Patent application title: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS
Howard Glenn Damude (Hockessin, DE, US)
Quinn Qun Zhu (West Chester, PA, US)
E.I. DU PONT DE NEMOURS AND COMPANY
IPC8 Class: AC11B100FI
Class name: Fatty compounds having an acid moiety which contains the carbonyl of a carboxylic acid, salt, ester, or amide group bonded directly to one end of an acyclic chain of at least seven (7) uninterrupted carbons, wherein any additional carbonyl in the acid moiety is (1) part of an aldehyde or ketone group, (2) bonded directly to a noncarbon atom which is between the additional carbonyl and the chain, or (3) attached indirectly to the chain via ionic bonding extraction directly from animal or plant source material (e.g., recovery from garbage, fish offal, slaughter house waste, whole fish, olive fruit, etc.) legume, nut, or seed source material (e.g., peanut, soya bean, rice bran, etc.)
Publication date: 2009-10-22
Patent application number: 20090264666
Isolated nucleic acid fragments and recombinant constructs comprising such
fragments encoding a delta-8 desaturase along with a method of making
long chain polyunsaturated fatty acids (PUFAs) using this delta-8
desaturase in plants and oleaginous yeast.
1. An isolated polynucleotide comprising:(a) a nucleotide sequence
encoding a polypeptide having delta-8 desaturase activity, wherein the
polypeptide has an amino acid sequence consisting essentially of SEQ ID
NOs:2 or 113; or,(b) a complement of the nucleotide sequence, wherein the
complement and the nucleotide sequence consist of the same number of
nucleotides and are 100% complementary.
39. Oil obtained from a seed obtained from a plant comprising a recombinant construct comprising an isolated polynucleotide comprising:(a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs:2; or,(b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
40. Oil obtained from a seed obtained from a plant made by a method for producing a transformed plant comprising transforming a plant cell with an isolated polynucleotide comprising:(a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs:2; or,(b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary and(c) regenerating a plant from the transformed plant cell.
41. The oil of claim 39, wherein the recombinant construct is operably linked to at least one regulatory sequence.
42. The oil of claim 39, wherein the plant is an oilseed plant.
43. Oil obtained from a seed obtained from a plant made by a method for producing a transformed plant comprising:a) transforming a plant cell with an isolated polynucleotide comprising with:(i) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs:2; or,(ii) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary,and b) regenerating a plant from the transformed plant cell.
44. The oil of claim 43, wherein the plant is a soybean plant.
45. Oil obtained from a seed obtained from an oilseed plant comprising a recombinant construct comprising:a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; andb) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, a delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase.
46. Oil obtained by a method for making long chain fatty acids in a plant cell comprising:a) transforming a cell with a recombinant construct comprising an isolated polynucleotide comprising:(i) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs:2; or,(ii) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary,and b) selecting those transformed cells that make long chain polyunsaturated fatty acids.
47. Oil obtained by a method for producing at least one polyunsaturated fatty acid in a soybean cell comprising:a) transforming a soybean cell with a recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, a delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase;b) regenerating a soybean plant from the transformed cell of step a); andc) selecting those seeds obtained from the plants of step b), wherein the seeds have an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed soybean plant.
This application claims the benefit of U.S. Provisional Application
No. 60/583,041, filed Jun. 25, 2004, and U.S. Provisional Application No.
60/624812, filed Nov. 4, 2004, the entire contents of which are herein
incorporated by reference.
FIELD OF THE INVENTION
This invention pertains to a polynucleotide sequence encoding a delta-8 desaturase and the use of this desaturase in making long chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
Lipids/fatty acids are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents such as chloroform, ether or benzene. Lipids have several important biological functions, serving as: (1) structural components of membranes; (2) storage and transport forms of metabolic fuels; (3) a protective coating on the surface of many organisms; and, (4) cell-surface components concerned in cell recognition, species specificity and tissue immunity. More specifically, polyunsaturated fatty acids (PUFAs) are important components of the plasma membrane of the cell, where they may be found in such forms as phospholipids and also can be found in triglycerides. PUFAs also serve as precursors to other molecules of importance in human beings and animals, including the prostacyclins, leukotrienes and prostaglandins. There are two main families of PUFAs (i.e., the omega-3 fatty acids and the omega-6 fatty acids).
The human body is capable of producing most of the PUFAs which it requires to function; however, eicosapentaenoic acid (EPA; 20:5, delta-5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6, delta-4,7,10,13,16,19) cannot be synthesized efficiently by the human body and thus must be supplied through the diet. Since the human body cannot produce adequate quantities of these PUFAs, they are called essential fatty acids. Because of their important roles in human health and nutrition, EPA and DHA are the subject of much interest as discussed herein.
DHA is a fatty acid of the omega-3 series according to the location of the last double bond in the methyl end. It is synthesized via alternating steps of desaturation and elongation. Production of DHA is important because of its beneficial effect on human health; for example, increased intake of DHA has been shown to be beneficial or have a positive effect in inflammatory disorders (e.g., rheumatoid arthritis), Type II diabetes, hypertension, atherosclerosis, depression, myocardial infarction, thrombosis, some cancers and for prevention of the onset of degenerative disorders such as Alzheimer's disease. Currently the major sources of DHA are oils from fish and algae.
EPA and arachidonic acid (AA or ARA; 20:4, delta-5,8,11,14) are both delta-5 essential fatty acids. EPA belongs to the omega-3 series with five double bonds in the acyl chain, is found in marine food, and is abundant in oily fish from the North Atlantic. Beneficial or positive effects of increased intake of EPA have been shown in patients with coronary heart disease, high blood pressure, inflammatory disorders, lung and kidney diseases, Type II diabetes, obesity, ulcerative colitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis, attention deficit/hyperactivity disorder and early stages of colorectal cancer (see, for example, the review of McColl, J., NutraCos 2(4):35-40 (2003)).
AA belongs to the omega-6 series with four double bonds. The lack of a double bond in the omega-3 position confers on AA different properties than those found in EPA. The eicosanoids produced from AA have strong inflammatory and platelet aggregating properties, whereas those derived from EPA have anti-inflammatory and anti-platelet aggregating properties. AA is recognized as the principal ω-6 fatty acid found in the human brain and an important component of breast milk and many infant formulas, based on its role in early neurological and visual development. AA can be obtained from some foods such as meat, fish, and eggs, but the concentration is low.
Gamma-linolenic acid (GLA; 18:3, delta-6,9,12) is another essential fatty acid found in mammals. GLA is the metabolic intermediate for very long chain omega-6 fatty acids and for various active molecules. In mammals, formation of long chain PUFAs is rate-limited by delta-6 desaturation. Many physiological and pathological conditions such as aging, stress, diabetes, eczema, and some infections have been shown to depress the delta-6 desaturation step. In addition, GLA is readily catabolized from the oxidation and rapid cell division associated with certain disorders, e.g., cancer or inflammation.
As described above, research has shown that various omega fatty acids reduce the risk of heart disease, have a positive effect on children's development and on certain mental illnesses, autoimmune diseases and joint complaints. However, although there are many health benefits associated with a diet supplemented with these fatty acids, it is recognized that different PUFAs exert different physiological effects in the body (e.g., most notably, the opposing physiological effects of GLA and AA). Thus, production of oils using recombinant means is expected to have several advantages over production from natural sources. For example, recombinant organisms having preferred characteristics for oil production can be used, since the naturally occurring fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways, thereby resulting in increased levels of production of desired PUFAs (or conjugated forms thereof) and decreased production of undesired PUFAs. Optionally, recombinant organisms can provide PUFAs in particular forms which may have specific uses; or, oil production can be manipulated such that the ratio of omega-3 to omega-6 fatty acids so produced is modified and/or a specific PUFA is produced without significant accumulation of other PUFA downstream or upstream products (e.g., production of oils comprising ARA and lacking GLA).
The mechanism of PUFA synthesis frequently occurs via the delta-6 desaturation pathway. For example, long chain PUFA synthesis in mammals proceeds predominantly by a delta-6 desaturation pathway, in which the first step is the delta-6 desaturation of LA and ALA to yield GLA and stearidonic acid (STA; 18:4, delta-6,9,12,15), respectively. Further fatty acid elongation and desaturation steps give rise to AA and EPA. Accordingly, genes encoding delta-6 desaturases, delta-6 elongase components (also identified as C18/20 elongases) and delta-5 desaturases have been cloned from a variety of organisms including higher plants, algae, mosses, fungi, nematodes and humans. Humans can synthesize long chain PUFAs from the essential fatty acids, linoleic acid (LA; 18:2, delta-9,12) and alpha-linolenic acid (ALA; 18:3, delta-9,12,15); LA and ALA must be obtained from the diet. However, biosynthesis of long chain PUFAs is somewhat limited and is regulated by dietary and hormonal changes.
WO 02/26946 (published Apr. 4, 2002) describes isolated nucleic acid molecules encoding FAD4, FAD5, FAD5-2 and FAD6 fatty acid desaturase family members which are expressed in long chain PUFA-producing organisms, e.g., Thraustochytrium, Pythium irregulare, Schizichytrium and Crypthecodinium. It is indicated that constructs containing the desaturase genes can be used in any expression system including plants, animals, and microorganisms for the production of cells capable of producing long chain PUFAs.
WO 98/55625 (published Dec. 19, 1998) describes the production of PUFAs by expression of polyketide-like synthesis genes in plants.
WO 98/46764 (published Oct. 22, 1998) describes compositions and methods for preparing long chain fatty acids in plants, plant parts and plant cells which utilize nucleic acid sequences and constructs encoding fatty acid desaturases, including delta-5 desaturases, delta-6 desaturases and delta-12 desaturases.
U.S. Pat. No. 6,075,183 (issued to Knutzon et al. on Jun. 13, 2000) describes methods and compositions for synthesis of long chain PUFAs in plants.
U.S. Pat. No. 6,459,018 (issued to Knutzon et al. on Oct. 1, 2002) describes a method for producing STA in plant seed utilizing a construct comprising a DNA sequence encoding a delta-6 desaturase.
Spychalla et al. (Proc. Natl. Acad. Sci. USA, 94:1142-1147 (1997)) describes the isolation and characterization of a cDNA from C. elegans that, when expressed in Arabidopsis, encodes a fatty acid desaturase which can catalyze the introduction of an omega-3 double bond into a range of 18- and 20-carbon fatty acids.
An alternate pathway for the biosynthesis of AA and EPA operates in some organisms (i.e., the delta-9 elongase/delta-8 desaturase pathway). Here LA and ALA are first elongated to eicosadienoic acid (EDA; 20:2, delta-11,14) and eicosatrienoic acid (EtrA; 20:3, delta-11,14,17), respectively, by a delta-9 elongase. Subsequent delta-8 and delta-5 desaturation of these products yields AA and EPA. The delta-8 pathway is present inter alia, in euglenoid species where it is the dominant pathway for formation of 20-carbon PUFAs.
WO 2000/34439 (published Jun. 15, 2000) discloses amino acid and nucleic acid sequences for delta-5 and delta-8 desaturase enzymes. Based on the information presented herein, it is apparent that the delta-8 nucleotide and amino acid sequences of WO 2000/34439 are not correct.
Wallis et al. (Archives of Biochemistry and Biophysics, 365(2):307-316 (May 15, 1999)) describes the cloning of a gene that appears to encode a delta-8 desaturase in Euglena gracilis. This appears to be the same sequence disclosed in WO 2000/34439.
Qi et al. (Nature Biotechnology, 22(6):739-45 (2004)) describes the production of long chain PUFAs using, among other things, a delta-8 desaturase from E. gracilis; however, the complete sequence of the delta-8 desaturase is not provided.
WO 2004/057001 (published Jul. 8, 2004) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from E. gracilis.
An expansive study of PUFAs from natural sources and from chemical synthesis are not sufficient for commercial needs. Therefore, it is of interest to find alternative means to allow production of commercial quantities of PUFAs. Biotechnology offers an attractive route for producing long chain PUFAs in a safe, cost efficient manner in microorganisms and plants.
With respect to microorganisms, many algae, bacteria, molds and yeast can synthesize oils in the ordinary course of cellular metabolism. Thus, oil production involves cultivating the microorganism in a suitable culture medium to allow for oil synthesis, followed by separation of the microorganism from the fermentation medium and treatment for recovery of the intracellular oil. Attempts have been made to optimize production of fatty acids by fermentive means involving varying such parameters as microorganisms used, media and conditions that permit oil production. However, these efforts have proved largely unsuccessful in improving yield of oil or the ability to control the characteristics of the oil composition produced. One class of microorganisms that has not been previously examined as a production platform for PUFAs (prior to work by the Applicants' Assignee), however, are the oleaginous yeasts. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)), and may offer a cost advantage compared to commercial micro-algae fermentation for production of omega-3 or omega-6 PUFAs. Whole yeast cells may also represent a convenient way of encapsulating omega-3 or omega-6 PUFA-enriched oils for use in functional foods and animal feed supplements.
WO 2004/101757 and WO 2004/101753 (published Nov. 25, 2004) concern the production of PUFAs in oleaginous yeasts and are Applicants'Assignee's copending applications.
WO 2004/071467 (published Aug. 26, 2004) concerns the production of PUFAs in plants, while WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are Applicants' Assignee's copending applications.
SUMMARY OF THE INVENTION
This invention concerns an isolated polynucleotide comprising:
(a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs: 2 or 113; or,
(b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
In a second embodiment, this invention concerns a recombinant construct comprising SEQ ID NOs:1 or 112 operably linked to at least one regulatory sequence.
In a third embodiment, this invention concerns a cell comprising the recombinant construct of the invention.
In a fourth embodiment, this invention concerns a method for transforming cells, plants and yeast with the recombinant construct of the invention.
In a fifth embodiment, this invention concerns seeds obtained from such plants and oil obtained from such seeds.
In a sixth embodiment, this invention concerns a method for making polyunsaturated fatty acids in a cell.
In a seventh embodiment, this invention concerns an oilseed plant comprising a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase.
In still another aspect, this invention concerns a method for producing at least one polyunsaturated fatty acid in a soybean cell comprising:
(a) transforming a soybean cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase.
(b) regenerating a soybean plant from the transformed cell of step (a); and
(c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed soybean plant.
In an eighth emodiment this invention concerns an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower.
In a ninth embodiment this invention concerns oilseed plants wherein the polyunsaturated fatty acid is selected from the group consisting of AA, EDA, EPA, ETA, EtrA, DGLA, DPA, DHA,
Further embodiments include seeds and oil obtained from the plants transformed with the isolated polynucleotides of the instant invention.
Additional embodiments concern food, feed and ingredients derived from the processing of the seeds obtained from the plants transformed with the isolated polynucleotides of the instant invention.
The following plasmids have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bear the following designations, accession numbers and dates of deposit.
TABLE-US-00001 Plasmid Accession Number Date of Deposit pKR681 ATCC PTA-6046 Jun. 4th, 2004 pKR685 ATCC PTA-6047 Jun. 4th, 2004 pY89-5 ATCC PTA-6048 Jun. 4th, 2004 pKR274 ATCC PTA-4988 Jan. 30th, 2003 PKR669 Jun. 13, 2005 PKR786 Jun. 13, 2005
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.
The sequence descriptions summarize the Sequences Listing attached hereto. The Sequence Listing contains one letter codes for nucleotide sequence characters and the single and three letter codes for amino acids as defined in the IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219(2):345-373 (1984).
FIG. 1 shows a chromatogram of the lipid profile of an Euglena gracilis cell extract as described in Example 10.
FIG. 2 shows an alignment of the claimed delta-8 desaturase polypeptide sequence from Euglena gracilis (SEQ ID NO:2), a version of a delta-8 desaturase with reduced activity (SEQ ID NO:4) and published non-functional versions of delta-8 desaturase sequences set forth in gi:5639724 (GenBank Accession No. AAD45877 and SEQ ID NO:6) and in WO 00/34439 or Wallis et al. (Archives of Biochem. Biophys, 365:307-316 (1999)) (SEQ ID NO:7). The method of alignment used corresponds to the "Clustal V method of alignment".
FIG. 3 provides plasmid maps for the following: (A) yeast expression vector pY89-5 as described in Example 5; and, (B) soybean expression vector pKR681 as described in Example 6.
FIG. 4 provides plasmid maps for the following: (A) soybean expression vector pKR685 as described in Example 8; and, (B) expression vector pKR274 as described in Example 9.
FIG. 5 provides plasmid maps for the following: (A) yeast expression vector pDMW240 as described in Example 1; (B) yeast expression vector pDMW255 as described in Example 1; (C) yeast expression vector pDMW261 as described in Example 1; and, (D) vector pKUNFmKF2 as described in Example 14.
FIG. 6 provides plasmid maps for the following: (A) yeast expression vector pDMW277 as described in Example 14; (B) vector pZF5T-PPC as described in Example 14; (C) yeast expression vector pDMW287 as described in Example 14; and, (D) yeast expression vector pDMW287F as described in Example 14.
FIG. 7 provides plasmid maps for the following: (A) vector pZUF17 as described in Example 15; (B) yeast expression vector pDMW237 as described in Example 15; (C) yeast expression vector pKUNT2 as described in Example 16; and, (D) yeast expression vector pDMW297 as described in Example 16.
FIG. 8 provides plasmid maps for the following: (A) soybean expression vector pKR682 as described in Example 17; (B) soybean expression vector pKR786 as described in Example 18; and, (C) soybean expression vector pKR669 as described in Example 19.
FIG. 9 is a representative PUFA biosynthetic pathway.
FIG. 10 shows a chromatogram of the lipid profile of a soybean embryo extract as described in Example 22.
SEQ ID NO:1 represents the 1271 bp of the Euglena gracilis sequence containing the ORF (nucleotides 4-1269 (Stop)) of the delta-8 desaturase gene.
SEQ ID NO:2 is the amino acid sequence encoded by nucleotides 4-1269 of SEQ ID NO:1.
SEQ ID NO:3 represents the 1271 bp of the Euglena gracilis sequence containing the ORF (nucleotides 4-1269 (Stop)) of the delta-8 desaturase gene containing a guanine for adenine substitution at position 835, as compared to the sequence of SEQ ID NO:1.
SEQ ID NO:4 is the deduced amino acid sequence encoded by nucleotides 4-1269 of SEQ ID NO:3, which contains an alanine for threonine substitution at position 278, when compared to the polypeptide sequence of SEQ ID NO:2.
SEQ ID NO:5 represents 1275 bp of the Euglena gracilis sequence set forth in gi:5639724 (GenBank Accession No. AAD45877), containing the ORF (nucleotides 14-1273 (Stop)) of a non-functional version of the delta-8 desaturase gene.
SEQ ID NO:6 is the deduced amino acid sequence encoded by nucleotides of SEQ ID NO:5 and set forth in gi:5639724.
SEQ ID NO:7 is the amino acid sequence of a non-functional version of the delta-8 desaturase disclosed in Wallis et al. (Archives of Biochem. Biophys., 365:307-316 (1999) and WO 00/34439).
SEQ ID NO:8 is the forward primer used for amplification of the delta-8 desaturase from Euglena gracilis in Example 3.
SEQ ID NO:9 is the reverse primer used for amplification of the delta-8 desaturase from Euglena gracilis in Example 3.
SEQ ID NO:10 is the forward primer used for sequencing a delta-8 desaturase clone as described in Example 3.
SEQ ID NO:11 is the reverse primer used for sequencing a delta-8 desaturase clone as described in Example 3.
SEQ ID NO:12 is the forward primer used for sequencing a delta-8 desaturase clone as described in Example 3.
SEQ ID NO:13 is the reverse primer used for sequencing a delta-8 desaturase clone as described in Example 3.
SEQ ID NO:14 is the multiple restriction enzyme site sequence introduced in front of the beta-conglycinin promoter as described in Example 6.
SEQ ID NO:15 is the forward primer used for amplification of the elongase.
SEQ ID NO:16 is the reverse primer used for amplification of the elongase.
SEQ ID NO:17 is the multiple restriction enzyme site sequence introduced upstream of the Kti promoter as described in Example 6.
SEQ ID NO:18 sets forth the sequence of the soy albumin transcription terminator with restriction enzyme sites as described in Example 6.
SEQ ID NO:19 is the primer oSalb-12 used for amplification of the albumin transcription terminator.
SEQ ID NO:20 is primer oSalb-13 used for amplification of the albumin transcription terminator.
SEQ ID NO:21 is primer GSP1 used for the amplification of the soybean annexin gene.
SEQ ID NO:22 is primer GSP2 used for the amplification of the soybean annexin gene.
SEQ ID NO:23 is primer GSP3 used for the amplification of soybean BD30.
SEQ ID NO:24 is primer GSP4 used for the amplification of soybean BD30.
SEQ ID NO:25 sets forth the soybean BD30 promoter sequence.
SEQ ID NO:26 sets forth the soybean Glycinin Gy1 promoter sequence.
SEQ ID NO:27 is the forward primer used for amplification of the soybean Glycinin Gy1 promoter sequence.
SEQ ID NO:28 is the reverse primer used for amplification of the soybean Glycinin Gy1 promoter sequence.
SEQ ID NO:29 sets forth the soybean annexin promoter sequence.
SEQ ID NO:30 is the forward primer used for amplification of the soybean annexin promoter sequence.
SEQ ID NO:31 is the reverse primer used for amplification of the soybean annexin promoter sequence.
SEQ ID NO:32 is the forward primer used for amplification of the soybean BD30 promoter sequence.
SEQ ID NO:33 is the reverse primer used for amplification of the soybean BD30 promoter sequence.
SEQ ID NO:34 is primer oKTi5 used for amplification of the Kti/NotI/Kti 3' cassette.
SEQ ID NO:35 is primer oKTi6 used for amplification of the Kti/NotI/Kti 3' cassette.
SEQ ID NO:36 is primer oSBD30-1 used for amplification of the soybean BD30 3' transcription terminator.
SEQ ID NO:37 is primer oSBD30-2 used for amplification of the soybean BD30 3' transcription terminator.
SEQ ID NO:38 is primer oCGR5-1 used for amplification of the M. alpina delta-6 desaturase.
SEQ ID NO:39 is primer oCGR5-2 used for amplification of the M. alpina delta-6 desaturase.
SEQ ID NO:40 is primer oSGly-1 used for amplification of the glycinin Gy1 promoter.
SEQ ID NO:41 is primer oSGly-2 used for amplification of the glycinin Gy1 promoter.
SEQ ID NO:42 is primer LegPro5' used for amplification of the legA2 promoter sequence.
SEQ ID NO:43 is primer LegPro3' used for amplification of the legA2 promoter sequence.
SEQ ID NO:44 is primer LegTerm5' used for amplification of the leg2A transcription terminator.
SEQ ID NO:45 is primer LegTerm3' used for amplification of the leg2A transcription terminator.
SEQ ID NO:46 is primer CGR4forward used for the amplification of the M. alpina desaturase.
SEQ ID NO:47 is primer CGR4reverse used for the amplification of the M. alpina desaturase.
SEQ ID NO:48 is the Euglena gracilis sequence, set forth in nucleotides 14-1275 of SEQ ID NO:5, optimized for codon usage in Yarrowia lipolytica.
SEQ ID NOs:49-74 correspond to primers D8-1A, D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, respectively, used for amplification as described in Example 1.
SEQ ID NOs:75-82 correspond to primers D8-1F, D8-3R, D8-4F, D8-6R, D8-7F, D8-9R, D8-10F and D8-13R, respectively, used for amplification as described in Example 1.
SEQ ID NO:83 is the 309 bp Nco/BgIII fragment described in Example 1.
SEQ ID NO:84 is the 321 bp BgIII/XhoI fragment described in Example 1.
SEQ ID NO:85 is the 264 bp XhoI/SacI fragment described in Example 1.
SEQ ID NO:86 is the 369 bp Sac1/Not1 fragment described in Example 1.
SEQ ID NO:87 is primer ODMW390 used for amplification as described in Example 1.
SEQ ID NO:88 is primer ODMW391 used for amplification as described in Example 1.
SEQ ID NO:89 is the chimeric gene described in Example 1.
SEQ ID NO:90 is the chimeric gene described in Example 1.
SEQ ID NO:91 is primer ODMW392 used for amplification as described in Example 1.
SEQ ID NO:92 is primer ODMW393 used for amplification as described in Example 1.
SEQ ID NO:93 is the synthetic delta-8 desaturase described in Example 1.
SEQ ID NO:94 is primer ODMW404 used for amplification as described in Example 14.
SEQ ID NO:95 is the Kpn/NotI fragment described in Example 14.
SEQ ID NOs:96-111 correspond to primers YL521, YL522, YL525, YL526, YL527, YL528, YL529, YL530, YL531, YL532, YL533, YL534, YL535, YL536, YL537 and YL538, respectively, used for amplification as described in Example 14.
SEQ ID NO:112 is the nucleotide sequence for the synthetic delta-8 desaturase codon-optimized for expression in Yarrowia lipolytica.
SEQ ID NO:113 is the amino acid sequence encoded by nucleotides 2-1270 of SEQ ID NO:112.
SEQ ID NO:114 is the DNA sequence (995 bp) of the Yarrowia lipolytica fructose-bisphosphate aldolase promoter containing a Yarrowia intron (FBAIN).
SEQ ID NO:118 is the nucleotide sequence for the synthetic delta-9 elongase codon-optimized for expression in Yarrowia lipolytica.
SEQ ID NO:119 is the DNA sequence of the Isochrysis galbana delta-9 elongase (792 bp), while SEQ ID NO:120 is the amino acid sequence of the Isochrysis galbana delta-9 elongase (263 AA).
SEQ ID NOs:121-136 correspond to primers IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A and IL3-8B, respectively, used for amplification as described in Example 15.
SEQ ID NOs:137-140 correspond to primers IL3-1F, IL3-4R, IL3-5F and IL3-8R, respectively, used for amplification as described in Example 15.
SEQ ID NO:141 is the 417 bp NcoI/PstI fragment described in Example 15.
SEQ ID NO:142 is the 377 bp PstI/Not1 fragment described in Example 15.
SEQ ID NO:146 is the DNA sequence of the Yarrowia lipolytica delta-12 desaturase (1936 bp), while SEQ ID NO:147 is the amino acid sequence of the Yarrowia lipolytica delta-12 desaturase (419 AA).
SEQ ID NO:149 is primer oIGsel1-1 used for amplifying a delta-9 elongase as described in Example 17.
SEQ ID NO:150 is primer oIGsel1-2 used for amplifying a delta-9 elongase as described in Example 17.
SEQ ID NO:151 is the fragment described in Example 18.
SEQ ID NOs:115, 116, 117, 143, 144, 145 and 148 are plasmids as identified in Table 1.
TABLE-US-00002 TABLE 1 Summary of Plasmid SEQ ID Numbers Plasmid SEQ ID NO Length pY54PC 115 8,502 bp pKUNFmkF2 116 7,145 bp pZF5T-PPC 117 5,553 bp pZUF17 143 8,165 bp pDMW237 144 7,879 pKUNT2 145 6,457 bp pDMW297 148 10,448 bp
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
In the context of this disclosure, a number of terms shall be utilized.
The term "fatty acids" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C12 to C22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C16 and C22. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (ω-6 or n-6) versus "omega-3 fatty acids" (ω-3 or n-3) are provided in WO2004/101757.
Fatty acids are described herein by a simple notation system of "X:Y", wherein the number before the colon indicates the number of carbon atoms in the fatty acid and the number after the colon is the number of double bonds that are present. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the "c" affix for the cis-configuration of the double bond [e.g., palmitic acid (16:0), stearic acid (18:0), oleic:acid (18:1, 9c), petroselinic acid (18:1, 6c), LA (18:2, 9c, 12c), GLA (18:3, 6c, 9c, 12c) and ALA (18:3, 9c, 12c, 15c)]. Unless otherwise specified 18:1, 18:2 and 18:3 refer to oleic, LA and linolenic fatty acids. If not specifically written as otherwise, double bonds are assumed to be of the cis configuration. For instance, the double bonds in 18:2 (9,12) would be assumed to be in the cis configuration.
A representative pathway is illustrated in FIG. 9, providing for the conversion of stearic acid through various intermediates to DHA, which demonstrates how both ω-3 and ω-6 fatty acids may be produced from a common source.
Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled "Shorthand Notation", the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of omega-3 and omega-6 fatty acids, the abbreviations that will be used throughout the remainder of the specification, and each compounds' chemical name.
TABLE-US-00003 TABLE 2 Nomenclature Of Polyunsaturated Fatty Acids Common Shorthand Name Abbreviation Chemical Name Notation Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6 γ-Linoleic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDA cis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLA cis-8,11,14-eicosatrienoic 20:3 ω-6 Linoleic Arachidonic AA or ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3 octadecatetraenoic Eicosatrienoic ETrA cis-11,14,17- 20:3 ω-3 eicosatrienoic Eicosatetraenoic ETA cis-8,11,14,17- 20:4 ω-3 eicosatetraenoic Eicosapentaenoic EPA cis-5,8,11,14,17- 20:5 ω-3 eicosapentaenoic Docosapentaenoic DPA cis-7,10,13,16,19- 22:5 ω-3 docosapentaenoic Docosahexaenoic DHA cis-4,7,10,13,16,19- 22:6 ω-3 docosahexaenoic
The term "essential fatty acid" refers to a particular PUFA that an organism must ingest in order to survive, being unable to synthesize the particular essential fatty acid de novo. For example, mammals can not synthesize the essential fatty acid LA. Other essential fatty acids include GLA, DGLA, ARA, EPA and DHA.
The term "fat" refers to a lipid substance that is solid at 25° C. and usually saturated.
The term "oil" refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. PUFAs are found in the oils of some algae, oleaginous yeasts and filamentous fungi. "Microbial oils" or "single cell oils" are those oils naturally produced by microorganisms during their lifespan. Such oils can contain long chain PUFAs.
The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see WO2005/003322). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane. More specifically, "PUFA biosynthetic pathway enzymes" refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase.
"Desaturase" is a polypeptide which can desaturate one or more fatty acids to produce a mono- or poly-unsaturated fatty acid or precursor which is of interest. Of particular interest herein are delta-8 desaturases that will desaturate a fatty acid between the 8th and 9th carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other useful fatty acid desaturases include, for example: 1.) delta-5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; 2.) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; 3.) delta-4 desaturases that catalyze the conversion of DPA to DHA; 4.) delta-12 desaturases that catalyze the conversion of oleic acid to LA; 5.) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; 6.) delta-17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; and 7.) delta-9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).
The term "elongase system" refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is 2 carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoA to yield CO2 and a β-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA.
For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA to β-ketoacyl-CoA) will be referred to generically as an "elongase". In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. For example, a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a C18/20 elongase will utilize a C18 substrate (e.g., GLA, STA) and a C20/22 elongase will utilize a C20 substrate (e.g., EPA). In like manner, a delta-9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively (see WO 2002/077213). It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C16/18 elongase and a C18/20 elongase).
The term "delta-9 elongase/delta-8 desaturase pathway" refers to a biosynthetic pathway for production of long chain PUFAs, said pathway minimally comprising a delta-9 elongase and a delta-8 desaturase and thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively. This pathway may be advantageous in some embodiments, as the biosynthesis of GLA and/or STA is excluded.
The terms "polynucleotide", "polynucleotide sequence", "nucleic acid sequence", "nucleic acid fragment" and "isolated nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5'-monophosphate form) are referred to by a single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
The terms "subfragment that is functionally equivalent" and "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.
The terms "homology", "homologous", "substantially similar" and "corresponding substantially" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions involves a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions involves the use of two final washes in 0.1×SSC, 0.1% SDS at 65° C.
"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. A "codon-optimized gene" is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ that plant is heterozygous at that locus.
"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B. Biochemistry of Plants 15:1-82 (1989).
"Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Mol. Biotechnol. 3:225-236 (1995)).
"3' non-coding sequences", "transcription terminator" or "termination sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell 1:671-680 (1989).
"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. A RNA transcript is referred to as the mature RNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a DNA that is complementary to, and synthesized from, a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described below.
"PCR" or "Polymerase Chain Reaction" is a technique for the synthesis of large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle".
The term "recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
The term "expression", as used herein, refers to the production of a functional end-product (e.g., a mRNA or a protein [either precursor or mature]).
The term "expression cassette" as used herein, refers to a discrete nucleic acid fragment into which a nucleic acid sequence or fragment can be moved.
"Mature" protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.
"Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.
"Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808 (2000)). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. Recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (WO 99/53050, published Oct. 21, 1999; WO 02/00904, published Jan. 3, 2002). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (WO 98/36083, published Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although genetic evidence has begun to unravel this complex situation (Elmayan et al., Plant Cell 10:1747-1757 (1998)).
The term "oleaginous" refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). Generally, the cellular oil content of these microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)).
The term "oleaginous yeast" refers to those microorganisms classified as yeasts that make oil. It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989)) and found in the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The "default parameters" are the parameters preset by the manufacturer of the program. For multiple alignments, they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10; and, for pairwise alignments, they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.
The present invention concerns an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NOs:2 or 113; or, (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
This delta-8 desaturase may be used alone or in combination with other desaturase and elongase components to produce various omega-6 and omega-3 PUFAs, including e.g., DGLA, ETA, ARA, EPA, DPA and/or DHA (FIG. 9). One skilled in the art will recognize the appropriate combinations of the delta-8 desaturase of the invention herein in conjunction with a delta-5 desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase, based on the particular host cell (and its native PUFA profile and/or desaturase and/or elongase profile), the availability of substrate, and the desired end product(s). In another embodiment, this invention concerns a recombinant construct comprising the polynucleotide of the invention operably linked to at least one regulatory sequence.
Plant Expression Systems, Cassettes and Vectors
As was noted above, a promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3') of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce mRNA which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3' transcription termination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cell to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.
The origin of the promoter chosen to drive expression of the coding sequence is not important as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired nucleic acid fragments in the desired host tissue at the right time. Either heterologous or non-heterologous (i.e., endogenous) promoters can be used to practice the invention. For example, suitable promoters include, but are not limited to: the alpha prime subunit of beta conglycinin promoter, Kunitz trypsin inhibitor 3 promoter, annexin promoter, Gly1 promoter, beta subunit of beta conglycinin promoter, P34/Gly Bd m 30K promoter, albumin promoter, Leg A1 promoter and Leg A2 promoter.
