WO2015005466A1

WO2015005466A1 - Method for producing lipid having high eicosapentaenoic acid content

Info

Publication number: WO2015005466A1
Application number: PCT/JP2014/068539
Authority: WO
Inventors: 順小川; 晃規安藤; 英治櫻谷; 昌清水; 茂平本
Original assignee: 国立大学法人京都大学; 日清ファルマ株式会社
Priority date: 2013-07-12
Filing date: 2014-07-11
Publication date: 2015-01-15
Also published as: JP2016165226A

Abstract

　A lipid-producing microorganism capable of efficiently producing EPA is provided. A mutant microorganism obtained by introducing a foreign ∆15 desaturase gene, or a foreign ∆12 desaturase gene and one or both of a foreign ∆15 desaturase gene and a foreign ∆17 desaturase gene, into a microorganism having an ω6 polyunsaturated fatty acid metabolic pathway, the eicosapentaenoic acid content in a fatty acid composition being at least 20% after culturing of the mutant microorganism for 10 days at 20°C or higher.

Description

Method for producing lipids with high content of eicosapentaenoic acid

The present invention relates to a method for producing a lipid containing a high content of eicosapentaenoic acid using a lipid-producing microorganism. More specifically, the present invention relates to a method for producing a lipid containing a high amount of eicosapentaenoic acid using a mutant microorganism into which a fatty acid desaturase gene has been introduced.

Polyunsaturated fatty acids are fatty acids having two or more unsaturated bonds, and are ω6 fatty acids such as linoleic acid (LA, 18: 2n-6), γ-linolenic acid (GLA, 18: 3n-6), and arachidonic acid. (ARA, 20: 4n-6), α-linolenic acid of ω3 fatty acids (ALA, 18: 3n-3), eicosapentaenoic acid (EPA, 20: 5n-3), docosahexaenoic acid (DHA, 22: 6n- 3) etc. exist. Polyunsaturated fatty acids are not only involved in the regulation of the fluidity of the membrane as the main component of the biological membrane, but are also important as precursors of the biological functional component. ARA and EPA are precursors such as prostaglandins, thromboxanes, and leukotrienes in higher animals, and DHA is a highly unsaturated fatty acid present in the brain in the largest amount. EPA has physiological actions such as platelet aggregation inhibitory action, blood neutral fat lowering action, anti-arteriosclerosis action, blood viscosity lowering action, blood pressure lowering action, anti-inflammatory action, antitumor action, etc., pharmaceuticals, foods, cosmetics It is used in various fields such as feed. In recent years, from the viewpoint of lifestyle-related disease prevention, active intake of ω3-fatty acids has been recommended, and this is a lipid molecular species whose demand has been remarkably expanding.

Living organisms DHA and EPA are biosynthesized from ALA in some organisms in addition to being taken from food. On the other hand, since humans cannot biosynthesize ALA, DHA and EPA are nutritionally essential fatty acids for humans. EPA is mainly contained in fish oil such as cod, herring, mackerel, salmon, sardine and krill, marine psychrotrophic bacteria such as Shewanella livingstonensis, and algae such as Labyrinthulomycetes. Yes. Methods for extracting or purifying EPA from these biological resources are known. The most common practice is EPA purification from fish oil. However, the EPA content in fish oil is low, and EPA derived from fish oil may have a fishy odor or a high content of erucic acid that causes heart disease, depending on the extraction or purification method. Have a problem.

In recent years, lipid producing microorganisms that accumulate lipids in cells have been attracting attention in relation to energy problems and the like, and methods for producing various lipids microbiologically have been developed. A filamentous fungus is a general term for microorganisms having a filamentous mycelium, and is a fungus that exists everywhere in the air, in the soil, and in water, including molds and mushrooms, but Mortierella (Mortierella) is one of them. It is known that filamentous fungi belonging to the genus have ω3 and ω6 highly unsaturated fatty acid metabolic pathways and produce EPA (Non-patent Document 1). Studies on methods for producing highly unsaturated fatty acids using Mortierella spp. Are ongoing. For example, Patent Document 1 discloses a method for obtaining EPA by culturing Mortierella microorganisms that produce EPA. Patent Document 2 discloses a method of producing ARA and EPA using a mutant strain obtained by subjecting Mortierella alpina to a mutation treatment. Patent Document 3 discloses a method for producing highly unsaturated fatty acids such as EPA using a transformed strain in which a gene of ω3 fatty acid unsaturated polypeptide isolated from Mortierella alpina is introduced into yeast. Yes. However, Mortierella spp. Microorganisms cannot produce EPA efficiently unless they are cultured under low-temperature conditions (20 ° C. or lower) at which the bacteria do not grow easily. Further, since Mortierella alpina-derived ω3 desaturase acts preferentially on fatty acids having a carbon chain length of 18, EPA having a carbon chain length of 20 is hardly synthesized. Therefore, it has been difficult to efficiently produce EPA by the conventional method.

JP-A-63-14697 Japanese Patent Laid-Open No. 11-243981 JP 2006-055104 A

The present invention relates to a mutant microorganism that can efficiently produce EPA at a room temperature of 20 ° C. or higher, and a method for producing a lipid containing EPA at a high concentration using the mutant microorganism.

The present inventors introduced and expressed a Δ15 desaturase gene derived from the genus Trichoderma sp. AM076 in a microorganism belonging to the genus Mortierella. As a result, the present inventors have found that the Δ15 desaturase encoded by the transgene is ω3 desaturation activity against highly unsaturated fatty acids having a carbon chain length of 18 on the lipid metabolism pathway of Mortierella microorganisms. And the mutant microorganism introduced with the gene can efficiently produce lipids containing EPA at a high concentration even at room temperature.

Furthermore, the present inventors have obtained a Δ12 desaturase derived from the basidiomycete Coprinus cinereus (hereinafter sometimes referred to as “C. cinereus”), a Δ15 desaturase derived from the Trichoderma genus or a filamentous form. Both Δ17 desaturase derived from the fungus Saproregnia diclina (hereinafter sometimes referred to as “S. diclina”) were introduced and expressed in Mortierella microorganisms. As a result, the present inventors are able to efficiently produce lipids containing a high concentration of EPA by suppressing the accumulation of oleic acid and arachidonic acid even when the mutant microorganism into which the gene is introduced is at room temperature. I found. EPA-containing lipids obtained from these mutant microorganisms are useful because they have a reduced amount of oleic acid and arachidonic acid and can efficiently purify EPA from EPA-containing lipids.

That is, the present invention is a method for producing a mutant microorganism,
introducing a foreign Δ15 desaturase gene into a parental microorganism having an ω6 polyunsaturated fatty acid metabolic pathway,
The mutant microorganism contains eicosapentaenoic acid in an amount of 20% by mass or more in the fatty acid composition after culturing at 20 ° C. or more for 10 days.
Provide a method.

The present invention also provides a method for producing a mutant microorganism,
Introducing an exogenous Δ12 desaturase gene and any one or more of an exogenous Δ15 desaturase gene and an exogenous Δ17 desaturase gene into a parental microorganism having a ω6 polyunsaturated fatty acid metabolic pathway Including
The mutant microorganism contains eicosapentaenoic acid in an amount of 20% by mass or more in the fatty acid composition after culturing at 20 ° C. or more for 10 days.
Provide a method.

The present invention also relates to a mutant microorganism in which a gene encoding an exogenous Δ15 desaturase is introduced into a microorganism having a ω6 highly unsaturated fatty acid metabolic pathway, wherein the fatty acid is cultured for 10 days at 20 ° C. or higher. Provided is a mutant microorganism having an eicosapentaenoic acid content of 20% or more in the composition.

The present invention also provides a microorganism having a ω6 polyunsaturated fatty acid metabolic pathway to any one or more of a foreign Δ12 desaturase gene, a foreign Δ15 desaturase gene, and a foreign Δ17 desaturase gene. Is provided, wherein the eicosapentaenoic acid content in the fatty acid composition after culturing for 10 days under the condition of 20 ° C. or higher is 20% or more.

Furthermore, the present invention provides a method for producing a lipid containing eicosapentaenoic acid, comprising culturing the mutant microorganism under a condition of 20 ° C. or higher.

Furthermore, the present invention provides a method for producing eicosapentaenoic acid, comprising purifying the lipid containing eicosapentaenoic acid produced by the above method.

Furthermore, the present invention provides a medicine, food or drink or feed containing eicosapentaenoic acid produced by the above method.

