Method Of Producing Microbial Oil Containing Fatty Acids Obtained From Stramenopile

Abstract

A method for producing a microbial oil includes the steps of: genetically modifying a labyrinthulid by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene; culturing the labyrinthulid, such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content; and collecting the microbial oil having the increased EPA content from the labyrinthulid. The increased EPA content is not less than 3.3% of a total fatty acid composition.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of copending application Ser. No. 16/208,047, filed Dec. 3, 2018, which is a Divisional of copending application Ser. No. 14/711,075, filed on May 13, 2015, which is a Divisional of application Ser. No. 13/877,225, filed on Aug. 1, 2013, and currently issued under U.S. Pat. No. 9,062,315, which is a 371 of PCT International Application No. PCT/JP2011/072650 filed on Sep. 30, 2011, which is based upon and claims the benefit of priority of the prior Japanese Patent Application No. 2011-179194, filed on Aug. 18, 2011, and Japanese Patent Application No. 2010-224225, filed on Oct. 1, 2010. The entire contents of each of the above documents are hereby incorporated by reference into the present application.

[0002] The sequence listing was submitted in the present application Ser. No. 17/526,464 in a computer readable form under the name of "P23609US03_replacement_sequence_listing.txt" and is hereby incorporated by reference into the present application. The electronic copy of the sequence listing in the computer readable form, the file size of which is 242 K bytes, was created on Jan. 29, 2016. The sequence listing submitted was also submitted in copending application Ser. No. 14/711,075 in a computer readable form under the name of "130412-revised_sequence_listing.txt" and is hereby incorporated by reference into the present application. The electronic copy of the sequence listing in the computer readable form, the file size of which is 242 K bytes, was created on Jan. 29, 2016.

TECHNICAL FIELD

[0003] The present invention relates to a method for transforming stramenopile whereby genes of stramenopile are disrupted and/or expression thereof is inhibited by genetic engineering. Particularly, the invention relates to a transformation method for disrupting genes associated with fatty acid biosynthesis and/or inhibiting expression thereof, a method for modifying the fatty acid composition of a stramenopile, a method for highly accumulating fatty acids in a stramenopile, a stramenopile having an enhanced unsaturated fatty acid content, a method for producing unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopile, a microbial oil comprising the fatty acid obtained from microorganisms belonging to stramenopile, especially from the class Labyrinthulomycetes, and a method of producing the microbial oil from the microorganisms, among others.

BACKGROUND ART

[0004] Polyunsaturated fatty acids (PUFA) represent an important component of animal and human nutrition. .omega.3 polyunsaturated fatty acids (also called n-3 polyunsaturated fatty acids) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have a wide range of roles in many aspects of health, including brain development in children, eye functions, syntheses of hormones and other signaling substances, and prevention of cardiovascular disease, cancer, and diabetes mellitus (Non-Patent Documents 1 and 2). These fatty acids therefore represent an important component of human nutrition. Accordingly, there is a need for polyunsaturated fatty acid production.

[0005] Meanwhile, microorganisms of the class Labyrinthulomycetes are known to produce polyunsaturated fatty acids. Concerning microorganisms of the family Thraustochytrium, there are reports of, for example, a polyunsaturated fatty acid-containing phospholipid producing method using Schizochytrium microorganisms (Patent Document 1), and Thraustochytrium microorganisms having a docosahexaenoic acid producing ability (Patent Document 2). For enhancement of food and/or feed by the unsaturated fatty acids, there is a strong demand for a simple economical process for producing these unsaturated fatty acids, particularly in the eukaryotic system.

[0006] With regard to the class Labyrinthulomycetes, there have been reported foreign gene introducing methods for specific strains of the genus Schizochytrium (the genus Auranthiochytrium (Non-Patent Document 4) in the current classification scheme (Non-Patent Document 3)) (Patent Documents 3 and 4). Further, a method that causes a change in fatty acid composition by means of transformation is known in which a polyketide synthase (PKS) gene is destroyed to change the resulting fatty acid composition (Non-Patent Document 5). However, there is no report directed to changing a fatty acid composition by manipulating the enzymes of the elongase/desaturase pathway. Under these circumstances, the present inventors found ways to change fatty acid compositions through introduction of elongase/desaturase genes into various species of Labyrinthulomycetes, and have filed a patent application therefor (Patent Document 5).

CITATION LIST

Patent Documents

[0007] Patent Document 1: JP-A-2007-143479 [0008] Patent Document 2: JP-A-2005-102680 [0009] Patent Document 3: JP-A-2006-304685 [0010] Patent Document 4: JP-A-2006-304686 [0011] Patent Document 5: WO2011/037207 [0012] Patent Document 6: WO1997/011094 [0013] Patent Document 7: US Patent Application US2005/0014231 [0014] Patent Document 8: JP-T-2007-532104 (the term "JP-T" as used herein means a published Japanese translation of a PCT patent application)

Non-Patent Documents

[0014] [0015] Non-Patent Document 1: Poulos A., Lipids, 30, 1-14(1995) [0016] Non-Patent Document 2: Horrocks L. A. and Yeo Y. K., Pharmacol Res., 40, 211-225 (1999) [0017] Non-Patent Document 3: Yokoyama R., Honda D., Mycoscience, 48, 199-211 (2007) [0018] Non-Patent Document 4: Lecture Summary for the 60th Conference of The Society for Biotechnology, Japan, p136 (2008) [0019] Non-Patent Document 5: Lippmeier J. C. et al., Lipids, 44(7), 621-630 (2009) [0020] Non-Patent Document 6: Tonon T. et al., FEBS Lett., 553, 440-444 (2003). [0021] Non-Patent Document 7: Thompson J. D. et al., Nucleic Acids Res., 22, 4673-4680 (1994) [0022] Non-Patent Document 8: Yazawa K., Lipids, 31, Supple. 297-300 (1996) [0023] Non-Patent Document 9: Jiang X. et al., Wei Sheng Wu Xue Bao., 48(2), 176-183 (2008) [0024] Non-Patent Document 10: PEREIRA S. L. et al., Biochem. J., 378, 665-671 (2004) [0025] Non-Patent Document 11: Prasher D. C. et al., Gene, 111(2), 229-233 (1992) [0026] Non-Patent Document 12: Chalfie M. et al., Science, 263, 802-805 (1994)

[0027] Non-Patent Document 13: Southern P. J., and Berg, P., J. Molec. Appl. Gen., 1, 327-339 (1982) [0028] Non-Patent Document 14: Saitou N. et al., Mol. Biol. Evol., 4, 406-425 (1987) [0029] Non-Patent Document 15: Ausubel F. M. et al., Current Protocols in Molecular Biology, Unit 13 (1994) [0030] Non-Patent Document 16: Guthrie C., Fink G. et al., Methods in Enzymology: Guide to Yeast Genetics and Molecular Biology, Volume 194 (1991) [0031] Non-Patent Document 17: Abe E., et al., J. Biochem, 142, 31561-31566 (2006) [0032] Non-Patent Document 18: Bio-Experiment Illustrated 2, Fundamentals of Gene Analysis, p117-128, Shujunsha, 1995 [0033] Non-Patent Document 19: Japan Society for Bioscience, Biotechnology, and Agrochemistry, 77, 2, 150-153 (2003) [0034] Non-Patent Document 20: Bio-Experiment Illustrated 2, Fundamentals of Gene Analysis, p63-68, Shujunsha, 1995 [0035] Non-Patent Document 21: Sanger, F. et al., Proc. Natl. Acad. Sci, 74, 5463 (1977) [0036] Non-Patent Document 22: Meyer, A., et al. J. Lipid Res., 45, 1899-1909 (2004) [0037] Non-Patent Document 23: Cigan and Donahue, 1987; Romanos et al., 1992 [0038] Non-Patent Document 24: Qiu, X., et al. J. Biol. Chem., 276, 31561-6 (2001) [0039] Non-Patent Document 25: DIG Application Manual [Japanese version] 8th, Roche Applied Science

SUMMARY OF THE INVENTION

Problems that the Invention is to Solve

[0040] The present invention is directed to improving the ability of a stramenopile to produce useful substances by way of transformation through disruption of stramenopile genes and/or inhibition of expression thereof by genetic engineering. By modifying the ability to produce useful substances through disruption of stramenopile genes associated with production of useful substances and/or inhibition of expression thereof by genetic engineering, the invention provides a modification method of a fatty acid composition produced by a stramenopile, a method for highly accumulating fatty acids in a stramenopile, an unsaturated fatty acid producing method, a stramenopile having an enhanced unsaturated fatty acid content, and production of unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopile. With the modification of a fatty acid composition produced by a stramenopile, and the method for highly accumulating fatty acids in a stramenopile, the present invention enables more efficient production of polyunsaturated fatty acids.

Means for Solving the Problems

[0041] The present inventors conducted intensive studies under the foregoing circumstances of the conventional techniques, and succeeded in transforming a stramenopile by way of disrupting stramenopile genes and/or inhibiting expression thereof by genetic engineering to greatly improve the ability of the stramenopile to produce an unsaturated fatty acid. The present inventors also found a method for modifying the fatty acid composition produced by a stramenopile through disruption of stramenopile genes or inhibition of expression thereof by genetic engineering, and a method for highly accumulating unsaturated fatty acids in the transformed stramenopile. The present invention was completed after further studies and development for practical applications.

[0042] The gist of the present invention includes the following stramenopile transformation methods (1) to (12). [0043] (1) A method for transforming stramenopile, the method including disrupting a stramenopile gene and/or inhibiting expression thereof by genetic engineering. In one embodiment, a method for producing a microbial oil includes the steps of: genetically modifying a labyrinthulid by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene; culturing the labyrinthulid, such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content; and collecting the microbial oil having the increased EPA content from the labyrinthulid. The increased EPA content is not less than 3.3% of a total fatty acid composition.

[0044] (2) The method according to (1), wherein the stramenopile belongs to the class Labyrinthulomycetes.

[0045] (3) The method according to (2), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

[0046] (4) The method according to (3), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.

[0047] (5) The method according to (4), wherein the microorganisms are Thraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364 (FERN BP-11298), Thraustochytrium roseum ATCC 28210, Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab (FERM BP-11421).

[0048] (6) The method according to any one of (1) to (5), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

[0049] (7) The method according to (6), wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

[0050] (8) The method according to (7), wherein the fatty acid chain elongase is a C20 elongase.

[0051] (9) The method according to (7), wherein the fatty acid desaturase is a 412 desaturase.

[0052] (10) The method according to any one of (1) to (9), wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

[0053] (11) The method according to any one of (1) to (10), wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

[0054] (12) The method according to any one of (1) to (11), further including introducing a gene associated with fatty acid desaturase.

[0055] (13) The method according to (12), wherein the gene associated with fatty acid desaturase is an .omega.3 desaturase.

[0056] Further, the gist of the present invention includes the following methods (14) to (26) for modifying the fatty acid composition of a stramenopile.

[0057] (14) A method for modifying the fatty acid composition of a stramenopile, the method including disrupting a stramenopile gene and/or inhibiting expression thereof by genetic engineering.

[0058] (15) The method according to (14), wherein the stramenopile belongs to the class Labyrinthulomycetes.

[0059] (16) The method according to (15), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

[0060] (17) The method according to (16), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.

[0061] (18) The method according to (17), wherein the microorganisms are Thraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364 (FERN BP-11298), Thraustochytrium roseum. ATCC 28210, Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab (FERM BP-11421).

[0062] (19) The method according to any one of (14) to (18), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

[0063] (20) The method according to (19), wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

[0064] (21) The method according to (20), wherein the fatty acid chain elongase is a C20 elongase.

[0065] (22) The method according to (21), wherein the fatty acid desaturase is a 412 desaturase.

[0066] (23) The method according to any one of (14) to (22), wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

[0067] (24) The method according to any one of (14) to (23), wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

[0068] (25) The method according to any one of (14) to (24), further including introducing a gene associated with fatty acid desaturase.

[0069] (26) The method according to (25), wherein the gene associated with fatty acid desaturase is an .omega.3 desaturase.

[0070] Further, the gist of the present invention includes the following methods (27) to (29) for highly accumulating fatty acids in a stramenopile.

[0071] (27) A method for highly accumulating a fatty acid in a stramenopile, wherein the method uses the method of any one of (14) to (26).

[0072] (28) The method according to (27), wherein the fatty acid is an unsaturated fatty acid.

[0073] (29) The method according to (28), wherein the unsaturated fatty acid is an unsaturated fatty acid of 18 to 22 carbon atoms.

[0074] Further, the gist of the present invention includes the following fatty acid (30).

[0075] (30) A fatty acid obtained from the stramenopile in which the fatty acid is highly accumulated by using the method of any one of (27) to (29).

[0076] Further, the gist of the present invention includes the following transformed stramenopiles (31) to (43).

[0077] (31) A stramenopile transformed for the modification of the fatty acid composition through disruption of its gene and/or inhibition of expression thereof by genetic engineering. In one embodiment, a labyrinthulid that has been genetically modified by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content. The increased EPA content is not less than 3.3% of a total fatty acid composition.

[0078] (32) The stramenopile according to (31), wherein the stramenopile belongs to the class Labyrinthulomycetes.

[0079] (33) The stramenopile according to (32), wherein the Labyrinthulomycetes are microorganisms belonging to the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium, Botryochytrium, Parietichytrium, or Sicyoidochytrium.

[0080] (34) The stramenopile according to (33), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.

[0081] (35) The stramenopile according to (34), wherein the microorganisms are Thraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364 (FERM BP-11298), Thraustochytrium roseum ATCC 28210, Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab (FERM BP-11421).

[0082] (36) The stramenopile according to any one of (31) to (35), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.

[0083] (37) The stramenopile according to (36), wherein the gene associated with fatty acid biosynthesis is a gene associated with polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase.

[0084] (38) The stramenopile according to (36), wherein the fatty acid chain elongase is a C20 elongase.

[0085] (39) The stramenopile according to (37), wherein the fatty acid desaturase is a 412 desaturase.

[0086] (40) The stramenopile according to any one of (31) to (39), wherein the method used to disrupt the stramenopile gene by genetic engineering is electroporation or a gene-gun technique introducing a loss-of-function gene or a DNA fragment from which a coding region of the gene is deleted.

[0087] (41) The stramenopile according to any one of (31) to (40), wherein the method used to inhibit expression of the stramenopile gene by genetic engineering is an antisense technique or RNA interference.

[0088] (42) The stramenopile according to any one of (31) to (41), further comprising introducing a gene associated with fatty acid desaturase is introduced.

[0089] (43) The stramenopile according to (42), wherein the gene associated with fatty acid desaturase is an .omega.3 desaturase.

Advantage of the Invention

[0090] The present invention improves the ability of a stramenopile to produce useful substances by way of transformation through disruption of stramenopile genes and/or inhibition of expression thereof by genetic engineering. By modifying the stramenopiles' ability to produce useful substances through disruption of stramenopile genes associated with production of useful substances and/or inhibition of expression thereof by genetic engineering, the invention provides a modification method of a fatty acid composition produced by a stramenopile, a method for highly accumulating fatty acids in a stramenopile, an unsaturated fatty acid producing method, a stramenopile having an enhanced unsaturated fatty acid content, and production of unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopile. With the modification of the fatty acid composition produced by a stramenopile, and the method for highly accumulating fatty acids in a stramenopile, the present invention enables more efficient production of polyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIG. 1 represents the result of RACE performed to amplify a T. aureum ATCC 34304-derived elongase gene in Example 2-2. [Brief Description of Reference Numerals] 1: 5'-RACE using a synthetic adapter-specific oligonucleotide and a denatured oligonucleotide elo-R; 2: 3'-RACE using a synthetic adapter-specific oligonucleotide and a denatured oligonucleotide elo-F; 3: 5'-RACE using only elo-R (negative control); 4: 3'-RACE using only elo-F (negative control); 5: 5'-RACE using only a synthetic adapter-specific oligonucleotide (negative control); 6: 3'-RACE using only a synthetic adapter-specific oligonucleotide (negative control).

[0092] FIG. 2 represents a molecular phylogenetic tree of T. aureum ATCC 34304-derived .DELTA.6/.DELTA.9 elongase and .DELTA.5/.DELTA.6 elongase (TaELO1 and TaELO2) of Example 2-3.

[0093] FIG. 3 represents the evaluation of transfectants with the introduced KONeor in Example 2-8. (A), an oligonucleotide primer set used for the evaluation of the transfectants by a PCR performed with template genomic DNA. [Brief Description of Reference Numerals] (1) Neor detection primers (SNeoF and SNeoR), (2) KO verification 1 (KO Pro F SmaI and KO Term R SmaI), (3) KO verification 2 (E2 KO ProF EcoRV and SNeoR), (4) KO verification 3 (SNeoF and E2 KO Term R EcoRV), (5) TaELO2 detection (E2 HindIII and E2 XbaI); (B), the result of agarose electrophoresis in the evaluation of the transfectants by a PCR performed with template genomic DNA. [Brief Description of Reference Numerals] 1, 5, 9, 13, 17: transfectants; 2, 6, 10, 14, 18: wild-type strains; 3, 7, 11, 15, 19: samples using KONeor as a template; 4, 8, 12, 16: no template. The numbers (1) to (5) above the lane numbers represent the oligonucleotide primer sets used.

[0094] FIG. 4 represents the result of confirming the copy numbers of TaELO2 by southern blotting in Example 2-9. [Brief Description of Reference Numerals] 1: genomic DNA (2.5 .mu.g), BamHI treatment; 2: BglII treatment; 3: EcoRI treatment; 4: EcoRV treatment; 5: HindIII treatment; 6: KpnI treatment; 7: SmaI treatment; 8: XbaI treatment; 9: positive control (a PCR product amplified with 1-ng E2 KO ProF EcoRV and E2 KO Term R EcoRV, containing TaELO2).

[0095] FIG. 5 represents the evaluation of TKONeor-introduced transfectants by southern blotting in Example 2-10. (A), a schematic view representing the southern blotting performed for the detection of a wild-type allele or a TKONeor-introduced mutant allele; (B), the result of southern blotting. [Brief Description of Reference Numerals] 1: T. aureum wild-type strain (2.5-.mu.g genomic DNA); 2, 3: TKONeor-introduced transfectants (2.5-.mu.g genomic DNA); 4: positive control (a PCR product amplified with 50-ng E2 KO ProF EcoRV and E2 KO Term R EcoRV, containing TaELO2).

[0096] FIG. 6 represents the PCR evaluation performed in Example 2-12 by using as a template the genomic DNA of the transfectant obtained by KOub600Hygr reintroduction. (A), the oligonucleotide primer set used. [Brief Description of Reference Numerals] (1) TaELO2 ORF detection (SNeoF and SNeoR), (2) KO verification (E2 KO Pro F EcoRV and ubi-hygro R); (B), the result of agarose electrophoresis in a PCR using the oligonucleotide primer set (1) for KO verification (arrows indicate transfectants for which amplification of a specific product was confirmed, and that were assumed to be TaELO2-deficient homozygotes); (C) the result of agarose electrophoresis in a PCR performed for the transfectants identified as TaELO2-deficient homozygotes using the oligonucleotide primer set (2) for TaELO2 ORF detection. [Brief Description of Reference Numerals] 1: sample using KOub600Hygr as a template; 2: wild-type strain.

[0097] FIG. 7 represents the southern blotting evaluation of the transfectants obtained by KOub600Hygr reintroduction in Example 2-12. (A), a schematic view representing the southern blotting performed for the detection of a wild-type allele, a KONeor-introduced mutant allele, and a KOub600Hygr-introduced mutant allele; (B), the result of southern blotting. [Brief Description of Reference Numerals] 1, 9: wild-type strains; 2-8 and 10-16: TaELO2-deficient homozygotes.

[0098] FIG. 8 represents the result of the southern blotting performed for the detection of TaELO2 in Example 2-12. [Brief Description of Reference Numerals] 1: wild-type strain; 2-5: T TaELO2-deficient homozygotes.

[0099] FIG. 9 represents the result of the RT-PCR agarose gel electrophoresis performed for the detection of TaELO2 mRNA in Example 2-12. [Brief Description of Reference Numerals] 1-4: TaELO2-deficient homozygotes; 5: wild-type strain; 6-9: TaELO2-deficient homozygotes, using total RNA as a template (negative control); 10: wild-type strain, using total RNA as a template (negative control); 11: sample using wild-type strain genomic DNA as a template (positive control).

[0100] FIG. 10 represents the result of the comparison of the fatty acid compositions of the wild-type strain and a TaELO2-deficient homozygote in Example 2-13.

[0101] FIG. 11 represents a plasmid containing the SV40 terminator sequence derived from a subcloned pcDNA 3.1 Myc-His vector.

[0102] FIG. 12 is a schematic view showing the primers used for fusion PCR, and the product. The end product is the fused sequence of a Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter and an artificial neomycin-resistant gene.

[0103] FIG. 13 represents a BglII cassette of the produced artificial neomycin-resistant gene.

[0104] FIG. 14 is a schematic view showing the primers used for fusion PCR, and the product. The end product is the fused sequence of a Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter and a pcDNA 3.1/Hygro-derived hygromycin-resistant gene.

[0105] FIG. 15 represents a BglII cassette of the produced pcDNA 3.1/Hygro-derived hygromycin-resistant gene.

[0106] FIG. 16 represents a plasmid containing a cloned Parietichytrium C20 elongase sequence.

[0107] FIG. 17 represents a plasmid with a BglII site inserted into the Parietichytrium C20 elongase sequence of the plasmid of FIG. 16.

[0108] FIG. 18 represents produced Parietichytrium C20 elongase gene targeting vectors (two vectors). The vectors have a neomycin-resistant gene (pRH85) or a hygromycin-resistant gene (pRH86) as a drug-resistance marker.

[0109] FIG. 19 is a schematic view representing the positions of the PCR primers used for the identification of the C20 elongase gene disrupted strain of Parietichytrium sarkarianum SEK364, and the expected products.

[0110] FIG. 20 represents the C20 elongase gene disruption evaluation performed by a PCR using the Parietichytrium sarkarianum SEK364 genomic DNA as a template. [Description of Reference Numerals]+/+: Parietichytrium sarkarianum SEK364 wild-type strain; +/-: Parietichytrium sarkarianum SEK364-derived C20 elongase gene first allele homologous recombinant; -/-: Parietichytrium sarkarianum SEK364-derived C20 elongase gene disrupted strain.

[0111] FIG. 21 represents the result of the comparison of the fatty acid compositions of the Parietichytrium sarkarianum SEK364 wild-type strain and the C20 elongase gene disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively. All values are given as mean value.+-.standard deviation.

[0112] FIG. 22 represents the proportions of the fatty acids of the C20 elongase gene disrupted strain relative to the Parietichytrium sarkarianum SEK364 wild-type strain taken as 100%.

[0113] FIG. 23 is a schematic view of the primers used for fusion PCR, and the product. The end product is the fused sequence of Thraustochytrium aureum ATCC 34304-derived 18S rDNA, Thraustochytrium aureum ATCC 34304-derived EF1.alpha. promoter, artificial neomycin-resistant gene, and Thraustochytrium aureum ATCC 34304-derived EF1.alpha. terminator.

[0114] FIG. 24 represents a plasmid obtained by partial cloning of the DNA fragment joined in FIG. 23. The plasmid contains a partial sequence on the 3'-end side of the EcoRI site of the Thraustochytrium aureum ATCC 34304-derived 18S rDNA, the Thraustochytrium aureum ATCC 34304-derived EF1.alpha. promoter, the artificial neomycin-resistant gene, and a partial sequence on the 5'-end side of the NcoI site of the Thraustochytrium aureum ATCC 34304-derived EF1.alpha. terminator.

[0115] FIG. 25 represents a produced Thraustochytrium aureum ATCC 34304 PKS pathway-associated gene orfA targeting vector. The vector has a neomycin-resistant gene as a drug-resistance marker.

[0116] FIG. 26 represents a plasmid containing the upstream sequence of Thraustochytrium aureum ATCC 34304 PKS pathway-associated gene orfA, a Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter, and a hygromycin-resistant gene.

[0117] FIG. 27 represents a produced Thraustochytrium aureum ATCC 34304 PKS pathway-associated gene orfA targeting vector. The vector has a hygromycin-resistant gene as a drug-resistance marker.

[0118] FIG. 28 is a schematic view representing the positions of the southern hybridization analysis probes used for the identification of the PKS pathway-associated gene orfA disrupted strain of Thraustochytrium aureum ATCC 34304, and the expected gene fragment sizes.

[0119] FIG. 29 represents the evaluation of PKS pathway-associated gene orfA disruption performed by southern hybridization using the Thraustochytrium aureum ATCC 34304 genomic DNA. [Description of Reference Numerals] T. au: Thraustochytrium aureum ATCC 34304 wild-type strain; +/-: Thraustochytrium aureum. ATCC 34304-derived PKS pathway-associated gene orfA first allele homologous recombinant; -/-: Thraustochytrium aureum ATCC 34304-derived PKS pathway-associated gene orfA disrupted strain.

[0120] FIG. 30 represents the result of the comparison of the fatty acid compositions of the Thraustochytrium aureum ATCC 34304 wild-type strain and the PKS pathway-associated gene orfA disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively. All values are given as mean value.+-.standard deviation.

[0121] FIG. 31 represents the proportions of the fatty acids of the PKS pathway-associated gene orfA disrupted strain relative to the Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

[0122] FIG. 32 is a schematic view representing the primers used for fusion PCR, and the product. The end product is the fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter and pTracer-CMV/Bsd/lacZ-derived blasticidin-resistant gene.

[0123] FIG. 33 represents a pTracer-CMV/Bsd/lacZ-derived blasticidin-resistant gene BglII cassette.

[0124] FIG. 34 is a schematic view representing the primers used for fusion PCR, and the product. The end product is the fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter and enhanced GFP gene (clontech).

[0125] FIG. 35 is a schematic view representing the primers used for fusion PCR, and the product. The end product is the fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter, enhanced GFP gene (clontech), and pcDNA3.1 Zeo(+)-derived zeocin-resistant gene.

[0126] FIG. 36 represents a produced enhanced GFP-zeocin-resistant fused gene BglII cassette.

[0127] FIG. 37 represents a plasmid containing a cloned Thraustochytrium aureum ATCC 34304 C20 elongase sequence and nearby sequences.

[0128] FIG. 38 represents a plasmid with the inserted BglII site after the complete deletion of the Thraustochytrium aureum ATCC 34304 C20 elongase sequence from the plasmid of FIG. 37.

[0129] FIG. 39 represents produced Thraustochytrium aureum ATCC 34304 C20 elongase gene targeting vectors (two vectors). The vectors have a blasticidin-resistant gene (pRH43) or an enhanced GFP-zeocin-resistant fused gene (pRH54) as a drug-resistance marker.

[0130] FIG. 40 is a schematic view representing the positions of the southern hybridization analysis probes used for the identification of the C20 elongase gene disrupted strain of the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disrupted strain, and the expected gene fragment sizes.

[0131] FIG. 41 represents the evaluation of C20 elongase gene disruption performed by southern hybridization using the Thraustochytrium aureum ATCC 34304 genomic DNA. [Description of Reference Numerals] T. au: Thraustochytrium aureum ATCC 34304 wild-type strain; -/-: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) and C20 elongase gene double disrupted strain.

[0132] FIG. 42 represents the result of the comparison of the fatty acid compositions of the Thraustochytrium aureum ATCC 34304 wild-type strain and the PKS pathway (orfA gene) and C20 elongase gene double disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively. All values are given as mean value.+-.standard deviation.

[0133] FIG. 43 represents the proportions of the fatty acids of the PKS pathway (orfA gene) and C20 elongase gene double disrupted strain relative to the Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

[0134] FIG. 44 is a schematic view representing the primers used for fusion PCR, and the product. The end product is the fused sequence of Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter, Saprolegnia diclina-derived? .omega.3 desaturase gene sequence, and Thraustochytrium aureum ATCC 34304-derived ubiquitin terminator.

[0135] FIG. 45 represents the plasmid containing a KpnI site replacing one of the BglII sites in the blasticidin-resistant gene BglII cassette of FIG. 33.

[0136] FIG. 46 represents a produced Saprolegnia diclina-derived .omega.3 desaturase gene expression plasmid. The plasmid has a blasticidin-resistant gene as a drug-resistance marker.

[0137] FIG. 47 is a schematic view representing the positions of the PCR primers used for the confirmation of the genome insertion of the Saprolegnia diclina-derived .omega.3 desaturase gene.

[0138] FIG. 48 represents the evaluation of the transfectant strain derived from the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disrupted strain. [Description of Reference Numerals] lanes 1 to 2: transfectants.

[0139] FIG. 49 represents the results of the comparison of the fatty acid compositions of the control Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disrupted strain and the .omega.3 desaturase gene introduced strain. Blank bar and solid bar indicate the fatty acid compositions of the control strain and the .omega.3 desaturase gene introduced strain, respectively. All values are given as mean value.+-.standard deviation.

[0140] FIG. 50 represents the proportions of the fatty acids of the .omega.3 desaturase gene introduced strain relative to the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disrupted strain taken as 100%.

[0141] FIG. 51 is a diagram representing a pRH59 cloning the sequence containing the Thraustochytrium aureum ATCC 34304-derived C20 elongase.

[0142] FIG. 52 is a diagram representing a pRH64 cloning the sequence containing a BglII site in the Thraustochytrium aureum ATCC 34304-derived C20 elongase.

[0143] FIG. 53 is a diagram representing a pRH65 containing aubiquitin promoter-, neomycin-resistant gene-, and SV40 terminator-containing sequence cloned into the Thraustochytrium aureum ATCC 34304-derived C20 elongase, and a pRH66 containing a ubiquitin promoter-, hygromycin-resistant gene-, and SV 40 terminator-containing sequence cloned into the Thraustochytrium aureum ATCC 34304-derived C20 elongase.

[0144] FIG. 54 represents the expected fragment sizes of the wild-type strain allele and knockout strains in a PCR.

[0145] FIG. 55 represents the detection results for the wild-type strain allele and knockout strains in a PCR.

[0146] FIG. 56 represents the fatty acid compositions of the wild-type strain and the C20 elongase knockout strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the strain, respectively.

[0147] FIG. 57 represents the result of the comparison of the fatty acid compositions of the wild-type strain and the knockout strain.

[0148] FIG. 58 represents a plasmid containing a sequence from 1,071 bp upstream of the .DELTA.4 desaturase gene to 1,500 bp within the .DELTA.4 desaturase gene of the cloned Thraustochytrium aureum ATCC 34304 strain.

[0149] FIG. 59 represents a plasmid containing a BglII site inserted into the deleted portion of the plasmid of FIG. 58 containing the 60 bp upstream of the .DELTA.4 desaturase gene and the 556-bp sequence containing the start codon within the .DELTA.4 desaturase gene (616 bp, SEQ ID NO: 205).

[0150] FIG. 60 represents produced Thraustochytrium aureum ATCC 34304 strain 44 desaturase gene targeting vectors (two vectors). The vectors have a blasticidin resistant gene (pTM6) or an enhanced GFP-zeocin-resistant fused gene (pTM8) as a drug-resistance marker.

[0151] FIG. 61 is a schematic view representing the positions of the PCR primers used for the identification of the .DELTA.4 desaturase gene disrupted strain of the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disrupted strain, and the expected product.

[0152] FIG. 62 represents the evaluation of 44 desaturase gene disruption performed by a PCR using the genomic DNA of the Thraustochytrium aureum ATCC 34304 strain as a template. [Description of Reference Numerals]+/+: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disrupted strain; +/-: 44 desaturase gene first allele homologous recombinant derived from Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disrupted strain; -/-: Thraustochytrium aureum. ATCC 34304-derived PKS pathway (orfA gene) and 44 desaturase gene double disrupted strain.

[0153] FIG. 63 represents the result of the comparison of the fatty acid compositions of the Thraustochytrium aureum ATCC 34304 wild-type strain, and the PKS pathway (orfA gene) and 44 desaturase gene double disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively.

[0154] FIG. 64 represents the proportions of the fatty acids of the PKS pathway (orfA gene) and 44 desaturase gene double disrupted strain relative to the Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

[0155] FIG. 65 represents the evaluation of C20 elongase gene disruption performed by a PCR using the genomic DNA of the Parietichytrium sp. SEK358 strain as a template. [Description of Reference Numerals]+/+: Parietichytrium sp. SEK358 wild-type strain; -/-: Parietichytrium sp. SEK358 strain-derived C20 elongase gene disrupted strain.

[0156] FIG. 66 represents the result of the comparison of the fatty acid compositions of the Parietichytrium sp. SEK358 wild-type strain, and the Parietichytrium sp. SEK358 strain-derived C20 elongase gene disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively.

[0157] FIG. 67 represents the proportions of the fatty acid compositions of the Parietichytrium sp. SEK358 strain-derived C20 elongase gene disrupted strain relative to the Parietichytrium sp. SEK358 wild-type strain taken as 100%. The diagonal line indicates that the fatty acid produced by the Parietichytrium sp. SEK358 wild-type strain is below the detection limit.

[0158] FIG. 68 represents the evaluation of C20 elongase gene disruption performed by a PCR using the genomic DNA of the Parietichytrium sp. SEK571 strain as a template. [Description of Reference Numerals]+/+: Parietichytrium sp. SEK571 wild-type strain; -/-: Parietichytrium sp. SEK571 strain-derived C20 elongase gene disrupted strain.

[0159] FIG. 69 represents the result of the comparison of the fatty acid compositions of the Parietichytrium sp. SEK571wild-type strain, and the Parietichytrium sp. SEK571 strain-derived C20 elongase gene disrupted strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively.

[0160] FIG. 70 represents the proportions of the fatty acids of the Parietichytrium sp. SEK571 strain-derived C20 elongase gene disrupted strain relative to the Parietichytrium sp. SEK571 wild-type strain taken as 100%.

[0161] FIG. 71 represents the multiple alignment of T.DELTA.12d with the putative amino acid sequences of the .DELTA.12 desaturase genes derived from Thalassiosira pseudonana, Micromonas sp, and Phaeodactylum tricornutum. [Description of Reference Numerals] Underlined portion: histidine box.

[0162] FIG. 72 represents a GC analysis chart for the T.DELTA.12d overexpressing strain of the budding yeast Saccharomyces cerevisiae, and the proportions of fatty acid compositions.

[0163] FIG. 73 is a diagram representing a T.DELTA.12d KO targeting vector construction scheme.

[0164] FIG. 74 represents a scheme for the preparation of a homologous recombination fragment for efficiently obtaining a homologous recombinant by a split marker method.

[0165] FIG. 75 represents the result of the amplification of the hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d gene by a PCR performed by using the genomic DNAs of the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain (two alleles are disrupted). [Description of Reference Numerals] M: .lamda.HindIII digest/.phi.X174 HincII digest; W: wild-type; S1 to S3: 1st allele knock-out strain; D1 to D3: 2nd allele knock-out strain.

[0166] FIG. 76 represents the result of the mRNA detection of the hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d gene by a RT-PCR for the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain. [Description of Reference Numerals] M: .lamda.HindIII digest/.phi.X174 HincII digest; W: wild-type; S1 to S3: 1st allele knock-out strain; D1 to D3: 2nd allele knock-out strain.

[0167] FIG. 77 represents the result of the southern blotting performed for the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain.

[0168] FIG. 78 represents the result of the growth rate comparison by the measurements of OD600 and dry cell weight for the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain.

[0169] FIG. 79 represents the proportions of the fatty acid compositions of the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain. [Description of Reference Numerals] Asterisk: significant difference at p<0.01 (n=3).

[0170] FIG. 80 represents the fatty acid level per dry cell in the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain. [Description of Reference Numerals] Asterisk: significant difference at p<0.01 (n=3).

[0171] FIG. 81 represents a plasmid containing a BamHI site inserted through modification of the Thraustochytrium aureum C20 elongase gene targeting vector (pRH43) of FIG. 39 with a blasticidin-resistant gene.

[0172] FIG. 82 represents a plasmid containing a KpnI site inserted through modification of the plasmid of FIG. 81.

[0173] FIG. 83 represents a produced Thraustochytrium aureum C20 elongase gene targeting and Saprolegnia diclina-derived .omega.3 desaturase expression vector. The vector has a blasticidin-resistant gene as a drug-resistance marker.

[0174] FIG. 84 is a schematic view representing the positions of the southern hybridization analysis probes used for the identification of the C20 elongase gene disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain of the Thraustochytrium aureum PKS pathway (orfA gene) disrupted strain, and the expected gene fragment sizes.

[0175] FIG. 85 represents the evaluation of the C20 elongase gene disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain by southern hybridization using the Thraustochytrium aureum ATCC 34304 genomic DNA. [Description of Reference Numerals] PKSKO: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disrupted strain; +/-: C20 elongase gene first allele homologous recombinant of the Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disrupted strain; -/-: Thraustochytrium aureum-derived PKS pathway (orfA gene) and C20 elongase gene double disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain.

[0176] FIG. 86 represents the result of the comparison of the fatty acid compositions of the Thraustochytrium aureum ATCC 34304 wild-type strain, and the PKS pathway (orfA gene) and C20 elongase gene double disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the gene disrupted strain, respectively.

[0177] FIG. 87 represents the proportions of the fatty acids of the PKS pathway (orfA gene) and C20 elongase gene double disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain relative to the Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

[0178] FIG. 88 represents a base plasmid used for Saprolegnia diclina-derived .omega.3 desaturase expression vector production.

[0179] FIG. 89 represents a plasmid containing a Saprolegnia diclina-derived .omega.3 desaturase expression KpnI cassette inserted into the plasmid of FIG. 88.

[0180] FIG. 90 represents a Saprolegnia diclina-derived .omega.3 desaturase expression vector produced by inserting a hygromycin-resistant gene as a drug-resistance marker into the plasmid of FIG. 89.

[0181] FIG. 91 is a schematic view representing the positions of the PCR primers used for the confirmation of the genome insertion of the Saprolegnia diclina-derived .omega.3 desaturase gene.

[0182] FIG. 92 represents the evaluation of the Parietichytrium sp. SEK571 C20 elongase gene disrupted strain-derived transfectant strain. [Description of Reference Numerals] Lanes 1 to 2: transfectants

[0183] FIG. 93 represents the result of the comparison of the fatty acid compositions of the Parietichytrium sp. SEK571 wild-type strain, and the C20 elongase gene disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the transfectant strain, respectively.

[0184] FIG. 94 represents the proportions of the fatty acids of the C20 elongase gene disrupted and Saprolegnia diclina-derived .omega.3 desaturase expressing strain relative to the Parietichytrium sp. SEK571wild-type strain taken as 100%.

[0185] FIG. 95 is a diagram representing a pRH70 cloning a sequence containing a Schizochytrium-derived C20 elongase gene.

[0186] FIG. 96 is a diagram representing a pRH71 cloning a sequence containing a BglII site within the Schizochytrium-derived C20 elongase.

[0187] FIG. 97 is a diagram representing a pRH73 cloning a sequence containing a ubiquitin promoter, a neomycin-resistant gene, and an SV40 terminator within the Schizochytrium-derived C20 elongase, and a pKS-SKO cloning a sequence containing a ubiquitin promoter, a hygromycin-resistant gene, and an SV40 terminator within the Schizochytrium-derived C20 elongase.

[0188] FIG. 98 represents the expected fragment sizes of the wild-type strain allele and the knockout strains in a PCR.

[0189] FIG. 99 represents the PCR detection result for the wild-type strain allele and the knockout strain.

[0190] FIG. 100 represents the fatty acid compositions of the wild-type strain and the C20 elongase knockout strain. Blank bar and solid bar indicate the fatty acid compositions of the wild-type strain and the strain, respectively.

[0191] FIG. 101 represents the result of the comparison of the fatty acid compositions of the wild-type strain and the knockout strain.

MODE FOR CARRYING OUT THE INVENTION

[0192] The recent studies of the physiological activity and the pharmacological effects of lipids have elucidated the conversion of unsaturated fatty acids into various chemical substances, and the roles of unsaturated fatty acids in the unsaturated fatty acid metabolism. Particularly considered important in relation to disease is the nutritionally preferred proportions of saturated fatty acids, monounsaturated fatty acids, and unsaturated fatty acids, and the proportions of fish oil-derived .omega.3 series (also known as the n-3 series) fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, and plant-derived .omega.6 series (also known as the n-6 series) fatty acids as represented by linoleic acid. Because animals are deficient in fatty acid desaturases (desaturases) or have low levels of fatty acid desaturases, some unsaturated fatty acids need to be ingested with food. Such fatty acids are called essential fatty acids (or vitamin F), which include linoleic acid (LA), .gamma.-linolenic acid (GLA), and arachidonic acid (AA or ARA).

[0193] Unsaturated fatty acid production involves enzymes called fatty acid desaturases (desaturases). The fatty acid desaturases (desaturases) are classified into two types: (1) those creating a double bond (also called an unsaturated bond) at a fixed position from the carbonyl group of a fatty acid (for example, 49 desaturase creates a double bond at the 9th position as counted from the carbonyl side), and (2) those creating a double bond at a specific position from the methyl end of a fatty acid (for example, .omega.3 desaturase creates a double bond at the 3rd position as counted from the methyl end). It is known that the biosynthesis of unsaturated fatty acid involves the creation of a double bond by the desaturase (unsaturation), and the repeated elongation of the chain length by several different elongases. For example, .DELTA.9 desaturase synthesizes oleic acid (OA) by unsaturating the stearic acid either synthesized in the body from palmitic acid or ingested directly from the outside of the body. .DELTA.6, .DELTA.5, and .DELTA.4 desaturases are fatty acid desaturases (desaturases) essential for the syntheses of polyunsaturated fatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0194] The Labyrinthulomycetes, a member of stramenopile, has two families: Thraustochytrium (Thraustochytriaceae) and Labyrinthulaceae. These microorganisms are known to accumulate polyunsaturated fatty acids such as arachidonic acid, EPA, DTA, DPA, and DHA.

[0195] The present invention is concerned with a stramenopile transformation method whereby stramenopile genes are disrupted and/or expression thereof is inhibited by genetic engineering. Specifically, the present invention developed and provides a transformation method for disrupting genes associated with fatty acid biosynthesis and/or inhibiting expression thereof, a method for modifying the fatty acid composition of a stramenopile with the use of the transformation method, a method for highly accumulating fatty acids in a stramenopile, a stramenopile having an enhanced unsaturated fatty acid content, and a method for producing unsaturated fatty acid from the unsaturated fatty acid content-enhanced stramenopile.

[0196] The present invention includes manipulating the enzymes of the stramenopile elongase/desaturase pathway to change the fatty acid composition produced by a stramenopile. Specifically, the present invention enables modification of the fatty acid composition produced by stramenopile through (1) disruption of a fatty acid chain elongase gene and/or inhibition of expression thereof, (2) disruption of a polyketide synthase gene and/or inhibition of expression thereof, (3) disruption of a fatty acid desaturase and/or inhibition of expression thereof, (3) disruption of two of or all of a polyketide synthase gene, a fatty acid chain elongase gene, and a fatty acid desaturase and/or inhibition of expression thereof, (4) disruption of a fatty acid chain elongase gene and/or inhibition of expression thereof, and introduction of a fatty acid desaturase gene, (5) disruption of a polyketide synthase gene and/or inhibition of expression thereof, and introduction of a fatty acid desaturase gene, (6) disruption of a fatty acid desaturase and/or inhibition of expression thereof, and introduction of a fatty acid desaturase gene, (6) disruption of two of or all of a polyketide synthase gene, a fatty acid chain elongase gene, and a fatty acid desaturase and/or inhibition of expression thereof, and introduction of a fatty acid desaturase gene.

[0197] The present invention is described below in more detail.

[0198] [Microorganism]

[0199] The microorganisms used in the fatty acid modification method of the present invention are not particularly limited, as long as the microorganisms are stramenopiles considered to undergo modification of the fatty acid composition through disruption of genes associated with fatty acid biosynthesis and/or inhibition of expression thereof. Particularly preferred microorganisms are those belonging to the class Labyrinthulomycetes. Examples of the Labyrinthulomycetes include those of the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Thraustochytrium, Ulkenia, Aurantiochytrium, Oblongichytrium, Botryochytrium, Parietichytrium, and Sicyoidochytrium.

[0200] Of note, Labyrinthuloides and Aplanochytrium are regarded as being synonymous among some scholars (Leander, Celeste A. & David Porter, Mycotaxon, vol. 76, 439-444 (2000)).

[0201] The Labyrinthulomycetes used in the present invention are preferably microorganisms belonging to the genus Thraustochytrium and the genus Parietichytrium, particularly preferably Thraustochytrium aureum, Parietichytrium sarkarianum, and Thraustochytrium roseum. Specific examples include strains of Thraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364 (FERM BP-11298), Thraustochytrium roseum ATCC 28210, Parietichytrium sp. SEK358 (FERM BP-11405), and Parietichytrium sp. SEK571 (FERM BP-11406). Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210 are deposited at the ATCC, and are commonly available. The Parietichytrium sarkarianum SEK364 strain was obtained from the surface water collected at the mouth of fukidougawa on Ishigakijima. The water (10 ml) was placed in a test tube, and left unattended at room temperature after adding pine pollens. After 7 days, the pine pollens were applied to a sterile agar medium (2 g glucose, 1 g peptone, 0.5 g yeast extract, 0.2 g chloramphenicol, 15 g agar, distilled water 100 mL, sea water 900 mL). Colonies appearing after 5 days were isolated and cultured. This was repeated several times to isolate the cells. This strain has been internationally deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM BP-11298; Sep. 24, 2010). The Parietichytrium sp. SEK358 strain was isolated from the cells cultured as above from the sea water sample collected at the mouth of Miyaragawa on Ishigakijima. This strain has been internationally deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM BP-11405; Aug. 11, 2011). The Parietichytrium sp. SEK571 strain was isolated from the cells cultured as above from the sea water sample collected at the mouth of Shiiragawa on Iriomotejima. This strain has been internationally deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM BP-11406; Aug. 11, 2011). The Schizochytrium sp. TY12Ab strain was isolated from the cells cultured as above from the dead leaves collected on the coast of Tanegashima. This strain has been internationally deposited, and is available from The National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM ABP-11421; Sep. 29, 2011). Then, the RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT (FERM BP-11421) was issued by International Patent Organism Depositary on Nov. 30, 2011

[Genes Associated with Fatty Acid Biosynthesis]

[0202] In the present invention, the genes associated with fatty acid biosynthesis are not particularly limited, as long as the genes are genes of enzymes associated with the fatty acid biosynthesis in stramenopile, particularly the Labyrinthulomycetes. Examples of such genes include polyketide synthase gene, fatty acid chain elongase gene, and fatty acid desaturase gene. In the present invention, one of or both of these genes are subject to the disruption or inhibition of expression by genetic engineering. Here, the target of the gene disruption and/or inhibition of expression is, for example, the open reading frame, when, for example, the fatty acid produced by the polyketide synthase in a stramenopile is not the desired fatty acid. In the case of the fatty acid chain elongase, the target is the gene associated with an enzyme that converts the desired fatty acid into an other fatty acid. For example, when eicosapentaenoic acid (EPA) is the desired product, the gene of the fatty acid chain elongase associated with the conversion of eicosapentaenoic acid into docosapentaenoic acid (DPA), specifically C20 elongase gene may be disrupted and/or expression thereof may be inhibited. In the case of the fatty acid desaturase, the target is the gene associated with the enzyme that converts the desired fatty acid into an other fatty acid. For example, when oleic acid is the desired product, the gene of the fatty acid desaturase associated with the conversion of oleic acid into linoleic acid, specifically 412 desaturase gene may be disrupted and/or expression thereof may be inhibited. Further, two of or all of the polyketide synthase gene, the fatty acid chain elongase gene, and the fatty acid desaturase may be disrupted and/or expression thereof may be inhibited according to the desired fatty acid.

[0203] Further, a gene associated with fatty acid biosynthesis may be introduced into a transfectant strain produced by disrupting a gene and/or inhibiting expression thereof by genetic engineering as above. Here, the introduced gene is a gene associated with the enzyme that performs biosynthesis of the desired fatty acid. For example, when eicosapentaenoic acid is the desired product, a gene of the fatty acid desaturase that converts arachidonic acid (AA) into eicosapentaenoic acid, specifically .omega.3 desaturase gene may be introduced.

[Polyketide Synthase and Fatty Acid Chain Elongase]

[0204] Polyketide synthase (PKS) is an enzyme that catalyzes the multiple condensation reactions of a starter substrate (acetyl-CoA, fatty acid CoA ester, benzoyl CoA, coumaroyl CoA) with an extender substrate (such as malonyl CoA), and the enzyme is generally known to be involved in the biosyntheses of secondary metabolites in organisms such as plants and fungi. Involvement in the biosynthesis of polyunsaturated fatty acid is also reported in some species of organisms. For example, the marine bacteria Shewanella produce eicosapentaenoic acid (EPA) with this enzyme (Non-Patent Document 8). In some species of stramenopile, the polyketide synthase is known to be involved in the biosynthesis of polyunsaturated fatty acid, and the gene sequence has been elucidated in the Labyrinthulomycetes. For example, as described in Patent Document 7, the polyketide synthase gene of the genus Schizochytrium of Labyrinthula has three open reading frames, OrfA, OrfB, and OrfC. Further, as described in Patent Document 8, the polyketide synthase gene of the genus Ulkenia of Labyrinthula is considered to have three open reading frames.

[0205] The fatty acid chain elongase of the present invention is not particularly limited, as long as it extends the chain length of a fatty acid. Preferred examples include C18 elongase gene, and C20 elongase gene. The C18 elongase gene and the C20 elongase gene extend fatty acids of 18 and 20 carbon atoms, respectively, in two-carbon units to produce fatty acids of 20 and 22 carbon atoms. These fatty acid chain elongases are found in a wide range of organisms, including stramenopiles, and in, for example, the genus Thraustochytrium of Labyrinthulomycetes, as reported in Non-Patent Document 9. The C18 elongase catalyzes the conversion of .gamma.-linolenic acid (GLA) to dihomo-.gamma.-linolenic acid (DGLA), and the conversion of stearidonic acid (STA) into eicosatetraenoic acid (ETA). The C20 elongase catalyzes the conversion of arachidonic acid (AA) into docosatetraenoic acid (DTA), and the conversion of eicosapentaenoic acid (EPA) into n-3 docosapentaenoic acid (DPA, 22:5n-3).

[0206] It follows from this that when the desired product is, for example, stearidonic acid (STA), a gene of the fatty acid chain elongase associated with the conversion of stearidonic acid into eicosatetraenoic acid (ETA), specifically C18 elongase gene may be disrupted and/or expression thereof may be inhibited. When the desired product is, for example, eicosapentaenoic acid (EPA), a gene of the fatty acid chain elongase associated with the conversion of the eicosapentaenoic acid into docosapentaenoic acid (DPA), specifically C20 elongase gene may be disrupted and/or expression thereof may be inhibited. Further, when the fatty acid biosynthesized with the polyketide synthase in a stramenopile is not the desired fatty acid, the polyketide synthase gene may be disrupted and/or expression thereof may be inhibited. As reported in Non-Patent Document 5, a strain of the genus Schizochytrium of Labyrinthula loses the ability to biosynthesize docosahexaenoic acid after the disruption of the polyketide synthase gene, and cannot grow in media unless supplemented with polyunsaturated fatty acid. In the present invention, however, some species of Labyrinthula, even with the disrupted polyketide synthase gene, are able to grow in media without adding polyunsaturated fatty acid, and the desired polyunsaturated fatty acid can thus be obtained by disrupting the gene or inhibiting gene expression in the manner described above.

[Fatty Acid Desaturase]

[0207] The fatty acid desaturase (desaturase) of the present invention is not particularly limited, as long as it functions as a fatty acid desaturase. The origin of the fatty acid desaturase gene is not particularly limited, and may be, for example, animals and plants. Examples of the preferred fatty acid desaturase genes include .DELTA.4 desaturase gene, .DELTA.5 desaturase gene, .DELTA.6 desaturase gene, .DELTA.12 desaturase gene, and .omega.3 desaturase gene, and these may be used either alone or in combination. The .DELTA.4 desaturase gene, .DELTA.5 desaturase gene, .DELTA.6 desaturase gene, and .DELTA.12 desaturase gene form an unsaturated bond at carbon 4, 5, 6, and 12, respectively, as counted from the carbon atom of the terminal carboxyl group (delta end) of the fatty acid. A specific example of these fatty acid desaturase genes is the microalgae-derived .DELTA.4 desaturase gene (Non-Patent Document 6). Specific examples of .DELTA.5 desaturase include T. aureum-derived .DELTA.5 desaturase, and .DELTA.5 desaturases derived from Thraustochytrium sp. ATCC 26185, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans, and Leishmania major. Examples of 412 desaturase include Pinguiochrysis pyriformis-derived .DELTA.12 desaturase, and fungus- and protozoa-derived .DELTA.12 desaturases. The .omega.3 desaturase forms a double bond at the third position as counted from the methyl terminal of the fatty acid carbon chain. Examples include Saprolegnia-derived .omega.3 desaturase (Non-Patent Document 10). The .DELTA.5 desaturase catalyzes, for example, the conversion of dihomo-.gamma.-linolenic acid (DGLA) to arachidonic acid (AA), and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA). .DELTA.6 desaturase catalyzes, for example, the conversion of linoleic acid (LA) to .gamma.-linolenic acid (GLA), and the conversion of .alpha.-linolenic acid (ALA) to stearidonic acid (STA). The .omega.3 desaturase catalyzes the conversion of arachidonic acid to eicosapentaenoic acid. Linoleic acid (LA) is produced from oleic acid (OA) by the action of .DELTA.12 desaturase.

[Product Unsaturated Fatty Acid]

[0208] The unsaturated fatty acid produced by the fatty acid desaturase expressed in a stramenopile is, for example, an unsaturated fatty acid of 18 to 22 carbon atoms. Preferred examples include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), though the preferred unsaturated fatty acids vary depending on the types of the fatty acid desaturase and the fatty acid substrate used. Other examples include .alpha.-linolenic acid (ALA), octadecatetraenoic acid (OTA, 18:4n-3), eicosatetraenoic acid (ETA, 20:4n-3), n-3 docosapentaenoic acid (DPA, 22:5n-3), tetracosapentaenoic acid (TPA, 24:5n-3), tetracosahexaenoic acid (THA, 24:6n-3), linoleic acid (LA), .gamma.-linolenic acid (GLA), eicosatrienoic acid (20:3n-6), arachidonic acid (AA), and n-6 docosapentaenoic acid (DPA, 22:5n-6).

[Gene Source of Enzyme Associated with Fatty Acid Biosynthesis]

[0209] The organisms that can be used as the gene sources of the polyketide synthase, fatty acid chain elongase, and/or fatty acid desaturase in the present invention are not limited to particular genuses, species, or strains, and may be any organisms having an ability to produce polyunsaturated fatty acids. For example, in the case of microorganisms, such organisms are readily available from microorganism depositary authorities. Examples of such microorganisms include the bacteria Moritella marina MP-1 strain (ATCC15381) of the genus Moritella. The following describes a method using this strain as an example of desaturase and elongase gene sources. The method, however, is also applicable to the isolation of the constituent desaturase and elongase genes from all biological species having the desaturase/elongase pathway.

[0210] Isolation of the desaturase and/or elongase gene from the MP-1 strain requires estimation of a conserved region in the amino acid sequence of the target enzyme gene. For example, in desaturase, it is known that a single cytochrome b5 domain and three histidine boxes are conserved across biological species, and that elongase has two conserved histidine boxes across biological species. More specifically, the conserved region of the target enzyme can be estimated by the multiple alignment comparison of the known amino acid sequences of the desaturase or elongase genes derived from various biological species using the clustal w program (Non-Patent Document 7). It is also possible to estimate conserved regions specific to desaturase and/or elongase having the same substrate specificity by the multiple alignment comparison of the amino acid sequences of desaturase or elongase genes having the same substrate specificity in the desaturase and/or elongase derived from known other organisms. Various degenerate oligonucleotide primers are then produced based on the estimated conserved regions, and the partial sequence of the target gene derived from the MP-1 strain is amplified using an MP-1 strain-derived cDNA library as a template, by using methods such as PCR and RACE. The resulting amplification product is cloned into a plasmid vector, and the base sequence is determined using an ordinary method. The sequence is then compared with a known enzyme gene to confirm isolation of a part of the target enzyme gene from the MP-1 strain. The full-length target enzyme gene can be obtained by hybridization screening using the obtained partial sequence as a probe, or by the RACE technique using the oligonucleotide primers produced from the partial sequence of the target gene.

[0211] The polyketide synthase can be cloned by using an ordinary method, using the PUFA PKS sequence of Patent Document 7 as a reference.

[Other Gene Sources]

[0212] Reference should be made to Non-Patent Document 11 or 12 for GFP (Green Fluorescent Protein), Patent Document 6 for EGFP (enhanced GFP), and Non-Patent Document 13 for neomycin-resistant gene.

[Disruption of Gene Associated with Fatty Acid Biosynthesis in Stramenopile]

[0213] The stramenopile gene associated with fatty acid biosynthesis may be disrupted by using conventional gene disruption methods used for microorganisms. An example of such a method is the transformation introducing a recombinant expression vector into a cell.

[0214] For example, for the disruption of a Thraustochytrium aureum gene, genomic DNA is extracted from a Thraustochytrium aureum by using an ordinary method, and a genome library is created. Then, genome walking primers are set using the DNA sequence of the target gene to be disrupted, and a PCR is run using the produced genome library as a template to obtain the upstream and downstream sequences of the target gene of Thraustochytrium aureum. These sequences are flanked on both sides to provide homologous recombination regions for gene disruption, and a drug marker gene is inserted therebetween for selection. The DNA is then linearized, and introduced into a Thraustochytrium aureum using a gene-gun technique, and the cells are cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA is extracted from cells that have acquired drug resistance, and strains that underwent homologous recombination are identified by PCR or southern hybridization. Because the Thraustochytrium aureum is a diploid, the procedures from the introduction of the linearized DNA into the cells using a gene-gun technique to the identification of homologous recombinant strains are repeated twice. In this way, a Thraustochytrium aureum with the disrupted target gene can be obtained. When two or more target genes are present, a strain with the disrupted multiple target genes can be obtained by repeating the foregoing procedures. Here, because the Thraustochytrium aureum is a diploid, two selection markers need to be prepared for each gene.

[0215] For the disruption of, for example, the C20 elongase of Parietichytrium sarkarianum, genomic DNA is extracted from a Parietichytrium species by using an ordinary method, and the genome is decoded. Then, a search is made for a gene sequence highly homologous to a known C20 elongase gene, and the gene sequence is amplified by PCR from the start codon to the stop codon. This is followed by insertion of a restriction enzyme site at substantially the center of the gene sequence by using a mutagenesis method, and insertion of a drug marker gene cassette to the restriction enzyme site for selection. The DNA is linearized, and introduced into a Parietichytrium sarkarianum SEK364 using a gene-gun technique. The cells are then cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA is extracted from cells that have acquired drug resistance, and strains that underwent homologous recombination are identified by PCR. Because the Parietichytrium sarkarianum SEK364 is a diploid, the procedures from the introduction of the linearized DNA into the cells using a gene-gun technique to the identification of the homologous recombinant strains are repeated twice. In this way, a Parietichytrium sarkarianum SEK364 with the disrupted C20 elongase gene can be obtained. Here, because the Parietichytrium sarkarianum SEK364 is a diploid, two selection markers need to be prepared. For the disruption of, for example, the .DELTA.4 desaturase of a Thraustochytrium aureum ATCC 34304-derived OrfA disrupted strain, genomic DNA is extracted from a Thraustochytrium aureum ATCC 34304 by using an ordinary method, and the genome is decoded. Then, a search is made for a gene sequence highly homologous to a known 44 desaturase, and the gene sequence is amplified by PCR from the upstream region to a region in the vicinity of the stop codon. By using a mutagenesis method, a restriction enzyme site is inserted at the same time as deleting a part of the ORF containing the start codon, and a drug marker gene cassette is inserted to the restriction enzyme site for selection. The DNA is linearized, and introduced into a Thraustochytrium aureum ATCC 34304-derived OrfA disrupted strain by using a gene-gun technique. The cells are then cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA is extracted from cells that have acquired drug resistance, and strains that underwent homologous recombination are identified by PCR. Because the Thraustochytrium aureum ATCC 34304 is a diploid, the procedures from the introduction of the linearized DNA using a gene-gun technique to the identification of homologous recombinant strains are repeated twice. In this way, a Thraustochytrium aureum ATCC 34304-derived OrfA disrupted strain with the disrupted .DELTA.4 desaturase gene can be obtained. Here, because the Thraustochytrium aureum ATCC 34304 is a diploid, two selection markers need to be prepared.

