Patent application title: Production Of Fatty Acid Derivatives
Inventors:
Derek L. Greenfield (South San Francisco, CA, US)
Derek L. Greenfield (South San Francisco, CA, US)
Andreas W. Schirmer (Hayward, CA, US)
Elizabeth J. Clarke (South San Francisco, CA, US)
Eli S. Groban (South San Francisco, CA, US)
Bernardo M. Da Costa (South San Francisco, CA, US)
Zhihao Hu (Zhajalgang, CN)
Kevin Holden (South San Francisco, CA, US)
Noah Helman (South San Francisco, CA, US)
IPC8 Class: AC12N1570FI
USPC Class:
1 1
Class name:
Publication date: 2021-11-11
Patent application number: 20210348173
Abstract:
The disclosure relates to recombinant host cells including strain
modifications effective to improve titer, yield and/or productivity of
fatty acid derivatives. The disclosure further relates to cell cultures
including the recombinant host cells for the fermentative production of
fatty acid derivatives and compositions thereof.Claims:
1.-26. (canceled)
27. A recombinant host cell comprising a genetically engineered polynucleotide sequence encoding a phosphoenolpyruvate carboxylase (ppc) polypeptide, wherein said recombinant host cell produces a fatty acid derivative composition at a higher titer, yield or productivity than a corresponding wild type host cell when cultured in a medium containing a carbon source under conditions effective to express said ppc polypeptide.
28. A cell culture comprising the recombinant host cell according to claim 27.
29. The cell culture of claim 28, wherein said medium comprises a fatty acid derivative composition.
30. The cell culture of claim 29, wherein the fatty acid derivative composition comprises at least one fatty acid derivative selected from the group consisting of fatty acid, a fatty ester, a fatty alcohol, a fatty aldehyde, an alkane, a terminal olefin, an internal olefin, and a ketone.
31. The cell culture of claim 28, wherein the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative.
32. The cell culture of claim 28, wherein the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative.
33. The cell culture of claim 29, wherein the fatty acid derivative composition comprises one or more of C8, C10, C12, C14, C16, and C18 fatty acid derivatives.
34. The cell culture of claim 29, wherein the fatty acid derivative composition comprises fatty acids.
35.-41. (canceled)
42. The cell culture of claim 29, wherein the fatty acid derivative composition comprises fatty acid derivatives having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty alcohol.
43. The cell culture of claim 29, wherein the fatty acid derivative composition comprises unsaturated fatty acid derivatives.
44. The cell culture of claim 29, wherein the fatty acid derivative composition comprises saturated fatty acid derivatives.
45. The cell culture of claim 29, wherein the fatty acid derivative composition comprises branched chain fatty acid derivatives.
46. The cell culture of claim 28, wherein said recombinant host cell has a titer that is at least about 5% greater than a titer of said corresponding wild type host cell when cultured under the same conditions as the recombinant host cell.
47. The cell culture of claim 46, wherein said recombinant host cell has a titer of from about 1 g/L to about 250 g/L.
48. The cell culture of claim 47, wherein said recombinant host cell has a titer of from about 90 g/L to about 120 g/L.
49. The cell culture of claim 28, wherein said recombinant host cell has a yield that is at least about 10% to about 40%.
50. The cell culture of claim 49, wherein said recombinant host cell has a yield of about 25%.
51. The cell culture of claim 28, wherein said productivity ranges from about 0.7 mg/L/hr to about 3 g/L/hr.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 61/619,324 filed Apr. 2, 2012, and is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 2, 2013, is named LS00042PCT_SL.txt and is 143,098 bytes in size.
FIELD
[0003] The disclosure relates to recombinant host cells including strain modifications effective to improve titer, yield and/or productivity of fatty acid derivatives. The disclosure further relates to cell cultures including the recombinant host cells for the fermentative production of fatty acid derivatives and compositions thereof.
BACKGROUND
[0004] Fatty acid derivatives including fatty aldehydes, fatty alcohols, hydrocarbons (alkanes and olefins), fatty esters (e.g., waxes, fatty acid esters, or fatty esters), and ketones denote important categories of industrial chemicals and fuels. These molecules and their derivatives have numerous applications including, but not limited to, use as surfactants, lubricants, plasticizers, solvents, emulsifiers, emollients, thickeners, flavors, fragrances, and fuels. Crude petroleum is currently a primary source of raw materials for producing petrochemicals and fuels. The two main classes of raw materials derived from petroleum are short chain olefins (e.g., ethylene and propylene) and aromatics (e.g., benzene and xylene isomers). These raw materials are derived from longer chain hydrocarbons in crude petroleum by cracking it at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials can be used to make petrochemicals such as monomers, solvents, detergents, and adhesives, which otherwise cannot be directly refined from crude petroleum. Petrochemicals, in turn, can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, gels, and the like. Particular specialty chemicals that can be produced from petrochemical raw materials include, but are not limited to, fatty acids, hydrocarbons, fatty aldehydes, fatty alcohols, esters, and ketones.
[0005] Hydrocarbons, for example, have many commercial uses. As such, shorter chain alkanes and alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenyl succinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers. Similarly, esters have many commercial uses. For example, biodiesel, an alternative fuel, is made of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are also made of esters. Esters are further used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
[0006] Aldehydes are used to produce a large number of specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and compounds used as hormones are aldehydes. Furthermore, many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction. Similarly, fatty alcohols have many commercial uses as well. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats. Fatty alcohols such as aliphatic alcohols include a chain of 8 to 22 carbon atoms. Fatty alcohols usually have an even number of carbon atoms and a single alcohol group (--OH) attached to the terminal carbon. Some are unsaturated and some are branched. They are widely used in industrial chemistry. Most fatty alcohols in nature are found as waxes which are esters with fatty acids and fatty alcohols. They are produced by bacteria, plants and animals. Currently, fatty alcohols are produced via catalytic hydrogenation of fatty acids produced from natural sources, such as coconut oil, palm oil, palm kernel oil, tallow and lard, or by chemical hydration of alpha-olefins produced from petrochemical feedstocks. Fatty alcohols derived from natural sources have varying chain lengths. The chain length of fatty alcohols is important and specific to particular applications. Dehydration of fatty alcohols to alpha-olefins can also be accomplished by chemical catalysis.
[0007] Due to the inherent challenges posed by exploring, extracting, transporting and refining petroleum for use in chemical- and fuel products, there is a need in the art for a an alternate source which can be produced economically and efficiently for the use of chemical- and fuel production. Moreover, the burning of petroleum-based fuels has become a serious hazard to the environment, especially in light of the ever increasing population inhabiting the planet. Thus, there is a need for a petroleum replacement that does not cause the type of environmental damage created by exploring, extracting, transporting and refining petroleum.
[0008] One option of producing renewable petroleum is by engineering host cells to produce renewable petroleum products. Biologically derived fuels and chemicals offer advantages over petroleum based fuels. Biologically derived chemicals such as hydrocarbons (e.g., alkanes, alkenes, or alkynes), fatty alcohols, esters, fatty acids, fatty aldehydes, and ketones are directly converted from biomass to the desired chemical product. However, in order for the use of biologically-derived fatty acid derivatives from fermentable sugars or biomass to be commercially viable as a source for production of renewable chemicals and fuels, the process must be optimized for efficient conversion and recovery of product. The development of biologically derived fuels and chemicals has been one focus of research and development in recent years. Still, there remains a considerable need for improvements in the relevant processes and products in order for biologically-derived fuels and chemicals to become a commercially viable option. Areas that need improvement include the energy efficiency of the production process and the final product yield. The current disclosure addresses this need.
SUMMARY
[0009] One aspect of the disclosure provides a recombinant host cell having a genetically engineered polynucleotide sequence, wherein the polynucleotide sequence codes for one or more polypeptides that have a specific enzymatic activity. The polynucleotide sequence is exogenous or endogenous to the host cell. As such, the disclosure provides a recombinant host cell having a genetically engineered polynucleotide sequence encoding one or more polypeptides, wherein the polypeptides have activity selected from the group including, but not limited to, 3-hydroxydecanoyl-[acp] dehydratase (E.C. 4.2.1.60) activity; .beta.-ketoacyl-ACP synthase I (E.C. 2.3.1.41) activity; .beta.-ketoacyl-ACP synthase II (E.C. 2.3.1.179) activity; [acp] S-malonyltransferase {malonyl-CoA-ACP transacylase} (E.C. 2.3.1.39) activity; 3-oxoacyl-{.beta.-ketoacyl}-ACP reductase (E.C. 1.1.1.100) activity; .beta.-ketoacyl-ACP synthase III (E.C. 2.3.1.180) activity; enoyl-ACP reductase (NADH) (E.C. 1.3.1.9) activity; enoyl-ACP reductase (NADPH) (E.C. 1.3.1.10) activity; 3-hydroxy-acyl-[acp] dehydratase (E.C. 4.2.1.59) activity; and trans-2, cis-3-decenoyl-ACP isomerase (E.C. 5.3.3.14) activity, wherein the recombinant host cell produce a fatty acid derivative composition at a higher titer, yield or productivity than a corresponding wild type host cell when cultured in a medium containing a carbon source under conditions effective to express the polynucleotide. In a related aspect, the recombinant host cell produces the fatty acid derivative composition at a higher titer, yield and/or productivity when the polypeptide is expressed in combination with at least one other polypeptide of the enzymatic activity. In another aspect, the recombinant host cell produces the fatty acid derivative composition at a higher titer, yield or productivity when the polypeptide is expressed in combination with at least five other polypeptides of the enzymatic activity. In yet another aspect, the recombinant host cell produces the fatty acid derivative composition at a higher titer, yield or productivity when expressed in combination with at least two or three or four or five or six or more polypeptides of the enzymatic activity. In another related aspect, the recombinant host cell includes one or more genetically engineered polynucleotide sequences that further code for a polypeptide that is an acyl carrier protein (ACP). ACP can be in expressed in combination with one or more of the polypeptides that code for any of the enzymatic activities, wherein the ACP further increases the titer, yield and/or productivity of the recombinant host cell when cultured under appropriate conditions. In yet another related aspect, a genetically engineered polynucleotide sequence further encodes a polypeptide that has accABCD activity (E.C. 6.4.1.2). accABCD can be in expressed in combination with one or more of the polypeptides that code for any of the enzymatic activities, wherein the accABCD further increases the titer, yield and/or productivity of the recombinant host cell when cultured under appropriate conditions.
[0010] Another aspect of the disclosure provides a recombinant host cell having a genetically engineered polynucleotide sequence encoding one or more polypeptides, wherein the polypeptides have enzymatic activity including, but not limited to, trans-2, cis-3-decenoyl-ACP isomerase activity (fabA or fabM); .beta.-ketoacyl-ACP synthase I (fabB); malonyl-CoA-ACP transacylase (fabD); .beta.-ketoacyl-ACP synthase I (fabF or fabB); .beta.-ketoacyl-ACP reductase (fabG); .beta.-ketoacyl-ACP synthase III (fabH); enoyl-ACP reductase (fabl or fabL or fabV or fabK); and 3-hydrox-acyl-[acp] dehydratase (fabA or fabZ); trans-2-enoyl-ACP reductase II (fabK). In a related aspect, the polypeptide is selected from fabA, fabB, fabD, fabF, fabG, fabH, fabl, fabL, fabV, fabZ, fabM, and fabK and or combinations thereof. In yet another related aspect, the polypeptide is selected from FabA from Salmonella typhimurium (NP 460041); FabB from Escherichia coli (NP_416826); FabD from Salmonella typhimurium (NP_460164); FabG from Salmonella typhimurium (NP_460165); FabH from Salmonella typhimurium (NP_460163); FabZ from Salmonella typhimurium (NP_459232); FabM from Streptococcus mutans (AAN59379); FabK from Streptococcus pneumoniae (AAF98273); FabV from Vibrio cholera (YP_001217283); FabF from Clostridium acetobutylicum (NP_350156); FabI from Bacillus subtillis subsp. subtilis str. 168 (NP_389054); FabL from Bacillus subtillis subsp. subtilis str. 168 (NP_388745); FabI from Acinetobacter sp. ADP1 (YP_047630); FabI from Marinobacter aquaeoli VT8 (YP_958813); FabI from Rhodococcus opacus B4 (YP_002784194); FabH from Acinetobacter sp. ADP1 (YP_046731); FabH from Marinobacter aquaeoli VT8 (YP_958649); and FabH from Rhodococcus opacus B4 (YP_00278448) or combinations thereof.
[0011] The disclosure further contemplates a recombinant host cell having a genetically engineered polynucleotide sequence encoding an ACP polypeptide, wherein the recombinant host cell produces a fatty acid derivative composition at a higher titer, yield or productivity than a corresponding wild type host cell when cultured in a medium containing a carbon source under conditions effective to express the ACP polypeptide. In a related aspect, the genetically engineered polynucleotide sequence further encodes a polypeptide that has phosphopantetheinyl transferase (E.C. 2.7.8.7) activity. Herein, the genetically engineered polynucleotide sequence includes a sfp gene coding encoding a phosphopantetheinyl transferase (E.C. 2.7.8.7). In a related aspect, a genetically engineered polynucleotide sequence further encodes a polypeptide that has accABCD activity (E.C. 6.4.1.2). ACP can be in expressed in combination with accABCD and/or a phosphopantetheinyl transferase, wherein the combination of any of the expressed polypeptides further leads to increases in the titer, yield and/or productivity of the recombinant host cell when cultured under appropriate conditions. In another related aspect, ACP is derived from the same organism as a terminal pathway enzyme expressed in the recombinant host cell, wherein the terminal enzyme cleaves any acyl-ACP species that is part of the fatty acid biosynthetic pathway. The ACP is exogenous or endogenous to the host cell.
[0012] The disclosure further encompasses a recombinant host cell including a genetically engineered polynucleotide sequence including a transposon, wherein insertion of the transposon into a yijP gene affects a second gene flanking the yijP gene, wherein the second gene codes for a polynucleotide that is up- or down regulated, and wherein the up- or down regulated polynucleotide codes for a polypeptide that affects production of a fatty acid derivative composition when the host cell is cultured in a medium containing a carbon source under conditions effective to express the polypeptide. The yijP gene can be flanked by genes on either side. In a related aspect, the insertion of the transposon into the yijP gene results in inactivation of the yijP gene or a polynucleotide thereof, which affects one or more of the genes flanking the yijP gene, wherein the flanking gene or genes code for a polypeptide that affects production of a fatty acid derivative composition when the host cell is cultured in a medium containing a carbon source under conditions effective to express the polypeptide. In one related aspect, the flanking gene includes polynucleotides including, but not limited to, ppc, yijO, frwD, pflC, pflD or argE.
[0013] Another aspect of the disclosure provides a recombinant host cell including a genetically engineered polynucleotide sequence encoding a phosphoenolpyruvate carboxylase (ppc) polypeptide, wherein the recombinant host cell produces a fatty acid derivative composition at a higher titer, yield or productivity than a corresponding wild type host cell when cultured in a medium containing a carbon source under conditions effective to express the ppc polypeptide.
[0014] Still, another aspect of the disclosure provides a cell culture that includes any of the recombinant host cells presented herein (supra). The recombinant host cell is cultured in a medium such that the recombinant host cell produces fatty acid derivative compositions according to the genetic engineering methods presented herein (supra). In a related aspect, the fatty acid derivative compositions produced by the recombinant host cells of the present disclosure include, but are not limited to, fatty acids, fatty esters, fatty alcohols, fatty aldehydes, alkanes, terminal olefins, internal olefins, and ketones. In another related aspect, the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative. In yet another related aspect, the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In a further related aspect, the fatty acid derivative composition comprises one or more of C8, C10, C12, C14, C16, and C18 fatty acid derivatives. The fatty acid derivative compositions produced by the cell cultures containing the recombinant host cells of the present disclosure include fatty acids, fatty aldehydes, fatty alcohols, fatty esters, alkanes, terminal olefins, internal olefins, and ketones. The disclosure further encompasses fatty acid derivative compositions that include fatty acid derivatives having a double bond at position 7 in the carbon chain between C7 and C8 from the reduced end of the fatty alcohol; fatty acid derivative compositions including unsaturated fatty acid derivatives; fatty acid derivative compositions including saturated fatty acid derivatives; and fatty acid derivative compositions including branched chain fatty acid derivatives.
[0015] The disclosure further contemplates a cell culture containing any of the recombinant host cells presented herein, wherein the recombinant host cells have a titer that is at least about 5% greater than the titer of the corresponding wild type host cells when cultured under the same conditions as the recombinant host cells. Herein, the recombinant host cells have a titer of from about 1 g/L to about 250 g/L, and more specifically from about 90 g/L to about 120 g/L. In a related aspect, the recombinant host cells have a yield that is at least about 10% to about 40%. In one aspect, the recombinant host cells have a yield of about 25%. Still encompassed herein is a cell culture containing any one of the recombinant host cells presented herein, wherein the productivity of the cell culture ranges from about 0.7 mg/L/hr to about 3 g/L/hr or higher.
[0016] Another aspect of the disclosure provides methods of making a recombinant host cell, including genetically engineering the recombinant host cell such that the cell expresses a polypeptide sequence that is encoded by one or more polynucleotide sequences under specific culture conditions, wherein the polynucleotide sequence codes for one or more polypeptides that have a specific enzymatic activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present disclosure is best understood when read in conjunction with the accompanying figures, which serve to illustrate the preferred embodiments. It is understood, however, that the disclosure is not limited to the specific embodiments disclosed in the figures.
[0018] FIG. 1 presents an exemplary biosynthetic pathway for use in production of acyl CoA as a precursor to fatty acid derivatives in a recombinant microorganism. The cycle is initiated by condensation of malonyl-ACP and acetyl-CoA.
[0019] FIG. 2 presents an exemplary fatty acid biosynthetic cycle, where malonyl-ACP is produced by the transacylation of malonyl-CoA to malonyl-ACP (catalyzed by malonyl-CoA:ACP transacylase (fabD)); then .beta.-ketoacyl-ACP synthase III (fabH) initiates condensation of malonyl-ACP with acetyl-CoA. Elongation cycles begin with the condensation of malonyl-ACP and an acyl-ACP catalyzed by .beta.-ketoacyl-ACP synthase I (fabB) and .beta.-ketoacyl-ACP synthase II (fabF) to produce a .beta.-keto-acyl-ACP, then the .beta.-keto-acyl-ACP is reduced by .beta.-ketoacyl-ACP reductase (fabG) to produce a .beta.-hydroxy-acyl-ACP, which is dehydrated to a trans-2-enoyl-acyl-ACP by .beta.-hydroxyacyl-ACP dehydratase (fabA or fabZ). FabA can also isomerize trans-2-enoyl-acyl-ACP to cis-3-enoyl-acyl-ACP, which can bypass fabl and can used by fabB (typically for up to an aliphatic chain length of C16) to produce .beta.-keto-acyl-ACP. The final step in each cycle is catalyzed by enoyl-ACP reductase (fabl) that converts trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described herein, termination of fatty acid synthesis occurs by thioesterase removal of the acyl group from acyl-ACP to release free fatty acids (FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which occur between acyl chains and ACP through sulfydryl bonds.
[0020] FIG. 3 illustrates the structure and function of the acetyl-CoA carboxylase (accABCD) enzyme complex. BirA biotinylates accB, the biotin carboxyl carrier protein, which is part of the acetyl-CoA carboxylase enzyme complex.
[0021] FIG. 4 presents an overview of an exemplary biosynthetic pathway for production of fatty alcohol starting with acyl-ACP, where the production of fatty aldehyde is catalyzed by the enzymatic activity of acyl-ACP reductase (AAR) or thioesterase (TE) and carboxylic acid reductase (Car). The fatty aldehyde is converted to fatty alcohol by aldehyde reductase (also referred to as alcohol dehydrogenase).
[0022] FIG. 5 presents an overview of two exemplary biosynthetic pathways for production of fatty esters starting with acyl-ACP, where the production of fatty esters is accomplished by a one-enzyme system or a three-enzyme-system.
[0023] FIG. 6 presents an overview of exemplary biosynthetic pathways for production of hydrocarbons starting with acyl-ACP; the production of internal olefins is catalyzed by the enzymatic activity of OleABCD; the production of alkanes is catalyzed by the enzymatic conversion of fatty aldehydes to alkanes by way of aldehyde decarbonylase (ADC); and the production of terminal olefins is catalyzed by the enzymatic conversion of fatty acids to terminal olefins by a decarboxylase.
[0024] FIG. 7 illustrates fatty acid derivative (Total Fatty Species) production by the MG1655 E. coli strain with the fadE gene attenuated (i.e., deleted) compared to fatty acid derivative production by E. coli MG1655. The data presented in FIG. 7 shows that attenuation of the fadE gene did not affect fatty acid derivative production
[0025] FIG. 8 shows malonyl-CoA levels in DAM1_i377 in log phase, expressing eight different C. glutamicum acetyl-CoA carboxylase (Acc) operon constructs.
[0026] FIG. 9 shows intracellular short chain-CoA levels in E. coli DAM1_i377 in log phase expressing ptrc1/3_accDACB-birA.+-.panK operon constructs. accDACB+birA is also referred to herein as accD+.
[0027] FIG. 10 shows fatty acid methyl ester (FAME) production in E. coli strain DV2 expressing ester synthase 9 from M. hydrocarbonoclasticus and components of an acetyl-CoA carboxylase complex from C. glutamicum.
[0028] FIG. 11 shows production of fatty alcohols by E. coli expressing the Synechococcus elongatus PCC7942 AAR together with the accD+ operon from C. glutamicum on a pCL plasmid. Triplicate samples are shown for the accD+ strains.
[0029] FIGS. 12A and 12B show data for production of Total Fatty Species (mg/L) from duplicate plate screens when plasmid pCL_P.sub.trc_tesA was transformed into each of the iFAB-containing strains shown in the figures and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32.degree. C. (FIG. 12A) and 37.degree. C. (FIG. 12B).
[0030] FIG. 13 shows FAME production of E. coli DAM1 with plasmid pDS57 and integrated fabHI operons. The fabH/I genes are from Marinobacter aquaeoli VT8 or from Acinetobacter baylyi ADP1. See Table 7 for a more details on the fabH/I operons in these strains.
[0031] FIG. 14 shows FAME production of E. coli DAM1 with plasmid pDS57 and different configurations of the C. glutamicum acc genes as well as integrated fabHI operons. The strains contain the fabH/I genes from Rhodococcus opacus or Acinetobacter baylyi ADP1. See Table 7 for more details on the fabH/I and acc operons.
[0032] FIG. 15 shows FAME and FFA titers of two E. coli DAM1 pDS57 strains with integrated fabH/I genes strains selected from FIG. 13 compared to the control strain E. coli DAM1 pDS57.
[0033] FIGS. 16A and 16B are a diagrammatic depiction of the iFAB138 locus, including a diagram of cat-loxP-P.sub.T5 cassette integrated in front of iFAB138 (FIG. 16A); and a diagram of the P.sub.T5_iFAB138 region (FIG. 16B).
[0034] FIG. 17 shows that strain V668, which has the rph and ilvG genes repaired, produced a higher level of FFA than EG149, which has neither of the genes repaired.
[0035] FIG. 18 is a diagrammatic depiction of a transposon cassette insertion in the yijP gene of strain LC535 (transposon hit 68F11). Promoters internal to the transposon cassette are shown, and may have effects on adjacent gene expression.
[0036] FIG. 19 illustrates fatty alcohol production in E. coli DV2 expressing Synechococcus elongatus acyl-ACP reductase (AAR) and coexpressing various cyanobacterial acyl carrier proteins (ACPs). Details regarding the source of the ACPs are provided in Table 12.
[0037] FIG. 20 illustrates fatty acid production in E. coli DV2 expressing leaderless E. coli thioesterase `tesA and coexpressing a cyanobacterial acyl carrier protein (cACP) and B. subtilis sfp.
DETAILED DESCRIPTION
[0038] General Overview
[0039] The disclosure is based, at least in part, on the discovery that modification of various aspects of the fatty acid biosynthetic pathway in a recombinant host cell facilitates enhanced production of fatty acid derivatives by the host cell. The disclosure relates to compositions of fatty acid derivatives having desired characteristics and methods for producing the same. Further, the disclosure relates to recombinant host cells (e.g., microorganisms), cultures of recombinant host cells, methods of making and using recombinant host cells, for example, use of cultured recombinant host cells in the fermentative production of fatty acid derivatives having desired characteristics.
[0040] More specifically, the production of a desired fatty acid derivative composition (e.g., acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones is enhanced by modifying the expression of one or more genes involved in a biosynthetic pathway for fatty acid, fatty ester, alkane, alkene, olefin, or fatty alcohol, production, degradation and/or secretion. The disclosure provides recombinant host cells which have been engineered to provide enhanced fatty acid biosynthesis relative to non-engineered or native host cells (e.g., wild type host cells that function as control cells), which is accomplished, for example, through strain improvements. As such, the disclosure identifies polynucleotides useful in the recombinant host cells, methods, and compositions of the disclosure. It will be generally recognized that absolute sequence identity to such polynucleotides is not necessary. For example, changes in a particular polynucleotide sequence can be made and the encoded polypeptide screened for activity. Such changes typically comprise conservative mutations and silent mutations (e.g., codon optimization). Genetically engineered or modified polynucleotides and encoded variant polypeptides can be screened for a desired function, including but not limited to, increased catalytic activity, increased stability, or decreased inhibition (e.g., decreased feedback inhibition), using methods known in the art.
[0041] The disclosure identifies enzymatic activities involved in various steps (i.e., reactions) of the fatty acid biosynthetic pathways described herein according to Enzyme Classification (EC) number, and provides exemplary polypeptides (e.g., enzymes) categorized by such EC numbers, and exemplary polynucleotides encoding such polypeptides. Such exemplary polypeptides and polynucleotides, which are identified herein by Accession Numbers and/or Sequence Identifier Numbers (SEQ ID NOs), are useful for engineering fatty acid pathways in parental host cells to obtain the recombinant host cells described herein. The polypeptides and polynucleotides described herein are exemplary and non-limiting. The sequences of homologues of exemplary polypeptides described herein are available to those of skill in the art through various databases (e.g., rhw Entrez databases provided by the National Center for Biotechnology Information (NCBI), the ExPasy databases provided by the Swiss Institute of Bioinformatics, the BRENDA database provided by the Technical University of Braunschweig, and the KEGG database provided by the Bioinformatics Center of Kyoto University and University of Tokyo, all which are available on the World Wide Web).
Definitions
[0042] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a recombinant host cell" includes two or more such recombinant host cells, reference to "a fatty alcohol" includes one or more fatty alcohols, or mixtures of fatty alcohols, reference to "a nucleic acid coding sequence" includes one or more nucleic acid coding sequences, reference to "an enzyme" includes one or more enzymes, and the like.
[0043] Accession Numbers: Sequence Accession numbers throughout this description were obtained from databases provided by the NCBI (National Center for Biotechnology Information) maintained by the National Institutes of Health, U.S.A. (which are identified herein as "NCBI Accession Numbers" or alternatively as "GenBank Accession Numbers"), and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute of Bioinformatics (which are identified herein as "UniProtKB Accession Numbers").
[0044] Enzyme Classification (EC) Numbers: EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), description of which is available on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reaction they catalyze.
[0045] As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups. The naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typically derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. The naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).
[0046] As used herein, the term "polynucleotide" refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms "polynucleotide," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and/or capped polynucleotides. The polynucleotide can be in any form, including but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
[0047] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term "recombinant polypeptide" refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.
[0048] As used herein, the terms "homolog," and "homologous" refer to a polynucleotide or a polypeptide comprising a sequence that is at least about 50% identical to the corresponding polynucleotide or polypeptide sequence. Preferably homologous polynucleotides or polypeptides have polynucleotide sequences or amino acid sequences that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence. As used herein the terms sequence "homology" and sequence "identity" are used interchangeably.
[0049] One of ordinary skill in the art is well aware of methods to determine homology between two or more sequences. Briefly, calculations of "homology" between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the length of a second sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences.
[0050] The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)). The percent homology between two amino acid sequences also can be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)). The percent homology between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can perform initial homology calculations and adjust the algorithm parameters accordingly. A preferred set of parameters (and the one that should be used if a practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Additional methods of sequence alignment are known in the biotechnology arts (see, e.g., Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul, et al., FEBS J., 272(20): 5101-5109 (2005)).
[0051] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions--6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature of the washes can be increased to 55.degree. C. for low stringency conditions); 2) medium stringency hybridization conditions--6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency hybridization conditions--6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2..times.SSC, 0.1% SDS at 65.degree. C.; and 4) very high stringency hybridization conditions--0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one or more washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
[0052] An "endogenous" polypeptide refers to a polypeptide encoded by the genome of the host cell (e.g., parental microbial cell) from which the recombinant cell is engineered or derived.
[0053] An "exogenous" polypeptide refers to a polypeptide which is not encoded by the genome of the parental microbial cell. A variant (i.e., mutant) polypeptide is an example of an exogenous polypeptide.
[0054] The term "heterologous" generally means derived from a different species or derived from a different organism. As used herein it refers to a nucleotide sequence or a polypeptide sequence that is not naturally present in a particular organism. Heterologous expression means that a protein or polypeptide is experimentally added to a cell that does not normally express that protein. As such, heterologous refers to the fact that a transferred protein was initially derived from a different cell type or a different species then the recipient. For example, a polynucleotide sequence endogenous to a plant cell can be introduced into a bacterial host cell by recombinant methods, and the plant polynucleotide is then a heterologous polynucleotide in a recombinant bacterial host cell.
[0055] As used herein, the term "fragment" of a polypeptide refers to a shorter portion of a full-length polypeptide or protein ranging in size from four amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the disclosure, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).
[0056] As used herein, the term "mutagenesis" refers to a process by which the genetic information of an organism is changed in a stable manner. Mutagenesis of a protein coding nucleic acid sequence produces a mutant protein. Mutagenesis also refers to changes in non-coding nucleic acid sequences that result in modified protein activity.
[0057] As used herein, the term "gene" refers to nucleic acid sequences encoding either an RNA product or a protein product, as well as operably-linked nucleic acid sequences affecting the expression of the RNA or protein (e.g., such sequences include but are not limited to promoter or enhancer sequences) or operably-linked nucleic acid sequences encoding sequences that affect the expression of the RNA or protein (e.g., such sequences include but are not limited to ribosome binding sites or translational control sequences).
[0058] Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
[0059] In the methods of the disclosure, an expression control sequence is operably linked to a polynucleotide sequence. By "operably linked" is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.
[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids," which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. The terms "plasmid" and "vector" are used interchangeably herein, in as much as a plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto. In some embodiments, a recombinant vector further comprises a promoter operably linked to the polynucleotide sequence. In some embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. The recombinant vector typically comprises at least one sequence including (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence. In certain embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region. The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.
[0061] Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. In certain embodiments, a polynucleotide sequence of the disclosure is operably linked to a promoter derived from bacteriophage T5.
[0062] In certain embodiments, the host cell is a yeast cell, and the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, Calif.), and picZ (Invitrogen Corp., San Diego, Calif.).
[0063] In other embodiments, the host cell is an insect cell, and the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. Cell Biol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)).
[0064] In yet another embodiment, the polynucleotide sequences described herein can be expressed in mammalian cells using a mammalian expression vector. Other suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," second edition, Cold Spring Harbor Laboratory, (1989).
[0065] The term "corresponding wild type host cell" as referred to herein, means a cell that functions as a control cell. For example, if a polypeptide in a recombinant host cell is up-regulated, then the same polypeptide would exist at a lower level in the control cell. Conversely, if a polypeptide in a recombinant host cell is down-regulated, then the same polypeptide would exist at a higher level in the control cell. Furthermore, the "recombinant or engineered host cell" is a microorganism used to produce one or more of fatty acid derivatives including, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones. In some embodiments, the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity.
[0066] As used herein "acyl-CoA" refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the 4'-phosphopantethionyl moiety of coenzyme A (CoA), which has the formula R--C(O)S-CoA, where R is any alkyl group having at least 4 carbon atoms.
[0067] As used herein "acyl-ACP" refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an acyl carrier protein (ACP). The phosphopantetheinyl moiety is post-translationally attached to a conserved serine residue on the ACP by the action of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyl transferase. In some embodiments an acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs. In some embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of these acyl-ACPs are substrates for enzymes that convert them to fatty acid derivatives.
[0068] As used herein, the term "fatty acid derivative" means a "fatty acid" or a "fatty acid derivative", which may be referred to as a "fatty acid or derivative thereof". The term "fatty acid" means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. A "fatty acid derivative" is a product made in part from the fatty acid biosynthetic pathway of the production host organism. "Fatty acid derivatives" includes products made in part from acyl-ACP or acyl-ACP derivatives. Exemplary fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.
[0069] A "fatty acid derivative composition" as referred to herein is produced by a recombinant host cell and typically comprises a mixture of fatty acid derivative. In some cases, the mixture includes more than one type of product (e.g., fatty acids and fatty alcohols, fatty acids and fatty acid esters or alkanes and olefins). In other cases, the fatty acid derivative compositions may comprise, for example, a mixture of fatty alcohols (or another fatty acid derivative) with various chain lengths and saturation or branching characteristics. In still other cases, the fatty acid derivative composition comprises a mixture of both more than one type of product and products with various chain lengths and saturation or branching characteristics.
[0070] As used herein, the term "fatty acid biosynthetic pathway" means a biosynthetic pathway that produces fatty acids and derivatives thereof. The fatty acid biosynthetic pathway may include additional enzymes or polypeptides with enzymatic activities besides the ones discussed herein to produce fatty acid derivatives having desired characteristics.
[0071] As used herein, "fatty aldehyde" means an aldehyde having the formula RCHO characterized by a carbonyl group (C.dbd.O). In some embodiments, the fatty aldehyde is any aldehyde made from a fatty alcohol. In certain embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the R group can have an R group bounded by any two of the above endpoints. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty aldehyde. In certain embodiments, the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty aldehyde.
[0072] As used herein, "fatty alcohol" means an alcohol having the formula ROH. In some embodiments, the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length. Alternatively, or in addition, the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the R group can have an R group bounded by any two of the above endpoints. For example, the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length. In some embodiments, the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty alcohol. In certain embodiments, the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty alcohol.
[0073] The R group of a fatty acid derivative, for example a fatty alcohol, can be a straight chain or a branched chain. Branched chains may have more than one point of branching and may include cyclic branches. In some embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 branched fatty acid, branched fatty aldehyde, or branched fatty alcohol. In particular embodiments, the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 branched fatty acid, branched fatty aldehyde, or branched fatty alcohol. In certain embodiments, the hydroxyl group of the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is in the primary (C1) position.
