JP2022047741A - Yeast containing Δ15 fatty acid desaturase expression cassette - Google Patents

Abstract

To provide technologies that can more efficiently produce fats and oils containing ω-3 fatty acid, which is represented by α-linolenic acid, DHA, EPA and the like, using cells such as yeast.SOLUTION: Provided is a method for producing fats and oils, which comprises culturing yeast containing the expression cassette of the Δ15 fatty acid desaturase described in the following (A) or (B): (A) a Δ15 fatty acid desaturase containing an amino acid sequence shown in any of three specific amino acid sequences; or (B) a Δ15 fatty acid desaturase containing an amino acid sequence having 80% or more identity with any of the three specific amino acid sequences of (A).SELECTED DRAWING: None

Description

The present invention relates to yeast and the like containing a Δ15 fatty acid desaturase expression cassette.

Fats and oils are closely related to our lives, and their uses are mainly classified into edible and industrial uses. For edible purposes, there are processed food fats and oils that are used as salad oil during cooking and by processing fat and oil raw materials into margarine, dressings, and the like. In industrial applications, it is used as it is as a fuel or lubricating oil, and is also used as an oil / fat chemical product such as shampoo, conditioner, and cosmetics after undergoing chemical conversion.

The raw materials for the fats and oils of the three main fats and oils (soybean oil, rapeseed oil and palm oil) are mainly vegetable oils obtained by pressing seeds such as rapeseed and soybean, and pulp such as palm and olive. Overseas, it is possible to supply oils and fats by cultivating oily plants that are the raw materials for oils and fats. On the other hand, in Japan, in order to improve the self-sufficiency rate of edible oils and fats, it is necessary to develop an oil and fat production system suitable for Japan.

At present, as an oil and fat production method, attention is being paid to the production of oil and fat using microorganisms instead of the oil and fat production from oil-based plants. Among them, fat and oil yeasts such as Lipomyces yeast have a high fat and oil storage capacity (Patent Document 1). However, most of the accumulated fats and oils are composed of saturated fatty acids or monounsaturated fatty acids, and the content of polyunsaturated fatty acids is extremely low.

Japanese Unexamined Patent Publication No. 2012-183012

Among polyunsaturated fatty acids, ω-3 fatty acids typified by α-linolenic acid, DHA, EPA, etc. have been found to be useful in improving lipid metabolism and have high added value. The present inventor has focused on producing ω-3 fatty acid using cells such as yeast.

Therefore, it is an object of the present invention to provide a technique capable of producing ω-3 fatty acid more efficiently.

As a result of diligent research in view of the above problems, the present inventor: Δ15 fatty acid desaturase described in (A) or (B) below: (A) Amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4. A yeast containing a Δ15 fatty acid desaturase containing, or (B) an expression cassette of a Δ15 fatty acid desaturase containing amino acid sequence B having at least 80% identity with amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4. If, it was found that the above problem can be solved. The present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.

Item 1. Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4 and 80% or more. A yeast comprising an expression cassette of a Δ15 fatty acid desaturase comprising amino acid sequence B having identity.

Item 2. Item 2. The yeast according to Item 1, wherein the identity is 90% or more.

Item 3. Item 2. The yeast according to Item 1 or 2, wherein the amino acid sequence A is the amino acid sequence shown in SEQ ID NO: 1 or 3.

Item 4. Item 6. The yeast according to any one of Items 1 to 3, wherein the amino acid sequence A is the amino acid sequence shown in SEQ ID NO: 1.

Item 5. Item 6. The yeast according to any one of Items 1 to 4, wherein the Δ15 fatty acid desaturase is a Δ15 fatty acid desaturase derived from an organism of the genus Spizellomyces, an organism of the genus Penicillium, or an organism of the genus Lobosporangium.

Item 6. Item 5. The yeast according to Item 5, wherein the Spizellomyces genus is Spizellomyces punctatus, the Penicillium genus is Penicillium italicum, and the Lobosporangium genus is Lobosporangium transversale.

Item 7. Item 6. The yeast according to any one of Items 1 to 6, further comprising an expression cassette of Δ12 fatty acid desaturase derived from the yeast Psudozyma antarctica.

Item 8. Item 2. The yeast according to any one of Items 1 to 7, which is an oil-and-fat yeast.

Item 9. Item 2. The yeast according to any one of Items 1 to 8, which is a yeast of the genus Lipomyces.

Item 10. Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4 and 80% or more. A polynucleotide comprising a coding sequence for a Δ15 fatty acid desaturase comprising an amino acid sequence B having the same identity, and a yeast-derived promoter.

Item 11. A cell comprising the polynucleotide according to Item 10.

Item 12. Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4 and 80% or more. A Δ15 fatty acid desaturase containing the amino acid sequence B having the same identity.