[0216] The C20 elongase gene sequence of Thraustochytrium aureum was used for disrupting the C20 elongase of Thraustochytrium roseum. Genomic DNA is extracted from a Thraustochytrium aureum by using an ordinary method, and the C20 elongase gene is amplified from the start codon to the stop codon by PCR. A restriction enzyme site is inserted to substantially the center of the gene sequence by using a mutagenesis method, and a drug marker gene cassette is inserted to the restriction enzyme site for selection. The DNA is linearized, and introduced into a Thraustochytrium roseum by using a gene-gun technique. The cells are then cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA is extracted from cells that have acquired drug resistance, and strains that underwent homologous recombination are identified by PCR. Because the Thraustochytrium roseum is a diploid, the procedures from the introduction of the linearized DNA using a gene-gun technique to the identification of the homologous recombinant strain are repeated twice. In this way, a Thraustochytrium roseum with the disrupted C20 elongase gene can be obtained. Here, because the Thraustochytrium roseum is a diploid, two selection markers need to be prepared.

[0217] Details of the disruption of stramenopile genes associated with fatty acid biosynthesis according to the present invention will be specifically described later in Examples. The stramenopile subject to transformation is not particularly limited, and those belonging to the class Labyrinthulomycetes can preferably be used, as described above.

[0218] For example, for the disruption of the C20 elongase of the Parietichytrium sp. SEK358 strain, the Parietichytrium C20 elongase gene targeting vector produced in Example 3-6 was used. The DNA is linearized, and introduced into a Parietichytrium sp. SEK358 strain by using a gene-gun technique. The cells are then cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA is extracted from cells that have acquired drug resistance, and strains that underwent homologous recombination were identified by PCR (see Example 9). For the disruption of, for example, the C20 elongase of the Parietichytrium sp. SEK571 strain, the Parietichytrium C20 elongase gene targeting vector produced in Example 3-6 was used. The DNA is linearized, and introduced into a Parietichytrium sp. SEK571 strain by using a gene-gun technique. The cells are then cultured for about 1 week on a drug-containing plate. By using an ordinary method, genomic DNA was extracted from cells that had acquired drug resistance, and the homologous recombinant strain was identified by PCR (see Example 10).

[0219] The expression vector is not particularly limited, and a recombinant expression vector with an inserted gene may be used. The vehicle used to produce the recombinant expression vector is not particularly limited, and, for example, a plasmid, a phage, and a cosmid may be used. A known method may be used for the production of the recombinant expression vector. The vector is not limited to specific types, and may be appropriately selected from vectors expressible in a host cell. Specifically, the expression vector may be one that is produced by incorporating the gene of the present invention into a plasmid or other vehicles with a promoter sequence appropriately selected according to the type of the host cell for reliable expression of the gene. The vector may be a cyclic or a linear vector. The expression vector preferably includes at least one selection marker. Examples of such selection markers include auxotrophic markers, drug-resistance markers, fluorescent protein markers, and fused markers of these. Examples of the auxotrophic markers include dihydrofolate reductase genes. Examples of the drug-resistance markers include neomycin-resistant genes, hygromycin-resistant genes, blasticidin-resistant genes, and zeocin-resistant genes. Examples of the fluorescent protein markers include GFPs, and enhanced GFPs (EGFPs). Examples of the fused markers include fused markers of fluorescent protein markers and drug-resistance markers, specifically, for example, GFP-fused zeocin-resistant genes. These selection markers allow for confirmation of whether the polynucleotide according to the present invention has been introduced into a host cell, or whether the polynucleotide is reliably expressed in the host cell. Alternatively, the fatty acid desaturase according to the present invention may be expressed as a fused polypeptide. For example, the fatty acid desaturase according to the present invention may be expressed as a GFP-fused polypeptide, using GFP as a marker.

[0220] Preferably, electroporation or a gene gun is used as the method of gene introduction for the gene disruption. In the present invention, the disruption of the gene associated with fatty acid biosynthesis changes the fatty acid composition of the cell from that before the gene disruption. Specifically, the fatty acid composition is modified by the disruption of the gene associated with fatty acid biosynthesis. A stramenopile with the disrupted fatty acid biosynthesis-related enzyme gene can produce the desired fatty acid in greater amounts when further introduced with a fatty acid desaturase gene. Preferably, an .omega.3 desaturase gene is introduced as the fatty acid desaturase gene.

[0221] The stramenopile transformation produces a stramenopile (microorganism) in which the composition of the fatty acid it produces is modified. The stramenopile with the disrupted gene associated with fatty acid biosynthesis can be used for, for example, the production of unsaturated fatty acids. Unsaturated fatty acid production is possible with the stramenopile that has been modified to change its produced fatty acid composition as above, and other conditions, including steps, equipment, and instruments are not particularly limited. The unsaturated fatty acid production includes the step of culturing a microorganism that has been modified to change its produced fatty acid composition by the foregoing modification method, and the microorganism is used with its medium to produce unsaturated fatty acids.

[0222] The cell culture conditions (including medium, culture temperature, and aeration conditions) may be appropriately set according to such factors as the type of the cell, and the type and amount of the unsaturated fatty acid to be produced. As used herein, the term "unsaturated fatty acids" encompasses substances containing unsaturated fatty acids, and attributes such as the content, purity, shape, and composition are not particularly limited. Specifically, in the present invention, the cell or its medium itself having a modified fatty acid composition may be regarded as unsaturated fatty acids. Further, a step of purifying the unsaturated fatty acids from such cells or media also may be included. A known method of purifying unsaturated fatty acids and other lipids (including conjugate lipids) may be used for the purification of the unsaturated fatty acids.

[Method of Highly Accumulating Unsaturated Fatty Acid in Stramenopile]

[0223] Accumulation of unsaturated fatty acids in stramenopile is realized by culturing the transformed stramenopile of the present invention. For example, the culture is performed using a common solid or liquid medium. The type of medium used is not particularly limited, as long as it is one commonly used for culturing Labyrinthulomycetes, and that contains, for example, a carbon source (such as glucose, fructose, saccharose, starch, and glycerine), a nitrogen source (such as a yeast extract, a corn steep liquor, polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate, ammonium nitrate, ammonium chloride, and sodium nitrate), and an inorganic salt (such as potassium phosphate) appropriately combined with other necessary components. Particularly preferably, a yeast extract/glucose medium (GY medium) is used. The prepared medium is adjusted to a pH of 3.0 to 8.0, and used after being sterilized with an autoclave or the like. The culture may be performed by aerated stirred culture, shake culture, or static culture at 10 to 40.degree. C., preferably 15 to 35.degree. C., for 1 to 14 days.

[0224] For the collection of the produced unsaturated fatty acids, the stramenopile is grown in a medium, and the intracellular lipids (oil and fat contents with the polyunsaturated fatty acids, or the polyunsaturated fatty acids) are released by processing the microorganism cells obtained from the medium. The lipids are then collected from the medium containing the released intracellular lipids. Specifically, the cultured stramenopile is collected by using a method such as centrifugation. The cells are then disrupted, and the intracellular fatty acids are extracted using a suitable organic solvent according to an ordinary method. Oil and fat with the enhanced polyunsaturated fatty acid content can be obtained in this manner.

[0225] In the present invention, the composition of the fatty acids produced by a stramenopile is modified by culturing a stramenopile transformed through disruption of genes associated with fatty acid biosynthesis, and/or inhibition of expression thereof, specifically disruption of the polyketide synthase, the fatty acid chain elongase, and/or the fatty acid desaturase gene, and/or inhibition of expression of these genes. Because the genes associated with fatty acid biosynthesis are disrupted and/or expression thereof is inhibited, the desired fatty acid can be accumulated in the stramenopile without being converted into other fatty acids. Further, by introducing the gene associated with fatty acid desaturase into a stramenopile transformed through gene disruption and/or inhibition of gene expression, the ability to convert the precursor fatty acid of the desired fatty acid into the desired fatty acid can be enhanced, and the desired fatty acid is accumulated.

[0226] The unsaturated fatty acids of the present invention encompass various drugs, foods, and industrial products, and the applicable areas of the unsaturated fatty acids are not particularly limited. Examples of the food containing oil and fat that contain the unsaturated fatty acids of the present invention include foods with health claims such as supplements, and food additives. Examples of the industrial products include feeds for non-human organisms, films, biodegradable plastics, functional fibers, lubricants, and detergents.

[0227] The present invention is described below in more detail based on examples. Note, however, that the present invention is in no way limited by the following examples.

Example 1

[0228] [Labyrinthulomycetes, Culture Method, and Preservation Method]

[0229] (1) Strains Used in the Present Invention

[0230] Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210 were obtained from ATCC. Parietichytrium sarkarianum SEK364 (FERN BP-11298), Parietichytrium sp. SEK358 (FERM BP-11405), and Parietichytrium sp. SEK571 (FERN BP-11406) were obtained from Konan University, Faculty of Science and Engineering. Schizochytrium sp. TY12Ab (FERN BP-11421) was obtained from University of Miyazaki, Faculty of Agriculture.

[0231] (2) Medium Composition

[0232] i. Agar Plate Medium Composition

[0233] PDA Agar Plate Medium

[0234] A 0.78% (w/v) potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd.), 1.75% (w/v) Sea Life (Marine Tech), and 1.21% (w/v) agar powder (nacalai tesque) were mixed, and sterilized with an autoclave at 121.degree. C. for 20 min. After sufficient cooling, ampicillin sodium (nacalai tesque) was added in a final concentration of 100 .mu.g/ml to prevent bacterial contamination. The medium was dispensed onto a petri dish, and allowed to stand on a flat surface to solidify.

[0235] ii. Liquid Medium Composition

[0236] GY Liquid Medium

[0237] 3.18% (w/v) glucose (nacalai tesque), 1.06% (w/v) dry yeast extract (nacalai tesque), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, and sterilized with an autoclave at 121.degree. C. for 20 min. Then, 100 .mu.g/ml ampicillin sodium (nacalai tesque) was added.

[0238] PD Liquid Medium

[0239] 0.48% (w/v) potato dextrose (Difco), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, and sterilized with an autoclave at 121.degree. C. for 20 min. Then, 100 .mu.g/ml ampicillin sodium (nacalai tesque) was added.

[0240] (3) Culture Method

[0241] i. Agar Plate Culture

[0242] Labyrinthula cells were inoculated using a platinum loop or a spreader, and static culture was performed at 25.degree. C. to produce colonies. Subcultures were produced by collecting the colonies with a platinum loop, suspending the collected colonies in a sterilized physiological saline, and applying the suspension using a platinum loop or a spreader. As required, the cells on the plate were inoculated in a liquid medium for conversion into a liquid culture.

[0243] ii. Liquid Culture

[0244] Labyrinthula cells were inoculated, and suspension culture was performed by stirring at 25.degree. C., 150 rpm in an Erlenmeyer flask or in a test tube. Subcultures were produced by adding a culture fluid to a new GY or PD liquid medium in a 1/200 to 1/10 volume after confirming proliferation from the logarithmic growth phase to the stationary phase. As required, the cell culture fluid was applied onto a PDA agar plate medium for conversion into an agar plate culture.

[0245] (4) Maintenance and Preservation Method of Labyrinthulomycetes

[0246] In addition to the subculture, cryopreservation was performed by producing a glycerol stock. Specifically, glycerol (nacalai tesque) was added in a final concentration of 15% (v/v) to the logarithmic growth phase to stationary phase of a cell suspension in a GY liquid medium, and the cells were conserved in a -80.degree. C. deep freezer.

Example 2

[0247] [Disruption of Thraustochytrium aureum C20 Elongase Gene]

[Example 2-1] Extraction of T. aureum ATCC 34304-Derived Total RNA, and mRNA Purification

[0248] A T. aureum ATCC 34304 culture fluid grown for 3 days using a GY liquid medium was centrifuged at 3,500.times.g for 15 min, and the cells were collected. After being suspended in sterilized physiological saline, the cells were washed by being recentrifuged. The cells were then rapidly frozen with liquid nitrogen, and ground into a powdery form with a mortar. Total RNA was extracted from the resulting cell disruption liquid, using Sepasol-RNA I Super (nacalai tesque). This was followed by purification of mRNA from the total RNA using the Oligotex.TM.-dT30 <Super> mRNA Purification Kit (Takara Bio) according to the manufacturer's protocol. The resulting total RNA and the mRNA were dissolved in a suitable amount of TE, and electrophoresed with a formalin-denatured gel (1% agarose/MOPS buffer). The result confirmed successful extraction of the total RNA, and purification of mRNA from the total RNA. It was also confirmed that the RNA was not degraded by the RNase. In order to minimize RNA degradation, all experimental procedures were performed with sanitary equipment such as rubber gloves and a mask. All instruments were RNase free, or were used after a diethylpyrocarbonate (nacalai tesque) treatment to deactivate the RNase. The solution used to dissolve the RNA was prepared by adding the recombinant RNase inhibitor RNaseOUT.TM. (invitrogen) to sterilized Milli Q water treated with diethylpyrocarbonate.

[Example 2-2] Isolation of T. aureum ATCC 34304-Derived Elongase Gene by RACE

[0249] Forward (elo-F;5'-TTY YTN CAY GTN TAY CAY CAY-3') (SEQ ID NO: 1), and reverse (elo-R;5'-GCR TGR TGR TAN ACR TGN ARR AA-3') (SEQ ID NO: 2) denatured oligonucleotides were synthesized, targeting the histidine box (His box) highly conserved in elongase genes. The oligonucleotides were synthesized with a DNA synthesizer (Applied Biosystems). Then, by addition of synthetic adapters to the 3'- and 5'-ends, 3'- and 5'-RACE cDNA libraries were produced by using the SMART.TM. RACE cDNA Amplification Kit (clontech) according to the manufacturer's protocol, respectively. By using these as templates, 3'- and 5'-RACE were performed using the synthetic adapter-specific oligonucleotides, and the denatured oligonucleotides elo-F and elo-R [PCR cycles: 94.degree. C. 1 min/94.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. 3 min, 30 cycles/72.degree. C. 10 min/4.degree. C. .infin.]. The result confirmed bands for the specifically amplified 3'- and 5'-RACE products (FIG. 1). The total RACE product amounts were subjected to electrophoresis with 1% agarose gel, and the isolated DNA fragments were cut out with a clean cutter or the like and extracted from the agarose gel according to the method described in Non-Patent Document 20. The DNA fragments were then TA cloned with a pGEM-T easy Vector (Promega), and the base sequences were determined by the method of Sanger et al. (Non-Patent Document 21). Specifically, the base sequences were determined by using a dye terminator method, using a BigDyeR Terminator v3.1 Cyele Sequencing Kit and a 3130 genetic analyzer (Applied Biosystems) according to the manufacturers' protocols.

[0250] As a result, two sequences, 190 bp and 210 bp, named elo1 (SEQ ID NO: 3) and elo2 (SEQ ID NO: 4) were successfully identified for the 3'-RACE product, and one sequence, 200 bp, named elo3 (SEQ ID NO: 5) was successfully identified for the 5'-RACE product. Because the elo1, elo2, and elo3 sequences had significant homology to the sequences of various elongase genes, the results suggested that these sequences were partial sequences of the T. aureum ATCC 34304-derived elongase gene. In an attempt to obtain cDNA sequences by RACE, oligonucleotide primers were redesigned for the elo1, elo2, and elo3. The oligonucleotide primers produced are as follows.

TABLE-US-00001 elo1 forward oligonucleotide primer (SEQ ID NO: 6) (elo1-F1; 5'-TAT GAT CGC CAA GTA CGC CCC-3') and reverse oligonucleotide primer (SEQ ID NO: 7) (elo1-R1; 5'-GAA CTG CGT CAT CTG CAG CGA-3') elo2 forward oligonucleotide primer (SEQ ID NO: 8) (elo2-F1; 5'-TCT CGC CCT CGA CCA CCA AC-3') and reverse oligonucleotide primer (SEQ ID NO: 9) (elo2-R1; 5'-CGG TGA CCG AGT TGA GGT AGC C-3') elo3 forward oligonucleotide primer (SEQ ID NO: 10) (elo3-F1; 5'-CAA CCC TTT CGG CCT CAA CAA G-3') and reverse oligonucleotide primer (SEQ ID NO: 11) (elo3-R1; 5'-TTC TTG AGG ATC ATC ATG AAC GTG TC-3')

[0251] By using these forward and reverse oligonucleotide primers, RACE and base sequence analysis of the amplification products were performed as above. As a result, specifically amplified 3'- and 5'-RACE products were obtained for elo1, and there was a complete match in the overlapping portion, identifying the sequence as a 1,139-bp elo1 cDNA sequence (SEQ ID NO: 12). Similarly, specifically amplified 3'- and 5'-RACE products were obtained for elo3, and there was a complete match in the overlapping portion, identifying the sequence as a 1,261-bp elo3 cDNA sequence (SEQ ID NO: 13).

[0252] It was found from the sequence analysis result that elo1 consisted of an 825-bp translated region (SEQ ID NO: 15) coding for 275 amino acid residues (SEQ ID NO: 14). It was also found from the result of a BLAST search that the sequence had significant homology to various elongase genes, and completely coincided with the sequence of a known T. aureum-derived putative 45 elongase gene (NCBI accession No. C5486301). On the other hand, it was assumed that the elo3 consisted of a 951-bp translated region (SEQ ID NO: 17) coding for 317 amino acid residues (SEQ ID NO: 16). It was also found from the result of a BLAST search that the sequence had significant homology to various elongase genes, and thus represented a T. aureum ATCC 34304-derived putative elongase gene. Note that the putative amino acid sequences of these genes contained His boxes highly conserved in elongase genes. From these results, elo1 and elo3 genes were identified as T. aureum ATCC 34304-derived putative elongase genes, and were named TaELO1 and TaELO2, respectively,

[Example 2-3] TaELO1 and TaELO2 Phylogenetic Analysis

[0253] Elongases are broadly classified into three groups on the basis of substrate specificity. [0254] 1. SFA/MUFA elongases (act on saturated fatty acids or monovalent unsaturated fatty acids) [0255] 2. PUFA-elongases (single-step) (act on polyvalent unsaturated fatty acids of certain chain lengths) [0256] 3. PUFA elongases (multi-step) (act on polyvalent unsaturated fatty acids of various chain lengths)

[0257] According to the elongase phylogenetic analysis conducted by Meyer et al. (Non-Patent Document 22), there is a good correlation between the substrate specificity and the phylogenetic relationships.

[0258] Accordingly, a phylogenetic analysis was performed for TaELO1, TaELO2, and various other elongase genes derived from other organisms, using the method of Meyer et al. Specifically, a molecular phylogenetic tree was created according to the neighbor-joining method (Non-Patent Document 14), using the CLUSTAL Wprogram (Non-Patent Document 7). It was found as a result that the TaELO1 and TaELO2 were classified into the PUFA-elongases (single-step) group, suggesting that these elongases act on polyvalent unsaturated fatty acids of certain chain lengths (FIG. 2).

[Example 2-4] TaELO1 and TaELO2 Expression in Budding Yeast Saccharomyces cerevisiae Host, and Fatty Acid Composition Analysis of Gene Introduced Strain

[0259] Expression vectors were constructed for TaELO1 and TaELO2 for their expression in budding yeast S. cerevisiae used as a host, as briefly described below. A set of oligonucleotide primer (E1 HindIII; 5'-ATA AGC TTA AAA TGT CTA GCA ACA TGA GCG CGT GGG GC-3') (SEQ ID NO: 18) and E1 XbaI; 5'-TGT CTA GAA CGC GCG GAC GGT CGC GAA A-3') (SEQ ID NO: 19) was produced using the sequence of the TaELO1 translated region. The E1 HindIII is a forward oligonucleotide primer, and has a restriction enzyme HindIII site (AAGCTT) at the 5'-end. The sequence in the vicinity of the TaELO1 start codon is modified by referring to a yeast consensus sequence ((A/Y) A (A/U) AAUGUCU; the start codon is underlined) (Non-Patent Document 23). The E1 XbaI is a reverse oligonucleotide primer, and has an XbaI site (TCTAGA) at the 5'-end.

[0260] In the same manner, a set of oligonucleotide primer (E2 HindIII; 5'-TAA AGC TTA AAA TGT CTA CGC GCA CCT CGA AGA GCG CTC C-3') (SEQ ID NO: 20) and E2 XbaI; 5'-CAT CTA GAC TCG GAC TTG GTG GGG GCG CTT G-3') (SEQ ID NO: 21) was produced using the sequence of the TaELO2 translated region. The E2 HindIII is a forward oligonucleotide primer, and has a restriction enzyme HindIII site at the 5'-end. The sequence in the vicinity of the TaELO2 start codon is modified by referring to a yeast consensus sequence. The E2 XbaI is a reverse oligonucleotide primer, and has an XbaI site at the 5'-end.

[0261] By using the two oligonucleotide primer sets, a PCR was performed using the 5'-RACE cDNA library of Example 2-2 as a template. The PCR amplified a 949-bp TaELO1 translated region (SEQ ID NO: 22) and a 967-bp TaELO2 translated region (SEQ ID NO: 23) having the restriction enzyme HindII and the restriction enzyme XbaI site at the 5'-end and the 3'-end, and modified to the yeast consensus sequence in the vicinity of the start codon. Note that a PrimeSTARR DNA polymerase (Takara Bio) of high proofreading activity was used as the PCR enzyme to avoid extension errors [PCR cycles: 98.degree. C. 2 min/98.degree. C. 5 sec, 60.degree. C. 5 sec, 72.degree. C. 1.5 min, 30 cycles/72.degree. C. 7 min/4.degree. C. cc].

[0262] After isolating the amplified PCR products with a 1% agarose gel, the DNA fragments were cut and extracted from the agarose gel. After treatment with restriction enzymes HindIII and XbaI, the product was purified again with an agarose gel. To construct a cyclic vector, the product was joined to a budding yeast expression vector pYES2/CT (invitrogen) with a DNA Ligation Kit <Mighty Mix> (Takara Bio) after linearizing the pYES2/CT vector with restriction enzymes HindIII and XbaI. This was followed by a base sequence analysis, which confirmed that no PCR extension error occurred and no mutation was introduced to the TaELO1 and TaELO2 translated region sequences introduced into the pYES2/CT. In this manner, a TaELO1 expression vector pYEELO1, and a TaELO2 expression vector pYEELO2 were successfully constructed.

[0263] The two expression vectors constructed above, and the pYES2/CT were introduced into the budding yeast S. cerevisiae by using the lithium acetate technique according to the methods described in Non-Patent Documents 15 and 16, and the transfectants were screened for. The resulting transfectants (pYEELO1 introduced strain, pYEELO2 introduced strain, and mock introduced strain) were cultured according to the method of Qiu et al. (Non-Patent Document 24), and the cell-derived fatty acids were extracted and methylesterificated. Note that each culture was performed in a medium supplemented with .alpha.-linolenic acid (ALA, C18:349, 12, 15) and linoleic acid (LA, C18:249, 12) added as 49 elongase substrates, stearidonic acid (STA, C18:446, 9, 12, 15) and .gamma.-linolenic acid (GLA, C18:346, 9, 12) added as 46 elongase substrates, and eicosapentaenoic acid (EPA, C20:545, 8, 11, 14, 17) and arachidonic acid (AA, C20:445, 8, 11, 14) added as 45 elongase substrates. Here, each supplement was added in a final concentration of 0.2 mM. This was followed by the gas chromatography (GC) analysis of the methylesterificated fatty acids according to the method of Abe et al. (Non-Patent Document 17). The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0264] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0265] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0266] Carrier gas: He (1.3 mL/min).

[0267] It was found as a result that, the pYEELO1 introduced strain had the 46 elongase activity not found in the host (mock introduced strain), converting the stearidonic acid (STA) into eicosatetraenoic acid (ETA, C20:4.DELTA.8, 11, 14, 17), and the .gamma.-linolenic acid (GLA) into dihomo-.gamma.-linolenic acid (DGLA, C20:3.DELTA.8, 11, 14). The pYEELO1 introduced strain also had the 49 elongase activity of converting the .alpha.-linolenic acid (ALA) into eicosatrienoic acid (ETrA, C20:3.DELTA.11, 14, 17), and the linoleic acid (LA) into eicosadienoic acid (EDA, C20:3.DELTA.11, 14), and the 45 elongase activity of converting the eicosapentaenoic acid (EPA) into .omega.3 docosapentaenoic acid (.omega.3 DPA, C22:5.DELTA.7, 10, 13, 16, 19), and the arachidonic acid (AA) into docosatetraenoic acid (DTA, C22:4.DELTA.7, 10, 13, 16) (Table 1).

[0268] As for the pYEELO2 introduced strain, it was found that this strain had the .DELTA.5 elongase activity not found in the host, converting EPA to .omega.3 DPA (C22:5.DELTA.7, 10, 13, 16, 19), and AA to DTA. The pYEELO2 introduced strain also had a weak 46 elongase activity, converting STA to ETA, and GLA to DGLA (Table 1). These results confirmed that the TaELO1 was a .DELTA.6/.DELTA.9/.DELTA.5 elongase, and the TaELO2 was a .DELTA.5/.DELTA.6elongase, contrary to the results expected from the TaELO1 and TaELO2 substrate specificity in the phylogenetic analysis described in Example 2-3 and FIG. 2.

TABLE-US-00002 TABLE 1 mock TaELO1 TaELO2 LA addition (0.2 mM) LA 30.5 23.5 36.3 EDA 0.2 8.9 0.2 Conversion 27.4 efficiency (%) GLA addition (0.2 mM) GLA 44.0 7.6 43.6 DGLA 0.2 29.0 0.8 Conversion 79.3 1.9 efficiency (%) ARA addition (0.2 mM) ARA 30.9 23.2 8.9 ADA -- 5.8 13.6 Conversion 20.1 60.3 efficiency (%) ALA addition (0.2 mM) ALA 49.1 25.8 47.1 ETrA 0.2 17.9 0.3 Conversion 41 efficiency (%) STA addition (0.2 mM) STA 46.2 8.3 40.5 ETA 0.3 28.1 1.7 Conversion 77.2 4.0 efficiency (%) EPA addition (0.2 mM) EPA 42.0 31.2 13.1 DPA 0.1 19.6 24.5 Conversion 25.3 65.1 efficiency (%) Conversion efficiency (%) = 100 .times. product (area)/substrate (area) + product (area) (n = 1)

[Example 2-5] Obtaining TaELO2 ORF Upstream and Downstream Regions by PCR Genome Walking

[0269] The TaELO2 ORF upstream and downstream regions as the homologous recombination sites in a targeting vector for disrupting TaELO2 were obtained by using the PCR genome walking technique, as briefly described below.

[0270] T. aureum ATCC 34304 cell grown for 3 days using a GY liquid medium was rapidly frozen with liquid nitrogen, and ground into a powdery form with a mortar. Then, genomic DNA was extracted according to the method described in Non-Patent Document 18, and dissolved in a suitable amount of TE. Genomic DNA levels and purity were assayed by O.D.260 and O.D.280 measurements. This was followed by construction of a genomic DNA library by adding a cassette sequence with restriction enzyme sites to the genomic DNA cut with various restriction enzymes, using a TaKaRa LA PCR.TM. in vitro Cloning Kit (Takara Bio) according to the manufacturer's protocol. Then, by using the genomic DNA library as a template, a nested PCR was performed according to the manufacturer's protocol, using the forward oligonucleotide primers E2 XbaI (Example 2-4; SEQ ID NO: 21) and elo3-F1 (Example 2-2; SEQ ID NO: 10) or the reverse oligonucleotide primers E2 HindIII (Example 2-4; SEQ ID NO: 20) and elo3-R1 (Example 2-2; SEQ ID NO: 11) produced from the TaELO2 sequence, and the oligonucleotide primers complementary to the cassette sequence (attached to the kit). As a result, a 1,122-bp TaELO2 ORF upstream sequence (SEQ ID NO: 24), and a 1,204-bp TaELO2 ORF downstream sequence (SEQ ID NO: 25) were successfully obtained.

[Example 2-6] Construction of TaELO2 Targeting Vector Using Selection Marker Neor

[0271] A DNA fragment joining TaELO2 ORF upstream sequence/artificial Neor/TaELO2 ORF downstream sequence was produced by fusion PCR. The following oligonucleotide primers were used.

TABLE-US-00003 KO Pro F SmaI (SEQ ID NO: 26) (31 mer: 5'-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3') Pro R (SEQ ID NO: 27) (25 mer: 5'-GGT CGC GTT TAC AAA GCA GCG CAG C-3') SNeo F (SEQ ID NO: 28) (52 mer; 5'-GCT GCG CTG CTT TGT AAA CGC GAC CAT GAT TGA ACA GGA CGG CCT TCA CGC T-3') SNeoR (SEQ ID NO: 29) (52 mer; 5'-TCG GGA GCC AGC CGG AAA CAG GTT CAA AAG AAC TCG TCC AGG AGG CGG TAG A-3') Term F (SEQ ID NO: 30) (23 mer: 5'-ACC TGT TTC CGG CTG GCT CCC GA-3') KO Term R SmaI (SEQ ID NO: 31) (27 mer: 5'-ATC CCG GGG CCG AGA ACG GGG TCG CCC-3')

[0272] The oligonucleotide primers KO Pro F SmaI/Pro R were used for the amplification of the TaELO2 ORF upstream sequence using the T. aureum ATCC 34304 genomic DNA of Example 2-5 as a template. The oligonucleotide primers SNeo F/SNeo R were used for the amplification of the artificial Neor using artificial Neor as a template. The oligonucleotide primers Term F/KO Term R SmaI were used for the amplification of the TaELO2 ORF downstream sequence using the T. aureum ATCC 34304 genomic DNA of Example 2-5 as a template. The PCR reaction was performed at a denature temperature of 98.degree. C. for 10 seconds, and the annealing and the extension reaction were performed while appropriately adjusted according to the primer Tm and the amplification product length.

[0273] As a result, a 2, 696-bp sequence (SEQ ID NO: 32) joining TaELO2 ORF upstream sequence/artificial Neor/TaELO2 ORF downstream sequence was successfully obtained, and the sequence after TA cloning with a pGEM-T easy Vector (Promega) was used as a knockout vector, named pTKONeor.

[Example 2-7] Introduction of TKONeor into T. aureum ATCC 34304

[0274] The TaELO2 targeting vector pTKONeor using artificial Neor as a selection marker (Example 2-6) was used as a template, and the TaELO2 ORF upstream sequence/artificial Neor/TaELO2 ORF downstream sequence was amplified using a set of oligonucleotide primers KO Pro F SmaI (Example 2-6, SEQ ID NO: 26)/KO Term R SmaI (Example 2-6, SEQ ID NO: 31), and PrimeSTAR HS DNA polymerase (Takara Bio) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 3 min, 30 cycles/68.degree. C. 10 min/4.degree. C. .infin.]. The DNA fragments were extracted after electrophoresis using a 1% agarose gel, and dissolved in a suitable amount of TE after ethanol precipitation. The DNA fragment levels and the purity were assayed by O.D.260 and O.D.280 measurements. In the following, the DNA fragment will be referred to as TKONeor.

[0275] This was followed by DNA penetration using the gene-gun technique. Specifically, T. aureum ATCC 34304 was cultured in a GY liquid medium from the middle to late stage of the logarithmic growth phase at 25.degree. C., 150 rpm, and the supernatant was removed by centrifugation at 3,500.times.g, 4.degree. C. for 10 min. The resulting cells were resuspended in a GY liquid medium in 100 times the concentration of the original culture fluid, and a 20-.mu.1 portion of the cell suspension was evenly applied as a thin layer of about a 3-cm diameter on a 5-cm diameter PDA agar plate medium containing 1 mg/ml G418 (nacalai tesque). After drying, penetration was performed using a PDS-1000/He system (BioRad) under the following conditions.

[0276] Target distance: 6 cm

[0277] Vacuum: 26 inches Hg

[0278] Micro carrier size: 0.6 .mu.m

[0279] Rupture disk (penetration pressure): 1,100 psi

[0280] Thereafter, a PD liquid medium (100 .mu.l) was dropped onto the PDA agar plate medium, and the cells were spread and statically cultured. As a result, transfectants with the conferred G418 resistance were obtained at the efficiency of 4.7.times.10.sup.1 cfu/.mu.g DNA.

[Example 2-8] PCR Using TKONeor-Introduced Transfectant Genomic DNA as a Template

[0281] Seven colonies of transfectants were collected with a toothpick, and inoculated in a GY liquid medium containing 0.5 mg/ml G418 (nacalai tesque). After multiple subculturing, genomic DNA was extracted from the cells using the method of Example 2-5, and dissolved in a suitable amount of TE after ethanol precipitation. The levels of extracted genomic DNA and the purity were assayed by O.D.260 and O.D.280 measurements. By using the genomic DNAs of the transfectants and the wild-type strain as templates, a PCR was performed with various oligonucleotide primer sets. The following oligonucleotide primer sets were used.

[0282] (1) Neor detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR (Example 2-6; SEQ ID NO: 29)

[0283] (2) KO verification 1: KO Pro F SmaI (Example 2-6; SEQ ID NO: 26) and KO Term R SmaI (Example 2-6; SEQ ID NO: 31)

[0284] (3) KO verification 2: E2 KO ProF EcoRV (30 mer: 5'-GGA TAT CCC CCG CGA GGC GAT GGC TGC TCC-3') (SEQ ID NO: 33) and SNeoR

[0285] (4) KO verification 3: SNeoF and E2 KO Term R EcoRV (30 mer: 5'-TGA TAT CGG GCC GCG CCC TGG GCC GTA GAT-3') (SEQ ID NO: 34)

[0286] (5) TaELO2 amplification: E2 HindIII (Example 2-4; SEQ ID NO: 20) and E2 XbaI (Example 2-4; SEQ ID NO:21) (FIG. 3A)

[0287] Six out of the seven clones analyzed were transfectants by random integration, and the homologous recombination replacement of TaELO2 ORF with Neor was confirmed in the remaining clone (FIG. 3B, lanes 9 and 13). It was also found that this was accompanied by the simultaneous TaELO2 ORF amplification (FIG. 3B, lane 17). These results suggested the possibility that the T. aureum ATCC 34304 was a diploid or higher ploidy, or the TaELO2 was a multicopy gene.

[Example 2-9] Confirmation of TaELO2 Copy Number by Southern Blotting

[0288] The following experiments were conducted according to the methods described in DIG Application Manual [Japanese version] 8th, Roche Applied Science (Non-Patent Document 25). Specifically, the genomic DNA of the wild-type strain was cut with various restriction enzymes, and electrophoresed in 2.5 .mu.g per lane using a 0.7% SeaKemR GTGR agarose (Takara Bio). This was transferred to a nylon membrane (Hybond.TM.-N+, GE Healthcare), and hybridized at 48.degree. C. for 16 hours with DIG-labeled probes produced by using a PCR DIG Probe Synthesis Kit (Roche Applied Science). The following oligonucleotide primer set was used for the production of the DIG-labeled probes.

TABLE-US-00004 TaELO2 det F (SEQ ID NO: 35) (25 mer: 5'-GTA CGT GCT CGG TGT GAT GCT GCT C-3') TaELO2 det R (SEQ ID NO: 36) (24 mer: 5'-GCG GCG TCC GAA CAG GTA GAG CAT-3') [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 65.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]

[0289] Detection of the hybridized probes was made by using a chromogenic method (NBT/BCIP solution).

[0290] As a result, a single band was detected in all lanes treated with the various restriction enzymes (FIG. 4), suggesting that the TaELO2 was a single copy gene. The result thus suggested that the T. aureum ATCC 34304 was a diploid or higher ploidy.

[Example 2-10] Evaluation of TKONeor-Introduced Transfectants by Southern Blotting

[0291] Southern blotting was performed by using the method of Example 2-9. Specifically, the genomic DNAs of the wild-type strain and the transfectants digested with EcoRV and PstI were subjected to southern blotting using a chromogenic method (NBT/BCIP solution), using DIG-labeled probes PCR amplified with a set of oligonucleotide primers uprobe F (35 mer: 5'-ATC CGC GTA TAT ATC CGT AAA CAA CGG AAC ATT CT-3') (SEQ ID NO: 37) and uprobe R (26 mer: 5'-CTT CGG GTG GAT CAG CGA GCG ACA GC-3') (SEQ ID NO: 38) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 65.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. Here, in contrast to about a 1.2-kbp DNA fragment detected for the wild-type allele, about a 2.5-kbp DNA fragment was detected for the mutant allele that underwent the homologous recombination replacement of TaELO2 ORF with Neor (FIG. 5A).

[0292] Because the wild-type allele band was simultaneously detected with the mutant allele band in the transfectants (FIG. 5B), the analysis result suggested that the T. aureum ATCC 34304 was a diploid or higher ploidy.

[Example 2-11] Construction of TaELO2 Targeting Vector Using Selection Marker Hygr

[0293] A TaELO2 targeting vector was constructed with a selection marker Hygr to disrupt the remaining wild-type allele.

[0294] First, a fusion PCR was performed to join Hygr to a T. aureum ATCC 34304-derived ubiquitin promoter sequence. The following oligonucleotide primers were used.

TABLE-US-00005 ubi-600p F (SEQ ID NO: 39) (27 mer: 5'-GCC GCA GCG CCT GGT GCA CCC GCC GGG-3') ubi-hygro R (SEQ ID NO: 40) (59 mer: 5'-TCG CGGG TGA GTT CAG GCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3') ubi-hygro F (SEQ ID NO: 41) (57 mer; 5'-AGC GAC CTA AGC AAC ACT AGGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3') hygro R (SEQ ID NO: 42) (29 mer; 5'-CTA TTC CTT TGC CCT CGG ACG AGT GCT GG-3')

[0295] The oligonucleotide primers ubi-600p F/ubi-hygro R were used for the amplification of the T. aureum ATCC 34304-derived ubiquitin promoter sequence using the T. aureum ATCC 34304 genomic DNA of Example 2-5 as a template. The oligonucleotide primers ubi-hygro F/hygro R were used for the amplification of the artificial Hygr using pcDNA 3.1 Zeo (Invitogen) as a template. The PCR reaction was performed at a denature temperature of 98.degree. C. for 10 seconds, and the annealing and the extension reaction were appropriately adjusted according to the primer Tm and the amplification product length.

[0296] As a result, a 1,636-bp (SEQ ID NO: 43) joining T. aureum ATCC 34304-derived ubiquitin promoter sequence/Hygr was successfully obtained, and the sequence after TA cloning with a pGEM-T easy Vector (Promega) was named pTub600Hygr.

[0297] By using the pTub600Hygr as a template, a PCR was performed with PrimeSTAR HS DNA polymerase (Takara Bio) to prepare a T. aureum ATCC 34304-derived ubiquitin promoter sequence/Hygr DNA fragment containing NheI and XbaI sites added to the 5' end and the 3' end, respectively. The PCR was run under the following conditions using a set of oligonucleotide primers ubi-600 pF NheI (33 mer: 5'-GTG CTA GCC GCA GCG CCT GGT GCA CCC GCC GGG-3') (SEQ ID NO: 44) and hygro R XbaI (37 mer: 5'-GTT CTA GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3') (SEQ ID NO: 45) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 3 min, 30 cycles/68.degree. C. 10 min/4.degree. C. co]. Separately, by using the pTKONeor of Example 2-6 as a template, a PCR was performed with PrimeSTAR HS DNA polymerase (Takara Bio) to prepare a linear vector that did not contain the Neor of the pTKONeor of Experiment Example 2-6 and to which NheI and XbaI sites were added to the 3' end and the 5' end, respectively. The PCR was run under the following conditions using a set of oligonucleotide primers KO vec F XbaI (37 mer: 5'-GTT CTA GAC CTG TTT CCG GCT GGC TCC CGA GCC ATG C-3') (SEQ ID NO: 46) and KO vec R NheI (40 mer: 5'-GTG CTA GCG GTC GCG TTT ACA AAG CAG CGC AGC AAC AGA A-3') (SEQ ID NO: 47) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 3 min, 30 cycles/68.degree. C. 10 min/4.degree. C. .infin.]. The both DNA fragments were digested with restriction enzymes NheI and XbaI, and purified with an agarose gel to construct a cyclic vector using a Ligation Convenience Kit (Nippon Gene).

[0298] The TaELO2 targeting vector using Hygr as a selection marker thus constructed used the pGEM-T easy Vector (Promega) as the platform, and contained a 3,537-bp insert sequence (SEQ ID NO: 48) of TaELO2 ORF upstream sequence/T. aureum ATCC 34304-derived ubiquitin promoter sequence/Hygr/TaELO2 ORF downstream sequence. This was named pTKOub600Hygr.

[Example 2-12] Reintroduction of KOub600Hygr, and Evaluation of Transfectants by PCR Using Genomic DNA as Template, and by Southern Blotting and RT-PCR

[0299] The constructed TaELO2 targeting vector pTKOub600Hygr (Example 2-11) using Hygr as a selection marker was used as a template, and the TaELO2 ORF upstream sequence/T. aureum ATCC 34304-derived ubiquitin promoter sequence/Hygr/TaELO2 ORF downstream sequence was amplified with a PrimeSTAR HS DNA polymerase (TakaraBio), using a set of oligonucleotide primers KO Pro F SmaI (Example 2-6, SEQ ID NO: 26)/KO Term R SmaI (Example 2-8, SEQ ID NO: 31) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 3.5 min, 30 cycles/68.degree. C. 10 min/4.degree. C. .infin.]. The resulting DNA fragment was named KOub600Hygr. This was introduced to the transfectants obtained in Example 2-6 by using the technique described therein, and statically cultured on a 1 mg/ml G418 (nacalai tesque)-containing PDA agar plate medium for 24 hours. The cells were collected, and statically cultured on a PDA agar plate medium supplemented with 1 mg/ml G418 (nacalai tesque) and 2 mg/ml hygromycin B (Wako Pure Chemical Industries, Ltd.). As a result, large numbers of transfectants were obtained (introduction efficiency: 1.02.times.10.sup.3 cfu/.mu.g DNA).

[0300] Fifty clones were collected, and subcultured multiple times in a GY liquid medium supplemented with 1 mg/ml G418 (nacalai tesque) and 2 mg/ml hygromycin B (Wako Pure Chemical Industries, Ltd.). Then, genomic DNA was extracted by using the same technique used in Example 2-5, and dissolved in a suitable amount of TE after ethanol precipitation. The levels of extracted genomic DNA and the purity were assayed by O.D.260 and O.D.280 measurements. By using the genomic DNAs of the resulting transfectants and the wild-type strain as templates, a PCR was performed with various oligonucleotide primer sets [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 10 min/4.degree. C. .infin.]. The following oligonucleotide primer sets were used.

[0301] (1) TaELO2 ORF detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR (Example 2-6; SEQ ID NO: 29)

[0302] (2) KO verification: E2 KO Pro F EcoRV (Example 2-8; SEQ ID NO: 33) and ubi-hygro R (Example 2-11; SEQ ID NO: 40) (FIG. 6A).

[0303] It was suggested that 14 out of the 50 clones analyzed were transfectants that underwent homologous recombination through TaELO2 ORF replacement (FIG. 6B, arrow). It was also confirmed that the TaELO2 ORF was not amplified in these clones (FIG. 6C).

[0304] This was followed by southern blotting using the same technique used in Example 2-10. Specifically, the genomic DNAs of the wild-type strain and the transfectants digested with EcoRV and PstI were subjected to southern blotting using a chromogenic method (NBT/BCIP solution), using DIG-labeled probes prepared with a set of oligonucleotide primers uprobe F (SEQ ID NO: 37) and uprobe R (SEQ ID NO: 38). Here, about a 1.2-kbp DNA fragment was detected for the wild-type allele. In contrast, about a 2.5-kbp DNA fragment was detected for the mutant allele that underwent the homologous recombination replacement of TaELO2 ORF with Neor, and about a 1.9-kbp DNA fragment was detected for the mutant allele that underwent the homologous recombination replacement of TaELO2 ORF with Hygr (FIG. 7A).

[0305] The analysis revealed that the wild-type allele band of about a 2.5 kbp was absent in the resulting transfectants, and a new band, about 1.9 kbp, was detected for the mutant allele in which the TaELO2 ORF was replaced with Hygr (FIG. 7B).

[0306] Southern blotting using a chromogenic method (NBT/BCIP solution) was also performed for the genomic DNAs of the wild-type strain and the transfectants (clones 1, 8, 9, and 10) digested with EcoRV, using TaELO2-detecting DIG-labeled probes prepared by PCR using a set of oligonucleotide primers TaELO2 probe F (30 mer: 5'-ATG GCG ACG CGC ACC TCG AAG AGC GCT CCG-3') (SEQ ID NO: 49) and TaELO2 probe R (30 mer: 5'-AGG ATC ATC ATG AAC GTG TCG CTC CAG TCG-3') (SEQ ID NO: 50) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 65.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. Here, TaELO2 was detected as about a 2.5-kbp DNA fragment (FIG. 7A).

[0307] The analysis revealed that in contrast to the wild-type strain in which the TaELO2 was detected (FIG. 8, lane 1), TaELO2 was not detected in any of the transfectants (FIG. 8, lanes 2 to 5).

[0308] To examine the TaELO2 disruption at the mRNA level, TaELO2 mRNA detection was performed by RT-PCR. Total RNA was extracted from the cells of the wild-type strain and the transfectants (clones 1, 8, 9, and 10) cultured for 3 days in GY liquid media, using Sepasol-RNA I Super (nacalai tesque) as in Example 2-1. The total RNA (50 .mu.g) was cleaned up using an RNeasyMini Kit (QIAGEN) according to the manufacturer's protocol, and treated at 37.degree. C. for 1 hour using 50 U Recombinant DNase I (Takara Bio) to degrade and remove the contaminated genomic DNA. By using the resulting total RNA as a template, a single-stranded cDNA library was created using oligo (dT) primer (Novagen) and PrimeScript Reverse Transcriptase (Takara Bio) according to the manufacturers' protocols. By using the resulting single-stranded cDNA library as a template, the TaELO2 ORF was amplified with a set of oligonucleotide primers E2 HindIII (Example 2-4; SEQ ID NO: 20) and E2 XbaI (Example 2-4; SEQ ID NO:21), and LA taq Hot Start Version (Takara Bio) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 10 min/4.degree. C. .infin.].

[0309] It was found as a result that the TaELO2 mRNA detected in the wild-type strain (FIG. 9, lane 5) was not detected in any of the transfectants (clones 1, 8, 9, and 10) (FIG. 9, lanes 1 to 4).

[0310] As demonstrated above, TaELO2-deficient homozygotes with the complete disruption of TaELO2 were successfully obtained. It was also found that the T. aureum ATCC 34304 was a diploid.

[Example 2-13] Comparison of Fatty Acid Compositions of Wild-Type Strain and TaELO2-Deficient Homozygote

[0311] The fatty acid compositions of the TaELO2-deficient homozygote and the wild-type strain of Example 2-12 were compared by the GC analysis of methylesterificated fatty acids. Specifically, the cells of the TaELO2-deficient homozygotes and the wild-type strain cultured for 5 days in GY liquid media were collected, and the fatty acids from these cells were extracted and methylesterificated by using the methods described in Example 2-4, and subjected to GC analysis. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0312] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.

[0313] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0314] Carrier gas: He (1.3 mL/min).

[0315] As a result, the level of the EPA as a TaELO2 substrate showed about a two-fold increase in the TaELO2-deficient homozygote compared to the wild-type strain, whereas the level of the downstream metabolite DHA was lower than in the wild-type strain (FIG. 10).

[0316] The present invention is the first example of the modification of fatty acid compositions through disruption of genes that form the desaturase/elongase pathways in Labyrinthula. Specifically, the present invention has elucidated the involvement of the desaturase/elongase pathways in the PUFA biosynthesis in the Labyrinthula T. aureum, and shows that modification of fatty acid composition is possible by knocking out the constitutive genes. In the future, it would be possible to perform molecular breeding of Labyrinthulomycetes that selectively produce industrially useful PUFAs in large quantities in a PUFA biosynthetic pathway artificially created from combinations of genetic modifications such as overexpression of foreign desaturase/elongase genes, and PUFA-PKS gene knockouts.

Example 3

[0317] [Disruption of Parietichytrium sarkarianum C20 Elongase Gene]

[Example 3-1] Subcloning of SV40 Terminator Sequence

[0318] An SV40 terminator sequence was amplified with PrimeSTAR HS DNA polymerase (Takara Bio), using a pcDNA 3.1 Myc-His vector as a template. The PCR primers used are as follows. RHO58 was set on the SV40 terminator sequence, and contains BglII and BamHI linker sequences. RHO52 was set on the SV40 terminator sequence, and contains a BglII sequence [RHO58: 34mer: 5'-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCG A-3' (SEQ ID NO: 51), RHO52: 24mer: 5'-ACG CAA TTA ATG TGA GAT CTA GCT-3' (SEQ ID NO: 52)]. The sequence was cloned into a pGEM-T easy vector (Promega) after being amplified under the following conditions [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min]. The sequence was confirmed with a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER) after being amplified with Escherichia coli, and was named pRH27.

[0319] A plasmid (pRH27) containing the subcloned SV40 terminator sequence (342 bp, SEQ ID NO: 53) is shown in FIG. 11.

[Example 3-2] Production of Artificial Neomycin-Resistant Gene Cassette

[0320] The Thraustochytrium aureum ATCC 34304 strain was cultured in GY medium, and cells at the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,500.times.g for 5 min to obtain a pellet. The pellet was then disrupted after being frozen with liquid nitrogen. The cell disruption liquid was extracted with phenol, and precipitated with ethanol. The precipitate was then dissolved in a TE solution. The nucleic acids dissolved in the TE solution were treated with RNase at 37.degree. C. for 30 min to degrade the RNA, and extracted again with phenol. After ethanol precipitation, the precipitate was dissolved in a TE solution. The DNA concentration was calculated by measuring A260/280.

[0321] By using this as a template, a ubiquitin promoter sequence (619 bp, SEQ ID NO: 54) was amplified using a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used are as follows. RHO53 was set on the ubiquitin promoter sequence, and contains a BglII linker sequence. The TKO' contains the ubiquitin promoter sequence and an artificial neomycin-resistant gene sequence [RHO53: 36mer: 5'-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3' (SEQ ID NO: 55), TKO': 58mer: 5'-CGT GAA GGC CGT CCT GTT CAA TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3' (SEQ ID NO: 56)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min].

[0322] An artificial neomycin-resistant gene sequence (826 bp, SEQ ID NO: 57) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (TakaraBio), using the artificial neomycin-resistant gene sequence as a template. The PCR primers used are as follows. TKO2 contains the ubiquitin promoter sequence and the artificial neomycin-resistant gene sequence. RHO57 contains the artificial neomycin-resistant gene sequence, and has a BglII linker sequence [TKO2: 54mer: 5'-AGC GAC CTA AGC AAC ACT AGC CAA CAT GAT TGA ACA GGA CGG CCT TCA CGC TGG-3' (SEQ ID NO: 58), RHO57: 26mer: 5'-CAG ATC TCA AAA GAA CTC GTC CAG GA-3' (SEQ ID NO: 59)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min].

[0323] By using SEQ ID NOS: 54 and 57 as templates, a fusion PCR was performed with RHO53 (SEQ ID NO: 55) and RHO57 (SEQ ID NO: 59) according to the method described in Non-Patent Document 19. The product was amplified under the following conditions by using an LA taq Hot start version (Takara Bio) as an enzyme, and digested with BglII [PCR cycles: 94.degree. C. 2 min/94.degree. C. 20 sec, 55.degree. C. 30 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.) (FIG. 12).

[0324] The fused product Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-artificial neomycin-resistant gene sequence (1,395 bp, SEQ ID NO: 60) was digested with BglII, and ligated to the BamHI site of the pRH27 of Example 3-1. The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER) and named pRH31.

[0325] The product artificial neomycin-resistant gene cassette (pRH31) is shown in FIG. 13.

[Example 3-3] Production of Hygromycin-Resistant Gene Cassette

[0326] By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, a ubiquitin promoter sequence (617 bp, SEQ ID NO: 61) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used are as follows. RHO53 was set on the ubiquitin promoter sequence, and contains a BglII linker sequence. KSO8 contains the ubiquitin promoter sequence and a hygromycin-resistant gene sequence [RHO53: 36mer: 5'-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3' (Example 3-2; SEQ ID NO: 55), KSO8: 58mer: 5'-TCG CGG TGA GTT CAG GCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3' (SEQ ID NO: 62)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min].

[0327] By using a pcDNA 3.1/Hygro (invitrogen) as a template, a hygromycin-resistant gene (1,058 bp, SEQ ID NO: 63) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used are as follows. KSO7 contains the ubiquitin promoter sequence and the hygromycin-resistant gene sequence. RHO56 contains the hygromycin-resistant gene, and has a BglII linker sequence [KSO7: 56mer: 5'-AGC GAC CIA AGC AAC ACT AGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3' (SEQ ID NO: 64), RHO56: 36mer: 5'-CAG ATC TCT ATT CCT TTG CCC TCG GAC GAG TGC TGG-3' (SEQ ID NO: 65)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min].

[0328] By using SEQ ID NOS: 61 and 63 as templates, a fusion PCR was performed with RHO53 (Example 3-2; SEQ ID NO: 55) and RHO56 (SEQ ID NO: 65) according to the method described in Non-Patent Document 19. The product was amplified under the following conditions using an LA taq Hot start version (Takara Bio) as an enzyme, and digested with BglII [PCR cycles: 94.degree. C. 2 min/94.degree. C. 20 sec, 55.degree. C. 30 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.)] (FIG. 14).

[0329] The fused product Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-pcDNA 3.1/Hygro (invitrogen)-derived hygromycin-resistant gene (1,625 bp, SEQ ID NO: 66) was digested with BglII, and ligated to the BamHI site of the pRH27 of Example 3-1 (FIG. 11). The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH32.

[0330] The product artificial neomycin-resistant gene cassette (pRH32) is shown in FIG. 15.

[Example 3-4] Cloning of Parietichytrium C20 Elongase Gene

[0331] The Parietichytrium sarkarianum SEK364 genomic DNA extracted by using the method of Example 3-2 was extracted, and the genome was decoded.

[0332] A forward oligonucleotide (PsTaELO2 F1; 5'-CCT TCG GCG CTC CTC TTA TGT ATG T-3') (SEQ ID NO: 67) and a reverse oligonucleotide (PsTaELO2 R2; 5'-CAA TGC AAG AGG CGA ACT GGG AGA G-3') (SEQ ID NO: 68) were synthesized by targeting a conserved region in the C20 elongase gene. The oligonucleotides PsTaELO2 F1 and PsTaELO2 R2 were then used for a PCR performed with an LA taq Hot start version (TaKaRa) using the Parietichytrium sarkarianum SEK364 genomic DNA prepared by using the method of Example 3-2 as a template [PCR cycles: 98.degree. C. 1 min/98.degree. C. 10 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. The resulting specific amplification product was gel purified, and the base sequence was analyzed by direct sequencing. The sequence showed significant homology with the sequence of a known C20 elongase gene, suggesting that the sequence was a partial sequence of the Parietichytrium sarkarianum SEK364-derived C20 elongase gene.

[0333] This was followed by cloning of the Parietichytrium sarkarianum SEK364-derived C20 elongase gene by 3'- and 5'-RACE, as in Example 2-2. First, forward oligonucleotide primers (PsRACE F1; 5'-TGG GGC TCT GGA ACC GCT GCT TAC G-3') (SEQ ID NO: 69) and (PsRACE F2; 5'-CTT CCA GCT CTC CCA GTT CGC CTC T-3') (SEQ ID NO: 70), and reverse oligonucleotide primers (PsRACE R1; 5'-CGG GTT GTT GAT GTT GAG CGA GGT G-3') (SEQ ID NO: 71) and (PsRACE R2; 5'-CCC ACG CCA TCC ACG AGC ACA CCA C-3') (SEQ ID NO: 72) were designed. This was followed by 3'- and 5'-RACE using a synthetic adapter-specific oligonucleotide and the oligonucleotide PsRACE F1 or PsRACE R1, using the cDNA library created with the SMART.TM. RACE cDNA Amplification Kit (Clontech) as a template [PCR cycles: 94.degree. C. 30 sec 5 cycles/94.degree. C. 30 sec, 70.degree. C. 30 sec, 72.degree. C. 3 min, 5 cycles/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 3 min, 25 cycles/4.degree. C. .infin.]. By using the resulting both RACE products as templates, a nested PCR was performed using a synthetic adapter-specific oligonucleotide, and the oligonucleotide PsRACE F2 or PsRACE R2 [PCR cycles: 94.degree. C. 1 min/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 3 min, 25 cycles/72.degree. C. 10 min/4.degree. C. .infin.]. The resulting specific product was gel purified, and the base sequence was analyzed after being TA cloned with a pGEM-T easy Vector (Promega). The result confirmed that the product was a Parietichytrium sarkarianum SEK364-derived C20 elongase gene.

[0334] A sequence (957 bp, SEQ ID NO: 73) containing the C20 elongase gene sequence was amplified with an LA taq Hot start version (Takara Bio), using the Parietichytrium genomic DNA extracted by using the method of Example 3-2 as a template. The PCR primers used are as follows. RHO153 contains a start codon, and has a BamHI site as a linker sequence. RHO154 contains a stop codon, and has a BamHI site as a linker sequence [RHO153: 32 mer: 5'-CCC GGA TCC ATG GCA GCT CGC GTG GAG AAA CA-3' (SEQ ID NO: 74), RHO154: 33 mer: 5'-CCC GGA TCC TTA CTG AGC CTT CTT GGA GGT CTC-3' (SEQ ID NO: 75)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 2 min].

[0335] The resulting DNA fragment was cloned into a pGEM-T easy vector, and amplified with Escherichia coli. Then, the sequence was confirmed with a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

[0336] The 936-bp Parietichytrium C20 elongase gene (SEQ ID NO: 76) was cloned, and named pRH80 (FIG. 16). The amino acid sequence is represented by SEQ ID NO: 77.

[Example 3-5] Production of Base Plasmid for Parietichytrium C20 Elongase Gene Targeting Vector Production

[0337] By using the pRH80 produced in Example 3-4 (FIG. 16) as a template, amplification was performed with a PrimeSTAR Max DNA Polymerase (Takara Bio), using a primer set of the reverse orientation prepared for the insertion of the BglII site in a portion halfway along the C20 elongase gene sequence. The PCR primers used were as follows, and the both primers have BglII linker sequences [RHO155: 26 mer: 5'-ACA AAG ATC TCG ACT GGA CCG ACA CC-3' (SEQ ID NO: 78), RHO156: 27 mer: 5'-AGT CGA GAT CTT TGT CAG GAG GTG GAC-3' (SEQ ID NO: 79)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 56.degree. C. 15 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min]. After the amplification under these conditions, the product was digested with BglII, and allowed to self-ligate. The ligated sample was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH83. The 935-bp C20 elongase gene sequence with the inserted BglII site is represented by SEQ ID NO: 80.

[0338] The produced base plasmid (pRH83) for Parietichytrium C20 elongase gene targeting vector production is shown in FIG. 17.

[Example 3-6] Production of Targeting Vectors (Artificial Neomycin-Resistant Gene and Hygromycin-Resistant Gene)

[0339] The pRH31 (FIG. 13) of Example 3-2 was digested with BglII, and a DNA fragment containing an artificial neomycin-resistant gene cassette was ligated to the BglII site of the pRH83 (FIG. 17) of Example 3-5. This was named pRH85.

[0340] The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and a DNA fragment containing a hygromycin-resistant gene cassette was ligated to the BglII site of the pRH83 (FIG. 17) of Example 3-5. This was named pRH86.

[0341] The two targeting vectors (pRH85 and 86) produced are shown in FIG. 18.

