Mutant ATF2 protein and use thereof

Abstract

To provide an ATF2 protein with enhanced isoamyl acetate productivity.SOLUTION: The mutant ATF2 protein disclosed herein contains at least one mutation in the amino acid sequence of a wild-type ATF2 protein of yeast and exhibits enhanced isoamyl acetate productivity compared with the wild-type ATF2 protein.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to mutant ATF2 proteins and uses thereof. [Background technology]

[0002] In sake brewing, yeast (Saccharomyces cerevisiae) produces not only alcohol but also many aroma compounds that form the ginjo aroma. The balance of the production amounts of these aroma compounds varies depending on the yeast strain. Therefore, the selection of the yeast is an important factor in product design.

[0003] Ethyl caproate, known as an apple-like aroma component, is a component of ginjo aroma in sake. Another component of ginjo aroma is isoamyl acetate, known to have a banana-like aroma. For this reason, yeast strains capable of producing these ginjo aroma components at high levels are being developed (Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 63-309175 [Patent Document 2] Japanese Patent Application Publication No. 62-006669 [Patent Document 3] Japanese Patent Application Laid-Open No. 2002-191355 Summary of the Invention [Problem to be solved by the invention]

[0005] It is known that the ATF2 gene is involved in the production of isoamyl acetate contained in sake. Therefore, yeast strains with improved isoamyl acetate productivity have been created by overexpressing the ATF2 gene. However, the present inventors have discovered a problem: even if the ATF2 gene is overexpressed, there is an upper limit to the amount of expression, and there is a limit to the improvement in the isoamyl acetate productivity of yeast strains.

[0006] Therefore, the present disclosure aims to provide an ATF2 protein with improved isoamyl acetate production ability, a gene encoding the ATF2 protein, a yeast containing the gene, a method for producing an alcohol-containing composition using the yeast, an extract, a food composition, and a cosmetic composition, as well as a method for producing the yeast. [Means for solving the problem]

[0007] To achieve the above object, the mutant ATF2 protein of the present disclosure comprises at least one mutation in the amino acid sequence of a wild-type yeast ATF2 protein, Compared to the wild-type ATF2 protein, the ability to produce isoamyl acetate is improved.

[0008] The mutant ATF2 gene of the present disclosure encodes the mutant ATF2 protein of the present disclosure.

[0009] The yeast of the present disclosure comprises a mutant ATF2 gene of the present disclosure.

[0010] A method for producing an alcohol-containing composition of the present disclosure includes brewing an alcohol-containing composition using the yeast of the present disclosure.

[0011] The alcohol-containing composition of the present disclosure can be obtained by the method for producing an alcohol-containing composition of the present disclosure.

[0012] The method for producing yeast of the present disclosure includes a mutation step of mutating a wild-type ATF2 gene in yeast into a mutant ATF2 gene, The mutant ATF2 gene is the mutant ATF2 gene of the present disclosure. [Effects of the Invention]

[0013] According to the present disclosure, there are provided an ATF2 protein with improved isoamyl acetate production ability, a gene encoding the ATF2 protein, a yeast containing the gene, a method for producing an alcohol-containing composition using the yeast, an extract, a food composition, and a cosmetic composition, and a method for producing the yeast. [Brief explanation of the drawings]

[0014]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6A

Figure 6B

[0015] <Definition> As used herein, "ATF2" refers to an enzyme that has alcohol acetyltransferase (AATF) activity and converts isoamyl alcohol to isoamyl acetate. An example of a wild-type yeast ATF2 protein (Aw protein) is a protein consisting of the amino acid sequence registered in Genbank under Accession No. AHY79519.1 (reference ATF2 protein (As protein), SEQ ID NO: 1). The Aw protein can also be found in Genbank under Accession No. AHY79519.1 (reference ATF2 protein (As protein), SEQ ID NO: 1). No.:DAA08272.1, GAA23550.1, GAX68483.1, AJP38949.1, EWG86048.1, CAE6484413.1, CAI5149645.1, CAI7134695. 1, AJR76269.1, AJS05359.1, CAI6537772.1, CAI4484870.1, CAI5274334.1, CAI4996414.1, EGA86689.1, CAI448670 7.1, CAI6533268.1, PTN21619.1, CAI5122473.1, CAI4884748.1, CAI5268485.1, AJR97486.1, AJS21986.1, CAI4522 121.1, CAI5265319.1, CAI5277444.1, CAI6689236.1, CAI6541768.1, PJP07685.1, CAI5008499.1, AJR81214.1, GFP 65339.1, AKB01509.1, CAI5277200.1, AAU09735.1, PTN15692.1, ONH72747.1, AJR80710.1, KOH50517.1, CAI449545 5.1, GMC31794.1, EHN06953.1, EGA82692.1, CAI7290657.1, CAI4484128.1, CAI4958699.1, AJR78753.1, CAI527366 8.1, AJS19990.1, CAI4488485.1, AJS28433.1, CAI4502001.1, AJS27455.1, CAI4488155.1, CAI4476236.1, AJS0882 4.1, AJS23483.1, AJR83202.1, AJS24981.1, CAI4513232.1, CAI4493159.1, AJR77264.1, AJR81713.1, CAI4495929.1, CAI4497927.1, AJS25979.1, AAP72993.1, CAI4507887.1, CAI4487095.1, CAI5277776.1, CAI4480295.1, GMC2 7539.1, CAI4508375.1, AJR99481.1, CAI4500089.1, AJS02923.1, CAI6526953.1, GES70550.1, AJS07347.1, CAI4 340895.1, CAI4494912.1, CAI5267991.1, AJR94592.1, CAD6626649.1, CAI4467811.1, AJR85200.1, CAI4488721.1, CAD6625169.1, CAD6625962.1, CAI7138860.1, CAI7152089.1, and CAI6663215.1. Specific examples of the Aw protein include proteins consisting of the amino acid sequences of SEQ ID NOs: 2 and 3.

[0016] As protein (SEQ ID NO: 1): AHY79519.1 [Saccharomyces cerevisiae YJM993] MEDIEGYEPHITQELIDRGHARRMGHLENYFAVLSRQKMYSNFTVYAELNKGVNKRQLMLVLKVLLQKYSTLAHTIIPKHYPHHEAYYSSEEYLSKPFPQHDFIKVISHLEFDDLIMNNQPEYREVMEKISEQFKKDDFKVTNRLIELISPVIIPLGNPKRPNWRLICLPGKDTDGFETWKNFVYVTNHCGSDGVSGSNFFKDLALLFCKIEEKGFDYDEEFIEDQVIIDYDRDYTEISKLPKPITDRIDYKPALTSLPKFFLTTFIYEHCNFKTSSESTLTARYSPSSNANASYNYLLHFSTKQVEQIRAQIKKNVHDGCTLTPFIQACFLVALYRLDKLFTKSLLEYGFDVAIPSNARRFLPNDEELRDSYKYGSNVGGSHYAYLISSFDIPEGDNDKFWSLVEYYYDRFLESYDNGDHLIGLGVLQLDFIVQNKNIDSLLANSYLHQQRGGAIISNTGLVSQDTTKPYYVRDLIFSQSAGALRFAFGLNVCSTNVNGMNMDMSVVQGTLRDRGEWESFCKLFYQTISEFASL

[0017] Aw protein (SEQ ID NO: 2): DAA08272.1 [Saccharomyces cerevisiae S288C] MEDIEGYEPHITQELIDRGHARRMGHLENYFAVLSRQKMYSNFTVYAELNKGVNKRQLMLVLKVLLQKYSTLAHTIIPKHYPHHEAYYSSEEYLSKPFPQHDFIKVISHLEFDDLIMNNQPEYREVMEKISEQFKKDDFKVTNRLIELISPVIIPLGNPKRPNWRLICLPGKDTDGFETWKNFVYVTNHCGSDGVSGSNFFKDLALLFCKIEEKGFDYDEEFIEDQVIIDYDRDYTEISKLPKPITDRIDYKPALTSLPKFFLTTFIYEHCNFKTSSESTLTARYSPSSNANASYNYLLHFSTKQVEQIRAQIKKNVHDGCTLTPFIQACFLVALYRLDKLFTKSLLEYGFDVAIPSNARRFLPNDEELRDSYKYGSNVGGSHYAYLISSFDIPEGDNDKFWSLVEYYYDRFLESYDNGDHLIGLGVLQLDFIVENKNIDSLLANSYLHQQRGGAIISNTGLVSQDTTKPYYVRDLIFSQSAGALRFAFGLNVCSTNVNGMNMDMSVVQGTLRDRGEWESFCKLFYQTIGEFASL

[0018] Aw protein (SEQ ID NO: 3): GAA23550.1 [Saccharomyces cerevisiae Kyokai no. 7] MEDIEGYEPHITQELIDRGHARRMGHLENYFAVLSRQKMYSNFTVYAELNKGVNKRQLMLVLKLLLQKYSTLAHTIIPKHYPHHEAYYSSEEYLSKPFPQHDFIKVISHLEFDDLIMNNQPEYREVMEKISEQFKKDDFKVTNRLIELISPVIIPVGNPKRPNWRLICLPGKDTDGFETWKNFVYVTNHCGSDGVSGSNFFKDLALLFCKIEEKGFDYDEEFIEDQVIIDYDRDYTEISKLPKPITDRIDYKPALTSLPKFFLTTFIYEHCNFKTSSESTLTARYSPSTNANASYNYLLHFSTKEVEQIRAQIKKNVHDGCTLTPFIQACFLVALYRLDKLFTKSLLEYGFDVAIPSNARRFLPNDEELRDSYKYGSNVGGSHYAYLISSFDIPEGDNDKFWSLVEYYYDRFLESYDNGDHLIGLGVLQLDFIVQNKNIDSLLANSYLHQQRGGAIISNTGLVSQDTTKPYYVRDLIFSQSAGALRFAFGLNVCSTNVNGMNMDMSVVQGTLRDRGEWESFCKLFYQTIGEFASL

[0019] As used herein, the term "wild-type ATF2 protein" (Aw protein) refers to an ATF2 protein in which, when the amino acid sequence of the As protein is used as a reference, the amino acid residues at a given position are the same as those in the As protein. The predetermined positions are positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, and / or 514 in the As protein, or positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and and / or 514, preferably at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, and 514, or at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514. In the As protein, the amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 are L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, and D514, respectively.

[0020] As used herein, "protein" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0021] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The nucleic acid includes, for example, DNA, RNA, and / or DNA / RNA composed of natural and / or unnatural nucleic acid residues. Examples of the unnatural nucleic acid residue include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue has been modified. When the sugar residue is modified, examples of the unnatural nucleic acid residue include constrained ethyl bicyclic nucleic acid (cEt, manufactured by Ionis Pharmaceuticals), LNA (trademark), Locked Nucleic Acid, and ENA (registered trademark, 2'-O,4'-C-Ethylenebridged Nucleic Acid). The nucleic acid may have, for example, a 5' cap at the 5' end. The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule. The polynucleotide may be composed of naturally occurring nucleotides, modified or artificial nucleotides, or a combination of both.

[0022] As used herein, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0023] As used herein, "exogenous" means something introduced into a host from outside the host.

[0024] As used herein, "isolated" means identified and separated and / or recovered from components of its natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0025] As used herein, an "expression vector" (vector) refers to in vitro orin vivo In the context of the present invention, the term refers to a recombinant plasmid, nucleic acid fragment, or virus that contains a nucleic acid to be delivered to a host cell.

[0026] As used herein, "yeast" refers to wild yeast or yeast used in brewing. The yeast may be, for example: Saccharomyces genus, Schizosaccharomyces genus, Candida genus, Yarrowia genus, Hansenula genus, Kluyveromyces genus, Pichia The genus and the like are exemplified, and preferably Saccharomyces The yeast is, for example, Saccharomyces Among the genus, Saccharomyces cerevisiae The yeast is particularly preferably, for example, Saccharomyces cerevisiae Examples of yeasts include those belonging to the family Genus Genus Genus, which may be haploid or diploid or higher, with diploid being preferred. The wild yeast can be isolated, for example, by the method described in JP 2014-212782 A. Examples of yeasts used in brewing include sake yeast (yeast for sake brewing). Examples of sake yeasts include yeasts sold by the Brewing Society of Japan (K-6, K-7, K-9, K-10, K-11, K-14, K-601, K-701, K-901, K-1001, K-1401, K-1501, K-1801, K-1601, K-1701, Kyokai No. 28, Kyokai No. 77, etc.), and yeasts for sake brewing developed by other public institutions, such as yeasts developed by the Aomori Prefectural Industrial Research Center (Mahoroba Hana Yeast, Yeast Type I, Yeast Type Ro, etc.). The sake yeast may be, for example, an available sake yeast as described above, or a yeast that has been further bred and improved by mutation treatment or the like, and a specific example is K-1801 (also known as Kyokai No. 1801 yeast) sold by the Brewing Society of Japan. The sake yeast may be any yeast that can be used in the production of sake made from rice, and may be, for example, yeast for shochu, baker's yeast, wine yeast, whiskey yeast, etc.

