PUFA Polyketide Synthase Systems and Uses Thereof

Abstract

Disclosed are the complete polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from Schizochytrium, and biologically active fragments and homologues thereof. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to proteins and domains thereof that comprise such PUFA PKS systems, to genetically modified organisms (plants and microorganisms) comprising such PUFA PKS systems, and to methods of making and using the PUFA PKS systems disclosed herein. This invention also relates to genetically modified plants and microorganisms and methods to efficiently produce lipids enriched in various polyunsaturated fatty acids (PUFAs) as well as other bioactive molecules by manipulation of a PUFA polyketide synthase (PKS) system.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No. 11/452,138, filed Jun. 12, 2006, which claims the benefit of priority under 35 U.S.C. .sctn.119(e) from U.S. Provisional Application No. 60/784,616, filed Mar. 21, 2006, and from U.S. Provisional Application No. 60/689,167, filed Jun. 10, 2005. U.S. application Ser. No. 11/452,138 is also a continuation-in-part of U.S. patent application Ser. No. 10/124,800, filed Apr. 16, 2002, which claims priority under 35 U.S.C. .sctn.119(e) from: U.S. Provisional Application Ser. No. 60/284,066, filed Apr. 16, 2001; U.S. Provisional Application Ser. No. 60/298,796, filed Jun. 15, 2001; and U.S. Provisional Application Ser. No. 60/323,269, filed Sep. 18, 2001. U.S. patent application Ser. No. 10/124,800 is also a continuation-in-part of U.S. application Ser. No. 09/231,899, filed Jan. 14, 1999, now U.S. Pat. No. 6,566,583. Each of the above-identified patent applications is incorporated herein by reference in its entirety for all purposes.

[0002] U.S. application Ser. No. 11/452,138 does not claim the benefit of priority from U.S. application Ser. No. 09/090,793, filed Jun. 4, 1998, now U.S. Pat. No. 6,140,486, although U.S. application Ser. No. 09/090,793 is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing submitted on a compact disc, in duplicate. Each of the two compact discs, which are identical to each other pursuant to 37 CFR .sctn.1.52(e)(4), contains the following file: "Sequence Listing", having a size in bytes of 301 KB, recorded on 12 Jun. 2006. The information contained on the compact disc is hereby incorporated by reference in its entirety pursuant to 37 CFR .sctn.1.77(b)(4).

FIELD OF THE INVENTION

[0004] This invention relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from Schizochytrium. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to such PUFA PKS systems, to genetically modified organisms comprising such PUFA PKS systems, and to methods of making and using such PUFA PKS systems disclosed herein. This invention also relates to PUFA PKS systems from non-bacterial and bacterial organisms identified using the Schizochytrium PUFA PKS systems described herein.

BACKGROUND OF THE INVENTION

[0005] Polyketide synthase (PKS) systems are generally known in the art as enzyme complexes related to fatty acid synthase (FAS) systems, but which are often highly modified to produce specialized products that typically show little resemblance to fatty acids. It has now been shown, however, that polyketide synthase systems exist in marine bacteria and certain eukaryotic organisms that are capable of synthesizing polyunsaturated fatty acids (PUFAs) from acetyl-CoA and malonyl-CoA. The PKS pathways for PUFA synthesis in Shewanella and another marine bacteria, Vibrio marinus, are described in detail in U.S. Pat. No. 6,140,486. The PKS pathways for PUFA synthesis in the eukaryotic Thraustochytrid, Schizochytrium is described in detail in U.S. Pat. No. 6,566,583. The PKS pathways for PUFA synthesis in eukaryotes such as members of Thraustochytriales, including the structural description of a PUFA PKS system in Schizochytrium and the identification of a PUFA PKS system in Thraustochytrium, including details regarding uses of these systems, are described in detail in U.S. Patent Application Publication No. 20020194641, published Dec. 19, 2002 (corresponding to U.S. patent application Ser. No. 10/124,800, filed Apr. 16, 2002). U.S. Patent Application Publication No. 20040235127, published Nov. 25, 2004 (corresponding to U.S. patent application Ser. No. 10/810,352, filed Mar. 24, 2004), discloses the structural description of a PUFA PKS system in Thraustochytrium, and further detail regarding the production of eicosapentaenoic acid (C20:5, .omega.-3) (EPA) and other PUFAs using such systems. U.S. Patent Application Publication No. 20050100995, published May 12, 2005 (corresponding to U.S. patent application Ser. No. 10/965,017, filed Oct. 13, 2004), discloses the structural and functional description of PUFA PKS systems in Shewanella olleyana and Shewanella japonica, and uses of such systems. These applications also disclose the genetic modification of organisms, including microorganisms and plants, with the genes comprising the PUFA PKS pathway and the production of PUFAs by such organisms. Furthermore, PCT Patent Publication No. WO 05/097982 describes a PUFA PKS system in Ulkenia, U.S. Patent Application Publication No. 20050014231 describes PUFA PKS genes and proteins from Thraustochytrium aureum.

[0006] Researchers have attempted to exploit polyketide synthase (PKS) systems that have been traditionally described in the literature as falling into one of three basic types, typically referred to as: Type I (modular or iterative), Type II, and Type III. For purposes of clarity, it is noted that the Type I modular PKS system has previously also been referred to as simply a "modular" PKS system, and the Type I iterative PKS system has previously also been referred to simply as a "Type I" PKS system. The Type II system is characterized by separable proteins, each of which carries out a distinct enzymatic reaction. The enzymes work in concert to produce the end product and each individual enzyme of the system typically participates several times in the production of the end product. This type of system operates in a manner analogous to the fatty acid synthase (FAS) systems found in plants and bacteria. Type I iterative PKS systems are similar to the Type II system in that the enzymes are used in an iterative fashion to produce the end product. The Type I iterative differs from Type II in that enzymatic activities, instead of being associated with separable proteins, occur as domains of larger proteins. This system is analogous to the Type I FAS systems found in animals and fungi.

[0007] In contrast to the Type II systems, in Type I modular PKS systems, each enzyme domain is used only once in the production of the end product. The domains are found in very large proteins and the product of each reaction is passed on to another domain in the PKS protein. Additionally, in the PKS systems described above, if a carbon-carbon double bond is incorporated into the end product, it is usually in the trans configuration.

[0008] Type III systems have been more recently discovered and belong to the plant chalcone synthase family of condensing enzymes. Type III PKSs are distinct from type I and type II PKS systems and utilize free CoA substrates in iterative condensation reactions to usually produce a heterocyclic end product.

[0009] Polyunsaturated fatty acids (PUFAs) are considered to be useful for nutritional, pharmaceutical, industrial, and other purposes. The current supply of PUFAs from natural sources and from chemical synthesis is not sufficient for commercial needs. A major current source for PUFAs is from marine fish; however, fish stocks are declining, and this may not be a sustainable resource. Additionally, contamination, from both heavy metals and toxic organic molecules, is a serious issue with oil derived from marine fish. Vegetable oils derived from oil seed crops are relatively inexpensive and do not have the contamination issues associated with fish oils. However, the PUFAs found in commercially developed plant oils are typically limited to linoleic acid (eighteen carbons with 2 double bonds, in the delta 9 and 12 positions--18:2 delta 9, 12) and linolenic acid (18:3 delta 9, 12, 15). In the conventional pathway (i.e., the "standard" pathway or "classical" pathway) for PUFA synthesis, medium chain-length saturated fatty acids (products of a fatty acid synthase (FAS) system) are modified by a series of elongation and desaturation reactions. The substrates for the elongation reaction are fatty acyl-CoA (the fatty acid chain to be elongated) and malonyl-CoA (the source of the 2 carbons added during each elongation reaction). The product of the elongase reaction is a fatty acyl-CoA that has two additional carbons in the linear chain. The desaturases create cis double bonds in the preexisting fatty acid chain by extraction of 2 hydrogens in an oxygen-dependant reaction. The substrates for the desaturases are either acyl-CoA (in some animals) or the fatty acid that is esterified to the glycerol backbone of a phospholipid (e.g. phosphatidylcholine).

[0010] Therefore, because a number of separate desaturase and elongase enzymes are required for fatty acid synthesis from linoleic and linolenic acids to produce the more unsaturated and longer chain PUFAs, engineering plant host cells for the expression of PUFAs such as EPA and docosahexaenoic acid (DHA) may require expression of several separate enzymes to achieve synthesis. Additionally, for production of useable quantities of such PUFAs, additional engineering efforts may be required. Therefore, it is of interest to obtain genetic material involved in PUPA biosynthesis from species that naturally produce these fatty acids (e.g., from a PUFA PKS system) and to express the isolated material alone or in combination in a heterologous system which can be manipulated to allow production of commercial quantities of PUFAs.

[0011] There have been many efforts to produce PUFAs in oil-seed crop plants by modification of the endogenously-produced fatty acids. Genetic modification of these plants with various individual genes for fatty acid elongases and desaturases has produced leaves or seeds containing measurable levels of PUFAs such as EPA, but also containing significant levels of mixed shorter-chain and less unsaturated PUFAs (Qi et al., Nature Biotech. 22:739 (2004); PCT Publication No. WO 04/071467; Abbadi et al., Plant Cell 16:1 (2004)); Napier and Sayanova, Proceedings of the Nutrition Society (2005), 64:387-393; Robert et al., Functional Plant Biology (2005) 32:473-479; or U.S. Patent Application Publication 2004/0172682.

[0012] Improvement in both microbial and plant production of PUFAs is a highly desirable commercial goal. Therefore, there remains a need in the art for a method to efficiently and effectively produce quantities of lipids (e.g., triacylglycerol (TAG) and phospholipid (PL)) enriched in desired PUFAs, particularly in commercially useful organisms such as microorganisms and oil-seed plants.

SUMMARY OF THE INVENTION

[0013] One embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a nucleic acid sequence selected from: SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5; (b) a nucleic acid sequence encoding an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; (c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; (d) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and (e) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.

[0014] In one aspect, the nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; (b) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and (c) a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.

[0015] In one aspect, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6. In another aspect, the nucleic acid molecule comprises a nucleic acid sequence selected from: SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.

[0016] In one aspect of this embodiment, the nucleic acid molecule of (a) comprises a nucleic acid sequence encoding the amino acid sequence encoded by a plasmid selected from: pKJ1126 (ATCC Accession No. ______), pJK306 (ATCC Accession No. ______), and pJK320 (ATCC Accession No. ______). In one aspect of this embodiment, the nucleic acid molecule of (b) comprises a nucleic acid sequence encoding the amino acid sequence encoded by a plasmid selected from: pJK1129 (ATCC Accession No. ______), and pJK324 (ATCC Accession No. ______). In another aspect of this embodiment, the nucleic acid molecule of (c) comprises a nucleic acid sequence encoding the amino acid sequence encoded by a plasmid selected from: pJK1131 (ATCC Accession No. ______) and pBR002 (ATCC Accession No. ______).

[0017] Another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a first nucleic acid sequence encoding a first amino acid sequence that has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:2, and wherein said first amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; (b) a first nucleic acid sequence encoding a first amino acid sequence that has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:4, and wherein said first amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and (c) a first nucleic acid sequence encoding a first amino acid sequence that has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:6, and wherein said first amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6. In one aspect of this embodiment, the first nucleic acid sequence is isolated from a Schizochytrium, such as, but not limited to, Schizochytrium ATCC 20888.

[0018] Yet another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a nucleic acid sequence of SEQ ID NO:9; (b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:10; and (c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the amino acid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 93 of SEQ ID NO:10 and a histidine at a position corresponding to amino acid 94 of SEQ ID NO:10. In one aspect of this embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the amino acid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 93 of SEQ ID NO:10 and a histidine at a position corresponding to amino acid 94 of SEQ ID NO:10. In one aspect, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:10.

[0019] Another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a nucleic acid sequence of SEQ ID NO:19; (b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:20; and (c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:20 or that is a fragment of SEQ ID NO:20, wherein the amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:20. In one aspect of this embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:20 or that is a fragment of SEQ ID NO:20, wherein the amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:20. In one aspect, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:20.

[0020] Yet another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from: (a) a nucleic acid sequence of SEQ ID NO:29; (b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30; and (c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 426-440 of SEQ ID NO:30. In one aspect, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 426-440 of SEQ ID NO:30. In one aspect, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30.

[0021] Another embodiment of the invention relates to a recombinant nucleic acid molecule comprising any of the nucleic acid molecules described above, operatively linked to at least one transcription control sequence.

[0022] Yet another embodiment of the invention relates to a recombinant cell transfected with any of the nucleic acid molecules described above. In one aspect, the recombinant cell is a microorganism. In another aspect, the recombinant cell is a plant cell.

[0023] Another embodiment of the present invention relates to an isolated nucleic acid molecule consisting essentially of a nucleic acid sequence that is fully complementary to any of the nucleic acid molecules described above.

[0024] Another embodiment of the present invention relates to a genetically modified microorganism that has been transformed with any of the nucleic acid molecules described above.

[0025] Yet another embodiment of the present invention relates to a genetically modified plant that has been transformed with any of the nucleic acid molecules described above.

[0026] Another embodiment of the present invention relates to a genetically modified microorganism that has been transformed with: (a) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; (b) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and (c) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6. In one aspect, the microorganism has been transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding SEQ ID NO:2, a nucleic acid molecule comprising a nucleic acid sequence encoding SEQ ID NO:4, and a nucleic acid molecule comprising a nucleic acid sequence encoding SEQ ID NO:6. In one aspect, the microorganism endogenously expresses a PUFA PKS system. In another aspect, the microorganism has been further transformed with a recombinant nucleic acid molecule encoding a phosphopantetheine transferase. The microorganism can include, but is not limited to, a Thraustochytriales microorganism, a bacterium or a yeast.

[0027] Yet another embodiment of the present invention relates to a method to produce a bioactive molecule, comprising culturing under conditions effective to produce said bioactive molecule a genetically modified organism described above. In one aspect, the bioactive molecule is a polyunsaturated fatty acid (PUFA).

[0028] Another embodiment of the present invention relates to a genetically modified plant or part of the plant, wherein said plant has been transformed with: (a) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; (b) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and (c) a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6. In one aspect, the plant has been further genetically modified to express a recombinant nucleic acid molecule encoding a phosphopantetheine transferase. In one aspect, the plant is a dicotyledonous plant, and in another aspect, the plant is a monocotyledonous plant. In another aspect, the plant is selected from: canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco.

[0029] In one aspect, the plant is an oilseed plant and the part of the plant is a mature oilseed. In one aspect, the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one PUFA selected from DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), and wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than about 10% of the total fatty acids produced by said plant. In one aspect, the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than 5% by weight of the total fatty acids produced by said plant. In another aspect, the fatty acids consisting of gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds, comprise less than 5% by weight of the total fatty acids produced by said plant. In another aspect, gamma-linolenic acid (GLA; 18:3, n-6) comprises less than 1% by weight of the total fatty acids produced by said plant.

[0030] Yet another embodiment of the present invention relates to a plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises detectable amounts of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), wherein the ratio of DPAn-6 to DHA is 1:1 or greater than 1:1.

[0031] Another embodiment of the present invention relates to a plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises detectable amounts of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), wherein the ratio of DPAn-6 to DHA is less than 1:1. In either of the two embodiments above, in one aspect, the total fatty acid profile in the plant or part of the plant contains less than 5% by weight in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0032] Yet another embodiment of the present invention relates to plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUPA) selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0033] Another embodiment of the present invention relates to a plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 1% of each of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0034] Another embodiment of the present invention relates to a plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 2% of gamma-linolenic acid (GLA; 18:3, n-6) and dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6).

[0035] Another embodiment of the present invention relates to seeds obtained from any of the plants or part of plants described above, a food product comprising such seeds, an oil obtained from such seeds, and a food product comprising such oil. Also included in the invention is an oil blend comprising such oil and another oil, such as, but not limited to, a microbial oil, a fish oil, and a vegetable oil.

[0036] Yet another embodiment of the present invention relates to an oil comprising the following fatty acids: DHA (C22:6n-3), DPAn-6 (C22:5n-6), oleic acid (C18:1), linolenic acid (C18:3), linoleic acid (C18:2), C16:0, C18.0, C20:0, C20:1n-9, C20:2n-6, C22:1n-9; wherein the oil comprises less than 0.5% of any of the following fatty acids: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0037] Another embodiment of the present invention relates to an oilseed plant that produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0038] Yet another embodiment of the present invention relates to an oilseed plant that produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid (PUFA) selected from DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 1% of gamma-linolenic acid (GLA; 18:3, n-6).

[0039] Another embodiment of the present invention relates to a method to produce a bioactive molecule, comprising growing under conditions effective to produce said bioactive molecule a genetically modified plant as described above. In one aspect, the bioactive molecule is a polyunsaturated fatty acid (PUFA).

[0040] Yet another embodiment of the present invention relates to a method to produce a plant that has a polyunsaturated fatty acid (PUFA) profile that differs from the naturally occurring plant, comprising genetically modifying said plant to express a PUFA PKS system comprising at least one of any of the nucleic acid molecules as described above.

[0041] Another embodiment of the present invention relates to a method to produce a recombinant microbe, comprising genetically modifying microbial cells to express at least one of any of the nucleic acid molecules as described above.

BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1 is a graphical representation of the domain structure of the Schizochytrium PUFA PKS system.

[0043] FIG. 2 shows a comparison of PKS domains from Schizochytrium and Shewanella.

[0044] FIG. 3 shows a GC FAME profile of control yeast and yeast expressing Orfs sA, sB, C and Het I.

[0045] FIG. 4 shows a GC FAME profile of the PUFA region from FIG. 3.

[0046] FIG. 5 shows GC FAME profiles of wild-type Arabidopsis and Arabidopsis Line 269 (plastid targeted).

[0047] FIG. 6 is a schematic diagram showing the construction of pSBS4107: Acyl-ACP transit peptide-HetI: Acyl-ACP transit peptide-ORFC.

[0048] FIG. 7 is a schematic diagram showing the construction of pSBS5720: Acyl-ACP transit peptide-ORFB.

[0049] FIG. 8 is a schematic diagram showing the construction of pSBS4757: Acyl-ACP transit peptide-ORFA.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention generally relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from Schizochytrium, to genetically modified organisms comprising Schizochytrium PUFA PKS systems, to methods of making and using such systems for the production of products of interest, including bioactive molecules, and to PUFA PKS systems identified using the structural information for the Schizochytrium PUFA PKS systems disclosed herein. In one preferred embodiment, the present invention relates to a method to produce PUFAs in an oil-seed plant that has been genetically modified to express a PUFA PKS system of the present invention. The oils produced by the plant contain at least one PUFA produced by the PUFA PKS system and are substantially free of the mixed shorter-chain and less unsaturated PUFAs that are fatty acid products produced by the modification of products of the FAS system.

[0051] As used herein, a PUFA PKS system (which may also be referred to as a PUFA synthase system or PUFA synthase) generally has the following identifying features: (1) it produces PUFAs, and particularly, long chain PUFAs, as a natural product of the system; and (2) it comprises several multifunctional proteins assembled into a complex that conducts both iterative processing of the fatty acid chain as well non-iterative processing, including trans-cis isomerization and enoyl reduction reactions in selected cycles. In addition, the ACP domains present in the PUFA synthase enzymes require activation by attachment of a cofactor (4-phosphopantetheine). Attachment of this cofactor is carried out by phosphopantetheinyl transferases (PPTase). If the endogenous PPTases of the host organism are incapable of activating the PUFA synthase ACP domains, then it is necessary to provide a PPTase that is capable of carrying out that function. The inventors have identified the Het I enzyme of Nostoc sp. as an exemplary and suitable PPTase for activating PUFA synthase ACP domains. Reference to a PUFA PKS system or a PUFA synthase refers collectively to all of the genes and their encoded products that work in a complex to produce PUFAs in an organism. Therefore, the PUFA PKS system refers specifically to a PKS system for which the natural products are PUFAs.

[0052] More specifically, first, a PUFA PKS system that forms the basis of this invention produces polyunsaturated fatty acids (PUFAs) and particularly, long chain PUFAs, as products (e.g., an organism that endogenously (naturally) contains such a PKS system makes PUFAs using this system). According to the present invention, PUFAs are fatty acids with a carbon chain length of at least 16 carbons, and more preferably at least 18 carbons, and more preferably at least 20 carbons, and more preferably 22 or more carbons, with at least 3 or more double bonds, and preferably 4 or more, and more preferably 5 or more, and even more preferably 6 or more double bonds, wherein all double bonds are in the cis configuration. Reference to long chain polyunsaturated fatty acids (LCPUFAs) herein more particularly refers to fatty acids of 18 and more carbon chain length, and preferably 20 and more carbon chain length, containing 3 or more double bonds. LCPUFAs of the omega-6 series include: gamma-linolenic acid (C18:3), di-homo-gammalinolenic acid (C20:3n-6), arachidonic acid (C20:4n-6), adrenic acid (also called docosatetraenoic acid or DTA) (C22:4n-6), and docosapentaenoic acid (C22:5n-6). The LCPUFAs of the omega-3 series include: alpha-linolenic acid (C18:3), eicosatrienoic acid (C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (C20:5n-3), docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid (C22:6n-3). The LCPUFAs also include fatty acids with greater than 22 carbons and 4 or more double bonds including but not limited to C28:8(n-3).

[0053] Second, a PUFA PKS system according to the present invention comprises several multifunctional proteins (and can include single function proteins, particularly for PUFA PKS systems from marine bacteria) that are assembled into a complex that conducts both iterative processing of the fatty acid chain as well non-iterative processing, including trans-cis isomerization and enoyl reduction reactions in selected cycles. These proteins can also be referred to herein as the core PUFA PKS enzyme complex or the core PUFA PKS system. The general functions of the domains and motifs contained within these proteins are individually known in the art and have been described in detail with regard to various PUFA PKS systems from marine bacteria and eukaryotic organisms (see, e.g., U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583; Metz et al., Science 293:290-293 (2001); U.S. Patent Application Publication No. 20020194641; U.S. Patent Application Publication No. 20040235127; and U.S. Patent Application Publication No. 20050100995). The domains may be found as a single protein (i.e., the domain and protein are synonymous) or as one of two or more (multiple) domains in a single protein, as mentioned above.

[0054] Before the discovery of a PUFA PKS system in marine bacteria (see U.S. Pat. No. 6,140,486), PKS systems were not known to possess this combination of iterative and selective enzymatic reactions, and they were not thought of as being able to produce carbon-carbon double bonds in the cis configuration. However, the PUFA PKS system described by the present invention has the capacity to introduce cis double bonds and the capacity to vary the reaction sequence in the cycle.

[0055] The present inventors propose to use these features of the PUFA PKS system to produce a range of bioactive molecules that could not be produced by the previously described (Type I iterative or modular, Type II, or Type III) PKS systems. These bioactive molecules include, but are not limited to, polyunsaturated fatty acids (PUFAs), antibiotics or other bioactive compounds, many of which will be discussed below. For example, using the knowledge of the PUFA PKS gene structures described herein, any of a number of methods can be used to alter the PUFA PKS genes, or combine portions of these genes with other synthesis systems, including other PKS systems, such that new products are produced. The inherent ability of this particular type of system to do both iterative and selective reactions will enable this system to yield products that would not be found if similar methods were applied to other types of PKS systems.

[0056] Preferably, a PUFA PKS system of the present invention comprises at least the following biologically active domains that are typically contained on three or more proteins: (a) at least one enoyl-ACP reductase (ER) domain; (b) multiple acyl carrier protein (ACP) domain(s) (e.g., at least from one to four, and preferably at least five ACP domains, and in some embodiments up to six, seven, eight, nine, or more than nine ACP domains); (c) at least two .beta.-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT) domain; (e) at least one .beta.-ketoacyl-ACP reductase (KR) domain; (f) at least two FabA-like .beta.-hydroxyacyl-ACP dehydrase (DH) domains; (g) at least one chain length factor (CLF) domain; (h) at least one malonyl-CoA:ACP acyltransferase (MAT) domain. In one embodiment, a PUFA PKS system according to the present invention also comprises at least one region containing a dehydratase (DH) conserved active site motif.

[0057] In a preferred embodiment, a Schizochytrium PUFA PKS system comprises at least the following biologically active domains: (a) two enoyl-ACP reductase (ER) domain; (b) nine acyl carrier protein (ACP) domains; (c) two .beta.-ketoacyl-ACP synthase (KS) domains; (d) one acyltransferase (AT) domain; (e) one .beta.-ketoacyl-ACP reductase (KR) domain; (f) two FabA-like .beta.-hydroxyacyl-ACP dehydrase (DH) domains; (g) one chain length factor (CLF) domain; and (h) one malonyl-CoA:ACP acyltransferase (MAT) domain. In one embodiment, a Schizochytrium PUFA PKS system according to the present invention also comprises at least one region or domain containing a dehydratase (DH) conserved active site motif that is not a part of a FabA-like DH domain. The structural and functional characteristics of these domains are generally individually known in the art and will be described in detail below with regard to the PUFA PKS systems of the present invention.

[0058] A PUFA PKS system can additionally include one or more accessory proteins, which are defined herein as proteins that are not considered to be part of the core PUFA PKS system as described above (i.e., not part of the PUFA synthase enzyme complex itself), but which may be, or are, necessary for PUFA production or at least for efficient PUFA production using the core PUFA synthase enzyme complex of the present invention, particularly in certain host organisms (e.g., plants). For example, in order to produce PUFAs, a PUFA PKS system must work with an accessory protein that transfers a 4'-phosphopantetheinyl moiety from Coenzyme A to the acyl carrier protein (ACP) domain(s). Therefore, a PUFA PKS system can be considered to include at least one 4'-phosphopantetheinyl transferase (PPTase) domain, or such a domain can be considered to be an accessory domain or protein to the PUFA PKS system. When genetically modifying organisms (e.g., microorganisms or plants) to express a PUFA PKS system according to the present invention, some host organisms may endogenously express accessory proteins that are needed to work with the PUFA PKS to produce PUFAs (e.g., PPTases). However, some organisms may be transformed with nucleic acid molecules encoding one or more accessory proteins described herein to enable and/or to enhance production of PUFAs by the organism, even if the organism endogenously produces a homologous accessory protein (i.e., some heterologous accessory proteins may operate more effectively or efficiently with the transformed PUFA synthase proteins than the host cells' endogenous accessory protein). The present invention provides an example of bacteria, yeast and plants that have been genetically modified with the PUFA PKS system of the present invention that includes an accessory PPTase. Structural and functional characteristics of PPTases will be described in more detail below.

[0059] According to the present invention, reference to a "standard" or "classical" pathway for the production of PUFAs refers to the fatty acid synthesis pathway where medium chain-length saturated fatty acids (products of a fatty acid synthase (FAS) system) are modified by a series of elongation and desaturation reactions. The substrates for the elongation reaction are fatty acyl-CoA (the fatty acid chain to be elongated) and malonyl-CoA (the source of the 2 carbons added during each elongation reaction). The product of the elongase reaction is a fatty acyl-CoA that has two additional carbons in the linear chain. The desaturases create cis double bonds in the preexisting fatty acid chain by extraction of 2 hydrogens in an oxygen-dependant reaction. Such pathways and the genes involved in such pathways are well-known in the literature.

[0060] As used herein, the term "lipid" includes phospholipids (PL); free fatty acids; esters of fatty acids; triacylglycerols (TAG); diacylglycerides; monoacylglycerides; phosphatides; waxes (esters of alcohols and fatty acids); sterols and sterol esters; carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art. The terms "polyunsaturated fatty acid" and "PUFA" include not only the free fatty acid form, but other forms as well, such as the TAG form and the PL form.

[0061] A PUFA PKS system described according to the present invention is a non-bacterial PUFA PKS system. In other words, the PUFA PKS system of the present invention is isolated from an organism that is not a bacteria, or is a homologue of or derived from a PUFA PKS system from an organism that is not a bacteria, such as a eukaryote or an archaebacterium. Eukaryotes are separated from prokaryotes based on the degree of differentiation of the cells, with eukaryotes being more differentiated than prokaryotes. In general, prokaryotes do not possess a nuclear membrane, do not exhibit mitosis during cell division, have only one chromosome, their cytoplasm contains 70S ribosomes, they do not possess any mitochondria, endoplasmic reticulum, chloroplasts, lysosomes or golgi apparatus, their flagella (if present) consists of a single fibril. In contrast, eukaryotes have a nuclear membrane, they do exhibit mitosis during cell division, they have many chromosomes, their cytoplasm contains 80S ribosomes, they do possess mitochondria, endoplasmic reticulum, chloroplasts (in algae), lysosomes and golgi apparatus, and their flagella (if present) consists of many fibrils. In general, bacteria are prokaryotes, while algae, fungi, protist, protozoa and higher plants are eukaryotes. The PUFA PKS systems of the marine bacteria (e.g., Shewanella and Vibrio marinus) are not the basis of the present invention, although the present invention does contemplate the use of domains from these bacterial PUFA PKS systems in conjunction with domains from the non-bacterial (e.g., Schizochytrium) PUFA PKS systems of the present invention. For example, according to the present invention, genetically modified organisms can be produced which incorporate non-bacterial PUFA PKS functional domains with bacterial PUFA PKS functional domains, as well as PKS functional domains or proteins from other PKS systems (Type I iterative or modular, Type II, or Type III) or FAS systems.

[0062] Schizochytrium is a Thraustochytrid marine microorganism that accumulates large quantities of triacylglycerols rich in DHA and docosapentaenoic acid (DPA; 22:5 .omega.-6); e.g., 30% DHA+DPA by dry weight (Barclay et al., J. Appl. Phycol. 6, 123 (1994)). In eukaryotes that synthesize 20- and 22-carbon PUFAs by an elongation/desaturation pathway, the pools of 18-, 20- and 22-carbon intermediates are relatively large so that in vivo labeling experiments using [.sup.14C]-acetate reveal clear precursor-product kinetics for the predicted intermediates (Gellerman et al., Biochim. Biophys. Acta 573:23 (1979)). Furthermore, radiolabeled intermediates provided exogenously to such organisms are converted to the final PUFA products. The present inventors have shown that [1-.sup.14C]-acetate was rapidly taken up by Schizochytrium cells and incorporated into fatty acids, but at the shortest labeling time (1 min), DHA contained 31% of the label recovered in fatty acids, and this percentage remained essentially unchanged during the 10-15 min of [.sup.14C]-acetate acetate incorporation and the subsequent 24 hours of culture growth (See U.S. Patent Application Publication No. 20020194641, supra). Similarly, DPA represented 10% of the label throughout the experiment. There is no evidence for a precursor-product relationship between 16- or 18-carbon fatty acids and the 22-carbon polyunsaturated fatty acids. These results are consistent with rapid synthesis of DHA from [.sup.14C]-acetate involving very small (possibly enzyme-bound) pools of intermediates. A cell-free homogenate derived from Schizochytrium cultures incorporated [1-.sup.14C]-malonyl-CoA into DHA, DPA, and saturated fatty acids. The same biosynthetic activities were retained by a 100,000.times.g supernatant fraction but were not present in the membrane pellet. Thus, DHA and DPA synthesis in Schizochytrium does not involve membrane-bound desaturases or fatty acid elongation enzymes like those described for other eukaryotes (Parker-Barnes et al., 2000, supra; Shanklin et al., 1998, supra). These fractionation data contrast with those obtained from the Shewanella enzymes (See Metz et al., 2001, supra) and may indicate use of a different (soluble) acyl acceptor molecule, such as CoA, by the Schizochytrium enzyme.

[0063] As described in U.S. Pat. No. 6,566,583, a cDNA library from Schizochytrium was constructed and approximately 8,000 random clones (ESTs) were sequenced. Within this dataset, only one moderately expressed gene (0.3% of all sequences) was identified as a fatty acid desaturase, although a second putative desaturase was represented by a single clone (0.01%). By contrast, sequences that exhibited homology to 8 of the 11 domains of the Shewanella PKS genes shown in FIG. 2 were all identified at frequencies of 0.2-0.5%. In U.S. Pat. No. 6,566,583, several cDNA clones showing homology to the Shewanella PKS genes were sequenced, and various clones were assembled into nucleic acid sequences representing two partial open reading frames and one complete open reading frame.

[0064] Nucleotides 390-4443 of the cDNA sequence containing the first partial open reading frame described in U.S. application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:69) match nucleotides 4677-8730 (plus the stop codon) of the sequence denoted herein as OrfA (SEQ ID NO:1). A cDNA clone described in U.S. application Ser. No. 09/231,899 as cDNA clone LIB3033-047-B5 comprises at least a portion of nucleotides 4677-8730 of SEQ ID NO:1 described herein, to the best of the present inventors' knowledge. Specifically, the sequence of the insert in cDNA clone LIB3033-047-B5 begins at nucleotide 6719 of SEQ ID NO:1 and extends to the end of the Orf (position 8730 of SEQ ID NO:1), plus about 71 nucleotides beyond the end of the Orf represented by SEQ ID NO:1. cDNA clone LIB3033-047-B5 (denoted cDNA clone LIB3033-047-B5 in the form of an E. coli plasmid vector containing "Orf6 homolog" partial gene sequence from Schizochytrium sp.) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on June 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of cDNA clone LIB3033-047-B5, and the amino acid sequence encoded by this cDNA clone are encompassed by the present invention. A second cDNA clone described in U.S. application Ser. No. 09/231,899 as cDNA clone LIB3033-046-E6 shared homology to the ACP domain of ORF6, contained 6 ACP repeats, and comprises at least a portion of nucleotides of SEQ ID NO:1 of the present invention. This cDNA clone did not have a poly-A-tail, and therefore, was a partial cDNA with additional regions of the cDNA found downstream of the sequence. The nucleotide sequence of cDNA clone LIB3033-046-E6, and the amino acid sequence encoded by this cDNA clone are encompassed by the present invention.

[0065] Nucleotides 1-4867 of the cDNA sequence containing the second partial open reading frame described in U.S. application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:71) matches nucleotides 1311-6177 (plus the stop codon) of the sequence denoted herein as OrfB (SEQ ID NO:3), with the exception of the nucleotide at position 2933 of SEQ ID NO:71 of the '899 application, which corresponds to the nucleotide at position 4243 of SEQ ID NO:3 set forth herein. This single nucleotide change (C to G) results in a single amino acid change in SEQ ID NO:4 disclosed herein, as compared to SEQ ID NO:72 of the '899 application. Specifically, the glutamine residue at position 978 of SEQ ID NO:72 in the '899 application is changed to a glutamate at position 1415 of SEQ ID NO:4. This amino acid occurs in the linker region between the AT domain and the ER domain of SEQ ID NO:4. A cDNA clone described in U.S. application Ser. No. 09/231,899 as cDNA clone LIB3033-046-D2 comprises nucleotides 1311-6177 of SEQ ID NO:3 described herein, plus about 382 additional nucleotides beyond the end of the Orf represented here as SEQ ID NO:3, to the best of the present inventors' knowledge. cDNA clone LIB3033-046-D2 (denoted cDNA clone LIB3033-046-D2 in the form of an E. coli plasmid vector containing "hglC/Orf7/Orf8/Orf9 homolog" gene from Schizochytrium) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of cDNA clone LIB3033-046-D2, and the amino acid sequence encoded by this cDNA clone are encompassed by the present invention.

[0066] Nucleotides 145-4653 of the cDNA sequence containing the complete open reading frame described in U.S. application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:76 and incorrectly designated as a partial open reading frame) matches nucleotides 1-2624 and 2675-4506 of the sequence denoted herein as OrfC (SEQ ID NO:5). Sequencing of the genomic DNA encoding OrfC revealed that there is an additional nucleotide at each of positions 2769, 2806 and 2818 of SEQ ID NO:76 of the '899 application which resulted in a frame shift and a short change in the amino acid sequence of the corresponding protein. Therefore, amino acid positions 924-939 of SEQ ID NO:73 of the '899 application represent an incorrect sequence. Positions 876-890 of SEQ ID NO:5 herein represent the correct amino acid sequence in this region. This sequence is located in the DH2 domain of OrfC (discussed below). A cDNA clone described in U.S. application Ser. No. 09/231,899 as cDNA clone LIB81-042-B9 comprises a portion of the 5' sequence of SEQ ID NO:5. To the best of the present inventors' knowledge, the sequence of the insert in LIB81-042-B9 contains 145 nucleotides upstream of the start codon of SEQ ID NO:5 and extends 2361 nucleotides into the Orf. cDNA clone LIB81-042-B9 (denoted cDNA clone LIB81-042-B9 in the form of an E. coli plasmid vector containing "0118 homolog" partial gene sequence from Schizochytrium sp.) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of cDNA clone LIB81-042-B9, and the amino acid sequence encoded by this cDNA clone are encompassed by the present invention. A second cDNA clone described in U.S. application Ser. No. 09/231,899 as cDNA clone LIB81-015-D5 aligns with Shewanella ORFS and also with Shewanella ORF9. The open reading frame of LIB81-015-D5 aligns with SEQ ID NO:5 beginning at nucleotide 2526 of SEQ ID NO:5 and extends to the end of the Orf (i.e., position 4506), plus about 115 bp including a poly A tail beyond SEQ ID NO:5. The nucleotide sequence of cDNA clone LIB81-015-D5, and the amino acid sequence encoded by this cDNA clone are encompassed by the present invention.

[0067] Further sequencing of cDNA and genomic clones by the present inventors allowed the identification of the full-length genomic sequence of each of OrfA, OrfB and OrfC in Schizochytrium, including in Schizochytrium sp. ATCC 20888 and the mutated daughter strain of ATCC 20888, denoted Schizochytrium sp., strain N230D. N230D was one of more than 1,000 randomly-chosen survivors of chemically mutagenised (NTG; 1-methyl-3-nitro-1-nitrosoguanidine) Schizochytrium ATCC 20888 screened for variations in fatty acid content. This particular strain was valued for its improved DHA productivity. Further, the complete identification of the domains with homology to those in Shewanella (see FIG. 2) were identified. It is noted that in Schizochytrium, the Orfs of the genomic DNA and cDNA are identical, due to the lack of introns in the organism genome, to the best of the present inventors' knowledge. Therefore, reference to a nucleotide sequence of Orfs from Schizochytrium can refer to genomic DNA or cDNA.

[0068] FIG. 1 is a graphical representation of the three open reading frames from the Schizochytrium PUFA PKS system, and includes the domain structure of this PUFA PKS system. The domain structure of each open reading frame is as follows:

Open Reading Frame A (OrfA):

[0069] The complete nucleotide sequence for OrfA is represented herein as SEQ ID NO:1. Nucleotides 4677-8730 of SEQ ID NO:1 correspond to nucleotides 390-4443 of the sequence denoted as SEQ ID NO:69 in U.S. application Ser. No. 09/231,899. Therefore, nucleotides 1-4676 of SEQ ID NO:1 represent additional sequence that was not disclosed in U.S. application Ser. No. 09/231,899. This novel region of SEQ ID NO:1 encodes the following domains in OrfA: (1) the ORFA-KS domain; (2) the ORFA-MAT domain; and (3) at least a portion of the ACP domain region (e.g., at least ACP domains 1-4). It is noted that nucleotides 1-389 of SEQ ID NO:69 in U.S. application Ser. No. 09/231,899 do not exactly match with the 389 nucleotides that are upstream of position 4677 in SEQ ID NO:1 disclosed herein. Therefore, positions 1-389 of SEQ ID NO:69 in U.S. application Ser. No. 09/231,899 appear to be incorrectly placed next to nucleotides 390-4443 of that sequence. Most of these first 389 nucleotides (about positions 60-389) are a match with an upstream portion of OrfA (SEQ ID NO:1) of the present invention and therefore, it is believed that an error occurred in the effort to prepare the contig of the cDNA constructs in U.S. application Ser. No. 09/231,899. The region in which the alignment error occurred in U.S. application Ser. No. 09/231,899 is within the region of highly repetitive sequence (i.e., the ACP region, discussed below), which probably created some confusion in the assembly of that sequence from various cDNA clones.

[0070] OrfA is a 8730 nucleotide sequence (not including the stop codon) which encodes a 2910 amino acid sequence, represented herein as SEQ ID NO:2. Within OrfA are twelve domains: (a) one .beta.-keto acyl-ACP synthase (KS) domain; (b) one malonyl-CoA:ACP acyltransferase (MAT) domain; (c) nine acyl carrier protein (ACP) domains; and (d) one ketoreductase (KR) domain.

[0071] A nucleotide sequence for OrfA has been deposited with GenBank as Accession No. AF378327 (amino acid sequence Accession No. AAK728879). The nucleotide sequence represented by GenBank Accession No. AF378327 differs from the sequence represented herein as SEQ ID NO:1 by the point nucleotide changes: (1) at position 1999 (A to G, resulting in an amino acid change from an asparagine to an aspartic acid at position 667 of SEQ ID NO:2); (2) at position 2003 (C to A, resulting in an amino acid change from a proline to a histidine at position 668 of SEQ ID NO:2); and (3) at position 2238 (A to C, resulting in no amino acid change at position 746 of SEQ ID NO:2). Each of the two amino acid changes from the amino acid sequence encoded by GenBank Accession No. AAK728879 are located in the MAT domain (SEQ ID NO:10) of SEQ ID NO:2.

[0072] Genomic DNA clones (plasmids) encoding OrfA from both Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytrium sp., strain N230D, have been isolated and sequenced. A genomic clone described herein as JK1126, isolated from Schizochytrium sp. ATCC 20888, comprises, to the best of the present inventors' knowledge, the nucleotide sequence spanning from position 1 to 4119 and from position 5498 to 8730 of SEQ ID NO:1, and encodes the corresponding amino acid sequence of SEQ ID NO:2. Indeed, it is expected that JK1126 comprises SEQ ID NO:1 in its entirety and encodes SEQ ID NO:2. Genomic clone pJK1126 (denoted pJK1126 OrfA genomic clone, in the form of an E. coli plasmid vector containing "OrfA" gene from Schizochytrium ATCC 20888) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK1126 OrfA genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0073] Two genomic clones described herein as pJK306 OrfA genomic clone and pJK320 OrfA genomic clone, isolated from Schizochytrium sp. N230D, together (overlapping clones) comprise, to the best of the present inventors' knowledge, the nucleotide sequence of SEQ ID NO:1, and encode the amino acid sequence of SEQ ID NO:2. Genomic clone pJK306 (denoted pJK306 OrfA genomic clone, in the form of an E. coli plasmid containing 5' portion of OrfA gene from Schizochytrium sp. N230D (2.2 kB overlap with pJK320)) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK306 OrfA genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention. Genomic clone pJK320 (denoted pJK320 OrfA genomic clone, in the form of an E. coli plasmid containing 3' portion of OrfA gene from Schizochytrium sp. N230D (2.2 kB overlap with pJK306)) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK320 OrfA genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0074] OrfA was compared with known sequences in a standard BLAST search (BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches, blastn for nucleic acid searches, and blastX for nucleic acid searches and searches of the translated amino acid sequence in all 6 open reading frames with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schaaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety)). At the nucleic acid level, OrfA has no significant homology to any known nucleotide sequence. At the amino acid level, the sequences with the greatest degree of homology to ORFA were: Nostoc sp. 7120 heterocyst glycolipid synthase (Accession No. NC.sub.--003272), which was 42% identical to ORFA over 1001 amino acid residues; and Moritella marinas (Vibrio marinas) ORF8 (Accession No. AB025342), which was 40% identical to ORFA over 993 amino acid residues.

[0075] The first domain in OrfA is a KS domain, also referred to herein as ORFA-KS. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1 and 40 of SEQ ID NO:1 (OrfA) to an ending point of between about positions 1428 and 1500 of SEQ ID NO:1 (based on homology to other PUFA PKS domains, the position of the domain spans from about position 1 to about position 1500; based on Pfam analysis, a KS core region spans from about position 40 to about position 1428). The nucleotide sequence containing the sequence encoding the ORFA-KS domain is represented herein as SEQ ID NO:7 (positions 1-1500 of SEQ ID NO:1). The amino acid sequence containing the KS domain spans from a starting point of between about positions 1 and 14 of SEQ ID NO:2 (ORFA) to an ending point of between about positions 476 and 500 of SEQ ID NO:2 (again, referring to the overall homology to PUFA PKS KS domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFA-KS domain is represented herein as SEQ ID NO:8 (positions 1-500 of SEQ ID NO:2). It is noted that the ORFA-KS domain contains an active site motif: DXAC* (*acyl binding site C.sub.215).

[0076] According to the present invention, a domain or protein having 3-keto acyl-ACP synthase (KS) biological activity (function) is characterized as the enzyme that carries out the initial step of the FAS (and PKS) elongation reaction cycle. The acyl group destined for elongation is linked to a cysteine residue at the active site of the enzyme by a thioester bond. In the multi-step reaction, the acyl-enzyme undergoes condensation with malonyl-ACP to form -keto acyl-ACP, CO.sub.2 and free enzyme. The KS plays a key role in the elongation cycle and in many systems has been shown to possess greater substrate specificity than other enzymes of the reaction cycle. For example, E. coli has three distinct KS enzymes--each with its own particular role in the physiology of the organism (Magnuson et al., Microbiol. Rev. 57, 522 (1993)). The two KS domains of the PUFA-PKS systems could have distinct roles in the PUFA biosynthetic reaction sequence.

[0077] As a class of enzymes, KS's have been well characterized. The sequences of many verified KS genes are know, the active site motifs have been identified and the crystal structures of several have been determined. Proteins (or domains of proteins) can be readily identified as belonging to the KS family of enzymes by homology to known KS sequences.

[0078] The second domain in OrfA is a MAT domain, also referred to herein as ORFA-MAT. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1723 and 1798 of SEQ ID NO:1 (OrfA) to an ending point of between about positions 2805 and 3000 of SEQ ID NO:1 (based on homology to other PUFA PKS domains, the position of the MAT domain spans from about position 1723 to about position 3000; based on Pfam analysis, a MAT core region spans from about position 1798 to about position 2805). The nucleotide sequence containing the sequence encoding the ORFA-MAT domain is represented herein as SEQ ID NO:9 (positions 1723-3000 of SEQ ID NO:1). The amino acid sequence containing the MAT domain spans from a starting point of between about positions 575 and 600 of SEQ ID NO:2 (ORFA) to an ending point of between about positions 935 and 1000 of SEQ ID NO:2 (again, referring to the overall homology to PUFA PKS MAT domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFA-MAT domain is represented herein as SEQ ID NO:10 (positions 575-1000 of SEQ ID NO:2). The MAT domain comprises an aspartate at position 93 and a histidine at position 94 (corresponding to positions 667 and 668, respectively, of SEQ ID NO:2). It is noted that the ORFA-MAT domain contains an active site motif: GHS*XG (*acyl binding site S.sub.706), represented herein as SEQ ID NO:11.

[0079] According to the present invention, a domain or protein having malonyl-CoA:ACP acyltransferase (MAT) biological activity (function) is characterized as one that transfers the malonyl moiety from malonyl-CoA to ACP. In addition to the active site motif (GxSxG), these enzymes possess an extended motif R and Q amino acids in key positions) that identifies them as MAT enzymes (in contrast to the AT domain of Schizochytrium Orf B). In some PKS systems (but not the PUFA PKS domain) MAT domains will preferentially load methyl- or ethyl-malonate on to the ACP group (from the corresponding CoA ester), thereby introducing branches into the linear carbon chain. MAT domains can be recognized by their homology to known MAT sequences and by their extended motif structure.

[0080] Domains 3-11 of OrfA are nine tandem ACP domains, also referred to herein as ORFA-ACP (the first domain in the sequence is ORFA-ACP1, the second domain is ORFA-ACP2, the third domain is ORFA-ACP3, etc.). The first ACP domain, ORFA-ACP1, is contained within the nucleotide sequence spanning from about position 3343 to about position 3600 of SEQ ID NO:1 (OrfA). The nucleotide sequence containing the sequence encoding the ORFA-ACP1 domain is represented herein as SEQ ID NO:12 (positions 3343-3600 of SEQ ID NO:1). The amino acid sequence containing the first ACP domain spans from about position 1115 to about position 1200 of SEQ ID NO:2. The amino acid sequence containing the ORFA-ACP1 domain is represented herein as SEQ ID NO:13 (positions 1115-1200 of SEQ ID NO:2). It is noted that the ORFA-ACP1 domain contains an active site motif: LGIDS* (*pantetheine binding motif S.sub.1157), represented herein by SEQ ID NO:14.