The annexin, or P34, promoter is described in WO 2004/071178 (published Aug. 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J Biol. Chem. 265(21):12486-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene. 211 (2):267-76 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter (Atanassova et al., Plant Mol. Biol. 37(2):275-85 (1989)).
Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before 10 days after pollination) and is largely quiescent in later stages. The expression profile of the annexin promoter is different from that of many seed-specific promoters, e.g., seed storage protein promoters, which often provide highest activity in later stages of development (Chen et al., Dev. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al., Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression, or suppression, of a gene in embryos is desired at an early developing stage. For example, it may be desirable to overexpress a gene regulating early embryo development or a gene involved in the metabolism prior to seed maturation.
Following identification of an appropriate promoter suitable for expression of a specific coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring
Harbor Laboratory Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
Once the recombinant construct has been made, it may then be introduced into a plant cell of choice by methods well known to those of ordinary skill in the art (e.g., transfection, transformation and electroporation). Oilseed plant cells are the preferred plant cells. The transformed plant cell is then cultured and regenerated under suitable conditions permitting expression of the long chain PUFA which is then optionally recovered and purified.
The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells.
Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.
The desired long chain PUFAs can be expressed in seed. Also within the scope of this invention are seeds or plant parts obtained from such transformed plants.
Plant parts include differentiated and undifferentiated tissues including, but not limited to: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the following: 1. The entire complement of genetic material (genes and non-coding sequences) is present in each cell of an organism, or virus or organelle. 2. A complete set of chromosomes inherited as a (haploid) unit from one parent.
Thus, this invention also concerns a method for transforming a cell, comprising transforming a cell with the recombinant construct of the invention and selecting those cells transformed with the recombinant construct of Claim 4.
Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the polynucleotide of the instant invention and regenerating a plant from the transformed plant cell.
Methods for transforming dicots (primarily by use of Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grant et al. Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65 (2000)). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (WO 92/17598), electroporation (Chowrira, G. M. et al. Mol. Biotechnol. 3:17-23 (1995); Christou, P. et al. Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection, or particle bombardment (McCabe, D. E. et. al. Bio/Technology 6:923 (1988); Christou et al. Plant Physiol. 87:671-674 (1988)).
There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic: San Diego, Calif. (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), generation of recombinant DNA fragments and recombinant expression constructs and the screening and isolating of clones. See, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: NY (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).
Examples of oilseed plants include, but are not limited to, soybean, Brassica species, sunflower, maize, cotton, flax, safflower.
Examples of polyunsaturated fatty acids having at least twenty carbon atoms and five or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.
In one embodiment this invention concerns an oilseed plant comprising: a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase.
Such desaturases are discussed in U.S. Pat. Nos. 6,075,183, 5,968,809, 6,136,574, 5,972,664, 6,051,754, 6,410,288 and WO 98/46763, WO 98/46764, WO 00/12720, WO 00/40705.
The choice of combination of cassettes used depends in part on the PUFA profile and/or desaturase profile of the oilseed plant cells to be transformed and the LC-PUFA which is to be expressed.
In another aspect, this invention concerns a method for making long chain polyunsaturated fatty acids in a plant cell comprising: (a) transforming a cell with the recombinant construct of the invention; and (b) selecting those transformed cells that make long chain polyunsaturated fatty acids.
In still another aspect, this invention concerns a method for producing at least one polyunsaturated fatty acid in a soybean cell comprising: (a) transforming a soybean cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4, a delta-5, delta-6, a delta-9, a delta-12, a delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and a C20 to C24 elongase. (b) regenerating a soybean plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed soybean plant.
Plant Seed Oils: Isolation and Hydrogenation
Methods of isolating seed oils are well known in the art: (Young et al., Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al., eds., Chapter 5 pp 253-257; Chapman & Hall: London (1994)). For example, soybean oil is produced using a series of steps involving the extraction and purification of an edible oil product from the oil-bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in the Table below.
TABLE-US-00004 TABLE 3 Generalized Steps For Soybean Oil And Byproduct Production Process Impurities Removed And/Or Step Process By-Products Obtained # 1 Soybean seed # 2 Oil extraction Meal # 3 Degumming Lecithin # 4 Alkali or physical refining Gums, free fatty acids, pigments # 5 Water washing Soap # 6 Bleaching Color, soap, metal # 7 (Hydrogenation) # 8 (Winterization) Stearine # 9 Deodorization Free fatty acids, tocopherols, sterols, volatiles # 10 Oil products
In general, soybean oil is produced using a series of steps involving the extraction and purification of an edible oil product from the oil bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in the diagram below.
TABLE-US-00005 Impurities Removed/ Process Byproducts Obtained ##STR00001## Meal ##STR00002## Lecithin ##STR00003## Gums, Free Fatty Acids, Pigments ##STR00004## Soap ##STR00005## Color, Soap, Metal ##STR00006## Stearine ##STR00007## FFA, Tocopherols, Sterols, Volatiles
More specifically, soybean seeds are cleaned, tempered, dehulled and flaked, thereby increasing the efficiency of oil extraction. Oil extraction is usually accomplished by solvent (e.g., hexane) extraction but can also be achieved by a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. The crude oil may be degummed by hydrating phospholipids and other polar and neutral lipid complexes that facilitate their separation from the nonhydrating, triglyceride fraction (soybean oil). The resulting lecithin gums may be further processed to make commercially important lecithin products used in a variety of food and industrial products as emulsification and release (i.e., antisticking) agents. Degummed oil may be further refined for the removal of impurities (primarily free fatty acids, pigments and residual gums). Refining is accomplished by the addition of a caustic agent that reacts with free fatty acid to form soap and hydrates phosphatides and proteins in the crude oil. Water is used to wash out traces of soap formed during refining. The soapstock byproduct may be used directly in animal feeds or acidulated to recover the free fatty acids. Color is removed through adsorption with a bleaching earth that removes most of the chlorophyll and carotenoid compounds. The refined oil can be hydrogenated, thereby resulting in fats with various melting properties and textures. Winterization (fractionation) may be used to remove stearine from the hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization (principally via steam distillation under vacuum) is the last step and is designed to remove compounds which impart odor or flavor to the oil. Other valuable byproducts such as tocopherols and sterols may be removed during the deodorization process. Deodorized distillate containing these byproducts may be sold for production of natural vitamin E and other high-value pharmaceutical products. Refined, bleached, (hydrogenated, fractionated) and deodorized oils and fats may be packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production and byproduct utilization can be found in Erickson, Practical Handbook of Soybean Processing and Utilization, The American Oil Chemists' Society and United Soybean Board (1995).
Soybean oil is liquid at room temperature because it is relatively low in saturated fatty acids when compared with oils such as coconut, palm, palm kernel and cocoa butter. Many processed fats (including spreads, confectionary fats, hard butters, margarines, baking shortenings, etc.) require varying degrees of solidity at room temperature and can only be produced from soybean oil through alteration of its physical properties. This is most commonly achieved through catalytic hydrogenation.
Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. High oleic soybean oil contains unsaturated oleic, LA and linolenic fatty acids and each of these can be hydrogenated. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifications increase the melting point resulting in a semi-liquid or solid fat at room temperature.
There are many variables which affect the hydrogenation reaction, which in turn alter the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation and reactor design are among the more important parameters that can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unsaturated ones. Very light or brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depends on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings (used in the manufacture of baking products, solid fats and shortenings used for commercial frying and roasting operations) and base stocks for margarine manufacture are among the myriad of possible oil and fat products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice. The American Oil Chemists' Society (1994).
Hydrogenated oils have also become controversial due to the presence of trans-fatty acid isomers that result from the hydrogenation process. Ingestion of large amounts of trans-isomers has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disease.
Compared to other vegetable oils, the oils of the invention are believed to function similarly to other oils in food applications from a physical standpoint. Partially hydrogenated oils, such as soybean oil, are widely used as ingredients for soft spreads, margarine and shortenings for baking and frying.
Examples of food products or food analogs into which altered seed oils or altered seeds of the invention may be incorporated include a meat product such as a processed meat product, a cereal food product, a snack food product, a baked goods product, a fried food product, a health food product, an infant formula, a beverage, a nutritional supplement, a dairy product, a pet food product, animal feed or an aquaculture food product. Food analogs can be made use processes well known to those skilled in the art. U.S. Pat. Nos. 6,355,296 B1 and 6,187,367 B1 describe emulsified meat analogs and emulsified meat extenders. U.S. Pat. No. 5,206,050 B1 describes soy protein curd useful for cooked food analogs (also can be used as a process to form a curd useful to make food analogs). U.S. Pat. No. 4,284,656 to Hwa describes a soy protein curd useful for food analogs. U.S. Pat. No. 3,988,485 to Hibbert et al. describes a meat-like protein food formed from spun vegetable protein fibers. U.S. Pat. No. 3,950,564 to Puski et al. describes a process of making a soy based meat substitute and U.S. Pat. No. 3,925,566 to Reinhart et al. describes a simulated meat product. For example, soy protein that has been processed to impart a structure, chunk or fiber for use as a food ingredient is called "textured soy protein" (TSP). TSPs are frequently made to resemble meat, seafood, or poultry in structure and appearance when hydrated.
There can be mentioned meat analogs, cheese analogs, milk analogs and the like.
Meat analogs made from soybeans contain soy protein or tofu and other ingredients mixed together to simulate various kinds of meats. These meat alternatives are sold as frozen, canned or dried foods. Usually, they can be used the same way as the foods they replace. Meat alternatives made from soybeans are excellent sources of protein, iron and B vitamins. Examples of meat analogs include, but are not limited to, ham analogs, sausage analogs, bacon analogs, and the like.
Food analogs can be classified as imitiation or substitutes depending on their functional and compositional characteristics. For example, an imitation cheese need only resemble the cheese it is designed to replace. However, a product can generally be called a substitute cheese only if it is nutritionally equivalent to the cheese it is replacing and meets the minimum compositional requirements for that cheese. Thus, substitute cheese will often have higher protein levels than imitation cheeses and be fortified with vitamins and minerals.
Milk analogs or nondairy food products include, but are not limited to, imitation milk, nondairy frozen desserts such as those made from soybeans and/or soy protein products.
Meat products encompass a broad variety of products. In the United States "meat" includes "red meats" produced from cattle, hogs and sheep. In addition to the red meats there are poultry items which include chickens, turkeys, geese, guineas, ducks and the fish and shellfish. There is a wide assortment of seasoned and processes meat products: fresh, cured and fried, and cured and cooked. Sausages and hot dogs are examples of processed meat products. Thus, the term "meat products" as used herein includes, but is not limited to, processed meat products.
A cereal food product is a food product derived from the processing of a cereal grain. A cereal grain includes any plant from the grass family that yields an edible grain (seed). The most popular grains are barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat and wild rice. Examples of a cereal food product include, but are not limited to, whole grain, crushed grain, grits, flour, bran, germ, breakfast cereals, extruded foods, pastas, and the like.
A baked goods product comprises any of the cereal food products mentioned above and has been baked or processed in a manner comparable to baking, i.e., to dry or harden by subjecting to heat. Examples of a baked good product include, but are not limited to bread, cakes, doughnuts, bread crumbs, baked snacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above, oils of the invention can be used as an ingredient.
A snack food product comprises any of the above or below described food products.
A fried food product comprises any of the above or below described food products that has been fried.
A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.
The beverage can be in a liquid or in a dry powdered form.
For example, there can be mentioned non-carbonated drinks; fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum® from Ross Products Division, Abbott Laboratories).
Infant formulas are liquids or reconstituted powders fed to infants and young children. They serve as substitutes for human milk. Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants. Although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive. Infant formula is becoming more and more increasingly close to breast milk.
A dairy product is a product derived from milk. A milk analog or nondairy product is derived from a source other than milk, for example, soymilk as was discussed above. These products include, but are not limited to, whole milk, skim milk, fermented milk products such as yoghurt or sour milk, cream, butter, condensed milk, dehydrated milk, coffee whitener, coffee creamer, ice cream, cheese, etc.
A pet food product is a product intended to be fed to a pet such as a dog, cat, bird, reptile, fish, rodent and the like. These products can include the cereal and health food products above, as well as meat and meat byproducts, soy protein products, grass and hay products, including but not limited to alfalfa, timothy, oat or brome grass, vegetables and the like.
Animal feed is a product intended to be fed to animals such as turkeys, chickens, cattle and swine and the like. As with the pet foods above, these products can include cereal and health food products, soy protein products, meat and meat byproducts, and grass and hay products as listed above.
Aqualculture feed is a product intended to be used in aquafarming which concerns the propagation, cultivation or farming of aquatic organisms, animals and/or plants in fresh or marine waters.
Microbial Biosynthesis of Fatty Acids
The process of de novo synthesis of palmitate (16:0) in oleaginous microorganisms is described in WO 2004/101757. This fatty acid is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases. For example, palmitate is converted to its unsaturated derivative [palmitoleic acid (16:1)] by the action of a delta-9 desaturase; similarly, palmitate is elongated to form stearic acid (18:0), which can be converted to its unsaturated derivative by a delta-9 desaturase to thereby yield oleic (18:1) acid.
Triacylglycerols (the primary storage unit for fatty acids) are formed by the esterification of two molecules of acyl-CoA to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid). The phosphate is then removed, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol. Triacylglycerol is formed upon the addition of a third fatty acid by the action of a diacylglycerol-acyl transferase.
Genes Involved in Omega Fatty Acid Production
Many microorganisms, including algae, bacteria, molds and yeasts, can synthesize PUFAs and omega fatty acids in the ordinary course of cellular metabolism. Particularly well-studied are fungi including Schizochytrium aggregatm, species of the genus Thraustochytrium and Morteriella alpina. Additionally, many dinoflagellates (Dinophyceaae) naturally produce high concentrations of PUFAs. As such, a variety of genes involved in oil production have been identified through genetic means and the DNA sequences of some of these genes are publicly available. See, for example: AY131238, Y055118, AY055117, AF296076, AF007561, L11421, NM--031344, AF465283, AF465281, AF110510, AF465282, AF419296, AB052086, AJ250735, AF126799, AF126798 (delta-6 desaturases); AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (delta-5 desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, AY332747, AAG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, X86736, AF240777, AB007640, AB075526, AP002063 (delta-12 desaturases); NP--441622, BAA18302, BAA02924, AAL36934 (delta-15 desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417, AF085500, AY504633, NM--069854, AF230693 (delta-9 desaturases); AF390174 (delta-9 elongase); and AX464731, NM--119617, NM--134255, NM--134383, NM--134382, NM--068396, NM--068392, NM--070713, NM--068746, NM--064685 (elongases).
Additionally, the patent literature provides many additional DNA sequences of genes (and/or details concerning several of the genes above and their methods of isolation) involved in PUFA production [e.g., U.S. Pat. No. 5,968,809 (delta-6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183 (delta-5 desaturases); WO 94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216 (delta-12 desaturases); WO 93/11245 (delta-15 desaturases); WO 91/13972 and U.S. Pat. No. 5,057,419 (delta-9 desaturases); U.S. 2003/0196217 A1 (delta-17 desaturase); and, WO 00/12720, WO 2002/077213 and U.S. 2002/0139974A1 (elongases)].
As will be obvious to one skilled in the art, the particular functionalities required to be introduced into a microbial host organism for production of a particular PUFA final product will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s). LA, GLA, EDA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA may all be produced in oleaginous yeasts, by introducing various combinations of the following PUFA enzyme functionalities: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase. One skilled in the art will be able to identify various candidate genes encoding each of the above enzymes, according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of microorganisms having the ability to produce PUFAs. The sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. In some embodiments, manipulation of genes endogenous to the host is preferred; for other purposes, it is necessary to introduce heterologous genes.
Although the particular source of the desaturase and elongase genes introduced into the host is not critical to the invention, considerations for choosing a specific polypeptide having desaturase or elongase activity include: 1.) the substrate specificity of the polypeptide; 2.) whether the polypeptide or a component thereof is a rate-limiting enzyme; 3.) whether the desaturase or elongase is essential for synthesis of a desired PUFA; and/or 4.) co-factors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell. For example, the polypeptide may have to compete for substrate with other enzymes in the host cell. Analyses of the KM and specific activity of the polypeptide are therefore considered in determining the suitability of a given polypeptide for modifying PUFA production in a given host cell. The polypeptide used in a particular host cell is one that can function under the biochemical conditions present in the intended host cell but otherwise can be any polypeptide having desaturase or elongase activity capable of modifying the desired PUFA.
In some cases, the host organism in which it is desirable to produce PUFAs will possess endogenous genes encoding some PUFA biosynthetic pathway enzymes. For example, oleaginous yeast can typically produce 18:2 fatty acids (and some have the additional capability of synthesizing 18:3 fatty acids); thus, oleaginous yeast typically possess native delta-12 desaturase activity and may also have delta-15 desaturases. In some embodiments, therefore, expression of the native desaturase enzyme is preferred over a heterologous (or "foreign") enzyme since: 1.) the native enzyme is optimized for interaction with other enzymes and proteins within the cell; and 2.) heterologous genes are unlikely to share the same codon preference in the host organism. Additionally, advantages are incurred when the sequence of the native gene is known, as it permits facile disruption of the endogenous gene by targeted disruption.
In many instances, however, the appropriate desaturases and elongases are not present in the host organism of choice to enable production of the desired PUFA products. Thus, it is necessary to introduce heterologous genes. In one embodiment of the present invention, work was conducted toward the goal of the development of an oleaginous yeast that accumulates oils enriched in long-chain omega-3 and/or omega-6 fatty acids. In order to express genes encoding the delta-9 elongase/delta-8 desaturase pathway for the biosynthesis of ARA and EPA in these organisms, it was therefore necessary to: (1) identify a suitable desaturase that functioned relatively efficiently in oleaginous yeast based on substrate-feeding trials; and, (2) subject the desaturase gene to codon-optimization techniques (infra) to further enhance the expression of the heterologous enzyme in the alternate oleaginous yeast host, to thereby enable maximal production of omega-3 and/or omega-6 fatty acids.
Optimization of Omega Fatty Acid Genes for Expression in Particular Organisms
Although the particular source of a PUFA desaturase or elongase is not critical in the invention herein, it will be obvious to one of skill in the art that heterologous genes will be expressed with variable efficiencies in an alternate host. Thus, omega-3 and/or omega-6 PUFA production may be optimized by selection of a particular desaturase or elongase whose level of expression in a heterologous host is preferred relative to the expression of an alternate desaturase or elongase in the host organism of interest. Furthermore, it may be desirable to modify the expression of particular PUFA biosynthetic pathway enzymes to achieve optimal conversion efficiency of each, according to the specific PUFA product composition of interest. A variety of genetic engineering techniques are available to optimize expression of a particular enzyme. Two such techniques include codon optimization and gene mutation, as described below. Genes produced by e.g., either of these two methods, having desaturase and/or elongase activity(s) would be useful in the invention herein for synthesis of omega-3 and/or omega-6 PUFAs.
Codon Optimization: As will be appreciated by one skilled in the art, it is frequently useful to modify a portion of the codons encoding a particular polypeptide that is to be expressed in a foreign host, such that the modified polypeptide uses codons that are preferred by the alternate host. Use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide.
In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having desaturase or elongase activity can be synthesized in whole or in part using the codons preferred in the host species. All (or portions) of the DNA also can be synthesized to remove any destabilizing sequences or regions of secondary structure that would be present in the transcribed mRNA. All (or portions) of the DNA also can be synthesized to alter the base composition to one more preferable in the desired host cell.
In the present invention, it was desirable to modify a portion of the codons encoding the polypeptide having delta-8 desaturase activity, to enhance the expression of the gene in the oleaginous yeast Yarrowia lipolytica. The nucleic acid sequence of the native gene (e.g., the Euglena gracilis delta-8 desaturase defined herein as Eg5) was modified to employ host-preferred codons. This wildtype desaturase has 421 amino acids (SEQ ID NO:2); in the codon-optimized gene created herein (SEQ ID NO:112), 207 bp of the 1263 bp coding region (corresponding to 192 codons) were codon-optimized and the translation initiation site was modified. The skilled artisan will appreciate that this optimization method will be equally applicable to other genes in the omega-3/omega-6 fatty acids biosynthetic pathway (see for example, WO 2004/101753, herein incorporated entirely by reference). Furthermore, modulation of the E. gracilis delta-8 desaturase is only exemplary; numerous other heterologous delta-8 desaturases from variable sources could be codon-optimized to improve their expression in an oleaginous yeast host. The present invention comprises the complete sequences of the synthetic codon-optimized gene as reported in the accompanying Sequence Listing, the complement of those complete sequences, and substantial portions of those sequences.
Gene Mutation: Methods for synthesizing sequences and bringing sequences together are well established in the literature. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), "gene shuffling" or other means can be employed to obtain mutations of naturally occurring desaturase or elongase genes (wherein such mutations may include deletions, insertions and point mutations, or combinations thereof). This would permit production of a polypeptide having desaturase or elongase activity, respectively, in vivo with more desirable physical and kinetic parameters for function in the host cell such as a longer half-life or a higher rate of production of a desired PUFA. Or, if desired, the regions of a polypeptide of interest (i.e., a desaturase or an elongase) important for enzymatic activity can be determined through routine mutagenesis, expression of the resulting mutant polypeptides and determination of their activities. An overview of these techniques are described in WO 2004/101757. All such mutant proteins and nucleotide sequences encoding them that are derived from the codon-optimized gene described herein are within the scope of the present invention.
Microbial Production of Omega-3 and/or Omega-6 Fatty Acids
Microbial production of omega-3 and/or omega-6 fatty acids has several advantages. For example: 1.) many microbes are known with greatly simplified oil compositions compared with those of higher organisms, making purification of desired components easier; 2.) microbial production is not subject to fluctuations caused by external variables, such as weather and food supply; 3.) microbially produced oil is substantially free of contamination by environmental pollutants; 4.) microbes can provide PUFAs in particular forms which may have specific uses; and 5.) microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrates for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways.
In addition to these advantages, production of omega-3 and/or omega-6 fatty acids from recombinant microbes provides the ability to alter the naturally occurring microbial fatty acid profile by providing new biosynthetic pathways in the host or by suppressing undesired pathways, thereby increasing levels of desired PUFAs, or conjugated forms thereof, and decreasing levels of undesired PUFAs. For example, it is possible to modify the ratio of omega-3 to omega-6 fatty acids so produced, produce either omega-3 or omega-6 fatty acids exclusively while eliminating production of the alternate omega fatty acid, or engineer production of a specific PUFA without significant accumulation of other PUFA downstream or upstream products (e.g., enable biosynthesis of ARA, EPA and/or DHA via the delta-9 elongase/delta-8 desaturase pathway, thereby avoiding synthesis of GLA and/or STA).
Microbial Expression Systems, Cassettes and Vectors
The genes and gene products described herein may be produced in heterologous microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica). Expression in recombinant microbial hosts may be useful for the production of various PUFA pathway intermediates, or for the modulation of PUFA pathways already existing in the host for the synthesis of new products heretofore not possible using the host.
Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of any of the gene products of the preferred desaturase and/or elongase sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes.
Accordingly, it is expected that introduction of chimeric genes encoding a PUFA biosynthetic pathway, under the control of the appropriate promoters will result in increased production of omega-3 and/or omega-6 fatty acids. It is contemplated that it will be useful to express various combinations of these PUFA desaturase and elongase genes together in a host microorganism. It will be obvious to one skilled in the art that the particular genes included within a particular expression cassette(s) will depend on the host cell, its ability to synthesize PUFAs using native desaturases and elongases, the availability of substrate and the desired end product(s). For example, it may be desirable for an expression cassette to be constructed comprising genes encoding one or more of the following enzymatic activities: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase. As such, the present invention encompasses a method of producing PUFAs comprising exposing a fatty acid substrate to the PUFA enzyme(s) described herein, such that the substrate is converted to the desired fatty acid product. Thus, each PUFA gene and corresponding enzyme product described herein (e.g., a wildtype, codon-optimized, synthetic and/or mutant enzyme having appropriate desaturase or elongase activity) can be used directly or indirectly for the production of PUFAs. Direct production of PUFAs occurs wherein the fatty acid substrate is converted directly into the desired fatty acid product without any intermediate steps or pathway intermediates. For example, production of ARA would occur in a host cell which produces or which is provided DGLA, by adding or introducing into said cell an expression cassette that provides delta-5 desaturase activity. Similarly, expression of the delta-8 desaturase of the invention permits the direct synthesis of DGLA and ETA (when provided EDA and ETrA, respectively, as substrate). Thus for example, the present invention is drawn to a method of producing either DGLA or ETA, respectively, comprising: a) providing an oleaginous yeast comprising: (i) a gene encoding a delta-8 desaturase polypeptide as set forth in SEQ ID NO:112; and (ii) a source of desaturase substrate consisting of either EDA or ETrA, respectively; and, b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding a delta-8 desaturase polypeptide is expressed and EDA is converted to DGLA or ETrA is converted to ETA, respectively; and, c) optionally recovering the DGLA or ETA, respectively, of step (b).
In contrast, multiple genes encoding the PUFA biosynthetic pathway may be used in combination, such that a series of reactions occur to produce a desired PUFA. For example, expression cassette(s) encoding elongase, delta-5 desaturase, delta-17 desaturase and delta-4 desaturase activity would enable a host cell that naturally produces GLA, to instead produce DHA (such that GLA is converted to DGLA by an elongase; DGLA may then be converted to ARA by a delta-5 desaturase; ARA is then converted to EPA by a delta-17 desaturase, which may in turn be converted to DPA by an elongase; and DPA would be converted to DHA by a delta-4 desaturase). In a related manner, expression of the delta-8 desaturase of the invention enables the indirection production of ARA, EPA, DPA and/or DHA as down-stream PUFAs, if subsequent desaturase and elongation reactions are catalyzed. In a preferred embodiment, wherein the host cell is an oleaginous yeast, expression cassettes encoding each of the enzymes necessary for PUFA biosynthesis will need to be introduced into the organism, since naturally produced PUFAs in these organisms are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA). Alternatively, substrate feeding may be required.
Vectors or DNA cassettes useful for the transformation of suitable microbial host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation and a region 3' of the DNA fragment that controls transcriptional termination. It is most preferred when both control regions are derived from genes from the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Initiation control regions or promoters which are useful to drive expression of desaturase and/or elongase ORFs in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of these genes in the selected host cell is suitable for the present invention. Expression in a microbial host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species. The transcriptional initiation regulatory regions can be obtained, for example, from: 1.) genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase (WO 2005/003310), phosphoglycerate mutase (WO 2005/003310), fructose-bisphosphate aldolase (WO 2005/049805), phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase (see U.S. Patent Application No. 60/610,060), etc.; or, 2.) regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, the translation elongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), etc. Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.
Nucleotide sequences surrounding the translational initiation codon `ATG` have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, as demonstrated in the invention herein in Yarrowia lipolytica, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.
The termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3'-regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: 1.) the nature of the relevant transcriptional promoter and terminator sequences; 2.) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; 3.) the final cellular location of the synthesized foreign protein; 4.) the efficiency of translation in the host organism; 5.) the intrinsic stability of the cloned gene protein within the host cell; and 6.) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the PUFA biosynthetic pathway enzymes.
Transformation of Microbial Hosts
Once the DNA encoding a desaturase or elongase polypeptide suitable for expression in an oleaginous yeast has been obtained, it is placed in a plasmid vector capable of autonomous replication in a host cell; or, it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
In the present invention, the preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired. Toward this end, it is desirable to identify a sequence within the genome that is present in multiple copies.
Schmid-Berger et al. (J. Bact. 176(9):2477-2482 (1994)) discovered the first retrotransposon-like element Ylt1 in Yarrowia lipolytica. This retrotransposon is characterized by the presence of long terminal repeats (LTRs; each approximately 700 bp in length) called zeta regions. Ylt1 and solo zeta elements were present in a dispersed manner within the genome in at least 35 copies/genome and 50-60 copies/genome, respectively; both elements were determined to function as sites of homologous recombination. Further, work by Juretzek et al. (Yeast 18:97-113 (2001)) demonstrated that gene expression could be dramatically increased by targeting plasmids into the repetitive regions of the yeast genome (using linear DNA with LTR zeta regions at both ends), as compared to the expression obtained using low-copy plasmid transformants. Thus, zeta-directed integration can be ideal as a means to ensure multiple integration of plasmid DNA into Y. lipolytica, thereby permitting high-level gene expression. Unfortunately, however, not all strains of Y. lipolytica possess zeta regions (e.g., the strain identified as ATCC #20362). When the strain lacks such regions, it is also possible to integrate plasmid DNA comprising expression cassettes into alternate loci to reach the desired copy number for the expression cassette. For example, preferred alternate loci include: the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP--503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12 desaturase gene locus (SEQ ID NO:23), the Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632).
Advantageously, the Ura3 gene can be used repeatedly in combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; "5-FOA") selection (infra), to readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.
Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other constructs to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
Constructs comprising the gene of interest may be introduced into a host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)).
For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified or is present on an extrachromosomal element having multiple copy numbers.
The transformed host cell can be identified by various selection techniques, as described in WO2004/101757. Preferred selection methods for use herein are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-FOA is used for selection of yeast Ura-mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5'-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura.sup.- mutant yeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147, 1997). More specifically, one can first knockout the native Ura3 gene to produce a strain having a Ura-phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene could be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+ phenotype. Subsequent integration would produce a new Ura3-strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in combination with 5-FOA selection) can be used as a selection marker in multiple rounds of transformation.
Following transformation, substrates suitable for the recombinantly expressed desaturases and/or elongases (and optionally other PUFA enzymes that are expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.
Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesis in Microbes
Methods for manipulating biochemical pathways are well known to those skilled in the art; and, it is expected that numerous manipulations will be possible to maximize omega-3 and/or omega-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This may require metabolic engineering directly within the PUFA biosynthetic pathway or additional manipulation of pathways that contribute carbon to the PUFA biosynthetic pathway.
In the case of manipulations within the PUFA biosynthetic pathway, it may be desirable to increase the production of LA to enable increased production of omega-6 and/or omega-3 fatty acids. Introducing and/or amplifying genes encoding delta-9 and/or delta-12 desaturases may accomplish this.
To maximize production of omega-6 unsaturated fatty acids, it is well known to one skilled in the art that production is favored in a host microorganism that is substantially free of ALA. Thus, preferably, the host is selected or obtained by removing or inhibiting delta-15 or omega-3 type desaturase activity that permits conversion of LA to ALA. The endogenous desaturase activity can be reduced or eliminated by, for example: 1.) providing a cassette for transcription of antisense sequences to the delta-15 desaturase transcription product; 2.) disrupting the delta-15 desaturase gene through insertion, substitution and/or deletion of all or part of the target gene; or 3.) using a host cell which naturally has [or has been mutated to have] low or no delta-15 desaturase activity. Inhibition of undesired desaturase pathways can also be accomplished through the use of specific desaturase inhibitors such as those described in U.S. Pat. No. 4,778,630.
Alternatively, it may be desirable to maximize production of omega-3 fatty acids (and minimize synthesis of omega-6 fatty acids). Thus, one could utilize a host microorganism wherein the delta-12 desaturase activity that permits conversion of oleic acid to LA is removed or inhibited, using any of the means described above (see also e.g., WO 2004/104167, herein incorporated entirely by reference). Subsequently, appropriate expression cassettes would be introduced into the host, along with appropriate substrates (e.g., ALA) for conversion to omega-3 fatty acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA, DHA).
Beyond the immediate PUFA biosynthetic pathway, it is expected that manipulation of several other enzymatic pathways leading to the biosynthesis of precursor fatty acids may contribute to the overall net biosynthesis of specific PUFAs. Identification and manipulation of these related pathways will be useful in the future.
Techniques to Up-Regulate Desirable Biosynthetic Pathways
Additional copies of desaturase and elongase genes may be introduced into the host to increase the output of omega-3 and/or omega-6 fatty acid biosynthetic pathways. Expression of the desaturase or elongase genes also can be increased at the transcriptional level through the use of a stronger promoter (either regulated or constitutive) to cause increased expression, by removing/deleting destabilizing sequences from either the mRNA or the encoded protein, or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141). Yet another approach to increase expression of the desaturase or elongase genes, as demonstrated in the instant invention, is to increase the translational efficiency of the encoded mRNAs by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism.
Techniques to Down-Regulate Undesirable Biosynthetic Pathways
Conversely, biochemical pathways competing with the omega-3 and/or omega-6 fatty acid biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means (e.g., antisense mRNA). For gene disruption, a foreign DNA fragment (typically a selectable marker gene) is inserted into the structural gene to be disrupted in order to interrupt its coding sequence and thereby functionally inactivate the gene. Transformation of the disruption cassette into the host cell results in replacement of the functional native gene by homologous recombination with the non-functional disrupted gene (see, for example: Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene 136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524 (1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277 (1996)).
Antisense technology is another method of down-regulating genes when the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA that encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
Although targeted gene disruption and antisense technology offer effective means of down-regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence-based (e.g., mutagenesis via UV radiation/chemical agents or use of transposable elements/transposons; see WO 2004/101757).
Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the methods described above. For example, the present invention provides methods whereby genes encoding key enzymes in the biosynthetic pathways are introduced into oleaginous yeasts for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express these genes in oleaginous yeasts that do not naturally possess omega-3 and/or omega-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.
Preferred Microbial Hosts for Recombinant Production of Omega-3 and/or Omega-6 Fatty Acids
Microbial host cells for production of omega fatty acids may include microbial hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, organic acids and alcohols, and/or hydrocarbons over a wide range of temperature and pH values.
Preferred microbial hosts, however, are oleaginous yeasts. These organisms are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica).
Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).