[0027] As used herein, "alcoholic beverages" refers to beverages with an alcohol content of 1% or more (1% by volume). Examples of the alcoholic beverages include sparkling alcoholic beverages, brewed alcoholic beverages, distilled alcoholic beverages, and blended alcoholic beverages. Examples of the sparkling alcoholic beverages include beer and happoshu. Examples of the brewed alcoholic beverages include sake, fruit liquor, and doburoku. Examples of the distilled alcoholic beverages include (pot distillation) shochu, whiskey, brandy, and spirits. Examples of the blended alcoholic beverages include mirin, sweet fruit liquor, and liqueur.

[0028] As used herein, "sake" refers to, for example, sake made primarily from rice, rice koji, and water and fermented with yeast, preferably sake as defined by the Liquor Tax Act of Japan. The "sake" does not, for example, fall under synthetic sake, other brewed alcoholic beverages, spirits, liqueurs, or miscellaneous alcoholic beverages as defined by the Liquor Tax Act. The alcoholic beverages do not include, for example, sake that has been rendered undrinkable by the addition of vinegar or salt in accordance with the Liquor Tax Act. The sake may or may not be designated as a specific name sake as defined by the Standards for Labeling of Sake Production Methods and Quality (National Tax Agency, "Standards for Labeling of Sake Production Methods and Quality" [online], [searched March 19, 2024], https: / / www.nta.go.jp / taxes / sake / hyoji / seishu / kokuji891122 / 03.htm). The sake may be, for example, ginjo sake, daiginjo sake, junmai sake, junmai ginjo sake, junmai daiginjo sake, special junmai sake, honjozo sake, or special honjozo sake. In this specification, the Liquor Tax Act, standards based on the Liquor Tax Act (regulations, notices, etc.), and their interpretations, etc., shall be in accordance with, for example, the filing date of this application (March 19, 2024).

[0029] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequences using appropriate sequence conversion software, etc.

[0030] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0031] <Mutant ATF2 protein> In one aspect, the present disclosure provides an ATF2 protein with improved isoamyl acetate production ability. The mutant ATF2 protein of the present disclosure (hereinafter also referred to as "Am protein") contains at least one mutation in the amino acid sequence of a wild-type yeast ATF2 protein (hereinafter also referred to as "Aw protein") and has improved isoamyl acetate production ability compared to the wild-type ATF2 protein.

[0032] As a result of extensive research, the present inventors have found that the ability of an ATF2 protein to produce isoamyl acetate can be improved by introducing a mutation into a specific position in the yeast Aw protein, and have thus completed the present disclosure. According to the present disclosure, a mutant ATF2 protein can be obtained from yeast ATF2 protein that has improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein.

[0033] The Am protein of the present disclosure may be, for example, an isolated Am protein or an Am protein present in mixture with other substances.

[0034] The Am protein of the present disclosure contains at least one mutation in the amino acid sequence of the Aw protein, and the upper limit of the number of mutations may be within a range such that the Am protein after mutation introduction has improved isoamyl acetate productivity compared to the Aw protein. Specific examples of the number of mutations include 1 to 160, 1 to 133, 1 to 107, 1 to 80, 1 to 53, 1 to 26, 1 to 21, 1 to 16, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0035] The improvement in isoamyl acetate productivity can be evaluated, for example, by preparing a transformant (test group) by introducing a gene encoding a target mutant ATF2 protein into Escherichia coli, and comparing the isoamyl acetate productivity of the transformant with that of a control transformant (control group) that does not contain the gene encoding the target mutant ATF2 protein but does contain a gene encoding a wild-type ATF2 protein, according to Example 1 described below. If the amount of isoamyl acetate produced in the test group is improved compared to the amount of isoamyl acetate produced in the control group, the target mutant ATF2 protein can be evaluated as having improved isoamyl acetate productivity.

[0036] The type of mutation in the mutated amino acid may be within the scope of modifying the function of the Aw protein, and preferably within the scope of improving the ability of the mutated Am protein to produce isoamyl acetate compared to the Aw protein. Therefore, the "mutation" can also be referred to as, for example, modification. Examples of the type of mutation include deletion, substitution, insertion, and / or addition of amino acids. The type of mutation introduced into the Am protein may be one or more.

[0037] The Am protein is a protein consisting of, for example, the amino acid sequence (Am) below, and has improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein.

[0038] (Am) The amino acid sequence (Am1), (Am2), or (Am3) below: (Am1) an amino acid sequence of a wild-type ATF2 protein (Aw protein) having a mutation in at least one amino acid residue selected from the group consisting of amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 in a reference ATF2 protein (As protein, SEQ ID NO: 1); (Am2) an amino acid sequence of (Am1) having one or several insertions, additions, substitutions and / or deletions, and maintaining the mutated amino acid residues of (Am1); (Am3) An amino acid sequence having 70% or more identity to the amino acid sequence of (Am1) and maintaining the mutated amino acid residues of (Am1).

[0039] In the amino acid sequence of (Am1), the Aw protein may be, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 1, 2, or 3.

[0040] In the amino acid sequence of (Am1), the amino acid residue mutation site is at least one selected from the group consisting of amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, and 514, or amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514. In the amino acid sequence of (Am1), the number of mutated amino acids at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, and 514 may be one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17). Furthermore, in the amino acid sequence of (Am1), the number of mutated amino acids at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 may be one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23). The variant amino acid refers to an amino acid residue in the amino acid sequence of (Am1) that is different from the amino acid residue at the corresponding position in the As protein when the amino acid sequence of (Am1) is aligned with the amino acid sequence of the As protein. The alignment can be performed using, for example, FASTA, BLAST, or the like with default parameters.

[0041] When the amino acid sequence of (Am1) contains one mutated amino acid, the position of the mutated amino acid is, for example, 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, or 514 in the As protein. The amino acid residues are those corresponding to the amino acid residues at positions 147, 290, 294, or 514, and because they can further improve the ability to produce isoamyl acetate, they are preferably those corresponding to the amino acid residues at positions 147, 290, 294, 479, 481, 482, 484, or 514. In the As protein, the amino acid residues at positions 147, 290, 294, 352, or 514 are located in a region of the ATF2 protein that is different from the AATF active site. As shown in Example 4 below, these mutations are presumed to contribute to improving the ability of the ATF2 protein to produce isoamyl acetate by, for example, changing the structure of the region containing the active site. Note that this presumption does not in any way limit the present disclosure.

[0042] When there are multiple mutated amino acids in the amino acid sequence of (Am1), the positions of the mutated amino acids may be, for example, amino acid residues corresponding to any two or more amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 398, 401, 410, 411, 468, and 514 in the As protein, or positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 in the As protein. Since this group can further improve the ability to produce isoamyl acetate, the mutations are preferably at amino acid residues corresponding to any two or more of the amino acid residues at positions 147, 250, 290, 301, 340, and 514, or 147, 250, 290, 301, 340, 481, 482, and 514, and more preferably at amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of positions 147 and 340, positions 250 and 301, and positions 290 and 514, or positions 147 and 340, positions 250 and 301, positions 290 and 514, and positions 481 and 482.

[0043] The positions of the mutated amino acids in the amino acid sequence of (Am1) are preferably L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, N398, F401, D410, R411, T468, and D51 in the As protein, because this can further improve the ability to produce isoamyl acetate. 4, or an amino acid residue corresponding to at least one amino acid residue selected from the group consisting of L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, and D514. When the amino acid sequence of (Am1) contains one mutated amino acid, the position of the mutated amino acid is more preferably at L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, N398, F401, D410, R411, T468, or D514 in the As protein, or at L27, E147, T187, F222, D250, E269, N290, S294, F The amino acid residues are those corresponding to the amino acid residues 301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, or D514, and more preferably those corresponding to the amino acid residues E147, N290, S294, or D514, or E147, N290, S294, S479, S481, A482, A484, or D514.Furthermore, when there are a plurality of mutated amino acids in the amino acid sequence of (Am1), the positions of the mutated amino acids are, for example, L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, N398, F401, D410, R411, T468, and D514, or amino acid residues corresponding to any two or more amino acid residues in L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, and D514, Since this can further improve the ability to produce isoamyl acetate, the mutations are preferably in amino acid residues corresponding to any two or more of the amino acid residues in E147, D250, N290, F301, K340, and D514, or E147, D250, N290, F301, K340, S481, A482, and D514, and more preferably in amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of E147 and K340, D250 and F301, and N290 and D514, or E147 and K340, D250 and F301, N290 and D514, and S481 and A482.

[0044] The type of mutation is preferably a substitution, for example, because it is expected that an Am protein having a mutated amino acid will have AATF activity and will likely have improved isoamyl acetate production ability. When the mutation is a substitution, it is preferable that the substitution is not a conservative substitution as described below. In particular, a substitution that changes the charge of the amino acid side chain or a substitution that changes the hydrophobicity of the amino acid side chain is preferred. By introducing such a substitution as a mutation, the Am protein's ability to produce isoamyl acetate can be improved, for example. Specific examples of the substitution include substitution of an amino acid residue having a hydrophilic (polar) side chain with an amino acid residue having a hydrophobic (nonpolar) side chain, substitution of an amino acid residue having a hydrophobic side chain with an amino acid residue having a hydrophilic side chain, substitution of a polar uncharged side chain amino acid residue with a polar charged side chain amino acid residue, substitution of a polar charged side chain amino acid residue with a polar uncharged side chain amino acid residue, substitution of an acidic amino acid residue with a basic amino acid residue, and substitution of a basic amino acid residue with an acidic amino acid residue.

[0045] Suitable amino acid residues for the substituted amino acid residue may be identified by site-directed random mutagenesis, for example, according to Example 1 described below. Specifically, when an amino acid residue corresponding to the amino acid residue at position 27 in the As protein is substituted, the substituted amino acid is, for example, N, T, V, or I. When an amino acid residue corresponding to the amino acid residue at position 294 in the As protein is substituted, the substituted amino acid is, for example, R, W, V, A, or G, preferably R, W, or V, and more preferably R. When an amino acid residue corresponding to the amino acid residue at position 398 in the As protein is substituted, the substituted amino acid is, for example, V, G, W, L, or T. When an amino acid residue corresponding to the amino acid residue at position 410 in the As protein is substituted, the substituted amino acid is, for example, L, A, K, or I.

[0046] As a specific example, the amino acid sequence of (Am1) in the Aw protein is L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, N398S, F401L, D410V, R411S, T468A, and D514G, or L27S, E147D, T187A, F222Y, D250V, E269D, The amino acid sequence has a mutation in the amino acid residue corresponding to at least one amino acid residue selected from the group consisting of N290K, S294R, E307K, K340R, D352N, N398S, F401L, D410V, R411S, T468A, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, G483H, A484G, and D514G. When the amino acid sequence of (Am1) has one mutated amino acid, the amino acid sequence of (Am1) can further improve the ability to produce isoamyl acetate. Therefore, preferably, the Aw protein has one mutated amino acid selected from the group consisting of L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, N398S, F401L, D410V, R411S, T468A, and D514G, or L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, D352N, N398S, F401L, D410V, R411S, T468A, and D514G. and more preferably, an amino acid sequence having a mutation in an amino acid residue corresponding to E147D, N290K, S294R, or D514G, or an amino acid sequence having a mutation in an amino acid residue corresponding to E147D, N290K, S294R, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, A484G, or D514G.When there are a plurality of mutated amino acids in the amino acid sequence of (Am1), the amino acid sequence of (Am1) can further improve, for example, the ability to produce isoamyl acetate, and therefore preferably, in the Aw protein, any two or more of E147D, N290K, K340R, and D514G, or E147D, N290K, K340R, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, A482T, A482L, and D514G. It is an amino acid sequence having mutations in amino acid residues corresponding to the above amino acid residues, more preferably an amino acid sequence having mutations in amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of E147D and K340R, and N290K and D514G, or E147D and K340R, N290K and D514G, S481L and A482P, S481L and A482V, S481L and A482T, and S481L and A482L.

