Microorganisms that produce eicosapentaenoic acid and method for producing eicosapentaenoic acid

Abstract

The present invention aims to provide a microorganism that efficiently produces EPA and a method for producing EPA using said microorganism. The present invention relates to a microorganism capable of producing docosahexanoic acid (DHA), which contains a protein (mutant OrfB) consisting of an amino acid sequence represented by SEQ ID NO: 2 in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 has been substituted with another amino acid residue, and which is capable of producing eicosapentaenoic acid (EPA).

Description

Technical Field

[0001] The present invention relates to a microorganism that produces eicosapentaenoic acid and a method for producing eicosapentaenoic acid using the microorganism.

Background Art

[0002] Polyunsaturated fatty acids (hereinafter referred to as PUFAs) are long-chain fatty acids having a plurality of unsaturated bonds in the molecule, such as docosahexaenoic acid (hereinafter referred to as DHA), eicosapentaenoic acid (hereinafter referred to as EPA), arachidonic acid (hereinafter referred to as ARA), and docosapentaenoic acid (hereinafter referred to as DPA). PUFAs are known to have various physiological functions such as prevention of arteriosclerosis and hyperlipidemia (Non-Patent Documents 1 and 2).

[0003] As biosynthetic pathways of PUFAs, two types are known: an aerobic pathway and an anaerobic pathway by polyunsaturated fatty acid polyketide synthase (hereinafter referred to as PUFA-PKS). The aerobic pathway is a pathway in which PUFAs are synthesized by introducing double bonds by a plurality of desaturating enzymes and elongating the carbon chain by a chain elongation enzyme to long-chain fatty acids such as palmitic acid synthesized by fatty acid synthase, and is a synthetic pathway known from ancient times possessed by many organisms (Non-Patent Document 3).

[0004] On the other hand, the anaerobic pathway by PUFA-PKS is a pathway for synthesizing PUFAs from acetyl-CoA and malonyl-CoA, and is known to be possessed by some marine bacteria and labyrinthulid eukaryotes (Non-Patent Documents 4 and 5).

[0005] PUFA-PKS is a complex enzyme composed of a plurality of proteins (hereinafter also referred to as a protein complex), and each protein has a plurality of functional domains involved in the synthesis of PUFAs.

[0006] The functional domains present in PUFA-PKS include the β-ketoacyl-acyl carrier protein synthase domain (hereinafter referred to as the KS domain), which is thought to be involved in the condensation of malonyl-ACP and acyl-ACP; the acyl carrier protein domain (hereinafter referred to as the ACP domain), which is thought to function as a site for fatty acid synthesis by binding to the acyl group via a thioester bond through a phosphopantetheinyl group; the ketoreductase domain (hereinafter referred to as the KR domain), which is thought to reduce the carbonyl group produced by the condensation; the DH domain, which is thought to dehydrate the hydroxyl group produced by the KR domain to form a double bond; and the carbon chain The fatty acid carbon chain is thought to be elongated by the coordinated action of multiple domains, including the chain elongation factor domain (hereinafter referred to as the CLF domain), which is involved in the elongation of the chain; the enoyl reductase domain (hereinafter referred to as the ER domain), which is thought to reduce the resulting double bond; the acyltransferase domain (hereinafter referred to as the AT domain) and the malonyl-CoA:acyltransferase domain (hereinafter referred to as the MAT domain), which are thought to be involved in the transfer of acyl groups; and the phosphopantetheine transferase domain (hereinafter referred to as the PPT domain), which is thought to activate the ACP domain.

[0007] It is known that PUFA-PKS produce different types of PUFAs depending on the species. For example, DHA is the main product of PUFA-PKS derived from Schizochytrium sp., Aurantiochytrium sp., and Moritella marina; EPA is the main product of PUFA-PKS derived from Shewanella oneidensis and Photobacterium profundum; and ARA is the main product of PUFA-PKS derived from Aureispira marina. Other PUFAs are produced in small amounts or in small quantities compared to the main product.

[0008] Thus, PUFA-PKS exhibits high product specificity, and numerous studies have been conducted to date to analyze its function. Non-patent documents 4 and 6 describe studies on cloning PUFA-PKS genes from Shewanella bacteria and stramenopil eukaryotes, expressing them in heterologous organisms, and producing PUFAs.

[0009] Non-patent document 7 discloses that studies using the pfaB gene, a constituent gene of PUFA-PKS derived from Moritella marina that produces DHA, and the pfaB gene, a constituent gene of PUFA-PKS derived from Shewanella pneumatophori that produces EPA, reveal that the pfaB gene encoding the AT domain is involved in the type of PUFA produced.

[0010] Non-patent document 8 discloses that introducing the DH domain of PUFA-PKS derived from the genus Thraustochytrium into Escherichia coli increases the production of fatty acids and the proportion of unsaturated fatty acids.

[0011] While methods for the industrial production of EPA include refining it from fish oil, these methods have the drawback of producing a large amount of by-products (Patent Document 2). [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] International Publication No. 2008 / 144473 [Patent Document 2] Japanese Patent Publication No. 2013-055893 [Non-patent literature]

[0013] [Non-Patent Document 1] Annu. Nutr. Metabol.,1991,35,128-131 [Non-Patent Document 2] J.Am.Clin.Nutr.,1994,13,658-664 [Non-Patent Document 3] Ann.Rev.Biochem.,1983,52,537-579 [Non-Patent Document 4] Science, 2001, 293, 290-293 [Non-Patent Document 5] PLoS One, 2011, 6, e20146 [Non-Patent Document 6] Plant Physiol.Biochem.,2009,47,472-478 [Non-Patent Document 7] FEMS Microbiol.Lett.,2009,295,170-176 [Non-Patent Document 8] Appl.Microbiol.Biotechnol.,2018,847-856 [Overview of the project] [Problems that the invention aims to solve]

[0014] As mentioned above, the industrial production method for EPA currently involves refining from fish oil, but this method produces many by-products and has poor production efficiency. Therefore, there is a need for more efficient EPA production methods.

[0015] Therefore, the present invention aims to provide microorganisms that efficiently produce EPA and a method for producing EPA using these microorganisms. [Means for solving the problem]

[0016] The inventors of the present invention have discovered that by expressing OrfB, which has a mutation introduced into a specific amino acid residue, in microorganisms capable of producing DHA, it is possible to produce PUFAs containing a high concentration of EPA, and have completed the present invention.

