS include cocoa, shea, mango, sal, illipe, kokum, and allanblackia. See section XII of this disclosure for a discussion of microalgal sterols.
TABLE-US-00006 TABLE 5a The fatty acid profiles of some commercial oilseed strains. Common Food Oils* C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 Corn oil <1.0 8.0-19.0 <0.5 0.5-4.0 19-50 38-65 <2.0 (Zea mays) Cottonseed oil <0.1 0.5-2.0 17-29 <1.5 1.0-4.0 13-44 40-63 0.1-2.1 (Gossypium barbadense) Canola <0.1 <0.2 <6.0 <1.0 <2.5 >50 <40 <14 (Brassica rapa, B. napus, B. juncea) Olive <0.1 6.5-20.0 .ltoreq.3.5 0.5-5.0 56-85 3.5-20.0 .ltoreq.1.2 (Olea europea) Peanut <0.1 <0.2 7.0-16.0 <1.0 1.3-6.5 35-72 13.0-43 <0.6 (Arachis hypogaea) Palm 0.5-5.9 32.0-47.0 2.0-8.0 34-44 7.2-12.0 (Elaeis guineensis) Safflower <0.1 <1.0 2.0-10.0 <0.5 1.0-10.0 7.0-16.0 72-81 <1.5 (Carthamus tinctorus) Sunflower <0.1 <0.5 3.0-10.0 <1.0 1.0-10.0 14-65 20-75 <0.5 (Helianthus annus) Soybean <0.1 <0.5 7.0-12.0 <0.5 2.0-5.5 19-30 48-65 5.0-10.0 (Glycine max) Solin-Flax <0.1 <0.5 2.0-9.0 <0.5 2.0-5.0 8.0-60 40-80 <5.0 (Linum usitatissimum) *Unless otherwise indicated, data taken from the U.S. Pharacopeia's Food and Chemicals Codex, 7th Ed. 2010-2011**
[0165] Accordingly, in an embodiment of the present invention, there is a method for increasing the amount of SOS in an oil (i.e. oil or fat) produced by a cell. The method comprises providing a cell and using classical and/or genetic engineering techniques (e.g., mutation, selection, strain-improvement, introduction of an exogenous gene and/or regulator element, or RNA-level modulation such as RNAi) to (i) increase the stearate in the oil, (ii) decrease the linoleate in the oil, and optionally (iii) increase the stereospecificity of the addition of oleate in the sn-2 position. The step of increasing the stearate can comprise decreasing desaturation by SAD (e.g., knockout, knockdown or use of regulatory elements) and increasing the conversion of palmitate to stearate (including overexpression of an endogenous or exogenous KASII and/or knockout or knockdown of FATA). Optionally, an exogenous FATA with greater stearate specificity then an endogenous FATA is expressed in the cell to increase stearate levels. Here, stearate-specificity of a FATA gene is a measure of the gene product's rate of cleavage of stearate over palmitate. The stearate-specific FATA gene insertion can be combined with a knockdown or knockout of the less-specific endogenous FATA gene. In this way, the ratio of stearate to palmitate can be increased, by 10%, 20%, 30%, 40%, 50%, 100% or more. The step of decreasing the linoleate can be via reduction of FADc/FAD2 activity including knockout and/or knockdown . The step of increasing the oleate at the sn-2 position can comprise expressing an exogenous oleate-preferring LPAAT such as an LPAAT having at least 75, 80, 85, 90, 85, 96, 97, 98, or 99% amino acid identity to an LPAAT disclosed herein.
[0166] In a specific embodiment, the cell (e.g, an oleaginous microalgal or other plastidic cell) produces an oil enriched in SOS (e.g., at least 50% SOS and in some cases 60% SOS). The cell is modified in at least four genes: (i) a .beta.-ketoacyl-ACP synthase II (KASII) is overexpressed, (ii) activity of an endogenous FATA acyl-ACP thioesterase is reduced (iii) a stearate-specific FATA acyl-ACP thioesterase is overexpressed, (iii) endogenous SAD activity is decreased, and (iv) endogenous FAD activity is decreased. Example 65 demonstrates this embodiment in a Prototheca moriformis microalga by disrupting the coding region of endogenous FATA and SAD2 through homologous recombination, overexpressing a .beta.-ketoacyl-ACP synthase II (KASII) gene, and activating FAD2 RNAi to decrease polyunsaturates.
[0167] In another specific embodiment, the cell (e.g, an oleaginous microalgal or other plastidic cell) produces an oil enriched in SOS (e.g., at least 50% SOS and in some cases 60% SOS). The cell is modified in at least four genes: (i) a .beta.-ketoacyl-ACP synthase II (KASII) is overexpressed, (ii) activity of an endogenous FATA acyl-ACP thioesterase is reduced (iii) a stearate-specific FATA acyl-ACP thioesterase is overexpressed, (iv) endogenous SAD activity is decreased, (v) endogenous FAD activity is decreased and (vi) an exogenous oleate-preferring LPAAT is expressed. See Examples 65 and 66. Optionally, these genes or regulatory elements have at least 75, 80, 85, 90, 85, 96, 97, 98, or 99% nucleic acid or amino acid identity to a gene or gene-product or regulatory element disclosed herein. Optionally, one or more of these genes is under control of a pH-sensitive or nitrogen-sensitive (pH-sensitive or pH-insensitive) promoter such as one having at least 75, 80, 85, 90, 85, 96, 97, 98, or 99% nucleic acid identity to one of those disclosed herein. Optionally, the cell oil is fractionated (see Example 64).
[0168] In an embodiment, fats produced by cells according to the invention are used to produce a confection, candy coating, or other food product. As a result, a food product like a chocolate or candy bar may have the "snap" (e.g., when broken) of a similar product produced using cocoa butter. The fat used may be in a beta polymorphic form or tend to a beta polymorphic form. In an embodiment, a method includes adding such a fat to a confection. Optionally, the fat can be a cocoa butter equivalent per EEC regulations, having greater than 65% SOS, less than 45% unsaturated fatty acid, less than 5% polyunsaturated fatty acids, less than 1% lauric acid, and less than 2% trans fatty acid. The fats can also be used as cocoa butter extenders, improvers, replacers, or anti-blooming agents, or as Shea butter replacers, including in food and personal care products. High SOS fats produced using the cells and methods disclosed here can be used in any application or formulation that calls for Shea butter or Shea fraction. However, unlike Shea butter, fats produced by the embodiments of the invention can have low amounts of unsaponifiables; e.g. less than 7, 5, 3, or 2% unsaponifiables. In addition, Shea butter tends to degrade quickly due to the presence of diacylglycerides whereas fats produced by the embodiments of the invention can have low amounts of diacylglycerides; e.g., less than 5, 4, 3, 2, 1, or 0.5% diacylglycerides.
[0169] In an embodiment of the invention there is a cell fat suitable as a shortening, and in particular, as a roll-in shortening. Thus, the shortening may be used to make pastries or other multi-laminate foods. The shortening can be produced using methods disclosed herein for producing engineered organisms and especially heterotrophic microalgae. In an embodiment, the shortening has a melting temperature of between 40 to 60.degree. C. and preferably between 45-55.degree. C. and can have a triglyceride profile with 15 to 20% medium chain fatty acids (C8 to C14), 45-50% long chain saturated fatty acids (C16 and higher), and 30-35% unsaturated fatty acids (preferably with more oleic than linoleic). The shortening may form .beta.' polymorphic crystals, optionally without passing through the .beta. polymorphic form. The shortening may be thixotropic. The shortening may have a solid fat content of less than 15% at 35.degree. C. In a specific embodiment, there is a cell oil suitable as a roll-in shortening produced by a recombinant microalga, where the oil has a yield stress between 400 and 700 or 500 and 600 Pa and a storage modulus of greater than 1.times.10.sup.5 Pa or 1.times.10.sup.6 Pa. (see Example 46)
[0170] A structured solid-liquid fat system can be produced using the structuring oils by blending them with an oil that is a liquid at room temperature (e.g., an oil high in tristearin or triolein). The blended system may be suitable for use in a food spread, mayonnaise, dressing, shortening; i.e. by forming an oil-water-oil emulsion. The structuring fats according to the embodiments described here, and especially those high in SOS, can be blended with other oils/fats to make a cocoa butter equivalent, replacer, or extender. For example, a cell fat having greater than 65% SOS can be blended with palm mid-fraction to make a cocoa butter equivalent.
[0171] In general, such high Sat-Unsat-Sat fats or fat systems can be used in a variety of other products including whipped toppings, margarines, spreads, salad dressings, baked goods (e.g. breads, cookies, crackers muffins, and pastries), cheeses, cream cheese, mayonnaise, etc.
[0172] In a specific embodiment, a Sat-Unsat-Sat fat described above is used to produce a margarine, spread, or the like. For example, a margarine can be made from the fat using any of the recipes or methods found in U.S. Pat. Nos. 7,118,773, 6,171,636, 4,447,462, 5,690,985, 5,888,575, 5,972,412, 6,171,636, or international patent publications WO9108677A1.
[0173] In an embodiment, a fat comprises a cell (e.g., from microalgal cells) fat optionally blended with another fat and is useful for producing a spread or margarine or other food product is produced by the genetically engineered cell and has glycerides derived from fatty acids which comprises:
[0174] (a) at least 10 weight % of C18 to C24 saturated fatty acids,
[0175] (b) which comprise stearic and/or arachidic and/or behenic and/or lignoceric acid and
[0176] (c) oleic and/or linoleic acid, while
[0177] (d) the ratio of saturated C18 acid/saturated (C20+C22+C24)-acids .gtoreq.1, preferably .gtoreq.5, more preferably .gtoreq.10,
[0178] which glycerides contain:
[0179] (e) .ltoreq.5 weight % of linolenic acid calculated on total fatty acid weight
[0180] (f) .ltoreq.5 weight % of trans fatty acids calculated on total fatty acid weight
[0181] (g) .ltoreq.75 weight %, preferably .ltoreq.60 weight % of oleic acid at the sn-2 position: which glycerides contain calculated on total glycerides weight
[0182] (h) .ltoreq.8 weight % HOH+HHO triglycerides
[0183] (i) .ltoreq.5 weight % of trisaturated triglycerides, and optionally one or more of the following properties:
[0184] (j) a solid fat content of .gtoreq.10% at 10.degree. C.
[0185] (k) a solid fat content .ltoreq.15% at 35.degree. C.,
[0186] (l) a solid fat content of .gtoreq.15% at 10.degree. C. and a solid fat content .ltoreq.25% at 35.degree. C.,
[0187] (m) the ratio of (HOH+HHO) and (HLH+HHL) triglycerides is >1, and preferably >2,
[0188] where H stands for C18-C24 saturated fatty acid, 0 for oleic acid, and L for linoleic acid.
[0189] Optionally, the solid content of the fat (% SFC) is 11 to 30 at 10.degree. C., 4 to 15 at 20.degree. C., 0.5 to 8 at 30.degree. C., and 0 to 4 at 35.degree. C. Alternately, the % SFC of the fat is 20 to 45 at 10.degree. C., 14 to 25 at 20.degree. C., 2 to 12 at 30.degree. C., and 0 to 5 at 35.degree. C. In related embodiment, the % SFC of the fat is 30 to 60 at 10.degree. C., 20 to 55 at 20.degree. C., 5 to 35 at 30.degree. C., and 0 to 15 at 35.degree. C. The C12-C16 fatty acid content can be <15 weight %. The fat can have <5 weight % disaturated diglycerides.
[0190] In related embodiments there is a spread, margarine or other food product made with the cell oil or cell oil blend. For example, the cell fat can be used to make an edible W/O (water/oil) emulsion spread comprising 70-20 wt. % of an aqueous phase dispersed in 30-80 wt. % of a fat phase which fat phase is a mixture of 50-99 wt. % of a vegetable triglyceride oil A and 1-50 wt. % of a structuring triglyceride fat B, which fat consists of 5-100 wt. % of a hardstock fat C and up to 95 wt. % of a fat D, where at least 45 wt. % of the hardstock fat C triglycerides consist of SatOSat triglycerides and where Sat denotes a fatty acid residue with a saturated C18-C24 carbon chain and O denotes an oleic acid residue and with the proviso that any hardstock fat C which has been obtained by fractionation, hydrogenation, esterification or interesterification of the fat is excluded. The hardstock fat can be a cell fat produced by a cell according to the methods disclosed herein. Accordingly, the hardstock fat can be a fat having a regiospecific profile having at least 50, 60, 70, 80, or 90% SOS. The W/O emulsion can be prepared to methods known in the art including in U.S. Pat. No. 7,118,773.
[0191] In related embodiment, the cell also expresses an endogenous hydrolyase enzyme that produces ricinoleic acid. As a result, the oil (e.g., a liquid oil or structured fat) produced may be more easily emulsified into a margarine, spread, or other food product or non-food product. For example, the oil produced may be emulsified using no added emulsifiers or using lower amounts of such emulsifiers. The U.S. patent application Ser. No. 13/365,253 discloses methods for expressing such hydroxylases in microalgae and other cells. In specific embodiments, a cell oil comprises at least 1, 2, or 5% SRS, where S is stearate and R is ricinoleic acid.
[0192] In an alternate embodiment, a cell oil that is a cocoa butter mimetic as described above (or other high sat-unsat-sat oil such as a Shea or Kolum mimetic) can be fractionated to remove trisaturates (e.g., tristearin and tripalmitin, SSP, and PPS). For example, it has been found that microalgae engineered to decrease SAD activity to increase SOS concentration make an oil that can be fractionated to remove trisaturated. See Example 47 and example 64. In specific embodiments, the melting temperature of the fractionated cell oil is similar to that of cocoa butter (about 30-32.degree. C.). The POP, POS and SOS levels can approximate cocoa butter at about 16, 38, and 23% respectively. For example, POP can be 16%.+-.20%, POS can be 38%.+-.20%, an SOS can be 23%.+-.20%. Or, POP can be 16%.+-.15%, POS can be 38%.+-.15%, an SOS can be 23%.+-.15%. Or, POP can be 16%.+-.10%, POS can be 38%.+-.10%, an SOS can be 23%.+-.10%. In addition, the tristearin levels can be less than 5% of the triacylglycerides.
[0193] In an embodiment, a method comprises obtaining a cell oil obtained from a genetically engineered (e.g., microalga or other microbe) cell that produces a starting oil with a TAG profile having at least 40, 50, or 60% SOS. Optionally, the cell comprises one or more of an overexpressed KASII gene, a SAD knockout or knockdown, or an exogenous C18-preferring FATA gene, an exogenous LPAAT, and a FAD2 knockout or knockdown. The oil is fractionated by dry fractionation or solvent fractionation to give an enriched oil (stearin fraction) that is increased in SOS and decreased in trisaturates relative to the starting oil. The enriched oil can have at least 60%, 70% or 80% SOS with no more than 5%, 4%, 3%, 2% or 1% trisaturates. The enriched oil can have a sn-2 profile having 85, 90, 95% or more oleate at the sn-2 position. For example, thefractionated oil can comprise at least 60% SOS, no more than 5% trisaturates and at least 85% oleate at the sn-2 position. Alternatively, the oil can comprise at least 70% SOS, no more than 4% trisaturates and at least 90% oleate at the sn-2 position or 80% SOS, no more than 4% trisaturates and at least 95% oleate at the sn-2 position. Optionally, the oil has essentially identical maximum heat-flow temperatures and/or the DSC-derived SFC curves to Kokum butter. The stearin fraction can be obtained by dry fractionation, solvent fractionation, or a combination of these. Optionally, the process includes a 2-step dry fractionation at a first temperature and a second temperature. The first termperature can be higher or lower than the second temperature. In a specific embodiment, the first temperature is effective at removing 00S and the second temperature is effective in removing trisaturates. Optionally, the stearin fraction is washed with a solvent (e.g. acetone) to remove the 00S after treatment at the first temperature. Optionally, the first temperature is about 24.degree. C. and the second temperature is about 29.degree. C.
VIII. HIGH MID-CHAIN OILS
[0194] In an embodiment of the present invention, the cell has recombinant nucleic acids operable to elevate the level of midchain fatty acids (e.g., C8:0, C10:0, C12:0, C14:0, or C16:0 fatty acids) in the cell or in the oil of the cell. One way to increase the levels of midchain fatty acids in the cell or in the oil of the cell is to engineer a cell to express an exogenous acyl-ACP thioesterase that has activity towards midchain fatty acyl-ACP substrates (e.g., one encoded by a FatB gene), either as a sole modification or in combination with one or more other genetic modifications. An additional genetic modification to increase the level of midchain fatty acids in the cell or oil of the cell is the expression of an exogenous lysophosphatidic acid acyltransferase gene encoding an active lysophosphatidic acid acyltransferase (LPAAT) that catalyzes the transfer of a mid-chain fatty-acyl group to the sn-2 position of a substituted acylglyceroester. For example, the LPAAT gene can have 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence identity or have 75, 80, 85 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleic acid sequence identity (or equivalent sequence to degeneracy of the genetic code) to the mid-chain preferring LPAATs disclosed in Examples 43-44 (SEQ ID NOs 77, 78, 79, 81,82, 84, and 85). In a specific related embodiment, both an exogenous acyl-ACP thioesterase and LPAAT are stably expressed in the cell. In an embodiment, recombinant nucleic acids are introduced into an oleaginous cell (and especially into a plastidic microbial cell) that cause expression of an exogenous mid-chain-specific thioesterase and an exogenous LPAAT that catalyzes the transfer of a mid-chain fatty-acyl group to the sn-2 position of a substituted acylglyceroester. As a result, the cell can be made to increase the percent of a midchain fatty acid in the TAGs that it produces by 10, 20 30, 40, 50, 60, 70, 80, 90-fold, or more. Introduction of the exogenous LPAAT can increase midchain fatty acids at the sn-2 position by 1.2, 1.5, 1.7, 2, 3, 4 fold or more compared to introducing an exogenous mid-chain preferring acyl-ACP thioesterase alone. In an embodiment, the mid-chain fatty acid is greater than 30, 40, 50 60, 70, 80, or 90% of the TAG fatty acids produced by the cell. In various embodiments, the mid-chain fatty acid is lauric, myristic, or palmitic. Examples 3, 43, and 44 describe expression of plant LPAATs in microalgal cells with resulting alterations in fatty acid profiles. As in the examples, the cells can also express an exogenous acyl-ACP thioesterase (which can also be from a plant) with a preference for a given fatty acyl-ACP chain length. For example, a microalgal cell can comprise exogenous genes encoding a LPAAT and an acyl-ACP thioesterase that preferentially cleave C8, C10, C12, C14, C8-C12, or C8-C10 fatty acids. In a specific embodiment, such a cell is capable of producing a cell oil with a fatty acid profile comprising 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99%, >20%, >30%, >40%, >50%, >60%, >70%, >80% or >90% C8, C10, C12, C14, C8-C12, or C8-C10 fatty acids. Other LPAATs can preferentially cleave C16 or C18 fatty acids (see Example 44). Further genetic manipulation of the fatty acid desaturase pathway (e.g., as described infra) can increase the stability of the oils.
[0195] Any of these cell oils can be interesterified. Interesterification can, for example, be used to lower the melting temperature or pour-point of the oil. In a specific embodiment, the cell oil comprises at least 50% of the sum of caprylic and capric acids and may be interesterified to reduce the pour point and/or kinematic viscosity. Such an oil (cell or interesterified) can optionally be a high stability oil comprising, for example, less than 2% polyunsaturated fatty acids.
[0196] Alternately, or in addition to expression of an exogenous LPAAT, the cell may comprise recombinant nucleic acids that are operable to express an exogenous KASI or KASIV enzyme and optionally to decrease or eliminate the activity of a KASII, which is particularly advantageous when a mid-chain-preferring acyl-ACP thioesterase is expressed. Example 37 describes the engineering of Prototheca cells to overexpress KASI or KASIV enzymes in conjunction with a mid-chain preferring acyl-ACP thioesterase to generate strains in which production of C10-C12 fatty acids is about 59% of total fatty acids. Mid-chain production can also be increased by suppressing the activity of KASI and/or KASII (e.g., using a knockout or knockdown). Example 38 details the chromosomal knockout of different alleles of Prototheca moriformis (UTEX 1435) KASI in conjunction with overexpression of a mid-chain preferring acyl-ACP thioesterase to achieve fatty acid profiles that are about 76% or 84% C10-C14 fatty acids. Example 39 provides recombinant cells and oils characterized by elevated midchain fatty acids as a result of expression of KASI RNA hairpin polynucleotides. In addition to any of these modifications, unsaturated or polyunsaturated fatty acid production can be suppressed (e.g., by knockout or knockdown) of a SAD or FAD enzyme.
[0197] In a particular embodiment, a recombinant cell produces TAG having 40% lauric acid or more. In another related embodiment, a recombinant cell produces TAG having a fatty acid profile of 40% or more of myristic, caprylic, capric, or palmitic acid. For example, an oleaginous recombinant clorophyte cell can produce 40% lauric or myristic acid in an oil that makes up 40, 50, or 60% or more of the cell's dry weight.
[0198] In a specific embodiment, a recombinant cell comprises nucleic acids operable to express a product of an exogenous gene encoding a lysophosphatidic acid acyltransferase that catalyzes the transfer of a mid-chain fatty-acyl group to the sn-2 position of a substituted acylglyceroester and nucleic acids operable to express a product of an acyl-ACP thioesterase exogenous gene encoding an active acyl-ACP thioesterase that catalyzes the cleavage of mid-chain fatty acids from ACP. As a result, in one embodiment, the oil produced can be characterized by a fatty acid profile elevated in C10 and C12 fatty acids and reduced in C16, C18, and C18:1 fatty acids as a result of the recombinant nucleic acids. See Example 3, in which overexpression of a Cuphea wrightii acyl-ACP thioesterase and a Cocos nucifera LPAAT gene increased the percentage of C12 fatty acids from about 0.04% in the untransformed cells to about 46% and increased the percentage of C10 fatty acids from about 0.01% in the untransformed cells to about 11%. For example, the FATB gene can have 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence identity or have 75, 80, 85 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleic acid sequence identity (or equivalent sequence to degeneracy of the genetic code) to SEQ ID NOs 10 or 11. In related embodiments, the increase in midchain fatty acid production is greater than 70%, from 75-85%, from 70-90%, from 90-200%, from 200-300%, from 300-400%, from 400-500%, or greater than 500%.
[0199] Average chain length can also be reduced by overexpression of a C18-specific acyl-ACP thioesterase. Recombinant nucleic acids operable to overexpress a C18 or other acyl-ACP thioesterase may be used alone or in combination with the other constructs described here to further reduce average chain length. Among other uses, the oils produced can be used as cocoa-butter/milk fat substitute. See Example 45 and the discussion of FIG. 17. In an embodiment, one of the above described high mid-chain producing cells is further engineered to produce a low polyunsaturated oil by knocking out or knocking down one or more fatty acyl desaturases, as described above in section IV. Accordingly, the oil produced can have the high stability characteristic mentioned in that section or in corresponding Examples. In a specific embodiment, the cell produces an oil comprising greater than 30% midchain fatty acids and 5% or less polyunsaturates. In a related embodiment, the cell produces an oil comprising greater than 40% midchain fatty acids and 4% or less polyunsaturates. In a further related embodiment, the cell produces an oil comprising greater than 50% midchain fatty acids and 3% or less polyunsaturates.
[0200] In a specific embodiment, the cell produces an oil characterized by a fatty acid profile in which the sum of lauric and myristic acids is at least 50%, 60% , 70%, or 75%. This can be accomplished using the techniques of Examples 37-39, 43-44, 52, and 60-61. For example, Example 52 describes a method for producing an oil that has a fatty acid profile in which the sum of lauric and myristic acids is about 79% using a recombinant cell with an exogenous plant FATB acyl-ACP thioesterase.
[0201] In another specific embodiment, the cell produces a cell oil characterized by a fatty acid profile in which capric acid (C10:0) is at least 30% and lauric acid (C12:0) is at least 30%. For example, the absolute level of capric acid and lauric acid in the cell oil can be balanced to within 5, 10, 15, 20 or 30%. This can be accomplished using the techniques of Examples 37-39, 43-44, 52, and 60-61. As in Example 60, exogenous plant FATB and KASI (or KASIV) genes can be combined to give balanced levels of capric and lauric. Optionally, an endogenous KASI gene can be knocked out and replaced with an exogenous KASI. In addition, two or more exogenous FATB genes can be used do reach a desired fatty acid profile. In a specific embodiment, a microalgal cell expresses at least one and optionally at least two exogenous FATB genes and an exogenous KASI/KASIV gene and produces an extractable cell oil with at least 30% C10 and at least 30% C12 fatty acids. For example, the cell can express a FATB acyl-ACP thioesterase having at least 70, 75, 80, 85, 90 or 95% amino acid sequence identity to the Cuphea hookeriana FATB2 (SEQ ID NO: 158) and a beta-ketoacyl ACP synthase having at least 70, 75, 80, 85, 90 or 95% amino acid sequence identity to the Cuphea wrightii KASA1 (SEQ ID NO: 159, with alternate transit peptide). Further, a second exogenous FATB gene/enzyme can be expressed. The second FATB can have at least 70, 75, 80, 85, 90 or 95% amino acid sequence identity to the Cuphea wrightii FATB2 acyl-ACP thioesterase (SEQ ID NO: 11.) For these purposes, plastid targeted peptides can be aligned with or with out the plastid targeting transit peptides, which are less conserved and more easily replaceable than the remaining enzyme domain sequence.
[0202] In an embodiment, the cell produces an oil comprising greater than 75% saturated fatty acids. Optionally, the cell produces an oil comprising greater than 75% saturated fatty acids with less than 25% capric acid, less than 50% lauric acid, and less than 5% palmitic acid. In related embodiments, the oil comprises at least 80%, 95% or 90% saturated fatty acids. Example 60 describes the production of such oil by microalgae comprising multiple exogenous FATB genes and replacement of an endogenous KASI gene with exogenous KASI or KASIV genes from plants.
[0203] Examples 60 and 62 also shows that selection of FATB and KAS genes can give rise to an oil with at least 50% total saturates with capric and lauric acids balanced to within 20% (or even to within 15%, or 10%).
[0204] High-mid chain oils in general, and those produced by strains similar to those of Example 60 and 62 can possess low kinematic viscosity. For example, the oil can have a kinematic viscosity as measured using ASTM D445 at 40.degree. C. of 25 cS.+-.20%, 25 cS.+-.10%, or 25 cS.+-.5%. Likewise, the oil can have a kinematic viscosity according to ASTM D445 at 100.degree. C. of 5.4 cS.+-.20%, 5.4 cS.+-.10%, or 5.4 cS.+-.5%. The oil can have a viscosity index as measured using ASTM 2280 of 160.+-.20%, 160.+-.10%, or 160.+-.5%.
[0205] In a specific example, an oil prepared using a strain similar to those reported in Example 60, produced an oil with greater than 30% C10:0 and greater than 30% C12:0 fatty acids. The oil had a kinematic viscocity by ASTM 445 of 24.61 cSt at 40.degree. C. and 5.36 cSt at 100.degree. C. with a viscosity index (ASTM 2270) of 159. To make this oil, a Cuphea hookeriana FATB2 acyl-ACP thioesterase was expressed with a Cuphea wrightii KASA1 gene (with a P. moriformis SAD transit peptide) in Prototheca moriformis under control of the UAPA1 and AMT03 promoters, respectively. Neomycin resitance was used at the selection marker and the construct with incorporated in the KAS1-1 site. Accordingly, in an embodiment, a host cell comprises an exogenous gene that expresses a protein having at least 70, 75, 80, 85, 90, or 95% amino acid sequence identity to SEQ ID NO: 158 and also expresses a protein having at least 70, 75, 80, 85, 90, or 95% amino acid sequence identity to SEQ ID NO: 159. The cell produces an oil comprising at least 30% C10:0 and/or at least 30% C12:0 fatty acids. Optionally, a cell oil can be extracted from the cell that has a kinematic viscosity as measured using ASTM D445 at 40.degree. C. of less than 30 cSt.
[0206] The high mid-chain oils or fatty acids derived from hydrolysis of these oils may be particularly useful in food, fuel and oleochemical applications including the production of lubricants and surfactants. For example, fatty acids derived from the cells can be esterified, cracked, reduced to an aldehyde or alcohol, aminated, sulfated, sulfonated, or subjected to other chemical process known in the art.
[0207] In some embodiments, the cell oil is interesterified and the kinematic viscosity of the interesterified cell oil is less than 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 centiStokes at 40.degree. C. In some embodiments, the kinematic viscosity is less than 3 centiStokes at 40.degree. C. In some embodiments, the pour point of an interesterified cell oil is less than, 5.degree. C., 0.degree. C., -10.degree. C., -12.degree. C., -15.degree. C., -20.degree. C., -25.degree. C., -30.degree. C., -35.degree. C., -40.degree. C., -45.degree. C., or -50.degree. C. In some embodiments, the pour point is less than -10.degree. C. In some embodiments, the pour point is less than -20.degree. C.
[0208] Example 53 describes the use of a plant FatB gene in algae to produce oils in microalgae with greater than 60% myristate. In an embodiment, a gene encoding a protein having at least 90, 95, 96, 97, 98, or 99% amino acid identity to SEQ ID NO:87 or SEQ ID NO:89 is used, optionally in combination with a mid-chain preferred LPAAT as described above.
[0209] As described in Example 62, we surprisingly discovered that the combination of a KASI gene with a FATB gene can shift the fatty acid profile of an oil produced by the cell in ways that neither gene can do on its own. Specifically, recombinant cells with exogenous plant myristate-preferring acyl-ACP thioesterases were discovered to shift their fatty acid profile to a greater percentage of laurate when a KASI gene was co-expressed. This is unexpected because KASI has an elongase activity yet the fatty acid profile was shifted to shorter chains. In other words, a cell expressing both the exogenous FATB and KASI gene produced an oil having a fatty acid profile that is shifted toward shorter fatty acid chains than a control cell with the FATB gene but without the KASI gene. Accordingly, an embodiment of the invention comprises constructing a recombinant cell or using the cell to make an oil, where the cell comprises an exogenous FATB with a given chain-length preference and a KASI gene, wherein the cell makes an oil with a shift in distribution toward shorter chains than is obtained without the KASI gene. Optionally, the FATB gene has a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical (or an equivalent sequence by virtue of degeneracy of the genetic code) or has an amino acid sequence that is least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the CcFATB2-UcFATB2 FATB of Example 62 (SEQ ID NO: 162), the Cuphea wrightii FATB2 (SEQ ID NO: 11), Cuphea palustris FATB2 (SEQ ID NO: 87; SEQ ID NO: 89), Cuphea hyssopifolia FATB1 (SEQ ID NO: 163), Cuphea hyssopifolia FATB3 (SEQ ID NO: 164), or Cuphea hookeriana FATB2 (SEQ ID NO: 158). Optionally, the KASI or KASIV gene has a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical (or an equivalent sequence by virtue of degeneracy of the genetic code) or has an amino acid sequence that is least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the Cuphea wrightii KASAI of Example 62 (SEQ ID NO: 159), the Cuphea hookeriana KASIV encoded by the sequence of SEQ ID NO:49, or the Cuphea pulch. KASIV encoded by SEQ ID NO: 48.
IX. HIGH OLEIC/PALMITIC OIL
[0210] In another embodiment, there is a high oleic oil with about 60% oleic acid, 25% palmitic acid and optionally 5% polyunsaturates or less. The high oleic oil can be produced using the methods disclosed in U.S. patent application Ser. No. 13/365,253, which is incorporated by reference in relevant part. For example, the cell can have nucleic acids operable to suppress an acyl-ACP thioesterase (e.g., knockout or knockdown of a gene encoding FATA) while also expressing a gene that increases KASII activity. The cell can have further modifications to inhibit expression of delta 12 fatty acid desaturase, including regulation of gene expression as described above. As a result, the polyunsaturates can be less than or equal to 5, 4, 3, 2, or 1 area%.
X. LOW SATURATE OIL
[0211] In an embodiment, a cell oil is produced from a recombinant cell. The oil produced has a fatty acid profile that has less that 4%, 3%, 2%, or 1% (area %), saturated fatty acids. In a specific embodiment, the oil has 0.1 to 3.5% saturated fatty acids. Certain of such oils can be used to produce a food with negligible amounts of saturated fatty acids. Optionally, these oils can have fatty acid profiles comprising at least 90% oleic acid or at least 90% oleic acid with at least 3% polyunsaturated fatty acids. In an embodiment, a cell oil produced by a recombinant cell comprises at least 90% oleic acid, at least 3% of the sum of linoleic and linolenic acid and has less than 3.5% saturated fatty acids. In a related embodiment, a cell oil produced by a recombinant cell comprises at least 90% oleic acid, at least 3% of the sum of linoleic and linolenic acid and has less than 3.5% saturated fatty acids, the majority of the saturated fatty acids being comprised of chain length 10 to 16. These oils may be produced by recombinant oleaginous cells including but not limited to those described here and in U.S. patent application Ser. No. 13/365,253. For example, overexpression of a KASII enzyme in a cell with a highly active SAD can produce a high oleic oil with less than or equal to 3.5% saturates. Optionally, an oleate-specific acyl-ACP thioesterase is also overexpressed and/or an endogenous thioesterase having a propensity to hydrolyze acyl chains of less than C18 knocked out or suppressed. The oleate-specific acyl-ACP thioesterase may be a transgene with low activity toward ACP-palmitate and ACP-stearate so that the ratio of oleic acid relative to the sum of palmitic acid and stearic acid in the fatty acid profile of the oil produced is greater than 3, 5, 7, or 10. Alternately, or in addition, a FATA gene may be knocked out or knocked down, as in Example 36 below. A FATA gene may be knocked out or knocked down and an exogenous KASII overexpressed. Another optional modification is to increase KASI and/or KASIII activity, which can further suppress the formation of shorter chain saturates. Optionally, one or more acyltransferases (e.g., an LPAAT) having specificity for transferring unsaturated fatty acyl moieties to a substituted glycerol is also overexpressed and/or an endogenous acyltransferase is knocked out or attenuated. An additional optional modification is to increase the activity of KCS enzymes having specificity for elongating unsaturated fatty acids and/or an endogenous KCS having specificity for elongating saturated fatty acids is knocked out or attenuated. Optionally, oleate is increased at the expense of linoleate production by knockout or knockdown of a delta 12 fatty acid desaturase; e.g., using the techniques of Section IV of this patent application. Optionally, the exogenous genes used can be plant genes; e.g., obtained from cDNA derived from mRNA found in oil seeds.
[0212] As described in Example 51, levels of saturated fats may also be reduced by introduction of an exogenous gene (e.g., a plant gene) that desaturates palmitic acid to palmitoleic acid. Examples of suitable genes for use in the oleaginous cells are found in the plants, including Macfadyena unguis (Cat's claw), Macadamia integrifolia (Macadamia nut) and Hippophae rhamnoides (sea buckthorn). Variant exogenous or endogenous SADs that desaturate palmitoyl-ACP can also be used and are further discussed in Example 51. Optionally, the PAD or SAD gene has at least 95% amino acid sequence identity to the gene product described in Example 51. This modification can be used alone, or in combination with oleate-increasing modifications such as those described immediately above, in section IX and in the Examples, including knockout or knockdown of one or more endogenous FATA alleles and/or overexpression of KASII. In one embodiment, an oleaginous cell such as an oleaginous microalgae has a combination of (i) a FATA knockout or knockdown with (ii) expression of an exogenous PAD gene (this could also be a variant SAD with PAD activity such as a L118W mutant or equivalent, see Examples 55-56) and/or a mutation in an endogenous SAD gene to give PAD activity. Such as cell may further comprise an overexpressed endogenous or exogenous KASII gene. In accordance with any of these embodiments of the invention, the oleaginous cell produces an oil having a fatty acid profile with 1-2, 2-3, 3-4, 5-6, 7-8, 9-10, 10-15, 15-20, 20-30, 30-40, 40-60, 60-70, 70-80, 80-90, or 90-100 area percent palmitoleic acid. In a specific embodiment, the cell produces greater than 50% oleic acid, greater than 1% palmitoleic acid, and 3.5 area% or less of saturated fatty acids. In another specific embodiment, a eukaryotic microalgal cell comprises an exogenous gene that desaturates palmitic acid to palmitoleic acid in operable linkage with regulatory elements operable in the microalgal cell. Due to expression and activity of the exogenous gene product, the cell produces a cell oil having a fatty acid profile in which the ratio of palmitoleic acid (C16:1) to palmitic acid (C16:0) is at least 0.05, 0.1 or 0.15, or 0.18. See Example 55 for examples of cells that produce such oils. Optionally, palmitoleic acid comprises 0.5% or more of the profile. Optionally, the cell oil comprises less than 3.5% saturated fatty acids.
[0213] In addition to the above genetic modifications, the low saturate oil can be a high-stability oil by virtue of low amounts of polyunsaturated fatty acids. Methods and characterizations of high-stability, low-polyunsaturated oils are described in the section above entitled Low Polyunsaturated Oils, including method to reduce the activity of endogenous .DELTA.12 fatty acid desaturase. In a specific embodiment, an oil is produced by a oleaginous microbial cell having a type II fatty acid synthetic pathway and has no more than 3.5% saturated fatty acids and also has no more than 3% polyunsaturated fatty acids. In another specific embodiment, the oil has no more than 3% saturated fatty acids and also has no more than 2% polyunsaturated fatty acids. In another specific embodiment, the oil has no more than 3% saturated fatty acids and also has no more than 1% polyunsaturated fatty acids. In another specific embodiment, a eukaryotic microalgal cell comprises an exogenous gene that desaturates palmitic acid to palmitoleic acid in operable linkage with regulatory elements operable in the microalgal cell. The cell further comprises a knockout or knockdown of a FAD gene. Due to the genetic modifications, the cell produces a cell oil having a fatty acid profile in which the ratio of palmitoleic acid (C16:1) to palmitic acid (C16:0) is greater than 0.1, with no more than 3% polyunsaturated fatty acids. Optionally, palmitoleic acid comprises 0.5% or more of the profile. Optionally, the cell oil comprises less than 3.5% saturated fatty acids.
[0214] The low saturate and low saturate/high stability oil can be blended with less expensive oils to reach a targeted saturated fatty acid level at less expense. For example, an oil with 1% saturated fat can be blended with an oil having 7% saturated fat (e.g. high-oleic sunflower oil) to give an oil having 3.5% or less saturated fat.
[0215] Oils produced according to embodiments of the present invention can be used in the transportation fuel, oleochemical, and/or food and cosmetic industries, among other applications. For example, transesterification of lipids can yield long-chain fatty acid esters useful as biodiesel. Other enzymatic and chemical processes can be tailored to yield fatty acids, aldehydes, alcohols, alkanes, and alkenes. In some applications, renewable diesel, jet fuel, or other hydrocarbon compounds are produced. The present disclosure also provides methods of cultivating microalgae for increased productivity and increased lipid yield, and/or for more cost-effective production of the compositions described herein. The methods described here allow for the production of oils from plastidic cell cultures at large scale; e.g., 1000, 10,000, 100,000 liters or more.
[0216] In an embodiment, an oil extracted from the cell has 3.5%, 3%, 2.5%, or 2% saturated fat or less and is incorporated into a food product. The finished food product has 3.5, 3, 2.5, or 2% saturated fat or less. For example, oils recovered from such recombinant microalgae can be used for frying oils or as an ingredient in a prepared food that is low in saturated fats. The oils can be used neat or blended with other oils so that the food has less than 0.5 g of saturated fat per serving, thus allowing a label stating zero saturated fat (per US regulation). In a specific embodiment, the oil has a fatty acid profile with at least 90% oleic acid, less than 3% saturated fat, and more oleic acid than linoleic acid.
[0217] As with the other oils disclosed in this patent application, the low-saturate oils described in this section, including those with increased levels palmitoleic acid, can have a microalgal sterol profile as described in Section XII of this application. For example, via expression of an exogenous PAD gene, an oil can be produced with a fatty acid profile characterized by a ratio of palmitoleic acid to palmitic acid of at least 0.1 and/or palmitoleic acid levels of 0.5% or more, as determined by FAME GC/FID analysis and a sterol profile characterized by an excess of ergosterol over .beta.-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol.
XI. COCOA BUTTER/MILK-FAT BLEND MIMETICS
[0218] In certain embodiments, the cell produces a cell oil that has a temperature-dependent solid fat content ("SFC-curve") that approximates a blend of cocoa butter and milk fat. Such oils may be used where the cocoa butter/milk fat blend could be used; for example, in chocolates other confections, ice cream or other frozen desserts, pastries, or dough, including for quickbreads, or other baked goods. The oils may inhibit blooming, enhance flavor, enhance texture, or reduce costs. In a specific example, the cell oil approximates. Accordingly, an embodiment of the invention is using a cell oil from a recombinant microalgal cell to replace a cocoa butter/milk fat blend in a recipe. In a related embodiment,
[0219] FIG. 17 shows a plot of % solid fat content for various oils as follows (a) P. moriformis RBD oil without lipid pathway engineering, (b) Brazilian cocoa butter+25% milk fat, (c) three replicates of P. moriformis RBD oil from a strain expressing hairpin nucleic acids that reduce levels of a SAD allele thus reducing oleic acid and increasing stearic acid in the TAG profile, (d) P. moriformis RBD oil from a strain overexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, see Example 45), (e) Malaysian cocoa butter+25% milk fat, and (f) Malaysian cocoa butter. The cocoa butter and cocoa butter milk fat values are literature values (Bailey's Industrial Oils and Fat Products, 6.sup.th ed.)
[0220] In an embodiment of the present invention, a cell oil that is similar in thermal properties to a 75% cocoa butter/25% milk fat blend is produced by a microalgal or other cell described above. The cell comprises recombinant nucleic acids operable to alter the fatty acid profile of triglycerides produced by the cell so as that the oil has a solid fat content (e.g., as determined by NMR) of 38%.+-.30% at 20.degree. C., 32%.+-.30% at 25.degree. C., 17%.+-.30% at 30.degree. C., and less than 5%.+-.30% at 35.degree. C. For the sake of clarity, .+-.10% refers to percent of the percent SFC (e.g., 30% of 5% SFC is 1.5% SFC so the range is 3.5 to 6.5% SFC at 35.degree. C.). In related embodiments, the oil has a solid fat content (e.g., as determined by NMR) of 38%.+-.20% at 20.degree. C., 32%.+-.20% at 25.degree. C., 17%.+-.20% at 30.degree. C., and less than 5%.+-.20% at 35.degree. C. or the oil has a solid fat content (e.g., as determined by NMR) of 38%.+-.10% at 20.degree. C., 32%.+-.10% at 25.degree. C., 17%.+-.10% at 30.degree. C., and less than 5%.+-.10% at 35.degree. C.
[0221] In a another embodiment a cell high oleic oil produced according to the methods of section IX or corresponding Examples, is converted into a structuring fat such as a cocoa butter equivalent, substitute, extender by enzymatic interesterification or transesterification with a source of saturated fatty acids (e.g. a hardstock fat or saturated fatty acid esters). For example, a 1,3-specific lipase can be used to add stearate, palmitate or both to a high oleic oil having greater than 80% oleic acid.
XII. MINOR OIL COMPONENTS
[0222] The oils produced according to the above methods in some cases are made using a microalgal host cell. As described above, the microalga can be, without limitation, fall in the classification of Chlorophyta, Trebouxiophyceae , Chlorellales, Chlorellaceae, or Chlorophyceae. It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and .beta.-sitosterol, when detected by GC-MS. However, it is believed that all sterols produced by Chlorella have C24.beta. stereochemistry. Thus, it is believed that the molecules detected as campesterol, stigmasterol, and .beta.-sitosterol, are actually 22,23-dihydrobrassicasterol, poriferasterol and clionasterol, respectively. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24.beta. stereochemistry and the absence of C24.alpha. stereochemistry in the sterols present. For example, the oils produced may contain 22, 23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in .beta.-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of .DELTA..sup.7-poriferasterol.
[0223] In one embodiment, the oils provided herein are not vegetable oils. Vegetable oils are oils extracted from plants and plant seeds. Vegetable oils can be distinguished from the non-plant oils provided herein on the basis of their oil content. A variety of methods for analyzing the oil content can be employed to determine the source of the oil or whether adulteration of an oil provided herein with an oil of a different (e.g. plant) origin has occurred. The determination can be made on the basis of one or a combination of the analytical methods. These tests include but are not limited to analysis of one or more of free fatty acids, fatty acid profile, total triacylglycerol content, diacylglycerol content, peroxide values, spectroscopic properties (e.g. UV absorption), sterol profile, sterol degradation products, antioxidants (e.g. tocopherols), pigments (e.g. chlorophyll), d13C values and sensory analysis (e.g. taste, odor, and mouth feel). Many such tests have been standardized for commercial oils such as the Codex Alimentarius standards for edible fats and oils.
[0224] Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, b-sitosterol, and stigmasterol are common plant sterols, with b-sitosterol being a principle plant sterol. For example, b-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).
[0225] Oil isolated from Prototheca moriformis strain UTEX1435 were separately clarified (CL), refined and bleached (RB), or refined, bleached and deodorized (RBD) and were tested for sterol content according to the procedure described in JAOCS vol. 60, no. 8, August 1983. Results of the analysis are shown below (units in mg/100 g) in Table 5b.
TABLE-US-00007 TABLE 5b Sterol profiles of oils from UTEX 1435. Refined, Clari- Refined & bleached, & Sterol Crude fied bleached deodorized 1 Ergosterol 384 398 293 302 (56%) (55%) (50%) (50%) 2 5,22-cholestadien-24- 14.6 18.8 14 15.2 methyl-3-ol (2.1%) (2.6%) (2.4%) (2.5%) (Brassicasterol) 3 24-methylcholest-5- 10.7 11.9 10.9 10.8 en-3-ol (Campersterol (1.6%) (1.6%) (1.8%) (1.8%) or 22,23- dihydrobrassicasterol) 4 5,22-cholestadien-24- 57.7 59.2 46.8 49.9 ethyl-3-ol (Stigmaserol (8.4%) (8.2%) (7.9%) (8.3%) or poriferasterol) 5 24-ethylcholest-5-en- 9.64 9.92 9.26 10.2 3-ol (.beta.-Sitosterol or (1.4%) (1.4%) (1.6%) (1.7%) clionasterol) 6 Other sterols 209 221 216 213 Total sterols 685.64 718.82 589.96 601.1
[0226] These results show three striking features. First, ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, .beta.-sitosterol, and stigmasterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% .beta.-sitosterol was found to be present. .beta.-sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of .beta.-sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol:.beta.-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
[0227] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% .beta.-sitosterol. In other embodiments the oil is free from .beta.-sitosterol. For any of the oils or cell-oils disclosed in this application, the oil can have the sterol profile of any column of Table 5b, above, with a sterol-by-sterol variation of 30%, 20%, 10% or less.
[0228] In some embodiments, the oil is free from one or more of .beta.-sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from .beta.-sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
[0229] In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.
[0230] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22, 23-dihydrobrassicasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% 22,23-dihydrobrassicasterol.
[0231] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5, 22-cholestadien-24-ethyl-3-ol. In some embodiments, the 5, 22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol.
[0232] In some embodiments, the oil content of an oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.
[0233] In some embodiments, the oil content contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4% or 5% brassicasterol. In some embodiments, the oil content contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.
[0234] In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.
[0235] In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% .beta.-sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% .beta.-sitosterol. In some embodiments, the oil content further comprises brassicasterol.
[0236] Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols b-sitosterol and stigmasterol. In contrast, the sterol profile of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen-Yen, Meinschein W. G., "Sterols as ecological indicators"; Geochimica et Cosmochimia Acta. Vol 43. pp 739-745).
[0237] In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than b-sitosterol and stigmasterol. In some embodiments of the microalgal oils, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.
[0238] In some embodiments the microalgal oils provided herein contain C28 sterols in excess of C29 sterols. In some embodiments of the microalgal oils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.
XIII. FUELS AND CHEMICALS
[0239] The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc.). The oils, triglycerides, fatty acids from the oils may be subjected to C--H activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.
[0240] The oils can be converted to alkanes (e.g., renewable diesel) or esters (e.g., methyl or ethyl esters for biodisesel produced by transesterification). The alkanes or esters may be used as fuel, as solvents or lubricants, or as a chemical feedstock. Methods for production of renewable diesel and biodiesel are well established in the art. See, for example, WO2011/150411.
[0241] In a specific embodiment of the present invention, a high-oleic or high-oleic-high stability oil described above is esterified. For example, the oils can be transesterified with methanol to an oil that is rich in methyl oleate. As described in Example 49, such formulations have been found to compare favorably with methyl oleate from soybean oil.
[0242] In another specific example, the oil is converted to C36 diacids or products of C36 diacids. Fatty acids produced from the oil can be polymerized to give a composition rich in C36 dimer acids. In a specific example, high-oleic oil is split to give a high-oleic fatty acid material which is polymerized to give a composition rich in C36-dimer acids. Optionally , the oil is high oleic high stability oil (e.g., greater than 60% oleic acid with less than 3% polyunsaturates, greater than 70% oleic acid with less than 2% polyunsaturates, or greater than 80% oleic acid with less than 1% polyunsaturates). It is believed that using a high oleic, high stability, starting material will give lower amounts of cyclic products, which may be desirable in some cases. After hydrolyzing the oil, one obtains a high concentration of oleic acid. In the process of making dimer acids, a high oleic acid stream will convert to a "cleaner" C36 dimer acid and not produce trimers acids (C54) and other more complex cyclic by-products which are obtained due to presence of C18:2 and C18:3 acids. For example, the oil can be hydrolyzed to fatty acids and the fatty acids purified and dimerized at 250.degree. C. in the presence of montmorillonite clay. See SRI Natural Fatty Acid, March 2009. A product rich in C36 dimers of oleic acid is recovered.
##STR00001##
[0243] Further, the C36 dimer acids can be esterified and hydrogenated to give diols. The diols can be polymerized by catalytic dehydration. Polymers can also be produced by transesterification of dimerdiols with dimethyl carbonate.
[0244] For the production of fuel in accordance with the methods of the invention lipids produced by cells of the invention are harvested, or otherwise collected, by any convenient means. Lipids can be isolated by whole cell extraction. The cells are first disrupted, and then intracellular and cell membrane/cell wall-associated lipids as well as extracellular hydrocarbons can be separated from the cell mass, such as by use of centrifugation. Intracellular lipids produced in oleaginous cells are, in some embodiments, extracted after lysing the cells. Once extracted, the lipids are further refined to produce oils, fuels, or oleochemicals.
[0245] Various methods are available for separating lipids from cellular lysates. For example, lipids and lipid derivatives such as fatty aldehydes, fatty alcohols, and hydrocarbons such as alkanes can be extracted with a hydrophobic solvent such as hexane (see Frenz et al. 1989, Enzyme Microb. Technol., 11:717). Lipids and lipid derivatives can also be extracted using liquefaction (see for example Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, Biomass Bioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al. 1995, Fuel 74(12):1735-1738); and supercritical CO.sub.2 extraction (see for example Mendes et al. 2003, Inorganica Chimica Acta 356:328-334). Miao and Wu describe a protocol of the recovery of microalgal lipid from a culture of Chlorella protothecoides in which the cells were harvested by centrifugation, washed with distilled water and dried by freeze drying. The resulting cell powder was pulverized in a mortar and then extracted with n-hexane. Miao and Wu, Biosource Technology (2006) 97:841-846.
[0246] Lipids and lipid derivatives can be recovered by extraction with an organic solvent. In some cases, the preferred organic solvent is hexane. Typically, the organic solvent is added directly to the lysate without prior separation of the lysate components. In one embodiment, the lysate generated by one or more of the methods described above is contacted with an organic solvent for a period of time sufficient to allow the lipid and/or hydrocarbon components to form a solution with the organic solvent. In some cases, the solution can then be further refined to recover specific desired lipid or hydrocarbon components. Hexane extraction methods are well known in the art.
[0247] Lipids produced by cells in vivo, or enzymatically modified in vitro, as described herein can be optionally further processed by conventional means. The processing can include "cracking" to reduce the size, and thus increase the hydrogen:carbon ratio, of hydrocarbon molecules. Catalytic and thermal cracking methods are routinely used in hydrocarbon and triglyceride oil processing. Catalytic methods involve the use of a catalyst, such as a solid acid catalyst. The catalyst can be silica-alumina or a zeolite, which result in the heterolytic, or asymmetric, breakage of a carbon-carbon bond to result in a carbocation and a hydride anion. These reactive intermediates then undergo either rearrangement or hydride transfer with another hydrocarbon. The reactions can thus regenerate the intermediates to result in a self-propagating chain mechanism. Hydrocarbons can also be processed to reduce, optionally to zero, the number of carbon-carbon double, or triple, bonds therein. Hydrocarbons can also be processed to remove or eliminate a ring or cyclic structure therein. Hydrocarbons can also be processed to increase the hydrogen:carbon ratio. This can include the addition of hydrogen ("hydrogenation") and/or the "cracking" of hydrocarbons into smaller hydrocarbons.
[0248] Thermal methods involve the use of elevated temperature and pressure to reduce hydrocarbon size. An elevated temperature of about 800.degree. C. and pressure of about 700 kPa can be used. These conditions generate "light," a term that is sometimes used to refer to hydrogen-rich hydrocarbon molecules (as distinguished from photon flux), while also generating, by condensation, heavier hydrocarbon molecules which are relatively depleted of hydrogen. The methodology provides homolytic, or symmetrical, breakage and produces alkenes, which may be optionally enzymatically saturated as described above.
[0249] Catalytic and thermal methods are standard in plants for hydrocarbon processing and oil refining. Thus hydrocarbons produced by cells as described herein can be collected and processed or refined via conventional means. See Hillen et al. (Biotechnology and Bioengineering, Vol. XXIV:193-205 (1982)) for a report on hydrocracking of microalgae-produced hydrocarbons. In alternative embodiments, the fraction is treated with another catalyst, such as an organic compound, heat, and/or an inorganic compound. For processing of lipids into biodiesel, a transesterification process is used as described below in this Section.
[0250] Hydrocarbons produced via methods of the present invention are useful in a variety of industrial applications. For example, the production of linear alkylbenzene sulfonate (LAS), an anionic surfactant used in nearly all types of detergents and cleaning preparations, utilizes hydrocarbons generally comprising a chain of 10-14 carbon atoms. See, for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517; 6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such as LAS, can be used in the manufacture of personal care compositions and detergents, such as those described in U.S. Pat. Nos. 5,942,479; 6,086,903; 5,833,999; 6,468,955; and 6,407,044.
[0251] Increasing interest is directed to the use of hydrocarbon components of biological origin in fuels, such as biodiesel, renewable diesel, and jet fuel, since renewable biological starting materials that may replace starting materials derived from fossil fuels are available, and the use thereof is desirable. There is an urgent need for methods for producing hydrocarbon components from biological materials. The present invention fulfills this need by providing methods for production of biodiesel, renewable diesel, and jet fuel using the lipids generated by the methods described herein as a biological material to produce biodiesel, renewable diesel, and jet fuel.
[0252] Traditional diesel fuels are petroleum distillates rich in paraffinic hydrocarbons. They have boiling ranges as broad as 370.degree. to 780.degree. F., which are suitable for combustion in a compression ignition engine, such as a diesel engine vehicle. The American Society of Testing and Materials (ASTM) establishes the grade of diesel according to the boiling range, along with allowable ranges of other fuel properties, such as cetane number, cloud point, flash point, viscosity, aniline point, sulfur content, water content, ash content, copper strip corrosion, and carbon residue. Technically, any hydrocarbon distillate material derived from biomass or otherwise that meets the appropriate ASTM specification can be defined as diesel fuel (ASTM D975), jet fuel (ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTM D6751).
[0253] After extraction, lipid and/or hydrocarbon components recovered from the microbial biomass described herein can be subjected to chemical treatment to manufacture a fuel for use in diesel vehicles and jet engines.
[0254] Biodiesel is a liquid which varies in color--between golden and dark brown--depending on the production feedstock. It is practically immiscible with water, has a high boiling point and low vapor pressure. Biodiesel refers to a diesel-equivalent processed fuel for use in diesel-engine vehicles. Biodiesel is biodegradable and non-toxic. An additional benefit of biodiesel over conventional diesel fuel is lower engine wear. Typically, biodiesel comprises C14-C18 alkyl esters. Various processes convert biomass or a lipid produced and isolated as described herein to diesel fuels. A preferred method to produce biodiesel is by transesterification of a lipid as described herein. A preferred alkyl ester for use as biodiesel is a methyl ester or ethyl ester.
[0255] Biodiesel produced by a method described herein can be used alone or blended with conventional diesel fuel at any concentration in most modern diesel-engine vehicles. When blended with conventional diesel fuel (petroleum diesel), biodiesel may be present from about 0.1% to about 99.9%. Much of the world uses a system known as the "B" factor to state the amount of biodiesel in any fuel mix. For example, fuel containing 20% biodiesel is labeled B20. Pure biodiesel is referred to as B100.
[0256] Biodiesel can be produced by transesterification of triglycerides contained in oil-rich biomass. Thus, in another aspect of the present invention a method for producing biodiesel is provided. In a preferred embodiment, the method for producing biodiesel comprises the steps of (a) cultivating a lipid-containing microorganism using methods disclosed herein (b) lysing a lipid-containing microorganism to produce a lysate, (c) isolating lipid from the lysed microorganism, and (d) transesterifying the lipid composition, whereby biodiesel is produced. Methods for growth of a microorganism, lysing a microorganism to produce a lysate, treating the lysate in a medium comprising an organic solvent to form a heterogeneous mixture and separating the treated lysate into a lipid composition have been described above and can also be used in the method of producing biodiesel. The lipid profile of the biodiesel is usually highly similar to the lipid profile of the feedstock oil.
[0257] Lipid compositions can be subjected to transesterification to yield long-chain fatty acid esters useful as biodiesel. Preferred transesterification reactions are outlined below and include base catalyzed transesterification and transesterification using recombinant lipases. In a base-catalyzed transesterification process, the triacylglycerides are reacted with an alcohol, such as methanol or ethanol, in the presence of an alkaline catalyst, typically potassium hydroxide. This reaction forms methyl or ethyl esters and glycerin (glycerol) as a byproduct.
[0258] Transesterification has also been carried out, as discussed above, using an enzyme, such as a lipase instead of a base. Lipase-catalyzed transesterification can be carried out, for example, at a temperature between the room temperature and 80.degree. C., and a mole ratio of the TAG to the lower alcohol of greater than 1:1, preferably about 3:1. Lipases suitable for use in transesterification include, but are not limited to, those listed in Table 9. Other examples of lipases useful for transesterification are found in, e.g., U.S. Pat. Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipases include, but are not limited to, lipases produced by microorganisms of Rhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida, and Humicola and pancreas lipase.
[0259] Subsequent processes may also be used if the biodiesel will be used in particularly cold temperatures. Such processes include winterization and fractionation. Both processes are designed to improve the cold flow and winter performance of the fuel by lowering the cloud point (the temperature at which the biodiesel starts to crystallize). There are several approaches to winterizing biodiesel. One approach is to blend the biodiesel with petroleum diesel. Another approach is to use additives that can lower the cloud point of biodiesel. Another approach is to remove saturated methyl esters indiscriminately by mixing in additives and allowing for the crystallization of saturates and then filtering out the crystals. Fractionation selectively separates methyl esters into individual components or fractions, allowing for the removal or inclusion of specific methyl esters. Fractionation methods include urea fractionation, solvent fractionation and thermal distillation.
[0260] Another valuable fuel provided by the methods of the present invention is renewable diesel, which comprises alkanes, such as C10:0, C12:0, C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel. High quality renewable diesel conforms to the ASTM D975 standard. The lipids produced by the methods of the present invention can serve as feedstock to produce renewable diesel. Thus, in another aspect of the present invention, a method for producing renewable diesel is provided. Renewable diesel can be produced by at least three processes: hydrothermal processing (hydrotreating); hydroprocessing; and indirect liquefaction. These processes yield non-ester distillates. During these processes, triacylglycerides produced and isolated as described herein, are converted to alkanes.
[0261] In one embodiment, the method for producing renewable diesel comprises (a) cultivating a lipid-containing microorganism using methods disclosed herein (b) lysing the microorganism to produce a lysate, (c) isolating lipid from the lysed microorganism, and (d) deoxygenating and hydrotreating the lipid to produce an alkane, whereby renewable diesel is produced. Lipids suitable for manufacturing renewable diesel can be obtained via extraction from microbial biomass using an organic solvent such as hexane, or via other methods, such as those described in U.S. Pat. No. 5,928,696. Some suitable methods may include mechanical pressing and centrifuging.
[0262] In some methods, the microbial lipid is first cracked in conjunction with hydrotreating to reduce carbon chain length and saturate double bonds, respectively. The material is then isomerized, also in conjunction with hydrotreating. The naptha fraction can then be removed through distillation, followed by additional distillation to vaporize and distill components desired in the diesel fuel to meet an ASTM D975 standard while leaving components that are heavier than desired for meeting the D975 standard. Hydrotreating, hydrocracking, deoxygenation and isomerization methods of chemically modifying oils, including triglyceride oils, are well known in the art. See for example European patent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1); EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos. 7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746; 5,885,440; 6,881,873.
[0263] In one embodiment of the method for producing renewable diesel, treating the lipid to produce an alkane is performed by hydrotreating of the lipid composition. In hydrothermal processing, typically, biomass is reacted in water at an elevated temperature and pressure to form oils and residual solids. Conversion temperatures are typically 300.degree. to 660.degree. F., with pressure sufficient to keep the water primarily as a liquid, 100 to 170 standard atmosphere (atm). Reaction times are on the order of 15 to 30 minutes. After the reaction is completed, the organics are separated from the water. Thereby a distillate suitable for diesel is produced.
[0264] In some methods of making renewable diesel, the first step of treating a triglyceride is hydroprocessing to saturate double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In some methods, hydrogenation and deoxygenation occur in the same reaction. In other methods deoxygenation occurs before hydrogenation. Isomerization is then optionally performed, also in the presence of hydrogen and a catalyst. Naphtha components are preferably removed through distillation. For examples, see U.S. Pat. No. 5,475,160 (hydrogenation of triglycerides); U.S. Pat. No. 5,091,116 (deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815 (hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).
[0265] One suitable method for the hydrogenation of triglycerides includes preparing an aqueous solution of copper, zinc, magnesium and lanthanum salts and another solution of alkali metal or preferably, ammonium carbonate. The two solutions may be heated to a temperature of about 20.degree. C. to about 85.degree. C. and metered together into a precipitation container at rates such that the pH in the precipitation container is maintained between 5.5 and 7.5 in order to form a catalyst. Additional water may be used either initially in the precipitation container or added concurrently with the salt solution and precipitation solution. The resulting precipitate may then be thoroughly washed, dried, calcined at about 300.degree. C. and activated in hydrogen at temperatures ranging from about 100.degree. C. to about 400.degree. C. One or more triglycerides may then be contacted and reacted with hydrogen in the presence of the above-described catalyst in a reactor. The reactor may be a trickle bed reactor, fixed bed gas-solid reactor, packed bubble column reactor, continuously stirred tank reactor, a slurry phase reactor, or any other suitable reactor type known in the art. The process may be carried out either batchwise or in continuous fashion. Reaction temperatures are typically in the range of from about 170.degree. C. to about 250.degree. C. while reaction pressures are typically in the range of from about 300 psig to about 2000 psig. Moreover, the molar ratio of hydrogen to triglyceride in the process of the present invention is typically in the range of from about 20:1 to about 700:1. The process is typically carried out at a weight hourly space velocity (WHSV) in the range of from about 0.1 hr.sup.-1 to about 5 hr.sup.-1. One skilled in the art will recognize that the time period required for reaction will vary according to the temperature used, the molar ratio of hydrogen to triglyceride, and the partial pressure of hydrogen. The products produced by the such hydrogenation processes include fatty alcohols, glycerol, traces of paraffins and unreacted triglycerides. These products are typically separated by conventional means such as, for example, distillation, extraction, filtration, crystallization, and the like.
[0266] Petroleum refiners use hydroprocessing to remove impurities by treating feeds with hydrogen. Hydroprocessing conversion temperatures are typically 300.degree. to 700.degree. F. Pressures are typically 40 to 100 atm. The reaction times are typically on the order of 10 to 60 minutes. Solid catalysts are employed to increase certain reaction rates, improve selectivity for certain products, and optimize hydrogen consumption.
[0267] Suitable methods for the deoxygenation of an oil includes heating an oil to a temperature in the range of from about 350.degree. F. to about 550.degree. F. and continuously contacting the heated oil with nitrogen under at least pressure ranging from about atmospheric to above for at least about 5 minutes.
[0268] Suitable methods for isomerization include using alkali isomerization and other oil isomerization known in the art.
[0269] Hydrotreating and hydroprocessing ultimately lead to a reduction in the molecular weight of the triglyceride feed. The triglyceride molecule is reduced to four hydrocarbon molecules under hydroprocessing conditions: a propane molecule and three heavier hydrocarbon molecules, typically in the C8 to C18 range.
[0270] Thus, in one embodiment, the product of one or more chemical reaction(s) performed on lipid compositions of the invention is an alkane mixture that comprises ASTM D975 renewable diesel. Production of hydrocarbons by microorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol (2005) 66: 486-496 and A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae, NREL/TP-580-24190, John Sheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).
[0271] The distillation properties of a diesel fuel is described in terms of T10-T90 (temperature at 10% and 90%, respectively, volume distilled). The T10-T90 of the material produced in Example 13 was 57.9.degree. C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10-T90 ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65.degree. C. using triglyceride oils produced according to the methods disclosed herein.
[0272] Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other T10 values, such as T10 between 180 and 295, between 190 and 270, between 210 and 250, between 225 and 245, and at least 290.
[0273] Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein can be employed to generate renewable diesel compositions with certain T90 values, such as T90 between 280 and 380, between 290 and 360, between 300 and 350, between 310 and 340, and at least 290.
[0274] The FBP of the material produced in Example 13 was 300.degree. C. Methods of hydrotreating, isomerization, and other covalent modification of oils disclosed herein, as well as methods of distillation and fractionation (such as cold filtration) disclosed herein, can be employed to generate renewable diesel compositions with other FBP values, such as FBP between 290 and 400, between 300 and 385, between 310 and 370, between 315 and 360, and at least 300.
[0275] Other oils provided by the methods and compositions of the invention can be subjected to combinations of hydrotreating, isomerization, and other covalent modification including oils with lipid profiles including (a) at least 1%-5%, preferably at least 4%, C8-C14; (b) at least 0.25%-1%, preferably at least 0.3%, C8; (c) at least 1%-5%, preferably at least 2%, C10; (d) at least 1%-5%, preferably at least 2%, C12; and (3) at least 20%-40%, preferably at least 30% C8-C14.
[0276] A traditional ultra-low sulfur diesel can be produced from any form of biomass by a two-step process. First, the biomass is converted to a syngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then, the syngas is catalytically converted to liquids. Typically, the production of liquids is accomplished using Fischer-Tropsch (FT) synthesis. This technology applies to coal, natural gas, and heavy oils. Thus, in yet another preferred embodiment of the method for producing renewable diesel, treating the lipid composition to produce an alkane is performed by indirect liquefaction of the lipid composition.
[0277] The present invention also provides methods to produce jet fuel. Jet fuel is clear to straw colored. The most common fuel is an unleaded/paraffin oil-based fuel classified as Aeroplane A-1, which is produced to an internationally standardized set of specifications. Jet fuel is a mixture of a large number of different hydrocarbons, possibly as many as a thousand or more. The range of their sizes (molecular weights or carbon numbers) is restricted by the requirements for the product, for example, freezing point or smoke point. Kerosene-type Aeroplane fuel (including Jet A and Jet A-1) has a carbon number distribution between about 8 and 16 carbon numbers. Wide-cut or naphtha-type Aeroplane fuel (including Jet B) typically has a carbon number distribution between about 5 and 15 carbons.
[0278] In one embodiment of the invention, a jet fuel is produced by blending algal fuels with existing jet fuel. The lipids produced by the methods of the present invention can serve as feedstock to produce jet fuel. Thus, in another aspect of the present invention, a method for producing jet fuel is provided. Herewith two methods for producing jet fuel from the lipids produced by the methods of the present invention are provided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).
[0279] Fluid Catalytic Cracking (FCC) is one method which is used to produce olefins, especially propylene from heavy crude fractions. The lipids produced by the method of the present invention can be converted to olefins. The process involves flowing the lipids produced through an FCC zone and collecting a product stream comprised of olefins, which is useful as a jet fuel. The lipids produced are contacted with a cracking catalyst at cracking conditions to provide a product stream comprising olefins and hydrocarbons useful as jet fuel.
[0280] In one embodiment, the method for producing jet fuel comprises (a) cultivating a lipid-containing microorganism using methods disclosed herein, (b) lysing the lipid-containing microorganism to produce a lysate, (c) isolating lipid from the lysate, and (d) treating the lipid composition, whereby jet fuel is produced. In one embodiment of the method for producing a jet fuel, the lipid composition can be flowed through a fluid catalytic cracking zone, which, in one embodiment, may comprise contacting the lipid composition with a cracking catalyst at cracking conditions to provide a product stream comprising C.sub.2-C.sub.5 olefins.
[0281] In certain embodiments of this method, it may be desirable to remove any contaminants that may be present in the lipid composition. Thus, prior to flowing the lipid composition through a fluid catalytic cracking zone, the lipid composition is pretreated. Pretreatment may involve contacting the lipid composition with an ion-exchange resin. The ion exchange resin is an acidic ion exchange resin, such as Amberlyst.TM.-15 and can be used as a bed in a reactor through which the lipid composition is flowed, either upflow or downflow. Other pretreatments may include mild acid washes by contacting the lipid composition with an acid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contacting is done with a dilute acid solution usually at ambient temperature and atmospheric pressure.
[0282] The lipid composition, optionally pretreated, is flowed to an FCC zone where the hydrocarbonaceous components are cracked to olefins. Catalytic cracking is accomplished by contacting the lipid composition in a reaction zone with a catalyst composed of finely divided particulate material. The reaction is catalytic cracking, as opposed to hydrocracking, and is carried out in the absence of added hydrogen or the consumption of hydrogen. As the cracking reaction proceeds, substantial amounts of coke are deposited on the catalyst. The catalyst is regenerated at high temperatures by burning coke from the catalyst in a regeneration zone. Coke-containing catalyst, referred to herein as "coked catalyst", is continually transported from the reaction zone to the regeneration zone to be regenerated and replaced by essentially coke-free regenerated catalyst from the regeneration zone. Fluidization of the catalyst particles by various gaseous streams allows the transport of catalyst between the reaction zone and regeneration zone. Methods for cracking hydrocarbons, such as those of the lipid composition described herein, in a fluidized stream of catalyst, transporting catalyst between reaction and regeneration zones, and combusting coke in the regenerator are well known by those skilled in the art of FCC processes. Exemplary FCC applications and catalysts useful for cracking the lipid composition to produce C.sub.2-C.sub.5 olefins are described in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporated in their entirety by reference.
[0283] Suitable FCC catalysts generally comprise at least two components that may or may not be on the same matrix. In some embodiments, both two components may be circulated throughout the entire reaction vessel. The first component generally includes any of the well-known catalysts that are used in the art of fluidized catalytic cracking, such as an active amorphous clay-type catalyst and/or a high activity, crystalline molecular sieve. Molecular sieve catalysts may be preferred over amorphous catalysts because of their much-improved selectivity to desired products. In some preferred embodiments, zeolites may be used as the molecular sieve in the FCC processes. Preferably, the first catalyst component comprises a large pore zeolite, such as a Y-type zeolite, an active alumina material, a binder material, comprising either silica or alumina and an inert filler such as kaolin.
[0284] In one embodiment, cracking the lipid composition of the present invention, takes place in the riser section or, alternatively, the lift section, of the FCC zone. The lipid composition is introduced into the riser by a nozzle resulting in the rapid vaporization of the lipid composition. Before contacting the catalyst, the lipid composition will ordinarily have a temperature of about 149.degree. C. to about 316.degree. C. (300.degree. F. to 600.degree. F.). The catalyst is flowed from a blending vessel to the riser where it contacts the lipid composition for a time of abort 2 seconds or less.
[0285] The blended catalyst and reacted lipid composition vapors are then discharged from the top of the riser through an outlet and separated into a cracked product vapor stream including olefins and a collection of catalyst particles covered with substantial quantities of coke and generally referred to as "coked catalyst." In an effort to minimize the contact time of the lipid composition and the catalyst which may promote further conversion of desired products to undesirable other products, any arrangement of separators such as a swirl arm arrangement can be used to remove coked catalyst from the product stream quickly. The separator, e.g. swirl arm separator, is located in an upper portion of a chamber with a stripping zone situated in the lower portion of the chamber. Catalyst separated by the swirl arm arrangement drops down into the stripping zone. The cracked product vapor stream comprising cracked hydrocarbons including light olefins and some catalyst exit the chamber via a conduit which is in communication with cyclones. The cyclones remove remaining catalyst particles from the product vapor stream to reduce particle concentrations to very low levels. The product vapor stream then exits the top of the separating vessel. Catalyst separated by the cyclones is returned to the separating vessel and then to the stripping zone. The stripping zone removes adsorbed hydrocarbons from the surface of the catalyst by counter-current contact with steam.
[0286] Low hydrocarbon partial pressure operates to favor the production of light olefins. Accordingly, the riser pressure is set at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35 to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressure of about 69 to 138 kPa (10 to 20 psia). This relatively low partial pressure for hydrocarbon is achieved by using steam as a diluent to the extent that the diluent is 10 to 55 wt-% of lipid composition and preferably about 15 wt-% of lipid composition. Other diluents such as dry gas can be used to reach equivalent hydrocarbon partial pressures.
[0287] The temperature of the cracked stream at the riser outlet will be about 510.degree. C. to 621.degree. C. (950.degree. F. to 1150.degree. F.). However, riser outlet temperatures above 566.degree. C. (1050.degree. F.) make more dry gas and more olefins. Whereas, riser outlet temperatures below 566.degree. C. (1050.degree. F.) make less ethylene and propylene. Accordingly, it is preferred to run the FCC process at a preferred temperature of about 566.degree. C. to about 630.degree. C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35 psia). Another condition for the process is the catalyst to lipid composition ratio which can vary from about 5 to about 20 and preferably from about 10 to about 15.
[0288] In one embodiment of the method for producing a jet fuel, the lipid composition is introduced into the lift section of an FCC reactor. The temperature in the lift section will be very hot and range from about 700.degree. C. (1292.degree. F.) to about 760.degree. C. (1400.degree. F.) with a catalyst to lipid composition ratio of about 100 to about 150. It is anticipated that introducing the lipid composition into the lift section will produce considerable amounts of propylene and ethylene.
[0289] In another embodiment of the method for producing a jet fuel using the lipid composition or the lipids produced as described herein, the structure of the lipid composition or the lipids is broken by a process referred to as hydrodeoxygenation (HDO). HDO means removal of oxygen by means of hydrogen, that is, oxygen is removed while breaking the structure of the material. Olefinic double bonds are hydrogenated and any sulfur and nitrogen compounds are removed. Sulfur removal is called hydrodesulphurization (HDS). Pretreatment and purity of the raw materials (lipid composition or the lipids) contribute to the service life of the catalyst.
[0290] Generally in the HDO/HDS step, hydrogen is mixed with the feed stock (lipid composition or the lipids) and then the mixture is passed through a catalyst bed as a co-current flow, either as a single phase or a two phase feed stock. After the HDO/MDS step, the product fraction is separated and passed to a separate isomerization reactor. An isomerization reactor for biological starting material is described in the literature (FI 100 248) as a co-current reactor.
[0291] The process for producing a fuel by hydrogenating a hydrocarbon feed, e.g., the lipid composition or the lipids herein, can also be performed by passing the lipid composition or the lipids as a co-current flow with hydrogen gas through a first hydrogenation zone, and thereafter the hydrocarbon effluent is further hydrogenated in a second hydrogenation zone by passing hydrogen gas to the second hydrogenation zone as a counter-current flow relative to the hydrocarbon effluent. Exemplary HDO applications and catalysts useful for cracking the lipid composition to produce C.sub.2-C.sub.5 olefins are described in U.S. Pat. No. 7,232,935, which is incorporated in its entirety by reference.
[0292] Typically, in the hydrodeoxygenation step, the structure of the biological component, such as the lipid composition or lipids herein, is decomposed, oxygen, nitrogen, phosphorus and sulfur compounds, and light hydrocarbons as gas are removed, and the olefinic bonds are hydrogenated. In the second step of the process, i.e. in the so-called isomerization step, isomerization is carried out for branching the hydrocarbon chain and improving the performance of the paraffin at low temperatures.
[0293] In the first step, i.e. HDO step, of the cracking process, hydrogen gas and the lipid composition or lipids herein which are to be hydrogenated are passed to a HDO catalyst bed system either as co-current or counter-current flows, said catalyst bed system comprising one or more catalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typically operated in a co-current manner. In case of a HDO catalyst bed system comprising two or more catalyst beds, one or more of the beds may be operated using the counter-current flow principle. In the HDO step, the pressure varies between 20 and 150 bar, preferably between 50 and 100 bar, and the temperature varies between 200 and 500.degree. C., preferably in the range of 300-400.degree. C. In the HDO step, known hydrogenation catalysts containing metals from Group VII and/or VIB of the Periodic System may be used. Preferably, the hydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a CoMo catalysts, the support being alumina and/or silica. Typically, NiMo/Al.sub.2O.sub.3 and CoMo/Al.sub.2O.sub.3 catalysts are used.
[0294] Prior to the HDO step, the lipid composition or lipids herein may optionally be treated by prehydrogenation under milder conditions thus avoiding side reactions of the double bonds. Such prehydrogenation is carried out in the presence of a prehydrogenation catalyst at temperatures of 50-400.degree. C. and at hydrogen pressures of 1-200 bar, preferably at a temperature between 150 and 250.degree. C. and at a hydrogen pressure between 10 and 100 bar. The catalyst may contain metals from Group VIII and/or VIB of the Periodic System. Preferably, the prehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMo catalyst, the support being alumina and/or silica.
[0295] A gaseous stream from the HDO step containing hydrogen is cooled and then carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulfur compounds, gaseous light hydrocarbons and other impurities are removed therefrom. After compressing, the purified hydrogen or recycled hydrogen is returned back to the first catalyst bed and/or between the catalyst beds to make up for the withdrawn gas stream. Water is removed from the condensed liquid. The liquid is passed to the first catalyst bed or between the catalyst beds.
[0296] After the HDO step, the product is subjected to an isomerization step. It is substantial for the process that the impurities are removed as completely as possible before the hydrocarbons are contacted with the isomerization catalyst. The isomerization step comprises an optional stripping step, wherein the reaction product from the HDO step may be purified by stripping with water vapor or a suitable gas such as light hydrocarbon, nitrogen or hydrogen. The optional stripping step is carried out in counter-current manner in a unit upstream of the isomerization catalyst, wherein the gas and liquid are contacted with each other, or before the actual isomerization reactor in a separate stripping unit utilizing counter-current principle.
[0297] After the stripping step the hydrogen gas and the hydrogenated lipid composition or lipids herein, and optionally an n-paraffin mixture, are passed to a reactive isomerization unit comprising one or several catalyst bed(s). The catalyst beds of the isomerization step may operate either in co-current or counter-current manner.
[0298] It is important for the process that the counter-current flow principle is applied in the isomerization step. In the isomerization step this is done by carrying out either the optional stripping step or the isomerization reaction step or both in counter-current manner. In the isomerization step, the pressure varies in the range of 20-150 bar, preferably in the range of 20-100 bar, the temperature being between 200 and 500.degree. C., preferably between 300 and 400.degree. C. In the isomerization step, isomerization catalysts known in the art may be used. Suitable isomerization catalysts contain molecular sieve and/or a metal from Group VII and/or a carrier. Preferably, the isomerization catalyst contains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pd or Ni and Al.sub.2O.sub.3 or SiO.sub.2. Typical isomerization catalysts are, for example, Pt/SAPO-11/Al.sub.2O.sub.3, Pt/ZSM-22/Al.sub.2O.sub.3, Pt/ZSM-23/Al.sub.2O.sub.3 and Pt/SAPO-11/SiO.sub.2. The isomerization step and the HDO step may be carried out in the same pressure vessel or in separate pressure vessels. Optional prehydrogenation may be carried out in a separate pressure vessel or in the same pressure vessel as the HDO and isomerization steps.
[0299] Thus, in one embodiment, the product of one or more chemical reactions is an alkane mixture that comprises HRJ-5. In another embodiment, the product of the one or more chemical reactions is an alkane mixture that comprises ASTM D1655 jet fuel. In some embodiments, the composition conforming to the specification of ASTM 1655 jet fuel has a sulfur content that is less than 10 ppm. In other embodiments, the composition conforming to the specification of ASTM 1655 jet fuel has a T10 value of the distillation curve of less than 205.degree. C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a final boiling point (FBP) of less than 300.degree. C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a flash point of at least 38.degree. C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a density between 775K/M.sup.3 and 840K/M.sup.3. In yet another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a freezing point that is below -47.degree. C. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a net Heat of Combustion that is at least 42.8 MJ/K. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a hydrogen content that is at least 13.4 mass %. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has a thermal stability, as tested by quantitative gravimetric JFTOT at 260.degree. C., which is below 3 mm of Hg. In another embodiment, the composition conforming to the specification of ASTM 1655 jet fuel has an existent gum that is below 7 mg/dl.
[0300] Thus, the present invention discloses a variety of methods in which chemical modification of microalgal lipid is undertaken to yield products useful in a variety of industrial and other applications. Examples of processes for modifying oil produced by the methods disclosed herein include, but are not limited to, hydrolysis of the oil, hydroprocessing of the oil, and esterification of the oil. Other chemical modification of microalgal lipid include, without limitation, epoxidation, oxidation, hydrolysis, sulfations, sulfonation, ethoxylation, propoxylation, amidation, and saponification. The modification of the microalgal oil produces basic oleochemicals that can be further modified into selected derivative oleochemicals for a desired function. In a manner similar to that described above with reference to fuel producing processes, these chemical modifications can also be performed on oils generated from the microbial cultures described herein. Examples of basic oleochemicals include, but are not limited to, soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogen compounds including fatty amides, fatty acid methyl esters, and glycerol. Examples of derivative oleochemicals include, but are not limited to, fatty nitriles, esters, dimer acids, quats (including betaines), surfactants, fatty alkanolamides, fatty alcohol sulfates, resins, emulsifiers, fatty alcohols, olefins, drilling muds, polyols, polyurethanes, polyacrylates, rubber, candles, cosmetics, metallic soaps, soaps, alpha-sulphonated methyl esters, fatty alcohol sulfates, fatty alcohol ethoxylates, fatty alcohol ether sulfates, imidazolines, surfactants, detergents, esters, quats (including betaines), ozonolysis products, fatty amines, fatty alkanolamides, ethoxysulfates, monoglycerides, diglycerides, triglycerides (including medium chain triglycerides), lubricants, hydraulic fluids, greases, dielectric fluids, mold release agents, metal working fluids, heat transfer fluids, other functional fluids, industrial chemicals (e.g., cleaners, textile processing aids, plasticizers, stabilizers, additives), surface coatings, paints and lacquers, electrical wiring insulation, and higher alkanes. Other derivatives include fatty amidoamines, amidoamine carboxylates, amidoamine oxides, amidoamine oxide carboxylates, amidoamine esters, ethanolamine amides, sulfonates, amidoamine sulfonates, diamidoamine dioxides, sulfonated alkyl ester alkoxylates, betaines, quarternized diamidoamine betaines, and sulfobetaines.
[0301] Hydrolysis of the fatty acid constituents from the glycerolipids produced by the methods of the invention yields free fatty acids that can be derivatized to produce other useful chemicals. Hydrolysis occurs in the presence of water and a catalyst which may be either an acid or a base. The liberated free fatty acids can be derivatized to yield a variety of products, as reported in the following: U.S. Pat. No. 5,304,664 (Highly sulfated fatty acids); U.S. Pat. No. 7,262,158 (Cleansing compositions); U.S. Pat. No. 7,115,173 (Fabric softener compositions); U.S. Pat. No. 6,342,208 (Emulsions for treating skin); U.S. Pat. No. 7,264,886 (Water repellent compositions); U.S. Pat. No. 6,924,333 (Paint additives); U.S. Pat. No. 6,596,768 (Lipid-enriched ruminant feedstock); and U.S. Pat. No. 6,380,410 (Surfactants for detergents and cleaners).
[0302] In some methods, the first step of chemical modification may be hydroprocessing to saturate double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In other methods, hydrogenation and deoxygenation may occur in the same reaction. In still other methods deoxygenation occurs before hydrogenation. Isomerization may then be optionally performed, also in the presence of hydrogen and a catalyst. Finally, gases and naphtha components can be removed if desired. For example, see U.S. Pat. No. 5,475,160 (hydrogenation of triglycerides); U.S. Pat. No. 5,091,116 (deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815 (hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).
[0303] In some embodiments of the invention, the triglyceride oils are partially or completely deoxygenated. The deoxygenation reactions form desired products, including, but not limited to, fatty acids, fatty alcohols, polyols, ketones, and aldehydes. In general, without being limited by any particular theory, the deoxygenation reactions involve a combination of various different reaction pathways, including without limitation: hydrogenolysis, hydrogenation, consecutive hydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation, and combined hydrogenation-hydrogenolysis reactions, resulting in at least the partial removal of oxygen from the fatty acid or fatty acid ester to produce reaction products, such as fatty alcohols, that can be easily converted to the desired chemicals by further processing. For example, in one embodiment, a fatty alcohol may be converted to olefins through FCC reaction or to higher alkanes through a condensation reaction.
[0304] One such chemical modification is hydrogenation, which is the addition of hydrogen to double bonds in the fatty acid constituents of glycerolipids or of free fatty acids. The hydrogenation process permits the transformation of liquid oils into semi-solid or solid fats, which may be more suitable for specific applications.
[0305] Hydrogenation of oil produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials provided herein, as reported in the following: U.S. Pat. No. 7,288,278 (Food additives or medicaments); U.S. Pat. No. 5,346,724 (Lubrication products); U.S. Pat. No. 5,475,160 (Fatty alcohols); U.S. Pat. No. 5,091,116 (Edible oils); U.S. Pat. No. 6,808,737 (Structural fats for margarine and spreads); U.S. Pat. No. 5,298,637 (Reduced-calorie fat substitutes); U.S. Pat. No. 6,391,815 (Hydrogenation catalyst and sulfur adsorbent); U.S. Pat. No. 5,233,099 and U.S. Pat. No. 5,233,100 (Fatty alcohols); U.S. Pat. No. 4,584,139 (Hydrogenation catalysts); U.S. Pat. No. 6,057,375 (Foam suppressing agents); and U.S. Pat. No. 7,118,773 (Edible emulsion spreads).
[0306] One skilled in the art will recognize that various processes may be used to hydrogenate carbohydrates. One suitable method includes contacting the carbohydrate with hydrogen or hydrogen mixed with a suitable gas and a catalyst under conditions sufficient in a hydrogenation reactor to form a hydrogenated product. The hydrogenation catalyst generally can include Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or any combination thereof, either alone or with promoters such as W, Mo, Au, Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof. Other effective hydrogenation catalyst materials include either supported nickel or ruthenium modified with rhenium. In an embodiment, the hydrogenation catalyst also includes any one of the supports, depending on the desired functionality of the catalyst. The hydrogenation catalysts may be prepared by methods known to those of ordinary skill in the art.
[0307] In some embodiments the hydrogenation catalyst includes a supported Group VIII metal catalyst and a metal sponge material (e.g., a sponge nickel catalyst). Raney nickel provides an example of an activated sponge nickel catalyst suitable for use in this invention. In other embodiment, the hydrogenation reaction in the invention is performed using a catalyst comprising a nickel-rhenium catalyst or a tungsten-modified nickel catalyst. One example of a suitable catalyst for the hydrogenation reaction of the invention is a carbon-supported nickel-rhenium catalyst.
[0308] In an embodiment, a suitable Raney nickel catalyst may be prepared by treating an alloy of approximately equal amounts by weight of nickel and aluminum with an aqueous alkali solution, e.g., containing about 25 weight % of sodium hydroxide. The aluminum is selectively dissolved by the aqueous alkali solution resulting in a sponge shaped material comprising mostly nickel with minor amounts of aluminum. The initial alloy includes promoter metals (i.e., molybdenum or chromium) in the amount such that about 1 to 2 weight % remains in the formed sponge nickel catalyst. In another embodiment, the hydrogenation catalyst is prepared using a solution of ruthenium (III) nitrosylnitrate, ruthenium (III) chloride in water to impregnate a suitable support material. The solution is then dried to form a solid having a water content of less than about 1% by weight. The solid may then be reduced at atmospheric pressure in a hydrogen stream at 300.degree. C. (uncalcined) or 400.degree. C. (calcined) in a rotary ball furnace for 4 hours. After cooling and rendering the catalyst inert with nitrogen, 5% by volume of oxygen in nitrogen is passed over the catalyst for 2 hours.
[0309] In certain embodiments, the catalyst described includes a catalyst support. The catalyst support stabilizes and supports the catalyst. The type of catalyst support used depends on the chosen catalyst and the reaction conditions. Suitable supports for the invention include, but are not limited to, carbon, silica, silica-alumina, zirconia, titania, ceria, vanadia, nitride, boron nitride, heteropolyacids, hydroxyapatite, zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene and any combination thereof.
[0310] The catalysts used in this invention can be prepared using conventional methods known to those in the art. Suitable methods may include, but are not limited to, incipient wetting, evaporative impregnation, chemical vapor deposition, wash-coating, magnetron sputtering techniques, and the like.
[0311] The conditions for which to carry out the hydrogenation reaction will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate reaction conditions. In general, the hydrogenation reaction is conducted at temperatures of 80.degree. C. to 250.degree. C., and preferably at 90.degree. C. to 200.degree. C., and most preferably at 100.degree. C. to 150.degree. C. In some embodiments, the hydrogenation reaction is conducted at pressures from 500 KPa to 14000 KPa.
[0312] The hydrogen used in the hydrogenolysis reaction of the current invention may include external hydrogen, recycled hydrogen, in situ generated hydrogen, and any combination thereof. As used herein, the term "external hydrogen" refers to hydrogen that does not originate from the biomass reaction itself, but rather is added to the system from another source.
[0313] In some embodiments of the invention, it is desirable to convert the starting carbohydrate to a smaller molecule that will be more readily converted to desired higher hydrocarbons. One suitable method for this conversion is through a hydrogenolysis reaction. Various processes are known for performing hydrogenolysis of carbohydrates. One suitable method includes contacting a carbohydrate with hydrogen or hydrogen mixed with a suitable gas and a hydrogenolysis catalyst in a hydrogenolysis reactor under conditions sufficient to form a reaction product comprising smaller molecules or polyols. As used herein, the term "smaller molecules or polyols" includes any molecule that has a smaller molecular weight, which can include a smaller number of carbon atoms or oxygen atoms than the starting carbohydrate. In an embodiment, the reaction products include smaller molecules that include polyols and alcohols. Someone of ordinary skill in the art would be able to choose the appropriate method by which to carry out the hydrogenolysis reaction.
[0314] In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may be converted to propylene glycol, ethylene glycol, and glycerol using a hydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo, W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or any combination thereof, either alone or with promoters such as Au, Ag, Cr, Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. The hydrogenolysis catalyst may also include a carbonaceous pyropolymer catalyst containing transition metals (e.g., chromium, molybdenum, tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals (e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium, iridium, and osmium). In certain embodiments, the hydrogenolysis catalyst may include any of the above metals combined with an alkaline earth metal oxide or adhered to a catalytically active support. In certain embodiments, the catalyst described in the hydrogenolysis reaction may include a catalyst support as described above for the hydrogenation reaction.
[0315] The conditions for which to carry out the hydrogenolysis reaction will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In general, they hydrogenolysis reaction is conducted at temperatures of 110.degree. C. to 300.degree. C., and preferably at 170.degree. C. to 220.degree. C., and most preferably at 200.degree. C. to 225.degree. C. In some embodiments, the hydrogenolysis reaction is conducted under basic conditions, preferably at a pH of 8 to 13, and even more preferably at a pH of 10 to 12. In some embodiments, the hydrogenolysis reaction is conducted at pressures in a range between 60 KPa and 16500 KPa, and preferably in a range between 1700 KPa and 14000 KPa, and even more preferably between 4800 KPa and 11000 KPa.
[0316] The hydrogen used in the hydrogenolysis reaction of the current invention can include external hydrogen, recycled hydrogen, in situ generated hydrogen, and any combination thereof.
[0317] In some embodiments, the reaction products discussed above may be converted into higher hydrocarbons through a condensation reaction in a condensation reactor. In such embodiments, condensation of the reaction products occurs in the presence of a catalyst capable of forming higher hydrocarbons. While not intending to be limited by theory, it is believed that the production of higher hydrocarbons proceeds through a stepwise addition reaction including the formation of carbon-carbon, or carbon-oxygen bond. The resulting reaction products include any number of compounds containing these moieties, as described in more detail below.
[0318] In certain embodiments, suitable condensation catalysts include an acid catalyst, a base catalyst, or an acid/base catalyst. As used herein, the term "acid/base catalyst" refers to a catalyst that has both an acid and a base functionality. In some embodiments the condensation catalyst can include, without limitation, zeolites, carbides, nitrides, zirconia, alumina, silica, aluminosilicates, phosphates, titanium oxides, zinc oxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandium oxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides, hydroxides, heteropolyacids, inorganic acids, acid modified resins, base modified resins, and any combination thereof. In some embodiments, the condensation catalyst can also include a modifier. Suitable modifiers include La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and any combination thereof. In some embodiments, the condensation catalyst can also include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe, Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys, and any combination thereof.
[0319] In certain embodiments, the catalyst described in the condensation reaction may include a catalyst support as described above for the hydrogenation reaction. In certain embodiments, the condensation catalyst is self-supporting. As used herein, the term "self-supporting" means that the catalyst does not need another material to serve as support. In other embodiments, the condensation catalyst in used in conjunction with a separate support suitable for suspending the catalyst. In an embodiment, the condensation catalyst support is silica.
[0320] The conditions under which the condensation reaction occurs will vary based on the type of starting material and the desired products. One of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In some embodiments, the condensation reaction is carried out at a temperature at which the thermodynamics for the proposed reaction are favorable. The temperature for the condensation reaction will vary depending on the specific starting polyol or alcohol. In some embodiments, the temperature for the condensation reaction is in a range from 80.degree. C. to 500.degree. C., and preferably from 125.degree. C. to 450.degree. C., and most preferably from 125.degree. C. to 250.degree. C. In some embodiments, the condensation reaction is conducted at pressures in a range between 0 Kpa to 9000 KPa, and preferably in a range between 0 KPa and 7000 KPa, and even more preferably between 0 KPa and 5000 KPa.
[0321] The higher alkanes formed by the invention include, but are not limited to, branched or straight chain alkanes that have from 4 to 30 carbon atoms, branched or straight chain alkenes that have from 4 to 30 carbon atoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenes that have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, and ketones. Suitable alkanes include, but are not limited to, butane, pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane, 3-methylpentane, 2,2,-dimethylbutane, 2,3-dimethylbutane, heptane, heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane, 2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane, decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene, tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane, nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane, doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, and isomers thereof. Some of these products may be suitable for use as fuels.
[0322] In some embodiments, the cycloalkanes and the cycloalkenes are unsubstituted. In other embodiments, the cycloalkanes and cycloalkenes are mono-substituted. In still other embodiments, the cycloalkanes and cycloalkenes are multi-substituted. In the embodiments comprising the substituted cycloalkanes and cycloalkenes, the substituted group includes, without limitation, a branched or straight chain alkyl having 1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to 12 carbon atoms, a phenyl, and any combination thereof. Suitable cycloalkanes and cycloalkenes include, but are not limited to, cyclopentane, cyclopentene, cyclohexane, cyclohexene, methyl-cyclopentane, methyl-cyclopentene, ethyl-cyclopentane, ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers and any combination thereof.
[0323] In some embodiments, the aryls formed are unsubstituted. In another embodiment, the aryls formed are mono-substituted. In the embodiments comprising the substituted aryls, the substituted group includes, without limitation, a branched or straight chain alkyl having 1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to 12 carbon atoms, a phenyl, and any combination thereof. Suitable aryls for the invention include, but are not limited to, benzene, toluene, xylene, ethyl benzene, para xylene, meta xylene, and any combination thereof.
[0324] The alcohols produced in the invention have from 4 to 30 carbon atoms. In some embodiments, the alcohols are cyclic. In other embodiments, the alcohols are branched. In another embodiment, the alcohols are straight chained. Suitable alcohols for the invention include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol, uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomers thereof.
[0325] The ketones produced in the invention have from 4 to 30 carbon atoms. In an embodiment, the ketones are cyclic. In another embodiment, the ketones are branched. In another embodiment, the ketones are straight chained. Suitable ketones for the invention include, but are not limited to, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, undecanone, dodecanone, tridecanone, tetradecanone, pentadecanone, hexadecanone, heptyldecanone, octyldecanone, nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone, tetraeicosanone, and isomers thereof.
[0326] Another such chemical modification is interesterification. Naturally produced glycerolipids do not have a uniform distribution of fatty acid constituents. In the context of oils, interesterification refers to the exchange of acyl radicals between two esters of different glycerolipids. The interesterification process provides a mechanism by which the fatty acid constituents of a mixture of glycerolipids can be rearranged to modify the distribution pattern. Interesterification is a well-known chemical process, and generally comprises heating (to about 200.degree. C.) a mixture of oils for a period (e.g., 30 minutes) in the presence of a catalyst, such as an alkali metal or alkali metal alkylate (e.g., sodium methoxide). This process can be used to randomize the distribution pattern of the fatty acid constituents of an oil mixture, or can be directed to produce a desired distribution pattern. This method of chemical modification of lipids can be performed on materials provided herein, such as microbial biomass with a percentage of dry cell weight as lipid at least 20%.
[0327] Directed interesterification, in which a specific distribution pattern of fatty acids is sought, can be performed by maintaining the oil mixture at a temperature below the melting point of some TAGs which might occur. This results in selective crystallization of these TAGs, which effectively removes them from the reaction mixture as they crystallize. The process can be continued until most of the fatty acids in the oil have precipitated, for example. A directed interesterification process can be used, for example, to produce a product with a lower calorie content via the substitution of longer-chain fatty acids with shorter-chain counterparts. Directed interesterification can also be used to produce a product with a mixture of fats that can provide desired melting characteristics and structural features sought in food additives or products (e.g., margarine) without resorting to hydrogenation, which can produce unwanted trans isomers.
[0328] Interesterification of oils produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials, or to produce products, as reported in the following: U.S. Pat. No. 6,080,853 (Nondigestible fat substitutes); U.S. Pat. No. 4,288,378 (Peanut butter stabilizer); U.S. Pat. No. 5,391,383 (Edible spray oil); U.S. Pat. No. 6,022,577 (Edible fats for food products); U.S. Pat. No. 5,434,278 (Edible fats for food products); U.S. Pat. No. 5,268,192 (Low calorie nut products); U.S. Pat. No. 5,258,197 (Reduce calorie edible compositions); U.S. Pat. No. 4,335,156 (Edible fat product); U.S. Pat. No. 7,288,278 (Food additives or medicaments); U.S. Pat. No. 7,115,760 (Fractionation process); U.S. Pat. No. 6,808,737 (Structural fats); U.S. Pat. No. 5,888,947 (Engine lubricants); U.S. Pat. No. 5,686,131 (Edible oil mixtures); and U.S. Pat. No. 4,603,188 (Curable urethane compositions).
[0329] In one embodiment in accordance with the invention, transesterification of the oil, as described above, is followed by reaction of the transesterified product with polyol, as reported in U.S. Pat. No. 6,465,642, to produce polyol fatty acid polyesters. Such an esterification and separation process may comprise the steps as follows: reacting a lower alkyl ester with polyol in the presence of soap; removing residual soap from the product mixture; water-washing and drying the product mixture to remove impurities; bleaching the product mixture for refinement; separating at least a portion of the unreacted lower alkyl ester from the polyol fatty acid polyester in the product mixture; and recycling the separated unreacted lower alkyl ester.
[0330] Transesterification can also be performed on microbial biomass with short chain fatty acid esters, as reported in U.S. Pat. No. 6,278,006. In general, transesterification may be performed by adding a short chain fatty acid ester to an oil in the presence of a suitable catalyst and heating the mixture. In some embodiments, the oil comprises about 5% to about 90% of the reaction mixture by weight. In some embodiments, the short chain fatty acid esters can be about 10% to about 50% of the reaction mixture by weight. Non-limiting examples of catalysts include base catalysts, sodium methoxide, acid catalysts including inorganic acids such as sulfuric acid and acidified clays, organic acids such as methane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid, and acidic resins such as Amberlyst 15. Metals such as sodium and magnesium, and metal hydrides also are useful catalysts.
[0331] Another such chemical modification is hydroxylation, which involves the addition of water to a double bond resulting in saturation and the incorporation of a hydroxyl moiety. The hydroxylation process provides a mechanism for converting one or more fatty acid constituents of a glycerolipid to a hydroxy fatty acid. Hydroxylation can be performed, for example, via the method reported in U.S. Pat. No. 5,576,027. Hydroxylated fatty acids, including castor oil and its derivatives, are useful as components in several industrial applications, including food additives, surfactants, pigment wetting agents, defoaming agents, water proofing additives, plasticizing agents, cosmetic emulsifying and/or deodorant agents, as well as in electronics, pharmaceuticals, paints, inks, adhesives, and lubricants. One example of how the hydroxylation of a glyceride may be performed is as follows: fat may be heated, preferably to about 30-50.degree. C. combined with heptane and maintained at temperature for thirty minutes or more; acetic acid may then be added to the mixture followed by an aqueous solution of sulfuric acid followed by an aqueous hydrogen peroxide solution which is added in small increments to the mixture over one hour; after the aqueous hydrogen peroxide, the temperature may then be increased to at least about 60.degree. C. and stirred for at least six hours; after the stirring, the mixture is allowed to settle and a lower aqueous layer formed by the reaction may be removed while the upper heptane layer formed by the reaction may be washed with hot water having a temperature of about 60.degree. C.; the washed heptane layer may then be neutralized with an aqueous potassium hydroxide solution to a pH of about 5 to 7 and then removed by distillation under vacuum; the reaction product may then be dried under vacuum at 100.degree. C. and the dried product steam-deodorized under vacuum conditions and filtered at about 50.degree. to 60.degree. C. using diatomaceous earth.
[0332] Hydroxylation of microbial oils produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials, or to produce products, as reported in the following: U.S. Pat. No. 6,590,113 (Oil-based coatings and ink); U.S. Pat. No. 4,049,724 (Hydroxylation process); U.S. Pat. No. 6,113,971 (Olive oil butter); U.S. Pat. No. 4,992,189 (Lubricants and lube additives); U.S. Pat. No. 5,576,027 (Hydroxylated milk); and U.S. Pat. No. 6,869,597 (Cosmetics).
[0333] Hydroxylated glycerolipids can be converted to estolides. Estolides consist of a glycerolipid in which a hydroxylated fatty acid constituent has been esterified to another fatty acid molecule. Conversion of hydroxylated glycerolipids to estolides can be carried out by warming a mixture of glycerolipids and fatty acids and contacting the mixture with a mineral acid, as described by Isbell et al., JAOCS 71(2):169-174 (1994). Estolides are useful in a variety of applications, including without limitation those reported in the following: U.S. Pat. No. 7,196,124 (Elastomeric materials and floor coverings); U.S. Pat. No. 5,458,795 (Thickened oils for high-temperature applications); U.S. Pat. No. 5,451,332 (Fluids for industrial applications); U.S. Pat. No. 5,427,704 (Fuel additives); and U.S. Pat. No. 5,380,894 (Lubricants, greases, plasticizers, and printing inks).
[0334] Another such chemical modification is olefin metathesis. In olefin metathesis, a catalyst severs the alkylidene carbons in an alkene (olefin) and forms new alkenes by pairing each of them with different alkylidine carbons. The olefin metathesis reaction provides a mechanism for processes such as truncating unsaturated fatty acid alkyl chains at alkenes by ethenolysis, cross-linking fatty acids through alkene linkages by self-metathesis, and incorporating new functional groups on fatty acids by cross-metathesis with derivatized alkenes.
[0335] In conjunction with other reactions, such as transesterification and hydrogenation, olefin metathesis can transform unsaturated glycerolipids into diverse end products. These products include glycerolipid oligomers for waxes; short-chain glycerolipids for lubricants; homo- and hetero-bifunctional alkyl chains for chemicals and polymers; short-chain esters for biofuel; and short-chain hydrocarbons for jet fuel. Olefin metathesis can be performed on triacylglycerols and fatty acid derivatives, for example, using the catalysts and methods reported in U.S. Pat. No. 7,119,216, US Patent Pub. No. 2010/0160506, and U.S. Patent Pub. No. 2010/0145086.
[0336] Olefin metathesis of bio-oils generally comprises adding a solution of Ru catalyst at a loading of about 10 to 250 ppm under inert conditions to unsaturated fatty acid esters in the presence (cross-metathesis) or absence (self-metathesis) of other alkenes. The reactions are typically allowed to proceed from hours to days and ultimately yield a distribution of alkene products. One example of how olefin metathesis may be performed on a fatty acid derivative is as follows: A solution of the first generation Grubbs Catalyst (dichloro[2(1-methylethoxy-.alpha.-O)phenyl]methylene-.alpha.-C] (tricyclohexyl-phosphine) in toluene at a catalyst loading of 222 ppm may be added to a vessel containing degassed and dried methyl oleate. Then the vessel may be pressurized with about 60 psig of ethylene gas and maintained at or below about 30.degree. C. for 3 hours, whereby approximately a 50% yield of methyl 9-decenoate may be produced.
[0337] Olefin metathesis of oils produced by the methods described herein can be performed in conjunction with one or more of the methods and/or materials, or to produce products, as reported in the following: Patent App. PCT/US07/081427 (.alpha.-olefin fatty acids) and U.S. patent application Ser. No. 12/281,938 (petroleum creams), Ser. No. 12/281,931 (paintball gun capsules), Ser. No. 12/653,742 (plasticizers and lubricants), Ser. No. 12/422,096 (bifunctional organic compounds), and Ser. No. 11/795,052 (candle wax).
[0338] Other chemical reactions that can be performed on microbial oils include reacting triacylglycerols with a cyclopropanating agent to enhance fluidity and/or oxidative stability, as reported in U.S. Pat. No. 6,051,539; manufacturing of waxes from triacylglycerols, as reported in U.S. Pat. No. 6,770,104; and epoxidation of triacylglycerols, as reported in "The effect of fatty acid composition on the acrylation kinetics of epoxidized triacylglycerols", Journal of the American Oil Chemists' Society, 79:1, 59-63, (2001) and Free Radical Biology and Medicine, 37:1, 104-114 (2004).
[0339] The generation of oil-bearing microbial biomass for fuel and chemical products as described above results in the production of delipidated biomass meal. Delipidated meal is a byproduct of preparing algal oil and is useful as animal feed for farm animals, e.g., ruminants, poultry, swine and aquaculture. The resulting meal, although of reduced oil content, still contains high quality proteins, carbohydrates, fiber, ash, residual oil and other nutrients appropriate for an animal feed. Because the cells are predominantly lysed by the oil separation process, the delipidated meal is easily digestible by such animals. Delipidated meal can optionally be combined with other ingredients, such as grain, in an animal feed. Because delipidated meal has a powdery consistency, it can be pressed into pellets using an extruder or expander or another type of machine, which are commercially available.
[0340] The invention, having been described in detail above, is exemplified in the following examples, which are offered to illustrate, but not to limit, the claimed invention.
XIV. EXAMPLES
Example 1
Fatty Acid Analysis by Fatty Acid Methyl Ester Detection
[0341] Lipid samples were prepared from dried biomass. 20-40 mg of dried biomass was resuspended in 2 mL of 5% H.sub.2SO.sub.4 in MeOH, and 200 ul of toluene containing an appropriate amount of a suitable internal standard (C19:0) was added. The mixture was sonicated briefly to disperse the biomass, then heated at 70-75.degree. C. for 3.5 hours. 2 mL of heptane was added to extract the fatty acid methyl esters, followed by addition of 2 mL of 6% K.sub.2CO.sub.3 (aq) to neutralize the acid. The mixture was agitated vigorously, and a portion of the upper layer was transferred to a vial containing Na.sub.2SO.sub.4 (anhydrous) for gas chromatography analysis using standard FAME GC/FID (fatty acid methyl ester gas chromatography flame ionization detection) methods. Fatty acid profiles reported below were determined by this method.
Example 2
Triacylglyceride Purification from Oil and Methods for Triacylglyceride Lipase Digestion
[0342] The triacylglyceride (TAG) fraction of each oil sample was isolated by dissolving .about.10 mg of oil in dichloromethane and loading it onto a Bond-Elut aminopropyl solid-phase extraction cartridge (500 mg) preconditioned with heptane. TAGs were eluted with dicholoromethane-MeOH (1:1) into a collection tube, while polar lipids were retained on the column. The solvent was removed with a stream of nitrogen gas. Tris buffer and 2 mg porcine pancreatic lipase (Type II, Sigma, 100-400 units/mg) were added to the TAG fraction, followed by addition of bile salt and calcium chloride solutions. The porcine pancreatic lipase cleaves sn-1 and sn-3 fatty acids, thereby generating 2-monoacylglycerides and free fatty acids. This mixture was heated with agitation at 40.degree. C. for three minutes, cooled briefly, then quenched with 6 N HCl. The mixture was then extracted with diethyl ether and the ether layer was washed with water then dried over sodium sulfate. The solvent was removed with a stream of nitrogen. To isolate the monoacylglyceride (MAG) fraction, the residue was dissolved in heptane and loaded onto a second aminopropyl solid phase extraction cartridge pretreated with heptane. Residual TAGs were eluted with diethyl ether-dichloromethane-heptane (1:9:40), diacylglycerides (DAGs) were eluted with ethyl acetate-heptane (1:4), and MAGs were eluted from the cartridge with dichloromethane-methanol (2:1). The resulting MAG, DAG, and TAG fractions were then concentrated to dryness with a stream of nitrogen and subjected to routine direct transesterification method of GC/FID analysis as described in Example 1.
Example 3
Engineering Microorganisms for Fatty Acid and SN-2 Profiles Increased in Lauric Acid Through Exogenous LPAAT Expression
[0343] This example describes the use of recombinant polynucleotides that encode a C. nucifera 1-acyl-sn-glycerol-3-phosphate acyltransferase (Cn LPAAT) enzyme to engineer a microorganism in which the fatty acid profile and the sn-2 profile of the transformed microorganism has been enriched in lauric acid.
[0344] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain A, was initially transformed with the plasmid construct pSZ1283 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. pSZ1283, described in PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696 hereby incorporated by reference, comprised the coding sequence of the Cuphea wrightii FATB2 (CwTE2) thioesterase (SEQ ID NO: 10), 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), to express the protein sequence given in SEQ ID NO: 3, under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. The CwTE2 protein coding sequence to express the protein sequence given in SEQ ID NO: 11, was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR. The protein coding regions of CwTE2 and suc2 were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/U52009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0345] Upon transformation of pSZ1283 into Strain A, positive clones were selected on agar plates with sucrose as the sole carbon source. Primary transformants were then clonally purified and a single transformant, Strain B, was selected for further genetic modification. This genetically engineered strain was transformed with plasmid construct pSZ2046 to interrupt the pLoop genomic locus of Strain B. Construct pSZ2046 comprised the coding sequence of the C. nucifera 1-acyl-sn-glycerol-3-phosphate acyltransferase (Cn LPAAT) enzyme (SEQ ID NO: 12), 5' (SEQ ID NO: 13) and 3' (SEQ ID NO: 14) homologous recombination targeting sequences (flanking the construct) to the pLoop genomic region for integration into the nuclear genome, and a neomycin resistance protein-coding sequence under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5), and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This NeoR expression cassette is listed as SEQ ID NO: 15 and served as a selectable marker. The Cn LPAAT protein coding sequence was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR. The protein coding regions of Cn LPAAT and NeoR were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. The amino acid sequence of Cn LPAAT is provided as SEQ ID NO: 16.
[0346] Upon transformation of pSZ2046 into Strain B, thereby generating Strain C, positive clones were selected on agar plates comprising G418 (Geneticin). Individual transformants were clonally purified and grown at pH 7.0 under conditions suitable for lipid production as detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass from each transformant and fatty acid profiles from these samples were analyzed using standard fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1.The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis UTEX 1435 (U1) grown on glucose as a sole carbon source, untransformed Strain B and five pSZ2046 positive transformants (Strain C, 1-5) are presented in Table 6.
TABLE-US-00008 TABLE 6 Effect of LPAAT expression on fatty acid profiles of transformed Prototheca moriformis (UTEX 1435) comprising a mid-chain preferring thioesterase. Area % Fatty Strain Strain Strain Strain Strain Strain acid U1 B C-1 C-2 C-3 C-4 C-5 C10:0 0.01 5.53 11.37 11.47 10.84 11.13 11.12 C12:0 0.04 31.04 46.63 46.47 45.84 45.80 45.67 C14:0 1.27 15.99 15.14 15.12 15.20 15.19 15.07 C16:0 27.20 12.49 7.05 7.03 7.30 7.20 7.19 C18:0 3.85 1.30 0.71 0.72 0.74 0.74 0.74 C18:1 58.70 24.39 10.26 10.41 10.95 11.31 11.45 C18:2 7.18 7.79 7.05 6.93 7.30 6.88 7.01 C10- 0.50 36.57 58.00 57.94 56.68 56.93 56.79 C12
[0347] As shown in Table 6, the fatty acid profile of Strain B expressing CwTE2 showed increased composition of C10:0, C12:0, and C14:0 fatty acids and a decrease in C16:0, C18:0, and C18:1 fatty acids relative to the fatty acid profile of the untransformed UTEX 1435 strain. The impact of additional genetic modification on the fatty acid profile of the transformed strains, namely the expression of CnLPAAT in Strain B, is a still further increase in the composition of C10:0 and C12:0 fatty acids, a still further decrease in C16:0, C18:0, and C18:1 fatty acids, but no significant effect on the C14:0 fatty acid composition. These data indicate that the CnLPAAT shows substrate preference in the context of a microbial host organism.
[0348] The untransformed P. moriformis Strain A is characterized by a fatty acid profile comprising less than 0.5% C12 fatty acids and less than 1% C10-C12 fatty acids. In contrast, the fatty acid profile of Strain B expressing a C. wrightii thioesterase comprised 31% C12:0 fatty acids, with C10-C12 fatty acids comprising greater than 36% of the total fatty acids. Further, fatty acid profiles of Strain C, expressing a higher plant thioesterase and a CnLPAAT enzyme, comprised between 45.67% and 46.63% C12:0 fatty acids, with C10-C12% fatty acids comprising between 71 and 73% of total fatty acids. The result of expressing an exogenous thioesterase was a 62-fold increase in the percentage of C12 fatty acid present in the engineered microbe. The result of expressing an exogenous thioesterase and exogenous LPAAT was a 92-fold increase in the percentage of C12 fatty acids present in the engineered microbe.
[0349] The TAG fraction of oil samples extracted from Strains A, B, and C were analyzed for the sn-2 profile of their triacylglycerides. The TAGs were extracted and processed as described in Example 2 and analyzed as in Examples 1 and 2. The fatty acid composition and the sn-2 profiles of the TAG fraction of oil extracted from Strains A, B, and C (expressed as Area % of total fatty acids) are presented in Table 7. Values not reported are indicated as "n.r."
TABLE-US-00009 TABLE 7 Effect of LPAAT expression on the fatty acid composition and the sn-2 profile of TAGs produced from transformed Prototheca moriformis (UTEX 1435) comprising a mid-chain preferring thioesterase. Strain Area Strain C % Strain A Strain B (pSZ1500 + fatty (untransformed) (pSZ1500) pSZ2046) acid FA sn-2 profile FA sn-2 profile FA sn-2 profile C10:0 n.r. n.r. 11.9 14.2 12.4 7.1 C12:0 n.r. n.r. 42.4 25 47.9 52.8 C14:0 1.0 0.6 12 10.4 13.9 9.1 C16:0 23.9 1.6 7.2 1.3 6.1 0.9 C18:0 3.7 0.3 n.r n.r. 0.8 0.3 C18:1 64.3 90.5 18.3 36.6 9.9 17.5 C18:2 4.5 5.8 5.8 10.8 6.5 10 C18:3 n.r. n.r. n.r. n.r. 1.1 1.6
[0350] As shown in Table 7, the fatty acid composition of triglycerides (TAGs) isolated from Strain B expressing CwTE2 was increased for C10:0, C12:0, and C14:0 fatty acids and decrease in C16:0 and C18:1 fatty acids relative to the fatty acid profile of TAGs isolated from untransformed Strain A. The impact of additional genetic modification on the fatty acid profile of the transformed strains, namely the expression of CnLPAAT, was a still further increase in the composition of C10:0 and C12:0 fatty acids, a still further decrease in C16:0, C18:0, and C18:1 fatty acids, but no significant effect on the C14:0 fatty acid composition. These data indicate that expression of the exogenous CnLPAAT improves the midchain fatty acid profile of transformed microbes.
[0351] The untransformed P. moriformis Strain A is characterized by an sn-2 profile of about 0.6% C14, about 1.6% C16:0, about 0.3% C18:0, about 90% C18:1, and about 5.8% C18:2. In contrast to Strain A, Strain B, expressing a C. wrightii thioesterase is characterized by an sn-2 profile that is higher in midchain fatty acids and lower in long chain fatty acids. C12 fatty acids comprised 25% of the sn-2 profile of Strain B. The impact of additional genetic modification on the sn-2 profile of the transformed strains, namely the expression of CnLPAAT, was still a further increase in C12 fatty acids (from 25% to 52.8%), a decrease in C18:1 fatty acids (from 36.6% to 17.5%), and a decrease in C10:0 fatty acids. (The sn-2 profile composition of C14:0 and C16:0 fatty acids was relatively similar for Strains B and C.)
[0352] These data demonstrate the utility and effectiveness of polynucleotides permitting exogenous LPAAT expression to alter the fatty acid profile of engineered microorganisms, and in particular in increasing the concentration of C10:0 and C12:0 fatty acids in microbial cells. These data further demonstrate the utility and effectiveness of polynucleotides permitting exogenous thioesterase and exogenous LPAAT expression to alter the sn-2 profile of TAGs produced by microbial cells, in particular in increasing the C12 composition of sn-2 profiles and decreasing the C18:1 composition of sn-2 profiles.
Example 4
Thermal Behavior of Oils Produced from Recombinant Microalgae
[0353] FIGS. 1-14 include fatty acid profiles and melting curves of refined, bleached and deodorized oils from genetically engineered Prototheca moriformis strains. In some cases, modifications of the melting curves are obtained via genetic engineering. For example, some of the oils produced have shallower or sharper melting transitions relative to control microalgal oils (i.e., those produced from strains lacking a given genetic modification) or relative to widely available plant oils. In addition, FIG. 12 shows scanning calorimetry for a high palmitic oil when tempered by holding at room temperature for several days (lower trace) and for the same oil after performing the first scan (upper trace). The scans ranged from -60.degree. C. to +50.degree. C. with a heating rate of 10.degree. C/minute. The differences between the two traces suggests that tempering of the oil caused a change in crystal structure within the oil.
[0354] Also of note, FIGS. 10 and 11 show stability testing of RBD-5 and RBD 6. Remarkably, RBD-6, an oil with less than 0.1% 18:2 and 18:3 fatty acids was substantially stable as measured by the oxidative stability index (AOCS Method Cd 12b-92) even after 36 hours of heating at 110.degree. C.
[0355] Table 8, below, gives details of the genetic engineering of the strains identified in FIGS. 1-13.
TABLE-US-00010 TABLE 8 Genetically engineered strains. RB Z Ulmus Americana thioesterase RBD-1 Cuphea wrightii FATB2 thioesterase driven by amt03 RBD-2 Ulmus americana thioesterase RBD-3 Native C. hookeriana C16:0-specific thioesterase with amt03 promoter RBD Y Ulmus Americana thioesterase with Btub promoter RBD X SAD2B knockout with native C wrightii FAT2B thioesterase, amt03 promoter RBD W SAD2B KO with Native C. wrightii FATB2 driven by amt03 at insertion site RBD-4 control strain RBD-5 FATA-1 knockout with Carthamus oleate sp. TE driven by amt03 promoter at insertion site RBD-6 FADc knockout with Carthamus tinctorius oleoyl thioesterase
Example 5
Characteristics of Processed Oil Produced from Engineered Microorganisms
[0356] Methods and effects of transforming Prototheca moriformis (UTEX 1435) with transformation vector pSZ1500 (SEQ ID NO: 17) have been previously described in PCT Application Nos. PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0357] A classically mutagenized (for higher oil production) derivative of Prototheca moriformis (UTEX 1435), Strain A, was transformed with pSZ1500 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. pSZ1500 comprised nucleotide sequence of the Carthamus tinctorius oleyl-thioesterase (CtOTE) gene, codon-optimized for expression in P. moriformis UTEX 1435. The pSZ1500 expression construct included 5' (SEQ ID NO: 18) and 3' (SEQ ID NO: 19) homologous recombination targeting sequences (flanking the construct) to the FADc genomic region for integration into the nuclear genome and a S. cerevisiae suc2 sucrose invertase coding region under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selection marker. The CtOTE coding region was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR, and the native transit peptide was replaced with the C. protothecoides stearoyl-ACP desaturase transit peptide (SEQ ID NO: 9). The protein coding regions of CtOTE and suc2 were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0358] Primary pSZ1500 transformants of Strain A were selected on agar plates containing sucrose as a sole carbon source, clonally purified, and a single engineered line, Strain D was selected for analysis. Strain D was grown as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Hexane extraction of the oil from the generated biomass was then performed using standard methods, and the resulting triglyceride oil was determined to be free of residual hexane. Other methods of extraction of oil from microalgae using an expeller press are described in PCT Application No. PCT/US2010/031108 and are hereby incorporated by reference.
[0359] Different lots of oil extracted from biomass of Strain D were refined, bleached, and deodorized using standard vegetable oil processing methods. These procedures generated oil samples RBD437, RBD469, RBD501, RBD 502, RBD503, and RBD529, which were subjected to analytical testing protocols according to methods defined through the American Oil Chemists' Society, the American Society for Testing and Materials, and the International Organization for Standardization. The results of these analyses are summarized below in Tables 9-14.
TABLE-US-00011 TABLE 9 Analytical results for oil sample RBD469. Method Number Test Description Results Units AOCS Ca 3a-46 Insoluble impurities <0.01 % AOCS Ca 5a-40 Free Fatty Acids (Oleic) 0.02 % AOCS Ca 5a-40 Acid Value 0.04 mg KOH/g AOCS CA 9f-57 Neutral oil 98.9 % D97 Cloud Point -15 deg C. D97 Pour Point -18 deg C. Karl Fischer Moisture 0.01 % AOCS Cc 13d-55 Chlorophyll <0.01 ppm (modified) Iodine Value 78.3 g I.sub.2/100 g AOCS Cd 8b-90 Peroxide Value 0.31 meq/kg ISO 6885 p-Anisidine Value 0.65 AOCS Cc 18-80 Dropping Melting point 6.2 deg C. (Mettler) AOCS Cd 11d-96 Tricylglicerides 98.6 % AOCS Cd 11d-96 Monoglyceride <0.01 % AOCS Cd 11d-96 Diglycerides 0.68 % AOCS Cd 20-91 Total Polar Compounds 2.62 % IUPAC, 2.507 and Oxidized & Polymerized 17.62 % 2.508 Triacylglycerides AOCS Cc 9b-55 Flash Point 244 deg C. AOCS Cc 9a-48 Smoke Point 232 deg C. AOCS Cd 12b-92 Oxidataive Stability Index 31.6 hours Rancimat (110.degree. C.) AOCS Ca 6a-40 Unsaponified Matter 2.28 %
[0360] RBD469 oil was analyzed for trace element content, solid fat content, and Lovibond color according to AOCS methods. Results of these analyses are presented below in Table 10, Table 10, and Table 11.
TABLE-US-00012 TABLE 10 ICP Elemental Analysis of RBD469 oil. Method Number Test Description Results in ppm AOCS Ca 20-99 and Phosphorus 1.09 AOCS Ca 17-01 Calcium 0.1 (modified) Magnesium 0.04 Iron <0.02 Sulfur 28.8 Copper <0.05 Potassium <0.50 Sodium <0.50 Silicon 0.51 Boron 0.06 Aluminum <0.20 Lead <0.20 Lithium <0.02 Nickel <0.20 Vanadium <0.05 Zinc <0.02 Arsenic <0.20 Mercury <0.20 Cadmium <0.03 Chromium <0.02 Manganese <0.05 Silver <0.05 Titanium <0.05 Selenium <0.50 UOP779 Chloride organic <1 UOP779 Chloride inorganic 7.24 AOCS Ba 4e-93 Nitrogen 6.7
TABLE-US-00013 TABLE 11 Solid Fat Content of RBD469 Oil Method Number Solid Fat Content Result AOCS Cd 12b-93 Solid Fat Content 10.degree. C. 0.13% AOCS Cd 12b-93 Solid Fat Content 15.degree. C. 0.13% AOCS Cd 12b-93 Solid Fat Content 20.degree. C. 0.28% AOCS Cd 12b-93 Solid Fat Content 25.degree. C. 0.14% AOCS Cd 12b-93 Solid Fat Content 30.degree. C. 0.08% AOCS Cd 12b-93 Solid Fat Content 35.degree. C. 0.25%
TABLE-US-00014 TABLE 12 Lovibond Color of RBD469 Oil Method Number Color Result Unit AOCS Cc 13j-97 red 2 Unit AOCS Cc 13j-97 yellow 27 Unit
[0361] RBD469 oil was subjected to transesterification to produce fatty acid methyl esters (FAMEs). The resulting FAME profile of RBD469 is shown in Table 12.
TABLE-US-00015 TABLE 13 FAME Profile of RBD469 Oil Fatty Acid Area % C10 0.01 C12:0 0.04 C14:0 0.64 C15:0 0.08 C16:0 8.17 C16:1 iso 0.39 C16:1 0.77 C17:0 0.08 C18:0 1.93 C18:1 85.88 C18:1 iso 0.05 C18:2 0.05 C20:0 0.3 C20:1 0.06 C20:1 0.44 C22:0 0.11 C23:0 0.03 C24:0 0.1 Total FAMEs Identified 99.13
[0362] The oil stability indexes (OSI) of 6 RBD oil samples without supplemented antioxidants and 3 RBD oil samples supplemented with antioxidants were analyzed according to the Oil Stability Index AOCS Method Cd 12b-92. Shown in Table 14 are the results of OSI AOCS Cd 12b-92 tests, conducted at 110.degree. C., performed using a Metrohm 873 Biodiesel Rancimat. Results, except where indicated with an asterisks (*), are the average of multiple OSI runs. Those samples not analyzed are indicated (NA).
TABLE-US-00016 TABLE 14 Oil Stability Index at 110.degree. C. of RBD oil samples with and without antioxidants. Antioxidant Antioxidant OSI (hours) for each RBD Sample added Concentration RBD437 RBD469 RBD502 RBD501 RBD503 RBD529 None 0 65.41 38.33 72.10 50.32 63.04 26.68 Tocopherol & 35 ppm/ 77.72 48.60 82.67 NA NA NA Ascorbyl 16.7 ppm Palmitate Tocopherol & 140 ppm/ 130.27 81.54* 211.49* NA NA NA Ascorbyl 66.7 ppm Palmitate Tocopherol & 1050 ppm/ >157* >144 242.5* NA NA NA Ascorbyl 500 ppm Palmitate Tocopherol 50 ppm NA 46.97 NA NA NA NA TBHQ 20 ppm 63.37 37.4 NA NA NA NA
[0363] The untransformed P. moriformis (UTEX 1435) acid profile comprises less than 60% C18:1 fatty acids and greater than 7% C18:2 fatty acids. In contrast, Strain D (comprising pSZ1500) exhibited fatty acid profiles with an increased composition of C18:1 fatty acids (to above 85%) and a decrease in C18:2 fatty acids (to less than 0.06%). Upon refining, bleaching, and degumming, RBD oils samples prepared from the oil made from strain E exhibited OSI values >26 hrs. With addition of antioxidants, the OSI of RBD oils prepared from oils of Strain D increased from 48.60 hours to greater than 242 hours. In other experiments, OSI values of over 400 hours were achieved. Additional properties of a low polyunsaturated oil according to embodiments of the invention are given in FIG. 16.
Example 6
Improving the Levels of Oleic Acid of Engineered Microbes Through Allelic Disruption of a Fatty Acid Desaturase and an Acyl-ACP Thioesterase
[0364] This example describes the use of a transformation vector to disrupt a FATA locus of a Prototheca moriformis strain previously engineered for high oleic acid and low linoleic acid production. The transformation cassette used in this example comprised a selectable marker and nucleotide sequences encoding a P. moriformis KASII enzyme to engineer microorganisms in which the fatty acid profile of the transformed microorganism has been altered for further increased oleic acid and lowered palmitic acid levels.
[0365] Strain D, described in Example 5 and in PCT/US2012/023696, is a classically mutagenized (for higher oil production) derivative of P. moriformis (UTEX 1435) subsequently transformed with the transformation construct pSZ1500 (SEQ ID NO: 17) according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strain was used as the host for transformation with construct pSZ2276 to increase expression of a KASII enzyme while concomitantly ablating an endogenous acyl-ACP thioesterase genetic locus to generate Strain E. The pSZ2276 transformation construct included 5' (SEQ ID NO: 20) and 3' (SEQ ID NO: 21) homologous recombination targeting sequences (flanking the construct) to the FATA1 genomic region for integration into the P. moriformis nuclear genome, an A. thaliana THIC protein coding region under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served as a selection marker. The P. moriformis KASII protein coding region was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR, and the native transit peptide of the KASII enzyme was replaced with the C. protothecoides stearoyl-ACP desaturase transit peptide (SEQ ID NO: 9). The codon-optimized sequence of PmKASII (Prototheca moriformis KASII) comprising a C. protothecoides S106 stearoyl-ACP desaturase transit peptide is provided the sequence listings as SEQ ID NO: 24. SEQ ID NO: 25 provides the protein translation of SEQ ID NO: 24. The protein coding regions of PmKASII and suc2 were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/U52009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0366] Primary pSZ2276 transformants of Strain D were selected on agar plates lacking thiamine, clonally purified, and a single engineered line, strain E was selected for analysis. Strain E was cultivated under heterotrophic lipid production conditions at pH5.0 and pH7.0 as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass from each transformant and fatty acid profiles from these samples were analyzed using standard fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1. The fatty acid profiles (expressed as Area % of total fatty acids) from the transgenic line arising from transformation with pSZ2276 into Strain D are shown in Table 15.
TABLE-US-00017 TABLE 15 Fatty acid profiles of Prototheca moriformis (UTEX 1435) Strains A, D, and E engineered for increased oleic acid and lowered linoleic acid levels. Transformation Area % Fatty Acid Strain Construct(s) pH C16:0 C18:0 C18:1 C18:2 C20:1 Strain None pH 5 26.6 3.3 60.5 6.7 0.07 A Strain None pH 7 28.3 4.1 58 6.5 0.06 A Strain pSZ1500 pH 5 17 3.6 77.1 0.01 0.14 D Strain pSZ1500 pH 7 19.5 5.3 72.6 0.01 0.09 D Strain pSZ1500 + pH 5 4.1 2.36 88.5 0.04 3.1 E pSZ2276 Strain pSZ1500 + pH 7 2.1 7.8 87.9 0.01 0.5 E pSZ2276
[0367] As shown in Table 15, targeted interruption of FADc alleles with a CtOTE expression cassette impacted the fatty acid profiles of transformed microorganisms. Fatty acid profiles of Strain D (comprising the pSZ1500 transformation vector) showed increased composition of C18:1 fatty acids with a concomitant decrease in C16:0 and C18:2 fatty acids relative to Strain A. Subsequent transformation of Strain D with pSZ2276 to overexpress a P. moriformis (UTEX 1435) KASII protein while concomitantly ablating a FATA genetic locus (thereby generating Strain E) resulted in still further impact on the fatty acid profiles of the transformed microorganisms. Fatty acid profiles of Strain E showed increased composition of C18:1 fatty acids, with a further decrease in C16:0 fatty acids relative to Strains A and D. Propagation of Strain E in culture conditions at pH 7, to induce expression from the Amt03 promoter, resulted in a fatty acid profile that was higher in C18:0 and C18:1 fatty acids and lower in C16:0 fatty acids, relative to the same strain cultured at pH 5.
[0368] These data demonstrate the utility of multiple genetic modifications to impact the fatty acid profile of a host organism for increased levels of oleic acid with concomitant decreased levels of linoleic acid and palmitic acid. Further, this example illustrates the use of recombinant polynucleotides to target gene interruption of an endogenous FATA allele with a cassette comprising a pH-regulatable promoter to control expression of an exogenous KASII protein-coding region in order to alter the fatty acid profile of a host microbe.
Example 7
Conditional Expression of a Fatty Acid Desaturase
[0369] This example describes the use of a transformation vector to conditionally express a delta 12 fatty acid desaturase (FADs) in a Prototheca moriformis strain previously engineered for high oleic acid and very low linoleic acid production in both seed and lipid productivity stages of propagation. Very low linoleic acid levels in cell oils are sought for use in certain applications. However, absence of linoleic acid during cell division phase ("seed stage") of a host microbe is disadvantageous. Linoleic acid may be supplemented to the seed medium to hasten cell division and not added during lipid production, but this addition imposes unwanted costs. To overcome this challenge, a transformation cassette was constructed for regulated expression of a FAD2 enzyme such that levels of linoleic acids sufficient for cell division could be achieved and oil with very low levels of linoleic acids could be produced during the oil production phase of culture of a microorganism. The transformation cassette used in this example comprised a selectable marker, a pH-regulatable promoter, and nucleotide sequences encoding a P. moriformis FAD2 enzyme to engineer microorganisms in which the fatty acid profile of the transformed microorganism has been altered for increased oleic acid production and regulatable linoleic acid production.
[0370] Strain D, described in Examples 5, 6, and in PCT/US2012/023696, is a classically mutagenized (for higher oil production) derivative of P. moriformis (UTEX 1435) subsequently transformed with the transformation construct pSZ1500 (SEQ ID NO: 17) according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strain was used as the host for transformation with construct pSZ2413 to introduce a pH-driven promoter for regulation of a P. moriformis FAD2 enzyme. The pSZ2413 transformation construct included 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the P. moriformis nuclear genome, an A. thaliana THIC protein coding region under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served as a selection marker. The P. moriformis FAD2 protein coding region was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR. The codon-optimized sequence of PmFAD2 is provided the sequence listings as SEQ ID NO: 26. SEQ ID NO: 27 provides the protein translation of SEQ ID NO: 26. The protein coding regions of PmFAD2 and suc2 were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0371] Primary pSZ2413 transformants of Strain D were selected on agar plates lacking thiamine, clonally purified, and isolates of the engineered line, Strain F were selected for analysis. These isolates were cultivated under heterotrophic lipid production conditions at pH7.0 (to activate expression of FAD2 from the PmAmt03 promoter) and at pH5.0, as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass from each transformant and fatty acid profiles from these samples were analyzed using standard fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1. The resulting profile of C18:2 fatty acids (expressed in Area %) from nine representative isolates of transgenic Strain F (F-1 through F-9) arising from transformation with pSZ2413 into Strain D are shown in Table 16.
TABLE-US-00018 TABLE 16 C18:2 fatty acid profiles of Prototheca moriformis (UTEX 1435) Strains A, D, and F. Area % C18:2 Strain Transformation Construct (s) pH 5.0 pH 7.0 A None 6.07 7.26 D pSZ1500 0.01 0.01 F-1 pSZ1500 + pSZ2413 0.37 5.29 F-2 pSZ1500 + pSZ2413 0.45 6.87 F-3 pSZ1500 + pSZ2413 0.50 6.79 F-4 pSZ1500 + pSZ2413 0.57 5.06 F-5 pSZ1500 + pSZ2413 0.57 7.58 F-6 pSZ1500 + pSZ2413 0.60 6.88 F-7 pSZ1500 + pSZ2413 0.62 6.52 F-8 pSZ1500 + pSZ2413 0.63 5.79 F-9 pSZ1500 + pSZ2413 0.77 4.53
[0372] As shown in Table 16 the impact of regulated expression of the PmFAD2 enzyme, effected though strain culture at different pH levels, is a clear increase in the composition of C18:2 fatty acids in the transformed microorganism. Linoleic acid comprises about 6% to about 7.3% of fatty acids of Strain A. In contrast, Strain D (comprising the pSZ1500 transformation vector to ablate both FAD2 alleles) is characterized by a fatty acid profile of 0.01% linoleic acid. Transformation of Strain D with pSZ2413 to generate Strain F results in a recombinant microbe in which the production of linoleic acid is regulated by the Amt03 promoter. Propagation of Strain F isolates in culture conditions at pH 7, to induce FAD2 expression from the Amt03 promoter, resulted in a fatty acid profile characterized by about 4.5% to about 7.5% linoleic acid. In contrast, propagation of Strain F isolates in culture conditions at pH 5 resulted in a fatty acid profile characterized by about 0.33 to about 0.77% linoleic acid.
[0373] These data demonstrate the utility of and effectiveness of recombinant polynucleotides permitting conditional expression of a FAD2 enzyme to alter the fatty acid profile of engineered microorganisms, and in particular in regulating the production of C18:2 fatty acids in microbial cells.
Example 8
Analysis of Regiospecific Profile
[0374] LC/MS TAG distribution analyses were carried out using a Shimadzu Nexera ultra high performance liquid chromatography system that included a SIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 in-line degasser, and a CTO-20A column oven, coupled to a Shimadzu LCMS 8030 triple quadrupole mass spectrometer equipped with an APCI source. Data was acquired using a Q3 scan of m/z 350-1050 at a scan speed of 1428 u/sec in positive ion mode with the CID gas (argon) pressure set to 230 kPa. The APCI, desolvation line, and heat block temperatures were set to 300, 250, and 200.degree. C., respectively, the flow rates of the nebulizing and drying gases were 3.0 L/min and 5.0 L/min, respectively, and the interface voltage was 4500 V. Oil samples were dissolved in dichloromethane-methanol (1:1) to a concentration of 5 mg/mL, and 0.8 .mu.L of sample was injected onto Shimadzu Shim-pack XR-ODS III (2.2 .mu.m, 2.0.times.200 mm) maintained at 30.degree. C. A linear gradient from 30% dichloromethane-2-propanol (1:1)/acetonitrile to 51% dichloromethane-2-propanol (1:1)/acetonitrile over 27 minutes at 0.48 mL/min was used for chromatographic separations.
Example 9
Engineering Microbes for Increased Production of SOS, POP, And POS Triacylglycerides
[0375] This example describes the use of recombinant polynucleotides that encode a C18:0-preferring Brassica napus thioesterase (BnOTE) enzyme to engineer a microorganism in which the triacylglyceride distribution of the transformed microorganism has been enriched in SOS, POS, and POP triacylglycerides.
[0376] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain A, was initially transformed with the plasmid construct pSZ1358 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. pSZ1358, described in PCT/US2012/023696, hereby incorporated by reference, comprised the coding sequence of the Brassica napus thioesterase (BnOTE) thioesterase (SEQ ID NO: 28), 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), to express the protein sequence given in SEQ ID NO: 3, under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. The BnOTE protein coding sequence to express the protein sequence given in SEQ ID NO: 29, was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3'UTR. The protein coding regions of BnOTE and suc2 were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0377] Primary pSZ1358 transformants of Strain A were selected on agar plates containing sucrose as a sole carbon source, clonally purified, and single engineered line, Strain G was selected for analysis. Strain G was cultivated under heterotrophic lipid production conditions at pH7.0 (to activate expression of BnOTE from the PmAmt03 promoter) as described in PCT/U52009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Oil samples obtained from Strain A and Strain G were analyzed for fatty acid composition using methods described in Examples 1 and 2, and, using the methods described in Example 8, for the regiospecificity of triacylglycerides in the oil. Fatty acid profiles of TAGs isolated from Strain A and G are shown in Table 17. Table 18 presents the regiospecificity profile of POP, POS, and SOS TAGs present in oil samples from Strain A and G.
TABLE-US-00019 TABLE 17 Effect of BnOTE expression on the fatty acid composition and the sn-2 profile of TAGs produced from transformed Prototheca moriformis. Strain G Area % Strain A (pSZ1358) Fatty acid FA profile FA profile C10:0 n.r. 0.5 C12:0 n.r. 0.5 C14:0 1.0 1.3 C16:0 23.9 25.8 C18:0 3.7 30.4 C18:1 64.3 30.2 C18:2 4.5 8.8 C18:3 .alpha. n.r. 0.4
TABLE-US-00020 TABLE 18 Effect of BnOTE expression on the regiospecific profile of POP, POS, and SOS TAGs produced from transformed Prototheca moriformis. Strain A (untransformed) Strain G (pSZ1358) Cocoa Butter Area Normalized Area Normalized Area Normalized TAG % Area % % Area % % Area % POP 13.09 76.8 10.6 23.5 17.9 22.1 POS 3.51 20.5 21.0 46.6 39.2 48.4 SOS 0.45 2.6 13.5 29.9 23.9 29.5 total 17.05 100 45.0 100 81.1 100
[0378] As shown in Table 17, the fatty acid composition of TAGs isolated from Strain G expressing BnOTE was markedly increased for C18:0 fatty acids (from 3.7% to 30.4%) and decreased in C18:1 fatty acids (from 64.3% to 30.2%) relative to the fatty acid profile of TAGs isolated from untransformed Strain A. The fatty acid composition of TAGs isolated from Strain A was characterized by about 23.9% palmitic acid, 3.7% stearic acid, and 64.3% oleic acid, a ratio for P:S:O of about 6.5:1:17.4. In contrast, the fatty acid composition of TAGs isolated from Strain G was characterized by about 25.8% palmitic acid, 30.4% stearic acid, and 30.2% oleic acid, a ratio for P:O:S of about 1:1.18:1.17.
[0379] The impact of expression of a C18:0 preferring thioesterase on the regiospecific profile of POP, POS, and SOS TAGs of oils produced from the transformed microorganism was an increase in all three TAGs as a proportion of the total TAGs present in the oil. As shown in Table 18, the sum of POP+POS+SOS TAGs accounted for 45% of the TAGs produced by Strain G, whereas POP, POS, and SOS TAGs summed to only about 17% of TAGs produced in Strain A. The percentages of POP, POS and SOS of strain G are compared to Cocoa butter in Table 18. As can be seen, ratios of POP, POS and SOS of Strain G are very similar to the ratios observed in cocoa butter.
[0380] These data demonstrate the utility and effectiveness of polynucleotides permitting exogenous thioesterase expression to alter the fatty acid and regiospecific profiles of TAGs of engineered microorganisms, in particular to increase the distribution of POP, POS, and SOS TAGs.
Examples 10-33
Engineering of Microorganisms
[0381] Examples 10-33 below describe the engineering of various microorganisms in accordance with the present invention. To alter the fatty acid profile of a microorganism, microorganisms can be genetically modified wherein endogenous or exogenous lipid biosynthesis pathway enzymes are expressed, overexpressed, or attenuated. Steps to genetically engineer a microbe to alter its fatty acid profile as to the degree of fatty acid unsaturation and to decrease or increase fatty acid chain length comprise the design and construction of a transformation vector (e.g., a plasmid), transformation of the microbe with one or more vectors, selection of transformed microbes (transformants), growth of the transformed microbe, and analysis of the fatty acid profile of the lipids produced by the engineered microbe.
[0382] Transgenes that alter the fatty acid profiles of host organisms can be expressed in numerous eukaryotic microbes. Examples of expression of transgenes in eukaryotic microbes including Chlamydomonas reinhardtii, Chlorella elhpsoidea, Chlorella saccarophila, Chlorella vulgaris, Chlorella kessleri, Chlorella sorokiniana, Haematococcus pluvialis, Gonium pectorals, Volvox carteri, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella salina, Closterium peracerosum-strigosum-littorale complex, Nannochloropsis sp., Thalassiosira pseudonana, Phaeodactylum tricornutum, Navicula saprophila, Cylindrotheca fusiformis, Cyclotella cryptica, Symbiodinium microadriacticum, Amphidinium sp., Chaetoceros sp., Mortierella alpina, and Yarrowia hpolytica can be found in the scientific literature. These expression techniques can be combined with the teachings of the present invention to produce engineered microorganisms with altered fatty acid profiles.
[0383] Transgenes that alter the fatty acid profiles of host organisms or alter the regiospecific distribution of glycerolipids produced by host organisms can also be expressed in numerous prokaryotic microbes. Examples of expression of transgenes in oleaginous microbes including Rhodococcus opacus can be found in the literature. These expression techniques can be combined with the teachings of the present invention to produce engineered microorganisms with altered fatty acid profiles.
TABLE-US-00021 TABLES 19A-D Codon preference listing. Haemato Amino Chlorella Chlorella Chlorella Chlorella Dunaliella Volvox coccus Acid Codon sorokiniana vulgaris ellipsoidea kessleri tertiolecta carteri pluvialis Ala GCG 0.20 0.25 0.15 0.14 0.09 0.25 0.21 Ala GCA 0.05 0.24 0.32 0.10 0.17 0.13 0.27 Ala GCT 0.12 0.16 0.26 0.18 0.31 0.26 0.17 Ala GCC 0.63 0.35 0.27 0.58 0.43 0.36 0.35 Arg AGG 0.03 0.09 0.10 0.09 0.26 0.08 0.14 Arg AGA 0.04 0.05 0.14 0.01 0.09 0.03 0.05 Arg CGG 0.06 0.19 0.09 0.06 0.06 0.17 0.15 Arg CGA 0.00 0.10 0.08 0.00 0.08 0.08 0.10 Arg CGT 0.06 0.09 0.37 0.14 0.12 0.22 0.13 Arg CGC 0.81 0.48 0.22 0.71 0.40 0.43 0.42 Asn AAT 0.04 0.16 0.43 0.06 0.27 0.23 0.21 Asn AAC 0.96 0.84 0.57 0.94 0.73 0.77 0.79 Asp GAT 0.13 0.25 0.47 0.12 0.40 0.35 0.27 Asp GAC 0.87 0.75 0.53 0.88 0.60 0.65 0.73 Cys TGT 0.06 0.13 0.43 0.09 0.20 0.17 0.27 Cys TGC 0.94 0.87 0.57 0.91 0.80 0.83 0.64 End TGA 0.00 0.72 0.14 0.14 0.36 0.24 0.70 End TAG 0.33 0.11 0.29 0.00 0.00 0.18 0.22 End TAA 0.67 0.17 4.00 0.86 0.64 0.59 0.09 Gln CAG 0.42 0.40 0.15 0.40 0.27 0.29 0.33 Gln CAA 0.04 0.04 0.21 0.40 0.27 0.07 0.10 Glu GAG 0.53 0.50 0.33 0.40 0.27 0.53 0.49 Glu GAA 0.02 0.06 0.31 0.40 0.27 0.11 0.07 Gly GGG 0.04 0.16 0.19 0.08 0.10 0.12 0.22 Gly GGA 0.02 0.11 0.13 0.07 0.13 0.12 0.11 Gly GGT 0.03 0.12 0.39 0.24 0.25 0.23 0.15 Gly GGC 0.91 0.61 0.29 0.96 0.51 0.53 0.52 His CAT 0.14 0.16 0.30 0.08 0.25 0.35 0.27 His CAC 0.86 0.84 0.70 0.93 0.75 0.65 0.73 Ile ATA 0.00 0.04 0.07 0.01 0.04 0.08 0.09 Ile ATT 0.15 0.30 0.63 0.29 0.31 0.35 0.29 Ile ATC 0.85 0.66 0.65 0.69 0.65 0.57 0.62 Leu TTG 0.03 0.07 0.03 0.05 0.14 0.14 0.16 Leu TTA 0.00 0.01 0.32 0.00 0.02 0.03 0.02 Leu CTG 0.72 0.61 0.34 0.61 0.60 0.45 0.53 Leu CTA 0.01 0.03 0.03 0.04 0.04 0.07 0.07 Leu CTT 0.04 0.08 0.16 0.06 0.06 0.14 0.09 Leu CTC 0.20 0.20 0.12 0.24 0.14 0.17 0.13 Lys AAG 0.98 0.94 0.54 0.98 0.90 0.90 0.84 Lys AAA 0.02 0.06 0.46 0.02 0.10 0.10 0.16 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.28 0.32 0.42 0.31 0.24 0.27 0.35 Phe TTC 0.72 0.68 0.58 0.69 0.76 0.73 0.65 Pro CCG 0.18 0.31 0.09 0.07 0.04 0.34 0.15 Pro CCA 0.06 0.17 0.36 0.07 0.04 0.20 0.24 Pro CCT 0.10 0.14 0.25 0.17 0.04 0.19 0.29 Pro CCC 0.66 0.38 0.29 0.69 0.04 0.27 0.32 Ser AGT 0.03 0.04 0.14 0.02 0.08 0.08 0.07 Ser AGC 0.27 0.38 0.18 0.18 0.31 0.27 0.31 Ser TCG 0.12 0.14 0.08 0.10 0.02 0.19 0.10 Ser TCA 0.03 0.08 0.14 0.08 0.09 0.09 0.14 Ser TCT 0.09 0.11 0.26 0.18 0.19 0.14 0.13 Ser TCC 0.47 0.24 0.20 0.44 0.30 0.24 0.24 Thr ACG 0.11 0.20 0.13 0.05 0.12 0.27 0.19 Thr ACA 0.01 0.20 0.32 0.07 0.20 0.12 0.23 Thr ACT 0.12 0.13 0.29 0.12 0.24 0.20 0.18 Thr ACC 0.76 0.47 0.26 0.76 0.44 0.41 0.40 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.07 0.15 0.43 0.27 0.28 0.24 0.19 Tyr TAC 0.93 0.85 0.57 0.73 0.72 0.76 0.81 Val GTG 0.71 0.54 0.37 0.60 0.54 0.46 0.62 Val GTA 0.00 0.05 0.25 0.03 0.09 0.07 0.09 Val GTT 0.11 0.14 0.24 0.09 0.14 0.17 0.09 Val GTC 0.18 0.27 0.14 0.28 0.23 0.30 0.21 Closterium peracerosum- strigosum- Amino littorale Dunaliella Dunaliella Gonium Phaeodactylum Chaetoceros Acid Codon complex viridis sauna pectorale tricornutum cornpressum Ala GCG 0.48 0.13 0.15 0.43 0.15 0.08 Ala GCA 0.10 0.27 0.20 0.09 0.10 0.37 Ala GCT 0.15 0.25 0.27 0.08 0.23 0.36 Ala GCC 0.26 0.35 0.39 0.41 0.52 0.18 Arg AGG 0.04 0.25 0.22 0.13 0.02 0.14 Arg AGA 0.00 0.06 0.05 0.00 0.04 0.29 Arg CGG 0.18 0.08 0.12 0.40 0.10 0.00 Arg CGA 0.00 0.06 0.06 0.05 0.12 0.19 Arg CGT 0.13 0.15 0.13 0.08 0.41 0.38 Arg CGC 0.64 0.39 0.43 0.35 0.31 0.00 Asn AAT 0.04 0.17 0.23 0.07 0.30 0.58 Asn AAC 0.96 0.83 0.77 0.93 0.65 0.42 Asp GAT 0.30 0.38 0.40 0.11 0.41 0.53 Asp GAC 0.70 0.62 0.60 0.89 0.59 0.47 Cys TGT 0.06 0.24 0.17 0.20 0.39 0.44 Cys TGC 0.94 0.76 0.83 0.90 0.61 0.56 End TGA 0.75 0.31 0.37 0.50 0.06 0.50 End TAG 0.00 0.15 0.14 0.00 0.13 0.00 End TAA 0.25 0.54 0.49 0.50 0.81 0.50 Gln CAG 0.53 0.36 0.32 0.31 0.23 0.16 Gln CAA 0.09 0.12 0.08 0.07 0.14 0.19 Glu GAG 0.31 0.44 0.51 0.56 0.21 0.28 Glu GAA 0.06 0.09 0.09 0.07 0.42 0.37 Gly GGG 0.31 0.14 0.10 0.18 0.08 0.12 Gly GGA 0.06 0.11 0.12 0.09 0.34 0.33 Gly GGT 0.09 0.22 0.22 0.07 0.30 0.39 Gly GGC 0.53 0.54 0.56 0.65 0.28 0.16 His CAT 0.33 0.25 0.25 0.43 0.28 0.84 His CAC 0.67 0.75 0.75 0.57 0.72 0.16 Ile ATA 0.03 0.03 0.03 0.07 0.03 0.12 Ile ATT 0.23 0.25 0.31 0.33 0.51 0.65 Ile ATC 0.74 0.72 0.66 0.59 0.46 0.23 Leu TTG 0.04 0.11 0.12 0.04 0.26 0.11 Leu TTA 0.00 0.01 0.01 0.00 0.02 0.14 Leu CTG 0.31 0.60 0.61 0.64 0.15 0.05 Leu CTA 0.01 0.05 0.04 0.01 0.05 0.08 Leu CTT 0.04 0.07 0.08 0.05 0.18 0.51 Leu CTC 0.60 0.16 0.14 0.26 0.34 0.11 Lys AAG 0.86 0.87 0.89 0.93 0.75 0.52 Lys AAA 0.14 0.13 0.11 0.07 0.25 0.48 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.09 0.25 0.29 0.10 0.44 0.65 Phe TTC 0.91 0.75 0.71 0.90 0.56 0.35 Pro CCG 0.28 0.10 0.08 0.53 0.29 0.05 Pro CCA 0.15 0.10 0.17 0.09 0.12 0.45 Pro CCT 0.12 0.10 0.30 0.04 0.20 0.33 Pro CCC 0.44 0.10 0.45 0.34 0.40 0.17 Ser AGT 0.04 0.09 0.06 0.02 0.12 0.14 Ser AGC 0.05 0.31 0.32 0.20 0.12 0.07 Ser TCG 0.22 0.04 0.06 0.42 0.19 0.08 Ser TCA 0.16 0.08 0.10 0.09 0.06 0.31 Ser TCT 0.05 0.17 0.15 0.07 0.15 0.23 Ser TCC 0.47 0.31 0.30 0.20 0.35 0.18 Thr ACG 0.30 0.16 0.13 0.42 0.23 0.10 Thr ACA 0.06 0.21 0.18 0.03 0.13 0.38 Thr ACT 0.22 0.18 0.23 0.08 0.19 0.27 Thr ACC 0.42 0.46 0.46 0.47 0.45 0.25 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.07 0.16 0.21 0.12 0.18 0.67 Tyr TAC 0.93 0.84 0.79 0.88 0.82 0.33 Val GTG 0.50 0.64 0.62 0.57 0.22 0.30 Val GTA 0.02 0.03 0.05 0.04 0.09 0.27 Val GTT 0.06 0.11 0.11 0.04 0.22 0.10 Val GTC 0.42 0.22 0.23 0.35 0.47 0.33 Symbio- Thal- Cylindro- Amphi- dinium Nanno- assiosira Amino theca dinium micro- chloro- Cyclotella Nayicula pseudo- C. Acid Codon fusiformis carterae adriacticum psis sp cryptica pelliculosa nana reinhardtii Ala GCG 0.07 0.17 0.22 0.24 0.11 0.00 0.11 0.35 Ala GCA 0.14 0.33 0.26 0.10 0.16 0.13 0.25 0.08 Ala GCT 0.35 0.29 0.20 0.17 0.45 0.44 0.33 0.13 Ala GCC 0.43 0.20 0.32 0.48 0.27 0.44 0.30 0.43 Arg AGG 0.09 0.15 0.27 0.00 0.09 0.05 0.18 0.05 Arg AGA 0.14 0.03 0.27 0.00 0.05 0.10 0.17 0.01 Arg CGG 0.06 0.08 0.09 0.00 0.04 0.05 0.06 0.20 Arg CGA 0.16 0.18 0.09 0.29 0.08 0.35 0.11 0.04 Arg CGT 0.34 0.18 0.09 0.14 0.47 0.20 0.34 0.09 Arg CGC 0.22 0.40 0.18 0.57 0.28 0.25 0.15 0.62 Asn AAT 0.42 0.37 0.21 0.00 0.25 0.47 0.43 0.09 Asn AAC 0.58 0.63 0.79 1.00 0.75 0.53 0.57 0.91 Asp GAT 0.54 0.54 0.50 0.20 0.52 0.20 0.56 0.14 Asp GAC 0.46 0.46 0.50 0.80 0.48 0.80 0.44 0.86 Cys TGT 0.44 0.75 0.50 0.00 0.29 0.10 0.54 0.10 Cys TGC 0.56 0.25 0.50 1.00 0.71 0.90 0.46 0.90 End TGA 0.13 0.50 1.00 0.00 0.10 0.00 0.31 0.27 End TAG 0.10 0.00 0.00 0.00 0.00 0.00 0.38 0.22 End TAA 0.77 0.50 0.00 1.00 0.90 1.00 0.31 0.52 Gln CAG 0.12 0.33 0.28 0.41 0.19 0.21 0.16 0.38 Gln CAA 0.25 0.15 0.17 0.00 0.17 0.28 0.19 0.04 Glu GAG 0.23 0.41 0.50 0.59 0.38 0.17 0.40 0.55 Glu GAA 0.39 0.10 0.06 0.00 0.26 0.34 0.26 0.03 Gly GGG 0.06 0.19 0.32 0.10 0.10 0.03 0.12 0.11 Gly GGA 0.47 0.10 0.12 0.05 0.45 0.28 0.51 0.06 Gly GGT 0.35 0.34 0.16 0.25 0.22 0.13 0.23 0.11 Gly GGC 0.12 0.37 0.40 0.60 0.24 0.56 0.14 0.72 His CAT 0.39 0.12 0.40 0.00 0.42 1.00 0.50 0.11 His CAC 0.61 0.88 0.60 1.00 0.58 0.00 0.50 0.89 Ile ATA 0.06 0.05 0.00 0.00 0.04 0.00 0.08 0.03 Ile ATT 0.42 0.53 0.38 0.14 0.53 0.73 0.38 0.22 Ile ATC 0.52 0.42 0.63 0.86 0.42 0.27 0.54 0.75 Leu TTG 0.26 0.35 0.39 0.22 0.20 0.16 0.29 0.04 Leu TTA 0.09 0.01 0.00 0.00 0.03 0.00 0.05 0.01 Leu CTG 0.09 0.22 0.39 0.09 0.06 0.12 0.08 0.73 Leu CTA 0.05 0.00 0.04 0.00 0.03 0.04 0.06 0.03 Leu CTT 0.37 0.31 0.13 0.04 0.39 0.36 0.20 0.05 Leu CTC 0.13 0.12 0.04 0.65 0.29 0.32 0.32 0.15 Lys AAG 0.60 0.93 0.85 1.00 0.70 0.83 0.76 0.95 Lys AAA 0.40 0.07 0.15 0.00 0.30 0.17 0.24 0.05 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.37 0.21 0.25 0.20 0.31 0.78 0.38 0.16 Phe TTC 0.63 0.79 0.75 0.80 0.69 0.22 0.62 0.84 Pro CCG 0.11 0.14 0.18 0.08 0.10 0.21 0.16 0.33 Pro CCA 0.33 0.42 0.09 0.08 0.16 0.29 0.31 0.08 Pro CCT 0.32 0.22 0.41 0.25 0.35 0.21 0.31 0.13 Pro CCC 0.24 0.22 0.32 0.58 0.39 0.29 0.23 0.47 Ser AGT 0.12 0.13 0.09 0.00 0.09 0.13 0.18 0.04 Ser AGC 0.09 0.24 0.14 0.13 0.08 0.28 0.11 0.35 Ser TCG 0.13 0.03 0.05 0.00 0.15 0.25 0.17 0.25 Ser TCA 0.12 0.25 0.05 0.00 0.12 0.08 0.12 0.05 Ser TCT 0.30 0.16 0.23 0.13 0.39 0.25 0.23 0.07 Ser TCC 0.24 0.19 0.45 0.75 0.18 0.03 0.19 0.25 Thr ACG 0.09 0.14 0.10 0.28 0.10 0.18 0.21 0.30 Thr ACA 0.15 0.28 0.10 0.00 0.15 0.09 0.19 0.08 Thr ACT 0.39 0.12 0.10 0.17 0.33 0.41 0.28 0.10 Thr ACC 0.37 0.47 0.70 0.56 0.43 0.32 0.32 0.52 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Tyr TAT 0.38 0.32 0.20 0.00 0.38 0.20 0.39 0.10 Tyr TAC 0.62 0.68 0.80 1.00 0.62 0.80 0.61 0.90 Val GTG 0.11 0.65 0.67 0.31 0.16 0.18 0.29 0.67 Val GTA 0.06 0.05 0.00 0.00 0.09 0.09 0.16 0.03 Val GTT 0.38 0.08 0.11 0.15 0.42 0.09 0.28 0.07 Val GTC 0.46 0.21 0.22 0.54 0.33 0.64 0.27 0.22 Amino Yarrowia Mortierella Rhodococcus Acid Codon lipolytica alpina opacus Ala GCG 0.08 0.14 0.35 Ala GCA 0.11 0.12 0.14 Ala GCT 0.35 0.29 0.09 Ala GCC 0.46 0.45 0.43 Arg AGG 0.05 0.05 0.05 Arg AGA 0.13 0.06 0.02 Arg CGG 0.12 0.06 0.26 Arg CGA 0.52 0.09 0.12 Arg CGT 0.11 0.32 0.11 Arg CGC 0.07 0.42 0.44 Asn AAT 0.17 0.15 0.21 Asn AAC 0.83 0.85 0.79 Asp GAT 0.35 0.42 0.24 Asp GAC 0.65 0.58 0.76 Cys TGT 0.46 0.13 0.26 Cys TGC 0.54 0.87 0.74 End TGA 0.16 0.05 0.72 End TAG 0.38 0.25 0.17 End TAA 0.46 0.70 0.11 Gln CAG 0.33 0.36 0.28 Gln CAA 0.08 0.06 0.06 Glu GAG 0.44 0.49 0.45 Glu GAA 0.14 0.09 0.22 Gly GGG 0.05 0.03 0.18 Gly GGA 0.28 0.29 0.15 Gly GGT 0.32 0.32 0.20 Gly GGC 0.34 0.36 0.48 His CAT 0.34 0.27 0.20 His CAC 0.66 0.73 0.80 Ile ATA 0.03 0.01 0.05
Ile ATT 0.44 0.33 0.14 Ile ATC 0.53 0.66 0.81 Leu TTG 0.09 0.27 0.09 Leu TTA 0.02 0.00 0.01 Leu CTG 0.37 0.26 0.41 Leu CTA 0.05 0.02 0.03 Leu CTT 0.18 0.12 0.06 Leu CTC 0.29 0.32 0.40 Lys AAG 0.84 0.91 0.80 Lys AAA 0.16 0.09 0.20 Met ATG 1.00 1.00 1.00 Phe TTT 0.38 0.39 0.09 Phe TTC 0.62 0.61 0.91 Pro CCG 0.10 0.07 0.52 Pro CCA 0.10 0.08 0.09 Pro CCT 0.32 0.36 0.07 Pro CCC 0.47 0.49 0.32 Ser AGT 0.07 0.05 0.08 Ser AGC 0.11 0.14 0.23 Ser TCG 0.16 0.32 0.33 Ser TCA 0.08 0.08 0.07 Ser TCT 0.28 0.12 0.05 Ser TCC 0.30 0.29 0.24 Thr ACG 0.11 0.17 0.28 Thr ACA 0.14 0.10 0.11 Thr ACT 0.26 0.23 0.07 Thr ACC 0.49 0.49 0.53 Trp TGG 1.00 1.00 1.00 Tyr TAT 0.18 0.20 0.18 Tyr TAC 0.82 0.80 0.82 Val GTG 0.33 0.22 0.37 Val GTA 0.05 0.02 0.05 Val GTT 0.26 0.27 0.10 Val GTC 0.36 0.49 0.49
TABLE-US-00022 TABLE 20 Lipid biosynthesis pathway proteins 3-Ketoacyl ACP synthase Cuphea hookeriana 3-ketoacyl-ACP synthase (GenBank Acc. No. AAC68861.1), Cuphea wrightii beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAB37271.1), Cuphea lanceolata beta-ketoacyl-ACP synthase IV (GenBank Acc. No. CAC59946.1), Cuphea wrightii beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAB37270.1), Ricinus communis ketoacyl-ACP synthase (GenBank Acc. No. XP_002516228), Gossypium hirsutum ketoacyl- ACP synthase (GenBank Acc. No. ADK23940.1), Glycine max plastid 3-keto-acyl-ACP synthase II-A (GenBank Acc No. AAW88763.1), Elaeis guineensis beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAF26738.2), Helianthus annuus plastid 3-keto-acyl-ACP synthase I (GenkBank Acc. No. ABM53471.1), Glycine max3-keto-acyl-ACP synthase I (GenBank Acc. No. NP_001238610.1), Helianthus annuus plastid 3-keto-acyl-ACP synthase II (GenBank Acc ABI18155.1), Brassica napus beta-ketoacyl-ACP synthetase 2 (GenBank Acc. No. AAF61739.1), Perilla frutescens beta-ketoacyl-ACP synthase II (GenBank Acc. No. AAC04692.1), Helianthus annus beta-ketoacyl-ACP synthase II (GenBank Accession No. ABI18155), Ricinus communis beta-ketoacyl-ACP synthase II (GenBank Accession No. AAA33872), Haematococcus pluvialis beta-ketoacyl acyl carrier protein synthase (GenBank Accession No. HM560033.1), Jatropha curcasbeta ketoacyl-ACP synthase I (GenBank Accession No. ABJ90468.1), Populus trichocarpa beta-ketoacyl-ACP synthase I (GenBank Accession No. XP_002303661.1), Coriandrum sativum beta-ketoacyl-ACP synthetase I (GenBank Accession No. AAK58535.1), Arabidopsis thaliana 3-oxoacyl-[acyl-carrier- protein] synthase I (GenBank Accession No. NP_001190479.1), Vitis vinifera 3-oxoacyl- [acyl-carrier-protein] synthase I (GenBank Accession No. XP_002272874.2) Fatty acyl-ACP Thioesterases Umbellularia californica fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49001), Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank Acc. No. Q39473), Umbellularia californica fatty acyl-ACP thioesterase (GenBank Acc. No. Q41635), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc. No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc. No. AAB71730), Elaeis guineensis fatty acyl- ACP thioesterase (GenBank Acc. No. ABD83939), Elaeis guineensis fatty acyl-ACP thioesterase (GenBank Acc. No. AAD42220), Populus tomentosa fatty acyl-ACP thioesterase (GenBank Acc. No. ABC47311), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank Acc. No. NP_172327), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank Acc. No. CAA85387), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank Acc. No. CAA85388), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc. No. Q9SQI3), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank Acc. No. CAA54060), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank Acc. No. AAC72882), Cuphea calophylla subsp. mesostemon fatty acyl-ACP thioesterase (GenBank Acc. No. ABB71581), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank Acc. No. CAC19933), Elaeis guineensis fatty acyl-ACP thioesterase (GenBank Acc. No. AAL15645), Cuphea hookeriana fatty acyl- ACP thioesterase (GenBank Acc. No. Q39513), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc. No. AAD01982), Vitis vinifera fatty acyl-ACP thioesterase (GenBank Acc. No. CAN81819), Garcinia mangostana fatty acyl-ACP thioesterase (GenBank Acc. No. AAB51525), Brassica juncea fatty acyl-ACP thioesterase (GenBank Acc. No. ABI18986), Madhuca longifolia fatty acyl-ACP thioesterase (GenBank Acc. No. AAX51637), Brassica napus fatty acyl-ACP thioesterase (GenBank Acc. No. ABH11710), Brassica napus fatty acyl-ACP thioesterase (GenBank Acc. No. CAA52070.1), Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No. EAY86877), Oryza sativa (japonica cultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No. NP_001068400), Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No. EAY99617), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49269), Ulmus Americana fatty acyl-ACP thioesterase (GenBank Acc. No. AAB71731), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank Acc. No. CAB60830), Cuphea palustris fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49180), Iris germanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858, Iris germanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858.1), Cuphea palustris fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49179), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc. No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc. No. AAB717291.1), Cuphea hookeriana fatty acyl-ACP thioesterase GenBank Acc. No. U39834), Umbelluaria californica fatty acyl-ACP thioesterase (GenBank Acc. No. M94159), Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank Acc. No. U31813), Ricinus communis fatty acyl-ACP thioesterase (GenBank Acc. No. ABS30422.1), Helianthus annuus acyl-ACP thioesterase (GenBank Accession No. AAL79361.1), Jatropha curcas acyl-ACP thioesterase (GenBank Accession No. ABX82799.3), Zea mays oleoyl-acyl carrier protein thioesterase, (GenBank Accession No. ACG40089.1), Haematococcus pluvialis fatty acyl- ACP thioesterase (GenBank Accession No. HM560034.1) Desaturase Enzymes Linum usitatissimum fatty acid desaturase 3C, (GenBank Acc. No. ADV92272.1), Ricinus communis omega-3 fatty acid desaturase, endoplasmic reticulum, putative, (GenBank Acc. No. EEF36775.1), Vernicia fordii omega-3 fatty acid desaturase, (GenBank Acc. No. AAF12821), Glycine max chloroplast omega 3 fatty acid desaturase isoform 2, (GenBank Acc. No. ACF19424.1), Prototheca moriformis FAD-D omega 3 desaturase (SEQ ID NO: 35), Prototheca moriformis linoleate desaturase (SEQ ID NO: 36), Carthamus tinctorius delta 12 desaturase, (GenBank Accession No. ADM48790.1), Gossypium hirsutum omega-6 desaturase, (GenBank Accession No. CAA71199.1), Glycine max microsomal desaturase (GenBank Accession No. BAD89862.1), Zea mays fatty acid desaturase (GenBank Accession No. ABF50053.1), Brassica napa linoleic acid desaturase (GenBank Accession No. AAA32994.1), Camelina sativa omega-3 desaturase (SEQ ID NO: 37), Prototheca moriformis delta 12 desaturase allele 2 (SEQ ID NO: 38, Camelina sativa omega-3 FAD7-1 (SEQ ID NO: 39), Helianthus annuus stearoyl-ACP desaturase, (GenBank Accession No. AAB65145.1), Ricinus communis stearoyl-ACP desaturase, (GenBank Accession No. AACG59946.1), Brassica juncea plastidic delta-9-stearoyl-ACP desaturase (GenBank Accession No. AAD40245.1), Glycine max stearoyl-ACP desaturase (GenBank Accession No. ACJ39209.1), Olea europaea stearoyl-ACP desaturase (GenBank Accession No. AAB67840.1), Vernicia fordii stearoyl-acyl-carrier protein desaturase, (GenBank Accession No. ADC32803.1), Descurainia sophia delta-12 fatty acid desaturase (GenBank Accession No. ABS86964.2), Euphorbia lagascae delta12-oleic acid desaturase (GenBank Acc. No. AAS57577.1), Chlorella vulgaris delta 12 fatty acid desaturase (GenBank Accession No. ACF98528), Chlorella vulgaris omega-3 fatty acid desaturase (GenBank Accession No. BAB78717), Haematococcus pluvialis omega-3 fatty acid desaturase (GenBank Accession No. HM560035.1), Haematococcus pluvialis stearoyl-ACP-desaturase GenBank Accession No. EF586860.1, Haematococcus pluvialis stearoyl-ACP-desaturase GenBank Accession No. EF523479.1 Oleate 12-hydroxylase Enzymes Ricinus communis oleate 12-hydroxylase (GenBank Acc. No. AAC49010.1), Physaria lindheimeri oleate 12-hydroxylase (GenBank Acc. No. ABQ01458.1), Physaria lindheimeri mutant bifunctional oleate 12-hydroxylase: desaturase (GenBank Acc. No. ACF17571.1), Physaria lindheimeri bifunctional oleate 12-hydroxylase: desaturase (GenBank Accession No. ACQ42234.1), Physaria lindheimeri bifunctional oleate 12- hydroxylase: desaturase (GenBank Acc. No. AAC32755.1), Arabidopsis lyrata subsp. Lyrata (GenBank Acc. No. XP_002884883.1) Glycerol-3-phosphate Enzymes Arabidopsis thaliana glycerol-3-phosphate acyltransferase BAA00575, Chlamydomonas reinhardtii glycerol-3-phosphate acyltransferase (GenBank Acc. No. EDP02129), Chlamydomonas reinhardtii glycerol-3-phosphate acyltransferase (GenBank Acc. No. Q886Q7), Cucurbita moschata acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase (GenBank Acc. No. BAB39688), Elaeis guineensis glycerol-3-phosphate acyltransferase, ((GenBank Acc. No. AAF64066), Garcina mangostana glycerol-3-phosphate acyltransferase (GenBank Acc. No. ABS86942), Gossypium hirsutum glycerol-3-phosphate acyltransferase (GenBank Acc. No. ADK23938), Jatropha curcas glycerol-3-phosphate acyltransferase (GenBank Acc. No. ADV77219), Jatropha curcas plastid glycerol-3- phosphate acyltransferase (GenBank Acc. No. ACR61638), Ricinus communis plastidial glycerol-phosphate acyltransferase (GenBank Acc. No. EEF43526), Vica faba glycerol-3- phosphate acyltransferase (GenBank Accession No. AAD05164), Zea mays glycerol-3- phosphate acyltransferase (GenBank Acc. No. ACG45812) Lysophosphatidic acid acyltransferase Enzymes Arabidopsis thaliana 1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No. AEE85783), Brassica juncea 1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No. ABQ42862), Brassica juncea 1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No. ABM92334), Brassica napus 1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No. CAB09138), Chlamydomonas reinhardtii lysophosphatidic acid acyltransferase (GenBank Accession No. EDP02300), Cocos nucifera lysophosphatidic acid acyltransferase (GenBank Acc. No. AAC49119), Limnanthes alba lysophosphatidic acid acyltransferase (GenBank Accession No. EDP02300), Limnanthes douglasii 1-acyl-sn-glycerol-3-phosphate acyltransferase (putative) (GenBank Accession No. CAA88620), Limnanthes douglasii acyl-CoA: sn-1-acylglycerol-3-phosphate acyltransferase (GenBank Accession No. ABD62751), Limnanthes douglasii 1-acylglycerol-3-phosphate O- acyltransferase (GenBank Accession No. CAA58239), Ricinus communis 1-acyl-sn-glycerol- 3-phosphate acyltransferase (GenBank Accession No. EEF39377) Diacylglycerol acyltransferase Enzymes Arabidopsis thaliana diacylglycerol acyltransferase (GenBank Acc. No. CAB45373),
Brassica juncea diacylglycerol acyltransferase (GenBank Acc. No. AAY40784), Elaeis guineensis putative diacylglycerol acyltransferase (GenBank Acc. No. AEQ94187), Elaeis guineensis putative diacylglycerol acyltransferase (GenBank Acc. No. AEQ94186), Glycine max acyl CoA: diacylglycerol acyltransferase (GenBank Acc. No. AAT73629), Helianthus annus diacylglycerol acyltransferase (GenBank Acc. No. ABX61081), Olea europaea acyl- CoA: diacylglycerol acyltransferase 1 (GenBank Acc. No. AAS01606), Ricinus communis diacylglycerol acyltransferase (GenBank Acc. No. AAR11479) Phospholipid diacylglycerol acyltransferase Enzymes Arabidopsis thaliana phospholipid: diacylglycerol acyltransferase (GenBank Acc. No. AED91921), Elaeis guineensis putative phospholipid: diacylglycerol acyltransferase (GenBank Acc. No. AEQ94116), Glycine max phospholipid: diacylglycerol acyltransferase 1-like (GenBank Acc. No. XP_003541296), Jatropha curcas phospholipid: diacylglycerol acyltransferase (GenBank Acc. No. AEZ56255), Ricinus communis phospholipid: diacylglycerol acyltransferase (GenBank Acc. No. ADK92410), Ricinus communis phospholipid: diacylglycerol acyltransferase (GenBank Acc. No. AEW99982)
Example 10
Engineering Chlorella Sorokiniana
[0384] Expression of recombinant genes in accordance with the present invention in Chlorella sorokiniana can be accomplished by modifying the methods and vectors taught by Dawson et al. as discussed herein. Briefly, Dawson et al., Current Microbiology Vol. 35 (1997) pp. 356-362, reported the stable nuclear transformation of Chlorella sorokiniana with plasmid DNA. Using the transformation method of microprojectile bombardment, Dawson introduced the plasmid pSV72-NRg, encoding the full Chlorella vulgaris nitrate reductase gene (NR, GenBank Accession No. U39931), into mutant Chlorella sorokiniana (NR-mutants). The NR-mutants are incapable of growth without the use of nitrate as a source of nitrogen. Nitrate reductase catalyzes the conversion of nitrate to nitrite. Prior to transformation, Chlorella sorokiniana NR-mutants were unable to grow beyond the microcolony stage on culture medium comprising nitrate (NO.sub.3.sup.-) as the sole nitrogen source. The expression of the Chlorella vulgaris NR gene product in NR-mutant Chlorella sorokiniana was used as a selectable marker to rescue the nitrate metabolism deficiency. Upon transformation with the pSV72-NRg plasmid, NR-mutant Chlorella sorokiniana stably expressing the Chlorella vulgaris NR gene product were obtained that were able to grow beyond the microcolony stage on agar plates comprising nitrate as the sole carbon source. Evaluation of the DNA of the stable transformants was performed by Southern analysis and evaluation of the RNA of the stable transformants was performed by RNase protection. Selection and maintenance of the transformed Chlorella sorokiniana (NR mutant) was performed on agar plates (pH 7.4) comprising 0.2 g/L MgSO.sub.4, 0.67 g/L KH.sub.2PO.sub.4, 3.5 g/L K.sub.2HPO.sub.4, 1.0 g/L Na.sub.3C.sub.6H.sub.5O.sub.7.H.sub.2O and 16.0 g/L agar, an appropriate nitrogen source (e.g., NO.sub.3), micronutrients, and a carbon source. Dawson also reported the propagation of Chlorella sorokiniana and Chlorella sorokiniana NR mutants in liquid culture medium. Dawson reported that the plasmid pSV72-NRg and the promoter and 3' UTR/terminator of the Chlorella vulgaris nitrate reductase gene were suitable to enable heterologous gene expression in Chlorella sorokiniana NR-mutants. Dawson also reported that expression of the Chlorella vulgaris nitrate reductase gene product was suitable for use as a selectable marker in Chlorella sorokiniana NR-mutants.
[0385] In an embodiment of the present invention, vector pSV72-NRg, comprising nucleotide sequence encoding the Chlorella vulgaris nitrate reductase (CvNR) gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Chlorella sorokiniana to reflect the codon bias inherent in nuclear genes of Chlorella sorokiniana in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the CvNR promoter upstream of the protein-coding sequence and operably linked to the CvNR 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chlorella sorokiniana genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Chlorella sorokiniana with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the CvNR gene product can be used as a selectable marker to rescue the nitrogen assimilation deficiency of Chlorella sorokiniana NR mutant strains and to select for Chlorella sorokiniana NR-mutants stably expressing the transformation vector. Growth media suitable for Chlorella sorokiniana lipid production include, but are not limited to 0.5 g/L KH.sub.2PO.sub.4, 0.5 g/L K.sub.2HPO.sub.4, 0.25 g/L MgSO.sub.4-7H2O, with supplemental micronutrients and the appropriate nitrogen and carbon sources (Patterson, Lipids Vol. 5:7 (1970), pp. 597-600). Evaluation of fatty acid profiles of Chlorella sorokiniana lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 11
Engineering Chlorella Vulgaris
[0386] Expression of recombinant genes in accordance with the present invention in Chlorella vulgaris can be accomplished by modifying the methods and vectors taught by Chow and Tung et al. as discussed herein. Briefly, Chow and Tung et al., Plant Cell Reports, Volume 18 (1999), pp. 778-780, reported the stable nuclear transformation of Chlorella vulgaris with plasmid DNA. Using the transformation method of electroporation, Chow and Tung introduced the plasmid pIG121-Hm (GenBank Accession No. AB489142) into Chlorella vulgaris. The nucleotide sequence of pIG121-Hm comprised sequence encoding a beta-glucuronidase (GUS) reporter gene product operably-linked to a CaMV 35S promoter upstream of the GUS protein-coding sequence and further operably linked to the 3' UTR/terminator of the nopaline synthase (nos) gene downstream of the GUS protein-coding sequence. The sequence of plasmid pIG121-Hm further comprised a hygromycin B antibiotic resistance cassette. This hygromycin B antibiotic resistance cassette comprised a CaMV 35S promoter operably linked to sequence encoding the hygromycin phosphotransferase (hpt, GenBank Accession No. BAH24259) gene product. Prior to transformation, Chlorella vulgaris was unable to be propagated in culture medium comprising 50 ug/ml hygromycin B. Upon transformation with the pIG121-Hm plasmid, transformants of Chlorella vulgaris were obtained that were propagated in culture medium comprising 50 ug/ml hygromycin B. The expression of the hpt gene product in Chlorella vulgaris enabled propagation of transformed Chlorella vulgaris in the presence of 50 ug/mL hygromycin B, thereby establishing the utility of the a hygromycin B resistance cassette as a selectable marker for use in Chlorella vulgaris. Detectable activity of the GUS reporter gene indicated that CaMV 35S promoter and nos 3'UTR are suitable for enabling heterologous gene expression in Chlorella vulgaris. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Selection and maintenance of transformed Chlorella vulgaris was performed on agar plates comprising YA medium (agar and 4 g/L yeast extract). The propagation of Chlorella vulgaris in liquid culture medium was conducted as discussed by Chow and Tung. Propagation of Chlorella vulgaris in media other than YA medium has been described (for examples, see Chader et al., Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26 and Illman et al., Enzyme and Microbial Technology, Vol. 27 (2000), pp. 631-635). Chow and Tung reported that the plasmid pIG121-Hm, the CaMV 35S promoter, and the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator are suitable to enable heterologous gene expression in Chlorella vulgaris. In addition, Chow and Tung reported the hygromycin B resistance cassette was suitable for use as a selectable marker in Chlorella vulgaris. Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Chlorella vulgaris have been discussed in Chader et al., Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26.