The mutant microorganism of the present invention is introduced with an exogenous gene for Δ15 desaturase that works at room temperature, and can produce EPA even under room temperature conditions of 20 ° C. or higher where microorganisms can easily grow. It suppresses the accumulation of arachidonic acid in cells and enables efficient EPA production. Furthermore, the mutant microorganism of the present invention in which the exogenous Δ12 desaturase and the exogenous Δ15 desaturase or the exogenous Δ17 desaturase are co-expressed suppresses not only arachidonic acid but also oleic acid accumulation, thereby improving efficiency. Enables good EPA production. Therefore, if these mutant microorganisms of the present invention are cultured under normal temperature conditions, lipids containing a high content of EPA can be produced efficiently. EPA is an important polyunsaturated fatty acid used in various fields such as pharmaceuticals, foods, cosmetics, and feeds, and the present invention that can be applied to production of EPA on an industrial scale is extremely useful.

M.M. Fatty acid biosynthesis pathway in alpina 1S-4. Structure of transformed binary vector pBIG35SSA2pΔ17m. The structure of the transformed binary vector pBIG35PP3pDES2m. Structure of the transformed binary vector pBIG35PP3pCopΔ12m. The structure of the transformed binary vector pBIG35PP3pCopΔ12mSSA2pΔ17m. Structure of the transformed binary vector pBIG35PP3pCopΔ12mSSA2pDES2m. S. M. dicilla derived Δ17 desaturase gene was introduced. Composition and production of fatty acids produced by alpina transformants. Trichoderma sp. M. introduced the AM076-derived Δ15 desaturase gene. Composition and production of fatty acids produced by alpina transformants. C. M. cinereaus-derived Δ12 desaturase gene was introduced. Composition and production of fatty acids produced by alpina transformants. M. introduced the Δ12 desaturase gene and the Δ17 desaturase gene. Composition and production of fatty acids produced by alpina transformants. M. introduced a Δ12 desaturase gene and a Δ15 desaturase gene. Composition and production of fatty acids produced by alpina transformants.

In the present specification, unless otherwise defined, the “one or more” used for amino acid sequence or nucleotide deletion, substitution, addition or insertion in an amino acid sequence or nucleotide sequence is, for example, 1 to 20, preferably May be 1 to 10, more preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, and still more preferably 1 to 2. In the present specification, “addition” of amino acids or nucleotides includes addition of one or more amino acids or nucleotides to one and both ends of the sequence.

In this specification, the identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 1993, 90, 5873) by Karlin and Altschul, or FASTA (Methods Enzymol., 1990, 183, 63). Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (J. Mol. Biol., 1990, 215, 403). When a nucleotide sequence is analyzed by BLASTN based on BLAST, parameters are set to, for example, Score = 100 and wordlength = 12. When an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. 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).

As used herein, “stringent conditions” refers to nucleotide sequences having high identity, such as 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity. This refers to a condition in which nucleotide sequences possessed hybridize and nucleotide sequences with lower identity do not hybridize. Specifically, “stringent conditions” in the present specification are the washing conditions for normal Southern hybridization, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, Conditions include 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, salt concentration and temperature corresponding to 0.1% SDS, and more preferably 2 to 3 times of washing conditions. .

In the present specification, the “ω6 highly unsaturated fatty acid metabolic pathway” refers to the production of ω6 highly unsaturated fatty acids such as γ-linolenic acid, dihomo-γ-linolenic acid and arachidonic acid (ARA) from linoleic acid. Metabolic pathway means “ω3 highly unsaturated fatty acid metabolic pathway”, which produces ω3 highly unsaturated fatty acids such as stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid (EPA) from α-linolenic acid. It refers to the metabolic pathway (see FIG. 1). In the present specification, “polyunsaturated fatty acid” refers to a long chain fatty acid having a carbon chain length of 18 or more and an unsaturated bond number of 2 or more.

In this specification, the term “inherent” used for a certain microorganism is used to indicate that a certain function or trait is possessed by a naturally occurring microorganism (wild type). The In contrast, the term “foreign” is used to denote a function or trait introduced from the outside, rather than originally present in the microorganism. For example, a gene introduced from the outside into a certain microorganism is a foreign gene. The foreign gene may be a gene derived from the same type of microorganism as the microorganism into which it has been introduced or a gene derived from a different organism.

The microorganism that becomes the parent strain of the mutant microorganism of the present invention (hereinafter sometimes referred to as “parental microorganism”) may be any microorganism that has the ω6 highly unsaturated fatty acid metabolic pathway, and preferably has the ω6 highly unsaturated fatty acid metabolic pathway. It is a lipid-producing bacterium (oleaginus microorganisms) that has ω6 highly unsaturated fatty acid.

Examples of microorganisms having a ω6 polyunsaturated fatty acid metabolic pathway that can be used as the parent microorganism of the mutant microorganism of the present invention include filamentous forms such as Mortierella, Mucor, and Umbelopsis. Preferred are Mortierella alpina, Mortierella chlamyspora, Mortierella elongata, Mortierella algera, Mortierella algora Mortierella Epigama (Mort) erella epigama), Mortierella acrotona, Mortierella minutissima, Mortierella lignaola, mortierella crotona , Mortierella humicola, Mortierella bainieri, Mortierella hyaline, Mortierella glovalpina (Mortierella gliala) Examples include, but are not limited to, Mortierella microorganisms such as obalpina), umbelopsis nana, and umbelopsis isabelina.

More preferably, the parental microorganism of the mutant microorganism of the present invention is a lipid-producing bacterium having an ω6 highly unsaturated fatty acid metabolic pathway and an ω3 highly unsaturated fatty acid metabolic pathway and producing ω6 and ω3 highly unsaturated fatty acids. More specifically, in addition to the ω6 polyunsaturated fatty acid metabolic pathway and the enzymes involved in it, it is a microorganism that has ω3 desaturase and inherently has the ability to produce EPA. Examples of such microorganisms include the above-mentioned Mortierella microorganisms, for example, Mortierella alpina (sometimes referred to as “M. alpina” in the present specification), Mortierella clonocystis, Mortierella nana, Mortierella humicola, Mortierella berlin, and globalpina. Furthermore, the parent microorganism of the mutant microorganism of the present invention includes mutants of the aforementioned Mortierella genus microorganism that have the ability to produce EPA. Examples of such microorganisms include M. pneumoniae. alpina 1S-4 (Agric. Biol. Chem., 1987, 51 (3), p. 785-790). Therefore, the parental microorganism of the mutant microorganism of the present invention includes a wild strain of Mortierella microorganism having EPA production ability and mutants thereof, preferably M. a wild-type strain of alpina and a mutant strain thereof capable of producing EPA, more preferably M. alpina. alpina 1S-4 and its EPA-producing mutants.

However, the ω3 desaturase possessed by the Mortierella spp. Is not active for EPA production at room temperature according to the present invention because the activity is low unless the temperature is low. Therefore, the parent microorganism of the mutant microorganism of the present invention further includes a strain lacking the ω3 desaturase of the genus Mortierella having the above-mentioned EPA production ability. Examples of the ω3 desaturase-deficient strain include M. alpina 1S-4 mutant alpina ST1358 (Biosci. Biotechnol. Biochem., 2010, vol. 74, p. 908-917). In the present specification, “deficient in ω3 desaturase” refers to a state in which the normal function inherent to ω3 desaturase cannot be fully exerted. For example, ω3 desaturase is not expressed at all. In a state where the expression level has decreased to such an extent that the normal function of the ω3 desaturase cannot be exhibited, in a state where the function of the ω3 desaturase gene product has been completely lost, or due to gene mutation, etc. The state in which the function of the ω3 desaturase gene product is reduced to such an extent that the normal function inherent to the ω3 desaturase cannot be exhibited. Methods for deleting ω3 desaturase include conventional methods such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (J. Gen. Microbiol., 1992, vol. 138, p. 997-1002), mutagen treatment such as 5-bromodeoxyuridine (BrdU), cisplatin, mitomycin C, or RNAi (Appl. Environ. Microbiol., 2005, vol. 71). , P. 5124-5128). Alternatively, a method of inducing mutation by irradiation, ultraviolet irradiation, high heat treatment or the like can be mentioned. However, methods for deleting ω3 desaturase are not limited to these methods as long as mutations having the above-described properties occur.

In one embodiment, the mutant microorganism of the present invention is produced by introducing a gene encoding an exogenous Δ15 desaturase into the parent microorganism. The Δ15 desaturase encoded by the introduced gene acts as a ω3 desaturase on a highly unsaturated fatty acid having a carbon chain length of 18 within the mutant microorganism of the present invention. Conversion of linoleic acid to α-linolenic acid and conversion of γ-linolenic acid to stearidonic acid in the biosynthetic pathway of unsaturated fatty acids can be promoted, and as a result, the amount of EPA biosynthesis in the microorganism can be increased.

