Patent Application
20190119689
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.
Polyunsaturated fatty acids (PUFA) represent an important component of animal and human nutrition. ω3 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.
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.
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).
Patent Documents
Patent Document 1: JP-A-2007-143479
Patent Document 2: JP-A-2005-102680
Patent Document 3: JP-A-2006-304685
Patent Document 4: JP-A-2006-304686
Patent Document 5: WO2011/037207
Patent Document 6: WO1997/011094
Patent Document 7: US Patent Application US2005/0014231
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 Document 1: Poulos A., Lipids, 30, 1-14(1995)
Non-Patent Document 2 : Horrocks L. A. and Yeo Y. K., Pharmacol Res., 40, 211-225 (1999)
Non-Patent Document 3: Yokoyama R., Honda D., Mycoscience, 48, 199-211 (2007)
Non-Patent Document 4: Lecture Summary for the 60th Conference of The Society for Biotechnology, Japan, p 136 (2008)
Non-Patent Document 5: Lippmeier J. C. et al., Lipids, 44(7), 621-630 (2009)
Non-Patent Document 6: Tonon T. et al., FEBS Lett., 553, 440-444 (2003).
Non-Patent Document 7 : Thompson J. D. et al., Nucleic Acids Res., 22, 4673-4680 (1994)
Non-Patent Document 8: Yazawa K., Lipids, 31, Supple. 297-300 (1996)
Non-Patent Document 9: Jiang X. et al., Wei Sheng Wu Xue Bao., 48(2), 176-183 (2008)
Non-Patent Document 10: PEREIRA S. L. et al., Biochem. J., 378, 665-671 (2004)
Non-Patent Document 11: Prasher D. C. et al., Gene, 111(2), 229-233 (1992)
Non-Patent Document 12: Chalfie M. et al., Science, 263, 802-805 (1994)
Non-Patent Document 13 : Southern P. J. , and Berg, P., J. Molec. Appl. Gen., 1, 327-339 (1982)
Non-Patent Document 14: Saitou N. et al., Mol. Biol. Evol., 4, 406-425 (1987)
Non-Patent Document 15: Ausubel F. M. et al., Current Protocols in Molecular Biology, Unit 13 (1994)
Non-Patent Document 16: Guthrie C., Fink G. et al., Methods in Enzymology: Guide to Yeast Genetics and Molecular Biology, Volume 194 (1991)
Non-Patent Document 17: Abe E., et al., J. Biochem, 142, 31561-31566 (2006)
Non-Patent Document 18: Bio-Experiment Illustrated 2, Fundamentals of Gene Analysis, p 117-128, Shujunsha, 1995
Non-Patent Document 19: Japan Society for Bioscience, Biotechnology, and Agrochemistry, 77, 2, 150-153 (2003)
Non-Patent Document 20: Bio-Experiment Illustrated 2, Fundamentals of Gene Analysis, p 63-68, Shujunsha, 1995
Non-Patent Document 21: Sanger, F. et al., Proc. Natl. Acad. Sci, 74, 5463 (1977)
Non-Patent Document 22: Meyer, A., et al. J. Lipid Res., 45, 1899-1909 (2004)
Non-Patent Document 23: Cigan and Donahue, 1987; Romanos et al., 1992
Non-Patent Document 24: Qiu, X., et al. J. Biol. Chem., 276, 31561-6 (2001)
Non-Patent Document 25: DIG Application Manual [Japanese version] 8th, Roche Applied Science
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
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.
The gist of the present invention includes the following stramenopile transformation methods (1) to (12).
(1) A method for transforming stramenopile, the method including disrupting a stramenopile gene and/or inhibiting expression thereof by genetic engineering.
(2) The method according to (1), wherein the stramenopile belongs to the class Labyrinthulomycetes.
(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.
(4) The method according to (3), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.
(5) The method according to (4), 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).
(6) The method according to any one of (1) to (5), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.
(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.
(8) The method according to (7), wherein the fatty acid chain elongase is a C20 elongase.
(9) The method according to (7), wherein the fatty acid desaturase is a Δ12 desaturase.
(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.
(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.
(12) The method according to any one of (1) to (11), further including introducing a gene associated with fatty acid desaturase.
(13) The method according to (12), wherein the gene associated with fatty acid desaturase is an ω3 desaturase.
Further, the gist of the present invention includes the following methods (14) to (26) for modifying the fatty acid composition of a stramenopile.
(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.
(15) The method according to (14), wherein the stramenopile belongs to the class Labyrinthulomycetes.
(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.
(17) The method according to (16), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.
(18) The method according to (17), 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).
(19) The method according to any one of (14) to (18), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.
(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.
(21) The method according to (20), wherein the fatty acid chain elongase is a C20 elongase.
(22) The method according to (21), wherein the fatty acid desaturase is a Δ12 desaturase.
(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.
(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.
(25) The method according to any one of (14) to (24), further including introducing a gene associated with fatty acid desaturase.
(26) The method according to (25), wherein the gene associated with fatty acid desaturase is an ω3 desaturase.
