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WO2016031947A1 - Procédé de production de lipides à haute teneur en acide eicosapentaénoïque - Google Patents

Procédé de production de lipides à haute teneur en acide eicosapentaénoïque Download PDF

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WO2016031947A1
WO2016031947A1 PCT/JP2015/074346 JP2015074346W WO2016031947A1 WO 2016031947 A1 WO2016031947 A1 WO 2016031947A1 JP 2015074346 W JP2015074346 W JP 2015074346W WO 2016031947 A1 WO2016031947 A1 WO 2016031947A1
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desaturase
seq
protein
gene
nucleotide sequence
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PCT/JP2015/074346
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Japanese (ja)
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順 小川
晃規 安藤
英治 櫻谷
昌 清水
茂 平本
昌卓 原田
有貴 竹本
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国立大学法人京都大学
日清ファルマ株式会社
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Publication of WO2016031947A1 publication Critical patent/WO2016031947A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a method for producing a lipid containing a high content of eicosapentaenoic acid using a lipid-producing microorganism. Specifically, eicosapentaenoic acid is increased by using a microorganism having the ability to produce ⁇ 9 polyunsaturated fatty acids or a mutant microorganism in which a fatty acid desaturase gene is introduced into a microorganism having a high content of arachidonic acid in the fatty acid composition.
  • the present invention relates to a method for producing a contained lipid.
  • Polyunsaturated fatty acids are fatty acids having two or more unsaturated bonds, and are ⁇ 6 fatty acids (also referred to as n-6 fatty acids) linoleic acid (LA, 18: 2n-6), ⁇ -linolenic acid (GLA, 18: 3n-6), arachidonic acid (ARA, 20: 4n-6), ⁇ -linolenic acid (ALA, 18: 3n-3) of ⁇ 3 fatty acid (also referred to as n-3 fatty acid), eicosapentaenoic acid ( EPA, 20: 5n-3), docosahexaenoic acid (DHA, 22: 6n-3), and the like.
  • ⁇ 6 fatty acids also referred to as n-6 fatty acids
  • LA linoleic acid
  • GLA ⁇ -linolenic acid
  • ARA arachidonic acid
  • ALA ⁇ -linolenic acid
  • ALA 18: 3n-3
  • EPA
  • 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.
  • 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.
  • active intake of ⁇ 3-fatty acids has been recommended, and this is a lipid molecular species whose demand has been remarkably expanding.
  • DHA and EPA are biosynthesized from ALA in some organisms in addition to being taken from food.
  • 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.
  • 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.
  • 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 ⁇ 3 and ⁇ 6 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.
  • 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 microorganism having a capability of producing an ⁇ 9 highly unsaturated fatty acid, in which Mortierella alpina (also referred to as Mortierella alpina, also referred to as M. alpina) is mutated. ing.
  • M.I A method for producing highly unsaturated fatty acids such as EPA using a transformed strain obtained by introducing a gene of ⁇ 3 fatty acid desaturated polypeptide isolated from alpina into yeast is disclosed.
  • 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.
  • M.M. Since the alpina-derived ⁇ 3 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 inventors of the present invention have introduced a ⁇ 17 desaturase gene, a ⁇ 5 desaturase gene, and a ⁇ 12 to a microorganism having the ability to produce ⁇ 9 highly unsaturated fatty acid, or a microorganism containing a high content of arachidonic acid in the fatty acid composition. It has been found that a mutant microorganism prepared by introducing any one or more selected from desaturase genes can efficiently produce lipids containing EPA at a high concentration even at room temperature. . Furthermore, the present inventors have found that by reducing the saturated fatty acid synthase activity of the mutant microorganism, the EPA concentration in the lipid produced in the mutant microorganism is further improved.
  • the EPA-containing lipids obtained from the above-mentioned mutant microorganisms are useful because the amount of highly unsaturated fatty acids other than EPA such as arachidonic acid is reduced and EPA can be efficiently purified using this as a raw material. is there.
  • the present invention provides an exogenous ⁇ 17 desaturase gene and an exogenous ⁇ 5 desaturase gene to microorganisms having the ability to produce ⁇ 9 polyunsaturated fatty acids or to microorganisms containing a high content of arachidonic acid in the fatty acid composition. And a mutant microorganism into which any one or more selected from the exogenous ⁇ 12 desaturase gene has been introduced, and the eicosapentaenoic acid content in the fatty acid composition after 20 days of cultivation at 20 ° C. or higher is 20% Provided is a mutant microorganism as described above.
  • 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
  • an exogenous ⁇ 17 desaturase gene working at normal temperature and an exogenous ⁇ 5 desaturase gene or an exogenous ⁇ 12 desaturase gene are introduced, and the microorganism is easy to grow.