[Example 3-7] Introduction of C20 Elongase Gene Targeting Vector

[0342] By using the two targeting vectors produced in Example 3-6 as templates, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using the RHO153 (Example 3-4; SEQ ID NO: 74) and RHO154 (Example 3-4; SEQ ID NO: 75) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was then calculated by measuring A260/280. The introduced fragment obtained from using the pRH85 (FIG. 18) of Example 3-6 as a template was 2,661 bp, and had the following sequence order: First half of Parietichytrium C20 elongase gene-SV40 terminator sequence-artificial neomycin-resistant gene sequence-ubiquitin promoter sequence-second half of Parietichytrium C20 elongase gene (SEQ ID NO: 81). The introduced fragment obtained from using the pRH86 (FIG. 18) of Example 3-6 as a template was 2,892 bp, and had the following sequence order: First half of Parietichytrium C20 elongase gene-SV40 terminator sequence-hygromycin-resistant gene sequence-ubiquitin promoter sequence-second half of Parietichytrium C20 elongase gene (SEQ ID NO: 82).

[0343] The Parietichytrium sarkarianum SEK364 strain was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,550 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied onto a PDA agar plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin). As a result, 10 to 20 drug resistant strains were obtained per penetration.

[Example 3-8] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0344] Genomic DNA was extracted from the Parietichytrium sarkarianum SEK364 strain, the C20 elongase gene hetero homologous recombinant, and the C20 elongase gene homo homologous recombinant (gene disrupted strain) by using the method described in Example 3-2, and the DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with an LA taq Hot start version (Takara Bio) to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected sizes of the amplification products are shown in FIG. 19. RHO184 and RHO185 were set on the upstream and downstream sides, respectively, of the C20 elongase. RHO142 and RHO143 were set on the artificial neomycin-resistant gene. RHO140 and RHO141 were set on the hygromycin-resistant gene [RHO140: 20 mer: 5'-GGT TGA CGG CAA TTT CGA TG-3' (SEQ ID NO: 83), RHO141: 22 mer: 5'-CCT CCT ACA TCG AAG CTG AAA G-3' (SEQ ID NO: 84), RHO142: 21 mer: 5'-CTT CTC GGG CTT TAT CGA CTG-3' (SEQ ID NO: 85), RHO143: 22 mer: 5'-TAA GGT CGG TCT TGA CAA ACA G-3' (SEQ ID NO: 86), RHO184: 24 mer: 5'-AGT AGT CCC CGA TTT GGT AGT TGA-3' (SEQ ID NO: 87), RHO185: 22 mer: 5'-GGC AGA GAG CAA AAA CAC GAG C-3' (SEQ ID NO: 88)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 4 min, 30 cycles/68.degree. C. 7 min].

[0345] C20 elongase knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele) and the artificial neomycin-resistant gene allele (NeoR allele) and the hygromycin-resistant gene allele (HygR allele) (FIG. 20).

[Example 3-9] Changes in Fatty Acid Composition by C20 Elongase Disruption

[0346] Parietichytrium sarkarianum SEK364, and the gene disrupted strains were cultured in GY media. Cells from the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,000 rpm for 10 min to form a pellet, suspended in 0.9% NaCl, and washed. The cells were further centrifuged at 4.degree. C., 3,000 rpm for 10 min, and the pellet was suspended in sterile water, and washed. After further centrifugation at 3,000 rpm for 10 min, the cells were freeze dried after removing the supernatant.

[0347] Then, 2 ml of methanolic KOH (7.5% KOH in 95% methanol) was added to the freeze dried cells, and, after being vortexed, the cells were ultrasonically disrupted (80.degree. C., 30 min). The cells were vortexed after adding sterile water (500 .mu.l), and vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was discarded. The cells were vortexed again after adding n-hexane (2 ml), and centrifuged at 3,000 rpm for 10 min. After discarding the upper layer, 6 N HCl (1 ml) was added to the remaining lower layer, and the mixture was vortexed. The mixture was vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. The collected upper layer was then concentrated and dried with nitrogen gas. The concentrated dry sample was incubated overnight at 80.degree. C. after adding 3 N methanolic HCl (2 ml).

[0348] The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml) was added. The mixture was vortexed after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. After adding a small amount of anhydrous sodium sulfate to the collected upper layer, the mixture was vortexed, and centrifuged at 3,000 rpm for 10 min. After collecting the upper layer, the upper layer was concentrated and dried with nitrogen gas. The concentrated dry sample was dissolved in n-hexane (0.5 ml), and 1 .mu.l of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0349] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0350] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0351] Carrier gas: He (1.3 mL/min).

[0352] As a result, knocking out the C20 elongase in the Parietichytrium sarkarianum SEK364 caused reduction of fatty acids of 22 or greater carbon chain length, and increased fatty acids of 20 carbon chain length (FIG. 21). FIG. 22 represents the proportions relative to the wild-type strain taken as 100%. FIG. 22 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: about 25.2%, DGLA: about 8.6%, ETA: about 0.6%, EPA: about 11.6%, n-6DPA: about 1.6%, and DHA: about 1.3%, which can also be described as the values of GC area such as n-6DPA/DTAL: 2.4, DHA/n-3DPA: 4.9, C20PUFA/C22PUFA: 11.9, and n-6PUFA/n-3PUFA 2.6. As can be seen from these results, the arachidonic acid increased about ten-fold, and the EPA showed about an eight-fold increase. The DPA and DHA reduced to about 1/4 and about 1/5, respectively.

Example 4

[0353] [Disruption of Thraustochytrium aureum PUFA PKS Pathway-Associated Gene OrfA]

[Example 4-1] Cloning of PUFA PKS Pathway-Associated Gene OrfA Upstream Sequence

[0354] Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304 by using the method described in Example 3-2, and the DNA concentration was calculated by measuring A260/280. By using this, a genome cassette library was produced with an LA PCR.TM. in vitro Cloning Kit (Takara Bio). A PCR lower primer [RHO20: 23mer: 5'-CGA TGA AAG GTC ACA GAA GAG TC-3' (SEQ ID NO: 89)] was set on the PUFA PKS pathway-associated gene OrfA described in Patent Document 7, and the DNA was amplified by using this primer in combination with the cassette primer attached to the kit [1st PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The 1st PCR amplification product was diluted 100 times, and amplified with the PCR lower primer [RHO20] and the nested primer attached to the kit [2nd PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

[0355] The 3,377-bp (SEQ ID NO: 91) DNA fragment containing the upstream 3,181 bp (SEQ ID NO: 90) of OrfA was cloned. The OrfA upstream DNA sequence was found to be 3,181 bp.

[Example 4-2] Cloning of PUFA PKS Pathway-Associated Gene OrfA Downstream Sequence

[0356] The genome cassette library produced in Example 4-1 was used as a template. The DNA was amplified by using the method described in Example 4-1, using a PCR upper primer [RHO21: 21mer: 5'-CAG GGC GAG CGA GTG TGG TTC-3' (SEQ ID NO: 92)] set on the PUFA PKS pathway-associated gene OrfA described in Patent Document 7. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). The 1, 204-bp DNA fragment (SEQ ID NO: 94) containing the downstream 1, 160 bp (SEQ ID NO: 93) of OrfA was cloned.

[0357] The DNA was amplified by using the method described in Example 4-1 using the PCR upper primer [RHO28: 20mer: 5'-TGA TGC CGA TGC TAC AAA AG-3' (SEQ ID NO: 95] produced on SEQ ID NO: 94. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

[0358] The 1,488-bp DNA fragment (SEQ ID NO: 96) containing the downstream sequence was cloned. The downstream DNA sequence of OrfA was found to be 2,551 bp in total (SEQ ID NO: 97).

[Example 4-3] Production of PUFA PKS Pathway-Associated Gene OrfA Targeting Vector

[0359] By using the genomic DNA of Thraustochytrium aureum ATCC 34304 as a template, an 18S rDNA sequence (1,835 bp, SEQ ID NO: 98) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are as follows. TMO30 was set on the 18S rDNA sequence. TMO31 contains the 18S rDNA sequence and an EF1.alpha. promoter sequence [TMO30: 30mer: 5'-CGA ATA TTC CTG GTT GAT CCT GCC AGT AGT-3' (SEQ ID NO: 99), TMO31: 46mer: 5'-GTA ACG GCT TTT TTT GAA TTG CAG GTT CAC TAC GCT TGT TAG AAA C-3' (SEQ ID NO: 100)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 2 min, 30 cycles/72.degree. C. 2 min]. Separately, by using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the EF1.alpha. promoter sequence (661 bp, SEQ ID NO: 101) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are as follows. TMO32 contains the 18S rDNA sequence and the EF1.alpha. promoter sequence. TMO33 contains the EF1.alpha. promoter sequence and an artificial neomycin-resistant gene sequence [TMO32: 46mer: 5'-GGT TTC CGT AGT GAA CCT GCA ATT CAA AAA AAG CCG TTA CTC ACA T-3' (SEQ ID NO: 102), TMO33: 46mer: 5'-GCG TGA AGG CCG TCC TGT TCA ATC ATC TAG CCT TCC TTT GCC GCT G-3' (SEQ ID NO: 103)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min].

[0360] By using the artificial neomycin-resistant gene as a template, the artificial neomycin-resistant gene sequence (835 bp, SEQ ID NO: 104) was amplified with a PrimeSTAR HS DNA polymerase (TakaraBio). The PCR primers used are as follows. TMO34 contains the EF1.alpha. promoter sequence and the artificial neomycin-resistant gene sequence. TMO35 contains the artificial neomycin-resistant gene sequence and the EF1.alpha. terminator sequence [TMO34: 45mer: 5'-CAT CGG CAA AGG AAG GCT AGA TGA TTG AAC AGG ACG GCC TTC ACG-3' (SEQ ID NO: 105), TMO 35: 46mer: 5'-GCG CAT AGC CGG CGC GGA TCT CAA AAG AAC TCG TCC AGG AGG CGG T-3' (SEQ ID NO: 106)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min].

[0361] Further, by using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the EF1.alpha. terminator sequence (1249 bp, SEQ ID NO: 107) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are as follows. TMO36 contains the artificial neomycin-resistant gene sequence and the EF1.alpha. terminator sequence. TMO37 was set within the EF1.alpha. terminator [TMO36: 46mer: 5'-TCC TGG ACG AGT TCT TTT GAG ATC CGC GCC GGC TAT GCG CCC GTG C-3' (SEQ ID NO: 108), TMO37: 30mer: 5'-CAC TGC AGC GAA AGA CGG GCC GTA AGG ACG-3' (SEQ ID NO: 109)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 2 min, 30 cycles/72.degree. C. 2 min].

[0362] By using SEQ ID NOS: 98, 101, 104, and 107 as templates, a fusion PCR was performed according to the method described in Non-Patent Document 19. An LA taq Hot start version (Takara Bio) was used as the enzyme. The TMO30 (SEQ ID NO: 99) and TMO33 (SEQ ID NO: 103) set, and the TMO34 (SEQ ID NO: 105) and TMO37 (SEQ ID NO: 109) set were used for the first amplification. The TMO30 (SEQ ID NO: 99) and TMO37 (SEQ ID NO: 109) set was used for the second amplification. The PCR reaction was performed at a denature temperature of 98.degree. C. for 10 seconds, and the annealing and the extension reaction were appropriately adjusted according to the primer Tm value and the amplification fragment length (FIG. 23).

[0363] The DNA fragment (FIG. 23, SEQ ID NO: 110, 4,453 bp) joined as above was cut at the EcoRI site of the T. aureum 18S rDNA, and the NcoI site of the T. aureum EF1.alpha. terminator, and ligated to a pGEM-T easy vector-derived vector. This was named pRH5 (FIG. 24).

[0364] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the DNA was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio), using PCR primers set in the upstream sequence found in Example 4-1 (SEQ ID NO: 90, and PUFA PKS pathway-associated gene OrfA described in Patent Document 7). The amplification yielded a 1,218-bp DNA fragment (SEQ ID NO: 111). This was used as the 5' homologous region of the targeting vector. The PCR primers used are as follows. An EcoRI site or a HindIII site was added as a linker sequence [RHO33: 32mer: 5'-CCC GAA TTC GGA CGA TGA CTG ACT GAC TGA TT-3' (SEQ ID NO: 112), RHO34: 28mer: 5'-CCC AAG CTT GTC TGC CTC GGC TCT TGG T-3' (SEQ ID NO: 113)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 57.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min].

[0365] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the DNA was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) using the PCR primers set in the downstream sequence (SEQ ID NO: 97) found in Example 4-2. The amplification yielded a 1,000-bp DNA fragment (SEQ ID NO: 114). This was used as the 3' homologous region of the targeting vector. The PCR primers used are as follows. A linker sequence NcoI site was added to the both primers [RHO29: 28mer: 5'-CCC CCA TGG TGT TGC TGT GGG ATT GGT C-3' (SEQ ID NO: 115), RHO30: 30mer: 5'-CCC CCA TGG CTC GGT TAC ATC TCT GAG GAA-3' (SEQ ID NO: 116)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 57.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min].

[0366] The amplified upstream sequence was joined to the EcoRI site and the HindIII site in the pRH5 of FIG. 24. The amplified downstream sequence was joined to the NcoI site. This vector was named pRH21.

[0367] The produced targeting vector (pRH21) using the artificial neomycin-resistant gene is shown in FIG. 25.

[Example 4-4] Production of PUFA PKS Pathway-Associated Gene OrfA Targeting Vector (Hygromycin-Resistant Gene)

[0368] By using the pRH32 (FIG. 15) of Example 3-3 as a template, a ubiqitin promoter-hygromycin-resistant gene fragment (1,632 bp, SEQ ID NO: 117) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used are as follows. RHO59 was set on the ubiquitin promoter, and a linker sequence HindIII site was added. RHO60 contains a hygromycin-resistant gene sequence stop codon, and has linker sequences SphI and SalI [RHO59: 36mer: 5'-CCC AAG CTT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3' (SEQ ID NO: 118), RHO60: 43mer: 5'-CCC GCA TGC GTC GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3' (SEQ ID NO: 119)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min].

[0369] The amplified fragment was joined to the HindIII and SphI sites of the pRH21 (FIG. 25) of Example 4-3 (FIG. 26, pRH30).

[0370] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the gene was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) using the PCR primers produced in the downstream sequence (SEQ ID NO: 97) found in Example 4-2. The amplification yielded a 1,000-bp DNA fragment (SEQ ID NO: 120). This was used as the 3' homologous region of the targeting vector. The PCR primers used are as follows. A linker sequence SalI site was added to the both primers [RHO61: 29mer: 5'-CCC GTC GAC GTG TTG CTG TGG GAT TGG TC-3' (SEQ ID NO: 121), RHO62: 29mer: 5'-CCC GTC GAC TCG GTT ACA TCT CTG AGG AA-3' (SEQ ID NO: 122)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 57.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min].

[0371] The amplified downstream sequence was joined to the SalI site of pRH30 (FIG. 26). This was named pRH33. The produced targeting vector (pRH33) using the hygromycin-resistant gene is shown in FIG. 27.

[Example 4-5] Introduction of PUFA PKS Pathway-Associated Gene OrfA Targeting Vector

[0372] By using the targeting vectors produced in Examples 4-3 and 4-4 as templates, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio) using the RHO30 (Example 4-3; SEQ ID NO: 116) and RHO33 (Example 4-3; SEQ ID NO: 112) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH21 (FIG. 25) of Example 4-3 as a template was 3,705 bp, and had the following sequence order: Thraustochytrium aureum OrfA gene upstream-EF1.alpha. promoter sequence-artificial neomycin-resistant gene sequence-Thraustochytrium aureum OrfA gene downstream (SEQ ID NO: 123). The introduced fragment obtained from using the pRH33 (FIG. 27) of Example 4-4 as a template was 3,826 bp, and had the following sequence order: Upstream of Thraustochytrium aureum OrfA gene-ubiquitin promoter sequence-hygromycin-resistant gene sequence-downstream of Thraustochytrium aureum OrfA gene (SEQ ID NO: 124).

[0373] The Thraustochytrium aureum ATCC 34304 strain was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with the introduced gene were applied onto a PDA agar plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin). As a result, 100 to 200 drug resistant strains were obtained per penetration.

[Example 4-6] Identification of PUFA PKS Pathway-Associated Gene OrfA Gene Targeting Homologous Recombinant

[0374] Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304, the hetero homologous recombinant, and the homo homologous recombinant (PKS pathway-associated gene disrupted strain) by using the method described in Example 3-2. The DNA concentration was calculated by measuring A260/280.

[0375] The genomic DNA was cut with restriction enzymes, and electrophoresed in about 2 to 3 .mu.g per well with a 0.7% SeaKem GTG agarose gel (Takara Bio). This was transferred to a nylon membrane, and hybridized at 54.degree. C. for 16 hours with the probes produced by using a DIG system (Roche Applied Science). The following primers were used for the probe production.

TABLE-US-00006 5' end (SEQ ID NO: 125) [RHO37: 22mer: 5'-GAA GCG TCC CGT AGA TGT GGT C-3', (SEQ ID NO: 126) RH038: 21mer: 5'-GCC CGA GAG GTC AAA GTA CGC-3'] 3' end (SEQ ID NO: 127) [RHO39: 20mer: 5'-GCG AGC CCA GGT CCA CTT GC-3', (SEQ ID NO: 128) RHO40: 22mer: 5'-CAG CCC GAT GAA AAA CTT GGT C-3'] PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. 2 min, 30 cycles/ 72.degree. C. 3 min

[0376] The restriction enzymes used and the probe positions are as shown in FIG. 28. Detection of the hybridized probes was made by using the chromogenic method (NBT/BCIP solution).

[0377] Bands of the sizes expected from the homologous recombination of the drug resistant genes were observed in the analyses of both the 5' end and the 3' end (FIG. 29).

[Example 4-7] Changes in Fatty Acid Composition by Disruption of PUFA PKS Pathway-Associated Gene OrfA

[0378] The Thraustochytrium aureum ATCC 34304 and the gene disrupted strain were cultured and freeze dried according to the methods of Example 3-9, and the fatty acids were methylesterificated, and GC analyzed.

[0379] FIG. 30 represents changes in fatty acid composition. FIG. 31 represents the proportions relative to the wild-type strain taken as 100%. FIG. 31 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 3.1%, DGLA: 0.2%, ETA: 0.04%, EPA: 6.8%, n-6DPA: 10.7%, and DHA: 22.6%, which can also be described as the values of GC area such as n-6DPA/DTA: 5.3, DHA/n-3DPA: 20.0, C20PUFA/C22PUFA: 0.3, and n-6PUFA/n-3PUFA: 0.5. As can be seen from these results, disrupting the PUFA PKS pathway-associated gene OrfA in the Thraustochytrium aureum tended to increase the DPA (C22: 5n-6) and decrease the DHA (C22: 6n-3).

Example 5

[0380] [Disruption of C20 Elongase Gene in Thraustochytrium aureum OrfA Disrupted Strain]

[Example 5-1] Cloning of Upstream Sequence of Thraustochytrium aureum C20 Elongase Gene

[0381] The genome cassette library produced in Example 4-1 was used as a template. A PCR lower primer [RHO71: 22mer: 5'-GGG AGC GCA GGG AAA ACG GTC T-3' (SEQ ID NO: 129)] was produced on the C20 elongase gene upstream sequence (SEQ ID NO: 24) of Example 2-5, and the gene was amplified by using this primer with the cassette primer attached to the kit used in Example 4-1 [1st PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The 1st PCR amplification product was diluted 100 times, and the gene was amplified by using the PCR lower primer [RHO72: 20mer: 5'-CCA GCC CAC GTC GTC GGA GC-3' (SEQ ID NO: 130)] with the nested primer attached to the kit used in Example 4-1 [2nd PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

[0382] The 2,297-bp DNA fragment (SEQ ID NO: 131) containing the upstream-3,277 bp to -981 bp region of the C20 elongase gene was cloned.

[Example 5-2] Cloning of C20 Elongase Gene Downstream Sequence

[0383] The genome cassette library produced in Example 4-1 was used as a template. A PCR upper primer [RHO87: 23 mer: 5'-GCC GCT CAT GCC CAC GCT CAA AC-3' (SEQ ID NO: 132)] was produced on the C20 elongase gene downstream sequence (SEQ ID NO: 25) of Example 2-5, and the gene was amplified by using this primer with the cassette primer attached to the kit used in Example 4-1 [1st PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The 1st PCR amplification product was diluted 100 times, and the gene was amplified by using the PCR lower primer [RHO73: 23 mer: 5'-CTT TCG GCT GCC AGG AAT CTA CG-3' (SEQ ID NO: 133)] with the nested primer attached to the kit used in Example 4-1 [2nd PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 56.degree. C. 30 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 5 min]. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

[0384] The 2,189-bp DNA fragment (SEQ ID NO: 134) containing the downstream 1,106 bp to 3,294 bp region of the C20 elongase gene was cloned.

[Example 5-3] Production of Blasticidin-Resistant Gene Cassette

[0385] A ubiquitin promoter sequence (618 bp, SEQ ID NO:135) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio), using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template. The PCR primers used are as follows. RHO53 was set on the ubiquitin promoter sequence, and contains a BglII linker sequence (Example 3-2, SEQ ID NO: 55). RHO48 contains the ubiquitin promoter sequence and a blasticidin-resistant gene sequence [RHO48: 58mer: 5'-CTT CTT GAG ACA AAG GCT TGG CCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3' (SEQ ID NO: 136)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min].

[0386] By using pTracer-CMV/Bsd/lacZ as a template, the blasticidin-resistant gene (432 bp, SEQ ID NO: 137) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer. The PCR primers used are as follows. RHO47 contains the ubiquitin promoter sequence and the blasticidin-resistant gene sequence. RHO49 contains the blasticidin-resistant gene sequence, and has a BglII linker sequence [RHO47: 54mer:5'-AGC GAC CTA AGC AAC ACT AGC CAA CAT GGC CAA GCC TTT GTC TCA AGA AGA ATC-3' (SEQ ID NO: 138), RHO49: 38mer: 5'-CCC AGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG-3' (SEQ ID NO: 139)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min].

[0387] By using SEQ ID NOS: 135 and 137 as templates, a fusion PCR was performed with RHO53 (Example 3-2, SEQ ID NO: 55) and RHO49 (SEQ ID NO: 139) according to the method described in Non-Patent Document 19. An LA taq Hot start version (Takara Bio) was used as the enzyme. After the amplification performed under the following conditions, the product was digested with BglII [PCR cycles: 94.degree. C. 2 min/94.degree. C. 20 sec, 55.degree. C. 30 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.)] (FIG. 32).

[0388] The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-pTracer-CMV/Bsd/lacZ-derived blasticidin-resistant gene (1,000 bp, SEQ ID NO: 140) was digested with BglII, and ligated to the BamHI site of the pRH27 (FIG. 11) of Example 3-1. The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH38.

[0389] The product blasticidin-resistant gene cassette (pRH38) is shown in FIG. 33.

[Example 5-4] Production of GFP-Fused Zeocin-Resistant Gene Cassette

[0390] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, a ubiquitin promoter sequence (812 bp, SEQ ID NO: 141) was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used are as follows. TMO38 was set on the ubiquitin promoter sequence. TMO39 contains the ubiquitin promoter sequence and an enhanced GFP gene sequence [TMO38: 29mer: 5'-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3' (SEQ ID NO: 142), TMO39: 41mer: 5'-TCC TCG CCC TTG CTC ACC ATG TTG GCT AGT GTT GCT TAG GT-3' (SEQ ID NO: 143)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min].

[0391] By using the enhanced GFP gene sequence (clontech) as a template, an enhanced GFP gene sequence (748 bp, SEQ ID NO: 144) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are as follows. TMO40 contains the ubiquitin promoter sequence and the enhanced GFP gene sequence. RHO91 contains the enhanced GFP sequence and a zeocin-resistant gene sequence [TMO40: 41mer: 5'-ACC TAA GCA ACA CTA GCC AAC ATG GTG AGC AAG GGC GAG GA-3' (SEQ ID NO: 145), RHO91: 58mer: 5'-GAA CGG CAC TGG TCA ACT TGG CGT CCA TGC CGA GAG TGA TCC CGG CGG CGG TCA CGA A-3' (SEQ ID NO: 146)] [PCR cycles: 98.degree. C. 10 sec/98.degree. C. 10 sec, 58.degree. C. 30 sec, 72.degree. C. 2 min, 30 cycles/72.degree. C. 2 min].

[0392] By using SEQ ID NOS: 141 and 144 as templates, a fusion PCR was performed with an LA taq Hot start version (Takara Bio) according to the method described in Non-Patent Document 19. TMO38 (SEQ ID NO: 142) and RHO91 (SEQ ID NO: 146) were used as primers, and the reaction was performed under the following conditions [PCR cycles: 94.degree. C. 2 min/94.degree. C. 20 sec, 55.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.)] (FIG. 34, 1,519 bp, SEQ ID NO: 147).

[0393] By using SEQ ID NO: 147 as a template, the ubiquitin promoter sequence-enhanced GFP gene sequence (1,319 bp, SEQ ID NO: 148) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The primers used are as follows. RHO53 (Example 3-2, SEQ ID NO: 55) contains the ubiquitin promoter sequence, and has a BglII site. RHO91 (SEQ ID NO: 146) contains the enhanced GFP sequence and the zeocin-resistant gene sequence [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min].

[0394] By using pcDNA3.1 Zeo(+) as a template, the zeocin-resistant gene sequence (408 bp, SEQ ID NO: 149) was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). RHO92 contains the enhanced GFP sequence and the zeocin-resistant gene sequence. RHO64 contains the zeocin-resistant gene sequence, and has a BglII site [RHO92: 54mer: 5'-CGC CGC CGG GAT CAC TCT CGG CAT GGA CGC CAA GTT GAC CAG TGC CGT TCC GGT-3' (SEQ ID NO: 150), RHO64: 38mer: 5'-CCC AGA TCT CAG TCC TGC TCC TCG GCC ACG AAG TGC AC-3' (SEQ ID NO: 151)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 1 min].

[0395] By using SEQ ID NOS: 148 and 149 as templates, a fusion PCR was performed with an LA taq Hot start version (Takara Bio) according to the method described in Non-Patent Document 19. RHO53 (Example 3-2, SEQ ID NO: 55) and RHO64 (SEQ ID NO: 151) were used as primers, and the reaction was performed under the following conditions [PCR cycles: 94.degree. C. 2 min/94.degree. C. 20 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.)] (FIG. 35).

[0396] The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-enhanced GFP gene-pcDNA3.1 Zeo(+)-derived zeocin-resistant gene (FIG. 35, 1,677 bp, SEQ ID NO: 152) was digested with BglII, and ligated to the BamHI site of the pRH27 (FIG. 11) of Example 3-1. The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH51.

[0397] The product GFP-fused zeocin-resistant gene cassette (pRH51) is shown in FIG. 36.

[Example 5-5] Production of Base Plasmid for C20 Elongase Gene Targeting Vector Production

[0398] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the C20 elongase gene and the nearby sequences (2,884 bp, SEQ ID NO: 153) were PCR amplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are as follows. The both primers contain EcoRI linker sequences. KSO9 was set upstream of the C20 elongase gene (SEQ ID NO: 24), and KSO10 downstream of the C20 elongase gene (SEQ ID NO: 25) [KSO9: 50mer: 5'-CCC GAA TTC ACT AGT GAT TCT CCC GGG TGG ACC TAG CGC GTG TGT CAC CT-3' (SEQ ID NO: 154), KSO10: 40mer: 5'-CCC GAA TTC GAT TAT CCC GGG GCC GAG AAC GGG GTC GCC C-3' (SEQ ID NO: 155)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 3.5 min, 30 cycles/68.degree. C. 10 min]. A PrimeSTAR HS DNA Polymerase (Takara Bio) was used as the enzyme. After the amplification, the product was digested with EcoRI, and cloned into the EcoRI site of the pBluescript(SK) (stratagene) vector. After amplification with Escherichia coli, the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER) (FIG. 37).

[0399] By using the plasmid of FIG. 37 as a template, amplification was performed with a PrimeSTAR Max DNA Polymerase (TakaraBio), using a primer set of the reverse orientation prepared for the deletion of the C20 elongase gene sequence portion and the insertion of a BglII site (1,939 bp, SEQ ID NO: 156). The PCR primers used are as follows. The both primers have BglII linker sequences [RHO69: 38mer: 5'-CCC AGA TCT ACC TGT TTC CGG CTG GCT CCC GAG CCA TG-3' (SEQ ID NO: 157), RHO70: 38mer: 5'-CCC AGA TCT GGT CGC GTT TAC AAA GCA GCG CAG CAA CA-3' (SEQ ID NO: 158)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. sec, 68.degree. C. 1.5 min, 30 cycles/68.degree. C. 1.5 min]. After the amplification performed under these conditions, the product was digested with BglII, and allowed to self ligate. The ligated sample was amplified with Escherichia coli, and the sequence was confirmed with a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH40.

[0400] The produced base plasmid (pRH40) for the production of the C20 elongase gene targeting vector is shown in FIG. 38.

[Example 5-6] Production of Targeting Vectors (Blasticidin-Resistant Gene and GFP-Fused Zeocin-Resistant Gene)

[0401] The pRH38 (FIG. 33) of Example 5-3 was digested with BglII, and the DNA fragment containing the blasticidin-resistant gene cassette was ligated to the BglII site of the pRH40 (FIG. 38) of Example 5-5. This was named pRH43.

[0402] The pRH51 (FIG. 36) of Example 5-4 was digested with BglII, and the DNA fragment containing the GFP-fused zeocin-resistant gene cassette was ligated to the BglII site of the pRH40 (FIG. 38) of Example 5-5. This was named pRH54.

[0403] The two targeting vectors (pRH43 and 54) produced are shown in FIG. 39.

[Example 5-7] Introduction of C20 Elongase Gene Targeting Vector into Thraustochytrium aureum OrfA Disrupted Strain

[0404] By using the two targeting vectors produced in Example 5-6 as templates, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using KSO11 and KSO12 as primers. KSO11 was set upstream of the Thraustochytrium aureum C20 elongase gene, and KSO12 downstream of the Thraustochytrium aureum C20 elongase gene [KSO11: 31mer: 5'-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3' (SEQ ID NO: 159), KSO12: 27mer: 5'-ATC CCG GGG CCG AGA ACG CCC TCG CCC-3' (SEQ ID NO: 160)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH43 (FIG. 39) of Example 5-6 as a template was 3,215 bp, and had the following sequence order: Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitin promoter-blasticidin-resistant gene sequence-SV40 terminator sequence-downstream of Thraustochytrium aureum C20 elongase gene (SEQ ID NO: 161). The introduced fragment obtained from using the pRH54 (FIG. 39) of Example 5-6 as a template was 3,887 bp, and had the following sequence order: Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitin promoter-enhanced GFP gene sequence-zeocin-resistant gene sequence-SV40 terminator sequence-downstream of Thraustochytrium aureum C20 elongase gene (SEQ ID NO: 162).

[0405] The disrupted strain of the PUFA PKS pathway-associated gene OrfA gene described in Example 4 was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with the introduced gene were applied onto a PDA agar plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin). As a result, 100 to 200 drug resistant strains were obtained per penetration.

[Example 5-8] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0406] Genomic DNA was extracted from the Thraustochytrium aureum and the C20 elongase gene disrupted strain of the Thraustochytrium aureum OrfA disrupted strain by using the method described in Example 3-2. The DNA concentration was calculated by measuring A260/280.

[0407] The genomic DNA was cut with restriction enzymes, and electrophoresed in about 2 to 3 .mu.g per well in a 0.7% SeaKem GTG agarose gel (Takara Bio). This was transferred to a nylon membrane, and hybridized at 51.degree. C. for 16 hours with the probes produced by using a DIG system (Roche Applied Science). The following primers were used for the probe production.

TABLE-US-00007 5' end [RHO94: 21mer: (SEQ ID NO: 163) 5'-ACG TCC GCT TCA AAC ACC TCG-3', RHO95: 24mer: (SEQ ID NO: 164) 5'-TCG GAA CAA CTG GAA CAA CTA AAG-3'] 3' end [RHO96: 22mer: (SEQ ID NO: 165) 5'-ATG TCG CTC TCC TTC TTC TCA G-3', RHO97: 21mer: (SEQ ID NO: 166) 5'-TCG GCT CCT GGA AAG TGC TCT-3'] PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 58.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min

[0408] The restriction enzymes used and the probe positions are as shown in FIG. 40. Detection of the hybridized probes was made by using a chromogenic method (NBT/BCIP solution).

[0409] Bands of the sizes expected from the homologous recombination of the drug resistant genes were observed in the analyses of both the 5' end and the 3' end (FIG. 41). It was found by the experiment that the Thraustochytrium aureum ATCC 34304 strain did not become auxotrophic even with the deletion of the PKS pathway-associated gene OrfA and the C20 elongase gene.

[Example 5-9] Changes in Fatty Acid Composition by Disruption of C20 Elongase Gene in Thraustochytrium aureum OrfA Disrupted Strain

[0410] The Thraustochytrium aureum ATCC 34304 and the gene disrupted strain were cultured and freeze dried according to the method of Example 3-9, and the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0411] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0412] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0413] Carrier gas: He (1.3 mL/min).

[0414] Changes in fatty acid composition are represented in FIG. 42. FIG. 43 represents the proportions relative to the wild-type strain taken as 100%. FIG. 43 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 19.5%, DGLA: 1.8%, ETA: 0.3%, EPA: 24.9%, n-6D PA: 5.9%, and DHA: 6.8%, which can also be described as the values of GC area such as n-6DPA/DTA: 12.6, DHA/n-3DPA: 11.7, C20PUFA/C22PUFA: 3.4, and n-6PUFA/n-3PUFA: 0.8.

[0415] As can be seen from these results, disrupting the C20 elongase gene in the Thraustochytrium aureum OrfA disrupted strain increased the C20:4n-6 (AA) about eight-fold, and the C20:5n3 (EPA) about four-fold, and decreased the C22:6n-3 (DHA) to about 1/5.

Example 6

[0416] [Expression of .omega.3 Desaturase Gene in Thraustochytrium aureum OrfA Disrupted Strain]

[Example 6-1] Cloning of Saprolegnia diclina-Derived .omega.3 Desaturase Gene and Production of Gene Expression Plasmid

[0417] Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304 by using the method of Example 3-2, and the DNA concentration was calculated by measuring A260/280. By using this as a template, the ubiquitin promoter sequence (longer) (812 bp, SEQ ID NO: 167) was amplified with an LA Taq with GC Buffer (Takara Bio, Buffer II was used). The PCR primers used are as follows. TMO42 was set on the ubiquitin promoter sequence, upstream of RHO53 (Example 3-2, SEQ ID NO: 55), and contains a KpnI linker sequence. TMO43 contains the ubiquitin promoter sequence and a Saprolegnia diclina-derived .omega.3 desaturase gene sequence [TMO42: 29mer: 5'-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3' (SEQ ID NO: 168), TMO43: 45mer: 5'-TTC GTC TTA TCC TCA GTC ATG TTG GCT AGT GTT GCT TAG GTC GCT-3' (SEQ ID NO: 169)] [PCR cycles: 96.degree. C. 2 min/98.degree. C. 20 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min].

[0418] Then, Saprolegnia diclina was cultured in a medium (adjusted with deionized water) containing D-Glucose (31.8 g) and yeast extract (10.6 g) per liter. Cells in the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,500.times.g for 5 min to form a pellet, and disrupted by being frozen with liquid nitrogen. After being extracted with phenol, the cell disruption liquid was precipitated with ethanol, and the precipitate was dissolved in a TE solution. The nucleic acids dissolved in the TE solution were treated with RNase at 37.degree. C. for 30 min to degrade RNA. After being reextracted with phenol, the product was precipitated with ethanol, and the precipitate was dissolved in a TE solution. The DNA purity and concentration were calculated by measuring A260/280. By using the resulting Saprolegnia diclina genomic DNA as a template, the Saprolegnia diclina-derived .omega.3 desaturase gene sequence (1,116 bp, SEQ ID NO: 170) was amplified with an LA Taq with GC Buffer (Takara Bio, Buffer II was used). The PCR primers used are as follows. TMO44 contains the ubiquitin promoter sequence and the Saprolegnia diclina-derived .omega.3 desaturase gene sequence. TMO45 contains the Saprolegnia diclina-derived .omega.3 desaturase gene sequence and the ubiquitin terminator [TMO44: 43mer: 5'-CCT AAG CAA CAC TAG CCA ACA TGA CTG AGG ATA AGA CGA AGG T-3' (SEQ ID NO: 171), TMO45: 40mer: 5'-ATA CTA CAG ATA GCT TAG TTT TAG TCC GAC TTG GCC TTG G-3' (SEQ ID NO: 172)] [PCR cycles: 96.degree. C. 2 min/98.degree. C. 20 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min 30 sec, 30 cycles/72.degree. C. 1 min 30 sec].

[0419] By using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the ubiquitin terminator sequence (614 bp, SEQ ID NO: 173) was amplified with an LA Taq with GC Buffer (Takara Bio, Buffer II was used). The primers used are as follows. TMO46 contains the Saprolegnia diclina-derived .omega.3 desaturase gene sequence and the ubiquitin terminator. TMO47 was designed on the ubiquitin terminator sequence, and contains a KpnI linker sequence [TMO46: 44mer: 5'-CCA AGG CCA AGT CGG ACT AAA ACT AAG CTA TCT GTA GTA TGT GC-3' (SEQ ID NO: 174), TMO47: 45mer: 5'-TCG GTA CCA CCG CGT AAT ACG ACT CAC TAT AGG GAG ACT GCA GTT-3' (SEQ ID NO: 175)] [PCR cycles: 96.degree. C. 2 min/98.degree. C. 20 sec, 60.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min].

[0420] By using SEQ ID NOS: 167, 170, and 173 as templates, a fusion PCR was performed with TMO42 (SEQ ID NO: 168) and TMO47 (SEQ ID NO: 175) according to the method described in Non-Patent Document 19. An LA Taq with GC Buffer (Takara Bio, Buffer II was used) was used as the enzyme, and the amplification was performed under the following conditions [PCR cycles: 96.degree. C. 2 min/98.degree. C. 20 sec, 55.degree. C. 30 sec, 68.degree. C. 3 min, 30 cycles/68.degree. C. 3 min (1.degree. C./10 sec from 55.degree. C. to 68.degree. C.] (FIG. 44, 2,463 bp, SEQ ID NO: 176).

[0421] By using the pRH38 (FIG. 33) of Example 5-3 as a template, a PCR was performed with RHO84 (SEQ ID NO: 177, the sequence is presented below) and RHO52 (Example 3-1, SEQ ID NO: 52). RHO84 was set on the ubiquitin promoter, and has a KpnI linker sequence. RHO52 was set on the SV40 terminator sequence, and has a BglII linker. An LA taq Hot start version was used as the enzyme, and, after the amplification performed under the following conditions, the product was cloned into a pGEM-T easy vector [RHO84: 36mer: 5'-CCC GGT ACC GCC GCA GCG CCT GGT GCA CCC GCC GGG-3' (SEQ ID NO: 177)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min 30 sec, 30 cycles/68.degree. C. 3 min]. After amplification with Escherichia coli, the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH45 (FIG. 45).

[0422] The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-Saprolegnia diclina-derived .omega.3 desaturase gene-Thraustochytrium aureum ATCC 34304-derived ubiquitin terminator (SEQ ID NO: 176; FIG. 44) was digested with KpnI, and ligated to the KpnI site of pRH45 (FIG. 45). The resulting plasmid was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH48.

[0423] The product Saprolegnia diclina-derived .omega.3 desaturase gene expression plasmid (pRH48) is shown in FIG. 46.

[Example 6-2] Introduction of Saprolegnia diclina-Derived .omega.3 Desaturase Expression Plasmid into Thraustochytrium aureum OrfA Disrupted Strain

[0424] By using the targeting vector produced in Example 6-1 as a template, DNA was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using TMO42 (SEQ ID NO: 168) and RHO52 (Example 3-1, SEQ ID NO: 52) as primers [PCR cycles: 94.degree. C. 30 sec, 72.degree. C. 1 min, 5 cycles/94.degree. C. 30 sec, 70.degree. C. 30 sec, 72.degree. C. 1 min, 5 cycles/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 1 min, 25 cycles/72.degree. C. 2 min]. The amplification product was collected from the 1.0% agarose gel, and precipitated with ethanol. The precipitate was then dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained by PCR was 3,777 bp, and had the following sequence order: ubiquitin promoter-.omega.3 desaturase gene-ubiquitin terminator-ubiquitin promoter-blasticidin-resistant gene sequence-SV40 terminator sequence (SEQ ID NO: 178).

[0425] The Thraustochytrium aureum OrfA disrupted strain produced in Example 4 was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with the introduced gene were applied to a PDA agar plate medium (containing 0.2 mg/ml blasticidin). As a result, 20 to 30 drug resistant strains were obtained per penetration.

[Example 6-3] Acquisition of Saprolegnia diclina-Derived .omega.3 Desaturase Gene Expression Strain

[0426] Genomic DNA was extracted from the Thraustochytrium aureum OrfA disrupted strain produced in Example 3 and the .omega.3 desaturase gene expressing strain by using the method described in Example 3-2. The DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with an LA taq Hot start version to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected size of the amplification product are shown in FIG. 47. TMO42 (Example 6-1, SEQ ID NO: 168) was set on the ubiquitin promoter. RHO49 (Example 5-3, SEQ ID NO: 139) was set on the blasticidin-resistant gene [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 4 min, 30 cycles/68.degree. C. 7 min].

[0427] The result of the amplification confirmed a band of the expected size (FIG. 48). That is, a strain was isolated that contained the introduced expression fragment stably introduced into its genome.

[Example 6-4] Changes in Fatty Acid Composition by .omega.3 Desaturase Expression in PUFA PKS Pathway Disrupted Strain

[0428] The Thraustochytrium aureum OrfA disrupted strain produced in Example 4, and the .omega.3 desaturase expressing strain produced in Example 6-3 were cultured by using the method described in Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0429] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0430] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0431] Carrier gas: He (1.3 mL/min).

[0432] The .omega.3 desaturase expressing strain had reduced levels of the n-6 series fatty acids, and there was a tendency for the n-3 series fatty acids to increase (FIG. 49). FIG. 50 represents the proportions relative to the wild-type strain taken as 100%. FIG. 50 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 1.5%, DGLA: 1.8%, ETA: 0.5%, EPA: 11.4%, n-6DPA: 1.2%, and DHA: 22.0%, which can also be described as the values of GC area such as n-6DPA/DTA: 0.6, DHA/n-3DPA: 5.7, C20PUFA/C22PUFA: 0.5, and n-6PUFA/n-3PUFA: 0.2.

[0433] As a result, the arachidonic acid was reduced to about 1/7, and the DPA to about 1/10. EPA and DHA increased by a factor of about 3.

Example 7

[0434] [Disruption of Thraustochytrium roseum C20 Elongase Gene]

[Example 7-1] Cloning of T. roseum-Derived C20 Elongase Gene

[0435] A forward denatured oligonucleotide (EL020F;5'-ATH GAR TWY TKB RTI TTY GTI CA-3') (SEQ ID NO: 179) and a reverse denatured oligonucleotide (EL020R;5'-TAR TRI SWR TAC ATI ADI AMR TG-3') (SEQ ID NO: 180) were synthesized by targeting a conserved region in the C20 elongase gene of the Thraustochytrium roseum ATCC 28210 strain. Then, a PCR was performed with an Advantage 2 Polymerase Mix (Clontech), using the T. roseum genomic DNA extracted by using the same technique described in the method of Example 2-5 as a template [PCR cycles: 94.degree. C. 1 min/94.degree. C. 30 sec, 55.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. The resulting specific product was isolated by 2% agarose gel electrophoresis, and purified. The DNA fragment was then TA cloned with a pGEM-T easy Vector (Promega), and the base sequence was analyzed. The sequence showed significant sequence identity with the sequence of a known T. aureum-derived C20 elongase gene, suggesting that the sequence was a partial sequence of the T. roseum-derived C20 elongase gene.

[0436] This was followed by cloning of the T. roseum-derived C20 elongase gene by 3'- and 5'-RACE, as in Example 2-2. First, the following oligonucleotide primers were designed.

[0437] Forward oligonucleotide primer (8 F1; 5'-CTG ACA AAG TTT CTC GAC TGG AGC GAC A-3') (SEQ ID NO: 181)

[0438] Reverse oligonucleotide primers (8 R1; 5'-TAC GCG GCG GTG CCC GAG CCC CAG-3') (SEQ ID NO: 182) and (8 R2; 5'-TGC CGA TCG TTG CGT GGT GGA ACA CCT G-3') (SEQ ID NO: 183)

[0439] This was followed by 3'- and 5'-RACE using a synthetic adapter-specific oligonucleotide, and the oligonucleotide 8 F1 or 8 R1, using the cDNA library created with a SMART.TM. RACE cDNA Amplification Kit (clontech) as a template [PCR cycles: 94.degree. C. 30 sec 5 cycles/94.degree. C. 30 sec, 70.degree. C. 30 sec, 72.degree. C. 3 min, 5 cycles/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 3 min, 25 cycles/4.degree. C. .infin.]. In the 5'RACE, a nested PCR was performed by using a synthetic adapter-specific oligonucleotide and the oligonucleotide 8 R2, using the RACE product as a template [PCR cycles: 94.degree. C. 1 min/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 3 min, 25 cycles/72.degree. C. 10 min/4.degree. C. .infin.]. The both specific products were gel purified, and the base sequence was analyzed after being TA cloned with a pGEM-T easy Vector (Promega). There was a complete match with the T. aureum. ATCC 34304-derived C20 elongase (TaELO2) (SEQ ID NO: 16) of Example 2-2.

[0440] Then, a forward oligonucleotide (8 ORF F; 5'-ATG GCG ACG CGC ACC TCG AA-3') (SEQ ID NO: 184) and a reverse oligonucleotide (8 ORF R; 5'-TTA CTC GGA CTT GGT GGG GGC G-3') (SEQ ID NO: 185) for amplifying a putative translated sequence were synthesized, and a PCR was performed with an Advantage GC 2 polymerase Mix (Clontech), using the T. roseum genomic DNA as a template [PCR cycles: 94.degree. C. 1 min/94.degree. C. 30 sec, 65.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. The resulting specific product was gel purified, and the base sequence was analyzed by direct sequencing. The T. roseum-derived C20 elongase gene was found to be identical to the TaELO2. As demonstrated above, the sequence had a complete match with the sequence of the Thraustochytrium aureum C20 elongase. The base sequence is represented by SEQ ID NO: 186, and the amino acid sequence by SEQ ID NO: 187.

[Example 7-2] Production of Base Plasmid for C20 Elongase Gene Targeting Vector Production

[0441] The Thraustochytrium aureum ATCC 34304 strain was cultured in a GY medium. Cells at the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,500.times.g for 5 min to form a pellet, and disrupted after being frozen with liquid nitrogen. After being extracted with phenol, the cell disruption liquid was precipitated with ethanol, and the precipitate was dissolved in a TE solution. The nucleic acids dissolved in the TE solution were treated with RNase at 37.degree. C. for 30 min to degrade the RNA. After being reextracted with phenol, the product was precipitated with ethanol, and the precipitate was dissolved in a TE solution. The DNA concentration was calculated by measuring A260/280. By using this as a template, the sequence (3,193 bp, SEQ ID NO: 188) containing the C20 elongase gene sequence was amplified with an LA taq Hot start version (Takara Bio) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 1 min, 30 cycles/68.degree. C. 2 min]. The E2 KO ProF EcoRV (SEQ ID NO: 33) and E2KO TermR EcoRV (SEQ ID NO: 34) of Example 2-8 were used as PCR primers. The resulting DNA fragment was cloned into a pGEM-T easy vector (Promega), amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH59 (FIG. 51).

[0442] By using the pRH59 (FIG. 51) as a template, amplification was performed with a PrimeSTAR Max DNA Polymerase (Takara Bio) using a primer set of the reverse orientation prepared for the insertion of the BglII site in a portion halfway along the C20 elongase gene sequence. The primers used are as follows. The both primers have BglII linker sequences [RHO120: 27 mer: 5'-GAC AAA GAT CTC GAC TGG AGC GAC CAC-3' (SEQ ID NO: 189), RHO121: 27 mer: 5'-GTC GAG ATC TTT TGT CAG GAG GTG CAC-3' (SEQ ID NO: 190)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 56.degree. C. 15 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min]. After the amplification performed under these conditions, the product was digested with BglII, and allowed to self ligate. The ligated sample was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH64. The C20 elongase gene sequence 951 bp with the inserted BglII site is represented by SEQ ID NO: 191.

[0443] The produced base plasmid (pRH64) for the production of the Thraustochytrium roseum C20 elongase gene targeting vector is shown in FIG. 52.

[Example 7-3] Production of Targeting Vectors (Artificial Neomycin-Resistant Gene and Hygromycin-Resistant Gene)

[0444] The pRH31 (FIG. 13) of Example 3-2 was digested with BglII, and the DNA fragment containing an artificial neomycin-resistant gene cassette was ligated to the BglII site of the pRH64 (FIG. 52) of Example 7-2. This was named pRH65.

[0445] The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and the DNA fragment containing a hygromycin-resistant gene cassette was ligated to the BglII site of the pRH64 (FIG. 52) of Example 7-2. This was named pRH66.

[0446] The two targeting vectors (pRH65 and 66) produced are shown in FIG. 53.

[Example 7-4] Introduction of C20 Elongase Gene Targeting Vector

[0447] By using the two targeting vectors produced in Example 7-3 as templates, the gene was amplified with a PrimeSTAR GXL polymerase (Takara Bio), using a forward primer containing a translation initiation site (RHO130: 5'-ATG GCG ACG CGC ACC TCG AAG AG-3') (SEQ ID NO: 192) and a reverse primer containing a translation termination site (RHO131: 5'-TTA CTC GGA CTT GCT GGG GGC GC) (SEQ ID NO: 193) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60 30 sec, 72.degree. C. 3 min, 30 cycles]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH65 (FIG. 53) of Example 7-3 as a template was 2,655 bp, and had the following sequence order: First half of Thraustochytrium aureum C20 elongase gene-SV40 terminator sequence-artificial neomycin-resistant gene sequence-ubiquitin promoter sequence-second half of Thraustochytrium aureum C20 elongase gene (SEQ ID NO: 194). The introduced fragment obtained from using the pRH66 (FIG. 53) of Example 7-3 as a template was 2,887 bp, and had the following sequence order: First half of Thraustochytrium aureum C20 elongase gene-ubiquitin promoter sequence-hygromycin-resistant gene sequence-SV40 terminator sequence-second half of Thraustochytrium aureum C20 elongase gene (SEQ ID NO: 195).

[0448] The Thraustochytrium roseum strain was cultured in a GY medium for 7 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique under the following conditions (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 900 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied to a PDA plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin).

[0449] As a result, about 20 drug resistant strains were obtained per penetration.

[Example 7-5] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0450] The Thraustochytrium roseum ATCC 28210 strain, the C20 elongase gene hetero homologous recombinant, and the C20 elongase gene homo homologous recombinant (gene disrupted strain) were cultured in GY media. The resulting cells were centrifuged at 4.degree. C., 3,000 rpm for 10 min to forma pellet, and lysed at 55.degree. C., 6 h/99.9.degree. C., 5 min after being suspended in a 20-.mu.1 SNET solution [20 mM Tris-HCl; pH 8.0, 5 mM NaCl, 0.3% SDS, 200 .mu.g/ml Proteinase K (nacalaitesque)]. The resulting cell lysate was diluted 10 times and used as a template in a PCR performed with a Mighty Amp DNA polymerase (Takara Bio) to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected sizes of the amplification products are shown in FIG. 54. RoseumF and RoseumR were set upstream and downstream of the C20 elongase, respectively. NeoF and NeoR were set on the artificial neomycin-resistant gene. HygF and HygR were set on the hygromycin-resistant gene [RoseumF: 26 mer: 5'-GCT CGG CTG GAA GTT GAG TAG TTT GC-3' (SEQ ID NO: 196), RoseumR: 24 mer: 5'-TCT TTC TTC GTC GAC GTC CCA CTG-3' (SEQ ID NO: 197), NeoF: 24 mer: 5'-ATG ATT GAA CAG GAC GGC CTT CAC-3' (SEQ ID NO: 198), NeoR: 24 mer: 5'-TCA AAA GAA CTC GTC CAG GAG GCG-3' (SEQ ID NO: 199), HygF: 24 mer: 5'-ATG AAA AAG CCT GAA CTC ACC GCG-3' (SEQ ID NO: 200), HygR: 25 mer: 5'-CTA TTC CTT TGC CCT CGG ACG AGT G-3' (SEQ ID NO: 201)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 60.degree. C. 15 sec, 68.degree. C. 4 min, 30 cycles].

[0451] C20 elongase knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele) but showed amplification of the artificial neomycin-resistant gene allele (NeoR allele) and hygromycin-resistant gene allele (HygR allele) (FIG. 55).

[Example 7-6] Changes in Fatty Acid Composition by C20 Elongase Disruption

[0452] The Thraustochytrium roseum ATCC 28210 strain and the gene disrupted strain were cultured in GY media. Cells at the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,000 rpm for 10 min to form a pellet, suspended in 0.9% NaCl, and washed. The cells were further centrifuged at 4.degree. C., 3,000 rpm for 10 min, and the pellet was suspended in sterile water, and washed. This was centrifuged at 3,000 rpm for 10 min, and freeze dried after removing the supernatant. Then, 2 ml-methanolic KOH (7.5% KOH in 95% methanol) was added to the freeze dried cells, and, after being vortexed, the cells were ultrasonically disrupted (80.degree. C., 30 min). The cells were vortexed after adding sterile water (500 .mu.l), and vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was discarded. The cells were vortexed again after adding n-hexane (2 ml), and centrifuged at 3,000 rpm for 10 min. After discarding the upper layer, 6 N HCl (1 ml) was added to the remaining lower layer, and the mixture was vortexed. The mixture was vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. The collected upper layer was then concentrated and dried with nitrogen gas. The concentrated dry sample was incubated overnight at 80.degree. C. after adding 3 N methanolic HCl (2 ml).

[0453] The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml) was added. The mixture was vortexed after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. After adding a small amount of anhydrous sodium sulfate to the collected upper layer, the mixture was vortexed, and centrifuged at 3,000 rpm for 10 min. After collecting the upper layer, the upper layer was concentrated and dried with nitrogen gas. The concentrated dry sample was dissolved in n-hexane (0.2 ml), and 2 .mu.l of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0454] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0455] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0456] Carrier gas: He (1.3 mL/min).

[0457] As a result, knocking out the C20 elongase in the Thraustochytrium roseum increased fatty acids of 20 carbon chain length (FIG. 56). FIG. 57 represents the proportions relative to the wild-type strain taken as 100%. FIG. 57 represents each proportion of the components based on the total amount of the fatty acids, which include AA: 5.5%, EPA: 8.5%, n-6DPA: 13.2%, and DHA: 43.9%.

[0458] As can be seen from these results, the arachidonic acid increased about 1.2-fold, EPA about 1.6-fold, DPA about 1.2-fold, and DHA about 1.5-fold.

Example 8

[0459] Disruption of 44 Desaturase Gene in Thraustochytrium aureum ATCC 34304 OrfA Disrupted Strain

[Example 8-1] Cloning of Sequence from 1,071 bp Upstream of 44 Desaturase Gene to 1,500 bp within 44 Desaturase Gene in Thraustochytrium aureum ATCC 34304 Strain

[0460] The genomic DNA of the Thraustochytrium aureum ATCC 34304 strain extracted by using the method described in Example 3-2 was decoded. Then, a search was made for a gene sequence highly homologous to a known 44 desaturase, and two PCR primers were designed by using the search result. TMO3 is a sequence located 1,071 to 1,049 bp upstream of the .DELTA.4 desaturase gene of the Thraustochytrium aureum ATCC 34304 strain. TMO4 is a sequence within the protein coding region, located 1,477 to 1,500 bp from the start codon [TMO3: 23 mer: 5'-GGC GGA GCG AAG TGT GAA AGT TA-3' (SEQ ID NO: 202), TMO4: 24 mer: 5'-GCG ACA GCA TCT TGA AAT AGG CAG-3' (SEQ ID NO: 203)]. By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 strain as a template, the sequence from 1,071 bp upstream of the .DELTA.4 desaturase gene to 1,500 bp within the .DELTA.4 desaturase gene of the Thraustochytrium aureum ATCC 34304 strain was amplified with the two primers, using an LA taq Hot start version (Takara Bio). The amplification was performed under the following conditions [PCR cycles: 98.degree. C. 2 min/98.degree. C. 20 sec, 60.degree. C. 30 sec, 72.degree. C. 3 min, 30 cycles/72.degree. C. 8 min]. The resulting DNA fragment was cloned into a pGEM-T easy vector, amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pTM1 (FIG. 58).

[Example 8-2] Production of Base Plasmid for 44 Desaturase Gene Targeting Vector Production

[0461] By using the pTM1 (FIG. 58) of Example 8-1 as a template, a primer set of the reverse orientation was prepared in a manner that allows the 60 bp upstream of the .DELTA.4 desaturase gene and a 556-bp sequence containing the start codon within the .DELTA.4 desaturase gene (616 bp, SEQ ID NO: 205) to be deleted, and a BglII site to occur in the deleted portion. TMO7 and TMO8 both contain BglII sequences. A PrimeSTAR Max DNA Polymerase (Takara Bio) was used for the amplification [TMO7: 25 mer: 5'-CAG GAG ATC TCC AAG TCG CGA TTC A-3' (SEQ ID NO: 206), TMO8: 26 mer: 5'-CTT GGA GAT CTC CTG CCC GTC CCG AA-3' (SEQ ID NO: 207)] [PCR cycles: 98.degree. C. 3 min/98.degree. C. 10 sec, 55.degree. C. 15 sec, 72.degree. C. 30 sec, 30 cycles/72.degree. C. 30 sec]. After the amplification performed under these conditions, the product was electrophoresed on an agarose gel, and purified. The resulting DNA fragment was introduced into Escherichia coli and amplified, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pTM2.

[0462] The product base plasmid (pTM2) for the .DELTA.4 desaturase gene targeting vector production is shown in FIG. 59.

[Example 8-3] Production of Targeting Vectors (Blasticidin-Resistant Gene and GFP-Fused Zeocin-Resistant Gene)

[0463] The pRH38 (FIG. 33) of Example 5-3 was digested with BglII, and the DNA fragment containing a blasticidin-resistant gene cassette was ligated to the BglII site of the pTM2 (FIG. 59) of Example 8-2. This was named pTM6.

[0464] The pRH51 (FIG. 36) of Example 5-4 was digested with BglII, and the DNA fragment containing a GFP-fused zeocin-resistant gene cassette was ligated to the BglII site of the pTM2 (FIG. 59) of Example 8-2. This was named pTM8.

[0465] The two targeting vectors (pTM6 and 8) produced are shown in FIG. 60.

[Example 8-4] Introduction of 44 Desaturase Gene Targeting Vector into Thraustochytrium aureum OrfA Disrupted Strain

[0466] By using the two targeting vectors produced in Example 8-3 as templates, the gene was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio), using TMO3 (Example 8-1; SEQ ID NO: 202) and TMO4 (Example 8-1; SEQ ID NO: 203) as primers [PCR cycles: 98.degree. C. 3 min/98.degree. C. 10 sec, 55.degree. C. 5 sec, 72.degree. C. 4 min, 30 cycles/72.degree. C. 3 min].

[0467] After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pTM6 (FIG. 60) of Example 8-3 as a template was 3,264 bp, and had the following sequence order: Upstream of Thraustochytrium aureum .DELTA.4 desaturase gene-SV40 terminator sequence-blasticidin-resistant gene sequence-ubiquitin promoter-sequence within Thraustochytrium aureum .DELTA.4 desaturase gene (SEQ ID NO: 208). The introduced fragment obtained from using the pTM8 (FIG. 60) of Example 8-3 as a template was 3,935 bp, and had the following sequence order: Upstream of Thraustochytrium aureum .DELTA.4 desaturase gene-SV40 terminator sequence-zeocin-resistant gene sequence-enhanced GFP gene sequence-ubiquitin promoter-sequence within Thraustochytrium aureum .DELTA.4 desaturase gene (SEQ ID NO: 209).

[0468] The gene disrupted strain of the PUFA PKS pathway-associated gene OrfA of Example 4 was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 by using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with the introduced gene was applied to a PDA agar plate medium (containing 20 mg/ml Zeocin or 0.2 mg/ml blasticidin). As a result, 100 to 200 drug resistant strains were obtained per penetration.

