JP2016165226A - Production method of lipid with high content of eicosapentaenoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide lipid producing microorganisms that can produce EPA efficiently.SOLUTION: The present invention provides a mutation microorganism obtained by introducing, into a microorganism having a metabolic pathway of ω6 highly unsaturated fatty acids, a foreign Δ15 desaturase gene, or a foreign Δ12 desaturase gene and either one or more of the foreign Δ15 desaturase gene and foreign Δ17 desaturase gene, and the content of eicosapentaenoic acid in the fatty acid composition after the culture under the condition of 20°C or more for 10 days is 20% or more.SELECTED DRAWING: None

Description

  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 with two or more unsaturated bonds, ω6 fatty acids linoleic acid (LA, 18: 2n-6), γ-linolenic acid (GLA, 18: 3n-6), arachidonic acid (ARA, 20: 4n-6), ω3-fatty acid α-linolenic acid (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 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, anti-tumor 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 omega-3 fatty acids has been recommended, and this is a lipid molecular species whose demand has increased significantly.

  Living 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 fish oil-derived EPA may have a fish 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, including fungi and mushrooms, which are present in air, soil, and water. 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 for 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 transformant in which a gene of ω3 fatty acid unsaturated polypeptide isolated from Mortierella alpina is introduced into yeast. Yes. However, Mortierella spp. Microorganisms cannot efficiently produce EPA unless they are cultured under low temperature conditions (20 ° C. or lower) at which the bacteria do not grow easily. Furthermore, Mortierella alpina-derived ω3 desaturase acts preferentially on fatty acids with a carbon chain length of 18, so EPA with a carbon chain length of 20 is difficult to synthesize. 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

Appl. Microbiol. Biotecnol., (1989), Vol.32, p.1-4

  The present invention relates to a mutant microorganism that can efficiently produce EPA at 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 filamentous fungus Trichoderma sp. AM076 in a microorganism of the genus Mortierella. As a result, the present inventors have found that the Δ15 desaturase encoded by the transgene is a ω3 desaturation activity against a highly unsaturated fatty acid having a carbon chain length of 18 on the lipid metabolic pathway of Mortierella microorganisms. And the mutant microorganism introduced with the gene can efficiently produce lipid 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 genus Trichoderma or a filamentous form. The fungus Saprolegnia diclina
The Δ17 desaturase derived from "S. diclina") was introduced into a Mortierella microorganism and expressed. 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 the amount of oleic acid and arachidonic acid accumulated is reduced and EPA can be efficiently purified from EPA-containing lipids.

That is, the present invention is 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, and the fatty acid after culturing 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 an ω6 polyunsaturated fatty acid metabolic pathway to one or more of an exogenous Δ12 desaturase gene, an exogenous Δ15 desaturase gene, and an exogenous 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, which comprises culturing the mutant microorganism under conditions of 20 ° C. or higher.
Furthermore, this invention provides the production method of eicosapentaenoic acid including refine | purifying the lipid containing the eicosapentaenoic acid produced by the said method.
Furthermore, this invention provides the pharmaceutical, food-drinks, or feed containing the eicosapentaenoic acid produced by the said method.

The mutant microorganism of the present invention is introduced with an exogenous Δ15 desaturase gene that works at room temperature, and is capable of producing EPA even under room temperature conditions of 20 ° C. or higher where microorganisms are likely to 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 amount of EPA can be efficiently produced. 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 on an industrial scale of EPA is extremely useful.

Fatty acid biosynthetic pathway in M. alpina 1S-4. Structure of the transformed binary vector pBIG35SSA2pΔ17m. 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. The composition and production amount of fatty acids produced by M. alpina transformants into which S. diclina-derived Δ17 desaturase gene was introduced. Composition and production amount of fatty acids produced by M. alpina transformants into which a Δ15 desaturase gene derived from Trichoderma sp. AM076 was introduced. Composition and production amount of fatty acids produced by M. alpina transformants into which a C. cinereus-derived Δ12 desaturase gene was introduced. Composition and production amount of fatty acids produced by M. alpina transformants into which Δ12 desaturase gene and Δ17 desaturase gene were introduced. Composition and production of fatty acids produced by M. alpina transformants into which the Δ12 desaturase gene and Δ15 desaturase gene were introduced.