  • an exogenous ⁇ 17 desaturase gene working at normal temperature and an exogenous ⁇ 5 desaturase gene or an exogenous ⁇ 12 desaturase gene are introduced, and the microorganism is easy to grow.
  • mutant microorganisms expressing at least both the ⁇ 17 desaturase gene and the ⁇ 5 desaturase gene eicosatetraenoic acid (ETA, 20: 4n-3) and dihomo- ⁇ -linolenic acid (DGLA) , 20: 3n-6) is suppressed, which enables production of high-purity EPA-containing lipids. 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 the production of EPA on an industrial scale is extremely useful.
  • 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.
  • “addition” of amino acids or nucleotides includes addition of one or more amino acids or nucleotides to one and both ends of the sequence.
  • 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. It means a condition in which nucleotide sequences (that is, complementary strands of one sequence and the other sequence) hybridize and nucleotide sequences having lower identity do not hybridize.
  • 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. .
  • PUFA polyunsaturated fatty acid
  • ⁇ 6 polyunsaturated fatty acid metabolic pathway refers to linoleic acid (LA, 18: 2n-6), ⁇ -linolenic acid (GLA, 18: 3n-6), dihomo- ⁇ -linolene.
  • DGLA ⁇ 6 polyunsaturated fatty acids leading to acid
  • ARA arachidonic acid
  • microorganism having the ability to produce ⁇ 9 polyunsaturated fatty acids means that the ⁇ 9 polyunsaturated fatty acid metabolism pathway is possessed, and the above-mentioned ⁇ 9 series in the total mass of fatty acids produced by the microorganisms. Any one of the microorganisms in which the content of any of the highly unsaturated fatty acids is improved as compared with the wild type strain of the microorganisms, or any of the above-mentioned ⁇ 9 highly unsaturated fatty acids in the total mass of fatty acids produced by the microorganisms A microorganism having a high content, preferably 5% by mass or more, more preferably 10% by mass or more.
  • the “microorganism having the ability to produce an ⁇ 9 polyunsaturated fatty acid” is a microorganism having an ability to produce mead acid and containing 10% by mass or more of mead acid in the total mass of the fatty acid.
  • microorganism containing a high content of arachidonic acid in the fatty acid composition means that the content of arachidonic acid in the total mass of fatty acids produced by the microorganism is improved compared to the wild strain of the microorganism. Or a microorganism having a high content of arachidonic acid in the total mass of fatty acids produced by the microorganism, preferably 30% by mass or more, more preferably 50% by mass or more.
  • the term “original” used for a certain microorganism is used to indicate that a certain function or character is possessed by the microorganism (wild type) that exists in nature.
  • the term “foreign” is used to denote a function or trait introduced from the outside, rather than originally present in the microorganism.
  • 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 is a microorganism that has the ability to produce ⁇ 9 polyunsaturated fatty acids or a microorganism that contains arachidonic acid in a high content in the fatty acid composition. I just need it.
  • the microorganism having the ability to produce ⁇ 9 highly unsaturated fatty acids has a ⁇ 9 highly unsaturated fatty acid metabolic pathway and a ⁇ 6 highly unsaturated fatty acid metabolic pathway, and the ⁇ 9 highly unsaturated fatty acid metabolic pathway is predominant. It is a production microbe (oleaginus microorganisms).
  • the ⁇ 9 polyunsaturated fatty acid metabolic pathway is dominant means that the progression of the ⁇ 6 polyunsaturated fatty acid metabolic pathway is stagnant as a result of the inhibition or cessation of the pathway from oleic acid to linoleic acid. The state in which the ⁇ 9 polyunsaturated fatty acid metabolic pathway is activated.
  • microorganisms capable of producing ⁇ 9 polyunsaturated fatty acids that can be used as parent microorganisms of the mutant microorganisms of the present invention or microorganisms containing a high content of arachidonic acid in the fatty acid composition include natural microorganisms having such properties, And microorganisms produced by artificial mutation treatment.
  • Microorganisms produced by artificial mutagenesis include mutations that improve the ability to produce ⁇ 9 polyunsaturated fatty acids or accumulation of arachidonic acid in cells relative to microorganisms that can be obtained from nature or from institutions. Examples include, but are not limited to, microorganisms that have been subjected to a mutation treatment so as to bring about mutations to be promoted.
  • microorganisms that can be subjected to the mutation treatment include, for example, the genus Mortierella, the genus Conidiobolus, the genus Phythium, the genus Phytophthora, the genus Penicillium, and Cladosporium. (Cladosporium), Mucor, Umbelopsis, Fusarium, Aspergillus, Rhodotorulae, Entomophra Microorganisms such as (Saprolegnia) genus Including but not limited to. Examples of the parent microorganism of the mutant microorganism of the present invention include M.I.
  • Examples include microorganisms having the ability to produce ⁇ 9 polyunsaturated fatty acids obtained by mutating alpina (for example, M. alpina SAM1861 (FERM BP-3590) and M. alpina SAM2086 (FERM BP-6032)).