[0017] This invention relates to the following: 1. A microorganism having the ability to produce DHA, comprising a protein (hereinafter referred to as mutant OrfB) consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue, and capable of producing eicosapentaenoic acid (hereinafter referred to as EPA). 2. A microorganism having the ability to produce DHA, comprising a protein (hereinafter referred to as mutant OrfB homolog) consisting of an amino acid sequence in which at least one of the amino acid residues corresponding to the amino acid residues at positions 6, 65, 230, 231, and 275 in SEQ ID NO: 2 is substituted with another amino acid residue when the amino acid sequence of a homologous protein of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (hereinafter referred to as OrfB homolog) is aligned with the amino acid sequence represented by SEQ ID NO: 2, and capable of producing EPA. 3. The microorganism according to 1 or 2 above, wherein the microorganism having the ability to produce DHA is a Labyrinthulomycetes microorganism. 4. The microorganism according to 3 above, wherein the Labyrinthulomycetes microorganism is a Labyrinthulomycetes microorganism belonging to the genus Aurantiochytrium, Thraustochytrium, Ulkenia, Parietichytrium, Labyrinthula, Aplanochytrium, Oblongichytrium, or Schizochytrium. 5. The microorganism according to 1 or 2 above, wherein the microorganism having the ability to produce DHA is a microorganism into which genes encoding the following domains (a) to (j) having the activity of synthesizing DHA in a microorganism having no DHA metabolic pathway are introduced. (a) KS domain (b) MAT domain (c) ACP domain (d) KR domain (e) Polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain (f) CLF domain (g) AT domain (h) FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain (i) ER domain (j) PPT domain 6. The microorganism according to 5 above, wherein the microorganism having no DHA metabolic pathway belongs to a microorganism belonging to the genus Escherichia, Bacillus, Corynebacterium, Yarrowia, Saccharomyces, Candida, or Pichia. 7. A method for producing EPA or an EPA-containing composition, comprising culturing the microorganism according to any one of 1 to 6 above in a medium, generating and accumulating EPA or an EPA-containing composition in the culture, and collecting EPA or an EPA-containing composition from the culture. 8. A method for producing EPA or an EPA-containing composition, using a microorganism capable of producing EPA of the following (I) or (II). (I) A microorganism having the ability to produce DHA, comprising a mutant OrfB consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue, and capable of producing EPA (II) A microorganism having the ability to produce DHA, comprising a mutant OrfB homolog consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 of SEQ ID NO: 2 is substituted with another amino acid residue when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by SEQ ID NO: 2, and capable of producing EPA

Advantages of the Invention

[0018] The microorganism of the present invention can efficiently produce EPA by introducing mutations into specific amino acid residues in microorganisms capable of producing DHA, thereby altering the specificity of the substrate and expressing a mutant OrfB. The EPA production method of the present invention allows for low-cost and highly efficient production of EPA by expressing a mutant OrfB in microorganisms capable of producing DHA at an industrial level, and can be applied to the industrial production of EPA. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows a schematic diagram of the structure of a PUFA-PKS of the genus Aurantiochytrium sp. [Figure 2] Figure 2 shows an example of the results of amino acid sequence alignment between OrfB and its homolog. [Modes for carrying out the invention]

[0020] In this invention, "polyunsaturated fatty acid (PUFA)" refers to a long-chain fatty acid with a carbon chain length of 18 or more carbon atoms and two or more unsaturated bonds. Furthermore, in this specification, "domain" refers to a portion of a protein consisting of a continuous amino acid sequence, which has a specific biological activity or function within the protein.

[0021] In this invention, "PUFA-PKS" is synonymous with PUFA synthase. PUFA synthase is a group of enzymes that synthesize specific long-chain unsaturated fatty acids using malonyl-CoA, etc., as a carbon source, and refers to those containing the KS, MAT, ACP, KR, PS-DH, CLF, AT, FabA-DH, ER, and PPTase domains (ACOS Lipid Library: PUFA synthase; Science, 2001, 293, 290-293; PLoS One, 2011, 6, e20146, etc.).

[0022] The KS domain is a domain found in proteins that make up the PUFA-PKS protein complex, and it is the domain involved in the condensation of malonyl ACP and acyl ACP.

[0023] MAT domains and AT domains are domains found in proteins that make up the PUFA-PKS protein complex, and are domains involved in the transfer of acyl groups.

[0024] The ACP domain is a domain found in proteins that constitute the PUFA-PKS protein complex. It is an essential domain for PUFA-PKS activity, as it binds to an acyl group via a thioester bond through a phosphopantetheinyl group and functions as a site for fatty acid synthesis.

[0025] A KR domain is a domain found in proteins that make up a protein complex possessing PUFA-PKS activity, and it is a domain involved in the reduction of ketone groups generated by condensation.

[0026] The DH domains, specifically the PS-DH domain and the FabA-DH domain, are domains found in proteins that constitute the PUFA-PKS protein complex. These domains are involved in the dehydration of hydroxyl groups generated by the reduction of ketone groups.

[0027] A CLF domain is a domain found in proteins that make up a protein complex possessing PUFA-PKS activity, and it is a domain involved in carbon chain elongation.

[0028] The ER domain, or malonyl-CoA:ACP acyltransferase domain, is a domain found in proteins that make up the PUFA-PKS protein complex, and is involved in the transfer of acyl groups.

[0029] PPTase refers to an enzyme that constitutes a protein complex with PUFA-PKS activity and is involved in the activation of the ACP domain.

[0030] In this specification, the identity of amino acid sequences and nucleotide sequences can be determined using the BLAST algorithm by Karlin and Altschul (Pro.Natl.Acad.Sci.USA, 1993, 90, 5873) or FASTA (Methods Enzymol., 1990, 183, 63). Based on this BLAST algorithm, programs called BLASTN and BLASTX have been developed (J.Mol.Biol., 1990, 215, 403). When analyzing nucleotide sequences using BLASTN based on BLAST, the parameters should be, for example, Score=100 and wordlength=12. When analyzing amino acid sequences using BLASTX based on BLAST, the parameters should be, for example, score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, the default parameters of each program should be used. The specific methods for these analysis methods are publicly known (see www.ncbi.nlm.nih.gov).

[0031] In this specification, "external" refers to something of a different species, not endogenous, and is used to mean introducing a gene according to the present invention into a host organism when the host organism before transformation does not have the gene to be introduced by the present invention, when the protein encoded by that gene is not substantially expressed, or when a different gene encodes the amino acid sequence of the protein, but does not express the activity of the endogenous protein after transformation.

[0032] [Microorganisms] The microorganism of the present invention is a microorganism capable of producing docosahexanoic acid (DHA), and is characterized by containing a protein (mutant OrfB) consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by Sequence ID No. 2 is substituted with another amino acid residue, and is capable of producing eicosapentaenoic acid (EPA).

[0033] The microorganisms capable of producing DHA include the following (1) and (2). (1) Microorganisms capable of metabolizing DHA. (2) A microorganism that has the ability to produce DHA by introducing genes encoding the KS domain, MAT domain, ACP domain, KR domain, PS-DH domain, CLF domain, AT domain, FabA-DH domain, ER domain, and PPT domain, which are domains that constitute PUFA-PKS and have the activity to biosynthesize DHA, into a host organism that does not have the ability to metabolize DHA.

