Microbial oil-producing labyrinthulae, microbial oils, and methods for making and using them - Patent Application 20070122997

Abstract

To provide Labyrinthulids that produce PUFA only by an elongase / desaturase pathway.SOLUTION: Labyrinthulids that have no producing ability or extremely weak producing ability of PUFA by an endogenous PUFA-PKS pathway, and has PUFA producing ability by an endogenous elongase / desaturase pathway. They are microorganisms that belong to either Parietichytrium or Schizochytrium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to microbial oils obtained from Labyrinthulea, to microbial oil-producing Labyrinthulea, and to uses of microbial oil-producing Labyrinthulea. More specifically, it relates to polyunsaturated fatty acids (PUFAs), their production, The present invention relates to Labyrinthuleae that have been genetically modified to alter their fatty acid composition, preferably Labyrinthuleae that produce PUFAs exclusively through the elongase / desaturase pathway, a method for producing PUFAs using them, and lipids (microbial oils) containing PUFAs produced using them, as well as uses thereof. [Background technology]

[0002] Labyrinthula ( Labyrinthulids ) is the Order Labyrinthula Labyrinthulales ), Thraustochytriales (Order Thraustochytriales ) including the Labyrinthula class (Class Labyrinthulomycetes ), which are known to be ubiquitous in the ocean. Microorganisms belonging to the Thraustochytriales order are also known as Thraustochytrids ( Thraustochytrids ) are sometimes collectively referred to as Labyrinthules have attracted industrial attention as oil-producing microorganisms, and the DHA produced by these Labyrinthules has been commercialized as a raw material for DHA-containing lipids, feed containing a high amount of DHA, etc. (Non-Patent Document 1). Specific examples include techniques for growing the genus Thraustochytrium and Schizochytrium (Patent Document 1), and techniques for utilizing Thraustochytriales and ω-3 HUFA (highly unsaturated fatty acids) extracted from Thraustochytriales (Patent Document 2).

[0003] It is generally known that PUFAs are biosynthesized via the elongase / desaturase pathway (sometimes called the standard pathway). However, it has been revealed that certain Labyrinthula species produce PUFAs via a different pathway, namely, a metabolic pathway using polyketide synthases (PKS) (Non-Patent Document 2). Hereinafter, in the present invention, this pathway will be referred to as the PUFA-PKS pathway or PKS pathway. The composition of the PUFAs produced via this pathway is characterized by the fact that most of them are DHA and DPAn-6.

[0004] Some scientists believe that Labyrinthulea (particularly Thraustochytrids) only have the PUFA-PKS pathway as a PUFA biosynthetic pathway, and do not have the elongase / desaturase pathway common in other organisms (Non-Patent Document 3). In fact, one example is a report showing that disruption of the genes in the PUFA-PKS pathway of Labyrinthulea renders the Labyrinthulea lethal, rendering them unable to grow unless PUFAs are added to the culture medium (Non-Patent Document 3). This result indicates that PUFAs are essential for the growth of the Labyrinthulea, and that disruption of the genes in the PUFA-PKS pathway, the only PUFA biosynthetic pathway, has resulted in a change in the organism's nature to require exogenous PUFAs for growth.

[0005] However, contrary to this common knowledge of those skilled in the art, as a result of our investigations, we have found that there are Labyrinthules that have both the PUFA-PKS pathway and the elongase / desaturase pathway as PUFA biosynthetic pathways. Specific details are described in Patent Document 3 and Non-Patent Document 5. Thraustochytrium aureumTaking ATCC34304 as an example, we demonstrated that this strain possesses the Δ12 desaturase gene, which is the entry enzyme of the elongase / desaturase pathway. Furthermore, we demonstrated that strains in which this gene was disrupted by homologous recombination significantly accumulated oleic acid, the substrate of Δ12 desaturase, compared to wild-type strains, and that the product, linoleic acid, and PUFAs downstream of the biosynthetic pathway were reduced. Furthermore, we demonstrated that because this strain is capable of producing PUFAs via the elongase / desaturase pathway, disruption of the PUFA-PKS pathway genes does not result in lethality. This report was the first to demonstrate that the elongase / desaturase pathway functions as a PUFA biosynthetic pathway in Labyrinthulea, and was also covered in Non-Patent Document 6.

[0006] As mentioned at the beginning, Labyrinthules that produce DHA as the major fatty acid are widely used industrially. In contrast, the creation of Labyrinthules that contain a desired PUFA other than DHA as the major fatty acid became possible only with the discovery of Labyrinthules that have both a PUFA-PKS pathway and an elongase / desaturase pathway, and the application of transformation techniques to these (Patent Document 3, Non-Patent Document 7). That is, by first disrupting the genes of the PUFA-PKS pathway by gene disruption through homologous recombination, and then appropriately disrupting or overexpressing the genes of the enzymes that make up the elongase / desaturase pathway, it is possible to create a strain that contains a specific PUFA other than DHA as the major fatty acid. A specific example is given in Example 12 of Patent Document 3. This Example includes: Thraustochytrium aureum The genes of the PUFA-PKS pathway of ATCC34304 were disrupted, followed by the C20 elongase gene. Saprolegnia diclina It is described that by introducing the ω3 desaturase gene of the origin, they succeeded in producing a strain in which arachidonic acid was increased by approximately 6 times, EPA by approximately 10 times, and DHA was reduced to approximately 1 / 16 times compared to the wild-type strain. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 3127161 [Patent Document 2] Patent No. 3669372 [Patent Document 3] WO2012 / 043826 [Patent Document 4] U.S. Patent Publication No. US2005 / 0014231 [Non-patent literature]

[0008] [Non-Patent Document 1] Zvi Cohen et al., eds., "Single Cell Oils Microbial and Algal Oils 2nd edition", (USA), AOCS Press, 2010, p.88 [Non-patent document 2] Metz JG, Roessler P, Faccioti D, et al. Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 2001;293:290-293 [Non-patent document 3] Ratledge C. Omega-3 biotechnology: Errors and omissions, Biotechnology Advances 30 (2012) 1746-1747 [Non-patent document 4] Lippmeier JC et al., Lipids, 44(7), 621-630(2009) [Non-patent document 5] Matsuda T, Sakaguchi K, Hamaguchi R, Kobayashi T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M. The analysis of delta12 fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of polyunsaturated fatty acids in Thraustochytrium aureum ATCC34304. J. Lipid Res. 53(6):1210-1222 (2012)

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

[0009] An objective of the present invention is to provide Labyrinthuleae that have been genetically modified to alter their fatty acid composition, preferably Labyrinthuleae that produce PUFAs exclusively through the elongase / desaturase pathway, a method for producing PUFAs using them, and lipids (microbial oils) containing PUFAs produced using them, as well as uses thereof. To generate strains containing specific PUFAs other than DHA as the primary fatty acid using Labyrinthulea species that possess both the PUFA-PKS pathway and the elongase / desaturase pathway, it was first necessary to somehow halt or suppress DHA production via the PUFA-PKS pathway. Specifically, methods such as disrupting the PUFA-PKS pathway genes by homologous recombination, obtaining mutant strains by UV irradiation or chemical treatment, or suppressing PUFA-PKS gene expression by RNAi (RNA interference) could be considered. Of these methods, gene disruption by homologous recombination is technically well established, as mentioned above. However, because the number of drug resistance genes that can be used as markers is generally limited, using markers to disrupt genes in the PUFA-PKS pathway reduces the number of markers available for further gene disruption or gene transfer. This reduces the number of gene disruption or gene transfers that can be performed after disrupting a gene in the PUFA-PKS pathway, posing an obstacle to the creation of strains with higher performance. Furthermore, although expression suppression by mutation or RNAi has been proven effective in other organisms, it is unclear whether these methods can be used to obtain the desired strains in Labyrinthula. The present invention aims to provide Labyrinthuleae that have no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway and have the ability to produce PUFAs through the endogenous elongase / desaturase pathway, a method for producing lipids containing PUFAs using them, and lipids containing PUFAs produced using them. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have discovered that there are Labyrinthules that have no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway, but have the ability to produce PUFAs through the endogenous elongase / desaturase pathway. This led to the completion of the present invention, which relates to microbial oils obtained from Labyrinthules, Labyrinthules that produce microbial oils, methods for using them, and uses of microbial oils.

[0011] The present invention relates to microbial oils described in (1) to (16) below. (1) A microbial oil that satisfies one or more of the following conditions (a) to (g), wherein the microorganism is a genetically modified Labyrinthula species (A) or (B) below that has no or very weak ability to produce PUFAs through an endogenous PUFA-PKS pathway and has the ability to produce PUFAs through an endogenous elongase / desaturase pathway. (a) ARA accounts for 10% or more of the total fatty acid composition, and the GC area ratio of ARA after gene modification is three times or more compared to before gene modification. (b) DGLA accounts for 1.9% or more of the total fatty acid composition, and the GC area ratio of DGLA after genetic modification is 4 times or more compared to before genetic modification. (c) ETA accounts for 0.35% or more of the total fatty acid composition, and the GC area ratio of ETA after genetic modification is 7 times or more compared to before genetic modification. (d) EPA accounts for 4% or more of the total fatty acid composition, and the GC area ratio of EPA after genetic modification is 7 times or more compared to before genetic modification. (e) n-6DPA is 0.20% or less of the total fatty acid composition. (f) DHA is 0.50% or less of the total fatty acid composition. (g) The total content of DHA and n-6DPA is 0.7% or less of the total fatty acid composition. (A) Labyrinthuleans whose fatty acid composition has been modified by disrupting or / and silencing the expression of PKS genes, fatty acid chain elongation enzyme genes, and / or fatty acid desaturase genes. (B) Labyrinthuleans whose fatty acid composition has been modified by introducing additional genes in addition to disrupting or / and silencing the expression of PKS genes, fatty acid chain elongation enzyme genes, and / or fatty acid desaturase genes.

[0012] (2) The microbial oil according to (1), wherein the n-6DPA / DTA value by GC area is 1.5 or less. (3) The microbial oil according to (1) or (2), wherein the DHA / n-3DPA value by GC area is 4 or less. (4) The microbial oil according to any one of (1) to (3), wherein the C20PUFA / C22PUFA value by GC area is 0.5 or more and 50 or less. (5) The microbial oil according to any one of (1) to (4), wherein the n-6PUFA / n-3PUFA value by GC area is 1.8 or more.

[0013] (6) The microbial oil according to any one of (1) to (5), wherein the fatty acid chain elongation enzyme gene is a C20 elongase gene. (7) The microbial oil according to any one of (1) to (6), wherein the fatty acid desaturase gene is a Δ4 desaturase gene or an ω3 desaturase gene. (8) The microbial oil according to any one of (1) to (7), wherein the gene to be introduced is a fatty acid chain elongation enzyme gene and / or a fatty acid desaturase gene. (9) A microbial oil according to any one of (1) to (8), wherein the method for disrupting or introducing a Labyrinthula gene is electroporation, gene gun method, or genome editing. (10) A microbial oil according to any one of (1) to (9), wherein the method for suppressing the expression of a Labyrinthula gene is the antisense method or RNA interference.

[0014] (11) The microbial oil according to any one of (1) to (10), wherein the Labyrinthulea having no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway and having the ability to produce PUFAs through the endogenous elongase / desaturase pathway is the Labyrinthulea of ​​(C) or (D) below: (C) Labyrinthulea having no or very weak ability to produce PUFAs through the PUFA-PKS pathway, (D) Labyrinthulea in which the PUFA-PKS that it originally possesses has been disrupted or the expression of which has been very weakly suppressed. (12) The (C) Labyrinthulae having no or very weak ability to produce PUFAs through the PUFA-PKS pathway, Parietichytrium Genus or Schizochytrium The microbial oil according to (11), which is a Labyrinthula belonging to the genus Labyrinthula. (13) Parietichytrium The Labyrinthula species belonging to the genus Parietichytrium sarkarianum belongs to, Schizochytrium The Labyrinthula species belonging to the genus Schizochytrium aggregatum The microbial oil according to (12), which is a microbial oil belonging to the Labyrinthula genus.

[0015] (14) Parietichytrium The Labyrinthula species belonging to the genus Parietichytrium sp.SEK358, FERM BP-11405, Parietichytrium sarkarianum SEK364, FERM BP-11298 or Parietichytrium sp. SEK571, FERM BP-11406, Schizochytrium The Labyrinthula species belonging to the genus Schizochytrium aggregatum The microbial oil according to (12), which is ATCC2829. (15) The Labyrinthula in which the (D) original PUFA-PKS is disrupted or the expression thereof is very weakly suppressed, Thraustochytrium The microbial oil according to (11), which is a Labyrinthula belonging to the genus Labyrinthula. (16) Thraustochytrium The Labyrinthula species belonging to the genus Thraustochytrium aureum The microbial oil according to (15), which is a microbial oil belonging to the Labyrinthula genus.

[0016] The present invention also relates to methods for using the microorganisms described in (17) to (21) below. (17) A method for using a microorganism to reduce the amount of DHA produced in microbial oil, the method comprising: genetically modifying the microorganism to alter the fatty acid composition of the Labyrinthulea so that the GC area ratio of DHA in the microbial oil is 1 / 5 or less of that before the genetic modification by disrupting or / and suppressing the expression of a PKS gene, a fatty acid chain elongase gene, and / or a fatty acid desaturase gene; the microorganism is a Labyrinthulea that has no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway and has the ability to produce PUFAs through the endogenous elongase / desaturase pathway; and the method comprises genetically modifying the Labyrinthulea to alter the fatty acid composition of the Labyrinthulea so that the GC area ratio of DHA in the microbial oil after the genetic modification is 1 / 5 or less of that before the genetic modification: (A) Labyrinthulea whose fatty acid composition has been altered by disrupting or / and suppressing the expression of a PKS gene, a fatty acid chain elongase gene, and / or a fatty acid desaturase gene. (B) Labyrinthula species in which the fatty acid composition has been modified by disrupting or / and suppressing the expression of PKS genes, fatty acid chain elongation enzyme genes, and / or fatty acid desaturase genes, as well as by introducing additional genes.

[0017] (18) The method for using a microorganism according to (17), wherein the microbial oil satisfies one or more of the following (a) to (g): (a) ARA accounts for 10% or more of the total fatty acid composition, and the GC area ratio of ARA after gene modification is three times or more compared to before gene modification. (b) DGLA accounts for 1.9% or more of the total fatty acid composition, and the GC area ratio of DGLA after genetic modification is 4 times or more compared to before genetic modification. (c) ETA accounts for 0.35% or more of the total fatty acid composition, and the GC area ratio of ETA after genetic modification is 7 times or more compared to before genetic modification. (d) EPA accounts for 4% or more of the total fatty acid composition, and the GC area ratio of EPA after genetic modification is 7 times or more compared to before genetic modification. (e) n-6DPA is 0.20% or less of the total fatty acid composition. (f) DHA is 0.50% or less of the total fatty acid composition. (g) The total content of DHA and n-6DPA is 0.7% or less of the total fatty acid composition.

[0018] (19) The method for using the microorganism according to (17), wherein the gene to be introduced is a fatty acid chain elongation enzyme gene and / or a fatty acid desaturase gene. (20) The method for using the microorganism according to (17), wherein the fatty acid chain elongation enzyme gene is a C20 elongase gene, and the fatty acid desaturase is a Δ4 desaturase gene or an ω3 desaturase gene. (21) The method for using the microorganism according to (17), wherein the Labyrinthules that have no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway and have the ability to produce PUFAs through the endogenous elongase / desaturase pathway are Labyrinthules of the following (C) or (D): (C) Labyrinthules that have no or very weak ability to produce PUFAs through the PUFA-PKS pathway. (D) Labyrinthules in which the original PUFA-PKS has been destroyed or its expression has been very weakly suppressed.

[0019] The present invention also relates to the following (22) food, feed, medicine, or industrial product, and (23) genetically modified Labyrinthules. (22) A food, feed, medicine, or industrial product comprising the microbial oil according to any one of (1) to (16). (23) Labyrinthules that produce the microbial oil according to any one of (1) to (16), that have been genetically modified to modify the fatty acid composition of the produced microbial oil, and that have no or very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway and have the ability to produce PUFAs via the endogenous elongase / desaturase pathway. [Brief explanation of the drawings]

[0020] [Figure 1]The elongase / desaturase pathway and the enzymes involved in this biosynthetic pathway are shown. (Brief explanation of symbols) C16:0: Palmitic acid, C18:0: Stearic acid, C18:1n-9: Oleic acid, C18:2n-6: Linoleic acid (LA), C18:3n-3: α-linolenic acid (ALA), C18:3n-6: γ-linolenic acid (GLA), C18:4n-3: Stearidonic acid (STA), C20:2n-6: Eicosadienoic acid (EDA), C20:3n-3: Eicosatrienoic acid (ETrA), C20:3n-6: Dihomo-γ-linolenic acid (DGLA) Dihomo-γ-Linolenic Acid), C20:4n-3: Eicosatetraenoic acid (ETA), C20:4n-6: Arachidonic acid (ARA), C20:5n-3: Eicosapentaenoic acid (EPA), C22:4n-6: Docosatetraenoic acid (DTA), C22:5n-3: n-3 Docosapentaenoic acid (DPA n-3), C22:5n-6: n-6 Docosapentaenoic acid (DPA n-6), C22:6n-3: Docosahexaenoic acid (DHA),Docosahexaenoic Acid), C16e:C16 elongase, Δ9d:Δ9 desaturase, Δ12d:Δ12 desaturase, Δ15d:Δ15 desaturase, Δ9e:Δ9 elongase, Δ6d:Δ6 desaturase, Δ8d:Δ8 desaturase, C18e:C18 elongase, Δ5d:Δ5 desaturase, C20e:C20 elongase, Δ17d:Δ17 desaturase, Δ4d:Δ4 desaturase Δ15 desaturase and Δ17 desaturase are sometimes called ω3 desaturases. [Figure 2] This represents a plasmid containing the SV40 terminator sequence derived from the subcloned pcDNA 3.1 Myc-His vector. [Figure 3] This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fused sequence of the ubiquitin promoter derived from Thraustochytrium aureum ATCC 34304 and an artificially synthesized neomycin resistance gene. [Figure 4] This shows the BglII cassette of the artificially synthesized neomycin resistance gene. [Figure 5] This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fusion sequence of the ubiquitin promoter derived from Thraustochytrium aureum ATCC 34304 and the hygromycin resistance gene derived from pcDNA 3.1 / Hygro. [Figure 6] This shows the BglII cassette of the hygromycin resistance gene derived from pcDNA3.1 / Hygro. [Figure 7]1 shows a plasmid containing the cloned Parietichytrium C20 elongase sequence. [Figure 8] This shows a plasmid in which a BglII site has been inserted into the Parietichytrium C20 elongase sequence of the plasmid shown in FIG. [Figure 9] These are the two Parietichytrium C20 elongase gene targeting vectors we constructed. They contain either the neomycin resistance gene (pRH85) or the hygromycin resistance gene (pRH86) as a drug resistance marker. [Figure 10] 1 shows a schematic diagram showing the positions of PCR primers used to identify a C20 elongase gene disruptant of Parietichytrium sarkarianum SEK364 and the predicted products.

[0021] [Figure 11] This shows the evaluation of C20 elongase gene disruption by PCR using Parietichytrium sarkarianum SEK364 genomic DNA as a template. (Keywords) + / +: Parietichytrium sarkarianum SEK364 wild-type strain + / -: Homologous recombinant of the first allele of the C20 elongase gene derived from Parietichytrium sarkarianum SEK364 - / -: C20 elongase gene-disrupted strain derived from Parietichytrium sarkarianum SEK364 [Figure 12] Comparison of fatty acid composition between the wild-type strain and the C20 elongase gene disruptant of Parietichytrium sarkarianum SEK364. Black and white bars represent the fatty acid composition of the wild-type strain and the gene disruptant, respectively. Values ​​are means ± standard deviation. [Figure 13] The fatty acid ratio of the C20 elongase gene disruptant is shown, relative to the ratio of the wild-type strain of Parietichytrium sarkarianum SEK364, which is set at 100%. [Figure 14]FIG. 1 shows a plasmid containing the cloned Parietichytrium sp. SEK571 Δ4 desaturase gene sequence and surrounding sequences. [Figure 15] This shows a plasmid in which the Parietichytrium sp. SEK571 Δ4 desaturase gene and a 600-bp sequence downstream of the Δ4 desaturase gene were deleted from the plasmid shown in Figure 14, and a BglII site was inserted. [Figure 16] This shows a plasmid in which a DNA fragment containing an artificially synthesized neomycin resistance gene cassette has been ligated into the BglII site of the plasmid shown in FIG. [Figure 17] Figure 1 shows the constructed Parietichytrium Δ4 desaturase gene targeting vector. It contains a neomycin resistance gene as a drug resistance marker. [Figure 18] A schematic diagram showing the positions of PCR primers used to identify Δ4 desaturase gene disruptants of Labyrinthulea species of the genus Parietichytrium, and the predicted products (primers were designed within the homologous recombination region). [Figure 19] 1 shows the results of evaluation of Δ4 desaturase gene disruption by PCR using Parietichytrium sarkarianum SEK364 genomic DNA as a template. [Figure 20] 1 shows charts of gas chromatographic analysis of the fatty acid compositions of the Parietichytrium sarkarianum SEK364 wild-type strain and its Δ4 desaturase gene disruptant. [Figure 21] 21 shows a partially enlarged view of FIG. 20. [Figure 22] This shows a comparison of the fatty acid compositions of the Parietichytrium sarkarianum SEK364 wild-type strain and its Δ4 desaturase gene disruptant. This table is a numerical representation of the chart in Figure 20. In the figure, < indicates that the number is less than or equal to the following number.