[0074] In certain embodiments, the branched fatty acid derivative is an iso-fatty acid derivative, for example an iso-fatty aldehyde, an iso-fatty alcohol, or an antesio-fatty acid derivative, an anteiso-fatty aldehyde, or an anteiso-fatty alcohol. In exemplary embodiments, the branched fatty acid derivative is selected from iso-C7:0, iso-C8:0, iso-C9:0, iso-C10:0, iso-C11:0, iso-C12:0, iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0, iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0, anteiso-C11:0, anteiso-C12:0, anteiso-C13:0, anteiso-C14:0, anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and an anteiso-C19:0 branched fatty alcohol.
[0075] The R group of a branched or unbranched fatty acid derivative can be saturated or unsaturated. If unsaturated, the R group can have one or more than one point of unsaturation. In some embodiments, the unsaturated fatty acid derivative is a monounsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated fatty acid derivative. In certain embodiments, the unsaturated fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In other embodiments, the unsaturated fatty acid derivative is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid derivative comprises a cis double bond.
[0076] As used herein, the term "clone" typically refers to a cell or group of cells descended from and essentially genetically identical to a single common ancestor, for example, the bacteria of a cloned bacterial colony arose from a single bacterial cell.
[0077] As used herein, the term "culture" typical refers to a liquid media comprising viable cells. In one embodiment, a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen. "Culturing" or "cultivation" refers to growing a population of recombinant host cells under suitable conditions in a liquid or solid medium. In particular embodiments, culturing refers to the fermentative bioconversion of a substrate to an end-product. Culturing media are well known and individual components of such culture media are available from commercial sources, e.g., under the Difco.TM. and BBL.TM. trademarks. In one non-limiting example, the aqueous nutrient medium is a "rich medium" comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium. The host cell of a culture can be additionally engineered to assimilate carbon efficiently and use cellulosic materials as carbon sources according to methods described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030; WO 2010127318. In addition, in some embodiments the host cell is engineered to express an invertase so that sucrose can be used as a carbon source.
[0078] As used herein, the term "under conditions effective to express a genetically engineered polynucleotide sequence" means any condition that allows a host cell to produce a desired fatty acid derivative. Suitable conditions include, for example, fermentation conditions.
[0079] As used herein, "modified" or an "altered level of" activity of a protein, for example an enzyme, in a recombinant host cell refers to a difference in one or more characteristics in the activity determined relative to the parent or native host cell. Typically differences in activity are determined between a recombinant host cell, having modified activity, and the corresponding wild-type host cell (e.g., comparison of a culture of a recombinant host cell relative to the corresponding wild-type host cell). Modified activities can be the result of, for example, modified amounts of protein expressed by a recombinant host cell (e.g., as the result of increased or decreased number of copies of DNA sequences encoding the protein, increased or decreased number of mRNA transcripts encoding the protein, and/or increased or decreased amounts of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., increased or decreased degradation of the protein). In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein. In certain instances, the coding sequences for the polypeptides described herein are codon optimized for expression in a particular host cell. For example, for expression in E. coli, one or more codons can be optimized as described in, e.g., Grosjean et al., Gene 18:199-209 (1982).
[0080] The term "regulatory sequences" as used herein typically refers to a sequence of bases in DNA, operably-linked to DNA sequences encoding a protein that ultimately controls the expression of the protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, modulators of transcription (such as enhancer elements), nucleotide sequences that affect RNA stability, and translational regulatory sequences (such as, ribosome binding sites (e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), initiation codons, termination codons).
[0081] The terms "altered level of expression" and "modified level of expression" are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present in a different concentration in an engineered host cell as compared to its concentration in a corresponding wild-type cell under the same conditions.
[0082] As used herein, the term "titer" refers to the quantity of fatty acid derivative produced per unit volume of host cell culture. In any aspect of the compositions and methods described herein, a fatty acid derivative is produced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative is produced at a titer of more than 100 g/L, more than 200 g/L, more than 300 g/L, or higher, such as 500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500 g/L, or 2000 g/L. The preferred titer of fatty acid derivative produced by a recombinant host cell according to the methods of the disclosure is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L. In one embodiment, the titer of fatty acid derivative produced by a recombinant host cell according to the methods of the disclosure is about 1 g/L to about 250 g/L and more particularly, 90 g/L to about 120 g/L. The titer may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
[0083] As used herein, the "yield of fatty acid derivative produced by a host cell" refers to the efficiency by which an input carbon source is converted to product (i.e., fatty alcohol or fatty aldehyde) in a host cell. Host cells engineered to produce fatty acid derivatives according to the methods of the disclosure have a yield of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% or a range bounded by any two of the foregoing values. In other embodiments, a fatty acid derivative or derivatives is produced at a yield of more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Alternatively, or in addition, the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less. Thus, the yield can be bounded by any two of the above endpoints. For example, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell according to the methods of the disclosure can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. In a particular embodiment, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell is about 10% to about 40%. In another particular embodiment, the yield of a fatty acid derivative or derivatives produced by the recombinant host cell is about 25%. The yield may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
[0084] As used herein, the term "productivity" refers to the quantity of a fatty acid derivative or derivatives produced per unit volume of host cell culture per unit time. In any aspect of the compositions and methods described herein, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell is at least 100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900 mg/L/hour, at least 2000 mg/L/hour, at least 2100 mg/L/hour, at least 2200 mg/L/hour, at least 2300 mg/L/hour, at least 2400 mg/L/hour, or at least 2500 mg/L/hour. For example, the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell according to the methods of the may be from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour. In one particular embodiment, the yield is about 0.7 mg/L/h to about 3 g/L/h. The productivity may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
[0085] As used herein, the term "total fatty species" and "total fatty acid product" may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis.
[0086] As used herein, the term "glucose utilization rate" means the amount of glucose used by the culture per unit time, reported as grams/liter/hour (g/L/hr). As used herein, the term "carbon source" refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO.sub.2). Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, such as glucose. In certain preferred embodiments, the carbon source is biomass. In other preferred embodiments, the carbon source is glucose. In other preferred embodiments the carbon source is sucrose.
[0087] As used herein, the term "biomass" refers to any biological material from which a carbon source is derived. In some embodiments, a biomass is processed into a carbon source, which is suitable for bioconversion. In other embodiments, the biomass does not require further processing into a carbon source. The carbon source can be converted into a biofuel. An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass. Another exemplary source of biomass is metabolic waste products, such as animal matter (e.g., cow manure). Further exemplary sources of biomass include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers. The term "biomass" also refers to sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
[0088] As used herein, the term "isolated," with respect to products (such as fatty acids and derivatives thereof) refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors. The fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and derivatives thereof can collect in an organic phase either intracellularly or extracellularly.
[0089] As used herein, the terms "purify," "purified," or "purification" mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. "Substantially purified" molecules are at least about 60% free (e.g., at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty acid derivatives in a sample. For example, when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivative in the sample is increased. The terms "purify," "purified," and "purification" are relative terms which do not require absolute purity. Thus, for example, when a fatty acid derivative is produced in recombinant host cells, a purified fatty acid derivative is a fatty acid derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).
[0090] Strain Improvements
[0091] In order generate a high titer, yield, and/or productivity of fatty acid derivatives, a number of modifications were made to the production host cells. FadR is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthetic pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. coli ACS enzyme FadD and the fatty acid transport protein FadL are components of a fatty acid uptake system. FadL mediates transport of fatty acids into the bacterial cell, and FadD mediates formation of acyl-CoA esters. When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters, which can bind to the transcription factor FadR and depress the expression of the fad genes that encode proteins responsible for fatty acid transport (FadL), activation (FadD), and (3-oxidation (FadA, FadB, FadE, and FadH). When alternative sources of carbon are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis, but are not substrates for .beta.-oxidation. Thus, acyl-CoA and acyl-ACP are both independent sources of fatty acids that can result in different end-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)).
[0092] There are conflicting speculations in the art as to the factors that can limit fatty acid biosynthesis in host cells, such as E. coli. One suggestion is that a limitation of the main precursors for fatty acid biosynthesis, for example, acetyl-CoA and malonyl-CoA can result in decreased synthesis of fatty acid derivatives. One approach to increasing the flux through fatty acid biosynthesis is to manipulate various enzymes in the pathway (see FIGS. 1 and 2). Example 3 describes studies which show construction of fab operons that encode enzymes in the biosynthetic pathway for conversion of malonyl-CoA into acyl-ACPs and integration into the chromosome of an E. coli host cell. Without wanting to be bound by theory, this may increase the flux of fatty acid biosynthesis. The supply of acyl-ACPs from acetyl-CoA via the acetyl-CoA carboxylase (acc) complex and fatty acid biosynthetic (fab) pathway is another step that may limit the rate of fatty acid derivative production (see FIG. 3). Example 2 shows the effect of overexpression of an optimized version of E. coli Corynebacterium glutamicum accABCD (.+-.birA) demonstrated that such genetic modifications can lead to increased production of acetyl-coA and malonyl-CoA in E. coli.
[0093] In another approach, mutations in the rph and ilvG genes in the E. coli host cell were shown to result in higher free fatty acid (FFA) production, which translated into higher production of fatty alcohol as shown in Example 4. In still another approach, transposon mutagenesis and high-throughput screening was carried out to find beneficial mutations that increase the titer or yield. As shown in Example 5, a transposon insertion in the yijP gene can improve the fatty alcohol yield in shake flask and fed-batch fermentations.
[0094] Generation of Fatty Acid Derivatives by Recombinant Host Cells
[0095] The present disclosure provides numerous examples of polypeptides (i.e., enzymes) having activities suitable for use in the fatty acid biosynthetic pathways described herein. Such polypeptides are collectively referred to herein as "fatty acid biosynthetic polypeptides" or "fatty acid biosynthetic enzymes". Non-limiting examples of fatty acid pathway polypeptides suitable for use in recombinant host cells of the disclosure are provided herein. In some embodiments, the disclosure includes a recombinant host cell including a polynucleotide sequence which encodes a fatty acid biosynthetic polypeptide. The polynucleotide sequence, which includes an open reading frame encoding a fatty acid biosynthetic polypeptide and operably-linked regulatory sequences, can be integrated into a chromosome of the recombinant host cells, incorporated in one or more plasmid expression systems resident in the recombinant host cell, or both. In one embodiment, a fatty acid biosynthetic polynucleotide sequence encodes a polypeptide which is endogenous to the parental host cell (i.e., the control cell) of the recombinant host cell that is being engineered. Some such endogenous polypeptides are overexpressed in the recombinant host cell. In another embodiment, the fatty acid biosynthetic polynucleotide sequence encodes an exogenous or heterologous polypeptide. In other words, the polypeptide encoded by the polynucleotide is exogenous to the parental host cell. In yet another embodiment, the genetically modified host cell overexpresses a gene encoding a polypeptide (protein) that increases the rate at which the host cell produces the substrate of a fatty acid biosynthetic enzyme, i.e., a fatty acyl-thioester substrate. In certain embodiments, the enzyme encoded by the expressed gene is directly involved in fatty acid biosynthesis. Such recombinant host cells may be further engineered to include a polynucleotide sequence encoding one or more fatty acid biosynthetic polypeptides (i.e., enzymes involved in fatty acid biosynthesis). Examples of such polypeptides are polpeptides or proteins having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids; or having thioesterase activity and carboxylic acid reductase (CAR) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; or having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; or having acyl-CoA reductase (AAR) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; or having acyl-CoA reductase (AAR) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; or having fatty alcohol forming acyl-CoA reductase (FAR) activity, wherein the recombinant host cell synthesizes fatty alcohols; or having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; or having acyl-CoA reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; or having ester synthase activity wherein the recombinant host cell synthesizes fatty esters (e.g., one enzyme system; see FIG. 5); or having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters (e.g., three enzyme system; see FIG. 5); or having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones; or having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; or having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins; or combinations thereof. In some embodiments, at least one polypeptide encoded by a fatty acid biosynthetic polynucleotide is an exogenous (or heterologous) polypeptide (e.g., a polypeptide originating from an organism other than the parental host cell, or a variant of a polypeptide native to the parental microbial cell) or an endogenous polypeptide (i.e., a polypeptide native to the parental host cell) wherein the endogenous polypeptide is overexpressed in the recombinant host cell.
[0096] Table 1 below provides a listing of exemplary proteins which can be expressed in recombinant host cells to facilitate production of particular fatty acid derivatives.
TABLE-US-00001 TABLE 1 Gene Designations Gene Source Designation Organism Enzyme Name Accession No. EC Number Exemplary Use 1. Fatty Acid Production Increase/Product Production Increase accA E. coli, Lactococci Acetyl-CoA AAC73296, 6.4.1.2 increase Malonyl-CoA carboxylase, NP_414727 production subunit A (carboxyltransferase alpha) accB E. coli, Lactococci Acetyl-CoA NP_417721 6.4.1.2 increase Malonyl-CoA carboxylase, production subunit B (BCCP: biotin carboxyl carrier protein) accC E. coli, Lactococci Acetyl-CoA NP_417722 6.4.1.2, increase Malonyl-CoA carboxylase, 6.3.4.14 production subunit C (biotin carboxylase) accD E. coli, Lactococci Acetyl-CoA NP_416819 6.4.1.2 increase Malonyl-CoA carboxylase, production subunit D (carboxyltransferase beta) fadD E. coli W3110 acyl-CoA AP_002424 2.3.1.86, increase Fatty acid synthase 6.2.1.3 production fabA E. coli K12 .beta.- NP_415474 4.2.1.60 increase fatty acyl- hydroxydecanoyl ACP/CoA production thioester dehydratase/ isomerase fabB E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 increase fatty acyl- carrier-protein] ACP/CoA production synthase I fabD E. coli K12 [acyl-carrier- AAC74176 2.3.1.39 increase fatty acyl- protein] S- ACP/CoA production malonyltransferase fabF E. coli K12 3-oxoacyl-[acyl- AAC74179 2.3.1.179 increase fatty acyl- carrier-protein] ACP/CoA production synthase II fabG E. coli K12 3-oxoacyl-[acyl- AAC74177 1.1.1.100 increase fatty acyl- carrier protein] ACP/CoA production reductase fabH E. coli K12 3-oxoacyl-[acyl- AAC74175 2.3.1.180 increase fatty acyl- carrier-protein] ACP/CoA production synthase III fabI E. coli K12 enoyl-[acyl- NP_415804 1.3.1.9 increase fatty acyl- carrier-protein] ACP/CoA production reductase fabR E. coli K12 Transcriptional NP_418398 none modulate unsaturated Repressor fatty acid production fabV Vibrio cholerae enoyl-[acyl- YP_001217283 1.3.1.9 increase fatty acyl- carrier-protein] ACP/CoA production reductase fabZ E. coli K12 (3R)- NP_414722 4.2.1.- increase fatty acyl- hydroxymyristol ACP/CoA production acyl carrier protein dehydratase fadE E. coli K13 acyl-CoA AAC73325 1.3.99.3, reduce fatty acid dehydrogenase 1.3.99.- degradation fadR E. coli transcriptional NP_415705 none Block or reverse fatty regulatory protein acid degradation 2. Chain Length Control tesA (with E. coli thioesterase- P0ADA1 3.1.2.-, C18 Chain Length or without leader sequence is 3.1.1.5 leader amino acids 1-26 sequence) tesA E. coli thioesterase AAC73596, 3.1.2.-, C18:1 Chain Length (without NP_415027 3.1.1.5 leader sequence) tesA (mutant E. coli thioesterase L109P 3.1.2.-, <C18 Chain Length of E. coli 3.1.1.5 thioesterase I complexed with octanoic acid) fatB1 Umbellularia californica thioesterase Q41635 3.1.2.14 C12:0 Chain Length fatB2 Cuphea hookeriana thioesterase AAC49269 3.1.2.14 C8:0-C10:0 Chain Length fatB3 Cuphea hookeriana thioesterase AAC72881 3.1.2.14 C14:0-C16:0 Chain Length fatB Cinnamomum camphora thioesterase Q39473 3.1.2.14 C14:0 Chain Length fatB Arabidopsis thaliana thioesterase CAA85388 3.1.2.14 C16:1 Chain Length fatA1 Helianthus annuus thioesterase AAL79361 3.1.2.14 C18:1 Chain Length atfata Arabidopsis thaliana thioesterase NP_189147, 3.1.2.14 C18:1 Chain Length NP_193041 fatA Brassica juncea thioesterase CAC39106 3.1.2.14 C18:1 Chain Length fatA Cuphea hookeriana thioesterase AAC72883 3.1.2.14 C18:1 Chain Length tes Photbacterium profundum thioesterase YP_130990 3.1.2.14 Chain Length tesB E. coli thioesterase NP_414986 3.1.2.14 Chain Length fadM E. coli thioesterase NP_414977 3.1.2.14 Chain Length yciA E. coli thioesterase NP_415769 3.1.2.14 Chain Length ybgC E. coli thioesterase NP_415264 3.1.2.14 Chain Length 3. Saturation Level Control* Sfa E. coli Suppressor of AAN79592, none increase fabA AAC44390 monounsaturated fatty acids fabA E. coli K12 .beta.- NP_415474 4.2.1.60 produce unsaturated hydroxydecanoyl fatty acids thioester dehydratase/ isomerase GnsA E. coli suppressors of the ABD18647.1 none increase unsaturated secG null fatty acid esters mutation GnsB E. coli suppressors of the AAC74076.1 none increase unsaturated secG null fatty acid esters mutation fabB E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 modulate unsaturated carrier-protein] fatty acid production synthase I des Bacillus subtilis D5 fatty acyl O34653 1.14.19 modulate unsaturated desaturase fatty acid production 4. Product Output: Wax Production AT3G51970 Arabidopsis thaliana long-chain- NP_190765 2.3.1.26 wax production alcohol O-fatty- acyltransferase ELO1 Pichia angusta Fatty acid BAD98251 2.3.1.- produce very long elongase chain length fatty acids plsC Saccharomyces cerevisiae acyltransferase AAA16514 2.3.1.51 wax production DAGAT/ Arabidopsis thaliana diacylglycerol AAF19262 2.3.1.20 wax production DGAT acyltransferase hWS Homo sapiens acyl-CoA wax AAX48018 2.3.1.20 wax production alcohol acyltransferase aft1 Acinetobacter sp. ADP1 bifunctional wax AAO17391 2.3.1.20 wax production ester synthase/acyl- CoA:diacylglycerol acyltransferase ES9 Marinobacter hydrocarbonoclasticus wax ester ABO21021 2.3.1.20 wax production synthase mWS Simmondsia chinensis wax ester AAD38041 2.3.1.- wax production synthase 5. Product Output: Fatty Alcohol Output thioesterases (see increase fatty above) acid/fatty alcohol production BmFAR Bombyxmori FAR (fatty BAC79425 1.1.1.- convert acyl-CoA to alcohol forming fatty alcohol acyl-CoA reductase) acr1 Acinetobacter sp. ADP1 acyl-CoA YP_047869 1.2.1.42 reduce fatty acyl-CoA reductase to fatty aldehydes yqhD E. coli W3110 alcohol AP_003562 1.1.-.- reduce fatty aldehydes dehydrogenase to fatty alcohols; increase fatty alcohol production alrA Acinetobacter sp. ADP1 alcohol CAG70252 1.1.-.- reduce fatty aldehydes dehydrogenase to fatty alcohols BmFAR Bombyxmori FAR (fatty BAC79425 1.1.1.- reduce fatty acyl-CoA alcohol forming to fatty alcohol acyl-CoA reductase) GTNG_1865 Geobacillusthermodenitrificans NG80-2 Long-chain YP_001125970 1.2.1.3 reduce fatty aldehydes aldehyde to fatty alcohols dehydrogenase AAR Synechococcus elongatus Acyl-ACP YP_400611 1.2.1.42 reduce fatty acyl- reductase ACP/CoA to fatty aldehydes carB Mycobacterium smegmatis carboxylic acid YP_889972 6.2.1.3, reduce fatty acids to reductase protein 1.2.1.42 fatty aldehyde FadD E. coli K12 acyl-CoA NP_416319 6.2.1.3 activates fatty acids to synthetase fatty acyl-CoAs atoB Erwiniacarotovora acetyl-CoA YP_049388 2.3.1.9 production of butanol acetyltransferase hbd Butyrivibriofibrisolvens Beta- BAD51424 1.1.1.157 production of butanol hydroxybutyryl- CoA dehydrogenase CPE0095 Clostridium perfringens crotonasebutyryl- BAB79801 4.2.1.55 production of butanol CoA dehydryogenase bcd Clostridium beijerinckii butyryl-CoA AAM14583 1.3.99.2 production of butanol dehydryogenase ALDH Clostridium beijerinckii coenzyme A- AAT66436 1.2.1.3 production of butanol acylating aldehyde dehydrogenase AdhE E. coli CFT073 aldehyde-alcohol AAN80172 1.1.1.1 production of butanol dehydrogenase 1.2.1.10 6. Fatty Alcohol Acetyl Ester Output thioesterases (see modify output above) acr1 Acinetobacter sp. ADP1 acyl-CoA YP_047869 1.2.1.42 modify output reductase yqhD E. Coli K12 alcohol AP_003562 1.1.-.- modify output dehydrogenase AAT Fragaria x ananassa alcohol O- AAG13130 2.3.1.84 modify output acetyltransferase 7. Product Export AtMRP5 Arabidopsis thaliana Arabidopsis NP_171908 none modify product export thaliana multidrug amount resistance- associated AmiS2 Rhodococcus sp. ABC transporter JC5491 none modify product export AmiS2 amount AtPGP1 Arabidopsis thaliana Arabidopsis NP_181228 none modify product export thaliana p amount glycoprotein 1 AcrA CandidatusProtochlamydiaamoebophila putative CAF23274 none modify product export UWE25 multidrug-efflux amount transport protein acrA AcrB CandidatusProtochlamydiaamoebophila probable CAF23275 none modify product export UWE25 multidrug-efflux amount transport protein, acrB TolC Francisellatularensis subsp. novicida Outer membrane ABD59001 none modify product export protein [Cell amount envelope biogenesis, AcrE Shigellasonnei Ss046 transmembrane YP_312213 none modify product export protein affects amount septum formation and cell
membrane permeability AcrF E. coli Acriflavine P24181 none modify product export resistance protein amount F tll1619 Thermosynechococcus elongatus [BP-1] multidrug efflux NP_682409.1 none modify product export transporter amount tll0139 Thermosynechococcus elongatus [BP-1] multidrug efflux NP_680930.1 none modify product export transporter amount 8. Fermentation replication increase output checkpoint efficiency genes umuD Shigellasonnei Ss046 DNA polymerase YP_310132 3.4.21.- increase output V, subunit efficiency umuC E. coli DNA polymerase ABC42261 2.7.7.7 increase output V, subunit efficiency pntA, pntB Shigellaflexneri NADH:NADPH P07001, 1.6.1.2 increase output transhydrogenase P0AB70 efficiency (alpha and beta subunits) 9. Other fabK Streptococcus pneumoniae trans-2-enoyl- AAF98273 1.3.1.9 Contributes to fatty ACP reductase II acid biosynthesis fabL Bacillus licheniformis DSM 13 enoyl-(acyl AAU39821 1.3.1.9 Contributes to fatty carrier protein) acid biosynthesis reductase fabM Streptococcus mutans trans-2, cis-3- DAA05501 4.2.1.17 Contributes to fatty decenoyl-ACP acid biosynthesis isomerase
[0097] Production of Fatty Acids
[0098] The recombinant host cells may include one or more polynucleotide sequences that comprise an open reading frame encoding a thioesterase, e.g., having an Enzyme Commission number of EC 3.1.1.5 or EC 3.1.2.- (for example, EC 3.1.2.14), together with operably-linked regulatory sequences that facilitate expression of the protein in the recombinant host cells. In the recombinant host cells, the open reading frame coding sequences and/or the regulatory sequences are modified relative to the corresponding wild-type gene encoding the thioesterase. The activity of the thioesterase in the recombinant host cell is modified relative to the activity of the thioesterase expressed from the corresponding wild-type gene in a corresponding host cell. In some embodiments, a fatty acid derivative composition including fatty acids is produced by culturing a recombinant cell in the presence of a carbon source under conditions effective to express the thioesterase. In related embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide having thioesterase activity, and one or more additional polynucleotides encoding polypeptides having other fatty acid biosynthetic enzyme activities. In some such instances, the fatty acid produced by the action of the thioesterase is converted by one or more enzymes having a different fatty acid biosynthetic enzyme activity to another fatty acid derivative, such as, for example, a fatty ester, fatty aldehyde, fatty alcohol, or a hydrocarbon.
[0099] The chain length of a fatty acid, or a fatty acid derivative made therefrom, can be selected for by modifying the expression of particular thioesterases. The thioesterase will influence the chain length of fatty acid derivatives produced. The chain length of a fatty acid derivative substrate can be selected for by modifying the expression of selected thioesterases (EC 3.1.2.14 or EC 3.1.1.5). Hence, host cells can be engineered to express, overexpress, have attenuated expression, or not express one or more selected thioesterases to increase the production of a preferred fatty acid derivative substrate. For example, C.sub.10 fatty acids can be produced by expressing a thioesterase that has a preference for producing C.sub.10 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C.sub.10 fatty acids (e.g., a thioesterase which prefers to produce C.sub.14 fatty acids). This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 10. In other instances, C.sub.14 fatty acids can be produced by attenuating endogenous thioesterases that produce non-C.sub.14 fatty acids and expressing the thioesterases that use C.sub.14-ACP. In some situations, C.sub.12 fatty acids can be produced by expressing thioesterases that use C.sub.12-ACP and attenuating thioesterases that produce non-C.sub.12 fatty acids. For example, C12 fatty acids can be produced by expressing a thioesterase that has a preference for producing C12 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C12 fatty acids. This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 12. The fatty acid derivatives are recovered from the culture medium with substantially all of the fatty acid derivatives produced extracellularly. The fatty acid derivative composition produced by a recombinant host cell can be analyzed using methods known in the art, for example, GC-FID, in order to determine the distribution of particular fatty acid derivatives as well as chain lengths and degree of saturation of the components of the fatty acid derivative composition. Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, or GC-MS subsequent to cell lysis. Additional non-limiting examples of thioesterases and polynucleotides encoding them for use in the fatty acid pathway are provided in PCT Publication Application No. WO2010/075483, expressly incorporated by reference herein.
[0100] Production of Fatty Aldehydes
[0101] In one embodiment, the recombinant host cell produces a fatty aldehyde. In some embodiments, a fatty acid produced by the recombinant host cell is converted into a fatty aldehyde. In some embodiments, the fatty aldehyde produced by the recombinant host cell is then converted into a fatty alcohol or a hydrocarbon. In some embodiments, native (endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases, are present in the host cell (e.g., E. coli) and are effective to convert fatty aldehydes to fatty alcohols. In other embodiments, a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed. In still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed. A native or recombinant host cell may comprise a polynucleotide encoding an enzyme having fatty aldehyde biosynthesis activity (e.g., a fatty aldehyde biosynthetic polypeptide or a fatty aldehyde biosynthetic polypeptide or enzyme). A fatty aldehyde is produced when the fatty aldehyde biosynthetic enzyme is expressed or overexpressed in the host cell. A recombinant host cell engineered to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol. In some embodiments, a fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity such as carboxylic acid reductase (CAR) activity. CarB, is an exemplary carboxylic acid reductase. In practicing the disclosure, a gene encoding a carboxylic acid reductase polypeptide may be expressed or overexpressed in the host cell. In some embodiments, the CarB polypeptide has the amino acid sequence of SEQ ID NO: 7. In other embodiments, the CarB polypeptide is a variant or mutant of SEQ ID NO: 7. Examples of carboxylic acid reductase (CAR) polypeptides and polynucleotides encoding them include, but are not limited to FadD9 (EC 6.2.1.-, UniProtKB Q50631, GenBank NP_217106, SEQ ID NO: 34), CarA (GenBank ABK75684), CarB (GenBank YP889972; SEQ ID NO: 33) and related polypeptides described in PCT Publication No. WO2010/042664 and U.S. Pat. No. 8,097,439, each of which is expressly incorporated by reference herein. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a thioesterase. In some embodiments, the fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a fatty aldehyde biosynthetic polypeptide, such as a polypeptide having acyl-ACP reductase (AAR) activity. Expression of acyl-ACP reductase in a recombinant host cell results in the production of fatty aldehydes and fatty alcohols (see FIG. 4). Native (endogenous) aldehyde reductases present in a recombinant host cell (e.g., E. coli), can convert fatty aldehydes into fatty alcohols. Exemplary acyl-ACP reductase polypeptides are described in PCT Publication Nos. WO2009/140695 and WO/2009/140696, both of which are expressly incorporated by reference herein. A composition comprising fatty aldehydes (a fatty aldehyde composition) is produced by culturing a host cell in the presence of a carbon source under conditions effective to express the fatty aldehyde biosynthetic enzyme. In some embodiments, the fatty aldehyde composition comprises fatty aldehydes and fatty alcohols. Typically, the fatty aldehyde composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.
[0102] Production of Fatty Alcohols
[0103] In some embodiments, the recombinant host cell includes a polynucleotide encoding a polypeptide (an enzyme) having fatty alcohol biosynthetic activity (a fatty alcohol biosynthetic polypeptide or a fatty alcohol biosynthetic enzyme), and a fatty alcohol is produced by the recombinant host cell. A composition comprising fatty alcohols (a fatty alcohol composition) may be produced by culturing the recombinant host cell in the presence of a carbon source under conditions effective to express a fatty alcohol biosynthetic enzyme. In some embodiments, the fatty alcohol composition comprises fatty alcohols, however, a fatty alcohol composition may comprise other fatty acid derivatives. Typically, the fatty alcohol composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium. In one approach, recombinant host cells have been engineered to produce fatty alcohols by expressing a thioesterase, which catalyzes the conversion of acyl-ACPs into free fatty acids (FFAs) and a carboxylic acid reductase (CAR), which converts free fatty acids into fatty aldehydes. Native (endogenous) aldehyde reductases present in the host cell (e.g., E. coli) can convert the fatty aldehydes into fatty alcohols. In some embodiments, native (endogenous) fatty aldehyde biosynthetic polypeptides, such as aldehyde reductases present in the host cell, may be sufficient to convert fatty aldehydes to fatty alcohols. However, in other embodiments, a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed and in still other embodiments, an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed. In some embodiments, the fatty alcohol is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity which converts a fatty aldehyde to a fatty alcohol. For example, an alcohol dehydrogenase (aldehyde reductase, e.g., EC 1.1.1.1), may be used in practicing the disclosure. As used herein, an alcohol dehydrogenase refers to a polypeptide capable of catalyzing the conversion of a fatty aldehyde to an alcohol (e.g., a fatty alcohol). One of ordinary skill in the art will appreciate that certain alcohol dehydrogenases are capable of catalyzing other reactions as well, and these non-specific alcohol dehydrogenases also are encompassed by the alcohol dehydrogenase. Examples of alcohol dehydrogenase polypeptides useful in accordance with the disclosure include, but are not limited to AlrA of Acinetobacter sp. M-1 (SEQ ID NO: 3) or AlrA homologs such as AlrAadp1 (SEQ ID NO: 4) and endogenous E. coli alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1]. Additional examples are described in International Patent Application Publication Nos. WO 2007/136762, WO2008/119082 and WO 2010/062480, each of which is expressly incorporated by reference herein. In certain embodiments, the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1).
[0104] In another approach, recombinant host cells have been engineered to produce fatty alcohols by expressing fatty alcohol forming acyl-CoA reductases or fatty acyl reductases (FARs) which convert fatty acyl-thioester substrates (e.g., fatty acyl-CoA or fatty acyl-ACP) to fatty alcohols. In some embodiments, the fatty alcohol is produced by expressing or overexpressing a polynucleotide encoding a polypeptide having fatty alcohol forming acyl-CoA reductase (FAR) activity in a recombinant host cell. Examples of FAR polypeptides useful in accordance with this embodiment are described in PCT Publication No. WO2010/062480 which is expressly incorporated by reference herein. Fatty alcohol may be produced via an acyl-CoA dependent pathway utilizing fatty acyl-ACP and fatty acyl-CoA intermediates and an acyl-CoA independent pathway utilizing fatty acyl-ACP intermediates but not a fatty acyl-CoA intermediate. In particular embodiments, the enzyme encoded by the over expressed gene is selected from a fatty acid synthase, an acyl-ACP thioesterase, a fatty acyl-CoA synthase and an acetyl-CoA carboxylase. In some embodiments, the protein encoded by the over expressed gene is endogenous to the host cell. In other embodiments, the protein encoded by the overexpressed gene is heterologous to the host cell. Fatty alcohols are also made in nature by enzymes that are able to reduce various acyl-ACP or acyl-CoA molecules to the corresponding primary alcohols. See also, U.S. Patent Publication Nos. 20100105963, and 20110206630 and U.S. Pat. No. 8,097,439, expressly incorporated by reference herein. Strategies to increase production of fatty alcohols by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and/or expression of exogenous fatty acid biosynthetic genes from different organisms in the production host such that fatty alcohol biosynthesis is increased.
[0105] Production of Esters
[0106] As used herein, the term "fatty ester" may be used with reference to an ester. A fatty ester as referred to herein can be any ester made from a fatty acid, for example a fatty acid ester. In some embodiments, a fatty ester contains an A side and a B side. As used herein, an "A side" of an ester refers to the carbon chain attached to the carboxylate oxygen of the ester. As used herein, a "B side" of an ester refers to the carbon chain comprising the parent carboxylate of the ester. In embodiments where the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is contributed by an alcohol, and the B side is contributed by a fatty acid. Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. Moreover, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol. The carbon chains comprising the A side or B side can be of any length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. When the fatty ester is a fatty acid methyl ester, the A side of the ester is 1 carbon in length. When the fatty ester is a fatty acid ethyl ester, the A side of the ester is 2 carbons in length. The B side of the ester can be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains can have one or more points of branching. In addition, the branched chains can include cyclic branches. Furthermore, the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation. In one embodiment, the fatty ester is produced biosynthetically. In this embodiment, first the fatty acid is activated. Non-limiting examples of "activated" fatty acids are acyl-CoA, acyl ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. In addition, acyl-CoA can be synthesized from a free fatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase. In some embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide, e.g., an enzyme having ester synthase activity, (ester synthase polypeptide or an ester synthase).