In another embodiment, the mutant microorganism of the present invention contains a gene encoding an exogenous Δ12 desaturase, a gene encoding an exogenous Δ15 desaturase, and an exogenous Δ17 desaturase in the parental microorganism. It is produced by introducing any one or more of genes encoding. The Δ12 desaturase encoded by the introduced gene promotes the conversion of oleic acid to linoleic acid in the mutant microorganism of the present invention and suppresses the accumulation of oleic acid in the microorganism. In addition, Δ17 desaturase acts on diunsaturated γ-linolenic acid in the biosynthetic pathway of polyunsaturated fatty acids by acting like a ω3 desaturase on polyunsaturated fatty acids having a carbon chain length of 20. It promotes the conversion to eicosatetraenoic acid and the conversion of arachidonic acid to eicosapentaenoic acid, and suppresses the accumulation of arachidonic acid in microorganisms.

In FIG. alpina 1S-4 fatty acid biosynthetic pathway. By promoting the conversion to ω3 highly unsaturated fatty acids such as α-linolenic acid and stearidonic acid, the biosynthetic amount of eicosapentaenoic acid that is an ω3 highly unsaturated fatty acid can be increased. Moreover, the biosynthesis amount of eicosapentaenoic acid can be further increased by suppressing the accumulation of oleic acid.

Δ15 desaturase is a protein exhibiting Δ15 desaturase activity. The Δ15 desaturase activity refers to an activity of desaturating a fatty acid between the 15th and 16th carbon atoms counted from the carboxyl terminus of the fatty acid molecule. If the enzyme acts on a polyunsaturated fatty acid having 18 carbon atoms, the ω3 position is desaturated when viewed from the methyl end, so that the function of the ω3 desaturase can be substituted. For example, Δ15 desaturase activity can include linoleic acid to α-linolenic acid conversion activity, γ-linolenic acid to stearidonic acid conversion activity. Preferably, the Δ15 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme that preferentially exhibits Δ15 desaturase activity with respect to a highly unsaturated fatty acid having a carbon chain length of 18. .

Δ12 desaturase is a protein exhibiting Δ12 desaturase activity. The Δ12 desaturase activity refers to the activity of desaturating fatty acids between the 12th and 13th carbon atoms counted from the carboxyl terminus of the fatty acid molecule. If the enzyme acts on a polyunsaturated fatty acid having 18 carbon atoms, the ω6 position is desaturated when viewed from the methyl end, so that the function of the ω6 desaturase can be substituted. For example, Δ12 desaturase activity can include conversion activity from oleic acid to linoleic acid. Preferably, the Δ12 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme that preferentially exhibits Δ12 desaturase activity with respect to a highly unsaturated fatty acid having a carbon chain length of 18. .

Δ17 desaturase is a protein exhibiting Δ17 desaturase activity. The Δ17 desaturase activity refers to an activity of desaturating a fatty acid between the 17th and 18th carbon atoms counted from the carboxyl terminus of the fatty acid molecule. If the enzyme acts on a polyunsaturated fatty acid having 20 carbon atoms, the ω3 position is desaturated when viewed from the methyl end, so that the function of the ω3 desaturase can be substituted. For example, Δ17 desaturase activity can include arachidonic acid to eicosapentaenoic acid conversion activity, dihomo-γ-linolenic acid to eicosatetraenoic acid conversion activity. Preferably, the Δ17 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme exhibiting Δ17 desaturase activity that acts preferentially on a highly unsaturated fatty acid having a carbon chain length of 20. It is.

Preferably, the Δ15 desaturase, Δ12 desaturase and Δ17 desaturase (hereinafter sometimes referred to as “desaturase used in the present invention”) used in the present invention are at room temperature. The enzyme activities are shown below. In the present specification, “showing enzyme activity at room temperature” means that the optimum temperature of enzyme activity is 20 ° C. or higher, preferably 20 to 40 ° C., or 70 ° C. of activity at the optimum temperature at 20 ° C. % Or more, preferably 80% or more.

Examples of the Δ15 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include, for example, derived from the genus Trichoderma, derived from the genus Pythium, derived from Caenorhabditis elegans (Biochemistry, 2000, 39 (39), p.11948-11195), Pichia pastoris (Yeast, 2008 Jan; 25 (1), p.21-7) Δ15 desaturase. The amino acid sequences of these enzymes are known (for example, J. Gen. Microbiol., 1991, vol. 137, p. 1825-1830, European Patent Publication No. EP 2500420A1). Preferably, the Δ15 desaturase used in the present invention is a Δ15 desaturase derived from Trichoderma sp. AM076 consisting of the amino acid sequence represented by SEQ ID NO: 3.

Accordingly, preferred examples of Δ15 desaturase used in the present invention include the following:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(B) an amino acid sequence represented by SEQ ID NO: 3, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ15 desaturase activity A protein having
(C) consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 3, and Δ15 desaturation A protein having enzymatic activity;
(D) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and has Δ15 desaturase activity Protein; or
(E) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more identical to the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2. A protein encoded by a polynucleotide comprising a nucleotide sequence having sex and having Δ15 desaturin activity.

Examples of the Δ12 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include, for example, Coprinus cinereus-derived Δ12 desaturase. The amino acid sequence of this enzyme is known (for example, FEBS Lett., 2007, 581, p.315-319) and is represented by SEQ ID NO: 6.

Accordingly, preferred examples of Δ12 desaturase used in the present invention include the following:
(A ′) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(B ′) an amino acid sequence represented by SEQ ID NO: 6, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ12 desaturase An active protein;
(C ′) consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 6, and Δ12 unsaturated A protein having an enzyme activity;
(D ′) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 52 to 1377 of SEQ ID NO: 5, and has Δ12 desaturase activity A protein having; or
(E ′) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more of the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5. A protein encoded by a polynucleotide having a nucleotide sequence having identity and having Δ12 desaturin activity.

Examples of the Δ17 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include Δ17 desaturase derived from the genus Saprolegnia or the genus Phytophthora. The amino acid sequences of these enzymes are known (for example, Biochem. J., 2004, 378, 665-671 doi: 10.1042 / BJ20031319, EP2010648B1). Preferably, the Δ17 desaturase used in the present invention is a Δ17 desaturase derived from Saprolegnia diclina having the amino acid sequence represented by SEQ ID NO: 9.

Accordingly, preferred examples of Δ17 desaturase used in the present invention include the following:
(A ″) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9;
(B ″) an amino acid sequence represented by SEQ ID NO: 9, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ17 desaturation A protein having enzymatic activity;
(C ″) consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 9, and Δ17 A protein having saturating enzyme activity;
(D ″) a Δ17 desaturase activity encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a complementary strand of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 Or a protein having
(E ″) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, more preferably 99% or more with the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 A protein encoded by a polynucleotide comprising a nucleotide sequence having the same identity and having Δ17 desaturase activity.

With regard to (b), (b ′), and (b ″) above, “mutations selected from deletion, substitution, insertion and addition of one or more amino acids” include the following:
(B1) deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 3, 6 or 9;
(B2) substitution of one or more amino acids with other amino acids in the amino acid sequence shown in SEQ ID NO: 3, 6 or 9;
(B3) insertion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 3, 6 or 9;
(B4) addition of one or more amino acids in total to one or both ends of the amino acid sequence shown in SEQ ID NO: 3, 6 or 9; or
(B5) a combination of (b1) to (b4) above, wherein the number of amino acids deleted, substituted, inserted and added is one or more in total,
Here, the positions of amino acid deletions, substitutions and insertions relative to the amino acid sequence are not particularly limited as long as the above-mentioned desaturase activity is retained in the mutated protein.

Furthermore, the desaturase used in the present invention is a protein represented by the above (a) to (e), (a ′) to (e ′), or (a ″) to (e ″). In amino acids, such as glycine and alanine, valine and leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc. It can also be a protein made. The position and the number of substitutions with similar amino acids are not particularly limited as long as the desired desaturase activity is retained in the substituted protein.

The gene encoding the desaturase used in the present invention may be a known amino acid sequence for each of the enzymes described above, (a) to (e), (a ′) to (e ′), or It can be obtained based on the amino acid sequence of the protein represented by (a ″) to (e ″). For example, the gene can be isolated by a conventional method from the above-described microorganism having the desaturase used in the present invention. Alternatively, it can be chemically synthesized based on the amino acid sequence of the desaturase used in the present invention described above.

