JP7101362B2

JP7101362B2 - Method for producing yeast with low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one

Info

Publication number: JP7101362B2
Application number: JP2016143331A
Authority: JP
Inventors: 敦子磯谷; 涼子神田; 優理子池田; 諒宇原; 力藤井; 好裕山脇; 克己松丸; 興若林; 豊久井上; 貴司中江
Original assignee: National Research Institute of Brewing
Current assignee: National Research Institute of Brewing
Priority date: 2016-07-21
Filing date: 2016-07-21
Publication date: 2022-07-15
Anticipated expiration: 2036-07-21
Also published as: JP2018011553A

Description

特許法第３０条第２項適用日本農芸化学会中四国支部第４４回講演会講演要旨集、第５８頁、日本農芸化学会中四国支部事務局〔刊行物等〕平成２８年１月２３日に日本農芸化学会中四国支部第４４回講演会にて発表Application of Article 30, Paragraph 2 of the Patent Law Japan Society for Bioscience and Biotechnology Chushikoku Branch 44th Lecture Abstracts, page 58, Japan Society for Bioscience and Biotechnology Chushikoku Branch Secretariat [Publications, etc.] January 23, 2016 Presented at the 44th Lecture Meeting of the Chushikoku Branch of the Japan Society for Bioscience and Biotechnology

本発明は、ジメチルトリスルフィドの前駆物質である1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オンの生成能が低い酵母の作出方法に関する。 The present invention relates to a method for producing yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one, which is a precursor of dimethyl trisulfide.

ジメチルトリスルフィド（DMTS）は清酒の貯蔵により生成する物質で、硫黄様、タマネギ様のにおいを呈する。清酒の劣化臭である老香の主要構成成分である（非特許文献１）。DMTSは、清酒以外の様々な飲料においても硫化物様のオフフレーバーの原因となりうる（非特許文献２、３）。近年清酒の人気は諸外国においても高まりを見せており、外国への輸出では輸送・貯蔵の期間が長期化することから、貯蔵中の清酒におけるDMTSの発生を抑制することはますます重要な課題となっている。 Dimethyl trisulfide (DMTS) is a substance produced by the storage of sake and has a sulfur-like and onion-like odor. It is a main component of old aroma, which is a deteriorated odor of sake (Non-Patent Document 1). DMTS can also cause sulfide-like off-flavors in various beverages other than sake (Non-Patent Documents 2 and 3). In recent years, the popularity of sake has been increasing in other countries as well, and since the transportation and storage period is prolonged when exporting to foreign countries, it is an increasingly important issue to control the generation of DMTS in sake during storage. It has become.

これまでの研究により、DMTSの前駆物質の一つである1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オン（DMTS-P1）の生成に酵母のMDE1遺伝子及びMRI1遺伝子が関与すること、これらの遺伝子を破壊することで清酒の貯蔵中に生成するDMTSを著しく抑制できることが明らかにされている（非特許文献４）。しかし、遺伝子組換え体を用いて清酒を製造するためには難しい課題が多く、実用化は難しいのが現状である。 Previous studies have shown that the yeast MDE1 and MRI1 genes are involved in the production of 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one (DMTS-P1), one of the precursors of DMTS. It has been clarified that by disrupting these genes, DMTS produced during the storage of sake can be significantly suppressed (Non-Patent Document 4). However, there are many difficult problems in producing sake using a genetically modified product, and it is difficult to put it into practical use.

日本醸造協会誌, 101, 125-131, 2006Journal of the Brewing Society of Japan, 101, 125-131, 2006 J. Agric. Food Chem. 48, 6196-6199, 2000J. Agric. Food Chem. 48, 6196-6199, 2000 J. Am. Soc. Brew. Chem. 56, 99-103, 1998J. Am. Soc. Brew. Chem. 56, 99-103, 1998 J. Biosci. Bioeng. 116, 475-479 (2013)J. Biosci. Bioeng. 116, 475-479 (2013)

従って、本発明の目的は、食品生産にも実用化可能なDMTS-P1低生産性酵母を取得するための新規な手段を提供することにある。 Therefore, an object of the present invention is to provide a novel means for obtaining a DMTS-P1 low-productivity yeast that can be put into practical use in food production.

MDE1遺伝子およびMRI1遺伝子に関係する表現型としては、MDE1破壊株がcaspofungin（Lesage, G. et al : Genetics, 167, 35-49 (2004)）やselenomethionine（Brockhorn, J. et al : PNAS, 105, 17682-17687 (2008)）に耐性を示すことなどの報告がある。しかしながら、本願発明者らが清酒酵母において検討したところ、これらの表現型は清酒酵母において再現されなかった。 Phenotypes related to the MDE1 and MRI1 genes include caspofungin (Lesage, G. et al: Genetics, 167, 35-49 (2004)) and selenomethionine (Brockhorn, J. et al: PNAS, 105). , 17682-17687 (2008)) has been reported to be resistant. However, when the inventors of the present application examined in sake yeast, these phenotypes were not reproduced in sake yeast.

一方、メチオニン再生経路で働く酵素に関する研究で、メチオニン要求性を示す酵母においてMEU1、MRI1、MDE1、UTR4等の遺伝子を破壊すると、メチオニン添加培地では増殖できるが5'-メチルチオアデノシン（MTA）添加培地では増殖能が低下することが報告されている（Pirkov, I. et al., FEBS Journal, 275, 4111-4120 (2008)）。本願発明者らは、この報告に着目して鋭意研究した結果、清酒酵母にメチオニン要求性を付与した上で変異誘発し、メチオニン添加培地よりもMTA添加培地での増殖が劣る株を選択することにより、MRI1遺伝子又はMDE1遺伝子に生じた変異によりDMTS-P1の生産能が大きく低下した株を選択した株の中から極めて高い確率で取得できること、取得されたDMTS-P1低生産性酵母株のメチオニン要求性を失わせることで醸造特性が回復し、DMTS生成ポテンシャル（強制劣化によるDMTS生成量）が低く清酒等のアルコール飲料の醸造に適した酵母株を得ることができること、また、上記の方法で取得されたDMTS-P1低生産性酵母の変異を利用し、セルフクローニング法により非組み換え体酵母に当該変異を導入することで、もとの非組み換え体酵母の醸造特性を維持した当該変異を有するセルフクローニング酵母株を作出できることを見出し、本願発明を完成した。 On the other hand, in a study on enzymes that act in the methionine regeneration pathway, when genes such as MEU1, MRI1, MDE1, and UTR4 are disrupted in yeast showing methionine requirement, it can grow in methionine-added medium, but 5'-methylthioadenosine (MTA) -added medium. Has been reported to reduce proliferative capacity (Pirkov, I. et al., FEBS Journal, 275, 4111-4120 (2008)). As a result of diligent research focusing on this report, the inventors of the present application select a strain that imparts methionine requirement to sake yeast, induces mutation, and grows inferior in MTA-added medium to methionine-added medium. Therefore, it is possible to obtain a strain in which the production capacity of DMTS-P1 is significantly reduced due to a mutation in the MRI1 gene or the MDE1 gene from the selected strains with an extremely high probability. By losing the requirement, the brewing characteristics are restored, the DMTS production potential (the amount of DMTS produced by forced deterioration) is low, and a yeast strain suitable for brewing alcoholic beverages such as sake can be obtained. By using the acquired mutation of DMTS-P1 low-productivity yeast and introducing the mutation into non-recombinant yeast by the self-cloning method, it has the mutation that maintains the brewing characteristics of the original non-recombinant yeast. We have found that a self-cloning yeast strain can be produced, and completed the present invention.