Historically, various strains of Y. lipolytica have been used for the manufacture and production of: isocitrate lyase (DD259637); lipases (SU1454852, WO2001083773, DD279267); polyhydroxyalkanoates (WO2001088144); citric acid (RU2096461, RU2090611, DD285372, DD285370, DD275480, DD227448, PL160027); erythritol (EP770683); 2-oxoglutaric acid (DD267999); γ-decalactone (U.S. Pat. No. 6,451,565, FR2734843); γ-dodecalatone (EP578388); and pyruvic acid (JP09252790).
Microbial Fermentation Processes for PUFA Production
The transformed microbial host cell is grown under conditions that optimize desaturase and elongase activities and produce the greatest and the most economical yield of the preferred PUFAs. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time of cell harvest. Microorganisms of interest, such as oleaginous yeast, are grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources may include, but are not limited to: monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats. Additionally, the carbon source may include one-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formate and carbon-containing amines) for which metabolic conversion into key biochemical intermediates has been demonstrated. Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources and will only be limited by the choice of the host organism. Although all of the above mentioned carbon sources and mixtures thereof are expected to be suitable in the present invention, preferred carbon sources are sugars and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.
Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) or organic source (e.g., urea or glutamate). In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art suitable for the growth of the microorganism and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Mn+2, Co+2, Zn+2, Mg+2) that promote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.
Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be "balanced" between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast. This approach is described in WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch and continuous) and considerations during growth.
Purification of Microbial PUFAs
The PUFAs may be found in the host microorganism as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cell through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).
In general, means for the purification of PUFAs may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. One is referred to the teachings of WO 2004/101757 for additional details.
DESCRIPTION OF PREFERRED EMBODIMENTS
The ultimate goal of the work described herein was the identification of a delta-8 desaturase suitable to enable expression of the delta-9 elongase/delta-8 desaturase pathway in plants and oleaginous yeast. Thus, initial work performed herein attempted to codon-optimize the delta-8 desaturase of Euglena gracilis (GenBank Accession No. AAD45877; WO 00/34439) for expression in Yarrowia lipolytica. Despite synthesis of three different codon-optimized genes (i.e., "D8S-1", "D8S-2" and "D8S-3"), none of the genes were capable of desaturating EDA to DGLA (Example 1). On the basis of these results, it was hypothesized that the previously published delta-8 desaturase sequences were incorrect.
Isolation of the delta-8 desaturase from Euglena gracilis directly, following mRNA isolation, cDNA synthesis and PCR (Examples 2 and 3) was attempted as described below. This resulted in two similar sequences, identified herein as Eg5 (SEQ ID NOs:1 and 2) and Eg12 (SEQ ID NOs:3 and 4), both of which possessed significant differences when compared to the previously published delta-8 desaturase sequences (Example 4). Eg5 and Eg12 were each cloned into a Saccharomyces cerevisiae yeast expression vector (Example 5) for functional analysis via substrate feeding trials (Example 11). This demonstrated that both Eg5 and Eg12 were able to desaturase EDA and ETrA to produce DGLA and ETA, respectively; Eg5 had significantly greater activity than Eg12.
Based on the confirmed delta-8 desaturase activity of Eg5 (SEQ ID NO:1 and 2), the sequence of Eg5 was codon-optimized for expression in Yarrowia lipolytica (Example 14), thereby resulting in the synthesis of a synthetic, functional codon-optimized delta-8 desaturase designated as "D8SF" (SEQ ID NOs:112 and 113). Co-expression of the codon-optimized delta-8 desaturase of the invention in conjunction with a codon-optimized delta-9 elongase (derived from Isochrysis galbana (GenBank Accession No. 390174)) in Y. lipolytica enabled synthesis of 6.4% DGLA, with no co-synthesis of GLA (Example 16).
A number of expression constructs were then created to enable synthesis of various PUFAs in soybean, using the confirmed delta-8 desaturase sequence of Eg5, the Yarrowia lipolytica codon-optimized Isochrysis galbana delta-9 elongase or the Mortierella alpina elongase, the Mortierella alpina delta-5 desaturase, the Fusarium delta-15 desaturase, and the Saprolegnia diclina delta-17 desaturase and combinations thereof (Examples 17 through 22). Expression of these constructs resulted in production of up to about 29.9% DGLA and up to about 29.4% EPA (Examples 21 and 22 respectively).
The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), "μl" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "M" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "pmole" mean micromole(s), "g" means gram(s), "μg" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s).
Transformation and Cultivation of Yarrowia lilpolytica
Yarrowia lipolytica strains ATCC #20362, #76982 and #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were usually grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
Transformation of Yarrowia lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 μl of resuspended cells, and maintained at 39° C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C. for 2 to 3 days.
For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Supplements of uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMU" selection media, prepared with 20 g/L agar).
Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar.
Fatty Acid Analysis of Yarrowia lipolytica
For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.
For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Synthesis and Expression of a Codon-Optimized Delta-8 Desaturase Gene in Yarrowia lipolytica
In order to express the delta-8 desaturase gene of Euglena gracilis (SEQ ID NOs:5 and 6, GenBank Accession No. AAD45877) in Yarrowia lipolytica, the codon usage of the delta-8 desaturase gene was optimized for expression in Y. lipolytica. A codon-optimized delta-8 desaturase gene (designated "D8S-1", SEQ ID NO:48) was designed, based on the published sequence of Euglena gracilis (SEQ ID NO:5), according to the Yarrowia codon usage pattern (WO 2004/101753), the consensus sequence around the `ATG` translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)). In addition to the modification of the translation initiation site, 200 bp of the 1260 bp coding region were modified (15.9%). None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:6) except the second amino acid from `K` to `E` to add the NcoI site around the translation initiation codon.
In Vitro Synthesis of a Codon-Optimized delta-8 Desaturase Gene for Yarrowia
The codon-optimized delta-8 desaturase gene was synthesized as follows. First, thirteen pairs of oligonucleotides were designed to extend the entire length of the codon-optimized coding region of the E. gracilis delta-8 desaturase gene (e.g., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, corresponding to SEQ ID NOs:49-74). Each pair of sense (A) and anti-sense (B) oligonucleotides were complementary, with the exception of a 4 bp overhang at each 5'-end. Additionally, primers D8-1A, D8-3B, D8-7A, D8-9B and D8-13B (SEQ ID NOs:49, 54, 60, 65 and 74) also introduced NcoI, BgIII, Xho1, SacI and Not1 restriction sites, respectively, for subsequent subcloning.
Each oligonucleotide (100 ng) was phosphorylated at 37° C. for 1 hr in a volume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense and antisense oligonucleotides was mixed and annealed in a thermocycler using the following parameters: 95° C. (2 min), 85° C. (2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min), and 4° C. (15 min). Thus, D8-1A (SEQ ID NO:49) was annealed to D8-1B (SEQ ID NO:50) to produce the double-stranded product "D8-1AB". Similarly, D8-2A (SEQ ID NO:51) was annealed to D8-2B (SEQ ID NO:52) to produce the double-stranded product "D8-2AB", etc.
Four separate pools of annealed, double-stranded oligonucleotides were then ligated together, as shown below: (a) Pool 1: comprised D8-1AB, D8-2AB and D8-3AB; (b) Pool 2: comprised D8-4AB, D8-5AB and D8-6AB; (c) Pool 3: comprised D8-7AB, D8-8AB, and D8-9AB; and, (d) Pool 4; comprised D8-10AB, D8-11AB, D8-12AB and D8-13AB. Each pool of annealed oligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNA ligase and the ligation reaction was incubated overnight at 16° C.
The product of each ligation reaction was then used as template to amplify the designed DNA fragment by PCR. Specifically, using the ligated "Pool 1" mixture (i.e., D8-1AB, D8-2AB and D8-3AB) as template, and oligonucleotides D8-1F (SEQ ID NO:75) and D8-3R (SEQ ID NO:76) as primers, the first portion of the codon-optimized delta-8 desaturase gene was amplified by PCR. The PCR amplification was carried out in a 50 μl total volume, comprising PCR buffer containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.). Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 40 sec. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The 309 bp PCR fragment was subcloned into the pGEM-T easy vector (Promega) to generate pT8(1-3).
Using the ligated "Pool 2" mixture (i.e., D8-4AB, D8-5AB and D8-6AB) as the template, and oligonucleotides D8-4F (SEQ ID NO:77) and D8-6R (SEQ ID NO:78) as primers, the second portion of the codon-optimized delta-8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(4-6). Using the ligated "Pool 3" mixture (i.e., D8-7AB, D8-8AB and D8-9AB) as the template and oligonucleotides D8-7F (SEQ ID NO: 79) and D8-9R (SEQ ID NO:80) as primers, the third portion of the codon-optimized delta-8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(7-9). Finally, using the "Pool 4" ligation mixture (i.e., D8-10AB, D8-11AB, D8-12AB and D8-13AB) as template, and oligonucleotides D8-10F (SEQ ID NO: 81) and D8-13R (SEQ ID NO:82) as primers, the fourth portion of the codon-optimized delta-8 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT8(10-13).
E. coli was transformed separately with pT8(1-3), pT8(4-6), pT8(7-9) and pT8(10-13) and the plasmid DNA was isolated from ampicillin-resistant transformants. Plasmid DNA was purified and digested with the appropriate restriction endonucleases to liberate the 309 bp NcoI/BgIII fragment of pT8(1-3) (SEQ ID NO:83), the 321 bp BgIII/XhoI fragment of pT8(4-6) (SEQ ID NO:84), the 264 bp XhoI/SacI fragment of pT8(7-9) (SEQ ID NO:85) and the 369 bp Sac1/Not1 fragment of pT8(10-13) (SEQ ID NO:86). These fragments were then combined and directionally ligated together with Nco1/Not1 digested pY54PC (SEQ ID NO:115; WO2004/101757) to generate pDMW240 (FIG. 5A). This resulted in a synthetic delta-8 desaturase gene ("D8S-1", SEQ ID NO:48) in pDMW240.
Compared with the published delta-8 desaturase amino acid sequence (SEQ ID NO:6) of E. gracilis, the second amino acid of D8S-1 was changed from `K` to `E` in order to add the NcoI site around the translation initiation codon. Another version of the synthesized gene, with the exact amino acid sequence as the published E. gracilis delta-8 desaturase sequence SEQ ID NO:6), was constructed by in vitro mutagenesis (Stratagene, San Diego, Calif.) using pDMW240 as a template and oligonucleotides ODMW390 (SEQ ID NO:87) and ODMW391 (SEQ ID NO:88) as primers. The resulting plasmid was designated pDMW255 (FIG. 5B). The synthetic delta-8 desaturase gene in pDMW255 was designated as "D8S-2" and the amino acid sequence is exactly the same as the sequence depicted in SEQ ID NO:5.
Yarrowia lipolytica strainATCC #76982(Leu-) was transformed with pDMW240 and pDMW255, respectively, as described in the General Methods. Yeast containing the recombinant constructs pDMW240 and pDMW255 (i.e., containing D8S-1 and D8S-2 respectively) were grown in MM supplemented with EDA, 20:2(11,14). Specifically, single colonies of transformant Y. lipolytica containing either pDMW240 or pDMW255 were grown in 3 mL MM at 30° C. to an OD600˜1.0. For substrate feeding, 100 μl of cells were then subcultured in 3 mL MM containing 10 μg of EDA substrate for about 24 hr at 30° C. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
Neither transformant produced DGLA from EDA and thus D8S-1 and D8S-2 were not functional and could not desaturate EDA. The chimeric D8S-1::XPR terminator and D8S-2::XPR terminator genes are shown in SEQ ID NOs:89 and 90, respectively.
A three amino acid difference between the protein sequence of the delta 8-desaturase deposited in GenBank (Accession No. AAD45877) and in WO 00/34439 or Wallis et al. (Archives of Biochem. Biophys, 365:307-316 (1999)) (SEQ ID NO:7 herein) was found. Specifically, three amino acids appeared to be missing in GenBank Accession No. AAD45877. Using pDMW255 as template and ODMW392 (SEQ ID NO:91) and ODMW393 (SEQ ID NO:92) as primers, 9 bp were added into the synthetic D8S-2 gene by in vitro mutagenesis (Stratagene, San Diego, Calif.), thus producing a protein that was identical to the sequence described in WO 00/34439 and Wallis et al. (supra) (SEQ ID NO:7). The resulting plasmid was called pDMW261 (FIG. 5C). The synthetic delta-8 desaturase gene in pDMW261 was designated as "D8S-3" (SEQ ID NO:93). Following transformation of the pDMW261 construct into Yarrowia, a similar feeding experiment using EDA was conducted, as described above. No desaturation of EDA to DGLA was observed with D8S-3.
Euglena gracilis Growth Conditions, Lipid Profile and mRNA Isolation
Euglena gracilis was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). From 10 mL of actively growing culture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining: 1 g of sodium acetate, 1 g of beef extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of Bacto® Tryptone (0123-17-3, Difco Laboratories) and 2 g of Bacto® Yeast Extract (0127-17-9, Difco Laboratories) in 970 mL of water. After filter sterilizing, 30 mL of Soil-Water Supernatant (Catalog #15-3790, Carolina Biological Supply Company, Burlington, N.C.) was aseptically added to produce the final Eg medium. Euglena gracilis cultures were grown at 23° C. with a 16 hr light, 8 hr dark cycle for 2 weeks with no agitation.
After 2 weeks, 10 mL of culture was removed for lipid analysis and centrifuged at 1,800×g for 5 min. The pellet was washed once with water and re-centrifuged. The resulting pellet was dried for 5 min under vacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20° C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Catalog #U-99-A, Nu-Chek Prep, Inc.) and the resulting chromatogram is shown in FIG. 1.
The remaining 2 week culture (240 mL) was pelleted by centrifugation at 1,800×g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet using the RNA STAT-60® reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water). In this way, 1 mg of total RNA (2 mg/mL) was obtained from the pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 85 μg of mRNA was obtained.
cDNA Synthesis and PCR of Euglena gracilis Delta-8 Desaturase
cDNA was Synthesized from 765 ng of mRNA (Example 2) Using the SuperScript® Choice System for cDNA synthesis (Invitrogen® Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primer according to the manufacturer's protocol. The synthesized cDNA was dissolved in 20 μL of water.
The Euglena gracilis delta-8 desaturase was amplified from cDNA with oligonucleotide primers Eg5-1 (SEQ ID NO:8) and Eg3-3 (SEQ ID NO:9) using the conditions described below.
cDNA (1 μL) from the reaction described above was combined with 50 μmol of Eg5-1, 50 μmol of Eg5-1, 1 μL of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 μL of 10×PCR buffer (Invitrogen), 1.5 μL of MgCl2 (50 mM, Invitrogen), 0.5 μL of Taq polymerase (Invitrogen) and water to 50 μL. The reaction conditions were 94° C. for 3 min followed by 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 1 min. The PCR was finished at 72° C. for 7 min and then held at 4° C. The PCR reaction was analyzed by agarose gel electrophoresis on 5 μL and a DNA band with molecular weight around 1.3 kB was observed. The remaining 45 μL of product was separated by agarose gel electrophoresis and the DNA band was purified using the Zymoclean® Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol. The resulting DNA was cloned into the pGEM®-T Easy Vector (Promega) following the manufacturer's protocol. Multiple clones were sequenced using T7 (SEQ ID NO:10), M13-28Rev (SEQ ID NO:11), Eg3-2 (SEQ ID NO:12) and Eg5-2 (SEQ ID NO:13).
Thus, two classes of DNA sequences were obtained, Eg5 (SEQ ID NO:1) and Eg12 (SEQ ID NO:3), that differed in only a few bp. Translation of Eg5 and Eg12 gave rise to protein sequences that differed in only one amino acid, SEQ ID NO:2 and 4, respectively. Thus, the DNA and protein sequences for Eg5 are set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively; the DNA and protein sequences for Eg12 are set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.
Comparison of the Polypeptide Sequences Set Forth in SEQ ID NOs:2 and 4 to Published Euglena gracilis Delta-8 Desaturase Sequences
An alignment of the protein sequences set forth in SEQ ID NO:2 and SEQ ID NO:4 with the protein sequence from GenBank Accession No. AAD45877 (gi: 5639724) and with the published protein sequences of Wallis et al. (Archives of Biochem. Biophys., 365:307-316 (1999); WO 00/34439) is shown in FIG. 2.
Amino acids conserved among all 4 sequences are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences. The putative cytochrome b5 domain is underlined. A putative His box is shown in bold.
Clearly, there are significant differences between the sequences of this invention and those described previously. Specifically, the N-terminus has multiple amino acid changes. As compared to SEQ ID NO:2, the published amino acid sequences have an extra serine between L9 and P10 and amino acids from position T12-T16 are completely different (`TIDGT` to `QLMEQ`). These changes result from multiple insertions in the DNA sequence of the published sequence and this causes 3 shifts in frame in this region. These changes are only 10 amino acids away from the putative cytochrome b5 domain (`HPGG`).
In addition to this, there are seven other single amino acid changes (S50 to F, S67 to F, W177 to C, L203 to P, S244 to C, T278 to A, S323 to P) with the change at W177 being only 4 amino acids away from the second putative His box (`HNAHH`). Surprisingly, the published GenBank protein sequence is missing 3 amino acids (S20, A21, W22) as compared to that for either SEQ ID NO:2, SEQ ID NO:4 or WO 00/34439. The DNA sequence shown in WO 00/34439 codes for a protein that is identical to AAD45877 (i.e., missing these 3 amino acids) and not for the protein sequence described in WO 00/34439. Interestingly, the protein sequence set forth in SEQ ID NO:4 has a single amino acid change as compared to SEQ ID NO:2 (T278 to A). In Table 4 percent identities between the functional delta-8 desaturase protein sequence from Euglena gracilis claimed in this invention (SEQ ID NO:2) and the published sequences (SEQ ID NOs:6 and 7) are shown.
TABLE-US-00006 TABLE 4 Percent Identity Of The Amino Acid Sequences Of Delta-8 Desaturase From Euglena gracilis And Homologous Polypeptides From Euglena gracilis % Identity to % Identity to SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 2 95.5 96.2 * "% Identity" is defined as the percentage of amino acids that are identical between the two proteins.
Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Cloning the Euglena gracilis Delta-8 Desaturase into a Yeast Expression Vector
The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson et al., Gene, 110:119-22 (1992)) contains sequences from the Saccharomyces cerevisiae 2μ endogenous plasmid, a LEU2 selectable marker and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK+. The S. cerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was cloned between the SaclI and SpeI sites of pRS425 in the same way as described in Jia et al. (Physiological Genomics, 3:83-92 (2000)) to produce PGPD-425. A NotI site was introduced into the BamHI site of pGPD-425 thus producing a NotI site flanked by BamHI sites, thereby resulting in plasmid pY-75. Eg5 (SEQ ID NO:1) and Eg12 (SEQ ID NO:3) were released from the PGEM®-T Easy vectors described in Example 2 by digestion with NotI and cloned into the NotI site of pY-75 to produce pY89-5 and pY89-12, respectively. In this way, the delta-8 desaturases (i.e., Eg5 [SEQ ID NO:1] and Eg12 [SEQ ID NO:3]) were cloned behind a strong constitutive promoter for expression in S. cerevisiae. A map of pY89-5 is shown in FIG. 3A.
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean Expression Vector and Co-Expression with a Mortierella alpina Elongase
A starting plasmid pKS123 (WO 02/08269, the contents of which are hereby incorporated by reference) contains the hygromycin B phosphotransferase gene (HPT) [Gritz, L. and Davies, J. Gene 25:179-188 (1983)], flanked by the T7 promoter and transcription terminator (T7prom/hpt/T7term cassette), and a bacterial origin of replication (ori) for selection and replication in bacteria (e.g., E. coli). In addition, pKS123 also contains the hygromycin B phosphotransferase gene, flanked by the 35S promoter (Odell et al., Nature 313:810-812 (1985)) and NOS 3' transcription terminator (Depicker et al., J. Mol. Appl. Genet. 1:561:570 (1982)) (35S/hpt/NOS3' cassette) for selection in plants such as soybean. pKS123 also contains a NotI restriction site, flanked by the promoter for the α' subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3' transcription termination region of the phaseolin gene (Doyle, J. J. et al. J. Biol. Chem. 261:9228-9238 (1986)) thus allowing for strong tissue-specific expression in the seeds of soybean of genes cloned into the NotI site.
Vector pKR72 is a derivative of pKS123, wherein the HindIII fragment containing the β-conglycinin/NotI/phaseolin cassette has been inverted and a sequence (SEQ ID NO:14) containing SbfI, FseI and BsiWI restriction enzyme sites was introduced between the HindIII and BamHI sites in front of the β-conglycinin promoter.
The gene for the Mortierella alpina elongase was amplified from pRPB2 (WO 00/12720) using primers RPB2forward (SEQ ID NO:15) and RPB2reverse (SEQ ID NO:16) which were designed to introduce NotI restriction enzyme sites at both ends of the elongase. The resulting PCR fragment was digested with NotI and cloned into the NotI site of pKR72 to produce pKR324.
Vector pKS121 (WO 02/00904) contains a NotI site flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell 1:1079-1093 (1989)) and the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965 (KTi/NotI/KTi3' cassette). Vector pKR457 is a derivative of pKS121 where the restriction sites upstream and downstream of the Kti/NotI/Kti3' cassette have been altered through a number of subcloning steps. Vector pKR457 also contains the Soy albumin transcription terminator downstream of the Kti terminator to lengthen and strengthen termination of transcription. In pKR457, the BamHI site upstream of the Kti promoter in pKS121 was removed and a new sequence (SEQ ID NO:17) added containing a BsiWI, SalI, SbfI and HindIII site with the BsiWI site being closest the 5' end of the Kti promoter.
In addition, the SalI site downstream of the Kti terminator in pKS121 was removed and a new sequence (SEQ ID NO:18) was added containing a XbaI (closest to 3' end of Kti terminator), a BamHI site, the soy albumin transcription terminator sequence, a BsiWI site and another BamHI site.
The albumin transcription terminator was previously amplified from soy genomic DNA using primer oSalb-12 (SEQ ID NO:19; designed to introduce BamHI, XbaI and BsiWI sites at the 3' end of the terminator), and primer oSalb-13 (SEQ ID NO:20; designed to introduce BamHI sites at the 5' end of the terminator). After PCR, sites at ends were modified by sub-cloning through various intermediate vectors to finally produce the sequence shown in SEQ ID NO:5.
Eg5 (SEQ ID NO:1) was released from the PGEM®-T Easy by digestion with NotI and cloned into the NotI site of pKR457 to produce pKR680. Plasmid pKR680 was then digested with BsiWI and the fragment containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI site of pKR324 (WO 2004/071467) to produce pKR681. Thus, the delta-8 desaturase (Eg5; SEQ ID NO:1) could be co-expressed with the Mortierella alpina elongase behind strong, seed-specific promoters. A map of pKR681 is shown in FIG. 3B.
Isolation of Soybean Seed-Specific Promoters
The soybean annexin and BD30 promoters were isolated with the Universal GenomeWalker system (Clontech) according to its user manual (PT3042-1). To make soybean GenomeWalker libraries, samples of soybean genomic DNA were digested with DraI, EcoRV, PvuII and StuI separately for two hrs. After DNA purification, the digested genomic DNAs were ligated to the GenomeWalker adaptors AP1 and AP2.
Two gene specific primers (i.e., GSP1 [SEQ ID NO:21] and GSP2 [SEQ ID NO:22]) were designed for the soybean annexin gene based on the 5' annexin cDNA coding sequences available in an EST database (E.I. duPont de Nemours and Co., Inc., Wilmington, Del.).
The AP1 and the GSP1 primers were used in the 1st round PCR using the conditions defined in the GenomeWalker system protocol. Cycle conditions were 94° C. for 4 min; 94° C. for 2 sec and 72° C. for 3 min, 7 cycles; 94° C. for 2 sec and 67° C. for 3 min, 32 cycles; 67° C. for 4 min. The products from the first run PCR were diluted 50-fold. One microliter of the diluted products were used as templates for the 2nd PCR with primers AP2 and GSP2. Cycle conditions were 94° C. for 4 min; 94° C. for 2 sec and 72° C. for 3 min, 5 cycles; 94° C. for 2 sec and 67° C. for 3 min, 20 cycles; 67° C. for 3 min. A 2.1 kB genomic fragment was amplified and isolated from the EcoRV-digested GenomeWalker library. The genomic fragment was digested with BamH I and Sal I and cloned into Bluescript KS.sup.+ vector for sequencing. The DNA sequence of this 2012 bp soybean annexin promoter fragment is set forth in SEQ ID NO:29. Based on this sequence, two oligonucleotides with either BamH I or NotI sites at the 5' ends were designed to re-amplify the promoter (i.e., SEQ ID NOs:30 and 31).
Two gene specific primers (GSP3 [SEQ ID NO:23] and GSP4 [SEQ ID NO:24]) were designed to amplify the soybean BD30 promoter based on the 5' BD30 cDNA coding sequences in GenBank (Accession No. J05560). The AP1 and the GSP3 primers were used in the 1st round PCR using the same conditions defined in the GenomeWalker system protocol; however, the cycle conditions used for soybean annexin promoter did not work well for the soybean BD30 promoter. A modified touchdown PCR protocol was used, wherein cycle conditions were: 94° C. for 4 min; 94° C. for 2 sec and 74° C. for 3 min, 6 cycles in which annealing temperature drops 1° C. every cycle; 94° C. for 2 sec and 69° C. for 3 min, 32 cycles; 69° C. for 4 min. The products from the 1st run PCR were diluted 50-fold. One microliter of the diluted products were used as templates for the 2nd PCR with primers AP2 and GSP4. Cycle conditions were: 94° C. for 4 min; 94° C. for 2 sec and 74° C. for 3 min, 6 cycles in which annealing temperature drops 1° C. every cycle; 94° C. for 2 sec and 69° C. for 3 min, 20 cycles; 69° C. for 3 min. A 1.5 kB genomic fragment was amplified and isolated from the PvuII-digested GenomeWalker library. The genomic fragment was digested with BamHI and SalI and cloned into Bluescript KS.sup.+ vector for sequencing. DNA sequencing determined that this genomic fragment contained a 1408 bp soybean BD30 promoter sequence (SEQ ID NO:25). Based on the sequence of the cloned soybean BD30 promoter, two oligonucleotides with either BamHI or Not I sites at the 5' ends were designed to re-amplify the BD30 promoter (i.e., SEQ ID NOs:32 and 33).
The re-amplified annexin and BD30 promoter fragments (supra) were digested with BamHI and NotI, purified and cloned into the BamHI and NotI sites of plasmid pZBL115 to produce pJS88 and pJS89, respectively. The pZBL115 plasmid contains the origin of replication from pBR322, the bacterial HPT hygromycin resistance gene driven by a T7 promoter and T7 terminator, and a 35S promoter-HPT-Nos3' gene to serve as a hygromycin resistant plant selection marker. The M. alpina delta-6 desaturase gene was cloned into the NotI site of pJS88 and pJS89, in the sense orientation, to make plant expression cassettes pJS92 and pJS93, respectively.
Based on the sequences of the soybean Glycinin Gy1 promoter sequence in GenBank (Accession No. X15121), the oligonucleotides set forth in SEQ ID NOs:27 and 28 were designed to amplify the soybean Glycinin Gy1 promoter (SEQ ID NO:26), wherein the primers had either BamHI or NotI sites at the 5' ends. The amplified soybean glycinin Gy1 promoter fragment was digested with BamHI and NotI, purified and cloned into the BamHI and NotI sites of plasmid pZBL115 (supra) to produce pZBL117.
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean Expression Vector and Co-Expression with EPA Biosynthetic Genes (Delta-8 Desaturase and Delta-17 Desaturase)
Plasmid pKR325 was generated from pKR72 (Example 5) by digestion with HindIII to remove the βcon/NotI/Phas3' cassette. Plasmid pKR680 (Example 5) was digested with BsiWI and the fragment containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI site of pKR325 to produce pKR683.
The KTi/NotI/KTi3' cassette from pKS121 was PCR-amplified using primers oKTi5 (SEQ ID NO:34) and oKTi6 (SEQ ID NO:35), designed to introduce an XbaI and BsiWI site at both ends of the cassette. The resulting PCR fragment was subcloned into the XbaI site of the cloning vector pUC19 to produce plasmid pKR124, thus adding a PstI and SbfI site at the 3' end of the Kti transcription terminator.
The SalI fragment of pJS93 containing soy BD30 promoter (WO 01/68887) was combined with the SalI fragment of pUC19 to produce pKR227, thus adding a PstI and SbfI site at the 5' end of the BD30 promoter.
The BD30 3' transcription terminator was PCR-amplified from soy genomic DNA using primer oSBD30-1 (SEQ ID NO:36; designed to introduce an NotI site at the 5' end of the terminator) and primer oSBD30-2 (SEQ ID NO:37; designed to introduce a BsiWI site at the 3' end of the terminator). The resulting PCR fragment was subcloned into the intermediate cloning vector pCR-Script AMP SK(+) (Stratagene) according the manufacturer's protocol to produce plasmid pKR251r. The EcoRI/NotI fragment from pKR251r, containing the BD30 3' transcription terminator, was cloned into the EcoRI/NotI fragment of intermediate cloning vector pKR227 to produce pKR256.
The annexin promoter from pJS92 (Example 7) was released by BamHI digestion and the ends were filled. The resulting fragment was ligated into the filled BsiWI fragment from the vector backbone of pKR124 in a direction which added a PstI and SbfI site at the 5' end of the annexin promoter to produce pKR265. The annexin promoter was released from pKR265 by digestion with SbfI and NotI and was cloned into the SbfllNotI fragment of pKR256 (containing the BD30 3' transcription terminator, an ampicillin resistance gene and a bacterial ori region) to produce pKR268.
The gene for the Saprolegnia diclina delta-17 desaturase was released from pKS203 (Pereira et al., Biochem. J. 378:665-671 (2004)) by partial digestion with NotI, and was cloned into the NotI site of pKR268 to produce pKR271. In this way, the delta-17 desaturase was cloned as an expression cassette behind the annexin promoter with the BD30 transcription terminator.
Plasmid pKR271 was then digested with PstI and the fragment containing the Saprolegnia diclina delta-17 desaturase was cloned into the SbfI site of pKR683 to produce pKR685. In this way, the delta-8 desaturase could be co-expressed with the S. diclina delta-17 desaturase behind strong, seed-specific promoters. A map of pKR685 is shown in FIG. 4A.
Assembling EPA Biosynthetic Pathway Genes for Expression in Somatic Soybean Embryos and Soybean Seeds (Delta-6 Desaturase, Elongase and Delta-5 Desaturase)
The M. alpina delta-6 desaturase (U.S. Pat. No. 5,968,809), M. alpina elongase (WO 00/12720) and M. alpina delta-5 desaturase (U.S. Pat. No. 6,075,183) were cloned into plasmid pKR274 (FIG. 4B) behind strong, seed-specific promoters allowing for high expression of these genes in somatic soybean embryos and soybean seeds. All of these promoters exhibit strong tissue specific expression in the seeds of soybean. Plasmid pKR274 also contains the hygromycin B phosphotransferase gene (Gritz, L. and Davies, J. Gene 25:179-188 (1983)) cloned behind the T7 RNA polymerase promoter and followed by the T7 terminator (T7prom/HPT/T7term cassette) for selection of the plasmid on hygromycin B in certain strains of E. coli (e.g., NovaBlue(DE3) (Novagen, Madison, Wis.), a strain that is lysogenic for lambda DE3 and carries the T7 RNA polymerase gene under lacUV5 control). In addition, plasmid pKR274 contains a bacterial origin of replication (ori) functional in E. coli from the vector pSP72 (Stratagene).
More specifically, the delta-6 desaturase was cloned behind the promoter for the α' subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) followed by the 3' transcription termination region of the phaseolin gene (Doyle, J. J. et al. J. Biol. Chem. 261:9228-9238 (1986)) (βcon/Mad6/Phas3' cassette).
The delta-5 desaturase was cloned behind the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell 1:1079-1093 (1989)), followed by the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965 (KTi/Mad5/KTi3' cassette).
The elongase was cloned behind the glycinin Gy1 promoter followed by the pea leguminA2 3' termination region (Gy1/Maelo/legA2 cassette).
The gene for the M. alpina delta-6 desaturase was PCR-amplified from pCGR5 (U.S. Pat. No. 5,968,809) using primers oCGR5-1 (SEQ ID NO:38) and oCGR5-2 (SEQ ID NO:39), which were designed to introduce NotI restriction enzyme sites at both ends of the delta-6 desaturase and an NcoI site at the start codon of the reading frame for the enzyme. The resulting PCR fragment was subcloned into the intermediate cloning vector pCR-Script AMP SK(+) (Stratagene) according the manufacturer's protocol to produce plasmid pKR159. The NotI fragment of pKR159, containing the M. alpina delta-6 desaturase gene, was cloned into NotI site of pZBL117 (Example 7) in the sense orientation to produce plant expression cassette pZBL119.
Vector pKR197 was constructed by combining the AscI fragment from plasmid pKS102 (WO 02/00905), containing the T7prom/hpt/T7term cassette and bacterial ori, with the Asci fragment of plasmid pKR72 (Example 5), containing the βcon/NotI/Phas cassette. Plasmid pKR159 was digested with NotI to release the M. alpina delta-6 desaturase, which was, in turn, cloned into the NotI site of the soybean expression vector pKR197 to produce pKR269.
The glycinin Gy1 promoter was amplified from pZBL119 using primer oSGly-1 (SEQ ID NO:40; designed to introduce an SbfI/PstI site at the 5' end of the promoter) and primer oSGly-2 (SEQ ID NO:41; designed to introduce a NotI site at the 3' end of the promoter). The resulting PCR fragment was subcloned into the intermediate cloning vector pCR-Script AMP SK(+) (Stratagene) according to the manufacturer's protocol to produce plasmid pSGly12.