Further, the gist of the present invention includes the following methods (27) to (29) for highly accumulating fatty acids in a stramenopile.
(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).
(28) The method according to (27), wherein the fatty acid is an unsaturated fatty acid.
(29) The method according to (28), wherein the unsaturated fatty acid is an unsaturated fatty acid of 18 to 22 carbon atoms.
Further, the gist of the present invention includes the following fatty acid (30).
(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).
Further, the gist of the present invention includes the following transformed stramenopiles (31) to (43).
(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.
(32) The stramenopile according to (31), wherein the stramenopile belongs to the class Labyrinthulomycetes.
(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.
(34) The stramenopile according to (33), wherein the microorganisms are Thraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytrium roseum, Parietichytrium sp., or Schizochytrium sp.
(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).
(36) The stramenopile according to any one of (31) to (35), wherein the stramenopile gene is a gene associated with fatty acid biosynthesis.
(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.
(38) The stramenopile according to (36), wherein the fatty acid chain elongase is a C20 elongase.
(39) The stramenopile according to (37), wherein the fatty acid desaturase is a Δ12 desaturase.
(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.
(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.
(42) The stramenopile according to any one of (31) to (41), further comprising introducing a gene associated with fatty acid desaturase is introduced.
(43) The stramenopile according to (42), wherein the gene associated with fatty acid desaturase is an ω3 desaturase.
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.
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 ω3 series (also known as the n-3 series) fatty acids such as eicosapentaenoic acid and docosahexaenoic acid, and plant-derived w6 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), γ-linolenic acid (GLA), and arachidonic acid (AA or ARA).
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, ω3 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, 49 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. Δ6, Δ5, and Δ4 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).
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.
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.
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.
The present invention is described below in more detail.
[Microorganism]
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.
Of note, Labyrinthuloides and Aplanochytrium are regarded as being synonymous among some scholars (Leander, Celeste A. & David Porter, Mycotaxon, vol. 76, 439-444 (2000)).
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]
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 Δ12 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.
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 ω3 desaturase gene may be introduced.
[Polyketide Synthase and Fatty Acid Chain Elongase]
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 bacteriaShewanella 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.
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 γ-linolenic acid (GLA) to dihomo-γ-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).
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]
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 Δ4 desaturase gene Δ5 desaturase gene, Δ6 desaturase gene, Δ12 desaturase gene, and ω3 desaturase gene, and these may be used either alone or in combination. The Δ4 desaturase gene, Δ5 desaturase gene, Δ6 desaturase gene, and Δ12 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 Δ4 desaturase gene (Non-Patent Document 6). Specific examples of Δ5 desaturase include T. aureum-derived Δ5 desaturase, and Δ5 desaturases derived from Thraustochytrium sp. ATCC 26185, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans, and Leishmania major. Examples of Δ12 desaturase include Pinguiochrysis pyriformis-derived Δ12 desaturase, and fungus- and protozoa-derived Δ12 desaturases. The ω3 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 ω3 desaturase (Non-Patent Document 10). The Δ5 desaturase catalyzes, for example, the conversion of dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA), and the conversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid (EPA). Δ6 desaturase catalyzes, for example, the conversion of linoleic acid (LA) to γ-linolenic acid (GLA), and the conversion of α-linolenic acid (ALA) to stearidonic acid (STA). The ω3 desaturase catalyzes the conversion of arachidonic acid to eicosapentaenoic acid. Linoleic acid (LA) is produced from oleic acid (OA) by the action of Δ12 desaturase.
[Product Unsaturated Fatty Acid]
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 α-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), γ-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]
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.
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.
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]
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]
The stramenopile gene associated with fatty acid biosynthesis maybe 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.
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.
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 Δ4 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 Δ4 desaturase gene can be obtained. Here, because the Thraustochytrium aureum ATCC 34304 is a diploid, two selection markers need to be prepared.
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.
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.
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).
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 maybe 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.
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 ω3 desaturase gene is introduced as the fatty acid desaturase gene.
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.
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]
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° C., preferably 15 to 35° C., for 1 to 14 days.
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.
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.
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.
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.
[Labyrinthulomycetes, Culture Method, and Preservation Method]
(1) Strains Used in the Present Invention
Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC 28210 were obtained from ATCC. Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK358 (FERM BP-11405), and Parietichytrium sp. SEK571 (FERM BP-11406) were obtained from Konan University, Faculty of Science and Engineering. Schizochytrium sp. TY12Ab (FERM BP-11421) was obtained from University of Miyazaki, Faculty of Agriculture.
(2) Medium Composition
i. Agar Plate Medium Composition
PDA Agar Plate Medium
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° C. for 20 min. After sufficient cooling, ampicillin sodium (nacalai tesque) was added in a final concentration of 100 μg/ml to prevent bacterial contamination. The medium was dispensed onto a petri dish, and allowed to stand on a flat surface to solidify.
ii. Liquid Medium Composition
GY Liquid Medium 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° C. for 20 min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.