[0022] [Figure 23]Figure 1 shows the evaluation of C20 elongase gene disruption by PCR using genomic DNA from the Parietichytrium sp. strain SEK358 as a template. (Keywords) + / +: Parietichytrium sp. SEK358 wild-type strain - / -: C20 elongase gene-disrupted strain derived from Parietichytrium sp. strain SEK358 [Figure 24] Comparison of fatty acid composition between the wild-type strain of Parietichytrium sp. SEK358 and a C20 elongase gene-disrupted strain derived from Parietichytrium sp. SEK358. The white and black bars represent the fatty acid compositions of the wild-type strain and the gene-disrupted strain, respectively. [Figure 25] The figure shows the fatty acid ratio of the C20 elongase gene disruptant derived from Parietichytrium sp. SEK358, with the wild-type strain of Parietichytrium sp. SEK358 set at 100%. Fatty acids below the detection limit in the wild-type strain of Parietichytrium sp. SEK358 are indicated by a diagonal line. [Figure 26] A schematic diagram showing the positions of PCR primers used to identify Δ4 desaturase gene disruptants of Labyrinthulea species of the genus Parietichytrium, and the predicted products (primers were designed outside the homologous recombination region). [Figure 27] 1 shows the results of evaluating disruption of the Δ4 desaturase gene by PCR using Parietichytrium sp. SEK358 genomic DNA as a template when primers were designed within the homologous recombination region. [Figure 28] 1 shows the results of evaluating disruption of the Δ4 desaturase gene by PCR using Parietichytrium sp. SEK358 genomic DNA as a template when primers were designed outside the homologous recombination region. [Figure 29] 1 shows charts of gas chromatographic analysis of the fatty acid compositions of the wild-type strain of Parietichytrium sp. SEK358 and its Δ4 desaturase gene disruptant. [Figure 30] 29A and 29B are enlarged views of the same. [Figure 31] This shows a comparison of the fatty acid compositions of the wild-type Parietichytrium sp. SEK358 strain and its Δ4 desaturase gene disruptant. This table is a numerical representation of the chart in Figure 29. In the figure, < indicates that the number is less than or equal to the following number. [Figure 32] Figure 1 shows the evaluation of C20 elongase gene disruption by PCR using genomic DNA from the Parietichytrium sp. strain SEK571 as a template. (Keywords) + / +: Parietichytrium sp. SEK571 wild-type strain - / -: C20 elongase gene-disrupted strain derived from Parietichytrium sp. strain SEK571

[0023] [Figure 33] Comparison of fatty acid composition between the wild-type strain of Parietichytrium sp. SEK571 and a C20 elongase gene-disrupted strain derived from Parietichytrium sp. SEK571. The white and black bars represent the fatty acid compositions of the wild-type strain and the gene-disrupted strain, respectively. [Figure 34] The fatty acid ratio of the C20 elongase gene-disrupted strain derived from Parietichytrium sp. SEK571 strain is shown, relative to the ratio of the wild-type strain of Parietichytrium sp. SEK571 strain, which is set at 100%. [Figure 35]The results of RACE for amplifying the elongase gene derived from T. aureum ATCC 34304 in Comparative Example 1-2 are shown. (Brief explanation of symbols) 1: 5'-RACE using a synthetic adapter-specific oligonucleotide and a degenerate oligonucleotide elo-R 2: 3'-RACE using a synthetic adapter-specific oligonucleotide and a degenerate oligonucleotide elo-F 3: 5'-RACE using elo-R alone (negative control) 4: 3'-RACE using elo-F alone (negative control) 5: 5'-RACE using a synthetic adapter-specific oligonucleotide alone (negative control) 6: 3'-RACE using a synthetic adapter-specific oligonucleotide alone (negative control) [Figure 36] Evaluation of transformants into which KONeor was introduced in Comparative Examples 1-7 is shown. (A) Oligonucleotide primer pairs used in evaluation of transformants by PCR using genomic DNA as a template are shown. (Brief explanation of symbols) (1) Neor detection primers (SNeoF and SNeoR) (2) KO confirmation 1 (KO Pro F SmaI and KO Term R SmaI) (3) KO confirmation 2 (E2 KO Pro F EcoRV and SNeoR) (4) KO confirmation 3 (SNeoF and E2 KO Term R EcoRV) (5) TaELO2 detection (E2 HindIII and E2 XbaI) (B) Agarose electrophoresis pattern of transformants evaluated by PCR using genomic DNA as a template. (Brief explanation of symbols) 1, 5, 9, 13, 17: transformant 2, 6, 10, 14, 18: wild-type strain 3, 7, 11, 15, 19: KONeor used as template 4, 8, 12, 16: no template The oligonucleotide primer pairs (1) to (5) used are listed above the lane numbers. [Figure 37]The results of confirming the copy number of TaELO2 by Southern blotting in Comparative Examples 1 to 8 are shown below. (Brief explanation of symbols) 1: Genomic DNA (2.5 μg), BamHI treatment, 2: BglII treatment, 3: EcoRI treatment, 4: EcoRV treatment, 5: HindIII treatment, 6: KpnI treatment, 7: SmaI treatment, 8: XbaI treatment, 9: Positive control (PCR product amplified with 1 ng of E2 KO ProF EcoRV and E2 KO Term R EcoRV, containing TaELO2). [Figure 38] The Southern blotting analysis of transformants into which TKONeor was introduced in Comparative Examples 1-9 is shown. (A) A schematic diagram of Southern blotting for detecting wild-type alleles or mutant alleles resulting from TKONeor introduction is shown. (B) The results of Southern blotting are shown. (Brief explanation of symbols) 1: T. aureum wild-type strain (2.5 μg of genomic DNA) 2, 3: TKONeor-introduced transformants (2.5 μg of genomic DNA) 4: Positive control (PCR product amplified with 50 ng of E2 KO ProF EcoRV and E2 KO Term R EcoRV, containing TaELO2) [Figure 39] The figures show evaluation by PCR using genomic DNA as a template of transformants obtained by reintroduction of KOub600Hygr in Comparative Examples 1-11. (A) The oligonucleotide primer pairs used are shown. (Brief explanation of symbols) (1) TaELO2 ORF detection (SNeoF and SNeoR) (2) KO confirmation (E2 KO Pro F EcoRV and ubi-hygro R) (B) Agarose electrophoresis of PCR using oligonucleotide primer pair (1) for KO confirmation. The arrow indicates a transformant in which amplification of a specific product was confirmed and which was presumed to be a TaELO2-deficient homozygote. (C) Agarose electrophoresis of PCR using oligonucleotide primer pair (2) for TaELO2 ORF detection in a transformant identified as a TaELO2-deficient homozygote. (Brief explanation of symbols) 1: KOub600Hygr used as a template 2: Wild-type strain [Figure 40]The Southern blotting analysis of transformants obtained by reintroducing KOub600Hygr in Comparative Examples 1-11 is shown. (A) A schematic diagram of Southern blotting for detecting wild-type alleles, mutant alleles due to KONeor introduction, and mutant alleles due to KOub600Hygr introduction is shown. (B) Southern blotting results are shown. (Brief explanation of symbols) 1, 9: wild-type strain 2-8 and 10-16: TaELO2-deficient homozygotes [Figure 41]

[0047] Figure 1 shows the results of Southern blotting to detect TaELO2 in Comparative Examples 1-11. (Brief explanation of symbols) 1: Wild-type strain 2-5: TaELO2-deficient homozygote [Figure 42] The results of agarose gel electrophoresis of RT-PCR for detecting TaELO2 mRNA in Comparative Examples 1-11 are shown. (Brief explanation of symbols) 1-4: TaELO2-deficient homozygote 5: Wild-type strain 6-9: TaELO2-deficient homozygote, total RNA used as template (negative control) 10: Wild-type strain, total RNA used as template (negative control) 11: Wild-type genomic DNA used as template (positive control)

[0024] [Figure 43] 1 shows the results of a comparison of fatty acid compositions between a wild-type strain and a TaELO2-deficient homozygote in Comparative Examples 1-12. [Figure 44] This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fused sequence of 18S rDNA derived from Thraustochytrium aureum ATCC 34304, the EF1α promoter derived from Thraustochytrium aureum ATCC 34304, the synthetic neomycin resistance gene, and the EF1α terminator derived from Thraustochytrium aureum ATCC 34304. [Figure 45]This shows a plasmid into which a portion of the DNA fragments ligated in Figure 42 was cloned. It contains a partial sequence 3' from the EcoRI site of 18S rDNA derived from Thraustochytrium aureum ATCC 34304, the EF1α promoter derived from Thraustochytrium aureum ATCC 34304, an artificially synthesized neomycin resistance gene, and a partial sequence 5' from the NcoI site of the EF1α terminator derived from Thraustochytrium aureum ATCC 34304. [Figure 46] This shows the targeting vector for the Thraustochytrium aureum ATCC 34304 PKS pathway-related gene orfA that was constructed. It contains the neomycin resistance gene as a drug resistance marker. [Figure 47] This represents a plasmid containing the upstream sequence of the Thraustochytrium aureum ATCC 34304 PKS pathway-related gene orfA, the Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter, and the hygromycin resistance gene. [Figure 48] This shows the targeting vector for the Thraustochytrium aureum ATCC 34304 PKS pathway-related gene orfA that was constructed. It contains the hygromycin resistance gene as a drug resistance marker. [Figure 49] 1 shows a schematic diagram showing the positions of probes for Southern hybridization analysis used to identify a PKS pathway-related gene orfA disruptant strain of Thraustochytrium aureum ATCC 34304, and the predicted sizes of gene fragments. [Figure 50]This shows the evaluation of disruption of the PKS pathway-related gene orfA by Southern hybridization using Thraustochytrium aureum ATCC 34304 genomic DNA. (Brief explanation of symbols) T.au: Thraustochytrium aureum ATCC 34304 wild-type strain + / -: Homologous recombinant of the first allele of the PKS pathway-related gene orfA derived from Thraustochytrium aureum ATCC 34304 - / -: Disrupted strain of the PKS pathway-related gene orfA derived from Thraustochytrium aureum ATCC 34304 [Figure 51] This shows a comparison of the fatty acid composition of the Thraustochytrium aureum ATCC 34304 wild-type strain and a PKS pathway-related gene orfA gene disruptant. The white and black bars represent the fatty acid compositions of the wild-type strain and the gene disruptant, respectively. Values ​​are the mean ± standard deviation. [Figure 52] The fatty acid ratio of the PKS pathway-related gene orfA gene disruptant is shown, relative to the ratio of the wild-type strain of Thraustochytrium aureum ATCC 34304, which is set at 100%.

[0025] [Figure 53] This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fusion sequence of the ubiquitin promoter derived from Thraustochytrium aureum ATCC 34304 and the blasticidin resistance gene derived from pTracer-CMV / Bsd / lacZ. [Figure 54] This shows the constructed pTracer-CMV / Bsd / lacZ-derived blasticidin resistance gene BglII cassette. [Figure 55] This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fused sequence of the ubiquitin promoter derived from Thraustochytrium aureum ATCC 34304 and the Enhanced GFP gene (Clontech). [Figure 56]This is a schematic diagram of the primers used in fusion PCR and the product. The final product is a fused sequence of the ubiquitin promoter derived from Thraustochytrium aureum ATCC 34304, the Enhanced GFP gene (Clontech), and the Zeocin resistance gene derived from pcDNA3.1 Zeo(+). [Figure 57] This shows the BglII cassette of the enhanced GFP-zeocin resistance fusion gene that was prepared. [Figure 58] Figure 1 shows a plasmid containing the cloned Thraustochytrium aureum ATCC 34304 C20 elongase sequence and surrounding sequences. [Figure 59] This shows a plasmid in which the Thraustochytrium aureum ATCC 34304 C20 elongase sequence was completely deleted from the plasmid shown in Figure 56 and a BglII site was inserted. [Figure 60] These figures show the two Thraustochytrium aureum ATCC 34304 C20 elongase gene targeting vectors we constructed. They carry either the blasticidin resistance gene (pRH43) or the enhanced GFP-zeocin resistance fusion gene (pRH54) as a drug resistance marker. [Figure 61] 1 shows a schematic diagram showing the positions of probes used in Southern hybridization analysis to identify a C20 elongase gene disruptant in the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene) disruptant, and the predicted size of the gene fragment. [Figure 62]This shows the evaluation of C20 elongase gene disruption by Southern hybridization using Thraustochytrium aureum ATCC 34304 genomic DNA. (Brief explanation of symbols) T.au: Thraustochytrium aureum ATCC 34304 wild-type strain. - / -: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) and C20 elongase gene double disruption strain.

[0026] [Figure 63] This figure shows a comparison of the fatty acid composition of the Thraustochytrium aureum ATCC 34304 wild-type strain and a double disruptant of the PKS pathway (orfA gene) and C20 elongase gene. The white and black bars represent the fatty acid composition of the wild-type strain and the gene disruptant, respectively. Values ​​are the mean ± standard deviation. [Figure 64] The fatty acid ratios of the double disruption strain of the PKS pathway (orfA gene) and C20 elongase gene are shown, relative to the ratio of the wild-type strain of Thraustochytrium aureum ATCC 34304, which is set at 100%. [Figure 65] This represents a plasmid containing the cloned 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. [Figure 66] This shows a plasmid in which 60 bp upstream of the Δ4 desaturase gene and a 556 bp sequence (616 bp, SEQ ID NO: 205) containing the initiation codon within the Δ4 desaturase gene were deleted from the plasmid shown in Figure 63, and a BglII site was inserted into the deleted region. [Figure 67] These figures show the two Δ4 desaturase gene targeting vectors constructed for the Thraustochytrium aureum ATCC 34304 strain. The vectors carry a blasticidin resistance gene (pTM6) or an enhanced GFP-zeocin resistance fusion gene (pTM8) as a drug resistance marker. [Figure 68]FIG. 1 shows a schematic diagram illustrating the positions of PCR primers used to identify a Δ4 desaturase gene-disrupted strain of Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene)-disrupted strain, and the predicted products. [Figure 69] The figure shows the evaluation of Δ4 desaturase gene disruption by PCR using Thraustochytrium aureum ATCC 34304 strain genomic DNA as a template. (Brief explanation of symbols) + / +: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disruptant + / -: Homologous recombinant of the first Δ4 desaturase gene allele derived from the Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) disruptant - / -: Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) and Δ4 desaturase gene double disruptant [Figure 70] This figure shows a comparison of the fatty acid compositions of the wild-type strain of Thraustochytrium aureum ATCC 34304 and a double-disruption strain of the PKS pathway (orfA gene) and the Δ4 desaturase gene. The white and black bars represent the fatty acid compositions of the wild-type strain and the gene-disruption strain, respectively. [Figure 71] The fatty acid ratios of the double disruption strain of the PKS pathway (orfA gene) and the Δ4 desaturase gene are shown, relative to the ratio of the wild-type strain of Thraustochytrium aureum ATCC 34304, which is set at 100%. DETAILED DESCRIPTION OF THE INVENTION

[0027] The impetus for this invention was the discovery of a new pattern in the biosynthetic pathway of polyunsaturated fatty acids (PUFAs) in microorganisms known as Labyrinthulomycetes. While PUFAs are generally known to be biosynthesized via the elongase / desaturase pathway, some organisms also synthesize them via a separate pathway known as the PUFA-PKS pathway. Previously, it was known that Labyrinthulomycetes existed in two types: (I) those that produce PUFAs exclusively via the PUFA-PKS pathway, and (II) those that produce PUFAs via both the elongase / desaturase pathway and the PUFA-PKS pathway. In this study, we discovered a new type, (III), that produces PUFAs exclusively via the elongase / desaturase pathway. In essence, we have discovered a new "pattern" of PUFA biosynthetic pathways. For example, (1) Labyrinthules that have no or very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway and have the ability to produce PUFAs via the endogenous elongase / desaturase pathway. The present invention relates to the above-mentioned (III) type of Labyrinthules that produce PUFAs exclusively via the elongase / desaturase pathway. It encompasses Labyrinthules that have been isolated, cultured, and amplified from wild-type strains that have the (III) type of PUFA biosynthetic pathway, as well as Labyrinthules that have "no or very weak" ability to produce PUFAs via the PUFA-PKS pathway. (2) The Labyrinthulea according to (1) above, characterized in that it does not have an endogenous PUFA-PKS pathway. This invention limits the above (1) to Labyrinthulea that does not have the genes or enzymes that constitute the PUFA-PKS pathway. (3) Labyrinthules according to (1) above, characterized in that the activity of the endogenous PUFA-PKS pathway is absent or extremely weak. This invention limits (1) above, and specifies a pattern in which information on the genes of the enzymes that make up the PUFA-PKS pathway is present in the genome but is not expressed (and therefore has no activity), or is expressed only extremely weakly. (4) A Labyrinthula according to any one of (1) to (3) above, characterized in that it loses its ability to produce DHA and / or DPA n-6 or the amount of DHA and / or DPA n-6 produced is significantly reduced due to disruption of the endogenous Δ4 desaturase gene. Type (III) Labyrinthules that produce PUFAs exclusively via the elongase / desaturase pathway differ from type (I) Labyrinthules that produce PUFAs exclusively via the PUFA-PKS pathway and type (II) Labyrinthules that produce PUFAs exclusively via the elongase / desaturase pathway and the PUFA-PKS pathway in that they do not have a PUFA-PKS pathway, and the present invention defines Labyrinthules that produce PUFAs exclusively via the elongase / desaturase pathway based on this perspective. In other words, disruption of the endogenous Δ4 desaturase gene as described above can be said to be a method for determining whether or not a species is the type that produces PUFAs exclusively via the elongase / desaturase pathway. (5) Labyrinthulae according to any one of (1) to (3) above, which have lost the ability to produce DHA and / or DPAn-6 or whose production of DHA and / or DPAn-6 is significantly reduced due to disruption of the endogenous C20 elongase gene. Type (III) Labyrinthules, which produce only via the elongase / desaturase pathway, differ from type (I) Labyrinthules, which produce only via the PUFA-PKS pathway, and type (II) Labyrinthules, which produce only via the elongase / desaturase pathway and a PUFA-PKS pathway, in that they do not have a PUFA-PKS pathway. Based on this perspective, the present invention defines Labyrinthules of the type that produce only via the elongase / desaturase pathway. In other words, disruption of the endogenous C20 elongase gene as described above can be said to be a method for determining whether or not a type produces only via the elongase / desaturase pathway. (6) Labyrinthulida are of the genus Parietychytrium ( Parietichytrium ) or Schizochytrium spp. ( Schizochytrium ) . The Labyrinthulea according to any one of (1) to (5) above, characterized in that it is a microorganism belonging to any one of the above. Parietichytrium Genus and SchizochytriumAlthough the Labyrinthulomycetes belonging to this genus were known before the filing of the present application, it was not known that they were type (III) Labyrinthulomycetes that produce PUFAs exclusively through the elongase / desaturase pathway. (7) Microorganisms Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK571 (FERM BP-11406) or Schizochytrium aggregatum The Labyrinthula according to (6) above, characterized in that it is ATCC28209. (8) A method for producing a lipid containing PUFA, which comprises culturing the Labyrinthula according to any one of (1) to (7) above in a medium and collecting lipid from the culture. (9) A method for producing lipids containing PUFAs, comprising using the Labyrinthulea as a host according to any one of (1) to (7) above, culturing the transformed Labyrinthulea in a medium for the purpose of modifying the fatty acid composition and / or accumulating fatty acids at a high level, and recovering the lipids from the culture. (10) A PUFA-containing lipid produced by the method of (8) or (9) above. According to the present invention, it is possible to provide a Labyrinthula that produces PUFAs solely through the elongase / desaturase pathway. It is also possible to create Labyrinthula equivalent to type (III), which produces polyunsaturated fatty acids using only the elongase / desaturase pathway, from type (II), which produces polyunsaturated fatty acids using the elongase / desaturase pathway and the PUFA-PKS pathway, by mutation or genetic recombination. Examples of such microorganisms include those belonging to the genus Thraustochytrium. By using these, it is possible to obtain polyunsaturated fatty acids similar to those of type (III) Labyrinthula.

[0028] [Microorganisms] Labyrinthules that have no or very weak ability to produce PUFAs via the PUFA-PKS pathway include Labyrinthules that produce PUFAs only via the elongase / desaturase pathway. Extremely weak PUFA production ability refers to Labyrinthules that are unable to produce PUFAs due to disruption of the genes in the elongase / desaturase pathway, and therefore cannot be cultured unless PUFAs are added to the medium. This refers to Labyrinthules in which the amount of DHA synthesized via the PUFA-PKS pathway is 1 / 100 or less of the DHA synthesized in vivo. Labyrinthules that have no or very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway and can produce PUFAs via the endogenous elongase / desaturase pathway are particularly preferred, but Parietichytrium Genus or Schizochytrium Among them, particularly preferred are Labyrinthula belonging to the genus Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK571 (FERM BP-11406) or Schizochytrium aggregate An example of such a strain is ATCC28209. Parietichytrium Sp. SEK358 was obtained as follows. First, 10 ml of surface water collected from the Miyara River estuary on Ishigaki Island was placed in a test tube, pine pollen was added, and the mixture was left at room temperature. After 7 days, the pine pollen was spread on a sterile agar medium (2 g glucose, 1 g peptone, 0.5 g yeast extract, 0.2 g chloramphenicol, 15 g agar, 100 ml distilled water, and 900 ml seawater). Five days later, colonies emerged. The process was repeated several times to isolate the fungus. This strain was deposited on August 11, 2011, with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Chuo-dai 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, Japan) under accession number FERM BP-11405 and is available from there. Parietichytrium SarkarianSEK364 was isolated from seawater samples collected from the estuary of the Fukidori River on Ishigaki Island by culturing the bacteria in the same manner as above. This strain was deposited internationally with the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture) under accession number FERM BP-11298 on September 24, 2010, and is available from there. Parietichytrium The strain was isolated from seawater samples collected from the estuary of the Ushiro River on Iriomote Island by culturing it in the same manner as above. This strain was deposited on August 11, 2011, at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture) under the accession number FERM BP-11406. Schizochytrium aggregate ATCC28209 has been deposited with ATCC and is available from there. Labyrinthulea in which the native PUFA-PKS has been disrupted or its expression has been very weakly suppressed include Labyrinthulea that produce PUFAs via the elongase / desaturase pathway + PUFA-PKS pathway. PUFAs produced by the PUFA-PKS pathway alone require the presence of the PUFA-PKS pathway, and disruption of the pathway results in PUFA auxotrophy. The difference is that Labyrinthulea that produce PUFAs via the elongase / desaturase pathway + PUFA-PKS pathway, which can produce PUFAs even when the PUFA-PKS pathway is disrupted, do not become PUFA auxotrophic. By utilizing the present invention to disrupt the Δ4 desaturase gene, it is possible to dramatically reduce the amounts of DHA and n-6DPA produced while maintaining the overall amount of PUFA produced. The properties possessed by the microorganisms obtained by the present invention can be combined in any desired manner to obtain desired properties.

[0029] [Microbial oil] The present invention relates to a microbial oil containing the fatty acid profile of the present invention. The microorganism of the present invention contains lipids at a lipid content of 15% by weight or more, preferably 30% by weight or more, more preferably 50% by weight or more, and even more preferably 70% by weight or more per gram of bacterial cells. The lipid components contain 30% by weight or more, preferably 50% by weight or more, and even more preferably 70% by weight or more fatty acids. The lipid acid components are accumulated as triglycerides at 70% by weight or more, preferably 80% by weight or more, and even more preferably 90% by weight or more. The microbial oil of the present invention is a "crude oil" or a "refined oil" containing at least about 35% by weight of a triacylglycerol fraction. "Crude oil" is oil extracted from microbial biomass without further processing. "Refined oil" is oil obtained by treating crude oil with standard refining, bleaching, and / or deodorizing processes. Microbial oils also include "finished oils," which are refined oils diluted with vegetable oil. "Microorganism" includes, but is not limited to, "microalgae," "labyrinthulophora," and taxonomic groups related to any of the deposited microorganisms described herein. The terms "labyrinthulophora," "microalgae ... Parietichytrium Genus”, “ Schizochytrium The terms "genus" and "genus Thraustochytrium" are based on current taxonomic classification, including available phylogenetic information, and are not intended to be limiting in the event that the taxonomic classification is revised after the filing date of this application. [Lipids] In the present invention, lipids refer to lipids produced by Labyrinthulea, and are mainly triglycerides, diglycerides, monoglycerides, phospholipids, free fatty acids, sterols, carotenoids, hydrocarbons, and the like.

[0030] [Fat] In the present invention, lipids refer to lipids produced by Labyrinthulea, and are mainly triglycerides, diglycerides, monoglycerides, phospholipids, free fatty acids, sterols, carotenoids, hydrocarbons, and the like.

[0031] [Highly unsaturated fatty acids] In the present invention, polyunsaturated fatty acids (PUFAs) are fatty acids having 18 or more carbon atoms and 3 or more double bonds, and more preferably 20 or more carbon atoms and 3 or more double bonds. Specifically, these include linoleic acid (LA, 18:2n-6), alpha-linolenic acid (ALA, 18:3n-3), gamma-linolenic acid (GLA, 18:3n-6), stearidonic acid (STA, 18:4n-3), dihomo-gamma-linolenic acid (DGLA, 20:3n-6), eicosatetraenoic acid (ETA, 20:4n-3), arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosatetraenoic acid (DTA, 22:4n-6), n-3 docosapentaenoic acid (n-3DPA, 22:5n-3), n-6 docosapentaenoic acid (n-6DPA, 22:5n-6), and docosahexaenoic acid (DHA, Examples include 22:6n-3). In this specification, arachidonic acid is also referred to as ARA. The total fatty acid composition refers to the fatty acids detected when a microorganism is cultured, freeze-dried, and the fatty acids are methyl-esterified and analyzed using GC, specifically the composition of fatty acids with carbon chains of 14 to 22. In the present invention, the GC area refers to the peak area in a GC chart. The proportion to the total fatty acid composition is the ratio of the peak area of ​​the target fatty acid to the total peak area of ​​the total fatty acid composition, and is expressed as a percentage. In this invention, C20 PUFA / C22 PUFA refers to the value obtained by dividing the total GC peak area of ​​highly unsaturated fatty acids with a carbon chain of 20 by the total GC peak area of ​​highly unsaturated fatty acids with a carbon chain of 22. In this invention, n-6 PUFA / n-3 PUFA refers to the value obtained by dividing the peak area in a GC chart of ω-6 fatty acids with a carbon chain of 20 or more by the peak area in a GC chart of ω-3 fatty acids with a carbon chain of 20 or more.

[0032] [PUFA biosynthetic pathway] Two different pathways are known for the biosynthesis of PUFAs. One is a pathway that produces highly unsaturated fatty acids (PUFAs) using polyketide synthases (PKS). In the present invention, this metabolic pathway is referred to as the PUFA-PKS pathway or PKS pathway. In the present invention, PKS genes refer to genes that encode proteins that constitute polyketide synthases. Polyketide synthases are enzymes that catalyze the reaction of multiple condensations of extended-chain substrates such as malonyl-CoA with starter substrates such as acetyl-CoA, and are generally known as enzymes involved in the biosynthesis of secondary metabolites in plants and fungi, but have also been reported to be involved in the biosynthesis of PUFAs in certain organisms. For example, marine bacteria Shewanella produces eicosapentaenoic acid (EPA) using this enzyme (Non-Patent Document 8). It is also known that polyketide synthases are involved in the biosynthesis of PUFAs in certain Labyrinthulea species. The other pathway, which starts with fatty acids such as palmitic acid and produces PUFAs such as EPA and DHA through repeated desaturation by desaturases (fatty acid desaturase enzymes) and chain elongation by elongases (fatty acid chain elongation enzymes), is referred to as the elongase / desaturase pathway in the present invention. Examples of enzymes that constitute this system include fatty acid synthesis-related enzymes such as C20 elongase and Δ4 desaturase.