[0047] The mutated amino acid in the amino acid sequence of (Am1) may be specified using the amino acid at position 294 in the As protein as a reference, instead of or in addition to the amino acid at the predetermined position. When the mutated amino acid is specified using the amino acid at position 294 in the As protein as a reference, the amino acid sequence of (Am1) has a mutation in an amino acid residue in the Aw protein that corresponds to an amino acid residue located within 10 Å, 9.5 Å, 9 Å, 8 Å, or 5 Å of the amino acid residue at position 294 in the As protein. When the amino acid residue at position 294 is used as a reference, examples of amino acid residues located within a predetermined distance include the amino acid residues shown in Table 1 below.

[0048] [Table 1]

[0049] In the amino acid sequence (Am2), "one or several" may be any number that maintains the mutated amino acids of (Am1), and preferably any number that results in a protein consisting of the amino acid sequence (Am2) with improved isoamyl acetate productivity compared to the Aw protein. The "one or several" may be, for example, 1 to 160, 1 to 133, 1 to 107, 1 to 80, 1 to 53, 1 to 26, 1 to 21, 1 to 16, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0050] In (Am2), the amino acid substitution may be, for example, a conservative substitution (the same applies hereinafter). The conservative substitution means substituting one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. The "substituting amino acid" and the "substituted amino acid" preferably have similar properties and / or functions. Specifically, they preferably have similar chemical properties such as hydrophobicity and hydrophilicity index (hydropathy), polarity, and charge, or physical properties such as secondary structure. Amino acids or amino acid derivatives having similar properties and / or functions are known in the art, for example. Specific examples of nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine; polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine; positively charged amino acids (basic amino acids) include arginine, histidine, and lysine; and negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0051] In the amino acid sequence (Am3), "identity" may be within a range in which the mutated amino acids in (Am1) are maintained, and preferably within a range in which a protein consisting of the amino acid sequence (Am3) has improved isoamyl acetate productivity compared to the Aw protein. The "identity" refers to, for example, the degree of identity when the compared sequences are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. The "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher. The identity can be calculated using analysis software such as BLAST or FASTA with default parameters (the same applies below).

[0052] In the Am protein, the mutated amino acid may be a mutation artificially introduced, or may be a mutation isolated from wild yeast, brewer's yeast, etc. In the former case, the phrase "having a mutation in an amino acid" can be interpreted as, for example, "an amino acid into which a mutation has been introduced," and the "mutated amino acid" can be interpreted as, for example, "an amino acid into which a mutation has been introduced."

[0053] When the mutated amino acid is an artificially introduced mutation, the Am protein can be introduced into an ATF2 protein, such as the Aw protein or the Am protein, using a method for introducing amino acid mutations into proteins. Specifically, the mutation can be introduced randomly (random mutagenesis method) or site-specifically (site-directed mutagenesis method). For example, the random mutation can be introduced by performing a PCR reaction on a polynucleotide encoding the ATF2 protein or an expression vector containing the same, using biased concentrations of deoxynucleotide triphosphates and in the presence of manganese. For example, the site-directed mutation can be introduced by polynucleotide (e.g., primer)-directed mutagenesis, which allows all types of base pair changes at any specific site in the polynucleotide encoding the ATF2 protein. Specifically, the directed mutagenesis can be performed by performing a PCR reaction using the base sequence of the polynucleotide encoding the ATF2 protein or an expression vector containing the same and a partially complementary polynucleotide containing one or more mismatched bases for the codon encoding the amino acid to be mutated. Examples of such mutations include homologous recombination (DNA shuffling), mutagenesis using a template nucleic acid molecule containing uracil, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using a repair-deficient host strain, deletion mutagenesis, mutagenesis by total gene synthesis, mutagenesis using double-strand break repair, mutagenesis by irradiation treatment with alpha rays, beta rays, gamma rays, X-rays, etc., mutagenesis by chemical treatment with mutagens such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU), mutagenesis by heavy ion beam treatment, and mutagenesis or introduction using genome editing technology.

[0054] When the mutated amino acid is a mutation isolated from wild yeast or brewer's yeast, the mutated amino acid can be isolated from the wild yeast or brewer's yeast by analyzing the amino acid sequence of the ATF2 protein and identifying the amino acid mutation.

[0055] The Am protein of the present disclosure can improve isoamyl acetate productivity, making it suitable for use as an additive protein in brewing, for example.

[0056] <Mutant ATF2 gene> In another aspect, the present disclosure provides a gene encoding the mutant ATF2 protein of the present disclosure. The gene of the present disclosure encodes the mutant ATF2 protein of the present disclosure. Because the gene of the present disclosure encodes the mutant ATF2 protein of the present disclosure, it can be suitably used, for example, to breed a yeast strain with improved isoamyl acetate productivity.

[0057] The gene of the present disclosure is composed of, for example, a nucleic acid (nucleic acid molecule) such as DNA or RNA. Therefore, the gene of the present disclosure can also be referred to as, for example, the nucleic acid or polynucleotide of the present disclosure.

[0058] In the gene of the present disclosure, a nucleic acid encoding the mutant ATF2 protein of the present disclosure can be designed, for example, by substituting corresponding codons based on the amino acid sequence of the Am protein. The base sequence of the nucleic acid may be codon-optimized.

[0059] The genes of the present disclosure can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques. In this case, the genes of the present disclosure can also be referred to as synthetic DNA, synthetic RNA, or synthetic DNA / RNA.

[0060] <Expression vector> The present disclosure provides an expression vector encoding the mutant ATF2 protein of the present disclosure. The expression vector of the present disclosure comprises the gene of the present disclosure. The expression vector of the present disclosure is useful for synthesizing (producing) the Am protein of the present disclosure by genetic engineering techniques. Furthermore, the expression vector of the present disclosure is useful for breeding yeast that expresses the Am protein of the present disclosure.

[0061] The expression vector can also be referred to as, for example, recombinant DNA.

[0062] The expression vector of the present disclosure is an expression vector containing a gene (polynucleotide) encoding the Am protein. Therefore, the gene encoding the Am protein of the present disclosure is inserted into, for example, an expression vector.

[0063] The expression vector of the present disclosure is not particularly limited in its configuration, as long as it contains a gene encoding the Am protein so that the gene encoding the Am protein can be expressed.

[0064] The expression vector of the present disclosure can be prepared, for example, by inserting a gene (polynucleotide) encoding an Am protein into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the vector is not particularly limited and can be determined appropriately depending on, for example, the type of host cell (host).

[0065] Examples of the host include non-human hosts such as microorganisms, animal cells, plant cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, avian cells, and mammalian cells. Examples of the prokaryotic organism include E. coli ( Escherichia coli ) and other Escherichia species, Pseudomonas putida ( Pseudomonas putida Examples of the eukaryote include bacteria such as Pseudomonas, Bacillus, and Mycobacterium, and actinomycetes such as Corynebacterium. Saccharomyces cerevisiaeExamples of the animal cells include yeasts such as yeasts of the genus Aspergillus, and filamentous fungi such as Aspergillus. Examples of the animal cells include COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero cells, and Hep-2 cells. Examples of the insect cells include Spodoptera frugiperda ( Spodoptera frugiperda ) (Sf) cells (e.g., Sf9, Sf21), Trichoplusia ni ( Trichoplusia ni ) cells (e.g., High Five cells), and Drosophila S2 cells.

[0066] The vector (basic vector) can be a non-viral vector or a viral vector. Examples of the viral vector include baculovirus; poxviruses such as vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoon pox virus, and swinepox virus; adenoviruses such as canine adenovirus; adeno-associated virus; herpes virus; and retrovirus. When the heat shock method is used to transform the host, examples of the vector include a binary vector. Examples of the vector include pQE70, pQE60, pQE-9, pBluescript, Phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as Escherichia coli, examples of the vector include the pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, pUC18, pUC19, pUC118, pUC119, pBluescript II SK(+), pBluescript II SK(-), pBluescript II KS(+), and pBluescript II KS(-). When transforming yeast, examples of the vector include pAUR101, pAUR112, pAUR123, pAUR135 (Takara Bio Inc.), pFastBac1, pWINEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL, pYepSec1, pMFa, and pYES2. When transforming insect cells, examples of the vector include pAc and pVL. When transforming mammalian cells, examples of the vector include pCDM8 and pMT2PC.

[0067] The expression vector of the present disclosure preferably has a regulatory sequence that regulates the expression of the gene encoding the Am protein and the expression of the Am protein encoded by the gene. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the expression vector of the present disclosure is not particularly limited. The regulatory sequence in the expression vector of the present disclosure may be located, for example, so long as it is capable of functionally regulating the expression of the gene encoding the Am protein and the expression of the Am protein encoded thereby, and can be located according to known methods. The regulatory sequence may be, for example, a sequence already included in the basic vector, or an additional regulatory sequence may be inserted into the basic vector, or the regulatory sequence included in the basic vector may be replaced with another regulatory sequence.

[0068] The expression vector of the present disclosure may further comprise, for example, a coding sequence for a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.

[0069] Insertion of DNA, insertion of the regulatory sequence, and / or insertion of the coding sequence of the selection marker into the vector may be carried out, for example, by a method using restriction enzymes and ligase, or by using a commercially available kit, etc.

[0070] <Transformants> In another aspect, the present disclosure provides a transformant comprising the gene of the present disclosure. The transformant of the present disclosure comprises the gene of the present disclosure. The transformant of the present disclosure is useful for synthesizing (producing) the Am protein or for producing a microorganism such as yeast with improved ability to produce isoamyl acetate.

[0071] In the transformant of the present disclosure, for example, the gene of the present disclosure exists as an exogenous molecule. In this case, the transformant of the present disclosure can be produced, for example, by introducing the gene of the present disclosure into the host. Alternatively, in the transformant of the present disclosure, for example, the gene of the present disclosure may exist as an endogenous molecule. In this case, the transformant of the present disclosure can be produced, for example, by applying genome editing technology to the ATF2 gene encoding the endogenous Aw protein to introduce a mutation into the gene. Furthermore, when the transformant of the present disclosure has a mutant ATF2 gene modified by genome editing technology, the transformant of the present disclosure can also be referred to as, for example, a microorganism of the present disclosure or a genome-edited microorganism of the present disclosure.

[0072] The method for introducing the gene is not particularly limited and can be carried out by a known method. The gene may be introduced, for example, using the expression vector. The introduction method can be appropriately selected depending on, for example, the type of the host. Examples of the introduction method include introduction using a gene gun such as a particle gun, the lithium acetate method, the spheroplast method, the protoplast method, the heat shock method, the calcium phosphate method, the polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid introduction, DEAE-dextran method, direct injection using microglass tubes or the like, the hydrodynamic method, the cationic liposome method, a method using an introduction adjuvant, and a method mediated by Agrobacterium. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction adjuvant include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, for example, among others, S . cerevisiae , E . coli , B . subtilis , C . glutamicum or Ps . putida A method via the like is preferred. The gene encoding the Am protein of the present disclosure may be introduced into the host using, for example, the expression vector of the present disclosure.

[0073] The genome editing method is not particularly limited and can be carried out by a known method. Introduction of a mutation using the genome editing technology can be carried out, for example, by introducing proteins and nucleic acids constituting the genome editing technology, or vectors encoding these, into the host. The protein can be, for example, CRISPR (Clustered Regularly Interspaced Short Palindromic Recombination) gene. Repeats) enzymes, specific examples of which include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4. Examples of the nucleic acid include crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, the nucleic acid is designed, for example, so that the base sequence that anneals to the target sequence in the crRNA is complementary to the base sequence encoding the ATF2 gene. One type of nucleic acid may be used alone, or two or more types may be used in combination. When using the genome editing technology, for example, the genome editing method can induce large deletions of the base sequence between target sequences by using two or more types of nucleic acids. Furthermore, by using two or more types of nucleic acids and further using a targeting vector (donor vector), the genome editing method can introduce, for example, nucleic acids derived from the targeting vector into the genome of the host.

