MICROORGANISM PRODUCING EICOSAPENTAENOIC ACID AND METHOD FOR PRODUCING EICOSAPENTAENOIC ACID

- KYOWA HAKKO BIO CO., LTD.

An object of the present invention is to provide a microorganism that efficiently produces EPA and a method for producing EPA using the microorganism. The present invention relates to a microorganism having an ability to produce docosahexaenoic acid (DHA), wherein the microorganism contains a protein composed 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 has been substituted with another amino acid residue (mutated OrfB), and is capable of producing eicosapentaenoic acid (EPA), and the like.

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Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patent application Ser. No. 17/266,984, filed Feb. 8, 2021, which is the U.S. national phase of International Patent Application No. PCT/JP2019/031652, filed Aug. 9, 2019, which claims the benefit of Japanese Patent Application No. 2018-151234, filed on Aug. 10, 2018, which are incorporated by reference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 91,166 bytes Extensible Markup Language (xml) file named “766641-ReplacementSequenceListing.xml,” created Mar. 21, 2023.

TECHNICAL FIELD

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

BACKGROUND ART

Long-chain fatty acids having a plurality of unsaturated bonds in a 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) are referred to as polyunsaturated fatty acids (hereinafter, referred to as PUFAs). PUFAs are known to have various physiological functions such as prevention of arteriosclerosis or hyperlipidemia (NPL 1 and NPL 2).

As the PUFA biosynthetic pathway, the following two types are known: an aerobic pathway and an anaerobic pathway by a polyunsaturated fatty acid polyketide synthase (hereinafter referred to as PUFA-PKS). The aerobic pathway is a pathway in which a PUFA is synthesized by introducing a double bond using a plurality of desaturases into a long-chain fatty acid such as palmitic acid synthesized using a fatty acid synthase or by elongating a carbon chain using a chain elongase, and is a synthetic pathway which is possessed by many organisms and has been known for many years (NPL 3).

On the other hand, the anaerobic pathway by a PUFA-PKS is a pathway for synthesizing a PUFA from malonyl-CoA, and some marine bacteria or eukaryotes of the Labyrinthulomycetes are known to have the pathway (NPL 4 and NPL 5).

The PUFA-PKS is a complex enzyme (hereinafter, also referred to as protein complex) composed of a plurality of proteins, and in each protein, a plurality of functional domains involved in the PUFA synthesis are present.

The functional domains present in the PUFA-PKS include a β-ketoacyl-acyl carrier protein synthase domain (hereinafter referred to as KS domain) believed to be involved in the condensation of malonyl-ACP and acyl-ACP, an acyl carrier protein domain (hereinafter, referred to as ACP domain) believed to function as a fatty acid synthesis site by binding to an acyl group via a phosphopantetheinyl group through a thioester bond, a ketoreductase domain (hereinafter referred to as KR domain) believed to reduce a carbonyl group generated by condensation, a DH domain believed to form a double bond by dehydration of a hydroxy group generated by the KR domain, a chain elongation factor domain (hereinafter, referred to as CLF domain) believed to be involved in the elongation of a carbon chain, an enoyl reductase domain (hereinafter referred to as ER domain) believed to reduce an obtained double bond, an acyltransferase domain (hereinafter, referred to as AT domain) and a malonyl-CoA:acyltransferase domain (hereinafter, referred to as MAT domain) believed to be involved in the transfer of an acyl group, and a phosphopantetheine transferase domain (hereinafter referred to as PPT domain) believed to activate an ACP domain, and it is considered that a carbon chain of a fatty acid is elongated by the plurality of domains working in cooperation with one another.

It is known that the PUFA-PKS produces a different type of PUFA depending on its type. For example, a PUFA-PKS derived from Schizochytrium sp., Aurantiochytrium sp., and Moritella marina produces DHA as a main product, a PUFA-PKS derived from Shewanella oneidensis and Photobacterium profundum produces EPA as a main product, and a PUFA-PKS derived from Aureispira marina produces ARA as a main product, and other PUFAs are hardly produced, or even if such other PUFAs are produced, they are produced in a small amount as compared with the main product.

The PUFA-PKS has high product specificity in this manner, however, many studies aiming at the functional analysis of the PUFA-PKS have been conducted so far. In NPLs 4 and 6, studies in which a PUFA-PKS gene is cloned from bacteria of the genus Shewanella or eukaryotes of the Stramenopiles and expressed in a heterogeneous organism to produce a PUFA have been conducted.

NPL 7 discloses that a pfaB gene encoding an AT domain is involved in the type of PUFA to be produced based on a study using a pfaB gene that is a structural gene of a PUFA-PKS derived from Moritella marina that produces DHA and a pfaB gene that constitutes a PUFA-PKS derived from Shewanella pneumatophori that produces EPA.

NPL 8 discloses that when a DH domain of a PUFA-PKS derived from the genus Thraustochytrium is introduced into E. coli, the production amount of fatty acids increases, and also the proportion of unsaturated fatty acids increases.

As a method for industrially producing EPA, a method of purifying EPA from a fish oil, or the like is known, but the method has a problem that there are a lot of by-products (PTL 2).

CITATION LIST Patent Literature

  • PTL 1: WO 2008/144473
  • PTL 2: JP-A-2013-055893

Non Patent Literature

  • NPL 1: Annu. Nutr. Metabol., 1991, 35, 128-131
  • NPL 2: J. Am. Coll. Nutr., 1994, 13, 658-664
  • NPL 3: Ann. Rev. Biochem., 1983, 52, 537-579
  • NPL 4: Science, 2001, 293, 290-293
  • NPL 5: PLoS One, 2011, 6, e20146
  • NPL 6: Plant Physiol. Biochem., 2009, 47, 472-478
  • NPL 7: FEMS Microbiol. Lett., 2009, 295, 170-176
  • NPL 8: Appl. Microbiol. Biotechnol., 2018, 847-856

SUMMARY OF INVENTION Technical Problem

As described above, as a method for industrially producing EPA, a method of purifying EPA from a fish oil, or the like is used, but the method has a problem that there are a lot of by-products and the production efficiency is low, and therefore, an efficient method for producing EPA has been awaited.

Accordingly, an object of the present invention is to provide a microorganism that efficiently produces EPA and a method for producing EPA using the microorganism.

Solution to Problem

The present inventors found that by expressing OrfB in which a mutation has been introduced into a specific amino acid residue in a microorganism having an ability to produce DHA, a PUFA containing EPA at a high concentration can be produced, and thus completed the present invention.

The present invention relates to the following.

1. A microorganism having an ability to produce DHA, wherein the microorganism contains a protein composed 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 has been substituted with another amino acid residue (hereinafter referred to as mutated OrfB), and the microorganism is capable of producing eicosapentaenoic acid (hereinafter referred to as EPA).

2. A microorganism having an ability to produce DHA, wherein the microorganism contains a protein composed of an amino acid sequence in which an amino acid residue corresponding to at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 in SEQ ID NO: 2 has been substituted with another amino acid residue (hereinafter referred to as mutated OrfB homolog) in an amino acid sequence of a homolog protein of a protein composed of the amino acid sequence represented by SEQ ID NO: 2 (hereinafter referred to as OrfB homolog) when the amino acid sequence of the OrfB homolog and the amino acid sequence represented by SEQ ID NO: 2 are aligned, and the microorganism is capable of producing EPA.

3. The microorganism according to the above 1 or 2, wherein the microorganism having an ability to produce DHA is a Labyrinthulomycetes microorganism.