[0034] In this specification, "host organism" refers to the original organism that is the subject of genetic modification and transformation. When the original organism that is the subject of transformation by gene introduction is a microorganism, it is also called the parent strain or host strain.

[0035] (1) Examples of microorganisms that possess DHA metabolic ability include microorganisms belonging to the Labyrinthula class. Examples of microorganisms belonging to the Labyrinthula class include microorganisms of the genera Aurantiochytrium, Thraustochytrium, Ulkenia, Parietichytrium, Labyrinthula, Aplanochytrium, Oblongichytrium, or Schizochytrium. Preferably, Aurantiochytrium limacinum and Thraustochytrium aureum are examples, but the microorganism is not limited to these as long as it inherently possesses a DHA metabolic pathway.

[0036] As for microorganisms that have the ability to metabolize DHA, for example, microorganisms belonging to the genus Aurantiochytrium are preferred, such as Aurantiochytrium SP OH4 strain (accession number FERM BP-11524), and mutant strains thereof that have the ability to produce DHA may also be used.

[0037] The aforementioned Aurantiochytrium sp. OH4 strain has been deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE), located at 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (postal code 305-8566). The date of receipt (deposit) is January 11, 2013, and the accession number is FERM BP-11524.

[0038] (2) Microorganisms that do not possess DHA metabolic ability refer to microorganisms that inherently do not possess the ability to produce DHA. Examples of microorganisms that do not possess DHA metabolic ability include bacteria, microalgae, fungi, protists, and protozoa.

[0039] Examples of bacteria include microorganisms belonging to one genus selected from the group consisting of Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and Aureispira.This includes Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus(Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp.D-0110, and Marine Aureispira The JCM23201 is a 1-piece unit.

[0040] Examples of microalgae include the Euglenophyceae (e.g., the genera Euglena and Peranema), the Chrysophyceae (e.g., the genus Ochromonas), the Dinobryaceae (e.g., the genera Dinobryon, Platychrysis, and Chrysochromulina), and the Dinophyceae (e.g., the genera Crypthecodinium, Gymnodini). [Genus Gymnodinium, Peridinium, Ceratium, Gyrodinium, and Oxyrrhis], Cryptophyceae [e.g., genera Cryptomonas and Rhodomonas], Xanthophyceae [e.g., genera Olisthodiscus], and flagellates Rhizochloridaceae and Aphanochaete Examples include algal species that undergo an amoeboid phase, such as the zoospores / gametes of Pascheri, Bumilleria stigeoclonium, and Vaucheria geminata, as well as the Eustigmatophyceae and Prymnesiopyceae [e.g., the genera Prymnesium and Diacronema].

[0041] Preferred species within these genera include, but are not limited to, Nannochloropsis oculata, Crypthecodinium cohnii, and Euglena gracilis.

[0042] Examples of fungi include the genus Saccharomyces [for example, yeasts including Saccharomyces cerevisiae and Saccharomyces carlsbergensis], or other yeasts such as the genera Yarrowia, Candida, Pichia, and Kluyveromyces, or other fungi such as fibrous fungi such as Aspergillus, Neurospora, and Penicillium.

[0043] The cell lines that can be used as host cells may be wild-type in the usual sense, or they may be trophic requirement mutants, antibiotic-resistant mutants, or transformed to possess various marker genes. Examples include strains that are resistant to antibiotics such as chloramphenicol, ampicillin, kanamycin, and tetracycline.

[0044] (2) For microorganisms that do not have the ability to metabolize DHA to acquire the ability to produce DHA, the genes encoding each domain that constitutes the PUFA-PKS having the activity to biosynthesize DHA (KS domain, MAT domain, ACP domain, KR domain, PS-DH domain, CLF domain, AT domain, FabA-DH domain, ER domain, and PPT domain) that are present in the microorganisms that have the ability to metabolize DHA as described above.

[0045] The domains constituting a PUFA-PKS are not limited to any particular domain, as long as they work together to produce DHA; examples include the domains found in known PUFA-PKS.

[0046] In this specification, "cooperating" means that when one protein is brought into coexistence with another protein, they work together to perform a specific reaction. In particular, in this specification, it means that when multiple domains necessary for PUFA-PKS activity are brought into coexistence, they work together with other domains to exhibit PUFA-PKS activity.

[0047] In this specification, "known PUFA-PKS" preferably refers to PUFA-PKS originally possessed by microorganisms belonging to genera selected from the group consisting of Aurantiochytrium, Thraustochytrium, Ulkenia, Parietichytrium, Labyrinthula, Aplanochytrium, Oblongichytrium, or Schizochytrium, and more preferably refers to PUFA-PKS originally possessed by microorganisms selected from the group consisting of Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium sp.ATCC 20888, and Thraustochytrium aureum ATCC 34304.

[0048] The fact that PUFA-PKS, consisting of each domain, possess DHA synthesis activity can be confirmed by creating microorganisms transformed with genes encoding each domain, culturing these microorganisms in a culture medium, allowing DHA to be produced and accumulated in the culture, and measuring the accumulated DHA in the culture by gas chromatography.

[0049] PUFA-PKS is a protein complex (complex enzyme) composed of multiple proteins having the domains described above, and OrfB is a protein that constitutes PUFA-PKS. Figure 1 shows a schematic diagram of the domain structure constituting the PUFA-PKS protein complex in a microorganism belonging to the genus Aurantiochytrium sp. OrfB contains one KS domain, a CLF domain, an AT domain, and an ER domain.

[0050] Examples of mutant OrfB include the proteins described in (a) or (b) below. (a) A protein having an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by Sequence ID No. 2 is replaced by another amino acid residue. (b) A protein comprising an amino acid sequence in which, when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by Sequence ID No. 2, at least one of the amino acid residues corresponding to the 6th, 65th, 230th, 231st and 275th amino acid residues of the amino acid sequence represented by Sequence ID No. 2 is replaced by another amino acid residue.

[0051] With respect to the protein (a) described above, it is preferable that in the amino acid sequence represented by Sequence ID No. 2, at least the 230th amino acid residue is substituted with another amino acid residue, and it is more preferable that, in addition to the 230th amino acid residue, at least one selected from the 6th, 65th, 231st, and 275th amino acid residues is substituted with another amino acid residue, and it is particularly preferable that the 6th and 230th amino acid residues, the 65th and 230th amino acid residues, the 6th, 65th, and 230th amino acid residues, or the 65th, 230th, 231st, and 275th amino acid residues are substituted with another amino acid residue.