Item 13. Item 12. An enzyme agent for modifying fats and oils, which contains the Δ15 fatty acid desaturase according to Item 12.

Item 14. A composition for producing fats and oils, which comprises the yeast according to any one of Items 1 to 9.

Item 15. A method for producing a fat or oil, which comprises recovering the fat or oil from at least one selected from the group consisting of the yeast culture according to any one of Items 1 to 9 and the composition for producing fat and oil according to Item 14.

Item 16. Yeast according to any one of Items 1 to 9 Extraction containing at least one oil and fat selected from the group consisting of the yeast culture according to any one of Items 1 to 9 and the composition for producing oil and fat according to Item 14. thing.

According to the present invention, it is possible to provide a technique capable of producing ω-3 fatty acid more efficiently.

In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".

1. 1. Yeast In one embodiment of the present invention, the Δ15 fatty acid desaturase according to (A) or (B) below: (A) a Δ15 fatty acid desaturase containing the amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4. Or (B) a Δ15 fatty acid desaturase containing an amino acid sequence B having 80% or more identity with the amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4 (in the present specification, the “desaturase of the present invention”). It relates to a fatty acid (which may also be referred to herein as "the yeast of the invention"), which comprises an expression cassette of). This will be described below.

1-1. The Δ15 fatty acid desaturase amino acid sequence A is preferably the amino acid sequence shown in SEQ ID NO: 1 or 3, and particularly preferably the amino acid sequence shown in SEQ ID NO: 1.

As used herein, the term "identity" of an amino acid sequence refers to the degree of coincidence of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), KarlinS, Altschul SF. It can be determined using "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90: 5873-7 (1993). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. The "identity" of the base sequence is also defined according to the above. Regarding the Δ15 fatty acid desaturase of (B) above, the degree of identity with respect to the amino acid sequence shown in any of SEQ ID NOs: 1 and 3 to 4 is preferably 85% or more, more preferably 90% or more, still more preferably 95%. The above is even more preferably 97% or more, and particularly preferably 99% or more.

The Δ15 fatty acid desaturase of the above (B) is preferably the protein according to the following (Ba):
(Ba) One or more amino acids are mutated (eg, substitution, deletion, addition, insertion, etc., preferably substitution, more preferably conservative) with respect to the amino acid sequence shown in any of SEQ ID NOs: 1 and 3-4. It is a Δ15 fatty acid desaturase consisting of a substituted amino acid sequence.

Regarding the above-mentioned protein (Ba), the plurality is, for example, 2 to 8, preferably 2 to 4, and more preferably 2.

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. Other amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine; aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitutions between amino acid residues are also conservative substitutions.

The desaturase of the present invention preferably reduces the linoleic acid content in the fatty acid composition of the yeast by expressing it in a Lipomyces yeast containing an expression cassette of the Δ12 fatty acid desaturase derived from the yeast Psudozyma antarctica described below (the desaturase of the present invention). For example, it has an activity capable of reducing to 50% or less, preferably 40% or less, more preferably 30% or less, further preferably 20% or less, still more preferably 15% or less). In addition, the desaturase of the present invention preferably expresses it in a yeast of the genus Lipomyces containing an expression cassette of the Δ12 fatty acid desaturase derived from the yeast Psudozyma antarctica described later, whereby the α-linolenic acid content in the fatty acid composition of the yeast is contained. Has an activity capable of increasing (for example, increasing to 15% or more, preferably 20% or more, more preferably 25% or more, still more preferably 30% or more, still more preferably 40% or more). As the yeast of the genus Lipomyces referred to here, it is preferable that no mutation is added to the fatty acid conversion pathway (particularly, the pathway for producing oleic acid and the pathway for converting oleic acid to other fatty acids (for example, linoleic acid)). The yeast of the genus Lipomyces is preferably L. starkeyi.

As used herein, the fatty acid composition of yeast can be measured according to known methods, for example using chromatography. "Can be decreased (or increased)" means that it can be decreased (or increased) to a certain level by adjusting the degree of expression, culture conditions, and the like.

The desaturase of the present invention is preferably a Δ15 fatty acid desaturase derived from an organism of the genus Spizellomyces (more preferably Spizellomyces punctatus), a genus Penicillium (more preferably Penicillium italicum), or an organism of the genus Lobosporangium (more preferably Lobosporangium transversale). .. As used herein, the term "derived from" an organism is used as long as it is originally possessed by the organism (that is, encoded by a wild-type genome), or the amino acid sequence is modified based thereto. , There are no particular restrictions. The degree of modification is particularly as long as it does not significantly impair the original activity (for example, maintains / has an activity of 70% or more, 80% or more, 90% or more, 95% or more, 99% or more of the original activity). Not limited.

The desaturase of the present invention may be added with a protein or peptide such as a known protein tag or signal sequence as long as it has the above-mentioned "activity". Examples of the protein tag include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.