[0387] In an embodiment of the present invention, pIG121-Hm, comprising the nucleotide sequence encoding the hygromycin B gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Chlorella vulgaris to reflect the codon bias inherent in nuclear genes of Chlorella vulgaris in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the CaMV 35S promoter upstream of the protein-coding sequence and operably linked to the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chlorella vulgaris genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Chlorella vulgaris with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the hygromycin B resistance gene product can be used as a marker to select for Chlorella vulgaris transformed with the transformation vector on, but not limited to, agar medium comprising hygromycin. Growth media suitable for Chlorella vulgaris lipid production include, but are not limited to BG11 medium (0.04 g/L KH.sub.2PO.sub.4, 0.075 g/L CaCl.sub.2, 0.036 g/L citric acid, 0.006 g/L Ammonium Ferric Citrate, 1 mg/L EDTA, and 0.02 g/L Na.sub.2CO.sub.3) supplemented with trace metals, and optionally 1.5 g/L NaNO3. Additional media suitable for culturing Chlorella vulgaris for lipid production include, for example, Watanabe medium (comprising 1.5 g/L KNO.sub.3, 1.25 g/L KH.sub.2PO.sub.4, 1.25 g l.sup.-1 MgSO.sub.4.7H.sub.2O, 20 mg l.sup.-1 FeSO.sub.4.7H.sub.2O with micronutrients and low-nitrogen medium (comprising 203 mg/l (NH.sub.4).sub.2HPO.sub.4, 2.236 g/l KCl, 2.465 g/l MgSO.sub.4, 1.361 g/l KH.sub.2PO.sub.4 and 10 mg/l FeSO.sub.4) as reported by Illman et al., Enzyme and Microbial Technology, Vol. 27 (2000), pp. 631-635. Evaluation of fatty acid profiles of Chlorella vulgaris lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 12
Engineering Chlorella Ellipsoidea
[0388] Expression of recombinant genes in accordance with the present invention in Chlorella ellipsoidea can be accomplished by modifying the methods and vectors taught by Chen et al. as discussed herein. Briefly, Chen et al., Current Genetics, Vol. 39:5 (2001), pp. 365-370, reported the stable transformation of Chlorella ellipsoidea with plasmid DNA. Using the transformation method of electroporation, Chen introduced the plasmid pBinU.OMEGA.NP-1 into Chlorella ellipsoidea. The nucleotide sequence of pBinU.OMEGA.NP-1 comprised sequence encoding the neutrophil peptide-1 (NP-1) rabbit gene product operably linked to a Zea mays Ubiquitin (ubi1) gene promoter upstream of the NP-1 protein-coding region and operably linked to the 3' UTR/terminator of the nopaline synthase (nos) gene downstream of the NP-1 protein-coding region. The sequence of plasmid pBinU.OMEGA.NP-1 further comprised a G418 antibiotic resistance cassette. This G418 antibiotic resistance cassette comprised sequence encoding the aminoglycoside 3'-phosphotransferase (aph 3') gene product. The aph 3' gene product confers resistance to the antibiotic G418. Prior to transformation, Chlorella ellipsoidea was unable to be propagated in culture medium comprising 30 ug/mL G418. Upon transformation with the pBinU.OMEGA.NP-1 plasmid, transformants of Chlorella ellipsoidea were obtained that were propagated in selective culture medium comprising 30 ug/mL G418. The expression of the aph 3' gene product in Chlorella ellipsoidea enabled propagation of transformed Chlorella ellipsoidea in the presence of 30 ug/mL G418, thereby establishing the utility of the G418 antibiotic resistance cassette as selectable marker for use in Chlorella ellipsoidea. Detectable activity of the NP-1 gene product indicated that the ubi 1 promoter and nos 3' UTR are suitable for enabling heterologous gene expression in Chlorella ellipsoidea. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Selection and maintenance of the transformed Chlorella ellipsoidea was performed on Knop medium (comprising 0.2 g/L K.sub.2HPO.sub.4, 0.2 g/L MgSO.sub.4.7H.sub.2O, 0.12 g/L KCl, and 10 mg/L FeCl3, pH 6.0-8.0 supplemented with 0.1% yeast extract and 0.2% glucose) with 15 ug/mL G418 (for liquid cultures) or with 30 ug/mL G418 (for solid cultures comprising 1.8% agar). Propagation of Chlorella ellipsoidea in media other than Knop medium has been reported (see Cho et al., Fisheries Science, Vol. 73:5 (2007), pp. 1050-1056, Jarvis and Brown, Current Genetics, Vol. 19 (1991), pp. 317-321 and Kim et al., Marine Biotechnology, Vol. 4 (2002), pp. 63-'73). Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Chlorella ellipsoidea have been reported (see Jarvis and Brown and Kim et al., Marine Biotechnology, Vol. 4 (2002), pp. 63-73). Chen reported that the plasmid pBinU.OMEGA.NP-1, the ubil promoter, and the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator are suitable to enable exogenous gene expression in Chlorella ellipsoidea. In addition, Chen reported that the G418 resistance cassette encoded on pBinU.OMEGA.NP-1 was suitable for use as a selectable marker in Chlorella ellipsoidea.
[0389] In an embodiment of the present invention, vector pBinU.OMEGA.NP-1, comprising the nucleotide sequence encoding the aph 3' gene product, conferring resistance to G418, for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Chlorella ellipsoidea to reflect the codon bias inherent in nuclear genes of Chlorella ellipsoidea in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Zea mays ubil promoter upstream of the protein-coding sequence and operably linked to the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chlorella ellipsoidea genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Chlorella ellipsoidea with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the aph 3' gene product can be used as a marker to select for Chlorella ellipsoidea transformed with the transformation vector on, but not limited to, Knop agar medium comprising G418. Growth media suitable for Chlorella ellipsoidea lipid production include, but are not limited to, Knop medium and those culture medium reported by Jarvis and Brown and Kim et al. Evaluation of fatty acid profiles of Chlorella ellipsoidea lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 13
Engineering Chlorella Kessleri
[0390] Expression of recombinant genes in accordance with the present invention in Chlorella kessleri can be accomplished by modifying the methods and vectors taught by El-Sheekh et al. as discussed herein. Briefly, El-Sheekh et al., Biologia Plantarium, Vol. 42:2 (1999), pp. 209-216, reported the stable transformation of Chlorella kessleri with plasmid DNA. Using the transformation method of microprojectile bombardment, El-Sheekh introduced the plasmid pBI121 (GenBank Accession No. AF485783) into Chlorella kessleri. Plasmid pBI121 comprised a kanamycin/neomycin antibiotic resistance cassette. This kanamycin/neomycin antibiotic resistance cassette comprised the Agrobacterium tumefaciens nopaline synthase (nos) gene promoter, sequence encoding the neomycin phosphotransferase II (nptII) gene product (GenBank Accession No. AAL92039) for resistance to kanamycin and G418, and the 3' UTR/terminator of the Agrobacterium tumefaciens nopaline synthase (nos) gene. pBI121 further comprised sequence encoding a beta-glucuronidase (GUS) reporter gene product operably linked to a CaMV 35S promoter and operably linked to a 3' UTR/terminator of the nos gene. Prior to transformation, Chlorella kessleri was unable to be propagated in culture medium comprising 15 ug/L kanamycin. Upon transformation with the pBI121plasmid, transformants of Chlorella kessleri were obtained that were propagated in selective culture medium comprising 15 mg/L kanamycin. The express ion of the nptII gene product in Chlorella kessleri enabled propagation in the presence of 15 mg/L kanamycin, thereby establishing the utility of the kanamycin/neomycin antibiotic resistance cassette as selectable marker for use in Chlorella kessleri. Detectable activity of the GUS gene product indicated that the CaMV 35S promoter and nos 3' UTR are suitable for enabling heterologous gene expression in Chlorella kessleri. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. As reported by El-Sheekh, selection and maintenance of transformed Chlorella kessleri was conducted on semisolid agar plates comprising YEG medium (1% yeast extract, 1% glucose) and 15 mg/L kanamycin. El-Sheekh also reported the propagation of Chlorella kessleri in YEG liquid culture media. Additional media suitable for culturing Chlorella kessleri for lipid production are disclosed in Sato et al., BBA Molecular and Cell Biology of Lipids, Vol. 1633 (2003), pp. 27-34). El-Sheekh reported that the plasmid pBI121, the CaMV promoter, and the nopaline synthase gene 3'UTR/terminator are suitable to enable heterologous gene expression in Chlorella kessleri. In addition, El-Sheekh reported that the kanamycin/neomycin resistance cassette encoded on pBI121 was suitable for use as a selectable marker in Chlorella kessleri.
[0391] In an embodiment of the present invention, vector pBI121, comprising the nucleotide sequence encoding the kanamycin/neomycin resistance gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Chlorella kessleri to reflect the codon bias inherent in nuclear genes of Chlorella kessleri in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the CaMV 35S promoter upstream of the protein-coding sequence and operably linked to the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chlorella kessleri genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Chlorella kessleri with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the nptII gene product can be used as a marker to select for Chlorella kessleri transformed with the transformation vector on, but not limited to, YEG agar medium comprising kanamycin or neomycin. Growth media suitable for Chlorella kessleri lipid production include, but are not limited to, YEG medium, and those culture media reported by Sato et al. Evaluation of fatty acid profiles of Chlorella kessleri lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 14
Engineering Dunaliella Tertiolecta
[0392] Expression of recombinant genes in accordance with the present invention in Dunaliella tertiolecta can be accomplished by modifying the methods and vectors taught by Walker et al. as discussed herein. Briefly, Walker et al., Journal of Applied Phycology, Vol. 17 (2005), pp. 363-368, reported stable nuclear transformation of Dunaliella tertiolecta with plasmid DNA. Using the transformation method of electroporation, Walker introduced the plasmid pDbleFLAG1.2 into Dunaliella tertiolecta. pDbleFLAG1.2 comprised sequence encoding a bleomycin antibiotic resistance cassette, comprising sequence encoding the Streptoalloteichus hindustanus Bleomycin binding protein (ble), for resistance to the antibiotic phleomycin, operably linked to the promoter and 3' UTR of the Dunaliella tertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene (rbcS1, GenBank Accession No. AY530155). Prior to transformation, Dunaliella tertiolecta was unable to be propagated in culture medium comprising 1 mg/L phleomycin. Upon transformation with the pDbleFLAG1.2 plasmid, transformants of Dunaliella tertiolecta were obtained that were propagated in selective culture medium comprising 1 mg/L phleomycin. The expression of the ble gene product in Dunaliella tertiolecta enabled propagation in the presence of 1 mg/L phleomycin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Dunaliella tertiolecta. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. As reported by Walker, selection and maintenance of transformed Dunaliella tertiolecta was conducted in Dunaliella medium (DM, as described by Provasoli et al., Archiv fur Mikrobiologie, Vol. 25 (1957), pp. 392-428) further comprising 4.5 g/L NaCl and 1 mg/L pheomycin. Additional media suitable for culturing Dunaliella tertiolecta for lipid production are discussed in Takagi et al., Journal of Bioscience and Bioengineering, Vol. 101:3 (2006), pp. 223-226 and in Massart and Hanston, Proceedings Venice 2010, Third International Symposium on Energy from Biomass and Waste. Walker reported that the plasmid pDbleFLAG1.2 and the promoter and 3' UTR of the Dunaliella tertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene are suitable to enable heterologous expression in Dunaliella tertiolecta. In addition, Walker reported that the bleomycin resistance cassette encoded on pDbleFLAG1.2 was suitable for use as a selectable marker in Dunaliella tertiolecta.
[0393] In an embodiment of the present invention, vector pDbleFLAG1.2, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Dunaliella tertiolecta to reflect the codon bias inherent in nuclear genes of Dunaliella tertiolecta in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the rbcS1 promoter upstream of the protein-coding sequence and operably linked to the rbcS1 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Dunaliella tertiolecta genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Dunaliella tertiolecta with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the ble gene product can be used as a marker to select for Dunaliella tertiolecta transformed with the transformation vector on, but not limited to, DM medium comprising pheomycin. Growth medium suitable for Dunaliella tertiolecta lipid production include, but are not limited to DM medium and those culture media described by Takagi et al. and Massart and Hanston. Evaluation of fatty acid profiles of Dunaliella tertiolecta lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 15
Engineering Volvox Carteri
[0394] Expression of recombinant genes in accordance with the present invention in Volvox carteri can be accomplished by modifying the methods and vectors taught by Hallman and Rappel et al. as discussed herein. Briefly, Hallman and Rappel et al., The Plant Journal, Volume 17 (1999), pp. 99-109, reported the stable nuclear transformation of Volvox carteri with plasmid DNA. Using the transformation method of microprojectile bombardment, Hallman and Rappel introduced the pzeoE plasmid into Volvox carteri. The pzeoE plasmid comprised sequence encoding a bleomycin antibiotic resistance cassette, comprising sequence encoding the Streptoalloteichus hindustanus Bleomycin binding protein (ble), for resistance to the antibiotic zeocin, operably linked to and the promoter and 3' UTR of the Volvox carteri beta-tubulin gene (GenBank Accession No. L24547). Prior to transformation, Volvox carteri was unable to be propagated in culture medium comprising 1.5 ug/ml zeocin. Upon transformation with the pzeoE plasmid, transformants of Volvox carteri were obtained that were propagated in selective culture medium comprising greater than 20 ug/ml zeocin. The expression of the ble gene product in Volvox carteri enabled propagation in the presence of 20 ug/ml zeocin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Volvox carteri. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. As reported by Hallman and Rappel, selection and maintenance of transformed Volvox carteri was conducted in Volvox medium (VM, as described by Provasoli and Pintner, The Ecology of Algae, Special Publication No. 2 (1959), Tyron, C. A. and Hartman, R. T., eds., Pittsburgh: University of Pittsburgh, pp. 88-96) with 1 mg/L pheomycin. Media suitable for culturing Volvox carteri for lipid production are also discussed by Starr in Starr R, C., Dev Biol Suppl., Vol. 4 (1970), pp. 59-100). Hallman and Rappel reported that the plasmid pzeoE and the promoter and 3' UTR of the Volvox carteri beta-tubulin gene are suitable to enable heterologous expression in Volvox carteri. In addition, Hallman and Rappel reported that the bleomycin resistance cassette encoded on pzeoE was suitable for use as a selectable marker in Volvox carteri. Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Volvox carteri and suitable for use as selective markers Volvox carteri in have been reported (for instance see Hallamann and Sumper, Proceedings of the National Academy of Sciences, Vol. 91 (1994), pp 11562-11566 and Hallman and Wodniok, Plant Cell Reports, Volume 25 (2006), pp. 582-581).
[0395] In an embodiment of the present invention, vector pzeoE, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 19, each protein-coding sequence codon-optimized for expression in Volvox carteri to reflect the codon bias inherent in nuclear genes of Volvox carteri in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Volvox carteri beta-tubulin promoter upstream of the protein-coding sequence and operably linked to the Volvox carteri beta-tubulin 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Volvox carteri genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Volvox carteri genome (referenced in the publication by Prochnik et al., Science, Vol. 329:5988 (2010), pp223-226). Stable transformation of Volvox carteri with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the ble gene product can be used as a marker to select for Volvox carteri transformed with the transformation vector on, but not limited to, VM medium comprising zeocin. Growth medium suitable for Volvox carteri lipid production include, but are not limited to VM medium and those culture media discussed by Starr. Evaluation of fatty acid profiles of Volvox carteri lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 16
Engineering Haematococcus Pluvialis
[0396] Expression of recombinant genes in accordance with the present invention in Haematococcus pluvialis can be accomplished by modifying the methods and vectors taught by Steinbrenner and Sandmann et al. as discussed herein. Briefly, Steinbrenner and Sandmann et al., Applied and Environmental Microbiology, Vol. 72:12 (2006), pp. 7477-7484, reported the stable nuclear transformation of Haematococcus pluvialis with plasmid DNA. Using the transformation method of microprojectile bombardment, Steinbrenner introduced the plasmid pPlat-pds-L504R into Haematococcus pluvialis. The plasmid pPlat-pds-L504R comprised a norflurazon resistance cassette, which comprised the promoter, protein-coding sequence, and 3'UTR of the Haematococcus pluvialis phytoene desaturase gene (Pds, GenBank Accession No. AY781170), wherein the protein-coding sequence of Pds was modified at position 504 (thereby changing a leucine to an arginine) to encode a gene product (Pds-L504R) that confers resistance to the herbicide norflurazon. Prior to transformation with pPlat-pds-L504R, Haematococcus pluvialis was unable to propagate on medium comprising 5 uM norflurazon. Upon transformation with the pPlat-pds-L504R plasmid, transformants of Haematococcus pluvialis were obtained that were propagated in selective culture medium comprising 5 uM norflurazon. The expression of the Pds-L504R gene product in Haematococcus pluvialis enabled propagation in the presence of 5 uM norflurazon, thereby establishing the utility of the norflurazon herbicide resistance cassette as selectable marker for use in Haematococcus pluvialis. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. As reported by Steinbrenner, selection and maintenance of transformed Haematococcus pluvialis was conducted on agar plates comprising OHA medium (OHM (0.41 g/L KNO.sub.3, 0.03 g/L Na.sub.2HPO.sub.4, 0.246 g/L MgSO.sub.4.7H.sub.2O, 0.11 g/L CaCl.sub.2.2H.sub.2O, 2.62 mg/L Fe.sub.(III)citrate.times.H.sub.2O, 0.011 mg/L CoCl.sub.2.6H.sub.2O, 0.012 mg/L CuSO.sub.4.5H.sub.2O, 0.075 mg/L Cr.sub.2O.sub.3, 0.98 mg/L MnCl.sub.2.4H.sub.2O, 0.12 mg/L Na.sub.2MoO.sub.4.times.2H.sub.2O, 0.005 mg/L SeO.sub.2 and 25 mg/L biotin, 17.5 mg/L thiamine, and 15 mg/L vitamin B12), supplemented with 2.42 g/L Tris-acetate, and 5 mM norflurazon. Propagation of Haematococcus pluvialis in liquid culture was performed by Steinbrenner and Sandmann using basal medium (basal medium as described by Kobayashi et al., Applied and Environmental Microbiology, Vol. 59 (1993), pp. 867-873). Steinbrenner and Sandmann reported that the pPlat-pds-L504R plasmid and promoter and 3' UTR of the Haematococcus pluvialis phytoene desaturase gene are suitable to enable heterologous expression in Haematococcus pluvialis. In addition, Steinbrenner and Sandmann reported that the norflurazon resistance cassette encoded on pPlat-pds-L504R was suitable for use as a selectable marker in Haematococcus pluvialis. Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Haematococcus pluvialis have been reported (see Kathiresan et al., Journal of Phycology, Vol. 45 (2009), pp 642-649).
[0397] In an embodiment of the present invention, vector pPlat-pds-L504R, comprising the nucleotide sequence encoding the Pds-L504R gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Haematococcus pluvialis to reflect the codon bias inherent in nuclear genes of Haematococcus pluvialis in accordance with Tables 19 A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Haematococcus pluvialis pds gene promoter upstream of the protein-coding sequence and operably linked to the Haematococcus pluvialis pds gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Haematococcus pluvialis genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Haematococcus pluvialis with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the Pds-L504R gene product can be used as a marker to select for Haematococcus pluvialis transformed with the transformation vector on, but not limited to, OHA medium comprising norflurazon. Growth media suitable for Haematococcus pluvialis lipid production include, but are not limited to basal medium and those culture media described by Kobayashi et al., Kathiresan et al, and Gong and Chen, Journal of Applied Phycology, Vol. 9:5 (1997), pp. 437-444). Evaluation of fatty acid profiles of Haematococcus pluvialis lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 17
Engineering Closterium Peracerosum-Strigosum-Littorale Complex
[0398] Expression of recombinant genes in accordance with the present invention in Closterium peracerosum-strigosum-littorals complex can be accomplished by modifying the methods and vectors taught by Abe et al. as discussed herein. Briefly, Abe et al., Plant Cell Physiology, Vol. 52:9 (2011), pp. 1676-1685, reported the stable nuclear transformation of Closterium peracerosum-strigosum-littorals complex with plasmid DNA. Using the transformation methods of microprojectile bombardment, Abe introduced the plasmid pSA106 into Closterium peracerosum-strigosum-littorals complex. Plasmid pSA106 comprised a bleomycin resistance cassette, comprising sequence encoding the Streptoalloteichus hindustanus Bleomycin binding protein gene (ble, GenBank Accession No. CAA37050) operably linked to the promoter and 3' UTR of the Closterium peracerosum-strigosum-littorals complex Chlorophyll a/b-binding protein gene (CAB, GenBank Accession No. AB363403). Prior to transformation with pSA106, Closterium peracerosum-strigosum-littorals complex was unable to propagate on medium comprising 3 ug/ml phleomycin. Upon transformation with pSA106, transformants of Closterium peracerosum-strigosum-littorals complex were obtained that were propagated in selective culture medium comprising 3 ug/ml phleomycin. The expression of the ble gene product in Closterium peracerosum-strigosum-littorals complex enabled propagation in the presence of 3 ug/ml phleomycin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Closterium peracerosum-strigosum-littorals complex. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. As reported by Abe, selection and maintenance of transformed Closterium peracerosum-strigosum-littorals complex was conducted first in top agar with C medium (0.1 g/L KNO.sub.3, 0.015 g/L Ca(NO.sub.3).sub.2.4H2O, 0.05 g/L glycerophosphate-Na2, 0.04 g/L MgSO.sub.4.7H.sub.2O, 0.5 g/L Tris (hydroxylmethyl) aminomethane, trace minerals, biotin, vitamins B.sub.1 and B.sub.12) and then subsequently isolated to agar plates comprising C medium supplemented with phleomycin. As reported by Abe, propagation of Closterium peracerosum-strigosum-littorals complex in liquid culture was performed in C medium. Additional liquid culture medium suitable for propagation of Closterium peracerosum-strigosum-littorale complex are discussed by Sekimoto et al., DNA Research, 10:4 (2003), pp. 147-153. Abe reported that the pSA106 plasmid and promoter and 3' UTR of the Closterium peracerosum-strigosum-littorals complex CAB gene are suitable to enable heterologous gene expression in Closterium peracerosum-strigosum-littorals complex. In addition, Abe reported that the bleomycin resistance cassette encoded on pSA106 was suitable for use as a selectable marker in Closterium peracerosum-strigosum-littorals complex. Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Closterium peracerosum-strigosum-littorale complex have been reported (see Abe et al., Plant Cell Physiology, Vol. 49 (2008), pp. 625-632).
[0399] In an embodiment of the present invention, vector pSA106, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Closterium peracerosum-strigosum-littorale complex to reflect the codon bias inherent in nuclear genes of Closterium peracerosum-strigosum-littorale complex in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Closterium peracerosum-strigosum-littorale complex CAB gene promoter upstream of the protein-coding sequence and operably linked to the Closterium peracerosum-strigosum-littorale complex CAB gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Closterium peracerosum-strigosum-littorale complex genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Closterium peracerosum-strigosum-littorale complex with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the ble gene product can be used as a marker to select for Closterium peracerosum-strigosum-littorale complex transformed with the transformation vector on, but not limited to, C medium comprising phleomycin. Growth media suitable for Closterium peracerosum-strigosum-littorale complex lipid production include, but are not limited to C medium and those culture media reported by Abe et al. and Sekimoto et al. Evaluation of fatty acid profiles of Closterium peracerosum-strigosum-littorale complex lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 18
Engineering Dunaliella Viridis
[0400] Expression of recombinant genes in accordance with the present invention in Dunaliella viridis can be accomplished by modifying the methods and vectors taught by Sun et al. as discussed herein. Briefly, Sun et al., Gene, Vol. 377 (2006), pp. 140-149, reported the stable transformation of Dunaliella viridis with plasmid DNA. Using the transformation method of electroporation, Sun introduced the plasmid pDVNR, encoding the full Dunaliella viridis nitrate reductase gene into mutant Dunaliella viridis (Dunaliella viridis NR-mutants.) The NR-mutants are incapable of growth without the use of nitrate as a source of nitrogen. Nitrate reductase catalyzes the conversion of nitrate to nitrite. Prior to transformation, Dunaliella viridis NR-mutants were unable to propagate in culture medium comprising nitrate (NO.sub.3.sup.-1 ) as the sole nitrogen source. The expression of the Dunaliella viridis NR gene product in NR-mutant Dunaliella viridis was used as a selectable marker to rescue the nitrate metabolism deficiency. Upon transformation with the pDVNR plasmid, NR-mutant Dunaliella viridis stably expressing the Dunaliella viridis NR gene product were obtained that were able to grow on agar plates comprising nitrate as the sole carbon source. Evaluation of the DNA of the stable transformants was performed by Southern analysis. Selection and maintenance of the transformed Dunaliella viridis (NR mutant) was performed on agar plates comprising 5 mM KNO.sub.3. Sun also reported the propagation of Dunaliella viridis and Dunaliella viridis NR mutants in liquid culture medium. Additional media suitable for propagation of Dunaliella viridis are reported by Gordillo et al., Journal of Applied Phycology, Vol. 10:2 (1998), pp. 135-144 and by Moulton and Burford, Hydrobiologia, Vols. 204-205:1 (1990), pp. 401-408. Sun reported that the plasmid pDVNR and the promoter and 3' UTR/terminator of the Dunaliella viridis nitrate reductase gene were suitable to enable heterologous expression in Dunaliella viridis NR-mutants. Sun also reported that expression of the Dunaliella viridis nitrate reductase gene product was suitable for use as a selectable marker in Dunaliella viridis NR-mutants.
[0401] In an embodiment of the present invention, vector pDVNR, comprising the nucleotide sequence encoding the Dunaliella viridis nitrate reductase (DvNR) gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Dunaliella viridis to reflect the codon bias inherent in nuclear genes of Dunaliella viridis in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the DvNR promoter upstream of the protein-coding sequence and operably linked to the DvNR 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Dunaliella viridis genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Dunaliella viridis NR mutants with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the DvNR gene product can be used as a selectable marker to rescue the nitrogen assimilation deficiency of Dunaliella viridis NR mutant strains and to select for Dunaliella viridis NR-mutants stably expressing the transformation vector. Growth media suitable for Dunaliella viridis lipid production include, but are not limited to those discussed by Sun et al., Moulton and Burford, and Gordillo et al. Evaluation of fatty acid profiles of Dunaliella viridis lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 19
Engineering Dunaliella Salina
[0402] Expression of recombinant genes in accordance with the present invention in Dunaliella salina can be accomplished by modifying the methods and vectors taught by Geng et al. as discussed herein. Briefly, Geng et al., Journal of Applied Phycology, Vol. 15 (2003), pp. 451-456, reported the stable transformation of Dunaliella salina with plasmid DNA. Using the transformation method of electroporation, Geng introduced the pU.OMEGA.HBsAg-CAT plasmid into Dunaliella salina. pU.OMEGA.HBsAg-CAT comprises a hepatitis B surface antigen (HBsAG) expression cassette comprising sequence encoding the hepatitis B surface antigen operably linked to a Zea mays ubil promoter upstream of the HBsAG protein-coding region and operably linked to the 3'UTR/terminator of the Agrobacterium tumefaciens nopaline synthase gene (nos) downstream of the HBsAG protein-coding region. pU.OMEGA.HBsAg-CAT further comprised a chloramphenicol resistance cassette, comprising sequence encoding the chloramphenicol acetyltransferase (CAT) gene product, conferring resistance to the antibiotic chloramphenicol, operably linked to the simian virus 40 promoter and enhancer. Prior to transformation with pU.OMEGA.HBsAg-CAT, Dunaliella salina was unable to propagate on medium comprising 60 mg/L chloramphenicol. Upon transformation with the pU.OMEGA.HBsAg-CAT plasmid, transformants of Dunaliella salina were obtained that were propagated in selective culture medium comprising 60 mg/L chloramphenicol. The expression of the CAT gene product in Dunaliella salina enabled propagation in the presence of 60 mg/L chloramphenicol, thereby establishing the utility of the chloramphenicol resistance cassette as selectable marker for use in Dunaliella salina. Detectable activity of the HBsAg gene product indicated that ubil promoter and nos 3'UTR/terminator are suitable for enabling gene expression in Dunaliella salina. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Geng reported that selection and maintenance of the transformed Dunaliella salina was performed on agar plates comprising Johnson's medium (J1, described by Borowitzka and Borowitzka (eds), Micro-algal Biotechnology. Cambridge University Press, Cambridge, pp. 460-461) with 60 mg/L chloramphenicol. Liquid propagation of Dunaliella salina was performed by Geng in J1 medium with 60 mg/L chloramphenicol. Propagation of Dunaliella salina in media other than J1 medium has been discussed (see Feng et al., Mol. Bio. Reports, Vol. 36 (2009), pp. 1433-1439 and Borowitzka et al., Hydrobiologia, Vols. 116-117:1 (1984), pp. 115-121). Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Dunaliella salina have been reported by Feng et al. Geng reported that the plasmid pU.OMEGA.HBsAg-CAT, the ubil promoter, and the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator are suitable to enable exogenous gene expression in Dunaliella salina. In addition, Geng reported that the CAT resistance cassette encoded on pU.OMEGA.HBsAg-CAT was suitable for use as a selectable marker in Dunaliella salina.
[0403] In an embodiment of the present invention, vector pU.OMEGA.HBsAg-CAT, comprising the nucleotide sequence encoding the CAT gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Dunaliella salina to reflect the codon bias inherent in nuclear genes of Dunaliella salina in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the ubil promoter upstream of the protein-coding sequence and operably linked to the Agrobacterium tumefaciens nopaline synthase gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Dunaliella salina genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Dunaliella salina with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the CAT gene product can be used as a selectable marker to select for Dunaliella salina transformed with the transformation vector in, but not limited to, J1 medium comprising chloramphenicol. Growth medium suitable for Dunaliella salina lipid production include, but are not limited to J1 medium and those culture media described by Feng et al. and Borowitzka et al. Evaluation of fatty acid profiles of Dunaliella salina lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 20
Engineering Gonium Pectoral
[0404] Expression of recombinant genes in accordance with the present invention in Gonium pectoral can be accomplished by modifying the methods and vectors taught by Lerche and Hallman et al. as discussed herein. Briefly, Lerche and Hallman et al., BMC Biotechnology, Volume 9:64, 2009, reported the stable nuclear transformation of Gonium pectorale with plasmid DNA. Using the transformation method of microprojectile bombardment, Lerche introduced the plasmid pPmr3 into Gonium pectorale. Plasmid pPmr3 comprised a paromomycin resistance cassette, comprising a sequence encoding the aminoglycoside 3'-phosphotransferase (aphVIII) gene product (GenBank Accession No. AAB03856) of Streptomyces rimosus for resistance to the antibiotic paromomycin, operably linked to the Volvox carteri hsp70A-rbcS3 hybrid promoter upstream of the aphVIII protein-coding region and operably linked to the 3' UTR/terminator of the Volvox carteri rbcS3 gene downstream of the aphVIII protein-coding region. Prior to transformation with pPmr3, Gonium pectorale was unable to propagate on medium comprising 0.06 ug/ml paromomycin. Upon transformation with pPmr3, transformants of Gonium pectorale were obtained that were propagated in selective culture medium comprising 0.75 and greater ug/ml paromomycin. The expression of the aphVIII gene product in Gonium pectorale enabled propagation in the presence of 0.75 and greater ug/ml paromomycin, thereby establishing the utility of the paromomycin antibiotic resistance cassette as selectable marker for use in Gonium pectorals. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Lerche and Hallman reported that selection and maintenance of the transformed Gonium pectorale was performed in liquid Jaworski's medium (20 mg/L Ca(NO.sub.3).sub.2.4H.sub.2O, 12.4 mg/L KH.sub.2PO.sub.4, 50 mg/L MgSO.sub.4.7H.sub.2O, 15.9 mg/L NaHCO.sub.3, 2.25 mg/L EDTA-FeNa, 2.25 mg/L EDTA Na.sub.2, 2.48 g/L H.sub.3BO.sub.3, 1.39 g/L MnCl.sub.2.4H.sub.2O, 1 mg/L (NH.sub.4).sub.6MO.sub.7O.sub.24.4H.sub.2O, 0.04 mg/L vitamin B12, 0.04 mg/L Thiamine-HCl, 0.04 mg/L biotin, 80 mg/L NaNO.sub.3, 36 mg/L Na.sub.4HPO.sub.4.12H.sub.2O) with 1.0 ug/ml paromomycin. Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Gonium pectorale are further discussed by Lerche and Hallman. Lerche and Hallman reported that the plasmid pPmr3, Volvox carteri hsp70A-rbcS3 hybrid promoter, and the 3' UTR/terminator of the Volvox carteri rbcS3 gene are suitable to enable exogenous gene expression in Gonium pectorals. In addition, Lerche and Hallman reported that the paromomycin resistance cassette encoded pPmr3 was suitable for use as a selectable marker in Gonium pectorale
[0405] In an embodiment of the present invention, vector pPmr3, comprising the nucleotide sequence encoding the aphVIII gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Gonium pectorale to reflect the codon bias inherent in nuclear genes of Gonium pectorale in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Volvox carteri hsp70A-rbcS3 hybrid promoter upstream of the protein-coding sequence and operably linked to the Volvox carteri rbcS3 gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Gonium pectorale genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Gonium pectorale with the transformation vector can be achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the aphVIII gene product can be used as a selectable marker to select for Gonium pectorale transformed with the transformation vector in, but not limited to, Jaworski's medium comprising paromomycin. Growth media suitable for Gonium pectorale lipid production include Jaworski's medium and media reported by Stein, American Journal of Botany, Vol. 45:9 (1958), pp. 664-672. Evaluation of fatty acid profiles of Gonium pectorale lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 21
Engineering Phaeodactylum Tricornutum
[0406] Expression of recombinant genes in accordance with the present invention in Phaeodactylum tricornutum can be accomplished by modifying the methods and vectors taught by Apt et al. as discussed herein. Briefly, Apt et al., Molecular and General Genetics, Vol. 252 (1996), pp. 572-579, reported the stable nuclear transformation of Phaeodactylum tricornutum with vector DNA. Using the transformation technique of microprojectile bombardment, Apt introduced the plasmid pfcpA into Phaeodactylum tricornutum. Plasmid pfcpA comprised a bleomycin resistance cassette, comprising sequence encoding the Streptoalloteichus hindustanus Bleomycin binding protein (ble), for resistance to the antibiotics phleomycin and zeocin, operably linked to the promoter of the Phaeodactylum tricornutum fucoxanthin chlorophyll a binding protein gene (fcpA) upstream of the ble protein-coding region and operably linked to the 3' UTR/terminator of the Phaeodactylum tricornutum fcpA gene at the 3' region, or downstream of the ble protein-coding region. Prior to transformation with pfcpA, Phaeodactylum tricornutum was unable to propagate on medium comprising 50 ug/ml zeocin. Upon transformation with pfcpA, transformants of Phaeodactylum tricornutum were obtained that were propagated in selective culture medium comprising 50 ug/ml zeocin. The expression of the ble gene product in Phaeodactylum tricornutum enabled propagation in the presence of 50 ug/ml zeocin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Phaeodactylum tricornutum. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Apt reported that selection and maintenance of the transformed Phaeodactylum tricornutum was performed on agar plates comprising LDM medium (as reported by Starr and Zeikus, Journal of Phycology, Vol. 29, Supplement, (1993)) with 50 mg/L zeocin. Apt reported liquid propagation of Phaeodactylum tricornutum transformants in LDM medium with 50 mg/L zeocin. Propagation of Phaeodactylum tricornutum in medium other than LDM medium has been discussed (by Zaslayskaia et al., Science, Vol. 292 (2001), pp. 2073-2075, and by Radokovits et al., Metabolic Engineering, Vol. 13 (2011), pp. 89-95). Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Phaeodactylum tricornutum have been reported in the same report by Apt et al., by Zaslayskaia et al., and by Radokovits et al.). Apt reported that the plasmid pfcpA, and the Phaeodactylum tricornutum fcpA promoter and 3' UTR/terminator are suitable to enable exogenous gene expression in Phaeodactylum tricornutum. In addition, Apt reported that the bleomycin resistance cassette encoded on pfcpA was suitable for use as a selectable marker in Phaeodactylum tricornutum.
[0407] In an embodiment of the present invention, vector pfcpA, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Phaeodactylum tricornutum to reflect the codon bias inherent in nuclear genes of Phaeodactylum tricornutum in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Phaeodactylum tricornutum fcpA gene promoter upstream of the protein-coding sequence and operably linked to the Phaeodactylum tricornutum fcpA gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Phaeodactylum tricornutum genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Phaeodactylum tricornutum genome (referenced in the publication by Bowler et al., Nature, Vol. 456 (2008), pp. 239-244). Stable transformation of Phaeodactylum tricornutum with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the ble gene product can be used as a marker to select for Phaeodactylum tricornutum transformed with the transformation vector in, but not limited to, LDM medium comprising paromomycin. Growth medium suitable for Phaeodactylum tricornutum lipid production include, but are not limited to f/2 medium as reported by Radokovits et al. Evaluation of fatty acid profiles of Phaeodactylum tricornutum lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 22
Engineering Chaetoceros sp.
[0408] Expression of recombinant genes in accordance with the present invention in Chaetoceros sp. can be accomplished by modifying the methods and vectors taught by Yamaguchi et al. as discussed herein. Briefly, Yamaguchi et al., Phsycological Research, Vol. 59:2 (2011), pp. 113-119, reported the stable nuclear transformation of Chaetoceros sp. with plasmid DNA. Using the transformation method of microprojectile bombardment, Yamaguchi introduced the plasmid pTpfcp/nat into Chaetoceros sp. pTpfcp/nat comprised a nourseothricin resistance cassette, comprising sequence encoding the nourseothricin acetyltransferase (nat) gene product (GenBank Accession No. AAC60439) operably linked to the Thalassiosira pseudonana fucoxanthin chlorophyll a/c binding protein gene (fcp) promoter upstream of the nat protein-coding region and operably linked to the Thalassiosira pseudonana fcp gene 3' UTR/terminator at the 3' region (downstream of the nat protein coding-sequence). The nat gene product confers resistance to the antibiotic nourseothricin. Prior to transformation with pTpfcp/nat, Chaetoceros sp. was unable to propagate on medium comprising 500 ug/ml nourseothricin. Upon transformation with pTpfcp/nat, transformants of Chaetoceros sp. were obtained that were propagated in selective culture medium comprising 500 ug/ml nourseothricin. The expression of the nat gene product in Chaetoceros sp. enabled propagation in the presence of 500 ug/ml nourseothricin, thereby establishing the utility of the nourseothricin antibiotic resistance cassette as selectable marker for use in Chaetoceros sp. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Yamaguchi reported that selection and maintenance of the transformed Chaetoceros sp. was performed on agar plates comprising f/2 medium (as reported by Guilard, R. R., Culture of Phytoplankton for feeding marine invertebrates, In Culture of Marine Invertebrate Animals, Smith and Chanley (eds) 1975, Plenum Press, New York, pp. 26-60) with 500 ug/ml nourseothricin. Liquid propagation of Chaetoceros sp. transformants, as performed by Yamaguchi, was carried out in f/2 medium with 500 mg/L nourseothricin. Propagation of Chaetoceros sp. in additional culture medium has been reported (for example in Napolitano et al., Journal of the World Aquaculture Society, Vol. 21:2 (1990), pp. 122-130, and by Volkman et al., Journal of Experimental Marine Biology and Ecology, Vol. 128:3 (1989), pp. 219-240). Additional plasmids, promoters, 3'UTR/terminators, and selectable markers suitable for enabling heterologous gene expression in Chaetoceros sp. have been reported in the same report by Yamaguchi et al. Yamaguchi reported that the plasmid pTpfcp/nat, and the Thalassiosira pseudonana fcp promoter and 3' UTR/terminator are suitable to enable exogenous gene expression in Chaetoceros sp. In addition, Yamaguchi reported that the nourseothricin resistance cassette encoded on pTpfcp/nat was suitable for use as a selectable marker in Chaetoceros sp.
[0409] In an embodiment of the present invention, vector pTpfcp/nat, comprising the nucleotide sequence encoding the nat gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in the closely-related Chaetoceros compressum to reflect the codon bias inherent in nuclear genes of Chaetoceros compressum in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Thalassiosira pseudonana fcp gene promoter upstream of the protein-coding sequence and operably linked to the Thalassiosira pseudonana fcp gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chaetoceros sp. genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Chaetoceros sp. with the transformation vector is achieved through well-known transformation including microprojectile bombardment or other known methods. Activity of the nat gene product can be used as a selectable marker to select for Chaetoceros sp. transformed with the transformation vector in, but not limited to, f/2 agar medium comprising nourseothricin. Growth medium suitable for Chaetoceros sp. lipid production include, but are not limited to, f/2 medium, and those culture media discussed by Napolitano et al. and Volkman et al. Evaluation of fatty acid profiles of Chaetoceros sp lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 23
Engineering Cylindrotheca Fusiformis
[0410] Expression of recombinant genes in accordance with the present invention in Cylindrotheca fusiformis can be accomplished by modifying the methods and vectors taught by Poulsen and Kroger et al. as discussed herein. Briefly, Poulsen and Kroger et al., FEBS Journal, Vol. 272 (2005), pp. 3413-3423, reported the transformation of Cylindrotheca fusiformis with plasmid DNA. Using the transformation method of microprojectile bombardment, Poulsen and Kroger introduced the pCF-ble plasmid into Cylindrotheca fusiformis. Plasmid pCF-ble comprised a bleomycin resistance cassette, comprising sequence encoding the Streptoalloteichus hindustanus Bleomycin binding protein (ble), for resistance to the antibiotics zeocin and phleomycin, operably linked to the Cylindrotheca fusiformis fucozanthin chlorophyll a/c binding protein gene (fcpA, GenBank Accession No. AY125580) promoter upstream of the ble protein-coding region and operably linked to the Cylindrotheca fusiformis fcpA gene 3'UTR/terminator at the 3' region (down-stream of the ble protein-coding region). Prior to transformation with pCF-ble, Cylindrotheca fusiformis was unable to propagate on medium comprising 1 mg/ml zeocin. Upon transformation with pCF-ble, transformants of Cylindrotheca fusiformis were obtained that were propagated in selective culture medium comprising 1 mg/ml zeocin. The expression of the ble gene product in Cylindrotheca fusiformis enabled propagation in the presence of 1 mg/ml zeocin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Cylindrotheca fusiformis. Poulsen and Kroger reported that selection and maintenance of the transformed Cylindrotheca fusiformis was performed on agar plates comprising artificial seawater medium with 1 mg/ml zeocin. Poulsen and Kroger reported liquid propagation of Cylindrotheca fusiformis transformants in artificial seawater medium with 1 mg/ml zeocin. Propagation of Cylindrotheca fusiformis in additional culture medium has been discussed (for example in Liang et al., Journal of Applied Phycology, Vol. 17:1 (2005), pp. 61-65, and by Orcutt and Patterson, Lipids, Vol. 9:12 (1974), pp. 1000-1003). Additional plasmids, promoters, and 3'UTR/terminators for enabling heterologous gene expression in Chaetoceros sp. have been reported in the same report by Poulsen and Kroger. Poulsen and Kroger reported that the plasmid pCF-ble and the Cylindrotheca fusiformis fcp promoter and 3' UTR/terminator are suitable to enable exogenous gene expression in Cylindrotheca fusiformis. In addition, Poulsen and Kroger reported that the bleomycin resistance cassette encoded on pCF-ble was suitable for use as a selectable marker in Cylindrotheca fusiformis.
[0411] In an embodiment of the present invention, vector pCF-ble, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Cylindrotheca fusiformis to reflect the codon bias inherent in nuclear genes of Cylindrotheca fusiformis in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Cylindrotheca fusiformis fcp gene promoter upstream of the protein-coding sequence and operably linked to the Cylindrotheca fusiformis fcp gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Cylindrotheca fusiformis genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Cylindrotheca fusiformis with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the ble gene product can be used as a selectable marker to select for Cylindrotheca fusiformis transformed with the transformation vector in, but not limited to, artificial seawater agar medium comprising zeocin. Growth media suitable for Cylindrotheca fusiformis lipid production include, but are not limited to, artificial seawater and those media reported by Liang et al. and Orcutt and Patterson. Evaluation of fatty acid profiles of Cylindrotheca fusiformis lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 24
Engineering Amphidinium sp.
[0412] Expression of recombinant genes in accordance with the present invention in Amphidinium sp. can be accomplished by modifying the methods and vectors taught by ten Lohuis and Miller et al. as discussed herein. Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol. 13:3 (1998), pp. 427-435, reported the stable transformation of Amphidinium sp. with plasmid DNA. Using the transformation technique of agitation in the presence of silicon carbide whiskers, ten Lohuis introduced the plasmid pMT NPT/GUS into Amphidinium sp. pMT NPT/GUS comprised a neomycin resistance cassette, comprising sequence encoding the neomycin phosphotransferase II (nptII) gene product (GenBank Accession No. AAL92039) operably linked to the Agrobacterium tumefaciens nopaline synthase (nos) gene promoter upstream, or 5' of the nptII protein-coding region and operably linked to the 3' UTR/terminator of the nos gene at the 3' region (down-stream of the nptII protein-coding region). The nptII gene product confers resistance to the antibiotic G418. The pMT NPT/GUS plasmid further comprised sequence encoding a beta-glucuronidase (GUS) reporter gene product operably-linked to a CaMV 35S promoter and further operably linked to the CaMV 35S 3' UTR/terminator. Prior to transformation with pMT NPT/GUS, Amphidinium sp. was unable to be propagated on medium comprising 3 mg/ml G418. Upon transformation with pMT NPT/GUS, transformants of Amphidinium sp. were obtained that were propagated in selective culture medium comprising 3 mg/ml G418. The expression of the nptII gene product in Amphidinium sp. enabled propagation in the presence of 3 mg/ml G418, thereby establishing the utility of the neomycin antibiotic resistance cassette as selectable marker for use in Amphidinium sp. Detectable activity of the GUS reporter gene indicated that CaMV 35S promoter and 3'UTR are suitable for enabling gene expression in Amphidinium sp. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. ten Lohuis and Miller reported liquid propagation of Amphidinium sp transformants in medium comprising seawater supplemented with F/2 enrichment solution (provided by the supplier Sigma) and 3 mg/ml G418 as well as selection and maintenance of Amphidinium sp. transformants on agar medium comprising seawater supplemented with F/2 enrichment solution and 3 mg/ml G418. Propagation of Amphidinium sp. in additional culture medium has been reported (for example in Mansour et al., Journal of Applied Phycology, Vol. 17:4 (2005) pp. 287-v300). An additional plasmid, comprising additional promoters, 3'UTR/terminators, and a selectable marker for enabling heterologous gene expression in Amphidinium sp. have been reported in the same report by ten Lohuis and Miller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUS and the promoter and 3' UTR/terminator of the nos and CaMV 35S genes are suitable to enable exogenous gene expression in Amphidinium sp. In addition, ten Lohuis and Miller reported that the neomycin resistance cassette encoded on pMT NPT/GUS was suitable for use as a selectable marker in Amphidinium sp.
[0413] In an embodiment of the present invention, vector pMT NPT/GUS, comprising the nucleotide sequence encoding the nptII gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Amphidinium sp. to reflect the codon bias inherent in nuclear genes of the closely-related species, Amphidinium carterae in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Agrobacterium tumefaciens nopaline synthase (nos) gene promoter upstream of the protein-coding sequence and operably linked to the nos 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Amphidinium sp. genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Amphidinium sp. with the transformation vector is achieved through well-known transformation techniques including silicon fibre-mediated microinjection or other known methods. Activity of the nptII gene product can be used as a selectable marker to select for Amphidinium sp. transformed with the transformation vector in, but not limited to, seawater agar medium comprising G418. Growth media suitable for Amphidinium sp. lipid production include, but are not limited to, artificial seawater and those media reported by Mansour et al. and ten Lohuis and Miller. Evaluation of fatty acid profiles of Amphidinium sp. lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 25
Engineering Symbiodinium Microadriacticum
[0414] Expression of recombinant genes in accordance with the present invention in Symbiodinium microadriacticum can be accomplished by modifying the methods and vectors taught by ten Lohuis and Miller et al. as discussed herein. Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol. 13:3 (1998), pp. 427-435, reported the stable transformation of Symbiodinium microadriacticum with plasmid DNA. Using the transformation technique of silicon fibre-mediated microinjection, ten Lohuis introduced the plasmid pMT NPT/GUS into Symbiodinium microadriacticum. pMT NPT/GUS comprised a neomycin resistance cassette, comprising sequence encoding the neomycin phosphotransferase II (nptII) gene product (GenBank Accession No. AAL92039) operably linked to the Agrobacterium tumefaciens nopaline synthase (nos) gene promoter upstream, or 5' of the nptII protein-coding region and operably linked to the 3' UTR/terminator of the nos gene at the 3' region (down-stream of the nptII protein-coding region). The nptII gene product confers resistance to the antibiotic G418. The pMT NPT/GUS plasmid further comprised sequence encoding a beta-glucuronidase (GUS) reporter gene product operably-linked to a CaMV 35S promoter and further operably linked to the CaMV 35S 3' UTR/terminator. Prior to transformation with pMT NPT/GUS, Symbiodinium microadriacticum was unable to be propagated on medium comprising 3 mg/ml G418. Upon transformation with pMT NPT/GUS, transformants of Symbiodinium microadriacticum were obtained that were propagated in selective culture medium comprising 3 mg/ml G418. The expression of the nptII gene product in Symbiodinium microadriacticum enabled propagation in the presence of 3 mg/ml G418, thereby establishing the utility of the neomycin antibiotic resistance cassette as selectable marker for use in Symbiodinium microadriacticum. Detectable activity of the GUS reporter gene indicated that CaMV 35S promoter and 3'UTR are suitable for enabling gene expression in Symbiodinium microadriacticum. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. ten Lohuis and Miller reported liquid propagation of Symbiodinium microadriacticum transformants in medium comprising seawater supplemented with F/2 enrichment solution (provided by the supplier Sigma) and 3 mg/ml G418 as well as selection and maintenance of Symbiodinium microadriacticum transformants on agar medium comprising seawater supplemented with F/2 enrichment solution and 3 mg/ml G418. Propagation of Symbiodinium microadriacticum in additional culture medium has been discussed (for example in Iglesias-Prieto et al., Proceedings of the National Academy of Sciences, Vol. 89:21 (1992) pp. 10302-10305). An additional plasmid, comprising additional promoters, 3'UTR/terminators, and a selectable marker for enabling heterologous gene expression in Symbiodinium microadriacticum have been discussed in the same report by ten Lohuis and Miller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUS and the promoter and 3' UTR/terminator of the nos and CaMV 35S genes are suitable to enable exogenous gene expression in Symbiodinium microadriacticum. In addition, ten Lohuis and Miller reported that the neomycin resistance cassette encoded on pMT NPT/GUS was suitable for use as a selectable marker in Symbiodinium microadriacticum.
[0415] In an embodiment of the present invention, vector pMT NPT/GUS, comprising the nucleotide sequence encoding the nptII gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected Table 20, each protein-coding sequence codon-optimized for expression in Symbiodinium microadriacticum to reflect the codon bias inherent in nuclear genes of Symbiodinium microadriacticum in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Agrobacterium tumefaciens nopaline synthase (nos) gene promoter upstream of the protein-coding sequence and operably linked to the nos 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Symbiodinium microadriacticum genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Symbiodinium microadriacticum with the transformation vector is achieved through well-known transformation techniques including silicon fibre-mediated microinjection or other known methods. Activity of the nptII gene product can be used as a selectable marker to select for Symbiodinium microadriacticum transformed with the transformation vector in, but not limited to, seawater agar medium comprising G418. Growth media suitable for Symbiodinium microadriacticum lipid production include, but are not limited to, artificial seawater and those media reported by Iglesias-Prieto et al. and ten Lohuis and Miller. Evaluation of fatty acid profiles of Symbiodinium microadriacticum lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 26
Engineering Nannochloropsis sp.
[0416] Expression of recombinant genes in accordance with the present invention in Nannochloropsis sp. W2J3B can be accomplished by modifying the methods and vectors taught by Kilian et al. as discussed herein. Briefly, Kilian et al., Proceedings of the National Academy of Sciences, Vol. 108:52 (2011) pp. 21265-21269, reported the stable nuclear transformation of Nannochloropsis with a transformation construct. Using the transformation method of electroporation, Kilian introduced the transformation construct C2 into Nannochloropsis sp. W2J3B. The C2 transformation construct comprised a bleomycin resistance cassette, comprising the coding sequence for the Streptoalloteichus hindustanus Bleomycin binding protein (ble), for resistance to the antibiotics phleomycin and zeocin, operably linked to and the promoter of the Nannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding protein gene VCP2 upstream of the ble protein-coding region and operably linked to the 3'UTR/terminator of the Nannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding gene VCP1 downstream of the ble protein-coding region. Prior to transformation with C2, Nannochloropsis sp. W2J3B was unable to propagate on medium comprising 2 ug/ml zeocin. Upon transformation with C2, transformants of Nannochloropsis sp. W2J3B were obtained that were propagated in selective culture medium comprising 2 ug/ml zeocin. The expression of the ble gene product in Nannochloropsis sp. W2J3B enabled propagation in the presence of 2 ug/ml zeocin, thereby establishing the utility of the bleomycin antibiotic resistance cassette as selectable marker for use in Nannochloropsis. Evaluation of the genomic DNA of the stable transformants was performed by PCR. Kilian reported liquid propagation of Nannochloropsis sp. W2J3B transformants in F/2 medium (reported by Guilard and Ryther, Canadian Journal of Microbiology, Vol. 8 (1962), pp. 229-239) comprising fivefold levels of trace metals, vitamins, and phosphate solution, and further comprising 2 ug/ml zeocin. Kilian also reported selection and maintenance of Nannochloropsis sp. W2J3B transformants on agar F/2 medium comprising artificial seawater 2 mg/ml zeocin. Propagation of Nannochloropsis in additional culture medium has been discussed (for example in Chiu et al., Bioresour Technol., Vol. 100:2 (2009), pp. 833-838 and Pal et al., Applied Microbiology and Biotechnology, Vol. 90:4 (2011), pp. 1429-1441.). Additional transformation constructs, comprising additional promoters and 3'UTR/terminators for enabling heterologous gene expression in Nannochloropsis sp. W2J3B and selectable markers for selection of transformants have been described in the same report by Kilian. Kilian reported that the transformation construct C2 and the promoter of the Nannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding protein gene VCP2 and 3' UTR/terminator of the Nannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding protein gene VCP1 are suitable to enable exogenous gene expression in Nannochloropsis sp. W2J3B. In addition, Kilian reported that the bleomycin resistance cassette encoded on C2 was suitable for use as a selectable marker in Nannochloropsis sp. W2J3B.
[0417] In an embodiment of the present invention, transformation construct C2, comprising the nucleotide sequence encoding the ble gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Nannochloropsis sp. W2J3B to reflect the codon bias inherent in nuclear genes of Nannochloropsis sp. in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Nannochloropsis sp. W2J3B VCP2 gene promoter upstream of the protein-coding sequence and operably linked to the Nannochloropsis sp. W2J3B VCP1 gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Nannochloropsis sp. W2J3B genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Nannochloropsis sp. W2J3B with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the ble gene product can be used as a selectable marker to select for Nannochloropsis sp. W2J3B transformed with the transformation vector in, but not limited to, F/2 medium comprising zeocin. Growth media suitable for Nannochloropsis sp. W2J3B lipid production include, but are not limited to, F/2 medium and those media reported by Chiu et al. and Pal et al. Evaluation of fatty acid profiles of Nannochloropsis sp. W2J3B lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 27
Engineering Cyclotella Cryptica
[0418] Expression of recombinant genes in accordance with the present invention in Cyclotella cryptica can be accomplished by modifying the methods and vectors taught by Dunahay et al. as discussed herein. Briefly, Dunahay et al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reported the stable transformation of Cyclotella cryptica with plasmid DNA. Using the transformation method of microprojectile bombardment, Dunahay introduced the plasmid pACCNPT5.1 into Cyclotella cryptica. Plasmid pACCNPT5.1 comprised a neomycin resistance cassette, comprising the coding sequence of the neomycin phosphotransferase II (nptII) gene product operably linked to the promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784) upstream of the nptII coding-region and operably linked to the 3'UTR/terminator of the Cyclotella cryptica ACCase gene at the 3' region (downstream of the nptII coding-region). The nptII gene product confers resistance to the antibiotic G418. Prior to transformation with pACCNPT5.1, Cyclotella cryptica was unable to propagate on 50% artificial seawater medium comprising 100 ug/ml G418. Upon transformation with pACCNPT5.1, transformants of Cyclotella cryptica were obtained that were propagated in selective 50% artificial seawater medium comprising 100 ug/ml G418. The expression of the nptII gene product in Cyclotella cryptica enabled propagation in the presence of 100 ug/ml G418, thereby establishing the utility of the neomycin antibiotic resistance cassette as selectable marker for use in Cyclotella cryptica. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Dunahay reported liquid propagation of Cyclotella cryptica in artificial seawater medium (ASW, as discussed by Brown, L., Phycologia, Vol. 21 (1982), pp. 408-410) supplemented with 1.07 mM sodium silicate and with 100 ug/ml G418. Dunahay also reported selection and maintenance of Cyclotella cryptica transformants on agar plates comprising ASW medium with 100 ug/ml G418. Propagation of Cyclotella cryptica in additional culture medium has been discussed (for example in Sriharan et al., Applied Biochemistry and Biotechnology, Vol. 28-29:1 (1991), pp. 317-326 and Pahl et al., Journal of Bioscience and Bioengineering, Vol. 109:3 (2010), pp. 235-239). Dunahay reported that the plasmid pACCNPT5.1 and the promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene are suitable to enable exogenous gene expression in Cyclotella cryptica. In addition, Dunahay reported that the neomycin resistance cassette encoded on pACCNPT5.1 was suitable for use as a selectable marker in Cyclotella cryptica.
[0419] In an embodiment of the present invention, vector pACCNPT5.1, comprising the nucleotide sequence encoding the nptII gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Cyclotella cryptica to reflect the codon bias inherent in nuclear genes of Cyclotella cryptica in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Cyclotella cryptica ACCase promoter upstream of the protein-coding sequence and operably linked to the Cyclotella cryptica ACCase 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Cyclotella cryptica genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Cyclotella cryptica with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the nptII gene product can be used as a marker to select for Cyclotella cryptica transformed with the transformation vector in, but not limited to, agar ASW medium comprising G418. Growth media suitable for Cyclotella cryptica lipid production include, but are not limited to, ASW medium and those media reported by Sriharan et al., 1991 and Pahl et al. Evaluation of fatty acid profiles of Cyclotella cryptica lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 28
Engineering Navicula Saprophila
[0420] Expression of recombinant genes in accordance with the present invention in Navicula saprophila can be accomplished by modifying the methods and vectors taught by Dunahay et al. as discussed herein. Briefly, Dunahay et al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reported the stable transformation of Navicula saprophila with plasmid DNA. Using the transformation method of microprojectile bombardment, Dunahay introduced the plasmid pACCNPT5.1 into Navicula saprophila. Plasmid pACCNPT5.1 comprised a neomycin resistance cassette, comprising the coding sequence of the neomycin phosphotransferase II (nptII) gene product operably linked to the promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784) upstream of the nptII coding-region and operably linked to the 3'UTR/terminator of the Cyclotella cryptica ACCase gene at the 3' region (downstream of the nptII coding-region). The nptII gene product confers resistance to the antibiotic G418. Prior to transformation with pACCNPT5.1, Navicula saprophila was unable to propagate on artificial seawater medium comprising 100 ug/ml G418. Upon transformation with pACCNPT5.1, transformants of Navicula saprophila were obtained that were propagated in selective artificial seawater medium comprising 100 ug/ml G418. The expression of the nptII gene product in Navicula saprophila enabled propagation in the presence of G418, thereby establishing the utility of the neomycin antibiotic resistance cassette as selectable marker for use in Navicula saprophila. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Dunahay reported liquid propagation of Navicula saprophila in artificial seawater medium (ASW, as discussed by Brown, L., Phycologia, Vol. 21 (1982), pp. 408-410) supplemented with 1.07 mM sodium silicate and with 100 ug/ml G418. Dunahay also reported selection and maintenance of Navicula saprophila transformants on agar plates comprising ASW medium with 100 ug/ml G418. Propagation of Navicula saprophila in additional culture medium has been discussed (for example in Tadros and Johansen, Journal of Phycology, Vol. 24:4 (1988), pp. 445-452 and Sriharan et al., Applied Biochemistry and Biotechnology, Vol. 20-21:1 (1989), pp. 281-291). Dunahay reported that the plasmid pACCNPT5.1 and the promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene are suitable to enable exogenous gene expression in Navicula saprophila. In addition, Dunahay reported that the neomycin resistance cassette encoded on pACCNPT5.1 was suitable for use as a selectable marker in Navicula saprophila.
[0421] In an embodiment of the present invention, vector pACCNPT5.1, comprising the nucleotide sequence encoding the nptII gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Navicula saprophila to reflect the codon bias inherent in nuclear genes of the closely-related Navicula pelliculosa in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Cyclotella cryptica ACCase gene promoter upstream of the protein-coding sequence and operably linked to the Cyclotella cryptica ACCase gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Navicula saprophila genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. Stable transformation of Navicula saprophila with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the nptII gene product can be used as a selectable marker to select for Navicula saprophila transformed with the transformation vector in, but not limited to, agar ASW medium comprising G418. Growth media suitable for Navicula saprophila lipid production include, but are not limited to, ASW medium and those media reported by Sriharan et al. 1989 and Tadros and Johansen. Evaluation of fatty acid profiles of Navicula saprophila lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 29
Engineering Thalassiosira Pseudonana
[0422] Expression of recombinant genes in accordance with the present invention in Thalassiosira pseudonana can be accomplished by modifying the methods and vectors taught by Poulsen et al. as discussed herein. Briefly, Poulsen et al., Journal of Phycology, Vol. 42 (2006), pp. 1059-1065, reported the stable transformation of Thalassiosira pseudonana with plasmid DNA. Using the transformation method of microprojectile bombardment, Poulsen introduced the plasmid pTpfcp/nat in to Thalassiosira pseudonana. pTpfcp/nat comprised a nourseothricin resistance cassette, comprising sequence encoding the nourseothricin acetyltransferase (nat) gene product (GenBank Accession No. AAC60439) operably linked to the Thalassiosira pseudonana fucoxanthin chlorophyll a/c binding protein gene (fcp) promoter upstream of the nat protein-coding region and operably linked to the Thalassiosira pseudonana fcp gene 3' UTR/terminator at the 3' region (downstream of the nat protein coding-sequence). The nat gene product confers resistance to the antibiotic nourseothricin. Prior to transformation with pTpfcp/nat, Thalassiosira pseudonana was unable to propagate on medium comprising 10 ug/ml nourseothricin. Upon transformation with pTpfcp/nat, transformants of Thalassiosira pseudonana were obtained that were propagated in selective culture medium comprising 100 ug/ml nourseothricin. The expression of the nat gene product in Thalassiosira pseudonana enabled propagation in the presence of 100 ug/ml nourseothricin, thereby establishing the utility of the nourseothricin antibiotic resistance cassette as selectable marker for use in Thalassiosira pseudonana. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Poulsen reported that selection and maintenance of the transformed Thalassiosira pseudonana was performed in liquid culture comprising modified ESAW medium (as discussed by Harrison et al., Journal of Phycology, Vol. 16 (1980), pp. 28-35) with 100 ug/ml nourseothricin. Propagation of Thalassiosira pseudonana in additional culture medium has been discussed (for example in Volkman et al., Journal of Experimental Marine Biology and Ecology, Vol. 128:3 (1989), pp. 219-240). An additional plasmid, comprising additional selectable markers suitable for use in Thalassiosira pseudonana has been discussed in the same report by Poulsen. Poulsen reported that the plasmid pTpfcp/nat, and the Thalassiosira pseudonana fcp promoter and 3' UTR/terminator are suitable to enable exogenous gene expression in Thalassiosira pseudonana. In addition, Poulsen reported that the nourseothricin resistance cassette encoded on pTpfcp/nat was suitable for use as a selectable marker in Thalassiosira pseudonana.
[0423] In an embodiment of the present invention, vector pTpfcp/nat, comprising the nucleotide sequence encoding the nat gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Thalassiosira pseudonana to reflect the codon bias inherent in nuclear genes of Thalassiosira pseudonana in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Thalassiosira pseudonana fcp gene promoter upstream of the protein-coding sequence and operably linked to the Thalassiosira pseudonana fcp gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Thalassiosira pseudonana genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Thalassiosira pseudonana genome (referenced in the publication by Armbrust et al., Science, Vol. 306: 5693 (2004): pp. 79-86). Stable transformation of Thalassiosira pseudonana with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the nat gene product can be used as a marker to select for Thalassiosira pseudonana transformed with the transformation vector in but not limited to, ESAW agar medium comprising nourseothricin. Growth media suitable for Thalassiosira pseudonana lipid production include, but are not limited to, ESAW medium, and those culture media discussed by Volkman et al. and Harrison et al. Evaluation of fatty acid profiles of Thalassiosira pseudonana lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 30
Engineering Chlamydomonas Reinhardtii
[0424] Expression of recombinant genes in accordance with the present invention in Chlamydomonas reinhardtii can be accomplished by modifying the methods and vectors taught by Cerutti et al. as discussed herein. Briefly, Cerutti et al., Genetics, Vol. 145:1 (1997), pp. 97-110, reported the stable nuclear transformation of Chlamydomonas reinhardtii with a transformation vector. Using the transformation method of microprojectile bombardment, Cerutti introduced transformation construct P[1030] into Chlamydomonas reinhardtii. Construct P[1030] comprised a spectinomycin resistance cassette, comprising sequence encoding the aminoglucoside 3''-adenyltransferase (aadA) gene product operably linked to the Chlamydomonas reinhardtii ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene (RbcS2, GenBank Accession No. X04472) promoter upstream of the aadA protein-coding region and operably linked to the Chlamydomonas reinhardtii RbcS2 gene 3' UTR/terminator at the 3' region (downstream of the aadA protein coding-sequence). The aadA gene product confers resistance to the antibiotic spectinomycin. Prior to transformation with P[1030], Chlamydomonas reinhardtii was unable to propagate on medium comprising 90 ug/ml spectinomycin. Upon transformation with P[1030], transformants of Chlamydomonas reinhardtii were obtained that were propagated in selective culture medium comprising 90 ug/ml spectinomycin, thereby establishing the utility of the spectinomycin antibiotic resistance cassette as a selectable marker for use in Chlamydomonas reinhardtii. Evaluation of the genomic DNA of the stable transformants was performed by Southern analysis. Cerutti reported that selection and maintenance of the transformed Chlamydomonas reinhardtii was performed on agar plates comprising Tris-acetate-phosphate medium (TAP, as described by Harris, The Chlamydomonas Sourcebook, Academic Press, San Diego, 1989) with 90 ug/ml spectinomycin. Cerutti additionally reported propagation of Chlamydomonas reinhardtii in TAP liquid culture with 90 ug/ml spectinomycin. Propagation of Chlamydomonas reinhardtii in alternative culture medium has been discussed (for example in Dent et al., African Journal of Microbiology Research, Vol. 5:3 (2011), pp. 260-270 and Yantao et al., Biotechnology and Bioengineering, Vol. 107:2 (2010), pp. 258-268). Additional constructs, comprising additional selectable markers suitable for use in Chlamydomonas reinhardtii as well as numerous regulatory sequences, including promoters and 3' UTRs suitable for promoting heterologous gene expression in Chlamydomonas reinhardtii are known in the art and have been discussed (for a review, see Radakovits et al., Eukaryotic Cell, Vol. 9:4 (2010), pp. 486-501). Cerutti reported that the transformation vector P[1030] and the Chlamydomonas reinhardtii promoter and 3' UTR/terminator are suitable to enable exogenous gene expression in Chlamydomonas reinhardtii. In addition, Cerutti reported that the spectinomycin resistance cassette encoded on P[1030] was suitable for use as a selectable marker in Chlamydomonas reinhardtii.
[0425] In an embodiment of the present invention, vector P[1030], comprising the nucleotide sequence encoding the aadA gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Chlamydomonas reinhardtii to reflect the codon bias inherent in nuclear genes of Chlamydomonas reinhardtii in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Chlamydomonas reinhardtii RbcS2 promoter upstream of the protein-coding sequence and operably linked to the Chlamydomonas reinhardtii RbcS2 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Chlamydomonas reinhardtii genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic site of an endogenous lipid biosynthesis pathway gene. One skilled in the art can identify such homology regions within the sequence of the Chlamydomonas reinhardtii genome (referenced in the publication by Merchant et al., Science, Vol. 318:5848 (2007), pp. 245-250). Stable transformation of Chlamydomonas reinhardtii with the transformation vector is achieved through well-known transformation techniques including microprojectile bombardment or other known methods. Activity of the aadA gene product can be used as a marker to select for Chlamydomonas reinhardtii transformed with the transformation vector on, but not limited to, TAP agar medium comprising spectinomycin. Growth media suitable for Chlamydomonas reinhardtii lipid production include, but are not limited to, ESAW medium, and those culture media discussed by Yantao et al. and Dent et al. Evaluation of fatty acid profiles of Chlamydomonas reinhardtii lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 31
Engineering Yarrowia Lipolytica
[0426] Expression of recombinant genes in accordance with the present invention in Yarrowia lipolytica can be accomplished by modifying the methods and vectors taught by Fickers et al. as discussed herein. Briefly, Fickers et al., Journal of Microbiological Methods, Vol. 55 (2003), pp. 727-737, reported the stable nuclear transformation of Yarrowia lipolytica with plasmid DNA. Using a lithium acetate transformation method, Fickers introduced the plasmid JMP123 into Yarrowia lipolytica. Plasmid JMP123 comprised a hygromycin B resistance cassette, comprising sequence encoding the hygromycin B phosphotransferase gene product (hph), operably-linked to the Yarrowia lipolytica LIP2 gene promoter (GenBank Accession No. AJ012632) upstream of the hph protein-coding region and operably linked to the Yarrowia lipolytica LIP2 gene 3'UTR/terminator downstream of the hph protein-coding region. Prior to transformation with JMP123, Yarrowia lipolytica were unable to propagate on medium comprising 100 ug/ml hygromycin. Upon transformation with JMP123, transformed Yarrowia lipolytica were obtained that were able to propagate on medium comprising 100 ug/ml hygromycin, thereby establishing the hygromycin B antibiotic resistance cassette as a selectable marker for use in Yarrowia lipolytica. The nucleotide sequence provided on JMP123 of the promoter and 3'UTR/terminator of the Yarrowia lipolytica LIP2 gene served as donor sequences for homologous recombination of the hph coding sequence into the LIP2 locus. Evaluation of the genomic DNA of the stable transformants was performed by Southern. Fickers reported that selection and maintenance of the transformed Yarrowia lipolytica was performed on agar plates comprising standard YPD medium (Yeast Extract Peptone Dextrose) with 100 ug/ml hygromycin. Liquid culturing of transformed Yarrowia lipolytica was performed in YPD medium with hygromycin. Other media and techniques used for culturing Yarrowia lipolytica have been reported and numerous other plasmids, promoters, 3' UTRs, and selectable markers for use in Yarrowia lipolytica have been reported (for example see Pignede et al., Applied and Environmental Biology, Vol. 66:8 (2000), pp. 3283-3289, Chuang et al., New Biotechnology, Vol. 27:4 (2010), pp. 277-282, and Barth and Gaillardin, (1996), In: K, W. (Ed.), Nonconventional Yeasts in Biotechnology. Sprinter-Verlag, Berlin-Heidelber, pp. 313-388). Fickers reported that the transformation vector JMP 123 and the Yarrowia lipolytica LIP2 gene promoter and 3' UTR/terminator are suitable to enable heterologous gene expression in Yarrowia lipolytica. In addition, Fickers reported that the hygromycin resistance cassette encoded on JMP123 was suitable for use as a selectable marker in Yarrowia lipolytica.
[0427] In an embodiment of the present invention, vector JMP123, comprising the nucleotide sequence encoding the hph gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Yarrowia lipolytica to reflect the codon bias inherent in nuclear genes of Yarrowia lipolytica in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Yarrowia lipolytica LIP2 gene promoter upstream of the protein-coding sequence and operably linked to the Yarrowia lipolytica LIP2 gene 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Yarrowia lipolytica genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Yarrowia lipolytica genome (referenced in the publication by Dujun et al., Nature, Vol. 430 (2004), pp. 35-44). Stable transformation of Yarrowia lipolytica with the transformation vector is achieved through well-known transformation techniques including lithium acetate transformation or other known methods. Activity of the hph gene product can be used as a marker to select for Yarrowia lipolytica transformed with the transformation vector on, but not limited to, YPD medium comprising hygromycin. Growth media suitable for Yarrowia lipolytica lipid production include, but are not limited to, YPD medium, and those culture media described by Chuang et al. Evaluation of fatty acid profiles of Yarrowia lipolytica lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 32
Engineering Mortierella Alpine
[0428] Expression of recombinant genes in accordance with the present invention in Mortierella alpine can be accomplished by modifying the methods and vectors taught by Mackenzie et al. as discussed herein. Briefly, Mackenzie et al., Applied and Environmental Microbiology, Vol. 66 (2000), pp. 4655-4661, reported the stable nuclear transformation of Mortierella alpina with plasmid DNA. Using a protoplast transformation method, MacKenzie introduced the plasmid pD4 into Mortierella alpina. Plasmid pD4 comprised a hygromycin B resistance cassette, comprising sequence encoding the hygromycin B phosphotransferase gene product (hpt), operably-linked to the Mortierella alpina histone H4.1 gene promoter (GenBank Accession No. AJ249812) upstream of the hpt protein-coding region and operably linked to the Aspergillus nidulans N-(5'-phophoribosyl)anthranilate isomerase (trpC) gene 3'UTR/terminator downstream of the hpt protein-coding region. Prior to transformation with pD4, Mortierella alpina were unable to propagate on medium comprising 300 ug/ml hygromycin. Upon transformation with pD4, transformed Mortierella alpina were obtained that were propagated on medium comprising 300 ug/ml hygromycin, thereby establishing the hygromycin B antibiotic resistance cassette as a selectable marker for use in Mortierella alpina. Evaluation of the genomic DNA of the stable transformants was performed by Southern. Mackenzie reported that selection and maintenance of the transformed Mortierella alpina was performed on PDA (potato dextrose agar) medium comprising hygromycin. Liquid culturing of transformed Mortierella alpina by Mackenzie was performed in PDA medium or in S2GYE medium (comprising 5% glucose, 0.5% yeast extract, 0.18% NH.sub.4SO.sub.4, 0.02% MgSO.sub.4-7H.sub.2O, 0.0001% FeCl.sub.3-6H.sub.2O, 0.1%, trace elements, 10 mM K.sub.2HPO.sub.4--NaH.sub.2PO.sub.4), with hygromycin. Other media and techniques used for culturing Mortierella alpina have been reported and other plasmids, promoters, 3' UTRs, and selectable markers for use in Mortierella alpina have been reported (for example see Ando et al., Applied and Environmental Biology, Vol. 75:17 (2009) pp. 5529-35 and Lu et al., Applied Biochemistry and Biotechnology, Vol. 164:7 (2001), pp. 979-90). Mackenzie reported that the transformation vector pD4 and the Mortierella alpina histone H4.1 promoter and A. nidulans trpC gene 3' UTR/terminator are suitable to enable heterologous gene expression in Mortierella alpina. In addition, Mackenzie reported that the hygromycin resistance cassette encoded on pD4 was suitable for use as a selectable marker in Mortierella alpina.
[0429] In an embodiment of the present invention, vector pD4, comprising the nucleotide sequence encoding the hpt gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Mortierella alpina to reflect the codon bias inherent in nuclear genes of Mortierella alpina in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the Mortierella alpina histone H4.1 gene promoter upstream of the protein-coding sequence and operably linked to the A. nidulans trpC 3'UTR/terminator at the 3' region, or downstream, of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Mortierella alpina genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Mortierella alpina genome (referenced in the publication by Wang et al., PLOS One, Vol. 6:12 (2011)). Stable transformation of Mortierella alpina with the transformation vector is achieved through well-known transformation techniques including protoplast transformation or other known methods. Activity of the hpt gene product can be used as a marker to select for Mortierella alpina transformed with the transformation vector on, but not limited to, PDA medium comprising hygromycin. Growth media suitable for Mortierella alpina lipid production include, but are not limited to, S2GYE medium, and those culture media described by Lu et al. and Ando et al. Evaluation of fatty acid profiles of Mortierella alpina lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 33
Engineering Rhodococcus Opacus PD630
[0430] Expression of recombinant genes in accordance with the present invention in Rhodococcus opacus PD630 can be accomplished by modifying the methods and vectors taught by Kalscheuer et al. as discussed herein. Briefly, Kalscheuer et al., Applied and Environmental Microbiology, Vol. 52 (1999), pp. 508-515, reported the stable transformation of Rhodococcus opacus with plasmid DNA. Using the transformation method of electroporation, Kalscheuer introduced the plasmid pNC9501 into Rhodococcus opacus PD630. Plasmid pNC9501 comprised a thiostrepton resistance (thio.sup.r) cassette, comprising the full nucleotide sequence of the Streptomyces azureus 23S rRNA A1067 methyltransferase gene, including the gene's promoter and 3' terminator sequence. Prior to transformation with pNC9501, Rhodococcus opacus was unable to propagate on medium comprising 1 mg/ml thiostrepton. Upon transformation of Rhodococcus opacus PD630 with pNC9501, transformants were obtained that propagated on culture medium comprising 1 mg/ml thiostrepton, thereby establishing the use of the thiostrepton resistance cassette as a selectable marker in Rhodococcus opacus PD630. A second plasmid described by Kalscheuer, pAK68, comprised the resistance thio.sup.r cassette as well as the gene sequences of the Ralstonia eutropha beta-ketothiolase (phaB), acetoacetyl-CoA reductase (phaA), and poly3-hydroxyalkanoic acid synthase (phaC) genes for polyhydroxyalkanoate biosynthesis, driven by the lacZ promoter. Upon pAK68 transformation of a Rhodococcus opacus PD630 strain deficient in polyhydroxyalkanoate biosynthesis, transformed Rhodococcus opacus PD630 were obtained that produced higher amounts of polyhydroxyalkanoates than the untransformed strain. Detectable activity of the introduced Ralstonia eutropha phaB, phaA, and phaC enzymes indicted that the regulatory elements encoded on the pAK68 plasmid were suitable for heterologous gene expression in Rhodococcus opacus PD630. Kalscheuer reported that selection and maintenance of the transformed Rhodococcus opacus PD630 was performed on standard Luria Broth (LB) medium, nutrient broth (NB), or mineral salts medium (MSM) comprising thiostrepton. Other media and techniques used for culturing Rhodococcus opacus PD630 have been described (for example see Kurosawa et al., Journal of Biotechnology, Vol. 147:3-4 (2010), pp. 212-218 and Alverez et al., Applied Microbial and Biotechnology, Vol. 54:2 (2000), pp. 218-223). Kalscheuer reported that the transformation vectors pNC9501 and pAK68, the promoters of the Streptomyces azureus 23S rRNA A1067 methyltransferase gene and lacZ gene are suitable to enable heterologous gene expression in Rhodococcus opacus PD630. In addition, Kalscheuer reported that the thio.sup.r cassette encoded on pNC9501 and pAK68 was suitable for use as a selectable marker in Rhodococcus opacus PD630.
[0431] In an embodiment of the present invention, vector pNC9501, comprising the nucleotide sequence encoding the thio.sup.r gene product for use as a selectable marker, is constructed and modified to further comprise a lipid biosynthesis pathway expression cassette sequence, thereby creating a transformation vector. The lipid biosynthesis pathway expression cassette encodes one or more lipid biosynthesis pathway proteins selected from Table 20, each protein-coding sequence codon-optimized for expression in Rhodococcus opacus PD630 to reflect the codon bias inherent in nuclear genes of Rhodococcus opacus in accordance with Tables 19A-D. For each lipid biosynthesis pathway protein of Table 20, the codon-optimized gene sequence can individually be operably linked to the lacZ gene promoter upstream of the protein-coding sequence. The transformation construct may additionally comprise homology regions to the Rhodococcus opacus PD630 genome for targeted genomic integration of the transformation vector. Homology regions may be selected to disrupt one or more genomic sites of endogenous lipid biosynthesis pathway genes. One skilled in the art can identify such homology regions within the sequence of the Rhodococcus opacus PD630 genome (referenced in the publication by Holder et al., PLOS Genetics, Vol. 7:9 (2011). Transformation of Rhodococcus opacus PD630 with the transformation vector is achieved through well-known transformation techniques including electroporation or other known methods. Activity of the Streptomyces azureus 23S rRNA A1067 methyltransferase gene product can be used as a marker to select for Rhodococcus opacus PD630 transformed with the transformation vector on, but not limited to, LB medium comprising thiostrepton. Growth media suitable Rhodococcus opacus PD630 lipid production include, but are not limited to those culture media discussed by Kurosawa et al. and Alvarez et al. Evaluation of fatty acid profiles of Rhodococcus opacus PD630 lipids can be assessed through standard lipid extraction and analytical methods described herein.
Example 34
Engineering Microalgae for Fatty Acid Auxotrophy
[0432] Strain B of Example 3, Prototheca moriformis (UTEX 1435) engineered to express a Cuphea wrightii thioesterase (CwTE2), was used as the host organism for further genetic modification to knockout both endogenous thioesterase alleles, FATA1-1 and FATA1-2. Here, a first transformation construct was generated to integrate a neomycin expression cassette into Strain B at the FATA1-1 locus. This construct, pSZ2226, included 5' (SEQ ID NO: 30) and 3' (SEQ ID NO: 31) homologous recombination targeting sequences (flanking the construct) to the FATA1-1 locus of the nuclear genome and a neomycin resistance protein-coding sequence under the control of the C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and the Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This NeoR expression cassette is listed as SEQ ID NO: 15 and served as a selectable marker.
[0433] Upon transformation of pSZ2226 into Strain B, individual transformants were selected on agar plates comprising sucrose and G418. A single isolate, Strain H, was selected for further genetic modification. A second transformation construct, pSZ2236, was generated to integrate polynucleotides enabling expression of a thiamine selectable marker into Strain H at the FATA1-2 locus. pSZ2236 included 5' (SEQ ID NO: 32) and 3' (SEQ ID NO: 33) homologous recombination targeting sequences (flanking the construct) to the FATA1-2 genomic region for integration into the P. moriformis (UTEX 1435) nuclear genome and an A. thaliana THIC protein coding region under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served as a selectable marker. Upon transformation of Strain H with pSZ2236 to generate Strain I, individual transformants, were selected on agar plates comprising free fatty acids. Strain I was able to propagate on agar plates and in medium lacking thiamine and supplemented with free fatty acids.
Example 35
Engineering Microorganisms for Increased Production of Stearic Acid
[0434] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain J, was transformed with the plasmid construct pSZ2281 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. pSZ2281 included polynucleotides encoding RNA hairpins (SAD2hpC, SEQ ID NO: 34) to down-regulate the expression of stearoyl-ACP desaturase, 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), to express the protein sequence given in SEQ ID NO: 3, under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. The polynucleotide sequence encoding the SAD2hpC RNA hairpin was under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6).
[0435] Upon transformation of Strain J with construct pSZ2281, thereby generating Strain K, positive clones were selected on agar plates containing sucrose as a sole carbon source. Individual transformants were clonally purified and propagated under heterotrophic conditions suitable for lipid production as those detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass and analyzed using standard fatty acid methyl ester gas chromatography flame ionization detection methods as described in Example 1 (also see PCT/US2012/023696). The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis UTEX Strain J propagated on glucose as a sole carbon source and three representative isolates of Strain K, propagated on sucrose as a sole carbon source, are presented in Table 21.
TABLE-US-00023 TABLE 21 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cells engineered to express a hairpin RNA construct targeting stearoyl ACP desaturase gene/gene products. Area % Fatty acid Strain J Strain K-1 Strain K-2 Strain K-3 Strain K-4 C8:0 0.02 C10:0 0.01 0.00 0.02 0.02 0.04 C12:0 0.03 0.05 0.05 0.05 0.08 C14:0 1.22 0.89 0.87 0.77 1.2 C16:0 26.75 29.23 28.96 27.55 28.06 C18:0 3.06 37.39 36.76 36.41 40.82 C18:1 59.62 23.90 24.76 26.92 22.02 C18:2 7.33 5.44 5.54 5.54 4.53 C18:3 0.14 C20:0 1.43
[0436] The data presented in Table 21 show a clear impact of the expression of SAD2 hairpin RNA construct on the C18:0 and C18:1 fatty acid profiles of the transformed organism. The fatty acid profiles of Strain K transformants comprising a SAD2 hairpin RNA construct demonstrated an increase in the percentage of saturated C18:0 fatty acids with a concomitant diminution of unsaturated C18:1 fatty acids. Fatty acid profiles of the untransformed strain comprise about 3% C18:0. Fatty acid profiles of the transformed strains comprise about 37% C18:0. These data illustrate the successful expression and use of polynucleotides enabling expression of a SAD RNA hairpin construct in Prototheca moriformis to alter the percentage of saturated fatty acids in the engineered host microbes, and in particular in increasing the concentration of C18:0 fatty acids and decreasing C18:1 fatty acids in microbial cells.
[0437] Also shown in Table 21, strain K-4 had a yet further elevated level of stearate. Strain K4 was created by inserting the construct of strains K1-K3 into the SAD2B locus. Thus, by knocking out one copy of the SAD gene and inhibiting the remaining copies at the RNA level, a further reduction in oleic acid and corresponding increase in stearate was obtained. Triglyceride analysis of RBD oil obtained from strain K4 showed about 12% POP, 27% POS and 18% SOS.
Example 36
Engineering Microorganisms for Increased Production of Oleic Acid Through Knockdown of an Endogenous Acyl-ACP Thioesterase
[0438] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain J, was transformed independently with each of the constructs pSZ2402-pSZ2407 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of the constructs pSZ2402-pSZ2407 included different polynucleotides encoding a hairpin RNA targeted against Prototheca moriformis FATA1 mRNA transcripts to down-regulate the expression of fatty acyl-ACP thioesterase, 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4) to express the protein sequence given in SEQ ID NO: 3 under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. Sequence listing identities for the polynucleotides corresponding to each hairpin are listed in Table 22. The polynucleotide sequence encoding each RNA hairpin was under the control of the C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6).
TABLE-US-00024 TABLE 22 Plasmid constructs used to transform Prototheca moriformis (UTEX 1435) Strain J. Plasmid construct Hairpin designation SEQ ID NO: pSZ2402 PmFATA-hpB SEQ ID NO: 40 pSZ2403 PmFATA-hpC SEQ ID NO: 41 pSZ2404 PmFATA-hpD SEQ ID NO: 42 pSZ2405 PmFATA-hpE SEQ ID NO: 43 pSZ2406 PmFATA-hpF SEQ ID NO: 44 pSZ2407 PmFATA-hpG SEQ ID NO: 45
[0439] Upon independent transformation of Strain J with each of the constructs listed in Table 22, positive clones were selected on agar plates containing sucrose as a sole carbon source. Individual transformants were clonally purified and propagated under heterotrophic conditions suitable for lipid production as those detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass and analyzed using standard fatty acid methyl ester gas chromatography flame ionization detection methods as described in Example 1 (also see PCT/US2012/023696). The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis (UTEX 1435) Strain J propagated on glucose as a sole carbon source and representative isolates of each transformation of Strain J, propagated on sucrose as a sole carbon source, are presented in Table 23.
TABLE-US-00025 TABLE 23 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cells engineered to express hairpin RNA constructs targeting fatty acyl-ACP thioesterase gene/gene products. Area % Fatty Acid Construct C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Strain J untransformed 0 0.05 1.32 26.66 3.1 59.07 7.39 PmFATA-hpB 0.04 0.07 1.36 24.88 2.24 61.92 6.84 0 0.08 1.33 25.34 2.39 61.72 6.5 0 0.07 1.29 25.44 2.26 61.7 6.69 0 0.06 1.33 25.1 2.37 61.56 6.87 PmFATA-hpC 0 0.08 1.18 22.03 1.71 63.8 8.63 0 0.07 1.21 24.5 2.23 62.32 7.19 0 0.08 1.29 24.93 2.24 62.02 7.01 0.05 0.06 1.29 25.45 2.26 61.81 6.76 PmFATA-hpD 0 0.02 0.68 15.8 1.88 72.64 6.96 0 0.03 0.78 17.56 1.7 71.8 6.03 0 0.03 0.92 19.04 2.03 68.82 7.05 0 0.04 1.27 23.14 2.25 65.27 6.07 PmFATA-hpE 0 0.03 0.79 18.55 2.13 69.66 6.77 0 0.04 1.11 21.01 1.74 65.18 8.55 0 0.03 1.08 21.11 1.54 64.76 8.87 0 0.03 1.17 21.93 1.71 63.89 8.77 PmFATA-hpF 0.03 0.04 0.34 8.6 1.69 78.08 8.87 0 0.03 0.49 10.2 1.52 76.97 8.78 0 0.03 1 20.47 2.22 66.34 7.45 0 0.03 1.03 21.61 1.88 65.39 7.76 PmFATA-hpG 0 0.03 1.03 20.57 2.36 64.73 8.75 0 0.03 1.2 24.39 2.47 61.9 7.49 0 0.04 1.29 24.14 2.29 61.41 8.22
[0440] The data presented in Table 23 show a clear impact of the expression of FATA hairpin RNA constructs on the C18:0 and C18:1 fatty acid profiles of the transformed organism. The fatty acid profiles of Strain J transformants comprising a FATA hairpin RNA construct demonstrated an increase in the percentage of C18:1 fatty acids with a concomitant diminution of C16:0 and C18:0 fatty acids. Fatty acid profiles of the untransformed Strain J are about 26.66% C16:0, 3% C18:0, and about 59% C18:1 fatty acids. In contrast, the fatty acid profiles of the transformed strains comprise as low as 8.6% C16:0 and 1.54% C18:0 and greater than 78% C18:1 fatty acids.
[0441] These data illustrate the utility and successful use of polynucleotide FATA RNA hairpin constructs in Prototheca moriformis to alter the fatty acids profile of engineered microbes, and in particular in increasing the concentration of C18:1 fatty acids and decreasing C18:0 and C16:0 fatty acids in microbial cells.
Example 37
Engineering Microorganisms for Increased Production of Mid-Chain Fatty Acids Through KASI or KASIV Overexpression
[0442] This example describes the use of recombinant polynucleotides that encode KASI or KASIV enzymes to engineer microorganisms in which the fatty acid profiles of the transformed microorganisms have been enriched in lauric acid, C10:0, and total saturated fatty acids.
[0443] Each of the constructs pSZD1132, pSZD1133, pSZD1134, or pSZD1201 was used independently to transform Strain B of Example 3, Prototheca moriformis (UTEX 1435) engineered to express a Cuphea wrightii thioesterase (CwTE2), according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of the above constructs included different polynucleotides encoding a KASI or KASIV enzyme, 5' (SEQ ID NO: 13) and 3' (SEQ ID NO: 14) homologous recombination targeting sequences (flanking the construct) to the pLoop genomic region for integration into the nuclear genome, and a neomycin resistance protein-coding sequence under the control of the C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and the Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This NeoR expression cassette is listed as SEQ ID NO: 15 and served as a selectable marker. Sequence listing identities for the polynucleotides corresponding to each construct are listed in Table 20. The polynucleotide sequence encoding each KAS enzyme was under the control of the P. moriformis UTEX 1435 Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). The protein coding regions of the KAS enzymes and neomycin resistance gene were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/U52009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0444] Upon transformation of individual plasmids into Strain B, positive clones were selected on agar plates comprising G418. Individual transformants were clonally purified and grown on sucrose as a sole carbon source at pH 7.0 under conditions suitable for lipid production as detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass from each transformant and fatty acid profiles from these samples were analyzed using standard fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1. The fatty acid profiles (expressed as Area % of total fatty acids) of Strain B and four positive transformants of each of pSZ2046 (Strains M-P, 1-4) are presented in Table 24.
TABLE-US-00026 TABLE 24 Plasmid constructs used to transform Prototheca moriformis (UTEX 1435) Strain B. Plasmid KASI/KASIV Transit construct source peptide SEQ ID NO: pSZD1134 Cuphea wrightii Native SEQ ID NO: 46 GenBank Accession No. U67317 pSZD1201 Cuphea wrightii PmSAD SEQ ID NO: 47 pSZD1132 Cuphea pulcherrima Native SEQ ID NO: 48 GenBank Accession No. AAC68860 pSZD1133 Cuphea hookeriana Native SEQ ID NO: 49
TABLE-US-00027 TABLE 25 Fatty acid profiles of Prototheca moriformis (UTEX 1435) Strain B engineered for increased C10, lauric acid, and total saturated fatty acids. Fatty Acid (Area %) % Plasmid C10- Saturates/ construct(s) No. C10 C12 C14 C16 C18:0 C18:1 C18:2 C12 Total pSZ1283 7.89 35.49 16.58 11.5 1.09 19.64 6.49 43.38 72.55 pSZ1283, 1 14.94 43.97 12.19 7.56 0.72 14.11 5.31 58.91 79.38 pSZD1134 pSZ1283, 2 10.27 39.61 15.35 9.61 0.94 17.1 5.88 49.88 75.78 pSZD1134 pSZ1283, 3 11.69 41.83 15.21 8.77 0.83 15.04 5.40 53.52 78.33 pSZD1134 D1134-20 4 10.76 40.77 15.32 9.19 0.88 16.06 5.76 51.53 76.92 pSZ1283, 1 10.77 40.31 15.21 9.43 0.88 16.18 5.97 51.08 76.6 pSZD1132 pSZ1283, 2 9.19 37.03 15.02 10.52 1.00 19.63 6.29 46.22 72.76 pSZD1132 pSZ1283, 3 8.97 36.09 15.01 10.77 1.05 20.38 6.39 45.06 71.89 pSZD1132 pSZ1283, 4 9.51 38.12 14.96 9.96 0.94 18.93 6.32 47.63 73.49 pSZD1132 pSZ1283, 1 13.06 46.21 9.84 7.12 0.75 16.7 5.22 59.27 76.98 pSZD1201 pSZ1283, 2 11.02 43.91 13.01 7.78 0.86 16.53 5.77 54.93 76.58 pSZD1201 pSZ1283, 3 11.59 45.14 12.41 7.61 0.82 15.72 5.65 56.73 77.57 pSZD1201 pSZ1283, 4 10.66 41.32 13.74 8.75 0.68 18.64 5.21 51.98 75.15 pSZD1201 pSZ1283, 1 6.90 36.08 15.15 11.02 1.00 21.74 6.77 42.98 70.15 pSZD1133 pSZ1283, 2 7.01 35.88 15.01 10.75 1.07 22.02 6.93 42.89 69.72 pSZD1133 pSZ1283, 3 10.65 41.94 12.38 8.48 0.85 18.28 6.15 52.59 74.3 pSZD1133 pSZ1283, 4 10.23 41.88 12.58 8.52 0.82 18.48 6.22 52.11 74.03 pSZD1133
[0445] The data presented in Table 25 show a clear impact of the exogenous expression of KASI and KASIV enzymes on the C10:0 and C12 fatty acid profiles of the transformed organism. The fatty acid profiles of Strain B, expressing the Cuphea wrightii thioesterase alone, comprised about 8% C10:0 and about 35.5% C12:0, with saturated fatty acids accounting for 72.55% of total fatty acids. In contrast, transformants of Strain B engineered to additionally express a Cuphea wrightii KASI with a P. moriformis stearoyl ACP desaturase transit peptide were characterized by a fatty acid profile of about 13% C10:0 and about 46% C12:0. Saturated fatty acids accounted for as high as 77% in transformants of Strain B co-expressing the C. wrightii KASI fusion protein. Similarly, transformants of Strain B engineered to express the C. wrightii KASI with the enzyme's native transit peptide were characterized by a fatty acid profile of about 15% C10, about 44% C12, and about 79% saturated fatty acids. The fatty acid profiles or many transformants of Strain B expressing either Cuphea pulcherrima KASIV or Cuphea hookeriana KASIV also displayed elevated C10% and C12% levels, compared to the fatty acid profile of Strain B itself.
[0446] These data demonstrate the utility and effectiveness of polynucleotides enabling expression of KASI and KASIV constructs in Prototheca moriformis (UTEX 1435) to alter the percentage of saturated fatty acids in the engineered host microbes, and in particular in increasing the concentration of C10:0 and C12:0 fatty acids in microbial cells.
Example 38
Engineering Microorganisms for Increased Production of Mid-Chain Fatty Acids Through KASI Knockout
[0447] This example describes the use of recombinant polynucleotides that disrupt different KASI alleles to engineer microorganisms in which the fatty acid profiles of the transformed microorganisms have been enriched in C10:0 and midchain fatty acids.
[0448] Constructs pSZ2302 and pSZ2304 were used to independently transform Strain B of Example 3, Prototheca moriformis (UTEX 1435) engineered to express a Cuphea wrightii thioesterase (CwTE2), according to biolistic transformation methods as described in PCT/U52009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. pSZ2302 included 5' (SEQ ID NO: 50) and 3' (SEQ ID NO: 51) homologous recombination targeting sequences (flanking the construct) to the KAS1 allele 1 genomic region for integration into the P. moriformis nuclear genome, anA. thaliana THIC protein coding region under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). pSZ2304 included 5' (SEQ ID NO: 52) and 3' (SEQ ID NO: 53) homologous recombination targeting sequences (flanking the construct) to the KAS1 allele 2 genomic region for integration into the P. moriformis nuclear genome, anA. thaliana THIC protein coding region under the control of the C. protothecoides actin promoter/5'UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6).This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served as a selection marker. The protein coding region of AtTHIC was codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0449] Upon independent transformation pSZ2302 and pSZ2304 into Strain B, thereby generating Strain Q and R, positive clones were selected on agar plates comprising thiamine. Individual transformants were clonally purified and cultivated on sucrose as a sole carbon source at pH 5.0 or pH 7.0 under heterotrophic conditions suitable for lipid production as detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass from each transformant and fatty acid profiles from these samples were analyzed using fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1. The fatty acid profiles (expressed as Area % of total fatty acids) of Strain B and positive pSZ2302 (Strain Q, 1-5) and pSZ2304 (Strain R, 1-5) transformants are presented in Tables 26 and 27.
TABLE-US-00028 TABLE 26 Fatty acid profiles of Prototheca moriformis (UTEX 1435) Strains B, Q, and R engineered for increased midchain fatty acids, cultured at pH 5.0. Trans- Fatty Acid (Area %) formation C10- Strain plasmid(s) C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C14 UTEX 1435 None 0.00 0.04 1.28 26.67 3.05 59.96 7.19 1.32 Strain B pSZ1283 0.01 0.09 1.09 21.60 2.21 65.15 7.94 1.19 Strain Q-1 pSZ1283, 0.08 1.21 7.52 38.71 1.38 38.32 8.75 8.81 pSZ2302 Strain Q-2 pSZ1283, 0.15 1.36 7.51 38.23 1.33 38.27 8.94 9.02 pSZ2302 Strain Q-3 pSZ1283, 0.16 1.43 7.49 38.88 1.30 37.58 8.73 9.08 pSZ2302 Strain Q-4 pSZ1283, 0.00 1.71 7.42 37.67 1.43 37.26 10.38 9.13 pSZ2302 Strain Q-5 pSZ1283, 0.13 1.21 7.36 38.81 1.31 38.07 8.71 8.7 pSZ2302 Strain R-1 pSZ1283, 0.19 1.78 8.47 40.11 1.34 33.46 9.98 10.44 pSZ2304 Strain R-2 pSZ1283, 0.90 8.00 7.78 28.96 1.15 30.26 17.14 16.68 pSZ2304 Strain R-3 pSZ1283, 0.26 3.58 7.77 34.98 1.56 32.86 14.60 11.61 pSZ2304 Strain R-4 pSZ1283, 1.64 13.50 7.61 21.38 0.90 36.13 14.73 22.75 pSZ2304 Strain R-5 pSZ1283, 1.03 9.63 7.56 25.61 1.00 31.70 18.23 18.22 pSZ2304
TABLE-US-00029 TABLE 27 Fatty acid profiles of Prototheca moriformis (UTEX 1435), Strains B, Q, and R engineered for increased midchain fatty acids, cultured at pH 7.0. Fatty Acid (Area %) Transformation C10- Strain plasmid(s) C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C14 UTEX None 0.01 0.04 1.34 27.94 3.24 57.46 7.88 1.39 1435 Strain B pSZ1283 4.72 29.57 15.56 12.63 1.20 27.65 7.39 49.85 Strain Q-1 pSZ1283, pSZ2302 16.00 50.61 9.52 5.33 0.54 11.79 5.28 76.13 Strain Q-2 pSZ1283, pSZ2302 16.32 49.79 9.82 5.52 0.54 12.28 4.87 75.93 Strain Q-3 pSZ1283, pSZ2302 15.08 47.58 10.23 5.93 0.56 15.12 4.50 72.89 Strain Q-4 pSZ1283, pSZ2302 14.27 47.30 10.44 6.17 0.56 15.50 4.59 72.01 Strain Q-5 pSZ1283, pSZ2302 14.75 47.28 10.32 6.04 0.59 15.50 4.65 72.35 Strain R-1 pSZ1283, pSZ2304 21.25 55.42 7.97 3.65 0.00 5.46 5.66 84.64 Strain R-2 pSZ1283, pSZ2304 13.00 55.05 10.88 5.78 0.28 7.90 6.29 78.93 Strain R-3 pSZ1283, pSZ2304 12.89 53.15 11.11 6.13 0.00 9.87 6.13 77.15 Strain R-4 pSZ1283, pSZ2304 12.80 51.64 13.86 6.69 0.00 7.51 6.70 78.3 Strain R-5 pSZ1283, pSZ2304 16.61 51.42 9.84 5.27 0.33 11.15 4.79 77.87
[0450] The data presented in Tables 26 and 27 show a clear impact of disruption of different KASI alleles on the fatty acid profiles of the transformed organisms. When cultivated at pH 5.0, the fatty acid profiles of Prototheca moriformis (UTEX 1435) and Prototheca moriformis (UTEX 1435) Strain B, expressing a Cuphea wrightii FATB2 thioesterase under control of a pH regulatable promoter were very similar. These profiles were characterized by about 1% C14:0, about 21-26% C16:0, about 2-3% C18:0, about 60-65% C18:1, about 7% C18:2, with C10-C14 fatty acids comprising about 1.19-1.3% of total fatty acids. In contrast, when cultivated at pH 5.0, Strain B further engineered to disrupt KASI allele 1 (Strain Q) or KASI allele 2 (Strain R) demonstrated altered fatty acid profiles that were characterized by increased levels of C12, increased levels of C14, decreased levels of C18, and decreased levels of C18:1 fatty acids compared to Strain B or UTEX 1435. The fatty acid profiles of isolates of Strains Q and R differed in that Strain R (allele 2 knockout) isolates had generally greater C12s and lower C16s and C18:1s than Strain Q (allele 1 knockout).
[0451] When cultivated at pH 7.0, the fatty acid profile of Prototheca moriformis (UTEX 1435) is distinct from that Prototheca moriformis (UTEX 1435) Strain B expressing a Cuphea wrightii FATB2 thioesterase under control of a pH regulatable promoter. When cultured at pH 7.0, Strain B was characterized by a fatty acid profile elevated in C10, C12, and C14 fatty acids (these comprised about 50% of the total fatty acids). When cultured at pH 7.0, Strain Q and Strain R demonstrated fatty acid profiles with still further increases in C10, C12, and C14 fatty acids and still further decreases in C18:0 and C18:1 fatty acids relative to that of Strain B. Again, differences in fatty acid profiles between Strain Q and R were observed with the profile of Strain R comprising greater percentage levels of C12 and lower levels of C18:1 than that of Strain Q.
[0452] These data illustrate the successful expression and use of polynucleotides enabling expression of KASI and KASIV constructs in Prototheca moriformis to alter the percentage of saturated fatty acids in the engineered host microbes, and in particular in increasing the concentration of C10:0 and C12:0 fatty acids and decreasing the concentration of C18:0 and C18:1 fatty acids in microbial cells. In addition, the data here indicate the different KASI alleles can be disrupted to result in altered fatty acid profiles of the transformed organisms.
Example 39
Engineering Microorganisms for Increased Production of Mid-Chain Fatty Acids Through KASI Knockdown
[0453] This example describes the use of recombinant polynucleotides that encode RNA hairpins to attenuate a KASI enzyme to engineer a microorganism in which the fatty acid profile of the transformed microorganism has been enriched in midchain fatty acids.
[0454] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain S, was transformed independently with each of the constructs pSZ2482-pSZ2485 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of the constructs pSZ2482-pSZ2485 included different polynucleotides encoding hairpin RNAs targeted against Prototheca moriformis (UTEX 1435) KASI mRNA transcripts to down-regulate the expression of fatty acyl-ACP thioesterase, 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4) to express the protein sequence given in SEQ ID NO: 3 under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. Sequence listing identities for the polynucleotides corresponding to each KASI hairpin are listed in Table 28. The polynucleotide sequence encoding each RNA hairpin was under the control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). The protein coding region of the suc2 expression cassette was codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
TABLE-US-00030 TABLE 28 Plasmid constructs used to transform Prototheca moriformis (UTEX 1435) Strain S. Transformation construct Hairpin SEQ ID NO: pSZ2482 KASI hairpin B SEQ ID NO: 54 pSZ2483 KASI hairpin C SEQ ID NO: 55 pSZ2484 KASI hairpin D SEQ ID NO: 56 pSZ2485 KASI hairpin E SEQ ID NO: 57
[0455] Upon independent transformation of Strain S with each of the constructs listed in Table 28, positive clones were selected on agar plates containing sucrose as a sole carbon source. Individual transformants were clonally purified and propagated under heterotrophic conditions suitable for lipid production as those detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass and analyzed using fatty acid methyl ester gas chromatography flame ionization detection methods as described in Example 1 (also see PCT/US2012/023696). The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis UTEX 1435 propagated on glucose as a sole carbon source and four representative isolates of each transformation of Strain S, propagated on sucrose as a sole carbon source, are presented in Table 29.
TABLE-US-00031 TABLE 29 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cells engineered to express hairpin RNA constructs targeting KASI gene/gene products. Fatty Acid (Area %) Strain Plasmid Number C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 UTEX 1435 none 1 0.00 0.04 1.45 27.97 3.18 58.35 6.78 0.60 Strain S pSZ2482 1 0.19 0.74 8.47 38.30 2.15 36.24 9.45 1.42 2 0.07 0.25 4.16 32.46 2.62 49.57 7.73 0.82 3 0.03 0.10 2.68 27.48 2.65 56.40 8.14 0.55 4 0.03 0.10 2.60 27.44 2.01 55.54 9.15 0.78 pSZ2483 1 0.00 0.06 1.94 30.58 1.55 53.26 9.31 0.76 2 0.20 0.05 1.76 28.01 2.31 56.61 8.70 0.60 3 0.00 0.06 1.60 24.38 2.65 58.25 9.93 1.15 4 0.00 0.04 1.56 26.65 2.96 60.06 6.92 0.52 pSZ2484 1 0.72 3.71 19.15 38.03 1.68 14.22 15.00 4.21 2 0.66 2.76 16.34 38.19 1.78 18.52 14.91 3.38 3 0.69 2.96 16.20 37.28 1.77 19.05 15.26 3.48 4 0.18 0.70 8.61 36.80 2.35 36.22 10.89 1.10 pSZ2485 1 0.00 0.04 1.41 25.34 3.16 60.12 7.78 0.48 2 0.03 0.04 1.41 23.85 2.19 61.23 8.75 0.67 3 0.00 0.04 1.41 24.41 2.23 60.64 8.69 0.67 4 0.00 0.04 1.41 24.51 2.16 60.85 8.91 0.66
[0456] The data presented in Table 29 show a clear impact of the expression of KAS hairpin RNA constructs on the fatty acid profiles of the transformed organisms. The fatty acid profiles of Strain S transformants comprising either pSZ2482 or pSZ2484 KASI hairpin RNA construct demonstrated an increase in the percentage of C10, C12, C14, and C16 fatty acids with a concomitant diminution of C18:0 and C18:1 fatty acids relative to the fatty acid profile of UTEX 1435.
[0457] These data illustrate the utility and successful use of polynucleotide KASI RNA hairpin constructs in Prototheca moriformis (UTEX 1435) to alter the fatty acids profile of engineered microbes, and in particular in increasing the concentration of midchain fatty acids and decreasing C18:0 and C18:1 fatty acids in microbial cells.
Example 40
Engineering Microorganisms for Increased Production of Stearic Acid Through Fatty Acid Elongase Overexpression
[0458] This example describes the use of recombinant polynucleotides that encode fatty acid elongases to engineer a microorganism in which the fatty acid profile of the transformed microorganism has been enriched in stearic acid, arachidic acid, and docosadienoic acid.
[0459] A classically mutagenized strain of Prototheca moriformis (UTEX 1435), Strain J, was transformed independently with each of the constructs pSZ2323, pSZ2324, or pSZ2328 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of the constructs included a protein coding region to overexpress an elongase, 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4) to express the protein sequence given in SEQ ID NO: 3 under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. Sequence listing identities for the polynucleotides corresponding to each elongase are listed in Table 30. The polynucleotide sequence encoding each elongase was under control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). The protein coding regions of the exogenous elongases and the suc2 expression cassette were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
TABLE-US-00032 TABLE 30 Plasmid constructs used to transform Prototheca moriformis (UTEX 1435) Strain J. Plasmid GenBank construct Elongase source Accession No. SEQ ID NO: pSZ2328 Marchantia polymorpha AAP74370 58, 59 pSZ2324 Trypanosoma brucei AAX70673 60, 61 pSZ2323 Saccharomyces cerevisiae P39540 62, 63
[0460] Upon independent transformation of Strain J with the constructs listed in Table 30, positive clones were selected on agar plates containing sucrose as a sole carbon source. Individual transformants were clonally purified and propagated under heterotrophic conditions suitable for lipid production as those detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass and analyzed using fatty acid methyl ester gas chromatography flame ionization detection methods as described in Example 1 (also see PCT/US2012/023696). The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis UTEX 1435 Strain J propagated on glucose as a sole carbon source and three representative isolates of each transformation of Strain J, propagated on sucrose as a sole carbon source are presented in Table 31.
TABLE-US-00033 TABLE 31 Fatty acid profiles of Prototheca moriformis (UTEX 1435) Strain J cells engineered to overexpress elongases. Fatty Acid Area % Plasmid C14: C16: C16: C18: C18: C18: C18: C20: C22: construct No. 0 0 1 0 1 2 3.alpha. 0 2n6 None 1 1.39 27.42 0.77 3.33 57.46 8.05 0.61 0.30 0.03 pSZ2328 1 1.25 19.23 0.85 8.26 57.54 9.34 0.79 0.73 0.94 pSZ2328 2 1.22 17.76 0.69 7.86 60.56 9.38 0.59 0.6 0.47 pSZ2328 3 1.26 18.37 0.92 7.83 58.77 10.01 0.72 0.64 0.52 pSZ2324 1 1.51 22.97 1.09 8.71 53.01 9.63 0.65 0.68 0.55 pSZ2324 2 1.29 20.6 0.92 7.53 56.97 9.92 0.73 0.64 0.43 pSZ2324 3 1.28 20.59 0.93 7.33 57.52 9.68 0.65 0.58 0.42 pSZ2323 1 1.65 27.27 0.67 3.56 56.68 8.72 0.33 0.36 0.00 pSZ2323 2 1.56 28.44 0.74 3.36 55.22 9.07 0.46 0.39 0.03 pSZ2323 3 1.64 28.7 0.75 3.34 55.29 8.59 0.49 0.36 0.02
[0461] The data presented in Table 31 show a clear impact of the expression of Marchantia polymorpha and Trypanosoma brucei enzymes on the C14, C16, C18:0, C20:0, and C22:2n6 fatty acid profiles of the transformed organisms. The fatty acid profile of untransformed Strain J was about 27.42% C16:0, about 3% C18:0, about 57.5% C18:1, about 0.3% C20:0 and about 0.03% C22:2n6 fatty acids. In contrast to that of Strain J, the fatty acid profiles of Strain J transformed with different plasmid constructs to express elongases comprised lower percentage levels of C16 and higher percentage levels of C18:0, C20:0, and C22:2n6 fatty acids. The result of overexpression of Marchantia polymorpha elongase was about a 2.5 fold increase in percentage levels of C18:0 fatty acids, a 2 fold increase in percentage levels of C20:0 fatty acids, and about a 15 to 30 fold increase in percentage levels of C22:2n6 fatty acids relative to the fatty acid profile of Strain J.
[0462] These data illustrate the successful use of polynucleotides encoding elongases for expression in Prototheca moriformis (UTEX 1435) to alter the fatty acid profile of engineered microbes, and in particular in increasing the concentration of C18:0, C20:0, and C22:2n6 fatty acids and decreasing C16:0 fatty acids in recombinant microbial cells.
Example 41
Engineering Microorganisms for Increased Production of Stearic Acid Through Acyl-ACP Thioesterase Overexpression
[0463] This example describes the use of recombinant polynucleotides that encode different C18:0-preferring acyl-ACP thioesterases to engineer microorganisms in which the fatty acid profiles of the transformed microorganisms have been enriched in stearic acid.
[0464] Classically mutagenized strains of Prototheca moriformis (UTEX 1435), Strain J or Strain A, were transformed independently with the constructs listed in Table 32 according to biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of the constructs included a protein coding region to overexpress a fatty acyl-ACP thioesterase with a C-terminal 3.times. FLAG.RTM. epitope tag, 5' (SEQ ID NO: 1) and 3' (SEQ ID NO: 2) homologous recombination targeting sequences (flanking the construct) to the 6S genomic region for integration into the nuclear genome, and a S. cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4) to express the protein sequence given in SEQ ID NO: 3 under the control of C. reinhardtii .beta.-tubulin promoter/5'UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO: 7 and served as a selectable marker. Sequence listing identities for the polynucleotides corresponding to each thioesterase are listed in Table 32. The polynucleotide sequence encoding each thioesterase was under control of the P. moriformis Amt03 promoter/5'UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3' UTR (SEQ ID NO: 6). The protein coding regions of the exogenous thioesterases and the suc2 expression cassette were codon optimized to reflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
TABLE-US-00034 TABLE 32 Plasmid constructs used to transform Prototheca moriformis (UTEX 1435) Strain A or Strain J. Acyl-ACP Thioesterase, Acyl-ACP Transit Plasmid GenBank Thioesterase Peptide construct Accession No. source source SEQ ID NO: pSZD581 FATA, Brassica napus native 64, 65 CAA52070 pSZD643 FATA, Brassica napus UTEX 66, 67 CAA52070 250 SAD pSZD645 FATA, C. tinctorius UTEX 68, 69 AAA33019 250 SAD pSZD644 FATA, Ricinis communis native 70, 71 ABS30422 pSZD1323 FATA, G. mangostana native 72, 73 AAB51523 pSZD1320 FATA Theobroma cacao native 74, 75
[0465] Upon independent transformation of Strain A or J with the constructs listed in Table 32, positive clones were selected on agar plates containing sucrose as a sole carbon source. Individual transformants were clonally purified and propagated under heterotrophic conditions suitable for lipid production as those detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were prepared from dried biomass and analyzed using fatty acid methyl ester gas chromatography flame ionization detection methods as described in Example 1 (also see PCT/US2012/023696). The fatty acid profiles (expressed as Area % of total fatty acids) of P. moriformis UTEX 1435 Strain J propagated on glucose as a sole carbon source and representative isolates of each transformation of Strain J, propagated on sucrose as a sole carbon source are presented in Table 33.
TABLE-US-00035 TABLE 33 Fatty acid profiles of Prototheca moriformis (UTEX 1435) Strain J cells engineered to overexpress exogenous acyl-ACP thioesterase enzymes. Plasmid Fatty Acid Area % Strain construct No. C14:0 C16:0 C18:0 C18:1 C18:2 C18:3.alpha. A None 1 1.08 25.48 3.23 59.70 8.25 0.70 J None 1 1.41 27.33 3.38 57.07 8.15 0.64 A pSZD581 1 1.02 26.60 14.47 44.80 10.05 0.65 2 1.08 28.24 13.57 43.89 10.07 0.68 3 0.97 24.70 9.13 50.85 11.27 0.82 A pSZD643 1 1.39 26.97 16.21 44.10 8.43 0.83 2 1.37 27.91 11.15 48.31 8.40 0.78 A pSZD645 1 0.90 23.39 8.35 50.69 13.34 0.96 A pSZD644 1 1.67 19.70 4.40 59.15 12.32 1.01 J pSZD1323 1 1.33 23.26 9.28 53.42 10.35 0.69 2 1.47 26.84 7.36 52.78 9.29 0.64 3 1.43 26.31 6.05 54.45 9.37 0.66 J pSZD1320 1 1.30 24.76 3.84 60.90 6.96 0.55 2 1.36 26.30 3.27 58.19 8.66 0.48 3 1.39 25.51 3.18 58.78 8.85 0.45
[0466] The data presented in Table 33 show a clear impact of the expression of exogenous acyl-ACP enzymes on the fatty acid profiles of the transformed microorganisms. The fatty acid profiles of untransformed Strain A and J were about 25% C16:0, about 3.3% C18:0, about 57 to 60% C18:1. In contrast, the fatty acid profiles of Strain A transformed with different plasmid constructs to express acyl-ACP enzymes comprised greater percentage levels of C18:0 and lower percentage levels of C18:1 fatty acids than that of Strain A. Expression of FATA enzymes from B. napus, C. tinctorius, R. communis and G. mangostana in Strain A or J enabled the accumulation of stearate levels in the transformed organisms. The result of overexpression of a Brassica napus acyl-ACP thioesterase was about a 2 to 5 fold increase in the percentage levels of C18:0 fatty acids of the fatty acid profile of the transformed organisms relative to the fatty acid profile of Strain A. Fatty acid profiles of cells engineered to overexpress a G. mangostana acyl-ACP FATA thioesterase with a C. protothecoides SAD1 transit peptide were characterized by about a 2 to 3 fold increase in the percentage levels of C18:0 fatty acids of the fatty acid profile of the transformed organism relative to the fatty acid profile of Strain J.
[0467] These data illustrate the utility and effective use of polynucleotides encoding fatty acyl-ACP thioesterases for expression in Prototheca moriformis (UTEX 1435) to alter the fatty acid profile of engineered microbes, and in particular in increasing the concentration of C18:0 and decreasing C18:1 fatty acids in recombinant microbial cells.
Example 42
Engineering Microorganisms for Increased Production of Erucic Acid Through Elongase or Beta-Ketoacyl-COA Synthase Overexpression
[0468] In an embodiment of the present invention, a recombinant polynucleotide transformation vector operable to express an exogenous elongase or beta-ketoacyl-CoA synthase in an optionally plastidic oleaginous microbe is constructed and employed to transform Prototheca moriformis (UTEX 1435) according to the biolistic transformation methods as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696 to obtain a cell increased for production of erucic acid. The transformation vector includes a protein coding region to overexpress an elongase or beta-ketoacyl-CoA synthase such as those listed in Table 5, promoter and 3'UTR control sequences to regulate expression of the exogenous gene, 5' and 3' homologous recombination targeting sequences targeting the recombinant polynucleotides for integration into the P. moriformis (UTEX 1435) nuclear genome, and nucleotides operable to express a selectable marker. The protein-coding sequences of the transformation vector are codon-optimized for expression in P. moriformis (UTEX 1435) as described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Recombinant polynucleotides encoding promoters, 3' UTRs, and selectable markers operable for expression in P. moriformis (UTEX 1435) are disclosed herein and in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.
[0469] Upon transformation of the transformation vector into P. moriformis (UTEX 1435) or a classically-mutagenized strain of P. moriformis (UTEX 1435), positive clones are selected on agar plates. Individual transformants are clonally purified and cultivated under heterotrophic conditions suitable for lipid production as detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Lipid samples are prepared from dried biomass from each transformant and fatty acid profiles from these samples are analyzed using fatty acid methyl ester gas chromatography flame ionization (FAME GC/FID) detection methods as described in Example 1. As a result of these manipulations, the cell may exhibit an increase in erucic acid of at least 5, 10, 15, or 20 fold.
Example 43
Generation Of Capric, Lauric, and Myristic Acid Rich Oils in Strain UTEX1435 by the Expression of Cuphea PSR23 LPAATs
[0470] We tested the effect of expression of two 1-acyl-sn-glycerol-3-phosphate acyltransferases (LPAATs) in a previously described P. moriformis (UTEX 1435) transgenic strain, expressing the acyl ACP thioesterase (FATB2) from Cuphea wrightii. The LPAAT2 and LPAAT3 genes from Cuphea PSR23 (CuPSR23) were identified by analysis of a combination of CuPSR23 genomic sequences and transcriptomic sequences derived from seed RNAs. The two LPAATs have not been previously described. The genes were codon optimized to reflect UTEX 1435 codon usage. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described.
[0471] Increased capric, lauric, and myristic accumulation in strain B by the expression of the Cuphea PSR23 1-acyl-sn-glycerol-3-phosphate acyltransferases (LPAAT2 and LPAAT3) [pSZ2299 and pSZ2300, respectively]: In this example, transgenic strains were generated via transformation of strain B with the constructs pSZ2299 or pSZ2300, encoding CuPSR23 LPAAT2 and LPAAT3, respectively. The transgenic strains were selected for resistance to the antibiotic G418. Construct pSZ2299 can be written as pLOOP5'::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT2-1:CvNR::pLOOP3'. Construct pSZ2300 can be written as pLOOP5'::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT3-1:CvNR::pLOOP3'. The sequence of the transforming DNA (pSZ2299 and pSZ2300) is provided below. The relevant restriction sites in the construct from 5'-3', BspQI, KpnI, XbaI, Mfe I, BamHI, EcoRI, SpeI, XhoI, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences at the 5' and 3' end of the construct represent genomic DNA from UTEX 1435 that target integration to the pLoop locus via homologous recombination. Proceeding in the 5' to 3' direction, the selection cassette has the C. reinhardtii .beta.-tubulin promoter driving expression of the NeoR gene (conferring resistance to G418) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for NeoR are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR is indicated by lowercase underlined text. The spacer region between the two cassettes is indicated by upper case text. The second cassette containing the codon optimized LPAAT2 gene (pSZ2299) or LPAAT3 gene (pSZ2300) from Cuphea PSR23 is driven by the Prototheca moriformis endogenous AMT3 promoter, and has the same Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. In this cassette, the AMT3 promoter in indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CuPSR23 LPAAT2 and LPAAT3 genes are indicated in uppercase italics, while the coding regions are indicated by lowercase italics. The 3' UTR is indicated by lowercase underlined text. The final constructs were sequenced to ensure correct reading frames and targeting sequences.
TABLE-US-00036 pSZ2299 Transforming Construct: (SEQ ID NO:90) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttc- aaaacttgat gaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgcc- agcttcgccc ccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgc- ccggcacggc tgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaata- gcgatgcggc aatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagg- gccccgatca agagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaat- ggtatgggaa ##STR00002## ccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttc- cgcctgtccg cccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcc- cgcctgtcct ggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctg- ctgggcgagg tgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgc- cgcctgcaca ccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggag- gccggcctgg tggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgc- atgcccgacg gcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggc- ttcatcgact gcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggc- ggcgagtggg ccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggac- gagttcttcT GAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgcca- cacttgctgc cttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttg- cgagttgcta gctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgc- aacttatcta cgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccg- cctgtattct cctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatgga- ggatccCGCG TCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATA- ACCACCTGAC GAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTG- ACAATGATCG ##STR00003## cctgatcatcaacctgttccaggcgctgtgcttcgtcctgatccgccccctgtccaagaacgcctaccgccgca- tcaaccgcgt gttcgcggagctgctgctgtccgagctgctgtgcctgttcgactggtgggcgggcgcgaagctgaagctgttca- ccgaccccga gacgttccgcctgatgggcaaggagcacgccctggtcatcatcaaccacatgaccgagctggactggatggtgg- gctgggtgat gggccagcacttcggctgcctgggctccatcatctccgtcgccaagaagtccacgaagttcctgcccgtgctgg- gctggtccat gtggttctccgagtacctgtacctggagcgctcctgggccaaggacaagtccaccctgaagtcccacatcgagc- gcctgatcga ctaccccctgcccttctggctggtcatcttcgtcgagggcacccgcttcacgcgcacgaagctgctggcggccc- agcagtacgc ggtctcctccggcctgcccgtcccccgcaacgtcctgatcccccgcacgaagggcttcgtctcctgcgtgtccc- acatgcgctc cttcgtccccgcggtgtacgacgtcacggtggcgttccccaagacgtcccccccccccacgctgctgaacctgt- tcgagggcca gtccatcatgctgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtcgcgg- agtggtgccg cgacaagttcgtcgagaaggacgccctgctggacaagcacaacgcggaggacacgttctccggccaggaggtgt- gccactccgg ctcccgccagctgaagtccctgctggtcgtgatctcctgggtcgtggtgacgacgttcggcgccctgaagttcc- tgcagtggtc ctcctggaagggcaaggcgttctccgccatcggcctgggcatcgtcaccctgctgatgcacgtgctgatcctgt- cctcccaggc cgagcgctccaaccccgccgaggtggcccaggccaagctgaagaccggcctgtccatctccaagaaggtgacgg- acaaggagaa cTGAttaattaactcgaggcagcagcaptcggatagtatcgacacactctggacgctggtcgtgtgatggactg- ttgccgccac acttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtac- gcgcttttgc gagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcat- cccaaccgca acttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtt- tgggctccgc ctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaac- acaaatggaa agcttgagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagct- tgttgagcga actgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacc- tcgtgcaaga actgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttc- ggtggcagta ctactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgcc- ttgcacttcg ctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcg- ctccgggagt caggaccggcggaaaataagaggcaacacactccgcttcttagctcttcg pSZ2300 Transforming Construct: (SEQ ID NO:91) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttc- aaaacttgat gaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgcc- agcttcgccc ccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgc- ccggcacggc tgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaata- gcgatgcggc aatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagg- gccccgatca agagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaat- ggtatgggaa ##STR00004## gccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgtt- ccgcctgtcc gcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgc- ccgcctgtcc tggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgct- gctgggcgag gtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcg- ccgcctgcac accctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatgga- ggccggcctg gtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccg- catgcccgac ggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccgg- cttcatcgac tgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctggg- cggcgagtgg gccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctgga- cgagttcttc TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgcc- acacttgctg ccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgctttt- gcgagttgct agctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccg- caacttatct acgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctcc- gcctgtattc tcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatgg- aggatccCGC GTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAAT- AACCACCTGA CGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGT- GACAATGATC ##STR00005## tgatcgtcaacctggtgcaggccgtctgcttcgtcctgatccgccccctgtccaagaacacgtaccgccgcatc- aaccgcgtgg tcgcggagctgctgtggctggagctggtgtggctgatcgactggtgggcgggcgtgaagatcaaggtcttcacg- gaccacgaga cgttccacctgatgggcaaggagcacgccctggtcatctgcaaccacaagtccgacatcgactggctggtcggc- tgggtcctgg gccagcgctccggctgcctgggctccaccctggcggtcatgaagaagtcctccaagttcctgcccgtcctgggc- tggtccatgt ggttctccgagtacctgttcctggagcgctcctgggccaaggacgagatcacgctgaagtccggcctgaaccgc- ctgaaggact accccctgcccttctggctggcgctgttcgtggagggcacgcgcttcacccgcgcgaagctgctggcggcgcag- cagtacgccg cgtcctccggcctgcccgtgccccgcaacgtgctgatcccccgcacgaagggcttcgtgtcctccgtgtcccac- atgcgctcct tcgtgcccgcgatctacgacgtcaccgtggccatccccaagacgtcccccccccccacgctgatccgcatgttc- aagggccagt cctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgccgtcgcgcag- tggtgccgcg acatcttcgtggagaaggacgcgctgctggacaagcacaacgccgaggacaccttctccggccaggagctgcag- gagaccggcc gccccatcaagtccctgctggtcgtcatctcctgggccgtcctggaggtgttcggcgccgtcaagttcctgcag- tggtcctccc tgctgtcctcctggaagggcctggcgttctccggcatcggcctgggcgtgatcaccctgctgatgcacatcctg- atcctgttct cccagtccgagcgctccacccccgccaaggtggcccccgcgaagcccaagaacgagggcgagtcctccaagacc- gagatggaga
aggagaagTGAttaattaactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtg- atggactgtt gccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatctt- gtgtgtacgc gcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatcccatccctcgtttcatatcg- cttgcatccc aaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagc- cttggtttgg gctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggga- tgggaacaca aatggaaagcttgagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtata- ccgagcttgt tgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaat- ccgaacctcg tgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgt- tcgcttcggt ggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgc- tcacgcctgc acttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacg- tcgtcgctcc gggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctcttcg
[0472] To determine the impact of the CuPSR23 LPAAT2 and LPAAT3 genes on mid-chain fatty acid accumulation, the above constructs containing the codon optimized CuPSR23 LPAAT2 or LPAAT3 genes driven by the UTEX 1453 AMT3 promoter were transformed into strain B.
[0473] Primary transformants were clonally purified and grown under standard lipid production conditions at pH7.0 (all the strains require growth at pH 7.0 to allow for maximal expression of the CuPSR23 LPAAT2 or LPAAT3 gene driven by the pH-regulated AMT3 promoter). The resulting profiles from a set of representative clones arising from these transformations are shown in Table 34, below. D1520 represents clones of Strain B with CuPSR23 LPAAT2 and D1521 represents clones of Strain B with CuPSR23 LPAAT3.
TABLE-US-00037 TABLE 34 Fatty acid profiles of Strain B and representative transgenic lines transformed with pSZ2299 and pSZ2300 DNA. Sample C10: C12: C14: C16: C18: C18: C18: ID 0 0 0 0 0 1 2 Strain B 4.83 28.54 15.64 12.64 1.3 27.99 7.75 D1520-A 8.59 35.09 16.55 11.96 1.69 19.49 5.59 D1520-B 8.13 33.93 16.46 12.44 1.57 20.66 5.96 D1520-C 7.6 33.1 16.21 12.65 1.5 21.41 6.48 D1520-D 7.35 32.54 16.03 12.79 1.67 22.16 6.41 D1520-E 7.28 32.21 16.2 12.99 1.73 22.39 6.28 D1521-A 6.14 31.5 15.98 12.96 1.96 22.52 8 D1521-B 6.17 31.38 15.98 12.87 2.08 22.54 7.92 D1521-C 5.99 31.31 15.75 12.79 2.23 22.45 8.36 D1521-D 5.95 31.05 15.71 12.84 2.48 22.69 8.32 D1521-E 5.91 30.58 15.85 13.22 1.97 23.55 7.84
[0474] The transgenic CuPSR23 LPAAT2 strains (D1520A-E) show a significant increase in the accumulation of C10:0, C12:0, and C14:0 fatty acids with a concomitant decrease in C18:1 and C18:2. The transgenic CuPSR23 LPAAT3 strains (D1521A-E) show a significant increase in the accumulation of C10:0, C12:0, and C14:0 fatty acids with a concomitant decrease in C18:1. The expression of the CuPSR23 LPAAT in these transgenic lines appears to be directly responsible for the increased accumulation of mid-chain fatty acids in general, and especially laurates. While the transgenic lines show a shift from longer chain fatty acids (C16:0 and above) to mid-chain fatty acids, the shift is targeted predominantly to C10:0 and C12:0 fatty acids with a slight effect on C14:0 fatty acids. The data presented also show that co-expression of the LPAAT2 and LPAAT3 genes from Cuphea PSR23 and the FATB2 from C. wrightii (expressed in the strain Strain B) have an additive effect on the accumulation of C12:0 fatty acids.
[0475] Our results suggest that the LPAAT enzymes from Cuphea PSR23 are active in the algal strains derived from UTEX 1435. These results also demonstrate that the enzyme functions in conjunction with the heterologous FatB2 acyl-ACP thioesterase enzyme expressed in Strain B, which is derived from Cuphea wrightii.
Example 44
Alteration of Fatty Acid Levels in Strain UTEX1435 by the Expression of Cuphea PSR23 LPAATX in Combination with Cuphea Wrightii FATB2
[0476] Here we demonstrate the effect of expression of a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) in a previously described P. moriformis (UTEX 1435) transgenic strain, Strain B. As described above, Strain B is a transgenic strain expressing the acyl ACP thioesterase (FATB2) from Cuphea wrightii, which accumulates C12:0 fatty acids between 40 to 49%. Further to Example 43, a third CuPSR23 LPAAT, LPAATx, was identified by analysis of a combination of CuPSR23 genomic sequences and transcriptomic sequences derived from seed RNAs. Expression of a mid-chain specific LPAAT should thus increase the percentage of TAGs that have a capric acid (C10:0 fatty acid), lauric acid (C12:0 fatty acid), or myristic acid (C14:0 fatty acid) at the sn-2 position, and should consequently elevate the overall levels of these fatty acids. In Example 43, LPAAT2 and LPAAT3 were shown to increase caprate, laurate, and myristate accumulation in strain B. LPAATx was introduced into strain B to determine its effect on fatty acid levels in this strain. The LPAATx gene was codon optimized to reflect UTEX 1435 codon usage. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described.
[0477] Decreased caprate, laurate, and myristate accumulation and increased palmitate and stearate accumulation in strain Strain B by the expression of the Cuphea PSR23 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAATx) [pSZ2575]: In this example, transgenic strains were generated via transformation of strain B with the construct pSZ2575 encoding CuPSR23 LPAATx. The transgenic strains were selected for resistance to the antibiotic G418. Construct pSZ2575 can be written as pLOOP5'::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAATx:CvNR ::pLOOP3'. The sequence of the transforming DNA is provided below (pSZ2575). The relevant restriction sites in the construct from 5'-3', BspQI, KpnI, XbaI, MfeI, BamHI, EcoRI, SpeI, XhoI, SacI, BspQ1, respectively, are indicated in lowercase, bold, and underlined. BspQ1 sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences at the 5' and 3' end of the construct represent genomic DNA from UTEX 1435 that target integration to the pLoop locus via homologous recombination. Proceeding in the 5' to 3' direction, the selection cassette has the C. reinhardtii, .beta.-tubulin promoter driving expression of the NeoR gene (conferring resistance to G418) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for NeoR are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR is indicated by lowercase underlined text. The spacer region between the two cassettes is indicated by upper case text. The second cassette containing the codon optimized LPAATx gene (pSZ2575) from Cuphea PSR23 is driven by the Prototheca moriformis endogenous AMT3 promoter, and has the same Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CuPSR23 LPAATx genes are indicated in uppercase italics, while the coding region is indicated by lowercase italics. The 3' UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.
[0478] 104961 pSZ2575 Transforming Construct
TABLE-US-00038 (SEQ ID NO: 92) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttc- aaaacttgat gaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgcc- agcttcgcc cccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcg- cccggca cggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtg- aatagcgatg cggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcc- cagggccc cgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactc- tgcaatggt ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## cctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgc- ctgtccgccca gggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcc- tgtcctggct ggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgg- gcgaggtgc ccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgc- ctgcacaccc tggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggcc- ggcctggtg gaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcat- gcccgacg gcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggc- ttcatcgactg cggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcg- gcgagtgg gccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctgga- cgagttcttcTG Acaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccac- acttgctgccttga cctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagt- tgctagctgcttgtgctattt gcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgt- cctgctatccctcagcg ctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaa- cctgtaaaccagcactg caatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatccCGCGTCTCGAACAGAGCGCGCA GAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAA TAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCA CACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGG ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ctagtATGgagatccccccccactgcctgtgctccccctcccccgccccctcccagctgtactacaagaagaag- aagcacgcc atcctgcagacccagaccccctaccgctaccgcgtgtcccccacctgcttcgcccccccccgcctgcgcaagca- gcacccctacc ccctgcccgtgctgtgctaccccaagctgctgcacttctcccagccccgctaccccctggtgcgctcccacctg- gccgaggccggc gtggcctaccgccccggctacgagctgctgggcaagatccgcggcgtgtgcttctacgccgtgaccgccgccgt- ggccctgctgct gttccagtgcatgctgctgctgcaccccttcgtgctgctgttcgaccccttcccccgcaaggcccaccacacca- tcgccaagctgtg gtccatctgctccgtgtccctgttctacaagatccacatcaagggcctggagaacctgccccccccccactccc- ccgccgtgtacgt gtccaaccaccagtccttcctggacatctacaccctgctgaccctgggccgcaccttcaagttcatctccaaga- ccgagatcttcctg taccccatcatcggctgggccatgtacatgctgggcaccatccccctgaagcgcctggactcccgctcccagct- ggacaccctga agcgctgcatggacctgatcaagaagggcgcctccgtgttcttcttccccgagggcacccgctccaaggacggc- aagctgggcg ccttcaagaagggcgccttctccatcgccgccaagtccaaggtgcccgtggtgcccatcaccctgatcggcacc- ggcaagatcat gccccccggctccgagctgaccgtgaaccccggcaccgtgcaggtgatcatccacaagcccatcgagggctccg- acgccgagg ccatgtgcaacgaggcccgcgccaccatctcccactccctggacgacTGAttaattaactcgaggcagcagcap- tcggatagt atcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatcc- ctgccgcttttatcaaa cagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaatacc- acccccagcatccccttcc ctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgct- cctgctcactgcccctcgc acagcttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcac- gggaagtagtgggat gggaacacaaatggaaagcttgagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaa- gffigtatac cgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatgg- atggaaaat ccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtg- agcgaact gttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtg- gcagatgagtg ctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattggg- caatggacga cgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctcttcg
[0479] To determine the impact of the CuPSR23 LPAATx gene on fatty acid accumulation, the above construct containing the codon optimized CuPSR23 LPAATx gene driven by the UTEX 1453 AMT3 promoter was transformed into strain B.
[0480] Primary transformants were clonally purified and grown under low nitrogen conditions at pH7.0; the strains require growth at pH 7.0 to allow for maximal expression of the CuPSR23 LPAATx and CwFATB2 genes driven by the pH-regulated AMT3 promoter. The resulting profiles from a set of representative clones arising from these transformations are shown in Table 35, below. D1542 represents clones of Strain B with CuPSR23 LPAATx.
TABLE-US-00039 TABLE 35 Fatty acid profiles of Strain B and representative transgenic lines transformed with pSZ2575. Sample ID C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Strain 4.77 28.63 15.48 12.65 1.28 28.20 7.57 B D1542- 1.19 13.25 10.48 21.34 4.49 32.07 14.78 A D1542- 1.15 14.01 10.62 20.61 3.99 32.12 15.24 B D1542- 1.21 13.69 10.83 20.40 3.59 33.54 15.05 C D1542- 1.56 16.83 11.51 18.44 2.94 33.97 12.74 D D1542- 2.15 18.58 11.94 18.22 3.17 32.63 11.62 E
[0481] The transgenic CuPSR23 LPAATx strains (D1542A-E) show a significant decrease in the accumulation of C10:0, C12:0, and C14:0 fatty acids relative to the parent, Strain B, with a concomitant increase in C16:0, C18:0, C18:1 and C18:2. The expression of the CuPSR23 LPAATx gene in these transgenic lines appears to be directly responsible for the decreased accumulation of mid-chain fatty acids (C10-C14) and the increased accumulation of C16:0 and C18 fatty acids, with the most pronounced increase observed in palmitates (C16:0). The data presented also show that despite the expression of the midchain specific FATB2 from C. wrightii (present in Strain B), the expression of CuPSR23 LPAATx appears to favor incorporation of longer chain fatty acids into TAGs.
[0482] Our results suggest that the LPAATx enzyme from Cuphea PSR23 is active in the algal strains derived from UTEX 1435. Contrary to Cuphea PSR23 LPAAT2 and LPAAT3, which increase mid-chain fatty acid levels, CuPSR23 LPAATx leads to increased C16:0 and C18:0 levels. These results demonstrate that the different LPAATs derived from CuPSR23 (LPAAT2, LPAAT3, and LPAATx) exhibit different fatty acid specificities in Strain B as judged by their effects on overall fatty acid levels.
Example 45
Reduction in Chain Length of Fatty Acid Profile as a Result of Overexpressing an Endogenous Microalgal FATA Acyl-ACP Thioesterase
[0483] Here, we demonstrate that over expression of the Prototheca moriformis endogenous thioesterases FATA1 in UTEX1435 results in a clear diminution of cell triglyceride C18:0 and C18:1 acyl chains with an increase in C16:0, C14:0.
[0484] Constructs used for the over expression of the P. moriformis FATA1 gene (pSZ2422, pSZ2421): To over express the PmFATA1 in P. moriformis STRAIN J, a codon optimized PmFATA1 gene was been transformed into STRAIN J. The Saccharomyces cerevisiae invertase gene was utilized as the selectable marker to confer the ability of growing on sucrose media. The construct pSZ2422 that have been expressed in STRAIN J can be written as: 6SA:: CrTUB2-ScSUC2-CvNR3':PmAMT3-Pm FATA1 (opt)-CvNR3'::6SB, and the construct pSZ2421 can be written as 6SA:: CrTUB2-ScSUC2-CvNR3':PmAMT3-S106SAD TP-Pm FATA1 (opt)-CvNR3'::6SB.
[0485] The sequence of the transforming DNA is provided below. Relevant restriction sites in the construct pSZ2422 are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from STRAIN J that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene (conferring the ability of STRAIN J to metabolize sucrose) is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous amt03 promoter of P. moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the PmFATA1 are indicated by uppercase, bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the STRAIN J 6S genomic region indicated by bold, lowercase text.
[0486] Relevant restriction sites in the construct pSZ2421 are the same as pSZ2422. In pSZ2421, the PmFATA1 is fused to the Chlorella protothecoides S106 stearoyl-ACP desaturase transit peptide and the transit peptide is located between initiator ATG of PmFATA1 and the Asc I site.
[0487] Nucleotide sequence of transforming DNA contained in pSZ2422
TABLE-US-00040 (SEQ ID NO: 93) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtc gctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaaga- ggagcatga gggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgc- accgaggc cgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatga- attgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgctt cgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgca- gaccggtgag agccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcat- cggcctcggcc ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## cgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgacc- ccaacggcc tgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacg- cccttgttctg gggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgact- ccggcgc cttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagc- gctgcgtggcca tctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcacc- gagtaccaga agaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaag- tggatcatgac cgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccg- cgttcgccaa cgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagt- cctactgggt gatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacg- gcacccacttcg aggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacacc- gacccgaccta cgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgct- cctccatgtccc tcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgag- ccgatcctg aacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgt- cgacctgtc caacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgt- tcgcggacctc tccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctcctt- cttcctggaccgc gggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagccctt- caagagcg agaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggc- gacgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcgac aagttccaggtgcgcgaggtcaagTGAcaattggcagcagcaptcggatagtatcgacacactctggacgctgg- tcgtgtgat ggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgt- ttgatcttgtgtgtacgcg cttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcg- cttgcatcccaaccgcaac ttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttg- ggctccgcctgtattctcc tggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggagg- atcccgcgtctcg aacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccac- ctgacgaatgcg cttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatga- tcggtggagctgatg ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## acttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtac- gcgatttgcgagttgctag ctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgca- acttatctacgctgtcctg ctatccctcagcgctgacctgacctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcc- tggtactgcaacctgt aaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctc- ttgttttccagaa ggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcga- atttaaaagctt ggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaa- acttgccgc tcaaaccgcgtacctctgattcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgag- cagtctgtaat tgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgcca- ctcgtacag cagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctcca- tgctctgagtg gccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcc- caccaccc ccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaa- accatacc acacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggg- gttgctaggga tcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc
[0488] To determine the impact on fatty acid profiles when the endogenous FATA1 gene have been over expressed in STRAIN J, both the P. moriformis FATA1 with native transit peptide and PmFATA1 fused to a Chlorella protothecoides SAD transit peptide were driven by the amt03 promoter and the resulting plasmids were transformed independently into STRAIN J.
[0489] Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at pH7.0 (all the plasmids require growth at pH 7.0 to allow for maximal PmFATA1 gene expression when driven by the pH regulated amt03 promoter). The resulting profiles from representative clones arising from transformations with pSZ2422 and pSZ2421 into STRAIN J are shown in the tables below.
[0490] In Table 36, below, the impact of over expressing native PmFATA1 is a clear diminution of C18:1 chain lengths with an increase in C16:0, C14:0, and possibly in C18:0. Considering the protein localization of processing, we also tried the PmFATA1 fused to a Chlorella protothecoides stearoyl-ACP desaturase transit peptide. Similar to the results we observed in the amt03-native PmFATA1 construct, the C16:0 and C14:0 levels are significantly higher than the parental strain J.
TABLE-US-00041 TABLE 36 Fatty acid profiles in Strain J and derivative transgenic lines transformed with pSZ2422 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH 7; Strain J; T374; 7.69 55.00 4.92 24.94 5.19 D1377-7 96well pH 7; Strain J; T374; 6.39 54.11 5.85 25.91 5.76 D1377-13 96well pH 7; Strain J; T374; 6.57 53.55 4.68 27.18 5.74 D1377-14 96well pH 7; Strain J; T374; 5.29 49.93 4.24 30.76 7.27 D1377-16 96well pH 7; Strain J; T374; 4.76 49.10 4.75 32.36 6.77 D1377-9 96well pH 7; Strain J; T374; 4.28 46.06 5.14 35.87 6.69 D1377-19 96well Ctrl-pH 7; Strain J 1.42 27.63 3.31 57.20 8.00
TABLE-US-00042 TABLE 37 Fatty acid profiles in STRAIN J and derivative transgenic lines transformed with pSZ2421 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH 7; STRAIN J; T374; 6.76 57.06 4.12 23.66 6.07 D1376-21 96well pH 7; STRAIN J; T374; 6.56 54.62 5.44 25.69 5.64 D1376-22 96well pH 7; STRAIN J; T374; 4.54 48.38 4.27 33.23 7.24 D1376-23 96well pH 7; STRAIN J; T374; 4.48 47.66 4.60 34.28 6.91 D1376-19 96well pH 7; STRAIN J; T374; 4.53 47.30 4.67 34.51 6.80 D1376-20 96well pH 7; STRAIN J; T374; 3.56 42.70 4.03 39.85 7.52 D1376-17 96well Ctrl-pH 7; STRAIN J 1.42 27.63 3.31 57.20 8.00
[0491] Thus, we conclude that percent myristic and lauric acid levels in the fatty acid profile of a microalgal cell can be increased by overexpression of a C18-preferring acyl-ACP thioesterase.
Example 46
Cell Oils Suitable for Use as Roll-In Shortenings
[0492] The nutritional and functional properties of edible fats have been traditionally associated with specific chemical compositions and crystallization conditions. Switching from one oil source to another is usually a difficult task since both the melting behavior and structure of the fat changes dramatically, leading to adverse changes in functionality. In recent history, we can recall the painful period when partially hydrogenated fats were replaced with palm oil and palm oil fractions. We examined how the yield stress, elastic modulus, polymorphism, microstructure and melting profile of two fats with vastly different chemical compositions can be matched. Oil A was produced from Prototheca moriformis cells expressing an exogenous invertase and an Ulmus americana acyl-ACP thioesterase with a Chlorella protothecoides plastid targeting sequence. Oil B was produced from Prototheca moriformis cells expressing an exogenous invertase and a Cuphea hookeriana acyl-ACP thioesterase. Oil A contained greater than 62% (w/w) medium chain fatty acids, or MCT (C8:0-C14:0), 23% (C16:0+C18:0) and 9% C18:1, while Oil B contained less than 2% C8:0-C14:0, 54% (C16:0+C18:0) and 29% C18:1. Oil A was thus a medium chain triglyceride rich fat, while Oil B resembled palm oil. Both oils had a solid fat content of .about.45% at 20.degree. C., and very similar SFC versus temperature profiles. DSC (dynamic scanning calorimetry) melting profiles showed two major peaks centered around .about.12-13.degree. C. and .about.28-35.degree. C. Both fats were in the beta-prime polymorphic form (as determined by X-ray diffraction) and displayed asymmetric, elongated crystallite morphology with characteristic features. The yield stresses and storage moduli (G') of Oil A and Oil B were 520-550 Pa, and 7.times.10.sup.6 Pa-1.8.times.10.sup.7 Pa, respectively. A yield stress in this region suggests a satisfactory plasticity, which combined with a high storage modulus makes for an ideal roll-in shortening. Thus, it is possible to alter the chemical composition of a food oil while retaining its lamination functionality.
[0493] Other suitable enzymes for use with the cells and methods of any of the above embodiments of the invention include those that have at least 70% amino acid identity with one of the proteins listed in the description above and that exhibit the corresponding desired enzymatic activity. In additional embodiments, the enzymatic activity is present in a sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with one of the above described nucleic acid sequences, all of which are hereby incorporated by reference as if fully set forth.
Example 47
Fractionation to Remove Trisaturates from a Tailored Microbial Oil that is a Cocoa Butter Mimetic
[0494] A refined bleached and deodorized oil was obtained from Strain K4 (see Example 35). The oil was heated to 70.degree. C. and cooled at 0.5.degree. C. per min to 36.degree. C. and held at 36.degree. C. for 1 hour. An approximately 2.5 ml sample was then centrifuged at 36.degree. C. for 1 hour at 4300. A liquid supernatant was recovered and analyzed using lipase and mass spectrometry. The sample was found to be depleted in tristearin (SSS), SSP, and PPS. The triacylglycerols of the sample were found to be very similar to that of cocoa butter and the liquid supernatant was even closer to that of cocoa butter in terms of low amounts of trisaturates. Further fractionation experiments are described in Example 64.
TABLE-US-00043 TABLE 38 TAG profile of oil from the K4 strain before and after fractionation as compared to cocoa butter. fractionation upper TAG K4 oil layer (liquid) cocoa butter OOL (+?) 0.12 0.12 0.00 POL 0.23 0.31 0.33 PLP 2.41 3.38 1.58 MOP 0.93 1.25 0.00 PPM (+MMS) 0.42 0.29 0.00 OOO 0.23 0.34 0.00 SOL 0.36 0.47 0.32 OOP 0.95 1.42 2.44 PLS 5.66 7.90 2.90 POP (+MSO) 11.80 15.20 17.93 PPP + MPS 2.22 1.07 0.36 OOS 1.19 1.68 3.02 SLS (+PLA) 3.96 5.11 1.77 POS 27.22 32.80 40.25 PPS (+SSM) 6.47 1.52 0.49 MaOO 0.00 0.00 0.36 SLA 0.31 0.34 0.00 SOS (+POA) 17.84 22.50 24.93 SSP (+PPA) 9.24 0.96 0.63 SOA (+POB) 1.39 1.68 1.51 SSS (+PSA) 5.25 0.23 0.33 SOB + LgOP 0.38 0.44 0.27 SSA 0.41 0.00 0.00 SOLg 0.41 0.00 0.00 PSLg + ASB 0.26 0.00 0.00 SOHx 0.12 0.51 0.00 SSLg 0.21 0.14 0.15 SUM area % 100.00 99.67 99.57
Example 48
Production of High-Stearate Triglyceride Oil in an Oleaginous Cell by Overexpression of KASII, Knockout of One SAD Allele and Repression of a Second SAD Allele
[0495] The oleaginous, non-photosynthetic alga, Prototheca moriformis, stores copious amounts of triacylglyceride oil under conditions where the nutritional carbon supply is in excess, but cell division is inhibited due to limitation of other essential nutrients. Bulk biosynthesis of fatty acids with carbon chain lengths up to C18 occurs in the plastids; fatty acids are then exported to the endoplasmic reticulum where elongation past C18 and incorporation into triacylglycerides (TAGs) is believed to occur. Lipids are stored in large cytoplasmic organelles called lipid bodies until environmental conditions change to favor growth, whereupon they are rapidly mobilized to provide energy and carbon molecules for anabolic metabolism. Wild-type P. moriformis storage lipid is mainly comprised of .about.60% oleic (C18:1), .about.25-30% palmitic (C16:0), and .about.5-8% linoleic (C18:2) acids, with minor amounts of stearic (C18:0), myristic (C14:0), .alpha.-linolenic (C18:3.alpha.), and palmitoleic (C16:1) acids. This fatty acid profile results from the relative activities and substrate affinities of the enzymes of the endogenous fatty acid biosynthetic pathway. P. moriformis is amenable to manipulation of fatty acid and lipid biosynthesis using molecular genetic tools, enabling the production of oils with fatty acid profiles that are very different to the wild-type composition. Herein we describe strains where we have modified the expression of stearoyl-ACP desaturase (SAD) and .beta.-ketoacyl-ACP synthase II (KASII) genes in order to generate strains with up to 57% stearate and as little as 7% palmitate. We identify additional strains with up to 55% stearate and as low as 2.4% linoleate when we perform similar modifications in conjunction with down-regulating the expression of the FATA thioesterase and the FAD2 fatty acid desaturase genes.
[0496] Soluble SADs are plastid-localized, di-iron enzymes which catalyze the desaturation of acyl carrier protein (ACP)-bound stearate to oleate (C18:1 cis-.DELTA..sup.9). Previously, we have established that hairpin constructs targeting the SAD1 or SAD2 transcripts activate the cellular RNA interference (RNAi) machinery, down-regulating SAD activity and resulting in elevated levels of C18:0 in the storage lipid. SAD activity is also reduced in strains where we disrupt one of the two alleles of SAD2, encoding the major SADs that are expressed during storage lipid biosynthesis. The Fatty Acid Desaturase 2 (FAD2) gene encodes an endoplasmic reticulum membrane-associated desaturase that converts oleate to linoleate (C18:2 cis-.DELTA..sup.9, cis-.DELTA..sup.12). Hairpin RNAi constructs targeting FAD2 reduce linoleate levels to 1-2%. KASII is a fatty acid synthase which specifically catalyzes the condensation of malonyl-ACP with palmitoyl (C16:0)-ACP to form .beta.-keto-stearoyl-ACP. We have shown that overexpression of KASII in P. moriformis causes C16:0 levels to decrease with a concomitant increase in C18:1 abundance. In the examples below we demonstrate that by down-regulating SAD gene expression using RNAi, disrupting an allele of the SAD2 gene, and overexpressing the KASII fatty acid synthase, we generate strains capable of accumulating stearate in excess of 50% of the total fatty acids, and with SOS as the major TAG species. SOS levels increase up to 47% in strains which combine SAD2 and FAD2 down-regulation with KASII overexpression.
[0497] Constructs used for SAD2 knockout/RNAi in S1920: A DNA construct, pSZ2282, was made to simultaneously disrupt the SAD2-1 allele and express a SAD2 hairpin construct in Strain J. A Saccharomyces cerevisiae SUC2 gene, encoding sucrose invertase, which was codon-optimized for expression in P. moriformis, was utilized as a selectable marker for transformation. The sequence of the transforming DNA is provided immediately below. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, AscI, MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the Chlamydomonas reinhardtii TUB2 promoter driving the expression of the Saccharomyces cerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, thereby permitting the strain to grow on sucrose) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for SUC2 are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the Chlorella vulgaris nitrate reductase (NR) gene is indicated by small capitals, followed by a spacer region indicated by lowercase text. A second C. reinhardtii TUB2 promoter sequence, indicated by lowercase boxed text, drives expression of the SAD2 hairpin C sequence. The sense and antisense strands are indicated with uppercase, bold italics, and are separated by the P. moriformis .DELTA..sup.12-fatty acid desaturase (FAD2) intron and the first 10 bases of the FAD2 second exon (uppercase italics). A second C. vulgaris NR 3' UTR is indicated by small capitals.
TABLE-US-00044 Nucleotide sequence of the transforming DNA from pSZ2282: (SEQ ID NO: 94) gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgtt- ccgccctcggtgca gcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgc- agagcatggggc agtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgccc- gaccccgagtcgc ccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctg- gtgggggacatg atcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgc- ggccgaccacc cgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctgg- ctgacggggc gcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaac- aacccttattt ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## gaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgt- ggtacgac gagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctg- gggccacg ccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgcc- ttctccggc tccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggc- catctggacc tacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtacca- gaagaacc ccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatc- atgaccgcgg ccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttc- gccaacgag ggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtccta- ctgggtgat gttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggca- cccacttcgag gccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccga- cccgacctac gggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctc- ctccatgtccc tcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgag- ccgatcct gaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacg- tcgacctg tccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgt- gttcgcgga cctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcct- ccttcttcctgg accgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccag- cccttca agagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaac- gacggcga cgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtgg- acaacctgt tctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACAC- TCTGGAC GCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAA- CAGCCTCA GTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAG- CATCCCCT TCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCT- GCTCCTGCT CACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTG- CAATGCT GATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgc- tgaaggt ctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccat- tagcgaagcgtccg gttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacag- cctagggatatc ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## TGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTFCCAG ##STR00070## ##STR00071## ##STR00072## ##STR00073## CAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGA- CCTGTGA ATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGC- TGCTTGTG CTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTA- CGCTGTCCT GCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTC- TCCTGGTA CTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGg- agctc cagccacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcg- ccagcgacga gggccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccg- cctacgccaaca tgatgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgc- aacctcttcgc cgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctca- acgcgcgctgga aggtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttc- cggaaactcgc cgagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgaga- tcatggtctagg gagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaa- tcggccaccccg cgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatc- cggccgcacat caaagggcccctccgccagagaagaagctcctttcccagcagactcctgaagagc
[0498] Identification and analysis of SAD2 knockout/knockdown strains: Construct D1283, derived from pSZ2282, was transformed into Strain J. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. The resulting fatty acid profiles from representative clones arising from transformation with pSZ2282 into Strain J are summarized in Table 39, below. D1283 transformants accumulated up to .about.42% C18:0 at the expense of C18:1, indicating that SAD activity was significantly reduced in these strains.
TABLE-US-00045 TABLE 39 Fatty acid profiles of D1283 [pSZ2282] primary transformants, compared to the wild-type parental strain, Strain J. Strain J D1283-4 D1283-7 D1283-19 D1283-27 D1283-40 D1283-24 Fatty C12:0 0.04 0.05 0.06 0.07 0.06 0.04 0.05 Acid C14:0 1.31 0.92 1.07 1.01 1.08 1.03 0.96 Area % C16:0 26.68 28.23 29.21 27.24 27.67 27.02 27.07 C16:1 0.78 0.05 0.06 0.08 0.33 0.14 0.12 C17:0 0.11 0.12 0.15 0.10 0.10 0.12 0.13 C18:0 3.15 41.98 40.94 34.20 26.26 23.18 22.82 C18:l 59.30 19.37 18.17 26.87 34.77 38.74 39.38 C18:2 7.47 6.22 7.43 7.42 7.31 7.25 7.38 C18:3.alpha. 0.57 0.93 1.03 0.75 0.71 0.72 0.51 C20:0 0.32 1.81 1.67 1.75 1.35 1.36 1.23 C20:l 0.00 0.10 0.00 0.12 0.00 0.12 0.11 C22:0 0.05 0.17 0.13 0.20 0.16 0.16 0.15 C24:0 0.00 0.00 0.00 0.10 0.00 0.00 0.00 sum C18 70.49 68.5 67.57 69.24 69.05 69.89 70.09 saturates 31.66 73.28 73.23 64.67 56.68 52.91 52.41 unsaturates 68.12 26.67 26.69 35.24 43.12 46.97 47.50
[0499] In Table 39, Stearate (C18:0) levels greater than the wild-type level are highlighted with bold text.
[0500] The fatty acid profiles of transformants D1283-4 and -7 were determined to be stable after more than 30 generations of growth in the absence of selection (growth on sucrose). The performance of selected strains in shake flask assays was then evaluated, and the fatty acid profiles and lipid titers are presented in Table 40, below. Strain X had the highest level of C18:0 (.about.44%) and the best lipid titer (.about.26%) relative to the Strain J parent, and so was selected for further fermentation development.
TABLE-US-00046 TABLE 40 Fatty acid profiles and lipid titers of SAD2 knockout/knock- down strains derived from D1283 primary transformants, compared to the wild-type parental strain, Strain J. Primary T342; D1283-4 T342; D1283-7 Strain J S T U V W X Fatty C14:0 1.59 1.61 1.58 1.55 1.81 1.84 1.34 Acid C16:0 30.47 29.41 28.58 29.24 28.77 29.09 28.47 Area % C16:l 0.82 0.05 0.07 0.05 0.07 0.05 0.06 C17:0 0.10 0.30 0.29 0.28 0.46 0.37 0.19 C18:0 3.58 42.85 41.86 43.38 39.99 41.41 44.42 C18:l 56.96 13.52 15.55 13.49 13.57 12.98 15.64 C18:2 5.50 8.01 7.85 7.65 10.37 9.47 5.72 C18:3.alpha. 0.37 0.78 0.73 0.82 0.95 0.91 0.64 C20:0 0.22 2.06 2.11 2.11 1.98 1.98 2.32 C22:0 0.05 0.32 0.34 0.33 0.33 0.32 0.35 C24:0 0.03 0.43 0.42 0.44 0.49 0.49 0.37 lipid titer 100 12.3 12.6 13.6 6.2 8.2 25.9 (% parent)
[0501] In Table 40, Stearate (C18:0) levels greater than the wild-type level are highlighted with bold text.
[0502] We optimized the performance of Strain X in 7-L fermentations, and found that we could match the .about.44% C18:0 level obtained in shake flasks, with lipid productivities that were .about.45% of the wild-type parent. The fatty acid profiles and lipid titers of representative strain K-4 fermentations are summarized in Table 41, below. Fermentation of Strain X under optimal conditions yielded nearly 44% C18:0, which was similar to the stearate level that accumulated in shake flask assays. Strain X produced high C18:0 levels at both flask and 7-L scale and had acceptable lipid productivity in 7-L fermentations; consequently this strain was selected as a base strain for additional modifications aimed at increasing C18:0 accumulation.
TABLE-US-00047 TABLE 41 Fatty acid profiles and lipid titers of strain X, compared to a control transgenic strain Strain Y. Strain Strain Y K-4 K-4 K-4 Fermentation 110088F14 120489F5 120531F8 120580F1 Fatty Acid C14:0 1.47 1.18 1.15 1.27 Area % C16:0 25.66 28.68 28.38 28.35 C16:1 0.71 0.11 0.09 0.06 C18:0 3.16 41.63 42.40 43.67 C18:1 62.24 20.78 19.38 17.63 C18:2 5.90 5.06 5.38 5.58 C18:3.alpha. 0.16 0.24 0.25 0.25 C20:0 0.24 1.36 1.99 2.11 C22:0 0.05 0.19 0.28 0.31 C24:0 0.05 0.34 0.29 0.31 sum C18 71.46 67.71 67.41 67.13 saturates 30.63 73.38 74.49 76.02 unsaturates 69.01 26.19 25.10 23.52 total lipid (g/L) 930 383 539 475
[0503] In Table 41, Stearate (C18:0) levels greater than the control are highlighted with bold text. Strain Y contains S. cerevisiae SUC2, encoding sucrose invertase, integrated at the 6S locus, and has a fatty acid profile that is indistinguishable from the Strain J wild-type parent.
[0504] Constructs used for KASII overexpression in Strain K-4: DNA construct pSZ2734 was made to overexpress a codon-optimized P. moriformis KASII gene in Strain X. The neoR gene from transposon Tn5, conferring resistance to aminoglycoside antibiotics, was used as a selectable marker for transformation. The sequence of the transforming DNA is provided immediately below. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, BglII, AflII HinDIII and SacI, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the 6S locus. Proceeding in the 5' to 3' direction, the C. reinhardtii TUB2 promoter driving the expression of neoR (encoding aminoglycoside phosphotransferase activity, thereby permitting the strain to grow on G418) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for neoR are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the C. vulgaris NR gene is indicated by small capitals, followed by a spacer region indicated by lowercase text. The P. moriformis SAD2-2 promoter sequence, indicated by boxed text, drives expression of the codon-optimized P. moriformis KASII gene. The region encoding the KASII plastid targeting sequence is indicated by uppercase italics. The sequence that encodes the mature P. moriformis KASII polypeptide is indicated with bold, uppercase italics, while a 3.times. FLAG epitope encoding sequence is in bold, underlined, uppercase italics. A second C. vulgaris NR 3' UTR is indicated by small capitals.
TABLE-US-00048 Nucleotide sequence of the transforming DNA from pSZ2734: (SEQ ID NO: 95) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgct gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagagga- gcatgagggagg actcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgag- gccgcctccaact ggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggtgtatgaattgtacagaa- caaccacgagc cttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccct- cgctgccgccgctt ctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatct- gaggacagtcggg gaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgtt- gtgcgccacccccca caccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagca- gtgcccagccgct ##STR00074## ##STR00075## ##STR00076## ##STR00077## tatcaATGatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgac- tgggcccag cagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagac- cgacctgtccg gcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgcc- gtgctggac gtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacct- ggcccccgc cgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgacc- accaggcca agcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcac- cagggc ctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacgg- cgacgcctg cctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgacc- gctaccagg acatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtac- ggcatcgcc gcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCG- GATAG TATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGT GAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAG TTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGC ATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCC CTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCA ATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcaga ggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgctt- ggttcttcgtccat tagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggt- cgaaacgttcac ##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099## GCAGACCGCCCACCAGCGCCCCCCCACCGAGGGCCACTGCTFCGGCGCCCGCCTGCCCACCGCCTCCC ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## TGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCC GCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTT GTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAA CTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTT GGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCAC GGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaattaagagctcttgttttccagaaggagttgctcctt- gag cctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaa- tgttggttcgtgcgt ctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcg- tacctctgctttc gcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaat- gtggaatcatctgcc ccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacc- tcacaatagttca taacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggt- gcttgcggagggc aggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctcccc- gggatgtgggcc caccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctga- attccttctgccg ctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgt- ggcggggcttgttc gagcttgaagagc
[0505] Overexpression of KASII in Strain X: Construct D1653 derived from pSZ2734 was transformed into Strain X as described previously. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. The resulting fatty acid profiles from representative clones arising from transformation of Strain X with D1653 are summarized in Table 42, below. Overexpression of KASII in the SAD2 knockout/knock-down Strain K-4 background resulted in multiple strains accumulating over 50% C18:0 and with substantially reduced levels of C16:0. We also observed that KASII over-expressing lines had lower overall ratios of saturated to unsaturated fatty acids compared to Strain X.
TABLE-US-00049 TABLE 42 Fatty acid profiles of D1653 [pSZ2734] primary transformants, compared to the Strain X base strain and the wild-type parental strain, Strain J. D165 D165 D165 D165 D165 D165 D165 D165 D165 D165 D165 D165 Strain J K-4 3-89 3-10A 3-2B 3-5B 3-7A 3-75 3-90 3-9B 3-72 3-6B 3-82 3-66 Fatty C12:0 0.04 0.06 0.27 0.13 0.20 0.19 0.24 0.13 0.12 0.27 0.16 0.18 0.25 0.22 Acid C14:0 1.44 1.06 1.55 1.65 1.79 1.67 1.70 1.53 1.50 1.74 1.57 1.64 1.48 1.56 Area C16:0 29.23 29.83 8.16 11.45 10.68 10.11 9.27 11.14 11.08 9.40 9.78 9.95 8.12 8.65 % C16:1 0.88 0.10 0.04 0.00 0.00 0.00 0.00 0.04 0.04 0.00 0.04 0.00 0.05 0.06 C18:0 2.97 40.17 54.25 53.87 53.61 53.46 53.32 53.32 53.15 52.43 52.20 51.23 50.52 50.02 C18:1 58.07 20.15 23.52 22.12 22.20 23.48 24.02 22.73 23.45 23.94 25.21 26.07 28.00 28.29 C18:2 6.25 5.25 6.75 6.05 6.42 6.25 6.56 6.19 5.96 6.88 6.28 6.31 6.59 6.31 C18:3.alpha. 0.50 0.68 0.79 0.88 0.78 0.79 0.79 0.85 0.82 0.86 0.78 0.78 0.78 0.83 C20:0 0.22 1.88 3.21 2.81 3.01 2.91 3.02 2.86 2.77 3.21 2.74 2.80 2.87 2.80 C20:1 0.02 0.07 0.19 0.21 0.34 0.27 0.28 0.12 0.11 0.41 0.14 0.30 0.28 0.26 C22:0 0.05 0.26 0.41 0.34 0.40 0.37 0.37 0.36 0.35 0.42 0.36 0.37 0.36 0.37 C24:0 0.04 0.27 0.49 0.38 0.42 0.41 0.45 0.38 0.36 0.46 0.39 0.37 0.41 0.41 sum C18 67.78 66.24 85.31 82.92 83.01 83.98 84.69 83.09 83.38 84.11 84.47 84.39 85.89 85.45 saturates 33.97 73.52 68.34 70.63 70.11 69.12 68.37 69.72 69.33 67.93 67.20 unsaturates 65.71 26.23 31.29 29.26 29.74 30.79 31.65 29.93 30.38 32.09 32.45
[0506] In Table 42, Stearate (C18:0) levels greater than the wild-type level are highlighted with bold text. Palmitate (C16:0) levels lower than Strain X or J are highlighted with bold. For three strains the ratio of saturated to unsaturated fatty acids is <2:1; these are highlighted with bold, italicized text.
[0507] Stable lines were isolated from the primary transformants shown in Table 42. The fatty acid profiles and lipid titers of shake flask cultures are presented in Table 43, below. The strains accumulated up to 55% C18:0, with as low as 7% C16:0, with comparable lipid titers to the Strain X parent. The saturates:unsaturates ratios were substantially reduced compared to Strain X. Strains AU and AV were selected for evaluation in 3-L high-density fermentations.
TABLE-US-00050 TABLE 43 Shake flask assays of strains derived from D1653, expressing KASII, driven by the PmSAD2-2 promoter, targeted to the 6S locus. 1653- 1653- 1653- 1653- D165 Primary 6B 9B 10A 72 3-89 Strain K-4 5664 AU BM BN BO BP BQ BR AV BS Fatty 10:0 .02 .04 .08 .09 .12 .06 .06 .08 .09 .12 .12 .12 Acid 12:0 .04 .09 .28 .29 .35 .20 .20 .23 .26 .32 .32 .33 Area 14:0 .42 .12 .81 .66 .73 .75 .72 .50 .61 .38 .43 .38 % 16:0 5.59 8.56 .39 .61 .44 .98 0.11 .26 .95 .81 .21 .63 16:1 .03 .10 .06 .05 .06 .06 .06 .04 .04 .03 .03 .03 18:0 .60 0.13 7.60 2.47 5.12 0.25 9.73 4.56 4.01 2.96 3.68 2.12 18:1 2.08 0.74 7.78 3.93 1.31 5.37 5.70 2.86 2.87 4.37 3.99 5.17 18:2 .16 .83 .98 .52 .72 .55 .64 .20 .24 .11 .83 .04 18:3.alpha. .40 .89 .21 .22 .49 .17 .07 .20 .29 .28 .24 .31 20:0 .18 .82 .62 .93 .75 .65 .66 .97 .72 .43 .10 .59 20:1 .04 .13 .37 .36 .39 .34 .34 .35 .34 .48 .41 .47 20:1 .07 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 20:1 .15 .08 .11 .09 .11 .10 .10 .09 .10 .12 .10 .12 22:0 .02 .20 .28 .30 .24 .29 .28 .30 .27 .32 .29 .35 24:0 .00 .03 .16 .29 .00 .03 .15 .16 .02 .05 .04 .07 Sum C18 1.23 7.58 4.57 5.13 5.63 4.34 4.13 5.81 5.40 6.71 6.73 6.63 Saturates 9.86 1.97 8.74 8.05 7.90 Unsaturates 9.91 7.76 1.07 1.73 1.87
[0508] In Table 43, Strain X is the parent strain; Strain J is the wild-type base strain. Stearate (C18:0) levels at least two-fold higher than in the wild-type strain are highlighted in bold. Palmitate levels that are less than in Strain J and Strain K-4 are highlighted bold. Bold italics indicate that the saturates:unsaturates ratio is <2:1.
[0509] The fatty acid profiles and performance metrics of strains AU and AV are detailed in Table 44, below. The fatty acid profile of the parent strain X, grown under the same fermentation conditions, is presented for comparison. The strains that over-express KASII accumulate about 11% more C18:0 than the strain K-4 parent. C16:0 is reduced to 7-9%, and levels of unsaturated fatty acids increase by 4-5%. The lipid titers of Strain AU and AV were comparable to K-4, indicating that KASII over-expression did not have deleterious effects on lipid production.
TABLE-US-00051 TABLE 44 End point fatty acid profiles of biomass from strain X, AU and AV fermentations. Strain K-4 AU AV Fermentation 120580F1 130097F3 130098F4 pH 5 5 5 C14:0 1.27 1.50 1.35 C16:0 28.35 8.88 7.33 C16:1 0.06 0.02 0.03 C18:0 43.67 56.88 57.24 C18:1 17.63 21.57 21.66 C18:2 5.58 6.06 6.94 C18:3.alpha. 0.25 0.29 0.22 C20:0 2.11 3.28 3.46 C22:0 0.31 0.40 0.40 C24:0 0.31 0.37 0.40 sum C18 67.13 84.80 86.06 saturates 76.02 71.31 70.18 unsaturates 23.52 27.94 28.85 total lipid (g/L) 475 529 418
[0510] The fermentations were cultured for 6 days using a fed-batch process. The Strain X fatty acid profile from fermentation 120580F1 was presented in Table 41, and is shown again in Table 44 for comparison with Strains AU and AV. All fermentations were carried out at 32.degree. C., pH 5, 30% dissolved oxygen (DO), 300 mM nitrogen [N], and 557.5 .mu.M iron. The sugar source was 70% sucrose (S70). Stearate (C18:0) levels higher than in the wild-type strain are indicated with bold. Palmitate (C16:0) levels that are less than in the wild-type are highlighted bold.
[0511] Lab scale oils were prepared from biomass derived from the shake flasks and fermentations described above. The TAG compositions of these oils were determined by LC/MS. SOS is the major TAG species in both Strain AU and AV, ranging from 33-35% in the biomass from shake flasks, and reaching 37% in the high-density fermentation biomass. The major palmitate-containing TAGs are substantially reduced, and trisaturate levels are less than half of those observed in Strain X oils. These results demonstrate that KASII over-expression in a high-stearate background significantly improves SOS accumulation, and reduces the accumulation of trisaturated TAGs.
[0512] Constructs used for FATA-1 disruption, KASII over-expression and FAD2 RNAi in Strain J: A DNA construct, pSZ2419, was made to simultaneously disrupt the FATA-1 allele, over-express P. moriformis KASII and express a FAD2 hairpin construct in Strain J. A version of the S. cerevisiae SUC2 gene, encoding sucrose invertase, which was codon-optimized for expression in P. moriformis, was utilized as a selectable marker for transformation. The sequence of the transforming DNA is provided immediately below. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, AscI, MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, AscI, ClaI, BglII, AflII, HinDIII, SacI, SpeI, and XhoI, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the FATA-1 locus. Proceeding in the 5' to 3' direction, the C. reinhardtii TUB2 promoter driving the expression of the S. cerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, thereby permitting the strain to grow on sucrose) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for SUC2 are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the C. vulgaris nitrate reductase (NR) gene is indicated by small capitals, followed by a spacer region indicated by lowercase text. The P. moriformis AMT3 promoter, indicated by lowercase boxed text, drives expression of the P. moriformis KASII gene. The region encoding the plastid targeting peptide from Chlorella protothecoides SAD1 is indicated by uppercase italics. The sequence that encodes the mature P. moriformis KASII polypeptide is indicated with bold, uppercase italics, while a 3.times. FLAG epitope encoding sequence is in bold, underlined, uppercase italics. A second C. vulgaris NR 3' UTR is indicated by small capitals. A second C. reinhardtii TUB2 promoter sequence, indicated by lowercase boxed text, drives expression of the P. moriformis FAD2 hairpin A sequence. The sense and antisense strands are indicated with uppercase, bold italics, and are separated by the FAD2 intron and the first 10 bases of the FAD2 second exon (uppercase italics). A third C. vulgaris NR 3' UTR is indicated by small capitals, followed by a second spacer region that is indicated by lowercase text.
TABLE-US-00052 Nucleotide sequence of the transforming DNA from pSZ2419: (SEQ ID NO: 96) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtc- agcaccgccagcc ggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactggcgt- ctcttccgcttct ctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccgcagcggcc- gctgcccatgcagc gccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacccgcccaagagttt- tgggagcagctt gagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtggaggtccggggctgacc- ggccgtcgcat tcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggccagaaacactgtccattgc- aagggcataggg atgcgttccttcacctctcatttctcatttctgaatccctccctgctcactctttctcctcctccttcccgttc- acgcagcattcggggtacc ##STR00122## ##STR00123## ##STR00124## ##STR00125## ggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcaccc- ccaacaagg gctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaac- ccgaacg acaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagccc- atcgccatc gccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttctt- caacgacacc atcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctccta- cagcctgga cggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccga- aggtcttctg gtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccg- acgacctga agtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatc- gaggtcccc accgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctc- cttcaaccag tacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaa- ggactactac gccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactggga- gtactccgcc ttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggc- caacccggaga cggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccacc- aacaccac gttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacg- ccgtcaac accacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgagga- gtacctccgc atgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaa- cccctacttc accaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggctt- gctggacc agaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaac- gccctgggc tccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAca- attgGCA GCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACT TGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGT ACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTC GTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTG CTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCT GTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgc gtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaat- aaccacctgacg aatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtga- caatgatcggtgg ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138## TTTCTCGGCGTFCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCA ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159## ##STR00160## GTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAG CCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAAT ACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTC CTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGC CTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGA ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## GCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCT ##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173## GCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTT GATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCA TCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAG CGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGT ACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATG GAaagctgtattgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccg- ctctaattgtggagg gggttcgaagacagggtggttggctggatggggaaacgctggtcgcgggattcgatcctgctgcttatatcctc- cctggaagcacac ccacgactctgaagaagaaaacgtgcacacacacaacccaaccggccgaatatttgcttccttatcccgggtcc- aagagagactgc gatgcccccctcaatcagcatcctcctccctgccgcttcaatcttccctgcttgcctgcgcccgcggtgcgccg- tctgcccgcccagtc agtcactcctgcacaggccccttgtgcgcagtgctcctgtaccctttaccgctccttccattctgcgaggcccc- ctattgaatgtattcg ttgcctgtgtggccaagcgggctgctgggcgcgccgccgtcgggcagtgctcggcgactttggcggaagccgat- tgttcttctgtaag ccacgcgcttgctgctttgggaagagaagggggggggtactgaatggatgaggaggagaaggaggggtattggt- attatctgagtt gggtgaagagc
[0513] Identification and analysis of FATA-1 knockout, KASII over-expression and FAD2 RNAi strains: Construct D1358, derived from pSZ2419, was transformed into Strain J as described previously. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. The resulting fatty acid profiles from representative clones arising from transformation of Strain J with D1358 are summarized in Table 45, below. The P. moriformis AMT3 promoter is repressed at pH 5 so the observed phenotypes did not reflect over-expression of P. moriformis KASII. Nevertheless, we observed that multiple strains had substantially reduced levels of C16:0 and 10-15% increases in C18:1, suggesting that the construct had disrupted the FATA-1 target gene, increasing the amount of palmitoyl-ACP available for extension by endogenous KASII. One line, D1358-13, was selected for further analysis. D1358-13 accumulated .about.17% C16:0, .about.75% C18:1 and less than 2% C18:2, indicating that we had successfully integrated at FATA-1 and down-regulated activity of the FAD2 .DELTA..sup.12-desaturase in this strain.
TABLE-US-00053 TABLE 45 Fatty acid profiles of D1358 [pSZ2419] primary transformants, compared to the wild-type parental strain, Strain J. Strain J D1358-13 D1358-19 D1358-11 D1358-9 D1358-30 D1358-28 D1358-6 D1358-8 D1358-10 D1358-3 Fatty C12:0 0.05 0.08 0.06 0.08 0.06 0.07 0.07 0.09 0.07 0.08 0.10 Acid C14:0 1.32 0.79 0.83 0.85 0.87 0.84 0.91 0.86 0.89 0.92 0.60 Area C16:0 26.66 17.43 18.84 20.03 16.27 18.4 19.1 18.18 15.6 16.42 11.24 % C16:1 0.84 0.74 0.79 0.97 0.60 0.77 1.17 0.75 0.56 0.61 0.57 C18:0 3.10 2.87 2.97 2.36 3.20 2.67 2.10 2.82 3.22 3.19 2.30 C18:1 59.07 74.78 69.54 68.78 71.48 69.55 69.02 68.93 70.44 69.64 75.27 C18:2 7.39 1.97 5.47 5.61 6.22 6.31 6.42 6.8 7.68 7.78 8.51 C18:3 .alpha. 0.55 0.23 0.59 0.51 0.26 0.39 0.46 0.38 0.24 0.27 0.24 C20:0 0.24 0.22 0.20 0.13 0.32 0.20 0.03 0.20 0.33 0.31 0.22 C20:1 0.11 0.40 0.29 0.37 0.23 0.33 0.33 0.39 0.36 0.27 0.40 C22:0 0.11 0.09 0.08 0.07 0.09 0.08 0.08 0.08 0.09 0.11 0.11 sum C18 70.11 79.85 78.57 77.26 81.16 78.92 78.00 78.93 81.58 80.88 86.32 saturates 31.48 21.48 22.98 23.52 20.81 22.26 22.29 22.23 20.20 21.03 14.57 unsaturates 67.96 78.12 76.68 76.24 78.79 77.35 77.4 77.25 79.28 78.57 84.99
[0514] In Table 45, Oleate (C18:1) levels greater than the wild-type level are highlighted with bold text. Palmitate (C16:0) levels less than the wild-type are highlighted with bold text. Levels of linoleate (C18:2) reduced by 1% or more compared to the Strain J parent are highlighted with bold text.
[0515] The fatty acid profiles of strains derived from transformant D1358-13 were determined to be stable after more than 60 generations of growth in the absence of selection (growth on sucrose). The performance of selected strains in shake flask assays was then evaluated, and the fatty acid profiles and lipid titers are presented in Table 46, below. Flask experiments were performed at pH 7, enabling activation of the PmAMT3 promoter driving expression of the KASII transgene. The combination of KASII over-expression and FATA-1 knockout leads to further reductions in palmitate levels and enhanced oleate accumulation compared to the phenotypes observed at pH 5 (Table 45). With more than 82% C18:1, less than 11% C16:0, less than 2% C18:2 and .about.83% of the wild-type lipid titer, Strain AA was determined to be the most appropriate strain from this set to serve as a host strain for subsequent modifications to elevate stearate levels. DNA blot analysis showed that S5003 has a simple insertion of construct D1358 [pSZ2419] at the FATA-1 locus.
TABLE-US-00054 TABLE 46 Fatty acid profiles and lipid titers of FATA-1 knockout, KASII over- expressing, FAD2 RNAi lines derived from D1358-13 primary transformants, compared to the wild-type parental strain, Strain J. Primary T389; D1358-13 Strain J AA AB AC AD AE AF AG AH AI AJ AK AL AM Fatty C12:0 0.05 0.08 0.09 0.11 0.19 0.11 0.14 0.10 0.12 0.08 0.11 0.09 0.20 0.20 Acid C14:0 1.34 0.96 0.98 1.03 1.04 0.96 1.02 0.98 1.03 0.98 1.01 1.00 1.03 1.02 Area C16:0 29.69 10.72 10.47 8.90 6.99 9.53 9.27 10.13 8.99 10.76 9.58 10.00 6.64 6.38 % C16:1 0.88 0.42 0.39 0.31 0.29 0.39 0.37 0.41 0.32 0.40 0.35 0.35 0.27 0.27 C18:0 2.78 2.92 3.00 3.16 2.71 2.88 2.85 2.91 3.21 3.03 3.10 3.20 2.77 2.71 C18:1 58.45 82.08 82.24 83.66 85.49 83.28 83.38 82.57 83.51 82.12 83.10 82.63 85.88 86.13 C18:2 5.83 1.89 1.88 1.80 2.01 1.83 1.89 1.89 1.77 1.73 1.75 1.76 1.94 1.96 C18:3.alpha. 0.42 0.23 0.23 0.25 0.35 0.27 0.29 0.27 0.25 0.22 0.24 0.23 0.34 0.36 C20:0 0.17 0.15 0.16 0.17 0.15 0.15 0.16 0.16 0.17 0.14 0.16 0.16 0.15 0.15 C20:1 0.05 0.23 0.24 0.27 0.36 0.28 0.29 0.26 0.27 0.21 0.25 0.24 0.38 0.39 sum C18 67.48 87.12 87.35 88.87 90.56 88.26 88.41 87.64 88.74 87.10 88.19 87.82 90.93 91.16 saturates 34.03 14.83 14.70 13.37 11.08 13.63 13.44 14.28 13.52 14.99 13.96 14.45 10.79 10.46 unsaturates 65.63 84.85 84.98 86.29 88.50 86.05 86.22 85.40 86.12 84.68 85.69 85.21 88.81 89.11 lipid titer 100.0 82.8 81.1 72.8 54.4 68.3 63.7 70.6 72.2 106.9 76.5 77.5 56.7 54.6 (% parent)
[0516] In Table 46, Stearate (C18:1) levels greater than the wild-type level are highlighted with bold text. Palmitate (C16:0) levels lower than the wild-type are highlighted with bold text. Linoleate (C18:2) levels that are lower than the wild-type are indicated with bold text.