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

5. The microorganism according to the above 1 or 2, wherein the microorganism having an ability to produce DHA is a microorganism in which genes encoding respective domains described in the following (a) to (j) having an activity of synthesizing DHA have been introduced into a microorganism that does not have a DHA metabolic pathway:

(a) a KS domain;

(b) a MAT domain;

(c) an ACP domain;

(d) a KR domain;

(e) a polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain;

(f) a CLF domain;

(g) a AT domain;

(h) a FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain;

(i) an ER domain; and

(j) a PPT domain.

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

7. A method for producing EPA or an EPA-containing composition, including culturing the microorganism according to any one of the above 1 to 6 in a culture medium so as to produce and accumulate EPA or an EPA-containing composition in a culture, and collecting EPA or the EPA-containing composition from the culture.

8. A method for producing EPA or an EPA-containing composition using the following microorganism (I) or (II) capable of producing EPA:

(I) a microorganism having an ability to produce DHA, wherein the microorganism contains mutated OrfB composed 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 has been substituted with another amino acid residue, and the microorganism is capable of producing EPA; or

(II) a microorganism having an ability to produce DHA, wherein the microorganism contains a mutated OrfB homolog composed of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231, and 275 in SEQ ID NO: 2 has been substituted with another amino acid residue in an amino acid sequence of an OrfB homolog when the amino acid sequence of the OrfB homolog and the amino acid sequence represented by SEQ ID NO: 2 are aligned, and the microorganism is capable of producing EPA.

Advantageous Effects of Invention

The microorganism of the present invention can efficiently produce EPA by expressing mutated OrfB in which a mutation has been introduced into a specific amino acid residue so as to change the specificity for a substrate in a microorganism having an ability to produce DHA. According to the method for producing EPA of the present invention, EPA can be produced at low cost with high efficiency by expressing mutated OrfB in a microorganism capable of producing DHA at an industrial level, and thus the method can be applied to the production of EPA at an industrial level.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic diagram of the structure of a PUFA-PKS of the genus Aurantiochytrium (Aurantiochytrium sp.).

FIG. 2 shows an example of the results of alignment of amino acid sequences of OrfB (SEQ ID NOs: 27-31) and an OrfB homolog (SEQ ID NO: 2).

 DESCRIPTION OF EMBODIMENTS

In the present invention, the “polyunsaturated fatty acid (PUFA)” refers to a long-chain fatty acid having a carbon chain length of 18 or more and having 2 or more unsaturated bonds. Further, the “domain” as used herein refers to a part composed of a continuous amino acid sequence in a protein, and is a region having a specific biological activity or function in the protein.

In the present invention, the “PUFA-PKS” has the same meaning as a PUFA synthase. The PUFA synthase is a group of enzymes that synthesize a specific long-chain unsaturated fatty acid using malonyl-CoA or the like as a carbon source, and refers to one containing the respective domains of KS, MAT, ACP, KR, PS-DH, CLF, AT, FabA-DH, ER, and PPTase (ACOS Lipid Library: PUFA synthase; Science, 2001, 293, 290-293; PLoS One, 2011, 6, e20146, etc.).

The KS domain is a domain included in a protein constituting a protein complex having a PUFA-PKS activity, and refers to a domain involved in the condensation of malonyl ACP and acyl ACP.

The MAT domain and the AT domain are domains included in a protein constituting a protein complex having a PUFA-PKS activity, and refer to domains involved in the transfer of an acyl group.

The ACP domain is a domain included in a protein constituting a protein complex having a PUFA-PKS activity, and refers to a domain that functions as a fatty acid synthesis site by binding to an acyl group via a phosphopantetheinyl group through a thioester bond, and is essential for a PUFA-PKS activity.

The KR domain is a domain included in a protein constituting a protein complex having a PUFA-PKS activity, and refers to a domain involved in the reduction of a ketone group generated by condensation.

The PS-DH domain and the FabA-DH domain, which are DH domains, are domains included in a protein constituting a protein complex having a PUFA-PKS activity, and refers to domains involved in the dehydration of a hydroxy group generated by the reduction of a ketone group.

The CLF domain is a domain included in a protein constituting a protein complex having a PUFA-PKS activity, and refers to a domain involved in the elongation of a carbon chain.

The ER domain is a domain included in a protein constituting a protein complex having a PUFA-PKS activity.

The PPTase is an enzyme that constitutes a protein complex having a PUFA-PKS activity, and refers to an enzyme involved in the activation of an ACP domain.

In this description, the identity of amino acid sequences or nucleotide sequences can be determined using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 1993, 90, 5873) or FASTA (Methods Enzymol., 1990, 183, 63) by Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (J. Mol. Biol., 1990, 215, 403). When analyzing a nucleotide sequence by BLASTN based on BLAST, parameters are set to, for example, as follows: score=100 and wordlength=12. Further, when analyzing an amino acid sequence by BLASTX based on BLAST, parameters are set to, for example, as follows: score=50 and wordlength=3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (see www.ncbi.nlm.nih.gov).

The “exogenous” as used herein refers to a substance that is not endogenous but is derived from a heterogeneous substance, and is used for meaning that a gene based on the present invention is introduced into a host organism when the host organism before transformation does not have a gene to be introduced according to the present invention, when a protein encoded by the gene is not substantially expressed, and when an amino acid sequence of the protein is encoded by a different gene, but the activity of an endogenous protein after transformation is not exerted.

[Microorganism]

The microorganism of the present invention is a microorganism having an ability to produce docosahexaenoic acid (DHA), and is characterized in that the microorganism contains a protein composed 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 has been substituted with another amino acid residue (mutated OrfB), and is capable of producing eicosapentaenoic acid (EPA).

As the microorganism having an ability to produce DHA, the following (1) and (2) are exemplified.

(1) a microorganism having a DHA metabolic activity

(2) a microorganism having an ability to produce DHA by introducing genes encoding a KS domain, a MAT domain, an ACP domain, a KR domain, a PS-DH domain, a CLF domain, an AT domain, a FabA-DH domain, an ER domain, and a PPT domain that are domains constituting a PUFA-PKS having an activity of biosynthesizing DHA into a host organism using a microorganism that does not have a DHA metabolic activity as the host organism

The “host organism” as used herein refers to an original organism to be subjected to genetic modification, transformation, or the like. When the original organism to be subjected to transformation by gene transfer is a microorganism, it is also referred to as a parent strain or a host strain.

As the microorganism (1) having a DHA metabolic activity, a microorganism belonging to the Labyrinthulomycetes is exemplified. Examples of the microorganism belonging to the Labyrinthulomycetes include microorganisms of the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium, or the genus Schizochytrium. Preferred examples thereof include Aurantiochytrium limacinum, Thraustochytrium aureum, and the like, however, the microorganism is not limited thereto as long as the microorganism has a DHA metabolic pathway by nature.

As the microorganism having a DHA metabolic activity, specifically, for example, a microorganism belonging to the genus Aurantiochytrium is preferred, and for example, Aurantiochytrium sp. OH4 strain (accession number FERM BP-11524) and the like are exemplified, and further, a microorganism that is a mutant thereof and has an ability to produce DHA may be used.

The Aurantiochytrium sp. OH4 strain was deposited in the National Institute of Technology and Evaluation (NITE), Patent Microorganisms Depositary Center, located at Central 6, 1-1, Higashi, Tsukuba, Ibaraki, Japan (zip code: 305-8566). The date of receipt (date of deposit) is January 11, Heisei 25 (AD 2013), and the accession number is FERM BP-11524.