PD Liquid Medium 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° C. for 20 min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.
(3) Culture Method
i. Agar Plate Culture
Labyrinthula cells were inoculated using a platinum loop or a spreader, and static culture was performed at 25° 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.
ii. Liquid Culture
Labyrinthula cells were inoculated, and suspension culture was performed by stirring at 25° 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.
(4) Maintenance and Preservation Method of Labyrinthulomycetes
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° C. deep freezer.
[Disruption of Thraustochytrium aureum C20 Elongase Gene]
A T. aureum. ATCC 34304 culture fluid grown for 3 days using a GY liquid medium was centrifuged at 3,500×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™-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™ (invitrogen) to sterilized Milli Q water treated with diethylpyrocarbonate.
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 SMARTTM 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° C. 1 min/94° C. 30 sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° C. 10 min/4° C. 00]. The result confirmed bands for the specifically amplified 3′- and 5′-RACE products (
As a result, two sequences, 190 bp and 210 bp, named elol (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 elol, 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 elol, elo2, and elo3. The oligonucleotide primers produced are as follows.
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 elol, and there was a complete match in the overlapping portion, identifying the sequence as a 1,139-bp elol 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).
It was found from the sequence analysis result that elol 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, elol and elo3 genes were identified as T. aureum ATCC 34304-derived putative elongase genes, and were named TaELO1 and TaELO2, respectively,
Elongases are broadly classified into three groups on the basis of substrate specificity.
1. SFA/MUFA elongases (act on saturated fatty acids or monovalent unsaturated fatty acids)
2. PUFA-elongases (single-step) (act on polyvalent unsaturated fatty acids of certain chain lengths)
3. PUFA elongases (multi-step) (act on polyvalent unsaturated fatty acids of various chain lengths)
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.
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 (
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.
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.
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° C. 2 min/98° C. 5 sec, 60° C. 5 sec, 72° C. 1.5 min, 30 cycles/72° C. 7 min/4° C. ∞].
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.
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 a-linolenic acid (ALA, C18:3Δ9, 12, 15) and linoleic acid (LA, C18:2Δ9, 12) added as 49 elongase substrates, stearidonic acid (STA, C18:4Δ6, 9, 12, 15) and γ-linolenic acid (GLA, C18:3Δ6, 9, 12) added as Δ6 elongase substrates, and eicosapentaenoic acid (EPA, C20:5Δ5, 8, 11, 14, 17) and arachidonic acid (AA, C20:4Δ5, 8, 11, 14) added as Δ5 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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C. →(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
It was found as a result that, the pYEELO1 introduced strain had the Δ6 elongase activity not found in the host (mock introduced strain), converting the stearidonic acid (STA) into eicosatetraenoic acid (ETA, C20:4Δ8, 11, 14, 17), and the γ-linolenic acid (GLA) into dihomo-γ-linolenic acid (DGLA, C20:3Δ8, 11, 14). The pYEELO1 introduced strain also had the Δ9 elongase activity of converting the a-linolenic acid (ALA) into eicosatrienoic acid (ETrA, C20:3Δ11, 14, 17), and the linoleic acid (LA) into eicosadienoic acid (EDA, C20:3Δ11, 14), and the 45 elongase activity of converting the eicosapentaenoic acid (EPA) into ω3 docosapentaenoic acid (ω3 DPA, C22:5Δ7, 10, 13, 16, 19), and the arachidonic acid (AA) into docosatetraenoic acid (DTA, C22:4Δ7, 10, 13, 16) (Table 1).
As for the pYEELO2 introduced strain, it was found that this strain had the 45 elongase activity not found in the host, converting EPA to ω3 DPA (C22:5Δ7, 10, 13, 16, 19), and AA to DTA. The pYEELO2 introduced strain also had a weak Δ6 elongase activity, converting STA to ETA, and GLA to DGLA (Table 1). These results confirmed that the TaELO1 was a Δ6/Δ9/Δ5 elongase, and the TaELO2 was a Δ5/Δ6elongase, contrary to the results expected from the TaELO1 and TaELO2 substrate specificity in the phylogenetic analysis described in Example 2-3 and
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.
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 PCRTM 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.
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.
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° 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.
Asa 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.
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) /K0 Term R SmaI (Example 2-6, SEQ ID NO: 31), and PrimeSTAR HS DNA polymerase (Takara Bio) [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3min, 30 cycles/68° C. 10 min/4° C. ∞]. 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.
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° C., 150 rpm, and the supernatant was removed by centrifugation at 3,500×g, 4° 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-μl 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.
Target distance: 6 cm
Vacuum: 26 inches Hg
Micro carrier size: 0.6 μm
Rupture disk (penetration pressure): 1,100 psi
Thereafter, a PD liquid medium (100 μ1) 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 4.7×101 cfu/μg DNA.
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.