[0081] The nucleotide and amino acid sequences of all nine ACP domains are highly conserved and therefore, the sequence for each domain is not represented herein by an individual sequence identifier. However, based on the information disclosed herein, one of skill in the art can readily determine the sequence containing each of the other eight ACP domains (see discussion below).

[0082] All nine ACP domains together span a region of OrfA of from about position 3283 to about position 6288 of SEQ ID NO:1, which corresponds to amino acid positions of from about 1095 to about 2096 of SEQ ID NO:2. The nucleotide sequence for the entire ACP region containing all nine domains is represented herein as SEQ ID NO:16. The region represented by SEQ ID NO:16 includes the linker segments between individual ACP domains. The repeat interval for the nine domains is approximately every 330 nucleotides of SEQ ID NO:16 (the actual number of amino acids measured between adjacent active site serines ranges from 104 to 116 amino acids). Each of the nine ACP domains contains a pantetheine binding motif LGIDS* (represented herein by SEQ ID NO:14), wherein S* is the pantetheine binding site serine (S). The pantetheine binding site serine (S) is located near the center of each ACP domain sequence. At each end of the ACP domain region and between each ACP domain is a region that is highly enriched for proline (P) and alanine (A), which is believed to be a linker region. For example, between ACP domains 1 and 2 is the sequence: APAPVKAAAPAAPVASAPAPA, represented herein as SEQ ID NO:15. The locations of the active site serine residues (i.e., the pantetheine binding site) for each of the nine ACP domains, with respect to the amino acid sequence of SEQ ID NO:2, are as follows: ACP1=S.sub.1157; ACP2=S.sub.1266; ACP3=S.sub.1377; ACP4=S.sub.1488; ACP5=S.sub.1604; ACP6=S.sub.1715; ACP7=S.sub.1819; ACP8=S.sub.1930; and ACP9=S.sub.2034. Given that the average size of ACP domain is about 85 amino acids, excluding the linker, and about 110 amino acids including the linker, with the active site serine being approximately in the center of the domain, one of skill in the art can readily determine the positions of each of the nine ACP domains in OrfA.

[0083] According to the present invention, a domain or protein having acyl carrier protein (ACP) biological activity (function) is characterized as being small polypeptides (typically, 80 to 100 amino acids long), that function as carriers for growing fatty acyl chains via a thioester linkage to a covalently bound co-factor of the protein. They occur as separate units or as domains within larger proteins. ACPs are converted from inactive apo-forms to functional holo-forms by transfer of the phosphopantetheinyl moiety of CoA to a highly conserved serine residue of the ACP. Acyl groups are attached to ACP by a thioester linkage at the free terminus of the phosphopantetheinyl moiety. ACPs can be identified by labeling with radioactive pantetheine and by sequence homology to known ACPs. The presence of variations of the above mentioned motif (LGIDS*) is also a signature of an ACP.

[0084] Domain 12 in OrfA is a KR domain, also referred to herein as ORFA-KR. This domain is contained within the nucleotide sequence spanning from a starting point of about position 6598 of SEQ ID NO:1 to an ending point of about position 8730 of SEQ ID NO:1. The nucleotide sequence containing the sequence encoding the ORFA-KR domain is represented herein as SEQ ID NO:17 (positions 6598-8730 of SEQ ID NO:1). The amino acid sequence containing the KR domain spans from a starting point of about position 2200 of SEQ ID NO:2 (ORFA) to an ending point of about position 2910 of SEQ ID NO:2. The amino acid sequence containing the ORFA-KR domain is represented herein as SEQ ID NO:18 (positions 2200-2910 of SEQ ID NO:2). Within the KR domain is a core region with homology to short chain aldehyde-dehydrogenases (KR is a member of this family). This core region spans from about position 7198 to about position 7500 of SEQ ID NO:1, which corresponds to amino acid positions 2400-2500 of SEQ ID NO:2.

[0085] According to the present invention, a domain or protein having ketoreductase activity, also referred to as 3-ketoacyl-ACP reductase (KR) biological activity (function), is characterized as one that catalyzes the pyridine-nucleotide-dependent reduction of 3-keto acyl forms of ACP. It is the first reductive step in the de novo fatty acid biosynthesis elongation cycle and a reaction often performed in polyketide biosynthesis. Significant sequence similarity is observed with one family of enoyl ACP reductases (ER), the other reductase of FAS (but not the ER family present in the PUFA PKS system), and the short-chain alcohol dehydrogenase family. Pfam analysis of the PUFA PKS region indicated above reveals the homology to the short-chain alcohol dehydrogenase family in the core region. Blast analysis of the same region reveals matches in the core area to known KR enzymes as well as an extended region of homology to domains from the other characterized PUFA PKS systems.

Open Reading Frame B (OrfB):

[0086] The complete nucleotide sequence for OrfB is represented herein as SEQ ID NO:3. Nucleotides 1311-6177 of SEQ ID NO:3 correspond to nucleotides 1-4867 of the sequence denoted as SEQ ID NO:71 in U.S. application Ser. No. 09/231,899, with the exception of the nucleotide at position 2933 of SEQ ID NO:71 of the `899 application or nucleotide 4243 of SEQ ID NO:3 herein, as discussed above. The cDNA sequence in U.S. application Ser. No. 09/231,899 contains about 345 additional nucleotides beyond the stop codon, including a polyA tail). Therefore, nucleotides 1-1310 of SEQ ID NO:1 represent additional sequence that was not disclosed in U.S. application Ser. No. 09/231,899. This novel region of SEQ ID NO:3 contains most of the KS domain encoded by OrfB.

[0087] OrfB is a 6177 nucleotide sequence (not including the stop codon) which encodes a 2059 amino acid sequence, represented herein as SEQ ID NO:4. Within OrfB are four domains: (a) one .beta.-keto acyl-ACP synthase (KS) domain; (b) one chain length factor (CLF) domain; (c) one acyl transferase (AT) domain; and, (d) one enoyl ACP-reductase (ER) domain.

[0088] A nucleotide sequence for OrfB has been deposited with GenBank as Accession No. AF378328 (amino acid sequence Accession No. AAK728880). The nucleotide sequence represented by GenBank Accession No. AF378328 differs from the nucleotide sequence represented herein as SEQ ID NO:3 by the point nucleotide changes: (1) at position 852 (T to C, resulting in no amino acid change at position 284 of SEQ ID NO:4); (2) at position 1110 (S to C, resulting in no amino acid change at position 370 of SEQ ID NO:4); (3) at position 1112 (Y to T, resulting in the resolution of an ambiguous amino acid call to a definite valine call at position 371 of SEQ ID NO:4); and (4) at position 4243 (C to G, resulting in a change from a glutamine to a glutamate at position 1415 of SEQ ID NO:4). The single amino acid change from the amino acid sequence encoded by GenBank Accession No. AAK728880 is located in the linker region located between the AT domain and the ER domain of SEQ ID NO:4.

[0089] Genomic DNA clones (plasmids) encoding OrfB from both Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytrium sp., strain N230D, have been isolated and sequenced. A genomic clone described herein as pJK1129, isolated from Schizochytrium sp. ATCC 20888, comprises, to the best of the present inventors' knowledge, the nucleotide sequence of SEQ ID NO:3, and encodes the amino acid sequence of SEQ ID NO:4. Genomic clone pJK1129 (denoted pJK1129 OrfB genomic clone, in the form of an E. coli plasmid vector containing "OrfB" gene from Schizochytrium ATCC 20888) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK1126 OrfB genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0090] A genomic clone described herein as pJK324 OrfB genomic clone, isolated from Schizochytrium sp. N230D, comprises, to the best of the present inventors' knowledge, the nucleotide sequence of SEQ ID NO:3, and encodes the amino acid sequence of SEQ ID NO:4. Genomic clone pJK324 (denoted pJK324 OrfB genomic clone, in the form of an E. coli plasmid containing the OrfB gene sequence from Schizochytrium sp. N230D) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK324 OrfB genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0091] OrfB was compared with known sequences in a standard BLAST search as described above. At the nucleic acid level, OrfB has no significant homology to any known nucleotide sequence. At the amino acid level, the sequences with the greatest degree of homology to ORFB were: Shewanella sp. hypothetical protein (Accession No. U73935), which was 53% identical to ORFB over 458 amino acid residues; Moritella marinus (Vibrio marinus) ORF11 (Accession No. AB025342), which was 53% identical to ORFB over 460 amino acid residues; Photobacterium profundum omega-3 polyunsaturated fatty acid synthase PfaD (Accession No. AF409100), which was 52% identical to ORFB over 457 amino acid residues; and Nostoc sp. 7120 hypothetical protein (Accession No. NC.sub.--003272), which was 53% identical to ORFB over 430 amino acid residues.

[0092] The first domain in OrfB is a KS domain, also referred to herein as ORFB-KS. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1 and 43 of SEQ ID NO:3 (OrfB) to an ending point of between about positions 1332 and 1350 of SEQ ID NO:3 (based on homology to other PUFA PKS domains, the position of the KS domain spans from about position 1 to about position 1350; based on Pfam analysis, a KS core region spans from about position 43 to about position 1332). The nucleotide sequence containing the sequence encoding the ORFB-KS domain is represented herein as SEQ ID NO:19 (positions 1-1350 of SEQ ID NO:3). The amino acid sequence containing the KS domain spans from a starting point of between about positions 1 and 15 of SEQ ID NO:4 (ORFB) to an ending point of between about positions 444 and 450 of SEQ ID NO:4 (again, referring to the overall homology to PUFA PKS KS domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFB-KS domain is represented herein as SEQ ID NO:20 (positions 1-450 of SEQ ID NO:4). This KS domain comprises a valine at position 371 of SEQ ID NO:20 (also position 371 of SEQ ID NO:20). It is noted that the ORFB-KS domain contains an active site motif: DXAC* (*acyl binding site C.sub.196). KS biological activity and methods of identifying proteins or domains having such activity is described above.

[0093] The second domain in OrfB is a CLF domain, also referred to herein as ORFB-CLF. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1378 and 1402 of SEQ ID NO:3 (OrfB) to an ending point of between about positions 2682 and 2700 of SEQ ID NO:3 (based on homology to other PUFA PKS domains, the position of the CLF domain spans from about position 1378 to about position 2700; based on Pfam analysis, a CLF core region spans from about position 1402 to about position 2682). The nucleotide sequence containing the sequence encoding the ORFB-CLF domain is represented herein as SEQ ID NO:21 (positions 1378-2700 of SEQ ID NO:3). The amino acid sequence containing the CLF domain spans from a starting point of between about positions 460 and 468 of SEQ ID NO:4 (ORFB) to an ending point of between about positions 894 and 900 of SEQ ID NO:4 (again, referring to the overall homology to PUFA PKS CLF domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFB-CLF domain is represented herein as SEQ ID NO:22 (positions 460-900 of SEQ ID NO:4). It is noted that the ORFB-CLF domain contains a KS active site motif without the acyl-binding cysteine.

[0094] According to the present invention, a domain or protein is referred to as a chain length factor (CLF) based on the following rationale. The CLF was originally described as characteristic of Type II (dissociated enzymes) PKS systems and was hypothesized to play a role in determining the number of elongation cycles, and hence the chain length, of the end product. CLF amino acid sequences show homology to KS domains (and are thought to form heterodimers with a KS protein), but they lack the active site cysteine. CLF's role in PKS systems is currently controversial. New evidence (C. Bisang et al., Nature 401, 502 (1999)) suggests a role in priming (providing the initial acyl group to be elongated) the PKS systems. In this role the CLF domain is thought to decarboxylate malonate (as malonyl-ACP), thus forming an acetate group that can be transferred to the KS active site. This acetate therefore acts as the `priming` molecule that can undergo the initial elongation (condensation) reaction. Homologues of the Type II CLF have been identified as `loading` domains in some modular PKS systems. A domain with the sequence features of the CLF is found in all currently identified PUFA PKS systems and in each case is found as part of a multidomain protein.

[0095] The third domain in OrfB is an AT domain, also referred to herein as ORFB-AT. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 2701 and 3598 of SEQ ID NO:3 (OrfB) to an ending point of between about positions 3975 and 4200 of SEQ ID NO:3 (based on homology to other PUFA PKS domains, the position of the AT domain spans from about position 2701 to about position 4200; based on Pfam analysis, an AT core region spans from about position 3598 to about position 3975). The nucleotide sequence containing the sequence encoding the ORFB-AT domain is represented herein as SEQ ID NO:23 (positions 2701-4200 of SEQ ID NO:3). The amino acid sequence containing the AT domain spans from a starting point of between about positions 901 and 1200 of SEQ ID NO:4 (ORFB) to an ending point of between about positions 1325 and 1400 of SEQ ID NO:4 (again, referring to the overall homology to PUFA PKS AT domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFB-AT domain is represented herein as SEQ ID NO:24 (positions 901-1400 of SEQ ID NO:4). It is noted that the ORFB-AT domain contains an active site motif of GxS*xG (*acyl binding site .sub.51140) that is characteristic of acyltransferse (AT) proteins.

[0096] An "acyltransferase" or "AT" refers to a general class of enzymes that can carry out a number of distinct acyl transfer reactions. The Schizochytrium domain shows good homology to a domain present in all of the other PUFA PKS systems currently examined and very weak homology to some acyltransferases whose specific functions have been identified (e.g. to malonyl-CoA:ACP acyltransferase, MAT). In spite of the weak homology to MAT, this AT domain is not believed to function as a MAT because it does not possess an extended motif structure characteristic of such enzymes (see MAT domain description, above). For the purposes of this disclosure, the functions of the AT domain in a PUFA PKS system include, but are not limited to: transfer of the fatty acyl group from the ORFA ACP domain(s) to water (i.e. a thioesterase--releasing the fatty acyl group as a free fatty acid), transfer of a fatty acyl group to an acceptor such as CoA, transfer of the acyl group among the various ACP domains, or transfer of the fatty acyl group to a lipophilic acceptor molecule (e.g. to lysophosphadic acid).

[0097] The fourth domain in OrfB is an ER domain, also referred to herein as ORFB-ER. This domain is contained within the nucleotide sequence spanning from a starting point of about position 4648 of SEQ ID NO:3 (OrfB) to an ending point of about position 6177 of SEQ ID NO:3. The nucleotide sequence containing the sequence encoding the ORFB-ER domain is represented herein as SEQ ID NO:25 (positions 4648-6177 of SEQ ID NO:3). The amino acid sequence containing the ER domain spans from a starting point of about position 1550 of SEQ ID NO:4 (ORFB) to an ending point of about position 2059 of SEQ ID NO:4. The amino acid sequence containing the ORFB-ER domain is represented herein as SEQ ID NO:26 (positions 1550-2059 of SEQ ID NO:4).

[0098] According to the present invention, this domain has enoyl reductase (ER) biological activity. The ER enzyme reduces the trans-double bond (introduced by the DH activity) in the fatty acyl-ACP, resulting in fully saturating those carbons. The ER domain in the PUFA-PKS shows homology to a newly characterized family of ER enzymes (Heath et al., Nature 406, 145 (2000)). Heath and Rock identified this new class of ER enzymes by cloning a gene of interest from Streptococcus pneumoniae, purifying a protein expressed from that gene, and showing that it had ER activity in an in vitro assay. The sequence of the Schizochytrium ER domain of OrfB shows homology to the S. pneumoniae ER protein. All of the PUFA PKS systems currently examined contain at least one domain with very high sequence homology to the Schizochytrium ER domain. The Schizochytrium PUFA PKS system contains two ER domains (one on OrfB and one on OrfC).

Open Reading Frame C (OrfC):

[0099] The complete nucleotide sequence for OrfC is represented herein as SEQ ID NO:5. Nucleotides 1-4506 of SEQ ID NO:5 (i.e., the entire open reading frame sequence, not including the stop codon) nearly correspond to nucleotides 145-4653 of the sequence denoted as SEQ ID NO:76 in U.S. application Ser. No. 09/231,899. The cDNA sequence in U.S. application Ser. No. 09/231,899 contains about 144 nucleotides upstream of the start codon for OrfC and about 110 nucleotides beyond the stop codon, including a polyA tail. In addition, as discussed above, nucleotides 145-4653 of the cDNA sequence containing the complete open reading frame described in U.S. application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:76) match nucleotides 1-2624 and 2675-4506 of the sequence denoted herein as OrfC (SEQ ID NO:5). OrfC is a 4506 nucleotide sequence (not including the stop codon) which encodes a 1502 amino acid sequence, represented herein as SEQ ID NO:6. Within OrfC are three domains: (a) two FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) domains; and (b) one enoyl ACP-reductase (ER) domain.

[0100] A nucleotide sequence for OrfC has been deposited with GenBank as Accession No. AF378329 (amino acid sequence Accession No. AAK728881). The nucleotide sequence represented by AF378329 differs from the nucleotide sequence represented herein as SEQ ID NO:5 by the point nucleotide insertions: (1) at position 2625 (an insertion of an A); (2) at position 2662 (an insertion of a C); and (3) at position 2674 (an insertion of an A). This resulted in a frame shift out of frame at position 2625 and then back into frame at position 2675. The amino acid sequence encoded by GenBank Accession No. AAK728881 differs from the amino acid sequence encoded by SEQ ID NO:5 (i.e., SEQ ID NO:6) in the region spanning from positions 876-891 of GenBank Accession No. AAK728881 or positions 876-890 of SEQ ID NO:6. This change in sequence occurs in the DH2 domain of OrfC (discussed below).

[0101] Genomic DNA clones (plasmids) encoding OrfC from both Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888, denoted Schizochytrium sp., strain N230D, have been isolated and sequenced. A genomic clone described herein as pJK1131, isolated from Schizochytrium sp. ATCC 20888, comprises, to the best of the present inventors' knowledge, the nucleotide sequence of SEQ ID NO:5, and encodes the amino acid sequence of SEQ ID NO:6. Genomic clone pJK1131 (denoted pJK1131 OrfC genomic clone, in the form of an E. coli plasmid vector containing "OrfC" gene from Schizochytrium ATCC 20888) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pJK1131 OrfC genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0102] A genomic clone described herein as pBR002 OrfC genomic clone, isolated from Schizochytrium sp. N230D, comprises, to the best of the present inventors' knowledge, the nucleotide sequence of SEQ ID NO:5, and encodes the amino acid sequence of SEQ ID NO:6. Genomic clone pBR002 (denoted pBR0O2 OrfC genomic clone, in the form of an E. coli plasmid vector containing the OrfC gene sequence from Schizochytrium sp. N230D) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on June 8, 2006, and assigned ATCC Accession No. ______. The nucleotide sequence of pBR002 OrfC genomic clone, and the amino acid sequence encoded by this plasmid are encompassed by the present invention.

[0103] OrfC was compared with known sequences in a standard BLAST search as described above. At the nucleic acid level, OrfC has no significant homology to any known nucleotide sequence. At the amino acid level (Blastp), the sequences with the greatest degree of homology to ORFC were: Moritella marinus (Vibrio marinus) ORF11 (Accession No. ABO25342), which is 45% identical to ORFC over 514 amino acid residues, Shewanella sp. hypothetical protein 8 (Accession No. U73935), which is 49% identical to ORFC over 447 amino acid residues, Nostoc sp. hypothetical protein (Accession No. NC.sub.--003272), which is 49% identical to ORFC over 430 amino acid residues, and Shewanella sp. hypothetical protein 7 (Accession No. U73935), which is 37% identical to ORFC over 930 amino acid residues.

[0104] The first domain in OrfC is a DH domain, also referred to herein as ORFC-DH1. This is one of two DH domains in OrfC, and therefore is designated DH1. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1 and 778 of SEQ ID NO:5 (OrfC) to an ending point of between about positions 1233 and 1350 of SEQ ID NO:5 (based on homology to other PUFA PKS domains, the position of the DH1 domain spans from about position 1 to about position 1350; based on Pfam analysis, a DH core region spans from about position 778 to about position 1233). The nucleotide sequence containing the sequence encoding the ORFC-DH1 domain is represented herein as SEQ ID NO:27 (positions 1-1350 of SEQ ID NO:5). The amino acid sequence containing the DH1 domain spans from a starting point of between about positions 1 and 260 of SEQ ID NO:6 (ORFC) to an ending point of between about positions 411 and 450 of SEQ ID NO:6 (again, referring to the overall homology to PUFA PKS DH domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFC-DH1 domain is represented herein as SEQ ID NO:28 (positions 1-450 of SEQ ID NO:6).

[0105] The characteristics of both the DH domains (see below for DH 2) in the PUFA PKS systems have been described in the preceding sections. This class of enzyme removes HOH from a .beta.-keto acyl-ACP and leaves a trans double bond in the carbon chain. The DH domains of the PUFA PKS systems show homology to bacterial DH enzymes associated with their FAS systems (rather than to the DH domains of other PKS systems). A subset of bacterial DH's, the FabA-like DH's, possesses cis-trans isomerase activity (Heath et al., J. Biol. Chem., 271, 27795 (1996)). It is the homologies to the FabA-like DH's that indicate that one or both of the DH domains is responsible for insertion of the cis double bonds in the PUFA PKS products.

[0106] The second domain in OrfC is a DH domain, also referred to herein as ORFC-DH2. This is the second of two DH domains in OrfC, and therefore is designated DH2. This domain is contained within the nucleotide sequence spanning from a starting point of between about positions 1351 and 2437 of SEQ ID NO:5 (OrfC) to an ending point of between about positions 2607 and 2847 of SEQ ID NO:5 (based on homology to other PUFA PKS domains, the position of the DH2 domain spans from about position 1351 to about position 2845; based on Pfam analysis, a DH core region spans from about position 2437 to about position 2847). The nucleotide sequence containing the sequence encoding the ORFC-DH2 domain is represented herein as SEQ ID NO:29 (positions 1351-2847 of SEQ ID NO:5). The amino acid sequence containing the DH2 domain spans from a starting point of between about positions 451 and 813 of SEQ ID NO:6 (ORFC) to an ending point of between about positions 869 and 949 of SEQ ID NO:6 (again, referring to the overall homology to PUFA PKS DH domains and to Pfam core regions, respectively). The amino acid sequence containing the ORFC-DH2 domain is represented herein as SEQ ID NO:30 (positions 451-949 of SEQ ID NO:6). This DH domain comprises the amino acids H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions 426 440 of SEQ ID NO:30. DH biological activity has been described above.

[0107] The third domain in OrfC is an ER domain, also referred to herein as ORFC-ER. This domain is contained within the nucleotide sequence spanning from a starting point of about position 2995 of SEQ ID NO:5 (OrfC) to an ending point of about position 4506 of SEQ ID NO:5. The nucleotide sequence containing the sequence encoding the ORFC-ER domain is represented herein as SEQ ID NO:31 (positions 2995-4506 of SEQ ID NO:5). The amino acid sequence containing the ER domain spans from a starting point of about position 999 of SEQ ID NO:6 (ORFC) to an ending point of about position 1502 of SEQ ID NO:6. The amino acid sequence containing the ORFC-ER domain is represented herein as SEQ ID NO:32 (positions 999-1502 of SEQ ID NO:6). ER biological activity has been described above.

Accessory Proteins

[0108] According to the present invention, a domain or protein having 4'-phosphopantetheinyl transferase (PPTase) biological activity (function) is characterized as the enzyme that transfers a 4'-phosphopantetheinyl moiety from Coenzyme A to the acyl carrier protein (ACP). This transfer to an invariant serine reside of the ACP activates the inactive apo-form to the holo-form. In both polyketide and fatty acid synthesis, the phosphopantetheine group forms thioesters with the growing acyl chains. The PPTases are a family of enzymes that have been well characterized in fatty acid synthesis, polyketide synthesis, and non-ribosomal peptide synthesis. The sequences of many PPTases are known, and crystal structures have been determined (e.g., Reuter K, Mofid M R, Marahiel M A, Ficner R. "Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily" EMBO J. 1999 Dec. 1; 18(23):6823-31) as well as mutational analysis of amino acid residues important for activity (Mofid M R, Finking R, Essen L O, Marahiel M A. "Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism" Biochemistry. 2004 Apr. 13: 43(14):4128-36).

[0109] The present inventors have identified two sequences (genes) in the Arabidopsis whole genome database that are likely to encode PPTases. These sequences (GenBank Accession numbers; AAG51443 and AAC05345) are currently listed as encoding "Unknown Proteins". They can be identified as putative PPTases based on the presence in the translated protein sequences of several signature motifs including; G(I/V)D and WxxKE(A/S)xxK (SEQ ID NO:33), (listed in Lambalot et al., 1996 as characteristic of all PPTases). In addition, these two putative proteins contain two additional motifs typically found in PPTases typically associated with PKS and non-ribosomal peptide synthesis systems; i.e., FN(I/L/V)SHS (SEQ ID NO:34) and (I/V/L)G(I/L/V)D(I/L/V) (SEQ ID NO:35). Furthermore, these motifs occur in the expected relative positions in the protein sequences. It is likely that homologues of the Arabidopsis genes are present in other plants, such as tobacco. Again, these genes can be cloned and expressed to see if the enzymes they encode can activate the Schizochytrium ORFA ACP domains, or alternatively, OrfA could be expressed directly in the transgenic plant (either targeted to the plastid or the cytoplasm).

[0110] Another heterologous PPTase which has been demonstrated by the inventors to recognize the OrfA ACP domains described herein as substrates is the Het I protein of Nostoc sp. PCC 7120 (formerly called Anabaena sp. PCC 7120).

[0111] One embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence from a non-bacterial PUFA PKS system, a homologue thereof, a fragment thereof, and/or a nucleic acid sequence that is complementary to any of such nucleic acid sequences. In one aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and biologically active fragments thereof; (b) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and biologically active fragments thereof; (c) a nucleic acid sequence encoding an amino acid sequence that is at least about 60% identical to at least 500 consecutive amino acids of said amino acid sequence of (a), wherein said amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; (d) a nucleic acid sequence encoding an amino acid sequence that is at least about 60% identical to said amino acid sequence of (b), wherein said amino acid sequence has a biological activity of at least one domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; or (e) a nucleic acid sequence that is fully complementary to the nucleic acid sequence of (a), (b), (c), or (d). In a further embodiment, nucleic acid sequences including a sequence encoding the active site domains or other functional motifs described above for several of the PUFA PKS domains are encompassed by the invention.

[0112] According to the present invention, an amino acid sequence that has a biological activity of at least one domain of a PUFA PKS system is an amino acid sequence that has the biological activity of at least one domain of the PUFA PKS system described in detail herein, as exemplified by the Schizochytrium PUFA PKS system. The biological activities of the various domains within the Schizochytrium PUFA PKS system have been described in detail above. Therefore, an isolated nucleic acid molecule of the present invention can encode the translation product of any PUFA PKS open reading frame, PUFA PKS domain, biologically active fragment thereof, or any homologue of a naturally occurring PUFA PKS open reading frame or domain which has biological activity. A homologue of given protein or domain is a protein or polypeptide that has an amino acid sequence which differs from the naturally occurring reference amino acid sequence (i.e., of the reference protein or domain) in that at least one or a few, but not limited to one or a few, amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or fragment), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol). Preferred homologues of a PUFA PKS protein or domain are described in detail below. It is noted that homologues can include synthetically produced homologues, naturally occurring allelic variants of a given protein or domain, or homologous sequences from organisms other than the organism from which the reference sequence was derived.

[0113] In general, the biological activity or biological action of a protein or domain refers to any function(s) exhibited or performed by the protein or domain that is ascribed to the naturally occurring form of the protein or domain as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). Biological activities of PUFA PKS systems and the individual proteins/domains that make up a PUFA PKS system have been described in detail elsewhere herein. Modifications of a protein or domain, such as in a homologue or mimetic (discussed below), may result in proteins or domains having the same biological activity as the naturally occurring protein or domain, or in proteins or domains having decreased or increased biological activity as compared to the naturally occurring protein or domain. Modifications which result in a decrease in expression or a decrease in the activity of the protein or domain, can be referred to as inactivation (complete or partial), down-regulation, or decreased action of a protein or domain. Similarly, modifications which result in an increase in expression or an increase in the activity of the protein or domain, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein or domain. A functional domain of a PUFA PKS system is a domain (i.e., a domain can be a portion of a protein) that is capable of performing a biological function (i.e., has biological activity).

[0114] In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the same chromosome. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.

[0115] Preferably, an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications provide the desired effect on PUFA PKS system biological activity as described herein. Protein homologues (e.g., proteins encoded by nucleic acid homologues) have been discussed in detail above.

[0116] A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene.

[0117] The minimum size of a nucleic acid molecule of the present invention is a size sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid (e.g., under moderate, high or very high stringency conditions) with the complementary sequence of a nucleic acid molecule useful in the present invention, or of a size sufficient to encode an amino acid sequence having a biological activity of at least one domain of a PUFA PKS system according to the present invention. As such, the size of the nucleic acid molecule encoding such a protein can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration). The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule of the present invention, in that the nucleic acid molecule can include a sequence sufficient to encode a biologically active fragment of a domain of a PUFA PKS system, an entire domain of a PUFA PKS system, several domains within an open reading frame (Orf) of a PUFA PKS system, an entire Orf of a PUFA PKS system, or more than one Orf of a PUFA PKS system.

[0118] In one embodiment of the present invention, an isolated nucleic acid molecule comprises or consists essentially of a nucleic acid sequence encoding an amino acid sequence selected from the group of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or biologically active fragments thereof. In one aspect, the nucleic acid sequence is selected from the group of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.

[0119] In one embodiment of the present invention, any of the above-described PUFA PKS amino acid sequences, as well as homologues of such sequences, can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal end of the given amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of" a given amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the given amino acid sequence or which would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the given amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of", when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a given amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the given amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the given amino acid sequence as it occurs in the natural gene.

[0120] The present invention also includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence having a biological activity of at least one domain of a PUFA PKS system. In one aspect, such a nucleic acid sequence encodes a homologue of any of the Schizochytrium PUFA PKS ORFs or domains, including: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein the homologue has a biological activity of at least one (or two, three, four or more) domain of a PUFA PKS system as described previously herein.

[0121] In one aspect of the invention, a homologue of a Schizochytrium PUFA PKS protein or domain encompassed by the present invention comprises an amino acid sequence that is at least about 60% identical to at least 500 consecutive amino acids of an amino acid sequence chosen from: SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein said amino acid sequence has a biological activity of at least one domain of a PUFA PKS system. In a further aspect, the amino acid sequence of the homologue is at least about 60% identical to at least about 600 consecutive amino acids, and more preferably to at least about 700 consecutive amino acids, and more preferably to at least about 800 consecutive amino acids, and more preferably to at least about 900 consecutive amino acids, and more preferably to at least about 1000 consecutive amino acids, and more preferably to at least about 1100 consecutive amino acids, and more preferably to at least about 1200 consecutive amino acids, and more preferably to at least about 1300 consecutive amino acids, and more preferably to at least about 1400 consecutive amino acids, and more preferably to at least about 1500 consecutive amino acids of any of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or to the full length of SEQ ID NO:6. In a further aspect, the amino acid sequence of the homologue is at least about 60% identical to at least about 1600 consecutive amino acids, and more preferably to at least about 1700 consecutive amino acids, and more preferably to at least about 1800 consecutive amino acids, and more preferably to at least about 1900 consecutive amino acids, and more preferably to at least about 2000 consecutive amino acids of any of SEQ ID NO:2 or SEQ ID NO:4, or to the full length of SEQ ID NO:4. In a further aspect, the amino acid sequence of the homologue is at least about 60% identical to at least about 2100 consecutive amino acids, and more preferably to at least about 2200 consecutive amino acids, and more preferably to at least about 2300 consecutive amino acids, and more preferably to at least about 2400 consecutive amino acids, and more preferably to at least about 2500 consecutive amino acids, and more preferably to at least about 2600 consecutive amino acids, and more preferably to at least about 2700 consecutive amino acids, and more preferably to at least about 2800 consecutive amino acids, and even more preferably, to the full length of SEQ ID NO:2.

[0122] In another aspect, a homologue of a Schizochytrium PUFA PKS protein or domain encompassed by the present invention comprises an amino acid sequence that is at least about 65% identical, and more preferably at least about 70% identical, and more preferably at least about 75% identical, and more preferably at least about 80% identical, and more preferably at least about 85% identical, and more preferably at least about 90% identical, and more preferably at least about 95% identical, and more preferably at least about 96% identical, and more preferably at least about 97% identical, and more preferably at least about 98% identical, and more preferably at least about 99% identical to an amino acid sequence chosen from: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, over any of the consecutive amino acid lengths described in the paragraph above, wherein the amino acid sequence has a biological activity of at least one domain of a PUFA PKS system.

[0123] In one aspect of the invention, a homologue of a Schizochytrium PUFA PKS protein or domain encompassed by the present invention comprises an amino acid sequence that is at least about 60% identical to an amino acid sequence chosen from: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein said amino acid sequence has a biological activity of at least one domain of a PUFA PKS system. In a further aspect, the amino acid sequence of the homologue is at least about 65% identical, and more preferably at least about 70% identical, and more preferably at least about 75% identical, and more preferably at least about 80% identical, and more preferably at least about 85% identical, and more preferably at least about 90% identical, and more preferably at least about 95% identical, and more preferably at least about 96% identical, and more preferably at least about 97% identical, and more preferably at least about 98% identical, and more preferably at least about 99% identical to an amino acid sequence chosen from: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, wherein the amino acid sequence has a biological activity of at least one domain of a PUFA PKS system.

[0124] According to the present invention, the term "contiguous" or "consecutive", with regard to nucleic acid or amino acid sequences described herein, means to be connected in an unbroken sequence. For example, for a first sequence to comprise 30 contiguous (or consecutive) amino acids of a second sequence, means that the first sequence includes an unbroken sequence of 30 amino acid residues that is 100% identical to an unbroken sequence of 30 amino acid residues in the second sequence. Similarly, for a first sequence to have "100% identity" with a second sequence means that the first sequence exactly matches the second sequence with no gaps between nucleotides or amino acids.

[0125] As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches, blastn for nucleic acid searches, and blastX for nucleic acid searches and searches of translated amino acids in all 6 open reading frames, all with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schaaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST). It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.

[0126] Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), "Blast 2 sequences--a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.

[0127] For blastn, using 0 BLOSUM62 matrix:

[0128] Reward for match=1

[0129] Penalty for mismatch=-2

[0130] Open gap (5) and extension gap (2) penalties

[0131] gap x_dropoff (50) expect (10) word size (11) filter (on)

[0132] For blastp, using 0 BLOSUM62 matrix:

[0133] Open gap (11) and extension gap (1) penalties

[0134] gap x_dropoff (50) expect (10) word size (3) filter (on).

[0135] In another embodiment of the invention, an amino acid sequence having the biological activity of at least one domain of a PUFA PKS system of the present invention includes an amino acid sequence that is sufficiently similar to a naturally occurring PUFA PKS protein or polypeptide that a nucleic acid sequence encoding the amino acid sequence is capable of hybridizing under moderate, high, or very high stringency conditions (described below) to (i.e., with) a nucleic acid molecule encoding the naturally occurring PUFA PKS protein or polypeptide (i.e., to the complement of the nucleic acid strand encoding the naturally occurring PUFA PKS protein or polypeptide). Preferably, an amino acid sequence having the biological activity of at least one domain of a PUFA PKS system of the present invention is encoded by a nucleic acid sequence that hybridizes under moderate, high or very high stringency conditions to the complement of a nucleic acid sequence that encodes a protein comprising an amino acid sequence represented by any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0136] In another embodiment of the invention, a nucleotide sequence of the present invention is a nucleotide sequence isolated from (obtainable from), identical to, or a homologue of, the nucleotide sequence from a Schizochytrium, wherein the nucleotide sequence from a Schizochytrium (including either strand of a DNA molecule from Schizochytrium) hybridizes under moderate, high, or very high stringency conditions to a nucleotide sequence encoding an amino acid sequence represented by any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. In one embodiment, the Schizochytrium is Schizochytrium ATCC 20888. In another embodiment, the Schizochytrium is a daughter strain of Schizochytrium 20888, including mutated strains thereof (e.g., N230D).

[0137] Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of PUFA PKS domains and proteins of the present invention, or of the nucleotide sequences encoding such amino acid sequences.

[0138] As used herein, hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

[0139] More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid. to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10.degree. C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6.times.SSC (0.9 M Na.sup.+) at a temperature of between about 20.degree. C. and about 35.degree. C. (lower stringency), more preferably, between about 28.degree. C. and about 40.degree. C. (more stringent), and even more preferably, between about 35.degree. C. and about 45.degree. C. (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6.times.SSC (0.9 M Na.sup.+) at a temperature of between about 30.degree. C. and about 45.degree. C., more preferably, between about 38.degree. C. and about 50.degree. C., and even more preferably, between about 45.degree. C. and about 55.degree. C., with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G+C content of about 40%. Alternatively, T.sub.m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25.degree. C. below the calculated T.sub.m of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20.degree. C. below the calculated T.sub.m of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6.times.SSC (50% formamide) at about 42.degree. C., followed by washing steps that include one or more washes at room temperature in about 2.times.SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37.degree. C. in about 0.1.times.-0.5.times.SSC, followed by at least one wash at about 68.degree. C. in about 0.1.times.-0.5.times.SSC).

[0140] Yet another embodiment of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence that is identical to, or that is a homologue of (as defined above) the nucleic acid sequence of a cDNA plasmid clone selected from LIB3033-046-D2 (ATCC Accession No. ______), LIB3033-047-B5 (ATCC Accession No. ______), or LIB81-042-B9 (ATCC Accession No. ______). In another embodiment, the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence that is identical to, or that is a homologue of (as defined above) the nucleic acid sequence of a genomic plasmid selected from: pJK1126 (ATCC Accession No. ______), pJK1129 (ATCC Accession No. ______), pJK1131 (ATCC Accession No. ______), pJK306 (ATCC Accession No. ______), pJK320 (ATCC Accession No. ______), pJK324 (ATCC Accession No. ______), or pBR002 (ATCC Accession No.) ______).

[0141] Yet another embodiment of the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence that encodes an amino acid sequence that is identical to, or that is a homologue of (as defined above) the amino acid sequence encoded by a cDNA plasmid clone selected from LIB3033-046-D2 (ATCC Accession No. ______), LIB3033-047-B5 (ATCC Accession No. ______), or LIB81-042-B9 (ATCC Accession No. ______). In another embodiment, the present invention includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a nucleic acid sequence that encodes an amino acid sequence that is identical to, or that is a homologue of (as defined above) the amino acid sequence encoded by a genomic plasmid selected from: pJK1126 (ATCC Accession No. ______), pJK1129 (ATCC Accession No. ______), pJK1131 (ATCC Accession No. ______), pJK306 (ATCC Accession No. ______), pJK320 (ATCC Accession No. ______), pJK324 (ATCC Accession No. ______), or pBR002 (ATCC Accession No. ______).

[0142] Another embodiment of the present invention includes a recombinant nucleic acid molecule comprising a recombinant vector and a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence having a biological activity of at least one domain or protein of a PUFA PKS system as described herein. Such nucleic acid sequences and domains or proteins are described in detail above. According to the present invention, a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and for introducing such a nucleic acid sequence into a host cell. The recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules of the present invention or which are useful for expression of the nucleic acid molecules of the present invention (discussed in detail below). The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant organism (e.g., a microbe or a plant). The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. A recombinant vector of the present invention can contain at least one selectable marker.

[0143] In one embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is an expression vector. As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest). In this embodiment, a nucleic acid sequence encoding the product to be produced (e.g., a PUFA PKS domain) is inserted into the recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell.

[0144] In another embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is a targeting vector. As used herein, the phrase "targeting vector" is used to refer to a vector that is used to deliver a particular nucleic acid molecule into a recombinant host cell, wherein the nucleic acid molecule is used to delete or inactivate an endogenous gene within the host cell or microorganism (i.e., used for targeted gene disruption or knock-out technology). Such a vector may also be known in the art as a "knock-out" vector. In one aspect of this embodiment, a portion of the vector, but more typically, the nucleic acid molecule inserted into the vector (i.e., the insert), has a nucleic acid sequence that is homologous to a nucleic acid sequence of a target gene in the host cell (i.e., a gene which is targeted to be deleted or inactivated). The nucleic acid sequence of the vector insert is designed to bind to the target gene such that the target gene and the insert undergo homologous recombination, whereby the endogenous target gene is deleted, inactivated or attenuated (i.e., by at least a portion of the endogenous target gene being mutated or deleted).

[0145] Typically, a recombinant nucleic acid molecule includes at least one nucleic acid molecule of the present invention operatively linked to one or more transcription control sequences. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to a transcription control sequence, but can be used interchangeably with the phrase "nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced.

[0146] Recombinant nucleic acid molecules of the present invention can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molecule of the present invention, including those which are integrated into the host cell chromosome, also contains secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein. Suitable signal segments include a signal segment that is naturally associated with the protein to be expressed or any heterologous signal segment capable of directing the secretion of the protein according to the present invention. In another embodiment, a recombinant molecule of the present invention comprises a leader sequence to enable an expressed protein to be delivered to and inserted into the membrane of a host cell. Suitable leader sequences include a leader sequence that is naturally associated with the protein, or any heterologous leader sequence capable of directing the delivery and insertion of the protein to the membrane of a cell.

[0147] The present inventors have found that the Schizochytrium PUFA PKS Orfs A and B are closely linked in the genome and region between the Orfs has been sequenced. The Orfs are oriented in opposite directions and 4244 base pairs separate the start (ATG) codons (i.e. they are arranged as follows: 3'OrfA5'-4244 bp-5'OrfB3'). Examination of the 4244 bp intergenic region did not reveal any obvious Orfs (no significant matches were found on a BlastX search). Both Orfs A and B are highly expressed in Schizochytrium, at least during the time of oil production, implying that active promoter elements are embedded in this intergenic region. These genetic elements are believed to have utility as a bi-directional promoter sequence for transgenic applications. For example, in a preferred embodiment, one could clone this region, place any genes of interest at each end and introduce the construct into Schizochytrium (or some other host in which the promoters can be shown to function). It is predicted that the regulatory elements, under the appropriate conditions, would provide for coordinated, high level expression of the two introduced genes. The complete nucleotide sequence for the regulatory region containing Schizochytrium PUFA PKS regulatory elements (e.g., a promoter) is represented herein as SEQ ID NO:36.

[0148] In a similar manner, OrfC is highly expressed in Schizochytrium during the time of oil production and regulatory elements are expected to reside in the region upstream of its start codon. A region of genomic DNA upstream of OrfC has been cloned and sequenced and is represented herein as (SEQ ID NO:37). This sequence contains the 3886 nt immediately upstream of the OrfC start codon. Examination of this region did not reveal any obvious Orfs (i.e., no significant matches were found on a BlastX search). It is believed that regulatory elements contained in this region, under the appropriate conditions, will provide for high-level expression of a gene placed behind them. Additionally, under the appropriate conditions, the level of expression may be coordinated with genes under control of the A-B intergenic region (SEQ ID NO:36).

[0149] Therefore, in one embodiment, a recombinant nucleic acid molecule useful in the present invention, as disclosed herein, can include a PUFA PKS regulatory region contained within SEQ ID NO:36 and/or SEQ ID NO:37. Such a regulatory region can include any portion (fragment) of SEQ ID NO:36 and/or SEQ ID NO:37 that has at least basal PUFA PKS transcriptional activity (at least basal promoter activity).

[0150] One or more recombinant molecules of the present invention can be used to produce an encoded product (e.g., a PUFA PKS domain, protein, or system) of the present invention. In one embodiment, an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transfecting a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transfect include, but are not limited to, any bacterial, fungal (e.g., yeast), insect, plant or animal cell that can be transfected. Host cells can be either untransfected cells or cells that are already transfected with at least one other recombinant nucleic acid molecule.

[0151] According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." However, in animal cells, transformation has acquired a second meaning which can refer to changes in the growth properties of cells in culture after they become cancerous, for example. Therefore, to avoid confusion, the term "transfection" is preferably used with regard to the introduction of exogenous nucleic acids into animal cells, and the term "transfection" will be used herein to generally encompass transfection of animal cells, plant cells and transformation of microbial cells, to the extent that the terms pertain to the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques include, but are not limited to, transformation, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.

[0152] It will be appreciated by one skilled in the art that use of recombinant DNA technologies can improve control of expression of transfected nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within the host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Additionally, the promoter sequence might be genetically engineered to improve the level of expression as compared to the native promoter. Recombinant techniques useful for controlling the expression of nucleic acid molecules include, but are not limited to, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts.

[0153] General discussion above with regard to recombinant nucleic acid molecules and transfection of host cells is intended to be applied to any recombinant nucleic acid molecule discussed herein, including those encoding any amino acid sequence having a biological activity of at least one domain from a PUFA PKS, those encoding amino acid sequences from other PKS systems, and those encoding other proteins or domains.

[0154] This invention also relates to PUFA PKS systems (and proteins or domains thereof) from microorganisms other than those described specifically herein that are homologous in structure, domain organization and/or function to a Schizochytrium PUFA PKS system (and proteins or domains thereof) as described herein. In one embodiment, the microorganism is a non-bacterial microorganism, and preferably, the microorganism is a eukaryotic microorganism. In addition, this invention relates to use of these microorganisms and the PUFA PKS systems or components thereof from these microorganisms in the various applications for a PUFA PKS system (e.g., genetically modified organisms and methods of producing bioactive molecules) according to the present invention. Such microorganisms have the following characteristics: (a) produces at least one PUFA; and (b) has an ability to produce increased PUFAs under dissolved oxygen conditions of less than about 5% of saturation in the fermentation medium, as compared to production of PUFAs by said microorganism under dissolved oxygen conditions of greater than 5% of saturation, more preferably 10% of saturation, more preferably greater than 15% of saturation and more preferably greater than 20% of saturation in the fermentation medium. A screening process for identification of microorganisms comprising a PUFA PKS system is described in detail in U.S. Patent Application Publication No. 20020194641, supra. The knowledge of the structure and function of the PUFA PKS proteins and domains described herein, and the nucleotide sequence encoding the same, are useful tools for the identification, confirmation, and/or isolation of homologues of such proteins or polynucleotides.

[0155] According to the present invention, the term "Thraustochytrid" refers to any members of the order Thraustochytriales, which includes the family Thraustochytriaceae, and the term "Labyrinthulid" refers to any member of the order Labyrinthulales, which includes the family Labyrinthulaceae. The members of the family Labyrinthulaceae have been considered to be members of the order Thraustochytriales, but in revisions of the taxonomy of such organisms, the family is now considered to be a member of the order Labyrinthulales, and both Labyrinthulales and Thraustochytriales are considered to be members of the phylum Labyrinthulomycota.

[0156] Developments have resulted in frequent revision of the taxonomy of the Thraustochytrids (thraustochytrids). Taxonomic theorists generally place Thraustochytrids with the algae or algae-like protists. However, because of taxonomic uncertainty, it would be best for the purposes of the present invention to consider the strains described in the present invention as Thraustochytrids to include the following organisms: Order: Thraustochytriales; Family: Thraustochytriaceae; Genera: Thraustochytrium (Species: sp., arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, striatum), Ulkenia (previously considered by some to be a member of Thraustochytrium) (Species: sp., amoeboidea, kerguelensis, minuta, profunda, radiata, sailens, sarkariana, schizochytrops, visurgensis, yorkensis), Schizochytrium (Species: sp., aggregatum, limnaceum, mangrovei, minutum, octosporum), Japonochytrium (Species: sp., marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis, profunda, stocchinoi), Althornia (Species: sp., crouchii), or Elina (Species: sp., marisalba, sinorifica).

[0157] Strains described in the present invention as Labyrinthulids include the following organisms: Order: Labyrinthulales, Family: Labyrinthulaceae, Genera: Labyrinthula (Species: sp., algeriensis, coenocystis, chattonii, macrocystis, macrocystis atlantica, macrocystis macrocystis, marina, minuta, roscoffensis, valkanovii, vitellina, vitellina pacifica, vitellina vitellina, zopfii), Labyrinthuloides (Species: sp., haliotidis, yorkensis), Labyrinthomyxa (Species: sp., marina), Diplophrys (Species: sp., archeri), Pyrrhosorus (Species: sp., marinus), Sorodiplophrys (Species: sp., stercorea) or Chlainydomyxa (Species: sp., labyrinthuloides, montana) (although there is currently not a consensus on the exact taxonomic placement of Pyrrhosorus, Sorodiplophrys or Chlamydomyxa).