[0517] Constructs used for SAD2 knockout/RNAi in S5003: Two DNA constructs, pSZ2283 and pSZ2697, were made to simultaneously disrupt the SAD2-1 allele and express a SAD2 hairpin construct in Strain AA. In each construct, the neoR gene from transposon Tn5, conferring resistance to aminoglycoside antibiotics, was used as a selectable marker for transformation. The sequence of the transforming DNA derived from pSZ2283 is provided immediately below. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the Chlamydomonas reinhardtii TUB2 promoter driving the expression of neoR (encoding aminoglycoside phosphotransferase activity, thereby permitting the strain to grow on G418) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for neoR are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the C. vulgaris NR gene is indicated by small capitals, followed by a spacer region indicated by lowercase text. A second C. reinhardtii TUB2 promoter sequence, indicated by lowercase boxed text, drives expression of the SAD2 hairpin C sequence. The sense and antisense strands are indicated with uppercase, bold italics, and are separated by the P. moriformis FAD2 intron and the first 10 bases of the FAD2 second exon (uppercase italics). A second C. vulgaris NR 3' UTR is indicated by small capitals.
TABLE-US-00055 Nucleotide sequence of the transforming DNA from pSZ2283: (SEQ ID NO: 97) gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgtt- ccgccctcggtgca gcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgc- agagcatggggc agtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgccc- gaccccgagtcgc ccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctg- gtgggggacatg atcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgc- ggccgaccacc cgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctgg- ctgacggggc gcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaac- aacccttattt ##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178## ggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccc- cgtgctgttc gtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccac- cggcgtgc cctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccag- gacctgct gtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggacc- ccgccacctg ccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccagg- acgacctg gacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgagga- cctggtg gtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcgg- ccgcctgggc gtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccga- ccgcttcc tggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGA- caattgGCAG CAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTT GCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGT ACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTC GTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTG CTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCT GTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgc gtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaat- aaccacctgacg aatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtga- caatgatcggtgg ##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186## GTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACC ##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191## GTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAAC AGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGA ATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTG TCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCG CCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGG ATGGGAACACAAATGGAaagctggagctccagccacggcaacaccgcgcgccttgcggccgagcacggcgacaa- gaacc tgagcaagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcctacacgcgcatcgtggacgag- ttcttccgcctc gaccccgagggcgccgtcgccgcctacgccaacatgatgcgcaagcagatcaccatgcccgcgcacctcatgga- cgacatgggcc acggcgaggccaacccgggccgcaacctcttcgccgacttctccgcggtcgccgagaagatcgacgtctacgac- gccgaggactac tgccgcatcctggagcacctcaacgcgcgctggaaggtggacgagcgccaggtcagcggccaggccgccgcgga- ccaggagtac gtcctgggcctgccccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgcgtcgcgcgcag- gcccgtcgcctt ctcctggatctccgggcgcgagatcatggtctagggagcgacgagtgtgcgtgcggggctggcgggagtgggac- gccctcctcgct cctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaacccccc- gatgataggttg cggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagc- agactcctgaag agc
[0518] The sequence of the transforming DNA derived from pSZ2697 is provided immediately below. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' NsiI, SpeI, BamHI, HinDIII, SacII, EcoRV, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, EcoRI and XbaI, respectively. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the SAD2 hairpin C sense and antisense strands are indicated with uppercase, bold italics, and are separated by the P. moriformis FAD2 intron and the first 10 bases of the FAD2 second exon (uppercase italics). The 3' UTR of the C. vulgaris NR gene is indicated by small capitals. The Chlorella sorokiniana Glutamate Dehydrogenase (GDH) promoter, driving the expression of neoR (encoding aminoglycoside phosphotransferase activity, thereby permitting the strain to grow on G418) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for neoR are indicated by uppercase italics, while the coding region is indicated with lowercase italics. A second C. vulgaris NR 3' UTR is indicated by small capitals, followed by a spacer region indicated by lowercase text.
TABLE-US-00056 Nucleotide sequence of the transforming DNA from pSZ2697: (SEQ ID NO: 98) atgcatgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtag- tcctcgacggaa acatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaat- gcaacgcgacacga ccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgt- tttctgggagcagttg ctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccc- taaatcgcaggc gatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcg- gcgcggaaccca gctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgt- atttggcgggcgt gctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggtt- ggcaggcatcgaaa cgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattggg- tgtggcgctcgcgt gcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccga- ctctcccgactagt ##STR00192## ##STR00193## ##STR00194## ##STR00195## GCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCT ##STR00196## ##STR00197## ##STR00198## ##STR00199## ##STR00200## GACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAG TGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACC CCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTA TCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATT CTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAA ##STR00201## ##STR00202## ##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215## ##STR00216## tagaatatcaATGatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggct- acgactggg cccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtg- aagaccgacct gtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcg- ccgccgtgc tggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcc- cacctggcc cccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgcccctt- cgaccaccag gccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgagga- gcacca gggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgaccc- acggcgac gcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggc- cgaccgctac caggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgct- gtacggca tcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCA- GCTCGGA TAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACC TGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGC GAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCT TGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTG CCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACT GCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcg cagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgc- gcttggttcttcg tccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctg- atggtcgaaacg ttcacagcctagggatatcgaattccgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccag- cgaggccccgctg ggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcc- cgagcgcctgga gctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggc- agccgcaggac tttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcc- cgacgagtacttt gtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacgg- cgtgcgcgacg acacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgac- ctgctgaaca agtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggc- atgaacccgca gacggacaacaacccttatttggggttcgtctacacctccttccaggagcgcgccaccaagtatctaga
[0519] Identification and analysis of SAD2 knockout/knockdown strains in the S5003 background: Constructs D1639, derived from pSZ2697, and D1682, derived from pSZ2283, were transformed into Strain AA as described previously. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 7. The resulting fatty acid profiles from representative clones arising from transformation are summarized in Table 47, below. D1639 transformants accumulated up to 56% C18:0, and D1682 transformants accumulated a maximum of about 35% C18:0. Most of the increases in stearate came at the expense of C18:1, indicating that SAD activity was significantly reduced by the SAD2 knockout/RNAi constructs in these strains. C16:0 levels varied from 6% to 14%; C18:2 ranged from 2-5%. Most strains maintained the low C16:0 and C18:2 phenotypes of the Strain AA parent. These fatty acid profiles demonstrate that down-regulating SAD2 expression using knockout/RNAi constructs, in a background with disrupted FATA-1, KASII over-expression and FAD2 RNAi, produces strains with high C18:0, low C16:0 and low C18:2 phenotypes. These strains will be useful for production of high stability, high stearate, high oleic oils, and oils which have high SOS content.
TABLE-US-00057 TABLE 47 Fatty acid profiles of D1639 [pSZ2697] and D1682 [pSZ2283] primary transformants, compared to the wild-type strain, Strain J, and the Strain AA parental base strain. Strain J AA D1682-4 D1682-17 D1682-7 D1682-6 D1639-2 D1639-5 D1639-10 D1639-19 Fatty C12:0 0.04 0.11 0.14 0.10 0.32 0.31 0.00 0.19 0.17 0.00 Acid C14:0 1.29 0.98 1.03 0.94 1.11 1.15 1.64 1.39 1.61 1.02 Area C16:0 27.50 7.75 8.68 10.41 5.70 5.96 7.54 9.90 14.39 12.02 % C16:1 0.71 0.30 0.06 0.07 0.07 0.10 0.00 0.00 0.00 0.00 C18:0 3.28 3.60 35.46 29.92 24.66 22.30 55.96 53.38 43.46 37.30 C18:1 57.80 84.14 48.39 52.49 61.04 63.60 23.70 26.79 32.93 42.81 C18:2 7.90 2.09 2.37 2.36 3.03 2.88 5.09 3.50 3.22 2.79 C18:3 .alpha. 0.57 0.32 0.50 0.65 0.66 0.58 1.59 0.98 1.01 0.85 C20:0 0.28 0.23 2.07 1.87 1.75 1.51 3.04 2.73 2.29 2.22 C20:1 0.18 0.35 0.54 0.49 0.78 0.83 0.37 0.33 0.30 0.40 C22:0 0.06 0.02 0.27 0.27 0.23 0.20 0.43 0.36 0.29 0.29 C24:0 0.09 0.02 0.33 0.26 0.34 0.26 0.64 0.45 0.32 0.31 sum C18 69.55 90.14 86.72 85.42 89.39 89.36 86.34 84.65 80.62 83.75 saturates 32.54 12.70 47.98 43.77 34.11 31.69 69.25 68.40 62.53 53.16 unsaturates 67.16 87.21 51.86 56.06 65.58 67.99 30.75 31.60 37.46 46.85
[0520] In Table 47, Stearate (C18:0) levels greater than the wild-type level are highlighted with bold text. Oleate (C18:1) levels that are higher than in the wild-type are indicated with bold text. Palmitate (C16:0) levels less than the wild-type level are highlighted with bold. Reduced levels of linoleate (C18:2) compared to the wild-type are highlighted with bold text.
[0521] Stable lines were isolated from a number of D1639 and D1682 transformants. Shake flask assays were carried out to evaluate the performance of four lines derived from D1639-5. Fatty acid profiles and relative lipid titers from the biomass are shown in Table 48, below.
TABLE-US-00058 TABLE 48 Shake flask assays of strains derived from D1639-5, expressing SAD2hpC, driven by the CrTUB2 promoter, targeted to the SAD2-1 locus. Primary T530; D1639-5 Strain J AA AW AX AY BL Fatty C10:0 0.01 0.00 0.07 0.08 0.05 0.04 Acid C12:0 0.02 0.11 0.19 0.22 0.25 0.23 Area % C14:0 1.52 1.10 1.35 1.32 1.30 1.43 C16:0 31.61 9.59 9.28 8.44 7.74 9.46 C16:1 1.04 0.34 0.03 0.02 0.01 0.01 C17:0 0.10 0.11 0.10 0.10 0.10 0.09 C18:0 2.98 4.36 53.01 53.52 55.32 52.09 C18:1 54.81 80.84 27.26 27.52 27.42 28.06 C18:2 6.88 2.42 3.55 3.52 2.38 3.45 C18:3.alpha. 0.53 0.33 0.97 1.03 0.82 1.06 C20:0 0.26 0.31 2.88 2.94 3.15 2.72 C20:1 0.05 0.34 0.38 0.38 0.40 0.37 C22:0 0.03 0.06 0.36 0.37 0.39 0.35 C24:0 0.07 0.08 0.53 0.54 0.53 0.60 sum C18 65.19 87.95 84.79 85.58 85.94 84.66 saturates 36.59 15.70 67.76 67.52 68.82 66.99 unsaturates 63.30 84.26 32.19 32.46 31.02 32.95 % wild-type 100.0 70.3 34.8 33.7 31.4 35.3 lipid titer
[0522] In Table 48, Strain AA is the parent strain; Strain J is the wild-type base strain. Stearate (C18:0) levels higher than in the wild-type strain are indicated with bold. Bold text indicates the increased level of oleate (C18:1) in Strain AA compared to the wild-type. Palmitate (C16:0) levels that are less than in the wild-type are highlighted bold. Linoleate (C18:2) levels that are less than in the wild-type are indicated with bold.
[0523] Lab scale oils were prepared from biomass collected from the Strain AW, AX and AY shake flasks. The TAG compositions of these oils were determined by LC/MS, and are shown in FIG. 21. SOS accumulation ranged from 42-47% in these strains. POS was the next most abundant TAG, at 16-17%. Linoleate-containing TAGs were reduced by more than 50% compared to the Strain AU and AV oils, described above. Strain AW,AX, and AY oils contained 12-13% trisaturated TAGs (S-S-S), similar to the amounts that accumulated in the Strain AU and AX oils. Modulation of SAD activity during oil production to prevent overproduction of saturated fatty acids may help to reduce accumulation of trisaturates.
Example 49
Properties of Methyl Oleate from High Oleic Microalgal Oils
[0524] Esterified oils high in methyl oleate are useful in a variety of applications such as cleaning and lubrication of machinery. For some of these applications, high thermal stability is desired. Thermal stability testing was performed on methylated oil prepared from high-oleic and high-stability-high oleic triglyceride oils prepared from heterotrophically grown oleaginous microalgae as described above. The oils were bleached and deodorized prior to methylation. Commercially available soya methyl ester was used as a control.
[0525] High Oleic (HO) oil was prepared from a high oil-yielding strain of Prototheca moriformis transformed with a plasmid that can be described as FatA_Btub:inv:nr::amt03-CwTE2:nr_FatA1. This plasmid was designed to homologously recombine in the FATA1 chromosomal site, thus ablating a FATA acyl-ACP thioesterase chromosomal allele, while expressing an exogenous acyl-ACP thioesterase from Cuphea wrightii (CwTE2, SEQ ID NO: 11) under control of the pH-regulatable amt3 promoter. The CwTE2 gene can be downregulated by cultivation at pH 5 during oil production to further elevate oleate production. Sucrose invertase was also expressed as a selection marker and to allow for cultivation of the strain on sucrose as a sole carbon source. The 3' UTR sequences are from the Chlorella vulgaris nitrate reductase gene. The resulting HO strain is denoted Stain Q. The fatty acid profile of the oil produced by Strain Q is listed below in Table 49.
TABLE-US-00059 TABLE 49 Fatty acid profile of high oleic oil from Strain Q. Fatty Acid Area % C10 0.01 C12:0 0.03 C14:0 0.43 C15:0 0.03 C16:0 7.27 C16:1 iso 0.81 C16:1 0.689 C17:0 0.06 C18:0 1.198 C18:1 80.15 C18:1 iso 0.08 C18:2 8.38 C18:3 ALPHA 0.25 C20:0 0.02 C20:1 0.38 C22:0 0.04 C24:0 0.03
[0526] A high-stability-high-oleic oil (HSAO) was also prepared from a high oil-yielding strain of Prototheca moriformis transformed with a plasmid that can be described as FADc5'_0 Btub:inv:nr::btub-CpSAD_CtOTE:nr_FADc3'. The resulting strain (Strain R) expresses sucrose invertase as a selectable marker and to allow for cultivation on sucrose as a sole carbon source. In addition, a FAD allele (encoding fatty acid desaturase responsible for the conversion of oleate to linoleate) is disrupted and an oleate-specific acyl-ACP thioesterase (Carthamus tinctorius OTE, see example 5) fused to the transit peptide from the SAD gene of Chlorella protothecoides is expressed under control of the beta tubulin promoter. The 3' UTR sequences are from the Chlorella vulgaris nitrate reductase gene. The fatty acid profile of the oil produced by Strain R after heterotrophic cultivation is listed below in Table 50. The fatty acid profile has greater than 85% oleate yet almost none of the major polyunsaturates, linoleic and linolenic acids.
TABLE-US-00060 TABLE 50 Fatty acid profile of high oleic oil from Strain R. Fatty Acid Area % C10 0.02 C12:0 0.07 C14:0 0.09 C15:0 0.05 C16:0 7.28 C16:1 0.70 C17:0 0.08 C18:0 2.15 C18:1 86.32 C20:0 0.30 C20:1 0.46 C22:0 0.08 C23:0 0.01 C24:0 0.06
[0527] The HO and HSAO oils were methylated by known biodiesel production techniques to make methyl-HO and methyl-HSAO esters. These methyl esters where then subjection to thermal testing according to the following procedure:
[0528] 1. Prepare equipment as shown in FIG. 1.
[0529] 2. Add 1 litre of water to test vessel and bring to an active boil on the hotplate.
[0530] 3. To each test product add 50ppm Cobalt (0.083 g of 6% Cobalt Napthenate in 100.0 gram sample) and mix thoroughly.
[0531] 4. Weigh out, in a watch glass, 7.0 g of 100% cotton gauze, (#50 Cheese Cloth).
[0532] 5. Evenly distribute 14.0 g of test product, as prepared in step 3, onto the gauze.
[0533] 6. Place thermocouple (thermometer) through the center of #15 stopper. Wrap cotton around the thermocouple.
[0534] 7. Place wrapped cotton into 24 mesh wire frame cylinder so that it occupies the upper 41/2 inches.
[0535] 8. Position cylinder with wrapped gauze into the 1 L tall form beaker. Secure the beaker in the boiling water and begin recording the temperature increase with time.
[0536] 9. Continue monitoring the temperature for 2 hours or until a 10 degree temperature drop in observed.
[0537] 10. Plot temperature vs time on a graph.
[0538] 11. Any sample which shows a temperature exceeding 100 degrees C. in 1 hour or 200 degrees C. in 2 hours should be regarded as a dangerous oxidation risk or one that is likely to spontaneously combust.
[0539] Results: The HO and HSAO methyl ester did not exhibit auto-oxidation as evidenced by a temperature rise. The control soya methyl ester sample did exhibit the potential for auto-oxidation. The time-temperature profiles are shown in FIG. 18.
[0540] In addition, methylated fatty acid from oil produced by Strain Q was found to have the following characteristics:
[0541] Flash Point (ASTM D93) of 182.degree. C.
[0542] Non-VOC
[0543] Kauri Butanol value (ASTM D1133) of 53.5
[0544] Viscosity at 40.degree. C. (ASTM D445) of 4.57 mm2/s
[0545] Acid Number (ASTM D664) of 0.17 mg KOH/g
[0546] Boiling range distribution (ASTM D2887) 325-362.degree. C.
Example 50
Further Properties of High Oleic (HO) and High-Stability-High-Oleic (HSAO) Microalgal Oils
[0547] The high oleic oil and the high-stability high-oleic algal oils can have the properties shown in FIG. 19 or these values .+-.20% for the measured parameters.
[0548] In one experiment, HSAO microalgal oil showed 512 hour stability measured by OSI at 110.degree. C. (estimated using 130.degree. C. data) with antioxidants of 0.5% phenyl-alpha-naphthylamine (PANA) and 500 ppm ascorbyl palmitate (AP).
Example 51
Production of Low Saturate Oil by Conversion of Palmitic to Palmitoleate
[0549] As described in the examples above, genetic manipulation of microalgae can decrease saturated fat levels, especially by increasing the production of oleic acid. However, in some cases, the acyl-ACP thioesterases expressed in the oleaginous cell liberate more than desirable amounts of palmitate. Here, we describe methods for converting palmitate (16:0) to palmitoleate (16:1) by overexpressing a palmitoyl-ACP desaturase (PAD) gene. The PAD gene can be obtained from natural sources such as Macfadyena unguis (Cat's claw), Macadamia integrifolia (Macadamia nut), Hippophae rhamnoides (sea buckthorn), or by creating a PAD via mutation of a stearoyl-ACP desaturase to have 16:1 activity. The Macfadyena unguis desaturase is denoted (MuPAD).
[0550] A high-oil-producing strain of Prototheca moriformis (Strain Z) is biolistically transformed with plasmid DNA constructs with a PAD gene. For example, one of the high oleic strains described in the Examples 6, 36, or 49 can further comprise an exogenous PAD gene. The constructs comprises sucrose invertase as a selectable marker and either the MuPAD or a SAD gene (e.g., Olea europaea stearoyl-ACP desaturase, GenBank Accession No. AAB67840.1) having the L118W mutation to shift substrate-specificity toward palmitate. See Cahoon, et al., Plant Physiol (1998) 117:593-598. Both the amt3 and beta tubulin (Btub) promoters are used. In addition, the native transit peptide of a plant PAD gene can be swapped with one known to be effective in microalgae (e.g., the transit peptide from the Chlorella vularis SAD gene).
[0551] The PAD gene can be expressed in a variety of strains including those with a FATA knockout or knockdown and/or a KASII knockin to produce high-oleic oil. Optionally, these strains can also produce high-stability (low polyunsaturate) oil by virtue of a FAD (delta 12 fatty acid desaturase) knockout, knockdown, or by placing FAD expression under control of a regulatable promoter and producing oil under conditions that downregulate FAD. In addition, useful base strains for the introduction of PAD gene activities might also include strains possessing KASII knockouts, and FATA Knockins, whereby levels of C16:0 palmitate are elevated.
[0552] As a result, lower levels of palmitic acid are found in the fatty acid profile of the microalgal oil as this is converted into cis-palmitoleic and cis-vaccenic acids. In some cases the total area percent of saturated fatty acids is less than equal to 3.5%, 3% or 2.5%.
[0553] Constructs for over expression of Macfadyena unguis C16:0 desaturase (MuPAD) follow:
[0554] 1) pSZ3142: 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S
[0555] Relevant Restriction Sites in the Construct pSZ3142 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene (conferring the ability of Strain Z to metabolize sucrose) is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous amt03 promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the MuPAD are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The Chlorella protothecoides S106 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00061 Nucleotide sequence of transforming DNA contained in pSZ3142: (SEQ ID NO: 99) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgct gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagagga- gcatgagggag gactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccga- ggccgcctccaa ctggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacag- aacaaccacg agccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgac- cctcgctgccgcc gcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccg- atctgaggacagt cggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgact- tgttgtgcgccac cccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagagg- acagcagtgccc ##STR00217## ##STR00218## ##STR00219## ##STR00220## ##STR00221## gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacg- acgagaag gacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggcca- cgccacgtc cgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccg- gctccatgg tggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctgg- acctacaaca ccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaac- cccgtgctg gccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgc- ggccaagtc ccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacg- agggcttcc tcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtg- atgttcatct ccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttc- gaggccttcga caaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacct- acgggagcg ccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatg- tccctcgtgcgc aagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcct- gaacatca gcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctg- tccaacag caccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcgg- acctctccctc tggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcct- ggaccgcggg aacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaa- gagcgag aacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcga- cgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcga ##STR00222## tggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtg- tttgatcttgtgtgtac gcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcata- tcgcttgcatcccaacc gcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttg- gtttgggctccgcctgt attctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaa- atggaggatccc gcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccaca- ataaccacctga cgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggt- gacaatgatcggt ##STR00223## ##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229## ##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237## agatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgcc- gccacacttgctgc cttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttg- cgagttgctagctgctt gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttat- ctacgctgtcctgcta tccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcct- ggtactgcaacctgta aaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctct- tgttttccagaa ggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcga- atttaaaagcttgg aatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaac- ttgccgctcaaa ccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagt- ctgtaattgcctca gaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtac- agcagaccatt atgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgag- tggccaccccccg gccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccg- cgatgggaag aatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacaca- aatatccttgg catcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgct- ccgagtccgcaaa cccttgtcgcgtggcggggcttgttcgagcttgaagagc
[0556] 2) pSZ3145: 6S::CrTUB2:ScSUC2:CvNR::PmAMT3:MuPAD:CvNR::6S
[0557] Relevant restriction sites in the construct pSZ3145 6S::CrTUB2:ScSUC2:CvNR::PmAMT3: MuPAD:CvNR::6S are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene (conferring the ability of Strain Z to metabolize sucrose) is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous amt03 promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the MuPAD are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00062 Nucleotide sequence of transforming DNA contained in pSZ3145: (SEQ ID NO: 100) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgct gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagagga- gcatgagggag gactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccga- ggccgcctccaa ctggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacag- aacaaccacg agccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgac- ccctcgctgccgcc gcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccg- atctgaggacagt cggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgact- tgttgtgcgccac cccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagagg- acagcagtgccc ##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242## gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacg- acgagaag gacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggcca- cgccacgtc cgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccg- gctccatgg tggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctgg- acctacaaca ccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaac- cccgtgctg gccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgc- ggccaagtc ccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacg- agggcttcc tcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtg- atgttcatct ccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttc- gaggccttcga caaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacct- acgggagcg ccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatg- tccctcgtgcgc aagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcct- gaacatca gcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctg- tccaacag caccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcgg- acctctccctc tggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcct- ggaccgcggg aacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaa- gagcgag aacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcga- cgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcga ##STR00243## tggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtg- tttgatcttgtgtgtac gcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcata- tcgcttgcatcccaacc gcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttg- gtttgggctccgcctgt attctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaa- atggaggatccc gcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccaca- ataaccacctga cgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggt- gacaatgatcggt ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258## cagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgcct- tgacctgtgaatat ccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgct- tgtgctatttgcgaata ccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgcta- tccctcagcgctgctc ctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgta- aaccagcactgcaat gctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctcttgttttccagaagga- gttgctccttga gcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttgga- atgttggttcgtgc gtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccg- cgtacctctgct ttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcaghtctgtaattgcctca- gaatgtggaatcatc tgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagccagaccattatg- ctacctcacaata gttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggcc- ctggtgcttgcgg agggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctc- tccccgggat gtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcg- gccctgaattc cttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaaccc- ttgtcgcgtggcg gggcttgttcgagcttgaagagc
[0558] 3) pSZ3137: 6S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S
[0559] Relevant restriction sites in the construct pSZ3137 6S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene (conferring the ability of Strain Z to metabolize sucrose) is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by C. reinhardtii (3-tubulin promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the MuPAD are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The Chlorella protothecoides S106 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00063 Nucleotide sequence of transforming DNA contained in pSZ3137: (SEQ ID NO: 101) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgct gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagagga- gcatgagggag gactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccga- ggccgcctccaa ctggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacag- aacaaccacg agccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgac- cctcgctgccgcc gcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccg- atctgaggacagt cggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgact- tgttgtgcgccac cccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagagg- acagcagtgccc ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacg- acgagaag gacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggcca- cgccacgtc cgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccg- gctccatgg tggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctgg- acctacaaca ccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaac- cccgtgctg gccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgc- ggccaagtc ccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacg- agggcttcc tcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtg- atgttcatct ccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttc- gaggccttcga caaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacct- acgggagcg ccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatg- tccctcgtgcgc aagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcct- gaacatca gcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctg- tccaacag caccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcgg- acctctccctc tggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcct- ggaccgcggg aacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaa- gagcgag aacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcga- cgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcga ##STR00264## tggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtg- tttgatcttgtgtgtac gcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcata- tcgcttgcatcccaacc gcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttg- gtttgggctccgcctgt attctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaa- atggaggatccc gcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccaca- ataaccacctga cgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggt- gacaatgatcggt ##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269## ##STR00270## agatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgcc- gccacacttgctgc cttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttg- cgagttgctagctgctt gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttat- ctacgctgtcctgcta tccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcct- ggtactgcaacctgta aaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctct- tgttttccagaa ggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcga- atttaaaagcttgg aatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaac- ttgccgctcaaa ccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagt- ctgtaattgcctca gaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtac- agcagaccatt atgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgag- tggccaccccccg gccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccg- cgatgggaag aatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacaca- aatatccttgg catcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgct- ccgagtccgcaaa cccttgtcgcgtggcggggcttgttcgagcttgaagagc
Example 52
Myristate Rich Oil Produced by Overexpressing a Cuphea Palustris Thioesterase
[0560] Here, we demonstrate that over expression of a Cuphea palustris thioesterase (Cpal FATB2, accession AAC49180) in UTEX1435 results in a large increase in C14:0 production (over 60% of the fatty acid profile).
[0561] Constructs used for the overexpression of the Cpal FATB2 gene were codon optimized for expression in P. moriformis as described herein. Cuphea palustris FATB2 is a C14 preferring thioesterase. Two constructs, both encoding the Cpal FATB2 gene, were prepared. The first construct, pSZ2479, can be written as 6SA::CrTUB2-ScSUC2-CvNR:PmAMT3-CpSAD1tpExt-CpalFATB2ExtA-CvNR::6SB. The FatB2 coding sequence is given as SEQ ID NO: 86 and the amino acid sequence is given as SEQ ID NO: 87. The second construct, pSZ2480 can be written as 6SA::CrTUB2-ScSUC2-CvNR:PmAMT3-CpSAD1tpExt-CpalFATB2FLAG ExtA-CvNR::6SB. The nucleic acid sequence and amino acid sequence are given as SEQ ID NO: 88 and SEQ ID NO: 89.
[0562] P. moriformis transformed with pSZ2480 produced high levels of myristic acid. The myristate content was 65.70 percent. This is a very large increase when compared to the myristate content of the wild-type oil produced by the base strain, which has a myristate content of approximately 1%.
[0563] The fatty acid profile of the high myristate strain is shown in the Table 51 below.
TABLE-US-00064 TABLE 51 Fatty acid profile of high myristate strain. Fatty Acid % C10:0 0.04 C12:0 1.19 C14:0 65.7 C16:0 13.55 C18:0 0.57 C18:1 12.2 C18:2 5.13 C20:0 0.05 C22:0 0.01 C24:0 0.01
Example 53
Production of Eicosenoic and Erucic Fatty Acids
[0564] In this example we demonstrate that expression of heterologous fatty acid elongase (FAE), also known as 3-ketoacyl-CoA synthase (KCS), genes from Cramble abyssinica (CaFAE, Accession No: AY793549), Lunaria annua (LaFAE, ACJ61777), and Cardamine graeca (CgFAE, ACJ61778) leads to production of very long chain monounsaturated fatty acids such as eicosenoic (20:1.sup..DELTA.11) and erucic (22:1.sup..DELTA.13) acids in classically mutagenized derivative of UTEX 1435, Strain Z. On the other hand a putative FAE gene from Tropaeolum majus (TmFAE, ABD77097) and two FAE genes from Brassica napus (BnFAE1, AAA96054 and BnFAE2, AAT65206), while resulting in modest increase in eicosenoic (20:1.sup..DELTA.11), produced no detectable erucic acid in STRAIN Z. Interestingly the unsaturated fatty acid profile obtained with heterologous expression of BnFAE1 in STRAIN Z resulted in noticeable increase in Docosadienoic acid (22:2n6). All the genes were codon optimized to reflect UTEX 1435 codon usage. These results suggest that CaFAE, LaFAE or CgFAE genes encode condensing enzymes involved in the biosynthesis of very long-chain utilizing monounsaturated and saturated acyl substrates, with specific capability for improving the eicosenoic and erucic acid content.
[0565] Construct used for the expression of the Cramble abyssinica fatty acid elongase (CaFAE) in P. moriformis (UTEX 1435 strain Z)-[IpSZ3070]: In this example STRAIN Z strains, transformed with the construct pSZ3070, were generated, which express sucrose invertase (allowing for their selection and growth on medium containing sucrose) and C. abyssinica FAE gene. Construct pSZ3070 introduced for expression in STRAIN Z can be written as 6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-CaFAE-Cvnr::6S.
[0566] The sequence of the transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, XbaI, MfeI, BamHI, EcoRI, SpeI, AfIII, SacI, BspQI, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from STRAIN Z that permit targeted integration at the 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the Saccharomyces cerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, thereby permitting the strain to grow on sucrose) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for SUC2 are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The Chlorella vulgaris nitrate reductase (NR) gene 3' UTR is indicated by lowercase underlined text followed by an endogenous AMT3 promoter of P. moriformis, indicated by boxed italicized text. The Initiator ATG and terminator TGA codons of the CaFAE are indicated by uppercase, bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the STRAIN Z 6S genomic region indicated by bold, lowercase text. The final construct was sequenced to ensure correct reading frames and targeting sequences.
TABLE-US-00065 Nucleotide sequence of transforming DNA contained in plasmid pSZ3070: (SEQ ID NO: 102) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgctgatgt ccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatg- agggaggactcctggt ccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctcc- aactggtcctccagca gccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaacgagccttg- tctaggcagaa tccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttc- tcccgcacgcttctttcca gcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactct- gatcagtctaaacccc cttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacct- cctcccagaccaattctgt ##STR00271## ##STR00272## ##STR00273## ##STR00274## ##STR00275## atgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacgg- cctgtggtacgacgag aaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctgggg- ccacgccacgtccgacg acctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctcc- atggtggtggactacaa caacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccc- cggagtccgaggagcagt acatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactcc- acccagttccgcgacccg aaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagat- ctactcctccgacgacctg aagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgat- cgaggtccccaccgagca ggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaacc- agtacttcgtcggcagcttc aacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgca- gaccttcttcaacaccgac ccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaaccc- ctggcgctcctccatgtcc ctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccga- gccgatcctgaacatca gcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctg- tccaacagcaccggca ccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctcc- ctctggttcaagggcctgga ggaccccgaggagtacctccgcatgggcttcgaggtgtccgctcctccttcttcctggaccgcgggaacagcaa- ggtgaagttcgtgaagga gaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactaca- aggtgtacggcttgctgg accagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccggg- aacgccctgggctccgtg ##STR00276## gtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatat- ccctgccgcttttatcaaacagcctc agtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccaccccca- gcatccccttccctcgtttcatatcgc ttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccc- tcgcacagccttggtttgggctccgcc tgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaaca- caaatggaggatcccgcgtctc gaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataacca- cctgacgaatgcgcttggtt cttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgg- agctgatggtcgaaacgttcac ##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288## ##STR00289## ggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttat- caaacagcctcagtgtgtttgatcttg tgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcg- tttcatatcgcttgcatcccaaccgca acttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtt- tgggctccgcctgtattctcctggtac tgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaa- ttaagagctcttgttttccaga aggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcg- aatttaaaagcttggaatg ttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgc- cgctcaaaccgcgtacc tctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattg- cctcagaatgtggaatcatc tgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgc- tacctcacaatagttca taacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggt- gcttgcggagggcaggt caaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccggga- tgtgggcccaccacc agcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattcctt- ctgccgctctgctacccg gtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgt- tcgagcttgaagagc
[0567] Constructs used for the expression of the FAE genes from higher plants in STRAIN Z: In addition to the CaFAE gene (pSZ3070), LaFAE (pSZ3071) from Lunaria annua, CgFAE (pSZ3072) from Cardamine graeca, TmFAE (pSZ3067) Tropaeolum majus and BnFAE1 (pSZ3068) and BnFAE2 (pSZ3069) genes from Brassica napus have been constructed for expression in STRAIN Z. These constructs can be described as:
[0568] pSZ3071--6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-LaFAE-Cvnr::6S
[0569] pSZ3072--6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-CgFAE-Cvnr::6S
[0570] pSZ3067--6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-TmFAE-Cvnr::6S
[0571] pSZ3068--6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-BnFAE1-Cvnr::6S
[0572] pSZ3069--6S::CrTUB2-ScSUC2-Cvnr:PmAmt03-BnFAE2-Cvnr::6S
[0573] All these constructs have the same vector backbone; selectable marker, promoters, and 3' utr as pSZ3070, differing only in the respective FAE genes. Relevant restriction sites in these constructs are also the same as in pSZ3070. The sequences of LaFAE, CgFAE, TmFAE, BnFAE1 and BnFAE2 are shown below. Relevant restriction sites as bold text including SpeI and AfIII are shown 5'-3' respectively.
[0574] To determine their impact on fatty acid profiles, the above constructs containing various heterologous FAE genes, driven by the PmAMT3 promoter, were transformed independently into STRAIN Z.
[0575] Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at pH7.0 (all the plasmids require growth at pH 7.0 to allow for maximal FAE gene expression when driven by the pH regulated PmAMT03 promoter). The resulting profiles from a set of representative clones arising from transformations with pSZ3070, pSZ3071, pSZ3072, pSZ3067, pSZ3068 and pSZ3069 into STRAIN Z are shown in Tables 52-57, respectively, below.
[0576] All the transgenic STRAIN Z strains expressing heterologous FAE genes show an increased accumulation of C20:1 and C22:1 fatty acid (see Tables 52-57). The increase in eicosenoic (20:1.sup..DELTA.11) and erucic (22:1.sup..DELTA.13) acids levels over the wildtype is consistently higher than the wildtype levels. Additionally, the unsaturated fatty acid profile obtained with heterologous expression of BnFAE1 in STRAIN Z resulted in noticeable increase in Docosadienoic acid (C22:2n6). Protein alignment of aforementioned FAE expressed in STRAIN Z is shown in FIG. 23.
TABLE-US-00066 TABLE 52 Unsaturated fatty acid profile in STRAIN Z and representative derivative transgenic lines transformed with pSZ3070 (CaFAE) DNA. Sample ID C18:1 C18:2 C18:3a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 51.49 9.13 0.65 4.35 1.24 0.11 0.00 D1828-20 STRAIN Z; T588; 55.59 7.65 0.50 3.78 0.85 0.00 0.13 D1828-23 STRAIN Z; T588; 54.70 7.64 0.50 3.44 0.85 0.09 0.00 D1828-43 STRAIN Z; T588; 52.43 7.89 0.59 2.72 0.73 0.00 0.00 D1828-12 STRAIN Z; T588; 56.02 7.12 0.52 3.04 0.63 0.10 0.11 D1828-19 Cntrl STRAIN Z 57.99 6.62 0.56 0.19 0.00 0.06 0.05 pH7 Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.00 0.05 0.05 pH5
TABLE-US-00067 TABLE 53 Unsaturated fatty acid profile in STRAIN Z and representative derivative transgenic lines transformed with pSZ3071 (LaFAE) DNA. Sample ID C18:1 C18:2 C18:3 a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 54.66 7.04 0.52 1.82 0.84 0.12 0.09 D1829-36 STRAIN Z; T588; 56.27 6.72 0.51 1.70 0.72 0.09 0.00 D1829-24 STRAIN Z; T588; 56.65 8.36 0.54 2.04 0.67 0.00 0.00 D1829-11 STRAIN Z; T588; 55.57 7.71 0.53 0.10 0.66 0.00 0.00 D1829-35 STRAIN Z; T588; 56.03 7.06 0.54 1.54 0.51 0.06 0.08 D1829-42 Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.00 0.06 0.05 pH7 Cntrl STRAIN Z 57.99 6.62 0.56 0.19 0.00 0.05 0.05 pH5
TABLE-US-00068 TABLE 54 Unsaturated fatty acid profile in STRAIN Z and representative derivative transgenic lines transformed with pSZ3072 (CgFAE) DNA. Sample ID C18:1 C18:2 C18:3 a C20:1 C22:1 C22:2n6 C22:5 STRAIN Z; T588; 57.74 7.79 0.52 1.61 0.25 0.11 0.05 D1830-47 STRAIN Z; T588; 58.06 7.39 0.55 1.64 0.22 0.07 0.06 D1830-16 STRAIN Z; T588; 57.77 6.86 0.51 1.34 0.19 0.09 0.00 D1830-12 STRAIN Z; T588; 58.45 7.54 0.49 1.65 0.19 0.06 0.00 D1830-37 STRAIN Z; T588; 57.10 7.28 0.56 1.43 0.19 0.07 0.00 D1830-44 Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.00 0.06 0.05 pH7 Cntrl STRAIN Z 57.99 6.62 0.56 0.19 0.00 0.05 0.05 pH5
TABLE-US-00069 TABLE 55 Unsaturated fatty acid profile in Strain AR and representative derivative transgenic lines transformed with pSZ3070 (TmFAE) DNA. No detectable Erucic (22:1) acid peaks were reported for these transgenic lines. Sample ID C18:1 C18:2 C18:3 a C20:1 C22:2n6 C22:5 STRAIN Z; T588; 59.97 7.44 0.56 0.57 0.00 0.00 D1825-47 STRAIN Z; T588; 58.77 7.16 0.51 0.50 0.09 0.11 D1825-35 STRAIN Z; T588; 60.40 7.82 0.47 0.44 0.07 0.07 D1825-27 STRAIN Z; T588; 58.07 7.32 0.54 0.41 0.05 0.05 D1825-14 STRAIN Z; T588; 58.66 7.74 0.46 0.39 0.08 0.00 D1825-40 Cntrl STRAIN Z 57.99 6.62 0.56 0.19 0.05 0.05 pH7 Cntrl STRAIN Z 57.70 7.08 0.54 0.11 0.06 0.05 pH5
TABLE-US-00070 TABLE 56 Unsaturated fatty acid profile in STRAIN Z and representative derivative transgenic lines transformed with pSZ3068 (BnFAE1) DNA. No detectable Erucic (22:1) acid peaks were reported for these transgenic lines. Sample ID C18:1 C18:2 C18:3 a C20:1 C22:2n6 C22:5 STRAIN Z; T588; 59.82 7.88 0.55 0.32 0.17 0.10 D1826-30 STRAIN Z; T588; 59.32 8.02 0.58 0.27 0.18 0.07 D1826-23 STRAIN Z; T588; 59.63 7.49 0.55 0.27 0.19 0.08 D1826-45 STRAIN Z; T588; 59.35 7.78 0.57 0.26 0.23 0.00 D1826-24 STRAIN Z; T588; 59.14 7.61 0.57 0.25 0.22 0.05 D1826-34 Cntrl STRAIN Z pH 7 57.81 7.15 0.59 0.19 0.04 0.06 Cntrl STRAIN Z pH 5 58.23 6.70 0.58 0.18 0.05 0.06
TABLE-US-00071 TABLE 57 Unsaturated fatty acid profile in STRAIN Z and representative derivative transgenic lines transformed with pSZ3069 (BnFAE2) DNA. No detectable Erucic (22:1) acid peaks were reported for these transgenic lines. Sample ID C18:1 C18:2 C18:3 a C20:1 C22:2n6 C22:5 STRAIN Z; T588; 60.59 8.20 0.57 0.34 0.00 0.07 D1827-6 STRAIN Z; T588; 59.62 6.44 0.52 0.30 0.07 0.00 D1827-42 STRAIN Z; T588; 59.71 7.99 0.59 0.30 0.06 0.00 D1827-48 STRAIN Z; T588; 60.66 8.21 0.59 0.29 0.04 0.00 D1827-43 STRAIN Z; T588; 60.26 7.99 0.57 0.28 0.04 0.00 D1827-3 Cntrl STRAIN Z pH 7 57.81 7.15 0.59 0.19 0.04 0.06 Cntrl STRAIN Z pH 5 58.23 6.70 0.58 0.18 0.05 0.06
Example 54
Elevating Total Unsaturated Fatty Acids Level by Expressing Heterologous Desaturase Genes
[0577] One of the approaches to generate a "zero SAT FAT" (e.g., total unsaturated fatty acids target at 97% or more/less than or equal to 3% saturated fat) strain is to express desaturase genes in a high oleic strain such as Strain N, which we found to produce about 85% C18:1 with total un-saturates around 93% in multiple fermentation runs. We investigated if the total saturates will be further diminished by expressing desaturase genes in Strain N.
[0578] In the examples below, we demonstrated the ability to reduce stearic and palmitic levels in wild type strain UTEX1435 by over expression of heterologous stearoyl-ACP desaturase genes, including desaturases from Olea europaea, Ricinus communis, and Chlorella protothecoides.
[0579] Construct used for the expression of the Olea europaea stearoyl-ACP desaturase: To introduce the O. europaea stearoyl-ACP desaturase (Accession No: AAB67840.1) into UTEX1435, Strain A, the Saccharomyces cerevisiae invertase gene was utilized as the selectable marker to confer the ability of growing on sucrose media. The construct that has been expressed in UTEX1435, Strain A can be written as 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:OeSAD:CvNR::65B and is termed pSZ1377.
[0580] Relevant restriction sites in the construct pSZ1377 are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the second C. reinhardtii .beta.-tubulin promoter driving the expression of the OeSAD, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the OeSAD are indicated by uppercase, bold italics, while the remainder of the stearoyl-ACP desaturase coding region is indicated by bold italics. The Chlorella protothecoides stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00072 Nucleotide sequence of transforming DNA contained in pSZ 1377: (SEQ ID NO: 108) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgctgatgtcca tcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagg- gaggactcctggtccaggg tcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactgg- tcctccagcagccgcagtcg ccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggc- agaatccctaccagtcat ggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttct- ttccagcaccgtgatggcgcg agccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccc- cttgcgcgttagtgttgcca tcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtc- acctttttggcgaaggcatcgg ##STR00290## ##STR00291## ##STR00292## ##STR00293## ccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaagg- acgccaagtggcacc tgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgac- ctgaccaactgggagg accagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaac- aacacctccggcttcttc aacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagta- catctcctacagcctg gacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgaccc- gaaggtcttctggtacg agccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgac- ctgaagtcctggaagc tggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccacc- gagcaggaccccagca agtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtc- ggcagcttcaacggcaccc acttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttc- aacaccgacccgacctac gggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctc- ctccatgtccctcgtgcgc aagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcct- gaacatcagcaacgc cggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaaca- gcaccggcaccctgga gttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggt- tcaagggcctggaggacc ccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtg- aagttcgtgaaggaga acccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaag- gtgtacggcttgctgga ccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccggga- acgccctgggctccgtg ##STR00294## agtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaata- tccctgccgcttttatcaaacag cctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacc- cccagcatccccttccctcgtttcat atcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcact- gcccctcgcacagccttggtttgg gctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggga- tgggaacacaaatggaggat cccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacacc- acaataaccacctgacgaa tgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgaca- atgatcggtggagctgatggtc ##STR00295## ##STR00296## ##STR00297## ##STR00298## ##STR00299## gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaa- cagcctcagtgtgtttgatcttgtg tgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtt- tcatatcgcttgcatcccaaccgca acttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtt- tgggctccgcctgtattctcctggt actgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt- aattaagagctcttgttttc cagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggaggggg- ttcgaatttaaaagcttggaa tgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaactt- gccgctcaaaccgcgtacct ctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgc- ctcagaatgtggaatcatctgc cccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctac- ctcacaatagttcataaca gtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttg- cggagggcaggtcaaccggc atggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcc- caccaccagcacaacctgc tggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgc- taccccggtgcttctgtccgaa gcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc
[0581] Construct used for the expression of the Ricinus communis stearoyl-ACP desaturase: To introduce the Ricinus communis stearoyl-ACP desaturase (Accession No: AAA74692.1) into UTEX1435, Strain A, the Saccharomyces cerevisiae invertase gene was utilized as the selectable marker to confer the ability of growing on sucrose media. The construct that has been expressed in UTEX1435, Strain A can be written as 6SA::CrTUB2:ScSUC2:CvNR::PmAMT03:CpSADtp:RcSAD:CvNR::6SB and is termed pSZ1454.
[0582] Relevant restriction sites in the construct pSZ1454 are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA that permit targeted integration at 6s nuclear chromosomal locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the endogenous AMT03 promoter driving the expression of the RcSAD, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the RcSAD are indicated by uppercase, bold italics, while the remainder of the stearoyl-ACP desaturase coding region is indicated by bold italics. The Chlorella protothecoides stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00073 Nucleotide sequence of transforming DNA contained in pSZ1454: (SEQ ID NO: 109) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtc gctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaaga- ggagcatga gggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgc- accgaggc cgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatga- attgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgctt cgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgca- gaccggtgag agccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcat- cggcctcggcc ##STR00300## ##STR00301## ##STR00302## ##STR00303## ##STR00304## cgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgacc- ccaacggcc tgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacg- cccttgttctg gggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgact- ccggcgc cttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagc- gctgcgtggcca tctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcacc- gagtaccaga agaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaag- tggatcatgac cgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccg- cgttcgccaa cgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagt- cctactgggt gatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacg- gcacccacttcg aggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacacc- gacccgaccta cgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgct- cctccatgtccc tcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgag- ccgatcctg aacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgt- cgacctgtc caacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgt- tcgcggacctc tccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctcctt- cttcctggaccgc gggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagccctt- caagagcg agaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggc- gacgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcgac ##STR00305## ggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgt- ttgatcttgtgtgtacgcg cttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcg- cttgcatcccaaccgcaac ttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttg- ggctccgcctgtattctcc tggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggagg- atcccgcgtctcg aacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccac- ctgacgaatgcg cttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatga- tcggtggagctgatg ##STR00306## ##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311## ##STR00312## ##STR00313## ##STR00314## ##STR00315## ##STR00316## ##STR00317## ##STR00318## ##STR00319## actgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgttt- gatcttgtgtgtacgcgctt ttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgctt- gcatcccaaccgcaactta tctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggc- tccgcctgtattctcctg gtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagc- ttaattaagagct cttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattg- tggagggggttc gaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggacc- atcagctcca aaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcata- ttgtgacgcttg agcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcg- aggacaccc gccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctcccca- acgagcacct ccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaat- ccccgacc ggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcc- caggcga gcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgctt- ctgtccgaagc aggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc
[0583] Construct used for the expression of the Chlorella protothecoides stearoyl-ACP desaturase: To introduce the Chlorella protothecoides stearoyl-ACP desaturase into UTEX1435, Strain Z, the Saccharomyces cerevisiae invertase gene was utilized as the selectable marker to confer the ability of growing on sucrose media. The construct that has been expressed in UTEX1435, Strain Z can be written as 6SA::CrTUB2:ScSUC2:CvNR::PmAMT03:CpSAD1:CvNR::6SB and is termed pSZ3144.
[0584] Relevant restriction sites in the construct pSZ3144 are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA that permit targeted integration at 6s locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii -tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the endogenous AMT03 promoter driving the expression of the CpSAD1, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the CpSAD1 are indicated by uppercase, bold italics, while the remainder of the stearoyl-ACP desaturase coding region is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6S genomic region indicated by bold, lowercase text.
TABLE-US-00074 Nucleotide sequence of transforming DNA contained in pSZ3144: (SEQ ID NO: 110) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtcgct gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagagga- gcatgagggag gactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccga- ggccgcctccaa ctggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacag- aacaaccacg agccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgac- cctcgctgccgcc gcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccg- atctgaggacagt cggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgact- tgttgtgcgccac cccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagagg- acagcagtgccc ##STR00320## ##STR00321## ##STR00322## ##STR00323## ##STR00324## gacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacg- acgagaag gacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggcca- cgccacgtc cgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccg- gctccatgg tggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctgg- acctacaaca ccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaac- cccgtgctg gccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgc- ggccaagtc ccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacg- agggcttcc tcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtg- atgttcatct ccatcaaccccggcgcccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccactt- cgaggccttcga caaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacct- acgggagcg ccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatg- tccctcgtgcgc aagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcct- gaacatca gcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctg- tccaacag caccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcgg- acctctccctc tggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcct- ggaccgcggg aacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaa- gagcgag aacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcga- cgtcgtgtcc accaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgtt- ctacatcga ##STR00325## tggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtg- tttgatcttgtgtgtac gcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcata- tcgcttgcatcccaacc gcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttg- gtttgggctccgcctgt attctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaa- atggaggatccc gcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccaca- ataaccacctga cgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggt- gacaatgatcggt ##STR00326## ##STR00327## ##STR00328## ##STR00329## ##STR00330## ##STR00331## ##STR00332## ##STR00333## ##STR00334## ##STR00335## ##STR00336## ##STR00337## ##STR00338## ##STR00339## ##STR00340## ccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttg- tgtgtacgcgcttttgc gagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcat- cccaaccgcaacttat ctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggct- ccgcctgtattctcctg gtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagc- ttaattaagag ctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaat- tgtggagggggttcg aatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggacca- tcagctccaaaa aacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattg- tgacgcttgagcag tctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggac- acccgccactcg tacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcac- ctccatgctctga gtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccgga- tcccaccaccc ccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaa- accataccac acaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggt- tgctagggatcgct ccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc
[0585] Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at either pH5.0 or pH7.0, depending on the promoters that drive the expression of the desaturase genes. Transgenic lines arising from the transformations with pSZ1377 (D583) were assayed in (low-nitrogen) lipid production media at pH5.0, because of the nature of the promoters and the fact that P. moriformis produces more lipid at pH5.0. Transgenic lines generated from the transformation of pSZ1454 (D648) and pSZ3144 (D1923) are assayed at pH 7.0 to allow for maximal desaturase gene expression when driven by the pH regulated PmAMT3 promoter. The resulting profiles from representative clones arising from transformations with D583, D648, and D1923 are shown in Tables 58, 59 and 60, respectively, below. The result of expression of OeSAD and CpSAD1 genes is a clear diminution of C18:0 chain lengths with an increase in C18:1. Also we noticed that there is a subtle increase in the level of C16:1, indicating these stearoyl-ACP desaturases may have broad specificity. The transformants resulted from the expression of RcSAD gene also diminishes in the level of C18:0, and elevation in C16:1. Notably, C16:1 could be increased from under 1% to over 1.5% or over 2%. However, there is also a drop in the level of C18:1 fatty acid and increase in C18:2, which may be caused by the growth defect of these transgenic lines.
TABLE-US-00075 TABLE 58 Lipid profile of representative clones arising from transformation with D583 (pSZ1377) DNA Sample ID C16:0 C16:1 C18:0 C18:1 C18:2 D583-25 19.20 1.53 1.15 64.08 11.76 D583-10 21.86 0.99 1.77 61.43 11.42 D583-3 21.94 0.95 1.85 62.22 10.53 D583-33 20.76 0.95 1.85 61.76 12.17 D583-26 20.18 0.92 1.89 62.56 11.97 D583-1 21.28 0.95 1.90 62.63 10.94 S1331 25.48 0.71 3.23 59.70 8.25
TABLE-US-00076 TABLE 59 Lipid profile of representative clones arising from transformation with D648 (pSZ1454) DNA Sample ID C16:0 C16:1 C18:0 C18:1 C18:2 D648-9 26.92 2.30 1.12 54.27 11.30 D648-28 26.54 2.50 1.32 52.58 12.90 D648-15 29.47 1.68 1.48 51.74 11.48 D648-12 27.39 1.41 1.66 54.45 11.58 D648-43 29.74 1.52 1.68 52.59 10.85 D648-7 26.98 1.62 1.69 54.51 11.39 S1331-pH 7 25.86 0.96 2.84 58.33 9.16
TABLE-US-00077 TABLE 60 Lipid profile of representative clones arising from transformation with D1923 (pSZ3144) DNA. Sample ID C14:0 C14.1 C16:0 C16:1 C18:0 C18:1 C18:2 Block 2; E2; pH7; 1.46 0.11 20.74 2.54 0.86 63.99 9.03 STRAIN Z; T613; D1923-2 Block 2; G12; pH7; 1.52 0.10 25.20 1.97 1.67 61.10 7.38 STRAIN Z; T613; D1923-36 Block 2; E8; pH7; 1.48 0.09 26.41 1.78 1.54 60.54 7.01 STRAIN Z; T613; D1923-8 Block 2; F3; 1.50 0.07 25.87 1.75 1.62 61.25 6.94 pH7STRAIN Z; T613; D1923-15 Block 2; F9; pH7; 1.47 0.07 27.02 1.73 1.84 60.15 6.55 STRAIN Z; T613; D1923-21 Block 2; F4; pH7; 1.44 0.07 24.30 1.71 1.41 62.79 7.29 STRAIN Z; T613; D1923-16 pH7 STRAIN Z 1.47 0.00 28.25 0.82 3.16 58.27 6.72
Example 55
Generation of Palmitoleic Acid by Introducing Mutated (L118W) Stearoyl-ACP Desaturases
[0586] To generate lower total saturates (Zero SAT FAT) strains, we have introduced both putative stearoyl-ACP desaturases (SAD) and palmitoyl-ACP desaturase (PAD) genes into Prototheca moriformis. We found that a single amino acid substitution (L118W) in P. moriformis SAD2-1 and Olea europaea SAD resulted in an increase in desaturation of palmitate moieties in the triglycerides produced by the cell. Oils with fatty acid profiles of over 5% palmitoleic acid were produced in the resulting transgenic lines. Therefore, these mutated SADs could be very useful to elevate palmitoleic as a route to lower total saturates, or to obtain palmitoleic acid containing oils. Oils with over 2, 3, 4, and 5 area% palmitoleic were obtained.
[0587] The Saccharomyces cerevisiae invertase gene (Accession no: NP 012104) was utilized as the selectable marker to introduce the Prototheca moriformis stearoyl-ACP desaturase PmSAD2-1 (L118W) and Olea europaea stearoyl-ACP desaturase OeSAD (L118W) into 6S nuclear chromosomal locus of P. moriformis strain Z by homologous recombination using previously described biolistic transformation methods.
[0588] The constructs that have we used to transform Strain Z can be written as:
[0589] 1)6SA::CrTUB2:ScSUC2:CvNR::PmUAPA1: PmSAD2-1(L118W)-CvNR::6SB (pSZ3305, D2066)
[0590] 2) 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2: PmSAD2-1(L118W)-CvNR::6SB (pSZ3299, D2060)
[0591] 3) 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp-OeSAD (L118W)-CvNR::6SB (pSZ3298, D2059)
[0592] Construct pSZ3305: 6SA::CrTUB2:ScSUC2:CvNR::PmUAPA1: PmSAD2-1(L118W)-CvNR::6SB The sequence of the pSZ3305 transforming DNA is provided below. Relevant restriction sites in pSZ3305 6SA::CrTUB2:ScSUC2:CvNR::PmUAPA1: PmSAD2-1(L118W)-CvNR::6SB are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Asc I, Mfe I, EcoRV, SpeI, AscI, ClaI, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent 6SA genomic DNA that permit targeted integration at 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the P. moriformis UAPA1 promoter, indicated by boxed italics text. The initiator ATG and terminator TGA codons of the PmSAD2-1 (L118W) are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6SB genomic region indicated by bold, lowercase text.
TABLE-US-00078 Nucleotide sequence of transforming DNA contained in pSZ3305: (SEQ ID NO: 111) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcgtc gctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaaga- ggagcatga gggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgc- accgaggc cgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatga- attgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgctt cgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgca- gaccggtgag agccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcat- cggcctcggcc ##STR00341## ##STR00342## ##STR00343## ##STR00344## ##STR00345## cctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgacccc- aacggcctg tggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcc- cttgttctgg ggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactc- cggcgcct tctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgc- tgcgtggccatc tggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccga- gtaccagaa gaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagt- ggatcatgacc gcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgc- gttcgccaac gagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtc- ctactgggtg atgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacgg- cacccacttcga ggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccg- acccgacctac gggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctc- ctccatgtccct cgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagc- cgatcctga acatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtc- gacctgtcc aacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgtt- cgcggacctct ccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttc- ttcctggaccgcg ggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttc- aagagcga gaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcg- acgtcgtgtcca ccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttc- tacatcgaca ##STR00346## gactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtt- tgatcttgtgtgtacgcgc ttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgc- ttgcatcccaaccgcaactt atctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttggg- ctccgcctgtattctcct ggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggagga- tcccgcgtctcg aacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccac- ctgacgaatgcg cttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatga- tcggtggagctgatg ##STR00347## ##STR00348## ##STR00349## ##STR00350## ##STR00351## ##STR00352## ##STR00353## ##STR00354## ##STR00355## ##STR00356## ##STR00357## ##STR00358## ##STR00359## tcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccc- tgccgcttttatcaaac agcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaatacca- cccccagcatccccttccct cgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcc- tgctcactgcccctcgca cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcac- gggaagtagtgggatg ggaacacaaatggaaagcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctca- gcctcgataacctc caaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcc- cagacttgtt gctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatct- gccctgttga aatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccct- gtgcgagccca tgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcata- acagtgacc atatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagg- gcaggtcaac cggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtg- ggcccacc accagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattc- cttctgccg ctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgt- ggcggggctt gttcgagcttgaagagc
[0593] Construct pSZ3299: 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2: PmSAD2-1(L118W)-CvNR::6SB The sequence of the pSZ3299 transforming DNA is provided in Sequence 56-2. Relevant restriction sites in pSZ3299 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:PmSAD2-1(L118W)-CvNR::65B are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, XbaI, Mfe I, EcoRV, SpeI, AscI, ClaI, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent 6SA genomic DNA that permit targeted integration at 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the C. reinhardtii .beta.-tubulin promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the PmSAD2-1 (L118W) are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6SB genomic region indicated by bold, lowercase text.
TABLE-US-00079 Nucleotide sequence of transforming DNA contained in pSZ3299: (SEQ ID NO: 112) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcg tcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaa- gaggagcat gagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgcc- gcaccgagg ccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatg- aattgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgct tcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgc- agaccggtg agagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggc- atcggcctc ##STR00360## ggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcaccc- ccaacaagg gctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaac- ccgaacgac accgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccat- cgccatcgc cccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttca- acgacacca tcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctac- agcctggac ggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaa- ggtcttctg gtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccg- acgacctga agtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatc- gaggtcccc accgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctc- cttcaacca gtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggca- aggactact acgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgg- gagtactcc gccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtacca- ggccaaccc ggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcg- ccaccaaca ccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtg- tacgccgtc aacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccga- ggagtacct ccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaagg- agaacccct acttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtac- ggcttgctg gaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgg- gaacgccct gggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagT- GAcaattgg cagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctg- ccttgacct gtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgc- tagctgctt gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttat- ctacgctgt cctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgta- ttctcctgg tactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatc- ccgcgtctc gaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataacca- cctgacgaa tgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgaca- atgatcggt ##STR00361## cgtggccgctcctggccgacgcgctgcctctcgtcctctggtggtgcacgccgtggcctccgaggctcctctgg- gcgtgcctc cctccgtgcagcgcccttctcccgtggtgtactccaagctggacaagcagcaccgcctgacgcctgagcgcctg- gagctggtg cagtccatgggccagttcgccgaggagcgcgtgctgcccgtgctgcaccccgtggacaagctgtggcagcccca- ggacttcct gcccgaccccgagtcccccgacttcgaggaccaggtggccgagctgcgcgcccgcgccaaggacctgcccgacg- agtacttcg tggtgctggtgggcgacatgatcaccgaggaggccctgcccacctacatggccatgctgaacacctgggacggc- gtgcgcgac gacaccggcgccgccgaccacccctgggcccgctggacccgccagtgggtggccgaggagaaccgccacggcga- cctgctgaa caagtactgctggctgaccggccgcgtgaacatgcgcgccgtggaggtgaccatcaacaacctgatcaagtccg- gcatgaacc cccagaccgacaacaacccctacctgggcttcgtgtacacctccttccaggagcgcgccaccaagtactcccac- ggcaacacc gcccgcctggccgccgagcacggcgacaagggcctgtccaagatctgcggcctgatcgcctccgacgagggccg- ccacgagat cgcctacacccgcatcgtggacgagttcttccgcctggaccccgagggcgccgtggccgcctacgccaacatga- tgcgcaagc agatcaccatgcccgcccacctgatggacgacatgggccacggcgaggccaaccccggccgcaacctgttcgcc- gacttctcc gccgtggccgagaagatcgacgtgtacgacgccgaggactactgccgcatcctggagcacctgaacgcccgctg- gaaggtgga cgagcgccaggtgtccggccaggccgccgccgaccaggagtacgtgctgggcctgccccagcgcttccgcaagc- tggccgaga agaccgccgccaagcgcaagcgcgtggcccgccgccccgtggccttctcctggatctccggccgcgagatcatg- gtgTGAatc gatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgtt- gccgccaca cttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacg- cgcttttgc gagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcat- cccaaccgc aacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggt- ttgggctcc gcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatggga- acacaaatg gaaagcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacc- tccaaagcc gctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagactt- gttgctcac tgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctg- ttgaaatcg ccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcg- agcccatgc caggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataaca- gtgaccata tttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggca- ggtcaaccg gcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtggg- cccaccacc agcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattcctt- ctgccgctc tgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggc- ggggcttgt tcgagcttgaagagc
[0594] Construct pSZ3298: 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp-OeSAD(L118W)-CvNR::6SB The sequence of the pSZ3299 transforming DNA is provided below. Relevant restriction sites in the construct pSZ3298 6SA::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp-OeSAD(L118W)-CvNR::6SB are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, XbaI, Mfe I, EcoRV, SpeI, AscI, ClaI, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent 6SA genomic DNA that permit targeted integration at 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the C. reinhardtii .beta.-tubulin promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the OeSAD (L118W) are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The Chlorella protothecoides S106 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6SB genomic region indicated by bold, lowercase text.
TABLE-US-00080 Nucleotide sequence of transforming DNA contained in pSZ3298: (SEQ ID NO: 113) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcg tcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaa- gaggagcat gagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgcc- gcaccgagg ccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatg- aattgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgct tcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgc- agaccggtg agagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggc- atcggcctc ##STR00362## ggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcaccc- ccaacaagg gctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaac- ccgaacgac accgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccat- cgccatcgc cccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttca- acgacacca tcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctac- agcctggac ggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaa- ggtcttctg gtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccg- acgacctga agtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatc- gaggtcccc accgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctc- cttcaacca gtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggca- aggactact acgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgg- gagtactcc gccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtacca- ggccaaccc ggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcg- ccaccaaca ccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtg- tacgccgtc aacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccga- ggagtacct ccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaagg- agaacccct acttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtac- ggcttgctg gaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgg- gaacgccct gggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagT- GAcaattgg cagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctg- ccttgacct gtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgc- tagctgctt gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttat- ctacgctgt cctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgta- ttctcctgg tactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatc- ccgcgtctc gaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataacca- cctgacgaa ##STR00363## tcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccgaggtgcacgtgc- aggtgaccc actccctggcccccgagaagcgcgagatcttcaactccctgaacaactgggcccaggagaacatcctggtgctg- ctgaaggac gtggacaagtgctggcagccctccgacttcctgcccgactccgcctccgagggcttcgacgagcaggtgatgga- gctgcgcaa gcgctgcaaggagatccccgacgactacttcatcgtgctggtgggcgacatgatcaccgaggaggccctgccca- cctaccaga ccatgctgaacacctgggacggcgtgcgcgacgagaccggcgcctccctgaccccctgggccatctggacccgc- gcctggacc gccgaggagaaccgccacggcgacctgctgaacaagtacctgtacctgtccggccgcgtggacatgaagcagat- cgagaagac catccagtacctgatcggctccggcatggacccccgcaccgagaacaacccctacctgggcttcatctacacct- ccttccagg agcgcgccaccttcatctcccacggcaacaccgcccgcctggccaaggagcacggcgacctgaagctggcccag- atctgcggc atcatcgccgccgacgagaagcgccacgagaccgcctacaccaagatcgtggagaagctgttcgagatcgaccc- cgacggcac cgtgctggccctggccgacatgatgcgcaagaaggtgtccatgcccgcccacctgatgtacgacggccaggacg- acaacctgt tcgagaacttctcctccgtggcccagcgcctgggcgtgtacaccgccaaggactacgccgacatcctggagttc- ctggtgggc cgctgggacatcgagaagctgaccggcctgtccggcgagggccgcaaggcccaggactacgtgtgcaccctgcc- cccccgcat ccgccgcctggaggagcgcgcccagtcccgcgtgaagaaggcctccgccacccccttctcctggatcttcggcc- gcgagatca acctgatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaag- TGAatcgat agatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgcc- gccacactt gctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgc- ttttgcgag ttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatccc- aaccgcaac ttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttg- ggctccgcc tgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaaca- caaatggaa agcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctcc- aaagccgct ctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgtt- gctcactgg gaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttg- aaatcgcca ccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagc- ccatgccag gcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtg- accatattt ctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggt- caaccggca tggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggccc- accaccagc acaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctg- ccgctctgc tacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggg- gcttgttcg agcttgaagagc
[0595] Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at pH5.0. The resulting profiles from representative clones arising from transformations with pSZ3305, pSZ3299 and pSZ3298 into Strain Z are shown in Tables 61-63 respectively. Thus, introductions of such mutations or genes can increase levels of palmitoleic acid and decrease levels of saturation in the fatty acid profiles of oils produced by recombinant microalgae. Oils were obtained with C16:1/C16:0 ratios of at least 0.1, 0.15, and 0.18.
TABLE-US-00081 TABLE 61 Fatty acid profiles in Strain Z and derivative transgenic lines transformed with pSZ3305 (D2066). C16:1: C16:0 Sample ID C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 ratio pH5; T657; 1.27 24.73 4.55 3.63 58.62 5.84 0.18 D2066-29 pH5; T657; 1.27 22.89 3.94 3.17 60.69 6.61 0.17 D2066-16 pH5; T657; 1.33 25.47 3.07 3.58 59.32 5.86 0.12 D2066-36 pH5; T657; 1.28 22.48 2.42 3.66 61.65 7.02 0.11 D2066-19 pH5;; T657; 1.29 26.25 2.26 3.99 59.27 5.50 0.09 D2066-12 pH5; T657; 1.33 24.49 2.26 3.24 61.42 6.01 0.09 D2066-21 pH5; Strain Z 1.40 27.70 0.89 3.91 57.34 7.05 0.03 (200:1)
TABLE-US-00082 TABLE 62 Fatty acid profiles in Strain Z and derivative transgenic lines transformed with pSZ3299 (D2060). C16:1: C16:0 Sample ID C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 ratio pH5; T655; D2060-9 1.35 24.67 2.73 3.21 60.34 6.22 0.11 pH5; T655; D2060-23 1.52 30.05 2.64 1.65 55.38 7.03 0.09 pH5; T655; D2060-21 1.29 23.54 2.43 2.94 62.25 6.18 0.10 pH5; T655; D2060-2 1.29 24.30 2.22 2.57 62.09 6.28 0.09 pH5; T655; D2060-12 1.37 27.67 1.90 2.84 59.69 5.41 0.07 pH5; T655; D2060-14 1.41 25.01 1.62 2.47 61.30 6.96 0.06 pH5 Strain Z 1.40 27.89 0.87 3.25 57.84 7.19 0.03
TABLE-US-00083 TABLE 63 Fatty acid profiles in Strain Z and derivative transgenic lines transformed with pSZ3298 (D2059). C16:1: C16:0 Sample ID C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 ratio pH5; T655; D2059-21 1.09 25.44 5.04 1.86 54.78 10.44 0.19 pH5; T655; D2059-19 1.28 23.11 2.71 2.19 60.66 8.64 0.12 pH5; T655; D2059-4 1.68 28.19 1.61 2.54 58.39 6.37 0.06 pH5; T655; D2059-23 1.37 23.25 1.45 2.92 62.15 7.44 0.06 pH5; T655; D2059-1 1.38 23.34 1.28 2.68 62.31 7.62 0.05 pH5 Strain Z 1.40 27.89 0.87 3.25 57.84 7.19 0.03
Example 56
Down Regulation of FATA and Over Expression of the Prototheca Moriformis KETO-Acyl-ACP Synthase II (PMKASII) Gene
[0596] A transgenic P. moriformis line was created with downregulation of an endogenous FATA1 gene combined with overexpression of an endogenous KASII gene. The resulting strain produced a triglyceride-rich oil that was enriched in oleate.
[0597] In the example below, we have followed up on previous work demonstrating that triacylglycerols in algae can be significantly enriched in levels of oleate (C18:1) utilizing molecular genetic approaches, such as down regulating endogenous FATA1 (a single FATA allele) and over-expression of endogenous KASII activity. In this example, we focus our efforts on combining these approaches into a single transgenic line. Constructs that disrupt a single copy of the FATA1 allele while simultaneously overexpressing the P. moriformis KASII gene (PmKASII). were introduced into a high oleic Prototheca moriformis Strain AO. Strain AO was derived from a high 18:1 producing mutant derived from UTEX 1435 using classical mutagenesis techniques. One of the resulting strains, termed Strain AP, produced an oil with a fatty acid profile having 85% C18:1 with total un-saturates around 93% in multiple fermentation runs. The strain AP also had high lipid productivity.
[0598] The Saccharomyces cerevisiae invertase gene (Accession no: NP 012104) was utilized as the selectable marker to introduce the PmKASII into the FATA1 nuclear chromosomal locus of P. moriformis strain AO by homologous recombination using biolistic transformation. To investigate the KASII activity when driven by different promoters, PmKASII was fused to several promoters: PmUAPA1, PmLDH1, and PmAMT3. Note that the integration constructs are all designed as reverse orientation to the FATA1 gene; this was found to give a greater likelihood of stable invertase expression. Therefore, the constructs that have been expressed in Strain AH can be written as:
[0599] 1)FATA1 3'::CrTUB2:ScSUC2:CvNR::PmUAPA1:PmKASII-CvNR::FATA1 5' (pSZ2533)
[0600] 2) FATA1 3'::CrTUB2:ScSUC2:CvNR::PmLDH1:PmKASII-CvNR::FATA1 5' (pSZ2532)
[0601] 3) FATA1 3'::CrTUB2:ScSUC2:CvNR::PmAMT3:PmKASII-CvNR::FATA1 5' (pSZ2750)
[0602] Strain AP is one of the transformants generated from pSZ2533. Relevant restriction sites in the construct pSZ2533 FATA13'::CrTUB2:ScSUC2:CvNR::PmUAPA1:PmKASII-CvNR::FATA1 5' are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Asc I, Mfe I, EcoRV, SpeI, AscI, ClaI, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent FATA1 3' genomic DNA that permit targeted integration at FATA1 locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii .beta.-tubulin promoter driving the expression of the yeast sucrose invertase gene is indicated by boxed text. The initiator ATG and terminator TGA for invertase are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the P. moriformis UAPA1 promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the PmKASII are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics. The Chlorella protothecoides S106 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the FATA1 5' genomic region indicated by bold, lowercase text.
TABLE-US-00084 Nucleotide sequence of transforming DNA contained in pSZ2533: (SEQ ID NO: 114) gctcttcacccaactcagataataccaatacccctccttctcctcctcatccattcagtacccccccccttctc- ttcccaaag cagcaagcgcgtggcttacagaagaacaatcggcttccgccaaagtcgccgagcactgcccgacggcggcgcgc- ccagcagcc cgcttggccacacaggcaacgaatacattcaatagggggcctcgcagaatggaaggagcggtaaagggtacagg- agcactgcg cacaaggggcctgtgcaggagtgactgactgggcgggcagacggcgcaccgcgggcgcaggcaagcagggaaga- ttgaagcgg cagggaggaggatgctgattgaggggggcatcgcagtctctcttggacccgggataaggaagcaaatattcggc- cggttgggt tgtgtgtgtgcacgttttcttcttcagagtcgtgggtgtgcttccagggaggatataagcagcaggatcgaatc- ccgcgacca ##STR00364## catgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacg- gcctgtggt acgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttg- ttctggggc cacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccgg- cgccttctc cggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcg- tggccatct ggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgag- taccagaag aaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtg- gatcatgac cgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccg- cgttcgcca acgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaag- tcctactgg gtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaa- cggcaccca cttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttca- acaccgacc cgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccc- tggcgctcc tccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacct- gaaggccga gccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaaca- gctacaacg tcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctcc- aagtccgtg ttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtc- cgcgtcctc cttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcg- tgaacaacc agcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctg- tacttcaac gacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgac- gggggtgga caacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattggcagcagcagctcggatagtat- cgacacact ctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctttt- atcaaacag cctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacc- cccagcatc cccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgc- tcctgctcc tgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagc- actgcaatg ctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaac- gctgaaggt ctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccat- tagcgaagc gtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgtt- cacagccta ##STR00365## gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccgccgccgccgccgacgccaacc- ccgcccgcc ccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagaccatcgagcagttctactcc- tccctgctg gagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacaccaccaccatcgccggcgagat- caagtccct gcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtgatcaagtacgtgtacatcgccg- gcaagcagg ccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccggcctggaccccgccctgtgcggc- gtgctgatc ggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgacccgcggcggcgtgcgcaagat- gaacccctt ctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatcggcttcatgggccccaact- actccatct ccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgccgcggcgacgccaacgtg- atgctggcc ggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaaggccctgtccaagcgcaa- cgacgagcc cgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagggcgccggcgtgctggtgc- tggaggagc tggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcgccgccacctccgacgcccac- cacatgacc gagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcctggcccccga- gcgcgtggg ctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccgcgccatccgcgccgtgatcc- cccaggact ccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccggcgccgtggaggccgtggcc- gccatccag gccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccggcgtggaccccgtggtgct- ggtgggccc ccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttcggcggccacaactcctgcg- tgatcttcc gcaagtacgacgagatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgac- gacgacaag TGAatcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatgg- actgttgcc gccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtg- tgtacgcgc ttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgc- ttgcatccc aaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagc- cttggtttg ggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggg- atgggaaca caaatggaaagcttaattaagagcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgat- aacctccaa agccgctctaattgtggagggggttcgaaccgaatgctgcgtgaacgggaaggaggaggagaaagagtgagcag- ggagggatt cgtgtcctctgatcatcatgcgattgattacgttgaatgcgacggccggtcagccccggacctccacgcaccgc- agaaatgag aaatgagaggtgaaggaacgcatccctatgcccttgcaatggacagtgtttctggccaccgccaccaagacttg- tgctcctcc aggaagatgcgcttgtcctccgccatcttgcagggctcaagctgctcccaaaactcttgggcgggttccggacg- gacggctac cgcgggtgcggccctgaccgccactgttcggaagcagcggcgctgcatgggcagcggccgctgcggtgcgccac- ggaccgcat gatccaccggaaaagcgcacgcgctggagcgcgcagaggaccacagagaagcggaagagacgccagtactggca- agcaggctg gtcggtgccatggcgcgctactaccctcgctatgactcgggtcctcggccggctggcggtgctgacaattcgtt- tagtggagc agcgactccattcagctaccagtcgaactcagtggcacagtgactccgctcttc
[0603] In addition to the construct pSZ2533, we also investigated the PmKASII activity when the KASII gene driven by other promoters, including PmLDH1, and PmAMT3. The plasmid pSZ2532 can be written as FATA1 3'::CrTUB2:ScSUC2:CvNR::PmLDH1:PmKASII-CvNR::FATA1 5', while the plasmid pSZ2750 can be written as FATA1 3'::CrTUB2:ScSUC2: CvNR::PmAMT3:PmKASII-CvNR::FATA1 5'. Since the sequences of these two plasmids are the same as pSZ2533 except for the promoter that drives the PmKASII, the following sequences only show the sequence of the PmLDH1 and PmAMT3 promoters.
TABLE-US-00085 Nucleotide sequence of PmLDH1 promoter that drive the expression of PmKASII in pSZ2532: (SEQ ID NO: 115) ##STR00366## Nucleotide sequence of PmAMT3 promoter that drive the expression of PmKASII in pSZ2750: (SEQ ID NO: 116) ##STR00367##
[0604] Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at either pH5.0 or pH7.0, depend on the promoters that driven the expression of the PmKASII gene. Transgenic lines arising from the transformations with pSZ2533 (D1636) and pSZ2532 (D1637) were assayed in lipid production media at pH5.0, because of the nature of the promoters and the fact that P. moriformis produces more lipid at pH5.0. Transgenic lines generated from the transformation of pSZ2750 (D1684) were assayed at pH 7.0 to allow for maximal PmKASII gene expression when driven by the pH regulated PmAMT3 promoter. The resulting profiles from representative clones arising from transformations with D1636 (pSZ2533), D1637 (pSZ2532), and D1684 (pSZ2750) are shown in Tables 64-66, respectively.
[0605] The impact of FATA1 knock-out and simultaneously overexpressing the P. moriformis KASII gene is a clear diminution of C16:0 chain lengths with a significant increase in C18:1. At pH5.0, it appears that PmUAPA1 is stronger than PmLDH1, the palmitate level in D1636 transformants is close to 3%, while none of the transformants in D1637 go below 7% at the same condition.
TABLE-US-00086 TABLE 64 Lipid profile of representative clones arising from transformation with D1636 (pSZ2533) DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH 5; T523; D1636-3 0.53 3.31 6.15 79.89 7.19 pH 5; T523; D1636-4 0.48 3.54 5.34 80.78 6.92 pH 5; T523; D1636-5 0.48 3.59 5.41 81.37 6.55 pH 5; T523; D1636-12 0.61 3.59 3.67 80.52 8.93 pH 5; T523; D1636-13 0.55 3.80 4.88 81.83 6.61 pH 5; T523; D1636-21 0.54 4.18 2.82 82.26 8.17 pH 5; Strain AO 0.89 17.28 2.69 70.53 6.86
TABLE-US-00087 TABLE 65 Lipid profile of representative clones arising from transformation with D1637 (pSZ2532) DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH 5; T523; D1637-6 0.46 7.64 3.43 80.08 6.33 pH 5; T523; D1637-12 0.66 8.49 1.90 77.06 9.59 pH 5; T523; D1637-13 0.47 8.59 3.18 79.39 6.54 pH 5; T523; D1637-15 0.60 9.60 2.51 76.41 8.85 pH 5; T523; D1637-7 0.61 11.16 2.21 75.82 8.04 pH 5; T523; D1637-8 0.93 11.29 3.61 74.84 6.61 pH 5; Strain AO 0.89 17.28 2.69 70.53 6.86
TABLE-US-00088 TABLE 66 Lipid profile of representative clones arising from transformation with D1684 (pSZ2750) DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH 7; T532; D1684-14 0.55 5.04 4.90 78.88 8.19 pH 7; T532; D1684-23 0.58 5.80 4.98 77.51 8.69 pH 7; T532; D1684-1 0.59 6.37 4.99 77.47 8.03 pH 7; T532; D1684-24 0.55 6.37 4.83 77.98 7.73 pH 7; T532; D1684-11 0.61 6.61 4.88 76.14 8.96 pH 7; T532; D1684-16 0.57 6.61 5.01 77.74 7.83 pH 7; Strain AO 0.84 20.12 3.52 66.86 6.77
Example 57
Generation of a High-Oleic High-Stability (HOHS) Oil-Producing Strain
[0606] Strain AP of Example 56 produces oil with about 85% oleic acid with total un-saturates around 93%. Here we show that that that the oxidative stability of the high-oleic oil can be improved by knock-down of a delta 12 fatty acid desaturase, thereby reducing linoleic acid production in the oleaginous cell.
[0607] We expressed a hairpin-RNA-producing construct in Strain AP targeting an endogenous FAD gene, PmFAD2. The resulting strains, including Strain AQ, produce >90% C18:1 and <1% C18:2 in fermenters. Most importantly, Strain AQ retains the same level of lipid productivity and sucrose hydrolyzing ability as its parental strain, Strain AP.
[0608] Generation of high oleic high stability oil producing strain AQ: Construct used for down regulating PmFAD2. To generate a strain that produces oil with high oxidative stability, the hairpin PmFAD2 was introduced into AP for down regulating PmFAD2 expression. Strain AQ is a stable line generated from the transformation of pSZ3372 DNA (6SA::PmHXT1:ScarMEL1:CvNR::CrTUB2: Hairpin PmFAD2:CvNR::6SB) into Strain AP. In this construct, the Saccharomyces carlbergensis MEL1 gene was utilized as the selectable marker to introduce the Hairpin PmFAD2 into the 6S nuclear chromosomal locus of P. moriformis strain AQ by homologous recombination using previously described transformation methods (biolistics).
[0609] The sequence of the pSZ3372 transforming DNA is provided below. Relevant restriction sites in pSZ3372 are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, SpeI, Mfe I, BamHI, EcoRV, SpeI, XhoI, SacI, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent 6SA genomic DNA that permits targeted integration at 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene is indicated by boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by the C. reinhardtii .beta.-tubulin promoter, indicated by boxed italics text. The hairpin PmFAD2 cassette includes the P. moriformis FAD2 exon1 (indicated by italics underlined text), the intron of PmFAD2 (italics lowercase text), and followed by the inverted PmFAD2 exon1 (indicated by italics underlined text). The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the 6SB genomic region indicated by bold, lowercase text.
TABLE-US-00089 Nucleotide sequence of transforming DNA contained in pSZ3372: (SEQ ID NO: 117) gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgc- tcgtgcgcg tcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaa- gaggagcat gagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgcc- gcaccgagg ccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatg- aattgtaca gaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagc- tgtccagcg accctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctgg- cgctgcgct tcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgc- agaccggtg agagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggc- atcggcctc ##STR00368## ccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaac- tggaacacg ttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacat- gggctacaa gtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagt- tccccaacg gcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtac- acgtgcgcc ggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaa- gtacgacaa ctgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctga- acaagacgg gccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaac- tcctggcgc atgtccggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactg- caagtacgc cggcttccactgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggct- ggaacgacc tggacaacctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatg- gtgaagtcc cccctgatcatcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcat- cgccatcaa ccaggactccaacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagg- gcgagatcc agatgtggtccggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgcccc- atgaacacg accctggaggagatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacct- gtgggcgaa ccgcgtcgacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgcca- ccgagcagt cctacaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcg- atcctgaac acgaccgtccccgcccacggcatcgcgttctaccgcctgcgcccctcctccTGAcaattggcagcagcagctcg- gatagtatc gacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctg- ccgctttta tcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcga- ataccaccc ccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctc- agcgctgct cctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgt- aaaccagca ctgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgc- agaggaacg ctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttct- tcgtccatt agcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtc- gaaacgttc ##STR00369## cactgtttcgagcgctcggcgcttcgtagcagcatgtacctggcctttgacatcgcggtcatgtccctgctcta- cgtcgcgtc gacgtacatcgaccctgcaccggtgcctacgtgggtcaagtacggcatcatgtggccgctctactggttcttcc- aggtgtgtt tgagggttttggttgcccgtattgaggtcctggtggcgcgcatggaggagaaggcgcctgtcccgctgaccccc- ccggctacc ctcccggcaccttccagggcgcgtacgggaagaaccagtagagcggccacatgatgccgtacttgacccacgta- ggcaccggt gcagggtcgatgtacgtcgacgcgacgtagagcagggacatgaccgcgatgtcaaaggccaggtacatgctgct- acgaagcgc cgagcgctcgaaacagtgcgcggggatggccttgcgcagcgtcccgatcgtgaacggaggcttctccacaggct- gcctgttcg tcttgatagccatctcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggac- tgttgccgc cacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtg- tacgcgctt ttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgctt- gcatcccaa ccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagcct- tggtttggg ctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggat- gggaacaca aatggaaagctgtagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacc- tccaaagcc gctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagactt- gttgctcac tgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctg- ttgaaatcg ccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcg- agcccatgc caggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataaca- gtgaccata tttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggca- ggtcaaccg gcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtggg- cccaccacc agcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattcctt- ctgccgctc tgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggc- ggggcttgt tcgagcttgaagagc
[0610] We introduced the hairpin PmFAD2 construct into strain AP. Transgenic lines arising from the transformations with pSZ3372 (D2082) were assayed in lipid production media at pH5.0, the resulting profiles from representative clones are shown in Table 67. Among more than 400 transformants we had screened, the strain AQ was isolated from the transformant D2082.1, which produced <1% C18:2 during the initial profile screening. Thus, this strain can be used to produce a triglyceride oil that is both high in oleic acid and low in polyunsaturates. Due to the low polyunsaturate levels, the oil is expected to have a high oxidative stability when tested via the AOCS Cd 12b-92 method (see Section IV of this patent application and corresponding examples).
TABLE-US-00090 TABLE 67 Lipid profile of representative clones arising from transformation with D2082 (pSZ3372) DNA. Sample ID C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. SAP_pH 5.0_glucose_day 5-T658; D2082-1 4.42 3.80 89.36 0.65 0.10 StrainAP_pH 5.0_glucose_day 5-T658; D2082-87 3.77 4.01 88.70 1.52 0.19 StrainAP_pH 5.0_glucose_day 5-T658; D2082-93 5.14 3.58 87.63 1.65 0.19 StrainAP_pH 5.0_glucose_day 5-T658; D2082-78 3.74 2.40 89.69 1.97 0.23 StrainAP_pH 5.0_glucose_day 5 4.10 3.77 83.55 6.41 0.40
Example 58
Generating High Oleic "Zero" Linoleic Strains by Knock-Out Prototheca Moriformis (PM) FAD2 AND FATA Genes and Over-Expression of PMKASII Gene
[0611] Triacylglycerols in microalgae can be significantly enriched in levels of oleate (C18:1) utilizing molecular genetic approaches, such as down regulating endogenous FATA1 and FADc genes and over-expression of endogenous KASII activity. In this example, we focus our efforts on combining these approaches into a single transgenic line. Constructs that disrupt a single copy of the FATA1 allele while simultaneously overexpressing the Prototheca moriformis KASII gene were introduced into different .DELTA.fad2 lines, termed Strain R and Strain D (see genealogy in FIG. 24). The resulting strains, such as Strain AS and Strain AZ produces around 90% C18:1 with <0.05% C18:2.
[0612] Strain D and Strain R are .DELTA.fad2 lines that produce oils comprised of 0% C18:2, and between 76% to 87% C18:1, depending upon whether they are grown in shake flasks or high cell density fermentations, respectively. To further elevate oleate levels in Strain D and Strain R, constructs that disrupt a single copy of the FATA1 allele while simultaneously overexpressing the P. moriformis KAS II gene were introduced in Strain D/Strain R via particle bombardment.
[0613] Construct to knock out FATA genes and over expression of PmKASII in S2530 background. Relevant restriction sites in the construct FATA1::CpACT-AtThic-nr:AMT03-S106SAD-PmKASII-nr::FATA1 (termed pSZ2276) are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UTEX1435 that permit targeted integration at FATA1 gene via homologous recombination. Proceeding in the 5' to 3' direction, the actin gene promoter from UTEX 250 driving the expression of the Arabidopsis thaliana THIC gene is indicated by the boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous AMT03 promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the P. moriformis KASII gene are indicated by uppercase, bold italics, while the remainder of the PmKASII coding region is indicated by bold italics. The Chlorella protothecoides UTEX 250 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX1435 FATA1 genomic region indicated by bold, lowercase text.
TABLE-US-00091 Nucleotide sequence of transforming DNA contained in pSZ2276: (SEQ ID NO: 118) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtc- agcaccgcc agccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactg- gcgtctctt ccgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccg- cagcggccg ctgcccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacc- cgcccaaga gttttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtgg- aggtccggg gctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggcc- agaaacact gtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactct- ttctcctcc ##STR00370## tggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtg- gtggtccag gccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa- ctccgagcg cgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgct- tccccaagt ccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac- ctgtccggc ggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct- gcgcaagga gtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcatca- cggaggaga tgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgccatc- atcccctcc aacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaacatcgg- caactccgc cgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacaccatcatgg- acctgtcca cgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtccccatc- taccaggcg ctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgagcaggccga- gcagggcgt ggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgacgggcatcg- tgtcccgcg gcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactgggacgacatc- ctggacatc tgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgccaacgacac- ggcccagtt cgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatgaacgagggcc- ccggccacg tgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcccttctacaccctg- ggccccctg acgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcggcgccctgggcac- cgccctgct gtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctaca- agatcgccg cccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttc- gagttccgc tggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgagacgctgcccgcgga- cggcgcgaa ggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggacatccgcaagtacg- ccgaggaga acggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgaggagttcaacatcgccaag- aagacgatc tccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTG- Acaattggc agcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgc- cttgacctg tgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgattttgcgagttgct- agctgcttg tgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatc- tacgctgtc ctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtat- tctcctggt actgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc- cgcgtctcg aacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccac- ctgacgaat gcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaa- tgatcggtg ##STR00371## gcccgcctgcccaccgcctcccgccgcgccgtgcgccgcgcctggtcccgcatcgcccgcgggcgcgccgccgc- cgccgccga cgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagacca- tcgagcagt tctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacaccacc- accatcgcc ggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtgatcaa- gtacgtgta catcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccggcctgg- accccgccc tgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgacccgc- ggcggcgtg cgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatcgg- cttcatggg ccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgcc- gcggcgacg ccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaag- gccctgtcc aagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgaggg- cgccggcgt gctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcgccg- ccacctccg acgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggagcgc- gcccgcctg gcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccgcgc- catccgcgc cgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccggcg- ccgtggagg ccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccggc- gtggacccc gtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttcgg- cggccacaa ctcctgcgtgatcttccgcaagtacgacgagatggactacaaggaccacgacggcgactacaaggaccacgaca- tcgactaca aggacgacgacgacaagTGAatcgatagatctcttaaggcagcagcagctcggatagtatcgacacactctgga- cgctggtcg tgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctca- gtgtgtttg atcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatcccctt- ccctcgttt catatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctc- actgcccct cgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgat- gcacgggaa gtagtgggatgggaacacaaatggaaagcttaattaagagctcttgttttccagaaggagttgctccttgagcc- tttcattct cagcctcgataacctccaaagccgctctaattgtggagggggttcgaagacagggtggttggctggatggggaa- acgctggtc gcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacaca- cacaaccca accggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctc- cctgccgct tcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggcccct- tgtgcgcag tgctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggcca- agcgggctg ctgggcgcgccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccacgcgctt- gctgctttg ggaagagaagggggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttgggtgaa- gagc
[0614] Construct to knock out FATA genes and over expression of PmKASII in S2532 background. Relevant restriction sites in the construct FATA1::CpACT-AtThic-nr:PmUAPA1-S106SAD-PmKASII-nr::FATA1 (termed pSZ2441) are indicated in lowercase, bold and underlining and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, BamH I, EcoR V, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UTEX1435 that permit targeted integration at FATA1 gene via homologous recombination. Proceeding in the 5' to 3' direction, the actin gene promoter from UTEX 250 driving the expression of the A. thaliana THIC gene is indicated by the boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous UAPA1 promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the P. moriformis KASII gene are indicated by uppercase, bold italics, while the remainder of the PmKASII coding region is indicated by bold italics. The Chlorella protothecoides UTEX 250 stearoyl-ACP desaturase transit peptide is located between initiator ATG and the Asc I site. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX1435 FATA1 genomic region indicated by bold, lowercase text.
TABLE-US-00092 Nucleotide sequence of transforming DNA contained in pSZ2441: (SEQ ID NO: 119) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtc- agcaccgcc agccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactg- gcgtctctt ccgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccg- cagcggccg ctgcccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacc- cgcccaaga gttttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtgg- aggtccggg gctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggcc- agaaacact gtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactct- ttctcctcc ##STR00372## tggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtg- gtggtccag gccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa- ctccgagcg cgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgct- tccccaagt ccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac- ctgtccggc ggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct- gcgcaagga gtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcatca- cggaggaga tgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgccatc- atcccctcc aacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaacatcgg- caactccgc cgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacaccatcatgg- acctgtcca cgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtccccatc- taccaggcg ctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgagcaggccga- gcagggcgt ggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgacgggcatcg- tgtcccgcg gcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactgggacgacatc- ctggacatc tgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgccaacgacac- ggcccagtt cgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatgaacgagggcc- ccggccacg tgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcccttctacaccctg- ggccccctg acgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcggcgccctgggcac- cgccctgct gtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctaca- agatcgccg cccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttc- gagttccgc tggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgagacgctgcccgcgga- cggcgcgaa ggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggacatccgcaagtacg- ccgaggaga acggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgaggagttcaacatcgccaag- aagacgatc tccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTG- Acaattggc agcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgc- cttgacctg tgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgct- agctgcttg tgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatc- tacgctgtc ctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtat- tctcctggt actgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc- cgcgtctcg aacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccac- ctgacgaat gcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaa- tgatcggtg ##STR00373## tgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgc- cgccgccgc cgccgacgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggcc- agaccatcg agcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctac- accaccacc atcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgt- gatcaagta cgtgtacatcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccg- gcctggacc ccgccctgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctg- acccgcggc ggcgtgcgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatgga- catcggctt catgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccaca- tccgccgcg gcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcc- tgcaaggcc ctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatggg- cgagggcgc cggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcg- gcgccgcca cctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctg- gagcgcgcc cgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagta- ccgcgccat ccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcg- ccggcgccg tggaggccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcc- cccggcgtg gaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcgg- cttcggcgg ccacaactcctgcgtgatcttccgcaagtacgacgagatggactacaaggaccacgacggcgactacaaggacc- acgacatcg actacaaggacgacgacgacaagTGAatcgatagatctcttaaggcagcagcagctcggatagtatcgacacac- tctggacgc tggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaaca- gcctcagtg tgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcat- ccccttccc tcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctc- ctgctcact gcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaat- gctgatgca cgggaagtagtgggatgggaacacaaatggaaagcttaattaagagctcttgttttccagaaggagttgctcct- tgagccttt cattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaagacagggtggttggctggat- ggggaaacg ctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtg- cacacacac aacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatc- ctcctccct gccgcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacag- gccccttgt gcgcagtgctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtg- tggccaagc gggctgctgggcgcgccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccac- gcgcttgct gctttgggaagagaagggggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttg- ggtgaagag c
[0615] Southern blot analysis of Strain AS and Strain AZ indicated that both are PmFATA double knock-out mutants. Since the PmFAD2 disruption cassettes contain a Carthamus tinctorius putative oleoyl-specific ACP-thioesterase (CtOTE), the absence of the endogenous FATA genes seems to be fully complemented by the expression of the CtOTE.
[0616] To determine the impact of FATA1 inactivation and over expression of PmKASII gene on lipid composition in .DELTA.fad2 lines Strain D/Strain R, the primary transformants of D1266/Strain D and D1415/Strain R were clonally purified and grown under standard lipid production conditions at both pH5.0 and pH7.0. The resulting profiles from the transgenic line arising from transformation with pSZ2276 into Strain D are shown in Table 68, and transgenic lines arising from transformation with pSZ2441 into Strain R are shown in Table 69.
[0617] As can be seen from Table 68, in Strain AZ at pH7.0, the combination of full activity of PmKASII driven by AMT03 and FATA1 knock results in very low levels of C16:0 (2%). Meanwhile, the Carthamus tinctorius thioesterase is also activated since it is also driven by AMT03 promoter. We observe 7.8% C18:0 when Strain AZ is cultivated at pH7. At pH5.0, decrease of the C16:0 level is largely contributed by the FATA1 inactivation, although PmKASII can be partially activated since we run the seed culture at pH6.8. The stearic level of Strain AZ is low at pH5.0 due to the low expression of the C. tinctorius TE. Overall, the oleic levels of Strain AZ exceed 85% (around 88%) at both pH7.0 and pH5.0.
TABLE-US-00093 TABLE 68 Fatty acid profiles in S1331, S2530 and S4266 at both pH 5.0 and pH 7.0 Strains C16:0 C18:0 C18:1 C18:2 C20:1 Strain A_pH 5 26.6 3.3 60.5 6.7 0.07 Strain A_pH 7 28.3 4.1 58 6.5 0.06 Strain D_pH 5 17 3.6 77.1 0.01 0.14 Strain D_pH 7 19.5 5.3 72.6 0.01 0.09 Strain AZ_pH 5 4.1 2.36 88.5 0.04 3.1 Strain AZ_pH 7 2.1 7.8 87.9 0.01 0.5
[0618] In the transgenic line Strain AS, both CrTUB2 and PmUAPA1 promoters are pH unbiased, hence, as reported in Table 69, the lipid profile at pH5.0 and pH7.0 are essentially same. Relative to Strain AZ, Strain AS produces much less stearic acid. Although the palmitic level in Strain AS is bit higher than that in Strain AZ, the oleic level in Strain AS is above 90%, which is the highest level we observed in the shake flask experiment.
TABLE-US-00094 TABLE 69 Fatty acid profiles in S1331, S2532 and S5204 at both pH 5.0 and pH 7.0 Strains C16:0 C18:0 C18:1 C18:2 Strain A_pH 5 26.6 3.3 60.5 6.7 Strain A_pH 7 28.3 4.1 58 6.5 Strain R_pH 5 23.3 2.1 72.1 0.01 Strain R_pH 7 23.4 2.3 71.9 0.01 Strain AS_pH 5 5.5 1.4 91.5 0.01 Strain AS_pH 7 5.6 1.6 91.3 0.01
Example 59
Complementation of FAD2 and FATA Knockout and KASII Overexpression Generates a Unique Oil with High C18-2 and Low C18-3 Levels
[0619] As described in Example 58, Strain AS was generated by knocking both copies of PmFATA1 in a Prototheca moriformis strain while simultaneously overexpressing PmKASII gene into a .DELTA.fad2 line (Strain R). Strain R is a FAD2 (also known as FADc) knockout strain generated by insertion of a oleate-specific C. tinctorius acyl-ACP thioesterase (GenBank Accession No: AAA33019.1) into a high-lipid producing strain derived from UTEX 1435, under the control of CrTUB2 promoter at the FAD2 locus. Strain AS and its parent, Strain R, have a disrupted endogenous PmFAD2-1 gene resulting in no .DELTA.12 specific desaturase activity manifested as 0% C18:2 (linoleic acid) levels in both nitrogen-rich seed and nitrogen-poor lipid production conditions. Lack of C18:2 in Stain AS (and its parent Strain R) resulted in growth defects which could be partially mitigated by exogenous addition of linoleic acid in the seed stage. However, for industrial applications, exogenous addition of linoleic acid is expensive. Complementation of Strain R (and a second .DELTA.fad2 strain) with PmFAD2-1 restored C18:2 levels back to wild type levels and also resulted in rescued growth characteristics during seed and lipid production without any linoleic supplementation.
[0620] In the present example we demonstrate that:
[0621] In trans expression of fatty acid desaturase-2 gene from Prototheca moriformis (PmFad2-1) under the control of a pH inducible PmAMT3 promoter results in functional complementation of PmFAD2-1 with restored growth and C18:2 levels in .DELTA.fad2, .DELTA.fata1 strain AS;
[0622] Complementation of Strain AS is conditional/inducible and occurs at pH 7.0 when the AMT3 promoter is actively driving the expression of PmFAD2-1 as opposed to pH 5.0 when the AMT3 promoter is inactive; and
[0623] Over expression of PmFAD2-1 at pH 7.0 results in strains with >20% C18:2 levels. The fatty acid profile of these high C18:2 strains mimic canola oil closely except that the new oil has 5 fold less C18:3 than the canola oil (10%). The elevated C18:2 levels are seen only in strains derived from Strain AS overexpressing PmFAD2-1 since overexpression of the same gene in wild-type (i.e., non-engineered) control Strain Z does not result in higher C18:2 levels.
[0624] Construct used for the expression of the Prototheca moriformis fatty acid desaturase 2 (PmFAD2-1) in .DELTA.fad2 strains Strain AS and Strain Z--[pSZ2721]. .DELTA.fad2 .DELTA.fata1 Strain AS and Strain Z were transformed with the construct pSZ2721. The sequence of the transforming DNA is provided below. Relevant restriction sites in the construct pSZ2721 (6S::CpACT-ScMEL1-CvNR::PmAMT3-PmFAD2-1-CvNR::6S) are indicated in lowercase, underlined and bold, and are from 5'-3' BspQ 1, KpnI, Xba I, Mfe I, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UEX 1435 that permits targeted integration of PmFAD2-1 at the 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the actin (ACT) gene promoter from UTEX 250 driving the expression of the Saccharomyces cerevisiae MEL1 gene is indicated by the boxed text. The initiator ATG and terminator TGA for ScMEL1 are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an endogenous AMT03 promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the PmFAD2-1 are indicated by uppercase, bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX 1435 6S genomic region indicated by bold, lowercase text. The final construct was sequenced to ensure correct reading frames and targeting sequences.
TABLE-US-00095 Nucleotide sequence of transforming DNA contained in plasmid pSZ2721: (SEQ ID NO: 120) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtc- agcaccgcc agccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactg- gcgtctctt ccgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccg- cagcggccg ctgcccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacc- cgcccaaga gttttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtgg- aggtccggg gctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggcc- agaaacact gtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactct- ttctcctcc ##STR00374## ccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaac- tggaacacg ttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacat- gggctacaa gtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagt- tccccaacg gcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtac- acgtgcgcc ggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaa- gtacgacaa ctgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctga- acaagacgg gccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaac- tcctggcgc atgtccggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactg- caagtacgc cggcttccactgctccatcatgaacatcctgaacaaggccgccccatgggccagaacgcgggcgtcggcggctg- gaacgacct ggacaacctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatgg- tgaagtccc ccctgatcatcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatc- gccatcaac caggactccaacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccaggg- cgagatcca gatgtggtccggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgcccca- tgaacacga ccctggaggagatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctg- tgggcgaac cgcgtcgacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccac- cgagcagtc ctacaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcga- tcctgaaca cgaccgtccccgcccacggcatcgcgttctaccgcctgcgcccctcctccTGAcaattggcagcagcagctcgg- atagtatcg acacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgc- cgcttttat caaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaa- taccacccc cagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctca- gcgctgctc ctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgta- aaccagcac tgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgca- gaggaacg ctgaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgct- tggttcttc gtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagct- gatggtcga ##STR00375## gcgcaaggccatccccgcccactgcttcgagcgctccgccctgcgctcctccatgtacctggccttcgacatcg- ccgtgatgt ccctgctgtacgtggcctccacctacatcgaccccgcccccgtgcccacctgggtgaagtacggcgtgatgtgg- cccctgtac tggttcttccagggcgccttcggcaccggcgtgtgggtgtgcgcccacgagtgcggccaccaggccttctcctc- ctcccaggc catcaacgacggcgtgggcctggtgttccactccctgctgctggtgccctactactcctggaagcactcccacc- gccgccacc actccaacaccggctgcctggacaaggacgaggtgttcgtgcccccccaccgcgccgtggcccacgagggcctg- gagtgggag gagtggctgcccatccgcatgggcaaggtgctggtgaccctgaccctgggctggcccctgtacctgatgttcaa- cgtggcctc ccgcccctacccccgcttcgccaaccacttcgacccctggtcccccatcttctccaagcgcgagcgcatcgagg- tggtgatct ccgacctggccctggtggccgtgctgtccggcctgtccgtgctgggccgcaccatgggctgggcctggctggtg- aagacctac gtggtgccctacctgatcgtgaacatgtggctggtgctgatcaccctgctgcagcacacccaccccgccctgcc- ccactactt cgagaaggactgggactggctgcgcggcgccatggccaccgtggaccgctccatgggcccccccttcatggaca- acatcctgc accacatctccgacacccacgtgctgcaccacctgttctccaccatcccccactaccacgccgaggaggcctcc- gccgccatc cgccccatcctgggcaagtactaccagtccgactcccgctgggtgggccgcgccctgtgggaggactggcgcga- ctgccgcta cgtggtgcccgacgcccccgaggacgactccgccctgtggttccacaagTAGatcgatagatctcttaaggcag- cagcagctc ggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtg- aatatccct gccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtg- ctatttgcg aataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcct- gctatccct cagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtac- tgcaacctg taaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaagagct- cttgttttc cagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggaggggg- ttcgaagac agggtggttggctggatggggaaacgctgctggtcgcgggattcgatcctgctgcttatatcctccctggaagc- acacccacg actctgaagaagaaaacgtgcacacacacaacccaaccggccgaatatttgcttccttatcccgggtccaagag- agactgcga tgcccccctcaatcagcatcctcctccctgccgcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtc- tgcccgccc agtcagtcactcctgcacaggccccttgtgcgcagtgctcctgtaccctttaccgctccttccattctgcgagg- ccccctatt gaatgtattcgttgcctgtgtggccaagcgggctgctgggcgcgccgccgtcgggcagtgctcggcgactttgg- cggaagccg attgttcttctgtaagccacgcgcttgctgctttgggaagagaagggggggggtactgaatggatgaggaggag- aaggagggg tattggtattatctgagttgggtgaagagc
[0625] To determine its impact on growth and fatty acid profiles, the above construct was transformed independently into a .DELTA.fad2 .DELTA.fata1 Strain AS or wild type Strain Z. Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at pH7.0 (AMT3 promoter active) and pH5.0 (AMT3 promoter inactive) for Strain AS transformants or at pH7.0 for Strain Z transformants. The resulting profiles from a set of representative clones arising from transformations are shown in Tables 70-73 respectively.
[0626] Expression of endogenous PmFad2-1 driven by AMT3 promoter at pH 7.0, in Strain AS resulted in .DELTA.12 specific desaturase activity with complete restoration of C18:2 fatty acid levels of the base strain A (Table 70). No such .DELTA.12 specific desaturase activity and thus no significant C18:2 restoration is detected when the lipid production is run at pH 5.0 when the AMT3 promoter is inactive (Table 71).
[0627] Interestingly, lipid production in complemented Strain AS strains at pH 7.0 results in several strains with 2 fold or more increase in C18:2 levels. The resulting strains produce an oil profile that is similar to Canola oil except that the new oil has less C18:3 levels than the commercially available canola oil (Table 72). The increase in C18:2 is not seen in wild type (Strain Z) strains transformed with the same AMT3 driven PmFAD2-1.
[0628] While we have seen other strains with high C18:2 levels, all of them were associated with growth defects in seed as well as lipid production media. Here, however, we have been able to increase the C18:2 levels in a targeted manner without any detrimental effect on the growth of resulting strains. While .DELTA.fad2 strain R and .DELTA.fad2 .DELTA.fata1 strain AS grow very poorly and hardly reach an OD750 of 10-20 in 42 hours, complemented Strain AS (D1673) lines grow very rapidly in the same time span and reach OD750 between 50-80.
[0629] Thus, it can be seen that we were able to produce cell oils with fatty acid profiles of less than 10% linolenic acid yet >20% linoleic acid (indeed we achieved <2% linolenic acid and >20% linoleic acid). It is surprising that C18:2 levels are elevated only in Strain AS, which has almost 90% C18:1 levels as compared to Strain Z with only 57% C18:1 levels, suggests excess availability of substrate C18:1 in the ER is a key to boost C18:2 levels. Since Prototheca has evolved to utilize C18:1 onto TAGS very efficiently, in wild type situations most likely the substrate leaves the ER very rapidly before being further desaturated by FAD2 enzymes. This limitation may be overcome in strains like Strain AS with very high C18:1 levels that likely stays available for desaturation by PmFAD2-1.
TABLE-US-00096 TABLE 70 Fatty acid profile in representative complemented (D1673) and parent Strain AS lines at pH 7.0 transformed with pSZ2721 (PmFAD2-1) DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3a AS; T533; D1673-16; pH 7.0 0.49 6.33 2.44 66.53 21.36 1.38 AS; T533; D1673-17; pH 7.0 0.44 6.02 2.25 68.97 19.53 1.36 AS; T533; D1673-02; pH 7.0 0.38 5.92 2.30 71.01 17.77 1.30 AS; T533; D1673-03; pH 7.0 0.38 5.83 2.31 71.31 17.45 1.29 AS; T533; D1673-10; pH 7.0 0.38 5.63 2.21 71.72 17.37 1.23 AS; pH 7.0 0.30 5.59 1.63 90.88 0.10 0.00 AT; pH 7.0 1.34 27.99 3.54 55.48 9.07 0.79
TABLE-US-00097 TABLE 71 Fatty acid profile in same representative complemented (D1673) and parent Strain AS lines at pH 5.0 transformed with pSZ2721 (PmFAD2-1) DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3a AS; T533; D1673-16; 0.47 5.16 1.76 90.94 0.06 0.18 pH 5.0 AS; T533; D1673-17; 0.45 4.97 1.72 91.32 0.05 0.00 pH 5.0 AS; T533; D1673-02; 0.46 5.20 1.75 90.94 0.05 0.18 pH 5.0 AS; T533; D1673-03; 0.41 4.93 1.65 89.92 1.56 0.16 pH 5.0 AS; T533; D1673-10; 0.45 4.97 1.69 89.96 1.35 0.16 pH 5.0 AS; pH 5.0 0.39 5.67 1.36 91.13 0.00 0.00 AT; pH 5.0 1.03 24.69 3.30 63.47 5.80 0.38
TABLE-US-00098 TABLE 72 Fatty acid profile of a stable D1673 line along with base strain Z and Canola oil. Sample ID C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. C20:1 pH 5 Strain Z 27.54 3.29 57.91 7.17 0.59 0.10 pH 7 Strain Z 27.92 3.09 58.30 6.71 0.59 0.07 pH 7; AS; T533; D1673.5.2-1 4.43 1.31 70.32 20.30 1.72 0.75 pH 7; AS; T533; D1673.5.2-2 4.55 1.26 67.53 22.17 1.82 1.22 pH 7; AS; T533; D1673.5.2-3 4.34 1.29 69.51 20.78 1.65 1.01 pH 7; AS; T533; D1673.5.2-4 4.81 1.26 68.08 21.53 1.77 1.06 pH 7; AS; T533; D1673.5.2-5 4.61 1.30 68.02 21.57 1.74 1.17 pH 7; AS; T533; D1673.5.2-6 4.36 1.30 68.88 21.16 1.68 1.10 pH 7; AS; T533; D1673.5.2-7 4.38 1.28 69.30 21.08 1.70 0.97 pH 7; AS; T533; D1673.5.2-8 4.87 1.27 68.44 20.87 1.83 1.14 Canola Oil 4.00 2.00 62.00 22.00 10.00 1.00
TABLE-US-00099 TABLE 73 Fatty acid profile in Strain Z at pH 5.0 and pH 7.0 and representative derivative transgenic lines at pH 7.0 transformed with pSZ2721 (PmFAD2-1) DNA. The lines are sorted by C18:2 levels. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3a Z; T573; D1791-23; pH 7.0 1.45 29.96 3.28 54.72 7.99 0.66 Z; T573; D1791-6; pH 7.0 1.73 30.25 2.48 55.01 7.74 0.69 Z; T573; D1791-17; pH 7.0 1.41 29.00 3.42 55.77 7.64 0.68 Z; T573; D1791-14; pH 7.0 1.48 29.82 3.45 55.22 7.56 0.67 Z; T573; D1791-8; pH 7.0 2.30 37.15 2.54 47.62 7.44 0.67 Z; T573; D1791-2; pH 7.0 1.38 29.29 3.45 56.10 7.12 0.63 Z; T573; D1791-10; pH 7.0 1.46 29.30 3.39 56.16 7.11 0.60 Z; T573; D1791-5; pH 7.0 1.45 29.45 3.36 56.15 7.02 0.61 Z; T573; D1791-11; pH 7.0 1.43 29.52 3.44 55.99 7.01 0.60 Z; T573; D1791-13; pH 7.0 1.41 28.96 3.46 56.47 7.01 0.62 Z; pH 7.0 1.41 27.76 3.45 57.71 7.17 0.58 Z; pH 5.0 1.49 28.19 3.27 58.04 6.65 0.57
Example 60
Combinatorial Expression of Mid-Chain Thioesterases and Ketoacyl Synthases to Generate Oils with Highly Elevated and Balanced C10:0 AND C12:0 Fatty Acid Levels
[0630] In this example we describe two molecular approaches to generate oils with highly elevated and balanced C10:0 and C12:0 fatty acids in a classically mutagenized high-oil-yielding derivative of UTEX 1435, Strain BA. Resulting transgenic strains co-express two distinct mid-chain specific thioesterases, the broad specificity C10:0-C14:0 Cuphea wrightii FATB2 thioesterase (expressed in Stain BA), and predominantly C10:0--specific Cuphea hookeriana FATB2 thioesterase (part of incoming vectors). In addition, D1550 transformants express C. wrightii KASIV elongase gene integrated at a neutral genomic site, Thi4b, (vector pSZ2424), while D1681 transformants--C. wrightii KASAI elongase as a part of an endogenous KASI disruption cassette (vector pSZ2746). The use of different KASI activities of plant origin in combination with the exogenous thioesterases resulted in a significant increase in overall C10-C12 levels as well as improved C10:0 specificity of the C. hookeriana thioesterase. The best strain synthesized about 85% total C10:0-C12:0 fatty acids with balanced levels of about 42% C10:0 and ca. 44% C12:0 fatty acids, respectively, less than 4% C14:0, and less than 1.5% C8:0. The results show that selection of FATB and KAS genes can give rise to an oil with at least 50% total saturates with capric and lauric acids balanced to within 20% (or even to within 15%, or 10%).
[0631] Relevant restriction sites in pSZ2424 are indicated in lowercase, bold and underlining text and are 5'-3' Pme I, Kpn I, Xba I, Mfe I, Eco RI, Spe I, Xho I, Hind III, SnaBI, Spe I, Asc I, Xho I, Eco RI, Sac I, BspQ I, respectively. Pme I and BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UTEX1435 that permit targeted integration at Thi4b locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii B-tubulin promoter driving the expression of the neomycin phosphotransferase gene (NeoR, conferring the ability of cells to grow on G418) is indicated by boxed text. The initiator ATG and terminator TGA for NeoR are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text. Next is the Amt03 promoter of Prototheca moriformis indicated by boxed lowercase text driving the expression of Cuphea hookeriana KASIV gene (ChKASIV) indicated in lowercase italics. The initiator ATG and terminator TGA for ChKASIV are indicated by uppercase, bold italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text. Next is the Amt03 promoter of Prototheca moriformis indicated by boxed lowercase text driving the expression of Cuphea hookeriana FATB2 gene (ChFATB2) fused to plastid transit peptide sequence derived from Prototheca moriformis FAD gene indicated in lowercase italics. The initiator ATG and terminator TGA for ChFATB2 are indicated by uppercase, bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX1435 Thi4b flanking sequence.
TABLE-US-00100 Nucleotide sequence of transforming DNA contained in pSZ2424: (SEQ ID NO: 121) gtttaaacccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttggcac- ggctctacacc ttcgagtacgccatgaggcggtgatggctgtggctgtgccccacttcgtccagggacggcaagtccatcatctg- catgcttggt gcgacgctacagcagtccctctgcagcagaggagcacgactttggccatttcacgcactcgagtgtacacaatt- catttttctta aagtaaatgactgctgattgaccagatgctgtaacgctgatttcgctccagatcgcacagtcacagattgcgac- catgttgctg cgtctgaaaatctggattccgaattcgaccctggcgctccatccatgcaacagatggcgacacttgttacaatt- cctgtcgccca tcggcatggagcaggtccacttagatccccgatcacccacgcgcatctcgctaatagtcattcattcgtgtctt- cgatcaaagtc aggtgagtatgcatggatcttggttgacgatgcggtatgggtttgcgccgctgactgcagggtctgtccaaggc- aagccaaccc agctcctctcctcgacaatactctcgcagacaaagccagccacttgccatccagattgccaataaactcaatca- tggcttctgtc atgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgcccc- gcccaccagcc ##STR00376## gactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgct- gttcgtgaaga ccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtg- ccctgcgcc gccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgct- gtcctcccac ctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctg- ccccttcgac caccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctgga- cgagga gcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtgg- tgacccacg gcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggc- gtggccgaccg ctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctgg- tgctgtacg gcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagca- gcagtcggat agtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaata- tccctgccgcttttatca aacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgctatttgcgaataccaccc- ccagcatcccctt ccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctg- ctcctgctcactgcccctc gcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatg- cacgggaagtagtgg ##STR00377## ##STR00378## tgcacctggctggtcgccgcgtgcatgcccacctccagcgacaacgacccccgctcgctgtcccacaagcgcct- gcgcctgagc cgccgccgccgcaccctgagctcgcactgctccctgcgcggcagcaccttccagtgcctggacccctgcaacca- gcagcgcttcc tgggcgacaacggcttcgcgtcgctgttcggctccaagcccctgcgcagcaaccgcggccacctgcgcctgggc- cgcacctcgc actccggcgaggtgatggccgtcgcgatgcagcccgcccaggaggtgagcaccaacaagaagcccgcgaccaag- cagcgcc gcgtggtcgtgaccggcatgggcgtcgtgacccccctgggccacgaccccgacgtgtattataacaacctgctg- gacggcatctc gggcatctccgagatcgagaacttcgactgcagccagttccccacccgcatcgccggcgagatcaagtcgttct- ccaccgacggc tgggtcgcgcccaagttcagcgagcgcatggacaagttcatgctgtatatgctgaccgccggcaagaaggcgct- ggccgacggc ggcatcaccgaggacgcgatgaaggagctgaacaagcgcaagtgcggcgtgctgatcggctcgggcctgggcgg- catgaag gtcttctccgacagcatcgaggccctgcgcacctcgtataagaagatctcccccttctgcgtgcccttcagcac- caccaacatgggc tcggcgatcctggcgatggacctgggctggatgggccccaactattccatcagcaccgcgtgcgccacctcgaa- cttctgcatcct gaacgcggccaaccacatcatcaagggcgaggcggacatgatgctgtgcggcggctccgacgccgcggtgctgc- ccgtcggc ctgggcggcttcgtggcctgccgcgcgctgagccagcgcaacaacgaccccaccaaggcctcgcgcccctggga- ctccaaccg cgacggcttcgtcatgggcgagggcgcgggcgtgctgctgctggaggagctggagcacgccaagaagcgcggcg- cgaccatc tatgccgagttcctgggcggcagcttcacctgcgacgcgtatcacatgaccgagccccaccccgagggcgccgg- cgtcatcctgt gcatcgagaaggcgctggcccagtcgggcgtgtcccgcgaggacgtgaactatatcaacgcgcacgccaccagc- acccccgc gggcgacatcaaggagtatcaggccctggcgcactgcttcggccagaactcggagctgcgcgtcaactccacca- agagcatga tcggccacctgctgggcggcgccggcggcgtggaggcggtcgccgtggtccaggcgatccgcaccggctggatc- caccccaac atcaacctggaggaccccgacgagggcgtggacgccaagctgctggtcggccccaagaaggagaagctgaaggt- gaaggtc ggcctgtcgaactccttcggcttcggcggccacaacagctcgatcctgttcgcgccctgcaacTGActcgaggc- agcagcagct cggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgt- gaatatccctgccgctt ttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgatttgcgagttgctagagcttgtgctatttgcg- aataccacccccagcatc cccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgc- tcctgctcctgctcactgc ccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgc- tgatgcacgggaagta ##STR00379## ##STR00380## gcctccgttcacgatcgggacgctgcgcaaggccatccccgcgcactgtttcgagcgctcggcgcttcgtgggc- gcgcccagctg cccgactggagccgcctgctgaccgccatcaccaccgtgttcgtgaagtccaagcgccccgacatgcacgaccg- caagtccaa gcgccccgacatgctggtggacagcttcggcctggagtccaccgtgcaggacggcctggtgttccgccagtcct- tctccatccgct cctacgagatcggcaccgaccgcaccgccagcatcgagaccctgatgaaccacctgcaggagacctccctgaac- cactgcaa gagcaccggcatcctgctggacggcttcggccgcaccctggagatgtgcaagcgcgacctgatctgggtggtga- ttaagatgca gatcaaggtgaaccgctaccccgcctggggcgacaccgtggagatcaacacccgcttcagccgcctgggcaaga- tcggcatgg gccgcgactggctgatctccgactgcaacaccggcgagatcctggtgcgcgccaccagcgcctacgccatgatg- aaccagaag acccgccgcctgtccaagctgccctacgaggtgcaccaggagatcgtgcccctgttcgtggacagccccgtgat- cgaggactccg acctgaaggtgcacaagttcaaggtgaagaccggcgacagcatccagaagggcctgacccccggctggaacgac- ctggacgt gaaccagcacgtgtccaacgtgaagtacatcggctggatcctggagagcatgcccaccgaggtgctggagaccc- aggagctgt gctccctggccctggagtaccgccgcgagtgcggccgcgactccgtgctggagagcgtgaccgccatggacccc- agcaaggtg ggcgtgcgctcccagtaccagcacctgctgcgcctggaggacggcaccgccatcgtgaacggcgccaccgagtg- gcgcccca agaacgccggcgccaacggcgccatctccaccggcaagaccagcaacggcaactccgtgtccatggactacaag- gaccacg acggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGActcgaggcagcagcagctcgg- atagta tcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccc- tgccgcttttatcaaac agcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaatacca- cccccagcatccccttccct cgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcc- tgctcactgcccctcgca cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcac- gggaagtagtgggatg ggaacacaaatggaaagctgtagaattctccagagctccagcgccatgccacgccttttgatggcttcaagtac- gataacggtgt tggattgtgcgtttgttgcgtagtgtgcatggcttagaataatgcagttggatttcttgctcacggcaatgtcg- gcttgtccgcag gttcaaccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacg- ccgaggtga gctatgtttaggccttgagtgaaaattgtcgtcgaagcatattcgcgctccgcgatagcatccaagcaaaatgt- caagtgcgttc cgatttgcgtccgcaggtcgatgttgtgatcgtcggtgccggatccgccggtctgtcctgcgcttacgagctga- ccaagcacccc gacgtccgggtacgcgagctgagattcgattggacataaactgaaaatgaaatcttttggagaaatgtaagggt- ctcaagcgg tgctcgattgcaagaaattggtcgtcccccactccgcaggtcgccatcatcgagcagggcgttgcacctggtgg- cggcgcctg gctggggggacagctgttctcggccatgtgtgtacgtagaagggtggatttcggatggtttcgttgcacagctg- tttgtcaatga tttgtcttagactattgccgatgtttctaaatgttttaggagctatgatatgtctgcaggcgactgaagagc
[0632] Relevant restriction sites in pSZ2746 are indicated in lowercase, bold and underlining text and are 5'-3' BspQ 1, Kpn I, Xba I, Mfe I, Hind III, AscI, Spe I, Xho I, Eco RI, Nde I, Sna BI, Xho I, Hind III, Sac I, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UTEX1435 that permit targeted integration (and knockout) at the KASI locus via homologous recombination. Proceeding in the 5' to 3' direction, the C. reinhardtii B-tubulin promoter driving the expression of the neomycin phosphotransferase gene (NeoR, conferring the ability of cells to grow on G418) is indicated by boxed text. The initiator ATG and terminator TGA for NeoR are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text. Next is the UAPA1 promoter of Prototheca moriformis indicated by boxed lowercase text driving the expression of Cuphea hookeriana FATB2 gene (ChFATB2) fused to plastid transit peptide sequence derived from Prototheca moriformis FAD gene indicated in lowercase italics. The initiator ATG and terminator TGA for ChFATB2 are indicated by uppercase, bold italics. The B. braunii cd191 3'UTR is indicated by lowercase underlined text. Next is the Amt03 promoter of Prototheca moriformis indicated by boxed lowercase text driving the expression of Cuphea wrightii KASAI gene indicated by lowercase italics fused to Prototheca moriformis SAD1 plastid transit peptide sequence. The C. wrightii KASAI sequence is in lowercase italics and is delineated by initiator ATG and terminator TGA. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX1435 KASI flanking sequence.
TABLE-US-00101 Nucleotide sequence of transforming DNA contained in pSZ2746: (SEQ ID NO: 122) gctcttcgctcaccgcgtgaattgctgtcccaaacgtaagcatcatcgtggctcggtcacgcgatcctggatcc- ggggatccta gaccgctggtggagagcgctgccgtcggattggtggcaagtaagattgcgcaggttggcgaagggagagaccaa- aaccgga ggctggaagcgggcacaacatcgtattattgcgtatagtagagcagtggcagtcgcatttcgaggtccgcaacg- gatctcgca agctcgctacgctcacagtaggagaaaggggaccactgcccctgccagaatggtcgcgaccctctccctcgccg- gccccgcct gcaacacgcagtgcgtatccggcaagcgggctgtcgccttcaaccgcccccatgttggcgtccgggctcgatca- ggtgcgctg aggggggtttggtgtgcccgcgcctctgggcccgtgtcggccgtgcggacgtggggccctgggcagtggatcag- cagggtttg cgtgcaaatgcctataccggcgattgaatagcgatgaacgggatacggttgcgctcactccatgcccatgcgac- cccgtttctg ##STR00381## ggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgc- cgccgtgttcc gcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggac- gaggccgcc cgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcga- ctggctgctg ctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccga- cgccatgcgc cgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcac- ccgcatgga ggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcc- tgaaggcc cgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacgg- ccgcttctcc ggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgc- cgaggagct gggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttct- accgcctgctg gacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatgg- actgttgccgcc acacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgt- acgcgcttttgcgagttgct agctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccg- caacttatctacgctgtcc tgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtatt- ctcctggtactgcaacct ##STR00382## ##STR00383## gggacgctgcgcaaggccatccccgcgcactgtttcgagcgctcggcgcttcgtgggcgcgcccagctgcccga- ctggagccgc ctgctgaccgccatcaccaccgtgttcgtgaagtccaagcgccccgacatgcacgaccgcaagtccaagcgccc- cgacatgctg gtggacagcttcggcctggagtccaccgtgcaggacggcctggtgttccgccagtccttctccatccgctccta- cgagatcggcac cgaccgcaccgccagcatcgagaccctgatgaaccacctgcaggagacctccctgaaccactgcaagagcaccg- gcatcctgc tggacggcttcggccgcaccctggagatgtgcaagcgcgacctgatctgggtggtgattaagatgcagatcaag- gtgaaccgcta ccccgcctggggcgacaccgtggagatcaacacccgcttcagccgcctgggcaagatcggcatgggccgcgact- ggctgatctc cgactgcaacaccggcgagatcctggtgcgcgccaccagcgcctacgccatgatgaaccagaagacccgccgcc- tgtccaag ctgccctacgaggtgcaccaggagatcgtgcccctgttcgtggacagccccgtgatcgaggactccgacctgaa- ggtgcacaag ttcaaggtgaagaccggcgacagcatccagaagggcctgacccccggctggaacgacctggacgtgaaccagca- cgtgtcca acgtgaagtacatcggctggatcctggagagcatgcccaccgaggtgctggagacccaggagctgtgctccctg- gccctggagt accgccgcgagtgcggccgcgactccgtgctggagagcgtgaccgccatggaccccagcaaggtgggcgtgcgc- tcccagtac cagcacctgctgcgcctggaggacggcaccgccatcgtgaacggcgccaccgagtggcgccccaagaacgccgg- cgccaac ggcgccatctccaccggcaagaccagcaacggcaactccgtgtccatggactacaaggaccacgacggcgacta- caaggacc acgacatcgactacaaggacgacgacgacaagTGActcgagagcgtccagcgtgtgggatgaagggtgcgatgg- aacggg gctgccgccccccctctgggcatctagctctgcaccgcacgccaggaagcccaagccaggccccgtcacactcc- ctcgctgaagtg ttccccccctgccccacactcatccaggtatcaacgccatcatgttctacgtccccgtcatcttcaactccctg- gggagcgggcgccgc ##STR00384## ##STR00385## atgtcggcgtgccccgcgatgactggcagggcccctggggcacgtcgctccggacggccagtcgccacccgcct- gaggtacgta ttccagtgcctggtggccagctgcatcgacccctgcgaccagtaccgcagcagcgccagcctgagcttcctggg- cgacaacggct tcgccagcctgttcggcagcaagcccttcatgagcaaccgcggccaccgccgcctgcgccgcgccagccacagc- ggcgaggc catggccgtggccctgcagcccgcccaggaggccggcaccaagaagaagcccgtgatcaagcagcgccgcgtgg- tggtgacc ggcatgggcgtggtgacccccctgggccacgagcccgacgtgttctacaacaacctgctggacggcgtgagcgg- catcagcga gatcgagaccttcgactgcacccagttccccacccgcatcgccggcgagatcaagagcttcagcaccgacggct- gggtggcccc caagctgagcaagcgcatggacaagttcatgctgtacctgctgaccgccggcaagaaggccctggccgacggcg- gcatcaccg acgaggtgatgaaggagctggacaagcgcaagtgcggcgtgctgatcggcagcggcatgggcggcatgaaggtg- ttcaacga cgccatcgaggccctgcgcgtgagctacaagaagatgaaccccttctgcgtgcccttcgccaccaccaacatgg- gcagcgccat gctggccatggacctgggctggatgggccccaactacagcatcagcaccgcctgcgccaccagcaacttctgca- tcctgaacgc cgccaaccacatcatccgcggcgaggccgacatgatgctgtgcggcggcagcgacgccgtgatcatccccatcg- gcctgggcg gcttcgtggcctgccgcgccctgagccagcgcaacagcgaccccaccaaggccagccgcccctgggacagcaac- cgcgacg gcttcgtgatgggcgagggcgccggcgtgctgctgctggaggagctggagcacgccaagaagcgcggcgccacc- atctacgcc gagttcctgggcggcagcttcacctgcgacgcctaccacatgaccgagccccaccccgagggcgccggcgtgat- cctgtgcatc gagaaggccctggcccaggccggcgtgagcaaggaggacgtgaactacatcaacgcccacgccaccagcaccag- cgccgg cgacatcaaggagtaccaggccctggcccgctgcttcggccagaacagcgagctgcgcgtgaacagcaccaaga- gcatgatc ggccacctgctgggcgccgccggcggcgtggaggccgtgaccgtggtgcaggccatccgcaccggctggattca- ccccaacct gaacctggaggaccccgacaaggccgtggacgccaagctgctggtgggccccaagaaggagcgcctgaacgtga- aggtggg cctgagcaacagcttcggcttcggcggccacaacagcagcatcctgttcgccccctgcaacgtgTGActcgagg- cagcagcag ctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacct- gtgaatatccctgccg cttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctat- ttgcgaataccacccccagc atccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgc- tgctcctgctcctgctcact gcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaat- gctgatgcacgggaa gtagtgggatgggaacacaaatggaaagcttgagctccacctgcatccgcctggcgctcgaggacgccggcgtc- tcgcccgac gaggtcaactacgtcaacgcgcacgccacctccaccctggtgggcgacaaggccgaggtgcgcgcggtcaagtc- ggtctttg gcgacatgaagggcatcaagatgaacgccaccaagtccatgatcgggcactgcctgggcgccgccggcggcatg- gaggccg tcgccacgctcatggccatccgcaccggctgggtgcaccccaccatcaaccacgacaaccccatcgccgaggtc- gacggcct ggacgtcgtcgccaacgccaaggcccagcacaaaatcaacgtcgccatctccaactccttcggcttcggcgggc- acaactcc gtcgtcgcctttgcgcccttccgcgagtaggcggagcgagcgcgcttggctgaggagggaggcggggtgcgagc- cctttggct gcgcgcgatactctccccgcacgagcagactccacgcgcctgaatctacttgtcaacgagcaaccgtgtgtttt- gtccgtggcc attcttattatttctccgactgtggccgtactctgtttggctgtgcaagcaccgaagagcc
[0633] Fatty acids profiles from representative shake flask cultures of stable lines derived from D1550 transformants are shown in Table 74. Two independent genetic lineages yielded strains with high and balanced levels of C10-C12:0 fatty acids.
TABLE-US-00102 TABLE 74 Fatty acid profiles in S5050 and derivative transgenic lines generated after transformation with pSZ2424 DNA. Fatty Acid (area %) Strain C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Total Saturates Strain BA 0.32 14.96 43.84 16.60 10.49 0.54 9.64 2.49 86.75 D1550-29.C4.A2 4.02 32.82 40.98 7.37 5.06 0.40 5.61 2.37 90.65 D1550-29.C4.A3 4.50 33.93 40.23 7.09 4.91 0.36 5.38 2.30 91.02 D1550-29.C4.A4 3.57 34.31 41.04 6.86 4.90 0.36 4.98 2.56 91.04 D1550-29.C4.A5 4.66 34.23 39.68 6.96 4.90 0.36 5.55 2.32 90.79 D1550-29.C6.E2 3.59 35.44 40.49 6.32 4.74 0.34 4.94 2.63 90.92 D1550-29.C6.E3 3.60 35.55 40.90 6.33 4.67 0.34 4.66 2.52 91.39 D1550-29.C6.E4 BB 3.97 35.85 40.23 6.26 4.65 0.34 4.83 2.51 91.30 D1550-29.C6.E5 4.02 35.19 39.89 6.59 4.79 0.34 5.12 2.60 90.82 D1550-29-1.14 3.30 39.62 40.04 5.16 4.04 0.30 3.49 2.67 92.46 D1550-29-1.2 3.12 39.50 40.22 5.13 3.86 0.29 3.42 2.82 92.12 D1550-29-1.12 3.26 39.36 39.91 5.13 4.15 0.30 3.73 2.77 92.11 D1550-29-1.17 3.25 39.21 40.21 5.22 4.11 0.30 3.70 2.67 92.30 D1550-29-1.39 4.12 38.44 39.23 5.83 4.25 0.30 3.96 2.46 92.17 D1550-29-1.35 BC 3.60 38.06 39.79 5.89 4.35 0.29 3.98 2.58 91.98 D1550-29-1.7 3.15 39.18 40.04 5.24 4.05 0.32 3.68 2.88 91.98 D1550-29-1.1 2.87 38.29 40.76 5.20 4.20 0.31 3.79 2.86 91.63
[0634] Next, we analyzed the performance of D1681 strains that were constructed using the KASI replacement strategy. Interestingly, unlike D1550 transformants, the D1681 strains demonstrated greater variability in fatty acid profiles (Table 75). In addition, the D1681 derived lines had lower C8:0 levels than what we observed in the D1550 derived transgenic lines suggesting a direct role of C. wrightii KASAI in improving C10:0 specificity of C. hookeriana FATB2 thioesterase.
TABLE-US-00103 TABLE 75 Fatty acid profiles in Strain BA and derivative transgenic lines generated after transformation with pSZ2746 DNA. Fatty Acid (area %) Total Strain C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Saturates Strain BA 0.89 13.17 40.52 17.53 11.60 0.59 11.38 2.95 84.30 D1681.3.7-2 BD 1.44 31.83 44.97 6.52 4.83 0.30 6.53 2.45 89.89 D1681.3.7-10 1.84 31.41 43.64 6.90 5.15 0.31 7.08 2.46 89.25 D1681.3.7-12 BE 1.85 31.64 43.50 6.76 5.08 0.31 7.16 2.49 89.14 D1681.3.4-1 1.29 31.61 45.92 6.65 4.49 0.29 6.20 2.46 90.25 D1681.3.4-6 BF 1.48 32.26 45.11 6.55 4.56 0.29 6.23 2.41 90.25 D1681.3.4-9 1.42 31.22 45.40 6.93 4.69 0.30 6.49 2.47 89.96 D1681.3.8-1 1.35 27.72 44.72 8.78 5.97 0.37 7.36 2.51 88.91 D1681.3.8-4 1.44 27.51 44.34 8.72 6.05 0.36 7.84 2.51 88.42 D1681-2.1-37 0.64 34.80 47.17 4.84 3.81 0.31 4.37 2.43 91.57 D1681-2.1-34 0.62 35.26 47.07 4.77 3.77 0.30 4.22 2.36 91.79 D1681-2.1-28 BG 0.64 35.99 46.80 4.65 3.68 0.29 4.02 2.34 92.05 D1681-2.1-12 0.67 34.78 47.21 4.94 3.79 0.34 4.30 2.35 91.73 D1681.2.4-1.3 BH 0.57 33.95 47.73 4.93 3.39 0.03 0.24 3.96 92.60 D1681.2.4-1.4 0.56 36.71 47.55 4.86 3.26 0.03 0.24 3.61 92.97 D1681.2.4-1.12 BI 1.89 34.72 44.70 6.32 4.03 0.02 0.27 5.18 91.68 D1681.2.4-1.2 1.73 36.43 44.25 5.57 4.09 0.03 0.32 4.55 92.10
[0635] Eight strains representing D1550 and D1681 families (from Tables 74-75) were subsequently evaluated in high cell density fermentations as shown in Table 76. Fermentations resulted in oils with a slightly improved mid-chain profile or the balance of C10-C12:0 fatty acid levels compared to the lab scale fermentation. Strain BE evaluated in two independent fermentations demonstrated superior profile reaching 85.2% C10-C12:0 fatty acid levels, 3.5% C14:0 levels, and ca. 1.2% C8:0 fatty acid levels, and accumulated over 92% total saturates.
TABLE-US-00104 TABLE 76 End-point fatty acid profiles in D1550 and D1681 derivative transgenic lines subjected to high cell density fermentation. Strain BB BC BG BH BI BF BE Run 130067 130196 130197 130291 130292 130253 130246 PF13029 Fatty C8:0 5.3 4.62 4.62 0.54 1.53 1.59 2.1 1.19 Acid C10:0 36.19 36.16 36.16 33.24 40 40.46 40.94 41.59 Profile C12:0 39.07 38.77 38.77 47.65 43.09 42.39 41.25 43.6 (Area %) C14:0 5.31 5.18 5.18 5 4.62 4.42 4.2 3.49 C16:0 3.72 3.9 3.9 3.4 2.83 2.63 2.8 2.28 C18:0 0.24 0.28 0.28 0.27 0.22 0.32 0.28 0.15 C18:1 6.12 6.79 6.79 5.88 4.95 5.34 5.8 4.76 C18:2 2.43 2.76 2.76 2.49 1.95 2.05 1.89 1.96 C10-C14 80.57 80.11 80.11 85.89 87.71 87.27 86.39 88.68 C10-C12 75.26 74.93 74.93 80.89 83.09 82.85 82.19 85.19 Total Saturates 89.83 88.91 88.91 90.1 92.29 91.81 91.57 92.3
Example 61
Tag Regiospecificity in UTEX1435 by Expression of Cuphea PSR23 LPAAT2 and LPAAT3 Genes
[0636] In Example 43, we demonstrated that the expression of 2 different 1-acyl-sn-glycerol-3-phosphate acyltransferases (LPAATs), the LPAAT2 and LPAAT3 genes from Cuphea PSR23 (CuPSR23) in the UTEX1435 derivative strain S2014 resulted in elevation of C10:0, C12:0 and C14:0 fatty acids levels. In this example we provide evidence that Cuphea PSR23 LPAAT2 exhibits high specificity towards incorporating C10:0 fatty acids at sn-2 position in TAGs. The Cuphea PSR23 LPAAT3 specifically incorporates C18:2 fatty acids at sn-2 position in TAGs.
[0637] Composition and properties of Prototheca moriformis (UTEX 1435) transgenic strain B, transforming vectors pSZ2299 and pSZ2300 that express CuPSR23 LPAAT2 and LPAAT3 genes, respectively, and their sequences were described previously.
[0638] To determine the impact of Cuphea PSR23 LPAAT genes on the resulting fatty acid profiles we have taken advantage of Strain B which synthesizes both mid chain and long chain fatty acids at relatively high levels. As shown in Table 77, the expression of the LPAAT2 gene (D1520) in Strain B resulted in increased C10-C12:0 levels (up to 12% in the best strain, D1520.3-7) suggesting that this LPAAT is specific for mid chain fatty acids. Alternatively, expression of the LPAAT3 gene resulted in a relatively modest increase, (up to 5% in the best strain, D1521.28-7) indicating it has little or no impact on mid-chain levels.
TABLE-US-00105 TABLE 77 Fatty acid profiles of Strain B and representative transgenic lines transformed with pSZ2299 (D1520) and pSZ2300 (D1521) DNA. Fatty Acid (area %) C10: C12: C14: C16: C18: C18: C18: C10- Total Strain C8:0 0 0 0 0 0 1 2 C12 Saturates Strain B 0.09 4.95 29.02 15.59 12.55 1.27 27.93 7.60 33.97 63.47 D1520.8-6 0.00 6.71 31.15 15.80 13.04 1.42 24.32 6.56 37.86 68.12 D1520.13-4 0.00 6.58 30.96 16.14 13.34 1.25 24.32 6.27 37.54 68.27 D1520.19-4 0.00 7.53 32.94 16.64 12.63 1.17 21.96 6.11 40.47 70.91 D1520.3-7 0.06 9.44 36.26 16.71 11.44 1.28 18.41 5.59 45.70 75.19 D1521.13-8 0.00 6.21 33.13 16.70 12.30 1.18 20.84 8.70 39.34 69.52 D1521.18-2 0.00 5.87 31.91 16.46 12.60 1.22 22.14 8.59 37.78 68.06 D1521.24-8 0.00 5.75 31.47 16.13 12.60 1.42 23.31 8.22 37.22 67.37 D1521.28-7 0.00 6.28 32.82 16.33 12.27 1.43 21.98 7.91 39.10 69.13
[0639] To determine if expression of the Cuphea PSR23 LPAAT genes affected regiospecificity of fatty acids at the sn-2 position, we analyzed TAGs from representative D1520 and D1521 strains utilizing the porcine pancreatic lipase method. See Example 2. As demonstrated in Table 78, the Cuphea PSR23 LPAAT2 gene shows remarkable specificity towards C10:0 fatty acids and appears to incorporate 50% more C10:0 fatty acids into the sn-2 position. The Cuphea PSR23 LPAAT3 gene appears to act exclusively on C18:2 fatty acids, resulting in redistribution of C18:2 fatty acids onto sn-2 position. Accordingly, microbial triglyceride oils with sn-2 profiles of greater than 15% or 20% C10:0 or C18:2 fatty acids are obtainable by introduction of an exogenous LPAAT gene having corresponding specificity.
TABLE-US-00106 TABLE 78 TAG and sn-2 fatty acid profiles in oils of parental S2014 strain and the progeny strains expressing Cuphea PSR23 LPAAT2 (BJ) and LPAAT3 (BK) genes. Strain Strain B Strain BI (D1520.3-7) Strain BK (D1521.13-8) Analysis TAG Profile sn-2 Profile TAG Profile sn-2 Profile TAG Profile sn-2 Profile Fatty C8:0 0 0 0.1 0 0 0 Acid C10:0 12 14.2 11 24.9 6.21 6.3 (area C12:0 42.8 25.1 40.5 24.3 33.13 19.5 %) C4:0 12.1 10.4 16.3 10 16.7 11.8 C6:0 7.3 1.3 10.2 1.4 12.3 3 C8:0 0.7 0.2 0.9 0.6 1.18 0.5 C8:1 18.5 36.8 15.4 29.2 20.84 36.3 C8:2 5.8 10.9 4.9 8.7 8.7 20.9 C18:3a 0.6 0.8 0.4 0.8 0.48 1.2 C10-C14 66.9 49.7 67.8 59.2 56.0 37.6 C10-C12 54.8 39.3 51.5 49.2 39.3 25.8
Example 62
Introduction of Heterologous Thioesterases into a Heterologous KAS-Expressing Prototheca Moriformis Strain
[0640] Here we demonstrate that heterologous fatty acyl-ACP thioesterases exhibit altered thioesterase specificity when combined with a heterologous plant KASI gene, Cuphea wrightii .beta.-ketoacyl-ACP synthase (KAS), CwKASA1, in P. moriformis (UTEX 1435) transgenic strain, S5818. S5818 is a transgenic strain expressing a thioesterase chimera from Cinnamomum camphora and Umbellularia californica, CcFATB2-UcFATB2 chimera B, at the 6S locus and additionally expressing the Cuphea wrightii KAS, CwKASA1, at the pLOOP locus. The addition of the CcFATB2-UcFATB2 chimera B and CwKASA1 genes leads to an S5818 fatty acid profile with 45% C12:0 and 14% C14:0. Five different constructs encoding thioesterases that were previously shown to exhibit predominantly C14:0 thioesterase activity and with less pronounced C12:0 thioesterase activity in P. moriformis were introduced into S5818 in an effort to increase C14:0 and C12:0 levels in this background. However, introduction of the five different C14:0 thioesterases into S5818 led to unexpected but significant increases in C12:0 fatty acid levels (>50% overall) with only modest increases in C14:0 fatty acid levels (<20% overall). This result suggests that the KASI-FATB thioesterase combination exhibits a unique activity not displayed when either gene is introduced separately. The results demonstrate that combination of heterologous KAS genes with heterologous thioesterases in oleaginous cells can be used to produce fatty acid profiles not exhibited by introduction of either gene alone. Furthermore, introduction of heterologous KASs may be an important and fruitful approach for revealing novel specificities of additional heterologous thioesterases.
[0641] Strain S5818 generation. S5818 was created by two successive transformations. The UTEX1435 base strain, S3150 (Strain Z above), was transformed with pSZ2448 (6 SA::CrTUB2-ScSUC2-CvNR:PmAMT3-CpSAD1tpExt-CcFATB2-UcFATB2-chimeraB-ExtA-C- vNR::6SB), encoding the CcFATB2-UcFATB2 chimera B thioesterase targeting the 6S locus, to yield strain S4954. S4954 produces .about.32% C12:0 and .about.16% C14:0 fatty acid levels (Table 62-1). S4954 was subsequently transformed with pSZ2229 (pLOOP::CrTUB2-NeoR-CvNR:PmAMT3-PmSADtp_CwKASAI-CvNR::pLOOP), encoding the C. wrightii KASA1 gene targeting the pLOOP locus, to yield strain S5818. S5818 produces .about.45% C12:0 and .about.14% C14:0 fatty acid levels (Table 79).
TABLE-US-00107 TABLE 79 Fatty acid profiles of S3150, S4954, and S5818. Sample ID C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Strain Z 0 0.05 1.49 28.83 3.24 57.87 6.27 S4954 0.17 31.52 16.39 9.81 1.19 32.14 7.19 S5818 0.34 45.16 13.77 8.54 0.81 24.63 5.38
[0642] Identification of C14:0 thioesterases. In an effort to increase C14:0 fatty acid levels, and to a lesser degree C12:0 fatty acid levels, several thioesterases that were found to exhibit C14:0 and C12:0 thioesterase activity in P. moriformis were cloned into vectors for introduction into S5818. The Cuphea hyssopifolia thioesterase ChsFATB3 was discovered by us as part of efforts to identify novel thioesterases by sequencing the mature, plant oilseeds of C. hyssopifolia. Although C. hyssopifolia seeds exhibit .about.84% C12:0 and .about.5% C14:0 fatty acid levels, the ChsFATB3 thioesterase we identified exhibits strong C14:0 thioesterase activity when expressed in S3150 (up to .about.34% C14:0). A version of ChsFATB3 in which we optimized the putative plastid-targeting transit peptide, named pSADD1tp_trimmed:ChsFATB3, similarly exhibited strong C14:0 thioesterase activity (.about.33% C14:0; Table 80).
TABLE-US-00108 TABLE 80 Fatty acid profiles of Cuphea hyssopifolia seeds and S3150 with introduction of ChsFATB3 or CpSAD1tp_trimmed:ChsFATB3. Sample ID C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Cuphea hyssopifolia seeds 0.24 6.53 83.69 5.13 1.10 0.12 0.00 1.74 S3150 0.00 0.00 0.05 1.49 28.83 3.24 57.87 6.27 S3150 + ChsFATB3 (T537; D1701-48) 0.00 0.00 8.09 33.66 26.46 1.57 23.75 5.3 S3150 + CpSAD1tp_trimmed:ChsFATB3 0.00 0.14 7.25 33.32 27.04 1.57 24.37 5.12 (T580; D1813-8)
[0643] Similarly, we also identified the Cuphea heterophylla thioesterase ChtFATB1a as part of our efforts to identify novel thioesterases by sequencing the mature, plant oil seeds of C. heterophylla. Although C. heterophylla seeds exhibit .about.44% C10:0, .about.40% C12:0 fatty acid levels, and only .about.4% C14:0, the transit peptide optimized version of the ChtFATB1a thioesterase we identified, CpSAD1tp_trimmed:ChtFATB1a, exhibits strong C14:0 thioesterase activity when expressed in S3150 (up to .about.35% C14:0; Table 81).
TABLE-US-00109 TABLE 81 Fatty acid profiles of Cuphea heterophylla seeds and S3150 with introduction of CpSAD1tp_trimmed:ChtFATB1a. Sample ID C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Cuphea heterophylla seeds 3.50 44.27 40.04 4.26 1.22 0.25 2.21 3.56 S3150 0.00 0.00 0.05 1.49 28.83 3.24 57.87 6.27 S3150 + CpSAD1tp_trimmed:ChtFATB1a 0.00 0.15 13.18 35.16 24.1 1.19 18.87 6.02 (T580; D1811-44)
[0644] A published Cuphea palustris C14:0 thioesterase, CpalFATB2, was also introduced into S5818 (vide infra).
[0645] Introduction of C14:0 thioesterases into S5818. Five constructs were generated using C14:0 thioesterases for introduction into S5818 (Table 82).
TABLE-US-00110 TABLE 82 Constructs engineered for introduction into S5818. D# pSZ# Construct D2104 pSZ3390 DAO1b::PmHXT1-ScarMel1-CvNR:PmUAPA1noSacI-CpSAD1tpExt-CpaIFATB2FLAGExtA-C- vNR::DAO1b D2202 pSZ3493 DAO1b5.sup.1::PmHXT1-ScarMEL1-CvNR:PmAMT3-ChsFATB3-CvNR::DAO1b3.sup.1 D2203 pSZ3494 DAO1b5.sup.1::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpSAD1tp_trimmed:ChsFATB3-CvNR:- :DAO1b3.sup.1 D2204 pSZ3495 DAO1b5.sup.1::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpSAD1tp_trimmed:ChtFATB1a-CvNR- ::DAO1b3.sup.1 D2235 pSZ3531 THI4A::PmHXT1-ScarMel1 - CpEF1a:PmUAPA1noSacI-CpSAD1tpExt-CpaIFATB2FLAGExtA-CvNR::THI4A
[0646] pSZ3390 and pSZ3531 introduce the CpalFATB2 thioesterase gene into the DAO1b and THI4A loci, respectively, under the control of the pH5-responsive UAPA1 promoter. pSZ3493, pSZ3494, and pSZ3495 introduce ChsFATB3, CpSAD1tp_trimmed:ChsFATB3, and CpSAD1tp trimmed:ChtFATB1a, respectively, into the DAO1b locus under the control of the pH7-responsive AMT3 promoter. Transgenic strains were selected for the ability to grow on melibiose. Cell culture, lipid production, and fatty acid analysis were all carried out as previously described. The transforming DNA for pSZ3390, pSZ3493, pSZ3494, pSZ3495, and pSZ3531 are provided below.
[0647] pSZ3390: pSZ3390 can be written as DAO1b:: PmHXT1-ScarMel1-CvNR:PmUAPA1noSacI-CpSAD1tpExt-CpalFATB2FLAGExtA-CvNR::DO- A1b. The relevant restriction sites in the construct from 5'-3', BspQI, KpnI, SpeI, SnaBI, XhoI, EcoRI, SpeI, HindIII, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences at the 5' and 3' end of the construct represent genomic DNA from UTEX 1435 that target integration to the DAO1b locus via homologous recombination. Proceeding in the 5' to 3' direction, the selection cassette has the P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene (conferring the ability to grow on melibiose) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3'UTR is indicated by lowercase, underlined text. The second cassette containing the CpSAD1tpExt-CpalFATB2FLAGExtA gene, fused to the heterologous Chlorella protothecoides SAD1 plastid-targeting transit peptide, is driven by the P. moriformis UAPA1 pH5-responsive promoter and has the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. In this cassette, the UAPA1 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CpSAD1tpExt-CpalFATB2FLAGExtA gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics. The 3' UTR is indicated by lowercase, underlined text.
TABLE-US-00111 pSZ3390 transforming construct: (SEQ ID NO: 123) gaagagcGCCCAATGTTTAAACagcccgcaccctcgttgatctgggagccctgcgcagccccttaaatcatctc- ag tcaggtttctgtgttcaactgagcctaaagggctttcgtcatgcgcacgagcacacgtatatcggccacgcagt- ttctcaaaagc ggtagaacagttcgcgagccctcgtaggtcgaaaacttgcgccagtactattaaattaaattaattgatcgaac- gagacgcga aacttttgcagaatgccaccgagtttgcccagagaatgggagtggcgccattcaccatccgcctgtgcccggct- tgattcgccg agacgatggacggcgagaccagggagcggcttgcgagccccgagccggtagcaggaacaatgatcgacaatctt- cctgtcc aattactggcaaccattagaaagagccggagcgcgttgaaagtctgcaatcgagtaatttttcgatacgtcggg- cctgctgaa ccctaaggctccggactttgtttaaggcgatccaagatgcacgcggccccaggcacgtatctcaagcacaaacc- ccagcctta gtttcgagactttgggagatagcgaccgatatctagtttggcattttgtatattaattacctcaagcaatggag- cgctctgatgcg gtgcagcgtcggctgcagcacctggcagtggcgctagggtcgccctatcgctcggaacctggtcagctggctcc- cgcctcctgc ##STR00386## ctccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggaca- actggaacac gttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggaca- tgggctacaa gtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagt- tccccaacggc atgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtacac- gtgcgccggcta ccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacg- acaactgct acaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaag- acgggccg ccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcct- ggcgcatgtccgg cgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacg- ccggcttcc actgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgac- ctggacaa cctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagt- cccccctgat catcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatca- accaggactcc aacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatcca- gatgtggtc cggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacga- ccctggagg agatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaac- cgcgtcgacaa ctccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcct- acaaggacg gcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacg- accgtccccg cccacggcatcgcgttctaccgcctgcgcccctcctccTGAtacgtactcgaggcagcagcagtcggatagtat- cgacacactc tggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctttta- tcaaacagcctcagtgt gtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatc- cccttccctcgtttcatatc gcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcc- cctcgcacagccttggtt tgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtg- ggatgggaacacaa ##STR00387## ##STR00388## ctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccc- tccccgtgcg cgctgccatcgccagcgaggtccccgtggccaccacctccccccgggcgcaccccaaggcgaacggcagcgcgg- tgtcgctg aagtcgggctccctggagacccaggaggacaagacgagcagctcgtccccccccccccgcacgttcatcaacca- gctgcccgt gtggagcatgctgctgtcggcggtgaccacggtcttcggcgtggccgagaagcagtggcccatgctggaccgca- agtccaagcg ccccgacatgctggtcgagcccctgggcgtggaccgcatcgtctacgacggcgtgagcttccgccagtcgttct- ccatccgcagct acgagatcggcgccgaccgcaccgcctcgatcgagacgctgatgaacatgttccaggagacctccctgaaccac- tgcaagatc atcggcctgctgaacgacggcttcggccgcacgcccgagatgtgcaagcgcgacctgatctgggtcgtgaccaa- gatgcagatc gaggtgaaccgctaccccacgtggggcgacaccatcgaggtcaacacgtgggtgagcgcctcgggcaagcacgg- catgggcc gcgactggctgatctccgactgccacaccggcgagatcctgatccgcgcgacgagcgtctgggcgatgatgaac- cagaagacc cgccgcctgtcgaagatcccctacgaggtgcgccaggagatcgagccccagttcgtcgactccgcccccgtgat- cgtggacgac cgcaagttccacaagctggacctgaagacgggcgacagcatctgcaacggcctgaccccccgctggacggacct- ggacgtga accagcacgtcaacaacgtgaagtacatcggctggatcctgcagtcggtccccaccgaggtgttcgagacgcag- gagctgtgcg gcctgaccctggagtaccgccgcgagtgcggccgcgactccgtgctggagagcgtcacggccatggacccctcg- aaggaggg cgaccgctccctgtaccagcacctgctgcgcctggaggacggcgcggacatcgtgaagggccgcaccgagtggc- gccccaag aacgccggcgccaagggcgccatcctgacgggcaagaccagcaacggcaactcgatctccatggactacaagga- ccacgac ggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGAaagcttgcagcagcagctcggat- agtatcg acacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgc- cgcttttatcaaacagc ctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccaccc- ccagcatccccttccctcgt ttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgc- tcactgcccctcgcacag ccttggtttgggctcccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgg- gaagtagtgggatgg gaacacaaatggaaagctggagctcagcgtctgcgtgttgggagctggagtcgtgggcttgacgacggcgctgc- agctgttgca ggatgtgcctggcgtgcgcgttcacgtcgtggctgagaaatatggcgacgaaacgttgacggctggggccggcg- ggctgtgg atgccatacgcattgggtacgcggccattggatgggattgataggcttatggagggataatagagtttttgccg- gatccaacgc atgtggatgcggtatcccggtgggctgaaagtgtggaaggatagtgcattggctattcacatgcactgcccacc- ccttttggca ggaaatgtgccggcatcgttggtgcaccgatggggaaaatcgacgttcgaccactacatgaagatttatacgtc- tgaagatgc agcgactgcgggtgcgaaacggatgacggtttggtcgtgtatgtcacagcatgtgctggatcttgcgggctaac- tccccctgcc acggcccattgcaggtgtcatgttgactggagggtacgacctttcgtccgtcaaattcccagaggaggacccgc- tctgggccg acattgtgcccactgaagagc
[0648] pSZ3493, pSZ3494, and pSZ3495: pSZ3493 can be written as DOA1b5'::PmHXT1-ScarMEL1-CvNR:PmAMT3-ChsFATB3-CvNR::DOA1b3'. pSZ3494 can be written as DOA1b5'::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpSAD1tp trimmed:ChsFATB3-CvNR::DOA1b3'. pSZ3495 can be written as DOA1b5'::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpSAD1tp_trimmed: ChtFATB1a-CvNR::DOA1b3'. The sequences of the three constructs differ only in the sequence of the thioesterase gene. The full transforming sequence for pSZ3493 is displayed in SEQ ID NO:124. The sequences of the CpSAD1tp_trimmed: ChsFATB3 and CpSAD1tp_trimmed:ChtFATBla genes alone, which take the place of ChsFATB3 from pSZ3493 in the pSZ3494 and pSZ3495 sequences, are displayed in SEQ ID NOs:125 and 126, respectively, along with flanking restriction sites.
[0649] The relevant restriction sites in the pSZ3493 construct from 5'-3', BspQI, KpnI, SpeI, SnaBI, XhoI, EcoRI, SpeI, XhoI, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences at the 5' and 3' end of the construct represent genomic DNA from UTEX 1435 that target integration to the DAO1b locus via homologous recombination. Proceeding in the 5' to 3' direction, the selection cassette has the P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene (conferring the ability to grow on melibiose) and the Chlorella vulgaris Nitrate reductase (NR) gene 3' UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3'UTR is indicated by lowercase, underlined text. The second cassette is comprised of the ChsFATB3 gene driven by the P. moriformis AMT3 pH7-responsive promoter and with the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the ChsFATB3 gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics. The 3' UTR is indicated by lowercase, underlined text.
TABLE-US-00112 pSZ3493 transforming construct: (SEQ ID NO: 124) gaagagcGCCCAATGTTTAAACagcccgcaccctcgttgatctgggagccctgcgcagccccttaaatcatctc- ag tcaggtttctgtgttcaactgagcctaaagggctttcgtcatgcgcacgagcacacgtatatcggccacgcagt- ttctcaaaagc ggtagaacagttcgcgagccctcgtaggtcgaaaacttgcgccagtactattaaattaaattaattgatcgaac- gagacgcga aacttttgcagaatgccaccgagtttgcccagagaatgggagtggcgccattcaccatccgcctgtgcccggct- tgattcgccg agacgatggacggcgagaccagggagcggcttgcgagccccgagccggtagcaggaacaatgatcgacaatctt- cctgtcc aattactggcaaccattagaaagagccggagcgcgttgaaagtctgcaatcgagtaatttttcgatacgtcggg- cctgctgaa ccctaaggctccggactttgtttaaggcgatccaagatgcacgcggccccaggcacgtatctcaagcacaaacc- ccagcctta gtttcgagactttgggagatagcgaccgatatctagtttggcattttgtatattaattacctcaagcaatggag- cgctctgatgcg gtgcagcgtcggctgcagcacctggcagtggcgctagggtcgccctatcgctcggaacctggtcagctggctcc- cgcctcctgc ##STR00389## ctccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggaca- actggaacac gttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggaca- tgggctacaa gtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagt- tccccaacggc atgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtacac- gtgcgccggcta ccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacg- acaactgct acaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaag- acgggccg ccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcct- ggcgcatgtccgg cgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacg- ccggcttcc actgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgac- ctggacaa cctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagt- cccccctgat catcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatca- accaggactcc aacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatcca- gatgtggtc cggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacga- ccctggagg agatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaac- cgcgtcgacaa ctccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcct- acaaggacg gcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacg- accgtccccg cccacggcatcgcgttctaccgcctgcgcccctcctccTGAtacgtactcgaggcagcagcagctcggatagta- tcgacacactc tggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctttta- tcaaacagcctcagtgt gtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatc- cccttccctcgtttcatatc gcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcc- cctcgcacagccttggtt Igggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtg- ggatgggaacacaa ##STR00390## ##STR00391## cggcacctcccccaagcccggcaagttcggcaactggcccacctccctgtccgtgcccttcaagtccaagtcca- accacaacgg cggcttccaggtgaaggccaacgcctccgcccgccccaaggccaacggctccgccgtgtccctgaagtccggct- ccctggacac ccaggaggacacctcctcctcctcctcccccccccgcaccttcatcaaccagctgcccgactggtccatgctgc- tgtccgccatcac caccgtgttcgtggccgccgagaagcagtggaccatgctggaccgcaagtccaagcgccccgacatgctgatgg- accccttcgg cgtggaccgcgtggtgcaggacggcgccgtgttccgccagtccttctccatccgctcctacgagatcggcgccg- accgcaccgcc tccatcgagaccctgatgaacatcttccaggagacctccctgaaccactgcaagtccatcggcctgctgaacga- cggcttcggcc gcacccccgagatgtgcaagcgcgacctgatctgggtggtgaccaagatgcacgtggaggtgaaccgctacccc- acctggggc gacaccatcgaggtgaacacctgggtgtccgagtccggcaagaccggcatgggccgcgactggctgatctccga- ctgccacac cggcgagatcctgatccgcgccacctccatgtgcgccatgatgaaccagaagacccgccgcttctccaagttcc- cctacgaggtg cgccaggagctggccccccacttcgtggactccgcccccgtgatcgaggactaccagaagctgcacaagctgga- cgtgaagac cggcgactccatctgcaacggcctgaccccccgctggaacgacctggacgtgaaccagcacgtgaacaacgtga- agtacatcg gctggatcctggagtccgtgcccaccgaggtgttcgagacccaggagctgtgcggcctgaccctggagtaccgc- cgcgagtgcg gccgcgactccgtgctggagtccgtgaccgccatggacccctccaaggagggcgaccgctccctgtaccagcac- ctgctgcgcc tggaggacggcgccgacatcgccaagggccgcaccaagtggcgccccaagaacgccggcaccaacggcgccatc- tccaccg gcaagacctccaacggcaactccatctccatggactacaaggaccacgacggcgactacaaggaccacgacatc- gactacaa ggacgacgacgacaagTGActcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtg- atggactgtt gccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatctt- gtgtgtacgcgcttttgcg agttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatc- ccaaccgcaacttatctac gctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgc- ctgtattctcctggtact gcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaAAGCTGTAT- AGGG ATAACAGGGTAATgagctcagcgtctgcgtgttgggagctggagtcgtgggcttgacgacggcgctgcagctgt- tg caggatgtgcctggcgtgcgcgttcacgtcgtggctgagaaatatggcgacgaaacgttgacggctggggccgg- cgggctgt ggatgccatacgcattgggtacgcggccattggatgggattgataggcttatggagggataatagagtttttgc- cggatccaac gcatgtggatgcggtatcccggtgggctgaaagtgtggaaggatagtgcattggctattcacatgcactgccca- ccccttttgg caggaaatgtgccggcatcgttggtgcaccgatggggaaaatcgacgttcgaccactacatgaagatttatacg- tctgaagat gcagcgactgcgggtgcgaaacggatgacggtttggtcgtgtatgtcacagcatgtgctggatcttgcgggcta- actccccctg ccacggcccattgcaggtgtcatgttgactggagggtacgacctttcgtccgtcaaattcccagaggaggaccc- gctctgggcc gacattgtgcccactgaagagc CpSAD1tp_trimmed:ChsFATB3 (from pSZ3494): (SEQ ID NO: 125) actagtAACAATGgccaccgcctccaccttctccgccttcaacgcccgctgcggcgacctgcgccgctccgccg- gctccgg cccccgccgccccgcccgccccctgcccgtgcgcgccgccatcaacgcctccgcccgccccaaggccaacggct- ccgccgtgt ccctgaagtccggctccctggacacccaggaggacacctcctcctcctcctcccccccccgcaccttcatcaac- cagctgcccgac tggtccatgctgctgtccgccatcaccaccgtgttcgtggccgccgagaagcagtggaccatgctggaccgcaa- gtccaagcgcc ccgacatgctgatggaccccttcggcgtggaccgcgtggtgcaggacggcgccgtgttccgccagtccttctcc- atccgctcctac gagatcggcgccgaccgcaccgcctccatcgagaccctgatgaacatcttccaggagacctccctgaaccactg- caagtccatc ggcctgctgaacgacggcttcggccgcacccccgagatgtgcaagcgcgacctgatctgggtggtgaccaagat- gcacgtgga ggtgaaccgctaccccacctggggcgacaccatcgaggtgaacacctgggtgtccgagtccggcaagaccggca- tgggccgc gactggctgatctccgactgccacaccggcgagatcctgatccgcgccacctccatgtgcgccatgatgaacca- gaagacccgc cgcttctccaagttcccctacgaggtgcgccaggagctggccccccacttcgtggactccgcccccgtgatcga- ggactaccaga agctgcacaagctggacgtgaagaccggcgactccatctgcaacggcctgaccccccgctggaacgacctggac- gtgaacca gcacgtgaacaacgtgaagtacatcggctggatcctggagtccgtgcccaccgaggtgttcgagacccaggagc- tgtgcggcct gaccctggagtaccgccgcgagtgcggccgcgactccgtgctggagtccgtgaccgccatggacccctccaagg- agggcgacc gctccctgtaccagcacctgctgcgcctggaggacggcgccgacatcgccaagggccgcaccaagtggcgcccc- aagaacgc cggcaccaacggcgccatctccaccggcaagacctccaacggcaactccatctccatggactacaaggaccacg- acggcgact acaaggaccacgacatcgactacaaggacgacgacgacaagTGActcgag CpSAD1tp_trimmed:ChtFATB1a (from pSZ3495): (SEQ ID NO: 126) actagtAACAATGgccaccgcctccaccttctccgccttcaacgcccgctgcggcgacctgcgccgctccgccg- gctccgg cccccgccgccccgcccgccccctgcccgtgcgcgccgccatcaacgcctccgcccaccccaaggccaacggct- ccgccgtga acctgaagtccggctccctggagacccaggaggacacctcctcctcctccccccccccccgcaccttcatcaag- cagctgcccga ctggggcatgctgctgtccaagatcaccaccgtgttcggcgccgccgagcgccagtggaagcgccccggcatgc- tggtggagcc cttcggcgtggaccgcatcttccaggacggcgtgttcttccgccagtccttctccatccgctcctacgagatcg- gcgccgaccgcac cgcctccatcgagaccctgatgaacatcttccaggagacctccctgaaccactgcaagtccatcggcctgctga- acgacggcttc
ggccgcacccccgagatgtgcaagcgcgacctgatctgggtggtgaccaagatccaggtggaggtgaaccgcta- ccccacctg gggcgacaccatcgaggtgaacacctgggtgtccgagtccggcaagaacggcatgggccgcgactggctgatct- ccgactgcc gcaccggcgagatcctgatccgcgccacctccgtgtgggccatgatgaaccgcaagacccgccgcctgtccaag- ttcccctacg aggtgcgccaggagatcgccccccacttcgtggactccgcccccgtgatcgaggacgacaagaagctgcacaag- ctggacgtg aagaccggcgactccatccgcaagggcctgaccccccgctggaacgacctggacgtgaaccagcacgtgaacaa- cgtgaagt acatcggctggatcctgaagtccgtgcccgccgaggtgttcgagacccaggagctgtgcggcgtgaccctggag- taccgccgcg agtgcggccgcgactccgtgctggagtccgtgaccgccatggacaccgccaaggagggcgaccgctccctgtac- cagcacctg ctgcgcctggaggacggcgccgacatcaccatcggccgcaccgagtggcgccccaagaacgccggcgccaacgg- cgccatct ccaccggcaagacctccaacgagaactccgtgtccatggactacaaggaccacgacggcgactacaaggaccac- gacatcga ctacaaggacgacgacgacaagTGActcgag
[0650] pSZ3531: pSZ3531 can be written as THI4A::PmHXT1-ScarMel1-CpEF1a:PmUAPA1noSacI-CpSAD1tpExt-CpalFATB2FLAGExtA- -CvNR::THI4A. The relevant restriction sites in the construct from 5'-3', BspQI, KpnI, SpeI, SnaBI, EcoRV, EcoRI, SpeI, HindIII, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences at the 5' and 3' end of the construct represent genomic DNA from UTEX 1435 that target integration to the THI4A locus via homologous recombination. Proceeding in the 5' to 3' direction, the selection cassette has the P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene (conferring the ability to grow on melibiose) and the Chlorella protothecoides EF1A gene 3' UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3'UTR is indicated by lowercase, underlined text. The second cassette containing the CpSAD1tpExt-CpalFATB2FLAGExtA gene, fused to the heterologous Chlorella protothecoides SAD1 plastid-targeting transit peptide, is driven by the P. moriformis UAPA1 pH5-responsive promoter and has the Chlorella vulgaris Nitrate Reductase (NR) gene 3' UTR. In this cassette, the UAPA1 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CpSAD1tpExt-CpalFATB2FLAGExtA gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics. The 3' UTR is indicated by lowercase, underlined text.
TABLE-US-00113 pSZ3531 transforming construct: (SEQ ID NO: 127) gaagagcGCCCAATGTTTAAACCcctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtt- t gcctccttggcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcgtcc- agagacggca agtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctgcagcagatgagcgtgactttggcca- tttcacgcact cgagtgtacacaatccatttttcttaaagcaaatgactgctgattgaccagatactgtaacgctgatttcgctc- cagatcgcaca gatagcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccatgcaacagat- ggcgacactt gttacaattcctgtcacccatcggcatggagcaggtccacttagattcccgatcacccacgcacatctcgctaa- tagtcattcgtt cgtgtcttcgatcaatctcaagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggc- tgcagggtctg cccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggtcacttgccttccagat- tgccaataa actcaattatggcctctgtcatgccatccatgggtctgatgaatggtcacgctcgtgtcctgaccgttccccag- cctctggcgtcc ##STR00392## ##STR00393## ggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgct- gctggacacg gccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctc- cggccgcgact ccgacggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaac- aactccttcc tgttcggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggag- gacgcccagt tcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcccgag- atctcctacc accgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactggggc- caggacctga ccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgcgcccc- gactcccgctg cccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctgaacaagg- ccgcccccat gggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaacctgacggacg- acgagg agaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaacaacctgaag- gcctcctccta ctccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtctggc- gctactacgtgt ccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtg- gcgctgct gaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctcca- agaagctgac ctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgca- acaagaccg ccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttc- ggccagaag atcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttctaccgcctgcg- cccctcctccT GATACAACTTATtacgtaacggagcgtcgtgcgggagggagtgtgccgagcggggagtcccggtctgtgcgagg- ccc ggcagctgacgctggcgagccgtacgccccgagggtccccctcccctgcaccctcttccccttccctctgacgg- ccgcgcctgttct ##STR00394## ##STR00395## catccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgccca- gcgaggcccct ccccgtgcgcgctgccatcgccagcgaggtccccgtggccaccacctccccccgggcgcaccccaaggcgaacg- gcagcgcg gtgtcgctgaagtcgggctccctggagacccaggaggacaagacgagcagctcgtccccccccccccgcacgtt- catcaacca gctgcccgtgtggagcatgctgctgtcggcggtgaccacggtcttcggcgtggccgagaagcagtggcccatgc- tggaccgcaa gtccaagcgccccgacatgctggtcgagcccctgggcgtggaccgcatcgtctacgacggcgtgagcttccgcc- agtcgttctcc atccgcagctacgagatcggcgccgaccgcaccgcctcgatcgagacgctgatgaacatgttccaggagacctc- cctgaaccac tgcaagatcatcggcctgctgaacgacggcttcggccgcacgcccgagatgtgcaagcgcgacctgatctgggt- cgtgaccaag atgcagatcgaggtgaaccgctaccccacgtggggcgacaccatcgaggtcaacacgtgggtgagcgcctcggg- caagcacg gcatgggccgcgactggctgatctccgactgccacaccggcgagatcctgatccgcgcgacgagcgtctgggcg- atgatgaacc agaagacccgccgcctgtcgaagatcccctacgaggtgcgccaggagatcgagccccagttcgtcgactccgcc- cccgtgatcg tggacgaccgcaagttccacaagctggacctgaagacgggcgacagcatctgcaacggcctgaccccccgctgg- acggacct ggacgtgaaccagcacgtcaacaacgtgaagtacatcggctggatcctgcagtcggtccccaccgaggtgttcg- agacgcagg agctgtgcggcctgaccctggagtaccgccgcgagtgcggccgcgactccgtgctggagagcgtcacggccatg- gacccctcg aaggagggcgaccgctccctgtaccagcacctgctgcgcctggaggacggcgcggacatcgtgaagggccgcac- cgagtggc gccccaagaacgccggcgccaagggcgccatcctgacgggcaagaccagcaacggcaactcgatctccatggac- tacaagg accacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagTGAaagcttgcagcagca- gctcgaagcttgcagcagcagctcg gatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtga- atatccctgccgctttt atcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcg- aataccacccccagcatcc ccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgct- cctgctcctgctcactgcc cctcgcacagccttggtttgggctcccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgc- tgatgcacgggaagta gtgggatgggaacacaaatggaanctggagctccagcgccatgccacgccattgatggcttcaagtacgattac- ggtgttgg attgtgtgtttgttgcgtagtgtgcatggtttagaataatacacttgatttcttgctcacggcaatctcggctt- gtccgcaggttcaa ccccatttcggagtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgagg- tgagctatg tttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagcacccaagcaaaatgtcaagtg- cgttccgattt gcgtccgcaggtcgatgttgtgatcgtcggcgccggatccgccggtctgtcctgcgcttacgagctgaccaagc- accctgacgt ccgggtacgcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcgcgaa- actgtgctc gattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgagcagggcgttgctcccggcggcggcg- cctggctgg ggggacagctgttctcggccatgtgtgtacgtagaaggatgaatttcagctggttttcgttgcacagctgtttg- tgcatgatttgtt tcagactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgactgaagagc
[0651] Increased C12:0 levels in strain S5818 by the expression of heterologous "C14:0-specific" thioesterases. In an effort to increase C14:0 fatty acid levels in S5818, several thioesterases that had previously displayed pronounced C14:0 thioesterase activity in P. moriformis were transformed into the S5818 background. Contrary to our expectations, we observed marked increases in C12:0 levels with decreases or only marginal increases in C14:0 levels. For example, introduction of the ChsFATB3 thioesterase (which leads to an increase in C14:0 levels of up to 34% in S3150) into S5818 causes C12:0 levels to rise to .about.77% (.DELTA.=+32% C12:0) and C14:0 levels to drop to .about.7% (.DELTA.=-7%). In addition, introduction of CpalFATB2 into S5818 at the DAO1b locus causes C12:0 levels to rise to .about.64% (.DELTA.=+19%) and C14:0 levels to drop to .about.12% (.DELTA.=-2%). The results for the top five transformants for each of the five constructs are displayed in Table 83.
[0652] Of note, S5818 expresses the C. wrightii KASAI gene from the pLOOP locus. As C. wrightii produces seed oil with 62% C12:0, we believe it likely that the CwKASA1 gene has evolved to be specific for production of C12:0 fatty acids when combined with C. wrightii thioesterases. Indeed, C. wrightii FATB2 encodes a thioesterase that exhibits C12:0 activity when introduced into P. moriformis. Thus, it is possible that the "C14:0" thioesterase genes identified in our transcriptome sequencing, namely ChsFATB3 and ChtFATB1a, exhibit C14:0 activity only when in combination with the P. moriformis endogenous KASI gene. These results further extend to CpalFATB2, which has been repeatedly shown to increase C14:0 levels in P. moriformis (data not shown). However, when ChsFATB3, ChtFATB1a, and CpalFATB2 are combined with a KASI gene from a Cuphea species that produces high C12:0 fatty acids, such as CwKASA1 from Cuphea wrightii, then a C12:0 activity of these thioesterases is revealed/exhibited. It should be further noted that C. hyssopifolia and C. heterophylla produce only low levels of C14:0 in oilseeds (5% and 4%, respectively) while producing relatively high levels of C12:0 (84% and 40%, respectively). Since the ChsFATB3 and ChtFATB1a thioesterases were identified from RNAs expressed in mature oil seeds, it is possible that these thioesterases indeed exhibited C12:0 activity in Cuphea seeds, significantly contributing to the high levels of C12:0 found therein.
[0653] Our results indicate that the combination of thioesterase and KAS is likely to be extremely important in determining the specificity of the thioesterase-KAS machinery in generating midchain fatty acids. Furthermore, introduction of heterologous KASs may be an important and fruitful approach for revealing novel specificities of additional heterologous thioesterases.
TABLE-US-00114 TABLE 83 Fatty acid profiles for the top 5 transformants for each of the pSZ3493, pSZ3494, pSZ3495, pSZ3390, and pSZ3531 constructs upon introduction into S5818. Sample ID pSZ#; construct Strain # C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 N/A S5818 0.34 45.16 13.77 8.54 0.81 24.63 5.38 pSZ3493; ChsFATB3 Block 6; C6; pH 7; S5818; T678; D2202-30 0.76 76.58 7.49 3.76 0.32 6.58 3.77 Block 6; D6; pH 7; S5818; T678; D2202-42 0.61 62.82 13.92 6.75 0.37 10.76 3.88 Block 6; D11; pH 7; S5818; T678; D2202-47 4.60 55.99 9.26 5.90 0.44 18.32 4.40 Block 6; A9; pH 7; S5818; T678; D2202-9 0.47 53.94 17.59 8.51 0.44 13.70 4.33 Block 6; D3; pH 7; S5818; T678; D2202-39 0.43 53.94 15.62 8.04 0.43 15.99 4.45 pSZ3494; Block 2; B8; pH 7; S5818; T678; D2203-20 0.43 56.76 14.15 7.60 0.56 14.23 5.09 CpSAD1tp_trimmed: Block 2; C1; pH 7; S5818; T678; D2203-25 0.46 54.82 17.08 7.81 0.46 13.67 4.85 ChsFATB3 Block 2; D1; pH 7; S5818; T678; D2203-37 0.43 54.47 11.51 8.14 0.95 18.58 4.99 Block 2; D7; pH 7; S5818; T678; D2203-43 0.43 52.86 18.70 8.91 0.58 13.18 4.45 Block 2; C11; pH 7; S5818; T678; D2203-35 0.44 52.81 19.54 8.87 0.54 12.57 4.29 pSZ3495; Block 2; G10; pH 7; S5818; T678; D2204-34 0.58 55.18 19.86 7.72 0.60 10.88 4.29 CpSAD1tp_trimmed: Block 2; H7; pH 7; S5818; T678; D2204-43 0.68 54.79 20.14 7.78 0.56 10.99 4.18 ChtFATB1a Block 2; H5; pH 7; S5818; T678; D2204-41 0.60 54.69 20.38 7.39 0.55 11.50 4.13 Block 2; G8; pH 7; S5818; T678; D2204-32 0.66 54.26 20.39 7.69 0.55 11.45 4.26 Block 2; F6; pH 7; S5818; T678; D2204-18 0.67 54.23 20.04 7.60 0.56 11.80 4.23 pSZ3390; CpSAD1tpExt- Block 4; A5; pH 7; S5818; T674; D2104-5 0.58 63.83 12.10 5.89 0.55 10.46 5.44 CpalFATB2FLAGExtA Block 4; A12; pH 7; S5818; T674; D2104-12 0.48 61.92 16.15 5.87 0.50 9.86 4.41 (DAO1b) Block 4; B9; pH 7; S5818; T674; D2104-21 0.41 54.31 18.41 7.26 0.51 13.80 4.49 Block 4; B5; pH 7; S5818; T674; D2104-17 0.37 53.56 16.54 7.25 0.58 16.34 4.42 Block 4; B11; pH 7; S5818; T674; D2104-23 0.41 52.44 17.99 7.60 0.54 15.38 4.66 pSZ3531; CpSAD1tpExt- Block 5B; A8; pH 7; S5818; T684; D2235-8 0.52 59.36 15.70 6.93 0.45 11.41 4.63 CpalFATB2FLAGExtA Block 5B; A12; pH 7; S5818; T684; D2235-12 0.44 55.60 16.98 6.98 0.53 14.21 4.59 (THI4A) Block 5B; B11; pH 7; S5818; T684; D2235-23 0.36 49.58 17.43 8.72 0.57 17.44 4.62 Block 5B; A4; pH 7; S5818; T684; D2235-4 0.35 49.43 18.63 8.22 0.62 17.29 4.54 Block 5B; A11; pH 7; S5818; T684; D2235-11 0.36 48.92 15.93 7.84 0.68 20.38 4.96
Example 63
A Suite of Regulatable Promoters to Conditionally Control Gene Expression Levels in Oleaginous Cells in Synchrony with Lipid Production
[0654] S5204 was generated by knocking out both copies of FATA1 in Prototheca moriformis (PmFATA1) while simultaneously overexpressing the endogenous PmKASII gene in a .DELTA.fad2 line, S2532. S2532 itself is a FAD2 (also known as FADc) double knockout strain that was previously generated by insertion of C. tinctorius ACP thioesterase (Accession No: AAA33019.1) into S1331, under the control of CrTUB2 promoter at the FAD2 locus. S5204 and its parent S2532 have a disrupted endogenous PmFAD2-1 gene resulting in no .DELTA.12 specific desaturase activity manifested as 0% C18:2 (linoleic acid) levels in both seed and lipid production stages. Lack of any C18:2 in S5204 (and its parent S2532) results in growth defects which can be partially mitigated by exogenous addition of linoleic acid in the seed stage. For industrial applications of a zero linoleic oil however, exogenous addition of linoleic acid entails additional cost. We have previously shown that complementation of S5204 (and other .DELTA.fad2 strains S2530 and S2532) with pH inducible AMT03p driven PmFAD2-1 restores C18:2 to wild-type levels at pH 7.0 and also results in rescued growth characteristics during seed stage without any linoleic supplementation. Additionally when the seed from pH 7.0 grown complemented lines is subsequently transferred into low-nitrogen lipid production flasks with pH adjusted to 5.0 (to control AMT03p driven FAD2 protein levels), the resulting final oil profile matches the parent S5204 or S2532 profile with zero linoleic levels but with rescued growth and productivity metrics. Thus in essence with AMT03p driven FAD2-1 we have developed a pH regulatable strain that potentially could be used to generate oils with varying linoleic levels depending on the desired application.