The microorganism (2) that does not have a DHA metabolic activity refers to a microorganism that does not have an ability to produce DHA by nature. Examples of the microorganism that does not have a DHA metabolic activity include a bacterium, a microalga, a fungus, a protist, and a protozoan.

Examples of the bacterium include microorganisms belonging to a genus selected from the group consisting of the genus Escherichia, the genus Serratia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, the genus Microbacterium, the genus Pseudomonas, and the genus Aureispira. Among these, a microorganism selected from the group consisting of 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 (manufactured by Stratagene Corporation), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas sp. D-0110, and Aureispira marina JCM 23201 is preferred.

Examples of the microalga include the class Euglenophyceae (for example, the genus Euglena and the genus Peranema), the class Chrysophyceae (for example, the genus Ochromonas), the class Dinobryaceae (for example, the genus Dinobryon, the genus Platychrysis, and the genus Chrysochromulina), the class Dinophyceae (for example, the genus Crypthecodinium, the genus Gymnodinium, the genus Peridinium, the genus Ceratium, the genus Gyrodinium, and the genus Oxyrrhis), the class Cryptophyceae (for example, the genus Cryptomonas and the genus Rhodomonas), the class Xanthophyceae (for example, the genus Olisthodiscus) (and including a variety of algae to pass into an amoeboid phase as in the case of a zoospore or a gamete of Rhizochloridaceae and Aphanochaete pascheri, Bumilleria stigeoclonium and Vaucheria geminata), the class Eustigmatophyceae, and the class Prymnesiopyceae (including, for example, the genus Prymnesium and the genus Diacronema).

Preferred species in such genera are not particularly limited, however, Nannochloropsis oculata, Crypthecodinium cohnii, and Euglena gracilis are exemplified.

Examples of the fungus include yeast including the genus Saccharomyces (for example, Saccharomyces cerevisiae and Saccharomyces carlsbergensis), or other yeast such as the genus Yarrowia, the genus Candida, the genus Pichia, the genus Kluyveromyces, or other fungi, for example, filamentous fungi such as the genus Aspergillus, the genus Neurospora, and the genus Penicillium, and the like.

A cell line that can be utilized as a host cell may be a wild type in the usual sense, or may be an auxotrophic mutant or an antibiotic resistant mutant, or may be transformed so as to have any of various marker genes. For example, a strain exhibiting resistance to an antibiotic such as chloramphenicol, ampicillin, kanamycin, or tetracycline is exemplified.

As the genes encoding respective domains constituting a PUFA-PKS having an activity of biosynthesizing DHA for allowing the microorganism (2) that does not have a DHA metabolic activity to acquire an ability to produce DHA, genes encoding respective domains (a KS domain, a MAT domain, an ACP domain, a KR domain, a PS-DH domain, a CLF domain, an AT domain, a FabA-DH domain, an ER domain, and a PPT domain) constituting a PUFA-PKS having an activity of biosynthesizing DHA possessed by the microorganism (1) having a DHA metabolic activity described above are preferred.

The respective domains constituting a PUFA-PKS are not limited as long as the domains produce DHA in cooperation with one another, however, for example, respective domains included in a known PUFA-PKS are exemplified.

The expression “in cooperation with one another” as used herein means that when a certain protein is allowed to coexist with another protein, the proteins carry out a specific reaction together. In particular, in this description, the expression refers to that when a plurality of domains necessary for a PUFA-PKS activity are allowed to coexist, the domain exhibits the PUFA-PKS activity together with the other domains.

In this description, as the “known PUFA-PKS”, preferably a PUFA-PKS originally possessed by a microorganism belonging to a genus selected from the group consisting of the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium, and the genus Schizochytrium, and more preferably a PUFA-PKS originally possessed by a microorganism selected from the group consisting of Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium sp. ATCC 20888, and Thraustochytrium aureum ATCC 34304 are exemplified.

It can be confirmed that the PUFA-PKS composed of respective domains has a DHA synthetic activity by creating a microorganism transformed with the genes encoding the respective domains, culturing the microorganism in a culture medium so as to produce and accumulate DHA in a culture, and measuring the DHA accumulated in the culture by gas chromatography.

The PUFA-PKS is a protein complex (complex enzyme) composed of a plurality of proteins having the above-mentioned domains, and OrfB is a protein constituting the PUFA-PKS. FIG. 1 shows a schematic diagram of the domain structure constituting the protein complex of the PUFA-PKS in a microorganism belonging to the genus Aurantiochytrium (Aurantiochytrium sp.). In OrfB, one KS domain, one CLF domain, one AT domain, and one ER domain are included.

As the mutated OrfB, a protein described in the following (a) or (b) is exemplified.

(a) a protein composed 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 has been substituted with another amino acid residue

(b) a protein composed 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 the amino acid sequence represented by SEQ ID NO: 2 has been substituted with another amino acid residue in the amino acid sequence of the OrfB homolog when the amino acid sequence and the amino acid sequence represented by SEQ ID NO: 2 are aligned

With respect to the above protein (a), in the amino acid sequence represented by SEQ ID NO: 2, it is preferred that at least the amino acid residue at position 230 has been substituted with another amino acid residue, it is more preferred that at least one selected from the amino acid residues at positions 6, 65, 231, and 275 has been further substituted with another amino acid residue in addition to the amino acid residue at position 230, and it is particularly preferred that the amino acid residues at positions 6 and 230, the amino acid residues at positions 65 and 230, the amino acid residues at positions 6, 65, and 230, or the amino acid residues at positions 65, 230, 231, and 275 have been substituted with another amino acid residue.

In addition, with respect to the above protein (b), in the amino acid sequence of the OrfB homolog, when the amino acid sequence of the OrfB homolog and the amino acid sequence represented by SEQ ID NO: 2 are aligned, it is preferred that at least an amino acid residue corresponding to the amino acid residue at position 230 in the amino acid sequence represented by SEQ ID NO: 2 has been substituted with another amino acid residue, it is more preferred that at least one selected from amino acid residues corresponding to the amino acid residues at positions 6, 65, 231, and 275 has been further substituted with another amino acid residue in addition to the amino acid residue corresponding to the amino acid residue at position 230, and it is particularly preferred that amino acid residues corresponding to the amino acid residues at positions 6 and 230, the amino acid residues at positions 65 and 230, the amino acid residues at positions 6, 65, and 230, or the amino acid residues at positions 65, 230, 231, and 275 have been substituted with another amino acid residue.

The OrfB homolog refers to a protein, which is composed of an amino acid sequence having a high homology with the amino acid sequence represented by SEQ ID NO: 2, in which a gene encoding the protein is considered to have the same evolutionary origin as a gene encoding the original protein because of similarity in structure and function to OrfB having the amino acid sequence represented by SEQ ID NO: 2, and which is possessed by an organism present in nature.

Specific examples of the OrfB homolog include PhoC derived from Photobacterium profundum represented by SEQ ID NO: 27, EpaC derived from Shewanella oneidensis represented by SEQ ID NO: 28, DhaC derived from Moritella marina represented by SEQ ID NO: 29, AraC derived from Aureispira marina represented by SEQ ID NO: 30, OrfB derived from Schizochytrium sp. (ATCC 20888) represented by SEQ ID NO: 31, and the like. An example of the results of alignment of the amino acid sequences of OrfB and the OrfB homolog is shown in FIG. 2.