  • Other examples of parental microorganisms include M. alpina 1S-4 (Agric. Bioi. Chem., 1987, 51 (3): 785-790), which has a mead acid production capacity of 5% by mass or more, preferably 10% by mass compared to that before mutation. %, Or an arachidonic acid-producing ability improved by 30% by mass or more, preferably 50% by mass or more, compared with that before mutation. alpina 1S-4 mutant.
  • Examples of the mutation treatment to the microorganisms listed above include conventional methods such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MMNG) (J Gen. Microbiol., 1992, 138: 997-1002), treatment with mutagen such as 5-bromodeoxyuridine (BrdU), cisplatin, mitomycin C, or RNAi (Appl. Environ. Microbiol., 2005, 71: 5124-). 5128).
  • EMS ethyl methanesulfonate
  • MMS methyl methanesulfonate
  • MMNG N-methyl-N-nitro-N-nitrosoguanidine
  • RNAi Appl. Environ. Microbiol., 2005, 71: 5124-). 5128.
  • a mutation for obtaining a microorganism having the ability to produce an ⁇ 9 polyunsaturated fatty acid a mutation that reduces or eliminates the ⁇ 12 desaturase activity is a preferred example.
  • a mutation for obtaining a microorganism containing a high content of arachidonic acid in the fatty acid composition a mutation that reduces the function of ⁇ 3 desaturase can be exemplified.
  • the present invention is not limited to these mutations as long as microorganisms having desired properties can be obtained as parent microorganisms of the mutant microorganisms of the present invention. In the mutation treatment, mutant microorganisms having various properties are generally obtained.
  • microorganisms having the ability to produce ⁇ 9 polyunsaturated fatty acids or microorganisms containing a high content of arachidonic acid in the fatty acid composition can be selected using the composition of the fatty acid produced as an index.
  • microorganisms may be selected using the production of ⁇ 9 polyunsaturated fatty acids such as n-9 octadienoic acid, n-9 eicodienoic acid, and mead acid as an index, preferably ⁇ -linolenic.
  • Microorganisms may be selected based on the fact that they do not produce ⁇ 6 highly unsaturated fatty acids such as acids, dihomo- ⁇ -linolenic acid, arachidonic acid or the like, and that the production amount is less than that of ⁇ 9 highly unsaturated fatty acids.
  • a microorganism may be selected using arachidonic acid production as an index. Further, the microorganism selected according to the above index is subjected to one or more mutation treatments again, and a microorganism having a higher production amount of ⁇ 9 polyunsaturated fatty acid or a microorganism containing a higher content of arachidonic acid is selected. May be.
  • the mutant microorganism of the present invention comprises a gene encoding an exogenous ⁇ 17 desaturase, a gene encoding an exogenous ⁇ 5 desaturase, and an exogenous ⁇ 12 desaturase. It is produced by introducing any one or more of the encoding genes.
  • the ⁇ 17 desaturase, ⁇ 5 desaturase and ⁇ 12 desaturase encoded by the introduced gene each exhibit an activity of introducing an unsaturated bond into a highly unsaturated fatty acid alone or in combination. As a result of the action of this activity and the fatty acid metabolic pathway derived from the parent microorganism, the biosynthetic amount of EPA in the mutant microorganism can be increased.
  • the mutant microorganism of the present invention comprises a gene encoding an exogenous ⁇ 17 desaturase, a gene encoding an exogenous ⁇ 5 desaturase, and an exogenous ⁇ 12 desaturation relative to the parental microorganism.
  • it is produced by reducing its saturated fatty acid synthase activity.
  • “reducing saturated fatty acid synthase activity” includes reducing or inhibiting the expression of saturated fatty acid synthase, reducing or inhibiting the enzymatic activity of saturated fatty acid synthase, and combinations thereof.
  • saturated fatty acids having 20 or more carbon atoms are not included in the ⁇ 9 polyunsaturated fatty acid pathway or the ⁇ 6 polyunsaturated fatty acid pathway, they are not always necessary when increasing the production of EPA. Therefore, by reducing the activity of a saturated fatty acid synthase that catalyzes a reaction that extends the carbon chain length of saturated fatty acid (for example, C18 ⁇ C20), the production efficiency of EPA is further improved and the effect of the present invention is further enhanced. be able to.
  • FIG. alpina 1S-4 fatty acid biosynthetic pathway.
  • the ⁇ 9 highly unsaturated fatty acid pathway (route surrounded by a dotted line in the figure) hardly progresses, and the ⁇ 6 highly unsaturated fatty acid pathway advances.
  • the present inventors performed mutation treatment on the 1S-4 strain to produce a microorganism having the ability to produce ⁇ 9 polyunsaturated fatty acids or a microorganism containing a high content of arachidonic acid in the fatty acid composition, which was used as the parent microorganism.