The legA2 promoter was amplified from pea genomic DNA using primer LegPro5' (SEQ ID NO:42; designed to introduce XbaI and BsiWI sites at the 5' end of the promoter) and primer LegPro3' (SEQ ID NO:43; designed to introduce a NotI site at the 3' end of the promoter). The legA2 transcription terminator was amplified from pea genomic DNA using primer LegTerm5' (SEQ ID NO:44; designed to introduce NotI site at the 5' end of the terminator) and primer LegTerm3' (SEQ ID NO:45; designed to introduce BsiWI and XbaI sites at the 3' end of the terminator). The resulting PCR fragments were then combined and re-amplified using primers LegPro5' and LegTerm3', thus forming a legA2/NotI/legA23' cassette. The legA2/NotI/legA23' cassette PCR fragment was subcloned into the intermediate cloning vector pCR-Script AMP SK(+) (Stratagene) according to the manufacturer's protocol to produce plasmid pKR140.
Plasmid pKR142 was constructed by cloning the BsiWI fragment of pKR140 (containing the legA2/NotI/legA23' cassette) into the BsiWI site of pKR124 (containing a bacterial ori and ampicillin resistance gene). The PstI/NotI fragment from plasmid pKR142 was then combined with the PstI/NotI fragment of plasmid pSGly12 (containing the glycininGy1 promoter) to produce pKR263. The gene for the M. alpina delta-5 desaturase was amplified from pCGR4 (U.S. Pat. No. 6,075,183) using primers CGR4foward (SEQ ID NO:46) and CGR4reverse (SEQ ID NO:47) which were designed to introduce NotI restriction enzyme sites at both ends of the desaturase. The resulting PCR fragment was digested with NotI and cloned into the NotI site of vector pKR124 (Example 6) to produce pKR136.
The NotI fragment containing the M. alpina elongase (Example 5) was cloned into the NotI site of vector pKR263 to produce pKR270. The Gy1/Maelo/legA2 cassette was released from plasmid pKR270 by digestion with BsiWI and SbfI and was cloned into the BsiWI/SbfI sites of plasmid pKR269 (containing the delta-6 desaturase, the T7prom/hpt/T7term cassette and the bacterial ori region). This was designated as plasmid pKR272. The KTi/Mad5/KTi3' cassette, released from pKR136 by digestion with BsiWI, was then cloned into the BsiWI site of pKR272 to produce pKR274 (FIG. 4B).
Assembling EPA Biosynthetic Pathway Genes for Expression in Somatic Soybean Embryos and Soybean Seeds (Delta-17 Desaturase and Delta-5 Desaturase)
In a manner similar to that described in Example 9, the delta-17 desaturase from S. diclina could be cloned into a soy expression vector along with the delta-5 desaturase from M. alpina. The annexin/delta17/BD30 cassette of pKR271 could be released by digestion with a suitable restriction enzyme such as PstI and cloned into a soy expression vector already carrying the M. alpina delta-5 desaturase behind a suitable promoter and a suitable selection marker such as hygromycin. The M. alpina delta-5 desaturase could be part of any suitable expression cassette described here. For instance, the NotI fragment containing the M. alpina delta-5 desaturase described above could be cloned into the NotI site of the Gy1/NotI/legA2 cassette of pKR263. This Gy1/delta5/legA2 cassette could then be cloned into a vector containing a suitable selectable marker for soy transformation. Such a vector could be co-transformed into soy with pKR681 (Example 6) and transformants expression genes from both plasmids selected. In this way, EPA could be produced using the delta-8 pathway independent of a delta-6 desaturase.
Functional Analysis of the Euglena gracilis Delta-8 Desaturase in Saccharomyces cerevisiae
Plasmids pY89-5 (comprising the Eg5 sequence; see FIG. 3A and ATCC PTA-6048), pY89-12 (identical to pY89-5, with the exception that the Eg12 sequence was inserted instead of Eg5) and pY-75 (Example 5, negative control cloning vector [lacking Eg5 or Eg12]) were transformed into Saccharomyces cerevisiae BY4741 (ATCC #201388) using standard lithium acetate transformation procedures. Transformants were selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformants from each plate were inoculated into 2 mL of DOB medium supplemented with CSM-leu (Qbiogene) and grown for 1 day at 30° C., after which 0.5 mL was transferred to the same medium supplemented with either EDA or EtrA to 1 mM. These were incubated overnight at 30° C., 250 rpm, pellets were obtained by centrifugation and dried under vacuum. Pellets were transesterified with 50 μL of TMSH and analyzed by GC as described in Example 1. Two clones for pY-75 (i.e., clones 75-1 and 75-2) and pY89-5 (i.e., clones 5-6-1 and 5-6-2) were analyzed, while two sets of clones for pY89-12 (i.e., clones 12-8-1, 12-8-2, 12-9-1 and 12-9-2) from two independent transformations were analyzed.
The lipid profile obtained by GC analysis of clones fed EDA are shown in Table 5; and the lipid profile obtained by GC analysis of clones fed EtrA are shown in Table 6.
TABLE-US-00007 TABLE 5 20:3 %20:2 Clone 16:0 16:1 18:0 18:1 20:2 (8, 11, 14) Converted 75-1 14 32 5 38 10 0 0 75-2 14 31 5 41 9 0 0 5-6-1 14 32 6 40 6 2 24 5-6-2 14 30 6 41 7 2 19 12-8-1 14 30 6 41 9 1 7 12-8-2 14 32 5 41 8 1 8 12-9-1 14 31 5 40 9 1 8 12-9-2 14 32 5 41 8 1 7
TABLE-US-00008 TABLE 6 20:3 20:4 %20:3 Clone 16:0 16:1 18:0 18:1 (11, 14, 17) (8, 11, 14, 17) Converted 75-1 12 25 5 33 24 0 0 75-2 12 24 5 36 22 1 5 5-6-1 13 25 6 34 15 7 32 5-6-2 13 24 6 34 17 6 27 12-8-1 12 24 5 34 22 2 8 12-8-2 12 25 5 35 20 2 9 12-9-1 12 24 5 34 22 2 9 12-9-2 12 25 6 35 20 2 9
The data in Tables 4 and 5 showed that the cloned Euglena delta-8 desaturase is able to desaturate EDA and EtrA. The sequence set forth in SEQ ID NO:4 has one amino acid change compared to the sequence set forth in SEQ ID NO:2 and has reduced delta-8 desaturase activity.
The small amount of 20:4(8,11,14,17) generated by clone 75-2 in Table 6 had a slightly different retention time than a standard for 20:4(8,11,14,17). This peak was more likely a small amount of a different fatty acid generated by the wild-type yeast in that experiment.
Cloning Other Delta-8 Desaturases or Elongases into Soybean Expression Vectors
In addition to the delta-8 desaturase from Euglena gracilis, other delta-8 desaturases can be cloned into the soybean expression vectors such as those described in Example 6 and Example 8. For instance, a suitable delta-8 desaturase from an organism other than Euglena gracilis can be cloned using methods similar to, but not limited to, the methods described in Example 2 and Example 3. PCR primers designed to introduce NotI sites at the 5' and 3' ends of the delta-8 desaturase can be used to amplify the gene. The resulting PCR product can then be digested with NotI and cloned into a soybean expression vector such as pKR457. Further sub-cloning into other vectors as described in Example 6 or Example 8 would yield vectors suitable for expression and co-expression of the delta-8 desaturase in soybean.
Likewise, in addition to the elongase from Mortierella alpina, other elongases can be cloned into the soybean expression vectors such as those described in Example 6 and Example 8. Specifically, elongases with specificity for linoleic acid or alpha-linolenic acid such as that from Isochrysis galbana (WO 2002/077213) can be used. For instance, a suitable elongase from an organism other than Mortierella alpina can be cloned using methods similar to, but limited not to, the methods described in Example 2 and Example 3. PCR primers designed to introduce NotI sites at the 5' and 3' ends of the elongase can be used to amplify the gene. The resulting PCR product can then be digested with NotI and cloned into soybean expression vectors such as pKR72 or pKR263. Further sub-cloning into other vectors as described in Example 6 or Example 8 would yield vectors suitable for expression and co-expression of the elongase in soybean.
Transformation of Somatic Soybean Embryo Cultures
Culture Conditions: Soybean embryogenic suspension cultures (cv. Jack) can be maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).
Soybean embryogenic suspension cultures can be transformed with the plasmids and DNA fragments described earlier by the method of particle gun bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations.
Soybean Embryogenic Suspension Culture Initiation: Soybean cultures are initiated twice each month with 5-7 days between each initiation.
Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm were placed on individual microscope slides. The small end of the seed is cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment: Either an Intact Plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene can be used for bombardment. Fragments from plasmids such pKR274 and pKR685 or pKR681 and/or other expression plasmids can be obtained by gel isolation of digested plasmids. In each case, 100 μg of plasmid DNA can be used in 0.5 mL of the specific enzyme mix described below. Plasmids could be digested with AscI (100 units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH 7.9), 100 μg/mL BSA, and 5 mM beta-mercaptoethanol at 37° C. for 1.5 hr. The resulting DNA fragments could be separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing EPA biosynthetic genes could be cut from the agarose gel. DNA can be purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol. Alternatively, whole plasmids or a combination of whole plasmid with fragment could be used.
A 50 μl aliquot of sterile distilled water containing 3 mg of gold particles can be added to 5 μl of a 1 μg/μl DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 μl 2.5M CaCl2 and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 μl 100% ethanol, the pellet is suspended by sonication in 40 μl of 100% ethanol. Five μl of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μl aliquot contained approximately 0.375 mg gold particles per bombardment (i.e., per disk).
Tissue Preparation and Bombardment with DNA: Approximately 150-200 mg of 7 day old embryonic suspension cultures are placed in an empty, sterile 60×15 mm petri dish and the dish is covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber is evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.
Selection of Transformed Embryos: Transformed embryos are selected either using hygromycin (when the hygromycin phosphotransferase, HPT, gene was used as the selectable marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene was used as the selectable marker). Specifically, following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing either a selection agent of 30 mg/L hygromycin or a selection agent of 100 ng/mL chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants: In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.
Embryo Maturation: Embryos can be cultured for 4-6 weeks at 26° C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 μE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos can be removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 11. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, could be screened at this stage. This would include (but not be limited to) alterations in: fatty acid profile, protein profile and content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.
Embryo Desiccation and Germination: Matured individual embryos can be desiccated by placing them into an empty, small petri dish (35×10 mm) for approximately 4-7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos can be planted into SB71-4 medium where they are left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then planted in Redi-Earth in 24-cell pack trays, covered with clear plastic domes. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets look hardy they are transplanted to 10'' pots of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds can be harvested, chipped and analyzed for fatty acids as described above.
TABLE-US-00009 SB 196 - FN Lite liquid proliferation medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO3 2.83 g (NH4)2SO4 0.463 g Asparagine 1.0 g Sucrose (1%) 10 g pH 5.8
TABLE-US-00010 FN Lite Stock Solutions Stock # 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na2 EDTA* 3.724 g 1.862 g FeSO4--7H2O 2.784 g 1.392 g *Add first, dissolve in dark bottle while stirring 2 MS Sulfate 100x stock MgSO4--7H2O 37.0 g 18.5 g MnSO4--H2O 1.69 g 0.845 g ZnSO4--7H2O 0.86 g 0.43 g CuSO4--5H2O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl2--2H2O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl2--6H2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x Stock KH2PO4 18.5 g 9.25 g H3BO3 0.62 g 0.31 g Na2MoO4--2H2O 0.025 g 0.0125 g SB1 solid medium (per liter) 1 pkg. MS salts (Catalog #11117-066, Gibco/BRL) 1 mL B5 vitamins 1000X stock 31.5 g sucrose 2 mL 2,4-D (20 mg/L final concentration) pH 5.7 8 g TC agar SB 166 solid medium (per liter) 1 pkg. MS salts (Catalog #11117-066, Gibco/BRL) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate 5 g activated charcoal pH 5.7 2 g gelrite SB 103 solid medium (per liter) 1 pkg. MS salts (Catalog #11117-066, Gibco/BRL) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate pH 5.7 2 g gelrite SB 71-4 solid medium (per liter) 1 bottle Gamborg's B5 salts with sucrose (Catalog #21153-036, Gibco/BRL) pH 5.7 5 g TC agar 2,4-D stock: obtained premade from Phytotech, Catalog #D 295; concentration is 1 mg/mL B5 Vitamins Stock (per 100 mL; store aliquots at -20° C.) 10 g myo-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine *If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate. Chlorsulfuron Stock 1 mg/mL in 0.01 N ammonium hydroxide
To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos, which produce secondary embryos, are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. Nature (London) 327:70-73 (1987); U.S. Pat. No. 4,945,050). A DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene which can be used to facilitate soybean transformation is a recombinant DNA construct composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. Nature 313:810-812 (1985)), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. Gene 25:179-188 (1983)) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptide and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation is then agitated for three min, spun in a microfuge for 10 sec and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one sec each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Further Modification of the Delta-8 Desaturase Gene Codon-Optimized for Yarrowia lipolytica
The amino acid sequence of the synthetic codon-optimized D8S-3 gene in pDMW261 (Example 1) was corrected according to the amino acid sequence of the functional Euglena delta-8 desaturase (SEQ ID NOs:1 and 2). Using pDMW261 as a template and oligonucleotides ODMW404 (SEQ ID NO:94) and D8-13R (SEQ ID NO:36), the DNA fragment encoding the synthetic D8S-3 desaturase gene was amplified. The resulting PCR fragment was purified with Bio101's Geneclean kit and subsequently digested with Kpn1 and Not1 (primer ODMW404 introduced a KpnI site while primer D8-13R introduced a NotI site). The Kpn1/Not1 fragment (SEQ ID NO:95) was cloned into Kpn1/Not1 digested pKUNFmKF2 (FIG. 5D; SEQ ID NO:116) to produce pDMW277 (FIG. 6A).
Oligonucleotides YL521 (SEQ ID NO:96) and YL522 (SEQ ID NO:97), which were designed to amplify and correct the 5' end of the D8S-3 gene, were used as primers in another PCR reaction where pDMW277 was used as the template. The primers introduced into the PCR fragment a Nco1 site and BgIII site at its 5' and 3' ends, respectively. The 318 bp PCR product was purified with Bio101's GeneClean kit and subsequently digested with Nco1 and BgIII. The digested fragment, along with the 954 bp BgIII/NotI fragment from pDMW277, was used to exchange the NcoI/NotI fragment of pZF5T-PPC (FIG. 6B; SEQ ID NO:117) to form pDMW287 (FIG. 6C). In addition to correcting the 5' end of the synthetic D8S-3 gene, this cloning reaction also placed the synthetic delta-8 desaturase gene under control of the Yarrowia lipolytica fructose-bisphosphate aldolase promoter containing a Yarrowia intron (FBAIN; SEQ ID NO:114; see WO 2005/049805).
The first reaction in a final series of site-directed mutagenesis reactions was then performed on pDMW287. The first set of primers, YL525 (SEQ ID NO:98) and YL526 (SEQ ID NO:99), was designed to correct amino acid from F to S (position #50) of the synthetic D8S-3 gene in pDMW287. The plasmid resulting from this mutagenesis reaction then became the template for the next site-directed mutagenesis reaction with YL527 (SEQ ID NO:100) and YL528 (SEQ ID NO:101) as primers. These primers were designed to correct the amino acid from F to S (position #67) of the D8S-3 gene and resulted in creation of plasmid pDMW287/YL527.
To complete the sequence corrections within the second quarter of the gene, the following reactions were carried out concurrently with the mutations on the first quarter of the gene. Using pDMW287 as template and oligonucleotides YL529 (SEQ ID NO:102) and YL530 (SEQ ID NO:103) as primers, an in vitro mutagenesis reaction was carried out to correct the amino acid from C to W (position #177) of the synthetic D8S-3 gene. The product (i.e., pDMW287NY529) of this mutagenesis reaction was used as the template in the following reaction using primers YL531 (SEQ ID NO:104) and YL532 (SEQ ID NO:105) to correct the amino acid from P to L (position #213). The product of this reaction was called pDMW287NYL529-31.
Concurrently with the mutations on the first and second quarter of the gene, reactions were similarly carried out on the 3' end of the gene. Each subsequent mutagenesis reaction used the plasmid product from the preceding reaction. Primers YL533 (SEQ ID NO:106) and YL534 (SEQ ID NO:107) were used on pDMW287 to correct the amino acid from C to S (position #244) to create pDMW287NYL533. Primers YL535 (SEQ ID NO:108) and YL536 (SEQ ID NO:109) were used to correct the amino acid A to T (position #280) in the synthetic D8S-3 gene of pDMW287/YL533 to form pDMW287/YL533-5. Finally, the amino acid P at position of #333 was corrected to S in the synthetic D8S-3 gene using pDMW287/YL533-5 as the template and YL537 (SEQ ID NO:110) and YL538 (SEQ ID NO:111) as primers. The resulting plasmid was named pDMW287/YL533-5-7.
The BgIII/XhoI fragment of pDMW287/YL529-31, and the XhoI/NotI fragment of pDMW287/YL533-5-7 was used to change the BgIII/NotI fragment of pDMW287/YL257 to produce pDMW287F (FIG. 6D) containing the completely corrected synthetic delta-8 desaturase gene, designated "D8SF" and set forth in SEQ ID NO:112. SEQ ID NO:113 sets forth the amino acid sequence encoded by nucleotides 2-1270 of SEQ ID NO:112, which is essentially the same as the sequence set forth in SEQ ID NO:2, except for an additional valine following the start methionine.
Synthesis and Functional Expression of a Codon-Optimized Delta-9 Elongase Gene in Yarrowia lipolytica
In order to express the delta-9 elongase/delta-8 desaturase pathway in Yarrowia lipolytica, it was necessary to obtain an appropriate delta-9 elongase that could be co-expressed with the synthetic codon-optimized delta-8 desaturase from Example 14. Thus, the codon usage of the delta-9 elongase gene of Isochrysis galbana (GenBank Accession No. AF390174) was optimized for expression in Y. lipolytica. According to the Yarrowia codon usage pattern, the consensus sequence around the ATG translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)), a codon-optimized delta-9 elongase gene was designed (SEQ ID NO:118), based on the DNA sequence of Isochrysis galbana; SEQ ID NO:119. In addition to modification of the translation initiation site, 126 bp of the 792 bp coding region were modified, and 123 codons were optimized. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (GenBank Accession No. AF390174; SEQ ID NO:120).
In Vitro Synthesis of a Codon-Optimized Delta-9 Elongase Gene for Yarrowia
The method used to synthesize the codon-optimized delta-9 elongase gene was the same as that used for synthesis of the delta-8 desaturase gene (Example 1). First, eight pairs of oligonucleotides were designed to extend the entire length of the codon-optimized coding region of the I. galbana delta-9 elongase gene (e.g., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A, IL3-8B, corresponding to SEQ ID NOs:121-136). Each pair of sense (A) and anti-sense (B) oligonucleotides were complementary, with the exception of a 4 bp overhang at each 5'-end. Additionally, primers IL3-1F, IL3-4R, IL3-5F and IL3-8R (SEQ ID NOs:137-140) also introduced NcoI, PstI, PstI and Not1 restriction sites, respectively, for subsequent subcloning.
Each oligonucleotide (100 ng) was phosphorylated at 37° C. for 1 hr in a volume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense and antisense oligonucleotides was mixed and annealed in a thermocycler using the following parameters: 95° C. (2 min), 85° C. (2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15 min). Thus, IL3-1A (SEQ ID NO:121) was annealed to IL3-1B (SEQ ID NO:122) to produce the double-stranded product "IL3-1AB". Similarly, IL3-2A (SEQ ID NO:123) was annealed to IL3-2B (SEQ ID NO:124) to produce the double-stranded product "IL3-2AB", etc.
Two separate pools of annealed, double-stranded oligonucleotides were then ligated together, as shown below: Pool 1 (comprising IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB); and, Pool 2 (comprising IL3-5AB, IL3-6AB, IL3-7AB and IL3-8AB). Each pool of annealed oligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNA ligase and the ligation reaction was incubated overnight at 16° C.
The product of each ligation reaction was then used as template to amplify the designed DNA fragment by PCR. Specifically, using the ligated "Pool 1" mixture (i.e., IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB) as template, and oligonucleotides IL3-1F and IL3-4R (SEQ ID NOs:137 and 138) as primers, the first portion of the codon-optimized delta-9 elongase gene was amplified by PCR (as described in Example 1). The 417 bp PCR fragment was subcloned into the PGEM-T easy vector (Promega) to generate pT9(1-4).
Using the ligated "Pool 2" mixture (i.e. IL3-5AB, IL3-6AB, IL3-7AB and IL3-8AB) as the template, and oligonucleotides IL3-5F and IL3-8R (SEQ ID NOs:139 and 140) as primers, the second portion of the codon-optimized delta-9 elongase gene was amplified similarly by PCR and cloned into the pGEM-T-easy vector to generate pT9(5-8).
E. coli was transformed separately with pT9(1-4) and pT9(5-8) and the plasmid DNA was isolated from ampicillin-resistant transformants. Plasmid DNA was purified and digested with the appropriate restriction endonucleases to liberate the 417 bp NcoI/PstI fragment of pT9(1-4) (SEQ ID NO:141) and the 377 bp PstI/NotI fragment of pT9(5-8) (SEQ ID NO:142). These two fragments were then combined and directionally ligated together with NcoI/NotI digested pZUF17 (SEQ ID NO:143; FIG. 7A) to generate pDMW237 (FIG. 7B; SEQ ID NO:144). The DNA sequence of the resulting synthetic delta-9 elongase gene ("IgD9e") in pDMW237 was exactly the same as the originally designed codon-optimized gene (i.e., SEQ ID NO:118) for Yarrowia.
Generation of Y. lipolytica Strain Y2031 (A Ura-Derivative of ATCC #20362)
Strain Y2031 was generated by integration of the TEF::Y.Δ12::Pex20 chimeric gene of plasmid pKUNT2 (FIG. 7C) into the Ura3 gene locus of Yarrowia lipolytica ATCC #20362, to thereby generate the Ura-genotype of strain Y2031.
Specifically, plasmid pKUNT2 contained the following components:
TABLE-US-00011 TABLE 7 Description of Plasmid pKUNT2 (SEQ ID NO: 145) RE Sites And Nucleotides Within SEQ ID Description Of NO: 145 Fragment And Chimeric Gene Components AscI/BsiWI 784 bp 5' part of Yarrowia Ura3 gene (3225-3015) (GenBank Accession No. AJ306421) SphI/PacI 516 bp 3' part of Yarrowia Ura3 gene (5933-13) (GenBank Accession No. AJ306421) EcoRI/BsiWI TEF::Y.Δ12::Pex20, comprising: (6380-8629) TEF: TEF promoter (GenBank Accession No. AF054508) Y.Δ12: Yarrowia delta-12 desaturase gene (SEQ ID NO: 146; see also WO 2004/104167) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613)
The pKUNT2 plasmid was digested with AscI/SphI, and then used for transformation of wild type Y. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media plates and maintained at 30° C. for 2 to 3 days. Specifically, FOA selection media comprised: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura-strains. Single colonies (5) of Ura-strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days.
The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC. GC analyses showed that there were about 45% LA in two Ura--strains (strains #2 and #3), compared to about 20% LA in the wild type ATCC #20362. Transformant strain #2 was designated as strain "Y2031".
Expression of the Codon-Optimized Delta-9 Elongase Gene in Y. lipolytica
Construct pDMW237 (comprising the chimeric FBAIN::IgD9e::Pex20 gene) was transformed into Yarrowia lipolytica strain Y2031, as described in the General Methods. Three transformants of Y2031 with pDMW237 were grown individually in MM media for two days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
The GC results showed that there were about 7.1%, 7.3% and 7.4% EDA produced in these transformants with pDMW237. These data demonstrated that the synthetic IgD9e could convert the C18:2 to EDA. The "percent (%) substrate conversion" or "conversion efficiency" of the codon-optimized gene was determined to be about 13%, wherein the conversion efficiency was calculated according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it. This term refers to the efficiency by which the particular enzyme can convert substrate to product.
Delta-9 Elongase/Delta-8 Desaturase Pathway Expression to Produce DGLA in Yarrowia lipolytica
The present Example describes DGLA biosynthesis and accumulation in Yarrowia lipolytica that was transformed to express the delta-9 elongase/delta-8 desaturase pathway. Thus, this required co-synthesis of the synthetic codon-optimized delta-9 elongase (SEQ ID NO:118; Example 15) and the synthetic codon-optimized delta-8 desaturase (SEQ ID NO:112; Example 14).
Specifically, the ClaI/PacI fragment comprising the chimeric FBAIN::D8SF::Pex16 gene of construct pDMW287F (FIG. 6D) was inserted into the ClaI/PacI sites of pDMW237 (FIG. 7B) to generate the construct pDMW297 (FIG. 7D). Thus, plasmid pDMW297 contained the following components:
TABLE-US-00012 TABLE 8 Description of Plasmid pDMW297(SEQ ID NO: 148) RE Sites And Nucleotides Within SEQ ID Description Of NO: 148 Fragment And Chimeric Gene Components EcoRI/ClaI ARS18 sequence (GenBank Accession No. A17608) (9053-10448) ClaI/PacI FBAIN::Δ8S::Pex16, comprising: (1-2590) FBAIN: FBAIN promoter (SEQ ID NO: 114) Δ8S: codon-optimized delta-8 desaturase gene (SEQ ID NO: 112), derived from Euglena gracilis (GenBank Accession No. AF139720) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene (GenBank Accession No. U75433) PacI/SalI Yarrowia Ura3 gene (GenBank Accession No. (2590-4082) AJ306421) SalI/BsiWI FBAIN::Δ9ES::Pex120, comprising: (4082-6257) FBAIN: FBAIN promoter (SEQ ID NO: 114) Δ9ES: codon-optimized delta-9 elongase gene (SEQ ID NO: 118), derived from Isochrysis galbana (GenBank Accession No. 390174) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613)
The pDMW297 plasmid was then used for transformation of strain Y2031 (Example 15) according to the General Methods. The transformant cells were plated onto MM selection media plates and maintained at 30° C. for 2 to 3 days. A total of 8 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
GC analyses showed that DGLA was produced in all of the transformants analyzed. One strain produced about 3.2%, 4 strains produced 4.3-4.5%, two strains produced 5.5-5.8% and one strain produced 6.4% DGLA (designated herein as strain "Y0489"). The "percent (%) substrate conversion" of the codon-optimized D8SF gene in strain Y0489 was determined to be 75% (using the formula of Example 15).
It will be obvious to one of skill in the art that other chimeric genes could be co-expressed with the D8SF and IgD9e genes in engineered Yarrowia to enable production of various other PUFAs. For example, in addition to the codon-optimized delta-9 elongase and delta-8 desaturase genes, one could readily express: (1) a delta-15 desaturase to enable production of ETA; (2) a delta-5 desaturase to enable production of ARA; (3) a delta-17 desaturase to enable production of ETA; (4) a delta-5 desaturase and a delta-17 desaturase to enable production of EPA; (6) a delta-5 desaturase, a delta-17 desaturase and a C20/22 elongase to enable production of DPA; or (7) a delta-5 desaturase, a delta-17 desaturase, a C20/22 elongase and a delta-4 desaturase to enable production of DHA (FIG. 9).
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean Expression Vector and Co-Expression with an Isochrysis galbana Elongase
The gene for the Isochrysis galbana elongase was amplified from pDMW237 (FIG. 7B; SEQ ID NO:144) using primers olGsel1-1 (SEQ ID NO:149) and olGsel1-2 (SEQ ID NO:150) which were designed to introduce NotI restriction enzyme sites at both ends of the elongase. The resulting PCR fragment was digested with NotI and cloned into the NotI site of pKR72 to give pKR607.
Plasmid pKR680 was digested with BsiWI and the fragment containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI site of pKR607 to give pKR682. Thus, the delta-8 desaturase (Eg5; SEQ ID NO:1) could be co-expressed with the Isochrysis galbana elongase behind strong, seed-specific promoters. A map of pKR682 is shown in FIG. 8A.
Assembling EPA Biosynthetic Pathway Genes with the Euglena gracilis Delta-8 Desaturase and Isochrysis galbana Elongase for Expression in Somatic Soybean Embryos and Soybean Seeds
An soybean expression vector (pKR786) containing the Euglena gracilis delta-8 desaturase, the Isochrysis galbana delta-9 elongase and the Mortierella alpina delta-5 desaturase (all under control of strong seed specific promoters) was constructed in the following way.
Through a number of sub-cloning steps, a sequence of DNA (SEQ ID NO:151) was effectively added into the SmaI site of vector pKR287 (WO 2004/071467 A2) to produce pKR767. In this way, a SbfI restriction site was added to the 3' end of the leg1A transcription terminator of the Gy1/Mad5/legA2 cassette.
The AscI fragment of pKR682 was cloned into the AscI site of pKR277 (WO 2004/071467 A2) to produce pKR769.
The Gy1/Mad5/legA2 cassette was released from pKR767 by digestion with SbfI and the resulting fragment was cloned into the SbfI site of pKR769 to produce pKR786. A map of pKR786 is shown in FIG. 8B.
Cloning the Fusarium Delta-15 Desaturase into a Soybean Expression Vector and Co-Expression with EPA Biosynthetic Genes (Delta-15 Desaturase, Delta-17 Desaturase)
The Kti3 promoter:Fm Δ15 desaturase ORF:Kti3 terminator cassette was released from plasmid pKR578 (WO 2005/047479) by digestion with BsiWI and was cloned into the BsiWI site of plasmid pKR226 (WO 2004/071467 A2), containing the ALS gene for selection, the T7prom/hpt/T7term cassette and the bacterial ori region, to produce pKR667.
Plasmid pKR271 was digested with PstI and the fragment containing the Saprolegnia diclina delta-17 desaturase was cloned into the SbfI site of pKR667 to produce pKR669. In this way, the delta-15 desaturase could be co-expressed with the Saprolegnia diclina delta-17 desaturase behind strong, seed-specific promoters. A map of pKR669 is shown in FIG. 8C.
Analysis of Somatic Soy Embryos Containing the Euglena gracilis Delta-8 Desaturase and Mortierella alpina Elongase Genes (pKR681)
Mature somatic soybean embryos are a good model for zygotic embryos. While in the globular embryo state in liquid culture, somatic soybean embryos contain very low amounts of triacylglycerol or storage proteins typical of maturing, zygotic soybean embryos. At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of zygotic soybean embryos at the developmental stage from which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed proteins, α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3, and seed lectin are essentially absent. Upon transfer to hormone-free media to allow differentiation to the maturing somatic embryo state, triacylglycerol becomes the most abundant lipid class. As well, mRNAs for α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3 and seed lectin become very abundant messages in the total mRNA population. On this basis, the somatic soybean embryo system behaves very similarly to maturing zygotic soybean embryos in vivo, and is thus a good and rapid model system for analyzing the phenotypic effects of modifying the expression of genes in the fatty acid biosynthesis pathway (Example 3 in WO 02/00904). Most importantly, the model system is also predictive of the fatty acid composition of seeds from plants derived from transgenic embryos.
Transgenic somatic soybean embryos containing the constructs described above were analyzed in a similar way. For this, fatty acid methyl esters were prepared from single, matured, somatic soy embryos by transesterification. Embryos were placed in a vial containing 50 μL of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 μL injected from hexane layer) are separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20° C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Catalog #U-99-A, Nu-Chek Prep, Inc.). Routinely, 6-10 embryos per event were analyzed by GC, using the methodology described above.
More specifically, embryo fatty acid profiles for ˜6 lines containing pKR681 are shown in Table 9. The best line (i.e., 1618-1-1-1) had embryos with an average DGLA content of 8.9% and an average ETA content of 3.1%. For lines 1618-1-8-1, 1618-3-6-1 and 1618-4-1-1, only the elongase appeared to be functioning. The best elongase line (i.e., 1618-4-1-1) had embryos with an average EDA content of 10.6% and an average EtrA content of 6.5%. Calculated % elongation, % desaturation and elongation and desaturation ratios are shown in Table 10. In line 1618-1-1-1, the delta-8 desaturase converts an average of 76.3% of the elongated C20 fatty acids to product with the best embryo converting 82.1% to product. The delta-8 desaturase appears to utilize EDA and EtrA equally well as the ratio of their respective % desaturations is around 1.0. In line 1618-4-1-1, the Mortiella alpina elongase converts an average of 23% of the C18 fatty axcids to product with the best embryo converting 30.2% to product. The elongase appears to have a slight preference for ALA as the ratio of their respective % elongations is around 0.6. Expression of only the elongase in these lines likely resulted from fragmentation of the construct during the transformation procedure or due to positional insertion effects differentially affecting expression of the delta-8.