Furthermore, a gene encoding an enzyme having a desired substrate specificity is further selected from the obtained genes encoding the desaturase used in the present invention by a general screening method. Can be used. For example, a gene encoding a Δ15 desaturase or Δ12 desaturase having a high substrate specificity for a highly unsaturated fatty acid having a carbon chain length of 18 or a substrate specific for a highly unsaturated fatty acid having a carbon chain length of 20 A gene encoding a Δ17 desaturase with high specificity can be selected and used in the present invention.

The gene encoding the Δ15 desaturase introduced into the mutant microorganism of the present invention includes the above-mentioned Trichoderma sp. (AM076) genus, Pysium genus, Caenorhabditis elegans genus, Pichia a gene encoding a Δ15 desaturase derived from P. pastoris, preferably Trichoderma sp. consisting of a nucleotide sequence represented by SEQ ID NO: 1. It is a gene encoding a Δ15 desaturase derived from AM076.

Examples of the gene encoding the Δ12 desaturase introduced into the mutant microorganism of the present invention include the above-described gene encoding the Δ12 desaturase derived from Coprinus cinereus comprising the nucleotide sequence represented by SEQ ID NO: 4.

Examples of the gene encoding the Δ17 desaturase introduced into the mutant microorganism of the present invention include the gene encoding the Δ17 desaturase derived from the genus Saprolegnia or the genus Phytophthora. Preferably, it is a gene encoding a Δ17 desaturase derived from Saprolegnia diclina consisting of the nucleotide sequence represented by SEQ ID NO: 7.

Furthermore, the gene encoding the Δ15 desaturase, Δ12 desaturase or Δ17 desaturase listed above is optimized for codon usage in accordance with the codon usage in the microorganism species into which the gene is introduced. It is preferable that Information on codons used by various microbial species can be obtained from Codon Usage Database (www.kazusa.or.jp/codon/). For example, when introducing a desaturase gene into a Mortierella microorganism, the codon usage frequency of the Mortierella microorganism (www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=64518) The nucleotide sequence of the gene can be modified with reference to to optimize the codon. Specifically, the Δ15 desaturase gene represented by SEQ ID NO: 1 is M.p. When modified in accordance with the frequency of alpina codon usage, a polynucleotide having the sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 can be obtained. The Δ12 desaturase gene represented by SEQ ID NO: 4 was designated as M. pylori. When modified in accordance with the frequency of alpina codon usage, a polynucleotide having the sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 can be obtained. The Δ17 desaturase gene represented by SEQ ID NO: 7 was designated as M. pylori. When modified in accordance with the frequency of alpina codon usage, a polynucleotide having the sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 can be obtained.

Accordingly, preferred examples of genes encoding Δ15 desaturase used in the present invention include the following:
(I) a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2;
(Ii) a nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2, comprising a nucleotide sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more nucleotides; A polynucleotide encoding a protein having Δ15 desaturase activity;
(Iii) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more identical to the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2. A polynucleotide encoding a protein comprising a nucleotide sequence having sex and having Δ15 desaturase activity;
(Iv) a polynucleotide that hybridizes with the nucleotide sequence shown in nucleotide numbers 53 to 1225 of SEQ ID NO: 2 under a stringent condition and encodes a protein having Δ15 desaturase activity.

Preferred examples of the gene encoding Δ12 desaturase used in the present invention include the following:
(I ′) a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5;
(Ii ′) consisting of a nucleotide sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more nucleotides in the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5; And a polynucleotide encoding a protein having Δ12 desaturase activity;
(Iii ′) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, more preferably 99% or more of the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5. A polynucleotide comprising a nucleotide sequence having identity and encoding a protein having Δ12 desaturase activity;
(Iv ′) a polynucleotide that hybridizes under stringent conditions with the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 and encodes a protein having Δ12 desaturase activity.

Preferred examples of the gene encoding the Δ17 desaturase used in the present invention include the following:
(I ″) a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 (ii ″) one or more nucleotides in the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 A polynucleotide consisting of a nucleotide sequence mutated selected from nucleotide deletions, substitutions, insertions and additions and encoding a protein having Δ17 desaturase activity (iii ″) nucleotide number of SEQ ID NO: 8 100% to 1176 nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more identity, and Polyn encoding a protein having Δ17 desaturase activity Creotide (iv ″) A polynucleotide that hybridizes under stringent conditions with the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 and encodes a protein having Δ17 desaturase activity.

The polynucleotides (ii) to (iv), (ii ′) to (iv ′) and (ii ″) to (iv ″) are composed of codons frequently used in Mortierella microorganisms. It is desirable. In the present invention, the polynucleotides (i) to (iv), (i ′) to (iv ′) and (i ″) to (iv ″) are preferably Mortierella microorganisms, more preferably M.M. Introduced into alpina.

The gene encoding the desaturase used in the present invention can be introduced into the parent microorganism using a vector. The type of vector used for the introduction is not particularly limited, and can be appropriately selected and used according to the parent microorganism, the cloning method, the purpose of gene expression, and the like. For example, when the parent microorganism is a microorganism belonging to the genus Mortierella, for example, a pD4 vector (Appl. Environ. Microbiol., November 2000, 66 (11), p. 4655-4661), a pDZeo vector (J. Biosci. Bioeng. , December 2005, 100 (6), p.617-622), pDura5 vector (Appl. Microbiol. Biotechnol., 2004, 65 (4), p.419-425), pDX vector (Curr. Genet., 2009, 55 (3), p. 349-356), pBIG3ura5 (Appl. Environ. Microbiol., 2009, 75, p. 5529-5535), etc., are introduced. If a vector capable of expressing a gene, but are not limited to.

Further, the above vector includes a promoter sequence or transcription termination signal sequence for expressing the incorporated desaturase gene, or a selection marker gene for selecting a transformant into which the desaturase gene has been introduced. It is preferable. As the promoter, a high expression promoter can be used when the parent microorganism is a Mortierella genus microorganism. Preferred high expression promoters for Mortierella microorganisms include M. Alpina-derived SSA2 promoter (SEQ ID NO: 10) and PP3 promoter (SEQ ID NO: 11), and promoters modified by adding substitutions, deletions, additions, etc. to the sequences of these promoters, but high expression of the introduced gene If it can be made, it will not be limited to these. Examples of selectable marker genes include drug resistance genes such as kanamycin resistance gene, streptomycin resistance gene, carboxin resistance gene, zeocin resistance gene, hygromycin resistance gene, amino acid requirements such as leucine, histidine, methionine, arginine, tryptophan, lysine, etc. Examples include genes that complement mutations, genes that complement nucleobase-requiring mutations such as uracil and adenine, and the like. Examples of preferred selectable marker genes include genes that complement uracil-requiring mutations. For example, M.M. A uracil auxotrophic mutant strain of alpina (Biosci Biotechnol Biochem., 2004, 68, p.277-285) has been developed. For such uracil-requiring strains, orotidine-5'-phosphate decarboxylase gene (ura3 gene) or orotidylate pyrophosphorylase gene (ura5 gene) can be used as a selectable marker gene. There are no particular limitations on the procedure for constructing the vector and the types of reagents used, such as restriction enzymes or ligation enzymes. A person skilled in the art can construct a vector according to ordinary knowledge or using commercially available products as appropriate.

The vector may contain any one of a gene encoding a Δ15 desaturase, a gene encoding a Δ12 desaturase, or a gene encoding a Δ17 desaturase. A plurality of genes, for example, Δ12 desaturase gene and Δ15 desaturase gene, Δ12 desaturase gene and Δ17 desaturase gene, or Δ12 desaturase gene and Δ15 desaturation An enzyme gene and a Δ17 desaturase gene may be included. Therefore, the mutant strain of the present invention may be introduced with a vector containing any one of the desaturase genes, may be introduced with a vector containing a plurality of the desaturase enzymes, Alternatively, two or more types of vectors containing different desaturase enzymes may be introduced.

The structure of an example of a transformed binary vector constructed for production of the mutant strain of the present invention is shown in FIGS. In the vector, a polynucleotide encoding a Δ17 desaturase, Δ15 desaturase or Δ12 desaturase is linked downstream of the PP3 promoter or SSA2 promoter, which are constitutively high expression promoters, and An sdhB terminator is incorporated as a terminator, and a ura5 gene is incorporated as a selection marker for the transformant.

In order to introduce the vector into the microorganism of the present invention, a known method such as an electroporation method, a particle gun (gene gun) method, a competent cell method, a protoplast method, or a calcium phosphate coprecipitation method can be used. . Furthermore, as a gene introduction method when a Mortierella genus microorganism is used as a parental microorganism, the ATMT method (Appl. Environ. Microbiol., 2009, vol. 75, p. 5529-5535), or a modified method of the ATMT method. However, the gene transfer method is not limited to these methods as long as a transformant that stably retains the target character can be obtained.