[0052] Furthermore, with respect to the protein in (b) above, when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by Sequence ID No. 2, it is preferable that at least the amino acid residue corresponding to the 230th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 is substituted with another amino acid residue, and it is more preferable that, in addition to the amino acid residue corresponding to the 230th amino acid residue, at least one selected from the amino acid residues corresponding to the 6th, 65th, 231st, and 275th amino acid residues is substituted with another amino acid residue, and it is particularly preferable that the amino acid residues corresponding to the 6th and 230th amino acid residues, the 65th and 230th amino acid residues, the 6th, 65th, and 230th amino acid residues, or the 65th, 230th, 231st, and 275th amino acid residues are substituted with another amino acid residue.

[0053] An OrfB homolog is a protein found in naturally occurring organisms that has an amino acid sequence with high homology to the amino acid sequence represented by Sequence ID No. 2, and whose structure and function are similar to OrfB having the amino acid sequence represented by Sequence ID No. 2, so that the gene encoding the protein is thought to have the same evolutionary origin as the gene encoding the original protein.

[0054] Specific examples of OrfB homologs include PhoC from Photobacterium profundum (represented by SEQ ID NO: 27), EpaC from Shewanella oneidensis (represented by SEQ ID NO: 28), DhaC from Moritella marina (represented by SEQ ID NO: 29), AraC from Aureispira marina (represented by SEQ ID NO: 30), and OrfB from Schizochytrium sp. (ATCC20888) (represented by SEQ ID NO: 31). An example of the amino acid sequence alignment results between OrfB and its homologs is shown in Figure 2.

[0055] Amino acid sequence alignments can be created using the known alignment program ClustalW [Nucelic Acids Research 22, 4673, (1994)]. ClustalW is available from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating alignments using ClustalW, default values ​​for parameters can be used, for example.

[0056] More preferably, mutant OrfB is a protein in which at least one of the following amino acid residues is substituted in the amino acid sequence of the protein described in (a) or (b) above. (i) The sixth amino acid residue in the amino acid sequence of Sequence ID No. 2, or the amino acid residue corresponding to that amino acid residue in the amino acid sequence of the OrfB homolog, is substituted with serine. (ii) The 65th amino acid residue in the amino acid sequence of SEQ ID NO: 2, or the amino acid residue corresponding to that amino acid residue in the amino acid sequence of the OrfB homolog, is substituted with leucine. (iii) The amino acid residue at position 230 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to said amino acid residue in the amino acid sequence of the OrfB homolog is substituted with leucine, L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine. (iv) The amino acid residue at position 231 of the amino acid sequence of SEQ ID NO: 2, or the amino acid residue corresponding to that amino acid residue in the amino acid sequence of the OrfB homolog, is substituted with threonine. (v) The amino acid residue at position 275 of the amino acid sequence of Sequence ID No. 2, or the amino acid residue corresponding to that amino acid residue in the amino acid sequence of the OrfB homolog, is substituted with glycine.

[0057] The amino acid residues after the above substitution may be mutually interchangeable amino acids. Examples of mutually interchangeable amino acids are shown below. Amino acids belonging to the same group are mutually interchangeable. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-hydroxyproline, 4-hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, tyrosine

[0058] The amino acids to be substituted as described above can be either natural or non-natural forms. Examples of natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0059] [Methods for creating microorganisms] Methods for expressing mutant OrfB or mutant OrfB homolog in microorganisms capable of producing DHA include, for example, (I) and (II) below. (I) A foreign gene encoding a mutant OrfB or a mutant OrfB homolog is introduced into a microorganism capable of producing DHA. (II) Introduce mutations into the gene encoding endogenous OrfB or an OrfB homolog in microorganisms capable of producing DHA.

[0060] Regarding (I) above, the introduction of a foreign gene encoding a mutant OrfB or a mutant OrfB homolog includes cases where the foreign gene exists in the cells of the host organism as an autonomously replicating plasmid, where the gene to be replaced in the cell is replaced with the corresponding foreign gene, and where the foreign gene encoding a mutant OrfB or a mutant OrfB homolog is incorporated into a region of the chromosomal DNA in the cell that is different from the gene encoding OrfB. When introducing a foreign gene, it is preferable to optimize the sequence by referring to the codon usage frequency of the host microorganism.

[0061] Regarding (II) above, for example, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Cloning, Third Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids By introducing site-directed mutations using site-directed mutagenesis methods described in Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc., mutations can be introduced into genes encoding endogenous OrfB or OrfB homologs.

[0062] In this specification, "gene" refers to DNA that may include a transcriptional regulatory region, a promoter region, and a terminator region in addition to the protein coding region. When using a prokaryotic organism such as bacteria as the parent strain of the host organism, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome-binding region, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases). In this DNA, a transcription termination factor is not necessarily required for the expression of the DNA, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0063] Genes introduced into host organisms can be introduced into host cells, for example, by inserting a recombinant gene downstream of the promoter of a suitable expression vector. Expression vectors can also include promoters, transcription termination signals, and selection marker genes for selecting transformants (e.g., drug resistance genes such as kanamycin resistance genes, streptomycin resistance genes, carboxyne resistance genes, zeosin resistance genes, hygromycin resistance genes, etc., genes that complement amino acid requirement mutations such as leucine, histidine, methionine, arginine, tryptophan, and lysine, etc., and genes that complement nucleoside base requirement mutations such as uracil and adenine). In the case of uracil-requiring strains, examples of marker genes include the orotidine-5'-phosphate decarboxylase gene (ura3 gene) or the orotidylate pyrophosphorylase gene (ura5 gene).

[0064] A promoter, whether structural or regulatory, is defined as a DNA base sequence that causes RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates mRNA synthesis at a high frequency and is preferably used. Promoters for the lac system, trp system, TAC or TRC system, major operators and promoter regions of λ phages, regulatory regions of fd coat proteins, glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamate decarboxylase A, serine hydroxymethyltransferase, etc., can be used depending on the properties of the host cell.

[0065] In addition to promoter and terminator sequences, other regulatory elements include, for example, selection markers, amplification signals, and replication origins. Preferred regulatory sequences include, for example, those described in "Gene Expression Technology: Methods in Enzymology 185," Academic Press (1990).

[0066] The vector is not particularly limited as long as it can express the target gene. The types of reagents used to construct the vector, such as restriction enzymes or ligation enzymes, are also not particularly limited, and commercially available products can be used as appropriate.

[0067] When using Labyrinthula microorganisms as host organisms, the promoters are not particularly limited as long as they function within the cells of Labyrinthula microorganisms. Examples include actin promoters, tubulin promoters, elongation factor Tu promoters, and glycolysis gene expression promoters.