すなわち、本発明は、
酵母親株より、メチオニン要求性を示す株を取得する工程、並びに
メチオニン要求性を示す株を変異処理した後、メチオニン含有培地及びメチオニンを含まず5’-メチルチオアデノシン（MTA）を含むMTA含有培地の両者にレプリカして培養し、MTA含有培地においてメチオニン含有培地よりも増殖能が低い株を選択する工程、
を含む、1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オンの生成能が低い酵母の作出方法であって、メチオニン含有培地及びMTA含有培地は、メチオニン及びMTA以外の硫黄源が制限された培地であって、メチオニン及びMTA以外の硫黄分濃度が0.005 mM未満の培地である、方法を提供する。
さらに、本発明は、MDE1遺伝子がコードするタンパク質の第５４番プロリンがロイシンになる変異、又はMRI1遺伝子がコードするタンパク質の第１９２番グリシンがアスパラギン酸になる変異を有する、1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オン低生産性酵母を提供する。該酵母は醸造酵母、例えば清酒酵母であり得る。
さらに、本発明は、上記本発明の方法により、1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オンの生成能が低い醸造酵母を作出し、該醸造酵母を用いてアルコール発酵を行なうこと、又は上記本発明の1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オン低生産性醸造酵母を用いてアルコール発酵を行なうことを含む、硫化物様のオフフレーバーが低減されたアルコール飲料の製造方法を提供する。さらに、本発明は、上記本発明の方法により、1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オンの生成能が低い清酒酵母を作出し、該清酒酵母を用いてアルコール発酵を行なうこと、又は上記本発明の1,2-ジヒドロキシ-5-(メチルスルフィニル)ペンタン-3-オン低生産性清酒酵母を用いてアルコール発酵を行なうことを含む、老香の発生が抑制された清酒の製造方法を提供する。

That is, the present invention
A step of obtaining a methionine-requiring strain from a yeast parent strain, and after mutating a methionine-requiring strain, a methionine-containing medium and a methionine-free MTA-containing medium containing 5'-methylthioadenosine (MTA). A step of replicating to both and culturing and selecting a strain having a lower growth ability than a methionine-containing medium in an MTA-containing medium.
It is a method for producing yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one, and the methionine-containing medium and the MTA-containing medium contain sulfur sources other than methionine and MTA. Provided is a restricted medium having a sulfur concentration other than methionine and MTA of less than 0.005 mM.
Furthermore, the present invention has a mutation in which proline No. 54 of the protein encoded by the MDE1 gene becomes leucine, or a mutation in which glycine No. 192 of the protein encoded by the MRI1 gene becomes aspartic acid. 5- (Methylsulfinyl) pentan-3-one provides a low-protein yeast. The yeast can be brewed yeast, for example sake yeast.
Furthermore, the present invention produces a brewed yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one by the above-mentioned method of the present invention, and alcoholic fermentation is carried out using the brewed yeast. Reduced sulfide-like off-flavors, including doing so or performing alcoholic fermentation with the 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one low productivity brewed yeast of the present invention. Provided is a method for producing alcoholic beverages. Furthermore, the present invention produces a sake yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one by the above-mentioned method of the present invention, and alcoholic fermentation is carried out using the sake yeast. Sake with suppressed generation of old aroma, including the above-mentioned 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one low-productivity sake yeast of the present invention, which comprises alcoholic fermentation. Providing a manufacturing method for.

本発明によれば、異種生物由来の配列をゲノム上に含まないDMTS-P1低生産性酵母を取得するための方法が提供される。MTA含有培地での増殖能の低下を指標として選択された酵母株は、極めて高い確率でMDE1遺伝子又はMRI1遺伝子の変異によるDMTS-P1低生産性を示す。醸造酵母を用いてDMTS-P1低生産性酵母を作出し、該酵母を用いてアルコール発酵を行なえば、清酒等アルコール飲料の貯蔵中のDMTS発生が抑制されるので、硫化物様のオフフレーバーの発生が抑制されたアルコール飲料を、清酒においては老香の発生が抑制された清酒を製造することができる。異種生物由来の配列をゲノム上に含まないため、食品産業で用いられる醸造酵母の育成に大いに貢献できる。 INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for obtaining a DMTS-P1 low-productivity yeast whose genome does not contain a sequence derived from a heterologous organism. Yeast strains selected with reduced proliferative potential in MTA-containing media have a very high probability of exhibiting low DMTS-P1 productivity due to mutations in the MDE1 or MRI1 genes. If DMTS-P1 low-productivity yeast is produced using brewed yeast and alcohol fermentation is performed using the yeast, DMTS generation during storage of alcoholic beverages such as sake is suppressed, so that sulfide-like off-flavor It is possible to produce an alcoholic beverage in which the generation is suppressed, and in the case of sake, a sake in which the generation of old aroma is suppressed. Since the genome does not contain sequences derived from heterologous organisms, it can greatly contribute to the growth of brewed yeast used in the food industry.