[0107] A fatty ester is produced by a reaction catalyzed by the ester synthase polypeptide expressed or overexpressed in the recombinant host cell. In some embodiments, a composition comprising fatty esters fatty ester is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express an ester synthase. In some embodiments, the fatty ester composition is recovered from the cell culture. Ester synthase polypeptides include, for example, an ester synthase polypeptide classified as EC 2.3.1.75, or any other polypeptide which catalyzes the conversion of an acyl-thioester to a fatty ester, including, without limitation, a thioesterase, an ester synthase, an acyl-CoA:alcohol transacylase, an acyltransferase, or a fatty acyl-CoA:fatty alcohol acyltransferase. For example, the polynucleotide may encode wax/dgat, a bifunctional ester synthase/acyl-CoA:diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp. Strain ADP, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus. In a particular embodiment, the ester synthase polypeptide is an Acinetobacter sp. diacylglycerol O-acyltransferase (wax-dgaT; UniProtKB Q8GGG1, GenBank AA017391) or Simmondsia chinensis wax synthase (UniProtKB Q9XGY6, GenBank AAD38041. In another embodiment, the ester synthase polypeptide is for example ES9 (a wax ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE51 (SEQ ID NO: 6); ES8 of Marinobacter hydrocarbonoclasticus DSM8789 (GenBank Accession No. AB021021; SEQ ID NO:7); GenBank AB021021, encoded by the ws2 gene; or ES376 (another wax ester synthase derived from Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE50, GenBank ABO21020, encoded by the ws1 gene. In a particular embodiment, the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant host cell. In some embodiments, a fatty acid ester is produced by a recombinant host cell engineered to express three fatty acid biosynthetic enzymes: a thioesterase enzyme, an acyl-CoA synthetase (fadD) enzyme and an ester synthase enzyme (e.g., three enzyme system; see FIG. 5). In other embodiments, a fatty acid ester is produced by a recombinant host cell engineered to express one fatty acid biosynthetic enzyme, an ester synthase enzyme (e.g., one enzyme system; see FIG. 5). Non-limiting examples of ester synthase polypeptides and polynucleotides encoding them suitable for use in these embodiments include those described in PCT Publication Nos. WO2007/136762 and WO2008/119082, and WO/2011/038134 (three enzyme system) and WO/2011/038132 (one enzyme system), each of which is expressly incorporated by reference herein. The recombinant host cell may produce a fatty ester, such as a fatty acid methyl ester, a fatty acid ethyl ester or a wax ester in the extracellular environment of the host cells.
[0108] Production of Hydrocarbons
[0109] This aspect of the disclosure is based, at least in part, on the discovery that altering the level of expression of a fatty aldehyde biosynthetic polypeptide, for example, an acyl-ACP reductase polypeptide (EC 6.4.1.2) and a hydrocarbon biosynthetic polypeptide, e.g., a decarbonylase in a recombinant host cell facilitates enhanced production of hydrocarbons by the recombinant host cell. In one embodiment, the recombinant host cell produces a hydrocarbon, such as an alkane or an alkene (e.g., a terminal olefin or an internal olefin) or a ketone. In some embodiments, a fatty aldehyde produced by a recombinant host cell is converted by decarbonylation, removing a carbon atom to form a hydrocarbon. In other embodiments, a fatty acid produced by a recombinant host cell is converted by decarboxylation, removing a carbon atom to form a terminal olefin. In some embodiments, an acyl-ACP intermediate is converted by decarboxylation, removing a carbon atom to form an internal olefin or a ketone (see FIG. 6). In some embodiments, the recombinant host cell comprises a polynucleotide encoding a polypeptide (an enzyme) having hydrocarbon biosynthetic activity (a hydrocarbon biosynthetic polypeptide or a hydrocarbon biosynthetic enzyme), and the hydrocarbon is produced by expression or overexpression of the hydrocarbon biosynthetic enzyme in a recombinant host cell. An alkane biosynthetic pathway from cyanobacteria consisting of an acyl-acyl carrier protein reductase (AAR) and an aldehyde decarbonylase (ADC), which together convert intermediates of fatty acid metabolism to alkanes and alkenes has been used to engineer recombinant host cells for the production of hydrocarbons (FIG. 6). The second of two reactions in the pathway through which saturated acyl-ACPs are converted to alkanes in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (ADC), a ferritin-like protein with a binuclear metal cofactor of unknown composition. In some embodiments, the hydrocarbon is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having hydrocarbon biosynthetic activity such as an aldehyde decarbonylase (ADC) activity (e.g., EC 4.1.99.5). Exemplary polynucleotides encoding an aldehyde decarbonylase useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication Nos. WO2008/119082 and WO2009/140695 which are expressly incorporated by reference herein and those sequences presented in Table 2 below. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments the recombinant host cell further comprises a polynucleotide encoding an acyl-ACP reductase. See, for example, Table 2 below.
TABLE-US-00002 TABLE 2 Exemplary Hydrocarbon Biosynthetic Polynucleotides and Polypeptides Polypeptide Nucleotide Protein name sequence sequence Sequence Decarbonylase SEQ ID SEQ ID Synechococcus (ADC) NO: 35 NO: 36 elongatus PCC7942 YP.sub.-400610 (Synpcc7942.sub.-1593) Acyl-ACP SEQ ID SEQ ID Synechococcus Reducatase NO: 37 NO: 38 elongatus PCC7942 (AAR) YP_400611 (Synpcc7942_1594) Decarbonylase SEQ ID SEQ ID Prochlorococcus mariunus (ADC) NO: 39 NO: 40 CCMP1986 PMM0532 Acyl-ACP SEQ ID SEQ ID Prochlorococcus marinus Reducatase NO: 41 NO: 42 CCMP1986 PMM0533 (AAR) (NP_892651)
[0110] In some embodiments, a composition comprising is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the Acyl-CoA reductase and decarbonylase polynucleotides. In some embodiments, the hydrocarbon composition comprises saturated and unsaturated hydrocarbons. However, a hydrocarbon composition may comprise other fatty acid derivatives. Typically, the hydrocarbon composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium. As used herein, an alkane refers to saturated hydrocarbons or compounds that consist only of carbon (C) and hydrogen (H), wherein these atoms are linked together by single bonds (i.e., they are saturated compounds). Olefins and alkenes refer to hydrocarbons containing at least one carbon-to-carbon double bond (i.e., they are unsaturated compounds). Terminal olefins, .alpha.-olefins, terminal alkenes, and 1-alkenes refer to the same compounds with reference to .alpha.-olefins or alkenes with a chemical formula CxH2x, distinguished from other olefins with a similar molecular formula by linearity of the hydrocarbon chain and the position of the double bond at the primary or alpha position. In some embodiments, a terminal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having decarboxylase activity as described, for example, in PCT Publication No. WO2009/085278 which is expressly incorporated by reference herein. In some embodiments the recombinant host cell further comprises a polynucleotide encoding a thioesterase. In other embodiments, a ketone is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having OleA activity as described, for example, in PCT Publication No. WO2008/147781, which is expressly incorporated by reference herein. In related embodiments, an internal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having OleCD or OleBCD activity together with a polypeptide having OleA activity as described, for example, in PCT Publication No. WO2008/147781, expressly incorporated by reference herein.
[0111] Recombinant Host Cells and Cell Cultures
[0112] Strategies to increase production of fatty acid derivatives by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and expression of exogenous fatty acid biosynthetic genes from different organisms in the production host. As used herein, a recombinant host cell or engineered host cell refers to a host cell whose genetic makeup has been altered relative to the corresponding wild-type host cell, for example, by deliberate introduction of new genetic elements and/or deliberate modification of genetic elements naturally present in the host cell. The offspring of such recombinant host cells also contain these new and/or modified genetic elements. In any of the aspects of the disclosure described herein, the host cell can be selected from the group consisting of a plant cell, insect cell, fungus cell (e.g., a filamentous fungus, such as Candida sp., or a budding yeast, such as Saccharomyces sp.), an algal cell and a bacterial cell. In one preferred embodiment, recombinant host cells are recombinant microorganisms. Examples of host cells that are microorganisms, include but are not limited to cells from the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell. In some embodiments, the host cell is an E. coli cell. In other embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell. In other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell. In yet other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell. In yet other embodiments, the host cell is an Actinomycetes cell. In some embodiments, the host cell is a Saccharomyces cerevisiae cell. In other embodiments, the host cell is a cell from a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, an engineered organism thereof, or a synthetic organism. In some embodiments, the host cell is light-dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or mixotrophic in the absence of light. In certain embodiments, the host cell is a cell from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
[0113] Production of Fatty Acid Derivative Compositions by Recombinant Host Cells
[0114] A large variety of fatty acid derivatives can be produced by recombinant host cells comprising strain improvements as described herein, including, but not limited to, fatty acids, acyl-CoA, fatty aldehydes, short and long chain alcohols, hydrocarbons (e.g., alkanes, alkenes or olefins, such as terminal or internal olefins), fatty alcohols, esters (e.g., wax esters, fatty acid esters (e.g., methyl or ethyl esters)), and ketones. In some embodiments of the present disclosure, the higher titer of fatty acid derivatives in a particular composition is a higher titer of a particular type of fatty acid derivative (e.g., fatty alcohols, fatty acid esters, or hydrocarbons) produced by a recombinant host cell culture relative to the titer of the same fatty acid derivatives produced by a control culture of a corresponding wild-type host cell. In such cases, the fatty acid derivative compositions may comprise, for example, a mixture of the fatty alcohols with a variety of chain lengths and saturation or branching characteristics. In other embodiments of the present disclosure, the higher titer of fatty acid derivatives in a particular compositions is a higher titer of a combination of different fatty acid derivatives (for example, fatty aldehydes and alcohols, or fatty acids and esters) relative to the titer of the same fatty acid derivative produced by a control culture of a corresponding wild-type host cell.
[0115] Engineering Host Cells
[0116] In some embodiments, a polynucleotide (or gene) sequence is provided to the host cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. In some embodiments, the recombinant vector includes at least one sequence including, but not limited to, (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence. The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein. Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, N.J.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide. Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident .lamda. prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," second edition, Cold Spring Harbor Laboratory, (1989). Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and PET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., pp. 60-89 (1990)). In certain embodiments, a polynucleotide sequence of the disclosure is operably linked to a promoter derived from bacteriophage T5. In one embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector. Vectors can be introduced into prokaryotic or eukaryotic cells via a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra). For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.
[0117] Host Cells
[0118] As used herein, an engineered or recombinant host cell is a cell used to produce a fatty acid derivative composition as further described herein. A host cell is referred to as an engineered host cell or a recombinant host cell if the expression of one or more polynucleotides or polypeptides in the host cell are altered or modified as compared to their expression in a corresponding wild-type host cell (e.g., control cell) under the same conditions. In any of the aspects of the disclosure described herein, the host cell can be selected from the group consisting of a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, engineered organisms thereof, or a synthetic organism. In some embodiments, the host cell is light dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity. Various host cells can be used to produce fatty acid derivatives, as described herein.
[0119] Mutants or Variants
[0120] In some embodiments, the polypeptide is a mutant or a variant of any of the polypeptides described herein. The terms mutant and variant as used herein refer to a polypeptide having an amino acid sequence that differs from a wild-type polypeptide by at least one amino acid. For example, the mutant can comprise one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the mutant polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. Preferred fragments or mutants of a polypeptide retain some or all of the biological function (e.g., enzymatic activity) of the corresponding wild-type polypeptide. In some embodiments, the fragment or mutant retains at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more of the biological function of the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant retains about 100% of the biological function of the corresponding wild-type polypeptide. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE.TM. software (DNASTAR, Inc., Madison, Wis.). In yet other embodiments, a fragment or mutant exhibits increased biological function as compared to a corresponding wild-type polypeptide. For example, a fragment or mutant may display at least a 10%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% improvement in enzymatic activity as compared to the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant displays at least 100% (e.g., at least 200%, or at least 500%) improvement in enzymatic activity as compared to the corresponding wild-type polypeptide. It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide function. Whether or not a particular substitution will be tolerated (i.e., will not adversely affect desired biological function, such as carboxylic acid reductase activity) can be determined as described in Bowie et al. (Science, 247: 1306-1310 (1990)). A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0121] Variants can be naturally occurring or created in vitro. In particular, such variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures. Methods of making variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates. For example, variants can be prepared by using random and site-directed mutagenesis. Random and site-directed mutagenesis are described in, for example, Arnold, Curr. Opin. Biotech., 4: 450-455 (1993). Random mutagenesis can be achieved using error prone PCR (see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCR Methods Applic., 2: 28-33 (1992)). In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Briefly, in such procedures, nucleic acids to be mutagenized (e.g., a polynucleotide sequence encoding a carboxylic reductase enzyme) are mixed with PCR primers, reaction buffer, MgCl.sub.2, MnCl.sub.2, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94.degree. C. for 1 min, 45.degree. C. for 1 min, and 72.degree. C. for 1 min. However, it will be appreciated that these parameters can be varied as appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector, and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated. Site-directed mutagenesis can be achieved using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science, 241: 53-57 (1988). Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., a polynucleotide sequence encoding a CAR polypeptide). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed. Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408. Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequences in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described in, for example, Stemmer, Proc. Natl. Acad. Sci., U.S.A., 91: 10747-10751 (1994). Variants can also be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such "mutator" strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence (e.g., a polynucleotide sequence encoding a CAR polypeptide) in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described in, for example, International Patent Application Publication No. WO1991/016427. Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains a completely and/or partially randomized native sequence. Recursive ensemble mutagenesis can also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89: 7811-7815 (1992). In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993). In some embodiments, variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250. Insertional mutagenesis is mutagenesis of DNA by the insertion of one or more bases. Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab, e.g., by transposon mutagenesis. When exogenous DNA is integrated into that of the host, the severity of any ensuing mutation depends entirely on the location within the host's genome wherein the DNA is inserted. For example, significant effects may be evident if a transposon inserts in the middle of an essential gene, in a promoter region, or into a repressor or an enhancer region. Transposon mutagenesis and high-throughput screening was done to find beneficial mutations that increase the titer or yield of a fatty acid derivative or derivatives.
[0122] Culture Recombinant Host Cells and Cell Cultures/Fermentation
[0123] As used herein, the term "fermentation" broadly refers to the conversion of organic materials into target substances by host cells, for example, the conversion of a carbon source by recombinant host cells into fatty acids or derivatives thereof by propagating a culture of the recombinant host cells in a media comprising the carbon source. As used herein, the term "conditions permissive for the production" means any conditions that allow a host cell to produce a desired product, such as a fatty acid or a fatty acid derivative. Similarly, the term "conditions in which the polynucleotide sequence of a vector is expressed" means any conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, including but not limited to temperature ranges, levels of aeration, feed rates and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro-aerobic). Exemplary culture media include broths or gels. Generally, the medium includes a carbon source that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source. For small scale production, the engineered host cells can be grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding a CAR polypeptide. For large scale production, the engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, and 1,000,000 L or larger; fermented; and induced to express a desired polynucleotide sequence. Alternatively, large scale fed-batch fermentation may be carried out. The fatty acid derivative compositions described herein are found in the extracellular environment of the recombinant host cell culture and can be readily isolated from the culture medium. A fatty acid derivative may be secreted by the recombinant host cell, transported into the extracellular environment or passively transferred into the extracellular environment of the recombinant host cell culture. The fatty acid derivative is isolated from a recombinant host cell culture using routine methods known in the art.
[0124] Products Derived from Recombinant Host Cells
[0125] As used herein, "fraction of modem carbon" or fM has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1. Bioproducts (e.g., the fatty acid derivatives produced in accordance with the present disclosure) comprising biologically produced organic compounds, and in particular, the fatty acid derivatives produced using the fatty acid biosynthetic pathway herein, have not been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or .sup.14C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference). The ability to distinguish bioproducts from petroleum based organic compounds is beneficial in tracking these materials in commerce. For example, organic compounds or chemicals comprising both biologically based and petroleum based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum based materials. Hence, the bioproducts herein can be followed or tracked in commerce on the basis of their unique carbon isotope profile. Bioproducts can be distinguished from petroleum based organic compounds by comparing the stable carbon isotope ratio (.sup.13C/.sup.12C) in each sample. The .sup.13C/.sup.12C ratio in a given bioproduct is a consequence of the .sup.13C/.sup.12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in .sup.13C/.sup.12C and the corresponding .delta..sup.13C values. Furthermore, lipid matter of C3 and C4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, .sup.13C shows large variations due to isotopic fractionation effects, the most significant of which for bioproducts is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO.sub.2). Two large classes of vegetation are those that incorporate the "C3" (or Calvin-Benson) photosynthetic cycle and those that incorporate the "C4" (or Hatch-Slack) photosynthetic cycle. In C3 plants, the primary CO.sub.2 fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound. C3 plants, such as hardwoods and conifers, are dominant in the temperate climate zones. In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The CO.sub.2 thus released is refixed by the C3 cycle. Examples of C4 plants are tropical grasses, corn, and sugar cane. Both C4 and C3 plants exhibit a range of .sup.13C/.sup.12C isotopic ratios, but typical values are about -7 to about -13 per mil for C4 plants and about -19 to about -27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)). Coal and petroleum fall generally in this latter range. The 13C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The "613C" values are expressed in parts per thousand (per mil), abbreviated, %0, and are calculated as follows:
.delta..sup.13C(.Salinity.)=[(.sup.13C/.sup.12C)sample-(.sup.13C/.sup.12- C)standard]/(.sup.13C/.sup.12C)standard.times.1000
[0126] Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is .delta..sup.13C. Measurements are made on CO.sub.2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45, and 46. The compositions described herein include bioproducts produced by any of the methods described herein, including, for example, fatty aldehyde and alcohol products. Specifically, the bioproduct can have a .delta..sup.13C of about -28 or greater, about -27 or greater, -20 or greater, -18 or greater, -15 or greater, -13 or greater, -10 or greater, or -8 or greater. For example, the bioproduct can have a .delta..sup.13C of about -30 to about -15, about -27 to about -19, about -25 to about -21, about -15 to about -5, about -13 to about -7, or about -13 to about -10. In other instances, the bioproduct can have a .delta..sup.13C of about -10, -11, -12, or -12.3. Bioproducts produced in accordance with the disclosure herein, can also be distinguished from petroleum based organic compounds by comparing the amount of .sup.14C in each compound. Because .sup.14C has a nuclear half-life of 5730 years, petroleum based fuels containing "older" carbon can be distinguished from bioproducts which contain "newer" carbon (see, e.g., Currie, "Source Apportionment of Atmospheric Particles", Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)). The basic assumption in radiocarbon dating is that the constancy of .sup.14C concentration in the atmosphere leads to the constancy of .sup.14C in living organisms. However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, .sup.14C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO.sub.2, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (.sup.14C/.sup.12C) of about 1.2.times.10-12, with an approximate relaxation "half-life" of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric .sup.14C since the onset of the nuclear age.) It is this latter biospheric .sup.14C time characteristic that holds out the promise of annual dating of recent biospheric carbon. .sup.14C can be measured by accelerator mass spectrometry (AMS), with results given in units of "fraction of modern carbon" (fM). fM is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C. As used herein, "fraction of modern carbon" or "fM" has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the .sup.14C/.sup.12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1. The compositions described herein include bioproducts that can have an fM .sup.14C of at least about 1. For example, the bioproduct of the disclosure can have an fM .sup.14C of at least about 1.01, an fM .sup.14C of about 1 to about 1.5, an fM .sup.14C of about 1.04 to about 1.18, or an fM .sup.14C of about 1.111 to about 1.124.
[0127] Another measurement of .sup.14C is known as the percent of modern carbon (pMC). For an archaeologist or geologist using .sup.14C dates, AD 1950 equals "zero years old". This also represents 100 pMC. "Bomb carbon" in the atmosphere reached almost twice the normal level in 1963 at the peak of thermo-nuclear weapons. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 107.5 pMC. This means that a fresh biomass material, such as corn, would give a .sup.14C signature near 107.5 pMC. Petroleum based compounds will have a pMC value of zero. Combining fossil carbon with present day carbon will result in a dilution of the present day pMC content. By presuming 107.5 pMC represents the .sup.14C content of present day biomass materials and 0 pMC represents the .sup.14C content of petroleum based products, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 107.5 pMC. If that material was diluted 50% with petroleum based products, it would give a radiocarbon signature of approximately 54 pMC. A biologically based carbon content is derived by assigning "100%" equal to 107.5 pMC and "0%" equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum based material. A bioproduct comprising one or more fatty acid derivatives as described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, a fatty acid derivative described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95. In yet other instances, a fatty acid derivative described herein can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
[0128] Screening Fatty Acid Derivative Compositions Produced by Recombinant Host Cells
[0129] To determine if conditions are sufficient to allow expression, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used. Recombinant host cell cultures are screened at the 96 well plate level, 1 liter and 5 liter tank level and in a 1000 L pilot plant using a GC/FID assay for "total fatty species".
[0130] Utility of Fatty Acid Derivative Compositions
[0131] A fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides. When they are not attached to other molecules, they are known as "free" fatty acids. Fatty acids are usually produced industrially by the hydrolysis of triglycerides, with the removal of glycerol. Palm, soybean, rapeseed, coconut oil and sunflower oil are currently the most common sources of fatty acids. The majority of fatty acids derived from such sources are used in human food products. Coconut oil and palm kernel oil (consist mainly of 12 and 14 carbon fatty acids). These are particularly suitable for further processing to surfactants for washing and cleansing agents as well as cosmetics. Palm, soybean, rapeseed, and sunflower oil, as well as animal fats such as tallow, contain mainly long-chain fatty acids (e.g., C18, saturated and unsaturated) which are used as raw materials for polymer applications and lubricants. Ecological and toxicological studies suggest that fatty acid-derived products based on renewable resources have more favorable properties than petrochemical-based substances. Fatty aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction. Fatty alcohols have many commercial uses. Worldwide annual sales of fatty alcohols and their derivatives are in excess of U.S. $1 billion. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats. The disclosure also provides a surfactant composition or a detergent composition comprising a fatty alcohol produced by any of the methods described herein. One of ordinary skill in the art will appreciate that, depending upon the intended purpose of the surfactant or detergent composition, different fatty alcohols can be produced and used. For example, when the fatty alcohols described herein are used as a feedstock for surfactant or detergent production, one of ordinary skill in the art will appreciate that the characteristics of the fatty alcohol feedstock will affect the characteristics of the surfactant or detergent composition produced. Hence, the characteristics of the surfactant or detergent composition can be selected for by producing particular fatty alcohols for use as a feedstock. A fatty alcohol-based surfactant and/or detergent composition described herein can be mixed with other surfactants and/or detergents well known in the art. In some embodiments, the mixture can include at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol. In other examples, a surfactant or detergent composition can be made that includes at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of a fatty alcohol that includes a carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons in length. Such surfactant or detergent compositions also can include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources such as plant oils or petroleum, which can be present in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol. Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl esters, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor, which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals. Hydrocarbons have many commercial uses. For example, shorter chain alkanes are used as fuels. Longer chain alkanes (e.g., from five to sixteen carbons) are used as transportation fuels (e.g., gasoline, diesel, or aviation fuel). Alkanes having more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as a paraffin wax. In addition, longer chain alkanes can be cracked to produce commercially valuable shorter chain hydrocarbons. Like short chain alkanes, short chain alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers. Ketones are used commercially as solvents. For example, acetone is frequently used as a solvent, but it is also a raw material for making polymers. Ketones are also used in lacquers, paints, explosives, perfumes, and textile processing. In addition, ketones are used to produce alcohols, alkenes, alkanes, imines, and enamines. Lubricants are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using raw materials refined from crude petroleum. Obtaining these specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals. The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.
EXAMPLES
Example 1
[0132] Production Host Modifications--Attenuation of Acyl-CoA Dehydrogenase
[0133] This example describes the construction of a genetically engineered host cell wherein the expression of a fatty acid degradation enzyme is attenuated.
[0134] The fadE gene of Escherichia coli MG1655 (an E. coli K strain) was deleted using the Lambda Red (also known as the Red-Driven Integration) system described by Datsenko et al., Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000), with the following modifications:
[0135] The following two primers were used to create the deletion of fadE:
TABLE-US-00003 Del-fadE-F (SEQ ID NO: 9) 5'-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATA TTGATTCCGGGGATCCGTCGACC; and Del-fadE-R (SEQ ID NO: 10) 5'-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTT CCTGTAGGCTGGAGCTGCTTC
[0136] The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 (described by Datsenko et al., supra) by PCR. The PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (described in Datsenko et al., supra) that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in a super optimal broth with catabolite repression (SOC) medium at 37.degree. C., the cells were plated on Luria agar plates containing 50 .mu.g/mL of Kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37.degree. C. Disruption of the fadE gene was confirmed by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.
[0137] The fadE deletion confirmation primers were:
TABLE-US-00004 (SEQ ID NO: 11) fadE-L2 5'-CGGGCAGGTGCTATGACCAGGAC; and (SEQ ID NO: 12) fadE-R1 5'-CGCGGCGTTGACCGGCAGCCTGG
[0138] After the fadE deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain with the fadE gene deleted and the KmR marker removed was named E. coli MG1655 .DELTA.fadE, or E. coli MG 1655 Dl. Fatty acid derivative (total fatty species) production by the MG1655 E. coli strain with the fadE gene deleted was compared to fatty acid derivative production by E. coli MG1655. The deletion of the fadE gene did not affect fatty acid derivative production (FIG. 7). A number of exemplary host cell strains are described herein, examples of which are described below in Table 3.
TABLE-US-00005 TABLE 3 Genetic Characterization of E. coli Strains Strain Genetic Characterization DV2 MG1655 F-, .lamda.-, ilvG-, rfb-50, rph-1, .DELTA.fhuA::FRT, .DELTA.fadE::FRT DV2.1 DV2 fabB::fabB[A329V] D178 DV2.1 entD::FRT_P.sub.T5_entD EG149 D178 .DELTA.insH-11::P.sub.LACUV5-iFAB138 V642 EG149 rph+ SL313 V642 lacIZ::P.sub.A1_`tesA/pDG109 V668 V642 ilvG.sup.+ LC397 V668 lacIZ::P.sub.TRC_`tesA(var)_kan SL571 V668 lacIZ::P.sub.TRC_`tesA(var)_FRT LC942 SL571 attTn7::P.sub.TRC_'tesA(var) DG16 LC942/pLC56 V940 LC397/pV171.1 D851 SL571 yijP::Tn5-cat/pV171.1 BD64 DV2 .DELTA.insH-11::P.sub.LACUV5-iFAB138 loxP_ P.sub.T5_fadR DAM1 DV2 attTn7::P.sub.TRC_tesA_fadD Shu.002 DV2 .DELTA.insH-11::P.sub.T5-iFAB138 loxP_P.sub.T5_ fadR Plasmids: pDG109, pLC56 and pV171.1 both are pCL_P.sub.trc_carB_tesA_alrA_fabB_fadR operon with variable expression of carB and tesA. iFAB138 is SEQ ID NO: 19.
Example 2
[0139] Increased Flux Through the Fatty Acid Synthesis Pathway--Acetyl CoA Carboxylase Mediated
[0140] Fatty Ester Production:
[0141] The main precursors for fatty acid biosynthesis are malonyl-CoA and acetyl-CoA (FIG. 1). It has been suggested that these precursors limit the rate of fatty acid biosynthesis in E. coli. In this example, synthetic acc operons [Corynebacterium glutamicum accABCD (.+-.birA)] were overexpressed and the genetic modifications led to increased acetyl-coA and malonyl-CoA production in E. coli. In one approach, in order to increase malonyl-CoA levels, an acetyl-CoA carboxylase enzyme complex from Corynebacterium glutamicum (C. glutamicum) was overexpressed in E. coli. Acetyl-CoA carboxylase (acc) consists of four discrete subunits, accA, accB, accC and accD (FIG. 3). The advantage of C. glutamicum acc is that two subunits are expressed as fusion proteins, accCB and accDA, respectively, which facilitates its balanced expression. Additionally, C. glutamicum birA, which biotinylates the accB subunit (FIG. 3) was overexpressed. Exemplary C. glutamicum birA DNA sequences are presented as SEQ ID NO: 55 and SEQ ID NO: 56. A C. glutamicum birA protein sequence is presented as SEQ ID NO: 57.
[0142] The synthetic operons of the C. glutamicum acc genes were cloned in the following way in OP80 (see WO2008/119082 as incorporated-by-reference herein) Ptrc1-accDACB, Ptrc3-accDACB, Ptrc1-accCBDA and Ptrc3-CBDA. Ptrc1 and Ptrc3 are derivatives of the commonly used Ptrc promoter, which allow attenuated transcription of target genes. Note that the native sequences were amplified from the chromosomal DNA as they showed favorable codon usage (only the codon for Arg6 in accCB was changed). The C. glutamicum birA gene was codon optimized and obtained by gene synthesis. It was cloned downstream of the acc genes in all four operon constructs. Below we refer to the operon configuration accDACB as accD- and the operon configuration accDACB+birA as accD+. The resulting plasmids were transformed into E. coli DAM1_i377, which contains integrated copies (i) of leaderless thioesterease `tesA and acyl-CoA synthetase fadD from E. coli and Ester synthase 9 (ES9) from Marinobacter hydrocarbonoclasticus (SEQ ID NO: 6). All genes are controlled by Ptrc promoters. The strains were grown in 5NBT media (described below) in shake flasks and were analyzed for malonyl-CoA using short chain-CoA assay described below. FIG. 8 shows that six of the eight C. glutamicum acc.+-.birA constructs showed elevated levels of malonyl-CoA in logarithmic phase demonstrating their functionality in E. coli. It was noted that coexpression of birA further increased malonyl-CoA levels in the ptrc1/3_accDACB strains, in particular with the plasmid containing the Ptrc3-accDACB-birA operon configuration (plasmid pAS119.50D; SEQ ID NO: 62).
[0143] In order to test the effect of combining panK and acc-birA overexpression, the optimized panK gene was cloned downstream of birA in ptrc1/3_accDACB-birA. Pantothenate kinase panK (or CoaA) catalyzes the first step in the biosynthesis of coenzyme A, an essential cofactor that is involved in many reactions, e.g., the formation of acetyl-CoA, the substrate for acetyl-CoA carboxylase. The resulting plasmids were transformed into DAM1_i377, grown in 5NBT (+TVS1) media in shake flasks, and the strains were analyzed for short-chain-CoAs using the method described below. As shown in FIG. 9, in log phase panK coexpression further increased malonyl-CoA levels and also increased acetyl-CoA levels demonstrating that panK can further increase the malonyl-CoA levels. The impact of coexpressing an acetyl-CoA carboxylase enzyme complex on fatty ester production was evaluated by expressing ester synthase 9 (SEQ ID NO: 6) with and without acc genes in another E. coli production host. More specifically, plasmids OP80 (vector control), pDS57 (with ES9), pDS57-accD- (with ES9 and accDACB) or pDS57-accD+(with ES9 and accDACB-birA; SEQ ID NO: 63) were transformed into E. coli strain DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin.
[0144] Two transformants of each plasmid were independently inoculated into LB medium supplemented with 100 mg/L of spectinomycin and grown for 5-8 hours at 32.degree. C. The cultures were diluted 30-fold into a minimal medium with the following composition: 0.5 g/L NaCl, 1 mM MgSO.sub.4.times.7 H.sub.2O, 0.1 mM CaCl.sub.2, 2 g/L NH.sub.4Cl, 3 g/L KH.sub.2PO.sub.4, 6 g/L Na.sub.2HPO.sub.4, 1 mg/L thiamine, 1.times. trace metal solution, 10 mg/L ferric citrate, 100 mM Bis-Tris (pH7.0), 30 g/L glucose and 100 mg/L spectinomycin. After over-night growth at 32.degree. C., the cultures were diluted 10-fold in quadruplicate into minimal medium of the same composition except that the media contained 1 g/L instead of 2 g/L NH.sub.4Cl and was supplemented with 1 mM IPTG and 2% (v/v) methanol. The resulting cultures were then grown at 32.degree. C. in a shaker. The production of fatty acid methyl esters (FAMEs) was analyzed by gas chromatography with flame ionization detector (GC-FID). The samples were extracted with butyl acetate in a ratio of 1:1 vol/vol. After vortexing, the samples were centrifuged, and the organic phase was analyzed by gas chromatography (GC). The analysis conditions were as follows: instrument: Trace GC Ultra, Thermo Electron Corporation with Flame ionization detector (FID) detector; column: DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane) CO1 UFM 1/0.1/501 DET from Thermo Electron Corporation, phase pH 5, FT: 0.4 .mu.m, length 5 m, id: 0.1 mm; inlet conditions: 250.degree. C. splitless, 3.8 m 1/25 split method used depending upon sample concentration with split flow of 75 mL/m; carrier gas, flow rate: Helium, 3.0 mL/m; block temperature: 330.degree. C.; oven temperature: 0.5 m hold at 50.degree. C., 100.degree. C./m to 330.degree. C., 0.5 m hold at 330.degree. C.; detector temperature: 300.degree. C.; injection volume: 2 .mu.L; run time/flow rate: 6.3 m/3.0 mL/m (splitless method), 3.8 m/1.5 mL/m (split 1/25 method), 3.04 m/1.2 mL/m (split 1/50 method). FAMEs produced are shown in FIG. 10. The expression of ES9 by itself in E. coli DV2 led to FAME production above the control DV2 OP80. Coexpression of the C. glutamicum acetyl-CoA carboxylase complex led to an approx. 1.5-fold increase in FAMEs and the additional expression of the C. glutamicum biotin protein ligase led to an approx. 5-fold increase in FAMEs. These results suggest that the increased supply of malonyl-CoA improves the ability of ES9 to convert intermediates of the fatty acid biosynthetic machinery to fatty acid methyl esters in E. coli.
[0145] Short-chain-CoA assay: 15 ml falcon tubes were prepared with 0.467 ml 10% TCA with crotonyl-CoA as internal standard and overlayed with 2 ml of silicone oil. The tubes were chilled on ice and fermentation broth equivalent to 1 ml OD600=31.2 was carefully layered on top of the silicone oil. The samples were centrifuged at 11,400 g at 4.degree. C. for four 4 min cycles. For each sample, a 400 ml aliquots of the TCA/cellular extract was removed and placed in a fresh Eppendorf tube for neutralization with 1 ml Octylamine (in CHCl3). After vortexing, the samples were centrifuged for 30 sec at 13,000 g. 200 ml of the top layer was filtered using a 0.2 um PTFE syringe filter and then subjected to LC-MS/MS analysis.