[Example 8-5] Identification of 44 Desaturase Gene Gene Targeting Homologous Recombinant

[0469] Genomic DNA was extracted from Thraustochytrium aureum, and the .DELTA.4 desaturase gene disrupted strain of the Thraustochytrium aureum OrfA disrupted strain by using the method of Example 3-2.

[0470] The DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with a Mighty Amp DNA polymerase (Takara Bio) to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected sizes of the amplification products are shown in FIG. 61. TMO1 was set upstream of the .DELTA.4 desaturase gene. TMO2 was set downstream of the .DELTA.4 desaturase gene. RHO198 and RHO49 (Example 5-3; SEQ ID NO: 139) were set on the blasticidin-resistant gene. RHO128 was set on the enhanced GFP gene. RHO64 (Example 5-4; SEQ ID NO: 151) was set on the zeocin-resistant gene [TMO1: 23 mer: 5'-AAA AGA ACA AGC CCT CTC CTG GA-3' (SEQ ID NO: 210), TMO2: 23 mer: 5'-GAG GTT TGT ATG TTC GGC GGT TT-3' (SEQ ID NO: 211), RHO198: 26 mer: 5'-TGG GGG ACC TTG TGC AGA ACT CGT GG-3' (SEQ ID NO: 212), RHO128: 22 mer: 5'-GAC CTA CGG CGT GCA GTG CTT C-3' (SEQ ID NO: 213)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 4 min 30 sec, 30 cycles/68.degree. C. 4 min].

[0471] .DELTA.4 desaturase gene knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele) but showed amplification of the blasticidin-resistant gene allele (BlaR allele) and zeocin-resistant gene allele (ZeoR allele) (FIG. 62). It was found by the experiment that the Thraustochytrium aureum ATCC 34304 strain did not become auxotrophic even with the deletion of the PKS pathway-associated gene OrfA and the .DELTA.4 desaturase gene.

[Example 8-6] Changes in Fatty Acid Composition by Disruption of 44 Desaturase Gene in Thraustochytrium aureum OrfA Disrupted Strain

[0472] The Thraustochytrium aureum ATCC 34304 and the gene disrupted strain were cultured by using the method of Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0473] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0474] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0475] Carrier gas: He (1.3 mL/min).

[0476] Changes in fatty acid composition are represented in FIG. 63. FIG. 64 represents the proportions relative to the wild-type strain taken as 100%. FIG. 64 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 8.4%, DGLA: 0.8%, ETA: 0.3%, EPA: 6.2%, n-6DPA: 0.2%, and DHA: 0.5%, which can also be described as the values of GC area such as n-6DPA/DTA: 0.02, DHA/n-3DPA: 0.03, C20PUFA/C22PUFA: 0.6, and n-6PUFA/n-3PUFA: 0.9.

[0477] As can be seen from the results, disrupting the .DELTA.4 desaturase gene in the Thraustochytrium aureum OrfA disrupted strain resulted in hardly peforming C22:5n-6 (DPA) and C22:6n-3 (DHA) biosyntheses, and C22:4n-6 (DTA) and C22:5n-3 (DPA) accumulated.

Example 9

[0478] Disruption of C20 Elongase Gene in Parietichytrium sp. SEK358 Strain [Example 9-1] Introduction of C20 Elongase Gene Targeting Vector into Parietichytrium sp. SEK358 Strain

[0479] By using the targeting vector produced with the pRH85 (FIG. 18) of Example 3-6 as a template, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using RHO153 (Example 3-4; SEQ ID NO: 74) and RHO154 (Example 3-4; SEQ ID NO: 75) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH85 (FIG. 18) of Example 3-6 as a template was 2,661 bp, and had the following sequence order: First half of Parietichytrium C20 elongase gene-SV40 terminator sequence-artificial neomycin-resistant gene sequence-ubiquitin promoter sequence-second half of Parietichytrium C20 elongase gene (Example 3-7; SEQ ID NO: 81). The Parietichytrium sp. SEK358 strain was cultured in a GY medium for 3 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 900 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied to a PDA agar plate medium containing 0.5 mg/ml G418. As a result, 10 to 30 drug resistant strains were obtained per penetration.

[Example 9-2] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0480] Genomic DNA was extracted from the Parietichytrium sp. SEK358 strain and the C20 elongase gene disrupted strain by using the method of Example 3-2. The DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with a Mighty Amp DNA polymerase (Takara Bio) to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected sizes of the amplification products are as described in Example 3-8 (FIG. 19).

[0481] RHO184 (Example 3-8; SEQ ID NO: 87) was set upstream of the C20 elongase. RHO185 (Example 3-8; SEQ ID NO: 88) was set downstream of the C20 elongase. RHO142 (Example 3-8; SEQ ID NO: 85) and RHO143 (Example 3-8; SEQ ID NO: 86) were set on the artificial neomycin-resistant gene [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 7 min].

[0482] C20 elongase knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele), but showed amplification of the artificial neomycin-resistant gene allele (NeoR allele) (FIG. 65).

[Example 9-3] Changes in Fatty Acid Composition by Disruption of C20 Elongase

[0483] The Parietichytrium sp. SEK358 strain and the gene disrupted strain were cultured by using the method of Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0484] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0485] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0486] Carrier gas: He (1.3 mL/min).

[0487] Changes in fatty acid composition are represented in FIG. 66. FIG. 67 represents the proportions relative to the wild-type strain taken as 100%. FIG. 67 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 21.4%, DGLA: 8.6%, ETA: 2.1%, EPA: 23.8%, n-6DPA: 0.5%, DHA: 0.9%, which can also be described as the values of GC area such as n-6DPA/DTA: 1.8, DHA/n-3DPA: 4.1, C20PUFA/C22PUFA: 29.6, and n-6PUFA/n-3PUFA: 1.1. As can be seen from the results, knocking out the C20 elongase in the Parietichytrium sp. SEK358 strain caused reduction of fatty acids of 22 or greater carbon chain length, and increased fatty acids of 20 carbon chain length. Specifically, the arachidonic acid increased about seven-fold, and the EPA about eleven-fold. The DPA and DHA reduced to about 1/15 and about 1/8, respectively.

Example 10

[0488] Disruption of C20 Elongase Gene in Parietichytrium sp. SEK571 Strain

[Example 10-1] Introduction of C20 Elongase Gene Targeting Vector into Parietichytrium sp. SEK571 Strain

[0489] By using the targeting vector produced with the pRH85 (FIG. 18) of Example 3-6 as a template, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using RHO153 (Example 3-4; SEQ ID NO: 74) and RHO154 (Example 3-4; SEQ ID NO: 75) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH85 (FIG. 18) of Example 3-6 as a template was 2,661 bp, and had the following sequence order: First half of Parietichytrium C20 elongase gene-SV40 terminator sequence-artificial neomycin-resistant gene sequence-ubiquitin promoter sequence-second half of Parietichytrium C20 elongase gene (Example 3-7; SEQ ID NO: 81). The Parietichytrium sp. SEK571 strain was cultured in a GY medium for 3 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was then introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1550 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied to a PDA agar plate medium containing 0.5 mg/ml G418. As a result, 5 to 15 drug resistant strains were obtained per penetration.

[Example 10-2] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0490] Genomic DNA was extracted from the Parietichytrium sp. SEK571 strain and the C20 elongase gene disrupted strain by using the method of Example 3-2, and the DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with a Mighty Amp DNA polymerase (Takara Bio) to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected sizes of the amplification products are as described in Example 3-8 (FIG. 19).

[0491] RHO184 (Example 3-8; SEQ ID NO: 87) was set upstream of the C20 elongase. RHO185 (Example 3-8; SEQ ID NO: 88) was set downstream of the C20 elongase. RHO142 (Example 3-8; SEQ ID NO: 85) and RHO143 (Example 3-8; SEQ ID NO: 86) were set on the artificial neomycin-resistant gene [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 7 min].

[0492] C20 elongase knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele), but showed amplification of the artificial neomycin-resistant gene allele (NeoR allele) (FIG. 68).

[Example 10-3] Changes in Fatty Acid Composition by C20 Elongase Disruption

[0493] The Parietichytrium sp. SEK571 strain and the gene disrupted strain were cultured by using the method of Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0494] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0495] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0496] Carrier gas: He (1.3 mL/min).

[0497] Changes in fatty acid composition are represented in FIG. 69. FIG. 70 represents the proportions relative to the wild-type strain taken as 100%. FIG. 70 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 13.2%, DGLA: 1.9%, ETA: 1.1%, EPA: 29.6%, n6DPA: 1.0%, and DHA: 1.2%, which can also be described as the values of GC area such as n-6DPA/DTA: 6.4, DHA/n-3DPA: 4.7, C20PUFA/C22PUFA: 14.6, and n-6PUFA/n-3PUFA: 0.7. As can be seen from the results, knocking out the C20 elongase in the Parietichytrium sp. SEK571 strain caused reduction of fatty acids of 22 or greater carbon chain length, and increased fatty acids of 20 carbon chain length. Specifically, the arachidonic acid increased about four-fold, and the EPA about eight-fold. The DPA and DHA both reduced to about 1/12.

Example 11

[0498] Disruption of Thraustochytrium aureum ATCC 34304-Derived 412 Desaturase Gene

[Example 11-1] Isolation of Thraustochytrium aureum ATCC 34304-Derived 412 Desaturase Gene

[0499] By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, a Thraustochytrium aureum ATCC 34304-derived .DELTA.12 desaturase gene was amplified by a PCR performed with a forward oligonucleotide primer T.omega.3-F1 (22 mer: 5'-ATG TGC AAG GTC GAT GGG ACA A-3') (SEQ ID NO: 214) and a reverse oligonucleotide primer T.omega.3-R1 (22 mer: 5'-TCA CAA ACA TCG CAG CCT TCG G-3') (SEQ ID NO: 215) (enzyme used: LA taq Hot Start Version, TaKaRa; PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 53.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.). As a result, a novel gene sequence having a 1,185-bp (SEQ ID NO: 217) ORF, encoding 395 amino acids (SEQ ID NO: 216) was obtained. In the amino acid sequence of the gene, three histidine boxes commonly conserved in desaturases, believed to construct the active site were conserved (FIG. 71). Further, because the gene showed high identity (41%, 44%, 41%) at the amino acid level with the Thalassiosira pseudonana-, Micromonas sp.-, and Phaeodactylum tricornutum-derived .DELTA.12 desaturases in a Blast search (FIG. 71), it was strongly suggested that the gene was a Thraustochytrium aureum ATCC 34304-derived .DELTA.12 desaturase gene. In the following, the gene will be referred to as T.DELTA.12d.

[Example 11-2] Expression of T.DELTA.12d using Budding Yeast Saccharomyces cerevisiae as Host, and Analysis of Fatty Acid Composition of Gene Introduced Strain

[0500] By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, a DNA fragment containing HindIII and Xba I sites added to the both ends of T.DELTA.12d was prepared in a PCR performed with a forward oligonucleotide primer T.omega.3-Hind3-F (30 mer: 5'-GGA AGC TTA TGT GCA AGG TCG ATG GGA CAA-3') (SEQ ID NO: 218) and a reverse oligonucleotide primer T.omega.3-XbaI-R (29 mer: 5'-TTC TAG ACT AGA GCT TTT TGG CCG CAC GC-3') (SEQ ID NO: 219) (enzyme used: LA taq Hot Start Version, TaKaRa; PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 53.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.). The DNA fragment was then incorporated in the HindIII/Xba I site of a pYES2/CT vector to construct a T.DELTA.12d expression vector pYESTD12. The pYESTD12 and pYES2/CT were then introduced into yeasts by using the lithium acetate method. In the GC analysis of the fatty acid composition of the T.DELTA.12d overexpressing strain (pYESTD12 introduced strain), novel peaks were confirmed at positions corresponding to the retention times of LA (C18:249,12) and C16:249,12, but not in the mock introduced strain (pYES2/CT introduced strain). FIG. 72 represents a GC analysis chart, and fatty acid levels per dry cell. On the other hand, no conversion activity for other fatty acids [LA, GLA (C18:346,9,12), C20:2411,14, DGLA (C20:348,11,14), ARA (C20:445,8,11,14), DTA (C22:447,10,13,16)] was confirmed in the T.DELTA.12d overexpressing strain. It became clear from these results that the T.DELTA.12d was a Thraustochytrium aureum ATCC 34304-derived 412 desaturase gene.

[Example 11-3] Construction of T.DELTA.12d Targeting Vector

[0501] By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, the upstream and downstream sequences (1,001 bp each) of the T.DELTA.12d ORF were amplified in a PCR performed under the following conditions (enzyme used: PrimeSTAR GXL, TaKaRa); PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 53.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.). The following forward and reverse oligonucleotide primers were used.

TABLE-US-00008 TD12d-up-F (SEQ ID NO: 220) (23 mer: 5'-AGT CAG CCC AGG CAC CGA TGA CG-3') and TD12d-up-R (SEQ ID NO: 221) (39 mer: 5'-AGC CAG AGC TAG ATC TCT TGT GCT CCT TTT CAA TCC TTT-3') TD12d-down-F (SEQ ID NO: 222) (39 mer: 5'-GGA GCA CAA GAG ATC TAG CTC TGG CTC AAG GGA CAC CGT-3') and TD12d-down-R (SEQ ID NO: 223) (24 mer: 5'-CAC AGA AAC TGC CTT CAC GGG TCT-3')

[0502] The resulting both DNA fragments were joined by fusion PCR with a Bgl II site inserted therebetween, and incorporated in a pGEM-T easy Vector (Promega). Then, the hygromycin-resistant gene cassette of Example 3-3, and the blasticidin-resistant gene cassette of Example 5-3 were incorporated at the Bgl II site of the resulting vector to construct T.DELTA.12d KO targeting vectors. These were named pTD12dKOHyg and pTD12dKOBla. The construction scheme of the T.DELTA.12d KO targeting vectors are shown in FIG. 73.

[Example 11-4] Introduction of T.DELTA.12d Targeting Vector to Thraustochytrium aureum ATCC 34304, and Acquisition of T.DELTA.12d Disrupted Strain

[0503] In order to obtain an efficient homologous recombinant by using a split marker method, two homologous recombination fragments were amplified by a PCR performed by using pTD12dKOHyg as a template [enzyme used: LA taq Hot Start Version, TaKaRa; PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. X min (X=1 min/kbp), 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.] (FIG. 74). The fragments were then introduced to the Thraustochytrium aureum ATCC 34304 by using the gene-gun technique. The following forward and reverse oligonucleotide primers were used for the amplification of the homologous recombination fragments.

TABLE-US-00009 (SEQ ID NO: 220) TD12d-up-F and Hyg-Knock-R (SEQ ID NO: 224) (24 mer: 5'-TGT TAT GCG GCC ATT GTC CGT CAG-3'), and Hyg-Knock-F (SEQ ID NO: 225) (24 mer: 5'-TGC GAT CGC TGC GGC CGA TCT TAG-3') and (SEQ ID NO: 223) TD12d-down-R

[0504] As a result, a homologous recombinant with the disrupted T.DELTA.12d first allele was obtained. Thereafter, by using pTD12dKOBla as a template, a homologous recombination fragment for disrupting the second allele was amplified by a PCR performed with the forward and reverse oligonucleotide primers TD12d-up-F (SEQ ID NO: 220) and TD12d-down-R (SEQ ID NO: 223) (enzyme used: LA taq Hot Start Version, TaKaRa) [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree. C. 3 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.]. The fragment was then introduced to the homologous recombinant containing the disrupted first allele. Complete disruption of T.DELTA.12d was verified by a PCR (using the genomic DNA below as a template) and a RT-PCR performed for the detection of hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d, or by southern blotting.

[0505] FIG. 75 represents the amplification results for the hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d amplified by a PCR performed by using the genomic DNAs of the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain (two alleles are disrupted) as templates.

[0506] As a result, amplification of the hygromycin-resistant gene and the blasticidin-resistant gene contained in the introduced homologous recombination fragment was confirmed in the T.DELTA.12d disrupted strain. However, no amplification of the disrupted T.DELTA.12d was confirmed. The following forward and reverse oligonucleotide primers were used for the amplification of the hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d.

TABLE-US-00010 Hyg-F (SEQ ID NO: 226) (26 mer: 5'-ATG AAA AAG CCT GAA CTC ACC GCG AC-3') and Hyg-R (SEQ ID NO: 227) (25 mer: 5'-CTA TTC CTT TGC CCT CGG ACG AGT G-3'), Bla-F (SEQ ID NO: 228) (27 mer: 5'-ATG GCC AAG CCT TTG TCT CAA GAA GAA-3'), and Bla-R (SEQ ID NO: 229) (30 mer: 5'-TTA GCC CTC CCA CAC ATA ACC AGA GGG CAG-3'), (SEQ ID NO: 214) Tw3-F1, and (SEQ ID NO: 215) Tw3-R1

[0507] FIG. 76 represents the results of the mRNA detection performed by RT-PCR for the hygromycin-resistant gene, blasticidin-resistant gene, and T.DELTA.12d in the wild-type strain, the T.DELTA.12d first allele disrupted strain, and the T.DELTA.12d disrupted strain. As a result, mRNA was detected for the hygromycin-resistant gene and the blasticidin-resistant gene contained in the introduced homologous recombination fragment in the T.DELTA.12d disrupted strain. However, mRNA was not detected for the disrupted T.DELTA.12d. Note that the primers used are the same primers as used for the PCR in which the genomic DNA was used as a template.

[0508] By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, two DIG-labeled probes were prepared, and southern blotting was performed with these probes. The following forward and reverse oligonucleotide primers were used for the preparation of the DIG-labeled probes.

TABLE-US-00011 KO up-probe-F1 (SEQ ID NO: 230) (23 mer: 5'-GGG GTC GGC CGG TGC AGC CTT AG-3') and KO up-probe-R1 (SEQ ID NO: 231) (24 mer: 5'-GGC GGT CAG CGA TCG GTC GGA CTC-3'), and KO down-probe-F3 (SEQ ID NO: 232) (23 mer: 5'-GCT TGC GGC TCC TGT TGG GTG AC-3') and KO down-probe-R3 (SEQ ID NO: 233) (23 mer: 5'-ACG CCT GGC TGC CCA CCA TAA AC-3')

[0509] As a result, the bands of the wild-type allele (upstream side 2,028 bp, downstream side 2,334 bp) disappeared in the T.DELTA.12d disrupted strain, and bands of the homologous recombination fragments (upstream side 5,880 bp and 5,253 bp; downstream side 1,496 bp and 2,334 bp) containing the hygromycin-resistant gene and the blasticidin-resistant gene were detected instead (FIG. 77).

[0510] The PCR using the genomic DNA as a template, the RT-PCR, and southern blotting made it clear that the T.DELTA.12d was disrupted.

[Example 11-5] Phenotypic Analysis of T.DELTA.12d Disrupted Strain

[0511] Cells cultured in a 250-ml GY liquid medium for 5 days were collected in 10 ml portions, and absorbance at OD 600 nm was measured (n=3). After the measurement, the cells were collected, and washed once with sterilized ultrapure water. After freeze drying, the dry cell weight was measured after 1-hour drying with a desiccator (n=3). As a result, no significant difference was observed in the proliferation among the wild-type strain, the first allele disrupted strain, and the T.DELTA.12d disrupted strain (FIG. 78). The wild-type strain, the first allele disrupted strain, and the T.DELTA.12d disrupted strain were GC analyzed for their fatty acid compositions.

[0512] As a result, large fatty acid composition changes were observed. Accumulation of C18:1n9 (OA) in the T.DELTA.12d disrupted strain was particularly prominent. FIG. 79 represents the proportion of each component in the fatty acid composition. FIG. 80 represents fatty acid levels per milligram of dry cells.

Example 12

[0513] Disruption of C20 Elongase Gene and Expression of .omega.3 Desaturase Gene in Thraustochytrium aureum ATCC 34304 OrfA Gene Disrupted Strain [Example 12-1] Production of C20 Elongase Gene Targeting and Saprolegnia diclina-Derived .omega.3 Desaturase Expression Vector (Blasticidin-Resistant Gene)

[0514] By using the pRH43 (FIG. 39) of Example 5-6 as a template, a primer set of the reverse orientation was prepared in a manner that allows the two restriction enzyme KpnI sites to be deleted, and a BamHI site to occur in the deleted portion. RHO189 and RHO190 both contain BamHI sequences. A PrimeSTAR Max DNA Polymerase (Takara Bio) was used for the amplification [RHO189: 28 mer: 5'-TTA GCG GGA TCC CAA TTC GCC CTA TAG T-3' (SEQ ID NO: 234), RHO190: 27 mer: 5'-AAT TGG GAT CCC GCT AAG TAT CTC CCG-3' (SEQ ID NO: 235)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 55.degree. C. 15 sec, 72.degree. C. 40 sec, 31 cycles/72.degree. C. 1 min]. After the amplification performed under these conditions, the product was electrophoresed on an agarose gel, and purified. The resulting DNA fragment was introduced into Escherichia coli and amplified, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH101 (FIG. 81).

[0515] By using the pRH101 as a template, a primer set of the reverse orientation was prepared in a manner that allows for insertion of a restriction enzyme KpnI site. RHO191 and RHO192 both contain KpnI sequences. A PrimeSTAR Max DNA Polymerase (Takara Bio) was used for the amplification [RHO191: 28 mer: 5'-AGA TCT GGT ACC GCA GCG CCT GGT GCA C-3' (SEQ ID NO: 236), RHO192: 27 mer: 5'-GCT GCG GTA CCA GAT CTG GTC GCG TTT-3' (SEQ ID NO: 237)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 55.degree. C. 15 sec, 72.degree. C. 40 sec, 31 cycles/72.degree. C. 1 min]. After the amplification performed under these conditions, the product was electrophoresed on an agarose gel, and purified. The resulting DNA fragment was introduced into Escherichia coli and amplified, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH102 (FIG. 82).

[0516] The pRH48 (FIG. 46) of Example 6-1 was digested with KpnI, and a DNA fragment containing a Saprolegnia diclina-derived .omega.3 desaturase expression cassette was ligated to the KpnI site of the pRH102 (FIG. 82). This was named pRH103.

[0517] The product C20 elongase gene targeting and Saprolegnia diclina-derived .omega.3 desaturase expression vector pRH103 is shown in FIG. 83.

[Example 12-2] Introduction of C20 Elongase Gene Targeting and Saprolegnia diclina-Derived .omega.3 Desaturase Expression Vector into Thraustochytrium aureum OrfA Disrupted Strain

[0518] By using the C20 elongase gene targeting vector pRH54 (FIG. 39) of Example 5-6 as a template, the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio) using KSO11 (Example 5-7; SEQ ID NO: 159) and KSO12 (Example 5-7; SEQ ID NO: 160) as primers [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 68.degree. C. 2 min, 30 cycles/68.degree. C. 2 min]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment was 3,887 bp, and had the following sequence order: Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitin promoter-Enhanced GFP gene sequence-zeocin-resistant gene sequence-SV40 terminator sequence-downstream of Thraustochytrium aureum C20 elongase gene (Example 5-7; SEQ ID NO: 162). The C20 elongase gene targeting and Saprolegnia diclina-derived .omega.3 desaturase expression vector pRH103 (FIG. 83) of Example 12-1 was digested with a restriction enzyme BamHI. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment was 5,611 bp, and had the following sequence order: Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitin promoter-Saprolegnia diclina-derived .omega.3 desaturase gene sequence-ubiquitin terminator-ubiquitin promoter-blasticidin-resistant gene sequence-SV40 terminator-downstream of Thraustochytrium aureum C20 elongase gene (SEQ ID NO: 238).

[0519] The PUFA PKS pathway-associated gene OrfA gene disrupted strain of Example 4 was cultured in a GY medium for 4 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was introduced into cells corresponding to OD600=1 to 1.5 by using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with the introduced gene were applied to a PDA agar plate medium (containing 20 mg/ml Zeocin or 0.2 mg/ml blasticidin). As a result, 20 to 60 drug resistant strains were obtained.

[Example 12-3] Introduction of Homologous Recombinant Containing C20 Elongase Gene Targeting and Saprolegnia diclina-Derived .omega.3 Desaturase Expression Vector Inserted in Genome

[0520] Genomic DNA was extracted from the Thraustochytrium aureum PUFA PKS pathway-associated gene OrfA disrupted strain, the C20 elongase gene first allele homologous recombinant of the Thraustochytrium aureum OrfA disrupted strain, and the disrupted strain by using the method described in Example 3-2. The DNA concentration was then calculated by measuring A260/280.

[0521] The genomic DNA was cut with restriction enzymes, and electrophoresed on a 0.7% SeaKem GTG agarose gel (Takara Bio) in about 2 to 3 .mu.g per well. This was transferred to a nylon membrane, and hybridized at 51.degree. C. for 16 hours with probes produced with a DIG system (Roche Applied Science). RHO94 (Example 5-8; SEQ ID NO: 163) and RHO95 (Example 5-8; SEQ ID NO: 164) were used for the production of the 5'-end probe. RHO96 (Example 5-8; SEQ ID NO:165) and RHO97 (Example 5-8; SEQ ID NO: 166) were used for the production of the 3'-end probe. The amplification was performed under the following conditions, and an LA taq Hot start version (Takara Bio) was used for the amplification [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 58.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 3 min]. The restriction enzymes used, and the probe positions are as shown in FIG. 84. Detection of the hybridized probes was made by using a chromogenic method (NBT/BCIP solution). Bands of the sizes expected from the homologous recombination of the drug resistant genes were observed in the analyses of both the 5' end and the 3' end (FIG. 85).

[Example 12-4] Disruption of C20 Elongase Gene in Thraustochytrium aureum OrfA. Disrupted Strain and Changes in Fatty Acid Composition by Saprolegnia diclina-Derived .omega.3 Desaturase Expression

[0522] The Thraustochytrium aureum ATCC 34304wild-type strain, and the Saprolegnia diclina-derived .omega.3 desaturase expressing strain with the double disruption of the PKS pathway (orfA gene) and the C20 elongase gene were cultured by using the method of Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0523] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0524] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0525] Carrier gas: He (1.3 mL/min).

[0526] Changes in fatty acid composition are represented in FIG. 86. FIG. 87 represents the proportions relative to the wild-type strain taken as 100%. FIG. 87 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 16.0%, DGLA: 2.2%, ETA: 0.3%, EPA: 21.6%, n-6DPA: 2.1%, and DHA: 2.2%, which can also be described as the values of GC area such as n-6DPA/DTA: 9.5, DHA/n-3DPA: 10.3, C20PUFA/C22PUFA: 8.5, and n-6PUFA/n-3PUFA: 0.8.

[0527] It was found as a result that disrupting the C20 elongase gene and expressing the Saprolegnia diclina-derived .omega.3 desaturase in the Thraustochytrium aureum OrfA disrupted strain increases the C20:4n-6 (AA) about six-fold and the C20:5n3 (EPA) about ten-fold, and decreases the C22:6n-3 (DHA) to about 1/16.

Example 13

[0528] Expression of .omega.3 Desaturase Gene in Parietichytrium sp. SEK571 C20 Elongase Gene Disrupted Strain

[Example 13-1] Production of Saprolegnia diclina-Derived .omega.3 Desaturase Expression Plasmid using Hygromycin as Drug-Resistance Marker

[0529] For the production of a Saprolegnia diclina-derived .omega.3 desaturase expression plasmid using hygromycin as a drug-resistance marker, a plasmid pRH107 (FIG. 88) was used as the base plasmid after partially modifying the restriction enzyme site by subcloning the Parietichytrium C20 elongase upstream sequence (904 bp, SEQ ID NO: 239) and Parietichytrium C20 elongase downstream sequence (721 bp, SEQ ID NO: 240) into a pGEM-T easy vector. For reference, the total pRH107 sequence is presented (4,592 bp, SEQ ID NO: 241). The sequence as the base of the expression plasmid production is not actively used for the introduction of cells in this experiment, and as such it is not necessarily required to use pRH107 as the base vector in similar experiments. In conducting a similar experiment, a cloning vector having a KpnI site and a BamHI site in proximity can be used instead. Here, the sequence between the KpnI site and the BamHI site should be as short as possible, because it is introduced into cells as a linker between the .omega.3 desaturase gene expression cassette and the drug resistant gene expression cassette. In this experiment example, the sequence corresponds to the Parietichytrium C20 elongase downstream sequence 37 bp (SEQ ID NO: 242).

[0530] The pRH48 (FIG. 46) of Example 6-1 was digested with KpnI, and the DNA fragment containing the Saprolegnia diclina-derived .omega.3 desaturase gene cassette was ligated to the KpnI site of pRH107 (FIG. 88). This was named pRH108 (FIG. 89).

[0531] The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and the DNA fragment containing the hygromycin-resistant gene cassette was ligated to the BamHI site of pRH108 (FIG. 89). This was named pRH109 (FIG. 90).

[Example 13-2] Introduction of Saprolegnia diclina-Derived .omega.3 Desaturase Expression Plasmid into Parietichytrium sp. SEK571 C20 Elongase Gene Disrupted Strain

[0532] By using the pRH109 (FIG. 90) produced in Example 13-1 as a template, the DNA was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), using TMO42 (Example 6-1, SEQ ID NO: 168) and RHO52 (Example 3-1, SEQ ID NO: 52) as primers [PCR cycles: 94.degree. C. 30 sec, 72.degree. C. 1 min, 5 cycles/94.degree. C. 30 sec, 70.degree. C. 30 sec, 72.degree. C. 1 min, 5 cycles/94.degree. C. 30 sec, 68.degree. C. 30 sec, 72.degree. C. 1 min, 25 cycles/72.degree. C. 2 min]. The amplification product was collected form a 1.0% agarose gel, and precipitated with ethanol. The precipitate was then dissolved in 0.1.times.TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained by the PCR was 4,448 bp, and had the following sequence order: Ubiquitin promoter-.omega.3 desaturase gene-ubiquitin terminator-ubiquitin promoter-hygromycin-resistant gene sequence-SV40 terminator sequence (SEQ ID NO: 243).

[0533] The Parietichytrium sp. SEK571 C20 elongase gene disrupted strain produced in Example 10 was cultured in a GY medium for 3 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was introduced into cells corresponding to OD600=1 to 1.5 by using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1550 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied to a PDA agar plate medium (containing 1.0 mg/ml hygromycin). As a result, 5 to 20 drug resistant strains were obtained per penetration.

[Example 13-3] Acquisition of Saprolegnia diclina-Derived .omega.3 Desaturase Gene Expressing Strain

[0534] Genomic DNA was extracted from the Parietichytrium sp. SEK571 C20 elongase gene disrupted strain produced in Example 10 and the .omega.3 desaturase gene expressing strain by using the method described in Example 3-2, and the DNA concentration was calculated by measuring A260/280. By using this as a template, a PCR was performed with an LA taq Hot start version to confirm the genome structure. The positions of the primers, combinations used for the amplification, and the expected size of the amplification product are shown in FIG. 91. RHO90 (27 mer: 5'-CGT TAG AAC GCG TAA TAC GAC TCA CTA-3' SEQ ID NO: 244) was set on the ubiquitin promoter, and RHO141 (Example 3-8, SEQ ID NO: 84) was set on the hygromycin-resistant gene [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 68.degree. C. 4 min, 30 cycles/68.degree. C. 7 min].

[0535] The result of amplification confirmed a band of the expected size (FIG. 92). That is, a strain was isolated that contained the introduced expression fragment stably introduced into its genome.

[Example 13-4] Changes in Fatty Acid Composition by .omega.3 Desaturase Expression in Parietichytrium sp. SEK571 C20 Elongase Gene Disrupted Strain

[0536] The Parietichytrium sp. SEK571 strain, and the .omega.3 desaturase gene expressing strain were cultured by using the method of Example 3-9. After freeze drying, the fatty acids were methylesterificated, and GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0537] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0538] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0539] Carrier gas: He (1.3 mL/min).

[0540] The .omega.3 desaturase expressing strain reduced levels of the n-6 series fatty acids, and there was a tendency for the n-3 series fatty acids to increase (FIG. 93). FIG. 94 represents the proportions relative to the wild-type strain taken as 100%. FIG. 94 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 1.5%, DGLA: 0.2%, ETA: 0.5%, EPA: 5.2%, n-6DPA: 0.3%, and DHA: 0.4%, which can also be described as the values of GC area such as DHA/n-3DPA: 1.9, C20PUFA/C22PUFA: 8.2, and n-6PUFA/n-3PUFA: 0.3. As a result, the arachidonic acid was reduced to about 1/2, and EPA increased by a factor of about 1.4.

Example 14

Disruption of Schizochytrium C20 Elongase Gene

[Example 14-1] Cloning of Schizochytrium-Derived C20 Elongase Gene

[0541] By using the genomic DNA extracted from Schizochytrium as a template, a Schizochytrium-derived C20 elongase gene was amplified by a PCR performed with a forward oligonucleotide primer RHO134 (32 mer: 5'-CCC GGA TCC ATG GTG GCC AGC GAG GTG CTC AG-3') (SEQ ID NO: 245) containing a BamHI site, and a reverse oligonucleotide primer RHO135 (34 mer: 5'-CCC GGA TCC TTA GTC GCG CTT GAG CTC AGC ATC C-3') (SEQ ID NO: 246) containing a BamHI site (enzyme used: LA taq Hot Start Version, TaKaRa; PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 53.degree. C. 30 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 7 min/4.degree. C. .infin.). The both specific products were gel purified, cloned into a pGEM-T easy vector (Promega), and amplified with Escherichia coli. The sequence was then confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH70 (FIG. 95). As a result of abase sequence analysis, a novel gene sequence having a 945-bp (SEQ ID NO: 248) ORF, encoding 315 amino acids (SEQ ID NO: 247) was obtained.

[Example 14-2] Production of Base Plasmid for ?C20 Elongase Gene Targeting Vector Production

[0542] By using the pRH70 (FIG. 95) produced in Example 14-1 as a template, the gene was amplified with a Prime STAR Max DNA Polymerase (Takara Bio), using a primer set of the reverse orientation prepared for insertion of a BglII site in a portion halfway along the C20 elongase gene sequence. The primers used are as follows. The both had BglII linker sequences [RHO136: 25 mer: 5'-CAT CGA GAT CTT CGT GTT TGT CCA C-3' (SEQ ID NO: 249), RHO137: 25 mer: 5'-ACG AAG ATC TCG ATG CGG GCG TCC C-3' (SEQ ID NO: 250)] [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 56.degree. C. 15 sec, 72.degree. C. 1 min, 30 cycles/72.degree. C. 1 min]. After the amplification performed under these conditions, the product was digested with BglII, and allowed to self ligate. The ligated sample was amplified with Escherichia coli, and the sequence was confirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH71. The C20 elongase gene sequence 945 bp with the inserted BglII site is represented by SEQ ID NO: 251.

[0543] The product base plasmid (pRH71) for the production of the Schizochytrium C20 elongase gene targeting vector is shown in FIG. 96.

[Example 14-3] Production of Targeting Vectors (Artificial Neomycin-Resistant Gene and Hygromycin-Resistant Gene)

[0544] The pRH31 (FIG. 13) of Example 2-2 was digested with BglII, and the DNA fragment containing an artificial neomycin-resistant gene cassette was ligated to the BglII site of the pRH71 (FIG. 96) of Example 14-2. This was named pRH73.

[0545] The pRH32 (FIG. 15) of Example 2-3 was digested with BglII, and the DNA fragment containing a hygromycin-resistant gene cassette was ligated to the BglII site of the pRH71 (FIG. 96) of Example 14-2. This was named pKS-SKO.

[0546] The two targeting vectors (pRH73 and pKS-SKO) produced are shown in FIG. 97.

[Example 14-4] Introduction of C20 Elongase Gene Targeting Vector

[0547] By using the two targeting vectors produced in Example 14-3 as templates, the gene was amplified with a Prime STAR GXL polymerase, using a forward primer (SorfF: 20 mer: 5'-AGA TGG TGG CCA GCG AGG TG-3') (SEQ ID NO: 252) containing a translation initiation site, and a reverse primer (SorfR: 25 mer: 5'-TTA GTC GCG CTT GAG CTC AGC ATC C-3') (SEQ ID NO: 253) containing a translation termination site [PCR cycles: 98.degree. C. 2 min/98.degree. C. 30 sec, 60 30 sec, 72.degree. C. 3 min, 30 cycles]. After being extracted with phenol-chloroform and then with chloroform, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1.times.TE. The DNA concentration was then calculated by measuring A260/280. The introduced fragment obtained from using the pRH73 (FIG. 97) of Example 14-3 as a template was 2,644 bp, and had the following sequence order: First half of Schizochytrium C20 elongase gene-SV40 terminator sequence-artificial neomycin-resistant gene sequence-ubiquitin promoter sequence-second half of Schizochytrium C20 elongase gene (SEQ ID NO: 254). The introduced fragment obtained from using the pKS-SKO (FIG. 97) of Example 14-3 as a template was 2,881 bp, and had the following sequence order: First half of Schizochytrium C20 elongase gene-ubiquitin promoter sequence-hygromycin-resistant gene sequence-SV40 terminator sequence-second half of Schizochytrium C20 elongase gene (SEQ ID NO: 255).

[0548] The Schizochytrium sp. TY12Ab strain was cultured in a GY medium for 7 days, and cells in the logarithmic growth phase were used for gene introduction. The DNA fragment (0.625 .mu.g) was introduced into cells corresponding to OD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 24-hour recovery time, the cells with the introduced gene were applied to a PDA plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin).

[0549] As a result, about 20 drug resistant strains were obtained per penetration.

[Example 14-5] Identification of C20 Elongase Gene Gene Targeting Homologous Recombinant

[0550] The Schizochytrium sp. TY12Ab strain (FERM BP-11421), the C20 elongase gene hetero homologous recombinant, and the C20 elongase gene homo homologous recombinant (gene disrupted strain) were cultured in GY media, and the resulting cells were centrifuged at 4.degree. C., 3,000 rpm for 10 min to form a pellet. The cells were then lysed at 55.degree. C., 6 h/99.9.degree. C., 5 min after being suspended in a 20-.mu.1 SNET solution [20 mM Tris-HCl; pH 8.0, 5 mM NaCl, 0.3% SDS, 200 .mu.g/ml Proteinase K (nacalai tesque)]. The resulting cell lysate was diluted 10 times and used as a template in a PCR performed with a Mighty Amp DNA polymerase (Takara Bio) to confirm the genome structure. The positions of the primers, and the expected size of the amplification product are shown in FIG. 98. The primers were used in the SorfF and SorfR combination used in Example 14-4 [PCR cycles: 98.degree. C. 2 min/98.degree. C. 10 sec, 60.degree. C. 15 sec, 68.degree. C. 4 min, 30 cycles].

[0551] C20 elongase knockout strains were obtained that showed no amplification of the wild-type allele (Wt allele), but showed amplification of the artificial neomycin-resistant gene allele (NeoR allele) and hygromycin-resistant gene allele (HygR allele) (FIG. 99).

[Example 14-6] Changes in Fatty Acid Composition by C20 Elongase Disruption

[0552] The Schizochytrium sp. TY12Ab strain and the gene disrupted strain were cultured in GY media. Cells at the late stage of the logarithmic growth phase were centrifuged at 4.degree. C., 3,000 rpm for 10 min to form a pellet, suspended in 0.9% NaCl, and washed. The cells were further centrifuged at 4.degree. C., 3,000 rpm for 10 min, and the pellet was suspended in sterile water, and washed. This was centrifuged at 3,000 rpm for 10 min, and freeze dried after removing the supernatant. Then, 2 ml-methanolic KOH (7.5% KOH in 95% methanol) was added to the freeze dried cells, and, after being vortexed, the cells were ultrasonically disrupted (80.degree. C., 30 min).

[0553] The cells were vortexed after adding sterile water (500 .mu.l), and vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was discarded. The cells were vortexed again after adding n-hexane (2 ml), and centrifuged at 3,000 rpm for 10 min. After discarding the upper layer, 6 N HCl (1 ml) was added to the remaining lower layer, and the mixture was vortexed. The mixture was vortexed again after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. The collected upper layer was then concentrated and dried with nitrogen gas. The concentrated dry sample was incubated overnight at 80.degree. C. after adding 3 N methanolic HCl (2 ml).

[0554] The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml) was added. The mixture was vortexed after adding n-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, and the upper layer was collected. The mixture was further vortexed after adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upper layer was collected. After adding a small amount of anhydrous sodium sulfate to the collected upper layer, the mixture was vortexed, and centrifuged at 3,000 rpm for 10 min. After collecting the upper layer, the upper layer was concentrated and dried with nitrogen gas. The concentrated dry sample was dissolved in n-hexane (0.2 ml), and 2 .mu.l of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:

[0555] Column: HR-SS-10 (30 m.times.0.25 mm; Shinwa Chemical Industries Ltd.)

[0556] Column temperature: 150.degree. C..fwdarw.(5.degree. C./min).fwdarw.220.degree. C. (10 min)

[0557] Carrier gas: He (1.3 mL/min).

[0558] As a result, knocking out the C20 elongase in the Schizochytrium sp. TY12Ab strain increased fatty acids of 20 carbon chain length (FIG. 100). FIG. 101 represents the proportions relative to the wild-type strain taken as 100%. FIG. 101 represents the proportion of each component based on the total amount of the fatty acids, which includes AA: 17.2%, EPA: 3.3%, n-6DPA: 13.7%, and DHA: 15.6%, which can also be described as the value of GC area such as DHA/n-3DPA: 38.8.

[0559] As can be seen from these results, the arachidonic acid increased about 1.7-fold, EPA about 1.3-fold, DPA (n-6) about 1.1-fold, and DHA about 0.9-fold. One of the EPA contents was increased to be 3.3% of a total fatty acid composition. It has been found that the EPA content can be 3.3% or more of a total fatty acid composition as shown by the above Examples.

INDUSTRIAL APPLICABILITY

[0560] The present invention provides a method for transforming stramenopile through disruption of stramenopile genes and/or inhibition of expression thereof, modification of the fatty acid composition produced by a stramenopile, and a method for highly accumulating fatty acids in a stramenopile. The present invention thus enables more efficient production of polyunsaturated fatty acids.

Sequence CWU 1

1

255121DNAArtificialprimermisc_feature(6)..(6)n is a, c, g, or tmisc_feature(12)..(12)n is a, c, g, or t 1ttyytncayg tntaycayca y 21223DNAArtificialprimermisc_feature(12)..(12)n is a, c, g, or tmisc_feature(18)..(18)n is a, c, g, or t 2gcrtgrtgrt anacrtgnar raa 233190DNAArtificialcDNA (DNA fragment contains elo1) 3cgccaccatc tttgctatct ggtttatgat cgccaagtac gccccgggcg gcgacgcata 60ctttagcgtc atcctgaact cgttcgtgca caccgtcatg tacgcgtact acttcttctc 120gtcgcagggc ttcgggttcg tcaagccgat caagccgtac atcacctcgc tgcagatgac 180gcagttcatg 1904210DNAArtificialcDNA (DNA fragment contains elo2) 4ccgacgacca gcacaccgag tgggtctcgt gcgtgcgctt ctcgccctcg accaccaacc 60cgctgatcgt gtcgtgcggc tgggacaagc tcgtcaaggt ctggaacctc tcgaactgca 120agcttcgggc caacctcatc ggccacgacg gctacctcaa ctcggtcacc gtcagcccgg 180acggctccct gtgcgcttcg ggcggcaagg 2105200DNAArtificialcDNA (DNA fragment contains elo3) 5aagctaacct gggcgtagtt tttcttgagg atcatcatga acgtgtcgct ccagtcgaga 60aactttgtca ggaggtgcac gaacacgaaa aactcgatgt tcgagtcgcg cgacttgttg 120aggccgaaag ggttgccgtt ggccaggtcg acctgcggcc agaggcccca caccatccag 180ccgcacaccg cgatttggac 200621DNAArtificialprimer 6tatgatcgcc aagtacgccc c 21721DNAArtificialprimer 7gaactgcgtc atctgcagcg a 21820DNAArtificialprimer 8tctcgccctc gaccaccaac 20922DNAArtificialprimer 9cggtgaccga gttgaggtag cc 221022DNAArtificialprimer 10caaccctttc ggcctcaaca ag 221126DNAArtificialprimer 11ttcttgagga tcatcatgaa cgtgtc 26121139DNAArtificialcDNA (elo1) 12ctaatacgac tcactatagg gcaagcagtg gtaacaacgc agagtacgcg gggaccccaa 60acgcccgacg acaaccaaga agacagccag ccgaacaatc ggacgaagat gacgagcaac 120atgagcgcgt ggggcgtcgc cgtcgaccag acgcagcagg tcgtcgacca gatcatgggc 180ggcgccgagc cgtacaagct gacagaaggg cgcatgacga acgtcgagac gatgctggcg 240atcgagtgcg gctacgccgc catgctgctg ttcctgaccc cgatcatgaa gcaggccgag 300aagcccttcg agctcaagtc cttcaagctc gcccacaacc tgttcctgtt cgtcctgtcc 360gcctacatgt gcctcgagac cgtccgccag gcctaccttg cgggctactc ggtgttcggc 420aacgacatgg agaagggcag cgagccgcac gcgcacggca tggcccaaat cgtgtggatc 480ttttacgtgt ccaaggcgta cgagttcgtg gacacgctga tcatgatcct gtgcaaaaag 540ttcaaccagg tctccgtcct gcacgtgtac caccacgcca ccatctttgc tatctggttt 600atgatcgcca agtacgcccc gggcggcgac gcatacttta gcgtcatcct gaactcgttc 660gtgcacaccg tcatgtacgc gtactacttc ttctcgtcgc agggcttcgg gttcgtcaag 720ccgatcaagc cgtacatcac ctcgctgcag atgacgcagt tcatggcgat gctcgtgcag 780tcgctgtacg actaccttta cccgtgcgac tacccgcagg ggctcgtcaa gctcctcggc 840gtgtacatgc tcaccctgct tgcgctcttc ggcaactttt tcgtgcagag ctacctcaag 900aagtcgaaca agcccaaggc caagtcggcc taagccgacc cgctcgccgg caaccgagca 960gcacctaggc gcatctcggc ccggaacctt ttcgacctgc tgtggagcgc gcgacgcgtt 1020tcgcgaccgt ccgcgcgttc ttgacactct ttgctctgtg tgtttcgcac ttgacaacct 1080ggaacagaca catacacgat acaaatcatc agaacagaca aaaaacaacc tcaaattat 1139131261DNAArtificialcDNA (elo3) 13ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagtacgcg gggaccccga 60acgtgtttct cccaggacgt gccgctgtcg ctcgctgatc cacccgaagc gcggtcggct 120ggcacggtcg ctcggctgga agttgagtag tttgctttct gttactgcgc tgctttgtaa 180acgcgaccat ggcgacgcgc acctcgaaga gcgctccggc ggtttccaag tcggccaagg 240ttgccgcgcc ggcgaagaag cggtcggtcg acaggagcga cggtttcttc cgcacgttca 300acctgtgcgc cctgtacggg tctgccctcg cctatgcgta caagcacggc ccggtggaca 360atgacggcca ggggctgtac tttcacaagt cgcccatgta cgcgttcgcc gtgtcggacg 420tcatgacctt cggcgcgccg ctgatgtacg tgctcggtgt gatgctgctc agcaggtaca 480tggcggacaa aaagcccctg actggcttca tcaagaccta catccagccc gtctacaacg 540tggtccaaat cgcggtgtgc ggctggatgg tgtggggcct ctggccgcag gtcgacctgg 600ccaacggcaa ccctttcggc ctcaacaagt cgcgcgactc gaacatcgag tttttcgtgt 660tcgtgcacct cctgacaaag tttctcgact ggagcgacac gttcatgatg atcctcaaga 720aaaactacgc ccaggttagc tttctgcagg tgttccacca cgcaacgatc ggcatggtgt 780ggtcgttcct tcttcagcgt ggctggggct cgggcaccgc cgcgtacggt gctttcatca 840actcggtcac gcacgtgatc atgtactcgc actactttgc cacctcgctc aacatcaaca 900acccgttcaa gcggtacatc acgagcttcc agctcgccca gtttgcaagc tgcatcgtgc 960atgccctact ggtgcttgcc ttcgaggagg tgtacccgct cgagtacgct tacctgcaga 1020tcagctacca catcatcatg ctctacctgt tcggacgccg catgaactgg agccccgagt 1080ggtgcaccgg tgagatcgac ggccttgacg ccccaagcgc ccccaccaag tccgagtaaa 1140cctgtttccg gctggctccc gagccatgct taccatgaat gaacctgcaa acagtctgag 1200gtccttgtgc aaaccgctca gtgggacgtc gacgaagaaa gaaacaatgt gtactcgtcc 1260c 126114275PRTT. aureum ATCC 34304misc_feature(275)..(275)Xaa can be any naturally occurring amino acid 14Met Thr Ser Asn Met Ser Ala Trp Gly Val Ala Val Asp Gln Thr Gln1 5 10 15Gln Val Val Asp Gln Ile Met Gly Gly Ala Glu Pro Tyr Lys Leu Thr 20 25 30Glu Gly Arg Met Thr Asn Val Glu Thr Met Leu Ala Ile Glu Cys Gly 35 40 45Tyr Ala Ala Met Leu Leu Phe Leu Thr Pro Ile Met Lys Gln Ala Glu 50 55 60Lys Pro Phe Glu Leu Lys Ser Phe Lys Leu Ala His Asn Leu Phe Leu65 70 75 80Phe Val Leu Ser Ala Tyr Met Cys Leu Glu Thr Val Arg Gln Ala Tyr 85 90 95Leu Ala Gly Tyr Ser Val Phe Gly Asn Asp Met Glu Lys Gly Ser Glu 100 105 110Pro His Ala His Gly Met Ala Gln Ile Val Trp Ile Phe Tyr Val Ser 115 120 125Lys Ala Tyr Glu Phe Val Asp Thr Leu Ile Met Ile Leu Cys Lys Lys 130 135 140Phe Asn Gln Val Ser Val Leu His Val Tyr His His Ala Thr Ile Phe145 150 155 160Ala Ile Trp Phe Met Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr 165 170 175Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr 180 185 190Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro 195 200 205Tyr Ile Thr Ser Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln 210 215 220Ser Leu Tyr Asp Tyr Leu Tyr Pro Cys Asp Tyr Pro Gln Gly Leu Val225 230 235 240Lys Leu Leu Gly Val Tyr Met Leu Thr Leu Leu Ala Leu Phe Gly Asn 245 250 255Phe Phe Val Gln Ser Tyr Leu Lys Lys Ser Asn Lys Pro Lys Ala Lys 260 265 270Ser Ala Xaa 27515825DNAArtificialcDNA (T. aureum ATCC 34304 elo1) 15atgacgagca acatgagcgc gtggggcgtc gccgtcgacc agacgcagca ggtcgtcgac 60cagatcatgg gcggcgccga gccgtacaag ctgacagaag ggcgcatgac gaacgtcgag 120acgatgctgg cgatcgagtg cggctacgcc gccatgctgc tgttcctgac cccgatcatg 180aagcaggccg agaagccctt cgagctcaag tccttcaagc tcgcccacaa cctgttcctg 240ttcgtcctgt ccgcctacat gtgcctcgag accgtccgcc aggcctacct tgcgggctac 300tcggtgttcg gcaacgacat ggagaagggc agcgagccgc acgcgcacgg catggcccaa 360atcgtgtgga tcttttacgt gtccaaggcg tacgagttcg tggacacgct gatcatgatc 420ctgtgcaaaa agttcaacca ggtctccgtc ctgcacgtgt accaccacgc caccatcttt 480gctatctggt ttatgatcgc caagtacgcc ccgggcggcg acgcatactt tagcgtcatc 540ctgaactcgt tcgtgcacac cgtcatgtac gcgtactact tcttctcgtc gcagggcttc 600gggttcgtca agccgatcaa gccgtacatc acctcgctgc agatgacgca gttcatggcg 660atgctcgtgc agtcgctgta cgactacctt tacccgtgcg actacccgca ggggctcgtc 720aagctcctcg gcgtgtacat gctcaccctg cttgcgctct tcggcaactt tttcgtgcag 780agctacctca agaagtcgaa caagcccaag gccaagtcgg cctaa 82516317PRTT. aureum ATCC 34304misc_feature(317)..(317)Xaa can be any naturally occurring amino acid 16Met Ala Thr Arg Thr Ser Lys Ser Ala Pro Ala Val Ser Lys Ser Ala1 5 10 15Lys Val Ala Ala Pro Ala Lys Lys Arg Ser Val Asp Arg Ser Asp Gly 20 25 30Phe Phe Arg Thr Phe Asn Leu Cys Ala Leu Tyr Gly Ser Ala Leu Ala 35 40 45Tyr Ala Tyr Lys His Gly Pro Val Asp Asn Asp Gly Gln Gly Leu Tyr 50 55 60Phe His Lys Ser Pro Met Tyr Ala Phe Ala Val Ser Asp Val Met Thr65 70 75 80Phe Gly Ala Pro Leu Met Tyr Val Leu Gly Val Met Leu Leu Ser Arg 85 90 95Tyr Met Ala Asp Lys Lys Pro Leu Thr Gly Phe Ile Lys Thr Tyr Ile 100 105 110Gln Pro Val Tyr Asn Val Val Gln Ile Ala Val Cys Gly Trp Met Val 115 120 125Trp Gly Leu Trp Pro Gln Val Asp Leu Ala Asn Gly Asn Pro Phe Gly 130 135 140Leu Asn Lys Ser Arg Asp Ser Asn Ile Glu Phe Phe Val Phe Val His145 150 155 160Leu Leu Thr Lys Phe Leu Asp Trp Ser Asp Thr Phe Met Met Ile Leu 165 170 175Lys Lys Asn Tyr Ala Gln Val Ser Phe Leu Gln Val Phe His His Ala 180 185 190Thr Ile Gly Met Val Trp Ser Phe Leu Leu Gln Arg Gly Trp Gly Ser 195 200 205Gly Thr Ala Ala Tyr Gly Ala Phe Ile Asn Ser Val Thr His Val Ile 210 215 220Met Tyr Ser His Tyr Phe Ala Thr Ser Leu Asn Ile Asn Asn Pro Phe225 230 235 240Lys Arg Tyr Ile Thr Ser Phe Gln Leu Ala Gln Phe Ala Ser Cys Ile 245 250 255Val His Ala Leu Leu Val Leu Ala Phe Glu Glu Val Tyr Pro Leu Glu 260 265 270Tyr Ala Tyr Leu Gln Ile Ser Tyr His Ile Ile Met Leu Tyr Leu Phe 275 280 285Gly Arg Arg Met Asn Trp Ser Pro Glu Trp Cys Thr Gly Glu Ile Asp 290 295 300Gly Leu Asp Ala Pro Ser Ala Pro Thr Lys Ser Glu Xaa305 310 31517951DNAArtificialcDNA (T. aureum ATCC 34304 elo3) 17atggcgacgc gcacctcgaa gagcgctccg gcggtttcca agtcggccaa ggttgccgcg 60ccggcgaaga agcggtcggt cgacaggagc gacggtttct tccgcacgtt caacctgtgc 120gccctgtacg ggtctgccct cgcctatgcg tacaagcacg gcccggtgga caatgacggc 180caggggctgt actttcacaa gtcgcccatg tacgcgttcg ccgtgtcgga cgtcatgacc 240ttcggcgcgc cgctgatgta cgtgctcggt gtgatgctgc tcagcaggta catggcggac 300aaaaagcccc tgactggctt catcaagacc tacatccagc ccgtctacaa cgtggtccaa 360atcgcggtgt gcggctggat ggtgtggggc ctctggccgc aggtcgacct ggccaacggc 420aaccctttcg gcctcaacaa gtcgcgcgac tcgaacatcg agtttttcgt gttcgtgcac 480ctcctgacaa agtttctcga ctggagcgac acgttcatga tgatcctcaa gaaaaactac 540gcccaggtta gctttctgca ggtgttccac cacgcaacga tcggcatggt gtggtcgttc 600cttcttcagc gtggctgggg ctcgggcacc gccgcgtacg gtgctttcat caactcggtc 660acgcacgtga tcatgtactc gcactacttt gccacctcgc tcaacatcaa caacccgttc 720aagcggtaca tcacgagctt ccagctcgcc cagtttgcaa gctgcatcgt gcatgcccta 780ctggtgcttg ccttcgagga ggtgtacccg ctcgagtacg cttacctgca gatcagctac 840cacatcatca tgctctacct gttcggacgc cgcatgaact ggagccccga gtggtgcacc 900ggtgagatcg acggccttga cgccccaagc gcccccacca agtccgagta a 9511838DNAArtificialprimer 18ataagcttaa aatgtctagc aacatgagcg cgtggggc 381928DNAArtificialprimer 19tgtctagaac gcgcggacgg tcgcgaaa 282040DNAArtificialprimer 20taaagcttaa aatgtctacg cgcacctcga agagcgctcc 402131DNAArtificialprimer 21catctagact cggacttggt gggggcgctt g 3122949DNAArtificialcDNA (TaELO1 coding region) 22ataagcttaa aatgacgagc aacatgagcg cgtggggcgt cgccgtcgac cagacgcagc 60aggtcgtcga ccagatcatg ggcggcgccg agccgtacaa gctgacagaa gggcgcatga 120cgaacgtcga gacgatgctg gcgatcgagt gcggctacgc cgccatgctg ctgttcctga 180ccccgatcat gaagcaggcc gagaagccct tcgagctcaa gtccttcaag ctcgcccaca 240acctgttcct gttcgtcctg tccgcctaca tgtgcctcga gaccgtccgc caggcctacc 300ttgcgggcta ctcggtgttc ggcaacgaca tggagaaggg cagcgagccg cacgcgcacg 360gcatggccca aatcgtgtgg atcttttacg tgtccaaggc gtacgagttc gtggacacgc 420tgatcatgat cctgtgcaaa aagttcaacc aggtctccgt cctgcacgtg taccaccacg 480ccaccatctt tgctatctgg tttatgatcg ccaagtacgc cccgggcggc gacgcatact 540ttagcgtcat cctgaactcg ttcgtgcaca ccgtcatgta cgcgtactac ttcttctcgt 600cgcagggctt cgggttcgtc aagccgatca agccgtacat cacctcgctg cagatgacgc 660agttcatggc gatgctcgtg cagtcgctgt acgactacct ttacccgtgc gactacccgc 720aggggctcgt caagctcctc ggcgtgtaca tgctcaccct gcttgcgctc ttcggcaact 780ttttcgtgca gagctacctc aagaagtcga acaagcccaa ggccaagtcg gcctaagccg 840acccgctcgc cggcaaccga gcagcaccta ggcgcatctc ggcccggaac cttttcgacc 900tgctgtggag cgcgcgacgc gtttcgcgac cgtccgcgcg ttctagaca 94923967DNAArtificialcDNA (TaELO2 coding region) 23taaagcttaa aatggcgacg cgcacctcga agagcgctcc ggcggtttcc aagtcggcca 60aggttgccgc gccggcgaag aagcggtcgg tcgacaggag cgacggtttc ttccgcacgt 120tcaacctgtg cgccctgtac gggtctgccc tcgcctatgc gtacaagcac ggcccggtgg 180acaatgacgg ccaggggctg tactttcaca agtcgcccat gtacgcgttc gccgtgtcgg 240acgtcatgac cttcggcgcg ccgctgatgt acgtgctcgg tgtgatgctg ctcagcaggt 300acatggcgga caaaaagccc ctgactggct tcatcaagac ctacatccag cccgtctaca 360acgtggtcca aatcgcggtg tgcggctgga tggtgtgggg cctctggccg caggtcgacc 420tggccaacgg caaccctttc ggcctcaaca agtcgcgcga ctcgaacatc gagtttttcg 480tgttcgtgca cctcctgaca aagtttctcg actggagcga cacgttcatg atgatcctca 540agaaaaacta cgcccaggtt agctttctgc aggtgttcca ccacgcaacg atcggcatgg 600tgtggtcgtt ccttcttcag cgtggctggg gctcgggcac cgccgcgtac ggtgctttca 660tcaactcggt cacgcacgtg atcatgtact cgcactactt tgccacctcg ctcaacatca 720acaacccgtt caagcggtac atcacgagct tccagctcgc ccagtttgca agctgcatcg 780tgcatgccct actggtgctt gccttcgagg aggtgtaccc gctcgagtac gcttacctgc 840agatcagcta ccacatcatc atgctctacc tgttcggacg ccgcatgaac tggagccccg 900agtggtgcac cggtgagatc gacggccttg acgccccaag cgcccccacc aagtccgagt 960ctagatg 967241122DNAArtificialcDNA (TaELO2 ORF upstream region) 24cgttagaacg cgtaatacga ctcactatag ggatatcccc cgcgaggcga tggctgctcc 60gacgacgtgg gctggcgacg tcgctcgcaa aggcgttccg caaccgcgcg ttccgctgta 120acgagaccgt tttccctgcg ctgctgggtg gacctagcgc gtgtgtcacc tgccggcccc 180cgttgcgtgc aaccgaattg atcgataata gaattacata acaaacaact tgctggatga 240gtacaagacc agcgtagtgt ggctgtggga cgttgaacgg agcgggtcct gtgacggcgc 300agaaaggaac tccgcccgag gtgaaacccc gatgcgcagg actctgcggc cacagcccct 360ccgccagtat tccactaaaa atccgccccc tttgacaaag atcgcaaccc cgtcccatca 420actcctcaca ataggctttc cactggcgga aacgtccccg gcacaggagt gcctcccgcg 480gttctgcgca tacggctgac cactacgcag cgcgatatcc tccatccgcg tatatatccg 540taaacaacgg aacattctcc ctctcaacga ggcgtggttt tcgaagccat gcctttcttc 600cttcctactt gccttccttc tttctttctt tctttccttc ttttgcaagc gtgcgcgaac 660ttgaaggtac tacttacact tgacagagag agatagagac ggcaattcga ccaagtactt 720tccacgattt tttttttttt tgttttggtc gctttcgttg gtcgtgcatg atggatggcc 780gggattttta caattggatg cgccaggctg ccacgcatgc cgtgacgctc gctcgcggcg 840actcatgatg cttgccagtg gcagtgcatc cagctcttcc ctctgctcgt cgtgtactca 900ctggcgatgc tctcggcgct cgttcaaggg ccatcgatcg atcgatcgat cgatcgatcg 960atcaatcacg tttggtggac tcggcagacc ccgaacgtgt ttctcccagg acgtgccgct 1020gtcgctcgct gatccacccg aagcgcggtc ggctggcacg gtcgctcggc tggaagttga 1080gtagtttgct ttctgttgct gcgctgcttt gtaaacgcga cc 1122251204DNAArtificialcDNA (TaELO2 downstream region) 25acctgtttcc ggctggctcc cgagccatgc ttaccatgaa tgaacctgca aacagtctga 60ggtccttgtg caaaccgctc agtgggacgt cgacgaagaa agaaacaatg tgtactcgtc 120ttgctctgct cccgcgccgt tttttatcgt tgttgagacc tctcgcgcag ttttgggaat 180caaccaaaac aagagcccgg cgtcagcgtt tgcttcgccc tcggctgcac tcgctcggca 240cgcaggtata actgggtgag taccaagccc cgcatttgtc tgtccgcgat ccgcgcacgc 300tgcgggtcag gacgacatcg cgctgcacgt cacagtgggt cccttttgac gtggctgcgg 360cgatgaggag gcttggctcg gcttcatggc aaggcaacag actcgcttcc aggacgcgca 420cgacgagcag cgctgctttg atcgaccttg cctgcgtcac cgcctcggct gctttgatcg 480atcgttgtca ccggccgagt gaccgcgaac gcattgcccg cacggctcgg ctcggctcgg 540accggaccgg ctcgccttgg cggcgcggcg cgatggcgac ccagacgcga ccggagccgc 600gcgcggagga caaggccatg ttcatcttcg ggctcgggta cgttgggagc aggctcgcca 660accagctggc ggaacagggg tggcgcgtcg cggggtcggt gagggagctc gggcgcgagg 720acgactttgc cgagttcgaa aagtccaagc tgagcggcaa ggtgcaggtg ttccgactcc 780cgcttgaggg cgaggacaac acgcccgctc gcgcgcggga gatacttagc gggtaccagc 840acctgctgtt cacggcgcca gtggaccgcg cccggaactg tgaccccttc ttgggcgacc 900ccgttctcgg ccccgtgatc gtcgagctag cagaggaggg ccgcatcgac tgggccggct 960atctctcaac cacttcggtc tacggcaacc acgacggcga gtgggtggac gagaccacgc 1020cgctcatgcc cacgctcaaa cgcggcgagc agcgcgtcat ggtggagcgc gccttcctgt 1080acgagtcggg cctcccggcc catatctttc ggctgccagg aatctacggc ccagggcgcg 1140gcccgatatc acgaattctc tccctatagt gagtcgtatt acgcgttcta acgacaatat 1200gtac 12042631DNAArtificialprimer 26ctcccgggtg gacctagcgc gtgtgtcacc t 312725DNAArtificialprimer 27ggtcgcgttt acaaagcagc gcagc 252852DNAArtificialprimer 28gctgcgctgc tttgtaaacg cgaccatgat tgaacaggac ggccttcacg ct 522952DNAArtificialprimer 29tcgggagcca