  • a mutant microorganism capable of efficiently producing EPA even under normal temperature conditions of 20 ° C. or higher can be obtained by performing the above-described gene transfer operation.
  • ⁇ 5 desaturase is a protein exhibiting ⁇ 5 desaturase activity.
  • the ⁇ 5 desaturase activity refers to an activity of desaturating a fatty acid between the fifth and sixth carbon atoms counted from the carboxyl end of the fatty acid molecule.
  • ⁇ 5 desaturase activity can include dihomo- ⁇ -linolenic acid to arachidonic acid conversion activity, eicosatetraenoic acid to eicosapentaenoic acid conversion activity.
  • the ⁇ 5 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme that preferentially exhibits ⁇ 5 desaturase activity with respect to a highly unsaturated fatty acid having a carbon chain length of 20. .
  • ⁇ 12 desaturase is a protein exhibiting ⁇ 12 desaturase activity.
  • the ⁇ 12 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 ⁇ 6 position is desaturated when viewed from the methyl end, so that the function of the ⁇ 6 desaturase can be substituted.
  • ⁇ 12 desaturase activity can include conversion activity from oleic acid to linoleic acid.
  • the ⁇ 12 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme that preferentially exhibits ⁇ 12 desaturase activity with respect to a highly unsaturated fatty acid having a carbon chain length of 18. .
  • ⁇ 17 desaturase is a protein exhibiting ⁇ 17 desaturase activity.
  • the ⁇ 17 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 ⁇ 3 position is desaturated when viewed from the methyl end, so that the function of the ⁇ 3 desaturase can be substituted.
  • ⁇ 17 desaturase activity can include arachidonic acid to eicosapentaenoic acid conversion activity, dihomo- ⁇ -linolenic acid to eicosatetraenoic acid conversion activity.
  • the ⁇ 17 desaturase encoded by the gene introduced into the mutant microorganism of the present invention is an enzyme exhibiting ⁇ 17 desaturase activity that acts preferentially on a highly unsaturated fatty acid having a carbon chain length of 20. It is.
  • the ⁇ 17 desaturase, ⁇ 5 desaturase and ⁇ 12 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.
  • “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 ⁇ 5 desaturase encoded by the gene to be introduced into the mutant microorganism of the present invention include, for example, haptoalgae (for example, Isochrysis galbana), japonicum (Thraustochytrium aureum), Trypanosomaaceae Leishmania major Origin, from the genus Plasinobacterium Ostreococcus (for example, Ostreococcus tauri, Ostreococcus lucimarineus), derived from Paramecium tetraurelia, and from Pavlova (for example, Pavlova salina or Rebecca salinase).
  • the amino acid sequences of these enzymes are known (for example, Japanese translation of PCT publication No.
  • the ⁇ 5 desaturase used in the present invention is a ⁇ 5 desaturase derived from Pavlova salina consisting of the amino acid sequence represented by SEQ ID NO: 3.
  • ⁇ 5 desaturase used in the present invention include the following.
  • (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.
  • Examples of the ⁇ 12 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include, for example, Coprinus cinereus-derived ⁇ 12 desaturase.
  • the amino acid sequence of this enzyme is known (for example, FEBS Lett., 2007, 581: 315-319) and is represented by SEQ ID NO: 6.
  • ⁇ 12 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 90% or more, preferably 95% or more, more preferably 98% or more
  • a poly (compound) comprising an amino acid sequence having an identity of 99% or more and a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 52 to 1377 of protein
  • d ′ SEQ ID NO: 5 having ⁇ 12 desaturase activity Encoded by a polynucleotide that hybridizes under stringent conditions with a nucleotide and ⁇ 12 desaturation fermentation 80% or more
  • Examples of the ⁇ 17 desaturase encoded by the gene introduced into the mutant microorganism of the present invention include ⁇ 17 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).
  • the ⁇ 17 desaturase used in the present invention is a ⁇ 17 desaturase derived from Saprolegnia diclina having the amino acid sequence represented by SEQ ID NO: 9.
  • ⁇ 17 desaturase used in the present invention include the following.
  • a poly (compound) comprising an amino acid sequence having an identity of 99% or more and a complementary strand sequence of the nucleotide sequence shown in nucleotide numbers 100 to 1176 of protein (d ′′) SEQ ID NO: 8 having ⁇ 17 desaturase activity Encoded by a polynucleotide that hybridizes under stringent conditions with nucleotides and 80% or more, preferably 90% or more
  • 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; (B5) A combination of (b1) to (b4) above, wherein the number of amino acids added, deleted, substituted, inserted and added is one or more in total.
  • the position of substitution and insertion is not particularly limited as long as the above-mentioned desaturase activity is retained in the mutated protein.