[0033] [Method for identifying PUFA biosynthetic pathways] A method for identifying the PUFA biosynthetic pathway of Labyrinthulomycetes is described below. However, this is merely an example, and it goes without saying that this method is not necessarily required for identification. As mentioned above, two pathways for PUFA biosynthesis are known in Labyrinthulomycetes. One is the PUFA-PKS pathway, which produces DHA and DPAn-6. This pathway is characterized by the fact that it produces almost no PUFAs other than DHA and DPAn-6, which is a major difference from the other PUFA biosynthetic pathway, the elongase / desaturase pathway. Another pathway, the elongase / desaturase pathway, is shown in Figure 1. Among the enzymes that make up this pathway, Δ4 desaturase is involved in converting DPAn-3 to DHA. This enzyme is also involved in converting DTA to DPAn-6. Therefore, disruption of the gene encoding this enzyme prevents these conversions, resulting in the accumulation of the enzyme's substrates, DPAn-3 and DTA, and conversely, a decrease in the enzyme's products, DHA and DPAn-6. If the products DHA and / or DPAn-6 are not detected or are significantly reduced in Labyrinthulomycetes in which the Δ4 desaturase gene has been disrupted, the Labyrinthulomycetes are judged to have no or very weak ability to produce PUFAs (especially DHA and / or DPAn-6) via the endogenous PUFA-PKS pathway. Instead of Δ4 desaturase, a C20 elongase can be selected and the gene for this enzyme disrupted. Specifically, C20 elongase is an enzyme involved in converting EPA to DPAn-3. It is also involved in converting ARA to DTA. Therefore, disruption of the gene for this enzyme prevents these conversions, resulting in the accumulation of its substrates, EPA and ARA, and conversely, the production of its products, DPAn-3 and DTA, decreases. Consequently, the production of DHA and DPAn-6, which are derived from DPAn-3 and DTA as substrates, also decreases. If the end products DHA and / or DPAn-6 are not detected or are significantly reduced in Labyrinthulomycetes in which the C20 elongase gene has been disrupted, the Labyrinthulomycetes are judged to have no or very weak ability to produce PUFAs (especially DHA and / or DPAn-6) via the endogenous PUFA-PKS pathway. Gene disruption is achieved by introducing into cells, using methods such as a gene gun, electroporation, or genome editing, an antibiotic resistance gene or the like that is designed to inactivate part or all of the target gene. Gene suppression is achieved by introducing into cells, using methods such as a gene gun, electroporation, or genome editing, an antisense gene designed to suppress the expression of the target gene, or a gene that expresses RNAi. Gene disruption and suppression are not limited to these methods as long as the expression of the target gene is inhibited. When disrupting or suppressing a PKS gene, there are no limitations as long as the enzyme activity can be eliminated or suppressed, but OrfA is preferably the target.

[0034] The present invention provides Labyrinthules that have no or very weak ability to produce PUFAs through the endogenous PUFA-PKS pathway, but have the ability to produce PUFAs through the endogenous elongase / desaturase pathway. The present invention also encompasses altering the fatty acid composition produced by Labyrinthulea by manipulating the genes for enzymes that make up the elongase / desaturase pathway of Labyrinthulea, and in particular, the fatty acid composition produced by Labyrinthulea can be altered by (1) disrupting or / and suppressing expression of a fatty acid chain elongase gene, (2) disrupting or / and suppressing expression of a fatty acid desaturase gene, (3) introducing a fatty acid chain elongase gene, (4) introducing a fatty acid desaturase gene, and (5) a combination of these. For example, if stearidonic acid (STA) is desired, the gene for the fatty acid chain elongase involved in converting stearidonic acid to eicosatetraenoic acid (ETA), specifically the C18 elongase gene, can be disrupted or / and suppressed expression. For example, if eicosapentaenoic acid (EPA) is desired, the gene encoding a fatty acid chain elongation enzyme involved in converting eicosapentaenoic acid to docosapentaenoic acid (DPA), specifically the C20 elongase gene, can be disrupted or / and expression suppressed. For example, if eicosapentaenoic acid is desired, the gene encoding a fatty acid desaturase that converts arachidonic acid (ARA) to eicosapentaenoic acid, specifically the ω3 desaturase gene, can be introduced. For Labyrinthuleans that produce fatty acids via the elongase / desaturase pathway plus the PUFA-PKS pathway, the fatty acid composition produced by the Labyrinthuleans can be modified by (1) disrupting or / and suppressing the expression of a fatty acid chain elongase gene, (2) disrupting or / and suppressing the expression of a fatty acid desaturase gene, (3) introducing a fatty acid chain elongase gene, (4) introducing a fatty acid desaturase gene, or (5) a combination of these, using a microorganism in which the PUFA-PKS pathway has been disrupted and / or suppressed. For example, if stearidonic acid (STA) is desired, the gene for the fatty acid chain elongase involved in converting stearidonic acid to eicosatetraenoic acid (ETA), specifically the C18 elongase gene, can be disrupted and / or suppressed. For example, if eicosapentaenoic acid (EPA) is desired, the gene for the fatty acid chain elongation enzyme involved in converting eicosapentaenoic acid to docosapentaenoic acid (DPA), specifically the C20 elongase gene, can be disrupted or / and its expression suppressed.Furthermore, for example, when eicosapentaenoic acid is desired, a gene for a fatty acid desaturase that converts arachidonic acid (ARA) to eicosapentaenoic acid, specifically an ω3 desaturase gene, may be introduced.

[0035] Transformation of Labyrinthulea can produce Labyrinthulea (microorganisms) with an altered fatty acid composition. These Labyrinthulea into which fatty acid biosynthesis-related genes have been introduced and / or disrupted can be used, for example, to produce unsaturated fatty acids. For the production of unsaturated fatty acids, Labyrinthulea that have no or very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway and have the ability to produce PUFAs via the endogenous elongase / desaturase pathway, or Labyrinthulea with the altered fatty acid composition described above, may be used; other steps and conditions, such as production equipment and tools, are not particularly limited. The production of unsaturated fatty acids includes culturing Labyrinthulea that have no or very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway and have the ability to produce PUFAs via the endogenous elongase / desaturase pathway, or microorganisms with an altered fatty acid composition produced by the above-described modification method, and unsaturated fatty acids are produced using the microorganisms and their culture media.

[0036] The culture conditions for the above cells (culture medium, culture temperature, aeration state, etc.) can be appropriately set depending on the type of cell, the type of unsaturated fatty acid of interest, its amount, etc. In addition, the unsaturated fatty acid in the present invention also refers to a substance containing unsaturated fatty acids, and its content, purity, shape, composition, etc. are not particularly limited. In other words, in the present invention, the cells with modified fatty acid composition or the culture medium itself may be considered as unsaturated fatty acids. Furthermore, a step of purifying the unsaturated fatty acids from the cells or culture medium may be further included. As a method for purifying unsaturated fatty acids, known methods for purifying lipids (including complex lipids) such as unsaturated fatty acids can be applied.

[0037] [Method for accumulating highly unsaturated fatty acids in labyrinthula] Accumulation of unsaturated fatty acids in Labyrinthulea can be achieved by culturing Labyrinthulea that lack or have only a very weak ability to produce PUFAs via the endogenous PUFA-PKS pathway but have the ability to produce PUFAs via the endogenous elongase / desaturase pathway, or Labyrinthulea transformed with such Labyrinthulea. For example, the culture can be performed in a conventional solid or liquid medium. The medium used can be, for example, a carbon source such as glucose, fructose, sucrose, starch, or glycerin; a nitrogen source such as yeast extract, corn steep liquor, polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate, ammonium nitrate, ammonium chloride, or sodium nitrate; an inorganic salt such as potassium phosphate; and other necessary components. While there are no particular limitations on the medium, as long as it is conventionally used for culturing Labyrinthulea, yeast extract-glucose medium (GY medium) is particularly preferred. After preparation, the pH of the medium is adjusted to 3.0–8.0 and sterilized by autoclaving or other methods before use. The culture may be carried out at 10 to 40°C, preferably 15 to 35°C, for 1 to 14 days by aeration and agitation culture, shaking culture, or static culture.

[0038] To recover the produced unsaturated fatty acids, Labyrinthulea are grown in a culture medium, and the microbial cells obtained from the culture medium are treated to release intracellular lipids (oil-containing substances containing highly unsaturated fatty acids or highly unsaturated fatty acids), and the lipids are recovered. That is, the Labyrinthulea cultured in this manner are recovered by centrifugation or the like, and if necessary, subjected to treatments such as drying and cell disruption, and then extracted using an appropriate organic solvent, supercritical carbon dioxide, liquefied dimethyl ether, or the like according to a standard method, to obtain lipids containing PUFAs. The microbial oil obtained in the present invention satisfies any of the following conditions. In the microbial oil obtained in the present invention, ARA accounts for 5% or more, 7% or more, 10% or more, or 15% of the total fatty acid composition. The high-ARA oils thus obtained can be used for nutritional supplements for infants, health foods for adults, and pharmaceutical applications. ARA can be 80% or less, 70% or less, 60% or less, or 50% or less of the total fatty acid composition. In the microbial oil obtained in the present invention, DGLA accounts for 2.5% or more, 5% or more, or 10% or more of the total fatty acid composition. The high-DGLA microbial oil obtained in this way can be used for pharmaceutical applications, including anti-inflammatory agents. DGLA can be 80% or less, 70% or less, 60% or less, or 50% or less of the total fatty acid composition. In the microbial oil obtained in the present invention, ETA accounts for 0.35% or more, 0.5% or more, 0.75% or more, or 1% or more of the total fatty acid composition. The high-ETA microbial oil obtained in this way can be used for pharmaceutical applications, including the treatment of arthritis. ETA can be 50% or less, 40% or less, 30% or less, or 20% or less of the total fatty acid composition. The microbial oil obtained in the present invention has an EPA content of 4% or more, 6% or more, 8% or more, 10% or more, or 12% or more of the total fatty acid composition. The microbial oil with a high EPA content obtained in this manner can be used for nutritional supplementation and pharmaceutical applications. EPA can be 80% or less, 70% or less, 60% or less, or 50% or less of the total fatty acid composition. The microbial oil obtained in the present invention has an n-6DPA content of 0.20% or less, 0.15% or less, 0.1% or less, or 0.05% or less of the total fatty acid composition. The low n-6DPA microbial oil obtained in this manner is less likely to inhibit the functions of other fatty acids. It is also advantageous when n-6DPA needs to be removed by purification. The n-6DPA content can be 0.001% or more, 0.005% or more, or 0.01% or more of the total fatty acid composition. The microbial oil obtained in the present invention has a DHA content of 0.50% or less, 0.3% or less, 0.2% or less, or 0.1% or less of the total fatty acid composition. The microbial oil with low DHA content obtained in this way is less likely to inhibit the functions of other fatty acids. This is also advantageous when DHA needs to be removed by purification.DHA can be 0.005% or more, 0.01% or more, or 0.05% or more of the total fatty acid composition. In the microbial oil obtained in the present invention, the total of DHA and n-6DPA is 0.7% or less, 0.8% or less, 0.9% or less, or 1.0% or less of the total fatty acid composition. The microbial oil thus obtained, with low DHA and n-6DPA, is less likely to inhibit the functions of other fatty acids and is stable to oxidation. This is also advantageous when removing DHA and n-6DPA by purification. The total of DHA and n-6DPA can be 0.05% or more, 0.1% or more, or 0.5% or more of the total fatty acid composition. The desired concentrations of these fatty acids can be arbitrarily combined, up to a total of 100%, to achieve the desired properties. In another embodiment, the microbial oil obtained by the present invention satisfies any of the following conditions. The microbial oil obtained by the present invention has an LA / DHA value of 0.6 or more, 0.7 or more, 0.8 or more, 0.9 to 10, 9 or less, 8 or less, 7 or less, or 6 or less in GC area. The microbial oil with a high LA / DHA value obtained in this way is stable to oxidation and is less likely to inhibit the function of LA due to DHA. The microbial oil obtained by the present invention has a GLA / DHA value of 0.35 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 to 10, 9 or less, 8 or less, or 7 or less in GC area. The microbial oil with a high GLA / DHA value obtained in this way is stable to oxidation and is less likely to inhibit the function of GLA due to DHA. The microbial oil obtained in the present invention has a DGLA / DHA ratio of 0.35 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 10 or less, 9 or less, 8 or less, or 7 or less, 6 or less, measured by GC area. The microbial oil with a high DGLA / DHA ratio thus obtained can be used for anti-inflammatory drugs, etc. The microbial oil obtained in the present invention has an ARA / DHA ratio of 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less, measured by GC area. The microbial oil with a high ARA / DHA ratio thus obtained can be used in infant formula. The EPA / DHA ratio of 0.35 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less, measured by GC area. The EPA typically used in pharmaceuticals is mainly in the form of an ester containing DHA, but the microbial oil with a high EPA / DHA value obtained in this way contains highly purified EPA and can be used in health foods, pharmaceuticals, etc. By arbitrarily combining the desired ratios of these fatty acids, the desired properties can be combined. The microbial oil obtained in the present invention has an LA / EPA value of 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.12 or less, 0.1 or less, or 0.08 or less, or 0.06 or less, measured by GC area. The microbial oil with a low LA / EPA value thus obtained can be used for infusions with low inflammatory effects. The microbial oil obtained in the present invention has a GLA / EPA value of 0.04 or more, 0.45 or more, 0.12 or less, 0.1 or less, or 0.8 or less, measured by GC area. The microbial oil with a low GLA / EPA value thus obtained can be used for infusions with low inflammatory effects. The microbial oil obtained in the present invention has a DTA / EPA value of 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.4 or less, 0.35 or less, or 0.3 or less, measured by GC area. The microbial oils with low DTA / EPA ratios obtained here are useful for separating high-purity EPA because DTA and EPA are close in GC. By arbitrarily combining the ratios of these fatty acids, desired properties can be achieved. The microbial oil obtained in the present invention has a DTA / ARA value of 0.01 or more, 0.03 or more, 0.05 or more, 0.07 or more, 0.1 or more, 0.45 or less, 0.4 or less, 0.35 or less, or 0.3 or less, measured by GC area. The microbial oil obtained here with a low DTA / ARA value can be used in infant formula. The microbial oil obtained in the present invention has a DTA / DGLA value of 0.01 or more, 0.05 or more, 0.1 or more, 0.15 or more, 0.2 or more, 1.45 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less, measured by GC area. The microbial oil obtained here with a low DTA / DGLA value can be used in pharmaceuticals and the like with anti-inflammatory effects. The microbial oil obtained in the present invention has an LA / n-6DPA value measured by GC area of ​​0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 20 or less, 18 or less, 16 or less, 14 or less, or 12 or less. The microbial oil obtained here with a high LA / n-6DPA value can be used as an edible oil. The microbial oil obtained in the present invention has a GLA / n-6DPA value measured by GC area of ​​0.2 or more, 0.4 or more, 0.6 or more, 0.8 or more, 10 or less, 8 or less, 6 or less, or 4 or less. The microbial oil obtained in the present invention with a high GLA / n-6DPA value can be used in health foods and supplements. The microbial oil obtained in the present invention has a DGLA / n-6DPA value measured by GC area of ​​0.35 or more, 0.5 or more, 0.75 or more, 1.0 or more, 30 or less, 27 or less, 25 or less, 22 or less, or 20 or less. The microbial oils obtained herein with high DGLA / n-6DPA values ​​can be used in drugs with anti-inflammatory effects. The microbial oils obtained herein have GC area ARA / n-6DPA values ​​of 0.7 or more, 1.0 or more, 2.0 or more, 3.0 or more, 60 or less, 50 or less, 40 or less, or 30 or less. The microbial oils obtained herein with high ARA / n-6DPA values ​​can be used in infant formula. The microbial oils obtained herein have GC area EPA / n-6DPA values ​​of 0.4 or more, 0.6 or more, 0.8 or more, 1 or more, 2 or more, 5 or more, 70 or less, 60 or less, 50 or less, 40 or less, or 30 or less. The microbial oils obtained herein with high EPA / n-6DPA values ​​can be used in health foods, supplements, etc.By combining these fatty acids in any desired ratio, desired properties can be achieved. The microbial oil obtained in the present invention has a DGLA / LA value measured by GC area of ​​1.4 or more, 2 or more, 3 or more, 10 or less, 9 or less, 8 or less, or 7 or less. The microbial oil obtained here can be used in foods and supplements with anti-inflammatory effects. The microbial oil obtained in the present invention has an ARA / LA value measured by GC area of ​​5.1 or more, 7 or more, 9 or more, 11 or more, 20 or less, 17 or less, 15 or less, or 12 or less. The microbial oil obtained here with a high ARA / LA value can be used in feed and infant formula. The microbial oil obtained in the present invention has an EPA / LA value measured by GC area of ​​5.5 or more, 7 or more, 9 or more, 11 or more, 13 or more, 30 or less, 25 or less, 22 or less, or 20 or less. The microbial oil obtained here with a high EPA / LA value can be used in medicines and health foods. The microbial oil obtained in the present invention has a DTA / LA value of 0.01 or more, 0.05 or more, 0.07 or more, 0.1 or more, 0.4 or less, 0.35 or less, 0.33 or less, 0.3 or less, or 0.28 or less in GC area. The microbial oil obtained here with a low DTA / LA value can be used in feed and food. The desired ratios of each of these fatty acids can be arbitrarily combined to achieve the desired properties. The microbial oil obtained in the present invention has a DGLA / GLA value of 4.5 or more, 5 or more, 6 or more, 7 or more, 20 or less, 17 or less, 15 or less, or 12 or less, as measured by GC area. The microbial oil obtained here with a high DGLA / GLA value can be used in anti-inflammatory pharmaceuticals, etc. The microbial oil obtained in the present invention has an ARA / GLA value of 9 or more, 10 or more, 12 or more, 13 or more, 14 or more, 30 or less, 28 or less, 26 or less, 24 or less, or 22 or less, as measured by GC area. The microbial oil obtained here with a high ARA / GLA value can be used in infant formula. The desired ratios of these fatty acids can be arbitrarily combined to achieve the desired properties. The microbial oil obtained in the present invention has an n-6DPA / DTA value of 0.001 or more, 0.01 or more, 0.02 or more, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less, measured by GC area. The microbial oil obtained here can be used in foods that prevent arteriosclerosis, etc. The microbial oil obtained in the present invention has a DHA / n-3DPA value of 0.001 or more, 0.01 or more, 0.02 or more, 4 or less, 4.5 or less, 4 or less, or 3.5 or less, measured by GC area. The microbial oil obtained here can be used in feed or health foods. The microbial oil obtained in the present invention has a C20PUFA / C22PUFA value of 0.5 or more, 0.7 or more, 1 or more, 1.2 or more, 50 or less, 45 or less, 40 or less, or 35 or less, measured by GC area. The microbial oil obtained here with a high C20PUFA / C22PUFA value can be used in health foods and supplements. The microbial oil obtained in the present invention has an n-6PUFA / n-3PUFA ratio of 1.8 or more, 2 or more, 2.5 or more, 3 or more, 100 or less, 80 or less, 70 or less, 60 or less, or 50 or less in GC area. The microbial oil obtained here with a high n-6PUFA / n-3PUFA ratio can be used as edible oil or feed. By arbitrarily combining the desired ratios of these fatty acids, desired properties can be combined. The desired ratios of each of these fatty acids can be arbitrarily combined to achieve the desired properties, and the desired ratios and concentrations of each of the fatty acids can be arbitrarily combined to achieve the desired properties, up to a total concentration of 100%.

[0039] The unsaturated fatty acids of the present invention also include various pharmaceuticals, foods, feeds, and industrial products, and their application fields are not particularly limited.Foods containing the unsaturated fatty acid-containing fats of the present invention include health foods such as supplements and food additives.Industrial products include feeds for organisms other than humans, films, biodegradable plastics, functional fibers, lubricating oils, detergents, etc.

[0040] The present invention will now be described in detail with reference to examples. In addition, it should be understood that the features of the invention described in the embodiments relating to each aspect of the invention in this specification may be combined in any manner to form new embodiments, and that such new embodiments may also be included in each aspect of the present invention. [Example]

[0041] [Labyrinthula species and their culture and preservation methods] (1) Strains used in the present invention Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium Sarkarian SEK364 (FERM BP-11298) and Parietichytrium sp. SEK571 (FERM BP-11406) was provided by the Faculty of Science and Engineering, Konan University. Thraustochytrium golden ATCC34304 was provided by ATCC. (2) Medium composition i. Agar plate medium composition PDA agar plate 0.78% (w / v) potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd.), 1.75% (w / v) Sea Life (Marine Tech Co., Ltd.), and 1.21% (w / v) agar powder (Nacalai Tesque) were mixed and sterilized in an autoclave at 121°C for 20 minutes. After sufficient cooling, sodium ampicillinate (Nacalai Tesque) was added to a final concentration of 100 μg / ml to prevent bacterial contamination, and the mixture was dispensed into petri dishes and left to solidify on a flat surface. 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 in an autoclave at 121°C for 20 minutes, after which 100 μg / ml sodium ampicillinate (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 in an autoclave at 121°C for 20 minutes, after which 100 μg / ml sodium ampicillinate (Nacalai Tesque) was added. (3)Culture method i. Agar plate culture Labyrinthula cells were inoculated using a platinum loop or spreader and allowed to stand at 25°C to allow colonies to appear. Subculture was performed by picking up colonies with a platinum loop, suspending them in sterile physiological saline, and then spreading the suspension using a platinum loop or spreader. If necessary, the cells on the plate were inoculated into liquid medium to convert to liquid culture. ii.Liquid culture Labyrinthulea cells were inoculated and suspension cultured in Erlenmeyer flasks or test tubes at 25°C with stirring at 150 rpm. Subculture was performed by adding 1 / 200 to 1 / 10 volume of the culture medium from the logarithmic to stationary phase, where growth had been confirmed, to fresh GY or PD liquid medium. If necessary, the cell culture medium was transferred to agar plate culture by spreading it on PDA agar plates. (4) Preservation and conservation of Labyrinthula In addition to subculture, cells were cryopreserved by preparing glycerol stocks. Specifically, glycerol (Nacalai Tesque) was added to a final concentration of 15% (v / v) to a cell suspension in GY liquid medium from the logarithmic to stationary phase, and the cells were stored in a deep freezer at -80°C. [Example]

[0042] [Parietichytrium Sarkarian Disruption of the C20 elongase gene of SEK364 and measurement of fatty acid composition of lipids produced by the transformant [Example 2-1] Subcloning of SV40 terminator sequence The SV40 terminator sequence was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with the pcDNA 3.1 myc-His vector (Invitrogen) as a template. The PCR primers used were as follows: RHO58 was designed on the SV40 terminator sequence and contained BglII and BamHI linker sequences; RHO52 was designed on the SV40 terminator sequence and contained a BglII sequence. [RHO58: 34-mer: 5'-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCG A-3' (SEQ ID NO: 1); RHO52: 24-mer: 5'-ACG CAA TTA ATG TGA GAT CTA GCT -3' (SEQ ID NO: 2)]. After amplification under the conditions described below, the fragment was cloned into the pGEM-T easy vector (Promega). PCR cycles: 98°C 2 min / 98°C 30 sec, 60°C 30 sec, 72°C 1 min, 30 cycles / 72°C 1 min. After amplification in E. coli, the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This fragment was named pRH27. The plasmid (pRH27) containing the subcloned SV40 terminator sequence (342 bp, SEQ ID NO: 3) is shown in FIG.

[0043] [Example 2-2] Preparation of an artificially synthesized neomycin resistance gene cassette Thraustochytrium golden ATCC 34304 strain was cultured in GY medium, and cells in the late logarithmic growth phase were pelleted by centrifugation at 3,500 x g for 5 minutes at 4°C. The pellet was then frozen in liquid nitrogen and disrupted. The cell lysate was extracted with phenol and precipitated with ethanol. The precipitate was dissolved in TE solution. The nucleic acids dissolved in TE solution were treated with RNase at 37°C for 30 minutes to degrade RNA, and then extracted again with phenol and precipitated with ethanol. The precipitate was dissolved in TE solution. The A260 / 280 ratio was measured and the DNA concentration was calculated. Using this as a template, the ubiquitin promoter sequence (619 bp, SEQ ID NO: 4) was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used were as follows: RHO53 was designed on the ubiquitin promoter sequence and contained a BglII linker sequence; TKO1 contained the ubiquitin promoter sequence and an artificially synthesized neomycin resistance gene sequence. [RHO53: 36mer: 5'- CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG -3' (SEQ ID NO: 5); TKO1: 58mer: 5'- CGT GAA GGC CGT CCT GTT CAA TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G -3' (SEQ ID NO: 6)]. [PCR cycles: 98℃ 2min / 98℃ 10 sec, 68℃ 1 min, 30 cycles / 68℃ 1min]. The artificial neomycin resistance gene sequence (826 bp, SEQ ID NO: 7) was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) with the artificial neomycin resistance gene sequence as a template. The PCR primers used were as follows: TKO2 contains a ubiquitin promoter sequence and the artificial neomycin resistance gene sequence; RHO57 contains the artificial neomycin resistance gene sequence and a BglII linker sequence. [TKO2: 54mer: 5'-AGC GAC CTA AGC AAC ACT AGC CAA CAT GAT TGA ACA GGA CGG CCT TCA CGC TGG -3' (SEQ ID NO: 8); RHO57: 26mer: 5'-CAG ATC TCA AAA GAA CTC GTC CAG GA -3' (SEQ ID NO: 9)]. [PCR cycles: 98℃ 2min / 98℃ 10 sec, 68℃ 1 min, 30 cycles / 68℃ 1min]. Fusion PCR was performed using SEQ ID NOS: 4 and 7 as templates and RHO53 (SEQ ID NOS: 5) and RHO57 (SEQ ID NOS: 9) according to the method described in Non-Patent Document 9. The enzyme used was LA Taq Hot Start Version (Takara Bio Inc.). Amplification was performed under the following PCR cycles: 94°C for 2 minutes, 94°C for 20 seconds, 55°C for 30 seconds, 68°C for 1 minute, 30 cycles, and 68°C for 1 minute (however, the temperature change from 55°C to 68°C was 1°C / 10 seconds), followed by digestion with BglII (Figure 3). Fusion was performed as above. Thraustochytrium golden The ATCC 34304-derived ubiquitin promoter-artificially synthesized neomycin resistance gene sequence (1395 bp, SEQ ID NO: 10) was digested with BglII and ligated into the BamHI site of pRH27 described in Example 2-1. The resulting plasmid was amplified in E. coli and its sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This plasmid was named pRH31. The constructed synthetic neomycin resistance gene cassette (pRH31) is shown in Figure 4.