TABLE-US-00013 TABLE 9 Accumulation Of Long Chain PUFAs In Lines Transformed With pKR681 Line 16:0 18:0 18:1 LA GLA ALA EDA DGLA EtrA ETA 1618-1-1-1 13.9 6.8 7.1 40.2 0.0 14.4 3.0 10.2 1.1 3.3 -2 14.5 10.0 6.2 38.9 0.0 11.0 4.1 10.5 1.2 3.0 -3 14.1 4.9 4.7 42.2 0.0 21.5 2.0 7.2 1.0 2.5 -4 14.1 7.1 6.2 42.7 0.0 13.3 3.3 9.1 1.1 2.8 -5 12.0 5.0 5.8 46.3 0.0 16.0 2.2 8.1 1.0 3.2 -6 12.1 4.7 5.7 42.0 0.0 20.9 1.7 8.5 0.9 3.5 Ave 13.5 6.4 6.0 42.1 0.0 16.2 2.7 8.9 1.0 3.1 1618-1-2-1 11.7 4.3 4.6 46.8 0.0 17.4 3.9 5.7 2.4 3.1 -2 12.2 6.0 4.7 45.5 0.0 14.9 5.1 5.8 2.9 2.9 -3 12.7 5.0 7.1 44.7 0.0 17.5 4.2 4.1 2.4 2.3 -4 12.4 6.3 6.8 43.0 0.0 13.9 6.6 4.9 3.9 2.3 -5 13.4 8.7 5.2 39.6 0.0 13.2 6.3 7.2 3.2 3.1 -6 12.8 5.5 6.2 45.7 0.0 15.6 4.3 5.2 2.2 2.5 Ave 12.5 6.0 5.8 44.2 0.0 15.4 5.1 5.5 2.8 2.7 1618-1-8-1 8.7 3.5 6.7 53.3 0.0 17.2 6.9 0.0 3.6 0.0 -2 9.2 2.9 12.0 49.0 0.0 18.9 4.7 0.0 3.3 0.0 -3 11.2 2.8 7.7 48.6 0.0 22.4 4.1 0.0 3.2 0.0 -4 12.0 3.6 13.6 46.7 0.0 16.0 4.8 0.0 3.2 0.0 -5 9.1 3.6 5.0 52.6 0.0 16.5 8.5 0.0 4.8 0.0 -6 9.3 2.8 12.7 47.2 0.0 20.0 4.6 0.0 3.4 0.0 Ave 9.9 3.2 9.6 49.6 0.0 18.5 5.6 0.0 3.6 0.0 1618-3-6-1 11.8 2.4 8.3 42.1 0.0 28.2 3.3 0.0 3.9 0.0 -2 10.6 4.2 12.8 43.7 0.0 17.6 6.7 0.0 4.3 0.0 -3 10.3 4.9 5.6 45.7 0.0 18.4 8.7 0.0 6.3 0.0 -4 11.8 5.2 21.2 39.5 0.0 15.2 4.4 0.0 2.8 0.0 -5 10.3 3.0 9.2 47.8 0.0 21.5 4.7 0.0 3.5 0.0 -6 9.4 2.5 9.4 47.9 0.0 23.2 4.0 0.0 3.7 0.0 Ave 10.7 3.7 11.1 44.4 0.0 20.7 5.3 0.0 4.1 0.0 1618-4-1-1 15.4 9.2 6.5 38.1 0.0 9.9 13.8 0.0 7.0 0.0 -2 11.1 5.6 5.7 43.3 0.0 16.8 10.2 0.0 7.4 0.0 -3 10.5 5.0 6.6 45.4 0.0 15.4 10.1 0.0 6.9 0.0 -4 10.2 5.8 6.5 45.1 0.0 12.9 12.3 0.0 7.2 0.0 -5 11.4 4.4 10.1 45.3 0.0 16.1 7.4 0.0 5.2 0.0 -6 10.7 5.2 13.6 42.9 0.0 12.6 9.6 0.0 5.3 0.0 Ave 11.5 5.9 8.2 43.4 0.0 14.0 10.6 0.0 6.5 0.0 Fatty acid compositions listed in Table 9 are expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1 = Oleic acid, LA = Linoleic acid, GLA = γ-Linoleic acid, ALA = alpha-Linolenic acid, EDA = Eicosadienoic acid, DGLA = Dihomo-γ-Linoleic, EtrA = Eicosatrienoic acid, ETA = Eicosa-tetraenoic acid.
TABLE-US-00014 TABLE 10 Comparison Of % Desaturation And % Elongation In Lines Transformed With pKR681 Ratio C20 Ratio EDA EtrA (EDA/EtrA) C18 % delta- LA ALA (LA/ALA) % delta- % delta- de;ta-8 Line % Elong 8 desat % Elong % Elong Elong 8 desat 8 desat seat 1618-1-1-1 24.4 76.6 24.7 23.5 1.1 77.1 75.0 1.0 -2 27.3 71.9 27.2 27.7 1.0 72.1 71.1 1.0 -3 16.6 76.4 17.9 14.0 1.3 78.1 72.0 1.1 -4 22.5 73.3 22.4 22.7 1.0 73.5 72.6 1.0 -5 18.9 77.7 18.3 20.7 0.9 78.4 76.0 1.0 -6 18.8 82.1 19.6 17.4 1.1 83.1 79.9 1.0 Ave 21.4 76.3 21.7 21.0 1.1 77.0 74.4 1.0 1618-1-2-1 19.1 58.1 17.0 24.0 0.7 59.0 56.6 1.0 -2 21.7 52.2 19.3 28.0 0.7 53.2 50.4 1.1 -3 17.3 49.2 15.8 21.0 0.8 49.5 48.5 1.0 -4 23.7 40.6 21.0 30.8 0.7 42.6 36.9 1.2 -5 27.3 51.8 25.5 32.2 0.8 53.3 48.6 1.1 -6 18.9 54.1 17.3 23.1 0.7 54.6 53.1 1.0 Ave 21.3 51.0 19.3 26.5 0.7 52.0 49.0 1.1 1618-1-8-1 13.0 11.5 17.5 0.7 -2 10.6 8.8 14.8 0.6 -3 9.4 7.9 12.5 0.6 -4 11.4 9.3 16.8 0.6 -5 16.2 13.9 22.7 0.6 -6 10.7 9.0 14.6 0.6 Ave 11.9 10.1 16.5 0.6 1618-3-6-1 9.3 7.4 12.0 0.6 -2 15.2 13.3 19.5 0.7 -3 19.0 15.9 25.6 0.6 -4 11.6 10.0 15.6 0.6 -5 10.6 9.0 14.0 0.6 -6 9.8 7.7 13.9 0.6 Ave 12.6 10.5 16.8 0.6 1618-4-1-1 30.2 26.6 41.4 0.6 -2 22.6 19.0 30.5 0.6 -3 21.9 18.2 31.0 0.6 -4 25.2 21.5 35.8 0.6 -5 17.1 14.1 24.4 0.6 -6 21.1 18.3 29.6 0.6 Ave 23.0 19.6 32.1 0.6
The C18% Elongation (C18% Elong) in Table 10 was calculated by dividing the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 9) by the sum of the wt. % for LA, ALA, EDA, DGLA, EtrA and ETA (Table 9) and multiplying by 100 to express as a %. The C20% A8 desaturation (C20% A8 desat. Table 10) was calculated by dividing the sum of the wt. % for DGLA and ETA (Table 9) by the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 9) and multiplying by 100 to express as a %. The individual elongations (LA % Elong or ALA % Elong) or A8 desaturations (EDA % Δ8 desat or EtrA % Δ8 desat) shown in Table 10 were calculated in a similar way but only using either the ω-6 substrates/products or the ω-3 substrates/products for each. The Ratio elongation for LA and ALA was obtained by dividing the LA % Elongation by the ALA % elongation. Similarly, the Ratio delta-8 desaturation was obtained by dividing the EDA % delta-8 desaturation by the EtrA % delta-8 desaturation.
Analysis of Somatic Soy Embryos Containing the Euglena gracilis Delta-8 Desaturase and Isochrysis galbana Elongase Genes (pKR682)
Embryo fatty acid profiles for 9 lines containing pKR682 are shown in Table 11. Calculated % elongation, % desaturation and elongation and desaturation ratios are shown in Table 12. The best line (1619-6-7) had embryos with an average DGLA content of 21.8% and an average ETA content of 4.1%. As can be seen from Table 12, in this line, the delta-8 desaturase converts an average of 91.6% of the elongated C20 fatty acids to product and, in the best embryo (1619-6-7-1), 95.1% of the elongated fatty acids are converted to product. As for pKR681, the delta-8 desaturase appears to utilize EDA and EtrA equally well with the ratio of their respective % desaturations being around 1.0 (Table 12). In these lines, the average % conversion of C18 fatty acids to C20 fatty acids ranges from 40.0% to 49.5%. As seen with pKR681, there are lines (1619-6-4, 1619-8-4) where only the elongase is functioning (Table 11). Again, this is likely due to positional effects or fragmentation of DNA. In lines where the delta-8 desaturase is not functioning, the best elongase line (1619-6-4) had embryos with an average EDA content of 24.1% and an average ETrA content of 8.7%. The best embryo analyzed had 27.4% EDA and 10.3% ETrA. Average elongation in this line is 49.5% with the best embryo (1619-6-4-6) having 58.9% elongation (Table 12). In these lines, the elongase appears to have no preference for LA or ALA as the ratio of their respective % elongations is around 1.0. Interestingly, in lines that also express the delta-8 desaturase, there seems to be a slight preference of the elongase for LA and the average elongation ratio is as high as 2.3 in line 1617-16-2-7. In many of the lines, a small amount of a fatty acid that runs with retention time identical to GLA is present when the delta-8 desaturase is functioning well.
TABLE-US-00015 TABLE 11 Accumulation Of Long Chain PUFAs In Lines Transformed With pKR682 16:0 18:0 18:1 LA GLA ALA EDA DGLA EtrA ETA 1617-16-2-7 20.1 2.7 5.9 18.0 2.0 12.8 5.1 26.5 2.4 4.4 -8 19.3 1.3 5.0 22.7 1.7 23.4 3.7 16.8 1.6 4.5 -9 20.4 2.4 4.7 13.7 2.3 15.3 5.2 26.2 3.5 6.3 -10 17.0 1.7 6.2 19.9 1.7 27.0 2.5 17.4 1.1 5.5 -11 16.4 1.3 5.1 21.5 3.3 28.2 2.8 15.2 2.1 4.1 -12 26.7 2.4 6.1 0.0 4.1 20.2 6.5 26.9 2.2 5.0 -13 17.5 1.5 5.8 21.6 2.6 20.3 3.9 19.9 1.8 5.1 -14 20.2 2.4 8.9 24.6 1.6 17.9 4.3 14.7 1.4 4.2 Ave 19.7 2.0 6.0 17.7 2.4 20.6 4.2 20.4 2.0 4.9 1619-6-4-1 18.5 1.7 9.4 25.0 0.0 8.2 27.4 0.0 9.7 0.0 -2 14.5 2.0 15.9 26.8 0.0 7.8 24.5 0.0 8.5 0.0 -3 23.6 3.8 12.2 19.3 0.0 8.7 23.8 0.0 8.8 0.0 -4 15.5 1.2 12.6 34.5 0.0 14.7 15.4 0.0 6.0 0.0 -5 15.2 1.6 15.7 25.5 0.0 6.7 26.4 0.0 8.9 0.0 -6 15.8 2.2 18.0 19.1 0.0 7.2 27.4 0.0 10.3 0.0 Ave 17.2 2.1 14.0 25.0 0.0 8.9 24.1 0.0 8.7 0.0 1619-6-5-1 22.1 2.1 6.2 23.9 3.9 10.5 4.2 21.1 1.3 4.8 -2 17.4 1.6 9.5 32.3 1.5 11.0 3.0 18.0 0.7 4.6 -3 17.5 2.6 9.9 32.9 0.5 11.3 5.8 15.3 0.6 3.3 -4 17.2 2.0 12.1 29.5 0.6 10.6 6.0 13.9 2.8 4.7 -5 24.2 3.1 7.1 25.4 2.0 10.7 5.2 16.8 1.9 3.4 -6 17.9 1.6 5.9 30.8 2.4 12.3 5.7 18.2 1.6 3.7 Ave 19.4 2.2 8.5 29.2 1.8 11.1 5.0 17.2 1.5 4.1 1619-6-7-1 19.1 1.7 4.8 32.0 1.1 21.7 1.0 16.3 0.0 2.3 -2 19.0 1.3 5.0 40.1 1.2 17.9 1.4 11.9 0.2 2.0 -3 17.8 1.2 6.2 26.6 2.0 13.1 2.1 24.6 0.4 5.9 -4 19.4 1.3 8.1 29.4 1.2 12.6 3.0 20.5 0.5 4.1 -5 19.9 1.4 9.2 19.6 3.1 8.8 2.1 29.9 0.5 5.4 -6 20.1 1.6 6.9 25.0 2.9 8.4 2.5 27.6 0.4 4.6 Ave 19.2 1.4 6.7 28.8 1.9 13.7 2.0 21.8 0.3 4.1 1619-7-3-1 15.4 1.9 9.4 34.7 0.6 12.1 11.9 6.7 4.3 3.0 -2 15.2 1.5 9.6 37.4 0.0 17.0 9.9 3.7 3.6 1.9 -3 17.0 3.0 14.5 26.6 0.5 9.9 11.0 10.5 3.1 3.9 -4 18.5 3.4 8.6 17.7 1.3 4.2 21.7 16.9 4.0 3.7 -5 16.5 2.4 10.2 25.8 0.8 7.0 15.1 12.6 4.7 5.0 -6 16.9 2.2 10.3 24.4 0.4 6.8 22.7 6.3 7.3 2.6 Ave 16.6 2.4 10.4 27.8 0.6 9.5 15.4 9.5 4.5 3.4 1619-7-7-1 21.2 1.5 12.8 17.6 1.3 8.8 6.8 23.1 2.1 4.8 -2 15.1 1.2 19.9 27.7 0.7 11.2 7.4 12.1 1.7 3.0 -3 17.4 2.1 16.4 23.8 0.6 9.1 10.4 15.2 1.9 3.2 -4 17.2 1.5 18.3 21.4 0.9 9.2 9.3 16.6 1.8 3.9 -5 16.4 1.0 13.4 24.2 1.2 15.9 6.5 16.2 2.1 3.3 -6 20.3 2.3 7.5 19.5 1.3 9.5 8.9 22.9 2.4 5.1 Ave 17.9 1.6 14.7 22.4 1.0 10.60 8.2 17.7 2.0 3.9 1619-7-8-1 19.2 1.8 5.7 21.7 2.1 11.1 12.1 17.5 4.0 4.9 -2 15.0 1.1 10.6 28.5 1.0 16.2 10.3 10.8 3.5 3.1 -3 17.0 1.6 11.3 20.6 1.2 8.6 12.4 17.8 4.3 5.2 -4 16.5 1.5 10.3 25.4 1.0 13.6 11.1 13.4 3.3 3.8 -5 16.0 1.3 10.0 29.0 0.8 12.6 4.7 19.0 1.2 5.5 -6 15.6 1.6 12.2 28.7 1.0 9.9 9.4 15.6 1.7 4.6 Ave 16.6 1.5 10.0 25.6 1.2 12.0 1.0.0 15.7 3.0 4.5 1619-8-1-1 20.4 1.7 5.8 24.4 2.7 15.0 4.3 20.3 1.5 3.9 -2 17.2 1.9 14.5 24.2 0.0 9.3 5.0 20.9 1.1 5.9 -3 16.4 1.8 13.0 23.5 1.4 11.6 6.9 19.9 1.3 4.1 -4 17.2 1.5 15.3 22.2 1.1 7.2 7.7 21.5 1.2 4.9 -5 19.4 1.3 9.9 21.7 2.8 13.8 5.1 20.2 1.7 4.1 -6 17.9 1.3 10.5 23.4 1.5 9.9 6.5 22.8 1.4 4.8 Ave 18.1 1.6 11.5 23.3 1.6 11.1 5.9 20.9 1.4 4.6 1619-8-4-1 15.7 1.1 6.7 50.5 0.0 18.1 6.2 0.0 1.8 0.0 -2 15.0 1.8 8.2 38.6 0.0 28.4 5.8 0.0 2.2 0.0 -3 18.1 2.9 7.2 35.6 0.0 32.2 2.7 0.0 1.3 0.0 -4 18.0 2.7 9.7 40.7 0.0 18.2 8.7 0.0 1.9 0.0 -5 16.0 1.5 6.9 50.4 0.0 20.8 3.0 0.0 1.4 0.0 -6 15.3 0.9 7.7 50.8 0.0 20.6 3.6 0.0 1.2 0.0 Ave 16.4 1.8 7.7 44.4 0.0 23.00 5.0 0.0 1.6 0.0 Fatty acid compositions listed in Table 11 are expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1 = Oleic acid, LA = Linoleic acid, GLA = γ-Linoleic acid, ALA = alpha-Linolenic acid, EDA = Eicosadienoic acid, DGLA = Dihomo-γ-Linoleic, EtrA = Eicosatrienoic acid, ETA = Eicosatetraenoic acid.
TABLE-US-00016 TABLE 12 Comparison Of % Desaturation And % Elongation In Lines Transformed With pKR682 Ratio C20 Ratio EDA EtrA (EDA/EtrA) C18 % delta- LA ALA (LA/ALA) % delta- % delta- de;ta-8 Line % Elong 8 desat % Elong % Elong Elong 8 desat 8 desat seat 1617-16-2-7 55.5 80.5 63.8 34.7 1.8 83.9 65.0 1.3 -8 36.6 80.2 47.4 20.8 2.3 82.1 73.6 1.1 -9 58.8 78.8 69.6 39.3 1.8 83.4 64.1 1.3 -10 36.0 86.6 49.9 19.5 2.6 87.5 83.9 1.0 -11 32.8 79.6 45.6 18.1 2.5 84.3 66.1 1.3 -12 66.7 78.6 100.0 26.1 3.8 80.6 69.5 1.2 -13 42.3 81.3 52.4 25.4 2.1 83.5 74.0 1.1 -14 36.6 76.7 43.5 23.8 1.8 77.4 74.4 1.0 Ave 45.7 80.3 59.0 26.0 2.3 82.8 71.3 1.2 1619-6-4-1 52.8 52.3 54.2 1.0 -2 48.8 47.7 52.1 0.9 -3 53.8 55.2 50.3 1.1 -4 30.3 30.8 29.1 1.1 -5 52.3 50.9 57.0 0.9 -6 58.9 58.9 58.9 1.0 Ave 49.5 49.3 50.3 1.0 1619-6-5-1 47.6 82.5 51.3 36.7 1.4 83.4 78.5 1.1 -2 37.8 85.8 39.4 32.3 1.2 85.5 86.9 1.0 -3 36.0 74.5 39.0 25.5 1.5 72.6 84.7 0.9 -4 40.6 67.7 40.2 41.7 1.0 69.6 62.5 1.1 -5 43.1 74.0 46.4 33.2 1.4 76.3 64.6 1.2 -6 40.4 74.9 43.7 30.1 1.5 76.0 70.1 1.1 Ave 40.9 76.6 43.3 33.2 1.3 77.3 74.6 1.0 1619-6-7-1 26.8 95.1 35.1 9.6 3.6 94.4 100.0 0.9 -2 21.2 89.5 25.0 11.1 2.3 89.4 90.1 1.0 -3 45.4 92.4 50.1 32.4 1.5 92.0 94.1 1.0 -4 40.1 87.6 44.4 26.5 1.7 87.2 89.9 1.0 -5 57.2 93.2 62.0 40.1 1.5 93.4 92.3 1.0 -6 51.2 91.9 54.6 37.4 1.5 91.8 92.9 1.0 Ave 40.3 91.6 45.2 26.2 2.0 91.4 93.2 1.0 1619-7-3-1 35.6 37.6 34.9 37.6 0.9 36.0 41.5 0.9 -2 26.1 29.4 26.8 24.5 1.1 27.3 34.6 0.8 -3 43.9 50.4 44.7 41.4 1.1 48.8 55.2 0.9 -4 67.8 44.5 68.5 64.6 1.1 43.8 48.1 0.9 -5 53.3 47.0 51.8 58.0 0.9 45.4 51.6 0.9 -6 55.5 23.0 54.3 59.2 0.9 21.8 26.5 0.8 Ave 47.0 38.7 46.8 47.6 1.0 37.2 42.9 0.9 1619-7-7-1 58.3 75.7 63.0 44.0 1.4 77.1 -2 38.4 62.4 41.3 29.8 1.4 61.9 86.9 1.0 -3 48.3 60.0 51.8 36.0 1.4 59.3 84.7 0.9 -4 50.7 64.9 54.6 38.2 1.4 64.2 62.5 1.1 -5 41.1 69.5 48.3 25.4 1.9 76.3 64.6 1.2 -6 57.5 71.1 62.0 44.0 1.4 76.0 70.1 1.1 Ave 49.0 67.2 53.5 36.2 1.5 77.3 74.6 1.0 1619-7-8-1 54.0 58.2 57.7 44.4 1.3 59.2 54.9 1.1 -2 38.2 50.1 42.6 28.8 1.5 51.2 46.6 1.1 -3 57.6 57.9 59.4 52.2 1.1 58.9 54.6 1.1 -4 44.8 54.5 49.1 34.5 1.4 54.8 53.5 1.0 -5 42.2 80.7 45.0 34.7 1.3 80.3 82.1 1.0 -6 44.7 64.6 46.5 38.8 1.2 62.4 73.0 0.9 Ave 46.9 61.0 50.1 38.9 1.3 61.1 60.8 1.0 1619-8-1-1 43.2 80.7 50.2 26.4 1.9 82.6 72.1 1.1 -2 49.5 81.5 51.7 42.7 1.2 80.7 84.7 1.0 -3 47.9 74.5 53.3 31.8 1.7 74.2 76.0 1.0 -4 54.6 74.1 56.8 45.8 1.2 73.7 79.6 0.9 -5 46.6 78.3 53.8 29.4 1.8 80.0 70.9 1.1 -6 51.5 77.8 55.6 38.3 1.5 77.8 77.9 1.0 Ave 48.9 77.9 53.6 35.7 1.6 78.2 76.9 1.0 1619-8-4-1 10.5 11.0 9.1 1.2 -2 10.7 13.1 7.1 1.8 -3 5.6 7.1 3.8 1.9 -4 15.3 17.7 9.5 1.8 -5 5.8 5.7 6.1 0.9 -6 6.3 6.6 5.4 1.2 Ave 9.0 10.2 6.8 1.5
The C18% Elongation (C18% Elong) in Table 12 was calculated by dividing the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 11) by the sum of the wt. % for LA, ALA, EDA, DGLA, EtrA and ETA (Table 11) and multiplying by 100 to express as a %. The C20% Δ8 desaturation (C20% Δ8 desat. Table 12) was calculated by dividing the sum of the wt. % for DGLA and ETA (Table 11) by the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 11) and multiplying by 100 to express as a %. The individual elongations (LA % Elong or ALA % Elong) or Δ8 desaturations (EDA % Δ8 desat or EtrA % Δ8 desat) shown in Table 12 were calculated in a similar way but only using either the ω-6 substrates/products or the ω-3 substrates/products for each. The Ratio elongation for LA and ALA was obtained by dividing the LA % Elongation by the ALA % elongation. Similarly, the Ratio delta-8 desaturation was obtained by dividing the EDA % delta-8 desaturation by the EtrA % delta-8 desaturation.
Analysis of Somatic Soy Embryos Containing the Euglena gracilis Delta-8 Desaturase, Isochrysis galbana Elongase and Other EPA Biosynthetic Genes (pKR786, pKR669)
Plasmid pKR786 and pKR669 were digested with AscI and the DNA fragments containing ALS selection and EPA biosynthetic genes were transformed into soy as described previously. Fatty acids from ten embryos for each line obtained containg pKR786 and pKR669 were analyzed by GC as described.
Ten embryos were analyzed for each individual transformation event. Fatty acids were identified by comparison of retention times to those for authentic standards. In this way, 169 events were analyzed. From the 169 lines analyzed, 25 were identified that produced EPA (average of 10 individual embryos) at a relative abundance greater than 10% of the total fatty acids. The ten best EPA-producing events are shown in Table 13 and Table 14. The results for 10 embryos from the best event are shown in Tables 15 and 16. The best line analyzed averaged 21.2% EPA with the best embryo of this line having 29.4% EPA (Table 16). A chromatogram for the embryo is shown in FIG. 10
Fatty acids in Table 13 and Table 14 are defined as X:Y where X is the fatty acid chain length and Y is the number of double bonds. In addition, fatty acids from Table 13 and Table 14 are further defined as follows where the number in parentheses corresponds to the position of the double bonds from the carboxyl end of the fatty acid: 18:1=18:1(9), 18:2=18:2(9,12), GLA=18:3(6,9,12), 18:3=18:3(9,12,15), STA=18:4(6,9,12,15), DGLA=20:3(8,11,14), ARA=20:4(5,8,11,14), ETA=20:4(8,11,14,17), EPA=20:5(5,8,11,14,17) and DPA=22:5(7,10,13,16,19). Fatty acids listed as "others" include: 18:2(6,9), 20:0, 20:1(11), 20:2(8,11) and 20:3 (5,11,14). Each of these fatty acids is present at a relative abundance of less than 2% of the total fatty acids. In all of the top lines, GLA is not present or is present at levels less than 0.2%.
TABLE-US-00017 TABLE 13 Accumulation Of Long Chain PUFAs In Lines Transformed With pKR786 And pKR669 (Averages of 10 embryos per line) Line 16:0 18:0 18:1 LA GLA ALA STA EDA DGLA ARA AFS 4314-2-1 17.0 2.6 15.6 16.8 0.1 20.4 0.3 2.7 1.2 0.1 AFS 4310-1-2 16.7 2.4 14.9 15.5 0.1 17.0 0.9 4.4 1.4 0.4 AFS 4310-5-6 15.7 3.0 15.7 17.6 0.0 10.1 0.7 7.4 1.7 0.2 AFS 4310-1-8 15.2 2.7 16.4 15.2 0.1 17.2 0.8 4.7 1.6 0.5 AFS 4314-6-1 14.0 3.1 12.3 17.0 0.1 8.5 0.7 11.2 2.4 0.4 AFS 4314-5-6 15.6 2.7 12.9 4.6 0.0 28.0 1.2 1.7 1.0 0.7 AFS 4310-7-5 17.3 1.9 9.6 16.0 0.0 22.9 0.8 2.4 2.1 1.9 AFS 4310-1-9 14.8 2.8 13.8 12.8 0.0 17.2 0.7 4.8 1.2 0.2 AFS 4314-3-4 16.1 2.5 12.6 14.9 0.1 18.6 0.3 3.4 1.4 0.2 AFS 4310-5-2 15.8 2.5 8.5 15.8 0.1 17.5 0.6 4.2 2.4 0.8 Fatty acid compositions listed in Table 13 are expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1 = Oleic acid, LA = Linoleic acid, GLA = γ-Linoleic acid, ALA = alpha-Linolenic acid, STA = Stearidonic acid, EDA = Eicosadienoic acid, DGLA = Dihomo-γ-Linoleic, ARA = Arachidonic acid.
TABLE-US-00018 TABLE 14 Accumulation Of Long Chain PUFAs In Lines Transformed With pKR786 And pKR669 (Averages of 10 embryos per line) Line EtrA 20:4(5, 11, 14, 17) ETA EPA DPA Other AFS 4314-2-1 2.4 2.1 4.0 13.6 0.1 1.0 AFS 4310-1-2 3.6 3.6 3.1 14.1 0.4 1.6 AFS 4310-5-6 3.8 2.8 4.2 15.1 0.4 1.4 AFS 4310-1-8 2.9 2.7 3.1 15.3 0.2 1.6 AFS 4314-6-1 4.8 3.0 4.8 15.6 0.2 1.9 AFS 4314-5-6 5.3 3.1 5.1 16.2 0.3 1.5 AFS 4310-7-5 2.4 1.6 4.0 16.6 0.1 0.5 AFS 4310-1-9 4.5 4.8 3.6 16.8 0.3 1.8 AFS 4314-3-4 3.3 3.2 4.6 16.9 0.4 1.6 AFS 4310-5-2 3.0 2.1 4.6 21.2 0.2 0.7 Fatty acid compositions listed in Table 14 are expressed as wt. %. EtrA = Eicosatrienoic acid, ETA = Eicosa-tetraenoic acid, EPA = Eicosa-pentaenoic acid, DPA = Docosa-pentaenoic acid
TABLE-US-00019 TABLE 15 Accumulation Of Long Chain PUFAs In Line AFS 4310-5-2 Transformed With pKR786 And pKR669 Embryo # 16:0 18:0 18:1 LA GLA ALA STA EDA DGLA ARA 1 16.9 2.1 8.4 20.4 0.0 18.1 0.0 2.0 1.9 0.5 2 16.3 1.8 5.3 15.6 0.1 22.3 0.4 2.2 1.9 0.9 3 17.4 3.6 10.9 14.4 0.1 21.4 0.4 5.3 2.1 0.2 4 18.3 2.6 7.9 11.5 0.2 20.4 0.9 6.9 2.9 2.0 5 13.8 3.8 11.1 14.5 0.1 15.0 0.7 7.7 3.0 1.0 6 15.3 3.0 11.8 15.5 0.0 18.6 0.9 4.9 1.6 0.5 7 14.3 2.0 7.6 16.4 0.2 15.9 0.6 3.2 2.1 0.3 8 15.7 2.0 5.0 17.4 0.2 12.5 0.6 3.0 3.1 1.0 9 15.1 2.2 8.1 16.3 0.2 17.0 1.1 2.6 2.8 1.3 10 15.1 1.5 8.5 15.8 0.2 13.7 0.8 4.3 2.3 0.5 Ave 15.8 2.5 8.5 15.8 0.1 17.5 0.6 4.2 2.4 0.8 Fatty acid compositions listed in Table 13 are expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1 = Oleic acid, LA = Linoleic acid, GLA = γ-Linoleic acid, ALA = alpha-Linolenic acid, STA = Stearidonic acid, EDA = Eicosadienoic acid, DGLA = Dihomo-γ-Linoleic, ARA = Arachidonic acid.