[0158] It is recognized that at the time of this invention, revision in the taxonomy of Thraustochytrids places the genus Labyrinthuloides in the family of Labyrinthulaceae and confirms the placement of the two families Thraustochytriaceae and Labyrinthulaceae within the Stramenopile lineage. It is noted that the Labyrinthulaceae are sometimes commonly called labyrinthulids or labyrinthula, or labyrinthuloides and the Thraustochytriaceae are commonly called thraustochytrids.

[0159] To produce significantly high yields of various bioactive molecules using the PUFA PKS system of the present invention, an organism, preferably a microorganism or a plant, can be genetically modified to affect the activity of a PUFA PKS system. In one aspect, such an organism can endogenously contain and express a PUFA PKS system, and the genetic modification can be a genetic modification of one or more of the functional domains of the endogenous PUFA PKS system, whereby the modification has some effect on the activity of the PUFA PKS system. In another aspect, such an organism can endogenously contain and express a PUFA PKS system, and the genetic modification can be an introduction of at least one exogenous nucleic acid sequence (e.g., a recombinant nucleic acid molecule), wherein the exogenous nucleic acid sequence encodes at least one biologically active domain or protein from the same or a second PKS system and/or a protein that affects the activity of said PUFA PKS system (e.g., a phosphopantetheinyl transferases (PPTase), discussed below). In yet another aspect, the organism does not necessarily endogenously (naturally) contain a PUFA PKS system, but is genetically modified to introduce at least one recombinant nucleic acid molecule encoding an amino acid sequence having the biological activity of at least one domain of a PUFA PKS system. In this aspect, PUFA PKS activity is affected by introducing or increasing PUFA PKS activity in the organism. Various embodiments associated with each of these aspects will be discussed in greater detail below.

[0160] Therefore, according to the present invention, one embodiment relates to a genetically modified microorganism, wherein the microorganism expresses a PKS system comprising at least one biologically active domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system. The at least one domain of the PUFA PKS system is encoded by a nucleic acid sequence described herein. The genetic modification affects the activity of the PKS system in the organism. The genetically modified microorganism can include any one or more of the above-identified nucleic acid sequences, and/or any of the other homologues of any of the Schizochytrium PUFA PKS ORFs or domains as described in detail above.

[0161] As used herein, a genetically modified microorganism can include a genetically modified bacterium, protist, microalgae, fungus, or other microbe, and particularly, any of the genera of the order Thraustochytriales (e.g., a Thraustochytrid) described herein. Such a genetically modified microorganism has a genome which is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (i.e., increased or modified PUFA PKS activity and/or production of a desired product using the PUFA PKS system or component thereof). Genetic modification of a microorganism can be accomplished using classical strain development and/or molecular genetic techniques. Such techniques known in the art and are generally disclosed for microorganisms, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. The reference Sambrook et al., ibid., is incorporated by reference herein in its entirety. A genetically modified microorganism can include a microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect within the microorganism.

[0162] Preferred microorganism host cells to modify according to the present invention include, but are not limited to, any bacteria, protist, microalga, fungus, or protozoa. In one aspect, preferred microorganisms to genetically modify include, but are not limited to, any microorganism of the order Thraustochytriales or any microorganism of the order Labyrinthulales. Particularly preferred host cells for use in the present invention could include microorganisms from a genus including, but not limited to: Thraustochytrium, Ulkenia, Schizochytrium, Japonochytrium, Aplanochytrium, Althornia, Elina, Labyrinthula, Labyrinthuloides, Labyrinthomyxa, Diplophrys, Pyrrhosorus, Sorodiplophrys or Chlamydomyxa. Other examples of suitable host microorganisms for genetic modification include, but are not limited to, yeast including Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeast such as Candida, Kluyveromyces, or other fungi, for example, filamentous fungi such as Aspergillus, Neurospora, Penicillium, etc. Bacterial cells also may be used as hosts. This includes Escherichia coli, which can be useful in fermentation processes. Alternatively, a host such as a Lactobacillus species or Bacillus species can be used as a host.

[0163] Another embodiment of the present invention relates to a genetically modified plant or part of a plant (e.g., wherein the plant has been genetically modified to express a PUPA PKS system described herein), which includes at least the core PUFA PKS enzyme complex and, in one embodiment, at least one PUFA PKS accessory protein, (e.g., a PPTase), so that the plant produces PUFAs. Preferably, the plant is an oil seed plant, wherein the oil seeds or oil in the oil seeds contain PUFAs produced by the PUFA PKS system. Such oils contain a detectable amount of at least one target or primary PUFA that is the product of the PUFA PKS system. Plants are not known to endogenously contain a PUFA PKS system, and therefore, the PUFA PKS systems of the present invention represent an opportunity to produce plants with unique fatty acid production capabilities. It is a particularly preferred embodiment of the present invention to genetically engineer plants to produce one or more PUFAs in the same plant, including, EPA, DHA, DPA, ARA, GLA, SDA and others. The present invention offers the ability to create any one of a number of "designer oils" in various ratios and forms. Moreover, the disclosure of the PUFA PKS genes from the particular marine organisms described herein offer the opportunity to more readily extend the range of PUFA production and successfully produce such PUFAs within temperature ranges used to grow most crop plants.

[0164] Methods for the genetic engineering of plants are well known in the art. For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.

[0165] The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumnefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by numerous references, including Gruber et al., supra, Miki et al., supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos. 4,940,838 and 5,464,763.

[0166] Another generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Sanford, J. C., Physiol. Plant 79:206 (1990), Klein et al., Biotechnology 10:268 (1992).

[0167] Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of Vllth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).

[0168] As used herein, a genetically modified plant can include any genetically modified plant including higher plants and particularly, any consumable plants or plants useful for producing a desired bioactive molecule of the present invention. "Plant parts", as used herein, include any parts of a plant, including, but not limited to, seeds (immature or mature), oils, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc. A genetically modified plant has a genome that is modified (i.e., mutated or changed) from its normal (i.e., wild-type or naturally occurring) form such that the desired result is achieved (e.g., PUFA PKS activity and production of PUFAs). Genetic modification of a plant can be accomplished using classical strain development and/or molecular genetic techniques. Methods for producing a transgenic plant, wherein a recombinant nucleic acid molecule encoding a desired amino acid sequence is incorporated into the genome of the plant, are known in the art. A preferred plant to genetically modify according to the present invention is preferably a plant suitable for consumption by animals, including humans.

[0169] Preferred plants to genetically modify according to the present invention (i.e., plant host cells) include, but are not limited to any higher plants, including both dicotyledonous and monocotyledonous plants, and particularly consumable plants, including crop plants and especially plants used for their oils. Such plants can include, for example: canola, soybeans, rapeseed, linseed, corn, safflowers, sunflowers and tobacco. Other preferred plants include those plants that are known to produce compounds used as pharmaceutical agents, flavoring agents, nutraceutical agents, functional food ingredients or cosmetically active agents or plants that are genetically engineered to produce these compounds/agents.

[0170] According to the present invention, a genetically modified microorganism or plant includes a microorganism or plant that has been modified using recombinant technology. As used herein, genetic modifications that result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) can be referred to as inactivation (complete or partial), deletion, interruption, blockage or down-regulation of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such gene, can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity or action). Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene.

[0171] The genetic modification of a microorganism or plant according to the present invention preferably affects the activity of the PKS system expressed by the plant, whether the PKS system is endogenous and genetically modified, endogenous with the introduction of recombinant nucleic acid molecules into the organism, or provided completely by recombinant technology. According to the present invention, to "affect the activity of a PKS system" includes any genetic modification that causes any detectable or measurable change or modification in the PKS system expressed by the organism as compared to in the absence of the genetic modification. A detectable change or modification in the PKS system can include, but is not limited to: the introduction of PKS system activity into an organism such that the organism now has measurable/detectable PKS system activity (i.e., the organism did not contain a PKS system prior to the genetic modification), the introduction into the organism of a functional domain from a different PKS system than a PKS system endogenously expressed by the organism such that the PKS system activity is modified (e.g., a bacterial PUFA PKS domain or a type I PKS domain is introduced into an organism that endogenously expresses a non-bacterial PUFA PKS system), a change in the amount of a bioactive molecule produced by the PKS system (e.g., the system produces more (increased amount) or less (decreased amount) of a given product as compared to in the absence of the genetic modification), a change in the type of a bioactive molecule produced by the PKS system (e.g., the system produces a new or different product, or a variant of a product that is naturally produced by the system), and/or a change in the ratio of multiple bioactive molecules produced by the PKS system (e.g., the system produces a different ratio of one PUFA to another PUFA, produces a completely different lipid profile as compared to in the absence of the genetic modification, or places various PUFAs in different positions in a triacylglycerol as compared to the natural configuration). Such a genetic modification includes any type of genetic modification and specifically includes modifications made by recombinant technology and by classical mutagenesis.

[0172] It should be noted that reference to increasing the activity of a functional domain or protein in a PUFA PKS system refers to any genetic modification in the organism containing the domain or protein (or into which the domain or protein is to be introduced) which results in increased functionality of the domain or protein system and can include higher activity of the domain or protein (e.g., specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the domain or protein system, and overexpression of the domain or protein. For example, gene copy number can be increased, expression levels can be increased by use of a promoter that gives higher levels of expression than that of the native promoter, or a gene can be altered by genetic engineering or classical mutagenesis to increase the activity of the domain or protein encoded by the gene.

[0173] Similarly, reference to decreasing the activity of a functional domain or protein in a PUFA PKS system refers to any genetic modification in the organism containing such domain or protein (or into which the domain or protein is to be introduced) which results in decreased functionality of the domain or protein and includes decreased activity of the domain or protein, increased inhibition or degradation of the domain or protein and a reduction or elimination of expression of the domain or protein. For example, the action of domain or protein of the present invention can be decreased by blocking or reducing the production of the domain or protein, "knocking out" the gene or portion thereof encoding the domain or protein, reducing domain or protein activity, or inhibiting the activity of the domain or protein. Blocking or reducing the production of a domain or protein can include placing the gene encoding the domain or protein under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the domain or protein (and therefore, of protein synthesis) could be turned off. Blocking or reducing the activity of domain or protein could also include using an excision technology approach similar to that described in U.S. Pat. No. 4,743,546, incorporated herein by reference. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal.

[0174] In one embodiment of the present invention, a genetic modification includes a modification of a nucleic acid sequence encoding an amino acid sequence that has a biological activity of at least one domain of a non-bacterial PUFA PKS system as described herein. Such a modification can be to an amino acid sequence within an endogenously (naturally) expressed non-bacterial PUFA PKS system, whereby a microorganism that naturally contains such a system is genetically modified by, for example, classical mutagenesis and selection techniques and/or molecular genetic techniques, include genetic engineering techniques. Genetic engineering techniques can include, for example, using a targeting recombinant vector to delete a portion of an endogenous gene, or to replace a portion of an endogenous gene with a heterologous sequence. Examples of heterologous sequences that could be introduced into a host genome include sequences encoding at least one functional domain from another PKS system, such as a different non-bacterial PUFA PKS system, a bacterial PUFA PKS system, a type I PKS system, a type II PKS system, or a modular PKS system. Other heterologous sequences to introduce into the genome of a host includes a sequence encoding a protein or functional domain that is not a domain of a PKS system, but which will affect the activity of the endogenous PKS system. For example, one could introduce into the host genome a nucleic acid molecule encoding a phosphopantetheinyl transferase (discussed below). Specific modifications that could be made to an endogenous PUPA PKS system are discussed in detail below.

[0175] In another aspect of this embodiment of the invention, the genetic modification can include: (1) the introduction of a recombinant nucleic acid molecule encoding an amino acid sequence having a biological activity of at least one domain of a non-bacterial PUFA PKS system; and/or (2) the introduction of a recombinant nucleic acid molecule encoding a protein or functional domain that affects the activity of a PUFA PKS system, into a host. The host can include: (1) a host cell that does not express any PKS system, wherein all functional domains of a PKS system are introduced into the host cell, and wherein at least one functional domain is from a non-bacterial PUFA PKS system; (2) a host cell that expresses a PKS system (endogenous or recombinant) having at least one functional domain of a non-bacterial PUFA PKS system, wherein the introduced recombinant nucleic acid molecule can encode at least one additional non-bacterial PUFA PKS domain function or another protein or domain that affects the activity of the host PKS system; and (3) a host cell that expresses a PKS system (endogenous or recombinant) which does not necessarily include a domain function from a non-bacterial PUFA PKS, and wherein the introduced recombinant nucleic acid molecule includes a nucleic acid sequence encoding at least one functional domain of a non-bacterial PUFA PKS system. In other words, the present invention intends to encompass any genetically modified organism (e.g., microorganism or plant), wherein the organism comprises at least one non-bacterial PUFA PKS domain function (either endogenously or by recombinant modification), and wherein the genetic modification has a measurable effect on the non-bacterial PUFA PKS domain function or on the PKS system when the organism comprises a functional PKS system.

[0176] Therefore, using the PUFA PKS systems of the present invention, gene mixing can be used to extend the range of PUFA products (and ratios thereof) to include EPA, DPA, DHA, ARA, GLA, SDA and others, as well as to produce a wide variety of bioactive molecules, including antibiotics, other pharmaceutical compounds, and other desirable products. The method to obtain these bioactive molecules includes not only the mixing of genes from various organisms but also various methods of genetically modifying the non-bacterial PUFA PKS genes disclosed herein. Knowledge of the genetic basis and domain structure of the non-bacterial PUFA PKS system of the present invention provides a basis for designing novel genetically modified organisms which produce a variety of bioactive molecules. Although mixing and modification of any PKS domains and related genes are contemplated by the present inventors, by way of example, various possible manipulations of the PUFA-PKS system are discussed in U.S. Patent Application Publication No. 20020194641, U.S. Patent Application Publication No. 20040235127, and U.S. Patent Application Publication No. 20050100995, supra with regard to genetic modification and bioactive molecule production.

[0177] The comparison of the Schizochytrium PUFA PKS architecture (domain organization) with other PUFA PKS system architecture illustrates nature's ability to alter domain order as well as incorporate new domains to create novel end products. In addition, the genes can now be manipulated in the laboratory to create new products. Proposed herein is the manipulation of PUFA PKS systems in either a directed or random way to influence the end products. For example, in a preferred embodiment, one could envision substituting one of the DH (FabA-like) domains of the PUFA-PKS system for a DH domain that did not posses isomerization activity, potentially creating a molecule with a mix of cis- and trans-double bonds. The current products of the Schizochytrium PUFA PKS system are DHA and DPA (C22:5 .omega.6). If one manipulated the system to produce C20 fatty acids, one would expect the products to be EPA and ARA (C20:4 .omega.6). This could provide a new source for ARA. One could also substitute domains from related PUFA-PKS systems that produced a different DHA to DPA ratio, for example, by using genes from Thraustochytrium 23B.

[0178] Additionally, one could envision specifically altering one of the ER domains (e.g. removing, or inactivating) in the Schizochytrium PUFA PKS system (other PUFA PKS systems described so far do not have two ER domains) to affect the end product profile. Similar strategies could be attempted in a directed manner for each of the distinct domains of the PUFA-PKS proteins using more or less sophisticated approaches. Of course one would not be limited to the manipulation of single domains. Finally, one could extend the approach by mixing domains from the PUFA-PKS system and other PKS or FAS systems (e.g., type I, type II, type III) to create an entire range of new end products. For example, one could introduce the PUFA-PKS DH domains into systems that do not normally incorporate cis double bonds into their end products.

[0179] Accordingly, encompassed by the present invention are methods to genetically modify microbial or plant cells by: genetically modifying at least one nucleic acid sequence in the organism that encodes an amino acid sequence having the biological activity of at least one functional domain of a PUFA PKS system according to the present invention, and/or expressing at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding such amino acid sequence. Various embodiments of such sequences, methods to genetically modify an organism, and specific modifications have been described in detail above. Typically, the method is used to produce a particular genetically modified organism that produces a particular bioactive molecule or molecules.

[0180] In one embodiment of the present invention, it is contemplated that a mutagenesis program could be combined with a selective screening process to obtain bioactive molecules of interest. This would include methods to search for a range of bioactive compounds. This search would not be restricted to production of those molecules with cis double bonds. The mutagenesis methods could include, but are not limited to: chemical mutagenesis, gene shuffling, switching regions of the genes encoding specific enzymatic domains, or mutagenesis restricted to specific regions of those genes, as well as other methods.

[0181] For example, high throughput mutagenesis methods could be used to influence or optimize production of the desired bioactive molecule. Once an effective model system has been developed, one could modify these genes in a high throughput manner. Utilization of these technologies can be envisioned on two levels. First, if a sufficiently selective screen for production of a product of interest (e.g., ARA) can be devised, it could be used to attempt to alter the system to produce this product (e.g., in lieu of, or in concert with, other strategies such as those discussed above). Additionally, if the strategies outlined above resulted in a set of genes that did produce the product of interest, the high throughput technologies could then be used to optimize the system. For example, if the introduced domain only functioned at relatively low temperatures, selection methods could be devised to peiinit removing that limitation. In one embodiment of the invention, screening methods are used to identify additional non-bacterial organisms having novel PKS systems similar to the PUFA PKS system of Schizochytrium, as described herein (see above). Homologous PKS systems identified in such organisms can be used in methods similar to those described herein for the Schizochytrium, as well as for an additional source of genetic material from which to create, further modify and/or mutate a PUFA PKS system for expression in that microorganism, in another microorganism, or in a higher plant, to produce a variety of compounds.

[0182] It is recognized that many genetic alterations, either random or directed, which one may introduce into a native (endogenous, natural) PUFA PKS system, will result in an inactivation of enzymatic functions. A preferred embodiment of the invention includes a system to select for only those modifications that do not block the ability of the PUFA PKS system to produce a product. For example, the FabB-strain of E. coli is incapable of synthesizing unsaturated fatty acids and requires supplementation of the medium with fatty acids that can substitute for its normal unsaturated fatty acids in order to grow (see Metz et al., 2001, supra). However, this requirement (for supplementation of the medium) can be removed when the strain is transformed with a functional PUFA-PKS system (i.e. one that produces a PUFA product in the E. coli host--see (Metz et al., 2001, supra, FIG. 2A). The transformed FabB-strain now requires a functional PUFA-PKS system (to produce the unsaturated fatty acids) for growth without supplementation. The key element in this example is that production of a wide range of unsaturated fatty acid will suffice (even unsaturated fatty acid substitutes such as branched chain fatty acids). Therefore, in another preferred embodiment of the invention, one could create a large number of mutations in one or more of the PUFA PKS genes disclosed herein, and then transform the appropriately modified FabB-strain (e.g. create mutations in an expression construct containing an ER domain and transform a FabB-strain having the other essential domains on a separate plasmid--or integrated into the chromosome) and select only for those transformants that grow without supplementation of the medium (i.e., that still possessed an ability to produce a molecule that could complement the FabB-defect). Additional screens could be developed to look for particular compounds (e.g. use of GC for fatty acids) being produced in this selective subset of an active PKS system. One could envision a number of similar selective screens for bioactive molecules of interest.

[0183] In one embodiment of invention, a genetically modified organism has a modification that changes at least one product produced by the endogenous PKS system, as compared to a wild-type organism.

[0184] In one embodiment, a genetically modified organism has been modified by transfecting the organism with a recombinant nucleic acid molecule encoding a protein that regulates the chain length of fatty acids produced by the PUFA PKS system. For example, the protein that regulates the chain length of fatty acids produced by the PUFA PKS system can be a chain length factor that directs the synthesis of C20 units or C22 units.

[0185] In another embodiment, a genetically modified organism expresses a PUFA PKS system comprising a genetic modification in a domain selected from the group consisting of a domain encoding .beta.-hydroxy acyl-ACP dehydrase (DH) and a domain encoding .beta.-ketoacyl-ACP synthase (KS), wherein the modification alters the ratio of long chain fatty acids produced by the PUFA PKS system as compared to in the absence of the modification. In one aspect of this embodiment, the modification is selected from the group consisting of a deletion of all or a part of the domain, a substitution of a homologous domain from a different organism for the domain, and a mutation of the domain.

[0186] In another embodiment, a genetically modified organism expresses a PUFA PKS system comprising a modification in an enoyl-ACP reductase (ER) domain, wherein the modification results in the production of a different compound as compared to in the absence of the modification. In one aspect of this embodiment, the modification is selected from the group consisting of a deletion of all or a part of the ER domain, a substitution of an ER domain from a different organism for the ER domain, and a mutation of the ER domain.

[0187] In one embodiment of the invention, the genetically modified organism produces a polyunsaturated fatty acid (PUFA) profile that differs from the naturally occurring organism without a genetic modification.

[0188] Many other genetic modifications useful for producing bioactive molecules will be apparent to those of skill in the art, given the present disclosure, and various other modifications have been discussed previously herein. The present invention contemplates any genetic modification related to a PUFA PKS system as described herein which results in the production of a desired bioactive molecule.

[0189] As described above, in one embodiment of the present invention, a genetically modified microorganism or plant includes a microorganism or plant which has an enhanced ability to synthesize desired bioactive molecules (products) or which has a newly introduced ability to synthesize specific products (e.g., to synthesize a specific antibiotic). According to the present invention, "an enhanced ability to synthesize" a product refers to any enhancement, or up-regulation, in a pathway related to the synthesis of the product such that the microorganism or plant produces an increased amount of the product (including any production of a product where there was none before) as compared to the wild-type microorganism or plant, cultured or grown, under the same conditions. Methods to produce such genetically modified organisms have been described in detail above. In one preferred embodiment, the present invention relates to a genetically modified plant or part of a plant (e.g., wherein the plant has been genetically modified to express a PUFA PKS system described herein), which includes at least the core PUFA PKS enzyme complex and, in one embodiment, at least one PUFA PKS accessory protein, (e.g., a PPTase), so that the plant produces PUFAs. Preferably, the plant is an oil seed plant, wherein the oil seeds or oil in the oil seeds contain PUFAs produced by the PUFA PKS system. Such oils contain a detectable amount of at least one target or primary PUFA that is the product of the PUFA PKS system.

[0190] The present inventors demonstrate herein the production of PUFAs in a plant that has been genetically modified to express the genes encoding a PUFA PKS system from Schizochytrium of the present invention and a PUFA PKS accessory enzyme, 4'-phosphopantetheinyl transferase (PPTase). The oils produced by these plants contain significant quantities of both DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), which are the predominant PUFAs (the primary PUFAs) produced by the Schizochytrium from which the PUFA PKS genes were derived. Significantly, oils from plants that produce PUFAs using the PUFA PKS pathway have a different fatty acid profile than plants that are genetically engineered to produce the same PUFAs by the "standard" pathway described above. In particular, oils from plants that have been genetically engineered to produce specific PUFAs by the PUFA PKS pathway are substantially free of the various intermediate products and side products that accumulate in oils that are produced as a result of the use of the standard PUFA synthesis pathway. This characteristic is discussed in detail below.

[0191] More particularly, efforts to produce long chain PUFAs in plants by the "standard" pathway have all taken the same basic approach, which is dictated by this synthesis pathway. These efforts relied on modification of the plants' endogenous fatty acids by introduction of genes encoding various elongases and desaturases. Plants typically produce 18 carbon fatty acids (e.g., oleic acid, linoleic acid, linolenic acid) via the Type II fatty acid synthase (FAS) in its plastids. Often, a single double bond is formed while that fatty acid is attached to ACP, and then the oleic acid (18:1) is cleaved from the ACP by the action of an acyl-ACP thioesterase. The free fatty acid is exported from the plastid and converted to an acyl-CoA. The 18:1 can be esterified to phosphatidylcholine (PC) and up to two more cis double bonds can be added. The newly introduced elongases can utilize substrates in the acyl-CoA pool to add carbons in two-carbon increments. Newly introduced desaturases can utilize either fatty acids esterified to PC, or those in the acyl-CoA pool, depending on the source of the enzyme. One consequence of this scheme for long chain PUFA production, however, is that intermediates or side products in the pathway accumulate, which often represent the majority of the novel fatty acids in the plant oil, rather than the target long chain PUFA.

[0192] For example, using the standard or classical pathway as described above, when the target PUFA product (i.e., the PUFA product that one is targeting for production, trying to produce, attempting to produce, by using the standard pathway) is DHA or EPA, for example (e.g., produced using elongases and desaturases that will produce the DHA or EPA from the products of the FAS system), a variety of intermediate products and side products will be produced in addition to the DHA or EPA, and these intermediate or side products frequently represent the majority of the products produced by the pathway, or are at least present in significant amounts in the lipids of the production organism. Such intermediate and side products include, but are not limited to, fatty acids having fewer carbons and/or fewer double bonds than the target, or primary PUFA, and can include unusual fatty acid side products that may have the same number of carbons as the target or primary PUFA, but which may have double bonds in unusual positions. By way of example, in the production of EPA using the standard pathway (e.g., see U.S. Patent Application Publication 2004/0172682), while the target PUFA of the pathway is EPA (i.e., due to the use of elongases and desaturases that specifically act on the products of the FAS system to produce EPA), the oils produced by the system include a variety of intermediate and side products including: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other intermediate or side products, such as 20:0; 20:1 (.DELTA.5); 20:1 (.DELTA.11): 20:2 (.DELTA.8,11); 20:2 (.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17). Intermediates of the system can also include long chain PUFAs that are not the target of the genetic modification (e.g., a standard pathway enzyme system for producing DHA can actually produce more EPA as an intermediate product than DHA).

[0193] In contrast, the PUFA PKS synthase of the present invention does not utilize the fatty acid products of FAS systems. Instead, it produces the final PUFA product (the primary PUFA product) from the same small precursor molecule that is utilized by FASB and elongases (malonyl-CoA). Therefore, intermediates in the synthesis cycle are not released in any significant amount, and the PUFA product (also referred to herein as the primary PUFA product) is efficiently transferred to phospholipids (PL) and triacylglycerol (TAG) fractions of the lipids. Indeed, a PUFA PKS system may produce two target or primary PUFA products (e.g., the PUFA PKS system from Schizochytrium produces both DHA and DPA n-6 as primary products), but DPA is not an inteimediate in the pathway to produce DHA. Rather, each is a separate product of the same PUFA PKS system. Therefore, the PUFA PKS genes of the present invention are an excellent means of producing oils containing PUFAs, and particularly, LCPUFAs in a heterologous host, such as a plant, wherein the oils are substantially free (defined below) of the intermediates and side products that contaminate oils produced by the "standard" PUFA pathway.

[0194] Therefore, it is an object of the present invention to produce, via the genetic manipulation of plants as described herein, polyunsaturated fatty acids and, by extension, oils obtained from such plants (e.g., obtained from the oil seeds of such plants) comprising these PUFAs. Examples of PUFAs that can be produced by the present invention include, but are not limited to, DHA (docosahexaenoic acid (C22:6, n-3)), ARA (eicosatetraenoic acid or arachidonic acid (C20:4, n-6)), DPA (docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA (eicosapentaenoic acid (C20:5, n-3)). The present invention allows for the production of commercially valuable lipids enriched in one or more desired (target or primary) PUFAs by the present inventors' development of genetically modified plants through the use of the polyketide synthase system of the present invention, as well as components thereof, that produces PUFAs.

[0195] According to the present invention, reference to a "primary PUFA", "target PUFA", "intended PUFA", or "desired PUFA" refers to the particular PUFA or PUFAs that are the intended product of the enzyme pathway that is used to produce the PUFA(s). For example, when using elongases and desaturases to modify products of the FAS system in the classical pathway for PUFA production, one can select particular combinations of elongases and desaturases that, when used together, will produce a target or desired PUFA (e.g., DHA or EPA). As discussed above, such target or desired PUFA produced by the standard pathway may not actually be a "primary" PUFA in terms of the amount of PUFA as a percentage of total fatty acids produced by the system, due to the formation of intermediates and side products that can actually represent the majority of products produced by the system. However, one may use the term "primary PUFA" even in that instance to refer to the target or intended PUFA product produced by the elongases or desaturases used in the system.

[0196] In contrast to the classical pathway for PUFA production, when using a PUFA PKS system, a given PUFA PKS system derived from a particular organism (or created from combining proteins and domains from PUFA PKS systems) will produce particular PUFA(s), such that selection of a PUFA PKS system from a particular organism will result in the production of specified target or primary PUFAs. For example, use of a PUFA PKS system from Schizochytrium according to the present invention will result in the production of DHA and DPAn-6 as the target or primary PUFAs. However, as discussed above, the use of various proteins and domains with proteins and domains from other PUPA PKS systems or other PKS systems (that produce bioactive molecules other than PUFAs) can be combined ("mixed and matched") to result in the production of different PUFA profiles.

[0197] When using a PUFA PKS system of the present invention, oils produced by the organism, such as a plant, are substantially free of intermediate or side products that are not the target or primary PUFA products and that are not naturally produced by the endogenous FAS system in the wild-type organism (e.g., wild-type plants produce some shorter or medium chain PUFAs, such as 18 carbon PUFAs, via the FAS system, but there will be new, or additional, fatty acids produced in the plant as a result of genetic modification with a PUFA PKS system). In other words, as compared to the profile of total fatty acids from the wild-type plant (not genetically modified) or the parent plant used as a recipient for the indicated genetic modification, the majority of additional fatty acids in the profile of total fatty acids produced by plants that have been genetically modified with the PUFA PKS system of the present invention (or a component thereof), comprise the target or intended PUFA products of the PUFA PKS system (i.e., the majority of additional fatty acids in the total fatty acids that are produced by the genetically modified plant are the target PUFA(s)).

[0198] According to the present invention, reference to "intermediate products" or "side products" of an enzyme system that produces PUFAs refers to any products, and particularly, fatty acid products, that are produced by the enzyme system as a result of the production of the target or primary PUFA of the system. Intermediate and side products are particularly significant in the standard pathway for PUFA synthesis and are substantially less significant in the PUFA PKS pathway, as discussed above. It is noted that a primary or target PUFA of one enzyme system may be an intermediate of a different enzyme system where the primary or target product is a different PUFA, and this is particularly true of products of the standard pathway of PUFA production, since the PUFA PKS system of the present invention substantially avoids the production of intermediates. For example, when using the standard pathway to produce EPA, fatty acids such as GLA, DGLA and SDA are produced as intermediate products in significant quantities (e.g., U.S. Patent Application Publication 2004/0172682 illustrates this point). Similarly, and also illustrated by U.S. Patent Application Publication 2004/0172682, when using the standard pathway to produce DHA, in addition to the fatty acids mentioned above, ETA and EPA (notably the target PUFA in the first example above) are produced in significant quantities and in fact, may be present in significantly greater quantities relative to the total fatty acid product than the target PUFA itself. This latter point is shown in U.S. Patent Application Publication 2004/0172682, where a plant that was engineered to produce DHA by the standard pathway produces more EPA as a percentage of total fatty acids than DHA.

[0199] Furthermore, to be "substantially free" of intermediate or side products of the system for synthesizing PUFAs, or to not have intermediate or side products present in substantial amounts, means that any intermediate or side product fatty acids that are produced in the genetically modified plant (and/or parts of plants and/or seed oil fraction) as a result of the enzyme system for producing PUFAS (i.e., that are not produced by the wild-type plant or the parent plant used as a recipient for the indicated genetic modification), are present in a quantity that is less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% by weight of the total fatty acids produced by the plant.

[0200] In a preferred embodiment, to be "substantially free" of intermediate or side products of the system for synthesizing PUFAs, or to not have intermediate or side products present in substantial amounts, means that any intermediate or side product fatty acids that are produced in the genetically modified plant (and/or parts of plants and/or seed oil fraction) as a result of the enzyme system for producing PUFAS (i.e., that are not produced by the wild-type plant or the parent plant used as a recipient for the indicated genetic modification), are present in a quantity that is less than about 10% by weight of the total additional fatty acids produced by the plant (additional fatty acids being those that are not produced by the wild-type plant or the parent plant used as a recipient for the indicated genetic modification), and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of the total additional fatty acids produced by the plant. Therefore, in contrast to the fatty acid profile of plants that have been genetically modified to produce PUFAs via the standard pathway, the majority of fatty acid products resulting from the genetic modification with a PUFA PKS system will be the target or intended fatty acid products.

[0201] When the target product of a PUFA PKS system is a long chain PUFA, such as DHA or DPA (n-6 or n-3) produced by the PUFA PKS system of the invention described herein, intermediate products and side products that are not present in substantial amounts in the total lipids of plants genetically modified with such PUFA PKS can include, but are not limited to: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other intermediate or side products, such as 20:0; 20:1 (.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2 (.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17). In addition, when the target product is a particular PUFA, such as DHA, the intermediate products and side products that are not present in substantial amounts in the total lipids of the genetically modified plants also include other PUFAs, including other PUFAs that are a natural product of a different PUFA PKS system, such as EPA in this example. It is to be noted that the PUFA PKS system of the present invention can also be used, if desired, to produce as a target PUFA a PUFA that can include GLA, SDA or DGLA (referring to embodiments where oils are produced using components of a PUFA PKS system described herein).

[0202] Using the knowledge of the genetic basis and domain structure of the PUFA PKS system described herein, the present inventors have designed and produced constructs encoding such a PUFA PKS system and have successfully produced transgenic plants expressing the PUFA PKS system. The transgenic plants produce oils containing PUFAs, and the oils are substantially free of intermediate products that accumulate in a standard PUFA pathway (see Example 3). The present inventors have also demonstrated the use of the constructs to produce PUFAs in another eukaryote, yeast, as a proof-of-concept experiment prior to the production of the transgenic plants (see Example 2). The examples demonstrate that transformation of both yeast and plants with a PUFA PKS system that produces DHA and DPAn-6 as the target PUFAs produces both of these PUFAs as the primary additional fatty acids in the total fatty acids of the plant (i.e., subtracting fatty acids that are produced in the wild-type plant), and in the yeast and further, that any other fatty acids that are not present in the fatty acids of the wild-type plant are virtually undetectable. Specific characteristics of genetically modified plants and parts and oils thereof of the present invention are described in detail elsewhere herein.

[0203] Accordingly, one embodiment of the present invention is a method to produce desired bioactive molecules (also referred to as products or compounds) by growing or culturing a genetically modified microorganism or a genetically modified plant of the present invention (described in detail above). Such a method includes the step of culturing in a fermentation medium or growing in a suitable environment, such as soil, a microorganism or plant, respectively, that has a genetic modification as described previously herein and in accordance with the present invention. In a preferred embodiment, method to produce bioactive molecules of the present invention includes the step of culturing under conditions effective to produce the bioactive molecule a genetically modified organism that expresses a PKS system comprising at least one biologically active domain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system as described herein.

[0204] In the method of production of desired bioactive compounds of the present invention, a genetically modified microorganism is cultured or grown in a suitable medium, under conditions effective to produce the bioactive compound. An appropriate, or effective, medium refers to any medium in which a genetically modified microorganism of the present invention, when cultured, is capable of producing the desired product. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients. Microorganisms of the present invention can be cultured in conventional fermentation bioreactors. The microorganisms can be cultured by any fermentation process which includes, but is not limited to, batch, fed-batch, cell recycle, and continuous fermentation. Preferred growth conditions for potential host microorganisms according to the present invention are well known in the art. The desired bioactive molecules produced by the genetically modified microorganism can be recovered from the fermentation medium using conventional separation and purification techniques. For example, the fermentation medium can be filtered or centrifuged to remove microorganisms, cell debris and other particulate matter, and the product can be recovered from the cell-free supernatant by conventional methods, such as, for example, ion exchange, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization. Alternatively, microorganisms producing the desired compound, or extracts and various fractions thereof, can be used without removal of the microorganism components from the product.

[0205] In the method for production of desired bioactive compounds of the present invention, a genetically modified plant is cultured in a fermentation medium or grown in a suitable medium such as soil. An appropriate, or effective, fermentation medium has been discussed in detail above. A suitable growth medium for higher plants includes any growth medium for plants, including, but not limited to, soil, sand, any other particulate media that support root growth (e.g. vermiculite, perlite, etc.) or Hydroponic culture, as well as suitable light, water and nutritional supplements which optimize the growth of the higher plant. The genetically modified plants of the present invention are engineered to produce significant quantities of the desired product through the activity of the PKS system that is genetically modified according to the present invention. The compounds can be recovered through purification processes which extract the compounds from the plant. In a preferred embodiment, the compound is recovered by harvesting the plant. In this embodiment, the plant can be consumed in its natural state or further processed into consumable products.

[0206] Bioactive molecules, according to the present invention, include any molecules (compounds, products, etc.) that have a biological activity, and that can be produced by a PKS system that comprises at least one amino acid sequence having a biological activity of at least one functional domain of a non-bacterial PUFA PKS system as described herein. Such bioactive molecules can include, but are not limited to: a polyunsaturated fatty acid (PUFA), an anti-inflammatory formulation, a chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, a drug for treatment of neurodegenerative disease, a drug for treatment of degenerative liver disease, an antibiotic, and a cholesterol lowering formulation. One advantage of the non-bacterial PUFA PKS system of the present invention is the ability of such a system to introduce carbon-carbon double bonds in the cis configuration, and molecules including a double bond at every third carbon. This ability can be utilized to produce a variety of compounds.

[0207] With respect to microorganisms, preferably, bioactive compounds of interest are produced by the genetically modified microorganism in an amount that is greater than about 0.05%, and preferably greater than about 0.1%, and more preferably greater than about 0.25%, and more preferably greater than about 0.5%, and more preferably greater than about 0.75%, and more preferably greater than about 1%, and more preferably greater than about 2.5%, and more preferably greater than about 5%, and more preferably greater than about 10%, and more preferably greater than about 15%, and even more preferably greater than about 20% of the dry weight of the microorganism. For lipid compounds, preferably, such compounds are produced in an amount that is greater than about 5% of the dry weight of the microorganism. For other bioactive compounds, such as antibiotics or compounds that are synthesized in smaller amounts, those strains possessing such compounds at of the dry weight of the microorganism are identified as predictably containing a novel. PKS system of the type described above. In some embodiments, particular bioactive molecules (compounds) are secreted by the microorganism, rather than accumulating. Therefore, such bioactive molecules are generally recovered from the culture medium and the concentration of molecule produced will vary depending on the microorganism and the size of the culture.

[0208] Preferably, a genetically modified organism (e.g., microorganism or plant) of the invention produces one or more polyunsaturated fatty acids including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), ALA (C18:3, n-3), and/or SDA (C18:4, n-3)), and more preferably, one or more long chain fatty acids, including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), or DTA (C22:4, n-6). In a particularly preferred embodiment, a genetically modified organism of the invention produces one or more polyunsaturated fatty acids including, but not limited to, EPA (C20:5, n-3), DHA (C22:6, n-3), and/or DPA (C22:5, n-6 or n-3).

[0209] Preferably, a genetically modified organism of the invention produces at least one PUFA (the target PUPA), wherein the total fatty acid profile in the organism (or a part of the organism that accumulates PUFAs, such as mature seeds or oil from such seeds, if the organism is an oil seed plant), comprises a detectable amount of this PUFA or PUFAs. Preferably, the PUFA is at least a 20 carbon PUFA and comprises at least 3 double bonds, and more preferably at least 4 double bonds, and even more preferably, at least 5 double bonds. In one embodiment, the PUFA is a PUFA that is not naturally produced by the organism (i.e., the wild-type organism in the absence of genetic modification or the parent organism used as a recipient for the indicated genetic modification).

[0210] Preferably, the total fatty acid profile in the organism (or part of the organism that accumulates PUFAs) comprises at least 0.1% of the target PUFA(s) by weight of the total fatty acids, and more preferably at least about 0.2%, and more preferably at least about 0.3%, and more preferably at least about 0.4%, and more preferably at least about 0.5%, and more preferably at least about 1%, and more preferably at least about 2%, and more preferably at least about 3%, and more preferably at least about 4%, and more preferably at least about 5%, and more preferably at least about 10%, and more preferably at least about 15%, and more preferably at least about 20%, and more preferably at least about 25%, and more preferably at least about 30%, and more preferably at least about 35%, and more preferably at least about 40%, and more preferably at least about 45%, and more preferably at least about 50%, and more preferably at least about 55%, and more preferably at least about 60%, and more preferably at least about 65%, and more preferably at least about 70%, and more preferably at least about 75%, and more preferably more that 75% of at least one polyunsaturated fatty acid (the target PUFA) by weight of the total fatty acids, or any percentage from 0.1% to 75%, or greater than 75% (up to 100% or about 100%), in 0.1% increments, of the target PUFA(s). As generally used herein, reference to a percentage amount of PUFA production is by weight of the total fatty acids produced by the organism, unless otherwise stated (e.g., in some cases, percentage by weight is relative to the total fatty acids produced by an enzyme complex, such as a PUFA PKS system). In one embodiment, total fatty acids produced by a plant are presented as a weight percent as determined by gas chromatography (GC) analysis of a fatty acid methyl ester (FAME) preparation.

[0211] As described above, it is an additional characteristic of the total fatty acids produced by a plant (and/or parts of plants or seed oil fraction) that has been genetically modified to express a PUFA PKS of the present invention that these total fatty acids produced by the plant comprise less than about 10% by weight of any fatty acids other than the target PUFA(s) that are produced by the enzyme complex that produces the target PUFA(s) (e.g., DHA and DPAn-6 are the target PUFAs if the entire PUFA PKS system of the invention is used). Preferably, any fatty acids that are produced by the enzyme complex that produces the target PUFA(s) other than the target PUFA(s) are present at less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% by weight of the total fatty acids produced by the plant.

[0212] In another embodiment, any fatty acids that are produced by the enzyme complex that produces the target PUFA(s) other than the target PUFA(s) are present at less than about 10% by weight of the total fatty acids that are produced by the enzyme complex that produces the target PUFA(s) in the plant (i.e., this measurement is limited to those total fatty acids that are produced by the enzyme complex that produces the target PUFAs), and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% by weight of the total fatty acids that are produced by the enzyme complex that produces the target PUFA(s) in the plant.

[0213] In another aspect of this embodiment of the invention, the total fatty acids produced by the plant (and/or parts of plants or seed oil fraction) contain less than (or do not contain any more than) 10% PUFAs having 18 or more carbons by weight of the total fatty acids produced by the plant, other than the target PUFA(s) or the PUFAs that are present in the wild-type plant (not genetically modified) or the parent plant used as a recipient for the indicated genetic modification. In further aspects, the total fatty acids produced by the plant (and/or parts of plants or seed oil fraction) contain less than 9% PUFAs having 18 or more carbons, or less than 8% PUFAs having 18 or more carbons, or less than 7% PUFAs having 18 or more carbons, or less than 6% PUFAs having 18 or more carbons, or less than 5% PUFAs having 18 or more carbons, or less than 4% PUFAs having 18 or more carbons, or less than 3% PUFAs having 18 or more carbons, or less than 2% PUFAs having 18 or more carbons, or less than 1% PUFAs having 18 or more carbons by weight of the total fatty acids produced by the plant, other than the target PUFA(s) or the PUFAs that are present in the wild-type plant (not genetically modified) or the parent plant used as a recipient for the indicated genetic modification.

[0214] In another aspect of this embodiment of the invention, the total fatty acids produced by the plant (and/or parts of plants or seed oil fraction) contain less than (or do not contain any more than) 10% PUFAs having 20 or more carbons by weight of the total fatty acids produced by the plant, other than the target PUFA(s) or the PUFAs that are present in the wild-type plant (not genetically modified) or the parent plant used as a recipient for the indicated genetic modification. In further aspects, the total fatty acids produced by the plant (and/or parts of plants or seed oil fraction) contain less than 9% PUFAs having 20 or more carbons, or less than 8% PUFAs having 20 or more carbons, or less than 7% PUFAs having 20 or more carbons, or less than 6% PUFAs having 20 or more carbons, or less than 5% PUFAs having 20 or more carbons, or less than 4% PUFAs having 20 or more carbons, or less than 3% PUFAs having 20 or more carbons, or less than 2% PUFAs having 20 or more carbons, or less than 1% PUFAs having 20 or more carbons by weight of the total fatty acids produced by the plant, other than the target PUFA(s) or the PUFAs that are present in the wild-type plant (not genetically modified) or the parent plant used as a recipient for the indicated genetic modification.

[0215] In one embodiment, the total fatty acids in the plant (and/or parts of plants or seed oil fraction) contain less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of a fatty acid selected from any one or more of: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids, such as 20:0; 20:1 (.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2 (.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17).

[0216] In another embodiment, the fatty acids that are produced by the enzyme system that produces the long chain PUFAs in the plant contain less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of a fatty acid selected from: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids, such as 20:0; 20:1 (.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2 (.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead acid (20:3; .DELTA.5,8,11); 20:4 (.DELTA.5,1,14,17).

[0217] In another embodiment, the fatty acids that are produced by the enzyme system that produces the long chain PUFAs in the plant contain less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0218] In another embodiment, the fatty acids that are produced by the enzyme system that produces the long chain PUFAs in the plant contain less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of each of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0219] In another embodiment, the fatty acids that are produced by the enzyme system that produces the long chain PUFAs in the plant contain less than about 10% by weight of the total fatty acids produced by the plant, and more preferably less than about 9%, and more preferably less than about 8%, and more preferably less than about 7%, and more preferably less than about 6%, and more preferably less than about 5%, and more preferably less than about 4%, and more preferably less than about 3%, and more preferably less than about 2%, and more preferably less than about 1% of any one or more of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

[0220] In one aspect of this embodiment of the invention, a genetically modified plant produces at least two target PUFAs (e.g., DHA and DPAn-6), and the total fatty acid profile in the plant, or the part of the plant that accumulates PUFAs (including oils from the oil seeds), comprises a detectable amount of these PUFAs. In this embodiment, the PUFAs are preferably each at least a 20 carbon PUFA and comprise at least 3 double bonds, and more preferably at least 4 double bonds, and even more preferably, at least 5 double bonds. Such PUFAs are most preferably chosen from DHA, DPAn-6 and EPA. In one aspect, the plant produces DHA and DPAn-6 (the products of a PUFA PKS system described herein), and the ratio of DHA to DPAn-6 is from about 1:10 to about 10:1, including any ratio in between. In a one embodiment, the ratio of DHA to DPA is from about 1:1 to about 3:1, and in another embodiment, about 2.5:1.

[0221] In another aspect of this embodiment of the invention, the plant produces the total fatty acid profile represented by FIG. 5.

[0222] The invention further includes any seeds produced by the plants described above, as well as any oils produced by the plants or seeds described above. The invention also includes any products produced using the plants, seed or oils described herein.

[0223] One embodiment of the present invention relates to a method to modify an endproduct containing at least one fatty acid, comprising adding to said endproduct an oil produced by a recombinant host cell that expresses at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one biologically active domain of a PUFA PKS system as described herein.

[0224] Preferably, the endproduct is selected from the group consisting of a food, a dietary supplement, a pharmaceutical formulation, a humanized animal milk, and an infant formula. Suitable pharmaceutical formulations include, but are not limited to, an anti-inflammatory formulation, a chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, a drug for treatment of neurodegenerative disease, a drug for treatment of degenerative liver disease, an antibiotic, and a cholesterol lowering formulation. In one embodiment, the endproduct is used to treat a condition selected from the group consisting of: chronic inflammation, acute inflammation, gastrointestinal disorder, cancer, cachexia, cardiac restenosis, neurodegenerative disorder, degenerative disorder of the liver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmune disease, preeclampsia, preterm birth, age related maculopathy, pulmonary disorder, and peroxisomal disorder.

[0225] Suitable food products include, but are not limited to, fine bakery wares, bread and rolls, breakfast cereals, processed and unprocessed cheese, condiments (ketchup, mayonnaise, etc.), dairy products (milk, yogurt), puddings and gelatine desserts, carbonated drinks, teas, powdered beverage mixes, processed fish products, fruit-based drinks, chewing gum, hard confectionery, frozen dairy products, processed meat products, nut and nut-based spreads, pasta, processed poultry products, gravies and sauces, potato chips and other chips or crisps, chocolate and other confectionery, soups and soup mixes, soya based products (milks, drinks, creams, whiteners), vegetable oil-based spreads, and vegetable-based drinks.

[0226] Yet another embodiment of the present invention relates to a method to produce a humanized animal milk. This method includes the steps of genetically modifying milk-producing cells of a milk-producing animal with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding at least one biologically active domain of a PUFA PKS system as described herein.

[0227] Methods to genetically modify a host cell and to produce a genetically modified non-human, milk-producing animal, are known in the art. Examples of host animals to modify include cattle, sheep, pigs, goats, yaks, etc., which are amenable to genetic manipulation and cloning for rapid expansion of a transgene expressing population. For animals, PKS-like transgenes can be adapted for expression in target organelles, tissues and body fluids through modification of the gene regulatory regions. Of particular interest is the production of PUFAs in the breast milk of the host animal.