[0044] [Example 2-3] Construction of a hygromycin resistance gene cassette Thraustochytrium goldenThe ubiquitin promoter sequence (617 bp, SEQ ID NO: 11) was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) with ATCC 34304 genomic DNA as a template. The PCR primers used were as follows: RHO53 was designed on the ubiquitin promoter sequence and contained a BglII linker sequence; KSO8 contained the ubiquitin promoter sequence and a hygromycin resistance gene sequence. [RHO53: 36mer: 5'- CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG -3' (described in Example 2-2, SEQ ID NO: 5); KSO8: 58mer: 5'- TCG CGG TGA GTT CAG GCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G -3' (SEQ ID NO: 12)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 68℃ 2 min, 30 cycles / 68℃ 2min]. The hygromycin resistance gene (1058 bp, SEQ ID NO: 13) was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) with pcDNA 3.1 / Hygro (Invitrogen) as a template. The PCR primers used were as follows: KSO7 contains the ubiquitin promoter sequence and the hygromycin resistance gene sequence; RHO56 contains the hygromycin resistance gene and a BglII linker sequence. [KSO7: 56mer: 5'-AGC GAC CTA AGC AAC ACT AGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GAC GTC TG -3' (SEQ ID NO: 14); RHO56: 36mer: 5'-CAG ATC TCT ATT CCT TTG CCC TCG GAC GAG TGC TGG -3' (SEQ ID NO: 15)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 68℃ 2 min, 30 cycles / 68℃ 2min]. Fusion PCR was performed using SEQ ID NOs: 11 and 13 as templates and RHO53 (described in Example 2-2, SEQ ID NO: 5) and RHO56 (SEQ ID NO: 15) according to the method described in Non-Patent Document 9. The enzyme used was LA Taq Hot Start Version (manufactured by Takara Bio Inc.), and amplification was performed under the following conditions, followed by digestion with BglII. [PCR cycles: 94°C for 2 min / 94°C for 20 sec, 55°C for 30 sec, 68°C for 1 min, 30 cycles / 68°C for 1 min, except that the temperature change from 55°C to 68°C was 1°C / 10 sec] (Figure 5). Fusion was performed as above. Thraustochytrium goldenThe hygromycin resistance gene (1625 bp, SEQ ID NO: 16) derived from ATCC 34304 ubiquitin promoter-pcDNA 3.1 / Hygro (Invitrogen) was digested with BglII and ligated into the BamHI site of pRH27 shown in Example 2-1, Figure 2. The resulting plasmid was amplified in E. coli and its sequence confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This plasmid was named pRH32. The constructed hygromycin resistance gene cassette (pRH32) is shown in FIG.

[0045] [Example 2-4] Parietichytrium Cloning of the C20 elongase gene of the genus By the method described in Example 2-2 Parietichytrium Sarkarian Genomic DNA of SEK364 was extracted and the genome was sequenced. The forward (PsTaELO2 F1; 5'-CCT TCG GCG CTC CTC TTA TGT ATG T -3') (SEQ ID NO: 17) and reverse (PsTaELO2 R2; 5'-CAA TGC AAG AGG CGA ACT GGG AGA G-3') (SEQ ID NO: 18) oligonucleotides were synthesized targeting the region conserved in the C20 elongase gene. Parietichytrium Sarkarian PCR was performed using oligonucleotides PsTaELO2 F1 and PsTaELO2 R2 with SEK364 genomic DNA as a template using LA Taq Hot start version (TaKaRa) [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 infinity]. The specific amplified product was gel-purified and its sequence analyzed by direct sequencing. It showed significant homology with the sequence of a known C20 elongase gene. Parietichytrium SarkarianIt was suggested that this was a partial sequence of the C20 elongase gene derived from SEK364. Subsequently, as in Comparative Example 1-2 described below, 3' and 5' RACE were performed. Parietichytrium Sarkarian The C20 elongase gene from SEK364 was cloned. First, forward oligonucleotide primers (PsRACE F1; 5'-TGG GGC TCT GGA ACC GCT GCT TAC G -3') (SEQ ID NO: 19) and (PsRACE F2; 5'-CTT CCA GCT CTC CCA GTT CGC CTC T -3') (SEQ ID NO: 20) and reverse oligonucleotide primers (PsRACE R1; 5'-CGG GTT GTT GAT GTT GAG CGA GGT G-3') (SEQ ID NO: 21) and (PsRACE R2; 5'-CCC ACG CCA TCC ACG AGC ACA CCA C-3') (SEQ ID NO: 22) were designed. Next, SMART TM Using a cDNA library prepared using the RACE cDNA Amplification Kit (Clontech) as a template, 3'- and 5'-RACE was performed using synthetic adaptor-specific oligonucleotides and the aforementioned oligonucleotides PsRACE F1 or PsRACE R1 [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 infinity]. Next, using both RACE products as templates, nested PCR was performed using synthetic adaptor-specific oligonucleotides and the aforementioned oligonucleotides 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 infinity]. The resulting specific products were gel-purified and TA cloned using pGEM-T easy Vector (Promega). The nucleotide sequence was analyzed. Parietichytrium Sarkarian It was confirmed that this was the C20 elongase gene derived from SEK364. Furthermore, the extract was prepared by the method described in Example 2-2. Parietichytrium Using genomic DNA from the genus Rho153 as a template, a sequence containing the C20 elongase gene sequence (957 bp, SEQ ID NO: 23) was amplified using LA Taq Hot Start version (Takara Bio). The PCR primers used were as follows: RHO153 contains an initiation codon and a BamHI site as a linker sequence; RHO154 contains a termination codon and 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: 24); RHO154: 33-mer: 5'- CCC GGA TCC TTA CTG AGC CTT CTT GGA GGT CTC -3' (SEQ ID NO: 25)]. [PCR cycles: 98℃ 2min / 98℃ 10 sec, 68℃ 1 min, 30 cycles / 68℃ 2min]. The resulting DNA fragment was cloned into pGEM-T easy vector and amplified in Escherichia coli, after which the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). Parietichytrium The 936 bp C20 elongase gene (SEQ ID NO: 26) was cloned into a vector designated pRH80 (FIG. 7). The amino acid sequence is shown in SEQ ID NO: 27.

[0046] [Example 2-5] Parietichytrium Construction of a base plasmid for the construction of a C20 elongase gene targeting vector Using pRH80 (Figure 7) prepared in Example 2-4 as a template, a primer set was prepared in reverse orientation to insert a BglII site into the middle of the C20 elongase gene sequence, and amplification was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). The PCR primers used were as follows, both of which contain a BglII linker sequence: [RHO155: 26-mer: 5'-ACA AAG ATC TCG ACT GGA CCG ACA CC -3' (SEQ ID NO: 28), RHO156: 27-mer: 5'-AGT CGA GAT CTT TGT CAG GAG GTG GAC -3' (SEQ ID NO: 29)]. [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 amplification under the above conditions, the fragment was digested with BglII and then self-ligated. The ligated sample was amplified in E. coli and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This fragment was named pRH83. The 935-bp C20 elongase gene sequence with the BglII site inserted is shown in SEQ ID NO: 30. Made Parietichytrium The base plasmid (pRH83) for the construction of the C20 elongase gene targeting vector is shown in Figure 8. [Example 2-6] Construction of targeting vector (artificially synthesized neomycin gene and hygromycin resistance gene) pRH31 (FIG. 4) described in Example 2-2 was digested with BglII, and a DNA fragment containing an artificially synthesized neomycin resistance gene cassette was ligated into the BglII site of pRH83 (FIG. 8) described in Example 2-5. This was named pRH85. pRH32 (FIG. 6) described in Example 2-3 was digested with BglII, and the DNA fragment containing the hygromycin resistance gene cassette was ligated into the BglII site of pRH83 (FIG. 8) described in Example 2-5. This was named pRH86. The two targeting vectors (pRH85 and 86) that were constructed are shown in FIG.

[0047] [Example 2-7] C20 elongase gene targeting vector introduction Using the two targeting vectors prepared in Example 2-6 as templates and RHO153 (described in Example 2-4, SEQ ID NO: 24) and RHO154 (described in Example 2-4, SEQ ID NO: 25) as primers, genes were amplified using PrimeSTAR Max DNA polymerase (Takara Bio Inc.). [PCR cycles: 98°C 2 min / 98°C 30 sec, 68°C 2 min, 30 cycles / 68°C 2 min]. After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. When pRH85 (Figure 9) described in Example 2-6 was used as a template, the introduced fragment obtained was 2661 bp. Parietichytrium Genus C20 elongase gene first half - SV40 terminator sequence - artificially synthesized neomycin resistance gene sequence - ubiquitin promoter sequence Parietichytrium The sequence is the second half of the C20 elongase gene (SEQ ID NO: 31). When pRH86 (FIG. 9) described in Example 2-6 was used as a template, the fragment obtained was 2892 bp. Parietichytrium Genus C20 elongase gene first half - SV40 terminator sequence - hygromycin resistance gene sequence - ubiquitin promoter sequence Parietichytrium The sequence is the second half of the C20 elongase gene (sequence number 32). Parietichytrium SarkarianThe SEK364 strain was cultured in GY medium for 4 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1–1.5 using the gene gun method (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mm Hg, rupture disk: 1,550 PSI). After a 24-hour recovery period, the transfected cells were plated onto PDA agar plates (containing 2 mg / ml G418 or 2 mg / ml hygromycin). As a result, 10–20 drug-resistant clones were obtained per bombardment.

[0048] [Example 2-8] Identification of C20 elongase gene gene targeting homologous recombinants Genomic DNA was extracted from Parietichytrium sarkarianum strain SEK364, a heterologous C20 elongase gene recombinant, and a homologous C20 elongase gene recombinant (gene disruptant) as described in Example 2-2. The A260 / 280 ratio was measured to calculate the DNA concentration. Using this as a template, PCR was performed to confirm the genome structure using LA Taq Hot Start Version (Takara Bio). The primer positions, amplification combinations, and predicted sizes of the amplified products are shown in Figure 10. RHO184 was located upstream of the C20 elongase gene, RHO185 downstream, RHO142 and RHO143 on the artificial neomycin resistance gene, and RHO140 and RHO141 on the hygromycin resistance gene. [RHO140: 20mer: 5'- GGT TGA CGG CAA TTT CGA TG -3' (SEQ ID NO: 33), RHO141: 22mer: 5'- CCT CCT ACA TCG AAG CTG AAA G -3' (SEQ ID NO: 34), RHO142: 21mer: 5'- CTT CTC GGG CTT TAT CGA CTG -3' (SEQ ID NO: 35), RHO143: 22mer: 5'- TAA GGT CGG TCT TGA CAA ACA G -3' (SEQ ID NO: 36), RHO184: 24mer: 5'- AGT AGT CCC CGA TTT GGT AGT TGA -3' (SEQ ID NO: 37), RHO185: 22mer: 5'- GGC AGA GAG CAA AAA CAC GAG C-3' (SEQ ID NO: 38)]. [PCR cycles: 98℃ 2min / 98℃ 10 sec, 68℃ 4 min, 30 cycles / 68℃ 7min]. A C20 elongase gene disruptant was obtained in which there was no amplification in the wild-type allele (Wt allele) but amplification in the artificially synthesized neomycin resistance gene allele (NeoR allele) and hygromycin resistance gene allele (HygR allele) (Figure 11). [Example 2-9] Changes in fatty acid composition due to C20 elongase gene disruption Parietichytrium sarkarianum SEK364 wild-type strain and its gene disruptant (C20 elongase gene knockout strain, C20 - / -) were cultured in GY medium. Cells in the late logarithmic growth phase were pelleted by centrifugation at 3,000 rpm at 4°C for 10 min, suspended in 0.9% NaCl, and washed. The pellet was then centrifuged at 3,000 rpm at 4°C for 10 min, and suspended and washed in sterile water. The pellet was then centrifuged again at 3,000 rpm for 10 min, and the supernatant was removed and lyophilized. Two ml of methanolic KOH (7.5% KOH in 95% methanol) was added to the lyophilized cells, vortexed, and then sonicated for 30 min at 80°C. Next, 500 μl of sterile water was added and vortexed, followed by 2 ml of n-hexane and vortexing. The mixture was then centrifuged at 3,000 rpm for 10 min and the upper layer was discarded. Another 2 ml of n-hexane was added and vortexed, followed by centrifugation at 3,000 rpm for 10 min and the upper layer was discarded. To the remaining lower layer, 1 ml of 6N HCl was added and vortexed, followed by 2 ml of n-hexane and vortexing. The mixture was then centrifuged at 3,000 rpm for 10 min and the upper layer was collected. Another 2 ml of n-hexane was added and vortexed, followed by centrifugation at 3,000 rpm for 10 min and the upper layer was collected. The collected upper layer was concentrated to dryness with nitrogen gas. To the concentrated and dried sample, 2 ml of 3N methanolic HCl was added and the mixture was incubated at 80°C overnight. The sample was cooled to room temperature and 1 ml of 0.9% NaCl was added. 2 ml of n-hexane was then added, the mixture was vortexed, and centrifuged at 3,000 rpm for 10 minutes. The upper layer was then collected. 2 ml of n-hexane was added again, the mixture was vortexed, and centrifuged at 3,000 rpm for 10 minutes. The upper layer was then collected. A small amount of anhydrous sodium sulfate was added to the collected upper layer, which was then vortexed and centrifuged at 3,000 rpm for 10 minutes. The collected upper layer was then concentrated to dryness using nitrogen gas. This concentrated and dried sample was dissolved in 0.5 ml of n-hexane, and 1 μl of the solution was subjected to GC analysis. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a column of HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.), column temperature of 150°C → (5°C / min) → 220°C (10 min), and carrier gas of He (1.3 mL / min). As a result, when the C20 elongase gene was knocked out in Parietichytrium sarkarianum SEK364, fatty acids with a carbon chain length of 22 or more decreased, and fatty acids with a carbon chain length of 20 increased (Figure 12). Figure 13 shows the percentages when the wild-type strain is taken as 100%. Of the total fatty acid composition, ARA was 25.22%, DGLA was 8.62%, ETA was 0.56%, EPA was 11.58%, n-6DPA was 1.64%, and DHA was 1.28%. In terms of GC area, the LA / DHA value was 5.8, the GLA / DHA value was 1.5, the DGLA / DHA value was 6.7, the ARA / DHA value was 19.7, and the EPA / DHA value was 9.0. The results show that the LA / EPA value is 0.64, the GLA / EPA value is 0.16, the DTA / EPA value is 0.06, the DTA / ARA value is 0.03, the DTA / DGLA value is 0.08, the LA / n-6DPA value is 4.5, the GLA / n-6DPA value is 1.2, the DGLA / n-6DPA value is 5.3, the ARA / n-6DPA value is 15.4, the EPA / n-6DPA value is 7.1, the DGLA / LA value is 1.2, the ARA / LA value is 3.4, the EPA / LA value is 1.6, the DTA / LA value is 0.09, the DGLA / GLA value is 4.5, the ARA / GLA value is 13.2, the n-6DPA / DTA value is 2.4, the DHA / n-3DPA value is 4.9, the C20PUFA / C22PUFA value is 11.94, and the n-6PUFA / n-3PUFA value is 2.67. As a result, arachidonic acid increased by approximately 10 times, EPA by approximately 8 times, and DGLA by approximately 16 times, while DPA decreased by approximately 1 / 4 and DHA by approximately 1 / 5. Thus, by selecting Parietichytrium sarkarianum SEK364, a Labyrinthulomycete lacking the PUFA-PKS pathway, we were able to create a strain that accumulates PUFAs other than DHA and DPA n-6 without disrupting genes in the PUFA-PKS pathway. This strain can be used as an EPA and / or ARA-producing strain, or a strain that produces the desired PUFA can be created by further disrupting or introducing genes for elongases or desaturases. [Example]

[0049] [Parietichytrium Sarkarian Disruption of the Δ4 desaturase gene of SEK364 and measurement of the fatty acid composition of lipids produced by the transformant [Example 3-1] Parietichytrium Cloning of the Δ4 desaturase gene of the genus By the method described in Example 2-2 Parietichytrium Genomic DNA was extracted from Rhodospirillum sp. SEK571. Using the extracted genomic DNA as a template, a sequence (5,003 bp, SEQ ID NO: 39) containing the Δ4 desaturase gene sequence was amplified using LA Taq Hot Start Version (Takara Bio Inc.). The PCR primers used were as follows: RHO241: 23-mer: 5'-GTT TGA GGA GCG AGG CAT TTC TT -3' (SEQ ID NO: 40), RHO242: 23-mer: 5'-AGT GCT CGT ACA ATG ACT GGC GT -3' (SEQ ID NO: 41). The resulting DNA fragment was cloned into pGEM-T easy vector, amplified in Escherichia coli, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This was designated pRH112 (SEQ ID NO: 42). Parietichytrium The plasmid (pRH112) containing the S. cerevisiae Δ4 desaturase gene sequence (1,542 bp, SEQ ID NO: 43) is shown in FIG.

[0050] [Example 3-2] Preparation of a base plasmid for constructing a Δ4 desaturase gene targeting vector Using pRH112 (Figure 14) prepared in Example 3-1 as a template, a primer set was prepared that deleted the Δ4 desaturase gene and 600 bp downstream of the Δ4 desaturase gene and created a BglII site at the deleted site. [RHO243: 26mer: 5'-GGC AAG ATC TAA CTT TCT GAG GCT CT -3' (SEQ ID NO: 44), RHO244: 26mer: 5'-AAG TTA GAT CTT GCC TAT TCC ACG AT -3' (SEQ ID NO: 45)]. PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) was used for amplification. The amplified sample was digested with BglII and then self-ligated. The ligated sample was amplified in E. coli and then sequenced using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This was designated pRH117. The constructed plasmid (pRH117) that served as the base for constructing the Δ4 desaturase gene targeting vector is shown in FIG.

[0051] [Example 3-3] Construction of Δ4 desaturase gene targeting vector pRH31 (Figure 4) described in Example 2-2 was digested with BglII, and the resulting DNA fragment containing the artificially synthesized neomycin resistance gene cassette was ligated into the BglII site of pRH117 (Figure 15) described in Example 3-2. This was designated pRH124 (Figure 16). Using pRH124 as a template, a primer set designed to delete the PstI site was prepared. [RHO261: 26-mer: 5'-GTG CAG ACG CAG AAG AAG ACT GAC AA -3' (SEQ ID NO: 46), RHO262: 25-mer: 5'-CTT CTG CGT CTG CAC GAG GAA TCG A -3' (SEQ ID NO: 47)]. PrimeSTAR Max DNA Polymerase (Takara Bio) was used for amplification. The PCR product was transformed into E. coli for amplification, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This was designated pRH126 (SEQ ID NO: 48). The constructed Δ4 desaturase gene targeting vector (pRH126) is shown in FIG.

[0052] [Example 3-4] Parietichytrium Sarkarian Introduction of a Δ4 desaturase gene targeting vector into SEK364 The targeting vector pRH126 (Figure 17) prepared in Example 3-3 was used as a template, and RHO241 (described in Example 3-1, SEQ ID NO: 40) and RHO242 (described in Example 3-1, SEQ ID NO: 41) were used as primers to amplify the gene using PrimeSTAR Max DNA polymerase (Takara Bio). After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. The A260 / 280 was measured to calculate the DNA concentration. The fragment obtained when pRH126 (Figure 17) described in Example 3-3 was used as a template was 4,562 bp. Parietichytrium SarkarianThe SEK364 strain was cultured in GY medium for 1-2 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1-2 using the gene gun method (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mm Hg, rupture disk: 1,550 PSI). After a 24-hour recovery period, the transfected cells were plated onto PDA agar plates containing 1 mg / ml G418. As a result, 0-2 drug-resistant strains were obtained per bombardment.

[0053] [Examples 3-5] Identification of Δ4 desaturase gene gene-targeted homologous recombinants By the method described in Example 2-2, Parietichytrium Sarkarian Genomic DNA was extracted from the SEK364 strain and the Δ4 desaturase gene disruptant, and the A260 / 280 was measured to calculate the DNA concentration. This was used as a template for PCR to confirm the genome structure using PrimeSTAR GXL DNA polymerase (Takara Bio Inc.). The positions of the primers used, the combinations used for amplification, and the predicted size of the amplified product are shown in Figure 18 (within the homologous region). A primer set designed within the homologous recombination region was used to Parietichytrium Sarkarian A 3,046-bp fragment was amplified in the SEK364 strain, and a 2,605-bp fragment was amplified in the Δ4 desaturase gene disruptant [RHO251: 20-mer: 5'-GTG GTC GAA GTG GAG TAT CT -3' (SEQ ID NO: 49), RHO252: 20-mer: 5'-ACT CGC CAT ACA ACT TTA CA -3' (SEQ ID NO: 50)]. As a result, a Δ4 desaturase gene-disrupted strain was obtained in which there was no amplification derived from the wild-type allele (Wt allele) but there was amplification derived from the Δ4 desaturase gene KO-type allele (NeoR allele) (Figure 19, lane 7: Δ4 desaturase KO mutant strain). [Examples 3-6] Changes in fatty acid composition due to disruption of the Δ4 desaturase gene According to the method described in Examples 2-9 Parietichytrium Sarkarian The wild-type SEK364 strain and its Δ4 desaturase gene disruptant were cultured and freeze-dried, and the fatty acids were methyl-esterified and analyzed by GC. For the culture, the GY liquid medium described in Example 1 was supplemented with a vitamin solution (1200 mg of vitamin B, 21 mg of vitamin B, 1200 mg of vitamin B, 1 mg of vitamin B). 12 The medium was supplemented with 0.1% EDTA disodium salt (1 mg dissolved in 100 mL of distilled water) and 0.2% trace element solution (30.0 g of EDTA disodium salt, 1.45 g of FeCl3·6H2O, 34.2 g of H2BO3, 4.3 g of MnCl2·4H2O, 1.335 g of ZnCl2, 0.13 g of CoCl2·6H2O, 0.26 g of NiSO4·6H2O, 0.01 g of CuSO4·5H2O, and 0.025 g of Na2MoO4·2H2O dissolved in 1 L of distilled water). GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a column of HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.), column temperature of 150°C → (2°C / min) → 220°C (10 min), and carrier gas of He (1.3 mL / min). The chart of the analysis results is shown in Figure 20, and its enlarged view is shown in Figure 21. The chart in Figure 20 has been converted into numerical values ​​and shown in the table in Figure 22. This table is Parietichytrium Sarkarian This table shows a comparison of the fatty acid composition of the SEK364 wild-type strain and its Δ4 desaturase gene disruptant. This table quantifies the chart in Figure 20 and shows that the total fatty acid composition is ARA 1.59%, DGLA 0.98%, ETA 0.05%, EPA 0.79%, n-6DPA 0.00%, and DHA 0.00%. The GC area shows an LA / EPA value of 5.09, a GLA / EPA value of 0.48, a DTA / EPA value of 7.44, a DTA / ARA value of 3.73, a DTA / DGLA value of 6.06, a DGLA / LA value of 0.24, an ARA / LA value of 0.39, an EPA / LA value of 0.20, a DTA / LA value of 1.46, a DGLA / GLA value of 2.57, an ARA / GLA value of 4.19, and an n-6DPA / DTA value of 0.00. As a result, Parietichytrium Sarkarian It was revealed that disruption of the Δ4 desaturase gene in the SEK364 strain resulted in almost no biosynthesis of DHA or DPA n-6, and conversely, increased production of their substrates, DPA n-3 and DTA. Thus, labyrinthuleans that do not have a PUFA-PKS pathway Parietichytrium Sarkarian By selecting SEK364, we were able to create a strain that accumulates PUFAs other than DHA and DPA n-6 without gene disruption in the PUFA-PKS pathway. This strain can be used to produce DPA n-3 and / or DTA, or by further gene disruption or gene introduction of elongase or desaturase, a strain that produces the desired PUFA can be created. [Example]

[0054] [Parietichytrium Disruption of the C20 elongase gene of Saccharomyces sp. SEK358 and measurement of fatty acid composition of lipids produced by the transformant. [Example 4-1] Parietichytrium Introduction of a C20 elongase gene targeting vector into the sp. strain SEK358 Using the targeting vector prepared using pRH85 (Figure 9) described in Example 2-6 as a template and RHO153 (described in Example 2-4, SEQ ID NO: 24) and RHO154 (described in Example 2-4, SEQ ID NO: 25) as primers, the gene was amplified using PrimeSTAR Max DNA polymerase (Takara Bio Inc.). [PCR cycles: 98°C 2 min / 98°C 30 sec, 68°C 2 min, 30 cycles / 68°C 2 min]. After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. The introduced fragment obtained when pRH85 (Figure 9) described in Example 2-6 was used as a template was 2661 bp. Parietichytrium Genus C20 elongase gene first half - SV40 terminator sequence - artificially synthesized neomycin resistance gene sequence - ubiquitin promoter sequence Parietichytrium The sequence is the second half of the C20 elongase gene (described in Examples 2-7, SEQ ID NO: 31). Parietichytrium The sp. SEK358 strain was cultured in GY medium for 3 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1–1.5 using a gene gun technique (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mm Hg, rupture disk: 900 PSI). After a 24-hour recovery period, the transfected cells were plated onto PDA agar plates containing 0.5 mg / ml G418. As a result, 10–30 drug-resistant strains were obtained per bombardment.