TABLE-US-00020 TABLE 16 Accumulation Of Long Chain PUFAs In Line AFS 4310-5-2 Transformed With pKR786 And pKR669 Embryo # EtrA 20:4(5, 11, 14, 17) ETA EPA DPA Other 1 1.9 2.1 4.5 21.1 0.0 0.0 2 2.5 2.0 5.1 22.8 0.3 0.6 3 2.9 1.0 4.7 14.7 0.0 0.6 4 5.6 1.9 4.1 13.6 0.1 1.0 5 3.7 2.4 3.3 18.5 0.1 1.2 6 4.1 2.5 3.6 16.4 0.1 1.1 7 2.7 2.6 5.6 25.7 0.3 0.6 8 2.0 2.3 4.6 29.4 0.6 0.7 9 1.8 2.0 4.2 24.0 0.3 0.9 10 2.9 2.2 5.9 25.6 0.2 0.6 Ave 3.0 2.1 4.6 21.2 0.2 0.7 Fatty acid compositions listed in Table 14 are expressed as wt. %. EtrA = Eicosatrienoic acid, ETA = Eicosa-tetraenoic acid, EPA = Eicosa-pentaenoic acid, DPA = Docosa-pentaenoic acid
15111271DNAEuglena gracilismisc_feature(4)..(1269)Eg5 delta-8 desaturase 1gaaatgaagt caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg 60tctgcctggg tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg 120gatgccactg atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc 180atgcccaaaa tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa 240gaggatttcc ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc 300ctctggtact catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg 360atggttcagt atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag 420atgggctggc tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac 480aacctcgtgg gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag 540gacagacaca atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac 600aacctccccc tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc 660aagctcattc agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt 720tggtgtttcc agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat 780cgctctcagt ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg 840ttccacttat tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag 900ctggttggcg gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag 960atcggggact cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg 1020aacattcggc gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag 1080caccatttgt ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa 1140cagctgtgcc agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc 1200atcctgctgc gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag 1260gctctataag g 12712421PRTEuglena gracilis 2Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu65 70 75 80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105 110Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile 115 120 125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn145 150 155 160Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile305 310 315 320Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His 325 330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345 350Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355 360 365His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile385 390 395 400Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 415Ala Gly Lys Ala Leu 42031271DNAEuglena gracilismisc_feature(4)..(1269)Eg12 delta-8 desaturase 3gaaatgaagt caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg 60tctgcctggg tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg 120gatgccactg atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc 180atgcccaaaa tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa 240gaggatttcc ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc 300ctctggtact catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg 360atggttcagt atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag 420atgggctggc tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac 480aacctcgtgg gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag 540gacagacaca atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac 600aacctccccc tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc 660aagctcattc agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt 720tggtgtttcc agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat 780cgctctcagt ataagaagga ggccattggc ctcgccctgc actggacctt gaaggccctg 840ttccacttat tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag 900ctggttggcg gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag 960atcggggact cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg 1020aacattcggc gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag 1080caccatttgt ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa 1140cagctgtgcc agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc 1200atcctgctgc gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag 1260gctctataag g 12714421PRTEuglena gracilis 4Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu65 70 75 80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105 110Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile 115 120 125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn145 150 155 160Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270His Trp Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile305 310 315 320Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His 325 330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345 350Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355 360 365His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile385 390 395 400Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 415Ala Gly Lys Ala Leu 42051275DNAEuglena gracilismisc_feature(14)..(1273)non-functional delta-8 desaturase 5attttttttc gaaatgaagt caaagcgcca agcgctatcc cccttacaat tgatggaaca 60aacatatgat gtggtcaatt tccaccctgg tggtgcggaa attatagaga attaccaagg 120aagggatgcc actgatgcct tcatggttat gcactttcaa gaagccttcg acaagctcaa 180gcgcatgccc aaaatcaatc ccagttttga gttgccaccc caggctgcag tgaatgaagc 240tcaagaggat ttccggaagc tccgagaaga gttgatcgca actggcatgt ttgatgcctc 300ccccctctgg tactcataca aaatcagcac cacactgggc cttggagtgc tgggttattt 360cctgatggtt cagtatcaga tgtatttcat tggggcagtg ttgcttggga tgcactatca 420acagatgggc tggctttctc atgacatttg ccaccaccag actttcaaga accggaactg 480gaacaacctc gtgggactgg tatttggcaa tggtctgcaa ggtttttccg tgacatgttg 540gaaggacaga cacaatgcac atcattcggc aaccaatgtt caagggcacg accctgatat 600tgacaacctc ccccccttag cctggtctga ggatgacgtc acacgggcgt caccgatttc 660ccgcaagctc attcagttcc agcagtacta tttcttggtc atctgtatct tgttgcggtt 720catttggtgt ttccagtgcg tgttgaccgt gcgcagtttg aaggacagag ataaccaatt 780ctatcgctct cagtataaga aggaggccat tggcctcgcc ctgcactgga ccttgaaggc 840cctgttccac ttattcttta tgcccagcat cctcacatcg ctgttggtgt ttttcgtttc 900ggagctggtt ggcggcttcg gcattgcgat cgtggtgttc atgaaccact acccactgga 960gaagatcggg gacccagtct gggatggcca tggattctcg gttggccaga tccatgagac 1020catgaacatt cggcgaggga ttatcacaga ttggtttttc ggaggcttga attaccagat 1080tgagcaccat ttgtggccga ccctccctcg ccacaacctg acagcggtta gctaccaggt 1140ggaacagctg tgccagaagc acaacctgcc gtatcggaac ccgctgcccc atgaagggtt 1200ggtcatcctg ctgcgctatc tggcggtgtt cgcccggatg gcggagaagc aacccgcggg 1260gaaggctcta taagg 12756419PRTEuglena gracilis 6Met Lys Ser Lys Arg Gln Ala Leu Ser Pro Leu Gln Leu Met Glu Gln1 5 10 15Thr Tyr Asp Val Val Asn Phe His Pro Gly Gly Ala Glu Ile Ile Glu 20 25 30Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met His Phe 35 40 45Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn Pro Ser 50 55 60Phe Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu Asp Phe65 70 75 80Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp Ala Ser 85 90 95Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu Gly Val 100 105 110Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile Gly Ala 115 120 125Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser His Asp 130 135 140Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn Leu Val145 150 155 160Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr Cys Trp 165 170 175Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln Gly His 180 185 190Asp Pro Asp Ile Asp Asn Leu Pro Pro Leu Ala Trp Ser Glu Asp Asp 195 200 205Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe Gln Gln 210 215 220Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp Cys Phe225 230 235 240Gln Cys Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn Gln Phe 245 250 255Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu His Trp 260 265 270Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile Leu Thr 275 280 285Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe Gly Ile 290 295 300Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile Gly Asp305 310 315 320Pro Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His Glu Thr 325 330 335Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly Gly Leu 340 345 350Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg His Asn 355 360 365Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys His Asn 370 375 380Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile Leu Leu385 390 395 400Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro Ala Gly 405 410 415Lys Ala Leu 7422PRTEuglena gracilis 7Met Lys Ser Lys Arg Gln Ala Leu Ser Pro Leu Gln Leu Met Glu Gln1 5 10 15Thr Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val 35 40 45Met His Phe Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile 50 55 60Asn Pro Ser Phe Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly 100 105 110Leu Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe 115 120 125Ile Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135 140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn145 150 155 160Asn Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val 165 170 175Thr Cys Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp Asn Leu Pro Pro Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp Cys Phe Gln Cys Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265 270Leu His Trp Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Pro Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375 380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val385 390 395 400Ile Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln 405 410 415Pro Ala Gly Lys Ala Leu 420819DNAArtificial SequencePrimer Eg5-1 8gaaatgaagt caaagcgcc 19919DNAArtificial SequencePrimer Eg3-3 9ccttatagag ccttccccg 191022DNAArtificial SequencePrimer T7 10gtaatacgac tcactatagg gc 221119DNAArtificial SequencePrimer M13-28Rev 11ggaaacagct atgaccatg 191219DNAArtificial SequencePrimer Eg3-2 12ttggcaatgg tctgcaagg 191319DNAArtificial SequencePrimer Eg5-2 13aatgttcatg gtctcatgg
191437DNAArtificial SequencePrimer 14ggatctcctg caggatctgg ccggccggat ctcgtac 371524DNAArtificial SequencePrimer RPB2forward 15gcggccgcat ggagtcgatt gcgc 241624DNAArtificial SequencePrimer RPB2reverse 16gcggccgctt actgcaactt cctt 241734DNAArtificial SequencePrimer 17aagcttgcat gcctgcaggt cgactcgacg tacg 3418280DNAArtificial Sequencesoybean albumin transcription terminator 18tctagaggat ccaaggccgc gaagttaaaa gcaatgttgt cacttgtcgt actaacacat 60gatgtgatag tttatgctag ctagctataa cataagctgt ctctgagtgt gttgtatatt 120aataaagatc atcactggtg aatggtgatc gtgtacgtac cctacttagt aggcaatgga 180agcacttaga gtgtgctttg tgcatggcct tgcctctgtt ttgagacttt tgtaatgttt 240tcgagtttaa atctttgcct ttgcgtacgt gggcggatcc 2801932DNAArtificial SequencePrimer oSalb-12 19tttggatcct ctagacgtac gcaaaggcaa ag 322036DNAArtificial SequencePrimer oSalb-13 20aaaggatcca aggccgcgaa gttaaaagca atgttg 362124DNAArtificial SequencePrimer GSP1 21gccccccatc ctttgaaagc ctgt 242234DNAArtificial SequencePrimer GSP2 22cgcggatccg agagcctcag catcttgagc agaa 342327DNAArtificial SequencePrimer GSP3 23ggtccaatat ggaacgatga gttgata 272435DNAArtificial SequencePrimer GSP4 24cgcggatccg ctggaactag aagagagacc taaga 35251408DNAGlycine maxmisc_featureBD30 promoter 25aactaaaaaa agctctcaaa ttacattttg agttgtttca ggttccattg ccttattgct 60aaaactccaa ctaaaataac aaatagcaca tgcaggtgca aacaacacgt tactctgatg 120aaggtgatgt gcctctagca gtctagctta tgaggctcgc tgcttatcaa cgattcatca 180ttccccaaga cgtgtacgca gattaaacaa tggacaaaac ttcaatcgat tatagaataa 240taattttaac agtgccgact tttttctgta aacaaaaggc cagaatcata tcgcacatca 300tcttgaatgc agtgtcgagt ttggaccatt tgagtacaaa gccaatattg aatgattttt 360cgattttaca tgtgtgaatc agacaaaagt gcatgcaatc acttgcaagt aaattaagga 420tactaatcta ttcctttcat tttatatgct ccacttttat ataaaaaaat atacattatt 480atatatgcat tattaattat tgcagtatta tgctattggt tttatggccc tgctaaataa 540cctaaatgag tctaactatt gcatatgaat caaatgaagg aagaatcatg atctaaacct 600gagtacccaa tgcaataaaa tgcgtcctat tacctaaact tcaaacacac attgccatcg 660gacgtataaa ttaatgcata taggttattt tgagaaaaga aaacatcaaa agctctaaaa 720cttcttttaa ctttgaaata agctgataaa aatacgcttt aaatcaactg tgtgctgtat 780ataagctgca atttcacatt ttaccaaacc gaaacaagaa tggtaacagt gaggcaaaaa 840tttgaaaaat gtcctacttc acattcacat caaattaatt acaactaaat aaataaacat 900cgtgattcaa gcagtaatga aagtcgaaat cagatagaat atacacgttt aacatcaatt 960gaattttttt ttaaatggat atatacaagt ttactatttt atatataatg aaaattcatt 1020ttgtgttagc acaaaactta cagaaagaga taaattttaa ataaagagaa ttatatccaa 1080ttttataatc caaaataatc aaattaaaga atattggcta gatagaccgg ctttttcact 1140gcccctgctg gataatgaaa attcatatca aaacaataca gaagttctag tttaataata 1200aaaaagttgg caaactgtca ttccctgttg gtttttaagc caaatcacaa ttcaattacg 1260tatcagaaat taatttaaac caaatatata gctacgaggg aacttcttca gtcattacta 1320gctagctcac taatcactat atatacgaca tgctacaagt gaagtgacca tatcttaatt 1380tcaaatcata aaattcttcc accaagtt 140826690DNAGlycine maxmisc_featureGlycinin Gy1 promoter 26tagcctaagt acgtactcaa aatgccaaca aataaaaaaa aagttgcttt aataatgcca 60aaacaaatta ataaaacact tacaacaccg gatttttttt aattaaaatg tgccatttag 120gataaatagt taatattttt aataattatt taaaaagccg tatctactaa aatgattttt 180atttggttga aaatattaat atgtttaaat caacacaatc tatcaaaatt aaactaaaaa 240aaaaataagt gtacgtggtt aacattagta cagtaatata agaggaaaat gagaaattaa 300gaaattgaaa gcgagtctaa tttttaaatt atgaacctgc atatataaaa ggaaagaaag 360aatccaggaa gaaaagaaat gaaaccatgc atggtcccct cgtcatcacg agtttctgcc 420atttgcaata gaaacactga aacacctttc tctttgtcac ttaattgaga tgccgaagcc 480acctcacacc atgaacttca tgaggtgtag cacccaaggc ttccatagcc atgcatactg 540aagaatgtct caagctcagc accctacttc tgtgacgttg tccctcattc accttcctct 600cttccctata aataaccacg cctcaggttc tccgcttcac aactcaaaca ttctcctcca 660ttggtcctta aacactcatc agtcatcacc 6902736DNAArtificial SequencePrimer 27cgcggatcct agcctaagta cgtactcaaa atgcca 362841DNAArtificial SequencePrimer 28gaattcgcgg ccgcggtgat gactgatgag tgtttaagga c 41292012DNAGlycine maxmisc_featureannexin promoter 29atcttaggcc cttgattata tggtgtttag atggattcac atgcaagttt ttatttcaat 60cccttttcct ttgaataact gaccaagaac aacaagaaaa aaaaaaaaag aaaaggatca 120ttttgaaagg atatttttcg ctcctattca aatactgtat ttttaccaaa aaaactgtat 180ttttcctaca ctctcaagct ttgtttttcg cttcgactct catgatttcc ttcatatgcc 240aatcactcta tttataaatg gcataaggta gtgtgaacaa ttgcaaagct tgtcatcaaa 300agcttgcaat gtacaaatta atgtttttca tgcctttcaa aattatctgc accccctagc 360tattaatcta acatctaagt aaggctagtg aattttttcg aatagtcatg cagtgcatta 420atttccccgt gactattttg gctttgactc caacactggc cccgtacatc cgtccctcat 480tacatgaaaa gaaatattgt ttatattctt aattaaaaat attgtccctt ctaaattttc 540atatagttaa ttattatatt acttttttct ctattctatt agttctattt tcaaattatt 600atttatgcat atgtaaagta cattatattt ttgctatata cttaaatatt tctaaattat 660taaaaaaaga ctgatatgaa aaatttattc tttttaaagc tatatcattt tatatatact 720ttttcttttc ttttctttca ttttctattc aatttaataa gaaataaatt ttgtaaattt 780ttatttatca atttataaaa atattttact ttatatgttt tttcacattt ttgttaaaca 840aatcatatca ttatgattga aagagaggaa attgacagtg agtaataagt gatgagaaaa 900aaatgtgtta tttcctaaaa aaaacctaaa caaacatgta tctactctct atttcatcta 960tctctcattt catttttctc tttatctctt tctttatttt tttatcatat catttcacat 1020taattatttt tactctcttt attttttctc tctatccctc tcttatttcc actcatatat 1080acactccaaa attggggcat gcctttatca ctactctatc tcctccacta aatcatttaa 1140atgaaactga aaagcattgg caagtctcct cccctcctca agtgatttcc aactcagcat 1200tggcatctga ttgattcagt atatctattg catgtgtaaa agtctttcca caatacataa 1260ctattaatta atcttaaata aataaaggat aaaatatttt tttttcttca taaaattaaa 1320atatgttatt ttttgtttag atgtatattc gaataaatct aaatatatga taatgatttt 1380ttatattgat taaacatata atcaatatta aatatgatat ttttttatat aggttgtaca 1440cataatttta taaggataaa aaatatgata aaaataaatt ttaaatattt ttatatttac 1500gagaaaaaaa aatattttag ccataaataa atgaccagca tattttacaa ccttagtaat 1560tcataaattc ctatatgtat atttgaaatt aaaaacagat aatcgttaag ggaaggaatc 1620ctacgtcatc tcttgccatt tgtttttcat gcaaacagaa agggacgaaa aaccacctca 1680ccatgaatca ctcttcacac catttttact agcaaacaag tctcaacaac tgaagccagc 1740tctctttccg tttcttttta caacactttc tttgaaatag tagtattttt ttttcacatg 1800atttattaac gtgccaaaag atgcttattg aatagagtgc acatttgtaa tgtactacta 1860attagaacat gaaaaagcat tgttctaaca cgataatcct gtgaaggcgt taactccaaa 1920gatccaattt cactatataa attgtgacga aagcaaaatg aattcacata gctgagagag 1980aaaggaaagg ttaactaaga agcaatactt ca 20123037DNAArtificial SequencePrimer 30cgcggatcca tcttaggccc ttgattatat ggtgttt 373143DNAArtificial SequencePrimer 31gaattcgcgg ccgctgaagt attgcttctt agttaacctt tcc 433241DNAArtificial SequencePrimer 32cgcggatcca actaaaaaaa gctctcaaat tacattttga g 413344DNAArtificial SequencePrimer 33gaattcgcgg ccgcaacttg gtggaagaat tttatgattt gaaa 443429DNAArtificial SequencePrimer oKTi5 34atctagacgt acgtcctcga agagaaggg 293522DNAArtificial SequencePrimer oKTi6 35ttctagacgt acggatataa tg 223617DNAArtificial SequencePrimer oSBD30-1 36tgcggccgca tgagccg 173732DNAArtificial SequencePrimer oSBD30-2 37acgtacggta ccatctgcta atattttaaa tc 323832DNAArtificial SequencePrimer oCGR5-1 38ttgcggccgc aaaccatggc tgctgctccc ag 323924DNAArtificial SequencePrimer oCGR5-2 39aagcggccgc ttactgcgcc ttac 244029DNAArtificial SequencePrimer oSGly-1 40ttcctgcagg ctagcctaag tacgtactc 294121DNAArtificial SequencePrimer oSGly-2 41aagcggccgc ggtgatgact g 214236DNAArtificial SequencePrimer LegPro5' 42tttctagacg tacgtccctt cttatctttg atctcc 364334DNAArtificial SequencePrimer LegPro3' 43gcggccgcag ttggatagaa tatatgtttg tgac 344441DNAArtificial SequencePrimer LegTerm5' 44ctatccaact gcggccgcat ttcgcaccaa atcaatgaaa g 414538DNAArtificial SequencePrimer LegTerm3' 45aatctagacg tacgtgaagg ttaaacatgg tgaatatg 384624DNAArtificial SequencePrimer CGR4forward 46gcggccgcat gggaacggac caag 244724DNAArtificial SequencePrimer CGR4reverse 47gcggccgcct actcttcctt ggga 24481270DNAArtificial SequenceD8S-1 Synthetic gene codon-optimized for expression in Yarrowia lipolytica 48ccatggagtc caagcgacag gctctgtctc ccctccagct gatggaacag acctacgacg 60tcgtgaactt ccaccctggt ggagctgaaa tcattgagaa ctaccaggga cgagatgcta 120ctgacgcctt catggttatg cactttcagg aagccttcga caagctcaag cgaatgccca 180agatcaaccc ctcctttgag ctgcctcccc aggctgccgt caacgaagct caggaggatt 240tccgaaagct ccgagaagag ctgatcgcca ctggcatgtt tgacgcctct cccctctggt 300actcgtacaa gatctccacc accctgggtc ttggcgtgct tggatacttc ctgatggtcc 360agtaccagat gtacttcatt ggtgctgtgc tgctcggtat gcactaccag caaatgggat 420ggctgtctca tgacatctgc caccaccaga ccttcaagaa ccgaaactgg aataacctcg 480tgggtctggt ctttggcaac ggactccagg gcttctccgt gacctgttgg aaggacagac 540acaacgccca tcattctgct accaacgttc agggtcacga tcccgacatt gataacctgc 600ctcccctcgc ctggtccgag gacgatgtca ctcgagcttc tcccatctcc cgaaagctca 660ttcagttcca acagtactat ttcctggtca tctgtattct cctgcgattc atctggtgtt 720tccagtgcgt gctgaccgtt cgatccctca aggaccgaga caaccagttc taccgatctc 780agtacaagaa agaggccatt ggactcgctc tgcactggac tctcaaggct ctgttccacc 840tcttctttat gccctccatc ctgacctcgc tcctggtgtt ctttgtttcc gagctcgtcg 900gtggcttcgg aattgccatc gtggtcttca tgaaccacta ccctctggag aagatcggtg 960atcccgtctg ggacggacat ggcttctctg tgggtcagat ccatgagacc atgaacattc 1020gacgaggcat cattactgac tggttctttg gaggcctgaa ctaccagatc gagcaccatc 1080tctggcccac cctgcctcga cacaacctca ctgccgtttc ctaccaggtg gaacagctgt 1140gccagaagca caacctcccc taccgaaacc ctctgcccca tgaaggtctc gtcatcctgc 1200tccgatacct ggccgtgttc gctcgaatgg ccgagaagca gcccgctggc aaggctctct 1260aagcggccgc 127049104DNAArtificial SequencePrimer D8-1A 49atggagtcca agcgacaggc tctgtctccc ctccagctga tggaacagac ctacgacgtc 60gtgaacttcc accctggtgg agctgaaatc attgagaact acca 10450104DNAArtificial SequencePrimer D8-1B 50tccctggtag ttctcaatga tttcagctcc accagggtgg aagttcacga cgtcgtaggt 60ctgttccatc agctggaggg gagacagagc ctgtcgcttg gact 10451102DNAArtificial SequencePrimer D8-2A 51gggacgagat gctactgacg ccttcatggt tatgcacttt caggaagcct tcgacaagct 60caagcgaatg cccaagatca acccctcctt tgagctgcct cc 10252102DNAArtificial SequencePrimer D8-2B 52ctggggaggc agctcaaagg aggggttgat cttgggcatt cgcttgagct tgtcgaaggc 60ttcctgaaag tgcataacca tgaaggcgtc agtagcatct cg 10253101DNAArtificial SequencePrimer D8-3A 53ccaggctgcc gtcaacgaag ctcaggagga tttccgaaag ctccgagaag agctgatcgc 60cactggcatg tttgacgcct ctcccctctg gtactcgtac a 10154101DNAArtificial SequencePrimer D8-3B 54atcttgtacg agtaccagag gggagaggcg tcaaacatgc cagtggcgat cagctcttct 60cggagctttc ggaaatcctc ctgagcttcg ttgacggcag c 10155101DNAArtificial SequencePrimer D8-4A 55ccaccaccct gggtcttggc gtgcttggat acttcctgat ggtccagtac cagatgtact 60tcattggtgc tgtgctgctc ggtatgcact accagcaaat g 10156101DNAArtificial SequencePrimer D8-4B 56atcccatttg ctggtagtgc ataccgagca gcacagcacc aatgaagtac atctggtact 60ggaccatcag gaagtatcca agcacgccaa gacccagggt g 10157104DNAArtificial SequencePrimer D8-5A 57ggatggctgt ctcatgacat ctgccaccac cagaccttca agaaccgaaa ctggaataac 60ctcgtgggtc tggtctttgg caacggactc cagggcttct ccgt 10458104DNAArtificial SequencePrimer D8-5B 58ggtcacggag aagccctgga gtccgttgcc aaagaccaga cccacgaggt tattccagtt 60tcggttcttg aaggtctggt ggtggcagat gtcatgagac agcc 10459101DNAArtificial SequencePrimer D8-6A 59gacctgttgg aaggacagac acaacgccca tcattctgct accaacgttc agggtcacga 60tcccgacatt gataacctgc ctcccctcgc ctggtccgag g 10160101DNAArtificial SequencePrimer D8-6B 60tcgtcctcgg accaggcgag gggaggcagg ttatcaatgt cgggatcgtg accctgaacg 60ttggtagcag aatgatgggc gttgtgtctg tccttccaac a 1016195DNAArtificial SequencePrimer D8-7A 61tcactcgagc ttctcccatc tcccgaaagc tcattcagtt ccaacagtac tatttcctgg 60tcatctgtat tctcctgcga ttcatctggt gtttc 956295DNAArtificial SequencePrimer D8-7B 62actggaaaca ccagatgaat cgcaggagaa tacagatgac caggaaatag tactgttgga 60actgaatgag ctttcgggag atgggagaag ctcga 956389DNAArtificial SequencePrimer D8-8A 63cagtgcgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta ccgatctcag 60tacaagaaag aggccattgg actcgctct 896489DNAArtificial SequencePrimer D8-8B 64gtgcagagcg agtccaatgg cctctttctt gtactgagat cggtagaact ggttgtctcg 60gtccttgagg gatcgaacgg tcagcacgc 896585DNAArtificial SequencePrimer D8-9A 65gcactggact ctcaaggctc tgttccacct cttctttatg ccctccatcc tgacctcgct 60cctggtgttc tttgtttccg agctc 856685DNAArtificial SequencePrimer D8-9B 66cgacgagctc ggaaacaaag aacaccagga gcgaggtcag gatggagggc ataaagaaga 60ggtggaacag agccttgaga gtcca 856791DNAArtificial SequencePrimer D8-10A 67gtcggtggct tcggaattgc catcgtggtc ttcatgaacc actaccctct ggagaagatc 60ggtgatcccg tctgggacgg acatggcttc t 916891DNAArtificial SequencePrimer D8-10B 68acagagaagc catgtccgtc ccagacggga tcaccgatct tctccagagg gtagtggttc 60atgaagacca cgatggcaat tccgaagcca c 916992DNAArtificial SequencePrimer D8-11A 69ctgtgggtca gatccatgag accatgaaca ttcgacgagg catcattact gactggttct 60ttggaggcct gaactaccag atcgagcacc at 927092DNAArtificial SequencePrimer D8-11B 70agagatggtg ctcgatctgg tagttcaggc ctccaaagaa ccagtcagta atgatgcctc 60gtcgaatgtt catggtctca tggatctgac cc 927193DNAArtificial SequencePrimer D8-12A 71ctctggccca ctctgcctcg acacaacctc actgccgttt cctaccaggt ggaacagctg 60tgccagaagc acaacctccc ctaccgaaac cct 937293DNAArtificial SequencePrimer D8-12B 72gcagagggtt tcggtagggg aggttgtgct tctggcacag ctgttccacc tggtaggaaa 60cggcagtgag gttgtgtcga ggcagagtgg gcc 937390DNAArtificial SequencePrimer D8-13A 73ctgccccatg aaggtctcgt catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc 60gagaagcagc ccgctggcaa ggctctctaa 907490DNAArtificial SequencePrimer D8-13B 74ccgcttagag agccttgcca gcgggctgct tctcggccat tcgagcgaac acggccaggt 60atcggagcag gatgacgaga ccttcatggg 907538DNAArtificial SequencePrimer D8-1F 75tttccatgga gtccaagcga caggctctgt ctcccctc 387637DNAArtificial SequencePrimer D8-3R 76tttagatctt gtacgagtac cagaggggag aggcgtc 377741DNAArtificial SequencePrimer D8-4F 77acaagatctc caccaccctg ggtcttggcg tgcttggata c 417843DNAArtificial SequencePrimer D8-6R 78tttctcgagt gacatcgtcc tcggaccagg cgaggggagg cag 437932DNAArtificial SequencePrimer D8-7F 79tcactcgagc ttctcccatc tcccgaaagc tc 328029DNAArtificial SequencePrimer D8-9R 80cgacgagctc ggaaacaaag aacaccagg 298139DNAArtificial SequencePrimer D8-10F 81tttgagctcg tcggtggctt cggaattgcc atcgtggtc 398236DNAArtificial SequencePrimer D8-13R 82tttgcggccg cttagagagc cttgccagcg ggctgc 3683309DNAArtificial Sequence309 bp NcoI/BglII fragment of pT8(1-3) 83catggagtcc aagcgacagg ctctgtctcc cctccagctg atggaacaga cctacgacgt 60cgtgaacttc caccctggtg gagctgaaat cattgagaac taccagggac gagatgctac 120tgacgccttc atggttatgc actttcagga agccttcgac aagctcaagc gaatgcccaa 180gatcaacccc tcctttgagc tgcctcccca ggctgccgtc aacgaagctc aggaggattt 240ccgaaagctc cgagaagagc tgatcgccac tggcatgttt gacgcctctc ccctctggta 300ctcgtacaa 30984321DNAArtificial Sequence321 bp BglII/XhoI fragment of pT8(4-6) 84gatctccacc accctgggtc ttggcgtgct tggatacttc ctgatggtcc agtaccagat 60gtacttcatt ggtgctgtgc tgctcggtat gcactaccag caaatgggat ggctgtctca 120tgacatctgc caccaccaga ccttcaagaa ccgaaactgg aataacctcg tgggtctggt 180ctttggcaac ggactccagg gcttctccgt gacctgttgg aaggacagac acaacgccca 240tcattctgct accaacgttc agggtcacga tcccgacatt gataacctgc ctcccctcgc 300ctggtccgag gacgatgtca c 32185264DNAArtificial Sequence264 bp XhoI/SacI fragment of pT8(7-9) 85tcgagcttct cccatctccc gaaagctcat tcagttccaa cagtactatt tcctggtcat 60ctgtattctc ctgcgattca tctggtgttt ccagtgcgtg ctgaccgttc gatccctcaa 120ggaccgagac aaccagttct
accgatctca gtacaagaaa gaggccattg gactcgctct 180gcactggact ctcaaggctc tgttccacct cttctttatg ccctccatcc tgacctcgct 240cctggtgttc tttgtttccg agct 26486369DNAArtificial Sequence369 bp Sac1/Not1 fragment of pT8(10-13) 86cgtcggtggc ttcggaattg ccatcgtggt cttcatgaac cactaccctc tggagaagat 60cggtgatccc gtctgggacg gacatggctt ctctgtgggt cagatccatg agaccatgaa 120cattcgacga ggcatcatta ctgactggtt ctttggaggc ctgaactacc agatcgagca 180ccatctctgg cccaccctgc ctcgacacaa cctcactgcc gtttcctacc aggtggaaca 240gctgtgccag aagcacaacc tcccctaccg aaaccctctg ccccatgaag gtctcgtcat 300cctgctccga tacctggccg tgttcgctcg aatggccgag aagcagcccg ctggcaaggc 360tctctaagc 3698734DNAArtificial SequencePrimer ODMW390 87aagaatcatt caccatgaag tccaagcgac aggc 348834DNAArtificial SequencePrimer ODMW391 88gcctgtcgct tggacttcat ggtgaatgat tctt 34891852DNAArtificial Sequencechimeric D8S-1::XPR terminator gene 89cgatcaggag agaccgggtt ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc 60caattgaccc caaattgacc cagtagcggg cccaaccccg gcgagagccc ccttcacccc 120acatatcaaa cctcccccgg ttcccacact tgccgttaag ggcgtagggt actgcagtct 180ggaatctacg cttgttcaga ctttgtacta gtttctttgt ctggccatcc gggtaaccca 240tgccggacgc aaaatagact actgaaaatt tttttgcttt gtggttggga ctttagccaa 300gggtataaaa gaccaccgtc cccgaattac ctttcctctt cttttctctc tctccttgtc 360aactcacacc cgaaatcgtt aagcatttcc ttctgagtat aagaatcatt caccatggag 420tccaagcgac aggctctgtc tcccctccag ctgatggaac agacctacga cgtcgtgaac 480ttccaccctg gtggagctga aatcattgag aactaccagg gacgagatgc tactgacgcc 540ttcatggtta tgcactttca ggaagccttc gacaagctca agcgaatgcc caagatcaac 600ccctcctttg agctgcctcc ccaggctgcc gtcaacgaag ctcaggagga tttccgaaag 660ctccgagaag agctgatcgc cactggcatg tttgacgcct ctcccctctg gtactcgtac 720aagatctcca ccaccctggg tcttggcgtg cttggatact tcctgatggt ccagtaccag 780atgtacttca ttggtgctgt gctgctcggt atgcactacc agcaaatggg atggctgtct 840catgacatct gccaccacca gaccttcaag aaccgaaact ggaataacct cgtgggtctg 900gtctttggca acggactcca gggcttctcc gtgacctgtt ggaaggacag acacaacgcc 960catcattctg ctaccaacgt tcagggtcac gatcccgaca ttgataacct gcctcccctc 1020gcctggtccg aggacgatgt cactcgagct tctcccatct cccgaaagct cattcagttc 1080caacagtact atttcctggt catctgtatt ctcctgcgat tcatctggtg tttccagtgc 1140gtgctgaccg ttcgatccct caaggaccga gacaaccagt tctaccgatc tcagtacaag 1200aaagaggcca ttggactcgc tctgcactgg actctcaagg ctctgttcca cctcttcttt 1260atgccctcca tcctgacctc gctcctggtg ttctttgttt ccgagctcgt cggtggcttc 1320ggaattgcca tcgtggtctt catgaaccac taccctctgg agaagatcgg tgatcccgtc 1380tgggacggac atggcttctc tgtgggtcag atccatgaga ccatgaacat tcgacgaggc 1440atcattactg actggttctt tggaggcctg aactaccaga tcgagcacca tctctggccc 1500accctgcctc gacacaacct cactgccgtt tcctaccagg tggaacagct gtgccagaag 1560cacaacctcc cctaccgaaa ccctctgccc catgaaggtc tcgtcatcct gctccgatac 1620ctggccgtgt tcgctcgaat ggccgagaag cagcccgctg gcaaggctct ctaagcggcc 1680gccaccgccg agattccggc ctcttcggcc gccaagcgac ccgggtggac gtctagaggt 1740acctagcaat taacagatag tttgccggtg ataattctct taacctccca cactcctttg 1800acataacgat ttatgtaacg aaactgaaat ttgaccagat attgtgtccg cg 1852901898DNAArtificial Sequencechimeric D8S-2::XPR terminator gene 90cgatcaggag agaccgggtt ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc 60caattgaccc caaattgacc cagtagcggg cccaaccccg gcgagagccc ccttcacccc 120acatatcaaa cctcccccgg ttcccacact tgccgttaag ggcgtagggt actgcagtct 180ggaatctacg cttgttcaga ctttgtacta gtttctttgt ctggccatcc gggtaaccca 240tgccggacgc aaaatagact actgaaaatt tttttgcttt gtggttggga ctttagccaa 300gggtataaaa gaccaccgtc cccgaattac ctttcctctt cttttctctc tctccttgtc 360aactcacacc cgaaatcgtt aagcatttcc ttctgagtat aagaatcatt caccatgaag 420tccaagcgac aggctctgtc tcccctccag ctgatggaac agacctacga cgtcgtgaac 480ttccaccctg gtggagctga aatcattgag aactaccagg gacgagatgc tactgacgcc 540ttcatggtta tgcactttca ggaagccttc gacaagctca agcgaatgcc caagatcaac 600ccctcctttg agctgcctcc ccaggctgcc gtcaacgaag ctcaggagga tttccgaaag 660ctccgagaag agctgatcgc cactggcatg tttgacgcct ctcccctctg gtactcgtac 720aagatctcca ccaccctggg tcttggcgtg cttggatact tcctgatggt ccagtaccag 780atgtacttca ttggtgctgt gctgctcggt atgcactacc agcaaatggg atggctgtct 840catgacatct gccaccacca gaccttcaag aaccgaaact ggaataacct cgtgggtctg 900gtctttggca acggactcca gggcttctcc gtgacctgtt ggaaggacag acacaacgcc 960catcattctg ctaccaacgt tcagggtcac gatcccgaca ttgataacct gcctcccctc 1020gcctggtccg aggacgatgt cactcgagct tctcccatct cccgaaagct cattcagttc 1080caacagtact atttcctggt catctgtatt ctcctgcgat tcatctggtg tttccagtgc 1140gtgctgaccg ttcgatccct caaggaccga gacaaccagt tctaccgatc tcagtacaag 1200aaagaggcca ttggactcgc tctgcactgg actctcaagg ctctgttcca cctcttcttt 1260atgccctcca tcctgacctc gctcctggtg ttctttgttt ccgagctcgt cggtggcttc 1320ggaattgcca tcgtggtctt catgaaccac taccctctgg agaagatcgg tgatcccgtc 1380tgggacggac atggcttctc tgtgggtcag atccatgaga ccatgaacat tcgacgaggc 1440atcattactg actggttctt tggaggcctg aactaccaga tcgagcacca tctctggccc 1500accctgcctc gacacaacct cactgccgtt tcctaccagg tggaacagct gtgccagaag 1560cacaacctcc cctaccgaaa ccctctgccc catgaaggtc tcgtcatcct gctccgatac 1620ctggccgtgt tcgctcgaat ggccgagaag cagcccgctg gcaaggctct ctaagcggcc 1680gccaccgcgg cccgagattc cggcctcttc ggccgccaag cgacccgggt ggacgtctag 1740aggtacctag caattaacag atagtttgcc ggtgataatt ctcttaacct cccacactcc 1800tttgacataa cgatttatgt aacgaaactg aaatttgacc agatattgtg tccgcggtgg 1860agctccagct tttgttccct ttagtgaggg ttaattaa 18989145DNAArtificial SequencePrimer ODMW392 91gaacagacct acgacgtctc cgcttgggtg aacttccacc ctggt 459245DNAArtificial SequencePrimer ODMW393 92accagggtgg aagttcaccc aagcggagac gtcgtaggtc tgttc 45931269DNAArtificial SequenceD8S-3 synthetic delta 8-desaturase gene codon-optimized for Yarrowia lipolytica in pDMW261 93atgaagtcca agcgacaggc tctgtctccc ctccagctga tggaacagac ctacgacgtc 60tccgcttggg tgaacttcca ccctggtgga gctgaaatca ttgagaacta ccagggacga 120gatgctactg acgccttcat ggttatgcac tttcaggaag ccttcgacaa gctcaagcga 180atgcccaaga tcaacccctc ctttgagctg cctccccagg ctgccgtcaa cgaagctcag 240gaggatttcc gaaagctccg agaagagctg atcgccactg gcatgtttga cgcctctccc 300ctctggtact cgtacaagat ctccaccacc ctgggtcttg gcgtgcttgg atacttcctg 360atggtccagt accagatgta cttcattggt gctgtgctgc tcggtatgca ctaccagcaa 420atgggatggc tgtctcatga catctgccac caccagacct tcaagaaccg aaactggaat 480aacctcgtgg gtctggtctt tggcaacgga ctccagggct tctccgtgac ctgttggaag 540gacagacaca acgcccatca ttctgctacc aacgttcagg gtcacgatcc cgacattgat 600aacctgcctc ccctcgcctg gtccgaggac gatgtcactc gagcttctcc catctcccga 660aagctcattc agttccaaca gtactatttc ctggtcatct gtattctcct gcgattcatc 720tggtgtttcc agtgcgtgct gaccgttcga tccctcaagg accgagacaa ccagttctac 780cgatctcagt acaagaaaga ggccattgga ctcgctctgc actggactct caaggctctg 840ttccacctct tctttatgcc ctccatcctg acctcgctcc tggtgttctt tgtttccgag 900ctcgtcggtg gcttcggaat tgccatcgtg gtcttcatga accactaccc tctggagaag 960atcggtgatc ccgtctggga cggacatggc ttctctgtgg gtcagatcca tgagaccatg 1020aacattcgac gaggcatcat tactgactgg ttctttggag gcctgaacta ccagatcgag 1080caccatctct ggcccaccct gcctcgacac aacctcactg ccgtttccta ccaggtggaa 1140cagctgtgcc agaagcacaa cctcccctac cgaaaccctc tgccccatga aggtctcgtc 1200atcctgctcc gatacctggc cgtgttcgct cgaatggccg agaagcagcc cgctggcaag 1260gctctctaa 12699429DNAArtificial SequencePrimer ODMW404 