[0068] When using a microorganism belonging to the genus Escherichia as the parent strain, the expression vectors include, for example, pColdI (Takara Bio Inc.), pET21a, pCOLADuet-1, pACYCDuet-1, pCDF-1b, pRSF-1b (all from Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Biosciences), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX-1 (Pro (Mega Corporation), pQE-30 (Qiagen Corporation), pET-3 (Novagen Corporation), pTrc99A (GE Healthcare Biosciences Corporation), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptII SK(+), pBluescriptII KS(-) (Stratagene), pTrS30 [Adjusted from Escherichia Koli JM109 / pTrS30 (Ferm BP-5407)], pTrS32 [Adjusted from Escherichia Koli JM109 / pTrS32 (Ferm BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL Examples include [MICROBIOLOGY, 2007, Vol.73, No.20, pp. 6378-6385], pPAC31 (International Publication No. 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), pPA1 (Japanese Patent Publication No. 63-233798), pHSG298 (manufactured by Takara Bio Inc.), and pUC18 (manufactured by Takara Bio Inc.).

[0069] The promoter used when employing the above expression vector is not particularly limited as long as it functions in the cells of microorganisms belonging to the genus Escherichia. Examples include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, and T7 promoter. Additionally, artificially designed and modified promoters such as a promoter with two Ptrps in series, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter are also acceptable.

[0070] When using Corynebacterium-type bacteria as the parent strain, examples of expression vectors include pCG1 (Japanese Patent Publication No. 57-134500), pCG2 (Japanese Patent Publication No. 58-35197), pCG4 (Japanese Patent Publication No. 57-183799), pCG11 (Japanese Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (both Japanese Patent Publication No. 58-105999), pCE51, pCE52, pCE53 [all from Molecular and General Genetics, 196, 175 (1984)].

[0071] The promoter used when employing the aforementioned expression vector is not particularly limited as long as it is a promoter that functions in the cells of Corynebacteria, for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].

[0072] When using a yeast strain as the parent strain, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp51 (ATCC37419), pHS19, and pHS15.

[0073] The promoter used when the aforementioned expression vector is used is not particularly limited as long as it is a promoter that functions in the cells of the yeast strain, and examples of promoters include the PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0074] Homologous recombination can be used to integrate recombinant genes into the chromosomes of a host organism. One example of homologous recombination is the use of a homologous recombination system that can be constructed by linking plasmid DNA containing a drug resistance gene that cannot autonomously replicate within the parent strain to be introduced, thereby introducing the recombinant gene. A frequently used homologous recombination method in Escherichia coli is the use of a lambda phage homologous recombination system to introduce the recombinant gene [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0075] Furthermore, by using selection methods that utilize the fact that E. coli becomes susceptible to sucrose due to Bacillus subtilis levansuclarase incorporated into the chromosome along with the recombinant gene, or by using selection methods that utilize the fact that E. coli becomes susceptible to streptomycin by incorporating the wild-type rpsL gene into E. coli having a streptomycin-resistant mutant rpsL gene [Mol.Microbiol.,55,137(2005), Biosci.Biotechnol.Biochem.,71,2905(2007)], it is possible to obtain microorganisms in which the target region on the chromosomal DNA of the parent strain has been replaced with recombinant DNA.

[0076] Furthermore, homologous recombination methods include, for example, the ATMT method via Agrobacterium [Appl. Environ. Microbiol., (2009), vol. 75, p. 5529-5535]. Moreover, if transformants that stably retain the desired trait can be obtained, this method includes, but is not limited to, improved ATMT methods.

[0077] Methods for introducing a gene to be introduced as a plasmid capable of autonomous replication in a host organism include, for example, the calcium ion method [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Publication No. 63-248394), and the electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0078] The fact that the microorganism obtained by the method described above is the target microorganism can be confirmed by culturing the microorganism and detecting the EPA accumulated in the culture by gas chromatography.

[0079] The microorganisms of the present invention preferably have an EPA / DHA ratio of 0.1 or higher, more preferably 0.2 or higher, and even more preferably 0.5 or higher, in the final product (PUFA) produced when cultured at 20°C for 48 hours, as measured by gas chromatography-mass spectrometry as described later in the examples.

[0080] [Method for producing EPA or EPA-containing compositions] The present invention includes a method for producing EPA or an EPA-containing composition (hereinafter referred to as the "production method of the present invention"), characterized by culturing the above-mentioned microorganisms in a culture medium, generating and accumulating EPA or an EPA-containing composition in the culture, and collecting EPA or an EPA-containing composition from the culture.

[0081] The EPA-containing composition may include, for example, EPA-containing oils or EPA-containing phospholipids, preferably EPA-containing oils. The culture of the microorganism is obtained by inoculating the microorganism into a suitable culture medium and culturing it according to a conventional method.

[0082] Any known culture medium containing a carbon source, nitrogen source, and inorganic salt can be used as the culture medium. For example, examples of carbon sources include carbohydrates such as glucose, fructose, and galactose, as well as oils and fats such as oleic acid and soybean oil, glycerol, and sodium acetate. These carbon sources can be used at a concentration of, for example, 20 to 300 g per liter of culture medium. In a particularly preferred embodiment, cultivation can be continued by feeding the carbon source after the initial carbon source has been consumed. By culturing under such conditions, the amount of carbon source consumed can be increased, thereby improving the production volume of the EPA-containing composition.

[0083] Examples of nitrogen sources include organic nitrogen such as yeast extract, corn steep liquor, polypeptone, monosodium glutamate, and urea, or inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, and ammonia. As inorganic salts, potassium phosphate and the like can be used in appropriate combinations.

[0084] The culture medium containing each of the above components is preferably used after adjusting the pH to within the range of 4.0 to 9.5 by adding an appropriate acid or base, and then sterilizing it by autoclaving. The culture temperature is generally 10 to 45°C, preferably 20 to 37°C. It is preferable to control the culture temperature to a temperature at which the EPA-containing composition can be produced. The pH during culture is generally 3.5 to 9.5, preferably 4.5 to 9.5. The particularly preferred pH varies depending on the purpose; for producing a large amount of oil and fat, the pH is 5.0 to 8.0.

[0085] The culture time can be, for example, 2 to 7 days, and the culture can be carried out by aeration and stirring. The method for separating the culture medium and microorganisms from the culture can be carried out by conventional methods known to those skilled in the art, such as centrifugation or filtration. After the microorganisms separated from the above culture are crushed by, for example, ultrasound or Dynomill, an EPA-containing composition can be obtained by solvent extraction with, for example, chloroform, hexane, or butanol.

[0086] The EPA-containing composition produced by the above manufacturing method can be concentrated by methods such as low-temperature solvent fractionation [Takahashi, Koretaro, Yukagaku, 40:931-941 (1991)] or by a method of freeing and removing short-chain fatty acids with hydrolytic enzymes such as lipase [Takahashi, Koretaro, Yukagaku, 40:931-941 (1991)] to obtain an EPA-containing composition with a high EPA content.