[0146] Description of Media Used in Experiments:
TABLE-US-00006 Media ID 4N-BT 5N-BT FA2 FA2.1 FA2.3 Concentration Ingredient 0.5 0.5 0.5 0.5 0.5 g/L NaCl 2 2 2 2 2 g/L NH.sub.4Cl 3 3 3 3 3 g/L KH.sub.2PO.sub.4 6 6 6 6 6 g/L Na.sub.2PO.sub.4 1 1 1 1 1 mM MgSO.sub.4 0.1 0.1 0.1 0.1 0.1 mM CaCl.sub.2 1 1 1 1 1 mg/L thiamine 0.2 0.2 0.1 0.1 0.1 M Bis-Tris pH7 0.1 0.1 0.05 0.1 0.1 % Triton X-100 1 1 1 1 1 x Trace Minerals 27 27 10 10 10 mg/L FeCl.sub.2.cndot.6H.sub.2O 40 50 30 30 35 g/L glucose
[0147] 1000 Fold Concentrated Trace Vitamins Solution
[0148] 0.06 g/L Riboflavin
[0149] 6 g/L Niacin
[0150] 5.4 g/L Pantothenic Acid
[0151] 1.4 g/L Pyridoxine
[0152] 0.06 g/L Biotin
[0153] 0.01 g/L Folic Acid
[0154] 1000 Fold Concentrated Trace Metal Solution
[0155] 2 mL/L Concentrated hydrochloric acid
[0156] 0.5 g/L boric acid
[0157] 1.9 g/L cupric sulfate, pentahydrate, USP
[0158] 1 g/L zinc chloride anhydrous
[0159] 2 g/L sodium molybdenate dehydrate
[0160] 2 g/L calcium chloride dehydrate
[0161] Fatty Alcohol Production:
[0162] The impact of coexpressing an acetyl-CoA carboxylase enzyme complex on Fatty alcohol production was evaluated by expressing the Acyl-ACP reductase (AAR) from Synechococcus elongatus (SEQ ID NO: 38) with and without acc genes in E. coli DV2. The accD+ operon configuration was selected as it gave the best results when coexpressed with ester synthase (see previous example). The accDABC-birA operon was cloned downstream from the aar gene in pLS9-185 (a pCL1920 derivative) using Infusion technology (Clontech Laboratories, Inc., Mountain View, Calif.). The resulting plasmid was transformed into E. coli DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin. Fatty alcohols produced are shown in FIG. 11. The coexpression of AAR and accD+ led to a ca. 1.5-fold increase in fatty alcohol titers as compared to the AAR only control (pLS9-185). The data were reproducible (triplicate samples were shown). These results demonstrate that increasing malonyl-CoA levels lead to improved fatty acid production when this acyl-ACP reductase is used. In addition, Example 3 describes co-expression of acc genes together with entire fab operons.
Example 3
[0163] Increased Flux Through the Fatty Acid Synthesis Pathway--iFABs
[0164] Fatty Acid Derivative Production:
[0165] Strategies to increase the flux through the fatty acid synthesis pathway in recombinant host cells include both overexpression of native E. coli fatty acid biosynthesis genes and expression of exogenous fatty acid biosynthesis genes from different organisms in E. coli. In this study, fatty acid biosynthesis genes from different organisms were combined in the genome of E. coli DV2 (Table 3) under the control of the lacUV5 promoter and integrated into the IS5-11 site. Sixteen strains containing iFABs 130-145 were evaluated. The detailed structure of iFABs 130-145 is presented in Tables 4 and 5.
TABLE-US-00007 TABLE 4 Components from Different Species used in iFABs 130-145 Abbreviation Full Description St_fabD Salmonella typhimurium fabD gene nSt_fabH Salmonella typhimurium FabH gene with the native RBS sSt_fabH Salmonella typhimurium fabH gene with a synthetic RBS Cac_fabF Clostridium acetobutylicum (ATCC824) fabF gene St_fabG Salmonella typhimurium fabG gene St_fabA Salmonella typhimurium fabA gene St_fabZ Salmonella typhimurium fabZ gene BS_fabI Bacillus subtilis fabI gene BS_FabL Bacillus subtilis fabL gene Vc_FabV Vibrio chorlerae fabV gene Ec_FabI Escherichia coli fabI gene
[0166] Each "iFAB" included various fab genes in the following order: 1) an enoyl-ACP reductase (BS_fabI, BS_FabL, Vc_FabV, or Ec_FabI); 2) a b-ketoacyl-ACP synthetase III (St fabH); 3) a malonyl-CoA-ACP transacylase (St_fabD); 4) a b-ketoacyl-ACP reductase (St fabG); 5) a 3-hydroxy-acyl-ACP dehydratase (St_fabA or St_fabZ); 6) a b-ketoacyl-ACP synthetase II (Cac_fabF). Note that St_fabA also has trans-2, cis-3-decenoyl-ACP isomerase activity and that Cac_fabF has b-ketoacyl-ACP synthetase II and b-ketoacyl-ACP synthetase I activities (Zhu et al., BMC Microbiology 9:119 (2009)). See Table 5, below for the specific composition of iFABs 130-145.
TABLE-US-00008 TABLE 5 Composition of iFABs 130-145 ifab BS_fabI BS_fabL Vc_fabV Ec_fabI nSt_fabH sSt_fabH St_fabD St_fabG St_fabA St_fabZ Cac_fabF ifab130 1 0 0 0 1 0 1 1 1 0 1 ifab131 1 0 0 0 1 0 1 1 0 1 1 ifab132 1 0 0 0 0 1 1 1 1 0 1 ifab133 1 0 0 0 0 1 1 1 0 1 1 ifab134 0 1 0 0 1 0 1 1 1 0 1 ifab135 0 1 0 0 1 0 1 1 0 1 1 ifab136 0 1 0 0 0 1 1 1 1 0 1 ifab137 0 1 0 0 0 1 1 1 0 1 1 ifab138 0 0 1 0 1 0 1 1 1 0 1 ifab139 0 0 1 0 1 0 1 1 0 1 1 ifab140 0 0 1 0 0 1 1 1 1 0 1 ifab141 0 0 1 0 0 1 1 1 0 1 1 ifab142 0 0 0 1 1 0 1 1 1 0 1 ifab143 0 0 0 1 1 0 1 1 0 1 1 ifab144 0 0 0 1 0 1 1 1 1 0 1 ifab145 0 0 0 1 0 1 1 1 0 1 1
[0167] The plasmid pCL_P.sub.trc_tesA was transformed into each of the strains and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32.degree. C. and 37.degree. C. Data for production of Total Fatty Species from duplicate plate screens is shown in FIGS. 12A and 12B. From this screen the best construct was determined to be DV2 with iFAB138. The sequence of iFAB138 in the genome of EG149 is presented as SEQ ID NO: 19.
[0168] Fatty Ester Production:
[0169] A full synthetic fab operon was integrated into the E. coli chromosome and evaluated for increased FAME production by expression in E. coli DAM1 pDS57. In addition, four synthetic acc operons from Corynebaterium glutamicum were coexpressed and evaluated for improved FAME productivity. Several strains were obtained that produced FAMES at a faster rate and higher titers. The sixteen different iFAB operons (Table 5) were put under the control of the lacUV5 promoter and integrated into the IS5-11 site of E. coli DAM1. These strains were named DAM1 ifab130 to 145. They were transformed either with pDS57 (containing ester synthase 377) or pDS57 co-expressing different versions of acc operons, see above) for evaluation of FAME production. Exemplary plasmids are described in Table 6.
TABLE-US-00009 TABLE 6 Plasmids containing Ester Synthase ES9 (from Marinobacter hydrocarbonclasticus) and Synthetic acc Operons (from Corynebactrium glutamicum) Plasmid Genes pTB.071 pDS57-accCBDA pTB.072 pDS57-accCBDA-birA pTB.073 pDS57-accDACB pTB.074 pDS57-accDACB-birA pDS57 = pCL_ptrc-ES9
[0170] The DAM1 ifab strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) (see above for medium description) and in fermenters at 32.degree. C. The best results were obtained in 96-well plates and in shake flasks, where several DAM1 ifab strains with pDS57-acc-birA plasmids showed higher FAME titers. In particular, DAM1 ifab131, ifab135, ifab137, ifab138 and ifab143 with pDS57-accDACB-birA showed 20-40% improved titers indicating that in these strains a higher flux through the fatty acid pathway was achieved, which resulted in a better product formation rate (these results were reproducible in several independent experiments).
[0171] Effect of overexpressing fabH and fabI on Fatty Acid Methyl Ester (FAME) Production:
[0172] Strategies to increase the flux through the fatty acid synthesis pathway in recombinant host cells include both overexpression of native fatty acid biosynthesis genes and expression of heterologous fatty acid biosynthesis genes. FabH and fabl are two fatty acid biosynthetic enzymes that have been shown to be feedback inhibited (Heath and Rock, JBC 271: 1833-1836 (1996)). A study was conducted to determine if FabH and FabI might be limiting the rate of FAME production. FabH and fabl homologues (from E. coli, B. subtilis, Acinetobacter baylyi ADP1, Marinobacter aquaeoli VT8, and Rhodococcus opacus) were overexpressed as a synthetic operon and evaluated in E. coli DAM1 pDS57 (a strain observed to be a good FAME producer). In one approach, fabHfabI operons were constructed from organisms that accumulate waxes (A. baylyi, M. aquaeoli) or triacylglycerides (R. opacus) and integrated into the chromosome of E. coli DAM1 pDS57. In a related approach, a synthetic acc operons from C. glutamicum were co-expressed (as described in Example 2, above). Eleven different fabHI operons were constructed (assembled in vitro) as summarized in Table 7. The fabHI operons were put under the control of IPTG inducible lacUV5 promoter and integrated into the IS5-11 site of E. coli DAM1. These strains were named as shown in the table below. They were transformed either with pDS57 (containing ester synthase 377) or pDS57 coexpressing different versions of acc operons for evaluation of FAME production.
TABLE-US-00010 TABLE 7 Genotype of Integrated fabHI Operons Strain Genotype of additional fab operon Plasmid stEP117 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) EcfabH (synRBS) Bsfabl::kan pDS57 stEP118 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) EcfabH (synRBS) BsfabL::kan pDS57 stEP127 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) EcfabH (EcRBS) Bsfabl::kan pDS57 stEP128 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) EcfabH (EcRBS) BsfabL::kan pDS57 stEP129 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) ADP1fabH (EcRBS) ADP1fabl::kan pDS57 stEP130 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) ADP1fabH (synRBS) ADP1fabl::kan pDS57 stEP131 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) VT8fabH1 (synRBS) VT8fabl::kan pDS57 stEP132 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) VT8fabH2 (synRBS) VT8fabl::kan pDS57 stEP133 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH1 (synRBS) VT8fabl::kan pDS57 stEP134 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH2 (synRBS) VT8fabl::kan pDS57 stEP151 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) Rofabl (synRBS) RofabH::kan pDS57 stEP153 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) ADP1fabH (EcRBS) ADP1fabl::kan pDS57-accCBDA stEP154 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) ADP1fabH (EcRBS) ADP1fabl::kan pDS57-accDACB stEP155 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) ADP1fabH (EcRBS) ADP1fabl::kan pDS57-accCBDA-birA stEP156 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) ADP1fabH (EcRBS) ADP1fabl::kan pDS57-accDACB-birA stEP157 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) EcfabH (synRBS) Bsfabl::kan pDS57-accCBDA stEP158 DAM1 .DELTA.insH::P.sub.LACUV5 (snyRBS) EcfabH (synRBS) Bsfabl::kan pDS57-accCBDA-birA stEP159 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) EcfabH (synRBS) Bsfabl::kan pDS57-accCBDA stEP160 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) EcfabH (synRBS) Bsfabl::kan pDS57-accCBDA-birA stEP161 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH1 (synRBS) VT8fabl::kan pDS57-accCBDA stEP162 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH1 (synRBS) VT8fabl::kan pDS57-accCBDA-birA stEP163 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH2 (synRBS) VT8fabl::kan pDS57-accCBDA stEP164 DAM1 .DELTA.insH::P.sub.LACUV5 (EcRBS) VT8fabH2 (synRBS) VT8fabl::kan pDS57-accCBDA-birA Bs: Bacillus subtilis; Ec: Escherichia coli; ADP1: Acinetobacter sp. ADP1; VT8: Marinobacter aquaeolei VT8; Ro: Rhodococcus opacus B4
[0173] The DAM1 ifabHI strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) and in fermenters at 32.degree. C. In a shake flask, a number of the ifabHI strains carrying pDS57 plasmid performed better than the control DAM1 pDS57 strain, reaching 10 to 15% higher FAME titers (FIG. 13). Additional increase in FAME titers was obtained when ifabHI strains were transformed with pDS57-acc-birA plasmids, in particular an increase of 50% in FAME titers was observed in strain StEP156 (DAM1 IS5-11::lacUV5(ecRBS)ADP1fabH (ecRBS)ADP1fabI pDS57-accDACB-birA) (FIG. 14).
[0174] Some of the strains with ifabHI were run in fermenters, where an increase in FAME titers, specific productivity and yield was also observed (FIG. 15), indicating that in these strains a higher flux through the fatty acid pathway was achieved, which resulted in a better product formation rate. In particular stEP129 (DAM15-11::UV5(ecRBS)ADP1fabH (ecRBS)ADP1fabI pDS57) showed higher FAME titers and yield in several independent fermentation runs. Other combinations of fabH and fabl may be used to achieve similar effects. Although FAME is exemplified here, this approach to alter fatty acid biosynthetic genes is a useful approach to increase production of any fatty acid derivative.
[0175] Effect of Inserting a Strong Promoter in Front of Operon FAB138 on Fatty Acid Methyl Ester (FAME) production:
[0176] The lacUV5 promoter of iFAB138 was replaced by a T5 promoter (SEQ ID NO: 2) leading to higher levels of expression of iFAB138, as confirmed by mRNA analysis. The expression of iFAB138 from the T5 promoter resulted in a higher titer, yield and productivity of fatty esters. Strain shu.002 (Table 3) is isogenic to strain BD64 (Table 3) except that it contains the T5 promoter controlling expression of the iFAB138 operon (SEQ ID NO: 19).
TABLE-US-00011 TABLE 8 Primers used to Generate iT5_138 Cassette and Verify its Insertion in New Strains SEQ Primer ID Name NO Sequence DG405 20 TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTT CTTTATGTAAAAAAAACgtttTAGGATGCATATGG CGGCC DG406 21 GATAAATCCACGAATTTTAGGTTTGATGATCATTG GTCTCCTCCTGCAGGTGCGTGTTCGTCGTCATCGC AATTG DG422 22 ACTCACCGCATTGGTGTAGTAAGGCGCACC DG423 23 TGAATGTCATCACGCAGTTCCCAGTCATCC EG744 24 CCATCTTCTTTGTACAGACGTTGACTGAACATG EG749 24 GCACCATAGCCGTAATCCCACAGGTTATAG oTREE047 26 TGTCATTAATGGTTAATAATGTTGA
[0177] Primers DG405 and DG406 (Table 8) were used to amplify a cat-loxP and T5 promoter cassette adding 50 bp homology to each end of the PCR product, such that it could be integrated into any strain replacing the lacUV5 promoter regulating expression of the iFAB138 operon. The cat-loxP-T5 promoter was transformed into BD64/pKD46 strain. Transformants were recovered on LB+chloramphenicol plates at 37.degree. C. overnight, patched to a fresh LB+chloramphenicol plate, and verified by colony PCR using primers DG422 and DG423. Plasmid pJW168 (Palmeros et al., Gene 247: 255-264 (2000)) was transformed into strain BD64 i-cat-loxP-T5_138 and selected on LB+carbenicillin plates at 32.degree. C. In order to remove the cat marker, expression of the cre-recombinase was induced by IPTG. The plasmid pJW168 was removed by growing cultures at 42.degree. C. Colonies were patched on LB+chloramphenicol and LB+carbenicillin to verify loss of pJW168 and removal of cat marker, respectively. The colony was also patched into LB as a positive control, all patched plates were incubated at 32.degree. C. The removal of the cat marker was confirmed by colony PCR using primers DG422 and DG423. The resulting PCR product was verified by sequencing with primers EG744, EG749 and oTREE047, the strain was called shu.002. FIG. 16 shows the iFAB138 locus: a diagram of the cat-loxP-P.sub.TS cassette integrated in front of FAB138 (FIG. 16A) and a diagram of the P.sub.T5_iFAB138 region (FIG. 16B). The sequence of the cat-loxP-T5 promoter integrated in front of iFAB138 with homology to integration site is presented as SEQ ID NO: 1 and the sequence of the iT5_FAB138 promoter region with homology to integration site is presented as SEQ ID NO: 2. There are a number of conditions that can lead to increased fatty acid flux. In this example increased fatty acid flux was achieved by altering the promoter strength of operon iFAB138. The expression of iFAB138 from the T5 promoter was beneficial, nonetheless, when this promoter change was combined with the insertion of yijP::Tn5 cassette further improvements were observed in titer, yield and productivity of fatty acid esters and other fatty acid derivatives (data not shown).
Example 4
[0178] Increasing the Amount of Free Fatty Acid (FFA) Product by Repairing the rph and ilvG Mutations
[0179] The ilvG and rph mutations were corrected in this strain resulting in higher production of FFA. Strains EG149 and V668 (Table 3) were transformed with pCL_P.sub.trc_tesA. Fermentation was run at 32.degree. C. in FA2 media for 40 hours to compare the FFA production of strains EG149 and V668 with pCL_P.sub.trc_tesA. Correcting the rph and ilvG mutations resulted in a 116% increase in the FFA production of the base strain with pCL_P.sub.trc_tesA. As seen in FIG. 17, V668/pCL_P.sub.trc_tesA produced more FFA than the EG149/pCL_P.sub.trc_tesA control. Since FFA is a precursor to the LS9 products, higher FFA production is a good indicator that the new strain can produce higher levels of LS9 products.
Example 5: Increased Production of Fatty Acid Derivatives by Transposon Mutagenesis--yijP
[0180] Fatty Alcohol Production:
[0181] To improve the titer, yield, productivity of fatty alcohol production by E. coli, transposon mutagenesis and high-throughput screening was carried out and beneficial mutations were sequenced. A transposon insertion in the yijP strain was shown to improve the strain's fatty alcohol yield in both shake flask and fed-batch fermentations. The SL313 strain produces fatty alcohols. The genotype of this strain is provided in Table 3. Transposon clones were then subjected to high-throughput screening to measure production of fatty alcohols. Briefly, colonies were picked into deep-well plates containing LB, grown overnight, inoculated into fresh LB and grown for 3 hours, inoculated into fresh FA2.1 media, grown for 16 hours, then extracted using butyl acetate. The crude extract was derivatized with BSTFA (N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using GC/FID. Spectinomycin (100 mg/L) was included in all media to maintain selection of the pDG109 plasmid. Hits were selected by choosing clones that produced a similar total fatty species as the control strain SL313, but that had a higher percent of fatty alcohol species and a lower percent of free fatty acids than the control. Strain 68F11 was identified as a hit and was validated in a shake flask fermentation using FA2.1 media. A comparison of transposon hit 68F11 to control strain SL313 indicated that 68F11 produces a higher percentage of fatty alcohol species than the control, while both strains produce similar titers of total fatty species. A single colony of hit 68F11, named LC535, was sequenced to identify the location of the transposon insertion. Briefly, genomic DNA was purified from a 10 mL overnight LB culture using the kit ZR Fungal/Bacterial DNA MiniPrep.TM. (Zymo Research Corporation, Irvine, Calif.) according to the manufacturer's instructions. The purified genomic DNA was sequenced outward from the transposon using primers internal to the transposon:
TABLE-US-00012 DG150 (SEQ ID NO: 27) 5'-GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG-3' DG131 (SEQ ID NO: 28) 5'-GAGCCAATATGCGAGAACACCCGAGAA-3'
[0182] Strain LC535 was determined to have a transposon insertion in the yijP gene (FIG. 18). yijP encodes a conserved inner membrane protein whose function is unclear. The yijP gene is in an operon and co-transcribed with the ppc gene, encoding phosphoenolpyruvate carboxylase, and the yijO gene, encoding a predicted DNA-binding transcriptional regulator of unknown function. Promoters internal to the transposon likely have effects on the level and timing of transcription of yijP, ppc and yijO, and may also have effects on adjacent genes frwD, pflC, pfld, and argE. Promoters internal to the transposon cassette are shown in FIG. 18, and may have effects on adjacent gene expression. Strain LC535 was evaluated in a fed-batch fermentation on two different dates. Both fermentations demonstrated that LC535 produced fatty alcohols with a higher yield than control SL313, and the improvement was 1.3-1.9% absolute yield based on carbon input. The yijP transposon cassette was further evaluated in a different strain V940, which produces fatty alcohol at a higher yield than strain SL313. The yijP::Tn5-cat cassette was amplified from strain LC535 using primers:
TABLE-US-00013 LC277 (SEQ ID NO: 29) 5'-CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCA G-3' LC278 (SEQ ID NO: 30) 5'-GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATCC AACG-3'
[0183] This linear DNA was electroporated into strain SL571 and integrated into the chromosome using the lambda red recombination system. Colonies were screened using primers outside the transposon region:
TABLE-US-00014 DG407 (SEQ ID NO: 31) 5'-AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG-3' DG408 (SEQ ID NO: 32) 5'-ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG-3'
[0184] A colony with the correct yijP transposon cassette was transformed with the production plasmid pV171.1 to produce strain D851. D851 was tested in a shake-flask fermentation against strain V940 that does not contain the yijP transposon cassette. The result of this fermentation showed that the yijP transposon cassette confers production of a higher percent of fatty alcohol by the D851 strain relative to the V940 strain and produces similar titers of total fatty species as the V940 control strain. Strain D851 was evaluated in a fed-batch fermentation on two different dates. Data from these fermentations is shown in Table 9 which illustrates that in 5-liter fed-batch fermentations, strains with the yijP::Tn5-cat transposon insertion had an increased total fatty species ("FAS") yield and an increase in percent fatty alcohol ("FALC"). The terms "total fatty species" and "total fatty acid product" may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and free fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO 2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis. As used herein, the term "fatty esters" includes beta hydroxy esters.
TABLE-US-00015 TABLE 9 Effect of yijP transposon insertion on titer and yield of FAS and FALC FAS FAS Percent FALC Strain Titer Yield FALC Yield V940 68 g/L 18.7% 95.0% 17.8% D851 70 g/L 19.4% 96.1% 18.6% V940 64 g/L 18.4% 91.9% 16.9% D851 67 g/L 19.0% 94.0% 17.8%
[0185] Tank Fermentation Method:
[0186] To assess production of fatty acid and fatty acid derivatives in tank a glycerol vial of desired strain was used to inoculate 20 mL LB+spectinomycin in shake flask and incubated at 32.degree. C. for approximately six hours. 4 mL of LB culture was used to inoculate 125 mL Low PFA Seed Media (below), which was then incubated at 32.degree. C. shaker overnight. 50 mL of the overnight culture was used to inoculate 1 L of Tank Media. Tanks were run at pH 7.2 and 30.5.degree. C. under pH stat conditions with a maximum feed rate of 16 g/L/hr glucose.
TABLE-US-00016 TABLE 10 Low P FA Seed Media: Component Concentration NH.sub.4Cl 2 g/L NaCl 0.5 g/L KH.sub.2PO.sub.4 1 g/L MgSO.sub.4--7H.sub.2O 0.25 g/L CaCl.sub.2--2H.sub.2O 0.015 g/L Glucose 20 g/L TM2 Trace Minerals solution 1 mL/L Ferric citrate 10 mg/L Bis Tris buffer (pH 7.0) 100 mM Spectinomycin 115 mg/L
TABLE-US-00017 TABLE 11 Tank Media Component Concentration (NH.sub.4).sub.2SO.sub.4 0.5 g/L KH.sub.2PO.sub.4 3.0 g/L Ferric Citrate 0.034 g/L TM2 Trace Minerals Solution 10 mL/L Casamino acids 5 g/L Post sterile additions MgSO.sub.4--7H.sub.2O 2.2 g/L Trace Vitamins Solution 1.25 mL/L Glucose 5 g/L Inoculum 50 mL/L
[0187] Further studies suggest that the improved titer and yield of FAS and FALC in strains with the yijP transposon insertion is due to reduction in the activity of phosphoenolpyruvate carboxylase (ppc). A ppc enzyme assay was carried out in-vitro in the following strains to evaluate this hypothesis.
[0188] 1) Appc=DG14 (LC942 .DELTA.ppc::cat-sacB/pLC56)
[0189] 2) wt-ppc=DG16 (LC942/pLC56)
[0190] 3) yijP::Tn5=DG18 (LC942 yijP::Tn5-cat/pLC56)
[0191] Ppc activity was measured in cells grown in a shake flask fermentation using a standard shake flask protocol in FA2.3 media (described above) and harvested 12-16 hours after induction. Approximately 5 mL of cells were centrifuged and the cell paste was suspended in BugBuster Protein Extraction Reagent (Novagen) with a protease inhibitor cocktail solution. The cell suspension was incubated with gentle shaking on a shaker for 20 min. Insoluble cell debris was removed by centrifugation at 16,000.times.g for 20 min at 4.degree. C. followed by transferring the supernatant to a new tube. Ppc activity in the cell lysate was determined by a coupling reaction with citrate synthase using following reaction mixture: 0.4 mM acetyl-CoA, 10 mM phosphoenolpyruvate, 0.5 mM monobromobimane, 5 mM MgCl.sub.2, 10 mM NaHCO.sub.3, and 10 units citrate synthase from porcine heart in 100 mM Tris-HCl (pH 8.0). The formation of CoA in the reaction with citrate synthase using oxaloacetate and acetyl-CoA was monitored photometrically using fluorescent derivatization of CoA with monobromobimane. The Ppc assay results showed that the yijP::Tn5-cat transposon cassette decreased the Ppc activity in the cell by 2.7 fold compared to wild type cells. The cells with deletion of ppc did not grow well and the activity was about 10 times lower than wild type cells. The results also indicate that the highest yield of fatty alcohol production requires a level of Ppc expression lower than the wild-type level. Proteomics data was also collected to assess the abundance of the Ppc protein in two strains with and without the yijP::Tn5-cat transposon cassette. Protein samples were collected from strains V940 and D851 grown in bioreactors under standard fatty alcohol production conditions (described above). Samples were taken at two different time points: 32 and 48 hours and prepared for analysis.
[0192] Sample collection and protein isolation was carried out as follows:
[0193] 20 ml of fermentation broth were collected from each bioreactor at each time point. Samples were quenched with ice-cold PBS and harvested by centrifugation (4500 rpm/10 min) at 4.degree. C. Cell pellet was washed with ice-cold PBS and centrifuged one more time and stored at -80.degree. C. for further processing.
[0194] Total protein extraction was performed using a French press protocol. Briefly, cell pellets were resuspended in 7 ml of ice-cold PBS and French pressed at 2000 psi twice to ensure complete lysing of the bacteria. Samples were centrifuged for 20 min at 10000 rpm at 4.degree. C. to separate non-lysed cells and cell debris from the protein fraction. Total protein concentration of clear lysate was determined using BCA Protein Assay Reagent. Samples were diluted to 2 mg proteins/ml concentration and frozen at -80.degree. C.
[0195] Samples were resuspended in the appropriate buffer and trypsinized overnight at 37.degree. C. and lyophilized. Fragmented protein samples were labeled with isotopically enriched methylpiperazine acetic acid at room temperature for 30 min. Labeled samples were separated using cation exchange liquid chromatography and subjected to mass spectroscopy analysis using an ion trap mass spectrometer. Raw data was normalized using background subtraction and bias correction.
[0196] Proteomics data showed a significant reduction in the relative abundance of Ppc protein in D851 strain when compared to V940 at 32 hours and 48 hours. D851 had about 15% of the Ppc levels of V940 at 32 hours and about 35% of the Ppc levels of V940 at 48 hours. These data show that the yijP::Tn5-cat transposon cassette results in a significant reduction in Ppc abundance in the cell. This suggests that the observed benefits to fatty alcohol production by strains harboring the yijP::Tn5-cat transposon hit is due to reducing the amount of Ppc protein.
[0197] These results suggest that altering ppc activity can improve the yield of fatty acid derivatives. There are a number of ways to alter the expression of the ppc gene, and the yijP transposon insertion is one way to accomplish this. Without wanting to be bound by theory, if the effect of reducing phosphoenolpyruvate carboxylase activity is to limit the flow of carbon through the TCA cycle, one could achieve similar results by decreasing the activity of citrate synthase (gltA) or slowing the TCA cycle by decreasing the activity of any of the enzymes involved in the TCA cycle.
Example 6
[0198] Increased Flux Through the Fatty Acid Synthesis Pathway--Acyl Carrier Protein (ACP) Mediated Fatty Alcohol Production
[0199] When terminal pathway enzymes from sources other than E. coli are expressed in E. coli as the heterologous host to convert fatty acyl-ACPs to products, limitations may exist in the recognition, affinity and/or turnover of the recombinant pathway enzyme towards the E. coli fatty acyl-ACPs. Note that although ACP proteins are conserved to some extent in all organisms, their primary sequence can differ significantly. To test this hypothesis the acp genes from several cyanobacteria were cloned downstream from the Synechococcus elongatus PCC7942 acyl-ACP reductase (AAR) present in pLS9-185, which is a pCL1920 derivative. In addition, the sfp gene (Accession no. X63158; SEQ ID NO: 53) from Bacillus subtilis, encoding a phosphopantetheinyl transferase with broad substrate specificity, was cloned downstream of the respective acp genes. This enzyme is involved in conversion of the inactive apo-ACP to the active holo-ACP. The plasmids constructed are described in Table 12.
TABLE-US-00018 TABLE 12 Plasmids Coexpressing Cyanobacterial ACP with and without B. subtilis sfp Downstream from S. elongatus PCC7942 AAR ACP SEQ Base ID NO. (DNA/ Without With plasmid ACP Source Polypeptide) sfp sfp pLS9-185 Synechococcus 49/50 pDS168 pDS168S elongatus 7942 pLS9-185 Synechocystis 45/46 pDS169 not sp. 6803 available pLS9-185 Prochlorococcus 47/48 pDS170 pDS170S marinus MED4 pLS9-185 Nostoc punctiforme 43/44 pDS171 pDS171S 73102 pLS9-185 Nostoc sp. 7120 51/52 pDS172 pDS172S
[0200] All the acp genes were cloned with a synthetic RBS into the EcoRI site immediately downstream of the aar gene in pLS9-185 using InFusion technology (Clontech Laboratories, Inc., Mountain View, Calif.). The EcoRI site was reconstructed downstream of the acp gene. Similarly, the B. subtilis sfp gene was InFusion cloned into this EcoRI site along with a synthetic RBS. All plasmids were transformed into E. coli MG1655 DV2 (Table 3). The control for these experiments was the expression of AAR alone (pLS9-185). The results from standard shake flask fermentation experiments are shown in FIG. 19. Significant improvement in fatty alcohol titers were observed in strains containing the plasmids pDS171S, pDS172S, pDS168 and pDS169 demonstrating that ACP overexpression can be beneficial for fatty alcohol production, in this case presumably by aiding in the recognition, affinity and/or turnover of acyl-ACPs by the heterologous terminal pathway enzyme. (See Table 12 for the source of the ACPs and presence or absence of sfp).
[0201] Fatty Acid Production:
[0202] In order to evaluate if the overexpression of an ACP can also increase free fatty acid production, one cyanobacterial ACP gene with sfp was amplified from pDS171s (Table 12) and cloned downstream from `tesA into a pCL vector. The resulting operon was under the control of the Ptrc3 promoter, which provides slightly lower transcription levels than the Ptrc wildtype promoter. The construct was cloned into E. coli DV2 and evaluated for fatty acid production. The control strain contained the identical plasmid but without cyanobacterial ACP and B. subtilis sfp. The results from a standard microtiter plate fermentation experiment are shown in FIG. 20. Significant improvement in fatty acid titer was observed in the strain coexpressing the heterologous ACP demonstrating that ACP overexpression can be beneficial for fatty acid production, in this case presumably by increasing the flux through the fatty acid biosynthetic pathway.