  • the desaturase used in the present invention is the protein represented by the above (a) to (e), (a ′) to (e ′), or (a ′′) to (e ′′), Substitutions between amino acids of similar nature (eg, 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) are further made. Protein.
  • 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 ′′).
  • 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.
  • 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.
  • a desaturase gene having a high unsaturated bond introduction activity under normal temperature conditions a gene encoding a ⁇ 12 desaturase having a high substrate specificity for a highly unsaturated fatty acid having a carbon chain length of 18, or a carbon chain length
  • a gene encoding ⁇ 5 desaturase or ⁇ 17 desaturase having high substrate specificity for 20 polyunsaturated fatty acids can be selected and used in the present invention.
  • Examples of the gene encoding the ⁇ 5 desaturase introduced into the mutant microorganism of the present invention include a gene encoding a ⁇ 5 desaturase derived from Pavlova salina consisting of the nucleotide sequence represented by SEQ ID NO: 1.
  • Examples of the gene encoding the ⁇ 12 desaturase introduced into the mutant microorganism of the present invention include the above-described gene encoding the ⁇ 12 desaturase derived from Coprinus cinereus comprising the nucleotide sequence represented by SEQ ID NO: 4.
  • Examples of the gene encoding the ⁇ 17 desaturase introduced into the mutant microorganism of the present invention include the gene encoding the ⁇ 17 desaturase derived from the genus Saprolegnia or the genus Phytophthora. Preferably, it is a gene encoding a ⁇ 17 desaturase derived from Saprolegnia diclina consisting of the nucleotide sequence represented by SEQ ID NO: 7.
  • genes encoding the ⁇ 5 desaturase, ⁇ 12 desaturase or ⁇ 17 desaturase listed above are optimized for codon usage in accordance with 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 Codon Usage Database (www.kazusa.or.jp/codon/).
  • the nucleotide sequence of the gene can be modified with reference to to optimize the codon.
  • the ⁇ 5 desaturase gene represented by SEQ ID NO: 1 is M.P.
  • a polynucleotide having the sequence represented by nucleotide numbers 7 to 1285 of SEQ ID NO: 2 can be obtained.
  • the ⁇ 12 desaturase gene represented by SEQ ID NO: 4 was designated as M. pylori.
  • a polynucleotide having the sequence represented by nucleotide numbers 52 to 1377 of SEQ ID NO: 5 can be obtained.
  • the ⁇ 17 desaturase gene represented by SEQ ID NO: 7 was designated as M. pylori.
  • a polynucleotide having the sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 can be obtained.
  • preferred examples of the gene encoding the ⁇ 5 desaturase used in the present invention include the following.
  • a polynucleotide comprising a mutated nucleotide sequence selected from substitution, insertion and addition, and encoding a protein having ⁇ 5 desaturase activity (iii) represented by nucleotide numbers 7 to 1285 of SEQ ID NO: 2 It comprises 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 to the nucleotide sequence, and ⁇ 5 desaturase activity
  • Preferable examples of the gene encoding ⁇ 12 desaturase used in the present invention include the following.
  • Polynucleotide (iii ′) consisting of a nucleotide sequence mutated selected from deletion, substitution, insertion and addition, and encoding a protein having ⁇ 12 desaturase activity nucleotide numbers 52 to 1377 of SEQ ID NO: 5
  • Preferable examples of the gene encoding ⁇ 17 desaturase used in the present invention include the following.
  • (I ′′) a polynucleotide consisting of the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8 (ii ′′) in the nucleotide sequence represented by nucleotide numbers 100 to 1176 of SEQ ID NO: 8,
  • Polynucleotide (iii ′′) consisting of a nucleotide sequence mutated selected from deletion, substitution, insertion and addition, and encoding a protein having ⁇ 17 desaturase activity, nucleotide numbers 100 to 1176 of SEQ ID NO: 8
  • Polyn encoding a protein having synthase activity A polynucleotide that hybridizes under stringent conditions with the nucleotide sequence shown in nucleotide numbers 100 to 1176 of nucleotide (iv ′′) SEQ ID NO: 8 and that encodes a protein having ⁇ 17 desaturase activity
  • the polynucleotides (i) to (iv), (i ′) to (iv ′) and (i ′′) to (iv ′′) are preferably Mortierella microorganisms, more preferably M. alpina, or M.I. It is introduced into a microorganism produced by subjecting alpina to a mutation treatment. More preferably, the polynucleotides (i) to (iv), (i ′) to (iv ′) and (i ′′) to (iv ′′) are more preferable than those before the mutation exemplified above as the parent microorganism. M. acid with improved ability to produce mead acid or arachidonic acid. Introduced into alpina 1S-4 mutant.
  • codons of the polynucleotides (ii) to (iv), (ii ′) to (iv ′) and (ii ′′) to (iv ′′) are replaced with other codons used in Mortierella microorganisms. It may be done. Table 1 below shows the codons and frequencies of Mortierella microorganisms.