1-2. Expression cassette The expression cassette of the desaturase of the present invention is not particularly limited as long as it contains the coding sequence of the desaturase of the present invention.

The coding sequence is not particularly limited as long as it is a polynucleotide consisting of a base sequence encoding the desaturase of the present invention.

In the present specification, polynucleotides such as DNA and RNA may be subjected to known chemical modifications as exemplified below. Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolases such as nucleases. Can be done. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is converted into -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned. Further, examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto. Further, BNA (LNA) or the like in which the conformation of the sugar portion is fixed to N-type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be preferably used. The coding sequence of the desaturase of the present invention includes, for example, the base sequence described in (C) or (D) below:
(C) The base sequence shown in any of SEQ ID NOs: 5 and 7 to 8, or (D) the base sequence having 80% or more identity with the base sequence shown in any of SEQ ID NOs: 5 and 7 to 8. Can be mentioned.

Regarding the base sequence of (D) above, the degree of identity with respect to the base sequence shown in any of SEQ ID NOs: 5 and 7 to 8 is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more. , More preferably 97% or more, and particularly preferably 99% or more.

The base sequence of the above (D) is preferably the base sequence described in the following (Da):
(Da) A base sequence in which one or more bases are mutated (for example, substitution, deletion, addition, insertion, etc.) with respect to the base sequence shown in any of SEQ ID NOs: 5 and 7-8.

Regarding the base sequence of (Da), the plurality is, for example, 2 to 8, preferably 2 to 4, and more preferably 2.

The expression cassette usually contains a promoter upstream of the coding sequence to express the protein encoded by the coding sequence. The promoter is not particularly limited, and for example, various pol II promoters can be used. The pol II promoter is not particularly limited, but is not particularly limited, for example, TDH3 promoter, TEF1 promoter, promoter, ADH1 promoter, TPI1 promoter, HXT7 promoter, PGK1 promoter, PYK1 promoter, GAL10 promoter, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter. , CAG promoter and the like. Among these, yeast-derived promoters such as TDH3 promoter, TEF1 promoter, promoter, ADH1 promoter, TPI1 promoter, HXT7 promoter, PGK1 promoter, PYK1 promoter, and GAL10 promoter (that is, promoters of endogenous genes of yeast) are preferable.

1-3. Yeast The yeast of the present invention is not particularly limited as long as the above-mentioned expression cassette is contained outside the genome and / or inside the genome.

As the yeast, fat and oil yeast is preferably mentioned. The oil-and-fat yeast is not particularly limited as long as it is a yeast having a high oil-and-fat accumulation property. For example, fat yeast has a high fat content, such as 20% (w / w) or higher, preferably 30% (w / w) or higher, more preferably 40% (w / w) or higher, and even more preferably 50% ( A yeast capable of achieving a fat content of w / w) or higher, more preferably 60% (w / w) or higher. Specific examples of the fat and oil yeast include Rhodosporodium yeast such as Rhodosporodium toruloides, Lipomyces yeast such as Lipomyces starkeyi, Cryptococcus yeast such as Cryptococcus albidus, Rhizopus yeast such as Rhizopus arrhizua, and Yarrowia lipolytica. Can be mentioned. Among these, yeasts of the genus Rhodosporodium, yeasts of the genus Lipomyces, yeasts of the genus Cryptococcus and the like are preferably mentioned, yeasts of the genus Lipomyces and the like are more preferable, and Lipomyces starkeyi and the like are more preferable.

Yeast may also have other mutations added. For example, yeast may be mutated in the fatty acid conversion pathway. More specifically, for example, for C16 / C18 fatty acid erongase, Δ9 desaturase, Δ9 erongase, Δ8 desaturase, Δ5 desaturase, Δ12 desaturase, Δ17 desaturase, etc., mutations such as introduction of an exogenous gene, modification of an endogenous gene or its promoter, etc. May be mutated in the fatty acid conversion pathway. This makes it possible to further increase the content of polyunsaturated fatty acids, preferably ω-3 fatty acids having high added value such as DHA and EPA.

In a preferred embodiment of the invention, the yeast of the invention further comprises an expression cassette of the Δ12 fatty acid desaturase derived from the yeast Psudozyma antarctica.

The Δ12 fatty acid desaturase is preferably the protein according to (i) or (ii) below:
(I) A protein containing the amino acid sequence shown in SEQ ID NO: 9, or (ii) a protein containing an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 9.

Regarding the protein of (ii) above, the degree of identity with respect to the amino acid sequence shown in SEQ ID NO: 9 is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more. , Especially preferably 99% or more.

The protein of (ii) above is preferably the protein described in (iia) below:
(Iia) Consists of an amino acid sequence in which one or more amino acids are mutated (for example, substitution, deletion, addition, insertion, etc., preferably substitution, more preferably conservative substitution) with respect to the amino acid sequence shown in SEQ ID NO: 9. It is a protein.