The amino acid sequence alignment can be created using a known alignment program ClustalW [Nucleic Acids Research 22, 4673, (1994)]. ClustalW can be utilized from www.ebi.ac.uk/clustalw/(European Bioinformatics Institute). As a parameter when creating an alignment using ClustalW, for example, default values are used.

As the mutated OrfB, more preferably, a protein in which at least one of the following substitutions of an amino acid residue in the amino acid sequence of the protein described in the above (a) or (b) is exemplified.

(i) a substitution of the amino acid residue at position 6 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding to the amino acid residue in the amino acid sequence of the OrfB homolog with serine

(ii) a substitution of the amino acid residue at position 65 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding to the amino acid residue in the amino acid sequence of the OrfB homolog with leucine

(iii) a substitution of the amino acid residue at position 230 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding to the amino acid residue in the amino acid sequence of the OrfB homolog with leucine, L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine

(iv) a substitution of the amino acid residue at position 231 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding to the amino acid residue in the amino acid sequence of the OrfB homolog with threonine

(v) a substitution of the amino acid residue at position 275 in the amino acid sequence of SEQ ID NO: 2 or an amino acid residue corresponding to the amino acid residue in the amino acid sequence of the OrfB homolog with glycine

The amino acid residue after the substitution may be a mutually substitutable amino acid. Hereinafter, examples of the mutually substitutable amino acid are shown. Amino acids included in the same group can be mutually substituted.

group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butyl glycine, t-butyl alanine, and cyclohexylalanine

group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, and 2-aminosuberic acid

group C: asparagine and glutamine

group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and 2,3-diaminopropionic acid

group E: proline, 3-hydroxyproline, and 4-hydroxyproline

group F: serine, threonine, and homoserine

group G: phenylalanine and tyrosine

The amino acid to be substituted, may be either a natural type or an unnatural type. Examples of the natural type amino acid 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, L-cysteine, and the like.

[Method for Creating Microorganism]

As a method for allowing a microorganism having an ability to produce DHA to express mutated OrfB or a mutated OrfB homolog, for example, the following (I) and (II) are exemplified.

(I) An exogenous gene encoding mutated OrfB or a mutated OrfB homolog is introduced into a microorganism having an ability to produce DHA.

(II) A mutation is introduced into a gene encoding endogenous OrfB or an OrfB homolog in a microorganism having an ability to produce DHA.

With respect to the above (I), the introduction of an exogenous gene encoding mutated OrfB or a mutated OrfB homolog includes a case where the gene is present in a cell of the host organism as an autonomously replicable plasmid, a case where a gene to be substituted in the cell is substituted with a corresponding exogenous gene, and a case where an exogenous gene encoding mutated OrfB or a mutated OrfB homolog is integrated into a region different from the gene encoding OrfB in a chromosomal DNA in the cell. Note that when an exogenous gene is introduced, it is preferred to optimize the sequence with reference to the codon usage frequency of a microorganism to be used as the host.

With respect to the above (II), a mutation can be introduced into a gene encoding endogenous OrfB or an OrfB homolog by introducing a site-directed mutation using a site-directed mutagenesis method described in, 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 Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), or the like.

The “gene” as used herein refers to a DNA that may contain a transcriptional regulatory region, a promoter region, and a terminator region, or the like in addition to a protein coding region. When a prokaryote such as a bacterium is used as a parent strain as a host organism, as the DNA, a plasmid in which a distance between the Shine-Dalgarno sequence that is a ribosome binding region and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases) is preferably used. In the DNA, a transcription termination factor is not always necessary for the expression of the DNA, but it is preferred to place the transcription termination sequence immediately downstream of the structural gene.

As the gene to be introduced into the host organism, for example, by preparing a recombinant gene in which the gene is inserted downstream of the promoter of an appropriate expression vector, the gene can be introduced into a host cell. The expression vector can also contain a promoter, a transcription termination signal, or a selection marker gene for selecting a transformant (for example, a drug resistance gene such as a kanamycin resistance gene, a streptomycin resistance gene, a carboxin resistance gene, a zeocin resistance gene, or a hygromycin resistance gene, a gene that complements an amino acid auxotrophic mutation such as a leucine, histidine, methionine, arginine, tryptophan, or lysine auxotrophic mutation, or the like, a gene that complements a nucleobase auxotrophic mutation such as an uracil or adenine auxotrophic mutation, or the like). In the case of an uracil auxotrophic strain, as the marker gene, for example, an orotidine-5′-phosphate decarboxylase gene (ura3 gene) or an orotidylate pyrophosphorylase gene (ura5 gene) is exemplified.

The promoter is defined as a base sequence of a DNA that initiates RNA synthesis by binding an RNA polymerase to the DNA regardless of whether it is a constitutive promoter or a regulatory promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency and is preferably used. A lac system, a trp system, a TAC or TRC system, major operator and promoter regions of a λ phage, a regulatory region of a fd coat protein, a promoter for a glycolytic enzyme (for example, 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase), glutamate decarboxylase A, or serine hydroxymethyltransferase, or the like can be used according to the properties of the host cell or the like.

In addition to the promoter and terminator sequences, as other regulatory elements, for example, a selection marker, an amplification signal, a replication origin, and the like are exemplified. As a preferred regulatory sequence, for example, sequences described in “Gene Expression Technology: Methods in Enzymology 185,” Academic Press (1990) are exemplified.

The vector is not particularly limited as long as a target gene can be expressed. The types of reagents for constructing the vector, for example, restriction enzymes or ligation enzymes, or the like are also not particularly limited, and commercially available products can be used as appropriate.

The promoter when a Labyrinthulomycetes microorganism is used as the host organism is not particularly limited as long as it is a promoter that functions in the cells of the Labyrinthulomycetes microorganism, and examples thereof include an actin promoter, a tubulin promoter, an elongation factor Tu promoter, and a glycolytic gene expression promoter.

When a microorganism belonging to the genus Escherichia is used as the parent strain, as the expression vector, for example, pColdI (manufactured by Takara Bio, Inc.), pET21a, pCOLADuet-1, pACYCDuet-1, pCDF-1b, pRSF-1b (all manufactured by Novagen, Inc.), PMAL-c2x (manufactured by New England Biolabs, Inc.), pGEX-4T-1 (manufactured by GE Healthcare Biosciences, Inc.), pTrcHis (manufactured by Invitrogen, Inc.), pSE280 (manufactured by Invitrogen, Inc.), pGEMEX-1 (manufactured by Promega, Inc.), PQE-30 (manufactured by Qiagen, Inc.), pET-3 (manufactured by Novagen, Inc.), pTrc99A (manufactured by GE Healthcare Biosciences, Inc.), pKYP10 (JP-A-558-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(+), pBluescript II KS(−) (manufactured by Stratagene Corporation), pTrS30 [prepared from Escherichia coli JM109/pTr30 (Ferm BP-5407)], pTrS32 [prepared from Escherichia coli JM109/pTrS32 (Ferm BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, pp. 6378-6385], pPAC31 (WO 98/12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio, Inc.), pUC118 (manufactured by Takara Bio, Inc.), pPA1 (JP-A-563-233798), pHSG298 (manufactured by Takara Bio, Inc.), and pUC18 (manufactured by Takara Bio, Inc.) are exemplified.