  • the replaced codon is desirably a codon that is frequently used in Mortierella microorganisms, and preferably has a frequency of 5% or more.
  • the present invention instead of the polynucleotides (i) to (iv), (i ′) to (iv ′) and (i ′′) to (iv ′′), at least a part of the codons (for example, 90% or less, preferably 50% or less, more preferably 20% or less, and even more preferably 10% or less), but a polynucleotide in which another codon encoding the same amino acid is substituted according to Table 1 below may be used. Good.
  • reducing the activity of the saturated fatty acid synthase reduces the accumulation of saturated fatty acids in the obtained mutant microorganism.
  • the amount of unsaturated fatty acid accumulated can be improved, and the accumulation of EPA can be improved.
  • saturated fatty acid synthases of microorganisms related to the production of saturated fatty acids are known. For example, M. of Mortierella sp.
  • the saturated fatty acid synthase of alpina 1S-4 strain is a saturated fatty acid chain lengthening enzyme described in Appl Microbiol Biotechnol (2008) 81: 497-503, which catalyzes the synthesis of C20 saturated fatty acid from C18 saturated fatty acid.
  • An enzyme (Mortierella alpina saturated fatty elongase; MAELO) is mentioned.
  • MAELO used in the present invention include the nucleotide sequence represented by SEQ ID NO: 10, or 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more of the nucleotide sequence.
  • RNA interference can be induced for the expression of saturated fatty acid synthase.
  • a partial sequence of the enzyme is designed so as to be complementary to the transcription product of the enzyme, and this is introduced with a constant expression promoter to induce shRNA in the microorganism, or the shRNA is directly introduced into the microorganism. By introducing, the expression of the enzyme is suppressed.
  • RNAi for decreasing the activity of MAELO examples include a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 11.
  • the gene encoding the desaturase used in the present invention can be introduced into the parent microorganism using a vector.
  • the above-described sequence for inducing RNAi (hereinafter also referred to as RNAi-inducing construct) 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.
  • a pD4 vector Appl. Environ.
  • a promoter sequence or transcription termination signal sequence for expressing the incorporated desaturase gene or RNAi-inducing construct, or a transformant introduced with the desaturase gene or RNAi-inducing construct is selected as the vector. It is preferable that a selection marker gene is included.
  • 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: 12), PP3 promoter (SEQ ID NO: 13), HSC promoter (SEQ ID NO: 14), his promoter (SEQ ID NO: 15), and substitution, deletion, addition, etc.
  • modified promoters can be mentioned, but the promoter is not limited thereto as long as the introduced gene can be expressed at a high level.
  • 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.
  • 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, and genes that complement nucleobase-requiring mutations such as uracil and adenine.
  • preferred selectable marker genes include genes that complement uracil-requiring mutations. For example
  • uracil auxotrophic mutant strain of alpina (Biosci Biotechnol Biochem., 2004, 68, p.277-285) has been developed.
  • orotidine-5'-phosphate decarboxylase gene (ura3 gene) or orotidylate pyrophosphorylase gene (ura5 gene) can be used as a selectable marker gene.
  • reagents 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 ⁇ 5 desaturase, a gene encoding a ⁇ 12 desaturase, or a gene encoding a ⁇ 17 desaturase.
  • a plurality of genes for example, ⁇ 5 desaturase gene and ⁇ 17 desaturase gene, ⁇ 12 desaturase gene and ⁇ 17 desaturase gene, or ⁇ 12 desaturase gene and ⁇ 5 desaturation
  • An enzyme gene and a ⁇ 17 desaturase gene may be included.
  • a vector containing any one of the desaturase genes may be introduced into the mutant microorganism of the present invention, or a vector containing a plurality of the desaturase enzymes may be introduced, Alternatively, two or more types of vectors containing different desaturase enzymes may be introduced. Furthermore, a vector containing an RNAi-inducing construct may be introduced into the mutant microorganism of the present invention. The RNAi-inducing construct may be incorporated in the same vector as the vector containing the desaturase described above, or may be incorporated in a vector different from the vector containing the desaturase mentioned above.
  • a ⁇ 17 desaturase, ⁇ 5 desaturase or ⁇ 12 desaturase, or saturated fatty acid chain length is downstream of the constitutive high expression promoter PP3 promoter, SSA2 promoter, his promoter or HSC promoter.
  • a polynucleotide encoding RNA that suppresses the expression of the extended enzyme MAELO (MAELORNAi) is linked, and an sdhB terminator is incorporated as a terminator, and a ura5 gene is incorporated as a selection marker for the transformant.
  • 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.