[0228] Each publication or reference cited herein is incorporated herein by reference in its entirety.

[0229] The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1

[0230] The following example demonstrates that Schizochytrium Orfs A, B and C encode a functional DHA/DPA synthesis enzyme via functional expression in E. coli.

General Preparation of E. coli Transformants

[0231] The three genes encoding the Schizochytrium PUFA PKS system that produce DHA and DPA (Orfs A, B & C; SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively) were cloned into a single E. coli expression vector (derived from pET21c (Novagen)). The genes are transcribed as a single message (by the T7 RNA-polymerase), and a ribosome-binding site cloned in front of each of the genes initiates translation. Modification of the Orf B coding sequence was needed to obtain production of a full-length Orf B protein in E. coli (see below). An accessory gene, encoding a PPTase (see below) was cloned into a second plasmid (derived from pACYC184, New England Biolabs).

[0232] The Orf B gene is predicted to encode a protein with a mass of .about.224 kDa. Initial attempts at expression of the gene in E. coli resulted in accumulation of a protein with an apparent molecular mass of .about.165 kDa (as judged by comparison to proteins of known mass during SDS-PAGE). Examination of the Orf B nucleotide sequence revealed a region containing 15 sequential serine codons--all of them being the TCT codon. The genetic code contains 6 different serine codons, and three of these are used frequently in E. coli. The inventors used four overlapping oligonucleotides in combination with a polymerase chain reaction protocol to resynthesize a small portion of the Orf B gene (a .about.195 base pair, BspHI to SacII restriction enzyme fragment) that contained the serine codon repeat region. In the synthetic Orf B fragment, a random mixture of the 3 serine codons commonly used by E. coli was used, and some other potentially problematic codons were changed as well (i.e., other codons rarely used by E. coli). The BspHI to SacII fragment present in the original Orf B was replaced by the resynthesized fragment (to yield Orf B*) and the modified gene was cloned into the relevant expression vectors. The modified OrfB* still encodes the amino acid sequence of SEQ ID NO:4. Expression of the modified Orf B* clone in E. coli resulted in the appearance of a .about.224 kDa protein, indicating that the full-length product of OrfB was produced. The sequence of the resynthesized Orf B* BspHI to SacII fragment is represented herein as SEQ ID NO:38. Referring to SEQ ID NO:38, the nucleotide sequence of the resynthesized BspHI to SacII region of Orf B is shown. The BspHI restriction site and the SacII restriction site are identified. The BspHI site starts at nucleotide 4415 of the Orf B CDS (SEQ ID NO:3) (note: there are a total of three BspHI sites in the Orf B CDS, while the SacII site is unique).

[0233] The ACP domains of the Orf A protein (SEQ ID NO:2 in Schizochytrium) must be activated by addition of phosphopantetheine group in order to function. The enzymes that catalyze this general type of reaction are called phosphopantetheine transferases (PPTases). E. coli contains two endogenous PPTases, but it was anticipated that they would not recognize the Orf A ACP domains from Schizochytrium. This was continued by expressing Orfs A, B* (see above) and C in E. coli without an additional PPTase. In this transformant, no DHA production was detected. The inventors tested two heterologous PPTases in the E. coli PUFA PKS expression system: (1) sfp (derived from Bacillus subtilis) and (2) Het I (from the cyanobacterium Nostoc strain 7120).

[0234] The sfp PPTase has been well characterized and is widely used due to its ability to recognize a broad range of substrates. Based on published sequence information (Nakana, et al., 1992, Molecular and General Genetics 232: 313-321), an expression vector for sfp was built by cloning the coding region, along with defined up- and downstream flanking DNA sequences, into a pACYC-184 cloning vector. Oligonucleotides were used to amplify the region of interest from genomic B. subtilus DNA. The oligonucleotides:

TABLE-US-00001 (forward; SEQ ID NO: 39) CGGGGTACCCGGGAGCCGCCTTGGCTTTGT; and (reverse; SEQ ID NO: 40) AAACTGCAGCCCGGGTCCAGCTGGCAGGCACCCTG,

were used to amplify the region of interest from genomic B. subtilus DNA. Convenient restriction enzyme sites were included in the oligonucleotides to facilitate cloning in an intermediate, high copy number vector and finally into the EcoRV site of pACYC184 to create the plasmid: pBR301. Examination of extracts of E. coli transformed with this plasmid revealed the presence of a novel protein with the mobility expected for sfp. Co-expression of the sfp construct in cells expressing the Orf A, B*, C proteins, under certain conditions, resulted in DHA production. This experiment demonstrated that sfp was able to activate the Schizochytrium Orf A ACP domains. In addition, the regulatory elements associated with the sfp gene were used to create an expression cassette into which other genes could be inserted. Specifically, the sfp coding region (along with three nucleotides immediately upstream of the ATG) in pBR301 was replaced with a 53 base pair section of DNA designed so that it contains several unique (for this construct) restriction enzyme sites. The initial restriction enzyme site in this region is NdeI. The ATG sequence embedded in this site is utilized as the initiation methionine codon for introduced genes. The additional restriction sites (BglLL, NotI, SmaI, PmelI, HindIII, SpeI and XhoI) were included to facilitate the cloning process. The functionality of this expression vector cassette was tested by using PCR to generate a version of sfp with a NdeI site at the 5' end and an XhoI site ate the 3' end. This fragment was cloned into the expression cassette and transferred into E. coli along with the Orf A, B* and C expression vector. Under appropriate conditions, these cells accumulated DHA, demonstrating that a functional sfp had been produced.

[0235] Het I is present in a cluster of genes in Nostoc known to be responsible for the synthesis of long chain hydroxy-fatty acids that are a component of a glyco-lipid layer present in heterocysts of that organism (Black and Wolk, 1994, J. Bacteriol. 176, 2282-2292; Campbell et al., 1997, Arch. Microbial. 167, 251-258). Het I activates the ACP domains of a protein, Hgl E, present in that cluster. The two ACP domains of Hgl E have a high degree of sequence homology to the ACP domains found in Schizochytrium Orf A. A Het I expression construct was made using PCR. Specifically, SEQ ID NO:41 represents the amino acid sequence of the Nostoc Het I protein. The endogenous start codon of Het I has not been identified (there is no methionine present in the putative protein). There are several potential alternative start codons (e.g., TTG and ATT) near the 5' end of the open reading frame. No methionine codons (ATG) are present in the sequence. A Het I expression construct was made by using PCR to replace the furthest 5' potential alternative start codon (TTG) with a methionine codon (ATG, as part of the above described NdeI restriction enzyme recognition site), and introducing an XhoI site at the 3' end of the coding sequence. The modified HetI coding sequence was then inserted into the NdeI and XhoI sites of the pACYC184 vector construct containing the sfp regulatory elements. Expression of this Het I construct in E. coli resulted in the appearance of a new protein of the size expected from the sequence data. Co-expression of Het I with Schizochytrium Orfs A, B*, C in E. coli under several conditions resulted in the accumulation of DHA and DPA in those cells. In all of the experiments in which sfp and Het I were compared, more DHA and DPA accumulated in the cells containing the Het I construct than in cells containing the sfp construct.

Production of DHA and DPA in E. coli Transformants

[0236] The two plasmids encoding: (1) the Schizochytrium PUFA PKS genes (Orfs A, B* and C) and (2) the PPTase (from sfp or from Het I) were transformed into E. coli strain BL21 which contains an inducible T7 RNA polymerase gene. Synthesis of the Schizochytrium proteins was induced by addition of IPTG to the medium, while PPTase expression was controlled by a separate regulatory element (see above). Cells were grown under various defined conditions and using either of the two heterologous PPTase genes. The cells were harvested and the fatty acids were converted to methyl-esters (FAME) and analyzed using gas-liquid chromatography.

[0237] Under several conditions, DHA and DPA were detected in E. coli cells expressing the Schizochytrium PUFA PKS genes, plus either of the two heterologous PPTases (data not shown). No DHA or DPA was detected in FAMEs prepared from control cells (i.e., cells transformed with a plasmid lacking one of the Oils). The ratio of DHA to DPA observed in E. coli approximates that of the endogenous DHA and DPA production observed in Schizochytrium. The highest level of PUFA (DHA plus DPA), representing .about.17% of the total FAME, was found in cells grown at 32.degree. C. in 765 medium (recipe available from the American Type Culture Collection) supplemented with 10% (by weight) glycerol. PUFA accumulation was also observed when cells were grown in Luria Broth supplemented with 5 or 10% glycerol, and when grown at 20.degree. C. Selection for the presence of the respective plasmids was maintained by inclusion of the appropriate antibiotics during the growth, and IPTG (to a final concentration of 0.5 mM) was used to induce expression of Orfs A, B* and C. Co-expression of Het I or sfp with Schizochytrium Orfs A, B*, C in E. coli under several conditions resulted in the accumulation of DHA and DPA in those cells. In all of the experiments in which sfp and Het I were compared, more DHA and DPA accumulated in the cells containing the Het I construct than in cells containing the sfp construct.

Example 2

[0238] The following example shows the expression of genes encoding the Schizochytrium PUFA synthase (sOrfA, sOrfB and native Orf C) along with Het I in baker's yeast (Saccharomyces cerevisiae).

[0239] The Schizochytrium PUFA synthase genes and Het I were expressed in yeast using materials obtained from Invitrogen (Invitrogen Corporation, Carlsbad, Calif.). The INVsc1 strain of Saccharomyces cerevisiae was used along with the following transformation vectors: pYESLeu (sOrfA), pYES3/CT (sOrfB), pYES2/CT (OrfC) and pYESHis (HetI). To accommodate yeast codon useage, the nucleotide sequences for OrfA (SEQ ID NO:1) and for OrfB (SEQ ID NO:3) were resynthesized. The nucleotide sequence for the resynthesized OrfA (contained in pYESLeu), designated sOrfA, is represented herein by SEQ ID NO:43. SEQ ID NO:43 still encodes the OrfA amino acid sequence of SEQ ID NO:2. The nucleotide sequence for the resynthesized OrfB (contained in pYES3/CT), designated sOrfB, is represented herein by SEQ ID NO:44. SEQ ID NO:44 still encodes the OrfB amino acid sequence of SEQ ID NO:4. The OrfC nucleotide sequence used in these experiments (contained in pYES2/CT) is the wild-type OrfC, represented by SEQ ID NO:5, and encoding SEQ ID NO:6.

[0240] Some of the vectors were modified to accommodate specific cloning requirements (e.g., restriction sites for cloning). Appropriate selection media were used (as specified by Invitrogen), depending on the particular experiment. The genes were cloned, in each case, behind a GAL1 promoter and expression was induced by re-suspension of washed cells in media containing galactose according to guidelines provide by Invitrogen. Cells were grown at 30.degree. C. and harvested (by centrifugation) after being transferred to the induction medium. The cell pellets were freeze dried and FAMEs were prepared using acidic methanol, extracted into hexane and analyzed by GC.

[0241] A comparison of the fatty acid profile from yeast cells expressing the Schizochytrium PUFA synthase system (sOrfA, sOrf B, OrfC and Het I) and one obtained from control cells (lacking the sOrfA gene) collected .about.20 hrs after induction, showed that two novel FAME peaks have appeared in the profile of the strain expressing the complete PUFA synthase system (FIG. 3). These two peaks were identified as DPAn-6 and DHA by comparison of the elution time with authentic standards and subsequently by MS analyses. As predicted from the characterization of the Schizochytrium PUPA synthase, aside from DHA and DPAn-6, no other novel peaks are evident in the profile. FIG. 4 shows the region of the GC chromatogram of FIG. 3 which contains the PUFA FAMEs. Both the control cells and the cell expressing the PUFA synthase contain a peak that elutes near the DHA FAME. This has been identified as C26:0 FAME and (based on literature references) is derived from sphingolipids. Although it elutes close to the DHA peak, the resolution is sufficient so that it does not interfere with the quantitation of DHA. The DPA n-6 peak is well separated from other endogenous yeast lipids in the FAME profile. In this particular example, the cells expressing the Schizochytrium PUFA synthase system accumulated 2.4% DHA and 2.0% DPA n-6 (as a percentage of the total FAMEs). The sum of DHA and DPA n-6=4.4% of the measured fatty acids in the cells. The ratio of DHA to DPA n-6 observed in the cells was .about.1.2:1.

Example 3

[0242] The following examples describes the expression of genes encoding the Schizochytrium PUFA synthase (Orf A, Orf B* and Orf C) along with Het I in Arabidopsis.

[0243] The Schizochytrium Orfs A, B* (see Example 1) and C along with Het I were cloned (separately or in various combinations including all 4 genes on one Super-construct) into the appropriate binary vectors for introduction of the genes into plants. Each gene was cloned behind a linin promoter and was followed by a linin terminator sequence (Chaudhary et al., 2001; PCT Publication Number No. WO 01/16340 A1). For localization of the PUFA synthase in the cytoplasm of plant cells, no additional protein encoding sequences were appended to the 5'end of the Orfs. For directing the proteins to the plastid, additional 5' sequences encoding a plastid targeting sequence derived from a Brassica napus acyl-ACP thioesterase were added to the Orfs. The amino acid sequence of the encoded targeting peptide is: MLKLSCNVTNHLHTFSFFSDSSLFIPVNRRTLAVS (SEQ ID NO:42). The nucleotide sequences encoding this peptide were placed in frame with the start methionine codons of each PUFA synthase Orf as well as the start codon of Het I.

[0244] More specifically, for one experiment described herein, the constructs and plants were prepared as follows:

Construction of pSBS4107: Acyl-ACP Transit Peptide-HetI: Acyl-ACP Transit Peptide-ORFC

[0245] This plant binary vector contained a double expression cassette which targeted the co-expression of HetI (SEQ ID NO:41) and ORFC (SEQ ID NO:6) to the plastid. The first expression cassette began with a signal peptide (SEQ ID NO:42) derived from an acyl-ACP thioesterase gene from Brassica juncea (GenBank Accession No. AJ294419) to target expression of the polypeptides to the plastid. The signal peptide was synthesized from two overlapping oligos with an engineered AfiIII site at the 5' end and an NcoI/SwaI/XmaI multiple cloning site at the 3' end. Immediately downstream was a sequence encoding for PPTase from Nostoc, encoded by HetI, to enable DHA to bind phosphopantetheine attachment sites. The second expression cassette also began with the acyl-ACP signal peptide followed immediately in-frame with a cDNA encoding ORFC (SEQ ID NO:5).

[0246] The backbone of this plasmid, pSBS4055, was based on the plant binary vector, pPZP200, described by Hajdukiewicz et al. (Plant Molecular Biology, 1994, 25:989-994). In place of the described multiple cloning site, a pat gene conferring host plant phosphinothricine resistance (Wohlleben et al., 1988, Gene 70:25-37) driven by the ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684), was inserted between the left and right border sequences. In addition to this cassette, two separate Linin promoter/terminators in tandem from Linum usitatissumum (Flax or Linseed) (Chaudhary et al., 2001; PCT Publication Number No. WO 01/16340 A1) were used to drive expression of ACP-HetI and ACP-ORFC. Standard restriction cloning was used to fuse the synthetic Acyl-ACP signal peptide in-frame with cDNAs encoding for either HetI or ORFC using NcoI/XmaI and NcoI/SwaI restriction endonuclease sites, respectively, to the 3' end of the Linin promoter. The result was plasmid pSBS4107: a DNA sequence encoding the Acyl-ACP signal peptide-HetI and Acyl-ACP signal peptide-ORFC polypeptides being placed in a binary vector under expression control of the linin promoter/terminator. The linin promoter controls the specific-temporal and tissue-specific expression of the transgene during seed development. The Acyl-ACP signal peptide targets the expression of the protein to the plastid (Loader et al., 1993, Plant Mol Biol 23:769-778). The complete plasmid map with annotated elements is shown in FIG. 6.

Construction of pSBS5720: Acyl-ACP Transit Peptide-ORFB

[0247] This plant binary vector contained an expression cassette which targeted the expression of ORFB (SEQ ID NO:4) to the plastid. Again, the expression cassette began with a signal peptide derived from an acyl-ACP thioesterase gene from Brassica juncea (SEQ ID NO:42) to target expression of the polypeptide to the plastid. The signal peptide was synthesized as above. Immediately downstream was a cDNA sequence encoding for ORFB (SEQ ID NO:3, except for the resynthesized BspHI to SacII region of Orf B, represented by SEQ ID NO:38; see Example 1 above).

[0248] The backbone of this plasmid, pSBS4055, was based on the plant binary vector, pPZP200, described by Hajdukiewicz et al. (Plant Molecular Biology, 1994, 25:989-994). In place of the described multiple cloning site, a phosphomannose isomerase (PMI) gene conferring host plant positive selection for mannose-6-phosphate driven by the ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck et al., 1993, Plant. Mol. Bio., 21:673-684), was inserted between the left and right border sequences. In addition to this cassette, a Linin promoter/terminator from Linum usitatissumum (Flax or Linseed) (Chaudhary et al., 2001; PCT Publication Number WO 01/16340 A1) was used to drive expression of ACP-ORFB. Standard restriction cloning was used to fuse the synthetic Acyl-ACP signal peptide in-frame with cDNAs encoding for ORFB, to the 3' end of the Linin promoter. The result was plasmid pSBS5720: a DNA sequence encoding the Acyl-ACP signal peptide-ORFB polypeptide being placed in a binary vector under expression control of the linin promoter/terminator. The linin promoter controls the specific-temporal and tissue-specific expression of the transgene during seed development. The Acyl-ACP signal peptide targets the expression of the protein to the plastid (Loader et al., 1993, Plant Mol Biol 23:769-778). The complete plasmid map with annotated elements is shown in FIG. 7.

Construction of pSBS4757: Acyl-ACP Transit Peptide-ORFA

[0249] This plant binary vector contained an expression cassette which targeted the expression of ORFA (SEQ ID NO:2) to the plastid. Again the expression cassette began with a signal peptide derived from an acyl-ACP thioesterase gene from Brassica juncea (SEQ ID NO:42) to target expression of the polypeptide to the plastid. The signal peptide was synthesized as above. Immediately downstream was a cDNA sequence encoding for ORFA (SEQ ID NO:1).

[0250] The backbone of this plasmid, pSBS4055, was based on the plant binary vector, pPZP200, described by Hajdukiewicz et al. (Plant Molecular Biology, 1994, 25:989-994). In place of the described multiple cloning site, a neomycin phosphotransferase (nptII) gene conferring host plant Kanamycin resistance driven by the mannopine synthase promoter/terminator, was inserted between the left and right border sequences. In addition, a Linin promoter/terminator from Linum usitatissumum (Flax or Linseed) (Chaudhary et al., 2001; PCT Publication Number WO 01/16340 A1) were used to drive expression of ACP-ORFA. Standard restriction cloning was used to fuse the synthetic Acyl-ACP signal peptide in-frame with a cDNA encoding for ORFA to the 3' end of the Linin promoter. The result was plasmid pSBS4757: a DNA sequence encoding the Acyl-ACP signal peptide-ORFA polypeptide being placed in a binary vector under expression control of the linin promoter/terminator. The linin promoter controls the specific-temporal and tissue-specific expression of the transgene during seed development. The Acyl-ACP signal peptide targets the expression of the protein to the plastid (Loader et al., 1993, Plant Mol Biol 23:769-778). The complete plasmid map with annotated elements is shown in FIG. 8.

[0251] Standard methods were used for introduction of the genes into Arabidopsis (floral dipping into suspension of Agrobacterium strains containing the appropriate vectors; see Clough and Bent, Plant J.; 16(6):735-43, 1998 Dec). Seeds obtained from those plants were plated on selective medium and allowed to germinate. Some of the plants that grew were taken to maturity and the seeds analyzed for PUFA content. Based on PUFA content some of those seeds were taken forward to the next generation. Pooled seeds obtained from those plants were analyzed for their fatty acid content. Analysis of a plant line transformed with the constructs specifically described above and denoted 269 is described in more detail below.

[0252] The top panel of FIG. 5 shows the typical fatty acid profile of wild type Arabidopsis seeds as represented by GC separation and FID detection of FAMEs prepared from a pooled seed sample. The predominant fatty acids of wild type Arabidopsis seeds as represented by GC separation and FID detection of FAMEs prepared from a pooled seed sample are: 16:0, 18:0, 16:1, 18:1, 20:1, 20:2 and 22:1. No DHA or DPA n-6 are present in the samples from wild type seed. The lower panel of FIG. 5 shows the fatty acid profile of a pooled seed sample from one of the transgenic Arabidopsis lines (line 269) expressing the Schizochytrium PUFA synthase genes and Het I gene. The proteins expressed from these transgenes contain plastid targeting sequences. Two FAME peaks are present in the profile from the transgenic plant seeds that are not present in the profile from wild type seeds. The elution pattern of these two peaks exactly corresponds to the elution of authentic DHA and DPA n-6 (using FAMEs prepared from Schizochytrium oil as standards, as well as a commercially purchased DHA standard from NuCheck Prep). In this particular example, the DHA peak represents 0.8% of total calculated FAMEs while the DPA n-6 peak represents 1.7%. The sum of novel PUFAs is 2.5% of total FAMEs. The appearance of DHA and DPA n-6 in the seed fatty acid profile demonstrates that introduced Schizochytrium PUFA synthase system functions when expressed in the plant cell and the proteins are targeted to the plastid.

[0253] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Sequence CWU 1

1

4418733DNASchizochytrium sp.CDS(1)..(8733) 1atg gcg gcc cgt ctg cag gag caa aag gga ggc gag atg gat acc cgc 48Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg1 5 10 15att gcc atc atc ggc atg tcg gcc atc ctc ccc tgc ggc acg acc gtg 96Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val 20 25 30cgc gag tcg tgg gag acc atc cgc gcc ggc atc gac tgc ctg tcg gat 144Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp 35 40 45ctc ccc gag gac cgc gtc gac gtg acg gcg tac ttt gac ccc gtc aag 192Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys 50 55 60acc acc aag gac aag atc tac tgc aag cgc ggt ggc ttc att ccc gag 240Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu65 70 75 80tac gac ttt gac gcc cgc gag ttc gga ctc aac atg ttc cag atg gag 288Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu 85 90 95gac tcg gac gca aac cag acc atc tcg ctt ctc aag gtc aag gag gcc 336Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala 100 105 110ctc cag gac gcc ggc atc gac gcc ctc ggc aag gaa aag aag aac atc 384Leu Gln Asp Ala Gly Ile Asp Ala Leu Gly Lys Glu Lys Lys Asn Ile 115 120 125ggc tgc gtg ctc ggc att ggc ggc ggc caa aag tcc agc cac gag ttc 432Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe 130 135 140tac tcg cgc ctt aat tat gtt gtc gtg gag aag gtc ctc cgc aag atg 480Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met145 150 155 160ggc atg ccc gag gag gac gtc aag gtc gcc gtc gaa aag tac aag gcc 528Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala 165 170 175aac ttc ccc gag tgg cgc ctc gac tcc ttc cct ggc ttc ctc ggc aac 576Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn 180 185 190gtc acc gcc ggt cgc tgc acc aac acc ttc aac ctc gac ggc atg aac 624Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn 195 200 205tgc gtt gtc gac gcc gca tgc gcc tcg tcc ctc atc gcc gtc aag gtc 672Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val 210 215 220gcc atc gac gag ctg ctc tac ggt gac tgc gac atg atg gtc acc ggt 720Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly225 230 235 240gcc acc tgc acg gat aac tcc atc ggc atg tac atg gcc ttc tcc aag 768Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys 245 250 255acc ccc gtg ttc tcc acg gac ccc agc gtg cgc gcc tac gac gaa aag 816Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys 260 265 270aca aag ggc atg ctc atc ggc gag ggc tcc gcc atg ctc gtc ctc aag 864Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys 275 280 285cgc tac gcc gac gcc gtc cgc gac ggc gat gag atc cac gct gtt att 912Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile 290 295 300cgc ggc tgc gcc tcc tcc agt gat ggc aag gcc gcc ggc atc tac acg 960Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr305 310 315 320ccc acc att tcg ggc cag gag gag gcc ctc cgc cgc gcc tac aac cgc 1008Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg 325 330 335gcc tgt gtc gac ccg gcc acc gtc act ctc gtc gag ggt cac ggc acc 1056Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr 340 345 350ggt act ccc gtt ggc gac cgc atc gag ctc acc gcc ttg cgc aac ctc 1104Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu 355 360 365ttt gac aag gcc tac ggc gag ggc aac acc gaa aag gtc gct gtg ggc 1152Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly 370 375 380agc atc aag tcc agc atc ggc cat ctc aag gcc gtc gcc ggt ctc gcc 1200Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala385 390 395 400ggt atg atc aag gtc atc atg gcg ctc aag cac aag act ctc ccg ggc 1248Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly 405 410 415acc atc aac gtc gac aac cca ccc aac ctc tac gac aac acg ccc atc 1296Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile 420 425 430aac gag tcc tcg ctc tac att aac acc atg aac cgc ccc tgg ttc ccg 1344Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro 435 440 445ccc cct ggt gtg ccc cgc cgc gcc ggc att tcg agc ttt ggc ttt ggt 1392Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly 450 455 460ggc gcc aac tac cac gcc gtc ctc gag gag gcc gag ccc gag cac acg 1440Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr465 470 475 480acc gcg tac cgc ctc aac aag cgc ccg cag ccc gtg ctc atg atg gcc 1488Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala 485 490 495gcc acg ccc gcg gcc ctc cag tcg ctc tgc gag gcc cag ctc aag gag 1536Ala Thr Pro Ala Ala Leu Gln Ser Leu Cys Glu Ala Gln Leu Lys Glu 500 505 510ttc gag gcc gcc atc aag gag aac gag acc gtc aag aac acc gcc tac 1584Phe Glu Ala Ala Ile Lys Glu Asn Glu Thr Val Lys Asn Thr Ala Tyr 515 520 525atc aag tgc gtc aag ttc ggc gag cag ttc aaa ttc cct ggc tcc atc 1632Ile Lys Cys Val Lys Phe Gly Glu Gln Phe Lys Phe Pro Gly Ser Ile 530 535 540ccg gcc aca aac gcg cgc ctc ggc ttc ctc gtc aag gat gct gag gat 1680Pro Ala Thr Asn Ala Arg Leu Gly Phe Leu Val Lys Asp Ala Glu Asp545 550 555 560gcc tgc tcc acc ctc cgt gcc atc tgc gcc caa ttc gcc aag gat gtc 1728Ala Cys Ser Thr Leu Arg Ala Ile Cys Ala Gln Phe Ala Lys Asp Val 565 570 575acc aag gag gcc tgg cgc ctc ccc cgc gag ggc gtc agc ttc cgc gcc 1776Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe Arg Ala 580 585 590aag ggc atc gcc acc aac ggc gct gtc gcc gcg ctc ttc tcc ggc cag 1824Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser Gly Gln 595 600 605ggc gcg cag tac acg cac atg ttt agc gag gtg gcc atg aac tgg ccc 1872Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn Trp Pro 610 615 620cag ttc cgc cag agc att gcc gcc atg gac gcc gcc cag tcc aag gtc 1920Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser Lys Val625 630 635 640gct gga agc gac aag gac ttt gag cgc gtc tcc cag gtc ctc tac ccg 1968Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu Tyr Pro 645 650 655cgc aag ccg tac gag cgt gag ccc gag cag gac cac aag aag atc tcc 2016Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys Ile Ser 660 665 670ctc acc gcc tac tcg cag ccc tcg acc ctg gcc tgc gct ctc ggt gcc 2064Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu Gly Ala 675 680 685ttt gag atc ttc aag gag gcc ggc ttc acc ccg gac ttt gcc gcc ggc 2112Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala Ala Gly 690 695 700cat tcg ctc ggt gag ttc gcc gcc ctc tac gcc gcg ggc tgc gtc gac 2160His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys Val Asp705 710 715 720cgc gac gag ctc ttt gag ctt gtc tgc cgc cgc gcc cgc atc atg ggc 2208Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile Met Gly 725 730 735ggc aag gac gca ccg gcc acc ccc aag ggc tgc atg gcc gcc gtc att 2256Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala Val Ile 740 745 750ggc ccc aac gcc gag aac atc aag gtc cag gcc gcc aac gtc tgg ctc 2304Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val Trp Leu 755 760 765ggc aac tcc aac tcg cct tcg cag acc gtc atc acc ggc tcc gtc gaa 2352Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser Val Glu 770 775 780ggt atc cag gcc gag agc gcc cgc ctc cag aag gag ggc ttc cgc gtc 2400Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe Arg Val785 790 795 800gtg cct ctt gcc tgc gag agc gcc ttc cac tcg ccc cag atg gag aac 2448Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met Glu Asn 805 810 815gcc tcg tcg gcc ttc aag gac gtc atc tcc aag gtc tcc ttc cgc acc 2496Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe Arg Thr 820 825 830ccc aag gcc gag acc aag ctc ttc agc aac gtc tct ggc gag acc tac 2544Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu Thr Tyr 835 840 845ccc acg gac gcc cgc gag atg ctt acg cag cac atg acc agc agc gtc 2592Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser Ser Val 850 855 860aag ttc ctc acc cag gtc cgc aac atg cac cag gcc ggt gcg cgc atc 2640Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala Arg Ile865 870 875 880ttt gtc gag ttc gga ccc aag cag gtg ctc tcc aag ctt gtc tcc gag 2688Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val Ser Glu 885 890 895acc ctc aag gat gac ccc tcg gtt gtc acc gtc tct gtc aac ccg gcc 2736Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn Pro Ala 900 905 910tcg ggc acg gat tcg gac atc cag ctc cgc gac gcg gcc gtc cag ctc 2784Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val Gln Leu 915 920 925gtt gtc gct ggc gtc aac ctt cag ggc ttt gac aag tgg gac gcc ccc 2832Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp Ala Pro 930 935 940gat gcc acc cgc atg cag gcc atc aag aag aag cgc act acc ctc cgc 2880Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr Leu Arg945 950 955 960ctt tcg gcc gcc acc tac gtc tcg gac aag acc aag aag gtc cgc gac 2928Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val Arg Asp 965 970 975gcc gcc atg aac gat ggc cgc tgc gtc acc tac ctc aag ggc gcc gca 2976Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly Ala Ala 980 985 990ccg ctc atc aag gcc ccg gag ccc gtt gtc gac gag gcc gcc aag cgc 3024Pro Leu Ile Lys Ala Pro Glu Pro Val Val Asp Glu Ala Ala Lys Arg 995 1000 1005gag gcc gag cgt ctc cag aag gag ctt cag gat gcc cag cgc cag 3069Glu Ala Glu Arg Leu Gln Lys Glu Leu Gln Asp Ala Gln Arg Gln 1010 1015 1020ctc gac gac gcc aag cgc gcc gcc gcc gag gcc aac tcc aag ctc 3114Leu Asp Asp Ala Lys Arg Ala Ala Ala Glu Ala Asn Ser Lys Leu 1025 1030 1035gcc gct gcc aag gag gag gcc aag acc gcc gct gct tcg gcc aag 3159Ala Ala Ala Lys Glu Glu Ala Lys Thr Ala Ala Ala Ser Ala Lys 1040 1045 1050ccc gca gtt gac act gct gtt gtc gaa aag cat cgt gcc atc ctc 3204Pro Ala Val Asp Thr Ala Val Val Glu Lys His Arg Ala Ile Leu 1055 1060 1065aag tcc atg ctc gcg gag ctc gat ggc tac gga tcg gtc gac gct 3249Lys Ser Met Leu Ala Glu Leu Asp Gly Tyr Gly Ser Val Asp Ala 1070 1075 1080tct tcc ctc cag cag cag cag cag cag cag acg gcc ccc gcc ccg 3294Ser Ser Leu Gln Gln Gln Gln Gln Gln Gln Thr Ala Pro Ala Pro 1085 1090 1095gtc aag gct gct gcg cct gcc gcc ccc gtt gcc tcg gcc cct gcc 3339Val Lys Ala Ala Ala Pro Ala Ala Pro Val Ala Ser Ala Pro Ala 1100 1105 1110ccg gct gtc tcg aac gag ctt ctt gag aag gcc gag act gtc gtc 3384Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val 1115 1120 1125atg gag gtc ctc gcc gcc aag acc ggc tac gag acc gac atg atc 3429Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile 1130 1135 1140gag gct gac atg gag ctc gag acc gag ctc ggc att gac tcc atc 3474Glu Ala Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile 1145 1150 1155aag cgt gtc gag atc ctc tcc gag gtc cag gcc atg ctc aat gtc 3519Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val 1160 1165 1170gag gcc aag gat gtc gat gcc ctc agc cgc act cgc act gtt ggt 3564Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly 1175 1180 1185gag gtt gtc aac gcc atg aag gcc gag atc gct ggc agc tct gcc 3609Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala 1190 1195 1200ccg gcg cct gct gcc gct gct ccg gct ccg gcc aag gct gcc cct 3654Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Lys Ala Ala Pro 1205 1210 1215gcc gcc gct gcg cct gct gtc tcg aac gag ctt ctc gag aag gcc 3699Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala 1220 1225 1230gag acc gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag 3744Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu 1235 1240 1245act gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc 3789Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly 1250 1255 1260att gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc 3834Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala 1265 1270 1275atg ctc aac gtc gag gcc aag gac gtc gac gct ctc agc cgc act 3879Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr 1280 1285 1290cgc act gtg ggt gag gtc gtc aac gcc atg aag gct gag atc gct 3924Arg Thr Val Gly Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala 1295 1300 1305ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc cca ggt ccg gct 3969Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Gly Pro Ala 1310 1315 1320gct gcc gcc cct gcg cct gcc gcc gcc gcc cct gct gtc tcg aac 4014Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn 1325 1330 1335gag ctt ctt gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc 4059Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala 1340 1345 1350gcc aag act ggc tac gag act gac atg atc gag tcc gac atg gag 4104Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu 1355 1360 1365ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc gag att 4149Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile 1370 1375 1380ctc tcc gag gtc cag gcc atg ctc aac gtc gag gcc aag gac gtc 4194Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val 1385 1390 1395gac gct ctc agc cgc acc cgc act gtt ggc gag gtc gtc gat gcc 4239Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala 1400 1405 1410atg aag gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc 4284Met Lys Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala 1415 1420 1425gct gct cct gct ccg gct gct gcc gcc cct gcg cct gcc gcc cct 4329Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Pro 1430 1435 1440gcg cct gct gtc tcg agc gag ctt ctc gag aag gcc gag act gtc 4374Ala Pro Ala Val Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val 1445 1450 1455gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act gac atg 4419Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met 1460 1465 1470atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac tcc 4464Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser 1475 1480 1485atc aag cgt gtc gag att ctc tcc gag gtc cag gcc atg ctc aac 4509Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn 1490 1495 1500gtc gag gcc aag gac gtc gac gct ctc agc cgc acc cgc act gtt 4554Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val 1505 1510

1515ggc gag gtc gtc gat gcc atg aag gcc gag atc gct ggt ggc tct 4599Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Gly Ser 1520 1525 1530gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc gcc 4644Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala 1535 1540 1545cct gcg cct gcc gcc cct gcg cct gcc gcc cct gcg cct gct gtc 4689Pro Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala Val 1550 1555 1560tcg agc gag ctt ctc gag aag gcc gag act gtc gtc atg gag gtc 4734Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val 1565 1570 1575ctc gcc gcc aag act ggc tac gag act gac atg att gag tcc gac 4779Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp 1580 1585 1590atg gag ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc 4824Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val 1595 1600 1605gag att ctc tcc gag gtt cag gcc atg ctc aac gtc gag gcc aag 4869Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys 1610 1615 1620gac gtc gac gct ctc agc cgc act cgc act gtt ggt gag gtc gtc 4914Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val 1625 1630 1635gat gcc atg aag gct gag atc gct ggc agc tcc gcc tcg gcg cct 4959Asp Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala Ser Ala Pro 1640 1645 1650gcc gcc gct gct cct gct ccg gct gct gcc gct cct gcg ccc gct 5004Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala 1655 1660 1665gcc gcc gcc cct gct gtc tcg aac gag ctt ctc gag aaa gcc gag 5049Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu 1670 1675 1680act gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act 5094Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr 1685 1690 1695gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc att 5139Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile 1700 1705 1710gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc atg 5184Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met 1715 1720 1725ctc aac gtc gag gcc aag gac gtc gat gcc ctc agc cgc acc cgc 5229Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg 1730 1735 1740act gtt ggc gag gtt gtc gat gcc atg aag gcc gag atc gct ggt 5274Thr Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly 1745 1750 1755ggc tct gcc ccg gcg cct gcc gcc gct gcc cct gct ccg gct gcc 5319Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala 1760 1765 1770gcc gcc cct gct gtc tcg aac gag ctt ctc gag aag gcc gag act 5364Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr 1775 1780 1785gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag acc gac 5409Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp 1790 1795 1800atg atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac 5454Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp 1805 1810 1815tcc atc aag cgt gtc gag att ctc tcc gag gtt cag gcc atg ctc 5499Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu 1820 1825 1830aac gtc gag gcc aag gac gtc gat gct ctc agc cgc act cgc act 5544Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr 1835 1840 1845gtt ggc gag gtc gtc gat gcc atg aag gct gag atc gcc ggc agc 5589Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Ser 1850 1855 1860tcc gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc 5634Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala 1865 1870 1875gct cct gcg ccc gct gcc gct gcc cct gct gtc tcg agc gag ctt 5679Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Ser Glu Leu 1880 1885 1890ctc gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag 5724Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys 1895 1900 1905act ggc tac gag act gac atg att gag tcc gac atg gag ctc gag 5769Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu 1910 1915 1920act gag ctc ggc att gac tcc atc aag cgt gtc gag atc ctc tcc 5814Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser 1925 1930 1935gag gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gat gcc 5859Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala 1940 1945 1950ctc agc cgc acc cgc act gtt ggc gag gtt gtc gat gcc atg aag 5904Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys 1955 1960 1965gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc 5949Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala 1970 1975 1980cct gct ccg gct gcc gcc gcc cct gct gtc tcg aac gag ctt ctt 5994Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu 1985 1990 1995gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag act 6039Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr 2000 2005 2010ggc tac gag acc gac atg atc gag tcc gac atg gag ctc gag acc 6084Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr 2015 2020 2025gag ctc ggc att gac tcc atc aag cgt gtc gag att ctc tcc gag 6129Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu 2030 2035 2040gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gac gct ctc 6174Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu 2045 2050 2055agc cgc act cgc act gtt ggc gag gtc gtc gat gcc atg aag gct 6219Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys Ala 2060 2065 2070gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gct cct 6264Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro 2075 2080 2085gcc tcg gct ggc gcc gcg cct gcg gtc aag att gac tcg gtc cac 6309Ala Ser Ala Gly Ala Ala Pro Ala Val Lys Ile Asp Ser Val His 2090 2095 2100ggc gct gac tgt gat gat ctt tcc ctg atg cac gcc aag gtg gtt 6354Gly Ala Asp Cys Asp Asp Leu Ser Leu Met His Ala Lys Val Val 2105 2110 2115gac atc cgc cgc ccg gac gag ctc atc ctg gag cgc ccc gag aac 6399Asp Ile Arg Arg Pro Asp Glu Leu Ile Leu Glu Arg Pro Glu Asn 2120 2125 2130cgc ccc gtt ctc gtt gtc gat gac ggc agc gag ctc acc ctc gcc 6444Arg Pro Val Leu Val Val Asp Asp Gly Ser Glu Leu Thr Leu Ala 2135 2140 2145ctg gtc cgc gtc ctc ggc gcc tgc gcc gtt gtc ctg acc ttt gag 6489Leu Val Arg Val Leu Gly Ala Cys Ala Val Val Leu Thr Phe Glu 2150 2155 2160ggt ctc cag ctc gct cag cgc gct ggt gcc gct gcc atc cgc cac 6534Gly Leu Gln Leu Ala Gln Arg Ala Gly Ala Ala Ala Ile Arg His 2165 2170 2175gtg ctc gcc aag gat ctt tcc gcg gag agc gcc gag aag gcc atc 6579Val Leu Ala Lys Asp Leu Ser Ala Glu Ser Ala Glu Lys Ala Ile 2180 2185 2190aag gag gcc gag cag cgc ttt ggc gct ctc ggc ggc ttc atc tcg 6624Lys Glu Ala Glu Gln Arg Phe Gly Ala Leu Gly Gly Phe Ile Ser 2195 2200 2205cag cag gcg gag cgc ttc gag ccc gcc gaa atc ctc ggc ttc acg 6669Gln Gln Ala Glu Arg Phe Glu Pro Ala Glu Ile Leu Gly Phe Thr 2210 2215 2220ctc atg tgc gcc aag ttc gcc aag gct tcc ctc tgc acg gct gtg 6714Leu Met Cys Ala Lys Phe Ala Lys Ala Ser Leu Cys Thr Ala Val 2225 2230 2235gct ggc ggc cgc ccg gcc ttt atc ggt gtg gcg cgc ctt gac ggc 6759Ala Gly Gly Arg Pro Ala Phe Ile Gly Val Ala Arg Leu Asp Gly 2240 2245 2250cgc ctc gga ttc act tcg cag ggc act tct gac gcg ctc aag cgt 6804Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser Asp Ala Leu Lys Arg 2255 2260 2265gcc cag cgt ggt gcc atc ttt ggc ctc tgc aag acc atc ggc ctc 6849Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys Thr Ile Gly Leu 2270 2275 2280gag tgg tcc gag tct gac gtc ttt tcc cgc ggc gtg gac att gct 6894Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val Asp Ile Ala 2285 2290 2295cag ggc atg cac ccc gag gat gcc gcc gtg gcg att gtg cgc gag 6939Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val Arg Glu 2300 2305 2310atg gcg tgc gct gac att cgc att cgc gag gtc ggc att ggc gca 6984Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly Ala 2315 2320 2325aac cag cag cgc tgc acg atc cgt gcc gcc aag ctc gag acc ggc 7029Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly 2330 2335 2340aac ccg cag cgc cag atc gcc aag gac gac gtg ctg ctc gtt tct 7074Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser 2345 2350 2355ggc ggc gct cgc ggc atc acg cct ctt tgc atc cgg gag atc acg 7119Gly Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr 2360 2365 2370cgc cag atc gcg ggc ggc aag tac att ctg ctt ggc cgc agc aag 7164Arg Gln Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys 2375 2380 2385gtc tct gcg agc gaa ccg gca tgg tgc gct ggc atc act gac gag 7209Val Ser Ala Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu 2390 2395 2400 aag gct gtg caa aag gct gct acc cag gag ctc aag cgc gcc ttt 7254Lys Ala Val Gln Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe 2405 2410 2415agc gct ggc gag ggc ccc aag ccc acg ccc cgc gct gtc act aag 7299Ser Ala Gly Glu Gly Pro Lys Pro Thr Pro Arg Ala Val Thr Lys 2420 2425 2430ctt gtg ggc tct gtt ctt ggc gct cgc gag gtg cgc agc tct att 7344Leu Val Gly Ser Val Leu Gly Ala Arg Glu Val Arg Ser Ser Ile 2435 2440 2445gct gcg att gaa gcg ctc ggc ggc aag gcc atc tac tcg tcg tgc 7389Ala Ala Ile Glu Ala Leu Gly Gly Lys Ala Ile Tyr Ser Ser Cys 2450 2455 2460gac gtg aac tct gcc gcc gac gtg gcc aag gcc gtg cgc gat gcc 7434Asp Val Asn Ser Ala Ala Asp Val Ala Lys Ala Val Arg Asp Ala 2465 2470 2475gag tcc cag ctc ggt gcc cgc gtc tcg ggc atc gtt cat gcc tcg 7479Glu Ser Gln Leu Gly Ala Arg Val Ser Gly Ile Val His Ala Ser 2480 2485 2490ggc gtg ctc cgc gac cgt ctc atc gag aag aag ctc ccc gac gag 7524Gly Val Leu Arg Asp Arg Leu Ile Glu Lys Lys Leu Pro Asp Glu 2495 2500 2505ttc gac gcc gtc ttt ggc acc aag gtc acc ggt ctc gag aac ctc 7569Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly Leu Glu Asn Leu 2510 2515 2520ctc gcc gcc gtc gac cgc gcc aac ctc aag cac atg gtc ctc ttc 7614Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met Val Leu Phe 2525 2530 2535agc tcg ctc gcc ggc ttc cac ggc aac gtc ggc cag tct gac tac 7659Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser Asp Tyr 2540 2545 2550gcc atg gcc aac gag gcc ctt aac aag atg ggc ctc gag ctc gcc 7704Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu Ala 2555 2560 2565aag gac gtc tcg gtc aag tcg atc tgc ttc ggt ccc tgg gac ggt 7749Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly 2570 2575 2580ggc atg gtg acg ccg cag ctc aag aag cag ttc cag gag atg ggc 7794Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly 2585 2590 2595gtg cag atc atc ccc cgc gag ggc ggc gct gat acc gtg gcg cgc 7839Val Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg 2600 2605 2610atc gtg ctc ggc tcc tcg ccg gct gag atc ctt gtc ggc aac tgg 7884Ile Val Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp 2615 2620 2625cgc acc ccg tcc aag aag gtc ggc tcg gac acc atc acc ctg cac 7929Arg Thr Pro Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His 2630 2635 2640cgc aag att tcc gcc aag tcc aac ccc ttc ctc gag gac cac gtc 7974Arg Lys Ile Ser Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val 2645 2650 2655atc cag ggc cgc cgc gtg ctg ccc atg acg ctg gcc att ggc tcg 8019Ile Gln Gly Arg Arg Val Leu Pro Met Thr Leu Ala Ile Gly Ser 2660 2665 2670ctc gcg gag acc tgc ctc ggc ctc ttc ccc ggc tac tcg ctc tgg 8064Leu Ala Glu Thr Cys Leu Gly Leu Phe Pro Gly Tyr Ser Leu Trp 2675 2680 2685gcc att gac gac gcc cag ctc ttc aag ggt gtc act gtc gac ggc 8109Ala Ile Asp Asp Ala Gln Leu Phe Lys Gly Val Thr Val Asp Gly 2690 2695 2700gac gtc aac tgc gag gtg acc ctc acc ccg tcg acg gcg ccc tcg 8154Asp Val Asn Cys Glu Val Thr Leu Thr Pro Ser Thr Ala Pro Ser 2705 2710 2715ggc cgc gtc aac gtc cag gcc acg ctc aag acc ttt tcc agc ggc 8199Gly Arg Val Asn Val Gln Ala Thr Leu Lys Thr Phe Ser Ser Gly 2720 2725 2730aag ctg gtc ccg gcc tac cgc gcc gtc atc gtg ctc tcc aac cag 8244Lys Leu Val Pro Ala Tyr Arg Ala Val Ile Val Leu Ser Asn Gln 2735 2740 2745ggc gcg ccc ccg gcc aac gcc acc atg cag ccg ccc tcg ctc gat 8289Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro Pro Ser Leu Asp 2750 2755 2760gcc gat ccg gcg ctc cag ggc tcc gtc tac gac ggc aag acc ctc 8334Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly Lys Thr Leu 2765 2770 2775ttc cac ggc ccg gcc ttc cgc ggc atc gat gac gtg ctc tcg tgc 8379Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu Ser Cys 2780 2785 2790acc aag agc cag ctt gtg gcc aag tgc agc gct gtc ccc ggc tcc 8424Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly Ser 2795 2800 2805gac gcc gct cgc ggc gag ttt gcc acg gac act gac gcc cat gac 8469Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp 2810 2815 2820ccc ttc gtg aac gac ctg gcc ttt cag gcc atg ctc gtc tgg gtg 8514Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val 2825 2830 2835cgc cgc acg ctc ggc cag gct gcg ctc ccc aac tcg atc cag cgc 8559Arg Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg 2840 2845 2850atc gtc cag cac cgc ccg gtc ccg cag gac aag ccc ttc tac att 8604Ile Val Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile 2855 2860 2865acc ctc cgc tcc aac cag tcg ggc ggt cac tcc cag cac aag cac 8649Thr Leu Arg Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His 2870 2875 2880gcc ctt cag ttc cac aac gag cag ggc gat ctc ttc att gat gtc 8694Ala Leu Gln Phe His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val 2885 2890 2895cag gct tcg gtc atc gcc acg gac agc ctt gcc ttc taa 8733Gln Ala Ser Val Ile Ala Thr Asp Ser Leu Ala Phe 2900 2905 291022910PRTSchizochytrium sp. 2Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg1 5 10 15Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val 20 25 30Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp 35 40 45Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys 50 55 60Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu65 70 75 80Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu 85 90 95Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala 100 105 110Leu Gln Asp Ala Gly Ile Asp Ala Leu Gly Lys Glu Lys Lys Asn Ile 115 120 125Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser

Ser His Glu Phe 130 135 140Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met145 150 155 160Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala 165 170 175Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn 180 185 190Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn 195 200 205Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val 210 215 220Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly225 230 235 240Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys 245 250 255Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys 260 265 270Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys 275 280 285Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile 290 295 300Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr305 310 315 320Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg 325 330 335Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr 340 345 350Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu 355 360 365Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly 370 375 380Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala385 390 395 400Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly 405 410 415Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile 420 425 430Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro 435 440 445Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly 450 455 460Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr465 470 475 480Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala 485 490 495Ala Thr Pro Ala Ala Leu Gln Ser Leu Cys Glu Ala Gln Leu Lys Glu 500 505 510Phe Glu Ala Ala Ile Lys Glu Asn Glu Thr Val Lys Asn Thr Ala Tyr 515 520 525Ile Lys Cys Val Lys Phe Gly Glu Gln Phe Lys Phe Pro Gly Ser Ile 530 535 540Pro Ala Thr Asn Ala Arg Leu Gly Phe Leu Val Lys Asp Ala Glu Asp545 550 555 560Ala Cys Ser Thr Leu Arg Ala Ile Cys Ala Gln Phe Ala Lys Asp Val 565 570 575Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe Arg Ala 580 585 590Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser Gly Gln 595 600 605Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn Trp Pro 610 615 620Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser Lys Val625 630 635 640Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu Tyr Pro 645 650 655Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys Ile Ser 660 665 670Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu Gly Ala 675 680 685Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala Ala Gly 690 695 700His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys Val Asp705 710 715 720Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile Met Gly 725 730 735Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala Val Ile 740 745 750Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val Trp Leu 755 760 765Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser Val Glu 770 775 780Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe Arg Val785 790 795 800Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met Glu Asn 805 810 815Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe Arg Thr 820 825 830Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu Thr Tyr 835 840 845Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser Ser Val 850 855 860Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala Arg Ile865 870 875 880Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val Ser Glu 885 890 895Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn Pro Ala 900 905 910Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val Gln Leu 915 920 925Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp Ala Pro 930 935 940Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr Leu Arg945 950 955 960Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val Arg Asp 965 970 975Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly Ala Ala 980 985 990Pro Leu Ile Lys Ala Pro Glu Pro Val Val Asp Glu Ala Ala Lys Arg 995 1000 1005Glu Ala Glu Arg Leu Gln Lys Glu Leu Gln Asp Ala Gln Arg Gln 1010 1015 1020Leu Asp Asp Ala Lys Arg Ala Ala Ala Glu Ala Asn Ser Lys Leu 1025 1030 1035Ala Ala Ala Lys Glu Glu Ala Lys Thr Ala Ala Ala Ser Ala Lys 1040 1045 1050Pro Ala Val Asp Thr Ala Val Val Glu Lys His Arg Ala Ile Leu 1055 1060 1065Lys Ser Met Leu Ala Glu Leu Asp Gly Tyr Gly Ser Val Asp Ala 1070 1075 1080Ser Ser Leu Gln Gln Gln Gln Gln Gln Gln Thr Ala Pro Ala Pro 1085 1090 1095Val Lys Ala Ala Ala Pro Ala Ala Pro Val Ala Ser Ala Pro Ala 1100 1105 1110Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val 1115 1120 1125Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile 1130 1135 1140Glu Ala Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile 1145 1150 1155Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val 1160 1165 1170Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly 1175 1180 1185Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala 1190 1195 1200Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Lys Ala Ala Pro 1205 1210 1215Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala 1220 1225 1230Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu 1235 1240 1245Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly 1250 1255 1260Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala 1265 1270 1275Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr 1280 1285 1290Arg Thr Val Gly Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala 1295 1300 1305Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Gly Pro Ala 1310 1315 1320Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn 1325 1330 1335Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala 1340 1345 1350Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu 1355 1360 1365Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile 1370 1375 1380Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val 1385 1390 1395Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala 1400 1405 1410Met Lys Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala 1415 1420 1425Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Pro 1430 1435 1440Ala Pro Ala Val Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val 1445 1450 1455Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met 1460 1465 1470Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser 1475 1480 1485Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn 1490 1495 1500Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val 1505 1510 1515Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Gly Ser 1520 1525 1530Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala 1535 1540 1545Pro Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala Val 1550 1555 1560Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val 1565 1570 1575Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp 1580 1585 1590Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val 1595 1600 1605Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys 1610 1615 1620Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val 1625 1630 1635Asp Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala Ser Ala Pro 1640 1645 1650Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala 1655 1660 1665Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu 1670 1675 1680Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr 1685 1690 1695Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile 1700 1705 1710Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met 1715 1720 1725Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg 1730 1735 1740Thr Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly 1745 1750 1755Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala 1760 1765 1770Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr 1775 1780 1785Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp 1790 1795 1800Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp 1805 1810 1815Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu 1820 1825 1830Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr 1835 1840 1845Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Ser 1850 1855 1860Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala 1865 1870 1875Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Ser Glu Leu 1880 1885 1890Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys 1895 1900 1905Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu 1910 1915 1920Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser 1925 1930 1935Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala 1940 1945 1950Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys 1955 1960 1965Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala 1970 1975 1980Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu 1985 1990 1995Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr 2000 2005 2010Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr 2015 2020 2025Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu 2030 2035 2040Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu 2045 2050 2055Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys Ala 2060 2065 2070Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro 2075 2080 2085Ala Ser Ala Gly Ala Ala Pro Ala Val Lys Ile Asp Ser Val His 2090 2095 2100Gly Ala Asp Cys Asp Asp Leu Ser Leu Met His Ala Lys Val Val 2105 2110 2115Asp Ile Arg Arg Pro Asp Glu Leu Ile Leu Glu Arg Pro Glu Asn 2120 2125 2130Arg Pro Val Leu Val Val Asp Asp Gly Ser Glu Leu Thr Leu Ala 2135 2140 2145Leu Val Arg Val Leu Gly Ala Cys Ala Val Val Leu Thr Phe Glu 2150 2155 2160Gly Leu Gln Leu Ala Gln Arg Ala Gly Ala Ala Ala Ile Arg His 2165 2170 2175Val Leu Ala Lys Asp Leu Ser Ala Glu Ser Ala Glu Lys Ala Ile 2180 2185 2190Lys Glu Ala Glu Gln Arg Phe Gly Ala Leu Gly Gly Phe Ile Ser 2195 2200 2205Gln Gln Ala Glu Arg Phe Glu Pro Ala Glu Ile Leu Gly Phe Thr 2210 2215 2220Leu Met Cys Ala Lys Phe Ala Lys Ala Ser Leu Cys Thr Ala Val 2225 2230 2235Ala Gly Gly Arg Pro Ala Phe Ile Gly Val Ala Arg Leu Asp Gly 2240 2245 2250Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser Asp Ala Leu Lys Arg 2255 2260 2265Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys Thr Ile Gly Leu 2270 2275 2280Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val Asp Ile Ala 2285 2290 2295Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val Arg Glu 2300 2305 2310Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly Ala 2315 2320 2325Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly 2330 2335 2340Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser 2345 2350 2355Gly Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr 2360 2365 2370Arg Gln Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys 2375 2380 2385Val Ser Ala Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu 2390 2395 2400Lys Ala Val Gln Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe 2405 2410 2415Ser Ala Gly Glu Gly Pro Lys Pro Thr Pro Arg Ala Val Thr Lys 2420 2425 2430Leu Val Gly Ser Val Leu Gly Ala Arg Glu Val Arg Ser Ser Ile 2435 2440 2445Ala Ala Ile Glu Ala Leu Gly Gly Lys Ala Ile Tyr Ser Ser Cys 2450 2455 2460Asp Val Asn Ser Ala Ala Asp Val Ala Lys Ala Val Arg Asp Ala 2465 2470 2475Glu Ser Gln Leu Gly Ala Arg Val Ser Gly Ile Val His Ala Ser 2480 2485 2490Gly Val Leu Arg Asp Arg Leu Ile Glu Lys Lys Leu Pro Asp Glu 2495 2500 2505Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly Leu Glu Asn Leu 2510 2515 2520Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met Val Leu Phe 2525 2530 2535Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser Asp Tyr 2540 2545 2550Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu Ala 2555 2560 2565Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly 2570 2575

2580Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly 2585 2590 2595Val Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg 2600 2605 2610Ile Val Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp 2615 2620 2625Arg Thr Pro Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His 2630 2635 2640Arg Lys Ile Ser Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val 2645 2650 2655Ile Gln Gly Arg Arg Val Leu Pro Met Thr Leu Ala Ile Gly Ser 2660 2665 2670Leu Ala Glu Thr Cys Leu Gly Leu Phe Pro Gly Tyr Ser Leu Trp 2675 2680 2685Ala Ile Asp Asp Ala Gln Leu Phe Lys Gly Val Thr Val Asp Gly 2690 2695 2700Asp Val Asn Cys Glu Val Thr Leu Thr Pro Ser Thr Ala Pro Ser 2705 2710 2715Gly Arg Val Asn Val Gln Ala Thr Leu Lys Thr Phe Ser Ser Gly 2720 2725 2730Lys Leu Val Pro Ala Tyr Arg Ala Val Ile Val Leu Ser Asn Gln 2735 2740 2745Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro Pro Ser Leu Asp 2750 2755 2760Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly Lys Thr Leu 2765 2770 2775Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu Ser Cys 2780 2785 2790Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly Ser 2795 2800 2805Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp 2810 2815 2820Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val 2825 2830 2835Arg Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg 2840 2845 2850Ile Val Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile 2855 2860 2865Thr Leu Arg Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His 2870 2875 2880Ala Leu Gln Phe His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val 2885 2890 2895Gln Ala Ser Val Ile Ala Thr Asp Ser Leu Ala Phe 2900 2905 291036180DNASchizochytrium sp.CDS(1)..(6180) 3atg gcc gct cgg aat gtg agc gcc gcg cat gag atg cac gat gaa aag 48Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys1 5 10 15cgc atc gcc gtc gtc ggc atg gcc gtc cag tac gcc gga tgc aaa acc 96Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr 20 25 30aag gac gag ttc tgg gag gtg ctc atg aac ggc aag gtc gag tcc aag 144Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys 35 40 45gtg atc agc gac aaa cga ctc ggc tcc aac tac cgc gcc gag cac tac 192Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr 50 55 60aaa gca gag cgc agc aag tat gcc gac acc ttt tgc aac gaa acg tac 240Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr65 70 75 80ggc acc ctt gac gag aac gag atc gac aac gag cac gaa ctc ctc ctc 288Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu 85 90 95aac ctc gcc aag cag gca ctc gca gag aca tcc gtc aaa gac tcg aca 336Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr 100 105 110cgc tgc ggc atc gtc agc ggc tgc ctc tcg ttc ccc atg gac aac ctc 384Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125cag ggt gaa ctc ctc aac gtg tac caa aac cat gtc gag aaa aag ctc 432Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140ggg gcc cgc gtc ttc aag gac gcc tcc cat tgg tcc gaa cgc gag cag 480Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln145 150 155 160tcc aac aaa ccc gag gcc ggt gac cgc cgc atc ttc atg gac ccg gcc 528Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175tcc ttc gtc gcc gaa gaa ctc aac ctc ggc gcc ctt cac tac tcc gtc 576Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val 180 185 190gac gca gca tgc gcc acg gcg ctc tac gtg ctc cgc ctc gcg cag gat 624Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205cat ctc gtc tcc ggc gcc gcc gac gtc atg ctc tgc ggt gcc acc tgc 672His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys 210 215 220ctg ccg gag ccc ttt ttc atc ctt tcg ggc ttt tcc acc ttc cag gcc 720Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala225 230 235 240atg ccc gtc ggc acg ggc cag aac gtg tcc atg ccg ctg cac aag gac 768Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp 245 250 255agc cag ggc ctc acc ccg ggt gag ggc ggc tcc atc atg gtc ctc aag 816Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys 260 265 270cgt ctc gat gat gcc atc cgc gac ggc gac cac atc tac ggc acc ctt 864Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu 275 280 285ctc ggc gcc aat gtc agc aac tcc ggc aca ggt ctg ccc ctc aag ccc 912Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro 290 295 300ctt ctc ccc agc gag aaa aag tgc ctc atg gac acc tac acg cgc att 960Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile305 310 315 320aac gtg cac ccg cac aag att cag tac gtc gag tgc cac gcc acc ggc 1008Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly 325 330 335acg ccc cag ggt gat cgt gtg gaa atc gac gcc gtc aag gcc tgc ttt 1056Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe 340 345 350gaa ggc aag gtc ccc cgt ttc ggt acc aca aag ggc aac ttt gga cac 1104Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His 355 360 365acc ctc gtc gca gcc ggc ttt gcc ggt atg tgc aag gtc ctc ctc tcc 1152Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser 370 375 380atg aag cat ggc atc atc ccg ccc acc ccg ggt atc gat gac gag acc 1200Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr385 390 395 400aag atg gac cct ctc gtc gtc tcc ggt gag gcc atc cca tgg cca gag 1248Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu 405 410 415acc aac ggc gag ccc aag cgc gcc ggt ctc tcg gcc ttt ggc ttt ggt 1296Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly 420 425 430ggc acc aac gcc cat gcc gtc ttt gag gag cat gac ccc tcc aac gcc 1344Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala 435 440 445gcc tgc acg ggc cac gac tcc att tct gcg ctc tcg gcc cgc tgc ggc 1392Ala Cys Thr Gly His Asp Ser Ile Ser Ala Leu Ser Ala Arg Cys Gly 450 455 460ggt gaa agc aac atg cgc atc gcc atc act ggt atg gac gcc acc ttt 1440Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe465 470 475 480ggc gct ctc aag gga ctc gac gcc ttc gag cgc gcc att tac acc ggc 1488Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Thr Gly 485 490 495gct cac ggt gcc atc cca ctc cca gaa aag cgc tgg cgc ttt ctc ggc 1536Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly 500 505 510aag gac aag gac ttt ctt gac ctc tgc ggc gtc aag gcc acc ccg cac 1584Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Ala Thr Pro His 515 520 525ggc tgc tac att gaa gat gtt gag gtc gac ttc cag cgc ctc cgc acg 1632Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe Gln Arg Leu Arg Thr 530 535 540ccc atg acc cct gaa gac atg ctc ctc cct cag cag ctt ctg gcc gtc 1680Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln Gln Leu Leu Ala Val545 550 555 560acc acc att gac cgc gcc atc ctc gac tcg gga atg aaa aag ggt ggc 1728Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly Met Lys Lys Gly Gly 565 570 575aat gtc gcc gtc ttt gtc ggc ctc ggc acc gac ctc gag ctc tac cgt 1776Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg 580 585 590cac cgt gct cgc gtc gct ctc aag gag cgc gtc cgc cct gaa gcc tcc 1824His Arg Ala Arg Val Ala Leu Lys Glu Arg Val Arg Pro Glu Ala Ser 595 600 605aag aag ctc aat gac atg atg cag tac att aac gac tgc ggc aca tcc 1872Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn Asp Cys Gly Thr Ser 610 615 620aca tcg tac acc tcg tac att ggc aac ctc gtc gcc acg cgc gtc tcg 1920Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser625 630 635 640tcg cag tgg ggc ttc acg ggc ccc tcc ttt acg atc acc gag ggc aac 1968Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn 645 650 655aac tcc gtc tac cgc tgc gcc gag ctc ggc aag tac ctc ctc gag acc 2016Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr 660 665 670ggc gag gtc gat ggc gtc gtc gtt gcg ggt gtc gat ctc tgc ggc agt 2064Gly Glu Val Asp Gly Val Val Val Ala Gly Val Asp Leu Cys Gly Ser 675 680 685gcc gaa aac ctt tac gtc aag tct cgc cgc ttc aag gtg tcc acc tcc 2112Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Thr Ser 690 695 700gat acc ccg cgc gcc agc ttt gac gcc gcc gcc gat ggc tac ttt gtc 2160Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala Asp Gly Tyr Phe Val705 710 715 720ggc gag ggc tgc ggt gcc ttt gtg ctc aag cgt gag act agc tgc acc 2208Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg Glu Thr Ser Cys Thr 725 730 735aag gac gac cgt atc tac gct tgc atg gat gcc atc gtc cct ggc aac 2256Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn 740 745 750gtc cct agc gcc tgc ttg cgc gag gcc ctc gac cag gcg cgc gtc aag 2304Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp Gln Ala Arg Val Lys 755 760 765ccg ggc gat atc gag atg ctc gag ctc agc gcc gac tcc gcc cgc cac 2352Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His 770 775 780ctc aag gac ccg tcc gtc ctg ccc aag gag ctc act gcc gag gag gaa 2400Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu785 790 795 800atc ggc ggc ctt cag acg atc ctt cgt gac gat gac aag ctc ccg cgc 2448Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp Asp Lys Leu Pro Arg 805 810 815aac gtc gca acg ggc agt gtc aag gcc acc gtc ggt gac acc ggt tat 2496Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val Gly Asp Thr Gly Tyr 820 825 830 gcc tct ggt gct gcc agc ctc atc aag gct gcg ctt tgc atc tac aac 2544Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala Leu Cys Ile Tyr Asn 835 840 845cgc tac ctg ccc agc aac ggc gac gac tgg gat gaa ccc gcc cct gag 2592Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp Glu Pro Ala Pro Glu 850 855 860gcg ccc tgg gac agc acc ctc ttt gcg tgc cag acc tcg cgc gct tgg 2640Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln Thr Ser Arg Ala Trp865 870 875 880ctc aag aac cct ggc gag cgt cgc tat gcg gcc gtc tcg ggc gtc tcc 2688Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala Val Ser Gly Val Ser 885 890 895gag acg cgc tcg tgc tat tcc gtg ctc ctc tcc gaa gcc gag ggc cac 2736Glu Thr Arg Ser Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His 900 905 910tac gag cgc gag aac cgc atc tcg ctc gac gag gag gcg ccc aag ctc 2784Tyr Glu Arg Glu Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu 915 920 925att gtg ctt cgc gcc gac tcc cac gag gag atc ctt ggt cgc ctc gac 2832Ile Val Leu Arg Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp 930 935 940aag atc cgc gag cgc ttc ttg cag ccc acg ggc gcc gcc ccg cgc gag 2880Lys Ile Arg Glu Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu945 950 955 960tcc gag ctc aag gcg cag gcc cgc cgc atc ttc ctc gag ctc ctc ggc 2928Ser Glu Leu Lys Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly 965 970 975gag acc ctt gcc cag gat gcc gct tct tca ggc tcg caa aag ccc ctc 2976Glu Thr Leu Ala Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu 980 985 990gct ctc agc ctc gtc tcc acg ccc tcc aag ctc cag cgc gag gtc gag 3024Ala Leu Ser Leu Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu 995 1000 1005ctc gcg gcc aag ggt atc ccg cgc tgc ctc aag atg cgc cgc gat 3069Leu Ala Ala Lys Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp 1010 1015 1020tgg agc tcc cct gct ggc agc cgc tac gcg cct gag ccg ctc gcc 3114Trp Ser Ser Pro Ala Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala 1025 1030 1035agc gac cgc gtc gcc ttc atg tac ggc gaa ggt cgc agc cct tac 3159Ser Asp Arg Val Ala Phe Met Tyr Gly Glu Gly Arg Ser Pro Tyr 1040 1045 1050tac ggc atc acc caa gac att cac cgc att tgg ccc gaa ctc cac 3204Tyr Gly Ile Thr Gln Asp Ile His Arg Ile Trp Pro Glu Leu His 1055 1060 1065gag gtc atc aac gaa aag acg aac cgt ctc tgg gcc gaa ggc gac 3249Glu Val Ile Asn Glu Lys Thr Asn Arg Leu Trp Ala Glu Gly Asp 1070 1075 1080cgc tgg gtc atg ccg cgc gcc agc ttc aag tcg gag ctc gag agc 3294Arg Trp Val Met Pro Arg Ala Ser Phe Lys Ser Glu Leu Glu Ser 1085 1090 1095cag cag caa gag ttt gat cgc aac atg att gaa atg ttc cgt ctt 3339Gln Gln Gln Glu Phe Asp Arg Asn Met Ile Glu Met Phe Arg Leu 1100 1105 1110gga atc ctc acc tca att gcc ttc acc aat ctg gcg cgc gac gtt 3384Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu Ala Arg Asp Val 1115 1120 1125ctc aac atc acg ccc aag gcc gcc ttt ggc ctc agt ctt ggc gag 3429Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser Leu Gly Glu 1130 1135 1140att tcc atg att ttt gcc ttt tcc aag aag aac ggt ctc atc tcc 3474Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu Ile Ser 1145 1150 1155gac cag ctc acc aag gat ctt cgc gag tcc gac gtg tgg aac aag 3519Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn Lys 1160 1165 1170gct ctg gcc gtt gaa ttt aat gcg ctg cgc gag gcc tgg ggc att 3564Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile 1175 1180 1185cca cag agt gtc ccc aag gac gag ttc tgg caa ggc tac att gtg 3609Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val 1190 1195 1200cgc ggc acc aag cag gat atc gag gcg gcc atc gcc ccg gac agc 3654Arg Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser 1205 1210 1215aag tac gtg cgc ctc acc atc atc aat gat gcc aac acc gcc ctc 3699Lys Tyr Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu 1220 1225 1230att agc ggc aag ccc gac gcc tgc aag gct gcg atc gcg cgt ctc 3744Ile Ser Gly Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu 1235 1240 1245ggt ggc aac att cct gcg ctt ccc gtg acc cag ggc atg tgc ggc 3789Gly Gly Asn Ile Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly 1250 1255 1260cac tgc ccc gag gtg gga cct tat acc aag gat atc gcc aag atc 3834His Cys Pro Glu Val Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile 1265 1270 1275cat gcc aac ctt gag ttc ccc gtt gtc gac ggc ctt gac ctc tgg 3879His Ala Asn Leu Glu Phe Pro Val Val Asp Gly Leu Asp Leu Trp 1280 1285 1290acc aca atc aac cag aag cgc ctc gtg cca cgc gcc acg ggc gcc 3924Thr Thr Ile Asn Gln Lys Arg Leu Val Pro Arg Ala Thr Gly Ala 1295 1300 1305aag gac gaa tgg gcc cct tct tcc ttt ggc gag tac gcc ggc cag 3969Lys Asp Glu Trp Ala Pro Ser Ser Phe

Gly Glu Tyr Ala Gly Gln 1310 1315 1320ctc tac gag aag cag gct aac ttc ccc caa atc gtc gag acc att 4014Leu Tyr Glu Lys Gln Ala Asn Phe Pro Gln Ile Val Glu Thr Ile 1325 1330 1335tac aag caa aac tac gac gtc ttt gtc gag gtt ggg ccc aac aac 4059Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu Val Gly Pro Asn Asn 1340 1345 1350cac cgt agc acc gca gtg cgc acc acg ctt ggt ccc cag cgc aac 4104His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly Pro Gln Arg Asn 1355 1360 1365cac ctt gct ggc gcc atc gac aag cag aac gag gat gct tgg acg 4149His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp Ala Trp Thr 1370 1375 1380acc atc gtc aag ctt gtg gct tcg ctc aag gcc cac ctt gtt cct 4194Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu Val Pro 1385 1390 1395ggc gtc acg atc tcg ccg ctg tac cac tcc aag ctt gtg gcg gag 4239Gly Val Thr Ile Ser Pro Leu Tyr His Ser Lys Leu Val Ala Glu 1400 1405 1410gct gag gct tgc tac gct gcg ctc tgc aag ggt gaa aag ccc aag 4284Ala Glu Ala Cys Tyr Ala Ala Leu Cys Lys Gly Glu Lys Pro Lys 1415 1420 1425aag aac aag ttt gtg cgc aag att cag ctc aac ggt cgc ttc aac 4329Lys Asn Lys Phe Val Arg Lys Ile Gln Leu Asn Gly Arg Phe Asn 1430 1435 1440agc aag gcg gac ccc atc tcc tcg gcc gat ctt gcc agc ttt ccg 4374Ser Lys Ala Asp Pro Ile Ser Ser Ala Asp Leu Ala Ser Phe Pro 1445 1450 1455cct gcg gac cct gcc att gaa gcc gcc atc tcg agc cgc atc atg 4419Pro Ala Asp Pro Ala Ile Glu Ala Ala Ile Ser Ser Arg Ile Met 1460 1465 1470aag cct gtc gct ccc aag ttc tac gcg cgt ctc aac att gac gag 4464Lys Pro Val Ala Pro Lys Phe Tyr Ala Arg Leu Asn Ile Asp Glu 1475 1480 1485cag gac gag acc cga gat ccg atc ctc aac aag gac aac gcg ccg 4509Gln Asp Glu Thr Arg Asp Pro Ile Leu Asn Lys Asp Asn Ala Pro 1490 1495 1500tct tct tct tct tct tct tct tct tct tct tct tct tct tct tct 4554Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 1505 1510 1515ccg tcg cct gct cct tcg gcc ccc gtg caa aag aag gct gct ccc 4599Pro Ser Pro Ala Pro Ser Ala Pro Val Gln Lys Lys Ala Ala Pro 1520 1525 1530gcc gcg gag acc aag gct gtt gct tcg gct gac gca ctt cgc agt 4644Ala Ala Glu Thr Lys Ala Val Ala Ser Ala Asp Ala Leu Arg Ser 1535 1540 1545gcc ctg ctc gat ctc gac agt atg ctt gcg ctg agc tct gcc agt 4689Ala Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser 1550 1555 1560gcc tcc ggc aac ctt gtt gag act gcg cct agc gac gcc tcg gtc 4734Ala Ser Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val 1565 1570 1575att gtg ccg ccc tgc aac att gcg gat ctc ggc agc cgc gcc ttc 4779Ile Val Pro Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe 1580 1585 1590atg aaa acg tac ggt gtt tcg gcg cct ctg tac acg ggc gcc atg 4824Met Lys Thr Tyr Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met 1595 1600 1605gcc aag ggc att gcc tct gcg gac ctc gtc att gcc gcc ggc cgc 4869Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly Arg 1610 1615 1620cag ggc atc ctt gcg tcc ttt ggc gcc ggc gga ctt ccc atg cag 4914Gln Gly Ile Leu Ala Ser Phe Gly Ala Gly Gly Leu Pro Met Gln 1625 1630 1635gtt gtg cgt gag tcc atc gaa aag att cag gcc gcc ctg ccc aat 4959Val Val Arg Glu Ser Ile Glu Lys Ile Gln Ala Ala Leu Pro Asn 1640 1645 1650ggc ccg tac gct gtc aac ctt atc cat tct ccc ttt gac agc aac 5004Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser Asn 1655 1660 1665ctc gaa aag ggc aat gtc gat ctc ttc ctc gag aag ggt gtc acc 5049Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val Thr 1670 1675 1680ttt gtc gag gcc tcg gcc ttt atg acg ctc acc ccg cag gtc gtg 5094Phe Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val Val 1685 1690 1695cgg tac cgc gcg gct ggc ctc acg cgc aac gcc gac ggc tcg gtc 5139Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala Asp Gly Ser Val 1700 1705 1710aac atc cgc aac cgt atc att ggc aag gtc tcg cgc acc gag ctc 5184Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu 1715 1720 1725gcc gag atg ttc atg cgt cct gcg ccc gag cac ctt ctt cag aag 5229Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu Gln Lys 1730 1735 1740ctc att gct tcc ggc gag atc aac cag gag cag gcc gag ctc gcc 5274Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu Ala 1745 1750 1755cgc cgt gtt ccc gtc gct gac gac atc gcg gtc gaa gct gac tcg 5319Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser 1760 1765 1770ggt ggc cac acc gac aac cgc ccc atc cac gtc att ctg ccc ctc 5364Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu 1775 1780 1785atc atc aac ctt cgc gac cgc ctt cac cgc gag tgc ggc tac ccg 5409Ile Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro 1790 1795 1800gcc aac ctt cgc gtc cgt gtg ggc gcc ggc ggt ggc att ggg tgc 5454Ala Asn Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys 1805 1810 1815ccc cag gcg gcg ctg gcc acc ttc aac atg ggt gcc tcc ttt att 5499Pro Gln Ala Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile 1820 1825 1830gtc acc ggc acc gtg aac cag gtc gcc aag cag tcg ggc acg tgc 5544Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys 1835 1840 1845gac aat gtg cgc aag cag ctc gcg aag gcc act tac tcg gac gta 5589Asp Asn Val Arg Lys Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val 1850 1855 1860tgc atg gcc ccg gct gcc gac atg ttc gag gaa ggc gtc aag ctt 5634Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys Leu 1865 1870 1875cag gtc ctc aag aag gga acc atg ttt ccc tcg cgc gcc aac aag 5679Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn Lys 1880 1885 1890ctc tac gag ctc ttt tgc aag tac gac tcg ttc gag tcc atg ccc 5724Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Glu Ser Met Pro 1895 1900 1905ccc gca gag ctt gcg cgc gtc gag aag cgc atc ttc agc cgc gcg 5769Pro Ala Glu Leu Ala Arg Val Glu Lys Arg Ile Phe Ser Arg Ala 1910 1915 1920ctc gaa gag gtc tgg gac gag acc aaa aac ttt tac att aac cgt 5814Leu Glu Glu Val Trp Asp Glu Thr Lys Asn Phe Tyr Ile Asn Arg 1925 1930 1935ctt cac aac ccg gag aag atc cag cgc gcc gag cgc gac ccc aag 5859Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu Arg Asp Pro Lys 1940 1945 1950ctc aag atg tcg ctg tgc ttt cgc tgg tac ctg agc ctg gcg agc 5904Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser Leu Ala Ser 1955 1960 1965cgc tgg gcc aac act gga gct tcc gat cgc gtc atg gac tac cag 5949Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp Tyr Gln 1970 1975 1980gtc tgg tgc ggt cct gcc att ggt tcc ttc aac gat ttc atc aag 5994Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile Lys 1985 1990 1995gga act tac ctt gat ccg gcc gtc gca aac gag tac ccg tgc gtc 6039Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val 2000 2005 2010gtt cag att aac aag cag atc ctt cgt gga gcg tgc ttc ttg cgc 6084Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg 2015 2020 2025cgt ctc gaa att ctg cgc aac gca cgc ctt tcc gat ggc gct gcc 6129Arg Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala 2030 2035 2040gct ctt gtg gcc agc atc gat gac aca tac gtc ccg gcc gag aag 6174Ala Leu Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys 2045 2050 2055ctg taa 6180Leu 42059PRTSchizochytrium sp. 4Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys1 5 10 15Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr 20 25 30Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys 35 40 45Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr 50 55 60Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr65 70 75 80Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu 85 90 95Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr 100 105 110Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln145 150 155 160Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val 180 185 190Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys 210 215 220Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala225 230 235 240Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp 245 250 255Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys 260 265 270Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu 275 280 285Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro 290 295 300Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile305 310 315 320Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly 325 330 335Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe 340 345 350Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His 355 360 365Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser 370 375 380Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr385 390 395 400Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu 405 410 415Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly 420 425 430Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala 435 440 445Ala Cys Thr Gly His Asp Ser Ile Ser Ala Leu Ser Ala Arg Cys Gly 450 455 460Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe465 470 475 480Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Thr Gly 485 490 495Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly 500 505 510Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Ala Thr Pro His 515 520 525Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe Gln Arg Leu Arg Thr 530 535 540Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln Gln Leu Leu Ala Val545 550 555 560Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly Met Lys Lys Gly Gly 565 570 575Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg 580 585 590His Arg Ala Arg Val Ala Leu Lys Glu Arg Val Arg Pro Glu Ala Ser 595 600 605Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn Asp Cys Gly Thr Ser 610 615 620Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser625 630 635 640Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn 645 650 655Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr 660 665 670Gly Glu Val Asp Gly Val Val Val Ala Gly Val Asp Leu Cys Gly Ser 675 680 685Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Thr Ser 690 695 700Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala Asp Gly Tyr Phe Val705 710 715 720Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg Glu Thr Ser Cys Thr 725 730 735Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn 740 745 750Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp Gln Ala Arg Val Lys 755 760 765Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His 770 775 780Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu785 790 795 800Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp Asp Lys Leu Pro Arg 805 810 815Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val Gly Asp Thr Gly Tyr 820 825 830Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala Leu Cys Ile Tyr Asn 835 840 845Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp Glu Pro Ala Pro Glu 850 855 860Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln Thr Ser Arg Ala Trp865 870 875 880Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala Val Ser Gly Val Ser 885 890 895Glu Thr Arg Ser Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His 900 905 910Tyr Glu Arg Glu Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu 915 920 925Ile Val Leu Arg Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp 930 935 940Lys Ile Arg Glu Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu945 950 955 960Ser Glu Leu Lys Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly 965 970 975Glu Thr Leu Ala Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu 980 985 990Ala Leu Ser Leu Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu 995 1000 1005Leu Ala Ala Lys Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp 1010 1015 1020Trp Ser Ser Pro Ala Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala 1025 1030 1035Ser Asp Arg Val Ala Phe Met Tyr Gly Glu Gly Arg Ser Pro Tyr 1040 1045 1050Tyr Gly Ile Thr Gln Asp Ile His Arg Ile Trp Pro Glu Leu His 1055 1060 1065Glu Val Ile Asn Glu Lys Thr Asn Arg Leu Trp Ala Glu Gly Asp 1070 1075 1080Arg Trp Val Met Pro Arg Ala Ser Phe Lys Ser Glu Leu Glu Ser 1085 1090 1095Gln Gln Gln Glu Phe Asp Arg Asn Met Ile Glu Met Phe Arg Leu 1100 1105 1110Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu Ala Arg Asp Val 1115 1120 1125Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser Leu Gly Glu 1130 1135 1140Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu Ile Ser 1145 1150 1155Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn Lys 1160 1165 1170Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile 1175 1180 1185Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val 1190 1195 1200Arg Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser 1205 1210

1215Lys Tyr Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu 1220 1225 1230Ile Ser Gly Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu 1235 1240 1245Gly Gly Asn Ile Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly 1250 1255 1260His Cys Pro Glu Val Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile 1265 1270 1275His Ala Asn Leu Glu Phe Pro Val Val Asp Gly Leu Asp Leu Trp 1280 1285 1290Thr Thr Ile Asn Gln Lys Arg Leu Val Pro Arg Ala Thr Gly Ala 1295 1300 1305Lys Asp Glu Trp Ala Pro Ser Ser Phe Gly Glu Tyr Ala Gly Gln 1310 1315 1320Leu Tyr Glu Lys Gln Ala Asn Phe Pro Gln Ile Val Glu Thr Ile 1325 1330 1335Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu Val Gly Pro Asn Asn 1340 1345 1350His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly Pro Gln Arg Asn 1355 1360 1365His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp Ala Trp Thr 1370 1375 1380Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu Val Pro 1385 1390 1395Gly Val Thr Ile Ser Pro Leu Tyr His Ser Lys Leu Val Ala Glu 1400 1405 1410Ala Glu Ala Cys Tyr Ala Ala Leu Cys Lys Gly Glu Lys Pro Lys 1415 1420 1425Lys Asn Lys Phe Val Arg Lys Ile Gln Leu Asn Gly Arg Phe Asn 1430 1435 1440Ser Lys Ala Asp Pro Ile Ser Ser Ala Asp Leu Ala Ser Phe Pro 1445 1450 1455Pro Ala Asp Pro Ala Ile Glu Ala Ala Ile Ser Ser Arg Ile Met 1460 1465 1470Lys Pro Val Ala Pro Lys Phe Tyr Ala Arg Leu Asn Ile Asp Glu 1475 1480 1485Gln Asp Glu Thr Arg Asp Pro Ile Leu Asn Lys Asp Asn Ala Pro 1490 1495 1500Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 1505 1510 1515Pro Ser Pro Ala Pro Ser Ala Pro Val Gln Lys Lys Ala Ala Pro 1520 1525 1530Ala Ala Glu Thr Lys Ala Val Ala Ser Ala Asp Ala Leu Arg Ser 1535 1540 1545Ala Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser 1550 1555 1560Ala Ser Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val 1565 1570 1575Ile Val Pro Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe 1580 1585 1590Met Lys Thr Tyr Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met 1595 1600 1605Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly Arg 1610 1615 1620Gln Gly Ile Leu Ala Ser Phe Gly Ala Gly Gly Leu Pro Met Gln 1625 1630 1635Val Val Arg Glu Ser Ile Glu Lys Ile Gln Ala Ala Leu Pro Asn 1640 1645 1650Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser Asn 1655 1660 1665Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val Thr 1670 1675 1680Phe Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val Val 1685 1690 1695Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala Asp Gly Ser Val 1700 1705 1710Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu 1715 1720 1725Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu Gln Lys 1730 1735 1740Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu Ala 1745 1750 1755Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser 1760 1765 1770Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu 1775 1780 1785Ile Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro 1790 1795 1800Ala Asn Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys 1805 1810 1815Pro Gln Ala Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile 1820 1825 1830Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys 1835 1840 1845Asp Asn Val Arg Lys Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val 1850 1855 1860Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys Leu 1865 1870 1875Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn Lys 1880 1885 1890Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Glu Ser Met Pro 1895 1900 1905Pro Ala Glu Leu Ala Arg Val Glu Lys Arg Ile Phe Ser Arg Ala 1910 1915 1920Leu Glu Glu Val Trp Asp Glu Thr Lys Asn Phe Tyr Ile Asn Arg 1925 1930 1935Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu Arg Asp Pro Lys 1940 1945 1950Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser Leu Ala Ser 1955 1960 1965Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp Tyr Gln 1970 1975 1980Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile Lys 1985 1990 1995Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val 2000 2005 2010Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg 2015 2020 2025Arg Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala 2030 2035 2040Ala Leu Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys 2045 2050 2055Leu 54509DNASchizochytrium sp.CDS(1)..(4509) 5atg gcg ctc cgt gtc aag acg aac aag aag cca tgc tgg gag atg acc 48Met Ala Leu Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr1 5 10 15aag gag gag ctg acc agc ggc aag acc gag gtg ttc aac tat gag gaa 96Lys Glu Glu Leu Thr Ser Gly Lys Thr Glu Val Phe Asn Tyr Glu Glu 20 25 30ctc ctc gag ttc gca gag ggc gac atc gcc aag gtc ttc gga ccc gag 144Leu Leu Glu Phe Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Pro Glu 35 40 45ttc gcc gtc atc gac aag tac ccg cgc cgc gtg cgc ctg ccc gcc cgc 192Phe Ala Val Ile Asp Lys Tyr Pro Arg Arg Val Arg Leu Pro Ala Arg 50 55 60gag tac ctg ctc gtg acc cgc gtc acc ctc atg gac gcc gag gtc aac 240Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn65 70 75 80aac tac cgc gtc ggc gcc cgc atg gtc acc gag tac gat ctc ccc gtc 288Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val 85 90 95aac gga gag ctc tcc gag ggc gga gac tgc ccc tgg gcc gtc ctg gtc 336Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val 100 105 110gag agt ggc cag tgc gat ctc atg ctc atc tcc tac atg ggc att gac 384Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp 115 120 125ttc cag aac cag ggc gac cgc gtc tac cgc ctg ctc aac acc acg ctc 432Phe Gln Asn Gln Gly Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu 130 135 140acc ttt tac ggc gtg gcc cac gag ggc gag acc ctc gag tac gac att 480Thr Phe Tyr Gly Val Ala His Glu Gly Glu Thr Leu Glu Tyr Asp Ile145 150 155 160cgc gtc acc ggc ttc gcc aag cgt ctc gac ggc ggc atc tcc atg ttc 528Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Gly Ile Ser Met Phe 165 170 175ttc ttc gag tac gac tgc tac gtc aac ggc cgc ctc ctc atc gag atg 576Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met 180 185 190cgc gat ggc tgc gcc ggc ttc ttc acc aac gag gag ctc gac gcc ggc 624Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Asp Ala Gly 195 200 205aag ggc gtc gtc ttc acc cgc ggc gac ctc gcc gcc cgc gcc aag atc 672Lys Gly Val Val Phe Thr Arg Gly Asp Leu Ala Ala Arg Ala Lys Ile 210 215 220cca aag cag gac gtc tcc ccc tac gcc gtc gcc ccc tgc ctc cac aag 720Pro Lys Gln Asp Val Ser Pro Tyr Ala Val Ala Pro Cys Leu His Lys225 230 235 240acc aag ctc aac gaa aag gag atg cag acc ctc gtc gac aag gac tgg 768Thr Lys Leu Asn Glu Lys Glu Met Gln Thr Leu Val Asp Lys Asp Trp 245 250 255gca tcc gtc ttt ggc tcc aag aac ggc atg ccg gaa atc aac tac aaa 816Ala Ser Val Phe Gly Ser Lys Asn Gly Met Pro Glu Ile Asn Tyr Lys 260 265 270ctc tgc gcg cgt aag atg ctc atg att gac cgc gtc acc agc att gac 864Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Ser Ile Asp 275 280 285cac aag ggc ggt gtc tac ggc ctc ggt cag ctc gtc ggt gaa aag atc 912His Lys Gly Gly Val Tyr Gly Leu Gly Gln Leu Val Gly Glu Lys Ile 290 295 300ctc gag cgc gac cac tgg tac ttt ccc tgc cac ttt gtc aag gat cag 960Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Lys Asp Gln305 310 315 320gtc atg gcc gga tcc ctc gtc tcc gac ggc tgc agc cag atg ctc aag 1008Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Met Leu Lys 325 330 335atg tac atg atc tgg ctc ggc ctc cac ctc acc acc gga ccc ttt gac 1056Met Tyr Met Ile Trp Leu Gly Leu His Leu Thr Thr Gly Pro Phe Asp 340 345 350ttc cgc ccg gtc aac ggc cac ccc aac aag gtc cgc tgc cgc ggc caa 1104Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln 355 360 365atc tcc ccg cac aag ggc aag ctc gtc tac gtc atg gag atc aag gag 1152Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu 370 375 380atg ggc ttc gac gag gac aac gac ccg tac gcc att gcc gac gtc aac 1200Met Gly Phe Asp Glu Asp Asn Asp Pro Tyr Ala Ile Ala Asp Val Asn385 390 395 400atc att gat gtc gac ttc gaa aag ggc cag gac ttt agc ctc gac cgc 1248Ile Ile Asp Val Asp Phe Glu Lys Gly Gln Asp Phe Ser Leu Asp Arg 405 410 415atc agc gac tac ggc aag ggc gac ctc aac aag aag atc gtc gtc gac 1296Ile Ser Asp Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp 420 425 430ttt aag ggc atc gct ctc aag atg cag aag cgc tcc acc aac aag aac 1344Phe Lys Gly Ile Ala Leu Lys Met Gln Lys Arg Ser Thr Asn Lys Asn 435 440 445ccc tcc aag gtt cag ccc gtc ttt gcc aac ggc gcc gcc act gtc ggc 1392Pro Ser Lys Val Gln Pro Val Phe Ala Asn Gly Ala Ala Thr Val Gly 450 455 460ccc gag gcc tcc aag gct tcc tcc ggc gcc agc gcc agc gcc agc gcc 1440Pro Glu Ala Ser Lys Ala Ser Ser Gly Ala Ser Ala Ser Ala Ser Ala465 470 475 480gcc ccg gcc aag cct gcc ttc agc gcc gat gtt ctt gcg ccc aag ccc 1488Ala Pro Ala Lys Pro Ala Phe Ser Ala Asp Val Leu Ala Pro Lys Pro 485 490 495gtt gcc ctt ccc gag cac atc ctc aag ggc gac gcc ctc gcc ccc aag 1536Val Ala Leu Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala Pro Lys 500 505 510gag atg tcc tgg cac ccc atg gcc cgc atc ccg ggc aac ccg acg ccc 1584Glu Met Ser Trp His Pro Met Ala Arg Ile Pro Gly Asn Pro Thr Pro 515 520 525tct ttt gcg ccc tcg gcc tac aag ccg cgc aac atc gcc ttt acg ccc 1632Ser Phe Ala Pro Ser Ala Tyr Lys Pro Arg Asn Ile Ala Phe Thr Pro 530 535 540ttc ccc ggc aac ccc aac gat aac gac cac acc ccg ggc aag atg ccg 1680Phe Pro Gly Asn Pro Asn Asp Asn Asp His Thr Pro Gly Lys Met Pro545 550 555 560ctc acc tgg ttc aac atg gcc gag ttc atg gcc ggc aag gtc agc atg 1728Leu Thr Trp Phe Asn Met Ala Glu Phe Met Ala Gly Lys Val Ser Met 565 570 575tgc ctc ggc ccc gag ttc gcc aag ttc gac gac tcg aac acc agc cgc 1776Cys Leu Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr Ser Arg 580 585 590agc ccc gct tgg gac ctc gct ctc gtc acc cgc gcc gtg tct gtg tct 1824Ser Pro Ala Trp Asp Leu Ala Leu Val Thr Arg Ala Val Ser Val Ser 595 600 605gac ctc aag cac gtc aac tac cgc aac atc gac ctc gac ccc tcc aag 1872Asp Leu Lys His Val Asn Tyr Arg Asn Ile Asp Leu Asp Pro Ser Lys 610 615 620ggt acc atg gtc ggc gag ttc gac tgc ccc gcg gac gcc tgg ttc tac 1920Gly Thr Met Val Gly Glu Phe Asp Cys Pro Ala Asp Ala Trp Phe Tyr625 630 635 640aag ggc gcc tgc aac gat gcc cac atg ccg tac tcg atc ctc atg gag 1968Lys Gly Ala Cys Asn Asp Ala His Met Pro Tyr Ser Ile Leu Met Glu 645 650 655atc gcc ctc cag acc tcg ggt gtg ctc acc tcg gtg ctc aag gcg ccc 2016Ile Ala Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys Ala Pro 660 665 670ctg acc atg gag aag gac gac atc ctc ttc cgc aac ctc gac gcc aac 2064Leu Thr Met Glu Lys Asp Asp Ile Leu Phe Arg Asn Leu Asp Ala Asn 675 680 685gcc gag ttc gtg cgc gcc gac ctc gac tac cgc ggc aag act atc cgc 2112Ala Glu Phe Val Arg Ala Asp Leu Asp Tyr Arg Gly Lys Thr Ile Arg 690 695 700aac gtc acc aag tgc act ggc tac agc atg ctc ggc gag atg ggc gtc 2160Asn Val Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Glu Met Gly Val705 710 715 720cac cgc ttc acc ttt gag ctc tac gtc gat gat gtg ctc ttt tac aag 2208His Arg Phe Thr Phe Glu Leu Tyr Val Asp Asp Val Leu Phe Tyr Lys 725 730 735ggc tcg acc tcg ttc ggc tgg ttc gtg ccc gag gtc ttt gcc gcc cag 2256Gly Ser Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ala Ala Gln 740 745 750gcc ggc ctc gac aac ggc cgc aag tcg gag ccc tgg ttc att gag aac 2304Ala Gly Leu Asp Asn Gly Arg Lys Ser Glu Pro Trp Phe Ile Glu Asn 755 760 765aag gtt ccg gcc tcg cag gtc tcc tcc ttt gac gtg cgc ccc aac ggc 2352Lys Val Pro Ala Ser Gln Val Ser Ser Phe Asp Val Arg Pro Asn Gly 770 775 780agc ggc cgc acc gcc atc ttc gcc aac gcc ccc agc ggc gcc cag ctc 2400Ser Gly Arg Thr Ala Ile Phe Ala Asn Ala Pro Ser Gly Ala Gln Leu785 790 795 800aac cgc cgc acg gac cag ggc cag tac ctc gac gcc gtc gac att gtc 2448Asn Arg Arg Thr Asp Gln Gly Gln Tyr Leu Asp Ala Val Asp Ile Val 805 810 815tcc ggc agc ggc aag aag agc ctc ggc tac gcc cac ggt tcc aag acg 2496Ser Gly Ser Gly Lys Lys Ser Leu Gly Tyr Ala His Gly Ser Lys Thr 820 825 830gtc aac ccg aac gac tgg ttc ttc tcg tgc cac ttt tgg ttt gac tcg 2544Val Asn Pro Asn Asp Trp Phe Phe Ser Cys His Phe Trp Phe Asp Ser 835 840 845gtc atg ccc gga agt ctc ggt gtc gag tcc atg ttc cag ctc gtc gag 2592Val Met Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu Val Glu 850 855 860gcc atc gcc gcc cac gag gat ctc gct ggc aag cac ggc att gcc aac 2640Ala Ile Ala Ala His Glu Asp Leu Ala Gly Lys His Gly Ile Ala Asn865 870 875 880ccc acc ttt gtg cac gcc ccg ggc aag atc agc tgg aag tac cgc ggc 2688Pro Thr Phe Val His Ala Pro Gly Lys Ile Ser Trp Lys Tyr Arg Gly 885 890 895cag ctc acg ccc aag agc aag aag atg gac tcg gag gtc cac atc gtg 2736Gln Leu Thr Pro Lys Ser Lys Lys Met Asp Ser Glu Val His Ile Val 900 905 910tcc gtg gac gcc cac gac ggc gtt gtc gac ctc gtc gcc gac ggc ttc 2784Ser Val Asp Ala His Asp Gly Val Val Asp Leu Val Ala Asp Gly Phe 915 920 925ctc tgg gcc gac agc ctc cgc gtc tac tcg gtg agc aac att cgc gtg 2832Leu Trp Ala Asp Ser Leu Arg Val Tyr Ser Val Ser Asn Ile Arg Val 930 935 940cgc atc gcc tcc ggt gag gcc cct gcc gcc gcc tcc tcc gcc gcc tct 2880Arg Ile Ala Ser Gly Glu Ala Pro Ala Ala Ala Ser Ser Ala Ala Ser945 950 955 960gtg ggc tcc tcg gct tcg tcc gtc gag cgc acg cgc tcg agc ccc gct 2928Val Gly Ser Ser Ala Ser Ser Val Glu Arg Thr Arg Ser Ser Pro Ala 965 970 975gtc gcc tcc ggc ccg gcc cag acc atc gac ctc aag cag ctc aag acc 2976Val Ala Ser Gly Pro Ala Gln Thr Ile Asp Leu Lys Gln Leu Lys Thr 980 985 990gag ctc ctc gag ctc gat gcc ccg ctc tac ctc tcg cag gac ccg acc 3024Glu Leu Leu Glu Leu Asp Ala Pro Leu Tyr Leu Ser Gln Asp Pro Thr