[0655] Prototheca moriformis undergoes rapid cell division during the first 24-30 hrs in fermenters before nitrogen runs out in the media and the cells switch to storing lipids. This initial cell division and growth in fermenters is critical for the overall strain productivity and, as reported above, FAD2 protein is crucial for sustaining vigorous growth characteristic of a particular strain. However when first generation, single insertion, genetically clean, PmFAD2-1 complemented strains (S4694 and S4695) were run in 7 L fermenters at pH 5.0 (with seed grown at pH 7.0), they did not perform on par with the original parent base strain (S1331) in terms of productivity. Western data suggested that AMT03p promoter driving PmFAD2-1 (as measured by FAD2 protein levels) is severely down regulated between 0-30 hrs in fermenters irrespective of fermenter pH (5.0 or 7.0). Work on fermentation conditions (batched vs unbatched/limited initial N, pH shift from 7 to 5 at different time points during production phase) suggested that initial batching (and excess amounts) of nitrogen during early lipid production was the likely cause of AMT03p promoter down regulation in fermenters. Indeed, this initial repression in AMT03 can be directly seen in transcript time-course during fermentation. A significant depression of Amt03 expression was observed early in the run, which corresponds directly with NH4 levels in the fermenter.
[0656] When the fermentations were performed with limited N, we were able to partially rescue the AMT03p promoter activity and while per cell productivity of S4694/S4695 was on par with the parent S1331, the overall productivity still lagged behind. These results suggest that a suboptimal or inactive AMT03p promoter and thus limitation of FAD2 protein in early fermentation stages inhibits any complemented strains from attaining their full growth potential and overall productivity. Here we identify new, improved promoter that allow differential gene activity during high-nitrogen growth and low-nitrogen lipid production phases.
[0657] In particular, we observed that:
[0658] In trans expression of the fatty acid desaturase-2 gene from Prototheca moriformis (PmFad2-1) under the control of down regulated promoter elements identified using a transcriptome based bioinformatics approach results in functional complementation of PmFAD2-1 with restored growth in .DELTA.fad2, .DELTA.fata1 strain S5204.
[0659] Complementation of S5204 manifested in a robust growth phenotype only occurs in seed and early fermentation stages when the new promoter elements are actively driving the expression of PmFAD2-1.
[0660] Once the cells enter the active lipid production phase (around the time when N runs out in the fermenter), the newly identified promoters are down regulated resulting in no additional FAD2 protein and the final oil profile of the complemented lines is same as the parent S5204 albeit with better growth characteristics.
[0661] These strains should potentially mitigate the problems that were encountered with AMT03p driven FAD2 in earlier complemented strains.
[0662] Importantly, we have identified down-regulatable promoters of varying strengths, some of which are relatively strong in the beginning with low-to-moderate levels provided during the remainder of the run. Thus depending on phenotype these promoters can be selected for fine-tuning the desired levels of transgenes.
[0663] Bioinformatics Methods: RNA was prepared from cells taken from 8 time points during a typical fermenter run. RNA was polyA-selected for run on an Illumina HiSeq. Illumina paired-end data (100 bp reads.times.2, .about.600 bp fragment size) was collected and processed for read quality using FastQC [www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Reads were run through a custom read-processing pipeline that de-duplicates, quality-trims, and length-trims reads.
[0664] Transcripts were assembled from Illumina paired-end reads using Oases/velvet [Velvet: algorithms for de novo short read assembly using de Bruijn graphs. D. R. Zerbino and E. Birney. Genome Research 18:821-829] and assessed by N50 and other metrics. The transcripts from all 8 time points were further collapsed using CD-Hit. [Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu and Weizhong Li, CD-HIT: accelerated for clustering the next generation sequencing data. Bioinformatics, (2012), 28 (23): 3150-3152. doi: 10.1093/bioinformatics/bts565; Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences", Weizhong Li & Adam Godzik Bioinformatics, (2006) 22:1658-9].
[0665] These transcripts were used as the base (reference assembly) for expression-level analysis. Reads from the 8 time points were analyzed using RSEM which provides raw read counts as well as a normalized value provided in Transcripts Per Million (TPM). [Li, Bo & Dewey, Colin N. (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome, BioMed Central: The Open Access Publisher. Retrieved at Oct. 10, 2012, from the website temoa: Open Educational Resources (OER) Portal at www.temoa.info/node/441614] The TPM was used to determine expression levels. Genes previously identified in screens for strong promoters were also used to gauge which levels should be considered as significantly high or low. This data was loaded into a Postgres database and visualized with Spotfire, along with integrated data that includes gene function and other characteristics such as categorization based on expression profile. This enabled rapid and targeted analysis of genes with significant changes in expression.
[0666] The promoters for genes, which we selected, were mapped onto a high-quality reference genome for S376 (our reference Prototheca moriformis strain). Briefly, PacBio long reads (.about.2 kb) were error-corrected by high-quality PacBio CCS reads (.about.600 bp) and assembled using the Allora assembler in SMRTPipe [pacbiodevnet.com]. This reference genome, in conjunction with transcriptome read mapping, was used to annotate the precise gene structures, promoter and UTR locations, and promoter elements within the region of interest, which then guided further sequencing and promoter element selection.
[0667] The criteria used for identifying new promoter elements were:
[0668] 1. Reasonable expression (e.g., >500, <100, or <50 transcripts per million [TPM]) of a downstream gene in seed and early lipid production stages (T0-T30 hrs)
[0669] 2. Severe down regulation of the gene above (e.g., >5-fold. 10-fold, or 15-fold) when the nitrogen gets depleted in the fermenters.
[0670] 3. pH neutrality of the promoter elements (e.g., less than a 2-fold change in TPM on going from pH 5.0 top 7.0 in cultivation conditions), or at least effective operation under pH5 conditions.
[0671] Using the above described criteria we identified several potentially down regulated promoter elements that were eventually used to drive PmFAD2-1 expression in S5204. A range of promoters was chosen that included some that started as being weak promoters and went down to extremely low levels, through those that started quite high and dropped only to moderately low levels. This was done because it was unclear a priori how much expression would be needed for FAD2 early on to support robust growth, and how little FAD2 would be required during the lipid production phase in order to achieve the zero linoleic phenotype.
[0672] The promoter elements that were selected for screening and their allelic forms were named after their downstream gene and are as follows:
[0673] 1. Carbamoyl phosphate synthase (PmCPS1p and PmCPS2p)
[0674] 2. Dipthine synthase (PmDPS1p and PmDPS2p)
[0675] 3. Inorganic pyrophosphatase (PmIPP1p)
[0676] 4. Adenosylhomocysteinase (PmAHC1p and PmAHC2p)
[0677] 5. Peptidyl-prolyl cis-trans isomerase (PmPPI1p and PmPPI2p)
[0678] 6. GMP Synthetase (PmGMPS1p and PmGMPS2p)
[0679] 7. Glutamate Synthase (PmGSp)
[0680] 8. Citrate Synthase (PmCS1p and PmCS2p)
[0681] 9. Gamma Glutamyl Hydrolase (PmGGH1p)
[0682] 10. Acetohydroxyacid Isomerase (PmAHI1p and PmAHI2p)
[0683] 11. Cysteine Endopeptidase (PmCEP1p)
[0684] 12. Fatty acid desaturase 2 (PmFAD2-1p and PmFad2-2p) [CONTROL]
[0685] The transcript profile of two representative genes viz. PmIPP (Inorganic Pyrophosphatase) and PmAHC, (Adenosylhomocysteinase) start off very strong (4000-5000 TPM) but once the cells enter active lipid production their levels fall off very quickly. While the transcript levels of PmIPP drop off to nearly 0 TPM, the levels of PmAHC drop to around 250 TPM and then stay steady for the rest of the fermentation. All the other promoters (based on their downstream gene transcript levels) showed similar downward expression profiles.
[0686] The elements were PCR amplified and wherever possible promoters from allelic genes were identified, cloned and named accordingly e.g. the promoter elements for 2 genes of Carbamoyl phosphate synthase were named PmCPS1p and PmCPS2p. As a comparator promoter elements from PmFAD2-1 and PmFAD2-2 were also amplified and used to drive PmFAD2-1 gene. While, in the present example, we used FAD2-1 expression and hence C18:2 levels to interrogate the newly identified down regulated promoters, in principle these promoter elements can be used to down regulate any gene of interest.
[0687] Construct used for the expression of the Prototheca moriformis fatty acid desaturase 2 (PmFAD2-1) under the expression of PmCPS1p in dfad2 strains S5204--[pSZ3377]: The .DELTA.fad2 .DELTA.fata1 S5204 strain was transformed with the construct pSZ3377. The sequence of the transforming DNA is provided below. Relevant restriction sites in the construct pSZ3377 (6S::PmHXT1p-ScMEL1-CvNR::PmCPS1p-PmFAD2-1-CvNR::6S) are indicated in lowercase, underlined and bold, and are from 5'-3' BspQ 1, KpnI, SpeI, SnaBI, EcoRV, SpeI, AflII, SacI, BspQ I, respectively. BspQI sites delimit the 5' and 3' ends of the transforming DNA. Bold, lowercase sequences represent genomic DNA from UTEX 1435 that permits targeted integration of the transforming DNA at the 6S locus via homologous recombination. Proceeding in the 5' to 3' direction, the Hexose transporter (HXT1) gene promoter from UTEX 1435 driving the expression of the Saccharomyces cerevisiae Melibiase (ScMEL1) gene is indicated by the boxed text. The initiator ATG and terminator TGA for ScMEL1 are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The Chlorella vulgaris nitrate reductase 3' UTR is indicated by lowercase underlined text followed by an UTEX 1435 CPS1p promoter of Prototheca moriformis, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the PmFAD2-1 are indicated by uppercase, bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3' UTR is again indicated by lowercase underlined text followed by the UTEX 1435 6S genomic region indicated by bold, lowercase text. The final construct was sequenced to ensure correct reading frames and targeting sequences.
TABLE-US-00115 Nucleotide sequence of transforming DNA contained in plasmid pSZ3377: (SEQ ID NO: 128) gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtc- agcaccgcca gccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactgg- cgtctcttc cgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccgc- agcggccgctg cccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacccgc- ccaagagt tttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtggag- gtccgggg ctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggcca- gaaacact gtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactct- ttctcctcctccttc ##STR00396## gcctgcatctccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatggg- ctgggacaact ggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctg- aaggacatgg gctacaagtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgag- cagaagttccc caacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcg- agtacacgtgc gccggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacct- gaagtacga caactgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccc- tgaacaaga cgggccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcg- aactcctggcgc atgtccggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactg- caagtacgc cggcttccactgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggct- ggaacgacct ggacaacctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatgg- tgaagtccc ccctgatcatcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatc- gccatcaaccag gactccaacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcga- gatccagat gtggtccggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatga- acacgaccct ggaggagatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtggg- cgaaccgcgtc gacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagca- gtcctacaa ggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctga- acacgaccgt ccccgcccacggcatcgcgttctaccgcctgcgcccctcctccTGAtacgtagcagcagcagtcggatagtatc- gacacactct ggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttat- caaacagcctcagtgtg tttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatcc- ccttccctcgtttcatatcgc ttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccc- tcgcacagccttggtttg ggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggg- atgggaacacaaat ##STR00397## cagcccgtggagaagccccccttcaccatcggcaccctgcgcaaggccatccccgcccactgcttcgagcgctc- cgccctgcgct cctccatgtacctggccttcgacatcgccgtgatgtccctgctgtacgtggcctccacctacatcgaccccgcc- cccgtgcccacctg ggtgaagtacggcgtgatgtggcccctgtactggttcttccagggcgccttcggcaccggcgtgtgggtgtgcg- cccacgagtgcg gccaccaggccttctcctcctcccaggccatcaacgacggcgtgggcctggtgttccactccctgctgctggtg- ccctactactcctg gaagcactcccaccgccgccaccactccaacaccggctgcctggacaaggacgaggtgttcgtgcccccccacc- gcgccgtgg cccacgagggcctggagtgggaggagtggctgcccatccgcatgggcaaggtgctggtgaccctgaccctgggc- tggcccctgt acctgatgttcaacgtggcctcccgcccctacccccgcttcgccaaccacttcgacccctggtcccccatcttc- tccaagcgcgagc gcatcgaggtggtgatctccgacctggccctggtggccgtgctgtccggcctgtccgtgctgggccgcaccatg- ggctgggcctgg ctggtgaagacctacgtggtgccctacctgatcgtgaacatgtggctggtgctgatcaccctgctgcagcacac- ccaccccgccct gccccactacttcgagaaggactgggactggctgcgcggcgccatggccaccgtggaccgctccatgggccccc- ccttcatgga caacatcctgcaccacatctccgacacccacgtgctgcaccacctgttctccaccatcccccactaccacgccg- aggaggcctcc gccgccatccgccccatcctgggcaagtactaccagtccgactcccgctgggtgggccgcgccctgtgggagga- ctggcgcgac tgccgctacgtggtgcccgacgcccccgaggacgactccgccctgtggttccacaagTAGatcgatcttaaggc- agcagcagct cggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgt- gaatatccctgccgctt ttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttg- cgaataccacccccagcatc ccatccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgct- cctgctcctgctcactgc ccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgc- tgatgcacgggaagta gtgggatgggaacacaaatggaaagcttaattaagagctcttgttttccagaaggagttgctccttgagccttt- cattctcagcctcg ataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctgg- aacaagccca gacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcg- cgcaatctgc cctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatc- tgccccctgtgc gagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaata- gttcataac agtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgctt- gcggagggca ggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccg- ggatgtgg gcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccc- tgaattcct tctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaaccctt- gtcgcgtggcg gggcttgttcgagcttgaagagc
[0688] The recombination between C. vulgaris nitrate reductase 3' UTR's in the construct pSZ3377 results in multiple copies of PmFAD2-1 in transgenic lines which would then manifest most likely as higher C18:2 levels at the end of fermentation. Since the goal was to create a strain with 0% terminal C18:2, we took precautions to avoid this recombination. In another version of the above plasmid ScMEL1 gene was followed by Chlorella protothecoides (UTEX 250) elongation factor 1a (CpEF1a) 3' UTR instead of C. vulgaris 3' UTR. The sequence of C. protothecoides (UTEX 250) elongation factor 1a (CpEF1a) 3' UTR used in construct pSZ3384 and other constructs with this 3' UTR (described below) is shown below. Plasmid pSZ3384 could be written as 6S::PmHXT1p-ScMEL1-CpEF1a::PmCPS1p-PmFAD2-1-CvNR::6S.
[0689] Nucleotide sequence of Chlorella protothecoides (UTEX 250) elongation factor 1a (CpEF1a) 3' UTR in pSZ3384:
TABLE-US-00116 (SEQ ID NO: 129) tacaacttattacgtaacggagcgtcgtgcgggagggagtgtgccgagcg gggagtcccggtctgtgcgaggcccggcagctgacgctggcgagccgtac gccccgagggtccccctcccctgcaccctcttccccttccctctgacggc cgcgcctgttcttgcatgttcagcgacgaggatatc
[0690] The C. protothecoides (UTEX 250) elongation factor 1a 3' UTR sequence is flanked by restriction sites SnaBI on 5' and EcoRV on 3' ends shown in lowercase bold underlined text. Note that the plasmids containing CpEF1a 3' UTR (pSZ3384 and others described below) after ScMEL1 stop codon contains 10 extra nucleotides before the 5' SnaBI site. These nucleotides are not present in the plasmids that contain C. vulgaris nitrate reductase 3' UTR after the S. ScMEL1 stop codon.
[0691] In addition to plasmids pSZ3377 and pSZ3384 expressing either a recombinative CvNR-Promoter-PmFAD2-1-CvNR or non-recombinative CpEF1a-Promoter-PmFAD2-1-CvNR expression unit described above, plasmids using other promoter elements mentioned above were constructed for expression in S5204. These constructs along with their transformation identifiers (D #) can be described as:
TABLE-US-00117 Plasmid ID D # Description pSZ3378 D2090 6SA::pPmHXT1-ScarIMEL1-CvNR:PmCPS2p-PmFad2-1-CvNR::6SB pSZ3385 D2097 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmCPS2p-PmFad2-1-CvNR::6SB pSZ3379 D2091 6SA::pPmHXT1-ScarIMEL1-CvNR:PmDPS1p-PmFad2-1-CvNR::6SB pSZ3386 D2098 6SA::pPmHXT1)-ScarIMEL1-CpEF1a:PmDPS1p-PmFad2-1-CvNR::6SB pSZ3380 D2092 6SA::pPmHXT1-ScarIMEL1-CvNR:PmDPS2p-PmFad2-1-CvNR::6SB pSZ3387 D2099 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmDPS2p-PmFad2-1-CvNR::6SB pSZ3480 D2259 6SA::pPmHXT1-ScarIMEL1-CvNR:PmIPP1p-PmFad2-1-CvNR::6SB pSZ3481 D2260 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmIPP1p-PmFad2-1-CvNR::6SB pSZ3509 D2434 6SA::pPmHXT1-ScarIMEL1-CvNR:PmAHC1p-PmFad2-1-CvNR::6SB pSZ3516 D2266 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmAHC1p-PmFad2-1-CvNR::6SB pSZ3510 D2435 6SA::pPmHXT1-ScarIMEL1-CvNR:PmAHC2p-PmFad2-1-CvNR::6SB pSZ3513 D2263 6SA::pPmHXT1-ScarIMEL1-CvNR:PmPPI1p-PmFad2-1-CvNR::6SB pSZ3689 D2440 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmPPI1p-PmFad2-1-CvNR::6SB pSZ3514 D2264 6SA::pPmHXT1-ScarIMEL1-CvNR:PmPPI2p-PmFad2-1-CvNR::6SB pSZ3518 D2268 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmPPI2p-PmFad2-1-CvNR::6SB pSZ3515 D2265 6SA::pPmHXT1-ScarIMEL1-CvNR:PmGMPS1p-PmFad2-1-CvNR::6SB pSZ3519 D2269 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmGMPS1p-PmFad2-1-CvNR::6SB pSZ3520 D2270 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmGMPS2p-PmFad2-1-CvNR::6SB pSZ3684 D2436 6SA::pPmHXT1-ScarIMEL1-CvNR:PmCS1p-PmFad2-1-CvNR::6SB pSZ3686 D2438 6SA::pPmHXT1-ScarIMEL1-CpEF1A:PmCS1p-PmFad2-1-CvNR::6SB pSZ3685 D2437 6SA::pPmHXT1-ScarIMEL1-CvNR:PmCS2p-PmFad2-1-CvNR::6SB pSZ3688 D2439 6SA::pPmHXT1-ScarIMEL1-CvNR:PmGGHp-PmFad2-1-CvNR::6SB pSZ3511 D2261 6SA::pPmHXT1-ScarIMEL1-CvNR:PmAHI2p-PmFad2-1-CvNR::6SB pSZ3517 D2267 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmAHI1p-PmFad2-1-CvNR::6SB pSZ3512 D2262 6SA::pPmHXT1-ScarIMEL1-CvNR:PmCEP1p-PmFad2-1-CvNR::6SB pSZ3375 D2087 6SA::pPmHXT1-ScarIMEL1-CvNR:PmFAD2-1p-PmFad2-1-CvNR::6SB pSZ3382 D2094 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmFAD2-1p-PmFad2-1-CvNR::6SB pSZ3376 D2088 6SA::pPmHXT1-ScarIMEL1-CvNR:PmFAD2-2p-PmFad2-1-CvNR::6SB pSZ3383 D2095 6SA::pPmHXT1-ScarIMEL1-CpEF1a:PmFAD2-2p-PmFad2-1-CvNR::6SB
[0692] The above constructs are the same as pSZ3377 or pSZ3384 except for the promoter element that drives PmFAD2-1. The sequences of different promoter elements used in the above constructs are shown below.
TABLE-US-00118 Nucleotide sequence of Carbamoyl phosphate synthase allele 2 promoter contained in plasmid pSZ3378 and pSZ3385 (PmCPS2p promoter sequence): (SEQ ID NO: 130) ##STR00398## Nucleotide sequence of Dipthine synthase allele 1 promoter contained in plasmid pSZ3379 and pSZ3386 (PmDPS1p promoter sequence): (SEQ ID NO: 131) ##STR00399## Nucleotide sequence of Dipthine synthase allele 2 promoter contained in plasmid pSZ3380 and pSZ3387 (PmDPS2p promoter sequence): (SEQ ID NO: 132) ##STR00400## Nucleotide sequence of Inorganic pyrophosphatase allele 1 promoter contained in plasmid pSZ3480 and pSZ3481 (PmIPP1p promoter sequence): (SEQ ID NO:133) ##STR00401## ##STR00402## Nucleotide sequence of Adenosylhomocysteinase allele 1 promoter contained in plasmid pSZ3509 and pSZ3516 (PmAHC1p promoter sequence): (SEQ ID NO: 134) ##STR00403## Nucleotide sequence of Adenosylhomocysteinase allele 2 promoter contained in plasmid pSZ3510 (PmAHC2p promoter sequence): (SEQ ID NO: 135) ##STR00404## Nucleotide sequence of Peptidyl-prolyl cis-trans isomerase allele 1 promoter contained in plasmid pSZ3513 and pSZ3689 (PmPPI1p promoter sequence): (SEQ ID NO: 136) ##STR00405## Nucleotide sequence of Peptidyl-prolyl cis-trans isomerase allele 2 promoter contained in plasmid pSZ3514 and pSZ3518 (PmPPI2p promoter sequence): (SEQ ID NO: 137) ##STR00406## Nucleotide sequence of GMP Synthetase allele 1 promoter contained in plasmid pSZ3515 and pSZ3519 (PmGMPS1p promoter sequence): (SEQ ID NO: 138) ##STR00407## Nucleotide sequence of GMP Synthetase allele 2 promoter contained in plasmid pSZ3520 (PmGMPS2p promoter sequence): (SEQ ID NO: 139) ##STR00408## Nucleotide sequence of Citrate synthase allele 1 promoter contained in plasmid pSZ3684 and pSZ3686 (PmCS1p promoter sequence): (SEQ ID NO: 140) ##STR00409## ##STR00410## Nucleotide sequence of Citrate synthase allele 2 promoter contained in plasmid pSZ3685 (PmCS2p promoter sequence): (SEQ ID NO: 141) ##STR00411## ##STR00412## Nucleotide sequence of Gamma Glutamyl Hydrolase allele 1 promoter contained in plasmid pSZ3688 (PmGGH1p promoter sequence): (SEQ ID NO: 142) ##STR00413## Nucleotide sequence of Acetohydroxyacid Isomerase allele 1 promoter contained in plasmid pSZ3517 (PmAHI1p promoter sequence): (SEQ ID NO: 143) ##STR00414## Nucleotide sequence of Acetohydroxyacid Isomerase allele 2 promoter contained in plasmid pSZ3511 (PmAHI2p promoter sequence): (SEQ ID NO: 144) ##STR00415## Nucleotide sequence of Cysteine Endopeptidase allele 1 promoter contained in plasmid pSZ3512 (PmCEP1 promoter sequence): (SEQ ID NO: 145) ##STR00416## Nucleotide sequence of Fatty acid desaturase 2 allele 1 promoter contained in plasmid pSZ3375 and 3382 (PmFAD2-1 promoter sequence): (SEQ ID NO: 146) ##STR00417## Nucleotide sequence of Fatty acid desaturase 2 allele 2 promoter contained in plasmid pSZ3376 and 3383 (PmFAD2-2 promoter sequence): (SEQ ID NO: 147) ##STR00418##
[0693] To determine their impact on growth and fatty acid profiles, the above-described constructs were independently transformed into a .DELTA.fad2 .DELTA.fata1 strain S5204. Primary transformants were clonally purified and grown under standard lipid production conditions at pH5.0 or at pH7.0. The resulting profiles from a set of representative clones arising from transformations are shown in Tables 84-114.
TABLE-US-00119 TABLE 84 Fatty acid profile in some representative complemented (D2087) and parent S5204 lines transformed with pSZ3375 DNA containing PmFAD2-1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH 7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH 5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH 7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH 5; S5204 0.39 5.67 1.36 91.13 0 0 pH 7; S5204; T665; D2087-22 0.38 4.43 1.78 83.93 7.58 0.81 pH 7; S5204; T665; D2087-16 0.41 4.92 1.94 83.21 7.55 0.84 pH 7; S5204; T665; D2087-17 0.40 4.82 1.78 83.51 7.52 0.79 pH 7; S5204; T665; D2087-26 1.30 8.06 2.54 79.03 7.30 0.82 pH 7; S5204; T665; D2087-29 1.13 7.88 2.45 79.48 7.26 0.79
TABLE-US-00120 TABLE 85 Fatty acid profile in some representative complemented (D) and parent S5204 lines transformed with pSZ3382 DNA containing PmFAD2-1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH 7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH 5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH 7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH 5; S5204 0.39 5.67 1.36 91.13 0 0 pH 7; S5204; T672; D2094-5 0.49 5.76 2.95 83.39 5.08 0.84 pH 7; S5204; T672; D2094-25 0.35 5.01 2.41 85.10 5.09 0.64 pH 7; S5204; T672; D2094-13 0.33 5.07 2.30 84.89 5.30 0.69 pH 7; S5204; T672; D2094-11 0.38 4.33 1.78 85.63 5.31 0.85 pH 7; S5204; T672; D2094-8 0.35 5.29 2.32 84.59 5.34 0.66
TABLE-US-00121 TABLE 86 Fatty acid profile in some representative complemented (D2088) and parent S5204 lines transformed with pSZ3376 DNA containing PmFAD2-2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH 7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH 5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH 7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH 5; S5204 0.39 5.67 1.36 91.13 0 0 pH 7; S5204; T665; D2088-16 1.11 8.18 2.92 78.13 6.96 0.87 pH 7; S5204; T665; D2088-20 1.06 7.78 2.95 78.65 6.95 0.84 pH 7; S5204; T665; D2088-29 0.91 7.13 2.87 79.63 6.93 0.78 pH 7; S5204; T665; D2088-6 1.18 8.29 2.98 77.90 6.91 0.88 pH 7; S5204; T665; D2088-18 1.10 7.98 3.09 78.42 6.78 0.81
TABLE-US-00122 TABLE 87 Fatty acid profile in some representative complemented (D) and parent S5204 lines transformed with pSZ3383 DNA containing PmFAD2-2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH7; S5204; T673; 0.30 5.43 2.45 85.10 4.62 0.68 D2095-47 pH7; S5204; T673; 0.38 5.16 2.48 84.46 5.41 0.68 D2095-14 pH7; S5204; T673; 0.43 4.60 2.54 84.82 5.47 0.58 D2095-16 pH7; S5204; T673; 0.34 5.41 2.57 84.21 5.49 0.66 D2095-6 pH7; S5204; T673; 0.42 5.30 2.49 83.97 5.57 0.68 D2095-39
TABLE-US-00123 TABLE 88 Fatty acid profile in representative complemented (D2089) and parent S5204 lines transformed with pSZ3377 DNA containing PmCPS1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T672; 0.35 4.73 2.29 88.94 1.79 0.39 D2089-40 pH7; S5204; T672; 0.51 4.85 2.96 87.55 2.05 0.41 D2089-2 pH7; S5204; T672; 0.56 5.00 3.04 87.24 2.07 0.36 D2089-14 pH7; S5204; T672; 0.38 5.04 2.39 88.02 2.39 0.44 D2089-7 pH7; S5204; T672; 0.38 5.00 2.37 87.93 2.42 0.43 D2089-18
TABLE-US-00124 TABLE 89 Fatty acid profile in some representative complemented (D2096) and parent S5204 lines transformed with pSZ3384 DNA containing PmCPS1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T673; 0.33 4.18 1.10 92.91 0.00 0.00 D2096-6 pH7; S5204; T673; 0.36 4.14 1.33 92.42 0.34 0.12 D2096-12 pH7; S5204; T673; 0.32 4.35 1.64 92.12 0.35 0.14 D2096-14 pH7; S5204; T673; 0.50 6.44 0.95 89.81 0.46 0.32 D2096-8 pH7; S5204; T673; 0.29 3.93 1.79 91.19 1.34 0.37 D2096-1
TABLE-US-00125 TABLE 90 Fatty acid profile in some representative complemented (D2090) and parent S5204 lines transformed with pSZ3378 DNA containing PmCPS2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T672; 0.33 4.73 1.84 91.24 0.00 0.00 D2090-5 pH7; S5204; T672; 0.42 4.99 2.01 91.06 0.00 0.00 D2090-29 pH7; S5204; T672; 0.43 4.31 1.87 90.44 0.78 0.16 D2090-22 pH7; S5204; T672; 0.32 3.77 2.43 89.72 1.68 0.35 D2090-1 pH7; S5204; T672; 0.49 5.01 1.97 88.48 1.84 0.38 D2090-32
TABLE-US-00126 TABLE 91 Fatty acid profile in some representative complemented (D2097) and parent S5204 lines transformed with pSZ3385 DNA containing PmCPS2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH5; S5204; T680; 0.50 5.73 1.97 87.12 2.61 0.76 D2097-1 pH5; S5204; T680; 0.75 8.20 2.46 85.73 0.89 0.53 D2097-2
TABLE-US-00127 TABLE 92 Fatty acid profile in some representative complemented (D2091) and parent S5204 lines transformed with pSZ3379 DNA containing PmDPS1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T672; 1.42 4.39 2.32 89.87 0.00 0.00 D2091-4 pH7; S5204; T672; 0.27 4.79 2.24 90.94 0.00 0.00 D2091-14 pH7; S5204; T672; 0.30 5.26 2.20 90.73 0.00 0.00 D2091-15 pH7; S5204; T672; 0.31 4.51 1.77 91.65 0.00 0.00 D2091-19 pH7; S5204; T672; 0.31 5.36 2.24 90.67 0.00 0.00 D2091-46
TABLE-US-00128 TABLE 93 Fatty acid profile in some representative complemented (D2098) and parent S5204 lines transformed with pSZ3386 DNA containing PmDPS1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T680; 0.34 4.89 1.56 92.08 0.00 0.00 D2098-39 pH7; S5204; T680; 0.30 4.31 1.61 92.34 0.30 0.00 D2098-7 pH7; S5204; T680; 0.33 3.89 1.58 92.65 0.36 0.00 D2098-3 pH7; S5204; T680; 0.32 4.18 1.64 92.34 0.36 0.11 D2098-25 pH7; S5204; T680; 0.32 4.36 1.50 92.10 0.37 0.12 D2098-13
TABLE-US-00129 TABLE 94 Fatty acid profile in some representative complemented (D2092) and parent S5204 lines transformed with pSZ3380 DNA containing PmDPS2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T672; 0.29 5.13 1.59 92.16 0.00 0.00 D2092-35 pH7; S5204; T672; 0.37 4.66 1.75 91.71 0.19 0.05 D2092-29 pH7; S5204; T672; 0.24 3.47 1.84 93.19 0.43 0.11 D2092-15 pH7; S5204; T672; 0.25 3.50 1.82 93.16 0.44 0.09 D2092-21 pH7; S5204; T672; 0.28 3.18 1.50 93.59 0.52 0.12 D2092-16
TABLE-US-00130 TABLE 95 Fatty acid profile in some representative complemented (D2099) and parent S5204 lines transformed with pSZ3387 DNA containing PmDPS2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH7; S5204; T680; 0.31 4.02 1.46 93.07 0.00 0.00 D2099-20 pH7; S5204; T680; 0.28 4.67 1.50 92.38 0.00 0.00 D2099-24 pH7; S5204; T680; 0.40 4.07 1.22 93.26 0.00 0.00 D2099-27 pH7; S5204; T680; 0.32 4.59 1.57 92.40 0.00 0.00 D2099-30 pH7; S5204; T680; 0.30 4.56 1.54 92.49 0.00 0.00 D2099-35
TABLE-US-00131 TABLE 96 Fatty acid profile in some representative complemented (D2259) and parent S5204 lines transformed with pSZ3480 DNA containing PmIPP1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH5; S5204; T711; 0.36 5.27 2.19 89.32 1.51 0.51 D2259-43 pH5; S5204; T711; 0.35 4.88 2.17 86.34 4.41 0.70 D2259-22 pH5; S5204; T711; 0.35 4.82 2.18 86.32 4.45 0.69 D2259-28 pH5; S5204; T711; 0.33 4.90 2.08 86.33 4.49 0.74 D2259-21 pH5; S5204; T711; 0.50 5.97 2.14 84.67 4.49 0.74 D2259-36
TABLE-US-00132 TABLE 97 Fatty acid profile in some representative complemented (D2260) and parent S5204 lines transformed with pSZ3481 DNA containing PmIPP1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.10 0.00 pH5; S5204 0.39 5.67 1.36 91.13 0.00 0.00 pH5; S5204; T711; 0.36 4.96 2.10 89.46 1.55 0.49 D2260-32 pH5; S5204; T711; 0.33 4.83 1.99 89.40 1.63 0.58 D2260-10 pH5; S5204; T711; 0.34 4.83 2.16 89.39 1.64 0.49 D2260-2 pH5; S5204; T711; 0.37 4.81 2.11 89.51 1.69 0.26 D2260-30 pH5; S5204; T711; 0.33 4.91 2.17 89.73 1.72 0.16 D2260-41
TABLE-US-00133 TABLE 98 Fatty acid profile in some representative complemented (D2434) and parent S5204 lines transformed with pSZ3509 DNA containing PmAHC1p driving PmFAD2-1. Sample ID C14.0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.33 4.45 1.55 81.55 8.51 1.38 T768; D2434-32 pH5; S5204; 0.62 7.27 1.58 78.65 9.44 1.49 T768; D2434-27 pH5; S5204; 0.38 5.81 1.79 79.63 10.01 1.18 T768; D2434-4 pH5; S5204; 0.5 5.93 1.5 78.7 10.25 1.56 T768; D2434-23 pH5; S5204; 0.51 6.08 1.6 78.79 10.25 1.36 T768; D2434-43
TABLE-US-00134 TABLE 99 Fatty acid profile in some representative complemented (D2266) and parent S5204 lines transformed with pSZ3516 DNA containing PmAHC1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; T718; 0.32 5.41 1.94 91.26 0.11 0.00 D2266-46 pH5; S5204; T718; 0.36 5.33 1.90 91.17 0.17 0.00 D2266-36 pH5; S5204; T718; 0.37 4.96 2.13 90.82 0.41 0.00 D2266-35 pH5; S5204; T718; 0.38 5.33 2.10 90.31 0.44 0.31 D2266-41 pH5; S5204; T718; 0.36 5.15 2.23 90.55 0.48 0.31 D2266-5
TABLE-US-00135 TABLE 100 Fatty acid profile in some representative complemented (D2435) and parent S5204 lines transformed with pSZ3510 DNA containing PmAHC2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; T768; 0.35 6.09 1.90 78.52 11.01 1.18 D2435-37 pH5; S5204; T768; 0.43 5.90 1.97 78.74 10.97 1.20 D2435-3 pH5; S5204; T768; 0.40 6.01 1.89 79.00 10.97 1.14 D2435-20 pH5; S5204; T768; 0.39 6.11 1.89 78.26 10.84 1.24 D2435-13 pH5; S5204; T768; 0.46 6.02 1.97 79.48 10.46 1.19 D2435-34
TABLE-US-00136 TABLE 101 Fatty acid profile in some representative complemented (D2263) and parent S5204 lines transformed with pSZ3513 DNA containing PmPPI1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; T718; 0.75 9.44 1.98 87.09 0.00 0.00 D2263-13 pH5; S5204; T718; 0.58 7.72 1.64 89.26 0.00 0.00 D2263-14 pH5; S5204; T718; 0.62 7.92 1.56 89.25 0.00 0.00 D2263-19 pH5; S5204; T718; 0.42 7.39 1.70 89.28 0.00 0.00 D2263-26 pH5; S5204; T718; 0.58 7.32 1.30 90.07 0.00 0.00 D2263-29
TABLE-US-00137 TABLE 102 Fatty acid profile in some representative complemented (D2440) and parent S5204 lines transformed with pSZ3689 DNA containing PmPPI1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.31 6.24 1.41 90.42 0.17 0.05 T770; D2440-23 pH5; S5204; 0.23 4.69 1.41 91.72 0.17 0.00 T770; D2440-32 pH5; S5204; 0.30 6.31 1.49 90.21 0.17 0.00 T770; D2440-38 pH5; S5204; 0.30 6.33 1.38 90.29 0.18 0.05 T770; D2440-7 pH5; S5204; 0.29 6.38 1.36 90.39 0.18 0.05 T770; D2440-36 pH5; S5204; 0.34 5.63 1.15 91.15 0.19 0.05 T770; D2440-8
TABLE-US-00138 TABLE 103 Fatty acid profile in some representative complemented (D2264) and parent S5204 lines transformed with pSZ3514 DNA containing PmPPI2p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH7; S6207; 0.49 6.15 1.61 90.82 0.00 0.00 T718; D2264-1 pH7; S6207; 0.38 5.36 1.51 91.58 0.00 0.00 T718; D2264-6 pH7; S6207; 0.45 6.09 1.46 91.10 0.00 0.00 T718; D2264-29 pH7; S6207; 0.40 5.42 2.28 89.86 0.90 0.00 T718; D2264-4 pH7; S6207; 0.40 5.37 2.02 90.18 1.04 0.00 T718; D2264-7
TABLE-US-00139 TABLE 104 Fatty acid profile in some representative complemented (D2268) and parent S5204 lines transformed with pSZ3518 DNA containing PmPPI2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.39 6.43 1.78 90.49 0.00 0.00 T720; D2268-1 pH5; S5204; 0.38 6.49 1.74 90.38 0.00 0.00 T720; D2268-2 pH5; S5204; 0.38 6.56 1.74 90.27 0.00 0.00 T720; D2268-3 pH5; S5204; 0.45 5.73 1.52 91.75 0.00 0.00 T720; D2268-4 pH5; S5204; 0.38 6.58 1.81 90.79 0.00 0.00 T720; D2268-5
TABLE-US-00140 TABLE 105 Fatty acid profile in some representative complemented (D2265) and parent S5204 lines transformed with pSZ3515 DNA containing PmGMPS1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.46 7.02 1.71 90.06 0.00 0.00 T718; D2265-16 pH5; S5204; 0.00 7.90 1.90 89.27 0.00 0.00 T718; D2265-43 pH5; S5204; 0.46 5.53 1.68 91.28 0.35 0.00 T718; D2265-14 pH5; S5204; 0.39 6.17 1.75 90.44 0.42 0.00 T718; D2265-4 pH5; S5204; 0.49 5.87 1.77 90.51 0.45 0.00 T718; D2265-9
TABLE-US-00141 TABLE 106 Fatty acid profile in some representative complemented (D2269) and parent S5204 lines transformed with pSZ3519 DNA containing PmGMPS1p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.38 6.73 1.68 90.24 0.00 0.00 T720; D2269-1 pH5; S5204; 0.36 6.76 1.71 90.17 0.00 0.00 T720; D2269-3 pH5; S5204; 0.42 6.57 1.71 90.32 0.00 0.00 T720; D2269-4 pH5; S5204; 0.59 8.81 1.93 87.97 0.00 0.00 T720; D2269-5 pH5; S5204; 0.50 7.29 1.73 89.29 0.00 0.00 T720; D2269-6
TABLE-US-00142 TABLE 107 Fatty acid profile in some representative complemented (D2270) and parent S5204 lines transformed with pSZ3520 DNA containing PmGMPS2p driving PmFAD2-1. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.37 6.80 1.74 90.18 0.00 0.00 T720; D2270-1 pH5; S5204; 0.46 6.76 1.83 89.90 0.00 0.00 T720; D2270-2 pH5; S5204; 0.41 6.69 1.70 90.22 0.00 0.00 T720; D2270-3 pH5; S5204; 0.43 7.44 1.72 89.31 0.00 0.00 T720; D2270-4 pH5; S5204; 0.44 6.98 1.78 89.79 0.00 0.00 T720; D2270-5
TABLE-US-00143 TABLE 108 Fatty acid profile in some representative complemented (D2436) and parent S5204 lines transformed with pSZ3684 DNA containing PmCS1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 7.59 1.57 88.88 0.18 0.00 0.00 T768; D2436-48 pH5; S5204; 6.37 1.50 85.00 3.97 1.04 0.00 T768; D2436-1 pH5; S5204; 9.40 1.86 81.13 4.11 1.21 0.00 T768; D2436-16 pH5; S5204; 6.07 1.77 84.78 4.26 0.94 0.00 T768; D2436-8 pH5; S5204; 5.97 1.62 85.28 4.50 0.98 0.00 T768; D2436-32
TABLE-US-00144 TABLE 109 Fatty acid profile in some representative complemented (D2438) and parent S5204 lines transformed with pSZ3686 DNA containing PmCS1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.50 5.96 1.69 89.87 1.30 0.00 T770; D2438-7 pH5; S5204; 0.41 6.05 1.86 87.88 2.46 0.00 T770; D2438-11 pH5; S5204; 0.41 5.75 1.93 88.35 2.50 0.00 T770; D2438-9 pH5; S5204; 0.45 6.18 1.85 87.86 2.59 0.00 T770; D2438-15 pH5; S5204; 0.40 5.92 1.97 87.80 2.59 0.00 T770; D2438-37
TABLE-US-00145 TABLE 110 Fatty acid profile in some representative complemented (D2437) and parent S5204 lines transformed with pSZ3685 DNA containing PmCSCp driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.00 4.83 1.98 90.43 1.17 0.53 T768; D2437-15 pH5; S5204; 0.45 6.03 1.81 88.69 1.88 0.31 T768; D2437-35 pH5; S5204; 0.39 4.96 2.00 88.58 3.24 0.00 T768; D2437-17 pH5; S5204; 0.90 9.55 2.07 82.29 3.37 1.24 T768; D2437-26 pH5; S5204; 0.53 10.76 1.55 79.62 4.46 1.12 T768; D2437-8
TABLE-US-00146 TABLE 111 Fatty acid profile in some representative complemented (D2439) and parent S5204 lines transformed with pSZ3688 DNA containing PmGGHp driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.31 6.79 1.47 89.97 0.00 0.00 T770; D2439-11 pH5; S5204; 0.27 4.19 0.94 92.91 0.08 0.00 T770; D2439-22 pH5; S5204; 0.39 6.02 1.26 90.91 0.16 0.00 T770; D2439-12 pH5; S5204; 0.64 6.50 1.10 89.53 0.20 0.00 T770; D2439-34 pH5; S5204; 0.33 5.25 1.45 89.98 1.08 0.51 T770; D2439-32
TABLE-US-00147 TABLE 112 Fatty acid profile in some representative complemented (D2261) and parent S5204 lines transformed with pSZ3511 DNA containing PmAHI2p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.45 5.06 2.02 89.35 1.73 0.63 T711; D2261-35 pH5; S5204; 0.46 5.12 2.19 88.92 2.16 0.19 T711; D2261-8 pH5; S5204; 0.37 5.12 2.15 88.62 2.30 0.45 T711; D2261-43 pH5; S5204; 0.42 5.27 2.14 88.23 2.39 0.30 T711; D2261-2 pH5; S5204; 0.41 5.14 2.23 88.44 2.39 0.45 T711; D2261-24
TABLE-US-00148 TABLE 113 Fatty acid profile in some representative complemented (D2267) and parent S5204 lines transformed with pSZ3517 DNA containing PmAHI1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.34 4.87 2.11 90.00 1.20 0.39 T720; D2267-3 pH5; S5204; 0.37 5.00 2.14 89.50 1.46 0.49 T720; D2267-20 pH5; S5204; 0.34 4.90 2.08 89.75 1.67 0.36 T720; D2267-36 pH5; S5204; 0.37 4.95 2.14 89.77 1.69 0.00 T720; D2267-15 pH5; S5204; 0.35 4.85 2.12 89.71 1.72 0.32 T720; D2267-2
TABLE-US-00149 TABLE 114 Fatty acid profile in some representative complemented (D2262) and parent S5204 lines transformed with pSZ3512 DNA containing PmCEP1p driving PmFAD2-1. C18:3 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 .alpha. pH7; S3150 1.71 29.58 3.13 56.53 6.43 0.68 pH5; S3150 1.56 27.70 2.98 59.49 5.95 0.53 pH7; S5204 0.30 5.59 1.63 90.88 0.1 0 pH5; S5204 0.39 5.67 1.36 91.13 0 0 pH5; S5204; 0.48 5.50 2.08 90.58 0.35 0.00 T711; D2262-3 pH5; S5204; 0.39 5.20 2.17 89.90 1.08 0.37 T711; D2262-33 pH5; S5204; 0.34 5.08 1.93 89.69 1.34 0.37 T711; D2262-24 pH5; S5204; 0.40 4.89 2.19 89.88 1.45 0.27 T711; D2262-32 pH5; S5204; 0.39 4.95 2.75 89.30 1.47 0.27 T711; D2262-34
[0694] Combined baseline expression of endogenous PmFAD2-1 and PmFAD2-2 in wild type Prototheca strains (like S3150, S1920 or S1331) manifests as 5-7% C18:2. S5204 overexpresses PmKASII which results in the elongation of C16:0 to C18:0. This increased pool of C18:0 is eventually desaturated by PmSAD2 resulting in elevated C18:1 levels. Additionally disruption of the both copies of PmFAD2 (viz. PmFAD2-1 and PmFAD2-2) in S5204 prevents further desaturation of C18:1 into C18:2 and results in a unique high oleic oil (C18:1) with 0% linoleic acid (C18:2). However as mentioned above any strain with 0% C18:2 grows very poorly and requires exogenous addition of linoleic acid to sustain growth/productivity. Complementation of a strain like S5204 with inducible PmAMT03p driven PmFAD2-1 can rescue the growth phenotype while preserving the terminal high C18:1 with 0% C18:2 levels. However data suggests that PmAMT03 shuts off in the early stages of fermentation thus severely compromising the ability of any complemented strain to achieve its full growth and productivity potential. The goal of this work was to identify promoter elements that would allow the complemented strains to grow efficiently in early stages of fermentation (TO-T30 hrs; irrespective of excess batched N in the fermenters) and then effectively shut off once the cells enter active lipid production (when N in the media gets depleted) so that the complemented strains would still finish with very high C18:1 and 0% C18:2 levels. As a comparator we also complemented S5204 with PmFAD2-1 being driven by either PmFAd2-1p or PmFAD2-2p promoter elements.
[0695] Complementation of S5204 with PmFAD2-1 driven by either PmFAD2-1p or PmFAD2-2p promoter elements results in complete restoration of the C18:2 levels using vectors either designed to amplify PmFAD2-1 copy number (e.g. pSZ3375 or pSZ3376) or the ones where PmFAD2-1 copy number is restricted to one (pSZ3382 or pSZ3383). Copy number of the PmFAD2-1 in these strains seems to have very marginal effect on the terminal C18:2 levels.
[0696] On the other hand expression of PmFAD2-1 driven by any of new promoter elements results in marked decrease in terminal C18:2 levels. The representative profiles from various strains expressing new promoters driving FAD2-1 are shown in Tables 84-114. This reduction in C18:2 levels is even more pronounced in strains where the copy number of PmFAD2-1 is limited to one. Promoter elements like PmDPS1 (D2091 & D2098), PmDPS2 (D2092 & D2099), PmPPI 1 (D2263 & D2440), PmPPI2 (D2264 & D2268), PmGMPS1 (D2265 & D2269), PmGMPS2 (D2270) resulted in strains with 0% or less than 0.5% terminal C18:2 levels in both single or multiple copy PmFAD2-1 versions. The rest of the promoters resulted in terminal C18:2 levels that ranged between 1-5%. One unexpected result was the data from PmAHC1p and PmAHC2p driving PmFAD2-1 in D2434 and D2435. Both these promoters resulted in very high levels of C18:2 (9-20%) in multiple copy FAD2-1 versions. The levels of terminal C18:2 in single copy version in D2266 was more in line with the transcriptomic data suggesting that PmAHC promoter activity and the corresponding PmAHC transcription is severely downregulated when cells are actively producing lipid in depleted nitrogen environment. A quick look at the transcriptome revealed that the initial transcription of PmAHC is very high (4000-5500 TPM) which then suddenly drops down to 250 TPM. Thus it is conceivable that in strains with multiple copies on PmFAD2-1 (D2434 and D2435), the massive amount of PmFAD2-1 protein produced earlier in the fermentation lingers and results in high C18:2 levels. In single copy PmFAD2-1 strains this is not the case and thus we do not see elevated C18:2 levels in D2266.
[0697] In complemented strains with 0% terminal C18:2 levels, the key question was whether they were complemented in the first place. In order to ascertain that, representative strains along with parent S5204 and previously AMT03p driven PmFAD2-1 complemented S2532 (viz S4695) strains were grown in seed medium in 96 well blocks. The cultures were seeded at 0.1 OD units per ml and the OD750 was checked at different time points. Compared to S5204, which grew very poorly, only S4695 and newly complemented strains grew to any meaningful OD's at 20 and 44 hrs (Table 115) demonstrating that the promoters identified above are active early on and switch off once cells enter the active lipid production phase.
TABLE-US-00150 TABLE 115 Growth characteristics of .DELTA.fad2 .DELTA.fata1 strain S5204, S4695 and representative complemented S5204 lines in seed medium sorted by OD750 at 44 hrs. Note that in 1 ml 96 well blocks after initial rapid division and growth, cells stop growing efficiently because of lack of nutrients, aeration etc. OD750 OD750 OD750 Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 C18:3.alpha. @20 hrs @44 hrs @68 hrs S5204 0.162 7.914 10.93 S5204 0.224 6.854 9.256 S4695 1.456 29.032 32.766 pH7; S5204; T672; D2091-46 0.31 5.36 2.24 90.67 0.00 0.00 1.38 33.644 33.226 pH5; S5204; T720; D2268-1 0.39 6.43 1.78 90.49 0.00 0.00 0.75 32.782 31.624 S5204; 1720; D2270-47 0.39 6.69 1.81 90.05 0.00 0.00 1.204 32.752 31.602 pH5; S5204; T720; D2270-39 0.39 6.87 1.81 89.94 0.00 0.00 1.012 32.552 33.138 pH7; S5204; T680; D2099-35 0.30 4.56 1.54 92.49 0.00 0.00 0.48 32.088 31.92 pH5; S5204; T720; D2270-44 0.51 6.85 1.74 90.06 0.00 0.00 1.468 31.802 30.61 pH5; S5204; T720; D2270-41 0.00 7.85 1.65 89.18 0.00 0.00 1.576 31.35 30.69 pH5; S5204; T720; D2270-17 0.46 6.78 1.71 90.24 0.00 0.00 1.79 30.732 24.768 pH7; S5204; T680; D2099-30 0.32 4.59 1.57 92.40 0.00 0.00 0.59 30.166 34.64 pH5; S5204; T720; D2268-40 0.42 6.66 1.86 90.02 0.00 0.00 0.764 29.62 29 pH5; S5204; T720; D2270-23 0.39 6.52 1.72 90.35 0.00 0.00 1.334 29.604 27.518 pH5; S5204; T720; D2270-42 0.61 6.59 1.53 90.28 0.00 0.00 2.042 28.986 32.184 pH7; S5204; T672; D2090-5 0.33 4.73 1.84 91.24 0.00 0.00 1.326 28.976 35.508 pH7; S5204; T672; D2091-15 0.30 5.26 2.20 90.73 0.00 0.00 0.826 28.824 32.848 pH7; S5204; T680; D2099-20 0.31 4.02 1.46 93.07 0.00 0.00 1.31 28.732 26.61 pH5; S5204; T720; D2269-19 0.42 6.51 1.61 90.43 0.00 0.00 1.278 28.65 31.362 pH5; S5204; T720; D2269-29 0.43 7.36 1.72 89.35 0.00 0.00 1.342 28.376 28.66 pH5; S5204; T720; D2270-19 0.39 6.81 1.75 90.05 0.00 0.00 2.142 28.376 25.934 pH5; S5204; T720; D2270-43 0.80 7.64 1.66 88.93 0.00 0.00 1.896 28.174 32.376 pH5; S5204; T720; D2270-46 0.45 6.75 1.72 90.02 0.00 0.00 1.644 28.122 30.464 pH5; S5204; T720; D2268-3 0.38 6.56 1.74 90.27 0.00 0.00 0.926 28.114 31.552 pH5; S5204; T720; D2268-12 0.00 5.68 1.84 91.53 0.00 0.00 1.414 28.106 30.644 pH5; S5204; T720; D2269-37 0.54 7.12 1.75 89.80 0.00 0.00 1.268 28.078 30.014 pH5; S5204; T720; D2270-31 0.46 6.94 1.74 89.71 0.00 0.00 1.224 28.064 29.344 pH5; S5204; T720; D2270-48 0.00 7.21 1.87 90.16 0.00 0.00 1.352 28 28.21 pH5; S5204; T720; D2269-8 0.33 6.67 1.64 90.34 0.00 0.00 0.96 27.912 27.564 pH5; S5204; T720; D2268-32 0.44 6.59 1.85 90.11 0.00 0.00 0.78 27.834 31.952 pH5; S5204; T720; D2269-47 0.42 6.83 1.82 89.85 0.00 0.00 1.17 27.76 29.648 pH7; S5204; T672; D2091-19 0.31 4.51 1.77 91.65 0.00 0.00 1.568 27.682 25.828 pH5; S5204; T720; D2270-38 0.39 6.65 1.83 90.11 0.00 0.00 1.74 27.606 31.104 pH5; S5204; T720; D2268-2 0.38 6.49 1.74 90.38 0.00 0.00 0.95 27.564 32.254 pH5; S5204; T720; D2269-35 0.38 7.04 1.68 89.82 0.00 0.00 1.19 27.482 29.186 pH5; S5204; T720; D2269-20 0.36 7.01 1.73 89.86 0.00 0.00 0.966 27.47 28.284 pH5; S5204; T720; D2269-13 0.39 6.76 1.89 89.98 0.00 0.00 0.936 27.39 33.464 pH7; S5204; T680; D2099-24 0.28 4.67 1.50 92.38 0.00 0.00 0.8 27.28 27.35 pH5; S5204; T720; D2268-11 0.38 6.56 1.85 90.56 0.00 0.00 1.136 27.254 32.508 pH5; S5204; T720; D2270-3 0.41 6.69 1.70 90.22 0.00 0.00 0.872 27.214 30.23 pH5; S5204; T720; D2269-33 0.39 6.36 1.67 90.59 0.00 0.00 0.956 27.194 30.568 pH5; S5204; T720; D2268-10 0.45 6.93 1.70 90.16 0.00 0.00 0.612 27.126 31.616 pH5; S5204; T720; D2269-43 0.36 6.55 1.84 90.25 0.00 0.00 0.998 27.086 29.618 pH5; S5204; T720; D2270-1 0.37 6.80 1.74 90.18 0.00 0.00 2.428 27.004 31.044 pH5; S5204; T720; D2268-4 0.45 5.73 1.52 91.75 0.00 0.00 0.736 26.948 28.796 pH5; S5204; T720; D2270-9 0.38 6.88 1.74 90.22 0.00 0.00 2.68 26.944 29.92 pH5; S5204; T720; D2269-26 0.41 6.85 1.68 90.03 0.00 0.00 0.896 26.794 31.31 pH5; S5204; T720; D2270-24 0.39 6.51 1.78 90.33 0.00 0.00 1.51 26.682 27.486 pH5; S5204; T720; D2269-18 0.41 7.04 1.71 89.83 0.00 0.00 1.024 26.58 29.794 pH5; S5204; T720; D2269-32 0.38 6.81 1.72 90.06 0.00 0.00 1.214 26.48 29.478 pH5; S5204; T720; D2268-31 0.33 6.68 1.76 90.20 0.00 0.00 0.808 26.432 31.294 pH5; S5204; T720; D2269-7 0.29 5.33 1.69 91.59 0.00 0.00 1.1 26.41 28.754 pH5; S5204; T720; D2268-6 0.39 6.62 1.70 90.28 0.00 0.00 0.626 26.372 30.822 pH7; S5204; T680; D2099-27 0.40 4.07 1.22 93.26 0.00 0.00 0.936 26.116 29.75 pH5; S5204; T720; D2269-39 0.48 6.88 1.82 89.67 0.00 0.00 2.218 26.106 30.8 pH5; S5204; T720; D2269-12 0.35 6.39 1.80 90.47 0.00 0.00 1.18 26.032 28.19 pH5; S5204; T720; D2269-42 0.39 6.99 1.67 89.91 0.00 0.00 2.132 25.924 27.854 pH5; S5204; T720; D2268-8 0.56 6.77 1.49 90.20 0.00 0.00 0.96 25.702 29.788 pH5; S5204; T720; D2270-37 0.44 7.33 1.71 89.69 0.00 0.00 0.916 25.612 34.034 pH5; S5204; T720; D2270-40 0.00 9.30 1.62 88.12 0.00 0.00 2.072 25.552 29.474 pH5; S5204; T720; D2270-14 0.43 7.40 1.71 89.73 0.00 0.00 1.916 25.526 27.908 pH5; S5204; T720; D2269-21 0.40 6.69 1.69 89.99 0.00 0.00 0.826 25.396 29 pH5; S5204; T718; D2265-16 0.46 7.02 1.71 90.06 0.00 0.00 0.9 25.332 32.018 pH5; S5204; T720; D2270-15 0.40 6.90 1.68 90.32 0.00 0.00 1.594 25.32 26.794 pH5; S5204; T720; D2269-40 0.00 7.00 1.66 90.15 0.00 0.00 1.804 25.286 29.468 pH5; S5204; T720; D2268-5 0.38 6.58 1.81 90.79 0.00 0.00 0.678 25.156 33.066 pH5; S5204; T720; D2270-18 0.45 6.20 1.45 91.09 0.00 0.00 2.646 25.126 27.536 pH5; S5204; T720; D2269-25 0.44 7.02 1.69 89.91 0.00 0.00 0.868 25.018 32.104 pH5; S5204; T720; D2269-30 0.45 6.77 1.78 90.00 0.00 0.00 0.718 24.978 29.868 pH5; S5204; T720; D2270-25 0.31 6.82 1.68 90.09 0.00 0.00 2.32 24.814 36.024 pH5; S5204; T720; D2270-21 0.52 7.23 1.70 89.99 0.00 0.00 1.92 24.58 25.398 pH5; S5204; T720; D2269-38 0.00 7.45 1.50 90.19 0.00 0.00 1.494 24.578 30.178 pH5; S5204; T720; D2268-9 0.48 5.94 1.51 90.83 0.00 0.00 0.73 24.344 30.83 pH5; S5204; T720; D2268-37 0.44 6.35 1.84 90.31 0.00 0.00 0.548 24.306 32.848 pH5; S5204; T720; D2269-28 0.41 7.12 1.66 89.73 0.00 0.00 0.808 24.288 31.27 pH5; S5204; T720; D2270-5 0.44 6.98 1.78 89.79 0.00 0.00 2.328 24.14 30.186 pH5; S5204; T720; D2269-23 0.44 6.99 1.71 89.43 0.00 0.00 0.876 24.076 29.494 pH5; S5204; T720; D2269-9 0.38 6.84 1.71 90.32 0.00 0.00 0.806 24 26.844 pH5; S5204; T720; D2269-24 0.55 7.31 1.71 89.68 0.00 0.00 1.09 23.97 29.642 pH5; S5204; T720; D2270-35 0.36 6.58 1.72 90.38 0.00 0.00 1.554 23.71 28.868 pH5; S5204; T720; D2269-15 0.00 5.69 1.36 91.86 0.00 0.00 1.246 23.584 28.196 pH5; S5204; T720; D2270-28 0.39 7.15 1.82 89.92 0.00 0.00 1.648 23.486 30.858 pH7; S5204; T680; D2098-39 0.34 4.89 1.56 92.08 0.00 0.00 1.08 23.46 31.888 pH5; S5204; T720; D2269-27 0.33 6.87 1.68 89.98 0.00 0.00 1.3 23.262 33.112 pH5; S5204; T718; D2265-43 0.00 7.90 1.90 89.27 0.00 0.00 0.832 23.23 30.052 pH5; S5204; T720; D2270-30 0.41 pH5; S5204; T720; D2269-22 0.39 7.12 1.72 89.63 0.00 0.00 1.08 22.634 27.532 pH5; S5204; T718; D2263-30 0.54 7.58 1.57 89.47 0.00 0.00 0.71 22.564 29.996 pH7; S5204; T672; D2091-47 0.32 5.22 2.23 90.45 0.00 0.00 0.938 22.486 32.046 pH5; S5204; T720; D2269-1 0.38 6.73 1.68 90.24 0.00 0.00 1.154 22.48 29.994 pH7; S5204; T673; D2096-6 0.33 4.18 1.10 92.91 0.00 0.00 0.91 22.446 28.714 pH5; S5204; T720; D2270-33 0.40 6.95 1.76 89.89 0.00 0.00 2.28 22.408 29.656 pH5; S5204; T718; D2263-14 0.58 7.72 1.64 89.26 0.00 0.00 0.306 22.35 32.294 pH5; S5204; T720; D2270-34 0.36 6.75 1.77 90.10 0.00 0.00 2.398 22.3 28.958 pH7; S5204; T672; D2090-29 0.42 4.99 2.01 91.06 0.00 0.00 1.16 22.112 30.376 pH5; S5204; T720; D2269-14 0.00 7.86 1.80 89.57 0.00 0.00 0.574 21.802 31.558 pH5; S5204; T718; D2263-29 0.58 7.32 1.30 90.07 0.00 0.00 0.418 21.746 30.426 pH5; S5204; T718; D2263-19 0.62 7.92 1.56 89.25 0.00 0.00 0.574 21.692 29.514 pH5; S5204; T720; D2269-10 0.39 6.82 1.70 90.05 0.00 0.00 1.104 21.622 25.264 pH5; S5204; T720; D2269-4 0.42 6.57 1.71 90.32 0.00 0.00 1.082 21.466 29.698 pH5; S5204; T720; D2270-4 0.43 7.44 1.72 89.31 0.00 0.00 1.758 21.446 32.656 pH5; S5204; T720; D2269-34 0.00 6.69 1.78 90.64 0.00 0.00 0.946 21.438 28.538 pH5; S5204; T720; D2270-16 0.39 7.08 1.71 89.70 0.00 0.00 1.592 21.422 27.72 pH5; S5204; T718; D2263-26 0.42 7.39 1.70 89.28 0.00 0.00 0.514 21.328 29.746 pH5; S5204; T720; D2269-3 0.36 6.76 1.71 90.17 0.00 0.00 0.668 21.242 29.74 pH5; S5204; T720; D2270-22 0.35 6.77 1.67 90.15 0.00 0.00 1.194 21.026 25.084 pH5; S5204; T720; D2270-26 0.41 6.81 1.82 89.66 0.00 0.00 1.606 20.948 32.142 pH5; S5204; T720; D2270-10 0.46 6.98 1.80 90.03 0.00 0.00 0.792 20.728 28.264 pH5; S5204; T720; D2269-16 0.51 6.17 1.50 90.64 0.00 0.00 0.922 20.502 30.132 pH5; S5204; T720; D2270-8 0.50 6.95 1.42 90.34 0.00 0.00 2.252 20.486 28.34 pH5; S5204; T720; D2270-2 0.46 6.76 1.83 89.90 0.00 0.00 0.97 20.366 31.758 pH5; S5204; T720; D2269-36 0.00 7.43 1.66 89.88 0.00 0.00 0.754 20.006 29.648 pH5; S5204; T720; D2269-31 0.72 9.29 1.86 86.92 0.00 0.00 2.062 19.002 27.61 pH5; S5204; T720; D2269-44 0.00 9.45 1.58 88.16 0.00 0.00 1.378 18.576 22.52 pH7; S5204; T672; D2091-14 0.27 4.79 2.24 90.94 0.00 0.00 0.93 18.1 30.434 pH5; S5204; T720; D2270-32 0.40 7.14 1.74 89.63 0.00 0.00 1.668 17.966 27.06 pH5; S5204; T720; D2270-11 0.82 9.24 1.93 87.35 0.00 0.00 1.178 15.998 28.196 pH5; S5204; T720; D2269-48 0.72 9.05 2.14 88.08 0.00 0.00 1.172 14.694 25.384 pH5; S5204; T720; D2269-17 0.66 9.08 2.12 87.12 0.00 0.00 0.84 14.488 25.886 pH5; S5204; T720; D2270-20 0.62 8.35 1.97 88.43 0.00 0.00 1.37 14.168 23.794 pH5; S5204; T718; D2263-13 0.75 9.44 1.98 87.09 0.00 0.00 0.64 13.854 29.466 pH5; S5204; T720; D2269-46 0.43 6.87 1.71 89.81 0.00 0.00 0.646 10.452 31.464 pH5; S5204; T720; D2269-5 0.59 8.81 1.93 87.97 0.00 0.00 0.654 9.37 25.786 pH7; S5204; T672; D2091-4 1.42 4.39 2.32 89.87 0.00 0.00 0.686 8.182 16.454 pH5; S5204; T720; D2269-6 0.50 7.29 1.73 89.29 0.00 0.00 0.79 7.978 21.346
pH5; S5204; T720; D2270-45 0.00 9.16 1.65 88.19 0.00 0.00 0.464 3.448 16.796 Blank 0 0 0
[0698] It is contemplated that these promoters, or variants thereof, discovered here can be used to regulate a fatty acid synthesis gene (e.g., any of the FATA, FATB, SAD, FAD2, KASI/IV, KASII, LPAAT or KCS genes disclosed herein) or other gene or gene-suppression element expressed in a cell including a microalgal cell. Variants can have for example 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or greater identity to the sequences disclosed here.
Example 64
Fractionation of a High SOS Oil to Increase SOS Concentration and Reduce Trisaturates
[0699] Microalgal oil was fractionated using dry fractionation and solvent fractionation techniques. The starting material was an oil that was high in SOS triglycerides. The oil was produced from Prototheca moriformis strain S7566, in which a the endogenous KASII gene was inserted into (and thereby knocking out) a SADII locus; additionally, the C18-preferring FATA1 gene from Garcinia mangostana was inserted and a FADII hairpin RNA was produced; as described above. After cultivation and extracted, the oil was refined, bleached and deodorized. The fatty acid profile of the oil is given in Table 115. The SOS TAG area% was about 62%. During the RBD processing, the total trisaturates (i.e. triglycerides with three fully saturated acyl chains such as SSS, PSS, PPS, PPP, etc.) in the oil decreased from 5.1% to 1.2%.
TABLE-US-00151 TABLE 116 Fatty acid profile of clarified oil from strain 7566. Strain S7566 Fatty Acid Area % C14:0 0.49 C16:0 3.12 C18:0 54.77 C18:l 35.88 C18:2 2.16 C18:3 .alpha. 0.23 C20:0 1.64 C22:0 0.19 C24:0 0.11 sum C18 93.05 saturates 60.69 unsaturates 38.55
[0700] The oil was fractionated using solvent (acetone or hexane) and dry fractionation. Acetone fractionation (1:1 oil-solvent, w/w; crystallization at 5.degree. C.) gave excellent recovery of an SOS-enriched stearin fraction, with relatively little SOS in the olein fraction. SOS was at 77%, with total trisaturates <1% for the stearin fraction.
[0701] Hexane fractionation(1:1 oil-solvent, w/w; crystallization at 5.degree. C.) gave a higher level (85%) of SOS, but also gave higher trisaturates (1.6%). Thus, using a single-step solvent fractionation, oils with over 75% SOS and less than 2% trisaturates were obtainable.
[0702] Dry fractionation was also successful in enriching SOS and decreasing trisaturates. The general approach was to remove trisaturates by crystallization at a higher temperature, then removing 00S at a lower temperature. The reverse order was also tried and yielded a superior result. It was also found that rinsing the SOS-enriched ("stearin") fraction with acetone helped in removing the olein fraction.
[0703] In one test, the oil was crystallized at 24.degree. C. and the stearin fraction was rinsed with acetone. Analysis showed that 00S levels decreased. The stearin fraction was heated and allowed to cool and crystallize overnight at 29.degree. C. The resulting liquid oil was separated from the crystallized trisaturates to afford a product with 84% SOS and <0.5% total trisaturates. Lipase-based sn-2 profile analysis of revealed that over 96% of that position was occupied by unsaturated fatty acids (93.3% oleate, 3.2% linolate, and 0.2% linolenate), while only 2.2% stearate was located there.
[0704] The DSC heating curve thermogram and DSC-derived solid fat content curve of the two step dry fractionated oil was compared to those of kokum butter. The two oils have essentially identical maximum heat-flow temperatures and the DSC-derived SFC curves are super-imposable. The oil could be expected to behave functionally similarly to kokum butter.
Example 65
Production of Microbial Oil with Over 60% SOS Content
[0705] Here, we demonstrate in the microalga Prototheca moriformis, that by disrupting an allele of the SAD2 gene, overexpressing KASII, knocking out endogenous FATA-1, overexpressing a more stearate-specific FATA (GarmFATA1 from Garcinia mangostana) relative to the endogenous FATA and activating FAD2 RNAi, we generate strains capable of accumulating over 60% SOS, useful as a structuring fat.
[0706] To reduce SAD activity, Strain S3150 was transformed with DNA constructs designed to recombine in the SAD2-1 and SAD2-2 alleles and express the selectable marker, Arabidopsis thialiana THIC (AtTHIC, codon-optimized for expression in P. moriformis). THIC encodes 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase, thereby allowing growth in the absence of added thiamine. Transformants were selected in the absence of exogenous thiamine.
[0707] To make the SAD2-1 ablation construct pSZ2601, the Arabidopsis thaliana THIC gene (AtTHIC, codon-optimized for expression in P. moriformis), was utilized as a selectable marker for transformation. The sequence of the transforming DNA is shown in SEQ ID NO:148. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, PmeI, KpnI, XbaI, MfeI, SacI, BspQI and PmeI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the Chlorella protothecoides ACT promoter (CpACT) driving the expression of the AtTHIC gene (encoding 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase activity, thereby permitting the strain to grow in the absence of exogenous thiamine) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals.
TABLE-US-00152 Nucleotide sequence of the transforming DNA from pSZ2601: (SEQ ID NO: 148) gaagagcgcccaatgtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACCGCTTCGTG ATCCACCGGGTGAACGTAGTCCTCGACGGAAACATCTGGTTCGGGCCTCCTGCTTGCACTCCCGCCC ATGCCGACAACCTTTCTGCTGTTACCACGACCCACAATGCAACGCGACACGACCGTGTGGGACTGAT CGGTTCACTGCACCTGCATGCAATTGTCACAAGCGCTTACTCCAATTGTATTCGTTTGTTTTCTGGGA GCAGTTGCTCGACCGCCCGCGTCCCGCAGGCAGCGATGACGTGTGCGTGGCCTGGGTGTTTCGTCG AAAGGCCAGCAACCCTAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACCAGAT CCGCCCCGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGAT TGGTGTCCGATACCTGGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGC GTGCTACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAGTT GATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGCGTGTCTGTTTTCGGCTGCACGAATT CAATAGTCGGATGGGCGACGGTAGAATTGGGTGTGGCGCTCGCGTGCATGCCTCGCCCCGTCGGGT GTCATGACCGGGACTGGAATCCCCCCTCGCGACCATCTTGCTAACGCTCCCGACTCTCCCGACCGCG ##STR00419## ##STR00420## ##STR00421## ##STR00422## ##STR00423## ##STR00424## ##STR00425## ##STR00426## ##STR00427## ##STR00428## ##STR00429## ##STR00430## ##STR00431## ##STR00432## gcccaactcctccctgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaaggagacga- cgaccaccc gcgccacgctgacgttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccc- tcctccccc gacttccagcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacga- ggagtccg gccacgtcctgaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactacgacacg- tccggccccc agaacgtcaacgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacg- ccccgct acacgcagatgtactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctg- gaccccg agttcgtccgctccgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagccc- atgatcgtg ggccgcaagttcctggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggt- ctacaagg tgcagtgggccaccatgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgc- gagtggat cctgcgcaactccgcggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcgg- agaacctg aactgggaggtgttccgcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcggg- cgtgctgc tgcgctacatccccctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtgg- tgcctggcct accacaaggagaacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctg- tccatcggc gacggcctgcgccccggctccatctacgacgccaacgacacggcccagttcgccgagctgctgacccagggcga- gctgacgc gccgcgcgtgggagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgag- aacatg cagaagcagctggagtggtgcaacgaggcgcccttctacaccctgggccccctgacgaccgacatcgcgcccgg- ctacgacc acatcacctccgccatcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaag- gagcacctg ggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggc- caagcag cacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttcgagttccgctggatggaccagttcgc- gctgtccctg gaccccatgacggcgatgtccttccacgacgagacgctgcccgcggacggcgcgaaggtcgcccacttctgctc- catgtgcgg ccccaagttctgctccatgaagatcacggaggacatccgcaagtacgccgaggagaacggctacggctccgccg- aggaggc catccgccagggcatggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacg- gcgaggt cggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattgGCAGCAGCAGCTCGGA- TAGTATC GACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTG- CCGCTTT TATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGC- GAATACCA CCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCC- TCAGCGCT GCTCCTG CTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAAC- CTGTAAACC AGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCGTAgagctcTAGGGAGCG- A CGAGTGTGCGTGCGGGGCTGGCGGGAGTGGGACGCCCTCCTCGCTCCTCTCTGTTCTGAACGGAAC AATCGGCCACCCCGCGCTACGCGCCACGCATCGAGCAACGAAGAAAACCCCCCGATGATAGGTTGC GGTGGCTGCCGGGATATAGATCCGGCCGCACATCAAAGGGCCCCTCCGCCAGAGAAGAAGCTCCTT TCCCAGCAGACTCCTTCTGCTGCCAAAACACTTCTCTGTCCACAGCAACACCAAAGGATGAACAGATC AACTTGCGTCTCCGCGTAGCTTCCTCGGCTAGCGTGCTTGCAACAGGTCCCTGCACTATTATCTTCCT GCTTTCCTCTGAATTATGCGGCAGGCGAGCGCTCGCTCTGGCGAGCGCTCCTTCGCGCCGCCCTCGC TGATCGAGTGTACAGTCAATGAATGGTCCTGGGCGAAGAACGAGGGAATTTGTGGGTAAAACAAG CATCGTCTCTCAGGCCCCGGCGCAGTGGCCGTTAAAGTCCAAGACCGTGACCAGGCAGCGCAGCGC GTCCGTGTGCGGGCCCTGCCTGGCGGCTCGGCGTGCCAGGCTCGAGAGCAGCTCCCTCAGGTCGCC TTGGACGGCCTCTGCGAGGCCGGTGAGGGCCTGCAGGAGCGCCTCGAGCGTGGCAGTGGCGGTCG TATCCGGGTCGCCGGTCACCGCCTGCGACTCGCCATCCgaagagcgtttaaac
[0708] The sequence of the transforming DNA from the SAD2-1 disruption construct, pSZ2607, is shown below in SEQ ID NO:149. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' PmeI, KpnI, XbaI, MfeI, SacI, BspQI and PmeI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the Chlorella protothecoides ACT promoter (CpACT) driving the expression of the AtTHIC gene (encoding 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase activity, thereby permitting the strain to grow in the absence of exogenous thiamine) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals.
TABLE-US-00153 Nucleotide sequence of the transforming DNA from pSZ2607: (SEQ ID NO: 149) gtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACCGCTTCGTGATCCACCGGGTG AACGTAGTCCTCGACGGAAACATCTGGTTCGGGCCTCCTGCTTGCACTCCCGCCCATGCCGACAACC TTTCTGCTGTTACCACGACCCACAATGCAACGCGACACGACCGTGTGGGACTGATCGGTTCACTGCA CCTGCATGCAATTGTCACAAGCGCTTACTCCAATTGTATTCGTTTGTTTTCTGGGAGCAGTTGCTCGA CCGCCCGCGTCCCGCAGGCAGCGATGACGTGTGCGTGGCCTGGGTGTTTCGTCGAAAGGCCAGCAA CCCTAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACCAGATCCGCCCCGATGC GGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCCGATA CCTGGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGG TTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAGTTGATGGGGTTGGC AGGCATCGAAACGCGCGTGCATGGTGTGCGTGTCTGTTTTCGGCTGCACGAATTCAATAGTCGGAT GGGCGACGGTAGAATTGGGTGTGGCGCTCGCGTGCATGCCTCGCCCCGTCGGGTGTCATGACCGG GACTGGAATCCCCCCTCGCGACCATCTTGCTAACGCTCCCGACTCTCCCGACCGCGCGCAGGATAGA ##STR00433## ##STR00434## ##STR00435## ##STR00436## ##STR00437## ##STR00438## ##STR00439## ##STR00440## ##STR00441## ##STR00442## ##STR00443## ##STR00444## ##STR00445## ##STR00446## gccgcgtccgtccactgcaccctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcc- caactcctccc tgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgcc- acgctgac gttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgact- tccagcccat cccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacg- tcctgaag gtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaa- cgtcaacgcc cacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgca- gatgtac tacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgt- ccgctccg aggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgc- aagttcct ggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagt- gggccac catgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgc- gcaactcc gcggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactg- ggaggtg ttccgcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcg- ctacatccc cctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctacc- acaaggag aacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcga- cggcctgcg ccccggctccatctacgacgccaacgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgcc- gcgcgtgg gagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaa- gcagct ggagtggtgcaacgaggcgcccttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccaca- tcacctccg ccatcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggc- ctgcccaac cgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcaccc- ccacgccc aggcgtgggacgacgcgctgtccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggac- cccatgacg gcgatgtccttccacgacgagacgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccc- caagttctgc tccatgaagatcacggaggacatccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccg- ccaggg catggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcg- gcgagat ctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACAC- ACTCTGG ACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCA- AACAGCCT CAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCC- AGCATCCC CTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTC- CTGCTCCTG CTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCAC- TGCAATGC TGATGCACGGGAAGTAGTGGGATGGGAAcAcAAATGGAGGATCGTAgagctcCAGCCACGGCAACACCGCGCG CCTTGCGGCCGAGCACGGCGACAAGAACCTGAGCAAGATCTGCGGGCTGATCGCCAGCGACGAGG GCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCCCGAGGGCGCCG TCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCATGGACGACATGG GCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCGGTCGCCGAGAAGATCG ACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTGGAAGGTGGACG AGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTCCTGGGCCTGCCCCAGCGCTTCC GGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCCGTCGCCTTCTCCT GGATCTCCGGGCGCGAGATCATGGTCTAGGGAGCGACGAGTGTGCGTGCGGGGCTGGCGGGAGT GGGACGCCCTCCTCGCTCCTCTCTGTTCTGAACGGAACAATCGGCCACCCCGCGCTACGCGCCACGC ATCGAGCAACGAAGAAAACCCCCCGATGATAGGTTGCGGTGGCTGCCGGGATATAGATCCGGCCGC ACATCAAAGGGCCCCTCCGCCAGAGAAGAAGCTCCTTTCCCAGCAGACTCCTgaagagcgtttaaac
[0709] The sequence of the transforming DNA from the SAD2-2 disruption construct,pSZ2622, is shown below in SEQ ID NO:150. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, PmeI, KpnI, XbaI, MfeI, SacI, BspQI and PmeI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. Proceeding in the 5' to 3' direction, the Chlorella protothecoides ACT promoter (CpACT) driving the expression of the AtTHIC gene (encoding 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase activity, thereby permitting the strain to grow in the absence of exogenous thiamine) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals.
TABLE-US-00154 Nucleotide sequence of the transforming DNA from pSZ2622: (SEQ ID NO: 150) gaagagcgcccaatgtttaaacGCCGGTCACCATCCGCATGCTCATATTACAGCGCACGCACCGCTTCGTGA TCCACCGGGTGAACGTAGTCCTCGACGGAAACATCTGGCTCGGGCCTCGTGCTGGCACTCCCTCCCA TGCCGACAACCTTTCTGCTGTCACCACGACCCACGATGCAACGCGACACGACCCGGTGGGACTGATC GGTTCACTGCACCTGCATGCAATTGTCACAAGCGCATACTCCAATCGTATCCGTTTGATTTCTGTGAA AACTCGCTCGACCGCCCGCGTCCCGCAGGCAGCGATGACGTGTGCGTGACCTGGGTGTTTCGTCGA AAGGCCAGCAACCCCAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGCTTGGACCAGATC CCCCACGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATT GGTGTCCGATACCTTGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCG TGCTACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACCGGCGCAGAGGGTGAGTTG ATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGTGTGTCTGTTTTCGGCTGCACAATTTCA ATAGTCGGATGGGCGACGGTAGAATTGGGTGTTGCGCTCGCGTGCATGCCTCGCCCCGTCGGGTGT CATGACCGGGACTGGAATCCCCCCTCGCGACCCTCCTGCTAACGCTCCCGACTCTCCCGCCCGCGCG ##STR00447## ##STR00448## ##STR00449## ##STR00450## ##STR00451## ##STR00452## ##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457## ##STR00458## ##STR00459## ##STR00460## ##STR00461## cccaactcctccctgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaaggagacgac- gaccacccg cgccacgctgacgttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccct- cctcccccg acttccagcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacgag- gagtccggc cacgtcctgaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactacgacacgtc- cggcccccag aacgtcaacgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacgcc- ccgctac acgcagatgtactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctgga- ccccga gttcgtccgctccgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagccca- tgatcgtgg gccgcaagttcctggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggtc- tacaaggt gcagtgggccaccatgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgcg- agtggatc ctgcgcaactccgcggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcgga- gaacctga actgggaggtgttccgcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcgggc- gtgctgct gcgctacatccccctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggt- gcctggccta ccacaaggagaacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctgt- ccatcggcg acggcctgcgccccggctccatctacgacgccaacgacacggcccagttcgccgagctgctgacccagggcgag- ctgacgcg ccgcgcgtgggagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgaga- acatgc agaagcagctggagtggtgcaacgaggcgcccttctacaccctgggccccctgacgaccgacatcgcgcccggc- tacgacca catcacctccgccatcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaagg- agcacctgg gcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggcc- aagcagc acccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttcgagttccgctggatggaccagttcgcg- ctgtccctg gaccccatgacggcgatgtccttccacgacgagacgctgcccgcggacggcgcgaaggtcgcccacttctgctc- catgtgcgg ccccaagttctgctccatgaagatcacggaggacatccgcaagtacgccgaggagaacggctacggctccgccg- aggaggc catccgccagggcatggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacg- gcgaggt cggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattgGCAGCAGCAGCTCGGA- TAGTATC GACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTG- CCGCTTT TATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGC- GAATACCA CCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCC- CTCAGCGCT GCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACC- TGTAAACC AGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGATCGTAgagctcCAGCCACGG- C AACACCGCGCGCCTGGCGGCCGAGCACGGCGACAAGGGCCTGAGCAAGATCTGCGGGCTGATCGC CAGCGACGAGGGCCGGCACGAGATCGCCTACACGCGCATCGTGGACGAGTTCTTCCGCCTCGACCC CGAGGGCGCCGTCGCCGCCTACGCCAACATGATGCGCAAGCAGATCACCATGCCCGCGCACCTCAT GGACGACATGGGCCACGGCGAGGCCAACCCGGGCCGCAACCTCTTCGCCGACTTCTCCGCCGTCGC CGAGAAGATCGACGTCTACGACGCCGAGGACTACTGCCGCATCCTGGAGCACCTCAACGCGCGCTG GAAGGTGGACGAGCGCCAGGTCAGCGGCCAGGCCGCCGCGGACCAGGAGTACGTTCTGGGCCTGC CCCAGCGCTTCCGGAAACTCGCCGAGAAGACCGCCGCCAAGCGCAAGCGCGTCGCGCGCAGGCCC GTCGCCTTCTCCTGGATCTCCGGACGCGAGATTATGGTCTAGGGAGGTACGAGCGCGCGCGAGGGA TTGGTGGGAGTGGGACGCGCTCGTCGCTCCTTTCTATTCTGAAGGGAAGATTGGCCACCCCGCTCCA CGCGCCACGCATCGAGCAACGAAGAAAACCCCCCGATGATAGGTTGCAGTGGCTGCCGAGATATAG ATCCGGCTGCACGTCAAAGGGCCCCTCGGCCAGAGAAGAAGCTCTTTTCCCAGCGACCGCAGACTCC Tgaagagcgtttaaac
[0710] Constructs D1557, D1565 and D1566, derived from pSZ2601, pSZ2607 and pSZ2622, respectively, were transformed into S3150 as described previously. Primary transformants were clonally purified and grown under low-nitrogen lipid production conditions at pH 5. The resulting fatty acid profiles from representative clones are summarized in Table 117. SAD2-1 disruption strains derived from D1557 and D1565 transformants accumulated up to 13.4% C18:0 at the expense of C18:1, indicating that SAD activity was significantly reduced in these strains. C18:0 levels only increased to 8.5% in SAD2-2 disruption strains, suggesting that the expression or activity of SAD2-2 was lower than that of SAD2-1. We also observed that C20:0 levels increased up to 1.1% in strains with elevated C18:0, demonstrating that C18:0 was an effective primer for fatty acid elongation reactions in the endoplasmic reticulum (ER).
TABLE-US-00155 TABLE 117 Fatty acid profiles from representative clones. Strain S3150 D1557-2 D1557-3 D1565-10 D1565-3 D1565-8 D1566-5 D1566-6 D1566-1 Fatty C14:0 1.30 1.14 1.20 1.08 1.12 1.11 1.18 1.12 1.21 Acid C16:0 28.71 29.32 29.74 28.84 29.34 29.11 29.21 29.13 28.46 Area C16:1 0.76 0.21 0.23 0.21 0.21 0.21 0.32 0.31 0.31 % C17:0 0.12 0.14 0.15 0.15 0.14 0.14 0.14 0.16 0.14 C18:0 2.93 13.42 11.92 14.29 14.14 14.04 8.47 8.47 7.68 C18:1 58.08 46.29 47.65 45.75 45.31 45.69 51.29 51.33 53.38 C18:2 6.81 7.15 6.96 7.09 7.18 7.19 7.25 7.34 6.92 C18:3 .alpha. 0.59 0.69 0.63 0.72 0.72 0.73 0.71 0.73 0.62 C20:0 0.24 0.93 0.84 1.10 1.09 1.04 0.75 0.77 0.63 C22:0 0.05 0.16 0.15 0.19 0.19 0.18 0.14 0.14 0.11 C24:0 0.06 0.16 0.16 0.20 0.20 0.20 0.17 0.17 0.14 sum C18 68.40 67.55 67.16 67.85 67.35 67.65 67.72 67.87 68.60 saturates 33.49 45.35 44.24 45.94 46.32 45.93 40.18 40.04 38.48 unsaturates 66.52 54.62 55.76 54.04 53.68 54.07 59.83 59.97 61.50
[0711] In order to increase C18:0 accumulation at the expense of C16:0 we generated DNA constructs which simultaneously ablated SAD2-1 and over-expressed a codon-optimized version of the endogenous .beta.-ketoacyl-ACP synthase II (PmKASII) gene. The sequence of the transforming DNA from the SAD2-1 ablation, PmKASII over-expression construct, pSZ2624, is shown below in SEQ ID NO:151. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' PmeI, SpeI, AscI, ClaI, SacI, AvrII, EcoRV, AflII, KpnI, XbaI, MfeI, BamHI, BspQI and PmeI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. The SAD2-1 5' integration flank contained the endogeneous SAD2-1 promoter, enabling the in situ activation of the PmKASII gene. Proceeding in the 5' to 3' direction, the region encoding the PmKASII plastid targeting sequence is indicated by lowercase, underlined italics. The sequence that encodes the mature PmKASII polypeptide is indicated with lowercase italics, while a 3.times. FLAG epitope encoding sequence is in bold italics. The initiator ATG and terminator TGA for PmKASII-FLAG are indicated by uppercase italics. Two spacer regions are represented by lowercase text. The CpACT promoter driving the expression of the AtTHIC gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3' UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals.
TABLE-US-00156 Nucleotide sequence of the transforming DNA from pSZ2624: (SEQ ID NO: 151) gtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACCGCTTCGTGATCCACCGGGTG AACGTAGTCCTCGACGGAAACATCTGGTTCGGGCCTCCTGCTTGCACTCCCGCCCATGCCGACAACC TTTCTGCTGTTACCACGACCCACAATGCAACGCGACACGACCGTGTGGGACTGATCGGTTCACTGCA CCTGCATGCAATTGTCACAAGCGCTTACTCCAATTGTATTCGTTTGTTTTCTGGGAGCAGTTGCTCGA CCGCCCGCGTCCCGCAGGCAGCGATGACGTGTGCGTGGCCTGGGTGTTTCGTCGAAAGGCCAGCAA CCCTAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACCAGATCCGCCCCGATGC GGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCCGATA CCTGGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGG TTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAGTTGATGGGGTTGGC AGGCATCGAAACGCGCGTGCATGGTGTGCGTGTCTGTTTTCGGCTGCACGAATTCAATAGTCGGAT GGGCGACGGTAGAATTGGGTGTGGCGCTCGCGTGCATGCCTCGCCCCGTCGGGTGTCATGACCGG GACTGGAATCCCCCCTCGCGACCATCTTGCTAACGCTCCCGACTCTCCCGACCGCGCGCAGGATAGA CTCTTGTTCAACCAATCGACAactagtATGcagaccgcccaccagcgcccccccaccgagggccactgcttcgg- cgcc cgcctgcccaccgcctcccgccgcgccgtgcgccgcgcctggtcccgcatcgcccgcgggcgcgccgccgccgc- cgccgacgcc aaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagaccatcga- gcagttcta ctcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacaccaccacca- tcgccggcga gatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtgatcaagtacg- tgtacatcg ccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccggcctggacccc- gccctgtgc ggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgacccgcggcgg- cgtgcgcaa gatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatcggcttca- tgggccccaact actccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgccgcggcgac- gccaacgtga tgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaaggccctgtcc- aagcgcaacg acgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagggcgccggcgtg- ctggtgct ggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcgccgccacctccg- acgccca ccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcc- tggcccccg agcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccgcgccatccgc- gccgtgatc ccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccggcgccgtgga- ggccgtggc cgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccggcgtggacc- ccgtggtgc tggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttcggcggccac- aactcctgc gtgatcttccgcaagtacgacgagatggactacaaggaccacgacggcgactacaaggaccacgacatcgacta- caagg acgacgacgacaagTGAatcgatAGATCTCTTAAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGC- TGGTC GTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTC- AGTGTGTT TGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCC- TTCCCTCG TTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTG- CTCACTGCC CCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCT- GATGCAC GGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTTAATTAAgagctccgcgtctcgaacagagcgcgcagagg- aacgctg aaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcg- tccattagcgaagc gtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaacgttc- acagcctaggt gatatccatcttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgta- gtgaccgccaat gtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggacc- aggcatcgcgag ##STR00462## ##STR00463## ##STR00464## ##STR00465## ##STR00466## ##STR00467## ##STR00468## ##STR00469## ##STR00470## ##STR00471## ##STR00472## ##STR00473## ##STR00474## ##STR00475## ##STR00476## ccaagctgcccaactcctccctgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaag- gagacgacg accacccgcgccacgctgacgttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccat- cgacccctc ctcccccgacttccagcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtgg- tgcacgagg agtccggccacgtcctgaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactac- gacacgtccg gcccccagaacgtcaacgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctg- ggcacgc cccgctacacgcagatgtactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgag- aagctgg accccgagttcgtccgctccgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctg- gagcccatg atcgtgggccgcaagttcctggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgagga- ggaggtct acaaggtgcagtgggccaccatgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgag- acgcgcg agtggatcctgcgcaactccgcggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggc- atcgcgga gaacctgaactgggaggtgttccgcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatcc- acgcggg cgtgctgctgcgctacatccccctgaccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacg- cgaagtggtg cctggcctaccacaaggagaacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacg- tcgccctgt ccatcggcgacggcctgcgccccggctccatctacgacgccaacgacacggcccagttcgccgagctgctgacc- cagggcga gctgacgcgccgcgcgtgggagaaggacgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaaga- tccccg agaacatgcagaagcagctggagtggtgcaacgaggcgcccttctacaccctgggccccctgacgaccgacatc- gcgcccgg ctacgaccacatcacctccgccatcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtga- cgcccaagg agcacctgggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggcc- gacctggc caagcagcacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttcgagttccgctggatggacc- agttcgcg ctgtccctggaccccatgacggcgatgtccttccacgacgagacgctgcccgcggacggcgcgaaggtcgccca- cttctgctcc atgtgcggccccaagttctgctccatgaagatcacggaggacatccgcaagtacgccgaggagaacggctacgg- ctccgccg aggaggccatccgccagggcatggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgag- cagcacg gcgaggtcggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattgACGGAGCGT- CGTGCG GGAGGGAGTGTGCCGAGCGGGGAGTCCCGGTCTGTGCGAGGCCCGGCAGCTGACGCTGGCGAGCCGTACGCCCC- GAG GGICCCCCTCCCCTGCACCCICTICCCCTICCCTCTGACGGCCGCGCCTGITCTTGCATGITCAGCGACggatc- cTAGGGA GCGACGAGTGTGCGTGCGGGGCTGGCGGGAGTGGGACGCCCTCCTCGCTCCTCTCTGTTCTGAACG GAACAATCGGCCACCCCGCGCTACGCGCCACGCATCGAGCAACGAAGAAAACCCCCCGATGATAGG TTGCGGTGGCTGCCGGGATATAGATCCGGCCGCACATCAAAGGGCCCCTCCGCCAGAGAAGAAGCT CCTTTCCCAGCAGACTCCTTCTGCTGCCAAAACACTTCTCTGTCCACAGCAACACCAAAGGATGAACA GATCAACTTGCGTCTCCGCGTAGCTTCCTCGGCTAGCGTGCTTGCAACAGGTCCCTGCACTATTATCT TCCTGCTTTCCTCTGAATTATGCGGCAGGCGAGCGCTCGCTCTGGCGAGCGCTCCTTCGCGCCGCCCT CGCTGATCGAGTGTACAGTCAATGAATGGTCCTGGGCGAAGAACGAGGGAATTTGTGGGTAAAACA AGCATCGTCTCTCAGGCCCCGGCGCAGTGGCCGTTAAAGTCCAAGACCGTGACCAGGCAGCGCAGC GCGTCCGTGTGCGGGCCCTGCCTGGCGGCTCGGCGTGCCAGGCTCGAGAGCAGCTCCCTCAGGTCG CCTTGGACGGCCTCTGCGAGGCCGGTGAGGGCCTGCAGGAGCGCCTCGAGCGTGGCAGTGGCGGT CGTATCCGGGTCGCCGGTCACCGCCTGCGACTCGCCATCCgaagagcgtttaaac
[0712] Using the methods of this example, by overexpressing KASII, and Garcinia mangostana FATA, and by reducing expression of endogenous SAD, FAD2, and FATA, we produced a strain of P. moriformis that produced and oil with greater than 55% SOS with Sat-O-Sat (where O is oleate and Sat is any saturated fatty acid) of about 70-75% and trisaturatated TAGs of less than 6.5%.
Example 66
Combining KASII, FATA and LPAAT Transgenes to Produce an Oil High in SOS
[0713] In Prototheca moriformis, we overexpressed the P. moriformis KASII, knocked out an endogenous SAD2 allele, knocked out the endogenous FATA allele, and overexpressed both a LPAAT from Brassica napus and a FATA gene from Garcinia mangostana ("GarmFAT1"). The resulting strain produced an oil with over 55% SOS, over 70% Sat-O-Sat, and less than 8% trisaturated TAGs.
[0714] A base strain was transformed with a linearized plasmid with flanking regions designed for homologous recombination at the SAD2 site. As in examples above, the construct ablated SAD2 and overexpressed P. moriformis KASII. A ThiC selection marker was used. This strain was further transformed with a construct designed to overexpress GarmFATA1 with a P. moriformis SASD1 plastid targeting peptide via homologous recombination at the 6S chromosomal site using invertase as a selection marker. The resulting strain, produced oil with about 62% stearate, 6% palmitate, 5% linoleate, 45% SOS and 20% trisaturates.
[0715] The sequence of the transforming DNA from the GarmFATA1 expression construct (pSZ3204) is shown below in SEQ ID NO:152. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, AflII, SacI and BspQI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the 6S locus. Proceeding in the 5' to 3' direction, the CrTUB2 promoter driving the expression of Saccharomyces cerevisiae SUC2 (ScSUC2) gene, enabling strains to utilize exogenous sucrose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScSUC2 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3' UTR of the CvNR gene is indicated by small capitals. A spacer region is represented by lowercase text. The P. moriformis SAD2-2 (PmSAD2-2) promoter driving the expression of the chimeric CpSAD1tp_GarmFATA1 FLAG gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding CpSAD1tp is represented by lowercase, underlined italics; the sequence encoding the GarmFATA1 mature polypeptide is indicated by lowercase italics; and the 3.times. FLAG epitope tag is represented by uppercase, bold italics. A second CvNR 3' UTR is indicated by small capitals.
TABLE-US-00157 Nucleotide sequence of the transforming DNA from pSZ3204: (SEQ ID NO: 152) gctcttcGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCGC CGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCA CTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGT CGCGGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCT CCAGCAGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACA GAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGC GAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGC GCGAGCCAGCGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCA GTCTAAACCCCCTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCG CCACCCCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCC ##STR00477## ##STR00478## ##STR00479## ##STR00480## ##STR00481## gcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaac- aagggctgg atgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaa- cgacacc gtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgc- catcgcccc gaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacg- acaccatcga cccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcc- tggacggcg gctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtc- ttctggtacg agccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgac- ctgaagtcc tggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggt- ccccaccga gcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttca- accagtacttc gtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggacta- ctacgccctg cagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactc- cgccttcgtg cccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaaccc- ggagacggag ctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacac- cacgttgac gaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtca- acaccacc cagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacct- ccgcatgggc ttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaaccccta- cttcaccaac cgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctgga- ccagaaca tcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctg- ggctccgtga acatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCA- GCAGCAG CTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCT- GTGAATA TCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGC- TTGTGCTA TTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGC- TGTCCTGCT ATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCC- TGGTACTG CAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcg- tctcga acagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacc- tgacgaatgcg cttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatga- tcggtggagctgat ##STR00482## ##STR00483## ##STR00484## ##STR00485## ##STR00486## ##STR00487## ##STR00488## ##STR00489## ##STR00490## ##STR00491## ##STR00492## ##STR00493## ##STR00494## ##STR00495## ##STR00496## ##STR00497## ##STR00498## actagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctc- cgggccccgg cgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctc- caaggtgaaccc cctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcc- tgtcctaca aggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctg- ctgcagg aggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaag- ctgcgcctga tctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcc- tggggccag ggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgc- cacctcca agtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtg- cactgcc cccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccc- tcccagtac tccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcgg- ctgggtgct ggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgcc- agcacgac gacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaa- cggctccgc caacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggaga- tcaaccgcg ##STR00499## ##STR00500## CGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCT- GCCGCTT TTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTG- CGAATACC ACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATC- CCTCAGCGC TGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAAC- CTGTAAAC CAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAAcAcAAATGGAaagcttaattaagagctcTTGT- TTTCC AGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGA GGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTC ACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTTCGCGCAA TCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGTAATTGCCTCAGAAT GTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGACACCCGCCACTC GTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAA CGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACCGG CATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTCTCCCCGGG ATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACAAATATCC TTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCT AGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTgaagagc
[0716] The resulting strain was further transformed with a construct designed to recombine at (and thereby disrupt) the endogenous FATA and also express the LPAAT from B. napus under control of the UAPA1 promoter and using alpha galactosidase as a selectable marker with selection on melbiose. The resulting strain showed increased production of SOS (about 57-60%) and Sat-O-Sat (about 70-76%) and lower amounts of trisaturates (4.8 to 7.6%).
[0717] Strains were generated in the high-C18:0 S6573 background in which we maximized SOS production and minimized the formation of trisaturated TAGs by targeting both the Brassica napus LPAT2(Bnl.13) gene and the PmFAD2hpA RNAi construct to the FATA-1 locus. The sequence of the transforming DNA from the PmFAD2hpA expression construct pSZ4164 is shown below in SEQ ID NO:153. Relevant restriction sites are indicated in lowercase, bold, and are from 5'-3' BspQI, KpnI, SpeI, SnaBI, BamHI, NdeI, NsiI, AflII, EcoRI, SpeI, BsiWI, XhoI, SacI and BspQI. Underlined sequences at the 5' and 3' flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the FATA-1 locus. Proceeding in the 5' to 3' direction, the PmHXT1 promoter driving the expression of Saccharomyces carlbergensis MEL1 (ScarMEL1) gene, enabling strains to utilize exogenous melibiose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScarMEL1 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3' UTR of the P. moriformis PGK gene is indicated by small capitals. A spacer region is represented by lowercase text. The P. moriformis UAPA1 promoter driving the expression of the BnLPAT2(Bnl.13) gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding BnLPAT2(Bnl.13) is represented by lowercase, underlined italics. The 3' UTR of the CvNR gene is indicated by small capitals. A second spacer region is represented by lowercase text. The C. reinhardtii CrTUB2 promoter driving the expression of the PmFAD2hpA hairpin sequence is indicated by lowercase, boxed text. The FAD2 exon 1 sequence in the forward orientation is indicated with lowercase italics; the FAD2 intron 1 sequence is represented with lowercase, bold italics; a short linker region is indicated with lowercase text, and the FAD2 exon 1 sequence in the reverse orientation is indicated with lowercase, underlined italics. A second CvNR 3' UTR is indicated by small capitals.
TABLE-US-00158 Nucleotide sequence of the transforming DNA from pSZ4164: (SEQ ID NO: 153) gctcttcCCAACTCAGATAATACCAATACCCCTCCTTCTCCTCCTCATCCATTCAGTACCCCCCCCCTTCTC TTCCCAAAGCAGCAAGCGCGTGGCTTACAGAAGAACAATCGGCTTCCGCCAAAGTCGCCGAGCACT GCCCGACGGCGGCGCGCCCAGCAGCCCGCTTGGCCACACAGGCAACGAATACATTCAATAGGGGG CCTCGCAGAATGGAAGGAGCGGTAAAGGGTACAGGAGCACTGCGCACAAGGGGCCTGTGCAGGA GTGACTGACTGGGCGGGCAGACGGCGCACCGCGGGCGCAGGCAAGCAGGGAAGATTGAAGCGGC AGGGAGGAGGATGCTGATTGAGGGGGGCATCGCAGTCTCTCTTGGACCCGGGATAAGGAAGCAAA TATTCGGCCGGTTGGGTTGTGTGTGTGCACGTTTTCTTCTTCAGAGTCGTGGGTGTGCTTCCAGGGA GGATATAAGCAGCAGGATCGAATCCCGCGACCAGCGTTTCCCCATCCAGCCAACCACCCTGTCggtac ##STR00501## ##STR00502## ##STR00503## ##STR00504## ##STR00505## ##STR00506## ##STR00507## ##STR00508## ##STR00509## ##STR00510## gtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgtt- cgcctgcgac gtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacaatgggctacaagt- acatcatcct ggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacggca- tgggccacg tcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggc- taccccggctc cctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaatacgacaactgcta- caacaa gggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggcc- gccccatct tctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatg- tccggcgacgt cacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggct- tccactgctc catcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggaca- acctgga ggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccc- tgatcatc ggcgcgaacgtgaacaacctgaaggcctcctcctactccatctgctcccaggcgtccgtcatcgccatcaacca- ggactccaac ggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagat- gtggtccg gccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgacc- ctggagga gatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaacc- gcgtcgacaa ctcccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcc- tacaaggac ggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacac- gaccgtcccc gcccacggcatcgcgttctaccgcctgcgcccctcctccTGAtacaacttattacgtaTTCTGACCGGCGCTGA- TGTGGCGCGG ACGCCGTCGTACTCTTTCAGACTTTACTCTTGAGGAATTGAACCTTTCTCGCTTGCTGGCATGTAAACATTGGC- GCAATTAA TTGTGTGATGAAGAAAGGGTGGCACAAGATGGATCGCGAATGTACGAGATCGACAACGATGGTGATTGTTATGA- GGGG CCAAACCTGGCTCAATCTTGTCGCATGTCCGGCGCAATGTGATCCAGCGGCGTGACTCTCGCAACCTGGTAGTG- TGTGGCG CACCGGGTCGCTTTGATTAAAACTGATCGCATTGCCATCCCGTCAACTCACAAGCCTACTCTAGCTCCCATTGC- GCACTCGG GCGCCCGGCTCGATCAATGTTCTGAGCGGAGGGCGAAGCGTCAGGAAATCGTCTCGGCAGCTGGAAGCGCATGG- AATGC GGAGCGGAGATCGAATCAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtc- gcacctcagc gcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacac- acgtgccacgttg ##STR00511## ##STR00512## ##STR00513## ##STR00514## ##STR00515## ##STR00516## ##STR00517## ##STR00518## ##STR00519## ##STR00520## ##STR00521## ##STR00522## ##STR00523## ctgctgcaggccatctgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggt- ggccgagacc ctgtggctggagctggtgtggatcgtggactggtgggccggcgtgaagatccaggtgttcgccgacaacgagac- cttcaacc gcatgggcaaggagcacgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcctg- gcccagcg ctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtcca- tgtggttctccg agtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgac- ttcccccgc cccttctggctggccctgttcgtggagggcacccgcttcaccgaggccaagctgaaggccgcccaggagtacgc- cgcctcctcc gagctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctc- cttcgtgcccg ccatctacgacatgaccgtggccatccccaagacctcccccccccccaccatgctgcgcctgttcaagggccag- ccctccgtggt gcacgtgcacatcaagtgccactccatgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcg- accagttcg tggccaaggacgccctgctggacaagcacatcgccgccgacaccttccccggccagcaggagcagaacatcggc- cgccccat caagtccctggccgtggtgctgtcctggtcctgcctgctgatcctgggcgccatgaagttcctgactggtccaa- cctgttctcctc ctggaagggcatcgccttctccgccctgggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcct- cccagtccgag cgctccacccccgccaggtggtgcccgccaagcccaaggacaaccacaacgactcggctcctcctcccagaccg- aggtgga gaagcagaagTGAatgcatGCAGCAGCAGCTCGGATGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACT- GTTG CCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTG- TGTGTACG CGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATAT- CGCTGCAT CCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCAC- AGCCTTGG TTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAG- TGGGAT GGGAACACAAATGGActtaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccat- gtcgtagtga ccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccg- gcaggaccaggca tcgcgagatacagcgcgagccagacacggagtgccgagctatgcgcacgctccaactagatatcatgtggatga- tgagcatgaatt ##STR00524## ##STR00525## ##STR00526## ##STR00527## gtggagaagcctccgttcacgatcgggacgctgcgcaaggccatccccgcgcactgtttcgagcgctcggcgct- tcgtagcag catgtacctggcctttgacatcgcggtcatgtccctgctctacgtcgcgtcgacgtacatcgaccctgcaccgg- tgcctacgtggg ##STR00528## ##STR00529## agtagagcggccacatgatgccgtacttgacccacgtaggcaccggtgcagggtcgatatacgtcgacacgaca- tagggca gggacatgaccgcgatgtcaaaggccaggtacatgctgctacgaagcgccgagcactcgaaacggtacgcgggg- atggcct tgcgcagcgtcccgatcgtgaacggaggcttctccacaggctgcctgttcgtcttgatagccatctcgagGCAG- CAGCAGCTCG GATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGA- ATATCCC TGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGT- GCTATTTG CGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTC- CTGCTATCC CTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGT- ACTGCAAC CTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGTAgagctc- ttgtttt ccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggg- gttcgaaCCGAA TGCTGCGTGAACGGGAAGGAGGAGGAGAAAGAGTGAGCAGGGAGGGATTCAGAAATGAGAAATG AGAGGTGAAGGAACGCATCCCTATGCCCTTGCAATGGACAGTGTTTCTGGCCACCGCCACCAAGACT TCGTGTCCTCTGATCATCATGCGATTGATTACGTTGAATGCGACGGCCGGTCAGCCCCGGACCTCCA CGCACCGGTGCTCCTCCAGGAAGATGCGCTTGTCCTCCGCCATCTTGCAGGGCTCAAGCTGCTCCCA AAACTCTTGGGCGGGTTCCGGACGGACGGCTACCGCGGGTGCGGCCCTGACCGCCACTGTTCGGAA GCAGCGGCGCTGCATGGGCAGCGGCCGCTGCGGTGCGCCACGGACCGCATGATCCACCGGAAAAG CGCACGCGCTGGAGCGCGCAGAGGACCACAGAGAAGCGGAAGAGACGCCAGTACTGGCAAGCAG GCTGGTCGGTGCCATGGCGCGCTACTACCCTCGCTATGACTCGGGTCCTCGGCCGGCTGGCGGTGCT GACAATTCGTTTAGTGGAGCAGCGACTCCATTCAGCTACCAGTCGAACTCAGTGGCACAGTGACTcc gctcttc
[0718] The described embodiments of the invention are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention. For example, the various triglyceride oils can be tailored in for a mixture of midchain and long chain fatty acids in order to adjust parameters such as polarity, solvency, and foam-height of the oils or chemicals made from the oils. In addition, where a knockout of a gene is called for, an equivalent result may be reached using knockdown techniques including mutation and expression of inhibitory substances such as RNAi or antisense.
TABLE-US-00159 SEQUENCE LISTING SEQ ID NO: 1 6S 5' genomic donor sequence GCTCTTCGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTTGGCCTTTTCG CCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGGTCTGCCTTGCGCCGGCTGAGCCA CTGCTTCGTCCGGGCGGCCAAGAGGAGCATGAGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGC GGCTCTGGGAGCGGGCCAGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGC AGCCGCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTACAGAACAACC ACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCTGGATGACGGCCTGCGAACAGCTG TCCAGCGACCCTCGCTGCCGCCGCTTCTCCCGCACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAG CGCCGCACGCTGGCGCTGCGCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCC CTTGCGCGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACCCCCCACA CCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGGCCTCGGCCTGCAGAGAGGAC AGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCACGCTCAGGTACC SEQ ID NO: 2 6S 3' genomic donor sequence GAGCTCCTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTCTCAGCCTCGATAACCTCCAAAG CCGCTCTAATTGTGGAGGGGGTTCGAATTTAAAAGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGC CCAGACTTGTTGCTCACTGGGAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCT CTGCTTTCGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCAGTCTGT AATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCAGGCATGTCGCGGGCGAGGA CACCCGCCACTCGTACAGCAGACCATTATGCTACCTCACAATAGTTCATAACAGTGACCATATTTCTC GAAGCTCCCCAACGAGCACCTCCATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCA GGTCAACCGGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAGAATCTC TCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGAGCGTCAAACCATACCACACA AATATCCTTGGCATCGGCCCTGAATTCCTTCTGCCGCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGG GGTTGCTAGGGATCGCTCCGAGTCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTGAAGAG C SEQ ID NO: 3 S. cereviseae invertase protein sequence MLLQAFLFLLAGFAAKISASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVW GTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTP ESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIETYSSDDLK SWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEA FDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQA NPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVF ADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKV YGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK SEQ ID NO: 4 S. cereviseae invertase protein coding sequence codon optimized for expression in P. moriformis (UTEX 1435) ATGctgctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaa cgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcc tgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgg gggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgc catcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacct ccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccg gagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaa ccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccaga agtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaag tcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcct gatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccg gcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggcc ttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacac cgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgc ccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggcc aacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctg gagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagca ccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttc gcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggt gtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctact tcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtg tacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacac ctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttct acatcgacaagttccaggtgcgcgaggtcaagTGA SEQ ID NO: 5 Chlamydomonas reinhardtii TUB2 (B-tub) promoter/5' UTR CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCAT GCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCC AGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCAT ATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGG GGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAAC SEQ ID NO: 6 Chlorella vulgaris nitrate reductase 3'UTR GCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACA CTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTG TGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCC CTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCT CCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCA ACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTT SEQ ID NO: 7 Nucleotide sequence of the codon-optimized expression cassette of S. cerevisiae suc2 gene with C. reinhardtii .beta.-tubulin promoter/5'UTR and C. vulgaris nitrate reductase 3' UTR CTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCAT GCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCC AGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCAT ATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGG GGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACGGCGCGCCATGCTGCTGCAGGCCTTCCT GTTCCTGCTGGCCGGCTTCGCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCC TGGTGCACTTCACCCCCAACAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGAC GCCAAGTGGCACCTGTACTTCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGG CCACGCCACGTCCGACGACCTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACG ACTCCGGCGCCTTCTCCGGCTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACC ATCGACCCGCGCCAGCGCTGCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACAT CTCCTACAGCCTGGACGGCGGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACT CCACCCAGTTCCGCGACCCGAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCC AAGTCCCAGGACTACAAGATCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGC GTTCGCCAACGAGGGCTTCCTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGC AGGACCCCAGCAAGTCCTACTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCC TTCAACCAGTACTTCGTCGGCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGT GGTGGACTTCGGCAAGGACTACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCG CCCTGGGCATCGCGTGGGCCTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCC TCCATGTCCCTCGTGCGCAAGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGAT CAACCTGAAGGCCGAGCCGATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACA CCACGTTGACGAAGGCCAACAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAG CTGGTGTACGCCGTCAACACCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTT CAAGGGCCTGGAGGACCCCGAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCC TGGACCGCGGGAACAGCAAGGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTG AACAACCAGCCCTTCAAGAGCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAA CATCCTGGAGCTGTACTTCAACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGA ACGCCCTGGGCTCCGTGAACATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTG CGCGAGGTCAAGTGACAATTGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTG TGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGC CTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATA CCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTC CTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGC CIGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGAT GGGAACACAAATGGAGGATCC SEQ ID NO: 8 Prototheca moriformis (UTEX 435) Amt03 promoter GGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAGGTCGTTGCTGCTGCTG GTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACGCTGGCGCCCGCGAGCC GGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAGAGCGCCCGGGTCAGTT GAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAATTGGACGTGCAGGTCC TGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGGTCCGTGTCATCCACTC TAAAGAGCTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACGCCTGAGACACTTGCCC AGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCCCCCGTGGCGAGCTGCC AGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCTCAGGTCATGGGAGGTG CAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCAGCTATTTCCTCTTCAC GAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTACGGGCCCTTCCCTCAAC CCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGTTTGGGACGGGCCGTCC CGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGCAATGGACTGCTCTGCA AAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAATCATTCGTCCTGATGGG GAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACTTTTGTGCACACATTCC ATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCACCTGTTTCCCGACCTC CTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCC SEQ ID NO: 9 Chlorella protothecoides (UTEX 250) stearoyl ACP desaturase transit peptide cDNA sequence codon optimized for expression in P. moriformis. ACTAGTATGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGC GGGCTCCGGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCC SEQ ID NO: 10 Cuphea wrightii FatB2 thioesterase nucleic acid sequence; Gen Bank Accession No. U56104 ATGGTGGTGGCCGCCGCCGCCAGCAGCGCCTTCTTCCCCGTGCCCGCCCCCCGCCCCACCCCCAAGCC CGGCAAGTTCGGCAACTGGCCCAGCAGCCTGAGCCAGCCCTTCAAGCCCAAGAGCAACCCCAACGGCC GCTTCCAGGTGAAGGCCAACGTGAGCCCCCACGGGCGCGCCCCCAAGGCCAACGGCAGCGCCGTGAGC CTGAAGTCCGGCAGCCTGAACACCCTGGAGGACCCCCCCAGCAGCCCCCCCCCCCGCACCTTCCTGAA CCAGCTGCCCGACTGGAGCCGCCTGCGCACCGCCATCACCACCGTGTTCGTGGCCGCCGAGAAGCAGT TCACCCGCCTGGACCGCAAGAGCAAGCGCCCCGACATGCTGGTGGACTGGTTCGGCAGCGAGACCATC GTGCAGGACGGCCTGGTGTTCCGCGAGCGCTTCAGCATCCGCAGCTACGAGATCGGCGCCGACCGCAC CGCCAGCATCGAGACCCTGATGAACCACCTGCAGGACACCAGCCTGAACCACTGCAAGAGCGTGGGCC TGCTGAACGACGGCTTCGGCCGCACCCCCGAGATGTGCACCCGCGACCTGATCTGGGTGCTGACCAAG ATGCAGATCGTGGTGAACCGCTACCCCACCTGGGGCGACACCGTGGAGATCAACAGCTGGTTCAGCCA GAGCGGCAAGATCGGCATGGGCCGCGAGTGGCTGATCAGCGACTGCAACACCGGCGAGATCCTGGTGC GCGCCACCAGCGCCTGGGCCATGATGAACCAGAAGACCCGCCGCTTCAGCAAGCTGCCCTGCGAGGTG CGCCAGGAGATCGCCCCCCACTTCGTGGACGCCCCCCCCGTGATCGAGGACAACGACCGCAAGCTGCA CAAGTTCGACGTGAAGACCGGCGACAGCATCTGCAAGGGCCTGACCCCCGGCTGGAACGACTTCGACG TGAACCAGCACGTGAGCAACGTGAAGTACATCGGCTGGATTCTGGAGAGCATGCCCACCGAGGTGCTG GAGACCCAGGAGCTGTGCAGCCTGACCCTGGAGTACCGCCGCGAGTGCGGCCGCGAGAGCGTGGTGGA GAGCGTGACCAGCATGAACCCCAGCAAGGTGGGCGACCGCAGCCAGTACCAGCACCTGCTGCGCCTGG AGGACGGCGCCGACATCATGAAGGGCCGCACCGAGTGGCGCCCCAAGAACGCCGGCACCAACCGCGCC ATCAGCACCTGA SEQ ID NO: 11 Cuphea wrightii FatB2 thioesterase amino acid sequence; Gen Bank Accession No. U56104 MVVAAAASSAFFPVPAPRPTPKPGKFGNWPSSLSQPFKPKSNPNGRFQVKANVSPHPKANGSAVSLKS GSLNTLEDPPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLDRKSKRPDMLVDWFGSETIVQD GLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCKSVGLLNDGFGRTPEMCTRDLIWVLTKMQI VVNRYPTWGDIVEINSWFSQSGKIGMGREWLISDCNTGEILVRATSAWAMMNQKTRRESKLPCEVRQE IAPHFVDAPPVIEDNDRKLHKFDVKTGDSICKGLTPGWNDFDVNQHVSNVKYIGWILESMPTEVLETQ ELCSLTLEYRRECGRESVVESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNRAIST SEQ ID NO: 12 Codon-optimized coding region of Cocus nucifera C12:0-preferring LPAAT from pSZ2046 ATGGACGCCTCCGGCGCCTCCTCCTTCCTGCGCGGCCGCTGCCTGGAGTCCTGCTTCAAGGCCTCCTT CGGCTACGTAATGTCCCAGCCCAAGGACGCCGCCGGCCAGCCCTCCCGCCGCCCCGCCGACGCCGACG ACTTCGTGGACGACGACCGCTGGATCACCGTGATCCTGTCCGTGGTGCGCATCGCCGCCTGCTTCCTG TCCATGATGGTGACCACCATCGTGTGGAACATGATCATGCTGATCCTGCTGCCCTGGCCCTACGCCCG CATCCGCCAGGGCAACCTGTACGGCCACGTGACCGGCCGCATGCTGATGTGGATTCTGGGCAACCCCA TCACCATCGAGGGCTCCGAGTTCTCCAACACCCGCGCCATCTACATCTGCAACCACGCCTCCCTGGTG GACATCTTCCTGATCATGTGGCTGATCCCCAAGGGCACCGTGACCATCGCCAAGAAGGAGATCATCTG GTATCCCCTGTTCGGCCAGCTGTACGTGCTGGCCAACCACCAGCGCATCGACCGCTCCAACCCCTCCG CCGCCATCGAGTCCATCAAGGAGGTGGCCCGCGCCGTGGTGAAGAAGAACCTGTCCCTGATCATCTTC CCCGAGGGCACCCGCTCCAAGACCGGCCGCCTGCTGCCCTTCAAGAAGGGCTTCATCCACATCGCCCT CCAGACCCGCCTGCCCATCGTGCCGATGGTGCTGACCGGCACCCACCTGGCCTGGCGCAAGAACTCCC TGCGCGTGCGCCCCGCCCCCATCACCGTGAAGTACTTCTCCCCCATCAAGACCGACGACTGGGAGGAG GAGAAGATCAACCACTACGTGGAGATGATCCACGCCCTGTACGTGGACCACCTGCCCGAGTCCCAGAA GCCCCTGGTGTCCAAGGGCCGCGACGCCTCCGGCCGCTCCAACTCCTGA SEQ ID NO: 13 pLoop 5' genomic donor sequence gctcttcgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcac gtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgc gctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgt gcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaa aataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatgg cttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccag ggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccc cacttgcactctgcaatggtatgggaaccacggggcagtcttgtgtgggtcgcgcctatcgcggtcgg cgaagaccgggaaggtacc SEQ ID NO: 14 pLoop 3' genomic donor sequence gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagc ttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatg gatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtcc aatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgtta aaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtg tcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctc cgggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctcttcc SEQ ID NO: 15 NeoR expression cassette including C. reinhardtii p-tubulin promoter/5'UTR and C. vulgaris nitrate reductase 3' UTR ##STR00530## gcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagacc atcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagac cgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccg gcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgag gtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgc catgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcg agcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggc ctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgac ccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcg gccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctg ggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgc cttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctoggatagtatcgacacact ctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgcc gcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgctt gtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgca acttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagcc ttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgca cgggaagtagtgggatgggaacacaaatggaggatcc
SEQ ID NO: 16 Cocos nucifera 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) MDASGASSFLRGRCLESCFKASFGYVMSQPKDAAGQPSRRPADADDFVDDDRWITVILSV VRIAACFLSMMVITIVWNMIMLILLPWPYARIRQGNLYGHVTGRMLMWILGNPITIEGSE FSNTRAIYICNHASLVDIFLIMMLIPKGIVTIAKKEIIWYPLFGQLYVLANHQRIDRSNP SAAIESIKEVARAVVKKNLSLIIFPEGTRSKTGRLLPFKKGFIHIALQTRLPIVPMVLTG THLAWRKNSLRVRPAPITVKYFSPIKTDDWEEEKINHYVEMIHALYVDHLPESQKPLVSK GRDASGRSNS SEQ ID NO: 17 pSZ1500 GGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGACCC ATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGT TTGCGCACTCCATAAACTCAAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTG CTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGT TCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGC ATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGC ACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGA GGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACC CCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAG TGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCT GCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCGGGTACCCTTTCTTGCGCTATGACACTTCC AGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACC CCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGC CAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACC ACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTC AGTCACAACCCGCAAACTCTAGAATATCAATGCTGCTGCAGGCCTTCCTGTTCCTGCTGGCCGGCTTC GCCGCCAAGATCAGCGCCTCCATGACGAACGAGACGTCCGACCGCCCCCTGGTGCACTTCACCCCCAA CAAGGGCTGGATGAACGACCCCAACGGCCTGTGGTACGACGAGAAGGACGCCAAGTGGCACCTGTACT TCCAGTACAACCCGAACGACACCGTCTGGGGGACGCCCTTGTTCTGGGGCCACGCCACGTCCGACGAC CTGACCAACTGGGAGGACCAGCCCATCGCCATCGCCCCGAAGCGCAACGACTCCGGCGCCTTCTCCGG CTCCATGGTGGTGGACTACAACAACACCTCCGGCTTCTTCAACGACACCATCGACCCGCGCCAGCGCT GCGTGGCCATCTGGACCTACAACACCCCGGAGTCCGAGGAGCAGTACATCTCCTACAGCCTGGACGGC GGCTACACCTTCACCGAGTACCAGAAGAACCCCGTGCTGGCCGCCAACTCCACCCAGTTCCGCGACCC GAAGGTCTTCTGGTACGAGCCCTCCCAGAAGTGGATCATGACCGCGGCCAAGTCCCAGGACTACAAGA TCGAGATCTACTCCTCCGACGACCTGAAGTCCTGGAAGCTGGAGTCCGCGTTCGCCAACGAGGGCTTC CTCGGCTACCAGTACGAGTGCCCCGGCCTGATCGAGGTCCCCACCGAGCAGGACCCCAGCAAGTCCTA CTGGGTGATGTTCATCTCCATCAACCCCGGCGCCCCGGCCGGCGGCTCCTTCAACCAGTACTTCGTCG GCAGCTTCAACGGCACCCACTTCGAGGCCTTCGACAACCAGTCCCGCGTGGTGGACTTCGGCAAGGAC TACTACGCCCTGCAGACCTTCTTCAACACCGACCCGACCTACGGGAGCGCCCTGGGCATCGCGTGGGC CTCCAACTGGGAGTACTCCGCCTTCGTGCCCACCAACCCCTGGCGCTCCTCCATGTCCCTCGTGCGCA AGTTCTCCCTCAACACCGAGTACCAGGCCAACCCGGAGACGGAGCTGATCAACCTGAAGGCCGAGCCG ATCCTGAACATCAGCAACGCCGGCCCCTGGAGCCGGTTCGCCACCAACACCACGTTGACGAAGGCCAA CAGCTACAACGTCGACCTGTCCAACAGCACCGGCACCCTGGAGTTCGAGCTGGTGTACGCCGTCAACA CCACCCAGACGATCTCCAAGTCCGTGTTCGCGGACCTCTCCCTCTGGTTCAAGGGCCTGGAGGACCCC GAGGAGTACCTCCGCATGGGCTTCGAGGTGTCCGCGTCCTCCTTCTTCCTGGACCGCGGGAACAGCAA GGTGAAGTTCGTGAAGGAGAACCCCTACTTCACCAACCGCATGAGCGTGAACAACCAGCCCTTCAAGA GCGAGAACGACCTGTCCTACTACAAGGTGTACGGCTTGCTGGACCAGAACATCCTGGAGCTGTACTTC AACGACGGCGACGTCGTGTCCACCAACACCTACTTCATGACCACCGGGAACGCCCTGGGCTCCGTGAA CATGACGACGGGGGTGGACAACCTGTTCTACATCGACAAGTTCCAGGTGCGCGAGGTCAAGTGACAAT TGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCA CACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTG TGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTT CCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTG CTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTG CAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAGGAT CCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCA TACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACAC ACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACAGCC TAGGGATATCGAATTCGGCCGACAGGACGCGCGTCAAAGGTGCTGGTCGTGTATGCCCTGGCCGGCAG GTCGTTGCTGCTGCTGGTTAGTGATTCCGCAACCCTGATTTTGGCGTCTTATTTTGGCGTGGCAAACG CTGGCGCCCGCGAGCCGGGCCGGCGGCGATGCGGTGCCCCACGGCTGCCGGAATCCAAGGGAGGCAAG AGCGCCCGGGTCAGTTGAAGGGCTTTACGCGCAAGGTACAGCCGCTCCTGCAAGGCTGCGTGGTGGAA TTGGACGTGCAGGTCCTGCTGAAGTTCCTCCACCGCCTCACCAGCGGACAAAGCACCGGTGTATCAGG TCCGTGTCATCCACTCTAAAGAACTCGACTACGACCTACTGATGGCCCTAGATTCTTCATCAAAAACG CCTGAGACACTTGCCCAGGATTGAAACTCCCTGAAGGGACCACCAGGGGCCCTGAGTTGTTCCTTCCC CCCGTGGCGAGCTGCCAGCCAGGCTGTACCTGTGATCGAGGCTGGCGGGAAAATAGGCTTCGTGTGCT CAGGTCATGGGAGGTGCAGGACAGCTCATGAAACGCCAACAATCGCACAATTCATGTCAAGCTAATCA GCTATTTCCTCTTCACGAGCTGTAATTGTCCCAAAATTCTGGTCTACCGGGGGTGATCCTTCGTGTAC GGGCCCTTCCCTCAACCCTAGGTATGCGCGCATGCGGTCGCCGCGCAACTCGCGCGAGGGCCGAGGGT TTGGGACGGGCCGTCCCGAAATGCAGTTGCACCCGGATGCGTGGCACCTTTTTTGCGATAATTTATGC AATGGACTGCTCTGCAAAATTCTGGCTCTGTCGCCAACCCTAGGATCAGCGGCGTAGGATTTCGTAAT CATTCGTCCTGATGGGGAGCTACCGACTACCCTAATATCAGCCCGACTGCCTGACGCCAGCGTCCACT TTIGTGCACACATTCCATTCGTGCCCAAGACATTTCATTGTGGTGCGAAGCGTCCCCAGTTACGCTCA CCTGTTTCCCGACCTCCTTACTGTTCTGTCGACAGAGCGGGCCCACAGGCCGGTCGCAGCCACTAGTA TGGCCACCGCATCCACTTTCTCGGCGTTCAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCC GGGCCCCGGCGCCCAGCGAGGCCCCTCCCCGTGCGCGGGCGCGCCGCCACCGGCGAGCAGCCCTCCGG CGTGGCCTCCCTGCGCGAGGCCGACAAGGAGAAGTCCCTGGGCAACCGCCTGCGCCTGGGCTCCCTGA CCGAGGACGGCCTGTCCTACAAGGAGAAGTTCGTGATCCGCTGCTACGAGGTGGGCATCAACAAGACC GCCACCATCGAGACCATCGCCAACCTGCTGCAGGAGGTGGGCGGCAACCACGCCCAGGGCGTGGGCTT CTCCACCGACGGCTTCGCCACCACCACCACCATGCGCAAGCTGCACCTGATCTGGGTGACCGCCCGCA TGCACATCGAGATCTACCGCTACCCCGCCTGGTCCGACGTGATCGAGATCGAGACCTGGGTGCAGGGC GAGGGCAAGGTGGGCACCCGCCGCGACTGGATCCTGAAGGACTACGCCAACGGCGAGGTGATCGGCCG CGCCACCTCCAAGTGGGTGATGATGAACGAGGACACCCGCCGCCTGCAGAAGGTGTCCGACGACGTGC GCGAGGAGTACCTGGTGTTCTGCCCCCGCACCCTGCGCCTGGCCTTCCCCGAGGAGAACAACAACTCC ATGAAGAAGATCCCCAAGCTGGAGGACCCCGCCGAGTACTCCCGCCTGGGCCTGGTGCCCCGCCGCTC CGACCTGGACATGAACAAGCACGTGAACAACGTGACCTACATCGGCTGGGCCCTGGAGTCCATCCCCC CCGAGATCATCGACACCCACGAGCTGCAGGCCATCACCCTGGACTACCGCCGCGAGTGCCAGCGCGAC GACATCGTGGACTCCCTGACCTCCCGCGAGCCCCTGGGCAACGCCGCCGGCGTGAAGTTCAAGGAGAT CAACGGCTCCGTGTCCCCCAAGAAGGACGAGCAGGACCTGTCCCGCTTCATGCACCTGCTGCGCTCCG CCGGCTCCGGCCTGGAGATCAACCGCTGCCGCACCGAGTGGCGCAAGAAGCCCGCCAAGCGCATGGAC TACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGTGAAT CGATAGATCTCTTAAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATG GACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAG TGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACC CCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCT ATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTA TTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAA CACAAATGGAAAGCTTAATTAAGAGCTCCCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAG GTGTTTGTGCCGCCGCACCGCGCAGTGGCGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCG CATGGGCAAGGTGCTGGTCACCCTGACCCTGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGC GGCCGTACCCGCGCTTCGCCAACCACTTTGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATC GAGGTGGTCATCTCCGACCTGGCGCTGGTGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCAT GGGCTGGGCCTGGCTGGTCAAGACCTACGTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCA TCACGCTGCTCCAGCACACGCACCCGGCGCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGC GGCGCCATGGCCACCGTGGACCGCTCCATGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTC CGACACCCACGTGCTGCACCACCTCTTCAGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCG CCATCAGGCCCATCCTGGGCAAGTACTACCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAG GACTGGCGCGACTGCCGCTACGTCGTCCCGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAA GTGAGTGAGTGA SEQ ID NO: 18 5' FADc genomic region donor DNA GGGCTGGTCTGAATCCTTCAGGCGGGTGTTACCCGAGAAAGAAAGGGTGCCGATTTCAAAGCAGACCC ATGTGCCGGGCCCTGTGGCCTGTGTTGGCGCCTATGTAGTCACCCCCCCTCACCCAATTGTCGCCAGT TTGCGCACTCCATAAACTCAAAACAGCAGCTTCTGAGCTGCGCTGTTCAAGAACACCTCTGGGGTTTG CTCACCCGCGAGGTCGACGCCCAGCATGGCTATCAAGACGAACAGGCAGCCTGTGGAGAAGCCTCCGT TCACGATCGGGACGCTGCGCAAGGCCATCCCCGCGCACTGTTTCGAGCGCTCGGCGCTTCGTAGCAGC ATGTACCTGGCCTTTGACATCGCGGTCATGTCCCTGCTCTACGTCGCGTCGACGTACATCGACCCTGC ACCGGTGCCTACGTGGGTCAAGTACGGCATCATGTGGCCGCTCTACTGGTTCTTCCAGGTGTGTTTGA GGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACC CCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCCTTCGGCACGGGTGTCTGGGTGTGCGCGCACGAG TGCGGCCACCAGGCCTTTTCCTCCAGCCAGGCCATCAACGACGGCGTGGGCCTGGTGTTCCACAGCCT GCTGCTGGTGCCCTACTACTCCTGGAAGCACTCGCACCG SEQ ID NO: 19 3' FADc genomic region donor DNA CCGCCACCACTCCAACACGGGGTGCCTGGACAAGGACGAGGTGTTTGTGCCGCCGCACCGCGCAGTGG CGCACGAGGGCCTGGAGTGGGAGGAGTGGCTGCCCATCCGCATGGGCAAGGTGCTGGTCACCCTGACC CIGGGCTGGCCGCTGTACCTCATGTTCAACGTCGCCTCGCGGCCGTACCCGCGCTTCGCCAACCACTT TGACCCGTGGTCGCCCATCTTCAGCAAGCGCGAGCGCATCGAGGTGGTCATCTCCGACCTGGCGCTGG TGGCGGTGCTCAGCGGGCTCAGCGTGCTGGGCCGCACCATGGGCTGGGCCTGGCTGGTCAAGACCTAC GTGGTGCCCTACCTGATCGTGAACATGTGGCTCGTGCTCATCACGCTGCTCCAGCACACGCACCCGGC GCTGCCGCACTACTTCGAGAAGGACTGGGACTGGCTGCGCGGCGCCATGGCCACCGTGGACCGCTCCA TGGGCCCGCCCTTCATGGACAACATCCTGCACCACATCTCCGACACCCACGTGCTGCACCACCTCTTC AGCACCATCCCGCACTACCACGCCGAGGAGGCCTCCGCCGCCATCAGGCCCATCCTGGGCAAGTACTA CCAGTCCGACAGCCGCTGGGTCGGCCGCGCCCTGTGGGAGGACTGGCGCGACTGCCGCTACGTCGTCC CGGACGCGCCCGAGGACGACTCCGCGCTCTGGTTCCACAAGTGAGTGAGTGA SEQ ID NO: 20 5' donor DNA sequence of Prototheca moriformis FATA1 knockout homologous recombination targeting construct GCTCTTCGGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGA ATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGA CCAGCCTGCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGC GCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTT CCGAACAGTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGG AGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAG GTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTT GGTGGCGGTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCT CATTTCTGAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGGGGTACC SEQ ID NO: 21 3' donor DNA sequence of Prototheca moriformis FATA1 knockout homologous recombination targeting construct GACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCICCCT GGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCT TCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCA ATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCC CCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTAT TCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCG GAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAAT GGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGTGAAGAGC SEQ ID NO: 22 Chlorella protothecoides actin promoter/5'UTR agtttaggtccagcgtccgtggggggggacgggctgggagcttgggccgggaagggcaagacgatgca gtccctctggggagtcacagccgactgtgtgtgttgcactgtgcggcccgcagcactcacacgcaaaa tgcctggccgacaggcaggccctgtccagtgcaacatccacggtccctctcatcaggctcaccttgct cattgacataacggaatgcgtaccgctctttcagatctgtccatccagagaggggagcaggctcccca ccgacgctgtcaaacttgcttcctgcccaaccgaaaacattattgtttgagggggggggggggggggc agattgcatggcgggatatctcgtgaggaacatcactgggacactgtggaacacagtgagtgcagtat gcagagcatgtatgctaggggtcagcgcaggaagggggcctttcccagtctcccatgccactgcaccg tatccacgactcaccaggaccagcttcttgatcggcttccgctcccgtggacaccagtgtgtagcctc tggactccaggtatgcgtgcaccgcaaaggccagccgatcgtgccgattcctggggtggaggatatga gtcagccaacttggggctcagagtgcacactggggcacgatacgaaacaacatctacaccgtgtcctc catgctgacacaccacagcttcgctccacctgaatgtgggcgcatgggcccgaatcacagccaatgtc gctgctgccataatgtgatccagaccctctccgcccagatgccgagcggatcgtgggcgctgaataga ttcctgtttcgatcactgtttgggtcctttccttttcgtctcggatgcgcgtctcgaaacaggctgcg tcgggctttcggatcccttttgctccctccgtcaccatcctgcgcgcgggcaagttgcttgaccctgg gctgtaccagggttggagggtattaccgcgtcaggccattcccagcccggattcaattcaaagtctgg gccaccaccctccgccgctctgtctgatcactccacattcgtgcatacactacgttcaagtcctgatc caggcgtgtctcgggacaaggtgtgcttgagtttgaatctcaaggacccactccagcacagctgctgg ttgaccccgccctcgcaa SEQ ID NO: 23 AtTHIC expression cassette comprising Chlorella protothecoides actin promoter/5'UTR, Arabidopsis thaliana THIC protein coding sequence codon-optimized for expression in Prototheca moriformis, and Chlorella vulgaris nitrate reductase 3' UTR agtttaggtccagcgtccgtggggggggacgggctgggagcttgggccgggaagggcaagacgatgca gtccctctggggagtcacagccgactgtgtgtgttgcactgtgcggcccgcagcactcacacgcaaaa tgcctggccgacaggcaggccctgtccagtgcaacatccacggtccctctcatcaggctcaccttgct cattgacataacggaatgcgtaccgctctttcagatctgtccatccagagaggggagcaggctcccca ccgacgctgtcaaacttgcttcctgcccaaccgaaaacattattgtttgagggggggggggggggggc agattgcatggcgggatatctcgtgaggaacatcactgggacactgtggaacacagtgagtgcagtat gcagagcatgtatgctaggggtcagcgcaggaagggggcctttcccagtctcccatgccactgcaccg tatccacgactcaccaggaccagcttcttgatcggcttccgctcccgtggacaccagtgtgtagcctc tggactccaggtatgcgtgcaccgcaaaggccagccgatcgtgccgattcctggggtggaggatatga gtcagccaacttggggctcagagtgcacactggggcacgatacgaaacaacatctacaccgtgtcctc catgctgacacaccacagcttcgctccacctgaatgtgggcgcatgggcccgaatcacagccaatgtc gctgctgccataatgtgatccagaccctctccgcccagatgccgagcggatcgtgggcgctgaataga ttcctgtttcgatcactgtttgggtcctttccttttcgtctcggatgcgcgtctcgaaacaggctgcg tcgggctttcggatcccttttgctccctccgtcaccatcctgcgcgcgggcaagttgcttgaccctgg gctgtaccagggttggagggtattaccgcgtcaggccattcccagcccggattcaattcaaagtctgg gccaccaccctccgccgctctgtctgatcactccacattcgtgcatacactacgttcaagtcctgatc caggcgtgtctcgggacaaggtgtgcttgagtttgaatctcaaggacccactccagcacagctgctgg ttgaccccgccctcgcaatctagaATGgccgcgtccgtccactgcaccctgatgtccgtggtctgcaa caacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtggtgg tccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccc cccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttcca gcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacgagg agtccggccacgtcctgaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgac aactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagctgcgcaaggagtg gatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatca tcacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtc gcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccg caagttcctggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggagg tctacaaggtgcagtgggccaccatgtggggcgccgacaccatcatggacctgtccacgggccgccac atccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtccccatctacca ggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcg agcaggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctg accgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggccta ccacaaggagaacttcgcctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcg ccctgtccatcggcgacggcctgcgccccggctccatctacgacgccaacgacacggcccagttcgcc gagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatgaacgaggg ccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgagg cgcccttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgcc atcggcgcggccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacct gggcctgcccaaccgcgacgacgtgaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccg acctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgtccaaggcgcgcttcgagttc cgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgagacgct gcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaaga tcacggaggacatccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccag ggcatggacgccatgtccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacggcga ggtcggcggcgagatctacctgcccgagtcctacgtcaaggccgcgcagaagTGAcaattggcagcag cagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctg ccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgc gcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgttt catatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctc ctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgta aaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc SEQ ID NO: 24 PmKASII (Prototheca moriformis KASII) comprising a C. protothecoides S106 stearoyl-ACP desaturase transit peptide ATGgccaccgcatccactttctcggcgttoaatgcccgctgcggcgacctgcgtcgctcggcgggctc cgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccgccgccgccgccgacgccaacc ccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccagaccatc
gagcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccac cggctacaccaccaccatcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgct gggccaagcgcgtggacgacgtgatcaagtacgtgtacatcgccggcaagcaggccctggagtccgcc ggcctgcccatcgaggccgccggcctggccggcgccggcctggaccccgccctgtgcggcgtgctgat cggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgacccgcggcggcgtgc gcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggac atcggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctggg cgccgccgaccacatccgccgcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatca tcccctccggcatcggcggcttcatcgcctgcaaggccctgtccaagcgcaacgacgagcccgagcgc gcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagggcgccggcgtgctggtgct ggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcgccgcca cctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgc gccctggagcgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccc cgccggcgacgtggccgagtaccgcgccatccgcgccgtgatcccccaggactccctgcgcatcaact ccaccaagtccatgatcggccacctgctgggcggcgccggcgccgtggaggccgtggccgccatccag gccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccggcgtggaccccgt ggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggct tcggcggccacaactcctgcgtgatcttccgcaagtacgacgagatggactacaaggaccacgacggc gactacaaggaccacgacatcgactacaaggacgacgacgacaagTGA SEQ ID NO: 25 PmKASII (Prototheca moriformis KASII) comprising a C. protothecoides S106 stearoylACP desaturase transit peptide MATASTESAFNARCGDLRRSAGSGPRRPARPLPVRGRAAAAADANPARPERRVVITGQGVVISLGQII EQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALESA GLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMD IGFMGPNYSISTACATGNYCILGAADHIRRGDANVMLAGGADAAIIPSGIGGFIACKALSKRNDEPER ASRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATILAELVGGAATSDAHHMTEPDPQGRGVRLCLER ALERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVIPQDSLRINSTKSMIGHLLGGAGAVEAVAAIQ ALRTGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLDVVLSNSFGFGGHNSCVIFRKYDEMDYKDHDG DYKDHDIDYKDDDDK SEQ ID NO: 26 Codon-optimized Prototheca moriformis (UTEX 1435) FAD2 protein- coding sequence ATGgccatcaagaccaaccgccagcccgtggagaagccccccttcaccatcggcaccctgcgcaaggc catccccgcccactgcttcgagcgctccgccctgcgctcctccatgtacctggccttcgacatcgccg tgatgtccctgctgtacgtggcctccacctacatcgaccccgcccccgtgcccacctgggtgaagtac ggcgtgatgtggcccctgtactggttcttccagggcgccttcggcaccggcgtgtgggtgtgcgccca cgagtgcggccaccaggccttctcctcctcccaggccatcaacgacggcgtgggcctggtgttccact ccctgctgctggtgccctactactcctggaagcactcccaccgccgccaccactccaacaccggctgc ctggacaaggacgaggtgttcgtgcccccccaccgcgccgtggcccacgagggcctggagtgggagga gtggctgcccatccgcatgggcaaggtgctggtgaccctgaccctgggctggcccctgtacctgatgt tcaacgtggcctcccgcccctacccccgcttcgccaaccacttcgacccctggtcccccatcttctcc aagcgcgagcgcatcgaggtggtgatctccgacctggccctggtggccgtgctgtccggcctgtccgt gctgggccgcaccatgggctgggcctggctggtgaagacctacgtggtgccctacctgatcgtgaaca tgtggctggtgctgatcaccctgctgcagcacacccaccccgccctgccccactacttcgagaaggac tgggactggctgcgcggcgccatggccaccgtggaccgctccatgggcccccccttcatggacaacat cctgcaccacatctccgacacccacgtgctgcaccacctgttctccaccatcccccactaccacgccg aggaggcctccgccgccatccgccccatcctgggcaagtactaccagtccgactcccgctgggtgggc cgcgccctgtgggaggactggcgcgactgccgctacgtggtgcccgacgcccccgaggacgactccgc cctgtggttccacaagTAG SEQ ID NO: 27 Amino acid sequence of Prototheca moriformis FAD2 MAIKTNRQPVEKPPFTIGILRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPIWVKY GVMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVFHSLLLVPYYSWKHSHRRHHSNTGC LDKDEVFVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMFNVASRPYPRFANHFDPWSPIFS KRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYLIVNMWLVLITLLQHTHPALPHYFEKD WDWLRGAMATVDRSMGPPFMDNILHHISDTHVLHHLFSTIPHYHAEEASAAIRPILGKYYQSDSRWVG RALWEDWRDCRYVVPDAPEDDSALWFHK SEQ ID NO: 28 Codon-optimized coding region of Brassica napus C18:0-preferring thioesterase from pSZ1358 ACTAGTATGCTGAAGCTGTCCTGCAACGTGACCAACAACCTGCACACCTTCTCCTTCTTCTCCGACTC CTCCCTGTTCATCCCCGTGAACCGCCGCACCATCGCCGTGTCCTCCGGGCGCGCCTCCCAGCTGCGCA AGCCCGCCCTGGACCCCCTGCGCGCCGTGATCTCCGCCGACCAGGGCTCCATCTCCCCCGTGAACTCC TGCACCCCCGCCGACCGCCTGCGCGCCGGCCGCCTGATGGAGGACGGCTACTCCTACAAGGAGAAGTT CATCGTGCGCTCCTACGAGGTGGGCATCAACAAGACCGCCACCGTGGAGACCATCGCCAACCTGCTGC AGGAGGTGGCCTGCAACCACGTGCAGAAGTGCGGCTTCTCCACCGACGGCTTCGCCACCACCCTGACC ATGCGCAAGCTGCACCTGATCTGGGTGACCGCCCGCATGCACATCGAGATCTACAAGTACCCCGCCTG GTCCGACGTGGTGGAGATCGAGACCTGGTGCCAGTCCGAGGGCCGCATCGGCACCCGCCGCGACTGGA TCCTGCGCGACTCCGCCACCAACGAGGTGATCGGCCGCGCCACCTCCAAGTGGGTGATGATGAACCAG GACACCCGCCGCCTGCAGCGCGTGACCGACGAGGTGCGCGACGAGTACCTGGTGTTCTGCCCCCGCGA GCCCCGCCTGGCCTTCCCCGAGGAGAACAACTCCTCCCTGAAGAAGATCCCCAAGCTGGAGGACCCCG CCCAGTACTCCATGCTGGAGCTGAAGCCCCGCCGCGCCGACCTGGACATGAACCAGCACGTGAACAAC GTGACCTACATCGGCTGGGTGCTGGAGTCCATCCCCCAGGAGATCATCGACACCCACGAGCTGCAGGT GATCACCCTGGACTACCGCCGCGAGTGCCAGCAGGACGACATCGTGGACTCCCTGACCACCTCCGAGA TCCCCGACGACCCCATCTCCAAGTTCACCGGCACCAACGGCTCCGCCATGTCCTCCATCCAGGGCCAC AACGAGTCCCAGTTCCTGCACATGCTGCGCCTGTCCGAGAACGGCCAGGAGATCAACCGCGGCCGCAC CCAGTGGCGCAAGAAGTCCTCCCGCATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACA TCGACTACAAGGACGACGACGACAAGTGAATCGAT SEQ ID NO: 29 Amino acid sequence of Brassica napus C18:0-preferring thioesterase (Accession No. CAA52070.1) MLKLSCNVTNNLHTFSFFSDSSLFIPVNRRTIAVSSSQLRKPALDPLRAVISADQGSISPVNSCTPAD RLRAGRLMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGESIDGFATTLIMRKLH LIWVTARMHIETYKYPAWSDVVEIETWCQSEGRIGIRRDWILRDSATNEVIGRATSKWVMMNQDTRRL QRVTDEVRDEYLVFCPREPRLAFPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTYIG WVLESIPQEIIDTHELQVITLDYRRECQQDDIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNESQF LHMLRLSENGQEINRGRTQWRKKSSR SEQ ID NO: 30 Prototheca moriformis FATA1 allele 1 5' homology donor region GGAGTCACTGTGCCACTGAGTTCGACTGGTAGCTGAATGGAGTCGCTGCTCCACTAAACGAATTGTCA GCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCGAGGGTAGTAGCGCGCCATGGCACCGACCAGCCT GCTTGCCAGTACTGGCGTCTCTTCCGCTTCTCTGTGGTCCTCTGCGCGCTCCAGCGCGTGCGCTTTTC CGGTGGATCATGCGGICCGTGGCGCACCGCAGCGGCCGCTGCCCATGCAGCGCCGCTGCTTCCGAACA GTGGCGGTCAGGGCCGCACCCGCGGTAGCCGTCCGTCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCT TGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCTTCCTGGAGGAGCACCGGTGCGTGGAGGTCCGGG GCTGACCGGCCGTCGCATTCAACGTAATCAATCGCATGATGATCAGAGGACACGAAGTCTTGGTGGCG GTGGCCAGAAACACTGTCCATTGCAAGGGCATAGGGATGCGTTCCTTCACCTCTCATTTCTCATTTCT GAATCCCTCCCTGCTCACTCTTTCTCCTCCTCCTTCCCGTTCACGCAGCATTCGG SEQ ID NO: 31 Prototheca moriformis FATA1 allele 1 3' homology donor region GACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGCTTATATCCTCCCT GGAAGCACACCCACGACTCTGAAGAAGAAAACGTGCACACACACAACCCAACCGGCCGAATATTTGCT TCCTTATCCCGGGTCCAAGAGAGACTGCGATGCCCCCCTCAATCAGCATCCTCCTCCCTGCCGCTTCA ATCTTCCCTGCTTGCCTGCGCCCGCGGTGCGCCGTCTGCCCGCCCAGTCAGTCACTCCTGCACAGGCC CCTTGTGCGCAGTGCTCCTGTACCCTTTACCGCTCCTTCCATTCTGCGAGGCCCCCTATTGAATGTAT TCGTTGCCTGTGTGGCCAAGCGGGCTGCTGGGCGCGCCGCCGTCGGGCAGTGCTCGGCGACTTTGGCG GAAGCCGATTGTTCTTCTGTAAGCCACGCGCTTGCTGCTTTGGGAAGAGAAGGGGGGGGGTACTGAAT GGATGAGGAGGAGAAGGAGGGGTATTGGTATTATCTGAGTTGGGT SEQ ID NO: 32 Prototheca moriformis FATA1 allele 2 5' homology donor region AATGGAGTCGCTGCTCCACTAATCGAATTGTCAGCACCGCCAGCCGGCCGAGGACCCGAGTCATAGCG AGGGTAGTAGCGCGCCATGGCACCGACCAGCCTGCTTGCCCGTACTGGCGTCTCTTCCGCTTCTCTGT GCTCCTCTACGCGCTCCGGCGCGTGCGCTTTTCCGGTGGATCATGCGGTCCGTGGCGCACCGCAGCGG CCGCTGCCCATGCAGCGCCGCTGCTTCCGAACAGTGGCTGTCAGGGCCGCACCCGCAGTAGCCGTCCG TCCGGAACCCGCCCAAGAGTTTTGGGAGCAGCTTGAGCCCTGCAAGATGGCGGAGGACAAGCGCATCT TCCTGGAGGAGCACCGGTGCGCGGAGGTCCGGGGCTGACCGGCCGTCGCATTCAACGTAATCAATCGC ATGATGATCACAGGACGCGACGTCTTGGTGGCGGTGGCCAGGGACACTGCCCATTGCACAGGCATAGG AATGCGTTCCTTCTCATTTCTCAGTTTTCTGAGCCCCTCCCTCTTCACTCTTTCTCCTCCTCCTCCCC TCTCACGCAGCATTCGTGG SEQ ID NO: 33 Prototheca moriformis FATA1 allele 2 3' homology donor region CACTAGTATCGATTTCGAACAGAGGAGAGGGTGGCTGGTAGTTGCGGGATGGCTGGTCGCCCGTCGAT CCTGCTGCTGCTATTGTCTCCTCCTGCACAAGCCCACCCACGACTCCGAAGAAGAAGAAGAAAACGCG CACACACACAACCCAACCGGCCGAATATTTGCTTCCTTATCCCGGGTCCAAGAGAGACGGCGATGCCC CCCTCAATCAGCCTCCTCCTCCCTGCCGCTCCAATCTTCCCTGCTTGCATGCGCCCGCGAGAGGCTGT CTGCGCGCCCCGTCAGTCACTCCCCGTGCAGACGCCTCGTGCTCGGTGCTCCTGTATCCTTTACCGCT CCTTTCATTCTGCGAGGCCCCCTGTTGAATGTATTCGTTGCCTGTGTGGCCAAGCGCGCTGCTGGGCG CGCCGCCGTCGGGCGGTGCTCGGCGACTCTGGCGGAAGCCGGTTGTTCTTCTGTAAGCCACGCGCTTG CTGCTTTTGGAAAAGAGGGGGGTTTACTGAATGGAGGAGGAGCAGGATAATTGGTAGTATCTGAGTTG TTG SEQ ID NO: 34 SAD2 hairpin C actagtGCGCTGGACGCGGCAGTGGGTGGCCGAGGAGAACCGGCACGGCGACCTGCTGAACAAGTACT GTTGGCTGACGGGGCGCGTCAACATGCGGGCCGTGGAGGTGACCATCAACAACCTGATCAAGAGCGGC ATGAACCCGCAGACGGACAACAACCCTTACTTGGGCTTCGTCTACACCTCCTTCCAGGAGCGCGCGAC CAAGTACAGCCACGGCAACACCGCGCGCCTTGCGGCCGAGCAGTGTGTTTGAGGGTTTTGGTTGCCCG TATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCC CGGCACCTTCCAGGGCGCGTACGggatccTGCTCGGCCGCAAGGCGCGCGGTGTTGCCGTGGCTGTAC TTGGTCGCGCGCTCCTGGAAGGAGGTGTAGACGAAGCCCAAGTAAGGGTTGTTGTCCGTCTGCGGGTT CATGCCGCTCTTGATCAGGTTGTTGATGGTCACCTCCACGGCCCGCATGTTGACGCGCCCCGTCAGCC AACAGTACTTGTTCAGCAGGTCGCCGTGCCGGTTCTCCTCGGCCACCCACTGCCGCGTCCAGCGCaag ctt SEQ ID NO: 35 Prototheca moriformis FAD-D omega 3 desaturase MSIQFALRAAYIKGICQRLSGRGAALGLSRDWIPGWILPRCWPASAAATAPPRARHQERAIHLTSGRR RHSALASDADERALPSNAPGLVMASQANYFRVRLLPEQEEGELESWSPNVRHTTLLCKPRAMLSKLQM RVMVGDRVIVTAIDPVNMTVHAPPFDPLPATRFLVAGEAADMDITVVLNKADLVPEEESAALAQEVAS WGPVVLTSTLTGRGLQELERQLGSTTAVLAGPSGAGKSSIINALARAARERPSDASVSNVPEEQVVGE DGRALANPPPFTLADIRNAIPKDCFRKSAAKSLAYLGDLSITGMAVLAYKINSPWLMPLYWFAQGTMF WALFVVGHDCGHQSFSTSKRLNDALAWLGALAAGTWTWALGVLPMLNLYLAPYVWLLVTYLHHHGPSD PREEMPWYRGREWSYMRGGLTTIDRDYGLFNKVHHDIGTHVVHH SEQ ID NO: 36 MFWALFVVGHDCGHQSFSTSKRLNDAVGLFVHSTIGVPYHGWRISHRTHHNNHGHVENDESWYPPTES GLKAMTDMGRQGRFHFPSMLFVYPFYLFWRSPGKTGSHFSPATDLFALWEAPLIRTSNACQLAWLGAL AAGTWALGVLPMLNLYLAPYVISVAWLDLVTYLHHHGPSDPREEMPWYRGREWSYMRGGLTTIDRDYG LFNKVHHDIGTHVVHHLFPQIPHYNLCRATKAAKKVLGPYYREPERCPLGLLPVHLLAPLLRSLGQDH FVDDAGSVLFYRRAEGINPWIQKLLPWLGGARRGADAQRDAAQ SEQ ID NO: 37 Camelina sativa omega-3 EAD7-2 MANLVLSECGIRPLPRIYTTPRSNFVSNNNKPIFKFRPFTSYKTSSSPLACSRDGFGKNWSLNVSVPL TTTTPIVDESPLKEEEEEKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKSMSYVLRDVAIVFALAAG ASYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNNPRLNNVVGHLLHSSILVPYHGWRISHRTH HQNHGHVENDESWHPMSEKIYQSLDKPTRFFRFTLPLVMLAYPFYLWARSPGKKGSHYHPESDLFLPK EKTDVLTSTACWTAMAALLICLNFVVGPVQMLKLYGIPYWINVMWLDFVTYLHHHGHEDKLPWYRGKE WSYLRGGLTTLDRDYGVINNIHHDIGTHVIHHLFPQIPHYHLVEATEAVKPVLGKYYREPDKSGPLPL HLLGILAKSIKEDHYVSDEGDVVYYKADPNMYGEIKVGAD SEQ ID NO: 38 Prototheca moriformis delta 12 desaturase allele 2 MAIKTNRQPVEKPPFTIGILRKAIPAHCFERSALRSSMYLAFDIAVMSLLYVASTYIDPAPVPIWVKY GIMWPLYWFFQGAFGTGVWVCAHECGHQAFSSSQAINDGVGLVFHSLLLVPYYSWKHSHRRHHSNTGC LDKDEVFVPPHRAVAHEGLEWEEWLPIRMGKVLVTLTLGWPLYLMFNVASRPYPRFANHFDPWSPIFS KRERIEVVISDLALVAVLSGLSVLGRTMGWAWLVKTYVVPYMIVNMWLVLITLLQHTHPALPHYFEKD WDWLRGAMATVDRSMGPPFMDSILHHISDTHVLHHLFSTIPHYHAEEASAAIRPILGKYYQSDSRWVG RALWEDWRDCRYVVPDAPEDDSALWFHK SEQ ID NO: 39 Camelina sativa omega-3 FAD7-1 MANLVLSECGIRPLPRIYTTPRSNFVSNNNKPIFKFRPLTSYKTSSPLFCSRDGFGRNWSLNVSVPLA TTTPIVDESPLEEEEEEEKQRFDPGAPPPFNLADIRAAIPKHCWVKNPWKSMSYVLRDVAIVFALAAG AAYLNNWIVWPLYWLAQGTMFWALFVLGHDCGHGSFSNNPRLNNVVGHLLHSSILVPYHGWRISHRTH HQNHGHVENDESWHPMSEKIYQSLDKPTRFFRFTLPLVMLAYPFYLWARSPGKKGSHYHPESDLFLPK EKTDVLTSTACWTAMAALLICLNFVVGPVQMLKLYGIPYWINVMWLDFVTYLHHHGHEDKLPWYRGKE WSYLRGGLTTLDRDYGVINNIHHDIGTHVIHHLFPQIPHYHLVEATEAVKPVLGKYYREPDKSGPLPL HLLGILAKSIKEDHYVSDEGDVVYYKADPNMYGEIKVGAD SEQ ID NO: 40 PmFATA-hpB actagtCATTCGGGGCAACGAGGTGGGCCCCICGCAGCGGCTGACGATCACGGCGGTGGCCAACATCC TGCAGGAGGCGGCGGGCAACCACGCGGTGGCCATGTGGGGCCGGAGCGTGTGTTTGAGGGTTTTGGTT GCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTGACCCCCCCGGCTAC CCTCCCGGCACCTTCCAGGGCGCGTACGggatccGCTCCGGCCCCACATGGCCACCGCGTGGTTGCCC GCCGCCTCCTGCAGGATGTTGGCCACCGCCGTGATCGTCAGCCGCTGCGAGGGGCCCACCTCGTTGCC CCGAATGaagctt SEQ ID NO: 41 PmFATA-hpC actagtGGAGGGTTTCGCGACGGACCCGGAGCTGCAGGAGGCGGGTCTCATCTTTGTGATGACGCGCA TGCAGATCCAGATGTACCGCTACCCGCGCTGGGGCGACCTGATGCAGGTGGAGACCTGGTTCCAGAGT GTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCC CGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccTCTGGAACCAGGTCT CCACCTGCATCAGGTCGCCCCAGCGCGGGTAGCGGTACATCTGGATCTGCATGCGCGTCATCACAAAG ATGAGACCCGCCTCCTGCAGCTCCGGGTCCGTCGCGAAACCCTCCaagctt SEQ ID NO: 42 PmFATA-hpD actagtCGGCGGGCAAGCTGGGCGCGCAGCGCGAGTGGGTGCTGCGCGACAAGCTGACCGGCGAGGCG CTGGGCGCGGCCACCTCGAGCTGGGTCATGATCAACATCCGCACGCGCCGGCCGTGCCGCATGCCGGG TGTGTTTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTC CCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccCCGGCATGCGGCAC GGCCGGCGCGTGCGGATGTTGATCATGACCCAGCTCGAGGTGGCCGCGCCCAGCGCCTCGCCGGTCAG CTTGTCGCGCAGCACCCACTCGCGCTGCGCGCCCAGCTTGCCCGCCGaagctt SEQ ID NO: 43 PmFATA-hpE actagtGTCCGCGTCAAGTCGGCCTTCTTCGCGCGCGAGCCGCCGCGCCTGGCGCTGCCGCCCGCGGT CACGCGTGCCAAGCTGCCCAACATCGCGACGCCGGCGCCGCTGCGCGGGCACCGCCAGGTCGCGCGCC GCACCGACATGGACATGAACGGGCACGTGAACAACGTGGCCTACCTGGCCTGGTGCCTGGAGTGTGTT TGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCTG ACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccTCCAGGCACCAGGCCAGGTA GGCCACGTTGTTCACGTGCCCGTTCATGTCCATGTCGGTGCGGCGCGCGACCTGGCGGTGCCCGCGCA GCGGCGCCGGCGTCGCGATGTTGGGCAGCTTGGCACGCGTGACCGCGGGCGGCAGCGCCAGGCGCGGC GGCTCGCGCGCGAAGAAGGCCGACTTGACGCGGACaagott SEQ ID NO: 44 PmFATA-hpF actagtCCGTGCCCGAGCACGTCTTCAGCGACTACCACCTCTACCAGATGGAGATCGACTTCAAGGCC GAGTGCCACGCGGGCGACGTCATCTCCTCCCAGGCCGAGCAGATCCCGCCCCAGGAGGCGCTCACGCA CAACGGCGCCGGCCGCAACCCCTCCTGCTTCGTCCATAGCATTCTGCGCGCCGAGACCGAGCGTGTGT TTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCTGTCCCGCT GACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccGCTCGGTCTCGGCGCGCAG AATGCTATGGACGAAGCAGGAGGGGTTGCGGCCGGCGCCGTTGTGCGTGAGCGCCTCCTGGGGCGGGA TCTGCTCGGCCTGGGAGGAGATGACGTCGCCCGCGTGGCACTCGGCCTTGAAGTCGATCTCCATCTGG TAGAGGTGGTAGTCGCTGAAGACGTGCTCGGGCACGGaagctt SEQ ID NO: 45 PmFATA-hpG
actagtTCGTCCGCGCGCGAACCACATGGTCGGCCCCCATCGACGCGCCCGCCGCCAAGCCGCCCAAG GCGAGCCACTGAGGACAGGGTGGTTGGCTGGATGGGGAAACGCTGGTCGCGGGATTCGATCCTGCTGC TTATATCCTCGTGTGTTTGAGGGTTTTGGTTGCCCGTATTGAGGTCCTGGTGGCGCGCATGGGGGAGA AGGCGCCTGTCCCGCTGACCCCCCCGGCTACCCTCCCGGCACCTTCCAGGGCGCGTACGggatccGAG GATATAAGCAGCAGGATCGAATCCCGCGACCAGCGTTTCCCCATCCAGCCAACCACCCTGTCCTCAGT GGCTCGCCTTGGGCGGCTTGGCGGCGGGCGCGTCGATGGGGGCCGACCATGTGGTTCGCGCGCGGACG Aaagctt SEQ ID NO: 46 Codon-optimized Cuphea wrightii KASAI ATGGCCGCCGCCGCCAGCATGGTGGCCAGCCCCTTCTGCACCTGGCTGGTGGCCAGCTGCATGAGCAC CAGCTTCGACAACGACCCCCGCAGCCCCAGCGTGAAGCGCTTCCCCCGCCGCAAGCGCGTGCTGAGCC AGCGCGGCAGCACCTACGTATTCCAGTGCCTGGTGGCCAGCTGCATCGACCCCTGCGACCAGTACCGC AGCAGCGCCAGCCTGAGCTTCCTGGGCGACAACGGCTTCGCCAGCCTGTTCGGCAGCAAGCCCTTCAT GAGCAACCGCGGCCACCGCCGCCTGCGCCGCGCCAGCCACAGCGGCGAGGCCATGGCCGIGGCCCTGC AGCCCGCCCAGGAGGCCGGCACCAAGAAGAAGCCCGTGATCAAGCAGCGCCGCGTGGTGGTGACCGGC ATGGGCGTGGTGACCCCCCTGGGCCACGAGCCCGACGTGTTCTACAACAACCTGCTGGACGGCGTGAG CGGCATCAGCGAGATCGAGACCTTCGACTGCACCCAGTTCCCCACCCGCATCGCCGGCGAGATCAAGA GCTTCAGCACCGACGGCTGGGTGGCCCCCAAGCTGAGCAAGCGCATGGACAAGTTCATGCTGTACCTG CTGACCGCCGGCAAGAAGGCCCTGGCCGACGGCGGCATCACCGACGAGGTGATGAAGGAGCTGGACAA GCGCAAGTGCGGCGTGCTGATCGGCAGCGGCATGGGCGGCATGAAGGTGTTCAACGACGCCATCGAGG CCCTGCGCGTGAGCTACAAGAAGATGAACCCCTTCTGCGTGCCCTTCGCCACCACCAACATGGGCAGC GCCATGCTGGCCATGGACCTGGGCTGGATGGGCCCCAACTACAGCATCAGCACCGCCTGCGCCACCAG CAACTTCTGCATCCTGAACGCCGCCAACCACATCATCCGCGGCGAGGCCGACATGATGCTGTGCGGCG GCAGCGACGCCGTGATCATCCCCATCGGCCTGGGCGGCTTCGTGGCCTGCCGCGCCCTGAGCCAGCGC AACAGCGACCCCACCAAGGCCAGCCGCCCCTGGGACAGCAACCGCGACGGCTTCGTGATGGGCGAGGG CGCCGGCGTGCTGCTGCTGGAGGAGCTGGAGCACGCCAAGAAGCGCGGCGCCACCATCTACGCCGAGT TCCTGGGCGGCAGCTTCACCTGCGACGCCTACCACATGACCGAGCCCCACCCCGAGGGCGCCGGCGTG ATCCTGTGCATCGAGAAGGCCCTGGCCCAGGCCGGCGTGAGCAAGGAGGACGTGAACTACATCAACGC CCACGCCACCAGCACCAGCGCCGGCGACATCAAGGAGTACCAGGCCCTGGCCCGCTGCTTCGGCCAGA ACAGCGAGCTGCGCGTGAACAGCACCAAGAGCATGATCGGCCACCTGCTGGGCGCCGCCGGCGGCGTG GAGGCCGTGACCGTGGTGCAGGCCATCCGCACCGGCTGGATTCACCCCAACCTGAACCTGGAGGACCC CGACAAGGCCGTGGACGCCAAGCTGCTGGTGGGCCCCAAGAAGGAGCGCCTGAACGTGAAGGTGGGCC TGAGCAACAGCTTCGGCTTCGGCGGCCACAACAGCAGCATCCTGTTCGCCCCCTGCAACGTGTGA SEQ ID NO: 47 Codon-optimized Cuphea wrightii KASAI with P. moriformis SAD transit peptide ATGGGCCGCGGTGTCTCCCTTCCCCGGCCCAGGGTCGCGGTGCGCGCCCAGTCGGCGAGTCAGGTTTT GGAGAGCTGTATTCCAGTGCCTGGTGGCCAGCTGCATCGACCCCTGCGACCAGTACCGCAGCAGCGCC AGCCTGAGCTTCCTGGGCGACAACGGCTTCGCCAGCCTGTTCGGCAGCAAGCCCTTCATGAGCAACCG CGGCCACCGCCGCCTGCGCCGCGCCAGCCACAGCGGCGAGGCCATGGCCGTGGCCCTGCAGCCCGCCC AGGAGGCCGGCACCAAGAAGAAGCCCGTGATCAAGCAGCGCCGCGTGGTGGTGACCGGCATGGGCGTG GTGACCCCCCTGGGCCACGAGCCCGACGTGTTCTACAACAACCTGCTGGACGGCGTGAGCGGCATCAG CGAGATCGAGACCTTCGACTGCACCCAGTTCCCCACCCGCATCGCCGGCGAGATCAAGAGCTTCAGCA CCGACGGCTGGGTGGCCCCCAAGCTGAGCAAGCGCATGGACAAGTTCATGCTGTACCTGCTGACCGCC GGCAAGAAGGCCCTGGCCGACGGCGGCATCACCGACGAGGTGATGAAGGAGCTGGACAAGCGCAAGTG CGGCGTGCTGATCGGCAGCGGCATGGGCGGCATGAAGGTGTTCAACGACGCCATCGAGGCCCTGCGCG TGAGCTACAAGAAGATGAACCCCTTCTGCGTGCCCTTCGCCACCACCAACATGGGCAGCGCCATGCTG GCCATGGACCTGGGCTGGATGGGCCCCAACTACAGCATCAGCACCGCCTGCGCCACCAGCAACTTCTG CATCCTGAACGCCGCCAACCACATCATCCGCGGCGAGGCCGACATGATGCTGTGCGGCGGCAGCGACG CCGTGATCATCCCCATCGGCCTGGGCGGCTTCGTGGCCTGCCGCGCCCTGAGCCAGCGCAACAGCGAC CCCACCAAGGCCAGCCGCCCCTGGGACAGCAACCGCGACGGCTTCGTGATGGGCGAGGGCGCCGGCGT GCTGCTGCTGGAGGAGCTGGAGCACGCCAAGAAGCGCGGCGCCACCATCTACGCCGAGTTCCTGGGCG GCAGCTTCACCTGCGACGCCTACCACATGACCGAGCCCCACCCCGAGGGCGCCGGCGTGATCCTGTGC ATCGAGAAGGCCCTGGCCCAGGCCGGCGTGAGCAAGGAGGACGTGAACTACATCAACGCCCACGCCAC CAGCACCAGCGCCGGCGACATCAAGGAGTACCAGGCCCTGGCCCGCTGCTTCGGCCAGAACAGCGAGC TGCGCGTGAACAGCACCAAGAGCATGATCGGCCACCTGCTGGGCGCCGCCGGCGGCGTGGAGGCCGTG ACCGTGGTGCAGGCCATCCGCACCGGCTGGATTCACCCCAACCTGAACCTGGAGGACCCCGACAAGGC CGTGGACGCCAAGCTGCTGGTGGGCCCCAAGAAGGAGCGCCTGAACGTGAAGGTGGGCCTGAGCAACA GCTTCGGCTTCGGCGGCCACAACAGCAGCATCCTGTTCGCCCCCTGCAACGTGTGA SEQ ID NO: 48 Codon-optimized Cuphea pulcherrima KASIV ATGCCCGCGGCCAGCTCGCTGCTGGCGTCCCCCCTGTGCACCTGGCTGCTGGCCGCGTGCATGAGCAC CTCGTTCCACCCCTCCGACCCCCTGCCCCCCAGCATCTCGTCCCCCCGCCGCCGCCTGAGCCGCCGCC GCATCCTGTCGCAGTGCGCCCCCCTGCCCTCCGCGAGCTCGGCCCTGCGCGGCTCCAGCTTCCACACC CTGGTGACCTCGTATCTGGCGTGCTTCGAGCCCTGCCACGACTATTATACCAGCGCCTCCCTGTTCGG CTCGCGCCCCATCCGCACCACCCGCCGCCACCGCCGCCTGAACCGCGCGAGCCCCICGCGCGAGGCGA TGGCGGTCGCCCTGCAGCCCGAGCAGGAGGTGACCACCAAGAAGAAGCCCTCCATCAAGCAGCGCCGC GTCGTGGTCACCGGCATGGGCGTGGTCACCCCCCTGGGCCACGACCCCGACGTGTTCTATAACAACCT GCTGGACGGCACCAGCGGCATCTCGGAGATCGAGACCTTCGACTGCGCGCAGTTCCCCACCCGCATCG CCGGCGAGATCAAGTCCTTCAGCACCGACGGCTGGGTCGCGCCCAAGCTGTCGAAGCGCATGGACAAG TTCATGCTGTATATGCTGACCGCCGGCAAGAAGGCGCTGACCGACGGCGGCATCACCGAGGACGTGAT GAAGGAGCTGGACAAGCGCAAGTGCGGCGTCCTGATCGGCTCCGCGATGGGCGGCATGAAGGTGTTCA ACGACGCGATCGAGGCCCTGCGCATCAGCTATAAGAAGATGAACCCCTTCTGCGTGCCCTTCGCGACC ACCAACATGGGCTCGGCCATGCTGGCGATGGACCTGGGCTGGATGGGCCCCAACTATTCCATCAGCAC CGCCTGCGCGACCTCGAACTTCTGCATCATGAACGCGGCCAACCACATCATCCGCGGCGAGGCGGACG TCATGCTGTGCGGCGGCTCCGACGCCGTGATCATCCCCATCGGCATGGGCGGCTTCGTCGCGTGCCGC GCCCTGAGCCAGCGCAACTCGGACCCCACCAAGGCGTCCCGCCCCTGGGACAGCAACCGCGACGGCTT CGTGATGGGCGAGGGCGCCGGCGTCCTGCTGCTGGAGGAGCTGGAGCACGCGAAGAAGCGCGGCGCCA CCATCTATGCGGAGTTCCTGGGCGGCTCGTTCACCTGCGACGCCTATCACATGACCGAGCCCCACCCC GACGGCGCCGGCGTGATCCTGTGCATCGAGAAGGCGCTGGCCCAGTCCGGCGTCAGCCGCGAGGACGT GAACTATATCAACGCGCACGCCACCTCGACCCCCGCGGGCGACATCAAGGAGTATCAGGCCCTGATCC ACTGCTTCGGCCAGAACCGCGAGCTGAAGGTCAACTCCACCAAGAGCATGATCGGCCACCTGCTGGGC GCGGCGGGCGGCGTGGAGGCGGTCTCGGTGGTCCAGGCCATCCGCACCGGCTGGATCCACCCCAACAT CAACCTGGAGAACCCCGACGAGGGCGTGGACACCAAGCTGCTGGTGGGCCCCAAGAAGGAGCGCCTGA ACGTCAAGGTGGGCCTGTCCAACAGCTTCGGCTTCGGCGGCCACAACTCGTCCATCCTGTTCGCGCCC TATATCTGA SEQ ID NO: 49 Codon-optimized Cuphea hookeriana KASIV ATGGTGGCCGCCGCCGCCTCCAGCGCCTTCTTCCCCGTGCCCGCCCCCGGCGCCTCCCCCAAGCCCGG CAAGTTCGGCAACTGGCCCTCCAGCCTGAGCCCCTCCTTCAAGCCCAAGTCCATCCCCAACGGCGGCT TCCAGGTGAAGGCCAACGACAGCGCCCACCCCAAGGCCAACGGCTCCGCCGTGAGCCTGAAGAGCGGC AGCCTGAACACCCAGGAGGACACCTCCTCCAGCCCCCCCCCCCGCACCTTCCTGCACCAGCTGCCCGA CTGGAGCCGCCTGCTGACCGCCATCACCACCGTGTTCGTGAAGTCCAAGCGCCCCGACATGCACGACC GCAAGTCCAAGCGCCCCGACATGCTGGTGGACAGCTTCGGCCTGGAGTCCACCGTGCAGGACGGCCTG GTGTTCCGCCAGTCCTTCTCCATCCGCTCCTACGAGATCGGCACCGACCGCACCGCCAGCATCGAGAC CCTGATGAACCACCTGCAGGAGACCTCCCTGAACCACTGCAAGAGCACCGGCATCCTGCTGGACGGCT TCGGCCGCACCCTGGAGATGTGCAAGCGCGACCTGATCTGGGTGGTGATCAAGATGCAGATCAAGGTG AACCGCTACCCCGCCTGGGGCGACACCGTGGAGATCAACACCCGCTTCAGCCGCCTGGGCAAGATCGG CATGGGCCGCGACTGGCTGATCTCCGACTGCAACACCGGCGAGATCCTGGTGCGCGCCACCAGCGCCT ACGCCATGATGAACCAGAAGACCCGCCGCCTGTCCAAGCTGCCCTACGAGGTGCACCAGGAGATCGTG CCCCTGTTCGTGGACAGCCCCGTGATCGAGGACTCCGACCTGAAGGTGCACAAGTTCAAGGTGAAGAC CGGCGACAGCATCCAGAAGGGCCTGACCCCCGGCTGGAACGACCTGGACGTGAACCAGCACGTGTCCA ACGTGAAGTACATCGGCTGGATCCTGGAGAGCATGCCCACCGAGGTGCTGGAGACCCAGGAGCTGTGC TCCCTGGCCCTGGAGTACCGCCGCGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGTGACCGCCATGGA CCCCAGCAAGGTGGGCGTGCGCTCCCAGTACCAGCACCTGCTGCGCCTGGAGGACGGCACCGCCATCG TGAACGGCGCCACCGAGTGGCGCCCCAAGAACGCCGGCGCCAACGGCGCCATCTCCACCGGCAAGACC AGCAACGGCAACTCCGTGTCCATGTGA SEQ ID NO: 50 Prototheca moriformis (UTEX 1435) KAS1 allele 1 5' donor sequence gctcttcctcaccgcgtgaattgctgtcccaaacgtaagcatcatcgtggctcggtcacgcgatcctg gatccggggatcctagaccgctggtggagagcgctgccgtcggattggtggcaagtaagattgcgcag gttggcgaagggagagaccaaaaccggaggctggaagcgggcacaacatcgtattattgcgtatagta gagcagtggcagtcgcatttcgaggtccgcaacggatctcgcaagctcgctacgctcacagtaggaga aaggggaccactgcccctgccagaatggtcgcgaccctctccctcgccggccccgcctgcaacacgca gtgcgtatccggcaagcgggctgtcgccttcaaccgcccccatgttggcgtccgggctcgatcaggtg cgctgaggggggtttggtgtgcccgcgcctctgggcccgtgtcggccgtgcggacgtggggccctggg cagtggatcagcagggtttgcgtgcaaatgcctataccggcgattgaatagcgatgaacgggatacgg ttgcgctcactccatgcccatgcgaccccgtttctgtccgccagccgtggtcgcccgggctgcgaagc gggaccccacccagcgcattgtgatcaccggaatgggcgtgggtacc SEQ ID NO: 51 Prototheca moriformis (UTEX 1435) KAS1 allele 1 3' donor sequence gagctccacctgcatccgcctggcgctcgaggacgccggcgtctcgcccgacgaggtcaactacgtca acgcgcacgccacctccaccctggtgggcgacaaggccgaggtgcgcgcggtcaagtcggtctttggc gacatgaagggcatcaagatgaacgccaccaagtccatgatcgggcactgcctgggcgccgccggcgg catggaggccgtcgccacgctcatggccatccgcaccggctgggtgcaccccaccatcaaccacgaca accccatcgccgaggtcgacggcctggacgtcgtcgccaacgccaaggcccagcacaaaatcaacgtc gccatctccaactccttcggcttcggcgggcacaactccgtcgtcgcctttgcgcccttccgcgagta ggcggagcgagcgcgcttggctgaggagggaggcggggtgcgagccctttggctgcgcgcgatactct ccccgcacgagcagactccacgcgcctgaatctacttgtcaacgagcaaccgtgtgttttgtccgtgg ccattcttattatttctccgactgtggccgtactctgtttggctgtgcaagcaccgaagagcc SEQ ID NO: 52 Prototheca moriformis (UTEX 1435) KAS1 allele 2 5' donor sequence gctcttcgcgcaagctcgctacgctcacagtaggagataggggaccactgcccctgccagaatggtcg cgaccctgtccctcgccggccccgcctgcaacacgcagtgcgtatccagcaagcgggttgtcgccttc aaccgcccccatgttggcgtccgggctcgatcaggtgcgctgaggggggtttggtgggcccgcgcctc tgggcccgtgtcggccgtgcggacgtggggcccggggtagtggatcagcaggggttgcatgcaaatgc ctataccggcgattgaatagcgatgaacgggatacggttgcgctcactccatgcccatgcgaccccgt ttctgtccgccagccgtggtcgcccgagctgcgaagcgggaccccacccagcgcattgtgatcaccgg aatgggcgtggcctccgtgtttggcaacgatgtcgagaccttttacgacaagcttctggaaggaacga gcggcgtggacctgatttccaggtgcgtaggtccttggatgaatgcgtctaggttgcgaggtgactgg ccaggaagcagcaggcttggggtttggtgttctgatttctggtaatttgaggtttcattataagattc tgtacggtcttgtttcggggtacc SEQ ID NO: 53 Prototheca moriformis (UTEX 1435) KAS1 allele 2 3' donor sequence gagctccacctgcatccgcctggcgctcgaggacgccggcgtctcgcccgacgaggtcaactacgtca acgcgcacgccacctccaccctggtgggcgacaaggccgaggtgcgcgcggtcaagtcggtctttggc gacatgaagggcatcaagatgaacgccaccaagtccatgatcgggcactgcctgggcgccgccggcgg catggaggccgtcgccacgctcatggccatccgcaccggctgggtgcaccccaccatcaaccacgaca accccatcgccgaggtcgacggcctggacgtcgtcgccaacgccaaggcccagcacaaaatcaacgtc gccatctccaactccttcggcttcggcgggcacaactccgtcgtcgcctttgcgcccttccgcgagta ggcggagcgagcgcgcttggctgaggagggaggcggggtgcgagccctttggctgcgcgcgatactct ccccgcacgagcagactccacgcgcctgaatctacttgtcaacgagcaaccgtgtgttttgtccgtgg ccattcttattatttctccgactgtggccgtactctgtttggctgtgcaagcaccgaagagcc SEQ ID NO: 54 Prototheca moriformis (UTEX 1435) KSI-hairpin B actagtcaTGGTCGCCCGGGCTGCGAAGCGGGACCCCACCCAGCGCATTGTGATCACCGGAATGGGCG TGGCCTCCGTGTTTGGCAACGATGTCGAGACCTTTTACAgtgtgtttgagggttttggttgcccgtat tgaggtcctggtggcgcgcatggaggagaaggcgcctgtcccgctgacccccccggctaccctcccgg caccttccagggcgcgtacgggatccTGTAAAAGGTCTCGACATCGTTGCCAAACACGGAGGCCACGC CCATTCCGGTGATCACAATGCGCTGGGTGGGGTCCCGCTTCGCAGCCCGGGCGACCAaagctt SEQ ID NO: 55 Prototheca moriformis (UTEX 1435) KSI-hairpin C actagtcaTTGACATCTCCGAGTTCCCGACCAAGTTTGCGGCGCAGATCACCGGCTTCTCCGTGGAGG ACTGCGTGGACAAGAAGAACGCGCGGCGGTACGACGACGCGCTGTCGTACGCGATGGTGGCCTCCAAG AAGGCCCTGCGCCAGGCGGGACTGGAGAAGGACAAGTGCCCCGAGGGCTACGGAGgtgtgtttgaggg ttttggttgcccgtattgaggtcctggtggcgcgcatggaggagaaggcgcctgtcccgctgaccccc ccggctaccctcccggcaccttccagggcgcgtacgggatccCTCCGTAGCCCTCGGGGCACTTGTCC TTCTCCAGTCCCGCCTGGCGCAGGGCCTTCTTGGAGGCCACCATCGCGTACGACAGCGCGTCGTCGTA CCGCCGCGCGTTCTTCTTGTCCACGCAGTCCTCCACGGAGAAGCCGGTGATCTGCGCCGCAAACTTGG TCGGGAACTCGGAGATGTCAAaagctt SEQ ID NO: 56 Prototheca moriformis (UTEX 1435) KSI-hairpin D actagtcaTGGGCGTGAGCACCTGCATCCGCCTGGCGCTCGAGGACGCCGGCGTCTCGCCCGACGAGG TCAACTACGTCAACGCGCACGCCACCTCCACCCTGGTGGGCGACAAGGCCGAGGTGCGCGCGGTCAAG TCGGTCTTTGGCGACATGAAGGGCATCAAGATgtgtgtttgagggttttggttgcccgtattgaggtc ctggtggcgcgcatggaggagaaggcgcctgtcccgctgacccccccggctaccctcccggcaccttc cagggcgcgtacgggatccATCTTGATGCCCTTCATGTCGCCAAAGACCGACTTGACCGCGCGCACCT CGGCCTTGTCGCCCACCAGGGTGGAGGTGGCGTGCGCGTTGACGTAGTTGACCTCGTCGGGCGAGACG CCGGCGTCCTCGAGCGCCAGGCGGATGCAGGTGCTCACGCCCAaagctt SEQ ID NO: 57 Prototheca moriformis (UTEX 1435) KSI-hairpin E actagtcaCAACCATCAACCACGACAACCCCATCGCCGAGGTCGACGGCCTGGACGTCGTCGCCAACG CCAAGGCCCAGCACAAAATCAACGTCGCCATCTCCAACTCCTTCGgtgtgtttgagggttttggttgc ccgtattgaggtcctggtggcgcgcatggaggagaaggcgcctgtcccgctgacccccccggctaccc tcccggcaccttccagggcgcgtacgggatccCGAAGGAGTTGGAGATGGCGACGTTGATTTTGTGCT GGGCCTTGGCGTTGGCGACGACGTCCAGGCCGTCGACCTCGGCGATGGGGTTGTCGTGGTTGATGGTa agctt SEQ ID NO: 58 Codon optimized M. polymorpha FAE3 (GenBank Accession No. AAP74370) ATGgactcccgcgcccagaaccgcgacggcggcgaggacgtgaagcaggagctgctgtccgccggcga cgacggcaaggtgccctgccccaccgtggccatcggcatccgccagcgcctgcccgacttcctgcagt ccgtgaacatgaagtacgtgaagctgggctaccactacctgatcacccacgccatgttcctgctgacc ctgcccgccttcttcctggtggccgccgagatcggccgcctgggccacgagcgcatctaccgcgagct gtggacccacctgcacctgaacctggtgtccatcatggcctgctcctccgccctggtggccggcgcca ccctgtacttcatgtcccgcccccgccccgtgtacctggtggagttcgcctgctaccgccccgacgag cgcctgaaggtgtccaaggacttcttcctggacatgtcccgccgcaccggcctgttctcctcctcctc catggacttccagaccaagatcacccagcgctccggcctgggcgacgagacctacctgccccccgcca tcctggcctccccccccaacccctgcatgcgcgaggcccgcgaggaggccgccatggtgatgttcggc gccctggacgagctgttcgagcagaccggcgtgaagcccaaggagatcggcgtgctggtggtgaactg ctccctgttcaaccccaccccctccatgtccgccatgatcgtgaaccactaccacatgcgcggcaaca tcaagtccctgaacctgggcggcatgggctgctccgccggcctgatctccatcgacctggcccgcgac ctgctgcaggtgcacggcaacacctacgccgtggtggtgtccaccgagaacatcaccctgaactggta cttcggcgacgaccgctccaagctgatgtccaactgcatcttccgcatgggcggcgccgccgtgctgc tgtccaacaagcgccgcgagcgccgccgcgccaagtacgagctgctgcacaccgtgcgcacccacaag ggcgccgacgacaagtgcttccgctgcgtgtaccaggaggaggactccaccggctccctgggcgtgtc cctgtcccgcgagctgatggccgtggccggcaacgccctgaaggccaacatcaccaccctgggccccc tggtgctgcccctgtccgagcagatcctgttcttcgcctccctggtggcccgcaagttcctgaacatg aagatgaagccctacatccccgacttcaagctggccttcgagcacttctgcatccacgccggcggccg cgccgtgctggacgagctggagaagaacctggacctgaccgagtggcacatggagccctcccgcatga ccctgtaccgcttcggcaacacctcctcctcctccctgtggtacgagctggcctacaccgaggcccag ggccgcgtgaagcgcggcgaccgcctgtggcagatcgccttcggctccggcttcaagtgcaactccgc cgtgtggcgcgcgctgcgcaccgtgaagccccccgtgaacaacgcctggtccgacgtgatcgaccgct tccccgtgaagctgccccagttcTGA SEQ ID NO: 59 M. polymorpha FAE3 (GenBank Accession No. AAP74370) MDSRAQNRDGGEDVKQELLSAGDDGKVPCPTVAIGIRQRLPDFLQSVNMKYVKLGYHYLITHAMFLLT LPAFFLVAAEIGRLGHERIYRELWTHLHLNLVSIMACSSALVAGATLYFMSRPRPVYLVEFACYRPDE RLKVSKDFFLDMSRRTGLFSSSSMDFQTKITQRSGLGDETYLPPAILASPPNPCMREAREEAAMVMFG ALDELFEQTGVKPKEIGVLVVNCSLFNPIPSMSAMIVNHYHMRGNIKSLNLGGMGCSAGLISIDLARD LLQVHGNIYAVVVSTENITLNWYFGDDRSKLMSNCIFRMGGAAVLLSNKRRERRRAKYELLHIVRTHK GADDKCFRCVYQEEDSIGSLGVSLSRELMAVAGNALKANITTLGPLVLPLSEQILFFASLVARKFLNM KMKPYIPDFKLAFEHFCIHAGGRAVLDELEKNLDLTEWHMEPSRMTLYRFGNISSSSLWYELAYTEAQ GRVKRGDRLWQIAFGSGFKCNSAVWRALRIVKPPVNNAWSDVIDRFPVKLPQF SEQ ID NO: 60 Trypanosoma brucei ELO3 (GenBank Accession No. AAX70673) ##STR00531## gtggatgctggaccacccctccgtgccctacatcgccggcgtgatgtacctgatcctggtgctgtacg tgcccaagtccatcatggcctcccagccccccctgaacctgcgcgccgccaacatcgtgtggaacctg ttcctgaccctgttctccatgtgcggcgcctactacaccgtgccctacctggtgaaggccttcatgaa ccccgagatcgtgatggccgcctccggcatcaagctggacgccaacacctcccccatcatcacccact ccggcttctacaccaccacctgcgccctggccgactccttctacttcaacggcgacgtgggcttctgg gtggccctgttcgccctgtccaagatccccgagatgatcgacaccgccttcctggtgttccagaagaa gcccgtgatcttcctgcactggtaccaccacctgaccgtgatgctgttctgctggttcgcctacgtgc agaagatctcctccggcctgtggttcgcctccatgaactactccgtgcactccatcatgtacctgtac tacttcgtgtgcgcctgcggccaccgccgcctggtgcgccccttcgcccccatcatcaccttcgtgca gatcttccagatggtggtgggcaccatcgtggtgtgctacacctacaccgtgaagcacgtgctgggcc
gctcctgcaccgtgaccgacttctccctgcacaccggcctggtgatgtacgtgtcctacctgctgctg ttctcccagctgttctaccgctcctacctgtccccccgcgacaaggcctccatcccccacgtggccgc ##STR00532## SEQ ID NO: 61 Trypanosoma brucei ELO3 (GenBank Accession No. AAX70673) MYPTHRDLILNNYSDIYRSPTCHYHTWHTLIHTPINELLFPNLPRECDFGYDIPYFRGQIDVFDGWSM IHFTSSNWCIPITVCLCYIMMIAGLKKYMGPRDGGRAPIQAKNYIIAWNLFLSFFSFAGVYYTVPYHL FDPENGLFAQGFYSTVCNNGAYYGNGNVGFFVWLFIYSKIFELVDIFFLLIRKNPVIFLHWYHHLTVL LYCWHAYSVRIGIGIWFATMNYSVHSVMYLYFAMTQYGPSTKKFAKKFSKFITTIQILQMVVGIIVTF AAMLYVTFDVPCYTSLANSVLGLMMYASYFVLFVQLYVSHYVSPKHVKQE SEQ ID NO: 62 Codon optimized Saccharomyces cerevisiae ELO1 (GenBank Accession No. P39540) ##STR00533## cttcttcaacatctacctgtgggactacttcaaccgcgccgtgggctgggccaccgccggccgcttcc agcccaaggacttcgagttcaccgtgggcaagcagcccctgtccgagccccgccccgtgctgctgttc atcgccatgtactacgtggtgatcttcggcggccgctccctggtgaagtcctgcaagcccctgaagct gcgcttcatctcccaggtgcacaacctgatgctgacctccgtgtccttcctgtggctgatcctgatgg tggagcagatgctgcccatcgtgtaccgccacggcctgtacttcgccgtgtgcaacgtggagtcctgg acccagcccatggagaccctgtactacctgaactacatgaccaagttcgtggagttcgccgacaccgt gctgatggtgctgaagcaccgcaagctgaccttcctgcacacctaccaccacggcgccaccgccctgc tgtgctacaaccagctggtgggctacaccgccgtgacctgggtgcccgtgaccctgaacctggccgtg cacgtgctgatgtactggtactacttcctgtccgcctccggcatccgcgtgtggtggaaggcctgggt gacccgcctgcagatcgtgcagttcatgctggacctgatcgtggtgtactacgtgctgtaccagaaga tcgtggccgcctacttcaagaacgcctgcaccccccagtgcgaggactgcctgggctccatgaccgcc atcgccgccggcgccgccatcctgacctcctacctgttcctgttcatctccttctacatcgaggtgta ##STR00534## SEQ ID NO: 63 Saccharomyces cerevisiae ELO1 (GenBank Accession No. P39540) MVSDWKNFCLEKASRFRPTIDRPFFNIYLWDYFNRAVGWATAGRFQPKDFEFTVGKQPLSEPRPVLLF IAMYYVVIFGGRSLVKSCKPLKLRFISQVHNLMLTSVSFLWLILMVEQMLPIVYRHGLYFAVCNVESW TQPMETLYYLNYMTKFVEFADTVLMVLKHRKLTFLHTYHHGATALLCYNQLVGYTAVTWVPVTLNLAV HVLMYWYYFLSASGIRVWWKAWVTRLQIVQFMLDLIVVYYVLYQKIVAAYFKNACTPQCEDCLGSMTA IAAGAAILTSYLFLFISFYIEVYKRGSASGKKKINKNN SEQ ID NO: 64 Codon optimized Brassica napus acyl-ACP thioesterase (GenBank Accession No. CAA52070) with 3X FLAG Tag ATGctgaagctgtcctgcaacgtgaccaacaacctgcacaccttctccttcttctccgactcctccct gttcatccccgtgaaccgccgcaccatcgccgtgtcctccgggcgcgcctcccagctgcgcaagcccg ccctggaccccctgcgcgccgtgatctccgccgaccagggctccatctcccccgtgaactcctgcacc cccgccgaccgcctgcgcgccggccgcctgatggaggacggctactcctacaaggagaagttcatcgt gcgctcctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggagg tggcctgcaaccacgtgcagaagtgcggcttctccaccgacggcttcgccaccaccctgaccatgcgc aagctgcacctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccga cgtggtggagatcgagacctggtgccagtccgagggccgcatcggcacccgccgcgactggatcctgc gcgactccgccaccaacgaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacc cgccgcctgcagcgcgtgaccgacgaggtgcgcgacgagtacctggtgttctgcccccgcgagccccg cctggccttccccgaggagaacaactcctccctgaagaagatccccaagctggaggaccccgcccagt actccatgctggagctgaagccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacc tacatcggctgggtgctggagtccatcccccaggagatcatcgacacccacgagctgcaggtgatcac cctggactaccgccgcgagtgccagcaggacgacatcgtggactccctgaccacctccgagatccccg acgaccccatctccaagttcaccggcaccaacggctccgccatgtcctccatccagggccacaacgag tcccagttcctgcacatgctgcgcctgtccgagaacggccaggagatcaaccgcggccgcacccagtg gcgcaagaagtoctoccgcATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACT ACAAGGACGACGACGACAAGTGA SEQ ID NO: 65 Brassica napus acyl-ACP thioesterase (Genbank Accession No. CAA52070) with 3X FLAG Tag (bold) ##STR00535## PADRLRAGRLMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTDGFATTLTMR KLHLIWVTARMHIETYKYPAWSDVVEIETWCQSEGRIGTRRDWILRDSATNEVIGRATSKWVMMNQDT RRLQRVTDEVRDEYLVFCPREPRLAFFEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVT YIGWVLESIPQEIIDTHELQVITLDYRRECQQDDIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNE SQFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 66 Codon optimized Brassica napus acyl-ACP thioesterase (GenBank Accession No. CAA52070), with UTEX 250 stearoyl-ACP desaturase (SAD) chloroplast transit peptide and 3X FLAG Tag ATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctc cgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgcctcccagctgcgcaagcccgccc tggaccccctgcgcgccgtgatctccgccgaccagggctccatctcccccgtgaactcctgcaccccc gccgaccgcctgcgcgccggccgcctgatggaggacggctactcctacaaggagaagttcatcgtgcg ctcctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgg cctgcaaccacgtgcagaagtgcggcttctccaccgacggcttcgccaccaccctgaccatgcgcaag ctgcacctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgt ggtggagatcgagacctggtgccagtccgagggccgcatcggcacccgccgcgactggatcctgcgcg actccgccaccaacgaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgc cgcctgcagcgcgtgaccgacgaggtgcgcgacgagtacctggtgttctgcccccgcgagccccgcct ggccttccccgaggagaacaactcctccctgaagaagatccccaagctggaggaccccgcccagtact ccatgctggagctgaagccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctac atcggctgggtgctggagtccatcccccaggagatcatcgacacccacgagctgcaggtgatcaccct ggactaccgccgcgagtgccagcaggacgacatcgtggactccctgaccacctccgagatccccgacg accccatctccaagttcaccggcaccaacggctccgccatgtcctccatccagggccacaacgagtcc cagttcctgcacatgctgcgcctgtccgagaacggccaggagatcaaccgcggccgcacccagtggcg caagaagtoctoccgcATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACA AGGACGACGACGACAAGTGA SEQ ID NO: 67 Brassica napus acyl-ACP thioesterase (GenBank Accession No. CAA52070) with UTEX 250 stearoyl-ACP desaturase (SAD) chloroplast transit peptide and 3X FLAG .RTM. Tag ##STR00536## ADRLRAGRLMEDGYSYKEKFIVRSYEVGINKTATVETIANLLQEVACNHVQKCGFSTDGFATTLTMRK LHLIWVTARMHIETYKYPAWSDVVEIETWCQSEGRIGIRRDWILRDSATNEVIGRATSKWVMMNQDTR RLQRVTDEVRDEYLVFCPREPRLAFPEENNSSLKKIPKLEDPAQYSMLELKPRRADLDMNQHVNNVTY IGWVLESIPQEIIDTHELQVITLDYRRECQQDDIVDSLTTSEIPDDPISKFTGTNGSAMSSIQGHNES QFLHMLRLSENGQEINRGRTQWRKKSSRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 68 Codon optimized C. tinctorius FATA (GenBank Accession No. AAA33019) with UTEX 250 stearoyl-ACP desaturase (SAD) chloroplast transit peptide and 3X FLAG .RTM. Tag ##STR00537## gcgtggcctccctgcgcgaggccgacaaggagaagtccctgggcaaccgcctgcgcctgggctccctg accgaggacggcctgtcctacaaggagaagttcgtgatccgctgctacgaggtgggcatcaacaagac cgccaccatcgagaccatcgccaacctgctgcaggaggtgggcggcaaccacgcccagggcgtgggct tctccaccgacggcttcgccaccaccaccaccatgcgcaagctgcacctgatctgggtgaccgcccgc atgcacatcgagatctaccgctaccccgcctggtccgacgtgatcgagatcgagacctgggtgcaggg cgagggcaaggtgggcacccgccgcgactggatcctgaaggactacgccaacggcgaggtgatcggcc gcgccacctccaagtgggtgatgatgaacgaggacacccgccgcctgcagaaggtgtccgacgacgtg cgcgaggagtacctggtgttctgcccccgcaccctgcgcctggccttccccgaggagaacaacaactc catgaagaagatccccaagctggaggaccccgccgagtactcccgcctgggcctggtgccccgccgct ccgacctggacatgaacaagcacgtgaacaacgtgacctacatcggctgggccctggagtccatcccc cccgagatcatcgacacccacgagctgcaggccatcaccctggactaccgccgcgagtgccagcgcga cgacatcgtggactccctgacctcccgcgagcccctgggcaacgccgccggcgtgaagttcaaggaga tcaacggctccgtgtcccccaagaaggacgagcaggacctgtcccgcttcatgcacctgctgcgctcc gccggctccggcctggagatcaaccgctgccgcaccgagtggcgcaagaagcccgccaagcgcATGGA ##STR00538## SEQ ID NO: 69 C. tinctorius FATA (GenBank Accession No. AAA33019) with UTEX 250 stearoyl-ACP desaturase (SAD) chloroplast transit peptide ##STR00539## TEDGLSYKEKFVIRCYEVGINKTATIETIANLLQEVGGNHAQGVGFSTDGFATTTTMRKLHLIWVTAR MHIETYRYPAWSDVIEIETWVQGEGKVGTRRDWILKDYANGEVIGRATSKWVMMNEDTRRLQKVSDDV REEYLVFCPRTLRLAFPEENNNSMKKIPKLEDPAEYSRLGLVPRRSDLDMNKHVNNVTYIGWALESIP PEIIDTHELQAITLDYRRECQRDDIVDSLTSREPLGNAAGVKFKEINGSVSPKKDEQDLSRFMHLLRS AGSGLEINRCRIEWRKKPAKRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 70 Codon optimized R. communis FATA (Genbank Accession No. ABS30422) with a 3xFLAG epitope tag ##STR00540## gacccacttcaacaaccgcccctacttcacccgccgcccctccatccccaccttcttctcctccaaga actcctccgcctccctgcaggccgtggtgtccgacatctcctccgtggagtccgccgcctgcgactcc ctggccaaccgcctgcgcctgggcaagctgaccgaggacggcttctcctacaaggagaagttcatcgt ggggcgcgcccgctcctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgc tgcaggaggtgggctgcaaccacgcccagtccgtgggcttctccaccgacggcttcgccaccaccacc tccatgcgcaagatgcacctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgc ctggtccgacgtggtggaggtggagacctggtgccagtccgagggccgcatcggcacccgccgcgact ggatcctgaccgactacgccaccggccagatcatcggccgcgccacctccaagtgggtgatgatgaac caggacacccgccgcctgcagaaggtgaccgacgacgtgcgcgaggagtacctggtgttctgcccccg cgagctgcgcctggccttccccgaggagaacaaccgctcctccaagaagatctccaagctggaggacc ccgcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaac aacgtgacctacatcggctgggtgctggagtccatcccccaggagatcatcgacacccacgagctgca gaccatcaccctggactaccgccgcgagtgccagcacgacgacatcgtggactccctgacctccgtgg agccctccgagaacctggaggccgtgtccgagctgcgcggcaccaacggctccgccaccaccaccgcc ggcgacgaggactgccgcaacttcctgcacctgctgcgcctgtccggcgacggcctggagatcaaccg cggccgcaccgagtggcgcaagaagtccgcccgcATGGACTACAAGGACCACGACGGCGACTACAAGG ##STR00541## SEQ ID NO: 71 R. communis FATA (Genbank Accession No. AB530422) with a 3xFLAG .RTM. epitope tag ##STR00542## SMRKMHLIWVTARMHIETYKYPAWSDVVEVETWCQSEGRIGIRRDWILTDYATGQIIGRATSKWVMMN QDTRRLQKVTDDVREEYLVFCPRELRLAFFEENNRSSKKISKLEDPAQYSKLGLVPRRADLDMNQHVN NVTYIGWVLESIPQEIIDTHELQTITLDYRRECQHDDIVDSLTSVEPSENLEAVSELRGTNGSATTTA GDEDCRNFLHLLRLSGDGLEINRGRIEWRKKSARMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 72 Codon optimized G. mangostana FATA1 (GenBank Accession No. AAB51523) with 3X FLAG .RTM. epitope tag ##STR00543## ccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtgg tgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggag aagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacct gctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccaccc ccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtacccc gcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcga ctggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatga accaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccc cgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggagga cccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtga acaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctg cagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccc cgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccg ccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaac cgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAAGGACCACGACGGCGACTACAA ##STR00544## SEQ ID NO: 73 G. mangostana FATA1 (GenBank Accession No. AAB51523) with 3X FLAG .RTM. epitope tag MLKLSSSRSPLARIPTRPRPNSIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKE KFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSIGGESTIPTMRKLRLIWVTARMHIETYKYP AWSDVVEIESWGQGEGKIGIRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCP RELRLAFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHEL QTITLDYRRECQHDDVVDSLTSPEPSEDAEAVFNHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEIN RGRTEWRKKFTRMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 74 Codon optimized Theobroma cacao FATA1 with 3X FLAG .RTM. epitope tag ##STR00545## cccccccgcccccttctccttccgctggcgcacccccgtggtggtgtcctgctccccctcctcccgcc ccaacctgtcccccctgcaggtggtgctgtccggccagcagcaggccggcatggagctggtggagtcc ggctccggctccctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaagga gaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacc tgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgacggcttcgccaccacc cgcaccatgcgcaagctgcacctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccc cgcctggtccgacgtgatcgagatcgagacctggtgccagtccgagggccgcatcggcacccgccgcg actggatcctgaaggacttcggcaccggcgaggtgatcggccgcgccacctccaagtgggtgatgatg aaccaggacacccgccgcctgcagaaggtgtccgacgacgtgcgcgaggagtacctggtgttctgccc ccgcgagctgcgcctggccttccccgaggagaacaacaactccctgaagaagatcgccaagctggacg actccttccagtactcccgcctgggcctgatgccccgccgcgccgacctggacatgaaccagcacgtg aacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagct gcagaccatcaccctggactaccgccgcgagtgccagcaggacgacgtggtggactccctgacctccc ccgagcaggtggagggcaccgagaaggtgtccgccatccacggcaccaacggctccgccgccgcccgc gaggacaagcaggactgccgccagttcctgcacctgctgcgcctgtcctccgacggccaggagatcaa ccgcggccgcaccgagtggcgcaagaagcccgcccgcATGGACTACAAGGACCACGACGGCGACTACA ##STR00546## SEQ ID NO: 75 Theobroma cacao FATA1 with 3X FLAG .RTM. epitope tag MLKLSSCNVTDQRQALAQCRFLAPPAPFSFRWRTPVVVSCSPSSRPNLSPLQVVLSGQQQAGMELVES GSGSLADRLRLGSLTEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTDGFATT RTMRKLHLIWVTARMHIETYKYPAWSDVIEIETWCQSEGRIGTRRDWILKDFGTGEVIGRATSKWVMM NQDTRRLQKVSDDVREEYLVFCPRELRLAFPEENNNSLKKIAKLDDSFQYSRLGLMPRRADLDMNQHV NNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQQDDVVDSLTSPEQVEGTEKVSAIHGTNGSAAAR EDKQDCRQFLHLLRLSSDGQEINRGRTEWRKKPARMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 76 23S rRNA for UTEX 1439, UTEX 1441, UTEX 1435, UTEX 1437 Prototheca moriformis TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGAATTAATAACTCGAAACCTAAG CGAAAGCAAGTCTTAATAGGGCGCTAATTTAACAAAACATTAAATAAAATCTAAAGTCATTTATTTTA GACCCGAACCTGAGTGATCTAACCATGGTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCCGAAC CGACCGATGTTGAAAAATCGGCGGATGAACTGTGGTTAGTGGTGAAATACCAGTCGAACTCAGAGCTA GCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAATATATCTCGTCTATCTAGGGGTAAAGCACTGTT TCGGTGCGGGCTATGAAAATGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACGATATATTA GTGAGACTATGGGGGATAAGCTCCATAGTCGAGAGGGAAACAGCCCAGACCACCAGTTAAGGCCCCAA AATGATAATGAAGTGGTAAAGGAGGTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCA TCCTTTAAAGAGTGCGTAATAGCTCACTG SEQ ID NO: 77 Cu P5R23 LPAAT2-1 MAIAAAAVIFLFGLIFFASGLIINLFQALCFVLIRPLSKNAYRRINRVFAELLLSELLCLFDWWAGAK LKLFTDPETFRLMGKEHALVIINHMTELDWMVGWVMGQHFGCLGSIISVAKKSTKFLPVLGWSMWFSE YLYLERSWAKDKSTLKSHIERLIDYPLPFWLVIFVEGTRFTRTKLLAAQQYAVSSGLPVPRNVLIPRT KGFVSCVSHMRSFVPAVYDVTVAFPKTSPPPTLLNLFEGQSIMLHVHIKRHAMKDLPESDDAVAEWCR DKFVEKDALLDKHNAEDTFSGQEVCHSGSRQLKSLLVVISWVVVTTFGALKFLQWSSWKGKAFSAIGL GIVTLLMHVLILSSQAERSNPAEVAQAKLKTGLSISKKVTDKEN SEQ ID NO: 78 CuPSR23 LPAAT3-1
MAIAAAAVIVPLSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVK IKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLGQRSGCLGSTLAVMKKSSKFLPVLGWSMWFSE YLFLERSWAKDEITLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRT KGFVSSVSHMRSFVPAIYDVIVAIPKTSPPPTLIRMFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCR DIFVEKDALLDKHNAEDTFSGQELQETGRPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSG IGLGVITLLMHILILFSQSERSTPAKVAPAKPKNEGESSKTEMEKEK SEQ ID NO: 79 Amino acid sequence for CuPSR23 LPPATx MEIPPHCLCSPSPAPSQLYYKKKKHAILQTQTPYRYRVSPTCFAPPRLRKQHPYPLPVLCYPKLLHFS QPRYPLVRSHLAEAGVAYRPGYELLGKIRGVCFYAVTAAVALLLFQCMLLLHPFVLLFDPFPRKAHHT IAKLWSICSVSLFYKIHIKGLENLPPPHSPAVYVSNHQSFLDIYTLLTLGRTFKFISKTEIFLYPIIG WAMYMLGTIPLKRLDSRSQLDTLKRCMDLIKKGASVFFFPEGTRSKDGKLGAFKKGAFSIAAKSKVPV VPITLIGTGKIMPPGSELTVNPGTVQVIIHKPIEGSDAEAMCNEARATISHSLDD SEQ ID NO: 80 cDNA sequence for CuPSR23 LPAATx coding region ATGGAGATCCCGCCTCACTGTCTCTGTTCGCCTTCGCCTGCGCCTTCGCAATTGTATTACAAGAAGAA GAAGCATGCCATTCTCCAAACTCAAACTCCCTATAGATATAGAGTTTCCCCGACATGCTTTGCCCCCC CCCGATTGAGGAAGCAGCATCCTTACCCTCTCCCTGTCCTCTGCTATCCAAAACTCCTCCACTTCAGC CAGCCTAGGTACCCTCTGGTTAGATCTCATTTGGCTGAAGCTGGTGTTGCTTATCGTCCAGGATACGA ATTATTAGGAAAAATAAGGGGAGTGTGTTTCTATGCTGTCACTGCTGCCGTTGCCTTGCTTCTATTTC AGTGCATGCTCCTCCTCCATCCCTTTGTGCTCCTCTTCGATCCATTTCCAAGAAAGGCTCACCATACC ATCGCCAAACTCTGGTCTATCTGCTCTGTTTCTCTTTTTTACAAGATTCACATCAAGGGTTTGGAAAA TCTTCCCCCACCCCACTCTCCTGCCGTCTATGTCTCTAATCATCAGAGTTTTCTCGACATCTATACTC TCCTCACTCTCGGTAGAACCTTCAAGTTCATCAGCAAGACTGAGATCTTTCTCTATCCAATTATCGGT TGGGCCATGTATATGTTGGGTACCATTCCTCTCAAGCGGTTGGACAGCAGAAGCCAATTGGACACTCT TAAGCGATGTATGGATCTCATCAAGAAGGGAGCATCCGTCTTTTTCTTCCCAGAGGGAACACGAAGTA AAGATGGGAAACTGGGTGCTTTCAAGAAAGGTGCATTCAGCATCGCAGCAAAAAGCAAGGTTCCTGTT GTGCCGATCACCCTTATTGGAACTGGCAAGATTATGCCACCTGGGAGCGAACTTACTGTCAATCCAGG AACTGTGCAAGTAATCATACATAAACCTATCGAAGGAAGTGATGCAGAAGCAATGTGCAATGAAGCTA GAGCCACGATTTCTCACTCACTTGATGATTAA SEQ ID NO: 81 cDNA sequence for CuPSR23 LPAAT 2-1 coding region ATGGCGATTGCAGCGGCAGCTGTCATCTTCCTCTTCGGCCTTATCTTCTTCGCCTCCGGCCTCATAAT CAATCTCTTCCAGGCGCTTTGCTTTGTCCTTATTCGGCCTCTTTCGAAAAACGCCTACMGGAGAATAA ACAGAGTTTTTGCAGAATTGTTGTTGTCGGAGCTTTTATGCCTATTCGATTGGTGGGCTGGTGCTAAG CTCAAATTATTTACCGACCCTGAAACCTTTCGCCTTATGGGCAAGGAACATGCTCTTGTCATAATTAA TCACATGACTGAACTTGACTGGATGGTTGGATGGGTTATGGGTCAGCATTTTGGTTGCCTTGGGAGCA TAATATCTGTTGCGAAGAAATCAACAAAATTTCTTCCGGTATTGGGGTGGTCAATGTGGTTTTCAGAG TACCTATATCTTGAGAGAAGCTGGGCCAAGGATAAAAGTACATTAAAGTCACATATCGAGAGGCTGAT AGACTACCCCCTGCCCTTCTGGTTGGTAATTTTTGTGGAAGGAACTCGGTTTACTCGGACAAAACTCT TGGCAGCCCAGCAGTATGCTGTCTCATCTGGGCTACCAGTGCCGAGAAATGTTTTGATCCCACGTACT AAGGGTTTTGTTTCATGTGTAAGTCACATGCGATCATTTGTTCCAGCAGTATATGATGTCACAGTGGC ATTCCCTAAGACTTCACCTCCACCAACGTTGCTAAATCTTTTCGAGGGTCAGTCCATAATGCTTCACG TTCACATCAAGCGACATGCAATGAAAGATTTACCAGAATCCGATGATGCAGTAGCAGAGTGGTGTAGA GACAAATTTGTGGAAAAGGATGCTTTGTTGGACAAGCATAATGCTGAGGACACTTTCAGTGGTCAAGA AGTTTGTCATAGCGGCAGCCGCCAGTTAAAGTCTCTTCTGGTGGTAATATCTTGGGTGGTTGTAACAA CATTTGGGGCTCTAAAGTTCCTTCAGTGGTCATCATGGAAGGGGAAAGCATTTTCAGCTATCGGGCTG GGCATCGTCACTCTACTTATGCACGTATTGATTCTATCCTCACAAGCAGAGCGGTCTAACCCTGCGGA GGTGGCACAGGCAAAGCTAAAGACCGGGTTGTCGATCTCAAAGAAGGTAACGGACAAGGAAAACTAG SEQ ID NO: 82 cDNA sequence for CuPSR23 LPAAx 3-1 coding region ATGGCGATTGCTGCGGCAGCTGTCATCGTCCCGCTCAGCCTCCTCTTCTTCGTCTCCGGCCTCATCGT CAATCTCGTACAGGCAGTTTGCTTTGTACTGATTAGGCCTCTGTCGAAAAACACTTACAGAAGAATAA ACAGAGTGGTTGCAGAATTGTTGTGGTTGGAGTTGGTATGGCTGATTGATTGGTGGGCTGGTGTCAAG ATAAAAGTATTCACGGATCATGAAACCTTTCACCTTATGGGCAAAGAACATGCTCTTGTCATTTGTAA TCACAAGAGTGACATAGACTGGCTGGTTGGGTGGGTTCTGGGACAGCGGTCAGGTTGCCTTGGAAGCA CATTAGCTGTTATGAAGAAATCATCAAAGTTTCTCCCGGTATTAGGGTGGTCAATGTGGTTCTCAGAG TATCTATTCCTTGAAAGAAGCTGGGCCAAGGATGAAATTACATTAAAGTCAGGTTTGAATAGGCTGAA AGACTATCCCTTACCCTTCTGGTTGGCACTTTTTGTGGAAGGAACTCGGTTCACTCGAGCAAAACTCT TGGCAGCCCAGCAGTATGCTGCCTCTTCGGGGCTACCTGTGCCGAGAAATGTTCTGATCCCGCGTACT AAGGGTTTTGTTTCTTCTGTGAGTCACATGCGATCATTTGTTCCAGCCATATATGATGTTACAGTGGC AATCCCAAAGACGTCACCTCCACCAACATTGATAAGAATGTTCAAGGGACAGTCCTCAGTGCTTCACG TCCACCTCAAGCGACACCTAATGAAAGATTTACCTGAATCAGATGATGCTGTTGCTCAGTGGTGCAGA GATATATTCGTCGAGAAGGATGCTTTGTTGGATAAGCATAATGCTGAGGACACTTTCAGTGGCCAAGA ACTTCAAGAAACTGGCCGCCCAATAAAGTCTCTTCTGGTTGTAATCTCTTGGGCGGTGTTGGAGGTAT TTGGAGCTGTGAAGTTTCTTCAATGGTCATCGCTGTTATCATCATGGAAGGGACTTGCATTTTCGGGA ATAGGACTGGGTGTCATCACGCTACTCATGCACATACTGATTTTATTCTCACAATCCGAGCGGTCTAC CCCTGCAAAAGTGGCACCAGCAAAGCCAAAGAATGAGGGAGAGTCCTCCAAGACGGAAATGGAAAAGG AAAAGTAG SEQ ID NO: 83 cDNA sequence for CuPSR23 LPAATx coding region codon optimized for Prototheca moriformis ATGgagatccccccccactgcctgtgctccccctcccccgccccctcccagctgtactacaagaagaa gaagcacgccatcctgcagacccagaccccctaccgctaccgcgtgtcccccacctgcttcgcccccc cccgcctgcgcaagcagcacccctaccccctgcccgtgctgtgctaccccaagctgctgcacttctcc cagccccgctaccccctggtgcgctcccacctggccgaggccggcgtggcctaccgccccggctacga gctgctgggcaagatccgcggcgtgtgcttctacgccgtgaccgccgccgtggccctgctgctgttcc agtgcatgctgctgctgcaccccttcgtgctgctgttcgaccccttcccccgcaaggcccaccacacc atcgccaagctgtggtccatctgctccgtgtccctgttctacaagatccacatcaagggcctggagaa cctgccccccccccactcccccgccgtgtacgtgtccaaccaccagtccttcctggacatctacaccc tgctgaccctgggccgcaccttcaagttcatctccaagaccgagatcttcctgtaccccatcatcggc tgggccatgtacatgctgggcaccatccccctgaagcgcctggactcccgctcccagctggacaccct gaagcgctgcatggacctgatcaagaagggcgcctccgtgttcttcttccccgagggcacccgctcca aggacggcaagctgggcgccttcaagaagggcgccttctccatcgccgccaagtccaaggtgcccgtg gtgcccatcaccctgatcggcaccggcaagatcatgccccccggctccgagctgaccgtgaaccccgg caccgtgcaggtgatcatccacaagcccatcgagggctccgacgccgaggccatgtgcaacgaggccc gcgccaccatctcccactccctggacgacTGA SEQ ID NO: 84 cDNA sequence for CuPSR23 LPAAT 2-1 coding region codon optimized for Prototheca moriformis ATGgcgatcgcggccgcggcggtgatcttcctgttcggcctgatcttcttcgcctccggcctgatcat caacctgttccaggcgctgtgcttcgtcctgatccgccccctgtccaagaacgcctaccgccgcatca accgcgtgttcgcggagctgctgctgtccgagctgctgtgcctgttcgactggtgggcgggcgcgaag ctgaagctgttcaccgaccccgagacgttccgcctgatgggcaaggagcacgccctggtcatcatcaa ccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctcca tcatctccgtcgccaagaagtccacgaagttcctgcccgtgctgggctggtccatgtggttctccgag tacctgtacctggagcgctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgat cgactaccccctgcccttctggctggtcatcttcgtcgagggcacccgcttcacgcgcacgaagctgc tggcggcccagcagtacgcggtctcctccggcctgcccgtcccccgcaacgtcctgatcccccgcacg aagggcttcgtctcctgcgtgtcccacatgcgctccttcgtccccgcggtgtacgacgtcacggtggc gttccccaagacgtcccccccccccacgctgctgaacctgttcgagggccagtccatcatgctgcacg tgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtcgcggagtggtgccgc gacaagttcgtcgagaaggacgccctgctggacaagcacaacgcggaggacacgttctccggccagga ggtgtgccactccggctcccgccagctgaagtccctgctggtcgtgatctcctgggtcgtggtgacga cgttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggcgttctccgccatcggcctg ggcatcgtcaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccga ggtggcccaggccaagctgaagaccggcctgtccatctccaagaaggtgacggacaaggagaacTGA SEQ ID NO: 85 cDNA sequence for CuPSR23 LPAAx 3-1 coding region codon optimized for Prototheca moriformis ATGgccatcgcggcggccgcggtgatcgtgcccctgtccctgctgttcttcgtgtccggcctgatcgt caacctggtgcaggccgtctgcttcgtcctgatccgccccctgtccaagaacacgtaccgccgcatca accgcgtggtcgcggagctgctgtggctggagctggtgtggctgatcgactggtgggcgggcgtgaag atcaaggtcttcacggaccacgagacgttccacctgatgggcaaggagcacgccctggtcatctgcaa ccacaagtccgacatcgactggctggtcggctgggtcctgggccagcgctccggctgcctgggctcca ccctggcggtcatgaagaagtcctccaagttcctgcccgtcctgggctggtccatgtggttctccgag tacctgttcctggagcgctcctgggccaaggacgagatcacgctgaagtccggcctgaaccgcctgaa ggactaccccctgcccttctggctggcgctgttcgtggagggcacgcgcttcacccgcgcgaagctgc tggcggcgcagcagtacgccgcgtcctccggcctgcccgtgccccgcaacgtgctgatcccccgcacg aagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgcgatctacgacgtcaccgtggc catccccaagacgtcccccccccccacgctgatccgcatgttcaagggccagtcctccgtgctgcacg tgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgccgtcgcgcagtggtgccgc gacatcttcgtggagaaggacgcgctgctggacaagcacaacgccgaggacaccttctccggccagga gctgcaggagaccggccgccccatcaagtccctgctggtcgtcatctcctgggccgtcctggaggtgt tcggcgccgtcaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggcgttctccggc atcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgttctcccagtccgagcgctccac ccccgccaaggtggcccccgcgaagcccaagaacgagggcgagtcctccaagaccgagatggagaagg agaagTGA SEQ ID NO: 86 Nucleic acid sequence encoding 14:0-ACP thioesterase, Cuphea palustris (Cpal FATB2, accession AAC49180) containing an extended heterologous transit peptide from C. protothecoides and a 41 amino acid N-terminal extension derived from the native Cpal FATB2 sequence in construct D1481 [pSZ2479] GCGCACCCCAAGGCGAACGGCAGCGCGGTGTCGCTGAAGTCGGGCTCCCIGGAGACCCAGGAGGACAA GACGAGCAGCTCGTCCCCCCCCCCCCGCACGTTCATCAACCAGCTGCCCGTGTGGAGCATGCTGCTGT CGGCGGTGACCACGGTCTTCGGCGTGGCCGAGAAGCAGTGGCCCATGCTGGACCGCAAGTCCAAGCGC CCCGACATGCTGGTCGAGCCCCTGGGCGTGGACCGCATCGTCTACGACGGCGTGAGCTTCCGCCAGTC GTTCTCCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCTCGATCGAGACGCTGATGAACATGT TCCAGGAGACCTCCCTGAACCACTGCAAGATCATCGGCCTGCTGAACGACGGCTTCGGCCGCACGCCC GAGATGTGCAAGCGCGACCTGATCTGGGTCGTGACCAAGATGCAGATCGAGGTGAACCGCTACCCCAC GTGGGGCGACACCATCGAGGTCAACACGTGGGTGAGCGCCTCGGGCAAGCACGGCATGGGCCGCGACT GGCTGATCTCCGACTGCCACACCGGCGAGATCCTGATCCGCGCGACGAGCGTCTGGGCGATGATGAAC CAGAAGACCCGCCGCCTGTCGAAGATCCCCTACGAGGTGCGCCAGGAGATCGAGCCCCAGTTCGTCGA CTCCGCCCCCGTGATCGTGGACGACCGCAAGTTCCACAAGCTGGACCTGAAGACGGGCGACAGCATCT GCAACGGCCTGACCCCCCGCTGGACGGACCTGGACGTGAACCAGCACGTCAACAACGTGAAGTACATC GGCTGGATCCTGCAGTCGGTCCCCACCGAGGTGTTCGAGACGCAGGAGCTGTGCGGCCTGACCCTGGA GTACCGCCGCGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGTCACGGCCATGGACCCCTCGAAGGAGG GCGACCGCTCCCTGTACCAGCACCTGCTGCGCCTGGAGGACGGCGCGGACATCGTGAAGGGCCGCACC GAGTGGCGCCCCAAGAACGCCGGCGCCAAGGGCGCCATCCTGACGGGCAAGACCAGCAACGGCAACTC GATCTCCTGA SEQ ID NO: 87 Amino acid sequence of 14:0-ACP thioesterase, Cuphea palustris (Cpal FATB2, accession AAC49180) containing an extended heterologous transit peptide from C. protothecoides and a 41 amino acid N- terminal extension derived from the native Cpal FATB2 sequence encoded by construct D1481 [pSZ2479] AHPKANGSAVSLKSGSLETQEDKTSSSSPPPRTFINQLPVWSMLLSAVTTVFGVAEKQWP MLDRKSKRPDMLVEPLGVDRIVYDGVSFRQSFSIRSYEIGADRTASIETLMNMFQETSLN HCKIIGLLNDGFGRTPEMCKRDLIWVVTKMQIEVNRYPTWGDTIEVNTWVSASGKHGMGR DWLISDCHTGEILIRATSVMAMMNQKTRRLSKIPYEVRQEIEPQFVDSAPVIVDDRKFHK LDLKTGDSICNGLTPRWTDLDVNQHVNNVKYIGWILQSVPTEVFETQELCGLTLEYRREC GRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIVKGRTEWRPKNAGAKGAILTGKTSN GNSIS SEQ ID No: 88 Nucleic acid sequence encoding 14:0-ACP thioesterase, Cuphea palustris (Cpal FATB2, accession AAC49180) containing an extended heterologous transit peptide from C. protothecoides, a 41 amino acid N-terminal extension derived from the native Cpal FATB2 sequence, and a C-terminal FLAG epitope tag in construct D1482 [pSZ2480] GCGCACCCCAAGGCGAACGGCAGCGCGGTGTCGCTGAAGTCGGGCTCCCTGGAGACCCAGGAGGACAA GACGAGCAGCTCGTCCCCCCCCCCCCGCACGTTCATCAACCAGCTGCCCGTGTGGAGCATGCTGCTGT CGGCGGTGACCACGGTCTTCGGCGTGGCCGAGAAGCAGTGGCCCATGCTGGACCGCAAGTCCAAGCGC CCCGACATGCTGGTCGAGCCCCTGGGCGTGGACCGCATCGTCTACGACGGCGTGAGCTTCCGCCAGTC GTTCTCCATCCGCAGCTACGAGATCGGCGCCGACCGCACCGCCTCGATCGAGACGCTGATGAACATGT TCCAGGAGACCTCCCTGAACCACTGCAAGATCATCGGCCTGCTGAACGACGGCTTCGGCCGCACGCCC GAGATGTGCAAGCGCGACCTGATCTGGGTCGTGACCAAGATGCAGATCGAGGTGAACCGCTACCCCAC GTGGGGCGACACCATCGAGGTCAACACGTGGGTGAGCGCCTCGGGCAAGCACGGCATGGGCCGCGACT GGCTGATCTCCGACTGCCACACCGGCGAGATCCTGATCCGCGCGACGAGCGTCTGGGCGATGATGAAC CAGAAGACCCGCCGCCTGTCGAAGATCCCCTACGAGGTGCGCCAGGAGATCGAGCCCCAGTTCGTCGA CTCCGCCCCCGTGATCGTGGACGACCGCAAGTTCCACAAGCTGGACCTGAAGACGGGCGACAGCATCT GCAACGGCCTGACCCCCCGCTGGACGGACCTGGACGTGAACCAGCACGTCAACAACGTGAAGTACATC GGCTGGATCCTGCAGTCGGTCCCCACCGAGGTGTTCGAGACGCAGGAGCTGTGCGGCCTGACCCTGGA GTACCGCCGCGAGTGCGGCCGCGACTCCGTGCTGGAGAGCGTCACGGCCATGGACCCCTCGAAGGAGG GCGACCGCTCCCTGTACCAGCACCTGCTGCGCCTGGAGGACGGCGCGGACATCGTGAAGGGCCGCACC GAGTGGCGCCCCAAGAACGCCGGCGCCAAGGGCGCCATCCTGACGGGCAAGACCAGCAACGGCAACTC GATCTCCatggactacaaggaccacgacggcgactacaaggaccacgacatcgactacaaggacgacg acgacaagtga SEQ ID NO: 89 Amino acid sequence of 14:0-ACP thioesterase, Cuphea palustris (Cpal FATB2, accession AAC49180) containing an extended heterologous transit peptide from C. protothecoides, a 41 amino acid N-terminal extension derived from the native Cpal FATB2 sequence, and a C- terminal FLAG epitope tag encoded by construct D1482 [pSZ2480] AHPKANGSAVSLKSGSLETQEDKTSSSSPPPRTFINQLPVWSMLLSAVTTVFGVAEKQWP MLDRKSKRPDMLVEPLGVDRIVYDGVSFRQSFSIRSYEIGADRTASIETLMNMFQETSLN HCKIIGLLNDGFGRTPEMCKRDLIWVVTKMQIEVNRYPTWGDTIEVNTWVSASGKHGMGR DWLISDCHTGEILIRATSVMAMMNQKTRRLSKIPYEVRQEIEPQFVDSAPVIVDDRKFHK LDLKTGDSICNGLTPRWTDLDVNQHVNNVKYIGWILQSVPTEVFETQELCGLTLEYRREC GRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIVKGRTEWRPKNAGAKGAILTGKTSN GNSISMDYKDHDGDYKDHDIDYKDDDDK SEQ ID NO: 154 Cuphea hyssopifolia FATB3 coding region, codon optimized for Prototheca moriformis gtggccgccgaggcctcctccgccctgttctccgtgcgcacccccggcacctcccccaagcccggcaa gttcggcaactggcccacctccctgtccgtgcccttcaagtccaagtccaaccacaacggcggcttcc aggtgaaggccaacgcctccgcccgccccaaggccaacggctccgccgtgtccctgaagtccggctcc ctggacacccaggaggacacctcctcctcctcctcccccccccgcaccttcatcaaccagctgcccga ctggtccatgctgctgtccgccatcaccaccgtgttcgtggccgccgagaagcagtggaccatgctgg accgcaagtccaagcgccccgacatgctgatggaccccttcggcgtggaccgcgtggtgcaggacggc gccgtgttccgccagtccttctccatccgctcctacgagatcggcgccgaccgcaccgcctccatcga gaccctgatgaacatcttccaggagacctccctgaaccactgcaagtccatcggcctgctgaacgacg gcttcggccgcacccccgagatgtgcaagcgcgacctgatctgggtggtgaccaagatgcacgtggag gtgaaccgctaccccacctggggcgacaccatcgaggtgaacacctgggtgtccgagtccggcaagac cggcatgggccgcgactggctgatctccgactgccacaccggcgagatcctgatccgcgccacctcca tgtgcgccatgatgaaccagaagacccgccgcttctccaagttcccctacgaggtgcgccaggagctg gccccccacttcgtggactccgcccccgtgatcgaggactaccagaagctgcacaagctggacgtgaa gaccggcgactccatctgcaacggcctgaccccccgctggaacgacctggacgtgaaccagcacgtga acaacgtgaagtacatcggctggatcctggagtccgtgcccaccgaggtgttcgagacccaggagctg tgcggcctgaccctggagtaccgccgcgagtgcggccgcgactccgtgctggagtccgtgaccgccat ggacccctccaaggagggcgaccgctccctgtaccagcacctgctgcgcctggaggacggcgccgaca tcgccaagggccgcaccaagtggcgccccaagaacgccggcaccaacggcgccatctccaccggcaag acctccaacggcaactccatctccatggactacaaggaccacgacggcgactacaaggaccacgacat cgactacaaggacgacgacgacaag SEQ ID NO: 155 Cuphea hyssopifolia FATB1 coding region, codon optimized for Prototheca moriformis gccaccgcctccaccttctccgccttcaacgcccgctgcggcgacctgcgccgctccgccggctccgg cccccgccgccccgcccgccccctgcccgtgcgcgccgccatcaacgcctccgcccaccccaaggcca acggctccgccgtgaacctgaagtccggctccctggagacccaggaggacacctcctcctcctccccc coccoccgcaccttcatcaagcagctgcccgactggggcatgctgctgtccaagatcaccaccgtgtt cggcgccgccgagcgccagtggaagcgcccoggcatgctggtggagccottoggcgtggaccgcatct tccaggacggcgtgttottccgccagtocttctccatccgctcctacgagatcggcgccgaccgcacc gcctccatcgagaccctgatgaacatcttccaggagacctocctgaaccactgcaagtccatcggcct gctgaacgacggcttoggccgcaccoccgagatgtgcaagcgcgacctgatctgggtggtgaccaaga tccaggtggaggtgaaccgctaccccacctggggcgacaccatcgaggtgaacacctgggtgtccgag tcoggcaagaacggcatgggccgcgactggctgatctccgactgccgcaccggcgagatcctgatccg
cgccacctccgtgtgggccatgatgaaccgcaagacccgccgcctgtccaagttccoctacgaggtgc gccaggagatcgccocccacttcgtggactccgccoccgtgatcgaggacgacaagaagctgcacaag ctggacgtgaagaccggcgactccatccgcaagggcctgaccocccgctggaacgacctggacgtgaa ccagcacgtgaacaacgtgaagtacatoggctggatcctgaagtccgtgcccgccgaggtgttcgaga cccaggagctgtgoggcgtgaccctggagtaccgccgcgagtgoggccgcgactccgtgctggagtcc gtgaccgccatggacaccgccaaggagggcgaccgctocctgtaccagcacctgctgcgcctggagga cggcgccgacatcaccatoggccgcaccgagtggcgccocaagaacgccggcgccaacggcgccatct ccaccggcaagacctccaacgagaactccgtgtccatggactacaaggaccacgacggcgactacaag gaccacgacatcgactacaaggacgacgacgacaag SEQ ID NO: 156 Garcinia mangostana FATA1 CDS MLKLSSSRSPLARIPTRPRPNSIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSLTEDGLSYKEKF IVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSIGGFSTIPTMRKLRLIWVTARMHIEIYKYPAWSD VVEIESWGQGEGKIGIRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRLA FPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVIYIGWVLESMPQEIIDTHELQIITLDYR RECQHDDVVDSLTSPEPSEDAEAVFNHNGINGSANVSANDHGCRNFLHLLRLSGNGLEINRGRIEWRKKP TR SEQ ID NO: 157 Brassic napus LPAAT CDS MAMAAAVIVPLGILFFISGLVVNLLQAVCYVLVRPMSKNTYRKINRVVAETLWLELVWIVDWWAGVKIQV FADDETFNRMGKEHALVVCNHRSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLE RNWAKDESTLQSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRIKGFVSAV SNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKCHSMKDLPEPEDEIAQWCRDQFVAKDAL LDKHIAADTFPGQKEQNIGRPIKSLAVVVSWACLLTLGAMKFLHWSNLFSSWKGIALSAFGLGIITLCMQ ILIRSSQSERSTPAKVAPAKPKDNHQSGPSSQTEVEEKQK SEQ ID NO: 158 Cuphea hookeriana FATB2 CDS MVAAAASSAFFPVPAPGASPKPGKEGNWPSSLSPSFKPKSIPNGGFQVKANDSAHPKANGSAVSLKSGSL NIQEDISSSPPPRTFLHQLPDWSRLLTAITIVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQ SFSIRSYEIGTDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKMQIKVNRYPAW GDIVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAYAMMNQKTRRLSKLPYEVHQEIVPLFVDSPV IEDSDLKVHKEKVKIGDSIQKGLIPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRREC GRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGANGAISIGKISNGNSVS SEQ ID NO: 159 Cuphea wrightii KASAI CDS with P moriformis SAD transit peptide (underlined) MASAAFTMSACPAMTGRAPGARRSGRPVATRLRYVFQCLVASCIDPCDQYRSSASLSFLGDNGFASLF GSKPFMSNRGHRRLRRASHSGEAMAVALQPAQEAGIKKKPVIKQRRVVVTGMGVVTPLGHEPDVFYNN LLDGVSGISEIETEDCTQFPTRIAGEIKSFSIDGWVAPKLSKRMDKFMLYLLTAGKKALADGGITDEV MKELDKRKCGVLIGSGMGGMKVFNDAIEALRVSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSIS TACATSNFCILNAANHIIRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNSDPIKASRPWDSNRDG FVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIEKALAQAGVSKED VNYINAHATSTSAGDIKEYQALARCFGQNSELRVNSTKSMIGHLLGAAGGVEAVTVVQAIRTGWIHPN LNLEDPDKAVDAKLLVGPKKERLNVKVGLSNSFGEGGHNSSILFAPCNV SEQ ID NO: 160 Native Protheca moriformis KASII amino acid sequence (native transit peptide is underlined) MQTAHQRPPTEGHCFGARLPTASRRAVRRAWSRIARAAAAADANPARPERRVVITGQGVVTSLGQTIE QFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQLDPYVPKRWAKRVDDVIKYVYIAGKQALESAG LPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGVEALTRGGVRKMNPFCIPFSISNMGGAMLAMDI GFMGPNYSISTACAIGNYCILGAADHIRRGDANVMLAGGADAAIIPSGIGGFIACKALSKRNDEPERA SRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATILAELVGGAATSDAHHMTEPDPQGRGVRLCLERA LERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVIPQDSLRINSTKSMIGHLLGGAGAVEAVAAIQA LRTGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLDVVLSNSFGEGGHNSCVIERKYDE SEQ ID NO: 161 Mature native Protheca moriformis KASII amino acid sequence (native transit peptide is underlined) AAAAADANPARPERRVVITGQGVVISLGQIIEQFYSSLLEGVSGISQIQKFDTTGYTTTIAGEIKSLQ LDPYVPKRWAKRVDDVIKYVYIAGKQALESAGLPIEAAGLAGAGLDPALCGVLIGTAMAGMTSFAAGV EALTRGGVRKMNPFCIPFSISNMGGAMLAMDIGFMGPNYSISTACAIGNYCILGAADHIRRGDANVML AGGADAAIIPSGIGGFIACKALSKRNDEPERASRPWDADRDGFVMGEGAGVLVLEELEHAKRRGATIL AELVGGAATSDAHHMTEPDPQGRGVRLCLERALERARLAPERVGYVNAHGTSTPAGDVAEYRAIRAVI PQDSLRINSTKSMIGHLLGGAGAVEAVAAIQALRIGWLHPNLNLENPAPGVDPVVLVGPRKERAEDLD VVLSNSFGFGGHNSCVIFRKYDE SEQ ID NO: 162 CcFATB2-UcFATB2 chimeric FATB PDWSMLFAVITTIFSAAEKQWTNLEWKPKPNPPQLLDDHFGPHGLVERRTFAIRSYEVGP DRSTSIVAVMNHLQEAALNHAKSVGILGDGFGTTLEMSKRDLIWVVRRTHVAVERYPTWG DIVEVECWIGASGNNGMRRDFLVRDCKTGEILTRCTSLSVLMNTRIRRLSTIPDEVRGEI GPAFIDNVAVKDDEIKKLQKLNDSTADYIQGGLIPRWNDLDVNQHVNNLKYVAWVFETVP DSIFESHHISSFTLEYRRECTRDSVLRSLTTVSGGSSEAGLVCDHLLQLEGGSEVLRART EWRPKLTDSFRGISVIPAEPRV SEQ ID NO: 163 Cuphea hyssopifolia FATB1 MVATNAAAFSAYTFFLTSPTHGYSSKRLADTQNGYPGTSLKSKSTPPPAAAAARNGALPLLASICKCP KKADGSMQLDSSLVEGFQFYIRSYEVGADQTVSIQTVLNYLQEAAINHVQSAGYFGDSFGATPEMTKR NLIWVITKMQVLVDRYPAWGDVVQVDTWICSSGKNSMQRDWFVRDLKTGDIITRASSVWVLMNRLTRK LSKIPEAVLEEAKLFVMNTAPTVDDNRKLPKLDGSSADYVLSGLTPRWSDLDMNQHVNNVKYIAWILE SVPQSIPETHKLSAITVEYRRECGKNSVLQSLTNVSGDGITCGNSIIECHHLLQLETGPEILLARTEW ISKEPGFRGAPIQAEKVYNNK* SEQ ID NO: 164 Cuphea hyssopifolia FATB3 MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASARPKANGSAVSLKSG SLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTMLDRKSKRPDMLMDPFGVDRVVQD GAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRDLIWVVTKMHV EVNRYPTWGDTIEVNTWVSESGKTGMGRDWLISDCHTGEILIRATSMCAMMNQKTRRESKFPYEVRQE LAPHFVDSAPVIEDYQKLHKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQE LCGLTLEYRRECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAISTG KTSNGNSIS*
Sequence CWU
1
1
1711726DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 1gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc
gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac
caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg cggccaagag
gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc
cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag
tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta cagaacaacc
acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg acggcctgcg
aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg
tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg
ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt gcagaccggt
gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca attctgtcac
ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc agccgctggg
ggttggcgga tgcacgctca 720ggtacc
7262749DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 2gagctccttg ttttccagaa
ggagttgctc cttgagcctt tcattctcag cctcgataac 60ctccaaagcc gctctaattg
tggagggggt tcgaatttaa aagcttggaa tgttggttcg 120tgcgtctgga acaagcccag
acttgttgct cactgggaaa aggaccatca gctccaaaaa 180acttgccgct caaaccgcgt
acctctgctt tcgcgcaatc tgccctgttg aaatcgccac 240cacattcata ttgtgacgct
tgagcagtct gtaattgcct cagaatgtgg aatcatctgc 300cccctgtgcg agcccatgcc
aggcatgtcg cgggcgagga cacccgccac tcgtacagca 360gaccattatg ctacctcaca
atagttcata acagtgacca tatttctcga agctccccaa 420cgagcacctc catgctctga
gtggccaccc cccggccctg gtgcttgcgg agggcaggtc 480aaccggcatg gggctaccga
aatccccgac cggatcccac cacccccgcg atgggaagaa 540tctctccccg ggatgtgggc
ccaccaccag cacaacctgc tggcccaggc gagcgtcaaa 600ccataccaca caaatatcct
tggcatcggc cctgaattcc ttctgccgct ctgctacccg 660gtgcttctgt ccgaagcagg
ggttgctagg gatcgctccg agtccgcaaa cccttgtcgc 720gtggcggggc ttgttcgagc
ttgaagagc 7493532PRTSaccharomyces
cerevisiae 3Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala
Lys 1 5 10 15 Ile
Ser Ala Ser Met Thr Asn Glu Thr Ser Asp Arg Pro Leu Val His
20 25 30 Phe Thr Pro Asn Lys
Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr 35
40 45 Asp Glu Lys Asp Ala Lys Trp His Leu
Tyr Phe Gln Tyr Asn Pro Asn 50 55
60 Asp Thr Val Trp Gly Thr Pro Leu Phe Trp Gly His Ala
Thr Ser Asp 65 70 75
80 Asp Leu Thr Asn Trp Glu Asp Gln Pro Ile Ala Ile Ala Pro Lys Arg
85 90 95 Asn Asp Ser Gly
Ala Phe Ser Gly Ser Met Val Val Asp Tyr Asn Asn 100
105 110 Thr Ser Gly Phe Phe Asn Asp Thr Ile
Asp Pro Arg Gln Arg Cys Val 115 120
125 Ala Ile Trp Thr Tyr Asn Thr Pro Glu Ser Glu Glu Gln Tyr
Ile Ser 130 135 140
Tyr Ser Leu Asp Gly Gly Tyr Thr Phe Thr Glu Tyr Gln Lys Asn Pro 145
150 155 160 Val Leu Ala Ala Asn
Ser Thr Gln Phe Arg Asp Pro Lys Val Phe Trp 165
170 175 Tyr Glu Pro Ser Gln Lys Trp Ile Met Thr
Ala Ala Lys Ser Gln Asp 180 185
190 Tyr Lys Ile Glu Ile Tyr Ser Ser Asp Asp Leu Lys Ser Trp Lys
Leu 195 200 205 Glu
Ser Ala Phe Ala Asn Glu Gly Phe Leu Gly Tyr Gln Tyr Glu Cys 210
215 220 Pro Gly Leu Ile Glu Val
Pro Thr Glu Gln Asp Pro Ser Lys Ser Tyr 225 230
235 240 Trp Val Met Phe Ile Ser Ile Asn Pro Gly Ala
Pro Ala Gly Gly Ser 245 250
255 Phe Asn Gln Tyr Phe Val Gly Ser Phe Asn Gly Thr His Phe Glu Ala
260 265 270 Phe Asp
Asn Gln Ser Arg Val Val Asp Phe Gly Lys Asp Tyr Tyr Ala 275
280 285 Leu Gln Thr Phe Phe Asn Thr
Asp Pro Thr Tyr Gly Ser Ala Leu Gly 290 295
300 Ile Ala Trp Ala Ser Asn Trp Glu Tyr Ser Ala Phe
Val Pro Thr Asn 305 310 315
320 Pro Trp Arg Ser Ser Met Ser Leu Val Arg Lys Phe Ser Leu Asn Thr
325 330 335 Glu Tyr Gln
Ala Asn Pro Glu Thr Glu Leu Ile Asn Leu Lys Ala Glu 340
345 350 Pro Ile Leu Asn Ile Ser Asn Ala
Gly Pro Trp Ser Arg Phe Ala Thr 355 360
365 Asn Thr Thr Leu Thr Lys Ala Asn Ser Tyr Asn Val Asp
Leu Ser Asn 370 375 380
Ser Thr Gly Thr Leu Glu Phe Glu Leu Val Tyr Ala Val Asn Thr Thr 385
390 395 400 Gln Thr Ile Ser
Lys Ser Val Phe Ala Asp Leu Ser Leu Trp Phe Lys 405
410 415 Gly Leu Glu Asp Pro Glu Glu Tyr Leu
Arg Met Gly Phe Glu Val Ser 420 425
430 Ala Ser Ser Phe Phe Leu Asp Arg Gly Asn Ser Lys Val Lys
Phe Val 435 440 445
Lys Glu Asn Pro Tyr Phe Thr Asn Arg Met Ser Val Asn Asn Gln Pro 450
455 460 Phe Lys Ser Glu Asn
Asp Leu Ser Tyr Tyr Lys Val Tyr Gly Leu Leu 465 470
475 480 Asp Gln Asn Ile Leu Glu Leu Tyr Phe Asn
Asp Gly Asp Val Val Ser 485 490
495 Thr Asn Thr Tyr Phe Met Thr Thr Gly Asn Ala Leu Gly Ser Val
Asn 500 505 510 Met
Thr Thr Gly Val Asp Asn Leu Phe Tyr Ile Asp Lys Phe Gln Val 515
520 525 Arg Glu Val Lys 530
41599DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 4atgctgctgc aggccttcct gttcctgctg
gccggcttcg ccgccaagat cagcgcctcc 60atgacgaacg agacgtccga ccgccccctg
gtgcacttca cccccaacaa gggctggatg 120aacgacccca acggcctgtg gtacgacgag
aaggacgcca agtggcacct gtacttccag 180tacaacccga acgacaccgt ctgggggacg
cccttgttct ggggccacgc cacgtccgac 240gacctgacca actgggagga ccagcccatc
gccatcgccc cgaagcgcaa cgactccggc 300gccttctccg gctccatggt ggtggactac
aacaacacct ccggcttctt caacgacacc 360atcgacccgc gccagcgctg cgtggccatc
tggacctaca acaccccgga gtccgaggag 420cagtacatct cctacagcct ggacggcggc
tacaccttca ccgagtacca gaagaacccc 480gtgctggccg ccaactccac ccagttccgc
gacccgaagg tcttctggta cgagccctcc 540cagaagtgga tcatgaccgc ggccaagtcc
caggactaca agatcgagat ctactcctcc 600gacgacctga agtcctggaa gctggagtcc
gcgttcgcca acgagggctt cctcggctac 660cagtacgagt gccccggcct gatcgaggtc
cccaccgagc aggaccccag caagtcctac 720tgggtgatgt tcatctccat caaccccggc
gccccggccg gcggctcctt caaccagtac 780ttcgtcggca gcttcaacgg cacccacttc
gaggccttcg acaaccagtc ccgcgtggtg 840gacttcggca aggactacta cgccctgcag
accttcttca acaccgaccc gacctacggg 900agcgccctgg gcatcgcgtg ggcctccaac
tgggagtact ccgccttcgt gcccaccaac 960ccctggcgct cctccatgtc cctcgtgcgc
aagttctccc tcaacaccga gtaccaggcc 1020aacccggaga cggagctgat caacctgaag
gccgagccga tcctgaacat cagcaacgcc 1080ggcccctgga gccggttcgc caccaacacc
acgttgacga aggccaacag ctacaacgtc 1140gacctgtcca acagcaccgg caccctggag
ttcgagctgg tgtacgccgt caacaccacc 1200cagacgatct ccaagtccgt gttcgcggac
ctctccctct ggttcaaggg cctggaggac 1260cccgaggagt acctccgcat gggcttcgag
gtgtccgcgt cctccttctt cctggaccgc 1320gggaacagca aggtgaagtt cgtgaaggag
aacccctact tcaccaaccg catgagcgtg 1380aacaaccagc ccttcaagag cgagaacgac
ctgtcctact acaaggtgta cggcttgctg 1440gaccagaaca tcctggagct gtacttcaac
gacggcgacg tcgtgtccac caacacctac 1500ttcatgacca ccgggaacgc cctgggctcc
gtgaacatga cgacgggggt ggacaacctg 1560ttctacatcg acaagttcca ggtgcgcgag
gtcaagtga 15995312DNAChlamydomonas reinhardtii
5ctttcttgcg ctatgacact tccagcaaaa ggtagggcgg gctgcgagac ggcttcccgg
60cgctgcatgc aacaccgatg atgcttcgac cccccgaagc tccttcgggg ctgcatgggc
120gctccgatgc cgctccaggg cgagcgctgt ttaaatagcc aggcccccga ttgcaaagac
180attatagcga gctaccaaag ccatattcaa acacctagat cactaccact tctacacagg
240ccactcgagc ttgtgatcgc actccgctaa gggggcgcct cttcctcttc gtttcagtca
300caacccgcaa ac
3126408DNAChlorella vulgaris 6gcagcagcag ctcggatagt atcgacacac tctggacgct
ggtcgtgtga tggactgttg 60ccgccacact tgctgccttg acctgtgaat atccctgccg
cttttatcaa acagcctcag 120tgtgtttgat cttgtgtgta cgcgcttttg cgagttgcta
gctgcttgtg ctatttgcga 180ataccacccc cagcatcccc ttccctcgtt tcatatcgct
tgcatcccaa ccgcaactta 240tctacgctgt cctgctatcc ctcagcgctg ctcctgctcc
tgctcactgc ccctcgcaca 300gccttggttt gggctccgcc tgtattctcc tggtactgca
acctgtaaac cagcactgca 360atgctgatgc acgggaagta gtgggatggg aacacaaatg
gaaagctt 40872333DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 7ctttcttgcg ctatgacact
tccagcaaaa ggtagggcgg gctgcgagac ggcttcccgg 60cgctgcatgc aacaccgatg
atgcttcgac cccccgaagc tccttcgggg ctgcatgggc 120gctccgatgc cgctccaggg
cgagcgctgt ttaaatagcc aggcccccga ttgcaaagac 180attatagcga gctaccaaag
ccatattcaa acacctagat cactaccact tctacacagg 240ccactcgagc ttgtgatcgc
actccgctaa gggggcgcct cttcctcttc gtttcagtca 300caacccgcaa acggcgcgcc
atgctgctgc aggccttcct gttcctgctg gccggcttcg 360ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg gtgcacttca 420cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag aaggacgcca 480agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg cccttgttct 540ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc gccatcgccc 600cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac aacaacacct 660ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc tggacctaca 720acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc tacaccttca 780ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc gacccgaagg 840tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc caggactaca 900agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc gcgttcgcca 960acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc cccaccgagc 1020aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc gccccggccg 1080gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc gaggccttcg 1140acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag accttcttca 1200acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac tgggagtact 1260ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc aagttctccc 1320tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag gccgagccga 1380tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc acgttgacga 1440aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag ttcgagctgg 1500tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac ctctccctct 1560ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag gtgtccgcgt 1620cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag aacccctact 1680tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac ctgtcctact 1740acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac gacggcgacg 1800tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc gtgaacatga 1860cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag gtcaagtgac 1920aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg tgtgatggac 1980tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt atcaaacagc 2040ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc ttgtgctatt 2100tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat cccaaccgca 2160acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc actgcccctc 2220gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg taaaccagca 2280ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga tcc 233381065DNAPrototheca
moriformis 8ggccgacagg acgcgcgtca aaggtgctgg tcgtgtatgc cctggccggc
aggtcgttgc 60tgctgctggt tagtgattcc gcaaccctga ttttggcgtc ttattttggc
gtggcaaacg 120ctggcgcccg cgagccgggc cggcggcgat gcggtgcccc acggctgccg
gaatccaagg 180gaggcaagag cgcccgggtc agttgaaggg ctttacgcgc aaggtacagc
cgctcctgca 240aggctgcgtg gtggaattgg acgtgcaggt cctgctgaag ttcctccacc
gcctcaccag 300cggacaaagc accggtgtat caggtccgtg tcatccactc taaagagctc
gactacgacc 360tactgatggc cctagattct tcatcaaaaa cgcctgagac acttgcccag
gattgaaact 420ccctgaaggg accaccaggg gccctgagtt gttccttccc cccgtggcga
gctgccagcc 480aggctgtacc tgtgatcgag gctggcggga aaataggctt cgtgtgctca
ggtcatggga 540ggtgcaggac agctcatgaa acgccaacaa tcgcacaatt catgtcaagc
taatcagcta 600tttcctcttc acgagctgta attgtcccaa aattctggtc taccgggggt
gatccttcgt 660gtacgggccc ttccctcaac cctaggtatg cgcgcatgcg gtcgccgcgc
aactcgcgcg 720agggccgagg gtttgggacg ggccgtcccg aaatgcagtt gcacccggat
gcgtggcacc 780ttttttgcga taatttatgc aatggactgc tctgcaaaat tctggctctg
tcgccaaccc 840taggatcagc ggcgtaggat ttcgtaatca ttcgtcctga tggggagcta
ccgactaccc 900taatatcagc ccgactgcct gacgccagcg tccacttttg tgcacacatt
ccattcgtgc 960ccaagacatt tcattgtggt gcgaagcgtc cccagttacg ctcacctgtt
tcccgacctc 1020cttactgttc tgtcgacaga gcgggcccac aggccggtcg cagcc
10659120DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 9actagtatgg ccaccgcatc cactttctcg
gcgttcaatg cccgctgcgg cgacctgcgt 60cgctcggcgg gctccgggcc ccggcgccca
gcgaggcccc tccccgtgcg cgggcgcgcc 120101236DNACuphea wrightii
10atggtggtgg ccgccgccgc cagcagcgcc ttcttccccg tgcccgcccc ccgccccacc
60cccaagcccg gcaagttcgg caactggccc agcagcctga gccagccctt caagcccaag
120agcaacccca acggccgctt ccaggtgaag gccaacgtga gcccccacgg gcgcgccccc
180aaggccaacg gcagcgccgt gagcctgaag tccggcagcc tgaacaccct ggaggacccc
240cccagcagcc cccccccccg caccttcctg aaccagctgc ccgactggag ccgcctgcgc
300accgccatca ccaccgtgtt cgtggccgcc gagaagcagt tcacccgcct ggaccgcaag
360agcaagcgcc ccgacatgct ggtggactgg ttcggcagcg agaccatcgt gcaggacggc
420ctggtgttcc gcgagcgctt cagcatccgc agctacgaga tcggcgccga ccgcaccgcc
480agcatcgaga ccctgatgaa ccacctgcag gacaccagcc tgaaccactg caagagcgtg
540ggcctgctga acgacggctt cggccgcacc cccgagatgt gcacccgcga cctgatctgg
600gtgctgacca agatgcagat cgtggtgaac cgctacccca cctggggcga caccgtggag
660atcaacagct ggttcagcca gagcggcaag atcggcatgg gccgcgagtg gctgatcagc
720gactgcaaca ccggcgagat cctggtgcgc gccaccagcg cctgggccat gatgaaccag
780aagacccgcc gcttcagcaa gctgccctgc gaggtgcgcc aggagatcgc cccccacttc
840gtggacgccc cccccgtgat cgaggacaac gaccgcaagc tgcacaagtt cgacgtgaag
900accggcgaca gcatctgcaa gggcctgacc cccggctgga acgacttcga cgtgaaccag
960cacgtgagca acgtgaagta catcggctgg attctggaga gcatgcccac cgaggtgctg
1020gagacccagg agctgtgcag cctgaccctg gagtaccgcc gcgagtgcgg ccgcgagagc
1080gtggtggaga gcgtgaccag catgaacccc agcaaggtgg gcgaccgcag ccagtaccag
1140cacctgctgc gcctggagga cggcgccgac atcatgaagg gccgcaccga gtggcgcccc
1200aagaacgccg gcaccaaccg cgccatcagc acctga
123611408PRTCuphea wrightii 11Met Val Val Ala Ala Ala Ala Ser Ser Ala Phe
Phe Pro Val Pro Ala 1 5 10
15 Pro Arg Pro Thr Pro Lys Pro Gly Lys Phe Gly Asn Trp Pro Ser Ser
20 25 30 Leu Ser
Gln Pro Phe Lys Pro Lys Ser Asn Pro Asn Gly Arg Phe Gln 35
40 45 Val Lys Ala Asn Val Ser Pro
His Pro Lys Ala Asn Gly Ser Ala Val 50 55
60 Ser Leu Lys Ser Gly Ser Leu Asn Thr Leu Glu Asp
Pro Pro Ser Ser 65 70 75
80 Pro Pro Pro Arg Thr Phe Leu Asn Gln Leu Pro Asp Trp Ser Arg Leu
85 90 95 Arg Thr Ala
Ile Thr Thr Val Phe Val Ala Ala Glu Lys Gln Phe Thr 100
105 110 Arg Leu Asp Arg Lys Ser Lys Arg
Pro Asp Met Leu Val Asp Trp Phe 115 120
125 Gly Ser Glu Thr Ile Val Gln Asp Gly Leu Val Phe Arg
Glu Arg Phe 130 135 140
Ser Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg Thr Ala Ser Ile Glu 145
150 155 160 Thr Leu Met Asn
His Leu Gln Asp Thr Ser Leu Asn His Cys Lys Ser 165
170 175 Val Gly Leu Leu Asn Asp Gly Phe Gly
Arg Thr Pro Glu Met Cys Thr 180 185
190 Arg Asp Leu Ile Trp Val Leu Thr Lys Met Gln Ile Val Val
Asn Arg 195 200 205
Tyr Pro Thr Trp Gly Asp Thr Val Glu Ile Asn Ser Trp Phe Ser Gln 210
215 220 Ser Gly Lys Ile Gly
Met Gly Arg Glu Trp Leu Ile Ser Asp Cys Asn 225 230
235 240 Thr Gly Glu Ile Leu Val Arg Ala Thr Ser
Ala Trp Ala Met Met Asn 245 250
255 Gln Lys Thr Arg Arg Phe Ser Lys Leu Pro Cys Glu Val Arg Gln
Glu 260 265 270 Ile
Ala Pro His Phe Val Asp Ala Pro Pro Val Ile Glu Asp Asn Asp 275
280 285 Arg Lys Leu His Lys Phe
Asp Val Lys Thr Gly Asp Ser Ile Cys Lys 290 295
300 Gly Leu Thr Pro Gly Trp Asn Asp Phe Asp Val
Asn Gln His Val Ser 305 310 315
320 Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser Met Pro Thr Glu Val
325 330 335 Leu Glu
Thr Gln Glu Leu Cys Ser Leu Thr Leu Glu Tyr Arg Arg Glu 340
345 350 Cys Gly Arg Glu Ser Val Val
Glu Ser Val Thr Ser Met Asn Pro Ser 355 360
365 Lys Val Gly Asp Arg Ser Gln Tyr Gln His Leu Leu
Arg Leu Glu Asp 370 375 380
Gly Ala Asp Ile Met Lys Gly Arg Thr Glu Trp Arg Pro Lys Asn Ala 385
390 395 400 Gly Thr Asn
Arg Ala Ile Ser Thr 405 12933DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
12atggacgcct ccggcgcctc ctccttcctg cgcggccgct gcctggagtc ctgcttcaag
60gcctccttcg gctacgtaat gtcccagccc aaggacgccg ccggccagcc ctcccgccgc
120cccgccgacg ccgacgactt cgtggacgac gaccgctgga tcaccgtgat cctgtccgtg
180gtgcgcatcg ccgcctgctt cctgtccatg atggtgacca ccatcgtgtg gaacatgatc
240atgctgatcc tgctgccctg gccctacgcc cgcatccgcc agggcaacct gtacggccac
300gtgaccggcc gcatgctgat gtggattctg ggcaacccca tcaccatcga gggctccgag
360ttctccaaca cccgcgccat ctacatctgc aaccacgcct ccctggtgga catcttcctg
420atcatgtggc tgatccccaa gggcaccgtg accatcgcca agaaggagat catctggtat
480cccctgttcg gccagctgta cgtgctggcc aaccaccagc gcatcgaccg ctccaacccc
540tccgccgcca tcgagtccat caaggaggtg gcccgcgccg tggtgaagaa gaacctgtcc
600ctgatcatct tccccgaggg cacccgctcc aagaccggcc gcctgctgcc cttcaagaag
660ggcttcatcc acatcgccct ccagacccgc ctgcccatcg tgccgatggt gctgaccggc
720acccacctgg cctggcgcaa gaactccctg cgcgtgcgcc ccgcccccat caccgtgaag
780tacttctccc ccatcaagac cgacgactgg gaggaggaga agatcaacca ctacgtggag
840atgatccacg ccctgtacgt ggaccacctg cccgagtccc agaagcccct ggtgtccaag
900ggccgcgacg cctccggccg ctccaactcc tga
93313563DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 13gctcttcgct aacggaggtc tgtcaccaaa
tggaccccgt ctattgcggg aaaccacggc 60gatggcacgt ttcaaaactt gatgaaatac
aatattcagt atgtcgcggg cggcgacggc 120ggggagctga tgtcgcgctg ggtattgctt
aatcgccagc ttcgcccccg tcttggcgcg 180aggcgtgaac aagccgaccg atgtgcacga
gcaaatcctg acactagaag ggctgactcg 240cccggcacgg ctgaattaca caggcttgca
aaaataccag aatttgcacg caccgtattc 300gcggtatttt gttggacagt gaatagcgat
gcggcaatgg cttgtggcgt tagaaggtgc 360gacgaaggtg gtgccaccac tgtgccagcc
agtcctggcg gctcccaggg ccccgatcaa 420gagccaggac atccaaacta cccacagcat
caacgccccg gcctatactc gaaccccact 480tgcactctgc aatggtatgg gaaccacggg
gcagtcttgt gtgggtcgcg cctatcgcgg 540tcggcgaaga ccgggaaggt acc
56314465DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
14gagctcagcg gcgacggtcc tgctaccgta cgacgttggg cacgcccatg aaagtttgta
60taccgagctt gttgagcgaa ctgcaagcgc ggctcaagga tacttgaact cctggattga
120tatcggtcca ataatggatg gaaaatccga acctcgtgca agaactgagc aaacctcgtt
180acatggatgc acagtcgcca gtccaatgaa cattgaagtg agcgaactgt tcgcttcggt
240ggcagtacta ctcaaagaat gagctgctgt taaaaatgca ctctcgttct ctcaagtgag
300tggcagatga gtgctcacgc cttgcacttc gctgcccgtg tcatgccctg cgccccaaaa
360tttgaaaaaa gggatgagat tattgggcaa tggacgacgt cgtcgctccg ggagtcagga
420ccggcggaaa ataagaggca acacactccg cttcttagct cttcc
465151533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 15ctttcttgcg ctatgacact tccagcaaaa
ggtagggcgg gctgcgagac ggcttcccgg 60cgctgcatgc aacaccgatg atgcttcgac
cccccgaagc tccttcgggg ctgcatgggc 120gctccgatgc cgctccaggg cgagcgctgt
ttaaatagcc aggcccccga ttgcaaagac 180attatagcga gctaccaaag ccatattcaa
acacctagat cactaccact tctacacagg 240ccactcgagc ttgtgatcgc actccgctaa
gggggcgcct cttcctcttc gtttcagtca 300caacccgcaa actctagaat atcaatgatc
gagcaggacg gcctccacgc cggctccccc 360gccgcctggg tggagcgcct gttcggctac
gactgggccc agcagaccat cggctgctcc 420gacgccgccg tgttccgcct gtccgcccag
ggccgccccg tgctgttcgt gaagaccgac 480ctgtccggcg ccctgaacga gctgcaggac
gaggccgccc gcctgtcctg gctggccacc 540accggcgtgc cctgcgccgc cgtgctggac
gtggtgaccg aggccggccg cgactggctg 600ctgctgggcg aggtgcccgg ccaggacctg
ctgtcctccc acctggcccc cgccgagaag 660gtgtccatca tggccgacgc catgcgccgc
ctgcacaccc tggaccccgc cacctgcccc 720ttcgaccacc aggccaagca ccgcatcgag
cgcgcccgca cccgcatgga ggccggcctg 780gtggaccagg acgacctgga cgaggagcac
cagggcctgg cccccgccga gctgttcgcc 840cgcctgaagg cccgcatgcc cgacggcgag
gacctggtgg tgacccacgg cgacgcctgc 900ctgcccaaca tcatggtgga gaacggccgc
ttctccggct tcatcgactg cggccgcctg 960ggcgtggccg accgctacca ggacatcgcc
ctggccaccc gcgacatcgc cgaggagctg 1020ggcggcgagt gggccgaccg cttcctggtg
ctgtacggca tcgccgcccc cgactcccag 1080cgcatcgcct tctaccgcct gctggacgag
ttcttctgac aattggcagc agcagctcgg 1140atagtatcga cacactctgg acgctggtcg
tgtgatggac tgttgccgcc acacttgctg 1200ccttgacctg tgaatatccc tgccgctttt
atcaaacagc ctcagtgtgt ttgatcttgt 1260gtgtacgcgc ttttgcgagt tgctagctgc
ttgtgctatt tgcgaatacc acccccagca 1320tccccttccc tcgtttcata tcgcttgcat
cccaaccgca acttatctac gctgtcctgc 1380tatccctcag cgctgctcct gctcctgctc
actgcccctc gcacagcctt ggtttgggct 1440ccgcctgtat tctcctggta ctgcaacctg
taaaccagca ctgcaatgct gatgcacggg 1500aagtagtggg atgggaacac aaatggagga
tcc 153316310PRTCocos nucifera 16Met Asp
Ala Ser Gly Ala Ser Ser Phe Leu Arg Gly Arg Cys Leu Glu 1 5
10 15 Ser Cys Phe Lys Ala Ser Phe
Gly Tyr Val Met Ser Gln Pro Lys Asp 20 25
30 Ala Ala Gly Gln Pro Ser Arg Arg Pro Ala Asp Ala
Asp Asp Phe Val 35 40 45
Asp Asp Asp Arg Trp Ile Thr Val Ile Leu Ser Val Val Arg Ile Ala
50 55 60 Ala Cys Phe
Leu Ser Met Met Val Thr Thr Ile Val Trp Asn Met Ile 65
70 75 80 Met Leu Ile Leu Leu Pro Trp
Pro Tyr Ala Arg Ile Arg Gln Gly Asn 85
90 95 Leu Tyr Gly His Val Thr Gly Arg Met Leu Met
Trp Ile Leu Gly Asn 100 105
110 Pro Ile Thr Ile Glu Gly Ser Glu Phe Ser Asn Thr Arg Ala Ile
Tyr 115 120 125 Ile
Cys Asn His Ala Ser Leu Val Asp Ile Phe Leu Ile Met Trp Leu 130
135 140 Ile Pro Lys Gly Thr Val
Thr Ile Ala Lys Lys Glu Ile Ile Trp Tyr 145 150
155 160 Pro Leu Phe Gly Gln Leu Tyr Val Leu Ala Asn
His Gln Arg Ile Asp 165 170
175 Arg Ser Asn Pro Ser Ala Ala Ile Glu Ser Ile Lys Glu Val Ala Arg
180 185 190 Ala Val
Val Lys Lys Asn Leu Ser Leu Ile Ile Phe Pro Glu Gly Thr 195
200 205 Arg Ser Lys Thr Gly Arg Leu
Leu Pro Phe Lys Lys Gly Phe Ile His 210 215
220 Ile Ala Leu Gln Thr Arg Leu Pro Ile Val Pro Met
Val Leu Thr Gly 225 230 235
240 Thr His Leu Ala Trp Arg Lys Asn Ser Leu Arg Val Arg Pro Ala Pro
245 250 255 Ile Thr Val
Lys Tyr Phe Ser Pro Ile Lys Thr Asp Asp Trp Glu Glu 260
265 270 Glu Lys Ile Asn His Tyr Val Glu
Met Ile His Ala Leu Tyr Val Asp 275 280
285 His Leu Pro Glu Ser Gln Lys Pro Leu Val Ser Lys Gly
Arg Asp Ala 290 295 300
Ser Gly Arg Ser Asn Ser 305 310 176676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
17gggctggtct gaatccttca ggcgggtgtt acccgagaaa gaaagggtgc cgatttcaaa
60gcagacccat gtgccgggcc ctgtggcctg tgttggcgcc tatgtagtca ccccccctca
120cccaattgtc gccagtttgc gcactccata aactcaaaac agcagcttct gagctgcgct
180gttcaagaac acctctgggg tttgctcacc cgcgaggtcg acgcccagca tggctatcaa
240gacgaacagg cagcctgtgg agaagcctcc gttcacgatc gggacgctgc gcaaggccat
300ccccgcgcac tgtttcgagc gctcggcgct tcgtagcagc atgtacctgg cctttgacat
360cgcggtcatg tccctgctct acgtcgcgtc gacgtacatc gaccctgcac cggtgcctac
420gtgggtcaag tacggcatca tgtggccgct ctactggttc ttccaggtgt gtttgagggt
480tttggttgcc cgtattgagg tcctggtggc gcgcatggag gagaaggcgc ctgtcccgct
540gacccccccg gctaccctcc cggcaccttc cagggcgcct tcggcacggg tgtctgggtg
600tgcgcgcacg agtgcggcca ccaggccttt tcctccagcc aggccatcaa cgacggcgtg
660ggcctggtgt tccacagcct gctgctggtg ccctactact cctggaagca ctcgcaccgg
720gtaccctttc ttgcgctatg acacttccag caaaaggtag ggcgggctgc gagacggctt
780cccggcgctg catgcaacac cgatgatgct tcgacccccc gaagctcctt cggggctgca
840tgggcgctcc gatgccgctc cagggcgagc gctgtttaaa tagccaggcc cccgattgca
900aagacattat agcgagctac caaagccata ttcaaacacc tagatcacta ccacttctac
960acaggccact cgagcttgtg atcgcactcc gctaaggggg cgcctcttcc tcttcgtttc
1020agtcacaacc cgcaaactct agaatatcaa tgctgctgca ggccttcctg ttcctgctgg
1080ccggcttcgc cgccaagatc agcgcctcca tgacgaacga gacgtccgac cgccccctgg
1140tgcacttcac ccccaacaag ggctggatga acgaccccaa cggcctgtgg tacgacgaga
1200aggacgccaa gtggcacctg tacttccagt acaacccgaa cgacaccgtc tgggggacgc
1260ccttgttctg gggccacgcc acgtccgacg acctgaccaa ctgggaggac cagcccatcg
1320ccatcgcccc gaagcgcaac gactccggcg ccttctccgg ctccatggtg gtggactaca
1380acaacacctc cggcttcttc aacgacacca tcgacccgcg ccagcgctgc gtggccatct
1440ggacctacaa caccccggag tccgaggagc agtacatctc ctacagcctg gacggcggct
1500acaccttcac cgagtaccag aagaaccccg tgctggccgc caactccacc cagttccgcg
1560acccgaaggt cttctggtac gagccctccc agaagtggat catgaccgcg gccaagtccc
1620aggactacaa gatcgagatc tactcctccg acgacctgaa gtcctggaag ctggagtccg
1680cgttcgccaa cgagggcttc ctcggctacc agtacgagtg ccccggcctg atcgaggtcc
1740ccaccgagca ggaccccagc aagtcctact gggtgatgtt catctccatc aaccccggcg
1800ccccggccgg cggctccttc aaccagtact tcgtcggcag cttcaacggc acccacttcg
1860aggccttcga caaccagtcc cgcgtggtgg acttcggcaa ggactactac gccctgcaga
1920ccttcttcaa caccgacccg acctacggga gcgccctggg catcgcgtgg gcctccaact
1980gggagtactc cgccttcgtg cccaccaacc cctggcgctc ctccatgtcc ctcgtgcgca
2040agttctccct caacaccgag taccaggcca acccggagac ggagctgatc aacctgaagg
2100ccgagccgat cctgaacatc agcaacgccg gcccctggag ccggttcgcc accaacacca
2160cgttgacgaa ggccaacagc tacaacgtcg acctgtccaa cagcaccggc accctggagt
2220tcgagctggt gtacgccgtc aacaccaccc agacgatctc caagtccgtg ttcgcggacc
2280tctccctctg gttcaagggc ctggaggacc ccgaggagta cctccgcatg ggcttcgagg
2340tgtccgcgtc ctccttcttc ctggaccgcg ggaacagcaa ggtgaagttc gtgaaggaga
2400acccctactt caccaaccgc atgagcgtga acaaccagcc cttcaagagc gagaacgacc
2460tgtcctacta caaggtgtac ggcttgctgg accagaacat cctggagctg tacttcaacg
2520acggcgacgt cgtgtccacc aacacctact tcatgaccac cgggaacgcc ctgggctccg
2580tgaacatgac gacgggggtg gacaacctgt tctacatcga caagttccag gtgcgcgagg
2640tcaagtgaca attggcagca gcagctcgga tagtatcgac acactctgga cgctggtcgt
2700gtgatggact gttgccgcca cacttgctgc cttgacctgt gaatatccct gccgctttta
2760tcaaacagcc tcagtgtgtt tgatcttgtg tgtacgcgct tttgcgagtt gctagctgct
2820tgtgctattt gcgaatacca cccccagcat ccccttccct cgtttcatat cgcttgcatc
2880ccaaccgcaa cttatctacg ctgtcctgct atccctcagc gctgctcctg ctcctgctca
2940ctgcccctcg cacagccttg gtttgggctc cgcctgtatt ctcctggtac tgcaacctgt
3000aaaccagcac tgcaatgctg atgcacggga agtagtggga tgggaacaca aatggaggat
3060cccgcgtctc gaacagagcg cgcagaggaa cgctgaaggt ctcgcctctg tcgcacctca
3120gcgcggcata caccacaata accacctgac gaatgcgctt ggttcttcgt ccattagcga
3180agcgtccggt tcacacacgt gccacgttgg cgaggtggca ggtgacaatg atcggtggag
3240ctgatggtcg aaacgttcac agcctaggga tatcgaattc ggccgacagg acgcgcgtca
3300aaggtgctgg tcgtgtatgc cctggccggc aggtcgttgc tgctgctggt tagtgattcc
3360gcaaccctga ttttggcgtc ttattttggc gtggcaaacg ctggcgcccg cgagccgggc
3420cggcggcgat gcggtgcccc acggctgccg gaatccaagg gaggcaagag cgcccgggtc
3480agttgaaggg ctttacgcgc aaggtacagc cgctcctgca aggctgcgtg gtggaattgg
3540acgtgcaggt cctgctgaag ttcctccacc gcctcaccag cggacaaagc accggtgtat
3600caggtccgtg tcatccactc taaagaactc gactacgacc tactgatggc cctagattct
3660tcatcaaaaa cgcctgagac acttgcccag gattgaaact ccctgaaggg accaccaggg
3720gccctgagtt gttccttccc cccgtggcga gctgccagcc aggctgtacc tgtgatcgag
3780gctggcggga aaataggctt cgtgtgctca ggtcatggga ggtgcaggac agctcatgaa
3840acgccaacaa tcgcacaatt catgtcaagc taatcagcta tttcctcttc acgagctgta
3900attgtcccaa aattctggtc taccgggggt gatccttcgt gtacgggccc ttccctcaac
3960cctaggtatg cgcgcatgcg gtcgccgcgc aactcgcgcg agggccgagg gtttgggacg
4020ggccgtcccg aaatgcagtt gcacccggat gcgtggcacc ttttttgcga taatttatgc
4080aatggactgc tctgcaaaat tctggctctg tcgccaaccc taggatcagc ggcgtaggat
4140ttcgtaatca ttcgtcctga tggggagcta ccgactaccc taatatcagc ccgactgcct
4200gacgccagcg tccacttttg tgcacacatt ccattcgtgc ccaagacatt tcattgtggt
4260gcgaagcgtc cccagttacg ctcacctgtt tcccgacctc cttactgttc tgtcgacaga
4320gcgggcccac aggccggtcg cagccactag tatggccacc gcatccactt tctcggcgtt
4380caatgcccgc tgcggcgacc tgcgtcgctc ggcgggctcc gggccccggc gcccagcgag
4440gcccctcccc gtgcgcgggc gcgccgccac cggcgagcag ccctccggcg tggcctccct
4500gcgcgaggcc gacaaggaga agtccctggg caaccgcctg cgcctgggct ccctgaccga
4560ggacggcctg tcctacaagg agaagttcgt gatccgctgc tacgaggtgg gcatcaacaa
4620gaccgccacc atcgagacca tcgccaacct gctgcaggag gtgggcggca accacgccca
4680gggcgtgggc ttctccaccg acggcttcgc caccaccacc accatgcgca agctgcacct
4740gatctgggtg accgcccgca tgcacatcga gatctaccgc taccccgcct ggtccgacgt
4800gatcgagatc gagacctggg tgcagggcga gggcaaggtg ggcacccgcc gcgactggat
4860cctgaaggac tacgccaacg gcgaggtgat cggccgcgcc acctccaagt gggtgatgat
4920gaacgaggac acccgccgcc tgcagaaggt gtccgacgac gtgcgcgagg agtacctggt
4980gttctgcccc cgcaccctgc gcctggcctt ccccgaggag aacaacaact ccatgaagaa
5040gatccccaag ctggaggacc ccgccgagta ctcccgcctg ggcctggtgc cccgccgctc
5100cgacctggac atgaacaagc acgtgaacaa cgtgacctac atcggctggg ccctggagtc
5160catccccccc gagatcatcg acacccacga gctgcaggcc atcaccctgg actaccgccg
5220cgagtgccag cgcgacgaca tcgtggactc cctgacctcc cgcgagcccc tgggcaacgc
5280cgccggcgtg aagttcaagg agatcaacgg ctccgtgtcc cccaagaagg acgagcagga
5340cctgtcccgc ttcatgcacc tgctgcgctc cgccggctcc ggcctggaga tcaaccgctg
5400ccgcaccgag tggcgcaaga agcccgccaa gcgcatggac tacaaggacc acgacggcga
5460ctacaaggac cacgacatcg actacaagga cgacgacgac aagtgaatcg atagatctct
5520taaggcagca gcagctcgga tagtatcgac acactctgga cgctggtcgt gtgatggact
5580gttgccgcca cacttgctgc cttgacctgt gaatatccct gccgctttta tcaaacagcc
5640tcagtgtgtt tgatcttgtg tgtacgcgct tttgcgagtt gctagctgct tgtgctattt
5700gcgaatacca cccccagcat ccccttccct cgtttcatat cgcttgcatc ccaaccgcaa
5760cttatctacg ctgtcctgct atccctcagc gctgctcctg ctcctgctca ctgcccctcg
5820cacagccttg gtttgggctc cgcctgtatt ctcctggtac tgcaacctgt aaaccagcac
5880tgcaatgctg atgcacggga agtagtggga tgggaacaca aatggaaagc ttaattaaga
5940gctcccgcca ccactccaac acggggtgcc tggacaagga cgaggtgttt gtgccgccgc
6000accgcgcagt ggcgcacgag ggcctggagt gggaggagtg gctgcccatc cgcatgggca
6060aggtgctggt caccctgacc ctgggctggc cgctgtacct catgttcaac gtcgcctcgc
6120ggccgtaccc gcgcttcgcc aaccactttg acccgtggtc gcccatcttc agcaagcgcg
6180agcgcatcga ggtggtcatc tccgacctgg cgctggtggc ggtgctcagc gggctcagcg
6240tgctgggccg caccatgggc tgggcctggc tggtcaagac ctacgtggtg ccctacctga
6300tcgtgaacat gtggctcgtg ctcatcacgc tgctccagca cacgcacccg gcgctgccgc
6360actacttcga gaaggactgg gactggctgc gcggcgccat ggccaccgtg gaccgctcca
6420tgggcccgcc cttcatggac aacatcctgc accacatctc cgacacccac gtgctgcacc
6480acctcttcag caccatcccg cactaccacg ccgaggaggc ctccgccgcc atcaggccca
6540tcctgggcaa gtactaccag tccgacagcc gctgggtcgg ccgcgccctg tgggaggact
6600ggcgcgactg ccgctacgtc gtcccggacg cgcccgagga cgactccgcg ctctggttcc
6660acaagtgagt gagtga
667618719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 18gggctggtct gaatccttca ggcgggtgtt
acccgagaaa gaaagggtgc cgatttcaaa 60gcagacccat gtgccgggcc ctgtggcctg
tgttggcgcc tatgtagtca ccccccctca 120cccaattgtc gccagtttgc gcactccata
aactcaaaac agcagcttct gagctgcgct 180gttcaagaac acctctgggg tttgctcacc
cgcgaggtcg acgcccagca tggctatcaa 240gacgaacagg cagcctgtgg agaagcctcc
gttcacgatc gggacgctgc gcaaggccat 300ccccgcgcac tgtttcgagc gctcggcgct
tcgtagcagc atgtacctgg cctttgacat 360cgcggtcatg tccctgctct acgtcgcgtc
gacgtacatc gaccctgcac cggtgcctac 420gtgggtcaag tacggcatca tgtggccgct
ctactggttc ttccaggtgt gtttgagggt 480tttggttgcc cgtattgagg tcctggtggc
gcgcatggag gagaaggcgc ctgtcccgct 540gacccccccg gctaccctcc cggcaccttc
cagggcgcct tcggcacggg tgtctgggtg 600tgcgcgcacg agtgcggcca ccaggccttt
tcctccagcc aggccatcaa cgacggcgtg 660ggcctggtgt tccacagcct gctgctggtg
ccctactact cctggaagca ctcgcaccg 71919732DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
19ccgccaccac tccaacacgg ggtgcctgga caaggacgag gtgtttgtgc cgccgcaccg
60cgcagtggcg cacgagggcc tggagtggga ggagtggctg cccatccgca tgggcaaggt
120gctggtcacc ctgaccctgg gctggccgct gtacctcatg ttcaacgtcg cctcgcggcc
180gtacccgcgc ttcgccaacc actttgaccc gtggtcgccc atcttcagca agcgcgagcg
240catcgaggtg gtcatctccg acctggcgct ggtggcggtg ctcagcgggc tcagcgtgct
300gggccgcacc atgggctggg cctggctggt caagacctac gtggtgccct acctgatcgt
360gaacatgtgg ctcgtgctca tcacgctgct ccagcacacg cacccggcgc tgccgcacta
420cttcgagaag gactgggact ggctgcgcgg cgccatggcc accgtggacc gctccatggg
480cccgcccttc atggacaaca tcctgcacca catctccgac acccacgtgc tgcaccacct
540cttcagcacc atcccgcact accacgccga ggaggcctcc gccgccatca ggcccatcct
600gggcaagtac taccagtccg acagccgctg ggtcggccgc gccctgtggg aggactggcg
660cgactgccgc tacgtcgtcc cggacgcgcc cgaggacgac tccgcgctct ggttccacaa
720gtgagtgagt ga
73220612DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 20gctcttcgga gtcactgtgc cactgagttc
gactggtagc tgaatggagt cgctgctcca 60ctaaacgaat tgtcagcacc gccagccggc
cgaggacccg agtcatagcg agggtagtag 120cgcgccatgg caccgaccag cctgcttgcc
agtactggcg tctcttccgc ttctctgtgg 180tcctctgcgc gctccagcgc gtgcgctttt
ccggtggatc atgcggtccg tggcgcaccg 240cagcggccgc tgcccatgca gcgccgctgc
ttccgaacag tggcggtcag ggccgcaccc 300gcggtagccg tccgtccgga acccgcccaa
gagttttggg agcagcttga gccctgcaag 360atggcggagg acaagcgcat cttcctggag
gagcaccggt gcgtggaggt ccggggctga 420ccggccgtcg cattcaacgt aatcaatcgc
atgatgatca gaggacacga agtcttggtg 480gcggtggcca gaaacactgt ccattgcaag
ggcataggga tgcgttcctt cacctctcat 540ttctcatttc tgaatccctc cctgctcact
ctttctcctc ctccttcccg ttcacgcagc 600attcggggta cc
61221528DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
21gacagggtgg ttggctggat ggggaaacgc tggtcgcggg attcgatcct gctgcttata
60tcctccctgg aagcacaccc acgactctga agaagaaaac gtgcacacac acaacccaac
120cggccgaata tttgcttcct tatcccgggt ccaagagaga ctgcgatgcc cccctcaatc
180agcatcctcc tccctgccgc ttcaatcttc cctgcttgcc tgcgcccgcg gtgcgccgtc
240tgcccgccca gtcagtcact cctgcacagg ccccttgtgc gcagtgctcc tgtacccttt
300accgctcctt ccattctgcg aggcccccta ttgaatgtat tcgttgcctg tgtggccaag
360cgggctgctg ggcgcgccgc cgtcgggcag tgctcggcga ctttggcgga agccgattgt
420tcttctgtaa gccacgcgct tgctgctttg ggaagagaag ggggggggta ctgaatggat
480gaggaggaga aggaggggta ttggtattat ctgagttggg tgaagagc
528221174DNAChlorella protothecoides 22agtttaggtc cagcgtccgt ggggggggac
gggctgggag cttgggccgg gaagggcaag 60acgatgcagt ccctctgggg agtcacagcc
gactgtgtgt gttgcactgt gcggcccgca 120gcactcacac gcaaaatgcc tggccgacag
gcaggccctg tccagtgcaa catccacggt 180ccctctcatc aggctcacct tgctcattga
cataacggaa tgcgtaccgc tctttcagat 240ctgtccatcc agagagggga gcaggctccc
caccgacgct gtcaaacttg cttcctgccc 300aaccgaaaac attattgttt gagggggggg
gggggggggc agattgcatg gcgggatatc 360tcgtgaggaa catcactggg acactgtgga
acacagtgag tgcagtatgc agagcatgta 420tgctaggggt cagcgcagga agggggcctt
tcccagtctc ccatgccact gcaccgtatc 480cacgactcac caggaccagc ttcttgatcg
gcttccgctc ccgtggacac cagtgtgtag 540cctctggact ccaggtatgc gtgcaccgca
aaggccagcc gatcgtgccg attcctgggg 600tggaggatat gagtcagcca acttggggct
cagagtgcac actggggcac gatacgaaac 660aacatctaca ccgtgtcctc catgctgaca
caccacagct tcgctccacc tgaatgtggg 720cgcatgggcc cgaatcacag ccaatgtcgc
tgctgccata atgtgatcca gaccctctcc 780gcccagatgc cgagcggatc gtgggcgctg
aatagattcc tgtttcgatc actgtttggg 840tcctttcctt ttcgtctcgg atgcgcgtct
cgaaacaggc tgcgtcgggc tttcggatcc 900cttttgctcc ctccgtcacc atcctgcgcg
cgggcaagtt gcttgaccct gggctgtacc 960agggttggag ggtattaccg cgtcaggcca
ttcccagccc ggattcaatt caaagtctgg 1020gccaccaccc tccgccgctc tgtctgatca
ctccacattc gtgcatacac tacgttcaag 1080tcctgatcca ggcgtgtctc gggacaaggt
gtgcttgagt ttgaatctca aggacccact 1140ccagcacagc tgctggttga ccccgccctc
gcaa 1174233529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
23agtttaggtc cagcgtccgt ggggggggac gggctgggag cttgggccgg gaagggcaag
60acgatgcagt ccctctgggg agtcacagcc gactgtgtgt gttgcactgt gcggcccgca
120gcactcacac gcaaaatgcc tggccgacag gcaggccctg tccagtgcaa catccacggt
180ccctctcatc aggctcacct tgctcattga cataacggaa tgcgtaccgc tctttcagat
240ctgtccatcc agagagggga gcaggctccc caccgacgct gtcaaacttg cttcctgccc
300aaccgaaaac attattgttt gagggggggg gggggggggc agattgcatg gcgggatatc
360tcgtgaggaa catcactggg acactgtgga acacagtgag tgcagtatgc agagcatgta
420tgctaggggt cagcgcagga agggggcctt tcccagtctc ccatgccact gcaccgtatc
480cacgactcac caggaccagc ttcttgatcg gcttccgctc ccgtggacac cagtgtgtag
540cctctggact ccaggtatgc gtgcaccgca aaggccagcc gatcgtgccg attcctgggg
600tggaggatat gagtcagcca acttggggct cagagtgcac actggggcac gatacgaaac
660aacatctaca ccgtgtcctc catgctgaca caccacagct tcgctccacc tgaatgtggg
720cgcatgggcc cgaatcacag ccaatgtcgc tgctgccata atgtgatcca gaccctctcc
780gcccagatgc cgagcggatc gtgggcgctg aatagattcc tgtttcgatc actgtttggg
840tcctttcctt ttcgtctcgg atgcgcgtct cgaaacaggc tgcgtcgggc tttcggatcc
900cttttgctcc ctccgtcacc atcctgcgcg cgggcaagtt gcttgaccct gggctgtacc
960agggttggag ggtattaccg cgtcaggcca ttcccagccc ggattcaatt caaagtctgg
1020gccaccaccc tccgccgctc tgtctgatca ctccacattc gtgcatacac tacgttcaag
1080tcctgatcca ggcgtgtctc gggacaaggt gtgcttgagt ttgaatctca aggacccact
1140ccagcacagc tgctggttga ccccgccctc gcaatctaga atggccgcgt ccgtccactg
1200caccctgatg tccgtggtct gcaacaacaa gaaccactcc gcccgcccca agctgcccaa
1260ctcctccctg ctgcccggct tcgacgtggt ggtccaggcc gcggccaccc gcttcaagaa
1320ggagacgacg accacccgcg ccacgctgac gttcgacccc cccacgacca actccgagcg
1380cgccaagcag cgcaagcaca ccatcgaccc ctcctccccc gacttccagc ccatcccctc
1440cttcgaggag tgcttcccca agtccacgaa ggagcacaag gaggtggtgc acgaggagtc
1500cggccacgtc ctgaaggtgc ccttccgccg cgtgcacctg tccggcggcg agcccgcctt
1560cgacaactac gacacgtccg gcccccagaa cgtcaacgcc cacatcggcc tggcgaagct
1620gcgcaaggag tggatcgacc gccgcgagaa gctgggcacg ccccgctaca cgcagatgta
1680ctacgcgaag cagggcatca tcacggagga gatgctgtac tgcgcgacgc gcgagaagct
1740ggaccccgag ttcgtccgct ccgaggtcgc gcggggccgc gccatcatcc cctccaacaa
1800gaagcacctg gagctggagc ccatgatcgt gggccgcaag ttcctggtga aggtgaacgc
1860gaacatcggc aactccgccg tggcctcctc catcgaggag gaggtctaca aggtgcagtg
1920ggccaccatg tggggcgccg acaccatcat ggacctgtcc acgggccgcc acatccacga
1980gacgcgcgag tggatcctgc gcaactccgc ggtccccgtg ggcaccgtcc ccatctacca
2040ggcgctggag aaggtggacg gcatcgcgga gaacctgaac tgggaggtgt tccgcgagac
2100gctgatcgag caggccgagc agggcgtgga ctacttcacg atccacgcgg gcgtgctgct
2160gcgctacatc cccctgaccg ccaagcgcct gacgggcatc gtgtcccgcg gcggctccat
2220ccacgcgaag tggtgcctgg cctaccacaa ggagaacttc gcctacgagc actgggacga
2280catcctggac atctgcaacc agtacgacgt cgccctgtcc atcggcgacg gcctgcgccc
2340cggctccatc tacgacgcca acgacacggc ccagttcgcc gagctgctga cccagggcga
2400gctgacgcgc cgcgcgtggg agaaggacgt gcaggtgatg aacgagggcc ccggccacgt
2460gcccatgcac aagatccccg agaacatgca gaagcagctg gagtggtgca acgaggcgcc
2520cttctacacc ctgggccccc tgacgaccga catcgcgccc ggctacgacc acatcacctc
2580cgccatcggc gcggccaaca tcggcgccct gggcaccgcc ctgctgtgct acgtgacgcc
2640caaggagcac ctgggcctgc ccaaccgcga cgacgtgaag gcgggcgtca tcgcctacaa
2700gatcgccgcc cacgcggccg acctggccaa gcagcacccc cacgcccagg cgtgggacga
2760cgcgctgtcc aaggcgcgct tcgagttccg ctggatggac cagttcgcgc tgtccctgga
2820ccccatgacg gcgatgtcct tccacgacga gacgctgccc gcggacggcg cgaaggtcgc
2880ccacttctgc tccatgtgcg gccccaagtt ctgctccatg aagatcacgg aggacatccg
2940caagtacgcc gaggagaacg gctacggctc cgccgaggag gccatccgcc agggcatgga
3000cgccatgtcc gaggagttca acatcgccaa gaagacgatc tccggcgagc agcacggcga
3060ggtcggcggc gagatctacc tgcccgagtc ctacgtcaag gccgcgcaga agtgacaatt
3120ggcagcagca gctcggatag tatcgacaca ctctggacgc tggtcgtgtg atggactgtt
3180gccgccacac ttgctgcctt gacctgtgaa tatccctgcc gcttttatca aacagcctca
3240gtgtgtttga tcttgtgtgt acgcgctttt gcgagttgct agctgcttgt gctatttgcg
3300aataccaccc ccagcatccc cttccctcgt ttcatatcgc ttgcatccca accgcaactt
3360atctacgctg tcctgctatc cctcagcgct gctcctgctc ctgctcactg cccctcgcac
3420agccttggtt tgggctccgc ctgtattctc ctggtactgc aacctgtaaa ccagcactgc
3480aatgctgatg cacgggaagt agtgggatgg gaacacaaat ggaggatcc
3529241476DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 24atggccaccg catccacttt ctcggcgttc
aatgcccgct gcggcgacct gcgtcgctcg 60gcgggctccg ggccccggcg cccagcgagg
cccctccccg tgcgcgggcg cgccgccgcc 120gccgccgacg ccaaccccgc ccgccccgag
cgccgcgtgg tgatcaccgg ccagggcgtg 180gtgacctccc tgggccagac catcgagcag
ttctactcct ccctgctgga gggcgtgtcc 240ggcatctccc agatccagaa gttcgacacc
accggctaca ccaccaccat cgccggcgag 300atcaagtccc tgcagctgga cccctacgtg
cccaagcgct gggccaagcg cgtggacgac 360gtgatcaagt acgtgtacat cgccggcaag
caggccctgg agtccgccgg cctgcccatc 420gaggccgccg gcctggccgg cgccggcctg
gaccccgccc tgtgcggcgt gctgatcggc 480accgccatgg ccggcatgac ctccttcgcc
gccggcgtgg aggccctgac ccgcggcggc 540gtgcgcaaga tgaacccctt ctgcatcccc
ttctccatct ccaacatggg cggcgccatg 600ctggccatgg acatcggctt catgggcccc
aactactcca tctccaccgc ctgcgccacc 660ggcaactact gcatcctggg cgccgccgac
cacatccgcc gcggcgacgc caacgtgatg 720ctggccggcg gcgccgacgc cgccatcatc
ccctccggca tcggcggctt catcgcctgc 780aaggccctgt ccaagcgcaa cgacgagccc
gagcgcgcct cccgcccctg ggacgccgac 840cgcgacggct tcgtgatggg cgagggcgcc
ggcgtgctgg tgctggagga gctggagcac 900gccaagcgcc gcggcgccac catcctggcc
gagctggtgg gcggcgccgc cacctccgac 960gcccaccaca tgaccgagcc cgacccccag
ggccgcggcg tgcgcctgtg cctggagcgc 1020gccctggagc gcgcccgcct ggcccccgag
cgcgtgggct acgtgaacgc ccacggcacc 1080tccacccccg ccggcgacgt ggccgagtac
cgcgccatcc gcgccgtgat cccccaggac 1140tccctgcgca tcaactccac caagtccatg
atcggccacc tgctgggcgg cgccggcgcc 1200gtggaggccg tggccgccat ccaggccctg
cgcaccggct ggctgcaccc caacctgaac 1260ctggagaacc ccgcccccgg cgtggacccc
gtggtgctgg tgggcccccg caaggagcgc 1320gccgaggacc tggacgtggt gctgtccaac
tccttcggct tcggcggcca caactcctgc 1380gtgatcttcc gcaagtacga cgagatggac
tacaaggacc acgacggcga ctacaaggac 1440cacgacatcg actacaagga cgacgacgac
aagtga 147625491PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Met Ala Thr Ala Ser Thr Phe Ser Ala Phe Asn Ala Arg Cys Gly Asp 1
5 10 15 Leu Arg Arg Ser
Ala Gly Ser Gly Pro Arg Arg Pro Ala Arg Pro Leu 20
25 30 Pro Val Arg Gly Arg Ala Ala Ala Ala
Ala Asp Ala Asn Pro Ala Arg 35 40
45 Pro Glu Arg Arg Val Val Ile Thr Gly Gln Gly Val Val Thr
Ser Leu 50 55 60
Gly Gln Thr Ile Glu Gln Phe Tyr Ser Ser Leu Leu Glu Gly Val Ser 65
70 75 80 Gly Ile Ser Gln Ile
Gln Lys Phe Asp Thr Thr Gly Tyr Thr Thr Thr 85
90 95 Ile Ala Gly Glu Ile Lys Ser Leu Gln Leu
Asp Pro Tyr Val Pro Lys 100 105
110 Arg Trp Ala Lys Arg Val Asp Asp Val Ile Lys Tyr Val Tyr Ile
Ala 115 120 125 Gly
Lys Gln Ala Leu Glu Ser Ala Gly Leu Pro Ile Glu Ala Ala Gly 130
135 140 Leu Ala Gly Ala Gly Leu
Asp Pro Ala Leu Cys Gly Val Leu Ile Gly 145 150
155 160 Thr Ala Met Ala Gly Met Thr Ser Phe Ala Ala
Gly Val Glu Ala Leu 165 170
175 Thr Arg Gly Gly Val Arg Lys Met Asn Pro Phe Cys Ile Pro Phe Ser
180 185 190 Ile Ser
Asn Met Gly Gly Ala Met Leu Ala Met Asp Ile Gly Phe Met 195
200 205 Gly Pro Asn Tyr Ser Ile Ser
Thr Ala Cys Ala Thr Gly Asn Tyr Cys 210 215
220 Ile Leu Gly Ala Ala Asp His Ile Arg Arg Gly Asp
Ala Asn Val Met 225 230 235
240 Leu Ala Gly Gly Ala Asp Ala Ala Ile Ile Pro Ser Gly Ile Gly Gly
245 250 255 Phe Ile Ala
Cys Lys Ala Leu Ser Lys Arg Asn Asp Glu Pro Glu Arg 260
265 270 Ala Ser Arg Pro Trp Asp Ala Asp
Arg Asp Gly Phe Val Met Gly Glu 275 280
285 Gly Ala Gly Val Leu Val Leu Glu Glu Leu Glu His Ala
Lys Arg Arg 290 295 300
Gly Ala Thr Ile Leu Ala Glu Leu Val Gly Gly Ala Ala Thr Ser Asp 305
310 315 320 Ala His His Met
Thr Glu Pro Asp Pro Gln Gly Arg Gly Val Arg Leu 325
330 335 Cys Leu Glu Arg Ala Leu Glu Arg Ala
Arg Leu Ala Pro Glu Arg Val 340 345
350 Gly Tyr Val Asn Ala His Gly Thr Ser Thr Pro Ala Gly Asp
Val Ala 355 360 365
Glu Tyr Arg Ala Ile Arg Ala Val Ile Pro Gln Asp Ser Leu Arg Ile 370
375 380 Asn Ser Thr Lys Ser
Met Ile Gly His Leu Leu Gly Gly Ala Gly Ala 385 390
395 400 Val Glu Ala Val Ala Ala Ile Gln Ala Leu
Arg Thr Gly Trp Leu His 405 410
415 Pro Asn Leu Asn Leu Glu Asn Pro Ala Pro Gly Val Asp Pro Val
Val 420 425 430 Leu
Val Gly Pro Arg Lys Glu Arg Ala Glu Asp Leu Asp Val Val Leu 435
440 445 Ser Asn Ser Phe Gly Phe
Gly Gly His Asn Ser Cys Val Ile Phe Arg 450 455
460 Lys Tyr Asp Glu Met Asp Tyr Lys Asp His Asp
Gly Asp Tyr Lys Asp 465 470 475
480 His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys 485
490 261107DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 26atggccatca agaccaaccg
ccagcccgtg gagaagcccc ccttcaccat cggcaccctg 60cgcaaggcca tccccgccca
ctgcttcgag cgctccgccc tgcgctcctc catgtacctg 120gccttcgaca tcgccgtgat
gtccctgctg tacgtggcct ccacctacat cgaccccgcc 180cccgtgccca cctgggtgaa
gtacggcgtg atgtggcccc tgtactggtt cttccagggc 240gccttcggca ccggcgtgtg
ggtgtgcgcc cacgagtgcg gccaccaggc cttctcctcc 300tcccaggcca tcaacgacgg
cgtgggcctg gtgttccact ccctgctgct ggtgccctac 360tactcctgga agcactccca
ccgccgccac cactccaaca ccggctgcct ggacaaggac 420gaggtgttcg tgccccccca
ccgcgccgtg gcccacgagg gcctggagtg ggaggagtgg 480ctgcccatcc gcatgggcaa
ggtgctggtg accctgaccc tgggctggcc cctgtacctg 540atgttcaacg tggcctcccg
cccctacccc cgcttcgcca accacttcga cccctggtcc 600cccatcttct ccaagcgcga
gcgcatcgag gtggtgatct ccgacctggc cctggtggcc 660gtgctgtccg gcctgtccgt
gctgggccgc accatgggct gggcctggct ggtgaagacc 720tacgtggtgc cctacctgat
cgtgaacatg tggctggtgc tgatcaccct gctgcagcac 780acccaccccg ccctgcccca
ctacttcgag aaggactggg actggctgcg cggcgccatg 840gccaccgtgg accgctccat
gggccccccc ttcatggaca acatcctgca ccacatctcc 900gacacccacg tgctgcacca
cctgttctcc accatccccc actaccacgc cgaggaggcc 960tccgccgcca tccgccccat
cctgggcaag tactaccagt ccgactcccg ctgggtgggc 1020cgcgccctgt gggaggactg
gcgcgactgc cgctacgtgg tgcccgacgc ccccgaggac 1080gactccgccc tgtggttcca
caagtag 110727368PRTPrototheca
moriformis 27Met Ala Ile Lys Thr Asn Arg Gln Pro Val Glu Lys Pro Pro Phe
Thr 1 5 10 15 Ile
Gly Thr Leu Arg Lys Ala Ile Pro Ala His Cys Phe Glu Arg Ser
20 25 30 Ala Leu Arg Ser Ser
Met Tyr Leu Ala Phe Asp Ile Ala Val Met Ser 35
40 45 Leu Leu Tyr Val Ala Ser Thr Tyr Ile
Asp Pro Ala Pro Val Pro Thr 50 55
60 Trp Val Lys Tyr Gly Val Met Trp Pro Leu Tyr Trp Phe
Phe Gln Gly 65 70 75
80 Ala Phe Gly Thr Gly Val Trp Val Cys Ala His Glu Cys Gly His Gln
85 90 95 Ala Phe Ser Ser
Ser Gln Ala Ile Asn Asp Gly Val Gly Leu Val Phe 100
105 110 His Ser Leu Leu Leu Val Pro Tyr Tyr
Ser Trp Lys His Ser His Arg 115 120
125 Arg His His Ser Asn Thr Gly Cys Leu Asp Lys Asp Glu Val
Phe Val 130 135 140
Pro Pro His Arg Ala Val Ala His Glu Gly Leu Glu Trp Glu Glu Trp 145
150 155 160 Leu Pro Ile Arg Met
Gly Lys Val Leu Val Thr Leu Thr Leu Gly Trp 165
170 175 Pro Leu Tyr Leu Met Phe Asn Val Ala Ser
Arg Pro Tyr Pro Arg Phe 180 185
190 Ala Asn His Phe Asp Pro Trp Ser Pro Ile Phe Ser Lys Arg Glu
Arg 195 200 205 Ile
Glu Val Val Ile Ser Asp Leu Ala Leu Val Ala Val Leu Ser Gly 210
215 220 Leu Ser Val Leu Gly Arg
Thr Met Gly Trp Ala Trp Leu Val Lys Thr 225 230
235 240 Tyr Val Val Pro Tyr Leu Ile Val Asn Met Trp
Leu Val Leu Ile Thr 245 250
255 Leu Leu Gln His Thr His Pro Ala Leu Pro His Tyr Phe Glu Lys Asp
260 265 270 Trp Asp
Trp Leu Arg Gly Ala Met Ala Thr Val Asp Arg Ser Met Gly 275
280 285 Pro Pro Phe Met Asp Asn Ile
Leu His His Ile Ser Asp Thr His Val 290 295
300 Leu His His Leu Phe Ser Thr Ile Pro His Tyr His
Ala Glu Glu Ala 305 310 315
320 Ser Ala Ala Ile Arg Pro Ile Leu Gly Lys Tyr Tyr Gln Ser Asp Ser
325 330 335 Arg Trp Val
Gly Arg Ala Leu Trp Glu Asp Trp Arg Asp Cys Arg Tyr 340
345 350 Val Val Pro Asp Ala Pro Glu Asp
Asp Ser Ala Leu Trp Phe His Lys 355 360
365 281191DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 28actagtatgc tgaagctgtc
ctgcaacgtg accaacaacc tgcacacctt ctccttcttc 60tccgactcct ccctgttcat
ccccgtgaac cgccgcacca tcgccgtgtc ctccgggcgc 120gcctcccagc tgcgcaagcc
cgccctggac cccctgcgcg ccgtgatctc cgccgaccag 180ggctccatct cccccgtgaa
ctcctgcacc cccgccgacc gcctgcgcgc cggccgcctg 240atggaggacg gctactccta
caaggagaag ttcatcgtgc gctcctacga ggtgggcatc 300aacaagaccg ccaccgtgga
gaccatcgcc aacctgctgc aggaggtggc ctgcaaccac 360gtgcagaagt gcggcttctc
caccgacggc ttcgccacca ccctgaccat gcgcaagctg 420cacctgatct gggtgaccgc
ccgcatgcac atcgagatct acaagtaccc cgcctggtcc 480gacgtggtgg agatcgagac
ctggtgccag tccgagggcc gcatcggcac ccgccgcgac 540tggatcctgc gcgactccgc
caccaacgag gtgatcggcc gcgccacctc caagtgggtg 600atgatgaacc aggacacccg
ccgcctgcag cgcgtgaccg acgaggtgcg cgacgagtac 660ctggtgttct gcccccgcga
gccccgcctg gccttccccg aggagaacaa ctcctccctg 720aagaagatcc ccaagctgga
ggaccccgcc cagtactcca tgctggagct gaagccccgc 780cgcgccgacc tggacatgaa
ccagcacgtg aacaacgtga cctacatcgg ctgggtgctg 840gagtccatcc cccaggagat
catcgacacc cacgagctgc aggtgatcac cctggactac 900cgccgcgagt gccagcagga
cgacatcgtg gactccctga ccacctccga gatccccgac 960gaccccatct ccaagttcac
cggcaccaac ggctccgcca tgtcctccat ccagggccac 1020aacgagtccc agttcctgca
catgctgcgc ctgtccgaga acggccagga gatcaaccgc 1080ggccgcaccc agtggcgcaa
gaagtcctcc cgcatggact acaaggacca cgacggcgac 1140tacaaggacc acgacatcga
ctacaaggac gacgacgaca agtgaatcga t 119129366PRTBrassica napus
29Met Leu Lys Leu Ser Cys Asn Val Thr Asn Asn Leu His Thr Phe Ser 1
5 10 15 Phe Phe Ser Asp
Ser Ser Leu Phe Ile Pro Val Asn Arg Arg Thr Ile 20
25 30 Ala Val Ser Ser Ser Gln Leu Arg Lys
Pro Ala Leu Asp Pro Leu Arg 35 40
45 Ala Val Ile Ser Ala Asp Gln Gly Ser Ile Ser Pro Val Asn
Ser Cys 50 55 60
Thr Pro Ala Asp Arg Leu Arg Ala Gly Arg Leu Met Glu Asp Gly Tyr 65
70 75 80 Ser Tyr Lys Glu Lys
Phe Ile Val Arg Ser Tyr Glu Val Gly Ile Asn 85
90 95 Lys Thr Ala Thr Val Glu Thr Ile Ala Asn
Leu Leu Gln Glu Val Ala 100 105
110 Cys Asn His Val Gln Lys Cys Gly Phe Ser Thr Asp Gly Phe Ala
Thr 115 120 125 Thr
Leu Thr Met Arg Lys Leu His Leu Ile Trp Val Thr Ala Arg Met 130
135 140 His Ile Glu Ile Tyr Lys
Tyr Pro Ala Trp Ser Asp Val Val Glu Ile 145 150
155 160 Glu Thr Trp Cys Gln Ser Glu Gly Arg Ile Gly
Thr Arg Arg Asp Trp 165 170
175 Ile Leu Arg Asp Ser Ala Thr Asn Glu Val Ile Gly Arg Ala Thr Ser
180 185 190 Lys Trp
Val Met Met Asn Gln Asp Thr Arg Arg Leu Gln Arg Val Thr 195
200 205 Asp Glu Val Arg Asp Glu Tyr
Leu Val Phe Cys Pro Arg Glu Pro Arg 210 215
220 Leu Ala Phe Pro Glu Glu Asn Asn Ser Ser Leu Lys
Lys Ile Pro Lys 225 230 235
240 Leu Glu Asp Pro Ala Gln Tyr Ser Met Leu Glu Leu Lys Pro Arg Arg
245 250 255 Ala Asp Leu
Asp Met Asn Gln His Val Asn Asn Val Thr Tyr Ile Gly 260
265 270 Trp Val Leu Glu Ser Ile Pro Gln
Glu Ile Ile Asp Thr His Glu Leu 275 280
285 Gln Val Ile Thr Leu Asp Tyr Arg Arg Glu Cys Gln Gln
Asp Asp Ile 290 295 300
Val Asp Ser Leu Thr Thr Ser Glu Ile Pro Asp Asp Pro Ile Ser Lys 305
310 315 320 Phe Thr Gly Thr
Asn Gly Ser Ala Met Ser Ser Ile Gln Gly His Asn 325
330 335 Glu Ser Gln Phe Leu His Met Leu Arg
Leu Ser Glu Asn Gly Gln Glu 340 345
350 Ile Asn Arg Gly Arg Thr Gln Trp Arg Lys Lys Ser Ser Arg
355 360 365
30599DNAPrototheca moriformis 30ggagtcactg tgccactgag ttcgactggt
agctgaatgg agtcgctgct ccactaaacg 60aattgtcagc accgccagcc ggccgaggac
ccgagtcata gcgagggtag tagcgcgcca 120tggcaccgac cagcctgctt gccagtactg
gcgtctcttc cgcttctctg tggtcctctg 180cgcgctccag cgcgtgcgct tttccggtgg
atcatgcggt ccgtggcgca ccgcagcggc 240cgctgcccat gcagcgccgc tgcttccgaa
cagtggcggt cagggccgca cccgcggtag 300ccgtccgtcc ggaacccgcc caagagtttt
gggagcagct tgagccctgc aagatggcgg 360aggacaagcg catcttcctg gaggagcacc
ggtgcgtgga ggtccggggc tgaccggccg 420tcgcattcaa cgtaatcaat cgcatgatga
tcagaggaca cgaagtcttg gtggcggtgg 480ccagaaacac tgtccattgc aagggcatag
ggatgcgttc cttcacctct catttctcat 540ttctgaatcc ctccctgctc actctttctc
ctcctccttc ccgttcacgc agcattcgg 59931521DNAPrototheca moriformis
31gacagggtgg ttggctggat ggggaaacgc tggtcgcggg attcgatcct gctgcttata
60tcctccctgg aagcacaccc acgactctga agaagaaaac gtgcacacac acaacccaac
120cggccgaata tttgcttcct tatcccgggt ccaagagaga ctgcgatgcc cccctcaatc
180agcatcctcc tccctgccgc ttcaatcttc cctgcttgcc tgcgcccgcg gtgcgccgtc
240tgcccgccca gtcagtcact cctgcacagg ccccttgtgc gcagtgctcc tgtacccttt
300accgctcctt ccattctgcg aggcccccta ttgaatgtat tcgttgcctg tgtggccaag
360cgggctgctg ggcgcgccgc cgtcgggcag tgctcggcga ctttggcgga agccgattgt
420tcttctgtaa gccacgcgct tgctgctttg ggaagagaag ggggggggta ctgaatggat
480gaggaggaga aggaggggta ttggtattat ctgagttggg t
52132563DNAPrototheca moriformis 32aatggagtcg ctgctccact aatcgaattg
tcagcaccgc cagccggccg aggacccgag 60tcatagcgag ggtagtagcg cgccatggca
ccgaccagcc tgcttgcccg tactggcgtc 120tcttccgctt ctctgtgctc ctctacgcgc
tccggcgcgt gcgcttttcc ggtggatcat 180gcggtccgtg gcgcaccgca gcggccgctg
cccatgcagc gccgctgctt ccgaacagtg 240gctgtcaggg ccgcacccgc agtagccgtc
cgtccggaac ccgcccaaga gttttgggag 300cagcttgagc cctgcaagat ggcggaggac
aagcgcatct tcctggagga gcaccggtgc 360gcggaggtcc ggggctgacc ggccgtcgca
ttcaacgtaa tcaatcgcat gatgatcaca 420ggacgcgacg tcttggtggc ggtggccagg
gacactgccc attgcacagg cataggaatg 480cgttccttct catttctcag ttttctgagc
ccctccctct tcactctttc tcctcctcct 540cccctctcac gcagcattcg tgg
56333547DNAPrototheca moriformis
33cactagtatc gatttcgaac agaggagagg gtggctggta gttgcgggat ggctggtcgc
60ccgtcgatcc tgctgctgct attgtctcct cctgcacaag cccacccacg actccgaaga
120agaagaagaa aacgcgcaca cacacaaccc aaccggccga atatttgctt ccttatcccg
180ggtccaagag agacggcgat gcccccctca atcagcctcc tcctccctgc cgctccaatc
240ttccctgctt gcatgcgccc gcgagaggct gtctgcgcgc cccgtcagtc actccccgtg
300cagacgcctc gtgctcggtg ctcctgtatc ctttaccgct cctttcattc tgcgaggccc
360cctgttgaat gtattcgttg cctgtgtggc caagcgcgct gctgggcgcg ccgccgtcgg
420gcggtgctcg gcgactctgg cggaagccgg ttgttcttct gtaagccacg cgcttgctgc
480ttttggaaaa gaggggggtt tactgaatgg aggaggagca ggataattgg tagtatctga
540gttgttg
54734615DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 34actagtgcgc tggacgcggc agtgggtggc
cgaggagaac cggcacggcg acctgctgaa 60caagtactgt tggctgacgg ggcgcgtcaa
catgcgggcc gtggaggtga ccatcaacaa 120cctgatcaag agcggcatga acccgcagac
ggacaacaac ccttacttgg gcttcgtcta 180cacctccttc caggagcgcg cgaccaagta
cagccacggc aacaccgcgc gccttgcggc 240cgagcagtgt gtttgagggt tttggttgcc
cgtatcgagg tcctggtggc gcgcatgggg 300gagaaggcgc ctgtcccgct gacccccccg
gctaccctcc cggcaccttc cagggcgcgt 360acgggatcct gctcggccgc aaggcgcgcg
gtgttgccgt ggctgtactt ggtcgcgcgc 420tcctggaagg aggtgtagac gaagcccaag
taagggttgt tgtccgtctg cgggttcatg 480ccgctcttga tcaggttgtt gatggtcacc
tccacggccc gcatgttgac gcgccccgtc 540agccaacagt acttgttcag caggtcgccg
tgccggttct cctcggccac ccactgccgc 600gtccagcgca agctt
61535452PRTPrototheca moriformis 35Met
Ser Ile Gln Phe Ala Leu Arg Ala Ala Tyr Ile Lys Gly Thr Cys 1
5 10 15 Gln Arg Leu Ser Gly Arg
Gly Ala Ala Leu Gly Leu Ser Arg Asp Trp 20
25 30 Thr Pro Gly Trp Thr Leu Pro Arg Cys Trp
Pro Ala Ser Ala Ala Ala 35 40
45 Thr Ala Pro Pro Arg Ala Arg His Gln Glu Arg Ala Ile His
Leu Thr 50 55 60
Ser Gly Arg Arg Arg His Ser Ala Leu Ala Ser Asp Ala Asp Glu Arg 65
70 75 80 Ala Leu Pro Ser Asn
Ala Pro Gly Leu Val Met Ala Ser Gln Ala Asn 85
90 95 Tyr Phe Arg Val Arg Leu Leu Pro Glu Gln
Glu Glu Gly Glu Leu Glu 100 105
110 Ser Trp Ser Pro Asn Val Arg His Thr Thr Leu Leu Cys Lys Pro
Arg 115 120 125 Ala
Met Leu Ser Lys Leu Gln Met Arg Val Met Val Gly Asp Arg Val 130
135 140 Ile Val Thr Ala Ile Asp
Pro Val Asn Met Thr Val His Ala Pro Pro 145 150
155 160 Phe Asp Pro Leu Pro Ala Thr Arg Phe Leu Val
Ala Gly Glu Ala Ala 165 170
175 Asp Met Asp Ile Thr Val Val Leu Asn Lys Ala Asp Leu Val Pro Glu
180 185 190 Glu Glu
Ser Ala Ala Leu Ala Gln Glu Val Ala Ser Trp Gly Pro Val 195
200 205 Val Leu Thr Ser Thr Leu Thr
Gly Arg Gly Leu Gln Glu Leu Glu Arg 210 215
220 Gln Leu Gly Ser Thr Thr Ala Val Leu Ala Gly Pro
Ser Gly Ala Gly 225 230 235
240 Lys Ser Ser Ile Ile Asn Ala Leu Ala Arg Ala Ala Arg Glu Arg Pro
245 250 255 Ser Asp Ala
Ser Val Ser Asn Val Pro Glu Glu Gln Val Val Gly Glu 260
265 270 Asp Gly Arg Ala Leu Ala Asn Pro
Pro Pro Phe Thr Leu Ala Asp Ile 275 280
285 Arg Asn Ala Ile Pro Lys Asp Cys Phe Arg Lys Ser Ala
Ala Lys Ser 290 295 300
Leu Ala Tyr Leu Gly Asp Leu Ser Ile Thr Gly Met Ala Val Leu Ala 305
310 315 320 Tyr Lys Ile Asn
Ser Pro Trp Leu Trp Pro Leu Tyr Trp Phe Ala Gln 325
330 335 Gly Thr Met Phe Trp Ala Leu Phe Val
Val Gly His Asp Cys Gly His 340 345
350 Gln Ser Phe Ser Thr Ser Lys Arg Leu Asn Asp Ala Leu Ala
Trp Leu 355 360 365
Gly Ala Leu Ala Ala Gly Thr Trp Thr Trp Ala Leu Gly Val Leu Pro 370
375 380 Met Leu Asn Leu Tyr
Leu Ala Pro Tyr Val Trp Leu Leu Val Thr Tyr 385 390
395 400 Leu His His His Gly Pro Ser Asp Pro Arg
Glu Glu Met Pro Trp Tyr 405 410
415 Arg Gly Arg Glu Trp Ser Tyr Met Arg Gly Gly Leu Thr Thr Ile
Asp 420 425 430 Arg
Asp Tyr Gly Leu Phe Asn Lys Val His His Asp Ile Gly Thr His 435
440 445 Val Val His His 450
36315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Met Phe Trp Ala Leu Phe Val Val Gly His Asp
Cys Gly His Gln Ser 1 5 10
15 Phe Ser Thr Ser Lys Arg Leu Asn Asp Ala Val Gly Leu Phe Val His
20 25 30 Ser Ile
Ile Gly Val Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr 35
40 45 His His Asn Asn His Gly His
Val Glu Asn Asp Glu Ser Trp Tyr Pro 50 55
60 Pro Thr Glu Ser Gly Leu Lys Ala Met Thr Asp Met
Gly Arg Gln Gly 65 70 75
80 Arg Phe His Phe Pro Ser Met Leu Phe Val Tyr Pro Phe Tyr Leu Phe
85 90 95 Trp Arg Ser
Pro Gly Lys Thr Gly Ser His Phe Ser Pro Ala Thr Asp 100
105 110 Leu Phe Ala Leu Trp Glu Ala Pro
Leu Ile Arg Thr Ser Asn Ala Cys 115 120
125 Gln Leu Ala Trp Leu Gly Ala Leu Ala Ala Gly Thr Trp
Ala Leu Gly 130 135 140
Val Leu Pro Met Leu Asn Leu Tyr Leu Ala Pro Tyr Val Ile Ser Val 145
150 155 160 Ala Trp Leu Asp
Leu Val Thr Tyr Leu His His His Gly Pro Ser Asp 165
170 175 Pro Arg Glu Glu Met Pro Trp Tyr Arg
Gly Arg Glu Trp Ser Tyr Met 180 185
190 Arg Gly Gly Leu Thr Thr Ile Asp Arg Asp Tyr Gly Leu Phe
Asn Lys 195 200 205
Val His His Asp Ile Gly Thr His Val Val His His Leu Phe Pro Gln 210
215 220 Ile Pro His Tyr Asn
Leu Cys Arg Ala Thr Lys Ala Ala Lys Lys Val 225 230
235 240 Leu Gly Pro Tyr Tyr Arg Glu Pro Glu Arg
Cys Pro Leu Gly Leu Leu 245 250
255 Pro Val His Leu Leu Ala Pro Leu Leu Arg Ser Leu Gly Gln Asp
His 260 265 270 Phe
Val Asp Asp Ala Gly Ser Val Leu Phe Tyr Arg Arg Ala Glu Gly 275
280 285 Ile Asn Pro Trp Ile Gln
Lys Leu Leu Pro Trp Leu Gly Gly Ala Arg 290 295
300 Arg Gly Ala Asp Ala Gln Arg Asp Ala Ala Gln
305 310 315 37448PRTCamelina sativa 37Met
Ala Asn Leu Val Leu Ser Glu Cys Gly Ile Arg Pro Leu Pro Arg 1
5 10 15 Ile Tyr Thr Thr Pro Arg
Ser Asn Phe Val Ser Asn Asn Asn Lys Pro 20
25 30 Ile Phe Lys Phe Arg Pro Phe Thr Ser Tyr
Lys Thr Ser Ser Ser Pro 35 40
45 Leu Ala Cys Ser Arg Asp Gly Phe Gly Lys Asn Trp Ser Leu
Asn Val 50 55 60
Ser Val Pro Leu Thr Thr Thr Thr Pro Ile Val Asp Glu Ser Pro Leu 65
70 75 80 Lys Glu Glu Glu Glu
Glu Lys Gln Arg Phe Asp Pro Gly Ala Pro Pro 85
90 95 Pro Phe Asn Leu Ala Asp Ile Arg Ala Ala
Ile Pro Lys His Cys Trp 100 105
110 Val Lys Asn Pro Trp Lys Ser Met Ser Tyr Val Leu Arg Asp Val
Ala 115 120 125 Ile
Val Phe Ala Leu Ala Ala Gly Ala Ser Tyr Leu Asn Asn Trp Ile 130
135 140 Val Trp Pro Leu Tyr Trp
Leu Ala Gln Gly Thr Met Phe Trp Ala Leu 145 150
155 160 Phe Val Leu Gly His Asp Cys Gly His Gly Ser
Phe Ser Asn Asn Pro 165 170
175 Arg Leu Asn Asn Val Val Gly His Leu Leu His Ser Ser Ile Leu Val
180 185 190 Pro Tyr
His Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn His 195
200 205 Gly His Val Glu Asn Asp Glu
Ser Trp His Pro Met Ser Glu Lys Ile 210 215
220 Tyr Gln Ser Leu Asp Lys Pro Thr Arg Phe Phe Arg
Phe Thr Leu Pro 225 230 235
240 Leu Val Met Leu Ala Tyr Pro Phe Tyr Leu Trp Ala Arg Ser Pro Gly
245 250 255 Lys Lys Gly
Ser His Tyr His Pro Glu Ser Asp Leu Phe Leu Pro Lys 260
265 270 Glu Lys Thr Asp Val Leu Thr Ser
Thr Ala Cys Trp Thr Ala Met Ala 275 280
285 Ala Leu Leu Ile Cys Leu Asn Phe Val Val Gly Pro Val
Gln Met Leu 290 295 300
Lys Leu Tyr Gly Ile Pro Tyr Trp Ile Asn Val Met Trp Leu Asp Phe 305
310 315 320 Val Thr Tyr Leu
His His His Gly His Glu Asp Lys Leu Pro Trp Tyr 325
330 335 Arg Gly Lys Glu Trp Ser Tyr Leu Arg
Gly Gly Leu Thr Thr Leu Asp 340 345
350 Arg Asp Tyr Gly Val Ile Asn Asn Ile His His Asp Ile Gly
Thr His 355 360 365
Val Ile His His Leu Phe Pro Gln Ile Pro His Tyr His Leu Val Glu 370
375 380 Ala Thr Glu Ala Val
Lys Pro Val Leu Gly Lys Tyr Tyr Arg Glu Pro 385 390
395 400 Asp Lys Ser Gly Pro Leu Pro Leu His Leu
Leu Gly Ile Leu Ala Lys 405 410
415 Ser Ile Lys Glu Asp His Tyr Val Ser Asp Glu Gly Asp Val Val
Tyr 420 425 430 Tyr
Lys Ala Asp Pro Asn Met Tyr Gly Glu Ile Lys Val Gly Ala Asp 435
440 445 38368PRTPrototheca
moriformis 38Met Ala Ile Lys Thr Asn Arg Gln Pro Val Glu Lys Pro Pro Phe
Thr 1 5 10 15 Ile
Gly Thr Leu Arg Lys Ala Ile Pro Ala His Cys Phe Glu Arg Ser
20 25 30 Ala Leu Arg Ser Ser
Met Tyr Leu Ala Phe Asp Ile Ala Val Met Ser 35
40 45 Leu Leu Tyr Val Ala Ser Thr Tyr Ile
Asp Pro Ala Pro Val Pro Thr 50 55
60 Trp Val Lys Tyr Gly Ile Met Trp Pro Leu Tyr Trp Phe
Phe Gln Gly 65 70 75
80 Ala Phe Gly Thr Gly Val Trp Val Cys Ala His Glu Cys Gly His Gln
85 90 95 Ala Phe Ser Ser
Ser Gln Ala Ile Asn Asp Gly Val Gly Leu Val Phe 100
105 110 His Ser Leu Leu Leu Val Pro Tyr Tyr
Ser Trp Lys His Ser His Arg 115 120
125 Arg His His Ser Asn Thr Gly Cys Leu Asp Lys Asp Glu Val
Phe Val 130 135 140
Pro Pro His Arg Ala Val Ala His Glu Gly Leu Glu Trp Glu Glu Trp 145
150 155 160 Leu Pro Ile Arg Met
Gly Lys Val Leu Val Thr Leu Thr Leu Gly Trp 165
170 175 Pro Leu Tyr Leu Met Phe Asn Val Ala Ser
Arg Pro Tyr Pro Arg Phe 180 185
190 Ala Asn His Phe Asp Pro Trp Ser Pro Ile Phe Ser Lys Arg Glu
Arg 195 200 205 Ile
Glu Val Val Ile Ser Asp Leu Ala Leu Val Ala Val Leu Ser Gly 210
215 220 Leu Ser Val Leu Gly Arg
Thr Met Gly Trp Ala Trp Leu Val Lys Thr 225 230
235 240 Tyr Val Val Pro Tyr Met Ile Val Asn Met Trp
Leu Val Leu Ile Thr 245 250
255 Leu Leu Gln His Thr His Pro Ala Leu Pro His Tyr Phe Glu Lys Asp
260 265 270 Trp Asp
Trp Leu Arg Gly Ala Met Ala Thr Val Asp Arg Ser Met Gly 275
280 285 Pro Pro Phe Met Asp Ser Ile
Leu His His Ile Ser Asp Thr His Val 290 295
300 Leu His His Leu Phe Ser Thr Ile Pro His Tyr His
Ala Glu Glu Ala 305 310 315
320 Ser Ala Ala Ile Arg Pro Ile Leu Gly Lys Tyr Tyr Gln Ser Asp Ser
325 330 335 Arg Trp Val
Gly Arg Ala Leu Trp Glu Asp Trp Arg Asp Cys Arg Tyr 340
345 350 Val Val Pro Asp Ala Pro Glu Asp
Asp Ser Ala Leu Trp Phe His Lys 355 360
365 39448PRTCamelina sativa 39Met Ala Asn Leu Val Leu
Ser Glu Cys Gly Ile Arg Pro Leu Pro Arg 1 5
10 15 Ile Tyr Thr Thr Pro Arg Ser Asn Phe Val Ser
Asn Asn Asn Lys Pro 20 25
30 Ile Phe Lys Phe Arg Pro Leu Thr Ser Tyr Lys Thr Ser Ser Pro
Leu 35 40 45 Phe
Cys Ser Arg Asp Gly Phe Gly Arg Asn Trp Ser Leu Asn Val Ser 50
55 60 Val Pro Leu Ala Thr Thr
Thr Pro Ile Val Asp Glu Ser Pro Leu Glu 65 70
75 80 Glu Glu Glu Glu Glu Glu Lys Gln Arg Phe Asp
Pro Gly Ala Pro Pro 85 90
95 Pro Phe Asn Leu Ala Asp Ile Arg Ala Ala Ile Pro Lys His Cys Trp
100 105 110 Val Lys
Asn Pro Trp Lys Ser Met Ser Tyr Val Leu Arg Asp Val Ala 115
120 125 Ile Val Phe Ala Leu Ala Ala
Gly Ala Ala Tyr Leu Asn Asn Trp Ile 130 135
140 Val Trp Pro Leu Tyr Trp Leu Ala Gln Gly Thr Met
Phe Trp Ala Leu 145 150 155
160 Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asn Asn Pro
165 170 175 Arg Leu Asn
Asn Val Val Gly His Leu Leu His Ser Ser Ile Leu Val 180
185 190 Pro Tyr His Gly Trp Arg Ile Ser
His Arg Thr His His Gln Asn His 195 200
205 Gly His Val Glu Asn Asp Glu Ser Trp His Pro Met Ser
Glu Lys Ile 210 215 220
Tyr Gln Ser Leu Asp Lys Pro Thr Arg Phe Phe Arg Phe Thr Leu Pro 225
230 235 240 Leu Val Met Leu
Ala Tyr Pro Phe Tyr Leu Trp Ala Arg Ser Pro Gly 245
250 255 Lys Lys Gly Ser His Tyr His Pro Glu
Ser Asp Leu Phe Leu Pro Lys 260 265
270 Glu Lys Thr Asp Val Leu Thr Ser Thr Ala Cys Trp Thr Ala
Met Ala 275 280 285
Ala Leu Leu Ile Cys Leu Asn Phe Val Val Gly Pro Val Gln Met Leu 290
295 300 Lys Leu Tyr Gly Ile
Pro Tyr Trp Ile Asn Val Met Trp Leu Asp Phe 305 310
315 320 Val Thr Tyr Leu His His His Gly His Glu
Asp Lys Leu Pro Trp Tyr 325 330
335 Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Leu
Asp 340 345 350 Arg
Asp Tyr Gly Val Ile Asn Asn Ile His His Asp Ile Gly Thr His 355
360 365 Val Ile His His Leu Phe
Pro Gln Ile Pro His Tyr His Leu Val Glu 370 375
380 Ala Thr Glu Ala Val Lys Pro Val Leu Gly Lys
Tyr Tyr Arg Glu Pro 385 390 395
400 Asp Lys Ser Gly Pro Leu Pro Leu His Leu Leu Gly Ile Leu Ala Lys
405 410 415 Ser Ile
Lys Glu Asp His Tyr Val Ser Asp Glu Gly Asp Val Val Tyr 420
425 430 Tyr Lys Ala Asp Pro Asn Met
Tyr Gly Glu Ile Lys Val Gly Ala Asp 435 440
445 40353DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 40actagtcatt cggggcaacg
aggtgggccc ctcgcagcgg ctgacgatca cggcggtggc 60caacatcctg caggaggcgg
cgggcaacca cgcggtggcc atgtggggcc ggagcgtgtg 120tttgagggtt ttggttgccc
gtattgaggt cctggtggcg cgcatggggg agaaggcgcc 180tgtcccgctg acccccccgg
ctaccctccc ggcaccttcc agggcgcgta cgggatccgc 240tccggcccca catggccacc
gcgtggttgc ccgccgcctc ctgcaggatg ttggccaccg 300ccgtgatcgt cagccgctgc
gaggggccca cctcgttgcc ccgaatgaag ctt 35341391DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
41actagtggag ggtttcgcga cggacccgga gctgcaggag gcgggtctca tctttgtgat
60gacgcgcatg cagatccaga tgtaccgcta cccgcgctgg ggcgacctga tgcaggtgga
120gacctggttc cagagtgtgt ttgagggttt tggttgcccg tattgaggtc ctggtggcgc
180gcatggggga gaaggcgcct gtcccgctga cccccccggc taccctcccg gcaccttcca
240gggcgcgtac gggatcctct ggaaccaggt ctccacctgc atcaggtcgc cccagcgcgg
300gtagcggtac atctggatct gcatgcgcgt catcacaaag atgagacccg cctcctgcag
360ctccgggtcc gtcgcgaaac cctccaagct t
39142393DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 42actagtcggc gggcaagctg ggcgcgcagc
gcgagtgggt gctgcgcgac aagctgaccg 60gcgaggcgct gggcgcggcc acctcgagct
gggtcatgat caacatccgc acgcgccggc 120cgtgccgcat gccgggtgtg tttgagggtt
ttggttgccc gtatcgaggt cctggtggcg 180cgcatggggg agaaggcgcc tgtcccgctg
acccccccgg ctaccctccc ggcaccttcc 240agggcgcgta cgggatcccc ggcatgcggc
acggccggcg cgtgcggatg ttgatcatga 300cccagctcga ggtggccgcg cccagcgcct
cgccggtcag cttgtcgcgc agcacccact 360cgcgctgcgc gcccagcttg cccgccgaag
ctt 39343517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
43actagtgtcc gcgtcaagtc ggccttcttc gcgcgcgagc cgccgcgcct ggcgctgccg
60cccgcggtca cgcgtgccaa gctgcccaac atcgcgacgc cggcgccgct gcgcgggcac
120cgccaggtcg cgcgccgcac cgacatggac atgaacgggc acgtgaacaa cgtggcctac
180ctggcctggt gcctggagtg tgtttgaggg ttttggttgc ccgtattgag gtcctggtgg
240cgcgcatggg ggagaaggcg cctgtcccgc tgaccccccc ggctaccctc ccggcacctt
300ccagggcgcg tacgggatcc tccaggcacc aggccaggta ggccacgttg ttcacgtgcc
360cgttcatgtc catgtcggtg cggcgcgcga cctggcggtg cccgcgcagc ggcgccggcg
420tcgcgatgtt gggcagcttg gcacgcgtga ccgcgggcgg cagcgccagg cgcggcggct
480cgcgcgcgaa gaaggccgac ttgacgcgga caagctt
51744519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 44actagtccgt gcccgagcac gtcttcagcg
actaccacct ctaccagatg gagatcgact 60tcaaggccga gtgccacgcg ggcgacgtca
tctcctccca ggccgagcag atcccgcccc 120aggaggcgct cacgcacaac ggcgccggcc
gcaacccctc ctgcttcgtc catagcattc 180tgcgcgccga gaccgagcgt gtgtttgagg
gttttggttg cccgtatcga ggtcctggtg 240gcgcgcatgg gggagaaggc gcctgtcccg
ctgacccccc cggctaccct cccggcacct 300tccagggcgc gtacgggatc cgctcggtct
cggcgcgcag aatgctatgg acgaagcagg 360aggggttgcg gccggcgccg ttgtgcgtga
gcgcctcctg gggcgggatc tgctcggcct 420gggaggagat gacgtcgccc gcgtggcact
cggccttgaa gtcgatctcc atctggtaga 480ggtggtagtc gctgaagacg tgctcgggca