  • a gene introduction method when a Mortierella genus microorganism is used as a parent microorganism, the AMT-mediated ATMT method (Appl. Environ. Microbiol., 2009, 75: 5529-5535) described in the Examples below. ) can be preferably exemplified, or a modification method of the ATMT method can be exemplified.
  • the gene transfer method is not limited to these methods as long as a transformant that stably retains the target character can be obtained.
  • 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.
  • the mutant microorganism of the present invention can be produced.
  • an exogenous ⁇ 17 desaturase gene and any one or more selected from the exogenous ⁇ 5 desaturase gene and the exogenous ⁇ 12 desaturase gene are introduced into the parent microorganism, and saturated fatty acid synthesis By adding a modification that reduces the activity of the enzyme, the mutant microorganism of the present invention can be produced.
  • the mutant microorganism of the present invention is produced at room temperature by the function of the desaturase encoded by the introduced gene and the ⁇ 9 highly unsaturated fatty acid metabolic pathway of the parent microorganism or the fatty acid metabolic pathway that highly expresses arachidonic acid. However, it exhibits ⁇ 3 desaturase activity against highly unsaturated fatty acids and can exhibit high EPA biosynthesis ability. Furthermore, when saturated fatty acid synthase is suppressed, higher EPA biosynthesis ability can be exhibited. 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.
  • 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.
  • 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.
  • the mutant microorganism of the present invention can be inoculated and cultured in a liquid medium or a solid medium.
  • a spore of a strain, a mycelium, or a preculture solution obtained by culturing in advance can be inoculated into the medium and cultured.
  • 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, organic acid, and various alcohols.
  • 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, 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.
  • the carbon source can be 0.1 to 40% by mass, preferably 1 to 25% by mass
  • the nitrogen source in the medium can be 0.01 to 10% by mass, preferably 0.1 to 10% by mass. . M.M.
  • 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.
  • GY glucose / yeast extract
  • SC medium or the like described later
  • known media for example, International Publication No. 98/29558
  • 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.
  • 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.
  • 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.
  • 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.
  • known literature for example, JP-A-6-153970
  • lipids are externally added to the medium.
  • a method of adding and culturing can also be employed.
  • lipids containing a high content of EPA are produced in the cells of the microorganism.
  • 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.
  • the culture solution is subjected to conventional means such as centrifugation and filtration to separate microbial cells.
  • 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.
  • the organic solvent include hexane, ether, ethyl acetate, butyl acetate, chloroform, cyclohexane, benzene, toluene, xylene, acetone, and other solvents that are highly soluble in highly unsaturated fatty acids and can be separated from water.
  • the target lipid can be extracted by distilling off the organic solvent from the extract under reduced pressure or the like.
  • 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.
  • EPA In the extracted lipid, various fatty acids that are compositing 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, and the like, an ester derivative of the target fatty acid can be easily obtained.
  • the ester derivative is preferably an ethyl ester derivative.
  • a lower alcohol containing an acid catalyst such as hydrochloric acid, sulfuric acid or BF 3 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.
  • 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.
  • 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.
  • the pharmaceutical dosage form include oral preparations such as tablets, capsules, granules, powders, syrups, dry syrups, liquids and suspensions; enteral preparations such as suppositories; instillations; injections; External preparations; 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 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
  • the content of EPA in the medicine or cosmetic 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 food or drink or feed contains EPA or a salt 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 health foods, functional foods and drinks, foods and drinks for specified health use, foods and drinks for sick people, livestock, aquaculture, racehorses, feed for appreciation animals, pet foods, and the like.
  • the form of the food or drink or feed is not particularly limited, and includes all forms that can contain EPA or a salt thereof.
  • 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.
  • 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, fermente
  • the above-mentioned food or drink or EPA includes EPA or a salt 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, taste ingredients) , Sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants, dyes, flavors, etc.) and the like, and can be prepared according to conventional methods.
  • the food / beverage products or feed based on this invention can be manufactured by mix
  • the content of EPA or a salt thereof in the above 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 30% by mass. % Range.
  • Czapek-Dox agar medium (3% (w / v) sucrose, 0.2% NaNO 3 , 0.1% KH 2 PO 4 , 0.05% KCl, 0.05% MgSO 4 .7H 2 O, 0.
  • ⁇ 17 desaturase gene (derived from Saprolegnia diclina) Similar to Reference Example 1, ⁇ 17 desaturase gene ( ⁇ 17m; 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 5 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 ⁇ 5 desaturase gene and ⁇ 12 desaturase
  • PBIG2RHPH2 provided by the company was modified and ligated to Appl. Environ.
  • the expression cassette is further linked to uracil-required marker gene (ura5) and tandem, and a binary vector for transformation, pBIG35hispPsD5m (for introducing ⁇ 5 desaturase gene: Reference Example 5) pBIG35PP3pCopD12m ( ⁇ 12 desaturase) For gene introduction: Reference Example 6), pBIG35SSA2pD17m ( ⁇ 17 desaturase gene introduction: Reference Example 7) was constructed.