The promoter when using the above-mentioned expression vector is not particularly limited as long as it is a promoter that functions in cells of a microorganism belonging to the genus Escherichia, and for example, a promoter derived from Escherichia coli, a phage, or the like such as a trp promoter (Ptrp), a lac promoter (Plac), a PL promoter, a PR promoter, a PSE promoter, or a T7 promoter is exemplified. Further, for example, an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series, a tac promoter, a trc promoter, a lacT7 promoter, or a letI promoter is exemplified.

When a coryneform bacterium is used as the parent strain, examples of the expression vector include pCG1 (JP-A-557-134500), pCG2 (JP-A-558-35197), pCG4 (JP-A-S57-183799), pCG11 (JP-A-557-134500), pCG116, pCE54, pCB101 (all in JP-A 105999), pCE51, pCE52, pCE53 [all in Molecular and General Genetics, 196, 175 (1984)], and the like.

The promoter when using the above-mentioned expression vector is not particularly limited as long as it is a promoter that functions in cells of a coryneform bacterium, and for example, a P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)] is exemplified.

When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp51 (ATCC 37419), pHS19, pHS15, and the like.

The promoter when using the above-mentioned expression vector is not particularly limited as long as it is a promoter that functions in cells of a yeast strain, and for example, promoters such as a PHOS promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal 1 promoter, a gal 10 promoter, a heat shock polypeptide promoter, an MFα1 promoter, and a CUP1 promoter are exemplified.

As a method of integrating a recombinant gene into the chromosome of a host organism, a homologous recombination method can be used. As the homologous recombination method, for example, a method of introducing a recombinant gene by utilizing a homologous recombination system that can be produced by ligating the gene to a plasmid DNA having a drug resistance gene which cannot be autonomously replicated in a parent strain into which the gene is desired to be introduced is exemplified. As a method utilizing homologous recombination frequently used in Escherichia coli, a method of introducing a recombinant gene by utilizing a homologous recombination system of a lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)] is exemplified.

Further, a microorganism in which a target region on the chromosomal DNA of a parent strain has been substituted with a recombinant DNA can be obtained using a selection method utilizing the fact that E. coli becomes sensitive to sucrose by Bacillus subtilis levansucrase integrated on the chromosome together with the recombinant gene, or a selection method utilizing the fact that E. coli becomes sensitive to streptomycin by integrating a 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)], or the like.

In addition, as the homologous recombination method, for example, an ATMT method mediated by an Agrobacterium [Appl. Environ. Microbiol., (2009), vol. 75, pp. 5529-5535] is exemplified. Further, an improved ATMT method or the like is included, and the method is not limited thereto as long as a transformant that stably carries a target trait can be obtained.

As a method of introducing a gene to be introduced as a plasmid autonomously replicable in a host organism, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], a protoplast method (JP-A-S63-248394), an electroporation method [Nucleic Acids Res., 16, 6127 (1988)], and the like are exemplified.

It can be confirmed that a microorganism obtained by the above-mentioned method is a target microorganism by culturing the microorganism and detecting EPA accumulated in the resulting culture by gas chromatography.

In the microorganism of the present invention, for example, the EPA/DHA ratio in the final product (PUFA) to be produced when the microorganism is cultured at 20° C. for 48 hours is preferably 0.1 or more, more preferably 0.2 or more, further more preferably 0.5 or more as measured by gas chromatography-mass spectrometry described later in Examples.

[Method for Producing EPA or EPA-Containing Composition]

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 microorganism created above in a culture medium so as to produce and accumulate EPA or an EPA-containing composition in a culture, and collecting EPA or the EPA-containing composition from the culture.

As the EPA-containing composition, for example, an EPA-containing oil or fat or an EPA-containing phospholipid, preferably an EPA-containing oil or fat is exemplified. The culture of the microorganism can be obtained by inoculating the microorganism into an appropriate culture medium and culturing the microorganism according to a conventional method.

As the culture medium, any known culture medium containing a carbon source, a nitrogen source, and an inorganic salt, or the like can be used. For example, as the carbon source, in addition to carbohydrates such as glucose, fructose, and galactose, oils and fats such as oleic acid and soybean oil, glycerol, sodium acetate, and the like can be exemplified. These carbon sources can be used, for example, at a concentration of 20 to 300 g per liter of the culture medium. According to a particularly preferred embodiment, the culture can be continuously carried out by feeding the carbon source after the initial carbon source is consumed. By carrying out the culture under such conditions, the amount of the carbon source to be consumed is increased, so that the production amount of the EPA-containing composition can be improved.

Further, examples of the nitrogen source include organic nitrogen such as yeast extract, corn steep liquor, polypeptone, sodium glutamate, and urea, and inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, and ammonia. As the inorganic salt, potassium phosphate or the like can be used in combination as appropriate.

The culture medium containing the above-mentioned respective components is preferably used after adjusting the pH within a range of 4.0 to 9.5 by adding an appropriate acid or base, followed by sterilization in an autoclave. The culture temperature is generally from 10 to 45° C., preferably from 20 to 37° C. The culture temperature is preferably controlled to a culture temperature at which an EPA-containing composition can be produced. The pH during culture is generally from 3.5 to 9.5, preferably from 4.5 to 9.5. The particularly preferred pH varies depending on the purpose, and is from 5.0 to 8.0 in order to produce a large amount of an oil or fat.

The culture time can be set to, for example, 2 to 7 days, and the culture can be carried out by aeration and agitation culture or the like. A method of separating the culture solution and the microorganism from the culture can be carried out by a conventional method known to those skilled in the art, for example, by centrifugation, filtration, or the like. The microorganism separated from the above culture is homogenized using, for example, ultrasonic waves, a dyno mill, or the like, followed by solvent extraction with, for example, chloroform, hexane, butanol, or the like, whereby the EPA-containing composition is obtained.

The EPA-containing composition produced by the above-mentioned production method is subjected to, for example, a method such as a low temperature solvent fractionation method [Koretaro Takahashi, Journal of Japan Oil Chemist's Society, 40: 931-941 (1991)] or a method of releasing and removing short-chain fatty acids with a hydrolase such as a lipase [Koretaro Takahashi, Journal of Japan Oil Chemist's Society, 40: 931-941 (1991)] so as to concentrate the EPA-containing composition, whereby the EPA-containing composition having a high EPA content can be obtained.

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 a hydrolysis method, EPA is separated and collecting by, for example, a urea addition method, a cooling separation method, high performance liquid chromatography, supercritical chromatography, or the like, whereby EPA can be produced.

Further, an EPA alkyl ester can be produced by separating and collecting the EPA alkyl ester from an EPA-containing composition. The EPA alkyl ester is not particularly limited as long as it is an EPA alkyl ester, but preferably an EPA ethyl ester is exemplified.

In order to separate and collect an EPA alkyl ester from an EPA-containing composition, for example, after preparing a mixed fatty acid alkyl ester containing an EPA alkyl ester from the EPA-containing composition by an alcoholysis method, the EPA alkyl ester can be separated and collected by, for example, a urea addition method, a cooling separation method, high performance liquid chromatography, supercritical chromatography, or the like.

EXAMPLES

Hereinafter, Examples will be shown, however, the present invention is not limited to the following Examples.

Example 1

Production of EPA Using E. Coli that Produces Mutated OrfB [1]

(1) Creation of Respective Expression Plasmids [Creation of OrfA Protein Expression Plasmid]

An expression plasmid pET21-orfA having a DNA (a DNA composed of the base sequence represented by SEQ ID NO: 4) encoding OrfA protein derived from Schizochytrium sp. (ATCC 20888) strain was obtained by a method similar to that of Hayashi et al. (Sci. Rep., 2016, 6, 35441).