[0087] EPA can be produced by separating and collecting EPA from an EPA-containing composition. For example, after preparing a mixed fatty acid containing EPA from an EPA-containing composition by hydrolysis, EPA can be produced by separating and collecting EPA using methods such as urea addition, cold separation, high-performance liquid chromatography, or supercritical chromatography.

[0088] Furthermore, EPA alkyl esters can be produced by separating and collecting them from an EPA-containing composition. The EPA alkyl ester is not particularly limited as long as it is an EPA alkyl ester, but EPA ethyl ester is preferred.

[0089] To separate and collect EPA alkyl esters from an EPA-containing composition, for example, a mixed fatty acid alkyl ester containing EPA alkyl esters can be prepared from the EPA-containing composition by alcoholization, and then the EPA alkyl esters can be separated and collected by methods such as urea addition, cold separation, high-performance liquid chromatography, or supercritical chromatography. [Examples]

[0090] Examples are shown below, but the present invention is not limited to these examples.

[0091] [Example 1] Production of EPA using E. coli producing mutant OrfB - 1 (1) Creation of each expression plasmid [Creation of OrfA protein expression plasmids] Using a method similar to that of Hayashi et al. (Sci.Rep.,2016,6,35441), we obtained the expression plasmid pET21-orfA, which contains DNA encoding the OrfA protein from the Schizochytrium sp. (ATCC20888) strain (DNA consisting of the nucleotide sequence represented by SEQ ID NO: 4).

[0092] [Creation of OrfC protein expression plasmids] Using genomic DNA from Auranctiochytrium sp.OH4 strain extracted by conventional methods as a template, PCR was performed using primers represented by SEQ ID NOs: 7 and 8 to obtain DNA fragments containing the DNA encoding the OrfC protein (DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3). The obtained DNA and the E. coli vector pCOLADuet-1 (Merck Millipore) were treated with restriction enzymes NdeI and MfeI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain the OrfC protein expression plasmid pCOLA-OH4_orfC derived from Auranctiochytrium sp.OH4 strain.

[0093] [Creation of HetI protein expression plasmids] Using a method similar to that of Hayashi et al. (Sci.Rep.,2016,6,35441), we obtained the expression plasmid pSTV-hetI, which contains DNA encoding the HetI protein (DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5) derived from the Nostoc sp. PCC7120 (ATCC27893) strain.

[0094] (2) Construction of a DNA library encoding mutant OrfB [Creation of wild-type OrfB expression plasmids] The OrfB expression plasmid pCDF-orfB1 (Sci.Rep.,2016,6,35441) derived from the Schizochytrium sp. (ATCC20888) strain was treated with AgeI to obtain an AgeI-treated fragment. The ends of this AgeI-treated fragment were blunted using the Blunting High kit (Toyobo Co., Ltd.), and then self-ligated. From this, pCDF-orfB1' was obtained, in which the AgeI recognition sequence downstream of the T7 terminator of pCDF-orfB1 was deleted.

[0095] Next, using the genomic DNA of Aurantiochytrium sp.OH4 strain extracted by conventional methods as a template, overlap extension PCR was performed using primers represented by SEQ ID NOs: 9, 10, 11, and 12 to amplify a DNA fragment containing the DNA encoding OrfB (DNA consisting of the base sequence represented by SEQ ID NO: 1). In the amplified DNA fragment, the 4713th base in the coding region was replaced from adenine to thymidine, and the NdeI recognition sequence (bases 4712-4717) was deleted. The obtained DNA fragment and pCDF-orfB1' were treated with restriction enzymes NdeI and EcoRI, respectively, and pCDF-OH4_orfB was obtained by ligation of the resulting restriction enzyme-treated fragments.

[0096] Next, using pCDF-OH4_orfB as a template, overlap extension PCR was performed using primers represented by SEQ ID NOs. 9, 12, 13, and 14 to amplify a DNA fragment containing the DNA encoding OrfB. In this DNA fragment, the 2625th base in the coding region was replaced from guanine to adenine, and an SphI recognition sequence was introduced at bases 2623-2628. The obtained DNA fragment and pCDF-orfB1' were treated with restriction enzymes NdeI and EcoRI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain plasmids pCDF-OH4_orfBs expressing wild-type OrfB from Auranctiochytrium sp.OH4 strain.

[0097] [Construction of a DNA library encoding mutant OrfB] Next, using pCDF-OH4_orfBs as a template and primers represented by SEQ ID NOs. 15 and 16, error-prone PCR was performed using TaKaRa Taq Hot Start Version (Takara Bio Inc.). In error-prone PCR, the concentration of MgCl in the PCR reaction solution was set to 5 mM to induce mutations.

[0098] DNA fragments obtained by error-prone PCR were purified and treated with restriction enzymes NdeI and AgeI, and then ligated with pCDF-OH4_orfBs that had undergone the same restriction enzyme treatment. From this, a DNA library encoding mutant OrfB was constructed.

[0099] (3) EPA productivity evaluation Using a method similar to that of Hayashi et al. (Sci.Rep., 2016, 6, 35441), we created an E. coli BLR(DE3)ΔfadE strain lacking the gene encoding acyl-CoA dehydrogenase FadE (a protein consisting of the amino acid sequence represented by SEQ ID NO: 6).

[0100] E. coli BLR(DE3)ΔfadE strain was transformed with DNA libraries encoding pET21-orfA, pCOLA-OH4_orfC, pSTV-hetI, and pCDF-OH4_orfBs or mutant OrfB.

[0101] The obtained E. coli were inoculated into 2 mL of Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, and streptomycin 20 mg / L, and incubated with shaking at 30°C for 16 hours.

[0102] One mL of the obtained culture medium was inoculated into a 200 mL vane flask containing 20 mL of newly prepared Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, streptomycin 20 mg / L, and 1 mM IPTG, and incubated at 230 rpm and 20°C for 48 hours.

[0103] After culturing, the culture medium was collected and lipids were extracted using the Bligh-Dyer method [Bligh, e. Gand Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917]. The fatty acids were then methylated with boron trifluoride-methanol solution and analyzed by gas chromatography-mass spectrometry. The amounts of DHA and EPA in the culture medium were calculated from the area of ​​the peaks corresponding to DHA methyl ester and EPA methyl ester obtained by gas chromatography-mass spectrometry, and the relative abundance of EPA and DHA was also calculated.

[0104] As a result, while E. coli producing wild-type OrfB did not produce EPA, strains of E. coli transformed with a DNA library encoding mutant OrfB that produced EPA were identified.

[0105] When the DNA encoding the mutant OrfB produced by the E. coli that produced the EPA was sequenced, it was found that the 230th L-phenylalanine in the OrfB amino acid sequence was replaced with L-leucine.