94cctggtacca tgaagtccaa gcgacaggc 29951272DNAArtificial Sequencechimeric gene 95catgaagtcc aagcgacagg ctctgtctcc cctccagctg atggaacaga cctacgacgt 60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg 120agatgctact gacgccttca tggttatgca ctttcaggaa gccttcgaca agctcaagcg 180aatgcccaag atcaacccct cctttgagct gcctccccag gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360gatggtccag taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca 420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc gaaactggaa 480taacctcgtg ggtctggtct ttggcaacgg actccagggc ttctccgtga cctgttggaa 540ggacagacac aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga 600taacctgcct cccctcgcct ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat 720ctggtgtttc cagtgcgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc tcaaggctct 840gttccacctc ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat cccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat 1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260ggctctctaa gc 12729680DNAArtificial SequencePrimer YL521 96tttccatggt gaagtccaag cgacaggctc tgcccctcac catcgacgga actacctacg 60acgtctccgc ttgggtgaac 809730DNAArtificial SequencePrimer YL522 97tggagatctt gtacgagtac cagaggggag 309837DNAArtificial SequencePrimer YL525 98ccttcatggt tatgcactct caggaagcct tcgacaa 379937DNAArtificial SequencePrimer YL526 99ttgtcgaagg cttcctgaga gtgcataacc atgaagg 3710038DNAArtificial SequencePrimer YL527 100ccaagatcaa cccctcctcc gagctgcctc cccaggct 3810138DNAArtificial SequencePrimer YL528 101agcctgggga ggcagctcgg aggaggggtt gatcttgg 3810237DNAArtificial SequencePrimer YL529 102gggcttctcc gtgacctggt ggaaggacag acacaac 3710337DNAArtificial SequencePrimer YL530 103gttgtgtctg tccttccacc aggtcacgga gaagccc 3710438DNAArtificial SequencePrimer YL531 104acattgataa cctgcctctg ctcgcctggt ccgaggac 3810538DNAArtificial SequencePrimer YL532 105gtcctcggac caggcgagca gaggcaggtt atcaatgt 3810638DNAArtificial SequencePrimer YL533 106tcatctggtg tttccagtct gtgctgaccg ttcgatcc 3810738DNAArtificial SequencePrimer YL534 107ggatcgaacg gtcagcacag actggaaaca ccagatga 3810839DNAArtificial SequencePrimer YL535 108ctgcactgga ctctcaagac cctgttccac ctcttcttt 3910939DNAArtificial SequencePrimer YL536 109aaagaagagg tggaacaggg tcttgagagt ccagtgcag 3911037DNAArtificial SequencePrimer YL537 110ctggagaaga tcggtgattc cgtctgggac ggacatg 3711137DNAArtificial SequencePrimer YL538 111catgtccgtc ccagacggaa tcaccgatct tctccag 371121272DNAArtificial SequenceD8SF synthetic delta-8 desaturase (codon-optimized for Yarrowia lipolytica) 112catggtgaag tccaagcgac aggctctgcc cctcaccatc gacggaacta cctacgacgt 60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg 120agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca agctcaagcg 180aatgcccaag atcaacccct cctccgagct gcctccccag gctgccgtca acgaagctca 240ggaggatttc cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc 300cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360gatggtccag taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca 420aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc gaaactggaa 480taacctcgtg ggtctggtct ttggcaacgg actccagggc ttctccgtga cctggtggaa 540ggacagacac aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga 600taacctgcct ctgctcgcct ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660aaagctcatt cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat 720ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta 780ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc tcaagaccct 840gttccacctc ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga 900gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960gatcggtgat tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat 1020gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact accagatcga 1080gcaccatctc tggcccaccc tgcctcgaca caacctcact gccgtttcct accaggtgga 1140acagctgtgc cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt 1200catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260ggctctctaa gc 1272113422PRTArtificial SequenceD8SF synthetic delta-8 desaturase (codon-optimized for Yarrowialipolytica) 113Met Val Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr1 5 10 15Thr Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val 35 40 45Met His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile 50 55 60Asn Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly 100 105 110Leu Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe 115 120 125Ile Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135 140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn145 150 155 160Asn Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265 270Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375 380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val385 390 395 400Ile Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln 405 410 415Pro Ala Gly Lys Ala Leu 420114995DNAYarrowia lipolytica 114agtgtacgca gtactataga ggaacaattg ccccggagaa gacggccagg ccgcctagat 60gacaaattca acaactcaca gctgactttc tgccattgcc actagggggg ggccttttta 120tatggccaag ccaagctctc cacgtcggtt gggctgcacc caacaataaa tgggtagggt 180tgcaccaaca aagggatggg atggggggta gaagatacga ggataacggg gctcaatggc 240acaaataaga acgaatactg ccattaagac tcgtgatcca gcgactgaca ccattgcatc 300atctaagggc ctcaaaacta cctcggaact gctgcgctga tctggacacc acagaggttc 360cgagcacttt aggttgcacc aaatgtccca ccaggtgcag gcagaaaacg ctggaacagc 420gtgtacagtt tgtcttaaca aaaagtgagg gcgctgaggt cgagcagggt ggtgtgactt 480gttatagcct ttagagctgc gaaagcgcgt atggatttgg ctcatcaggc cagattgagg 540gtctgtggac acatgtcatg ttagtgtact tcaatcgccc cctggatata gccccgacaa 600taggccgtgg cctcattttt ttgccttccg cacatttcca ttgctcggta cccacacctt 660gcttctcctg cacttgccaa ccttaatact ggtttacatt gaccaacatc ttacaagcgg 720ggggcttgtc tagggtatat ataaacagtg gctctcccaa tcggttgcca gtctcttttt 780tcctttcttt ccccacagat tcgaaatcta aactacacat cacacaatgc ctgttactga 840cgtccttaag cgaaagtccg gtgtcatcgt cggcgacgat gtccgagccg tgagtatcca 900cgacaagatc agtgtcgaga cgacgcgttt tgtgtaatga cacaatccga aagtcgctag 960caacacacac tctctacaca aactaaccca gctct 9951158502DNAArtificial SequencePlasmid pY54PC 115ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180gtggagctcc agcttttgtt ccctttagtg agggttaatt aatcgagctt ggcgtaatca 240tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 300gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 360gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct gcattaatga 420atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 480actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 540gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 600cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 660ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 720ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 780ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 840agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 900cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 960aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 1020gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 1080agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 1140ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 1200cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 1260tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 1320aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 1380tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 1440atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 1500cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 1560gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 1620gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 1680tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 1740tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 1800tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 1860aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 1920atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 1980tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 2040catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 2100aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 2160tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 2220gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 2280tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 2340tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg 2400ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 2460cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 2520gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 2580ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 2640ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 2700ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 2760attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 2820aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt 2880gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 2940ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 3000cggccagtga attgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg 3060aggtcgacgg tatcgataag cttgatatcg aattcatgtc acacaaaccg atcttcgcct 3120caaggaaacc taattctaca tccgagagac tgccgagatc cagtctacac tgattaattt 3180tcgggccaat aatttaaaaa aatcgtgtta tataatatta tatgtattat atatatacat 3240catgatgata ctgacagtca tgtcccattg ctaaatagac agactccatc tgccgcctcc 3300aactgatgtt ctcaatattt aaggggtcat ctcgcattgt ttaataataa acagactcca 3360tctaccgcct ccaaatgatg ttctcaaaat atattgtatg aacttatttt tattacttag 3420tattattaga caacttactt gctttatgaa aaacacttcc tatttaggaa acaatttata 3480atggcagttc gttcatttaa caatttatgt agaataaatg ttataaatgc gtatgggaaa 3540tcttaaatat ggatagcata aatgatatct gcattgccta attcgaaatc aacagcaacg 3600aaaaaaatcc cttgtacaac ataaatagtc atcgagaaat atcaactatc aaagaacagc 3660tattcacacg ttactattga gattattatt ggacgagaat cacacactca actgtctttc 3720tctcttctag aaatacaggt acaagtatgt actattctca ttgttcatac ttctagtcat 3780ttcatcccac atattccttg gatttctctc caatgaatga cattctatct tgcaaattca 3840acaattataa taagatatac caaagtagcg gtatagtggc aatcaaaaag cttctctggt 3900gtgcttctcg tatttatttt tattctaatg atccattaaa ggtatatatt tatttcttgt 3960tatataatcc ttttgtttat tacatgggct ggatacataa aggtattttg atttaatttt 4020ttgcttaaat tcaatccccc ctcgttcagt gtcaactgta atggtaggaa attaccatac 4080ttttgaagaa gcaaaaaaaa tgaaagaaaa aaaaaatcgt atttccaggt tagacgttcc 4140gcagaatcta gaatgcggta tgcggtacat tgttcttcga acgtaaaagt tgcgctccct 4200gagatattgt acatttttgc ttttacaagt acaagtacat cgtacaacta tgtactactg 4260ttgatgcatc cacaacagtt tgttttgttt ttttttgttt tttttttttc taatgattca 4320ttaccgctat gtatacctac ttgtacttgt agtaagccgg gttattggcg ttcaattaat 4380catagactta tgaatctgca cggtgtgcgc tgcgagttac ttttagctta tgcatgctac 4440ttgggtgtaa tattgggatc tgttcggaaa tcaacggatg ctcaaccgat ttcgacagta 4500ataatttgaa tcgaatcgga gcctaaaatg aacccgagta tatctcataa aattctcggt 4560gagaggtctg tgactgtcag tacaaggtgc cttcattatg ccctcaacct taccatacct 4620cactgaatgt agtgtacctc taaaaatgaa atacagtgcc aaaagccaag gcactgagct 4680cgtctaacgg acttgatata caaccaatta aaacaaatga aaagaaatac agttctttgt 4740atcatttgta acaattaccc tgtacaaact aaggtattga aatcccacaa tattcccaaa 4800gtccacccct ttccaaattg tcatgcctac aactcatata ccaagcacta acctaccaaa 4860caccactaaa accccacaaa atatatctta ccgaatatac agtaacaagc taccaccaca 4920ctcgttgggt gcagtcgcca gcttaaagat atctatccac atcagccaca actcccttcc 4980tttaataaac cgactacacc cttggctatt gaggttatga gtgaatatac tgtagacaag 5040acactttcaa gaagactgtt tccaaaacgt accactgtcc tccactacaa acacacccaa 5100tctgcttctt ctagtcaagg ttgctacacc ggtaaattat aaatcatcat ttcattagca 5160gggcagggcc ctttttatag agtcttatac actagcggac cctgccggta gaccaacccg 5220caggcgcgtc agtttgctcc ttccatcaat gcgtcgtaga aacgacttac tccttcttga 5280gcagctcctt gaccttgttg gcaacaagtc tccgacctcg gaggtggagg aagagcctcc 5340gatatcggcg gtagtgatac cagcctcgac ggactccttg acggcagcct caacagcgtc 5400accggcgggc ttcatgttaa gagagaactt gagcatcatg gcggcagaca gaatggtggc 5460aatggggttg accttctgct tgccgagatc gggggcagat ccgtgacagg gctcgtacag 5520accgaacgcc tcgttggtgt cgggcagaga agccagagag gcggagggca gcagacccag 5580agaaccgggg atgacggagg cctcgtcgga gatgatatcg ccaaacatgt tggtggtgat 5640gatgatacca ttcatcttgg agggctgctt gatgaggatc atggcggccg agtcgatcag 5700ctggtggttg agctcgagct gggggaattc gtccttgagg actcgagtga cagtctttcg 5760ccaaagtcga gaggaggcca gcacgttggc cttgtcaaga gaccacacgg gaagaggggg 5820gttgtgctga agggccagga aggcggccat tcgggcaatt cgctcaacct caggaacgga 5880gtaggtctcg gtgtcggaag cgacgccaga tccgtcatcc tcctttcgct ctccaaagta 5940gatacctccg acgagctctc ggacaatgat gaagtcggtg ccctcaacgt ttcggatggg 6000ggagagatcg gcgagcttgg gcgacagcag ctggcagggt cgcaggttgg cgtacaggtt 6060caggtccttt cgcagcttga ggagaccctg ctcgggtcgc acgtcggttc gtccgtcggg 6120agtggtccat acggtgttgg cagcgcctcc gacagcaccg agcataatag agtcagcctt 6180tcggcagatg tcgagagtag cgtcggtgat gggctcgccc tccttctcaa tggcagctcc 6240tccaatgagt cggtcctcaa acacaaactc ggtgccggag gcctcagcaa cagacttgag 6300caccttgacg gcctcggcaa tcacctcggg gccacagaag tcgccgccga gaagaacaat 6360cttcttggag tcagtcttgg tcttcttagt ttcgggttcc attgtggatg tgtgtggttg 6420tatgtgtgat gtggtgtgtg gagtgaaaat ctgtggctgg caaacgctct tgtatatata 6480cgcacttttg cccgtgctat gtggaagact aaacctccga agattgtgac tcaggtagtg 6540cggtatcggc tagggaccca aaccttgtcg atgccgatag cgctatcgaa cgtaccccag 6600ccggccggga gtatgtcgga ggggacatac gagatcgtca agggtttgtg gccaactggt 6660aaataaatga tgactcaggc gacgacggaa ttcctgcagc ccatcgatgc agaattcagg 6720agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac 6780cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 6840aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 6900cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 6960gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 7020aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 7080cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaccatgg ctgctgctcc 7140cagtgtgagg acgtttactc gggccgaggt tttgaatgcc gaggctctga atgagggcaa 7200gaaggatgcc gaggcaccct tcttgatgat catcgacaac aaggtgtacg atgtccgcga 7260gttcgtccct gatcatcccg gtggaagtgt gattctcacg cacgttggca aggacggcac 7320tgacgtcttt gacacttttc accccgaggc tgcttgggag actcttgcca acttttacgt 7380tggtgatatt gacgagagcg accgcgatat caagaatgat gactttgcgg ccgaggtccg 7440caagctgcgt accttgttcc agtctcttgg ttactacgat tcttccaagg catactacgc 7500cttcaaggtc tcgttcaacc tctgcatctg gggtttgtcg acggtcattg tggccaagtg 7560gggccagacc tcgaccctcg ccaacgtgct ctcggctgcg cttttgggtc tgttctggca 7620gcagtgcgga tggttggctc acgacttttt gcatcaccag gtcttccagg accgtttctg 7680gggtgatctt ttcggcgcct tcttgggagg tgtctgccag ggcttctcgt cctcgtggtg 7740gaaggacaag cacaacactc accacgccgc ccccaacgtc cacggcgagg atcccgacat 7800tgacacccac cctctgttga cctggagtga gcatgcgttg gagatgttct cggatgtccc 7860agatgaggag ctgacccgca tgtggtcgcg tttcatggtc ctgaaccaga cctggtttta 7920cttccccatt ctctcgtttg cccgtctctc ctggtgcctc cagtccattc tctttgtgct 7980gcctaacggt caggcccaca agccctcggg cgcgcgtgtg cccatctcgt tggtcgagca 8040gctgtcgctt gcgatgcact ggacctggta cctcgccacc atgttcctgt tcatcaagga 8100tcccgtcaac atgctggtgt actttttggt gtcgcaggcg gtgtgcggaa acttgttggc 8160gatcgtgttc tcgctcaacc acaacggtat gcctgtgatc tcgaaggagg aggcggtcga 8220tatggatttc ttcacgaagc agatcatcac gggtcgtgat gtccacccgg gtctatttgc 8280caactggttc acgggtggat tgaactatca gatcgagcac cacttgttcc cttcgatgcc 8340tcgccacaac ttttcaaaga tccagcctgc tgtcgagacc ctgtgcaaaa agtacaatgt 8400ccgataccac accaccggta tgatcgaggg aactgcagag gtctttagcc gtctgaacga 8460ggtctccaag gctacctcca agatgggtaa ggcgcagtaa gc 85021167145DNAArtificial SequencePlasmid pKUNFmkF2 116catggcgtcc acttcggctc tgcccaagca gaaccctgcg cttagacgca ccgtcacctc 60aactactgtg acggattctg agtctgccgc cgtctctcct tcagactctc cccgccactc 120ggcctcttcc acatcgctct cgtccatgtc cgaggttgat atcgccaagc ccaagtccga 180gtatggtgtc atgctcgaca cctacggcaa ccagttcgag gttcccgact ttaccatcaa 240ggacatctac aatgccatcc ctaagcactg cttcaagcgc tccgctctca agggatacgg 300ttatatcctc cgcgacattg tcctcctgac taccactttc agcatctggt acaactttgt 360gacccccgaa tatatcccct ccacccccgc ccgcgctggt ctgtgggccg tgtacaccgt 420tcttcagggt cttttcggta ctggtctctg ggttattgcc catgagtgcg gtcacggtgc 480tttctccgat tctcgcatca tcaacgacat tactggctgg gttcttcact cttccctcct 540tgtcccctac ttcagctggc aaatctccca ccgaaagcac cacaaggcca ctggcaacat 600ggagcgtgac atggtcttcg ttccccgaac ccgcgagcag caggctactc gtctcggaaa 660gatgacccac gagctcgctc atcttactga gnnnntcgtn ggctggccca actacctcat 720caccaatgtt accggccaca actaccacga gcgccagcgt gagggtcgcg gcaagggcaa 780gcataacggc ctcggcggtg gtgttaacca cttcgatccc cgcagccctc tgtacgagaa 840cagtgacgct aagctcatcg tcctcagcga tattggtatc ggtctgatgg ccactgctct 900gtacttcctc gttcagaagt tcggtttcta caacatggcc atctggtact ttgttcccta 960cctctgggtt aaccactggc tcgttgccat caccttcctc cagcacaccg accctaccct 1020tccccactac accaacgacg agtggaactt cgtccgtggt gccgctgcta ccattgaccg 1080tgagatgggc ttcatcggcc gccaccttct ccacggcatc atcgagactc atgtcctcca 1140ccactacgtc agcagcatcc ccttctacaa cgcggacgag gccaccgagg ccattaagcc 1200catcatgggc aagcactacc gggctgatgt ccaggatggt cctcgtggct tcatccgcgc 1260catgtaccgc agtgcgcgta tgtgccagtg ggttgagccc agcgctggtg ccgagggtgc 1320tggtaagggt gttctgttct tccgcaaccg caacaacgtg ggcacccccc ccgctgttat 1380caagcccgtt gcttaagtag gcgcggccgc tatttatcac tctttacaac ttctacctca 1440actatctact ttaataaatg aatatcgttt attctctatg attactgtat atgcgttcct 1500ctaagacaaa tcgaaaccag catgtgatcg aatggcatac aaaagtttct tccgaagttg 1560atcaatgtcc tgatagtcag gcagcttgag aagattgaca caggtggagg ccgtagggaa 1620ccgatcaacc tgtctaccag cgttacgaat ggcaaatgac gggttcaaag ccttgaatcc 1680ttgcaatggt gccttggata ctgatgtcac aaacttaaga agcagccgct tgtcctcttc 1740ctcgatcgat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 1800aacgtacgaa gtcgtcaatg atgtcgatat gggttttgat catgcacaca taaggtccga 1860ccttatcggc aagctcaatg agctccttgg tggtggtaac atccagagaa gcacacaggt 1920tggttttctt ggctgccacg agcttgagca ctcgagcggc aaaggcggac ttgtggacgt 1980tagctcgagc ttcgtaggag ggcattttgg tggtgaagag gagactgaaa taaatttagt 2040ctgcagaact ttttatcgga accttatctg gggcagtgaa gtatatgtta tggtaatagt 2100tacgagttag ttgaacttat agatagactg gactatacgg ctatcggtcc aaattagaaa 2160gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa aatgtgatca tgatgaaagc 2220cagcaatgac gttgcagctg atattgttgt cggccaaccg cgccgaaaac gcagctgtca 2280gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat ccaagcacac tcatagttgg 2340agtcgtactc caaaggcggc aatgacgagt cagacagata ctcgtcgacc ttttccttgg 2400gaaccaccac cgtcagccct tctgactcac gtattgtagc caccgacaca ggcaacagtc 2460cgtggatagc agaatatgtc ttgtcggtcc atttctcacc aactttaggc gtcaagtgaa 2520tgttgcagaa gaagtatgtg ccttcattga gaatcggtgt tgctgatttc aataaagtct 2580tgagatcagt ttggcgcgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 2640tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2700gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 2760ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2820ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 2880acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 2940tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3000ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 3060ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3120ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 3180actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 3240gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 3300tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 3360caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3420atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3480acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 3540ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 3600ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 3660tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 3720tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 3780gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 3840tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 3900tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 3960ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4020tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 4080ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 4140gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 4200ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 4260cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4320ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 4380ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 4440gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 4500ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 4560gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa 4620ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 4680tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 4740atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 4800attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 4860actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 4920tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 4980gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 5040cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 5100tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 5160cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 5220tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc 5280gaattgggcc cgacgtcgca tgcagtggtg gtattgtgac tggggatgta gttgagaata 5340agtcatacac aagtcagctt tcttcgagcc tcatataagt ataagtagtt caacgtatta 5400gcactgtacc cagcatctcc gtatcgagaa acacaacaac atgccccatt ggacagatca 5460tgcggataca caggttgtgc agtatcatac atactcgatc agacaggtcg tctgaccatc 5520atacaagctg aacaagcgct ccatacttgc acgctctcta tatacacagt taaattacat 5580atccatagtc taacctctaa cagttaatct tctggtaagc ctcccagcca gccttctggt 5640atcgcttggc ctcctcaata ggatctcggt tctggccgta cagacctcgg ccgacaatta 5700tgatatccgt tccggtagac atgacatcct caacagttcg gtactgctgt ccgagagcgt 5760ctcccttgtc gtcaagaccc accccggggg tcagaataag ccagtcctca gagtcgccct 5820taattaattt gaatcgaatc gatgagccta aaatgaaccc gagtatatct cataaaattc 5880tcggtgagag gtctgtgact gtcagtacaa ggtgccttca ttatgccctc aaccttacca 5940tacctcactg aatgtagtgt acctctaaaa atgaaataca gtgccaaaag ccaaggcact 6000gagctcgtct aacggacttg atatacaacc aattaaaaca aatgaaaaga aatacagttc 6060tttgtatcat ttgtaacaat taccctgtac aaactaaggt attgaaatcc cacaatattc 6120ccaaagtcca cccctttcca aattgtcatg cctacaactc atataccaag cactaaccta 6180ccgtttaaac agtgtacgca gatctactat agaggaacat ttaaattgcc ccggagaaga 6240cggccaggcc gcctagatga caaattcaac aactcacagc tgactttctg ccattgccac 6300tagggggggg cctttttata tggccaagcc aagctctcca cgtcggttgg gctgcaccca 6360acaataaatg ggtagggttg caccaacaaa gggatgggat ggggggtaga agatacgagg 6420ataacggggc tcaatggcac aaataagaac gaatactgcc attaagactc gtgatccagc 6480gactgacacc attgcatcat ctaagggcct caaaactacc tcggaactgc tgcgctgatc 6540tggacaccac agaggttccg agcactttag gttgcaccaa atgtcccacc aggtgcaggc 6600agaaaacgct ggaacagcgt gtacagtttg tcttaacaaa aagtgagggc gctgaggtcg 6660agcagggtgg tgtgacttgt tatagccttt agagctgcga aagcgcgtat ggatttggct 6720catcaggcca gattgagggt ctgtggacac atgtcatgtt agtgtacttc aatcgccccc 6780tggatatagc cccgacaata ggccgtggcc tcattttttt gccttccgca catttccatt 6840gctcgatacc cacaccttgc ttctcctgca cttgccaacc
ttaatactgg tttacattga 6900ccaacatctt acaagcgggg ggcttgtcta gggtatatat aaacagtggc tctcccaatc 6960ggttgccagt ctcttttttc ctttctttcc ccacagattc gaaatctaaa ctacacatca 7020cagaattccg agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt 7080aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct 7140ggtac 71451175553DNAArtificial SequencePlasmid pZF5T-PPC 117ggccgcattg atgattggaa acacacacat gggttatatc taggtgagag ttagttggac 60agttatatat taaatcagct atgccaacgg taacttcatt catgtcaacg aggaaccagt 120gactgcaagt aatatagaat ttgaccacct tgccattctc ttgcactcct ttactatatc 180tcatttattt cttatataca aatcacttct tcttcccagc atcgagctcg gaaacctcat 240gagcaataac atcgtggatc tcgtcaatag agggcttttt ggactccttg ctgttggcca 300ccttgtcctt gctgtctggc tcattctgtt tcaacgcctt ttaattaatc gagcttggcg 360taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 420atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 480ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 540taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 660aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 720aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 780ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 840acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 960tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1020tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1080gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1140agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1200tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1260agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1320tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1380acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1440tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1560tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1620acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1680tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1740ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1800agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1860tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1920acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1980agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2040actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2100tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2160gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2220ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2280tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2340aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2460tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2520gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2580gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2640acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2700agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2760ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2820ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2880taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2940aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 3000actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3060gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 3120aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 3180ccctcgaggt cgacgtttaa acagtgtacg cagtactata gaggaacatc gattgccccg 3240gagaagacgg ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca 3300ttgccactag gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct 3360gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga 3420tacgaggata acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg 3480atccagcgac tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc 3540gctgatctgg acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg 3600tgcaggcaga aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct 3660gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga 3720tttggctcat caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat 3780cgccccctgg atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat 3840ttccattgct cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt 3900acattgacca acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct 3960cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta 4020cacatcacac aatgcctgtt actgacgtcc ttaagcgaaa gtccggtgtc atcgtcggcg 4080acgatgtccg agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt 4140aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct 4200ccatgggaac ggaccaagga aaaaccttca cctgggaaga gctggcggcc cataacacca 4260aggacgacct actcttggcc atccgcggca gggtgtacga tgtcacaaag ttcttgagcc 4320gccatcctgg tggagtggac actctcctgc tcggagctgg ccgagatgtt actccggtct 4380ttgagatgta tcacgcgttt ggggctgcag atgccattat gaagaagtac tatgtcggta 4440cactggtctc gaatgagctg cccatcttcc cggagccaac ggtgttccac aaaaccatca 4500agacgagagt cgagggctac tttacggatc ggaacattga tcccaagaat agaccagaga 4560tctggggacg atacgctctt atctttggat ccttgatcgc ttcctactac gcgcagctct 4620ttgtgccttt cgttgtcgaa cgcacatggc ttcaggtggt gtttgcaatc atcatgggat 4680ttgcgtgcgc acaagtcgga ctcaaccctc ttcatgatgc gtctcacttt tcagtgaccc 4740acaaccccac tgtctggaag attctgggag ccacgcacga ctttttcaac ggagcatcgt 4800acctggtgtg gatgtaccaa catatgctcg gccatcaccc ctacaccaac attgctggag 4860cagatcccga cgtgtcgacg tctgagcccg atgttcgtcg tatcaagccc aaccaaaagt 4920ggtttgtcaa ccacatcaac cagcacatgt ttgttccttt cctgtacgga ctgctggcgt 4980tcaaggtgcg cattcaggac atcaacattt tgtactttgt caagaccaat gacgctattc 5040gtgtcaatcc catctcgaca tggcacactg tgatgttctg gggcggcaag gctttctttg 5100tctggtatcg cctgattgtt cccctgcagt atctgcccct gggcaaggtg ctgctcttgt 5160tcacggtcgc ggacatggtg tcgtcttact ggctggcgct gaccttccag gcgaaccacg 5220ttgttgagga agttcagtgg ccgttgcctg acgagaacgg gatcatccaa aaggactggg 5280cagctatgca ggtcgagact acgcaggatt acgcacacga ttcgcacctc tggaccagca 5340tcactggcag cttgaactac caggctgtgc accatctgtt ccccaacgtg tcgcagcacc 5400attatcccga tattctggcc atcatcaaga acacctgcag cgagtacaag gttccatacc 5460ttgtcaagga tacgttttgg caagcatttg cttcacattt ggagcacttg cgtgttcttg 5520gactccgtcc caaggaagag taggcagcta agc 5553118792DNAArtificial SequenceIgD9e synthetic delta-9 elongase (codon-optimized for Yarrowia lipolytica) 118atggctctgg ccaacgacgc tggcgagcga atctgggctg ccgtcaccga tcccgaaatc 60ctcattggca ccttctccta cctgctcctg aagcctctcc tgcgaaactc tggtctcgtg 120gacgagaaga aaggagccta ccgaacctcc atgatctggt acaacgtcct cctggctctc 180ttctctgccc tgtccttcta cgtgactgcc accgctctcg gctgggacta cggtactgga 240gcctggctgc gaagacagac cggtgatact ccccagcctc tctttcagtg tccctctcct 300gtctgggact ccaagctgtt cacctggact gccaaggcct tctactattc taagtacgtg 360gagtacctcg acaccgcttg gctggtcctc aagggcaagc gagtgtcctt tctgcaggcc 420ttccatcact ttggagctcc ctgggacgtc tacctcggca ttcgactgca caacgagggt 480gtgtggatct tcatgttctt taactcgttc attcacacca tcatgtacac ctactatgga 540ctgactgccg ctggctacaa gttcaaggcc aagcctctga tcactgccat gcagatttgc 600cagttcgtcg gtggctttct cctggtctgg gactacatca acgttccctg cttcaactct 660gacaagggca agctgttctc ctgggctttc aactacgcct acgtcggatc tgtctttctc 720ctgttctgtc acttctttta ccaggacaac ctggccacca agaaatccgc taaggctggt 780aagcagcttt ag 792119792DNAIsochrysis galbanamisc_featuredelta-9 elongase 119atggccctcg caaacgacgc gggagagcgc atctgggcgg ctgtgaccga cccggaaatc 60ctcattggca ccttctcgta cttgctactc aaaccgctgc tccgcaattc cgggctggtg 120gatgagaaga agggcgcata caggacgtcc atgatctggt acaacgttct gctggcgctc 180ttctctgcgc tgagcttcta cgtgacggcg accgccctcg gctgggacta tggtacgggc 240gcgtggctgc gcaggcaaac cggcgacaca ccgcagccgc tcttccagtg cccgtccccg 300gtttgggact cgaagctctt cacatggacc gccaaggcat tctattactc caagtacgtg 360gagtacctcg acacggcctg gctggtgctc aagggcaaga gggtctcctt tctccaggcc 420ttccaccact ttggcgcgcc gtgggatgtg tacctcggca ttcggctgca caacgagggc 480gtatggatct tcatgttttt caactcgttc attcacacca tcatgtacac ctactacggc 540ctcaccgccg ccgggtataa gttcaaggcc aagccgctca tcaccgcgat gcagatctgc 600cagttcgtgg gcggcttcct gttggtctgg gactacatca acgtcccctg cttcaactcg 660gacaaaggga agttgttcag ctgggctttc aactatgcat acgtcggctc ggtcttcttg 720ctcttctgcc actttttcta ccaggacaac ttggcaacga agaaatcggc caaggcgggc 780aagcagctct ag 792120263PRTIsochrysis galbana 120Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr1 5 10 15Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro 20 25 30Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg 35 40 45Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu 50 55 60Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly65 70 75 80Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln 85 90 95Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr Trp Thr Ala Lys 100 105 110Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125Val Leu Lys Gly Lys Arg Val Ser Phe Leu Gln Ala Phe His His Phe 130 135 140Gly Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly145 150 155 160Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr 165 170 175Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro 180 185 190Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu 195 200 205Val Trp Asp Tyr Ile Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys 210 215 220Leu Phe Ser Trp Ala Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu225 230 235 240Leu Phe Cys His Phe Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser 245 250 255Ala Lys Ala Gly Lys Gln Leu 260121101DNAArtificial SequencePrimer IL3-1A 121gccaacgacg ctggcgagcg aatctgggct gccgtcaccg atcccgaaat cctcattggc 60accttctcct acctgctcct gaagcctctc ctgcgaaact c 101122101DNAArtificial SequencePrimer IL3-1B 122accagagttt cgcaggagag gcttcaggag caggtaggag aaggtgccaa tgaggatttc 60gggatcggtg acggcagccc agattcgctc gccagcgtcg t 101123100DNAArtificial SequencePrimer IL3-2A 123tggtctcgtg gacgagaaga aaggagccta ccgaacctcc atgatctggt acaacgtcct 60cctggctctc ttctctgccc tgtccttcta cgtgactgcc 100124100DNAArtificial SequencePrimer IL3-2B 124cggtggcagt cacgtagaag gacagggcag agaagagagc caggaggacg ttgtaccaga 60tcatggaggt tcggtaggct cctttcttct cgtccacgag 100125100DNAArtificial SequencePrimer IL3-3A 125accgctctcg gctgggacta cggtactgga gcctggctgc gaagacagac cggtgatact 60ccccagcctc tctttcagtg tccctctcct gtctgggact 100126100DNAArtificial SequencePrimer IL3-3B 126ttggagtccc agacaggaga gggacactga aagagaggct ggggagtatc accggtctgt 60cttcgcagcc aggctccagt accgtagtcc cagccgagag 100127100DNAArtificial SequencePrimer IL3-4A 127ccaagctgtt cacctggact gccaaggcct tctactattc taagtacgtg gagtacctcg 60acaccgcttg gctggtcctc aagggcaagc gagtgtcctt 100128100DNAArtificial SequencePrimer IL3-4B 128cagaaaggac actcgcttgc ccttgaggac cagccaagcg gtgtcgaggt actccacgta 60cttagaatag tagaaggcct tggcagtcca ggtgaacagc 10012989DNAArtificial SequencePrimer IL3-5A 129ttccatcact ttggagctcc ctgggacgtc tacctcggca ttcgactgca caacgagggt 60gtgtggatct tcatgttctt taactcgtt 8913089DNAArtificial SequencePrimer IL3-5B 130aatgaacgag ttaaagaaca tgaagatcca cacaccctcg ttgtgcagtc gaatgccgag 60gtagacgtcc cagggagctc caaagtgat 8913191DNAArtificial SequencePrimer IL3-6A 131cattcacacc atcatgtaca cctactatgg actgactgcc gctggctaca agttcaaggc 60caagcctctg atcactgcca tgcagatttg c 9113291DNAArtificial SequencePrimer IL3-6B 132actggcaaat ctgcatggca gtgatcagag gcttggcctt gaacttgtag ccagcggcag 60tcagtccata gtaggtgtac atgatggtgt g 9113394DNAArtificial SequencePrimer IL3-7A 133cagttcgtcg gtggctttct cctggtctgg gactacatca acgttccctg cttcaactct 60gacaagggca agctgttctc ctgggctttc aact 9413494DNAArtificial SequencePrimer IL3-7B 134gcgtagttga aagcccagga gaacagcttg cccttgtcag agttgaagca gggaacgttg 60atgtagtccc agaccaggag aaagccaccg acga 9413591DNAArtificial SequencePrimer IL3-8A 135acgcctacgt cggatctgtc tttctcctgt tctgtcactt cttttaccag gacaacctgg 60ccaccaagaa atccgctaag gctggtaagc a 9113691DNAArtificial SequencePrimer IL3-8B 136aagctgctta ccagccttag cggatttctt ggtggccagg ttgtcctggt aaaagaagtg 60acagaacagg agaaagacag atccgacgta g 9113741DNAArtificial SequencePrimer IL3-1F 137tttccatggc tctggccaac gacgctggcg agcgaatctg g 4113836DNAArtificial SequencePrimer IL3-4R 138tttctgcaga aaggacactc gcttgccctt gaggac 3613941DNAArtificial SequencePrimer IL3-5F 139tttctgcagg ccttccatca ctttggagct ccctgggacg t 4114042DNAArtificial SequencePrimer IL3-8R 140tttgcggccg ctaaagctgc ttaccagcct tagcggattt ct 42141417DNAArtificial Sequence417 bp NcoI/PstI fragment pT9(1-4) 141catggctctg gccaacgacg ctggcgagcg aatctgggct gccgtcaccg atcccgaaat 60cctcattggc accttctcct acctgctcct gaagcctctc ctgcgaaact ctggtctcgt 120ggacgagaag aaaggagcct accgaacctc catgatctgg tacaacgtcc tcctggctct 180cttctctgcc ctgtccttct acgtgactgc caccgctctc ggctgggact acggtactgg 240agcctggctg cgaagacaga ccggtgatac tccccagcct ctctttcagt gtccctctcc 300tgtctgggac tccaagctgt tcacctggac tgccaaggcc ttctactatt ctaagtacgt 360ggagtacctc gacaccgctt ggctggtcct caagggcaag cgagtgtcct ttctgca 417142377DNAArtificial Sequence377 bp PstI/Not1 fragment pT9(5-8) 142ggccttccat cactttggag ctccctggga cgtctacctc ggcattcgac tgcacaacga 60gggtgtgtgg atcttcatgt tctttaactc gttcattcac accatcatgt acacctacta 120tggactgact gccgctggct acaagttcaa ggccaagcct ctgatcactg ccatgcagat 180ttgccagttc gtcggtggct ttctcctggt ctgggactac atcaacgttc cctgcttcaa 240ctctgacaag ggcaagctgt tctcctgggc tttcaactac gcctacgtcg gatctgtctt 300tctcctgttc tgtcacttct tttaccagga caacctggcc accaagaaat ccgctaaggc 360tggtaagcag ctttagc 3771438165DNAArtificial SequencePlasmid pZUF17 143gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