gccggaaaca ggttcaaaag aactcgtcca ggaggcggta ga 523023DNAArtificialprimer 30acctgtttcc ggctggctcc cga 233127DNAArtificialprimer 31atcccggggc cgagaacggg gtcgccc 27322696DNAArtificialcDNA (TaELO2 ORF upstream/Neor/ TaELO2 ORF downstream) 32ctcccgggtg gacctagcgc gtgtgtcacc tgccggcccc cgttgcgtgc aaccgaattg 60atcgataata gaattacata acaaacaact tgctggatga gtacaagacc agcgtagtgt 120ggctgtggga cgttgaacgg agcgggtcct gtgatggcgc agaaaggaac tccgcccgag 180gtgaaacccc gatgcgcagg actctgcggc cacagcccct ccgccagtat tccactaaaa 240atccgccccc tttgacaaag atcgcaaccc cgtcccatca actcctcaca ataggctttc 300cactggcgga aacgtccccg gcacaggagt gcctcccgcg gttctgcgca tacggctgac 360cactacgcag cgcgatatcc tccatccgcg tatatatccg taaacaacgg aacattctcc 420ctctcaacga ggcgtggttt tcgaagccat gcctttcttc cttcctactt gccttccttc 480tttctttctt tctttccttc ttttgcaagc gtgcgcgaac ttgaaggtac tacttacact 540tgacagagag agatagagac ggcaattcga ccaagtactt tccacgattt tttttttttt 600tgttttggtc gctttcgttg gtcgtgcatg atggatggcc gggattttta caattggatg 660cgccaggctg ccacgcatgc cgtgacgctt gctcgcggcg actcatgatg cttgccagtg 720gcagtgcatc cagctcttcc ctctgctcgt cgtgtactca ctggcgatgc tctcggcgct 780cgttcaaggg ccatcgatcg atcgatcgat cgatcgatcg atcaatcacg tttggtggac 840tcggcagacc ccgaacgtgt ttctcccagg acgcgccgct gtcgctcgct gatccacccg 900aagcgcggtc ggctggcacg gtcgctcggc tggaagttga gtagtttgct ttctgttgct 960gcgctgcttt gtaaacgcga ccatgattga acaggacggc cttcacgctg gctcgcccgc 1020tgcttgggtg gaacggctgt tcggctacga ctgggctcag cagacgatcg gctgctcgga 1080cgcggccgtg ttccgcctta gcgcgcaggg ccggccggtc ctgtttgtca agaccgacct 1140tagcggcgcc ctcaacgagc tccaggacga agctgcccgc ctcagctggc ttgccacgac 1200gggggttccg tgcgccgctg tgctcgacgt cgtcaccgaa gccggccgcg actggctgct 1260cctcggggaa gtgcccggcc aggacctcct cagcagccac ctcgcgcccg ctgagaaggt 1320gtccatcatg gccgacgcca tgcgccgcct gcacaccctc gaccccgcca cctgcccctt 1380cgaccaccag gcgaagcaca ggatcgaacg cgcccgcacg cggatggagg ctggcctcgt 1440cgaccaagac gacctcgacg aggagcacca gggcctcgcg ccggcggaac tgttcgccag 1500gcttaaggct aggatgccgg acggcgagga cctcgtggtc acgcacggcg acgcctgcct 1560ccccaacatc atggtcgaga acggccgctt ctcgggcttt atcgactgcg ggcgcctggg 1620cgtggcggac cgctaccaag acatcgcgct cgccacgcgg gacatcgccg aggagcttgg 1680cggcgagtgg gccgaccgct ttctcgtgct ctacggcatc gccgccccgg acagccagag 1740gattgcgttc taccgcctcc tggacgagtt cttttgaacc tgtttccggc tggctcccga 1800gccatgctta ccatgaatga acctgcaaac agtctgaggt ccttgtgcaa accgctcagt 1860gggacgtcga cgaagaaaga aacaatgtgt actcgtcttg ctctgctccc gcgccgtttt 1920ttatcgttgt tgagacctct cgcgcagttt tgggaatcaa ccaaaacaag agcccggcgt 1980cagcgtttgc ttcgccctcg gctgcactcg ctcggcacgc aggtataact gggtgagtac 2040caagccccgc atttgtctgt ccgcgatccg cgcacgctgc gggtcaggac gacatcgcgc 2100tgcacgtcac agtgggtccc ttttgacgtg gctgcggcga tgaggaggct tggctcggct 2160tcatggcaag gcaacagact cgcttccggg acgcgcacga cgagcagcgc tgctttgatc 2220gaccttgcct gcgtcaccgc ctcggctgct ttgatcgatc gttgtcaccg gccgagtgac 2280cgcgaacgca ttgcccgcac ggctcggctc ggcccggacc ggaccggctc gccttggcgg 2340cgcggcgcga tggcgaccca gacgcggccg gagccgcgcg cggaggacaa ggccatgttc 2400atcttcgggc tcgggtacgt tgggagcagg ctcgccaacc agctggcgga acaggggtgg 2460cgcgtcgcgg ggtcggtgag ggagctcggg cgcgaggacg actttgccga gttcgaaaag 2520tccaagctga gcggcaaggt gcaggtgttc cgactcccgc ttgagggcga ggacaacacg 2580cccgctcgcg cgcgggagat acttagcggg taccagcacc tgctgttcac ggcgccagtg 2640gaccgcgccc ggaactgtga ccccttcttg ggcgaccccg ttctcggccc cgggat 26963330DNAArtificialprimer 33ggatatcccc cgcgaggcga tggctgctcc 303430DNAArtificialprimer 34tgatatcggg ccgcgccctg ggccgtagat 303525DNAArtificialprimer 35gtacgtgctc ggtgtgatgc tgctc 253624DNAArtificialprimer 36gcggcgtccg aacaggtaga gcat 243735DNAArtificialprimer 37atccgcgtat atatccgtaa acaacggaac attct 353826DNAArtificialprimer 38cttcgggtgg atcagcgagc gacagc 263927DNAArtificialprimer 39gccgcagcgc ctggtgcacc cgccggg 274059DNAArtificialprimer 40tcgcgggtga gttcaggctt tttcatgttg gctagtgttg cttaggtcgc ttgctgctg 594157DNAArtificialprimer 41agcgacctaa gcaacactag gccaacatga aaaagcctga actcaccgcg acgtctg 574229DNAArtificialprimer 42ctattccttt gccctcggac gagtgctgg 29431636DNAArtificialcDNA (T. aureum ATCC 34304 ubiruitin promoter/ Hygr) 43gctagccgca gcgcctggtg cacccgccgg gcgttggttg tgtgtgctat ttactatgcc 60taccgagaga gagagcggag cggatgcata ggaaatcggg ccacgcggga gggccatgcg 120ttcgccccac acgccactta taccacgccc gctctctctc cggccggcag gcagcgcata 180actataccga cgctggcagg cttggtagca actggcaggg acaactcgcg cgcgggtccc 240ggtcgttcga tgtgccaacc cgagagaatc cagccagcag ggcggttggc ctcatcgccc 300acctgctatg gtgcagcgaa ccaactcccg aagcggccgg ttccgcgatt ccctcttctg 360aattctgaat tctgaactga ttccggagga gaaccctctg gaagcgcggg ttgcctctcc 420agttctgccg aactagacag gggagtgagc atgatgagtg accctgacgc gtgagctgag 480ctggttgctg gaatatagtc gctgaacgct gggctgtgtc acgcgtccac ttcgggcaga 540ccccaaacga caagcagaac aagcaacacc agcagcagca agcgacctaa gcaacactag 600ccaacatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 660tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 720tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 780aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 840acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 900cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 960tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 1020aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 1080tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 1140atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 1200tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 1260aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 1320tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 1380cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 1440ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 1500ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 1560atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 1620caaaggaata gtctag 16364433DNAArtificialprimer 44gtgctagccg cagcgcctgg tgcacccgcc ggg 334537DNAArtificialprimer 45gttctagact attcctttgc cctcggacga gtgctgg 374637DNAArtificialprimer 46gttctagacc tgtttccggc tggctcccga gccatgc 374740DNAArtificialprimer 47gtgctagcgg tcgcgtttac aaagcagcgc agcaacagaa 40483537DNAArtificialcDNA (TaELO2 ORF upstream region/T. aureum ATCC 34304 ubiquitin promotor/ Hygr /TaELO2 ORF downstream region) 48ctcccgggtg gacctagcgc gtgtgtcacc tgccggcccc cgttgcgtgc aaccgaattg 60atcgataata gaattacata acaaacaact tgctggatga gtacaagacc agcgtagtgt 120ggctgtggga cgttgaacgg agcgggtcct gtgacggcgc agaaaggaac tccgcccgag 180gtgaaacccc gatgcgcagg actctgcggc cacagcccct ccgccagtat tccactaaaa 240atccgccccc tttgacaaag atcgcaaccc cgtcccatca actcctcaca ataggctttc 300cactggcgga aacgtccccg gcacaggagt gcctcccgcg gttctgcgca tacggctgac 360cactacgcag cgcgatatcc tccatccgcg tatatatccg taaacaacgg aacattctcc 420ctctcaacga ggcgtggttt tcgaagccat gcctttcttc cttcctactt gccttccttc 480tttctttctt tctttctttc ttttgtaagc gtgcgcgaac ttgaaggtac tacttacact 540tgacagagag agatagagac ggcaattcga ccaagtactt tccacgattt tttttttttt 600tgttttggtc gctttcgttg gtcgtgcatg atggatggcc gggattttta caattggatg 660cgccaggctg ccacgcatgc cgtgacgctc gctcgcggcg actcatggtg cttgccagtg 720gcagtgcatc cagctcttcc ctctgctcgt cgtgtactca ctggcgatgc tctcggcgct 780cgttcaaggg ccatcgatcg atcgatcgat cgatcgatcg atcaatcacg tttggtggac 840tcggcagacc ccgaacgtgt ttctcccagg acgtgccgct gtcgctcgct gatccacccg 900aagcgcggtc ggctggcacg gtcgctcggc tggaagttga gtagtttgct ttctgttgct 960gcgctgcttt gtaaacgcga ccgctagccg cagcgcctgg tgcacccgcc gggcgttggt 1020tgtgtgtgct atttactatg cctaccgaga gagagagcgg agcggatgca taggaaatcg 1080ggccacgcgg gagggccatg cgttcgcccc acacgccact tataccacgc ccgctctctc 1140tccggccggc aggcagcgca taactatacc gacgctggca ggcttggtag caactggcag 1200ggacaactcg cgcgcgggtc ccggtcgttc gatgtgccaa cccgagagaa tccagccagc 1260agggcggttg gcctcatcgc ccacctgcta tggtgcagcg aaccaactcc cgaagcggcc 1320ggttccgcga ttccctcttc tgaattctga attctgaact gattccggag gagaaccctc 1380tggaagcgcg ggttgcctct ccagttctgc cgaactagac aggggagtga gcatgatgag 1440tgaccctgac gcgtgagctg agctggttgc tggaatatag tcgctgaacg ctgggctgtg 1500tcacgcgtcc acttcgggca gaccccaaac gacaagcaga acaagcaaca ccagcagcag 1560caagcgacct aagcaacact agccaacatg aaaaagcctg aactcaccgc gacgtctgtc 1620gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1680gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1740agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1800ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1860tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1920ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1980gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 2040tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 2100gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 2160cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 2220acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 2280atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 2340aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 2400gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 2460cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 2520agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 2580cgccccagca ctcgtccgag ggcaaaggaa tagtctagac ctgtttccgg ctggctcccg 2640agccatgctt accatgaatg aacctgcaaa cagtctgagg tccttgtgca aaccgctcag 2700tgggacgtcg acgaagaaag aaacaatgtg tactcgtctt gctctgctcc cgcgccgttt 2760tttatcgttg ttgagacctc tcgcgcagtt ttgggaatca accaaaacaa gagcccggcg 2820tcagcgtttg cttcgccctc ggctgcactc gctcggcacg caggtataac tgggtgagta 2880ccaagccccg catttgtctg tccgcgatcc gcgcacgctg cgggtcagga cgacatcgcg 2940ctgcacgtca cagtgggtcc cttttgacgt ggctgcggcg atgaggaggc ttggctcggc 3000ttcatggcaa ggcaacagac tcgcttccgg gacgcgcacg acgagcagcg ctgctttgat 3060cgaccttgcc tgcgtcaccg cctcggctgc tttgatcgat cgttgtcacc ggccgagtga 3120ccgcgaacgc attgcccgca cggctcggct cggcccggac cggaccggct cgccttggcg 3180gcgcggcgcg atggcgaccc agacgcggcc ggagccgcgc gcggaggaca aggccatgtt 3240catcttcggg ctcgggtacg ttgggagcag gctcgccaac cagctggcgg aacaggggtg 3300gcgcgtcgcg gggtcggtga gggagctcgg gcgcgaggac gactttgccg agttcgaaaa 3360gtccaagctg agcggcaagg tgcaggtgtt ccgactcccg cttgagggcg aggacaacac 3420gcccgctcgc gcgcgggaga tacttagcgg gtaccagcac ctgctgttca cggcgccagt 3480ggaccgcgcc cggaactgtg accccttctt gggcgacccc gttctcggcc ccgggat 35374930DNAArtificialprimer 49atggcgacgc gcacctcgaa gagcgctccg 305030DNAArtificialprimer 50aggatcatca tgaacgtgtc gctccagtcg 305134DNAArtificialprimer 51cagatctgga tccgcgaaat gaccgaccaa gcga 345224DNAArtificialprimer 52acgcaattaa tgtgagatct agct 2453342DNAArtificialSV40 terminator 53cagatctgga tccgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 60gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 120tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 180attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 240tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 300tgtataccgt cgacctctag ctagatctca cattaattgc gt 34254619DNAArtificialubiquitin promoter 54cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttggtgtgc tcttcttgcc 60tccgagagag agagcggagc ggatgcatag gaaatcgggc cacgcgggag ggccatgcgt 120tcgccccaca cgccactttc cacgcccgct ctctctccgg ccggcaggca gcgcataact 180ctccgacgct ggcaggctgg tagcaactgg cagggacaac tcgcgcgcgg gtcccggtcg 240ttcgatgtgc caacccgaga gaatccagcc agcagggcgg ttggcctcat cgcccacctg 300ctatggtgca gcgaaccaac tcccgaagcg gccggttctg cgattccctc ttctgaattc 360tgaattctga actgattccg gaggagaacc ctctggaagc gcgggttgcc tctccagttc 420tgccgaacta gacaggggag tgagcagaga gtgaccctga cgcgggagcg agctggttgc 480tggaaaagtc gcgaacgctg ggctgtgtca cgcgtccact tcgggcagac cccaaacgac 540aagcagaaca agcaacacca gcagcagcaa gcgacctaag caacactagc caacatgatt 600gaacaggacg gccttcacg 6195536DNAArtificialprimer 55cccagatctg ccgcagcgcc tggtgcaccc gccggg 365658DNAArtificialprimer 56cgtgaaggcc gtcctgttca atcatgttgg ctagtgttgc ttaggtcgct tgctgctg 5857826DNAArtificialNeomycin resistance gene (Neor) 57agcgacctaa gcaacactag ccaacatgat tgaacaggac ggccttcacg ctggctcgcc 60cgctgcttgg gtggaacggc tgttcggcta cgactgggct cagcagacga tcggctgctc 120ggacgcggcc gtgttccgcc ttagcgcgca gggccggccg gtcctgtttg tcaagaccga 180ccttagcggc gccctcaacg agctccagga cgaagctgcc cgcctcagct ggcttgccac 240gacgggggtt ccgtgcgccg ctgtgctcga cgtcgtcacc gaagccggcc gcgactggct 300gctcctcggg gaagtgcccg gccaggacct cctcagcagc cacctcgcgc ccgctgagaa 360ggtgtccatc atggccgacg ccatgcgccg cctgcacacc ctcgaccccg ccacctgccc 420cttcgaccac caggcgaagc acaggatcga acgcgcccgc acgcggatgg aggctggcct 480cgtcgaccaa gacgacctcg acgaggagca ccagggcctc gcgccggcgg aactgttcgc 540caggcttaag gctaggatgc cggacggcga ggacctcgtg gtcacgcacg gcgacgcctg 600cctccccaac atcatggtcg agaacggccg cttctcgggc tttatcgact gcgggcgcct 660gggcgtggcg gaccgctacc aagacatcgc gctcgccacg cgggacatcg ccgaggagct 720tggcggcgag tgggccgacc gctttctcgt gctctacggc atcgccgccc cggacagcca 780gaggattgcg ttctaccgcc tcctggacga gttcttttga gatctg 8265854DNAArtificialprimer 58agcgacctaa gcaacactag ccaacatgat tgaacaggac ggccttcacg ctgg 545926DNAArtificialprimer 59cagatctcaa aagaactcgt ccagga 26601395DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/Neor) 60cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttggtgtgc tcttcttgcc 60tccgagagag agagcggagc ggatgcatag gaaatcgggc cacgcgggag ggccatgcgt 120tcgccccaca cgccactttc cacgcccgct ctctctccgg ccggcaggca gcgcataact 180ctccgacgct ggcaggctgg tagcaactgg cagggacaac tcgcgcgcgg gtcccggtcg 240ttcgatgtgc caacccgaga gaatccagcc agcagggcgg ttggcctcat cgcccacctg 300ctatggtgca gcgaaccaac tcccgaagcg gccggttctg cgattccctc ttctgaattc 360tgaattctga actgattccg gaggagaacc ctctggaagc gcgggttgcc tctccagttc 420tgccgaacta gacaggggag tgagcagaga gtgaccctga cgcgggagcg agctggttgc 480tggaaaagtc gcgaacgctg ggctgtgtca cgcgtccact tcgggcagac cccaaacgac 540aagcagaaca agcaacacca gcagcagcaa gcgacctaag caacactagc caacatgatt 600gaacaggacg gccttcacgc tggctcgccc gctgcttggg tggaacggct gttcggctac 660gactgggctc agcagacgat cggctgctcg gacgcggccg tgttccgcct tagcgcgcag 720ggccggccgg tcctgtttgt caagaccgac cttagcggcg ccctcaacga gctccaggac 780gaagctgccc gcctcagctg gcttgccacg acgggggttc cgtgcgccgc tgtgctcgac 840gtcgtcaccg aagccggccg cgactggctg ctcctcgggg aagtgcccgg ccaggacctc 900ctcagcagcc acctcgcgcc cgctgagaag gtgtccatca tggccgacgc catgcgccgc 960ctgcacaccc tcgaccccgc cacctgcccc ttcgaccacc aggcgaagca caggatcgaa 1020cgcgcccgca cgcggatgga ggctggcctc gtcgaccaag acgacctcga cgaggagcac 1080cagggcctcg cgccggcgga actgttcgcc aggcttaagg ctaggatgcc ggacggcgag 1140gacctcgtgg tcacgcacgg cgacgcctgc ctccccaaca tcatggtcga gaacggccgc 1200ttctcgggct ttatcgactg cgggcgcctg ggcgtggcgg accgctacca agacatcgcg 1260ctcgccacgc gggacatcgc cgaggagctt ggcggcgagt gggccgaccg ctttctcgtg 1320ctctacggca tcgccgcccc ggacagccag aggattgcgt tctaccgcct cctggacgag 1380ttcttttgag atctg 139561617DNAArtificialubiquitin promoter 61cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttgtgtgct cttcttgcct 60ccgagagaga gagcggagcg gatgcatagg aaatcgggcc acgcgggagg gccatgcgtt 120cgccccacac gccactttcc acgcccgctc tctctccggc cggcaggcag cgcataactc 180tccgacgctg gcaggctggt agcaactggc agggacaact cgcgcgcggg tcccggtcgt 240tcgatgtgcc aacccgagag aatccagcca gcagggcggt tggcctcatc gcccacctgc 300tatggtgcag cgaaccaact cccgaagcgg ccggttctgc gattccctct tctgaattct 360gaattctgaa ctgattccgg aggagaaccc tctggaagcg cgggttgcct ctccagttct 420gccgaactag acaggggagt gagcagagag tgaccctgac gcggagcgag ctggttgctg 480gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc gggcagaccc caaacgacaa 540gcagaacaag caacaccagc agcagcaagc gacctaagca acactagcca acatgaaaaa 600gcctgaactc accgcga 6176258DNAArtificialprimer 62tcgcggtgag ttcaggcttt ttcatgttgg ctagtgttgc ttaggtcgct tgctgctg 58631058DNAArtificialHygromycin resistance gene (Hygr) 63agcgacctaa gcaacactag ccaacatgaa aaagcctgaa ctcaccgcga cgtctgtcga 60gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 120agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 180ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct 240cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc 300ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 360gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg 420gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg

atttcatatg 480cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 540gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 600gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 660agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 720cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 780gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga 840ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 900atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 960aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 1020ccccagcact cgtccgaggg caaaggaata gagatctg 10586456DNAArtificialprimer 64agcgacctaa gcaacactag ccaacatgaa aaagcctgaa ctcaccgcga cgtctg 566536DNAArtificialprimer 65cagatctcta ttcctttgcc ctcggacgag tgctgg 36661625DNAArtificialfusion DNA (Thraustochytrium aureum ATCC 34304 ubiquitin promoter-pcDNA 3.1/ Hygr) 66cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttgtgtgct cttcttgcct 60ccgagagaga gagcggagcg gatgcatagg aaatcgggcc acgcgggagg gccatgcgtt 120cgccccacac gccactttcc acgcccgctc tctctccggc cggcaggcag cgcataactc 180tccgacgctg gcaggctggt agcaactggc agggacaact cgcgcgcggg tcccggtcgt 240tcgatgtgcc aacccgagag aatccagcca gcagggcggt tggcctcatc gcccacctgc 300tatggtgcag cgaaccaact cccgaagcgg ccggttctgc gattccctct tctgaattct 360gaattctgaa ctgattccgg aggagaaccc tctggaagcg cgggttgcct ctccagttct 420gccgaactag acaggggagt gagcagagag tgaccctgac gcggagcgag ctggttgctg 480gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc gggcagaccc caaacgacaa 540gcagaacaag caacaccagc agcagcaagc gacctaagca acactagcca acatgaaaaa 600gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 660cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 720gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 780ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt 840cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 900gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc 960tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 1020atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca 1080aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 1140ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 1200tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 1260ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 1320gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg 1380ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 1440cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 1500tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 1560agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag 1620atctg 16256725DNAArtificialprimer 67ccttcggcgc tcctcttatg tatgt 256825DNAArtificialprimer 68caatgcaaga ggcgaactgg gagag 256925DNAArtificialprimer 69tggggctctg gaaccgctgc ttacg 257025DNAArtificialprimer 70cttccagctc tcccagttcg cctct 257125DNAArtificialprimer 71cgggttgttg atgttgagcg aggtg 257225DNAArtificialprimer 72cccacgccat ccacgagcac accac 2573957DNAArtificialcDNA (Parietichytrium genomic DNA contains C20 elongase coding region) 73cccggatcca tggcagctcg cgtggagaaa cagcaggcac ctgcgaaggc cgccaagaag 60gtggggtcgc gtgtggaccg cagtgatggg ttctttcgca ctttcaacct ctgtgcgctg 120tacggaagcg cgttcgcgta cgcttacaac aatgggccag tggacaacga cggcaagggc 180ttgtactttt caaagtctcc attctacgca ttcctcgtct cggacgccat gaccttcggc 240gctcctctta tgtatgtaat tgctgtcatg gcactcagcc gatacatggc agacaagcag 300cccctcactg gcttcattaa aagctacatt cagccagttt acaacattgt gcaaatcgtg 360gtgtgctcgt ggatggcgtg gggccttttg ccacaggtgg acatcttcaa cctcaaccca 420ttcggtctca acaagcagcg tgatgccaac atcgagttct ttgtcatggt ccacctcctg 480acaaagttcc tcgactggac cgacaccttc atcatgattt tcaagaagaa ctatgcacag 540gtctcttttc tccaggtgtt ccaccatgcc accatcggaa tggtgtggtc cttcctcctc 600cagcgcggct ggggctctgg aaccgctgct tacggagcgt tcatcaactc ggtcacccat 660gtcatcatgt acactcatta ctttgtcacc tcgctcaaca tcaacaaccc gttcaagagg 720tacatcaccg gcttccagct ctcccagttc gcctcttgca ttgtacatgc tctcctcgtc 780cttgccttcg aggaggtgta ccccctcgag tacgcttacc ttcagatcag ctaccacatc 840atcatgctct acctcttcgg caggagaatg aactggagcc ctctctggtg cactggcgag 900gtcgacgggc ttgacgtcaa cgtcgagacc tccaagaagg ctcagtaagg atccggg 9577432DNAArtificialprimer 74cccggatcca tggcagctcg cgtggagaaa ca 327533DNAArtificialprimer 75cccggatcct tactgagcct tcttggaggt ctc 3376936DNAArtificialgenomic DNA (Parietichytrium C20 elongase gene) 76atggcagctc gcgtggagaa acagcaggca cctgcgaagg ccgccaagaa ggtggggtcg 60cgtgtggacc gcagtgatgg gttctttcgc actttcaacc tctgtgcgct gtacggaagc 120gcgttcgcgt acgcttacaa caatgggcca gtggacaacg acggcaaggg cttgtacttt 180tcaaagtctc cattctacgc attcctcgtc tcggacgcca tgaccttcgg cgctcctctt 240atgtatgtaa ttgctgtcat ggcactcagc cgatacatgg cagacaagca gcccctcact 300ggcttcatta aaagctacat tcagccagtt tacaacattg tgcaaatcgt ggtgtgctcg 360tggatggcgt ggggcctttt gccacaggtg gacatcttca acctcaaccc attcggtctc 420aacaagcagc gtgatgccaa catcgagttc tttgtcatgg tccacctcct gacaaagttc 480ctcgactgga ccgacacctt catcatgatt ttcaagaaga actatgcaca ggtctctttt 540ctccaggtgt tccaccatgc caccatcgga atggtgtggt ccttcctcct ccagcgcggc 600tggggctctg gaaccgctgc ttacggagcg ttcatcaact cggtcaccca tgtcatcatg 660tacactcatt actttgtcac ctcgctcaac atcaacaacc cgttcaagag gtacatcacc 720ggcttccagc tctcccagtt cgcctcttgc attgtacatg ctctcctcgt ccttgccttc 780gaggaggtgt accccctcga gtacgcttac cttcagatca gctaccacat catcatgctc 840tacctcttcg gcaggagaat gaactggagc cctctctggt gcactggcga ggtcgacggg 900cttgacgtca acgtcgagac ctccaagaag gctcag 93677312PRTParietichytrium 77Met Ala Ala Arg Val Glu Lys Gln Gln Ala Pro Ala Lys Ala Ala Lys1 5 10 15Lys Val Gly Ser Arg Val Asp Arg Ser Asp Gly Phe Phe Arg Thr Phe 20 25 30Asn Leu Cys Ala Leu Tyr Gly Ser Ala Phe Ala Tyr Ala Tyr Asn Asn 35 40 45Gly Pro Val Asp Asn Asp Gly Lys Gly Leu Tyr Phe Ser Lys Ser Pro 50 55 60Phe Tyr Ala Phe Leu Val Ser Asp Ala Met Thr Phe Gly Ala Pro Leu65 70 75 80Met Tyr Val Ile Ala Val Met Ala Leu Ser Arg Tyr Met Ala Asp Lys 85 90 95Gln Pro Leu Thr Gly Phe Ile Lys Ser Tyr Ile Gln Pro Val Tyr Asn 100 105 110Ile Val Gln Ile Val Val Cys Ser Trp Met Ala Trp Gly Leu Leu Pro 115 120 125Gln Val Asp Ile Phe Asn Leu Asn Pro Phe Gly Leu Asn Lys Gln Arg 130 135 140Asp Ala Asn Ile Glu Phe Phe Val Met Val His Leu Leu Thr Lys Phe145 150 155 160Leu Asp Trp Thr Asp Thr Phe Ile Met Ile Phe Lys Lys Asn Tyr Ala 165 170 175Gln Val Ser Phe Leu Gln Val Phe His His Ala Thr Ile Gly Met Val 180 185 190Trp Ser Phe Leu Leu Gln Arg Gly Trp Gly Ser Gly Thr Ala Ala Tyr 195 200 205Gly Ala Phe Ile Asn Ser Val Thr His Val Ile Met Tyr Thr His Tyr 210 215 220Phe Val Thr Ser Leu Asn Ile Asn Asn Pro Phe Lys Arg Tyr Ile Thr225 230 235 240Gly Phe Gln Leu Ser Gln Phe Ala Ser Cys Ile Val His Ala Leu Leu 245 250 255Val Leu Ala Phe Glu Glu Val Tyr Pro Leu Glu Tyr Ala Tyr Leu Gln 260 265 270Ile Ser Tyr His Ile Ile Met Leu Tyr Leu Phe Gly Arg Arg Met Asn 275 280 285Trp Ser Pro Leu Trp Cys Thr Gly Glu Val Asp Gly Leu Asp Val Asn 290 295 300Val Glu Thr Ser Lys Lys Ala Gln305 3107826DNAArtificialprimer 78acaaagatct cgactggacc gacacc 267927DNAArtificialprimer 79agtcgagatc tttgtcagga ggtggac 2780935DNAArtificialBglII inserted C20 elongase 80atggcagctc gcgtggagaa acagcaggca cctgcgaagg ccgccaagaa ggtggggtcg 60cgtgtggacc gcagtgatgg gttctttcgc actttcaacc tctgtgcgct gtacggaagc 120gcgttcgcgt acgcttacaa caatgggcca gtggacaacg acggcaaggg cttgtacttt 180tcaaagtctc cattctacgc attcctcgtc tcggacgcca tgaccttcgg cgctcctctt 240atgtatgtaa ttgctgtcat ggcactcagc cgatacatgg cagacaagca gcccctcact 300ggcttcatta aaagctacat tcagccagtt tacaacattg tgcaaatcgt ggtgtgctcg 360tggatggcgt ggggcctttt gccacaggtg gacatcttca acctcaaccc attcggtctc 420aacaagcagc gtgatgccaa catcgagttc tttgtcatgg tccacctcct gacaaagatc 480tcgactggac cgacaccttc atcatgattt tcaagaagaa ctatgcacag gtctcttttc 540tccaggtgtt ccaccatgcc accatcggaa tggtgtggtc cttcctcctc cagcgcggct 600ggggctctgg aaccgctgct tacggagcgt tcatcaactc ggtcacccat gtcatcatgt 660acactcatta ctttgtcacc tcgctcaaca tcaacaaccc gttcaagagg tacatcaccg 720gcttccagct ctcccagttc gcctcttgca ttgtacatgc tctcctcgtc cttgccttcg 780aggaggtgta ccccctcgag tacgcttacc ttcagatcag ctaccacatc atcatgctct 840acctcttcgg caggagaatg aactggagcc ctctctggtg cactggcgag gtcgacgggc 900ttgacgtcaa cgtcgagacc tccaagaagg ctcag 935812661DNAArtificialfusion DNA (Parietichytrium C20 elongase 5' region/SV40 terminator/Neor/ubiquitin promoter/Parietichytrium C20 elongase 3' region) 81cccggatcca tggcagctcg cgtggagaaa cagcaggcac ctgcgaaggc cgccaagaag 60gtggggtcgc gtgtggaccg cagtgatggg ttctttcgca ctttcaacct ctgtgcgctg 120tacggaagcg cgttcgcgta cgcttacaac aatgggccag tggacaacga cggcaagggc 180ttgtactttt caaagtctcc attctacgca ttcctcgtct cggacgccat gaccttcggc 240gctcctctta tgtatgtaat tgctgtcatg gcactcagcc gatacatggc agacaagcag 300cccctcactg gcttcattaa aagctacatt cagccagttt acaacattgt gcaaatcgtg 360gtgtgctcgt ggatggcgtg gggccttttg ccacaggtgg acatcttcaa cctcaaccca 420ttcggtctca acaagcagcg tgatgccaac atcgagttct ttgtcatggt ccacctcctg 480acaaagatct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg 540gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 600ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc 660agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag 720cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc 780gcttggtcgg tcatttcgcg gatctcaaaa gaactcgtcc aggaggcggt agaacgcaat 840cctctggctg tccggggcgg cgatgccgta gagcacgaga aagcggtcgg cccactcgcc 900gccaagctcc tcggcgatgt cccgcgtggc gagcgcgatg tcttggtagc ggtccgccac 960gcccaggcgc ccgcagtcga taaagcccga gaagcggccg ttctcgacca tgatgttggg 1020gaggcaggcg tcgccgtgcg tgaccacgag gtcctcgccg tccggcatcc tagccttaag 1080cctggcgaac agttccgccg gcgcgaggcc ctggtgctcc tcgtcgaggt cgtcttggtc 1140gacgaggcca gcctccatcc gcgtgcgggc gcgttcgatc ctgtgcttcg cctggtggtc 1200gaaggggcag gtggcggggt cgagggtgtg caggcggcgc atggcgtcgg ccatgatgga 1260caccttctca gcgggcgcga ggtggctgct gaggaggtcc tggccgggca cttccccgag 1320gagcagccag tcgcggccgg cttcggtgac gacgtcgagc acagcggcgc acggaacccc 1380cgtcgtggca agccagctga ggcgggcagc ttcgtcctgg agctcgttga gggcgccgct 1440aaggtcggtc ttgacaaaca ggaccggccg gccctgcgcg ctaaggcgga acacggccgc 1500gtccgagcag ccgatcgtct gctgagccca gtcgtagccg aacagccgtt ccacccaagc 1560agcgggcgag ccagcgtgaa ggccgtcctg ttcaatcatg ttggctagtg ttgcttaggt 1620cgcttgctgc tgctggtgtt gcttgttctg cttgtcgttt ggggtctgcc cgaagtggac 1680gcgtgacaca gcccagcgtt cgcgactttt ccagcaacca gctcgctccg cgtcagggtc 1740actctctgct cactcccctg tctagttcgg cagaactgga gaggcaaccc gcgcttccag 1800agggttctcc tccggaatca gttcagaatt cagaattcag aagagggaat cgcagaaccg 1860gccgcttcgg gagttggttc gctgcaccat agcaggtggg cgatgaggcc aaccgccctg 1920ctggctggat tctctcgggt tggcacatcg aacgaccggg acccgcgcgc gagttgtccc 1980tgccagttgc taccagcctg ccagcgtcgg agagttatgc gctgcctgcc ggccggagag 2040agagcgggcg tggaaagtgg cgtgtggggc gaacgcatgg ccctcccgcg tggcccgatt 2100tcctatgcat ccgctccgct ctctctctcg gaggcaagaa gagcacacca acaacgcccg 2160gcgggtgcac caggcgctgc ggcagatcca gatctcgact ggaccgacac cttcatcatg 2220attttcaaga agaactatgc acaggtctct tttctccagg tgttccacca tgccaccatc 2280ggaatggtgt ggtccttcct cctccagcgc ggctggggct ctggaaccgc tgcttacgga 2340gcgttcatca actcggtcac ccatgtcatc atgtacactc attactttgt cacctcgctc 2400aacatcaaca acccgttcaa gaggtacatc accggcttcc agctctccca gttcgcctct 2460tgcattgtac atgctctcct cgtccttgcc ttcgaggagg tgtaccccct cgagtacgct 2520taccttcaga tcagctacca catcatcatg ctctacctct tcggcaggag aatgaactgg 2580agccctctct ggtgcactgg cgaggtcgac gggcttgacg tcaacgtcga gacctccaag 2640aaggctcagt aaggatccgg g 2661822892DNAArtificialfusion DNA (Parietichytrium C20 elongase 5' region/SV40 terminator/Hygr/ubiquitin promoter/Parietichytrium C20 elongase 3' region) 82cccggatcca tggcagctcg cgtggagaaa cagcaggcac ctgcgaaggc cgccaagaag 60gtggggtcgc gtgtggaccg cagtgatggg ttctttcgca ctttcaacct ctgtgcgctg 120tacggaagcg cgttcgcgta cgcttacaac aatgggccag tggacaacga cggcaagggc 180ttgtactttt caaagtctcc attctacgca ttcctcgtct cggacgccat gaccttcggc 240gctcctctta tgtatgtaat tgctgtcatg gcactcagcc gatacatggc agacaagcag 300cccctcactg gcttcattaa aagctacatt cagccagttt acaacattgt gcaaatcgtg 360gtgtgctcgt ggatggcgtg gggccttttg ccacaggtgg acatcttcaa cctcaaccca 420ttcggtctca acaagcagcg tgatgccaac atcgagttct ttgtcatggt ccacctcctg 480acaaagatct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg 540gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 600ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc 660agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag 720cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc 780gcttggtcgg tcatttcgcg gatctctatt cctttgccct cggacgagtg ctggggcgtc 840ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc 900gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac 960cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa 1020gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc 1080gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt 1140tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta 1200tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga 1260cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac 1320tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata 1380tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc 1440tcgtctggct aagatcggcc gcagcgatcg catccatggc ctccgcgacc ggctgcagaa 1500cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga 1560tgcaataggt caggctctcg ctgaattccc caatgtcaag cacttccgga atcgggagcg 1620cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat 1680ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc 1740cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga 1800cagacgtcgc ggtgagttca ggctttttca tgttggctag tgttgcttag gtcgcttgct 1860gctgctggtg ttgcttgttc tgcttgtcgt ttggggtctg cccgaagtgg acgcgtgaca 1920cagcccagcg ttcgcgactt ttccagcaac cagctcgctc cgcgtcaggg tcactctctg 1980ctcactcccc tgtctagttc ggcagaactg gagaggcaac ccgcgcttcc agagggttct 2040cctccggaat cagttcagaa ttcagaattc agaagaggga atcgcagaac cggccgcttc 2100gggagttggt tcgctgcacc atagcaggtg ggcgatgagg ccaaccgccc tgctggctgg 2160attctctcgg gttggcacat cgaacgaccg ggacccgcgc gcgagttgtc cctgccagtt 2220gctaccagcc tgccagcgtc ggagagttat gcgctgcctg ccggccggag agagagcggg 2280cgtggaaagt ggcgtgtggg gcgaacgcat ggccctcccg cgtggcccga tttcctatgc 2340atccgctccg ctctctctct cggaggcaag aagagcacac aacaacgccc ggcgggtgca 2400ccaggcgctg cggcagatcc agatctcgac tggaccgaca ccttcatcat gattttcaag 2460aagaactatg cacaggtctc ttttctccag gtgttccacc atgccaccat cggaatggtg 2520tggtccttcc tcctccagcg cggctggggc tctggaaccg ctgcttacgg agcgttcatc 2580aactcggtca cccatgtcat catgtacact cattactttg tcacctcgct caacatcaac 2640aacccgttca agaggtacat caccggcttc cagctctccc agttcgcctc ttgcattgta 2700catgctctcc tcgtccttgc cttcgaggag gtgtaccccc tcgagtacgc ttaccttcag 2760atcagctacc acatcatcat gctctacctc ttcggcagga gaatgaactg gagccctctc 2820tggtgcactg gcgaggtcga cgggcttgac gtcaacgtcg agacctccaa gaaggctcag 2880taaggatccg gg 28928320DNAArtificialprimer 83ggttgacggc aatttcgatg 208422DNAArtificialprimer 84cctcctacat cgaagctgaa ag 228521DNAArtificialprimer 85cttctcgggc tttatcgact g 218622DNAArtificialprimer 86taaggtcggt cttgacaaac ag 228724DNAArtificialprimer 87agtagtcccc gatttggtag ttga 248822DNAArtificialprimer 88ggcagagagc aaaaacacga gc 228923DNAArtificialprimer 89cgatgaaagg tcacagaaga gtc 23903181DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA upstream) 90aagcttttgc tctgcggctc tgcttgttcg aagccaacgc gcctcgcgaa

gtatctgcaa 60tctgcactcc tccggagagt aagtacgtaa gtacgtgcgt ggtgcgcgcg gattgcggtg 120acgaaagaga gggttgggtt ggagatgctg cggcatgccg ggcgactcga gcagcatgtc 180gccgcgagag gacctggaaa gctttcggtt tggtccgctg ccgaggcgag gctggcagag 240tactgcgggc ggagctctcg agggaatatg ctcctcaaag acggcgtgcg cgtttgtgcc 300cccgaatccg aatgcggaga gtcctgcgcg tttcggcccg ccgcgcgtat ccggccacgg 360gagcgccgct gtgacgacga ggggatcaat ctgggtgcca gagtcaacgc ccagggtcgg 420ggggatcacg ccgcgctcca ttgcaaggag aaccttgcac attcccgcaa agccggccgc 480gacgagggtg tgtccgaagt tgcctttcgt ggacccgatc cgggggcttg cgccctcaaa 540gcaggctttg acggcttgga gctcgacggt gtcgccctgc ggcgtgccgg tggcgtggca 600ctcgacgtac tggacgtctc ggggcggcac gccgacgagc tcgtaggtgg ctttcaagca 660ggcctcctcg cttggctggt gcggcttgag aggaagcccg cagcctgcgt tgctcaagct 720ggccccaaga agcgtcccgt agatgtggtc tccgtcgcgc tcggcgtccg cgaggcgctt 780gagcaccatc accgagccgc cctcgccggg cgtcagccct tgcgtgtccc gatgaaacgg 840catcgagaca ccgttctcac cgactggcat cgcgtggaac gtgctaaacc cagtcaggat 900gaagaagggc tctgggaagc acgtcgctcc gcacagcatc aagtcagcct cgcccgagag 960gaggtggtcc tgagcgagtc gcagaacgta aagggccgag gcgcaggcgg cgtcgagcga 1020gtagtgcagc gggccgaggc cgagctgtcc ggcgacgaag gaggctgggt cgcggtgggt 1080cctcgggtcc ccgggcagcg ggtgaagcgc tctggttcgc gtcgaccagg gcgtttggtc 1140cgcgaagcaa tgcttgccaa tccgcctctc agcatgggct tggtaaaggt tgagcagctc 1200gccttgcagg ttgtccatcg ggaaggacag gcagccgctg acaatgccgc agcgcttgag 1260ctgcgctggg tcgaacttgc cgccgtcgct gcgcctgtcc tgcgcgtctt gaagcgcagc 1320cgcggcgagg ccgaggagca ggtcgtgctc gttgtcgact ttgggatcga tgcatccgta 1380cctctcgttg cagaacgtat cggcgtactt tgacctctcg ggcgcatagt gctcttctcg 1440tcgtgctgac ccgaggcgat cgtctgagat acaggcagag ttgattttgc cgttcatgag 1500cgtgtcccag aacgcttcct tgccgcggca ccctgcatac tcgaccgcca tgcccacgac 1560agcgatccgc gtgtcagggc atgggtccgc gcgcgccacc gagacgccct cgtcatttct 1620cccgccctgg ttcatctctt tctgagcctt gtggcctctt gctttcgatt tcgatcggca 1680gctccaacgc gcagctcgat ggccgattgc ttgctaaggc ggcgtgcaga caaccgctgc 1740tcgaggttct gggcaagccg aggttcgccg cagggttcgt ggaggcactg ggatgttgtt 1800ttgcggcagc tgcagcgctt gcggagcgag cggcgcagga cgacgttttc cgcgttcgcg 1860cgaagctgcc tctcggctat tgtgacccgc cgccgacgct ggcagagacg tcgccgtcgg 1920ccaccgcacc tcgagatcaa ttcgcagagg ctggcagagc cggtagcatt gcggcgtggc 1980attgcgtgtg ccatgtgcat gtgtggcaaa caaatccagc caacctccga gtcgggcaga 2040ccgaccgtgt gagttctcgc tgttgactga tctcttgatt gagcccaata atgatcacgg 2100cctgagatcc ttcgcgctga gagatgcatg cgggcgctcg ttcctgggtt ggcgacccaa 2160cggcgagtca cgtcgcccac tccgccacgc cccacacatg gccgccgatc cctcccgcca 2220cacgaacggc gggccaagat cgcacgcctc cgtcggacga tgactgactg actgattggc 2280tgacgacggc cgccctcgtg cgcggcgtcg ggcgtcgtcg caaaccaggc aggcaggcag 2340gaaggaagga aggaagggcc aggccctggt gcgaaacgct ggcctgctcc gctgcaagcc 2400aagccgcgct cgcaggtgta cttccgagtc ctcgcgatga ttaggcaagc ctgagcgagc 2460acgtaagctg cactgcggct gttcaaccag agagagagtt ggctctcttg cgtcaaggcg 2520gcgcgcagcc cacttgcgtc gcggctgagg gcccctggag gggaggaagg aggccggcga 2580gcggcgagtg gcggccctca ctggcaccag gtcgcaggag gccaggcagc ccgccacgga 2640caggaatcct cagggcgcag cagcgcacta cgtagtgcag agacgcagag cgggccggat 2700ccgcagtgcg gtcgcgccac cccgccgcgc agctcgctcg cggacggggt ccgtggccgc 2760gcgaaaacgg acacggtgtg ggagcggaca tgggatcgag aacgccgttc gccctgctcg 2820cgctgccagc agcaggagcc gtccgaagga cgagcggccg gccgcctgtc ccccctccgc 2880gcactcgaag cgcgcccggc agcgccccat tgcgtgcgcg gatggcgtct tggctggtcc 2940ctctcgaggc gcttgctcgt gctcgccacg ccttgtccgc ctcctcgctg agcaagcgat 3000gagctgagca cggaccgcct gcaagtgcaa gtgttcttgt gctgcagggc gccgaagaat 3060tggattctgg cccatgatca gtttgattgg gccgagggag ggagggaggc tgggcgagtg 3120ggcgacacca gcaagccgga ctgcgagagg ggcggggcag gatgtgagcg caggaaagtg 3180a 3181913377DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA upstream genomic DNA fragment) 91gaattcgatt aatacgactc actataggga gaagcttttg ctctgcggct ctgcttgttc 60gaagccaacg cgcctcgcga agtatctgca atctgcactc ctccggagag taagtacgta 120agtacgtgcg tggtgcgcgc ggattgcggt gacgaaagag agggttgggt tggagatgct 180gcggcatgcc gggcgactcg agcagcatgt cgccgcgaga ggacctggaa agctttcggt 240ttggtccgct gccgaggcga ggctggcaga gtactgcggg cggagctctc gagggaatat 300gctcctcaaa gacggcgtgc gcgtttgtgc ccccgaatcc gaatgcggag agtcctgcgc 360gtttcggccc gccgcgcgta tccggccacg ggagcgccgc tgtgacgacg aggggatcaa 420tctgggtgcc agagtcaacg cccagggtcg gggggatcac gccgcgctcc attgcaagga 480gaaccttgca cattcccgca aagccggccg cgacgagggt gtgtccgaag ttgcctttcg 540tggacccgat ccgggggctt gcgccctcaa agcaggcttt gacggcttgg agctcgacgg 600tgtcgccctg cggcgtgccg gtggcgtggc actcgacgta ctggacgtct cggggcggca 660cgccgacgag ctcgtaggtg gctttcaagc aggcctcctc gcttggctgg tgcggcttga 720gaggaagccc gcagcctgcg ttgctcaagc tggccccaag aagcgtcccg tagatgtggt 780ctccgtcgcg ctcggcgtcc gcgaggcgct tgagcaccat caccgagccg ccctcgccgg 840gcgtcagccc ttgcgtgtcc cgatgaaacg gcatcgagac accgttctca ccgactggca 900tcgcgtggaa cgtgctaaac ccagtcagga tgaagaaggg ctctgggaag cacgtcgctc 960cgcacagcat caagtcagcc tcgcccgaga ggaggtggtc ctgagcgagt cgcagaacgt 1020aaagggccga ggcgcaggcg gcgtcgagcg agtagtgcag cgggccgagg ccgagctgtc 1080cggcgacgaa ggaggctggg tcgcggtggg tcctcgggtc cccgggcagc gggtgaagcg 1140ctctggttcg cgtcgaccag ggcgtttggt ccgcgaagca atgcttgcca atccgcctct 1200cagcatgggc ttggtaaagg ttgagcagct cgccttgcag gttgtccatc gggaaggaca 1260ggcagccgct gacaatgccg cagcgcttga gctgcgctgg gtcgaacttg ccgccgtcgc 1320tgcgcctgtc ctgcgcgtct tgaagcgcag ccgcggcgag gccgaggagc aggtcgtgct 1380cgttgtcgac tttgggatcg atgcatccgt acctctcgtt gcagaacgta tcggcgtact 1440ttgacctctc gggcgcatag tgctcttctc gtcgtgctga cccgaggcga tcgtctgaga 1500tacaggcaga gttgattttg ccgttcatga gcgtgtccca gaacgcttcc ttgccgcggc 1560accctgcata ctcgaccgcc atgcccacga cagcgatccg cgtgtcaggg catgggtccg 1620cgcgcgccac cgagacgccc tcgtcatttc tcccgccctg gttcatctct ttctgagcct 1680tgtggcctct tgctttcgat ttcgatcggc agctccaacg cgcagctcga tggccgattg 1740cttgctaagg cggcgtgcag acaaccgctg ctcgaggttc tgggcaagcc gaggttcgcc 1800gcagggttcg tggaggcact gggatgttgt tttgcggcag ctgcagcgct tgcggagcga 1860gcggcgcagg acgacgtttt ccgcgttcgc gcgaagctgc ctctcggcta ttgtgacccg 1920ccgccgacgc tggcagagac gtcgccgtcg gccaccgcac ctcgagatca attcgcagag 1980gctggcagag ccggtagcat tgcggcgtgg cattgcgtgt gccatgtgca tgtgtggcaa 2040acaaatccag ccaacctccg agtcgggcag accgaccgtg tgagttctcg ctgttgactg 2100atctcttgat tgagcccaat aatgatcacg gcctgagatc cttcgcgctg agagatgcat 2160gcgggcgctc gttcctgggt tggcgaccca acggcgagtc acgtcgccca ctccgccacg 2220ccccacacat ggccgccgat ccctcccgcc acacgaacgg cgggccaaga tcgcacgcct 2280ccgtcggacg atgactgact gactgattgg ctgacgacgg ccgccctcgt gcgcggcgtc 2340gggcgtcgtc gcaaaccagg caggcaggca ggaaggaagg aaggaagggc caggccctgg 2400tgcgaaacgc tggcctgctc cgctgcaagc caagccgcgc tcgcaggtgt acttccgagt 2460cctcgcgatg attaggcaag cctgagcgag cacgtaagct gcactgcggc tgttcaacca 2520gagagagagt tggctctctt gcgtcaaggc ggcgcgcagc ccacttgcgt cgcggctgag 2580ggcccctgga ggggaggaag gaggccggcg agcggcgagt ggcggccctc actggcacca 2640ggtcgcagga ggccaggcag cccgccacgg acaggaatcc tcagggcgca gcagcgcact 2700acgtagtgca gagacgcaga gcgggccgga tccgcagtgc ggtcgcgcca ccccgccgcg 2760cagctcgctc gcggacgggg tccgtggccg cgcgaaaacg gacacggtgt gggagcggac 2820atgggatcga gaacgccgtt cgccctgctc gcgctgccag cagcaggagc cgtccgaagg 2880acgagcggcc ggccgcctgt cccccctccg cgcactcgaa gcgcgcccgg cagcgcccca 2940ttgcgtgcgc ggatggcgtc ttggctggtc cctctcgagg cgcttgctcg tgctcgccac 3000gccttgtccg cctcctcgct gagcaagcga tgagctgagc acggaccgcc tgcaagtgca 3060agtgttcttg tgctgcaggg cgccgaagaa ttggattctg gcccatgatc agtttgattg 3120ggccgaggga gggagggagg ctgggcgagt gggcgacacc agcaagccgg actgcgagag 3180gggcggggca ggatgtgagc gcaggaaagt gacgcaagtg catccggcca tcattgggcc 3240atcattgggc catcattggt gttttgggcc gcgctttgcg gatcgtccgg ccgatcaggt 3300acgaggccac gaacctacgt cgtttgccgc gctcaggctg gttggttgca cttggactct 3360tctgtgacct ttcatcg 33779221DNAArtificialprimer 92cagggcgagc gagtgtggtt c 21931160DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream) 93tggctctcga ccaaagccga gtagagtact ctactcagta ctcttttcac ataccggcag 60gcagtgttgc tgtgggattg gtccgggggc tcttctgcac gcggcctccg tcgcgcgcag 120aaatgccccg tcactggctg cccaggaggc agccgaatcc ctctagctag ctagctaggc 180tagagcgtct tttccgtagt ttttcacaaa gccagtatca catggataac gaacgaaggt 240ttcgggctcg cgctcgcagg cgttaggacg aagttgatcg ccccacgtca cttcaaacga 300gtgaaccaag atcacgttgc atctgctcgc aagatcttct tcttccacgc cgcatcgatg 360cgatggattt caaactcttt tcagggcttt taggtgagta tggcagcgct gtttgcgtgg 420cagcgctgtt tgcgtggttg tactctctaa aggtgcttcc acgcatgcgc gcacaaaggg 480gcatggcatg gttggcggcg cactctggcc ctcatttgaa gcagactatc gaagggtcca 540gttggtactg cggcaggtcc ggcgagagca agcgcggcgg tcgctcccac tcgtccctgc 600acagttgctg gactggcgac ggctggcgca cctgactacg agaagactcg agacgcacag 660aggtagtcag ggacgaccga ccgcaaagca caaaccgctc caaaacggcc gcaccaggca 720gggcagtaaa ctaaaaacga atgtacctcc atcgcgcgta tctgccgagc ctcctcccac 780gcttcggctg ggcttgattc accagtgtcc gcaagctgaa ccgaccgtct tcgatgtcat 840gaagcttggc gcggcattag tcagacgacg cggcacgcca ggattctgtc ggtttctggg 900aaatgggcat ctatatagct gattccctct gtcatgaggc ggccttgttc tggccctggg 960ccgccgttcg gatgatctat gatgtcgttg tacgcataaa gcttgtcgaa aacgtcggcc 1020atgtcttcct cagagatgta accgagcggc gcgtcgtggc gattgatgcc gatgctacaa 1080aagccgccga gttagctcga atgtcagatg cattgcgggc tggcccgcat ggcgcgggcg 1140cagcagcgag aggttctaga 1160941204DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 94cagggcgagc gagtgtggtt ctgaacaagg ctctttcgtt ttgatggctc tcgaccaaag 60ccgagtagag tactctactc agtactcttt tcacataccg gcaggcagtg ttgctgtggg 120attggtccgg gggctcttct gcacgcggcc tccgtcgcgc gcagaaatgc cccgtcactg 180gctgcccagg aggcagccga atccctctag ctagctagct aggctagagc gtcttttccg 240tagtttttca caaagccagt atcacatgga taacgaacga aggtttcggg ctcgcgctcg 300caggcgttag gacgaagttg atcgccccac gtcacttcaa acgagtgaac caagatcacg 360ttgcatctgc tcgcaagatc ttcttcttcc acgccgcatc gatgcgatgg atttcaaact 420cttttcaggg cttttaggtg agtatggcag cgctgtttgc gtggcagcgc tgtttgcgtg 480gttgtactct ctaaaggtgc ttccacgcat gcgcgcacaa aggggcatgg catggttggc 540ggcgcactct ggccctcatt tgaagcagac tatcgaaggg tccagttggt actgcggcag 600gtccggcgag agcaagcgcg gcggtcgctc ccactcgtcc ctgcacagtt gctggactgg 660cgacggctgg cgcacctgac tacgagaaga ctcgagacgc acagaggtag tcagggacga 720ccgaccgcaa agcacaaacc gctccaaaac ggccgcacca ggcagggcag taaactaaaa 780acgaatgtac ctccatcgcg cgtatctgcc gagcctcctc ccacgcttcg gctgggcttg 840attcaccagt gtccgcaagc tgaaccgacc gtcttcgatg tcatgaagct tggcgcggca 900ttagtcagac gacgcggcac gccaggattc tgtcggtttc tgggaaatgg gcatctatat 960agctgattcc ctctgtcatg aggcggcctt gttctggccc tgggccgccg ttcggatgat 1020ctatgatgtc gttgtacgca taaagcttgt cgaaaacgtc ggccatgtct tcctcagaga 1080tgtaaccgag cggcgcgtcg tggcgattga tgccgatgct acaaaagccg ccgagttagc 1140tcgaatgtca gatgcattgc gggctggccc gcatggcgcg ggcgcagcag cgagaggttc 1200taga 12049520DNAArtificialprimer 95tgatgccgat gctacaaaag 20961488DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 96aagcttgtac ggtgaaaagc cctttggcgc agcccgaaac aagtcttgct tctcctgccc 60cgtcaaactc gcaaactctg gcagcaactc ccgcacgctc tgtaccacgg cgaacccaag 120ggcaggcacg cggtgaaacg acttgcatgc ttgcacaaca acccccttgc cgacgtcgac 180gcggtcgcct tcggagagcc caaacacagc gaacgccgga tccgcctgcg cctctgcatg 240cgcctctgca tgcgcctcga catgcgcctc ggcctccgtg cctgcttgcc gggccggcgg 300ggcagcagga agtgcgtggc cgaggtccat cgcatcaaag gctcgcttcg cggcgtgaaa 360ggcctcgagc gcctccgccg gcaagtacac cttggtcttg cacttgagca tgctcctgat 420ccgcgcgtag aggaagacgg ccgcgcagtg gtccaggtgc ccgtgcgaca agaacacgtg 480ctccgccctc gccgcggcct tgtccggctc gtccccgagc gacccgcagt cgaactgcaa 540gcagacccgc gagcccaggt ccacttgcag cgccgtgccg cagccggccc tcgactgccc 600cgtcacgcgg acgtgcgagg ccatctcccg ccgcgagcct ggagcgccag agcctcctgc 660tgctgccgtg ccgcctcggg gggcgcgagg agggtctcgc ctgatgcagc gcgcggggcc 720gacgcagcag cgcgggtgga ggaagactgc gctgtgggcg gcggccctcg ggctgctgct 780cttgtggctc ctgtccgtgc gctcgttcgt gcacggcgtg gcggacaggg aggcggacgc 840cgtcgccccg cgcgagggcc ccagggcgcc ggcgccaaag aggactggcg ggaggaatga 900tatgcccgct gagcctgccg ctggtaggcc cgcgcacagc tcgcctcgag ggacgcccga 960cggcaacgcg gtcgagtgct ccacgaccaa gggcccgttc cgcgtggtcc tcacgcctag 1020cctagcgccg aacgggacca agtttttcat cgggctggtg gaagcaggct atttcgacca 1080aggcatcgcc ttctttcgcg tcaacaaggc catcacgcag ttcgggatca ccaagcgaag 1140gccacgcgat gaggatccgt tcgtgcagtt cagaggcggg gcccagcgcg acgagaaccc 1200tttcggtggc gtggaggatg acgaggagag tgtccatcgc aggcacatgc acccgtggcg 1260gcgcggcacg attgcctcga taggcggctt ccactttgtt gtcacgatcc gcggggacaa 1320aaagtaagtt cttgaatgtt gtgaagtgcg ccaactcgcg ttcggagcgg acctggaccg 1380atattcagca atctagaacc tctcgctgct gcgcccgcgc catgcgggcc agcccgcaat 1440gcatctgaca ttcgagctaa ctcggcggct tttgtagcat cggcatca 1488972551DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 97tggctctcga ccaaagccga gtagagtact ctactcagta ctcttttcac ataccggcag 60gcagtgttgc tgtgggattg gtccgggggc tcttctgcac gcggcctccg tcgcgcgcag 120aaatgccccg tcactggctg cccaggaggc agccgaatcc ctctagctag ctagctaggc 180tagagcgtct tttccgtagt ttttcacaaa gccagtatca catggataac gaacgaaggt 240ttcgggctcg cgctcgcagg cgttaggacg aagttgatcg ccccacgtca cttcaaacga 300gtgaaccaag atcacgttgc atctgctcgc aagatcttct tcttccacgc cgcatcgatg 360cgatggattt caaactcttt tcagggcttt taggtgagta tggcagcgct gtttgcgtgg 420cagcgctgtt tgcgtggttg tactctctaa aggtgcttcc acgcatgcgc gcacaaaggg 480gcatggcatg gttggcggcg cactctggcc ctcatttgaa gcagactatc gaagggtcca 540gttggtactg cggcaggtcc ggcgagagca agcgcggcgg tcgctcccac tcgtccctgc 600acagttgctg gactggcgac ggctggcgca cctgactacg agaagactcg agacgcacag 660aggtagtcag ggacgaccga ccgcaaagca caaaccgctc caaaacggcc gcaccaggca 720gggcagtaaa ctaaaaacga atgtacctcc atcgcgcgta tctgccgagc ctcctcccac 780gcttcggctg ggcttgattc accagtgtcc gcaagctgaa ccgaccgtct tcgatgtcat 840gaagcttggc gcggcattag tcagacgacg cggcacgcca ggattctgtc ggtttctggg 900aaatgggcat ctatatagct gattccctct gtcatgaggc ggccttgttc tggccctggg 960ccgccgttcg gatgatctat gatgtcgttg tacgcataaa gcttgtcgaa aacgtcggcc 1020atgtcttcct cagagatgta accgagcggc gcgtcgtggc gattgatgcc gatgctacaa 1080aagccgccga gttagctcga atgtcagatg cattgcgggc tggcccgcat ggcgcgggcg 1140cagcagcgag aggttctaga ttgctgaata tcggtccagg tccgctccga acgcgagttg 1200gcgcacttca caacattcaa gaacttactt tttgtccccg cggatcgtga caacaaagtg 1260gaagccgcct atcgaggcaa tcgtgccgcg ccgccacggg tgcatgtgcc tgcgatggac 1320actctcctcg tcatcctcca cgccaccgaa agggttctcg tcgcgctggg ccccgcctct 1380gaactgcacg aacggatcct catcgcgtgg ccttcgcttg gtgatcccga actgcgtgat 1440ggccttgttg acgcgaaaga aggcgatgcc ttggtcgaaa tagcctgctt ccaccagccc 1500gatgaaaaac ttggtcccgt tcggcgctag gctaggcgtg aggaccacgc ggaacgggcc 1560cttggtcgtg gagcactcga ccgcgttgcc gtcgggcgtc cctcgaggcg agctgtgcgc 1620gggcctacca gcggcaggct cagcgggcat atcattcctc ccgccagtcc tctttggcgc 1680cggcgccctg gggccctcgc gcggggcgac ggcgtccgcc tccctgtccg ccacgccgtg 1740cacgaacgag cgcacggaca ggagccacaa gagcagcagc ccgagggccg ccgcccacag 1800cgcagtcttc ctccacccgc gctgctgcgt cggccccgcg cgctgcatca ggcgagaccc 1860tcctcgcgcc ccccgaggcg gcacggcagc agcaggaggc tctggcgctc caggctcgcg 1920gcgggagatg gcctcgcacg tccgcgtgac ggggcagtcg agggccggct gcggcacggc 1980gctgcaagtg gacctgggct cgcgggtctg cttgcagttc gactgcgggt cgctcgggga 2040cgagccggac aaggccgcgg cgagggcgga gcacgtgttc ttgtcgcacg ggcacctgga 2100ccactgcgcg gccgtcttcc tctacgcgcg gatcaggagc atgctcaagt gcaagaccaa 2160ggtgtacttg ccggcggagg cgctcgaggc ctttcacgcc gcgaagcgag cctttgatgc 2220gatggacctc ggccacgcac ttcctgctgc cccgccggcc cggcaagcag gcacggaggc 2280cgaggcgcat gtcgaggcgc atgcagaggc gcatgcagag gcgcaggcgg atccggcgtt 2340cgctgtgttt gggctctccg aaggcgaccg cgtcgacgtc ggcaaggggg ttgttgtgca 2400agcatgcaag tcgtttcacc gcgtgcctgc ccttgggttc gccgtggtac agagcgtgcg 2460ggagttgctg ccagagtttg cgagtttgac ggggcaggag aagcaagact tgtttcgggc 2520tgcgccaaag ggcttttcac cgtacaagct t 2551981835DNAArtificial18S rDNA (T. aureum ATCC 34304) 98cgaatattcc tggttgatcc tgccagtagt catacgctta tctcaaagat taagccatgc 60atgtctaagt ataaaggctt atactctgaa actgcgaacg gctcattata tcagttatag 120tttctttgat agtgtttttt ctacatggat acttgtggca aatctagaaa caatacatgc 180gtacaggcct gactttgggg gagggctgca tttatttgac ttaagccaat acccctcggg 240gttgttttgg tgattcagaa taactgagcg aatcgcatag ctttcgggcg gcgatgaatc 300atttcaagtt tctgccccat cagctgtcga tggtagggta taggcctacc atggctgtca 360cgggtgacgg agaattaggg ttcgattccg gagagggagc ctgagagacg gctaccacat 420ccaaggaagg cagcaggcgc gtaaattact caatgttgac tcgacgaagt agtgacgaga 480attaacaatg cggagcgctc agcgttttgc aattggaatg agagcaatgt aaaagcctca 540tcgaggatcc attggagggc aagtctggtg ccagcagccg cggtaattcc agctccaata 600gcgtatacta aagttgttgc agttaaaaag ctcgtagttg aacctctggt agggccgacc 660ttggcgcgcg gtgaatgccg cgtcgtttag aagcgtcgtg cccggccatc ctcccccggt 720cttttgggct gggggtcgtt tactgtaaaa aaaatagagt gttccaagca gggggtaata 780tcccggtata tagtagtatg gaataatgag ataggacttt ggtactattt tgttggtttg 840catgccaagg taatgattaa gagggacagt tgggggtatt cgtatttaga tgtcagaggt 900gaaattcttg gattttcgaa agacgaacta ctgcgaaagc atttaccaag gatgttttca 960ttaatcaaga acgaaagtta ggggatcgaa gatgattaga taccatcgta gtcttaaccg 1020taaactatgc cgacttgcga ttgtccggcg tcgcttttag atgacctggg cagcagcaca 1080tgagaaatca aagtctttgg gttccggggg gagtatggtc gcaaggctga aacttaaagg 1140aattgacgga agggcaccac caggagtgga gcctgcggct taatttgact caacacggga 1200aaacttacca ggtccggaca taggaaggat tgacagattg agagctcttt cttgattcta 1260tgggtggtgg