[0106] (4) Obtaining further variant OrfB Furthermore, using DNA encoding a mutant OrfB, which consists of an amino acid sequence in which L-phenylalanine at position 230 is replaced with L-leucine, as a template, error-prone PCR was performed in the same manner as described above, and the mutant was introduced into E. coli BLR(DE3)ΔfadE strain in the same manner as described above to confirm the productivity of EPA.

[0107] As a result, a strain was identified that showed even greater EPA productivity than the mutant OrfB strain obtained above, which consists of an amino acid sequence in which the 230th L-phenylalanine is replaced with L-leucine.

[0108] When the DNA encoding the mutant OrfB expressed by E. coli with improved EPA productivity was sequenced, it was found that in addition to the substitution of L-phenylalanine at position 230 of the OrfB amino acid sequence with L-leucine, L-asparagine at position 6 was substituted with L-serine, and L-phenylalanine at position 65 was substituted with L-leucine.

[0109] Table 1 summarizes the results of measuring EPA, DHA, and DPA in the culture medium described above.

[0110] [Table 1]

[0111] As shown in Table 1, it was found that EPA can be produced more efficiently by using E. coli that produce mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is replaced with L-leucine, or mutant OrfB in which the 6th amino acid residue is replaced with L-serine, the 65th amino acid residue with L-leucine, and the 230th amino acid residue with L-leucine, compared to using E. coli that produces wild-type OrfB.

[0112] [Example 2] Production of EPA using E. coli producing mutant OrfB - 2 (1) Creation of each expression plasmid Using the pCDF-OH4_orfB obtained in Example 1(2) as a template, PCR was performed using primers represented by SEQ ID NOs: 9 and 17 to amplify DNA fragments containing the DNA encoding the N-terminal region of the KS domain of OrfB.

[0113] Furthermore, using pCDF-OH4_orfB as a template, PCR was performed using the primer represented by SEQ ID NO: 16 and the primers represented by SEQ ID NOs: 18, 19, 20, 21, or 22 to amplify DNA fragments containing DNA encoding the C-terminal region of the KS domain of a mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine.

[0114] Overlap extension PCR was performed using DNA fragments encoding the N-terminal or C-terminal region of the KS domain, along with primers represented by SEQ ID NOs: 9 and 16, to obtain DNA fragments containing the full-length encoding of the KS domain of a mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine.

[0115] The DNA fragment and pCDF-OH4_orfBs were treated with restriction enzymes NdeI and AgeI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-F230W, pCDF-OH4_orfB-F230N, pCDF-OH4_orfB-F230G, pCDF-OH4_orfB-F230D, and pCDF-OH4_orfB-F230A.

[0116] Furthermore, plasmid pCDF-OH4_orfB-F230L was obtained from E. coli acquired in Example 1(3), containing DNA encoding a mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is substituted with L-leucine.

[0117] (2) EPA manufacturing E. coli BLR(DE3)ΔfadE strain was transformed with pET21-orfA, pCOLA-OH4_orfC, and pSTV-hetI, as well as expression plasmids of wild-type OrfB or six mutant OrfB strains (pCDF-OH4_orfBs, pCDF-OH4_orfB-F230L, pCDF-OH4_orfB-F230W, pCDF-OH4_orfB-F230N, pCDF-OH4_orfB-F230G, pCDF-OH4_orfB-F230D, or pCDF-OH4_orfB-F230A).

[0118] The obtained E. coli were inoculated into 2 mL of Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, and streptomycin 20 mg / L, and incubated with shaking at 30°C for 16 hours.

[0119] One mL of the obtained culture medium was inoculated into a 200 mL vane flask containing 20 mL of newly prepared Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, streptomycin 20 mg / L, and 1 mM IPTG, and incubated at 230 rpm and 20°C for 48 hours.

[0120] After culturing, the culture medium was collected, lipids were extracted using the Bligh-Dyer method, fatty acids were methylated with boron trifluoride-methanol solution, and the mixture was analyzed by gas chromatography-mass spectrometry.

[0121] Table 2 shows the results of measuring EPA, DHA, and DPA in the culture medium.

[0122] [Table 2]

[0123] As shown in Table 2, it was found that EPA can be produced more efficiently when using E. coli that produce mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is replaced with L-leucine, or when using E. coli that produce mutant OrfB in which the 230th amino acid residue of the OrfB amino acid sequence is replaced with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine, compared to when using E. coli that produce wild-type OrfB.

[0124] [Example 3] Production of EPA using E. coli producing mutant OrfB - 3 (1) Creation of each expression plasmid [Construction of pCDF-OH4_orfB-N6S-F230L] Using pCDF-OH4_orfB-F230L as a template, PCR was performed using primers represented by SEQ ID NOs. 23 and 16 to obtain DNA fragments containing DNA encoding the KS domain of OrfB. The obtained DNA fragments and pCDF-OH4_orfB-F230L were treated with restriction enzymes NdeI and AgeI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-N6S-F230L.

[0125] pCDF-OH4_orfB-N6S-F230L has DNA encoding an amino acid sequence in which the 6th amino acid residue of the OrfB amino acid sequence from the Auranctiochytrium sp.OH4 strain is substituted with L-serine and the 230th amino acid residue is substituted with L-leucine.

[0126] [Construction of pCDF-OH4_orfB-F65L-F230L] Using pCDF-OH4_orfB-F230L as a template, overlap extension PCR was performed using primers represented by SEQ ID NOs: 24, 25, 26, and 16 to obtain DNA fragments containing DNA encoding the KS domain of OrfB. The obtained DNA fragments and pCDF-OH4_orfB-F230L were treated with restriction enzymes NdeI and AgeI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-F65L-F230L.

[0127] pCDF-OH4_orfB-F65L-F230L has DNA encoding an amino acid sequence in which the 65th amino acid residue and the 230th amino acid residue of the OrfB amino acid sequence derived from the Auranctiochytrium sp.OH4 strain are substituted with L-leucine.

[0128] [Construction of pCDF-OH4_orfB-N6S-F65L-F230L] Plasmids were extracted from Escherichia coli producing OrfB obtained in Example 1(4), in which the 230th L-phenylalanine was replaced with L-leucine, the 6th L-asparagine was replaced with L-serine, and the 65th L-phenylalanine was replaced with L-leucine, to obtain pCDF-OF4_orfB-N6S-F65L-F230L.

[0129] pCDF-OF4_orfB-N6S-F65L-F230L has DNA encoding an amino acid sequence in which the 6th amino acid residue of the OrfB amino acid sequence from Auranctiochytrium sp.OH4 strain is substituted with L-serine, the 65th amino acid residue with L-leucine, and the 230th amino acid residue with L-leucine.

[0130] (2) EPA manufacturing E. coli BLR(DE3)ΔfadE strain was transformed with pET21-orfA, pCOLA-OH4_orfC, and pSTV-hetI, as well as expression plasmids of wild-type OrfB or four mutant OrfB strains (pCDF-OH4_orfBs, pCDF-OH4_orfB-F230L, pCDF-OH4_orfB-N6S-F230L, pCDF-OH4_orfB-F65L-F230L, or pCDF-OH4_orfB-N6S-F65L-F230L).