cggaagctt 51945415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
45actagttcgt ccgcgcgcga accacatggt cggcccccat cgacgcgccc gccgccaagc
60cgcccaaggc gagccactga ggacagggtg gttggctgga tggggaaacg ctggtcgcgg
120gattcgatcc tgctgcttat atcctcgtgt gtttgagggt tttggttgcc cgtattgagg
180tcctggtggc gcgcatgggg gagaaggcgc ctgtcccgct gacccccccg gctaccctcc
240cggcaccttc cagggcgcgt acgggatccg aggatataag cagcaggatc gaatcccgcg
300accagcgttt ccccatccag ccaaccaccc tgtcctcagt ggctcgcctt gggcggcttg
360gcggcgggcg cgtcgatggg ggccgaccat gtggttcgcg cgcggacgaa agctt
415461629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46atggccgccg ccgccagcat ggtggccagc
cccttctgca cctggctggt ggccagctgc 60atgagcacca gcttcgacaa cgacccccgc
agccccagcg tgaagcgctt cccccgccgc 120aagcgcgtgc tgagccagcg cggcagcacc
tacgtattcc agtgcctggt ggccagctgc 180atcgacccct gcgaccagta ccgcagcagc
gccagcctga gcttcctggg cgacaacggc 240ttcgccagcc tgttcggcag caagcccttc
atgagcaacc gcggccaccg ccgcctgcgc 300cgcgccagcc acagcggcga ggccatggcc
gtggccctgc agcccgccca ggaggccggc 360accaagaaga agcccgtgat caagcagcgc
cgcgtggtgg tgaccggcat gggcgtggtg 420acccccctgg gccacgagcc cgacgtgttc
tacaacaacc tgctggacgg cgtgagcggc 480atcagcgaga tcgagacctt cgactgcacc
cagttcccca cccgcatcgc cggcgagatc 540aagagcttca gcaccgacgg ctgggtggcc
cccaagctga gcaagcgcat ggacaagttc 600atgctgtacc tgctgaccgc cggcaagaag
gccctggccg acggcggcat caccgacgag 660gtgatgaagg agctggacaa gcgcaagtgc
ggcgtgctga tcggcagcgg catgggcggc 720atgaaggtgt tcaacgacgc catcgaggcc
ctgcgcgtga gctacaagaa gatgaacccc 780ttctgcgtgc ccttcgccac caccaacatg
ggcagcgcca tgctggccat ggacctgggc 840tggatgggcc ccaactacag catcagcacc
gcctgcgcca ccagcaactt ctgcatcctg 900aacgccgcca accacatcat ccgcggcgag
gccgacatga tgctgtgcgg cggcagcgac 960gccgtgatca tccccatcgg cctgggcggc
ttcgtggcct gccgcgccct gagccagcgc 1020aacagcgacc ccaccaaggc cagccgcccc
tgggacagca accgcgacgg cttcgtgatg 1080ggcgagggcg ccggcgtgct gctgctggag
gagctggagc acgccaagaa gcgcggcgcc 1140accatctacg ccgagttcct gggcggcagc
ttcacctgcg acgcctacca catgaccgag 1200ccccaccccg agggcgccgg cgtgatcctg
tgcatcgaga aggccctggc ccaggccggc 1260gtgagcaagg aggacgtgaa ctacatcaac
gcccacgcca ccagcaccag cgccggcgac 1320atcaaggagt accaggccct ggcccgctgc
ttcggccaga acagcgagct gcgcgtgaac 1380agcaccaaga gcatgatcgg ccacctgctg
ggcgccgccg gcggcgtgga ggccgtgacc 1440gtggtgcagg ccatccgcac cggctggatt
caccccaacc tgaacctgga ggaccccgac 1500aaggccgtgg acgccaagct gctggtgggc
cccaagaagg agcgcctgaa cgtgaaggtg 1560ggcctgagca acagcttcgg cttcggcggc
cacaacagca gcatcctgtt cgccccctgc 1620aacgtgtga
1629471552DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
47atgggccgcg gtgtctccct tccccggccc agggtcgcgg tgcgcgccca gtcggcgagt
60caggttttgg agagctgtat tccagtgcct ggtggccagc tgcatcgacc cctgcgacca
120gtaccgcagc agcgccagcc tgagcttcct gggcgacaac ggcttcgcca gcctgttcgg
180cagcaagccc ttcatgagca accgcggcca ccgccgcctg cgccgcgcca gccacagcgg
240cgaggccatg gccgtggccc tgcagcccgc ccaggaggcc ggcaccaaga agaagcccgt
300gatcaagcag cgccgcgtgg tggtgaccgg catgggcgtg gtgacccccc tgggccacga
360gcccgacgtg ttctacaaca acctgctgga cggcgtgagc ggcatcagcg agatcgagac
420cttcgactgc acccagttcc ccacccgcat cgccggcgag atcaagagct tcagcaccga
480cggctgggtg gcccccaagc tgagcaagcg catggacaag ttcatgctgt acctgctgac
540cgccggcaag aaggccctgg ccgacggcgg catcaccgac gaggtgatga aggagctgga
600caagcgcaag tgcggcgtgc tgatcggcag cggcatgggc ggcatgaagg tgttcaacga
660cgccatcgag gccctgcgcg tgagctacaa gaagatgaac cccttctgcg tgcccttcgc
720caccaccaac atgggcagcg ccatgctggc catggacctg ggctggatgg gccccaacta
780cagcatcagc accgcctgcg ccaccagcaa cttctgcatc ctgaacgccg ccaaccacat
840catccgcggc gaggccgaca tgatgctgtg cggcggcagc gacgccgtga tcatccccat
900cggcctgggc ggcttcgtgg cctgccgcgc cctgagccag cgcaacagcg accccaccaa
960ggccagccgc ccctgggaca gcaaccgcga cggcttcgtg atgggcgagg gcgccggcgt
1020gctgctgctg gaggagctgg agcacgccaa gaagcgcggc gccaccatct acgccgagtt
1080cctgggcggc agcttcacct gcgacgccta ccacatgacc gagccccacc ccgagggcgc
1140cggcgtgatc ctgtgcatcg agaaggccct ggcccaggcc ggcgtgagca aggaggacgt
1200gaactacatc aacgcccacg ccaccagcac cagcgccggc gacatcaagg agtaccaggc
1260cctggcccgc tgcttcggcc agaacagcga gctgcgcgtg aacagcacca agagcatgat
1320cggccacctg ctgggcgccg ccggcggcgt ggaggccgtg accgtggtgc aggccatccg
1380caccggctgg attcacccca acctgaacct ggaggacccc gacaaggccg tggacgccaa
1440gctgctggtg ggccccaaga aggagcgcct gaacgtgaag gtgggcctga gcaacagctt
1500cggcttcggc ggccacaaca gcagcatcct gttcgccccc tgcaacgtgt ga
1552481641DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 48atgcccgcgg ccagctcgct gctggcgtcc
cccctgtgca cctggctgct ggccgcgtgc 60atgagcacct cgttccaccc ctccgacccc
ctgcccccca gcatctcgtc cccccgccgc 120cgcctgagcc gccgccgcat cctgtcgcag
tgcgcccccc tgccctccgc gagctcggcc 180ctgcgcggct ccagcttcca caccctggtg
acctcgtatc tggcgtgctt cgagccctgc 240cacgactatt ataccagcgc ctccctgttc
ggctcgcgcc ccatccgcac cacccgccgc 300caccgccgcc tgaaccgcgc gagcccctcg
cgcgaggcga tggcggtcgc cctgcagccc 360gagcaggagg tgaccaccaa gaagaagccc
tccatcaagc agcgccgcgt cgtggtcacc 420ggcatgggcg tggtcacccc cctgggccac
gaccccgacg tgttctataa caacctgctg 480gacggcacca gcggcatctc ggagatcgag
accttcgact gcgcgcagtt ccccacccgc 540atcgccggcg agatcaagtc cttcagcacc
gacggctggg tcgcgcccaa gctgtcgaag 600cgcatggaca agttcatgct gtatatgctg
accgccggca agaaggcgct gaccgacggc 660ggcatcaccg aggacgtgat gaaggagctg
gacaagcgca agtgcggcgt cctgatcggc 720tccgcgatgg gcggcatgaa ggtgttcaac
gacgcgatcg aggccctgcg catcagctat 780aagaagatga accccttctg cgtgcccttc
gcgaccacca acatgggctc ggccatgctg 840gcgatggacc tgggctggat gggccccaac
tattccatca gcaccgcctg cgcgacctcg 900aacttctgca tcatgaacgc ggccaaccac
atcatccgcg gcgaggcgga cgtcatgctg 960tgcggcggct ccgacgccgt gatcatcccc
atcggcatgg gcggcttcgt cgcgtgccgc 1020gccctgagcc agcgcaactc ggaccccacc
aaggcgtccc gcccctggga cagcaaccgc 1080gacggcttcg tgatgggcga gggcgccggc
gtcctgctgc tggaggagct ggagcacgcg 1140aagaagcgcg gcgccaccat ctatgcggag
ttcctgggcg gctcgttcac ctgcgacgcc 1200tatcacatga ccgagcccca ccccgacggc
gccggcgtga tcctgtgcat cgagaaggcg 1260ctggcccagt ccggcgtcag ccgcgaggac
gtgaactata tcaacgcgca cgccacctcg 1320acccccgcgg gcgacatcaa ggagtatcag
gccctgatcc actgcttcgg ccagaaccgc 1380gagctgaagg tcaactccac caagagcatg
atcggccacc tgctgggcgc ggcgggcggc 1440gtggaggcgg tctcggtggt ccaggccatc
cgcaccggct ggatccaccc caacatcaac 1500ctggagaacc ccgacgaggg cgtggacacc
aagctgctgg tgggccccaa gaaggagcgc 1560ctgaacgtca aggtgggcct gtccaacagc
ttcggcttcg gcggccacaa ctcgtccatc 1620ctgttcgcgc cctatatctg a
1641491251DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
49atggtggccg ccgccgcctc cagcgccttc ttccccgtgc ccgcccccgg cgcctccccc
60aagcccggca agttcggcaa ctggccctcc agcctgagcc cctccttcaa gcccaagtcc
120atccccaacg gcggcttcca ggtgaaggcc aacgacagcg cccaccccaa ggccaacggc
180tccgccgtga gcctgaagag cggcagcctg aacacccagg aggacacctc ctccagcccc
240cccccccgca ccttcctgca ccagctgccc gactggagcc gcctgctgac cgccatcacc
300accgtgttcg tgaagtccaa gcgccccgac atgcacgacc gcaagtccaa gcgccccgac
360atgctggtgg acagcttcgg cctggagtcc accgtgcagg acggcctggt gttccgccag
420tccttctcca tccgctccta cgagatcggc accgaccgca ccgccagcat cgagaccctg
480atgaaccacc tgcaggagac ctccctgaac cactgcaaga gcaccggcat cctgctggac
540ggcttcggcc gcaccctgga gatgtgcaag cgcgacctga tctgggtggt gatcaagatg
600cagatcaagg tgaaccgcta ccccgcctgg ggcgacaccg tggagatcaa cacccgcttc
660agccgcctgg gcaagatcgg catgggccgc gactggctga tctccgactg caacaccggc
720gagatcctgg tgcgcgccac cagcgcctac gccatgatga accagaagac ccgccgcctg
780tccaagctgc cctacgaggt gcaccaggag atcgtgcccc tgttcgtgga cagccccgtg
840atcgaggact ccgacctgaa ggtgcacaag ttcaaggtga agaccggcga cagcatccag
900aagggcctga cccccggctg gaacgacctg gacgtgaacc agcacgtgtc caacgtgaag
960tacatcggct ggatcctgga gagcatgccc accgaggtgc tggagaccca ggagctgtgc
1020tccctggccc tggagtaccg ccgcgagtgc ggccgcgact ccgtgctgga gagcgtgacc
1080gccatggacc ccagcaaggt gggcgtgcgc tcccagtacc agcacctgct gcgcctggag
1140gacggcaccg ccatcgtgaa cggcgccacc gagtggcgcc ccaagaacgc cggcgccaac
1200ggcgccatct ccaccggcaa gaccagcaac ggcaactccg tgtccatgtg a
125150659DNAPrototheca moriformis 50gctcttcctc accgcgtgaa ttgctgtccc
aaacgtaagc atcatcgtgg ctcggtcacg 60cgatcctgga tccggggatc ctagaccgct
ggtggagagc gctgccgtcg gattggtggc 120aagtaagatt gcgcaggttg gcgaagggag
agaccaaaac cggaggctgg aagcgggcac 180aacatcgtat tattgcgtat agtagagcag
tggcagtcgc atttcgaggt ccgcaacgga 240tctcgcaagc tcgctacgct cacagtagga
gaaaggggac cactgcccct gccagaatgg 300tcgcgaccct ctccctcgcc ggccccgcct
gcaacacgca gtgcgtatcc ggcaagcggg 360ctgtcgcctt caaccgcccc catgttggcg
tccgggctcg atcaggtgcg ctgagggggg 420tttggtgtgc ccgcgcctct gggcccgtgt
cggccgtgcg gacgtggggc cctgggcagt 480ggatcagcag ggtttgcgtg caaatgccta
taccggcgat tgaatagcga tgaacgggat 540acggttgcgc tcactccatg cccatgcgac
cccgtttctg tccgccagcc gtggtcgccc 600gggctgcgaa gcgggacccc acccagcgca
ttgtgatcac cggaatgggc gtgggtacc 65951607DNAPrototheca moriformis
51gagctccacc tgcatccgcc tggcgctcga ggacgccggc gtctcgcccg acgaggtcaa
60ctacgtcaac gcgcacgcca cctccaccct ggtgggcgac aaggccgagg tgcgcgcggt
120caagtcggtc tttggcgaca tgaagggcat caagatgaac gccaccaagt ccatgatcgg
180gcactgcctg ggcgccgccg gcggcatgga ggccgtcgcc acgctcatgg ccatccgcac
240cggctgggtg caccccacca tcaaccacga caaccccatc gccgaggtcg acggcctgga
300cgtcgtcgcc aacgccaagg cccagcacaa aatcaacgtc gccatctcca actccttcgg
360cttcggcggg cacaactccg tcgtcgcctt tgcgcccttc cgcgagtagg cggagcgagc
420gcgcttggct gaggagggag gcggggtgcg agccctttgg ctgcgcgcga tactctcccc
480gcacgagcag actccacgcg cctgaatcta cttgtcaacg agcaaccgtg tgttttgtcc
540gtggccattc ttattatttc tccgactgtg gccgtactct gtttggctgt gcaagcaccg
600aagagcc
60752636DNAPrototheca moriformis 52gctcttcgcg caagctcgct acgctcacag
taggagatag gggaccactg cccctgccag 60aatggtcgcg accctgtccc tcgccggccc
cgcctgcaac acgcagtgcg tatccagcaa 120gcgggttgtc gccttcaacc gcccccatgt
tggcgtccgg gctcgatcag gtgcgctgag 180gggggtttgg tgggcccgcg cctctgggcc
cgtgtcggcc gtgcggacgt ggggcccggg 240gtagtggatc agcaggggtt gcatgcaaat
gcctataccg gcgattgaat agcgatgaac 300gggatacggt tgcgctcact ccatgcccat
gcgaccccgt ttctgtccgc cagccgtggt 360cgcccgagct gcgaagcggg accccaccca
gcgcattgtg atcaccggaa tgggcgtggc 420ctccgtgttt ggcaacgatg tcgagacctt
ttacgacaag cttctggaag gaacgagcgg 480cgtggacctg atttccaggt gcgtaggtcc
ttggatgaat gcgtctaggt tgcgaggtga 540ctggccagga agcagcaggc ttggggtttg
gtgttctgat ttctggtaat ttgaggtttc 600attataagat tctgtacggt cttgtttcgg
ggtacc 63653607DNAPrototheca moriformis
53gagctccacc tgcatccgcc tggcgctcga ggacgccggc gtctcgcccg acgaggtcaa
60ctacgtcaac gcgcacgcca cctccaccct ggtgggcgac aaggccgagg tgcgcgcggt
120caagtcggtc tttggcgaca tgaagggcat caagatgaac gccaccaagt ccatgatcgg
180gcactgcctg ggcgccgccg gcggcatgga ggccgtcgcc acgctcatgg ccatccgcac
240cggctgggtg caccccacca tcaaccacga caaccccatc gccgaggtcg acggcctgga
300cgtcgtcgcc aacgccaagg cccagcacaa aatcaacgtc gccatctcca actccttcgg
360cttcggcggg cacaactccg tcgtcgcctt tgcgcccttc cgcgagtagg cggagcgagc
420gcgcttggct gaggagggag gcggggtgcg agccctttgg ctgcgcgcga tactctcccc
480gcacgagcag actccacgcg cctgaatcta cttgtcaacg agcaaccgtg tgttttgtcc
540gtggccattc ttattatttc tccgactgtg gccgtactct gtttggctgt gcaagcaccg
600aagagcc
60754335DNAPrototheca moriformis 54actagtcatg gtcgcccggg ctgcgaagcg
ggaccccacc cagcgcattg tgatcaccgg 60aatgggcgtg gcctccgtgt ttggcaacga
tgtcgagacc ttttacagtg tgtttgaggg 120ttttggttgc ccgtattgag gtcctggtgg
cgcgcatgga ggagaaggcg cctgtcccgc 180tgaccccccc ggctaccctc ccggcacctt
ccagggcgcg tacgggatcc tgtaaaaggt 240ctcgacatcg ttgccaaaca cggaggccac
gcccattccg gtgatcacaa tgcgctgggt 300ggggtcccgc ttcgcagccc gggcgaccaa
agctt 33555503DNAPrototheca moriformis
55actagtcatt gacatctccg agttcccgac caagtttgcg gcgcagatca ccggcttctc
60cgtggaggac tgcgtggaca agaagaacgc gcggcggtac gacgacgcgc tgtcgtacgc
120gatggtggcc tccaagaagg ccctgcgcca ggcgggactg gagaaggaca agtgccccga
180gggctacgga ggtgtgtttg agggttttgg ttgcccgtat tgaggtcctg gtggcgcgca
240tggaggagaa ggcgcctgtc ccgctgaccc ccccggctac cctcccggca ccttccaggg
300cgcgtacggg atccctccgt agccctcggg gcacttgtcc ttctccagtc ccgcctggcg
360cagggccttc ttggaggcca ccatcgcgta cgacagcgcg tcgtcgtacc gccgcgcgtt
420cttcttgtcc acgcagtcct ccacggagaa gccggtgatc tgcgccgcaa acttggtcgg
480gaactcggag atgtcaaaag ctt
50356457DNAPrototheca moriformis 56actagtcatg ggcgtgagca cctgcatccg
cctggcgctc gaggacgccg gcgtctcgcc 60cgacgaggtc aactacgtca acgcgcacgc
cacctccacc ctggtgggcg acaaggccga 120ggtgcgcgcg gtcaagtcgg tctttggcga
catgaagggc atcaagatgt gtgtttgagg 180gttttggttg cccgtattga ggtcctggtg
gcgcgcatgg aggagaaggc gcctgtcccg 240ctgacccccc cggctaccct cccggcacct
tccagggcgc gtacgggatc catcttgatg 300cccttcatgt cgccaaagac cgacttgacc
gcgcgcacct cggccttgtc gcccaccagg 360gtggaggtgg cgtgcgcgtt gacgtagttg
acctcgtcgg gcgagacgcc ggcgtcctcg 420agcgccaggc ggatgcaggt gctcacgccc
aaagctt 45757345DNAPrototheca moriformis
57actagtcaca accatcaacc acgacaaccc catcgccgag gtcgacggcc tggacgtcgt
60cgccaacgcc aaggcccagc acaaaatcaa cgtcgccatc tccaactcct tcggtgtgtt
120tgagggtttt ggttgcccgt attgaggtcc tggtggcgcg catggaggag aaggcgcctg
180tcccgctgac ccccccggct accctcccgg caccttccag ggcgcgtacg ggatcccgaa
240ggagttggag atggcgacgt tgattttgtg ctgggccttg gcgttggcga cgacgtccag
300gccgtcgacc tcggcgatgg ggttgtcgtg gttgatggta agctt
345581590DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 58atggactccc gcgcccagaa ccgcgacggc
ggcgaggacg tgaagcagga gctgctgtcc 60gccggcgacg acggcaaggt gccctgcccc
accgtggcca tcggcatccg ccagcgcctg 120cccgacttcc tgcagtccgt gaacatgaag
tacgtgaagc tgggctacca ctacctgatc 180acccacgcca tgttcctgct gaccctgccc
gccttcttcc tggtggccgc cgagatcggc 240cgcctgggcc acgagcgcat ctaccgcgag
ctgtggaccc acctgcacct gaacctggtg 300tccatcatgg cctgctcctc cgccctggtg
gccggcgcca ccctgtactt catgtcccgc 360ccccgccccg tgtacctggt ggagttcgcc
tgctaccgcc ccgacgagcg cctgaaggtg 420tccaaggact tcttcctgga catgtcccgc
cgcaccggcc tgttctcctc ctcctccatg 480gacttccaga ccaagatcac ccagcgctcc
ggcctgggcg acgagaccta cctgcccccc 540gccatcctgg cctccccccc caacccctgc
atgcgcgagg cccgcgagga ggccgccatg 600gtgatgttcg gcgccctgga cgagctgttc
gagcagaccg gcgtgaagcc caaggagatc 660ggcgtgctgg tggtgaactg ctccctgttc
aaccccaccc cctccatgtc cgccatgatc 720gtgaaccact accacatgcg cggcaacatc
aagtccctga acctgggcgg catgggctgc 780tccgccggcc tgatctccat cgacctggcc
cgcgacctgc tgcaggtgca cggcaacacc 840tacgccgtgg tggtgtccac cgagaacatc
accctgaact ggtacttcgg cgacgaccgc 900tccaagctga tgtccaactg catcttccgc
atgggcggcg ccgccgtgct gctgtccaac 960aagcgccgcg agcgccgccg cgccaagtac
gagctgctgc acaccgtgcg cacccacaag 1020ggcgccgacg acaagtgctt ccgctgcgtg
taccaggagg aggactccac cggctccctg 1080ggcgtgtccc tgtcccgcga gctgatggcc
gtggccggca acgccctgaa ggccaacatc 1140accaccctgg gccccctggt gctgcccctg
tccgagcaga tcctgttctt cgcctccctg 1200gtggcccgca agttcctgaa catgaagatg
aagccctaca tccccgactt caagctggcc 1260ttcgagcact tctgcatcca cgccggcggc
cgcgccgtgc tggacgagct ggagaagaac 1320ctggacctga ccgagtggca catggagccc
tcccgcatga ccctgtaccg cttcggcaac 1380acctcctcct cctccctgtg gtacgagctg
gcctacaccg aggcccaggg ccgcgtgaag 1440cgcggcgacc gcctgtggca gatcgccttc
ggctccggct tcaagtgcaa ctccgccgtg 1500tggcgcgcgc tgcgcaccgt gaagcccccc
gtgaacaacg cctggtccga cgtgatcgac 1560cgcttccccg tgaagctgcc ccagttctga
159059529PRTMetrosideros polymorpha
59Met Asp Ser Arg Ala Gln Asn Arg Asp Gly Gly Glu Asp Val Lys Gln 1
5 10 15 Glu Leu Leu Ser
Ala Gly Asp Asp Gly Lys Val Pro Cys Pro Thr Val 20
25 30 Ala Ile Gly Ile Arg Gln Arg Leu Pro
Asp Phe Leu Gln Ser Val Asn 35 40
45 Met Lys Tyr Val Lys Leu Gly Tyr His Tyr Leu Ile Thr His
Ala Met 50 55 60
Phe Leu Leu Thr Leu Pro Ala Phe Phe Leu Val Ala Ala Glu Ile Gly 65
70 75 80 Arg Leu Gly His Glu
Arg Ile Tyr Arg Glu Leu Trp Thr His Leu His 85
90 95 Leu Asn Leu Val Ser Ile Met Ala Cys Ser
Ser Ala Leu Val Ala Gly 100 105
110 Ala Thr Leu Tyr Phe Met Ser Arg Pro Arg Pro Val Tyr Leu Val
Glu 115 120 125 Phe
Ala Cys Tyr Arg Pro Asp Glu Arg Leu Lys Val Ser Lys Asp Phe 130
135 140 Phe Leu Asp Met Ser Arg
Arg Thr Gly Leu Phe Ser Ser Ser Ser Met 145 150
155 160 Asp Phe Gln Thr Lys Ile Thr Gln Arg Ser Gly
Leu Gly Asp Glu Thr 165 170
175 Tyr Leu Pro Pro Ala Ile Leu Ala Ser Pro Pro Asn Pro Cys Met Arg
180 185 190 Glu Ala
Arg Glu Glu Ala Ala Met Val Met Phe Gly Ala Leu Asp Glu 195
200 205 Leu Phe Glu Gln Thr Gly Val
Lys Pro Lys Glu Ile Gly Val Leu Val 210 215
220 Val Asn Cys Ser Leu Phe Asn Pro Thr Pro Ser Met
Ser Ala Met Ile 225 230 235
240 Val Asn His Tyr His Met Arg Gly Asn Ile Lys Ser Leu Asn Leu Gly
245 250 255 Gly Met Gly
Cys Ser Ala Gly Leu Ile Ser Ile Asp Leu Ala Arg Asp 260
265 270 Leu Leu Gln Val His Gly Asn Thr
Tyr Ala Val Val Val Ser Thr Glu 275 280
285 Asn Ile Thr Leu Asn Trp Tyr Phe Gly Asp Asp Arg Ser
Lys Leu Met 290 295 300
Ser Asn Cys Ile Phe Arg Met Gly Gly Ala Ala Val Leu Leu Ser Asn 305
310 315 320 Lys Arg Arg Glu
Arg Arg Arg Ala Lys Tyr Glu Leu Leu His Thr Val 325
330 335 Arg Thr His Lys Gly Ala Asp Asp Lys
Cys Phe Arg Cys Val Tyr Gln 340 345
350 Glu Glu Asp Ser Thr Gly Ser Leu Gly Val Ser Leu Ser Arg
Glu Leu 355 360 365
Met Ala Val Ala Gly Asn Ala Leu Lys Ala Asn Ile Thr Thr Leu Gly 370
375 380 Pro Leu Val Leu Pro
Leu Ser Glu Gln Ile Leu Phe Phe Ala Ser Leu 385 390
395 400 Val Ala Arg Lys Phe Leu Asn Met Lys Met
Lys Pro Tyr Ile Pro Asp 405 410
415 Phe Lys Leu Ala Phe Glu His Phe Cys Ile His Ala Gly Gly Arg
Ala 420 425 430 Val
Leu Asp Glu Leu Glu Lys Asn Leu Asp Leu Thr Glu Trp His Met 435
440 445 Glu Pro Ser Arg Met Thr
Leu Tyr Arg Phe Gly Asn Thr Ser Ser Ser 450 455
460 Ser Leu Trp Tyr Glu Leu Ala Tyr Thr Glu Ala
Gln Gly Arg Val Lys 465 470 475
480 Arg Gly Asp Arg Leu Trp Gln Ile Ala Phe Gly Ser Gly Phe Lys Cys
485 490 495 Asn Ser
Ala Val Trp Arg Ala Leu Arg Thr Val Lys Pro Pro Val Asn 500
505 510 Asn Ala Trp Ser Asp Val Ile
Asp Arg Phe Pro Val Lys Leu Pro Gln 515 520
525 Phe 60906DNATrypanosoma brucei 60atgctgatga
acttcggcgg ctcctacgac gcctacatca acaacttcca gggcaccttc 60ctggccgagt
ggatgctgga ccacccctcc gtgccctaca tcgccggcgt gatgtacctg 120atcctggtgc
tgtacgtgcc caagtccatc atggcctccc agccccccct gaacctgcgc 180gccgccaaca
tcgtgtggaa cctgttcctg accctgttct ccatgtgcgg cgcctactac 240accgtgccct
acctggtgaa ggccttcatg aaccccgaga tcgtgatggc cgcctccggc 300atcaagctgg
acgccaacac ctcccccatc atcacccact ccggcttcta caccaccacc 360tgcgccctgg
ccgactcctt ctacttcaac ggcgacgtgg gcttctgggt ggccctgttc 420gccctgtcca
agatccccga gatgatcgac accgccttcc tggtgttcca gaagaagccc 480gtgatcttcc
tgcactggta ccaccacctg accgtgatgc tgttctgctg gttcgcctac 540gtgcagaaga
tctcctccgg cctgtggttc gcctccatga actactccgt gcactccatc 600atgtacctgt
actacttcgt gtgcgcctgc ggccaccgcc gcctggtgcg ccccttcgcc 660cccatcatca
ccttcgtgca gatcttccag atggtggtgg gcaccatcgt ggtgtgctac 720acctacaccg
tgaagcacgt gctgggccgc tcctgcaccg tgaccgactt ctccctgcac 780accggcctgg
tgatgtacgt gtcctacctg ctgctgttct cccagctgtt ctaccgctcc 840tacctgtccc
cccgcgacaa ggcctccatc ccccacgtgg ccgccgagat caagaagaag 900gagtga
90661322PRTTrypanosoma brucei 61Met Tyr Pro Thr His Arg Asp Leu Ile Leu
Asn Asn Tyr Ser Asp Ile 1 5 10
15 Tyr Arg Ser Pro Thr Cys His Tyr His Thr Trp His Thr Leu Ile
His 20 25 30 Thr
Pro Ile Asn Glu Leu Leu Phe Pro Asn Leu Pro Arg Glu Cys Asp 35
40 45 Phe Gly Tyr Asp Ile Pro
Tyr Phe Arg Gly Gln Ile Asp Val Phe Asp 50 55
60 Gly Trp Ser Met Ile His Phe Thr Ser Ser Asn
Trp Cys Ile Pro Ile 65 70 75
80 Thr Val Cys Leu Cys Tyr Ile Met Met Ile Ala Gly Leu Lys Lys Tyr
85 90 95 Met Gly
Pro Arg Asp Gly Gly Arg Ala Pro Ile Gln Ala Lys Asn Tyr 100
105 110 Ile Ile Ala Trp Asn Leu Phe
Leu Ser Phe Phe Ser Phe Ala Gly Val 115 120
125 Tyr Tyr Thr Val Pro Tyr His Leu Phe Asp Pro Glu
Asn Gly Leu Phe 130 135 140
Ala Gln Gly Phe Tyr Ser Thr Val Cys Asn Asn Gly Ala Tyr Tyr Gly 145
150 155 160 Asn Gly Asn
Val Gly Phe Phe Val Trp Leu Phe Ile Tyr Ser Lys Ile 165
170 175 Phe Glu Leu Val Asp Thr Phe Phe
Leu Leu Ile Arg Lys Asn Pro Val 180 185
190 Ile Phe Leu His Trp Tyr His His Leu Thr Val Leu Leu
Tyr Cys Trp 195 200 205
His Ala Tyr Ser Val Arg Ile Gly Thr Gly Ile Trp Phe Ala Thr Met 210
215 220 Asn Tyr Ser Val
His Ser Val Met Tyr Leu Tyr Phe Ala Met Thr Gln 225 230
235 240 Tyr Gly Pro Ser Thr Lys Lys Phe Ala
Lys Lys Phe Ser Lys Phe Ile 245 250
255 Thr Thr Ile Gln Ile Leu Gln Met Val Val Gly Ile Ile Val
Thr Phe 260 265 270
Ala Ala Met Leu Tyr Val Thr Phe Asp Val Pro Cys Tyr Thr Ser Leu
275 280 285 Ala Asn Ser Val
Leu Gly Leu Met Met Tyr Ala Ser Tyr Phe Val Leu 290
295 300 Phe Val Gln Leu Tyr Val Ser His
Tyr Val Ser Pro Lys His Val Lys 305 310
315 320 Gln Glu 62933DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 62atggtgtccg actggaagaa
cttctgcctg gagaaggcct cccgcttccg ccccaccatc 60gaccgcccct tcttcaacat
ctacctgtgg gactacttca accgcgccgt gggctgggcc 120accgccggcc gcttccagcc
caaggacttc gagttcaccg tgggcaagca gcccctgtcc 180gagccccgcc ccgtgctgct
gttcatcgcc atgtactacg tggtgatctt cggcggccgc 240tccctggtga agtcctgcaa
gcccctgaag ctgcgcttca tctcccaggt gcacaacctg 300atgctgacct ccgtgtcctt
cctgtggctg atcctgatgg tggagcagat gctgcccatc 360gtgtaccgcc acggcctgta
cttcgccgtg tgcaacgtgg agtcctggac ccagcccatg 420gagaccctgt actacctgaa
ctacatgacc aagttcgtgg agttcgccga caccgtgctg 480atggtgctga agcaccgcaa
gctgaccttc ctgcacacct accaccacgg cgccaccgcc 540ctgctgtgct acaaccagct
ggtgggctac accgccgtga cctgggtgcc cgtgaccctg 600aacctggccg tgcacgtgct
gatgtactgg tactacttcc tgtccgcctc cggcatccgc 660gtgtggtgga aggcctgggt
gacccgcctg cagatcgtgc agttcatgct ggacctgatc 720gtggtgtact acgtgctgta
ccagaagatc gtggccgcct acttcaagaa cgcctgcacc 780ccccagtgcg aggactgcct
gggctccatg accgccatcg ccgccggcgc cgccatcctg 840acctcctacc tgttcctgtt
catctccttc tacatcgagg tgtacaagcg cggctccgcc 900tccggcaaga agaagatcaa
caagaacaac tga 93363310PRTSaccharomyces
cerevisiae 63Met Val Ser Asp Trp Lys Asn Phe Cys Leu Glu Lys Ala Ser Arg
Phe 1 5 10 15 Arg
Pro Thr Ile Asp Arg Pro Phe Phe Asn Ile Tyr Leu Trp Asp Tyr
20 25 30 Phe Asn Arg Ala Val
Gly Trp Ala Thr Ala Gly Arg Phe Gln Pro Lys 35
40 45 Asp Phe Glu Phe Thr Val Gly Lys Gln
Pro Leu Ser Glu Pro Arg Pro 50 55
60 Val Leu Leu Phe Ile Ala Met Tyr Tyr Val Val Ile Phe
Gly Gly Arg 65 70 75
80 Ser Leu Val Lys Ser Cys Lys Pro Leu Lys Leu Arg Phe Ile Ser Gln
85 90 95 Val His Asn Leu
Met Leu Thr Ser Val Ser Phe Leu Trp Leu Ile Leu 100
105 110 Met Val Glu Gln Met Leu Pro Ile Val
Tyr Arg His Gly Leu Tyr Phe 115 120
125 Ala Val Cys Asn Val Glu Ser Trp Thr Gln Pro Met Glu Thr
Leu Tyr 130 135 140
Tyr Leu Asn Tyr Met Thr Lys Phe Val Glu Phe Ala Asp Thr Val Leu 145
150 155 160 Met Val Leu Lys His
Arg Lys Leu Thr Phe Leu His Thr Tyr His His 165
170 175 Gly Ala Thr Ala Leu Leu Cys Tyr Asn Gln
Leu Val Gly Tyr Thr Ala 180 185
190 Val Thr Trp Val Pro Val Thr Leu Asn Leu Ala Val His Val Leu
Met 195 200 205 Tyr
Trp Tyr Tyr Phe Leu Ser Ala Ser Gly Ile Arg Val Trp Trp Lys 210
215 220 Ala Trp Val Thr Arg Leu
Gln Ile Val Gln Phe Met Leu Asp Leu Ile 225 230
235 240 Val Val Tyr Tyr Val Leu Tyr Gln Lys Ile Val
Ala Ala Tyr Phe Lys 245 250
255 Asn Ala Cys Thr Pro Gln Cys Glu Asp Cys Leu Gly Ser Met Thr Ala
260 265 270 Ile Ala
Ala Gly Ala Ala Ile Leu Thr Ser Tyr Leu Phe Leu Phe Ile 275
280 285 Ser Phe Tyr Ile Glu Val Tyr
Lys Arg Gly Ser Ala Ser Gly Lys Lys 290 295
300 Lys Ile Asn Lys Asn Asn 305 310
641179DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 64atgctgaagc tgtcctgcaa cgtgaccaac aacctgcaca
ccttctcctt cttctccgac 60tcctccctgt tcatccccgt gaaccgccgc accatcgccg
tgtcctccgg gcgcgcctcc 120cagctgcgca agcccgccct ggaccccctg cgcgccgtga
tctccgccga ccagggctcc 180atctcccccg tgaactcctg cacccccgcc gaccgcctgc
gcgccggccg cctgatggag 240gacggctact cctacaagga gaagttcatc gtgcgctcct
acgaggtggg catcaacaag 300accgccaccg tggagaccat cgccaacctg ctgcaggagg
tggcctgcaa ccacgtgcag 360aagtgcggct tctccaccga cggcttcgcc accaccctga
ccatgcgcaa gctgcacctg 420atctgggtga ccgcccgcat gcacatcgag atctacaagt
accccgcctg gtccgacgtg 480gtggagatcg agacctggtg ccagtccgag ggccgcatcg
gcacccgccg cgactggatc 540ctgcgcgact ccgccaccaa cgaggtgatc ggccgcgcca
cctccaagtg ggtgatgatg 600aaccaggaca cccgccgcct gcagcgcgtg accgacgagg
tgcgcgacga gtacctggtg 660ttctgccccc gcgagccccg cctggccttc cccgaggaga
acaactcctc cctgaagaag 720atccccaagc tggaggaccc cgcccagtac tccatgctgg
agctgaagcc ccgccgcgcc 780gacctggaca tgaaccagca cgtgaacaac gtgacctaca
tcggctgggt gctggagtcc 840atcccccagg agatcatcga cacccacgag ctgcaggtga
tcaccctgga ctaccgccgc 900gagtgccagc aggacgacat cgtggactcc ctgaccacct
ccgagatccc cgacgacccc 960atctccaagt tcaccggcac caacggctcc gccatgtcct
ccatccaggg ccacaacgag 1020tcccagttcc tgcacatgct gcgcctgtcc gagaacggcc
aggagatcaa ccgcggccgc 1080acccagtggc gcaagaagtc ctcccgcatg gactacaagg
accacgacgg cgactacaag 1140gaccacgaca tcgactacaa ggacgacgac gacaagtga
117965392PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 65Met Leu Lys Leu Ser Cys
Asn Val Thr Asn Asn Leu His Thr Phe Ser 1 5
10 15 Phe Phe Ser Asp Ser Ser Leu Phe Ile Pro Val
Asn Arg Arg Thr Ile 20 25
30 Ala Val Ser Ser Gly Arg Ala Ser Gln Leu Arg Lys Pro Ala Leu
Asp 35 40 45 Pro
Leu Arg Ala Val Ile Ser Ala Asp Gln Gly Ser Ile Ser Pro Val 50
55 60 Asn Ser Cys Thr Pro Ala
Asp Arg Leu Arg Ala Gly Arg Leu Met Glu 65 70
75 80 Asp Gly Tyr Ser Tyr Lys Glu Lys Phe Ile Val
Arg Ser Tyr Glu Val 85 90
95 Gly Ile Asn Lys Thr Ala Thr Val Glu Thr Ile Ala Asn Leu Leu Gln
100 105 110 Glu Val
Ala Cys Asn His Val Gln Lys Cys Gly Phe Ser Thr Asp Gly 115
120 125 Phe Ala Thr Thr Leu Thr Met
Arg Lys Leu His Leu Ile Trp Val Thr 130 135
140 Ala Arg Met His Ile Glu Ile Tyr Lys Tyr Pro Ala
Trp Ser Asp Val 145 150 155
160 Val Glu Ile Glu Thr Trp Cys Gln Ser Glu Gly Arg Ile Gly Thr Arg
165 170 175 Arg Asp Trp
Ile Leu Arg Asp Ser Ala Thr Asn Glu Val Ile Gly Arg 180
185 190 Ala Thr Ser Lys Trp Val Met Met
Asn Gln Asp Thr Arg Arg Leu Gln 195 200
205 Arg Val Thr Asp Glu Val Arg Asp Glu Tyr Leu Val Phe
Cys Pro Arg 210 215 220
Glu Pro Arg Leu Ala Phe Pro Glu Glu Asn Asn Ser Ser Leu Lys Lys 225
230 235 240 Ile Pro Lys Leu
Glu Asp Pro Ala Gln Tyr Ser Met Leu Glu Leu Lys 245
250 255 Pro Arg Arg Ala Asp Leu Asp Met Asn
Gln His Val Asn Asn Val Thr 260 265
270 Tyr Ile Gly Trp Val Leu Glu Ser Ile Pro Gln Glu Ile Ile
Asp Thr 275 280 285
His Glu Leu Gln Val Ile Thr Leu Asp Tyr Arg Arg Glu Cys Gln Gln 290
295 300 Asp Asp Ile Val Asp
Ser Leu Thr Thr Ser Glu Ile Pro Asp Asp Pro 305 310
315 320 Ile Ser Lys Phe Thr Gly Thr Asn Gly Ser
Ala Met Ser Ser Ile Gln 325 330
335 Gly His Asn Glu Ser Gln Phe Leu His Met Leu Arg Leu Ser Glu
Asn 340 345 350 Gly
Gln Glu Ile Asn Arg Gly Arg Thr Gln Trp Arg Lys Lys Ser Ser 355
360 365 Arg Met Asp Tyr Lys Asp
His Asp Gly Asp Tyr Lys Asp His Asp Ile 370 375
380 Asp Tyr Lys Asp Asp Asp Asp Lys 385
390 661176DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 66atggccaccg catccacttt ctcggcgttc
aatgcccgct gcggcgacct gcgtcgctcg 60gcgggctccg ggccccggcg cccagcgagg
cccctccccg tgcgcgggcg cgcctcccag 120ctgcgcaagc ccgccctgga ccccctgcgc
gccgtgatct ccgccgacca gggctccatc 180tcccccgtga actcctgcac ccccgccgac
cgcctgcgcg ccggccgcct gatggaggac 240ggctactcct acaaggagaa gttcatcgtg
cgctcctacg aggtgggcat caacaagacc 300gccaccgtgg agaccatcgc caacctgctg
caggaggtgg cctgcaacca cgtgcagaag 360tgcggcttct ccaccgacgg cttcgccacc
accctgacca tgcgcaagct gcacctgatc 420tgggtgaccg cccgcatgca catcgagatc
tacaagtacc ccgcctggtc cgacgtggtg 480gagatcgaga cctggtgcca gtccgagggc
cgcatcggca cccgccgcga ctggatcctg 540cgcgactccg ccaccaacga ggtgatcggc
cgcgccacct ccaagtgggt gatgatgaac 600caggacaccc gccgcctgca gcgcgtgacc
gacgaggtgc gcgacgagta cctggtgttc 660tgcccccgcg agccccgcct ggccttcccc
gaggagaaca actcctccct gaagaagatc 720cccaagctgg aggaccccgc ccagtactcc
atgctggagc tgaagccccg ccgcgccgac 780ctggacatga accagcacgt gaacaacgtg
acctacatcg gctgggtgct ggagtccatc 840ccccaggaga tcatcgacac ccacgagctg
caggtgatca ccctggacta ccgccgcgag 900tgccagcagg acgacatcgt ggactccctg
accacctccg agatccccga cgaccccatc 960tccaagttca ccggcaccaa cggctccgcc
atgtcctcca tccagggcca caacgagtcc 1020cagttcctgc acatgctgcg cctgtccgag
aacggccagg agatcaaccg cggccgcacc 1080cagtggcgca agaagtcctc ccgcatggac
tacaaggacc acgacggcga ctacaaggac 1140cacgacatcg actacaagga cgacgacgac
aagtga 117667391PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Met Ala Thr Ala Ser Thr Phe Ser Ala Phe Asn Ala Arg Cys Gly Asp 1
5 10 15 Leu Arg Arg Ser
Ala Gly Ser Gly Pro Arg Arg Pro Ala Arg Pro Leu 20
25 30 Pro Val Arg Gly Arg Ala Ser Gln Leu
Arg Lys Pro Ala Leu Asp Pro 35 40
45 Leu Arg Ala Val Ile Ser Ala Asp Gln Gly Ser Ile Ser Pro
Val Asn 50 55 60
Ser Cys Thr Pro Ala Asp Arg Leu Arg Ala Gly Arg Leu Met Glu Asp 65
70 75 80 Gly Tyr Ser Tyr Lys
Glu Lys Phe Ile Val Arg Ser Tyr Glu Val Gly 85
90 95 Ile Asn Lys Thr Ala Thr Val Glu Thr Ile
Ala Asn Leu Leu Gln Glu 100 105
110 Val Ala Cys Asn His Val Gln Lys Cys Gly Phe Ser Thr Asp Gly
Phe 115 120 125 Ala
Thr Thr Leu Thr Met Arg Lys Leu His Leu Ile Trp Val Thr Ala 130
135 140 Arg Met His Ile Glu Ile
Tyr Lys Tyr Pro Ala Trp Ser Asp Val Val 145 150
155 160 Glu Ile Glu Thr Trp Cys Gln Ser Glu Gly Arg
Ile Gly Thr Arg Arg 165 170
175 Asp Trp Ile Leu Arg Asp Ser Ala Thr Asn Glu Val Ile Gly Arg Ala
180 185 190 Thr Ser
Lys Trp Val Met Met Asn Gln Asp Thr Arg Arg Leu Gln Arg 195
200 205 Val Thr Asp Glu Val Arg Asp
Glu Tyr Leu Val Phe Cys Pro Arg Glu 210 215
220 Pro Arg Leu Ala Phe Pro Glu Glu Asn Asn Ser Ser
Leu Lys Lys Ile 225 230 235
240 Pro Lys Leu Glu Asp Pro Ala Gln Tyr Ser Met Leu Glu Leu Lys Pro
245 250 255 Arg Arg Ala
Asp Leu Asp Met Asn Gln His Val Asn Asn Val Thr Tyr 260
265 270 Ile Gly Trp Val Leu Glu Ser Ile
Pro Gln Glu Ile Ile Asp Thr His 275 280
285 Glu Leu Gln Val Ile Thr Leu Asp Tyr Arg Arg Glu Cys
Gln Gln Asp 290 295 300
Asp Ile Val Asp Ser Leu Thr Thr Ser Glu Ile Pro Asp Asp Pro Ile 305
310 315 320 Ser Lys Phe Thr
Gly Thr Asn Gly Ser Ala Met Ser Ser Ile Gln Gly 325
330 335 His Asn Glu Ser Gln Phe Leu His Met
Leu Arg Leu Ser Glu Asn Gly 340 345
350 Gln Glu Ile Asn Arg Gly Arg Thr Gln Trp Arg Lys Lys Ser
Ser Arg 355 360 365
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp 370
375 380 Tyr Lys Asp Asp Asp
Asp Lys 385 390 681155DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
68atggccaccg catccacttt ctcggcgttc aatgcccgct gcggcgacct gcgtcgctcg
60gcgggctccg ggccccggcg cccagcgagg cccctccccg tgcgcgggcg cgccgccacc
120ggcgagcagc cctccggcgt ggcctccctg cgcgaggccg acaaggagaa gtccctgggc
180aaccgcctgc gcctgggctc cctgaccgag gacggcctgt cctacaagga gaagttcgtg
240atccgctgct acgaggtggg catcaacaag accgccacca tcgagaccat cgccaacctg
300ctgcaggagg tgggcggcaa ccacgcccag ggcgtgggct tctccaccga cggcttcgcc
360accaccacca ccatgcgcaa gctgcacctg atctgggtga ccgcccgcat gcacatcgag
420atctaccgct accccgcctg gtccgacgtg atcgagatcg agacctgggt gcagggcgag
480ggcaaggtgg gcacccgccg cgactggatc ctgaaggact acgccaacgg cgaggtgatc
540ggccgcgcca cctccaagtg ggtgatgatg aacgaggaca cccgccgcct gcagaaggtg
600tccgacgacg tgcgcgagga gtacctggtg ttctgccccc gcaccctgcg cctggccttc
660cccgaggaga acaacaactc catgaagaag atccccaagc tggaggaccc cgccgagtac
720tcccgcctgg gcctggtgcc ccgccgctcc gacctggaca tgaacaagca cgtgaacaac
780gtgacctaca tcggctgggc cctggagtcc atcccccccg agatcatcga cacccacgag
840ctgcaggcca tcaccctgga ctaccgccgc gagtgccagc gcgacgacat cgtggactcc
900ctgacctccc gcgagcccct gggcaacgcc gccggcgtga agttcaagga gatcaacggc
960tccgtgtccc ccaagaagga cgagcaggac ctgtcccgct tcatgcacct gctgcgctcc
1020gccggctccg gcctggagat caaccgctgc cgcaccgagt ggcgcaagaa gcccgccaag
1080cgcatggact acaaggacca cgacggcgac tacaaggacc acgacatcga ctacaaggac
1140gacgacgaca agtga
115569384PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Met Ala Thr Ala Ser Thr Phe Ser Ala Phe Asn
Ala Arg Cys Gly Asp 1 5 10
15 Leu Arg Arg Ser Ala Gly Ser Gly Pro Arg Arg Pro Ala Arg Pro Leu
20 25 30 Pro Val
Arg Gly Arg Ala Ala Thr Gly Glu Gln Pro Ser Gly Val Ala 35
40 45 Ser Leu Arg Glu Ala Asp Lys
Glu Lys Ser Leu Gly Asn Arg Leu Arg 50 55
60 Leu Gly Ser Leu Thr Glu Asp Gly Leu Ser Tyr Lys
Glu Lys Phe Val 65 70 75
80 Ile Arg Cys Tyr Glu Val Gly Ile Asn Lys Thr Ala Thr Ile Glu Thr
85 90 95 Ile Ala Asn
Leu Leu Gln Glu Val Gly Gly Asn His Ala Gln Gly Val 100
105 110 Gly Phe Ser Thr Asp Gly Phe Ala
Thr Thr Thr Thr Met Arg Lys Leu 115 120
125 His Leu Ile Trp Val Thr Ala Arg Met His Ile Glu Ile
Tyr Arg Tyr 130 135 140
Pro Ala Trp Ser Asp Val Ile Glu Ile Glu Thr Trp Val Gln Gly Glu 145
150 155 160 Gly Lys Val Gly
Thr Arg Arg Asp Trp Ile Leu Lys Asp Tyr Ala Asn 165
170 175 Gly Glu Val Ile Gly Arg Ala Thr Ser
Lys Trp Val Met Met Asn Glu 180 185
190 Asp Thr Arg Arg Leu Gln Lys Val Ser Asp Asp Val Arg Glu
Glu Tyr 195 200 205
Leu Val Phe Cys Pro Arg Thr Leu Arg Leu Ala Phe Pro Glu Glu Asn 210
215 220 Asn Asn Ser Met Lys
Lys Ile Pro Lys Leu Glu Asp Pro Ala Glu Tyr 225 230
235 240 Ser Arg Leu Gly Leu Val Pro Arg Arg Ser
Asp Leu Asp Met Asn Lys 245 250
255 His Val Asn Asn Val Thr Tyr Ile Gly Trp Ala Leu Glu Ser Ile
Pro 260 265 270 Pro
Glu Ile Ile Asp Thr His Glu Leu Gln Ala Ile Thr Leu Asp Tyr 275
280 285 Arg Arg Glu Cys Gln Arg
Asp Asp Ile Val Asp Ser Leu Thr Ser Arg 290 295
300 Glu Pro Leu Gly Asn Ala Ala Gly Val Lys Phe
Lys Glu Ile Asn Gly 305 310 315
320 Ser Val Ser Pro Lys Lys Asp Glu Gln Asp Leu Ser Arg Phe Met His
325 330 335 Leu Leu
Arg Ser Ala Gly Ser Gly Leu Glu Ile Asn Arg Cys Arg Thr 340
345 350 Glu Trp Arg Lys Lys Pro Ala
Lys Arg Met Asp Tyr Lys Asp His Asp 355 360
365 Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp
Asp Asp Asp Lys 370 375 380
701194DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 70atgctgaagg tgccctgctg caacgccacc
gaccccatcc agtccctgtc ctcccagtgc 60cgcttcctga cccacttcaa caaccgcccc
tacttcaccc gccgcccctc catccccacc 120ttcttctcct ccaagaactc ctccgcctcc
ctgcaggccg tggtgtccga catctcctcc 180gtggagtccg ccgcctgcga ctccctggcc
aaccgcctgc gcctgggcaa gctgaccgag 240gacggcttct cctacaagga gaagttcatc
gtggggcgcg cccgctccta cgaggtgggc 300atcaacaaga ccgccaccgt ggagaccatc
gccaacctgc tgcaggaggt gggctgcaac 360cacgcccagt ccgtgggctt ctccaccgac
ggcttcgcca ccaccacctc catgcgcaag 420atgcacctga tctgggtgac cgcccgcatg
cacatcgaga tctacaagta ccccgcctgg 480tccgacgtgg tggaggtgga gacctggtgc
cagtccgagg gccgcatcgg cacccgccgc 540gactggatcc tgaccgacta cgccaccggc
cagatcatcg gccgcgccac ctccaagtgg 600gtgatgatga accaggacac ccgccgcctg
cagaaggtga ccgacgacgt gcgcgaggag 660tacctggtgt tctgcccccg cgagctgcgc
ctggccttcc ccgaggagaa caaccgctcc 720tccaagaaga tctccaagct ggaggacccc
gcccagtact ccaagctggg cctggtgccc 780cgccgcgccg acctggacat gaaccagcac
gtgaacaacg tgacctacat cggctgggtg 840ctggagtcca tcccccagga gatcatcgac
acccacgagc tgcagaccat caccctggac 900taccgccgcg agtgccagca cgacgacatc
gtggactccc tgacctccgt ggagccctcc 960gagaacctgg aggccgtgtc cgagctgcgc
ggcaccaacg gctccgccac caccaccgcc 1020ggcgacgagg actgccgcaa cttcctgcac
ctgctgcgcc tgtccggcga cggcctggag 1080atcaaccgcg gccgcaccga gtggcgcaag
aagtccgccc gcatggacta caaggaccac 1140gacggcgact acaaggacca cgacatcgac
tacaaggacg acgacgacaa gtga 119471397PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
71Met Leu Lys Val Pro Cys Cys Asn Ala Thr Asp Pro Ile Gln Ser Leu 1
5 10 15 Ser Ser Gln Cys
Arg Phe Leu Thr His Phe Asn Asn Arg Pro Tyr Phe 20
25 30 Thr Arg Arg Pro Ser Ile Pro Thr Phe
Phe Ser Ser Lys Asn Ser Ser 35 40
45 Ala Ser Leu Gln Ala Val Val Ser Asp Ile Ser Ser Val Glu
Ser Ala 50 55 60
Ala Cys Asp Ser Leu Ala Asn Arg Leu Arg Leu Gly Lys Leu Thr Glu 65
70 75 80 Asp Gly Phe Ser Tyr
Lys Glu Lys Phe Ile Val Gly Arg Ala Arg Ser 85
90 95 Tyr Glu Val Gly Ile Asn Lys Thr Ala Thr
Val Glu Thr Ile Ala Asn 100 105
110 Leu Leu Gln Glu Val Gly Cys Asn His Ala Gln Ser Val Gly Phe
Ser 115 120 125 Thr
Asp Gly Phe Ala Thr Thr Thr Ser Met Arg Lys Met His Leu Ile 130
135 140 Trp Val Thr Ala Arg Met
His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp 145 150
155 160 Ser Asp Val Val Glu Val Glu Thr Trp Cys Gln
Ser Glu Gly Arg Ile 165 170
175 Gly Thr Arg Arg Asp Trp Ile Leu Thr Asp Tyr Ala Thr Gly Gln Ile
180 185 190 Ile Gly
Arg Ala Thr Ser Lys Trp Val Met Met Asn Gln Asp Thr Arg 195
200 205 Arg Leu Gln Lys Val Thr Asp
Asp Val Arg Glu Glu Tyr Leu Val Phe 210 215
220 Cys Pro Arg Glu Leu Arg Leu Ala Phe Pro Glu Glu
Asn Asn Arg Ser 225 230 235
240 Ser Lys Lys Ile Ser Lys Leu Glu Asp Pro Ala Gln Tyr Ser Lys Leu
245 250 255 Gly Leu Val
Pro Arg Arg Ala Asp Leu Asp Met Asn Gln His Val Asn 260
265 270 Asn Val Thr Tyr Ile Gly Trp Val
Leu Glu Ser Ile Pro Gln Glu Ile 275 280
285 Ile Asp Thr His Glu Leu Gln Thr Ile Thr Leu Asp Tyr
Arg Arg Glu 290 295 300
Cys Gln His Asp Asp Ile Val Asp Ser Leu Thr Ser Val Glu Pro Ser 305
310 315 320 Glu Asn Leu Glu
Ala Val Ser Glu Leu Arg Gly Thr Asn Gly Ser Ala 325
330 335 Thr Thr Thr Ala Gly Asp Glu Asp Cys
Arg Asn Phe Leu His Leu Leu 340 345
350 Arg Leu Ser Gly Asp Gly Leu Glu Ile Asn Arg Gly Arg Thr
Glu Trp 355 360 365
Arg Lys Lys Ser Ala Arg Met Asp Tyr Lys Asp His Asp Gly Asp Tyr 370
375 380 Lys Asp His Asp Ile
Asp Tyr Lys Asp Asp Asp Asp Lys 385 390
395 721128DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 72atgctgaagc tgtcctcctc ccgctccccc
ctggcccgca tccccacccg cccccgcccc 60aactccatcc ccccccgcat catcgtggtg
tcctcctcct cctccaaggt gaaccccctg 120aagaccgagg ccgtggtgtc ctccggcctg
gccgaccgcc tgcgcctggg ctccctgacc 180gaggacggcc tgtcctacaa ggagaagttc
atcgtgcgct gctacgaggt gggcatcaac 240aagaccgcca ccgtggagac catcgccaac
ctgctgcagg aggtgggctg caaccacgcc 300cagtccgtgg gctactccac cggcggcttc
tccaccaccc ccaccatgcg caagctgcgc 360ctgatctggg tgaccgcccg catgcacatc
gagatctaca agtaccccgc ctggtccgac 420gtggtggaga tcgagtcctg gggccagggc
gagggcaaga tcggcacccg ccgcgactgg 480atcctgcgcg actacgccac cggccaggtg
atcggccgcg ccacctccaa gtgggtgatg 540atgaaccagg acacccgccg cctgcagaag
gtggacgtgg acgtgcgcga cgagtacctg 600gtgcactgcc cccgcgagct gcgcctggcc
ttccccgagg agaacaactc ctccctgaag 660aagatctcca agctggagga cccctcccag
tactccaagc tgggcctggt gccccgccgc 720gccgacctgg acatgaacca gcacgtgaac
aacgtgacct acatcggctg ggtgctggag 780tccatgcccc aggagatcat cgacacccac
gagctgcaga ccatcaccct ggactaccgc 840cgcgagtgcc agcacgacga cgtggtggac
tccctgacct cccccgagcc ctccgaggac 900gccgaggccg tgttcaacca caacggcacc
aacggctccg ccaacgtgtc cgccaacgac 960cacggctgcc gcaacttcct gcacctgctg
cgcctgtccg gcaacggcct ggagatcaac 1020cgcggccgca ccgagtggcg caagaagccc
acccgcatgg actacaagga ccacgacggc 1080gactacaagg accacgacat cgactacaag
gacgacgacg acaagtga 112873375PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
73Met Leu Lys Leu Ser Ser Ser Arg Ser Pro Leu Ala Arg Ile Pro Thr 1
5 10 15 Arg Pro Arg Pro
Asn Ser Ile Pro Pro Arg Ile Ile Val Val Ser Ser 20
25 30 Ser Ser Ser Lys Val Asn Pro Leu Lys
Thr Glu Ala Val Val Ser Ser 35 40
45 Gly Leu Ala Asp Arg Leu Arg Leu Gly Ser Leu Thr Glu Asp
Gly Leu 50 55 60
Ser Tyr Lys Glu Lys Phe Ile Val Arg Cys Tyr Glu Val Gly Ile Asn 65
70 75 80 Lys Thr Ala Thr Val
Glu Thr Ile Ala Asn Leu Leu Gln Glu Val Gly 85
90 95 Cys Asn His Ala Gln Ser Val Gly Tyr Ser
Thr Gly Gly Phe Ser Thr 100 105
110 Thr Pro Thr Met Arg Lys Leu Arg Leu Ile Trp Val Thr Ala Arg
Met 115 120 125 His
Ile Glu Ile Tyr Lys Tyr Pro Ala Trp Ser Asp Val Val Glu Ile 130
135 140 Glu Ser Trp Gly Gln Gly
Glu Gly Lys Ile Gly Thr Arg Arg Asp Trp 145 150
155 160 Ile Leu Arg Asp Tyr Ala Thr Gly Gln Val Ile
Gly Arg Ala Thr Ser 165 170
175 Lys Trp Val Met Met Asn Gln Asp Thr Arg Arg Leu Gln Lys Val Asp
180 185 190 Val Asp
Val Arg Asp Glu Tyr Leu Val His Cys Pro Arg Glu Leu Arg 195
200 205 Leu Ala Phe Pro Glu Glu Asn
Asn Ser Ser Leu Lys Lys Ile Ser Lys 210 215
220 Leu Glu Asp Pro Ser Gln Tyr Ser Lys Leu Gly Leu
Val Pro Arg Arg 225 230 235
240 Ala Asp Leu Asp Met Asn Gln His Val Asn Asn Val Thr Tyr Ile Gly
245 250 255 Trp Val Leu
Glu Ser Met Pro Gln Glu Ile Ile Asp Thr His Glu Leu 260
265 270 Gln Thr Ile Thr Leu Asp Tyr Arg
Arg Glu Cys Gln His Asp Asp Val 275 280
285 Val Asp Ser Leu Thr Ser Pro Glu Pro Ser Glu Asp Ala
Glu Ala Val 290 295 300
Phe Asn His Asn Gly Thr Asn Gly Ser Ala Asn Val Ser Ala Asn Asp 305
310 315 320 His Gly Cys Arg
Asn Phe Leu His Leu Leu Arg Leu Ser Gly Asn Gly 325
330 335 Leu Glu Ile Asn Arg Gly Arg Thr Glu
Trp Arg Lys Lys Pro Thr Arg 340 345
350 Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
Ile Asp 355 360 365
Tyr Lys Asp Asp Asp Asp Lys 370 375
741197DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 74atgctgaagc tgtcctcctg caacgtgacc gaccagcgcc
aggccctggc ccagtgccgc 60ttcctggccc cccccgcccc cttctccttc cgctggcgca
cccccgtggt ggtgtcctgc 120tccccctcct cccgccccaa cctgtccccc ctgcaggtgg
tgctgtccgg ccagcagcag 180gccggcatgg agctggtgga gtccggctcc ggctccctgg
ccgaccgcct gcgcctgggc 240tccctgaccg aggacggcct gtcctacaag gagaagttca
tcgtgcgctg ctacgaggtg 300ggcatcaaca agaccgccac cgtggagacc atcgccaacc
tgctgcagga ggtgggctgc 360aaccacgccc agtccgtggg ctactccacc gacggcttcg
ccaccacccg caccatgcgc 420aagctgcacc tgatctgggt gaccgcccgc atgcacatcg
agatctacaa gtaccccgcc 480tggtccgacg tgatcgagat cgagacctgg tgccagtccg
agggccgcat cggcacccgc 540cgcgactgga tcctgaagga cttcggcacc ggcgaggtga
tcggccgcgc cacctccaag 600tgggtgatga tgaaccagga cacccgccgc ctgcagaagg
tgtccgacga cgtgcgcgag 660gagtacctgg tgttctgccc ccgcgagctg cgcctggcct
tccccgagga gaacaacaac 720tccctgaaga agatcgccaa gctggacgac tccttccagt
actcccgcct gggcctgatg 780ccccgccgcg ccgacctgga catgaaccag cacgtgaaca
acgtgaccta catcggctgg 840gtgctggagt ccatgcccca ggagatcatc gacacccacg
agctgcagac catcaccctg 900gactaccgcc gcgagtgcca gcaggacgac gtggtggact
ccctgacctc ccccgagcag 960gtggagggca ccgagaaggt gtccgccatc cacggcacca
acggctccgc cgccgcccgc 1020gaggacaagc aggactgccg ccagttcctg cacctgctgc
gcctgtcctc cgacggccag 1080gagatcaacc gcggccgcac cgagtggcgc aagaagcccg
cccgcatgga ctacaaggac 1140cacgacggcg actacaagga ccacgacatc gactacaagg
acgacgacga caagtga 119775398PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 75Met Leu Lys Leu Ser Ser
Cys Asn Val Thr Asp Gln Arg Gln Ala Leu 1 5
10 15 Ala Gln Cys Arg Phe Leu Ala Pro Pro Ala Pro
Phe Ser Phe Arg Trp 20 25
30 Arg Thr Pro Val Val Val Ser Cys Ser Pro Ser Ser Arg Pro Asn
Leu 35 40 45 Ser
Pro Leu Gln Val Val Leu Ser Gly Gln Gln Gln Ala Gly Met Glu 50
55 60 Leu Val Glu Ser Gly Ser
Gly Ser Leu Ala Asp Arg Leu Arg Leu Gly 65 70
75 80 Ser Leu Thr Glu Asp Gly Leu Ser Tyr Lys Glu
Lys Phe Ile Val Arg 85 90
95 Cys Tyr Glu Val Gly Ile Asn Lys Thr Ala Thr Val Glu Thr Ile Ala
100 105 110 Asn Leu
Leu Gln Glu Val Gly Cys Asn His Ala Gln Ser Val Gly Tyr 115
120 125 Ser Thr Asp Gly Phe Ala Thr
Thr Arg Thr Met Arg Lys Leu His Leu 130 135
140 Ile Trp Val Thr Ala Arg Met His Ile Glu Ile Tyr
Lys Tyr Pro Ala 145 150 155
160 Trp Ser Asp Val Ile Glu Ile Glu Thr Trp Cys Gln Ser Glu Gly Arg
165 170 175 Ile Gly Thr
Arg Arg Asp Trp Ile Leu Lys Asp Phe Gly Thr Gly Glu 180
185 190 Val Ile Gly Arg Ala Thr Ser Lys
Trp Val Met Met Asn Gln Asp Thr 195 200
205 Arg Arg Leu Gln Lys Val Ser Asp Asp Val Arg Glu Glu
Tyr Leu Val 210 215 220
Phe Cys Pro Arg Glu Leu Arg Leu Ala Phe Pro Glu Glu Asn Asn Asn 225
230 235 240 Ser Leu Lys Lys
Ile Ala Lys Leu Asp Asp Ser Phe Gln Tyr Ser Arg 245
250 255 Leu Gly Leu Met Pro Arg Arg Ala Asp
Leu Asp Met Asn Gln His Val 260 265
270 Asn Asn Val Thr Tyr Ile Gly Trp Val Leu Glu Ser Met Pro
Gln Glu 275 280 285
Ile Ile Asp Thr His Glu Leu Gln Thr Ile Thr Leu Asp Tyr Arg Arg 290
295 300 Glu Cys Gln Gln Asp
Asp Val Val Asp Ser Leu Thr Ser Pro Glu Gln 305 310
315 320 Val Glu Gly Thr Glu Lys Val Ser Ala Ile
His Gly Thr Asn Gly Ser 325 330
335 Ala Ala Ala Arg Glu Asp Lys Gln Asp Cys Arg Gln Phe Leu His
Leu 340 345 350 Leu
Arg Leu Ser Ser Asp Gly Gln Glu Ile Asn Arg Gly Arg Thr Glu 355
360 365 Trp Arg Lys Lys Pro Ala
Arg Met Asp Tyr Lys Asp His Asp Gly Asp 370 375
380 Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
Asp Asp Lys 385 390 395
76573DNAPrototheca moriformis 76tgttgaagaa tgagccggcg acttaaaata
aatggcaggc taagagaatt aataactcga 60aacctaagcg aaagcaagtc ttaatagggc
gctaatttaa caaaacatta aataaaatct 120aaagtcattt attttagacc cgaacctgag
tgatctaacc atggtcagga tgaaacttgg 180gtgacaccaa gtggaagtcc gaaccgaccg
atgttgaaaa atcggcggat gaactgtggt 240tagtggtgaa ataccagtcg aactcagagc
tagctggttc tccccgaaat gcgttgaggc 300gcagcaatat atctcgtcta tctaggggta
aagcactgtt tcggtgcggg ctatgaaaat 360ggtaccaaat cgtggcaaac tctgaatact
agaaatgacg atatattagt gagactatgg 420gggataagct ccatagtcga gagggaaaca
gcccagacca ccagttaagg ccccaaaatg 480ataatgaagt ggtaaaggag gtgaaaatgc
aaatacaacc aggaggttgg cttagaagca 540gccatccttt aaagagtgcg taatagctca
ctg 57377384PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
77Met Ala Ile Ala Ala Ala Ala Val Ile Phe Leu Phe Gly Leu Ile Phe 1
5 10 15 Phe Ala Ser Gly
Leu Ile Ile Asn Leu Phe Gln Ala Leu Cys Phe Val 20
25 30 Leu Ile Arg Pro Leu Ser Lys Asn Ala
Tyr Arg Arg Ile Asn Arg Val 35 40
45 Phe Ala Glu Leu Leu Leu Ser Glu Leu Leu Cys Leu Phe Asp
Trp Trp 50 55 60
Ala Gly Ala Lys Leu Lys Leu Phe Thr Asp Pro Glu Thr Phe Arg Leu 65
70 75 80 Met Gly Lys Glu His
Ala Leu Val Ile Ile Asn His Met Thr Glu Leu 85
90 95 Asp Trp Met Val Gly Trp Val Met Gly Gln
His Phe Gly Cys Leu Gly 100 105
110 Ser Ile Ile Ser Val Ala Lys Lys Ser Thr Lys Phe Leu Pro Val
Leu 115 120 125 Gly
Trp Ser Met Trp Phe Ser Glu Tyr Leu Tyr Leu Glu Arg Ser Trp 130
135 140 Ala Lys Asp Lys Ser Thr
Leu Lys Ser His Ile Glu Arg Leu Ile Asp 145 150
155 160 Tyr Pro Leu Pro Phe Trp Leu Val Ile Phe Val
Glu Gly Thr Arg Phe 165 170
175 Thr Arg Thr Lys Leu Leu Ala Ala Gln Gln Tyr Ala Val Ser Ser Gly
180 185 190 Leu Pro
Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val 195
200 205 Ser Cys Val Ser His Met Arg
Ser Phe Val Pro Ala Val Tyr Asp Val 210 215
220 Thr Val Ala Phe Pro Lys Thr Ser Pro Pro Pro Thr
Leu Leu Asn Leu 225 230 235
240 Phe Glu Gly Gln Ser Ile Met Leu His Val His Ile Lys Arg His Ala
245 250 255 Met Lys Asp
Leu Pro Glu Ser Asp Asp Ala Val Ala Glu Trp Cys Arg 260
265 270 Asp Lys Phe Val Glu Lys Asp Ala
Leu Leu Asp Lys His Asn Ala Glu 275 280
285 Asp Thr Phe Ser Gly Gln Glu Val Cys His Ser Gly Ser
Arg Gln Leu 290 295 300
Lys Ser Leu Leu Val Val Ile Ser Trp Val Val Val Thr Thr Phe Gly 305
310 315 320 Ala Leu Lys Phe
Leu Gln Trp Ser Ser Trp Lys Gly Lys Ala Phe Ser 325
330 335 Ala Ile Gly Leu Gly Ile Val Thr Leu
Leu Met His Val Leu Ile Leu 340 345
350 Ser Ser Gln Ala Glu Arg Ser Asn Pro Ala Glu Val Ala Gln
Ala Lys 355 360 365
Leu Lys Thr Gly Leu Ser Ile Ser Lys Lys Val Thr Asp Lys Glu Asn 370
375 380 78387PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
78Met Ala Ile Ala Ala Ala Ala Val Ile Val Pro Leu Ser Leu Leu Phe 1
5 10 15 Phe Val Ser Gly
Leu Ile Val Asn Leu Val Gln Ala Val Cys Phe Val 20
25 30 Leu Ile Arg Pro Leu Ser Lys Asn Thr
Tyr Arg Arg Ile Asn Arg Val 35 40
45 Val Ala Glu Leu Leu Trp Leu Glu Leu Val Trp Leu Ile Asp
Trp Trp 50 55 60
Ala Gly Val Lys Ile Lys Val Phe Thr Asp His Glu Thr Phe His Leu 65
70 75 80 Met Gly Lys Glu His
Ala Leu Val Ile Cys Asn His Lys Ser Asp Ile 85
90 95 Asp Trp Leu Val Gly Trp Val Leu Gly Gln
Arg Ser Gly Cys Leu Gly 100 105
110 Ser Thr Leu Ala Val Met Lys Lys Ser Ser Lys Phe Leu Pro Val
Leu 115 120 125 Gly
Trp Ser Met Trp Phe Ser Glu Tyr Leu Phe Leu Glu Arg Ser Trp 130
135 140 Ala Lys Asp Glu Ile Thr
Leu Lys Ser Gly Leu Asn Arg Leu Lys Asp 145 150
155 160 Tyr Pro Leu Pro Phe Trp Leu Ala Leu Phe Val
Glu Gly Thr Arg Phe 165 170
175 Thr Arg Ala Lys Leu Leu Ala Ala Gln Gln Tyr Ala Ala Ser Ser Gly
180 185 190 Leu Pro
Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val 195
200 205 Ser Ser Val Ser His Met Arg
Ser Phe Val Pro Ala Ile Tyr Asp Val 210 215
220 Thr Val Ala Ile Pro Lys Thr Ser Pro Pro Pro Thr
Leu Ile Arg Met 225 230 235
240 Phe Lys Gly Gln Ser Ser Val Leu His Val His Leu Lys Arg His Leu
245 250 255 Met Lys Asp
Leu Pro Glu Ser Asp Asp Ala Val Ala Gln Trp Cys Arg 260
265 270 Asp Ile Phe Val Glu Lys Asp Ala
Leu Leu Asp Lys His Asn Ala Glu 275 280
285 Asp Thr Phe Ser Gly Gln Glu Leu Gln Glu Thr Gly Arg
Pro Ile Lys 290 295 300
Ser Leu Leu Val Val Ile Ser Trp Ala Val Leu Glu Val Phe Gly Ala 305
310 315 320 Val Lys Phe Leu
Gln Trp Ser Ser Leu Leu Ser Ser Trp Lys Gly Leu 325
330 335 Ala Phe Ser Gly Ile Gly Leu Gly Val
Ile Thr Leu Leu Met His Ile 340 345
350 Leu Ile Leu Phe Ser Gln Ser Glu Arg Ser Thr Pro Ala Lys
Val Ala 355 360 365
Pro Ala Lys Pro Lys Asn Glu Gly Glu Ser Ser Lys Thr Glu Met Glu 370
375 380 Lys Glu Lys 385
79327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Met Glu Ile Pro Pro His Cys Leu Cys Ser Pro
Ser Pro Ala Pro Ser 1 5 10
15 Gln Leu Tyr Tyr Lys Lys Lys Lys His Ala Ile Leu Gln Thr Gln Thr
20 25 30 Pro Tyr
Arg Tyr Arg Val Ser Pro Thr Cys Phe Ala Pro Pro Arg Leu 35
40 45 Arg Lys Gln His Pro Tyr Pro
Leu Pro Val Leu Cys Tyr Pro Lys Leu 50 55
60 Leu His Phe Ser Gln Pro Arg Tyr Pro Leu Val Arg
Ser His Leu Ala 65 70 75
80 Glu Ala Gly Val Ala Tyr Arg Pro Gly Tyr Glu Leu Leu Gly Lys Ile
85 90 95 Arg Gly Val
Cys Phe Tyr Ala Val Thr Ala Ala Val Ala Leu Leu Leu 100
105 110 Phe Gln Cys Met Leu Leu Leu His
Pro Phe Val Leu Leu Phe Asp Pro 115 120
125 Phe Pro Arg Lys Ala His His Thr Ile Ala Lys Leu Trp
Ser Ile Cys 130 135 140
Ser Val Ser Leu Phe Tyr Lys Ile His Ile Lys Gly Leu Glu Asn Leu 145
150 155 160 Pro Pro Pro His
Ser Pro Ala Val Tyr Val Ser Asn His Gln Ser Phe 165
170 175 Leu Asp Ile Tyr Thr Leu Leu Thr Leu
Gly Arg Thr Phe Lys Phe Ile 180 185
190 Ser Lys Thr Glu Ile Phe Leu Tyr Pro Ile Ile Gly Trp Ala
Met Tyr 195 200 205
Met Leu Gly Thr Ile Pro Leu Lys Arg Leu Asp Ser Arg Ser Gln Leu 210
215 220 Asp Thr Leu Lys Arg
Cys Met Asp Leu Ile Lys Lys Gly Ala Ser Val 225 230
235 240 Phe Phe Phe Pro Glu Gly Thr Arg Ser Lys
Asp Gly Lys Leu Gly Ala 245 250
255 Phe Lys Lys Gly Ala Phe Ser Ile Ala Ala Lys Ser Lys Val Pro
Val 260 265 270 Val
Pro Ile Thr Leu Ile Gly Thr Gly Lys Ile Met Pro Pro Gly Ser 275
280 285 Glu Leu Thr Val Asn Pro
Gly Thr Val Gln Val Ile Ile His Lys Pro 290 295
300 Ile Glu Gly Ser Asp Ala Glu Ala Met Cys Asn
Glu Ala Arg Ala Thr 305 310 315
320 Ile Ser His Ser Leu Asp Asp 325
80984DNAArtificial SequenceDescription of Artificial Sequence Synthetic
polynucleotide 80atggagatcc cgcctcactg tctctgttcg ccttcgcctg
cgccttcgca attgtattac 60aagaagaaga agcatgccat tctccaaact caaactccct
atagatatag agtttccccg 120acatgctttg cccccccccg attgaggaag cagcatcctt
accctctccc tgtcctctgc 180tatccaaaac tcctccactt cagccagcct aggtaccctc
tggttagatc tcatttggct 240gaagctggtg ttgcttatcg tccaggatac gaattattag
gaaaaataag gggagtgtgt 300ttctatgctg tcactgctgc cgttgccttg cttctatttc
agtgcatgct cctcctccat 360ccctttgtgc tcctcttcga tccatttcca agaaaggctc
accataccat cgccaaactc 420tggtctatct gctctgtttc tcttttttac aagattcaca
tcaagggttt ggaaaatctt 480cccccacccc actctcctgc cgtctatgtc tctaatcatc
agagttttct cgacatctat 540actctcctca ctctcggtag aaccttcaag ttcatcagca
agactgagat ctttctctat 600ccaattatcg gttgggccat gtatatgttg ggtaccattc
ctctcaagcg gttggacagc 660agaagccaat tggacactct taagcgatgt atggatctca
tcaagaaggg agcatccgtc 720tttttcttcc cagagggaac acgaagtaaa gatgggaaac
tgggtgcttt caagaaaggt 780gcattcagca tcgcagcaaa aagcaaggtt cctgttgtgc
cgatcaccct tattggaact 840ggcaagatta tgccacctgg gagcgaactt actgtcaatc
caggaactgt gcaagtaatc 900atacataaac ctatcgaagg aagtgatgca gaagcaatgt
gcaatgaagc tagagccacg 960atttctcact cacttgatga ttaa
984811155DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 81atggcgattg cagcggcagc
tgtcatcttc ctcttcggcc ttatcttctt cgcctccggc 60ctcataatca atctcttcca
ggcgctttgc tttgtcctta ttcggcctct ttcgaaaaac 120gcctacmgga gaataaacag
agtttttgca gaattgttgt tgtcggagct tttatgccta 180ttcgattggt gggctggtgc
taagctcaaa ttatttaccg accctgaaac ctttcgcctt 240atgggcaagg aacatgctct
tgtcataatt aatcacatga ctgaacttga ctggatggtt 300ggatgggtta tgggtcagca
ttttggttgc cttgggagca taatatctgt tgcgaagaaa 360tcaacaaaat ttcttccggt
attggggtgg tcaatgtggt tttcagagta cctatatctt 420gagagaagct gggccaagga
taaaagtaca ttaaagtcac atatcgagag gctgatagac 480taccccctgc ccttctggtt
ggtaattttt gtggaaggaa ctcggtttac tcggacaaaa 540ctcttggcag cccagcagta
tgctgtctca tctgggctac cagtgccgag aaatgttttg 600atcccacgta ctaagggttt
tgtttcatgt gtaagtcaca tgcgatcatt tgttccagca 660gtatatgatg tcacagtggc
attccctaag acttcacctc caccaacgtt gctaaatctt 720ttcgagggtc agtccataat
gcttcacgtt cacatcaagc gacatgcaat gaaagattta 780ccagaatccg atgatgcagt
agcagagtgg tgtagagaca aatttgtgga aaaggatgct 840ttgttggaca agcataatgc
tgaggacact ttcagtggtc aagaagtttg tcatagcggc 900agccgccagt taaagtctct
tctggtggta atatcttggg tggttgtaac aacatttggg 960gctctaaagt tccttcagtg
gtcatcatgg aaggggaaag cattttcagc tatcgggctg 1020ggcatcgtca ctctacttat
gcacgtattg attctatcct cacaagcaga gcggtctaac 1080cctgcggagg tggcacaggc
aaagctaaag accgggttgt cgatctcaaa gaaggtaacg 1140gacaaggaaa actag
1155821164DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
82atggcgattg ctgcggcagc tgtcatcgtc ccgctcagcc tcctcttctt cgtctccggc
60ctcatcgtca atctcgtaca ggcagtttgc tttgtactga ttaggcctct gtcgaaaaac
120acttacagaa gaataaacag agtggttgca gaattgttgt ggttggagtt ggtatggctg
180attgattggt gggctggtgt caagataaaa gtattcacgg atcatgaaac ctttcacctt
240atgggcaaag aacatgctct tgtcatttgt aatcacaaga gtgacataga ctggctggtt
300gggtgggttc tgggacagcg gtcaggttgc cttggaagca cattagctgt tatgaagaaa
360tcatcaaagt ttctcccggt attagggtgg tcaatgtggt tctcagagta tctattcctt
420gaaagaagct gggccaagga tgaaattaca ttaaagtcag gtttgaatag gctgaaagac
480tatcccttac ccttctggtt ggcacttttt gtggaaggaa ctcggttcac tcgagcaaaa
540ctcttggcag cccagcagta tgctgcctct tcggggctac ctgtgccgag aaatgttctg
600atcccgcgta ctaagggttt tgtttcttct gtgagtcaca tgcgatcatt tgttccagcc
660atatatgatg ttacagtggc aatcccaaag acgtcacctc caccaacatt gataagaatg
720ttcaagggac agtcctcagt gcttcacgtc cacctcaagc gacacctaat gaaagattta
780cctgaatcag atgatgctgt tgctcagtgg tgcagagata tattcgtcga gaaggatgct
840ttgttggata agcataatgc tgaggacact ttcagtggcc aagaacttca agaaactggc
900cgcccaataa agtctcttct ggttgtaatc tcttgggcgg tgttggaggt atttggagct
960gtgaagtttc ttcaatggtc atcgctgtta tcatcatgga agggacttgc attttcggga
1020ataggactgg gtgtcatcac gctactcatg cacatactga ttttattctc acaatccgag
1080cggtctaccc ctgcaaaagt ggcaccagca aagccaaaga atgagggaga gtcctccaag
1140acggaaatgg aaaaggaaaa gtag
116483984DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 83atggagatcc ccccccactg cctgtgctcc
ccctcccccg ccccctccca gctgtactac 60aagaagaaga agcacgccat cctgcagacc
cagaccccct accgctaccg cgtgtccccc 120acctgcttcg cccccccccg cctgcgcaag
cagcacccct accccctgcc cgtgctgtgc 180taccccaagc tgctgcactt ctcccagccc
cgctaccccc tggtgcgctc ccacctggcc 240gaggccggcg tggcctaccg ccccggctac
gagctgctgg gcaagatccg cggcgtgtgc 300ttctacgccg tgaccgccgc cgtggccctg
ctgctgttcc agtgcatgct gctgctgcac 360cccttcgtgc tgctgttcga ccccttcccc
cgcaaggccc accacaccat cgccaagctg 420tggtccatct gctccgtgtc cctgttctac
aagatccaca tcaagggcct ggagaacctg 480cccccccccc actcccccgc cgtgtacgtg
tccaaccacc agtccttcct ggacatctac 540accctgctga ccctgggccg caccttcaag
ttcatctcca agaccgagat cttcctgtac 600cccatcatcg gctgggccat gtacatgctg
ggcaccatcc ccctgaagcg cctggactcc 660cgctcccagc tggacaccct gaagcgctgc
atggacctga tcaagaaggg cgcctccgtg 720ttcttcttcc ccgagggcac ccgctccaag
gacggcaagc tgggcgcctt caagaagggc 780gccttctcca tcgccgccaa gtccaaggtg
cccgtggtgc ccatcaccct gatcggcacc 840ggcaagatca tgccccccgg ctccgagctg
accgtgaacc ccggcaccgt gcaggtgatc 900atccacaagc ccatcgaggg ctccgacgcc
gaggccatgt gcaacgaggc ccgcgccacc 960atctcccact ccctggacga ctga
984841155DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
84atggcgatcg cggccgcggc ggtgatcttc ctgttcggcc tgatcttctt cgcctccggc
60ctgatcatca acctgttcca ggcgctgtgc ttcgtcctga tccgccccct gtccaagaac
120gcctaccgcc gcatcaaccg cgtgttcgcg gagctgctgc tgtccgagct gctgtgcctg
180ttcgactggt gggcgggcgc gaagctgaag ctgttcaccg accccgagac gttccgcctg
240atgggcaagg agcacgccct ggtcatcatc aaccacatga ccgagctgga ctggatggtg
300ggctgggtga tgggccagca cttcggctgc ctgggctcca tcatctccgt cgccaagaag
360tccacgaagt tcctgcccgt gctgggctgg tccatgtggt tctccgagta cctgtacctg
420gagcgctcct gggccaagga caagtccacc ctgaagtccc acatcgagcg cctgatcgac
480taccccctgc ccttctggct ggtcatcttc gtcgagggca cccgcttcac gcgcacgaag
540ctgctggcgg cccagcagta cgcggtctcc tccggcctgc ccgtcccccg caacgtcctg
600atcccccgca cgaagggctt cgtctcctgc gtgtcccaca tgcgctcctt cgtccccgcg
660gtgtacgacg tcacggtggc gttccccaag acgtcccccc cccccacgct gctgaacctg
720ttcgagggcc agtccatcat gctgcacgtg cacatcaagc gccacgccat gaaggacctg
780cccgagtccg acgacgccgt cgcggagtgg tgccgcgaca agttcgtcga gaaggacgcc
840ctgctggaca agcacaacgc ggaggacacg ttctccggcc aggaggtgtg ccactccggc
900tcccgccagc tgaagtccct gctggtcgtg atctcctggg tcgtggtgac gacgttcggc
960gccctgaagt tcctgcagtg gtcctcctgg aagggcaagg cgttctccgc catcggcctg
1020ggcatcgtca ccctgctgat gcacgtgctg atcctgtcct cccaggccga gcgctccaac
1080cccgccgagg tggcccaggc caagctgaag accggcctgt ccatctccaa gaaggtgacg
1140gacaaggaga actga
1155851164DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 85atggccatcg cggcggccgc ggtgatcgtg
cccctgtccc tgctgttctt cgtgtccggc 60ctgatcgtca acctggtgca ggccgtctgc
ttcgtcctga tccgccccct gtccaagaac 120acgtaccgcc gcatcaaccg cgtggtcgcg
gagctgctgt ggctggagct ggtgtggctg 180atcgactggt gggcgggcgt gaagatcaag
gtcttcacgg accacgagac gttccacctg 240atgggcaagg agcacgccct ggtcatctgc
aaccacaagt ccgacatcga ctggctggtc 300ggctgggtcc tgggccagcg ctccggctgc
ctgggctcca ccctggcggt catgaagaag 360tcctccaagt tcctgcccgt cctgggctgg
tccatgtggt tctccgagta cctgttcctg 420gagcgctcct gggccaagga cgagatcacg
ctgaagtccg gcctgaaccg cctgaaggac 480taccccctgc ccttctggct ggcgctgttc
gtggagggca cgcgcttcac ccgcgcgaag 540ctgctggcgg cgcagcagta cgccgcgtcc
tccggcctgc ccgtgccccg caacgtgctg 600atcccccgca cgaagggctt cgtgtcctcc
gtgtcccaca tgcgctcctt cgtgcccgcg 660atctacgacg tcaccgtggc catccccaag
acgtcccccc cccccacgct gatccgcatg 720ttcaagggcc agtcctccgt gctgcacgtg
cacctgaagc gccacctgat gaaggacctg 780cccgagtccg acgacgccgt cgcgcagtgg
tgccgcgaca tcttcgtgga gaaggacgcg 840ctgctggaca agcacaacgc cgaggacacc
ttctccggcc aggagctgca ggagaccggc 900cgccccatca agtccctgct ggtcgtcatc
tcctgggccg tcctggaggt gttcggcgcc 960gtcaagttcc tgcagtggtc ctccctgctg
tcctcctgga agggcctggc gttctccggc 1020atcggcctgg gcgtgatcac cctgctgatg
cacatcctga tcctgttctc ccagtccgag 1080cgctccaccc ccgccaaggt ggcccccgcg
aagcccaaga acgagggcga gtcctccaag 1140accgagatgg agaaggagaa gtga
1164861098DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
86gcgcacccca aggcgaacgg cagcgcggtg tcgctgaagt cgggctccct ggagacccag
60gaggacaaga cgagcagctc gtcccccccc ccccgcacgt tcatcaacca gctgcccgtg
120tggagcatgc tgctgtcggc ggtgaccacg gtcttcggcg tggccgagaa gcagtggccc
180atgctggacc gcaagtccaa gcgccccgac atgctggtcg agcccctggg cgtggaccgc
240atcgtctacg acggcgtgag cttccgccag tcgttctcca tccgcagcta cgagatcggc
300gccgaccgca ccgcctcgat cgagacgctg atgaacatgt tccaggagac ctccctgaac
360cactgcaaga tcatcggcct gctgaacgac ggcttcggcc gcacgcccga gatgtgcaag
420cgcgacctga tctgggtcgt gaccaagatg cagatcgagg tgaaccgcta ccccacgtgg
480ggcgacacca tcgaggtcaa cacgtgggtg agcgcctcgg gcaagcacgg catgggccgc
540gactggctga tctccgactg ccacaccggc gagatcctga tccgcgcgac gagcgtctgg
600gcgatgatga accagaagac ccgccgcctg tcgaagatcc cctacgaggt gcgccaggag
660atcgagcccc agttcgtcga ctccgccccc gtgatcgtgg acgaccgcaa gttccacaag
720ctggacctga agacgggcga cagcatctgc aacggcctga ccccccgctg gacggacctg
780gacgtgaacc agcacgtcaa caacgtgaag tacatcggct ggatcctgca gtcggtcccc
840accgaggtgt tcgagacgca ggagctgtgc ggcctgaccc tggagtaccg ccgcgagtgc
900ggccgcgact ccgtgctgga gagcgtcacg gccatggacc cctcgaagga gggcgaccgc
960tccctgtacc agcacctgct gcgcctggag gacggcgcgg acatcgtgaa gggccgcacc
1020gagtggcgcc ccaagaacgc cggcgccaag ggcgccatcc tgacgggcaa gaccagcaac
1080ggcaactcga tctcctga
109887365PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Ala His Pro Lys Ala Asn Gly Ser Ala Val Ser
Leu Lys Ser Gly Ser 1 5 10
15 Leu Glu Thr Gln Glu Asp Lys Thr Ser Ser Ser Ser Pro Pro Pro Arg
20 25 30 Thr Phe
Ile Asn Gln Leu Pro Val Trp Ser Met Leu Leu Ser Ala Val 35
40 45 Thr Thr Val Phe Gly Val Ala
Glu Lys Gln Trp Pro Met Leu Asp Arg 50 55
60 Lys Ser Lys Arg Pro Asp Met Leu Val Glu Pro Leu
Gly Val Asp Arg 65 70 75
80 Ile Val Tyr Asp Gly Val Ser Phe Arg Gln Ser Phe Ser Ile Arg Ser
85 90 95 Tyr Glu Ile
Gly Ala Asp Arg Thr Ala Ser Ile Glu Thr Leu Met Asn 100
105 110 Met Phe Gln Glu Thr Ser Leu Asn
His Cys Lys Ile Ile Gly Leu Leu 115 120
125 Asn Asp Gly Phe Gly Arg Thr Pro Glu Met Cys Lys Arg
Asp Leu Ile 130 135 140
Trp Val Val Thr Lys Met Gln Ile Glu Val Asn Arg Tyr Pro Thr Trp 145
150 155 160 Gly Asp Thr Ile
Glu Val Asn Thr Trp Val Ser Ala Ser Gly Lys His 165
170 175 Gly Met Gly Arg Asp Trp Leu Ile Ser
Asp Cys His Thr Gly Glu Ile 180 185
190 Leu Ile Arg Ala Thr Ser Val Trp Ala Met Met Asn Gln Lys
Thr Arg 195 200 205
Arg Leu Ser Lys Ile Pro Tyr Glu Val Arg Gln Glu Ile Glu Pro Gln 210
215 220 Phe Val Asp Ser Ala
Pro Val Ile Val Asp Asp Arg Lys Phe His Lys 225 230
235 240 Leu Asp Leu Lys Thr Gly Asp Ser Ile Cys
Asn Gly Leu Thr Pro Arg 245 250
255 Trp Thr Asp Leu Asp Val Asn Gln His Val Asn Asn Val Lys Tyr
Ile 260 265 270 Gly
Trp Ile Leu Gln Ser Val Pro Thr Glu Val Phe Glu Thr Gln Glu 275
280 285 Leu Cys Gly Leu Thr Leu
Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser 290 295
300 Val Leu Glu Ser Val Thr Ala Met Asp Pro Ser
Lys Glu Gly Asp Arg 305 310 315
320 Ser Leu Tyr Gln His Leu Leu Arg Leu Glu Asp Gly Ala Asp Ile Val
325 330 335 Lys Gly
Arg Thr Glu Trp Arg Pro Lys Asn Ala Gly Ala Lys Gly Ala 340
345 350 Ile Leu Thr Gly Lys Thr Ser
Asn Gly Asn Ser Ile Ser 355 360
365 881167DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 88gcgcacccca aggcgaacgg cagcgcggtg
tcgctgaagt cgggctccct ggagacccag 60gaggacaaga cgagcagctc gtcccccccc
ccccgcacgt tcatcaacca gctgcccgtg 120tggagcatgc tgctgtcggc ggtgaccacg
gtcttcggcg tggccgagaa gcagtggccc 180atgctggacc gcaagtccaa gcgccccgac
atgctggtcg agcccctggg cgtggaccgc 240atcgtctacg acggcgtgag cttccgccag
tcgttctcca tccgcagcta cgagatcggc 300gccgaccgca ccgcctcgat cgagacgctg
atgaacatgt tccaggagac ctccctgaac 360cactgcaaga tcatcggcct gctgaacgac
ggcttcggcc gcacgcccga gatgtgcaag 420cgcgacctga tctgggtcgt gaccaagatg
cagatcgagg tgaaccgcta ccccacgtgg 480ggcgacacca tcgaggtcaa cacgtgggtg
agcgcctcgg gcaagcacgg catgggccgc 540gactggctga tctccgactg ccacaccggc
gagatcctga tccgcgcgac gagcgtctgg 600gcgatgatga accagaagac ccgccgcctg
tcgaagatcc cctacgaggt gcgccaggag 660atcgagcccc agttcgtcga ctccgccccc
gtgatcgtgg acgaccgcaa gttccacaag 720ctggacctga agacgggcga cagcatctgc
aacggcctga ccccccgctg gacggacctg 780gacgtgaacc agcacgtcaa caacgtgaag
tacatcggct ggatcctgca gtcggtcccc 840accgaggtgt tcgagacgca ggagctgtgc
ggcctgaccc tggagtaccg ccgcgagtgc 900ggccgcgact ccgtgctgga gagcgtcacg
gccatggacc cctcgaagga gggcgaccgc 960tccctgtacc agcacctgct gcgcctggag
gacggcgcgg acatcgtgaa gggccgcacc 1020gagtggcgcc ccaagaacgc cggcgccaag
ggcgccatcc tgacgggcaa gaccagcaac 1080ggcaactcga tctccatgga ctacaaggac
cacgacggcg actacaagga ccacgacatc 1140gactacaagg acgacgacga caagtga
116789388PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Ala His Pro Lys Ala Asn Gly Ser Ala Val Ser Leu Lys Ser Gly Ser 1
5 10 15 Leu Glu Thr Gln
Glu Asp Lys Thr Ser Ser Ser Ser Pro Pro Pro Arg 20
25 30 Thr Phe Ile Asn Gln Leu Pro Val Trp
Ser Met Leu Leu Ser Ala Val 35 40
45 Thr Thr Val Phe Gly Val Ala Glu Lys Gln Trp Pro Met Leu
Asp Arg 50 55 60
Lys Ser Lys Arg Pro Asp Met Leu Val Glu Pro Leu Gly Val Asp Arg 65
70 75 80 Ile Val Tyr Asp Gly
Val Ser Phe Arg Gln Ser Phe Ser Ile Arg Ser 85
90 95 Tyr Glu Ile Gly Ala Asp Arg Thr Ala Ser
Ile Glu Thr Leu Met Asn 100 105
110 Met Phe Gln Glu Thr Ser Leu Asn His Cys Lys Ile Ile Gly Leu
Leu 115 120 125 Asn
Asp Gly Phe Gly Arg Thr Pro Glu Met Cys Lys Arg Asp Leu Ile 130
135 140 Trp Val Val Thr Lys Met
Gln Ile Glu Val Asn Arg Tyr Pro Thr Trp 145 150
155 160 Gly Asp Thr Ile Glu Val Asn Thr Trp Val Ser
Ala Ser Gly Lys His 165 170
175 Gly Met Gly Arg Asp Trp Leu Ile Ser Asp Cys His Thr Gly Glu Ile
180 185 190 Leu Ile
Arg Ala Thr Ser Val Trp Ala Met Met Asn Gln Lys Thr Arg 195
200 205 Arg Leu Ser Lys Ile Pro Tyr
Glu Val Arg Gln Glu Ile Glu Pro Gln 210 215
220 Phe Val Asp Ser Ala Pro Val Ile Val Asp Asp Arg
Lys Phe His Lys 225 230 235
240 Leu Asp Leu Lys Thr Gly Asp Ser Ile Cys Asn Gly Leu Thr Pro Arg
245 250 255 Trp Thr Asp
Leu Asp Val Asn Gln His Val Asn Asn Val Lys Tyr Ile 260
265 270 Gly Trp Ile Leu Gln Ser Val Pro
Thr Glu Val Phe Glu Thr Gln Glu 275 280
285 Leu Cys Gly Leu Thr Leu Glu Tyr Arg Arg Glu Cys Gly
Arg Asp Ser 290 295 300
Val Leu Glu Ser Val Thr Ala Met Asp Pro Ser Lys Glu Gly Asp Arg 305
310 315 320 Ser Leu Tyr Gln
His Leu Leu Arg Leu Glu Asp Gly Ala Asp Ile Val 325
330 335 Lys Gly Arg Thr Glu Trp Arg Pro Lys
Asn Ala Gly Ala Lys Gly Ala 340 345
350 Ile Leu Thr Gly Lys Thr Ser Asn Gly Asn Ser Ile Ser Met
Asp Tyr 355 360 365
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp 370
375 380 Asp Asp Asp Lys 385
905428DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 90gctcttccgc taacggaggt ctgtcaccaa
atggaccccg tctattgcgg gaaaccacgg 60cgatggcacg tttcaaaact tgatgaaata
caatattcag tatgtcgcgg gcggcgacgg 120cggggagctg atgtcgcgct gggtattgct
taatcgccag cttcgccccc gtcttggcgc 180gaggcgtgaa caagccgacc gatgtgcacg
agcaaatcct gacactagaa gggctgactc 240gcccggcacg gctgaattac acaggcttgc
aaaaatacca gaatttgcac gcaccgtatt 300cgcggtattt tgttggacag tgaatagcga
tgcggcaatg gcttgtggcg ttagaaggtg 360cgacgaaggt ggtgccacca ctgtgccagc
cagtcctggc ggctcccagg gccccgatca 420agagccagga catccaaact acccacagca
tcaacgcccc ggcctatact cgaaccccac 480ttgcactctg caatggtatg ggaaccacgg
ggcagtcttg tgtgggtcgc gcctatcgcg 540gtcggcgaag accgggaagg taccctttct
tgcgctatga cacttccagc aaaaggtagg 600gcgggctgcg agacggcttc ccggcgctgc
atgcaacacc gatgatgctt cgaccccccg 660aagctccttc ggggctgcat gggcgctccg
atgccgctcc agggcgagcg ctgtttaaat 720agccaggccc ccgattgcaa agacattata
gcgagctacc aaagccatat tcaaacacct 780agatcactac cacttctaca caggccactc
gagcttgtga tcgcactccg ctaagggggc 840gcctcttcct cttcgtttca gtcacaaccc
gcaaactcta gaatatcaat gatcgagcag 900gacggcctcc acgccggctc ccccgccgcc
tgggtggagc gcctgttcgg ctacgactgg 960gcccagcaga ccatcggctg ctccgacgcc
gccgtgttcc gcctgtccgc ccagggccgc 1020cccgtgctgt tcgtgaagac cgacctgtcc
ggcgccctga acgagctgca ggacgaggcc 1080gcccgcctgt cctggctggc caccaccggc
gtgccctgcg ccgccgtgct ggacgtggtg 1140accgaggccg gccgcgactg gctgctgctg
ggcgaggtgc ccggccagga cctgctgtcc 1200tcccacctgg cccccgccga gaaggtgtcc
atcatggccg acgccatgcg ccgcctgcac 1260accctggacc ccgccacctg ccccttcgac
caccaggcca agcaccgcat cgagcgcgcc 1320cgcacccgca tggaggccgg cctggtggac
caggacgacc tggacgagga gcaccagggc 1380ctggcccccg ccgagctgtt cgcccgcctg
aaggcccgca tgcccgacgg cgaggacctg 1440gtggtgaccc acggcgacgc ctgcctgccc
aacatcatgg tggagaacgg ccgcttctcc 1500ggcttcatcg actgcggccg cctgggcgtg
gccgaccgct accaggacat cgccctggcc 1560acccgcgaca tcgccgagga gctgggcggc
gagtgggccg accgcttcct ggtgctgtac 1620ggcatcgccg cccccgactc ccagcgcatc
gccttctacc gcctgctgga cgagttcttc 1680tgacaattgg cagcagcagc tcggatagta
tcgacacact ctggacgctg gtcgtgtgat 1740ggactgttgc cgccacactt gctgccttga
cctgtgaata tccctgccgc ttttatcaaa 1800cagcctcagt gtgtttgatc ttgtgtgtac
gcgcttttgc gagttgctag ctgcttgtgc 1860tatttgcgaa taccaccccc agcatcccct
tccctcgttt catatcgctt gcatcccaac 1920cgcaacttat ctacgctgtc ctgctatccc
tcagcgctgc tcctgctcct gctcactgcc 1980cctcgcacag ccttggtttg ggctccgcct
gtattctcct ggtactgcaa cctgtaaacc 2040agcactgcaa tgctgatgca cgggaagtag
tgggatggga acacaaatgg aggatcccgc 2100gtctcgaaca gagcgcgcag aggaacgctg
aaggtctcgc ctctgtcgca cctcagcgcg 2160gcatacacca caataaccac ctgacgaatg
cgcttggttc ttcgtccatt agcgaagcgt 2220ccggttcaca cacgtgccac gttggcgagg
tggcaggtga caatgatcgg tggagctgat 2280ggtcgaaacg ttcacagcct agggatatcg
aattcggccg acaggacgcg cgtcaaaggt 2340gctggtcgtg tatgccctgg ccggcaggtc
gttgctgctg ctggttagtg attccgcaac 2400cctgattttg gcgtcttatt ttggcgtggc
aaacgctggc gcccgcgagc cgggccggcg 2460gcgatgcggt gccccacggc tgccggaatc
caagggaggc aagagcgccc gggtcagttg 2520aagggcttta cgcgcaaggt acagccgctc
ctgcaaggct gcgtggtgga attggacgtg 2580caggtcctgc tgaagttcct ccaccgcctc
accagcggac aaagcaccgg tgtatcaggt 2640ccgtgtcatc cactctaaag agctcgacta
cgacctactg atggccctag attcttcatc 2700aaaaacgcct gagacacttg cccaggattg
aaactccctg aagggaccac caggggccct 2760gagttgttcc ttccccccgt ggcgagctgc
cagccaggct gtacctgtga tcgaggctgg 2820cgggaaaata ggcttcgtgt gctcaggtca
tgggaggtgc aggacagctc atgaaacgcc 2880aacaatcgca caattcatgt caagctaatc
agctatttcc tcttcacgag ctgtaattgt 2940cccaaaattc tggtctaccg ggggtgatcc
ttcgtgtacg ggcccttccc tcaaccctag 3000gtatgcgcgc atgcggtcgc cgcgcaactc
gcgcgagggc cgagggtttg ggacgggccg 3060tcccgaaatg cagttgcacc cggatgcgtg
gcaccttttt tgcgataatt tatgcaatgg 3120actgctctgc aaaattctgg ctctgtcgcc
aaccctagga tcagcggcgt aggatttcgt 3180aatcattcgt cctgatgggg agctaccgac
taccctaata tcagcccgac tgcctgacgc 3240cagcgtccac ttttgtgcac acattccatt
cgtgcccaag acatttcatt gtggtgcgaa 3300gcgtccccag ttacgctcac ctgtttcccg
acctccttac tgttctgtcg acagagcggg 3360cccacaggcc ggtcgcagcc actagtatgg
cgatcgcggc cgcggcggtg atcttcctgt 3420tcggcctgat cttcttcgcc tccggcctga
tcatcaacct gttccaggcg ctgtgcttcg 3480tcctgatccg ccccctgtcc aagaacgcct
accgccgcat caaccgcgtg ttcgcggagc 3540tgctgctgtc cgagctgctg tgcctgttcg
actggtgggc gggcgcgaag ctgaagctgt 3600tcaccgaccc cgagacgttc cgcctgatgg
gcaaggagca cgccctggtc atcatcaacc 3660acatgaccga gctggactgg atggtgggct
gggtgatggg ccagcacttc ggctgcctgg 3720gctccatcat ctccgtcgcc aagaagtcca
cgaagttcct gcccgtgctg ggctggtcca 3780tgtggttctc cgagtacctg tacctggagc
gctcctgggc caaggacaag tccaccctga 3840agtcccacat cgagcgcctg atcgactacc
ccctgccctt ctggctggtc atcttcgtcg 3900agggcacccg cttcacgcgc acgaagctgc
tggcggccca gcagtacgcg gtctcctccg 3960gcctgcccgt cccccgcaac gtcctgatcc
cccgcacgaa gggcttcgtc tcctgcgtgt 4020cccacatgcg ctccttcgtc cccgcggtgt
acgacgtcac ggtggcgttc cccaagacgt 4080cccccccccc cacgctgctg aacctgttcg
agggccagtc catcatgctg cacgtgcaca 4140tcaagcgcca cgccatgaag gacctgcccg
agtccgacga cgccgtcgcg gagtggtgcc 4200gcgacaagtt cgtcgagaag gacgccctgc
tggacaagca caacgcggag gacacgttct 4260ccggccagga ggtgtgccac tccggctccc
gccagctgaa gtccctgctg gtcgtgatct 4320cctgggtcgt ggtgacgacg ttcggcgccc
tgaagttcct gcagtggtcc tcctggaagg 4380gcaaggcgtt ctccgccatc ggcctgggca
tcgtcaccct gctgatgcac gtgctgatcc 4440tgtcctccca ggccgagcgc tccaaccccg
ccgaggtggc ccaggccaag ctgaagaccg 4500gcctgtccat ctccaagaag gtgacggaca
aggagaactg attaattaac tcgaggcagc 4560agcagctcgg atagtatcga cacactctgg
acgctggtcg tgtgatggac tgttgccgcc 4620acacttgctg ccttgacctg tgaatatccc
tgccgctttt atcaaacagc ctcagtgtgt 4680ttgatcttgt gtgtacgcgc ttttgcgagt
tgctagctgc ttgtgctatt tgcgaatacc 4740acccccagca tccccttccc tcgtttcata
tcgcttgcat cccaaccgca acttatctac 4800gctgtcctgc tatccctcag cgctgctcct
gctcctgctc actgcccctc gcacagcctt 4860ggtttgggct ccgcctgtat tctcctggta
ctgcaacctg taaaccagca ctgcaatgct 4920gatgcacggg aagtagtggg atgggaacac
aaatggaaag cttgagctca gcggcgacgg 4980tcctgctacc gtacgacgtt gggcacgccc
atgaaagttt gtataccgag cttgttgagc 5040gaactgcaag cgcggctcaa ggatacttga
actcctggat tgatatcggt ccaataatgg 5100atggaaaatc cgaacctcgt gcaagaactg
agcaaacctc gttacatgga tgcacagtcg 5160ccagtccaat gaacattgaa gtgagcgaac
tgttcgcttc ggtggcagta ctactcaaag 5220aatgagctgc tgttaaaaat gcactctcgt
tctctcaagt gagtggcaga tgagtgctca 5280cgccttgcac ttcgctgccc gtgtcatgcc
ctgcgcccca aaatttgaaa aaagggatga 5340gattattggg caatggacga cgtcgtcgct
ccgggagtca ggaccggcgg aaaataagag 5400gcaacacact ccgcttctta gctcttcg
5428915436DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
91gctcttccgc taacggaggt ctgtcaccaa atggaccccg tctattgcgg gaaaccacgg
60cgatggcacg tttcaaaact tgatgaaata caatattcag tatgtcgcgg gcggcgacgg
120cggggagctg atgtcgcgct gggtattgct taatcgccag cttcgccccc gtcttggcgc
180gaggcgtgaa caagccgacc gatgtgcacg agcaaatcct gacactagaa gggctgactc
240gcccggcacg gctgaattac acaggcttgc aaaaatacca gaatttgcac gcaccgtatt
300cgcggtattt tgttggacag tgaatagcga tgcggcaatg gcttgtggcg ttagaaggtg
360cgacgaaggt ggtgccacca ctgtgccagc cagtcctggc ggctcccagg gccccgatca
420agagccagga catccaaact acccacagca tcaacgcccc ggcctatact cgaaccccac
480ttgcactctg caatggtatg ggaaccacgg ggcagtcttg tgtgggtcgc gcctatcgcg
540gtcggcgaag accgggaagg taccctttct tgcgctatga cacttccagc aaaaggtagg
600gcgggctgcg agacggcttc ccggcgctgc atgcaacacc gatgatgctt cgaccccccg
660aagctccttc ggggctgcat gggcgctccg atgccgctcc agggcgagcg ctgtttaaat
720agccaggccc ccgattgcaa agacattata gcgagctacc aaagccatat tcaaacacct
780agatcactac cacttctaca caggccactc gagcttgtga tcgcactccg ctaagggggc
840gcctcttcct cttcgtttca gtcacaaccc gcaaactcta gaatatcaat gatcgagcag
900gacggcctcc acgccggctc ccccgccgcc tgggtggagc gcctgttcgg ctacgactgg
960gcccagcaga ccatcggctg ctccgacgcc gccgtgttcc gcctgtccgc ccagggccgc
1020cccgtgctgt tcgtgaagac cgacctgtcc ggcgccctga acgagctgca ggacgaggcc
1080gcccgcctgt cctggctggc caccaccggc gtgccctgcg ccgccgtgct ggacgtggtg
1140accgaggccg gccgcgactg gctgctgctg ggcgaggtgc ccggccagga cctgctgtcc
1200tcccacctgg cccccgccga gaaggtgtcc atcatggccg acgccatgcg ccgcctgcac
1260accctggacc ccgccacctg ccccttcgac caccaggcca agcaccgcat cgagcgcgcc
1320cgcacccgca tggaggccgg cctggtggac caggacgacc tggacgagga gcaccagggc
1380ctggcccccg ccgagctgtt cgcccgcctg aaggcccgca tgcccgacgg cgaggacctg
1440gtggtgaccc acggcgacgc ctgcctgccc aacatcatgg tggagaacgg ccgcttctcc
1500ggcttcatcg actgcggccg cctgggcgtg gccgaccgct accaggacat cgccctggcc
1560acccgcgaca tcgccgagga gctgggcggc gagtgggccg accgcttcct ggtgctgtac
1620ggcatcgccg cccccgactc ccagcgcatc gccttctacc gcctgctgga cgagttcttc
1680tgacaattgg cagcagcagc tcggatagta tcgacacact ctggacgctg gtcgtgtgat
1740ggactgttgc cgccacactt gctgccttga cctgtgaata tccctgccgc ttttatcaaa
1800cagcctcagt gtgtttgatc ttgtgtgtac gcgcttttgc gagttgctag ctgcttgtgc
1860tatttgcgaa taccaccccc agcatcccct tccctcgttt catatcgctt gcatcccaac
1920cgcaacttat ctacgctgtc ctgctatccc tcagcgctgc tcctgctcct gctcactgcc
1980cctcgcacag ccttggtttg ggctccgcct gtattctcct ggtactgcaa cctgtaaacc
2040agcactgcaa tgctgatgca cgggaagtag tgggatggga acacaaatgg aggatcccgc
2100gtctcgaaca gagcgcgcag aggaacgctg aaggtctcgc ctctgtcgca cctcagcgcg
2160gcatacacca caataaccac ctgacgaatg cgcttggttc ttcgtccatt agcgaagcgt
2220ccggttcaca cacgtgccac gttggcgagg tggcaggtga caatgatcgg tggagctgat
2280ggtcgaaacg ttcacagcct agggatatcg aattcggccg acaggacgcg cgtcaaaggt
2340gctggtcgtg tatgccctgg ccggcaggtc gttgctgctg ctggttagtg attccgcaac
2400cctgattttg gcgtcttatt ttggcgtggc aaacgctggc gcccgcgagc cgggccggcg
2460gcgatgcggt gccccacggc tgccggaatc caagggaggc aagagcgccc gggtcagttg
2520aagggcttta cgcgcaaggt acagccgctc ctgcaaggct gcgtggtgga attggacgtg
2580caggtcctgc tgaagttcct ccaccgcctc accagcggac aaagcaccgg tgtatcaggt
2640ccgtgtcatc cactctaaag agctcgacta cgacctactg atggccctag attcttcatc
2700aaaaacgcct gagacacttg cccaggattg aaactccctg aagggaccac caggggccct
2760gagttgttcc ttccccccgt ggcgagctgc cagccaggct gtacctgtga tcgaggctgg
2820cgggaaaata ggcttcgtgt gctcaggtca tgggaggtgc aggacagctc atgaaacgcc
2880aacaatcgca caattcatgt caagctaatc agctatttcc tcttcacgag ctgtaattgt
2940cccaaaattc tggtctaccg ggggtgatcc ttcgtgtacg ggcccttccc tcaaccctag
3000gtatgcgcgc atgcggtcgc cgcgcaactc gcgcgagggc cgagggtttg ggacgggccg
3060tcccgaaatg cagttgcacc cggatgcgtg gcaccttttt tgcgataatt tatgcaatgg
3120actgctctgc aaaattctgg ctctgtcgcc aaccctagga tcagcggcgt aggatttcgt
3180aatcattcgt cctgatgggg agctaccgac taccctaata tcagcccgac tgcctgacgc
3240cagcgtccac ttttgtgcac acattccatt cgtgcccaag acatttcatt gtggtgcgaa
3300gcgtccccag ttacgctcac ctgtttcccg acctccttac tgttctgtcg acagagcggg
3360cccacaggcc ggtcgcagcc actagtatgg ccatcgcggc ggccgcggtg atcgtgcccc
3420tgtccctgct gttcttcgtg tccggcctga tcgtcaacct ggtgcaggcc gtctgcttcg
3480tcctgatccg ccccctgtcc aagaacacgt accgccgcat caaccgcgtg gtcgcggagc
3540tgctgtggct ggagctggtg tggctgatcg actggtgggc gggcgtgaag atcaaggtct
3600tcacggacca cgagacgttc cacctgatgg gcaaggagca cgccctggtc atctgcaacc
3660acaagtccga catcgactgg ctggtcggct gggtcctggg ccagcgctcc ggctgcctgg
3720gctccaccct ggcggtcatg aagaagtcct ccaagttcct gcccgtcctg ggctggtcca
3780tgtggttctc cgagtacctg ttcctggagc gctcctgggc caaggacgag atcacgctga
3840agtccggcct gaaccgcctg aaggactacc ccctgccctt ctggctggcg ctgttcgtgg
3900agggcacgcg cttcacccgc gcgaagctgc tggcggcgca gcagtacgcc gcgtcctccg
3960gcctgcccgt gccccgcaac gtgctgatcc cccgcacgaa gggcttcgtg tcctccgtgt
4020cccacatgcg ctccttcgtg cccgcgatct acgacgtcac cgtggccatc cccaagacgt
4080cccccccccc cacgctgatc cgcatgttca agggccagtc ctccgtgctg cacgtgcacc
4140tgaagcgcca cctgatgaag gacctgcccg agtccgacga cgccgtcgcg cagtggtgcc
4200gcgacatctt cgtggagaag gacgcgctgc tggacaagca caacgccgag gacaccttct
4260ccggccagga gctgcaggag accggccgcc ccatcaagtc cctgctggtc gtcatctcct
4320gggccgtcct ggaggtgttc ggcgccgtca agttcctgca gtggtcctcc ctgctgtcct
4380cctggaaggg cctggcgttc tccggcatcg gcctgggcgt gatcaccctg ctgatgcaca
4440tcctgatcct gttctcccag tccgagcgct ccacccccgc caaggtggcc cccgcgaagc
4500ccaagaacga gggcgagtcc tccaagaccg agatggagaa ggagaagtga ttaattaact
4560cgaggcagca gcagctcgga tagtatcgac acactctgga cgctggtcgt gtgatggact
4620gttgccgcca cacttgctgc cttgacctgt gaatatccct gccgctttta tcaaacagcc
4680tcagtgtgtt tgatcttgtg tgtacgcgct tttgcgagtt gctagctgct tgtgctattt
4740gcgaatacca cccccagcat ccccttccct cgtttcatat cgcttgcatc ccaaccgcaa
4800cttatctacg ctgtcctgct atccctcagc gctgctcctg ctcctgctca ctgcccctcg
4860cacagccttg gtttgggctc cgcctgtatt ctcctggtac tgcaacctgt aaaccagcac
4920tgcaatgctg atgcacggga agtagtggga tgggaacaca aatggaaagc ttgagctcag
4980cggcgacggt cctgctaccg tacgacgttg ggcacgccca tgaaagtttg tataccgagc
5040ttgttgagcg aactgcaagc gcggctcaag gatacttgaa ctcctggatt gatatcggtc
5100caataatgga tggaaaatcc gaacctcgtg caagaactga gcaaacctcg ttacatggat
5160gcacagtcgc cagtccaatg aacattgaag tgagcgaact gttcgcttcg gtggcagtac
5220tactcaaaga atgagctgct gttaaaaatg cactctcgtt ctctcaagtg agtggcagat
5280gagtgctcac gcctgcactt cgctgcccgt gtcatgccct gcgccccaaa atttgaaaaa
5340agggatgaga ttattgggca atggacgacg tcgtcgctcc gggagtcagg accggcggaa
5400aataagaggc aacacactcc gcttcttagc tcttcg
5436925257DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 92gctcttccgc taacggaggt ctgtcaccaa
atggaccccg tctattgcgg gaaaccacgg 60cgatggcacg tttcaaaact tgatgaaata
caatattcag tatgtcgcgg gcggcgacgg 120cggggagctg atgtcgcgct gggtattgct
taatcgccag cttcgccccc gtcttggcgc 180gaggcgtgaa caagccgacc gatgtgcacg
agcaaatcct gacactagaa gggctgactc 240gcccggcacg gctgaattac acaggcttgc
aaaaatacca gaatttgcac gcaccgtatt 300cgcggtattt tgttggacag tgaatagcga
tgcggcaatg gcttgtggcg ttagaaggtg 360cgacgaaggt ggtgccacca ctgtgccagc
cagtcctggc ggctcccagg gccccgatca 420agagccagga catccaaact acccacagca
tcaacgcccc ggcctatact cgaaccccac 480ttgcactctg caatggtatg ggaaccacgg
ggcagtcttg tgtgggtcgc gcctatcgcg 540gtcggcgaag accgggaagg taccctttct
tgcgctatga cacttccagc aaaaggtagg 600gcgggctgcg agacggcttc ccggcgctgc
atgcaacacc gatgatgctt cgaccccccg 660aagctccttc ggggctgcat gggcgctccg
atgccgctcc agggcgagcg ctgtttaaat 720agccaggccc ccgattgcaa agacattata
gcgagctacc aaagccatat tcaaacacct 780agatcactac cacttctaca caggccactc
gagcttgtga tcgcactccg ctaagggggc 840gcctcttcct cttcgtttca gtcacaaccc
gcaaactcta gaatatcaat gatcgagcag 900gacggcctcc acgccggctc ccccgccgcc
tgggtggagc gcctgttcgg ctacgactgg 960gcccagcaga ccatcggctg ctccgacgcc
gccgtgttcc gcctgtccgc ccagggccgc 1020cccgtgctgt tcgtgaagac cgacctgtcc
ggcgccctga acgagctgca ggacgaggcc 1080gcccgcctgt cctggctggc caccaccggc
gtgccctgcg ccgccgtgct ggacgtggtg 1140accgaggccg gccgcgactg gctgctgctg
ggcgaggtgc ccggccagga cctgctgtcc 1200tcccacctgg cccccgccga gaaggtgtcc
atcatggccg acgccatgcg ccgcctgcac 1260accctggacc ccgccacctg ccccttcgac
caccaggcca agcaccgcat cgagcgcgcc 1320cgcacccgca tggaggccgg cctggtggac
caggacgacc tggacgagga gcaccagggc 1380ctggcccccg ccgagctgtt cgcccgcctg
aaggcccgca tgcccgacgg cgaggacctg 1440gtggtgaccc acggcgacgc ctgcctgccc
aacatcatgg tggagaacgg ccgcttctcc 1500ggcttcatcg actgcggccg cctgggcgtg
gccgaccgct accaggacat cgccctggcc 1560acccgcgaca tcgccgagga gctgggcggc
gagtgggccg accgcttcct ggtgctgtac 1620ggcatcgccg cccccgactc ccagcgcatc
gccttctacc gcctgctgga cgagttcttc 1680tgacaattgg cagcagcagc tcggatagta
tcgacacact ctggacgctg gtcgtgtgat 1740ggactgttgc cgccacactt gctgccttga
cctgtgaata tccctgccgc ttttatcaaa 1800cagcctcagt gtgtttgatc ttgtgtgtac
gcgcttttgc gagttgctag ctgcttgtgc 1860tatttgcgaa taccaccccc agcatcccct
tccctcgttt catatcgctt gcatcccaac 1920cgcaacttat ctacgctgtc ctgctatccc
tcagcgctgc tcctgctcct gctcactgcc 1980cctcgcacag ccttggtttg ggctccgcct
gtattctcct ggtactgcaa cctgtaaacc 2040agcactgcaa tgctgatgca cgggaagtag
tgggatggga acacaaatgg aggatcccgc 2100gtctcgaaca gagcgcgcag aggaacgctg
aaggtctcgc ctctgtcgca cctcagcgcg 2160gcatacacca caataaccac ctgacgaatg
cgcttggttc ttcgtccatt agcgaagcgt 2220ccggttcaca cacgtgccac gttggcgagg
tggcaggtga caatgatcgg tggagctgat 2280ggtcgaaacg ttcacagcct agggatatcg
aattcggccg acaggacgcg cgtcaaaggt 2340gctggtcgtg tatgccctgg ccggcaggtc
gttgctgctg ctggttagtg attccgcaac 2400cctgattttg gcgtcttatt ttggcgtggc
aaacgctggc gcccgcgagc cgggccggcg 2460gcgatgcggt gccccacggc tgccggaatc
caagggaggc aagagcgccc gggtcagttg 2520aagggcttta cgcgcaaggt acagccgctc
ctgcaaggct gcgtggtgga attggacgtg 2580caggtcctgc tgaagttcct ccaccgcctc
accagcggac aaagcaccgg tgtatcaggt 2640ccgtgtcatc cactctaaag agctcgacta
cgacctactg atggccctag attcttcatc 2700aaaaacgcct gagacacttg cccaggattg
aaactccctg aagggaccac caggggccct 2760gagttgttcc ttccccccgt ggcgagctgc
cagccaggct gtacctgtga tcgaggctgg 2820cgggaaaata ggcttcgtgt gctcaggtca
tgggaggtgc aggacagctc atgaaacgcc 2880aacaatcgca caattcatgt caagctaatc
agctatttcc tcttcacgag ctgtaattgt 2940cccaaaattc tggtctaccg ggggtgatcc
ttcgtgtacg ggcccttccc tcaaccctag 3000gtatgcgcgc atgcggtcgc cgcgcaactc
gcgcgagggc cgagggtttg ggacgggccg 3060tcccgaaatg cagttgcacc cggatgcgtg
gcaccttttt tgcgataatt tatgcaatgg 3120actgctctgc aaaattctgg ctctgtcgcc
aaccctagga tcagcggcgt aggatttcgt 3180aatcattcgt cctgatgggg agctaccgac
taccctaata tcagcccgac tgcctgacgc 3240cagcgtccac ttttgtgcac acattccatt
cgtgcccaag acatttcatt gtggtgcgaa 3300gcgtccccag ttacgctcac ctgtttcccg
acctccttac tgttctgtcg acagagcggg 3360cccacaggcc ggtcgcagcc actagtatgg
agatcccccc ccactgcctg tgctccccct 3420cccccgcccc ctcccagctg tactacaaga
agaagaagca cgccatcctg cagacccaga 3480ccccctaccg ctaccgcgtg tcccccacct
gcttcgcccc cccccgcctg cgcaagcagc 3540acccctaccc cctgcccgtg ctgtgctacc
ccaagctgct gcacttctcc cagccccgct 3600accccctggt gcgctcccac ctggccgagg
ccggcgtggc ctaccgcccc ggctacgagc 3660tgctgggcaa gatccgcggc gtgtgcttct
acgccgtgac cgccgccgtg gccctgctgc 3720tgttccagtg catgctgctg ctgcacccct
tcgtgctgct gttcgacccc ttcccccgca 3780aggcccacca caccatcgcc aagctgtggt
ccatctgctc cgtgtccctg ttctacaaga 3840tccacatcaa gggcctggag aacctgcccc
ccccccactc ccccgccgtg tacgtgtcca 3900accaccagtc cttcctggac atctacaccc
tgctgaccct gggccgcacc ttcaagttca 3960tctccaagac cgagatcttc ctgtacccca
tcatcggctg ggccatgtac atgctgggca 4020ccatccccct gaagcgcctg gactcccgct
cccagctgga caccctgaag cgctgcatgg 4080acctgatcaa gaagggcgcc tccgtgttct
tcttccccga gggcacccgc tccaaggacg 4140gcaagctggg cgccttcaag aagggcgcct
tctccatcgc cgccaagtcc aaggtgcccg 4200tggtgcccat caccctgatc ggcaccggca
agatcatgcc ccccggctcc gagctgaccg 4260tgaaccccgg caccgtgcag gtgatcatcc
acaagcccat cgagggctcc gacgccgagg 4320ccatgtgcaa cgaggcccgc gccaccatct
cccactccct ggacgactga ttaattaact 4380cgaggcagca gcagctcgga tagtatcgac
acactctgga cgctggtcgt gtgatggact 4440gttgccgcca cacttgctgc cttgacctgt
gaatatccct gccgctttta tcaaacagcc 4500tcagtgtgtt tgatcttgtg tgtacgcgct
tttgcgagtt gctagctgct tgtgctattt 4560gcgaatacca cccccagcat ccccttccct
cgtttcatat cgcttgcatc ccaaccgcaa 4620cttatctacg ctgtcctgct atccctcagc
gctgctcctg ctcctgctca ctgcccctcg 4680cacagccttg gtttgggctc cgcctgtatt
ctcctggtac tgcaacctgt aaaccagcac 4740tgcaatgctg atgcacggga agtagtggga
tgggaacaca aatggaaagc ttgagctcag 4800cggcgacggt cctgctaccg tacgacgttg
ggcacgccca tgaaagtttg tataccgagc 4860ttgttgagcg aactgcaagc gcggctcaag
gatacttgaa ctcctggatt gatatcggtc 4920caataatgga tggaaaatcc gaacctcgtg
caagaactga gcaaacctcg ttacatggat 4980gcacagtcgc cagtccaatg aacattgaag
tgagcgaact gttcgcttcg gtggcagtac 5040tactcaaaga atgagctgct gttaaaaatg
cactctcgtt ctctcaagtg agtggcagat 5100gagtgctcac gccttgcact tcgctgcccg
tgtcatgccc tgcgccccaa aatttgaaaa 5160aagggatgag attattgggc aatggacgac
gtcgtcgctc cgggagtcag gaccggcgga 5220aaataagagg caacacactc cgcttcttag
ctcttcg 5257936714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
93gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg
1080gccggcttcg ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg
1140gtgcacttca cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag
1200aaggacgcca agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg
1260cccttgttct ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc
1320gccatcgccc cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac
1380aacaacacct ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc
1440tggacctaca acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc
1500tacaccttca ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc
1560gacccgaagg tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc
1620caggactaca agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc
1680gcgttcgcca acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc
1740cccaccgagc aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc
1800gccccggccg gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc
1860gaggccttcg acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag
1920accttcttca acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac
1980tgggagtact ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc
2040aagttctccc tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag
2100gccgagccga tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc
2160acgttgacga aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag
2220ttcgagctgg tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac
2280ctctccctct ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag
2340gtgtccgcgt cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag
2400aacccctact tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac
2460ctgtcctact acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac
2520gacggcgacg tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc
2580gtgaacatga cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag
2640gtcaagtgac aattggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg
2700tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt
2760atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc
2820ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat
2880cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc
2940actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg
3000taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggagga
3060tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct gtcgcacctc
3120agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg tccattagcg
3180aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat gatcggtgga
3240gctgatggtc gaaacgttca cagcctaggg atatcgaatt cggccgacag gacgcgcgtc
3300aaaggtgctg gtcgtgtatg ccctggccgg caggtcgttg ctgctgctgg ttagtgattc
3360cgcaaccctg attttggcgt cttattttgg cgtggcaaac gctggcgccc gcgagccggg
3420ccggcggcga tgcggtgccc cacggctgcc ggaatccaag ggaggcaaga gcgcccgggt
3480cagttgaagg gctttacgcg caaggtacag ccgctcctgc aaggctgcgt ggtggaattg
3540gacgtgcagg tcctgctgaa gttcctccac cgcctcacca gcggacaaag caccggtgta
3600tcaggtccgt gtcatccact ctaaagaact cgactacgac ctactgatgg ccctagattc
3660ttcatcaaaa acgcctgaga cacttgccca ggattgaaac tccctgaagg gaccaccagg
3720ggccctgagt tgttccttcc ccccgtggcg agctgccagc caggctgtac ctgtgatcga
3780ggctggcggg aaaataggct tcgtgtgctc aggtcatggg aggtgcagga cagctcatga
3840aacgccaaca atcgcacaat tcatgtcaag ctaatcagct atttcctctt cacgagctgt
3900aattgtccca aaattctggt ctaccggggg tgatccttcg tgtacgggcc cttccctcaa
3960ccctaggtat gcgcgcatgc ggtcgccgcg caactcgcgc gagggccgag ggtttgggac
4020gggccgtccc gaaatgcagt tgcacccgga tgcgtggcac cttttttgcg ataatttatg
4080caatggactg ctctgcaaaa ttctggctct gtcgccaacc ctaggatcag cggcgtagga
4140tttcgtaatc attcgtcctg atggggagct accgactacc ctaatatcag cccgactgcc
4200tgacgccagc gtccactttt gtgcacacat tccattcgtg cccaagacat ttcattgtgg
4260tgcgaagcgt ccccagttac gctcacctgt ttcccgacct ccttactgtt ctgtcgacag
4320agcgggccca caggccggtc gcagccacta gtatggcccc cacctccctg ctggcctcca
4380ccggcgtgtc ctccgcctcc ctgtggtcct ccgcccgctc ctccgcctgc gccttccccg
4440tggaccacgc cgtgcgcggc gccccccagc gccccctgcc catgcagcgc cgctgcttcc
4500gcaccgtggc cgtgcgcggg cgcgccgccg cccccgccgt ggccgtgcgc cccgagcccg
4560cccaggagtt ctgggagcag ctggagccct gcaagatggc cgaggacaag cgcatcttcc
4620tggaggagca ccgcatccgc ggcaacgagg tgggcccctc ccagcgcctg accatcaccg
4680ccgtggccaa catcctgcag gaggccgccg gcaaccacgc cgtggccatg tggggccgct
4740cctccgaggg cttcgccacc gaccccgagc tgcaggaggc cggcctgatc ttcgtgatga
4800cccgcatgca gatccagatg taccgctacc cccgctgggg cgacctgatg caggtggaga
4860cctggttcca gaccgccggc aagctgggcg cccagcgcga gtgggtgctg cgcgacaagc
4920tgaccggcga ggccctgggc gccgccacct cctcctgggt gatgatcaac atccgcaccc
4980gccgcccctg ccgcatgccc gagctggtgc gcgtgaagtc cgccttcttc gcccgcgagc
5040ccccccgcct ggccctgccc cccgccgtga cccgcgccaa gctgcccaac atcgccaccc
5100ccgcccccct gcgcggccac cgccaggtgg cccgccgcac cgacatggac atgaacggcc
5160acgtgaacaa cgtggcctac ctggcctggt gcctggaggc cgtgcccgag cacgtgttct
5220ccgactacca cctgtaccag atggagatcg acttcaaggc cgagtgccac gccggcgacg
5280tgatctcctc ccaggccgag cagatccccc cccaggaggc cctgacccac aacggcgccg
5340gccgcaaccc ctcctgcttc gtgcactcca tcctgcgcgc cgagaccgag ctggtgcgcg
5400cccgcaccac ctggtccgcc cccatcgacg cccccgccgc caagcccccc aaggcctccc
5460acatggacta caaggaccac gacggcgact acaaggacca cgacatcgac tacaaggacg
5520acgacgacaa gtgaatcgat agatctctta aggcagcagc agctcggata gtatcgacac
5580actctggacg ctggtcgtgt gatggactgt tgccgccaca cttgctgcct tgacctgtga
5640atatccctgc cgcttttatc aaacagcctc agtgtgtttg atcttgtgtg tacgcgcttt
5700tgcgagttgc tagctgcttg tgctatttgc gaataccacc cccagcatcc ccttccctcg
5760tttcatatcg cttgcatccc aaccgcaact tatctacgct gtcctgctat ccctcagcgc
5820tgctcctgct cctgctcact gcccctcgca cagccttggt ttgggctccg cctgtattct
5880cctggtactg caacctgtaa accagcactg caatgctgat gcacgggaag tagtgggatg
5940ggaacacaaa tggaaagctt aattaagagc tcttgttttc cagaaggagt tgctccttga
6000gcctttcatt ctcagcctcg ataacctcca aagccgctct aattgtggag ggggttcgaa
6060tttaaaagct tggaatgttg gttcgtgcgt ctggaacaag cccagacttg ttgctcactg
6120ggaaaaggac catcagctcc aaaaaacttg ccgctcaaac cgcgtacctc tgctttcgcg
6180caatctgccc tgttgaaatc gccaccacat tcatattgtg acgcttgagc agtctgtaat
6240tgcctcagaa tgtggaatca tctgccccct gtgcgagccc atgccaggca tgtcgcgggc
6300gaggacaccc gccactcgta cagcagacca ttatgctacc tcacaatagt tcataacagt
6360gaccatattt ctcgaagctc cccaacgagc acctccatgc tctgagtggc caccccccgg
6420ccctggtgct tgcggagggc aggtcaaccg gcatggggct accgaaatcc ccgaccggat
6480cccaccaccc ccgcgatggg aagaatctct ccccgggatg tgggcccacc accagcacaa
6540cctgctggcc caggcgagcg tcaaaccata ccacacaaat atccttggca tcggccctga
6600attccttctg ccgctctgct acccggtgct tctgtccgaa gcaggggttg ctagggatcg
6660ctccgagtcc gcaaaccctt gtcgcgtggc ggggcttgtt cgagcttgaa gagc
6714945279DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 94gctcttcggg tcgccgcgct gcctcgcgtc
ccctggtggt gcgcgcggtc gccagcgagg 60ccccgctggg cgttccgccc tcggtgcagc
gcccctcccc cgtggtctac tccaagctgg 120acaagcagca ccgcctgacg cccgagcgcc
tggagctggt gcagagcatg gggcagtttg 180cggaggagag ggtgctgccc gtgctgcacc
ccgtggacaa gctgtggcag ccgcaggact 240ttttgcccga ccccgagtcg cccgacttcg
aggatcaggt ggcggagctg cgcgcgcgcg 300ccaaggacct gcccgacgag tactttgtgg
tgctggtggg ggacatgatc acggaggagg 360cgctgccgac ctacatggcc atgctcaaca
cgctggacgg cgtgcgcgac gacacgggcg 420cggccgacca cccgtgggcg cgctggacgc
ggcagtgggt ggccgaggag aaccggcacg 480gcgacctgct gaacaagtac tgctggctga
cggggcgcgt caacatgcgg gccgtggagg 540tgaccatcaa caacctgatc aagagcggca
tgaacccgca gacggacaac aacccttatt 600tggggttcgt ctacacctcc ttccaggagc
gcgccaccaa gtaggtaccc tttcttgcgc 660tatgacactt ccagcaaaag gtagggcggg
ctgcgagacg gcttcccggc gctgcatgca 720acaccgatga tgcttcgacc ccccgaagct
ccttcggggc tgcatgggcg ctccgatgcc 780gctccagggc gagcgctgtt taaatagcca
ggcccccgat tgcaaagaca ttatagcgag 840ctaccaaagc catattcaaa cacctagatc
actaccactt ctacacaggc cactcgagct 900tgtgatcgca ctccgctaag ggggcgcctc
ttcctcttcg tttcagtcac aacccgcaaa 960cggcgcgcca tgctgctgca ggccttcctg
ttcctgctgg ccggcttcgc cgccaagatc 1020agcgcctcca tgacgaacga gacgtccgac
cgccccctgg tgcacttcac ccccaacaag 1080ggctggatga acgaccccaa cggcctgtgg
tacgacgaga aggacgccaa gtggcacctg 1140tacttccagt acaacccgaa cgacaccgtc
tgggggacgc ccttgttctg gggccacgcc 1200acgtccgacg acctgaccaa ctgggaggac
cagcccatcg ccatcgcccc gaagcgcaac 1260gactccggcg ccttctccgg ctccatggtg
gtggactaca acaacacctc cggcttcttc 1320aacgacacca tcgacccgcg ccagcgctgc
gtggccatct ggacctacaa caccccggag 1380tccgaggagc agtacatctc ctacagcctg
gacggcggct acaccttcac cgagtaccag 1440aagaaccccg tgctggccgc caactccacc
cagttccgcg acccgaaggt cttctggtac 1500gagccctccc agaagtggat catgaccgcg
gccaagtccc aggactacaa gatcgagatc 1560tactcctccg acgacctgaa gtcctggaag
ctggagtccg cgttcgccaa cgagggcttc 1620ctcggctacc agtacgagtg ccccggcctg
atcgaggtcc ccaccgagca ggaccccagc 1680aagtcctact gggtgatgtt catctccatc
aaccccggcg ccccggccgg cggctccttc 1740aaccagtact tcgtcggcag cttcaacggc
acccacttcg aggccttcga caaccagtcc 1800cgcgtggtgg acttcggcaa ggactactac
gccctgcaga ccttcttcaa caccgacccg 1860acctacggga gcgccctggg catcgcgtgg
gcctccaact gggagtactc cgccttcgtg 1920cccaccaacc cctggcgctc ctccatgtcc
ctcgtgcgca agttctccct caacaccgag 1980taccaggcca acccggagac ggagctgatc
aacctgaagg ccgagccgat cctgaacatc 2040agcaacgccg gcccctggag ccggttcgcc
accaacacca cgttgacgaa ggccaacagc 2100tacaacgtcg acctgtccaa cagcaccggc
accctggagt tcgagctggt gtacgccgtc 2160aacaccaccc agacgatctc caagtccgtg
ttcgcggacc tctccctctg gttcaagggc 2220ctggaggacc ccgaggagta cctccgcatg
ggcttcgagg tgtccgcgtc ctccttcttc 2280ctggaccgcg ggaacagcaa ggtgaagttc
gtgaaggaga acccctactt caccaaccgc 2340atgagcgtga acaaccagcc cttcaagagc
gagaacgacc tgtcctacta caaggtgtac 2400ggcttgctgg accagaacat cctggagctg
tacttcaacg acggcgacgt cgtgtccacc 2460aacacctact tcatgaccac cgggaacgcc
ctgggctccg tgaacatgac gacgggggtg 2520gacaacctgt tctacatcga caagttccag
gtgcgcgagg tcaagtgaca attggcagca 2580gcagctcgga tagtatcgac acactctgga
cgctggtcgt gtgatggact gttgccgcca 2640cacttgctgc cttgacctgt gaatatccct
gccgctttta tcaaacagcc tcagtgtgtt 2700tgatcttgtg tgtacgcgct tttgcgagtt
gctagctgct tgtgctattt gcgaatacca 2760cccccagcat ccccttccct cgtttcatat
cgcttgcatc ccaaccgcaa cttatctacg 2820ctgtcctgct atccctcagc gctgctcctg
ctcctgctca ctgcccctcg cacagccttg 2880gtttgggctc cgcctgtatt ctcctggtac
tgcaacctgt aaaccagcac tgcaatgctg 2940atgcacggga agtagtggga tgggaacaca
aatggaggat cccgcgtctc gaacagagcg 3000cgcagaggaa cgctgaaggt ctcgcctctg
tcgcacctca gcgcggcata caccacaata 3060accacctgac gaatgcgctt ggttcttcgt
ccattagcga agcgtccggt tcacacacgt 3120gccacgttgg cgaggtggca ggtgacaatg
atcggtggag ctgatggtcg aaacgttcac 3180agcctaggga tatcgaattc ctttcttgcg
ctatgacact tccagcaaaa ggtagggcgg 3240gctgcgagac ggcttcccgg cgctgcatgc
aacaccgatg atgcttcgac cccccgaagc 3300tccttcgggg ctgcatgggc gctccgatgc
cgctccaggg cgagcgctgt ttaaatagcc 3360aggcccccga ttgcaaagac attatagcga
gctaccaaag ccatattcaa acacctagat 3420cactaccact tctacacagg ccactcgagc
ttgtgatcgc actccgctaa gggggcgcct 3480cttcctcttc gtttcagtca caacccgcaa
acactagtgc gctggacgcg gcagtgggtg 3540gccgaggaga accggcacgg cgacctgctg
aacaagtact gttggctgac ggggcgcgtc 3600aacatgcggg ccgtggaggt gaccatcaac
aacctgatca agagcggcat gaacccgcag 3660acggacaaca acccttactt gggcttcgtc
tacacctcct tccaggagcg cgcgaccaag 3720tacagccacg gcaacaccgc gcgccttgcg
gccgagcagt gtgtttgagg gttttggttg 3780cccgtatcga ggtcctggtg gcgcgcatgg
gggagaaggc gcctgtcccg ctgacccccc 3840cggctaccct cccggcacct tccagggcgc
gtacgggatc ctgctcggcc gcaaggcgcg 3900cggtgttgcc gtggctgtac ttggtcgcgc
gctcctggaa ggaggtgtag acgaagccca 3960agtaagggtt gttgtccgtc tgcgggttca
tgccgctctt gatcaggttg ttgatggtca 4020cctccacggc ccgcatgttg acgcgccccg
tcagccaaca gtacttgttc agcaggtcgc 4080cgtgccggtt ctcctcggcc acccactgcc
gcgtccagcg caagcttgca gcagcagctc 4140ggatagtatc gacacactct ggacgctggt
cgtgtgatgg actgttgccg ccacacttgc 4200tgccttgacc tgtgaatatc cctgccgctt
ttatcaaaca gcctcagtgt gtttgatctt 4260gtgtgtacgc gcttttgcga gttgctagct
gcttgtgcta tttgcgaata ccacccccag 4320catccccttc cctcgtttca tatcgcttgc
atcccaaccg caacttatct acgctgtcct 4380gctatccctc agcgctgctc ctgctcctgc
tcactgcccc tcgcacagcc ttggtttggg 4440ctccgcctgt attctcctgg tactgcaacc
tgtaaaccag cactgcaatg ctgatgcacg 4500ggaagtagtg ggatgggaac acaaatggaa
agctggagct ccagccacgg caacaccgcg 4560cgccttgcgg ccgagcacgg cgacaagaac
ctgagcaaga tctgcgggct gatcgccagc 4620gacgagggcc ggcacgagat cgcctacacg
cgcatcgtgg acgagttctt ccgcctcgac 4680cccgagggcg ccgtcgccgc ctacgccaac
atgatgcgca agcagatcac catgcccgcg 4740cacctcatgg acgacatggg ccacggcgag
gccaacccgg gccgcaacct cttcgccgac 4800ttctccgcgg tcgccgagaa gatcgacgtc
tacgacgccg aggactactg ccgcatcctg 4860gagcacctca acgcgcgctg gaaggtggac
gagcgccagg tcagcggcca ggccgccgcg 4920gaccaggagt acgtcctggg cctgccccag
cgcttccgga aactcgccga gaagaccgcc 4980gccaagcgca agcgcgtcgc gcgcaggccc
gtcgccttct cctggatctc cgggcgcgag 5040atcatggtct agggagcgac gagtgtgcgt
gcggggctgg cgggagtggg acgccctcct 5100cgctcctctc tgttctgaac ggaacaatcg
gccaccccgc gctacgcgcc acgcatcgag 5160caacgaagaa aaccccccga tgataggttg
cggtggctgc cgggatatag atccggccgc 5220acatcaaagg gcccctccgc cagagaagaa
gctcctttcc cagcagactc ctgaagagc 5279956580DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
95gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggtgt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgatcgagc aggacggcct ccacgccggc
1080tcccccgccg cctgggtgga gcgcctgttc ggctacgact gggcccagca gaccatcggc
1140tgctccgacg ccgccgtgtt ccgcctgtcc gcccagggcc gccccgtgct gttcgtgaag
1200accgacctgt ccggcgccct gaacgagctg caggacgagg ccgcccgcct gtcctggctg
1260gccaccaccg gcgtgccctg cgccgccgtg ctggacgtgg tgaccgaggc cggccgcgac
1320tggctgctgc tgggcgaggt gcccggccag gacctgctgt cctcccacct ggcccccgcc
1380gagaaggtgt ccatcatggc cgacgccatg cgccgcctgc acaccctgga ccccgccacc
1440tgccccttcg accaccaggc caagcaccgc atcgagcgcg cccgcacccg catggaggcc
1500ggcctggtgg accaggacga cctggacgag gagcaccagg gcctggcccc cgccgagctg
1560ttcgcccgcc tgaaggcccg catgcccgac ggcgaggacc tggtggtgac ccacggcgac
1620gcctgcctgc ccaacatcat ggtggagaac ggccgcttct ccggcttcat cgactgcggc
1680cgcctgggcg tggccgaccg ctaccaggac atcgccctgg ccacccgcga catcgccgag
1740gagctgggcg gcgagtgggc cgaccgcttc ctggtgctgt acggcatcgc cgcccccgac
1800tcccagcgca tcgccttcta ccgcctgctg gacgagttct tctgacaatt ggcagcagca
1860gctcggatag tatcgacaca ctctggacgc tggtcgtgtg atggactgtt gccgccacac
1920ttgctgcctt gacctgtgaa tatccctgcc gcttttatca aacagcctca gtgtgtttga
1980tcttgtgtgt acgcgctttt gcgagttgct agctgcttgt gctatttgcg aataccaccc
2040ccagcatccc cttccctcgt ttcatatcgc ttgcatccca accgcaactt atctacgctg
2100tcctgctatc cctcagcgct gctcctgctc ctgctcactg cccctcgcac agccttggtt
2160tgggctccgc ctgtattctc ctggtactgc aacctgtaaa ccagcactgc aatgctgatg
2220cacgggaagt agtgggatgg gaacacaaat ggaggatccc gcgtctcgaa cagagcgcgc
2280agaggaacgc tgaaggtctc gcctctgtcg cacctcagcg cggcatacac cacaataacc
2340acctgacgaa tgcgcttggt tcttcgtcca ttagcgaagc gtccggttca cacacgtgcc
2400acgttggcga ggtggcaggt gacaatgatc ggtggagctg atggtcgaaa cgttcacagc
2460ctagggatat cctgaagaat gggaggcagg tgttgttgat tatgagtgtg taaaagaaag
2520gggtagagag ccgtcctcag atccgactac tatgcaggta gccgctcgcc catgcccgcc
2580tggctgaata ttgatgcatg cccatcaagg caggcaggca tttctgtgca cgcaccaagc
2640ccacaatctt ccacaacaca cagcatgtac caacgcacgc gtaaaagttg gggtgctgcc
2700agtgcgtcat gccaggcatg atgtgctcct gcacatccgc catgatctcc tccatcgtct
2760cgggtgtttc cggcgcctgg tccgggagcc gttccgccag atacccagac gccacctccg
2820acctcacggg gtacttttcg agcgtctgcc ggtagtcgac gatcgcgtcc accatggagt
2880agccgaggcg ccggaactgg cgtgacggag ggaggagagg gaggagagag aggggggggg
2940ggggggggga tgattacacg ccagtctcac aacgcatgca agacccgttt gattatgagt
3000acaatcatgc actactagat ggatgagcgc caggcataag gcacaccgac gttgatggca
3060tgagcaactc ccgcatcata tttcctattg tcctcacgcc aagccggtca ccatccgcat
3120gctcatatta cagcgcacgc accgcttcgt gatccaccgg gtgaacgtag tcctcgacgg
3180aaacatctgg ctcgggcctc gtgctggcac tccctcccat gccgacaacc tttctgctgt
3240caccacgacc cacgatgcaa cgcgacacga cccggtggga ctgatcggtt cactgcacct
3300gcatgcaatt gtcacaagcg catactccaa tcgtatccgt ttgatttctg tgaaaactcg
3360ctcgaccgcc cgcgtcccgc aggcagcgat gacgtgtgcg tgacctgggt gtttcgtcga
3420aaggccagca accccaaatc gcaggcgatc cggagattgg gatctgatcc gagcttggac
3480cagatccccc acgatgcggc acgggaactg catcgactcg gcgcggaacc cagctttcgt
3540aaatgccaga ttggtgtccg ataccttgat ttgccatcag cgaaacaaga cttcagcagc
3600gagcgtattt ggcgggcgtg ctaccagggt tgcatacatt gcccatttct gtctggaccg
3660ctttaccggc gcagagggtg agttgatggg gttggcaggc atcgaaacgc gcgtgcatgg
3720tgtgtgtgtc tgttttcggc tgcacaattt caatagtcgg atgggcgacg gtagaattgg
3780gtgttgcgct cgcgtgcatg cctcgccccg tcgggtgtca tgaccgggac tggaatcccc
3840cctcgcgacc ctcctgctaa cgctcccgac tctcccgccc gcgcgcagga tagactctag
3900ttcaaccaat cgacaactag tatgcagacc gcccaccagc gcccccccac cgagggccac
3960tgcttcggcg cccgcctgcc caccgcctcc cgccgcgccg tgcgccgcgc ctggtcccgc
4020atcgcccgcg ggcgcgccgc cgccgccgcc gacgccaacc ccgcccgccc cgagcgccgc
4080gtggtgatca ccggccaggg cgtggtgacc tccctgggcc agaccatcga gcagttctac
4140tcctccctgc tggagggcgt gtccggcatc tcccagatcc agaagttcga caccaccggc
4200tacaccacca ccatcgccgg cgagatcaag tccctgcagc tggaccccta cgtgcccaag
4260cgctgggcca agcgcgtgga cgacgtgatc aagtacgtgt acatcgccgg caagcaggcc
4320ctggagtccg ccggcctgcc catcgaggcc gccggcctgg ccggcgccgg cctggacccc
4380gccctgtgcg gcgtgctgat cggcaccgcc atggccggca tgacctcctt cgccgccggc
4440gtggaggccc tgacccgcgg cggcgtgcgc aagatgaacc ccttctgcat ccccttctcc
4500atctccaaca tgggcggcgc catgctggcc atggacatcg gcttcatggg ccccaactac
4560tccatctcca ccgcctgcgc caccggcaac tactgcatcc tgggcgccgc cgaccacatc
4620cgccgcggcg acgccaacgt gatgctggcc ggcggcgccg acgccgccat catcccctcc
4680ggcatcggcg gcttcatcgc ctgcaaggcc ctgtccaagc gcaacgacga gcccgagcgc
4740gcctcccgcc cctgggacgc cgaccgcgac ggcttcgtga tgggcgaggg cgccggcgtg
4800ctggtgctgg aggagctgga gcacgccaag cgccgcggcg ccaccatcct ggccgagctg
4860gtgggcggcg ccgccacctc cgacgcccac cacatgaccg agcccgaccc ccagggccgc
4920ggcgtgcgcc tgtgcctgga gcgcgccctg gagcgcgccc gcctggcccc cgagcgcgtg
4980ggctacgtga acgcccacgg cacctccacc cccgccggcg acgtggccga gtaccgcgcc
5040atccgcgccg tgatccccca ggactccctg cgcatcaact ccaccaagtc catgatcggc
5100cacctgctgg gcggcgccgg cgccgtggag gccgtggccg ccatccaggc cctgcgcacc
5160ggctggctgc accccaacct gaacctggag aaccccgccc ccggcgtgga ccccgtggtg
5220ctggtgggcc cccgcaagga gcgcgccgag gacctggacg tggtgctgtc caactccttc
5280ggcttcggcg gccacaactc ctgcgtgatc ttccgcaagt acgacgagat ggactacaag
5340gaccacgacg gcgactacaa ggaccacgac atcgactaca aggacgacga cgacaagtga
5400atcgatagat ctcttaaggc agcagcagct cggatagtat cgacacactc tggacgctgg
5460tcgtgtgatg gactgttgcc gccacacttg ctgccttgac ctgtgaatat ccctgccgct
5520tttatcaaac agcctcagtg tgtttgatct tgtgtgtacg cgcttttgcg agttgctagc
5580tgcttgtgct atttgcgaat accaccccca gcatcccctt ccctcgtttc atatcgcttg
5640catcccaacc gcaacttatc tacgctgtcc tgctatccct cagcgctgct cctgctcctg
5700ctcactgccc ctcgcacagc cttggtttgg gctccgcctg tattctcctg gtactgcaac
5760ctgtaaacca gcactgcaat gctgatgcac gggaagtagt gggatgggaa cacaaatgga
5820aagcttaatt aagagctctt gttttccaga aggagttgct ccttgagcct ttcattctca
5880gcctcgataa cctccaaagc cgctctaatt gtggaggggg ttcgaattta aaagcttgga
5940atgttggttc gtgcgtctgg aacaagccca gacttgttgc tcactgggaa aaggaccatc
6000agctccaaaa aacttgccgc tcaaaccgcg tacctctgct ttcgcgcaat ctgccctgtt
6060gaaatcgcca ccacattcat attgtgacgc ttgagcagtc tgtaattgcc tcagaatgtg
6120gaatcatctg ccccctgtgc gagcccatgc caggcatgtc gcgggcgagg acacccgcca
6180ctcgtacagc agaccattat gctacctcac aatagttcat aacagtgacc atatttctcg
6240aagctcccca acgagcacct ccatgctctg agtggccacc ccccggccct ggtgcttgcg
6300gagggcaggt caaccggcat ggggctaccg aaatccccga ccggatccca ccacccccgc
6360gatgggaaga atctctcccc gggatgtggg cccaccacca gcacaacctg ctggcccagg
6420cgagcgtcaa accataccac acaaatatcc ttggcatcgg ccctgaattc cttctgccgc
6480tctgctaccc ggtgcttctg tccgaagcag gggttgctag ggatcgctcc gagtccgcaa
6540acccttgtcg cgtggcgggg cttgttcgag cttgaagagc
6580968087DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 96gctcttcgga gtcactgtgc cactgagttc
gactggtagc tgaatggagt cgctgctcca 60ctaaacgaat tgtcagcacc gccagccggc
cgaggacccg agtcatagcg agggtagtag 120cgcgccatgg caccgaccag cctgcttgcc
agtactggcg tctcttccgc ttctctgtgg 180tcctctgcgc gctccagcgc gtgcgctttt
ccggtggatc atgcggtccg tggcgcaccg 240cagcggccgc tgcccatgca gcgccgctgc
ttccgaacag tggcggtcag ggccgcaccc 300gcggtagccg tccgtccgga acccgcccaa
gagttttggg agcagcttga gccctgcaag 360atggcggagg acaagcgcat cttcctggag
gagcaccggt gcgtggaggt ccggggctga 420ccggccgtcg cattcaacgt aatcaatcgc
atgatgatca gaggacacga agtcttggtg 480gcggtggcca gaaacactgt ccattgcaag
ggcataggga tgcgttcctt cacctctcat 540ttctcatttc tgaatccctc cctgctcact
ctttctcctc ctccttcccg ttcacgcagc 600attcggggta ccctttcttg cgctatgaca
cttccagcaa aaggtagggc gggctgcgag 660acggcttccc ggcgctgcat gcaacaccga
tgatgcttcg accccccgaa gctccttcgg 720ggctgcatgg gcgctccgat gccgctccag
ggcgagcgct gtttaaatag ccaggccccc 780gattgcaaag acattatagc gagctaccaa
agccatattc aaacacctag atcactacca 840cttctacaca ggccactcga gcttgtgatc
gcactccgct aagggggcgc ctcttcctct 900tcgtttcagt cacaacccgc aaacggcgcg
ccatgctgct gcaggccttc ctgttcctgc 960tggccggctt cgccgccaag atcagcgcct
ccatgacgaa cgagacgtcc gaccgccccc 1020tggtgcactt cacccccaac aagggctgga
tgaacgaccc caacggcctg tggtacgacg 1080agaaggacgc caagtggcac ctgtacttcc
agtacaaccc gaacgacacc gtctggggga 1140cgcccttgtt ctggggccac gccacgtccg
acgacctgac caactgggag gaccagccca 1200tcgccatcgc cccgaagcgc aacgactccg
gcgccttctc cggctccatg gtggtggact 1260acaacaacac ctccggcttc ttcaacgaca
ccatcgaccc gcgccagcgc tgcgtggcca 1320tctggaccta caacaccccg gagtccgagg
agcagtacat ctcctacagc ctggacggcg 1380gctacacctt caccgagtac cagaagaacc
ccgtgctggc cgccaactcc acccagttcc 1440gcgacccgaa ggtcttctgg tacgagccct
cccagaagtg gatcatgacc gcggccaagt 1500cccaggacta caagatcgag atctactcct
ccgacgacct gaagtcctgg aagctggagt 1560ccgcgttcgc caacgagggc ttcctcggct
accagtacga gtgccccggc ctgatcgagg 1620tccccaccga gcaggacccc agcaagtcct
actgggtgat gttcatctcc atcaaccccg 1680gcgccccggc cggcggctcc ttcaaccagt
acttcgtcgg cagcttcaac ggcacccact 1740tcgaggcctt cgacaaccag tcccgcgtgg
tggacttcgg caaggactac tacgccctgc 1800agaccttctt caacaccgac ccgacctacg
ggagcgccct gggcatcgcg tgggcctcca 1860actgggagta ctccgccttc gtgcccacca
acccctggcg ctcctccatg tccctcgtgc 1920gcaagttctc cctcaacacc gagtaccagg
ccaacccgga gacggagctg atcaacctga 1980aggccgagcc gatcctgaac atcagcaacg
ccggcccctg gagccggttc gccaccaaca 2040ccacgttgac gaaggccaac agctacaacg
tcgacctgtc caacagcacc ggcaccctgg 2100agttcgagct ggtgtacgcc gtcaacacca
cccagacgat ctccaagtcc gtgttcgcgg 2160acctctccct ctggttcaag ggcctggagg
accccgagga gtacctccgc atgggcttcg 2220aggtgtccgc gtcctccttc ttcctggacc
gcgggaacag caaggtgaag ttcgtgaagg 2280agaaccccta cttcaccaac cgcatgagcg
tgaacaacca gcccttcaag agcgagaacg 2340acctgtccta ctacaaggtg tacggcttgc
tggaccagaa catcctggag ctgtacttca 2400acgacggcga cgtcgtgtcc accaacacct
acttcatgac caccgggaac gccctgggct 2460ccgtgaacat gacgacgggg gtggacaacc
tgttctacat cgacaagttc caggtgcgcg 2520aggtcaagtg acaattggca gcagcagctc
ggatagtatc gacacactct ggacgctggt 2580cgtgtgatgg actgttgccg ccacacttgc
tgccttgacc tgtgaatatc cctgccgctt 2640ttatcaaaca gcctcagtgt gtttgatctt
gtgtgtacgc gcttttgcga gttgctagct 2700gcttgtgcta tttgcgaata ccacccccag
catccccttc cctcgtttca tatcgcttgc 2760atcccaaccg caacttatct acgctgtcct
gctatccctc agcgctgctc ctgctcctgc 2820tcactgcccc tcgcacagcc ttggtttggg
ctccgcctgt attctcctgg tactgcaacc 2880tgtaaaccag cactgcaatg ctgatgcacg
ggaagtagtg ggatgggaac acaaatggag 2940gatcccgcgt ctcgaacaga gcgcgcagag
gaacgctgaa ggtctcgcct ctgtcgcacc 3000tcagcgcggc atacaccaca ataaccacct
gacgaatgcg cttggttctt cgtccattag 3060cgaagcgtcc ggttcacaca cgtgccacgt
tggcgaggtg gcaggtgaca atgatcggtg 3120gagctgatgg tcgaaacgtt cacagcctag
ggatatcgaa ttcggccgac aggacgcgcg 3180tcaaaggtgc tggtcgtgta tgccctggcc
ggcaggtcgt tgctgctgct ggttagtgat 3240tccgcaaccc tgattttggc gtcttatttt
ggcgtggcaa acgctggcgc ccgcgagccg 3300ggccggcggc gatgcggtgc cccacggctg
ccggaatcca agggaggcaa gagcgcccgg 3360gtcagttgaa gggctttacg cgcaaggtac
agccgctcct gcaaggctgc gtggtggaat 3420tggacgtgca ggtcctgctg aagttcctcc
accgcctcac cagcggacaa agcaccggtg 3480tatcaggtcc gtgtcatcca ctctaaagaa
ctcgactacg acctactgat ggccctagat 3540tcttcatcaa aaacgcctga gacacttgcc
caggattgaa actccctgaa gggaccacca 3600ggggccctga gttgttcctt ccccccgtgg
cgagctgcca gccaggctgt acctgtgatc 3660gaggctggcg ggaaaatagg cttcgtgtgc
tcaggtcatg ggaggtgcag gacagctcat 3720gaaacgccaa caatcgcaca attcatgtca
agctaatcag ctatttcctc ttcacgagct 3780gtaattgtcc caaaattctg gtctaccggg
ggtgatcctt cgtgtacggg cccttccctc 3840aaccctaggt atgcgcgcat gcggtcgccg
cgcaactcgc gcgagggccg agggtttggg 3900acgggccgtc ccgaaatgca gttgcacccg
gatgcgtggc accttttttg cgataattta 3960tgcaatggac tgctctgcaa aattctggct
ctgtcgccaa ccctaggatc agcggcgtag 4020gatttcgtaa tcattcgtcc tgatggggag
ctaccgacta ccctaatatc agcccgactg 4080cctgacgcca gcgtccactt ttgtgcacac
attccattcg tgcccaagac atttcattgt 4140ggtgcgaagc gtccccagtt acgctcacct
gtttcccgac ctccttactg ttctgtcgac 4200agagcgggcc cacaggccgg tcgcagccac
tagtatggcc accgcatcca ctttctcggc 4260gttcaatgcc cgctgcggcg acctgcgtcg
ctcggcgggc tccgggcccc ggcgcccagc 4320gaggcccctc cccgtgcgcg ggcgcgccgc
cgccgccgcc gacgccaacc ccgcccgccc 4380cgagcgccgc gtggtgatca ccggccaggg
cgtggtgacc tccctgggcc agaccatcga 4440gcagttctac tcctccctgc tggagggcgt
gtccggcatc tcccagatcc agaagttcga 4500caccaccggc tacaccacca ccatcgccgg
cgagatcaag tccctgcagc tggaccccta 4560cgtgcccaag cgctgggcca agcgcgtgga
cgacgtgatc aagtacgtgt acatcgccgg 4620caagcaggcc ctggagtccg ccggcctgcc
catcgaggcc gccggcctgg ccggcgccgg 4680cctggacccc gccctgtgcg gcgtgctgat
cggcaccgcc atggccggca tgacctcctt 4740cgccgccggc gtggaggccc tgacccgcgg
cggcgtgcgc aagatgaacc ccttctgcat 4800ccccttctcc atctccaaca tgggcggcgc
catgctggcc atggacatcg gcttcatggg 4860ccccaactac tccatctcca ccgcctgcgc
caccggcaac tactgcatcc tgggcgccgc 4920cgaccacatc cgccgcggcg acgccaacgt
gatgctggcc ggcggcgccg acgccgccat 4980catcccctcc ggcatcggcg gcttcatcgc
ctgcaaggcc ctgtccaagc gcaacgacga 5040gcccgagcgc gcctcccgcc cctgggacgc
cgaccgcgac ggcttcgtga tgggcgaggg 5100cgccggcgtg ctggtgctgg aggagctgga
gcacgccaag cgccgcggcg ccaccatcct 5160ggccgagctg gtgggcggcg ccgccacctc
cgacgcccac cacatgaccg agcccgaccc 5220ccagggccgc ggcgtgcgcc tgtgcctgga
gcgcgccctg gagcgcgccc gcctggcccc 5280cgagcgcgtg ggctacgtga acgcccacgg
cacctccacc cccgccggcg acgtggccga 5340gtaccgcgcc atccgcgccg tgatccccca
ggactccctg cgcatcaact ccaccaagtc 5400catgatcggc cacctgctgg gcggcgccgg
cgccgtggag gccgtggccg ccatccaggc 5460cctgcgcacc ggctggctgc accccaacct
gaacctggag aaccccgccc ccggcgtgga 5520ccccgtggtg ctggtgggcc cccgcaagga
gcgcgccgag gacctggacg tggtgctgtc 5580caactccttc ggcttcggcg gccacaactc
ctgcgtgatc ttccgcaagt acgacgagat 5640ggactacaag gaccacgacg gcgactacaa
ggaccacgac atcgactaca aggacgacga 5700cgacaagtga atcgatagat ctcttaaggc
agcagcagct cggatagtat cgacacactc 5760tggacgctgg tcgtgtgatg gactgttgcc
gccacacttg ctgccttgac ctgtgaatat 5820ccctgccgct tttatcaaac agcctcagtg
tgtttgatct tgtgtgtacg cgcttttgcg 5880agttgctagc tgcttgtgct atttgcgaat
accaccccca gcatcccctt ccctcgtttc 5940atatcgcttg catcccaacc gcaacttatc
tacgctgtcc tgctatccct cagcgctgct 6000cctgctcctg ctcactgccc ctcgcacagc
cttggtttgg gctccgcctg tattctcctg 6060gtactgcaac ctgtaaacca gcactgcaat
gctgatgcac gggaagtagt gggatgggaa 6120cacaaatgga aagcttaatt aagagctcct
ttcttgcgct atgacacttc cagcaaaagg 6180tagggcgggc tgcgagacgg cttcccggcg
ctgcatgcaa caccgatgat gcttcgaccc 6240cccgaagctc cttcggggct gcatgggcgc
tccgatgccg ctccagggcg agcgctgttt 6300aaatagccag gcccccgatt gcaaagacat
tatagcgagc taccaaagcc atattcaaac 6360acctagatca ctaccacttc tacacaggcc
actcgagctt gtgatcgcac tccgctaagg 6420gggcgcctct tcctcttcgt ttcagtcaca
acccgcaaac actagtatgg ctatcaagac 6480gaacaggcag cctgtggaga agcctccgtt
cacgatcggg acgctgcgca aggccatccc 6540cgcgcactgt ttcgagcgct cggcgcttcg
tagcagcatg tacctggcct ttgacatcgc 6600ggtcatgtcc ctgctctacg tcgcgtcgac
gtacatcgac cctgcaccgg tgcctacgtg 6660ggtcaagtac ggcatcatgt ggccgctcta
ctggttcttc caggtgtgtt tgagggtttt 6720ggttgcccgt attgaggtcc tggtggcgcg
catggaggag aaggcgcctg tcccgctgac 6780ccccccggct accctcccgg caccttccag
ggcgcgtacg ggaagaacca gtagagcggc 6840cacatgatgc cgtacttgac ccacgtaggc
accggtgcag ggtcgatgta cgtcgacgcg 6900acgtagagca gggacatgac cgcgatgtca
aaggccaggt acatgctgct acgaagcgcc 6960gagcgctcga aacagtgcgc ggggatggcc
ttgcgcagcg tcccgatcgt gaacggaggc 7020ttctccacag gctgcctgtt cgtcttgata
gccatctcga ggcagcagca gctcggatag 7080tatcgacaca ctctggacgc tggtcgtgtg
atggactgtt gccgccacac ttgctgcctt 7140gacctgtgaa tatccctgcc gcttttatca
aacagcctca gtgtgtttga tcttgtgtgt 7200acgcgctttt gcgagttgct agctgcttgt
gctatttgcg aataccaccc ccagcatccc 7260cttccctcgt ttcatatcgc ttgcatccca
accgcaactt atctacgctg tcctgctatc 7320cctcagcgct gctcctgctc ctgctcactg
cccctcgcac agccttggtt tgggctccgc 7380ctgtattctc ctggtactgc aacctgtaaa
ccagcactgc aatgctgatg cacgggaagt 7440agtgggatgg gaacacaaat ggaaagctgt
attgttttcc agaaggagtt gctccttgag 7500cctttcattc tcagcctcga taacctccaa
agccgctcta attgtggagg gggttcgaag 7560acagggtggt tggctggatg gggaaacgct
ggtcgcggga ttcgatcctg ctgcttatat 7620cctccctgga agcacaccca cgactctgaa
gaagaaaacg tgcacacaca caacccaacc 7680ggccgaatat ttgcttcctt atcccgggtc
caagagagac tgcgatgccc ccctcaatca 7740gcatcctcct ccctgccgct tcaatcttcc
ctgcttgcct gcgcccgcgg tgcgccgtct 7800gcccgcccag tcagtcactc ctgcacaggc
cccttgtgcg cagtgctcct gtacccttta 7860ccgctccttc cattctgcga ggccccctat
tgaatgtatt cgttgcctgt gtggccaagc 7920gggctgctgg gcgcgccgcc gtcgggcagt
gctcggcgac tttggcggaa gccgattgtt 7980cttctgtaag ccacgcgctt gctgctttgg
gaagagaagg gggggggtac tgaatggatg 8040aggaggagaa ggaggggtat tggtattatc
tgagttgggt gaagagc 8087974479DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
97gctcttcggg tcgccgcgct gcctcgcgtc ccctggtggt gcgcgcggtc gccagcgagg
60ccccgctggg cgttccgccc tcggtgcagc gcccctcccc cgtggtctac tccaagctgg
120acaagcagca ccgcctgacg cccgagcgcc tggagctggt gcagagcatg gggcagtttg
180cggaggagag ggtgctgccc gtgctgcacc ccgtggacaa gctgtggcag ccgcaggact
240ttttgcccga ccccgagtcg cccgacttcg aggatcaggt ggcggagctg cgcgcgcgcg
300ccaaggacct gcccgacgag tactttgtgg tgctggtggg ggacatgatc acggaggagg
360cgctgccgac ctacatggcc atgctcaaca cgctggacgg cgtgcgcgac gacacgggcg
420cggccgacca cccgtgggcg cgctggacgc ggcagtgggt ggccgaggag aaccggcacg
480gcgacctgct gaacaagtac tgctggctga cggggcgcgt caacatgcgg gccgtggagg
540tgaccatcaa caacctgatc aagagcggca tgaacccgca gacggacaac aacccttatt
600tggggttcgt ctacacctcc ttccaggagc gcgccaccaa gtaggtaccc tttcttgcgc
660tatgacactt ccagcaaaag gtagggcggg ctgcgagacg gcttcccggc gctgcatgca
720acaccgatga tgcttcgacc ccccgaagct ccttcggggc tgcatgggcg ctccgatgcc
780gctccagggc gagcgctgtt taaatagcca ggcccccgat tgcaaagaca ttatagcgag
840ctaccaaagc catattcaaa cacctagatc actaccactt ctacacaggc cactcgagct
900tgtgatcgca ctccgctaag ggggcgcctc ttcctcttcg tttcagtcac aacccgcaaa
960ctctagaata tcaatgatcg agcaggacgg cctccacgcc ggctcccccg ccgcctgggt
1020ggagcgcctg ttcggctacg actgggccca gcagaccatc ggctgctccg acgccgccgt
1080gttccgcctg tccgcccagg gccgccccgt gctgttcgtg aagaccgacc tgtccggcgc
1140cctgaacgag ctgcaggacg aggccgcccg cctgtcctgg ctggccacca ccggcgtgcc
1200ctgcgccgcc gtgctggacg tggtgaccga ggccggccgc gactggctgc tgctgggcga
1260ggtgcccggc caggacctgc tgtcctccca cctggccccc gccgagaagg tgtccatcat
1320ggccgacgcc atgcgccgcc tgcacaccct ggaccccgcc acctgcccct tcgaccacca
1380ggccaagcac cgcatcgagc gcgcccgcac ccgcatggag gccggcctgg tggaccagga
1440cgacctggac gaggagcacc agggcctggc ccccgccgag ctgttcgccc gcctgaaggc
1500ccgcatgccc gacggcgagg acctggtggt gacccacggc gacgcctgcc tgcccaacat
1560catggtggag aacggccgct tctccggctt catcgactgc ggccgcctgg gcgtggccga
1620ccgctaccag gacatcgccc tggccacccg cgacatcgcc gaggagctgg gcggcgagtg
1680ggccgaccgc ttcctggtgc tgtacggcat cgccgccccc gactcccagc gcatcgcctt
1740ctaccgcctg ctggacgagt tcttctgaca attggcagca gcagctcgga tagtatcgac
1800acactctgga cgctggtcgt gtgatggact gttgccgcca cacttgctgc cttgacctgt
1860gaatatccct gccgctttta tcaaacagcc tcagtgtgtt tgatcttgtg tgtacgcgct
1920tttgcgagtt gctagctgct tgtgctattt gcgaatacca cccccagcat ccccttccct
1980cgtttcatat cgcttgcatc ccaaccgcaa cttatctacg ctgtcctgct atccctcagc
2040gctgctcctg ctcctgctca ctgcccctcg cacagccttg gtttgggctc cgcctgtatt
2100ctcctggtac tgcaacctgt aaaccagcac tgcaatgctg atgcacggga agtagtggga
2160tgggaacaca aatggaggat cccgcgtctc gaacagagcg cgcagaggaa cgctgaaggt
2220ctcgcctctg tcgcacctca gcgcggcata caccacaata accacctgac gaatgcgctt
2280ggttcttcgt ccattagcga agcgtccggt tcacacacgt gccacgttgg cgaggtggca
2340ggtgacaatg atcggtggag ctgatggtcg aaacgttcac agcctaggga tatcgaattc
2400ctttcttgcg ctatgacact tccagcaaaa ggtagggcgg gctgcgagac ggcttcccgg
2460cgctgcatgc aacaccgatg atgcttcgac cccccgaagc tccttcgggg ctgcatgggc
2520gctccgatgc cgctccaggg cgagcgctgt ttaaatagcc aggcccccga ttgcaaagac
2580attatagcga gctaccaaag ccatattcaa acacctagat cactaccact tctacacagg
2640ccactcgagc ttgtgatcgc actccgctaa gggggcgcct cttcctcttc gtttcagtca
2700caacccgcaa acactagtgc gctggacgcg gcagtgggtg gccgaggaga accggcacgg
2760cgacctgctg aacaagtact gttggctgac ggggcgcgtc aacatgcggg ccgtggaggt
2820gaccatcaac aacctgatca agagcggcat gaacccgcag acggacaaca acccttactt
2880gggcttcgtc tacacctcct tccaggagcg cgcgaccaag tacagccacg gcaacaccgc
2940gcgccttgcg gccgagcagt gtgtttgagg gttttggttg cccgtatcga ggtcctggtg
3000gcgcgcatgg gggagaaggc gcctgtcccg ctgacccccc cggctaccct cccggcacct
3060tccagggcgc gtacgggatc ctgctcggcc gcaaggcgcg cggtgttgcc gtggctgtac
3120ttggtcgcgc gctcctggaa ggaggtgtag acgaagccca agtaagggtt gttgtccgtc
3180tgcgggttca tgccgctctt gatcaggttg ttgatggtca cctccacggc ccgcatgttg
3240acgcgccccg tcagccaaca gtacttgttc agcaggtcgc cgtgccggtt ctcctcggcc
3300acccactgcc gcgtccagcg caagcttgca gcagcagctc ggatagtatc gacacactct
3360ggacgctggt cgtgtgatgg actgttgccg ccacacttgc tgccttgacc tgtgaatatc
3420cctgccgctt ttatcaaaca gcctcagtgt gtttgatctt gtgtgtacgc gcttttgcga
3480gttgctagct gcttgtgcta tttgcgaata ccacccccag catccccttc cctcgtttca
3540tatcgcttgc atcccaaccg caacttatct acgctgtcct gctatccctc agcgctgctc
3600ctgctcctgc tcactgcccc tcgcacagcc ttggtttggg ctccgcctgt attctcctgg
3660tactgcaacc tgtaaaccag cactgcaatg ctgatgcacg ggaagtagtg ggatgggaac
3720acaaatggaa agctggagct ccagccacgg caacaccgcg cgccttgcgg ccgagcacgg
3780cgacaagaac ctgagcaaga tctgcgggct gatcgccagc gacgagggcc ggcacgagat
3840cgcctacacg cgcatcgtgg acgagttctt ccgcctcgac cccgagggcg ccgtcgccgc
3900ctacgccaac atgatgcgca agcagatcac catgcccgcg cacctcatgg acgacatggg
3960ccacggcgag gccaacccgg gccgcaacct cttcgccgac ttctccgcgg tcgccgagaa
4020gatcgacgtc tacgacgccg aggactactg ccgcatcctg gagcacctca acgcgcgctg
4080gaaggtggac gagcgccagg tcagcggcca ggccgccgcg gaccaggagt acgtcctggg
4140cctgccccag cgcttccgga aactcgccga gaagaccgcc gccaagcgca agcgcgtcgc
4200gcgcaggccc gtcgccttct cctggatctc cgggcgcgag atcatggtct agggagcgac
4260gagtgtgcgt gcggggctgg cgggagtggg acgccctcct cgctcctctc tgttctgaac
4320ggaacaatcg gccaccccgc gctacgcgcc acgcatcgag caacgaagaa aaccccccga
4380tgataggttg cggtggctgc cgggatatag atccggccgc acatcaaagg gcccctccgc
4440cagagaagaa gctcctttcc cagcagactc ctgaagagc
4479985243DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 98atgcatgccg gtcaccaccc gcatgctcgt
actacagcgc acgcaccgct tcgtgatcca 60ccgggtgaac gtagtcctcg acggaaacat
ctggttcggg cctcctgctt gcactcccgc 120ccatgccgac aacctttctg ctgttaccac
gacccacaat gcaacgcgac acgaccgtgt 180gggactgatc ggttcactgc acctgcatgc
aattgtcaca agcgcttact ccaattgtat 240tcgtttgttt tctgggagca gttgctcgac
cgcccgcgtc ccgcaggcag cgatgacgtg 300tgcgtggcct gggtgtttcg tcgaaaggcc
agcaacccta aatcgcaggc gatccggaga 360ttgggatctg atccgagttt ggaccagatc
cgccccgatg cggcacggga actgcatcga 420ctcggcgcgg aacccagctt tcgtaaatgc
cagattggtg tccgatacct ggatttgcca 480tcagcgaaac aagacttcag cagcgagcgt
atttggcggg cgtgctacca gggttgcata 540cattgcccat ttctgtctgg accgctttac
tggcgcagag ggtgagttga tggggttggc 600aggcatcgaa acgcgcgtgc atggtgtgcg
tgtctgtttt cggctgcacg aattcaatag 660tcggatgggc gacggtagaa ttgggtgtgg
cgctcgcgtg catgcctcgc cccgtcgggt 720gtcatgaccg ggactggaat cccccctcgc
gaccatcttg ctaacgctcc cgactctccc 780gactagtgcg ctggacgcgg cagtgggtgg
ccgaggagaa ccggcacggc gacctgctga 840acaagtactg ttggctgacg gggcgcgtca
acatgcgggc cgtggaggtg accatcaaca 900acctgatcaa gagcggcatg aacccgcaga
cggacaacaa cccttacttg ggcttcgtct 960acacctcctt ccaggagcgc gcgaccaagt
acagccacgg caacaccgcg cgccttgcgg 1020ccgagcagtg tgtttgaggg ttttggttgc
ccgtatcgag gtcctggtgg cgcgcatggg 1080ggagaaggcg cctgtcccgc tgaccccccc
ggctaccctc ccggcacctt ccagggcgcg 1140tacgggatcc tgctcggccg caaggcgcgc
ggtgttgccg tggctgtact tggtcgcgcg 1200ctcctggaag gaggtgtaga cgaagcccaa
gtaagggttg ttgtccgtct gcgggttcat 1260gccgctcttg atcaggttgt tgatggtcac
ctccacggcc cgcatgttga cgcgccccgt 1320cagccaacag tacttgttca gcaggtcgcc
gtgccggttc tcctcggcca cccactgccg 1380cgtccagcgc aagcttgcag cagcagctcg
gatagtatcg acacactctg gacgctggtc 1440gtgtgatgga ctgttgccgc cacacttgct
gccttgacct gtgaatatcc ctgccgcttt 1500tatcaaacag cctcagtgtg tttgatcttg
tgtgtacgcg cttttgcgag ttgctagctg 1560cttgtgctat ttgcgaatac cacccccagc
atccccttcc ctcgtttcat atcgcttgca 1620tcccaaccgc aacttatcta cgctgtcctg
ctatccctca gcgctgctcc tgctcctgct 1680cactgcccct cgcacagcct tggtttgggc
tccgcctgta ttctcctggt actgcaacct 1740gtaaaccagc actgcaatgc tgatgcacgg
gaagtagtgg gatgggaaca caaatggaaa 1800gctggagctc aaagatatca acttaattaa
ccaaggtacc cgcctgcaac gcaagggcag 1860ccacagccgc tcccacccgc cgctgaaccg
acacgtgctt gggcgcctgc cgcctgcctg 1920ccgcatgctt gtgctggtga ggctgggcag
tgctgccatg ctgattgagg cttggttcat 1980cgggtggaag cttatgtgtg tgctgggctt
gcatgccggg caatgcgcat ggtggcaaga 2040gggcggcagc acttgctgga gctgccgcgg
tgcctccagg tggttcaatc gcggcagcca 2100gagggatttc agatgatcgc gcgtacaggt
tgagcagcag tgtcagcaaa ggtagcagtt 2160tgccagaatg atcggttcag ctgttaatca
atgccagcaa gagaaggggt caagtgcaaa 2220cacgggcatg ccacagcacg ggcaccgggg
agtggaatgg caccaccaag tgtgtgcgag 2280ccagcatcgc cgcctggctg tttcagctac
aacggcagga gtcatccaac gtaaccatga 2340gctgatcaac actgcaatca tcgggcgggc
gtgatgcaag catgcctggc gaagacacat 2400ggtgtgcgga tgctgccggc tgctgcctgc
tgcgcacgcc gttgagttgg cagcaggctc 2460agccatgcac tggatggcag ctgggctgcc
actgcaatgt ggtggatagg atgcaagtgg 2520agcgaatacc aaaccctctg gctgcttgct
gggttgcatg gcatcgcacc atcagcagga 2580gcgcatgcga agggactggc cccatgcacg
ccatgccaaa ccggagcgca ccgagtgtcc 2640acactgtcac caggcccgca agctttgcag
aaccatgctc atggacgcat gtagcgctga 2700cgtcccttga cggcgctcct ctcgggtgtg
ggaaacgcaa tgcagcacag gcagcagagg 2760cggcggcagc agagcggcgg cagcagcggc
gggggccacc cttcttgcgg ggtcgcgccc 2820cagccagcgg tgatgcgctg atcccaaacg
agttcacatt catttgcatg cctggagaag 2880cgaggctggg gcctttgggc tggtgcagcc
cgcaatggaa tgcgggaccg ccaggctagc 2940agcaaaggcg cctcccctac tccgcatcga
tgttccatag tgcattggac tgcatttggg 3000tggggcggcc ggctgtttct ttcgtgttgc
aaaacgcgcc agctcagcaa cctgtcccgt 3060gggtcccccg tgccgatgaa atcgtgtgca
cgccgatcag ctgattgccc ggctcgcgaa 3120gtaggcgccc tcctttctgc tcgccctctc
tccgtcccgc ctctagaata tcaatgatcg 3180agcaggacgg cctccacgcc ggctcccccg
ccgcctgggt ggagcgcctg ttcggctacg 3240actgggccca gcagaccatc ggctgctccg
acgccgccgt gttccgcctg tccgcccagg 3300gccgccccgt gctgttcgtg aagaccgacc
tgtccggcgc cctgaacgag ctgcaggacg 3360aggccgcccg cctgtcctgg ctggccacca
ccggcgtgcc ctgcgccgcc gtgctggacg 3420tggtgaccga ggccggccgc gactggctgc
tgctgggcga ggtgcccggc caggacctgc 3480tgtcctccca cctggccccc gccgagaagg
tgtccatcat ggccgacgcc atgcgccgcc 3540tgcacaccct ggaccccgcc acctgcccct
tcgaccacca ggccaagcac cgcatcgagc 3600gcgcccgcac ccgcatggag gccggcctgg
tggaccagga cgacctggac gaggagcacc 3660agggcctggc ccccgccgag ctgttcgccc
gcctgaaggc ccgcatgccc gacggcgagg 3720acctggtggt gacccacggc gacgcctgcc
tgcccaacat catggtggag aacggccgct 3780tctccggctt catcgactgc ggccgcctgg
gcgtggccga ccgctaccag gacatcgccc 3840tggccacccg cgacatcgcc gaggagctgg
gcggcgagtg ggccgaccgc ttcctggtgc 3900tgtacggcat cgccgccccc gactcccagc
gcatcgcctt ctaccgcctg ctggacgagt 3960tcttctgaca attggcagca gcagctcgga
tagtatcgac acactctgga cgctggtcgt 4020gtgatggact gttgccgcca cacttgctgc
cttgacctgt gaatatccct gccgctttta 4080tcaaacagcc tcagtgtgtt tgatcttgtg
tgtacgcgct tttgcgagtt gctagctgct 4140tgtgctattt gcgaatacca cccccagcat
ccccttccct cgtttcatat cgcttgcatc 4200ccaaccgcaa cttatctacg ctgtcctgct
atccctcagc gctgctcctg ctcctgctca 4260ctgcccctcg cacagccttg gtttgggctc
cgcctgtatt ctcctggtac tgcaacctgt 4320aaaccagcac tgcaatgctg atgcacggga
agtagtggga tgggaacaca aatggaggat 4380cccgcgtctc gaacagagcg cgcagaggaa
cgctgaaggt ctcgcctctg tcgcacctca 4440gcgcggcata caccacaata accacctgac
gaatgcgctt ggttcttcgt ccattagcga 4500agcgtccggt tcacacacgt gccacgttgg
cgaggtggca ggtgacaatg atcggtggag 4560ctgatggtcg aaacgttcac agcctaggga
tatcgaattc cgggtcgccg cgctgcctcg 4620cgtcccctgg tggtgcgcgc ggtcgccagc
gaggccccgc tgggcgttcc gccctcggtg 4680cagcgcccct cccccgtggt ctactccaag
ctggacaagc agcaccgcct gacgcccgag 4740cgcctggagc tggtgcagag catggggcag
tttgcggagg agagggtgct gcccgtgctg 4800caccccgtgg acaagctgtg gcagccgcag
gactttttgc ccgaccccga gtcgcccgac 4860ttcgaggatc aggtggcgga gctgcgcgcg
cgcgccaagg acctgcccga cgagtacttt 4920gtggtgctgg tgggggacat gatcacggag
gaggcgctgc cgacctacat ggccatgctc 4980aacacgctgg acggcgtgcg cgacgacacg
ggcgcggccg accacccgtg ggcgcgctgg 5040acgcggcagt gggtggccga ggagaaccgg
cacggcgacc tgctgaacaa gtactgctgg 5100ctgacggggc gcgtcaacat gcgggccgtg
gaggtgacca tcaacaacct gatcaagagc 5160ggcatgaacc cgcagacgga caacaaccct
tatttggggt tcgtctacac ctccttccag 5220gagcgcgcca ccaagtatct aga
5243996804DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
99gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg
1080gccggcttcg ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg
1140gtgcacttca cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag
1200aaggacgcca agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg
1260cccttgttct ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc
1320gccatcgccc cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac
1380aacaacacct ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc
1440tggacctaca acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc
1500tacaccttca ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc
1560gacccgaagg tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc
1620caggactaca agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc
1680gcgttcgcca acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc
1740cccaccgagc aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc
1800gccccggccg gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc
1860gaggccttcg acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag
1920accttcttca acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac
1980tgggagtact ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc
2040aagttctccc tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag
2100gccgagccga tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc
2160acgttgacga aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag
2220ttcgagctgg tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac
2280ctctccctct ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag
2340gtgtccgcgt cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag
2400aacccctact tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac
2460ctgtcctact acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac
2520gacggcgacg tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc
2580gtgaacatga cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag
2640gtcaagtgac aattggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg
2700tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt
2760atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc
2820ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat
2880cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc
2940actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg
3000taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggagga
3060tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct gtcgcacctc
3120agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg tccattagcg
3180aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat gatcggtgga
3240gctgatggtc gaaacgttca cagcctaggg atatcgaatt cggccgacag gacgcgcgtc
3300aaaggtgctg gtcgtgtatg ccctggccgg caggtcgttg ctgctgctgg ttagtgattc
3360cgcaaccctg attttggcgt cttattttgg cgtggcaaac gctggcgccc gcgagccggg
3420ccggcggcga tgcggtgccc cacggctgcc ggaatccaag ggaggcaaga gcgcccgggt
3480cagttgaagg gctttacgcg caaggtacag ccgctcctgc aaggctgcgt ggtggaattg
3540gacgtgcagg tcctgctgaa gttcctccac cgcctcacca gcggacaaag caccggtgta
3600tcaggtccgt gtcatccact ctaaagaact cgactacgac ctactgatgg ccctagattc
3660ttcatcaaaa acgcctgaga cacttgccca ggattgaaac tccctgaagg gaccaccagg
3720ggccctgagt tgttccttcc ccccgtggcg agctgccagc caggctgtac ctgtgatcga
3780ggctggcggg aaaataggct tcgtgtgctc aggtcatggg aggtgcagga cagctcatga
3840aacgccaaca atcgcacaat tcatgtcaag ctaatcagct atttcctctt cacgagctgt
3900aattgtccca aaattctggt ctaccggggg tgatccttcg tgtacgggcc cttccctcaa
3960ccctaggtat gcgcgcatgc ggtcgccgcg caactcgcgc gagggccgag ggtttgggac
4020gggccgtccc gaaatgcagt tgcacccgga tgcgtggcac cttttttgcg ataatttatg
4080caatggactg ctctgcaaaa ttctggctct gtcgccaacc ctaggatcag cggcgtagga
4140tttcgtaatc attcgtcctg atggggagct accgactacc ctaatatcag cccgactgcc
4200tgacgccagc gtccactttt gtgcacacat tccattcgtg cccaagacat ttcattgtgg
4260tgcgaagcgt ccccagttac gctcacctgt ttcccgacct ccttactgtt ctgtcgacag
4320agcgggccca caggccggtc gcagccacta gtatggccac cgcatccact ttctcggcgt
4380tcaatgcccg ctgcggcgac ctgcgtcgct cggcgggctc cgggccccgg cgcccagcga
4440ggcccctccc cgtgcgcggg cgcgccgcca ccctgcgctc cggcctgcgc gacgtggaga