[0055] [Example 4-2] Identification of C20 elongase gene gene targeting homologous recombinants By the method described in Example 2-2, Parietichytrium Genomic DNA was extracted from the sp. SEK358 strain and the C20 elongase gene disruptant, and the A260 / 280 was measured to calculate the DNA concentration. Using this as a template, PCR was performed to confirm the genome structure using MightyAmp DNA polymerase (Takara Bio Inc.). The positions of the primers used, the combinations used for amplification, and the expected sizes of the amplified products are described in Example 2-8 and shown in Figure 10. RHO184 (described in Example 2-8, SEQ ID NO: 37) was designed upstream of the C20 elongase, RHO185 (described in Example 2-8, SEQ ID NO: 38) was designed downstream, and RHO142 (described in Example 2-8, SEQ ID NO: 35) and RHO143 (described in Example 2-8, SEQ ID NO: 36) were designed on an artificially synthesized neomycin resistance gene. [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 2 min, 30 cycles / 68°C 7 min]. A C20 elongase gene disruptant was obtained in which there was no amplification in the wild-type allele (Wt allele) but amplification in the artificially synthesized neomycin resistance gene allele (NeoR allele) (Figure 23).

[0056] [Example 4-3] Changes in fatty acid composition due to C20 elongase gene disruption By the method described in Examples 2-9 Parietichytrium The wild-type strain of S. sp. SEK358 and its gene-disrupted strain (C20 elongase gene knockout strain, C20 KO) were cultured and lyophilized, and the fatty acids were methyl-esterified and analyzed by GC. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a HR-SS-10 (30 m x 0.25 mm; Shinwa Kako Co., Ltd.) column, with a column temperature of 150°C → (5°C / min) → 220°C (10 min), and a carrier gas of He (1.3 mL / min). The changes in fatty acid composition are shown in Figure 24. Figure 25 shows the percentage of the wild-type strain (100%). The total fatty acid composition was ARA (ARA) 21.35%, DGLA 8.64%, ETA 2.14%, EPA 23.83%, n-6DPA 0.46%, and DHA 0.94%. GC area showed LA / DHA value of 4.6, GLA / DHA value of 2.8, DGLA / DHA value of 9.19, ARA / DHA value of 22.7, EPA / DHA value of 25.4, LA / EPA value of 0.18, GLA / EPA value of 0.11, DTA / EPA value of 0.01, DTA / ARA value of 0.01, and DTA / DGLA value of 0.03. The results show that the LA / n-6DPA value is 9.3, the GLA / n-6DPA value is 5.7, the DGLA / n-6DPA value is 18.8, the R / n-6DPA value is 46.4, the EPA / n-6DPA value is 51.8, the DGLA / LA value is 2.0, the ARA / LA value is 5.0, the EPA / LA value is 5.6, the DTA / LA value is 0.06, the DGLA / GLA value is 3.3, the ARA / GLA value is 8.2, the n-6DPA / DTA value is 1.8, the DHA / n-3DPA value is 4.1, the C20PUFA / C22PUFA value is 29.61, and the n-6PUFA / n-3PUFA value is 1.1. As a result, knockout of the C20 elongase gene in Parietichytrium sp. SEK358 strain resulted in a decrease in fatty acids with a carbon chain length of 22 or more, and an increase in fatty acids with a carbon chain length of 20. Specifically, arachidonic acid increased by approximately 7 times, EPA by approximately 11 times, DPA by approximately 1 / 15, and DHA by approximately 1 / 8. Thus, labyrinthuleans that do not have a PUFA-PKS pathway Parietichytrium By selecting sp. SEK358, we were able to create a strain that accumulates PUFAs other than DHA and DPA n-6 without gene disruption in the PUFA-PKS pathway. This strain can be used as an EPA and / or ARA producer, or by further gene disruption or gene introduction of elongase or desaturase, a strain that produces the desired PUFA can be created. [Example]

[0057] [Parietichytrium Disruption of the Δ4 desaturase gene of Saccharomyces sp. SEK358 and measurement of fatty acid composition of lipids produced by the transformant [Example 5-1] Parietichytrium Introduction of a Δ4 desaturase gene targeting vector into sp. SEK358 The targeting vector prepared using pRH126 (Figure 17) described in Example 3-3 was used as a template, and RHO241 (described in Example 3-1, SEQ ID NO: 40) and RHO242 (described in Example 3-1, SEQ ID NO: 41) were used as primers to amplify the gene using PrimeSTAR Max DNA polymerase (Takara Bio). After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. The A260 / 280 was measured to calculate the DNA concentration. The fragment obtained when pRH126 (Figure 17) described in Example 3-3 was used as a template was 4,562 bp. ParietichytriumThe sp. SEK358 strain was cultured in GY medium for 1-2 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1-2 using the gene gun method (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mm Hg, rupture disk: 1,550 PSI). After a 24-hour recovery period, the transfected cells were plated onto PDA agar plates containing 1 mg / ml G418. As a result, 0-2 drug-resistant strains were obtained per bombardment.

[0058] [Example 5-2] Identification of Δ4 desaturase gene gene targeting homologous recombinants By the method described in Example 2-2, Parietichytrium Genomic DNA was extracted from the sp. SEK358 strain and the Δ4 desaturase gene disruptant, and the A260 / 280 was measured to calculate the DNA concentration. This was used as a template for PCR to confirm the genome structure using PrimeSTAR GXL DNA polymerase (Takara Bio Inc.). The positions of the primers used, the combinations used for amplification, and the predicted sizes of the amplified products are shown in Figure 18 (within the homologous region) and Figure 26 (outside the homologous region). A primer set designed within the homologous recombination region was used to Parietichytrium A 3,046 bp fragment was amplified in the sp. SEK358 strain, and a 2,605 bp fragment was amplified in the Δ4 desaturase gene disruptant [RHO251: 20 mer: 5'-GTG GTC GAA GTG GAG TAT CT -3' (SEQ ID NO: 49), RHO252: 20 mer: 5'-ACT CGC CAT ACA ACT TTA CA -3' (SEQ ID NO: 50)]. A primer set designed outside the homologous recombination region was used. Parietichytriumstrain SEK358, a 5,231-bp fragment is amplified, and a 4,790-bp fragment is amplified in the Δ4 desaturase gene disruptant [HGO32: 25-mer: 5'-CGG AGC TCG GAG AAC AAC ATA GAA G -3' (SEQ ID NO: 51), HGO33: 23-mer: 5'-GTG CAA CCA GGT GGC AAG ATT GT -3' (SEQ ID NO: 52)]. As a result, a Δ4 desaturase gene-disrupted strain was obtained in which there was no amplification derived from the wild-type allele (Wt allele) but there was amplification derived from the Δ4 desaturase gene KO allele (NeoR allele) (Figure 27, lane 4; Figure 28, lane 4: Δ4 desaturase KO mutant strain).

[0059] [Example 5-3] Changes in fatty acid composition due to disruption of the Δ4 desaturase gene According to the method described in Examples 2-9 Parietichytrium The wild-type strain of S. sp. SEK358 and its Δ4 desaturase gene disruptant (SEK358 delta4 des. KO mutant strain) were cultured and lyophilized, and the fatty acids were methyl-esterified and analyzed by GC. The culture was performed in the GY liquid medium described in Example 1 supplemented with a vitamin solution (1200 mg of vitamin B, 21 mg of vitamin B, 1 mg of vitamin B). 12 The medium was supplemented with 0.1% EDTA disodium salt (1 mg dissolved in 100 mL of distilled water) and 0.2% trace element solution (30.0 g of EDTA disodium salt, 1.45 g of FeCl3·6H2O, 34.2 g of H2BO3, 4.3 g of MnCl2·4H2O, 1.335 g of ZnCl2, 0.13 g of CoCl2·6H2O, 0.26 g of NiSO4·6H2O, 0.01 g of CuSO4·5H2O, and 0.025 g of Na2MoO4·2H2O dissolved in 1 L of distilled water). GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a column of HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.), column temperature of 150°C → (2°C / min) → 220°C (10 min), and carrier gas of He (1.3 mL / min). The chart of the analysis results is shown in Figure 29, and its enlarged view is shown in Figure 30. The chart in Figure 29 has been converted into numerical values ​​and shown in the table in Figure 31. This table is Parietichytrium The figure shows a comparison of the fatty acid composition of the wild-type sp. SEK358 strain and its Δ4 desaturase gene disruptant, with ARA 3.03%, DGLA 1.35%, ETA 0.03%, EPA 1.10%, n-6DPA 0.00%, and DHA 0.00% of the total fatty acid composition. The GC area shows an LA / EPA ratio of 4.2, a GLA / EPA ratio of 0.71, a DTA / EPA ratio of 7.19, a DTA / ARA ratio of 2.60, a DTA / DGLA ratio of 5.85, a DGLA / LA ratio of 0.29, an ARA / LA ratio of 0.66, an EPA / LA ratio of 0.24, a DTA / LA ratio of 1.71, a DGLA / GLA ratio of 1.72, an ARA / GLA ratio of 3.87, a C20PUFA / C22PUFA ratio of 0.42, and an n-6PUFA / n-3PUFA ratio of 2.0. As a result, Parietichytrium It was revealed that disruption of the Δ4 desaturase gene in the sp. strain SEK358 resulted in almost no biosynthesis of DHA or DPA n-6, and conversely, increased production of their substrates, DPA n-3 and DTA. Thus, labyrinthuleans that do not have a PUFA-PKS pathway Parietichytrium By selecting sp. SEK358, we were able to create a strain that accumulates PUFAs other than DHA and DPA n-6 without gene disruption in the PUFA-PKS pathway. This strain can be used to produce DPA n-3 and / or DTA, or by further gene disruption or gene introduction of elongase or desaturase, a strain that produces the desired PUFA can be created. [Example]

[0060] [Parietichytrium Disruption of the C20 elongase gene of Saccharomyces sp. SEK571 and measurement of fatty acid composition of lipids produced by the transformant. [Example 6-1] Parietichytrium Introduction of a C20 elongase gene targeting vector into the sp. strain SEK571 Using the targeting vector prepared using pRH85 (Figure 9) described in Example 2-6 as a template and RHO153 (described in Example 2-4, SEQ ID NO: 24) and RHO154 (described in Example 2-4, SEQ ID NO: 25) as primers, the gene was amplified using PrimeSTAR Max DNA polymerase (Takara Bio Inc.). [PCR cycles: 98°C 2 min / 98°C 30 sec, 68°C 2 min, 30 cycles / 68°C 2 min]. After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. The introduced fragment obtained when pRH85 (Figure 9) described in Example 2-6 was used as a template was 2661 bp. Parietichytrium Genus C20 elongase gene first half - SV40 terminator sequence - artificially synthesized neomycin resistance gene sequence - ubiquitin promoter sequence Parietichytrium The sequence is the second half of the C20 elongase gene (described in Example 2-7, SEQ ID NO: 31). Parietichytrium The sp. SEK571 strain was cultured in GY medium for 3 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1–1.5 using the gene gun method (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1550 PSI). After a 24-hour recovery period, the transfected cells were plated onto PDA agar plates containing 0.5 mg / ml G418. As a result, 5–15 drug-resistant strains were obtained per bombardment.

[0061] [Example 6-2] Identification of C20 elongase gene gene targeting homologous recombinants By the method described in Example 2-2, ParietichytriumGenomic DNA was extracted from the strain SEK571 and the C20 elongase gene disruptant, and the A260 / 280 was measured to calculate the DNA concentration. Using this as a template, PCR was performed to confirm the genome structure using MightyAmp DNA polymerase (Takara Bio Inc.). The positions of the primers used, the combinations used for amplification, and the expected sizes of the amplified products are described in Example 2-8 and shown in Figure 10. RHO184 (described in Example 2-8, SEQ ID NO: 37) was designed upstream of the C20 elongase, RHO185 (described in Example 2-8, SEQ ID NO: 38) was designed downstream, and RHO142 (described in Example 2-8, SEQ ID NO: 35) and RHO143 (described in Example 2-8, SEQ ID NO: 36) were designed on an artificially synthesized neomycin resistance gene. [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 2 min, 30 cycles / 68°C 7 min]. A C20 elongase gene disruptant was obtained in which there was no amplification in the wild-type allele (Wt allele) but amplification in the artificially synthesized neomycin resistance gene allele (NeoR allele) (Figure 32).

[0062] [Example 6-3] Changes in fatty acid composition due to C20 elongase gene disruption By the method described in Examples 2-9 Parietichytrium The strain SEK571 and its gene disruptant (C20 elongase gene knockout strain, C20 KO) were cultured and lyophilized, and the fatty acids were methyl esterified and analyzed by GC. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a HR-SS-10 (30 m x 0.25 mm; Shinwa Kako Co., Ltd.) column at 150°C → (5°C / min) → 220°C (10 min), with a carrier gas of He (1.3 mL / min). The changes in fatty acid composition are shown in Figure 33. Figure 34 shows the percentages when the wild-type strain is set at 100%. The total fatty acid composition is ARA 13.24%, DGLA 1.93%, ETA 1.14%, EPA 29.58%, n-6DPA 0.96%, and DHA 1.17%. In GC area, the LA / DHA value was 1.5, the GLA / DHA value was 0.9, the DGLA / DHA value was 1.65, the ARA / DHA value was 11.3, the EPA / DHA value was 25.3, the LA / EPA value was 0.06, the GLA / EPA value was 0.04, the DTA / EPA value was 0.01, the DTA / ARA value was 0.01, the DTA / DGLA value was 0.08, the LA / n-6DPA value was 1.8, the GLA / n-6DPA value was 1.1, the DGLA / n-6DPA value was 2.0, the ARA / n-6DPA value was 13.8, the EPA / n-6DPA value was 30.8, the DGLA / LA value was 1.1, the ARA / LA value was 7.8, the EPA / LA value was 17.4. The DTA / LA value is 0.09, DGLA / GLA value is 1.8, ARA / GLA value is 12.4, n-6DPA / DTA value is 6.4, DHA / n-3DPA value is 4.7, C20PUFA / C22PUFA value is 18.1, and n-6PUFA / n-3PUFA value is 0.51. As a result, Parietichytrium When the C20 elongase gene was knocked out in the sp. SEK571 strain, the amount of fatty acids with a carbon chain length of 22 or more decreased, while the amount of fatty acids with a carbon chain length of 20 increased. Specifically, the amount of arachidonic acid increased by approximately four times, the amount of EPA increased by approximately eight times, and the amounts of DPA and DHA decreased by approximately 1 / 12. In this way, Parietichytrium It was revealed that sp. SEK571 has no or very weak ability to produce PUFAs via the PUFA-PKS pathway, and by selecting such Labyrinthulea species, we were able to create a strain that accumulates PUFAs other than DHA and DPA n-6 without gene disruption in the PUFA-PKS pathway. This strain can be used as an EPA and / or ARA producer, or by further gene disruption or gene introduction of elongases or desaturases, a strain that produces the desired PUFA can be created.

[0063] [Comparative Example 1] [ Thraustochytrium goldenDisruption of the C20 elongase gene of ATCC34304 and measurement of the fatty acid composition of lipids produced by the transformant [Comparative Example 1-1] T . golden Extraction of total RNA from ATCC34304 and purification of mRNA On day 3 of culture in GY liquid medium T . golden The ATCC 34304 culture medium was centrifuged at 3,500 xg for 15 minutes to collect the bacterial cells. The obtained bacterial cells were washed by suspending them in sterile saline and centrifuging again, then quickly frozen in liquid nitrogen and ground to a powder in a mortar. Total RNA was extracted from the obtained cell lysate using Sepasol RNA I Super (Nacalai Tesque). Subsequently, Oligotex TM -dT30 <super>Using the mRNA Purification Kit (Takara Bio), mRNA was purified from total RNA according to the attached manual. The obtained total RNA and mRNA were dissolved in an appropriate amount of TE and subjected to electrophoresis using formalin-denatured gel (1% agarose / MOPS buffer). The results confirmed that total RNA was successfully extracted, mRNA was purified from total RNA, and the RNA was not degraded by RNase. To minimize RNA degradation, rubber gloves and masks were worn throughout the experimental procedures, and all equipment used was RNase-free or had been treated with diethyl pyrocarbonate (Nacalai Tesque) to inactivate RNase. When dissolving RNA, sterilized Milli-Q water treated with diethyl pyrocarbonate was dissolved in a solution containing the recombinant RNase inhibitor RNaseOUT. TM (manufactured by Invitrogen) was added to the solution.

[0064] [Comparative Example 1-2] RACE method T . golden Isolation of the elongase gene from ATCC34304 Degenerate oligonucleotides were synthesized in the forward (elo-F; 5'- TTY YTN CAY GTN TAY CAY CAY -3') (SEQ ID NO: 53) and reverse (elo-R; 5'- GCR TGR TGR TAN ACR TGN ARR AA -3') (SEQ ID NO: 54) directions, targeting the histidine box (His box) highly conserved in elongase genes. The oligonucleotides were synthesized using a DNA synthesizer (Applied Biosystems). Next, SMART TM Using the RACE cDNA Amplification Kit (Clontech) and the accompanying manual, 3'- and 5'-RACE cDNA libraries were prepared by adding synthetic adapters to the 3' and 5' ends. Using these as templates, 3'- and 5'-RACE was performed using synthetic adapter-specific oligonucleotides and the degenerate oligonucleotides elo-F and elo-R described above [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 infinity], and bands corresponding to specifically amplified 3'- and 5'-RACE products were confirmed (Figure 35). The entire RACE product was then subjected to electrophoresis on a 1% agarose gel. The separated DNA fragments were excised using a clean cutter and extracted from the agarose gel according to the method described in Non-Patent Document 10. Next, the DNA fragments were subjected to TA cloning using pGEM-T easy Vector (Promega), and their nucleotide sequences were determined by the method of Sanger et al. (Non-Patent Document 11). Specifically, the BigDye® Terminator v3.1 Cycle Sequencing Kit and a 3130 Genetic Analyzer (Applied Biosystems) were used to determine the nucleotide sequences using the dye terminator method according to the attached manual. As a result, two sequences of 190 bp and 210 bp, designated elo1 (SEQ ID NO: 55) and elo2 (SEQ ID NO: 56), were successfully identified in the 3'-RACE product, and one sequence of 200 bp, designated elo3 (SEQ ID NO: 57), was successfully identified in the 5'-RACE product. These sequences of elo1, elo2, and elo3 show significant homology with the sequences of various elongase genes. T . golden These results suggest that the cDNA sequences are partial sequences of the elongase gene derived from ATCC 34304. Furthermore, we designed oligonucleotide primers for elo1, elo2, and elo3, and attempted to obtain the cDNA sequences by RACE. The oligonucleotide primers used are shown below. elo1 forward oligonucleotide primer (elo1-F1; 5'- TAT GAT CGC CAA GTA CGC CCC -3') (SEQ ID NO: 58) and reverse oligonucleotide primer (elo1-R1; 5'- GAA CTG CGT CAT CTG CAG CGA -3') (SEQ ID NO: 59), elo2 forward oligonucleotide primer (elo2-F1; 5'- TCT CGC CCT CGA CCA CCA AC -3') (SEQ ID NO: 60) and reverse oligonucleotide primer (elo2-R1; 5'- CGG TGA CCG AGT TGA GGT AGC C -3') (SEQ ID NO: 61), elo3 forward oligonucleotide primer (elo3-F1; 5'- CAA CCC TTT CGG CCT CAA CAA G -3') (SEQ ID NO: 62) and reverse oligonucleotide primer (elo3-R1; 5'- TTC TTG AGG ATC ATC ATG AAC GTG TC -3'). (SEQ ID NO: 63). Using the forward and reverse oligonucleotide primers, RACE and sequencing of the amplified products were performed as described above. Specifically amplified 3'- and 5'-RACE products for elo1 were obtained, and the overlapping regions were found to be identical, revealing a 1,139-bp elo1 cDNA sequence (SEQ ID NO: 64). Similarly, specifically amplified 3'- and 5'-RACE products for elo3 were obtained, and the overlapping regions were found to be identical, revealing a 1,261-bp elo3 cDNA sequence (SEQ ID NO: 65). Sequence analysis revealed that elo1 consists of an 825-bp translated region (SEQ ID NO: 67) encoding 275 amino acid residues (SEQ ID NO: 66). BLAST search revealed significant homology with various elongase genes, as well as with known elongase genes. T . golden The sequence of elo3 was found to be completely identical to that of the putative Δ5 elongase gene (NCBI accession number CS486301). On the other hand, elo3 is predicted to consist of a 951 bp translated region (SEQ ID NO: 69) encoding 317 amino acid residues (SEQ ID NO: 68). BLAST searches showed significant homology with various elongase genes. T . golden These genes were confirmed to be putative elongase genes derived from ATCC 34304. Furthermore, the deduced amino acid sequences of both genes contained His boxes, which are highly conserved in elongase genes. Based on these results, the elo1 and elo3 genes were T . golden These putative elongase genes were derived from ATCC 34304 and named TaELO1 and TaELO2, respectively.

[0065] [Comparative Example 1-3] Budding yeast Saccharomyces yeast Expression of TaELO1 and TaELO2 in the host and analysis of the fatty acid composition of the transgenic strain TaELO1 and TaELO2 were isolated from budding yeast S . yeast To express TaELO1 in the host, expression vectors for each were constructed. An outline of the vectors is shown below. Based on the sequence of the TaELO1 translation region, a pair of oligonucleotide primers (E1 HindIII; 5'-ATA AGC TTA AAA TGT CTA GCA ACA TGA GCG CGT GGG GC -3') (SEQ ID NO: 70) and (E1 XbaI; 5'-TGT CTA GAA CGC GCG GAC GGT CGC GAA A -3') (SEQ ID NO: 71) were prepared. E1 HindIII is a forward oligonucleotide primer and contains a restriction enzyme HindIII site (AAGCTT) at the 5' end. The yeast consensus sequence ((A / Y)A (A / U)A AUGUST The sequence near the start codon of TaELO1 was modified with reference to UCU (the underlined part is the start codon) (Non-Patent Document 12). E1 XbaI is a reverse oligonucleotide primer and has an XbaI site (TCTAGA) at the 5' end. Similarly, a pair of oligonucleotide primers (E2 HindIII; 5'-TAA AGC TTA AAA TGT CTA CGC GCA CCT CGA AGA GCG CTC C -3') (SEQ ID NO: 72) and (E2 XbaI; 5'-CAT CTA GAC TCG GAC TTG GTG GGG GCG CTT G -3') (SEQ ID NO: 73) were constructed based on the sequence of the TaELO2 coding region. E2 HindIII is a forward oligonucleotide primer and contains a HindIII restriction enzyme site at the 5' end. Furthermore, the sequence near the start codon of TaELO2 was modified based on the yeast consensus sequence. E2 XbaI is a reverse oligonucleotide primer and contains an XbaI site at the 5' end. Using these two oligonucleotide primer pairs and the 5'-RACE cDNA library described in Comparative Example 1-2 as a template, PCR was performed to amplify the 949-bp TaELO1 translated region (SEQ ID NO: 74) and the 967-bp TaELO2 translated region (SEQ ID NO: 75), which had a HindII restriction site at the 5' end and an XbaI restriction site at the 3' end and had the yeast consensus sequence modified near the start codon. PrimeSTAR® DNA polymerase (Takara Bio Inc.), which has 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 infinity]. Next, the amplified PCR product was separated on a 1% agarose gel, and the DNA fragment was excised and extracted from the agarose gel. After further treatment with the restriction enzymes HindIII and XbaI, the DNA fragment was purified again using agarose gel. The resulting DNA fragment was then ligated to the budding yeast expression vector pYES2 / CT (Invitrogen) linearized by treatment with the restriction enzymes HindIII and XbaI using a DNA Ligation Kit.<Mighty Mix> A circularized vector was constructed by ligation using a PCR primer (Takara Bio). Nucleotide sequence analysis confirmed that no mutations due to PCR extension errors had been introduced into the TaELO1 and TaELO2 translation regions introduced into pYES2 / CT. Based on these results, we successfully constructed the TaELO1 expression vector pYEELO1 and the TaELO2 expression vector pYEELO2. The two constructed expression vectors and pYES2 / CT were transformed into budding yeast by the lithium acetate method according to the methods described in Non-Patent Documents 13 and 14. S . yeast The resulting transformants (pYEELO1-introduced strain, pYEELO2-introduced strain, and mock-introduced strain) were then cultured according to the method of Qiu et al. (Non-Patent Document 15), and fatty acids derived from the cells were extracted and methyl esterified. However, the medium was supplemented with α-linolenic acid (ALA, C18:3Δ9,12,15) and linoleic acid (LA, C18:2Δ9,12) as substrates for Δ9 elongase, stearidonic acid (STA, C18:4Δ6,9,12,15) and γ-linolenic acid (GLA, C18:3Δ6,9,12) as substrates for Δ6 elongase, and eicosapentaenoic acid (EPA, C20:5Δ5,8,11,14,17) and arachidonic acid (AA, C20:4Δ5,8,11,14) as substrates for Δ5 elongase, each at a final concentration of 0.2 mM. Subsequently, gas chromatography (GC) analysis of the methyl-esterified fatty acids was performed according to the method of Abe et al. (Non-Patent Document 16). GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a column of HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Industry Co., Ltd.), column temperature: 150°C → (5°C / min) → 220°C (10 min), and carrier gas: He (1.3 mL / min). The pYEELO1-transfected strain exhibited Δ6 elongase activity, which converted stearidonic acid (STA) to eicosatetraenoic acid (ETA, C20:4Δ8,11,14,17) and γ-linolenic acid (GLA) to dihomo-γ-linolenic acid (DGLA, C20:3Δ8,11,14), respectively, and was not present in the host (mock-transfected strain). On the other hand, it also exhibited Δ9 elongase activity, which converted α-linolenic acid (ALA) to eicosatetraenoic acid (ETrA, C20:3Δ11,14,17) and linoleic acid (LA) to eicosadienoic acid (EDA, C20:3Δ11,14), respectively, and ω3 docosapentaenoic acid (EPA, C22:5Δ7,10,13,14). 16, 19) and arachidonic acid (ARA) to docosatetraenoic acid (DTA, C22:4Δ7, 10, 13, 16), respectively (Table 1). Furthermore, the pYEELO2-transfected strain showed Δ5 elongase (=C20 elongase) activity, which converts EPA to ω3 DPA (C22:5Δ7, 10, 13, 16, 19) and ARA to DTA, but also showed slight Δ6 elongase activity, which converts STA to ETA and GLA to DGLA (Table 1). These results confirmed that TaELO1 is a Δ6 / Δ9 / Δ5 elongase and TaELO2 is a Δ5 / Δ6 elongase.