tgcatggccg ttcttagttg gtggagtgat ttgtctggtt aattccgtta 1320acgaacgaga ccacagccta ctaaatagtg gccgttatgg cgacatagcg gtgaacttct 1380tagagggaca tttcgggtat accggaagga agtttgtggc aataacaggt ctgtgatgcc 1440cttagatgtt ctgggccgca cgcgcgctac actgatcggt tcaacgagta tttgtttttt 1500tctcattttg ggagggggca gagtccttgg ccggaaggtc tgggtaatct tttgaatgcc 1560gatcgtgatg gggctagatt tttgcaatta ttaatctcca acgaggaatt cctagtagac 1620gcaagtcatc agcttgcatc gattacgtcc ctgccctttg tacacaccgc ccgtcgcacc 1680taccgattga acgatccggt gagaccttgg gattctgttg tggctgattc attttggctg 1740cgatgggaga acttgagcaa accttatcgt ttagaggaag gtgaagtcgt aacaaggttt 1800ccgtagtgaa cctgcaattc aaaaaaagcc gttac 18359930DNAArtificialprimer 99cgaatattcc tggttgatcc tgccagtagt 3010046DNAArtificialprimer 100gtaacggctt tttttgaatt gcaggttcac tacgcttgtt agaaac 46101661DNAArtificialEF1 alpha promoter (T. aureum ATCC 34304) 101ggtttccgta gtgaacctgc aattcaaaaa aagccgttac tcacatcagg ccgccactca 60tccgggcgaa agcttcgcgc attcgtcctc gtcacctcgg gtcccctgtg tcgtgacgga 120aagcgcgacg agacgcggcc gcagcagaga gccccggggg cccgcgtcac ggggggcctg 180gcggcggtcc tccttaagcc aaaccgaggg ttagggctcc aggctgttcg gcggggtcgc 240gggcgcggtg gacgcgcggg gccgcctagc acctcctagc gcgcgactac caggatagcc 300cccgcgagtg cgcagggcgg tccgcggggc ggagggcggc ccagcagcgc ggcgcggcgg 360gcgggtgcgg ctgcgtaagg tggcggcggg cgcgggcggt tagtgttggt gttaggtcgc 420ggcggggctg tgttccgggc atccgcctta cggcggtgca tactggttgg ctgggaggcg 480gtttgcgggg ttagataggc ggccaaggtg agctgcgttg ggcggataaa tccgtggagg 540cgctcgttga cggcgcggca gagacggaac gcggagcagc acggagtagc aagcaggagt 600agcaggagta gcaagcagcg gcaaaggaag gctagatgat tgaacaggac ggccttcacg 660c 66110246DNAArtificialprimer 102ggtttccgta gtgaacctgc aattcaaaaa aagccgttac tcacat 4610346DNAArtificialprimer 103gcgtgaaggc cgtcctgttc aatcatctag ccttcctttg ccgctg 46104835DNAArtificialNeomycin resistance gene (Neor) 104catcggcaaa ggaaggctag atgattgaac aggacggcct tcacgctggc tcgcccgctg 60cttgggtgga acggctgttc ggctacgact gggctcagca gacgatcggc tgctcggacg 120cggccgtgtt ccgccttagc gcgcagggcc ggccggtcct gtttgtcaag accgacctta 180gcggcgccct caacgagctc caggacgaag ctgcccgcct cagctggctt gccacgacgg 240gggttccgtg cgccgctgtg ctcgacgtcg tcaccgaagc cggccgcgac tggctgctcc 300tcggggaagt gcccggccag gacctcctca gcagccacct cgcgcccgct gagaaggtgt 360ccatcatggc cgacgccatg cgccgcctgc acaccctcga ccccgccacc tgccccttcg 420accaccaggc gaagcacagg atcgaacgcg cccgcacgcg gatggaggct ggcctcgtcg 480accaagacga cctcgacgag gagcaccagg gcctcgcgcc ggcggaactg ttcgccaggc 540ttaaggctag gatgccggac ggcgaggacc tcgtggtcac gcacggcgac gcctgcctcc 600ccaacatcat ggtcgagaac ggccgcttct cgggctttat cgactgcggg cgcctgggcg 660tggcggaccg ctaccaagac atcgcgctcg ccacgcggga catcgccgag gagcttggcg 720gcgagtgggc cgaccgcttt ctcgtgctct acggcatcgc cgccccggac agccagagga 780ttgcgttcta ccgcctcctg gacgagttct tttgagatcc gcgccggcta tgcgc 83510545DNAArtificialprimer 105catcggcaaa ggaaggctag atgattgaac aggacggcct tcacg 4510646DNAArtificialprimer 106gcgcatagcc ggcgcggatc tcaaaagaac tcgtccagga ggcggt 461071249DNAArtificialEF1 alpha terminator (T. aureum ATCC 34304) 107tcctggacga gttcttttga gatccgcgcc ggctatgcgc ccgtgctcga ctgccacact 60gcccacattg cctgcaagtt cgctgagctc cagaacaaga tggaccgccg ctcgggcaag 120attctcgagg agacccccaa gttcatcaag tcgggtggac tctgccatgg tcaagatgta 180tcccctccaa gcgcatgtgc gtcgagtcct tcaccgagta cccgccgctc ggccgctttg 240ccgtgcgcga catgcgcgtc accgtcgctg tcggcgtcat caagtccgtc accaagggcg 300acaaataaat tctacgaaag atttttttcc tcaagaagcg ccctaaagtt gacccctagc 360agcgacgact gtgtgtgccg ttgtgagtcg agttgcgatg tcgtgcagcg cccgtcgcgt 420cccatgctcg cgcgcgactc cgtctctgct tttcatctca agtcaagagt gggaagttcc 480cttgctttat ctcactattt agaggtcgct cacggctgct ggttcctcgt cgcatgtagc 540acagcctcgt ccaatcgcag cctgcaccac cccgctcgcc tgggaaaatg cgctcagcgg 600attcgcactg gcactcctct cctcggacag gtgcgatgtg gaagcggtca catcctcggc 660gccctcggcc acgccagcat ctgcgcaatc gctctcctcg ttctcagccg caaccgcagg 720caggccgacg tcgtttacct cggaatccac cgagcatttc gagcccatcg cgctggcgtc 780cacctcgatc ataccttctc catcgccgtc cgctgcggct tccgattctt ctgctgccgc 840aaccgcgacg tcggcccccg tctcctccgt tctttccgat gccggcgcag tggccgcgcc 900ctctgctcga accggctcgt gttcagcgtc agggcctgcg cttgagctcg ggcggctctt 960ccgagtgatc cggccccgcg aggcaaggaa tcggcggctc tggagtgtcg gggcagccgc 1020tctcactgcc ggtctttggc tggctgcctg tcctgcctcg cgttggcctt tgcttttgcc 1080taggctttcg ccttggtgac ggcgtttgcc tgctgcggcg acttggcgcg gccgcggaat 1140agcgcctcaa agtcctgctc gaggcgcccc agctctgact tgatttgcga ggtcccggtg 1200gcatgagctc cgctgccctc gtccttacgg cccgtctttc gctgcagtg 124910846DNAArtificialprimer 108tcctggacga gttcttttga gatccgcgcc ggctatgcgc ccgtgc 4610930DNAArtificialprimer 109cactgcagcg aaagacgggc cgtaaggacg 301104453DNAArtificialfusion DNA (T. aureum ATCC 34304 18S rDNA/T. aureum ATCC 34304 EF1 alpha promoter/Neor/T. aureum ATCC 34304 EF1 alpha terminator) 110cgaatattcc tggttgatcc tgccagtagt catacgctta tctcaaagat taagccatgc 60atgtctaagt ataaaggctt atactctgaa actgcgaacg gctcattata tcagttatag 120tttctttgat agtgtttttt ctacatggat acttgtggca aatctagaaa caatacatgc 180gtacaggcct gactttgggg gagggctgca tttatttgac ttaagccaat acccctcggg 240gttgttttgg tgattcagaa taactgagcg aatcgcatag ctttcgggcg gcgatgaatc 300atttcaagtt tctgccccat cagctgtcga tggtagggta taggcctacc atggctgtca 360cgggtgacgg agaattaggg ttcgattccg gagagggagc ctgagagacg gctaccacat 420ccaaggaagg cagcaggcgc gtaaattact caatgttgac tcgacgaagt agtgacgaga 480attaacaatg cggagcgctc agcgttttgc aattggaatg agagcaatgt aaaagcctca 540tcgaggatcc attggagggc aagtctggtg ccagcagccg cggtaattcc agctccaata 600gcgtatacta aagttgttgc agttaaaaag ctcgtagttg aacctctggt agggccgacc 660ttggcgcgcg gtgaatgccg cgtcgtttag aagcgtcgtg cccggccatc ctcccccggt 720cttttgggct gggggtcgtt tactgtaaaa aaaatagagt gttccaagca gggggtaata 780tcccggtata tagtagtatg gaataatgag ataggacttt ggtactattt tgttggtttg 840catgccaagg taatgattaa gagggacagt tgggggtatt cgtatttaga tgtcagaggt 900gaaattcttg gattttcgaa agacgaacta ctgcgaaagc atttaccaag gatgttttca 960ttaatcaaga acgaaagtta ggggatcgaa gatgattaga taccatcgta gtcttaaccg 1020taaactatgc cgacttgcga ttgtccggcg tcgcttttag atgacctggg cagcagcaca 1080tgagaaatca aagtctttgg gttccggggg gagtatggtc gcaaggctga aacttaaagg 1140aattgacgga agggcaccac caggagtgga gcctgcggct taatttgact caacacggga 1200aaacttacca ggtccggaca taggaaggat tgacagattg agagctcttt cttgattcta 1260tgggtggtgg tgcatggccg ttcttagttg gtggagtgat ttgtctggtt aattccgtta 1320acgaacgaga ccacagccta ctaaatagtg gccgttatgg cgacatagcg gtgaacttct 1380tagagggaca tttcgggtat accggaagga agtttgtggc aataacaggt ctgtgatgcc 1440cttagatgtt ctgggccgca cgcgcgctac actgatcggt tcaacgagta tttgtttttt 1500tctcattttg ggagggggca gagtccttgg ccggaaggtc tgggtaatct tttgaatgcc 1560gatcgtgatg gggctagatt tttgcaatta ttaatctcca acgaggaatt cctagtagac 1620gcaagtcatc agcttgcatc gattacgtcc ctgccctttg tacacaccgc ccgtcgcacc 1680taccgattga acgatccggt gagaccttgg gattctgttg tggctgattc attttggctg 1740cgatgggaga acttgagcaa accttatcgt ttagaggaag gtgaagtcgt aacaaggttt 1800ccgtagtgaa cctgcaattc aaaaaaagcc gttactcaca tcaggccgcc actcatccgg 1860gcgaaagctt cgcgcattcg tcctcgtcac ctcgggtccc ctgtgtcgtg acggaaagcg 1920cgacgagacg cggccgcagc agagagcccc gggggcccgc gtcacggggg gcctggcggc 1980ggtcctcctt aagccaaacc gagggttagg gctccaggct gttcggcggg gtcgcgggcg 2040cggtggacgc gcggggccgc ctagcacctc ctagcgcgcg actaccagga tagcccccgc 2100gagtgcgcag ggcggtccgc ggggcggagg gcggcccagc agcgcggcgc ggcgggcggg 2160tgcggctgcg taaggtggcg gcgggcgcgg gcggttagtg ttggtgttag gtcgcggcgg 2220ggctgtgttc cgggcatccg ccttacggcg gtgcatactg gttggctggg aggcggtttg 2280cggggttaga taggcggcca aggtgagctg cgttgggcgg ataaatccgt ggaggcgctc 2340gttgacggcg cggcagagac ggaacgcgga gcagcacgga gtagcaagca ggagtagcag 2400gagtagcaag catggcaaag gaaggctaga tgattgaaca ggacggcctt cacgctggct 2460cgcccgctgc ttgggtggaa cggctgttcg gctacgactg ggctcagcag acgatcggct 2520gctcggacgc ggccgtgttc cgccttagcg cgcagggccg gccggtcctg tttgtcaaga 2580ccgaccttag cggcgccctc aacgagctcc aggacgaagc tgcccgcctc agctggcttg 2640ccacgacggg ggttccgtgc gccgctgtgc tcgacgtcgt caccgaagcc ggccgcgact 2700ggctgctcct cggggaagtg cccggccagg acctcctcag cagccacctc gcgcccgctg 2760agaaggtgtc catcatggcc gacgccatgc gccgcctgca caccctcgac cccgccacct 2820gccccttcga ccaccaggcg aagcacagga tcgaacgcgc ccgcacgcgg atggaggctg 2880gcctcgtcga ccaagacgac ctcgacgagg agcaccaggg cctcgcgccg gcggaactgt 2940tcgccaggct taaggctagg atgccggacg gcgaggacct cgtggtcacg cacggcgacg 3000cctgcctccc caacatcatg gtcgagaacg gccgcttctc gggctttatc gactgcgggc 3060gcctgggcgt ggcggaccgc taccaagaca tcgcgctcgc cacgcgggac atcgccgagg 3120agcttggcgg cgagtgggcc gaccgctttc tcgtgctcta cggcatcgcc gccccggaca 3180gccagaggat tgcgttctac cgcctcctgg acgagttctt ttgagatccg cgccggctat 3240gcgcccgtgc tcgactgcca cactgcccac attgcctgca agttcgctga gctccagaac 3300aagatggacc gccgctcggg caagattctc gaggagaccc ccaagttcat caagtcgggt 3360ggactctgcc atggtcaaga tgtatcccct ccaagcgcat gtgcgtcgag tccttcaccg 3420agtacccgcc gctcggccgc tttgccgtgc gcgacatgcg cgtcaccgtc gctgtcggcg 3480tcatcaagtc cgtcaccaag ggcgacaaat aaattctacg aaagattttt ttcctcaaga 3540agcgccctaa agttgacccc tagcagcgac gactgtgtgt gccgttgtga gtcgagttgc 3600gatgtcgtgc agcgcccgtc gcgtcccatg ctcgcgcgcg actccgtctc tgcttttcat 3660ctcaagtcaa gagtgggaag ttcccttgct ttatctcact atttagaggt cgctcacggc 3720tgctggttcc tcgtcgcatg tagcacagcc tcgtccaatc gcagcctgca ccaccccgct 3780cgcctgggaa aatgcgctca gcggattcgc actggcactc ctctcctcgg acaggtgcga 3840tgtggaagcg gtcacatcct cggcgccctc ggccacgcca gcatctgcgc aatcgctctc 3900ctcgttctca gccgcaaccg caggcaggcc gacgtcgttt acctcggaat ccaccgagca 3960tttcgagccc atcgcgctgg cgtccacctc gatcatacct tctccatcgc cgtccgctgc 4020ggcttccgat tcttctgctg ccgcaaccgc gacgtcggcc cccgtctcct ccgttctttc 4080cgatgccggc gcagtggccg cgccctctgc tcgaaccggc tcgtgttcag cgtcagggcc 4140tgcgcttgag ctcgggcggc tcttccgagt gatccggccc cgcgaggcaa ggaatcggcg 4200gctctggagt gtcggggcag ccgctctcac tgccggtctt tggctggctg cctgtcctgc 4260ctcgcgttgg cctttgcttt tgcctaggct ttcgccttgg tgacggcgtt tgcctgctgc 4320ggcgacttgg cgcggccgcg gaatagcgcc tcaaagtcct gctcgaggcg ccccagctct 4380gacttgattt gcgaggtccc ggtggcatga gctccgctgc cctcgtcctt acggcccgtc 4440tttcgctgca gtg 44531111218DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA upstream genomic DNA fragment) 111cccgaattcg gacgatgact gactgactga ttggctgacg acggccgccc tcgtgcgcgg 60cgtcgggcgt cgtcgcaaac caggcaggca ggcaggaagg aaggaaggaa gggccaggcc 120ctggtgcgaa acgctggcct gctccgctgc aagccaagcc gcgctcgcag gtgtacttcc 180gagtcctcgc gatgattagg caagcctgag cgagcacgta agctgcactg cggctgttca 240accagagaga gagttggctc tcttgcgtca aggcggcgcg cagcccactt gcgtcgcggc 300tgagggcccc tggaggggag gaaggaggcc ggcgagcggc gagtggcggc cctcactggc 360accaggtcgc aggaggccag gcagcccgcc acggacagga atcctcaggg cgcagcagcg 420cactacgtag tgcagagacg cagagcgggc cggatccgca gtgcggtcgc gccaccccgc 480cgcgcagctc gctcgcggac ggggtccgtg gccgcgcgaa aacggacacg gtgtgggagc 540ggacatggga tcgagaacgc cgttcgccct gctcgcgctg ccagcagcag gagccgtccg 600aaggacgagc ggccggccgc ctgtcccccc tccgcgcact cgaagcgcgc ccggcagcgc 660cccattgcgt gcgcggatgg cgtcttggct ggtccctctc gaggcgcttg ctcgtgctcg 720ccacgccttg tccgcctcct cgctgagcaa gcgatgagct gagcacggac cgcctgcaag 780tgcaagtgtt cttgtgctgc agggcgccga agaattggat tctggcccat gatcagtttg 840attgggccga gggagggagg gaggctgggc gagtgggcga caccagcaag ccggactgcg 900agaggggcgg ggcaggatgt gagcgcagga aagtgacgca agtgcatccg gccatcattg 960ggccatcatt gggccatcat tggtgttttg ggccgcgctt tgcggatcgt ccggccgatc 1020aggtacgagg ccacgaacct acgtcgtttg ccgcgctcag gctggttggt tgcacttgga 1080ctcttctgtg acctttcatc gtgtgcaggc aaactcgatt tgcagacccg agacacggcg 1140aaggatccgt gctgcaaacg caagtggagt gcgtcgagag caccgccgag accaagagcc 1200gaggcagaca agcttggg 121811232DNAArtificialprimer 112cccgaattcg gacgatgact gactgactga tt 3211328DNAArtificialprimer 113cccaagcttg tctgcctcgg ctcttggt 281141000DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 114cccccatggt gttgctgtgg gattggtccg ggggctcttc tgcacgcggc ctccgtcgcg 60cgcagaaatg ccccgtcact ggctgcccag gaggcagccg aatccctcta gctagctagc 120taggctagag cgtcttttcc gtagtttttc acaaagccag tatcacatgg ataacgaacg 180aaggtttcgg gctcgcgctc gcaggcgtta ggacgaagtt gatcgcccca cgtcacttca 240aacgagtgaa ccaagatcac gttgcatctg ctcgcaagat cttcttcttc cacgccgcat 300cgatgcgatg gatttcaaac tcttttcagg gcttttaggt gagtatggca gcgctgtttg 360cgtggcagcg ctgtttgcgt ggttgtactc tctaaaggtg cttccacgca tgcgcgcaca 420aaggggcatg gcatggttgg cggcgcactc tggccctcat ttgaagcaga ctatcgaagg 480gtccagttgg tactgcggca ggtccggcga gagcaagcgc ggcggtcgct cccactcgtc 540cctgcacagt tgctggactg gcgacggctg gcgcacctga ctacgagaag actcgagacg 600cacagaggta gtcagggacg accgaccgca aagcacaaac cgctccaaaa cggccgcacc 660aggcagggca gtaaactaaa aacgaatgta cctccatcgc gcgtatctgc cgagcctcct 720cccacgcttc ggctgggctt gattcaccag tgtccgcaag ctgaaccgac cgtcttcgat 780gtcatgaagc ttggcgcggc attagtcaga cgacgcggca cgccaggatt ctgtcggttt 840ctgggaaatg ggcatctata tagctgattc cctctgtcat gaggcggcct tgttctggcc 900ctgggccgcc gttcggatga tctatgatgt cgttgtacgc ataaagcttg tcgaaaacgt 960cggccatgtc ttcctcagag atgtaaccga gccatggggg 100011528DNAArtificialprimer 115cccccatggt gttgctgtgg gattggtc 2811630DNAArtificialprimer 116cccccatggc tcggttacat ctctgaggaa 301171632DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiqitin promoter/Hygr) 117cccaagcttg ccgcagcgcc tggtgcaccc gccgggcgtt gttgtgtgct cttcttgcct 60ccgagagaga gagcggagcg gatgcatagg aaatcgggcc acgcgggagg gccatgcgtt 120cgccccacac gccactttcc acgcccgctc tctctccggc cggcaggcag cgcataactc 180tccgacgctg gcaggctggt agcaactggc agggacaact cgcgcgcggg tcccggtcgt 240tcgatgtgcc aacccgagag aatccagcca gcagggcggt tggcctcatc gcccacctgc 300tatggtgcag cgaaccaact cccgaagcgg ccggttctgc gattccctct tctgaattct 360gaattctgaa ctgattccgg aggagaaccc tctggaagcg cgggttgcct ctccagttct 420gccgaactag acaggggagt gagcagagag tgaccctgac gcggagcgag ctggttgctg 480gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc gggcagaccc caaacgacaa 540gcagaacaag caacaccagc agcagcaagc gacctaagca acactagcca acatgaaaaa 600gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 660cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 720gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 780ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt 840cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 900gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc 960tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 1020atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca 1080aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 1140ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 1200tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 1260ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 1320gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg 1380ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 1440cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 1500tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 1560agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagtc 1620gacgcatgcg gg 163211836DNAArtificialprimer 118cccaagcttg ccgcagcgcc tggtgcaccc gccggg 3611943DNAArtificialprimer 119cccgcatgcg tcgactattc ctttgccctc ggacgagtgc tgg 431201000DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 120cccgtcgacg tgttgctgtg ggattggtcc gggggctctt ctgcacgcgg cctccgtcgc 60gcgcagaaat gccccgtcac tggctgccca ggaggcagcc gaatccctct agctagctag 120ctaggctaga gcgtcttttc cgtagttttt cacaaagcca gtatcacatg gataacgaac 180gaaggtttcg ggctcgcgct cgcaggcgtt aggacgaagt tgatcgcccc acgtcacttc 240aaacgagtga accaagatca cgttgcatct gctcgcaaga tcttcttctt ccacgccgca 300tcgatgcgat ggatttcaaa ctcttttcag ggcttttagg tgagtatggc agcgctgttt 360gcgtggcagc gctgtttgcg tggttgtact ctctaaaggt gcttccacgc atgcgcgcac 420aaaggggcat ggcatggttg gcggcgcact ctggccctca tttgaagcag actatcgaag 480ggtccagttg gtactgcggc aggtccggcg agagcaagcg cggcggtcgc tcccactcgt 540ccctgcacag ttgctggact ggcgacggct ggcgcacctg actacgagaa gactcgagac 600gcacagaggt agtcagggac gaccgaccgc aaagcacaaa ccgctccaaa acggccgcac 660caggcagggc agtaaactaa aaacgaatgt acctccatcg cgcgtatctg ccgagcctcc 720tcccacgctt cggctgggct tgattcacca gtgtccgcaa gctgaaccga ccgtcttcga 780tgtcatgaag cttggcgcgg cattagtcag acgacgcggc acgccaggat tctgtcggtt 840tctgggaaat gggcatctat atagctgatt ccctctgtca tgaggcggcc ttgttctggc 900cctgggccgc cgttcggatg atctatgatg tcgttgtacg cataaagctt gtcgaaaacg 960tcggccatgt cttcctcaga gatgtaaccg agtcgacggg 100012129DNAArtificialprimer 121cccgtcgacg tgttgctgtg ggattggtc 2912229DNAArtificialprimer 122cccgtcgact cggttacatc tctgaggaa 291233705DNAArtificialfusion DNA (T. aureum OrfA upstream/EF1 alpha

promoter/Neor/T. aureum OrfA downstream) 123cccccatggc tcggttacat ctctgaggaa gacatggccg acgttttcga caagctttat 60gcgtacaacg acatcataga tcatccgaac ggcggcccag ggccagaaca aggccgcctc 120atgacagagg gaatcagcta tatagatgcc catttcccag aaaccgacag aatcctggcg 180tgccgcgtcg tctgactaat gccgcgccaa gcttcatgac atcgaagacg gtcggttcag 240cttgcggaca ctggtgaatc aagcccagcc gaagcgtggg aggaggctcg gcagatacgc 300gcgatggagg tacattcgtt tttagtttac tgccctgcct ggtgcggccg ttttggagcg 360gtttgtgctt tgcggtcggt cgtccctgac tacctctgtg cgtctcgagt cttctcgtag 420tcaggtgcgc cagccgtcgc cagtccagca actgtgcagg gacgagtggg agcgaccgcc 480gcgcttgctc tcgccggacc tgccgcagta ccaactggac ccttcgatag tctgcttcaa 540atgagggcca gagtgcgccg ccaaccatgc catgcccctt tgtgcgcgca tgcgtggaag 600cacctttaga gagtacaacc acgcaaacag cgctgccacg caaacagcgc tgccatactc 660acctaaaagc cctgaaaaga gtttgaaatc catcgcatcg atgcggcgtg gaagaagaag 720atcttgcgag cagatgcaac gtgatcttgg ttcactcgtt tgaagtgacg tggggcgatc 780aacttcgtcc taacgcctgc gagcgcgagc ccgaaacctt cgttcgttat ccatgtgata 840ctggctttgt gaaaaactac ggaaaagacg ctctagccta gctagctagc tagagggatt 900cggctgcctc ctgggcagcc agtgacgggg catttctgcg cgcgacggag gccgcgtgca 960gaagagcccc cggaccaatc ccacagcaac accatggcag agtcgcccga cttgatgaac 1020ttgggggtct cctcgagaat cttgcccgag cggcggtcca tcttgttctg gagctcagcg 1080aacttgcagg caatgtgggc agtgtggcgg tcgagcacgg gcgcatagcc ggcgcggatc 1140tcaaaagaac tcgtccagga ggcggtagaa cgcaatcctc tggctgtccg gggcggcgat 1200gccgtagagc acgagaaagc ggtcggccca ctcgccgcca agctcctcgg cgatgtcccg 1260cgtggcgagc gcgatgtctt ggtagcggtc cgccacgccc aggcgcccgc agtcgataaa 1320gcccgagaag cggccgttct cgaccatgat gttggggagg caggcgtcgc cgtgcgtgac 1380cacgaggtcc tcgccgtccg gcatcctagc cttaagcctg gcgaacagtt ccgccggcgc 1440gaggccctgg tgctcctcgt cgaggtcgtc ttggtcgacg aggccagcct ccatccgcgt 1500gcgggcgcgt tcgatcctgt gcttcgcctg gtggtcgaag gggcaggtgg cggggtcgag 1560ggtgtgcagg cggcgcatgg cgtcggccat gatggacacc ttctcagcgg gcgcgaggtg 1620gctgctgagg aggtcctggc cgggcacttc cccgaggagc agccagtcgc ggccggcttc 1680ggtgacgacg tcgagcacag cggcgcacgg aacccccgtc gtggcaagcc agctgaggcg 1740ggcagcttcg tcctggagct cgttgagggc gccgctaagg tcggtcttga caaacaggac 1800cggccggccc tgcgcgctaa ggcggaacac ggccgcgtcc gagcagccga tcgtctgctg 1860agcccagtcg tagccgaaca gccgttccac ccaagcagcg ggcgagccag cgtgaaggcc 1920gtcctgttca atcatctagc cttcctttgc cgctgcttgc tactcctgct actcctgctt 1980gttacttcgt gttgctccgc gttccgtctc tgccgcgccg tccacgagcg cctccacgga 2040tttatccgcc caacgcggct caccttggcc gcctatctaa ccccgcaaac cgcctcccag 2100ccaaccattg cgccgccgta aggcggattc ccagaacaca gccccgccgc gacctaaccc 2160aacctaaccg cccgcgcccg ccgccacctt acgcagccgc acccgcccgc cgcgccgcgc 2220tgctgggccg ccctcgcccc gcagaccgcc ctgcgcgctc gcgggggcta tcctggtagt 2280cgcgcgctag gaggtgctag gcggccccgt gcttccacct cgcccgcgac cccgccgaac 2340agcctggagc cctaaccctc ggtttggctt aaggaggact gccgccaggc cccccgtgac 2400gcgggccccc ggggctctct gctgcggccg cgtctcgtcg cactttccgt cccgacacag 2460gggacccgag gtgacgagga cgaatgcgcg aagcttgtct gcctcggctc ttggtctcgg 2520cggtgctctc gacgcactcc acttgcgttt gcagcacgga tccttcgccg tgtctcgggt 2580ctgcaaatcg agtttgcctg cacacgatga aaggtcacag aagagtccaa gtgcaaccaa 2640ccagcctgag cgcggcaaac gacgtaggtt cgtggcctcg tacctgatcg gccggacgat 2700ccgcaaagcg cggcccaaaa caccaatgat ggcccaatga tggcccaatg atggccggat 2760gcacttgcgt cactttcctg cgctcacatc ctgccccgcc cctctcgcag tccggcttgc 2820tggtgtcgcc cactcgccca gcctccctcc ctccctcggc ccaatcaaac tgatcatggg 2880ccagaatcca attcttcggc gccctgcagc acaagaacac ttgcacttgc aggcggtccg 2940tgctcagctc atcgcttgct cagcgaggag gcggacaagg cgtggcgagc acgagcaagc 3000gcctcgagag ggaccagcca agacgccatc cgcgcacgca atggggcgct gccgggcgcg 3060cttcgagtgc gcggaggggg gacaggcggc cggccgctcg tccttcggac ggctcctgct 3120gctggcagcg cgagcagggc gaacggcgtt ctcgatccca tgtccgctcc cacaccgtgt 3180ccgttttcgc gcggccacgg accccgtccg cgagcgagct gcgcggcggg gtggcgcgac 3240cgcactgcgg atccggcccg ctctgcgtct ctgcactacg tagtgcgctg ctgcgccctg 3300aggattcctg tccgtggcgg gctgcctggc ctcctgcgac ctggtgccag tgagggccgc 3360cactcgccgc tcgccggcct ccttcctccc ctccaggggc cctcagccgc gacgcaagtg 3420ggctgcgcgc cgccttgacg caagagagcc aactctctct ctggttgaac agccgcagtg 3480cagcttacgt gctcgctcag gcttgcctaa tcatcgcgag gactcggaag tacacctgcg 3540agcgcggctt ggcttgcagc ggagcaggcc agcgtttcgc accagggcct ggcccttcct 3600tccttccttc ctgcctgcct gcctggtttg cgacgacgcc cgacgccgcg cacgagggcg 3660gccgtcgtca gccaatcagt cagtcagtca tcgtccgaat tcggg 37051243826DNAArtificialfusion DNA (T. aureum OrfA upstream/ubiquitin promoter/Hygr/T. aureum OrfA downstream) 124cccccatggc tcggttacat ctctgaggaa gacatggccg acgttttcga caagctttat 60gcgtacaacg acatcataga tcatccgaac ggcggcccag ggccagaaca aggccgcctc 120atgacagagg gaatcagcta tatagatgcc catttcccag aaaccgacag aatcctggcg 180tgccgcgtcg tctgactaat gccgcgccaa gcttcatgac atcgaagacg gtcggttcag 240cttgcggaca ctggtgaatc aagcccagcc gaagcgtggg aggaggctcg gcagatacgc 300gcgatggagg tacattcgtt tttagtttac tgccctgcct ggtgcggccg ttttggagtg 360gtttgtgctt tgcggtcggt cgtccctgac tacctctgtg cgtctcgagt cttctcgtag 420tcaggtgcgc cagccgtcgc cagtccagca actgtgcagg gacgagtggg agcgaccgcc 480gcgcttgctc tcgccggacc tgccgcagta ccaactggac ccttcgatag tctgcttcaa 540atgagggcca gagtgcgccg ccaaccatgc catgcccctt tgtgcgcgca tgcgtggaag 600cacctttaga gagtacaacc acgcaaacag cgctgccacg caaacagcgc tgccacgcaa 660acagcgctgc catactcacc taaaagccct gaaaagagtt tgaaatccat cgcgtcgatg 720cggcgtggaa gaagaagatc ttgcgagcag acgcaacgtg atcttggttc actcgtttga 780agtgacgcgg gacgatcaac ttcgtcctaa cgcctgcgag cgcgagcccg aaaccttcgt 840tcgttatcca tgtgatactg gctttgtgaa aaactacgga aaagacgcta gctagaggga 900ttcggctgcc tccttgggca gccagtgacg gggcatttct gcgcgcgacg gaggccgcgt 960gcaaaagagc ccccggacca atcccacagc aacacgtcga ctattccttt gccctcggac 1020gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga 1080cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga 1140ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct 1200gatagagttg gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa 1260gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc 1320tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt 1380caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt 1440gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct 1500gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat 1560cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga 1620atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg 1680cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct 1740gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt 1800ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac 1860catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag 1920cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga 1980tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcatgttg gctagtgttg 2040cttaggtcgc ttgctgctgc tggtgttgct tgttctgctt gtcgtttggg gtctgcccga 2100agtggacgcg tgacacagcc cagcgttcgc gacttttcca gcaaccagct cgctcccgcg 2160tcagggtcac tctctgctca ctcccctgtc tagttcggca gaactggaga ggcaacccgc 2220gcttccagag ggttctcctc cggaatcagt tcagaattca gaattcagaa gagggaatcg 2280cagaaccggc cgcttcggga gttggttcgc tgcaccatag caggtgggcg atgaggccaa 2340ccgccctgct ggctggattc tctcgggttg gcacatcgaa cgaccgggac ccgcgcgcga 2400gttgtccctg ccagttgcta ccagcctgcc agcgtcggag agttatgcgc tgcctgccgg 2460ccggagagag agcgggcgtg gaaagtggcg tgtggggcga acgcatggcc ctcccgcgtg 2520gcccgatttc ctatgcatcc gctccgctct ctctctcgga ggcaagaaga gcacaccaac 2580aacgcccggc gggtgcacca ggcgctgcgg caagcttgtc tgcctcggct cttggtctcg 2640gcggtgctct cgacgcactc cacttgcgtt tgcagcacgg atccttcgcc gtgtctcggg 2700tctgcaaatc gagtttgcct gcacacgatg aaaggtcaca gaagagtcca agtgcaacca 2760accagcctga gcgcggcaaa cgacgtaggt tcgtggcctc gtacctgatc ggccggacga 2820tccgcaaagc gcggcccaaa acaccaatga tggcccaatg atggcccaat gatggccgga 2880tgcacttgcg tcactttcct gcgctcacat cctgccccgc ccctctcgca gtccggcttg 2940ctggtgtcgc ccactcgccc agcctccctc cctccctcgg cccaatcaaa ctgatcatgg 3000gccagaatcc aattcttcgg cgccctgcag cacaagaaca cttgcacttg caggcggtcc 3060gtgctcagct catcgcttgc tcagcgagga ggcggacaag gcgtggcgag cacgagcaag 3120cgcctcgaga gggaccagcc aagacgccat ccgcgcacgc aatggggcgc tgccgggcgc 3180gcttcgagtg cgcggagggg ggacaggcgg ccggccgctc gtccttcgga cggctcctgc 3240tgctggcagc gcgagcaggg cgaacggcgt tctcgatccc atgtccgctc ccacaccgtg 3300tccgttttcg cgcggccacg gaccccgtcc gcgagcgagc tgcgcggcgg ggtggcgcga 3360ccgcactgcg gatccggccc gctctgcgtc tctgcactac gtagtgcgct gctgcgccct 3420gaggattcct gtccgtggcg ggctgcctgg cctcctgcga cctggtgcca gtgagggccg 3480ccactcgccg ctcgccggcc tccttcctcc cctccagggg ccctcagccg cgacgcaagt 3540gggctgcgcg ccgccttgac gcaagagagc caactctctc tctggttgaa cagccgcagt 3600gcagcttacg tgctcgctca ggcttgccta atcatcgcga ggactcggaa gtacacctgc 3660gagcgcggct tggcttgcag cggagcaggc cagcgtttcg caccagggcc tggcccttcc 3720ttccttcctt cctgcctgcc tgcctggttt gcgacgacgc ccgacgccgc gcacgagggc 3780ggccgtcgtc agccaatcag tcagtcagtc atcgtccgaa ttcggg 382612522DNAArtificialprimer 125gaagcgtccc gtagatgtgg tc 2212621DNAArtificialprimer 126gcccgagagg tcaaagtacg c 2112720DNAArtificialprimer 127gcgagcccag gtccacttgc 2012822DNAArtificialprimer 128cagcccgatg aaaaacttgg tc 2212922DNAArtificialprimer 129gggagcgcag ggaaaacggt ct 2213020DNAArtificialprimer 130ccagcccacg tcgtcggagc 201312297DNAArtificialgenomic DNA (T. aureum ATCC 34304 C20 elongase upstream genomic DNA fragment) 131ggccggggca gcccgcccag cacgccgctg cgctgctttc ggtcatgcga acctggctcc 60ccacagcaat gctgcgcggt cgctgcgcct cttgaggctc ggcgacgttg gcccggtttg 120gggcaccctg acgttgcacg aacgtccgct gcatctcagg cgcactcgga tcgacaactg 180tgcaaccggt cagcctttcg cggcagattg ggcacttgcc gcgctcgcgt atccgcgtgg 240cgcattcttc gcacacgcag gcgtgccggc agggaagcag gagggtgttt atcgtggcgt 300ccatgtagac cttgcacagc cgcggctcac tttccctcgg cgcagtcccg tgcccaacgt 360cggggccggg cgccggcgcc ggcgagggcg tcggttctgg gatgggatca ggatccgccg 420aggctgcaga ttgctgtgcg ggggtgccgg ggcgcggccc attagcaccg tcctgcggaa 480tatccaggag ggtgctcatc acggaagcca tgtccgggcg ctggctgccg tcttcgtgcg 540tgcaacgatc caccaggtca aggagcaccc gagccacgtt ttggcgggtg cgaaaagcgg 600cttggtcaac gcagcgctcc gggtcgaagc cgttgtcgcg catccagtac ggaagcaggt 660cgaggccgcg gcggcaggga aagcaaggcg gcttgccgga cacaagctcg ccgagcacga 720cgccaaaggc gtaaacgtcc acggggcgat tgtactcgac atgggtggcg ccctccaggt 780cggagatctc gggcgccatg tagccaagcg tgcccacttg tgtcatcgtt tgcatggtgt 840ggagcgtggt actggcggcc accttggaca cgccaaagtc cgtccagcac agccttaggc 900ccgcgccctt gttcaggttg accagagcgt tgtcgctctt ggatgtctct gtgcagcacg 960ccagcagcgt gcagcgccct gaggccgtgc gccgcctggt atgccagcgc ctcgcgagcg 1020ctgccgtcca aaagcggcgc gctccccgaa tcatcgcgga gctggatggc cttcttgagc 1080gacatttcca tgcggggcat gatgatcgca aacctgccgc cgctctgtgg ctcgagcgcg 1140gtcgccagga ccgtgagcac gttctcgtgc gttgcgctcg ccatccgcga ggcctcagcg 1200cgaaagtcat cgaggacgcc gagcgtctgc ccgggccgcg gtaccttgac agcgcagcgc 1260ccgaacggcg ccacgtccgc ttcaaacacc tcgccaaagg cgccttgtcc gagcagctcg 1320ccccagccgc agccgcgacc actcgatctc gggcacgcgt gccatcgacc cttgcagcgt 1380ggccttgcca agtcacagtc cagcgcgcag ttcagtgtct gccgcgccag gtccaccacg 1440atcaattcat ccgagtcggc tgcgaactgg acaagtgcca tttgggtgcg ggcaacgcgc 1500aagatcaccc agcactggca tgaagaccat gaatgaatga atgaccgtgc gcgagtgacc 1560gaccaacacg agtccagccg actccttctt cttctccttc ttcttcttct tcttcttctc 1620gtagcgggcg tcaacagcat caatcaggca tggcggcatt cactctgcgc gatggatggc 1680acgagcgctg gaggtgatga acgcactgcc cggattggct ctcggtcact gtcagcacat 1740gatgcctgtg cttgcgcgga gcgcgctatg tctcgttctg tgtcaagaca caggcgcaac 1800tcttgatgga ttcttgaagc gcatgtaact gaagtctgac agactcggaa gtccattgtg 1860aacaatgttg ttccacaatt gctccaattg ttccgattat tccacaattg ttgttccaat 1920tgttccaatt gttccgatta ttccgattat tccactttag ttgttccagt tgttccgatt 1980gttccacaat tgttgttccg attattccag ttgttccagt tgttccaatt attccaattg 2040ttccagttcc ttactcttga catcggggga ataacgggtg tgtatttagg ggttcggcga 2100aagcagaatg gccgaacgta acagcggaga ggaacctctt tagcggggtt tgcgtatcgg 2160ggaaaccagg tgttgtgctg gcgaggagga tcccccgcga ggcgatggct gctccgacga 2220cgtgggctgg cgacgtcgct cgcaaaggcg ttccgcaacc gcgcgttccg ctgtaacgag 2280accgttttcc ctgcgct 229713223DNAArtificialprimer 132gccgctcatg cccacgctca aac 2313323DNAArtificialprimer 133ctttcggctg ccaggaatct acg 231342189DNAArtificialgenomic DNA (T. aureum ATCC 34304 OrfA downstream genomic DNA fragment) 134ctttcggctg ccaggaatct acggcccagg gcgcggcccg atctcacgaa ttcgcaaggg 60ccaggcccgc agtatcgtca aggagggcca ggtcttctcg cgggcgcacg tcgacgatat 120caccggtgcg atccgcgctt cgctggccaa cccaaacccg ggccgcgcct acaacgtttg 180cgacgacgag cctgcaatga accatgtcgt gacagagttt gcctgcgaac tcatggacgt 240cccgcccccg aagcgcgaag actttgacaa ggtgcgcgag accatgtcaa gcatgtcgct 300ctccttcttc tcagagagca agcgggtctt caacaagcgg ctcaaggaag agctgcggta 360cgcgctattg tacccgacct accgcgaagg gatcaaagcc caactggagg aggagcttgc 420caacggctgg acgctcatcg acgcctcggg tgcttctgct ggaaccgact cccctgcctc 480gcccaaagcg cccgccccca tcgccgcctc aagtgacgag tcgagcgggc agagcgcgac 540agcggccgag ccggtgcgcc ggcgcaggcg ccccgagcgc aaggcgctcc cgcctgctgg 600gccgagtggg ccgtcggtct tgcagagggt ttctcgggca atttatgggc cgttcagttg 660gctcctcggt cgcctgtttg ggccactttc gagccgcgct gtcggcttgt ttcgcggctg 720ggcgcactgg ctgttgcgtc tcgtggggct gcgcgcatcc gcgccgggcg gcggccgtac 780aacctgcctc cttgttgaca acggctcgct caaaccagag ccttttcgcc agctgcgcgt 840gcacgcggcg aacctcgaag agtctcttag gagcgacgcg cgtgccccac atcccgtgca 900ggtggtggcc gtcagcgcga ggtacagcga ccgcatcgac gcctcccttc tggacggcaa 960gcccggcgtc gccctcgccg ggttcctgag ttccttcaag gccgacgccg agtcgcagcc 1020agcaaccagc gaggttggcc gcatcatcgc gctcccctac tttctgggcc caagcaagac 1080ggccacgtcg tatgttgctt cccagctcgc agagcacttt ccaggagccg agcgcaccat 1140tgccgctccg ctcgtgtcgc gggacggcgc cattgcgcag ctcctcgctg acatggtcca 1200tgacgtcgct cgggcgcgcg cgctgcaggc cccgtacgcg gtagttctcg tcgaccacgg 1260gtccccgagc cgagcggtca accgcgttcg gcgggccatc gctgcgcgga tgcgccgccg 1320ccttggcccg aacgcgcgct gcgttgtcga ctgctccatg gagcgccgcg agggcgacgc 1380tttcgccttc aacgagcctc tgctagagtc ggttttcacc aagggtggtc tcgactctgg 1440cgacgtcatt ctcgcgatgg catttttggc gcctggtcgc cacgctggcg agggcggcga 1500tatcgcggag atccttgacg aggctatcgc aaagtcggct ggcaagctgc gcgttcacca 1560aacgcggttg attggtgacg tggacaggaa cggtacgcag atttgcgccc tcctcaagaa 1620caggccgctt gccgcgctgt aacggcaaga gcatccacaa ttcctgacct gagcaaacca 1680gcccacgcga gagaccgaac acgtcaagcc gatgaggcgc agaaaacaaa gaaaaaaagc 1740aaaaagaaca aaaacccaag gcaaaatgat ggcaattttc ttggtatgga aagccgatga 1800tcgccgagtg tcgctggcta tttgctctgg tggggcatcg agctcgatga ccgaaatcca 1860ccaattatct gcgtgtcaat catttggagc ataagacccg ggaaggcctt gagcaagcga 1920agaaaccggc gcgtgttcac acgatagtac gagacgtcgc tctctgcgcg gatctcaatc 1980tgagccttct tgtctccgcg gatgaaagtg ttcatgtccc cgacaagggc gccgcgccca 2040acccctcgtt tgggctgcgc cgcgctactg gaaatggtga ttccgcgaaa cgtgcccgat 2100tcgcctttct caacagggct caccgtgaca gaaccctcag cgacaagaac gatgccgtca 2160atcttttcgc cgggcgaggc tttctgcag 2189135618DNAArtificialgenomic DNA (T. aureum ATCC 34304 ubiquitin promoter) 135cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttggtgtgc tcttcttgcc 60tccgagagag agagcggagc ggatgcatag gaaatcgggc cacgcgggag ggccatgcgt 120tcgccccaca cgccactttc cacgcccgct ctctctccgg ccggcaggca gcgcataact 180ctccgacgct ggcaggctgg tagcaactgg cagggacaac tcgcgcgcgg gtcccggtcg 240ttcgatgtgc caacccgaga gaatccagcc agcagggcgg ttggcctcat cgcccacctg 300ctatggtgca gcgaaccaac tcccgaagcg gccggttctg cgattccctc ttctgaattc 360tgaattctga actgattccg gaggagaacc ctctggaagc gcgggttgcc tctccagttc 420tgccgaacta gacaggggag tgagcagaga gtgaccctga cgcggagcga gctggttgct 480ggaaaagtcg cgaacgctgg gctgtgtcac gcgtccactt cgggcagtcc ccaaacgaca 540agcagaacaa gcaacaccag cagcagcaag cgacctaagc aacactagcc aacatggcca 600agcctttgtc tcaagaag 61813658DNAArtificialprimer 136cttcttgaga caaaggcttg gccatgttgg ctagtgttgc ttaggtcgct tgctgctg 58137432DNAArtificialBlasticidin resistance gene (Blar) 137agcgacctaa gcaacactag ccaacatggc caagcctttg tctcaagaag aatccaccct 60cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc 120cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac 180tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa 240cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg 300gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga 360tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg 420ctaagatctg gg 43213854DNAArtificialprimer 138agcgacctaa gcaacactag ccaacatggc caagcctttg tctcaagaag aatc 5413938DNAArtificialprimer 139cccagatctt agccctccca cacataacca gagggcag 381401000DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/pTracer-CMV/Bsd/lacZ Blar) 140cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttggtgtgc tcttcttgcc 60tccgagagag agagcggagc ggatgcatag gaaatcgggc cacgcgggag ggccatgcgt 120tcgccccaca cgccactttc cacgcccgct ctctctccgg ccggcaggca gcgcataact 180ctccgacgct ggcaggctgg tagcaactgg