Regarding the above-mentioned (iia) protein, the plurality is, for example, 2 to 8, preferably 2 to 4, and more preferably 2.

The Δ12 fatty acid desaturase preferably reduces the oleic acid content in the fatty acid of the yeast by expressing it in a yeast of the genus Lipomyces (for example, 12% or less, preferably 11% or less, more preferably 10% or less, It has an activity capable of reducing it to 9% or less, more preferably 8% or less). Further, the Δ12 fatty acid desaturase preferably increases the polyunsaturated fatty acid content in the fatty acid of the yeast by expressing it in a yeast of the genus Lipomyces (for example, 50% or more, preferably 60% or more, more preferably. Has an activity that can be increased to 65% or more, more preferably 70% or more). As the yeast of the genus Lipomyces referred to here, it is preferable that no mutation is added to the fatty acid conversion pathway (particularly, the pathway for producing oleic acid and the pathway for converting oleic acid to other fatty acids (for example, linoleic acid)). The yeast of the genus Lipomyces is preferably L. starkeyi.

The expression cassette of the Δ12 fatty acid desaturase is not particularly limited as long as it contains the coding sequence of the Δ12 fatty acid desaturase.

The coding sequence is not particularly limited as long as it is a polynucleotide consisting of a base sequence encoding the Δ12 fatty acid desaturase. The coding sequence of the Δ12 fatty acid desaturase is, for example, the base sequence described in (X) or (Y) below:
Examples thereof include the base sequence shown in (X) SEQ ID NO: 10 and the base sequence having 80% or more identity with the base sequence shown in (Y) SEQ ID NO: 10.

Regarding the base sequence of (Y) above, the degree of identity with respect to the base sequence shown in SEQ ID NO: 10 is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97%. As mentioned above, it is particularly preferably 99% or more.

The base sequence of (Y) above is preferably the base sequence described in (Ya) below:
(Ya) A base sequence in which one or more bases are mutated (for example, substitution, deletion, addition, insertion, etc.) with respect to the base sequence shown in SEQ ID NO: 10.

Regarding the base sequence of (Ya) above, the plurality is, for example, 2 to 8, preferably 2 to 4, and more preferably 2.

In addition, the expression cassette of the Δ12 fatty acid desaturase is the same as the expression cassette of the desaturase of the present invention described above.

The yeast of the present invention is prepared according to or in accordance with a known method, for example, by an operation of introducing the above expression cassette prepared according to a genetic engineering technique such as PCR, restriction enzyme cleavage, DNA ligation technique, etc. into cells, a transformation method, or the like. can do.

2. Polynucleotides, Cells The present invention relates to polynucleotides (polynucleotides of the invention) comprising the coding sequence of the desaturase of the invention and yeast-derived promoters, in one aspect of the invention. The present invention also relates to a cell (cell of the present invention) containing the polynucleotide of the present invention in one aspect thereof. These will be described below. For matters not described in this section, the description in the above section is used.

The polynucleotides of the invention may optionally contain other elements (eg, multicloning sites (MCS), drug resistance genes, origins of replication, etc.). For example, between the promoter and the coding sequence of the desaturase of the invention (preferably adjacent to one or both), on the 3'side (preferably adjacent) of the coding sequence of the desaturase of the invention, the MCS. Examples of the arrangement are mentioned. The MCS is not particularly limited as long as it contains a plurality of (for example, 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites.

The polynucleotide of the present invention may constitute a vector. The type of vector is not particularly limited, and examples thereof include pUC-based vectors.

The cell of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention outside the genome and / or within the genome.

The cells are preferably yeast. The yeast is the same as the description in "1. Yeast" above.

The polynucleotide of the present invention can be prepared according to or according to a known method, for example, according to a genetic engineering technique such as PCR, restriction enzyme cleavage, DNA ligation technique, or the like. In addition, the cells of the present invention can be produced according to or according to a known method, for example, by an operation of introducing the polynucleotide of the present invention into cells, a transformation method, or the like.

3. 3. Δ15 fatty acid desaturase, its use The present invention relates to the desaturase of the present invention and an enzyme agent for modifying fats and oils containing the desaturase in one aspect thereof. This will be described below. For matters not described in this section, the description in the above section is used.

The desaturase of the present invention may be chemically modified as long as it has the above-mentioned "activity".

The desaturase of the present invention may have a C-terminal of any of a carboxyl group (-COOH), a carboxylate (-COO- ⁾ , an amide (-CONH ₂ ) or an ester (-COOR).

Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example, phenyl. , C _6-12 aryl groups such as α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl, phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. ₁₄ Alkyl group; Pivaloyloxymethyl group etc. are used.