[0066] [Table 1]

[0067] [Comparative Example 1-4] Obtaining the upstream and downstream regions of TaELO2 ORF by PCR genome walking method In the targeting vector for disrupting TaELO2, we used PCR genome walking to obtain the upstream and downstream regions of the TaELO2 ORF, which serve as homologous recombination sites. The outline of this procedure is as follows. On day 3 of culture in GY liquid medium T . golden ATCC 34304 cells were rapidly frozen in liquid nitrogen and crushed to a powder in a mortar. Genomic DNA was extracted according to the method described in Non-Patent Document 17 and then dissolved in an appropriate amount of TE. The quantity and purity of genomic DNA were assayed by measuring OD260 and OD280. Next, TaKaRa LA PCR TM A genomic DNA library was constructed using the in vitro Cloning Kit (Takara Bio Inc.) by adding a cassette sequence containing restriction enzyme sites to genomic DNA digested with various restriction enzymes according to the attached protocol. Next, using the constructed genomic DNA library as a template, nested PCR was performed using forward oligonucleotide primers E2 XbaI (see Comparative Example 1-3, SEQ ID NO: 73) and elo3-F1 (see Comparative Example 1-2, SEQ ID NO: 62) or reverse oligonucleotide primers E2 HindIII (see Comparative Example 1-3, SEQ ID NO: 72) and elo3-R1 (see Comparative Example 1-2, SEQ ID NO: 63), both of which were constructed based on the TaELO2 sequence, along with oligonucleotide primers complementary to the cassette sequences provided in the kit, according to the attached protocol. As a result, a 1,122-bp TaELO2 ORF upstream sequence (SEQ ID NO: 76) and a 1,204-bp TaELO2 ORF downstream sequence (SEQ ID NO: 77) were successfully isolated.

[0068] [Comparative Example 1-5] Construction of TaELO2 targeting vector using Neor as a selection marker A DNA fragment linking the upstream sequence of TaELO2 ORF, the artificially synthesized Neor, and the downstream sequence of TaELO2 ORF was generated by fusion PCR. The oligonucleotide primers used are shown below. ·KO Pro F SmaI (31 mer: 5'- CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3') (SEQ ID NO: 78) ·Pro R (25 mer: 5'-GGT CGC GTT TAC AAA GCA GCG CAG C -3') (SEQ ID NO: 79) ·SNeo F (52 mer; 5'- GCT GCG CTG CTT TGT AAA CGC GAC CAT GAT TGA ACA GGA CGG CCT TCA CGC T -3') (SEQ ID NO: 80) ·SNeo R (52 mer; 5'-TCG GGA GCC AGC CGG AAA CAG GTT CAA AAG AAC TCG TCC AGG AGG CGG TAG A-3') (SEQ ID NO: 81) ·Term F (23 mer: 5'- ACC TGT TTC CGG CTG GCT CCC GA -3') (SEQ ID NO:82) KO Term R SmaI (27mer: 5'- ATC CCG GGG CCG AGA ACG GGG TCG CCC -3') (SEQ ID NO: 83) Among these oligonucleotide primers, KO Pro F SmaI / Pro R was the same as that described in Comparative Example 1-4. T . golden Amplification of the upstream sequence of TaELO2 ORF using ATCC 34304 genomic DNA as a template. SNeo F / SNeo R are amplification of artificially synthesized NeoR using artificially synthesized NeoR as a template. Term F / KO Term R SmaI are the same as those described in Comparative Example 1-4. T . golden This was used to amplify the downstream sequence of the TaELO2 ORF using ATCC 34304 genomic DNA as a template. The PCR reaction conditions were a denaturation temperature of 98°C for 10 seconds, and the annealing and extension reactions were adjusted appropriately depending on the Tm of the primers and the length of the amplified product. As a result, we succeeded in linking a 2,696 bp (sequence number 84) TaELO2 ORF upstream sequence / artificially synthesized Neor / TaELO2 ORF downstream sequence, and TA-cloned this using pGEM-T easy Vector (Promega) to create a knockout vector named pTKONeor.

[0069] [Comparative Examples 1-6] T . golden Introduction of TKONeor into ATCC34304 The TaELO2 targeting vector pTKONeor, which uses the synthetic Neor construct prepared in Comparative Examples 1-5 as a selectable marker, was used as a template. The TaELO2 ORF upstream sequence / synthetic Neor / TaELO2 ORF downstream sequence was amplified using a pair of oligonucleotide primers, KO Pro F SmaI (Comparative Examples 1-5, SEQ ID NO: 78) / KO Term R SmaI (Comparative Examples 1-5, SEQ ID NO: 83), and PrimeSTAR® HS DNA polymerase (Takara Bio Inc.) [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 3 min, 30 cycles / 68°C 10 min / 4°C infinity]. The DNA fragment was extracted after electrophoresis on a 1% agarose gel, precipitated with ethanol, and dissolved in an appropriate amount of TE. The quantity and purity of the DNA fragment were assayed by measuring OD260 and OD280. The resulting DNA fragment is hereafter referred to as TKONeor. Next, DNA was injected using a gene gun. T . golden ATCC 34304 was cultured at 25°C and 150 rpm until mid- to late-logarithmic growth phase. The culture was then centrifuged at 3,500 x g for 10 min at 4°C and the supernatant was removed. The resulting cells were then resuspended in GY liquid medium to a concentration 100-fold greater than the original culture. A 20 μl aliquot of the cell suspension was spread thinly and evenly over a 3 cm diameter area on a 5 cm diameter PDA agar plate containing 1 mg / ml G418 (Nacalai Tesque) and allowed to dry. The plate was then bombarded using a PDS-1000 / He system (BioRad) under the following conditions: target distance - 6 cm, vacuum - 26 inches Hg, microcarrier size - 0.6 μm, and rupture disk (bombardment pressure) - 1,100 psi. 100 μl of PD liquid medium was then added dropwise to the PDA agar plate, and the cells were redistributed and subjected to static culture. The result is 4.7 x 10 1 Transformants conferred with G418 resistance were obtained at an efficiency of cfu / μg DNA.

[0070] [Comparative Example 1-7] PCR using genomic DNA of a transformant into which TKONeor has been introduced as a template Seven colonies of the transformants were picked with a toothpick and then inoculated into GY liquid medium containing 0.5 mg / ml G418 (Nacalai Tesque). After multiple subcultures, genomic DNA was extracted from the cells using the method described in Comparative Example 1-4, precipitated with ethanol, and dissolved in an appropriate amount of TE. The amount and purity of the extracted genomic DNA were assayed by measuring OD260 and OD280. Next, PCR was performed using various oligonucleotide primer pairs as templates with the genomic DNA of the obtained transformants and wild-type strain. The oligonucleotide primer pairs used were: (1) Neo detection—SNeo F (described in Comparative Examples 1-5, SEQ ID NO: 80) and SNeo R (described in Comparative Examples 1-5, SEQ ID NO: 81) (2) KO Confirmation 1 - KO Pro F SmaI (described in Comparative Examples 1-5, SEQ ID NO: 78) and KO Term R SmaI (described in Comparative Examples 1-5, SEQ ID NO: 83) (3) KO Confirmation 2 - E2 KO ProF EcoRV (30mer: 5'- GGA TAT CCC CCG CGA GGC GAT GGC TGC TCC -3') (SEQ ID NO: 85) and SNeo R (described in Comparative Examples 1-5, SEQ ID NO: 81) (4) KO Confirmation 3 - Sneo F (described in Comparative Examples 1-5; SEQ ID NO: 80) and E2 KO Term R EcoRV (30 mer: 5'- TGA TAT CGG GCC GCG CCC TGG GCC GTA GAT -3') (SEQ ID NO: 86) (5) TaELO2 Amplification—E2 HindIII (described in Comparative Examples 1-3, SEQ ID NO: 72) and E2 XbaI (described in Comparative Examples 1-3, SEQ ID NO: 73) (Figure 36A). As a result, six of the seven clones analyzed were transformants due to random integration, but one clone was confirmed to have had the TaELO2 ORF replaced with Neor by homologous recombination (Fig. 36B, lanes 9 and 13). At the same time, however, it was found that the TaELO2 ORF was amplified (Fig. 36B, lane 17). T . golden It was suggested that ATCC 34304 may be more than diploid or that TaELO2 may be a multicopy gene.

[0071] [Comparative Example 1-8] Confirmation of TaELO2 copy number by Southern blotting The following experiment was carried out according to the method described in "DIG Manual [Japanese Edition] 8th Edition, Roche Applied Science" (Non-Patent Document 18). Specifically, wild-type strain genomic DNA was digested with various restriction enzymes, and then 2.5 μg of 0.7% SeaKem® GTG was added per lane. TM The mixture was subjected to electrophoresis using agarose (manufactured by Takara Bio Inc.). TM The DNA was transferred to a PCR tube (GE Healthcare) and hybridized with a DIG-labeled probe prepared using a PCR DIG Probe Synthesis Kit (Roche Applied Science) at 48°C for 16 hours. The pair of oligonucleotide primers used to prepare the DIG-labeled probe was TaELO2 det F (25-mer: 5'-GTA CGT GCT CGG TGT GAT GCT GCT C -3') (SEQ ID NO: 87) and TaELO2 det R (24-mer: 5'-GCG GCG TCC GAA CAG GTA GAG CAT-3') (SEQ ID NO: 88) [PCR cycles: 98°C for 2 minutes, 98°C for 30 seconds, 65°C for 30 seconds, 72°C for 1 minute, 30 cycles of 72°C for 7 minutes, and 4°C for infinity]. The hybridized probe was detected using a colorimetric method (NBT / BCIP solution). As a result, a single band was detected in all lanes treated with various restriction enzymes (Figure 37), indicating that TaELO2 is a single-copy gene. T . golden This suggests that ATCC 34304 is more than diploid.

[0072] [Comparative Example 1-9] Evaluation of transformants into which TKONeor gene was introduced by Southern blotting Southern blotting was performed as described in Comparative Examples 1 to 8. Specifically, a pair of oligonucleotide primers, uprobe F (35-mer: 5'-ATC CGC GTA TAT ATC CGT AAA CAA CGG AAC ATT CT-3') (SEQ ID NO: 89) and uprobe R (26-mer: 5'-CTT CGG GTG GAT CAG CGA GCG ACA GC-3') (SEQ ID NO: 90), was used to amplify the DIG-labeled probe by PCR [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 infinity]. Southern blotting was performed using a colorimetric method (NBT / BCIP solution) on the genomic DNA of the wild-type strain and the transformant digested with EcoRV and PstI. In this case, a DNA fragment of approximately 1.2 kbp was detected in the wild-type allele, whereas a DNA fragment of approximately 2.5 kbp was detected in the mutant allele in which the TaELO2 ORF was replaced by Neor by homologous recombination (Figure 38A). As a result of the analysis, bands of the mutant allele and wild-type allele were detected in the transformants (Figure 38B). T . golden ATCC 34304 was shown to be more than diploid.

[0073] [Comparative Example 1-10] Construction of TaELO2 targeting vector using Hygr as a selection marker To disrupt the remaining wild-type allele, a TaELO2 targeting vector was constructed using Hygr as a selection marker. First, by fusion PCR T . golden The ubiquitin promoter sequence derived from ATCC 34304 was ligated to Hygr. The oligonucleotide primers used are shown below. ubi-600p F (27mer: 5'- GCC GCA GCG CCT GGT GCA CCC GCC GGG-3') (SEQ ID NO: 91) ·ubi-hygro R (59 mer: 5'-TCG CGGG TGA GTT CAG GCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3') (SEQ ID NO: 92) ·ubi-hygro F (57 mer; 5'-AGC GAC CTA AGC AAC ACT AGGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3') (SEQ ID NO: 93) ·hygro R (29 mer; 5'-CTA TTC CTT TGC CCT CGG ACG AGT GCT GG-3') (SEQ ID NO: 94) Among these oligonucleotide primers, ubi-600p F / ubi-hygro R was used as the primer for the ubiquitous oligonucleotides described in Comparative Example 1-4. T . golden ATCC 34304 genomic DNA was used as a template T . golden It was used to amplify the ubiquitin promoter sequence derived from ATCC 34304. ubi-hygro F / hygro R was used to amplify artificially synthesized Hygr using pcDNA 3.1 Zeo (Invitrogen) as a template. The PCR reaction conditions were a denaturation temperature of 98°C for 10 seconds, and the annealing and extension reactions were adjusted appropriately depending on the Tm of the primers and the length of the amplified product. As a result, a 1,636 bp fragment (SEQ ID NO: 95) T . golden We successfully ligated the ATCC 34304-derived ubiquitin promoter sequence with Hygr, and TA-cloned this using pGEM-T easy Vector (Promega), which was named pTub600Hygr. Subsequently, using pTub600Hygr as a template and PrimeSTAR HS DNA polymerase (Takara Bio), PCR was performed using a pair 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: 96) and hygro R XbaI (37 mer: 5'-GTT CTA GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3') (SEQ ID NO: 97) [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 3 min, 30 cycles / 68°C 10 min / 4°C infinity] to add an NheI site to the 5' end and an XbaI site to the 3' end. T . golden A ubiquitin promoter sequence / Hygr DNA fragment derived from ATCC 34304 was prepared. Furthermore, using pTKONeor described in Comparative Examples 1-5 as a template, PCR was performed using a pair of oligonucleotide primers, KOvec F XbaI (37-mer: 5'-GTT CTA GAC CTG TTT CCG GCT GGC TCC CGA GCC ATG C-3') (SEQ ID NO: 98) and KOvec R NheI (40-mer: 5'-GTG CTA GCG GTC GCG TTT ACA AAG CAG CGC AGC AAC AGA A-3') (SEQ ID NO: 99) [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 3 min, 30 cycles / 68°C 10 min / 4°C infinity] to prepare a linear vector in which Neor was removed from pTKONeor described in Comparative Examples 1-5 and an NheI site was added to the 3' end and an XbaI site was added to the 5' end. Both DNA fragments were digested with restriction enzymes NheI and XbaI, purified using agarose gel, and a circular vector was constructed using a Ligation Convenience Kit (Nippon Gene). The constructed TaELO2 targeting vector, which uses Hygr as a selection marker, has a pGEM-T easy Vector (Promega) as a base skeleton and contains a 3,537 bp (SEQ ID NO: 100) TaELO2 ORF upstream sequence / T . golden This vector contained the ATCC 34304-derived ubiquitin promoter sequence, Hygr, and TaELO2 ORF downstream sequence and was named pTKOub600Hygr.

[0074] [Comparative Example 1-11] Reintroduction of KOub600Hygr and evaluation of transformants by PCR, Southern blotting, and RT-PCR using genomic DNA as a template The constructed TaELO2 targeting vector pTKOub600Hygr (described in Comparative Examples 1-10) using Hygr as a selection marker was used as a template, and the TaELO2 ORF upstream sequence / T . golden The ATCC 34304-derived ubiquitin promoter sequence / Hygr / TaELO2 ORF downstream sequence was amplified [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 3.5 min, 30 cycles / 68°C 10 min / 4°C infinity], and the resulting DNA fragment was designated KOub600Hygr. This was then introduced into the transformant obtained in Comparative Example 1-7 in the same manner. After static culture for 24 hours on PDA agar plates containing 1 mg / ml G418 (Nacalai Tesque), the cells were harvested and further cultured on PDA agar plates containing 1 mg / ml G418 (Nacalai Tesque) and 2 mg / ml hygromycin B (Wako Pure Chemical Industries, Ltd.). A large number of transformants were obtained (transfection efficiency: 1.02 x 10 3 cfu / μg DNA). Fifty of these clones were picked and subcultured multiple times in GY liquid medium containing 1 mg / ml G418 (Nacalai Tesque) and 2 mg / ml hygromycin B (Wako Pure Chemical Industries, Ltd.). Genomic DNA was extracted using the same method as in Comparative Examples 1-4, precipitated with ethanol, and dissolved in an appropriate amount of TE. The quantity and purity of the extracted genomic DNA were assayed by measuring OD260 and OD280. Next, PCR was performed using various oligonucleotide primer pairs [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 1 min, 30 cycles / 68°C 10 min / 4°C infinity] using the genomic DNA of the obtained transformants and wild-type strain as templates. The oligonucleotide primer pairs used were: (1) TaELO2 ORF detection—Sneo F (described in Comparative Examples 1-5, SEQ ID NO: 80) and SNeo R (described in Comparative Examples 1-5, SEQ ID NO: 81), (2) KO Confirmation—E2 KO Pro F EcoRV (described in Comparative Examples 1-7, SEQ ID NO: 85) and ubi-hygro R (described in Comparative Examples 1-10, SEQ ID NO: 92) (Figure 39A). As a result, 14 of the 50 clones analyzed were suggested to be transformants that had undergone homologous recombination, resulting in the replacement of the TaELO2 ORF (Fig. 39B, arrows), and it was confirmed that the TaELO2 ORF was not amplified in these clones (Fig. 39C). Subsequently, Southern blotting was performed using the method described in Comparative Examples 1-9. Specifically, a DIG-labeled probe prepared using a pair of oligonucleotide primers, uprobe F (SEQ ID NO: 89) and uprobe R (SEQ ID NO: 90), was used to perform Southern blotting using a colorimetric method (NBT / BCIP solution) on genomic DNA of the wild-type strain and the transformant digested with EcoRV and PstI. In this case, a DNA fragment of approximately 1.2 kbp was detected in the wild-type allele, a DNA fragment of approximately 2.5 kbp was detected in the mutant allele in which the TaELO2 ORF was replaced by Neor by homologous recombination, and a DNA fragment of approximately 1.9 kbp was detected in the mutant allele in which the TaELO2 ORF was replaced by Hygr by homologous recombination (Figure 40A). As a result of the analysis, the approximately 2.5 kbp band corresponding to the wild-type allele disappeared in the obtained transformants, and instead a new approximately 1.9 kbp band corresponding to the mutant allele in which the TaELO2 ORF had been replaced by Hygr was detected (Figure 40B). Similarly, a pair of oligonucleotide primers, TaELO2 probe F (30-mer: 5'-ATG GCG ACG CGC ACC TCG AAG AGC GCT CCG-3') (SEQ ID NO: 101) and TaELO2 probe R (30-mer: 5'-AGG ATC ATC ATG AAC GTG TCG CTC CAG TCG-3') (SEQ ID NO: 102), was used to prepare a DIG-labeled probe for TaELO2 detection by PCR [PCR cycles: 98°C for 2 min, 98°C for 30 sec, 65°C for 30 sec, 72°C for 1 min, 30 cycles, 72°C for 7 min, 4°C for infinity]. Southern blotting using a colorimetric method (NBT / BCIP solution) was performed on EcoRV-digested genomic DNA from the wild-type strain and transformants (clones 1, 8, 9, and 10). In this case, TaELO2 is detected as a DNA fragment of approximately 2.5 kbp (Figure 38A). The analysis revealed that TaELO2 was detected in the wild-type strain (Fig. 41, lane 1), but was not detected at all in the transformants (Fig. 41, lanes 2-5). To further verify TaELO2 disruption at the mRNA level, RT-PCR was performed to detect TaELO2 mRNA. Total RNA was extracted from wild-type strains and transformants (clones 1, 8, 9, and 10) cultured in GY liquid medium on day 3 using Sepasol RNA I Super (Nacalai Tesque) as in Comparative Example 1-1. Subsequently, 50 μg of total RNA was cleaned using the RNeasy Mini Kit (QIAGEN) according to the attached protocol. Contaminating genomic DNA was then removed by treatment with 50 U of Recombinant DNase I (Takara Bio) at 37°C for 1 hour. Subsequently, a single-stranded cDNA library was prepared using the resulting total RNA as a template, oligo(dT) primer (Novagen) and PrimeScript Reverse Transcriptase (Takara Bio) according to the attached manual. Furthermore, using the obtained single-stranded cDNA library as a template, a pair of oligonucleotide primers E2 HindIII (described in Comparative Examples 1-3, SEQ ID NO: 72) and E2 XbaI (described in Comparative Examples 1-3, SEQ ID NO: 73) and LA Taq Hot Start Version (manufactured by Takara Bio Inc.) were used to amplify the TaELO2 ORF [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 1 min, 30 cycles / 68°C 10 min / 4°C infinity]. As a result, it was found that TaELO2 mRNA was detected in the wild-type strain (Fig. 42, lane 5), but was not detected at all in the transformants (clones 1, 8, 9, and 10) (Fig. 42, lanes 1-4). These results demonstrate that we have successfully obtained TaELO2-deficient homozygotes in which TaELO2 is completely disrupted. T . golden ATCC 34304 was found to be diploid.

[0075] [Comparative Example 1-12] Comparison of fatty acid composition between wild-type strain and TaELO2-deficient homozygote The fatty acid compositions of the TaELO2-deficient homozygote and wild-type strains obtained in Comparative Example 1-11 were analyzed by GC analysis of methyl-esterified fatty acids. Specifically, the TaELO2-deficient homozygote and wild-type strain cells were harvested after 5 days of culture in GY liquid medium. The fatty acids from the cells were extracted, methyl-esterified, and analyzed by GC as described in Comparative Example 1-3. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with an HR-SS-10 column (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.) at a column temperature of 150°C → (5°C / min) → 220°C (10 min), and a carrier gas of He (1.3 mL / min). As a result, in TaELO2-deficient homozygotes (TaELO2 KO), the amount of EPA, the substrate of TaELO2, was increased to about twice that of the wild type, and the amount of DHA, the downstream metabolic product, was observed to be reduced (Figure 43). As mentioned above, T. golden ATCC34304 Parietichytrium As with the Labyrinthula genus, it was confirmed that the C20 elongase gene knockout increased EPA, the substrate of C20 elongase, compared to the wild-type strain, while decreasing downstream metabolic products such as DHA. Parietichytrium Unlike the Labyrinthula genus, even if the C20 elongase gene is knocked out, T . golden The DHA content in ATCC34304 did not decrease significantly. Specifically, the DHA content in the wild-type strain was 54.38%, while that in the C20 elongase gene knockout strain was only slightly reduced to 48.77%. A similar trend was observed for DPA n-6. As described in Comparative Example 1-2, T. golden In addition to the TaELO2 KO'd in this study, TaELO1 also exists in ATCC34304, and both were shown to have Δ5 elongase activity (= C20 elongase activity) in Comparative Examples 1-3. However, it has also been revealed that the Δ5 elongase activity of TaELO1 is considerably lower than that of TaELO2, and it is difficult to explain the reason why DHA and DPA n-6 did not decrease significantly in the TaELO2-deficient homozygote (TaELO2 KO) in this study by the Δ5 elongase activity of TaELO1 (= C20 elongase activity). From the above, Thraustochytrium golden It was suggested that in ATCC34304, DHA and DPA n-6 may be produced by other biosynthetic pathways in addition to the elongase / desaturase pathway. When such Labyrinthulea are selected, it is not possible to produce strains that accumulate PUFAs other than DHA and DPA n-6 even if the C20 elongase gene is knocked out, unlike in Examples 2, 4, and 6. Therefore, to produce such strains, it is necessary to knock out genes related to the DHA and DPA n-6 biosynthetic pathway other than the elongase / desaturase pathway.

[0076] Comparative Example 2 [ Thraustochytrium golden Disruption of PUFA-PKS genes in ATCC34304 and measurement of fatty acid composition of lipids produced by the transformant [Comparative Example 2-1] PUFA-PKS pathway-related genes: Cloning of the upstream sequence of OrfA By the method described in Example 2-2 Thraustochytrium golden After extracting genomic DNA from ATCC 34304, A260 / 280 was measured and the DNA concentration was calculated. TM A genomic cassette library was prepared using an in vitro Cloning Kit (Takara Bio Inc.). A PCR lower primer [RHO20: 23mer: 5'-CGA TGA AAG GTC ACA GAA GAG TC -3' (SEQ ID NO: 103)] was designed on OrfA, a PUFA-PKS pathway-related gene described in Patent Document 4, and DNA was amplified in combination with the cassette primers provided with the kit [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]. Next, the first PCR product was diluted 100-fold, and DNA was amplified using the PCR lower primer [RHO20] and the nested primers provided with the kit (second 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 the pGEM-T easy vector, amplified in E. coli, and sequenced using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). A 3,377 bp (SEQ ID NO: 105) DNA fragment containing 3,181 bp (SEQ ID NO: 104) upstream of OrfA was cloned, revealing 3,181 bp of DNA sequence information upstream of OrfA.