cagggacaac tcgcgcgcgg gtcccggtcg 240ttcgatgtgc caacccgaga gaatccagcc agcagggcgg ttggcctcat cgcccacctg 300ctatggtgca gcgaaccaac tcccgaagcg gccggttctg cgattccctc ttctgaattc 360tgaattctga actgattccg gaggagaacc ctctggaagc gcgggttgcc tctccagttc 420tgccgaacta gacaggggag tgagcagaga gtgaccctga cgcggagcga gctggttgct 480ggaaaagtcg cgaacgctgg gctgtgtcac gcgtccactt cgggcagtcc ccaaacgaca 540agcagaacaa gcaacaccag cagcagcaag cgacctaagc aacactagcc aacatggcca 600agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca 660tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct 720tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg 780gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga 840acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg 900ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat 960tgctgccctc tggttatgtg tgggagggct aagatctggg 1000141812DNAArtificialcDNA (T.aureum ATCC 34304 ubiquitin promoter) 141tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt tgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagctg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gatctaagca 780acactagcca acatggtgag caagggcgag ga 81214229DNAArtificialprimer 142tcggtacccg ttagaacgcg taatacgac 2914341DNAArtificialprimer 143tcctcgccct tgctcaccat gttggctagt gttgcttagg t 41144748DNAArtificialEnhanced GFP gene (Enhanced GFP DNA fragment) 144acctaagcaa cactagccaa catggtgagc aagggcgagg agctgttcac cggggtggtg 60cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 120ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 180ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 240cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 300gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 360aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 420gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 480atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 540gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 600gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 660gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 720atggacgcca agttgaccag tgccgttc 74814541DNAArtificialprimer 145acctaagcaa cactagccaa catggtgagc aagggcgagg a 4114658DNAArtificialprimer 146gaacggcact ggtcaacttg gcgtccatgc cgagagtgat cccggcggcg gtcacgaa 581471519DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/ Enhanced GFP) 147tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt tgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagctg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780acactagcca acatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 840gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 900gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 960ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 1020gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 1080cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 1140ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1200atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1260aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1320gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1380cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1440gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgcc 1500aagttgacca gtgccgttc 15191481319DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/ Enhanced GFP) 148cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttgtgtgct cttcttgcct 60ccgagagaga gagcggagcg gatgcatagg aaatcgggcc acgcgggagg gccatgcgtt 120tgccccacac gccactttcc acgcccgctc tctctccggc cggcaggcag cgcataactc 180tccgacgctg gcaggctggt agcaactggc agggacaact cgcgcgcggg tcccggtcgt 240tcgatgtgcc aacccgagag aatccagcca gcagggcggt tggcctcatc gcccacctgc 300tatggtgcag cgaaccaact cccgaagctg ccggttctgc gattccctct tctgaattct 360gaattctgaa ctgattccgg aggagaaccc tctggaagcg cgggttgcct ctccagttct 420gccgaactag acaggggagt gagcagagag tgaccctgac gcggagcgag ctggttgctg 480gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc gggcagaccc caaacgacaa 540gcagaacaag caacaccagc agcagcaagc gacctaagca acactagcca acatggtgag 600caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 660aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 720gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 780caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 840cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 900cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 960catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 1020gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 1080ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 1140ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 1200cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 1260gttcgtgacc gccgccggga tcactctcgg catggacgcc aagttgacca gtgccgttc 1319149408DNAArtificialcDNA (Zeor) 149cgccgccggg atcactctcg gcatggacgc caagttgacc agtgccgttc cggtgctcac 60cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 120cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 180ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga 240cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 300ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 360cggcaactgc gtgcacttcg tggccgagga gcaggactga gatctggg 40815054DNAArtificialprimer 150cgccgccggg atcactctcg gcatggacgc caagttgacc agtgccgttc cggt 5415138DNAArtificialprimer 151cccagatctc agtcctgctc ctcggccacg aagtgcac 381521677DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/Enhanced GFP/pcDNA3.1 Zeo(+) Zeor) 152cccagatctg ccgcagcgcc tggtgcaccc gccgggcgtt gttgtgtgct cttcttgcct 60ccgagagaga gagcggagcg gatgcatagg aaatcgggcc acgcgggagg gccatgcgtt 120tgccccacac gccactttcc acgcccgctc tctctccggc cggcaggcag cgcataactc 180tccgacgctg gcaggctggt agcaactggc agggacaact cgcgcgcggg tcccggtcgt 240tcgatgtgcc aacccgagag aatccagcca gcagggcggt tggcctcatc gcccacctgc 300tatggtgcag cgaaccaact cccgaagctg ccggttctgc gattccctct tctgaattct 360gaattctgaa ctgattccgg aggagaaccc tctggaagcg cgggttgcct ctccagttct 420gccgaactag acaggggagt gagcagagag tgaccctgac gcggagcgag ctggttgctg 480gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc gggcagaccc caaacgacaa 540gcagaacaag caacaccagc agcagcaagc gacctaagca acactagcca acatggtgag 600caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 660aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 720gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 780caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 840cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 900cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 960catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 1020gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 1080ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 1140ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 1200cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 1260gttcgtgacc gccgccggga tcactctcgg catggacgcc aagttgacca gtgccgttcc 1320ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt 1380ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt 1440catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg 1500cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc 1560ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg 1620cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgag atctggg 16771532884DNAArtificialgenomic DNA (T. aureum ATCC 34304 C20 elongase upstream/C20 elongase/C20 elongase downstream) 153cccgaattca ctagtgattc tcccgggtgg acctagcgcg tgtgtcacct gccggccccc 60gttgcgtgca accgaattga tcgataatag aattacataa caaacaactt gctggatgag 120tacaagacca gcgtagtgtg gctgtgggac gttgaacgga gcgggtcctg tgatggcgca 180gaaaggaact ccgcccgagg tgaaaccccg atgcgcagga ctctgcggcc acagcccctc 240cgccagtatt ccactaaaaa tccgccccct ttgacaaaga tcgcaacccc gtcccatcaa 300ctcctcacaa taggctttcc actggcggaa acgtccccgg cacaggagtg cctcccgcgg 360ttctgcgcat acggctgacc actacgcagc gcgatatcct ccatccgcgt atatatccgt 420aaacaacgga acattctccc tctcaacgag gcgtggtttt cgaagtcatg cctttcttcc 480ttcctacttt ccttccttct ttctttcttt ctttccttct tttgcaagcg tgcgcgaact 540tgaaggtact acttacactt gacagagaga gatagagacg gcaattcgac caagtacttt 600ccacgatttt tttttttttt gttttggtcg ctttcgttgg tcgtgcatga tggatggccg 660ggatttttac aattggatgc gccaggctgc cacgcatgcc gtgacgcttg ctcgcggcga 720ctcatgatgc ttgccagtgg cagtgcatcc agctcttccc tctgctcgtc gtgtactcac 780tggcgatgct ctcggcgctc gttcaagggc catcgatcga tcgatcgatc gatcgatcga 840tcaatcacgt ttggtggact cggcagaccc cgaacgtgtt tctcccagga cgcgccgctg 900tcgctcgcta atccacccga agcgcggtcg gctggcacgg tcgctcggct ggaagttgag 960tagtttgctt tctgttgctg cgctgctttg taaacgcgac catggcgacg cgcacctcga 1020agagcgctcc ggcggtttcc aagtcggcca aggttgccgc gccggcgaag aagcggtcgg 1080tcgacaggag cgacggtttc ttccgcacgt tcaacctgtg cgccctgtac gggtctgccc 1140tcgcctatgc gtacaagcac ggcccggtgg acaatgacgg ccaggggctg tactttcaca 1200agtcgcccat gtacgcgttc gccgtgtcgg acgtcatgac cttcggcgcg ccgctgatgt 1260acgtgctcgg tgtgatgctg ctcagcaggt acatggcgga caaaaagccc ctgactggct 1320tcatcaagac ctacatccag cccgtctaca acgtggtcca aatcgcggtg tgcggctgga 1380tggtgtgggg cctctggccg caggtcgacc tggccaacgg caaccctttc ggcctcaaca 1440agtcgcgcga ctcgaacatc gagtttttcg tgttcgtgca cctcctgaca aagtttctcg 1500actggagcga cacgttcatg atgatcctca agaaaaacta cgcccaggtt agctttctgc 1560aggtgttcca ccacgcaacg atcggcatgg tgtggtcgtt ccttcttcag cgtggctggg 1620gctcgggcac cgccgcgtac ggtgctttca tcaactcggt cacgcacgtg atcatgtact 1680cgcactactt tgccacctcg ctcaacatca acaacccgtt caagcggtac atcacgagct 1740tccagctcgc ccagtttgca agctgcatcg tgcatgccct actggtgctt gccttcgagg 1800aggtgtaccc gctcgagtac gcttacctgc agatcagcta ccacatcatc atgctctacc 1860tgttcggacg ccgcatgaac tggagccccg agtggtgcac cggtgagatc gacggccttg 1920acgccccaag cgcccccacc aagtccgagt aaacctgttt ccggctggct cccgagccat 1980gcttaccatg aatgaacctg caaacagtct gaggtccttg tgcaaaccgc tcagtgggac 2040gtcgacgaag aaagaaacaa tgtgtactcg tcttgctctg ctcccgcgcc gttttttatc 2100gttgttgaga cctctcgcgc agttttggga atcaaccaaa acaagagccc ggcgtcagcg 2160tttgcttcgc cctcggctgc actcgctcgg cacgcaggta taactgggtg agtaccaagc 2220cccgcatttg tctgtccgcg atccgcgcac gctgcgggtc aggacgacat cgcgctgcac 2280gtcacagtgg gtcccttttg acgtggctgc ggcgatgagg aggcttggct cggcttcatg 2340gcaaggcaac agactcgctt ccgggacgcg cacgacgagc agcgctgctt tgatcgacct 2400tgcctgcgtc accgcctcgg ctgctttgat cgatcgttgt caccggccga gtgaccgcga 2460acgcattgcc cgcacggctc ggctcggccc ggaccggacc ggctcgcctt ggcggcgcgg 2520cgcgatggcg acccagacgc ggccggagcc gcgcgcggag gacaaggcca tgttcatctt 2580cgggctcggg tacgttggga gcaggctcgc caaccagctg gcggaacagg ggtggcgcgt 2640cgcggggtcg gtgagggagc tcgggcgcga ggacgacttt gccgagttcg aaaagtccaa 2700gctgagcggc aaggtgcagg tgttccgact cccgcttgag ggcgaggaca acacgcccgc 2760tcgcgcgcgg gagatactta gcgggtacca gcacctgctg ttcacggcgc cagtggaccg 2820cgcccggaac tgtgacccct tcttgggcga ccccgttctc ggccccggga taatcgaatt 2880cggg 288415450DNAArtificialprimer 154cccgaattca ctagtgattc tcccgggtgg acctagcgcg tgtgtcacct 5015540DNAArtificialprimer 155cccgaattcg attatcccgg ggccgagaac ggggtcgccc 401561939DNAArtificialfusion DNA (T. aureum ATCC 34304 C20 elongase upstream/C20 elongase downstream) 156cccgaattca ctagtgattc tcccgggtgg acctagcgcg tgtgtcacct gccggccccc 60gttgcgtgca accgaattga tcgataatag aattacataa caaacaactt gctggatgag 120tacaagacca gcgtagtgtg gctgtgggac gttgaacgga gcgggtcctg tgatggcgca 180gaaaggaact ccgcccgagg tgaaaccccg atgcgcagga ctctgcggcc acagcccctc 240cgccagtatt ccactaaaaa tccgccccct ttgacaaaga tcgcaacccc gtcccatcaa 300ctcctcacaa taggctttcc actggcggaa acgtccccgg cacaggagtg cctcccgcgg 360ttctgcgcat acggctgacc actacgcagc gcgatatcct ccatccgcgt atatatccgt 420aaacaacgga acattctccc tctcaacgag gcgtggtttt cgaagtcatg cctttcttcc 480ttcctacttt ccttccttct ttctttcttt ctttccttct tttgcaagcg tgcgcgaact 540tgaaggtact acttacactt gacagagaga gatagagacg gcaattcgac caagtacttt 600ccacgatttt tttttttttt gttttggtcg ctttcgttgg tcgtgcatga tggatggccg 660ggatttttac aattggatgc gccaggctgc cacgcatgcc gtgacgcttg ctcgcggcga 720ctcatgatgc ttgccagtgg cagtgcatcc agctcttccc tctgctcgtc gtgtactcac 780tggcgatgct ctcggcgctc gttcaagggc catcgatcga tcgatcgatc gatcgatcga 840tcaatcacgt ttggtggact cggcagaccc cgaacgtgtt tctcccagga cgcgccgctg 900tcgctcgcta atccacccga agcgcggtcg gctggcacgg tcgctcggct ggaagttgag 960tagtttgctt tctgttgctg cgctgctttg taaacgcgac cagatctacc tgtttccggc 1020tggctcccga gccatgctta ccatgaatga acctgcaaac agtctgaggt ccttgtgcaa 1080accgctcagt gggacgtcga cgaagaaaga aacaatgtgt actcgtcttg ctctgctccc 1140gcgccgtttt ttatcgttgt tgagacctct cgcgcagttt tgggaatcaa ccaaaacaag 1200agcccggcgt cagcgtttgc ttcgccctcg gctgcactcg ctcggcacgc aggtataact 1260gggtgagtac caagccccgc atttgtctgt ccgcgatccg cgcacgctgc gggtcaggac 1320gacatcgcgc tgcacgtcac agtgggtccc ttttgacgtg gctgcggcga tgaggaggct 1380tggctcggct tcatggcaag gcaacagact cgcttccggg acgcgcacga cgagcagcgc 1440tgctttgatc gaccttgcct gcgtcaccgc ctcggctgct ttgatcgatc gttgtcaccg 1500gccgagtgac cgcgaacgca ttgcccgcac ggctcggctc ggcccggacc ggaccggctc 1560gccttggcgg cgcggcgcga tggcgaccca gacgcggccg gagccgcgcg cggaggacaa 1620ggccatgttc atcttcgggc tcgggtacgt tgggagcagg ctcgccaacc agctggcgga 1680acaggggtgg cgcgtcgcgg ggtcggtgag ggagctcggg cgcgaggacg actttgccga 1740gttcgaaaag tccaagctga gcggcaaggt gcaggtgttc cgactcccgc ttgagggcga 1800ggacaacacg cccgctcgcg cgcgggagat acttagcggg taccagcacc tgctgttcac 1860ggcgccagtg gaccgcgccc ggaactgtga ccccttcttg ggcgaccccg ttctcggccc 1920cgggataatc gaattcggg 193915738DNAArtificialprimer 157cccagatcta cctgtttccg gctggctccc gagccatg 3815838DNAArtificialprimer 158cccagatctg gtcgcgttta caaagcagcg cagcaaca 3815931DNAArtificialprimer 159ctcccgggtg gacctagcgc gtgtgtcacc t 3116027DNAArtificialprimer 160atcccggggc cgagaacgcc ctcgccc 271613215DNAArtificialfusion DNA (T. aureum C20 elongase upstream/ ubiquitin promoter/Blar/SV40 terminator/T. aureum C20 elongase downstream) 161ctcccgggtg gacctagcgc gtgtgtcacc tgccggcccc cgttgcgtgc aaccgaattg 60atcgataata gaattacata acaaacaact tgctggatga gtacaagacc agcgtagtgt 120ggctgtggga cgttgaacgg agcgggtcct gtgatggcgc agaaaggaac tccgcccgag 180gtgaaacccc gatgcgcagg actctgcggc cacagcccct ccgccagtat tccactaaaa 240atccgccccc tttgacaaag atcgcaaccc cgtcccatca actcctcaca ataggctttc 300cactggcgga aacgtccccg gcacaggagt gcctcccgcg gttctgcgca tgcggctgac 360cactacgcag cgcgatatcc tccatccgcg tatatatccg taaacaacgg aacattctcc 420ctctcaacga ggcgtggttt tcgaagtcat gcctttcttc cttcctactt tccttccttc 480tttctttctt tctttccttc ttttgcaagc gtgcgcgaac ttgaaggtac tacttacact 540tgacagagag agatagagac ggcaattcga ccaagtactt tccacgattt tttttttttt 600tgttttggtc gctttcgttg gtcgtgcatg atggatggcc gggattttta caattggatg 660cgccaggctg ccacgcatgc cgtgacgctt gctcgcggcg actcatgatg cttgccagtg 720gcagtgcatc cagctcttcc ctctgctcgt cgtgtactca ctggcgatgc tctcggcgct 780cgttcaaggg ccatcgatcg atcgatcgat cgatcgatcg atcaatcacg tttggtggac 840tcggcagacc ccgaacgtgt ttctcccagg

acgcgccgct gtcgctcgct aatccacccg 900aagcgcggtc ggctggcacg gtcgctcggc tggaagttga gtagtttgct ttctgttgct 960gcgctgcttt gtaaacgcga ccagatctgg atctgccgca gcgcctggtg cacccgccgg 1020gcgttgttgt gtgctcttct tgcctccgag agagagagcg gagcggatgc ataggaaatc 1080gggccacgcg ggagggccat gcgttcgccc cacacgccac tttccacgcc cgctctctct 1140ccggccggca ggcagcgcat aactctccga cgctggcagg ctggtagcaa ctggcaggga 1200caactcgcgc gcgggtcccg gtcgttcgat gtgccaaccc gagagaatcc agccagcagg 1260gcggttggcc tcatcgccca cctgctatgg tgcagcgaac caactcccga agcggccggt 1320tctgcgattc cctcttctga attctgaatt ctgaactgat tccggaggag aaccctctgg 1380aagcgcgggt tgcctctcca gttctgccga actagacagg ggagtgagca gagagtgacc 1440ctgacgcgga gcgagctggt tgctggaaaa gtcgcgaacg ctgggctgtg tcacgcgtcc 1500acttcgggca gtccccaaac gacaagcaga acaagcaaca ccagcagcag caagcgacct 1560aagcaacact agccaacatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa 1620gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag 1680ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac 1740cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt 1800gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac 1860aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc 1920cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagatc 1980cgcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 2040gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 2100cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 2160aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 2220cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 2280acctctagct agatctacct gtttccggct ggctcccgag ccatgcttac catgaatgaa 2340cctgcaaaca gtctgaggtc cttgtgcaaa ccgctcagtg ggacgtcgac gaagaaagaa 2400acaatgtgta ctcgtcttgc tctgctcccg cgccgttttt tatcgttgtt gagacctctc 2460gcgcagtttt gggaatcaac caaaacaaga gcccggcgtc agcgtttgct tcgccctcgg 2520ctgcactcgc tcggcacgca ggtataactg ggtgagtacc aagccccgca tttgtctgtc 2580cgcgatccgc gcacgctgcg ggtcaggacg acatcgcgct gcacgtcaca gtgggtccct 2640tttgacgtgg ctgcggcgat gaggaggctt ggctcggctt catggcaagg caacagactc 2700gcttccggga cgcgcacgac gagcagcgct gctttgatcg accttgcctg cgtcaccgcc 2760tcggctgctt tgatcgatcg ttgtcaccgg ccgagtgacc gcgaacgcat tgcccgcacg 2820gctcggctcg gcccggaccg gaccggctcg ccttggcggc gcggcgcgat ggcgacccag 2880acgcggccgg agccgcgcgc ggaggacaag gccatgttca tcttcgggct cgggtacgtt 2940gggagcaggc tcgccaacca gctggcggaa caggggtggc gcgtcgcggg gtcggtgagg 3000gagctcgggc gcgaggacga ctttgccgag ttcgaaaagt ccaagctgag cggcaaggtg 3060caggtgttcc aactcccgct tgagggcgag gacaacacgc ccgctcgcgc gcgggagata 3120cttagcgggt accagcacct gctgttcacg gcgccagtgg accgcgcccg gaactgtgac 3180cccttcttgg gcgaccccgt tctcggcccc gggat 32151623887DNAArtificialfusion DNA (T. aureum C20 elongase upstream/ ubiquitin promoter/Enhanced GFP/Zeor/SV40 terminator/T. aureum C20 elogase downstream) 162ctcccgggtg gacctagcgc gtgtgtcacc tgccggcccc cgttgcgtgc aaccgaattg 60atcgataata gaattacata acaaacaact tgctggatga gtacaagacc agcgtagtgt 120ggctgtggga cgttgaacgg agcgggtcct gtgatggcgc agaaaggaac tccgcccgag 180gtgaaacccc gatgcgcagg actctgcggc cacagcccct ccgccagtat tccactaaaa 240atccgccccc tttgacaaag atcgcaaccc cgtcccatca actcctcaca ataggctttc 300cactggcgga aacgtccccg gcacaggagt gcctcccgcg gttctgcgca tacggctgac 360cactacgcag cgcgatatcc tccatccgcg tatatatccg taaacaacgg aacattctcc 420ctctcaacga ggcgtggttt tcgaagtcat gcctttcttc cttcctactt tccttccttc 480tttctttctt tctttccttc ttttgcaagc gtgcgcgaac ttgaaggtac tacttacact 540tgacagagag agatagagac ggcaattcga ccaagtactt tccacgattt tttttttttt 600tgttttggtc gctttcgttg gtcgtgcatg atggatggcc gggattttta caattggatg 660cgccaggctg ccacgcatgc cgtgacgctt gctcgcggcg actcatgatg cttgccagtg 720gcagtgcatc cagctcttcc ctctgctcgt cgtgtactca ctggcgatgc tctcggcgct 780cgttcaaggg ccatcgatcg atcgatcgat cgatcgatcg atcaatcacg tttggtggac 840tcggcagacc ccgaacgtgt ttctcccagg acgcgccgct gtcgctcgct aatccacccg 900aagcgcggtc ggctggcacg gtcgctcggc tggaagttga gtagtttgct ttctgttgct 960gcgctgcttt gtaaacgcga ccagatctgc cgcagcgcct ggtgcacccg ccgggcgttg 1020ttgtgtgctc ttcttgcctc cgagagagag agcggagcgg atgcatagga aatcgggcca 1080cgcgggaggg ccatgcgttt gccccacacg ccactttcca cgcccgctct ctctccggcc 1140ggcaggcagc gcataactct ccgacgctgg caggctggta gcaactggca gggacaactc 1200gcgcgcgggt cccggtcgtt cgatgtgcca acccgagaga atccagccag cagggcggtt 1260ggcctcatcg cccacctgct atggtgcagc gaaccaactc ccgaagctgc cggttctgcg 1320attccctctt ctgaattctg aattctgaac tgattccgga ggagaaccct ctggaagcgc 1380gggttgcctc tccagttctg ccgaactaga caggggagtg agcagagagt gaccctgacg 1440cggagcgagc tggttgctgg aaaagtcgcg aacgctgggc tgtgtcacgc gtccacttcg 1500ggcagacccc aaacgacaag cagaacaagc aacaccagca gcagcaagcg atctaagcaa 1560cactagccaa catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 1620tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 1680atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 1740cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 1800accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 1860gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 1920gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 1980tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 2040agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 2100tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 2160ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 2220atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgcca 2280agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct 2340ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc 2400gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc 2460tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 2520ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 2580ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 2640aggactgaga tccgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 2700gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 2760tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 2820attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 2880tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 2940tgtataccgt cgacctctag ctagatctac ctgtttccgg ctggctcccg agccatgctt 3000accatgaatg aacctgcaaa cagtctgagg tccttgtgca aaccgctcag tgggacgtcg 3060acgaagaaag aaacaatgtg tactcgtctt gctctgctcc cgcgccgttt tttatcgttg 3120ttgagacctc tcgcgcagtt ttgggaatca accaaaacaa gagcccggcg tcagcgtttg 3180cttcgccctc ggctgcactc gctcggcacg caggtataac tgggtgagta ccaagccccg 3240catttgtctg tccgcgatcc gcgcacgctg cgggtcagga cgacatcgcg ctgcacgtca 3300cagtgggtcc cttttgacgt ggctgcggcg atgaggaggc ttggctcggc ttcatggcaa 3360ggcaacagac tcgcttccgg gacgcgcacg acgagcagcg ctgctttgat cgaccttgcc 3420tgcgtcaccg cctcggctgc tttgatcgat cgttgtcacc ggccgagtga ccgcgaacgc 3480attgcccgca cggctcggct cggcccggac cggaccggct cgccttggcg gcgcggcgcg 3540atggcgaccc agacgcggcc ggagccgcgc gcggaggaca aggccatgtt catcttcggg 3600ctcgggtacg ttgggagcag gctcgccaac cagctggcgg aacaggggtg gcgcgtcgcg 3660gggtcggtga gggagctcgg gcgcgaggac gactttgccg agttcgaaaa gtccaagctg 3720agcggcaagg tgcaggtgtt ccgactcccg cttgagggcg aggacaacac gcccgctcgc 3780gcgcgggaga tacttagcgg gtaccagcac ctgctgttca cggcgccagt ggaccgcgcc 3840cggaactgtg accccttctt gggcgacccc gttctcggcc ccgggat 388716321DNAArtificialprimer 163acgtccgctt caaacacctc g 2116424DNAArtificialprimer 164tcggaacaac tggaacaact aaag 2416522DNAArtificialprimer 165atgtcgctct ccttcttctc ag 2216621DNAArtificialprimer 166tcggctcctg gaaagtgctc t 21167812DNAArtificialubiquitin promoter 167tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780acactagcca acatgactga ggataagacg aa 81216829DNAArtificialprimer 168tcggtacccg ttagaacgcg taatacgac 2916945DNAArtificialprimer 169ttcgtcttat cctcagtcat gttggctagt gttgcttagg tcgct 451701116DNAArtificialcDNA (Saprolegnia diclina omega3 desaturase) 170cctaagcaac actagccaac atgactgagg ataagacgaa ggtcgagttc ccgacgctca 60cggagctcaa gcactcgatc ccgaacgcgt gctttgagtc gaacctcggc ctctcgctct 120actacacggc ccgcgcgatc ttcaacgcgt cggcctcggc ggcgctgctc tacgcggcgc 180gctcgacgcc gttcattgcc gataacgttc tgctccacgc gctcgtttgc gccacctaca 240tctacgtgca gggcgtcatc ttctggggct tcttcacggt cggccacgac tgcggccact 300cggccttctc gcgctaccac agcgtcaact ttatcatcgg ctgcatcatg cactctgcga 360ttttgacgcc gttcgagagc tggcgcgtga cgcaccgcca ccaccacaag aacacgggca 420acattgataa ggacgagatc ttttacccgc accggtcggt caaggacctc caggacgtgc 480gccaatgggt ctacacgctc ggcggtgcgt ggtttgtcta cttgaaggtc gggtatgccc 540cgcgcacgat gagccacttt gacccgtggg acccgctcct ccttcgccgc gcgtcggccg 600tcatcgtgtc gctcggcgtc tgggccgcct tcttcgccgc gtacgcgtac ctcacatact 660cgctcggctt tgccgtcatg ggcctctact actatgcgcc gctctttgtc tttgcttcgt 720tcctcgtcat tacgaccttc ttgcaccaca acgacgaagc gacgccgtgg tacggcgact 780cggagtggac gtacgtcaag ggcaacctct cgagcgtcga ccgctcgtac ggcgcgttcg 840tggacaacct gagccaccac attggcacgc accaggtcca ccacttgttc ccgatcattc 900cgcactacaa gctcaacgaa gccaccaagc actttgcggc cgcgtacccg cacctcgtgc 960gcaagaacga cgagcccatc atctcggcct tcttcaagac cgcgcacctc tttgtcaact 1020acggcgctgt gcccgagacg gcgcagatct tcacgctcaa agagtcggcc gcggccgcca 1080aggccaagtc ggactaaact aagctatctg tagtat 111617143DNAArtificialprimer 171cctaagcaac actagccaac atgactgagg ataagacgaa ggt 4317240DNAArtificialprimer 172atactacaga tagcttagtt ttagtccgac ttggccttgg 40173614DNAArtificialubiquitin terminator 173ccaaggccaa gtcggactaa actaagctat ctgtagtatg tgctatactc gaatcatgct 60gccctgtacg tacctaccta tatctgattg agcgtgctgc gtcgaccata gacgcgggaa 120cgcgggccag cctaccacgt tgccgccgcc ggtatccacg ggcacgccaa agcattggtc 180gataacgctc tgcccagggc ttcctggcga ggacccgagg ccaacatgca tgcatgtgct 240atcagcggtc atcatcgccc tcatcagcgc gcatcggcga gctcgcgcac gaacggcaag 300cgcccaactc aactcactta ctcacactat ggtcttgccg ttggcggttg cttagctaat 360gcgtgacgtc actctgcctc caacatcgcg aggcagagtc gcgagcagtg cagaggccac 420ggcggacgcc aacaaagcgc caaccagcgc aacgcaccag cgggtctgtg ggcgtagctc 480gagcgggcgt cttcaagagc cgccgtggag ccgacgcccc tgcgaagggc tcgagtgcaa 540gcggggccgt tgagccgcgt ggtaggaaca actgcagtct ccctatagtg agtcgtatta 600cgcggtggta ccga 61417444DNAArtificialprimer 174ccaaggccaa gtcggactaa aactaagcta tctgtagtat gtgc 4417545DNAArtificialprimer 175tcggtaccac cgcgtaatac gactcactat agggagactg cagtt 451762463DNAArtificialfusion DNA (T. aureum ATCC 34304 ubiquitin promoter/Saprolegnia diclina omega3 desaturase/T. aureum ATCC 34304 ubiquitin terminator) 176tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780acactagcca acatgactga ggataagacg aaggtcgagt tcccgacgct cacggagctc 840aagcactcga tcccgaacgc gtgctttgag tcgaacctcg gcctctcgct ctactacacg 900gcccgcgcga tcttcaacgc gtcggcctcg gcggcgctgc tctacgcggc gcgctcgacg 960ccgttcattg ccgataacgt tctgctccac gcgctcgttt gcgccaccta catctacgtg 1020cagggcgtca tcttctgggg cttcttcacg gtcggccacg actgcggcca ctcggccttc 1080tcgcgctacc acagcgtcaa ctttatcatc ggctgcatca tgcactctgc gattttgacg 1140ccgttcgaga gctggcgcgt gacgcaccgc caccaccaca agaacacggg caacattgat 1200aaggacgaga tcttttaccc gcaccggtcg gtcaaggacc tccaggacgt gcgccaatgg 1260gtctacacgc tcggcggtgc gtggtttgtc tacttgaagg tcgggtatgc cccgcgcacg 1320atgagccact ttgacccgtg ggacccgctc ctccttcgcc gcgcgtcggc cgtcatcgtg 1380tcgctcggcg tctgggccgc cttcttcgcc gcgtacgcgt acctcacata ctcgctcggc 1440tttgccgtca tgggcctcta ctactatgcg ccgctctttg tctttgcttc gttcctcgtc 1500attacgacct tcttgcacca caacgacgaa gcgacgccgt ggtacggcga ctcggagtgg 1560acgtacgtca agggcaacct ctcgagcgtc gaccgctcgt acggcgcgtt cgtggacaac 1620ctgagccacc acattggcac gcaccaggtc caccacttgt tcccgatcat tccgcactac 1680aagctcaacg aagccaccaa gcactttgcg gccgcgtacc cgcacctcgt gcgcaagaac 1740gacgagccca tcatctcggc cttcttcaag accgcgcacc tctttgtcaa ctacggcgct 1800gtgcccgaga cggcgcagat cttcacgctc aaagagtcgg ccgcggccgc caaggccaag 1860tcggactaaa ctaagctatc tgtagtatgt gctatactcg aatcatgctg ccctgtacgt 1920acctacctat atctgattga gcgtgctgcg tcgaccatag acgcgggaac gcgggccagc 1980ctaccacgtt gccgccgccg gtatccacgg gcacgccaaa gcattggtcg ataacgctct 2040gcccagggct tcctggcgag gacccgaggc caacatgcat gcatgtgcta tcagcggtca 2100tcatcgccct catcagcgcg catcggcgag ctcgcgcacg aacggcaagc gcccaactca 2160actcacttac tcacactatg gtcttgccgt tggcggttgc ttagctaatg cgtgacgtca 2220ctctgcctcc aacatcgcga ggcagagtcg cgagcagtgc agaggccacg gcggacgcca 2280acaaagcgcc aaccagcgca acgcaccagc gggtctgtgg gcgtagctcg agcgggcgtc 2340ttcaagagcc gccgtggagc cgacgcccct gcgaagggct cgagtgcaag cggggccgtt 2400gagccgcgtg gtaggaacaa ctgcagtctc cctatagtga gtcgtattac gcggtggtac 2460cga 246317736DNAArtificialprimer 177cccggtaccg ccgcagcgcc tggtgcaccc gccggg 361783777DNAArtificialfusion DNA (ubiquitin promoter/omega 3 desaturase/ubiquitin terminator/ubiquitin promoter/Blar/SV40 terminator) 178tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780acactagcca acatgactga ggataagacg aaggtcgagt tcccgacgct cacggagctc 840aagcactcga tcccgaacgc gtgctttgag tcgaacctcg gcctctcgct ctactacacg 900gcccgcgcga tcttcaacgc gtcggcctcg gcggcgctgc tctacgcggc gcgctcgacg 960ccgttcattg ccgataacgt tctgctccac gcgctcgttt gcgccaccta catctacgtg 1020cagggcgtca tcttctgggg cttcttcacg gtcggccacg actgcggcca ctcggccttc 1080tcgcgctacc acagcgtcaa ctttatcatc ggctgcatca tgcactctgc gattttgacg 1140ccgttcgaga gctggcgcgt gacgcaccgc caccaccaca agaacacggg caacattgat 1200aaggacgaga tcttttaccc gcaccggtcg gtcaaggacc tccaggacgt gcgccaatgg 1260gtctacacgc tcggcggtgc gtggtttgtc tacttgaagg tcgggtatgc cccgcgcacg 1320atgagccact ttgacccgtg ggacccgctc ctccttcgcc gcgcgtcggc cgtcatcgtg 1380tcgctcggcg tctgggccgc cttcttcgcc gcgtacgcgt acctcacata ctcgctcggc 1440tttgccgtca tgggcctcta ctactatgcg ccgctctttg tctttgcttc gttcctcgtc 1500attacgacct tcttgcacca caacgacgaa gcgacgccgt ggtacggcga ctcggagtgg 1560acgtacgtca agggcaacct ctcgagcgtc gaccgctcgt acggcgcgtt cgtggacaac 1620ctgagccacc acattggcac gcaccaggtc caccacttgt tcccgatcat tccgcactac 1680aagctcaacg aagccaccaa gcactttgcg gccgcgtacc cgcacctcgt gcgcaagaac 1740gacgagccca tcatctcggc cttcttcaag accgcgcacc tctttgtcaa ctacggcgct 1800gtgcccgaga cggcgcagat cttcacgctc aaagagtcgg ccgcggccgc caaggccaag 1860tcggactaaa ctaagctatc tgtagtatgt gctatactcg aatcatgctg ccctgtacgt 1920acctacctat atctgattga gcgtgctgcg tcgaccatag acgcgggaac gcgggccagc 1980ctaccacgtt gccgccgccg gtatccacgg gcacgccaaa gcattggtcg ataacgctct 2040gcccagggct tcctggcgag gacccgaggc

caacatgcat gcatgtgcta tcagcggtca 2100tcatcgccct catcagcgcg catcggcgag ctcgcgcacg aacggcaagc gcccaactca 2160actcacttac tcacactatg gtcttgccgt tggcggttgc ttagctaatg cgtgacgtca 2220ctctgcctcc aacatcgcga ggcagagtcg cgagcagtgc agaggccacg gcggacgcca 2280acaaagcgcc aaccagcgca acgcaccagc gggtctgtgg gcgtagctcg agcgggcgtc 2340ttcaagagcc gccgtggagc cgacgcccct gcgaagggct cgagtgcaag cggggccgtt 2400gagccgcgtg gtaggaacaa ctgcagtctc cctatagtga gtcgtattac gcggtggtac 2460cgccgcagcg cctggtgcac ccgccgggcg ttgttgtgtg ctcttcttgc ctccgagaga 2520gagagcggag cggatgcata ggaaatcggg ccacgcggga gggccatgcg ttcgccccac 2580acgccacttt ccacgcccgc tctctctccg gccggcaggc agcgcataac tctccgacgc 2640tggcaggctg gtagcaactg gcagggacaa ctcgcgcgcg ggtcccggtc gttcgatgtg 2700ccaacccgag agaatccagc cagcagggcg gttggcctca tcgcccacct gctatggtgc 2760agcgaaccaa ctcccgaagc ggccggttct gcgattccct cttctgaatt ctgaattctg 2820aactgattcc ggaggagaac cctctggaag cgcgggttgc ctctccagtt ctgccgaact 2880agacagggga gtgagcagag agtgaccctg acgcggagcg agctggttgc tggaaaagtc 2940gcgaacgctg ggctgtgtca cgcgtccact tcgggcagtc cccaaacgac aagcagaaca 3000agcaacacca gcagcagcaa gcgacctaag caacactagc caacatggcc aagcctttgt 3060ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct 3120ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg 3180tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg 3240ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca 3300tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag 3360ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct 3420ctggttatgt gtgggagggc taagatccgc gaaatgaccg accaagcgac gcccaacctg 3480ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 3540ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 3600caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 3660ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 3720gtatcttatc atgtctgtat accgtcgacc tctagctaga tctcacatta attgcgt 377717923DNAArtificialprimermisc_feature(15)..(15)n is inosine.misc_feature(21)..(21)n is inosine. 179athgartwyt kbrtnttygt nca 2318023DNAArtificialprimermisc_feature(6)..(6)n is inosine.misc_feature(15)..(15)n is inosine.misc_feature(18)..(18)n is inosine. 180tartrnswrt acatnadnam rtg 2318128DNAArtificialprimer 181ctgacaaagt ttctcgactg gagcgaca 2818224DNAArtificialprimer 182tacgcggcgg tgcccgagcc ccag 2418328DNAArtificialprimer 183tgccgatcgt tgcgtggtgg aacacctg 2818420DNAArtificialprimer 184atggcgacgc gcacctcgaa 2018522DNAArtificialprimer 185ttactcggac ttggtggggg cg 22186951DNAThraustochytrium aureumgenomic C20 elongase 186atggcgacgc gcacctcgaa gagcgctccg gcggtttcca agtcggccaa ggttgccgcg 60ccggcgaaga agcggtcggt cgacaggagc gacggtttct tccgcacgtt caacctgtgc 120gccctgtacg ggtctgccct cgcctatgcg tacaagcacg gcccggtgga caatgacggc 180caggggctgt actttcacaa gtcgcccatg tacgcgttcg ccgtgtcgga cgtcatgacc 240ttcggcgcgc cgctgatgta cgtgctcggt gtgatgctgc tcagcaggta catggcggac 300aaaaagcccc tgactggctt catcaagacc tacatccagc ccgtctacaa cgtggtccaa 360atcgcggtgt gcggctggat ggtgtggggc ctctggccgc aggtcgacct ggccaacggc 420aaccctttcg gcctcaacaa gtcgcgcgac tcgaacatcg agtttttcgt gttcgtgcac 480ctcctgacaa agtttctcga ctggagcgac acgttcatga tgatcctcaa gaaaaactac 540gcccaggtta gctttctgca ggtgttccac cacgcaacga tcggcatggt gtggtcgttc 600cttcttcagc gtggctgggg ctcgggcacc gccgcgtacg gtgctttcat caactcggtc 660acgcacgtga tcatgtactc gcactacttt gccacctcgc tcaacatcaa caacccgttc 720aagcggtaca tcacgagctt ccagctcgcc cagtttgcaa gctgcatcgt gcatgcccta 780ctggtgcttg ccttcgagga ggtgtacccg ctcgagtacg cttacctgca gatcagctac 840cacatcatca tgctctacct gttcggacgc cgcatgaact ggagccccga gtggtgcacc 900ggtgagatcg acggccttga cgccccaagc gcccccacca agtccgagta a 951187317PRTThraustochytrium aureummisc_feature(317)..(317)Xaa can be any naturally occurring amino acidC20 elongase 187Met Ala Thr Arg Thr Ser Lys Ser Ala Pro Ala Val Ser Lys Ser Ala1 5 10 15Lys Val Ala Ala Pro Ala Lys Lys Arg Ser Val Asp Arg Ser Asp Gly 20 25 30Phe Phe Arg Thr Phe Asn Leu Cys Ala Leu Tyr Gly Ser Ala Leu Ala 35 40 45Tyr Ala Tyr Lys His Gly Pro Val Asp Asn Asp Gly Gln Gly Leu Tyr 50 55 60Phe His Lys Ser Pro Met Tyr Ala Phe Ala Val Ser Asp Val Met Thr65 70 75 80Phe Gly Ala Pro Leu Met Tyr Val Leu Gly Val Met Leu Leu Ser Arg 85 90 95Tyr Met Ala Asp Lys Lys Pro Leu Thr Gly Phe Ile Lys Thr Tyr Ile 100 105 110Gln Pro Val Tyr Asn Val Val Gln Ile Ala Val Cys Gly Trp Met Val 115 120 125Trp Gly Leu Trp Pro Gln Val Asp Leu Ala Asn Gly Asn Pro Phe Gly 130 135 140Leu Asn Lys Ser Arg Asp Ser Asn Ile Glu Phe Phe Val Phe Val His145 150 155 160Leu Leu Thr Lys Phe Leu Asp Trp Ser Asp Thr Phe Met Met Ile Leu 165 170 175Lys Lys Asn Tyr Ala Gln Val Ser Phe Leu Gln Val Phe His His Ala 180 185 190Thr Ile Gly Met Val Trp Ser Phe Leu Leu Gln Arg Gly Trp Gly Ser 195 200 205Gly Thr Ala Ala Tyr Gly Ala Phe Ile Asn Ser Val Thr His Val Ile 210 215 220Met Tyr Ser His Tyr Phe Ala Thr Ser Leu Asn Ile Asn Asn Pro Phe225 230 235 240Lys Arg Tyr Ile Thr Ser Phe Gln Leu Ala Gln Phe Ala Ser Cys Ile 245 250 255Val His Ala Leu Leu Val Leu Ala Phe Glu Glu Val Tyr Pro Leu Glu 260 265 270Tyr Ala Tyr Leu Gln Ile Ser Tyr His Ile Ile Met Leu Tyr Leu Phe 275 280 285Gly Arg Arg Met Asn Trp Ser Pro Glu Trp Cys Thr Gly Glu Ile Asp 290 295 300Gly Leu Asp Ala Pro Ser Ala Pro Thr Lys Ser Glu Xaa305 310 3151883193DNAArtificialgenomic DNA (Thraustochytrium aureum genomic DNA contains C20 elongase coding region) 188ggatatcccc cgcgaggcga tggctgctcc gacgacgtgg gctggcgacg tcgctcgcaa 60aggcgttccg caaccgcgcg ttccgctgta acgagaccgt tttccctgcg ctgctgggtg 120gacctagcgc gtgtgtcacc tgccggcccc cgttgcgtgc aaccgaattg atcgataata 180gaattacata acaaacaact tgctggatga gtacaagacc agcgtagtgt ggctgtggga 240cgttgaacgg agcgggtcct gtgacggcgc agaaaggaac tccgcccgag gtgaaacccc 300gatgcgcagg actctgcggc cacagcccct ccgccagtat tccactaaaa atccgccccc 360tttgacaaag atcgcaaccc cgtcccatca actcctcaca ataggctttc cactggcgga 420aacgtccccg gcacaggagt gcctcccgcg gttctgcgca tacggctgac cactacgcag 480cgcgatatcc tccatccgcg tatatatccg taaacaacgg aacattctcc ctctcaacga 540ggcgtggttt tcgaagccat gcctttcttc cttcctactt gccttccttc tttctttctt 600tctttctttc ttttgcaagc gtgcgcgaac ttgaaggtac tacttacact tgacagagag 660agatagagac ggcaattcga ccaagtactt tccacgattt tttttttttt gttttggtcg 720ctttcgttgg tcgtgcatga tggatggccg ggatttttac aattggatgc gccaggctgc 780cacgcatgcc gtgacgctcg ctcgcggcga ctcatgatgc ttgccagtgg cagtgcatcc 840agctcttccc tctgctcgtc gtgtactcac tggcgatgct ctcggcgctc gttcaggggc 900catcgaccga tcgatcgatc gatcgatcga tcaatcacgt tcggtggact cggcagaccc 960cgaacgtgtt tctcccagga cgtgccgctg tcgctcgctg atccacccga agcgcggtcg 1020gctggcacgg tcgctcggct ggaagttgag tagtttgctt tctgttgctg cgctgctttg 1080taaacgcgac catggcgacg cgcacctcga agagcgctcc ggcggtttcc aagtcggcca 1140aggttgccgc gccggcgaag aagcggtcgg tcgacaggag cgacggtttc ttccgcacgt 1200tcaacctgtg cgccctgtac gggtctgccc tcgcctatgc gtacaagcac ggcccggtgg 1260acaatgacgg ccaggggctg tactttcaca agtcgcccat gtacgcgttc gccgtgtcgg 1320acgtcatgac cttcggcgcg ccgctgatgt acgtgctcgg tgtgatgctg ctcagcaggt 1380acatggcgga caaaaagccc ctgactggct tcatcaagac ctacatccag cccgtctaca 1440acgtggtcca aatcgcggtg tgcggctgga tggtgtgggg cctctggccg caggtcgacc 1500tggccaacgg caaccctttc ggcctcaaca agtcgcgcga ctcgaacatc gagtttttcg 1560tgttcgtgca cctcctgaca aagtttctcg actggagcga cacgttcatg atgatcctca 1620agaaaaacta cgcccaggtt agctttctgc aggtgttcca ccacgcaacg atcggcatgg 1680tgtggtcgtt ccttcttcag cgtggctggg gctcgggcac cgccgcgtac ggtgctttca 1740tcaactcggt cacgcacgtg atcatgtact cgcactactt tgccacctcg ctcaacatca 1800acaacccgtt caagcggtac atcacgagct tccagctcgc ccagtttgca agctgcatcg 1860tgcatgccct actggtgctt gccttcgagg aggtgtaccc gctcgagtac gcttacctgc 1920agatcagcta ccacatcatc atgctctacc tgttcggacg ccgcatgaac tggagccccg 1980agtggtgcac cggtgagatc gacggccttg acgccccaag cgcccccacc aagtccgagt 2040aaacctgttt ccggctggct cccgagccat gcttaccatg aatgaacctg caaacagtct 2100gaggtccttg tgcaaaccgc tcagtgggac gtcgacgaag aaagaaacaa tgtgtactcg 2160tcttgctctg ctcccgcgcc gttttttatc gttgttgaga cctctcgcgc agttttggga 2220atcaaccaaa acaagagccc ggcgtcagcg tttgcttcgc cctcggctgc actcgctcgg 2280cacgcaggta taactgggtg agtaccaagc cccgcatttg tctgtccgcg atccgcgcac 2340gctgcgggtc aggacgacat cgcgctgcac gtcacagtgg gtcccttttg acgtggctgc 2400ggcgatgagg aggcttggct cggcttcatg gcaaggcaac agactcgctt ccaggacgcg 2460cacgacgagc agcgctgctt tgatcgacct tgcctgcgtc accgcctcgg ctgctttgat 2520cgatcgttgt caccggccga gtgaccgcga acgcattgcc cgcacggctc ggctcggctc 2580ggaccggacc ggctcgcctt ggcggcgcgg cgcgatggcg acccagacgc gaccggagcc 2640gcgcgcggag gacaaggcca tgtacatctt cgggctcggg tacgttggga gcaggctcgc 2700caaccagctg gcggaacagg ggtggcgcgt cgcggggtcg gtgagggagc tcgggcgcga 2760ggacgacttt gccgagttcg aaaagtccaa gctgagcggc aaggtgcagg tgttccaact 2820cccgcttgag ggcgaggaca acacgcccgc tcgcgcgcgg gagatactta gcgggtacca 2880gcgcctgctg ttcacggcgc cagtggaccg cgcccggaac tgtgacccct tcttgggcga 2940ccccgttctc ggccccgtga tcgtcgagct agcagaggag ggccgcatcg actgggccgg 3000ctatctctca accacttcgg tctacggcaa ccacgacggc gagtgggtgg acgagaccac 3060gccgctcatg cccacgctca aacgcggcga gcagcgcgtc atggtggagc gcgccttcct 3120gtacgagtcg ggcctcccgg cccatatctt tcggctgcca ggaatctacg gcccagggcg 3180cggcccgata tca 319318927DNAArtificialprimer 189gacaaagatc tcgactggag cgaccac 2719027DNAArtificialprimer 190gtcgagatct tttgtcagga ggtgcac 27191951DNAArtificialBglII inserted C20 elongase 191atggcgacgc gcacctcgaa gagcgctccg gcggtttcca agtcggccaa ggttgccgcg 60ccggcgaaga agcggtcggt cgacaggagc gacggtttct tccgcacgtt caacctgtgc 120gccctgtacg ggtctgccct cgcctatgcg tacaagcacg gcccggtgga caatgacggc 180caggggctgt actttcacaa gtcgcccatg tacgcgttcg ccgtgtcgga cgtcatgacc 240ttcggcgcgc cgctgatgta cgtgctcggt gtgatgctgc tcagcaggta catggcggac 300aaaaagcccc tgactggctt catcaagacc tacatccagc ccgtctacaa cgtggtccaa 360atcgcggtgt gcggctggat ggtgtggggc ctctggccgc aggtcgacct ggccaacggc 420aaccctttcg gcctcaacaa gtcgcgcgac tcgaacatcg agtttttcgt gttcgtgcac 480ctcctgacaa agatctcgac tggagcgaca cgttcatgat gatcctcaag aaaaactacg 540cccaggttag ctttctgcag gtgttccacc acgcaacgat cggcatggtg tggtcgttcc 600ttcttcagcg tggctggggc tcgggcaccg ccgcgtacgg tgctttcatc aactcggtca 660cgcacgtgat catgtactcg cactactttg ccacctcgct caacatcaac aacccgttca 720agcggtacat cacgagcttc cagctcgccc agtttgcaag ctgcatcgtg catgccctac 780tggtgcttgc cttcgaggag gtgtacccgc tcgagtacgc ttacctgcag atcagctacc 840acatcatcat gctctacctg ttcggacgcc gcatgaactg gagccccgag tggtgcaccg 900gtgagatcga cggccttgac gccccaagcg cccccaccaa gtccgagtaa a 95119223DNAArtificialprimer 192atggcgacgc gcacctcgaa gag 2319323DNAArtificialprimer 193ttactcggac ttgctggggg cgc 231942655DNAArtificialfusion DNA(Thraustochytrium aureum C20 elongase 5' region/SV40 terminator/Neor/ubiquitin promoter/ Thraustochytrium aureum C20 elongase 3' region) 194atggcgacgc gcacctcgaa gagcgctccg gcggtttcca agtcggccaa ggttgccgcg 60ccggcgaaga agcggtcggt cgacaggagc gacggtttct tccgcacgtt caacctgtgc 120gccctgtacg ggtctgccct cgcctatgcg tacaagcacg gcccggtgga caatgacggc 180caggggctgt actttcacaa gtcgcccatg tacgcgttcg ccgtgtcgga cgtcatgacc 240ttcggcgcgc cgctgatgta cgtgctcggt gtgatgctgc tcagcaggta catggcggac 300aaaaagcccc tgactggctt catcaagacc tacatccagc ccgtctacaa cgtggtccaa 360atcgcggtgt gcggctggat ggtgtggggc ctctggccgc aggtcgacct ggccaacggc 420aaccctttcg gcctcaacaa gtcgcgcgac tcgaacatcg agtttttcgt gttcgtgcac 480ctcctgacaa agatctagct agaggtcgac ggtatacaga catgataaga tacattgatg 540agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg 600atgctattgc tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag 660aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt 720ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg 780ggcgtcgctt ggtcggtcat ttcgcggatc tcaaaagaac tcgtccagga ggcggtagaa 840cgcaatcctc tggctgtccg gggcggcgat gccgtagagc acgagaaagc ggtcggccca 900ctcgccgcca agctcctcgg cgatgtcccg cgtggcgagc gcgatgtctt ggtagcggtc 960cgccacgccc aggcgcccgc agtcgataaa gcccgagaag cggccgttct cgaccatgat 1020gttggggagg caggcgtcgc cgtgcgtgac cacgaggtcc tcgccgtccg gcatcctagc 1080cttaagcctg gcgaacagtt ccgccggcgc gaggccctgg tgctcctcgt cgaggtcgtc 1140ttggtcgacg aggccagcct ccatccgcgt gcgggcgcgt tcgatcctgt gcttcgcctg 1200gtggtcgaag gggcaggtgg cggggtcgag ggtgtgcagg cggcgcatgg cgtcggccat 1260gatggacacc ttctcagcgg gcgcgaggtg gctgctgagg aggtcctggc cgggcacttc 1320cccgaggagc agccagtcgc ggccggcttc ggtgacgacg tcgagcacag cggcgcacgg 1380aacccccgtc gtggcaagcc agctgaggcg ggcagcttcg tcctggagct cgttgagggc 1440gccgctaagg tcggtcttga caaacaggac cggccggccc tgcgcgctaa ggcggaacac 1500ggccgcgtcc gagcagccga tcgtctgctg agcccagtcg tagccgaaca gccgttccac 1560ccaagcagcg ggcgagccag cgtgaaggcc gtcctgttca atcatgttgg ctagtgttgc 1620ttaggtcgct tgctgctgct ggtgttgctt gttctgcttg tcgtttgggg tctgcccgaa 1680gtggacgcgt gacacagccc agcgttcgcg acttttccag caaccagctc gctccgcgtc 1740agggtcactc tctgctcact cccctgtcta gttcggcaga actggagagg caacccgcgc 1800ttccagaggg ttctcctccg gaatcagttc agaattcaga attcagaaga gggaatcgca 1860gaaccggccg cttcgggagt tggttcgctg caccatagca ggtgggcgat gaggccaacc 1920gccctgctgg ctggattctc tcgggttggc acatcgaacg accgggaccc gcgcgcgagt 1980tgtccctgcc agttgctacc agcctgccag cgtcggagag ttatgcgctg cctgccggcc 2040ggagagagag cgggcgtgga aagtggcgtg tggggcgaac gcatggccct cccgcgtggc 2100ccgatttcct atgcatccgc tccgctctct ctctcggagg caagaagagc acaccaacaa 2160cgcccggcgg gtgcaccagg cgctgcggca gatccagatc tcgactggag cgacacgttc 2220atgatgatcc tcaagaaaaa ctacgcccag gttagctttc tgcaggtgtt ccaccacgca 2280acgatcggca tggtgtggtc gttccttctt cagcgtggct ggggctcggg caccgccgcg 2340tacggtgctt tcatcaactc ggtcacgcac gtgatcatgt actcgcacta ctttgccacc 2400tcgctcaaca tcaacaaccc gttcaagtgg tacatcacga gcttccagct cgcccagttt 2460gcaagctgca tcgtgcatgc cctactggtg cttgccttcg aggaggtgta cccgctcgag 2520tacgcttacc tgcagatcag ctaccacatc atcatgctct acctgttcgg acgccgcatg 2580aactggagcc ccgagtggtg caccggtgag atcgacggcc ttgacgcccc aagcgccccc 2640accaagtccg agtaa 26551952886DNAArtificialfusion DNA(Thraustochytrium aureum C20 elongase 5' region/ubiquitin promoter/Hygr/SV40 terminator/Thraustochytrium aureum C20 elongase 3' region) 195atggcgacgc gcacctcgaa gagcgctccg gcggtttcca agtcggccaa ggttgccgcg 60ccggcgaaga agcggtcggt cgacaggagc gacggtttct tccgcacgtt caacctgtgc 120gccctgtacg ggtctgccct cgcctatgcg tacaagcacg gcccggtgga caatgacggc 180caggggctgt actttcacaa gtcgcccatg tacgcgttcg ccgtgtcgga cgtcatgacc 240ttcggcgcgc cgctgatgta cgtgctcggt gtgatgctgc tcagcaggta catggcggac 300aaaaagcccc tgactggctt catcaagacc tacatccagc ccgtctacaa cgtggtccaa 360atcgcggtgt gcggctggat ggtgtggggc ctctggccgc aggtcgacct ggccaacggc 420aaccctttcg gcctcaacaa gtcgcgcgac tcgaacatcg agtttttcgt gttcgtgcac 480ctcctgacaa agatctggat ctgccgcagc gcctggtgca cccgccgggc gttgttgtgt 540gctcttcttg cctccgagag agagagcgga gcggatgcat aggaaatcgg gccacgcggg 600agggccatgc gttcgcccca cacgccactt tccacgcccg ctctctctcc ggccggcagg 660cagcgcataa ctctccgacg ctggcaggct ggtagcaact ggcagggaca actcgcgcgc 720gggtcccggt cgttcgatgt gccaacccga gagaatccag ccagcagggc ggttggcctc 780atcgcccacc tgctatggtg cagcgaacca actcccgaag cggccggttc tgcgattccc 840tcttctgaat tctgaattct gaactgattc cggaggagaa ccctctggaa gcgcgggttg 900cctctccagt tctgccgaac tagacagggg agtgagcaga gagtgaccct gacgcggagc 960gagctggttg ctggaaaagt cgcgaacgct gggctgtgtc acgcgtccac ttcgggcaga 1020ccccaaacga caagcagaac aagcaacacc agcagcagca agcgacctaa gcaacactag 1080ccaacatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 1140tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 1200tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 1260aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 1320acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 1380cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 1440tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 1500aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 1560tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 1620atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 1680tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 1740aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg

aggccgtggt 1800tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 1860cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 1920ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 1980ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 2040atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 2100caaaggaata gagatccgcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 2160tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 2220cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt 2280gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 2340agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 2400tgtctgtata ccgtcgacct cyagctagat ctcgactgga gcgacacgtt catgatgatc 2460ctcaagaaaa actacgccca ggttagcttt ctgcaggtgt tccaccacgc aacgatcggc 2520atggtgtggt cgttccttct tcagcgtggc tggggctcgg gcaccgccgc gtacggtgct 2580ttcatcaact cggtcacgca cgtgatcatg tactcgcact actttgccac ctcgctcaac 2640atcaacaacc cgttcaagtg gtacatcacg agcttccagc tcgcccagtt tgcaagctgc 2700atcgtgcatg ccctactggt gcttgccttc gaggaggtgt acccgctcga gtacgcttac 2760ctgcagatca gctaccacat catcatgctc tacctgttcg gacgccgcat gaactggagc 2820cccgagtggt gcaccggtga gatcgacggc cttgacgccc caagcgcccc caccaagtcc 2880gagtaa 288619626DNAArtificialprimer 196gctcggctgg aagttgagta gtttgc 2619724DNAArtificialprimer 197tctttcttcg tcgacgtccc actg 2419824DNAArtificialprimer 198atgattgaac aggacggcct tcac 2419924DNAArtificialprimer 199tcaaaagaac tcgtccagga ggcg 2420024DNAArtificialprimer 200atgaaaaagc ctgaactcac cgcg 2420125DNAArtificialprimer 201ctattccttt gccctcggac gagtg 2520223DNAArtificialprimer 202ggcggagcga agtgtgaaag tta 2320324DNAArtificialprimer 203gcgacagcat cttgaaatag gcag 242042571DNAThraustochytrium aureumGenomic delta 4 desaturase upstream/T. aureum delta 4 desaturase 204ggcggagcga agtgtgaaag ttacaaccca gttactgccc attcccggga aaagttgcgc 60agctcacgcg gttcgctttt ctggtggcct ggcgacgttc gccgcttgcc ggatactccc 120tcgtgccccc gcgccaggtt tgcccgctgt cgctcgagga gtggactcgc gagtcgcgac 180agcagcagca ccaaggggga tggatcctcg ttgacagcac caagatgctc tctgcctttc 240aggtgaaatc gatcgatcaa ttgatcaatc aagatcattg gaagcaaatg ggaagcaaat 300gcgaaggggg aagaccctcg gtctctgctc gggaacccga cacgaggctg agggcgcgct 360tctacaggtt gtgcagcggc cgcactgcga gcttgcgccg ggccaaggcg ctcgccagaa 420ttgctgcgtc tgccgcctcg ggatcagcca ctcggttttt cgtcatcagg gtccaccttc 480aacctggaag tggactcggc aagtcggcag atccactccg gaattccaag atccccggtc 540gatcggtgct ggtgcgaatt aggatggacc caggctatgt gagagtcgga gggtggcggt 600tgtctccacc gtgacagcgc gcgtgtggtg agtaacgcga agcgcgtggt ggagaaatgg 660ggggagattc gtaggacgcg atgcgctcgt cactgagggt gcgccggtga cgaagcttcg 720gacccagatt ccgtcggtat ggctcgtgtt cgcacacctt caggaacccg catgacgaga 780ccactggagt tttcaacgtc acgaagccgc tctgtgtgac gagaattggc ttgcgagtga 840cgtgaggcgc cgagcatgtc gttggtttgc ctcttcacaa cagaatcaga cgactgggag 900gctgcacgag gctaaggcca agggcactca ctgactcgga cgtgaagcag aagcagaagc 960agagcgctcg acggcacgtg gcggcagacc ggcttcggga cgggcaggag acgcaaggcg 1020cgcaacacta gggggctgga cgtggaccac tggctaagga gcgctggaaa gatgacggtc 1080gggtttgacg aaacggtgac tatggacacg gtccgcaacc acaacatgcc ggacgacgcc 1140tggtgcgcga tccacggcac cgtgtacgac atcaccaagt tcagcaaggt gcaccccggc 1200ggggacatca tcatgctggc cgctggcaag gaggccacca tcctgttcga gacctaccac 1260atcaagggcg tcccggacgc ggtgctgcgc aagtacaagg tcggcaagct cccccagggc 1320aagaagggcg aaacgagcca cgtgcccacc gggctcgact cggcctccta ctactcgtgg 1380gacagcgagt tttacagggt gctccgcgag cgcgtcgcca agaagctggc cgagcccggc 1440ctcatgcagc gcgcgcgcat ggagctctgg gccaaggcga tcttcctcct ggcaggtttc 1500tggggctccc tttacgccat gtgcgtgcta gacccgcacg gcggtgccat ggtagccgcc 1560gttacgctcg gcgtgttcgc tgcctttgtc ggaacttgca tccagcacga cggcagccac 1620ggcgccttct ccaagtcgcg attcatgaac aaggcggcgg gctggaccct cgacatgatc 1680ggcgcgagcg cgatgacctg ggagatgcag cacgttcttg gccaccaccc gtacaccaac 1740ctcatcgaga tggagaacgg tttggccaag gtcaagggcg ccgacgtcga cccgaagaag 1800gtcgaccagg agagcgaccc ggacgtcttc agtacgtacc cgatgcttcg cctgcacccg 1860tggcaccgcc agcggtttta ccacaagttc cagcacctgt acgccccgtt tatctttggg 1920tttatgacga ttaacaaggt gatttcccag gatgtcgggg ttgtgctgcg caagcgcctg 1980ttccagatcg acgccaactg ccggtatggc agcccctggt acgtggcccg cttctggatc 2040atgaagctcc tcaccacgct ctacatggtg gcgcttccca tgtacatgca ggggcctgct 2100cagggcttga agcttttctt catggcccac ttcacctgcg gagaggtcct cgccaccatg 2160tttattgtca accacatcat cgagggcgtc agctacgctt ccaaggacgc ggtcaagggc 2220gtcatggctc cgccgcgcac tgtgcacggt gtcaccccga tgcaggtgac gcaaaaggcg 2280ctcagtgcgg ccgagtcgac caagtcggac gccgacaaga cgaccatgat ccccctcaac 2340gactgggccg ctgtgcagtg ccagacctct gtgaactggg ctgtcgggtc gtggttttgg 2400aaccactttt cgggcggcct caaccaccag attgagcacc actgcttccc caaaaccccc 2460acacggtcaa cgtctacatc tcgggcatcg tcaaggagac ctgcgaagaa tacggcgtgc 2520cgtaccaggc tgagatcagc ctcttctctg cctatttcaa gatgctgtcg c 2571205616DNAThraustochytrium aureumgenomic delta 4 desaturase upstream/T. aureum delta 4 desaturase 205cgcaaggcgc gcaacactag ggggctggac gtggaccact ggctaaggag cgctggaaag 60atgacggtcg ggtttgacga aacggtgact atggacacgg tccgcaacca caacatgccg 120gacgacgcct ggtgcgcgat ccacggcacc gtgtacgaca tcaccaagtt cagcaaggtg 180caccccggcg gggacatcat catgctggcc gctggcaagg aggccaccat cctgttcgag 240acctaccaca tcaagggcgt cccggacgcg gtgctgcgca agtacaaggt cggcaagctc 300ccccagggca agaagggcga aacgagccac gtgcccaccg ggctcgactc ggcctcctac 360tactcgtggg acagcgagtt ttacagggtg ctccgcgagc gcgtcgccaa gaagctggcc 420gagcccggcc tcatgcagcg cgcgcgcatg gagctctggg ccaaggcgat cttcctcctg 480gcaggtttct ggggctccct ttacgccatg tgcgtgctag acccgcacgg cggtgccatg 540gtagccgccg ttacgctcgg cgtgttcgct gcctttgtcg gaacttgcat ccagcacgac 600ggcagccacg gcgcct 61620625DNAArtificialprimer 206caggagatct ccaagtcgcg attca 2520726DNAArtificialprimer 207cttggagatc tcctgcccgt cccgaa 262083264DNAArtificialfusion DNA (T. aureum delta 4 desaturase upstream/SV40 terminator /BlaR/ubiquitin promoter/T. aureum delta 4 desaturase) 208ggcggagcga agtgtgaaag ttacaaccca gttactgccc attcccggga aaagttgcgc 60agctcacgcg gttcgctttt ctggtggcct ggcgacgttc gccgcttgcc ggatactccc 120tcgtgccccc gcgccaggtt tgcccgctgt cgctcgagga gtggactcgc gagtcgcgac 180agcagcagca ccaaggggga tggatcctcg ttgacagcac caagatgctc tctgcctttc 240aggtgaaatc gatcgatcaa ttgatcaatc aagatcattg gaagcaaatg ggaagcaaat 300gcgaaggggg aagaccctcg gtctctgctc gggaacccga cacgaggctg agggcgcgct 360tctacaggtt gtgcagcggc cgcactgcga gcttgcgccg ggccaaggcg ctcgccagaa 420ttgctgcgtc tgccgcctcg ggatcagcca ctcggttttt cgtcatcagg gtccaccttc 480aacctggaag tggactcggc aagtcggcag atccactccg gaattccaag atccccggtc 540gatcggtgct ggtgcgaatt aggatggacc caggctatgt gagagtcgga gggtggcggt 600tgtctccacc gtgacagcgc gcgtgtggtg agtaacgcga agcgcgtggt ggagaaatgg 660ggggagattc gtaggacgcg atgcgctcgt cactgagggt gcgccggtga cgaagcttcg 720gacccagatt ccgtcggtat ggctcgtgtt cgcacacctt caggaacccg catgacgaga 780ccactggagt tttcaacgtc acgaagccgc tctgtgtgac gagaattggc ttgcgagtga 840cgtgaggcgc cgagcatgtc gttggtttgc ctcttcacaa cagaatcaga cgactgggag 900gctgcacgag gctaaggcca agggcactca ctgactcgga cgtgaagcag aagcagaagc 960agagcgctcg acggcacgtg gcggcagacc ggcttcggga cgggcaggag atctagctag 1020aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag 1080aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 1140cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc 1200gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga 1260aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt 1320cgcggatctt agccctccca cacataacca gagggcagca attcacgaat cccaactgcc 1380gtcggctgtc catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc 1440acctgtcggc accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg 1500atacaagtca ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca 1560caaggtcccc cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag 1620agagctgcgc tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt 1680gctctttcaa tgagggtgga ttcttcttga gacaaaggct tggccatgtt ggctagtgtt 1740gcttaggtcg cttgctgctg ctggtgttgc ttgttctgct tgtcgtttgg ggactgcccg 1800aagtggacgc gtgacacagc ccagcgttcg cgacttttcc agcaaccagc tcgctccgcg 1860tcagggtcac tctctgctca ctcccctgtc tagttcggca gaactggaga ggcaacccgc 1920gcttccagag ggttctcctc cggaatcagt tcagaattca gaattcagaa gagggaatcg 1980cagaaccggc cgcttcggga gttggttcgc tgcaccatag caggtgggcg atgaggccaa 2040ccgccctgct ggctggattc tctcgggttg gcacatcgaa cgaccgggac ccgcgcgcga 2100gttgtccctg ccagttgcta ccagcctgcc agcgtcggag agttatgcgc tgcctgccgg 2160ccggagagag agcgggcgtg gaaagtggcg tgtggggcga acgcatggcc ctcccgcgtg 2220gcccgatttc ctatgcatcc gctccgctct ctctctcgga ggcaagaaga gcacaccaac 2280aacgcccggc gggtgcacca ggcgctgcgg cagatccaga tctccaagtc gcgattcatg 2340aacaaggcgg cgggctggac cctcgacatg atcggcgcga gcgcgatgac ctgggagatg 2400cagcacgttc ttggccacca cccgtacacc aacctcatcg agatggagaa cggtttggcc 2460aaggtcaagg gcgccgacgt cgacccgaag aaggtcgacc aggagagcga cccggacgtc 2520ttcagtacgt acccgatgct tcgcctgcac ccgtggcacc gccagcggtt ttaccacaag 2580ttccagcacc tgtacgcccc gtttatcttt gggtttatga cgattaacaa ggtgatttcc 2640caggatgtcg gggttgtgct gcgcaagcgc ctgttccaga tcgacgccaa ctgccggtat 2700ggcagcccct ggtacgtggc ccgcttctgg atcatgaagc tcctcaccac gctctacatg 2760gtggcgcttc ccatgtacat gcaggggcct gctcagggct tgaagctttt cttcatggcc 2820cacttcacct gcggagaggt cctcgccacc atgtttattg tcaaccacat catcgagggc 2880gtcagctacg cttccaagga cgcggtcaag ggcgtcatgg ctccgccgcg cactgtgcac 2940ggtgtcaccc cgatgcaggt gacgcaaaag gcgctcagtg cggccgagtc gaccaagtcg 3000gacgccgaca agacgaccat gatccccctc aacgactggg ccgctgtgca gtgccagacc 3060tctgtgaact gggctgtcgg gtcgtggttt tggaaccact tttcgggcgg cctcaaccac 3120cagattgagc accactgctt ccccaaaacc cccacacggt caacgtctac atctcgggca 3180tcgtcaagga gacctgcgaa gaatacggcg tgccgtacca ggctgagatc agcctcttct 3240ctgcctattt caagatgctg tcgc 32642093935DNAArtificialfusion DNA (T. aureum delta 4 desaturase upstream/SV40 terminator/ZeoR/Enhanced GFP/ubiquitin promoter/T. aureum delta 4 desaturase) 209ggcggagcga agtgtgaaag ttacaaccca gttactgccc attcccggga aaagttgcgc 60agctcacgcg gttcgctttt ctggtggcct ggcgacgttc gccgcttgcc ggatactccc 120tcgtgccccc gcgccaggtt tgcccgctgt cgctcgagga gtggactcgc gagtcgcgac 180agcagcagca ccaaggggga tggatcctcg ttgacagcac caagatgctc tctgcctttc 240aggtgaaatc gatcgatcaa ttgatcaatc aagatcattg gaagcaaatg ggaagcaaat 300gcgaaggggg aagaccctcg gtctctgctc gggaacccga cacgaggctg agggcgcgct 360tctacaggtt gtgcagcggc cgcactgcga gcttgcgccg ggccaaggcg ctcgccagaa 420ttgctgcgtc tgccgcctcg ggatcagcca ctcggttttt cgtcatcagg gtccaccttc 480aacctggaag tggactcggc aagtcggcag atccactccg gaattccaag atccccggtc 540gatcggtgct ggtgcgaatt aggatggacc caggctatgt gagagtcgga gggtggcggt 600tgtctccacc gtgacagcgc gcgtgtggtg agtaacgcga agcgcgtggt ggagaaatgg 660ggggagattc gtaggacgcg atgcgctcgt cactgagggt gcgccggtga cgaagcttcg 720gacccagatt ccgtcggtat ggctcgtgtt cgcacacctt caggaacccg catgacgaga 780ccactggagt tttcaacgtc acgaagccgc tctgtgtgac gagaattggc ttgcgagtga 840cgtgaggcgc cgagcatgtc gttggtttgc ctcttcacaa cagaatcaga cgactgggag 900gctgcacgag gctaaggcca agggcactca ctgactcgga cgtgaagcag aagcagaagc 960agagcgctcg acggcacgtg gcggcagacc ggcttcggga cgggcaggag atctagctag 1020aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag 1080aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 1140cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc 1200gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga 1260aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt 1320cgcggatctc agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc 1380agggcgaact cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg 1440tcccggaagt tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc 1500acccacaccc aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac 1560agggtcacgt cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac 1620ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga 1680acggcactgg tcaacttggc gtccatgccg agagtgatcc cggcggcggt cacgaactcc 1740agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc 1800aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag 1860tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg 1920atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc 1980agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg 2040ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc 2100ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg 2160tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc 2220acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc 2280ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg 2340tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc 2400accatgttgg ctagtgttgc ttagatcgct tgctgctgct ggtgttgctt gttctgcttg 2460tcgtttgggg tctgcccgaa gtggacgcgt gacacagccc agcgttcgcg acttttccag 2520caaccagctc gctccgcgtc agggtcactc tctgctcact cccctgtcta gttcggcaga 2580actggagagg caacccgcgc ttccagaggg ttctcctccg gaatcagttc agaattcaga 2640attcagaaga gggaatcgca gaaccggcag cttcgggagt tggttcgctg caccatagca 2700ggtgggcgat gaggccaacc gccctgctgg ctggattctc tcgggttggc acatcgaacg 2760accgggaccc gcgcgcgagt tgtccctgcc agttgctacc agcctgccag cgtcggagag 2820ttatgcgctg cctgccggcc ggagagagag cgggcgtgga aagtggcgtg tggggcaaac 2880gcatggccct cccgcgtggc ccgatttcct atgcatccgc tccgctctct ctctcggagg 2940caagaagagc acacaacaac gcccggcggg tgcaccaggc gctgcggcag atctccaagt 3000cgcgattcat gaacaaggcg gcgggctgga ccctcgacat gatcggcgcg agcgcgatga 3060cctgggagat gcagcacgtt cttggccacc acccgtacac caacctcatc gagatggaga 3120acggtttggc caaggtcaag ggcgccgacg tcgacccgaa gaaggtcgac caggagagcg 3180acccggacgt cttcagtacg tacccgatgc ttcgcctgca cccgtggcac cgccagcggt 3240tttaccacaa gttccagcac ctgtacgccc cgtttatctt tgggtttatg acgattaaca 3300aggtgatttc ccaggatgtc ggggttgtgc tgcgcaagcg cctgttccag atcgacgcca 3360actgccggta tggcagcccc tggtacgtgg cccgcttctg gatcatgaag ctcctcacca 3420cgctctacat ggtggcgctt cccatgtaca tgcaggggcc tgctcagggc ttgaagcttt 3480tcttcatggc ccacttcacc tgcggagagg tcctcgccac catgtttatt gtcaaccaca 3540tcatcgaggg cgtcagctac gcttccaagg acgcggtcaa gggcgtcatg gctccgccgc 3600gcactgtgca cggtgtcacc ccgatgcagg tgacgcaaaa ggcgctcagt gcggccgagt 3660cgaccaagtc ggacgccgac aagacgacca tgatccccct caacgactgg gccgctgtgc 3720agtgccagac ctctgtgaac tgggctgtcg ggtcgtggtt ttggaaccac ttttcgggcg 3780gcctcaacca ccagattgag caccactgct tccccaaaac ccccacacgg tcaacgtcta 3840catctcgggc atcgtcaagg agacctgcga agaatacggc gtgccgtacc aggctgagat 3900cagcctcttc tctgcctatt tcaagatgct gtcgc 393521023DNAArtificialprimer 210aaaagaacaa gccctctcct gga 2321123DNAArtificialprimer 211gaggtttgta tgttcggcgg ttt 2321226DNAArtificialprimer 212tgggggacct tgtgcagaac tcgtgg 2621322DNAArtificialprimer 213gacctacggc gtgcagtgct tc 2221422DNAArtificialprimer 214atgtgcaagg tcgatgggac aa 2221522DNAArtificialprimer 215tcacaaacat cgcagccttc gg 22216395PRTThraustochytrium aureum ATCC 34304misc_feature(395)..(395)Xaa can be any naturally occurring amino acid 216Met Cys Lys Val Asp Gly Thr Asn Arg Ala Ser Ser Ala Gln Ala Gln1 5 10 15Ala Glu Gln Glu Lys Leu Pro Thr Ile Gly Glu Leu Arg Lys Ala Val 20 25 30Pro Ala His Cys Phe Glu Lys Ser Thr Leu Lys Ser Leu Phe Phe Val 35 40 45Ala Arg Asp Leu Ala Phe Cys Ser Ala Ile Gly Tyr Ala Ala Trp Glu 50 55 60Tyr Ile Pro Val Glu Trp Ser Ile Lys Ala Ile Ala Leu Trp Thr Leu65 70 75 80Tyr Ala Ile Val Gln Gly Thr Val Ala Thr Gly Val Trp Val Leu Gly 85 90 95His Glu Gly Gly His Gly Gly Ile Ser Ser Tyr Ser Ile Val Asn Asp 100 105 110Thr Val Gly Tyr Val Leu His Ser Ile Leu Leu Val Pro Tyr Phe Ser 115 120 125Trp Gln Asp Ser His Arg Arg His His Ala Arg Cys Asn His Leu Leu 130 135 140Asp Gly Glu Ser His Asn Pro Asp Leu Lys Arg Lys Val Tyr Lys Met145 150 155 160Tyr Glu Lys Ile Leu Asp Thr Val Gly Glu Asp Ala Phe Val Ile Met 165 170 175Gln Ile Val Leu His Leu Val Leu Gly Trp Pro Met Tyr Leu Leu Met 180 185 190His Ala Thr Gly Ser Arg Arg Ser Pro Val Thr Gly Gln Lys Tyr Thr 195 200 205Lys Lys Pro Asn His Phe Asn Trp Gly Ala Ser Asn Glu Gln Tyr Pro 210 215 220Ala Lys Leu Arg Phe Lys Ile Phe Leu Ser Ser Leu Gly Val Ile

Ala225 230 235 240Thr Leu Ala Gly Ile Ala Val Leu Ala Asn Lys Leu Gly Ala Ala Lys 245 250 255Val Ser Leu Met Tyr Phe Gly Pro Tyr Leu Val Val Asn Ala Trp Leu 260 265 270Val Gly Tyr Thr Trp Leu Gln His Thr Asp Gln Asp Ala Pro His Tyr 275 280 285Gly Glu Asp Glu Trp Thr Trp Ile Lys Gly Ala Met Thr Thr Ile Asp 290 295 300Arg Pro Tyr Pro Trp Ile Val Asp Glu Leu His His His Ile Gly Thr305 310 315 320Thr His Val Cys His His Leu Phe Ser Asp Met Pro His Tyr Lys Ala 325 330 335Gln Glu Ala Thr Glu Ala Leu Lys Pro Val Leu Gly Lys His Tyr Arg 340 345 350Phe Asp Pro Thr Pro Leu Ala Gln Ala Met Trp Asn Thr Ala Arg Asp 355 360 365Cys His Tyr Val Glu Gly Leu Asp Gly Val Gln Tyr Pro Gln Ser Ile 370 375 380Ile Ala Glu Lys Arg Ala Ala Lys Lys Leu Xaa385 390 3952171185DNAArtificialgenomic DNA (T. aureum ATCC34304 delta 4 desaturase DNA) 217atgtgcaagg tcgatgggac aaaccgggcg agctcggctc aagcccaggc agagcaggaa 60aagctgccca ccatcggcga gctgcgcaag gctgtgcccg cgcactgttt cgaaaagtcg 120acgttgaaga gcctgttctt cgtggctcgt gacctggcgt tttgcagcgc catcgggtac 180gcggcctggg agtacatccc cgtcgagtgg tcaatcaagg ccatcgccct gtggaccctg 240tacgccatag tgcagggcac cgtggcgacc ggggtctggg ttctgggcca cgaaggcgga 300cacggaggga tctcgagcta ctctattgtc aacgatactg tcgggtacgt gctgcactcg 360atcctgctcg tgccgtactt ttcctggcag gacagccaca ggcgccacca cgcgcggtgc 420aaccacctcc tggacgggga gtcgcacaac ccggacctca agcgcaaggt ttacaagatg 480tacgaaaaga tcctcgacac ggtgggcgag gacgcctttg tgatcatgca gatcgtcctt 540caccttgtct tagggtggcc catgtacctg ctgatgcacg cgaccgggtc tcgccgcagc 600cccgtgactg ggcaaaagta caccaaaaag cccaatcact tcaactgggg tgcgagcaac 660gagcagtacc cggccaagtt gcgcttcaag atttttctgt cctcgcttgg cgtgatcgcg 720acgctcgcag ggatcgccgt gctggccaac aagctcggcg ccgccaaggt ctcgctcatg 780tactttggcc cctacctcgt ggtgaatgcc tggctcgtgg gatacacctg gctccagcac 840accgaccagg acgccccgca ctatggcgag gacgagtgga cctggatcaa gggcgccatg 900acgacgatcg accgccccta cccctggatt gtggacgagc tccaccacca catcggcacg 960acgcacgttt gccaccacct gttttccgac atgccgcact acaaggccca ggaagccacc 1020gaggcgctca agccggtgct cggcaagcac taccgcttcg acccgacccc gctggcgcag 1080gccatgtgga acaccgctcg cgactgccac tacgtcgagg gcctcgacgg agtgcagtac 1140ccgcagtcaa tcatcgccga gaagcgtgcg gccaaaaagc tctag 118521830DNAArtificialprimer 218ggaagcttat gtgcaaggtc gatgggacaa 3021929DNAArtificialprimer 219ttctagacta gagctttttg gccgcacgc 2922023DNAArtificialprimer 220agtcagccca ggcaccgatg acg 2322139DNAArtificialprimer 221agccagagct agatctcttg tgctcctttt caatccttt 3922239DNAArtificialprimer 222ggagcacaag agatctagct ctggctcaag ggacaccgt 3922324DNAArtificialprimer 223cacagaaact gccttcacgg gtct 2422424DNAArtificialprimer 224tgttatgcgg ccattgtccg tcag 2422524DNAArtificialprimer 225tgcgatcgct gcggccgatc ttag 2422626DNAArtificialprimer 226atgaaaaagc ctgaactcac cgcgac 2622725DNAArtificialprimer 227ctattccttt gccctcggac gagtg 2522827DNAArtificialprimer 228atggccaagc ctttgtctca agaagaa 2722930DNAArtificialprimer 229ttagccctcc cacacataac cagagggcag 3023023DNAArtificialprimer 230ggggtcggcc ggtgcagcct tag 2323124DNAArtificialprimer 231ggcggtcagc gatcggtcgg actc 2423223DNAArtificialprimer 232gcttgcggct cctgttgggt gac 2323323DNAArtificialprimer 233acgcctggct gcccaccata aac 2323428DNAArtificialprimer 234ttagcgggat cccaattcgc cctatagt 2823527DNAArtificialprimer 235aattgggatc ccgctaagta tctcccg 2723628DNAArtificialprimer 236agatctggta ccgcagcgcc tggtgcac 2823727DNAArtificialprimer 237gctgcggtac cagatctggt cgcgttt 272385611DNAArtificialfusion DNA (Thraustochytrium aureum C20 elongase upstream/ubiquitin promoter/ 3 desaturase/ubiquitin terminator/ubiquitin promoter/BlaR/SV40 terminator/T.aureum C20 elongase downstream) 238tcccccgggc tgcaggaatt cactagtgat tctcccgggt ggacctagcg cgtgtgtcac 60ctgccggccc ccgttgcgtg caaccgaatt gatcgataat agaattacat aacaaacaac 120ttgctggatg agtacaagac cagcgtagtg tggctgtggg acgttgaacg gagcgggtcc 180tgtgatggcg cagaaaggaa ctccgcccga ggtgaaaccc cgatgcgcag gactctgcgg 240ccacagcccc tccgccagta ttccactaaa aatccgcccc ctttgacaaa gatcgcaacc 300ccgtcccatc aactcctcac aataggcttt ccactggcgg aaacgtcccc ggcacaggag 360tgcctcccgc ggttctgcgc atgcggctga ccactacgca gcgcgatatc ctccatccgc 420gtatatatcc gtaaacaacg gaacattctc cctctcaacg aggcgtggtt ttcgaagtca 480tgcctttctt ccttcctact ttccttcctt ctttctttct ttctttcctt cttttgcaag 540cgtgcgcgaa cttgaaggta ctacttacac ttgacagaga gagatagaga cggcaattcg 600accaagtact ttccacgatt tttttttttt ttgttttggt cgctttcgtt ggtcgtgcat 660gatggatggc cgggattttt acaattggat gcgccaggct gccacgcatg ccgtgacgct 720tgctcgcggc gactcatgat gcttgccagt ggcagtgcat ccagctcttc cctctgctcg 780tcgtgtactc actggcgatg ctctcggcgc tcgttcaagg gccatcgatc gatcgatcga 840tcgatcgatc gatcaatcac gtttggtgga ctcggcagac cccgaacgtg tttctcccag 900gacgcgccgc tgtcgctcgc taatccaccc gaagcgcggt cggctggcac ggtcgctcgg 960ctggaagttg agtagtttgc tttctgttgc tgcgctgctt tgtaaacgcg accagatctg 1020gtacccgtta gaacgcgtaa tacgactcac tatagggaga gtcgactgag cacaactctg 1080ctgcgagcgg gcctcgagag cgtttgcttc gagccgcgga gcaaggggga tggatcgctc 1140atgcggtcgt gcggccctcg gtcacccggt gggtcctgca ctgacgcatc tgttctgatc 1200agacacacga acgaacaaac cgaggagccg cagcgcctgg tgcacccgcc gggcgttgtt 1260gtgtgctctt cttgcctccg agagagagag cggagcggat gcataggaaa tcgggccacg 1320cgggagggcc atgcgttcgc cccacacgcc actttccacg cccgctctct ctccggccgg 1380caggcagcgc ataactctcc gacgctggca ggctggtagc aactggcagg gacaactcgc 1440gcgcgggtcc cggtcgttcg atgtgccaac ccgagagaat ccagccagca gggcggttgg 1500cctcatcgcc cacctgctat ggtgcagcga accaactccc gaagcggccg gttctgcgat 1560tccctcttct gaattctgaa ttctgaactg attccggagg agaaccctct ggaagcgcgg 1620gttgcctctc cagttctgcc gaactagaca ggggagtgag cagagagtga ccctgacgcg 1680gagcgagctg gttgctggaa aagtcgcgaa cgctgggctg tgtcacgcgt ccacttcggg 1740cagaccccaa acgacaagca gaacaagcaa caccagcagc agcaagcgac ctaagcaaca 1800ctagccaaca tgactgagga taagacgaag gtcgagttcc cgacgctcac ggagctcaag 1860cactcgatcc cgaacgcgtg ctttgagtcg aacctcggcc tctcgctcta ctacacggcc 1920cgcgcgatct tcaacgcgtc ggcctcggcg gcgctgctct acgcggcgcg ctcgacgccg 1980ttcattgccg ataacgttct gctccacgcg ctcgtttgcg ccacctacat ctacgtgcag 2040ggcgtcatct tctggggctt cttcacggtc ggccacgact gcggccactc ggccttctcg 2100cgctaccaca gcgtcaactt tatcatcggc tgcatcatgc actctgcgat tttgacgccg 2160ttcgagagct ggcgcgtgac gcaccgccac caccacaaga acacgggcaa cattgataag 2220gacgagatct tttacccgca ccggtcggtc aaggacctcc aggacgtgcg ccaatgggtc 2280tacacgctcg gcggtgcgtg gtttgtctac ttgaaggtcg ggtatgcccc gcgcacgatg 2340agccactttg acccgtggga cccgctcctc cttcgccgcg cgtcggccgt catcgtgtcg 2400ctcggcgtct gggccgcctt cttcgccgcg tacgcgtacc tcacatactc gctcggcttt 2460gccgtcatgg gcctctacta ctatgcgccg ctctttgtct ttgcttcgtt cctcgtcatt 2520acgaccttct tgcaccacaa cgacgaagcg acgccgtggt acggcgactc ggagtggacg 2580tacgtcaagg gcaacctctc gagcgtcgac cgctcgtacg gcgcgttcgt ggacaacctg 2640agccaccaca ttggcacgca ccaggtccac cacttgttcc cgatcattcc gcactacaag 2700ctcaacgaag ccaccaagca ctttgcggcc gcgtacccgc acctcgtgcg caagaacgac 2760gagcccatca tctcggcctt cttcaagacc gcgcacctct ttgtcaacta cggcgctgtg 2820cccgagacgg cgcagatctt cacgctcaaa gagtcggccg cggccgccaa ggccaagtcg 2880gactaaacta agctatctgt agtatgtgct atactcgaat catgctgccc tgtacgtacc 2940tacctatatc tgattgagcg tgctgcgtcg accatagacg cgggaacgcg ggccagccta 3000ccacgttgcc gccgccggta tccacgggca cgccaaagca ttggtcgata acgctctgcc 3060cagggcttcc tggcgaggac ccgaggccaa catgcatgca tgtgctatca gcggtcatca 3120tcgccctcat cagcgcgcat cggcgagctc gcgcacgaac ggcaagcgcc caactcaact 3180cacttactca cactatggtc ttgccgttgg cggttgctta gctaatgcgt gacgtcactc 3240tgcctccaac atcgcgaggc agagtcgcga gcagtgcaga ggccacggcg gacgccaaca 3300aagcgccaac cagcgcaacg caccagcggg tctgtgggcg tagctcgagc gggcgtcttc 3360aagagccgcc gtggagccga cgcccctgcg aagggctcga gtgcaagcgg ggccgttgag 3420ccgcgtggta ggaacaactg cagtctccct atagtgagtc gtattacgcg gtggtaccgc 3480agcgcctggt gcacccgccg ggcgttgttg tgtgctcttc ttgcctccga gagagagagc 3540ggagcggatg cataggaaat cgggccacgc gggagggcca tgcgttcgcc ccacacgcca 3600ctttccacgc ccgctctctc tccggccggc aggcagcgca taactctccg acgctggcag 3660gctggtagca actggcaggg acaactcgcg cgcgggtccc ggtcgttcga tgtgccaacc 3720cgagagaatc cagccagcag ggcggttggc ctcatcgccc acctgctatg gtgcagcgaa 3780ccaactcccg aagcggccgg ttctgcgatt ccctcttctg aattctgaat tctgaactga 3840ttccggagga gaaccctctg gaagcgcggg ttgcctctcc agttctgccg aactagacag 3900gggagtgagc agagagtgac cctgacgcgg agcgagctgg ttgctggaaa agtcgcgaac 3960gctgggctgt gtcacgcgtc cacttcgggc agtccccaaa cgacaagcag aacaagcaac 4020accagcagca gcaagcgacc taagcaacac tagccaacat ggccaagcct ttgtctcaag 4080aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag 4140actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg 4200tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg 4260cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga 4320gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag 4380tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt 4440atgtgtggga gggctaagat ccgcgaaatg accgaccaag cgacgcccaa cctgccatca 4500cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 4560gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 4620aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 4680aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 4740tatcatgtct gtataccgtc gacctctagc tagatctacc tgtttccggc tggctcccga 4800gccatgctta ccatgaatgg acctgcaaac agtctgaggt ccttgtgcaa accgctcagt 4860gggacgtcga cgaagaaaga aacaatgtgt actcgtcttg ctctgctccc gcgccgtttt 4920ttatcgttgt tgagacctct cgcgcagttt tgggaatcaa ccaaaacaag agcccggcgt 4980cagcgtttgc ttcgccctcg gctgcactcg ctcggcacgc aggtataact gggtgagtac 5040caagccccgc atttgtctgt ccgcgatccg cgcacgctgc gggtcaggac gacatcgcgc 5100tgcacgtcac agtgggtccc ttttgacgtg gctgcggcga tgaggaggct tggctcggct 5160tcatggcaag gcaacagact cgcttccggg acgcgcacga cgagcagcgc tgctttgatc 5220gaccttgcct gcgtcaccgc ctcggctgct ttgatcgatc gttgtcaccg gccgagtgac 5280cgcgaacgca ttgcccgcac ggctcggctc ggcccggacc ggaccggctc gccttggcgg 5340cgcggcgcga tggcgaccca gacgcggccg gagccgcgcg cggaggacaa ggccatgttc 5400atcttcgggc tcgggtacgt tgggagcagg ctcgccaacc agctggcgga acaggggtgg 5460cgcgtcgcgg ggtcggtgag ggagctcggg cgcgaggacg actttgccga gttcgaaaag 5520tccaagctga gcggcaaggt gcaggtgttc caactcccgc ttgagggcga ggacaacacg 5580cccgctcgcg cgcgggagat acttagcggg a 5611239904DNAArtificialGenomic DNA (Parietichytrium C20 elongase upstream) 239gacgtcgatt cccggaagag agaggacttg taaggaactt ttgtgtaaaa agatgtaaaa 60agatggaaag tattcaacgc gttggcgtga cgcctcactc acggttgcac gggcagagtc 120aggcgtggtg agtggtgact ccaaaagaaa gaaagaaaga aggagggctt tcgtttcttg 180cttgagatca agattgaaag tttttctgaa ttttgaattc tttttttttg gcggtctgac 240tcgtgtgttt gtgccaagtt cgaaaagcat tgcagtcttg ccacgtgaac acgagaacca 300gcattctttg atttctttgg actggaaaag acgagactca tgcgctaaag gagagaagct 360gtctcggggg gtccaatcat gtggaaatgt gtgagtgtgt aattggcggt tccatgcctc 420gcctagagag tcgggtagac ggctttgcca gtctgcagcg gagtcatcgg accacgtatc 480cggaaactcg tgtgtctccg atgtctcagc ctctctctct cgacaacttt gtttctaata 540ttttctaatt gtcgtgatcg tcgtgacagg tgagcatagg tgagcccgca tcatcatcga 600tcggtgggtg tctctgacgg gggttgggac tccgatgaac tttgaaaaga gacgtggtag 660tacaagtatg taataaacac cggtacatat catgaaggtt acgcttgcta ggctactgga 720agaggaaagt ggagcttaga ctttacgaga tgaagggtgt agcgccttga gtgtggcgct 780gacgggtctg caaatcctga aacgccggat tggttgcgtg gtcgagctga aaacgacaga 840acggtggtcc agtgcagtag tccccgattt ggtagttgac caaaagttga gagaaacgga 900gagg 904240721DNAArtificialGenomic DNA (Parietichytrium C20 elongase downstream) 240taccgacctt gtactcgagg agttgttgtg cgcgcggatc cgagcgcaaa agtggacgtc 60ggtgagagac aggacaatgt ttggtagcag agcagcagtt cgcgctttgc aaagcagcgg 120cttgcgactt gggagcacag cgcggagggc ctctcaccat gggctgtttt cgctggaagg 180cacggcgccc agagtgcacc cggaggcgtg gattgcgcat aacgcagttg tcgtgggcga 240tgtagaaatc ggggccaggt cgagcgtgtg gtttggggcc tgcattcgcg gtgaccgcga 300cttgatatcg atcggggaag agacaaacat tcaggacggg agtgtgctgc acacggatgc 360aggcgtccct atgaagatac atgatcgcgt caccatcgga cacatggtca tgctgcacgg 420ctgcacggtg cattctgggt ctctgatcgg cattggggcg acaatactaa acaagtaggt 480ttctatgaag tgaggaaggg ggaaggaatt cggttgtgtg tttcctgact gtgcaccgct 540tctctgcagg gccgtcatcg ggaagaattg cctgattggt gcgaacgctc taatcacgga 600agggaaagtc atcccggacg gaagtctagt gatgggccgc aaccaggtgg ttcgacagct 660caccgagaag gagatcgagg gaattcagcg cactgcggct ggctatgtgc agaaccaagg 720g 7212414592DNAArtificialfusion DNA (pGEM-T easy vector/ Parietichytrium C20 elongase upstream / Parietichytrium C20 elongase downstream / pGEM-T easy vector) 241gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60tgacgtcgat tcccggaaga gagaggactt gtaaggaact tttgtgtaaa aagatgtaaa 120aagatggaaa gtattcaacg cgttggcgtg acgcctcact cacggttgca cgggcagagt 180caggcgtggt gagtggtgac tccaaaagaa agaaagaaag aaggagggct ttcgtttctt 240gcttgagatc aagattgaaa gtttttctga attttgaatt cttttttttt ggcggtctga 300ctcgtgtgtt tgtgccaagt tcgaaaagca ttgcagtctt gccacgtgaa cacgagaacc 360agcattcttt gatttctttg gactggaaaa gacgagactc atgcgctaaa ggagagaagc 420tgtctcgggg ggtccaatca tgtggaaatg tgtgagtgtg taattggcgg ttccatgcct 480cgcctagaga gtcgggtaga cggctttgcc agtctgcagc ggagtcatcg gaccacgtat 540ccggaaactc gtgtgtctcc gatgtctcag cctctctctc tcgacaactt tgtttctaat 600attttctaat tgtcgtgatc gtcgtgacag gtgagcatag gtgagcccgc atcatcatcg 660atcggtgggt gtctctgacg ggggttggga ctccgatgaa ctttgaaaag agacgtggta 720gtacaagtat gtaataaaca ccggtacata tcatgaaggt tacgcttgct aggctactgg 780aagaggaaag tggagcttag actttacgag atgaagggtg tagcgccttg agtgtggcgc 840tgacgggtct gcaaatcctg aaacgccgga ttggttgcgt ggtcgagctg aaaacgacag 900aacggtggtc cagtgcagta gtccccgatt tggtagttga ccaaaagttg agagaaacgg 960agaggtaccg accttgtact cgaggagttg ttgtgcgcgc ggatccgagc gcaaaagtgg 1020acgtcggtga gagacaggac aatgtttggt agcagagcag cagttcgcgc tttgcaaagc 1080agcggcttgc gacttgggag cacagcgcgg agggcctctc accatgggct gttttcgctg 1140gaaggcacgg cgcccagagt gcacccggag gcgtggattg cgcataacgc agttgtcgtg 1200ggcgatgtag aaatcggggc caggtcgagc gtgtggtttg gggcctgcat tcgcggtgac 1260cgcgacttga tatcgatcgg ggaagagaca aacattcagg acgggagtgt gctgcacacg 1320gatgcaggcg tccctatgaa gatacatgat cgcgtcacca tcggacacat ggtcatgctg 1380cacggctgca cggtgcattc tgggtctctg atcggcattg gggcgacaat actaaacaag 1440taggtttcta tgaagtgagg aagggggaag gaattcggtt gtgtgtttcc tgactgtgca 1500ccgcttctct gcagggccgt catcgggaag aattgcctga ttggtgcgaa cgctctaatc 1560acggaaggga aagtcatccc ggacggaagt ctagtgatgg gccgcaacca ggtggttcga 1620cagctcaccg agaaggagat cgagggaatt cagcgcactg cggctggcta tgtgcagaac 1680caagggccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt 1740ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 1800caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 1860cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 1920gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 1980ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 2040ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 2100agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2160taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2220cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2280tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2340gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2400gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2460tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2520gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2580cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2640aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2700tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2760ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2820attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2880ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2940tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 3000aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 3060acgctcaccg gctccagatt tatcagcaat aaaccagcca

gccggaaggg ccgagcgcag 3120aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 3180agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3240ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3300agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3360tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3420tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3480attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3540taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3600aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3660caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3720gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3780cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3840tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3900acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat 3960tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 4020taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 4080gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 4140caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 4200aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 4260atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 4320aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 4380cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt 4440tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 4500gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 4560acggccagtg aattgtaata cgactcacta ta 459224237DNAArtificialGenomic DNA (Parietichytrium C20 elongase downstream) 242accgaccttg tactcgagga gttgttgtgc gcgcgga 372434448DNAArtificialfusion DNA fusion DNA (ubiquitin promoter/omega 3 desaturase/ubiquitin terminator/ubiquitin promoter/HygR/ SV40 terminator) 243tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780acactagcca acatgactga ggataagacg aaggtcgagt tcccgacgct cacggagctc 840aagcactcga tcccgaacgc gtgctttgag tcgaacctcg gcctctcgct ctactacacg 900gcccgcgcga tcttcaacgc gtcggcctcg gcggcgctgc tctacgcggc gcgctcgacg 960ccgttcattg ccgataacgt tctgctccac gcgctcgttt gcgccaccta catctacgtg 1020cagggcgtca tcttctgggg cttcttcacg gtcggccacg actgcggcca ctcggccttc 1080tcgcgctacc acagcgtcaa ctttatcatc ggctgcatca tgcactctgc gattttgacg 1140ccgttcgaga gctggcgcgt gacgcaccgc caccaccaca agaacacggg caacattgat 1200aaggacgaga tcttttaccc gcaccggtcg gtcaaggacc tccaggacgt gcgccaatgg 1260gtctacacgc tcggcggtgc gtggtttgtc tacttgaagg tcgggtatgc cccgcgcacg 1320atgagccact ttgacccgtg ggacccgctc ctccttcgcc gcgcgtcggc cgtcatcgtg 1380tcgctcggcg tctgggccgc cttcttcgcc gcgtacgcgt acctcacata ctcgctcggc 1440tttgccgtca tgggcctcta ctactatgcg ccgctctttg tctttgcttc gttcctcgtc 1500attacgacct tcttgcacca caacgacgaa gcgacgccgt ggtacggcga ctcggagtgg 1560acgtacgtca agggcaacct ctcgagcgtc gaccgctcgt acggcgcgtt cgtggacaac 1620ctgagccacc acattggcac gcaccaggtc caccacttgt tcccgatcat tccgcactac 1680aagctcaacg aagccaccaa gcactttgcg gccgcgtacc cgcacctcgt gcgcaagaac 1740gacgagccca tcatctcggc cttcttcaag accgcgcacc tctttgtcaa ctacggcgct 1800gtgcccgaga cggcgcagat cttcacgctc aaagagtcgg ccgcggccgc caaggccaag 1860tcggactaaa ctaagctatc tgtagtatgt gctatactcg aatcatgctg ccctgtacgt 1920acctacctat atctgattga gcgtgctgcg tcgaccatag acgcgggaac gcgggccagc 1980ctaccacgtt gccgccgccg gtatccacgg gcacgccaaa gcattggtcg ataacgctct 2040gcccagggct tcctggcgag gacccgaggc caacatgcat gcatgtgcta tcagcggtca 2100tcatcgccct catcagcgcg catcggcgag ctcgcgcacg aacggcaagc gcccaactca 2160actcacttac tcacactatg gtcttgccgt tggcggttgc ttagctaatg cgtgacgtca 2220ctctgcctcc aacatcgcga ggcagagtcg cgagcagtgc agaggccacg gcggacgcca 2280acaaagcgcc aaccagcgca acgcaccagc gggtctgtgg gcgtagctcg agcgggcgtc 2340ttcaagagcc gccgtggagc cgacgcccct gcgaagggct cgagtgcaag cggggccgtt 2400gagccgcgtg gtaggaacaa ctgcagtctc cctatagtga gtcgtattac gcggtggtac 2460cgaccttgta ctcgaggagt tgttgtgcgc gcggatctgg atctgccgca gcgcctggtg 2520cacccgccgg gcgttgttgt gtgctcttct tgcctccgag agagagagcg gagcggatgc 2580ataggaaatc gggccacgcg ggagggccat gcgttcgccc cacacgccac tttccacgcc 2640cgctctctct ccggccggca ggcagcgcat aactctccga cgctggcagg ctggtagcaa 2700ctggcaggga caactcgcgc gcgggtcccg gtcgttcgat gtgccaaccc gagagaatcc 2760agccagcagg gcggttggcc tcatcgccca cctgctatgg tgcagcgaac caactcccga 2820agcggccggt tctgcgattc cctcttctga attctgaatt ctgaactgat tccggaggag 2880aaccctctgg aagcgcgggt tgcctctcca gttctgccga actagacagg ggagtgagca 2940gagagtgacc ctgacgcgga gcgagctggt tgctggaaaa gtcgcgaacg ctgggctgtg 3000tcacgcgtcc acttcgggca gaccccaaac gacaagcaga acaagcaaca ccagcagcag 3060caagcgacct aagcaacact agccaacatg aaaaagcctg aactcaccgc gacgtctgtc 3120gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 3180gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 3240agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 3300ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 3360tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 3420ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 3480gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 3540tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 3600gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 3660cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 3720acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 3780atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 3840aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 3900gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 3960cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 4020agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 4080cgccccagca ctcgtccgag ggcaaaggaa tagagatccg cgaaatgacc gaccaagcga 4140cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 4200tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 4260agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 4320gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 4380aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag atctgagatt 4440aattgcgt 444824427DNAArtificialprimer 244cgttagaacg cgtaatacga ctcacta 2724532DNAArtificialprimer 245cccggatcca tggtggccag cgaggtgctc ag 3224634DNAArtificialprimer 246cccggatcct tagtcgcgct tgagctcagc atcc 34247314PRTSchizochytrium 247Met Val Ala Ser Glu Val Leu Ser Ala Pro Lys Ala Ala Ala Asp Ala1 5 10 15Ala Ala Lys Pro Lys Gln Ala Arg Arg Pro Val Lys Val Asp Arg Asp 20 25 30Asp Ala Phe Phe Arg Thr Phe Asn Leu Gly Ala Leu Tyr Cys Ser Ala 35 40 45Leu Tyr Tyr Ala Ile Gln Val Gly Pro Val Asp Asn Asp Gly Lys Gly 50 55 60Leu Tyr Phe Ala Lys Asn Lys Phe Tyr Gln Ile Met Leu Ser Asp Ala65 70 75 80Val Val Phe Gly Ala Pro Val Leu Tyr Val Leu Ala Val Met Gly Leu 85 90 95Ser Arg Phe Met Val Asn Lys Lys Pro Leu Thr Ala Phe Leu Arg Ala 100 105 110Tyr Val Gln Pro Leu Tyr Asn Val Val Gln Ile Val Val Cys Ala Trp 115 120 125Met Val Tyr Gly Ile Met Pro Gln Val Asp Ile Leu Asn Gly Asn Pro 130 135 140Phe Gly Leu Asn Thr Lys Arg Asp Ala Arg Ile Glu Phe Phe Val Phe145 150 155 160Val His Tyr Leu Thr Lys Phe Leu Asp Trp Thr Asp Thr Phe Ile Met 165 170 175Ile Leu Ser Lys Ser Tyr His Gln Val Ser Phe Leu Gln Val Phe His 180 185 190His Ala Thr Ile Gly Met Val Trp Gly Phe Leu Leu Gln Arg Gly Trp 195 200 205Gly Ser Gly Thr Cys Ala Tyr Gly Ala Phe Ile Asn Ser Val Thr His 210 215 220Val Leu Met Tyr Ser His Tyr Leu Trp Thr Ser Phe Gly Phe Lys Asn225 230 235 240Pro Leu Lys Lys Trp Leu Thr Lys Phe Gln Leu Ala Gln Phe Ala Ser 245 250 255Cys Ile Val His Ala Leu Leu Val Leu Ala Phe Glu Glu Ala Tyr Pro 260 265 270Leu Glu Phe Ala Phe Met Gln Ile Ser Tyr His Ile Ile Met Leu Tyr 275 280 285Leu Phe Gly Lys Arg Met Ser Trp Ala Pro Leu Trp Cys Thr Gly Met 290 295 300Thr Asp Met Asp Ala Glu Leu Lys Arg Asp305 310248945DNASchizochytriumcDNA (genomic DNA contains C20 elongase coding region) 248atggtggcca gcgaggtgct cagcgccccc aaggccgcgg ccgacgccgc ggccaagccc 60aagcaggcgc gtcgcccggt caaggtggac cgcgacgatg cattcttccg cacctttaac 120ctgggggcac tctactgcag cgcactctac tacgccatcc aggttggccc cgtcgacaat 180gacggcaagg gcctctactt tgccaagaac aagttctacc agatcatgct ctccgacgcg 240gtcgtctttg gcgcccccgt cctctacgtc ctcgccgtca tgggtctctc ccgcttcatg 300gtcaacaaga agcccctcac cgccttcctc cgcgcctacg tgcagccgct ctacaacgtc 360gtgcagatcg tcgtgtgcgc ctggatggtc tacggcatca tgccccaggt cgatatcctc 420aacgggaacc ccttcggcct caacaccaag cgggacgccc gcatcgagtt cttcgtgttt 480gtccactacc tcaccaagtt tcttgactgg accgacacct tcatcatgat cctctccaag 540agctaccacc aggtctcctt cctgcaggtc ttccaccacg ccaccatcgg catggtctgg 600ggctttcttc tgcagcgcgg ctggggatcg ggcacctgtg cttacggcgc cttcatcaac 660tcggtcaccc acgtcctcat gtactcgcac tacctctgga cctcctttgg cttcaagaac 720ccgctcaaga agtggctcac caagttccag ctcgcgcagt ttgcctcgtg cattgtccac 780gccctcctgg tccttgcctt cgaggaggcc tacccgctcg agtttgcttt catgcagatc 840agctaccaca ttatcatgct ctaccttttt ggcaagcgca tgagctgggc cccgctttgg 900tgcacgggga tgactgatat ggatgctgag ctcaagcgcg actaa 94524924DNAArtificialprimer 249catcgagatc ttcgtgtttg tcca 2425025DNAArtificialprimer 250acgaagatct cgatgcgggc gtccc 25251945DNAArtificialSchizochytrium derived BglII inserted C20 elongase 251atggtggccg gcgaggtgct cagcgccccc aaggccgcgg ccgacgccgc ggccaagccc 60aagcaggcgc gccgcccggt caaggtggac cgtgacgatg cattcttccg cacctttaac 120ctgggggcac tctactgcag cgcactctac tacgccatcc aggttggtcc cgtcgacaat 180gacggcaagg gcctctactt tgccaagaac aagttctacc agatcatgct ctccgacgcg 240gtcgtctttg gcgcccccgt cctctacgtc ctcgccgtca tgggcctctc ccgcttcatg 300gtcaacaaga agcccctcac cgccttcctc cgcgcctacg tgcagccgct ctacaacgtc 360gtgcagatcg tcgtgtgcgc ctggatggtc tacggcatca tgccccaggt cgatatcctc 420aacgggaacc ccttcggcct caacaccaag cgggacgccc gcatcgagat cttcgtgttt 480gtccactacc tcaccaagtt tcttgactgg accgacacct tcatcatgat cctctccaag 540agctaccacc aggtctcctt cctgcaggtc ttccaccacg ccaccatcgg catggtctgg 600ggctttcttc tgcagcgcgg ctggggatcg ggcacctgtg cttacggcgc cttcatcaac 660tcggtcaccc acgtcctcat gtactcgcac tacctctgga cctcctttgg cttcaagaac 720ccgctcaaga agtggctcac caagttccag ctcgcgcagt ttgcctcgtg cattgtccac 780gccctcctgg tccttgcctt cgaggaggcc tacccgctcg agtttgcttt catgcagatc 840agctaccaca ttatcatgct ctaccttttt ggcaagcgca tgagctgggc cccgctttgg 900tgcacgggga tgactgatat ggatgctgag ctcaagcgcg actaa 94525220DNAArtificialprimer 252agatggtggc cagcgaggtg 2025325DNAArtificialprimer 253ttagtcgcgc ttgagctcag catcc 252542644DNAArtificialfusion DNA (Schizochytrium C20 elongase 5' region/SV40 terminator/Neor/ubiquitin promoter/Schizochytrium C20 elongase 3' region) 254atggtggccg gcgaggtgct cagcgccccc aaggccgcgg ccgacgccgc ggccaagccc 60aagcaggcgc gccgcccggt caaggtggac cgtgacgatg cattcttccg cacctttaac 120ctgggggcac tctactgcag cgcactctac tacgccatcc aggttggtcc cgtcgacaat 180gacggcaagg gcctctactt tgccaagaac aagttctacc agatcatgct ctccgacgcg 240gtcgtctttg gcgcccccgt cctctacgtc ctcgccgtca tgggcctctc ccgcttcatg 300gtcaacaaga agcccctcac cgccttcctc cgcgcctacg tgcagccgct ctacaacgtc 360gtgcagatcg tcgtgtgcgc ctggatggtc tacggcatca tgccccaggt cgatatcctc 420aacgggaacc ccttcggcct caacaccaag cgggacgccc gcatcgagat ctgccgcagc 480gcctggtgca cccgccgggc gttgttggtg tgctcttctt gcctccgaga gagagagcgg 540agcggatgca taggaaatcg ggccacgcgg gagggccatg cgttcgcccc acacgccact 600ttccacgccc gctctctctc cggccggcag gcagcgcata actctccgac gctggcaggc 660tggtagcaac tggcagggac aactcgcgcg cgggtcccgg tcgttcgatg tgccaacccg 720agagaatcca gccagcaggg cggttggcct catcgcccac ctgctatggt gcagcgaacc 780aactcccgaa gcggccggtt ctgcgattcc ctcttctgaa ttctgaattc tgaactgatt 840ccggaggaga accctctgga agcgcgggtt gcctctccag ttctgccgaa ctagacaggg 900gagtgagcag agagtgaccc tgacgcggag cgagctggtt gctggaaaag tcgcgaacgc 960tgggctgtgt cacgcgtcca cttcgggcag accccaaacg acaagcagaa caagcaacac 1020cagcagcagc aagcgaccta agcaacacta gccaacatga ttgaacagga cggccttcac 1080gctggctcgc ccgctgcttg ggtggaacgg ctgttcggct acgactgggc tcagcagacg 1140atcggctgct cggacgcggc cgtgttccgc cttagcgcgc agggccggcc ggtcctgttt 1200gtcaagaccg accttagcgg cgccctcaac gagctccagg acgaagctgc ccgcctcagc 1260tggcttgcca cgacgggggt tccgtgcgcc gctgtgctcg acgtcgtcac cgaagccggc 1320cgcgactggc tgctcctcgg ggaagtgccc ggccaggacc tcctcagcag ccacctcgcg 1380cccgctgaga aggtgtccat catggccgac gccatgcgcc gcctgcacac cctcgacccc 1440gccacctgcc ccttcgacca ccaggcgaag cacaggatcg aacgcgcccg cacgcggatg 1500gaggctggcc tcgtcgacca agacgacctc gacgaggagc accagggcct cgcgccggcg 1560gaactgttcg ccaggcttaa ggctaggatg ccggacggcg aggacctcgt ggtcacgcac 1620ggcgacgcct gcctccccaa catcatggtc gagaacggcc gcttctcggg ctttatcgac 1680tgcgggcgcc tgggcgtggc ggaccgctac caagacatcg cgctcgccac gcgggacatc 1740gccgaggagc ttggcggcga gtgggccgac cgctttctcg tgctctacgg catcgccgcc 1800ccggacagcc agaggattgc gttctaccgc ctcctggacg agttcttttg agatccgcga 1860aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 1920ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 1980cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 2040ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 2100tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 2160tagctagatc ttcgtgtttg tccactacct caccaagttt cttgactgga ccgacacctt 2220catcatgatc ctctccaaga gctaccacca ggtctccttc ctgcaggtct tccaccacgc 2280caccatcggc atggtctggg gctttcttct gcagcgcggc tggggatcgg gcacctgtgc 2340ttacggcgcc ttcatcaact cggtcaccca cgtcctcatg tactcgcact acctctggac 2400ctcctttggc ttcaagaacc cgctcaagaa gtggctcacc aagttccagc tcgcgcagtt 2460tgcctcgtgc attgtccacg ccctcctggt ccttgccttc gaggaggcct acccgctcga 2520gtttgctttc atgcagatca gctaccacat tatcatgctc tacctttttg gcaagcgcat 2580gagctgggcc ccgctttggt gcacggggat gactgatatg gatgctgagc tcaagcgcga 2640ctaa 26442552881DNAArtificialfusion DNA (Schizochytrium C20 elongase 5' region/ubiquitin promoter/Hygr/SV40 terminator/Schizochytrium C20 elongase 3' region) 255atggtggccg gcgaggtgct cagcgccccc aaggccgcgg ccgacgccgc ggccaagccc 60aagcaggcgc gccgcccggt caaggtggac cgtgacgatg cattcttccg cacctttaac 120ctgggggcac tctactgcag cgcactctac tacgccatcc aggttggtcc cgtcgacaat 180gacggcaagg gcctctactt tgccaagaac aagttctacc agatcatgct ctccgacgcg 240gtcgtctttg gcgcccccgt cctctacgtc ctcgccgtca tgggcctctc ccgcttcatg 300gtcaacaaga agcccctcac cgccttcctc cgcgcctacg tgcagccgct ctacaacgtc 360gtgcagatcg tcgtgtgcgc ctggatggtc tacggcatca tgccccaggt cgatatcctc 420aacgggaacc ccttcggcct caacaccaag cgggacgccc gcatcgagat ctagctrgag 480gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa 540tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 600ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag atccccgcgc 660tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag 720gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg 780cggatctcta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga 840gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc 900cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat 960catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat 1020acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct 1080gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg 1140aatccccgaa

catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca 1200ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg 1260cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca 1320cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag 1380tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg 1440ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc agttcggttt 1500caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag gtcaggctct 1560cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat gcaaagtgcc 1620gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc aggacatatc 1680cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag agctgcatca 1740ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc gcggtgagtt 1800caggcttttt catgttggct agtgttgctt aggtcgcttg ctgctgctgg tgttgcttgt 1860tctgcttgtc gtttggggtc tgcccgaagt ggacgcgtga cacagcccag cgttcgcgac 1920ttttccagca accagctcgc tccgcgtcag ggtcactctc tgctcactcc cctgtctagt 1980tcggcagaac tggagaggca acccgcgctt ccagagggtt ctcctccgga atcagttcag 2040aattcagaat tcagaagagg gaatcgcaga accggccgct tcgggagttg gttcgctgca 2100ccatagcagg tgggcgatga ggccaaccgc cctgctggct ggattctctc gggttggcac 2160atcgaacgac cgggacccgc gcgcgagttg tccctgccag ttgctaccag cctgccagcg 2220tcggagagtt atgcgctgcc tgccggccgg agagagagcg ggcgtggaaa gtggcgtgtg 2280gggcgaacgc atggccctcc cgcgtggccc gatttcctat gcatccgctc cgctctctct 2340ctcggaggca agaagagcac acaacaacgc ccggcgggtg caccaggcgc tgcggcagat 2400ccagatcttc gtgtttgtcc actacctcac caagtttctt gactggaccg acaccttcat 2460catgatcctc tccaagagct accaccaggt ctccttcctg caggtcttcc accacgccac 2520catcggcatg gtctggggct ttcttctgca gcgcggctgg ggatcgggca cctgtgctta 2580cggcgccttc atcaactcgg tcacccacgt cctcatgtac tcgcactacc tctggacctc 2640ctttggcttc aagaacccgc tcaagaagtg gctcaccaag ttccagctcg cgcagtttgc 2700ctcgtgcatt gtccacgccc tcctggtcct tgccttcgag gaggcctacc cgctcgagtt 2760tgctttcatg cagatcagct accacattat catgctctac ctttttggca agcgcatgag 2820ctgggccccg ctttggtgca cggggatgac tgatatggat gctgagctca agcgcgacta 2880a 2881

Claims

1. A method for producing a microbial oil, comprising: genetically modifying a labyrinthulid by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene; culturing the labyrinthulid, such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content; and collecting the microbial oil having the increased EPA content from the labyrinthulid, wherein the increased EPA content is not less than 3.3% of a total fatty acid composition.

2. The method for producing the microbial oil according to claim 1, wherein the disrupted and/or silenced gene is a polyketide synthase (PKS) gene, a fatty acid elongase gene and/or a fatty acid desaturase gene, wherein the transformed another gene is a fatty acid elongase gene and/or a fatty acid desaturase gene.

3. The method for producing the microbial oil according to claim 2, wherein the polyketide synthase (PKS) gene is OrfA, wherein the fatty acid elongase gene is a C20 elongase gene, and/or wherein the fatty acid desaturase gene is a .DELTA.4 desaturase gene and/or an .omega.3 desaturase gene.

4. The method for producing the microbial oil according to claim 1, wherein the genetically modified labyrinthulid is able to grow in media which do not contain PUFA.

5. The method for producing the microbial oil according to claim 1, wherein the step of disrupting or transforming the gene of a labyrinthulid utilizes electroporation or a gene gun method, and/or wherein the step of silencing the gene utilizes an antisense method or RNA interference.

6. The method for producing the microbial oil according to claim 1, wherein the labyrinthulid belonging to the genus of Thraustochytrium, Parietichytrium, Schizochytrium, or Ulkenia.

7. The method for producing the microbial oil according to claim 1, wherein the labyrinthulid is Thraustochytrium aureum, Thraustochytrium roseum, Parietichytrium sarkarianum, Parietichytrium sp., or Schizochytrium sp.

8. The method for producing the microbial oil according to claim 1, wherein the labyrinthulid is Thraustochytrium aureum (ATCC 34304), Thraustochytrium roseum (ATCC 28210), Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab (FERM BP-11421).

9. A labyrinthulid that has been genetically modified by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content, wherein the increased EPA content is not less than 3.3% of a total fatty acid composition.

10. The labyrinthulid according to claim 9, wherein the disrupted and/or silenced gene is a polyketide synthase (PKS) gene, a fatty acid elongase gene and/or a fatty acid desaturase gene, and wherein the transformed another gene is a fatty acid elongase gene and/or a fatty acid desaturase gene.

11. The labyrinthulid according to claim 10, wherein the polyketide synthase (PKS) gene is OrfA, wherein the fatty acid elongase gene is a C20 elongase gene, and/or wherein the fatty acid desaturase gene is a .DELTA.4 desaturase gene and/or an .omega.3 desaturase gene.

12. The labyrinthulid according to claim 9, wherein the labyrinthulid is able to grow in media which do not contain PUFA.

13. The labyrinthulid according to claim 9, wherein the labyrinthulid belongs to the genus of Thraustochytrium, Parietichytrium, Schizochytrium, or Ulkenia.

14. The labyrinthulid according to claim 9, wherein the labyrinthulid is Thraustochytrium aureum, Thraustochytrium roseum, Parietichytrium sarkarianum, Parietichytrium sp., or Schizochytrium sp.

15. The labyrinthulid according to claim 9, wherein the labyrinthulid is Thraustochytrium aureum (ATCC 34304), Thraustochytrium roseum (ATCC 28210), Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab (FERM BP-11421).