In the desaturase of the present invention, a carboxyl group (or carboxylate) other than the C-terminal may be amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Further, in the desaturase of the present invention, the amino group of the N-terminal amino acid residue is protected by a protective group (for example, a C _1-6 acyl group such as C _1-6 alkanoyl such as a formyl group or an acetyl group). , N-terminal glutamine residue that can be cleaved in vivo and oxidized with pyroglutamine, substituents on the side chain of amino acids in the molecule (eg-OH, -SH, amino group, imidazole group, etc. An indol group, a guanidino group, etc.) is protected by a suitable protective group (for example, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as a formyl group or an acetyl group), or a sugar chain is bonded. Complex proteins such as so-called glycoproteins are also included.

The desaturases of the present invention may be in the form of pharmaceutically acceptable salts with acids or bases. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be adopted. Examples of acidic salts are inorganic acid salts such as hydrochlorides, hydrobromide, sulfates, nitrates, phosphates; acetates, propionates, tartrates, fumarates, maleates, apples. Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparagates and glutamates can be mentioned. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.

The desaturase of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.

The oil / fat reforming enzyme agent may contain other components, if necessary. Examples of other components include excipients, buffers, suspensions, stabilizers, preservatives, preservatives, saline solutions, fats and oils, and the like. As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. As the buffer, phosphate, citrate, acetate and the like can be used. Propylene glycol, ascorbic acid and the like can be used as the stabilizer. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The content of the desaturase of the present invention in the enzyme preparation for oil and fat modification is not particularly limited, and is, for example, 0.01 to 100 mg / mL.

Various fats and oils (for example, glycerin fatty acid ester) containing linoleic acid and / or linoleic acid ester can be used as the fat and oil as a substrate of the oil and fat reforming enzyme agent, and for example, soybean oil, rapeseed oil, rice oil, etc. Vegetable fats such as corn oil, cottonseed oil, palm oil, palm kernel oil, palm oil, cacao fat, monkey fat, animal and vegetable fats such as fish oil, lard, beef fat, milk fat, and their fractionated fats, hardened oils, and ester exchanges. Examples thereof include triacylglycerides including oils and synthetic fats and oils such as MCT, trilaurin, triolein, tripalmitin and tristea resynthesized from fatty acids and glycerin. Further, various diacyl glycerin esters and monoacyl glycerin esters can also be used.

The enzyme agent for modifying fats and oils is preferably used for converting linoleic acid and / or linoleic acid constituting the linoleic acid ester into α-linolenic acid.

Five. Oil and fat production composition, method for producing oil and fat In one aspect of the present invention, the composition for oil and fat production containing the yeast of the present invention (in the present specification, referred to as "the composition for oil and fat production of the present invention"). There is also.).

Further, in one aspect of the present invention, a method for producing a fat or oil, which comprises recovering the fat or oil from at least one selected from the group consisting of the culture of the yeast of the present invention and the composition for producing the fat and oil of the present invention. (In the present specification, it may be referred to as "the oil and fat production method of the present invention").

These will be described below. For matters not described in this section, the description in the above section is used.

The composition for producing fats and oils of the present invention is not particularly limited as long as it contains the yeast of the present invention. The composition for producing fats and oils of the present invention can be, for example, a culture of yeast of the present invention or a suspension of yeast of the present invention.

The culture can be carried out by a conventionally known method using a culture solution containing a carbon source. As the carbon source, sugars, sugar alcohols and acidic sugars or biomass containing these can be used without particular limitation. Here, in the present invention, "biomass" means a renewable material containing the above carbon source.

Examples of saccharides include monosaccharides, oligosaccharides and polysaccharides. Oligosaccharides refer to di to ten saccharides, which may be homo-oligosaccharides or hetero-oligosaccharides. Further, the polysaccharide refers to a saccharide having a larger number of monosaccharide units than the oligosaccharide, and these may be homopolysaccharides or heteropolysaccharides. Specifically, examples of monosaccharides include pentoses such as L-arabinose, D-xylose and D-ribose, hexoses such as D-glucose, D-galactose, D-fractose and D-mannose, and 6- such as L-ramnose. Deoxyhexose and the like can be mentioned. Examples of oligosaccharides include disaccharides such as sucrose, maltose, lactose, cellobiose, trehalose and melibiose, and trisaccharides such as raffinose. Examples of the polysaccharide include starch, cellulose, glycogen, dextran, mannan, xylan and the like. The above saccharides may be used alone or in combination as appropriate. The above combination also includes starch hydrolysates and the like. Further, as the saccharide, a raw material containing saccharide as a main component, for example, molasses, okara, etc. can also be used.

Examples of sugar alcohols include D-sorbitol, D-mannitol, galactitol, maltitol and the like. Examples of the acidic sugar include glucuronic acid and galacturonic acid.