  In the present specification, unless otherwise defined, the “one or more” used for amino acid 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, further 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 the amino acid sequence and nucleotide sequence 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 the nucleotide sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. Further, 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 techniques for 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, the “stringent conditions” in the present specification are the normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, More preferably, conditions such as washing at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS once, more preferably 2-3 times, and the like can be mentioned.

  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 “original” used for a certain microorganism is used to indicate that a certain function or trait is possessed by the microorganism (wild type) that exists in nature. 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 is the parent strain of the mutant microorganism of the present invention (hereinafter sometimes referred to as “parental microorganism”) may be any microorganism that has an ω6 highly unsaturated fatty acid metabolic pathway, and preferably has an ω6 highly unsaturated fatty acid metabolic pathway. These are oleaginous microorganisms that produce omega-6 highly unsaturated fatty acids.

  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. Preferably, Mortierella alpina, Mortierella chlamydospora, Mortierella elongata, Mortierella exigua, Mortierella agro tier hyella ), Mortierella epigama, Mortierella acrotona, Mortierella minutissima, Mortierella lignicola, Mortierella chronosti (Mortierella clonocystis), Mortierella nana, Mortierella humicola, Mortierella bainieri, Mortierella hyaline, Mortierella globalpina (Mortierella global) Examples include, but are not limited to, Mortierella microorganisms such as Umbelopsis nana and Umbelopsis isabellina.

  More preferably, the parent 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 an ω3 desaturase and inherently has the ability to produce EPA. Examples of such microorganisms include the aforementioned Mortierella microorganisms such as Mortierella alpina (hereinafter sometimes referred to as “M. alpina” in the present specification), Mortierella clonocystis, Mortierella nana, Mortierella humicola, Mortierella bainieri, Mortierella hyaline. And Mortierella globalpina. Furthermore, parental microorganisms of the mutant microorganism of the present invention include mutants of the aforementioned Mortierella genus microorganisms that have EPA production ability. Examples of such microorganisms include M. alpina 1S-4 (Agric. Biol. Chem., 1987, 51 (3), p. 785-790). Therefore, the parental microorganism of the mutant microorganism of the present invention is preferably a wild strain of Mortierella spp. Having an EPA producing ability and a mutant thereof, preferably a wild strain of M. alpina and a mutant having an EPA producing ability, more preferably. Includes M. alpina 1S-4 and its mutants.

  However, the ω3 desaturase of the Mortierella microorganism has low activity unless it is at a low temperature, and is therefore not necessary for EPA production at room temperature according to the present invention. Therefore, the parental microorganism of the mutant microorganism of the present invention further includes a strain lacking the ω3 desaturase of the genus Mortierella having the EPA-producing ability. Examples of the ω3 desaturase-deficient strain include M. alpina ST1358, which is an M. alpina 1S-4 mutant (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 the extent that the normal function of the ω3 desaturase cannot be exerted, the function of the ω3 desaturase gene product has been completely lost, or due to genetic 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. As a method for deleting ω3 desaturase, 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), treatment with mutagen 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 microorganisms can be increased.

In another embodiment, the mutant microorganism of the present invention comprises a gene encoding a foreign Δ12 desaturase, a gene encoding a foreign Δ15 desaturase, and a foreign Δ
It is produced by introducing any one or more genes encoding 17 desaturase. 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. Moreover, Δ17 desaturase acts from dihomo-γ-linolenic acid in the biosynthetic pathway of polyunsaturated fatty acids by acting like a ω3 desaturase on polyunsaturated fatty acids with 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.