実施例１において取得したメチオニン要求性変異株MR-01、及びMR-01株よりMTA培地での増殖能の低下を指標として選択した変異株9株を液体培養し、培養上清中のDMTS-P1の生成量を調べた結果である。Nine mutant strains selected from the methionine-requiring mutant strain MR-01 obtained in Example 1 and the MR-01 strain using the decrease in proliferation ability in the MTA medium as an index were liquid-cultured, and DMTS- in the culture supernatant was obtained. This is the result of investigating the amount of P1 produced. 実施例１においてK701株の1倍体から取得したMRI1遺伝子変異株LMU-H9株（1倍体）、実施例２においてセルフクローニングにより作出したLMU-H9株のMRI1変異をホモに持つ30-5株（2倍体）、及びセルフクローニングの親株K901株（2倍体）を用いた小仕込み試験において、炭酸ガス減量を調べた結果である。30-5 having the MRI1 gene mutant LMU-H9 (polyploid) obtained from the haploid of the K701 strain in Example 1 and the MRI1 mutation of the LMU-H9 strain produced by self-cloning in Example 2 as homozygous. This is the result of investigating the weight loss of carbon dioxide in a small preparation test using a strain (diploid) and a self-cloning parent strain K901 strain (diploid). 実施例３においてMTA培地での増殖能の低下を指標として選択した変異株を液体培養し、培養上清中のDMTS-P1の生成量を調べた結果である（n=2）。This is the result of liquid culturing the mutant strain selected in Example 3 with the decrease in proliferation ability in the MTA medium as an index, and examining the amount of DMTS-P1 produced in the culture supernatant (n = 2).

本発明によるDMTS-P1低生産酵母の作出方法に供する酵母株は、好ましくは醸造酵母である。醸造酵母の具体例としては、清酒酵母、ビール酵母、ワイン酵母、焼酎酵母などが挙げられる。中でも本発明においては、親株として清酒酵母を特に好ましく用いることができる。 The yeast strain used in the method for producing DMTS-P1 low-producing yeast according to the present invention is preferably brewed yeast. Specific examples of brewed yeast include sake yeast, brewer's yeast, wine yeast, shochu yeast and the like. Above all, in the present invention, sake yeast can be particularly preferably used as the parent strain.

まず、酵母親株より、メチオニン要求性を示す株を取得する。メチオニン要求性を示す酵母株は鉛イオンPb²⁺の存在下で暗褐色～黒色に発色することが知られており（Gregory, J. Cost et al : YEAST, 12, 939-941 (1996)）、鉛添加培地上でのコロニー色を指標としてメチオニン要求性を示す株を選択することができる。培地の鉛（Pb²⁺）濃度は0.3～3 mM程度で良い。変異処理した酵母親株又は変異処理しない酵母親株を鉛添加培地に播き、暗褐色～黒色に発色したコロニーを回収すればよい。変異処理の具体例としては、紫外線照射、放射線照射等の物理的変異処理、及びエチルメタンスルフォン酸（EMS）等の変異剤で処理する化学的変異処理等が挙げられるが、これらに限定されない。親株の変異処理は行っても行わなくても良い。 First, a strain showing methionine requirement is obtained from the yeast parent strain. Yeast strains exhibiting methionine requirement are known to develop a dark brown to black color in the presence of lead ion Pb ²⁺ (Gregory, J. Cost et al: YEAST, 12, 939-941 (1996)). , Strains showing methionine requirement can be selected using the colony color on the lead-added medium as an index. The lead (Pb ²⁺ ) concentration in the medium may be about 0.3 to 3 mM. The mutated yeast parent strain or the unmutated yeast parent strain may be sown in a lead-added medium, and colonies that develop a dark brown to black color may be collected. Specific examples of the mutation treatment include, but are not limited to, physical mutation treatment such as ultraviolet irradiation and radiation irradiation, and chemical mutation treatment treated with a mutation agent such as ethylmethanesulphonic acid (EMS). Mutation treatment of the parent strain may or may not be performed.

次いで、得られたメチオニン要求性酵母株を、メチオニン含有培地と、メチオニンを含まず5’-メチルチオアデノシン（MTA）を含むMTA含有培地の両者で培養し、MTA含有培地においてメチオニン含有培地よりも増殖能が低い株を選択する。当該工程においても、メチオニン要求性株の変異処理は行っても行わなくてもよい。得られたメチオニン要求性酵母株を変異処理して、又は変異処理せずに、YPDプレート等の通常の酵母用寒天培地に播種して培養後、各コロニーをメチオニン含有培地及びMTA含有培地の両方に播種して培養し、メチオニン含有培地に比べてMTA含有培地での増殖が劣るものを選択すればよい。また、MTA含有培地を用いてメチオニン要求性酵母株をナイスタチン処理し、増殖の劣るものを濃縮してからメチオニン含有培地及びMTA含有培地に播種することも可能である。ナイスタチンはポリエンマクロライド系の抗生物質であり、増殖する酵母に取り込まれて酵母を死滅させる。増殖能が低いものは死滅率が低くなるため、結果として増殖の悪い酵母細胞が濃縮される。 Next, the obtained methionine-requiring yeast strain was cultured in both a methionine-containing medium and an MTA-containing medium containing 5'-methylthioadenosine (MTA) without methionine, and grown in the MTA-containing medium more than the methionine-containing medium. Select a strain with low ability. In this step as well, the mutation treatment of the methionine-requiring strain may or may not be performed. After seeding and culturing the obtained methionine-requiring yeast strain on a normal yeast agar medium such as a YPD plate with or without mutation treatment, each colony is cultivated in both a methionine-containing medium and an MTA-containing medium. It may be sown and cultured, and a medium having inferior growth in the MTA-containing medium as compared with the methionine-containing medium may be selected. It is also possible to treat a methionine-requiring yeast strain with an MTA-containing medium by nystatin treatment, concentrate the inferior growth medium, and then inoculate the methionine-containing medium and the MTA-containing medium. Nystatin is a polyene macrolide antibiotic that is taken up by growing yeast and kills it. Those with low proliferative capacity have a low mortality rate, resulting in the enrichment of yeast cells with poor proliferation.

メチオニン含有培地及びMTA含有培地は、酵母用の最少培地にメチオニン及びMTAを添加してそれぞれ調製することができる。メチオニン及びMTAの濃度は0.1mM～10mM程度でよい。最少培地は、硫黄源が制限されたものを好ましく用いることができる。すなわち、メチオニン含有培地及びMTA含有培地は、メチオニン及びMTA以外の硫黄源が制限された培地であることが好ましい。ここでいう「硫黄源が制限された」とは、メチオニン及びMTA以外の硫黄分濃度が5 mM未満、例えば0.005 mM未満であることをいう。メチオニン及びMTA以外の硫黄源が制限された培地をこの工程で用いることで、メチオニン含有培地及びMTA含有培地での増殖の差がより明瞭になるので、増殖能の差を判別しやすくなる。 The methionine-containing medium and the MTA-containing medium can be prepared by adding methionine and MTA to the minimum medium for yeast, respectively. The concentration of methionine and MTA may be about 0.1 mM to 10 mM. As the minimum medium, a medium having a limited sulfur source can be preferably used. That is, the methionine-containing medium and the MTA-containing medium are preferably media in which sulfur sources other than methionine and MTA are restricted. The term "restricted sulfur source" as used herein means that the concentration of sulfur other than methionine and MTA is less than 5 mM, for example, less than 0.005 mM. By using a medium in which a sulfur source other than methionine and MTA is restricted in this step, the difference in growth between the methionine-containing medium and the MTA-containing medium becomes clearer, and it becomes easier to discriminate the difference in growth ability.