(1) Neor detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR (Example 2-6; SEQ ID NO: 29)
(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)
(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 (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) (5) TaELO2 amplification: E2 HindIII (Example 2-4; SEQ ID NO: 20) and E2 XbaI (Example 2-4; SEQ ID NO:21) (
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 (
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 μg per lane using a 0.7% SeaKemR GTGR agarose (Takara Bio). This was transferred to a nylon membrane (Hybond™-N+, GE Healthcare), and hybridized at 48° 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.
[PCR cycles: 98° C. 2 min/98° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]
Detection of the hybridized probes was made by using a chromogenic method (NBT/BCIP solution).
As a result, a single band was detected in all lanes treated with the various restriction enzymes (
Southern Blotting
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° C. 2 min/98° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. Do]. 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 (
Because the wild-type allele band was simultaneously detected with the mutant allele band in the transfectants (
A TaELO2 targeting vector was constructed with a selection marker Hygr to disrupt the remaining wild-type allele.
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.
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° C. for 10 seconds, and the annealing and the extension reaction were appropriately adjusted according to the primer Tm and the amplification product length.
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.
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-600p F 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° C. 2 min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C. 10 min/4° C. 00]. 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° C. 2 min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C. 10 min/4° C. ∞]. 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 Convinience Kit (Nippon Gene).
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.
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° C. 2 min/98° C. 10 sec, 68° C. 3.5 min, 30 cycles/68° C. 10 min/4° C. ∞]. 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: 1.02×103 cfu/μg DNA).
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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 10 min/4° C. ∞]. The following oligonucleotide primer sets were used.
(1) TaELO2 ORF detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR (Example 2-6; SEQ ID NO: 29)
(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) (
It was suggested that 14 out of the 50 clones analyzed were transfectants that underwent homologous recombination through TaELO2 ORF replacement (
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 (
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 (
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° C. 2 min/98° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. Here, TaELO2 was detected as about a 2.5-kbp DNA fragment (
The analysis revealed that in contrast to the wild-type strain in which the TaELO2 was detected (
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 μg) was cleaned up using an RNeasyMini Kit (QIAGEN) according to the manufacturer's protocol, and treated at 37° 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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 10 min/4° C. ∞].
It was found as a result that the TaELO2 mRNA detected in the wild-type strain (
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.
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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
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 (
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.
[Disruption of Parietichytrium sarkarianum C20 Elongase Gene]
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: 34 mer: 5′-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCG A-3′ (SEQ ID NO: 51), RHO52: 24 mer: 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° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° 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.
A plasmid (pRH27) containing the subcloned SV40 terminator sequence (342 bp, SEQ ID NO: 53) is shown in
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° C., 3,500×g for 5min 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° 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.
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: 36 mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (SEQ ID NO: 55), TKO1: 58 mer: 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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].
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 [TK02: 54 mer: 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: 26 mer: 5′-CAG ATC TCA AAA GAA CTC GTC CAG GA-3′ (SEQ ID NO: 59)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].
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° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min (1° C./10 sec from 55° C. to 68° C.) (
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.
The product artificial neomycin-resistant gene cassette (pRH31) is shown in
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. K508 contains the ubiquitin promoter sequence and a hygromycin-resistant gene sequence [RHO53: 36 mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (Example 3-2; SEQ ID NO: 55), KSO8: 58 mer: 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° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min].
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: 56 mer: 5′-AGC GAC CTA AGC AAC ACT AGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3′ (SEQ ID NO: 64), RHO56: 36 mer: 5′-CAG ATC TCT ATT CCT TTG CCC TCG GAC GAG TGC TGG-3′ (SEQ ID NO: 65) ] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min].
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° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min (1° C./10 sec from 55° C. to 68° C.) ] (
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 (
The product artificial neomycin-resistant gene cassette (pRH32) is shown in
The Parietichytrium sarkarianum SEK364 genomic DNA extracted by using the method of Example 3-2 was extracted, and the genome was decoded.
A forward oligonucleotide (PsTaELO2 F1; 5′-CCT TCG GCG CTC CTC TTA TGT ATG-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° C. 1 min/98° C. 10 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. 00]. 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.
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™ RACE cDNA Amplification Kit (Clontech) as a template [PCR cycles: 94° C. 30 sec 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 3 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/4° C. Do]. 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° C. 1 min/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/72° C. 10 min/4° C. ∞]. 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.
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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 2 min].
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).
The 936-bp Parietichytrium C20 elongase gene (SEQ ID NO: 76) was cloned, and named pRH80 (
By using the pRH80 produced in Example 3-4 (
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° C. 2 min/98° C. 10 sec, 56° C. 15 sec, 72° C. 1 min, 30 cycles/72° 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.
The produced base plasmid (pRH83) for Parietichytrium C20 elongase gene targeting vector production is shown in
The pRH31 (
The pRH32 (
The two targeting vectors (pRH85 and 86) produced are shown in
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° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° 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×TE. The DNA concentration was then calculated by measuring A260/280. The introduced fragment obtained from using the pRH85 (
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 μg) 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.
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
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) (
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° 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° 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.