  FIG. 1 shows the biosynthetic pathway of fatty acids of M. alpina 1S-4. By promoting the conversion to ω3 highly unsaturated fatty acids such as α-linolenic acid and stearidonic acid, the biosynthetic amount of eicosapentaenoic acid, which is a 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 that exhibits Δ15 desaturase activity. The Δ15 desaturase activity refers to the activity of desaturating fatty acids 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 an activity of desaturating a fatty acid 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, Δ encoded by a gene introduced into the mutant microorganism of the present invention
The 12 desaturase 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 that exhibits 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 used in the present invention are used.
Desaturating enzymes (hereinafter sometimes referred to as “unsaturating enzymes used in the present invention”) exhibit their enzymatic activity at room temperature. In the present specification, “showing enzyme activity at room temperature” means that the optimum temperature for enzyme activity is 20 ° C. or higher, preferably 20 to 40 ° C., or 70% of activity at 20 ° C. at the optimum temperature. % 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, the genus Trichoderma, the genus Pythium, and the genus Caenorhabditis elegans (Biochemistry, 2000, 39 (39), p.11948-11954), Δ15 desaturase derived from Pichia pastoris (Yeast, 2008 Jan; 25 (1), p.21-7). The amino acid sequences of these enzymes are known (for example, J. Gen. Microbiol., 1991, vol.137, p.1825-1830, European Patent Publication EP 2500420 A1). 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 the Δ15 desaturase used in the present invention include the following.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 (b) the amino acid sequence shown in SEQ ID NO: 3 is subjected to mutation selected from deletion, substitution, insertion and addition of one or more amino acids. 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% of the amino acid sequence shown in SEQ ID NO: 3 (c). A polynucleotide comprising a complementary sequence of a nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 and a protein (d) having an amino acid sequence having at least% identity and having Δ15 desaturase activity Encoded by polynucleotides that hybridize under mild conditions and have Δ15 desaturase activity That 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 a property and having Δ15 desaturase activity

Examples of the Δ12 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include Δ12 desaturase derived from Coprinus cinereus. 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 the Δ12 desaturase used in the present invention include the following.
(A ′) a protein comprising the amino acid sequence shown in SEQ ID NO: 6 (b ′) a mutation selected from deletion, substitution, insertion and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 6 The amino acid sequence and Δ
A protein having 12 desaturase activity (c ′) 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 shown by SEQ ID NO: 6. A protein (d ′) comprising an amino acid sequence and having a Δ12 desaturase activity 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 A protein encoded by a polynucleotide and having Δ12 desaturase activity, or
(E ′) 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. Protein encoded by a polynucleotide comprising a nucleotide sequence having identity and having Δ12 desaturase 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 consisting of the amino acid sequence represented by SEQ ID NO: 9.

Accordingly, preferred examples of the Δ17 desaturase used in the present invention include the following.
(A ″) a protein comprising the amino acid sequence shown in SEQ ID NO: 9 (b ″) selected from deletion, substitution, insertion and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 9 The amino acid sequence is mutated and Δ
17 Protein having desaturase activity (c ″) It has 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 a polynucleotide comprising a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 of protein (d ″) having Δ17 desaturase activity and having a Δ17 desaturase activity under stringent conditions A protein encoded by a hybridizing polynucleotide and having Δ17 desaturase activity, or
(E ″) 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and 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 desaturin activity

Regarding the above-mentioned (b), (b ′) and (b ″), examples of the “mutation selected from the 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) (b1) to (b4) above Mutation in which the number of amino acids added, deleted, substituted, inserted and added is one or more in total. The positions of amino acid deletions, substitutions and insertions relative to the amino acid sequence are the protein after the mutation. As long as the above-mentioned desaturase activity is maintained, there is no particular limitation.

  Furthermore, the desaturase used in the present invention is the protein represented by the above-mentioned (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 with 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 that encodes a highly reliable Δ17 desaturase can be selected and used in the present invention.

Examples of the gene encoding Δ15 desaturase introduced into the mutant microorganism of the present invention include the above-mentioned Trichoderma sp. (AM076) genus, Pythium genus, Nematode (Caenorhabditis elegans) genus, Pichia A gene encoding a Δ15 desaturase derived from pastoris can be mentioned, and a gene encoding a Δ15 desaturase derived from Trichoderma sp. AM076 consisting of the nucleotide sequence represented by SEQ ID NO: 1 is preferred.

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 consisting of 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 described above. Preferably, it is a gene encoding Δ17 desaturase derived from Saprolegnia diclina consisting of the nucleotide sequence shown in SEQ ID NO: 7.