メチオニン含有培地及びMTA含有培地での増殖能の評価は、反復して行っても良い。すなわち、MTA含有培地での増殖能が低い株を候補株として選択した後、メチオニン含有培地及びMTA含有培地で候補株を培養し、増殖能に差があることが再度確認された株をMTA含有培地での増殖能が低い株として選択してもよい。 The evaluation of the proliferative ability in the methionine-containing medium and the MTA-containing medium may be repeated. That is, after selecting a strain having a low growth ability in an MTA-containing medium as a candidate strain, the candidate strain was cultured in a methionine-containing medium and an MTA-containing medium, and the strain in which the difference in the growth ability was confirmed again was contained in the MTA. It may be selected as a strain having a low growth ability in a medium.

上記の工程で取得された、MTA含有培地での増殖能が低い株は、極めて高い確率でMDE1遺伝子又はMRI1遺伝子の機能欠損変異を有し、DMTS-P1低生産性を示す。実際にDMTS-P1低生産性であることを確認するためには、例えば、該酵母株及び親株を液体培地中で一定期間（例えば数日間、ないしは１週間程度）培養し、培養上清中のDMTS-P1濃度を測定し、親株とDMTS-P1濃度を比較すればよい。MDE1遺伝子又はMRI1遺伝子に変異が生じているか否かは、各遺伝子の配列をシークエンシングにより決定し、親株の各遺伝子の配列と比較すればよい。野生型の醸造酵母のMDE1遺伝子及びMRI1遺伝子の一例として、清酒酵母きょうかい701号のMDE1遺伝子の配列を配列番号１（コード領域の塩基配列）、配列番号２（アミノ酸配列）、配列番号３（コード領域＋前後各300bp程度のゲノム領域）に、MRI1遺伝子の配列を配列番号４（コード領域の塩基配列）、配列番号５（アミノ酸配列）、配列番号６（コード領域＋前後各300bp程度のゲノム領域）にそれぞれ示す。また、配列番号５にはMDE1遺伝子の隣接する各300bp程度を含むゲノム領域を、配列番号６にはMRI1遺伝子の隣接する各300bp程度を含むゲノム領域をそれぞれ示す。 The strains with low proliferative ability in the MTA-containing medium obtained in the above step have an extremely high probability of having a function-deficient mutation in the MDE1 gene or the MRI1 gene, and show low productivity of DMTS-P1. In order to confirm that the DMTS-P1 productivity is actually low, for example, the yeast strain and the parent strain are cultured in a liquid medium for a certain period of time (for example, for several days or about one week), and then in the culture supernatant. The DMTS-P1 concentration may be measured and the DMTS-P1 concentration may be compared with that of the parent strain. Whether or not the MDE1 gene or the MRI1 gene is mutated may be determined by sequencing the sequence of each gene and compared with the sequence of each gene of the parent strain. As an example of the MDE1 gene and MRI1 gene of wild-type brewed yeast, the sequence of the MDE1 gene of sake yeast genome No. 701 is sequenced in SEQ ID NO: 1 (base sequence of coding region), SEQ ID NO: 2 (amino acid sequence), and SEQ ID NO: 3 (sequence number 3). The sequence of the MRI1 gene is placed in the coding region + genome region of about 300 bp each before and after), SEQ ID NO: 4 (base sequence of the coding region), SEQ ID NO: 5 (amino acid sequence), and SEQ ID NO: 6 (coding region + genome of about 300 bp each before and after). Area). Further, SEQ ID NO: 5 shows a genomic region containing about 300 bp of each adjacent MDE1 gene, and SEQ ID NO: 6 shows a genomic region containing about 300 bp of each adjacent MRI1 gene.

下記実施例では、MDE1遺伝子においては、第161位のシトシンがチミンになり（161C→T）、タンパク質の第54番プロリンがロイシンになる（54Pro→Leu）置換変異が同定され、MRI1遺伝子においては、第575位のグアニンがアデニンになり（575G→A）、タンパク質の第192番グリシンがアスパラギン酸になる（192Gly→Asp）置換変異が同定されている。このような一塩基置換の他にも、異なる部位での少数の塩基の置換や欠失等が生じる可能性があるが、配列を決定すれば容易に変異を特定できる。変異の特定は、MTA含有培地での増殖能が低い株を選択する工程の後、又は選択された株を液体培養してDMTS-P1の生産能を確認した後に任意で行われ得る。 In the following examples, in the MDE1 gene, a substitution mutation was identified in which cytosine at position 161 becomes timine (161C → T) and proline 54 of the protein becomes leucine (54Pro → Leu), and in the MRI1 gene, a substitution mutation was identified. , Guanine at position 575 becomes adenine (575G → A), and protein No. 192 glycine becomes aspartic acid (192Gly → Asp). Substitution mutations have been identified. In addition to such single base substitutions, substitutions or deletions of a small number of bases at different sites may occur, but mutations can be easily identified by determining the sequence. Mutation identification can optionally be performed after the step of selecting strains with low growth potential in MTA-containing medium, or after culturing the selected strains in liquid to confirm the productivity of DMTS-P1.

清酒酵母等の醸造酵母では、メチオニン要求株は、大吟醸仕込みなどもろみの栄養分が少ない条件では増殖や発酵の遅れが生じる可能性がある。そのため、醸造酵母、特に清酒酵母で上記の通りにDMTS-P1低生産変異株を取得した場合には、メチオニン要求性を喪失させることが望ましい。 In brewed yeasts such as sake yeast, methionine-requiring strains may cause delays in growth and fermentation under conditions such as mashing with low nutrient content. Therefore, it is desirable to lose the methionine requirement when a DMTS-P1 low-production mutant is obtained in brewed yeast, especially sake yeast, as described above.