Sequence CWU
1
1
6411232DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 1ttgtccatct ttatataatt tgggggtagg
gtgttcttta tgtaaaaaaa acgttttagg 60atgcatatgg cggccgcata acttcgtata
gcatacatta tacgaagtta tctagagttg 120catgcctgca ggtccgctta ttatcactta
ttcaggcgta gcaaccaggc gtttaagggc 180accaataact gccttaaaaa aattacgccc
cgccctgcca ctcatcgcag tactgttgta 240attcattaag cattctgccg acatggaagc
catcacaaac ggcatgatga acctgaatcg 300ccagcggcat cagcaccttg tcgccttgcg
tataatattt gcccatggtg aaaacggggg 360cgaagaagtt gtccatattg gccacgttta
aatcaaaact ggtgaaactc acccagggat 420tggctgagac gaaaaacata ttctcaataa
accctttagg gaaataggcc aggttttcac 480cgtaacacgc cacatcttgc gaatatatgt
gtagaaactg ccggaaatcg tcgtggtatt 540cactccagag cgatgaaaac gtttcagttt
gctcatggaa aacggtgtaa caagggtgaa 600cactatccca tatcaccagc tcaccgtctt
tcattgccat acggaattcc ggatgagcat 660tcatcaggcg ggcaagaatg tgaataaagg
ccggataaaa cttgtgctta tttttcttta 720cggtctttaa aaaggccgta atatccagct
gaacggtctg gttataggta cattgagcaa 780ctgactgaaa tgcctcaaaa tgttctttac
gatgccattg ggatatatca acggtggtat 840atccagtgat ttttttctcc attttagctt
ccttagctcc tgaaaatctc gataactcaa 900aaaatacgcc cggtagtgat cttatttcat
tatggtgaaa gttggaacct cttacgtgcc 960gatcaacgtc tcattttcgc caaaagttgg
cccagggctt cccggtatca acagggacac 1020caggatttat ttattctgcg aagtgatctt
ccgtcacagg tatttattcg actctagata 1080acttcgtata gcatacatta tacgaagtta
tggatccagc ttatcgatac cgtcaaacaa 1140atcataaaaa atttatttgc tttcaggaaa
atttttctgt ataatagatt caattgcgat 1200gacgacgaac acgcacctgc aggaggagac
ca 12322232DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 2ttgtccatct ttatataatt tgggggtagg gtgttcttta tgtaaaaaaa
acgttttagg 60atgcatatgg cggccgcata acttcgtata gcatacatta tacgaagtta
tggatccagc 120ttatcgatac cgtcaaacaa atcataaaaa atttatttgc tttcaggaaa
atttttctgt 180ataatagatt caattgcgat gacgacgaac acgcacctgc aggaggagac
ca 2323340PRTAcinetobacter sp. 3Met Ser Asn His Gln Ile Arg
Ala Tyr Ala Ala Met Gln Ala Gly Glu1 5 10
15Gln Val Val Pro Tyr Gln Phe Asp Ala Gly Glu Leu Lys
Ala His Gln 20 25 30Val Glu
Val Lys Val Glu Tyr Cys Gly Leu Cys His Ser Asp Leu Ser 35
40 45Val Ile Asn Asn Glu Trp Gln Ser Ser Val
Tyr Pro Ala Val Ala Gly 50 55 60His
Glu Ile Ile Gly Thr Ile Ile Ala Leu Gly Ser Glu Ala Lys Gly65
70 75 80Leu Lys Leu Gly Gln Arg
Val Gly Ile Gly Trp Thr Ala Glu Thr Cys 85
90 95Gln Ala Cys Asp Pro Cys Ile Gly Gly Asn Gln Val
Leu Cys Thr Gly 100 105 110Glu
Lys Lys Ala Thr Ile Ile Gly His Ala Gly Gly Phe Ala Asp Lys 115
120 125Val Arg Ala Gly Trp Gln Trp Val Ile
Pro Leu Pro Asp Asp Leu Asp 130 135
140Pro Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Leu Asp145
150 155 160Pro Leu Leu Lys
His Lys Ile Gln Ala Thr His His Val Gly Val Ile 165
170 175Gly Ile Gly Gly Leu Gly His Ile Ala Ile
Lys Leu Leu Lys Ala Trp 180 185
190Gly Cys Glu Ile Thr Ala Phe Ser Ser Asn Pro Asp Lys Thr Glu Glu
195 200 205Leu Lys Ala Asn Gly Ala Asp
Gln Val Val Asn Ser Arg Asp Ala Gln 210 215
220Ala Ile Lys Gly Thr Arg Trp Lys Leu Ile Ile Leu Ser Thr Ala
Asn225 230 235 240Gly Thr
Leu Asn Val Lys Ala Tyr Leu Asn Thr Leu Ala Pro Lys Gly
245 250 255Ser Leu His Phe Leu Gly Val
Thr Leu Glu Pro Ile Pro Val Ser Val 260 265
270Gly Ala Ile Met Gly Gly Ala Lys Ser Val Thr Ser Ser Pro
Thr Gly 275 280 285Ser Pro Leu Ala
Leu Arg Gln Leu Leu Gln Phe Ala Ala Arg Lys Asn 290
295 300Ile Ala Pro Gln Val Glu Leu Phe Pro Met Ser Gln
Leu Asn Glu Ala305 310 315
320Ile Glu Arg Leu His Ser Gly Gln Ala Arg Tyr Arg Ile Val Leu Lys
325 330 335Ala Asp Phe Asp
3404314PRTAcinetobacter sp. 4Met Ala Thr Thr Asn Val Ile His Ala Tyr
Ala Ala Met Gln Ala Gly1 5 10
15Glu Ala Leu Val Pro Tyr Ser Phe Asp Ala Gly Glu Leu Gln Pro His
20 25 30Gln Val Glu Val Lys Val
Glu Tyr Cys Gly Leu Cys His Ser Asp Val 35 40
45Ser Val Leu Asn Asn Glu Trp His Ser Ser Val Tyr Pro Val
Val Ala 50 55 60Gly His Glu Val Ile
Gly Thr Ile Thr Gln Leu Gly Ser Glu Ala Lys65 70
75 80Gly Leu Lys Ile Gly Gln Arg Val Gly Ile
Gly Trp Thr Ala Glu Ser 85 90
95Cys Gln Ala Cys Asp Gln Cys Ile Ser Gly Gln Gln Val Leu Cys Thr
100 105 110Gly Glu Asn Thr Ala
Thr Ile Ile Gly His Ala Gly Gly Phe Ala Asp 115
120 125Lys Val Arg Ala Gly Trp Gln Trp Val Ile Pro Leu
Pro Asp Glu Leu 130 135 140Asp Pro Thr
Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe145
150 155 160Asp Pro Ile Leu Lys His Gln
Ile Gln Ala Ile His His Val Ala Val 165
170 175Ile Gly Ile Gly Gly Leu Gly His Met Ala Ile Lys
Leu Leu Lys Ala 180 185 190Trp
Gly Cys Glu Ile Thr Ala Phe Ser Ser Asn Pro Asn Lys Thr Asp 195
200 205Glu Leu Lys Ala Met Gly Ala Asp His
Val Val Asn Ser Arg Asp Asp 210 215
220Ala Glu Ile Lys Ser Gln Gln Gly Lys Phe Asp Leu Leu Leu Ser Thr225
230 235 240Val Asn Val Pro
Leu Asn Trp Asn Ala Tyr Leu Asn Thr Leu Ala Pro 245
250 255Asn Gly Thr Phe His Phe Leu Gly Val Val
Met Glu Pro Ile Pro Val 260 265
270Pro Val Gly Ala Leu Leu Gly Gly Ala Lys Ser Leu Thr Ala Ser Pro
275 280 285Thr Gly Ser Pro Ala Ala Leu
Arg Lys Leu Leu Glu Phe Ala Ala Arg 290 295
300Lys Asn Ile Ala Pro Gln Ile Glu Met Tyr305
31051020DNAEscherichia coli 5atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg
gcgaactgga agtttatgag 60tacgatcccg gtgagctgag gccacaagat gttgaagtgc
aggtggatta ctgcgggatc 120tgccattccg atctgtcgat gatcgataac gaatggggat
tttcacaata tccgctggtt 180gccgggcatg aggtgattgg gcgcgtggtg gcactcggga
gcgccgcgca ggataaaggt 240ttgcaggtcg gtcagcgtgt cgggattggc tggacggcgc
gtagctgtgg tcactgcgac 300gcctgtatta gcggtaatca gatcaactgc gagcaaggtg
cggtgccgac gattatgaat 360cgcggtggct ttgccgagaa gttgcgtgcg gactggcaat
gggtgattcc actgccagaa 420aatattgata tcgagtccgc cgggccgctg ttgtgcggcg
gtatcacggt ctttaaacca 480ctgttgatgc accatatcac tgctaccagc cgcgttgggg
taattggtat tggcgggctg 540gggcatatcg ctataaaact tctgcacgca atgggatgcg
aggtgacagc ctttagttct 600aatccggcga aagagcagga agtgctggcg atgggtgccg
ataaagtggt gaatagccgc 660gatccgcagg cactgaaagc actggcgggg cagtttgatc
tcattatcaa caccgtcaac 720gtcagcctcg actggcagcc ctattttgag gcgctgacct
atggcggtaa tttccatacg 780gtcggtgcgg ttctcacgcc gctgtctgtt ccggccttta
cgttaattgc gggcgatcgc 840agcgtctctg gttctgctac cggcacgcct tatgagctgc
gtaagctgat gcgttttgcc 900gcccgcagca aggttgcgcc gaccaccgaa ctgttcccga
tgtcgaaaat taacgacgcc 960atccagcatg tgcgcgacgg taaggcgcgt taccgcgtgg
tgttgaaagc cgatttttga 10206473PRTMarinobacter hydrocarbonoclasticus
6Met Lys Arg Leu Gly Thr Leu Asp Ala Ser Trp Leu Ala Val Glu Ser1
5 10 15Glu Asp Thr Pro Met His
Val Gly Thr Leu Gln Ile Phe Ser Leu Pro 20 25
30Glu Gly Ala Pro Glu Thr Phe Leu Arg Asp Met Val Thr
Arg Met Lys 35 40 45Glu Ala Gly
Asp Val Ala Pro Pro Trp Gly Tyr Lys Leu Ala Trp Ser 50
55 60Gly Phe Leu Gly Arg Val Ile Ala Pro Ala Trp Lys
Val Asp Lys Asp65 70 75
80Ile Asp Leu Asp Tyr His Val Arg His Ser Ala Leu Pro Arg Pro Gly
85 90 95Gly Glu Arg Glu Leu Gly
Ile Leu Val Ser Arg Leu His Ser Asn Pro 100
105 110Leu Asp Phe Ser Arg Pro Leu Trp Glu Cys His Val
Ile Glu Gly Leu 115 120 125Glu Asn
Asn Arg Phe Ala Leu Tyr Thr Lys Met His His Ser Met Ile 130
135 140Asp Gly Ile Ser Gly Val Arg Leu Met Gln Arg
Val Leu Thr Thr Asp145 150 155
160Pro Glu Arg Cys Asn Met Pro Pro Pro Trp Thr Val Arg Pro His Gln
165 170 175Arg Arg Gly Ala
Lys Thr Asp Lys Glu Ala Ser Val Pro Ala Ala Val 180
185 190Ser Gln Ala Met Asp Ala Leu Lys Leu Gln Ala
Asp Met Ala Pro Arg 195 200 205Leu
Trp Gln Ala Gly Asn Arg Leu Val His Ser Val Arg His Pro Glu 210
215 220Asp Gly Leu Thr Ala Pro Phe Thr Gly Pro
Val Ser Val Leu Asn His225 230 235
240Arg Val Thr Ala Gln Arg Arg Phe Ala Thr Gln His Tyr Gln Leu
Asp 245 250 255Arg Leu Lys
Asn Leu Ala His Ala Ser Gly Gly Ser Leu Asn Asp Ile 260
265 270Val Leu Tyr Leu Cys Gly Thr Ala Leu Arg
Arg Phe Leu Ala Glu Gln 275 280
285Asn Asn Leu Pro Asp Thr Pro Leu Thr Ala Gly Ile Pro Val Asn Ile 290
295 300Arg Pro Ala Asp Asp Glu Gly Thr
Gly Thr Gln Ile Ser Phe Met Ile305 310
315 320Ala Ser Leu Ala Thr Asp Glu Ala Asp Pro Leu Asn
Arg Leu Gln Gln 325 330
335Ile Lys Thr Ser Thr Arg Arg Ala Lys Glu His Leu Gln Lys Leu Pro
340 345 350Lys Ser Ala Leu Thr Gln
Tyr Thr Met Leu Leu Met Ser Pro Tyr Ile 355 360
365Leu Gln Leu Met Ser Gly Leu Gly Gly Arg Met Arg Pro Val
Phe Asn 370 375 380Val Thr Ile Ser Asn
Val Pro Gly Pro Glu Gly Thr Leu Tyr Tyr Glu385 390
395 400Gly Ala Arg Leu Glu Ala Met Tyr Pro Val
Ser Leu Ile Ala His Gly 405 410
415Gly Ala Leu Asn Ile Thr Cys Leu Ser Tyr Ala Gly Ser Leu Asn Phe
420 425 430Gly Phe Thr Gly Cys
Arg Asp Thr Leu Pro Ser Met Gln Lys Leu Ala 435
440 445Val Tyr Thr Gly Glu Ala Leu Asp Glu Leu Glu Ser
Leu Ile Leu Pro 450 455 460Pro Lys Lys
Arg Ala Arg Thr Arg Lys465 4707455PRTMarinobacter
hydrocarbonoclasticus 7Met Thr Pro Leu Asn Pro Thr Asp Gln Leu Phe Leu
Trp Leu Glu Lys1 5 10
15Arg Gln Gln Pro Met His Val Gly Gly Leu Gln Leu Phe Ser Phe Pro
20 25 30Glu Gly Ala Pro Asp Asp Tyr
Val Ala Gln Leu Ala Asp Gln Leu Arg 35 40
45Gln Lys Thr Glu Val Thr Ala Pro Phe Asn Gln Arg Leu Ser Tyr
Arg 50 55 60Leu Gly Gln Pro Val Trp
Val Glu Asp Glu His Leu Asp Leu Glu His65 70
75 80His Phe Arg Phe Glu Ala Leu Pro Thr Pro Gly
Arg Ile Arg Glu Leu 85 90
95Leu Ser Phe Val Ser Ala Glu His Ser His Leu Met Asp Arg Glu Arg
100 105 110Pro Met Trp Glu Val His
Leu Ile Glu Gly Leu Lys Asp Arg Gln Phe 115 120
125Ala Leu Tyr Thr Lys Val His His Ser Leu Val Asp Gly Val
Ser Ala 130 135 140Met Arg Met Ala Thr
Arg Met Leu Ser Glu Asn Pro Asp Glu His Gly145 150
155 160Met Pro Pro Ile Trp Asp Leu Pro Cys Leu
Ser Arg Asp Arg Gly Glu 165 170
175Ser Asp Gly His Ser Leu Trp Arg Ser Val Thr His Leu Leu Gly Leu
180 185 190Ser Asp Arg Gln Leu
Gly Thr Ile Pro Thr Val Ala Lys Glu Leu Leu 195
200 205Lys Thr Ile Asn Gln Ala Arg Lys Asp Pro Ala Tyr
Asp Ser Ile Phe 210 215 220His Ala Pro
Arg Cys Met Leu Asn Gln Lys Ile Thr Gly Ser Arg Arg225
230 235 240Phe Ala Ala Gln Ser Trp Cys
Leu Lys Arg Ile Arg Ala Val Cys Glu 245
250 255Ala Tyr Gly Thr Thr Val Asn Asp Val Val Thr Ala
Met Cys Ala Ala 260 265 270Ala
Leu Arg Thr Tyr Leu Met Asn Gln Asp Ala Leu Pro Glu Lys Pro 275
280 285Leu Val Ala Phe Val Pro Val Ser Leu
Arg Arg Asp Asp Ser Ser Gly 290 295
300Gly Asn Gln Val Gly Val Ile Leu Ala Ser Leu His Thr Asp Val Gln305
310 315 320Asp Ala Gly Glu
Arg Leu Leu Lys Ile His His Gly Met Glu Glu Ala 325
330 335Lys Gln Arg Tyr Arg His Met Ser Pro Glu
Glu Ile Val Asn Tyr Thr 340 345
350Ala Leu Thr Leu Ala Pro Ala Ala Phe His Leu Leu Thr Gly Leu Ala
355 360 365Pro Lys Trp Gln Thr Phe Asn
Val Val Ile Ser Asn Val Pro Gly Pro 370 375
380Ser Arg Pro Leu Tyr Trp Asn Gly Ala Lys Leu Glu Gly Met Tyr
Pro385 390 395 400Val Ser
Ile Asp Met Asp Arg Leu Ala Leu Asn Met Thr Leu Thr Ser
405 410 415Tyr Asn Asp Gln Val Glu Phe
Gly Leu Ile Gly Cys Arg Arg Thr Leu 420 425
430Pro Ser Leu Gln Arg Met Leu Asp Tyr Leu Glu Gln Gly Leu
Ala Glu 435 440 445Leu Glu Leu Asn
Ala Gly Leu 450 4558457PRTAlcanivorax borkumensis 8Met
Lys Ala Leu Ser Pro Val Asp Gln Leu Phe Leu Trp Leu Glu Lys1
5 10 15Arg Gln Gln Pro Met His Val
Gly Gly Leu Gln Leu Phe Ser Phe Pro 20 25
30Glu Gly Ala Gly Pro Lys Tyr Val Ser Glu Leu Ala Gln Gln
Met Arg 35 40 45Asp Tyr Cys His
Pro Val Ala Pro Phe Asn Gln Arg Leu Thr Arg Arg 50 55
60Leu Gly Gln Tyr Tyr Trp Thr Arg Asp Lys Gln Phe Asp
Ile Asp His65 70 75
80His Phe Arg His Glu Ala Leu Pro Lys Pro Gly Arg Ile Arg Glu Leu
85 90 95Leu Ser Leu Val Ser Ala
Glu His Ser Asn Leu Leu Asp Arg Glu Arg 100
105 110Pro Met Trp Glu Ala His Leu Ile Glu Gly Ile Arg
Gly Arg Gln Phe 115 120 125Ala Leu
Tyr Tyr Lys Ile His His Ser Val Met Asp Gly Ile Ser Ala 130
135 140Met Arg Ile Ala Ser Lys Thr Leu Ser Thr Asp
Pro Ser Glu Arg Glu145 150 155
160Met Ala Pro Ala Trp Ala Phe Asn Thr Lys Lys Arg Ser Arg Ser Leu
165 170 175Pro Ser Asn Pro
Val Asp Met Ala Ser Ser Met Ala Arg Leu Thr Ala 180
185 190Ser Ile Ser Lys Gln Ala Ala Thr Val Pro Gly
Leu Ala Arg Glu Val 195 200 205Tyr
Lys Val Thr Gln Lys Ala Lys Lys Asp Glu Asn Tyr Val Ser Ile 210
215 220Phe Gln Ala Pro Asp Thr Ile Leu Asn Asn
Thr Ile Thr Gly Ser Arg225 230 235
240Arg Phe Ala Ala Gln Ser Phe Pro Leu Pro Arg Leu Lys Val Ile
Ala 245 250 255Lys Ala Tyr
Asn Cys Thr Ile Asn Thr Val Val Leu Ser Met Cys Gly 260
265 270His Ala Leu Arg Glu Tyr Leu Ile Ser Gln
His Ala Leu Pro Asp Glu 275 280
285Pro Leu Ile Ala Met Val Pro Met Ser Leu Arg Gln Asp Asp Ser Thr 290
295 300Gly Gly Asn Gln Ile Gly Met Ile
Leu Ala Asn Leu Gly Thr His Ile305 310
315 320Cys Asp Pro Ala Asn Arg Leu Arg Val Ile His Asp
Ser Val Glu Glu 325 330
335Ala Lys Ser Arg Phe Ser Gln Met Ser Pro Glu Glu Ile Leu Asn Phe
340 345 350Thr Ala Leu Thr Met Ala
Pro Thr Gly Leu Asn Leu Leu Thr Gly Leu 355 360
365Ala Pro Lys Trp Arg Ala Phe Asn Val Val Ile Ser Asn Ile
Pro Gly 370 375 380Pro Lys Glu Pro Leu
Tyr Trp Asn Gly Ala Gln Leu Gln Gly Val Tyr385 390
395 400Pro Val Ser Ile Ala Leu Asp Arg Ile Ala
Leu Asn Ile Thr Leu Thr 405 410
415Ser Tyr Val Asp Gln Met Glu Phe Gly Leu Ile Ala Cys Arg Arg Thr
420 425 430Leu Pro Ser Met Gln
Arg Leu Leu Asp Tyr Leu Glu Gln Ser Ile Arg 435
440 445Glu Leu Glu Ile Gly Ala Gly Ile Lys 450
455970DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 9aaaaacagca
acaatgtgag ctttgttgta attatattgt aaacatattg attccgggga 60tccgtcgacc
701068DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 10aaacggagcc tttcggctcc
gttattcatt tacgcggctt caactttcct gtaggctgga 60gctgcttc
681123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 11cgggcaggtg ctatgaccag gac
231223DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 12cgcggcgttg
accggcagcc tgg
231360DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 13ccttggcatt ggcaatttga
gaattcgagg aggaaaacta aatgaccatt tcctcacctt 601460DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 14ttttgttcgg gcccaagctt ttattgcaaa cgcagatgcg tgatttcacc
cgcattcagc 601560DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 15cgggcccaag
cttcgaattc ttattgcaaa cgcagatgcg tgatttcacc cgcattcagc
601660DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 16gaatagcgcc gtcgacgagg
aggaaaacta aatgaccatt tcctcacctt tgattgacgt 601760DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 17tgatgatgat gatggtcgac ttattgcaaa cgcagatgcg tgatttcacc
cgcattcagc 60184250DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 18atgaccattt
cctcaccttt gattgacgtc gccaaccttc cagacatcaa caccactgcc 60ggcaagatcg
ccgaccttaa ggctcgccgc gcggaagccc atttccccat gggtgaaaag 120gcagtagaga
aggtccacgc tgctggacgc ctcactgccc gtgagcgctt ggattactta 180ctcgatgagg
gctccttcat cgagaccgat cagctggctc gccaccgcac caccgctttc 240ggcctgggcg
ctaagcgtcc tgcaaccgac ggcatcgtga ccggctgggg caccattgat 300ggacgcgaag
tctgcatctt ctcgcaggac ggcaccgtat tcggtggcgc gcttggtgag 360gtgtacggcg
aaaagatgat caagatcatg gagctggcaa tcgacaccgg ccgcccattg 420atcggtcttt
acgaaggcgc tggcgctcgt attcaggacg gcgctgtctc cctggacttc 480atttcccaga
ccttctacca aaacattcag gcttctggcg ttatcccaca gatctccgtc 540atcatgggcg
catgtgcagg tggcaacgct tacggcccag ctctgaccga cttcgtggtc 600atggtggaca
agacctccaa gatgttcgtt accggcccag acgtgatcaa gaccgtcacc 660ggcgaggaaa
tcacccagga agagcttggc ggagcaacca cccacatggt gaccgctggt 720aactcccact
acaccgctgc gaccgatgag gaagcactgg attgggtaca ggacctggtg 780tccttcctcc
catccaacaa tcgctcctac gcaccgatgg aagacttcga cgaggaagaa 840ggcggcgttg
aagaaaacat caccgctgac gatctgaagc tcgacgagat catcccagat 900tccgcgaccg
ttccttacga cgtccgcgat gtcatcgaat gcctcaccga cgatggcgaa 960tacctggaaa
tccaggcaga ccgcgcagaa aacgttgtta ttgcattcgg ccgcatcgaa 1020ggccagtccg
ttggctttgt tgccaaccag ccaacccagt tcgctggctg cctggacatc 1080gactcctctg
agaaggcagc tcgcttcgtc cgcacctgcg acgcgttcaa catcccaatc 1140gtcatgcttg
tcgacgtccc cggcttcctc ccaggcgcag gccaggagta cggtggcatt 1200ctgcgtcgtg
gcgcaaagct gctctacgca tacggcgaag caaccgttcc aaagatcacc 1260gtcaccatgc
gtaaggctta cggcggagcg tactgcgtga tgggttccaa gggcttgggc 1320tctgacatca
accttgcatg gccaaccgca cagatcgccg tcatgggcgc tgctggcgca 1380gttggattca
tctaccgcaa ggagctcatg gcagctgatg ccaagggcct cgataccgta 1440gctctggcta
agtccttcga gcgcgagtat gaagaccaca tgctcaaccc gtaccacgct 1500gcagaacgtg
gcctgatcga cgccgtgatc ctgccaagcg aaacccgcgg acagatttcc 1560cgcaaccttc
gcctgctcaa gcacaagaac gtcactcgcc ctgctcgcaa gcacggcaac 1620atgccactgt
aaggaggaaa actaaatgtc agtcgagact cgcaagatca ccaaggttct 1680tgtcgctaac
cgtggtgaga ttgcaatccg cgtgttccgt gcagctcgag atgaaggcat 1740cggatctgtc
gccgtctacg cagagccaga tgcagatgca ccattcgtgt catatgcaga 1800cgaggctttt
gccctcggtg gccaaacatc cgctgagtcc taccttgtca ttgacaagat 1860catcgatgcg
gcccgcaagt ccggcgccga cgccatccac cccggctacg gcttcctcgc 1920agaaaacgct
gacttcgcag aagcagtcat caacgaaggc ctgatctgga ttggaccttc 1980acctgagtcc
atccgctccc tcggcgacaa ggtcaccgct cgccacatcg cagataccgc 2040caaggctcca
atggctcctg gcaccaagga accagtaaaa gacgcagcag aagttgtggc 2100tttcgctgaa
gaattcggtc tcccaatcgc catcaaggca gctttcggtg gcggcggacg 2160tggcatgaag
gttgcctaca agatggaaga agtcgctgac ctcttcgagt ccgcaacccg 2220tgaagcaacc
gcagcgttcg gccgcggcga gtgcttcgtg gagcgctacc tggacaaggc 2280acgccacgtt
gaggctcagg tcatcgccga taagcacggc aacgttgttg tcgccggaac 2340ccgtgactgc
tccctgcagc gccgtttcca gaagctcgtc gaagaagcac cagcaccatt 2400cctcaccgat
gaccagcgcg agcgtctcca ctcctccgcg aaggctatct gtaaggaagc 2460tggctactac
ggtgcaggca ccgttgagta cctcgttggc tccgacggcc tgatctcctt 2520cctcgaggtc
aacacccgcc tccaggtgga acacccagtc accgaagaga ccaccggcat 2580cgacctggtc
cgcgaaatgt tccgcatcgc agaaggccac gagctctcca tcaaggaaga 2640tccagctcca
cgcggccacg cattcgagtt ccgcatcaac ggcgaagacg ctggctccaa 2700cttcatgcct
gcaccaggca agatcaccag ctaccgcgag ccacagggcc caggcgtccg 2760catggactcc
ggtgtcgttg aaggttccga aatctccgga cagttcgact ccatgctggc 2820aaagctgatc
gtttggggcg acacccgcga gcaggctctc cagcgctccc gccgtgcact 2880tgcagagtac
gttgtcgagg gcatgccaac cgttatccca ttccaccagc acatcgtgga 2940aaacccagca
ttcgtgggca acgacgaagg cttcgagatc tacaccaagt ggatcgaaga 3000ggtttgggat
aacccaatcg caccttacgt tgacgcttcc gagctcgacg aagatgagga 3060caagacccca
gcacagaagg ttgttgtgga gatcaacggc cgtcgcgttg aggttgcact 3120cccaggcgat
ctggcactcg gtggcaccgc tggtcctaag aagaaggcca agaagcgtcg 3180cgcaggtggt
gcaaaggctg gcgtatccgg cgatgcagtg gcagctccaa tgcagggcac 3240tgtcatcaag
gtcaacgtcg aagaaggcgc tgaagtcaac gaaggcgaca ccgttgttgt 3300cctcgaggct
atgaagatgg aaaaccctgt gaaggctcat aagtccggaa ccgtaaccgg 3360ccttactgtc
gctgcaggcg agggtgtcaa caagggcgtt gttctcctcg agatcaagta 3420atctagagga
ggaaaactaa atgaatgttg acattagccg ctctcgtgaa ccgttgaacg 3480tggaactgtt
gaaagaaaaa ctgctgcaga acggtgattt cggtcaagtg atctacgaga 3540aggtcaccgg
ctctaccaat gcggacctgc tggctctggc gggcagcggc gctccaaact 3600ggaccgtcaa
gactgttgaa tttcaggacc acgcccgtgg ccgtctgggt cgtccgtgga 3660gcgcaccgga
gggttcccaa accatcgtca gcgttctggt ccaactgagc attgatcagg 3720tggaccgtat
tggtacgatc ccgctggccg caggcttggc tgttatggat gcgctgaatg 3780atctgggcgt
ggagggtgca ggcctgaaat ggccgaacga tgttcagatc cacggtaaga 3840agttgtgcgg
tattctggtt gaagcaaccg gcttcgactc cactccgacc gtggttatcg 3900gttggggtac
gaatatctcg ttgacgaaag aagagctgcc ggtcccgcac gcgaccagcc 3960tggccctgga
gggtgttgaa gttgaccgta cgacgttcct gattaacatg ctgacccatc 4020tgcatacccg
tctggatcag tggcagggtc cgtctgtgga ctggctggat gactatcgcg 4080cggtttgtag
cagcattggc caagatgtgc gtgtcctgct gcctggtgac aaagagctgc 4140tgggcgaggc
gattggcgtg gcgaccggtg gtgagatccg tgtgcgcgac gccagcggca 4200cggtccacac
gctgaatgcg ggtgaaatca cgcatctgcg tttgcaataa
4250195659DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 19atgatcatca aacctaaaat
tcgtggattt atctgtacaa caacgcaccc agtgggttgt 60gaagcgaacg taaaagaaca
aattgcctac acaaaagcac aaggtccgat caaaaacgca 120cctaagcgcg tgttggttgt
cggatcgtct agcggctatg gtctgtcatc acgcatcgct 180gcggcgtttg gcggtggtgc
ggcgacgatc ggcgtatttt tcgaaaagcc gggcactgac 240aaaaaaccag gtactgcggg
tttctacaat gcagcagcgt ttgacaagct agcgcatgaa 300gcgggcttgt acgcaaaaag
cctgaacggc gatgcgttct cgaacgaagc gaagcaaaaa 360gcgattgagc tgattaagca
agacctcggc cagattgatt tggtggttta ctcgttggct 420tctccagtgc gtaagatgcc
agacacgggt gagctagtgc gctctgcact aaaaccgatc 480ggcgaaacgt acacctctac
cgcggtagat accaataaag atgtgatcat tgaagccagt 540gttgaacctg cgaccgagca
agaaatcgct gacactgtca ccgtgatggg cggtcaagat 600tgggaactgt ggatccaagc
actggaagag gcgggtgttc ttgctgaagg ttgcaaaacc 660gtggcgtaca gctacatcgg
tactgaattg acttggccaa tttactggga tggcgcttta 720ggccgtgcca agatggacct
agatcgcgca gcgacagcgc tgaacgaaaa gctggcagcg 780aaaggtggta ccgcgaacgt
tgcagttttg aaatcagtgg tgactcaagc aagctctgcg 840attcctgtga tgccgctcta
catcgcaatg gtgttcaaga agatgcgtga acagggcgtg 900catgaaggct gtatggagca
gatctaccgc atgttcagtc aacgtctgta caaagaagat 960ggttcagcgc cggaagtgga
tgatcacaat cgtctgcgtt tggatgactg ggaactgcgt 1020gatgacattc agcagcactg
ccgtgatctg tggccacaaa tcaccacaga gaacctgcgt 1080gagctgaccg attacgacat
gtacaaagaa gagttcatca agctgtttgg ctttggcatt 1140gaaggcattg attacgatgc
tgacgtcaat ccagaagtcg aatttgatgt gattgatatc 1200gagtaattta gtgactgagc
gtacatgtat acgaagatta ttggtactgg cagctatctg 1260cccgaacaag tgcggactaa
cgccgatctg gaaaaaatgg ttgagacctc tgacgagtgg 1320attgtcactc gtacaggtat
tcgtaaacgc catatcgccg cgccgaatga aactgtcgcg 1380acgatgggct ttaccgctgc
gaatcgcgcg attgagatgg cggggatcga taaagaccaa 1440attggcttga ttgtggtggc
taccacatca gcaacgcatg catttccaag cgcggcatgt 1500cagattcaaa gtatgctcgg
tattaaaggt tgcccggcgt ttgatgtcgc ggcagcgtgc 1560gcaggtttca cctacgcgtt
aagcatcgcc gaccagtacg ttaaatccgg cgcggttaaa 1620cacgcgctgg tggtcggttc
cgatgtatta gcccgcactt gcgatcctgg cgatcgcggt 1680acgatcatta ttttcggcga
tggcgcaggc gcggccgtac tgagcgcttc tgaagaaccg 1740ggtattatct ccactcatct
tcatgccgat ggccgttacg gtgaattact gaccctgccg 1800aatgccgatc gcgtaaatcc
ggataacccg atttacctga caatggcggg caatgaagtc 1860tttaaagtgg cggtcactga
actggcgcat attgtcgatg agacgctggc ggctaataac 1920ctggatcgct cagaactcga
ttggctggtg ccgcatcagg ctaacctgcg tatcattagc 1980gcgacagcga aaaaactcgg
catgtcgatg gacaatgtcg tcgtcacgct ggacaggcac 2040ggcaatacct ccgcggcttc
tgtgccgtgc gcgctggatg aagccgtgcg tgacgggcga 2100attaaagccg gtcagctggt
attgcttgaa gccttcgggg gtggattcac ctggggctcc 2160gcgctgattc gtttctagta
taaggattta aacatgacgc aatttgcatt tgtgttcccc 2220ggtcagggtt ctcagagcgt
tgggatgttg gccgagatgg cggcaaatta ccctatcgta 2280gaagaaacgt ttgctgaagc
ttctgcggct ctgggatatg atctgtgggc gctcacccag 2340caaggtccag cggaagaact
gaataaaacc tggcagacgc agccggcgtt attaaccgct 2400tccgtcgcgc tttggcgcgt
ttggcagcag cagggcggta aaatgcctgc gttaatggca 2460ggtcacagcc tgggcgaata
ttccgcgctg gtttgcgctg gcgtcatcaa ctttgctgat 2520gccgttcgtc tggtggaaat
gcgcggtaaa ttcatgcagg aagcggttcc ggaaggcact 2580ggcggcatgt ctgcgatcat
cgggctggat gatgcctcta ttgctaaagc ctgtgaagaa 2640tctgccgaag ggcaggttgt
ttcgccggtt aactttaact cgccgggaca ggtggttatc 2700gccgggcata aagaggcggt
agaacgtgcg ggcgcagcct gtaaagccgc tggcgcgaaa 2760cgcgcgctgc cgctgccggt
gagcgtaccg tcgcactgcg cgctgatgaa accagcggca 2820gataagctgg cggttgaatt
agccaaaatt acctttagcg cgccaacggt gccggtagtg 2880aacaacgttg acgtgaaatg
tgaaaccgat gccgccgcta tccgcgatgc gctggttcgc 2940cagttgtaca atccggtaca
gtggacgaag agcgtggaat ttatcgcggc gcagggcgtt 3000gaacatcttt atgaagtggg
tccaggtaaa gtcctcactg gtctgacgaa acgtattgtc 3060gacaccctga cagcgtcggc
gctgaacgag ccggcggcgc tgtctgcggc acttacgcaa 3120taaaagagga aaaccatgag
ctttgaagga aagattgcgc tggtgactgg tgcaagccgt 3180ggcataggcc gcgcaattgc
agagactctc gttgcccgcg gcgcgaaagt tatcgggact 3240gcgaccagtg aaaatggtgc
gaagaacatt agcgactatt taggtgctaa cgggaaaggt 3300ttgatgttga atgtgaccga
tcctgcatct attgaatctg ttctggaaaa tattcgcgca 3360gaatttggtg aagtggatat
cctggttaat aatgccggta tcactcgtga taatctgttg 3420atgcgaatga aagatgatga
gtggaacgat attatcgaaa ccaacttatc atccgttttc 3480cgcctgtcaa aagcggtaat
gcgcgctatg atgaaaaagc gttgtggtcg cattatcact 3540attggttctg tggttggtac
catgggaaat gcaggtcagg caaactacgc tgcggcgaaa 3600gcgggcctga tcggtttcag
taaatcactg gcgcgtgaag ttgcgtcccg tggtattact 3660gtcaatgttg tggctccggg
ttttattgaa acggacatga cgcgtgcgct gtctgacgat 3720cagcgtgcgg gtatcctggc
gcaggtgcct gcgggtcgcc tcggcggcgc tcaggaaatc 3780gccagtgcgg ttgcattttt
agcctctgac gaagcgagtt acatcactgg tgagactctg 3840cacgtcaacg gcggaatgta
catggtttaa ttttaaggtt tacataaaac atggtagata 3900aacgcgaatc ctatacaaaa
gaagaccttc ttgcctctgg tcgtggtgaa ctgtttggcg 3960ctaaagggcc gcaactccct
gcaccgaaca tgctgatgat ggaccgcgtc gttaagatga 4020ccgaaacggg cggcaatttc
gacaaaggct atgtcgaagc cgagctggat atcaatccgg 4080atctatggtt cttcggatgc
cactttatcg gcgatccggt gatgcccggt tgtctgggtc 4140tggatgctat gtggcaattg
gtgggattct acctgggctg gttgggcggc gaaggcaaag 4200gccgcgctct gggcgtgggc
gaagtgaaat ttaccggcca ggttctgccg acagccagga 4260aagtcaccta tcgtattcat
ttcaaacgta tcgtaaaccg tcgcctgatc atgggcctgg 4320cggacggtga ggttctggtg
gatggtcgcc tgatctatac cgcacacgat ttgaaagtcg 4380gtttgttcca ggatacttcc
gcgttctaaa aggaggcaac aaaatgaatc gccgcgttgt 4440cattacgggt attggtgcag
tgacgccggt gggtaacaac gctgatagct tctggtgcag 4500catcaaagag ggtaaatgtg
gcattgacaa gatcaaagcg tttgacgcaa ccgatttcaa 4560agttaagctg gctgccgaag
tgaaggactt caccccggag gactttatcg acaagcgtga 4620ggcgaaccgt atggaccgtt
ttagccagtt tgcgatcgtt gcggcggatg aggcaatcaa 4680ggacagcaaa ctggacctgg