[0074] <Method for producing mutant ATF2 protein> The present disclosure provides a method for producing the Am protein of the present disclosure. The method for producing the Am protein of the present disclosure is not particularly limited, and examples thereof include production by genetic engineering techniques. In this case, the method for producing the Am protein of the present disclosure includes an expression step of expressing a gene encoding the Am protein. The method for producing the Am protein of the present disclosure makes it possible to produce an Am protein with improved ability to produce isoamyl acetate.

[0075] The gene encoding the Am protein may be expressed, for example, using the expression vector of the present disclosure. The method for expressing the gene encoding the Am protein is not particularly limited, and any known method can be used, for example, a host or a cell-free protein synthesis system.

[0076] In the former case, it is preferable to use the host into which a gene encoding the Am protein or an expression vector containing the gene has been introduced, and to express the gene encoding the Am protein in the host by culturing the host. In this way, for example, by introducing the gene encoding the Am protein or an expression vector containing the gene into a host, a transformant that synthesizes the Am protein of the present disclosure can be produced, and the Am protein can be synthesized by culturing the transformant.

[0077] The method for culturing the host is not particularly limited and can be appropriately determined depending on the type of the host. The medium used for culturing is not particularly limited and can be appropriately determined depending on the type of the host.

[0078] In the latter case, it is preferable to express the gene encoding the Am protein in a cell-free protein synthesis system. In this case, an expression vector may be used to express the gene encoding the Am protein. The cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which the gene encoding the Am protein has been introduced, and for example, a commercially available reagent kit can be used.

[0079] The method for producing an Am protein of the present disclosure may include, for example, a recovery step of recovering the Am protein. The recovery step can be performed using, for example, a known protein purification method. The purification method is not particularly limited, and examples thereof include salting out; density gradient centrifugation using cesium chloride, sucrose, iodixanol, etc.; and various column chromatography methods such as ion exchange columns, hydrophobic columns, and gel filtration columns. The solvent used in the various purification steps is not particularly limited, and examples thereof include water, buffer solutions, etc. The pH of the solvent is, for example, 6 to 8.

[0080] The Am protein obtained by the production method of the present disclosure may be used, for example, as a crudely purified protein (unpurified protein) as is, or as a partially purified protein, or as a single purified protein.

[0081] In addition, in the production method of the present disclosure, the obtained Am protein may be powdered by, for example, freeze-drying, vacuum drying, spray drying, etc. In this case, in the production method of the present disclosure, for example, the Am protein may be dissolved in advance in a buffer solution such as acetate buffer, citrate buffer, imidazole buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD buffer (e.g., PIPES, MES, MOPS, etc.).

[0082] <yeast> In another aspect, the present disclosure provides a yeast having improved ability to produce isoamyl acetate. The yeast of the present disclosure comprises the mutant ATF2 gene of the present disclosure. When the yeast is diploid, the yeast exhibits superior ability to produce isoamyl acetate compared to yeast having a homozygous Aw protein-encoding gene. When the yeast is haploid, the yeast exhibits superior ability to produce isoamyl acetate compared to haploid yeast having the Aw protein-encoding gene.

[0083] When the yeast of the present disclosure is haploid, the yeast has, for example, a gene encoding one Am protein. When the yeast of the present disclosure is diploid or higher, the yeast may have, for example, a gene encoding one Am protein and a gene encoding one Aw protein, or may have genes encoding two or more Am proteins. However, since this can further improve the ability to produce isoamyl acetate, it is preferable that the yeast has genes encoding two or more Am proteins.

[0084] <Yeast extract> In another aspect, the present disclosure provides a yeast extract having improved ability to produce isoamyl acetate. The yeast extract of the present disclosure is the yeast extract of the present disclosure.

[0085] The yeast extract of the present disclosure can be produced, for example, by subjecting the yeast of the present disclosure to an extraction treatment. The extraction treatment can be, for example, a known extraction treatment for microorganisms, and specific examples include disruption treatments such as mechanical disruption and ultrasonic disruption, and solvent extraction treatments such as ethanol.

[0086] <Yeast production method> In another aspect, the present disclosure provides a method for producing yeast with improved isoamyl acetate productivity. The method for producing yeast of the present disclosure includes a mutation step of mutating a wild-type ATF2 gene of yeast into a mutant ATF2 gene, wherein the mutant ATF2 gene is the mutant ATF2 gene of the present disclosure. According to the method for producing yeast of the present disclosure, yeast with improved isoamyl acetate productivity can be produced.

[0087] In the mutation step, a wild-type ATF2 gene is mutated into a mutant ATF2 gene in yeast. For example, when the yeast is diploid, the yeast (parent strain) used in the mutation step may be a yeast having a homozygous wild-type ATF2 gene, or a yeast having a heterozygous wild-type ATF2 gene and a mutant ATF2 gene. The wild-type ATF2 gene is a gene encoding the wild-type ATF2 protein. For example, when the yeast is haploid, the yeast used in the mutation step is a yeast having a wild-type ATF2 gene.

[0088] The method for introducing the mutation in the mutation step is not particularly limited and may be, for example, a known genetic engineering technique such as genome (gene) editing, gene recombination, or site-directed mutagenesis, or a non-genetic engineering technique using a mutagen or the like. The type of mutation is not particularly limited and may be, for example, a base deletion, substitution, insertion, or addition, or a combination thereof. The ATF2 gene is located, for example, at the ATF2 locus. Therefore, in the mutation step, for example, a mutation is introduced into the ATF2 gene located at the ATF2 locus (the same applies hereinafter). The trait conferred by the mutant ATF2 gene, i.e., an increase in the production of isoamyl acetate, is, for example, a dominant trait. Therefore, when the parent strain is haploid, in the mutation step, for example, one ATF2 gene is mutated into a mutant ATF2 gene. Furthermore, when the parent strain is diploid and has two ATF2 genes, the mutation step preferably involves mutating at least one of the two ATF2 genes to a mutant ATF2 gene, i.e., mutating the ATF2 gene to a heterozygous or homozygous state. Furthermore, when the parent strain is diploid and has the ATF2 gene and the mutant ATF2 gene in a heterozygous state, the mutation step preferably involves mutating one ATF2 gene to a mutant ATF2 gene, i.e., mutating the ATF2 gene to a homozygous state.

[0089] The mutagenesis step is a step of introducing a mutation into the ATF2 gene. The method for introducing a mutation in the mutagenesis step can be, for example, the same as that described for the method for introducing a mutation in the mutagenesis step. When the parent strain is haploid, the mutagenesis step involves, for example, introducing a mutation into one ATF2 gene. When the parent strain is diploid and has two ATF2 genes, the mutagenesis step involves, for example, introducing a mutation into at least one of the two ATF2 genes. When the parent strain is diploid and has the wild-type ATF2 gene and the mutant ATF2 gene in a heterozygous state, the mutagenesis step involves, for example, introducing a mutation into the wild-type ATF2 gene, or introducing mutations into the wild-type ATF2 gene and the mutant ATF2 gene.

[0090] Next, in the selection step, yeast having the mutant ATF2 gene is selected from the obtained yeast. In the selection step, for example, the nucleotide sequence of the ATF2 gene after the mutation is introduced is decoded for a parent strain into which a mutation has been introduced, and the amino acid sequence is decoded from the nucleotide sequence. The selection step can be carried out, for example, by confirming whether a mutation has been introduced into the amino acid residue at the predetermined position in the amino acid encoded by the ATF2 gene after the mutation. Specifically, when the parent strain is haploid, for example, yeast having an ATF2 gene in which a mutation has been introduced at the predetermined position in one ATF2 gene is selected as a candidate yeast having improved isoamyl acetate production ability. Furthermore, when the parent strain is diploid, for example, yeast having an ATF2 gene in which a mutation has been introduced at the predetermined position in at least one of two ATF2 genes is selected as a candidate yeast having improved isoamyl acetate production ability. In the selection, the candidate yeast may contain, for example, a mutant amino acid at an amino acid residue other than the predetermined position.

[0091] In the selection step, further selection may be performed based on the ability of the selected candidate yeast to produce isoamyl acetate. In this case, in the selection step, for example, the candidate yeast and the yeast before the introduction of the mutation are cultured and the amount of isoamyl acetate produced by each yeast is compared, according to Example 2 described below. Then, in the selection step, for example, if the amount of isoamyl acetate produced by the candidate yeast is increased compared to the yeast before the introduction of the mutation, the candidate yeast is selected as a yeast with improved ability to produce isoamyl acetate.

[0092] In this way, the yeast production method of the present disclosure can produce yeast with improved ability to produce isoamyl acetate, for example.

[0093] <Method of producing alcohol-containing composition> In another aspect, the present disclosure provides a method for producing an alcohol-containing composition. The method for producing an alcohol-containing composition of the present disclosure is characterized by including a production step of producing an alcohol-containing composition using the yeast of the present disclosure. According to the method for producing an alcohol-containing composition of the present disclosure, for example, when the yeast is diploid, the content of isoamyl acetate in the resulting alcohol-containing composition can be increased compared to when the alcohol-containing composition is produced using yeast that has a homozygous Aw protein-encoding gene. When the yeast is haploid, the content of isoamyl acetate in the resulting alcohol-containing composition can be increased compared to when the alcohol-containing composition is produced using a haploid yeast that has a gene that encodes the Aw protein.

[0094] The alcohol-containing composition may be, for example, a food composition, a cosmetic composition, or an alcoholic beverage.

[0095] The production process can be carried out, for example, by alcohol fermentation using yeast, and can be appropriately determined depending on the alcohol-containing composition. The alcohol is, for example, ethanol. The alcohol-containing composition may be, for example, so-called bioethanol.

[0096] When the alcohol-containing composition is an alcoholic beverage, the production process includes, for example, a fermentation step of fermenting raw materials for the alcoholic beverage. The raw materials and fermentation conditions are not particularly limited and can be determined appropriately depending on, for example, the type of alcoholic beverage. When the alcoholic beverage is sake, the raw materials are, for example, rice. Furthermore, the fermentation conditions are not particularly limited and can be the same as the fermentation conditions used in conventional sake yeast.

[0097] When the alcoholic beverage is a fruit wine or sweet fruit wine, the raw material is, for example, fruit, fruit and water, or fruit or fruit and water to which sugars have been added. The fermentation conditions are not particularly limited, and the same conditions as those for conventional fermentation using wine yeast can be used.

[0098] When the alcoholic beverage is beer, the raw materials are, for example, malt and water, etc. The fermentation conditions are not particularly limited, and the same conditions as those used for conventional fermentation using beer yeast can be used.

[0099] When the alcoholic beverage is shochu, the raw materials are, for example, grains or potatoes, koji made from these grains, and water. The fermentation conditions are not particularly limited, and the same conditions as those used for conventional shochu yeast fermentation can be used.

[0100] When the alcoholic beverage is whiskey, the raw materials are, for example, germinated grains and water. The fermentation conditions are not particularly limited, and the same conditions as those used for conventional fermentation using whiskey yeast or beer yeast can be used.

[0101] The production step preferably includes, for example, a filling step of filling the alcohol-containing composition into a container. In the filling step, after filling the container, it is preferable to seal the container by, for example, sealing the opening of the container. When the production method for the alcohol-containing composition of the present disclosure includes the filling step, a bottled alcoholic beverage can be produced. In this case, the production method for the alcohol-containing composition of the present disclosure can also be referred to as, for example, a "production method for a bottled alcohol-containing composition."

[0102] The container is not particularly limited, and examples thereof include glass containers such as glass bottles and glass cups, metal containers such as aluminum cans, paper cartons for alcoholic beverages, and composite plastic containers such as pouch containers or bottle containers. The container may be labeled with, for example, the type of alcoholic beverage and its contents.

[0103] <Alcohol-containing composition> In another aspect, the present disclosure provides an alcohol-containing composition. The alcohol-containing composition of the present disclosure is obtained by the method for producing an alcohol-containing composition of the present disclosure. The alcohol-containing composition of the present disclosure is produced using the yeast of the present disclosure. Therefore, for example, when the yeast is diploid, the alcohol-containing composition of the present disclosure exhibits a superior isoamyl acetate content compared to an alcohol-containing composition produced using a yeast having a homozygous Aw protein-encoding gene, and when the yeast is haploid, the alcohol-containing composition exhibits a superior isoamyl acetate content compared to an alcohol-containing composition produced using a haploid yeast having a Aw protein-encoding gene.