親株として１倍体の酵母を用いた場合には、２倍体化を行なう。まず、DMTS-P1低生産変異株を性の異なるメチオニン要求性をもたない1倍体酵母と掛けあわせ、得られた2倍体から1倍体を取得する。得られた1倍体の中には、DMTS-P1低生産変異株と性は異なるが、MDE1遺伝子又はMRI1遺伝子に同じ変異が入った株が得られる。この株をDMTS-P1低生産変異株と掛け合わせることで、ホモに変異が入った2倍体が得られる。この時、メチオニン要求性や変異により生じる可能性がある発酵能の低下等も原因遺伝子が相補され、高い確率で欠点が減少する。従って、２倍体化した株の中から、メチオニン要求性が失われた株を効率よく取得することができる。メチオニン非含有培地で２倍体化した株を培養し、増殖可能となった株を選択すればよい。 When haploid yeast is used as the parent strain, haploidization is performed. First, the DMTS-P1 low-production mutant is crossed with a haploid yeast having different sex and does not have methionine requirement, and a haploid is obtained from the obtained diploid. Among the obtained haploids, a strain having the same mutation in the MDE1 gene or the MRI1 gene can be obtained, although the sex is different from that of the DMTS-P1 low-production mutant. By multiplying this strain with a DMTS-P1 low-production mutant strain, a diploid with a homozygous mutation can be obtained. At this time, the causal gene is complemented with the decrease in fermentative ability that may be caused by methionine requirement or mutation, and the defects are reduced with high probability. Therefore, it is possible to efficiently obtain a strain having lost methionine requirement from the diploid strains. The diploid strain may be cultured in a methionine-free medium, and the strain capable of growing may be selected.

親株として２倍体の酵母を用いた場合には、メチオニン非含有培地で増殖できるリバータントを取得すればよい。例えば、徐々にメチオニン濃度を低下させてDMTS-P1低生産変異株を継代培養し、最終的にメチオニン非添加の培地でも増殖可能となった株を選択すればよい。 When diploid yeast is used as the parent strain, a revertant that can grow in a methionine-free medium may be obtained. For example, a DMTS-P1 low-producing mutant strain may be subcultured by gradually lowering the methionine concentration, and finally a strain that can grow even in a medium without methionine may be selected.

上記の通りに作出した酵母DMTS-P1低生産変異株の変異を、セルフクローニング法により所望の酵母に導入することにより、DMTS-P1低生産性酵母株を作出することも可能である。この場合、変異を導入する酵母としてメチオニン要求性を示す酵母株を使用する必要がないため、醸造特性が損なわれるリスクがない。 It is also possible to produce a DMTS-P1 low-productivity yeast strain by introducing the mutation of the yeast DMTS-P1 low-production mutant strain produced as described above into a desired yeast by a self-cloning method. In this case, since it is not necessary to use a yeast strain exhibiting methionine requirement as the yeast for introducing the mutation, there is no risk of impairing the brewing characteristics.

本発明において、セルフクローニング法とは、同一種由来の核酸が移植され、異種由来の核酸がゲノム上に残らない組換えDNA技術をいう。異種由来の核酸には、形質転換体の選抜のための薬剤耐性等のマーカー遺伝子、及び遺伝子組換え用プラスミドベクターに由来する核酸が包含される。食品産業で用いられる実用酵母の育成においては、そのような遺伝子導入のためだけに必要な外来DNA配列がゲノム上に残らない手法が望ましい。
酵母のセルフクローニング技術はこの分野で良く知られており、例えばAritomi et al., Biosci. Biotechnol. Biochem., 68(1), 206-214, 2004や特開2003-144164等に記載されている。これらの方法では、形質転換体の選抜のための薬剤耐性マーカーと、マーカー除去株の選抜のための生育抑制マーカーとを含むプラスミドベクターを利用して、ゲノム中の正常遺伝子を変異遺伝子に置き換える。以下、当該技術を用いたセルフクローニング法によるDMTS-P1低生産性酵母株の作出方法について説明する。 In the present invention, the self-cloning method refers to a recombinant DNA technique in which nucleic acids derived from the same species are transplanted and nucleic acids derived from different species do not remain on the genome. The heterologous nucleic acid includes a marker gene such as drug resistance for selection of a transformant, and a nucleic acid derived from a plasmid vector for gene recombination. In the cultivation of practical yeast used in the food industry, it is desirable to use a method in which the foreign DNA sequence required only for such gene transfer does not remain on the genome.
Yeast self-cloning techniques are well known in this field and are described, for example, in Aritomi et al., Biosci. Biotechnol. Biochem., 68 (1), 206-214, 2004 and JP-A-2003-144164. .. These methods utilize a plasmid vector containing a drug resistance marker for selection of transformants and a growth inhibitory marker for selection of marker-removed strains to replace normal genes in the genome with mutant genes. Hereinafter, a method for producing a DMTS-P1 low-productivity yeast strain by a self-cloning method using the technique will be described.

まず、上記のようにMTA含有培地での増殖能低下を指標として取得した酵母DMTS-P1低生産変異株より、MDE1遺伝子又はMRI1遺伝子上に生じた変異を含む領域（変異型領域）を増幅し、形質転換体選抜用の薬剤耐性マーカー及びマーカー除去株選抜用の生育抑制マーカーを含むセルフクローニング用のプラスミドベクターに組み込む。増幅させる変異型領域は、変異部位を含んでいればサイズは問わず、変異を有するMDE1遺伝子又はMRI1遺伝子の一部領域でも全長でもよいが、酵母ゲノムへの導入及び異種由来配列の除去の過程では相同組換え機構を利用することから、相同組換えの効率を考慮し、500bp程度以上の領域を増幅して用いることが好ましい。また、形質転換用プラスミドベクターは、酵母に導入したい変異型領域内で1か所を切断しリニア化して酵母細胞に導入するため、ベクターを切断せず、変異部位に影響しない部位において変異型領域内を1か所だけで切断する制限酵素サイトを有するように増幅領域を選択する。 First, the region containing the mutation generated on the MDE1 gene or the MRI1 gene (mutant region) was amplified from the yeast DMTS-P1 low-production mutant obtained as an index of the decrease in proliferation ability in the MTA-containing medium as described above. , Incorporate into a plasmid vector for self-cloning containing a drug resistance marker for selection of transformants and a growth inhibitory marker for selection of marker-removed strains. The mutant region to be amplified may be a partial region or the full length of the MDE1 gene or the MRI1 gene having a mutation, regardless of the size as long as it contains the mutation site, but the process of introduction into the yeast genome and removal of the heterologous sequence. Since the homologous recombination mechanism is used, it is preferable to amplify and use a region of about 500 bp or more in consideration of the efficiency of homologous recombination. In addition, since the plasmid vector for transformation is introduced into yeast cells by cutting one site in the mutant region to be introduced into yeast and linearizing it, the vector is not cut and the mutant region is not affected by the mutation site. Select the amplification region to have a restriction enzyme site that cleaves the inside in only one place.