agtcgattga taagaatcgt ttcggcgtca ttgttggtag 4740cggcattggc ggcatcggca
ccattgagaa gcaggatgaa aagctgatta ccaaaggtcc 4800gggtcgtgtg agccctatga
ctattccgat gatcattgcg aatatggcaa gcggtaatct 4860ggcgattcgt tatggcgcta
aaggtatttg cacgaccatt gtcaccgcat gtgcgagcgc 4920gaacaacagc attggtgagt
ccttccgtaa cattaagttt ggttatagcg acgttatgat 4980ctctggtggt agcgaagcag
gtatcacccc gttgagcctg gcgggttttg cctcgatgaa 5040ggccgtgacc aaatctgagg
acccgaagcg cgccagcatc ccgttcgata aggatcgcag 5100cggttttgtg atgggcgagg
gcagcggtat cgttatcttg gaagagttgg agcacgcgct 5160gaagcgtggt gccaaaatct
atgccgagat cgttggctat ggtgcgacct gcgacgcata 5220tcatatcacg agcccagcgc
cgaatggtga aggtggtgca cgtgcaatga aactggcaat 5280ggaagaagat aatgtccgcc
cagaggacat ttcctatatc aacgcgcacg gtacgagcac 5340ggcgtacaat gacagcttcg
aaacccaagc gatcaagacg gtcctgggtg aatacgccta 5400caaagtgccg gtgtctagca
ccaagagcat gaccggccac ctgctgggcg ctggcggtgc 5460agtcgaagcg attatctgtg
ccaaagctat tgaagagggt ttcattccgc cgaccatcgg 5520ctacaaagag gcggatccgg
aatgcgacct ggattacgtt cctaacgagg gccgtaatgc 5580agaagtcaac tacgttctgt
ccaacagcct gggcttcggt ggccataatg cgactctgct 5640gttcaaaaag tacaaatga
56592075DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 20ttgtccatct ttatataatt tgggggtagg gtgttcttta tgtaaaaaaa
acgttttagg 60atgcatatgg cggcc
752175DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 21gataaatcca
cgaattttag gtttgatgat cattggtctc ctcctgcagg tgcgtgttcg 60tcgtcatcgc
aattg
752230DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 22actcaccgca ttggtgtagt
aaggcgcacc 302330DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 23tgaatgtcat cacgcagttc ccagtcatcc
302433DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 24ccatcttctt
tgtacagacg ttgactgaac atg
332530DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 25gcaccatagc cgtaatccca
caggttatag 302625DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 26tgtcattaat ggttaataat gttga
252740DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 27gcagttattg
gtgcccttaa acgcctggtt gctacgcctg
402827DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 28gagccaatat gcgagaacac ccgagaa
272948DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 29cgctgaacgt attgcaggcc gagttgctgc accgctcccg ccaggcag
483051DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 30ggaattgcca
cggtgcggca ggctccatac gcgaggccag gttatccaac g
513135DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 31aatcaccagc actaaagtgc
gcggttcgtt acccg 353234DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 32atctgccgtg gattgcagag tctattcagc tacg
34331168PRTMycobacterium smegmatis 33Met Thr Ile Glu Thr
Arg Glu Asp Arg Phe Asn Arg Arg Ile Asp His1 5
10 15Leu Phe Glu Thr Asp Pro Gln Phe Ala Ala Ala
Arg Pro Asp Glu Ala 20 25
30Ile Ser Ala Ala Ala Ala Asp Pro Glu Leu Arg Leu Pro Ala Ala Val
35 40 45Lys Gln Ile Leu Ala Gly Tyr Ala
Asp Arg Pro Ala Leu Gly Lys Arg 50 55
60Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thr Thr Ala Lys Leu65
70 75 80Leu Pro Arg Phe Asp
Thr Ile Thr Tyr Arg Gln Leu Ala Gly Arg Ile 85
90 95Gln Ala Val Thr Asn Ala Trp His Asn His Pro
Val Asn Ala Gly Asp 100 105
110Arg Val Ala Ile Leu Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp
115 120 125Ile Ala Leu Leu Glu Leu Gly
Ala Val Ser Val Pro Leu Gln Thr Ser 130 135
140Ala Pro Val Ala Gln Leu Gln Pro Ile Val Ala Glu Thr Glu Pro
Lys145 150 155 160Val Ile
Ala Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu Val
165 170 175Glu Ser Gly Pro Ala Pro Ser
Arg Leu Val Val Phe Asp Tyr Ser His 180 185
190Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala Ala Lys Gly
Lys Leu 195 200 205Ala Gly Thr Gly
Val Val Val Glu Thr Ile Thr Asp Ala Leu Asp Arg 210
215 220Gly Arg Ser Leu Ala Asp Ala Pro Leu Tyr Val Pro
Asp Glu Ala Asp225 230 235
240Pro Leu Thr Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
245 250 255Gly Ala Met Tyr Pro
Glu Ser Lys Thr Ala Thr Met Trp Gln Ala Gly 260
265 270Ser Lys Ala Arg Trp Asp Glu Thr Leu Gly Val Met
Pro Ser Ile Thr 275 280 285Leu Asn
Phe Met Pro Met Ser His Val Met Gly Arg Gly Ile Leu Cys 290
295 300Ser Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe
Ala Ala Arg Ser Asp305 310 315
320Leu Ser Thr Phe Leu Glu Asp Leu Ala Leu Val Arg Pro Thr Gln Leu
325 330 335Asn Phe Val Pro
Arg Ile Trp Asp Met Leu Phe Gln Glu Tyr Gln Ser 340
345 350Arg Leu Asp Asn Arg Arg Ala Glu Gly Ser Glu
Asp Arg Ala Glu Ala 355 360 365Ala
Val Leu Glu Glu Val Arg Thr Gln Leu Leu Gly Gly Arg Phe Val 370
375 380Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser
Ala Glu Met Lys Ser Trp385 390 395
400Val Glu Asp Leu Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser
Thr 405 410 415Glu Ala Gly
Ala Val Phe Ile Asp Gly Gln Ile Gln Arg Pro Pro Val 420
425 430Ile Asp Tyr Lys Leu Val Asp Val Pro Asp
Leu Gly Tyr Phe Ala Thr 435 440
445Asp Arg Pro Tyr Pro Arg Gly Glu Leu Leu Val Lys Ser Glu Gln Met 450
455 460Phe Pro Gly Tyr Tyr Lys Arg Pro
Glu Ile Thr Ala Glu Met Phe Asp465 470
475 480Glu Asp Gly Tyr Tyr Arg Thr Gly Asp Ile Val Ala
Glu Leu Gly Pro 485 490
495Asp His Leu Glu Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
500 505 510Gln Gly Glu Phe Val Thr
Val Ser Lys Leu Glu Ala Val Phe Gly Asp 515 520
525Ser Pro Leu Val Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala
Arg Ser 530 535 540Tyr Leu Leu Ala Val
Val Val Pro Thr Glu Glu Ala Leu Ser Arg Trp545 550
555 560Asp Gly Asp Glu Leu Lys Ser Arg Ile Ser
Asp Ser Leu Gln Asp Ala 565 570
575Ala Arg Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp Phe Leu
580 585 590Val Glu Thr Thr Pro
Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile 595
600 605Arg Lys Leu Ala Arg Pro Lys Leu Lys Ala His Tyr
Gly Glu Arg Leu 610 615 620Glu Gln Leu
Tyr Thr Asp Leu Ala Glu Gly Gln Ala Asn Glu Leu Arg625
630 635 640Glu Leu Arg Arg Asn Gly Ala
Asp Arg Pro Val Val Glu Thr Val Ser 645
650 655Arg Ala Ala Val Ala Leu Leu Gly Ala Ser Val Thr
Asp Leu Arg Ser 660 665 670Asp
Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser 675
680 685Phe Ser Asn Leu Leu His Glu Ile Phe
Asp Val Asp Val Pro Val Gly 690 695
700Val Ile Val Ser Pro Ala Thr Asp Leu Ala Gly Val Ala Ala Tyr Ile705
710 715 720Glu Gly Glu Leu
Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His 725
730 735Gly Arg Asp Ala Thr Glu Val Arg Ala Arg
Asp Leu Ala Leu Gly Lys 740 745
750Phe Ile Asp Ala Lys Thr Leu Ser Ala Ala Pro Gly Leu Pro Arg Ser
755 760 765Gly Thr Glu Ile Arg Thr Val
Leu Leu Thr Gly Ala Thr Gly Phe Leu 770 775
780Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val
Asp785 790 795 800Gly Lys
Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg
805 810 815Ala Arg Leu Asp Ala Thr Phe
Asp Thr Gly Asp Ala Thr Leu Leu Glu 820 825
830His Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile Ala
Gly Asp 835 840 845Lys Gly Glu Ala
Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu 850
855 860Ala Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala
Leu Val Asn His865 870 875
880Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr Ala
885 890 895Glu Leu Ile Arg Ile
Ala Leu Thr Thr Thr Ile Lys Pro Tyr Val Tyr 900
905 910Val Ser Thr Ile Gly Val Gly Gln Gly Ile Ser Pro
Glu Ala Phe Val 915 920 925Glu Asp
Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp 930
935 940Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp
Ala Gly Glu Val Leu945 950 955
960Leu Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg
965 970 975Cys Asp Met Ile
Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu 980
985 990Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu
Val Ala Thr Gly Ile 995 1000
1005Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn Arg Gln
1010 1015 1020Arg Ala His Tyr Asp Gly
Leu Pro Val Glu Phe Ile Ala Glu Ala 1025 1030
1035Ile Ser Thr Ile Gly Ser Gln Val Thr Asp Gly Phe Glu Thr
Phe 1040 1045 1050His Val Met Asn Pro
Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr 1055 1060
1065Val Asp Trp Leu Ile Glu Ala Gly Tyr Pro Val His Arg
Val Asp 1070 1075 1080Asp Tyr Ala Thr
Trp Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala 1085
1090 1095Leu Pro Glu Arg Gln Arg Gln Ala Ser Leu Leu
Pro Leu Leu His 1100 1105 1110Asn Tyr
Gln Gln Pro Ser Pro Pro Val Cys Gly Ala Met Ala Pro 1115
1120 1125Thr Asp Arg Phe Arg Ala Ala Val Gln Asp
Ala Lys Ile Gly Pro 1130 1135 1140Asp
Lys Asp Ile Pro His Val Thr Ala Asp Val Ile Val Lys Tyr 1145
1150 1155Ile Ser Asn Leu Gln Met Leu Gly Leu
Leu 1160 1165341168PRTMycobacterium tuberculosis 34Met
Ser Ile Asn Asp Gln Arg Leu Thr Arg Arg Val Glu Asp Leu Tyr1
5 10 15Ala Ser Asp Ala Gln Phe Ala
Ala Ala Ser Pro Asn Glu Ala Ile Thr 20 25
30Gln Ala Ile Asp Gln Pro Gly Val Ala Leu Pro Gln Leu Ile
Arg Met 35 40 45Val Met Glu Gly
Tyr Ala Asp Arg Pro Ala Leu Gly Gln Arg Ala Leu 50 55
60Arg Phe Val Thr Asp Pro Asp Ser Gly Arg Thr Met Val
Glu Leu Leu65 70 75
80Pro Arg Phe Glu Thr Ile Thr Tyr Arg Glu Leu Trp Ala Arg Ala Gly
85 90 95Thr Leu Ala Thr Ala Leu
Ser Ala Glu Pro Ala Ile Arg Pro Gly Asp 100
105 110Arg Val Cys Val Leu Gly Phe Asn Ser Val Asp Tyr
Thr Thr Ile Asp 115 120 125Ile Ala
Leu Ile Arg Leu Gly Ala Val Ser Val Pro Leu Gln Thr Ser 130
135 140Ala Pro Val Thr Gly Leu Arg Pro Ile Val Thr
Glu Thr Glu Pro Thr145 150 155
160Met Ile Ala Thr Ser Ile Asp Asn Leu Gly Asp Ala Val Glu Val Leu
165 170 175Ala Gly His Ala
Pro Ala Arg Leu Val Val Phe Asp Tyr His Gly Lys 180
185 190Val Asp Thr His Arg Glu Ala Val Glu Ala Ala
Arg Ala Arg Leu Ala 195 200 205Gly
Ser Val Thr Ile Asp Thr Leu Ala Glu Leu Ile Glu Arg Gly Arg 210
215 220Ala Leu Pro Ala Thr Pro Ile Ala Asp Ser
Ala Asp Asp Ala Leu Ala225 230 235
240Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Ala
Met 245 250 255Tyr Arg Glu
Ser Gln Val Met Ser Phe Trp Arg Lys Ser Ser Gly Trp 260
265 270Phe Glu Pro Ser Gly Tyr Pro Ser Ile Thr
Leu Asn Phe Met Pro Met 275 280
285Ser His Val Gly Gly Arg Gln Val Leu Tyr Gly Thr Leu Ser Asn Gly 290
295 300Gly Thr Ala Tyr Phe Val Ala Lys
Ser Asp Leu Ser Thr Leu Phe Glu305 310
315 320Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Cys Phe
Val Pro Arg Ile 325 330
335Trp Asp Met Val Phe Ala Glu Phe His Ser Glu Val Asp Arg Arg Leu
340 345 350Val Asp Gly Ala Asp Arg
Ala Ala Leu Glu Ala Gln Val Lys Ala Glu 355 360
365Leu Arg Glu Asn Val Leu Gly Gly Arg Phe Val Met Ala Leu
Thr Gly 370 375 380Ser Ala Pro Ile Ser
Ala Glu Met Thr Ala Trp Val Glu Ser Leu Leu385 390
395 400Ala Asp Val His Leu Val Glu Gly Tyr Gly
Ser Thr Glu Ala Gly Met 405 410
415Val Leu Asn Asp Gly Met Val Arg Arg Pro Ala Val Ile Asp Tyr Lys
420 425 430Leu Val Asp Val Pro
Glu Leu Gly Tyr Phe Gly Thr Asp Gln Pro Tyr 435
440 445Pro Arg Gly Glu Leu Leu Val Lys Thr Gln Thr Met
Phe Pro Gly Tyr 450 455 460Tyr Gln Arg
Pro Asp Val Thr Ala Glu Val Phe Asp Pro Asp Gly Phe465
470 475 480Tyr Arg Thr Gly Asp Ile Met
Ala Lys Val Gly Pro Asp Gln Phe Val 485
490 495Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
Gln Gly Glu Phe 500 505 510Ile
Ala Val Ser Lys Leu Glu Ala Val Phe Gly Asp Ser Pro Leu Val 515
520 525Arg Gln Ile Phe Ile Tyr Gly Asn Ser
Ala Arg Ala Tyr Pro Leu Ala 530 535
540Val Val Val Pro Ser Gly Asp Ala Leu Ser Arg His Gly Ile Glu Asn545
550 555 560Leu Lys Pro Val
Ile Ser Glu Ser Leu Gln Glu Val Ala Arg Ala Ala 565
570 575Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
Phe Ile Ile Glu Thr Thr 580 585
590Pro Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile Arg Lys Leu Ala
595 600 605Arg Pro Gln Leu Lys Lys Phe
Tyr Gly Glu Arg Leu Glu Arg Leu Tyr 610 615
620Thr Glu Leu Ala Asp Ser Gln Ser Asn Glu Leu Arg Glu Leu Arg
Gln625 630 635 640Ser Gly
Pro Asp Ala Pro Val Leu Pro Thr Leu Cys Arg Ala Ala Ala
645 650 655Ala Leu Leu Gly Ser Thr Ala
Ala Asp Val Arg Pro Asp Ala His Phe 660 665
670Ala Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser Leu Ala
Asn Leu 675 680 685Leu His Glu Ile
Phe Gly Val Asp Val Pro Val Gly Val Ile Val Ser 690
695 700Pro Ala Ser Asp Leu Arg Ala Leu Ala Asp His Ile
Glu Ala Ala Arg705 710 715
720Thr Gly Val Arg Arg Pro Ser Phe Ala Ser Ile His Gly Arg Ser Ala
725 730 735Thr Glu Val His Ala
Ser Asp Leu Thr Leu Asp Lys Phe Ile Asp Ala 740
745 750Ala Thr Leu Ala Ala Ala Pro Asn Leu Pro Ala Pro
Ser Ala Gln Val 755 760 765Arg Thr
Val Leu Leu Thr Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu 770
775 780Ala Leu Glu Trp Leu Asp Arg Met Asp Leu Val
Asn Gly Lys Leu Ile785 790 795
800Cys Leu Val Arg Ala Arg Ser Asp Glu Glu Ala Gln Ala Arg Leu Asp
805 810 815Ala Thr Phe Asp
Ser Gly Asp Pro Tyr Leu Val Arg His Tyr Arg Glu 820
825 830Leu Gly Ala Gly Arg Leu Glu Val Leu Ala Gly
Asp Lys Gly Glu Ala 835 840 845Asp
Leu Gly Leu Asp Arg Val Thr Trp Gln Arg Leu Ala Asp Thr Val 850
855 860Asp Leu Ile Val Asp Pro Ala Ala Leu Val
Asn His Val Leu Pro Tyr865 870 875
880Ser Gln Leu Phe Gly Pro Asn Ala Ala Gly Thr Ala Glu Leu Leu
Arg 885 890 895Leu Ala Leu
Thr Gly Lys Arg Lys Pro Tyr Ile Tyr Thr Ser Thr Ile 900
905 910Ala Val Gly Glu Gln Ile Pro Pro Glu Ala
Phe Thr Glu Asp Ala Asp 915 920
925Ile Arg Ala Ile Ser Pro Thr Arg Arg Ile Asp Asp Ser Tyr Ala Asn 930
935 940Gly Tyr Ala Asn Ser Lys Trp Ala
Gly Glu Val Leu Leu Arg Glu Ala945 950
955 960His Glu Gln Cys Gly Leu Pro Val Thr Val Phe Arg
Cys Asp Met Ile 965 970
975Leu Ala Asp Thr Ser Tyr Thr Gly Gln Leu Asn Leu Pro Asp Met Phe
980 985 990Thr Arg Leu Met Leu Ser
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser 995 1000
1005Phe Tyr Glu Leu Asp Ala His Gly Asn Arg Gln Arg
Ala His Tyr 1010 1015 1020Asp Gly Leu
Pro Val Glu Phe Val Ala Glu Ala Ile Cys Thr Leu 1025
1030 1035Gly Thr His Ser Pro Asp Arg Phe Val Thr Tyr
His Val Met Asn 1040 1045 1050Pro Tyr
Asp Asp Gly Ile Gly Leu Asp Glu Phe Val Asp Trp Leu 1055
1060 1065Asn Ser Pro Thr Ser Gly Ser Gly Cys Thr
Ile Gln Arg Ile Ala 1070 1075 1080Asp
Tyr Gly Glu Trp Leu Gln Arg Phe Glu Thr Ser Leu Arg Ala 1085
1090 1095Leu Pro Asp Arg Gln Arg His Ala Ser
Leu Leu Pro Leu Leu His 1100 1105
1110Asn Tyr Arg Glu Pro Ala Lys Pro Ile Cys Gly Ser Ile Ala Pro
1115 1120 1125Thr Asp Gln Phe Arg Ala
Ala Val Gln Glu Ala Lys Ile Gly Pro 1130 1135
1140Asp Lys Asp Ile Pro His Leu Thr Ala Ala Ile Ile Ala Lys
Tyr 1145 1150 1155Ile Ser Asn Leu Arg
Leu Leu Gly Leu Leu 1160 116535696DNASynechococcus
elongatus 35atgccgcagc ttgaagccag ccttgaactg gactttcaaa gcgagtccta
caaagacgct 60tacagccgca tcaacgcgat cgtgattgaa ggcgaacaag aggcgttcga
caactacaat 120cgccttgctg agatgctgcc cgaccagcgg gatgagcttc acaagctagc
caagatggaa 180cagcgccaca tgaaaggctt tatggcctgt ggcaaaaatc tctccgtcac
tcctgacatg 240ggttttgccc agaaattttt cgagcgcttg cacgagaact tcaaagcggc
ggctgcggaa 300ggcaaggtcg tcacctgcct actgattcaa tcgctaatca tcgagtgctt
tgcgatcgcg 360gcttacaaca tctacatccc agtggcggat gcttttgccc gcaaaatcac
ggagggggtc 420gtgcgcgacg aatacctgca ccgcaacttc ggtgaagagt ggctgaaggc
gaattttgat 480gcttccaaag ccgaactgga agaagccaat cgtcagaacc tgcccttggt
ttggctaatg 540ctcaacgaag tggccgatga tgctcgcgaa ctcgggatgg agcgtgagtc
gctcgtcgag 600gactttatga ttgcctacgg tgaagctctg gaaaacatcg gcttcacaac
gcgcgaaatc 660atgcgtatgt ccgcctatgg ccttgcggcc gtttga
69636231PRTSynechococcus elongatus 36Met Pro Gln Leu Glu Ala
Ser Leu Glu Leu Asp Phe Gln Ser Glu Ser1 5
10 15Tyr Lys Asp Ala Tyr Ser Arg Ile Asn Ala Ile Val
Ile Glu Gly Glu 20 25 30Gln
Glu Ala Phe Asp Asn Tyr Asn Arg Leu Ala Glu Met Leu Pro Asp 35
40 45Gln Arg Asp Glu Leu His Lys Leu Ala
Lys Met Glu Gln Arg His Met 50 55
60Lys Gly Phe Met Ala Cys Gly Lys Asn Leu Ser Val Thr Pro Asp Met65
70 75 80Gly Phe Ala Gln Lys
Phe Phe Glu Arg Leu His Glu Asn Phe Lys Ala 85
90 95Ala Ala Ala Glu Gly Lys Val Val Thr Cys Leu
Leu Ile Gln Ser Leu 100 105
110Ile Ile Glu Cys Phe Ala Ile Ala Ala Tyr Asn Ile Tyr Ile Pro Val
115 120 125Ala Asp Ala Phe Ala Arg Lys
Ile Thr Glu Gly Val Val Arg Asp Glu 130 135
140Tyr Leu His Arg Asn Phe Gly Glu Glu Trp Leu Lys Ala Asn Phe
Asp145 150 155 160Ala Ser
Lys Ala Glu Leu Glu Glu Ala Asn Arg Gln Asn Leu Pro Leu
165 170 175Val Trp Leu Met Leu Asn Glu
Val Ala Asp Asp Ala Arg Glu Leu Gly 180 185
190Met Glu Arg Glu Ser Leu Val Glu Asp Phe Met Ile Ala Tyr
Gly Glu 195 200 205Ala Leu Glu Asn
Ile Gly Phe Thr Thr Arg Glu Ile Met Arg Met Ser 210
215 220Ala Tyr Gly Leu Ala Ala Val225
230371029DNASynechococcus elongatus 37atggcattcg gtcttatcgg tcatctcacc
agtttggagc aggcccgcga cgtttctcgc 60aggatgggct acgacgaata cgccgatcaa
ggattggagt tttggagtag cgctcctcct 120caaatcgttg atgaaatcac agtcaccagt
gccacaggca aggtgattca cggtcgctac 180atcgaatcgt gtttcttgcc ggaaatgctg
gcggcgcgcc gcttcaaaac agccacgcgc 240aaagttctca atgccatgtc ccatgcccaa
aaacacggca tcgacatctc ggccttgggg 300ggctttacct cgattatttt cgagaatttc
gatttggcca gtttgcggca agtgcgcgac 360actaccttgg agtttgaacg gttcaccacc
ggcaatactc acacggccta cgtaatctgt 420agacaggtgg aagccgctgc taaaacgctg
ggcatcgaca ttacccaagc gacagtagcg 480gttgtcggcg cgactggcga tatcggtagc
gctgtctgcc gctggctcga cctcaaactg 540ggtgtcggtg atttgatcct gacggcgcgc
aatcaggagc gtttggataa cctgcaggct 600gaactcggcc ggggcaagat tctgcccttg
gaagccgctc tgccggaagc tgactttatc 660gtgtgggtcg ccagtatgcc tcagggcgta
gtgatcgacc cagcaaccct gaagcaaccc 720tgcgtcctaa tcgacggggg ctaccccaaa
aacttgggca gcaaagtcca aggtgagggc 780atctatgtcc tcaatggcgg ggtagttgaa
cattgcttcg acatcgactg gcagatcatg 840tccgctgcag agatggcgcg gcccgagcgc
cagatgtttg cctgctttgc cgaggcgatg 900ctcttggaat ttgaaggctg gcatactaac
ttctcctggg gccgcaacca aatcacgatc 960gagaagatgg aagcgatcgg tgaggcatcg
gtgcgccacg gcttccaacc cttggcattg 1020gcaatttga
102938342PRTSynechococcus elongatus
38Met Ala Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu Gln Ala Arg1
5 10 15Asp Val Ser Arg Arg Met
Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu 20 25
30Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu
Ile Thr Val 35 40 45Thr Ser Ala
Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys 50
55 60Phe Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys
Thr Ala Thr Arg65 70 75
80Lys Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile
85 90 95Ser Ala Leu Gly Gly Phe
Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu 100
105 110Ala Ser Leu Arg Gln Val Arg Asp Thr Thr Leu Glu
Phe Glu Arg Phe 115 120 125Thr Thr
Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130
135 140Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile Thr
Gln Ala Thr Val Ala145 150 155
160Val Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu
165 170 175Asp Leu Lys Leu
Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180
185 190Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly
Arg Gly Lys Ile Leu 195 200 205Pro
Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210
215 220Ser Met Pro Gln Gly Val Val Ile Asp Pro
Ala Thr Leu Lys Gln Pro225 230 235
240Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly Ser Lys
Val 245 250 255Gln Gly Glu
Gly Ile Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260
265 270Phe Asp Ile Asp Trp Gln Ile Met Ser Ala
Ala Glu Met Ala Arg Pro 275 280
285Glu Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290
295 300Glu Gly Trp His Thr Asn Phe Ser
Trp Gly Arg Asn Gln Ile Thr Ile305 310
315 320Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg
His Gly Phe Gln 325 330
335Pro Leu Ala Leu Ala Ile 34039717DNAProchlorococcus mariunus
39atgcaaacac tcgaatctaa taaaaaaact aatctagaaa attctattga tttacccgat
60tttactactg attcttacaa agacgcttat agcaggataa atgcaatagt tattgaaggt
120gaacaagagg ctcatgataa ttacatttcc ttagcaacat taattcctaa cgaattagaa
180gagttaacta aattagcgaa aatggagctt aagcacaaaa gaggctttac tgcatgtgga
240agaaatctag gtgttcaagc tgacatgatt tttgctaaag aattcttttc caaattacat
300ggtaattttc aggttgcgtt atctaatggc aagacaacta catgcctatt aatacaggca
360attttaattg aagcttttgc tatatccgcg tatcacgttt acataagagt tgctgatcct
420ttcgcgaaaa aaattaccca aggtgttgtt aaagatgaat atcttcattt aaattatgga
480caagaatggc taaaagaaaa tttagcgact tgtaaagatg agctaatgga agcaaataag
540gttaaccttc cattaatcaa gaagatgtta gatcaagtct cggaagatgc ttcagtacta
600gctatggata gggaagaatt aatggaagaa ttcatgattg cctatcagga cactctcctt
660gaaataggtt tagataatag agaaattgca agaatggcaa tggctgctat agtttaa
71740238PRTProchlorococcus mariunus 40Met Gln Thr Leu Glu Ser Asn Lys Lys
Thr Asn Leu Glu Asn Ser Ile1 5 10
15Asp Leu Pro Asp Phe Thr Thr Asp Ser Tyr Lys Asp Ala Tyr Ser
Arg 20 25 30Ile Asn Ala Ile
Val Ile Glu Gly Glu Gln Glu Ala His Asp Asn Tyr 35
40 45Ile Ser Leu Ala Thr Leu Ile Pro Asn Glu Leu Glu
Glu Leu Thr Lys 50 55 60Leu Ala Lys
Met Glu Leu Lys His Lys Arg Gly Phe Thr Ala Cys Gly65 70
75 80Arg Asn Leu Gly Val Gln Ala Asp
Met Ile Phe Ala Lys Glu Phe Phe 85 90
95Ser Lys Leu His Gly Asn Phe Gln Val Ala Leu Ser Asn Gly
Lys Thr 100 105 110Thr Thr Cys
Leu Leu Ile Gln Ala Ile Leu Ile Glu Ala Phe Ala Ile 115
120 125Ser Ala Tyr His Val Tyr Ile Arg Val Ala Asp
Pro Phe Ala Lys Lys 130 135 140Ile Thr
Gln Gly Val Val Lys Asp Glu Tyr Leu His Leu Asn Tyr Gly145
150 155 160Gln Glu Trp Leu Lys Glu Asn
Leu Ala Thr Cys Lys Asp Glu Leu Met 165
170 175Glu Ala Asn Lys Val Asn Leu Pro Leu Ile Lys Lys
Met Leu Asp Gln 180 185 190Val
Ser Glu Asp Ala Ser Val Leu Ala Met Asp Arg Glu Glu Leu Met 195
200 205Glu Glu Phe Met Ile Ala Tyr Gln Asp
Thr Leu Leu Glu Ile Gly Leu 210 215
220Asp Asn Arg Glu Ile Ala Arg Met Ala Met Ala Ala Ile Val225
230 235411044DNAProchlorococcus mariunus 41atggcatttg
ggcttatagg tcattcaact agttttgaag atgcaaaaag aaaggcttca 60ttattgggct
ttgatcatat tgcggatggt gatttagatg tttggtgcac agctccacct 120caactagttg
aaaatgtaga ggttaaaagt gctataggta tatcaattga aggttcttat 180attgattcat
gtttcgttcc tgaaatgctt tcaagattta aaacggcaag aagaaaagta 240ttaaatgcaa
tggaattagc tcaaaaaaaa ggtattaata ttaccgcttt gggggggttc 300acttctatca
tctttgaaaa ttttaatctc cttcaacata agcagattag aaacacttca 360ctagagtggg
aaaggtttac aactggtaat actcatactg cgtgggttat ttgcaggcaa 420ttagagatga
atgctcctaa aataggtatt gatcttaaaa gcgcaacagt tgctgtagtt 480ggtgctactg
gagatatagg cagtgctgtt tgtcgatggt taatcaataa aacaggtatt 540ggggaacttc
ttttggtagc taggcaaaag gaacccttgg attctttgca aaaggaatta 600gatggtggaa
ctatcaaaaa tctagatgaa gcattgcctg aagcagatat tgttgtatgg 660gtagcaagta
tgccaaagac aatggaaatc gatgctaata atcttaaaca accatgttta 720atgattgatg
gaggttatcc aaagaatcta gatgaaaaat ttcaaggaaa taatatacat 780gttgtaaaag
gaggtatagt aagattcttc aatgatatag gttggaatat gatggaacta 840gctgaaatgc
aaaatcccca gagagaaatg tttgcatgct ttgcagaagc aatgatttta 900gaatttgaaa
aatgtcatac aaactttagc tggggaagaa ataatatatc tctcgagaaa 960atggagttta
ttggagctgc ttctgtaaag catggcttct ctgcaattgg cctagataag 1020catccaaaag
tactagcagt ttga
104442347PRTProchlorococcus mariunus 42Met Ala Phe Gly Leu Ile Gly His
Ser Thr Ser Phe Glu Asp Ala Lys1 5 10
15Arg Lys Ala Ser Leu Leu Gly Phe Asp His Ile Ala Asp Gly
Asp Leu 20 25 30Asp Val Trp
Cys Thr Ala Pro Pro Gln Leu Val Glu Asn Val Glu Val 35
40 45Lys Ser Ala Ile Gly Ile Ser Ile Glu Gly Ser
Tyr Ile Asp Ser Cys 50 55 60Phe Val
Pro Glu Met Leu Ser Arg Phe Lys Thr Ala Arg Arg Lys Val65
70 75 80Leu Asn Ala Met Glu Leu Ala
Gln Lys Lys Gly Ile Asn Ile Thr Ala 85 90
95Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asn
Leu Leu Gln 100 105 110His Lys
Gln Ile Arg Asn Thr Ser Leu Glu Trp Glu Arg Phe Thr Thr 115
120 125Gly Asn Thr His Thr Ala Trp Val Ile Cys
Arg Gln Leu Glu Met Asn 130 135 140Ala
Pro Lys Ile Gly Ile Asp Leu Lys Ser Ala Thr Val Ala Val Val145
150 155 160Gly Ala Thr Gly Asp Ile
Gly Ser Ala Val Cys Arg Trp Leu Ile Asn 165
170 175Lys Thr Gly Ile Gly Glu Leu Leu Leu Val Ala Arg
Gln Lys Glu Pro 180 185 190Leu
Asp Ser Leu Gln Lys Glu Leu Asp Gly Gly Thr Ile Lys Asn Leu 195
200 205Asp Glu Ala Leu Pro Glu Ala Asp Ile
Val Val Trp Val Ala Ser Met 210 215
220Pro Lys Thr Met Glu Ile Asp Ala Asn Asn Leu Lys Gln Pro Cys Leu225
230 235 240Met Ile Asp Gly
Gly Tyr Pro Lys Asn Leu Asp Glu Lys Phe Gln Gly 245
250 255Asn Asn Ile His Val Val Lys Gly Gly Ile
Val Arg Phe Phe Asn Asp 260 265
270Ile Gly Trp Asn Met Met Glu Leu Ala Glu Met Gln Asn Pro Gln Arg
275 280 285Glu Met Phe Ala Cys Phe Ala
Glu Ala Met Ile Leu Glu Phe Glu Lys 290 295
300Cys His Thr Asn Phe Ser Trp Gly Arg Asn Asn Ile Ser Leu Glu
Lys305 310 315 320Met Glu
Phe Ile Gly Ala Ala Ser Val Lys His Gly Phe Ser Ala Ile
325 330 335Gly Leu Asp Lys His Pro Lys
Val Leu Ala Val 340 34543255DNANostoc
punctiforme 43atgagccaaa cggaactttt tgaaaaggtc aagaaaatcg tcatcgaaca
actgagtgtt 60gaagatgctt ccaaaatcac tccacaagct aagtttatgg aagatttagg
agctgattcc 120ctggatactg ttgaactcgt gatggctttg gaagaagaat ttgatatcga
aattcccgac 180gaagctgccg agcagattgt atcggttcaa gacgcagtag attacatcaa
taacaaagtt 240gctgcatcag cttaa
2554484PRTNostoc punctiforme 44Met Ser Gln Thr Glu Leu Phe
Glu Lys Val Lys Lys Ile Val Ile Glu1 5 10
15Gln Leu Ser Val Glu Asp Ala Ser Lys Ile Thr Pro Gln
Ala Lys Phe 20 25 30Met Glu
Asp Leu Gly Ala Asp Ser Leu Asp Thr Val Glu Leu Val Met 35
40 45Ala Leu Glu Glu Glu Phe Asp Ile Glu Ile
Pro Asp Glu Ala Ala Glu 50 55 60Gln