[Creation of OrfC Protein Expression Plasmid]

PCR was carried out using the genomic DNA of Aurantiochytrium sp. OH4 strain extracted by a conventional method as a template and primers represented by SEQ ID NOS: 7 and 8, whereby a DNA fragment containing a DNA (a DNA composed of the base sequence represented by SEQ ID NO: 3) encoding OrfC protein was obtained. The obtained DNA and an E. coli vector pCOLADuet-1 (manufactured by Merck Millipore Corporation) were each treated with restriction enzymes NdeI and MfeI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby an OrfC protein expression plasmid pCOLA-OH4_orfC derived from Aurantiochytrium sp. OH4 strain was obtained.

[Creation of HetI Protein Expression Plasmid]

An expression plasmid pSTV-hetI having a DNA (a DNA composed of the base sequence represented by SEQ ID NO: 5) encoding HetI protein derived from Nostoc sp. PCC7120 (ATCC 27893) strain was obtained by a method similar to that of Hayashi et al. (Sci. Rep., 2016, 6, 35441).

(2) Construction of DNA Library Encoding Mutated OrfB [Creation of Wild-Type OrfB Expression Plasmid]

An OrfB expression plasmid pCDF-orfB1 (Sci. Rep., 2016, 6, 35441) derived from Schizochytrium sp. (ATCC 20888) strain was treated with AgeI, whereby an AgeI-treated fragment was obtained, and the ends of the AgeI-treated fragment were blunted using Blunting high kit (manufactured by Toyobo Co., Ltd.), and then self-ligated. In this manner, pCDF-orfB1′ in which the AgeI recognition sequence downstream of the T7 terminator of pCDF-orfB1 was deleted was obtained.

Subsequently, overlap extension PCR was carried out using the genomic DNA of Aurantiochytrium sp. OH4 strain extracted by a conventional method as a template and primers represented by SEQ ID NOS: 9, 10, 11, and 12, whereby a DNA fragment containing a DNA (a DNA composed of the base sequence represented by SEQ ID NO: 1) encoding OrfB was amplified. In the amplified DNA fragment, the base at position 4713 in the cording region has been changed from adenine to thymidine, and the NdeI recognition sequence (the base sequence at positions 4712 to 4717) has been deleted. The obtained DNA fragment and pCDF-orfB1′ were each treated with restriction enzymes NdeI and EcoRI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby pCDF-OH4_orfB was obtained.

Subsequently, overlap extension PCR was carried out using pCDF-OH4_orfB as a template and primers represented by SEQ ID NOS: 9, 12, 13, and 14, whereby a DNA fragment containing a DNA encoding OrfB was amplified. In the DNA fragment, the base at position 2625 in the cording region has been changed from guanine to adenine, and a SphI recognition sequence has been introduced into the base sequence at positions 2623 to 2628. The obtained DNA fragment and pCDF-orfB1′ were each treated with restriction enzymes NdeI and EcoRI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby a plasmid pCDF-OH4_orfBs that expresses wild-type OrfB derived from Aurantiochytrium sp. OH4 strain was obtained.

[Construction of DNA Library Encoding Mutated OrfB]

Subsequently, error-prone PCR was carried out with TAKARA TAQ™ Hot Start Version (manufactured by Takara Bio, Inc.) using pCDF-OH4_orfBs as a template and primers represented by SEQ ID NOS: 15 and 16. In the error-prone PCR, in order to induce a mutation, the concentration of MgCl in the PCR reaction solution was set to 5 mM.

The DNA fragment obtained by the error-prone PCR was purified, and then treated with restriction enzymes NdeI and AgeI, and ligated to pCDF-OH4_orfBs having been treated with the same restriction enzymes. In this manner, a DNA library encoding mutated OrfB was constructed.

(3) Evaluation of Productivity of EPA

E. coli BLR(DE3)ΔfadE strain in which a gene encoding acyl-CoA dehydrogenase FadE (a protein composed of the amino acid sequence represented by SEQ ID NO: 6) has been deleted was created by a method similar to that of Hayashi et al. (Sci. Rep., 2016, 6, 35441).

E. coli BLR(DE3)ΔfadE strain was transformed using pET21-orfA, pCOLA-OH4_orfC, and pSTV-hetI, and pCDF-OH4_orfBs or the DNA library encoding mutated OrfB.

The obtained E. coli was inoculated into 2 mL of TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, and 20 mg/L streptomycin, and subjected to shaking culture at 30° C. for 16 hours.

1 mL of the obtained culture solution was inoculated into a 200-mL flask equipped with a blade containing 20 mL of newly prepared TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, 20 mg/L streptomycin, and 1 mM IPTG, and the E. coli was cultured at 230 rpm and 20° C. for 48 hours.

After culture, the culture solution was collected, and a lipid was extracted by a Bligh-Dyer method [Bligh, e. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917], and then, a fatty acid was methylated using a boron trifluoride-methanol solution, and analyzed by gas chromatography-mass spectrometry. The abundance of DHA or EPA in the culture solution was calculated from the area of the peak corresponding to DHA methyl ester or EPA methyl ester in the gas chromatography-mass spectrometry, and further, the abundance ratio of EPA to DHA was also calculated.

As a result, the E. coli that produces wild-type OrfB did not produce EPA, whereas among the E. coli strains transformed with the DNA library encoding mutated OrfB, a strain that produced EPA was confirmed.

When the base sequence of the DNA encoding the mutated OrfB produced by the E. coli that produced EPA was determined, L-phenylalanine at position 230 in the amino acid sequence of OrfB was substituted with L-leucine.

(4) Acquisition of Mutated OrfB with Additional Mutation

In addition, error-prone PCR was carried out in the same manner as described above using the DNA encoding mutated OrfB composed of an amino acid sequence in which L-phenylalanine at position 230 has been substituted with L-leucine as a template, and the resulting fragment was introduced into E. coli BLR(DE3)ΔfadE strain in the same manner as described above, and the productivity of EPA was confirmed.

As a result, a strain in which the productivity of EPA was further improved as compared with the E. coli that produces mutated OrfB composed of an amino acid sequence in which L-phenylalanine at position 230 has been substituted with L-leucine obtained above was confirmed.

When the base sequence of the DNA encoding the mutated OrfB expressed by the E. coli in which the productivity of EPA was further improved was determined, in the amino acid sequence of OrfB, L-asparagine at position 6 was substituted with L-serine, and L-phenylalanine at position 65 was substituted with L-leucine in addition to the substitution of L-phenylalanine at position 230 with L-leucine.

A summary of the results of measuring EPA, DHA, and DPA in the culture solution is shown in Table 1.

TABLE 1 Mutation site in OrfB protein EPA DHA DPA (ω-6) DPA (ω-3) EPA/ produced by [μg/mL/ [μg/mL/ [μg/mL/ [μg/mL/ DHA E. coli OD] OD] OD] OD] ratio Non (wild type) N.D. 1.10 ± 0.04 0.26 ± 0.01 N.D. 0 F230L 0.10 ± 0.01 0.68 ± 0.07 0.06 ± 0.00 0.06 ± 0.01 0.15 N6S/F65L/F230L 0.38 ± 0.05 1.12 ± 0.14 0.09 ± 0.01 0.12 ± 0.02 0.34

As shown in Table 1, it was found that by using the E. coli that produces mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-leucine or mutated OrfB in which the amino acid residue at position 6 was substituted with L-serine, the amino acid residue at position 65 was substituted with L-leucine, and the amino acid residue at position 230 was substituted with L-leucine, EPA can be efficiently produced as compared with the case where the E. coli that produces wild-type OrfB was used.