4500ccgtgaagaa gaccttctcc cccgcccgcg aggtgcacgt gcaggtgacc cactccatgg
4560ccccccagaa gatcgagatc ttcaaggcca tggaggactg ggccgagaac aacatcctgg
4620tgcacctgaa gaacgtggag aagtgccccc agccccagga cttcctgccc gaccccgcct
4680ccgacgagtt ccacgaccag atcaaggagc tgcgcgagcg cgccaaggag atccccgacg
4740actacttcgt ggtgctggtg ggcgacatga tcaccgagga ggccctgccc acctaccaga
4800ccatgctgaa cacctgggac ggcgtgcgcg acgagaccgg cgcctccccc acctcctggg
4860ccatctggac ccgcgcctgg accgccgagg agaaccgcca cggcgacccc ctgaacaagt
4920acctgtacct gtccggccgc gtggacatga agcagatcga gaagaccatc cagtacctga
4980tcggctccgg catggacccc cgcaccgaga actcccccta cctgggcttc atctacacct
5040ccttccagga gcgcgccacc ttcatctccc acggcaacac cgcccgcctg gcccgcgacc
5100acggcgactt caagctggcc cagatctgcg gcaccatcgc ctccgacgag aagcgccacg
5160agaccgccta caccaagatc gtggagaagc tgttcgagat cgaccccgac ggcaccgtgc
5220tggccttcgg cgacatgatg aagaagaaga tctccatgcc cgaccacttc atgtacgacg
5280gccgcgacga caacctgttc gaccacttct cctccgtggc ccagcgcctg ggcgtgtaca
5340ccgccaagga ctacgccgac atcctggagc acctggtggg ccgctggaag gtggagaagc
5400tgaccggcct gtccgccgag ggccagaagg cccaggacta cgtgtgcggc ctgccccccc
5460gcatccgccg cctggaggag cgcgcccaga tccgcgccaa gcaggccccc cgcctgccct
5520tctcctggat ctacgaccgc gaggtgcagc tgatggacta caaggaccac gacggcgact
5580acaaggacca cgacatcgac tacaaggacg acgacgacaa gtgaatcgat agatctctta
5640aggcagcagc agctcggata gtatcgacac actctggacg ctggtcgtgt gatggactgt
5700tgccgccaca cttgctgcct tgacctgtga atatccctgc cgcttttatc aaacagcctc
5760agtgtgtttg atcttgtgtg tacgcgcttt tgcgagttgc tagctgcttg tgctatttgc
5820gaataccacc cccagcatcc ccttccctcg tttcatatcg cttgcatccc aaccgcaact
5880tatctacgct gtcctgctat ccctcagcgc tgctcctgct cctgctcact gcccctcgca
5940cagccttggt ttgggctccg cctgtattct cctggtactg caacctgtaa accagcactg
6000caatgctgat gcacgggaag tagtgggatg ggaacacaaa tggaaagctt aattaagagc
6060tcttgttttc cagaaggagt tgctccttga gcctttcatt ctcagcctcg ataacctcca
6120aagccgctct aattgtggag ggggttcgaa tttaaaagct tggaatgttg gttcgtgcgt
6180ctggaacaag cccagacttg ttgctcactg ggaaaaggac catcagctcc aaaaaacttg
6240ccgctcaaac cgcgtacctc tgctttcgcg caatctgccc tgttgaaatc gccaccacat
6300tcatattgtg acgcttgagc agtctgtaat tgcctcagaa tgtggaatca tctgccccct
6360gtgcgagccc atgccaggca tgtcgcgggc gaggacaccc gccactcgta cagcagacca
6420ttatgctacc tcacaatagt tcataacagt gaccatattt ctcgaagctc cccaacgagc
6480acctccatgc tctgagtggc caccccccgg ccctggtgct tgcggagggc aggtcaaccg
6540gcatggggct accgaaatcc ccgaccggat cccaccaccc ccgcgatggg aagaatctct
6600ccccgggatg tgggcccacc accagcacaa cctgctggcc caggcgagcg tcaaaccata
6660ccacacaaat atccttggca tcggccctga attccttctg ccgctctgct acccggtgct
6720tctgtccgaa gcaggggttg ctagggatcg ctccgagtcc gcaaaccctt gtcgcgtggc
6780ggggcttgtt cgagcttgaa gagc
68041006792DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 100gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcgaatt cggccgacag gacgcgcgtc 3300aaaggtgctg gtcgtgtatg ccctggccgg
caggtcgttg ctgctgctgg ttagtgattc 3360cgcaaccctg attttggcgt cttattttgg
cgtggcaaac gctggcgccc gcgagccggg 3420ccggcggcga tgcggtgccc cacggctgcc
ggaatccaag ggaggcaaga gcgcccgggt 3480cagttgaagg gctttacgcg caaggtacag
ccgctcctgc aaggctgcgt ggtggaattg 3540gacgtgcagg tcctgctgaa gttcctccac
cgcctcacca gcggacaaag caccggtgta 3600tcaggtccgt gtcatccact ctaaagaact
cgactacgac ctactgatgg ccctagattc 3660ttcatcaaaa acgcctgaga cacttgccca
ggattgaaac tccctgaagg gaccaccagg 3720ggccctgagt tgttccttcc ccccgtggcg
agctgccagc caggctgtac ctgtgatcga 3780ggctggcggg aaaataggct tcgtgtgctc
aggtcatggg aggtgcagga cagctcatga 3840aacgccaaca atcgcacaat tcatgtcaag
ctaatcagct atttcctctt cacgagctgt 3900aattgtccca aaattctggt ctaccggggg
tgatccttcg tgtacgggcc cttccctcaa 3960ccctaggtat gcgcgcatgc ggtcgccgcg
caactcgcgc gagggccgag ggtttgggac 4020gggccgtccc gaaatgcagt tgcacccgga
tgcgtggcac cttttttgcg ataatttatg 4080caatggactg ctctgcaaaa ttctggctct
gtcgccaacc ctaggatcag cggcgtagga 4140tttcgtaatc attcgtcctg atggggagct
accgactacc ctaatatcag cccgactgcc 4200tgacgccagc gtccactttt gtgcacacat
tccattcgtg cccaagacat ttcattgtgg 4260tgcgaagcgt ccccagttac gctcacctgt
ttcccgacct ccttactgtt ctgtcgacag 4320agcgggccca caggccggtc gcagccacta
gtatggccct gaagctgaac gccatcaact 4380tccagtcccc caagtgctcc tccttcggcc
tgccccccgt ggtgtccctg cgctccccca 4440agctgtccgt ggccgccacc ctgcgctccg
gcctgcgcga cgtggagacc gtgaagaaga 4500ccttctcccc cgcccgcgag gtgcacgtgc
aggtgaccca ctccatggcc ccccagaaga 4560tcgagatctt caaggccatg gaggactggg
ccgagaacaa catcctggtg cacctgaaga 4620acgtggagaa gtgcccccag ccccaggact
tcctgcccga ccccgcctcc gacgagttcc 4680acgaccagat caaggagctg cgcgagcgcg
ccaaggagat ccccgacgac tacttcgtgg 4740tgctggtggg cgacatgatc accgaggagg
ccctgcccac ctaccagacc atgctgaaca 4800cctgggacgg cgtgcgcgac gagaccggcg
cctcccccac ctcctgggcc atctggaccc 4860gcgcctggac cgccgaggag aaccgccacg
gcgaccccct gaacaagtac ctgtacctgt 4920ccggccgcgt ggacatgaag cagatcgaga
agaccatcca gtacctgatc ggctccggca 4980tggacccccg caccgagaac tccccctacc
tgggcttcat ctacacctcc ttccaggagc 5040gcgccacctt catctcccac ggcaacaccg
cccgcctggc ccgcgaccac ggcgacttca 5100agctggccca gatctgcggc accatcgcct
ccgacgagaa gcgccacgag accgcctaca 5160ccaagatcgt ggagaagctg ttcgagatcg
accccgacgg caccgtgctg gccttcggcg 5220acatgatgaa gaagaagatc tccatgcccg
accacttcat gtacgacggc cgcgacgaca 5280acctgttcga ccacttctcc tccgtggccc
agcgcctggg cgtgtacacc gccaaggact 5340acgccgacat cctggagcac ctggtgggcc
gctggaaggt ggagaagctg accggcctgt 5400ccgccgaggg ccagaaggcc caggactacg
tgtgcggcct gcccccccgc atccgccgcc 5460tggaggagcg cgcccagatc cgcgccaagc
aggccccccg cctgcccttc tcctggatct 5520acgaccgcga ggtgcagctg atggactaca
aggaccacga cggcgactac aaggaccacg 5580acatcgacta caaggacgac gacgacaagt
gaatcgatag atctcttaag gcagcagcag 5640ctcggatagt atcgacacac tctggacgct
ggtcgtgtga tggactgttg ccgccacact 5700tgctgccttg acctgtgaat atccctgccg
cttttatcaa acagcctcag tgtgtttgat 5760cttgtgtgta cgcgcttttg cgagttgcta
gctgcttgtg ctatttgcga ataccacccc 5820cagcatcccc ttccctcgtt tcatatcgct
tgcatcccaa ccgcaactta tctacgctgt 5880cctgctatcc ctcagcgctg ctcctgctcc
tgctcactgc ccctcgcaca gccttggttt 5940gggctccgcc tgtattctcc tggtactgca
acctgtaaac cagcactgca atgctgatgc 6000acgggaagta gtgggatggg aacacaaatg
gaaagcttaa ttaagagctc ttgttttcca 6060gaaggagttg ctccttgagc ctttcattct
cagcctcgat aacctccaaa gccgctctaa 6120ttgtggaggg ggttcgaatt taaaagcttg
gaatgttggt tcgtgcgtct ggaacaagcc 6180cagacttgtt gctcactggg aaaaggacca
tcagctccaa aaaacttgcc gctcaaaccg 6240cgtacctctg ctttcgcgca atctgccctg
ttgaaatcgc caccacattc atattgtgac 6300gcttgagcag tctgtaattg cctcagaatg
tggaatcatc tgccccctgt gcgagcccat 6360gccaggcatg tcgcgggcga ggacacccgc
cactcgtaca gcagaccatt atgctacctc 6420acaatagttc ataacagtga ccatatttct
cgaagctccc caacgagcac ctccatgctc 6480tgagtggcca ccccccggcc ctggtgcttg
cggagggcag gtcaaccggc atggggctac 6540cgaaatcccc gaccggatcc caccaccccc
gcgatgggaa gaatctctcc ccgggatgtg 6600ggcccaccac cagcacaacc tgctggccca
ggcgagcgtc aaaccatacc acacaaatat 6660ccttggcatc ggccctgaat tccttctgcc
gctctgctac ccggtgcttc tgtccgaagc 6720aggggttgct agggatcgct ccgagtccgc
aaacccttgt cgcgtggcgg ggcttgttcg 6780agcttgaaga gc
67921016051DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
101gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg
1080gccggcttcg ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg
1140gtgcacttca cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag
1200aaggacgcca agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg
1260cccttgttct ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc
1320gccatcgccc cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac
1380aacaacacct ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc
1440tggacctaca acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc
1500tacaccttca ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc
1560gacccgaagg tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc
1620caggactaca agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc
1680gcgttcgcca acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc
1740cccaccgagc aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc
1800gccccggccg gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc
1860gaggccttcg acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag
1920accttcttca acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac
1980tgggagtact ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc
2040aagttctccc tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag
2100gccgagccga tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc
2160acgttgacga aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag
2220ttcgagctgg tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac
2280ctctccctct ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag
2340gtgtccgcgt cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag
2400aacccctact tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac
2460ctgtcctact acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac
2520gacggcgacg tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc
2580gtgaacatga cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag
2640gtcaagtgac aattggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg
2700tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt
2760atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc
2820ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat
2880cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc
2940actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg
3000taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggagga
3060tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct gtcgcacctc
3120agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg tccattagcg
3180aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat gatcggtgga
3240gctgatggtc gaaacgttca cagcctaggg atatcgaatt cctttcttgc gctatgacac
3300ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg gcgctgcatg caacaccgat
3360gatgcttcga ccccccgaag ctccttcggg gctgcatggg cgctccgatg ccgctccagg
3420gcgagcgctg tttaaatagc caggcccccg attgcaaaga cattatagcg agctaccaaa
3480gccatattca aacacctaga tcactaccac ttctacacag gccactcgag cttgtgatcg
3540cactccgcta agggggcgcc tcttcctctt cgtttcagtc acaacccgca aacactagta
3600tggccaccgc atccactttc tcggcgttca atgcccgctg cggcgacctg cgtcgctcgg
3660cgggctccgg gccccggcgc ccagcgaggc ccctccccgt gcgcgggcgc gccgccaccc
3720tgcgctccgg cctgcgcgac gtggagaccg tgaagaagac cttctccccc gcccgcgagg
3780tgcacgtgca ggtgacccac tccatggccc cccagaagat cgagatcttc aaggccatgg
3840aggactgggc cgagaacaac atcctggtgc acctgaagaa cgtggagaag tgcccccagc
3900cccaggactt cctgcccgac cccgcctccg acgagttcca cgaccagatc aaggagctgc
3960gcgagcgcgc caaggagatc cccgacgact acttcgtggt gctggtgggc gacatgatca
4020ccgaggaggc cctgcccacc taccagacca tgctgaacac ctgggacggc gtgcgcgacg
4080agaccggcgc ctcccccacc tcctgggcca tctggacccg cgcctggacc gccgaggaga
4140accgccacgg cgaccccctg aacaagtacc tgtacctgtc cggccgcgtg gacatgaagc
4200agatcgagaa gaccatccag tacctgatcg gctccggcat ggacccccgc accgagaact
4260ccccctacct gggcttcatc tacacctcct tccaggagcg cgccaccttc atctcccacg
4320gcaacaccgc ccgcctggcc cgcgaccacg gcgacttcaa gctggcccag atctgcggca
4380ccatcgcctc cgacgagaag cgccacgaga ccgcctacac caagatcgtg gagaagctgt
4440tcgagatcga ccccgacggc accgtgctgg ccttcggcga catgatgaag aagaagatct
4500ccatgcccga ccacttcatg tacgacggcc gcgacgacaa cctgttcgac cacttctcct
4560ccgtggccca gcgcctgggc gtgtacaccg ccaaggacta cgccgacatc ctggagcacc
4620tggtgggccg ctggaaggtg gagaagctga ccggcctgtc cgccgagggc cagaaggccc
4680aggactacgt gtgcggcctg cccccccgca tccgccgcct ggaggagcgc gcccagatcc
4740gcgccaagca ggccccccgc ctgcccttct cctggatcta cgaccgcgag gtgcagctga
4800tggactacaa ggaccacgac ggcgactaca aggaccacga catcgactac aaggacgacg
4860acgacaagtg aatcgataga tctcttaagg cagcagcagc tcggatagta tcgacacact
4920ctggacgctg gtcgtgtgat ggactgttgc cgccacactt gctgccttga cctgtgaata
4980tccctgccgc ttttatcaaa cagcctcagt gtgtttgatc ttgtgtgtac gcgcttttgc
5040gagttgctag ctgcttgtgc tatttgcgaa taccaccccc agcatcccct tccctcgttt
5100catatcgctt gcatcccaac cgcaacttat ctacgctgtc ctgctatccc tcagcgctgc
5160tcctgctcct gctcactgcc cctcgcacag ccttggtttg ggctccgcct gtattctcct
5220ggtactgcaa cctgtaaacc agcactgcaa tgctgatgca cgggaagtag tgggatggga
5280acacaaatgg aaagcttaat taagagctct tgttttccag aaggagttgc tccttgagcc
5340tttcattctc agcctcgata acctccaaag ccgctctaat tgtggagggg gttcgaattt
5400aaaagcttgg aatgttggtt cgtgcgtctg gaacaagccc agacttgttg ctcactggga
5460aaaggaccat cagctccaaa aaacttgccg ctcaaaccgc gtacctctgc tttcgcgcaa
5520tctgccctgt tgaaatcgcc accacattca tattgtgacg cttgagcagt ctgtaattgc
5580ctcagaatgt ggaatcatct gccccctgtg cgagcccatg ccaggcatgt cgcgggcgag
5640gacacccgcc actcgtacag cagaccatta tgctacctca caatagttca taacagtgac
5700catatttctc gaagctcccc aacgagcacc tccatgctct gagtggccac cccccggccc
5760tggtgcttgc ggagggcagg tcaaccggca tggggctacc gaaatccccg accggatccc
5820accacccccg cgatgggaag aatctctccc cgggatgtgg gcccaccacc agcacaacct
5880gctggcccag gcgagcgtca aaccatacca cacaaatatc cttggcatcg gccctgaatt
5940ccttctgccg ctctgctacc cggtgcttct gtccgaagca ggggttgcta gggatcgctc
6000cgagtccgca aacccttgtc gcgtggcggg gcttgttcga gcttgaagag c
60511027041DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 102gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcgaatt cggccgacag gacgcgcgtc 3300aaaggtgctg gtcgtgtatg ccctggccgg
caggtcgttg ctgctgctgg ttagtgattc 3360cgcaaccctg attttggcgt cttattttgg
cgtggcaaac gctggcgccc gcgagccggg 3420ccggcggcga tgcggtgccc cacggctgcc
ggaatccaag ggaggcaaga gcgcccgggt 3480cagttgaagg gctttacgcg caaggtacag
ccgctcctgc aaggctgcgt ggtggaattg 3540gacgtgcagg tcctgctgaa gttcctccac
cgcctcacca gcggacaaag caccggtgta 3600tcaggtccgt gtcatccact ctaaagaact
cgactacgac ctactgatgg ccctagattc 3660ttcatcaaaa acgcctgaga cacttgccca
ggattgaaac tccctgaagg gaccaccagg 3720ggccctgagt tgttccttcc ccccgtggcg
agctgccagc caggctgtac ctgtgatcga 3780ggctggcggg aaaataggct tcgtgtgctc
aggtcatggg aggtgcagga cagctcatga 3840aacgccaaca atcgcacaat tcatgtcaag
ctaatcagct atttcctctt cacgagctgt 3900aattgtccca aaattctggt ctaccggggg
tgatccttcg tgtacgggcc cttccctcaa 3960ccctaggtat gcgcgcatgc ggtcgccgcg
caactcgcgc gagggccgag ggtttgggac 4020gggccgtccc gaaatgcagt tgcacccgga
tgcgtggcac cttttttgcg ataatttatg 4080caatggactg ctctgcaaaa ttctggctct
gtcgccaacc ctaggatcag cggcgtagga 4140tttcgtaatc attcgtcctg atggggagct
accgactacc ctaatatcag cccgactgcc 4200tgacgccagc gtccactttt gtgcacacat
tccattcgtg cccaagacat ttcattgtgg 4260tgcgaagcgt ccccagttac gctcacctgt
ttcccgacct ccttactgtt ctgtcgacag 4320agcgggccca caggccggtc gcagccacta
gtatgacctc catcaacgtg aagctgctgt 4380accactacgt gatcaccaac ctgttcaacc
tgtgcttctt ccccctgacc gccatcgtgg 4440ccggcaaggc ctcccgcctg accatcgacg
acctgcacca cctgtactac tcctacctgc 4500agcacaacgt gatcaccatc gcccccctgt
tcgccttcac cgtgttcggc tccatcctgt 4560acatcgtgac ccgccccaag cccgtgtacc
tggtggagta ctcctgctac ctgcccccca 4620cccagtgccg ctcctccatc tccaaggtga
tggacatctt ctaccaggtg cgcaaggccg 4680accccttccg caacggcacc tgcgacgact
cctcctggct ggacttcctg cgcaagatcc 4740aggagcgctc cggcctgggc gacgagaccc
acggccccga gggcctgctg caggtgcccc 4800cccgcaagac cttcgccgcc gcccgcgagg
agaccgagca ggtgatcgtg ggcgccctga 4860agaacctgtt cgagaacacc aaggtgaacc
ccaaggacat cggcatcctg gtggtgaact 4920cctccatgtt caaccccacc ccctccctgt
ccgccatggt ggtgaacacc ttcaagctgc 4980gctccaacgt gcgctccttc aacctgggcg
gcatgggctg ctccgccggc gtgatcgcca 5040tcgacctggc caaggacctg ctgcacgtgc
acaagaacac ctacgccctg gtggtgtcca 5100ccgagaacat cacctacaac atctacgccg
gcgacaaccg ctccatgatg gtgtccaact 5160gcctgttccg cgtgggcggc gccgccatcc
tgctgtccaa caagccccgc gaccgccgcc 5220gctccaagta cgagctggtg cacaccgtgc
gcacccacac cggcgccgac gacaagtcct 5280tccgctgcgt gcagcagggc gacgacgaga
acggcaagac cggcgtgtcc ctgtccaagg 5340acatcaccga ggtggccggc cgcaccgtga
agaagaacat cgccaccctg ggccccctga 5400tcctgcccct gtccgagaag ctgctgttct
tcgtgacctt catggccaag aagctgttca 5460aggacaaggt gaagcactac tacgtgcccg
acttcaagct ggccatcgac cacttctgca 5520tccacgccgg cggccgcgcc gtgatcgacg
tgctggagaa gaacctgggc ctggccccca 5580tcgacgtgga ggcctcccgc tccaccctgc
accgcttcgg caacacctcc tcctcctcca 5640tctggtacga gctggcctac atcgaggcca
agggccgcat gaagaagggc aacaaggtgt 5700ggcagatcgc cctgggctcc ggcttcaagt
gcaactccgc cgtgtgggtg gccctgtcca 5760acgtgaaggc ctccaccaac tccccctggg
agcactgcat cgaccgctac cccgtgaaga 5820tcgactccga ctccgccaag tccgagaccc
gcgcccagaa cggccgctcc tgacttaagg 5880cagcagcagc tcggatagta tcgacacact
ctggacgctg gtcgtgtgat ggactgttgc 5940cgccacactt gctgccttga cctgtgaata
tccctgccgc ttttatcaaa cagcctcagt 6000gtgtttgatc ttgtgtgtac gcgcttttgc
gagttgctag ctgcttgtgc tatttgcgaa 6060taccaccccc agcatcccct tccctcgttt
catatcgctt gcatcccaac cgcaacttat 6120ctacgctgtc ctgctatccc tcagcgctgc
tcctgctcct gctcactgcc cctcgcacag 6180ccttggtttg ggctccgcct gtattctcct
ggtactgcaa cctgtaaacc agcactgcaa 6240tgctgatgca cgggaagtag tgggatggga
acacaaatgg aaagcttaat taagagctct 6300tgttttccag aaggagttgc tccttgagcc
tttcattctc agcctcgata acctccaaag 6360ccgctctaat tgtggagggg gttcgaattt
aaaagcttgg aatgttggtt cgtgcgtctg 6420gaacaagccc agacttgttg ctcactggga
aaaggaccat cagctccaaa aaacttgccg 6480ctcaaaccgc gtacctctgc tttcgcgcaa
tctgccctgt tgaaatcgcc accacattca 6540tattgtgacg cttgagcagt ctgtaattgc
ctcagaatgt ggaatcatct gccccctgtg 6600cgagcccatg ccaggcatgt cgcgggcgag
gacacccgcc actcgtacag cagaccatta 6660tgctacctca caatagttca taacagtgac
catatttctc gaagctcccc aacgagcacc 6720tccatgctct gagtggccac cccccggccc
tggtgcttgc ggagggcagg tcaaccggca 6780tggggctacc gaaatccccg accggatccc
accacccccg cgatgggaag aatctctccc 6840cgggatgtgg gcccaccacc agcacaacct
gctggcccag gcgagcgtca aaccatacca 6900cacaaatatc cttggcatcg gccctgaatt
ccttctgccg ctctgctacc cggtgcttct 6960gtccgaagca ggggttgcta gggatcgctc
cgagtccgca aacccttgtc gcgtggcggg 7020gcttgttcga gcttgaagag c
70411031530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
103actagtatga cctccatcaa cgtgaagctg ctgtaccact acgtgatcac caacttcttc
60aacctgtgct tcttccccct gaccgccatc ctggccggca aggcctcccg cctgaccacc
120aacgacctgc accacttcta ctcctacctg cagcacaacc tgatcaccct gaccctgctg
180ttcgccttca ccgtgttcgg ctccgtgctg tacttcgtga cccgccccaa gcccgtgtac
240ctggtggact actcctgcta cctgcccccc cagcacctgt ccgccggcat ctccaagacc
300atggagatct tctaccagat ccgcaagtcc gaccccctgc gcaacgtggc cctggacgac
360tcctcctccc tggacttcct gcgcaagatc caggagcgct ccggcctggg cgacgagacc
420tacggccccg agggcctgtt cgagatcccc ccccgcaaga acctggcctc cgcccgcgag
480gagaccgagc aggtgatcaa cggcgccctg aagaacctgt tcgagaacac caaggtgaac
540cccaaggaga tcggcatcct ggtggtgaac tcctccatgt tcaaccccac cccctccctg
600tccgccatgg tggtgaacac cttcaagctg cgctccaaca tcaagtcctt caacctgggc
660ggcatgggct gctccgccgg cgtgatcgcc atcgacctgg ccaaggacct gctgcacgtg
720cacaagaaca cctacgccct ggtggtgtcc accgagaaca tcacccagaa catctacacc
780ggcgacaacc gctccatgat ggtgtccaac tgcctgttcc gcgtgggcgg cgccgccatc
840ctgctgtcca acaagcccgg cgaccgccgc cgctccaagt accgcctggc ccacaccgtg
900cgcacccaca ccggcgccga cgacaagtcc ttcggctgcg tgcgccagga ggaggacgac
960tccggcaaga ccggcgtgtc cctgtccaag gacatcaccg gcgtggccgg catcaccgtg
1020cagaagaaca tcaccaccct gggccccctg gtgctgcccc tgtccgagaa gatcctgttc
1080gtggtgacct tcgtggccaa gaagctgctg aaggacaaga tcaagcacta ctacgtgccc
1140gacttcaagc tggccgtgga ccacttctgc atccacgccg gcggccgcgc cgtgatcgac
1200gtgctggaga agaacctggg cctgtccccc atcgacgtgg aggcctcccg ctccaccctg
1260caccgcttcg gcaacacctc ctcctcctcc atctggtacg agctggccta catcgaggcc
1320aagggccgca tgaagaaggg caacaaggcc tggcagatcg ccgtgggctc cggcttcaag
1380tgcaactccg ccgtgtgggt ggccctgcgc aacgtgaagg cctccgccaa ctccccctgg
1440gagcactgca tccacaagta ccccgtgcag atgtactccg gctcctccaa gtccgagacc
1500cgcgcccaga acggccgctc ctgacttaag
15301041533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 104actagtatga cctccatcaa cgtgaagctg
ctgtaccact acgtgctgac caacttcttc 60aacctgtgcc tgttccccct gaccgccttc
cccgccggca aggcctccca gctgaccacc 120aacgacctgc accacctgta ctcctacctg
caccacaacc tgatcaccgt gaccctgctg 180ttcgccttca ccgtgttcgg ctccatcctg
tacatcgtga cccgccccaa gcccgtgtac 240ctggtggact actcctgcta cctgcccccc
cgccacctgt cctgcggcat ctcccgcgtg 300atggagatct tctacgagat ccgcaagtcc
gacccctccc gcgaggtgcc cttcgacgac 360ccctcctccc tggagttcct gcgcaagatc
caggagcgct ccggcctggg cgacgagacc 420tacggccccc agggcctggt gcacgacatg
cccctgcgca tgaacttcgc cgccgcccgc 480gaggagaccg agcaggtgat caacggcgcc
ctggagaagc tgttcgagaa caccaaggtg 540aacccccgcg agatcggcat cctggtggtg
aactcctcca tgttcaaccc caccccctcc 600ctgtccgcca tggtggtgaa caccttcaag
ctgcgctcca acatcaagtc cttctccctg 660ggcggcatgg gctgctccgc cggcatcatc
gccatcgacc tggccaagga cctgctgcac 720gtgcacaaga acacctacgc cctggtggtg
tccaccgaga acatcaccca ctccacctac 780accggcgaca accgctccat gatggtgtcc
aactgcctgt tccgcatggg cggcgccgcc 840atcctgctgt ccaacaaggc cggcgaccgc
cgccgctcca agtacaagct ggcccacacc 900gtgcgcaccc acaccggcgc cgacgaccag
tccttccgct gcgtgcgcca ggaggacgac 960gaccgcggca agatcggcgt gtgcctgtcc
aaggacatca ccgccgtggc cggcaagacc 1020gtgaccaaga acatcgccac cctgggcccc
ctggtgctgc ccctgtccga gaagttcctg 1080tacgtggtgt ccctgatggc caagaagctg
ttcaagaaca agatcaagca cacctacgtg 1140cccgacttca agctggccat cgaccacttc
tgcatccacg ccggcggccg cgccgtgatc 1200gacgtgctgg agaagaacct ggccctgtcc
cccgtggacg tggaggcctc ccgctccacc 1260ctgcaccgct tcggcaacac ctcctcctcc
tccatctggt acgagctggc ctacatcgag 1320gccaagggcc gcatgaagaa gggcaacaag
gtgtggcaga tcgccatcgg ctccggcttc 1380aagtgcaact ccgccgtgtg ggtggccctg
tgcaacgtga agccctccgt gaactccccc 1440tgggagcact gcatcgaccg ctaccccgtg
gagatcaact acggctcctc caagtccgag 1500acccgcgccc agaacggccg ctcctgactt
aag 15331051524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
105actagtatgt ccggcaccaa ggccacctcc gtgtccgtgc ccctgcccga cttcaagcag
60tccgtgaacc tgaagtacgt gaagctgggc taccactact ccatcaccca cgccatgtac
120ctgttcctga cccccctgct gctgatcatg tccgcccaga tctccacctt ctccatccag
180gacttccacc acctgtacaa ccacctgatc ctgcacaacc tgtcctccct gatcctgtgc
240atcgccctgc tgctgttcgt gctgaccctg tacttcctga cccgccccac ccccgtgtac
300ctgctgaact tctcctgcta caagcccgac gccatccaca agtgcgaccg ccgccgcttc
360atggacacca tccgcggcat gggcacctac accgaggaga acatcgagtt ccagcgcaag
420gtgctggagc gctccggcat cggcgagtcc tcctacctgc cccccaccgt gttcaagatc
480cccccccgcg tgtacgacgc cgaggagcgc gccgaggccg agatgctgat gttcggcgcc
540gtggacggcc tgttcgagaa gatctccgtg aagcccaacc agatcggcgt gctggtggtg
600aactgcggcc tgttcaaccc catcccctcc ctgtcctcca tgatcgtgaa ccgctacaag
660atgcgcggca acgtgttctc ctacaacctg ggcggcatgg gctgctccgc cggcgtgatc
720tccatcgacc tggccaagga cctgctgcag gtgcgcccca actcctacgc cctggtggtg
780tccctggagt gcatctccaa gaacctgtac ctgggcgagc agcgctccat gctggtgtcc
840aactgcctgt tccgcatggg cggcgccgcc atcctgctgt ccaacaagat gtccgaccgc
900tggcgctcca agtaccgcct ggtgcacacc gtgcgcaccc acaagggcac cgaggacaac
960tgcttctcct gcgtgacccg caaggaggac tccgacggca agatcggcat ctccctgtcc
1020aagaacctga tggccgtggc cggcgacgcc ctgaagacca acatcaccac cctgggcccc
1080ctggtgctgc ccatgtccga gcagctgctg ttcttcgcca ccctggtggg caagaaggtg
1140ttcaagatga agctgcagcc ctacatcccc gacttcaagc tggccttcga gcacttctgc
1200atccacgccg gcggccgcgc cgtgctggac gagctggaga agaacctgaa gctgtcctcc
1260tggcacatgg agccctcccg catgtccctg taccgcttcg gcaacacctc ctcctcctcc
1320ctgtggtacg agctggccta ctccgaggcc aagggccgca tcaagaaggg cgaccgcgtg
1380tggcagatcg ccttcggctc cggcttcaag tgcaactccg ccgtgtggaa ggccctgcgc
1440aacgtgaacc ccgccgagga gaagaacccc tggatggacg agatccacct gttccccgtg
1500gaggtgcccc tgaactgact taag
15241061530DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 106actagtatga cctccatcaa cgtgaagctg
ctgtaccact acgtgatcac caacctgttc 60aacctgtgct tcttccccct gaccgccatc
gtggccggca aggcctacct gaccatcgac 120gacctgcacc acctgtacta ctcctacctg
cagcacaacc tgatcaccat cgcccccctg 180ctggccttca ccgtgttcgg ctccgtgctg
tacatcgcca cccgccccaa gcccgtgtac 240ctggtggagt actcctgcta cctgcccccc
acccactgcc gctcctccat ctccaaggtg 300atggacatct tcttccaggt gcgcaaggcc
gacccctccc gcaacggcac ctgcgacgac 360tcctcctggc tggacttcct gcgcaagatc
caggagcgct ccggcctggg cgacgagacc 420cacggccccg agggcctgct gcaggtgccc
ccccgcaaga ccttcgcccg cgcccgcgag 480gagaccgagc aggtgatcat cggcgccctg
gagaacctgt tcaagaacac caacgtgaac 540cccaaggaca tcggcatcct ggtggtgaac
tcctccatgt tcaaccccac cccctccctg 600tccgccatgg tggtgaacac cttcaagctg
cgctccaacg tgcgctcctt caacctgggc 660ggcatgggct gctccgccgg cgtgatcgcc
atcgacctgg ccaaggacct gctgcacgtg 720cacaagaaca cctacgccct ggtggtgtcc
accgagaaca tcacctacaa catctacgcc 780ggcgacaacc gctccatgat ggtgtccaac
tgcctgttcc gcgtgggcgg cgccgccatc 840ctgctgtcca acaagccccg cgaccgccgc
cgctccaagt acgagctggt gcacaccgtg 900cgcacccaca ccggcgccga cgacaagtcc
ttccgctgcg tgcagcaggg cgacgacgag 960aacggccaga ccggcgtgtc cctgtccaag
gacatcaccg acgtggccgg ccgcaccgtg 1020aagaagaaca tcgccaccct gggccccctg
atcctgcccc tgtccgagaa gctgctgttc 1080ttcgtgacct tcatgggcaa gaagctgttc
aaggacgaga tcaagcacta ctacgtgccc 1140gacttcaagc tggccatcga ccacttctgc
atccacgccg gcggcaaggc cgtgatcgac 1200gtgctggaga agaacctggg cctggccccc
atcgacgtgg aggcctcccg ctccaccctg 1260caccgcttcg gcaacacctc ctcctcctcc
atctggtacg agctggccta catcgagccc 1320aagggccgca tgaagaaggg caacaaggtg
tggcagatcg ccctgggctc cggcttcaag 1380tgcaactccg ccgtgtgggt ggccctgaac
aacgtgaagg cctccaccaa ctccccctgg 1440gagcactgca tcgaccgcta ccccgtgaag
atcgactccg actccggcaa gtccgagacc 1500cgcgtgccca acggccgctc ctgacttaag
15301071599DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
107actagtatgg agcgcaccaa ctccatcgag atggaccagg agcgcctgac cgccgagatg
60gccttcaagg actcctcctc cgccgtgatc cgcatccgcc gccgcctgcc cgacttcctg
120acctccgtga agctgaagta cgtgaagctg ggcctgcaca actccttcaa cttcaccacc
180ttcctgttcc tgctgatcat cctgcccctg accggcaccg tgctggtgca gctgaccggc
240ctgaccttcg agaccttctc cgagctgtgg tacaaccacg ccgcccagct ggacggcgtg
300acccgcctgg cctgcctggt gtccctgtgc ttcgtgctga tcatctacgt gaccaaccgc
360tccaagcccg tgtacctggt ggacttctcc tgctacaagc ccgaggacga gcgcaagatg
420tccgtggact ccttcctgaa gatgaccgag cagaacggcg ccttcaccga cgacaccgtg
480cagttccagc agcgcatctc caaccgcgcc ggcctgggcg acgagaccta cctgccccgc
540ggcatcacct ccaccccccc caagctgaac atgtccgagg cccgcgccga ggccgaggcc
600gtgatgttcg gcgccctgga ctccctgttc gagaagaccg gcatcaagcc cgccgaggtg
660ggcatcctga tcgtgtcctg ctccctgttc aaccccaccc cctccctgtc cgccatgatc
720gtgaaccact acaagatgcg cgaggacatc aagtcctaca acctgggcgg catgggctgc
780tccgccggcc tgatctccat cgacctggcc aacaacctgc tgaaggccaa ccccaactcc
840tacgccgtgg tggtgtccac cgagaacatc accctgaact ggtacttcgg caacgaccgc
900tccatgctgc tgtgcaactg catcttccgc atgggcggcg ccgccatcct gctgtccaac
960cgccgccagg accgctccaa gtccaagtac gagctggtga acgtggtgcg cacccacaag
1020ggctccgacg acaagaacta caactgcgtg taccagaagg aggacgagcg cggcaccatc
1080ggcgtgtccc tggcccgcga gctgatgtcc gtggccggcg acgccctgaa gaccaacatc
1140accaccctgg gccccatggt gctgcccctg tccggccagc tgatgttctc cgtgtccctg
1200gtgaagcgca agctgctgaa gctgaaggtg aagccctaca tccccgactt caagctggcc
1260ttcgagcact tctgcatcca cgccggcggc cgcgccgtgc tggacgaggt gcagaagaac
1320ctggacctgg aggactggca catggagccc tcccgcatga ccctgcaccg cttcggcaac
1380acctcctcct cctccctgtg gtacgagatg gcctacaccg aggccaaggg ccgcgtgaag
1440gccggcgacc gcctgtggca gatcgccttc ggctccggct tcaagtgcaa ctccgccgtg
1500tggaaggccc tgcgcgtggt gtccaccgag gagctgaccg gcaacgcctg ggccggctcc
1560atcgagaact accccgtgaa gatcgtgcag tgacttaag
15991085988DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 108gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcgaatt cctttcttgc gctatgacac 3300ttccagcaaa aggtagggcg ggctgcgaga
cggcttcccg gcgctgcatg caacaccgat 3360gatgcttcga ccccccgaag ctccttcggg
gctgcatggg cgctccgatg ccgctccagg 3420gcgagcgctg tttaaatagc caggcccccg
attgcaaaga cattatagcg agctaccaaa 3480gccatattca aacacctaga tcactaccac
ttctacacag gccactcgag cttgtgatcg 3540cactccgcta agggggcgcc tcttcctctt
cgtttcagtc acaacccgca aacactagta 3600tggccaccgc atccactttc tcggcgttca
atgcccgctg cggcgacctg cgtcgctcgg 3660cgggctccgg gccccggcgc ccagcgaggc
ccctccccgt gcgcgggcgc gccgaggtgc 3720acgtgcaggt gacccactcc ctggcccccg
agaagcgcga gatcttcaac tccctgaaca 3780actgggccca ggagaacatc ctggtgctgc
tgaaggacgt ggacaagtgc tggcagccct 3840ccgacttcct gcccgactcc gcctccgagg
gcttcgacga gcaggtgatg gagctgcgca 3900agcgctgcaa ggagatcccc gacgactact
tcatcgtgct ggtgggcgac atgatcaccg 3960aggaggccct gcccacctac cagaccatgc
tgaacaccct ggacggcgtg cgcgacgaga 4020ccggcgcctc cctgaccccc tgggccatct
ggacccgcgc ctggaccgcc gaggagaacc 4080gccacggcga cctgctgaac aagtacctgt
acctgtccgg ccgcgtggac atgaagcaga 4140tcgagaagac catccagtac ctgatcggct
ccggcatgga cccccgcacc gagaacaacc 4200cctacctggg cttcatctac acctccttcc
aggagcgcgc caccttcatc tcccacggca 4260acaccgcccg cctggccaag gagcacggcg
acctgaagct ggcccagatc tgcggcatca 4320tcgccgccga cgagaagcgc cacgagaccg
cctacaccaa gatcgtggag aagctgttcg 4380agatcgaccc cgacggcacc gtgctggccc
tggccgacat gatgcgcaag aaggtgtcca 4440tgcccgccca cctgatgtac gacggccagg
acgacaacct gttcgagaac ttctcctccg 4500tggcccagcg cctgggcgtg tacaccgcca
aggactacgc cgacatcctg gagttcctgg 4560tgggccgctg ggacatcgag aagctgaccg
gcctgtccgg cgagggccgc aaggcccagg 4620actacgtgtg caccctgccc ccccgcatcc
gccgcctgga ggagcgcgcc cagtcccgcg 4680tgaagaaggc ctccgccacc cccttctcct
ggatcttcgg ccgcgagatc aacctgatgg 4740actacaagga ccacgacggc gactacaagg
accacgacat cgactacaag gacgacgacg 4800acaagtgaat cgatagatct cttaaggcag
cagcagctcg gatagtatcg acacactctg 4860gacgctggtc gtgtgatgga ctgttgccgc
cacacttgct gccttgacct gtgaatatcc 4920ctgccgcttt tatcaaacag cctcagtgtg
tttgatcttg tgtgtacgcg cttttgcgag 4980ttgctagctg cttgtgctat ttgcgaatac
cacccccagc atccccttcc ctcgtttcat 5040atcgcttgca tcccaaccgc aacttatcta
cgctgtcctg ctatccctca gcgctgctcc 5100tgctcctgct cactgcccct cgcacagcct
tggtttgggc tccgcctgta ttctcctggt 5160actgcaacct gtaaaccagc actgcaatgc
tgatgcacgg gaagtagtgg gatgggaaca 5220caaatggaaa gcttaattaa gagctcttgt
tttccagaag gagttgctcc ttgagccttt 5280cattctcagc ctcgataacc tccaaagccg
ctctaattgt ggagggggtt cgaatttaaa 5340agcttggaat gttggttcgt gcgtctggaa
caagcccaga cttgttgctc actgggaaaa 5400ggaccatcag ctccaaaaaa cttgccgctc
aaaccgcgta cctctgcttt cgcgcaatct 5460gccctgttga aatcgccacc acattcatat
tgtgacgctt gagcagtctg taattgcctc 5520agaatgtgga atcatctgcc ccctgtgcga
gcccatgcca ggcatgtcgc gggcgaggac 5580acccgccact cgtacagcag accattatgc
tacctcacaa tagttcataa cagtgaccat 5640atttctcgaa gctccccaac gagcacctcc
atgctctgag tggccacccc ccggccctgg 5700tgcttgcgga gggcaggtca accggcatgg
ggctaccgaa atccccgacc ggatcccacc 5760acccccgcga tgggaagaat ctctccccgg
gatgtgggcc caccaccagc acaacctgct 5820ggcccaggcg agcgtcaaac cataccacac
aaatatcctt ggcatcggcc ctgaattcct 5880tctgccgctc tgctacccgg tgcttctgtc
cgaagcaggg gttgctaggg atcgctccga 5940gtccgcaaac ccttgtcgcg tggcggggct
tgttcgagct tgaagagc 59881096807DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
109gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg
1080gccggcttcg ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg
1140gtgcacttca cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag
1200aaggacgcca agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg
1260cccttgttct ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc
1320gccatcgccc cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac
1380aacaacacct ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc
1440tggacctaca acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc
1500tacaccttca ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc
1560gacccgaagg tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc
1620caggactaca agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc
1680gcgttcgcca acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc
1740cccaccgagc aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc
1800gccccggccg gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc
1860gaggccttcg acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag
1920accttcttca acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac
1980tgggagtact ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc
2040aagttctccc tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag
2100gccgagccga tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc
2160acgttgacga aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag
2220ttcgagctgg tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac
2280ctctccctct ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag
2340gtgtccgcgt cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag
2400aacccctact tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac
2460ctgtcctact acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac
2520gacggcgacg tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc
2580gtgaacatga cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag
2640gtcaagtgac aattggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg
2700tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt
2760atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc
2820ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat
2880cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc
2940actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg
3000taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggagga
3060tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct gtcgcacctc
3120agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg tccattagcg
3180aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat gatcggtgga
3240gctgatggtc gaaacgttca cagcctaggg atatcgaatt cggccgacag gacgcgcgtc
3300aaaggtgctg gtcgtgtatg ccctggccgg caggtcgttg ctgctgctgg ttagtgattc
3360cgcaaccctg attttggcgt cttattttgg cgtggcaaac gctggcgccc gcgagccggg
3420ccggcggcga tgcggtgccc cacggctgcc ggaatccaag ggaggcaaga gcgcccgggt
3480cagttgaagg gctttacgcg caaggtacag ccgctcctgc aaggctgcgt ggtggaattg
3540gacgtgcagg tcctgctgaa gttcctccac cgcctcacca gcggacaaag caccggtgta
3600tcaggtccgt gtcatccact ctaaagaact cgactacgac ctactgatgg ccctagattc
3660ttcatcaaaa acgcctgaga cacttgccca ggattgaaac tccctgaagg gaccaccagg
3720ggccctgagt tgttccttcc ccccgtggcg agctgccagc caggctgtac ctgtgatcga
3780ggctggcggg aaaataggct tcgtgtgctc aggtcatggg aggtgcagga cagctcatga
3840aacgccaaca atcgcacaat tcatgtcaag ctaatcagct atttcctctt cacgagctgt
3900aattgtccca aaattctggt ctaccggggg tgatccttcg tgtacgggcc cttccctcaa
3960ccctaggtat gcgcgcatgc ggtcgccgcg caactcgcgc gagggccgag ggtttgggac
4020gggccgtccc gaaatgcagt tgcacccgga tgcgtggcac cttttttgcg ataatttatg
4080caatggactg ctctgcaaaa ttctggctct gtcgccaacc ctaggatcag cggcgtagga
4140tttcgtaatc attcgtcctg atggggagct accgactacc ctaatatcag cccgactgcc
4200tgacgccagc gtccactttt gtgcacacat tccattcgtg cccaagacat ttcattgtgg
4260tgcgaagcgt ccccagttac gctcacctgt ttcccgacct ccttactgtt ctgtcgacag
4320agcgggccca caggccggtc gcagccacta gtatggccac cgcatccact ttctcggcgt
4380tcaatgcccg ctgcggcgac ctgcgtcgct cggcgggctc cgggccccgg cgcccagcga
4440ggcccctccc cgtgcgcggg cgcgccgcct ccaccctgaa gtccggctcc aaggaggtgg
4500agaacctgaa gaagcccttc atgccccccc gcgaggtgca cgtgcaggtg acccactcca
4560tgccccccca gaagatcgag atcttcaagt ccctggacaa ctgggccgag gagaacatcc
4620tggtgcacct gaagcccgtg gagaagtgct ggcagcccca ggacttcctg cccgaccccg
4680cctccgacgg cttcgacgag caggtgcgcg agctgcgcga gcgcgccaag gagatccccg
4740acgactactt cgtggtgctg gtgggcgaca tgatcaccga ggaggccctg cccacctacc
4800agaccatgct gaacaccctg gacggcgtgc gcgacgagac cggcgcctcc cccacctcct
4860gggccatctg gacccgcgcc tggaccgccg aggagaaccg ccacggcgac ctgctgaaca
4920agtacctgta cctgtccggc cgcgtggaca tgcgccagat cgagaagacc atccagtacc
4980tgatcggctc cggcatggac ccccgcaccg agaactcccc ctacctgggc ttcatctaca
5040cctccttcca ggagcgcgcc accttcatct cccacggcaa caccgcccgc caggccaagg
5100agcacggcga catcaagctg gcccagatct gcggcaccat cgccgccgac gagaagcgcc
5160acgagaccgc ctacaccaag atcgtggaga agctgttcga gatcgacccc gacggcaccg
5220tgctggcctt cgccgacatg atgcgcaaga agatctccat gcccgcccac ctgatgtacg
5280acggccgcga cgacaacctg ttcgaccact tctccgccgt ggcccagcgc ctgggcgtgt
5340acaccgccaa ggactacgcc gacatcctgg agttcctggt gggccgctgg aaggtggaca
5400agctgaccgg cctgtccgcc gagggccaga aggcccagga ctacgtgtgc cgcctgcccc
5460cccgcatccg ccgcctggag gagcgcgccc agggccgcgc caaggaggcc cccaccatgc
5520ccttctcctg gatcttcgac cgccaggtga agctgatgga ctacaaggac cacgacggcg
5580actacaagga ccacgacatc gactacaagg acgacgacga caagtgaatc gatagatctc
5640ttaaggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg tgtgatggac
5700tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt atcaaacagc
5760ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc ttgtgctatt
5820tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat cccaaccgca
5880acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc actgcccctc
5940gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg taaaccagca
6000ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggaaag cttaattaag
6060agctcttgtt ttccagaagg agttgctcct tgagcctttc attctcagcc tcgataacct
6120ccaaagccgc tctaattgtg gagggggttc gaatttaaaa gcttggaatg ttggttcgtg
6180cgtctggaac aagcccagac ttgttgctca ctgggaaaag gaccatcagc tccaaaaaac
6240ttgccgctca aaccgcgtac ctctgctttc gcgcaatctg ccctgttgaa atcgccacca
6300cattcatatt gtgacgcttg agcagtctgt aattgcctca gaatgtggaa tcatctgccc
6360cctgtgcgag cccatgccag gcatgtcgcg ggcgaggaca cccgccactc gtacagcaga
6420ccattatgct acctcacaat agttcataac agtgaccata tttctcgaag ctccccaacg
6480agcacctcca tgctctgagt ggccaccccc cggccctggt gcttgcggag ggcaggtcaa
6540ccggcatggg gctaccgaaa tccccgaccg gatcccacca cccccgcgat gggaagaatc
6600tctccccggg atgtgggccc accaccagca caacctgctg gcccaggcga gcgtcaaacc
6660ataccacaca aatatccttg gcatcggccc tgaattcctt ctgccgctct gctacccggt
6720gcttctgtcc gaagcagggg ttgctaggga tcgctccgag tccgcaaacc cttgtcgcgt
6780ggcggggctt gttcgagctt gaagagc
68071106744DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 110gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcgaatt cggccgacag gacgcgcgtc 3300aaaggtgctg gtcgtgtatg ccctggccgg
caggtcgttg ctgctgctgg ttagtgattc 3360cgcaaccctg attttggcgt cttattttgg
cgtggcaaac gctggcgccc gcgagccggg 3420ccggcggcga tgcggtgccc cacggctgcc
ggaatccaag ggaggcaaga gcgcccgggt 3480cagttgaagg gctttacgcg caaggtacag
ccgctcctgc aaggctgcgt ggtggaattg 3540gacgtgcagg tcctgctgaa gttcctccac
cgcctcacca gcggacaaag caccggtgta 3600tcaggtccgt gtcatccact ctaaagaact
cgactacgac ctactgatgg ccctagattc 3660ttcatcaaaa acgcctgaga cacttgccca
ggattgaaac tccctgaagg gaccaccagg 3720ggccctgagt tgttccttcc ccccgtggcg
agctgccagc caggctgtac ctgtgatcga 3780ggctggcggg aaaataggct tcgtgtgctc
aggtcatggg aggtgcagga cagctcatga 3840aacgccaaca atcgcacaat tcatgtcaag
ctaatcagct atttcctctt cacgagctgt 3900aattgtccca aaattctggt ctaccggggg
tgatccttcg tgtacgggcc cttccctcaa 3960ccctaggtat gcgcgcatgc ggtcgccgcg
caactcgcgc gagggccgag ggtttgggac 4020gggccgtccc gaaatgcagt tgcacccgga
tgcgtggcac cttttttgcg ataatttatg 4080caatggactg ctctgcaaaa ttctggctct
gtcgccaacc ctaggatcag cggcgtagga 4140tttcgtaatc attcgtcctg atggggagct
accgactacc ctaatatcag cccgactgcc 4200tgacgccagc gtccactttt gtgcacacat
tccattcgtg cccaagacat ttcattgtgg 4260tgcgaagcgt ccccagttac gctcacctgt
ttcccgacct ccttactgtt ctgtcgacag 4320agcgggccca caggccggtc gcagccacta
gtatggccac cgcctccacc ttctccgcct 4380tcaacgcccg ctgcggcgac ctgcgccgct
ccgccggctc cggcccccgc cgccccgccc 4440gccccctgcc cgtgcgcgcc gccatcgcct
ccgaggtgcc cgtggccacc acctcccccc 4500gccccggccc caccgtgtac tccaagctgg
acaaggccca caccctgacc cccgagcgca 4560tggagctgat caacggcatg tccgccttcg
ccgaggagcg catcctgccc gtgctgcagc 4620ccgtggagaa gctgtggcag ccccaggacc
tgctgcccga ccccgagtcc cccgacttcc 4680tggaccaggt ggccgagctg cgcgcccgcg
ccgccaacgt gcccgacgac tacttcgtgg 4740tgctggtggg cgacatgatc accgaggagg
ccctgcccac ctacatggcc atgctgaaca 4800ccctggacgg cgtgcgcgac gagaccggcg
ccgccgacca cccctggggc cgctggaccc 4860gccagtgggt ggccgaggag aaccgccacg
gcgacctgct gaacaagtac tgctggctga 4920ccggccgcgt gaacatgaag gccatcgagg
tgaccatcca gaacctgatc ggctccggca 4980tgaaccccaa gaccgagaac aacccctacc
tgggcttcgt gtacacctcc ttccaggagc 5040gcgccaccaa gtactcccac ggcaacaccg
cccgcctggc cgcccagtac ggcgacgcca 5100ccctgtccaa ggtgtgcggc gtgatcgccg
ccgacgaggg ccgccacgag atcgcctaca 5160cccgcatcgt ggaggagttc ttccgcctgg
accccgaggg cgccatgtcc gcctacgccg 5220acatgatgcg caagcagatc accatgcccg
cccacctgat ggacgaccag cagcacggca 5280cccgcaacac cggccgcaac ctgttcgccg
acttctccgc cgtgaccgag aagctggacg 5340tgtacgacgc cgaggactac tgcaagatcc
tggagcacct gaactcccgc tggaagatcg 5400ccgaccgcac cgtgtccggc gacgccggcg
ccgaccagga gtacgtgctg cgcctgccct 5460cccgcttccg caagctggcc gagaagtccg
ccgccaagcg cgccaagacc aagcccaagc 5520ccgtggcctt ctcctggctg tccggccgcg
aggtgatggt gtgaatcgat agatctctta 5580aggcagcagc agctcggata gtatcgacac
actctggacg ctggtcgtgt gatggactgt 5640tgccgccaca cttgctgcct tgacctgtga
atatccctgc cgcttttatc aaacagcctc 5700agtgtgtttg atcttgtgtg tacgcgcttt
tgcgagttgc tagctgcttg tgctatttgc 5760gaataccacc cccagcatcc ccttccctcg
tttcatatcg cttgcatccc aaccgcaact 5820tatctacgct gtcctgctat ccctcagcgc
tgctcctgct cctgctcact gcccctcgca 5880cagccttggt ttgggctccg cctgtattct
cctggtactg caacctgtaa accagcactg 5940caatgctgat gcacgggaag tagtgggatg
ggaacacaaa tggaaagctt aattaagagc 6000tcttgttttc cagaaggagt tgctccttga
gcctttcatt ctcagcctcg ataacctcca 6060aagccgctct aattgtggag ggggttcgaa
tttaaaagct tggaatgttg gttcgtgcgt 6120ctggaacaag cccagacttg ttgctcactg
ggaaaaggac catcagctcc aaaaaacttg 6180ccgctcaaac cgcgtacctc tgctttcgcg
caatctgccc tgttgaaatc gccaccacat 6240tcatattgtg acgcttgagc agtctgtaat
tgcctcagaa tgtggaatca tctgccccct 6300gtgcgagccc atgccaggca tgtcgcgggc
gaggacaccc gccactcgta cagcagacca 6360ttatgctacc tcacaatagt tcataacagt
gaccatattt ctcgaagctc cccaacgagc 6420acctccatgc tctgagtggc caccccccgg
ccctggtgct tgcggagggc aggtcaaccg 6480gcatggggct accgaaatcc ccgaccggat
cccaccaccc ccgcgatggg aagaatctct 6540ccccgggatg tgggcccacc accagcacaa
cctgctggcc caggcgagcg tcaaaccata 6600ccacacaaat atccttggca tcggccctga
attccttctg ccgctctgct acccggtgct 6660tctgtccgaa gcaggggttg ctagggatcg
ctccgagtcc gcaaaccctt gtcgcgtggc 6720ggggcttgtt cgagcttgaa gagc
67441116667DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
111gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaacgg cgcgccatgc tgctgcaggc cttcctgttc ctgctggccg
1080gcttcgccgc caagatcagc gcctccatga cgaacgagac gtccgaccgc cccctggtgc
1140acttcacccc caacaagggc tggatgaacg accccaacgg cctgtggtac gacgagaagg
1200acgccaagtg gcacctgtac ttccagtaca acccgaacga caccgtctgg gggacgccct
1260tgttctgggg ccacgccacg tccgacgacc tgaccaactg ggaggaccag cccatcgcca
1320tcgccccgaa gcgcaacgac tccggcgcct tctccggctc catggtggtg gactacaaca
1380acacctccgg cttcttcaac gacaccatcg acccgcgcca gcgctgcgtg gccatctgga
1440cctacaacac cccggagtcc gaggagcagt acatctccta cagcctggac ggcggctaca
1500ccttcaccga gtaccagaag aaccccgtgc tggccgccaa ctccacccag ttccgcgacc
1560cgaaggtctt ctggtacgag ccctcccaga agtggatcat gaccgcggcc aagtcccagg
1620actacaagat cgagatctac tcctccgacg acctgaagtc ctggaagctg gagtccgcgt
1680tcgccaacga gggcttcctc ggctaccagt acgagtgccc cggcctgatc gaggtcccca
1740ccgagcagga ccccagcaag tcctactggg tgatgttcat ctccatcaac cccggcgccc
1800cggccggcgg ctccttcaac cagtacttcg tcggcagctt caacggcacc cacttcgagg
1860ccttcgacaa ccagtcccgc gtggtggact tcggcaagga ctactacgcc ctgcagacct
1920tcttcaacac cgacccgacc tacgggagcg ccctgggcat cgcgtgggcc tccaactggg
1980agtactccgc cttcgtgccc accaacccct ggcgctcctc catgtccctc gtgcgcaagt
2040tctccctcaa caccgagtac caggccaacc cggagacgga gctgatcaac ctgaaggccg
2100agccgatcct gaacatcagc aacgccggcc cctggagccg gttcgccacc aacaccacgt
2160tgacgaaggc caacagctac aacgtcgacc tgtccaacag caccggcacc ctggagttcg
2220agctggtgta cgccgtcaac accacccaga cgatctccaa gtccgtgttc gcggacctct
2280ccctctggtt caagggcctg gaggaccccg aggagtacct ccgcatgggc ttcgaggtgt
2340ccgcgtcctc cttcttcctg gaccgcggga acagcaaggt gaagttcgtg aaggagaacc
2400cctacttcac caaccgcatg agcgtgaaca accagccctt caagagcgag aacgacctgt
2460cctactacaa ggtgtacggc ttgctggacc agaacatcct ggagctgtac ttcaacgacg
2520gcgacgtcgt gtccaccaac acctacttca tgaccaccgg gaacgccctg ggctccgtga
2580acatgacgac gggggtggac aacctgttct acatcgacaa gttccaggtg cgcgaggtca
2640agtgacaatt ggcagcagca gctcggatag tatcgacaca ctctggacgc tggtcgtgtg
2700atggactgtt gccgccacac ttgctgcctt gacctgtgaa tatccctgcc gcttttatca
2760aacagcctca gtgtgtttga tcttgtgtgt acgcgctttt gcgagttgct agctgcttgt
2820gctatttgcg aataccaccc ccagcatccc cttccctcgt ttcatatcgc ttgcatccca
2880accgcaactt atctacgctg tcctgctatc cctcagcgct gctcctgctc ctgctcactg
2940cccctcgcac agccttggtt tgggctccgc ctgtattctc ctggtactgc aacctgtaaa
3000ccagcactgc aatgctgatg cacgggaagt agtgggatgg gaacacaaat ggaggatccc
3060gcgtctcgaa cagagcgcgc agaggaacgc tgaaggtctc gcctctgtcg cacctcagcg
3120cggcatacac cacaataacc acctgacgaa tgcgcttggt tcttcgtcca ttagcgaagc
3180gtccggttca cacacgtgcc acgttggcga ggtggcaggt gacaatgatc ggtggagctg
3240atggtcgaaa cgttcacagc ctagggatat catagcgact gctacccccc gaccatgtgc
3300cgaggcagaa attatataca agaagcagat cgcaattagg cacatcgctt tgcattatcc
3360acacactatt catcgctgct gcggcaaggc tgcagagtgt atttttgtgg cccaggagct
3420gagtccgaag tcgacgcgac gagcggcgca ggatccgacc cctagacgag ctctgtcatt
3480ttccaagcac gcagctaaat gcgctgagac cgggtctaaa tcatccgaaa agtgtcaaaa
3540tggccgattg ggttcgccta ggacaatgcg ctgcggattc gctcgagtcc gctgccggcc
3600aaaaggcggt ggtacaggaa ggcgcacggg gccaaccctg cgaagccggg ggcccgaacg
3660ccgaccgccg gccttcgatc tcgggtgtcc ccctcgtcaa tttcctctct cgggtgcagc
3720cacgaaagtc gtgacgcagg tcacgaaatc cggttacgaa aaacgcaggt cttcgcaaaa
3780acgtgagggt ttcgcgtctc gccctagcta ttcgtatcgc cgggtcagac ccacgtgcag
3840aaaagccctt gaataacccg ggaccgtggt taccgcgccg cctgcaccag ggggcttata
3900taagcccaca ccacacctgt ctcaccacgc atttctccaa ctcgcgactt ttcggaagaa
3960attgttatcc acctagtata gactgccacc tgcaggacct tgtgtcttgc agtttgtatt
4020ggtcccggcc gtcgagctcg acagatctgg gctagggttg gcctggccgc tcggcactcc
4080cctttagccg cgcgcatccg cgttccagag gtgcgattcg gtgtgtggag cattgtcatg
4140cgcttgtggg ggtcgttccg tgcgcggcgg gtccgccatg ggcgccgacc tgggccctag
4200ggtttgtttt cgggccaagc gagcccctct cacctcgtcg cccccccgca ttccctctct
4260cttgcagcca ctagtatggc ctccgctgtg accttcgcct gcgctcctcc tcgcaggcgc
4320gccggtgccg tggccgctcc tggccgacgc gctgcctctc gtcctctggt ggtgcacgcc
4380gtggcctccg aggctcctct gggcgtgcct ccctccgtgc agcgcccttc tcccgtggtg
4440tactccaagc tggacaagca gcaccgcctg acgcctgagc gcctggagct ggtgcagtcc
4500atgggccagt tcgccgagga gcgcgtgctg cccgtgctgc accccgtgga caagctgtgg
4560cagccccagg acttcctgcc cgaccccgag tcccccgact tcgaggacca ggtggccgag
4620ctgcgcgccc gcgccaagga cctgcccgac gagtacttcg tggtgctggt gggcgacatg
4680atcaccgagg aggccctgcc cacctacatg gccatgctga acacctggga cggcgtgcgc
4740gacgacaccg gcgccgccga ccacccctgg gcccgctgga cccgccagtg ggtggccgag
4800gagaaccgcc acggcgacct gctgaacaag tactgctggc tgaccggccg cgtgaacatg
4860cgcgccgtgg aggtgaccat caacaacctg atcaagtccg gcatgaaccc ccagaccgac
4920aacaacccct acctgggctt cgtgtacacc tccttccagg agcgcgccac caagtactcc
4980cacggcaaca ccgcccgcct ggccgccgag cacggcgaca agggcctgtc caagatctgc
5040ggcctgatcg cctccgacga gggccgccac gagatcgcct acacccgcat cgtggacgag
5100ttcttccgcc tggaccccga gggcgccgtg gccgcctacg ccaacatgat gcgcaagcag
5160atcaccatgc ccgcccacct gatggacgac atgggccacg gcgaggccaa ccccggccgc
5220aacctgttcg ccgacttctc cgccgtggcc gagaagatcg acgtgtacga cgccgaggac
5280tactgccgca tcctggagca cctgaacgcc cgctggaagg tggacgagcg ccaggtgtcc
5340ggccaggccg ccgccgacca ggagtacgtg ctgggcctgc cccagcgctt ccgcaagctg
5400gccgagaaga ccgccgccaa gcgcaagcgc gtggcccgcc gccccgtggc cttctcctgg
5460atctccggcc gcgagatcat ggtgtgaatc gatagatctc ttaaggcagc agcagctcgg
5520atagtatcga cacactctgg acgctggtcg tgtgatggac tgttgccgcc acacttgctg
5580ccttgacctg tgaatatccc tgccgctttt atcaaacagc ctcagtgtgt ttgatcttgt
5640gtgtacgcgc ttttgcgagt tgctagctgc ttgtgctatt tgcgaatacc acccccagca
5700tccccttccc tcgtttcata tcgcttgcat cccaaccgca acttatctac gctgtcctgc
5760tatccctcag cgctgctcct gctcctgctc actgcccctc gcacagcctt ggtttgggct
5820ccgcctgtat tctcctggta ctgcaacctg taaaccagca ctgcaatgct gatgcacggg
5880aagtagtggg atgggaacac aaatggaaag cttaattaag agctcttgtt ttccagaagg
5940agttgctcct tgagcctttc attctcagcc tcgataacct ccaaagccgc tctaattgtg
6000gagggggttc gaatttaaaa gcttggaatg ttggttcgtg cgtctggaac aagcccagac
6060ttgttgctca ctgggaaaag gaccatcagc tccaaaaaac ttgccgctca aaccgcgtac
6120ctctgctttc gcgcaatctg ccctgttgaa atcgccacca cattcatatt gtgacgcttg
6180agcagtctgt aattgcctca gaatgtggaa tcatctgccc cctgtgcgag cccatgccag
6240gcatgtcgcg ggcgaggaca cccgccactc gtacagcaga ccattatgct acctcacaat
6300agttcataac agtgaccata tttctcgaag ctccccaacg agcacctcca tgctctgagt
6360ggccaccccc cggccctggt gcttgcggag ggcaggtcaa ccggcatggg gctaccgaaa
6420tccccgaccg gatcccacca cccccgcgat gggaagaatc tctccccggg atgtgggccc
6480accaccagca caacctgctg gcccaggcga gcgtcaaacc ataccacaca aatatccttg
6540gcatcggccc tgaattcctt ctgccgctct gctacccggt gcttctgtcc gaagcagggg
6600ttgctaggga tcgctccgag tccgcaaacc cttgtcgcgt ggcggggctt gttcgagctt
6660gaagagc
66671125991DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 112gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcgaatt cctttcttgc gctatgacac 3300ttccagcaaa aggtagggcg ggctgcgaga
cggcttcccg gcgctgcatg caacaccgat 3360gatgcttcga ccccccgaag ctccttcggg
gctgcatggg cgctccgatg ccgctccagg 3420gcgagcgctg tttaaatagc caggcccccg
attgcaaaga cattatagcg agctaccaaa 3480gccatattca aacacctaga tcactaccac
ttctacacag gccactcgag cttgtgatcg 3540cactccgcta agggggcgcc tcttcctctt
cgtttcagtc acaacccgca aacactagta 3600tggcctccgc tgtgaccttc gcctgcgctc
ctcctcgcag gcgcgccggt gccgtggccg 3660ctcctggccg acgcgctgcc tctcgtcctc
tggtggtgca cgccgtggcc tccgaggctc 3720ctctgggcgt gcctccctcc gtgcagcgcc
cttctcccgt ggtgtactcc aagctggaca 3780agcagcaccg cctgacgcct gagcgcctgg
agctggtgca gtccatgggc cagttcgccg 3840aggagcgcgt gctgcccgtg ctgcaccccg
tggacaagct gtggcagccc caggacttcc 3900tgcccgaccc cgagtccccc gacttcgagg
accaggtggc cgagctgcgc gcccgcgcca 3960aggacctgcc cgacgagtac ttcgtggtgc
tggtgggcga catgatcacc gaggaggccc 4020tgcccaccta catggccatg ctgaacacct
gggacggcgt gcgcgacgac accggcgccg 4080ccgaccaccc ctgggcccgc tggacccgcc
agtgggtggc cgaggagaac cgccacggcg 4140acctgctgaa caagtactgc tggctgaccg
gccgcgtgaa catgcgcgcc gtggaggtga 4200ccatcaacaa cctgatcaag tccggcatga
acccccagac cgacaacaac ccctacctgg 4260gcttcgtgta cacctccttc caggagcgcg
ccaccaagta ctcccacggc aacaccgccc 4320gcctggccgc cgagcacggc gacaagggcc
tgtccaagat ctgcggcctg atcgcctccg 4380acgagggccg ccacgagatc gcctacaccc
gcatcgtgga cgagttcttc cgcctggacc 4440ccgagggcgc cgtggccgcc tacgccaaca
tgatgcgcaa gcagatcacc atgcccgccc 4500acctgatgga cgacatgggc cacggcgagg
ccaaccccgg ccgcaacctg ttcgccgact 4560tctccgccgt ggccgagaag atcgacgtgt
acgacgccga ggactactgc cgcatcctgg 4620agcacctgaa cgcccgctgg aaggtggacg
agcgccaggt gtccggccag gccgccgccg 4680accaggagta cgtgctgggc ctgccccagc
gcttccgcaa gctggccgag aagaccgccg 4740ccaagcgcaa gcgcgtggcc cgccgccccg
tggccttctc ctggatctcc ggccgcgaga 4800tcatggtgtg aatcgataga tctcttaagg
cagcagcagc tcggatagta tcgacacact 4860ctggacgctg gtcgtgtgat ggactgttgc
cgccacactt gctgccttga cctgtgaata 4920tccctgccgc ttttatcaaa cagcctcagt
gtgtttgatc ttgtgtgtac gcgcttttgc 4980gagttgctag ctgcttgtgc tatttgcgaa
taccaccccc agcatcccct tccctcgttt 5040catatcgctt gcatcccaac cgcaacttat
ctacgctgtc ctgctatccc tcagcgctgc 5100tcctgctcct gctcactgcc cctcgcacag
ccttggtttg ggctccgcct gtattctcct 5160ggtactgcaa cctgtaaacc agcactgcaa
tgctgatgca cgggaagtag tgggatggga 5220acacaaatgg aaagcttaat taagagctct
tgttttccag aaggagttgc tccttgagcc 5280tttcattctc agcctcgata acctccaaag
ccgctctaat tgtggagggg gttcgaattt 5340aaaagcttgg aatgttggtt cgtgcgtctg
gaacaagccc agacttgttg ctcactggga 5400aaaggaccat cagctccaaa aaacttgccg
ctcaaaccgc gtacctctgc tttcgcgcaa 5460tctgccctgt tgaaatcgcc accacattca
tattgtgacg cttgagcagt ctgtaattgc 5520ctcagaatgt ggaatcatct gccccctgtg
cgagcccatg ccaggcatgt cgcgggcgag 5580gacacccgcc actcgtacag cagaccatta
tgctacctca caatagttca taacagtgac 5640catatttctc gaagctcccc aacgagcacc
tccatgctct gagtggccac cccccggccc 5700tggtgcttgc ggagggcagg tcaaccggca
tggggctacc gaaatccccg accggatccc 5760accacccccg cgatgggaag aatctctccc
cgggatgtgg gcccaccacc agcacaacct 5820gctggcccag gcgagcgtca aaccatacca
cacaaatatc cttggcatcg gccctgaatt 5880ccttctgccg ctctgctacc cggtgcttct
gtccgaagca ggggttgcta gggatcgctc 5940cgagtccgca aacccttgtc gcgtggcggg
gcttgttcga gcttgaagag c 59911135988DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
113gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtacccttt cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct
780tcccggcgct gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc
840atgggcgctc cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc
900aaagacatta tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta
960cacaggccac tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt
1020cagtcacaac ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg
1080gccggcttcg ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg
1140gtgcacttca cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag
1200aaggacgcca agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg
1260cccttgttct ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc
1320gccatcgccc cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac
1380aacaacacct ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc
1440tggacctaca acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc
1500tacaccttca ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc
1560gacccgaagg tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc
1620caggactaca agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc
1680gcgttcgcca acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc
1740cccaccgagc aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc
1800gccccggccg gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc
1860gaggccttcg acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag
1920accttcttca acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac
1980tgggagtact ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc
2040aagttctccc tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag
2100gccgagccga tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc
2160acgttgacga aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag
2220ttcgagctgg tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac
2280ctctccctct ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag
2340gtgtccgcgt cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag
2400aacccctact tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac
2460ctgtcctact acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac
2520gacggcgacg tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc
2580gtgaacatga cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag
2640gtcaagtgac aattggcagc agcagctcgg atagtatcga cacactctgg acgctggtcg
2700tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc tgccgctttt
2760atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc
2820ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata tcgcttgcat
2880cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct gctcctgctc
2940actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta ctgcaacctg
3000taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac aaatggagga
3060tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct gtcgcacctc
3120agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg tccattagcg
3180aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat gatcggtgga
3240gctgatggtc gaaacgttca cagcctaggg atatcgaatt cctttcttgc gctatgacac
3300ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg gcgctgcatg caacaccgat
3360gatgcttcga ccccccgaag ctccttcggg gctgcatggg cgctccgatg ccgctccagg
3420gcgagcgctg tttaaatagc caggcccccg attgcaaaga cattatagcg agctaccaaa
3480gccatattca aacacctaga tcactaccac ttctacacag gccactcgag cttgtgatcg
3540cactccgcta agggggcgcc tcttcctctt cgtttcagtc acaacccgca aacactagta
3600tggccaccgc atccactttc tcggcgttca atgcccgctg cggcgacctg cgtcgctcgg
3660cgggctccgg gccccggcgc ccagcgaggc ccctccccgt gcgcgggcgc gccgaggtgc
3720acgtgcaggt gacccactcc ctggcccccg agaagcgcga gatcttcaac tccctgaaca
3780actgggccca ggagaacatc ctggtgctgc tgaaggacgt ggacaagtgc tggcagccct
3840ccgacttcct gcccgactcc gcctccgagg gcttcgacga gcaggtgatg gagctgcgca
3900agcgctgcaa ggagatcccc gacgactact tcatcgtgct ggtgggcgac atgatcaccg
3960aggaggccct gcccacctac cagaccatgc tgaacacctg ggacggcgtg cgcgacgaga
4020ccggcgcctc cctgaccccc tgggccatct ggacccgcgc ctggaccgcc gaggagaacc
4080gccacggcga cctgctgaac aagtacctgt acctgtccgg ccgcgtggac atgaagcaga
4140tcgagaagac catccagtac ctgatcggct ccggcatgga cccccgcacc gagaacaacc
4200cctacctggg cttcatctac acctccttcc aggagcgcgc caccttcatc tcccacggca
4260acaccgcccg cctggccaag gagcacggcg acctgaagct ggcccagatc tgcggcatca
4320tcgccgccga cgagaagcgc cacgagaccg cctacaccaa gatcgtggag aagctgttcg
4380agatcgaccc cgacggcacc gtgctggccc tggccgacat gatgcgcaag aaggtgtcca
4440tgcccgccca cctgatgtac gacggccagg acgacaacct gttcgagaac ttctcctccg
4500tggcccagcg cctgggcgtg tacaccgcca aggactacgc cgacatcctg gagttcctgg
4560tgggccgctg ggacatcgag aagctgaccg gcctgtccgg cgagggccgc aaggcccagg
4620actacgtgtg caccctgccc ccccgcatcc gccgcctgga ggagcgcgcc cagtcccgcg
4680tgaagaaggc ctccgccacc cccttctcct ggatcttcgg ccgcgagatc aacctgatgg
4740actacaagga ccacgacggc gactacaagg accacgacat cgactacaag gacgacgacg
4800acaagtgaat cgatagatct cttaaggcag cagcagctcg gatagtatcg acacactctg
4860gacgctggtc gtgtgatgga ctgttgccgc cacacttgct gccttgacct gtgaatatcc
4920ctgccgcttt tatcaaacag cctcagtgtg tttgatcttg tgtgtacgcg cttttgcgag
4980ttgctagctg cttgtgctat ttgcgaatac cacccccagc atccccttcc ctcgtttcat
5040atcgcttgca tcccaaccgc aacttatcta cgctgtcctg ctatccctca gcgctgctcc
5100tgctcctgct cactgcccct cgcacagcct tggtttgggc tccgcctgta ttctcctggt
5160actgcaacct gtaaaccagc actgcaatgc tgatgcacgg gaagtagtgg gatgggaaca
5220caaatggaaa gcttaattaa gagctcttgt tttccagaag gagttgctcc ttgagccttt
5280cattctcagc ctcgataacc tccaaagccg ctctaattgt ggagggggtt cgaatttaaa
5340agcttggaat gttggttcgt gcgtctggaa caagcccaga cttgttgctc actgggaaaa
5400ggaccatcag ctccaaaaaa cttgccgctc aaaccgcgta cctctgcttt cgcgcaatct
5460gccctgttga aatcgccacc acattcatat tgtgacgctt gagcagtctg taattgcctc
5520agaatgtgga atcatctgcc ccctgtgcga gcccatgcca ggcatgtcgc gggcgaggac
5580acccgccact cgtacagcag accattatgc tacctcacaa tagttcataa cagtgaccat
5640atttctcgaa gctccccaac gagcacctcc atgctctgag tggccacccc ccggccctgg
5700tgcttgcgga gggcaggtca accggcatgg ggctaccgaa atccccgacc ggatcccacc
5760acccccgcga tgggaagaat ctctccccgg gatgtgggcc caccaccagc acaacctgct
5820ggcccaggcg agcgtcaaac cataccacac aaatatcctt ggcatcggcc ctgaattcct
5880tctgccgctc tgctacccgg tgcttctgtc cgaagcaggg gttgctaggg atcgctccga
5940gtccgcaaac ccttgtcgcg tggcggggct tgttcgagct tgaagagc
59881146696DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 114gctcttcacc caactcagat aataccaata
cccctccttc tcctcctcat ccattcagta 60ccccccccct tctcttccca aagcagcaag
cgcgtggctt acagaagaac aatcggcttc 120cgccaaagtc gccgagcact gcccgacggc
ggcgcgccca gcagcccgct tggccacaca 180ggcaacgaat acattcaata gggggcctcg
cagaatggaa ggagcggtaa agggtacagg 240agcactgcgc acaaggggcc tgtgcaggag
tgactgactg ggcgggcaga cggcgcaccg 300cgggcgcagg caagcaggga agattgaagc
ggcagggagg aggatgctga ttgagggggg 360catcgcagtc tctcttggac ccgggataag
gaagcaaata ttcggccggt tgggttgtgt 420gtgtgcacgt tttcttcttc agagtcgtgg
gtgtgcttcc agggaggata taagcagcag 480gatcgaatcc cgcgaccagc gtttccccat
ccagccaacc accctgtcgg taccctttct 540tgcgctatga cacttccagc aaaaggtagg
gcgggctgcg agacggcttc ccggcgctgc 600atgcaacacc gatgatgctt cgaccccccg
aagctccttc ggggctgcat gggcgctccg 660atgccgctcc agggcgagcg ctgtttaaat
agccaggccc ccgattgcaa agacattata 720gcgagctacc aaagccatat tcaaacacct
agatcactac cacttctaca caggccactc 780gagcttgtga tcgcactccg ctaagggggc
gcctcttcct cttcgtttca gtcacaaccc 840gcaaacggcg cgccatgctg ctgcaggcct
tcctgttcct gctggccggc ttcgccgcca 900agatcagcgc ctccatgacg aacgagacgt
ccgaccgccc cctggtgcac ttcaccccca 960acaagggctg gatgaacgac cccaacggcc
tgtggtacga cgagaaggac gccaagtggc 1020acctgtactt ccagtacaac ccgaacgaca
ccgtctgggg gacgcccttg ttctggggcc 1080acgccacgtc cgacgacctg accaactggg
aggaccagcc catcgccatc gccccgaagc 1140gcaacgactc cggcgccttc tccggctcca
tggtggtgga ctacaacaac acctccggct 1200tcttcaacga caccatcgac ccgcgccagc
gctgcgtggc catctggacc tacaacaccc 1260cggagtccga ggagcagtac atctcctaca
gcctggacgg cggctacacc ttcaccgagt 1320accagaagaa ccccgtgctg gccgccaact
ccacccagtt ccgcgacccg aaggtcttct 1380ggtacgagcc ctcccagaag tggatcatga
ccgcggccaa gtcccaggac tacaagatcg 1440agatctactc ctccgacgac ctgaagtcct
ggaagctgga gtccgcgttc gccaacgagg 1500gcttcctcgg ctaccagtac gagtgccccg
gcctgatcga ggtccccacc gagcaggacc 1560ccagcaagtc ctactgggtg atgttcatct
ccatcaaccc cggcgccccg gccggcggct 1620ccttcaacca gtacttcgtc ggcagcttca
acggcaccca cttcgaggcc ttcgacaacc 1680agtcccgcgt ggtggacttc ggcaaggact
actacgccct gcagaccttc ttcaacaccg 1740acccgaccta cgggagcgcc ctgggcatcg
cgtgggcctc caactgggag tactccgcct 1800tcgtgcccac caacccctgg cgctcctcca
tgtccctcgt gcgcaagttc tccctcaaca 1860ccgagtacca ggccaacccg gagacggagc
tgatcaacct gaaggccgag ccgatcctga 1920acatcagcaa cgccggcccc tggagccggt
tcgccaccaa caccacgttg acgaaggcca 1980acagctacaa cgtcgacctg tccaacagca
ccggcaccct ggagttcgag ctggtgtacg 2040ccgtcaacac cacccagacg atctccaagt
ccgtgttcgc ggacctctcc ctctggttca 2100agggcctgga ggaccccgag gagtacctcc
gcatgggctt cgaggtgtcc gcgtcctcct 2160tcttcctgga ccgcgggaac agcaaggtga
agttcgtgaa ggagaacccc tacttcacca 2220accgcatgag cgtgaacaac cagcccttca
agagcgagaa cgacctgtcc tactacaagg 2280tgtacggctt gctggaccag aacatcctgg
agctgtactt caacgacggc gacgtcgtgt 2340ccaccaacac ctacttcatg accaccggga
acgccctggg ctccgtgaac atgacgacgg 2400gggtggacaa cctgttctac atcgacaagt
tccaggtgcg cgaggtcaag tgacaattgg 2460cagcagcagc tcggatagta tcgacacact
ctggacgctg gtcgtgtgat ggactgttgc 2520cgccacactt gctgccttga cctgtgaata
tccctgccgc ttttatcaaa cagcctcagt 2580gtgtttgatc ttgtgtgtac gcgcttttgc
gagttgctag ctgcttgtgc tatttgcgaa 2640taccaccccc agcatcccct tccctcgttt
catatcgctt gcatcccaac cgcaacttat 2700ctacgctgtc ctgctatccc tcagcgctgc
tcctgctcct gctcactgcc cctcgcacag 2760ccttggtttg ggctccgcct gtattctcct
ggtactgcaa cctgtaaacc agcactgcaa 2820tgctgatgca cgggaagtag tgggatggga
acacaaatgg aggatcccgc gtctcgaaca 2880gagcgcgcag aggaacgctg aaggtctcgc
ctctgtcgca cctcagcgcg gcatacacca 2940caataaccac ctgacgaatg cgcttggttc
ttcgtccatt agcgaagcgt ccggttcaca 3000cacgtgccac gttggcgagg tggcaggtga
caatgatcgg tggagctgat ggtcgaaacg 3060ttcacagcct agggatatca tagcgactgc
taccccccga ccatgtgccg aggcagaaat 3120tatatacaag aagcagatcg caattaggca
catcgctttg cattatccac acactattca 3180tcgctgctgc ggcaaggctg cagagtgtat
ttttgtggcc caggagctga gtccgaagtc 3240gacgcgacga gcggcgcagg atccgacccc
tagacgagct ctgtcatttt ccaagcacgc 3300agctaaatgc gctgagaccg ggtctaaatc
atccgaaaag tgtcaaaatg gccgattggg 3360ttcgcctagg acaatgcgct gcggattcgc
tcgagtccgc tgccggccaa aaggcggtgg 3420tacaggaagg cgcacggggc caaccctgcg
aagccggggg cccgaacgcc gaccgccggc 3480cttcgatctc gggtgtcccc ctcgtcaatt
tcctctctcg ggtgcagcca cgaaagtcgt 3540gacgcaggtc acgaaatccg gttacgaaaa
acgcaggtct tcgcaaaaac gtgagggttt 3600cgcgtctcgc cctagctatt cgtatcgccg
ggtcagaccc acgtgcagaa aagcccttga 3660ataacccggg accgtggtta ccgcgccgcc
tgcaccaggg ggcttatata agcccacacc 3720acacctgtct caccacgcat ttctccaact
cgcgactttt cggaagaaat tgttatccac 3780ctagtataga ctgccacctg caggaccttg
tgtcttgcag tttgtattgg tcccggccgt 3840cgagctcgac agatctgggc tagggttggc
ctggccgctc ggcactcccc tttagccgcg 3900cgcatccgcg ttccagaggt gcgattcggt
gtgtggagca ttgtcatgcg cttgtggggg 3960tcgttccgtg cgcggcgggt ccgccatggg
cgccgacctg ggccctaggg tttgttttcg 4020ggccaagcga gcccctctca cctcgtcgcc
cccccgcatt ccctctctct tgcagccttg 4080ccactagtat ggccaccgca tccactttct
cggcgttcaa tgcccgctgc ggcgacctgc 4140gtcgctcggc gggctccggg ccccggcgcc
cagcgaggcc cctccccgtg cgcgggcgcg 4200ccgccgccgc cgccgacgcc aaccccgccc
gccccgagcg ccgcgtggtg atcaccggcc 4260agggcgtggt gacctccctg ggccagacca
tcgagcagtt ctactcctcc ctgctggagg 4320gcgtgtccgg catctcccag atccagaagt
tcgacaccac cggctacacc accaccatcg 4380ccggcgagat caagtccctg cagctggacc
cctacgtgcc caagcgctgg gccaagcgcg 4440tggacgacgt gatcaagtac gtgtacatcg
ccggcaagca ggccctggag tccgccggcc 4500tgcccatcga ggccgccggc ctggccggcg
ccggcctgga ccccgccctg tgcggcgtgc 4560tgatcggcac cgccatggcc ggcatgacct
ccttcgccgc cggcgtggag gccctgaccc 4620gcggcggcgt gcgcaagatg aaccccttct
gcatcccctt ctccatctcc aacatgggcg 4680gcgccatgct ggccatggac atcggcttca
tgggccccaa ctactccatc tccaccgcct 4740gcgccaccgg caactactgc atcctgggcg
ccgccgacca catccgccgc ggcgacgcca 4800acgtgatgct ggccggcggc gccgacgccg
ccatcatccc ctccggcatc ggcggcttca 4860tcgcctgcaa ggccctgtcc aagcgcaacg
acgagcccga gcgcgcctcc cgcccctggg 4920acgccgaccg cgacggcttc gtgatgggcg
agggcgccgg cgtgctggtg ctggaggagc 4980tggagcacgc caagcgccgc ggcgccacca
tcctggccga gctggtgggc ggcgccgcca 5040cctccgacgc ccaccacatg accgagcccg
acccccaggg ccgcggcgtg cgcctgtgcc 5100tggagcgcgc cctggagcgc gcccgcctgg
cccccgagcg cgtgggctac gtgaacgccc 5160acggcacctc cacccccgcc ggcgacgtgg
ccgagtaccg cgccatccgc gccgtgatcc 5220cccaggactc cctgcgcatc aactccacca
agtccatgat cggccacctg ctgggcggcg 5280ccggcgccgt ggaggccgtg gccgccatcc
aggccctgcg caccggctgg ctgcacccca 5340acctgaacct ggagaacccc gcccccggcg
tggaccccgt ggtgctggtg ggcccccgca 5400aggagcgcgc cgaggacctg gacgtggtgc
tgtccaactc cttcggcttc ggcggccaca 5460actcctgcgt gatcttccgc aagtacgacg
agatggacta caaggaccac gacggcgact 5520acaaggacca cgacatcgac tacaaggacg
acgacgacaa gtgaatcgat agatctctta 5580aggcagcagc agctcggata gtatcgacac
actctggacg ctggtcgtgt gatggactgt 5640tgccgccaca cttgctgcct tgacctgtga
atatccctgc cgcttttatc aaacagcctc 5700agtgtgtttg atcttgtgtg tacgcgcttt
tgcgagttgc tagctgcttg tgctatttgc 5760gaataccacc cccagcatcc ccttccctcg
tttcatatcg cttgcatccc aaccgcaact 5820tatctacgct gtcctgctat ccctcagcgc
tgctcctgct cctgctcact gcccctcgca 5880cagccttggt ttgggctccg cctgtattct
cctggtactg caacctgtaa accagcactg 5940caatgctgat gcacgggaag tagtgggatg
ggaacacaaa tggaaagctt aattaagagc 6000tcttgttttc cagaaggagt tgctccttga
gcctttcatt ctcagcctcg ataacctcca 6060aagccgctct aattgtggag ggggttcgaa
ccgaatgctg cgtgaacggg aaggaggagg 6120agaaagagtg agcagggagg gattcagaaa
tgagaaatga gaggtgaagg aacgcatccc 6180tatgcccttg caatggacag tgtttctggc
caccgccacc aagacttcgt gtcctctgat 6240catcatgcga ttgattacgt tgaatgcgac
ggccggtcag ccccggacct ccacgcaccg 6300gtgctcctcc aggaagatgc gcttgtcctc
cgccatcttg cagggctcaa gctgctccca 6360aaactcttgg gcgggttccg gacggacggc
taccgcgggt gcggccctga ccgccactgt 6420tcggaagcag cggcgctgca tgggcagcgg
ccgctgcggt gcgccacgga ccgcatgatc 6480caccggaaaa gcgcacgcgc tggagcgcgc
agaggaccac agagaagcgg aagagacgcc 6540agtactggca agcaggctgg tcggtgccat
ggcgcgctac taccctcgct atgactcggg 6600tcctcggccg gctggcggtg ctgacaattc
gtttagtgga gcagcgactc cattcagcta 6660ccagtcgaac tcagtggcac agtgactccg
ctcttc 6696115993DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
115gatatctccc tccgtctctg cactctggcg cccctcctcc gtctcgtgga ctgacggacg
60agagtctggg cgccgctttt ctatccacac cgccctttcc gcatcgaaga caccacccat
120cgtgccgcca ggtcttcccc aatcacccgc cctgtggtcc tctctcccag ccgtgtttgg
180tcgctgcgtc cacatttttc cattcgtgcc ccacgatcct cgcccatctt ggcgccttgg
240ataggcaccc ttttttcagc acgccctggt gtgtagcaca acctgacctc tctctaccgc
300atcgcctccc tcccacacct cagttgactc cctcgtcgca cgttgcaccc gcaagctccc
360catttcatcc tattgacaat cgcacactgt acatgtatgc tcattatttt gcaaaaaaac
420agggggtcgg ttcactcctg gcagacgacg cggtgctgcc gcgcgccgct gaggcggcgt
480cgcgacggca acacccatcg caccgcacgt cgacgagtca acccaccctg ctcaacggtg
540atctccccat cgcgacaccc cccgtgaccg tactatgtgc gtccatacgc aacatgaaaa
600ggaccttggt ccccggaggc ggcgagctcg taatcccgag gttggccccg cttccgctgg
660acacccatcg catcttccgg ctcgcccgct gtcgagcaag cgccctcgtg cgcgcaaccc
720ttgtggtgcc tgcccgcaga gccgggcata aaggcgagca ccacacccga accagtccaa
780tttgctttct gcattcactc accaactttt acatccacac atcgtactac cacacctgcc
840cagtcgggtt tgatttctat tgcaaaggtg cgggggggtt ggcgcactgc gtgggttgtg
900cagccggccg ccgcggctgt acccagcgat caggtagctt gggctgtatc ttctcaagca
960ttaccttgtc ctgggcgtag gtttgccact agt
9931161083DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 116gatatcgaat tcggccgaca ggacgcgcgt
caaaggtgct ggtcgtgtat gccctggccg 60gcaggtcgtt gctgctgctg gttagtgatt
ccgcaaccct gattttggcg tcttattttg 120gcgtggcaaa cgctggcgcc cgcgagccgg
gccggcggcg atgcggtgcc ccacggctgc 180cggaatccaa gggaggcaag agcgcccggg
tcagttgaag ggctttacgc gcaaggtaca 240gccgctcctg caaggctgcg tggtggaatt
ggacgtgcag gtcctgctga agttcctcca 300ccgcctcacc agcggacaaa gcaccggtgt
atcaggtccg tgtcatccac tctaaagaac 360tcgactacga cctactgatg gccctagatt
cttcatcaaa aacgcctgag acacttgccc 420aggattgaaa ctccctgaag ggaccaccag
gggccctgag ttgttccttc cccccgtggc 480gagctgccag ccaggctgta cctgtgatcg
aggctggcgg gaaaataggc ttcgtgtgct 540caggtcatgg gaggtgcagg acagctcatg
aaacgccaac aatcgcacaa ttcatgtcaa 600gctaatcagc tatttcctct tcacgagctg
taattgtccc aaaattctgg tctaccgggg 660gtgatccttc gtgtacgggc ccttccctca
accctaggta tgcgcgcatg cggtcgccgc 720gcaactcgcg cgagggccga gggtttggga
cgggccgtcc cgaaatgcag ttgcacccgg 780atgcgtggca ccttttttgc gataatttat
gcaatggact gctctgcaaa attctggctc 840tgtcgccaac cctaggatca gcggcgtagg
atttcgtaat cattcgtcct gatggggagc 900taccgactac cctaatatca gcccgactgc
ctgacgccag cgtccacttt tgtgcacaca 960ttccattcgt gcccaagaca tttcattgtg
gtgcgaagcg tccccagtta cgctcacctg 1020tttcccgacc tccttactgt tctgtcgaca
gagcgggccc acaggccggt cgcagccact 1080agt
10831175662DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
117gctcttcgcc gccgccactc ctgctcgagc gcgcccgcgc gtgcgccgcc agcgccttgg
60ccttttcgcc gcgctcgtgc gcgtcgctga tgtccatcac caggtccatg aggtctgcct
120tgcgccggct gagccactgc ttcgtccggg cggccaagag gagcatgagg gaggactcct
180ggtccagggt cctgacgtgg tcgcggctct gggagcgggc cagcatcatc tggctctgcc
240gcaccgaggc cgcctccaac tggtcctcca gcagccgcag tcgccgccga ccctggcaga
300ggaagacagg tgaggggggt atgaattgta cagaacaacc acgagccttg tctaggcaga
360atccctacca gtcatggctt tacctggatg acggcctgcg aacagctgtc cagcgaccct
420cgctgccgcc gcttctcccg cacgcttctt tccagcaccg tgatggcgcg agccagcgcc
480gcacgctggc gctgcgcttc gccgatctga ggacagtcgg ggaactctga tcagtctaaa
540cccccttgcg cgttagtgtt gccatccttt gcagaccggt gagagccgac ttgttgtgcg
600ccacccccca caccacctcc tcccagacca attctgtcac ctttttggcg aaggcatcgg
660cctcggcctg cagagaggac agcagtgccc agccgctggg ggttggcgga tgcacgctca
720ggtaccgcgg tgagaatcga aaatgcatcg tttctaggtt cggagacggt caattccctg
780ctccggcgaa tctgtcggtc aagctggcca gtggacaatg ttgctatggc agcccgcgca
840catgggcctc ccgacgcggc catcaggagc ccaaacagcg tgtcagggta tgtgaaactc
900aagaggtccc tgctgggcac tccggcccca ctccgggggc gggacgccag gcattcgcgg
960tcggtcccgc gcgacgagcg aaatgatgat tcggttacga gaccaggacg tcgtcgaggt
1020cgagaggcag cctcggacac gtctcgctag ggcaacgccc cgagtccccg cgagggccgt
1080aaacattgtt tctgggtgtc ggagtgggca ttttgggccc gatccaatcg cctcatgccg
1140ctctcgtctg gtcctcacgt tcgcgtacgg cctggatccc ggaaagggcg gatgcacgtg
1200gtgttgcccc gccattggcg cccacgtttc aaagtccccg gccagaaatg cacaggaccg
1260gcccggctcg cacaggccat gctgaacgcc cagatttcga cagcaacacc atctagaata
1320atcgcaacca tccgcgtttt gaacgaaacg aaacggcgct gtttagcatg tttccgacat
1380cgtgggggcc gaagcatgct ccggggggag gaaagcgtgg cacagcggta gcccattctg
1440tgccacacgc cgacgaggac caatccccgg catcagcctt catcgacggc tgcgccgcac
1500atataaagcc ggacgcctaa ccggtttcgt ggttatgact agtatgttcg cgttctactt
1560cctgacggcc tgcatctccc tgaagggcgt gttcggcgtc tccccctcct acaacggcct
1620gggcctgacg ccccagatgg gctgggacaa ctggaacacg ttcgcctgcg acgtctccga
1680gcagctgctg ctggacacgg ccgaccgcat ctccgacctg ggcctgaagg acatgggcta
1740caagtacatc atcctggacg actgctggtc ctccggccgc gactccgacg gcttcctggt
1800cgccgacgag cagaagttcc ccaacggcat gggccacgtc gccgaccacc tgcacaacaa
1860ctccttcctg ttcggcatgt actcctccgc gggcgagtac acgtgcgccg gctaccccgg
1920ctccctgggc cgcgaggagg aggacgccca gttcttcgcg aacaaccgcg tggactacct
1980gaagtacgac aactgctaca acaagggcca gttcggcacg cccgagatct cctaccaccg
2040ctacaaggcc atgtccgacg ccctgaacaa gacgggccgc cccatcttct actccctgtg
2100caactggggc caggacctga ccttctactg gggctccggc atcgcgaact cctggcgcat
2160gtccggcgac gtcacggcgg agttcacgcg ccccgactcc cgctgcccct gcgacggcga
2220cgagtacgac tgcaagtacg ccggcttcca ctgctccatc atgaacatcc tgaacaaggc
2280cgcccccatg ggccagaacg cgggcgtcgg cggctggaac gacctggaca acctggaggt
2340cggcgtcggc aacctgacgg acgacgagga gaaggcgcac ttctccatgt gggccatggt
2400gaagtccccc ctgatcatcg gcgcgaacgt gaacaacctg aaggcctcct cctactccat
2460ctactcccag gcgtccgtca tcgccatcaa ccaggactcc aacggcatcc ccgccacgcg
2520cgtctggcgc tactacgtgt ccgacacgga cgagtacggc cagggcgaga tccagatgtg
2580gtccggcccc ctggacaacg gcgaccaggt cgtggcgctg ctgaacggcg gctccgtgtc
2640ccgccccatg aacacgaccc tggaggagat cttcttcgac tccaacctgg gctccaagaa
2700gctgacctcc acctgggaca tctacgacct gtgggcgaac cgcgtcgaca actccacggc
2760gtccgccatc ctgggccgca acaagaccgc caccggcatc ctgtacaacg ccaccgagca
2820gtcctacaag gacggcctgt ccaagaacga cacccgcctg ttcggccaga agatcggctc
2880cctgtccccc aacgcgatcc tgaacacgac cgtccccgcc cacggcatcg cgttctaccg
2940cctgcgcccc tcctcctgac aattggcagc agcagctcgg atagtatcga cacactctgg
3000acgctggtcg tgtgatggac tgttgccgcc acacttgctg ccttgacctg tgaatatccc
3060tgccgctttt atcaaacagc ctcagtgtgt ttgatcttgt gtgtacgcgc ttttgcgagt
3120tgctagctgc ttgtgctatt tgcgaatacc acccccagca tccccttccc tcgtttcata
3180tcgcttgcat cccaaccgca acttatctac gctgtcctgc tatccctcag cgctgctcct
3240gctcctgctc actgcccctc gcacagcctt ggtttgggct ccgcctgtat tctcctggta
3300ctgcaacctg taaaccagca ctgcaatgct gatgcacggg aagtagtggg atgggaacac
3360aaatggagga tcccgcgtct cgaacagagc gcgcagagga acgctgaagg tctcgcctct
3420gtcgcacctc agcgcggcat acaccacaat aaccacctga cgaatgcgct tggttcttcg
3480tccattagcg aagcgtccgg ttcacacacg tgccacgttg gcgaggtggc aggtgacaat
3540gatcggtgga gctgatggtc gaaacgttca cagcctaggg atatcgaatt cctttcttgc
3600gctatgacac ttccagcaaa aggtagggcg ggctgcgaga cggcttcccg gcgctgcatg
3660caacaccgat gatgcttcga ccccccgaag ctccttcggg gctgcatggg cgctccgatg
3720ccgctccagg gcgagcgctg tttaaatagc caggcccccg attgcaaaga cattatagcg
3780agctaccaaa gccatattca aacacctaga tcactaccac ttctacacag gccactcgag
3840cttgtgatcg cactccgcta agggggcgcc tcttcctctt cgtttcagtc acaacccgca
3900aacactagta tggctatcaa gacgaacagg cagcctgtgg agaagcctcc gttcacgatc
3960gggacgctgc gcaaggccat ccccgcgcac tgtttcgagc gctcggcgct tcgtagcagc
4020atgtacctgg cctttgacat cgcggtcatg tccctgctct acgtcgcgtc gacgtacatc
4080gaccctgcac cggtgcctac gtgggtcaag tacggcatca tgtggccgct ctactggttc
4140ttccaggtgt gtttgagggt tttggttgcc cgtattgagg tcctggtggc gcgcatggag
4200gagaaggcgc ctgtcccgct gacccccccg gctaccctcc cggcaccttc cagggcgcgt
4260acgggaagaa ccagtagagc ggccacatga tgccgtactt gacccacgta ggcaccggtg
4320cagggtcgat gtacgtcgac gcgacgtaga gcagggacat gaccgcgatg tcaaaggcca
4380ggtacatgct gctacgaagc gccgagcgct cgaaacagtg cgcggggatg gccttgcgca
4440gcgtcccgat cgtgaacgga ggcttctcca caggctgcct gttcgtcttg atagccatct
4500cgaggcagca gcagctcgga tagtatcgac acactctgga cgctggtcgt gtgatggact
4560gttgccgcca cacttgctgc cttgacctgt gaatatccct gccgctttta tcaaacagcc
4620tcagtgtgtt tgatcttgtg tgtacgcgct tttgcgagtt gctagctgct tgtgctattt
4680gcgaatacca cccccagcat ccccttccct cgtttcatat cgcttgcatc ccaaccgcaa
4740cttatctacg ctgtcctgct atccctcagc gctgctcctg ctcctgctca ctgcccctcg
4800cacagccttg gtttgggctc cgcctgtatt ctcctggtac tgcaacctgt aaaccagcac
4860tgcaatgctg atgcacggga agtagtggga tgggaacaca aatggaaagc tgtagagctc
4920ttgttttcca gaaggagttg ctccttgagc ctttcattct cagcctcgat aacctccaaa
4980gccgctctaa ttgtggaggg ggttcgaatt taaaagcttg gaatgttggt tcgtgcgtct
5040ggaacaagcc cagacttgtt gctcactggg aaaaggacca tcagctccaa aaaacttgcc
5100gctcaaaccg cgtacctctg ctttcgcgca atctgccctg ttgaaatcgc caccacattc
5160atattgtgac gcttgagcag tctgtaattg cctcagaatg tggaatcatc tgccccctgt
5220gcgagcccat gccaggcatg tcgcgggcga ggacacccgc cactcgtaca gcagaccatt
5280atgctacctc acaatagttc ataacagtga ccatatttct cgaagctccc caacgagcac
5340ctccatgctc tgagtggcca ccccccggcc ctggtgcttg cggagggcag gtcaaccggc
5400atggggctac cgaaatcccc gaccggatcc caccaccccc gcgatgggaa gaatctctcc
5460ccgggatgtg ggcccaccac cagcacaacc tgctggccca ggcgagcgtc aaaccatacc
5520acacaaatat ccttggcatc ggccctgaat tccttctgcc gctctgctac ccggtgcttc
5580tgtccgaagc aggggttgct agggatcgct ccgagtccgc aaacccttgt cgcgtggcgg
5640ggcttgttcg agcttgaaga gc
56621187963DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 118gctcttcgga gtcactgtgc cactgagttc
gactggtagc tgaatggagt cgctgctcca 60ctaaacgaat tgtcagcacc gccagccggc
cgaggacccg agtcatagcg agggtagtag 120cgcgccatgg caccgaccag cctgcttgcc
agtactggcg tctcttccgc ttctctgtgg 180tcctctgcgc gctccagcgc gtgcgctttt
ccggtggatc atgcggtccg tggcgcaccg 240cagcggccgc tgcccatgca gcgccgctgc
ttccgaacag tggcggtcag ggccgcaccc 300gcggtagccg tccgtccgga acccgcccaa
gagttttggg agcagcttga gccctgcaag 360atggcggagg acaagcgcat cttcctggag
gagcaccggt gcgtggaggt ccggggctga 420ccggccgtcg cattcaacgt aatcaatcgc
atgatgatca gaggacacga agtcttggtg 480gcggtggcca gaaacactgt ccattgcaag
ggcataggga tgcgttcctt cacctctcat 540ttctcatttc tgaatccctc cctgctcact
ctttctcctc ctccttcccg ttcacgcagc 600attcggggta ccagtttagg tccagcgtcc
gtgggggggg acgggctggg agcttgggcc 660gggaagggca agacgatgca gtccctctgg
ggagtcacag ccgactgtgt gtgttgcact 720gtgcggcccg cagcactcac acgcaaaatg
cctggccgac aggcaggccc tgtccagtgc 780aacatccacg gtccctctca tcaggctcac
cttgctcatt gacataacgg aatgcgtacc 840gctctttcag atctgtccat ccagagaggg
gagcaggctc cccaccgacg ctgtcaaact 900tgcttcctgc ccaaccgaaa acattattgt
ttgagggggg gggggggggg gcagattgca 960tggcgggata tctcgtgagg aacatcactg
ggacactgtg gaacacagtg agtgcagtat 1020gcagagcatg tatgctaggg gtcagcgcag
gaagggggcc tttcccagtc tcccatgcca 1080ctgcaccgta tccacgactc accaggacca
gcttcttgat cggcttccgc tcccgtggac 1140accagtgtgt agcctctgga ctccaggtat
gcgtgcaccg caaaggccag ccgatcgtgc 1200cgattcctgg ggtggaggat atgagtcagc
caacttgggg ctcagagtgc acactggggc 1260acgatacgaa acaacatcta caccgtgtcc
tccatgctga cacaccacag cttcgctcca 1320cctgaatgtg ggcgcatggg cccgaatcac
agccaatgtc gctgctgcca taatgtgatc 1380cagaccctct ccgcccagat gccgagcgga
tcgtgggcgc tgaatagatt cctgtttcga 1440tcactgtttg ggtcctttcc ttttcgtctc
ggatgcgcgt ctcgaaacag gctgcgtcgg 1500gctttcggat cccttttgct ccctccgtca
ccatcctgcg cgcgggcaag ttgcttgacc 1560ctgggctgta ccagggttgg agggtattac
cgcgtcaggc cattcccagc ccggattcaa 1620ttcaaagtct gggccaccac cctccgccgc
tctgtctgat cactccacat tcgtgcatac 1680actacgttca agtcctgatc caggcgtgtc
tcgggacaag gtgtgcttga gtttgaatct 1740caaggaccca ctccagcaca gctgctggtt
gaccccgccc tcgcaatcta gaatggccgc 1800gtccgtccac tgcaccctga tgtccgtggt
ctgcaacaac aagaaccact ccgcccgccc 1860caagctgccc aactcctccc tgctgcccgg
cttcgacgtg gtggtccagg ccgcggccac 1920ccgcttcaag aaggagacga cgaccacccg
cgccacgctg acgttcgacc cccccacgac 1980caactccgag cgcgccaagc agcgcaagca
caccatcgac ccctcctccc ccgacttcca 2040gcccatcccc tccttcgagg agtgcttccc
caagtccacg aaggagcaca aggaggtggt 2100gcacgaggag tccggccacg tcctgaaggt
gcccttccgc cgcgtgcacc tgtccggcgg 2160cgagcccgcc ttcgacaact acgacacgtc
cggcccccag aacgtcaacg cccacatcgg 2220cctggcgaag ctgcgcaagg agtggatcga
ccgccgcgag aagctgggca cgccccgcta 2280cacgcagatg tactacgcga agcagggcat
catcacggag gagatgctgt actgcgcgac 2340gcgcgagaag ctggaccccg agttcgtccg
ctccgaggtc gcgcggggcc gcgccatcat 2400cccctccaac aagaagcacc tggagctgga
gcccatgatc gtgggccgca agttcctggt 2460gaaggtgaac gcgaacatcg gcaactccgc
cgtggcctcc tccatcgagg aggaggtcta 2520caaggtgcag tgggccacca tgtggggcgc
cgacaccatc atggacctgt ccacgggccg 2580ccacatccac gagacgcgcg agtggatcct
gcgcaactcc gcggtccccg tgggcaccgt 2640ccccatctac caggcgctgg agaaggtgga
cggcatcgcg gagaacctga actgggaggt 2700gttccgcgag acgctgatcg agcaggccga
gcagggcgtg gactacttca cgatccacgc 2760gggcgtgctg ctgcgctaca tccccctgac
cgccaagcgc ctgacgggca tcgtgtcccg 2820cggcggctcc atccacgcga agtggtgcct
ggcctaccac aaggagaact tcgcctacga 2880gcactgggac gacatcctgg acatctgcaa
ccagtacgac gtcgccctgt ccatcggcga 2940cggcctgcgc cccggctcca tctacgacgc
caacgacacg gcccagttcg ccgagctgct 3000gacccagggc gagctgacgc gccgcgcgtg
ggagaaggac gtgcaggtga tgaacgaggg 3060ccccggccac gtgcccatgc acaagatccc
cgagaacatg cagaagcagc tggagtggtg 3120caacgaggcg cccttctaca ccctgggccc
cctgacgacc gacatcgcgc ccggctacga 3180ccacatcacc tccgccatcg gcgcggccaa
catcggcgcc ctgggcaccg ccctgctgtg 3240ctacgtgacg cccaaggagc acctgggcct
gcccaaccgc gacgacgtga aggcgggcgt 3300catcgcctac aagatcgccg cccacgcggc
cgacctggcc aagcagcacc cccacgccca 3360ggcgtgggac gacgcgctgt ccaaggcgcg
cttcgagttc cgctggatgg accagttcgc 3420gctgtccctg gaccccatga cggcgatgtc
cttccacgac gagacgctgc ccgcggacgg 3480cgcgaaggtc gcccacttct gctccatgtg
cggccccaag ttctgctcca tgaagatcac 3540ggaggacatc cgcaagtacg ccgaggagaa
cggctacggc tccgccgagg aggccatccg 3600ccagggcatg gacgccatgt ccgaggagtt
caacatcgcc aagaagacga tctccggcga 3660gcagcacggc gaggtcggcg gcgagatcta
cctgcccgag tcctacgtca aggccgcgca 3720gaagtgacaa ttggcagcag cagctcggat
agtatcgaca cactctggac gctggtcgtg 3780tgatggactg ttgccgccac acttgctgcc
ttgacctgtg aatatccctg ccgcttttat 3840caaacagcct cagtgtgttt gatcttgtgt
gtacgcgctt ttgcgagttg ctagctgctt 3900gtgctatttg cgaataccac ccccagcatc
cccttccctc gtttcatatc gcttgcatcc 3960caaccgcaac ttatctacgc tgtcctgcta
tccctcagcg ctgctcctgc tcctgctcac 4020tgcccctcgc acagccttgg tttgggctcc
gcctgtattc tcctggtact gcaacctgta 4080aaccagcact gcaatgctga tgcacgggaa
gtagtgggat gggaacacaa atggaggatc 4140ccgcgtctcg aacagagcgc gcagaggaac
gctgaaggtc tcgcctctgt cgcacctcag 4200cgcggcatac accacaataa ccacctgacg
aatgcgcttg gttcttcgtc cattagcgaa 4260gcgtccggtt cacacacgtg ccacgttggc
gaggtggcag gtgacaatga tcggtggagc 4320tgatggtcga aacgttcaca gcctagggat
atcgaattcg gccgacagga cgcgcgtcaa 4380aggtgctggt cgtgtatgcc ctggccggca
ggtcgttgct gctgctggtt agtgattccg 4440caaccctgat tttggcgtct tattttggcg
tggcaaacgc tggcgcccgc gagccgggcc 4500ggcggcgatg cggtgcccca cggctgccgg
aatccaaggg aggcaagagc gcccgggtca 4560gttgaagggc tttacgcgca aggtacagcc
gctcctgcaa ggctgcgtgg tggaattgga 4620cgtgcaggtc ctgctgaagt tcctccaccg
cctcaccagc ggacaaagca ccggtgtatc 4680aggtccgtgt catccactct aaagaactcg
actacgacct actgatggcc ctagattctt 4740catcaaaaac gcctgagaca cttgcccagg
attgaaactc cctgaaggga ccaccagggg 4800ccctgagttg ttccttcccc ccgtggcgag
ctgccagcca ggctgtacct gtgatcgagg 4860ctggcgggaa aataggcttc gtgtgctcag
gtcatgggag gtgcaggaca gctcatgaaa 4920cgccaacaat cgcacaattc atgtcaagct
aatcagctat ttcctcttca cgagctgtaa 4980ttgtcccaaa attctggtct accgggggtg
atccttcgtg tacgggccct tccctcaacc 5040ctaggtatgc gcgcatgcgg tcgccgcgca
actcgcgcga gggccgaggg tttgggacgg 5100gccgtcccga aatgcagttg cacccggatg
cgtggcacct tttttgcgat aatttatgca 5160atggactgct ctgcaaaatt ctggctctgt
cgccaaccct aggatcagcg gcgtaggatt 5220tcgtaatcat tcgtcctgat ggggagctac
cgactaccct aatatcagcc cgactgcctg 5280acgccagcgt ccacttttgt gcacacattc
cattcgtgcc caagacattt cattgtggtg 5340cgaagcgtcc ccagttacgc tcacctgttt
cccgacctcc ttactgttct gtcgacagag 5400cgggcccaca ggccggtcgc agccactagt
atgcagaccg cccaccagcg cccccccacc 5460gagggccact gcttcggcgc ccgcctgccc
accgcctccc gccgcgccgt gcgccgcgcc 5520tggtcccgca tcgcccgcgg gcgcgccgcc
gccgccgccg acgccaaccc cgcccgcccc 5580gagcgccgcg tggtgatcac cggccagggc
gtggtgacct ccctgggcca gaccatcgag 5640cagttctact cctccctgct ggagggcgtg
tccggcatct cccagatcca gaagttcgac 5700accaccggct acaccaccac catcgccggc
gagatcaagt ccctgcagct ggacccctac 5760gtgcccaagc gctgggccaa gcgcgtggac
gacgtgatca agtacgtgta catcgccggc 5820aagcaggccc tggagtccgc cggcctgccc
atcgaggccg ccggcctggc cggcgccggc 5880ctggaccccg ccctgtgcgg cgtgctgatc
ggcaccgcca tggccggcat gacctccttc 5940gccgccggcg tggaggccct gacccgcggc
ggcgtgcgca agatgaaccc cttctgcatc 6000cccttctcca tctccaacat gggcggcgcc
atgctggcca tggacatcgg cttcatgggc 6060cccaactact ccatctccac cgcctgcgcc
accggcaact actgcatcct gggcgccgcc 6120gaccacatcc gccgcggcga cgccaacgtg
atgctggccg gcggcgccga cgccgccatc 6180atcccctccg gcatcggcgg cttcatcgcc
tgcaaggccc tgtccaagcg caacgacgag 6240cccgagcgcg cctcccgccc ctgggacgcc
gaccgcgacg gcttcgtgat gggcgagggc 6300gccggcgtgc tggtgctgga ggagctggag
cacgccaagc gccgcggcgc caccatcctg 6360gccgagctgg tgggcggcgc cgccacctcc
gacgcccacc acatgaccga gcccgacccc 6420cagggccgcg gcgtgcgcct gtgcctggag
cgcgccctgg agcgcgcccg cctggccccc 6480gagcgcgtgg gctacgtgaa cgcccacggc
acctccaccc ccgccggcga cgtggccgag 6540taccgcgcca tccgcgccgt gatcccccag
gactccctgc gcatcaactc caccaagtcc 6600atgatcggcc acctgctggg cggcgccggc
gccgtggagg ccgtggccgc catccaggcc 6660ctgcgcaccg gctggctgca ccccaacctg
aacctggaga accccgcccc cggcgtggac 6720cccgtggtgc tggtgggccc ccgcaaggag
cgcgccgagg acctggacgt ggtgctgtcc 6780aactccttcg gcttcggcgg ccacaactcc
tgcgtgatct tccgcaagta cgacgagatg 6840gactacaagg accacgacgg cgactacaag
gaccacgaca tcgactacaa ggacgacgac 6900gacaagtgaa tcgatagatc tcttaaggca
gcagcagctc ggatagtatc gacacactct 6960ggacgctggt cgtgtgatgg actgttgccg
ccacacttgc tgccttgacc tgtgaatatc 7020cctgccgctt ttatcaaaca gcctcagtgt
gtttgatctt gtgtgtacgc gcttttgcga 7080gttgctagct gcttgtgcta tttgcgaata
ccacccccag catccccttc cctcgtttca 7140tatcgcttgc atcccaaccg caacttatct
acgctgtcct gctatccctc agcgctgctc 7200ctgctcctgc tcactgcccc tcgcacagcc
ttggtttggg ctccgcctgt attctcctgg 7260tactgcaacc tgtaaaccag cactgcaatg
ctgatgcacg ggaagtagtg ggatgggaac 7320acaaatggaa agcttaatta agagctcttg
ttttccagaa ggagttgctc cttgagcctt 7380tcattctcag cctcgataac ctccaaagcc
gctctaattg tggagggggt tcgaagacag 7440ggtggttggc tggatgggga aacgctggtc
gcgggattcg atcctgctgc ttatatcctc 7500cctggaagca cacccacgac tctgaagaag
aaaacgtgca cacacacaac ccaaccggcc 7560gaatatttgc ttccttatcc cgggtccaag
agagactgcg atgcccccct caatcagcat 7620cctcctccct gccgcttcaa tcttccctgc
ttgcctgcgc ccgcggtgcg ccgtctgccc 7680gcccagtcag tcactcctgc acaggcccct
tgtgcgcagt gctcctgtac cctttaccgc 7740tccttccatt ctgcgaggcc ccctattgaa
tgtattcgtt gcctgtgtgg ccaagcgggc 7800tgctgggcgc gccgccgtcg ggcagtgctc
ggcgactttg gcggaagccg attgttcttc 7860tgtaagccac gcgcttgctg ctttgggaag
agaagggggg gggtactgaa tggatgagga 7920ggagaaggag gggtattggt attatctgag
ttgggtgaag agc 79631197887DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
119gctcttcgga gtcactgtgc cactgagttc gactggtagc tgaatggagt cgctgctcca
60ctaaacgaat tgtcagcacc gccagccggc cgaggacccg agtcatagcg agggtagtag
120cgcgccatgg caccgaccag cctgcttgcc agtactggcg tctcttccgc ttctctgtgg
180tcctctgcgc gctccagcgc gtgcgctttt ccggtggatc atgcggtccg tggcgcaccg
240cagcggccgc tgcccatgca gcgccgctgc ttccgaacag tggcggtcag ggccgcaccc
300gcggtagccg tccgtccgga acccgcccaa gagttttggg agcagcttga gccctgcaag
360atggcggagg acaagcgcat cttcctggag gagcaccggt gcgtggaggt ccggggctga
420ccggccgtcg cattcaacgt aatcaatcgc atgatgatca gaggacacga agtcttggtg
480gcggtggcca gaaacactgt ccattgcaag ggcataggga tgcgttcctt cacctctcat
540ttctcatttc tgaatccctc cctgctcact ctttctcctc ctccttcccg ttcacgcagc
600attcggggta ccagtttagg tccagcgtcc gtgggggggg acgggctggg agcttgggcc
660gggaagggca agacgatgca gtccctctgg ggagtcacag ccgactgtgt gtgttgcact
720gtgcggcccg cagcactcac acgcaaaatg cctggccgac aggcaggccc tgtccagtgc
780aacatccacg gtccctctca tcaggctcac cttgctcatt gacataacgg aatgcgtacc
840gctctttcag atctgtccat ccagagaggg gagcaggctc cccaccgacg ctgtcaaact
900tgcttcctgc ccaaccgaaa acattattgt ttgagggggg gggggggggg gcagattgca
960tggcgggata tctcgtgagg aacatcactg ggacactgtg gaacacagtg agtgcagtat
1020gcagagcatg tatgctaggg gtcagcgcag gaagggggcc tttcccagtc tcccatgcca
1080ctgcaccgta tccacgactc accaggacca gcttcttgat cggcttccgc tcccgtggac
1140accagtgtgt agcctctgga ctccaggtat gcgtgcaccg caaaggccag ccgatcgtgc
1200cgattcctgg ggtggaggat atgagtcagc caacttgggg ctcagagtgc acactggggc
1260acgatacgaa acaacatcta caccgtgtcc tccatgctga cacaccacag cttcgctcca
1320cctgaatgtg ggcgcatggg cccgaatcac agccaatgtc gctgctgcca taatgtgatc
1380cagaccctct ccgcccagat gccgagcgga tcgtgggcgc tgaatagatt cctgtttcga
1440tcactgtttg ggtcctttcc ttttcgtctc ggatgcgcgt ctcgaaacag gctgcgtcgg
1500gctttcggat cccttttgct ccctccgtca ccatcctgcg cgcgggcaag ttgcttgacc
1560ctgggctgta ccagggttgg agggtattac cgcgtcaggc cattcccagc ccggattcaa
1620ttcaaagtct gggccaccac cctccgccgc tctgtctgat cactccacat tcgtgcatac
1680actacgttca agtcctgatc caggcgtgtc tcgggacaag gtgtgcttga gtttgaatct
1740caaggaccca ctccagcaca gctgctggtt gaccccgccc tcgcaatcta gaatggccgc
1800gtccgtccac tgcaccctga tgtccgtggt ctgcaacaac aagaaccact ccgcccgccc
1860caagctgccc aactcctccc tgctgcccgg cttcgacgtg gtggtccagg ccgcggccac
1920ccgcttcaag aaggagacga cgaccacccg cgccacgctg acgttcgacc cccccacgac
1980caactccgag cgcgccaagc agcgcaagca caccatcgac ccctcctccc ccgacttcca
2040gcccatcccc tccttcgagg agtgcttccc caagtccacg aaggagcaca aggaggtggt
2100gcacgaggag tccggccacg tcctgaaggt gcccttccgc cgcgtgcacc tgtccggcgg
2160cgagcccgcc ttcgacaact acgacacgtc cggcccccag aacgtcaacg cccacatcgg
2220cctggcgaag ctgcgcaagg agtggatcga ccgccgcgag aagctgggca cgccccgcta
2280cacgcagatg tactacgcga agcagggcat catcacggag gagatgctgt actgcgcgac
2340gcgcgagaag ctggaccccg agttcgtccg ctccgaggtc gcgcggggcc gcgccatcat
2400cccctccaac aagaagcacc tggagctgga gcccatgatc gtgggccgca agttcctggt
2460gaaggtgaac gcgaacatcg gcaactccgc cgtggcctcc tccatcgagg aggaggtcta
2520caaggtgcag tgggccacca tgtggggcgc cgacaccatc atggacctgt ccacgggccg
2580ccacatccac gagacgcgcg agtggatcct gcgcaactcc gcggtccccg tgggcaccgt
2640ccccatctac caggcgctgg agaaggtgga cggcatcgcg gagaacctga actgggaggt
2700gttccgcgag acgctgatcg agcaggccga gcagggcgtg gactacttca cgatccacgc
2760gggcgtgctg ctgcgctaca tccccctgac cgccaagcgc ctgacgggca tcgtgtcccg
2820cggcggctcc atccacgcga agtggtgcct ggcctaccac aaggagaact tcgcctacga
2880gcactgggac gacatcctgg acatctgcaa ccagtacgac gtcgccctgt ccatcggcga
2940cggcctgcgc cccggctcca tctacgacgc caacgacacg gcccagttcg ccgagctgct
3000gacccagggc gagctgacgc gccgcgcgtg ggagaaggac gtgcaggtga tgaacgaggg
3060ccccggccac gtgcccatgc acaagatccc cgagaacatg cagaagcagc tggagtggtg
3120caacgaggcg cccttctaca ccctgggccc cctgacgacc gacatcgcgc ccggctacga
3180ccacatcacc tccgccatcg gcgcggccaa catcggcgcc ctgggcaccg ccctgctgtg
3240ctacgtgacg cccaaggagc acctgggcct gcccaaccgc gacgacgtga aggcgggcgt
3300catcgcctac aagatcgccg cccacgcggc cgacctggcc aagcagcacc cccacgccca
3360ggcgtgggac gacgcgctgt ccaaggcgcg cttcgagttc cgctggatgg accagttcgc
3420gctgtccctg gaccccatga cggcgatgtc cttccacgac gagacgctgc ccgcggacgg
3480cgcgaaggtc gcccacttct gctccatgtg cggccccaag ttctgctcca tgaagatcac
3540ggaggacatc cgcaagtacg ccgaggagaa cggctacggc tccgccgagg aggccatccg
3600ccagggcatg gacgccatgt ccgaggagtt caacatcgcc aagaagacga tctccggcga
3660gcagcacggc gaggtcggcg gcgagatcta cctgcccgag tcctacgtca aggccgcgca
3720gaagtgacaa ttggcagcag cagctcggat agtatcgaca cactctggac gctggtcgtg
3780tgatggactg ttgccgccac acttgctgcc ttgacctgtg aatatccctg ccgcttttat
3840caaacagcct cagtgtgttt gatcttgtgt gtacgcgctt ttgcgagttg ctagctgctt
3900gtgctatttg cgaataccac ccccagcatc cccttccctc gtttcatatc gcttgcatcc
3960caaccgcaac ttatctacgc tgtcctgcta tccctcagcg ctgctcctgc tcctgctcac
4020tgcccctcgc acagccttgg tttgggctcc gcctgtattc tcctggtact gcaacctgta
4080aaccagcact gcaatgctga tgcacgggaa gtagtgggat gggaacacaa atggaggatc
4140ccgcgtctcg aacagagcgc gcagaggaac gctgaaggtc tcgcctctgt cgcacctcag
4200cgcggcatac accacaataa ccacctgacg aatgcgcttg gttcttcgtc cattagcgaa
4260gcgtccggtt cacacacgtg ccacgttggc gaggtggcag gtgacaatga tcggtggagc
4320tgatggtcga aacgttcaca gcctagggat atcatagcga ctgctacccc ccgaccatgt
4380gccgaggcag aaattatata caagaagcag atcgcaatta ggcacatcgc tttgcattat
4440ccacacacta ttcatcgctg ctgcggcaag gctgcagagt gtatttttgt ggcccaggag
4500ctgagtccga agtcgacgcg acgagcggcg caggatccga cccctagacg agctctgtca
4560ttttccaagc acgcagctaa atgcgctgag accgggtcta aatcatccga aaagtgtcaa
4620aatggccgat tgggttcgcc taggacaatg cgctgcggat tcgctcgagt ccgctgccgg
4680ccaaaaggcg gtggtacagg aaggcgcacg gggccaaccc tgcgaagccg ggggcccgaa
4740cgccgaccgc cggccttcga tctcgggtgt ccccctcgtc aatttcctct ctcgggtgca
4800gccacgaaag tcgtgacgca ggtcacgaaa tccggttacg aaaaacgcag gtcttcgcaa
4860aaacgtgagg gtttcgcgtc tcgccctagc tattcgtatc gccgggtcag acccacgtgc
4920agaaaagccc ttgaataacc cgggaccgtg gttaccgcgc cgcctgcacc agggggctta
4980tataagccca caccacacct gtctcaccac gcatttctcc aactcgcgac ttttcggaag
5040aaattgttat ccacctagta tagactgcca cctgcaggac cttgtgtctt gcagtttgta
5100ttggtcccgg ccgtcgagct cgacagatct gggctagggt tggcctggcc gctcggcact
5160cccctttagc cgcgcgcatc cgcgttccag aggtgcgatt cggtgtgtgg agcattgtca
5220tgcgcttgtg ggggtcgttc cgtgcgcggc gggtccgcca tgggcgccga cctgggccct
5280agggtttgtt ttcgggccaa gcgagcccct ctcacctcgt cgcccccccg cattccctct
5340ctcttgcagc cactagtatg gccaccgcat ccactttctc ggcgttcaat gcccgctgcg
5400gcgacctgcg tcgctcggcg ggctccgggc cccggcgccc agcgaggccc ctccccgtgc
5460gcgggcgcgc cgccgccgcc gccgacgcca accccgcccg ccccgagcgc cgcgtggtga
5520tcaccggcca gggcgtggtg acctccctgg gccagaccat cgagcagttc tactcctccc
5580tgctggaggg cgtgtccggc atctcccaga tccagaagtt cgacaccacc ggctacacca
5640ccaccatcgc cggcgagatc aagtccctgc agctggaccc ctacgtgccc aagcgctggg
5700ccaagcgcgt ggacgacgtg atcaagtacg tgtacatcgc cggcaagcag gccctggagt
5760ccgccggcct gcccatcgag gccgccggcc tggccggcgc cggcctggac cccgccctgt
5820gcggcgtgct gatcggcacc gccatggccg gcatgacctc cttcgccgcc ggcgtggagg
5880ccctgacccg cggcggcgtg cgcaagatga accccttctg catccccttc tccatctcca
5940acatgggcgg cgccatgctg gccatggaca tcggcttcat gggccccaac tactccatct
6000ccaccgcctg cgccaccggc aactactgca tcctgggcgc cgccgaccac atccgccgcg
6060gcgacgccaa cgtgatgctg gccggcggcg ccgacgccgc catcatcccc tccggcatcg
6120gcggcttcat cgcctgcaag gccctgtcca agcgcaacga cgagcccgag cgcgcctccc
6180gcccctggga cgccgaccgc gacggcttcg tgatgggcga gggcgccggc gtgctggtgc
6240tggaggagct ggagcacgcc aagcgccgcg gcgccaccat cctggccgag ctggtgggcg
6300gcgccgccac ctccgacgcc caccacatga ccgagcccga cccccagggc cgcggcgtgc
6360gcctgtgcct ggagcgcgcc ctggagcgcg cccgcctggc ccccgagcgc gtgggctacg
6420tgaacgccca cggcacctcc acccccgccg gcgacgtggc cgagtaccgc gccatccgcg
6480ccgtgatccc ccaggactcc ctgcgcatca actccaccaa gtccatgatc ggccacctgc
6540tgggcggcgc cggcgccgtg gaggccgtgg ccgccatcca ggccctgcgc accggctggc
6600tgcaccccaa cctgaacctg gagaaccccg cccccggcgt ggaccccgtg gtgctggtgg
6660gcccccgcaa ggagcgcgcc gaggacctgg acgtggtgct gtccaactcc ttcggcttcg
6720gcggccacaa ctcctgcgtg atcttccgca agtacgacga gatggactac aaggaccacg
6780acggcgacta caaggaccac gacatcgact acaaggacga cgacgacaag tgaatcgata
6840gatctcttaa ggcagcagca gctcggatag tatcgacaca ctctggacgc tggtcgtgtg
6900atggactgtt gccgccacac ttgctgcctt gacctgtgaa tatccctgcc gcttttatca
6960aacagcctca gtgtgtttga tcttgtgtgt acgcgctttt gcgagttgct agctgcttgt
7020gctatttgcg aataccaccc ccagcatccc cttccctcgt ttcatatcgc ttgcatccca
7080accgcaactt atctacgctg tcctgctatc cctcagcgct gctcctgctc ctgctcactg
7140cccctcgcac agccttggtt tgggctccgc ctgtattctc ctggtactgc aacctgtaaa
7200ccagcactgc aatgctgatg cacgggaagt agtgggatgg gaacacaaat ggaaagctta
7260attaagagct cttgttttcc agaaggagtt gctccttgag cctttcattc tcagcctcga
7320taacctccaa agccgctcta attgtggagg gggttcgaag acagggtggt tggctggatg
7380gggaaacgct ggtcgcggga ttcgatcctg ctgcttatat cctccctgga agcacaccca
7440cgactctgaa gaagaaaacg tgcacacaca caacccaacc ggccgaatat ttgcttcctt
7500atcccgggtc caagagagac tgcgatgccc ccctcaatca gcatcctcct ccctgccgct
7560tcaatcttcc ctgcttgcct gcgcccgcgg tgcgccgtct gcccgcccag tcagtcactc
7620ctgcacaggc cccttgtgcg cagtgctcct gtacccttta ccgctccttc cattctgcga
7680ggccccctat tgaatgtatt cgttgcctgt gtggccaagc gggctgctgg gcgcgccgcc
7740gtcgggcagt gctcggcgac tttggcggaa gccgattgtt cttctgtaag ccacgcgctt
7800gctgctttgg gaagagaagg gggggggtac tgaatggatg aggaggagaa ggaggggtat
7860tggtattatc tgagttgggt gaagagc
78871207072DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 120gctcttcgga gtcactgtgc cactgagttc
gactggtagc tgaatggagt cgctgctcca 60ctaaacgaat tgtcagcacc gccagccggc
cgaggacccg agtcatagcg agggtagtag 120cgcgccatgg caccgaccag cctgcttgcc
agtactggcg tctcttccgc ttctctgtgg 180tcctctgcgc gctccagcgc gtgcgctttt
ccggtggatc atgcggtccg tggcgcaccg 240cagcggccgc tgcccatgca gcgccgctgc
ttccgaacag tggcggtcag ggccgcaccc 300gcggtagccg tccgtccgga acccgcccaa
gagttttggg agcagcttga gccctgcaag 360atggcggagg acaagcgcat cttcctggag
gagcaccggt gcgtggaggt ccggggctga 420ccggccgtcg cattcaacgt aatcaatcgc
atgatgatca gaggacacga agtcttggtg 480gcggtggcca gaaacactgt ccattgcaag
ggcataggga tgcgttcctt cacctctcat 540ttctcatttc tgaatccctc cctgctcact
ctttctcctc ctccttcccg ttcacgcagc 600attcggggta ccagtttagg tccagcgtcc
gtgggggggg acgggctggg agcttgggcc 660gggaagggca agacgatgca gtccctctgg
ggagtcacag ccgactgtgt gtgttgcact 720gtgcggcccg cagcactcac acgcaaaatg
cctggccgac aggcaggccc tgtccagtgc 780aacatccacg gtccctctca tcaggctcac
cttgctcatt gacataacgg aatgcgtacc 840gctctttcag atctgtccat ccagagaggg
gagcaggctc cccaccgacg ctgtcaaact 900tgcttcctgc ccaaccgaaa acattattgt
ttgagggggg gggggggggg gcagattgca 960tggcgggata tctcgtgagg aacatcactg
ggacactgtg gaacacagtg agtgcagtat 1020gcagagcatg tatgctaggg gtcagcgcag
gaagggggcc tttcccagtc tcccatgcca 1080ctgcaccgta tccacgactc accaggacca
gcttcttgat cggcttccgc tcccgtggac 1140accagtgtgt agcctctgga ctccaggtat
gcgtgcaccg caaaggccag ccgatcgtgc 1200cgattcctgg ggtggaggat atgagtcagc
caacttgggg ctcagagtgc acactggggc 1260acgatacgaa acaacatcta caccgtgtcc
tccatgctga cacaccacag cttcgctcca 1320cctgaatgtg ggcgcatggg cccgaatcac
agccaatgtc gctgctgcca taatgtgatc 1380cagaccctct ccgcccagat gccgagcgga
tcgtgggcgc tgaatagatt cctgtttcga 1440tcactgtttg ggtcctttcc ttttcgtctc
ggatgcgcgt ctcgaaacag gctgcgtcgg 1500gctttcggat cccttttgct ccctccgtca
ccatcctgcg cgcgggcaag ttgcttgacc 1560ctgggctgta ccagggttgg agggtattac
cgcgtcaggc cattcccagc ccggattcaa 1620ttcaaagtct gggccaccac cctccgccgc
tctgtctgat cactccacat tcgtgcatac 1680actacgttca agtcctgatc caggcgtgtc
tcgggacaag gtgtgcttga gtttgaatct 1740caaggaccca ctccagcaca gctgctggtt
gaccccgccc tcgcaatcta gaatgttcgc 1800gttctacttc ctgacggcct gcatctccct
gaagggcgtg ttcggcgtct ccccctccta 1860caacggcctg ggcctgacgc cccagatggg
ctgggacaac tggaacacgt tcgcctgcga 1920cgtctccgag cagctgctgc tggacacggc
cgaccgcatc tccgacctgg gcctgaagga 1980catgggctac aagtacatca tcctggacga
ctgctggtcc tccggccgcg actccgacgg 2040cttcctggtc gccgacgagc agaagttccc
caacggcatg ggccacgtcg ccgaccacct 2100gcacaacaac tccttcctgt tcggcatgta
ctcctccgcg ggcgagtaca cgtgcgccgg 2160ctaccccggc tccctgggcc gcgaggagga
ggacgcccag ttcttcgcga acaaccgcgt 2220ggactacctg aagtacgaca actgctacaa
caagggccag ttcggcacgc ccgagatctc 2280ctaccaccgc tacaaggcca tgtccgacgc
cctgaacaag acgggccgcc ccatcttcta 2340ctccctgtgc aactggggcc aggacctgac
cttctactgg ggctccggca tcgcgaactc 2400ctggcgcatg tccggcgacg tcacggcgga
gttcacgcgc cccgactccc gctgcccctg 2460cgacggcgac gagtacgact gcaagtacgc
cggcttccac tgctccatca tgaacatcct 2520gaacaaggcc gcccccatgg gccagaacgc
gggcgtcggc ggctggaacg acctggacaa 2580cctggaggtc ggcgtcggca acctgacgga
cgacgaggag aaggcgcact tctccatgtg 2640ggccatggtg aagtcccccc tgatcatcgg
cgcgaacgtg aacaacctga aggcctcctc 2700ctactccatc tactcccagg cgtccgtcat
cgccatcaac caggactcca acggcatccc 2760cgccacgcgc gtctggcgct actacgtgtc
cgacacggac gagtacggcc agggcgagat 2820ccagatgtgg tccggccccc tggacaacgg
cgaccaggtc gtggcgctgc tgaacggcgg 2880ctccgtgtcc cgccccatga acacgaccct
ggaggagatc ttcttcgact ccaacctggg 2940ctccaagaag ctgacctcca cctgggacat
ctacgacctg tgggcgaacc gcgtcgacaa 3000ctccacggcg tccgccatcc tgggccgcaa
caagaccgcc accggcatcc tgtacaacgc 3060caccgagcag tcctacaagg acggcctgtc
caagaacgac acccgcctgt tcggccagaa 3120gatcggctcc ctgtccccca acgcgatcct
gaacacgacc gtccccgccc acggcatcgc 3180gttctaccgc ctgcgcccct cctcctgaca
attggcagca gcagctcgga tagtatcgac 3240acactctgga cgctggtcgt gtgatggact
gttgccgcca cacttgctgc cttgacctgt 3300gaatatccct gccgctttta tcaaacagcc
tcagtgtgtt tgatcttgtg tgtacgcgct 3360tttgcgagtt gctagctgct tgtgctattt
gcgaatacca cccccagcat ccccttccct 3420cgtttcatat cgcttgcatc ccaaccgcaa
cttatctacg ctgtcctgct atccctcagc 3480gctgctcctg ctcctgctca ctgcccctcg
cacagccttg gtttgggctc cgcctgtatt 3540ctcctggtac tgcaacctgt aaaccagcac
tgcaatgctg atgcacggga agtagtggga 3600tgggaacaca aatggaggat cccgcgtctc
gaacagagcg cgcagaggaa cgctgaaggt 3660ctcgcctctg tcgcacctca gcgcggcata
caccacaata accacctgac gaatgcgctt 3720ggttcttcgt ccattagcga agcgtccggt
tcacacacgt gccacgttgg cgaggtggca 3780ggtgacaatg atcggtggag ctgatggtcg
aaacgttcac agcctaggga tatcgaattc 3840ggccgacagg acgcgcgtca aaggtgctgg
tcgtgtatgc cctggccggc aggtcgttgc 3900tgctgctggt tagtgattcc gcaaccctga
ttttggcgtc ttattttggc gtggcaaacg 3960ctggcgcccg cgagccgggc cggcggcgat
gcggtgcccc acggctgccg gaatccaagg 4020gaggcaagag cgcccgggtc agttgaaggg
ctttacgcgc aaggtacagc cgctcctgca 4080aggctgcgtg gtggaattgg acgtgcaggt
cctgctgaag ttcctccacc gcctcaccag 4140cggacaaagc accggtgtat caggtccgtg
tcatccactc taaagaactc gactacgacc 4200tactgatggc cctagattct tcatcaaaaa
cgcctgagac acttgcccag gattgaaact 4260ccctgaaggg accaccaggg gccctgagtt
gttccttccc cccgtggcga gctgccagcc 4320aggctgtacc tgtgatcgag gctggcggga
aaataggctt cgtgtgctca ggtcatggga 4380ggtgcaggac agctcatgaa acgccaacaa
tcgcacaatt catgtcaagc taatcagcta 4440tttcctcttc acgagctgta attgtcccaa
aattctggtc taccgggggt gatccttcgt 4500gtacgggccc ttccctcaac cctaggtatg
cgcgcatgcg gtcgccgcgc aactcgcgcg 4560agggccgagg gtttgggacg ggccgtcccg
aaatgcagtt gcacccggat gcgtggcacc 4620ttttttgcga taatttatgc aatggactgc
tctgcaaaat tctggctctg tcgccaaccc 4680taggatcagc ggcgtaggat ttcgtaatca
ttcgtcctga tggggagcta ccgactaccc 4740taatatcagc ccgactgcct gacgccagcg
tccacttttg tgcacacatt ccattcgtgc 4800ccaagacatt tcattgtggt gcgaagcgtc
cccagttacg ctcacctgtt tcccgacctc 4860cttactgttc tgtcgacaga gcgggcccac
aggccggtcg cagccactag tatggccatc 4920aagaccaacc gccagcccgt ggagaagccc
cccttcacca tcggcaccct gcgcaaggcc 4980atccccgccc actgcttcga gcgctccgcc
ctgcgctcct ccatgtacct ggccttcgac 5040atcgccgtga tgtccctgct gtacgtggcc
tccacctaca tcgaccccgc ccccgtgccc 5100acctgggtga agtacggcgt gatgtggccc
ctgtactggt tcttccaggg cgccttcggc 5160accggcgtgt gggtgtgcgc ccacgagtgc
ggccaccagg ccttctcctc ctcccaggcc 5220atcaacgacg gcgtgggcct ggtgttccac
tccctgctgc tggtgcccta ctactcctgg 5280aagcactccc accgccgcca ccactccaac
accggctgcc tggacaagga cgaggtgttc 5340gtgccccccc accgcgccgt ggcccacgag
ggcctggagt gggaggagtg gctgcccatc 5400cgcatgggca aggtgctggt gaccctgacc
ctgggctggc ccctgtacct gatgttcaac 5460gtggcctccc gcccctaccc ccgcttcgcc
aaccacttcg acccctggtc ccccatcttc 5520tccaagcgcg agcgcatcga ggtggtgatc
tccgacctgg ccctggtggc cgtgctgtcc 5580ggcctgtccg tgctgggccg caccatgggc
tgggcctggc tggtgaagac ctacgtggtg 5640ccctacctga tcgtgaacat gtggctggtg
ctgatcaccc tgctgcagca cacccacccc 5700gccctgcccc actacttcga gaaggactgg
gactggctgc gcggcgccat ggccaccgtg 5760gaccgctcca tgggcccccc cttcatggac
aacatcctgc accacatctc cgacacccac 5820gtgctgcacc acctgttctc caccatcccc
cactaccacg ccgaggaggc ctccgccgcc 5880atccgcccca tcctgggcaa gtactaccag
tccgactccc gctgggtggg ccgcgccctg 5940tgggaggact ggcgcgactg ccgctacgtg
gtgcccgacg cccccgagga cgactccgcc 6000ctgtggttcc acaagtagat cgatagatct
cttaaggcag cagcagctcg gatagtatcg 6060acacactctg gacgctggtc gtgtgatgga
ctgttgccgc cacacttgct gccttgacct 6120gtgaatatcc ctgccgcttt tatcaaacag
cctcagtgtg tttgatcttg tgtgtacgcg 6180cttttgcgag ttgctagctg cttgtgctat
ttgcgaatac cacccccagc atccccttcc 6240ctcgtttcat atcgcttgca tcccaaccgc
aacttatcta cgctgtcctg ctatccctca 6300gcgctgctcc tgctcctgct cactgcccct
cgcacagcct tggtttgggc tccgcctgta 6360ttctcctggt actgcaacct gtaaaccagc
actgcaatgc tgatgcacgg gaagtagtgg 6420gatgggaaca caaatggaaa gcttaattaa
gagctcttgt tttccagaag gagttgctcc 6480ttgagccttt cattctcagc ctcgataacc
tccaaagccg ctctaattgt ggagggggtt 6540cgaagacagg gtggttggct ggatggggaa
acgctggtcg cgggattcga tcctgctgct 6600tatatcctcc ctggaagcac acccacgact
ctgaagaaga aaacgtgcac acacacaacc 6660caaccggccg aatatttgct tccttatccc
gggtccaaga gagactgcga tgcccccctc 6720aatcagcatc ctcctccctg ccgcttcaat
cttccctgct tgcctgcgcc cgcggtgcgc 6780cgtctgcccg cccagtcagt cactcctgca
caggcccctt gtgcgcagtg ctcctgtacc 6840ctttaccgct ccttccattc tgcgaggccc
cctattgaat gtattcgttg cctgtgtggc 6900caagcgggct gctgggcgcg ccgccgtcgg
gcagtgctcg gcgactttgg cggaagccga 6960ttgttcttct gtaagccacg cgcttgctgc
tttgggaaga gaaggggggg ggtactgaat 7020ggatgaggag gagaaggagg ggtattggta
ttatctgagt tgggtgaaga gc 70721218834DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
121gtttaaaccc ctcaactgcg acgctgggaa ccttctccgg gcaggcgatg tgcgtgggtt
60tgcctccttg gcacggctct acaccttcga gtacgccatg aggcggtgat ggctgtggct
120gtgccccact tcgtccaggg acggcaagtc catcatctgc atgcttggtg cgacgctaca
180gcagtccctc tgcagcagag gagcacgact ttggccattt cacgcactcg agtgtacaca
240attcattttt cttaaagtaa atgactgctg attgaccaga tgctgtaacg ctgatttcgc
300tccagatcgc acagtcacag attgcgacca tgttgctgcg tctgaaaatc tggattccga
360attcgaccct ggcgctccat ccatgcaaca gatggcgaca cttgttacaa ttcctgtcgc
420ccatcggcat ggagcaggtc cacttagatc cccgatcacc cacgcgcatc tcgctaatag
480tcattcattc gtgtcttcga tcaaagtcag gtgagtatgc atggatcttg gttgacgatg
540cggtatgggt ttgcgccgct gactgcaggg tctgtccaag gcaagccaac ccagctcctc
600tcctcgacaa tactctcgca gacaaagcca gccacttgcc atccagattg ccaataaact
660caatcatggc ttctgtcatg ccatccatgg gtctgatgaa tggtcacgct cgtgtcctga
720ccgttcccca gcctctggcg tcccctgccc cgcccaccag cccacgccgc gcggcagtcg
780ctgccaaggc tgtctcggag gtaccctttc ttgcgctatg acacttccag caaaaggtag
840ggcgggctgc gagacggctt cccggcgctg catgcaacac cgatgatgct tcgacccccc
900gaagctcctt cggggctgca tgggcgctcc gatgccgctc cagggcgagc gctgtttaaa
960tagccaggcc cccgattgca aagacattat agcgagctac caaagccata ttcaaacacc
1020tagatcacta ccacttctac acaggccact cgagcttgtg atcgcactcc gctaaggggg
1080cgcctcttcc tcttcgtttc agtcacaacc cgcaaactct agaatatcaa tgatcgagca
1140ggacggcctc cacgccggct cccccgccgc ctgggtggag cgcctgttcg gctacgactg
1200ggcccagcag accatcggct gctccgacgc cgccgtgttc cgcctgtccg cccagggccg
1260ccccgtgctg ttcgtgaaga ccgacctgtc cggcgccctg aacgagctgc aggacgaggc
1320cgcccgcctg tcctggctgg ccaccaccgg cgtgccctgc gccgccgtgc tggacgtggt
1380gaccgaggcc ggccgcgact ggctgctgct gggcgaggtg cccggccagg acctgctgtc
1440ctcccacctg gcccccgccg agaaggtgtc catcatggcc gacgccatgc gccgcctgca
1500caccctggac cccgccacct gccccttcga ccaccaggcc aagcaccgca tcgagcgcgc
1560ccgcacccgc atggaggccg gcctggtgga ccaggacgac ctggacgagg agcaccaggg
1620cctggccccc gccgagctgt tcgcccgcct gaaggcccgc atgcccgacg gcgaggacct
1680ggtggtgacc cacggcgacg cctgcctgcc caacatcatg gtggagaacg gccgcttctc
1740cggcttcatc gactgcggcc gcctgggcgt ggccgaccgc taccaggaca tcgccctggc
1800cacccgcgac atcgccgagg agctgggcgg cgagtgggcc gaccgcttcc tggtgctgta
1860cggcatcgcc gcccccgact cccagcgcat cgccttctac cgcctgctgg acgagttctt
1920ctgacaattg gcagcagcag ctcggatagt atcgacacac tctggacgct ggtcgtgtga
1980tggactgttg ccgccacact tgctgccttg acctgtgaat atccctgccg cttttatcaa
2040acagcctcag tgtgtttgat cttgtgtgta cgcgcttttg cgagttgcta gctgcttgtg
2100ctatttgcga ataccacccc cagcatcccc ttccctcgtt tcatatcgct tgcatcccaa
2160ccgcaactta tctacgctgt cctgctatcc ctcagcgctg ctcctgctcc tgctcactgc
2220ccctcgcaca gccttggttt gggctccgcc tgtattctcc tggtactgca acctgtaaac
2280cagcactgca atgctgatgc acgggaagta gtgggatggg aacacaaatg gaaagctgta
2340tagggataag aattcggccg acaggacgcg cgtcaaaggt gctggtcgtg tatgccctgg
2400ccggcaggtc gttgctgctg ctggttagtg attccgcaac cctgattttg gcgtcttatt
2460ttggcgtggc aaacgctggc gcccgcgagc cgggccggcg gcgatgcggt gccccacggc
2520tgccggaatc caagggaggc aagagcgccc gggtcagttg aagggcttta cgcgcaaggt
2580acagccgctc ctgcaaggct gcgtggtgga attggacgtg caggtcctgc tgaagttcct
2640ccaccgcctc accagcggac aaagcaccgg tgtatcaggt ccgtgtcatc cactctaaag
2700aactcgacta cgacctactg atggccctag attcttcatc aaaaacgcct gagacacttg
2760cccaggattg aaactccctg aagggaccac caggggccct gagttgttcc ttccccccgt
2820ggcgagctgc cagccaggct gtacctgtga tcgaggctgg cgggaaaata ggcttcgtgt
2880gctcaggtca tgggaggtgc aggacagctc atgaaacgcc aacaatcgca caattcatgt
2940caagctaatc agctatttcc tcttcacgag ctgtaattgt cccaaaattc tggtctaccg
3000ggggtgatcc ttcgtgtacg ggcccttccc tcaaccctag gtatgcgcgc atgcggtcgc
3060cgcgcaactc gcgcgagggc cgagggtttg ggacgggccg tcccgaaatg cagttgcacc
3120cggatgcgtg gcaccttttt tgcgataatt tatgcaatgg actgctctgc aaaattctgg
3180ctctgtcgcc aaccctagga tcagcggcgt aggatttcgt aatcattcgt cctgatgggg
3240agctaccgac taccctaata tcagcccgac tgcctgacgc cagcgtccac ttttgtgcac
3300acattccatt cgtgcccaag acatttcatt gtggtgcgaa gcgtccccag ttacgctcac
3360ctgtttcccg acctccttac tgttctgtcg acagagcggg cccacaggcc ggtcgcagcc
3420actagtgcga ccgccagctg catggtggcg tcgcccttct gcacctggct ggtcgccgcg
3480tgcatgccca cctccagcga caacgacccc cgctcgctgt cccacaagcg cctgcgcctg
3540agccgccgcc gccgcaccct gagctcgcac tgctccctgc gcggcagcac cttccagtgc
3600ctggacccct gcaaccagca gcgcttcctg ggcgacaacg gcttcgcgtc gctgttcggc
3660tccaagcccc tgcgcagcaa ccgcggccac ctgcgcctgg gccgcacctc gcactccggc
3720gaggtgatgg ccgtcgcgat gcagcccgcc caggaggtga gcaccaacaa gaagcccgcg
3780accaagcagc gccgcgtggt cgtgaccggc atgggcgtcg tgacccccct gggccacgac
3840cccgacgtgt attataacaa cctgctggac ggcatctcgg gcatctccga gatcgagaac
3900ttcgactgca gccagttccc cacccgcatc gccggcgaga tcaagtcgtt ctccaccgac
3960ggctgggtcg cgcccaagtt cagcgagcgc atggacaagt tcatgctgta tatgctgacc
4020gccggcaaga aggcgctggc cgacggcggc atcaccgagg acgcgatgaa ggagctgaac
4080aagcgcaagt gcggcgtgct gatcggctcg ggcctgggcg gcatgaaggt cttctccgac
4140agcatcgagg ccctgcgcac ctcgtataag aagatctccc ccttctgcgt gcccttcagc
4200accaccaaca tgggctcggc gatcctggcg atggacctgg gctggatggg ccccaactat
4260tccatcagca ccgcgtgcgc cacctcgaac ttctgcatcc tgaacgcggc caaccacatc
4320atcaagggcg aggcggacat gatgctgtgc ggcggctccg acgccgcggt gctgcccgtc
4380ggcctgggcg gcttcgtggc ctgccgcgcg ctgagccagc gcaacaacga ccccaccaag
4440gcctcgcgcc cctgggactc caaccgcgac ggcttcgtca tgggcgaggg cgcgggcgtg
4500ctgctgctgg aggagctgga gcacgccaag aagcgcggcg cgaccatcta tgccgagttc
4560ctgggcggca gcttcacctg cgacgcgtat cacatgaccg agccccaccc cgagggcgcc
4620ggcgtcatcc tgtgcatcga gaaggcgctg gcccagtcgg gcgtgtcccg cgaggacgtg
4680aactatatca acgcgcacgc caccagcacc cccgcgggcg acatcaagga gtatcaggcc
4740ctggcgcact gcttcggcca gaactcggag ctgcgcgtca actccaccaa gagcatgatc
4800ggccacctgc tgggcggcgc cggcggcgtg gaggcggtcg ccgtggtcca ggcgatccgc
4860accggctgga tccaccccaa catcaacctg gaggaccccg acgagggcgt ggacgccaag
4920ctgctggtcg gccccaagaa ggagaagctg aaggtgaagg tcggcctgtc gaactccttc
4980ggcttcggcg gccacaacag ctcgatcctg ttcgcgccct gcaactgact cgaggcagca
5040gcagctcgga tagtatcgac acactctgga cgctggtcgt gtgatggact gttgccgcca
5100cacttgctgc cttgacctgt gaatatccct gccgctttta tcaaacagcc tcagtgtgtt
5160tgatcttgtg tgtacgcgct tttgcgagtt gctagctgct tgtgctattt gcgaatacca
5220cccccagcat ccccttccct cgtttcatat cgcttgcatc ccaaccgcaa cttatctacg
5280ctgtcctgct atccctcagc gctgctcctg ctcctgctca ctgcccctcg cacagccttg
5340gtttgggctc cgcctgtatt ctcctggtac tgcaacctgt aaaccagcac tgcaatgctg
5400atgcacggga agtagtggga tgggaacaca aatggaaagc ttcacatacg taggccgaca
5460ggacgcgcgt caaaggtgct ggtcgtgtat gccctggccg gcaggtcgtt gctgctgctg
5520gttagtgatt ccgcaaccct gattttggcg tcttattttg gcgtggcaaa cgctggcgcc
5580cgcgagccgg gccggcggcg atgcggtgcc ccacggctgc cggaatccaa gggaggcaag
5640agcgcccggg tcagttgaag ggctttacgc gcaaggtaca gccgctcctg caaggctgcg
5700tggtggaatt ggacgtgcag gtcctgctga agttcctcca ccgcctcacc agcggacaaa
5760gcaccggtgt atcaggtccg tgtcatccac tctaaagaac tcgactacga cctactgatg
5820gccctagatt cttcatcaaa aacgcctgag acacttgccc aggattgaaa ctccctgaag
5880ggaccaccag gggccctgag ttgttccttc cccccgtggc gagctgccag ccaggctgta
5940cctgtgatcg aggctggcgg gaaaataggc ttcgtgtgct caggtcatgg gaggtgcagg
6000acagctcatg aaacgccaac aatcgcacaa ttcatgtcaa gctaatcagc tatttcctct
6060tcacgagctg taattgtccc aaaattctgg tctaccgggg gtgatccttc gtgtacgggc
6120ccttccctca accctaggta tgcgcgcatg cggtcgccgc gcaactcgcg cgagggccga
6180gggtttggga cgggccgtcc cgaaatgcag ttgcacccgg atgcgtggca ccttttttgc
6240gataatttat gcaatggact gctctgcaaa attctggctc tgtcgccaac cctaggatca
6300gcggcgtagg atttcgtaat cattcgtcct gatggggagc taccgactac cctaatatca
6360gcccgactgc ctgacgccag cgtccacttt tgtgcacaca ttccattcgt gcccaagaca
6420tttcattgtg gtgcgaagcg tccccagtta cgctcacctg tttcccgacc tccttactgt
6480tctgtcgaca gagcgggccc acaggccggt cgcagccact agtatggcta tcaagacgaa
6540caggcagcct gtggagaagc ctccgttcac gatcgggacg ctgcgcaagg ccatccccgc
6600gcactgtttc gagcgctcgg cgcttcgtgg gcgcgcccag ctgcccgact ggagccgcct
6660gctgaccgcc atcaccaccg tgttcgtgaa gtccaagcgc cccgacatgc acgaccgcaa
6720gtccaagcgc cccgacatgc tggtggacag cttcggcctg gagtccaccg tgcaggacgg
6780cctggtgttc cgccagtcct tctccatccg ctcctacgag atcggcaccg accgcaccgc
6840cagcatcgag accctgatga accacctgca ggagacctcc ctgaaccact gcaagagcac
6900cggcatcctg ctggacggct tcggccgcac cctggagatg tgcaagcgcg acctgatctg
6960ggtggtgatt aagatgcaga tcaaggtgaa ccgctacccc gcctggggcg acaccgtgga
7020gatcaacacc cgcttcagcc gcctgggcaa gatcggcatg ggccgcgact ggctgatctc
7080cgactgcaac accggcgaga tcctggtgcg cgccaccagc gcctacgcca tgatgaacca
7140gaagacccgc cgcctgtcca agctgcccta cgaggtgcac caggagatcg tgcccctgtt
7200cgtggacagc cccgtgatcg aggactccga cctgaaggtg cacaagttca aggtgaagac
7260cggcgacagc atccagaagg gcctgacccc cggctggaac gacctggacg tgaaccagca
7320cgtgtccaac gtgaagtaca tcggctggat cctggagagc atgcccaccg aggtgctgga
7380gacccaggag ctgtgctccc tggccctgga gtaccgccgc gagtgcggcc gcgactccgt
7440gctggagagc gtgaccgcca tggaccccag caaggtgggc gtgcgctccc agtaccagca
7500cctgctgcgc ctggaggacg gcaccgccat cgtgaacggc gccaccgagt ggcgccccaa
7560gaacgccggc gccaacggcg ccatctccac cggcaagacc agcaacggca actccgtgtc
7620catggactac aaggaccacg acggcgacta caaggaccac gacatcgact acaaggacga
7680cgacgacaag tgactcgagg cagcagcagc tcggatagta tcgacacact ctggacgctg
7740gtcgtgtgat ggactgttgc cgccacactt gctgccttga cctgtgaata tccctgccgc
7800ttttatcaaa cagcctcagt gtgtttgatc ttgtgtgtac gcgcttttgc gagttgctag
7860ctgcttgtgc tatttgcgaa taccaccccc agcatcccct tccctcgttt catatcgctt
7920gcatcccaac cgcaacttat ctacgctgtc ctgctatccc tcagcgctgc tcctgctcct
7980gctcactgcc cctcgcacag ccttggtttg ggctccgcct gtattctcct ggtactgcaa
8040cctgtaaacc agcactgcaa tgctgatgca cgggaagtag tgggatggga acacaaatgg
8100aaagctgtag aattctccag agctccagcg ccatgccacg ccttttgatg gcttcaagta
8160cgataacggt gttggattgt gcgtttgttg cgtagtgtgc atggcttaga ataatgcagt
8220tggatttctt gctcacggca atgtcggctt gtccgcaggt tcaaccccat ttcggagtct
8280caggtcagcc gcgcaatgac cagccgctac ttcaaggact tgcacgacaa cgccgaggtg
8340agctatgttt aggccttgag tgaaaattgt cgtcgaagca tattcgcgct ccgcgatagc
8400atccaagcaa aatgtcaagt gcgttccgat ttgcgtccgc aggtcgatgt tgtgatcgtc
8460ggtgccggat ccgccggtct gtcctgcgct tacgagctga ccaagcaccc cgacgtccgg
8520gtacgcgagc tgagattcga ttggacataa actgaaaatg aaatcttttg gagaaatgta
8580agggtctcaa gcggtgctcg attgcaagaa attggtcgtc ccccactccg caggtcgcca
8640tcatcgagca gggcgttgca cctggtggcg gcgcctggct ggggggacag ctgttctcgg
8700ccatgtgtgt acgtagaagg gtggatttcg gatggtttcg ttgcacagct gtttgtcaat
8760gatttgtctt agactattgc cgatgtttct aaatgtttta ggagctatga tatgtctgca
8820ggcgactgaa gagc
88341228329DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 122gctcttcgct caccgcgtga attgctgtcc
caaacgtaag catcatcgtg gctcggtcac 60gcgatcctgg atccggggat cctagaccgc
tggtggagag cgctgccgtc ggattggtgg 120caagtaagat tgcgcaggtt ggcgaaggga
gagaccaaaa ccggaggctg gaagcgggca 180caacatcgta ttattgcgta tagtagagca
gtggcagtcg catttcgagg tccgcaacgg 240atctcgcaag ctcgctacgc tcacagtagg
agaaagggga ccactgcccc tgccagaatg 300gtcgcgaccc tctccctcgc cggccccgcc
tgcaacacgc agtgcgtatc cggcaagcgg 360gctgtcgcct tcaaccgccc ccatgttggc
gtccgggctc gatcaggtgc gctgaggggg 420gtttggtgtg cccgcgcctc tgggcccgtg
tcggccgtgc ggacgtgggg ccctgggcag 480tggatcagca gggtttgcgt gcaaatgcct
ataccggcga ttgaatagcg atgaacggga 540tacggttgcg ctcactccat gcccatgcga
ccccgtttct gtccgccagc cgtggtcgcc 600cgggctgcga agcgggaccc cacccagcgc
attgtgatca ccggaatggg cgtggggtac 660cctttcttgc gctatgacac ttccagcaaa
aggtagggcg ggctgcgaga cggcttcccg 720gcgctgcatg caacaccgat gatgcttcga
ccccccgaag ctccttcggg gctgcatggg 780cgctccgatg ccgctccagg gcgagcgctg
tttaaatagc caggcccccg attgcaaaga 840cattatagcg agctaccaaa gccatattca
aacacctaga tcactaccac ttctacacag 900gccactcgag cttgtgatcg cactccgcta
agggggcgcc tcttcctctt cgtttcagtc 960acaacccgca aactctagaa tatcaatgat
cgagcaggac ggcctccacg ccggctcccc 1020cgccgcctgg gtggagcgcc tgttcggcta
cgactgggcc cagcagacca tcggctgctc 1080cgacgccgcc gtgttccgcc tgtccgccca
gggccgcccc gtgctgttcg tgaagaccga 1140cctgtccggc gccctgaacg agctgcagga
cgaggccgcc cgcctgtcct ggctggccac 1200caccggcgtg ccctgcgccg ccgtgctgga
cgtggtgacc gaggccggcc gcgactggct 1260gctgctgggc gaggtgcccg gccaggacct
gctgtcctcc cacctggccc ccgccgagaa 1320ggtgtccatc atggccgacg ccatgcgccg
cctgcacacc ctggaccccg ccacctgccc 1380cttcgaccac caggccaagc accgcatcga
gcgcgcccgc acccgcatgg aggccggcct 1440ggtggaccag gacgacctgg acgaggagca
ccagggcctg gcccccgccg agctgttcgc 1500ccgcctgaag gcccgcatgc ccgacggcga
ggacctggtg gtgacccacg gcgacgcctg 1560cctgcccaac atcatggtgg agaacggccg
cttctccggc ttcatcgact gcggccgcct 1620gggcgtggcc gaccgctacc aggacatcgc
cctggccacc cgcgacatcg ccgaggagct 1680gggcggcgag tgggccgacc gcttcctggt
gctgtacggc atcgccgccc ccgactccca 1740gcgcatcgcc ttctaccgcc tgctggacga
gttcttctga caattggcag cagcagctcg 1800gatagtatcg acacactctg gacgctggtc
gtgtgatgga ctgttgccgc cacacttgct 1860gccttgacct gtgaatatcc ctgccgcttt
tatcaaacag cctcagtgtg tttgatcttg 1920tgtgtacgcg cttttgcgag ttgctagctg
cttgtgctat ttgcgaatac cacccccagc 1980atccccttcc ctcgtttcat atcgcttgca
tcccaaccgc aacttatcta cgctgtcctg 2040ctatccctca gcgctgctcc tgctcctgct
cactgcccct cgcacagcct tggtttgggc 2100tccgcctgta ttctcctggt actgcaacct
gtaaaccagc actgcaatgc tgatgcacgg 2160gaagtagtgg gatgggaaca caaatggaaa
gctgtatagg gataaaagct tatagcgact 2220gctacccccc gaccatgtgc cgaggcagaa
attatataca agaagcagat cgcaattagg 2280cacatcgctt tgcattatcc acacactatt
catcgctgct gcggcaaggc tgcagagtgt 2340atttttgtgg cccaggagct gagtccgaag
tcgacgcgac gagcggcgca ggatccgacc 2400cctagacgag cactgtcatt ttccaagcac
gcagctaaat gcgctgagac cgggtctaaa 2460tcatccgaaa agtgtcaaaa tggccgattg
ggttcgccta ggacaatgcg ctgcggattc 2520gctcgagtcc gctgccggcc aaaaggcggt
ggtacaggaa ggcgcacggg gccaaccctg 2580cgaagccggg ggcccgaacg ccgaccgccg
gccttcgatc tcgggtgtcc ccctcgtcaa 2640tttcctctct cgggtgcagc cacgaaagtc
gtgacgcagg tcacgaaatc cggttacgaa 2700aaacgcaggt cttcgcaaaa acgtgagggt
ttcgcgtctc gccctagcta ttcgtatcgc 2760cgggtcagac ccacgtgcag aaaagccctt
gaataacccg ggaccgtggt taccgcgccg 2820cctgcaccag ggggcttata taagcccaca
ccacacctgt ctcaccacgc atttctccaa 2880ctcgcgactt ttcggaagaa attgttatcc
acctagtata gactgccacc tgcaggacct 2940tgtgtcttgc agtttgtatt ggtcccggcc
gtcgagcacg acagatctgg gctagggttg 3000gcctggccgc tcggcactcc cctttagccg
cgcgcatccg cgttccagag gtgcgattcg 3060gtgtgtggag cattgtcatg cgcttgtggg
ggtcgttccg tgcgcggcgg gtccgccatg 3120ggcgccgacc tgggccctag ggtttgtttt
cgggccaagc gagcccctct cacctcgtcg 3180cccccccgca ttccctctct cttgcagcca
ctagtatggc tatcaagacg aacaggcagc 3240ctgtggagaa gcctccgttc acgatcggga
cgctgcgcaa ggccatcccc gcgcactgtt 3300tcgagcgctc ggcgcttcgt gggcgcgccc
agctgcccga ctggagccgc ctgctgaccg 3360ccatcaccac cgtgttcgtg aagtccaagc
gccccgacat gcacgaccgc aagtccaagc 3420gccccgacat gctggtggac agcttcggcc
tggagtccac cgtgcaggac ggcctggtgt 3480tccgccagtc cttctccatc cgctcctacg
agatcggcac cgaccgcacc gccagcatcg 3540agaccctgat gaaccacctg caggagacct
ccctgaacca ctgcaagagc accggcatcc 3600tgctggacgg cttcggccgc accctggaga
tgtgcaagcg cgacctgatc tgggtggtga 3660ttaagatgca gatcaaggtg aaccgctacc
ccgcctgggg cgacaccgtg gagatcaaca 3720cccgcttcag ccgcctgggc aagatcggca
tgggccgcga ctggctgatc tccgactgca 3780acaccggcga gatcctggtg cgcgccacca
gcgcctacgc catgatgaac cagaagaccc 3840gccgcctgtc caagctgccc tacgaggtgc
accaggagat cgtgcccctg ttcgtggaca 3900gccccgtgat cgaggactcc gacctgaagg
tgcacaagtt caaggtgaag accggcgaca 3960gcatccagaa gggcctgacc cccggctgga
acgacctgga cgtgaaccag cacgtgtcca 4020acgtgaagta catcggctgg atcctggaga
gcatgcccac cgaggtgctg gagacccagg 4080agctgtgctc cctggccctg gagtaccgcc
gcgagtgcgg ccgcgactcc gtgctggaga 4140gcgtgaccgc catggacccc agcaaggtgg
gcgtgcgctc ccagtaccag cacctgctgc 4200gcctggagga cggcaccgcc atcgtgaacg
gcgccaccga gtggcgcccc aagaacgccg 4260gcgccaacgg cgccatctcc accggcaaga
ccagcaacgg caactccgtg tccatggact 4320acaaggacca cgacggcgac tacaaggacc
acgacatcga ctacaaggac gacgacgaca 4380agtgactcga gagcgtccag cgtgtgggat
gaagggtgcg atggaacggg gctgccgccc 4440cccctctggg catctagctc tgcaccgcac
gccaggaagc ccaagccagg ccccgtcaca 4500ctccctcgct gaagtgttcc ccccctgccc
cacactcatc caggtatcaa cgccatcatg 4560ttctacgtcc ccgtcatctt caactccctg
gggagcgggc gccgcgcgtc gctgctgaac 4620accatcatca tcaacgccgt caactttgtt
aattaagaat tcggccgaca ggacgcgcgt 4680caaaggtgct ggtcgtgtat gccctggccg
gcaggtcgtt gctgctgctg gttagtgatt 4740ccgcaaccct gattttggcg tcttattttg
gcgtggcaaa cgctggcgcc cgcgagccgg 4800gccggcggcg atgcggtgcc ccacggctgc
cggaatccaa gggaggcaag agcgcccggg 4860tcagttgaag ggctttacgc gcaaggtaca
gccgctcctg caaggctgcg tggtggaatt 4920ggacgtgcag gtcctgctga agttcctcca
ccgcctcacc agcggacaaa gcaccggtgt 4980atcaggtccg tgtcatccac tctaaagaac
tcgactacga cctactgatg gccctagatt 5040cttcatcaaa aacgcctgag acacttgccc
aggattgaaa ctccctgaag ggaccaccag 5100gggccctgag ttgttccttc cccccgtggc
gagctgccag ccaggctgta cctgtgatcg 5160aggctggcgg gaaaataggc ttcgtgtgct
caggtcatgg gaggtgcagg acagctcatg 5220aaacgccaac aatcgcacaa ttcatgtcaa
gctaatcagc tatttcctct tcacgagctg 5280taattgtccc aaaattctgg tctaccgggg
gtgatccttc gtgtacgggc ccttccctca 5340accctaggta tgcgcgcatg cggtcgccgc
gcaactcgcg cgagggccga gggtttggga 5400cgggccgtcc cgaaatgcag ttgcacccgg
atgcgtggca ccttttttgc gataatttat 5460gcaatggact gctctgcaaa attctggctc
tgtcgccaac cctaggatca gcggcgtagg 5520atttcgtaat cattcgtcct gatggggagc
taccgactac cctaatatca gcccgactgc 5580ctgacgccag cgtccacttt tgtgcacaca
ttccattcgt gcccaagaca tttcattgtg 5640gtgcgaagcg tccccagtta cgctcacctg
tttcccgacc tccttactgt tctgtcgaca 5700gagcgggccc acaggccggt cgcagcccat
atggcttccg cggcattcac catgtcggcg 5760tgccccgcga tgactggcag ggcccctggg
gcacgtcgct ccggacggcc agtcgccacc 5820cgcctgaggt acgtattcca gtgcctggtg
gccagctgca tcgacccctg cgaccagtac 5880cgcagcagcg ccagcctgag cttcctgggc
gacaacggct tcgccagcct gttcggcagc 5940aagcccttca tgagcaaccg cggccaccgc
cgcctgcgcc gcgccagcca cagcggcgag 6000gccatggccg tggccctgca gcccgcccag
gaggccggca ccaagaagaa gcccgtgatc 6060aagcagcgcc gcgtggtggt gaccggcatg
ggcgtggtga cccccctggg ccacgagccc 6120gacgtgttct acaacaacct gctggacggc
gtgagcggca tcagcgagat cgagaccttc 6180gactgcaccc agttccccac ccgcatcgcc
ggcgagatca agagcttcag caccgacggc 6240tgggtggccc ccaagctgag caagcgcatg
gacaagttca tgctgtacct gctgaccgcc 6300ggcaagaagg ccctggccga cggcggcatc
accgacgagg tgatgaagga gctggacaag 6360cgcaagtgcg gcgtgctgat cggcagcggc
atgggcggca tgaaggtgtt caacgacgcc 6420atcgaggccc tgcgcgtgag ctacaagaag
atgaacccct tctgcgtgcc cttcgccacc 6480accaacatgg gcagcgccat gctggccatg
gacctgggct ggatgggccc caactacagc 6540atcagcaccg cctgcgccac cagcaacttc
tgcatcctga acgccgccaa ccacatcatc 6600cgcggcgagg ccgacatgat gctgtgcggc
ggcagcgacg ccgtgatcat ccccatcggc 6660ctgggcggct tcgtggcctg ccgcgccctg
agccagcgca acagcgaccc caccaaggcc 6720agccgcccct gggacagcaa ccgcgacggc
ttcgtgatgg gcgagggcgc cggcgtgctg 6780ctgctggagg agctggagca cgccaagaag
cgcggcgcca ccatctacgc cgagttcctg 6840ggcggcagct tcacctgcga cgcctaccac
atgaccgagc cccaccccga gggcgccggc 6900gtgatcctgt gcatcgagaa ggccctggcc
caggccggcg tgagcaagga ggacgtgaac 6960tacatcaacg cccacgccac cagcaccagc
gccggcgaca tcaaggagta ccaggccctg 7020gcccgctgct tcggccagaa cagcgagctg
cgcgtgaaca gcaccaagag catgatcggc 7080cacctgctgg gcgccgccgg cggcgtggag
gccgtgaccg tggtgcaggc catccgcacc 7140ggctggattc accccaacct gaacctggag
gaccccgaca aggccgtgga cgccaagctg 7200ctggtgggcc ccaagaagga gcgcctgaac
gtgaaggtgg gcctgagcaa cagcttcggc 7260ttcggcggcc acaacagcag catcctgttc
gccccctgca acgtgtgact cgaggcagca 7320gcagctcgga tagtatcgac acactctgga
cgctggtcgt gtgatggact gttgccgcca 7380cacttgctgc cttgacctgt gaatatccct
gccgctttta tcaaacagcc tcagtgtgtt 7440tgatcttgtg tgtacgcgct tttgcgagtt
gctagctgct tgtgctattt gcgaatacca 7500cccccagcat ccccttccct cgtttcatat
cgcttgcatc ccaaccgcaa cttatctacg 7560ctgtcctgct atccctcagc gctgctcctg
ctcctgctca ctgcccctcg cacagccttg 7620gtttgggctc cgcctgtatt ctcctggtac
tgcaacctgt aaaccagcac tgcaatgctg 7680atgcacggga agtagtggga tgggaacaca
aatggaaagc ttgagctcca cctgcatccg 7740cctggcgctc gaggacgccg gcgtctcgcc
cgacgaggtc aactacgtca acgcgcacgc 7800cacctccacc ctggtgggcg acaaggccga
ggtgcgcgcg gtcaagtcgg tctttggcga 7860catgaagggc atcaagatga acgccaccaa
gtccatgatc gggcactgcc tgggcgccgc 7920cggcggcatg gaggccgtcg ccacgctcat
ggccatccgc accggctggg tgcaccccac 7980catcaaccac gacaacccca tcgccgaggt
cgacggcctg gacgtcgtcg ccaacgccaa 8040ggcccagcac aaaatcaacg tcgccatctc
caactccttc ggcttcggcg ggcacaactc 8100cgtcgtcgcc tttgcgccct tccgcgagta
ggcggagcga gcgcgcttgg ctgaggaggg 8160aggcggggtg cgagcccttt ggctgcgcgc
gatactctcc ccgcacgagc agactccacg 8220cgcctgaatc tacttgtcaa cgagcaaccg
tgtgttttgt ccgtggccat tcttattatt 8280tctccgactg tggccgtact ctgtttggct
gtgcaagcac cgaagagcc 83291236750DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
123gaagagcgcc caatgtttaa acagcccgca ccctcgttga tctgggagcc ctgcgcagcc
60ccttaaatca tctcagtcag gtttctgtgt tcaactgagc ctaaagggct ttcgtcatgc
120gcacgagcac acgtatatcg gccacgcagt ttctcaaaag cggtagaaca gttcgcgagc
180cctcgtaggt cgaaaacttg cgccagtact attaaattaa attaattgat cgaacgagac
240gcgaaacttt tgcagaatgc caccgagttt gcccagagaa tgggagtggc gccattcacc
300atccgcctgt gcccggcttg attcgccgag acgatggacg gcgagaccag ggagcggctt
360gcgagccccg agccggtagc aggaacaatg atcgacaatc ttcctgtcca attactggca
420accattagaa agagccggag cgcgttgaaa gtctgcaatc gagtaatttt tcgatacgtc
480gggcctgctg aaccctaagg ctccggactt tgtttaaggc gatccaagat gcacgcggcc
540ccaggcacgt atctcaagca caaaccccag ccttagtttc gagactttgg gagatagcga
600ccgatatcta gtttggcatt ttgtatatta attacctcaa gcaatggagc gctctgatgc
660ggtgcagcgt cggctgcagc acctggcagt ggcgctaggg tcgccctatc gctcggaacc
720tggtcagctg gctcccgcct cctgctcagc ctcttccggt accgcggtga gaatcgaaaa
780tgcatcgttt ctaggttcgg agacggtcaa ttccctgctc cggcgaatct gtcggtcaag
840ctggccagtg gacaatgttg ctatggcagc ccgcgcacat gggcctcccg acgcggccat
900caggagccca aacagcgtgt cagggtatgt gaaactcaag aggtccctgc tgggcactcc
960ggccccactc cgggggcggg acgccaggca ttcgcggtcg gtcccgcgcg acgagcgaaa
1020tgatgattcg gttacgagac caggacgtcg tcgaggtcga gaggcagcct cggacacgtc
1080tcgctagggc aacgccccga gtccccgcga gggccgtaaa cattgtttct gggtgtcgga
1140gtgggcattt tgggcccgat ccaatcgcct catgccgctc tcgtctggtc ctcacgttcg
1200cgtacggcct ggatcccgga aagggcggat gcacgtggtg ttgccccgcc attggcgccc
1260acgtttcaaa gtccccggcc agaaatgcac aggaccggcc cggctcgcac aggccatgct
1320gaacgcccag atttcgacag caacaccatc tagaataatc gcaaccatcc gcgttttgaa
1380cgaaacgaaa cggcgctgtt tagcatgttt ccgacatcgt gggggccgaa gcatgctccg
1440gggggaggaa agcgtggcac agcggtagcc cattctgtgc cacacgccga cgaggaccaa
1500tccccggcat cagccttcat cgacggctgc gccgcacata taaagccgga cgcctaaccg
1560gtttcgtggt tatgactagt atgttcgcgt tctacttcct gacggcctgc atctccctga
1620agggcgtgtt cggcgtctcc ccctcctaca acggcctggg cctgacgccc cagatgggct
1680gggacaactg gaacacgttc gcctgcgacg tctccgagca gctgctgctg gacacggccg
1740accgcatctc cgacctgggc ctgaaggaca tgggctacaa gtacatcatc ctggacgact
1800gctggtcctc cggccgcgac tccgacggct tcctggtcgc cgacgagcag aagttcccca
1860acggcatggg ccacgtcgcc gaccacctgc acaacaactc cttcctgttc ggcatgtact
1920cctccgcggg cgagtacacg tgcgccggct accccggctc cctgggccgc gaggaggagg
1980acgcccagtt cttcgcgaac aaccgcgtgg actacctgaa gtacgacaac tgctacaaca
2040agggccagtt cggcacgccc gagatctcct accaccgcta caaggccatg tccgacgccc
2100tgaacaagac gggccgcccc atcttctact ccctgtgcaa ctggggccag gacctgacct
2160tctactgggg ctccggcatc gcgaactcct ggcgcatgtc cggcgacgtc acggcggagt
2220tcacgcgccc cgactcccgc tgcccctgcg acggcgacga gtacgactgc aagtacgccg
2280gcttccactg ctccatcatg aacatcctga acaaggccgc ccccatgggc cagaacgcgg
2340gcgtcggcgg ctggaacgac ctggacaacc tggaggtcgg cgtcggcaac ctgacggacg
2400acgaggagaa ggcgcacttc tccatgtggg ccatggtgaa gtcccccctg atcatcggcg
2460cgaacgtgaa caacctgaag gcctcctcct actccatcta ctcccaggcg tccgtcatcg
2520ccatcaacca ggactccaac ggcatccccg ccacgcgcgt ctggcgctac tacgtgtccg
2580acacggacga gtacggccag ggcgagatcc agatgtggtc cggccccctg gacaacggcg
2640accaggtcgt ggcgctgctg aacggcggct ccgtgtcccg ccccatgaac acgaccctgg
2700aggagatctt cttcgactcc aacctgggct ccaagaagct gacctccacc tgggacatct
2760acgacctgtg ggcgaaccgc gtcgacaact ccacggcgtc cgccatcctg ggccgcaaca
2820agaccgccac cggcatcctg tacaacgcca ccgagcagtc ctacaaggac ggcctgtcca
2880agaacgacac ccgcctgttc ggccagaaga tcggctccct gtcccccaac gcgatcctga
2940acacgaccgt ccccgcccac ggcatcgcgt tctaccgcct gcgcccctcc tcctgatacg
3000tactcgaggc agcagcagct cggatagtat cgacacactc tggacgctgg tcgtgtgatg
3060gactgttgcc gccacacttg ctgccttgac ctgtgaatat ccctgccgct tttatcaaac
3120agcctcagtg tgtttgatct tgtgtgtacg cgcttttgcg agttgctagc tgcttgtgct
3180atttgcgaat accaccccca gcatcccctt ccctcgtttc atatcgcttg catcccaacc
3240gcaacttatc tacgctgtcc tgctatccct cagcgctgct cctgctcctg ctcactgccc
3300ctcgcacagc cttggtttgg gctccgcctg tattctcctg gtactgcaac ctgtaaacca
3360gcactgcaat gctgatgcac gggaagtagt gggatgggaa cacaaatgga aagctgtaga
3420attcatagcg actgctaccc cccgaccatg tgccgaggca gaaattatat acaagaagca
3480gatcgcaatt aggcacatcg ctttgcatta tccacacact attcatcgct gctgcggcaa
3540ggctgcagag tgtatttttg tggcccagga gctgagtccg aagtcgacgc gacgagcggc
3600gcaggatccg acccctagac gagcactgtc attttccaag cacgcagcta aatgcgctga
3660gaccgggtct aaatcatccg aaaagtgtca aaatggccga ttgggttcgc ctaggacaat
3720gcgctgcgga ttcgctcgag tccgctgccg gccaaaaggc ggtggtacag gaaggcgcac
3780ggggccaacc ctgcgaagcc gggggcccga acgccgaccg ccggccttcg atctcgggtg
3840tccccctcgt caatttcctc tctcgggtgc agccacgaaa gtcgtgacgc aggtcacgaa
3900atccggttac gaaaaacgca ggtcttcgca aaaacgtgag ggtttcgcgt ctcgccctag
3960ctattcgtat cgccgggtca gacccacgtg cagaaaagcc cttgaataac ccgggaccgt
4020ggttaccgcg ccgcctgcac cagggggctt atataagccc acaccacacc tgtctcacca
4080cgcatttctc caactcgcga cttttcggaa gaaattgtta tccacctagt atagactgcc
4140acctgcagga ccttgtgtct tgcagtttgt attggtcccg gccgtcgagc acgacagatc
4200tgggctaggg ttggcctggc cgctcggcac tcccctttag ccgcgcgcat ccgcgttcca
4260gaggtgcgat tcggtgtgtg gagcattgtc atgcgcttgt gggggtcgtt ccgtgcgcgg
4320cgggtccgcc atgggcgccg acctgggccc tagggtttgt tttcgggcca agcgagcccc
4380tctcacctcg tcgccccccc gcattccctc tctcttgcag ccactagtaa caatggccac
4440cgcatccact ttctcggcgt tcaatgcccg ctgcggcgac ctgcgtcgct cggcgggctc
4500cgggccccgg cgcccagcga ggcccctccc cgtgcgcgct gccatcgcca gcgaggtccc