Furthermore, the genes encoding Δ15 desaturase, Δ12 desaturase or Δ17 desaturase listed above are optimized for codon usage according to the frequency of 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 the Codon Usage Database (www.kazusa.or.jp/codon/). For example, when introducing desaturase genes into Mortierella microorganisms, the codon usage of Mortierella microorganisms (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, by modifying the Δ15 desaturase gene represented by SEQ ID NO: 1 in accordance with the codon usage of M. alpina, a polynucleotide having the sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 is obtained. Can do. When the Δ12 desaturase gene represented by SEQ ID NO: 4 is modified according to the codon usage of M. alpina, a polynucleotide having the sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 can be obtained. When the Δ17 desaturase gene represented by SEQ ID NO: 7 is modified in accordance with the codon usage of M. alpina, a polynucleotide having the sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 can be obtained.

Accordingly, preferred examples of the gene 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) deletion of one or more nucleotides in the nucleotide sequence represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 A polynucleotide comprising a mutated nucleotide sequence selected from substitution, insertion and addition and encoding a protein having Δ15 desaturase activity (iii) represented by nucleotide numbers 53 to 1225 of SEQ ID NO: 2 A nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more, still more preferably 99% or more identity with the nucleotide sequence, and Δ15 desaturase activity A polynucleotide encoding a protein having 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

Preferable 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 ′) one or more nucleotides of the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 A polynucleotide consisting of a nucleotide sequence mutated selected from deletion, substitution, insertion and addition, and encoding a protein having Δ12 desaturase activity (iii ′) nucleotide numbers 52 to 1377 of SEQ ID NO: 5 And a nucleotide sequence having an identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more, and Δ12 unsaturated A polynucleotide encoding a protein having oxidase activity (iv ' ) A polynucleotide that hybridizes with the nucleotide sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 under a stringent condition and encodes a protein having Δ12 desaturase activity

Preferable examples of the gene encoding 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 nucleotide sequences represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 A polynucleotide comprising a nucleotide sequence mutated selected from nucleotide deletions, substitutions, insertions and additions and which encodes a protein having Δ17 desaturase activity (iii ″), the 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 Polynucleoside encoding a protein with Δ17 desaturase activity Chide (iv ″) A polynucleotide that hybridizes with the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8 under a stringent condition 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 Introduced to M. 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., but the introduced gene can be expressed in the parental microorganism. If it is a vector, it will not be limited to these.

  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 the PP3 and SSA2 promoters from M. alpina, and promoters modified by adding substitutions, deletions, additions, etc. to the sequences of these promoters. The present invention is not limited to these as long as the expressed gene can be highly expressed. 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, a uracil auxotrophic mutant of M. 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. Multiple 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 this vector, a Δ17 desaturase, Δ15 desaturase or Δ12 downstream of the PP3 promoter or SSA2 promoter which is a constitutive high expression promoter.
A polynucleotide encoding desaturase is linked, and sdhB terminator is incorporated as a terminator, and ura5 gene is incorporated as a selectable marker for transformants.

  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 transfer method when a Mortierella genus microorganism is used as a parental microorganism, an ATMT method (Appl. Environ. Microbiol., 2009, vol. 75, p. .5529-5535) can be preferably exemplified, or a modification method of the ATMT method can be exemplified. 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 desaturase described above may be introduced directly 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.

Through 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 against 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, lipids containing a high amount of EPA produced by the mutant microorganism of the present invention have a reduced accumulated amount of oleic acid and arachidonic acid. Therefore, by purifying the produced lipid, high purity EPA can be efficiently obtained. It becomes possible to produce.

  Accordingly, a further embodiment of the present invention is a method for producing lipids 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 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 can be 0.01 to 10% by mass, preferably 0.1 to 10% by mass in the medium. . When culturing M. alpina or a mutant strain thereof, the following Czapek medium, Czapek-dox medium, glucose / yeast extract (hereinafter also referred to as “GY”) medium, SC medium, or the like 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 mutant microorganism of the present invention is cultured at an optimal 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., still more preferably about 20 to 40 ° C., still more preferably about 20 to 30 ° C. It can be cultured. For example, M. alpina or a mutant strain 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 to 40 ° C., more preferably about 20 to 30 ° C.

  The culture period is 2 to 20 days, preferably 2 to 14 days. Known methods for culturing Mortierella microorganisms can also be referred to (for example, JP-A-6-153970).