Example 2

Production of EPA Using E. Coli that Produces Mutated OrfB [2]

(1) Creation of Respective Expression Plasmids

PCR was carried out using pCDF-OH4_orfB obtained in Example 1 (2) as a template and primers represented by SEQ ID NOS: 9 and 17, whereby a DNA fragment containing a DNA encoding an N-terminal region of the KS domain of OrfB was amplified.

Further, PCR was carried out using pCDF-OH4_orfB as a template and a primer represented by SEQ ID NO: 16 and a primer represented by SEQ ID NO: 18, 19, 20, 21, or 22, whereby a DNA fragment containing a DNA encoding a C-terminal region of the KS domain of mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine was amplified.

Overlap extension PCR was carried out using the obtained DNA fragment encoding an N-terminal region or a C-terminal region of the KS domain and primers represented by SEQ ID NOS: 9 and 16, whereby a DNA fragment containing a DNA encoding the full length of the KS domain of mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine was obtained.

The DNA fragment and pCDF-OH4_orfBs were each treated with restriction enzymes NdeI and AgeI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby pCDF-OH4_orfB-F230W, pCDF-OH4_orfB-F230N, pCDF-OH4_orfB-F230G, pCDF-OH4_orfB-F230D, and pCDF-OH4_orfB-F230A were obtained.

Further, by using the E. coli obtained in Example 1 (3), a plasmid pCDF-OH4_orfB-F230L having a DNA encoding mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-leucine was obtained.

(2) Production of EPA

E. coli BLR(DE3)ΔfadE strain was transformed using pET21-orfA, pCOLA-OH4_orfC, and pSTV-hetI, and the expression plasmid for wild-type OrfB or any of the 6 types of mutated OrfB (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).

The obtained E. coli was inoculated into 2 mL of TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, and 20 mg/L streptomycin, and subjected to shaking culture at 30° C. for 16 hours.

1 mL of the obtained culture solution was inoculated into a 200-mL flask equipped with a blade containing 20 mL of newly prepared TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, 20 mg/L streptomycin, and 1 mM IPTG, and the E. coli was cultured at 230 rpm and 20° C. for 48 hours.

After culture, the culture solution was collected, and a lipid was extracted by a Bligh-Dyer method, and then, a fatty acid was methylated using a boron trifluoride-methanol solution, and analyzed by gas chromatography-mass spectrometry.

The results of measuring EPA, DHA, and DPA in the culture solution are shown in Table 2.

TABLE 2 Mutation site in OrfB protein EPA DHA DPA (ω-6) DPA (ω-3) EPA/ produced by [μg/mL/ [μg/mL/ [μg/mL/ [μg/mL/ DHA E. coli OD] OD] OD] OD] ratio Non (wild type) N.D.  0.93 ± 0.08 0.46 ± 0.04 N.D. 0 F230L 0.68 ± 0.10  3.6 ± 0.5 0.32 ± 0.05 0.31 ± 0.05 0.19 F230W N.D. 0.029 ± 0.002 N.D. N.D. 0 F230N 0.39 ± 0.04  3.36 ± 0.12 0.84 ± 0.02 N.D. 0.12 F230G 0.55 ± 0.13  3.04 ± 0.5 0.78 ± 0.06 N.D. 0.18 F230D 0.77 ± 0.07  4.4 ± 0.3 1.01 ± 0.07 N.D. 0.18 F230A 0.31 ± 0.04  2.8 ± 0.4 0.69 ± 0.1 N.D. 0.11

As shown in Table 2, it was found that even if using the E. coli that produces mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid, or L-alanine, EPA can be efficiently produced as compared with the case where the E. coli that produces wild-type OrfB was used in the same manner as in the case where mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-leucine was used.

Example 3

Production of EPA Using E. Coli that Produces Mutated OrfB [3]

(1) Creation of Respective Expression Plasmids

[Creation of pCDF-OH4_orfB-N6S-F230L]

PCR was carried out using pCDF-OH4_orfB-F230L as a template and primers represented by SEQ ID NOS: 23 and 16, whereby a DNA fragment containing a DNA encoding the KS domain of OrfB was obtained. The obtained DNA fragment and pCDF-OH4_orfB-F230L were each treated with restriction enzymes NdeI and AgeI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby pCDF-OH4_orfB-N6S-F230L was obtained.

The pCDF-OH4_orfB-N6S-F230L has a DNA encoding an amino acid sequence in which in the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain, the amino acid residue at position 6 was substituted with L-serine and the amino acid residue at position 230 was substituted with L-leucine.

[Creation of pCDF-OH4_orfB-F65L-F230L]

Overlap extension PCR was carried out using pCDF-OH4_orfB-F230L as a template and primers represented by SEQ ID NOS: 24, 25, 26, and 16, whereby a DNA fragment containing a DNA encoding the KS domain of OrfB was obtained. The obtained DNA fragment and pCDF-OH4_orfB-F230L were each treated with restriction enzymes NdeI and AgeI, and the resulting restriction enzyme-treated fragments were ligated to each other, whereby pCDF-OH4_orfB-F65L-F230L was obtained.

The pCDF-OH4_orfB-F65L-F230L has a DNA encoding an amino acid sequence in which in the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain, the amino acid residue at position 65 was substituted with L-leucine and the amino acid residue at position 230 was substituted with L-leucine.

[Creation of pCDF-OH4_orfB-N6S-F65L-F230L]

A plasmid was extracted from the E. coli that produces OrfB in which L-phenylalanine at position 230 was substituted with L-leucine, L-asparagine at position 6 was substituted with L-serine, and L-phenylalanine at position 65 was substituted with L-leucine obtained in Example 1 (4), whereby pCDF-OH4_orfB-N6S-F65L-F230L was obtained.

The pCDF-OH4_orfB-N6S-F65L-F230L has a DNA encoding an amino acid sequence in which in the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain, the amino acid residue at position 6 was substituted with L-serine, the amino acid residue at position 65 was substituted with L-leucine, and the amino acid residue at position 230 was substituted with L-leucine.

(2) Production of EPA

E. coli BLR(DE3)ΔfadE strain was transformed using pET21-orfA, pCOLA-OH4_orfC, and pSTV-hetI, and the expression plasmid for wild-type OrfB or any of the 4 types of mutated OrfB (pCDF-OH4_orfBs, pCDF-OH4_orfB-F230L, pCDF-OH4_orfB-N6S-F230L, pCDF-OH4_orfB-F65L-F230L, or pCDF-OH4_orfB-N6S-F65L-F230).

The obtained E. coli was inoculated into 2 mL of TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, and 20 mg/L streptomycin, and subjected to shaking culture at 30° C. for 16 hours.

1 mL of the obtained culture solution was inoculated into a 200-mL flask equipped with a blade containing 20 mL of newly prepared TERRIFIC BROTH™ medium (manufactured by Becton, Dickinson and Company) containing 100 mg/L ampicillin, 20 mg/L kanamycin, 30 mg/L chloramphenicol, 20 mg/L streptomycin, and 1 mM IPTG, and the E. coli was cultured at 230 rpm and 20° C. for 48 hours.