[0104] The alcohol-containing composition of the present disclosure may be stored, for example, in a container.

[0105] <Food composition> In another aspect, the present disclosure provides a food composition. The food composition of the present disclosure comprises the yeast of the present disclosure or an extract thereof. Therefore, for example, when the yeast is diploid, the food composition of the present disclosure exhibits a superior isoamyl acetate content compared to a food composition produced using a yeast having a homozygous Aw protein-encoding gene, and when the yeast is haploid, the food composition exhibits a superior isoamyl acetate content compared to a food composition produced using a haploid yeast having a Aw protein-encoding gene. The food composition of the present disclosure may be obtained by the method for producing an alcohol-containing composition of the present disclosure.

[0106] The food composition of the present disclosure may be, for example, a food composition containing the yeast of the present disclosure or an extract thereof. Examples of the food composition include bread, vinegar, cooking sake, etc. The food composition may also include, for example, a beverage.

[0107] The food composition of the present disclosure can be produced, for example, by a known production method depending on the type of food composition.

[0108] <Cosmetic composition> In another aspect, the present disclosure provides a cosmetic composition. The cosmetic composition of the present disclosure comprises the yeast of the present disclosure or an extract thereof. Therefore, for example, when the yeast is diploid, the cosmetic composition of the present disclosure exhibits a superior isoamyl acetate content compared to a cosmetic composition produced using a yeast that has a homozygous Aw protein-encoding gene, and when the yeast is haploid, the cosmetic composition exhibits a superior isoamyl acetate content compared to a cosmetic composition produced using a haploid yeast that has the Aw protein-encoding gene. The cosmetic composition of the present disclosure may be obtained by the method for producing an alcohol-containing composition of the present disclosure.

[0109] The cosmetic composition of the present disclosure may be, for example, a cosmetic composition containing the yeast of the present disclosure or an extract thereof. Examples of the cosmetic composition include aqueous cosmetics and oil-based cosmetics.

[0110] The cosmetic composition of the present disclosure can be prepared, for example, by mixing it with other components that constitute the cosmetic composition. [Example]

[0111] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to the attached protocols. In the following description, "mol / l" may also be abbreviated as "M."

[0112] [Example 1] By introducing random and site-directed random mutations into the ATF2 gene, we obtained mutant strains with higher isoamyl acetate production activity than the wild-type enzyme.

[0113] (1) Preparation of expression vector The yeast is Kodo yeast ( Saccharomyces cerevisiae ) was used. Using the chromosomal DNA of Kodo yeast as a template, a primer set (ATF2_NdeI_F (SEQ ID NO: 4) and ATF2cds_R02 (SEQ ID NO: 5)) was used to amplify a nucleic acid containing the gene encoding wild-type ATF2 (As protein (SEQ ID NO: 1)) by PCR. Inverse PCR was performed using pUC118 and primers iv_pUC_NdeI_R (SEQ ID NO: 6) and iv_pUC118_NdeI_F (SEQ ID NO: 7). After PCR, the vector was digested with NdeI and self-ligated to prepare pUC118_NdeI. The amplified fragment containing the wild-type ATF2 gene was cloned into the NdeI-XbaI site of pUC118_NdeI to create pkdATF2_pUC_WT (expression vector 1).

[0114] (2) Random mutation Using the cloned expression vector (expression vector 1) as a template, a primer set (if_ATF2cds_F04 (SEQ ID NO: 8) and if_ATF2cds_R01 (SEQ ID NO: 9)) was used to obtain an amplified fragment in which random mutations were introduced into the wild-type ATF2 gene by PCR. The random mutations were performed using a kit (Diversify™ PCR Random Mutagenesis Kit (Takara Bio Inc., Z0703N)). The kit varied the concentration of dNTPs and Mn 2+ By coexisting with this, base incorporation errors can be induced in Taq polymerase, allowing random mutations to be introduced into the ATF2 gene.

[0115] Next, using the expression vector 1 and primers iv_pUC_NdeI_R02 (SEQ ID NO: 10) and iv_ATF2dw_F01 (SEQ ID NO: 11), a pkdATF2_pUC amplified fragment (expression vector 2) was obtained by PCR. The obtained mutated fragment was cloned into the expression vector 2 to obtain pkdATF2_pUC_RD (expression vector 3). The cloning was performed using a cloning kit (In-Fusion™ HD Cloning Kit (Takara Bio Inc., Z9648N)). The nucleotide sequences of the primers used for PCR are shown in Table 2 below.

[0116] [Table 2]

[0117] (3) Preparation of crude enzyme solution The Escherichia coli used was the DH5α strain. The culture medium used for the Escherichia coli was LB medium (1.0% Bactotryptone, 0.5% yeast extract, and 1.0% (w / v) NaCl) containing 100 μg / ml ampicillin. The expression vector 3 prepared in Example 1(2) above was introduced into the Escherichia coli to obtain a transformant. The resulting transformant was cultured at 25°C for 2 days. The resulting culture was then centrifuged (20,400 × g, 5 minutes) to recover 1.5 ml of bacterial cells. 0.5 ml of lysozyme solution (20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.2% Triton X-100, 0.1% lysozyme) was added to the recovered bacterial cells and incubated at 37°C for 2 hours. The mixture was then centrifuged (20,400 × g, 5 minutes) to recover the supernatant as a crude enzyme solution.

[0118] (4) Measurement of isoamyl acetate AATFase activity was measured using a modified version of the method described in Japanese Patent Application Laid-Open No. 2002-191355. Specifically, 50 μl of the crude enzyme solution was added to a test solution containing 25 mM Tris-HCl (pH 8.0), 46 mM isoamyl alcohol, 1 mM acetyl-CoA, 0.1% Triton X-100, 1 mM DTT, 0.1 M NaCl, and 20% glycerol to prepare a 1.0 ml enzyme reaction solution. The resulting enzyme reaction solution was reacted in a 10 ml vial at 25°C for 1 hour. The reaction was then terminated by adding 0.6 g of NaCl to the enzyme reaction solution. The isoamyl acetate produced by the enzyme reaction was measured using gas chromatography-mass spectrometry (GC / MS, Agilent GC7890A, Agilent MS5975C) according to the standard analytical method of the National Research Institute of Brewing (see Reference 1 below). The amount of isoamyl acetate produced was analyzed in the same manner, except that a transformant obtained using the expression vector 1 (wild-type ATF2 protein) was used as a control. The GC / MS measurement conditions are as follows. The measurement results of isoamyl acetate obtained by the enzymatic reaction are shown in Figure 1. Reference 1: Brewery Society of Japan, National Research Institute of Brewing, Standard Analysis Methods Commentary 2017.

[0119] (GC / MS measurement conditions) Equipment: Agilent GC7890A, Agilent MS5975C Column: DB-WAX 0.25 mmφ×30 m, film thickness 0.25 μm Column temperature: 40°C (5 min) → 5°C / min → 120°C → 10°C / min → 220°C (5 min) (total 36 min) Carrier gas: Helium Inlet temperature: 250℃ Ion source temperature: 230℃ Mass range: m / z 35-500 Ionization method: EI Ionization voltage: 70 eV

[0120] FIG. 1 shows the isoamyl acetate production activity of transformants containing mutant ATF2 genes obtained by random mutation. In FIG. 1, the horizontal axis shows the relative OD600 value when the average OD600 value of the control culture solution is set to 1, and the vertical axis shows the relative amount of isoamyl acetate produced when the average amount of isoamyl acetate produced in the control enzyme reaction solution is set to 1. As shown in FIG. 1, strains incorporating the randomly mutated ATF2 gene were identified that produced higher amounts of isoamyl acetate than the wild-type. One hundred and seventy-six strains incorporating the randomly mutated ATF2 gene were screened, and 31 strains with higher activity than the wild-type ATF2 protein were obtained. Of the strains incorporating the randomly mutated ATF2 gene screened, 20 strains with the highest activity were selected. Expression vectors were extracted from the 20 selected strains, and the nucleotide and amino acid sequences of the mutant ATF2 genes were confirmed. As a result, the following mutants were obtained: L27S, in which the 27th leucine of the 535 amino acids constituting the ATF2 protein was replaced with serine; T187A, F222Y, E269D, S294G, E307K, N398S, F401L, D410V, R411S, T468A, E147D-K340R (two substitutions), D250V-F301X (X is any amino acid), and N290K-D514G (two substitutions). The results of comparing the isoamyl acetate production of the random mutants with that of wild-type ATF2 are shown in Table 3 below.

[0121] [Table 3]

[0122] Table 3 shows the ratio of the amount of isoamyl acetate produced in the randomly mutated strains to that produced by the wild-type ATF2. As shown in Table 3, the amount of isoamyl acetate produced in the randomly mutated strains was found to be higher than that produced by the wild-type ATF2.

[0123] (5) Site-specific random mutation Of the substitution variants obtained by random mutation, site-specific random mutations were introduced at four positions: L27, S294, D398, and N410. Site-specific random mutations were performed by PCR using a 32-mix primer set (SEQ ID NOS: 12-14 and 16 in Table 4 below) in which the base sequence encoding the amino acid residue at each position was set to "NNS" (N: A, C, G, or T; S: G or C), which can encode 20 amino acids. The amplified fragments into which the site-specific random mutations were introduced were introduced into expression vector 2 to obtain each expression vector. The L27 substitution variant, N398 substitution variant, and D410 substitution variant were produced by the megaprimer method. Using expression vector 1 as a template and a primer set tailored to each substitution, amplified fragments (megaprimers) containing each substitution were obtained by PCR. For the L27 substitution mutant, the primer set was if_ATF2cds_F04 (SEQ ID NO: 8) and kdATF2_L027X_R (SEQ ID NO: 12), and the resulting amplified fragment was designated Megaprimer 1. For the N398 substitution mutant, the primer set was kdATF2_N398X (SEQ ID NO: 13) and if_ATF2cds_R01 (SEQ ID NO: 9), and the resulting amplified fragment was designated Megaprimer 2. For the D410 substitution mutant, the primer set was kdATF2_D410X (SEQ ID NO: 14) and if_ATF2cds_R01 (SEQ ID NO: 9), and the resulting amplified fragment was designated Megaprimer 3. For the L27 substitution mutant, full-length ATF2 was obtained by PCR using Expression Vector 1 as a template, Megaprimer 1, and if_ATF2cds_R01 (SEQ ID NO: 9). Similarly, full-length ATF2 was obtained by PCR using megaprimer 2 and if_ATF2cds_F04 (SEQ ID NO: 8) for the N398 substitution variant, and megaprimer 3 and if_ATF2cds_F04 (SEQ ID NO: 8) for the D410 substitution variant. The amplified fragments obtained for each substitution variant were cloned into expression vector 2 to obtain an expression vector for the L27 substitution variant (pkdATF2_pUC_L027X), an expression vector for the N398 substitution variant (pkdATF2_pUC_N398X), and an expression vector for the D410 substitution variant (pkdATF2_pUC_D410X).The cloning was performed using a cloning kit (In-Fusion™ HD Cloning Kit (Takara Bio Inc., Z9648N)). The S294 substitution was prepared by simultaneously ligating two fragments amplified using expression vector 1 as a template with primers if_ATF2cds_F04 (SEQ ID NO: 8) and iv_kdATF2_S294X_R (SEQ ID NO: 15) and primers kdATF2_S294X (SEQ ID NO: 16) and if_ATF2cds_R01 (SEQ ID NO: 9) into expression vector 2 to prepare an expression vector (pkdATF2_pUC_S294X). The nucleotide sequences of the primers used for PCR are shown in Table 4 below.

[0124] [Table 4]

[0125] (6) Measurement of isoamyl acetate in site-directed randomly mutated strains A crude enzyme solution was prepared using the resulting expression vector in the same manner as in Example 1(3). The isoamyl acetate-producing activity of the resulting crude enzyme solution was then measured. Furthermore, 46 strains were selected for each substitution at each site. Among the transformants containing the selected substitutions, those containing mutant ATF2 proteins with higher isoamyl acetate-producing activity than the wild-type ATF2 protein were selected. The expression vectors were extracted and sequenced to determine the nucleotide and amino acid sequences encoding the mutant ATF2 proteins. The amount of isoamyl acetate produced was analyzed in the same manner as in Example 1(4), except that a transformant obtained using the expression vector 1 was used as a control. The results of measuring the isoamyl acetate-producing activity of the crude enzyme solution are shown in Figure 2 and Tables 5 to 8 below.