  • ura5 uracil-required marker gene
  • pBIG35hispPsD5m for introducing ⁇ 5 desaturase gene: Reference Example 5
  • pBIG35PP3pCopD12m ⁇ 12 desaturase
  • Reference Example 6 pBIG35SSA2pD17m ( ⁇ 17 desaturase gene introduction: Reference Example 7) was constructed.
  • MNNG-treated spores were inoculated into GY medium and cultured at 12 ° C. for 2 to 3 days.
  • the culture solution was filtered through a glass filter (pore size 20-30 ⁇ m) to obtain spores before germination. This was inoculated on a GY agar medium and cultured at 28 ° C. for 2 days. About the appearing colony, 100 colonies were randomly picked up per one mutation treatment experiment, and liquid culture of the spore suspension was performed.
  • the spore suspension was inoculated into a 10 mL Erlenmeyer flask containing 2 mL of GY medium and cultured with reciprocal shaking (120 strokes / min).
  • the cells obtained by centrifugation are washed with distilled water and dried at 100 ° C., and the cell fat composition is determined by gas liquid chromatography (GLC) (5% Advanced DS on 80 / 100 mesh Chromsorb W, 3 mm ⁇ 2 m, column temperature: 190 ° C.).
  • the fatty acid composition of the cells obtained from a total of 1300 colonies was measured.
  • the ST1358 strain producing about 55% arachidonic acid in the fatty acid composition was selected using the measured amount of arachidonic acid produced by the cells as an index.
  • Agrobacterium (Agrobacterium tumefaciens C58C1, provided by Kyoto Prefectural University) was electrolyzed with the ⁇ 5 desaturase gene, the ⁇ 12 desaturase gene, the ⁇ 17 desaturase gene and the MAELO RNAi introduction binary vector prepared in Reference Example 8. The cells were transformed by poration, and cultured on LB-Mg agar medium at 28 ° C.
  • 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. 100 ⁇ L of the above Agrobacterium suspension was added to an equal amount of the above M.I.
  • 5-FOA 5-fluoroorotic acid
  • ⁇ 12 The desaturase gene, ⁇ 17 desaturase gene, and MAELORNAi-introduced strain were determined. This operation was performed three times in order to select transformants that stably maintain the character.
  • Production Example 2 M. as host strain
  • the ⁇ 5 desaturase gene, the ⁇ 12 desaturase gene, the ⁇ 17 desaturation were the same as in Production Example 1 except that the ST1358 strain (Reference Example 9) was used instead of the alpina 1S-4 uracil-requiring mutant.
  • a synthase gene and MAELORNAi-introduced strain were produced.
  • Test Example 1 Fatty acid composition and production amount in transgenic strains The modified strains obtained in Production Examples 1 to 3 were each aerobically cultured at 300 rpm for 10 days at 28 ° C in 10 mL GY medium. As a control, M.M. Alpina 1S-4 wild type was cultured in the same manner. 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, 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.
  • the accumulation of eicosatetraenoic acid (ETA) was suppressed to 1% or less, and arachidonic acid (ARA) was also 5% or less in the transformed strain of ST1358. It was suppressed to 10% or less in the transformant of JT180.
  • the amount of fatty acid produced by transformed strains of ST1358 strain and JT180 strain is Alpina 1S-4 increased compared to the wild strain, and was equal to or higher than its own host strain.

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Abstract

L'invention concerne un micro-organisme produisant des lipides, qui peut produire, efficacement, de l'acide eicosapentaénoïque (EPA). Ce micro-organisme mutant est obtenu par l'introduction d'un gène de Δ17-désaturase étranger et d'un ou de plusieurs parmi un gène de Δ5-désaturase étranger et d'un gène de Δ12-désaturase étranger dans un micro-organisme pouvant produire des acides gras ω9 hautement insaturés ou dans un micro-organisme présentant une haute teneur en acide arachidonique dans une composition d'acides gras et, après 10 jours de culture à 20°C ou plus, la teneur en acide eicosapentaénoïque dans la composition d'acide gras est de 20 % ou plus.
PCT/JP2015/074346 2014-08-29 2015-08-28 Procédé de production de lipides à haute teneur en acide eicosapentaénoïque WO2016031947A1 (fr)

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CN109266698A (zh) * 2018-05-17 2019-01-25 梁云 被孢霉属微生物油脂中脂肪酸组合物成分调整的方法
WO2019208803A1 (fr) * 2018-04-26 2019-10-31 日本水産株式会社 Huile microbienne et procédé de production d'huile microbienne

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JP2014045740A (ja) * 2012-09-03 2014-03-17 Kyoto Univ 外来不飽和化酵素遺伝子導入による脂質生産微生物での高度不飽和脂肪酸の生産

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