The amount of the carbon source in the medium is not particularly limited, but is usually about 3 to 15% (w / w).

The medium may contain a nitrogen source, an inorganic substance and other nutrients in addition to the carbon source. As the nitrogen source, an inorganic organic nitrogen compound such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, sodium nitrate and urea can be used. Further, as the nitrogen source, nitrogen-containing natural products such as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean meal or its digested product can also be used. Inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, ferrous chloride, manganese sulfate, calcium chloride, calcium carbonate, zinc sulfate, copper sulfate, boric acid / molybdic acid. Ammonium, potassium iodide, etc. can be used.

One aspect of the culture conditions is as follows. Culturing is performed under aerobic conditions such as shaking culture or deep stirring culture. The culture temperature is generally preferably 20 to 35 ° C., but other temperature conditions may be used as long as the bacteria grow. The pH of the culture medium during culture is usually 4.0 to 7.2. The culture period is not particularly limited, and is, for example, 2 to 10 days.

The obtained culture and the cells in the culture contain fats and oils such as α-linolenic acid, oleic acid, palmitic acid, stearic acid, linoleic acid, and palmitoleic acid.

Recovery of fats and oils from the culture can be performed according to or according to a known method. For example, it can be recovered by squeezing, a French press, a ball mill, or the like.

The fat and oil accumulated in the cells can be recovered, for example, by removing the liquid fraction from the culture as needed and obtaining the fat and oil-containing extract from the obtained cells according to a known method or in accordance with the known method. .. The liquid fraction can be removed by operations such as centrifugation and static sedimentation, or by an apparatus such as a separator, a decanter, and a filter.

For the fats and oils secreted extracellularly, for example, by adding a solvent to the liquid fraction obtained by removing the cells from the culture or, if necessary, the cultures, and dissolving the fats and oils in the solvent. Can be recovered. As the solvent, an organic solvent that dissolves fats and oils and is immiscible or poorly mixed with water and is liquid at room temperature, for example, lower halogenated alkanes (chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane), n-hexane. , Ethyl ether, ethyl acetate, aromatic hydrocarbons (benzene, toluene, xylene) and the like are preferably used. The amount of the extraction solvent added is not particularly limited as long as it can sufficiently recover the fats and oils produced and accumulated in the culture or its liquid fraction.

The recovered fats and oils (extracts containing fats and oils) have a high ω-3 fatty acid content in the fatty acid composition even without undergoing a fatty acid conversion treatment. The content is, for example, 15% or more, preferably 20% or more, more preferably 25% or more, still more preferably 30% or more, still more preferably 40% or more.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Reference example 1. Preparation of expression vector for PaΔ12d
pKS-18S-Pact1-nat1-Tact1-Ptdh3-LsD12d1-Ttdh3-18S plasmid (north lysine resistance gene under the control of LsACT1 gene promoter and terminator, L. starkeyi-derived LsD12d1 gene LsTDH3 gene promoter and ter A construct for expression in the nator. This construct is inserted into the 18S rDNA region in the genomic DNA of L. starkeyi), and PaΔ12d (Δ12 fatty acid desaturase derived from yeast Psudozyma antarctica) (amino acid sequence is SEQ ID NO: 9). An expression plasmid (shown, whose coding sequence is shown by SEQ ID NO: 10) was prepared.

pKS-18S-Pact1-nat1-Tact1-Ptdh3-LsD12d1-Ttdh3-18S is pKS-18S-hph (Oguro Y, Yamazaki H, Shida Y, Ogasawara W, Takagi M, Takaku H (2015) Multicopy integration and expression of heterologous Based on genes in the oleaginous yeast, Lipomyces starkeyi. Biosci Biotechnol Biochem 79, 512-515), artificially synthesized Ptdh3-LsD12d1-Ttdh3 sequence (SEQ ID NO: 11) is restricted to pKS-18S-hph enzymes (PmeI and AvrII). And inserted using DNA ligase. In addition, the selection marker for pKS-18S-hph was replaced from the hygromycin resistance gene hph to the north leolicin resistance gene.

First, using pKS-18S-Pact1-nat1-Tact1-Ptdh3-LsD12d1-Ttdh3-18S as the template DNA, primer set 3 (THD3_terF: 5'-GTGTGCGGTTGATGGTCTTCTATCTTCC-3'(SEQ ID NO: 12), and TDH_proR: 5'-TGCGAATGTGGATTAGAGTAAGATAGATA. PCR was performed by -3'(SEQ ID NO: 13)). DpnI was added to the obtained PCR solution, and the template DNA was digested by treating at 37 ° C. for 3 hours, and then the DNA was purified by gel extraction.