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° C., 30 min). The cells were vortexed after adding sterile water (500 μ1), 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° C. after adding 3 N methanolic HCl (2 ml).
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 (2ml), 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 μl of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
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).
[Disruption of Thraustochytrium aureum PUFA PKS Pathway-Associated Gene OrfA]
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 PCRTM in vitro Cloning Kit (Takara Bio). A PCR lower primer [RHO20: 23 mer: 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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4min, 30 cycles/72° 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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30 cycles/72° 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).
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.
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: 21 mer: 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.
The DNA was amplified by using the method described in Example 4-1 using the PCR upper primer [RHO28: 20 mer: 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).
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).
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 EF1α promoter sequence [TMO30: 30 mer: 5′-CGA ATA TTC CTG GTT GAT CCT GCC AGT AGT-3′ (SEQ ID NO: 99), TMO31: 46 mer: 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° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min]. Separately, by using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the EF1α 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 EF1α promoter sequence. TMO33 contains the EF1α promoter sequence and an artificial neomycin-resistant gene sequence [TMO32: 46 mer: 5′-GGT TTC CGT AGT GAA CCT GCA ATT CAA AAA AAG CCG TTA CTC ACA T-3′ (SEQ ID NO: 102), TMO33: 46 mer: 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° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].
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 EF1α promoter sequence and the artificial neomycin-resistant gene sequence. TMO35 contains the artificial neomycin-resistant gene sequence and the EF1α terminator sequence [TMO34: 45 mer: 5′-CAT CGG CAA AGG AAG GCT AGA TGA TTG AAC AGG ACG GCC TTC ACG-3′ (SEQ ID NO: 105), TMO 35: 46 mer: 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° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].
Further, by using the Thraustochytrium aureum ATCC 34304 genomic DNA as a template, the EF1α 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 EF1α terminator sequence. TMO37 was set within the EF1α terminator [TMO36: 46 mer: 5′-TCC TGG ACG AGT TCT TTT GAG ATC CGC GCC GGC TAT GCG CCC GTG C-3′ (SEQ ID NO: 108), TMO37: 30 mer: 5′-CAC TGC AGC GAA AGA CGG GCC GTA AGG ACG-3′ (SEQ ID NO: 109)] [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min].
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° 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 (
The DNA fragment (
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: 32 mer: 5′-CCC GAA TTC GGA CGA TGA CTG ACT GAC TGA TT-3′ (SEQ ID NO: 112), RHO34: 28 mer: 5′-CCC AAG CTT GTC TGC CTC GGC TCT TGG T-3′ (SEQ ID NO: 113)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min].
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: 28 mer: 5′-CCC CCA TGG TGT TGC TGT GGG ATT GGT C-3′ (SEQ ID NO: 115), RHO30: 30 mer: 5′-CCC CCA TGG CTC GGT TAC ATC TCT GAG GAA-3′ (SEQ ID NO: 116)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min].
The amplified upstream sequence was joined to the EcoRI site and the HindIII site in the pRH5 of
The produced targeting vector (pRH21) using the artificial neomycin-resistant gene is shown in
By using the pRH32 (
The amplified fragment was joined to the HindIII and SphI sites of the pRH21 (
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: 29 mer: 5′-CCC GTC GAC GTG TTG CTG TGG GAT TGG TC-3′ (SEQ ID NO: 121), RHO62: 29 mer: 5′-CCC GTC GAC TCG GTT ACA TCT CTG AGG AA -3′ (SEQ ID NO: 122) ] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min].
The amplified downstream sequence was joined to the SalI site of pRH30 (
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° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° 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×TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH21 (
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 μg) 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.
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.
The genomic DNA was cut with restriction enzymes, and electrophoresed in about 2 to 3 μg per well with a 0.7% SeaKem GTG agarose gel (Takara Bio). This was transferred to a nylon membrane, and hybridized at 54 ° 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.
PCR cycles: 98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 2 min, 30 cycles/72° C. 3 min
The restriction enzymes used and the probe positions are as shown in
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 (
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.
[Disruption of C20 Elongase Gene in Thraustochytrium aureum OrfA Disrupted Strain]
The genome cassette library produced in Example 4-1 was used as a template. A PCR lower primer [RHO71: 22 mer: 5′-GGG AGC GCA GGG AAAACG 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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The 1st PCR amplification product was diluted 100 times, and the gene was amplified by using the PCR lower primer [RHO72: 20 mer: 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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5min]. 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 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.
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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30 cycles/72° 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° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30 cycles/72° 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).
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.
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: 58 mer: 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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].
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: 54 mer :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: 38 mer: 5′-CCC AGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG-3′ (SEQ ID NO: 139) ] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].
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° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min (1° C./10 sec from 55° C. to 68° C.) ] (
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 (
The product blasticidin-resistant gene cassette (pRH38) is shown in
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: 29 mer: 5′-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3′ (SEQ ID NO: 142), TMO39: 41 mer: 5′-TCC TCG CCC TTG CTC ACC ATG TTG GCT AGT GTT GCT TAG GT-3′ (SEQ ID NO: 143) ] [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].