Ile Val Ser Val Gln Asp Ala Val Asp Tyr Ile Asn Asn Lys Val65
70 75 80Ala Ala Ser
Ala45234DNASynechocystis sp. 45atgaatcagg aaatttttga aaaagtaaaa
aaaatcgtcg tggaacagtt ggaagtggat 60cctgacaaag tgacccccga tgccaccttt
gccgaagatt taggggctga ttccctcgat 120acagtggaat tggtcatggc cctggaagaa
gagtttgata ttgaaattcc cgatgaagtg 180gcggaaacca ttgataccgt gggcaaagcc
gttgagcata tcgaaagtaa ataa 2344677PRTSynechocystis sp. 46Met Asn
Gln Glu Ile Phe Glu Lys Val Lys Lys Ile Val Val Glu Gln1 5
10 15Leu Glu Val Asp Pro Asp Lys Val
Thr Pro Asp Ala Thr Phe Ala Glu 20 25
30Asp Leu Gly Ala Asp Ser Leu Asp Thr Val Glu Leu Val Met Ala
Leu 35 40 45Glu Glu Glu Phe Asp
Ile Glu Ile Pro Asp Glu Val Ala Glu Thr Ile 50 55
60Asp Thr Val Gly Lys Ala Val Glu His Ile Glu Ser Lys65
70 7547243DNAProchlorococcus marinus
47atgtcacaag aagaaatcct tcaaaaagta tgctctattg tttctgagca actaagtgtt
60gaatcagccg aagtaaaatc tgattcaaac tttcaaaatg atttaggtgc agactcccta
120gacaccgtag agctagttat ggctcttgaa gaagcatttg atatcgagat acctgatgaa
180gcagctgaag gtatcgcaac agtaggagat gctgttaaat tcatcgaaga aaaaaaaggt
240taa
2434880PRTProchlorococcus marinus 48Met Ser Gln Glu Glu Ile Leu Gln Lys
Val Cys Ser Ile Val Ser Glu1 5 10
15Gln Leu Ser Val Glu Ser Ala Glu Val Lys Ser Asp Ser Asn Phe
Gln 20 25 30Asn Asp Leu Gly
Ala Asp Ser Leu Asp Thr Val Glu Leu Val Met Ala 35
40 45Leu Glu Glu Ala Phe Asp Ile Glu Ile Pro Asp Glu
Ala Ala Glu Gly 50 55 60Ile Ala Thr
Val Gly Asp Ala Val Lys Phe Ile Glu Glu Lys Lys Gly65 70
75 8049243DNASynechococcus elongatus
49atgagccaag aagacatctt cagcaaagtc aaagacattg tggctgagca gctgagtgtg
60gatgtggctg aagtcaagcc agaatccagc ttccaaaacg atctgggagc ggactcgctg
120gacaccgtgg aactggtgat ggctctggaa gaggctttcg atatcgaaat ccccgatgaa
180gccgctgaag gcattgcgac cgttcaagac gccgtcgatt tcatcgctag caaagctgcc
240tag
2435080PRTSynechococcus elongatus 50Met Ser Gln Glu Asp Ile Phe Ser Lys
Val Lys Asp Ile Val Ala Glu1 5 10
15Gln Leu Ser Val Asp Val Ala Glu Val Lys Pro Glu Ser Ser Phe
Gln 20 25 30Asn Asp Leu Gly
Ala Asp Ser Leu Asp Thr Val Glu Leu Val Met Ala 35
40 45Leu Glu Glu Ala Phe Asp Ile Glu Ile Pro Asp Glu
Ala Ala Glu Gly 50 55 60Ile Ala Thr
Val Gln Asp Ala Val Asp Phe Ile Ala Ser Lys Ala Ala65 70
75 8051255DNANostoc sp. 51atgagccaat
cagaaacttt tgaaaaagtc aaaaaaattg ttatcgaaca actaagtgtg 60gagaaccctg
acacagtaac tccagaagct agttttgcca acgatttaca ggctgattcc 120ctcgatacag
tagaactagt aatggctttg gaagaagaat ttgatatcga aattcccgat 180gaagccgcag
agaaaattac cactgttcaa gaagcggtgg attacatcaa taaccaagtt 240gccgcatcag
cttaa
2555284PRTNostoc sp. 52Met Ser Gln Ser Glu Thr Phe Glu Lys Val Lys Lys
Ile Val Ile Glu1 5 10
15Gln Leu Ser Val Glu Asn Pro Asp Thr Val Thr Pro Glu Ala Ser Phe
20 25 30Ala Asn Asp Leu Gln Ala Asp
Ser Leu Asp Thr Val Glu Leu Val Met 35 40
45Ala Leu Glu Glu Glu Phe Asp Ile Glu Ile Pro Asp Glu Ala Ala
Glu 50 55 60Lys Ile Thr Thr Val Gln
Glu Ala Val Asp Tyr Ile Asn Asn Gln Val65 70
75 80Ala Ala Ser Ala53675DNABacillus subtilis
53atgaagattt acggaattta tatggaccgc ccgctttcac aggaagaaaa tgaacggttc
60atgactttca tatcacctga aaaacgggag aaatgccgga gattttatca taaagaagat
120gctcaccgca ccctgctggg agatgtgctc gttcgctcag tcataagcag gcagtatcag
180ttggacaaat ccgatatccg ctttagcacg caggaatacg ggaagccgtg catccctgat
240cttcccgacg ctcatttcaa catttctcac tccggccgct gggtcattgg tgcgtttgat
300tcacagccga tcggcataga tatcgaaaaa acgaaaccga tcagccttga gatcgccaag
360cgcttctttt caaaaacaga gtacagcgac cttttagcaa aagacaagga cgagcagaca
420gactattttt atcatctatg gtcaatgaaa gaaagcttta tcaaacagga aggcaaaggc
480ttatcgcttc cgcttgattc cttttcagtg cgcctgcatc aggacggaca agtatccatt
540gagcttccgg acagccattc cccatgctat atcaaaacgt atgaggtcga tcccggctac
600aaaatggctg tatgcgccgc acaccctgat ttccccgagg atatcacaat ggtctcgtac
660gaagagcttt tataa
67554224PRTBacillus subtilis 54Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg
Pro Leu Ser Gln Glu Glu1 5 10
15Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30Arg Arg Phe Tyr His Lys
Glu Asp Ala His Arg Thr Leu Leu Gly Asp 35 40
45Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp
Lys Ser 50 55 60Asp Ile Arg Phe Ser
Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp65 70
75 80Leu Pro Asp Ala His Phe Asn Ile Ser His
Ser Gly Arg Trp Val Ile 85 90
95Gly Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110Pro Ile Ser Leu Glu
Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr 115
120 125Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr
Asp Tyr Phe Tyr 130 135 140His Leu Trp
Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly145
150 155 160Leu Ser Leu Pro Leu Asp Ser
Phe Ser Val Arg Leu His Gln Asp Gly 165
170 175Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro
Cys Tyr Ile Lys 180 185 190Thr
Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His 195
200 205Pro Asp Phe Pro Glu Asp Ile Thr Met
Val Ser Tyr Glu Glu Leu Leu 210 215
22055867DNACorynebacterium glutamicum 55ttgggcgtgt cgcccttaaa gcgcgctttt
cgacgcgacc ccactacatt ggcttccatg 60aacgttgaca tttcacgatc cagagagccg
ctaaacgttg agctcctgaa ggaaaaattg 120ctccaaaacg gtgactttgg ccaggtcatt
tacgaaaaag tgacaggctc cactaatgct 180gacttgctgg cacttgcagg ttctggcgct
ccaaactgga cggtgaaaac tgtcgagttt 240caagatcatg cgcgtgggcg actcggccgc
ccgtggtctg cccctgaggg ttcccaaaca 300atcgtgtctg tgctcgttca actatctatt
gatcaagtgg accggattgg cactattcca 360ctcgcggcgg gactcgctgt catggatgcg
ttgaatgacc tcggtgtgga aggtgccgga 420ctgaaatggc ccaacgatgt tcaaatccac
ggcaagaaac tctgcggcat cctggtggaa 480gccaccggct ttgattccac cccaacagtt
gtcatcggtt ggggcactaa tatcagcctg 540actaaagagg agcttcctgt tcctcatgca
acttccctcg cattggaagg tgttgaagtc 600gacagaacca cattccttat taatatgctc
acacatctgc atactcgact ggaccagtgg 660cagggtccaa gtgtggattg gctcgatgat
taccgtgcgg tatgttccag tattggccaa 720gatgttcgag tgcttctacc tggggataaa
gaactcttag gtgaagcgat cggtgtcgcg 780actggcggag aaattcgtgt tcgcgatgct
tcgggcaccg ttcacaccct caacgccggt 840gaaattacgc accttcgcct gcagtaa
86756810DNACorynebacterium glutamicum
56atgaatgttg acattagccg ctctcgtgaa ccgttgaacg tggaactgtt gaaagaaaaa
60ctgctgcaga acggtgattt cggtcaagtg atctacgaga aggtcaccgg ctctaccaat
120gcggacctgc tggctctggc gggcagcggc gctccaaact ggaccgtcaa gactgttgaa
180tttcaggacc acgcccgtgg ccgtctgggt cgtccgtgga gcgcaccgga gggttcccaa
240accatcgtca gcgttctggt ccaactgagc attgatcagg tggaccgtat tggtacgatc
300ccgctggccg caggcttggc tgttatggat gcgctgaatg atctgggcgt ggagggtgca
360ggcctgaaat ggccgaacga tgttcagatc cacggtaaga agttgtgcgg tattctggtt
420gaagcaaccg gcttcgactc cactccgacc gtggttatcg gttggggtac gaatatctcg
480ttgacgaaag aagagctgcc ggtcccgcac gcgaccagcc tggccctgga gggtgttgaa
540gttgaccgta cgacgttcct gattaacatg ctgacccatc tgcatacccg tctggatcag
600tggcagggtc cgtctgtgga ctggctggat gactatcgcg cggtttgtag cagcattggc
660caagatgtgc gtgtcctgct gcctggtgac aaagagctgc tgggcgaggc gattggcgtg
720gcgaccggtg gtgagatccg tgtgcgcgac gccagcggca cggtccacac gctgaatgcg
780ggtgaaatca cgcatctgcg tttgcaataa
81057269PRTCorynebacterium glutamicum 57Met Asn Val Asp Ile Ser Arg Ser
Arg Glu Pro Leu Asn Val Glu Leu1 5 10
15Leu Lys Glu Lys Leu Leu Gln Asn Gly Asp Phe Gly Gln Val
Ile Tyr 20 25 30Glu Lys Val
Thr Gly Ser Thr Asn Ala Asp Leu Leu Ala Leu Ala Gly 35
40 45Ser Gly Ala Pro Asn Trp Thr Val Lys Thr Val
Glu Phe Gln Asp His 50 55 60Ala Arg
Gly Arg Leu Gly Arg Pro Trp Ser Ala Pro Glu Gly Ser Gln65
70 75 80Thr Ile Val Ser Val Leu Val
Gln Leu Ser Ile Asp Gln Val Asp Arg 85 90
95Ile Gly Thr Ile Pro Leu Ala Ala Gly Leu Ala Val Met
Asp Ala Leu 100 105 110Asn Asp
Leu Gly Val Glu Gly Ala Gly Leu Lys Trp Pro Asn Asp Val 115
120 125Gln Ile His Gly Lys Lys Leu Cys Gly Ile
Leu Val Glu Ala Thr Gly 130 135 140Phe
Asp Ser Thr Pro Thr Val Val Ile Gly Trp Gly Thr Asn Ile Ser145
150 155 160Leu Thr Lys Glu Glu Leu
Pro Val Pro His Ala Thr Ser Leu Ala Leu 165
170 175Glu Gly Val Glu Val Asp Arg Thr Thr Phe Leu Ile
Asn Met Leu Thr 180 185 190His
Leu His Thr Arg Leu Asp Gln Trp Gln Gly Pro Ser Val Asp Trp 195
200 205Leu Asp Asp Tyr Arg Ala Val Cys Ser
Ser Ile Gly Gln Asp Val Arg 210 215
220Val Leu Leu Pro Gly Asp Lys Glu Leu Leu Gly Glu Ala Ile Gly Val225
230 235 240Ala Thr Gly Gly
Glu Ile Arg Val Arg Asp Ala Ser Gly Thr Val His 245
250 255Thr Leu Asn Ala Gly Glu Ile Thr His Leu
Arg Leu Gln 260 265581632DNACorynebacterium
glutamicum 58atgaccattt cctcaccttt gattgacgtc gccaaccttc cagacatcaa
caccactgcc 60ggcaagatcg ccgaccttaa ggctcgccgc gcggaagccc atttccccat
gggtgaaaag 120gcagtagaga aggtccacgc tgctggacgc ctcactgccc gtgagcgctt
ggattactta 180ctcgatgagg gctccttcat cgagaccgat cagctggctc gccaccgcac
caccgctttc 240ggcctgggcg ctaagcgtcc tgcaaccgac ggcatcgtga ccggctgggg
caccattgat 300ggacgcgaag tctgcatctt ctcgcaggac ggcaccgtat tcggtggcgc
gcttggtgag 360gtgtacggcg aaaagatgat caagatcatg gagctggcaa tcgacaccgg
ccgcccattg 420atcggtcttt acgaaggcgc tggcgctcgt attcaggacg gcgctgtctc
cctggacttc 480atttcccaga ccttctacca aaacattcag gcttctggcg ttatcccaca
gatctccgtc 540atcatgggcg catgtgcagg tggcaacgct tacggcccag ctctgaccga
cttcgtggtc 600atggtggaca agacctccaa gatgttcgtt accggcccag acgtgatcaa
gaccgtcacc 660ggcgaggaaa tcacccagga agagcttggc ggagcaacca cccacatggt
gaccgctggt 720aactcccact acaccgctgc gaccgatgag gaagcactgg attgggtaca
ggacctggtg 780tccttcctcc catccaacaa tcgctcctac gcaccgatgg aagacttcga
cgaggaagaa 840ggcggcgttg aagaaaacat caccgctgac gatctgaagc tcgacgagat
catcccagat 900tccgcgaccg ttccttacga cgtccgcgat gtcatcgaat gcctcaccga
cgatggcgaa 960tacctggaaa tccaggcaga ccgcgcagaa aacgttgtta ttgcattcgg
ccgcatcgaa 1020ggccagtccg ttggctttgt tgccaaccag ccaacccagt tcgctggctg
cctggacatc 1080gactcctctg agaaggcagc tcgcttcgtc cgcacctgcg acgcgttcaa
catcccaatc 1140gtcatgcttg tcgacgtccc cggcttcctc ccaggcgcag gccaggagta
cggtggcatt 1200ctgcgtcgtg gcgcaaagct gctctacgca tacggcgaag caaccgttcc
aaagatcacc 1260gtcaccatgc gtaaggctta cggcggagcg tactgcgtga tgggttccaa
gggcttgggc 1320tctgacatca accttgcatg gccaaccgca cagatcgccg tcatgggcgc
tgctggcgca 1380gttggattca tctaccgcaa ggagctcatg gcagctgatg ccaagggcct
cgataccgta 1440gctctggcta agtccttcga gcgcgagtat gaagaccaca tgctcaaccc
gtaccacgct 1500gcagaacgtg gcctgatcga cgccgtgatc ctgccaagcg aaacccgcgg
acagatttcc 1560cgcaaccttc gcctgctcaa gcacaagaac gtcactcgcc ctgctcgcaa
gcacggcaac 1620atgccactgt aa
163259543PRTCorynebacterium glutamicum 59Met Thr Ile Ser Ser
Pro Leu Ile Asp Val Ala Asn Leu Pro Asp Ile1 5
10 15Asn Thr Thr Ala Gly Lys Ile Ala Asp Leu Lys
Ala Arg Arg Ala Glu 20 25
30Ala His Phe Pro Met Gly Glu Lys Ala Val Glu Lys Val His Ala Ala
35 40 45Gly Arg Leu Thr Ala Arg Glu Arg
Leu Asp Tyr Leu Leu Asp Glu Gly 50 55
60Ser Phe Ile Glu Thr Asp Gln Leu Ala Arg His Arg Thr Thr Ala Phe65
70 75 80Gly Leu Gly Ala Lys
Arg Pro Ala Thr Asp Gly Ile Val Thr Gly Trp 85
90 95Gly Thr Ile Asp Gly Arg Glu Val Cys Ile Phe
Ser Gln Asp Gly Thr 100 105
110Val Phe Gly Gly Ala Leu Gly Glu Val Tyr Gly Glu Lys Met Ile Lys
115 120 125Ile Met Glu Leu Ala Ile Asp
Thr Gly Arg Pro Leu Ile Gly Leu Tyr 130 135
140Glu Gly Ala Gly Ala Arg Ile Gln Asp Gly Ala Val Ser Leu Asp
Phe145 150 155 160Ile Ser
Gln Thr Phe Tyr Gln Asn Ile Gln Ala Ser Gly Val Ile Pro
165 170 175Gln Ile Ser Val Ile Met Gly
Ala Cys Ala Gly Gly Asn Ala Tyr Gly 180 185
190Pro Ala Leu Thr Asp Phe Val Val Met Val Asp Lys Thr Ser
Lys Met 195 200 205Phe Val Thr Gly
Pro Asp Val Ile Lys Thr Val Thr Gly Glu Glu Ile 210
215 220Thr Gln Glu Glu Leu Gly Gly Ala Thr Thr His Met
Val Thr Ala Gly225 230 235
240Asn Ser His Tyr Thr Ala Ala Thr Asp Glu Glu Ala Leu Asp Trp Val
245 250 255Gln Asp Leu Val Ser
Phe Leu Pro Ser Asn Asn Arg Ser Tyr Ala Pro 260
265 270Met Glu Asp Phe Asp Glu Glu Glu Gly Gly Val Glu
Glu Asn Ile Thr 275 280 285Ala Asp
Asp Leu Lys Leu Asp Glu Ile Ile Pro Asp Ser Ala Thr Val 290
295 300Pro Tyr Asp Val Arg Asp Val Ile Glu Cys Leu
Thr Asp Asp Gly Glu305 310 315
320Tyr Leu Glu Ile Gln Ala Asp Arg Ala Glu Asn Val Val Ile Ala Phe
325 330 335Gly Arg Ile Glu
Gly Gln Ser Val Gly Phe Val Ala Asn Gln Pro Thr 340
345 350Gln Phe Ala Gly Cys Leu Asp Ile Asp Ser Ser
Glu Lys Ala Ala Arg 355 360 365Phe
Val Arg Thr Cys Asp Ala Phe Asn Ile Pro Ile Val Met Leu Val 370
375 380Asp Val Pro Gly Phe Leu Pro Gly Ala Gly
Gln Glu Tyr Gly Gly Ile385 390 395
400Leu Arg Arg Gly Ala Lys Leu Leu Tyr Ala Tyr Gly Glu Ala Thr
Val 405 410 415Pro Lys Ile
Thr Val Thr Met Arg Lys Ala Tyr Gly Gly Ala Tyr Cys 420
425 430Val Met Gly Ser Lys Gly Leu Gly Ser Asp
Ile Asn Leu Ala Trp Pro 435 440
445Thr Ala Gln Ile Ala Val Met Gly Ala Ala Gly Ala Val Gly Phe Ile 450
455 460Tyr Arg Lys Glu Leu Met Ala Ala
Asp Ala Lys Gly Leu Asp Thr Val465 470
475 480Ala Leu Ala Lys Ser Phe Glu Arg Glu Tyr Glu Asp
His Met Leu Asn 485 490
495Pro Tyr His Ala Ala Glu Arg Gly Leu Ile Asp Ala Val Ile Leu Pro
500 505 510Ser Glu Thr Arg Gly Gln
Ile Ser Arg Asn Leu Arg Leu Leu Lys His 515 520
525Lys Asn Val Thr Arg Pro Ala Arg Lys His Gly Asn Met Pro
Leu 530 535
540601776DNACorynebacterium glutamicum 60atgtcagtcg agactcgcaa gatcaccaag
gttcttgtcg ctaaccgtgg tgagattgca 60atccgcgtgt tccgtgcagc tcgagatgaa
ggcatcggat ctgtcgccgt ctacgcagag 120ccagatgcag atgcaccatt cgtgtcatat
gcagacgagg cttttgccct cggtggccaa 180acatccgctg agtcctacct tgtcattgac
aagatcatcg atgcggcccg caagtccggc 240gccgacgcca tccaccccgg ctacggcttc
ctcgcagaaa acgctgactt cgcagaagca 300gtcatcaacg aaggcctgat ctggattgga
ccttcacctg agtccatccg ctccctcggc 360gacaaggtca ccgctcgcca catcgcagat
accgccaagg ctccaatggc tcctggcacc 420aaggaaccag taaaagacgc agcagaagtt
gtggctttcg ctgaagaatt cggtctccca 480atcgccatca aggcagcttt cggtggcggc
ggacgtggca tgaaggttgc ctacaagatg 540gaagaagtcg ctgacctctt cgagtccgca
acccgtgaag caaccgcagc gttcggccgc 600ggcgagtgct tcgtggagcg ctacctggac
aaggcacgcc acgttgaggc tcaggtcatc 660gccgataagc acggcaacgt tgttgtcgcc
ggaacccgtg actgctccct gcagcgccgt 720ttccagaagc tcgtcgaaga agcaccagca
ccattcctca ccgatgacca gcgcgagcgt 780ctccactcct ccgcgaaggc tatctgtaag
gaagctggct actacggtgc aggcaccgtt 840gagtacctcg ttggctccga cggcctgatc
tccttcctcg aggtcaacac ccgcctccag 900gtggaacacc cagtcaccga agagaccacc
ggcatcgacc tggtccgcga aatgttccgc 960atcgcagaag gccacgagct ctccatcaag
gaagatccag ctccacgcgg ccacgcattc 1020gagttccgca tcaacggcga agacgctggc
tccaacttca tgcctgcacc aggcaagatc 1080accagctacc gcgagccaca gggcccaggc
gtccgcatgg actccggtgt cgttgaaggt 1140tccgaaatct ccggacagtt cgactccatg
ctggcaaagc tgatcgtttg gggcgacacc 1200cgcgagcagg ctctccagcg ctcccgccgt
gcacttgcag agtacgttgt cgagggcatg 1260ccaaccgtta tcccattcca ccagcacatc
gtggaaaacc cagcattcgt gggcaacgac 1320gaaggcttcg agatctacac caagtggatc
gaagaggttt gggataaccc aatcgcacct 1380tacgttgacg cttccgagct cgacgaagat
gaggacaaga ccccagcaca gaaggttgtt 1440gtggagatca acggccgtcg cgttgaggtt
gcactcccag gcgatctggc actcggtggc 1500accgctggtc ctaagaagaa ggccaagaag
cgtcgcgcag gtggtgcaaa ggctggcgta 1560tccggcgatg cagtggcagc tccaatgcag
ggcactgtca tcaaggtcaa cgtcgaagaa 1620ggcgctgaag tcaacgaagg cgacaccgtt
gttgtcctcg aggctatgaa gatggaaaac 1680cctgtgaagg ctcataagtc cggaaccgta
accggcctta ctgtcgctgc aggcgagggt 1740gtcaacaagg gcgttgttct cctcgagatc
aagtaa 177661591PRTCorynebacterium glutamicum
61Met Ser Val Glu Thr Arg Lys Ile Thr Lys Val Leu Val Ala Asn Arg1
5 10 15Gly Glu Ile Ala Ile Arg
Val Phe Arg Ala Ala Arg Asp Glu Gly Ile 20 25
30Gly Ser Val Ala Val Tyr Ala Glu Pro Asp Ala Asp Ala
Pro Phe Val 35 40 45Ser Tyr Ala
Asp Glu Ala Phe Ala Leu Gly Gly Gln Thr Ser Ala Glu 50
55 60Ser Tyr Leu Val Ile Asp Lys Ile Ile Asp Ala Ala
Arg Lys Ser Gly65 70 75
80Ala Asp Ala Ile His Pro Gly Tyr Gly Phe Leu Ala Glu Asn Ala Asp
85 90 95Phe Ala Glu Ala Val Ile
Asn Glu Gly Leu Ile Trp Ile Gly Pro Ser 100
105 110Pro Glu Ser Ile Arg Ser Leu Gly Asp Lys Val Thr
Ala Arg His Ile 115 120 125Ala Asp
Thr Ala Lys Ala Pro Met Ala Pro Gly Thr Lys Glu Pro Val 130
135 140Lys Asp Ala Ala Glu Val Val Ala Phe Ala Glu
Glu Phe Gly Leu Pro145 150 155
160Ile Ala Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly Met Lys Val
165 170 175Ala Tyr Lys Met
Glu Glu Val Ala Asp Leu Phe Glu Ser Ala Thr Arg 180
185 190Glu Ala Thr Ala Ala Phe Gly Arg Gly Glu Cys
Phe Val Glu Arg Tyr 195 200 205Leu
Asp Lys Ala Arg His Val Glu Ala Gln Val Ile Ala Asp Lys His 210
215 220Gly Asn Val Val Val Ala Gly Thr Arg Asp
Cys Ser Leu Gln Arg Arg225 230 235
240Phe Gln Lys Leu Val Glu Glu Ala Pro Ala Pro Phe Leu Thr Asp
Asp 245 250 255Gln Arg Glu
Arg Leu His Ser Ser Ala Lys Ala Ile Cys Lys Glu Ala 260
265 270Gly Tyr Tyr Gly Ala Gly Thr Val Glu Tyr
Leu Val Gly Ser Asp Gly 275 280
285Leu Ile Ser Phe Leu Glu Val Asn Thr Arg Leu Gln Val Glu His Pro 290
295 300Val Thr Glu Glu Thr Thr Gly Ile
Asp Leu Val Arg Glu Met Phe Arg305 310
315 320Ile Ala Glu Gly His Glu Leu Ser Ile Lys Glu Asp
Pro Ala Pro Arg 325 330
335Gly His Ala Phe Glu Phe Arg Ile Asn Gly Glu Asp Ala Gly Ser Asn
340 345 350Phe Met Pro Ala Pro Gly
Lys Ile Thr Ser Tyr Arg Glu Pro Gln Gly 355 360
365Pro Gly Val Arg Met Asp Ser Gly Val Val Glu Gly Ser Glu
Ile Ser 370 375 380Gly Gln Phe Asp Ser
Met Leu Ala Lys Leu Ile Val Trp Gly Asp Thr385 390
395 400Arg Glu Gln Ala Leu Gln Arg Ser Arg Arg
Ala Leu Ala Glu Tyr Val 405 410
415Val Glu Gly Met Pro Thr Val Ile Pro Phe His Gln His Ile Val Glu
420 425 430Asn Pro Ala Phe Val
Gly Asn Asp Glu Gly Phe Glu Ile Tyr Thr Lys 435
440 445Trp Ile Glu Glu Val Trp Asp Asn Pro Ile Ala Pro
Tyr Val Asp Ala 450 455 460Ser Glu Leu
Asp Glu Asp Glu Asp Lys Thr Pro Ala Gln Lys Val Val465
470 475 480Val Glu Ile Asn Gly Arg Arg
Val Glu Val Ala Leu Pro Gly Asp Leu 485
490 495Ala Leu Gly Gly Thr Ala Gly Pro Lys Lys Lys Ala
Lys Lys Arg Arg 500 505 510Ala
Gly Gly Ala Lys Ala Gly Val Ser Gly Asp Ala Val Ala Ala Pro 515
520 525Met Gln Gly Thr Val Ile Lys Val Asn
Val Glu Glu Gly Ala Glu Val 530 535
540Asn Glu Gly Asp Thr Val Val Val Leu Glu Ala Met Lys Met Glu Asn545
550 555 560Pro Val Lys Ala
His Lys Ser Gly Thr Val Thr Gly Leu Thr Val Ala 565
570 575Ala Gly Glu Gly Val Asn Lys Gly Val Val
Leu Leu Glu Ile Lys 580 585
5906210025DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 62cactatacca attgagatgg
gctagtcaat gataattact agtccttttc ctttgagttg 60tgggtatctg taaattctgc
tagacctttg ctggaaaact tgtaaattct gctagaccct 120ctgtaaattc cgctagacct
ttgtgtgttt tttttgttta tattcaagtg gttataattt 180atagaataaa gaaagaataa
aaaaagataa aaagaataga tcccagccct gtgtataact 240cactacttta gtcagttccg
cagtattaca aaaggatgtc gcaaacgctg tttgctcctc 300tacaaaacag accttaaaac
cctaaaggcg tcggcatccg cttacagaca agctgtgacc 360gtctccggga gctgcatgtg
tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag 420cagatcaatt cgcgcgcgaa
ggcgaagcgg catgcattta cgttgacacc atcgaatggt 480gcaaaacctt tcgcggtatg
gcatgatagc gcccggaaga gagtcaattc agggtggtga 540atgtgaaacc agtaacgtta
tacgatgtcg cagagtatgc cggtgtctct tatcagaccg 600tttcccgcgt ggtgaaccag
gccagccacg tttctgcgaa aacgcgggaa aaagtggaag 660cggcgatggc ggagctgaat
tacattccca accgcgtggc acaacaactg gcgggcaaac 720agtcgttgct gattggcgtt
gccacctcca gtctggccct gcacgcgccg tcgcaaattg 780tcgcggcgat taaatctcgc
gccgatcaac tgggtgccag cgtggtggtg tcgatggtag 840aacgaagcgg cgtcgaagcc
tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca 900gtgggctgat cattaactat
ccgctggatg accaggatgc cattgctgtg gaagctgcct 960gcactaatgt tccggcgtta
tttcttgatg tctctgacca gacacccatc aacagtatta 1020ttttctccca tgaagacggt
acgcgactgg gcgtggagca tctggtcgca ttgggtcacc 1080agcaaatcgc gctgttagcg
ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg 1140gctggcataa atatctcact
cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact 1200ggagtgccat gtccggtttt
caacaaacca tgcaaatgct gaatgagggc atcgttccca 1260ctgcgatgct ggttgccaac
gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt 1320ccgggctgcg cgttggtgcg
gatatctcgg tagtgggata cgacgatacc gaagacagct 1380catgttatat cccgccgtta
accaccatca aacaggattt tcgcctgctg gggcaaacca 1440gcgtggaccg cttgctgcaa
ctctctcagg gccaggcggt gaagggcaat cagctgttgc 1500ccgtctcact ggtgaaaaga
aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc 1560gcgcgttggc cgattcatta
atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 1620agtgagcgca acgcaattaa
tgtaagttag cgcgaattga tctggtttga cagcttatca 1680tcgactgcac ggtgcaccaa
tgcttctggc gtcaggcagc catcggaagc tgtggtatgg 1740ctgtgcaggt cgtaaatcac
tgcataattc gtgtcgctca aggcgcactc ccgttctgga 1800taatgttttt tgcgccgaca
tcataacggt tctggcaaat attctgttga caattaatca 1860tccggctcgt ataaagtgtg
gaattgtgag cggataacaa tttcacacag gaaacagcgc 1920cgctgagaaa aagcgaagcg
gcactgctct ttaacaattt atcagacaat ctgtgtgggc 1980actcgaccgg aattatcgat
taactttatt attaaaaatt aaagaggtat atattaatgt 2040atcgattaaa taaggaggaa
taaaccatga ccatttcctc acctttgatt gacgtcgcca 2100accttccaga catcaacacc
actgccggca agatcgccga ccttaaggct cgccgcgcgg 2160aagcccattt ccccatgggt
gaaaaggcag tagagaaggt ccacgctgct ggacgcctca 2220ctgcccgtga gcgcttggat
tacttactcg atgagggctc cttcatcgag accgatcagc 2280tggctcgcca ccgcaccacc
gctttcggcc tgggcgctaa gcgtcctgca accgacggca 2340tcgtgaccgg ctggggcacc
attgatggac gcgaagtctg catcttctcg caggacggca 2400ccgtattcgg tggcgcgctt
ggtgaggtgt acggcgaaaa gatgatcaag atcatggagc 2460tggcaatcga caccggccgc
ccattgatcg gtctttacga aggcgctggc gctcgtattc 2520aggacggcgc tgtctccctg
gacttcattt cccagacctt ctaccaaaac attcaggctt 2580ctggcgttat cccacagatc
tccgtcatca tgggcgcatg tgcaggtggc aacgcttacg 2640gcccagctct gaccgacttc
gtggtcatgg tggacaagac ctccaagatg ttcgttaccg 2700gcccagacgt gatcaagacc
gtcaccggcg aggaaatcac ccaggaagag cttggcggag 2760caaccaccca catggtgacc
gctggtaact cccactacac cgctgcgacc gatgaggaag 2820cactggattg ggtacaggac
ctggtgtcct tcctcccatc caacaatcgc tcctacgcac 2880cgatggaaga cttcgacgag
gaagaaggcg gcgttgaaga aaacatcacc gctgacgatc 2940tgaagctcga cgagatcatc
ccagattccg cgaccgttcc ttacgacgtc cgcgatgtca 3000tcgaatgcct caccgacgat
ggcgaatacc tggaaatcca ggcagaccgc gcagaaaacg 3060ttgttattgc attcggccgc
atcgaaggcc agtccgttgg ctttgttgcc aaccagccaa 3120cccagttcgc tggctgcctg
gacatcgact cctctgagaa ggcagctcgc ttcgtccgca 3180cctgcgacgc gttcaacatc
ccaatcgtca tgcttgtcga cgtccccggc ttcctcccag 3240gcgcaggcca ggagtacggt
ggcattctgc gtcgtggcgc aaagctgctc tacgcatacg 3300gcgaagcaac cgttccaaag
atcaccgtca ccatgcgtaa ggcttacggc ggagcgtact 3360gcgtgatggg ttccaagggc
ttgggctctg acatcaacct tgcatggcca accgcacaga 3420tcgccgtcat gggcgctgct
ggcgcagttg gattcatcta ccgcaaggag ctcatggcag 3480ctgatgccaa gggcctcgat
accgtagctc tggctaagtc cttcgagcgc gagtatgaag 3540accacatgct caacccgtac
cacgctgcag aacgtggcct gatcgacgcc gtgatcctgc 3600caagcgaaac ccgcggacag
atttcccgca accttcgcct gctcaagcac aagaacgtca 3660ctcgccctgc tcgcaagcac
ggcaacatgc cactgtaagg aggaaaacta aatgtcagtc 3720gagactcgca agatcaccaa
ggttcttgtc gctaaccgtg gtgagattgc aatccgcgtg 3780ttccgtgcag ctcgagatga
aggcatcgga tctgtcgccg tctacgcaga gccagatgca 3840gatgcaccat tcgtgtcata
tgcagacgag gcttttgccc tcggtggcca aacatccgct 3900gagtcctacc ttgtcattga
caagatcatc gatgcggccc gcaagtccgg cgccgacgcc 3960atccaccccg gctacggctt
cctcgcagaa aacgctgact tcgcagaagc agtcatcaac 4020gaaggcctga tctggattgg
accttcacct gagtccatcc gctccctcgg cgacaaggtc 4080accgctcgcc acatcgcaga
taccgccaag gctccaatgg ctcctggcac caaggaacca 4140gtaaaagacg cagcagaagt
tgtggctttc gctgaagaat tcggtctccc aatcgccatc 4200aaggcagctt tcggtggcgg
cggacgtggc atgaaggttg cctacaagat ggaagaagtc 4260gctgacctct tcgagtccgc
aacccgtgaa gcaaccgcag cgttcggccg cggcgagtgc 4320ttcgtggagc gctacctgga
caaggcacgc cacgttgagg ctcaggtcat cgccgataag 4380cacggcaacg ttgttgtcgc
cggaacccgt gactgctccc tgcagcgccg tttccagaag 4440ctcgtcgaag aagcaccagc
accattcctc accgatgacc agcgcgagcg tctccactcc 4500tccgcgaagg ctatctgtaa
ggaagctggc tactacggtg caggcaccgt tgagtacctc 4560gttggctccg acggcctgat
ctccttcctc gaggtcaaca cccgcctcca ggtggaacac 4620ccagtcaccg aagagaccac
cggcatcgac ctggtccgcg aaatgttccg catcgcagaa 4680ggccacgagc tctccatcaa
ggaagatcca gctccacgcg gccacgcatt cgagttccgc 4740atcaacggcg aagacgctgg
ctccaacttc atgcctgcac caggcaagat caccagctac 4800cgcgagccac agggcccagg
cgtccgcatg gactccggtg tcgttgaagg ttccgaaatc 4860tccggacagt tcgactccat
gctggcaaag ctgatcgttt ggggcgacac ccgcgagcag 4920gctctccagc gctcccgccg
tgcacttgca gagtacgttg tcgagggcat gccaaccgtt 4980atcccattcc accagcacat
cgtggaaaac ccagcattcg tgggcaacga cgaaggcttc 5040gagatctaca ccaagtggat
cgaagaggtt tgggataacc caatcgcacc ttacgttgac 5100gcttccgagc tcgacgaaga
tgaggacaag accccagcac agaaggttgt tgtggagatc 5160aacggccgtc gcgttgaggt
tgcactccca ggcgatctgg cactcggtgg caccgctggt 5220cctaagaaga aggccaagaa
gcgtcgcgca ggtggtgcaa aggctggcgt atccggcgat 5280gcagtggcag ctccaatgca
gggcactgtc atcaaggtca acgtcgaaga aggcgctgaa 5340gtcaacgaag gcgacaccgt
tgttgtcctc gaggctatga agatggaaaa ccctgtgaag 5400gctcataagt ccggaaccgt
aaccggcctt actgtcgctg caggcgaggg tgtcaacaag 5460ggcgttgttc tcctcgagat
caagtaatct agaggaggaa aactaaatga atgttgacat 5520tagccgctct cgtgaaccgt
tgaacgtgga actgttgaaa gaaaaactgc tgcagaacgg 5580tgatttcggt caagtgatct
acgagaaggt caccggctct accaatgcgg acctgctggc 5640tctggcgggc agcggcgctc
caaactggac cgtcaagact gttgaatttc aggaccacgc 5700ccgtggccgt ctgggtcgtc
cgtggagcgc accggagggt tcccaaacca tcgtcagcgt 5760tctggtccaa ctgagcattg