Alternatively, the gene encoding the above desaturase may be directly introduced into the genome of the parent microorganism. Together with the desaturase gene, the above promoter sequence, transcription termination signal sequence or selectable marker gene may be introduced together. Furthermore, a plurality of genes encoding different desaturases may be introduced together. A homologous recombination method is mentioned as a method of introducing a gene directly into the genome.

According to the above procedure, a gene encoding a foreign Δ15 desaturase, a gene encoding a foreign Δ12 desaturase, a gene encoding a foreign Δ15 desaturase, and a foreign Δ17 By introducing any one or more genes encoding a saturating enzyme, the mutant microorganism of the present invention can be produced. The mutant microorganism of the present invention exhibits ω3 desaturase activity for highly unsaturated fatty acids and exhibits high EPA biosynthesis ability even at room temperature by the action of the desaturase encoded by the introduced gene. Can do. Therefore, by culturing the mutant microorganism of the present invention, a lipid containing a high content of EPA is produced in the cells of the microorganism. Furthermore, since lipids containing a high amount of EPA produced by the mutant microorganism of the present invention have a reduced amount of oleic acid and arachidonic acid, high purity EPA can be efficiently obtained by purifying the produced lipid. It becomes possible to produce.

Therefore, a further embodiment of the present invention is a method for producing a lipid containing EPA, comprising culturing the mutant microorganism of the present invention. Another further embodiment of the present invention is a method for producing EPA, comprising purifying a lipid containing EPA produced by the mutant microorganism of the present invention.

In the method for producing lipids containing EPA of the present invention, the mutant microorganism of the present invention can be inoculated and cultured in a liquid medium or a solid medium. For example, when the mutant microorganism is a fungus, a spore of a strain, a mycelium, or a preculture solution obtained by culturing in advance can be inoculated into the medium and cultured. Examples of the carbon source of the medium include, but are not limited to, glucose, fructose, xylose, saccharose, maltose, soluble starch, corn starch, glycerol, mannitol, lipid, alkane, alkene and the like. As a nitrogen source, in addition to natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, soy protein, defatted soybean, cottonseed dregs and wheat bran, organic nitrogen sources such as urea, In addition, inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate are included, but not limited thereto. Furthermore, lipids such as soybean oil, coconut and corn oil may be added. In addition, as a trace nutrient source, inorganic salts such as phosphate, magnesium sulfate, iron sulfate, and copper sulfate, vitamins, and the like can be appropriately added. These medium components are not particularly limited as long as the concentration does not impair the growth of the mutant microorganism of the present invention. For example, the carbon source can be 0.1 to 40% by mass, preferably 1 to 25% by mass, and the nitrogen source in the medium can be 0.01 to 10% by mass, preferably 0.1 to 10% by mass. . M.M. When alpina or a mutant thereof is cultured, a Czapek medium, a Czapek-dox medium, a glucose / yeast extract (hereinafter also referred to as “GY”) medium, an SC medium, or the like described later can be used. Alternatively, known media (for example, International Publication No. 98/29558) can be referred to for the culture medium for Mortierella microorganisms. The pH of the medium can be 4-10, preferably 6-9. The culture can be an aeration and agitation culture, a shaking culture or a stationary culture.

The culture of the mutant microorganism of the present invention is performed at an optimum growth temperature. For example, the mutant microorganism of the present invention has a temperature of about 5 to 60 ° C., preferably about 10 to 50 ° C., more preferably about 10 to 40 ° C., more preferably about 20 to 40 ° C., still more preferably about 20 to 30 ° C. It can be cultured. For example, M.M. alpina or a mutant thereof can be cultured at about 10 to 40 ° C, preferably about 20 to 40 ° C, more preferably about 20 to 30 ° C. In order for the mutant microorganism of the present invention to efficiently produce EPA, the culture temperature is 20 ° C. or higher, preferably about 20-40 ° C., more preferably about 20-30 ° C.

The culture period is 2 to 20 days, preferably 2 to 14 days. With respect to the culture method of Mortierella microorganisms, known literature (for example, JP-A-6-153970) can also be referred to.

By culturing the mutant microorganism of the present invention under the above conditions, lipids containing a high content of EPA are produced in the cells of the microorganism. When the mutant microorganism of the present invention is cultured for 10 days under a condition of 20 ° C. or higher, the EPA content in the total fatty acid composition of the lipid contained in the microorganism is 20% by mass or more. The fatty acid composition in microbial cells can be measured by gas chromatography analysis.

After completion of the culture, the culture solution is subjected to conventional means such as centrifugation and filtration to separate microbial cells. For example, the culture solution is centrifuged or filtered to remove the liquid, and the separated cells are washed and then dried by lyophilization, air drying or the like to obtain dried cells. The desired lipid can be extracted from the dried cells by a known method such as organic solvent extraction. Examples of the organic solvent include hexane, ether, ethyl acetate, butyl acetate, chloroform, cyclohexane, benzene, toluene, xylene, and the like, which are highly soluble in highly unsaturated fatty acids and can be separated from water. Or these organic solvents can also be used in combination. The target lipid can be extracted by distilling off the organic solvent from the extract under reduced pressure or the like. Alternatively, lipids can be extracted from wet cells without drying the cells. The obtained lipid may be further purified by appropriately using general methods such as degumming, deoxidation, deodorization, decolorization, column treatment, distillation and the like.

In the extracted lipid, various fatty acids that are mixed substances are contained in addition to the target EPA. Therefore, EPA with higher purity can be obtained by further purifying the lipid. EPA can be separated directly from lipids, but it is preferable to separate the desired ester derivative of EPA after once converting the fatty acid in the lipid into an ester derivative with a lower alcohol. Since the ester derivative can be separated by using various separation and purification operations depending on the number of carbon atoms, the number of double bonds, the difference in position, etc., an ester derivative of the target fatty acid can be easily obtained. However, since arachidonic acid having the same carbon number and one double bond number as that of EPA is difficult to separate from EPA, it is preferable that the EPA-containing lipid contains less arachidonic acid. The ester derivative is preferably an ethyl ester derivative. For the esterification, a lower alcohol containing an acid catalyst such as hydrochloric acid, sulfuric acid or BF3, or a base catalyst such as sodium methoxide or potassium hydroxide can be used. The desired ester derivative of EPA can be separated from the obtained ester derivative by column chromatography, low temperature crystallization method, urea addition fractionation method or the like alone or in combination. The separated ester derivative of EPA is hydrolyzed with an alkali, and then extracted with an organic solvent such as ether or ethyl acetate, whereby EPA can be purified. EPA may be purified in the form of a salt.

When the production of lipids containing high EPA according to the present invention is performed on an industrial scale, for example, the mutant microorganism of the present invention is cultured on a large scale in a tank or the like, filtered with a filter press or the like, and the cells are collected and dried. The cells can be crushed with a ball mill or the like, and the lipids can be extracted with an organic solvent. In addition, many methods for extracting and using components in microorganisms on an industrial scale and methods for purifying EPA from lipids are known, and these can be appropriately modified and used in the method of the present invention.

EPA obtained by the present invention can be used for the production of pharmaceuticals, cosmetics, foods, feeds, etc. for human or non-human animals. Examples of the pharmaceutical dosage form include oral preparations such as tablets, capsules, granules, powders, syrups, dry syrups, liquids and suspensions; enteral preparations such as inhalants and suppositories; Injections; topical agents; transdermal, transmucosal, nasal agents; inhalants; patch agents and the like. Examples of the form of the cosmetic include any form that cosmetics can usually take such as cream, emulsion, lotion, suspension, gel, powder, pack, sheet, patch, stick, cake and the like.

The above pharmaceutical products or cosmetics contain EPA, or a salt or ester thereof as an active ingredient. The pharmaceutical or cosmetic is also a pharmaceutically acceptable carrier or a cosmetically acceptable carrier such as an excipient, a disintegrant, a binder, a lubricant, a surfactant, a pH adjuster, a dispersant, It may contain emulsifiers, preservatives, antioxidants, colorants, alcohol, water, water-soluble polymers, fragrances, sweeteners, corrigents, acidulants, and other active ingredients as necessary. For example, it may contain medicinal ingredients, cosmetic ingredients and the like. The said pharmaceutical or cosmetics can be manufactured by mix | blending the said carrier and another active ingredient with EPA, its salt, or ester according to a dosage form, and preparing according to a conventional method. The content of EPA, or a salt or ester thereof in the pharmaceutical or cosmetic composition varies depending on the dosage form, but is usually in the range of 0.1 to 99% by mass, preferably 1 to 80% by mass.