薬剤耐性マーカーとしてはYAP1（セルレニン、シクロヘキシミド耐性）、AUR1-C（オーレオバシジン耐性）など、生育抑制マーカーとしてはGIN11等の公知のマーカーを用いることができる。そのようなマーカー遺伝子を用いた酵母のセルフクローニング用プラスミドは、上掲のAritomi et al., Biosci. Biotechnol. Biochem., 68(1), 206-214, 2004や特開2003-144164等に記載され公知であり、またpAUR135等の市販品も存在する。いずれのものを用いても良い。 Known markers such as YAP1 (cerulenin and cycloheximide resistance) and AUR1-C (aureobasidedin resistance) can be used as drug resistance markers, and GIN11 and the like can be used as growth inhibitory markers. A yeast self-cloning plasmid using such a marker gene is described in Aritomi et al., Biosci. Biotechnol. Biochem., 68 (1), 206-214, 2004 and JP-A-2003-144164 described above. It is well known, and there are also commercially available products such as pAUR135. Any one may be used.

変異型領域を組み込んだセルフクローニング用プラスミドベクターは、変異型領域内の1か所だけで切断する制限酵素により切断してリニア化し、酵母細胞内に導入する。ここで用いる酵母としては、メチオニン要求性を示さない通常の2倍体の非組み換え体酵母、例えば清酒酵母等の醸造酵母を好ましく用いることができる。ここでいう「非組み換え体」とは、典型的には、野生型ないしは天然の若しくは人為的に誘発された変異を有する酵母であるが、異種由来の配列を含まない酵母も包含され、例えば何らかの特性が付与されたセルフクローニング株も包含される。 The self-cloning plasmid vector incorporating the mutant region is cleaved with a restriction enzyme that cleaves at only one location in the mutant region, linearized, and introduced into yeast cells. As the yeast used here, a normal diploid non-recombinant yeast that does not show methionine requirement, for example, a brewed yeast such as sake yeast can be preferably used. The term "non-recombinant" as used herein is typically a yeast having a wild-type or natural or artificially induced mutation, but also includes yeasts that do not contain heterologous sequences, such as some. Self-cloning strains with imparted properties are also included.

プラスミドをリニア化したDNAコンストラクトは、２つに分断された変異型領域（そのうちの一方に変異部位が含まれる）が両末端に配置され、その間に薬剤耐性マーカー、生育抑制マーカー、及びプラスミド由来配列が含まれた構造をとる。このDNAコンストラクトは、両末端に分断して配置された変異型領域と、酵母ゲノム上の変異を有しないもとの遺伝子領域との間での相同組換えにより、その遺伝子領域の内部に組み込まれる。これにより、酵母細胞のゲノム中では、マーカー遺伝子及びプラスミド由来配列を介して正常型遺伝子配列と変異型遺伝子配列が縦列して存在する状態になる。まず、薬剤耐性により、DNAコンストラクトがゲノム中に組み込まれた酵母を選択する。 The plasmid-linearized DNA construct has a mutant region divided into two (one of which contains the mutant site) located at both ends, with drug resistance markers, growth inhibitory markers, and plasmid-derived sequences in between. Takes a structure that includes. This DNA construct is integrated into the gene region by homologous recombination between the mutant region divided at both ends and the original gene region having no mutation on the yeast genome. .. As a result, the normal gene sequence and the mutant gene sequence are present in a column in the yeast cell genome via the marker gene and the plasmid-derived sequence. First, depending on drug resistance, yeast with a DNA construct integrated into the genome is selected.

次いで、ゲノム中で縦列して存在する正常型遺伝子配列と変異型遺伝子配列との間で相同組換えが生じると、薬剤耐性マーカー及び生育抑制マーカーを含むプラスミド配列が脱落し、正常型遺伝子及び変異型遺伝子のいずれかがゲノム上に残される。そのため、DNAコンストラクトがゲノム中に組み込まれた酵母細胞について、生育抑制マーカーを発現させる条件下で（例えば、生育抑制マーカーがガラクトース誘導性過剰発現プロモーターの制御下にある場合には、ガラクトース培地上で）さらに選択をかけると、プラスミド配列が残存する酵母細胞は生育抑制マーカーの作用により生育することができず、一方、相同組換えが生じてプラスミド配列が脱落した酵母細胞は生育できるので、プラスミド配列が脱落し正常型遺伝子又は変異型遺伝子のみがゲノム中に残存する酵母株を得ることができる。ゲノム中に残存した遺伝子が変異型配列の遺伝子であるかどうかは、変異部位近傍のシークエンシングを行なって確認すればよい。 Then, when homologous recombination occurs between the normal gene sequence and the mutant gene sequence existing in tandem in the genome, the plasmid sequence containing the drug resistance marker and the growth inhibitory marker is dropped, and the normal gene and the mutation are lost. One of the type genes is left on the genome. Therefore, for yeast cells in which the DNA construct has been integrated into the genome, under conditions that express a growth inhibitory marker (for example, when the growth inhibitory marker is under the control of a galactose-induced overexpression promoter, on a galactose medium). ) When further selection is made, yeast cells in which the plasmid sequence remains cannot grow due to the action of the growth inhibitory marker, while yeast cells in which the plasmid sequence has been shed due to homologous recombination can grow, so that the plasmid sequence can be grown. It is possible to obtain a yeast strain in which only the normal type gene or the mutant type gene remains in the genome. Whether or not the gene remaining in the genome is a gene with a mutant sequence may be confirmed by performing sequencing in the vicinity of the mutation site.

2倍体の酵母を親株としてセルフクローニングを行なった場合、1回のセルフクローニング操作によって得られる形質転換体は、通常は一方のアリルのみ変異型配列で置き換えられたヘテロ変異株である。このヘテロ変異株に対して上記のDNAコンストラクトを導入し、薬剤耐性マーカーと生育抑制マーカーを利用したスクリーニングを行なうことで、両アリルが変異型配列で置き換えられたホモ変異株を得ることができる。 When self-cloning is performed using diploid yeast as the parent strain, the transformant obtained by one self-cloning operation is usually a heterozygous mutant in which only one allele is replaced with a mutant sequence. By introducing the above DNA construct into this heterozygous mutant and performing screening using a drug resistance marker and a growth inhibitory marker, a homozygous mutant in which both alleles are replaced with a mutant sequence can be obtained.

MTA含有培地での増殖能低下を指標として取得した酵母DMTS-P1低生産変異株、及び該変異株の変異部位を利用したセルフクローニング株は、DMTS-P1の生産性が低く、DMTS生成ポテンシャルも低い。そのため、醸造酵母を用いてこれらの株を作出し、該株を用いてアルコール発酵を行なえば、アルコール飲料貯蔵中のDMTSの発生が抑制されるので、硫化物様のオフフレーバーの発生が抑制されたアルコール飲料を、清酒酵母の場合は老香の発生が抑制された清酒を生産することができる。 The yeast DMTS-P1 low-producing mutant strain obtained using the decrease in growth ability in the MTA-containing medium and the self-cloning strain using the mutant site of the mutant strain have low DMTS-P1 productivity and DMTS production potential. low. Therefore, if these strains are produced using brewed yeast and alcohol fermentation is performed using the strains, the generation of DMTS during the storage of alcoholic beverages is suppressed, and the generation of sulfide-like off-flavors is suppressed. In the case of sake yeast, it is possible to produce sake with suppressed generation of old scent.