[0077] [Comparative Example 2-2] PUFA-PKS pathway related genes: Cloning of downstream sequences of OrfA The genomic cassette library prepared in Comparative Example 2-1 was used as a template. A PCR upper primer [RHO21: 21mer: 5'-CAG GGC GAG CGA GTG TGG TTC -3' (SEQ ID NO: 106)] was designed on OrfA, a PUFA PKS pathway-related gene described in Patent Document 4, and DNA was amplified using the method described in Comparative Example 2-1. The resulting DNA fragment was cloned into pGEM-T easy vector, amplified in E. coli, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). A 1,204 bp DNA fragment (SEQ ID NO: 108) containing 1,160 bp downstream of OrfA (SEQ ID NO: 107) was cloned. A PCR upper primer [RHO28: 20mer: 5'-TGA TGC CGA TGC TAC AAA AG-3' (SEQ ID NO: 109)] was again prepared on SEQ ID NO: 94, and DNA was amplified by the method described in Comparative Example 2-1. The resulting DNA fragment was cloned into pGEM-T easy vector and amplified in Escherichia coli, after which the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). Furthermore, a 1,488 bp DNA fragment (SEQ ID NO: 110) containing the downstream sequence was cloned, revealing a total of 2,551 bp (SEQ ID NO: 111) of DNA sequence information downstream of OrfA.

[0078] [Comparative Example 2-3] PUFA-PKS pathway-related genes: Construction of OrfA targeting vector Thraustochytrium golden The 18S rDNA sequence (1,835 bp, SEQ ID NO: 112) was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with ATCC 34304 genomic DNA as a template. The PCR primers used were as follows: TMO30 was designed on the 18S rDNA sequence; TMO31 contained the 18S rDNA sequence and the EF1α promoter sequence. [TMO30: 30mer: 5'-CGA ATA TTC CTG GTT GAT CCT GCC AGT AGT -3' (SEQ ID NO: 113); TMO31: 46mer: 5'-GTA ACG GCT TTT TTT GAA TTG CAG GTT CAC TAC GCT TGT TAG AAA C -3' (SEQ ID NO: 114)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 2 min, 30 cycles / 72℃ 2min]. Also, Thraustochytrium golden The EF1α promoter sequence (661 bp, SEQ ID NO: 115) was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with ATCC 34304 genomic DNA as a template. The PCR primers used were as follows: TMO32 contains the 18S rDNA sequence and the EF1α promoter sequence; TMO33 contains the EF1α promoter sequence and an artificial neomycin resistance gene sequence. [TMO32: 46mer: 5'- GGT TTC CGT AGT GAA CCT GCA ATT CAA AAA AAG CCG TTA CTC ACA T -3' (SEQ ID NO: 116); TMO33: 46mer: 5'- GCG TGA AGG CCG TCC TGT TCA ATC ATC TAG CCT TCC TTT GCC GCT G-3' (SEQ ID NO: 117)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 1min]. The artificial neomycin resistance gene sequence (835 bp, SEQ ID NO: 118) was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with the artificial neomycin resistance gene as a template. The PCR primers used were as follows: TMO34 contains the EF1α promoter sequence and the artificial neomycin resistance gene sequence; TMO35 contains the artificial neomycin resistance gene sequence and the EF1α terminator sequence. [TMO34: 45mer: 5'-CAT CGG CAA AGG AAG GCT AGA TGA TTG AAC AGG ACG GCC TTC ACG -3' (SEQ ID NO: 119); TMO 35: 46mer: 5'-GCG CAT AGC CGG CGC GGA TCT CAA AAG AAC TCG TCC AGG AGG CGG T -3' (SEQ ID NO: 120)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 1min]. Also, Thraustochytrium golden The EF1α terminator sequence (1249 bp, SEQ ID NO: 121) was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with ATCC 34304 genomic DNA as a template. The PCR primers used were as follows: TMO36 contains an artificially synthesized neomycin resistance gene sequence and the EF1α terminator sequence; TMO37 was designed within the EF1α terminator sequence. [TMO36: 46mer: 5'-TCC TGG ACG AGT TCT TTT GAG ATC CGC GCC GGC TAT GCG CCC GTG C -3' (SEQ ID NO: 122), TMO37: 30mer: 5'-CAC TGC AGC GAA AGA CGG GCC GTA AGG ACG -3' (SEQ ID NO: 123)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 2 min, 30 cycles / 72℃ 2min]. Fusion PCR was performed using SEQ ID NOs: 112, 115, 118, and 121 as templates according to the method described in Non-Patent Document 9. LA Taq Hot Start Version (Takara Bio) was used as the enzyme. The first round of amplification used a set of TMO30 (SEQ ID NO: 113) and TMO33 (SEQ ID NO: 117), and a set of TMO34 (SEQ ID NO: 119) and TMO37 (SEQ ID NO: 123). The second round of amplification used a set of TMO30 (SEQ ID NO: 113) and TMO37 (SEQ ID NO: 123). PCR reaction conditions included a denaturation temperature of 98°C for 10 seconds, and annealing and extension reactions were adjusted appropriately depending on the Tm values ​​of the primers and the length of the amplified fragment (Figure 42). The DNA fragment ligated as described above (Figure 44, SEQ ID NO: 124, 4453 bp) T . golden EcoRI site in 18S rDNA, and T . golden The EF1α terminator was excised at the NcoI site and ligated into a vector derived from the pGEM-T easy vector, which was named pRH5 (Figure 45). Thraustochytrium golden Using ATCC 34304 genomic DNA as a template, PCR primers were designed within the upstream sequence (SEQ ID NO: 104) identified in Comparative Example 2-1 and the PUFA-PKS pathway-related gene OrfA described in Patent Document 4, and the DNA was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio Inc.). This amplification yielded a 1218 bp DNA fragment (SEQ ID NO: 125). This was used as the 5' homologous region of the targeting vector. The PCR primers used were as follows, each containing an EcoRI or HindIII site as a linker sequence: [RHO33: 32mer: 5'- CCC GAA TTC GGA CGA TGA CTG ACT GAC TGA TT -3' (SEQ ID NO: 126), RHO34: 28mer: 5'- CCC AAG CTT GTC TGC CTC GGC TCT TGG T -3' (SEQ ID NO: 127)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 57℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 3min]. Thraustochytrium golden Using ATCC 34304 genomic DNA as a template, PCR primers were designed within the downstream sequence (SEQ ID NO: 111) identified in Comparative Example 2-2, and DNA was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). This amplification yielded a 1,000 bp DNA fragment (SEQ ID NO: 128). This was used as the 3' homologous region of the targeting vector. The PCR primers used were as follows, both of which contained a linker sequence containing an NcoI site: [RHO29: 28mer: 5'-CCC CCA TGG TGT TGC TGT GGG ATT GGT C -3' (SEQ ID NO: 129), RHO30: 30mer: 5'-CCC CCA TGG CTC GGT TAC ATC TCT GAG GAA -3' (SEQ ID NO: 130)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 57℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 3min]. The amplified upstream sequence was ligated into the EcoRI and HindIII sites of pRH5 shown in Figure 43. The amplified downstream sequence was ligated into the NcoI site. This vector was named pRH21. The targeting vector (pRH21) using the artificially synthesized neomycin resistance gene is shown in FIG.

[0079] [Comparative Example 2-4] PUFA-PKS pathway-related genes: Construction of OrfA targeting vector (hygromycin resistance gene) Using pRH32 (Figure 6) described in Example 2-3 as a template, a ubiquitin promoter-hygromycin resistance gene fragment (1632 bp, SEQ ID NO: 131) was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used were as follows: RHO59 was designed on the ubiquitin promoter and contained a HindIII linker sequence; RHO60 contained the hygromycin resistance gene sequence including a stop codon and SphI and SalI linker sequences. [RHO59: 36mer: 5'-CCC AAG CTT GCC GCA GCG CCT GGT GCA CCC GCC GGG -3' (SEQ ID NO: 132); RHO60: 43mer: 5'-CCC GCA TGC GTC GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G -3' (SEQ ID NO: 133)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 68℃ 2 min, 30 cycles / 68℃ 2min]. The amplified fragment was ligated to the HindIII and SphI sites of pRH21 (FIG. 46) described in Comparative Example 2-3 (FIG. 47, pRH30). Thraustochytrium golden Using ATCC 34304 genomic DNA as a template, the gene was amplified using PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio) with PCR primers engineered in the downstream sequence (SEQ ID NO: 111) identified in Comparative Example 2-2. This amplification yielded a 1,000 bp DNA fragment (SEQ ID NO: 134). This was used as the 3' homologous region of the targeting vector. The PCR primers used were as follows, both of which contained a linker sequence containing a SalI site: [RHO61: 29mer: 5'- CCC GTC GAC GTG TTG CTG TGG GAT TGG TC -3' (SEQ ID NO: 135), RHO62: 29mer: 5'- CCC GTC GAC TCG GTT ACA TCT CTG AGG AA -3' (SEQ ID NO: 136)]. [PCR cycles: 98℃ 2min / 98℃ 30 sec, 57℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 3min]. The amplified downstream sequence was ligated into the SalI site of pRH30 (Figure 45). This was named pRH33. The targeting vector (pRH33) using the constructed hygromycin resistance gene is shown in Figure 48.

[0080] [Comparative Example 2-5] PUFA-PKS pathway related genes: OrfA targeting vector introduction Using the targeting vectors prepared in Comparative Examples 2-3 and 2-4 as templates and RHO30 (described in Comparative Example 2-3, SEQ ID NO: 130) and RHO33 (described in Comparative Example 2-3, SEQ ID NO: 126) as primers, the gene was amplified with PrimeSTAR Max DNA polymerase (Takara Bio Inc.). [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 phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. When pRH21 (Figure 46) described in Comparative Example 2-3 was used as a template, the introduced fragment obtained was 3705 bp. Thraustochytrium golden Upstream of the OrfA gene - EF1α promoter sequence - Artificially synthesized neomycin resistance gene sequence Thraustochytrium golden The sequence is the downstream of the OrfA gene (SEQ ID NO: 137). When pRH33 (FIG. 46) described in Comparative Example 2-4 was used as a template, the fragment obtained was 3826 bp. Thraustochytrium golden Upstream of the OrfA gene - ubiquitin promoter sequence - hygromycin resistance gene sequence Thraustochytrium golden The sequence is downstream of the OrfA gene (SEQ ID NO: 138). Thraustochytrium golden ATCC 34304 strain was cultured in GY medium for 4 days, and cells in the logarithmic growth phase were used for gene transfection. 0.625 μg of DNA fragments were transfected into cells equivalent to an OD600 of 1–1.5 using the gene gun method (microcarrier: 0.6 micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4–6 hour recovery period, the transfected cells were plated onto PDA agar plates (containing 2 mg / ml G418 or 2 mg / ml hygromycin). As a result, 100–200 drug-resistant clones were obtained per bombardment.

[0081] [Comparative Example 2-6] PUFA-PKS pathway-related genes: Identification of OrfA gene-targeting homologous recombinants By the method described in Example 2-2 Thraustochytrium golden Genomic DNA was extracted from ATCC 34304, heterologous recombinants, and homologous recombinants (PKS pathway-related gene disruptant strains), and the A260 / 280 was measured to calculate the DNA concentration. Genomic DNA was digested with restriction enzymes and electrophoresed on a 0.7% SeaKem GTG agarose gel (Takara Bio) at approximately 2-3 μg per well. This was then transferred to a nylon membrane and hybridized with a probe prepared using the DIG system (Roche Applied Science) at 54°C for 16 hours. The primers used to prepare the probe were as follows: The 5' end [RHO37: 22mer: 5'-GAA GCG TCC CGT AGA TGT GGT C -3' (SEQ ID NO: 139), RHO38: 21mer: 5'-GCC CGA GAG GTC AAA GTA CGC -3' (SEQ ID NO: 140)], and the 3' end [RHO39: 20mer: 5'-GCG AGC CCA GGT CCA CTT GC -3' (SEQ ID NO: 141), RHO40: 22mer: 5'-CAG CCC GAT GAA AAA CTT GGT C -3' (SEQ ID NO: 142)] [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 and probe positions used are shown in Figure 49. The hybridized probe was detected using a color development method (NBT / BCIP solution). In both the 5' and 3' side analyses, bands were observed at the sizes predicted when the drug resistance gene underwent homologous recombination (Fig. 50).

[0082] [Comparative Example 2-7] By the method described in Examples 2-9 Thraustochytrium golden ATCC 34304 and the gene disruptant were cultured and lyophilized, and the fatty acids were methyl-esterified and analyzed by GC. The changes in fatty acid composition are shown in Figure 51. Figure 52 shows the percentages when the wild-type strain is taken as 100%. Of the total fatty acid composition, ARA 3.10%, DGLA 0.23%, ETA 0.04%, EPA 6.82%, n-6DPA 10.66%, and DHA 22.58% are shown. In GC area, the LA / DHA value was 0.05, the GLA / DHA value was 0.03, the DGLA / DHA value was 0.01, the ARA / DHA value was 0.1, the EPA / DHA value was 0.3, The results show that the LA / EPA value is 0.16, the GLA / EPA value is 0.11, the DTA / EPA value is 0.29, the DTA / ARA value is 0.65, the DTA / DGLA value is 8.7, the LA / n-6DPA value is 0.1, the GLA / n-6DPA value is 0.07, the DGLA / n-6DPA value is 0.02, the ARA / n-6DPA value is 0.3, the EPA / n-6DPA value is 0.6, the DGLA / LA value is 0.2, the ARA / LA value is 2.9, the EPA / LA value is 6.4, the DTA / LA value is 1.9, the DGLA / GLA value is 0.3, the ARA / GLA value is 4.0, the n-6DPA / DTA value is 5.3, the DHA / n-3DPA value is 20.0, the C20PUFA / C22PUFA value is 0.3, and the n-6PUFA / n-3PUFA value is 0.52. As a result, Thraustochytrium golden When OrfA, a gene related to the PUFA PKS pathway, was disrupted in this plant, DPA (C22: 5n-6) tended to increase, while DHA (C22: 6n-3) tended to decrease. Schizochytrium genus ( Schizochytrium ) and Aurantiochytrium spp. ( Aurantium chytrium It is known that disruption of the genes in the PUFA-PKS pathway makes the bacteria auxotrophic for exogenous PUFAs, and they cannot grow without the supply of exogenous PUFAs (Non-Patent Document 4). Thraustochytrium golden Unlike these strains, ATCC34304 could be cultivated without the addition of exogenous PUFAs due to the disruption of genes related to the PUFA-PKS pathway. Furthermore, disruption of genes related to the PUFA-PKS pathway reduced DHA by only about two-thirds compared to the wild-type strain, while slightly increased DPA (C22: 5n-6). From these results, Thraustochytrium golden It was suggested that in ATCC34304, DHA and DPA n-6 may be produced not only by the PUFA-PKS pathway but also by other biosynthetic pathways. Thraustochytrium golden The reason why ATCC34304 can be cultured without adding exogenous PUFAs to the medium is presumably because endogenous PUFAs are supplied by biosynthetic pathways other than the PUFA-PKS pathway.

[0083] Comparative Example 3 [ Thraustochytrium golden Disruption of PUFA-PKS and C20 elongase genes in ATCC34304 and measurement of fatty acid composition of lipids produced by the transformants [Comparative Example 3-1] Thraustochytrium golden Cloning of upstream sequences of the C20 elongase gene The genome cassette library prepared in Comparative Example 2-1 was used as a template. A PCR lower primer [RHO71: 22mer: 5'-GGG AGC GCA GGG AAA ACG GTC T -3' (SEQ ID NO: 143)] was prepared on the upstream sequence of the C20 elongase gene described in Comparative Example 1-4 (SEQ ID NO: 76). This primer was combined with the cassette primers provided with the kit described in Comparative Example 2-1 to amplify the gene [first 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 first PCR amplification product was then diluted 100-fold, and the gene was amplified using a PCR lower primer [RHO72: 20mer: 5'-CCA GCC CAC GTC GTC GGA GC -3' (SEQ ID NO: 144)] and the nested primers provided with the kit described in Comparative Example 2-1 [second 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 the pGEM-T easy vector, amplified in Escherichia coli, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). A 2297 bp DNA fragment (SEQ ID NO: 145) containing the region from −3277 bp to −981 bp upstream of the C20 elongase gene was cloned.

[0084] [Comparative Example 3-2] Cloning of downstream sequence of C20 elongase gene The genome cassette library prepared in Comparative Example 2-1 was used as a template. A PCR upper primer [RHO87: 23mer: 5'-GCC GCT CAT GCC CAC GCT CAA AC -3' (SEQ ID NO: 146)] was prepared on the downstream sequence of the C20 elongase gene described in Comparative Example 1-4 (SEQ ID NO: 77). This was combined with the cassette primers provided with the kit described in Comparative Example 2-1 to amplify the gene [first 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 first PCR amplification product was then diluted 100-fold, and the gene was amplified using a PCR lower primer [RHO73: 23mer: 5'-CTT TCG GCT GCC AGG AAT CTA CG -3' (SEQ ID NO: 147)] and the nested primers provided with the kit described in Comparative Example 2-1 [second 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 the pGEM-T easy vector, amplified in Escherichia coli, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). A 2,189 bp DNA fragment (SEQ ID NO: 148) containing the region from 1,106 bp to 3,294 bp downstream of the C20 elongase gene was cloned.

[0085] [Comparative Example 3-3] Preparation of a blasticidin resistance gene cassette Thraustochytrium golden The ubiquitin promoter sequence (618 bp, SEQ ID NO: 149) was amplified from ATCC 34304 using genomic DNA as a template with PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used were as follows: RHO53 was designed on the ubiquitin promoter sequence and contained a BglII linker sequence (Example 2-2, SEQ ID NO: 5); RHO48 contained the ubiquitin promoter sequence and a blasticidin resistance gene sequence. [RHO48: 58mer: 5'-CTT CTT GAG ACA AAG GCT TGG CCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G -3' (SEQ ID NO: 150)]. [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 1 min, 30 cycles / 68°C 1 min]. The blasticidin resistance gene (432 bp, SEQ ID NO: 151) was amplified using pTracer-CMV / Bsd / lacZ (Invitrogen) as a template with PrimeSTAR HS DNA polymerase with GC Buffer. The PCR primers used were as follows: RHO47 contains the ubiquitin promoter sequence and the blasticidin resistance gene sequence; RHO49 contains the blasticidin resistance gene sequence and a BglII linker sequence. [RHO47: 54mer: 5'-AGC GAC CTA AGC AAC ACT AGC CAA CAT GGC CAA GCC TTT GTC TCA AGA AGA ATC -3' (SEQ ID NO: 152); RHO49: 38mer: 5'-CCC AGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG -3' (SEQ ID NO: 153)]. [PCR cycles: 98℃ 2min / 98℃ 10 sec, 68℃ 1 min, 30 cycles / 68℃ 1min]. Fusion PCR was performed using SEQ ID NOs: 149 and 151 as templates and RHO53 (Example 2-2, SEQ ID NO: 5) and RHO49 (SEQ ID NO: 153) according to the method described in Non-Patent Document 9. The enzyme used was LA Taq Hot Start Version (Takara Bio Inc.), and amplification was performed under the following conditions, followed by digestion with BglII. [PCR cycles: 94°C for 2 min / 94°C for 20 sec, 55°C for 30 sec, 68°C for 1 min, 30 cycles / 68°C for 1 min, except that the temperature change from 55°C to 68°C was 1°C / 10 sec] (Figure 53). Fusion was performed as above. Thraustochytrium golden The ATCC 34304-derived ubiquitin promoter-pTracer-CMV / Bsd / lacZ-derived blasticidin resistance gene (1,000 bp, SEQ ID NO: 154) was digested with BglII and ligated into the BamHI site of pRH27 (Figure 2) described in Example 2-1. The resulting plasmid was amplified in E. coli and its sequence confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This plasmid was named pRH38. The constructed blasticidin resistance gene cassette (pRH38) is shown in FIG.

[0086] [Comparative Example 3-4] Preparation of GFP-fused Zeocin resistance gene cassette Thraustochytrium golden The ubiquitin promoter sequence (812 bp, SEQ ID NO: 155) was amplified from ATCC 34304 using genomic DNA as a template with PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primers used were as follows: TMO38 was designed on the ubiquitin promoter sequence; TMO39 contained the ubiquitin promoter sequence and the Enhanced GFP gene sequence. [TMO38: 29mer: 5'-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC -3' (SEQ ID NO: 156), TMO39: 41mer: 5'-TCC TCG CCC TTG CTC ACC ATG TTG GCT AGT GTT GCT TAG GT -3' (SEQ ID NO: 157)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 1 min, 30 cycles / 72℃ 1min]. The Enhanced GFP gene sequence (748 bp, SEQ ID NO: 158) was amplified using PrimeSTAR HS DNA polymerase (Takara Bio) with the Enhanced GFP gene sequence (Clontech) as a template. The PCR primers used were as follows: TMO40 contains the ubiquitin promoter sequence and the Enhanced GFP gene sequence; RHO91 contains the Enhanced GFP sequence and the Zeocin resistance gene sequence. [TMO40: 41mer: 5'-ACC TAA GCA ACA CTA GCC AAC ATG GTG AGC AAG GGC GAG GA -3' (SEQ ID NO: 159); RHO91: 58mer: 5'-GAA CGG CAC TGG TCA ACT TGG CGT CCA TGC CGA GAG TGA TCC CGG CGG CGG TCA CGA A-3' (SEQ ID NO: 160)]. [PCR cycles: 98℃ 10 sec / 98℃ 10 sec, 58℃ 30 sec, 72℃ 2 min, 30 cycles / 72℃ 2min]. Fusion PCR was performed using SEQ ID NOs: 156 and 158 as templates with LA Taq Hot Start version (Takara Bio Inc.) according to the method described in Non-Patent Document 9. Primers used were TMO38 (SEQ ID NO: 156) and RHO91 (SEQ ID NO: 160), and the PCR conditions were: 94°C 2 min / 94°C 20 sec, 55°C 30 sec, 68°C 2 min, 30 cycles / 68°C 2 min (however, 1°C / 10 sec from 55°C to 68°C) (Figure 55, 1519 bp, SEQ ID NO: 161). Using SEQ ID NO: 161 as a template, the ubiquitin promoter sequence-Enhanced GFP gene sequence (1,319 bp, SEQ ID NO: 162) was amplified with PrimeSTAR HS DNA polymerase (Takara Bio Inc.). The primers used are as follows: RHO53 (Example 2-2, SEQ ID NO: 5) contains the ubiquitin promoter sequence and a BglII site; RHO91 (SEQ ID NO: 160) contains the Enhanced GFP sequence and Zeocin resistance gene sequence. [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 2 min, 30 cycles / 68°C 2 min]. The Zeocin® resistance gene sequence (408 bp, SEQ ID NO: 163) was amplified using pcDNA3.1 Zeo(+) as a template with PrimeSTAR HS DNA polymerase (Takara Bio). RHO92 contains an enhanced GFP sequence and the Zeocin® resistance gene sequence. RHO64 contains the Zeocin® resistance gene sequence and a BglII site. [RHO92: 54mer: 5'- CGC CGC CGG GAT CAC TCT CGG CAT GGA CGC CAA GTT GAC CAG TGC CGT TCC GGT -3' (SEQ ID NO: 164), RHO64: 38mer: 5'- CCC AGA TCT CAG TCC TGC TCC TCG GCC ACG AAG TGC AC -3' (SEQ ID NO: 165)]. [PCR cycles: 98℃ 2 min / 98℃ 10 sec, 68℃ 1 min, 30 cycles / 68℃ 1min]. Fusion PCR was performed using SEQ ID NOs: 162 and 163 as templates with LA taq Hot start version (Takara Bio) according to the method described in Non-Patent Document 9. Primers used were RHO53 (Example 2-2, SEQ ID NO: 5) and RHO64 (SEQ ID NO: 165), and the PCR conditions were: 94°C 2 min / 94°C 20 sec, 68°C 2 min, 30 cycles / 68°C 2 min (however, 1°C / 10 sec from 55°C to 68°C) (Figure 56). Fusion was performed as above. Thraustochytrium golden The ATCC 34304-derived ubiquitin promoter-Enhanced GFP gene-pcDNA3.1 Zeo(+)-derived Zeocin resistance gene (Figure 56, 1677 bp, SEQ ID NO: 166) was digested with BglII and ligated into the BamHI site of pRH27 (Figure 2) described in Example 2-1. The resulting plasmid was amplified in E. coli and its sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This plasmid was named pRH51. The GFP-fused zeocin resistance gene cassette (pRH51) thus constructed is shown in FIG.