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: 41 mer: 5′-ACC TAA GCA ACA CTA GCC AAC ATG GTG AGC AAG GGC GAG GA -3′ (SEQ ID NO: 145), RHO91: 58 mer: 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° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min].
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° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min (1° C./10 sec from 55° C. to 68° C.)] (
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° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30 cycles/68° C. 2 min].
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: 54 mer: 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: 38 mer: 5′-CCC AGA TCT CAG TCC TGC TCC TCG GCC ACG AAG TGC AC-3′ (SEQ ID NO: 151)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 1 min].
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° C. 2 min/94° C. 20 sec, 68° C. 2 min, 30 cycles/68° C. 2 min (1° C./10sec from 55° C. to 68° C.)] (
The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-enhanced GFP gene-pcDNA3.1 Zeo(+) -derived zeocin-resistant gene (
The product GFP-fused zeocin-resistant gene cassette (pRH51) is shown in
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 KS010 downstream of the C20 elongase gene (SEQ ID NO: 25) [K509: 50 mer: 5′-CCC GAA TTC ACT AGT GAT TCT CCC GGG TGG ACC TAG CGC GTG TGT CAC CT-3′ (SEQ ID NO: 154), KS010: 40 mer: 5′-CCC GAA TTC GAT TAT CCC GGG GCC GAG AAC GGG GTC GCC C-3′ (SEQ ID NO: 155)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3.5 min, 30 cycles/68° 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) (
By using the plasmid of
The produced base plasmid (pRH40) for the production of the C20 elongase gene targeting vector is shown in
The pRH38 (
The pRH51 (
The two targeting vectors (pRH43 and 54) produced are shown in
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: 31 mer: 5′-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3′ (SEQ ID NO: 159), KSO12: 27 mer: 5′-ATC CCG GGG CCG AGA ACG CCC TCG CCC-3′ (SEQ ID NO: 160)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° 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×TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH43 (
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 μg) 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.
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.
The genomic DNA was cut with restriction enzymes, and electrophoresed in about 2 to 3 μg per well in a 0.7% SeaKem GTG agarose gel (Takara Bio). This was transferred to a nylon membrane, and hybridized at 51° 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.
PCR cycles: 98° C. 2 min/98° C. 30 sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3min
The restriction enzymes used and the probe positions are as shown in
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 (
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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
Changes in fatty acid composition are represented in
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 ⅕.
[Expression of ω3 Desaturase Gene in Thraustochytrium aureum OrfA Disrupted Strain]
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 ω3 desaturase gene sequence [TMO42: 29 mer: 5′-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3′ (SEQ ID NO: 168), TMO43: 45 mer: 5′-TTC GTC TTA TCC TCA GTC ATG TTG GCT AGT GTT GCT TAG GTC GCT-3′ (SEQ ID NO: 169)] [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].
Then, Saprolegnia diclinawas 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° C., 3,500×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° 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 ω3 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 ω3 desaturase gene sequence. TMO45 contains the Saprolegnia diclina-derived ω3 desaturase gene sequence and the ubiquitin terminator [TMO44: 43 mer: 5′-CCT AAG CAA CAC TAG CCA ACA TGA CTG AGG ATA AGA CGA AGG T-3′ (SEQ ID NO: 171), TMO45: 40 mer: 5′-ATA CTA CAG ATA GCT TAG TTT TAG TCC GAC TTG GCC TTG G-3′ (SEQ ID NO: 172) ] [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min 30 sec, 30 cycles/72° C. 1 min 30 sec].
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 ω3 desaturase gene sequence and the ubiquitin terminator. TMO47 was designed on the ubiquitin terminator sequence, and contains a KpnI linker sequence [TMO46: 44 mer: 5′-CCA AGG CCA AGT CGG ACT AAA ACT AAG CTA TCT GTA GTA TGT GC-3′ (SEQ ID NO: 174), TMO47: 45 mer: 5′-TCG GTA CCA CCG CGT AAT ACG ACT CAC TAT AGG GAG ACT GCA GTT-3′ (SEQ ID NO: 175)] [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].
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° C. 2 min/98° C. 20 sec, 55° C. 30 sec, 68° C. 3 min, 30 cycles/68° C. 3 min (1° C./10 sec from 55° C. to 68° C.] (
By using the pRH38 (
The fused Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter-Saprolegnia diclina-derived ω3 desaturase gene-Thraustochytrium aureum ATCC 34304-derived ubiquitin terminator (SEQ ID NO: 176;
The product Saprolegnia diclina-derived ω3 desaturase gene expression plasmid (pRH48) is shown in
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° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 1 min, 25 cycles/72° 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×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-ω3 desaturase gene-ubiquitin terminator-ubiquitin promoter-blasticidin-resistant gene sequence-SV40 terminator sequence (SEQ ID NO: 178).
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 μg) 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.