The above-mentioned food and drink or feed contains EPA, or a salt or ester thereof as an active ingredient. These foods and drinks or feeds are intended to have effects such as platelet aggregation inhibitory action, blood neutral fat lowering action, anti-arteriosclerosis action, blood viscosity lowering action, blood pressure lowering action, anti-inflammatory action, antitumor action, etc. It may be a health food, a functional food / beverage product, a food / beverage product for specific health use, a food / beverage product for a sick person, a livestock, a racehorse, a feed for an appreciation animal, a pet food or the like.

The form of the above food or drink or feed is not particularly limited, and includes all forms in which EPA, or a salt or ester thereof can be blended. For example, the form of the food or drink may be solid, semi-solid or liquid, or various types such as tablets, chewable tablets, powders, capsules, granules, drinks, gels, syrups, liquid foods for enteral nutrition A form is mentioned. Specific examples of the form of food and drink include tea drinks such as green tea, oolong tea and tea, coffee drinks, soft drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, fruit juice drinks, lactic acid bacteria drinks, fermented milk drinks, Powdered beverages, cocoa beverages, alcoholic beverages, beverages such as purified water, butter, jam, sprinkles, margarine spreads, mayonnaise, shortening, custard cream, dressings, breads, cooked rice, noodles, pasta, miso soup, tofu , Milk, yogurt, soups or sauces, confectionery (for example, biscuits and cookies, chocolate, candy, cake, ice cream, chewing gum, tablets). Since the said feed can be utilized with the composition and form substantially the same as food / beverage products, the description regarding the food / beverage products in this specification can be applied similarly about feed.

The above-mentioned food or drink or feed is EPA, or a salt or ester thereof, and other food or drink materials used in the production of food or drink or feed, various nutrients, various vitamins, minerals, amino acids, various oils and fats, various additives (for example, It can be produced by blending flavoring ingredients, sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, pigments, flavors, etc., and preparing them according to conventional methods. it can. Or the food / beverage products or feed based on this invention can be manufactured by mix | blending EPA, its salt, or ester with the food / beverage products or feed normally eaten. The content of EPA or a salt or ester thereof in the food or drink or feed varies depending on the form of the food, but is usually 0.01 to 80% by mass, preferably 0.1 to 50% by mass, more preferably 1 to It is in the range of 30% by mass.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples.

(Reagents and media)
LB agar medium containing 50 μg / mL ampicillin or kanamycin, Czapek-Dox agar medium (3% sucrose, 0.2% NaNO ₃ , 0.1% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO _4. 7H ₂ O, 0.001% FeSO ₄ · 7H ₂ O, 2% agar, pH 6.0), GY medium (2% (w / v) glucose, 1% yeast extract), LB-Mg agar medium (1% Tryptone, 0.5% yeast extract, 85 mM NaCl, 0.5 mM MgSO ₄ .7H ₂ O, 0.5 mM NaOH, 1.5% agar, pH 7.0), minimal medium (MM) (10 mM K ₂ HPO ₄ , 10 mM KH ₂ PO ₄ , 2.5 mM NaCl, 2 mM MgSO ₄ .7H ₂ O, 0.7 mM CaCl ₂ , 9 μM FeSO ₄ .7H ₂ O, 4 mM (NH ₄ ) ₂ SO ₄ , 10 mM glucose, pH 7.0), induction medium (IM) (0.5% (w / v) glycerol in MM, 200 μM acetosyringone, 40 mM 2- (N-morpholino) ethanesulfonic acid (MES) ) And adjusted to pH 5.3), SC medium (5.0 g Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco), 1.7 g (NH ₄ ) ₂ SO ₄ , 20 g glucose, 20 g agar, 20 mg adenine, 30 mg tyrosine, 1.0 mg methionine, 2.0 mg arginine, 2.0 mg histidine, 4.0 mg lysine, 4.0 mg tryptophan, 5.0 mg threonine, 6.0 mg isoleucine, 6.0 mg leucine, 6.0 mg / L phenylalanine), Cef Otaxime, spectinomycin, Nile blue A (Sigma) was used. Restriction enzymes, ligation enzymes, and the like were used from Takara Bio or New England BioLabs.

(Reference Example 1) Codon optimization of Δ15 desaturase gene (DES2m; derived from Trichoderma sp. AM076) Δ15 desaturase gene (DES2m; SEQ ID NO: 1) derived from the filamentous fungus Trichoderma sp. (Trichoderma sp. AM076) Codons of M. Alpina codon usage frequency (www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=64518) was optimized. SpeI and BamHI sites were constructed before and after CDS of the obtained gene sequence, and total synthesis was performed (Life Technologies). The nucleotide sequence after optimization of the gene and construction of the restriction enzyme cleavage site is shown in SEQ ID NO: 2. The gene was cloned into the SpMA-RQ (ampR) plasmid.

(Reference Example 2) Codon optimization of Δ12 desaturase gene (CopΔ12m; derived from Coprinus cinereus) Similar to Reference Example 1, Δ12 desaturase gene (CopΔ12m; SEQ ID NO: derived from Coprinus cinereus) 4) the codon of M. SpeI and BamHI sites were constructed before and after CDS of the resulting gene sequence based on the frequency of alpina codon usage, and total synthesis was performed (Life Technologies). The nucleotide sequence after optimization of the gene and construction of the restriction enzyme cleavage site is shown in SEQ ID NO: 5. The gene was cloned into the SpMA-RQ (ampR) plasmid.

(Reference Example 3) Codon optimization of Δ17 desaturase gene (derived from Saprolegnia diclina) Similar to Reference Example 1, Δ17 desaturase gene (Δ17m; SEQ ID NO: 7) derived from filamentous fungus Saproregnia (Saprolegnia diclina) Codons of M. SpeI and BamHI sites were constructed before and after CDS of the resulting gene sequence based on the frequency of alpina codon usage, and total synthesis was performed (Life Technologies). The nucleotide sequence after optimization of the gene and construction of the restriction enzyme cleavage site is shown in SEQ ID NO: 8. The gene was cloned into the SpMA-RQ (ampR) plasmid.

(Reference Examples 4 to 8) Construction of Binary Vector for Gene Introduction Each plasmid prepared in Reference Examples 1 to 3 was treated with SpeI and BamHI restriction enzymes, and the obtained Δ17 desaturase gene and Δ15 desaturase The gene and a fragment of the Δ12 desaturase gene were modified from a plasmid pBIG35 (a pBIG2RHPH2 provided by Kyoto Prefectural University) containing the PP3 promoter (SEQ ID NO: 11) or SSA2 promoter (SEQ ID NO: 10), which are constitutively high expression promoters. Appl. Environ. Microbiol., 2009, vol. 75, p. 5529-5535) to construct an expression cassette. The expression cassette is further ligated to uracil-required marker gene (ura5) and tandem, and a binary vector for transformation, pBIG35SSA2pΔ17m (Δ17 desaturase gene: Reference Example 4), pBIG35PP3pDES2m (Δ15 desaturase gene) : Reference Example 5), pBIG35PP3pCopΔ12m (Δ12 desaturase gene: Reference Example 6), pBIG35PP3pCopΔ12mSSA2pΔ17m (Δ12 desaturase gene and Δ17 desaturase gene: Reference Example 7), and pBIG35PP3pCopΔ12mSSA2pDES2 gene (Δ12 desaturated enzyme) And Δ15 desaturase gene: Reference Example 8) was constructed. 2 to 6 show the structures of the vectors of Reference Examples 4 to 8, respectively.