  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 at 20 ° C. or higher, the EPA content in the 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 compositing substances are contained in addition to the target EPA. Therefore, the lipid can be further purified to obtain EPA with higher purity. EPA can be separated directly from lipids, but it is preferable to once convert the fatty acid in the lipid to an ester derivative with a lower alcohol, and then separate the desired EPA ester derivative. 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, and the like, an ester derivative of the target fatty acid can be easily obtained. However, since arachidonic acid having the same carbon number and different double bond number as 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 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. From the obtained ester derivative, the target EPA ester derivative can be separated 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 salt form.

  When producing EPA-rich lipids according to the present invention 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 recovered 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.

  The EPA obtained in 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 said pharmaceutical or cosmetics contain EPA or its salt 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 medicine or cosmetic can be produced by blending the carrier or other active ingredient with EPA or a salt thereof according to the dosage form and preparing it according to a conventional method. Although content of EPA in the said pharmaceutical or cosmetics changes with the dosage forms, it is 0.1-99 mass% normally, Preferably it is the range of 1-80 mass%.

  The said food-drinks or feed contains EPA or its salt 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 food / drink or feed is not particularly limited, and includes all forms in which EPA or a salt 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, purified water beverages, 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 food or drink or EPA includes EPA or a salt thereof, and other food or drink materials used for the production of food or drink, feed, various nutrients, various vitamins, minerals, amino acids, various fats and oils, various additives (for example, taste ingredients , Sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, dyes, flavors, etc., and the like, and can be produced according to conventional methods. Or the food-drinks or feed which concerns on this invention can be manufactured by mix | blending EPA or its salt with the food-drinks or feed normally eaten. Although content of EPA or its salt in the said food / beverage products or feed changes with forms of foodstuff, it is 0.01-80 mass% normally, Preferably it is 0.1-50 mass%, More preferably, it is 1-30 mass. % Range.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

(Reagent and medium)
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% KC1, 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 aceto Siringon, 40 mM 2- (N-morpholino) ethanesulfonic acid (MES) added to adjust 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.Omg / L phenylalanine), cefotaxime, spectinomycin, Nile blue A (Sigma )It was used. Restriction enzymes, ligation enzymes, etc. were manufactured by Takara Bio or New England BioLabs.

(Reference Example 1) Codon optimization of Δ15 desaturase gene (DES2m; derived from Trichoderma sp. AM076) Δ15 desaturase gene (DE) derived from Trichoderma sp. AM076
The codon of S2m; SEQ ID NO: 1) was optimized with reference to the codon usage frequency of M. alpina (www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=64518). 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) The codon of 4) was optimized based on the codon usage of M. alpina, 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: 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 Saprolegnia diclina Were co-optimized based on the codon usage of M. alpina, 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: 8. The gene was cloned into the SpMA-RQ (ampR) plasmid.

(Reference Examples 4 to 8) Construction of Binary Vector for Gene Introduction Treated with plasmids prepared in Reference Examples 1 to 3 with SpeI and BamHI restriction enzymes, and obtained Δ17 desaturase gene and Δ15 desaturase The gene and a fragment of the Δ12 desaturase gene were modified from plasmid pBIG35 containing PP3 promoter or SSA2 promoter, which are constitutively high expression promoters (pBIG2RHPH2 provided by Kyoto Prefectural University, Appl. Environ. Microbiol., 2009, vol.75, p.5529-5535) to construct an expression cassette. The expression cassette is further linked in tandem with a marker gene (ura5) requiring uracil, and a binary vector for transformation, pBIG35SSA2pΔ17m (Δ17 desaturase gene:
Reference Example 4), pBIG35PP3pDES2m (Δ15 desaturase gene: Reference Example 5), pBIG35PP3pCop
Δ12m (Δ12 desaturase gene: Reference Example 6), pBIG35PP3pCopΔ12mSSA2pΔ17m (Δ12
Desaturase gene and Δ17 desaturase gene: Reference Example 7), and pBIG35PP3pCop
Δ12mSSA2pDES2m (Δ12 desaturase gene and Δ15 desaturase gene: Reference Example 8)
Built. The structures of the vectors of Reference Examples 4 to 8 are shown in FIGS.