Genomic DNA was extracted from the Thraustochytrium aureum OrfA disrupted strain produced in Example 3 and the ω3 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
The result of the amplification confirmed a band of the expected size (
The Thraustochytrium aureum OrfA disrupted strain produced in Example 4, and the ω3 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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
The ω3 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 (
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.
[Disruption of Thraustochytrium roseum C20 Elongase Gene]
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° C. 1 min/94° C. 30 sec, 55° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. 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.
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.
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™ RACE cDNA Amplification Kit (clontech) as a template [PCR cycles: 94° C. 30 sec 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 3 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/4° C. ∞]. 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° C. 1 min/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/72° C. 10 min/4° C. ∞]. 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.
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° C. 1 min/94° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. 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.
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° C., 3,500×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° 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° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° 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 (
By using the pRH59 (
The produced base plasmid (pRH64) for the production of the Thraustochytrium roseum C20 elongase gene targeting vector is shown in
The pRH31 (
The pRH32 (
The two targeting vectors (pRH65 and 66) produced are shown in
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° C. 2 min/98° C. 30 sec, 60 30 sec, 72° 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×TE. The DNA concentration was calculated by measuring A260/280. The introduced fragment obtained from using the pRH65 (
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 μg) 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).
As a result, about 20 drug resistant strains were obtained per penetration.
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° C., 3,000 rpm for 10 min to form a pellet, and lysed at 55° C., 6 h/99.9° C., 5 min after being suspended in a 20-μ1 SNET solution [20 mM Tris-HCl; pH 8.0, 5 mM NaCl, 0. 3% SDS, 200 μg/m1 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
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) (
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° 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° 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° C., 30 min). The cells were vortexed after adding sterile water (500 μ1), 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° C. after adding 3 N methanolic HCl (2 ml).
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 (2ml), 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 μ1 of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
As a result, knocking out the C20 elongase in the Thraustochytrium roseum increased fatty acids of 20 carbon chain length (
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.
Disruption of 44 Desaturase Gene in Thraustochytrium aureum ATCC 34304 OrfA Disrupted Strain
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 Δ4 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 Δ4 desaturase gene to 1,500 bp within the Δ4 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° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° 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 (
By using the pTM1 (
The product base plasmid (pTM2) for the Δ4 desaturase gene targeting vector production is shown in
The pRH38 (
The pRH51 (
The two targeting vectors (pTM6 and 8) produced are shown in
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° C. 3 min/98° C. 10 sec, 55° C. 5 sec, 72° C. 4 min, 30 cycles/72° 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×TE. The DNA concentration was calculated by measuring A260/280 . The introduced fragment obtained from using the pTM6 (
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 μg) 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.
Genomic DNA was extracted from Thraustochytrium aureum, and the Δ4 desaturase gene disrupted strain of the Thraustochytrium aureum OrfA 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 shown in
Δ4 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) (
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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
Changes in fatty acid composition are represented in
As can be seen from the results, disrupting the Δ4 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.
Disruption of C20 Elongase Gene in Parietichytrium sp. SEK358 Strain
By using the targeting vector produced with the pRH85 (
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 (
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° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30 cycles/68° C. 7 min].
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) (
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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.␣(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
Changes in fatty acid composition are represented in FIG. 66.
Disruption of C20 Elongase Gene in Parietichytrium sp. SEK571 Strain
By using the targeting vector produced with the pRH85 (
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 (
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° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30 cycles/68° C. 7 min].
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) (
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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
Changes in fatty acid composition are represented in
Disruption of Thraustochytrium aureum ATCC 34304-Derived Δ12 Desaturase Gene
By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as a template, a Thraustochytrium aureum ATCC 34304-derived Δ12 desaturase gene was amplified by a PCR performed with a forward oligonucleotide primer Tw3-F1 (22 mer: 5′-ATG TGC AAG GTC GAT GGG ACA A -3′) (SEQ ID NO: 214) and a reverse oligonucleotide primer Tω3-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° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞). 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 (
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 TΔ12d was prepared in a PCR performed with a forward oligonucleotide primer Tw3-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 Tw3-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° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. Go). The DNA fragment was then incorporated in the HindII/Xba I site of a pYES2/CT vector to construct a TΔ12d 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 TΔ12d overexpressing strain (pYESTD12 introduced strain), novel peaks were confirmed at positions corresponding to the retention times of LA (C18:2Δ9, 12) and C16:2Δ9, 12, but not in the mock introduced strain (pYES2/CT introduced strain).
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 TΔ12d ORF were amplified in a PCR performed under the following conditions (enzyme used: PrimeSTAR GXL, TaKaRa); PCR cycles: 98° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞). The following forward and reverse oligonucleotide primers were used.