995 1000 1005agc ggc cag ctc aag aag cac acc gac gtg gcc tcc ggc cag gcc 3069Ser Gly Gln Leu Lys Lys His Thr Asp Val Ala Ser Gly Gln Ala 1010 1015 1020acc atc gtg cag ccc tgc acg ctc ggc gac ctc ggt gac cgc tcc 3114Thr Ile Val Gln Pro Cys Thr Leu Gly Asp Leu Gly Asp Arg Ser 1025 1030 1035ttc atg gag acc tac ggc gtc gtc gcc ccg ctg tac acg ggc gcc 3159Phe Met Glu Thr Tyr Gly Val Val Ala Pro Leu Tyr Thr Gly Ala 1040 1045 1050atg gcc aag ggc att gcc tcg gcg gac ctc gtc atc gcc gcc ggc 3204Met Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly 1055 1060 1065aag cgc aag atc ctc ggc tcc ttt ggc gcc ggc ggc ctc ccc atg 3249Lys Arg Lys Ile Leu Gly Ser Phe Gly Ala Gly Gly Leu Pro Met 1070 1075 1080cac cac gtg cgc gcc gcc ctc gag aag atc cag gcc gcc ctg cct 3294His His Val Arg Ala Ala Leu Glu Lys Ile Gln Ala Ala Leu Pro 1085 1090 1095cag ggc ccc tac gcc gtc aac ctc atc cac tcg cct ttt gac agc 3339Gln Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser 1100 1105 1110aac ctc gag aag ggc aac gtc gat ctc ttc ctc gag aag ggc gtc 3384Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val 1115 1120 1125act gtg gtg gag gcc tcg gca ttc atg acc ctc acc ccg cag gtc 3429Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val 1130 1135 1140gtg cgc tac cgc gcc gcc ggc ctc tcg cgc aac gcc gac ggt tcg 3474Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Asn Ala Asp Gly Ser 1145 1150 1155gtc aac atc cgc aac cgc atc atc ggc aag gtc tcg cgc acc gag 3519Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu 1160 1165 1170ctc gcc gag atg ttc atc cgc ccg gcc ccg gag cac ctc ctc gag 3564Leu Ala Glu Met Phe Ile Arg Pro Ala Pro Glu His Leu Leu Glu 1175 1180 1185aag ctc atc gcc tcg ggc gag atc acc cag gag cag gcc gag ctc 3609Lys Leu Ile Ala Ser Gly Glu Ile Thr Gln Glu Gln Ala Glu Leu 1190 1195 1200gcg cgc cgc gtt ccc gtc gcc gac gat atc gct gtc gag gct gac 3654Ala Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp 1205 1210 1215tcg ggc ggc cac acc gac aac cgc ccc atc cac gtc atc ctc ccg 3699Ser Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro 1220 1225 1230ctc atc atc aac ctc cgc aac cgc ctg cac cgc gag tgc ggc tac 3744Leu Ile Ile Asn Leu Arg Asn Arg Leu His Arg Glu Cys Gly Tyr 1235 1240 1245ccc gcg cac ctc cgc gtc cgc gtt ggc gcc ggc ggt ggc gtc ggc 3789Pro Ala His Leu Arg Val Arg Val Gly Ala Gly Gly Gly Val Gly 1250 1255 1260tgc ccg cag gcc gcc gcc gcc gcg ctc acc atg ggc gcc gcc ttc 3834Cys Pro Gln Ala Ala Ala Ala Ala Leu Thr Met Gly Ala Ala Phe 1265 1270 1275atc gtc acc ggc act gtc aac cag gtc gcc aag cag tcc ggc acc 3879Ile Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr 1280 1285 1290tgc gac aac gtg cgc aag cag ctc tcg cag gcc acc tac tcg gat 3924Cys Asp Asn Val Arg Lys Gln Leu Ser Gln Ala Thr Tyr Ser Asp 1295 1300 1305atc tgc atg gcc ccg gcc gcc gac atg ttc gag gag ggc gtc aag 3969Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys 1310 1315 1320ctc cag gtc ctc aag aag gga acc atg ttc ccc tcg cgc gcc aac 4014Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn 1325 1330 1335aag ctc tac gag ctc ttt tgc aag tac gac tcc ttc gac tcc atg 4059Lys Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Asp Ser Met 1340 1345 1350cct cct gcc gag ctc gag cgc atc gag aag cgt atc ttc aag cgc 4104Pro Pro Ala Glu Leu Glu Arg Ile Glu Lys Arg Ile Phe Lys Arg 1355 1360 1365gca ctc cag gag gtc tgg gag gag acc aag gac ttt tac att aac 4149Ala Leu Gln Glu Val Trp Glu Glu Thr Lys Asp Phe Tyr Ile Asn 1370 1375 1380ggt ctc aag aac ccg gag aag atc cag cgc gcc gag cac gac ccc 4194Gly Leu Lys Asn Pro Glu Lys Ile Gln Arg Ala Glu His Asp Pro 1385 1390 1395aag ctc aag atg tcg ctc tgc ttc cgc tgg tac ctt ggt ctt gcc 4239Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Gly Leu Ala 1400 1405 1410agc cgc tgg gcc aac atg ggc gcc ccg gac cgc gtc atg gac tac 4284Ser Arg Trp Ala Asn Met Gly Ala Pro Asp Arg Val Met Asp Tyr 1415 1420 1425cag gtc tgg tgt ggc ccg gcc att ggc gcc ttc aac gac ttc atc 4329Gln Val Trp Cys Gly Pro Ala Ile Gly Ala Phe Asn Asp Phe Ile 1430 1435 1440aag ggc acc tac ctc gac ccc gct gtc tcc aac gag tac ccc tgt 4374Lys Gly Thr Tyr Leu Asp Pro Ala Val Ser Asn Glu Tyr Pro Cys 1445 1450 1455gtc gtc cag atc aac ctg caa atc ctc cgt ggt gcc tgc tac ctg 4419Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly Ala Cys Tyr Leu 1460 1465 1470cgc cgt ctc aac gcc ctg cgc aac gac ccg cgc att gac ctc gag 4464Arg Arg Leu Asn Ala Leu Arg Asn Asp Pro Arg Ile Asp Leu Glu 1475 1480 1485acc gag gat gct gcc ttt gtc tac gag ccc acc aac gcg ctc taa 4509Thr Glu Asp Ala Ala Phe Val Tyr Glu Pro Thr Asn Ala Leu 1490 1495 150061502PRTSchizochytrium sp. 6Met Ala Leu Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr1 5 10 15Lys Glu Glu Leu Thr Ser Gly Lys Thr Glu Val Phe Asn Tyr Glu Glu 20 25 30Leu Leu Glu Phe Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Pro Glu 35 40 45Phe Ala Val Ile Asp Lys Tyr Pro Arg Arg Val Arg Leu Pro Ala Arg 50 55 60Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn65 70 75 80Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val 85 90 95Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val 100 105 110Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp 115 120 125Phe Gln Asn Gln Gly Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu 130 135 140Thr Phe Tyr Gly Val Ala His Glu Gly Glu Thr Leu Glu Tyr Asp Ile145 150 155 160Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Gly Ile Ser Met Phe 165 170 175Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met 180 185 190Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Asp Ala Gly 195 200 205Lys Gly Val Val Phe Thr Arg Gly Asp Leu Ala Ala Arg Ala Lys Ile 210 215 220Pro Lys Gln Asp Val Ser Pro Tyr Ala Val Ala Pro Cys Leu His Lys225 230 235 240Thr Lys Leu Asn Glu Lys Glu Met Gln Thr Leu Val Asp Lys Asp Trp 245 250 255Ala Ser Val Phe Gly Ser Lys Asn Gly Met Pro Glu Ile Asn Tyr Lys 260 265 270Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Ser Ile Asp 275 280 285His Lys Gly Gly Val Tyr Gly Leu Gly Gln Leu Val Gly Glu Lys Ile 290 295 300Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Lys Asp Gln305 310 315 320Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Met Leu Lys 325 330 335Met Tyr Met Ile Trp Leu Gly Leu His Leu Thr Thr Gly Pro Phe Asp 340 345 350Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln 355 360 365Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu 370 375 380Met Gly Phe Asp Glu Asp Asn Asp Pro Tyr Ala Ile Ala Asp Val Asn385 390 395 400Ile Ile Asp Val Asp Phe Glu Lys Gly Gln Asp Phe Ser Leu Asp Arg 405 410 415Ile Ser Asp Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp 420 425 430Phe Lys Gly Ile Ala Leu Lys Met Gln Lys Arg Ser Thr Asn Lys Asn 435 440 445Pro Ser Lys Val Gln Pro Val Phe Ala Asn Gly Ala Ala Thr Val Gly 450 455 460Pro Glu Ala Ser Lys Ala Ser Ser Gly Ala Ser Ala Ser Ala Ser Ala465 470 475 480Ala Pro Ala Lys Pro Ala Phe Ser Ala Asp Val Leu Ala Pro Lys Pro 485 490 495Val Ala Leu Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala Pro Lys 500 505 510Glu Met Ser Trp His Pro Met Ala Arg Ile Pro Gly Asn Pro Thr Pro 515 520 525Ser Phe Ala Pro Ser Ala Tyr Lys Pro Arg Asn Ile Ala Phe Thr Pro 530 535 540Phe Pro Gly Asn Pro Asn Asp Asn Asp His Thr Pro Gly Lys Met Pro545 550 555 560Leu Thr Trp Phe Asn Met Ala Glu Phe Met Ala Gly Lys Val Ser Met 565 570 575Cys Leu Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr Ser Arg 580 585 590Ser Pro Ala Trp Asp Leu Ala Leu Val Thr Arg Ala Val Ser Val Ser 595 600 605Asp Leu Lys His Val Asn Tyr Arg Asn Ile Asp Leu Asp Pro Ser Lys 610 615 620Gly Thr Met Val Gly Glu Phe Asp Cys Pro Ala Asp Ala Trp Phe Tyr625 630 635 640Lys Gly Ala Cys Asn Asp Ala His Met Pro Tyr Ser Ile Leu Met Glu 645 650 655Ile Ala Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys Ala Pro 660 665 670Leu Thr Met Glu Lys Asp Asp Ile Leu Phe Arg Asn Leu Asp Ala Asn 675 680 685Ala Glu Phe Val Arg Ala Asp Leu Asp Tyr Arg Gly Lys Thr Ile Arg 690 695 700Asn Val Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Glu Met Gly Val705 710 715 720His Arg Phe Thr Phe Glu Leu Tyr Val Asp Asp Val Leu Phe Tyr Lys 725 730 735Gly Ser Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ala Ala Gln 740 745 750Ala Gly Leu Asp Asn Gly Arg Lys Ser Glu Pro Trp Phe Ile Glu Asn 755 760 765Lys Val Pro Ala Ser Gln Val Ser Ser Phe Asp Val Arg Pro Asn Gly 770 775 780Ser Gly Arg Thr Ala Ile Phe Ala Asn Ala Pro Ser Gly Ala Gln Leu785 790 795 800Asn Arg Arg Thr Asp Gln Gly Gln Tyr Leu Asp Ala Val Asp Ile Val 805 810 815Ser Gly Ser Gly Lys Lys Ser Leu Gly Tyr Ala His Gly Ser Lys Thr 820 825 830Val Asn Pro Asn Asp Trp Phe Phe Ser Cys His Phe Trp Phe Asp Ser 835 840 845Val Met Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu Val Glu 850 855 860Ala Ile Ala Ala His Glu Asp Leu Ala Gly Lys His Gly Ile Ala Asn865 870 875 880Pro Thr Phe Val His Ala Pro Gly Lys Ile Ser Trp Lys Tyr Arg Gly 885 890 895Gln Leu Thr Pro Lys Ser Lys Lys Met Asp Ser Glu Val His Ile Val 900 905 910Ser Val Asp Ala His Asp Gly Val Val Asp Leu Val Ala Asp Gly Phe 915 920 925Leu Trp Ala Asp Ser Leu Arg Val Tyr Ser Val Ser Asn Ile Arg Val 930 935 940Arg Ile Ala Ser Gly Glu Ala Pro Ala Ala Ala Ser Ser Ala Ala Ser945 950 955 960Val Gly Ser Ser Ala Ser Ser Val Glu Arg Thr Arg Ser Ser Pro Ala 965 970 975Val Ala Ser Gly Pro Ala Gln Thr Ile Asp Leu Lys Gln Leu Lys Thr 980 985 990Glu Leu Leu Glu Leu Asp Ala Pro Leu Tyr Leu Ser Gln Asp Pro Thr 995 1000 1005Ser Gly Gln Leu Lys Lys His Thr Asp Val Ala Ser Gly Gln Ala 1010 1015 1020Thr Ile Val Gln Pro Cys Thr Leu Gly Asp Leu Gly Asp Arg Ser 1025 1030 1035Phe Met Glu Thr Tyr Gly Val Val Ala Pro Leu Tyr Thr Gly Ala 1040 1045 1050Met Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly 1055 1060 1065Lys Arg Lys Ile Leu Gly Ser Phe Gly Ala Gly Gly Leu Pro Met 1070 1075 1080His His Val Arg Ala Ala Leu Glu Lys Ile Gln Ala Ala Leu Pro 1085 1090 1095Gln Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser 1100 1105 1110Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val 1115 1120 1125Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val 1130 1135 1140Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Asn Ala Asp Gly Ser 1145 1150 1155Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu 1160 1165 1170Leu Ala Glu Met Phe Ile Arg Pro Ala Pro Glu His Leu Leu Glu 1175 1180 1185Lys Leu Ile Ala Ser Gly Glu Ile Thr Gln Glu Gln Ala Glu Leu 1190 1195 1200Ala Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp 1205 1210 1215Ser Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro 1220 1225 1230Leu Ile Ile Asn Leu Arg Asn Arg Leu His Arg Glu Cys Gly Tyr 1235 1240 1245Pro Ala His Leu Arg Val Arg Val Gly Ala Gly Gly Gly Val Gly 1250 1255 1260Cys Pro Gln Ala Ala Ala Ala Ala Leu Thr Met Gly Ala Ala Phe 1265 1270 1275Ile Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr 1280 1285 1290Cys Asp Asn Val Arg Lys Gln Leu Ser Gln Ala Thr Tyr Ser Asp 1295 1300 1305Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys 1310 1315 1320Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn 1325 1330 1335Lys Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Asp Ser Met 1340 1345 1350Pro Pro Ala Glu Leu Glu Arg Ile Glu Lys Arg Ile Phe Lys Arg 1355 1360 1365Ala Leu Gln Glu Val Trp Glu Glu Thr Lys Asp Phe Tyr Ile Asn 1370 1375 1380Gly Leu Lys Asn Pro Glu Lys Ile Gln Arg Ala Glu His Asp Pro 1385 1390 1395Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Gly Leu Ala 1400 1405 1410Ser Arg Trp Ala Asn Met Gly Ala Pro Asp Arg Val Met Asp Tyr 1415 1420 1425Gln Val Trp Cys Gly Pro Ala Ile Gly Ala Phe Asn Asp Phe Ile 1430 1435 1440Lys Gly Thr Tyr Leu Asp Pro Ala Val Ser Asn Glu Tyr Pro Cys 1445 1450 1455Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly Ala Cys Tyr Leu 1460 1465 1470Arg Arg Leu Asn Ala Leu Arg Asn Asp Pro Arg Ile Asp Leu Glu 1475 1480 1485Thr Glu Asp Ala Ala Phe Val Tyr Glu Pro Thr Asn Ala Leu 1490 1495 150071500DNASchizochytrium sp.CDS(1)..(1500) 7atg gcg gcc cgt ctg cag gag caa aag gga ggc gag atg gat acc cgc 48Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg1 5 10 15att gcc atc atc ggc atg tcg gcc atc ctc ccc tgc ggc acg acc gtg 96Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val 20 25 30cgc gag tcg tgg gag acc atc cgc gcc ggc atc gac tgc ctg tcg gat 144Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp 35 40 45ctc ccc gag gac cgc gtc gac gtg acg gcg tac ttt gac ccc gtc aag 192Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys 50 55 60acc acc aag gac aag atc tac tgc aag cgc ggt ggc ttc att ccc gag 240Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu65

70 75 80tac gac ttt gac gcc cgc gag ttc gga ctc aac atg ttc cag atg gag 288Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu 85 90 95gac tcg gac gca aac cag acc atc tcg ctt ctc aag gtc aag gag gcc 336Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala 100 105 110ctc cag gac gcc ggc atc gac gcc ctc ggc aag gaa aag aag aac atc 384Leu Gln Asp Ala Gly Ile Asp Ala Leu Gly Lys Glu Lys Lys Asn Ile 115 120 125ggc tgc gtg ctc ggc att ggc ggc ggc caa aag tcc agc cac gag ttc 432Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe 130 135 140tac tcg cgc ctt aat tat gtt gtc gtg gag aag gtc ctc cgc aag atg 480Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met145 150 155 160ggc atg ccc gag gag gac gtc aag gtc gcc gtc gaa aag tac aag gcc 528Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala 165 170 175aac ttc ccc gag tgg cgc ctc gac tcc ttc cct ggc ttc ctc ggc aac 576Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn 180 185 190gtc acc gcc ggt cgc tgc acc aac acc ttc aac ctc gac ggc atg aac 624Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn 195 200 205tgc gtt gtc gac gcc gca tgc gcc tcg tcc ctc atc gcc gtc aag gtc 672Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val 210 215 220gcc atc gac gag ctg ctc tac ggt gac tgc gac atg atg gtc acc ggt 720Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly225 230 235 240gcc acc tgc acg gat aac tcc atc ggc atg tac atg gcc ttc tcc aag 768Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys 245 250 255acc ccc gtg ttc tcc acg gac ccc agc gtg cgc gcc tac gac gaa aag 816Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys 260 265 270aca aag ggc atg ctc atc ggc gag ggc tcc gcc atg ctc gtc ctc aag 864Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys 275 280 285cgc tac gcc gac gcc gtc cgc gac ggc gat gag atc cac gct gtt att 912Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile 290 295 300cgc ggc tgc gcc tcc tcc agt gat ggc aag gcc gcc ggc atc tac acg 960Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr305 310 315 320ccc acc att tcg ggc cag gag gag gcc ctc cgc cgc gcc tac aac cgc 1008Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg 325 330 335gcc tgt gtc gac ccg gcc acc gtc act ctc gtc gag ggt cac ggc acc 1056Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr 340 345 350ggt act ccc gtt ggc gac cgc atc gag ctc acc gcc ttg cgc aac ctc 1104Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu 355 360 365ttt gac aag gcc tac ggc gag ggc aac acc gaa aag gtc gct gtg ggc 1152Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly 370 375 380agc atc aag tcc agc atc ggc cat ctc aag gcc gtc gcc ggt ctc gcc 1200Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala385 390 395 400ggt atg atc aag gtc atc atg gcg ctc aag cac aag act ctc ccg ggc 1248Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly 405 410 415acc atc aac gtc gac aac cca ccc aac ctc tac gac aac acg ccc atc 1296Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile 420 425 430aac gag tcc tcg ctc tac att aac acc atg aac cgc ccc tgg ttc ccg 1344Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro 435 440 445ccc cct ggt gtg ccc cgc cgc gcc ggc att tcg agc ttt ggc ttt ggt 1392Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly 450 455 460ggc gcc aac tac cac gcc gtc ctc gag gag gcc gag ccc gag cac acg 1440Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr465 470 475 480acc gcg tac cgc ctc aac aag cgc ccg cag ccc gtg ctc atg atg gcc 1488Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala 485 490 495gcc acg ccc gcg 1500Ala Thr Pro Ala 5008500PRTSchizochytrium sp. 8Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg1 5 10 15Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val 20 25 30Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp 35 40 45Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys 50 55 60Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu65 70 75 80Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu 85 90 95Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala 100 105 110Leu Gln Asp Ala Gly Ile Asp Ala Leu Gly Lys Glu Lys Lys Asn Ile 115 120 125Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe 130 135 140Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met145 150 155 160Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala 165 170 175Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn 180 185 190Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn 195 200 205Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val 210 215 220Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly225 230 235 240Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys 245 250 255Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys 260 265 270Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys 275 280 285Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile 290 295 300Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr305 310 315 320Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg 325 330 335Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr 340 345 350Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu 355 360 365Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly 370 375 380Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala385 390 395 400Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly 405 410 415Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile 420 425 430Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro 435 440 445Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly 450 455 460Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr465 470 475 480Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala 485 490 495Ala Thr Pro Ala 50091278DNASchizochytrium sp.CDS(1)..(1278) 9gat gtc acc aag gag gcc tgg cgc ctc ccc cgc gag ggc gtc agc ttc 48Asp Val Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe1 5 10 15cgc gcc aag ggc atc gcc acc aac ggc gct gtc gcc gcg ctc ttc tcc 96Arg Ala Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser 20 25 30ggc cag ggc gcg cag tac acg cac atg ttt agc gag gtg gcc atg aac 144Gly Gln Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn 35 40 45tgg ccc cag ttc cgc cag agc att gcc gcc atg gac gcc gcc cag tcc 192Trp Pro Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser 50 55 60aag gtc gct gga agc gac aag gac ttt gag cgc gtc tcc cag gtc ctc 240Lys Val Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu65 70 75 80tac ccg cgc aag ccg tac gag cgt gag ccc gag cag gac cac aag aag 288Tyr Pro Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys 85 90 95atc tcc ctc acc gcc tac tcg cag ccc tcg acc ctg gcc tgc gct ctc 336Ile Ser Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu 100 105 110ggt gcc ttt gag atc ttc aag gag gcc ggc ttc acc ccg gac ttt gcc 384Gly Ala Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala 115 120 125gcc ggc cat tcg ctc ggt gag ttc gcc gcc ctc tac gcc gcg ggc tgc 432Ala Gly His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys 130 135 140gtc gac cgc gac gag ctc ttt gag ctt gtc tgc cgc cgc gcc cgc atc 480Val Asp Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile145 150 155 160atg ggc ggc aag gac gca ccg gcc acc ccc aag ggc tgc atg gcc gcc 528Met Gly Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala 165 170 175gtc att ggc ccc aac gcc gag aac atc aag gtc cag gcc gcc aac gtc 576Val Ile Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val 180 185 190tgg ctc ggc aac tcc aac tcg cct tcg cag acc gtc atc acc ggc tcc 624Trp Leu Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser 195 200 205gtc gaa ggt atc cag gcc gag agc gcc cgc ctc cag aag gag ggc ttc 672Val Glu Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe 210 215 220cgc gtc gtg cct ctt gcc tgc gag agc gcc ttc cac tcg ccc cag atg 720Arg Val Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met225 230 235 240gag aac gcc tcg tcg gcc ttc aag gac gtc atc tcc aag gtc tcc ttc 768Glu Asn Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe 245 250 255cgc acc ccc aag gcc gag acc aag ctc ttc agc aac gtc tct ggc gag 816Arg Thr Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu 260 265 270acc tac ccc acg gac gcc cgc gag atg ctt acg cag cac atg acc agc 864Thr Tyr Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser 275 280 285agc gtc aag ttc ctc acc cag gtc cgc aac atg cac cag gcc ggt gcg 912Ser Val Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala 290 295 300cgc atc ttt gtc gag ttc gga ccc aag cag gtg ctc tcc aag ctt gtc 960Arg Ile Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val305 310 315 320tcc gag acc ctc aag gat gac ccc tcg gtt gtc acc gtc tct gtc aac 1008Ser Glu Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn 325 330 335ccg gcc tcg ggc acg gat tcg gac atc cag ctc cgc gac gcg gcc gtc 1056Pro Ala Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val 340 345 350cag ctc gtt gtc gct ggc gtc aac ctt cag ggc ttt gac aag tgg gac 1104Gln Leu Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp 355 360 365gcc ccc gat gcc acc cgc atg cag gcc atc aag aag aag cgc act acc 1152Ala Pro Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr 370 375 380ctc cgc ctt tcg gcc gcc acc tac gtc tcg gac aag acc aag aag gtc 1200Leu Arg Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val385 390 395 400cgc gac gcc gcc atg aac gat ggc cgc tgc gtc acc tac ctc aag ggc 1248Arg Asp Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly 405 410 415gcc gca ccg ctc atc aag gcc ccg gag ccc 1278Ala Ala Pro Leu Ile Lys Ala Pro Glu Pro 420 42510426PRTSchizochytrium sp. 10Asp Val Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe1 5 10 15Arg Ala Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser 20 25 30Gly Gln Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn 35 40 45Trp Pro Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser 50 55 60Lys Val Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu65 70 75 80Tyr Pro Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys 85 90 95Ile Ser Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu 100 105 110Gly Ala Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala 115 120 125Ala Gly His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys 130 135 140Val Asp Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile145 150 155 160Met Gly Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala 165 170 175Val Ile Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val 180 185 190Trp Leu Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser 195 200 205Val Glu Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe 210 215 220Arg Val Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met225 230 235 240Glu Asn Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe 245 250 255Arg Thr Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu 260 265 270Thr Tyr Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser 275 280 285Ser Val Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala 290 295 300Arg Ile Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val305 310 315 320Ser Glu Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn 325 330 335Pro Ala Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val 340 345 350Gln Leu Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp 355 360 365Ala Pro Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr 370 375 380Leu Arg Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val385 390 395 400Arg Asp Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly 405 410 415Ala Ala Pro Leu Ile Lys Ala Pro Glu Pro 420 425115PRTSchizochytrium sp.MISC_FEATURE(4)..(4)X = any amino acid 11Gly His Ser Xaa Gly1 512258DNASchizochytrium sp.CDS(1)..(258) 12gct gtc tcg aac gag ctt ctt gag aag gcc gag act gtc gtc atg gag 48Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu1 5 10 15gtc ctc gcc gcc aag acc ggc tac gag acc gac atg atc gag gct gac 96Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ala Asp 20 25 30atg gag ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc gag 144Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu 35 40 45atc ctc tcc gag gtc cag gcc atg ctc aat gtc gag gcc aag gat gtc 192Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val 50 55 60gat gcc ctc agc cgc act cgc act gtt ggt gag gtt gtc aac gcc atg 240Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asn Ala Met65 70 75 80aag gcc gag atc gct ggc

258Lys Ala Glu Ile Ala Gly 851386PRTSchizochytrium sp. 13Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu1 5 10 15Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ala Asp 20 25 30Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu 35 40 45Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val 50 55 60Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asn Ala Met65 70 75 80Lys Ala Glu Ile Ala Gly 85145PRTSchizochytrium sp. 14Leu Gly Ile Asp Ser1 51521PRTSchizochytrium sp. 15Ala Pro Ala Pro Val Lys Ala Ala Ala Pro Ala Ala Pro Val Ala Ser1 5 10 15Ala Pro Ala Pro Ala 20163006DNASchizochytrium sp. 16gcccccgccc cggtcaaggc tgctgcgcct gccgcccccg ttgcctcggc ccctgccccg 60gctgtctcga acgagcttct tgagaaggcc gagactgtcg tcatggaggt cctcgccgcc 120aagaccggct acgagaccga catgatcgag gctgacatgg agctcgagac cgagctcggc 180attgactcca tcaagcgtgt cgagatcctc tccgaggtcc aggccatgct caatgtcgag 240gccaaggatg tcgatgccct cagccgcact cgcactgttg gtgaggttgt caacgccatg 300aaggccgaga tcgctggcag ctctgccccg gcgcctgctg ccgctgctcc ggctccggcc 360aaggctgccc ctgccgccgc tgcgcctgct gtctcgaacg agcttctcga gaaggccgag 420accgtcgtca tggaggtcct cgccgccaag actggctacg agactgacat gatcgagtcc 480gacatggagc tcgagactga gctcggcatt gactccatca agcgtgtcga gatcctctcc 540gaggttcagg ccatgctcaa cgtcgaggcc aaggacgtcg acgctctcag ccgcactcgc 600actgtgggtg aggtcgtcaa cgccatgaag gctgagatcg ctggtggctc tgccccggcg 660cctgccgccg ctgccccagg tccggctgct gccgcccctg cgcctgccgc cgccgcccct 720gctgtctcga acgagcttct tgagaaggcc gagaccgtcg tcatggaggt cctcgccgcc 780aagactggct acgagactga catgatcgag tccgacatgg agctcgagac cgagctcggc 840attgactcca tcaagcgtgt cgagattctc tccgaggtcc aggccatgct caacgtcgag 900gccaaggacg tcgacgctct cagccgcacc cgcactgttg gcgaggtcgt cgatgccatg 960aaggccgaga tcgctggtgg ctctgccccg gcgcctgccg ccgctgctcc tgctccggct 1020gctgccgccc ctgcgcctgc cgcccctgcg cctgctgtct cgagcgagct tctcgagaag 1080gccgagactg tcgtcatgga ggtcctcgcc gccaagactg gctacgagac tgacatgatc 1140gagtccgaca tggagctcga gaccgagctc ggcattgact ccatcaagcg tgtcgagatt 1200ctctccgagg tccaggccat gctcaacgtc gaggccaagg acgtcgacgc tctcagccgc 1260acccgcactg ttggcgaggt cgtcgatgcc atgaaggccg agatcgctgg tggctctgcc 1320ccggcgcctg ccgccgctgc tcctgctccg gctgctgccg cccctgcgcc tgccgcccct 1380gcgcctgccg cccctgcgcc tgctgtctcg agcgagcttc tcgagaaggc cgagactgtc 1440gtcatggagg tcctcgccgc caagactggc tacgagactg acatgattga gtccgacatg 1500gagctcgaga ccgagctcgg cattgactcc atcaagcgtg tcgagattct ctccgaggtt 1560caggccatgc tcaacgtcga ggccaaggac gtcgacgctc tcagccgcac tcgcactgtt 1620ggtgaggtcg tcgatgccat gaaggctgag atcgctggca gctccgcctc ggcgcctgcc 1680gccgctgctc ctgctccggc tgctgccgct cctgcgcccg ctgccgccgc ccctgctgtc 1740tcgaacgagc ttctcgagaa agccgagact gtcgtcatgg aggtcctcgc cgccaagact 1800ggctacgaga ctgacatgat cgagtccgac atggagctcg agactgagct cggcattgac 1860tccatcaagc gtgtcgagat cctctccgag gttcaggcca tgctcaacgt cgaggccaag 1920gacgtcgatg ccctcagccg cacccgcact gttggcgagg ttgtcgatgc catgaaggcc 1980gagatcgctg gtggctctgc cccggcgcct gccgccgctg cccctgctcc ggctgccgcc 2040gcccctgctg tctcgaacga gcttctcgag aaggccgaga ctgtcgtcat ggaggtcctc 2100gccgccaaga ctggctacga gaccgacatg atcgagtccg acatggagct cgagaccgag 2160ctcggcattg actccatcaa gcgtgtcgag attctctccg aggttcaggc catgctcaac 2220gtcgaggcca aggacgtcga tgctctcagc cgcactcgca ctgttggcga ggtcgtcgat 2280gccatgaagg ctgagatcgc cggcagctcc gccccggcgc ctgccgccgc tgctcctgct 2340ccggctgctg ccgctcctgc gcccgctgcc gctgcccctg ctgtctcgag cgagcttctc 2400gagaaggccg agaccgtcgt catggaggtc ctcgccgcca agactggcta cgagactgac 2460atgattgagt ccgacatgga gctcgagact gagctcggca ttgactccat caagcgtgtc 2520gagatcctct ccgaggttca ggccatgctc aacgtcgagg ccaaggacgt cgatgccctc 2580agccgcaccc gcactgttgg cgaggttgtc gatgccatga aggccgagat cgctggtggc 2640tctgccccgg cgcctgccgc cgctgcccct gctccggctg ccgccgcccc tgctgtctcg 2700aacgagcttc ttgagaaggc cgagaccgtc gtcatggagg tcctcgccgc caagactggc 2760tacgagaccg acatgatcga gtccgacatg gagctcgaga ccgagctcgg cattgactcc 2820atcaagcgtg tcgagattct ctccgaggtt caggccatgc tcaacgtcga ggccaaggac 2880gtcgacgctc tcagccgcac tcgcactgtt ggcgaggtcg tcgatgccat gaaggctgag 2940atcgctggtg gctctgcccc ggcgcctgcc gccgctgctc ctgcctcggc tggcgccgcg 3000cctgcg 3006172133DNASchizochytrium sp.CDS(1)..(2133) 17ttt ggc gct ctc ggc ggc ttc atc tcg cag cag gcg gag cgc ttc gag 48Phe Gly Ala Leu Gly Gly Phe Ile Ser Gln Gln Ala Glu Arg Phe Glu1 5 10 15ccc gcc gaa atc ctc ggc ttc acg ctc atg tgc gcc aag ttc gcc aag 96Pro Ala Glu Ile Leu Gly Phe Thr Leu Met Cys Ala Lys Phe Ala Lys 20 25 30gct tcc ctc tgc acg gct gtg gct ggc ggc cgc ccg gcc ttt atc ggt 144Ala Ser Leu Cys Thr Ala Val Ala Gly Gly Arg Pro Ala Phe Ile Gly 35 40 45gtg gcg cgc ctt gac ggc cgc ctc gga ttc act tcg cag ggc act tct 192Val Ala Arg Leu Asp Gly Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser 50 55 60gac gcg ctc aag cgt gcc cag cgt ggt gcc atc ttt ggc ctc tgc aag 240Asp Ala Leu Lys Arg Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys65 70 75 80acc atc ggc ctc gag tgg tcc gag tct gac gtc ttt tcc cgc ggc gtg 288Thr Ile Gly Leu Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val 85 90 95gac att gct cag ggc atg cac ccc gag gat gcc gcc gtg gcg att gtg 336Asp Ile Ala Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val 100 105 110cgc gag atg gcg tgc gct gac att cgc att cgc gag gtc ggc att ggc 384Arg Glu Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly 115 120 125gca aac cag cag cgc tgc acg atc cgt gcc gcc aag ctc gag acc ggc 432Ala Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly 130 135 140aac ccg cag cgc cag atc gcc aag gac gac gtg ctg ctc gtt tct ggc 480Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser Gly145 150 155 160ggc gct cgc ggc atc acg cct ctt tgc atc cgg gag atc acg cgc cag 528Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr Arg Gln 165 170 175atc gcg ggc ggc aag tac att ctg ctt ggc cgc agc aag gtc tct gcg 576Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys Val Ser Ala 180 185 190agc gaa ccg gca tgg tgc gct ggc atc act gac gag aag gct gtg caa 624Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu Lys Ala Val Gln 195 200 205aag gct gct acc cag gag ctc aag cgc gcc ttt agc gct ggc gag ggc 672Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe Ser Ala Gly Glu Gly 210 215 220ccc aag ccc acg ccc cgc gct gtc act aag ctt gtg ggc tct gtt ctt 720Pro Lys Pro Thr Pro Arg Ala Val Thr Lys Leu Val Gly Ser Val Leu225 230 235 240ggc gct cgc gag gtg cgc agc tct att gct gcg att gaa gcg ctc ggc 768Gly Ala Arg Glu Val Arg Ser Ser Ile Ala Ala Ile Glu Ala Leu Gly 245 250 255ggc aag gcc atc tac tcg tcg tgc gac gtg aac tct gcc gcc gac gtg 816Gly Lys Ala Ile Tyr Ser Ser Cys Asp Val Asn Ser Ala Ala Asp Val 260 265 270gcc aag gcc gtg cgc gat gcc gag tcc cag ctc ggt gcc cgc gtc tcg 864Ala Lys Ala Val Arg Asp Ala Glu Ser Gln Leu Gly Ala Arg Val Ser 275 280 285ggc atc gtt cat gcc tcg ggc gtg ctc cgc gac cgt ctc atc gag aag 912Gly Ile Val His Ala Ser Gly Val Leu Arg Asp Arg Leu Ile Glu Lys 290 295 300aag ctc ccc gac gag ttc gac gcc gtc ttt ggc acc aag gtc acc ggt 960Lys Leu Pro Asp Glu Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly305 310 315 320ctc gag aac ctc ctc gcc gcc gtc gac cgc gcc aac ctc aag cac atg 1008Leu Glu Asn Leu Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met 325 330 335gtc ctc ttc agc tcg ctc gcc ggc ttc cac ggc aac gtc ggc cag tct 1056Val Leu Phe Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser 340 345 350gac tac gcc atg gcc aac gag gcc ctt aac aag atg ggc ctc gag ctc 1104Asp Tyr Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu 355 360 365gcc aag gac gtc tcg gtc aag tcg atc tgc ttc ggt ccc tgg gac ggt 1152Ala Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly 370 375 380ggc atg gtg acg ccg cag ctc aag aag cag ttc cag gag atg ggc gtg 1200Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly Val385 390 395 400cag atc atc ccc cgc gag ggc ggc gct gat acc gtg gcg cgc atc gtg 1248Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg Ile Val 405 410 415ctc ggc tcc tcg ccg gct gag atc ctt gtc ggc aac tgg cgc acc ccg 1296Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp Arg Thr Pro 420 425 430tcc aag aag gtc ggc tcg gac acc atc acc ctg cac cgc aag att tcc 1344Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His Arg Lys Ile Ser 435 440 445gcc aag tcc aac ccc ttc ctc gag gac cac gtc atc cag ggc cgc cgc 1392Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val Ile Gln Gly Arg Arg 450 455 460gtg ctg ccc atg acg ctg gcc att ggc tcg ctc gcg gag acc tgc ctc 1440Val Leu Pro Met Thr Leu Ala Ile Gly Ser Leu Ala Glu Thr Cys Leu465 470 475 480ggc ctc ttc ccc ggc tac tcg ctc tgg gcc att gac gac gcc cag ctc 1488Gly Leu Phe Pro Gly Tyr Ser Leu Trp Ala Ile Asp Asp Ala Gln Leu 485 490 495ttc aag ggt gtc act gtc gac ggc gac gtc aac tgc gag gtg acc ctc 1536Phe Lys Gly Val Thr Val Asp Gly Asp Val Asn Cys Glu Val Thr Leu 500 505 510acc ccg tcg acg gcg ccc tcg ggc cgc gtc aac gtc cag gcc acg ctc 1584Thr Pro Ser Thr Ala Pro Ser Gly Arg Val Asn Val Gln Ala Thr Leu 515 520 525aag acc ttt tcc agc ggc aag ctg gtc ccg gcc tac cgc gcc gtc atc 1632Lys Thr Phe Ser Ser Gly Lys Leu Val Pro Ala Tyr Arg Ala Val Ile 530 535 540gtg ctc tcc aac cag ggc gcg ccc ccg gcc aac gcc acc atg cag ccg 1680Val Leu Ser Asn Gln Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro545 550 555 560ccc tcg ctc gat gcc gat ccg gcg ctc cag ggc tcc gtc tac gac ggc 1728Pro Ser Leu Asp Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly 565 570 575aag acc ctc ttc cac ggc ccg gcc ttc cgc ggc atc gat gac gtg ctc 1776Lys Thr Leu Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu 580 585 590tcg tgc acc aag agc cag ctt gtg gcc aag tgc agc gct gtc ccc ggc 1824Ser Cys Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly 595 600 605tcc gac gcc gct cgc ggc gag ttt gcc acg gac act gac gcc cat gac 1872Ser Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp 610 615 620ccc ttc gtg aac gac ctg gcc ttt cag gcc atg ctc gtc tgg gtg cgc 1920Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val Arg625 630 635 640cgc acg ctc ggc cag gct gcg ctc ccc aac tcg atc cag cgc atc gtc 1968Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg Ile Val 645 650 655cag cac cgc ccg gtc ccg cag gac aag ccc ttc tac att acc ctc cgc 2016Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile Thr Leu Arg 660 665 670tcc aac cag tcg ggc ggt cac tcc cag cac aag cac gcc ctt cag ttc 2064Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His Ala Leu Gln Phe 675 680 685cac aac gag cag ggc gat ctc ttc att gat gtc cag gct tcg gtc atc 2112His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val Gln Ala Ser Val Ile 690 695 700gcc acg gac agc ctt gcc ttc 2133Ala Thr Asp Ser Leu Ala Phe705 71018711PRTSchizochytrium sp. 18Phe Gly Ala Leu Gly Gly Phe Ile Ser Gln Gln Ala Glu Arg Phe Glu1 5 10 15Pro Ala Glu Ile Leu Gly Phe Thr Leu Met Cys Ala Lys Phe Ala Lys 20 25 30Ala Ser Leu Cys Thr Ala Val Ala Gly Gly Arg Pro Ala Phe Ile Gly 35 40 45 Val Ala Arg Leu Asp Gly Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser 50 55 60Asp Ala Leu Lys Arg Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys65 70 75 80Thr Ile Gly Leu Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val 85 90 95Asp Ile Ala Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val 100 105 110Arg Glu Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly 115 120 125Ala Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly 130 135 140Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser Gly145 150 155 160Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr Arg Gln 165 170 175Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys Val Ser Ala 180 185 190Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu Lys Ala Val Gln 195 200 205Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe Ser Ala Gly Glu Gly 210 215 220Pro Lys Pro Thr Pro Arg Ala Val Thr Lys Leu Val Gly Ser Val Leu225 230 235 240Gly Ala Arg Glu Val Arg Ser Ser Ile Ala Ala Ile Glu Ala Leu Gly 245 250 255Gly Lys Ala Ile Tyr Ser Ser Cys Asp Val Asn Ser Ala Ala Asp Val 260 265 270Ala Lys Ala Val Arg Asp Ala Glu Ser Gln Leu Gly Ala Arg Val Ser 275 280 285Gly Ile Val His Ala Ser Gly Val Leu Arg Asp Arg Leu Ile Glu Lys 290 295 300Lys Leu Pro Asp Glu Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly305 310 315 320Leu Glu Asn Leu Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met 325 330 335Val Leu Phe Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser 340 345 350Asp Tyr Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu 355 360 365Ala Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly 370 375 380Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly Val385 390 395 400Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg Ile Val 405 410 415Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp Arg Thr Pro 420 425 430Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His Arg Lys Ile Ser 435 440 445Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val Ile Gln Gly Arg Arg 450 455 460Val Leu Pro Met Thr Leu Ala Ile Gly Ser Leu Ala Glu Thr Cys Leu465 470 475 480Gly Leu Phe Pro Gly Tyr Ser Leu Trp Ala Ile Asp Asp Ala Gln Leu 485 490 495Phe Lys Gly Val Thr Val Asp Gly Asp Val Asn Cys Glu Val Thr Leu 500 505 510Thr Pro Ser Thr Ala Pro Ser Gly Arg Val Asn Val Gln Ala Thr Leu 515 520 525Lys Thr Phe Ser Ser Gly Lys Leu Val Pro Ala Tyr Arg Ala Val Ile 530 535 540Val Leu Ser Asn Gln Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro545 550 555 560Pro Ser Leu Asp Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly 565 570 575Lys Thr Leu Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu 580 585 590Ser Cys Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly 595 600 605Ser Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp 610 615 620Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val Arg625 630 635 640Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg Ile Val 645 650 655Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile Thr Leu Arg 660 665 670Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His Ala Leu Gln Phe 675