(Production Example 1) Production 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 in Reference Example 4 The binary vector pBIG35SSA2pΔ17m for introducing an enzyme gene was introduced into M. alpina 1S-4 (uracil auxotrophic strain) which is a host strain by the ATMT method via Agrobacterium according to the following procedure.
A culture obtained by culturing M. alpina 1S-4 strain (uracil auxotrophic strain) on 0.05 mg / mL uracil-containing Czapek-Dox agar medium was collected and filtered with Miracloth (Calbiochem). A spore suspension of alpina was freshly prepared.
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 was subjected to 28 ° C. on LB-Mg agar medium. And cultured 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 induced and cultured on a rotary shaker for 8-12 hours at 28 ° C. until the OD ₆₆₀ was 0.4 to 3.7.
100 μL of the above Agrobacterium suspension was mixed with an equal amount of the above M. alpina suspension (10 ⁸ mL ⁻¹ ), and a nitrocellulose membrane (70 mm diameter; hardened low-ash grade 50, Whatman) It was applied on the coculture medium (same composition as IM, but containing 5 mM glucose and 1.5% agar instead of 10 mM glucose) 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 uracil-free SC agar but cannot grow on GY agar 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 Δ15 desaturase gene introduction 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 manner as in Production Example 1, except that the binary vector pBIG35PP3pCopΔ12m for reference example 12 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. Δ12
A desaturase gene and a Δ17 desaturase gene-introduced strain were prepared.

(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 introduction of Δ15 desaturase gene were used. Δ12
A desaturase gene and a Δ15 desaturase gene-introduced strain were prepared.

  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 1-5 were each in 10 mL GY medium (2% glucose, 1% yeast extract) at 28 ° C for 1 week, 300 rpm. Aerobically cultured. From each culture solution, the introduced M. alpina cells were collected by suction filtration and dried at 120 ° C. for 3 hours. To the dried cells, add 1 ml of a dichloromethane solution containing an internal standard (saturated fatty acid having 23 carbon atoms that cannot be biosynthesized by M. alpina) at a concentration of 0.5 mg / ml, and 2 ml of methanolic hydrochloric acid. 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 (0.25mm × 60m), column temperature 180 ° C, vaporization chamber temperature 250 ° C, detector temperature 250 ° C, carrier gas He, makeup Gas N ₂ , H ₂ flow rate 40 mL / min, Air flow rate 400 mL / min, split ratio 50, 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. The Δ15 desaturase gene-introduced strain of Production Example 2 also reduced arachidonic acid accumulation and 48% per total fatty acid
The accumulation of EPA was seen. 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 introduction strain of Δ12 desaturase and Δ17 desaturase in Production Example 4, in addition to arachidonic acid, oleic acid was reduced, and more than 50% of EPA accumulated per total EPA fatty acid. It was seen. In addition, the Δ12 desaturase and Δ15 desaturase gene-introduced strains of Production Example 5 also showed a decrease in oleic acid and arachidonic acid, and an accumulation of EPA of 40% or more per EPA total fatty acid. It was.