Next, Psudozyma antarctica was cultured in YPD (1% yeast extract, 2% polypeptone, 2% glucose) at 30 ° C for 2 days, and then total RNA was collected and cDNA was synthesized. Extraction of total RNA and synthesis of cDNA were performed using NucleoSpin RNA of Takara Bio Inc. and PrimeScript II 1st strand cDNA Synthesis Kit. Primer set 4 (Tdh3p_PaD12d_F: 5'-TAATCCACATTCGCAATGTCGTCTGCAGTGGCTGCCAACG-3'(SEQ ID NO: 14) and PaD12d_ Tdh3t_R: 5'-CCATCAACCGCACACTCACTCGGACATGGCGATGCCAG-3' (SEQ ID NO: 15)) using this cDNA as a template. , The gene of interest was obtained by gel extraction.

These purified DNA fragments were fused using Clontech's In-Fusion cloning system and introduced into Escherichia coli DH5α. The plasmid was extracted from the transformant obtained on LB + ampicillin 100 μg / ml medium, and the target plasmid pKS-18S-Pact1-nat1-Tact1-Ptdh3-PaD12d-Ttdh3-18S was obtained by restriction enzyme treatment and sequence analysis. It was confirmed that it was produced.

Reference example 2. Preparation of Lipomyces yeast expressing PaΔ12d Primer set 5 (Li18S_F: 5'-GCTTCTTCGGAAGCTCTTTGGTGATTCATG-3'(SEQ ID NO: 16) and Li18S_R: 5'-CGACTATATCTTAAGCCGCACAACGGCCC-3'(SEQ ID NO: 17) using the prepared expression plasmid as a template. ) Was used for PCR. The resulting DNA solution was used for transformation of L. starkey iΔlig4 + LsElo1.

Example 1. Preparation of Δ15d expression vector
Δ15 fatty acid desaturase derived from Spizellomyces punctatus (SpD15d) (amino acid sequence: SEQ ID NO: 1), Δ15 fatty acid desaturase derived from Fusarium verticillioides (FvD15d) (amino acid sequence: SEQ ID NO: 2), Δ15 fatty acid desaturase derived from Penicillium italicum (PiD15d) (amino acid sequence: sequence) Expression vectors for No. 3) and Δ15 fatty acid desaturase (LtD15d) derived from Lobosporangium transversale (amino acid sequence: SEQ ID NO: 4) were prepared. Specifically, it was carried out as follows.

pKS-18S-P _ACT1 -KanR-T _ACT1 -P _TDH3 -D15d-T _TDH3 -18S was prepared. The meaning of each abbreviation is as follows: 18S: Lipomyces starkeyi-derived 18S rRNA region, P _ACT1 : L. starkeyi-derived ACT1 gene promoter sequence, KanR: canamycin resistance gene, T _ACT1 : L. starkeyi-derived ACT1 gene terminator. Sequence, P _TDH3 : Promoter sequence of TDH3 gene derived from L. starkeyi, D15d: Δ15 fatty acid desaturase gene, T _TDH3 : Terminator sequence of TDH3 gene derived from L. starkeyi. D15d is homologous for InFusion PCR at the 5'end and 3'end of the coding sequence of the Δ15 fatty acid desaturase gene (SpD15d: SEQ ID NO: 5, FvD15d: SEQ ID NO: 6, PiD15d: SEQ ID NO: 7, LtD15d: SEQ ID NO: 8). It is a sequence to which a sequence is added (SpD15d: SEQ ID NO: 18, FvD15d: SEQ ID NO: 19, PiD15d: SEQ ID NO: 20, LtD15d: SEQ ID NO: 21).

A plasmid was prepared in which the sNAT gene of the pKS-18S-sNAT1-LsFAD2 plasmid (Matsuzawa et al. Applied Microbiol BIotechnol 2020 doi: 10.1007 / s00253-020-10401-9) was replaced with the kanamycin resistance gene. Using this plasmid as a template, primer set (Primer 1: 5'-TGCGAATGTGGATTAGAGTA-3'(SEQ ID NO: 22), Primer 2: 5'-GTGTGCGGTTGATGGTCTTC-3' (SEQ ID NO: 23)) and KOD -Plus- (TOYOBO) PCR was performed using. PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio Inc.). The artificially synthesized D15d gene and the above PCR product were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.). Escherichia coli ECOS DH5α (Nippon Gene) was transformed with the above In-Fusion HD Cloning Kit reaction product, and Escherichia coli was applied to LB (LB + Amp) agar medium containing ampicillin.

Eight colonies growing on the LB + Amp agar medium were picked up for each synthetic gene, and Emerald Amp PCR Master Mix (Takara Bio Inc.) and primer set (Primer 3: 5'-GTGTGCGGTTGATGGTCTTC-3'(SEQ ID NO: 24)). , Primer 4: 5'-TCGGTATTATAGAATACGGC-3'(SEQ ID NO: 25)) was used to confirm that the D15d gene was cloned by colony PCR (cPCR). -The Escherichia coli clone in which cloning of the D15d gene was confirmed by cPCR was liquid-cultured in LB + Amp medium, and the desired plasmid DNA was prepared using the NucleoSpin plasmid Easy Pure kit (Takara Bio Inc.).