[0126] Figure 2 shows the isoamyl acetate production activity of mutants in which amino acid substitutions were made by site-directed random mutagenesis. In Figure 2, the horizontal axis shows the relative OD600 value when the average OD600 value of the control culture solution is set to 1, and the vertical axis shows the relative amount of isoamyl acetate produced when the average amount of isoamyl acetate produced in the control enzyme reaction solution is set to 1. The amino acid residues shown in Figure 2 indicate the amino acid residues after substitution. As shown in Figure 2, transformants expressing mutant ATF2 proteins that produce higher amounts of isoamyl acetate than the wild-type ATF2 protein were confirmed.

[0127] [Table 5]

[0128] [Table 6]

[0129] [Table 7]

[0130] [Table 8]

[0131] Tables 5 to 8 show the ratios of the isoamyl acetate production amounts of the L27 substitution mutant, S294 substitution mutant, L398 substitution mutant, and D410 substitution mutant to the wild-type ATF2, respectively. As shown in Tables 5 to 8, it was confirmed that the isoamyl acetate production activity of the L27 substitution mutant, N398 substitution mutant, and D410 substitution mutant was approximately 1.1 to 1.2 times higher than that of the wild-type ATF2. No amino acids common to the strains with high isoamyl acetate production activity were identified, suggesting that amino acid substitutions at these sites are important. Furthermore, it was confirmed that the S294 substitution mutant ATF2 protein had approximately 1.3 to 1.5 times higher isoamyl acetate production activity than the wild-type ATF2 protein. It was confirmed that the strains with high isoamyl acetate production activity were particularly substituted with arginine.

[0132] (7) Three-dimensional structure prediction The wild-type ATF2 protein was subjected to three-dimensional structure prediction using AlphaFold2 (DeepMind Technologies). The results of the three-dimensional structure prediction are shown in FIG.

[0133] Figure 3 shows the results of the predicted three-dimensional structure of ATF2. In Figure 3, (A) to (C) show the three-dimensional structure of the ATF2 protein at different angles. Also, in Figure 3, amino acid residues predicted to be important for activity are enclosed by dashed lines. Amino acid residues mutated by random mutagenesis are enclosed by solid lines. Amino acid residues mutated by site-directed random mutagenesis are circled. As shown in Figure 3(A), L27 was predicted to be located near the binding site for the coenzyme acetyl-CoA. As shown in Figure 3(B), E147, N290, S294, and D514 were predicted to be located nearby in the three-dimensional structure, but in positions different from the active site of the ATF2 protein. Furthermore, S294 was predicted to be located inside the ATF2 protein. As shown in Figure 3(C), D398 and N410 were predicted to be located near the entrance and middle of the α-helix, respectively. These findings indicate that many of the substitutions isolated by random mutagenesis are located at positions different from the enzymatic active site of the ATF2 protein.

[0134] [Example 2] The mutant ATF2 protein obtained in Example 1 was expressed in yeast, and it was confirmed that the resulting transformant had high isoamyl acetate-producing activity.

[0135] (1) Preparation of ATF2 expression vector for Kodo yeast An ATF2 protein expression vector for Kodō yeast, as shown in Figure 4A, was constructed. pAUR112 (Takara Bio Inc., 3601) was used as the base vector. Using the chromosomal DNA of Kodō yeast as a template, a pgk1 promoter fragment was amplified by PCR using the primer set (pAUR-propgk (SEQ ID NO: 17) and Ppgk-atf2R (SEQ ID NO: 18)). Using the chromosomal DNA of Kodō yeast as a template, a primer set (Ppgk-atf2F (SEQ ID NO: 19) and ter-pAUR (SEQ ID NO: 20)) was used to amplify the ATF2 gene ORF (atf2 ORF) and atf2 terminator fragment by PCR. The amplified pgk1 promoter fragment, atf2 ORF, and atf2 terminator fragments were cloned into pAUR112 that had been digested with the Sal1 and Xho1 restriction enzymes. The cloning was performed using a cloning kit (In-Fusion™ HD Cloning Kit (Takara Bio Inc., Z9648N)). The resulting expression vector was designated as the ATF2 expression vector for Kodo yeast (pAUR112-Ppgk-Atf2(KODO)).

[0136] (2) Preparation of ATF2 expression vector for K901 An ATF2 expression vector for K901 (Kyoto yeast strain) was constructed as shown in Figure 4A. The base vector was the pAUR112 described above. Using the chromosomal DNA of Komichi yeast as a template, a pgk1 promoter fragment was amplified by PCR using the primer set (pAUR-propgk (SEQ ID NO: 17) and Ppgk-atf2R (SEQ ID NO: 18)). Using the chromosomal DNA of K901 as a template, a primer set (Ppgk-atf2F (SEQ ID NO: 19) and ter-pAUR (SEQ ID NO: 20)) was used to amplify the ORF of the ATF2 gene (atf2 ORF) and the atf2 terminator fragment by PCR. The amplified pgk1 promoter fragment, atf2 ORF, and atf2 terminator fragment were cloned into pAUR112 that had been digested with restriction enzymes Sal1 and Xho1. The cloning was performed using the cloning kit described above. The resulting expression vector was designated as the ATF2 expression vector for K901 (pAUR112-Ppgk-Atf2(K901)).

[0137] (3) Preparation of ATF2(S294R) expression vector for Kodo yeast An ATF2(S294R) expression vector for Kodo yeast was constructed by introducing S294R, which has high isoamyl acetate production activity, from the mutant ATF2 gene obtained in Example 1, as shown in Figure 4B. The base vector used was pAUR112. Using the Kodo yeast ATF2 expression vector (pAUR112-Ppgk1-Atf2(KODO)) as a template and a primer set (pAUR-propgk (SEQ ID NO: 17) and if_kdATF2_S294R_R (SEQ ID NO: 21)), a mutant ATF2 gene fragment (atf2_S294R mutation fragment) containing the S294R substitution was amplified by PCR. Furthermore, using the pAUR112-Ppgk1-Atf2(KODO) as a template, a primer set (if_kdATF2_N296_F (SEQ ID NO: 22) and ter-pAUR (SEQ ID NO: 20)) was used to amplify the polynucleotide (atf2C) encoding the C-terminal region of the ATF2 gene and the atf2 terminator fragment by PCR. The amplified atf2_S294R mutant fragment and the atf2C and atf2 terminator fragments were cloned into pAUR112 that had been digested with restriction enzymes Sal1 and Xho1. The cloning was performed using the cloning kit described above. The resulting expression vector was designated the Kodo yeast ATF2(S294R) expression vector (pAUR112-Ppgk-Atf2_S294R(KODO)).

[0138] (4) Preparation of ATF2(S294R) expression vector for K901 An ATF2(S294R) expression vector for K901 was constructed by introducing S294R, which has high isoamyl acetate production activity, from the mutant ATF2 gene obtained in Example 1, as shown in Figure 4C. The base vector used was pAUR112. Using the pAUR112-Ppgk1-Atf2(K901) as a template and a primer set (pAUR-propgk (SEQ ID NO: 17) and if_kdATF2_S294R_R_K901 (SEQ ID NO: 23)), a mutant ATF2 gene fragment (atf2_S294R mutation fragment) containing the S294R substitution was amplified by PCR. Using the pAUR112-Ppgk1-Atf2(K901) as a template and a primer set (if_kdATF2_N296_F_K901 (SEQ ID NO: 24) and ter-pAUR (SEQ ID NO: 20)), a polynucleotide encoding the C-terminal region of the ATF2 gene (atf2C) and the atf2 terminator fragment were amplified by PCR. The amplified atf2_S294R mutant fragment and the atf2C and atf2 terminator fragments were cloned into pAUR112 that had been digested with restriction enzymes Sal1 and Xho1. The cloning was performed using the cloning kit described above. The resulting expression vector was designated the ATF2(S294R) expression vector for K901 (pAUR112-Ppgk-Atf2_S294R(K901)). The primers used for PCR are listed in Table 9 below.

[0139] [Table 9]

[0140] (5) Yeast transformation The yeast is Saccharomyces cerevisiaeBY4742 (National BioResource Project (NBRP) BY6314) was used. Each of the expression vectors prepared in Example 2(1) to (4) and the empty vector pAUR112 was introduced into yeast using electroporation (Gene Pulser Xcell® (BIO-RAD) 1.5 kV, 25 μF, 200 Ω) for transformation. The resulting transformants were cultured at 30°C for 4 days on YPD agar medium (Becton Dickinson, 242720) containing 0.5 μg / ml of aureobasidin A (Takara Bio Inc., Z0499N). After the culture, formed colonies were isolated and transformants were selected.

[0141] (6) Preparation of crude enzyme solution A loopful of the selected colony was inoculated into 2 ml of YPD liquid medium containing 0.5 μg / ml aureobasidin A. The inoculated medium was cultured at 30°C for 3 days with shaking (160 rpm). The turbidity (OD: 660 nm) of the resulting liquid medium was measured, and a portion of the liquid medium was inoculated into 2 ml of YPD10 liquid medium (YPD with a 10% glucose concentration) containing 0.5 μg / ml aureobasidin A so that the OD660 was 0.01 / ml, and cultured. The culture was statically cultured at 30°C for 2 days. The liquid medium after the culture was transferred to a tube and centrifuged (9,100 × g, 5 minutes). After the centrifugation, the supernatant was removed. After the supernatant was removed, glass beads (0.5 mm) were added to the bacterial cells, followed by the addition of 0.5 ml of extraction buffer (25 mM imidazole-HCl buffer (pH 7.5), 0.1% Triton X-100, 0.1 M NaCl, 1 mM DTT, and 20% glycerol). After the addition, the bacterial cells were disrupted using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.). The disruption was performed at 1°C for a total of 10 cycles, each cycle consisting of 2,500 rpm for 30 seconds and 0 rpm for 30 seconds. The resulting disrupted bacterial cell solution was centrifuged (9,100 × g, 5 minutes), and 0.5 ml of the supernatant was collected. This supernatant was used as the first crude enzyme solution. 0.5 ml of the extraction buffer was added again to the residue after the supernatant was collected. The bacterial cells were disrupted using the Multi-Beads Shocker under the same conditions as the previous disruption. The resulting disrupted bacterial cell solution was centrifuged (9,100 × g, 5 minutes), and 0.5 ml of the supernatant was collected. The resulting supernatant was used as the second crude enzyme solution. The first and second crude enzyme solutions were mixed, and a total of 1 ml of crude enzyme solution was collected. The protein content of the crude enzyme solution was measured using a Bio-Rad protein assay (BIO-RAD, 5000006).

[0142] (7) Measurement of isoamyl acetate A reaction solution was prepared by dissolving 50 mM isoamyl alcohol and 1 mM acetyl-CoA lithium salt in the extraction buffer. 1 ml of the reaction solution and 0.05 ml of the crude enzyme solution were added to a 10 ml vial, and the enzyme reaction was carried out at 25°C for 1 hour. After the enzyme reaction, 0.6 g of NaCl was added to stop the enzyme reaction. 0.05 ml of an internal standard solution (n-pentanol 200 mg / L) was added to the enzyme reaction solution, and isoamyl acetate was measured by gas chromatography-mass spectrometry under the same conditions as in Example 1(4). The average production amount of isoamyl acetate is shown in Table 10 below.

[0143] [Table 10]

[0144] Table 10 shows the average isoamyl acetate production of transformants into which each expression vector was introduced. As shown in Table 10, the Kodo yeast ATF2(S294R) expression vector group, which contains a mutant ATF2 gene with the S294R substitution, was confirmed to produce 3.1-fold more isoamyl acetate than the Kodo yeast ATF2 expression vector group. Furthermore, the K901 ATF2(S294R) expression vector group was confirmed to produce 1.2-fold more isoamyl acetate than the K901 ATF2 expression vector group. In other words, it was found that introduction of the S294R high-expression vector increased isoamyl acetate production. These results demonstrate that yeast can also produce isoamyl acetate with the same improved isoamyl acetate productivity as E. coli.

[0145] [Example 3] The yeast transformants obtained in Example 2 were cultured in koji medium, and it was confirmed that the yeast into which the Kodo yeast ATF2(S294R) expression vector had been introduced produced increased amounts of isoamyl acetate compared to other transformants.