(Production Example 1) Preparation of Δ17 desaturase gene-introduced strain Based on the report by Ando et al. (Appl. Environ. Microbiol., 2009, vol. 75, p. 5529-5535), Δ17 desaturation of Reference Example 4 The binary vector pBIG35SSA2pΔ17m for introducing an enzyme gene was transformed into the host strain M.I. by the ATMT method via Agrobacterium. It was introduced into alpina 1S-4 (uracil auxotrophic strain) by the following procedure.
(1) M.M. A culture obtained by culturing alpina 1S-4 strain (uracil auxotrophic strain) on Czapek-Dox agar medium containing 0.05 mg / mL uracil is collected and filtered with Miracloth (Calbiochem). A spore suspension of alpina was freshly prepared.
(2) Agrobacterium (Agrobacterium tumefaciens C58C1, provided by Kyoto Prefectural University) was transformed with the binary vector for Δ17 desaturase gene introduction prepared in Reference Example 4 by electroporation, and then on LB-Mg agar medium. And incubated at 28 ° C. for 48 hours. Agrobacterium containing the vector was confirmed by PCR. Agrobacterium containing the vector was cultured in minimal medium (MM) for 2 days, centrifuged at 5,800 × g, and fresh IM was added to prepare a suspension. The suspension was induction-cultured on a rotary shaker for 8-12 hours at 28 ° C. until the OD ₆₆₀ was 0.4 to 3.7.
(3) 100 μL of the above Agrobacterium suspension was added to an equal amount of the above M.I. Alpina suspension (10 ⁸ mL ⁻¹ ) and mixed with a nitrocellulose membrane (70 mm diameter; hardened low-ash grade 50, Whatman) (same composition as IM, except for 10 mM glucose) (Containing 5 mM glucose and 1.5% agar), and cultured at 23 ° C. for 2 to 5 days. After co-culture, the membrane was transferred to an SC agar medium containing uracil-free, 50 g / mL cefotaxime and 50 g / mL spectinomycin, 0.03% Nile blue A (Sigma), and cultured at 28 ° C. for 5 days. Mycelia from visible fungal colonies were transferred to fresh uracil-free SC medium. A Δ17 desaturase gene-introduced strain that stably grows cells that can grow on a uracil-free SC agar medium but cannot grow on a GY agar medium containing 5-fluoroorotic acid (5-FOA) It was judged. This operation was performed three times in order to select transformants that stably maintain the character.

(Production Example 2) Preparation of Δ15 desaturase gene-introduced strain Δ15 desaturase in the same procedure as in Production Example 1 except that the binary vector pBIG35PP3pDES2m for reference gene Δ15 in Reference Example 5 was used. A transgenic strain was prepared.

(Production Example 3) Preparation of Δ12 desaturase gene-introduced strain Δ12 desaturase in the same procedure as in Production Example 1 except that the binary vector pBIG35PP3pCopΔ12m for Δ12 desaturase gene introduction in Reference Example 6 was used. A transgenic strain was prepared.

(Production Example 4) Preparation of Δ12, Δ17 desaturase gene-introduced strain Same as Production Example 1 except that the Δ12 desaturase gene of Reference Example 7 and the binary vector pBIG35PP3pCopΔ12mSSA2pΔ17m for introduction of Δ17 desaturase gene were used. The Δ12 desaturase gene and Δ17 desaturase gene-introduced strains were prepared by the above procedure.

(Production Example 5) Preparation of Δ12, Δ15 desaturase gene-introduced strain Same as Production Example 1 except that the Δ12 desaturase gene of Reference Example 8 and the binary vector pBIG35PP3pCopΔ12mSSA2pDES2m for Δ15 desaturase gene introduction were used. The Δ12 desaturase gene and Δ15 desaturase gene-introduced strains were prepared by the above procedure.

The mutant strains produced in Production Examples 1 to 5 are summarized below.

(Test Example 1) Fatty acid composition and production amount in transgenic strains The mutant strains obtained in Production Examples 1 to 5 were each in 10 mL GY medium (2% glucose, 1% yeast extract) at 28 ° C for 1 week at 300 rpm. Aerobically cultured. From each culture solution, gene transfer was performed by suction filtration. Alpina cells were collected and dried at 120 ° C. for 3 hours. Add 1 mL of a dichloromethane solution containing an internal standard (saturated fatty acid having 23 carbon atoms that M. alpina cannot biosynthesize) at a concentration of 0.5 mg / mL to dry cells, and 2 mL of methanolic hydrochloric acid, and warm bath at 55 ° C. for 2 hours The fatty acid was methyl esterified. Thereafter, 1 mL of distilled water and 4 mL of hexane were added, and the hexane layer was extracted and centrifuged under reduced pressure to recover the fatty acid methyl ester. Each sample was dissolved in chloroform, and the fatty acid composition in the sample was measured by gas liquid chromatography (GLC). GLC uses Shimadzu GC-2010, GL Sciences capillary column TC70 (0.25 mm × 60 m), column temperature 180 ° C., vaporization chamber temperature 250 ° C., detector temperature 250 ° C., carrier gas He, makeup It was performed under the conditions of upgas N ₂ , H ₂ flow rate 40 mL / min, Air flow rate 400 mL / min, split ratio 50, and analysis time 30 min. The amount of extracted fatty acid was quantified based on the amount of fatty acid of the internal standard from the peak area value of the GLC chart.

As a result, the Δ17 desaturase gene-introduced strain of Production Example 1 showed reduced arachidonic acid accumulation and 47% eicosapentaenoic acid (EPA) accumulation per total fatty acid. In the Δ15 desaturase gene-introduced strain of Production Example 2, arachidonic acid accumulation was reduced and 48% of EPA was accumulated per total fatty acid. On the other hand, in the Δ12 desaturase gene-introduced strain of Production Example 3, although oleic acid reduction and arachidonic acid accumulation improvement were observed, EPA accumulation did not improve. In the strain introduced with the gene for Δ12 desaturase and Δ17 desaturase in Production Example 4, in addition to the reduction of arachidonic acid, a decrease in oleic acid was observed, and an accumulation of EPA of 50% or more per total fatty acid was observed. It was. In addition, in the strain introduced with the gene for Δ12 desaturase and Δ15 desaturase in Production Example 5, oleic acid and arachidonic acid were reduced, and more than 40% of EPA was accumulated per total fatty acid. .

(Test Example 2) Fatty acid composition and production change over time in the desaturase gene-introduced strain (Δ17 desaturase gene-introduced strain)
Δ17 desaturase gene introduced M. obtained in Production Example 1 alpina mutant strains (# 1, # 2, # 4, # 6, # 9, # 19, # 21 and # 24) in 4 mL GY medium (2% glucose, 1% yeast extract), 3 at 28 ° C., The aerobic culture was performed at 120 rpm for 7, 10, 14 days, and the composition and production amount of fatty acids in the cells were analyzed over time by the same procedure as in Test Example 1. The results are shown in FIG. Wild strains (M. alpina 1S-4), control strains (strains in which a vector containing only a marker gene was inserted into a uracil-requiring strain), and host strains (uracil-requiring strains) did not produce EPA under these culture conditions. However, all the Δ17 desaturase gene-introduced strains had improved EPA production and reduced arachidonic acid. The percentage of EPA in total fatty acids reached a maximum at about 10 days in culture, and up to 47% (0.9 mg / mL) of EPA per total fatty acid was produced on day 14 of culture, while arachidonic acid Accumulation was reduced to 2% (strain # 9). In addition, the amount of fatty acid produced on the 10th day of culture produced more fatty acids than many control strains in many transgenic strains. From this, it was considered that EPA was efficiently produced in the Δ17 desaturase gene-introduced strain.

(Δ15 desaturase gene introduced strain)
Δ15 desaturase gene-introduced M. coli obtained in Production Example 2 alpina mutants (# 2, # 3, # 9, # 13, # 14, and # 20) in 4 mL GY medium (2% glucose, 1% yeast extract), 3, 7, 10, 14 at 28 ° C. The cells were aerobically cultured at 120 rpm for one day, and the composition and production amount of fatty acids in the cells were analyzed over time by the same procedure as in Test Example 1. The results are shown in FIG. The Δ15 desaturase gene-introduced strains all had improved EPA production and reduced arachidonic acid compared to the control strain (a strain in which a vector containing only the marker gene was inserted into the uracil-requiring strain). The proportion of EPA in total fatty acids is almost maximal on the 14th day of culture, and up to 48% (0.6 mg / mL) of EPA per total fatty acid is produced in 14 days of culture, while the accumulation of arachidonic acid is 6%. % (Strain # 20). Moreover, as for the amount of fatty acid produced, on the seventh day of culture, many gene-introduced strains produced more fatty acids than the control strains. From this, it was considered that EPA was efficiently produced in the Δ15 desaturase gene-introduced strain.

(Δ12 desaturase gene-introduced strain)
Δ12 desaturase gene-introduced M. obtained in Production Example 3 alpina mutants (# 1, # 2, # 7, # 22, # 23, and # 24) in 4 mL GY medium (2% glucose, 1% yeast extract), 3, 7, 10, 14 at 28 ° C. The cells were aerobically cultured at 120 rpm for one day, and the composition and production amount of fatty acids in the cells were analyzed over time by the same procedure as in Test Example 1. The results are shown in FIG. In the Δ12 desaturase gene-introduced strain, the EPA production amount was hardly improved compared to the control strain (a strain in which a vector containing only the marker gene was inserted into the uracil-requiring strain) and the host strain (uracil-requiring strain). Lipid production and the proportion of arachidonic acid in the total fatty acid composition increased, while the amount of oleic acid accumulated in the control strain decreased. For example, in the strain # 7, the lipid production increased by 10% or more compared to the host strain, and the ratio of arachidonic acid in the total fatty acid composition was 15% on the 7th day of culture and 10th day of the culture compared to the control strain. Improved to 20% or more (53% of total fatty acids). On the other hand, the amount of oleic acid was 30% in the wild strain and 12% in the control strain on the 7th day of culture, whereas it was 6% or less on the 7th day of culture in the Δ12 desaturase gene-introduced strain. It decreased to 2% on the 10th and 14th days of culture.