After culture, the culture solution was collected, and a lipid was extracted by a Bligh-Dyer method, and then, a fatty acid was methylated using a boron trifluoride-methanol solution, and analyzed by gas chromatography-mass spectrometry.

The results of measuring EPA, DHA, and DPA in the culture solution are shown in Table 3.

TABLE 3 Mutation site in OrfB protein EPA DHA DPA (ω-6) EPA/ produced by [μg/mL/ [μg/mL/ [μg/mL/ DHA E. coli OD] OD] OD] ratio Non (wild type) N.D. 3.66 ± 0.08 1.57 ± 0.04 0 F230L 1.04 ± 0.16 5.65 ± 0.89 0.53 ± 0.10 0.18 N6S/F230L 1.16 ± 0.14 5.36 ± 0.75 0.54 ± 0.15 0.22 F65L/F230L 1.59 ± 0.07 5.94 ± 0.12 0.48 ± 0.03 0.27 N6S/F65L/F230L 1.91 ± 0.18 6.36 ± 0.42 0.56 ± 0.03 0.30

As shown in Table 3, it was found that when using the E. coli that produces mutated OrfB in which in the amino acid sequence of OrfB, the amino acid residue at position 6 and/or the amino acid residue at position 65 were/was substituted with L-serine and L-leucine, respectively, in addition to the amino acid residue at position 230, EPA can be more efficiently produced as compared with the case where the E. coli that produces mutated OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB was substituted with L-leucine was used.

Example 4

Production of EPA Using E. Coli that Produces Mutated OrfB [4]

Error-prone PCR was carried out in the same manner as in Example 1 (2) using the DNA encoding mutated OrfB composed of an amino acid sequence in which L-phenylalanine at position 230 has been substituted with L-leucine and L-phenylalanine at position 65 has been substituted with L-leucine obtained in Example 3 as a template, and the resulting fragment was introduced into E. coli BLR(DE3)ΔfadE strain in the same manner as in Example 1 (3), and the productivity of EPA was confirmed.

As a result, a strain in which the productivity of EPA was further improved as compared with the E. coli that produces mutated OrfB composed of an amino acid sequence in which L-phenylalanine at position 230 has been substituted with L-leucine and L-phenylalanine at position 65 has been substituted with L-leucine was confirmed.

When the base sequence of the DNA encoding the mutated OrfB expressed by the E. coli in which the productivity of EPA was improved was determined, in the amino acid sequence of OrfB, L-isoleucine at position 231 was substituted with L-threonine and L-aspartic acid at position 275 was substituted with L-glycine in addition to the substitution of L-phenylalanine at position 230 with L-leucine and the substitution of L-phenylalanine at position 65 with L-leucine.

A summary of the results of measuring EPA, DHA, and DPA in the culture solution is shown in Table 4.

TABLE 4 Mutation site in OrfB protein EPA DHA DPA (ω-6) DPA (ω-3) EPA/ produced by [μg/mL/ [μg/mL/ [μg/mL/ [μg/mL/ DHA E. coli OD] OD] OD] OD] ratio Non (wild type) N.D. 1.67 ± 0.39 1.77 ± 0.17 N.D. 0 F65L/F230L 1.09 ± 0.07 4.07 ± 0.21 0.36 ± 0.03 0.31 ± 0.02 0.27 F65L/F230L/ 1.92 ± 0.25 1.79 ± 0.21 0.04 ± 0.01 0.21 ± 0.02 1.07 I231T/D275G

As shown in Table 4, it was found that by using the E. coli that produces mutated OrfB in which in the amino acid sequence of OrfB, the amino acid residue at position 231 was substituted with L-threonine and the amino acid residue at position 275 was substituted with glycine in addition to the amino acid residue at position 230 and the amino acid residue at position 65, EPA can be more efficiently produced as compared with the case where the E. coli that produces mutated OrfB in which in the amino acid sequence of OrfB, the amino acid residue at position 230 and the amino acid residue at position 65 were each substituted with L-leucine was used.

The present invention has been described in detail with reference to the specific aspects, but it is obvious for those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. The present application is based on a Japanese Patent Application (Patent Application No. 2018-151234) filed on Aug. 10, 2018, which is incorporated by reference in its entirety. Also, all references cited herein are incorporated in their entirety.

Claims

1. A microorganism having an ability to produce docosahexaenoic acid (DHA), wherein the microorganism contains a mutated OrfB homolog protein that is a homolog of a protein composed of an amino acid represented by SEQ ID NO: 2, except that at least one of the amino acid residues corresponding to amino acids at positions 6, 65, 230, 231, and 275 in SEQ ID NO: 2 has been substituted with another amino acid residue, and wherein the mutated OrfB homolog protein gives the microorganism an ability to produce eicosapentaenoic acid (EPA).

2. The microorganism according to claim 1, wherein the microorganism having an ability to produce DHA is a Labyrinthulomycetes microorganism.

3. The microorganism according to claim 2, wherein the Labyrinthulomycetes microorganism is a Labyrinthulomycetes microorganism belonging to the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium, or the genus Schizochytrium.

4. The microorganism according to claim 1, wherein the microorganism having an ability to produce DHA is a microorganism in which genes encoding respective domains described in the following (a) to (j) having an activity of synthesizing DHA have been introduced into a microorganism that does not have a DHA metabolic pathway:

(a) a β-ketoacyl-ACP synthase (hereinafter referred to as KS) domain;
(b) a malonyl-CoA:ACP acyltransferase (hereinafter referred to as MAT) domain;
(c) an acyl carrier protein (ACP) domain;
(d) a ketoreductase (hereinafter referred to as KR) domain;
(e) a polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain;
(f) a chain elongation factor (hereinafter, referred to as CLF) domain;
(g) an acyltransferase (hereinafter referred to as AT) domain;
(h) a FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain;
(i) an enoyl-ACP reductase (hereinafter referred to as ER) domain; and
(j) a phosphopantetheine transferase (hereinafter referred to as PPT) domain.

5. The microorganism according to claim 4, wherein the microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genus Escherichia, the genus Bacillus, the genus Corynebacterium, the genus Yarrowia, the genus Saccharomyces, the genus Candida, or the genus Pichia.

6. A method for producing EPA or an EPA-containing composition, comprising:

culturing the microorganism according to claim 1 in a culture medium so as to produce and accumulate EPA or an EPA-containing composition in a culture, and
collecting EPA or the EPA-containing composition from the culture.

7. The microorganism according to claim 1, wherein the mutated OrfB homolog protein, in the absence of the mutation wherein at least one of the amino acid residues corresponding to amino acids at positions 6, 65, 230, 231, and 275 in SEQ ID NO: 2 has been substituted with another amino acid residue, does not give the microorganism an ability to produce EPA.

Patent History
Publication number: 20230235271
Type: Application
Filed: Feb 15, 2023
Publication Date: Jul 27, 2023
Applicant: KYOWA HAKKO BIO CO., LTD. (Tokyo)
Inventors: Tohru DAIRI (Sapporo), Yasuharu SATOH (Sapporo), Shohei HAYASHI (Sapporo), Mai NAKA (Sapporo), Tetsuro UJIHARA (Tokyo)
Application Number: 18/169,487
Classifications
International Classification: C12N 1/10 (20060101); C12N 15/70 (20060101); C12P 7/64 (20060101);