[0131] The obtained E. coli were inoculated into 2 mL of Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, and streptomycin 20 mg / L, and incubated with shaking at 30°C for 16 hours.

[0132] One mL of the obtained culture medium was inoculated into a 200 mL vane flask containing 20 mL of newly prepared Terrific Broth medium (Becton, Dickinson & Co.) containing ampicillin 100 mg / L, kanamycin 20 mg / L, chloramphenicol 30 mg / L, streptomycin 20 mg / L, and 1 mM IPTG, and incubated at 230 rpm and 20°C for 48 hours.

[0133] After culturing, the culture medium was collected, lipids were extracted using the Bligh-Dyer method, fatty acids were methylated with boron trifluoride-methanol solution, and the mixture was analyzed by gas chromatography-mass spectrometry.

[0134] Table 3 shows the results of measuring EPA, DHA, and DPA in the culture medium.

[0135] [Table 3]

[0136] As shown in Table 3, it was found that using E. coli that produces a mutant OrfB in which the 230th amino acid residue of OrfB, as well as the 6th and / or 65th amino acid residues, are substituted with L-serine and L-leucine, respectively, allows for more efficient EPA production compared to using E. coli that produces a mutant OrfB in which the 230th amino acid residue of OrfB is substituted with L-leucine.

[0137] [Example 4] Production of EPA using E. coli producing mutant OrfB - 4 Using the mutant OrfB DNA obtained in Example 3, which consists of an amino acid sequence in which L-phenylalanine at position 230 is replaced with L-leucine and L-phenylalanine at position 65 is replaced with L-leucine, as a template, error-prone PCR was performed in the same manner as in Example 1(2), and the mutant was introduced into E. coli BLR(DE3)ΔfadE strain in the same manner as in Example 1(3) to confirm the productivity of EPA.

[0138] As a result, a strain was identified that showed even greater EPA productivity than the E. coli strain producing mutant OrfB, which has an amino acid sequence in which the 230th L-phenylalanine is replaced with L-leucine and the 65th L-phenylalanine is replaced with L-leucine.

[0139] When the DNA encoding the mutant OrfB expressed by E. coli with improved EPA productivity was sequenced, it was found that in the amino acid sequence of OrfB, L-phenylalanine at position 230 was replaced with L-leucine, L-phenylalanine at position 65 was replaced with L-leucine, in addition to L-isoleucine at position 231 being replaced with L-threonine, and L-aspartic acid at position 275 being replaced with L-glycine.

[0140] Table 4 summarizes the results of measuring EPA, DHA, and DPA in the culture medium described above.

[0141] [Table 4]

[0142] As shown in Table 4, it was found that using E. coli that produces a mutant OrfB in which the 230th and 65th amino acid residues of OrfB, as well as the 231st amino acid residue, are substituted with L-threonine and the 275th amino acid residue is substituted with glycine, allows for more efficient EPA production compared to using E. coli that produces a mutant OrfB in which the 230th and 65th amino acid residues of OrfB are substituted with L-leucine.

[0143] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2018-151234, filed on 10 August 2018, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole.

Claims

1. A microorganism capable of producing docosahexaenoic acid (hereinafter referred to as DHA), comprising a protein (hereinafter referred to as mutant OrfB) consisting of an amino acid sequence in which the 230th phenylalanine in the amino acid sequence represented by Sequence ID No. 2 is substituted with leucine, asparagine, glycine, aspartic acid, or alanine, and capable of producing eicosapentaenoic acid (hereinafter referred to as EPA).

2. A microorganism capable of producing DHA, wherein the amino acid sequence of the homologous protein of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (hereinafter referred to as the OrfB homolog) contains a protein consisting of an amino acid sequence in which, when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by SEQ ID NO: 2, the amino acid residue corresponding to position 230 of SEQ ID NO: 2 is substituted with leucine, asparagine, glycine, aspartic acid, or alanine (hereinafter referred to as the mutant OrfB homolog), and the microorganism is capable of producing EPA, wherein the amino acid sequence of the OrfB homolog is the amino acid sequence represented by any one of SEQ ID NOs: 27 to 31.

3. The microorganism according to claim 1 or 2, wherein the microorganism capable of producing DHA is a Labyrinthula microorganism.

4. The microorganism according to claim 3, wherein the Labyrinthula microorganism is a Labyrinthula microorganism belonging to the genera Aurantiochytrium, Thraustochytrium, Ulkenia, Parietichytrium, Labyrinthula, Aplanochytrium, Oblongichytrium, or Schizochytrium.

5. The microorganism according to claim 1 or 2, wherein the microorganism capable of producing DHA is a microorganism into which genes encoding the following domains (a) to (j), which have activity to synthesize DHA, have been introduced into a microorganism that does not have a DHA metabolic pathway. (a) β-ketoacyl-ACP synthase (hereinafter referred to as KS) domain (b) Malonyl-CoA:ACP acyltransferase (MAT) domain (c) ACP domain (d) Ketoreductase (hereinafter referred to as KR) domain (e) Polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain (f) Chain elongation factor (CLF) domain (g) Acyltransferase (hereinafter referred to as AT) domain (h) FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain (i) Enoyl ACP-reductase (hereinafter referred to as ER) domain (j) Phosphopantetheine transferase (hereinafter referred to as PPT) domain

6. The microorganism according to claim 5, wherein the microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genera Escherichia, Bacillus, Corynebacterium, Yarrowia, Saccharomyces, Candida, or Pichia.

7. A method for producing EPA or an EPA-containing composition, comprising culturing a microorganism described in any one of claims 1 to 6 in a culture medium, generating and accumulating EPA or an EPA-containing composition in the culture, and collecting EPA or an EPA-containing composition from the culture.

8. A method for producing EPA or an EPA-containing composition using microorganisms capable of producing EPA as described in (I) or (II) below. (I) Microorganisms capable of producing DHA, which include mutant OrfB having an amino acid sequence in which the 230th phenylalanine in the amino acid sequence represented by Sequence ID No. 2 is replaced with leucine, asparagine, glycine, aspartic acid, or alanine, and which are capable of producing EPA. (II) A microorganism capable of producing DHA, wherein, in the amino acid sequence of the OrfB homolog, when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by SEQ ID NO: 2, the amino acid residue corresponding to position 230 of SEQ ID NO: 2 is substituted with leucine, asparagine, glycine, aspartic acid, or alanine, and the microorganism capable of producing EPA, wherein the amino acid sequence of the OrfB homolog is the amino acid sequence represented by any one of SEQ ID NOs: 27 to 31.

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