680 685His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val Gln Ala Ser Val Ile 690 695 700Ala Thr Asp Ser Leu Ala Phe705 710191350DNASchizochytrium sp.CDS(1)..(1350) 19atg gcc gct cgg aat gtg agc gcc gcg cat gag atg cac gat gaa aag 48Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys1 5 10 15cgc atc gcc gtc gtc ggc atg gcc gtc cag tac gcc gga tgc aaa acc 96Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr 20 25 30aag gac gag ttc tgg gag gtg ctc atg aac ggc aag gtc gag tcc aag 144Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys 35 40 45gtg atc agc gac aaa cga ctc ggc tcc aac tac cgc gcc gag cac tac 192Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr 50 55 60aaa gca gag cgc agc aag tat gcc gac acc ttt tgc aac gaa acg tac 240Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr65 70 75 80ggc acc ctt gac gag aac gag atc gac aac gag cac gaa ctc ctc ctc 288Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu 85 90 95aac ctc gcc aag cag gca ctc gca gag aca tcc gtc aaa gac tcg aca 336Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr 100 105 110cgc tgc ggc atc gtc agc ggc tgc ctc tcg ttc ccc atg gac aac ctc 384Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125cag ggt gaa ctc ctc aac gtg tac caa aac cat gtc gag aaa aag ctc 432Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140ggg gcc cgc gtc ttc aag gac gcc tcc cat tgg tcc gaa cgc gag cag 480Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln145 150 155 160tcc aac aaa ccc gag gcc ggt gac cgc cgc atc ttc atg gac ccg gcc 528Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175tcc ttc gtc gcc gaa gaa ctc aac ctc ggc gcc ctt cac tac tcc gtc 576Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val 180 185 190gac gca gca tgc gcc acg gcg ctc tac gtg ctc cgc ctc gcg cag gat 624Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205cat ctc gtc tcc ggc gcc gcc gac gtc atg ctc tgc ggt gcc acc tgc 672His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys 210 215 220ctg ccg gag ccc ttt ttc atc ctt tcg ggc ttt tcc acc ttc cag gcc 720Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala225 230 235 240atg ccc gtc ggc acg ggc cag aac gtg tcc atg ccg ctg cac aag gac 768Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp 245 250 255agc cag ggc ctc acc ccg ggt gag ggc ggc tcc atc atg gtc ctc aag 816Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys 260 265 270cgt ctc gat gat gcc atc cgc gac ggc gac cac atc tac ggc acc ctt 864Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu 275 280 285ctc ggc gcc aat gtc agc aac tcc ggc aca ggt ctg ccc ctc aag ccc 912Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro 290 295 300ctt ctc ccc agc gag aaa aag tgc ctc atg gac acc tac acg cgc att 960Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile305 310 315 320aac gtg cac ccg cac aag att cag tac gtc gag tgc cac gcc acc ggc 1008Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly 325 330 335acg ccc cag ggt gat cgt gtg gaa atc gac gcc gtc aag gcc tgc ttt 1056Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe 340 345 350gaa ggc aag gtc ccc cgt ttc ggt acc aca aag ggc aac ttt gga cac 1104Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His 355 360 365acc ctc gtc gca gcc ggc ttt gcc ggt atg tgc aag gtc ctc ctc tcc 1152Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser 370 375 380atg aag cat ggc atc atc ccg ccc acc ccg ggt atc gat gac gag acc 1200Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr385 390 395 400aag atg gac cct ctc gtc gtc tcc ggt gag gcc atc cca tgg cca gag 1248Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu 405 410 415acc aac ggc gag ccc aag cgc gcc ggt ctc tcg gcc ttt ggc ttt ggt 1296Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly 420 425 430ggc acc aac gcc cat gcc gtc ttt gag gag cat gac ccc tcc aac gcc 1344Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala 435 440 445gcc tgc 1350Ala Cys 45020450PRTSchizochytrium sp. 20Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys1 5 10 15Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr 20 25 30Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys 35 40 45Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr 50 55 60Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr65 70 75 80Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu 85 90 95Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr 100 105 110Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln145 150 155 160Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val 180 185 190Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys 210 215 220Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala225 230 235 240Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp 245 250 255Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys 260 265 270Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu 275 280 285Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro 290 295 300Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile305 310 315 320Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly 325 330 335Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe 340 345 350Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His 355 360 365Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser 370 375 380Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr385 390 395 400Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu 405 410 415Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly 420 425 430Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala 435 440 445Ala Cys 450211323DNASchizochytrium sp.CDS(1)..(1323) 21tcg gcc cgc tgc ggc ggt gaa agc aac atg cgc atc gcc atc act ggt 48Ser Ala Arg Cys Gly Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly1 5 10 15atg gac gcc acc ttt ggc gct ctc aag gga ctc gac gcc ttc gag cgc 96Met Asp Ala Thr Phe Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg 20 25 30gcc att tac acc ggc gct cac ggt gcc atc cca ctc cca gaa aag cgc 144Ala Ile Tyr Thr Gly Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg 35 40 45tgg cgc ttt ctc ggc aag gac aag gac ttt ctt gac ctc tgc ggc gtc 192Trp Arg Phe Leu Gly Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val 50 55 60aag gcc acc ccg cac ggc tgc tac att gaa gat gtt gag gtc gac ttc 240Lys Ala Thr Pro His Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe65 70 75 80cag cgc ctc cgc acg ccc atg acc cct gaa gac atg ctc ctc cct cag 288Gln Arg Leu Arg Thr Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln 85 90 95cag ctt ctg gcc gtc acc acc att gac cgc gcc atc ctc gac tcg gga 336Gln Leu Leu Ala Val Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly 100 105 110atg aaa aag ggt ggc aat gtc gcc gtc ttt gtc ggc ctc ggc acc gac 384Met Lys Lys Gly Gly Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp 115 120 125ctc gag ctc tac cgt cac cgt gct cgc gtc gct ctc aag gag cgc gtc 432Leu Glu Leu Tyr Arg His Arg Ala Arg Val Ala Leu Lys Glu Arg Val 130 135 140cgc cct gaa gcc tcc aag aag ctc aat gac atg atg cag tac att aac 480Arg Pro Glu Ala Ser Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn145 150 155 160gac tgc ggc aca tcc aca tcg tac acc tcg tac att ggc aac ctc gtc 528Asp Cys Gly Thr Ser Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val 165 170 175gcc acg cgc gtc tcg tcg cag tgg ggc ttc acg ggc ccc tcc ttt acg 576Ala Thr Arg Val Ser Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr 180 185 190atc acc gag ggc aac aac tcc gtc tac cgc tgc gcc gag ctc ggc aag 624Ile Thr Glu Gly Asn Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys 195 200 205tac ctc ctc gag acc ggc gag gtc gat ggc gtc gtc gtt gcg ggt gtc 672Tyr Leu Leu Glu Thr Gly Glu Val Asp Gly Val Val Val Ala Gly Val 210 215 220gat ctc tgc ggc agt gcc gaa aac ctt tac gtc aag tct cgc cgc ttc 720Asp Leu Cys Gly Ser Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe225 230 235 240aag gtg tcc acc tcc gat acc ccg cgc gcc agc ttt gac gcc gcc gcc 768Lys Val Ser Thr Ser Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala 245 250 255gat ggc tac ttt gtc ggc gag ggc tgc ggt gcc ttt gtg ctc aag cgt 816Asp Gly Tyr Phe Val Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg 260 265 270gag act agc tgc acc aag gac gac cgt atc tac gct tgc atg gat gcc 864Glu Thr Ser Cys Thr Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala 275 280 285atc gtc cct ggc aac gtc cct agc gcc tgc ttg cgc gag gcc ctc gac 912Ile Val Pro Gly Asn Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp 290 295 300cag gcg cgc gtc aag ccg ggc gat atc gag atg ctc gag ctc agc gcc 960Gln Ala Arg Val Lys Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala305 310 315 320gac tcc gcc cgc cac ctc aag gac ccg tcc gtc ctg ccc aag gag ctc 1008Asp Ser Ala Arg His Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu 325 330 335act gcc gag gag gaa atc ggc ggc ctt cag acg atc ctt cgt gac gat 1056Thr Ala Glu Glu Glu Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp 340 345 350gac aag ctc ccg cgc aac gtc gca acg ggc agt gtc aag gcc acc gtc 1104Asp Lys Leu Pro Arg Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val 355 360 365ggt gac acc ggt tat gcc tct ggt gct gcc agc ctc atc aag gct gcg 1152Gly Asp Thr Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala 370 375 380ctt tgc atc tac aac cgc tac ctg ccc agc aac ggc gac gac tgg gat 1200Leu Cys Ile Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp385 390 395 400gaa ccc gcc cct gag gcg ccc tgg gac agc acc ctc ttt gcg tgc cag 1248Glu Pro Ala Pro Glu Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln 405 410 415acc tcg cgc gct tgg ctc aag aac cct ggc gag cgt cgc tat gcg gcc 1296Thr Ser Arg Ala Trp Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala 420 425 430gtc tcg ggc gtc tcc gag acg cgc tcg 1323Val Ser Gly Val Ser Glu Thr Arg Ser 435 44022441PRTSchizochytrium sp. 22Ser Ala Arg Cys Gly Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly1 5 10 15Met Asp Ala Thr Phe Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg 20 25 30Ala Ile Tyr Thr Gly Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg 35 40 45Trp Arg Phe Leu Gly Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val 50 55 60Lys Ala Thr Pro His Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe65 70 75 80Gln Arg Leu Arg Thr Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln 85 90 95Gln Leu Leu Ala Val Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly 100 105 110Met Lys Lys Gly Gly Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp 115 120 125Leu Glu Leu Tyr Arg His Arg Ala Arg Val Ala Leu Lys Glu Arg Val 130 135 140Arg Pro Glu Ala Ser Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn145 150 155 160Asp Cys Gly Thr Ser Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val 165 170 175Ala Thr Arg Val Ser Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr 180 185 190Ile Thr Glu Gly Asn Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys 195 200 205Tyr Leu Leu Glu Thr Gly Glu Val Asp Gly Val Val Val Ala Gly Val 210 215 220Asp Leu Cys Gly Ser Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe225 230 235 240Lys Val Ser Thr Ser Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala 245 250 255Asp Gly Tyr Phe Val Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg 260 265 270Glu Thr Ser Cys Thr Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala 275 280 285Ile Val Pro Gly Asn Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp 290 295 300Gln Ala Arg Val Lys Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala305 310 315 320Asp Ser Ala Arg His Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu 325 330 335Thr Ala Glu Glu Glu Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp 340 345 350Asp Lys Leu Pro Arg Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val 355 360 365Gly Asp Thr Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala 370 375 380Leu Cys Ile Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp385 390 395 400Glu Pro Ala Pro Glu Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln 405 410 415Thr Ser Arg Ala Trp Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala 420 425 430Val Ser Gly Val Ser Glu Thr Arg Ser 435 440231500DNASchizochytrium sp.CDS(1)..(1500) 23tgc tat tcc gtg ctc ctc tcc gaa gcc gag ggc cac tac gag cgc gag 48Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His Tyr Glu Arg Glu1 5 10 15aac cgc atc tcg ctc gac gag gag gcg ccc aag ctc att gtg ctt cgc 96Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu Ile Val Leu Arg 20 25 30gcc gac tcc cac gag gag atc ctt ggt cgc ctc gac aag atc cgc gag 144Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp Lys Ile Arg Glu 35 40 45cgc ttc ttg cag ccc acg ggc

gcc gcc ccg cgc gag tcc gag ctc aag 192Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu Ser Glu Leu Lys 50 55 60gcg cag gcc cgc cgc atc ttc ctc gag ctc ctc ggc gag acc ctt gcc 240Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly Glu Thr Leu Ala65 70 75 80cag gat gcc gct tct tca ggc tcg caa aag ccc ctc gct ctc agc ctc 288Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu Ala Leu Ser Leu 85 90 95gtc tcc acg ccc tcc aag ctc cag cgc gag gtc gag ctc gcg gcc aag 336Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu Leu Ala Ala Lys 100 105 110ggt atc ccg cgc tgc ctc aag atg cgc cgc gat tgg agc tcc cct gct 384Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp Trp Ser Ser Pro Ala 115 120 125ggc agc cgc tac gcg cct gag ccg ctc gcc agc gac cgc gtc gcc ttc 432Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala Ser Asp Arg Val Ala Phe 130 135 140atg tac ggc gaa ggt cgc agc cct tac tac ggc atc acc caa gac att 480Met Tyr Gly Glu Gly Arg Ser Pro Tyr Tyr Gly Ile Thr Gln Asp Ile145 150 155 160cac cgc att tgg ccc gaa ctc cac gag gtc atc aac gaa aag acg aac 528His Arg Ile Trp Pro Glu Leu His Glu Val Ile Asn Glu Lys Thr Asn 165 170 175cgt ctc tgg gcc gaa ggc gac cgc tgg gtc atg ccg cgc gcc agc ttc 576Arg Leu Trp Ala Glu Gly Asp Arg Trp Val Met Pro Arg Ala Ser Phe 180 185 190aag tcg gag ctc gag agc cag cag caa gag ttt gat cgc aac atg att 624Lys Ser Glu Leu Glu Ser Gln Gln Gln Glu Phe Asp Arg Asn Met Ile 195 200 205gaa atg ttc cgt ctt gga atc ctc acc tca att gcc ttc acc aat ctg 672Glu Met Phe Arg Leu Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu 210 215 220gcg cgc gac gtt ctc aac atc acg ccc aag gcc gcc ttt ggc ctc agt 720Ala Arg Asp Val Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser225 230 235 240ctt ggc gag att tcc atg att ttt gcc ttt tcc aag aag aac ggt ctc 768Leu Gly Glu Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu 245 250 255atc tcc gac cag ctc acc aag gat ctt cgc gag tcc gac gtg tgg aac 816Ile Ser Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn 260 265 270aag gct ctg gcc gtt gaa ttt aat gcg ctg cgc gag gcc tgg ggc att 864Lys Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile 275 280 285cca cag agt gtc ccc aag gac gag ttc tgg caa ggc tac att gtg cgc 912Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val Arg 290 295 300ggc acc aag cag gat atc gag gcg gcc atc gcc ccg gac agc aag tac 960Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser Lys Tyr305 310 315 320gtg cgc ctc acc atc atc aat gat gcc aac acc gcc ctc att agc ggc 1008Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu Ile Ser Gly 325 330 335aag ccc gac gcc tgc aag gct gcg atc gcg cgt ctc ggt ggc aac att 1056Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu Gly Gly Asn Ile 340 345 350cct gcg ctt ccc gtg acc cag ggc atg tgc ggc cac tgc ccc gag gtg 1104Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly His Cys Pro Glu Val 355 360 365gga cct tat acc aag gat atc gcc aag atc cat gcc aac ctt gag ttc 1152Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile His Ala Asn Leu Glu Phe 370 375 380ccc gtt gtc gac ggc ctt gac ctc tgg acc aca atc aac cag aag cgc 1200Pro Val Val Asp Gly Leu Asp Leu Trp Thr Thr Ile Asn Gln Lys Arg385 390 395 400ctc gtg cca cgc gcc acg ggc gcc aag gac gaa tgg gcc cct tct tcc 1248Leu Val Pro Arg Ala Thr Gly Ala Lys Asp Glu Trp Ala Pro Ser Ser 405 410 415ttt ggc gag tac gcc ggc cag ctc tac gag aag cag gct aac ttc ccc 1296Phe Gly Glu Tyr Ala Gly Gln Leu Tyr Glu Lys Gln Ala Asn Phe Pro 420 425 430caa atc gtc gag acc att tac aag caa aac tac gac gtc ttt gtc gag 1344Gln Ile Val Glu Thr Ile Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu 435 440 445gtt ggg ccc aac aac cac cgt agc acc gca gtg cgc acc acg ctt ggt 1392Val Gly Pro Asn Asn His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly 450 455 460ccc cag cgc aac cac ctt gct ggc gcc atc gac aag cag aac gag gat 1440Pro Gln Arg Asn His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp465 470 475 480gct tgg acg acc atc gtc aag ctt gtg gct tcg ctc aag gcc cac ctt 1488Ala Trp Thr Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu 485 490 495gtt cct ggc gtc 1500Val Pro Gly Val 50024500PRTSchizochytrium sp. 24Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His Tyr Glu Arg Glu1 5 10 15Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu Ile Val Leu Arg 20 25 30Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp Lys Ile Arg Glu 35 40 45Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu Ser Glu Leu Lys 50 55 60Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly Glu Thr Leu Ala65 70 75 80Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu Ala Leu Ser Leu 85 90 95Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu Leu Ala Ala Lys 100 105 110Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp Trp Ser Ser Pro Ala 115 120 125Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala Ser Asp Arg Val Ala Phe 130 135 140Met Tyr Gly Glu Gly Arg Ser Pro Tyr Tyr Gly Ile Thr Gln Asp Ile145 150 155 160His Arg Ile Trp Pro Glu Leu His Glu Val Ile Asn Glu Lys Thr Asn 165 170 175Arg Leu Trp Ala Glu Gly Asp Arg Trp Val Met Pro Arg Ala Ser Phe 180 185 190Lys Ser Glu Leu Glu Ser Gln Gln Gln Glu Phe Asp Arg Asn Met Ile 195 200 205Glu Met Phe Arg Leu Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu 210 215 220Ala Arg Asp Val Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser225 230 235 240Leu Gly Glu Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu 245 250 255Ile Ser Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn 260 265 270Lys Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile 275 280 285Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val Arg 290 295 300Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser Lys Tyr305 310 315 320Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu Ile Ser Gly 325 330 335Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu Gly Gly Asn Ile 340 345 350Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly His Cys Pro Glu Val 355 360 365Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile His Ala Asn Leu Glu Phe 370 375 380Pro Val Val Asp Gly Leu Asp Leu Trp Thr Thr Ile Asn Gln Lys Arg385 390 395 400Leu Val Pro Arg Ala Thr Gly Ala Lys Asp Glu Trp Ala Pro Ser Ser 405 410 415Phe Gly Glu Tyr Ala Gly Gln Leu Tyr Glu Lys Gln Ala Asn Phe Pro 420 425 430Gln Ile Val Glu Thr Ile Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu 435 440 445Val Gly Pro Asn Asn His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly 450 455 460Pro Gln Arg Asn His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp465 470 475 480Ala Trp Thr Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu 485 490 495Val Pro Gly Val 500251530DNASchizochytrium sp.CDS(1)..(1530) 25ctg ctc gat ctc gac agt atg ctt gcg ctg agc tct gcc agt gcc tcc 48Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser Ala Ser1 5 10 15ggc aac ctt gtt gag act gcg cct agc gac gcc tcg gtc att gtg ccg 96Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val Ile Val Pro 20 25 30ccc tgc aac att gcg gat ctc ggc agc cgc gcc ttc atg aaa acg tac 144Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe Met Lys Thr Tyr 35 40 45ggt gtt tcg gcg cct ctg tac acg ggc gcc atg gcc aag ggc att gcc 192Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala 50 55 60tct gcg gac ctc gtc att gcc gcc ggc cgc cag ggc atc ctt gcg tcc 240Ser Ala Asp Leu Val Ile Ala Ala Gly Arg Gln Gly Ile Leu Ala Ser65 70 75 80ttt ggc gcc ggc gga ctt ccc atg cag gtt gtg cgt gag tcc atc gaa 288Phe Gly Ala Gly Gly Leu Pro Met Gln Val Val Arg Glu Ser Ile Glu 85 90 95aag att cag gcc gcc ctg ccc aat ggc ccg tac gct gtc aac ctt atc 336Lys Ile Gln Ala Ala Leu Pro Asn Gly Pro Tyr Ala Val Asn Leu Ile 100 105 110cat tct ccc ttt gac agc aac ctc gaa aag ggc aat gtc gat ctc ttc 384His Ser Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe 115 120 125ctc gag aag ggt gtc acc ttt gtc gag gcc tcg gcc ttt atg acg ctc 432Leu Glu Lys Gly Val Thr Phe Val Glu Ala Ser Ala Phe Met Thr Leu 130 135 140acc ccg cag gtc gtg cgg tac cgc gcg gct ggc ctc acg cgc aac gcc 480Thr Pro Gln Val Val Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala145 150 155 160gac ggc tcg gtc aac atc cgc aac cgt atc att ggc aag gtc tcg cgc 528Asp Gly Ser Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg 165 170 175 acc gag ctc gcc gag atg ttc atg cgt cct gcg ccc gag cac ctt ctt 576Thr Glu Leu Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu 180 185 190cag aag ctc att gct tcc ggc gag atc aac cag gag cag gcc gag ctc 624Gln Lys Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu 195 200 205gcc cgc cgt gtt ccc gtc gct gac gac atc gcg gtc gaa gct gac tcg 672Ala Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser 210 215 220ggt ggc cac acc gac aac cgc ccc atc cac gtc att ctg ccc ctc atc 720Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu Ile225 230 235 240atc aac ctt cgc gac cgc ctt cac cgc gag tgc ggc tac ccg gcc aac 768Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro Ala Asn 245 250 255ctt cgc gtc cgt gtg ggc gcc ggc ggt ggc att ggg tgc ccc cag gcg 816Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys Pro Gln Ala 260 265 270gcg ctg gcc acc ttc aac atg ggt gcc tcc ttt att gtc acc ggc acc 864Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile Val Thr Gly Thr 275 280 285gtg aac cag gtc gcc aag cag tcg ggc acg tgc gac aat gtg cgc aag 912Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys 290 295 300cag ctc gcg aag gcc act tac tcg gac gta tgc atg gcc ccg gct gcc 960Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val Cys Met Ala Pro Ala Ala305 310 315 320gac atg ttc gag gaa ggc gtc aag ctt cag gtc ctc aag aag gga acc 1008Asp Met Phe Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr 325 330 335atg ttt ccc tcg cgc gcc aac aag ctc tac gag ctc ttt tgc aag tac 1056Met Phe Pro Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr 340 345 350gac tcg ttc gag tcc atg ccc ccc gca gag ctt gcg cgc gtc gag aag 1104Asp Ser Phe Glu Ser Met Pro Pro Ala Glu Leu Ala Arg Val Glu Lys 355 360 365cgc atc ttc agc cgc gcg ctc gaa gag gtc tgg gac gag acc aaa aac 1152Arg Ile Phe Ser Arg Ala Leu Glu Glu Val Trp Asp Glu Thr Lys Asn 370 375 380ttt tac att aac cgt ctt cac aac ccg gag aag atc cag cgc gcc gag 1200Phe Tyr Ile Asn Arg Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu385 390 395 400cgc gac ccc aag ctc aag atg tcg ctg tgc ttt cgc tgg tac ctg agc 1248Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser 405 410 415ctg gcg agc cgc tgg gcc aac act gga gct tcc gat cgc gtc atg gac 1296Leu Ala Ser Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp 420 425 430tac cag gtc tgg tgc ggt cct gcc att ggt tcc ttc aac gat ttc atc 1344Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile 435 440 445aag gga act tac ctt gat ccg gcc gtc gca aac gag tac ccg tgc gtc 1392Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val 450 455 460gtt cag att aac aag cag atc ctt cgt gga gcg tgc ttc ttg cgc cgt 1440Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg Arg465 470 475 480ctc gaa att ctg cgc aac gca cgc ctt tcc gat ggc gct gcc gct ctt 1488Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala Ala Leu 485 490 495gtg gcc agc atc gat gac aca tac gtc ccg gcc gag aag ctg 1530Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys Leu 500 505 51026510PRTSchizochytrium sp. 26Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser Ala Ser1 5 10 15Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val Ile Val Pro 20 25 30Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe Met Lys Thr Tyr 35 40 45Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala 50 55 60Ser Ala Asp Leu Val Ile Ala Ala Gly Arg Gln Gly Ile Leu Ala Ser65 70 75 80Phe Gly Ala Gly Gly Leu Pro Met Gln Val Val Arg Glu Ser Ile Glu 85 90 95Lys Ile Gln Ala Ala Leu Pro Asn Gly Pro Tyr Ala Val Asn Leu Ile 100 105 110His Ser Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe 115 120 125Leu Glu Lys Gly Val Thr Phe Val Glu Ala Ser Ala Phe Met Thr Leu 130 135 140Thr Pro Gln Val Val Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala145 150 155 160Asp Gly Ser Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg 165 170 175Thr Glu Leu Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu 180 185 190Gln Lys Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu 195 200 205Ala Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser 210 215 220Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu Ile225 230 235 240Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro Ala Asn 245 250 255Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys Pro Gln Ala 260 265 270Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile Val Thr Gly Thr 275 280 285Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys 290 295 300Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val Cys Met Ala Pro Ala Ala305 310 315 320Asp Met Phe Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr 325 330 335Met Phe Pro Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr 340 345 350Asp Ser Phe Glu Ser Met Pro Pro Ala Glu Leu Ala Arg Val Glu Lys 355 360 365Arg Ile Phe Ser Arg Ala Leu Glu Glu Val Trp Asp Glu Thr Lys Asn 370 375 380Phe Tyr Ile Asn Arg Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu385 390 395 400Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser

405 410 415Leu Ala Ser Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp 420 425 430Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile 435 440 445Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val 450 455 460Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg Arg465 470 475 480Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala Ala Leu 485 490 495Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys Leu 500 505 510271350DNASchizochytrium sp.CDS(1)..(1350) 27atg gcg ctc cgt gtc aag acg aac aag aag cca tgc tgg gag atg acc 48Met Ala Leu Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr1 5 10 15aag gag gag ctg acc agc ggc aag acc gag gtg ttc aac tat gag gaa 96Lys Glu Glu Leu Thr Ser Gly Lys Thr Glu Val Phe Asn Tyr Glu Glu 20 25 30ctc ctc gag ttc gca gag ggc gac atc gcc aag gtc ttc gga ccc gag 144Leu Leu Glu Phe Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Pro Glu 35 40 45ttc gcc gtc atc gac aag tac ccg cgc cgc gtg cgc ctg ccc gcc cgc 192Phe Ala Val Ile Asp Lys Tyr Pro Arg Arg Val Arg Leu Pro Ala Arg 50 55 60gag tac ctg ctc gtg acc cgc gtc acc ctc atg gac gcc gag gtc aac 240Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn65 70 75 80aac tac cgc gtc ggc gcc cgc atg gtc acc gag tac gat ctc ccc gtc 288Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val 85 90 95aac gga gag ctc tcc gag ggc gga gac tgc ccc tgg gcc gtc ctg gtc 336Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val 100 105 110gag agt ggc cag tgc gat ctc atg ctc atc tcc tac atg ggc att gac 384Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp 115 120 125ttc cag aac cag ggc gac cgc gtc tac cgc ctg ctc aac acc acg ctc 432Phe Gln Asn Gln Gly Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu 130 135 140acc ttt tac ggc gtg gcc cac gag ggc gag acc ctc gag tac gac att 480Thr Phe Tyr Gly Val Ala His Glu Gly Glu Thr Leu Glu Tyr Asp Ile145 150 155 160cgc gtc acc ggc ttc gcc aag cgt ctc gac ggc ggc atc tcc atg ttc 528Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Gly Ile Ser Met Phe 165 170 175ttc ttc gag tac gac tgc tac gtc aac ggc cgc ctc ctc atc gag atg 576Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met 180 185 190cgc gat ggc tgc gcc ggc ttc ttc acc aac gag gag ctc gac gcc ggc 624Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Asp Ala Gly 195 200 205aag ggc gtc gtc ttc acc cgc ggc gac ctc gcc gcc cgc gcc aag atc 672Lys Gly Val Val Phe Thr Arg Gly Asp Leu Ala Ala Arg Ala Lys Ile 210 215 220cca aag cag gac gtc tcc ccc tac gcc gtc gcc ccc tgc ctc cac aag 720Pro Lys Gln Asp Val Ser Pro Tyr Ala Val Ala Pro Cys Leu His Lys225 230 235 240acc aag ctc aac gaa aag gag atg cag acc ctc gtc gac aag gac tgg 768Thr Lys Leu Asn Glu Lys Glu Met Gln Thr Leu Val Asp Lys Asp Trp 245 250 255gca tcc gtc ttt ggc tcc aag aac ggc atg ccg gaa atc aac tac aaa 816Ala Ser Val Phe Gly Ser Lys Asn Gly Met Pro Glu Ile Asn Tyr Lys 260 265 270ctc tgc gcg cgt aag atg ctc atg att gac cgc gtc acc agc att gac 864Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Ser Ile Asp 275 280 285cac aag ggc ggt gtc tac ggc ctc ggt cag ctc gtc ggt gaa aag atc 912His Lys Gly Gly Val Tyr Gly Leu Gly Gln Leu Val Gly Glu Lys Ile 290 295 300ctc gag cgc gac cac tgg tac ttt ccc tgc cac ttt gtc aag gat cag 960Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Lys Asp Gln305 310 315 320gtc atg gcc gga tcc ctc gtc tcc gac ggc tgc agc cag atg ctc aag 1008Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Met Leu Lys 325 330 335atg tac atg atc tgg ctc ggc ctc cac ctc acc acc gga ccc ttt gac 1056Met Tyr Met Ile Trp Leu Gly Leu His Leu Thr Thr Gly Pro Phe Asp 340 345 350ttc cgc ccg gtc aac ggc cac ccc aac aag gtc cgc tgc cgc ggc caa 1104Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln 355 360 365atc tcc ccg cac aag ggc aag ctc gtc tac gtc atg gag atc aag gag 1152Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu 370 375 380atg ggc ttc gac gag gac aac gac ccg tac gcc att gcc gac gtc aac 1200Met Gly Phe Asp Glu Asp Asn Asp Pro Tyr Ala Ile Ala Asp Val Asn385 390 395 400atc att gat gtc gac ttc gaa aag ggc cag gac ttt agc ctc gac cgc 1248Ile Ile Asp Val Asp Phe Glu Lys Gly Gln Asp Phe Ser Leu Asp Arg 405 410 415atc agc gac tac ggc aag ggc gac ctc aac aag aag atc gtc gtc gac 1296Ile Ser Asp Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp 420 425 430ttt aag ggc atc gct ctc aag atg cag aag cgc tcc acc aac aag aac 1344Phe Lys Gly Ile Ala Leu Lys Met Gln Lys Arg Ser Thr Asn Lys Asn 435 440 445ccc tcc 1350Pro Ser 45028450PRTSchizochytrium sp. 28Met Ala Leu Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr1 5 10 15Lys Glu Glu Leu Thr Ser Gly Lys Thr Glu Val Phe Asn Tyr Glu Glu 20 25 30Leu Leu Glu Phe Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Pro Glu 35 40 45Phe Ala Val Ile Asp Lys Tyr Pro Arg Arg Val Arg Leu Pro Ala Arg 50 55 60Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn65 70 75 80Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val 85 90 95Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val 100 105 110Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp 115 120 125Phe Gln Asn Gln Gly Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu 130 135 140Thr Phe Tyr Gly Val Ala His Glu Gly Glu Thr Leu Glu Tyr Asp Ile145 150 155 160Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Gly Ile Ser Met Phe 165 170 175Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met 180 185 190Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Asp Ala Gly 195 200 205Lys Gly Val Val Phe Thr Arg Gly Asp Leu Ala Ala Arg Ala Lys Ile 210 215 220Pro Lys Gln Asp Val Ser Pro Tyr Ala Val Ala Pro Cys Leu His Lys225 230 235 240Thr Lys Leu Asn Glu Lys Glu Met Gln Thr Leu Val Asp Lys Asp Trp 245 250 255Ala Ser Val Phe Gly Ser Lys Asn Gly Met Pro Glu Ile Asn Tyr Lys 260 265 270Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Ser Ile Asp 275 280 285His Lys Gly Gly Val Tyr Gly Leu Gly Gln Leu Val Gly Glu Lys Ile 290 295 300Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Lys Asp Gln305 310 315 320Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Met Leu Lys 325 330 335Met Tyr Met Ile Trp Leu Gly Leu His Leu Thr Thr Gly Pro Phe Asp 340 345 350Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln 355 360 365Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu 370 375 380Met Gly Phe Asp Glu Asp Asn Asp Pro Tyr Ala Ile Ala Asp Val Asn385 390 395 400Ile Ile Asp Val Asp Phe Glu Lys Gly Gln Asp Phe Ser Leu Asp Arg 405 410 415Ile Ser Asp Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp 420 425 430Phe Lys Gly Ile Ala Leu Lys Met Gln Lys Arg Ser Thr Asn Lys Asn 435 440 445Pro Ser 450291497DNASchizochytrium sp.CDS(1)..(1497) 29aag gtt cag ccc gtc ttt gcc aac ggc gcc gcc act gtc ggc ccc gag 48Lys Val Gln Pro Val Phe Ala Asn Gly Ala Ala Thr Val Gly Pro Glu1 5 10 15gcc tcc aag gct tcc tcc ggc gcc agc gcc agc gcc agc gcc gcc ccg 96Ala Ser Lys Ala Ser Ser Gly Ala Ser Ala Ser Ala Ser Ala Ala Pro 20 25 30gcc aag cct gcc ttc agc gcc gat gtt ctt gcg ccc aag ccc gtt gcc 144Ala Lys Pro Ala Phe Ser Ala Asp Val Leu Ala Pro Lys Pro Val Ala 35 40 45ctt ccc gag cac atc ctc aag ggc gac gcc ctc gcc ccc aag gag atg 192Leu Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala Pro Lys Glu Met 50 55 60tcc tgg cac ccc atg gcc cgc atc ccg ggc aac ccg acg ccc tct ttt 240Ser Trp His Pro Met Ala Arg Ile Pro Gly Asn Pro Thr Pro Ser Phe65 70 75 80gcg ccc tcg gcc tac aag ccg cgc aac atc gcc ttt acg ccc ttc ccc 288Ala Pro Ser Ala Tyr Lys Pro Arg Asn Ile Ala Phe Thr Pro Phe Pro 85 90 95ggc aac ccc aac gat aac gac cac acc ccg ggc aag atg ccg ctc acc 336Gly Asn Pro Asn Asp Asn Asp His Thr Pro Gly Lys Met Pro Leu Thr 100 105 110tgg ttc aac atg gcc gag ttc atg gcc ggc aag gtc agc atg tgc ctc 384Trp Phe Asn Met Ala Glu Phe Met Ala Gly Lys Val Ser Met Cys Leu 115 120 125ggc ccc gag ttc gcc aag ttc gac gac tcg aac acc agc cgc agc ccc 432Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr Ser Arg Ser Pro 130 135 140gct tgg gac ctc gct ctc gtc acc cgc gcc gtg tct gtg tct gac ctc 480Ala Trp Asp Leu Ala Leu Val Thr Arg Ala Val Ser Val Ser Asp Leu145 150 155 160aag cac gtc aac tac cgc aac atc gac ctc gac ccc tcc aag ggt acc 528Lys His Val Asn Tyr Arg Asn Ile Asp Leu Asp Pro Ser Lys Gly Thr 165 170 175atg gtc ggc gag ttc gac tgc ccc gcg gac gcc tgg ttc tac aag ggc 576Met Val Gly Glu Phe Asp Cys Pro Ala Asp Ala Trp Phe Tyr Lys Gly 180 185 190gcc tgc aac gat gcc cac atg ccg tac tcg atc ctc atg gag atc gcc 624Ala Cys Asn Asp Ala His Met Pro Tyr Ser Ile Leu Met Glu Ile Ala 195 200 205ctc cag acc tcg ggt gtg ctc acc tcg gtg ctc aag gcg ccc ctg acc 672Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys Ala Pro Leu Thr 210 215 220atg gag aag gac gac atc ctc ttc cgc aac ctc gac gcc aac gcc gag 720Met Glu Lys Asp Asp Ile Leu Phe Arg Asn Leu Asp Ala Asn Ala Glu225 230 235 240ttc gtg cgc gcc gac ctc gac tac cgc ggc aag act atc cgc aac gtc 768Phe Val Arg Ala Asp Leu Asp Tyr Arg Gly Lys Thr Ile Arg Asn Val 245 250 255acc aag tgc act ggc tac agc atg ctc ggc gag atg ggc gtc cac cgc 816Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Glu Met Gly Val His Arg 260 265 270ttc acc ttt gag ctc tac gtc gat gat gtg ctc ttt tac aag ggc tcg 864Phe Thr Phe Glu Leu Tyr Val Asp Asp Val Leu Phe Tyr Lys Gly Ser 275 280 285acc tcg ttc ggc tgg ttc gtg ccc gag gtc ttt gcc gcc cag gcc ggc 912Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ala Ala Gln Ala Gly 290 295 300ctc gac aac ggc cgc aag tcg gag ccc tgg ttc att gag aac aag gtt 960Leu Asp Asn Gly Arg Lys Ser Glu Pro Trp Phe Ile Glu Asn Lys Val305 310 315 320ccg gcc tcg cag gtc tcc tcc ttt gac gtg cgc ccc aac ggc agc ggc 1008Pro Ala Ser Gln Val Ser Ser Phe Asp Val Arg Pro Asn Gly Ser Gly 325 330 335cgc acc gcc atc ttc gcc aac gcc ccc agc ggc gcc cag ctc aac cgc 1056Arg Thr Ala Ile Phe Ala Asn Ala Pro Ser Gly Ala Gln Leu Asn Arg 340 345 350cgc acg gac cag ggc cag tac ctc gac gcc gtc gac att gtc tcc ggc 1104Arg Thr Asp Gln Gly Gln Tyr Leu Asp Ala Val Asp Ile Val Ser Gly 355 360 365agc ggc aag aag agc ctc ggc tac gcc cac ggt tcc aag acg gtc aac 1152Ser Gly Lys Lys Ser Leu Gly Tyr Ala His Gly Ser Lys Thr Val Asn 370 375 380ccg aac gac tgg ttc ttc tcg tgc cac ttt tgg ttt gac tcg gtc atg 1200Pro Asn Asp Trp Phe Phe Ser Cys His Phe Trp Phe Asp Ser Val Met385 390 395 400ccc gga agt ctc ggt gtc gag tcc atg ttc cag ctc gtc gag gcc atc 1248Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu Val Glu Ala Ile 405 410 415gcc gcc cac gag gat ctc gct ggc aag cac ggc att gcc aac ccc acc 1296Ala Ala His Glu Asp Leu Ala Gly Lys His Gly Ile Ala Asn Pro Thr 420 425 430ttt gtg cac gcc ccg ggc aag atc agc tgg aag tac cgc ggc cag ctc 1344Phe Val His Ala Pro Gly Lys Ile Ser Trp Lys Tyr Arg Gly Gln Leu 435 440 445acg ccc aag agc aag aag atg gac tcg gag gtc cac atc gtg tcc gtg 1392Thr Pro Lys Ser Lys Lys Met Asp Ser Glu Val His Ile Val Ser Val 450 455 460gac gcc cac gac ggc gtt gtc gac ctc gtc gcc gac ggc ttc ctc tgg 1440Asp Ala His Asp Gly Val Val Asp Leu Val Ala Asp Gly Phe Leu Trp465 470 475 480gcc gac agc ctc cgc gtc tac tcg gtg agc aac att cgc gtg cgc atc 1488Ala Asp Ser Leu Arg Val Tyr Ser Val Ser Asn Ile Arg Val Arg Ile 485 490 495gcc tcc ggt 1497Ala Ser Gly 30499PRTSchizochytrium sp. 30Lys Val Gln Pro Val Phe Ala Asn Gly Ala Ala Thr Val Gly Pro Glu1 5 10 15Ala Ser Lys Ala Ser Ser Gly Ala Ser Ala Ser Ala Ser Ala Ala Pro 20 25 30Ala Lys Pro Ala Phe Ser Ala Asp Val Leu Ala Pro Lys Pro Val Ala 35 40 45Leu Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala Pro Lys Glu Met 50 55 60Ser Trp His Pro Met Ala Arg Ile Pro Gly Asn Pro Thr Pro Ser Phe65 70 75 80Ala Pro Ser Ala Tyr Lys Pro Arg Asn Ile Ala Phe Thr Pro Phe Pro 85 90 95Gly Asn Pro Asn Asp Asn Asp His Thr Pro Gly Lys Met Pro Leu Thr 100 105 110Trp Phe Asn Met Ala Glu Phe Met Ala Gly Lys Val Ser Met Cys Leu 115 120 125Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr Ser Arg Ser Pro 130 135 140Ala Trp Asp Leu Ala Leu Val Thr Arg Ala Val Ser Val Ser Asp Leu145 150 155 160Lys His Val Asn Tyr Arg Asn Ile Asp Leu Asp Pro Ser Lys Gly Thr 165 170 175Met Val Gly Glu Phe Asp Cys Pro Ala Asp Ala Trp Phe Tyr Lys Gly 180 185 190Ala Cys Asn Asp Ala His Met Pro Tyr Ser Ile Leu Met Glu Ile Ala 195 200 205Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys Ala Pro Leu Thr 210 215 220Met Glu Lys Asp Asp Ile Leu Phe Arg Asn Leu Asp Ala Asn Ala Glu225 230 235 240Phe Val Arg Ala Asp Leu Asp Tyr Arg Gly Lys Thr Ile Arg Asn Val 245 250 255Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Glu Met Gly Val His Arg 260 265 270Phe Thr Phe Glu Leu Tyr Val Asp Asp Val Leu Phe Tyr Lys Gly Ser 275 280 285Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ala Ala Gln Ala Gly 290 295 300Leu Asp Asn Gly Arg Lys Ser Glu Pro Trp Phe Ile Glu Asn Lys Val305 310 315 320Pro Ala Ser Gln Val Ser Ser Phe Asp Val Arg Pro Asn Gly Ser Gly 325 330 335Arg Thr Ala Ile Phe Ala Asn Ala Pro Ser Gly Ala Gln Leu Asn Arg 340 345 350Arg Thr Asp Gln Gly Gln Tyr Leu Asp Ala Val Asp Ile Val Ser Gly 355 360

365Ser Gly Lys Lys Ser Leu Gly Tyr Ala His Gly Ser Lys Thr Val Asn 370 375 380Pro Asn Asp Trp Phe Phe Ser Cys His Phe Trp Phe Asp Ser Val Met385 390 395 400Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu Val Glu Ala Ile 405 410 415Ala Ala His Glu Asp Leu Ala Gly Lys His Gly Ile Ala Asn Pro Thr 420 425 430Phe Val His Ala Pro Gly Lys Ile Ser Trp Lys Tyr Arg Gly Gln Leu 435 440 445Thr Pro Lys Ser Lys Lys Met Asp Ser Glu Val His Ile Val Ser Val 450 455 460Asp Ala His Asp Gly Val Val Asp Leu Val Ala Asp Gly Phe Leu Trp465 470 475 480Ala Asp Ser Leu Arg Val Tyr Ser Val Ser Asn Ile Arg Val Arg Ile 485 490 495Ala Ser Gly311512DNASchizochytrium sp.CDS(1)..(1512) 31gcc ccg ctc tac ctc tcg cag gac ccg acc agc ggc cag ctc aag aag 48Ala Pro Leu Tyr Leu Ser Gln Asp Pro Thr Ser Gly Gln Leu Lys Lys1 5 10 15cac acc gac gtg gcc tcc ggc cag gcc acc atc gtg cag ccc tgc acg 96His Thr Asp Val Ala Ser Gly Gln Ala Thr Ile Val Gln Pro Cys Thr 20 25 30ctc ggc gac ctc ggt gac cgc tcc ttc atg gag acc tac ggc gtc gtc 144Leu Gly Asp Leu Gly Asp Arg Ser Phe Met Glu Thr Tyr Gly Val Val 35 40 45gcc ccg ctg tac acg ggc gcc atg gcc aag ggc att gcc tcg gcg gac 192Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Asp 50 55 60ctc gtc atc gcc gcc ggc aag cgc aag atc ctc ggc tcc ttt ggc gcc 240Leu Val Ile Ala Ala Gly Lys Arg Lys Ile Leu Gly Ser Phe Gly Ala65 70 75 80ggc ggc ctc ccc atg cac cac gtg cgc gcc gcc ctc gag aag atc cag 288Gly Gly Leu Pro Met His His Val Arg Ala Ala Leu Glu Lys Ile Gln 85 90 95gcc gcc ctg cct cag ggc ccc tac gcc gtc aac ctc atc cac tcg cct 336Ala Ala Leu Pro Gln Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro 100 105 110ttt gac agc aac ctc gag aag ggc aac gtc gat ctc ttc ctc gag aag 384Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys 115 120 125ggc gtc act gtg gtg gag gcc tcg gca ttc atg acc ctc acc ccg cag 432Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln 130 135 140gtc gtg cgc tac cgc gcc gcc ggc ctc tcg cgc aac gcc gac ggt tcg 480Val Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Asn Ala Asp Gly Ser145 150 155 160gtc aac atc cgc aac cgc atc atc ggc aag gtc tcg cgc acc gag ctc 528Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu 165 170 175gcc gag atg ttc atc cgc ccg gcc ccg gag cac ctc ctc gag aag ctc 576Ala Glu Met Phe Ile Arg Pro Ala Pro Glu His Leu Leu Glu Lys Leu 180 185 190atc gcc tcg ggc gag atc acc cag gag cag gcc gag ctc gcg cgc cgc 624Ile Ala Ser Gly Glu Ile Thr Gln Glu Gln Ala Glu Leu Ala Arg Arg 195 200 205gtt ccc gtc gcc gac gat atc gct gtc gag gct gac tcg ggc ggc cac 672Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser Gly Gly His 210 215 220acc gac aac cgc ccc atc cac gtc atc ctc ccg ctc atc atc aac ctc 720Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu Ile Ile Asn Leu225 230 235 240cgc aac cgc ctg cac cgc gag tgc ggc tac ccc gcg cac ctc cgc gtc 768Arg Asn Arg Leu His Arg Glu Cys Gly Tyr Pro Ala His Leu Arg Val 245 250 255cgc gtt ggc gcc ggc ggt ggc gtc ggc tgc ccg cag gcc gcc gcc gcc 816Arg Val Gly Ala Gly Gly Gly Val Gly Cys Pro Gln Ala Ala Ala Ala 260 265 270gcg ctc acc atg ggc gcc gcc ttc atc gtc acc ggc act gtc aac cag 864Ala Leu Thr Met Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln 275 280 285gtc gcc aag cag tcc ggc acc tgc gac aac gtg cgc aag cag ctc tcg 912Val Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys Gln Leu Ser 290 295 300cag gcc acc tac tcg gat atc tgc atg gcc ccg gcc gcc gac atg ttc 960Gln Ala Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe305 310 315 320gag gag ggc gtc aag ctc cag gtc ctc aag aag gga acc atg ttc ccc 1008Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro 325 330 335tcg cgc gcc aac aag ctc tac gag ctc ttt tgc aag tac gac tcc ttc 1056Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe 340 345 350gac tcc atg cct cct gcc gag ctc gag cgc atc gag aag cgt atc ttc 1104Asp Ser Met Pro Pro Ala Glu Leu Glu Arg Ile Glu Lys Arg Ile Phe 355 360 365aag cgc gca ctc cag gag gtc tgg gag gag acc aag gac ttt tac att 1152Lys Arg Ala Leu Gln Glu Val Trp Glu Glu Thr Lys Asp Phe Tyr Ile 370 375 380aac ggt ctc aag aac ccg gag aag atc cag cgc gcc gag cac gac ccc 1200Asn Gly Leu Lys Asn Pro Glu Lys Ile Gln Arg Ala Glu His Asp Pro385 390 395 400aag ctc aag atg tcg ctc tgc ttc cgc tgg tac ctt ggt ctt gcc agc 1248Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Gly Leu Ala Ser 405 410 415cgc tgg gcc aac atg ggc gcc ccg gac cgc gtc atg gac tac cag gtc 1296Arg Trp Ala Asn Met Gly Ala Pro Asp Arg Val Met Asp Tyr Gln Val 420 425 430tgg tgt ggc ccg gcc att ggc gcc ttc aac gac ttc atc aag ggc acc 1344Trp Cys Gly Pro Ala Ile Gly Ala Phe Asn Asp Phe Ile Lys Gly Thr 435 440 445tac ctc gac ccc gct gtc tcc aac gag tac ccc tgt gtc gtc cag atc 1392Tyr Leu Asp Pro Ala Val Ser Asn Glu Tyr Pro Cys Val Val Gln Ile 450 455 460aac ctg caa atc ctc cgt ggt gcc tgc tac ctg cgc cgt ctc aac gcc 1440Asn Leu Gln Ile Leu Arg Gly Ala Cys Tyr Leu Arg Arg Leu Asn Ala465 470 475 480ctg cgc aac gac ccg cgc att gac ctc gag acc gag gat gct gcc ttt 1488Leu Arg Asn Asp Pro Arg Ile Asp Leu Glu Thr Glu Asp Ala Ala Phe 485 490 495gtc tac gag ccc acc aac gcg ctc 1512Val Tyr Glu Pro Thr Asn Ala Leu 50032504PRTSchizochytrium sp. 32Ala Pro Leu Tyr Leu Ser Gln Asp Pro Thr Ser Gly Gln Leu Lys Lys1 5 10 15His Thr Asp Val Ala Ser Gly Gln Ala Thr Ile Val Gln Pro Cys Thr 20 25 30Leu Gly Asp Leu Gly Asp Arg Ser Phe Met Glu Thr Tyr Gly Val Val 35 40 45 Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Asp 50 55 60Leu Val Ile Ala Ala Gly Lys Arg Lys Ile Leu Gly Ser Phe Gly Ala65 70 75 80Gly Gly Leu Pro Met His His Val Arg Ala Ala Leu Glu Lys Ile Gln 85 90 95Ala Ala Leu Pro Gln Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro 100 105 110Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys 115 120 125Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln 130 135 140Val Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Asn Ala Asp Gly Ser145 150 155 160Val Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu 165 170 175Ala Glu Met Phe Ile Arg Pro Ala Pro Glu His Leu Leu Glu Lys Leu 180 185 190Ile Ala Ser Gly Glu Ile Thr Gln Glu Gln Ala Glu Leu Ala Arg Arg 195 200 205Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser Gly Gly His 210 215 220Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu Ile Ile Asn Leu225 230 235 240Arg Asn Arg Leu His Arg Glu Cys Gly Tyr Pro Ala His Leu Arg Val 245 250 255Arg Val Gly Ala Gly Gly Gly Val Gly Cys Pro Gln Ala Ala Ala Ala 260 265 270Ala Leu Thr Met Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln 275 280 285Val Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys Gln Leu Ser 290 295 300Gln Ala Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe305 310 315 320Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro 325 330 335Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe 340 345 350Asp Ser Met Pro Pro Ala Glu Leu Glu Arg Ile Glu Lys Arg Ile Phe 355 360 365Lys Arg Ala Leu Gln Glu Val Trp Glu Glu Thr Lys Asp Phe Tyr Ile 370 375 380Asn Gly Leu Lys Asn Pro Glu Lys Ile Gln Arg Ala Glu His Asp Pro385 390 395 400Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Gly Leu Ala Ser 405 410 415Arg Trp Ala Asn Met Gly Ala Pro Asp Arg Val Met Asp Tyr Gln Val 420 425 430Trp Cys Gly Pro Ala Ile Gly Ala Phe Asn Asp Phe Ile Lys Gly Thr 435 440 445Tyr Leu Asp Pro Ala Val Ser Asn Glu Tyr Pro Cys Val Val Gln Ile 450 455 460Asn Leu Gln Ile Leu Arg Gly Ala Cys Tyr Leu Arg Arg Leu Asn Ala465 470 475 480Leu Arg Asn Asp Pro Arg Ile Asp Leu Glu Thr Glu Asp Ala Ala Phe 485 490 495Val Tyr Glu Pro Thr Asn Ala Leu 500339PRTArtificial sequencemotif 33Trp Xaa Xaa Lys Glu Xaa Xaa Xaa Lys1 5346PRTArtificial sequencemotif 34Phe Asn Xaa Ser His Ser1 5355PRTArtificial sequencemotif 35Xaa Gly Xaa Asp Xaa1 5364244DNASchizochytrium sp. 36tttctctctc tcgagctgtt gctgctgctg ctgctgctgc tgcttccttg ctggttctca 60cgtccgttcg atcaagcgct cgctcgctcg accgatcggt gcgtgcgtgc gtgcgtgagt 120cttgttgcca ggcagccgca ggctgtctgt ctgtttgtgt agttttaccc tcggggttcg 180gggtctgcct gcctcccgct cccgcccgcc gccgcccgta tccaccccgc tcgcctccgc 240ccatcgggcc tcgcctcctc gcgccgcacg catcgcgcgc atcgcatgca tcatgctgcc 300acgcacgggg ggacgcgcgc cccgcgtccc ccgccgccgc cgtcgtcgtc tggcgatgcc 360gtcgccgccc tccttccttc cctcgcctcc tcttcctccc gagcccccct gtcttccttc 420gcccccgcag cggcgcgcag gaagcgagga gagcggggag gagagaagaa aagaaaagaa 480aagaaaagaa aataacagcg ccgtctcgcg cagacgcgcg cggccgcgtg cgaggcggcg 540tgatggggct tctcgtggcg cggctgcggc ctggcccggc ctcgcctttg aggtgcaggc 600tttgggagag aagagtggga cgcggagaag ataagatggt gccatggcgc aggacggaga 660ggttgctgaa acttcttcga gcggcacagg cgatggcgag agaccgacag ctgccggcgc 720ggaggggatg gatacctccc gaggctggca tggacgagct ggccgcgcgg atctggctgg 780ccgcgcggcg gtgggtccgg aggcgcgagg ttggttttct tcatacctga taccatacgg 840tattcattct tcctctccag gaaggaagca agtcacatag agtatcacta gcctaatgat 900ggactctatg ttttagggca cgtcggagca gaaggcgcga gcgattcgaa tgcgagcgat 960agatacagca cagagacctt gccggcgacg cggatgcagg cgagcacgca cgcaccgcac 1020gcacggcagc ggtgcacgcg ctcctcggca gatgcacggt tctgcgccgc gcctttacat 1080tttttgattt taggtggtgt gcctgccact ttgaacatca tccacaagtc aacgcagcat 1140caagaggcaa gcaagtacat acatccattc gaattcaagt tcaagagacg cagcaacagc 1200cgccgctccg ctcaagctgc agctagctgg ctgacagggc tcgctggctg tagtggaaaa 1260ttccattcac ttttctgcat ccgcggccag caggcccgta cgcacgttct ctcgtttgtt 1320tgttcgttcg tgcgtgcgtg cgtgcgtccc agctgcctgt ctaatctgcc gcgcgatcca 1380acgaccctcg gtcgtcgccg caagcgaaac ccgacgccga cctggccaat gccgcaagaa 1440tgctaagcgc gcagcaatgc tgagagtaat cttcagccca ccaagtcatt atcgctgccc 1500aagtctccat cgcagccaca ttcaggcttt ctctctctct ccctccctct ctttctgccg 1560ggagagaagg aaagacccgc cgccgccgcc tctgcgcctg tgacgggctg tccgttgtaa 1620gccctcttag acagttccta ggtgccgggc gccgccgcgc ctccgtcgca ggcacacgta 1680ggcggccacg ggttcccccc gcaccttcca caccttcttc ccccgcagcc ggaccgcgcg 1740ccgtctgctt acgcacttcg cgcggccgcc gcccgcgaac ccgagcgcgt gctgtgggcg 1800ccgtcttccg gccgcgtcgg aggtcgtccc cgcgccgcgc tactccgggt cctgtgcggt 1860acgtacttaa tattaacagt gggacctcgc acaggacctg acggcagcac agacgtcgcc 1920gcctcgcatc gctggggacg caggcgaggc atcccggcgc ggccccgcac cggggaggct 1980gcggggcggc ctcttccggc cggcggccgc atcaggcgga tgacgcaaga gccctcgcag 2040tcgctcgctc gcgggagcgc agcgcggcgc cagcgtggcc aagctcccgc cccttctggc 2100tggctgcatg cctgcctgcc tgcctgcctg cgtgcgtgcg tgcgtgcgtg ccttcgtgcg 2160tgcctgcctt cgtgcgtgcg tgcgtgagtg cggcggaaga gggatcatgc gaggatcaat 2220cacccgccgc acctcgactt ttgaagaagc cgcgatgcga tgcgatgcga tgcgatgcga 2280cgcgataccg tgcgaggcta cgaagcgagt ctggccggcc gtcatacaac gcacgttttc 2340gagaaggagg gctggcggag gcgtgcatgc cggcgaccat tgcgaacgcg gcgtctcgtg 2400gctggcgaag gtgcctggag gatctaacga tcgctgctat gatgctatag ctgtgctgat 2460ccccggtcca ttccaccacg tctgtgcctg ccgcctgacc tgcgcttggc tttccttcaa 2520gttctcctcc gccgggcctt caggaccgag acgagacctg cagctgcagc tagactcgcg 2580ctcgctcgcg gaggattcgc cggccgccgg gccggacggg actcgcgagg tcacacggcc 2640gccggcgatc gcgatggctg tgctgacgta ctcgtgcgtg gcagccgtac gtcagcgacg 2700ccgcctccgt attgtggatt cgttagttgg ttgttggttg atttgttgat taattttttt 2760gttcgtaggc ttggttatag ctaatagttt agtttatact ggtgctcttc ggtgctgatt 2820tagctcgact tgggtccaca ccactgcccc tctactgtga atggatcaat ggacgcacga 2880cgggccgacg aaagtgcgcg agtgaggtaa cctaagcaac ggcggtcttc agaggggacg 2940cacgccctcc gtcgcagtca gtccagacag gcagaaaagc gtcttaggga ccacgcacgc 3000acgcacgcac gcacgcacgc ccgcacgcac gctccctccc tcgcgtgcct atttttttag 3060gcttccttcc gcacgggcct acctctcgct ccctcgcctc gccgcaccag gcggcagcag 3120cgatacctgc cggtgccgcc tccgtcacgc gctcagccgc agctcagccc agccgcgagc 3180tagggtttgt tcgtcctgaa ttgtttgatt tgatttgatt tgatttgatc cgatccgatc 3240cgatctgatc tgatttgctt tgctttgctt tgtctccctc ccggcgcgga ccaagcgtcc 3300gtctgcgcgc cgcagcttcc cttcttctcc cagccctcct tctgctcccg cctctcgcgc 3360aagcacgcag cttcgccgcc gcatccggtc ggtcggtcgg tcgatcgacc cgcctgccgc 3420tgctgctgtg gccgggcttt tctccatcgg cgactctttc ttctccatac gtcctactac 3480gtacatacat actgccggct tcctcctctt ccagcgcggc gacggcggca ggctgcgacg 3540tcgtcgccgc cgcgggcgcc gcgcgcgccg ccgccgccgc ccgcgtcgca gggcctcgtc 3600gccgccgccg ctccgctccg ctccgaggcc gcgagagggc cgcggcggcg cgatggatgg 3660atggatggat ggatggatgg atggattttg ttgatcgatg gcggcgcatg ggcggagatg 3720agcgaggacg agcgcgcgag cgcggcagcc ggattcgcag ggcctcgctc gcctcgcgcc 3780cgctgccgcg cccgccttgc gagcctgcgc cgcgagcgag cgagcgagcg agcggggctt 3840tctttgtctc gcgcgccgct tggcctcgtg tgtcttgtgc ttgcgtagcg ggcgccgcgg 3900tggaagatgg ctcattcaat cgacccattc acgcacgcac tccggcgcgc agagaaggcc 3960gaggaggagc agcaagcaaa ccaaaagctc tcgcgctcgc ggtctcgggc tcgagcggtc 4020tcggagagag agtcttgcgg cgaccaccgg cagcagcagc agcagcagca gcgctgtcga 4080gcacgagcac gagcacgagc acgagcacga gcattcgagc aagaggacag acacggttgt 4140cagcgcctag ctcgctcgat acagaaagag gcgggttggg cgtaaaaaaa aaggagcacg 4200caagccgcca gccagccagc tagctagcca gcctgcctgc caaa 4244373886DNASchizochytrium sp.misc_feature(2115)..(2115)n = a, c, g, or t 37gatcttgatt gccaagctct ggattgtcga ttccgatgaa tcgagctctt tgttgtcgag 60ctctggcttg ccgagctttc agaaatagac aaaattgccg agttcctgat tgcggggctc 120tcgattgcca aggtctggtg gattctcgaa ctctcgattg tcaaaatctt ggtcgtctcg 180tcggattctt tcctgatttg ttttgtcaag accttgagat tgtgcaaaac cttgatcgtt 240gacaaaccct tgatcgacag cagcctttca tcacgctcag ctcttgtcat tgattatatt 300ccccctgaca gccaacacct tgatgcaggg tctcaacctt gatttttgga ggccatcatc 360agcatcacgc cccggcactc accctcaaca ttcgacagcc aacgcttttt tttcttcgac 420taggatctga gaataaaagc aggtcaccac gaccgtaggc caacgcgaca accatggaaa 480taaagtgaca acgaacgact tgcaagttta aatgtaaaga gcagcaattg cccgcccaca 540gacaaatgaa agcaggcgcc gagtcttatt tgaggaggtg ggcctgtggc aatgggcgaa 600agaaaatcaa ggacaaggag agcaggttac gtaccggtat actggtatac gtacatggat 660ggttcttggc aagttgacgg gatgtgtgcg agtgaccgtg gtagttaacg aaagagccgc 720aagggcaagg aaagcaagag aatgcagact tttccacagg atggatgggt ccgcagcttg 780ccgcatgatg aaacgctgta tttcacctgg cacgtggtgg cgcacgcgcc cacatatgat 840cgcggcggcg ggtgtattat acattttccc cctcaggtct actgccatcc ctccatgcgt 900cgctcgtgcg aacgacgcaa gcctttcgca tcgtgcagcc tctttctggt aaggcaagag 960ctaaacccaa acctaaacga aagaacattt ttacctctct ctctctccca ttggtcgcgt 1020gcgctccgcc gctcgctcct cctcctgcca gtgtcgcgcc ctaacttccc ccctccctcc 1080ctccctccct ccctccctct ctcctgccac cgcccctctc tccgcgctgc gtgcggtgct 1140gccctggacc aatggcatgc tgctgcacgc tcggcggatg acgcaagccg cttcgcaatt 1200tccggatcag atctcggcgg ggcgtgcgcc gcggggtcac tgcggacctg ccgcggcccc 1260tgcttctttc acatccatca tgtcctccaa acctccgcct cctccacgca cgtacgcacg 1320cccgctcgca cgcgcgcact gccgctgcga aagcaagcgc ccgcccgccg cccggcgacg 1380ggaaggcggc cgcggtctcc ctccgcggtt gcctcgctcc cgcgcggggc tgggcgggca 1440gcagaaggcg ggtggcggcg gcggcttccg tcttcgtcag cggcctacgt cggcggcggc 1500gcgcgagact acgcatgccc ttgcgtcatg cgctcgcagg tagccgccgc gggcctagcg 1560tttccgctgg cgccgcgcct