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 TΔ12d KO targeting vectors. These were named pTD12dKOHyg and pTD12dKOBla. The construction scheme of the TΔ12d KO targeting vectors are shown in
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° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. X min (X=1 min/kbp), 30 cycles/72° C. 7 min/4° C. 00] (
TD12d-up-F (SEQ ID NO: 220) and Hyg-Knock-R (24 mer: 5′-TGT TAT GCG GCC ATT GTC CGT CAG-3′) (SEQ ID NO: 224), and Hyg-Knock-F (24 mer: 5′-TGC GAT CGC TGC GGC CGA TCT TAG-3′) (SEQ ID NO: 225) and TD12d-down-R (SEQ ID NO: 223)
As a result, a homologous recombinant with the disrupted TΔ12d 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° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° C. 7 min/4° C. ∞]. The fragment was then introduced to the homologous recombinant containing the disrupted first allele. Complete disruption of TΔ12d 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 TΔ12d, or by southern blotting.
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 TΔ12d disrupted strain. However, no amplification of the disrupted TΔ12d was confirmed. The following forward and reverse oligonucleotide primers were used for the amplification of the hygromycin-resistant gene, blasticidin-resistant gene, and TΔ12d.
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.
As a result, the bands of the wild-type allele (upstream side 2,028 bp, downstream side 2,334 bp) disappeared in the TΔ12d 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 (
The PCR using the genomic DNA as a template, the RT-PCR, and southern blotting made it clear that the TΔ12d was disrupted.
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 TΔ12d disrupted strain (
As a result, large fatty acid composition changes were observed. Accumulation of C18:1n9 (OA) in the TΔ12d disrupted strain was particularly prominent.
Disruption of C20 Elongase Gene and Expression of ω3 Desaturase Gene in Thraustochytrium aureum ATCC 34304 OrfA Gene Disrupted Strain
By using the pRH43 (
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° C. 2 min/98° C. 10 sec, 55° C. 15 sec, 72° C. 40 sec, 31 cycles/72° 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 (
The pRH48 (
The product C20 elongase gene targeting and Saprolegnia diclina-derived ω3 desaturase expression vector pRH103 is shown in
By using the C20 elongase gene targeting vector pRH54 (
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 μg) 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.
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.
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 μg per well. This was transferred to a nylon membrane, and hybridized at 51° 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° C. 2 min/98° C. 30 sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min]. The restriction enzymes used, and the probe positions are as shown in
The Thraustochytrium aureum ATCC 34304 wild-type strain, and the Saprolegnia diclina-derived ω3 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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C(10 min)
Carrier gas: He (1.3 mL/min).
Changes in fatty acid composition are represented in
It was found as a result that disrupting the C20 elongase gene and expressing the Saprolegnia diclina-derived ω3 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.
Expression of ω3 Desaturase Gene in Parietichytrium sp. SEK571 C20 Elongase Gene Disrupted Strain
For the production of a Saprolegnia diclina-derived ω3 desaturase expression plasmid using hygromycin as a drug-resistance marker, a plasmid pRH107 (
The pRH48 (
The pRH32 (
By using the pRH109 (
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 μg) 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.
Genomic DNA was extracted from the Parietichytrium sp. SEK571 C20 elongase gene disrupted strain produced in Example 10 and the ω3 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
The result of amplification confirmed a band of the expected size (
The Parietichytrium sp. SEK571 strain, and the ω3 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:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
The ω3 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 (
Disruption of Schizochytrium C20 Elongase Gene
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° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞). 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 (
By using the pRH70 (
The product base plasmid (pRH71) for the production of the Schizochytrium C20 elongase gene targeting vector is shown in FIG.
96.
The pRH31 (
The pRH32 (
The two targeting vectors (pRH73 and pKS-SKO) produced are shown in
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° C. 2 min/98° C. 30 sec, 60 30 sec, 72° 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×TE. The DNA concentration was then calculated by measuring A260/280. The introduced fragment obtained from using the pRH73 (
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 μg) 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).
As a result, about 20 drug resistant strains were obtained per penetration.
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° C., 3,000 rpm for 10 min to form a pellet. The cells were then lysed at 55° C., 6 h/99.9° C., 5 min after being suspended in a 20-μ1 SNET solution [20 mM Tris-HCl; pH 8.0, 5 mM NaCl, 0.3% SDS, 200 μg/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
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) (
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° 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° 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° C., 30 min).
The cells were vortexed after adding sterile water (500 μl), 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° C. after adding 3 N methanolic HCl (2 ml).
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 μl of the sample was GC analyzed. The GC analysis was performed with a gas chromatograph GC-2014 (Shimadzu Corporation) under the following conditions:
Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)
Column temperature: 150° C.→(5° C./min)→220° C. (10 min)
Carrier gas: He (1.3 mL/min).
As a result, knocking out the C20 elongase in the Schizochytrium sp. TY12Ab strain increased fatty acids of 20 carbon chain length (
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-224225 | Oct 2010 | JP | national |
| 2011/179194 | Aug 2011 | JP | national |
This application 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. The sequence listing submitted was submitted in copending application Ser. No. 14/711,075 in a computer readable form under the name of “130412-revised_sequence_listing.txt” 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.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14711075 | May 2015 | US |
| Child | 16208047 | US | |
| Parent | 13877225 | Aug 2013 | US |
| Child | 14711075 | US |