4560cgtggccacc acctcccccc gggcgcaccc caaggcgaac ggcagcgcgg tgtcgctgaa
4620gtcgggctcc ctggagaccc aggaggacaa gacgagcagc tcgtcccccc ccccccgcac
4680gttcatcaac cagctgcccg tgtggagcat gctgctgtcg gcggtgacca cggtcttcgg
4740cgtggccgag aagcagtggc ccatgctgga ccgcaagtcc aagcgccccg acatgctggt
4800cgagcccctg ggcgtggacc gcatcgtcta cgacggcgtg agcttccgcc agtcgttctc
4860catccgcagc tacgagatcg gcgccgaccg caccgcctcg atcgagacgc tgatgaacat
4920gttccaggag acctccctga accactgcaa gatcatcggc ctgctgaacg acggcttcgg
4980ccgcacgccc gagatgtgca agcgcgacct gatctgggtc gtgaccaaga tgcagatcga
5040ggtgaaccgc taccccacgt ggggcgacac catcgaggtc aacacgtggg tgagcgcctc
5100gggcaagcac ggcatgggcc gcgactggct gatctccgac tgccacaccg gcgagatcct
5160gatccgcgcg acgagcgtct gggcgatgat gaaccagaag acccgccgcc tgtcgaagat
5220cccctacgag gtgcgccagg agatcgagcc ccagttcgtc gactccgccc ccgtgatcgt
5280ggacgaccgc aagttccaca agctggacct gaagacgggc gacagcatct gcaacggcct
5340gaccccccgc tggacggacc tggacgtgaa ccagcacgtc aacaacgtga agtacatcgg
5400ctggatcctg cagtcggtcc ccaccgaggt gttcgagacg caggagctgt gcggcctgac
5460cctggagtac cgccgcgagt gcggccgcga ctccgtgctg gagagcgtca cggccatgga
5520cccctcgaag gagggcgacc gctccctgta ccagcacctg ctgcgcctgg aggacggcgc
5580ggacatcgtg aagggccgca ccgagtggcg ccccaagaac gccggcgcca agggcgccat
5640cctgacgggc aagaccagca acggcaactc gatctccatg gactacaagg accacgacgg
5700cgactacaag gaccacgaca tcgactacaa ggacgacgac gacaagtgaa agcttgcagc
5760agcagctcgg atagtatcga cacactctgg acgctggtcg tgtgatggac tgttgccgcc
5820acacttgctg ccttgacctg tgaatatccc tgccgctttt atcaaacagc ctcagtgtgt
5880ttgatcttgt gtgtacgcgc ttttgcgagt tgctagctgc ttgtgctatt tgcgaatacc
5940acccccagca tccccttccc tcgtttcata tcgcttgcat cccaaccgca acttatctac
6000gctgtcctgc tatccctcag cgctgctcct gctcctgctc actgcccctc gcacagcctt
6060ggtttgggct cccgcctgta ttctcctggt actgcaacct gtaaaccagc actgcaatgc
6120tgatgcacgg gaagtagtgg gatgggaaca caaatggaaa gctggagctc agcgtctgcg
6180tgttgggagc tggagtcgtg ggcttgacga cggcgctgca gctgttgcag gatgtgcctg
6240gcgtgcgcgt tcacgtcgtg gctgagaaat atggcgacga aacgttgacg gctggggccg
6300gcgggctgtg gatgccatac gcattgggta cgcggccatt ggatgggatt gataggctta
6360tggagggata atagagtttt tgccggatcc aacgcatgtg gatgcggtat cccggtgggc
6420tgaaagtgtg gaaggatagt gcattggcta ttcacatgca ctgcccaccc cttttggcag
6480gaaatgtgcc ggcatcgttg gtgcaccgat ggggaaaatc gacgttcgac cactacatga
6540agatttatac gtctgaagat gcagcgactg cgggtgcgaa acggatgacg gtttggtcgt
6600gtatgtcaca gcatgtgctg gatcttgcgg gctaactccc cctgccacgg cccattgcag
6660gtgtcatgtt gactggaggg tacgaccttt cgtccgtcaa attcccagag gaggacccgc
6720tctgggccga cattgtgccc actgaagagc
67501246838DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 124gaagagcgcc caatgtttaa acagcccgca
ccctcgttga tctgggagcc ctgcgcagcc 60ccttaaatca tctcagtcag gtttctgtgt
tcaactgagc ctaaagggct ttcgtcatgc 120gcacgagcac acgtatatcg gccacgcagt
ttctcaaaag cggtagaaca gttcgcgagc 180cctcgtaggt cgaaaacttg cgccagtact
attaaattaa attaattgat cgaacgagac 240gcgaaacttt tgcagaatgc caccgagttt
gcccagagaa tgggagtggc gccattcacc 300atccgcctgt gcccggcttg attcgccgag
acgatggacg gcgagaccag ggagcggctt 360gcgagccccg agccggtagc aggaacaatg
atcgacaatc ttcctgtcca attactggca 420accattagaa agagccggag cgcgttgaaa
gtctgcaatc gagtaatttt tcgatacgtc 480gggcctgctg aaccctaagg ctccggactt
tgtttaaggc gatccaagat gcacgcggcc 540ccaggcacgt atctcaagca caaaccccag
ccttagtttc gagactttgg gagatagcga 600ccgatatcta gtttggcatt ttgtatatta
attacctcaa gcaatggagc gctctgatgc 660ggtgcagcgt cggctgcagc acctggcagt
ggcgctaggg tcgccctatc gctcggaacc 720tggtcagctg gctcccgcct cctgctcagc
ctcttccggt accgcggtga gaatcgaaaa 780tgcatcgttt ctaggttcgg agacggtcaa
ttccctgctc cggcgaatct gtcggtcaag 840ctggccagtg gacaatgttg ctatggcagc
ccgcgcacat gggcctcccg acgcggccat 900caggagccca aacagcgtgt cagggtatgt
gaaactcaag aggtccctgc tgggcactcc 960ggccccactc cgggggcggg acgccaggca
ttcgcggtcg gtcccgcgcg acgagcgaaa 1020tgatgattcg gttacgagac caggacgtcg
tcgaggtcga gaggcagcct cggacacgtc 1080tcgctagggc aacgccccga gtccccgcga
gggccgtaaa cattgtttct gggtgtcgga 1140gtgggcattt tgggcccgat ccaatcgcct
catgccgctc tcgtctggtc ctcacgttcg 1200cgtacggcct ggatcccgga aagggcggat
gcacgtggtg ttgccccgcc attggcgccc 1260acgtttcaaa gtccccggcc agaaatgcac
aggaccggcc cggctcgcac aggccatgct 1320gaacgcccag atttcgacag caacaccatc
tagaataatc gcaaccatcc gcgttttgaa 1380cgaaacgaaa cggcgctgtt tagcatgttt
ccgacatcgt gggggccgaa gcatgctccg 1440gggggaggaa agcgtggcac agcggtagcc
cattctgtgc cacacgccga cgaggaccaa 1500tccccggcat cagccttcat cgacggctgc
gccgcacata taaagccgga cgcctaaccg 1560gtttcgtggt tatgactagt atgttcgcgt
tctacttcct gacggcctgc atctccctga 1620agggcgtgtt cggcgtctcc ccctcctaca
acggcctggg cctgacgccc cagatgggct 1680gggacaactg gaacacgttc gcctgcgacg
tctccgagca gctgctgctg gacacggccg 1740accgcatctc cgacctgggc ctgaaggaca
tgggctacaa gtacatcatc ctggacgact 1800gctggtcctc cggccgcgac tccgacggct
tcctggtcgc cgacgagcag aagttcccca 1860acggcatggg ccacgtcgcc gaccacctgc
acaacaactc cttcctgttc ggcatgtact 1920cctccgcggg cgagtacacg tgcgccggct
accccggctc cctgggccgc gaggaggagg 1980acgcccagtt cttcgcgaac aaccgcgtgg
actacctgaa gtacgacaac tgctacaaca 2040agggccagtt cggcacgccc gagatctcct
accaccgcta caaggccatg tccgacgccc 2100tgaacaagac gggccgcccc atcttctact
ccctgtgcaa ctggggccag gacctgacct 2160tctactgggg ctccggcatc gcgaactcct
ggcgcatgtc cggcgacgtc acggcggagt 2220tcacgcgccc cgactcccgc tgcccctgcg
acggcgacga gtacgactgc aagtacgccg 2280gcttccactg ctccatcatg aacatcctga
acaaggccgc ccccatgggc cagaacgcgg 2340gcgtcggcgg ctggaacgac ctggacaacc
tggaggtcgg cgtcggcaac ctgacggacg 2400acgaggagaa ggcgcacttc tccatgtggg
ccatggtgaa gtcccccctg atcatcggcg 2460cgaacgtgaa caacctgaag gcctcctcct
actccatcta ctcccaggcg tccgtcatcg 2520ccatcaacca ggactccaac ggcatccccg
ccacgcgcgt ctggcgctac tacgtgtccg 2580acacggacga gtacggccag ggcgagatcc
agatgtggtc cggccccctg gacaacggcg 2640accaggtcgt ggcgctgctg aacggcggct
ccgtgtcccg ccccatgaac acgaccctgg 2700aggagatctt cttcgactcc aacctgggct
ccaagaagct gacctccacc tgggacatct 2760acgacctgtg ggcgaaccgc gtcgacaact
ccacggcgtc cgccatcctg ggccgcaaca 2820agaccgccac cggcatcctg tacaacgcca
ccgagcagtc ctacaaggac ggcctgtcca 2880agaacgacac ccgcctgttc ggccagaaga
tcggctccct gtcccccaac gcgatcctga 2940acacgaccgt ccccgcccac ggcatcgcgt
tctaccgcct gcgcccctcc tcctgatacg 3000tactcgaggc agcagcagct cggatagtat
cgacacactc tggacgctgg tcgtgtgatg 3060gactgttgcc gccacacttg ctgccttgac
ctgtgaatat ccctgccgct tttatcaaac 3120agcctcagtg tgtttgatct tgtgtgtacg
cgcttttgcg agttgctagc tgcttgtgct 3180atttgcgaat accaccccca gcatcccctt
ccctcgtttc atatcgcttg catcccaacc 3240gcaacttatc tacgctgtcc tgctatccct
cagcgctgct cctgctcctg ctcactgccc 3300ctcgcacagc cttggtttgg gctccgcctg
tattctcctg gtactgcaac ctgtaaacca 3360gcactgcaat gctgatgcac gggaagtagt
gggatgggaa cacaaatgga aagctgtaga 3420attcggccga caggacgcgc gtcaaaggtg
ctggtcgtgt atgccctggc cggcaggtcg 3480ttgctgctgc tggttagtga ttccgcaacc
ctgattttgg cgtcttattt tggcgtggca 3540aacgctggcg cccgcgagcc gggccggcgg
cgatgcggtg ccccacggct gccggaatcc 3600aagggaggca agagcgcccg ggtcagttga
agggctttac gcgcaaggta cagccgctcc 3660tgcaaggctg cgtggtggaa ttggacgtgc
aggtcctgct gaagttcctc caccgcctca 3720ccagcggaca aagcaccggt gtatcaggtc
cgtgtcatcc actctaaaga actcgactac 3780gacctactga tggccctaga ttcttcatca
aaaacgcctg agacacttgc ccaggattga 3840aactccctga agggaccacc aggggccctg
agttgttcct tccccccgtg gcgagctgcc 3900agccaggctg tacctgtgat cgaggctggc
gggaaaatag gcttcgtgtg ctcaggtcat 3960gggaggtgca ggacagctca tgaaacgcca
acaatcgcac aattcatgtc aagctaatca 4020gctatttcct cttcacgagc tgtaattgtc
ccaaaattct ggtctaccgg gggtgatcct 4080tcgtgtacgg gcccttccct caaccctagg
tatgcgcgca tgcggtcgcc gcgcaactcg 4140cgcgagggcc gagggtttgg gacgggccgt
cccgaaatgc agttgcaccc ggatgcgtgg 4200cacctttttt gcgataattt atgcaatgga
ctgctctgca aaattctggc tctgtcgcca 4260accctaggat cagcggcgta ggatttcgta
atcattcgtc ctgatgggga gctaccgact 4320accctaatat cagcccgact gcctgacgcc
agcgtccact tttgtgcaca cattccattc 4380gtgcccaaga catttcattg tggtgcgaag
cgtccccagt tacgctcacc tgtttcccga 4440cctccttact gttctgtcga cagagcgggc
ccacaggccg gtcgcagcca ctagtatggt 4500ggccgccgag gcctcctccg ccctgttctc
cgtgcgcacc cccggcacct cccccaagcc 4560cggcaagttc ggcaactggc ccacctccct
gtccgtgccc ttcaagtcca agtccaacca 4620caacggcggc ttccaggtga aggccaacgc
ctccgcccgc cccaaggcca acggctccgc 4680cgtgtccctg aagtccggct ccctggacac
ccaggaggac acctcctcct cctcctcccc 4740cccccgcacc ttcatcaacc agctgcccga
ctggtccatg ctgctgtccg ccatcaccac 4800cgtgttcgtg gccgccgaga agcagtggac
catgctggac cgcaagtcca agcgccccga 4860catgctgatg gaccccttcg gcgtggaccg
cgtggtgcag gacggcgccg tgttccgcca 4920gtccttctcc atccgctcct acgagatcgg
cgccgaccgc accgcctcca tcgagaccct 4980gatgaacatc ttccaggaga cctccctgaa
ccactgcaag tccatcggcc tgctgaacga 5040cggcttcggc cgcacccccg agatgtgcaa
gcgcgacctg atctgggtgg tgaccaagat 5100gcacgtggag gtgaaccgct accccacctg
gggcgacacc atcgaggtga acacctgggt 5160gtccgagtcc ggcaagaccg gcatgggccg
cgactggctg atctccgact gccacaccgg 5220cgagatcctg atccgcgcca cctccatgtg
cgccatgatg aaccagaaga cccgccgctt 5280ctccaagttc ccctacgagg tgcgccagga
gctggccccc cacttcgtgg actccgcccc 5340cgtgatcgag gactaccaga agctgcacaa
gctggacgtg aagaccggcg actccatctg 5400caacggcctg accccccgct ggaacgacct
ggacgtgaac cagcacgtga acaacgtgaa 5460gtacatcggc tggatcctgg agtccgtgcc
caccgaggtg ttcgagaccc aggagctgtg 5520cggcctgacc ctggagtacc gccgcgagtg
cggccgcgac tccgtgctgg agtccgtgac 5580cgccatggac ccctccaagg agggcgaccg
ctccctgtac cagcacctgc tgcgcctgga 5640ggacggcgcc gacatcgcca agggccgcac
caagtggcgc cccaagaacg ccggcaccaa 5700cggcgccatc tccaccggca agacctccaa
cggcaactcc atctccatgg actacaagga 5760ccacgacggc gactacaagg accacgacat
cgactacaag gacgacgacg acaagtgact 5820cgaggcagca gcagctcgga tagtatcgac
acactctgga cgctggtcgt gtgatggact 5880gttgccgcca cacttgctgc cttgacctgt
gaatatccct gccgctttta tcaaacagcc 5940tcagtgtgtt tgatcttgtg tgtacgcgct
tttgcgagtt gctagctgct tgtgctattt 6000gcgaatacca cccccagcat ccccttccct
cgtttcatat cgcttgcatc ccaaccgcaa 6060cttatctacg ctgtcctgct atccctcagc
gctgctcctg ctcctgctca ctgcccctcg 6120cacagccttg gtttgggctc cgcctgtatt
ctcctggtac tgcaacctgt aaaccagcac 6180tgcaatgctg atgcacggga agtagtggga
tgggaacaca aatggaaagc tgtataggga 6240taacagggta atgagctcag cgtctgcgtg
ttgggagctg gagtcgtggg cttgacgacg 6300gcgctgcagc tgttgcagga tgtgcctggc
gtgcgcgttc acgtcgtggc tgagaaatat 6360ggcgacgaaa cgttgacggc tggggccggc
gggctgtgga tgccatacgc attgggtacg 6420cggccattgg atgggattga taggcttatg
gagggataat agagtttttg ccggatccaa 6480cgcatgtgga tgcggtatcc cggtgggctg
aaagtgtgga aggatagtgc attggctatt 6540cacatgcact gcccacccct tttggcagga
aatgtgccgg catcgttggt gcaccgatgg 6600ggaaaatcga cgttcgacca ctacatgaag
atttatacgt ctgaagatgc agcgactgcg 6660ggtgcgaaac ggatgacggt ttggtcgtgt
atgtcacagc atgtgctgga tcttgcgggc 6720taactccccc tgccacggcc cattgcaggt
gtcatgttga ctggagggta cgacctttcg 6780tccgtcaaat tcccagagga ggacccgctc
tgggccgaca ttgtgcccac tgaagagc 68381251303DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
125actagtaaca atggccaccg cctccacctt ctccgccttc aacgcccgct gcggcgacct
60gcgccgctcc gccggctccg gcccccgccg ccccgcccgc cccctgcccg tgcgcgccgc
120catcaacgcc tccgcccgcc ccaaggccaa cggctccgcc gtgtccctga agtccggctc
180cctggacacc caggaggaca cctcctcctc ctcctccccc ccccgcacct tcatcaacca
240gctgcccgac tggtccatgc tgctgtccgc catcaccacc gtgttcgtgg ccgccgagaa
300gcagtggacc atgctggacc gcaagtccaa gcgccccgac atgctgatgg accccttcgg
360cgtggaccgc gtggtgcagg acggcgccgt gttccgccag tccttctcca tccgctccta
420cgagatcggc gccgaccgca ccgcctccat cgagaccctg atgaacatct tccaggagac
480ctccctgaac cactgcaagt ccatcggcct gctgaacgac ggcttcggcc gcacccccga
540gatgtgcaag cgcgacctga tctgggtggt gaccaagatg cacgtggagg tgaaccgcta
600ccccacctgg ggcgacacca tcgaggtgaa cacctgggtg tccgagtccg gcaagaccgg
660catgggccgc gactggctga tctccgactg ccacaccggc gagatcctga tccgcgccac
720ctccatgtgc gccatgatga accagaagac ccgccgcttc tccaagttcc cctacgaggt
780gcgccaggag ctggcccccc acttcgtgga ctccgccccc gtgatcgagg actaccagaa
840gctgcacaag ctggacgtga agaccggcga ctccatctgc aacggcctga ccccccgctg
900gaacgacctg gacgtgaacc agcacgtgaa caacgtgaag tacatcggct ggatcctgga
960gtccgtgccc accgaggtgt tcgagaccca ggagctgtgc ggcctgaccc tggagtaccg
1020ccgcgagtgc ggccgcgact ccgtgctgga gtccgtgacc gccatggacc cctccaagga
1080gggcgaccgc tccctgtacc agcacctgct gcgcctggag gacggcgccg acatcgccaa
1140gggccgcacc aagtggcgcc ccaagaacgc cggcaccaac ggcgccatct ccaccggcaa
1200gacctccaac ggcaactcca tctccatgga ctacaaggac cacgacggcg actacaagga
1260ccacgacatc gactacaagg acgacgacga caagtgactc gag
13031261282DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 126actagtaaca atggccaccg cctccacctt
ctccgccttc aacgcccgct gcggcgacct 60gcgccgctcc gccggctccg gcccccgccg
ccccgcccgc cccctgcccg tgcgcgccgc 120catcaacgcc tccgcccacc ccaaggccaa
cggctccgcc gtgaacctga agtccggctc 180cctggagacc caggaggaca cctcctcctc
ctcccccccc ccccgcacct tcatcaagca 240gctgcccgac tggggcatgc tgctgtccaa
gatcaccacc gtgttcggcg ccgccgagcg 300ccagtggaag cgccccggca tgctggtgga
gcccttcggc gtggaccgca tcttccagga 360cggcgtgttc ttccgccagt ccttctccat
ccgctcctac gagatcggcg ccgaccgcac 420cgcctccatc gagaccctga tgaacatctt
ccaggagacc tccctgaacc actgcaagtc 480catcggcctg ctgaacgacg gcttcggccg
cacccccgag atgtgcaagc gcgacctgat 540ctgggtggtg accaagatcc aggtggaggt
gaaccgctac cccacctggg gcgacaccat 600cgaggtgaac acctgggtgt ccgagtccgg
caagaacggc atgggccgcg actggctgat 660ctccgactgc cgcaccggcg agatcctgat
ccgcgccacc tccgtgtggg ccatgatgaa 720ccgcaagacc cgccgcctgt ccaagttccc
ctacgaggtg cgccaggaga tcgcccccca 780cttcgtggac tccgcccccg tgatcgagga
cgacaagaag ctgcacaagc tggacgtgaa 840gaccggcgac tccatccgca agggcctgac
cccccgctgg aacgacctgg acgtgaacca 900gcacgtgaac aacgtgaagt acatcggctg
gatcctgaag tccgtgcccg ccgaggtgtt 960cgagacccag gagctgtgcg gcgtgaccct
ggagtaccgc cgcgagtgcg gccgcgactc 1020cgtgctggag tccgtgaccg ccatggacac
cgccaaggag ggcgaccgct ccctgtacca 1080gcacctgctg cgcctggagg acggcgccga
catcaccatc ggccgcaccg agtggcgccc 1140caagaacgcc ggcgccaacg gcgccatctc
caccggcaag acctccaacg agaactccgt 1200gtccatggac tacaaggacc acgacggcga
ctacaaggac cacgacatcg actacaagga 1260cgacgacgac aagtgactcg ag
12821276696DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
127gaagagcgcc caatgtttaa acccctcaac tgcgacgctg ggaaccttct ccgggcaggc
60gatgtgcgtg ggtttgcctc cttggcacgg ctctacaccg tcgagtacgc catgaggcgg
120tgatggctgt gtcggttgcc acttcgtcca gagacggcaa gtcgtccatc ctctgcgtgt
180gtggcgcgac gctgcagcag tccctctgca gcagatgagc gtgactttgg ccatttcacg
240cactcgagtg tacacaatcc atttttctta aagcaaatga ctgctgattg accagatact
300gtaacgctga tttcgctcca gatcgcacag atagcgacca tgttgctgcg tctgaaaatc
360tggattccga attcgaccct ggcgctccat ccatgcaaca gatggcgaca cttgttacaa
420ttcctgtcac ccatcggcat ggagcaggtc cacttagatt cccgatcacc cacgcacatc
480tcgctaatag tcattcgttc gtgtcttcga tcaatctcaa gtgagtgtgc atggatcttg
540gttgacgatg cggtatgggt ttgcgccgct ggctgcaggg tctgcccaag gcaagctaac
600ccagctcctc tccccgacaa tactctcgca ggcaaagccg gtcacttgcc ttccagattg
660ccaataaact caattatggc ctctgtcatg ccatccatgg gtctgatgaa tggtcacgct
720cgtgtcctga ccgttcccca gcctctggcg tcccctgccc cgcccaccag cccacgccgc
780gcggcagtcg ctgccaaggc tgtctcggag gtaccgcggt gagaatcgaa aatgcatcgt
840ttctaggttc ggagacggtc aattccctgc tccggcgaat ctgtcggtca agctggccag
900tggacaatgt tgctatggca gcccgcgcac atgggcctcc cgacgcggcc atcaggagcc
960caaacagcgt gtcagggtat gtgaaactca agaggtccct gctgggcact ccggccccac
1020tccgggggcg ggacgccagg cattcgcggt cggtcccgcg cgacgagcga aatgatgatt
1080cggttacgag accaggacgt cgtcgaggtc gagaggcagc ctcggacacg tctcgctagg
1140gcaacgcccc gagtccccgc gagggccgta aacattgttt ctgggtgtcg gagtgggcat
1200tttgggcccg atccaatcgc ctcatgccgc tctcgtctgg tcctcacgtt cgcgtacggc
1260ctggatcccg gaaagggcgg atgcacgtgg tgttgccccg ccattggcgc ccacgtttca
1320aagtccccgg ccagaaatgc acaggaccgg cccggctcgc acaggccatg ctgaacgccc
1380agatttcgac agcaacacca tctagaataa tcgcaaccat ccgcgttttg aacgaaacga
1440aacggcgctg tttagcatgt ttccgacatc gtgggggccg aagcatgctc cggggggagg
1500aaagcgtggc acagcggtag cccattctgt gccacacgcc gacgaggacc aatccccggc
1560atcagccttc atcgacggct gcgccgcaca tataaagccg gacgcctaac cggtttcgtg
1620gttatgacta gtatgttcgc gttctacttc ctgacggcct gcatctccct gaagggcgtg
1680ttcggcgtct ccccctccta caacggcctg ggcctgacgc cccagatggg ctgggacaac
1740tggaacacgt tcgcctgcga cgtctccgag cagctgctgc tggacacggc cgaccgcatc
1800tccgacctgg gcctgaagga catgggctac aagtacatca tcctggacga ctgctggtcc
1860tccggccgcg actccgacgg cttcctggtc gccgacgagc agaagttccc caacggcatg
1920ggccacgtcg ccgaccacct gcacaacaac tccttcctgt tcggcatgta ctcctccgcg
1980ggcgagtaca cgtgcgccgg ctaccccggc tccctgggcc gcgaggagga ggacgcccag
2040ttcttcgcga acaaccgcgt ggactacctg aagtacgaca actgctacaa caagggccag
2100ttcggcacgc ccgagatctc ctaccaccgc tacaaggcca tgtccgacgc cctgaacaag
2160acgggccgcc ccatcttcta ctccctgtgc aactggggcc aggacctgac cttctactgg
2220ggctccggca tcgcgaactc ctggcgcatg tccggcgacg tcacggcgga gttcacgcgc
2280cccgactccc gctgcccctg cgacggcgac gagtacgact gcaagtacgc cggcttccac
2340tgctccatca tgaacatcct gaacaaggcc gcccccatgg gccagaacgc gggcgtcggc
2400ggctggaacg acctggacaa cctggaggtc ggcgtcggca acctgacgga cgacgaggag
2460aaggcgcact tctccatgtg ggccatggtg aagtcccccc tgatcatcgg cgcgaacgtg
2520aacaacctga aggcctcctc ctactccatc tactcccagg cgtccgtcat cgccatcaac
2580caggactcca acggcatccc cgccacgcgc gtctggcgct actacgtgtc cgacacggac
2640gagtacggcc agggcgagat ccagatgtgg tccggccccc tggacaacgg cgaccaggtc
2700gtggcgctgc tgaacggcgg ctccgtgtcc cgccccatga acacgaccct ggaggagatc
2760ttcttcgact ccaacctggg ctccaagaag ctgacctcca cctgggacat ctacgacctg
2820tgggcgaacc gcgtcgacaa ctccacggcg tccgccatcc tgggccgcaa caagaccgcc
2880accggcatcc tgtacaacgc caccgagcag tcctacaagg acggcctgtc caagaacgac
2940acccgcctgt tcggccagaa gatcggctcc ctgtccccca acgcgatcct gaacacgacc
3000gtccccgccc acggcatcgc gttctaccgc ctgcgcccct cctcctgata caacttatta
3060cgtaacggag cgtcgtgcgg gagggagtgt gccgagcggg gagtcccggt ctgtgcgagg
3120cccggcagct gacgctggcg agccgtacgc cccgagggtc cccctcccct gcaccctctt
3180ccccttccct ctgacggccg cgcctgttct tgcatgttca gcgacgagga tatcgaattc
3240atagcgactg ctaccccccg accatgtgcc gaggcagaaa ttatatacaa gaagcagatc
3300gcaattaggc acatcgcttt gcattatcca cacactattc atcgctgctg cggcaaggct
3360gcagagtgta tttttgtggc ccaggagctg agtccgaagt cgacgcgacg agcggcgcag
3420gatccgaccc ctagacgagc actgtcattt tccaagcacg cagctaaatg cgctgagacc
3480gggtctaaat catccgaaaa gtgtcaaaat ggccgattgg gttcgcctag gacaatgcgc
3540tgcggattcg ctcgagtccg ctgccggcca aaaggcggtg gtacaggaag gcgcacgggg
3600ccaaccctgc gaagccgggg gcccgaacgc cgaccgccgg ccttcgatct cgggtgtccc
3660cctcgtcaat ttcctctctc gggtgcagcc acgaaagtcg tgacgcaggt cacgaaatcc
3720ggttacgaaa aacgcaggtc ttcgcaaaaa cgtgagggtt tcgcgtctcg ccctagctat
3780tcgtatcgcc gggtcagacc cacgtgcaga aaagcccttg aataacccgg gaccgtggtt
3840accgcgccgc ctgcaccagg gggcttatat aagcccacac cacacctgtc tcaccacgca
3900tttctccaac tcgcgacttt tcggaagaaa ttgttatcca cctagtatag actgccacct
3960gcaggacctt gtgtcttgca gtttgtattg gtcccggccg tcgagcacga cagatctggg
4020ctagggttgg cctggccgct cggcactccc ctttagccgc gcgcatccgc gttccagagg
4080tgcgattcgg tgtgtggagc attgtcatgc gcttgtgggg gtcgttccgt gcgcggcggg
4140tccgccatgg gcgccgacct gggccctagg gtttgttttc gggccaagcg agcccctctc
4200acctcgtcgc ccccccgcat tccctctctc ttgcagccac tagtaacaat ggccaccgca
4260tccactttct cggcgttcaa tgcccgctgc ggcgacctgc gtcgctcggc gggctccggg
4320ccccggcgcc cagcgaggcc cctccccgtg cgcgctgcca tcgccagcga ggtccccgtg
4380gccaccacct ccccccgggc gcaccccaag gcgaacggca gcgcggtgtc gctgaagtcg
4440ggctccctgg agacccagga ggacaagacg agcagctcgt cccccccccc ccgcacgttc
4500atcaaccagc tgcccgtgtg gagcatgctg ctgtcggcgg tgaccacggt cttcggcgtg
4560gccgagaagc agtggcccat gctggaccgc aagtccaagc gccccgacat gctggtcgag
4620cccctgggcg tggaccgcat cgtctacgac ggcgtgagct tccgccagtc gttctccatc
4680cgcagctacg agatcggcgc cgaccgcacc gcctcgatcg agacgctgat gaacatgttc
4740caggagacct ccctgaacca ctgcaagatc atcggcctgc tgaacgacgg cttcggccgc
4800acgcccgaga tgtgcaagcg cgacctgatc tgggtcgtga ccaagatgca gatcgaggtg
4860aaccgctacc ccacgtgggg cgacaccatc gaggtcaaca cgtgggtgag cgcctcgggc
4920aagcacggca tgggccgcga ctggctgatc tccgactgcc acaccggcga gatcctgatc
4980cgcgcgacga gcgtctgggc gatgatgaac cagaagaccc gccgcctgtc gaagatcccc
5040tacgaggtgc gccaggagat cgagccccag ttcgtcgact ccgcccccgt gatcgtggac
5100gaccgcaagt tccacaagct ggacctgaag acgggcgaca gcatctgcaa cggcctgacc
5160ccccgctgga cggacctgga cgtgaaccag cacgtcaaca acgtgaagta catcggctgg
5220atcctgcagt cggtccccac cgaggtgttc gagacgcagg agctgtgcgg cctgaccctg
5280gagtaccgcc gcgagtgcgg ccgcgactcc gtgctggaga gcgtcacggc catggacccc
5340tcgaaggagg gcgaccgctc cctgtaccag cacctgctgc gcctggagga cggcgcggac
5400atcgtgaagg gccgcaccga gtggcgcccc aagaacgccg gcgccaaggg cgccatcctg
5460acgggcaaga ccagcaacgg caactcgatc tccatggact acaaggacca cgacggcgac
5520tacaaggacc acgacatcga ctacaaggac gacgacgaca agtgaaagct tgcagcagca
5580gctcggatag tatcgacaca ctctggacgc tggtcgtgtg atggactgtt gccgccacac
5640ttgctgcctt gacctgtgaa tatccctgcc gcttttatca aacagcctca gtgtgtttga
5700tcttgtgtgt acgcgctttt gcgagttgct agctgcttgt gctatttgcg aataccaccc
5760ccagcatccc cttccctcgt ttcatatcgc ttgcatccca accgcaactt atctacgctg
5820tcctgctatc cctcagcgct gctcctgctc ctgctcactg cccctcgcac agccttggtt
5880tgggctcccg cctgtattct cctggtactg caacctgtaa accagcactg caatgctgat
5940gcacgggaag tagtgggatg ggaacacaaa tggaaagctg gagctccagc gccatgccac
6000gccctttgat ggcttcaagt acgattacgg tgttggattg tgtgtttgtt gcgtagtgtg
6060catggtttag aataatacac ttgatttctt gctcacggca atctcggctt gtccgcaggt
6120tcaaccccat ttcggagtct caggtcagcc gcgcaatgac cagccgctac ttcaaggact
6180tgcacgacaa cgccgaggtg agctatgttt aggacttgat tggaaattgt cgtcgacgca
6240tattcgcgct ccgcgacagc acccaagcaa aatgtcaagt gcgttccgat ttgcgtccgc
6300aggtcgatgt tgtgatcgtc ggcgccggat ccgccggtct gtcctgcgct tacgagctga
6360ccaagcaccc tgacgtccgg gtacgcgagc tgagattcga ttagacataa attgaagatt
6420aaacccgtag aaaaatttga tggtcgcgaa actgtgctcg attgcaagaa attgatcgtc
6480ctccactccg caggtcgcca tcatcgagca gggcgttgct cccggcggcg gcgcctggct
6540ggggggacag ctgttctcgg ccatgtgtgt acgtagaagg atgaatttca gctggttttc
6600gttgcacagc tgtttgtgca tgatttgttt cagactattg ttgaatgttt ttagatttct
6660taggatgcat gatttgtctg catgcgactg aagagc
66961285851DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 128gctcttcgga gtcactgtgc cactgagttc
gactggtagc tgaatggagt cgctgctcca 60ctaaacgaat tgtcagcacc gccagccggc
cgaggacccg agtcatagcg agggtagtag 120cgcgccatgg caccgaccag cctgcttgcc
agtactggcg tctcttccgc ttctctgtgg 180tcctctgcgc gctccagcgc gtgcgctttt
ccggtggatc atgcggtccg tggcgcaccg 240cagcggccgc tgcccatgca gcgccgctgc
ttccgaacag tggcggtcag ggccgcaccc 300gcggtagccg tccgtccgga acccgcccaa
gagttttggg agcagcttga gccctgcaag 360atggcggagg acaagcgcat cttcctggag
gagcaccggt gcgtggaggt ccggggctga 420ccggccgtcg cattcaacgt aatcaatcgc
atgatgatca gaggacacga agtcttggtg 480gcggtggcca gaaacactgt ccattgcaag
ggcataggga tgcgttcctt cacctctcat 540ttctcatttc tgaatccctc cctgctcact
ctttctcctc ctccttcccg ttcacgcagc 600attcggggta ccgcggtgag aatcgaaaat
gcatcgtttc taggttcgga gacggtcaat 660tccctgctcc ggcgaatctg tcggtcaagc
tggccagtgg acaatgttgc tatggcagcc 720cgcgcacatg ggcctcccga cgcggccatc
aggagcccaa acagcgtgtc agggtatgtg 780aaactcaaga ggtccctgct gggcactccg
gccccactcc gggggcggga cgccaggcat 840tcgcggtcgg tcccgcgcga cgagcgaaat
gatgattcgg ttacgagacc aggacgtcgt 900cgaggtcgag aggcagcctc ggacacgtct
cgctagggca acgccccgag tccccgcgag 960ggccgtaaac attgtttctg ggtgtcggag
tgggcatttt gggcccgatc caatcgcctc 1020atgccgctct cgtctggtcc tcacgttcgc
gtacggcctg gatcccggaa agggcggatg 1080cacgtggtgt tgccccgcca ttggcgccca
cgtttcaaag tccccggcca gaaatgcaca 1140ggaccggccc ggctcgcaca ggccatgctg
aacgcccaga tttcgacagc aacaccatct 1200agaataatcg caaccatccg cgttttgaac
gaaacgaaac ggcgctgttt agcatgtttc 1260cgacatcgtg ggggccgaag catgctccgg
ggggaggaaa gcgtggcaca gcggtagccc 1320attctgtgcc acacgccgac gaggaccaat
ccccggcatc agccttcatc gacggctgcg 1380ccgcacatat aaagccggac gcctaaccgg
tttcgtggtt atgactagta tgttcgcgtt 1440ctacttcctg acggcctgca tctccctgaa
gggcgtgttc ggcgtctccc cctcctacaa 1500cggcctgggc ctgacgcccc agatgggctg
ggacaactgg aacacgttcg cctgcgacgt 1560ctccgagcag ctgctgctgg acacggccga
ccgcatctcc gacctgggcc tgaaggacat 1620gggctacaag tacatcatcc tggacgactg
ctggtcctcc ggccgcgact ccgacggctt 1680cctggtcgcc gacgagcaga agttccccaa
cggcatgggc cacgtcgccg accacctgca 1740caacaactcc ttcctgttcg gcatgtactc
ctccgcgggc gagtacacgt gcgccggcta 1800ccccggctcc ctgggccgcg aggaggagga
cgcccagttc ttcgcgaaca accgcgtgga 1860ctacctgaag tacgacaact gctacaacaa
gggccagttc ggcacgcccg agatctccta 1920ccaccgctac aaggccatgt ccgacgccct
gaacaagacg ggccgcccca tcttctactc 1980cctgtgcaac tggggccagg acctgacctt
ctactggggc tccggcatcg cgaactcctg 2040gcgcatgtcc ggcgacgtca cggcggagtt
cacgcgcccc gactcccgct gcccctgcga 2100cggcgacgag tacgactgca agtacgccgg
cttccactgc tccatcatga acatcctgaa 2160caaggccgcc cccatgggcc agaacgcggg
cgtcggcggc tggaacgacc tggacaacct 2220ggaggtcggc gtcggcaacc tgacggacga
cgaggagaag gcgcacttct ccatgtgggc 2280catggtgaag tcccccctga tcatcggcgc
gaacgtgaac aacctgaagg cctcctccta 2340ctccatctac tcccaggcgt ccgtcatcgc
catcaaccag gactccaacg gcatccccgc 2400cacgcgcgtc tggcgctact acgtgtccga
cacggacgag tacggccagg gcgagatcca 2460gatgtggtcc ggccccctgg acaacggcga
ccaggtcgtg gcgctgctga acggcggctc 2520cgtgtcccgc cccatgaaca cgaccctgga
ggagatcttc ttcgactcca acctgggctc 2580caagaagctg acctccacct gggacatcta
cgacctgtgg gcgaaccgcg tcgacaactc 2640cacggcgtcc gccatcctgg gccgcaacaa
gaccgccacc ggcatcctgt acaacgccac 2700cgagcagtcc tacaaggacg gcctgtccaa
gaacgacacc cgcctgttcg gccagaagat 2760cggctccctg tcccccaacg cgatcctgaa
cacgaccgtc cccgcccacg gcatcgcgtt 2820ctaccgcctg cgcccctcct cctgatacgt
agcagcagca gctcggatag tatcgacaca 2880ctctggacgc tggtcgtgtg atggactgtt
gccgccacac ttgctgcctt gacctgtgaa 2940tatccctgcc gcttttatca aacagcctca
gtgtgtttga tcttgtgtgt acgcgctttt 3000gcgagttgct agctgcttgt gctatttgcg
aataccaccc ccagcatccc cttccctcgt 3060ttcatatcgc ttgcatccca accgcaactt
atctacgctg tcctgctatc cctcagcgct 3120gctcctgctc ctgctcactg cccctcgcac
agccttggtt tgggctccgc ctgtattctc 3180ctggtactgc aacctgtaaa ccagcactgc
aatgctgatg cacgggaagt agtgggatgg 3240gaacacaaat ggagatatcg cgaggggtct
gcctgggcca gccgctccct ctaaacacgg 3300gacgcgtggt ccaattcggg cttcgggacc
ctttggcggt ttgaacgcca gggatggggc 3360gcccgcgagc ctggggaccc cggcaacggc
ttccccagag cctgccttgc aatctcgcgc 3420gtcctctccc tcagcacgtg gcggttccac
gtgtggtcgg gcttcccgga ctagctcgcg 3480tcgtgaccta gcttaatgaa cccagccggg
cctgtagcac cgcctaagag gttttgatta 3540tttcattata ccaatctatt cgccactagt
atggccatca agaccaaccg ccagcccgtg 3600gagaagcccc ccttcaccat cggcaccctg
cgcaaggcca tccccgccca ctgcttcgag 3660cgctccgccc tgcgctcctc catgtacctg
gccttcgaca tcgccgtgat gtccctgctg 3720tacgtggcct ccacctacat cgaccccgcc
cccgtgccca cctgggtgaa gtacggcgtg 3780atgtggcccc tgtactggtt cttccagggc
gccttcggca ccggcgtgtg ggtgtgcgcc 3840cacgagtgcg gccaccaggc cttctcctcc
tcccaggcca tcaacgacgg cgtgggcctg 3900gtgttccact ccctgctgct ggtgccctac
tactcctgga agcactccca ccgccgccac 3960cactccaaca ccggctgcct ggacaaggac
gaggtgttcg tgccccccca ccgcgccgtg 4020gcccacgagg gcctggagtg ggaggagtgg
ctgcccatcc gcatgggcaa ggtgctggtg 4080accctgaccc tgggctggcc cctgtacctg
atgttcaacg tggcctcccg cccctacccc 4140cgcttcgcca accacttcga cccctggtcc
cccatcttct ccaagcgcga gcgcatcgag 4200gtggtgatct ccgacctggc cctggtggcc
gtgctgtccg gcctgtccgt gctgggccgc 4260accatgggct gggcctggct ggtgaagacc
tacgtggtgc cctacctgat cgtgaacatg 4320tggctggtgc tgatcaccct gctgcagcac
acccaccccg ccctgcccca ctacttcgag 4380aaggactggg actggctgcg cggcgccatg
gccaccgtgg accgctccat gggccccccc 4440ttcatggaca acatcctgca ccacatctcc
gacacccacg tgctgcacca cctgttctcc 4500accatccccc actaccacgc cgaggaggcc
tccgccgcca tccgccccat cctgggcaag 4560tactaccagt ccgactcccg ctgggtgggc
cgcgccctgt gggaggactg gcgcgactgc 4620cgctacgtgg tgcccgacgc ccccgaggac
gactccgccc tgtggttcca caagtagatc 4680gatcttaagg cagcagcagc tcggatagta
tcgacacact ctggacgctg gtcgtgtgat 4740ggactgttgc cgccacactt gctgccttga
cctgtgaata tccctgccgc ttttatcaaa 4800cagcctcagt gtgtttgatc ttgtgtgtac
gcgcttttgc gagttgctag ctgcttgtgc 4860tatttgcgaa taccaccccc agcatcccct
tccctcgttt catatcgctt gcatcccaac 4920cgcaacttat ctacgctgtc ctgctatccc
tcagcgctgc tcctgctcct gctcactgcc 4980cctcgcacag ccttggtttg ggctccgcct
gtattctcct ggtactgcaa cctgtaaacc 5040agcactgcaa tgctgatgca cgggaagtag
tgggatggga acacaaatgg aaagcttaat 5100taagagctct tgttttccag aaggagttgc
tccttgagcc tttcattctc agcctcgata 5160acctccaaag ccgctctaat tgtggagggg
gttcgaattt aaaagcttgg aatgttggtt 5220cgtgcgtctg gaacaagccc agacttgttg
ctcactggga aaaggaccat cagctccaaa 5280aaacttgccg ctcaaaccgc gtacctctgc
tttcgcgcaa tctgccctgt tgaaatcgcc 5340accacattca tattgtgacg cttgagcagt
ctgtaattgc ctcagaatgt ggaatcatct 5400gccccctgtg cgagcccatg ccaggcatgt
cgcgggcgag gacacccgcc actcgtacag 5460cagaccatta tgctacctca caatagttca
taacagtgac catatttctc gaagctcccc 5520aacgagcacc tccatgctct gagtggccac
cccccggccc tggtgcttgc ggagggcagg 5580tcaaccggca tggggctacc gaaatccccg
accggatccc accacccccg cgatgggaag 5640aatctctccc cgggatgtgg gcccaccacc
agcacaacct gctggcccag gcgagcgtca 5700aaccatacca cacaaatatc cttggcatcg
gccctgaatt ccttctgccg ctctgctacc 5760cggtgcttct gtccgaagca ggggttgcta
gggatcgctc cgagtccgca aacccttgtc 5820gcgtggcggg gcttgttcga gcttgaagag c
5851129186DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
129tacaacttat tacgtaacgg agcgtcgtgc gggagggagt gtgccgagcg gggagtcccg
60gtctgtgcga ggcccggcag ctgacgctgg cgagccgtac gccccgaggg tccccctccc
120ctgcaccctc ttccccttcc ctctgacggc cgcgcctgtt cttgcatgtt cagcgacgag
180gatatc
186130305DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 130gcgaggggtc tgcctgggcc agccgctccc
tctgaacacg ggacgcgtgg tccaattcgg 60gcttcgggac cctttggcgg tttgaacgcc
tgggagaggg cgcccgcgag cctggggacc 120ccggcaacgg cttccccaga gcctgccttg
caatctcgcg cgtcctctcc ctcagcacgt 180ggcggttcca cgtgtggtcg ggcgtcccgg
actagctcac gtcgtgacct agcttaatga 240acccagccgg gcctgcagca ccaccttaga
ggttttgatt atttgattag accaatctat 300tcacc
305131305DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
131ggcgaataga ttggtataat gaaataatca aaacctctta ggcggtgcta caggcccggc
60tgggttcatt aagctaggtc acgacgcgag ctagtccggg aagcccgacc acacgtggaa
120ccgccacgtg ctgagggaga ggacgcgcga gattgcaagg caggctctgg ggaagccgtt
180gccggggtcc ccaggctcgc gggcgcccca tccctggcgt tcaaaccgcc aaagggtccc
240gaagcccgaa ttggaccacg cgtcccgtgt ttagagggag cggctggccc aggcagaccc
300ctcgc
305132305DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 132ggtgaataga ttggtctaat caaataatca
aaacctctaa ggtggtgctg caggcccggc 60tgggttcatt aagctaggtc acgacgtgag
ctagtccggg acgcccgacc acacgtggaa 120ccgccacgtg ctgagggaga ggacgcgcga
gattgcaagg caggctctgg ggaagccgtt 180gccggggtcc ccaggctcgc gggcgccctc
tcccaggcgt tcaaaccgcc aaagggtccc 240gaagcccgaa ttggaccacg cgtcccgtgt
tcagagggag cggctggccc aggcagaccc 300ctcgc
3051331322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
133gtgatgggtt ctttagacga tccagcccag gatcatgtgt tgcccacatg gagcctatcc
60acgctggcct agaaggcaag cacatttcaa ggtgaaccca cgtccatgga gcgatggcgc
120caatatctcg cctctagacc aagcggttct caccccaact gcgtcatttg tatgtatggc
180tgcaaagttg tcggtacgat agaggccgcc aacctggcgg cgagggcgag gagctggttg
240ccgatctgtg cccaagcatg tgtcggagct cggctgtctc ggcagcgagc tcctgtgcaa
300ggggcttgca tcgagaatgt caggcgatag acactgcacg ttggggacac ggaggtgccc
360ctgtggcgtg tcctggatgc cctcgggtcc gtcgcgagaa gctctggcga ccagcacccg
420gccacaaccg cagcaggcgt tcacccacaa gaatcttcca gatcgtgatg cgcatgtatc
480gtgacacgat tggcgaggtc cgcaggacgc acacggactc gtccactcat cagaactggt
540cagggcaccc atctgcgtcc cttttcagga accacccacc gctgccaggc accttcgcca
600gcggcggact ccacacagag aatgccttgc tgtgagagac catggccggc aagtgctgtc
660ggatctgccc gcatacggtc agtccccagc acaaggaagc caagagtaca ggctgttggt
720gtcgatggag gagtggccgt tcccacaagt agtgagcggc agctgctcaa cggcttcccc
780ctgttcatct tggcaaagcc agtgacttcc tacaagtatg tgatgcagat cggcactgca
840atctgtcggc atgcgtacag aacatcggct cgccagggca gcgttgctcg ctctggatga
900gctgcttggg aggaatcatc ggcacacgcc cgtgccgtgc ccgcgccccg cgcccgtcgg
960gaaaggcccc cggttaggac actgccgcgt cagccagtcg tgggatcgat cggacgtggc
1020gaatcctcgc ccggacaccc tcatcacacc ccacatttcc ctgcaagcaa tcttgccgac
1080aaaatagtca agatccattg ggtttaggga acacgtgcga gactgggcag ctgtatctgt
1140ccttgccccg cgtcaaattc ctgggcgtga cgcagtcaca ggagaatcta ttagaccctg
1200gacttgcagc tcagtcatgg gcgtgagtgg ctaaagcacc taggtcaggc gagtaccgcc
1260ccttccccag gattcactct tctgcgattg acgttgagcc tgcatcgggc tgcttcgtca
1320cc
1322134841DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 134tcggagctaa agcagagact ggacaagact
tgcgttcgca tactggtgac acagaatagc 60tcccatctat tcatacgcct ttgggaaaag
gaacgagcct tgtggcctct gcattgctgc 120ctgctttgag gccgaggacg gtgcgggacg
ctcagatcca tcagcgatcg ccccaccctc 180agagcacctc cgatccaagg caatactatc
aggcaaagtt tccaaattca aacattccaa 240aatcacgcca gggactggat cacacacgca
gatcagcgcc gttttgctct ttgcctacgg 300gcgactgtgc cacttgtcga cccctggtga
cgggagggac cacgcctgcg gttggcatcc 360acttcgacgg acccagggac ggtttctcat
gccaaacctg agatttgagc acccagatga 420gcacattatg cgttttagga tgcctgagca
gcgggcgtgc aggaatctgg tctcgccaga 480ttcaccgaag atgcgcccat cggagcgagg
cgagggcttt gtgaccacgc aaggcagtgt 540gaggcaaaca catagggaca cctgcgtctt
tcaatgcaca gacatctatg gtgcccatgt 600atataaaatg ggctacttct gagtcaaacc
aacgcaaact gcgctatggc aaggccggcc 660aaggttggaa tcccggtctg tctggatttg
agtttgtggg ggctatcacg tgacaatccc 720tgggattggg cggcagcagc gcacggcctg
ggtggcaatg gcgcactaat actgctgaaa 780gcacggctct gcatcccttt ctcttgacct
gcgattggtc cttttcgcaa gcgtgatcat 840c
841135841DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
135tcggagctaa agcagaaact gaacaagact tgcgttcgca tacttgtgac actgaatagg
60ttcaatctat tcatacgcct ttgggaaact gaacgagcct tgtggcctct gcattgctgc
120ctgctttgag gccgaggacg gcgcggaacg cacagatcca tcagcgatcg ccccaccctc
180agagtacatc cgatccaagg caatactatc aggcaaagtt tccaaattca aacattccaa
240aattacgtca gggactggat cacacacgca gatcagcgcc gttttgctct ttgcctacgg
300gcgactgtgc cacttgtcga cgcctggtga cgggagggac cacgcctgcg gttggcatcc
360acttcgacgg acccagggac ggtctcacat gccaaacctg agatttgagc accaagatga
420gcacattatg cgtttttgga tgcctgagca gcgggcgtgc aggaatctgg tctcgccaga
480ttcaccgaag atgcggccat cggagcgagg cgagggctgt gtggccacgc caggcagtgt
540gaggcaaaca cacagggaca tctgcttctt tcgatgcaca gacatctatg ttgcccgtgc
600atataaaatg ggctacttct gaatcaaacc aacgcaaact tcgctatggc aaggccggcc
660aaggttggaa tcccggtctg tctggatttg agtttgtggg ggctatcacg tgacaatccc
720tgggattggg cggcagcagc gcacggcctg gatggcaatg gcgcactaat actgctgaaa
780gcacggctct gcatcccttt ctcttgacct gcgattggtc cttttcgcaa gcgtgatcat
840c
841136512DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 136caccgatcac tccgtcgccg cccaagagaa
atcaacctcg atggagggcg aggtggatca 60gaggtattgg ttatcgttcg ttcttagtct
caatcaatcg tacaccttgc agttgcccga 120gtttctccac acatacagca cctcccgctc
ccagcccatt cgagcgaccc aatccgggcg 180atcccagcga tcgtcgtcgc ttcagtgctg
accggtggaa agcaggagat ctcgggcgag 240caggaccaca tccagcccag gatcttcgac
tggctcagag ctgaccctca cgcggcacag 300caaaagtagc acgcacgcgt tatgcaaact
ggttacaacc tgtccaacag tgttgcgacg 360ttgactggct acattgtctg tctgtcgcga
gtgcgcctgg gcccttacgg tgggacactg 420gaactccgcc ccgagtcgaa cacctagggc
gacgcccgca gcttggcatg acagctctcc 480ttgtgttcta aataccttgc gcgtgtggga
ga 512137516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
137atccaccgat cactccgtcg ccgcccaaga gaattcaacc tcgatggagg gcaaggtgga
60tcagaggtat tggttatcgt tcgctattag tctcaatcaa tcgtgcacct tgcagttgct
120cgagtttctc cacacataca gcacctcccg ctcccagccc attcgagcga cccaatccgg
180gcgatcccag cgatcgtcgt cgcttcagtg ctgaccggtg gaaagcagga gatctcgggc
240gagcaggacc acatccagca caggatcttc gactggctca gagctgaccc tcacgcggca
300cagcaaaagt agcccgcacg cgttatgcaa acaggttaca acctgtccaa cactgttgcg
360acgttgactg gctacattgt ctgtctgtcg cgagtacgcc tggaccctta cggtgggaca
420ctggaactcc gccccgagtc gaacacctag ggcgacgccc gcagcttggc atgacagctc
480tccttgtatt ctaaatacct cgcgcgtgtg ggagaa
516138335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 138atgatgcgcg tgtacgacta tcaaggaaga
aagaggactt aatttcttac cttctaacca 60ccatattctt tttgctggat gcttgctcgt
ctcgatgaca attgtgaacc tcttgtgtga 120ccctgaccct gctgcaaggc tctccgaccg
cacgcaaggc gcagccggcg cgtccggagg 180cgatcggatc caatccagtc gtcctcccgc
agcccgggca cgtttgccca tgcaggccct 240tccacaccgc tcaagagact cccgaacacc
gcccactcgg cactcgcttc ggctgccgag 300tgcgcgtttg agtttgccct gccacagaag
acacc 335139335DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
139atgatgcgcg tgtacgacta tcaaggaaga aagaggactt aatttcttac cttctaacca
60ccatattctt tttgctggat gcttgctcgt ctcgatgaca attgtgaacc tcttgtgtga
120ccctgaccct gctgcaaggc tctccgaccg cacgcaaggc gcagccggcg cgtccggagg
180cgatcggatc caatccagtc gtcctcccgc agcccgggca cgtttgccca tgcaggccct
240tccacaccgc tcaagagact cccgaacacc gcccactcgg cactcgcttc ggctgccgag
300tgcgcgtttg agtttgccct gccacaggag acatc
3351401097DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 140cccgggcgag ctgtacgcct acggagcgag
gcctggtgtg accgttgcga tctcgccagc 60agacgtcgcg gagcctcgtc ccaaaggccc
tttctgatcg agcttgtcgt ccactggacg 120ctttaagttg cgcgcgcgat gggataaccg
agctgatctg cactcagatt ttggtttgtt 180ttcgcgcatg gtgcagcgag gggaggtact
acgctggggt acgagatcct ccggattccc 240agaccgtgtt gccggcattt acccggtcat
cgccagcgat tcgggacgac aaggccttat 300cctgtgctga gacgctcgag cacgtttata
aaattgtggg taccgcggta tgcacagcgt 360tcaacacgcg ccacgccgaa attggttggt
gggggagcac gtatgggact gacgtatggc 420cagcagcgaa cactcaccga acaagtgcca
atgtatacct tgcatcaatg atgctccggc 480agcttcgatt gactgtctcg aaaaagtgtg
agcaagcaga tcatgtggcc gctctgtcgc 540gcagcacctg acgcattcga cacccacggc
aatgcccagg ccagggaata gagagtaaga 600caactcccat tgttcagcaa aacattgcac
tgcagtgcct tcacaactat acaatgaatg 660ggagggaata tgggctctgc atgggacagc
ttagctggga cattcggcta ctgaacaaga 720aaaccccacg agaaccaatt ggcgaaacct
gccgggagga ggtgatcgtt tctgtaaatg 780gcttacgcat tcccccccgg cggctcacga
ggggtgtggt gaaccctgcc agctgatcaa 840gtgcttgctg acgtcggcca gggaggtgta
tgtgattggg ccgtggggcg tgagttatcc 900taccgccgga cccgcgaagt cacatgacga
atggccgtgc gggatgacga gagcacgact 960cgctctttct tcgccggccc ggcttcatgg
aggacaataa taaagggtgg ccaccggcaa 1020cagccctcca tacctgaacc gattccagac
ccaaacctct tgaattttga gggatccagt 1080tcaccggtat agtcacg
10971411105DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
141atccccgggc gagctgtacg cctacggagc gaggcctggt gtgaccgttg cgatctcgcc
60agcagacgtc gcggagcctc gtcccaaagg ccctttctga tcgagcttgt cgtccactgg
120acgctttaag ttgcgcgcgc gatgggataa ccgagctgat ctgcactcag attttggttt
180gttttcgcgc atggtgcagc gaggggaggt actacgctgg ggtacgagat cctccggatt
240cccagaccgt gttgccggca tttacccggt catcgccagc gattcgggac gacaaggcct
300tatcctgtgc tgagacgctc gagcacgttt ataaaattgt ggtcaccgtg gtacgcacag
360cgtccaacac gcgccacgcc gaaattcgtt ggtgggggag cacgtatcgg actgacgtat
420ggccagcagc gaacactcac caaacaggtg ccaatgtata gcttgcatca atgatgctct
480ggcagcttcg attgactgtc tcgaaaaagt gtgtgcaaac agattatgtg gccgctctgt
540ggccgcgcag cacctgacgc actcgacacc cacggcaatg cccaggccaa ggaacagaga
600gtaagacaac tcccattgtt cagtaaaaca ttgcactgca gtgccttcac aaacatacaa
660cgaatgggag ggaatatggg cttcgaatgg gacagcttag ctgggacatt cggttactga
720acaagaaaac cccacgagaa ccaactggcg aaacctgccg ggaggaggtg atcgtttttg
780taaatggctt acgcattccc cccccggcgg ctcacggggg gtgtggtgaa ccctgccagc
840tgatcaagtg cttgctgacg tcggccaggg aggtgtatgt gatttggccg tggggcgtga
900gttatcctac cgccggaccc gcgaagtcac atgacgaatg gccgtgcggg atgacgagag
960cagggctcgc tctttcttcg ccggcccggc ttcatggagg acaataataa agggtggcca
1020ccggcaacag ccctccatac ctgaaccgat tccagaccca aacctcttga attttgaggg
1080atccagttca ccggtatagt cacga
1105142754DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 142gcgagtggtt ttgctgccgg gaagggagtg
gggagcgtcg agcgagggac gcggcgctcg 60aggcgcacgt cgtctgtcaa cgcgcgcggc
cctcgcggcc cgcggcccca cccagctcta 120atcatcgaaa actaagaggc tccacacgcc
tgtcgtagaa tgcatgggat tcgccagtag 180accacgatct gcgccgaaga agctggtcta
cccgacgttt tttgttgctc ctttattctg 240aatgatatga agatagtgtg cgcagtgcca
cgcataggca tcaggagcaa gggaggacgg 300gtcaacttga aagaaccaaa ccatccatcc
gagaaatgcg catcatcttt gtagtaccat 360caaacgcctt ggccaatgtc ttctgcatgg
acaacacaac ctgctcctgg ccacacggtc 420gacttggagc gccccatgcg cccaggtcgc
cacgacccgc ggcccagcgc gcggcgattc 480gcctcacgag atcccggcgg acccggcacg
cccgcgggcc gacggtgcgc ttggcgatgc 540tgctcattaa cccacggccg tcacccgatc
cacatgctct ttttcaacac atccacattg 600gaatagagct ctaccagggt gagtactgca
ttctttgggg ctgggaggac cccactcgac 660acctggtcct tcatcggccg aaagcccgaa
cctgagcgct tccccgcccc gttcctcatc 720cccgactttc cgatggccca ttgcagtttc
aaac 754143318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
143atctgggtgg aggactggga gtaagatgta aggatattaa ttaaacattc tagtttgttg
60atggcacaac agtcaatgca tttcagtcgt cttgctcctt ataacctatg cgtgtgccat
120cgccggccat gcacctgtgg cgtggtaccg accatcgggg agaggcccga gattcggagg
180tacctcccgc cctgggcgag cccttcacgt gacggcacaa gtcccttgca tcggcccgcg
240agcacggaat acagagcccc gtgcccccca cgggccctca catcatccac tccattgttc
300ttgccacacc gatcagca
318144316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 144tgggtggagg actgggaaga agatgtaagg
atatcaattt aacattctag tttgttgatg 60gcacaacagt cactgaatac cgggcgtctg
gctgctaaaa tagccggagc gtgtgccatc 120gccggccatg catctgtggc gtggtaccga
ccatcaggga gaggcccgag attcggaggt 180acctcccgcc ctgggcgagc ccttcacgtg
acggcacaag tcccttgcat cggcccgcga 240gcacggaata cagagccccg tgctccccac
gggccctcac atcatccact ccattgttct 300tgccacaccg atcagc
316145350DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
145ataacgaggc acaatgatcg atatttctat cgaacaactg tatttagccc tgtacgtacc
60ccgctcttgg gccagcccgt ccgtgcttgc cttcggaaaa ttgcatggcg cctcatgcaa
120actcgcgctc tcacagcaga tctcgcccag ctcccgggag agcaatcgcg ggtggggccc
180ggggcgaatc caggacgcgc cccgcggggc cgctccactc gccagggcca atgggcggct
240tatagtcctg gcatgggctc tgcatgcaca gtatcgcagt ttgggcgagg tgttgccccc
300gcgatttcga atacgcgacg cccggtactc gtgcgagaac agggttcttg
350146818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 146atcgcgatgg tgcgcactcg tgcgcaatga
atatggggtc acgcggtgga cgaacgcgga 60gggggcctgg ccgaatctag gcttgcattc
ctcagatcac tttctgccgg cggtccgggg 120tttgcgcgtc gcgcaacgct ccgtctccct
agccgctgcg caccgcgcgt gcgacgcgaa 180ggtcattttc cagaacaacg accatggctt
gtcttagcga tcgctcgaat gactgctagt 240gagtcgtacg ctcgacccag tcgctcgcag
gagaacgcgg caactgccga gcttcggctt 300gccagtcgtg actcgtatgt gatcaggaat
cattggcatt ggtagcatta taattcggct 360tccgcgctgt ttatgggcat ggcaatgtct
catgcagtcg accttagtca accaattctg 420ggtggccagc tccgggcgac cgggctccgt
gtcgccgggc accacctcct gccatgagta 480acagggccgc cctctcctcc cgacgttggc
ccactgaata ccgtgtcttg gggccctaca 540tgatgggctg cctagtcggg cgggacgcgc
aactgcccgc gcaatctggg acgtggtctg 600aatcctccag gcgggtttcc ccgagaaaga
aagggtgccg atttcaaagc agagccatgt 660gccgggccct gtggcctgtg ttggcgccta
tgtagtcacc ccccctcacc caattgtcgc 720cagtttgcgc aatccataaa ctcaaaactg
cagcttctga gctgcgctgt tcaagaacac 780ctctggggtt tgctcacccg cgaggtcgac
gcccagca 818147819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
147atcacgatgg tgcgcattcg tgcaaagtga atatggggtc acgcggtgga cgaacgcgga
60gggggcatga ccgaatctag gctcgcattc ctcagatcac ttcatgccgg cggtccgggg
120tttgcgcgtc gcgcaaggct acgtctccct agccgctgcg caccacgcgt gcgacgcgga
180ggccatcttc cggagcaacg accatggatt gtcttagcga tcgcacgaat gagtgctagt
240gagtcgtacg ctcgacccag tcgctcgcag gagaaggcgg cagctgccga gcttcggctt
300accagtcgtg actcgtatgt gatcaggaat cattggcatt ggtagcatta taattcggct
360tccgcgctgc gtatgggcat ggcaatgtct catgcagtcg atcttagtca accaattttg
420ggtggccagg tccgggcgac cgggctccgt gtcgccgggc accacctcct gccaggagta
480gcagggccgc cctctcgtcc cgacgttggc ccactgaata ccgtggcttc gagccctaca
540tgatgggctg cctagtcggg cgggacgcgc aactgcccgc gcgatctggg ggctggtctg
600aatccttcag gcgggtgtta cccgagaaag aaagggtgcc gatttcaaag cagacccatg
660tgccgggccc tgtggcctgt gttggcgcct atgtagtcac cccccctcac ccaattgtcg
720ccagtttgcg cactccataa actcaaaaca gcagcttctg agctgcgctg ttcaagaaca
780cctctggggt ttgctcaccc gcgaggtcga cgcccagca
8191485104DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 148gaagagcgcc caatgtttaa acgccggtca
ccacccgcat gctcgtacta cagcgcacgc 60accgcttcgt gatccaccgg gtgaacgtag
tcctcgacgg aaacatctgg ttcgggcctc 120ctgcttgcac tcccgcccat gccgacaacc
tttctgctgt taccacgacc cacaatgcaa 180cgcgacacga ccgtgtggga ctgatcggtt
cactgcacct gcatgcaatt gtcacaagcg 240cttactccaa ttgtattcgt ttgttttctg
ggagcagttg ctcgaccgcc cgcgtcccgc 300aggcagcgat gacgtgtgcg tggcctgggt
gtttcgtcga aaggccagca accctaaatc 360gcaggcgatc cggagattgg gatctgatcc
gagtttggac cagatccgcc ccgatgcggc 420acgggaactg catcgactcg gcgcggaacc
cagctttcgt aaatgccaga ttggtgtccg 480atacctggat ttgccatcag cgaaacaaga
cttcagcagc gagcgtattt ggcgggcgtg 540ctaccagggt tgcatacatt gcccatttct
gtctggaccg ctttactggc gcagagggtg 600agttgatggg gttggcaggc atcgaaacgc
gcgtgcatgg tgtgcgtgtc tgttttcggc 660tgcacgaatt caatagtcgg atgggcgacg
gtagaattgg gtgtggcgct cgcgtgcatg 720cctcgccccg tcgggtgtca tgaccgggac
tggaatcccc cctcgcgacc atcttgctaa 780cgctcccgac tctcccgacc gcgcgcagga
tagactcttg ttcaaccaat cgacaggtac 840cagtttaggt ccagcgtccg tgggggggga
cgggctggga gcttgggccg ggaagggcaa 900gacgatgcag tccctctggg gagtcacagc
cgactgtgtg tgttgcactg tgcggcccgc 960agcactcaca cgcaaaatgc ctggccgaca
ggcaggccct gtccagtgca acatccacgg 1020tccctctcat caggctcacc ttgctcattg
acataacgga atgcgtaccg ctctttcaga 1080tctgtccatc cagagagggg agcaggctcc
ccaccgacgc tgtcaaactt gcttcctgcc 1140caaccgaaaa cattattgtt tgaggggggg
gggggggggg cagattgcat ggcgggatat 1200ctcgtgagga acatcactgg gacactgtgg
aacacagtga gtgcagtatg cagagcatgt 1260atgctagggg tcagcgcagg aagggggcct
ttcccagtct cccatgccac tgcaccgtat 1320ccacgactca ccaggaccag cttcttgatc
ggcttccgct cccgtggaca ccagtgtgta 1380gcctctggac tccaggtatg cgtgcaccgc
aaaggccagc cgatcgtgcc gattcctggg 1440tggaggatat gagtcagcca acttggggct
cagagtgcac actggggcac gatacgaaac 1500aacatctaca ccgtgtcctc catgctgaca
caccacagct tcgctccacc tgaatgtggg 1560cgcatgggcc cgaatcacag ccaatgtcgc
tgctgccata atgtgatcca gaccctctcc 1620gcccagatgc cgagcggatc gtgggcgctg
aatagattcc tgtttcgatc actgtttggg 1680tcctttcctt ttcgtctcgg atgcgcgtct
cgaaacaggc tgcgtcgggc tttcggatcc 1740cttttgctcc ctccgtcacc atcctgcgcg
cgggcaagtt gcttgaccct gggctgatac 1800cagggttgga gggtattacc gcgtcaggcc
attcccagcc cggattcaat tcaaagtctg 1860ggccaccacc ctccgccgct ctgtctgatc
actccacatt cgtgcataca ctacgttcaa 1920gtcctgatcc aggcgtgtct cgggacaagg
tgtgcttgag tttgaatctc aaggacccac 1980tccagcacag ctgctggttg accccgccct
cgcaatctag aatggccgcg tccgtccact 2040gcaccctgat gtccgtggtc tgcaacaaca
agaaccactc cgcccgcccc aagctgccca 2100actcctccct gctgcccggc ttcgacgtgg
tggtccaggc cgcggccacc cgcttcaaga 2160aggagacgac gaccacccgc gccacgctga
cgttcgaccc ccccacgacc aactccgagc 2220gcgccaagca gcgcaagcac accatcgacc
cctcctcccc cgacttccag cccatcccct 2280ccttcgagga gtgcttcccc aagtccacga
aggagcacaa ggaggtggtg cacgaggagt 2340ccggccacgt cctgaaggtg cccttccgcc
gcgtgcacct gtccggcggc gagcccgcct 2400tcgacaacta cgacacgtcc ggcccccaga
acgtcaacgc ccacatcggc ctggcgaagc 2460tgcgcaagga gtggatcgac cgccgcgaga
agctgggcac gccccgctac acgcagatgt 2520actacgcgaa gcagggcatc atcacggagg
agatgctgta ctgcgcgacg cgcgagaagc 2580tggaccccga gttcgtccgc tccgaggtcg
cgcggggccg cgccatcatc ccctccaaca 2640agaagcacct ggagctggag cccatgatcg
tgggccgcaa gttcctggtg aaggtgaacg 2700cgaacatcgg caactccgcc gtggcctcct
ccatcgagga ggaggtctac aaggtgcagt 2760gggccaccat gtggggcgcc gacaccatca
tggacctgtc cacgggccgc cacatccacg 2820agacgcgcga gtggatcctg cgcaactccg
cggtccccgt gggcaccgtc cccatctacc 2880aggcgctgga gaaggtggac ggcatcgcgg
agaacctgaa ctgggaggtg ttccgcgaga 2940cgctgatcga gcaggccgag cagggcgtgg
actacttcac gatccacgcg ggcgtgctgc 3000tgcgctacat ccccctgacc gccaagcgcc
tgacgggcat cgtgtcccgc ggcggctcca 3060tccacgcgaa gtggtgcctg gcctaccaca
aggagaactt cgcctacgag cactgggacg 3120acatcctgga catctgcaac cagtacgacg
tcgccctgtc catcggcgac ggcctgcgcc 3180ccggctccat ctacgacgcc aacgacacgg
cccagttcgc cgagctgctg acccagggcg 3240agctgacgcg ccgcgcgtgg gagaaggacg
tgcaggtgat gaacgagggc cccggccacg 3300tgcccatgca caagatcccc gagaacatgc
agaagcagct ggagtggtgc aacgaggcgc 3360ccttctacac cctgggcccc ctgacgaccg
acatcgcgcc cggctacgac cacatcacct 3420ccgccatcgg cgcggccaac atcggcgccc
tgggcaccgc cctgctgtgc tacgtgacgc 3480ccaaggagca cctgggcctg cccaaccgcg
acgacgtgaa ggcgggcgtc atcgcctaca 3540agatcgccgc ccacgcggcc gacctggcca
agcagcaccc ccacgcccag gcgtgggacg 3600acgcgctgtc caaggcgcgc ttcgagttcc
gctggatgga ccagttcgcg ctgtccctgg 3660accccatgac ggcgatgtcc ttccacgacg
agacgctgcc cgcggacggc gcgaaggtcg 3720cccacttctg ctccatgtgc ggccccaagt
tctgctccat gaagatcacg gaggacatcc 3780gcaagtacgc cgaggagaac ggctacggct
ccgccgagga ggccatccgc cagggcatgg 3840acgccatgtc cgaggagttc aacatcgcca
agaagacgat ctccggcgag cagcacggcg 3900aggtcggcgg cgagatctac ctgcccgagt
cctacgtcaa ggccgcgcag aagtgacaat 3960tggcagcagc agctcggata gtatcgacac
actctggacg ctggtcgtgt gatggactgt 4020tgccgccaca cttgctgcct tgacctgtga
atatccctgc cgcttttatc aaacagcctc 4080agtgtgtttg atcttgtgtg tacgcgcttt
tgcgagttgc tagctgcttg tgctatttgc 4140gaataccacc cccagcatcc ccttccctcg
tttcatatcg cttgcatccc aaccgcaact 4200tatctacgct gtcctgctat ccctcagcgc
tgctcctgct cctgctcact gcccctcgca 4260cagccttggt ttgggctccg cctgtattct
cctggtactg caacctgtaa accagcactg 4320caatgctgat gcacgggaag tagtgggatg
ggaacacaaa tggaggatcg tagagctcta 4380gggagcgacg agtgtgcgtg cggggctggc
gggagtggga cgccctcctc gctcctctct 4440gttctgaacg gaacaatcgg ccaccccgcg
ctacgcgcca cgcatcgagc aacgaagaaa 4500accccccgat gataggttgc ggtggctgcc
gggatataga tccggccgca catcaaaggg 4560cccctccgcc agagaagaag ctcctttccc
agcagactcc ttctgctgcc aaaacacttc 4620tctgtccaca gcaacaccaa aggatgaaca
gatcaacttg cgtctccgcg tagcttcctc 4680ggctagcgtg cttgcaacag gtccctgcac
tattatcttc ctgctttcct ctgaattatg 4740cggcaggcga gcgctcgctc tggcgagcgc
tccttcgcgc cgccctcgct gatcgagtgt 4800acagtcaatg aatggtcctg ggcgaagaac
gagggaattt gtgggtaaaa caagcatcgt 4860ctctcaggcc ccggcgcagt ggccgttaaa
gtccaagacc gtgaccaggc agcgcagcgc 4920gtccgtgtgc gggccctgcc tggcggctcg
gcgtgccagg ctcgagagca gctccctcag 4980gtcgccttgg acggcctctg cgaggccggt
gagggcctgc aggagcgcct cgagcgtggc 5040agtggcggtc gtatccgggt cgccggtcac
cgcctgcgac tcgccatccg aagagcgttt 5100aaac
51041495110DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
149gtttaaacgc cggtcaccac ccgcatgctc gtactacagc gcacgcaccg cttcgtgatc
60caccgggtga acgtagtcct cgacggaaac atctggttcg ggcctcctgc ttgcactccc
120gcccatgccg acaacctttc tgctgttacc acgacccaca atgcaacgcg acacgaccgt
180gtgggactga tcggttcact gcacctgcat gcaattgtca caagcgctta ctccaattgt
240attcgtttgt tttctgggag cagttgctcg accgcccgcg tcccgcaggc agcgatgacg
300tgtgcgtggc ctgggtgttt cgtcgaaagg ccagcaaccc taaatcgcag gcgatccgga
360gattgggatc tgatccgagt ttggaccaga tccgccccga tgcggcacgg gaactgcatc
420gactcggcgc ggaacccagc tttcgtaaat gccagattgg tgtccgatac ctggatttgc
480catcagcgaa acaagacttc agcagcgagc gtatttggcg ggcgtgctac cagggttgca
540tacattgccc atttctgtct ggaccgcttt actggcgcag agggtgagtt gatggggttg
600gcaggcatcg aaacgcgcgt gcatggtgtg cgtgtctgtt ttcggctgca cgaattcaat
660agtcggatgg gcgacggtag aattgggtgt ggcgctcgcg tgcatgcctc gccccgtcgg
720gtgtcatgac cgggactgga atcccccctc gcgaccatct tgctaacgct cccgactctc
780ccgaccgcgc gcaggataga ctcttgttca accaatcgac aggtaccagt ttaggtccag
840cgtccgtggg gggggacggg ctgggagctt gggccgggaa gggcaagacg atgcagtccc
900tctggggagt cacagccgac tgtgtgtgtt gcactgtgcg gcccgcagca ctcacacgca
960aaatgcctgg ccgacaggca ggccctgtcc agtgcaacat ccacggtccc tctcatcagg
1020ctcaccttgc tcattgacat aacggaatgc gtaccgctct ttcagatctg tccatccaga
1080gaggggagca ggctccccac cgacgctgtc aaacttgctt cctgcccaac cgaaaacatt
1140attgtttgag gggggggggg ggggggcaga ttgcatggcg ggatatctcg tgaggaacat
1200cactgggaca ctgtggaaca cagtgagtgc agtatgcaga gcatgtatgc taggggtcag
1260cgcaggaagg gggcctttcc cagtctccca tgccactgca ccgtatccac gactcaccag
1320gaccagcttc ttgatcggct tccgctcccg tggacaccag tgtgtagcct ctggactcca
1380ggtatgcgtg caccgcaaag gccagccgat cgtgccgatt cctgggtgga ggatatgagt
1440cagccaactt ggggctcaga gtgcacactg gggcacgata cgaaacaaca tctacaccgt
1500gtcctccatg ctgacacacc acagcttcgc tccacctgaa tgtgggcgca tgggcccgaa
1560tcacagccaa tgtcgctgct gccataatgt gatccagacc ctctccgccc agatgccgag
1620cggatcgtgg gcgctgaata gattcctgtt tcgatcactg tttgggtcct ttccttttcg
1680tctcggatgc gcgtctcgaa acaggctgcg tcgggctttc ggatcccttt tgctccctcc
1740gtcaccatcc tgcgcgcggg caagttgctt gaccctgggc tgataccagg gttggagggt
1800attaccgcgt caggccattc ccagcccgga ttcaattcaa agtctgggcc accaccctcc
1860gccgctctgt ctgatcactc cacattcgtg catacactac gttcaagtcc tgatccaggc
1920gtgtctcggg acaaggtgtg cttgagtttg aatctcaagg acccactcca gcacagctgc
1980tggttgaccc cgccctcgca atctagaatg gccgcgtccg tccactgcac cctgatgtcc
2040gtggtctgca acaacaagaa ccactccgcc cgccccaagc tgcccaactc ctccctgctg
2100cccggcttcg acgtggtggt ccaggccgcg gccacccgct tcaagaagga gacgacgacc
2160acccgcgcca cgctgacgtt cgaccccccc acgaccaact ccgagcgcgc caagcagcgc
2220aagcacacca tcgacccctc ctcccccgac ttccagccca tcccctcctt cgaggagtgc
2280ttccccaagt ccacgaagga gcacaaggag gtggtgcacg aggagtccgg ccacgtcctg
2340aaggtgccct tccgccgcgt gcacctgtcc ggcggcgagc ccgccttcga caactacgac
2400acgtccggcc cccagaacgt caacgcccac atcggcctgg cgaagctgcg caaggagtgg
2460atcgaccgcc gcgagaagct gggcacgccc cgctacacgc agatgtacta cgcgaagcag
2520ggcatcatca cggaggagat gctgtactgc gcgacgcgcg agaagctgga ccccgagttc
2580gtccgctccg aggtcgcgcg gggccgcgcc atcatcccct ccaacaagaa gcacctggag
2640ctggagccca tgatcgtggg ccgcaagttc ctggtgaagg tgaacgcgaa catcggcaac
2700tccgccgtgg cctcctccat cgaggaggag gtctacaagg tgcagtgggc caccatgtgg
2760ggcgccgaca ccatcatgga cctgtccacg ggccgccaca tccacgagac gcgcgagtgg
2820atcctgcgca actccgcggt ccccgtgggc accgtcccca tctaccaggc gctggagaag
2880gtggacggca tcgcggagaa cctgaactgg gaggtgttcc gcgagacgct gatcgagcag
2940gccgagcagg gcgtggacta cttcacgatc cacgcgggcg tgctgctgcg ctacatcccc
3000ctgaccgcca agcgcctgac gggcatcgtg tcccgcggcg gctccatcca cgcgaagtgg
3060tgcctggcct accacaagga gaacttcgcc tacgagcact gggacgacat cctggacatc
3120tgcaaccagt acgacgtcgc cctgtccatc ggcgacggcc tgcgccccgg ctccatctac
3180gacgccaacg acacggccca gttcgccgag ctgctgaccc agggcgagct gacgcgccgc
3240gcgtgggaga aggacgtgca ggtgatgaac gagggccccg gccacgtgcc catgcacaag
3300atccccgaga acatgcagaa gcagctggag tggtgcaacg aggcgccctt ctacaccctg
3360ggccccctga cgaccgacat cgcgcccggc tacgaccaca tcacctccgc catcggcgcg
3420gccaacatcg gcgccctggg caccgccctg ctgtgctacg tgacgcccaa ggagcacctg
3480ggcctgccca accgcgacga cgtgaaggcg ggcgtcatcg cctacaagat cgccgcccac
3540gcggccgacc tggccaagca gcacccccac gcccaggcgt gggacgacgc gctgtccaag
3600gcgcgcttcg agttccgctg gatggaccag ttcgcgctgt ccctggaccc catgacggcg
3660atgtccttcc acgacgagac gctgcccgcg gacggcgcga aggtcgccca cttctgctcc
3720atgtgcggcc ccaagttctg ctccatgaag atcacggagg acatccgcaa gtacgccgag
3780gagaacggct acggctccgc cgaggaggcc atccgccagg gcatggacgc catgtccgag
3840gagttcaaca tcgccaagaa gacgatctcc ggcgagcagc acggcgaggt cggcggcgag
3900atctacctgc ccgagtccta cgtcaaggcc gcgcagaagt gacaattggc agcagcagct
3960cggatagtat cgacacactc tggacgctgg tcgtgtgatg gactgttgcc gccacacttg
4020ctgccttgac ctgtgaatat ccctgccgct tttatcaaac agcctcagtg tgtttgatct
4080tgtgtgtacg cgcttttgcg agttgctagc tgcttgtgct atttgcgaat accaccccca
4140gcatcccctt ccctcgtttc atatcgcttg catcccaacc gcaacttatc tacgctgtcc
4200tgctatccct cagcgctgct cctgctcctg ctcactgccc ctcgcacagc cttggtttgg
4260gctccgcctg tattctcctg gtactgcaac ctgtaaacca gcactgcaat gctgatgcac
4320gggaagtagt gggatgggaa cacaaatgga ggatcgtaga gctccagcca cggcaacacc
4380gcgcgccttg cggccgagca cggcgacaag aacctgagca agatctgcgg gctgatcgcc
4440agcgacgagg gccggcacga gatcgcctac acgcgcatcg tggacgagtt cttccgcctc
4500gaccccgagg gcgccgtcgc cgcctacgcc aacatgatgc gcaagcagat caccatgccc
4560gcgcacctca tggacgacat gggccacggc gaggccaacc cgggccgcaa cctcttcgcc
4620gacttctccg cggtcgccga gaagatcgac gtctacgacg ccgaggacta ctgccgcatc
4680ctggagcacc tcaacgcgcg ctggaaggtg gacgagcgcc aggtcagcgg ccaggccgcc
4740gcggaccagg agtacgtcct gggcctgccc cagcgcttcc ggaaactcgc cgagaagacc
4800gccgccaagc gcaagcgcgt cgcgcgcagg cccgtcgcct tctcctggat ctccgggcgc
4860gagatcatgg tctagggagc gacgagtgtg cgtgcggggc tggcgggagt gggacgccct
4920cctcgctcct ctctgttctg aacggaacaa tcggccaccc cgcgctacgc gccacgcatc
4980gagcaacgaa gaaaaccccc cgatgatagg ttgcggtggc tgccgggata tagatccggc
5040cgcacatcaa agggcccctc cgccagagaa gaagctcctt tcccagcaga ctcctgaaga
5100gcgtttaaac
51101505129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 150gaagagcgcc caatgtttaa acgccggtca
ccatccgcat gctcatatta cagcgcacgc 60accgcttcgt gatccaccgg gtgaacgtag
tcctcgacgg aaacatctgg ctcgggcctc 120gtgctggcac tccctcccat gccgacaacc
tttctgctgt caccacgacc cacgatgcaa 180cgcgacacga cccggtggga ctgatcggtt
cactgcacct gcatgcaatt gtcacaagcg 240catactccaa tcgtatccgt ttgatttctg
tgaaaactcg ctcgaccgcc cgcgtcccgc 300aggcagcgat gacgtgtgcg tgacctgggt
gtttcgtcga aaggccagca accccaaatc 360gcaggcgatc cggagattgg gatctgatcc
gagcttggac cagatccccc acgatgcggc 420acgggaactg catcgactcg gcgcggaacc
cagctttcgt aaatgccaga ttggtgtccg 480ataccttgat ttgccatcag cgaaacaaga
cttcagcagc gagcgtattt ggcgggcgtg 540ctaccagggt tgcatacatt gcccatttct
gtctggaccg ctttaccggc gcagagggtg 600agttgatggg gttggcaggc atcgaaacgc
gcgtgcatgg tgtgtgtgtc tgttttcggc 660tgcacaattt caatagtcgg atgggcgacg
gtagaattgg gtgttgcgct cgcgtgcatg 720cctcgccccg tcgggtgtca tgaccgggac
tggaatcccc cctcgcgacc ctcctgctaa 780cgctcccgac tctcccgccc gcgcgcagga
tagactctag ttcaaccaat cgacaggtac 840cagtttaggt ccagcgtccg tgggggggga
cgggctggga gcttgggccg ggaagggcaa 900gacgatgcag tccctctggg gagtcacagc
cgactgtgtg tgttgcactg tgcggcccgc 960agcactcaca cgcaaaatgc ctggccgaca
ggcaggccct gtccagtgca acatccacgg 1020tccctctcat caggctcacc ttgctcattg
acataacgga atgcgtaccg ctctttcaga 1080tctgtccatc cagagagggg agcaggctcc
ccaccgacgc tgtcaaactt gcttcctgcc 1140caaccgaaaa cattattgtt tgaggggggg
gggggggggg cagattgcat ggcgggatat 1200ctcgtgagga acatcactgg gacactgtgg
aacacagtga gtgcagtatg cagagcatgt 1260atgctagggg tcagcgcagg aagggggcct
ttcccagtct cccatgccac tgcaccgtat 1320ccacgactca ccaggaccag cttcttgatc
ggcttccgct cccgtggaca ccagtgtgta 1380gcctctggac tccaggtatg cgtgcaccgc
aaaggccagc cgatcgtgcc gattcctggg 1440tggaggatat gagtcagcca acttggggct
cagagtgcac actggggcac gatacgaaac 1500aacatctaca ccgtgtcctc catgctgaca
caccacagct tcgctccacc tgaatgtggg 1560cgcatgggcc cgaatcacag ccaatgtcgc
tgctgccata atgtgatcca gaccctctcc 1620gcccagatgc cgagcggatc gtgggcgctg
aatagattcc tgtttcgatc actgtttggg 1680tcctttcctt ttcgtctcgg atgcgcgtct
cgaaacaggc tgcgtcgggc tttcggatcc 1740cttttgctcc ctccgtcacc atcctgcgcg
cgggcaagtt gcttgaccct gggctgatac 1800cagggttgga gggtattacc gcgtcaggcc
attcccagcc cggattcaat tcaaagtctg 1860ggccaccacc ctccgccgct ctgtctgatc
actccacatt cgtgcataca ctacgttcaa 1920gtcctgatcc aggcgtgtct cgggacaagg
tgtgcttgag tttgaatctc aaggacccac 1980tccagcacag ctgctggttg accccgccct
cgcaatctag aatggccgcg tccgtccact 2040gcaccctgat gtccgtggtc tgcaacaaca
agaaccactc cgcccgcccc aagctgccca 2100actcctccct gctgcccggc ttcgacgtgg
tggtccaggc cgcggccacc cgcttcaaga 2160aggagacgac gaccacccgc gccacgctga
cgttcgaccc ccccacgacc aactccgagc 2220gcgccaagca gcgcaagcac accatcgacc
cctcctcccc cgacttccag cccatcccct 2280ccttcgagga gtgcttcccc aagtccacga
aggagcacaa ggaggtggtg cacgaggagt 2340ccggccacgt cctgaaggtg cccttccgcc
gcgtgcacct gtccggcggc gagcccgcct 2400tcgacaacta cgacacgtcc ggcccccaga
acgtcaacgc ccacatcggc ctggcgaagc 2460tgcgcaagga gtggatcgac cgccgcgaga
agctgggcac gccccgctac acgcagatgt 2520actacgcgaa gcagggcatc atcacggagg
agatgctgta ctgcgcgacg cgcgagaagc 2580tggaccccga gttcgtccgc tccgaggtcg
cgcggggccg cgccatcatc ccctccaaca 2640agaagcacct ggagctggag cccatgatcg
tgggccgcaa gttcctggtg aaggtgaacg 2700cgaacatcgg caactccgcc gtggcctcct
ccatcgagga ggaggtctac aaggtgcagt 2760gggccaccat gtggggcgcc gacaccatca
tggacctgtc cacgggccgc cacatccacg 2820agacgcgcga gtggatcctg cgcaactccg
cggtccccgt gggcaccgtc cccatctacc 2880aggcgctgga gaaggtggac ggcatcgcgg
agaacctgaa ctgggaggtg ttccgcgaga 2940cgctgatcga gcaggccgag cagggcgtgg
actacttcac gatccacgcg ggcgtgctgc 3000tgcgctacat ccccctgacc gccaagcgcc
tgacgggcat cgtgtcccgc ggcggctcca 3060tccacgcgaa gtggtgcctg gcctaccaca
aggagaactt cgcctacgag cactgggacg 3120acatcctgga catctgcaac cagtacgacg
tcgccctgtc catcggcgac ggcctgcgcc 3180ccggctccat ctacgacgcc aacgacacgg
cccagttcgc cgagctgctg acccagggcg 3240agctgacgcg ccgcgcgtgg gagaaggacg
tgcaggtgat gaacgagggc cccggccacg 3300tgcccatgca caagatcccc gagaacatgc
agaagcagct ggagtggtgc aacgaggcgc 3360ccttctacac cctgggcccc ctgacgaccg
acatcgcgcc cggctacgac cacatcacct 3420ccgccatcgg cgcggccaac atcggcgccc
tgggcaccgc cctgctgtgc tacgtgacgc 3480ccaaggagca cctgggcctg cccaaccgcg
acgacgtgaa ggcgggcgtc atcgcctaca 3540agatcgccgc ccacgcggcc gacctggcca
agcagcaccc ccacgcccag gcgtgggacg 3600acgcgctgtc caaggcgcgc ttcgagttcc
gctggatgga ccagttcgcg ctgtccctgg 3660accccatgac ggcgatgtcc ttccacgacg
agacgctgcc cgcggacggc gcgaaggtcg 3720cccacttctg ctccatgtgc ggccccaagt
tctgctccat gaagatcacg gaggacatcc 3780gcaagtacgc cgaggagaac ggctacggct
ccgccgagga ggccatccgc cagggcatgg 3840acgccatgtc cgaggagttc aacatcgcca
agaagacgat ctccggcgag cagcacggcg 3900aggtcggcgg cgagatctac ctgcccgagt
cctacgtcaa ggccgcgcag aagtgacaat 3960tggcagcagc agctcggata gtatcgacac
actctggacg ctggtcgtgt gatggactgt 4020tgccgccaca cttgctgcct tgacctgtga
atatccctgc cgcttttatc aaacagcctc 4080agtgtgtttg atcttgtgtg tacgcgcttt
tgcgagttgc tagctgcttg tgctatttgc 4140gaataccacc cccagcatcc ccttccctcg
tttcatatcg cttgcatccc aaccgcaact 4200tatctacgct gtcctgctat ccctcagcgc
tgctcctgct cctgctcact gcccctcgca 4260cagccttggt ttgggctccg cctgtattct
cctggtactg caacctgtaa accagcactg 4320caatgctgat gcacgggaag tagtgggatg
ggaacacaaa tggaggatcg tagagctcca 4380gccacggcaa caccgcgcgc ctggcggccg
agcacggcga caagggcctg agcaagatct 4440gcgggctgat cgccagcgac gagggccggc
acgagatcgc ctacacgcgc atcgtggacg 4500agttcttccg cctcgacccc gagggcgccg
tcgccgccta cgccaacatg atgcgcaagc 4560agatcaccat gcccgcgcac ctcatggacg
acatgggcca cggcgaggcc aacccgggcc 4620gcaacctctt cgccgacttc tccgccgtcg
ccgagaagat cgacgtctac gacgccgagg 4680actactgccg catcctggag cacctcaacg
cgcgctggaa ggtggacgag cgccaggtca 4740gcggccaggc cgccgcggac caggagtacg
ttctgggcct gccccagcgc ttccggaaac 4800tcgccgagaa gaccgccgcc aagcgcaagc
gcgtcgcgcg caggcccgtc gccttctcct 4860ggatctccgg acgcgagatt atggtctagg
gaggtacgag cgcgcgcgag ggattggtgg 4920gagtgggacg cgctcgtcgc tcctttctat
tctgaaggga agattggcca ccccgctcca 4980cgcgccacgc atcgagcaac gaagaaaacc
ccccgatgat aggttgcagt ggctgccgag 5040atatagatcc ggctgcacgt caaagggccc
ctcggccaga gaagaagctc ttttcccagc 5100gaccgcagac tcctgaagag cgtttaaac
51291517194DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
151gtttaaacgc cggtcaccac ccgcatgctc gtactacagc gcacgcaccg cttcgtgatc
60caccgggtga acgtagtcct cgacggaaac atctggttcg ggcctcctgc ttgcactccc
120gcccatgccg acaacctttc tgctgttacc acgacccaca atgcaacgcg acacgaccgt
180gtgggactga tcggttcact gcacctgcat gcaattgtca caagcgctta ctccaattgt
240attcgtttgt tttctgggag cagttgctcg accgcccgcg tcccgcaggc agcgatgacg
300tgtgcgtggc ctgggtgttt cgtcgaaagg ccagcaaccc taaatcgcag gcgatccgga
360gattgggatc tgatccgagt ttggaccaga tccgccccga tgcggcacgg gaactgcatc
420gactcggcgc ggaacccagc tttcgtaaat gccagattgg tgtccgatac ctggatttgc
480catcagcgaa acaagacttc agcagcgagc gtatttggcg ggcgtgctac cagggttgca
540tacattgccc atttctgtct ggaccgcttt actggcgcag agggtgagtt gatggggttg
600gcaggcatcg aaacgcgcgt gcatggtgtg cgtgtctgtt ttcggctgca cgaattcaat
660agtcggatgg gcgacggtag aattgggtgt ggcgctcgcg tgcatgcctc gccccgtcgg
720gtgtcatgac cgggactgga atcccccctc gcgaccatct tgctaacgct cccgactctc
780ccgaccgcgc gcaggataga ctcttgttca accaatcgac aactagtatg cagaccgccc
840accagcgccc ccccaccgag ggccactgct tcggcgcccg cctgcccacc gcctcccgcc
900gcgccgtgcg ccgcgcctgg tcccgcatcg cccgcgggcg cgccgccgcc gccgccgacg
960ccaaccccgc ccgccccgag cgccgcgtgg tgatcaccgg ccagggcgtg gtgacctccc
1020tgggccagac catcgagcag ttctactcct ccctgctgga gggcgtgtcc ggcatctccc
1080agatccagaa gttcgacacc accggctaca ccaccaccat cgccggcgag atcaagtccc
1140tgcagctgga cccctacgtg cccaagcgct gggccaagcg cgtggacgac gtgatcaagt
1200acgtgtacat cgccggcaag caggccctgg agtccgccgg cctgcccatc gaggccgccg
1260gcctggccgg cgccggcctg gaccccgccc tgtgcggcgt gctgatcggc accgccatgg
1320ccggcatgac ctccttcgcc gccggcgtgg aggccctgac ccgcggcggc gtgcgcaaga
1380tgaacccctt ctgcatcccc ttctccatct ccaacatggg cggcgccatg ctggccatgg
1440acatcggctt catgggcccc aactactcca tctccaccgc ctgcgccacc ggcaactact
1500gcatcctggg cgccgccgac cacatccgcc gcggcgacgc caacgtgatg ctggccggcg
1560gcgccgacgc cgccatcatc ccctccggca tcggcggctt catcgcctgc aaggccctgt
1620ccaagcgcaa cgacgagccc gagcgcgcct cccgcccctg ggacgccgac cgcgacggct
1680tcgtgatggg cgagggcgcc ggcgtgctgg tgctggagga gctggagcac gccaagcgcc
1740gcggcgccac catcctggcc gagctggtgg gcggcgccgc cacctccgac gcccaccaca
1800tgaccgagcc cgacccccag ggccgcggcg tgcgcctgtg cctggagcgc gccctggagc
1860gcgcccgcct ggcccccgag cgcgtgggct acgtgaacgc ccacggcacc tccacccccg
1920ccggcgacgt ggccgagtac cgcgccatcc gcgccgtgat cccccaggac tccctgcgca
1980tcaactccac caagtccatg atcggccacc tgctgggcgg cgccggcgcc gtggaggccg
2040tggccgccat ccaggccctg cgcaccggct ggctgcaccc caacctgaac ctggagaacc
2100ccgcccccgg cgtggacccc gtggtgctgg tgggcccccg caaggagcgc gccgaggacc
2160tggacgtggt gctgtccaac tccttcggct tcggcggcca caactcctgc gtgatcttcc
2220gcaagtacga cgagatggac tacaaggacc acgacggcga ctacaaggac cacgacatcg
2280actacaagga cgacgacgac aagtgaatcg atagatctct taaggcagca gcagctcgga
2340tagtatcgac acactctgga cgctggtcgt gtgatggact gttgccgcca cacttgctgc
2400cttgacctgt gaatatccct gccgctttta tcaaacagcc tcagtgtgtt tgatcttgtg
2460tgtacgcgct tttgcgagtt gctagctgct tgtgctattt gcgaatacca cccccagcat
2520ccccttccct cgtttcatat cgcttgcatc ccaaccgcaa cttatctacg ctgtcctgct
2580atccctcagc gctgctcctg ctcctgctca ctgcccctcg cacagccttg gtttgggctc
2640cgcctgtatt ctcctggtac tgcaacctgt aaaccagcac tgcaatgctg atgcacggga
2700agtagtggga tgggaacaca aatggaaagc ttaattaaga gctccgcgtc tcgaacagag
2760cgcgcagagg aacgctgaag gtctcgcctc tgtcgcacct cagcgcggca tacaccacaa
2820taaccacctg acgaatgcgc ttggttcttc gtccattagc gaagcgtccg gttcacacac
2880gtgccacgtt ggcgaggtgg caggtgacaa tgatcggtgg agctgatggt cgaaacgttc
2940acagcctagg tgatatccat cttaaggatc taagtaagat tcgaagcgct cgaccgtgcc
3000ggacggactg cagccccatg tcgtagtgac cgccaatgta agtgggctgg cgtttccctg
3060tacgtgagtc aacgtcactg cacgcgcacc accctctcga ccggcaggac caggcatcgc
3120gagatacagc gcgagccaga cacggagtgc cgagctatgc gcacgctcca actaggtacc
3180agtttaggtc cagcgtccgt ggggggggac gggctgggag cttgggccgg gaagggcaag
3240acgatgcagt ccctctgggg agtcacagcc gactgtgtgt gttgcactgt gcggcccgca
3300gcactcacac gcaaaatgcc tggccgacag gcaggccctg tccagtgcaa catccacggt
3360ccctctcatc aggctcacct tgctcattga cataacggaa tgcgtaccgc tctttcagat
3420ctgtccatcc agagagggga gcaggctccc caccgacgct gtcaaacttg cttcctgccc
3480aaccgaaaac attattgttt gagggggggg gggggggggc agattgcatg gcgggatatc
3540tcgtgaggaa catcactggg acactgtgga acacagtgag tgcagtatgc agagcatgta
3600tgctaggggt cagcgcagga agggggcctt tcccagtctc ccatgccact gcaccgtatc
3660cacgactcac caggaccagc ttcttgatcg gcttccgctc ccgtggacac cagtgtgtag
3720cctctggact ccaggtatgc gtgcaccgca aaggccagcc gatcgtgccg attcctgggt
3780ggaggatatg agtcagccaa cttggggctc agagtgcaca ctggggcacg atacgaaaca
3840acatctacac cgtgtcctcc atgctgacac accacagctt cgctccacct gaatgtgggc
3900gcatgggccc gaatcacagc caatgtcgct gctgccataa tgtgatccag accctctccg
3960cccagatgcc gagcggatcg tgggcgctga atagattcct gtttcgatca ctgtttgggt
4020cctttccttt tcgtctcgga tgcgcgtctc gaaacaggct gcgtcgggct ttcggatccc
4080ttttgctccc tccgtcacca tcctgcgcgc gggcaagttg cttgaccctg ggctgatacc
4140agggttggag ggtattaccg cgtcaggcca ttcccagccc ggattcaatt caaagtctgg
4200gccaccaccc tccgccgctc tgtctgatca ctccacattc gtgcatacac tacgttcaag
4260tcctgatcca ggcgtgtctc gggacaaggt gtgcttgagt ttgaatctca aggacccact
4320ccagcacagc tgctggttga ccccgccctc gcaatctaga atggccgcgt ccgtccactg
4380caccctgatg tccgtggtct gcaacaacaa gaaccactcc gcccgcccca agctgcccaa
4440ctcctccctg ctgcccggct tcgacgtggt ggtccaggcc gcggccaccc gcttcaagaa
4500ggagacgacg accacccgcg ccacgctgac gttcgacccc cccacgacca actccgagcg
4560cgccaagcag cgcaagcaca ccatcgaccc ctcctccccc gacttccagc ccatcccctc
4620cttcgaggag tgcttcccca agtccacgaa ggagcacaag gaggtggtgc acgaggagtc
4680cggccacgtc ctgaaggtgc ccttccgccg cgtgcacctg tccggcggcg agcccgcctt
4740cgacaactac gacacgtccg gcccccagaa cgtcaacgcc cacatcggcc tggcgaagct
4800gcgcaaggag tggatcgacc gccgcgagaa gctgggcacg ccccgctaca cgcagatgta
4860ctacgcgaag cagggcatca tcacggagga gatgctgtac tgcgcgacgc gcgagaagct
4920ggaccccgag ttcgtccgct ccgaggtcgc gcggggccgc gccatcatcc cctccaacaa
4980gaagcacctg gagctggagc ccatgatcgt gggccgcaag ttcctggtga aggtgaacgc
5040gaacatcggc aactccgccg tggcctcctc catcgaggag gaggtctaca aggtgcagtg
5100ggccaccatg tggggcgccg acaccatcat ggacctgtcc acgggccgcc acatccacga
5160gacgcgcgag tggatcctgc gcaactccgc ggtccccgtg ggcaccgtcc ccatctacca
5220ggcgctggag aaggtggacg gcatcgcgga gaacctgaac tgggaggtgt tccgcgagac
5280gctgatcgag caggccgagc agggcgtgga ctacttcacg atccacgcgg gcgtgctgct
5340gcgctacatc cccctgaccg ccaagcgcct gacgggcatc gtgtcccgcg gcggctccat
5400ccacgcgaag tggtgcctgg cctaccacaa ggagaacttc gcctacgagc actgggacga
5460catcctggac atctgcaacc agtacgacgt cgccctgtcc atcggcgacg gcctgcgccc
5520cggctccatc tacgacgcca acgacacggc ccagttcgcc gagctgctga cccagggcga
5580gctgacgcgc cgcgcgtggg agaaggacgt gcaggtgatg aacgagggcc ccggccacgt
5640gcccatgcac aagatccccg agaacatgca gaagcagctg gagtggtgca acgaggcgcc
5700cttctacacc ctgggccccc tgacgaccga catcgcgccc ggctacgacc acatcacctc
5760cgccatcggc gcggccaaca tcggcgccct gggcaccgcc ctgctgtgct acgtgacgcc
5820caaggagcac ctgggcctgc ccaaccgcga cgacgtgaag gcgggcgtca tcgcctacaa
5880gatcgccgcc cacgcggccg acctggccaa gcagcacccc cacgcccagg cgtgggacga
5940cgcgctgtcc aaggcgcgct tcgagttccg ctggatggac cagttcgcgc tgtccctgga
6000ccccatgacg gcgatgtcct tccacgacga gacgctgccc gcggacggcg cgaaggtcgc
6060ccacttctgc tccatgtgcg gccccaagtt ctgctccatg aagatcacgg aggacatccg
6120caagtacgcc gaggagaacg gctacggctc cgccgaggag gccatccgcc agggcatgga
6180cgccatgtcc gaggagttca acatcgccaa gaagacgatc tccggcgagc agcacggcga
6240ggtcggcggc gagatctacc tgcccgagtc ctacgtcaag gccgcgcaga agtgacaatt
6300gacggagcgt cgtgcgggag ggagtgtgcc gagcggggag tcccggtctg tgcgaggccc
6360ggcagctgac gctggcgagc cgtacgcccc gagggtcccc ctcccctgca ccctcttccc
6420cttccctctg acggccgcgc ctgttcttgc atgttcagcg acggatccta gggagcgacg
6480agtgtgcgtg cggggctggc gggagtggga cgccctcctc gctcctctct gttctgaacg
6540gaacaatcgg ccaccccgcg ctacgcgcca cgcatcgagc aacgaagaaa accccccgat
6600gataggttgc ggtggctgcc gggatataga tccggccgca catcaaaggg cccctccgcc
6660agagaagaag ctcctttccc agcagactcc ttctgctgcc aaaacacttc tctgtccaca
6720gcaacaccaa aggatgaaca gatcaacttg cgtctccgcg tagcttcctc ggctagcgtg
6780cttgcaacag gtccctgcac tattatcttc ctgctttcct ctgaattatg cggcaggcga
6840gcgctcgctc tggcgagcgc tccttcgcgc cgccctcgct gatcgagtgt acagtcaatg
6900aatggtcctg ggcgaagaac gagggaattt gtgggtaaaa caagcatcgt ctctcaggcc
6960ccggcgcagt ggccgttaaa gtccaagacc gtgaccaggc agcgcagcgc gtccgtgtgc
7020gggccctgcc tggcggctcg gcgtgccagg ctcgagagca gctccctcag gtcgccttgg
7080acggcctctg cgaggccggt gagggcctgc aggagcgcct cgagcgtggc agtggcggtc
7140gtatccgggt cgccggtcac cgcctgcgac tcgccatccg aagagcgttt aaac
71941527081DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 152gctcttcgcc gccgccactc ctgctcgagc
gcgcccgcgc gtgcgccgcc agcgccttgg 60ccttttcgcc gcgctcgtgc gcgtcgctga
tgtccatcac caggtccatg aggtctgcct 120tgcgccggct gagccactgc ttcgtccggg
cggccaagag gagcatgagg gaggactcct 180ggtccagggt cctgacgtgg tcgcggctct
gggagcgggc cagcatcatc tggctctgcc 240gcaccgaggc cgcctccaac tggtcctcca
gcagccgcag tcgccgccga ccctggcaga 300ggaagacagg tgaggggggt atgaattgta
cagaacaacc acgagccttg tctaggcaga 360atccctacca gtcatggctt tacctggatg
acggcctgcg aacagctgtc cagcgaccct 420cgctgccgcc gcttctcccg cacgcttctt
tccagcaccg tgatggcgcg agccagcgcc 480gcacgctggc gctgcgcttc gccgatctga
ggacagtcgg ggaactctga tcagtctaaa 540cccccttgcg cgttagtgtt gccatccttt
gcagaccggt gagagccgac ttgttgtgcg 600ccacccccca caccacctcc tcccagacca
attctgtcac ctttttggcg aaggcatcgg 660cctcggcctg cagagaggac agcagtgccc
agccgctggg ggttggcgga tgcacgctca 720ggtacccttt cttgcgctat gacacttcca
gcaaaaggta gggcgggctg cgagacggct 780tcccggcgct gcatgcaaca ccgatgatgc
ttcgaccccc cgaagctcct tcggggctgc 840atgggcgctc cgatgccgct ccagggcgag
cgctgtttaa atagccaggc ccccgattgc 900aaagacatta tagcgagcta ccaaagccat
attcaaacac ctagatcact accacttcta 960cacaggccac tcgagcttgt gatcgcactc
cgctaagggg gcgcctcttc ctcttcgttt 1020cagtcacaac ccgcaaactc tagaatatca
atgctgctgc aggccttcct gttcctgctg 1080gccggcttcg ccgccaagat cagcgcctcc
atgacgaacg agacgtccga ccgccccctg 1140gtgcacttca cccccaacaa gggctggatg
aacgacccca acggcctgtg gtacgacgag 1200aaggacgcca agtggcacct gtacttccag
tacaacccga acgacaccgt ctgggggacg 1260cccttgttct ggggccacgc cacgtccgac
gacctgacca actgggagga ccagcccatc 1320gccatcgccc cgaagcgcaa cgactccggc
gccttctccg gctccatggt ggtggactac 1380aacaacacct ccggcttctt caacgacacc
atcgacccgc gccagcgctg cgtggccatc 1440tggacctaca acaccccgga gtccgaggag
cagtacatct cctacagcct ggacggcggc 1500tacaccttca ccgagtacca gaagaacccc
gtgctggccg ccaactccac ccagttccgc 1560gacccgaagg tcttctggta cgagccctcc
cagaagtgga tcatgaccgc ggccaagtcc 1620caggactaca agatcgagat ctactcctcc
gacgacctga agtcctggaa gctggagtcc 1680gcgttcgcca acgagggctt cctcggctac
cagtacgagt gccccggcct gatcgaggtc 1740cccaccgagc aggaccccag caagtcctac
tgggtgatgt tcatctccat caaccccggc 1800gccccggccg gcggctcctt caaccagtac
ttcgtcggca gcttcaacgg cacccacttc 1860gaggccttcg acaaccagtc ccgcgtggtg
gacttcggca aggactacta cgccctgcag 1920accttcttca acaccgaccc gacctacggg
agcgccctgg gcatcgcgtg ggcctccaac 1980tgggagtact ccgccttcgt gcccaccaac
ccctggcgct cctccatgtc cctcgtgcgc 2040aagttctccc tcaacaccga gtaccaggcc
aacccggaga cggagctgat caacctgaag 2100gccgagccga tcctgaacat cagcaacgcc
ggcccctgga gccggttcgc caccaacacc 2160acgttgacga aggccaacag ctacaacgtc
gacctgtcca acagcaccgg caccctggag 2220ttcgagctgg tgtacgccgt caacaccacc
cagacgatct ccaagtccgt gttcgcggac 2280ctctccctct ggttcaaggg cctggaggac
cccgaggagt acctccgcat gggcttcgag 2340gtgtccgcgt cctccttctt cctggaccgc
gggaacagca aggtgaagtt cgtgaaggag 2400aacccctact tcaccaaccg catgagcgtg
aacaaccagc ccttcaagag cgagaacgac 2460ctgtcctact acaaggtgta cggcttgctg
gaccagaaca tcctggagct gtacttcaac 2520gacggcgacg tcgtgtccac caacacctac
ttcatgacca ccgggaacgc cctgggctcc 2580gtgaacatga cgacgggggt ggacaacctg
ttctacatcg acaagttcca ggtgcgcgag 2640gtcaagtgac aattggcagc agcagctcgg
atagtatcga cacactctgg acgctggtcg 2700tgtgatggac tgttgccgcc acacttgctg
ccttgacctg tgaatatccc tgccgctttt 2760atcaaacagc ctcagtgtgt ttgatcttgt
gtgtacgcgc ttttgcgagt tgctagctgc 2820ttgtgctatt tgcgaatacc acccccagca
tccccttccc tcgtttcata tcgcttgcat 2880cccaaccgca acttatctac gctgtcctgc
tatccctcag cgctgctcct gctcctgctc 2940actgcccctc gcacagcctt ggtttgggct
ccgcctgtat tctcctggta ctgcaacctg 3000taaaccagca ctgcaatgct gatgcacggg
aagtagtggg atgggaacac aaatggagga 3060tcccgcgtct cgaacagagc gcgcagagga
acgctgaagg tctcgcctct gtcgcacctc 3120agcgcggcat acaccacaat aaccacctga
cgaatgcgct tggttcttcg tccattagcg 3180aagcgtccgg ttcacacacg tgccacgttg
gcgaggtggc aggtgacaat gatcggtgga 3240gctgatggtc gaaacgttca cagcctaggg
atatcctgaa gaatgggagg caggtgttgt 3300tgattatgag tgtgtaaaag aaaggggtag
agagccgtcc tcagatccga ctactatgca 3360ggtagccgct cgcccatgcc cgcctggctg
aatattgatg catgcccatc aaggcaggca 3420ggcatttctg tgcacgcacc aagcccacaa
tcttccacaa cacacagcat gtaccaacgc 3480acgcgtaaaa gttggggtgc tgccagtgcg
tcatgccagg catgatgtgc tcctgcacat 3540ccgccatgat ctcctccatc gtctcgggtg
tttccggcgc ctggtccggg agccgttccg 3600ccagataccc agacgccacc tccgacctca
cggggtactt ttcgagcgtc tgccggtagt 3660cgacgatcgc gtccaccatg gagtagccga
ggcgccggaa ctggcgtgac ggagggagga 3720gagggaggag agagaggggg gggggggggg
gggatgatta cacgccagtc tcacaacgca 3780tgcaagaccc gtttgattat gagtacaatc
atgcactact agatggatga gcgccaggca 3840taaggcacac cgacgttgat ggcatgagca
actcccgcat catatttcct attgtcctca 3900cgccaagccg gtcaccatcc gcatgctcat
attacagcgc acgcaccgct tcgtgatcca 3960ccgggtgaac gtagtcctcg acggaaacat
ctggctcggg cctcgtgctg gcactccctc 4020ccatgccgac aacctttctg ctgtcaccac
gacccacgat gcaacgcgac acgacccggt 4080gggactgatc ggttcactgc acctgcatgc
aattgtcaca agcgcatact ccaatcgtat 4140ccgtttgatt tctgtgaaaa ctcgctcgac
cgcccgcgtc ccgcaggcag cgatgacgtg 4200tgcgtgacct gggtgtttcg tcgaaaggcc
agcaacccca aatcgcaggc gatccggaga 4260ttgggatctg atccgagctt ggaccagatc
ccccacgatg cggcacggga actgcatcga 4320ctcggcgcgg aacccagctt tcgtaaatgc
cagattggtg tccgatacct tgatttgcca 4380tcagcgaaac aagacttcag cagcgagcgt
atttggcggg cgtgctacca gggttgcata 4440cattgcccat ttctgtctgg accgctttac
cggcgcagag ggtgagttga tggggttggc 4500aggcatcgaa acgcgcgtgc atggtgtgtg
tgtctgtttt cggctgcaca atttcaatag 4560tcggatgggc gacggtagaa ttgggtgttg
cgctcgcgtg catgcctcgc cccgtcgggt 4620gtcatgaccg ggactggaat cccccctcgc
gaccctcctg ctaacgctcc cgactctccc 4680gcccgcgcgc aggatagact ctagttcaac
caatcgacaa ctagtatggc caccgcatcc 4740actttctcgg cgttcaatgc ccgctgcggc
gacctgcgtc gctcggcggg ctccgggccc 4800cggcgcccag cgaggcccct ccccgtgcgc
gggcgcgcca tccccccccg catcatcgtg 4860gtgtcctcct cctcctccaa ggtgaacccc
ctgaagaccg aggccgtggt gtcctccggc 4920ctggccgacc gcctgcgcct gggctccctg
accgaggacg gcctgtccta caaggagaag 4980ttcatcgtgc gctgctacga ggtgggcatc
aacaagaccg ccaccgtgga gaccatcgcc 5040aacctgctgc aggaggtggg ctgcaaccac
gcccagtccg tgggctactc caccggcggc 5100ttctccacca cccccaccat gcgcaagctg
cgcctgatct gggtgaccgc ccgcatgcac 5160atcgagatct acaagtaccc cgcctggtcc
gacgtggtgg agatcgagtc ctggggccag 5220ggcgagggca agatcggcac ccgccgcgac
tggatcctgc gcgactacgc caccggccag 5280gtgatcggcc gcgccacctc caagtgggtg
atgatgaacc aggacacccg ccgcctgcag 5340aaggtggacg tggacgtgcg cgacgagtac
ctggtgcact gcccccgcga gctgcgcctg 5400gccttccccg aggagaacaa ctcctccctg
aagaagatct ccaagctgga ggacccctcc 5460cagtactcca agctgggcct ggtgccccgc
cgcgccgacc tggacatgaa ccagcacgtg 5520aacaacgtga cctacatcgg ctgggtgctg
gagtccatgc cccaggagat catcgacacc 5580cacgagctgc agaccatcac cctggactac
cgccgcgagt gccagcacga cgacgtggtg 5640gactccctga cctcccccga gccctccgag
gacgccgagg ccgtgttcaa ccacaacggc 5700accaacggct ccgccaacgt gtccgccaac
gaccacggct gccgcaactt cctgcacctg 5760ctgcgcctgt ccggcaacgg cctggagatc
aaccgcggcc gcaccgagtg gcgcaagaag 5820cccacccgca tggactacaa ggaccacgac
ggcgactaca aggaccacga catcgactac 5880aaggacgacg acgacaagtg aatcgataga
tctcttaagg cagcagcagc tcggatagta 5940tcgacacact ctggacgctg gtcgtgtgat
ggactgttgc cgccacactt gctgccttga 6000cctgtgaata tccctgccgc ttttatcaaa
cagcctcagt gtgtttgatc ttgtgtgtac 6060gcgcttttgc gagttgctag ctgcttgtgc
tatttgcgaa taccaccccc agcatcccct 6120tccctcgttt catatcgctt gcatcccaac
cgcaacttat ctacgctgtc ctgctatccc 6180tcagcgctgc tcctgctcct gctcactgcc
cctcgcacag ccttggtttg ggctccgcct 6240gtattctcct ggtactgcaa cctgtaaacc
agcactgcaa tgctgatgca cgggaagtag 6300tgggatggga acacaaatgg aaagcttaat
taagagctct tgttttccag aaggagttgc 6360tccttgagcc tttcattctc agcctcgata
acctccaaag ccgctctaat tgtggagggg 6420gttcgaattt aaaagcttgg aatgttggtt
cgtgcgtctg gaacaagccc agacttgttg 6480ctcactggga aaaggaccat cagctccaaa
aaacttgccg ctcaaaccgc gtacctctgc 6540tttcgcgcaa tctgccctgt tgaaatcgcc
accacattca tattgtgacg cttgagcagt 6600ctgtaattgc ctcagaatgt ggaatcatct
gccccctgtg cgagcccatg ccaggcatgt 6660cgcgggcgag gacacccgcc actcgtacag
cagaccatta tgctacctca caatagttca 6720taacagtgac catatttctc gaagctcccc
aacgagcacc tccatgctct gagtggccac 6780cccccggccc tggtgcttgc ggagggcagg
tcaaccggca tggggctacc gaaatccccg 6840accggatccc accacccccg cgatgggaag
aatctctccc cgggatgtgg gcccaccacc 6900agcacaacct gctggcccag gcgagcgtca
aaccatacca cacaaatatc cttggcatcg 6960gccctgaatt ccttctgccg ctctgctacc
cggtgcttct gtccgaagca ggggttgcta 7020gggatcgctc cgagtccgca aacccttgtc
gcgtggcggg gcttgttcga gcttgaagag 7080c
70811538286DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
153gctcttccca actcagataa taccaatacc cctccttctc ctcctcatcc attcagtacc
60cccccccttc tcttcccaaa gcagcaagcg cgtggcttac agaagaacaa tcggcttccg
120ccaaagtcgc cgagcactgc ccgacggcgg cgcgcccagc agcccgcttg gccacacagg
180caacgaatac attcaatagg gggcctcgca gaatggaagg agcggtaaag ggtacaggag
240cactgcgcac aaggggcctg tgcaggagtg actgactggg cgggcagacg gcgcaccgcg
300ggcgcaggca agcagggaag attgaagcgg cagggaggag gatgctgatt gaggggggca
360tcgcagtctc tcttggaccc gggataagga agcaaatatt cggccggttg ggttgtgtgt
420gtgcacgttt tcttcttcag agtcgtgggt gtgcttccag ggaggatata agcagcagga
480tcgaatcccg cgaccagcgt ttccccatcc agccaaccac cctgtcggta ccgcggtgag
540aatcgaaaat gcatcgtttc taggttcgga gacggtcaat tccctgctcc ggcgaatctg
600tcggtcaagc tggccagtgg acaatgttgc tatggcagcc cgcgcacatg ggcctcccga
660cgcggccatc aggagcccaa acagcgtgtc agggtatgtg aaactcaaga ggtccctgct
720gggcactccg gccccactcc gggggcggga cgccaggcat tcgcggtcgg tcccgcgcga
780cgagcgaaat gatgattcgg ttacgagacc aggacgtcgt cgaggtcgag aggcagcctc
840ggacacgtct cgctagggca acgccccgag tccccgcgag ggccgtaaac attgtttctg
900ggtgtcggag tgggcatttt gggcccgatc caatcgcctc atgccgctct cgtctggtcc
960tcacgttcgc gtacggcctg gatcccggaa agggcggatg cacgtggtgt tgccccgcca
1020ttggcgccca cgtttcaaag tccccggcca gaaatgcaca ggaccggccc ggctcgcaca
1080ggccatgctg aacgcccaga tttcgacagc aacaccatct agaataatcg caaccatccg
1140cgttttgaac gaaacgaaac ggcgctgttt agcatgtttc cgacatcgtg ggggccgaag
1200catgctccgg ggggaggaaa gcgtggcaca gcggtagccc attctgtgcc acacgccgac
1260gaggaccaat ccccggcatc agccttcatc gacggctgcg ccgcacatat aaagccggac
1320gcctaaccgg tttcgtggtt atgactagta tgttcgcgtt ctacttcctg acggcctgca
1380tctccctgaa gggcgtgttc ggcgtctccc cctcctacaa cggcctgggc ctgacgcccc
1440agatgggctg ggacaactgg aacacgttcg cctgcgacgt ctccgagcag ctgctgctgg
1500acacggccga ccgcatctcc gacctgggcc tgaaggacat gggctacaag tacatcatcc
1560tggacgactg ctggtcctcc ggccgcgact ccgacggctt cctggtcgcc gacgagcaga
1620agttccccaa cggcatgggc cacgtcgccg accacctgca caacaactcc ttcctgttcg
1680gcatgtactc ctccgcgggc gagtacacgt gcgccggcta ccccggctcc ctgggccgcg
1740aggaggagga cgcccagttc ttcgcgaaca accgcgtgga ctacctgaag tacgacaact
1800gctacaacaa gggccagttc ggcacgcccg agatctccta ccaccgctac aaggccatgt
1860ccgacgccct gaacaagacg ggccgcccca tcttctactc cctgtgcaac tggggccagg
1920acctgacctt ctactggggc tccggcatcg cgaactcctg gcgcatgtcc ggcgacgtca
1980cggcggagtt cacgcgcccc gactcccgct gcccctgcga cggcgacgag tacgactgca
2040agtacgccgg cttccactgc tccatcatga acatcctgaa caaggccgcc cccatgggcc
2100agaacgcggg cgtcggcggc tggaacgacc tggacaacct ggaggtcggc gtcggcaacc
2160tgacggacga cgaggagaag gcgcacttct ccatgtgggc catggtgaag tcccccctga
2220tcatcggcgc gaacgtgaac aacctgaagg cctcctccta ctccatctac tcccaggcgt
2280ccgtcatcgc catcaaccag gactccaacg gcatccccgc cacgcgcgtc tggcgctact
2340acgtgtccga cacggacgag tacggccagg gcgagatcca gatgtggtcc ggccccctgg
2400acaacggcga ccaggtcgtg gcgctgctga acggcggctc cgtgtcccgc cccatgaaca
2460cgaccctgga ggagatcttc ttcgactcca acctgggctc caagaagctg acctccacct
2520gggacatcta cgacctgtgg gcgaaccgcg tcgacaactc cacggcgtcc gccatcctgg
2580gccgcaacaa gaccgccacc ggcatcctgt acaacgccac cgagcagtcc tacaaggacg
2640gcctgtccaa gaacgacacc cgcctgttcg gccagaagat cggctccctg tcccccaacg
2700cgatcctgaa cacgaccgtc cccgcccacg gcatcgcgtt ctaccgcctg cgcccctcct
2760cctgatacaa cttattacgt attctgaccg gcgctgatgt ggcgcggacg ccgtcgtact
2820ctttcagact ttactcttga ggaattgaac ctttctcgct tgctggcatg taaacattgg
2880cgcaattaat tgtgtgatga agaaagggtg gcacaagatg gatcgcgaat gtacgagatc
2940gacaacgatg gtgattgtta tgaggggcca aacctggctc aatcttgtcg catgtccggc
3000gcaatgtgat ccagcggcgt gactctcgca acctggtagt gtgtgcgcac cgggtcgctt
3060tgattaaaac tgatcgcatt gccatcccgt caactcacaa gcctactcta gctcccattg
3120cgcactcggg cgcccggctc gatcaatgtt ctgagcggag ggcgaagcgt caggaaatcg
3180tctcggcagc tggaagcgca tggaatgcgg agcggagatc gaatcaggat cccgcgtctc
3240gaacagagcg cgcagaggaa cgctgaaggt ctcgcctctg tcgcacctca gcgcggcata
3300caccacaata accacctgac gaatgcgctt ggttcttcgt ccattagcga agcgtccggt
3360tcacacacgt gccacgttgg cgaggtggca ggtgacaatg atcggtggag ctgatggtcg
3420aaacgttcac agcctagcat agcgactgct accccccgac catgtgccga ggcagaaatt
3480atatacaaga agcagatcgc aattaggcac atcgctttgc attatccaca cactattcat
3540cgctgctgcg gcaaggctgc agagtgtatt tttgtggccc aggagctgag tccgaagtcg
3600acgcgacgag cggcgcagga tccgacccct agacgagctc tgtcattttc caagcacgca
3660gctaaatgcg ctgagaccgg gtctaaatca tccgaaaagt gtcaaaatgg ccgattgggt
3720tcgcctagga caatgcgctg cggattcgct cgagtccgct gccggccaaa aggcggtggt
3780acaggaaggc gcacggggcc aaccctgcga agccgggggc ccgaacgccg accgccggcc
3840ttcgatctcg ggtgtccccc tcgtcaattt cctctctcgg gtgcagccac gaaagtcgtg
3900acgcaggtca cgaaatccgg ttacgaaaaa cgcaggtctt cgcaaaaacg tgagggtttc
3960gcgtctcgcc ctagctattc gtatcgccgg gtcagaccca cgtgcagaaa agcccttgaa
4020taacccggga ccgtggttac cgcgccgcct gcaccagggg gcttatataa gcccacacca
4080cacctgtctc accacgcatt tctccaactc gcgacttttc ggaagaaatt gttatccacc
4140tagtatagac tgccacctgc aggaccttgt gtcttgcagt ttgtattggt cccggccgtc
4200gagctcgaca gatctgggct agggttggcc tggccgctcg gcactcccct ttagccgcgc
4260gcatccgcgt tccagaggtg cgattcggtg tgtggagcat tgtcatgcgc ttgtgggggt
4320cgttccgtgc gcggcgggtc cgccatgggc gccgacctgg gccctagggt ttgttttcgg
4380gccaagcgag cccctctcac ctcgtcgccc ccccgcattc cctctctctt gcagcccata
4440tggccatggc cgccgccgtg atcgtgcccc tgggcatcct gttcttcatc tccggcctgg
4500tggtgaacct gctgcaggcc atctgctacg tgctgatccg ccccctgtcc aagaacacct
4560accgcaagat caaccgcgtg gtggccgaga ccctgtggct ggagctggtg tggatcgtgg
4620actggtgggc cggcgtgaag atccaggtgt tcgccgacaa cgagaccttc aaccgcatgg
4680gcaaggagca cgccctggtg gtgtgcaacc accgctccga catcgactgg ctggtgggct
4740ggatcctggc ccagcgctcc ggctgcctgg gctccgccct ggccgtgatg aagaagtcct
4800ccaagttcct gcccgtgatc ggctggtcca tgtggttctc cgagtacctg ttcctggagc
4860gcaactgggc caaggacgag tccaccctga agtccggcct gcagcgcctg aacgacttcc
4920cccgcccctt ctggctggcc ctgttcgtgg agggcacccg cttcaccgag gccaagctga
4980aggccgccca ggagtacgcc gcctcctccg agctgcccgt gccccgcaac gtgctgatcc
5040cccgcaccaa gggcttcgtg tccgccgtgt ccaacatgcg ctccttcgtg cccgccatct
5100acgacatgac cgtggccatc cccaagacct cccccccccc caccatgctg cgcctgttca
5160agggccagcc ctccgtggtg cacgtgcaca tcaagtgcca ctccatgaag gacctgcccg
5220agtccgacga cgccatcgcc cagtggtgcc gcgaccagtt cgtggccaag gacgccctgc
5280tggacaagca catcgccgcc gacaccttcc ccggccagca ggagcagaac atcggccgcc
5340ccatcaagtc cctggccgtg gtgctgtcct ggtcctgcct gctgatcctg ggcgccatga
5400agttcctgca ctggtccaac ctgttctcct cctggaaggg catcgccttc tccgccctgg
5460gcctgggcat catcaccctg tgcatgcaga tcctgatccg ctcctcccag tccgagcgct
5520ccacccccgc caaggtggtg cccgccaagc ccaaggacaa ccacaacgac tccggctcct
5580cctcccagac cgaggtggag aagcagaagt gaatgcatgc agcagcagct cggatagtat
5640cgacacactc tggacgctgg tcgtgtgatg gactgttgcc gccacacttg ctgccttgac
5700ctgtgaatat ccctgccgct tttatcaaac agcctcagtg tgtttgatct tgtgtgtacg
5760cgcttttgcg agttgctagc tgcttgtgct atttgcgaat accaccccca gcatcccctt
5820ccctcgtttc atatcgcttg catcccaacc gcaacttatc tacgctgtcc tgctatccct
5880cagcgctgct cctgctcctg ctcactgccc ctcgcacagc cttggtttgg gctccgcctg
5940tattctcctg gtactgcaac ctgtaaacca gcactgcaat gctgatgcac gggaagtagt
6000gggatgggaa cacaaatgga cttaaggatc taagtaagat tcgaagcgct cgaccgtgcc
6060ggacggactg cagccccatg tcgtagtgac cgccaatgta agtgggctgg cgtttccctg
6120tacgtgagtc aacgtcactg cacgcgcacc accctctcga ccggcaggac caggcatcgc
6180gagatacagc gcgagccaga cacggagtgc cgagctatgc gcacgctcca actagatatc
6240atgtggatga tgagcatgaa ttcctttctt gcgctatgac acttccagca aaaggtaggg
6300cgggctgcga gacggcttcc cggcgctgca tgcaacaccg atgatgcttc gaccccccga
6360agctccttcg gggctgcatg ggcgctccga tgccgctcca gggcgagcgc tgtttaaata
6420gccaggcccc cgattgcaaa gacattatag cgagctacca aagccatatt caaacaccta
6480gatcactacc acttctacac aggccactcg agcttgtgat cgcactccgc taagggggcg
6540cctcttcctc ttcgtttcag tcacaacccg caaacactag tatggctatc aagacgaaca
6600ggcagcctgt ggagaagcct ccgttcacga tcgggacgct gcgcaaggcc atccccgcgc
6660actgtttcga gcgctcggcg cttcgtagca gcatgtacct ggcctttgac atcgcggtca
6720tgtccctgct ctacgtcgcg tcgacgtaca tcgaccctgc accggtgcct acgtgggtca
6780agtacggcat catgtggccg ctctactggt tcttccaggt gtgtttgagg gttttggttg
6840cccgtattga ggtcctggtg gcgcgcatgg aggagaaggc gcctgtcccg ctgacccccc
6900cggctaccct cccggcacct tccagggcgc gtacgggaag aaccagtaga gcggccacat
6960gatgccgtac ttgacccacg taggcaccgg tgcagggtcg atgtacgtcg acgcgacgta
7020gagcagggac atgaccgcga tgtcaaaggc caggtacatg ctgctacgaa gcgccgagcg
7080ctcgaaacag tgcgcgggga tggccttgcg cagcgtcccg atcgtgaacg gaggcttctc
7140cacaggctgc ctgttcgtct tgatagccat ctcgaggcag cagcagctcg gatagtatcg
7200acacactctg gacgctggtc gtgtgatgga ctgttgccgc cacacttgct gccttgacct
7260gtgaatatcc ctgccgcttt tatcaaacag cctcagtgtg tttgatcttg tgtgtacgcg
7320cttttgcgag ttgctagctg cttgtgctat ttgcgaatac cacccccagc atccccttcc
7380ctcgtttcat atcgcttgca tcccaaccgc aacttatcta cgctgtcctg ctatccctca
7440gcgctgctcc tgctcctgct cactgcccct cgcacagcct tggtttgggc tccgcctgta
7500ttctcctggt actgcaacct gtaaaccagc actgcaatgc tgatgcacgg gaagtagtgg
7560gatgggaaca caaatggaaa gctgtagagc tcttgttttc cagaaggagt tgctccttga
7620gcctttcatt ctcagcctcg ataacctcca aagccgctct aattgtggag ggggttcgaa
7680ccgaatgctg cgtgaacggg aaggaggagg agaaagagtg agcagggagg gattcagaaa
7740tgagaaatga gaggtgaagg aacgcatccc tatgcccttg caatggacag tgtttctggc
7800caccgccacc aagacttcgt gtcctctgat catcatgcga ttgattacgt tgaatgcgac
7860ggccggtcag ccccggacct ccacgcaccg gtgctcctcc aggaagatgc gcttgtcctc
7920cgccatcttg cagggctcaa gctgctccca aaactcttgg gcgggttccg gacggacggc
7980taccgcgggt gcggccctga ccgccactgt tcggaagcag cggcgctgca tgggcagcgg
8040ccgctgcggt gcgccacgga ccgcatgatc caccggaaaa gcgcacgcgc tggagcgcgc
8100agaggaccac agagaagcgg aagagacgcc agtactggca agcaggctgg tcggtgccat
8160ggcgcgctac taccctcgct atgactcggg tcctcggccg gctggcggtg ctgacaattc
8220gtttagtgga gcagcgactc cattcagcta ccagtcgaac tcagtggcac agtgactccg
8280ctcttc
82861541317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 154gtggccgccg aggcctcctc cgccctgttc
tccgtgcgca cccccggcac ctcccccaag 60cccggcaagt tcggcaactg gcccacctcc
ctgtccgtgc ccttcaagtc caagtccaac 120cacaacggcg gcttccaggt gaaggccaac
gcctccgccc gccccaaggc caacggctcc 180gccgtgtccc tgaagtccgg ctccctggac
acccaggagg acacctcctc ctcctcctcc 240cccccccgca ccttcatcaa ccagctgccc
gactggtcca tgctgctgtc cgccatcacc 300accgtgttcg tggccgccga gaagcagtgg
accatgctgg accgcaagtc caagcgcccc 360gacatgctga tggacccctt cggcgtggac
cgcgtggtgc aggacggcgc cgtgttccgc 420cagtccttct ccatccgctc ctacgagatc
ggcgccgacc gcaccgcctc catcgagacc 480ctgatgaaca tcttccagga gacctccctg
aaccactgca agtccatcgg cctgctgaac 540gacggcttcg gccgcacccc cgagatgtgc
aagcgcgacc tgatctgggt ggtgaccaag 600atgcacgtgg aggtgaaccg ctaccccacc
tggggcgaca ccatcgaggt gaacacctgg 660gtgtccgagt ccggcaagac cggcatgggc
cgcgactggc tgatctccga ctgccacacc 720ggcgagatcc tgatccgcgc cacctccatg
tgcgccatga tgaaccagaa gacccgccgc 780ttctccaagt tcccctacga ggtgcgccag
gagctggccc cccacttcgt ggactccgcc 840cccgtgatcg aggactacca gaagctgcac
aagctggacg tgaagaccgg cgactccatc 900tgcaacggcc tgaccccccg ctggaacgac
ctggacgtga accagcacgt gaacaacgtg 960aagtacatcg gctggatcct ggagtccgtg
cccaccgagg tgttcgagac ccaggagctg 1020tgcggcctga ccctggagta ccgccgcgag
tgcggccgcg actccgtgct ggagtccgtg 1080accgccatgg acccctccaa ggagggcgac
cgctccctgt accagcacct gctgcgcctg 1140gaggacggcg ccgacatcgc caagggccgc
accaagtggc gccccaagaa cgccggcacc 1200aacggcgcca tctccaccgg caagacctcc
aacggcaact ccatctccat ggactacaag 1260gaccacgacg gcgactacaa ggaccacgac
atcgactaca aggacgacga cgacaag 13171551260DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
155gccaccgcct ccaccttctc cgccttcaac gcccgctgcg gcgacctgcg ccgctccgcc
60ggctccggcc cccgccgccc cgcccgcccc ctgcccgtgc gcgccgccat caacgcctcc
120gcccacccca aggccaacgg ctccgccgtg aacctgaagt ccggctccct ggagacccag
180gaggacacct cctcctcctc cccccccccc cgcaccttca tcaagcagct gcccgactgg
240ggcatgctgc tgtccaagat caccaccgtg ttcggcgccg ccgagcgcca gtggaagcgc
300cccggcatgc tggtggagcc cttcggcgtg gaccgcatct tccaggacgg cgtgttcttc
360cgccagtcct tctccatccg ctcctacgag atcggcgccg accgcaccgc ctccatcgag
420accctgatga acatcttcca ggagacctcc ctgaaccact gcaagtccat cggcctgctg
480aacgacggct tcggccgcac ccccgagatg tgcaagcgcg acctgatctg ggtggtgacc
540aagatccagg tggaggtgaa ccgctacccc acctggggcg acaccatcga ggtgaacacc
600tgggtgtccg agtccggcaa gaacggcatg ggccgcgact ggctgatctc cgactgccgc
660accggcgaga tcctgatccg cgccacctcc gtgtgggcca tgatgaaccg caagacccgc
720cgcctgtcca agttccccta cgaggtgcgc caggagatcg ccccccactt cgtggactcc
780gcccccgtga tcgaggacga caagaagctg cacaagctgg acgtgaagac cggcgactcc
840atccgcaagg gcctgacccc ccgctggaac gacctggacg tgaaccagca cgtgaacaac
900gtgaagtaca tcggctggat cctgaagtcc gtgcccgccg aggtgttcga gacccaggag
960ctgtgcggcg tgaccctgga gtaccgccgc gagtgcggcc gcgactccgt gctggagtcc
1020gtgaccgcca tggacaccgc caaggagggc gaccgctccc tgtaccagca cctgctgcgc
1080ctggaggacg gcgccgacat caccatcggc cgcaccgagt ggcgccccaa gaacgccggc
1140gccaacggcg ccatctccac cggcaagacc tccaacgaga actccgtgtc catggactac
1200aaggaccacg acggcgacta caaggaccac gacatcgact acaaggacga cgacgacaag
1260156352PRTGarcinia mangostana 156Met Leu Lys Leu Ser Ser Ser Arg Ser
Pro Leu Ala Arg Ile Pro Thr 1 5 10
15 Arg Pro Arg Pro Asn Ser Ile Pro Pro Arg Ile Ile Val Val
Ser Ser 20 25 30
Ser Ser Ser Lys Val Asn Pro Leu Lys Thr Glu Ala Val Val Ser Ser
35 40 45 Gly Leu Ala Asp
Arg Leu Arg Leu Gly Ser Leu Thr Glu Asp Gly Leu 50
55 60 Ser Tyr Lys Glu Lys Phe Ile Val
Arg Cys Tyr Glu Val Gly Ile Asn 65 70
75 80 Lys Thr Ala Thr Val Glu Thr Ile Ala Asn Leu Leu
Gln Glu Val Gly 85 90
95 Cys Asn His Ala Gln Ser Val Gly Tyr Ser Thr Gly Gly Phe Ser Thr
100 105 110 Thr Pro Thr
Met Arg Lys Leu Arg Leu Ile Trp Val Thr Ala Arg Met 115
120 125 His Ile Glu Ile Tyr Lys Tyr Pro
Ala Trp Ser Asp Val Val Glu Ile 130 135
140 Glu Ser Trp Gly Gln Gly Glu Gly Lys Ile Gly Thr Arg
Arg Asp Trp 145 150 155
160 Ile Leu Arg Asp Tyr Ala Thr Gly Gln Val Ile Gly Arg Ala Thr Ser
165 170 175 Lys Trp Val Met
Met Asn Gln Asp Thr Arg Arg Leu Gln Lys Val Asp 180
185 190 Val Asp Val Arg Asp Glu Tyr Leu Val
His Cys Pro Arg Glu Leu Arg 195 200
205 Leu Ala Phe Pro Glu Glu Asn Asn Ser Ser Leu Lys Lys Ile
Ser Lys 210 215 220
Leu Glu Asp Pro Ser Gln Tyr Ser Lys Leu Gly Leu Val Pro Arg Arg 225
230 235 240 Ala Asp Leu Asp Met
Asn Gln His Val Asn Asn Val Thr Tyr Ile Gly 245
250 255 Trp Val Leu Glu Ser Met Pro Gln Glu Ile
Ile Asp Thr His Glu Leu 260 265
270 Gln Thr Ile Thr Leu Asp Tyr Arg Arg Glu Cys Gln His Asp Asp
Val 275 280 285 Val
Asp Ser Leu Thr Ser Pro Glu Pro Ser Glu Asp Ala Glu Ala Val 290
295 300 Phe Asn His Asn Gly Thr
Asn Gly Ser Ala Asn Val Ser Ala Asn Asp 305 310
315 320 His Gly Cys Arg Asn Phe Leu His Leu Leu Arg
Leu Ser Gly Asn Gly 325 330
335 Leu Glu Ile Asn Arg Gly Arg Thr Glu Trp Arg Lys Lys Pro Thr Arg
340 345 350
157390PRTBrassic napus 157Met Ala Met Ala Ala Ala Val Ile Val Pro Leu Gly
Ile Leu Phe Phe 1 5 10
15 Ile Ser Gly Leu Val Val Asn Leu Leu Gln Ala Val Cys Tyr Val Leu
20 25 30 Val Arg Pro
Met Ser Lys Asn Thr Tyr Arg Lys Ile Asn Arg Val Val 35
40 45 Ala Glu Thr Leu Trp Leu Glu Leu
Val Trp Ile Val Asp Trp Trp Ala 50 55
60 Gly Val Lys Ile Gln Val Phe Ala Asp Asp Glu Thr Phe
Asn Arg Met 65 70 75
80 Gly Lys Glu His Ala Leu Val Val Cys Asn His Arg Ser Asp Ile Asp
85 90 95 Trp Leu Val Gly
Trp Ile Leu Ala Gln Arg Ser Gly Cys Leu Gly Ser 100
105 110 Ala Leu Ala Val Met Lys Lys Ser Ser
Lys Phe Leu Pro Val Ile Gly 115 120
125 Trp Ser Met Trp Phe Ser Glu Tyr Leu Phe Leu Glu Arg Asn
Trp Ala 130 135 140
Lys Asp Glu Ser Thr Leu Gln Ser Gly Leu Gln Arg Leu Asn Asp Phe 145
150 155 160 Pro Arg Pro Phe Trp
Leu Ala Leu Phe Val Glu Gly Thr Arg Phe Thr 165
170 175 Glu Ala Lys Leu Lys Ala Ala Gln Glu Tyr
Ala Ala Ser Ser Glu Leu 180 185
190 Pro Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val
Ser 195 200 205 Ala
Val Ser Asn Met Arg Ser Phe Val Pro Ala Ile Tyr Asp Met Thr 210
215 220 Val Ala Ile Pro Lys Thr
Ser Pro Pro Pro Thr Met Leu Arg Leu Phe 225 230
235 240 Lys Gly Gln Pro Ser Val Val His Val His Ile
Lys Cys His Ser Met 245 250
255 Lys Asp Leu Pro Glu Pro Glu Asp Glu Ile Ala Gln Trp Cys Arg Asp
260 265 270 Gln Phe
Val Ala Lys Asp Ala Leu Leu Asp Lys His Ile Ala Ala Asp 275
280 285 Thr Phe Pro Gly Gln Lys Glu
Gln Asn Ile Gly Arg Pro Ile Lys Ser 290 295
300 Leu Ala Val Val Val Ser Trp Ala Cys Leu Leu Thr
Leu Gly Ala Met 305 310 315
320 Lys Phe Leu His Trp Ser Asn Leu Phe Ser Ser Trp Lys Gly Ile Ala
325 330 335 Leu Ser Ala
Phe Gly Leu Gly Ile Ile Thr Leu Cys Met Gln Ile Leu 340
345 350 Ile Arg Ser Ser Gln Ser Glu Arg
Ser Thr Pro Ala Lys Val Ala Pro 355 360
365 Ala Lys Pro Lys Asp Asn His Gln Ser Gly Pro Ser Ser
Gln Thr Glu 370 375 380
Val Glu Glu Lys Gln Lys 385 390 158415PRTCuphea
hookeriana 158Met Val Ala Ala Ala Ala Ser Ser Ala Phe Phe Pro Val Pro Ala
Pro 1 5 10 15 Gly
Ala Ser Pro Lys Pro Gly Lys Phe Gly Asn Trp Pro Ser Ser Leu
20 25 30 Ser Pro Ser Phe Lys
Pro Lys Ser Ile Pro Asn Gly Gly Phe Gln Val 35
40 45 Lys Ala Asn Asp Ser Ala His Pro Lys
Ala Asn Gly Ser Ala Val Ser 50 55
60 Leu Lys Ser Gly Ser Leu Asn Thr Gln Glu Asp Thr Ser
Ser Ser Pro 65 70 75
80 Pro Pro Arg Thr Phe Leu His Gln Leu Pro Asp Trp Ser Arg Leu Leu
85 90 95 Thr Ala Ile Thr
Thr Val Phe Val Lys Ser Lys Arg Pro Asp Met His 100
105 110 Asp Arg Lys Ser Lys Arg Pro Asp Met
Leu Val Asp Ser Phe Gly Leu 115 120
125 Glu Ser Thr Val Gln Asp Gly Leu Val Phe Arg Gln Ser Phe
Ser Ile 130 135 140
Arg Ser Tyr Glu Ile Gly Thr Asp Arg Thr Ala Ser Ile Glu Thr Leu 145
150 155 160 Met Asn His Leu Gln
Glu Thr Ser Leu Asn His Cys Lys Ser Thr Gly 165
170 175 Ile Leu Leu Asp Gly Phe Gly Arg Thr Leu
Glu Met Cys Lys Arg Asp 180 185
190 Leu Ile Trp Val Val Ile Lys Met Gln Ile Lys Val Asn Arg Tyr
Pro 195 200 205 Ala
Trp Gly Asp Thr Val Glu Ile Asn Thr Arg Phe Ser Arg Leu Gly 210
215 220 Lys Ile Gly Met Gly Arg
Asp Trp Leu Ile Ser Asp Cys Asn Thr Gly 225 230
235 240 Glu Ile Leu Val Arg Ala Thr Ser Ala Tyr Ala
Met Met Asn Gln Lys 245 250
255 Thr Arg Arg Leu Ser Lys Leu Pro Tyr Glu Val His Gln Glu Ile Val
260 265 270 Pro Leu
Phe Val Asp Ser Pro Val Ile Glu Asp Ser Asp Leu Lys Val 275
280 285 His Lys Phe Lys Val Lys Thr
Gly Asp Ser Ile Gln Lys Gly Leu Thr 290 295
300 Pro Gly Trp Asn Asp Leu Asp Val Asn Gln His Val
Ser Asn Val Lys 305 310 315
320 Tyr Ile Gly Trp Ile Leu Glu Ser Met Pro Thr Glu Val Leu Glu Thr
325 330 335 Gln Glu Leu
Cys Ser Leu Ala Leu Glu Tyr Arg Arg Glu Cys Gly Arg 340
345 350 Asp Ser Val Leu Glu Ser Val Thr
Ala Met Asp Pro Ser Lys Val Gly 355 360
365 Val Arg Ser Gln Tyr Gln His Leu Leu Arg Leu Glu Asp
Gly Thr Ala 370 375 380
Ile Val Asn Gly Ala Thr Glu Trp Arg Pro Lys Asn Ala Gly Ala Asn 385
390 395 400 Gly Ala Ile Ser
Thr Gly Lys Thr Ser Asn Gly Asn Ser Val Ser 405
410 415 159525PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 159Met Ala Ser Ala Ala Phe
Thr Met Ser Ala Cys Pro Ala Met Thr Gly 1 5
10 15 Arg Ala Pro Gly Ala Arg Arg Ser Gly Arg Pro
Val Ala Thr Arg Leu 20 25
30 Arg Tyr Val Phe Gln Cys Leu Val Ala Ser Cys Ile Asp Pro Cys
Asp 35 40 45 Gln
Tyr Arg Ser Ser Ala Ser Leu Ser Phe Leu Gly Asp Asn Gly Phe 50
55 60 Ala Ser Leu Phe Gly Ser
Lys Pro Phe Met Ser Asn Arg Gly His Arg 65 70
75 80 Arg Leu Arg Arg Ala Ser His Ser Gly Glu Ala
Met Ala Val Ala Leu 85 90
95 Gln Pro Ala Gln Glu Ala Gly Thr Lys Lys Lys Pro Val Ile Lys Gln
100 105 110 Arg Arg
Val Val Val Thr Gly Met Gly Val Val Thr Pro Leu Gly His 115
120 125 Glu Pro Asp Val Phe Tyr Asn
Asn Leu Leu Asp Gly Val Ser Gly Ile 130 135
140 Ser Glu Ile Glu Thr Phe Asp Cys Thr Gln Phe Pro
Thr Arg Ile Ala 145 150 155
160 Gly Glu Ile Lys Ser Phe Ser Thr Asp Gly Trp Val Ala Pro Lys Leu
165 170 175 Ser Lys Arg
Met Asp Lys Phe Met Leu Tyr Leu Leu Thr Ala Gly Lys 180
185 190 Lys Ala Leu Ala Asp Gly Gly Ile
Thr Asp Glu Val Met Lys Glu Leu 195 200
205 Asp Lys Arg Lys Cys Gly Val Leu Ile Gly Ser Gly Met
Gly Gly Met 210 215 220
Lys Val Phe Asn Asp Ala Ile Glu Ala Leu Arg Val Ser Tyr Lys Lys 225
230 235 240 Met Asn Pro Phe
Cys Val Pro Phe Ala Thr Thr Asn Met Gly Ser Ala 245
250 255 Met Leu Ala Met Asp Leu Gly Trp Met
Gly Pro Asn Tyr Ser Ile Ser 260 265
270 Thr Ala Cys Ala Thr Ser Asn Phe Cys Ile Leu Asn Ala Ala
Asn His 275 280 285
Ile Ile Arg Gly Glu Ala Asp Met Met Leu Cys Gly Gly Ser Asp Ala 290
295 300 Val Ile Ile Pro Ile
Gly Leu Gly Gly Phe Val Ala Cys Arg Ala Leu 305 310
315 320 Ser Gln Arg Asn Ser Asp Pro Thr Lys Ala
Ser Arg Pro Trp Asp Ser 325 330
335 Asn Arg Asp Gly Phe Val Met Gly Glu Gly Ala Gly Val Leu Leu
Leu 340 345 350 Glu
Glu Leu Glu His Ala Lys Lys Arg Gly Ala Thr Ile Tyr Ala Glu 355
360 365 Phe Leu Gly Gly Ser Phe
Thr Cys Asp Ala Tyr His Met Thr Glu Pro 370 375
380 His Pro Glu Gly Ala Gly Val Ile Leu Cys Ile
Glu Lys Ala Leu Ala 385 390 395
400 Gln Ala Gly Val Ser Lys Glu Asp Val Asn Tyr Ile Asn Ala His Ala
405 410 415 Thr Ser
Thr Ser Ala Gly Asp Ile Lys Glu Tyr Gln Ala Leu Ala Arg 420
425 430 Cys Phe Gly Gln Asn Ser Glu
Leu Arg Val Asn Ser Thr Lys Ser Met 435 440
445 Ile Gly His Leu Leu Gly Ala Ala Gly Gly Val Glu
Ala Val Thr Val 450 455 460
Val Gln Ala Ile Arg Thr Gly Trp Ile His Pro Asn Leu Asn Leu Glu 465
470 475 480 Asp Pro Asp
Lys Ala Val Asp Ala Lys Leu Leu Val Gly Pro Lys Lys 485
490 495 Glu Arg Leu Asn Val Lys Val Gly
Leu Ser Asn Ser Phe Gly Phe Gly 500 505
510 Gly His Asn Ser Ser Ile Leu Phe Ala Pro Cys Asn Val
515 520 525 160467PRTPrototheca
moriformis 160Met Gln Thr Ala His Gln Arg Pro Pro Thr Glu Gly His Cys Phe
Gly 1 5 10 15 Ala
Arg Leu Pro Thr Ala Ser Arg Arg Ala Val Arg Arg Ala Trp Ser
20 25 30 Arg Ile Ala Arg Ala
Ala Ala Ala Ala Asp Ala Asn Pro Ala Arg Pro 35
40 45 Glu Arg Arg Val Val Ile Thr Gly Gln
Gly Val Val Thr Ser Leu Gly 50 55
60 Gln Thr Ile Glu Gln Phe Tyr Ser Ser Leu Leu Glu Gly
Val Ser Gly 65 70 75
80 Ile Ser Gln Ile Gln Lys Phe Asp Thr Thr Gly Tyr Thr Thr Thr Ile
85 90 95 Ala Gly Glu Ile
Lys Ser Leu Gln Leu Asp Pro Tyr Val Pro Lys Arg 100
105 110 Trp Ala Lys Arg Val Asp Asp Val Ile
Lys Tyr Val Tyr Ile Ala Gly 115 120
125 Lys Gln Ala Leu Glu Ser Ala Gly Leu Pro Ile Glu Ala Ala
Gly Leu 130 135 140
Ala Gly Ala Gly Leu Asp Pro Ala Leu Cys Gly Val Leu Ile Gly Thr 145
150 155 160 Ala Met Ala Gly Met
Thr Ser Phe Ala Ala Gly Val Glu Ala Leu Thr 165
170 175 Arg Gly Gly Val Arg Lys Met Asn Pro Phe
Cys Ile Pro Phe Ser Ile 180 185
190 Ser Asn Met Gly Gly Ala Met Leu Ala Met Asp Ile Gly Phe Met
Gly 195 200 205 Pro
Asn Tyr Ser Ile Ser Thr Ala Cys Ala Thr Gly Asn Tyr Cys Ile 210
215 220 Leu Gly Ala Ala Asp His
Ile Arg Arg Gly Asp Ala Asn Val Met Leu 225 230
235 240 Ala Gly Gly Ala Asp Ala Ala Ile Ile Pro Ser
Gly Ile Gly Gly Phe 245 250
255 Ile Ala Cys Lys Ala Leu Ser Lys Arg Asn Asp Glu Pro Glu Arg Ala
260 265 270 Ser Arg
Pro Trp Asp Ala Asp Arg Asp Gly Phe Val Met Gly Glu Gly 275
280 285 Ala Gly Val Leu Val Leu Glu
Glu Leu Glu His Ala Lys Arg Arg Gly 290 295
300 Ala Thr Ile Leu Ala Glu Leu Val Gly Gly Ala Ala
Thr Ser Asp Ala 305 310 315
320 His His Met Thr Glu Pro Asp Pro Gln Gly Arg Gly Val Arg Leu Cys
325 330 335 Leu Glu Arg
Ala Leu Glu Arg Ala Arg Leu Ala Pro Glu Arg Val Gly 340
345 350 Tyr Val Asn Ala His Gly Thr Ser
Thr Pro Ala Gly Asp Val Ala Glu 355 360
365 Tyr Arg Ala Ile Arg Ala Val Ile Pro Gln Asp Ser Leu
Arg Ile Asn 370 375 380
Ser Thr Lys Ser Met Ile Gly His Leu Leu Gly Gly Ala Gly Ala Val 385
390 395 400 Glu Ala Val Ala
Ala Ile Gln Ala Leu Arg Thr Gly Trp Leu His Pro 405
410 415 Asn Leu Asn Leu Glu Asn Pro Ala Pro
Gly Val Asp Pro Val Val Leu 420 425
430 Val Gly Pro Arg Lys Glu Arg Ala Glu Asp Leu Asp Val Val
Leu Ser 435 440 445
Asn Ser Phe Gly Phe Gly Gly His Asn Ser Cys Val Ile Phe Arg Lys 450
455 460 Tyr Asp Glu 465
161431PRTPrototheca moriformis 161Ala Ala Ala Ala Ala Asp Ala Asn Pro
Ala Arg Pro Glu Arg Arg Val 1 5 10
15 Val Ile Thr Gly Gln Gly Val Val Thr Ser Leu Gly Gln Thr
Ile Glu 20 25 30
Gln Phe Tyr Ser Ser Leu Leu Glu Gly Val Ser Gly Ile Ser Gln Ile
35 40 45 Gln Lys Phe Asp
Thr Thr Gly Tyr Thr Thr Thr Ile Ala Gly Glu Ile 50
55 60 Lys Ser Leu Gln Leu Asp Pro Tyr
Val Pro Lys Arg Trp Ala Lys Arg 65 70
75 80 Val Asp Asp Val Ile Lys Tyr Val Tyr Ile Ala Gly
Lys Gln Ala Leu 85 90
95 Glu Ser Ala Gly Leu Pro Ile Glu Ala Ala Gly Leu Ala Gly Ala Gly
100 105 110 Leu Asp Pro
Ala Leu Cys Gly Val Leu Ile Gly Thr Ala Met Ala Gly 115
120 125 Met Thr Ser Phe Ala Ala Gly Val
Glu Ala Leu Thr Arg Gly Gly Val 130 135
140 Arg Lys Met Asn Pro Phe Cys Ile Pro Phe Ser Ile Ser
Asn Met Gly 145 150 155
160 Gly Ala Met Leu Ala Met Asp Ile Gly Phe Met Gly Pro Asn Tyr Ser
165 170 175 Ile Ser Thr Ala
Cys Ala Thr Gly Asn Tyr Cys Ile Leu Gly Ala Ala 180
185 190 Asp His Ile Arg Arg Gly Asp Ala Asn
Val Met Leu Ala Gly Gly Ala 195 200
205 Asp Ala Ala Ile Ile Pro Ser Gly Ile Gly Gly Phe Ile Ala
Cys Lys 210 215 220
Ala Leu Ser Lys Arg Asn Asp Glu Pro Glu Arg Ala Ser Arg Pro Trp 225
230 235 240 Asp Ala Asp Arg Asp
Gly Phe Val Met Gly Glu Gly Ala Gly Val Leu 245
250 255 Val Leu Glu Glu Leu Glu His Ala Lys Arg
Arg Gly Ala Thr Ile Leu 260 265
270 Ala Glu Leu Val Gly Gly Ala Ala Thr Ser Asp Ala His His Met
Thr 275 280 285 Glu
Pro Asp Pro Gln Gly Arg Gly Val Arg Leu Cys Leu Glu Arg Ala 290
295 300 Leu Glu Arg Ala Arg Leu
Ala Pro Glu Arg Val Gly Tyr Val Asn Ala 305 310
315 320 His Gly Thr Ser Thr Pro Ala Gly Asp Val Ala
Glu Tyr Arg Ala Ile 325 330
335 Arg Ala Val Ile Pro Gln Asp Ser Leu Arg Ile Asn Ser Thr Lys Ser
340 345 350 Met Ile
Gly His Leu Leu Gly Gly Ala Gly Ala Val Glu Ala Val Ala 355
360 365 Ala Ile Gln Ala Leu Arg Thr
Gly Trp Leu His Pro Asn Leu Asn Leu 370 375
380 Glu Asn Pro Ala Pro Gly Val Asp Pro Val Val Leu
Val Gly Pro Arg 385 390 395
400 Lys Glu Arg Ala Glu Asp Leu Asp Val Val Leu Ser Asn Ser Phe Gly
405 410 415 Phe Gly Gly
His Asn Ser Cys Val Ile Phe Arg Lys Tyr Asp Glu 420
425 430 162322PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 162Pro Asp Trp Ser Met
Leu Phe Ala Val Ile Thr Thr Ile Phe Ser Ala 1 5
10 15 Ala Glu Lys Gln Trp Thr Asn Leu Glu Trp
Lys Pro Lys Pro Asn Pro 20 25
30 Pro Gln Leu Leu Asp Asp His Phe Gly Pro His Gly Leu Val Phe
Arg 35 40 45 Arg
Thr Phe Ala Ile Arg Ser Tyr Glu Val Gly Pro Asp Arg Ser Thr 50
55 60 Ser Ile Val Ala Val Met
Asn His Leu Gln Glu Ala Ala Leu Asn His 65 70
75 80 Ala Lys Ser Val Gly Ile Leu Gly Asp Gly Phe
Gly Thr Thr Leu Glu 85 90
95 Met Ser Lys Arg Asp Leu Ile Trp Val Val Arg Arg Thr His Val Ala
100 105 110 Val Glu
Arg Tyr Pro Thr Trp Gly Asp Thr Val Glu Val Glu Cys Trp 115
120 125 Ile Gly Ala Ser Gly Asn Asn
Gly Met Arg Arg Asp Phe Leu Val Arg 130 135
140 Asp Cys Lys Thr Gly Glu Ile Leu Thr Arg Cys Thr
Ser Leu Ser Val 145 150 155
160 Leu Met Asn Thr Arg Thr Arg Arg Leu Ser Thr Ile Pro Asp Glu Val
165 170 175 Arg Gly Glu
Ile Gly Pro Ala Phe Ile Asp Asn Val Ala Val Lys Asp 180
185 190 Asp Glu Ile Lys Lys Leu Gln Lys
Leu Asn Asp Ser Thr Ala Asp Tyr 195 200
205 Ile Gln Gly Gly Leu Thr Pro Arg Trp Asn Asp Leu Asp
Val Asn Gln 210 215 220
His Val Asn Asn Leu Lys Tyr Val Ala Trp Val Phe Glu Thr Val Pro 225
230 235 240 Asp Ser Ile Phe
Glu Ser His His Ile Ser Ser Phe Thr Leu Glu Tyr 245
250 255 Arg Arg Glu Cys Thr Arg Asp Ser Val
Leu Arg Ser Leu Thr Thr Val 260 265
270 Ser Gly Gly Ser Ser Glu Ala Gly Leu Val Cys Asp His Leu
Leu Gln 275 280 285
Leu Glu Gly Gly Ser Glu Val Leu Arg Ala Arg Thr Glu Trp Arg Pro 290
295 300 Lys Leu Thr Asp Ser
Phe Arg Gly Ile Ser Val Ile Pro Ala Glu Pro 305 310
315 320 Arg Val 163361PRTCuphea hyssopifolia
163Met Val Ala Thr Asn Ala Ala Ala Phe Ser Ala Tyr Thr Phe Phe Leu 1
5 10 15 Thr Ser Pro Thr
His Gly Tyr Ser Ser Lys Arg Leu Ala Asp Thr Gln 20
25 30 Asn Gly Tyr Pro Gly Thr Ser Leu Lys
Ser Lys Ser Thr Pro Pro Pro 35 40
45 Ala Ala Ala Ala Ala Arg Asn Gly Ala Leu Pro Leu Leu Ala
Ser Ile 50 55 60
Cys Lys Cys Pro Lys Lys Ala Asp Gly Ser Met Gln Leu Asp Ser Ser 65
70 75 80 Leu Val Phe Gly Phe
Gln Phe Tyr Ile Arg Ser Tyr Glu Val Gly Ala 85
90 95 Asp Gln Thr Val Ser Ile Gln Thr Val Leu
Asn Tyr Leu Gln Glu Ala 100 105
110 Ala Ile Asn His Val Gln Ser Ala Gly Tyr Phe Gly Asp Ser Phe
Gly 115 120 125 Ala
Thr Pro Glu Met Thr Lys Arg Asn Leu Ile Trp Val Ile Thr Lys 130
135 140 Met Gln Val Leu Val Asp
Arg Tyr Pro Ala Trp Gly Asp Val Val Gln 145 150
155 160 Val Asp Thr Trp Thr Cys Ser Ser Gly Lys Asn
Ser Met Gln Arg Asp 165 170
175 Trp Phe Val Arg Asp Leu Lys Thr Gly Asp Ile Ile Thr Arg Ala Ser
180 185 190 Ser Val
Trp Val Leu Met Asn Arg Leu Thr Arg Lys Leu Ser Lys Ile 195
200 205 Pro Glu Ala Val Leu Glu Glu
Ala Lys Leu Phe Val Met Asn Thr Ala 210 215
220 Pro Thr Val Asp Asp Asn Arg Lys Leu Pro Lys Leu
Asp Gly Ser Ser 225 230 235
240 Ala Asp Tyr Val Leu Ser Gly Leu Thr Pro Arg Trp Ser Asp Leu Asp
245 250 255 Met Asn Gln
His Val Asn Asn Val Lys Tyr Ile Ala Trp Ile Leu Glu 260
265 270 Ser Val Pro Gln Ser Ile Pro Glu
Thr His Lys Leu Ser Ala Ile Thr 275 280
285 Val Glu Tyr Arg Arg Glu Cys Gly Lys Asn Ser Val Leu
Gln Ser Leu 290 295 300
Thr Asn Val Ser Gly Asp Gly Ile Thr Cys Gly Asn Ser Ile Ile Glu 305
310 315 320 Cys His His Leu
Leu Gln Leu Glu Thr Gly Pro Glu Ile Leu Leu Ala 325
330 335 Arg Thr Glu Trp Ile Ser Lys Glu Pro
Gly Phe Arg Gly Ala Pro Ile 340 345
350 Gln Ala Glu Lys Val Tyr Asn Asn Lys 355
360 164417PRTCuphea hyssopifolia 164Met Val Ala Ala Glu Ala
Ser Ser Ala Leu Phe Ser Val Arg Thr Pro 1 5
10 15 Gly Thr Ser Pro Lys Pro Gly Lys Phe Gly Asn
Trp Pro Thr Ser Leu 20 25
30 Ser Val Pro Phe Lys Ser Lys Ser Asn His Asn Gly Gly Phe Gln
Val 35 40 45 Lys
Ala Asn Ala Ser Ala Arg Pro Lys Ala Asn Gly Ser Ala Val Ser 50
55 60 Leu Lys Ser Gly Ser Leu
Asp Thr Gln Glu Asp Thr Ser Ser Ser Ser 65 70
75 80 Ser Pro Pro Arg Thr Phe Ile Asn Gln Leu Pro
Asp Trp Ser Met Leu 85 90
95 Leu Ser Ala Ile Thr Thr Val Phe Val Ala Ala Glu Lys Gln Trp Thr
100 105 110 Met Leu
Asp Arg Lys Ser Lys Arg Pro Asp Met Leu Met Asp Pro Phe 115
120 125 Gly Val Asp Arg Val Val Gln
Asp Gly Ala Val Phe Arg Gln Ser Phe 130 135
140 Ser Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg Thr
Ala Ser Ile Glu 145 150 155
160 Thr Leu Met Asn Ile Phe Gln Glu Thr Ser Leu Asn His Cys Lys Ser
165 170 175 Ile Gly Leu
Leu Asn Asp Gly Phe Gly Arg Thr Pro Glu Met Cys Lys 180
185 190 Arg Asp Leu Ile Trp Val Val Thr
Lys Met His Val Glu Val Asn Arg 195 200
205 Tyr Pro Thr Trp Gly Asp Thr Ile Glu Val Asn Thr Trp
Val Ser Glu 210 215 220
Ser Gly Lys Thr Gly Met Gly Arg Asp Trp Leu Ile Ser Asp Cys His 225
230 235 240 Thr Gly Glu Ile
Leu Ile Arg Ala Thr Ser Met Cys Ala Met Met Asn 245
250 255 Gln Lys Thr Arg Arg Phe Ser Lys Phe
Pro Tyr Glu Val Arg Gln Glu 260 265
270 Leu Ala Pro His Phe Val Asp Ser Ala Pro Val Ile Glu Asp
Tyr Gln 275 280 285
Lys Leu His Lys Leu Asp Val Lys Thr Gly Asp Ser Ile Cys Asn Gly 290
295 300 Leu Thr Pro Arg Trp
Asn Asp Leu Asp Val Asn Gln His Val Asn Asn 305 310
315 320 Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser
Val Pro Thr Glu Val Phe 325 330
335 Glu Thr Gln Glu Leu Cys Gly Leu Thr Leu Glu Tyr Arg Arg Glu
Cys 340 345 350 Gly
Arg Asp Ser Val Leu Glu Ser Val Thr Ala Met Asp Pro Ser Lys 355
360 365 Glu Gly Asp Arg Ser Leu
Tyr Gln His Leu Leu Arg Leu Glu Asp Gly 370 375
380 Ala Asp Ile Ala Lys Gly Arg Thr Lys Trp Arg
Pro Lys Asn Ala Gly 385 390 395
400 Thr Asn Gly Ala Ile Ser Thr Gly Lys Thr Ser Asn Gly Asn Ser Ile
405 410 415 Ser
165542PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(22)..(22)Any amino acidMOD_RES(24)..(33)Any amino
acidMOD_RES(92)..(92)Ile or LeuMOD_RES(93)..(93)Any amino
acidMOD_RES(128)..(130)Any amino acidMOD_RES(142)..(142)Any amino
acidMOD_RES(145)..(145)Any amino acidMOD_RES(148)..(148)Any amino
acidMOD_RES(153)..(153)Any amino acidMOD_RES(158)..(158)Any amino
acidMOD_RES(182)..(182)Any amino acidMOD_RES(184)..(184)Any amino
acidMOD_RES(192)..(192)Any amino acidMOD_RES(202)..(202)Any amino
acidMOD_RES(206)..(206)Any amino acidMOD_RES(249)..(249)Any amino
acidMOD_RES(295)..(295)Any amino acidMOD_RES(310)..(310)Any amino
acidMOD_RES(322)..(322)Any amino acidMOD_RES(350)..(350)Any amino
acidMOD_RES(352)..(353)Any amino acidMOD_RES(356)..(356)Any amino
acidMOD_RES(359)..(359)Any amino acidMOD_RES(373)..(373)Any amino
acidMOD_RES(380)..(380)Any amino acidMOD_RES(399)..(400)Any amino
acidMOD_RES(490)..(490)Any amino acidMOD_RES(529)..(529)Any amino acid
165Met Glu Arg Thr Asn Ser Ile Glu Met Asp Gln Glu Arg Leu Thr Ala 1
5 10 15 Glu Met Ala Phe
Lys Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Xaa Leu Pro Asp Phe Met Thr Ser Ile
Asn Val Lys Leu Leu Tyr His 35 40
45 Tyr Val Ile Thr Asn Leu Phe Asn Leu Cys Phe Phe Pro Leu
Thr Ala 50 55 60
Ile Val Ala Gly Lys Ala Ser Arg Leu Thr Ile Asx Asp Leu His His 65
70 75 80 Leu Tyr Ser Tyr Leu
Gln His Asn Leu Ile Thr Xaa Xaa Leu Leu Phe 85
90 95 Ala Phe Thr Val Phe Gly Ser Ile Leu Tyr
Ile Val Thr Arg Pro Lys 100 105
110 Pro Val Tyr Leu Val Asp Tyr Ser Cys Tyr Leu Pro Pro Thr His
Xaa 115 120 125 Xaa
Xaa Ser Ile Ser Lys Val Met Asp Ile Phe Tyr Gln Xaa Arg Lys 130
135 140 Xaa Asp Pro Xaa Arg Asn
Gly Thr Xaa Asp Asp Ser Ser Xaa Leu Asp 145 150
155 160 Phe Leu Arg Lys Ile Gln Glu Arg Ser Gly Leu
Gly Asp Glu Thr Tyr 165 170
175 Gly Pro Glu Gly Leu Xaa Gln Xaa Pro Pro Arg Lys Asn Phe Ala Xaa
180 185 190 Ala Arg
Glu Glu Thr Glu Gln Val Ile Xaa Gly Ala Leu Xaa Asn Leu 195
200 205 Phe Glu Asn Thr Lys Val Asn
Pro Lys Glu Ile Gly Ile Leu Val Val 210 215
220 Asn Ser Ser Met Phe Asn Pro Thr Pro Ser Leu Ser
Ala Met Val Val 225 230 235
240 Asn Thr Phe Lys Leu Arg Ser Asn Xaa Lys Ser Phe Asn Leu Gly Gly
245 250 255 Met Gly Cys
Ser Ala Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu 260
265 270 Leu His Val His Lys Asn Thr Tyr
Ala Leu Val Val Ser Thr Glu Asn 275 280
285 Ile Thr Tyr Asn Ile Tyr Xaa Gly Asp Asn Arg Ser Met
Met Val Ser 290 295 300
Asn Cys Leu Phe Arg Xaa Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys 305
310 315 320 Pro Xaa Asp Arg
Arg Arg Ser Lys Tyr Glu Leu Val His Thr Val Arg 325
330 335 Thr His Thr Gly Ala Asp Asp Lys Ser
Phe Arg Cys Val Xaa Gln Xaa 340 345
350 Xaa Asp Glu Xaa Gly Lys Xaa Gly Val Ser Leu Ser Lys Asp
Ile Thr 355 360 365
Ala Val Ala Gly Xaa Thr Val Lys Lys Asn Ile Xaa Thr Leu Gly Pro 370
375 380 Leu Val Leu Pro Leu
Ser Glu Lys Leu Leu Phe Phe Val Thr Xaa Xaa 385 390
395 400 Ala Lys Lys Leu Phe Lys Asp Lys Ile Lys
His Tyr Tyr Val Pro Asp 405 410
415 Phe Lys Leu Ala Ile Asp His Phe Cys Ile His Ala Gly Gly Arg
Ala 420 425 430 Val
Ile Asp Val Leu Glu Lys Asn Leu Gly Leu Ser Pro Ile Asp Val 435
440 445 Glu Ala Ser Arg Ser Thr
Leu His Arg Phe Gly Asn Thr Ser Ser Ser 450 455
460 Ser Ile Trp Tyr Glu Leu Ala Tyr Ile Glu Ala
Lys Gly Arg Met Lys 465 470 475
480 Lys Gly Asn Lys Val Trp Gln Ile Ala Xaa Gly Ser Gly Phe Lys Cys
485 490 495 Asn Ser
Ala Val Trp Val Ala Leu Arg Asn Val Lys Ala Ser Thr Asn 500
505 510 Ser Pro Trp Glu His Cys Ile
Asp Arg Tyr Pro Val Lys Ile Asp Ser 515 520
525 Xaa Ser Ser Lys Ser Glu Thr Arg Ala Gln Asn Gly
Arg Ser 530 535 540
166505PRTBrassica napus 166Met Thr Ser Ile Asn Val Lys Leu Leu Tyr His
Tyr Val Ile Thr Asn 1 5 10
15 Leu Phe Asn Leu Cys Phe Phe Pro Leu Thr Ala Ile Val Ala Gly Lys
20 25 30 Ala Tyr
Leu Thr Ile Asp Asp Leu His His Leu Tyr Tyr Ser Tyr Leu 35
40 45 Gln His Asn Leu Ile Thr Ile
Ala Pro Leu Leu Ala Phe Thr Val Phe 50 55
60 Gly Ser Val Leu Tyr Ile Ala Thr Arg Pro Lys Pro
Val Tyr Leu Val 65 70 75
80 Glu Tyr Ser Cys Tyr Leu Pro Pro Thr His Cys Arg Ser Ser Ile Ser
85 90 95 Lys Val Met
Asp Ile Phe Phe Gln Val Arg Lys Ala Asp Pro Ser Arg 100
105 110 Asn Gly Thr Cys Asp Asp Ser Ser
Trp Leu Asp Phe Leu Arg Lys Ile 115 120
125 Gln Glu Arg Ser Gly Leu Gly Asp Glu Thr His Gly Pro
Glu Gly Leu 130 135 140
Leu Gln Val Pro Pro Arg Lys Thr Phe Ala Arg Ala Arg Glu Glu Thr 145
150 155 160 Glu Gln Val Ile
Ile Gly Ala Leu Glu Asn Leu Phe Lys Asn Thr Asn 165
170 175 Val Asn Pro Lys Asp Ile Gly Ile Leu
Val Val Asn Ser Ser Met Phe 180 185
190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe
Lys Leu 195 200 205
Arg Ser Asn Val Arg Ser Phe Asn Leu Gly Gly Met Gly Cys Ser Ala 210
215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230
235 240 Asn Thr Tyr Ala Leu Val Val Ser Thr Glu
Asn Ile Thr Tyr Asn Ile 245 250
255 Tyr Ala Gly Asp Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe
Arg 260 265 270 Val
Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Arg Asp Arg Arg 275
280 285 Arg Ser Lys Tyr Glu Leu
Val His Thr Val Arg Thr His Thr Gly Ala 290 295
300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Asn Gly 305 310 315
320 Gln Thr Gly Val Ser Leu Ser Lys Asp Ile Thr Asp Val Ala Gly Arg
325 330 335 Thr Val
Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340
345 350 Ser Glu Lys Leu Leu Phe Phe
Val Thr Phe Met Gly Lys Lys Leu Phe 355 360
365 Lys Asp Glu Ile Lys His Tyr Tyr Val Pro Asp Phe
Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Lys Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Asn
Leu Gly Leu Ala Pro Ile Asp Val Glu Ala Ser Arg Ser 405
410 415 Thr Leu His Arg Phe Gly Asn Thr
Ser Ser Ser Ser Ile Trp Tyr Glu 420 425
430 Leu Ala Tyr Ile Glu Pro Lys Gly Arg Met Lys Lys Gly
Asn Lys Val 435 440 445
Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp 450
455 460 Val Ala Leu Asn
Asn Val Lys Ala Ser Thr Asn Ser Pro Trp Glu His 465 470
475 480 Cys Ile Asp Arg Tyr Pro Val Lys Ile
Asp Ser Asp Ser Gly Lys Ser 485 490
495 Glu Thr Arg Val Pro Asn Gly Arg Ser 500
505 167528PRTBrassica napus 167Met Glu Arg Thr Asn Ser Ile
Glu Met Asp Gln Glu Arg Leu Thr Ala 1 5
10 15 Glu Met Ala Phe Lys Asp Ser Ser Ser Ala Val
Ile Arg Ile Arg Arg 20 25
30 Arg Leu Pro Asp Phe Leu Thr Ser Val Lys Leu Lys Tyr Val Lys
Leu 35 40 45 Gly
Leu His Asn Ser Phe Asn Phe Thr Thr Phe Leu Phe Leu Leu Ile 50
55 60 Ile Leu Pro Leu Thr Gly
Thr Val Leu Val Gln Leu Thr Gly Leu Thr 65 70
75 80 Phe Glu Thr Phe Ser Glu Leu Trp Tyr Asn His
Ala Ala Gln Leu Asp 85 90
95 Gly Val Thr Arg Leu Ala Cys Leu Val Ser Leu Cys Phe Val Leu Ile
100 105 110 Ile Tyr
Val Thr Asn Arg Ser Lys Pro Val Tyr Leu Val Asp Phe Ser 115
120 125 Cys Tyr Lys Pro Glu Asp Glu
Arg Lys Met Ser Val Asp Ser Phe Leu 130 135
140 Lys Met Thr Glu Gln Asn Gly Ala Phe Thr Asp Asp
Thr Val Gln Phe 145 150 155
160 Gln Gln Arg Ile Ser Asn Arg Ala Gly Leu Gly Asp Glu Thr Tyr Leu
165 170 175 Pro Arg Gly
Ile Thr Ser Thr Pro Pro Lys Leu Asn Met Ser Glu Ala 180
185 190 Arg Ala Glu Ala Glu Ala Val Met
Phe Gly Ala Leu Asp Ser Leu Phe 195 200
205 Glu Lys Thr Gly Ile Lys Pro Ala Glu Val Gly Ile Leu
Ile Val Ser 210 215 220
Cys Ser Leu Phe Asn Pro Thr Pro Ser Leu Ser Ala Met Ile Val Asn 225
230 235 240 His Tyr Lys Met
Arg Glu Asp Ile Lys Ser Tyr Asn Leu Gly Gly Met 245
250 255 Gly Cys Ser Ala Gly Leu Ile Ser Ile
Asp Leu Ala Asn Asn Leu Leu 260 265
270 Lys Ala Asn Pro Asn Ser Tyr Ala Val Val Val Ser Thr Glu
Asn Ile 275 280 285
Thr Leu Asn Trp Tyr Phe Gly Asn Asp Arg Ser Met Leu Leu Cys Asn 290
295 300 Cys Ile Phe Arg Met
Gly Gly Ala Ala Ile Leu Leu Ser Asn Arg Arg 305 310
315 320 Gln Asp Arg Ser Lys Ser Lys Tyr Glu Leu
Val Asn Val Val Arg Thr 325 330
335 His Lys Gly Ser Asp Asp Lys Asn Tyr Asn Cys Val Tyr Gln Lys
Glu 340 345 350 Asp
Glu Arg Gly Thr Ile Gly Val Ser Leu Ala Arg Glu Leu Met Ser 355
360 365 Val Ala Gly Asp Ala Leu
Lys Thr Asn Ile Thr Thr Leu Gly Pro Met 370 375
380 Val Leu Pro Leu Ser Gly Gln Leu Met Phe Ser
Val Ser Leu Val Lys 385 390 395
400 Arg Lys Leu Leu Lys Leu Lys Val Lys Pro Tyr Ile Pro Asp Phe Lys
405 410 415 Leu Ala
Phe Glu His Phe Cys Ile His Ala Gly Gly Arg Ala Val Leu 420
425 430 Asp Glu Val Gln Lys Asn Leu
Asp Leu Glu Asp Trp His Met Glu Pro 435 440
445 Ser Arg Met Thr Leu His Arg Phe Gly Asn Thr Ser
Ser Ser Ser Leu 450 455 460
Trp Tyr Glu Met Ala Tyr Thr Glu Ala Lys Gly Arg Val Lys Ala Gly 465
470 475 480 Asp Arg Leu
Trp Gln Ile Ala Phe Gly Ser Gly Phe Lys Cys Asn Ser 485
490 495 Ala Val Trp Lys Ala Leu Arg Val
Val Ser Thr Glu Glu Leu Thr Gly 500 505
510 Asn Ala Trp Ala Gly Ser Ile Glu Asn Tyr Pro Val Lys
Ile Val Gln 515 520 525
168506PRTCrambe abyssinica 168Met Thr Ser Ile Asn Val Lys Leu Leu Tyr
His Tyr Val Ile Thr Asn 1 5 10
15 Leu Phe Asn Leu Cys Phe Phe Pro Leu Thr Ala Ile Val Ala Gly
Lys 20 25 30 Ala
Ser Arg Leu Thr Ile Asp Asp Leu His His Leu Tyr Tyr Ser Tyr 35
40 45 Leu Gln His Asn Val Ile
Thr Ile Ala Pro Leu Phe Ala Phe Thr Val 50 55
60 Phe Gly Ser Ile Leu Tyr Ile Val Thr Arg Pro
Lys Pro Val Tyr Leu 65 70 75
80 Val Glu Tyr Ser Cys Tyr Leu Pro Pro Thr Gln Cys Arg Ser Ser Ile
85 90 95 Ser Lys
Val Met Asp Ile Phe Tyr Gln Val Arg Lys Ala Asp Pro Phe 100
105 110 Arg Asn Gly Thr Cys Asp Asp
Ser Ser Trp Leu Asp Phe Leu Arg Lys 115 120
125 Ile Gln Glu Arg Ser Gly Leu Gly Asp Glu Thr His
Gly Pro Glu Gly 130 135 140
Leu Leu Gln Val Pro Pro Arg Lys Thr Phe Ala Ala Ala Arg Glu Glu 145
150 155 160 Thr Glu Gln
Val Ile Val Gly Ala Leu Lys Asn Leu Phe Glu Asn Thr 165
170 175 Lys Val Asn Pro Lys Asp Ile Gly
Ile Leu Val Val Asn Ser Ser Met 180 185
190 Phe Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn
Thr Phe Lys 195 200 205
Leu Arg Ser Asn Val Arg Ser Phe Asn Leu Gly Gly Met Gly Cys Ser 210
215 220 Ala Gly Val Ile
Ala Ile Asp Leu Ala Lys Asp Leu Leu His Val His 225 230
235 240 Lys Asn Thr Tyr Ala Leu Val Val Ser
Thr Glu Asn Ile Thr Tyr Asn 245 250
255 Ile Tyr Ala Gly Asp Asn Arg Ser Met Met Val Ser Asn Cys
Leu Phe 260 265 270
Arg Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Arg Asp Arg
275 280 285 Arg Arg Ser Lys
Tyr Glu Leu Val His Thr Val Arg Thr His Thr Gly 290
295 300 Ala Asp Asp Lys Ser Phe Arg Cys
Val Gln Gln Gly Asp Asp Glu Asn 305 310
315 320 Gly Lys Thr Gly Val Ser Leu Ser Lys Asp Ile Thr
Glu Val Ala Gly 325 330
335 Arg Thr Val Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu Pro
340 345 350 Leu Ser Glu
Lys Leu Leu Phe Phe Val Thr Phe Met Ala Lys Lys Leu 355
360 365 Phe Lys Asp Lys Val Lys His Tyr
Tyr Val Pro Asp Phe Lys Leu Ala 370 375
380 Ile Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val
Ile Asp Val 385 390 395
400 Leu Glu Lys Asn Leu Gly Leu Ala Pro Ile Asp Val Glu Ala Ser Arg
405 410 415 Ser Thr Leu His
Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr 420
425 430 Glu Leu Ala Tyr Ile Glu Ala Lys Gly
Arg Met Lys Lys Gly Asn Lys 435 440
445 Val Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser
Ala Val 450 455 460
Trp Val Ala Leu Ser Asn Val Lys Ala Ser Thr Asn Ser Pro Trp Glu 465
470 475 480 His Cys Ile Asp Arg
Tyr Pro Val Lys Ile Asp Ser Asp Ser Ala Lys 485
490 495 Ser Glu Thr Arg Ala Gln Asn Gly Arg Ser
500 505 169506PRTCardamine graeca 169Met
Thr Ser Ile Asn Val Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu
Phe Pro Leu Thr Ala Phe Pro Ala Gly Lys 20
25 30 Ala Ser Gln Leu Thr Thr Asn Asp Leu His
His Leu Tyr Ser Tyr Leu 35 40
45 His His Asn Leu Ile Thr Val Thr Leu Leu Phe Ala Phe Thr
Val Phe 50 55 60
Gly Ser Ile Leu Tyr Ile Val Thr Arg Pro Lys Pro Val Tyr Leu Val 65
70 75 80 Asp Tyr Ser Cys Tyr
Leu Pro Pro Arg His Leu Ser Cys Gly Ile Ser 85
90 95 Arg Val Met Glu Ile Phe Tyr Glu Ile Arg
Lys Ser Asp Pro Ser Arg 100 105
110 Glu Val Pro Phe Asp Asp Pro Ser Ser Leu Glu Phe Leu Arg Lys
Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Gly Pro Gln Gly Leu 130
135 140 Val His Asp Met Pro Leu
Arg Met Asn Phe Ala Ala Ala Arg Glu Glu 145 150
155 160 Thr Glu Gln Val Ile Asn Gly Ala Leu Glu Lys
Leu Phe Glu Asn Thr 165 170
175 Lys Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met
180 185 190 Phe Asn
Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys 195
200 205 Leu Arg Ser Asn Ile Lys Ser
Phe Ser Leu Gly Gly Met Gly Cys Ser 210 215
220 Ala Gly Ile Ile Ala Ile Asp Leu Ala Lys Asp Leu
Leu His Val His 225 230 235
240 Lys Asn Thr Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr His Ser
245 250 255 Thr Tyr Thr
Gly Asp Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe 260
265 270 Arg Met Gly Gly Ala Ala Ile Leu
Leu Ser Asn Lys Ala Gly Asp Arg 275 280
285 Arg Arg Ser Lys Tyr Lys Leu Ala His Thr Val Arg Thr
His Thr Gly 290 295 300
Ala Asp Asp Gln Ser Phe Arg Cys Val Arg Gln Glu Asp Asp Asp Arg 305
310 315 320 Gly Lys Ile Gly
Val Cys Leu Ser Lys Asp Ile Thr Ala Val Ala Gly 325
330 335 Lys Thr Val Thr Lys Asn Ile Ala Thr
Leu Gly Pro Leu Val Leu Pro 340 345
350 Leu Ser Glu Lys Phe Leu Tyr Val Val Ser Leu Met Ala Lys
Lys Leu 355 360 365
Phe Lys Asn Lys Ile Lys His Thr Tyr Val Pro Asp Phe Lys Leu Ala 370
375 380 Ile Asp His Phe Cys
Ile His Ala Gly Gly Arg Ala Val Ile Asp Val 385 390
395 400 Leu Glu Lys Asn Leu Ala Leu Ser Pro Val
Asp Val Glu Ala Ser Arg 405 410
415 Ser Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp
Tyr 420 425 430 Glu
Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Lys 435
440 445 Val Trp Gln Ile Ala Ile
Gly Ser Gly Phe Lys Cys Asn Ser Ala Val 450 455
460 Trp Val Ala Leu Cys Asn Val Lys Pro Ser Val
Asn Ser Pro Trp Glu 465 470 475
480 His Cys Ile Asp Arg Tyr Pro Val Glu Ile Asn Tyr Gly Ser Ser Lys
485 490 495 Ser Glu
Thr Arg Ala Gln Asn Gly Arg Ser 500 505
170505PRTLunaria annua 170Met Thr Ser Ile Asn Val Lys Leu Leu Tyr His Tyr
Val Ile Thr Asn 1 5 10
15 Phe Phe Asn Leu Cys Phe Phe Pro Leu Thr Ala Ile Leu Ala Gly Lys
20 25 30 Ala Ser Arg
Leu Thr Thr Asn Asp Leu His His Phe Tyr Ser Tyr Leu 35
40 45 Gln His Asn Leu Ile Thr Leu Thr
Leu Leu Phe Ala Phe Thr Val Phe 50 55
60 Gly Ser Val Leu Tyr Phe Val Thr Arg Pro Lys Pro Val
Tyr Leu Val 65 70 75
80 Asp Tyr Ser Cys Tyr Leu Pro Pro Gln His Leu Ser Ala Gly Ile Ser
85 90 95 Lys Thr Met Glu
Ile Phe Tyr Gln Ile Arg Lys Ser Asp Pro Leu Arg 100
105 110 Asn Val Ala Leu Asp Asp Ser Ser Ser
Leu Asp Phe Leu Arg Lys Ile 115 120
125 Gln Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Gly Pro Glu
Gly Leu 130 135 140
Phe Glu Ile Pro Pro Arg Lys Asn Leu Ala Ser Ala Arg Glu Glu Thr 145
150 155 160 Glu Gln Val Ile Asn
Gly Ala Leu Lys Asn Leu Phe Glu Asn Thr Lys 165
170 175 Val Asn Pro Lys Glu Ile Gly Ile Leu Val
Val Asn Ser Ser Met Phe 180 185
190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys
Leu 195 200 205 Arg
Ser Asn Ile Lys Ser Phe Asn Leu Gly Gly Met Gly Cys Ser Ala 210
215 220 Gly Val Ile Ala Ile Asp
Leu Ala Lys Asp Leu Leu His Val His Lys 225 230
235 240 Asn Thr Tyr Ala Leu Val Val Ser Thr Glu Asn
Ile Thr Gln Asn Ile 245 250
255 Tyr Thr Gly Asp Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg
260 265 270 Val Gly
Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg Arg 275
280 285 Arg Ser Lys Tyr Arg Leu Ala
His Thr Val Arg Thr His Thr Gly Ala 290 295
300 Asp Asp Lys Ser Phe Gly Cys Val Arg Gln Glu Glu
Asp Asp Ser Gly 305 310 315
320 Lys Thr Gly Val Ser Leu Ser Lys Asp Ile Thr Gly Val Ala Gly Ile
325 330 335 Thr Val Gln
Lys Asn Ile Thr Thr Leu Gly Pro Leu Val Leu Pro Leu 340
345 350 Ser Glu Lys Ile Leu Phe Val Val
Thr Phe Val Ala Lys Lys Leu Leu 355 360
365 Lys Asp Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys
Leu Ala Val 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Asn Leu
Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser 405
410 415 Thr Leu His Arg Phe Gly Asn Thr Ser
Ser Ser Ser Ile Trp Tyr Glu 420 425
430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn
Lys Ala 435 440 445
Trp Gln Ile Ala Val Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp 450
455 460 Val Ala Leu Arg Asn
Val Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470
475 480 Cys Ile His Lys Tyr Pro Val Gln Met Tyr
Ser Gly Ser Ser Lys Ser 485 490
495 Glu Thr Arg Ala Gln Asn Gly Arg Ser 500
505 171503PRTTropaeolum majus 171Met Ser Gly Thr Lys Ala Thr Ser
Val Ser Val Pro Leu Pro Asp Phe 1 5 10
15 Lys Gln Ser Val Asn Leu Lys Tyr Val Lys Leu Gly Tyr
His Tyr Ser 20 25 30
Ile Thr His Ala Met Tyr Leu Phe Leu Thr Pro Leu Leu Leu Ile Met
35 40 45 Ser Ala Gln Ile
Ser Thr Phe Ser Ile Gln Asp Phe His His Leu Tyr 50
55 60 Asn His Leu Ile Leu His Asn Leu
Ser Ser Leu Ile Leu Cys Ile Ala 65 70
75 80 Leu Leu Leu Phe Val Leu Thr Leu Tyr Phe Leu Thr
Arg Pro Thr Pro 85 90
95 Val Tyr Leu Leu Asn Phe Ser Cys Tyr Lys Pro Asp Ala Ile His Lys
100 105 110 Cys Asp Arg
Arg Arg Phe Met Asp Thr Ile Arg Gly Met Gly Thr Tyr 115
120 125 Thr Glu Glu Asn Ile Glu Phe Gln
Arg Lys Val Leu Glu Arg Ser Gly 130 135
140 Ile Gly Glu Ser Ser Tyr Leu Pro Pro Thr Val Phe Lys
Ile Pro Pro 145 150 155
160 Arg Val Tyr Asp Ala Glu Glu Arg Ala Glu Ala Glu Met Leu Met Phe
165 170 175 Gly Ala Val Asp
Gly Leu Phe Glu Lys Ile Ser Val Lys Pro Asn Gln 180
185 190 Ile Gly Val Leu Val Val Asn Cys Gly
Leu Phe Asn Pro Ile Pro Ser 195 200
205 Leu Ser Ser Met Ile Val Asn Arg Tyr Lys Met Arg Gly Asn
Val Phe 210 215 220
Ser Tyr Asn Leu Gly Gly Met Gly Cys Ser Ala Gly Val Ile Ser Ile 225
230 235 240 Asp Leu Ala Lys Asp
Leu Leu Gln Val Arg Pro Asn Ser Tyr Ala Leu 245
250 255 Val Val Ser Leu Glu Cys Ile Ser Lys Asn
Leu Tyr Leu Gly Glu Gln 260 265
270 Arg Ser Met Leu Val Ser Asn Cys Leu Phe Arg Met Gly Gly Ala
Ala 275 280 285 Ile
Leu Leu Ser Asn Lys Met Ser Asp Arg Trp Arg Ser Lys Tyr Arg 290
295 300 Leu Val His Thr Val Arg
Thr His Lys Gly Thr Glu Asp Asn Cys Phe 305 310
315 320 Ser Cys Val Thr Arg Lys Glu Asp Ser Asp Gly
Lys Ile Gly Ile Ser 325 330
335 Leu Ser Lys Asn Leu Met Ala Val Ala Gly Asp Ala Leu Lys Thr Asn
340 345 350 Ile Thr
Thr Leu Gly Pro Leu Val Leu Pro Met Ser Glu Gln Leu Leu 355
360 365 Phe Phe Ala Thr Leu Val Gly
Lys Lys Val Phe Lys Met Lys Leu Gln 370 375
380 Pro Tyr Ile Pro Asp Phe Lys Leu Ala Phe Glu His
Phe Cys Ile His 385 390 395
400 Ala Gly Gly Arg Ala Val Leu Asp Glu Leu Glu Lys Asn Leu Lys Leu
405 410 415 Ser Ser Trp
His Met Glu Pro Ser Arg Met Ser Leu Tyr Arg Phe Gly 420
425 430 Asn Thr Ser Ser Ser Ser Leu Trp
Tyr Glu Leu Ala Tyr Ser Glu Ala 435 440
445 Lys Gly Arg Ile Lys Lys Gly Asp Arg Val Trp Gln Ile
Ala Phe Gly 450 455 460
Ser Gly Phe Lys Cys Asn Ser Ala Val Trp Lys Ala Leu Arg Asn Val 465
470 475 480 Asn Pro Ala Glu
Glu Lys Asn Pro Trp Met Asp Glu Ile His Leu Phe 485
490 495 Pro Val Glu Val Pro Leu Asn
500
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