[0146] (1) Yeast cultivation The medium used was a koji soup medium supplemented with aureobasidin A at 0.5 μg / ml. The koji soup medium was prepared by adding 5.7 L of water to 1 kg of dried koji (Tokushima Koji Co., Ltd., 1-60), saccharifying at 55°C for 16 hours, and then filtering the filtrate. Water was added to the filtrate to a sucrose reading of 10°C. Furthermore, 0.3 g of KH2PO4 and 0.3 g of NaCl were added per liter. For preculture, one loopful of yeast containing each expression vector obtained in Example 2 was inoculated into 2 ml of the koji soup medium. The inoculated medium was cultured at 30°C for 3 days with shaking (160 rpm). The turbidity (OD: 660 nm) of the resulting liquid medium was measured, and a portion of the koji soup medium was inoculated into 50 ml of the koji soup medium to achieve an OD660 of 0.01, followed by expansion. The expansion was performed by static culture at 15°C. The koji medium was sampled at predetermined culture days (7th, 10th, or 14th days) based on the start of the culture (day 0). The turbidity (OD: 660 nm) of the sampled medium was measured to confirm that each transformant had similar growth ability. Each sample was centrifuged, and the isoamyl acetate in the supernatant was measured by GC / MS in the same manner as in Example 1(4).

[0147] Next, the average production amount of isoamyl acetate in the sampled medium on the 10th day of the culture is shown in Table 11 below.

[0148] [Table 11]

[0149] Table 11 shows the average amount of isoamyl acetate produced in yeast into which each expression vector was introduced. As shown in Table 11, the Komichi yeast ATF2(S294R) expression vector group showed an approximately 2.2-fold increase in isoamyl acetate production compared to the Komichi yeast ATF2 expression vector group. It was also confirmed that the K901 ATF2(S294R) expression vector group showed an approximately 1.1-fold increase in isoamyl acetate production compared to the K901 ATF2 expression vector group.

[0150] [Example 4] In the ATF2 protein, amino acid residues present in the vicinity of amino acid residue S294 were predicted.

[0151] The amino acid sequence of the Kodo yeast ATF2 protein (SEQ ID NO: 1) and the mutant ATF2 protein (S294R) were predicted using AlphaFold2 (DeepMind Technologies). The three-dimensional structures of the two proteins were then compared. The results are shown in Figure 5.

[0152] Figure 5 shows the predicted three-dimensional structures of the Kodo yeast ATF2 protein and the mutant ATF2 protein (S294R). In Figure 5, (A) shows the predicted three-dimensional structures of the Kodo yeast ATF2 protein (S294) and the mutant ATF2 protein (S294R(R294)), as well as a predicted diagram overlaying these structures. (B) shows the relative positions of R294 and S481 in the mutant ATF2 protein (S294R). As shown in Figure 5(A), the predicted overall three-dimensional structure of the mutant ATF2 protein (S294R) was almost identical to that of the Kodo yeast ATF2 protein. On the other hand, as shown in Figure 5(B), the distance between the side chains at amino acids 294 and 481 in the mutant ATF2 protein (S294R) was shortened, suggesting that an interaction occurs.

[0153] In the mutants obtained by random mutagenesis in Example 1, substitutions of multiple amino acid residues near the amino acid residue at position 294 demonstrated improved isoamyl acetate production. Considering the structural changes in the predicted three-dimensional structure, it is predicted that introducing mutations into amino acid residues near position 294 of the ATF2 protein, thereby shortening the distance between the side chains of the amino acid residues at positions 294 and 481, will similarly improve isoamyl acetate production. These mutations shorten the distance between the side chains of the amino acid residues at positions 294 and 481, shifting the position of the loop structure containing the amino acid residue at position 481, thereby facilitating access of acetyl-CoA and substrates to the active center, thereby contributing to improved isoamyl acetate production by the ATF2 protein. Therefore, amino acid residues located within a specified distance from the amino acid residue at position 294 in the ATF2 protein of Kodo yeast were extracted. The results are shown in Table 12 below.

[0154] [Table 12]

[0155] As shown in Table 12, the mutants contained multiple amino acid residues that had been substituted in the mutants obtained by the random mutagenesis in Example 1. Furthermore, the mutants contained multiple substitution sites that improved the ability to produce isoamyl acetate in Example 5 described below. Based on these findings, it is expected that a mutant ATF2 protein with improved ability to produce isoamyl acetate can be obtained by modifying amino acid residues located within a certain distance from position 294 of ATF2.

[0156] [Example 5] It was confirmed that the isoamyl acetate-producing activity of the ATF2 protein was improved by substituting the amino acid residues D352, S479, S481, A482, G483, A484, and S481-A482.

[0157] (1) Site-specific random mutation First, D352 substitution, S479 substitution, S481 substitution, A482 substitution, G483 substitution, A484 substitution, and S481-A482 substitution were prepared. Site-specific random mutagenesis was performed in the same manner as in Example 1(5) above, except that the primers in Table 13 below were used instead of the 32-mix primers (SEQ ID NOs: 12 to 14 and 16) in Example 1(5) above.

[0158] Specifically, the D352 substitution was obtained by amplifying pkdATF2_pUC_WT (expression vector 1) as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_F351_Rv01 (sequence number 25), or primers if_kdATF2_D352X_Fw01 (sequence number 26) and if_ATF2cds_R01 (sequence number 9). The two amplified fragments obtained were simultaneously cloned into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2D352X).

[0159] The S479 substitution was obtained by amplifying the expression vector 1 as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_S479X_Rv01 (sequence number 27), or primers if_kdATF2_S481_Fw01 (sequence number 28) and if_ATF2cds_R01 (sequence number 9), and cloning the resulting two amplified fragments simultaneously into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2S479X).

[0160] The S481 substitution was obtained by amplifying the expression vector 1 as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_Q480_Rv01 (sequence number 29), or primers if_kdATF2_S481X_Fw01 (sequence number 30) and if_ATF2cds_R01 (sequence number 9), and cloning the resulting two amplified fragments simultaneously into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2S481X).

[0161] The A482 substitution was obtained by amplifying the expression vector 1 as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_S481_Rv03 (sequence number 31), or primers if_kdATF2_A482X_Fw01 (sequence number 32) and if_ATF2cds_R01 (sequence number 9), and cloning the resulting two amplified fragments simultaneously into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2A482X).

[0162] The G483 substitution was obtained by amplifying the expression vector 1 as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_S481_Rv03 (sequence number 31), or primers if_kdATF2_G483X_Fw01 (sequence number 33) and if_ATF2cds_R01 (sequence number 9), and simultaneously ligating the resulting two amplified fragments into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2G483X).

[0163] The A484 substitution was prepared by using the expression vector 1 as a template and primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_G483_Rv01 (sequence number 34), or primers if_kdATF2_A484X_Fw01 (sequence number 35) and if_ATF2cds_R01 (sequence number 9), and simultaneously cloning the two amplified fragments into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2A484X).

[0164] The S481-A482 substitution was obtained by amplifying the S481L substitution from the plasmid pUCNdeI_kdATF2S481L as a template using primers if_ATF2cds_F04 (SEQ ID NO: 8) and if_kdATF2_S481L_Rv02 (SEQ ID NO: 36), or primers if_kdATF2_S481L-A482X_Fw01 (SEQ ID NO: 37) and if_ATF2cds_R01 (SEQ ID NO: 9). The two amplified fragments were simultaneously cloned into expression vector 2 to obtain the expression vector (pUCNdeI_kdATF2S481L-A482X). The nucleotide sequences of the primers used in PCR are shown in Table 13 below. The cloning kit was used for cloning each substitution.

[0165] [Table 13]

[0166] (2) Preparation of crude enzyme solution A crude enzyme solution was prepared in the same manner as in Example 1(3), except that the expression vector prepared in Example 5(1) was used instead of the expression vector 3 prepared in Example 1(2).

[0167] (3) Measurement of isoamyl acetate The crude enzyme solution prepared in Example 5(2) was used to measure the amount of isoamyl acetate produced, as in Example 1(4). Among the transformants containing the substitution products, those containing mutant ATF2 proteins with isoamyl acetate production activity equivalent to or greater than that of a crude enzyme solution containing wild-type ATF2 protein (control) were selected. Expression vectors were extracted and sequenced to determine the nucleotide and amino acid sequences encoding the mutant ATF2 proteins. The results of measuring the isoamyl acetate production activity of each crude enzyme solution are shown in Figure 6 and Tables 14 to 20.

[0168] Figure 6 shows the isoamyl acetate production activity of the D352 substitution mutant, S479 substitution mutant, S481 substitution mutant, A482 substitution mutant, G483 substitution mutant, A484 substitution mutant, and S481-A482 substitution mutant. In Figure 6, the horizontal axis shows the relative OD600 value when the average OD600 value of the control culture solution is set to 1, and the vertical axis shows the relative value of the amount of isoamyl acetate produced when the average amount of isoamyl acetate produced in the control enzyme reaction solution is set to 1. The amino acid residues shown in Figure 6 indicate the amino acid residues after substitution. As shown in Figure 6, transformants expressing mutant ATF2 proteins that produce higher amounts of isoamyl acetate than the wild-type ATF2 protein were confirmed.

[0169] [Table 14]

[0170] [Table 15]

[0171] [Table 16]

[0172] [Table 17]

[0173] [Table 18]

[0174] [Table 19]

[0175] [Table 20]

[0176] Tables 14 to 20 show the ratios of isoamyl acetate production amounts in the D352 substitution mutant, S479 substitution mutant, S481 substitution mutant, A482 substitution mutant, G483 substitution mutant, A484 substitution mutant, and S481-A482 substitution mutant, respectively, relative to the wild-type ATF2 protein. As shown in Tables 14 to 20, it was confirmed that the isoamyl acetate production activity of the D352 substitution mutant, S479 substitution mutant, S481 substitution mutant, A482 substitution mutant, G483 substitution mutant, A484 substitution mutant, and S481-A482 substitution mutant was approximately 1.04 to 1.9 times higher than that of the wild-type ATF2 protein.

[0177] [Example 6] The codon encoding the amino acid at position 294 of the S294R substitution mutant was changed, and it was confirmed that the ability to produce isoamyl acetate was equivalent.

[0178] (1) Preparation of expression vector In the site-directed random mutagenesis of S294X in Example 1(5) above, two expression vectors encoding the S294R substitution were obtained: one in which the codon encoding arginine (R) at position 294 is "cgg" (pUCNdeI_kdATF2S294R(cgg)), and the other in which the codon encoding arginine (R) at position 294 is "agg" (pUCNdeI_kdATF2S294R(agg)). Therefore, we investigated whether the isoamyl acetate production activity would change if the codon encoding arginine (R) was "aga" or "cgc." An expression vector in which the codon at site 294 is "aga" was prepared by amplifying the expression vector 1 as a template using primers if_ATF2cds_F04 (sequence number 8) and if_kdATF2_S294R_R (sequence number 21), or primers if_kdATF2_N296_F (sequence number 22) and if_ATF2cds_R01 (sequence number 9). The two amplified fragments obtained were simultaneously cloned into expression vector 2 to produce an expression vector (pUCNdeI_kdATF2S294R(aga)). An expression vector in which the codon at position 294 is "cgc" was prepared by amplifying the expression vector 1 as a template with primers if_ATF2cds_F04 (SEQ ID NO: 8) and if_kdATF2_A293_Rv01 (SEQ ID NO: 38), or primers if_kdATF2_S294R(cgc)_Fw01 (SEQ ID NO: 39) and if_ATF2cds_R01 (SEQ ID NO: 9). The two amplified fragments obtained were simultaneously cloned into expression vector 2 to prepare an expression vector (pUCNdeI_kdATF2S294R(cgc)). The nucleotide sequences of the primers used in PCR are shown in Table 21 below. The cloning kit was used.

[0179] [Table 21]

[0180] (2) Preparation of crude enzyme solution A crude enzyme solution was prepared in the same manner as in Example 1(3), except that the expression vector prepared in Example 6(1) was used instead of the expression vector 3 prepared in Example 1(2).

[0181] (3) Measurement of isoamyl acetate The crude enzyme solutions prepared in Example 6(2) were used to measure the amount of isoamyl acetate produced in the same manner as in Example 1(4). The results of measuring the isoamyl acetate production activity of each crude enzyme solution are shown in Table 22 below.