下記実施例では、MTA含有培地での増殖能の低下を指標とした方法により取得される変異の一例として、MDE1遺伝子においては、第161位のシトシンがチミンになり（161C→T）、タンパク質の第54番プロリンがロイシンになる（54Pro→Leu）置換変異が同定され、MRI1遺伝子においては、第575位のグアニンがアデニンになり（575G→A）、タンパク質の第192番グリシンがアスパラギン酸になる（192Gly→Asp）置換変異が同定されている。これらの変異を有する酵母株は、上記した本発明の方法によるほか、野生型の酵母株のゲノムから該当領域を増幅し、遺伝子工学分野の常法により増幅断片に一塩基置換を導入し、これをセルフクローニング法により酵母に導入することによって作出することも可能である。良好な醸造特性を有する酵母を用いてこれらの変異を導入した酵母株も、硫化物様のオフフレーバーの発生が抑制されたアルコール飲料、老香の発生が抑制された清酒の生産に好ましく用いることができる。 In the following example, as an example of the mutation obtained by the method using the decrease in proliferation ability in the MTA-containing medium as an index, in the MDE1 gene, the 161st-position citocin becomes timine (161C → T), and the protein A substitution mutation was identified in which proline 54 becomes leucine (54Pro → Leu), and in the MRI1 gene, guanine at position 575 becomes adenine (575G → A) and protein 192 glycine becomes asparagic acid. (192Gly → Asp) Substitution mutations have been identified. For yeast strains having these mutations, in addition to the method of the present invention described above, the relevant region is amplified from the genome of the wild-type yeast strain, and a one-base substitution is introduced into the amplified fragment by a conventional method in the field of genetic engineering. Can also be produced by introducing the strain into yeast by the self-cloning method. Yeast strains into which these mutations have been introduced using yeast with good brewing properties should also be preferably used for the production of alcoholic beverages in which the generation of sulfide-like off-flavour is suppressed and sake in which the generation of old aroma is suppressed. Can be done.

以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。DMTS-P1及びDMTSの分析は以下の通りに行った。 Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples. The analysis of DMTS-P1 and DMTS was performed as follows.

＜DMTS-P1の分析（LC/MS）＞
試料（培養上清又は清酒）1 mLに、内部標準として20 mg/Lの1,2-dihydroxy-5-(methyl(d3)sulfinyl)pentan-3-one (methyl(d3)DMTS-P1)を25μL添加し、蒸留水で2mLにメスアップした。これを約1mLの陽イオン交換樹脂（Dowex50WX4）に通液し、蒸留水6 mLで洗浄した。通過液を合併し、凍結乾燥後蒸留水1 mLに溶解し、もしくは合併した通過液をそのままフィルターろ過してLC/MS試料とした。LC/MSの条件は以下のとおり。
装置：島津製作所社製高速液体クロマトグラフ質量分析計LCMS-8040
カラム：BDS Hypersil C18 (3.0×150mm)
移動相：超純水、0.3mL/min
インジェクション：5μL
イオン化モード：ESI (+)
インターフェイス電圧：4.5 kV
ネブライザーガス流量：1.5 L/min
ドライイングガス流量：10.0 L/min
ブロックヒーター温度：500℃
試料導入部温度：300℃
MRM条件
DMTS-P1：プレカーサーイオン；m/z 181, モニターイオン；m/z 117
Methyl(d3)DMTS-P1：プレカーサーイオン；m/z 184, モニターイオン；m/z 117 <Analysis of DMTS-P1 (LC / MS)>
20 mg / L of 1,2-dihydroxy-5- (methyl (d3) sulfinyl) pentan-3-one (methyl (d3) DMTS-P1) as an internal standard is added to 1 mL of a sample (culture supernatant or sake). 25 μL was added, and the volume was increased to 2 mL with distilled water. This was passed through about 1 mL of a cation exchange resin (Dowex50WX4) and washed with 6 mL of distilled water. The passing liquid was combined, frozen and dried, and then dissolved in 1 mL of distilled water, or the combined passing liquid was filtered as it was to obtain an LC / MS sample. The conditions for LC / MS are as follows.
Equipment: Shimadzu High Performance Liquid Chromatograph Mass Spectrometer LCMS-8040
Column: BDS Hypersil C18 (3.0 × 150mm)
Mobile phase: ultrapure water, 0.3 mL / min
Injection: 5 μL
Ionization mode: ESI (+)
Interface voltage: 4.5 kV
Nebulizer gas flow rate: 1.5 L / min
Drying gas flow rate: 10.0 L / min
Block heater temperature: 500 ℃
Sample introduction temperature: 300 ° C
MRM condition
DMTS-P1: Precursor ion; m / z 181, Monitor ion; m / z 117
Methyl (d3) DMTS-P1: Precursor ion; m / z 184, Monitor ion; m / z 117

＜DMTSの分析（GC/MS）＞
stir bar sorptive extraction (SBSE)により行った。エタノール濃度10％となるように加水した清酒試料10mlに、塩化ナトリウム2g、および内部標準としてdimethyl(d6)trisulfide (DMTS-d6) を1 μg/Lとなるように添加し、攪拌子（Twister、Gerstel社製）を入れて700 rpm、30分間攪拌し、香気成分の抽出を行った。香気成分を吸着したTwisterをGerstel社製の加熱脱着装置（TDSA）に装着した。
加熱脱着条件
TDSA：20℃（1min）→60℃/min→230℃（4min）
CIS4：-150℃（0.7min）→12℃/sec→250℃（10min）
GC/MS条件
装置：Agilent社製 GC6890およびMSD5973
カラム：HP-INNOWax（30 m×0.25 mm×0.25 μm）
オーブン温度：40℃（5min）→5℃/min→120℃→15℃/min→240℃（10min）
キャリアガス：He, 1.0 mL/min
インジェクション：スプリットレス
SIMモード
モニターイオン：DMTS; m/z 126, DMTS-d6; m/z 132 <DMTS analysis (GC / MS)>
Performed by stir bar sorptive extraction (SBSE). To 10 ml of sake sample hydrated to an ethanol concentration of 10%, add 2 g of sodium chloride and dimethyl (d6) trisulfide (DMTS-d6) as an internal standard to 1 μg / L, and add a stirrer (Twister,). (Made by Gerstel) was added and stirred at 700 rpm for 30 minutes to extract aroma components. The Twister that adsorbed the aroma component was attached to a heat desorption device (TDSA) manufactured by Gerstel.
Heat desorption conditions
TDSA: 20 ℃ (1min) → 60 ℃ / min → 230 ℃ (4min)
CIS4: -150 ℃ (0.7min) → 12 ℃ / sec → 250 ℃ (10min)
GC / MS Conditioner: Agilent GC6890 and MSD5973
Column: HP-INNOWax (30 m x 0.25 mm x 0.25 μm)
Oven temperature: 40 ℃ (5min) → 5 ℃ / min → 120 ℃ → 15 ℃ / min → 240 ℃ (10min)
Carrier gas: He, 1.0 mL / min
Injection: splitless
SIM mode monitor ion: DMTS; m / z 126, DMTS-d6; m / z 132