atcaggtgga ccgtattggt acgatcccgc tggccgcagg 5820cttggctgtt atggatgcgc
tgaatgatct gggcgtggag ggtgcaggcc tgaaatggcc 5880gaacgatgtt cagatccacg
gtaagaagtt gtgcggtatt ctggttgaag caaccggctt 5940cgactccact ccgaccgtgg
ttatcggttg gggtacgaat atctcgttga cgaaagaaga 6000gctgccggtc ccgcacgcga
ccagcctggc cctggagggt gttgaagttg accgtacgac 6060gttcctgatt aacatgctga
cccatctgca tacccgtctg gatcagtggc agggtccgtc 6120tgtggactgg ctggatgact
atcgcgcggt ttgtagcagc attggccaag atgtgcgtgt 6180cctgctgcct ggtgacaaag
agctgctggg cgaggcgatt ggcgtggcga ccggtggtga 6240gatccgtgtg cgcgacgcca
gcggcacggt ccacacgctg aatgcgggtg aaatcacgca 6300tctgcgtttg caataaaagc
ttgtttaaac ggtctccagc ttggctgttt tggcggatga 6360gagaagattt tcagcctgat
acagattaaa tcagaacgca gaagcggtct gataaaacag 6420aatttgcctg gcggcagtag
cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg 6480aaacgccgta gcgccgatgg
tagtgtgggg tctccccatg cgagagtagg gaactgccag 6540gcatcaaata aaacgaaagg
ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 6600gtcggtgaac gctctcctga
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 6660tttcacaccg catatggtgc
actctcagta caatctgctc tgatgccgca tagttaagcc 6720agccccgaca cccgccaaca
cccgctgacg agcttagtaa agccctcgct agattttaat 6780gcggatgttg cgattacttc
gccaactatt gcgataacaa gaaaaagcca gcctttcatg 6840atatatctcc caatttgtgt
agggcttatt atgcacgctt aaaaataata aaagcagact 6900tgacctgata gtttggctgt
gagcaattat gtgcttagtg catctaacgc ttgagttaag 6960ccgcgccgcg aagcggcgtc
ggcttgaacg aattgttaga cattatttgc cgactacctt 7020ggtgatctcg cctttcacgt
agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa 7080gcgatcttct tcttgtccaa
gataagcctg tctagcttca agtatgacgg gctgatactg 7140ggccggcagg cgctccattg
cccagtcggc agcgacatcc ttcggcgcga ttttgccggt 7200tactgcgctg taccaaatgc
gggacaacgt aagcactaca tttcgctcat cgccagccca 7260gtcgggcggc gagttccata
gcgttaaggt ttcatttagc gcctcaaata gatcctgttc 7320aggaaccgga tcaaagagtt
cctccgccgc tggacctacc aaggcaacgc tatgttctct 7380tgcttttgtc agcaagatag
ccagatcaat gtcgatcgtg gctggctcga agatacctgc 7440aagaatgtca ttgcgctgcc
attctccaaa ttgcagttcg cgcttagctg gataacgcca 7500cggaatgatg tcgtcgtgca
caacaatggt gacttctaca gcgcggagaa tctcgctctc 7560tccaggggaa gccgaagttt
ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc 7620aagccttacg gtcaccgtaa
ccagcaaatc aatatcactg tgtggcttca ggccgccatc 7680cactgcggag ccgtacaaat
gtacggccag caacgtcggt tcgagatggc gctcgatgac 7740gccaactacc tctgatagtt
gagtcgatac ttcggcgatc accgcttccc tcatgatgtt 7800taactttgtt ttagggcgac
tgccctgctg cgtaacatcg ttgctgctcc ataacatcaa 7860acatcgaccc acggcgtaac
gcgcttgctg cttggatgcc cgaggcatag actgtacccc 7920aaaaaaacag tcataacaag
ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc 7980ggtcaaggtt ctggaccagt
tgcgtgagcg catacgctac ttgcattaca gcttacgaac 8040cgaacaggct tatgtccact
gggttcgtgc cttcatccgt ttccacggtg tgcgtcaccc 8100ggcaaccttg ggcagcagcg
aagtcgaggc atttctgtcc tggctggcga acgagcgcaa 8160ggtttcggtc tccacgcatc
gtcaggcatt ggcggccttg ctgttcttct acggcaaggt 8220gctgtgcacg gatctgccct
ggcttcagga gatcggaaga cctcggccgt cgcggcgctt 8280gccggtggtg ctgaccccgg
atgaagtggt tcgcatcctc ggttttctgg aaggcgagca 8340tcgtttgttc gcccagcttc
tgtatggaac gggcatgcgg atcagtgagg gtttgcaact 8400gcgggtcaag gatctggatt
tcgatcacgg cacgatcatc gtgcgggagg gcaagggctc 8460caaggatcgg gccttgatgt
tacccgagag cttggcaccc agcctgcgcg agcaggggaa 8520ttaattccca cgggttttgc
tgcccgcaaa cgggctgttc tggtgttgct agtttgttat 8580cagaatcgca gatccggctt
cagccggttt gccggctgaa agcgctattt cttccagaat 8640tgccatgatt ttttccccac
gggaggcgtc actggctccc gtgttgtcgg cagctttgat 8700tcgataagca gcatcgcctg
tttcaggctg tctatgtgtg actgttgagc tgtaacaagt 8760tgtctcaggt gttcaatttc
atgttctagt tgctttgttt tactggtttc acctgttcta 8820ttaggtgtta catgctgttc
atctgttaca ttgtcgatct gttcatggtg aacagctttg 8880aatgcaccaa aaactcgtaa
aagctctgat gtatctatct tttttacacc gttttcatct 8940gtgcatatgg acagttttcc
ctttgatatg taacggtgaa cagttgttct acttttgttt 9000gttagtcttg atgcttcact
gatagataca agagccataa gaacctcaga tccttccgta 9060tttagccagt atgttctcta
gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc 9120attgagatca tacttacttt
gcatgtcact caaaaatttt gcctcaaaac tggtgagctg 9180aatttttgca gttaaagcat
cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc 9240tgatgtaatg gttgttggta
ttttgtcacc attcattttt atctggttgt tctcaagttc 9300ggttacgaga tccatttgtc
tatctagttc aacttggaaa atcaacgtat cagtcgggcg 9360gcctcgctta tcaaccacca
atttcatatt gctgtaagtg tttaaatctt tacttattgg 9420tttcaaaacc cattggttaa
gccttttaaa ctcatggtag ttattttcaa gcattaacat 9480gaacttaaat tcatcaaggc
taatctctat atttgccttg tgagttttct tttgtgttag 9540ttcttttaat aaccactcat
aaatcctcat agagtatttg ttttcaaaag acttaacatg 9600ttccagatta tattttatga
atttttttaa ctggaaaaga taaggcaata tctcttcact 9660aaaaactaat tctaattttt
cgcttgagaa cttggcatag tttgtccact ggaaaatctc 9720aaagccttta accaaaggat
tcctgatttc cacagttctc gtcatcagct ctctggttgc 9780tttagctaat acaccataag
cattttccct actgatgttc atcatctgag cgtattggtt 9840ataagtgaac gataccgtcc
gttctttcct tgtagggttt tcaatcgtgg ggttgagtag 9900tgccacacag cataaaatta
gcttggtttc atgctccgtt aagtcatagc gactaatcgc 9960tagttcattt gctttgaaaa
caactaattc agacatacat ctcaattggt ctaggtgatt 10020ttaat
100256311469DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 63cactatacca attgagatgg gctagtcaat gataattact agtccttttc
ctttgagttg 60tgggtatctg taaattctgc tagacctttg ctggaaaact tgtaaattct
gctagaccct 120ctgtaaattc cgctagacct ttgtgtgttt tttttgttta tattcaagtg
gttataattt 180atagaataaa gaaagaataa aaaaagataa aaagaataga tcccagccct
gtgtataact 240cactacttta gtcagttccg cagtattaca aaaggatgtc gcaaacgctg
tttgctcctc 300tacaaaacag accttaaaac cctaaaggcg tcggcatccg cttacagaca
agctgtgacc 360gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
cgcgaggcag 420cagatcaatt cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc
atcgaatggt 480gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
agggtggtga 540atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
tatcagaccg 600tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
aaagtggaag 660cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
gcgggcaaac 720agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
tcgcaaattg 780tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
tcgatggtag 840aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
caacgcgtca 900gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
gaagctgcct 960gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
aacagtatta 1020ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
ttgggtcacc 1080agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
cgtctggctg 1140gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
gaaggcgact 1200ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
atcgttccca 1260ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
attaccgagt 1320ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
gaagacagct 1380catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
gggcaaacca 1440gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
cagctgttgc 1500ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
gcctctcccc 1560gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
gaaagcgggc 1620agtgagcgca acgcaattaa tgtaagttag cgcgaattga tctggtttga
cagcttatca 1680tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
tgtggtatgg 1740ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
ccgttctgga 1800taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
gagctgttga 1860caattaatca tccggctcgt ataatgtgtg gaattgtgag cggataacaa
tttcacacag 1920gaaacagcgc cgctgagaaa aagcgaagcg gcactgctct ttaacaattt
atcagacaat 1980ctgtgtgggc actcgaccgg aattatcgat taactttatt attaaaaatt
aaagaggtat 2040atattaatgt atcgattaaa taaggaggaa taaaccatga aacgtctcgg
aaccctggac 2100gcctcctggc tggcggttga atctgaagac accccgatgc atgtgggtac
gcttcagatt 2160ttctcactgc cggaaggcgc accagaaacc ttcctgcgtg acatggtcac
tcgaatgaaa 2220gaggccggcg atgtggcacc accctgggga tacaaactgg cctggtctgg
tttcctcggg 2280cgcgtgatcg ccccggcctg gaaagtcgat aaggatatcg atctggatta
tcacgtccgg 2340cactcagccc tgcctcgccc cggcggggag cgcgaactgg gtattctggt
atcccgactg 2400cactctaacc ccctggattt ttcccgccct ctttgggaat gccacgttat
tgaaggcctg 2460gagaataacc gttttgccct ttacaccaaa atgcaccact cgatgattga
cggcatcagc 2520ggcgtgcgac tgatgcagag ggtgctcacc accgatcccg aacgctgcaa
tatgccaccg 2580ccctggacgg tacgcccaca ccaacgccgt ggtgcaaaaa ccgacaaaga
ggccagcgtg 2640cccgcagcgg tttcccaggc aatggacgcc ctgaagctcc aggcagacat
ggcccccagg 2700ctgtggcagg ccggcaatcg cctggtgcat tcggttcgac acccggaaga
cggactgacc 2760gcgcccttca ctggaccggt ttcggtgctc aatcaccggg ttaccgcgca
gcgacgtttt 2820gccacccagc attatcaact ggaccggctg aaaaacctgg cccatgcttc
cggcggttcc 2880ttgaacgaca tcgtgcttta cctgtgtggc accgcattgc ggcgctttct
ggctgagcag 2940aacaatctgc cagacacccc gctgacggct ggtataccgg tgaatatccg
gccggcagac 3000gacgagggta cgggcaccca gatcagtttt atgattgcct cgctggccac
cgacgaagct 3060gatccgttga accgcctgca acagatcaaa acctcgaccc gacgggccaa
ggagcacctg 3120cagaaacttc caaaaagtgc cctgacccag tacaccatgc tgctgatgtc
accctacatt 3180ctgcaattga tgtcaggtct cggggggagg atgcgaccag tcttcaacgt
gaccatttcc 3240aacgtgcccg gcccggaagg cacgctgtat tatgaaggag cccggcttga
ggccatgtat 3300ccggtatcgc taatcgctca cggcggcgcc ctgaacatca cctgcctgag
ctatgccgga 3360tcgctgaatt tcggttttac cggctgtcgg gatacgctgc cgagcatgca
gaaactggcg 3420gtttataccg gtgaagctct ggatgagctg gaatcgctga ttctgccacc
caagaagcgc 3480gcccgaaccc gcaagtaact cgaggaggaa aactaaatga ccatttcctc
acctttgatt 3540gacgtcgcca accttccaga catcaacacc actgccggca agatcgccga
ccttaaggct 3600cgccgcgcgg aagcccattt ccccatgggt gaaaaggcag tagagaaggt
ccacgctgct 3660ggacgcctca ctgcccgtga gcgcttggat tacttactcg atgagggctc
cttcatcgag 3720accgatcagc tggctcgcca ccgcaccacc gctttcggcc tgggcgctaa
gcgtcctgca 3780accgacggca tcgtgaccgg ctggggcacc attgatggac gcgaagtctg
catcttctcg 3840caggacggca ccgtattcgg tggcgcgctt ggtgaggtgt acggcgaaaa
gatgatcaag 3900atcatggagc tggcaatcga caccggccgc ccattgatcg gtctttacga
aggcgctggc 3960gctcgtattc aggacggcgc tgtctccctg gacttcattt cccagacctt
ctaccaaaac 4020attcaggctt ctggcgttat cccacagatc tccgtcatca tgggcgcatg
tgcaggtggc 4080aacgcttacg gcccagctct gaccgacttc gtggtcatgg tggacaagac
ctccaagatg 4140ttcgttaccg gcccagacgt gatcaagacc gtcaccggcg aggaaatcac
ccaggaagag 4200cttggcggag caaccaccca catggtgacc gctggtaact cccactacac
cgctgcgacc 4260gatgaggaag cactggattg ggtacaggac ctggtgtcct tcctcccatc
caacaatcgc 4320tcctacgcac cgatggaaga cttcgacgag gaagaaggcg gcgttgaaga
aaacatcacc 4380gctgacgatc tgaagctcga cgagatcatc ccagattccg cgaccgttcc
ttacgacgtc 4440cgcgatgtca tcgaatgcct caccgacgat ggcgaatacc tggaaatcca
ggcagaccgc 4500gcagaaaacg ttgttattgc attcggccgc atcgaaggcc agtccgttgg
ctttgttgcc 4560aaccagccaa cccagttcgc tggctgcctg gacatcgact cctctgagaa
ggcagctcgc 4620ttcgtccgca cctgcgacgc gttcaacatc ccaatcgtca tgcttgtcga
cgtccccggc 4680ttcctcccag gcgcaggcca ggagtacggt ggcattctgc gtcgtggcgc
aaagctgctc 4740tacgcatacg gcgaagcaac cgttccaaag atcaccgtca ccatgcgtaa
ggcttacggc 4800ggagcgtact gcgtgatggg ttccaagggc ttgggctctg acatcaacct
tgcatggcca 4860accgcacaga tcgccgtcat gggcgctgct ggcgcagttg gattcatcta
ccgcaaggag 4920ctcatggcag ctgatgccaa gggcctcgat accgtagctc tggctaagtc
cttcgagcgc 4980gagtatgaag accacatgct caacccgtac cacgctgcag aacgtggcct
gatcgacgcc 5040gtgatcctgc caagcgaaac ccgcggacag atttcccgca accttcgcct
gctcaagcac 5100aagaacgtca ctcgccctgc tcgcaagcac ggcaacatgc cactgtaagg
aggaaaacta 5160aatgtcagtc gagactcgca agatcaccaa ggttcttgtc gctaaccgtg
gtgagattgc 5220aatccgcgtg ttccgtgcag ctcgagatga aggcatcgga tctgtcgccg
tctacgcaga 5280gccagatgca gatgcaccat tcgtgtcata tgcagacgag gcttttgccc
tcggtggcca 5340aacatccgct gagtcctacc ttgtcattga caagatcatc gatgcggccc
gcaagtccgg 5400cgccgacgcc atccaccccg gctacggctt cctcgcagaa aacgctgact
tcgcagaagc 5460agtcatcaac gaaggcctga tctggattgg accttcacct gagtccatcc
gctccctcgg 5520cgacaaggtc accgctcgcc acatcgcaga taccgccaag gctccaatgg
ctcctggcac 5580caaggaacca gtaaaagacg cagcagaagt tgtggctttc gctgaagaat
tcggtctccc 5640aatcgccatc aaggcagctt tcggtggcgg cggacgtggc atgaaggttg
cctacaagat 5700ggaagaagtc gctgacctct tcgagtccgc aacccgtgaa gcaaccgcag
cgttcggccg 5760cggcgagtgc ttcgtggagc gctacctgga caaggcacgc cacgttgagg
ctcaggtcat 5820cgccgataag cacggcaacg ttgttgtcgc cggaacccgt gactgctccc
tgcagcgccg 5880tttccagaag ctcgtcgaag aagcaccagc accattcctc accgatgacc
agcgcgagcg 5940tctccactcc tccgcgaagg ctatctgtaa ggaagctggc tactacggtg
caggcaccgt 6000tgagtacctc gttggctccg acggcctgat ctccttcctc gaggtcaaca
cccgcctcca 6060ggtggaacac ccagtcaccg aagagaccac cggcatcgac ctggtccgcg
aaatgttccg 6120catcgcagaa ggccacgagc tctccatcaa ggaagatcca gctccacgcg
gccacgcatt 6180cgagttccgc atcaacggcg aagacgctgg ctccaacttc atgcctgcac
caggcaagat 6240caccagctac cgcgagccac agggcccagg cgtccgcatg gactccggtg
tcgttgaagg 6300ttccgaaatc tccggacagt tcgactccat gctggcaaag ctgatcgttt
ggggcgacac 6360ccgcgagcag gctctccagc gctcccgccg tgcacttgca gagtacgttg
tcgagggcat 6420gccaaccgtt atcccattcc accagcacat cgtggaaaac ccagcattcg
tgggcaacga 6480cgaaggcttc gagatctaca ccaagtggat cgaagaggtt tgggataacc
caatcgcacc 6540ttacgttgac gcttccgagc tcgacgaaga tgaggacaag accccagcac
agaaggttgt 6600tgtggagatc aacggccgtc gcgttgaggt tgcactccca ggcgatctgg
cactcggtgg 6660caccgctggt cctaagaaga aggccaagaa gcgtcgcgca ggtggtgcaa
aggctggcgt 6720atccggcgat gcagtggcag ctccaatgca gggcactgtc atcaaggtca
acgtcgaaga 6780aggcgctgaa gtcaacgaag gcgacaccgt tgttgtcctc gaggctatga
agatggaaaa 6840ccctgtgaag gctcataagt ccggaaccgt aaccggcctt actgtcgctg
caggcgaggg 6900tgtcaacaag ggcgttgttc tcctcgagat caagtaatct agaggaggaa
aactaaatga 6960atgttgacat tagccgctct cgtgaaccgt tgaacgtgga actgttgaaa
gaaaaactgc 7020tgcagaacgg tgatttcggt caagtgatct acgagaaggt caccggctct
accaatgcgg 7080acctgctggc tctggcgggc agcggcgctc caaactggac cgtcaagact
gttgaatttc 7140aggaccacgc ccgtggccgt ctgggtcgtc cgtggagcgc accggagggt
tcccaaacca 7200tcgtcagcgt tctggtccaa ctgagcattg atcaggtgga ccgtattggt
acgatcccgc 7260tggccgcagg cttggctgtt atggatgcgc tgaatgatct gggcgtggag
ggtgcaggcc 7320tgaaatggcc gaacgatgtt cagatccacg gtaagaagtt gtgcggtatt
ctggttgaag 7380caaccggctt cgactccact ccgaccgtgg ttatcggttg gggtacgaat
atctcgttga 7440cgaaagaaga gctgccggtc ccgcacgcga ccagcctggc cctggagggt
gttgaagttg 7500accgtacgac gttcctgatt aacatgctga cccatctgca tacccgtctg
gatcagtggc 7560agggtccgtc tgtggactgg ctggatgact atcgcgcggt ttgtagcagc
attggccaag 7620atgtgcgtgt cctgctgcct ggtgacaaag agctgctggg cgaggcgatt
ggcgtggcga 7680ccggtggtga gatccgtgtg cgcgacgcca gcggcacggt ccacacgctg
aatgcgggtg 7740aaatcacgca tctgcgtttg caataagttt aaacggtctc cagcttggct
gttttggcgg 7800atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
gtctgataaa 7860acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
cgaactcaga 7920agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
tagggaactg 7980ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
tttatctgtt 8040gtttgtcggt gaacgctctc ctgacgcctg atgcggtatt ttctccttac
gcatctgtgc 8100ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
cgcatagtta 8160agccagcccc gacacccgcc aacacccgct gacgagctta gtaaagccct
cgctagattt 8220taatgcggat gttgcgatta cttcgccaac tattgcgata acaagaaaaa
gccagccttt 8280catgatatat ctcccaattt gtgtagggct tattatgcac gcttaaaaat
aataaaagca 8340gacttgacct gatagtttgg ctgtgagcaa ttatgtgctt agtgcatcta
acgcttgagt 8400taagccgcgc cgcgaagcgg cgtcggcttg aacgaattgt tagacattat
ttgccgacta 8460ccttggtgat ctcgcctttc acgtagtgga caaattcttc caactgatct
gcgcgcgagg 8520ccaagcgatc ttcttcttgt ccaagataag cctgtctagc ttcaagtatg
acgggctgat 8580actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc
gcgattttgc 8640cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc
tcatcgccag 8700cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca
aatagatcct 8760gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca
acgctatgtt 8820ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc
tcgaagatac 8880ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta
gctggataac 8940gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg
agaatctcgc 9000tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc
cgcgttgttt 9060catcaagcct tacggtcacc gtaaccagca aatcaatatc actgtgtggc
ttcaggccgc 9120catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga
tggcgctcga 9180tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct
tccctcatga 9240tgtttaactt tgttttaggg cgactgccct gctgcgtaac atcgttgctg
ctccataaca 9300tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc
atagactgta 9360ccccaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta
ccaccgctgc 9420gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat
tacagcttac 9480gaaccgaaca ggcttatgtc cactgggttc gtgccttcat ccgtttccac
ggtgtgcgtc 9540acccggcaac cttgggcagc agcgaagtcg aggcatttct gtcctggctg
gcgaacgagc 9600gcaaggtttc ggtctccacg catcgtcagg cattggcggc cttgctgttc
ttctacggca 9660aggtgctgtg cacggatctg ccctggcttc aggagatcgg aagacctcgg
ccgtcgcggc 9720gcttgccggt ggtgctgacc ccggatgaag tggttcgcat cctcggtttt
ctggaaggcg 9780agcatcgttt gttcgcccag cttctgtatg gaacgggcat gcggatcagt
gagggtttgc 9840aactgcgggt caaggatctg gatttcgatc acggcacgat catcgtgcgg
gagggcaagg 9900gctccaagga tcgggccttg atgttacccg agagcttggc acccagcctg
cgcgagcagg 9960ggaattaatt cccacgggtt ttgctgcccg caaacgggct gttctggtgt
tgctagtttg 10020ttatcagaat cgcagatccg gcttcagccg gtttgccggc tgaaagcgct
atttcttcca 10080gaattgccat gattttttcc ccacgggagg cgtcactggc tcccgtgttg
tcggcagctt 10140tgattcgata agcagcatcg cctgtttcag gctgtctatg tgtgactgtt
gagctgtaac 10200aagttgtctc aggtgttcaa tttcatgttc tagttgcttt gttttactgg
tttcacctgt 10260tctattaggt gttacatgct gttcatctgt tacattgtcg atctgttcat
ggtgaacagc 10320tttgaatgca ccaaaaactc gtaaaagctc tgatgtatct atctttttta
caccgttttc 10380atctgtgcat atggacagtt ttccctttga tatgtaacgg tgaacagttg
ttctactttt 10440gtttgttagt cttgatgctt cactgataga tacaagagcc ataagaacct
cagatccttc 10500cgtatttagc cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc
catgagaacg 10560aaccattgag atcatactta ctttgcatgt cactcaaaaa ttttgcctca
aaactggtga 10620gctgaatttt tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta
tgtaggtagg 10680aatctgatgt aatggttgtt ggtattttgt caccattcat ttttatctgg
ttgttctcaa 10740gttcggttac gagatccatt tgtctatcta gttcaacttg gaaaatcaac
gtatcagtcg 10800ggcggcctcg cttatcaacc accaatttca tattgctgta agtgtttaaa
tctttactta 10860ttggtttcaa aacccattgg ttaagccttt taaactcatg gtagttattt
tcaagcatta 10920acatgaactt aaattcatca aggctaatct ctatatttgc cttgtgagtt
ttcttttgtg 10980ttagttcttt taataaccac tcataaatcc tcatagagta tttgttttca
aaagacttaa 11040catgttccag attatatttt atgaattttt ttaactggaa aagataaggc
aatatctctt 11100cactaaaaac taattctaat ttttcgcttg agaacttggc atagtttgtc
cactggaaaa 11160tctcaaagcc tttaaccaaa ggattcctga tttccacagt tctcgtcatc
agctctctgg 11220ttgctttagc taatacacca taagcatttt ccctactgat gttcatcatc
tgagcgtatt 11280ggttataagt gaacgatacc gtccgttctt tccttgtagg gttttcaatc
gtggggttga 11340gtagtgccac acagcataaa attagcttgg tttcatgctc cgttaagtca
tagcgactaa 11400tcgctagttc atttgctttg aaaacaacta attcagacat acatctcaat
tggtctaggt 11460gattttaat
11469641173PRTMycobacterium smegmatis 64Met Thr Ser Asp Val His
Asp Ala Thr Asp Gly Val Thr Glu Thr Ala1 5
10 15Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu
Leu Tyr Ala Thr 20 25 30Asp
Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala 35
40 45Ala His Lys Pro Gly Leu Arg Leu Ala
Glu Ile Leu Gln Thr Leu Phe 50 55
60Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu65
70 75 80Ala Thr Asp Glu Gly
Gly Arg Thr Val Thr Arg Leu Leu Pro Arg Phe 85
90 95Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg
Val Gln Ala Val Ala 100 105
110Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp Ala
115 120 125Val Ala Thr Ile Gly Phe Ala
Ser Pro Asp Tyr Leu Thr Leu Asp Leu 130 135
140Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn
Ala145 150 155 160Pro Val
Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg Ile
165 170 175Leu Thr Val Ser Ala Glu Tyr
Leu Asp Leu Ala Val Glu Ser Val Arg 180 185
190Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His
Pro Glu 195 200 205Val Asp Asp His
Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu Ala 210
215 220Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile
Ala Asp Glu Gly225 230 235
240Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg
245 250 255Leu Ala Met Ile Leu
Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly 260
265 270Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp
Thr Met Ser Phe 275 280 285Ile Thr
Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro Leu 290
295 300Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr
Ala Val Gln Asn Gly305 310 315
320Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe Glu
325 330 335Asp Leu Ala Leu
Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg Val 340
345 350Ala Asp Met Leu Tyr Gln His His Leu Ala Thr
Val Asp Arg Leu Val 355 360 365Thr
Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu 370
375 380Leu Arg Glu Gln Val Leu Gly Gly Arg Val
Ile Thr Gly Phe Val Ser385 390 395
400Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr
Leu 405 410 415Gly Ala His
Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala Val 420
425 430Thr Arg Asp Gly Val Ile Val Arg Pro Pro
Val Ile Asp Tyr Lys Leu 435 440
445Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr Pro 450
455 460Arg Gly Glu Leu Leu Val Arg Ser
Gln Thr Leu Thr Pro Gly Tyr Tyr465 470
475 480Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg
Asp Gly Tyr Tyr 485 490
495His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr
500 505 510Val Asp Arg Arg Asn Asn
Val Leu Lys Leu Ala Gln Gly Glu Phe Val 515 520
525Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala Ala Leu
Val Arg 530 535 540Gln Ile Phe Val Tyr
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val545 550
555 560Val Val Pro Thr Pro Glu Ala Leu Glu Gln
Tyr Asp Pro Ala Ala Leu 565 570
575Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala Glu
580 585 590Leu Gln Ser Tyr Glu
Val Pro Ala Asp Phe Ile Val Glu Thr Glu Pro 595
600 605Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly
Lys Leu Leu Arg 610 615 620Pro Asn Leu
Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala625
630 635 640Asp Ile Ala Ala Thr Gln Ala
Asn Gln Leu Arg Glu Leu Arg Arg Ala 645
650 655Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln
Ala Ala Ala Thr 660 665 670Ile
Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr Asp 675
680 685Leu Gly Gly Asp Ser Leu Ser Ala Leu
Thr Leu Ser Asn Leu Leu Ser 690 695
700Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro Ala705
710 715 720Thr Asn Leu Ala
Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala 725
730 735Gly Asp Arg Arg Pro Ser Phe Thr Thr Val
His Gly Ala Asp Ala Thr 740 745
750Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu
755 760 765Thr Leu Arg Ala Ala Pro Gly
Leu Pro Lys Val Thr Thr Glu Pro Arg 770 775
780Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu
Thr785 790 795 800Leu Gln
Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile Thr
805 810 815Ile Val Arg Gly Arg Asp Asp
Ala Ala Ala Arg Ala Arg Leu Thr Gln 820 825
830Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu
Leu Ala 835 840 845Asp Arg His Leu
Arg Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu 850
855 860Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala
Glu Val Asp Leu865 870 875
880Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg Gln
885 890 895Leu Phe Gly Pro Asn
Val Val Gly Thr Ala Glu Val Ile Lys Leu Ala 900
905 910Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser
Thr Val Ser Val 915 920 925Ala Met
Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr Val 930
935 940Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala
Asn Gly Tyr Gly Asn945 950 955
960Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys
965 970 975Gly Leu Pro Val
Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro 980
985 990Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met
Phe Thr Arg Leu Leu 995 1000
1005Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile
1010 1015 1020Gly Asp Gly Glu Arg Pro
Arg Ala His Tyr Pro Gly Leu Thr Val 1025 1030
1035Asp Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln
Arg 1040 1045 1050Glu Gly Tyr Val Ser
Tyr Asp Val Met Asn Pro His Asp Asp Gly 1055 1060
1065Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala
Gly His 1070 1075 1080Pro Ile Asp Arg
Val Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe 1085
1090 1095Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys Arg
Arg Ala Gln Thr 1100 1105 1110Val Leu
Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro Leu 1115
1120 1125Arg Gly Ala Pro Glu Pro Thr Glu Val Phe
His Ala Ala Val Arg 1130 1135 1140Thr
Ala Lys Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu Ala 1145
1150 1155Leu Ile Asp Lys Tyr Ile Arg Asp Leu
Arg Glu Phe Gly Leu Ile 1160 1165
1170
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