[0182] [Table 22]

[0183] Table 22 shows the ratio of the amount of isoamyl acetate produced relative to the wild-type ATF2 depending on the codon difference in S294R. As shown in Table 22, for each codon, the isoamyl acetate production activity was about 1.9-fold higher than that of the wild-type ATF2 protein, confirming that there was no significant difference.

[0184] [Example 7] It was confirmed that the S294V substitution mutant had higher isoamyl acetate activity than the wild-type ATF2 protein.

[0185] (1) Preparation of expression vector An expression vector (pUCNdeI_kdATF2S294V) in which the codon encoding valine (V) at position 294 is "gtg" was obtained as an expression vector encoding the S294V substitution, which was substituted by site-directed random mutation of S294X in Example 1(5) above.

[0186] (2) Preparation of crude enzyme solution A crude enzyme solution was prepared in the same manner as in Example 1(3), except that the expression vector prepared in Example 7(1) was used instead of the expression vector 3 prepared in Example 1(2).

[0187] (3) Measurement of isoamyl acetate The crude enzyme solutions prepared in Example 7(2) were used to measure the amount of isoamyl acetate produced in the same manner as in Example 1(4). The results of measuring the isoamyl acetate production activity of each crude enzyme solution are shown in Table 23 below.

[0188] [Table 23]

[0189] Table 23 shows the ratio of the amount of isoamyl acetate produced in the S294V substitution mutant to that of the wild-type ATF2. As shown in Table 23, it was confirmed that the isoamyl acetate production activity was about 1.3 times higher than that of the wild-type ATF2 protein.

[0190] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0191] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Mutant ATF2 protein> (Appendix 1) It contains at least one mutation in the amino acid sequence of the wild-type ATF2 protein of yeast, A mutant ATF2 protein having improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein. (Appendix 2) The mutant ATF2 protein according to Appendix 1, which comprises the amino acid sequence (Am) below and has improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein: (Am) The amino acid sequence (Am1), (Am2), or (Am3) below: (Am1) an amino acid sequence of a wild-type ATF2 protein (Aw protein) having a mutation in at least one amino acid residue selected from the group consisting of amino acid residues at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 in a reference ATF2 protein (As protein, SEQ ID NO: 1); (Am2) an amino acid sequence of (Am1) having 1 to 107 insertions, additions, substitutions and / or deletions, and maintaining the mutated amino acid residues of (Am1); (Am3) An amino acid sequence having 70% or more identity to the amino acid sequence of (Am1) and maintaining the mutated amino acid residues of (Am1). (Appendix 3) The amino acid sequence of (Am1) is a mutant ATF2 protein described in Appendix 2, having a mutation in an amino acid residue in the Aw protein corresponding to an amino acid residue at position 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, or 514 in the As protein. (Appendix 4) A mutant ATF2 protein described in Appendix 2 or 3, wherein the amino acid sequence of (Am1) has a mutation in an amino acid residue in the Aw protein corresponding to the amino acid residue at position 147, 290, 294, 352, 479, 481, 482, 484, or 514 in the As protein. (Appendix 5) A mutant ATF2 protein described in any of Appendices 2 to 4, wherein the amino acid sequence of (Am1) has mutations in the Aw protein at amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of positions 147 and 340, positions 250 and 301, positions 290 and 514, and positions 481 and 482 in the As protein. (Appendix 6) The amino acid sequence of (Am1) is a mutant ATF2 protein described in Appendix 2, having a mutation in the Aw protein at an amino acid residue corresponding to at least one amino acid residue selected from the group consisting of L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, and D514 in the As protein. (Appendix 7) A mutant ATF2 protein described in Appendix 6, wherein the amino acid sequence of (Am1) has mutations in the Aw protein at amino acid residues corresponding to the amino acid residues L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, or D514 in the As protein. (Appendix 8) A mutant ATF2 protein described in Appendix 6 or 7, wherein the amino acid sequence of (Am1) has mutations in the amino acid residues in the Aw protein corresponding to the amino acid residues E147, N290, S294, D352, S479, S481, A482, A484, or D514 in the As protein. (Appendix 9) A mutant ATF2 protein described in any of Appendices 2, 6, 7, and 8, wherein the amino acid sequence of (Am1) has mutations in the Aw protein at amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of E147 and K340, D250 and F301, N290 and D514, and S481 and A482 in the As protein. (Appendix 10) The amino acid sequence of (Am1) is a mutant ATF2 protein described in Appendix 2, having, in the Aw protein, a mutation of an amino acid residue corresponding to at least one amino acid residue selected from the group consisting of L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, D352N, N398S, F401L, D410V, R411S, T468A, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, G483H, A484G, and D514G in the As protein. (Appendix 11) The amino acid sequence of (Am1) is a mutant ATF2 protein described in Appendix 10, having mutations in the Aw protein at amino acid residues corresponding to the following amino acid residues in the As protein: L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, D352N, N398S, F401L, D410V, R411S, T468A, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, G483H, A484G, or D514G. (Appendix 12) A mutant ATF2 protein described in Appendix 10 or 11, wherein the amino acid sequence of (Am1) in the Aw protein has mutations in amino acid residues corresponding to E147D, N290K, S294R, D352N, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, A484G, or D514G in the As protein. (Appendix 13) A mutant ATF2 protein described in any of Appendices 2 and 10 to 12, wherein the amino acid sequence of (Am1) has mutations in the Aw protein at amino acid residues corresponding to at least one set of amino acid residues selected from the group consisting of E147D and K340R, N290K and D514G, S481L and A482P, S481L and A482V, S481L and A482T, and S481L and A482L in the As protein. (Appendix 14) A mutant ATF2 protein described in any of Appendices 1 to 13, wherein the amino acid sequence of (Am1) has a mutation in an amino acid residue in the Aw protein that corresponds to an amino acid residue located within 10 Å of the amino acid residue at position 294 in the As protein. (Appendix 15) A mutant ATF2 protein according to Appendix 14, wherein the amino acid residues present within 10 Å of the amino acid residue at position 294 correspond to at least one amino acid residue selected from the group consisting of amino acid residues at positions 147, 150, 292, 293, 294, 295, 296, 479, 481, 482, 483, 506, 507, 508, 509, and 515 in the As protein. (Appendix 16) the mutant ATF2 protein according to Appendix 14 or 15, wherein the amino acid residues present within 10 Å of the amino acid residue at position 294 correspond to at least one amino acid residue selected from the group consisting of E147, S150, N292, A293, S294, Y295, N296, S479, S481, A482, G483, S506, V507, V508, Q509, and R515 in the As protein. <Mutant ATF2 gene> (Appendix 17) A mutant ATF2 gene encoding a mutant ATF2 protein according to any one of appendices 1 to 16. <Transformants> (Appendix 18) A transformant comprising the mutant ATF2 gene described in Appendix 17. <yeast> (Appendix 19) A yeast comprising a mutant ATF2 gene according to appendix 17. <Method of producing alcohol-containing composition> (Appendix 20) A method for producing an alcohol-containing composition, comprising the step of producing an alcohol-containing composition using the yeast described in Appendix 19. (Appendix 21) The method of claim 20, wherein the alcohol-containing composition is an alcoholic beverage. (Appendix 22) The method of producing according to Appendix 21, wherein the alcoholic beverage is sake. (Appendix 23) 23. The method of any one of claims 20 to 22, comprising filling the alcohol-containing composition into a container. <Alcohol-containing composition> (Appendix 24) An alcohol-containing composition obtained by the method for producing alcohol according to any one of Appendices 20 to 23. (Appendix 25) 25. The alcohol-containing composition according to claim 24, wherein the alcohol-containing composition is an alcoholic beverage. (Appendix 26) The alcohol-containing composition according to claim 25, wherein the alcoholic beverage is sake. (Appendix 27) 27. The alcohol-containing composition of any one of claims 24 to 26, stored in a container. <Extract> (Appendix 28) An extract of the transformant of Appendix 18 or the yeast of Appendix 19. <Food composition> (Appendix 29) A food composition comprising the transformant or an extract thereof according to Appendix 18, or the yeast or an extract thereof according to Appendix 19. (Appendix 30) 29. The food composition of claim 29, wherein the food is bread, vinegar, or cooking sake. <Cosmetic composition> (Appendix 31) A cosmetic composition comprising the transformant or an extract thereof described in Appendix 18, or the yeast or an extract thereof described in Appendix 19. <Yeast production method> (Appendix 32) A mutation step of mutating a wild-type ATF2 gene into a mutant ATF2 gene in yeast, A method for producing a yeast, wherein the mutant ATF2 gene is the mutant ATF2 gene described in Appendix 17. (Appendix 33) The mutation step comprises: a mutation introduction step of introducing a mutation into the wild-type ATF2 gene of the yeast; and a selection step of selecting a yeast having the mutant ATF2 gene from the obtained yeast. [Industrial Applicability]

[0192] As described above, the present disclosure provides an ATF2 protein with improved isoamyl acetate productivity, which is extremely useful in, for example, the brewing industry.

Claims

1. It contains at least one mutation in the amino acid sequence of the wild-type yeast ATF2 protein, A mutant ATF2 protein having improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein.

2. The mutant ATF2 protein according to claim 1, which comprises the amino acid sequence (Am) below and has improved ability to produce isoamyl acetate compared to the wild-type ATF2 protein: (Am) The amino acid sequence (Am1), (Am2), or (Am3) below: (Am1) an amino acid sequence of a wild-type ATF2 protein (Aw protein) having a mutation in at least one amino acid residue corresponding to the amino acid residue at positions 27, 147, 187, 222, 250, 269, 290, 294, 301, 307, 340, 352, 398, 401, 410, 411, 468, 479, 481, 482, 483, 484, and 514 in the reference ATF2 protein (As protein, SEQ ID NO: 1); (Am2) an amino acid sequence of (Am1) having 1 to 53 insertions, additions, substitutions and / or deletions, and maintaining the mutated amino acid residues of (Am1); (Am3) An amino acid sequence having 90% or more identity to the amino acid sequence of (Am1) and maintaining the mutated amino acid residues of (Am1).

3. The mutant ATF2 protein of claim 2, wherein the amino acid sequence of (Am1) has a mutation in an amino acid residue in the Aw protein that corresponds to the amino acid residue at positions 147, 290, 294, 352, 479, 481, 482, 484, or 514 in the As protein.

4. The mutant ATF2 protein of claim 2, wherein the amino acid sequence of (Am1) in the Aw protein has a mutation in an amino acid residue corresponding to at least one amino acid residue selected from the group consisting of L27, E147, T187, F222, D250, E269, N290, S294, F301, E307, K340, D352, N398, F401, D410, R411, T468, S479, S481, A482, G483, A484, and D514 in the As protein.

5. The mutant ATF2 protein of claim 2, wherein the amino acid sequence of (Am1) has mutations in the amino acid residues in the Aw protein that correspond to the amino acid residues E147, N290, S294, D352, S479, S481, A482, A484, or D514 in the As protein.

6. The amino acid sequence of (Am1) in the Aw protein has a mutation of an amino acid residue corresponding to at least one amino acid residue selected from the group consisting of L27S, E147D, T187A, F222Y, D250V, E269D, N290K, S294R, E307K, K340R, D352N, N398S, F401L, D410V, R411S, T468A, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, G483H, A484G, and D514G in the As protein. The mutant ATF2 protein of claim 2.

7. The mutant ATF2 protein according to claim 2, wherein the amino acid sequence of (Am1) in the Aw protein has mutations at amino acid residues corresponding to E147D, N290K, S294R, D352N, S479A, S481L, S481R, S481H, S481W, S481M, A482C, A482P, A482V, A484G, or D514G in the As protein.

8. A mutant ATF2 gene encoding the mutant ATF2 protein according to any one of claims 1 to 7.

9. A yeast comprising the mutant ATF2 gene according to claim 8.

10. A method for producing an alcohol-containing composition, comprising the step of producing an alcohol-containing composition using the yeast according to claim 9.

11. An alcohol-containing composition obtained by the method for producing alcohol according to claim 10.

12. 10. An extract of the yeast according to claim 9.

13. A food composition comprising the yeast or an extract thereof according to claim 9.

14. A cosmetic composition comprising the yeast or an extract thereof according to claim 9.

15. A mutation step of mutating a wild-type ATF2 gene into a mutant ATF2 gene in yeast, The method for producing yeast, wherein the mutant ATF2 gene is the mutant ATF2 gene according to claim 8 .

Images