Example 2. Preparation of Lipomyces genus yeast expressing exogenous Δ15d Primer set using the pKS-18S-P _ACT1 -KanR-T _ACT1 -P _TDH3 -D15d-T _TDH3 -18S plasmid (Example 1) cloned from each D15d gene as a template. PCR was performed using (Primer 5: 5'-GCTTCTTCGGAAGCTCTTTGGTGATTCATG-3'(SEQ ID NO: 26), Primer 6: 5'-CGACTATATCTTAAGCCGCACAACGGCCCA-3' (SEQ ID NO: 27)) and KOD -Plus- (TOYOBO), and DNA was performed. Fragments were prepared. The prepared DNA solution was introduced into the lslig4Δ + PaD12d + LsElo1 strain (Psudozyma antarctica-derived D12d and Lipomyces starkeyi-derived Elo1 overexpressing strain, Reference Example 2). Transformation was performed according to the paper by Oguro et al. (Biosci Biotechnol Biochem 2015 79: 512-515). After transformation, the cells were planted in YPD (2% pepton, 1% yeast extract, 2% glucose) + 50 μg / mL Zeocin + 50 μg / mL Nourseothricin + 50 μg / mL G418 agar medium and about 1 at 30 ° C. It was cultured for a week. Three colonies were picked up for each D15d gene.

Example 3. Analysis of fat production characteristics Example 3 After culturing the obtained colonies in a GY (5% glucose, 0.5% yeast extract) liquid medium at 140 rpm and 20 ° C for 4 days, the paper (Matsuzawa et al. 2020 Appl Microbiol Biotechnol 104: The fatty acid was methylated by the method of 2537-2544), and the fatty acid composition was analyzed by gas chromatography-mass spectrometer (GC-MS, Shimadzu QP2010 SE). In this analysis, helium gas was used as the carrier gas, and each fatty acid was separated by Agilent Technologies DB-225MS capillary column (30 m × 0.25 mm id).

The results are shown in Table 1. It was found that expressing the exogenous Δ15d of the above example in yeast improves the content of ω-3 fatty acid such as α-linolenic acid in the fatty acid composition.

Claims (16)

Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3-4 and 80% or more. A yeast comprising an expression cassette of a Δ15 fatty acid desaturase comprising amino acid sequence B having identity. The yeast according to claim 1, wherein the identity is 90% or more. The yeast according to claim 1 or 2, wherein the amino acid sequence A is the amino acid sequence shown in SEQ ID NO: 1 or 3. The yeast according to any one of claims 1 to 3, wherein the amino acid sequence A is the amino acid sequence shown in SEQ ID NO: 1. The yeast according to any one of claims 1 to 4, wherein the Δ15 fatty acid desaturase is a Δ15 fatty acid desaturase derived from an organism of the genus Spizellomyces, an organism of the genus Penicillium, or an organism of the genus Lobosporangium. The yeast according to claim 5, wherein the Spizellomyces genus is Spizellomyces punctatus, the Penicillium genus is Penicillium italicum, and the Lobosporangium genus is Lobosporangium transversale. The yeast according to any one of claims 1 to 6, further comprising an expression cassette of the Δ12 fatty acid desaturase derived from the yeast Psudozyma antarctica. The yeast according to any one of claims 1 to 7, which is an oil-and-fat yeast. The yeast according to any one of claims 1 to 8, which is a yeast of the genus Lipomyces. Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4 and 80% or more. A polynucleotide comprising a coding sequence for a Δ15 fatty acid desaturase comprising an amino acid sequence B having the same identity, and a yeast-derived promoter. A cell comprising the polynucleotide according to claim 10. Δ15 fatty acid desaturase according to (A) or (B) below:
(A) Δ15 fatty acid desaturase containing amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4, or (B) amino acid sequence A shown in any of SEQ ID NOs: 1 and 3 to 4 and 80% or more. A Δ15 fatty acid desaturase containing the amino acid sequence B having the same identity. An enzyme agent for modifying fats and oils, which contains the Δ15 fatty acid desaturase according to claim 12. A composition for producing fats and oils, which comprises the yeast according to any one of claims 1 to 9. A method for producing a fat or oil, which comprises recovering the fat or oil from at least one selected from the group consisting of the yeast culture according to any one of claims 1 to 9 and the composition for producing fat and oil according to claim 14. .. Yeast according to any one of claims 1 to 9 At least one selected from the group consisting of the yeast culture according to any one of claims 1 to 9 and the composition for producing fats and oils according to claim 14. Oil-containing extract.