(Test Example 2) Time-dependent changes in fatty acid composition and production in desaturase gene-introduced strains (Δ17 desaturase gene-introduced strain)
Δ17 desaturase gene-introduced M. alpina mutants obtained in Production Example 1 (# 1, # 2, # 4
, # 6, # 9, # 19, # 21 and # 24) in 4 mL GY medium (2% glucose, 1% yeast extract), aerobic at 28 rpm for 3, 7, 10, 14 days at 120 rpm The fatty acid composition and production amount in the cells were analyzed over time in 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 any EPA under these culture conditions However, all of 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 almost on day 10 of culture, and on day 14 of culture, a maximum of 47% (0.9 mg / mL) of EPA was produced per total fatty acid, while accumulation of arachidonic acid 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. alpina mutants obtained in Production Example 2 (# 2, # 3, # 9
, # 13, # 14, and # 20) in 4 mL GY medium (2% glucose, 1% yeast extract), aerobically cultured at 120 rpm for 3, 7, 10, 14 days at 28 ° C. and tested In the same procedure as in Example 1, the composition and production amount of fatty acids in the cells were analyzed over time. 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 percentage of EPA in total fatty acids is almost the maximum 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. alpina mutants obtained in Production Example 3 (# 1, # 2, # 7
, # 22, # 23, and # 24) in 4 mL GY medium (2% glucose, 1% yeast extract), aerobically cultured at 28 rpm for 3, 7, 10, 14 days at 120 rpm, tested In the same procedure as in Example 1, the composition and production amount of fatty acids in the cells were analyzed over time. The results are shown in FIG. In the Δ12 desaturase gene-introduced strain, EPA production 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 fatty acid composition increased, while the amount of oleic acid accumulated in the control strain decreased. For example, in strain # 7, the lipid production increased by 10% or more compared to the host strain, and the ratio of arachidonic acid in the 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% for the wild type and 12% for the control strain on the 7th day of culture, whereas it was 6% or less on the 7th day of culture for 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. alpina mutant strains obtained in Production Example 4 (# 6, # 11
, # 16, # 17, # 21, # 23, # 25, # 27, # 33, # 37, # 40, # 45, # 46, and # 48) in 4 mL GY medium (2% glucose, 1 % Yeast extract), aerobically cultured at 120 rpm for 4-10 days at 28 ° C., 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 strains into which the Δ12 desaturase gene and Δ17 desaturase gene were introduced, both EPA production increased 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, suggesting that EPA was being produced efficiently. On the 14th day of cultivation, 51% (0.3 mg / mL) of EPA was produced 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. alpina mutant strains obtained in Production Example 5 (# 4, # 7
, # 8, # 11, and # 16) were cultured in 4 mL GY medium (2% glucose, 1% yeast extract) aerobically at 28 rpm for 4, 10, 14 days at 120 rpm. Test Example 1 In the same procedure as above, the composition and production amount of fatty acids in the cells were analyzed over time. The results are shown in FIG. In the strains into which the Δ12 desaturase gene and Δ15 desaturase gene were introduced, both were control strains (
EPA production is improved compared to uracil-requiring strains with a vector containing only the marker gene), and both arachidonic acid and oleic acid are reduced, and EPA is considered to be produced efficiently. It was. On the 14th day of culturing, a maximum of 43% (0.13 mg / mL) EPA was produced per total fatty acid, and arachidonic acid accumulation was reduced to 12% and oleic acid accumulation to 5% (strain # 7).

Claims (14)

A mutant microorganism in which an exogenous Δ15 desaturase gene is introduced into a microorganism having a ω6 polyunsaturated fatty acid metabolic pathway, and the eicosapentaenoic acid content in the fatty acid composition after culturing for 10 days at 20 ° C. or higher A mutant microorganism that is 20% by mass or more. To microorganisms having ω6 highly unsaturated fatty acid metabolic pathway, exogenous Δ12 desaturase gene,
A mutant microorganism into which any one or more of an exogenous Δ15 desaturase gene and an exogenous Δ17 desaturase gene has been introduced, and the fatty acid composition in the fatty acid composition after culturing for 10 days at 20 ° C. or higher. A mutant microorganism having an icosapentaenoic acid content of 20% by mass or more.   The mutant microorganism according to claim 1 or 2, wherein the microorganism is a microorganism belonging to the genus Mortierella.   The mutant microorganism according to any one of claims 1 to 3, wherein the microorganism is Mortierella alpina. The mutant microorganism according to any one of claims 1 to 4, wherein the Δ15 desaturase gene is derived from Trichoderma sp. AM076. The Δ15 desaturase gene is a gene encoding the following protein:
The mutant microorganism according to any one of the above:
(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 mutated 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) is 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 2 to 6, wherein the Δ12 desaturase gene is derived from Coprinus cinereus. The Δ12 desaturase gene is a gene encoding the following protein:
The mutant microorganism according to any one of the above:
(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 Δ
A protein having 12 desaturase activity;
(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 2 to 8, wherein the Δ17 desaturase gene is derived from Saprolegnia diclina. The mutant microorganism according to any one of claims 2 to 8, 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 Δ
17 protein having desaturase 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 ″) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the complementary sequence of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of SEQ ID NO: 8, and Δ17 desaturase activity Or a protein having
(E ″) a protein encoded by a polynucleotide comprising a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8, and having Δ17 desaturase activity.   A method for producing a lipid containing eicosapentaenoic acid, comprising culturing the mutant microorganism according to any one of claims 1 to 10 under a condition of 20 ° C or higher.   A method for producing eicosapentaenoic acid, comprising purifying a lipid containing eicosapentaenoic acid produced by the method according to claim 11.   A medicament comprising eicosapentaenoic acid produced by the method of claim 12.   The food-drinks or feed containing the eicosapentaenoic acid produced by the method of Claim 12.