(Δ17, Δ12 desaturase gene-introduced strain)
Δ12, Δ17 desaturase gene introduced M.M. obtained in Production Example 4 4 mL for alpina mutant strains (# 6, # 11, # 16, # 17, # 21, # 23, # 25, # 27, # 33, # 37, # 40, # 45, # 46, and # 48) Composition and production of fatty acids in the cells in the same manner as in Test Example 1 after culturing aerobically at 120 rpm for 4, 10, 14 days at 28 ° C in GY medium (2% glucose, 1% yeast extract) The amount was analyzed over time. The results are shown in FIG. In the strains into which the Δ12 desaturase gene and the Δ17 desaturase gene were introduced, both EPA production was improved compared to the control strain (a strain in which a vector containing only the marker gene was inserted into the uracil-requiring strain), Both arachidonic acid and oleic acid were reduced, and it was considered that EPA was produced efficiently. On the 14th day of cultivation, EPA was produced up to 51% (0.3 mg / mL) per total fatty acid, and arachidonic acid accumulation was reduced to 2.5% and oleic acid accumulation to 4% (strain # 17). .

(Δ15, Δ12 desaturase gene-introduced strain)
Δ12, Δ15 desaturase gene introduced M.M. obtained in Production Example 5 Alpina mutant strains (# 4, # 7, # 8, # 11, and # 16) were favored in 120 mL for 4-10, 14 days at 28 ° C. in 4 mL GY medium (2% glucose, 1% yeast extract). The culture was carried out and the composition and production amount of fatty acids in the cells were analyzed over time in the same procedure as in Test Example 1. The results are shown in FIG. In the strains into which the Δ12 desaturase gene and Δ15 desaturase gene were introduced, both the EPA production amount was improved and arachidone was improved compared to the control strain (a strain in which a vector containing only the marker gene was inserted into the uracil-requiring strain). Both acid and oleic acid were reduced, and it was considered that EPA was produced efficiently. On the 14th day of cultivation, a maximum of 43% (0.13 mg / mL) EPA was produced per total fatty acid, and the accumulation of arachidonic acid was reduced to 12% and the accumulation of oleic acid to 5% (strain # 7).

Claims

A method for producing a mutant microorganism, comprising:
introducing a foreign Δ15 desaturase gene into a parental microorganism having an ω6 polyunsaturated fatty acid metabolic pathway,
The mutant microorganism contains eicosapentaenoic acid in an amount of 20% by mass or more in the fatty acid composition after culturing at 20 ° C. or more for 10 days.
Method.
A method for producing a mutant microorganism, comprising:
Introducing an exogenous Δ12 desaturase gene and any one or more of an exogenous Δ15 desaturase gene and an exogenous Δ17 desaturase gene into a parental microorganism having a ω6 polyunsaturated fatty acid metabolic pathway Including
The mutant microorganism contains eicosapentaenoic acid in an amount of 20% by mass or more in the fatty acid composition after culturing at 20 ° C. or more for 10 days.
Method.
The method according to claim 1 or 2, wherein the parent microorganism is a microorganism belonging to the genus Mortierella.
The method according to any one of claims 1 to 3, wherein the parental microorganism is Mortierella alpina.
The method according to any one of claims 1 to 4, wherein the Δ15 desaturase gene is derived from Trichoderma sp. AM076.
The method according to any one of claims 1 to 4, wherein the Δ15 desaturase gene is a gene encoding the following protein:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(B) an amino acid sequence represented by SEQ ID NO: 3, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ15 desaturase activity A protein having
(C) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 and having Δ15 desaturase activity;
(D) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and has Δ15 desaturase activity Protein; or
(E) a protein encoded by a polynucleotide comprising a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and having Δ15 desaturase activity.
The method according to any one of claims 2 to 6, wherein the Δ12 desaturase gene is derived from Coprinus cinereus.
The method according to any one of claims 2 to 6, wherein the Δ12 desaturase gene is a gene encoding the following protein:
(A ′) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(B ′) an amino acid sequence represented by SEQ ID NO: 6, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ12 desaturase An active protein;
(C ′) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 6 and having Δ12 desaturase activity;
(D ′) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 52 to 1377 of SEQ ID NO: 5, and has Δ12 desaturase activity A protein having; or
(E ′) a protein encoded by a polynucleotide comprising a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 and having Δ12 desaturase activity.
The method according to any one of claims 2 to 8, wherein the Δ17 desaturase gene is derived from Saprolegnia diclina.
The method according to any one of claims 2 to 8, wherein the Δ17 desaturase gene is a gene encoding the lower protein:
(A ″) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9;
(B ″) an amino acid sequence represented by SEQ ID NO: 9, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ17 desaturation A protein having enzymatic activity;
(C ″) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 9 and having Δ17 desaturase activity;
(D ″) a Δ17 desaturase activity encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a complementary strand of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 Or a protein having
(E ″) a protein encoded by a polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 and having Δ17 desaturase activity.
Eicosapentaenoic acid content in fatty acid composition after culturing for 10 days at 20 ° C or higher under the condition that a foreign Δ15 desaturase gene is introduced into a parental microorganism having ω6 polyunsaturated fatty acid metabolic pathway A mutant microorganism having a mass of 20% by mass or more.
An exogenous Δ12 desaturase gene and one or more of an exogenous Δ15 desaturase gene and an exogenous Δ17 desaturase gene were introduced into the parental microorganism having the ω6 polyunsaturated fatty acid metabolic pathway. A mutant microorganism, wherein the eicosapentaenoic acid content in the fatty acid composition after culturing for 10 days at 20 ° C. or higher is 20% by mass or more.
The mutant microorganism according to claim 11 or 12, wherein the parent microorganism is a microorganism belonging to the genus Mortierella.
The mutant microorganism according to any one of claims 11 to 13, wherein the parent microorganism is Mortierella alpina.
The mutant microorganism according to any one of claims 11 to 14, wherein the Δ15 desaturase gene is derived from Trichoderma sp. AM076.
The mutant microorganism according to any one of claims 11 to 14, wherein the Δ15 desaturase gene is a gene encoding the following protein:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(B) an amino acid sequence represented by SEQ ID NO: 3, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ15 desaturase activity A protein having
(C) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 and having Δ15 desaturase activity;
(D) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and has Δ15 desaturase activity Protein; or
(E) a protein encoded by a polynucleotide comprising a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and having Δ15 desaturase activity.
The mutant microorganism according to any one of claims 12 to 16, wherein the Δ12 desaturase gene is derived from Coprinus cinereus.
The mutant microorganism according to any one of claims 12 to 16, wherein the Δ12 desaturase gene is a gene encoding the following protein:
(A ′) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6;
(B ′) an amino acid sequence represented by SEQ ID NO: 6, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ12 desaturase An active protein;
(C ′) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 6 and having Δ12 desaturase activity;
(D ′) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 52 to 1377 of SEQ ID NO: 5, and has Δ12 desaturase activity A protein having; or
(E ′) a protein encoded by a polynucleotide comprising a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 and having Δ12 desaturase activity.
The mutant microorganism according to any one of claims 12 to 18, wherein the Δ17 desaturase gene is derived from Saprolegnia diclina.
The mutant microorganism according to any one of claims 12 to 18, wherein the Δ17 desaturase gene is a gene encoding the protein below:
(A ″) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9;
(B ″) an amino acid sequence represented by SEQ ID NO: 9, comprising an amino acid sequence subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids, and Δ17 desaturation A protein having enzymatic activity;
(C ″) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 9 and having Δ17 desaturase activity;
(D ″) a Δ17 desaturase activity encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a complementary strand of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 Or a protein having
(E ″) a protein encoded by a polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 and having Δ17 desaturase activity.
A mutant microorganism produced by the method according to any one of claims 1 to 10, or the mutant microorganism according to any one of claims 11 to 20 is cultured under conditions of 20 ° C or higher. A method for producing a lipid containing icosapentaenoic acid.
A method for producing eicosapentaenoic acid, comprising purifying a lipid containing eicosapentaenoic acid produced by the method according to claim 21.