aagcccccgg cgcgcacggt attgccgcga taccgtacgg 1620ccaagaccgc cgcagacgtc ggccctctcg cggccagcca gccagcagcg cagcggagga 1680agagcgcgca ggcgcggcgg gagggcggcc gcggagcagc gcagagcggg gcggagcagc 1740gcggagcaga acgggcagac tcggagcggg cagggcgggc agagctttgg ggtttaagga 1800ccgggttacc ggcgaagtga gcggctgcgg ggagcggctg tgggaggggt gagtacgcaa 1860gcacgatgcg agcgagagag agacgctgcc gcgaatcaag aaggtaggcg cgctgcgagg 1920cgcggcggcg gagcggagcg agggagaggg agagggagag agagggaggg agacgtcgcc 1980gcggcggggc ctggcctggc ctggtttggc ttggtcagcg cggccttgtc cgagcgtgca 2040gctggagttg ggtggattca tttggatttt cttttgtttt tgtttttctc tctttcccgg 2100aaagtgttgg ccggncggtg ttctttgttt tgatttcttc aaaagttttg gtggttggtt 2160ctctctcttg gctctctgtc aggcggtccg gtccacgccc cggcctctcc tctcctctcc 2220tctcctctcc tctccgtgcg tatacgtacg tacgtttgta tacgtacata catcccgccc 2280gccgtgccgg cgagggtttg ctcagcctgg agcaatgcga tgcgatgcga tgcgatgcga 2340cgcgacgcga cgcgagtcac tggttcgcgc tgtggctgtg gcttgcttgc ttacttgctt 2400tcgagctctc ccgctttctt ctttccttct cacgccacca ccaacgaaag aagatcggcc 2460ccggcacgcc gctgagaagg gctggcggcg atgacggcac gcgcgcccgc tgccacgttg 2520gcgctcgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc tgctgcttct 2580gcgcgcaggc tttgccacga ggccggcgtg ctggccgctg ccgcttccag tccgcgtgga 2640gagatcgaat gagagataaa ctggatggat tcatcgaggg atgaatgaac gatggttgga 2700tgcctttttc ctttttcagg tccacagcgg gaagcaggag cgcgtgaatc tgccgccatc 2760cgcatacgtc tgcatcgcat cgcatcgcat gcacgcatcg ctcgccggga gccacagacg 2820ggcgacaggg cggccagcca gccaggcagc cagccaggca ggcaccagag ggccagagag 2880cgcgcctcac gcacgcgccg cagtgcgcgc atcgctcgca gtgcagacct tgattccccg 2940cgcggatctc cgcgagcccg aaacgaagag cgccgtacgg gcccatccta gcgtcgcctc 3000gcaccgcatc gcatcgcatc gcgttcccta gagagtagta ctcgacgaag gcaccatttc 3060cgcgctcctc ttcggcgcga tcgaggcccc cggcgccgcg acgatcgcgg cggccgcggc 3120gctggcggcg gccctggcgc tcgcgctggc ggccgccgcg ggcgtctggc cctggcgcgc 3180gcgggcgccg caggaggagc ggcagcggct gctcgccgcc agagaagagc gcgccgggcc 3240cggggaggga cggggaggag aaggagaagg cgcgcaaggc ggccccgaaa gagaagaccc 3300tggacttgaa cgcgaagaag aagaagaagg agaagaagtt gaagaagaag aagaagaagg 3360agaggaagtt gaagaagacg aggagcaggc gcgttccaag gcgcgttctc ttccggaggc 3420gcgttccagc tgcggcggcg gggcgggctg cggggcgggc gcgggcgcgg gtgcgggcag 3480aggggacgcg cgcgcggagg cggagggggc cgagcgggag cccctgctgc tgcggggcgc 3540ccgggccgca ggtgtggcgc gcgcgacgac ggaggcgacg acgccagcgg ccgcgacgac 3600aaggccggcg gcgtcggcgg gcggaaggcc ccgcgcggag caggggcggg agcaggacaa 3660ggcgcaggag caggagcagg gccgggagcg ggagcgggag cgggcggcgg agcccgaggc 3720agaacccaat cgagatccag agcgagcaga ggccggccgc gagcccgagc ccgcgccgca 3780gatcactagt accgctgcgg aatcacagca gcagcagcag cagcagcagc agcagcagca 3840gcagcagcag ccacgagagg gagataaaga aaaagcggca gagacg 388638192DNASchizochytrium sp. 38tcatgaagcc ggttgctccg aagttctacg cgcgtctcaa cattgacgag caggacgaga 60cccgtgatcc gatcctcaac aaggacaacg cgccgtcttc cagctctagc tcctcttcca 120gctcttccag ctcttccagc ccgtcgccag ctccgtccgc cccagtgcaa aagaaggctg 180ctccggccgc gg 1923930DNAArtificialoligonucleotide primer 39cggggtaccc gggagccgcc ttggctttgt 304035DNAArtificialoligonucleotide primer 40aaactgcagc ccgggtccag ctggcaggca ccctg 3541237PRTNostoc sp. 41Leu Leu Gln His Thr Trp Leu Pro Lys Pro Pro Asn Leu Thr Leu Leu1 5 10 15Ser Asp Glu Val His Leu Trp Arg Ile Pro Leu Asp Gln Pro Glu Ser 20 25 30Gln Leu Gln Asp Leu Ala Ala Thr Leu Ser Ser Asp Glu Leu Ala Arg 35 40 45Ala Asn Arg Phe Tyr Phe Pro Glu His Arg Arg Arg Phe Thr Ala Gly 50 55 60Arg Gly Ile Leu Arg Ser Ile Leu Gly Gly Tyr Leu Gly Val Glu Pro65 70 75 80Gly Gln Val Lys Phe Asp Tyr Glu Ser Arg Gly Lys Pro Ile Leu Gly 85 90 95Asp Arg Phe Ala Glu Ser Gly Leu Leu Phe Asn Leu Ser His Ser Gln 100 105 110Asn Leu Ala Leu Cys Ala Val Asn Tyr Thr Arg Gln Ile Gly Ile Asp 115 120 125Leu Glu Tyr Leu Arg Pro Thr Ser Asp Leu Glu Ser Leu Ala Lys Arg 130 135 140Phe Phe Leu Pro Arg Glu Tyr Glu Leu Leu Arg Ser Leu Pro Asp Glu145 150 155 160Gln Lys Gln Lys Ile Phe Phe Arg Tyr Trp Thr Cys Lys Glu Ala Tyr 165 170 175Leu Lys Ala Thr Gly Asp Gly Ile Ala Lys Leu Glu Glu Ile Glu Ile 180 185 190Ala Leu Thr Pro Thr Glu Pro Ala Lys Leu Gln Thr Ala Pro Ala Trp 195 200 205Ser Leu Leu Glu Leu Val Pro Asp Asp Asn Cys Val Ala Ala Val Ala 210 215 220Val Ala Gly Phe Gly Trp Gln Pro Lys Phe Trp His Tyr225 230 2354235PRTBrassica napus 42Met Leu Lys Leu Ser Cys Asn Val Thr Asn His Leu His Thr Phe Ser1 5 10 15Phe Phe Ser Asp Ser Ser Leu Phe Ile Pro Val Asn Arg Arg Thr Leu 20 25 30Ala Val Ser 35438730DNAArtificialsynthetic DNA 43atggctgcta ggttgcaaga acaaaaaggt ggtgagatgg atactagaat tgctatcatt 60ggaatgtctg ctattttgcc atgtggtact actgttagag aatcttggga aactattaga 120gctggtattg attgtttgtc tgatttgcct gaagatagag ttgatgttac tgcttacttt 180gatccagtta aaactactaa agataaaatc tattgtaaga gaggtggttt cattccagaa 240tatgattttg atgctagaga atttggtttg aatatgtttc agatggaaga ttctgatgct 300aatcaaacta tttctttgtt gaaagttaaa gaagcattgc aagatgctgg catcgatgct 360ttgggtaaag agaagaagaa tattggttgt gttttgggta ttggtggtgg tcaaaaatct 420tctcatgaat tttactcaag attgaattat gttgttgttg agaaggtatt gagaaaaatg 480ggtatgccag aagaagatgt taaagttgct gttgaaaaat acaaagctaa ttttccagag 540tggagattgg attcttttcc aggtttcttg ggaaatgtta ctgcaggaag atgtactaat 600acttttaatc ttgatggcat gaattgtgtt gttgatgctg cttgtgcttc ttctttgatt 660gctgttaaag ttgctattga tgaattgttg tacggtgatt gtgatatgat ggttactggt 720gctacttgta ctgataattc tattggaatg tacatggctt tttctaaaac tccagttttc 780tctactgatc catctgttag agcttatgat gaaaaaacta aaggaatgtt gattggtgaa 840ggttctgcta tgttggtttt gaaaagatat gctgatgctg ttagagatgg tgatgaaatt 900catgctgtta ttagaggttg tgcttcttct tctgatggta aagctgctgg tatctatact 960ccaactattt ctggtcaaga agaagcattg agaagagctt ataatagagc ttgtgttgat 1020ccagctactg ttactttggt tgaaggtcat ggtactggta ctccagttgg tgatagaatt 1080gaattgactg ctttgagaaa tttgtttgat aaagcatatg gtgaaggtaa tactgaaaaa 1140gttgctgttg gttctattaa atcttctatt ggtcatttga aagctgttgc tggtttggct 1200ggaatgatta aagttatcat ggctttgaaa cataaaactt tgccaggaac tattaatgtt 1260gataatccac caaacttgta cgataatact ccaattaacg aatcttcttt gtacattaat 1320actatgaata gaccttggtt tccaccacca ggtgttccaa gaagagctgg tatttcttct 1380tttggttttg gtggtgctaa ttatcatgct gttttggaag aagctgaacc agaacatact 1440actgcttata ggttgaacaa aagaccacaa ccagttttga tgatggctgc tactccagct 1500gctttgcaat ctttgtgtga agctcaattg aaagaatttg aagctgctat taaagaaaac 1560gaaactgtta aaaatactgc ttatattaaa tgtgttaaat ttggtgaaca attcaaattc 1620cctggtagta ttccagctac taatgctagg ttgggtttct tggttaaaga tgctgaagat 1680gcttgttcta ctttgagagc tatttgtgct caatttgcta aagatgttac taaagaagca 1740tggagattgc caagagaagg tgtttctttt agagctaaag gtattgctac taatggtgct 1800gttgctgctt tgttttctgg tcaaggtgct caatatactc atatgttttc tgaagttgct 1860atgaattggc cacaattcag acaatctatt gctgctatgg atgctgctca atctaaagtt 1920gctggttctg ataaagattt tgaaagagtt tctcaagttt tgtatccaag aaaaccatac 1980gagagagaac cagagcaaga tcataagaag atttctttga ctgcttattc tcaaccatct 2040actttggctt gtgctttggg tgcttttgaa atttttaaag aagctggttt tactccagat 2100tttgctgctg gtcattcttt gggtgaattt gctgctttgt acgctgctgg ttgtgttgat 2160agagatgaat tgtttgaatt ggtttgtaga agagctagaa ttatgggtgg taaagatgct 2220ccagctactc caaaaggttg catggctgct gttattggtc caaatgctga aaatattaaa 2280gttcaagctg ctaatgtttg gttaggaaat tctaattctc catctcaaac tgttattact 2340ggttctgttg aaggtattca agctgaatct gctaggttgc aaaaagaagg ttttagagtt 2400gttccattgg cttgtgaatc tgcttttcat tctccacaga tggaaaatgc ttcttctgct 2460tttaaagatg ttatctctaa agtttctttt agaactccaa aagctgaaac taaattgttt 2520tctaatgttt ctggtgaaac ttatccaact gatgctagag aaatgttgac tcaacatatg 2580acttcttctg ttaaattttt gactcaagtt agaaatatgc atcaagctgg tgctagaatt 2640tttgttgaat tcggtccaaa acaagttttg tctaaattgg tttctgaaac tttgaaagat 2700gatccatctg ttgttactgt ttctgttaat ccagcttctg gtactgattc tgatattcaa 2760ttgagagatg ctgctgttca attggttgtt gctggtgtta atttgcaagg ttttgataaa 2820tgggatgctc cagatgctac tagaatgcaa gctattaaaa aaaaaagaac tactttgaga 2880ttgtctgctg ctacttatgt ttctgataaa actaagaaag ttagagatgc tgctatgaat 2940gatggtagat gtgttactta cttgaaaggt gctgctccat tgattaaagc tccagaacca 3000gttgttgatg aagctgctaa aagagaagct gaaagattgc aaaaagaatt gcaagatgct 3060caaagacaat tggatgatgc taaaagagct gctgctgaag ctaattctaa attggctgct 3120gctaaagaag aagctaaaac tgctgctgct tctgctaaac cagctgttga tactgctgtt 3180gttgaaaaac atagagctat tttgaaatct atgttggctg aattggatgg ttatggttct 3240gttgatgctt cttctttgca acaacaacaa caacaacaaa ctgctccagc tccagttaaa 3300gctgctgctc cagctgctcc agttgcttct gctccagcac ccgcagttag caacgaactc 3360ttagaaaaag ccgagacagt agtgatggaa gttcttgcag ctaaaacggg gtacgaaaca 3420gatatgattg aagcagatat ggaacttgaa actgaactgg gcattgattc gattaaacgc 3480gtggaaattc tgtcagaagt gcaagctatg ttaaatgttg aagcgaaaga tgttgatgca 3540ctgtcacgca cacgcaccgt gggcgaagta gtgaacgcca tgaaagcaga aattgcaggc 3600tcctcagcac ccgcgccggc cgcagcagca ccagcccccg caaaagccgc ccccgcagcg 3660gcggctccag ccgtttcaaa cgaattactc gaaaaagcag aaaccgtagt gatggaagtc 3720cttgccgcca aaacgggtta tgagaccgat atgatcgaaa gcgatatgga attagaaacc 3780gaattaggga ttgatagtat taaacgcgta gaaattctgt ccgaagtaca agctatgctg 3840aatgtagaag caaaagatgt agatgcgtta agccgcacac gcactgttgg tgaagttgtg 3900aatgctatga aagctgaaat tgcaggaggt tcagcaccgg ccccagcagc cgcagcccca 3960ggtccagcag cagccgcacc ggcccccgcc gccgccgcac cggcagtatc aaacgagttg 4020ttagagaaag cggaaaccgt tgtgatggaa gtacttgccg cgaagacagg ttacgagacc 4080gatatgatcg aaagtgacat ggaattagaa accgaattgg gcattgatag cattaaacgc 4140gtagaaattt tatccgaagt tcaagccatg ttaaatgttg aagccaaaga tgtggatgcg 4200ttatcccgca cgcgtaccgt cggagaagta gtggacgcta tgaaagcaga gattgcagga 4260ggaagtgcac cggctccagc agcagcagca cccgccccag cggcagcggc gccggcaccg 4320gccgctccgg ccccagccgt tagttcagaa ctcctcgaaa aagcagaaac tgttgtcatg 4380gaagtattag ctgcaaaaac aggttacgag acggatatga ttgaaagcga tatggaatta 4440gaaaccgaat taggcattga ttcaattaaa cgtgttgaaa tcttaagtga agtccaagcc 4500atgcttaatg ttgaagccaa agatgtagat gcattatctc gcacgcgtac agtgggtgaa 4560gttgtcgatg cgatgaaagc agaaatcgcg ggaggatcag cgccagcccc ggcagcagca 4620gcccccgcgc ccgccgcggc cgcacctgcg ccggccgccc cagcccctgc agcaccggcc 4680ccagcagtgt cgtcggaatt actcgaaaaa gctgaaacgg tcgttatgga agtacttgct 4740gcaaagacgg gctatgaaac ggatatgatt gaatcggata tggaattaga aacagaactt 4800ggtattgact ctattaaacg cgtggaaatt ctgagcgaag tacaggcaat gttaaacgta 4860gaagccaaag atgtagacgc tttgtcacgc acacggacgg taggagaagt tgtggatgcg 4920atgaaagctg aaattgccgg ttcaagtgct agcgcccctg ctgccgccgc ccctgcccct 4980gccgccgcag caccggcccc ggcagccgca gctccagcag ttagtaacga attactcgaa 5040aaagcagaaa cggtggtcat ggaagtgtta gcagcaaaaa ctggatatga aacggacatg 5100attgaaagcg atatggaatt agaaacagaa ctgggaattg atagtattaa acgtgttgag 5160attttatctg aggttcaagc tatgctgaat gttgaagcga aagatgtaga cgcactgtct 5220cggacccgca cagtaggtga agtggtggac gcgatgaaag cagaaatcgc aggtggaagt 5280gctccggccc cggcggcagc cgcacccgcg cccgcggccg cagccccagc agttagcaac 5340gaattactcg agaaagcaga aactgtagtg atggaagtgt tagccgcaaa aacgggttat 5400gaaacggata tgattgaaag cgatatggaa ctggaaaccg aactgggcat tgattctatt 5460aaacgtgtcg aaatcttatc ggaagtccaa gcaatgctga acgtagaggc aaaggatgtt 5520gatgccctgt cacgtacccg taccgtaggt gaagttgtag atgccatgaa agctgaaatc 5580gcaggcagta gcgccccggc accagccgcc gccgcccccg cgccggcagc cgccgcaccc 5640gcgccagccg cagctgctcc agctgtatct agtgagctgc tcgaaaaagc agaaaccgtg 5700gttatggaag tgctcgccgc taaaacagga tatgaaaccg atatgattga aagcgatatg 5760gaattagaaa ccgaactggg tattgatagt attaagcgtg ttgaaatttt gtcagaagtt 5820caagctatgt tgaatgtaga agccaaagat gtagacgctt taagtcggac gcgtactgtt 5880ggagaagtcg tagacgccat gaaagcagag attgcaggcg gaagtgcacc ggccccggca 5940gcagcagccc cagcaccagc ggccgccgct cctgcagtgt caaacgaact tctggaaaaa 6000gctgaaaccg tcgtcatgga agtgctggct gcaaaaactg gatatgaaac agacatgatt 6060gaatcagata tggaactcga aaccgaactg gggattgata gcattaaacg tgtggaaatt 6120ttatcggagg tacaagcaat gttaaatgtg gaagcaaaag atgtggatgc actgagccgt 6180actcgtactg ttggtgaggt cgtggatgcg atgaaagcag aaattgctgg agggagtgcg 6240cctgccccgg ccgccgccgc acccgcgtct gccggtgctg cccccgctgt caaaattgat 6300tctgttcatg gtgctgattg tgatgatttg tctttgatgc atgctaaagt tgttgatatt 6360agaagaccag atgaattgat tttggaaaga ccagaaaata gaccagtttt ggttgttgat 6420gatggttctg aattgacttt ggctttggtt agagttttgg gtgcttgtgc tgttgttttg 6480acttttgaag gtttgcaatt ggctcaaaga gctggtgctg ctgctattag acatgttttg 6540gctaaagatt tgtctgctga atctgctgaa aaagctatta aagaagctga acaaagattt 6600ggtgctttgg gtggttttat ctctcaacaa gctgaaagat ttgaaccagc tgaaattttg 6660ggttttactt tgatgtgtgc taaatttgct aaagcatctt tgtgcactgc tgttgctggt 6720ggtagaccag ctttcattgg tgttgctagg ttggatggta ggttgggttt tacttctcaa 6780ggaacttctg atgctttgaa aagagctcaa agaggtgcta tttttggttt gtgcaagact 6840attggtttgg aatggtctga atctgatgtt ttctcaagag gtgttgatat tgctcaaggt 6900atgcatccag aagatgctgc tgttgctatt gttagagaaa tggcttgtgc tgatattaga 6960attagagaag ttggtattgg tgctaatcaa caaagatgta ctattagagc tgctaaattg 7020gaaactggaa atccacaaag acaaattgct aaagatgatg ttttgttggt ttctggtggt 7080gctagaggaa ttactccatt gtgcattaga gaaattacta gacaaattgc tggtggaaag 7140tatattttgt tgggtaggtc taaagtttct gcttctgaac cagcttggtg tgctggtatt 7200actgatgaaa aagctgttca aaaagctgct actcaagaat tgaaaagagc tttttctgct 7260ggtgaaggtc caaaaccaac tccaagagct gttactaaat tggttggttc tgttttgggt 7320gctagagaag ttaggtcttc tattgctgct attgaagcat tgggtggaaa agctatctat 7380tcttcttgtg atgttaattc tgctgctgat gttgctaaag ctgttagaga tgctgaatct 7440caattgggtg ctagagtttc tggtattgtt catgcttctg gtgttttgag agataggttg 7500attgaaaaaa aattgccaga tgaatttgat gctgtttttg gtactaaagt tactggtttg 7560gaaaatttgt tggctgctgt tgatagagct aatttgaaac atatggtttt gttttcttct 7620ttggctggtt ttcatggtaa tgttggtcaa tctgattatg ctatggctaa cgaagcattg 7680aacaaaatgg gtttggaatt ggctaaagat gtttctgtta aatctatttg ttttggtcct 7740tgggatggtg gtatggttac tccacaattg aaaaaacaat ttcaagaaat gggtgttcaa 7800attattccaa gagaaggtgg tgctgatact gttgctagaa ttgttttggg ttcttctcca 7860gctgaaattt tggttggtaa ttggagaact ccatctaaaa aagttggttc tgatactatt 7920actttgcaca gaaaaatttc tgctaaatct aatccatttt tggaagatca tgtcattcaa 7980ggtagaagag ttttgccaat gactttggct attggttctt tggctgaaac ttgtttgggt 8040ttgtttcctg gatattcttt gtgggctatt gatgatgctc aattgtttaa aggtgttact 8100gttgatggtg atgttaattg tgaagttact ttgactccat ctactgctcc ttctggtaga 8160gttaatgttc aagctacttt gaaaactttt tcttctggta aattggttcc agcttataga 8220gctgttattg ttttgtctaa tcaaggtgct ccaccagcta atgctactat gcaaccacca 8280tctttggatg ctgatccagc tttgcaaggt tctgtttatg atggaaagac tttgtttcat 8340ggtccagctt ttagaggtat tgatgatgtt ttgtcttgta ctaaatctca attggttgct 8400aaatgttctg ctgttccagg ttctgatgct gctagaggtg aatttgctac tgatactgat 8460gctcatgatc catttgttaa tgatttggct tttcaagcta tgttggtttg ggttagaaga 8520actttgggtc aagctgcttt gccaaattct attcaaagaa ttgttcaaca cagaccagtt 8580ccacaagata aaccatttta tattactttg agatctaatc aatctggtgg tcattctcaa 8640cataaacatg ctttgcaatt tcataacgaa caaggtgatt tgttcattga tgttcaagca 8700tctgttattg ctactgattc tttggctttt 8730446177DNAArtificialSynthetic DNA 44atggctgcta gaaatgtttc tgctgctcat gaaatgcatg atgaaaaaag aattgctgtt 60gttggtatgg ctgttcaata tgctggttgt aagactaaag atgaattttg ggaagttttg 120atgaatggta aagttgaatc taaagttatc tctgataaaa gattgggttc taattaccga 180gctgaacatt acaaggctga aagatccaaa tacgctgata ctttttgtaa cgaaacttat 240ggtactttgg atgaaaacga aattgataac gaacatgaat tgttgttgaa tttggctaaa 300caagcattgg ctgaaacttc tgttaaagat tctactagat gtggtattgt ttctggttgt 360ttgtcttttc ctatggataa tttgcaaggt gaattgttga atgtctatca aaatcatgtt 420gagaagaaat tgggtgctag agtttttaaa gatgcttctc attggtctga aagagaacaa 480tctaacaaac cagaagctgg tgatagaaga attttcatgg acccagcttc ttttgttgct 540gaagaattga atttgggtgc tttgcattat tctgttgatg ctgcttgtgc tactgcttta 600tacgttttga gattggctca agatcatttg gtttctggtg ctgctgatgt tatgttgtgt 660ggtgctactt gtttgccaga accattcttt atcttgtctg gtttttctac ttttcaagct 720atgccagttg gtactggtca aaatgtttct atgccattgc ataaagattc tcaaggtttg 780actccaggtg aaggtggttc tatcatggtt ttgaaaagat tggatgatgc tattagagat 840ggtgatcata tctatggtac tttgttgggt gctaatgttt ctaattctgg cactggtttg 900ccattgaaac cattgttgcc atctgaaaaa aaatgtttga tggatactta tactagaatt 960aatgttcatc cacataaaat tcaatatgtt gaatgtcatg ctactggtac tccacaaggt 1020gatagggttg aaattgatgc tgttaaagca tgttttgaag gaaaagttcc aagatttggt 1080actactaaag gaaactttgg tcatactttg gttgctgctg gttttgctgg aatgtgcaaa 1140gttttgttgt ctatgaaaca tggtatcatt ccaccaactc caggtattga tgatgaaact 1200aagatggacc cattggttgt ttctggtgaa gctattcctt ggccagaaac taatggtgaa 1260ccaaaaagag ctggtttgtc tgcttttggt tttggtggta ctaatgctca tgctgttttt 1320gaagaacatg atccatctaa tgctgcttgt actggtcatg attctatttc tgctttgtct 1380gctagatgtg gtggtgaatc taatatgaga attgctatta ctggtatgga tgctactttt 1440ggtgctttga aaggtttgga tgcttttgaa agagccatct acactggtgc tcatggtgct 1500attccattgc cagaaaagag atggagattt ttgggcaaag ataaagattt cttggatttg 1560tgtggtgtta aagctactcc acatggttgt tatattgaag atgttgaagt tgattttcaa 1620agattgagaa ctccaatgac tccagaagat atgttgttgc cacaacaatt gttggctgtt 1680actactattg atagagctat tttggattct ggtatgaaaa aaggtggtaa tgttgctgtt

1740tttgttggtt tgggtaccga tttggaattg tacagacata gagctagagt tgctttgaaa 1800gaaagagtta gaccagaagc atctaaaaaa ttgaatgata tgatgcagta cattaatgat 1860tgtggcacct ctacttctta tacttcttat attggtaatt tggttgctac tagagtttct 1920tctcaatggg gttttactgg tccatctttt actattactg aagggaataa ctctgtttat 1980agatgtgctg aattgggaaa gtatttgttg gaaactggtg aagttgatgg tgttgttgtt 2040gctggtgttg atttgtgtgg ttctgctgaa aacttatacg ttaaatcaag aagattcaaa 2100gtttctactt ctgatactcc aagagcttct tttgatgctg ctgctgatgg ttactttgtt 2160ggtgaaggtt gtggtgcttt tgttttgaaa agagaaactt cttgtactaa agatgataga 2220atctatgctt gcatggatgc tattgttcca ggtaatgttc catctgcttg tttgagagaa 2280gcattggatc aagctagagt taaaccaggt gatattgaaa tgttggaatt gtctgctgat 2340tctgctagac atttgaaaga tccatctgtt ttgccaaaag aattgactgc tgaagaagaa 2400attggtggtt tgcaaactat tttgagagat gatgataaat tgccaagaaa tgttgctact 2460ggttctgtta aagctactgt tggtgatact ggttatgctt ctggtgctgc ttctttgatt 2520aaagctgctt tgtgcatcta taataggtat ttgccatcta atggtgatga ttgggatgaa 2580ccagctccag aagctccttg ggattctact ttgtttgctt gtcaaacttc aagagcttgg 2640ttgaaaaatc ctggagagag aagatatgct gctgtttctg gtgtttctga aactaggtct 2700tgttattctg ttttgttgtc tgaagctgaa ggtcattatg aaagagaaaa tagaatttct 2760ttggatgaag aagctccaaa attgattgtt ttgagagctg attctcatga agaaattttg 2820ggtaggttgg ataaaattag agaaagattt ttgcaaccaa ctggtgctgc tccaagagaa 2880tctgaattga aagctcaagc tagaagaatt ttcttggaat tgttgggtga aactttggct 2940caagatgctg cttcttctgg ttctcaaaaa ccattggctt tgtctttggt ttctactcca 3000tctaaattgc aaagagaagt tgaattggct gctaaaggta ttccaagatg tttgaaaatg 3060agaagagatt ggtcttctcc agctggttca agatatgctc cagaaccatt ggcttctgat 3120agagttgctt tcatgtacgg tgaaggaagg tctccatact atggaatcac tcaagatatt 3180catagaattt ggccagaatt gcatgaagtt attaacgaaa aaactaatag gttgtgggct 3240gaaggtgata gatgggttat gccaagagct tcttttaaat ctgaattgga atctcaacaa 3300caagaatttg atagaaatat gattgaaatg tttaggttgg gtattttgac ttctattgct 3360tttactaatt tggctagaga tgttttgaat attactccaa aagctgcttt tggtttgtct 3420ttgggtgaaa tttctatgat ttttgctttt tctaaaaaaa atggtttgat ttctgatcaa 3480ttgactaaag atttgagaga atctgatgtt tggaacaaag cattggctgt tgaattcaat 3540gctttgagag aagcatgggg tattccacaa tctgttccaa aagatgaatt ttggcaaggt 3600tatattgtta gaggtactaa acaagatatt gaagctgcta ttgctccaga ttccaaatac 3660gttaggttga ctatcattaa tgatgctaat actgctttga tttctggtaa accagatgct 3720tgtaaagctg ctattgctag gttgggtggt aatattccag ctttgccagt tactcaagga 3780atgtgtggtc attgtccaga agttggtcca tatactaaag atattgctaa aattcatgct 3840aatttggaat ttccagttgt tgatggtttg gatttgtgga ctactattaa tcaaaaaaga 3900ttggttccaa gagctactgg tgctaaagat gaatgggctc catcttcttt tggtgaatat 3960gctggtcaac tttacgaaaa acaagctaat tttccacaaa ttgttgaaac tatctacaaa 4020caaaattatg atgtttttgt tgaggttggt ccaaacaacc ataggtctac tgctgttaga 4080actactttgg gtccacaaag aaatcatttg gctggtgcta ttgataaaca aaacgaagat 4140gcttggacta ctattgttaa attggttgct tctttgaaag ctcatttggt tccaggtgtt 4200actatttctc cattgtatca ttctaaattg gttgctgaag ctgaagcatg ttatgctgct 4260ctgtgcaaag gagaaaaacc taagaagaac aaatttgtta gaaaaattca attgaatggt 4320aggttcaatt ctaaagctga tccaatttct tctgctgatt tggcttcttt tccaccagct 4380gatccagcta ttgaagctgc tatttcttca agaattatga aaccagttgc tccaaaattt 4440tatgctaggt tgaatattga tgaacaagac gaaacaagag atccaatttt gaacaaagat 4500aatgctccat ctagttcatc tagttcctct tcatctagtt cttcatctag ttctccatct 4560ccagctcctt ctgctccagt tcaaaaaaaa gctgctccag ctgctgaaac taaagctgtt 4620gcttctgctg atgctttgag atctgctttg ttggatttgg attctatgtt ggctttgtct 4680tctgcttctg cttctggtaa tttggttgaa actgctccat ctgatgcttc tgttattgtt 4740ccaccatgta atattgctga tttgggttca agagctttta tgaaaactta tggtgtttct 4800gctccattgt acactggtgc tatggctaaa ggtattgctt ctgctgattt ggttattgct 4860gctggtagac aaggcatttt ggcttctttt ggtgctggtg gtttgccaat gcaagttgtt 4920agagaatcta ttgaaaaaat tcaagctgct ttgccaaatg gtccatatgc tgttaatttg 4980attcattctc catttgattc taatttggaa aaaggtaatg ttgatttgtt tttggaaaaa 5040ggtgttactt ttgttgaagc atctgctttt atgactttga ctccacaagt tgttaggtac 5100agagctgctg gtttgactag aaatgctgat ggttctgtta atattagaaa tagaattatc 5160ggaaaggttt caagaactga attggctgaa atgtttatga gacctgcccc agaacacttg 5220ttgcaaaaat tgattgcttc tggtgaaatt aatcaagaac aagctgaatt ggctagaaga 5280gttccagttg ctgatgatat tgctgttgaa gctgattctg gtggtcatac tgataataga 5340ccaattcatg ttatcttgcc attgattatt aatttgagag acagattgca tagagaatgt 5400ggttatccag ctaatttgag agttagagtt ggtgctggtg gtggtattgg ttgtccacaa 5460gctgctttgg ctacttttaa tatgggtgct tctttcattg ttactggcac tgttaatcaa 5520gttgctaaac aatctggtac ttgtgataat gttagaaaac aattggctaa agctacttat 5580tctgatgttt gcatggctcc agctgctgat atgtttgaag aaggtgttaa attgcaagtt 5640ttgaagaaag ggacaatgtt tccatcaaga gctaataagt tatacgaatt gttttgcaag 5700tatgattctt ttgaatctat gccaccagct gaattggcta gagttgaaaa aagaattttc 5760tcaagagctt tggaagaagt ttgggatgaa actaaaaatt tttacattaa taggttgcac 5820aatccagaaa aaattcaaag agctgaaaga gatccaaaat tgaaaatgtc tttgtgtttt 5880agatggtatt tgtctttggc ttcaagatgg gctaatactg gtgcttctga tagagttatg 5940gattatcaag tttggtgtgg tccagctatt ggttctttta atgatttcat taaaggcacc 6000tacttggacc cagctgttgc taacgaatat ccatgcgttg ttcaaattaa caaacaaatt 6060ttgagaggtg cttgtttcct cagaagattg gaaattttga gaaatgctag gttgtctgat 6120ggtgctgctg ctttggttgc ttctattgat gatacttatg ttccagctga aaaattg 6177

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 SEQ ID NO:3, and SEQ ID NO:5; b) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; d) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and e) a nucleic acid sequence encoding an amino aid sequence that is at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.

2-7. (canceled)

8. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a first nucleic acid sequence encoding a first amino acid sequence that has .beta.-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:2, and wherein said first amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2; b) a first nucleic acid sequence encoding a first amino acid sequence that has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:4, and wherein said first amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and c) a first nucleic acid sequence encoding a first amino acid sequence that has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:6, and wherein said first amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.

9-10. (canceled)

11. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence of SEQ ID NO:9; b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:10; and c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the amino acid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 93 of SEQ ID NO:10 and a histidine at a position corresponding to amino acid 94 of SEQ ID NO:10.

12-13. (canceled)

14. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence of SEQ ID NO:19; b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:20; and c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:20 or that is a fragment of SEQ ID NO:20, wherein the amino acid sequence has .beta.-keto acyl-ACP synthase (KS) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:20.

15-16. (canceled)

17. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence of SEQ ID NO:29; b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30; and c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the amino acid sequence has FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH) activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 426-440 of SEQ ID NO:30.

18-19. (canceled)

20. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 1, operatively linked to at least one transcription control sequence.

21. A recombinant cell transfected with the nucleic acid molecule of claim 1.

22. The recombinant cell of claim 21, wherein said recombinant cell is a microorganism.

23. The recombinant cell of claim 21, wherein said recombinant cell is a plant cell.

24. An isolated nucleic acid molecule consisting essentially of a nucleic acid sequence that is fully complementary to the nucleic acid molecule of claim 1.

25. A genetically modified microorganism that has been transformed with the nucleic acid molecule of claim 1.

26. A genetically modified plant or part of a plant that has been transformed with the nucleic acid molecule of claim 1.

27-28. (canceled)

29. The genetically modified microorganism of claim 25, wherein said microorganism endogenously expresses a PUFA PKS system.

30. The genetically modified microorganism of claim 25, wherein said microorganism has been further transformed with a recombinant nucleic acid molecule encoding a phosphopantetheine transferase.

31. The genetically modified microorganism of claim 25, wherein said microorganism is a Thraustochytriales microorganism.

32. The genetically modified microorganism of claim 25, wherein said microorganism is a bacterium.

33. The genetically modified microorganism of claim 25, wherein said microorganism is a yeast.

34. A method to produce a bioactive molecule, comprising culturing under conditions effective to produce said bioactive molecule a genetically modified organism according to claim 25.

35-36. (canceled)

37. The genetically modified plant or part of a plant of claim 26, wherein said plant has been further genetically modified to express a recombinant nucleic acid molecule encoding a phosphopantetheine transferase.

38-39. (canceled)

40. The genetically modified plant or part of a plant of claim 26, wherein said plant is selected from the group consisting of: canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco.

41. The genetically modified plant or part of a plant of claim 26, wherein the plant is an oilseed plant and wherein the part of the plant is a mature oilseed.

42. The genetically modified plant or part of a plant of claim 26, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one PUFA selected from the group consisting of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), and wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than about 10% of the total fatty acids produced by said plant.

43. The genetically modified plant or part of a plant of claim 42, wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than 5% by weight of the total fatty acids produced by said plant.

44. The genetically modified plant or part of a plant of claim 42, wherein the fatty acids consisting of gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds, comprise less than 5% by weight of the total fatty acids produced by said plant.

45. The genetically modified plant or part of a plant of claim 42, wherein gamma-linolenic acid (GLA; 18:3, n-6) comprises less than 1% by weight of the total fatty acids produced by said plant.

46. A plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises detectable amounts of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6).

47. The plant or a part of the plant of claim 46, wherein the ratio of DPAn-6 to DHA is less than 1:1.

48. The plant or a part of the plant of claim 46, wherein the total fatty acid profile in the plant or part of the plant contains less than 5% by weight in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

49. The plant or a part of the plant of claim 46, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

50. The plant or a part of the plant of claim 46, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

51. The plant or a part of the plant of claim 46, wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 2% of gamma-linolenic acid (GLA; 18:3, n-6) and dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6).

52. Seeds obtained from the plant or part of the plant of claim 46.

53. A food product comprising the seeds of claim 52.

54. An oil obtained from seeds of the plant of claim 46.

55. A food product comprising the oil of claim 54.

56. An oil blend comprising the oil of claim 54 and another oil.

57-59. (canceled)

60. The oil of claim 54, comprising the following fatty acids: DHA (C22:6n-3), DPAn-6 (C22:5n-6), oleic acid (C18:1), linolenic acid (C18:3), linoleic acid (C18:2), C16:0, C18.0, C20:0, C20:1n-9, C20:2n-6, C22:1n-9; wherein the oil comprises less than 0.5% of any of the following fatty acids: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

61. The plant of claim 26, wherein said plant produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds.

62. The plant of claim 61, where said plant produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 1% of gamma-linolenic acid (GLA; 18:3, n-6).

63. A method to produce a bioactive molecule, comprising growing under conditions effective to produce said bioactive molecule a genetically modified plant according to claim 26.

64-66. (canceled)

67. The plant or a part of the plant of claim 46, wherein the ratio of DPAn-6 to DHA is 1:1 or greater than 1:1.