[0087] [Comparative Example 3-5] Preparation of a base plasmid for the construction of a C20 elongase gene targeting vector Thraustochytrium golden Using ATCC 34304 genomic DNA as a template, the C20 elongase gene and its surrounding sequences were amplified by PCR using PrimeSTAR HS DNA polymerase (Takara Bio Inc.) (2884 bp, SEQ ID NO: 167). The PCR primers used were as follows; both contained EcoRI linker sequences. KSO9 was designed to be located upstream of the C20 elongase gene (SEQ ID NO: 76), and KSO10 was designed to be located downstream of the C20 elongase gene (SEQ ID NO: 77): [KSO9: 50mer: 5'-CCC GAA TTC ACT AGT GAT TCT CCC GGG TGG ACC TAG CGC GTG TGT CAC CT -3' (SEQ ID NO: 168), KSO10: 40mer: 5'-CCC GAA TTC GAT TAT CCC GGG GCC GAG AAC GGG GTC GCC C -3' (SEQ ID NO: 169)]. PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 3.5 min, 30 cycles / 68°C 10 min. PrimeSTAR HS DNA Polymerase (Takara Bio) was used. After amplification, the fragment was digested with EcoRI and cloned into the EcoRI site of pBluescript(SK) (Stratagene) vector. After amplification in E. coli, the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter) (Figure 58). Using the plasmid shown in Figure 58 as a template, a primer set was prepared in reverse orientation to delete the C20 elongase gene sequence and insert a BglII site (1939 bp, SEQ ID NO: 170). Amplification was performed using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). The PCR primers used were as follows, both of which contain a BglII linker sequence: RHO69: 38mer: 5'-CCC AGA TCT ACC TGT TTC CGG CTG GCT CCC GAG CCA TG -3' (SEQ ID NO: 171), RHO70: 38mer: 5'-CCC AGA TCT GGT CGC GTT TAC AAA GCA GCG CAG CAA CA -3' (SEQ ID NO: 172). [PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 1.5 min, 30 cycles / 68°C 1.5 min] After amplification under the above conditions, the fragment was digested with BglII and self-ligated. The ligated sample was amplified in E. coli and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This fragment was named pRH40. The base plasmid (pRH40) for constructing the C20 elongase gene targeting vector is shown in Figure 59.

[0088] [Comparative Example 3-6] Construction of targeting vectors (blasticidin resistance gene and GFP-fused zeocin resistance gene) pRH38 (Fig. 52) described in Comparative Example 3-3 was digested with BglII, and the DNA fragment containing the blasticidin resistance gene cassette was ligated into the BglII site of pRH40 (Fig. 59) described in Comparative Example 3-5. This was named pRH43. pRH51 (Fig. 55) described in Comparative Example 3-4 was digested with BglII, and the DNA fragment containing the GFP-fused Zeocin resistance gene cassette was ligated into the BglII site of pRH40 (Fig. 57) described in Comparative Example 3-5. This was named pRH54. The two targeting vectors (pRH43 and 54) that were constructed are shown in FIG.

[0089] [Comparative Example 3-7] Thraustochytrium golden Introduction of C20 elongase gene targeting vector into OrfA disruptant The two types of targeting vectors prepared in Comparative Example 3-6 were used as templates, and genes were amplified using PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.) with KSO11 and KSO12 as primers. Thraustochytrium golden Upstream of the C20 elongase gene, KSO12 Thraustochytrium golden The primers were placed downstream of the C20 elongase gene. [KSO11: 31mer: 5'-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T -3' (SEQ ID NO: 173), KSO12: 27mer: 5'-ATC CCG GGG CCG AGA ACG CCC TCG CCC -3' (SEQ ID NO: 174)]. [PCR cycles: 98°C 2 min / 98°C 30 sec, 68°C 2 min, 30 cycles / 68°C 2 min]. After phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. The inserted fragment obtained when pRH43 (Figure 60) described in Comparative Example 3-6 was used as a template was 3215 bp. Thraustochytrium golden C20 elongase gene upstream - ubiquitin promoter - blasticidin resistance gene sequence - SV40 terminator sequence Thraustochytrium golden The sequence is downstream of the C20 elongase gene (SEQ ID NO: 175). When pRH54 (Figure 60) described in Comparative Example 3-6 was used as a template, the fragment obtained was 3887 bp. Thraustochytrium golden C20 elongase gene upstream - ubiquitin promoter - Enhanced GFP gene sequence - Zeocin resistance gene sequence - SV40 terminator sequence Thraustochytrium golden The sequence is downstream of the C20 elongase gene (sequence number 176). The PUFA-PKS pathway-related gene: OrfA gene disruptant described in Comparative Example 2 was cultured in GY medium for 4 days, and cells in the logarithmic growth phase were used for gene transfer. 0.625 μg of DNA fragments were introduced into cells equivalent to an OD600 of 1 to 1.5 using the gene gun method (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 period, the transfected cells were plated on PDA agar plates (containing 2 mg / ml G418 or 2 mg / ml hygromycin). As a result, 100 to 200 drug-resistant strains were obtained per bombardment.

[0090] [Comparative Example 3-8] Identification of C20 elongase gene gene targeting homologous recombinants By the method described in Example 2-2, Thraustochytrium golden and Thraustochytrium golden Genomic DNA was extracted from the OrfA-disrupted strain in which the C20 elongase gene was disrupted, and the A260 / 280 was measured to calculate the DNA concentration. Genomic DNA was digested with restriction enzymes and electrophoresed on a 0.7% SeaKem GTG agarose gel (Takara Bio) at approximately 2-3 μg per well. This was then transferred to a nylon membrane and hybridized with a probe prepared using the DIG system (Roche Applied Science) at 51°C for 16 hours. The primers used to prepare the probe were as follows: The 5' end [RHO94: 21mer: 5'-ACG TCC GCT TCA AAC ACC TCG -3' (SEQ ID NO: 177), RHO95: 24mer: 5'-TCG GAA CAA CTG GAA CAA CTA AAG -3' (SEQ ID NO: 178)], and the 3' end [RHO96: 22mer: 5'-ATG TCG CTC TCC TTC TTC TCA G-3' (SEQ ID NO: 179), RHO97: 21mer: 5'-TCG GCT CCT GGA AAG TGC TCT -3' (SEQ ID NO: 180)]. [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 and probe positions used are shown in Figure 61. The hybridized probe was detected using a color development method (NBT / BCIP solution). In both the 5' and 3' analysis, bands were observed at the size predicted when the drug resistance gene underwent homologous recombination (Figure 62). Thraustochytrium golden It was found that the ATCC 34304 strain did not become auxotrophic even when the PKS pathway-related genes, OrfA and the C20 elongase gene, were deleted.

[0091] [Comparative Example 3-9] Thraustochytrium golden Changes in fatty acid composition due to disruption of the C20 elongase gene in OrfA disruptant strains By the method described in Examples 2-9 Thraustochytrium golden ATCC 34304 and the gene disruptant were cultured and lyophilized, and the fatty acids were methyl-esterified and analyzed by GC. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.) column at 150°C → (5°C / min) → 220°C (10 min), with a carrier gas of He (1.3 mL / min). The changes in fatty acid composition are shown in Figure 63. Figure 64 shows the percentages when the wild-type strain is taken as 100%. Of the total fatty acid composition, ARA 19.50%, DGLA 1.81%, ETA 0.31%, EPA 24.92%, n-6DPA 5.90%, and DHA 6.78% are shown. In terms of GC area, the LA / DHA value was 0.25, the GLA / DHA value was 0.07, the DGLA / DHA value was 0.27, the ARA / DHA value was 2.88, the EPA / DHA value was 3.68, the LA / EPA value was 0.07, the GLA / EPA value was 0.02, the DTA / EPA value was 0.02, and the DTA / ARA value was 0.02. The values ​​for DTA / DGLA are 0.26, LA / n-6DPA are 0.29, GLA / n-6DPA are 0.08, DGLA / n-6DPA are 0.31, ARA / n-6DPA are 3.31, EPA / n-6DPA are 4.22, DGLA / LA are 1.06, ARA / LA are 11.40, EPA / LA are 14.57, DTA / LA are 0.27, DGLA / GLA are 4.02, ARA / GLA are 43.33, n-6DPA / DTA are 12.55, DHA / n-3DPA are 11.69, C20PUFA / C22PUFA are 3.39, and n-6PUFA / n-3PUFA are 0.85. As a result, Thraustochytrium golden When the C20 elongase gene was disrupted in the OrfA disruptant, C20: 4n-6 (ARA) increased approximately 8-fold, C20: 5n3 (EPA) increased approximately 4-fold, and C22: 6n-3 (DHA) decreased approximately 5-fold. Thus, it has both an endogenous elongase / desaturase pathway and an endogenous PUFA-PKS pathway. Thraustochytrium golden It became clear from ATCC34304 that in order to generate a strain with significantly reduced DHA and DPA n-6 production compared to the wild-type strain, it was necessary to disrupt both the genes for enzymes that make up the elongase / desaturase pathway (e.g., the C20 elongase gene) and the genes related to the PUFA-PKS pathway.

[0092] Comparative Example 4 [ Thraustochytrium golden Disruption of the PUFA-PKS and Δ4 desaturase genes of ATCC34304 and measurement of the fatty acid composition of lipids produced by the transformants [Comparative Example 4-1] Thraustochytrium golden Cloning of a sequence from 1071 bp upstream of the Δ4 desaturase gene to 1500 bp within the Δ4 desaturase gene of ATCC 34304 strain Extracted by the method described in Example 2-2 Thraustochytrium golden The genomic DNA of the ATCC 34304 strain was sequenced and a search was conducted for gene sequences with high homology to known Δ4 desaturases. Based on the search results, two PCR primers were designed. TMO3 is Thraustochytrium golden The sequence was located 1071 to 1049 bp upstream of the Δ4 desaturase gene of the ATCC 34304 strain, and TMO4 was located 1477 to 1500 bp from the initiation codon in the protein-coding region [TMO3: 23 mer: 5'- GGC GGA GCG AAG TGT GAA AGT TA -3' (SEQ ID NO: 181), TMO4: 24 mer: 5'- GCG ACA GCA TCT TGA AAT AGG CAG -3' (SEQ ID NO: 182)]. These two primers were used to amplify the sequences. Thraustochytrium golden The genomic DNA of the ATCC 34304 strain was used as a template, and the LA Taq Hot Start version (manufactured by Takara Bio) was used. Thraustochytrium golden A sequence from 1071 bp upstream of the Δ4 desaturase gene to 1500 bp within the Δ4 desaturase gene of the ATCC 34304 strain (2571 bp, SEQ ID NO: 183) was amplified. The amplification conditions were as follows: [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 the pGEM-T easy vector, amplified in E. coli, and the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This was designated pTM1 (Figure 65).

[0093] [Comparative Example 4-2] Preparation of a base plasmid for constructing a Δ4 desaturase gene targeting vector Using pTM1 (Figure 65) prepared in Comparative Example 4-1 as a template, 60 bp upstream of the Δ4 desaturase gene and a 556-bp sequence (616 bp, SEQ ID NO: 184) containing the initiation codon within the Δ4 desaturase gene were deleted, and a primer set was prepared in reverse orientation to create a BglII site at the deleted site. Both TMO7 and TMO8 contain a BglII sequence. PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) was used for amplification. [TMO7: 25-mer: 5'-CAG GAG ATC TCC AAG TCG CGA TTC A -3' (SEQ ID NO: 185), TMO8: 26-mer: 5'-CTT GGA GAT CTC CTG CCC GTC CCG AA -3' (SEQ ID NO: 186)]. PCR cycles: 98°C 3 min / 98°C 10 sec, 55°C 15 sec, 72°C 30 sec, 30 cycles / 72°C 30 sec. After amplification under these conditions, the fragment was purified by agarose gel electrophoresis. The resulting DNA fragment was transformed into E. coli and amplified. The sequence was confirmed using a Dye Terminator Cycle Sequencing Kit (Beckman Coulter). This fragment was named pTM2. The constructed plasmid (pTM2) that served as the base for constructing the Δ4 desaturase gene targeting vector is shown in Figure 66.

[0094] [Comparative Example 4-3] Construction of targeting vectors (blasticidin resistance gene and GFP-fused zeocin resistance gene) pRH38 (Fig. 54) described in Comparative Example 3-3 was digested with BglII, and the DNA fragment containing the blasticidin resistance gene cassette was ligated into the BglII site of pTM2 (Fig. 66) described in Comparative Example 4-2. This was named pTM6. pRH51 (Fig. 57) described in Comparative Example 3-4 was digested with BglII, and the DNA fragment containing the GFP-fused Zeocin resistance gene cassette was ligated into the BglII site of pTM2 (Fig. 66) described in Comparative Example 4-2. This was named pTM8. The two targeting vectors (pTM6 and 8) that were constructed are shown in Figure 67.

[0095] [Comparative Example 4-4] Thraustochytrium golden Introduction of a Δ4 desaturase gene targeting vector into the OrfA disruptant Using the two targeting vectors prepared in Comparative Example 4-3 as templates and TMO3 (described in Comparative Example 4-1, SEQ ID NO: 181) and TMO4 (described in Comparative Example 4-1, SEQ ID NO: 182) as primers, the gene was amplified with PrimeSTAR HS DNA polymerase (Takara Bio Inc.). [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 phenol-chloroform extraction and chloroform extraction, the DNA was precipitated with ethanol, and the precipitate was dissolved in 0.1x TE. A260 / 280 was measured to calculate the DNA concentration. The fragment introduced when pTM6 (Figure 67) described in Comparative Example 4-3 was used as a template was 3264 bp. Thraustochytrium golden Δ4 desaturase gene upstream - SV40 terminator sequence - blasticidin resistance gene sequence - ubiquitin promoter Thraustochytrium golden The sequence is the same as that in the Δ4 desaturase gene (SEQ ID NO: 187). When pTM8 (FIG. 67) described in Comparative Example 4-3 was used as a template, the fragment obtained was 3935 bp. Thraustochytrium golden Δ4 desaturase gene upstream - SV40 terminator sequence - Zeocin resistance gene sequence - Enhanced GFP gene sequence - ubiquitin promoter Thraustochytrium golden The sequence is that of the Δ4 desaturase gene (SEQ ID NO: 188). The PUFA PKS pathway-related gene: OrfA gene disruptant described in Comparative Example 2 was cultured in GY medium for 4 days, and cells in the logarithmic growth phase were used for gene transfer. 0.625 μg of DNA fragments were introduced into cells equivalent to an OD600 of 1 to 1.5 using the gene gun method (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 period, the transfected cells were plated onto PDA agar plates (containing 20 mg / ml Zeocin or 0.2 mg / ml Blasticidin). As a result, 100 to 200 drug-resistant strains were obtained per bombardment.

[0096] [Comparative Example 4-5] Identification of Δ4 desaturase gene gene targeting homologous recombinants By the method described in Example 2-2, Thraustochytrium golden and Thraustochytrium golden Genomic DNA was extracted from the OrfA-disrupted strain in which the Δ4 desaturase gene had been disrupted, and the A260 / 280 was measured to calculate the DNA concentration. Using this as a template, PCR was performed to confirm the genome structure using Mighty Amp DNA polymerase (Takara Bio Inc.). The positions of the primers used, the combinations used for amplification, and the predicted sizes of the amplified products are shown in Figure 68. TMO1 was located upstream of the Δ4 desaturase gene, TMO2 downstream of the Δ4 desaturase gene, RHO198 (SEQ ID NO: 191) and RHO49 (described in Comparative Example 3-3; SEQ ID NO: 153) on the blasticidin resistance gene, RHO128 on the enhanced GFP gene, and RHO64 (described in Comparative Example 3-4; SEQ ID NO: 165) on the zeocin resistance gene. TMO1: 23-mer: 5'-AAA AGA ACA AGC CCT CTC CTG GA -3' (SEQ ID NO: 189), TMO2: 23-mer: 5'-GAG GTT TGT ATG TTC GGC GGT TT -3' (SEQ ID NO: 190), RHO198: 26-mer: 5'-TGG GGG ACC TTG TGC AGA ACT CGT GG -3' (SEQ ID NO: 191), RHO128: 22-mer: 5'-GAC CTA CGG CGT GCA GTG CTT C -3' (SEQ ID NO: 192). PCR cycles: 98°C 2 min / 98°C 10 sec, 68°C 4 min 30 sec, 30 cycles / 68°C 4 min. A Δ4 desaturase gene disruptant was obtained in which the wild-type allele (Wt allele) was not amplified, but the blasticidin resistance gene allele (BlaR allele) and the zeocin resistance gene allele (ZeoR allele) were amplified (Figure 69). Thraustochytrium golden It was found that the ATCC 34304 strain did not become auxotrophic even when the PKS pathway-related genes, OrfA and the Δ4 desaturase gene, were deleted.

[0097] [Comparative Example 4-6] Thraustochytrium golden Changes in fatty acid composition due to disruption of the Δ4 desaturase gene in OrfA disruptant strains By the method described in Examples 2-9 Thraustochytrium golden ATCC 34304 and the gene disruptant were cultured and lyophilized, and the fatty acids were methyl-esterified and analyzed by GC. GC analysis was performed using a gas chromatograph GC-2014 (Shimadzu Corporation) with a HR-SS-10 (30 m x 0.25 mm; Shinwa Chemical Co., Ltd.) column at 150°C → (5°C / min) → 220°C (10 min), with a carrier gas of He (1.3 mL / min). The changes in fatty acid composition are shown in Figure 70. Figure 71 shows the percentages when the wild-type strain is taken as 100%. Of the total fatty acid composition, ARA 6.35%, DGLA 0.90%, ETA 0.28%, EPA 6.22%, n-6DPA 0.21%, and DHA 0.51% are shown. In terms of GC area, the LA / DHA value was 8.76, the GLA / DHA value was 1.59, the DGLA / DHA value was 1.76, the ARA / DHA value was 12.45, the EPA / DHA value was 12.20, the LA / EPA value was 0.72, the GLA / EPA value was 0.13, the DTA / EPA value was 1.77, and the DTA / ARA value was 1.73. The DTA / DGLA value is 12.23, LA / n-6DPA value is 21.29, GLA / n-6DPA value is 3.86, DGLA / n-6DPA value is 4.29, ARA / n-6DPA value is 30.24, EPA / n-6DPA value is 29.62, DGLA / LA value is 0.20, ARA / LA value is 1.42, EPA / LA value is 1.39, DTA / LA value is 2.46, DGLA / GLA value is 1.11, ARA / GLA value is 7.84, n-6DPA / DTA value is 0.02, DHA / n-3DPA value is 0.03, C20PUFA / C22PUFA value is 0.50, and n-6PUFA / n-3PUFA value is 0.81. As a result, Thraustochytrium golden When the Δ4 desaturase gene was disrupted in the OrfA disruptant, biosynthesis of C22:5n-6 (DPA) and C22:6n-3 (DHA) was almost completely stopped, and C22:4n-6 (DTA) and C22:5n-3 (DPA), which are substrates for Δ4 desaturase, accumulated. Thus, it has both an endogenous elongase / desaturase pathway and an endogenous PUFA-PKS pathway. Thraustochytrium golden ATCC34304 revealed that in order to generate a strain that is largely unable to biosynthesize DHA and DPA n-6, it was necessary to disrupt both the genes for the enzymes that make up the elongase / desaturase pathway (e.g., the Δ4 desaturase gene) and the PUFA-PKS genes.

[0098] By using the microbial oil obtained in this way, it is possible to obtain microbial oil with a fatty acid composition that increases the composition ratio of PUFAs other than DHA and n-6 ​​DPA. By genetically modifying microorganisms that produce a large amount of DHA, it is possible to produce any PUFA. Furthermore, by producing microbial oil with particularly low levels of DHA and n-6 ​​DPA, it is possible to produce microbial oil with a low burden during the refining process. Furthermore, by introducing elongase and desaturase into microorganisms in this way, it is possible to obtain microorganisms that produce microbial oil. [Industrial Applicability]

[0099] We have discovered a novel "pattern" for the biosynthetic pathway of polyunsaturated fatty acids (PUFAs) in microorganisms called Labyrinthulae. By providing Labyrinthulae that produce PUFAs exclusively through the elongase / desaturase pathway, we hope to mass-produce PUFAs using only the elongase / desaturase pathway.< / super>

Claims

1. A method for producing microbial oil from Labyrinthulea that has been genetically modified to alter its fatty acid composition, wherein the Labyrinthulea belongs to the genus Parietichytrium or Schizochytrium and has no or very weak ability to produce highly unsaturated fatty acids (PUFAs) through an endogenous PUFA-PKS pathway and has the ability to produce PUFAs through an endogenous elongase / desaturase pathway, and the fatty acid composition of the Labyrinthulea has been altered by disrupting and / or suppressing the expression of a fatty acid desaturase gene in the Labyrinthulea, and the microbial oil obtained from the Labyrinthulea satisfies the following (a) and (b): (a) A microbial oil in which DHA accounts for 0.50% or less of the total fatty acid composition. (b) A microbial oil in which the sum of DHA and n-6DPA is 0.7% or less of the total fatty acid composition.

2. The method for producing a microbial oil according to claim 1, wherein the method for disrupting the gene of Labyrinthula is electroporation, gene gun method or genome editing.

3. The method for producing microbial oil according to claim 1, wherein the method for suppressing the expression of a Labyrinthula gene is an antisense method or RNA interference.

4. The method for producing a microbial oil according to any one of claims 1 to 3, wherein the Labyrinthula belonging to the genus Parietichytrium belongs to Parietichytrium sarkarianum, and the Labyrinthula belonging to the genus Schizochytrium belongs to Schizochytrium aggregatum.

5. The method for producing a microbial oil according to any one of claims 1 to 3, wherein the Labyrinthula belonging to the genus Parietichytrium is Parietichytrium sp. SEK358, FERM BP-11405, Parietichytrium sarkarianum SEK364, FERM BP-11298 or Parietichytrium sp. SEK571, FERM BP-11406, and the Labyrinthula belonging to the genus Schizochytrium is Schizochytrium aggregatum ATCC28209.

6. A method for modifying the fatty acid composition of Labyrinthulea, wherein the Labyrinthulea belongs to the genus Parietichytrium or Schizochytrium and has no or very weak ability to produce highly unsaturated fatty acids (PUFAs) through an endogenous PUFA-PKS pathway and has the ability to produce PUFAs through an endogenous elongase / desaturase pathway, and the fatty acid composition of the Labyrinthulea has been modified by disrupting and / or suppressing the expression of a fatty acid desaturase gene in the Labyrinthulea, and the microbial oil obtained from the Labyrinthulea is a microbial oil that satisfies the following (a) and (b): (a) A microbial oil in which DHA accounts for 0.50% or less of the total fatty acid composition. (b) A microbial oil in which the sum of DHA and n-6DPA is 0.7% or less of the total fatty acid composition.

7. The method for modifying a fatty acid composition according to claim 6, wherein the method for disrupting the Labyrinthula gene is carried out by electroporation, gene gun method or genome editing.

8. 7. The method for modifying a fatty acid composition according to claim 6, wherein the method for suppressing the expression of a Labyrinthule gene is an antisense method or RNA interference.

9. 9. The method for modifying a fatty acid composition according to any one of claims 6 to 8, wherein the fatty acid desaturase gene is a Δ4 desaturase gene.

10. 10. The method for modifying a fatty acid composition according to any one of claims 6 to 9, wherein the Labyrinthulea belonging to the genus Parietichytrium belongs to Parietichytrium sarkarianum, and the Labyrinthulea belonging to the genus Schizochytrium belongs to Schizochytrium aggregatum.

11. The method for modifying a fatty acid composition according to any one of claims 6 to 9, wherein the Labyrinthulea belonging to the genus Parietichytrium is Parietichytrium sp. SEK358, FERM BP-11405, Parietichytrium sarkarianum SEK364, FERM BP-11298, or Parietichytrium sp. SEK571, FERM BP-11406, and the Labyrinthulea belonging to the genus Schizochytrium is Schizochytrium aggregatum ATCC28209.

12. A method for producing a microorganism that produces a microbial oil that satisfies the following (a) and (b), wherein the microorganism is a Labyrinthula belonging to the genus Parietichytrium or Schizochytrium that has no or very weak ability to produce highly unsaturated fatty acids (PUFAs) through an endogenous PUFA-PKS pathway and has the ability to produce PUFAs through an endogenous elongase / desaturase pathway, and the method for producing a microorganism that produces a microbial oil that satisfies the following (a) and (b) by disrupting and / or suppressing the expression of a fatty acid desaturase gene. (a) DHA accounts for 0.50% or less of the total fatty acid composition. (b) The total amount of DHA and n-6DPA is 0.7% or less of the total fatty acid composition.

13. The method for producing a microorganism according to claim 12, wherein the method for disrupting a Labyrinthula gene is electroporation, a gene gun method, or genome editing.

14. 13. The method for producing a microorganism according to claim 12, wherein the method for suppressing the expression of a Labyrinthule gene is an antisense method or RNA interference.

15. 15. The method for producing a microorganism according to any one of claims 12 to 14, wherein the fatty acid desaturase gene is a Δ4 desaturase gene.

16. 16. The method for producing a microorganism according to any one of claims 12 to 15, wherein the Labyrinthulea belonging to the genus Parietichytrium belongs to Parietichytrium sarkarianum, and the Labyrinthulea belonging to the genus Schizochytrium belongs to Schizochytrium aggregatum.

17. The method for producing a microorganism according to any one of claims 12 to 15, wherein the Labyrinthulea belonging to the genus Parietichytrium is Parietichytrium sp. SEK358, FERM BP-11405, Parietichytrium sarkarianum SEK364, FERM BP-11298, or Parietichytrium sp. SEK571, FERM BP-11406, and the Labyrinthulea belonging to the genus Schizochytrium is Schizochytrium aggregatum ATCC28209.

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