cgtcaatacg ggataatacc 1740gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt 4260agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag 4380gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca 4440cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca 4500gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc 4560tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga 4740tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg 5460ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata 5700ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc 5760gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg 5880ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg 6000gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga 6060caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata 6360tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc 6540cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat 6600gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga 6780taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct 6960gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca 7200tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga 7320ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac 8040atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact 8100cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160gttgc 81651447879DNAArtificial SequencePlasmid pDMW237 144ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080ctctccatgg ctctggccaa cgacgctggc gagcgaatct gggctgccgt caccgatccc 7140gaaatcctca ttggcacctt ctcctacctg ctcctgaagc ctctcctgcg aaactctggt 7200ctcgtggacg agaagaaagg agcctaccga acctccatga tctggtacaa cgtcctcctg 7260gctctcttct ctgccctgtc cttctacgtg actgccaccg ctctcggctg ggactacggt 7320actggagcct ggctgcgaag acagaccggt gatactcccc agcctctctt tcagtgtccc 7380tctcctgtct gggactccaa gctgttcacc tggactgcca aggccttcta ctattctaag 7440tacgtggagt acctcgacac cgcttggctg gtcctcaagg gcaagcgagt gtcctttctg 7500caggccttcc atcactttgg agctccctgg gacgtctacc tcggcattcg actgcacaac 7560gagggtgtgt ggatcttcat gttctttaac tcgttcattc acaccatcat gtacacctac 7620tatggactga ctgccgctgg ctacaagttc aaggccaagc ctctgatcac tgccatgcag 7680atttgccagt tcgtcggtgg ctttctcctg gtctgggact acatcaacgt tccctgcttc 7740aactctgaca agggcaagct gttctcctgg gctttcaact acgcctacgt cggatctgtc 7800tttctcctgt tctgtcactt cttttaccag gacaacctgg ccaccaagaa atccgctaag 7860gctggtaagc agctttagc 78791456457DNAArtificial SequencePlasmid pKUNT2 145tttgaatcga atcgatgagc ctaaaatgaa cccgagtata tctcataaaa ttctcggtga 60gaggtctgtg actgtcagta caaggtgcct tcattatgcc ctcaacctta ccatacctca 120ctgaatgtag tgtacctcta aaaatgaaat acagtgccaa aagccaaggc actgagctcg 180tctaacggac ttgatataca accaattaaa acaaatgaaa agaaatacag ttctttgtat 240catttgtaac aattaccctg tacaaactaa ggtattgaaa tcccacaata ttcccaaagt 300ccaccccttt ccaaattgtc atgcctacaa ctcatatacc aagcactaac ctaccgttta 360aacaccacta aaaccccaca aaatatatct taccgaatat acagatctgc gacgacggaa 420ttcctgcagc ccatctgcag aattcaggag agaccgggtt ggcggcgtat ttgtgtccca 480aaaaacagcc ccaattgccc caattgaccc caaattgacc cagtagcggg cccaaccccg 540gcgagagccc ccttcacccc
acatatcaaa cctcccccgg ttcccacact tgccgttaag 600ggcgtagggt actgcagtct ggaatctacg cttgttcaga ctttgtacta gtttctttgt 660ctggccatcc gggtaaccca tgccggacgc aaaatagact actgaaaatt tttttgcttt 720gtggttggga ctttagccaa gggtataaaa gaccaccgtc cccgaattac ctttcctctt 780cttttctctc tctccttgtc aactcacacc cgaaatcgtt aagcatttcc ttctgagtat 840aagaatcatt caccatggat tcgaccacgc agaccaacac cggcaccggc aaggtggccg 900tgcagccccc cacggccttc attaagccca ttgagaaggt gtccgagccc gtctacgaca 960cctttggcaa cgagttcact cctccagact actctatcaa ggatattctg gatgccattc 1020cccaggagtg ctacaagcgg tcctacgtta agtcctactc gtacgtggcc cgagactgct 1080tctttatcgc cgtttttgcc tacatggcct acgcgtacct gcctcttatt ccctcggctt 1140ccggccgagc tgtggcctgg gccatgtact ccattgtcca gggtctgttt ggcaccggtc 1200tgtgggttct tgcccacgag tgtggccact ctgctttctc cgactctaac accgtcaaca 1260acgtcaccgg atgggttctg cactcctcca tgctggtccc ttactacgcc tggaagctga 1320cccactccat gcaccacaag tccactggtc acctcacccg tgatatggtg tttgtgccca 1380aggaccgaaa ggagtttatg gagaaccgag gcgcccatga ctggtctgag cttgctgagg 1440acgctcccct catgaccctc tacggcctca tcacccagca ggtgtttgga tggcctctgt 1500atctgctgtc ttacgttacc ggacagaagt accccaagct caacaaatgg gctgtcaacc 1560acttcaaccc caacgccccg ctgtttgaga agaaggactg gttcaacatc tggatctcta 1620acgtcggtat tggtatcacc atgtccgtca tcgcatactc catcaaccga tggggcctgg 1680cttccgtcac cctctactac ctgatcccct acctgtgggt caaccactgg ctcgtggcca 1740tcacctacct gcagcacacc gaccccactc tgccccacta ccacgccgac cagtggaact 1800tcacccgagg agccgccgcc accatcgacc gagagtttgg cttcatcggc tccttctgct 1860tccatgacat catcgagacc cacgttctgc accactacgt gtctcgaatt cccttctaca 1920acgcccgaat cgccactgag aagatcaaga aggtcatggg caagcactac cgacacgacg 1980acaccaactt catcaagtct ctttacactg tcgcccgaac ctgccagttt gttgaaggta 2040aggaaggcat tcagatgttt agaaacgtca atggagtcgg agttgctcct gacggcctgc 2100cttctaaaaa gtaggcggcc gcaagtgtgg atggggaagt gagtgcccgg ttctgtgtgc 2160acaattggca atccaagatg gatggattca acacagggat atagcgagct acgtggtggt 2220gcgaggatat agcaacggat atttatgttt gacacttgag aatgtacgat acaagcactg 2280tccaagtaca atactaaaca tactgtacat actcatactc gtacccgggc aacggtttca 2340cttgagtgca gtggctagtg ctcttactcg tacagtgtgc aatactgcgt atcatagtct 2400ttgatgtata tcgtattcat tcatgttagt tgcgtacgaa gtcgtcaatg atgtcgatat 2460gggttttgat catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg 2520tggtggtaac atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca 2580ctcgagcggc aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg 2640tggtgaagag gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg 2700gggcagtgaa gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg 2760gactatacgg ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt 2820tgccgacaaa aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt 2880cggccaaccg cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg 2940tcaaagtgat ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt 3000cagacagata ctcgtcgacc ttttccttgg gaaccaccac cgtcagccct tctgactcac 3060gtattgtagc caccgacaca ggcaacagtc cgtggatagc agaatatgtc ttgtcggtcc 3120atttctcacc aactttaggc gtcaagtgaa tgttgcagaa gaagtatgtg ccttcattga 3180gaatcggtgt tgctgatttc aataaagtct tgagatcagt ttggcgcgcc agctgcatta 3240atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3300gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3360ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3420aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3480ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 3540aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3600gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3660tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3720tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3780gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3840cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3900cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3960agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4020caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4080ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4140aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4200tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4260agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4320gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4380accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4440tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 4500tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 4560acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 4620atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 4680aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 4740tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 4800agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 4860gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 4920ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 4980atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5040tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5100tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5160tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5220tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag 5280cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 5340ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 5400tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 5460gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 5520tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 5580agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 5640gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 5700gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa 5760gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 5820aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 5880agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca tgcagtggtg 5940gtattgtgac tggggatgta gttgagaata agtcatacac aagtcagctt tcttcgagcc 6000tcatataagt ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa 6060acacaacaac atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac 6120atactcgatc agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc 6180acgctctcta tatacacagt taaattacat atccatagtc taacctctaa cagttaatct 6240tctggtaagc ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt 6300tctggccgta cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct 6360caacagttcg gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg 6420tcagaataag ccagtcctca gagtcgccct taattaa 64571461936DNAYarrowia lipolyticaCDS(283)..(1539)delta-12 desaturase 146cgtagttata tacaagaggt agatgcgtgc tggtgttaga ggggctctca ggattaggag 60gaaaatttga cattggccct caacatataa cctcgggtgt gcctctgttt accctcagct 120tttgcttgtc cccaagtcag tcacgccagg ccaaaaaggt tggtggattg acagggagaa 180aaaaaaaagc ctagtgggtt taaactcgag gtaagacatt gaaatatata ccggtcggca 240tcctgagtcc ctttctcgta ttccaacaga ccgaccatag aa atg gat tcg acc 294 Met Asp Ser Thr 1acg cag acc aac acc ggc acc ggc aag gtg gcc gtg cag ccc ccc acg 342Thr Gln Thr Asn Thr Gly Thr Gly Lys Val Ala Val Gln Pro Pro Thr5 10 15 20gcc ttc att aag ccc att gag aag gtg tcc gag ccc gtc tac gac acc 390Ala Phe Ile Lys Pro Ile Glu Lys Val Ser Glu Pro Val Tyr Asp Thr 25 30 35ttt ggc aac gag ttc act cct cca gac tac tct atc aag gat att ctg 438Phe Gly Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile Lys Asp Ile Leu 40 45 50gat gcc att ccc cag gag tgc tac aag cgg tcc tac gtt aag tcc tac 486Asp Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser Tyr Val Lys Ser Tyr 55 60 65tcg tac gtg gcc cga gac tgc ttc ttt atc gcc gtt ttt gcc tac atg 534Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile Ala Val Phe Ala Tyr Met 70 75 80gcc tac gcg tac ctg cct ctt att ccc tcg gct tcc ggc cga gct gtg 582Ala Tyr Ala Tyr Leu Pro Leu Ile Pro Ser Ala Ser Gly Arg Ala Val85 90 95 100gcc tgg gcc atg tac tcc att gtc cag ggt ctg ttt ggc acc ggt ctg 630Ala Trp Ala Met Tyr Ser Ile Val Gln Gly Leu Phe Gly Thr Gly Leu 105 110 115tgg gtt ctt gcc cac gag tgt ggc cac tct gct ttc tcc gac tct aac 678Trp Val Leu Ala His Glu Cys Gly His Ser Ala Phe Ser Asp Ser Asn 120 125 130acc gtc aac aac gtc acc gga tgg gtt ctg cac tcc tcc atg ctg gtc 726Thr Val Asn Asn Val Thr Gly Trp Val Leu His Ser Ser Met Leu Val 135 140 145cct tac tac gcc tgg aag ctg acc cac tcc atg cac cac aag tcc act 774Pro Tyr Tyr Ala Trp Lys Leu Thr His Ser Met His His Lys Ser Thr 150 155 160ggt cac ctc acc cgt gat atg gtg ttt gtg ccc aag gac cga aag gag 822Gly His Leu Thr Arg Asp Met Val Phe Val Pro Lys Asp Arg Lys Glu165 170 175 180ttt atg gag aac cga ggc gcc cat gac tgg tct gag ctt gct gag gac 870Phe Met Glu Asn Arg Gly Ala His Asp Trp Ser Glu Leu Ala Glu Asp 185 190 195gct ccc ctc atg acc ctc tac ggc ctc atc acc cag cag gtg ttt gga 918Ala Pro Leu Met Thr Leu Tyr Gly Leu Ile Thr Gln Gln Val Phe Gly 200 205 210tgg cct ctg tat ctg ctg tct tac gtt acc gga cag aag tac ccc aag 966Trp Pro Leu Tyr Leu Leu Ser Tyr Val Thr Gly Gln Lys Tyr Pro Lys 215 220 225ctc aac aaa tgg gct gtc aac cac ttc aac ccc aac gcc ccg ctg ttt 1014Leu Asn Lys Trp Ala Val Asn His Phe Asn Pro Asn Ala Pro Leu Phe 230 235 240gag aag aag gac tgg ttc aac atc tgg atc tct aac gtc ggt att ggt 1062Glu Lys Lys Asp Trp Phe Asn Ile Trp Ile Ser Asn Val Gly Ile Gly245 250 255 260atc acc atg tcc gtc atc gca tac tcc atc aac cga tgg ggc ctg gct 1110Ile Thr Met Ser Val Ile Ala Tyr Ser Ile Asn Arg Trp Gly Leu Ala 265 270 275tcc gtc acc ctc tac tac ctg atc ccc tac ctg tgg gtc aac cac tgg 1158Ser Val Thr Leu Tyr Tyr Leu Ile Pro Tyr Leu Trp Val Asn His Trp 280 285 290ctc gtg gcc atc acc tac ctg cag cac acc gac ccc act ctg ccc cac 1206Leu Val Ala Ile Thr Tyr Leu Gln His Thr Asp Pro Thr Leu Pro His 295 300 305tac cac gcc gac cag tgg aac ttc acc cga gga gcc gcc gcc acc atc 1254Tyr His Ala Asp Gln Trp Asn Phe Thr Arg Gly Ala Ala Ala Thr Ile 310 315 320gac cga gag ttt ggc ttc atc ggc tcc ttc tgc ttc cat gac atc atc 1302Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe Cys Phe His Asp Ile Ile325 330 335 340gag acc cac gtt ctg cac cac tac gtg tct cga att ccc ttc tac aac 1350Glu Thr His Val Leu His His Tyr Val Ser Arg Ile Pro Phe Tyr Asn 345 350 355gcc cga atc gcc act gag aag atc aag aag gtc atg ggc aag cac tac 1398Ala Arg Ile Ala Thr Glu Lys Ile Lys Lys Val Met Gly Lys His Tyr 360 365 370cga cac gac gac acc aac ttc atc aag tct ctt tac act gtc gcc cga 1446Arg His Asp Asp Thr Asn Phe Ile Lys Ser Leu Tyr Thr Val Ala Arg 375 380 385acc tgc cag ttt gtt gaa ggt aag gaa ggc att cag atg ttt aga aac 1494Thr Cys Gln Phe Val Glu Gly Lys Glu Gly Ile Gln Met Phe Arg Asn 390 395 400gtc aat gga gtc gga gtt gct cct gac ggc ctg cct tct aaa aag 1539Val Asn Gly Val Gly Val Ala Pro Asp Gly Leu Pro Ser Lys Lys405 410 415tagagctaga aatgttattt gattgtgttt taactgaaca gcaccgagcc cgaggctaag 1599ccaagcgaag ccgaggggtt gtgtagtcca tggacgtaac gagtaggcga tatcaccgca 1659ctcggcactg cgtgtctgcg ttcatgggcg aagtcacatt acgctgacaa ccgttgtagt 1719ttccctttag tatcaatact gttacaagta ccggtctcgt actcgtactg atacgaatct 1779gtgggaagaa gtcaccctta tcagaccttc atactgatgt ttcggatatc aatagaactg 1839gcatagagcc gttaaagaag tttcacttaa tcactccaac cctcctactt gtagattcaa 1899gcagatcgat aagatggatt tgatggtcag tgctagc 1936147419PRTYarrowia lipolytica 147Met Asp Ser Thr Thr Gln Thr Asn Thr Gly Thr Gly Lys Val Ala Val1 5 10 15Gln Pro Pro Thr Ala Phe Ile Lys Pro Ile Glu Lys Val Ser Glu Pro 20 25 30Val Tyr Asp Thr Phe Gly Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile 35 40 45Lys Asp Ile Leu Asp Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser Tyr 50 55 60Val Lys Ser Tyr Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile Ala Val65 70 75 80Phe Ala Tyr Met Ala Tyr Ala Tyr Leu Pro Leu Ile Pro Ser Ala Ser 85 90 95Gly Arg Ala Val Ala Trp Ala Met Tyr Ser Ile Val Gln Gly Leu Phe 100 105 110Gly Thr Gly Leu Trp Val Leu Ala His Glu Cys Gly His Ser Ala Phe 115 120 125Ser Asp Ser Asn Thr Val Asn Asn Val Thr Gly Trp Val Leu His Ser 130 135 140Ser Met Leu Val Pro Tyr Tyr Ala Trp Lys Leu Thr His Ser Met His145 150 155 160His Lys Ser Thr Gly His Leu Thr Arg Asp Met Val Phe Val Pro Lys 165 170 175Asp Arg Lys Glu Phe Met Glu Asn Arg Gly Ala His Asp Trp Ser Glu 180 185 190Leu Ala Glu Asp Ala Pro Leu Met Thr Leu Tyr Gly Leu Ile Thr Gln 195 200 205Gln Val Phe Gly Trp Pro Leu Tyr Leu Leu Ser Tyr Val Thr Gly Gln 210 215 220Lys Tyr Pro Lys Leu Asn Lys Trp Ala Val Asn His Phe Asn Pro Asn225 230 235 240Ala Pro Leu Phe Glu Lys Lys Asp Trp Phe Asn Ile Trp Ile Ser Asn 245 250 255Val Gly Ile Gly Ile Thr Met Ser Val Ile Ala Tyr Ser Ile Asn Arg 260 265 270Trp Gly Leu Ala Ser Val Thr Leu Tyr Tyr Leu Ile Pro Tyr Leu Trp 275 280 285Val Asn His Trp Leu Val Ala Ile Thr Tyr Leu Gln His Thr Asp Pro 290 295 300Thr Leu Pro His Tyr His Ala Asp Gln Trp Asn Phe Thr Arg Gly Ala305 310 315 320Ala Ala Thr Ile Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe Cys Phe 325 330 335His Asp Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Arg Ile 340 345 350Pro Phe Tyr Asn Ala Arg Ile Ala Thr Glu Lys Ile Lys Lys Val Met 355 360 365Gly Lys His Tyr Arg His Asp Asp Thr Asn Phe Ile Lys Ser Leu Tyr 370 375 380Thr Val Ala Arg Thr Cys Gln Phe Val Glu Gly Lys Glu Gly Ile Gln385 390 395 400Met Phe Arg Asn Val Asn Gly Val Gly Val Ala Pro Asp Gly Leu Pro 405 410 415Ser Lys Lys 14810448DNAArtificial SequencePlasmid pDMW297 148cgattgcccc ggagaagacg gccaggccgc ctagatgaca aattcaacaa ctcacagctg 60actttctgcc attgccacta ggggggggcc tttttatatg gccaagccaa gctctccacg 120tcggttgggc tgcacccaac aataaatggg tagggttgca ccaacaaagg gatgggatgg 180ggggtagaag atacgaggat aacggggctc aatggcacaa ataagaacga atactgccat 240taagactcgt gatccagcga ctgacaccat tgcatcatct aagggcctca aaactacctc 300ggaactgctg cgctgatctg gacaccacag aggttccgag cactttaggt tgcaccaaat 360gtcccaccag gtgcaggcag aaaacgctgg aacagcgtgt acagtttgtc ttaacaaaaa 420gtgagggcgc tgaggtcgag cagggtggtg tgacttgtta tagcctttag agctgcgaaa 480gcgcgtatgg atttggctca tcaggccaga ttgagggtct gtggacacat gtcatgttag 540tgtacttcaa tcgccccctg gatatagccc cgacaatagg ccgtggcctc atttttttgc 600cttccgcaca tttccattgc tcggtaccca caccttgctt ctcctgcact tgccaacctt 660aatactggtt tacattgacc aacatcttac aagcgggggg cttgtctagg gtatatataa 720acagtggctc tcccaatcgg ttgccagtct cttttttcct ttctttcccc acagattcga 780aatctaaact acacatcaca caatgcctgt tactgacgtc cttaagcgaa agtccggtgt 840catcgtcggc gacgatgtcc gagccgtgag tatccacgac aagatcagtg tcgagacgac 900gcgttttgtg taatgacaca atccgaaagt cgctagcaac acacactctc tacacaaact 960aacccagctc tccatggtga agtccaagcg acaggctctg cccctcacca tcgacggaac 1020tacctacgac gtctccgctt gggtgaactt ccaccctggt ggagctgaaa tcattgagaa 1080ctaccaggga cgagatgcta ctgacgcctt catggttatg cactctcagg aagccttcga 1140caagctcaag cgaatgccca agatcaaccc ctcctccgag ctgcctcccc aggctgccgt 1200caacgaagct caggaggatt tccgaaagct ccgagaagag ctgatcgcca ctggcatgtt 1260tgacgcctct cccctctggt actcgtacaa gatctccacc accctgggtc ttggcgtgct 1320tggatacttc ctgatggtcc agtaccagat gtacttcatt ggtgctgtgc tgctcggtat 1380gcactaccag caaatgggat ggctgtctca tgacatctgc caccaccaga ccttcaagaa 1440ccgaaactgg aataacctcg tgggtctggt ctttggcaac ggactccagg gcttctccgt 1500gacctggtgg aaggacagac acaacgccca tcattctgct accaacgttc agggtcacga 1560tcccgacatt gataacctgc
ctctgctcgc ctggtccgag gacgatgtca ctcgagcttc 1620tcccatctcc cgaaagctca ttcagttcca acagtactat ttcctggtca tctgtattct 1680cctgcgattc atctggtgtt tccagtctgt gctgaccgtt cgatccctca aggaccgaga 1740caaccagttc taccgatctc agtacaagaa agaggccatt ggactcgctc tgcactggac 1800tctcaagacc ctgttccacc tcttctttat gccctccatc ctgacctcgc tcctggtgtt 1860ctttgtttcc gagctcgtcg gtggcttcgg aattgccatc gtggtcttca tgaaccacta 1920ccctctggag aagatcggtg attccgtctg ggacggacat ggcttctctg tgggtcagat 1980ccatgagacc atgaacattc gacgaggcat cattactgac tggttctttg gaggcctgaa 2040ctaccagatc gagcaccatc tctggcccac cctgcctcga cacaacctca ctgccgtttc 2100ctaccaggtg gaacagctgt gccagaagca caacctcccc taccgaaacc ctctgcccca 2160tgaaggtctc gtcatcctgc tccgatacct ggccgtgttc gctcgaatgg ccgagaagca 2220gcccgctggc aaggctctct aagcggccgc attgatgatt ggaaacacac acatgggtta 2280tatctaggtg agagttagtt ggacagttat atattaaatc agctatgcca acggtaactt 2340cattcatgtc aacgaggaac cagtgactgc aagtaatata gaatttgacc accttgccat 2400tctcttgcac tcctttacta tatctcattt atttcttata tacaaatcac ttcttcttcc 2460cagcatcgag ctcggaaacc tcatgagcaa taacatcgtg gatctcgtca atagagggct 2520ttttggactc cttgctgttg gccaccttgt ccttgctgtc tggctcattc tgtttcaacg 2580ccttttaatt aagtcataca caagtcagct ttcttcgagc ctcatataag tataagtagt 2640tcaacgtatt agcactgtac ccagcatctc cgtatcgaga aacacaacaa catgccccat 2700tggacagatc atgcggatac acaggttgtg cagtatcata catactcgat cagacaggtc 2760gtctgaccat catacaagct gaacaagcgc tccatacttg cacgctctct atatacacag 2820ttaaattaca tatccatagt ctaacctcta acagttaatc ttctggtaag cctcccagcc 2880agccttctgg tatcgcttgg cctcctcaat aggatctcgg ttctggccgt acagacctcg 2940gccgacaatt atgatatccg ttccggtaga catgacatcc tcaacagttc ggtactgctg 3000tccgagagcg tctcccttgt cgtcaagacc caccccgggg gtcagaataa gccagtcctc 3060agagtcgccc ttaggtcggt tctgggcaat gaagccaacc acaaactcgg ggtcggatcg 3120ggcaagctca atggtctgct tggagtactc gccagtggcc agagagccct tgcaagacag 3180ctcggccagc atgagcagac ctctggccag cttctcgttg ggagagggga ctaggaactc 3240cttgtactgg gagttctcgt agtcagagac gtcctccttc ttctgttcag agacagtttc 3300ctcggcacca gctcgcaggc cagcaatgat tccggttccg ggtacaccgt gggcgttggt 3360gatatcggac cactcggcga ttcggtgaca ccggtactgg tgcttgacag tgttgccaat 3420atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc ttaagagcaa gttccttgag 3480ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg tcgatatggg ttttgatcat 3540gcacacataa ggtccgacct tatcggcaag ctcaatgagc tccttggtgg tggtaacatc 3600cagagaagca cacaggttgg ttttcttggc tgccacgagc ttgagcactc gagcggcaaa 3660ggcggacttg tggacgttag ctcgagcttc gtaggagggc attttggtgg tgaagaggag 3720actgaaataa atttagtctg cagaactttt tatcggaacc ttatctgggg cagtgaagta 3780tatgttatgg taatagttac gagttagttg aacttataga tagactggac tatacggcta 3840tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg tcgcctttgc cgacaaaaat 3900gtgatcatga tgaaagccag caatgacgtt gcagctgata ttgttgtcgg ccaaccgcgc 3960cgaaaacgca gctgtcagac ccacagcctc caacgaagaa tgtatcgtca aagtgatcca 4020agcacactca tagttggagt cgtactccaa aggcggcaat gacgagtcag acagatactc 4080gtcgactcag gcgacgacgg aattcctgca gcccatctgc agaattcagg agagaccggg 4140ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccggagaag acggccaggc 4200cgcctagatg acaaattcaa caactcacag ctgactttct gccattgcca ctaggggggg 4260gcctttttat atggccaagc caagctctcc acgtcggttg ggctgcaccc aacaataaat 4320gggtagggtt gcaccaacaa agggatggga tggggggtag aagatacgag gataacgggg 4380ctcaatggca caaataagaa cgaatactgc cattaagact cgtgatccag cgactgacac 4440cattgcatca tctaagggcc tcaaaactac ctcggaactg ctgcgctgat ctggacacca 4500cagaggttcc gagcacttta ggttgcacca aatgtcccac caggtgcagg cagaaaacgc 4560tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg cgctgaggtc gagcagggtg 4620gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta tggatttggc tcatcaggcc 4680agattgaggg tctgtggaca catgtcatgt tagtgtactt caatcgcccc ctggatatag 4740ccccgacaat aggccgtggc ctcatttttt tgccttccgc acatttccat tgctcggtac 4800ccacaccttg cttctcctgc acttgccaac cttaatactg gtttacattg accaacatct 4860tacaagcggg gggcttgtct agggtatata taaacagtgg ctctcccaat cggttgccag 4920tctctttttt cctttctttc cccacagatt cgaaatctaa actacacatc acacaatgcc 4980tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc ggcgacgatg tccgagccgt 5040gagtatccac gacaagatca gtgtcgagac gacgcgtttt gtgtaatgac acaatccgaa 5100agtcgctagc aacacacact ctctacacaa actaacccag ctctccatgg ctctggccaa 5160cgacgctggc gagcgaatct gggctgccgt caccgatccc gaaatcctca ttggcacctt 5220ctcctacctg ctcctgaagc ctctcctgcg aaactctggt ctcgtggacg agaagaaagg 5280agcctaccga acctccatga tctggtacaa cgtcctcctg gctctcttct ctgccctgtc 5340cttctacgtg actgccaccg ctctcggctg ggactacggt actggagcct ggctgcgaag 5400acagaccggt gatactcccc agcctctctt tcagtgtccc tctcctgtct gggactccaa 5460gctgttcacc tggactgcca aggccttcta ctattctaag tacgtggagt acctcgacac 5520cgcttggctg gtcctcaagg gcaagcgagt gtcctttctg caggccttcc atcactttgg 5580agctccctgg gacgtctacc tcggcattcg actgcacaac gagggtgtgt ggatcttcat 5640gttctttaac tcgttcattc acaccatcat gtacacctac tatggactga ctgccgctgg 5700ctacaagttc aaggccaagc ctctgatcac tgccatgcag atttgccagt tcgtcggtgg 5760ctttctcctg gtctgggact acatcaacgt tccctgcttc aactctgaca agggcaagct 5820gttctcctgg gctttcaact acgcctacgt cggatctgtc tttctcctgt tctgtcactt 5880cttttaccag gacaacctgg ccaccaagaa atccgctaag gctggtaagc agctttagcg 5940gccgcaagtg tggatgggga agtgagtgcc cggttctgtg tgcacaattg gcaatccaag 6000atggatggat tcaacacagg gatatagcga gctacgtggt ggtgcgagga tatagcaacg 6060gatatttatg tttgacactt gagaatgtac gatacaagca ctgtccaagt acaatactaa 6120acatactgta catactcata ctcgtacccg ggcaacggtt tcacttgagt gcagtggcta 6180gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt atatcgtatt 6240cattcatgtt agttgcgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 6300agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 6360gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6420gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6480ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6540aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6600ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6660gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6720cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6780gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6840tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6900cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6960cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7020gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 7080agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7140cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 7200tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 7260tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 7320ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 7380cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 7440cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 7500accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 7560ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7620ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 7680tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 7740acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 7800tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 7860actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7920ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 7980aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 8040ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 8100cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 8160aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 8220actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 8280cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 8340ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 8400tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 8460cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 8520tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 8580tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 8640cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 8700ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 8760gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg 8820ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 8880cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 8940cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa 9000ttgggtaccg ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg 9060tcacacaaac cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga 9120tccagtctac actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat 9180tatatgtatt atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag 9240acagactcca tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt 9300gtttaataat aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta 9360tgaacttatt tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt 9420cctatttagg aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa 9480tgttataaat gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc 9540taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa 9600atatcaacta tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga 9660atcacacact caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct 9720cattgttcat acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat 9780gacattctat cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg 9840gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta 9900aaggtatata tttatttctt gttatataat ccttttgttt attacatggg ctggatacat 9960aaaggtattt tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg 10020taatggtagg aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc 10080gtatttccag gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac attgttcttc 10140gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac 10200atcgtacaac tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt 10260tttttttttt tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc 10320gggttattgg cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt 10380acttttagct tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga 10440tgctcaat 1044814928DNAArtificial SequencePrimer oIGsel1-1 149agcggccgca ccatggctct ggccaacg 2815025DNAArtificial SequencePrimer oIGsel1-2 150tgcggccgct aaagctgctt accag 2515169DNAArtificial SequencePrimer 151catggtcaat caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60cggatcccc 69
Patent applications by Howard Glenn Damude, Hockessin, DE US
Patent applications by Quinn Qun Zhu, West Chester, PA US
Patent applications by E.I. DU PONT DE NEMOURS AND COMPANY
Patent applications in class Legume, nut, or seed source material (e.g., peanut, soya bean, rice bran, etc.)
Patent applications in all subclasses Legume, nut, or seed source material (e.g., peanut, soya bean, rice bran, etc.)