実施例１：MTA培地での増殖能低下を指標としたDMTS-P1低生産変異酵母株のスクリーニング（その１）
(１) メチオニン要求性変異株のスクリーニング
清酒酵母はMet要求性を持たないため、まず清酒きょうかい701号（K701）の1倍体株であるK701-α9株より、Pb添加培地上でのコロニー色を指標として（Gregory, J. Cost et al., YEAST, 12, 939-941 (1996)）Met要求性変異株のスクリーニングを行なった。 Example 1: Screening of a DMTS-P1 low-producing mutant yeast strain using a decrease in proliferation ability in an MTA medium as an index (Part 1)
(1) Screening of methionine-requiring mutant strains Since sake yeast does not have Met-requiring strain, first, colonies on Pb-added medium from the K701-α9 strain, which is a ploid strain of Sake Kyokai No. 701 (K701). Met-requiring mutants were screened using color as an index (Gregory, J. Cost et al., YEAST, 12, 939-941 (1996)).

K701-α9株をEMS処理し、Pb添加培地（硝酸鉛添加、培地の鉛（Pb²⁺）濃度は3mM）上に播種し、30℃で４～５日間培養後、暗褐色～黒色に発色したコロニーを回収した。約14,000コロニーからのスクリーニングの結果、７株のメチオニン要求性変異株を取得した。この７株より、増殖試験で増殖が良好であり、かつ、リバータント率（先祖返りしてメチオニン要求を失う率）が低い株を４株取得した。この４株のうち、MTA資化能が最も良いMR-01株をその後のスクリーニング工程に付した。 The K701-α9 strain was treated with EMS, seeded on a Pb-added medium (lead nitrate added, lead (Pb ²⁺ ) concentration in the medium was 3 mM), cultured at 30 ° C for 4 to 5 days, and then developed a dark brown to black color. The colonies were collected. As a result of screening from about 14,000 colonies, 7 methionine-requiring mutants were obtained. From these 7 strains, 4 strains having good growth in the growth test and a low revertant rate (rate of throwing back and losing methionine requirement) were acquired. Of these four strains, the MR-01 strain with the best MTA assimilation ability was submitted to the subsequent screening process.

(２) MTA培地及びMet培地での増殖差を指標としたDMTS-P1低生産変異株のスクリーニング
目的とするmde1もしくはmri1変異株のスクリーニングは、MTAもしくはMetを添加し、それ以外の硫黄源を制限した最少培地（MTA培地、Met培地）での増殖差を指標として行った。 (2) Screening of DMTS-P1 low-production mutants using the growth difference between MTA medium and Met medium For screening of mde1 or mri1 mutants for the purpose, add MTA or Met and use other sulfur sources. The growth difference in the restricted minimum medium (MTA medium, Met medium) was used as an index.

上記で得られたMet要求性変異株MR-01株を培養して増殖させた後、EMS処理して変異を誘発させた。変異処理後の酵母細胞をYPDプレートに播種して培養後、出てきたコロニーをMTA培地およびMet培地にレプリカし、Met培地に比べてMTA培地での増殖が劣る株を候補株とした。MTA培地及びMet培地の組成は表１に示す通りであり、MTA及びMet以外の硫黄源を制限した組成とした（MTA及びMet以外の硫黄分濃度は約1.2μM未満）。 The Met-requiring mutant MR-01 strain obtained above was cultured and propagated, and then EMS-treated to induce mutation. After seeding the yeast cells after the mutation treatment on a YPD plate and culturing, the colonies that emerged were replicated in MTA medium and Met medium, and the strain that grew poorly in MTA medium compared to Met medium was selected as a candidate strain. The composition of the MTA medium and the Met medium is as shown in Table 1, and the composition was set to limit the sulfur sources other than MTA and Met (sulfur concentration other than MTA and Met is less than about 1.2 μM).

候補株について、スポット法（初期菌体数が同じになるよう調整した菌体懸濁液を寒天培地上に滴下し、一定期間後、菌体増殖の違いを観察する方法）により増殖差を確認し、差がみられた株を選択した。これらをYPD培地で一晩前培養後、菌体数が1×10⁶cells/mLとなるようにYPD培地に植菌し、30℃で1週間静置培養して、培養液の上清のDMTS-P1濃度をLC-MSにより測定した。 For the candidate strains, the growth difference was confirmed by the spot method (a method in which a cell suspension adjusted to have the same initial number of cells was dropped onto an agar medium and the difference in cell growth was observed after a certain period of time). Then, the strains showing the difference were selected. After culturing these in YPD medium overnight, inoculate them into YPD medium so that the number of cells is 1 × 10 ⁶ cells / mL, and incubate at 30 ° C for 1 week to obtain the supernatant of the culture solution. DMTS-P1 concentration was measured by LC-MS.

MR-01株由来の約24,000コロニーからのスクリーニングの結果、MTA培地での増殖能が低い株が19株得られた。そのうちの9株で親株よりも有意にDMTS-P1の生成量が低下しており、9株中の3株ではDMTS-P1の生成がほとんど見られなかった（図１）。DNAシーケンスの結果、3株のうちの2株（LMU-H1株、LMU-H9株）については、それぞれMDE1およびMRI1のORF内にアミノ酸置換を伴う変異が確認された（MDE1, 161C→T, 54Pro→Leu; MRI1, 575G→A, 192Gly→Asp）。 As a result of screening from about 24,000 colonies derived from the MR-01 strain, 19 strains having low growth ability in MTA medium were obtained. Nine of these strains produced significantly lower DMTS-P1 than the parent strain, and three of the nine strains produced almost no DMTS-P1 (Fig. 1). As a result of DNA sequencing, mutations with amino acid substitutions were confirmed in the ORFs of MDE1 and MRI1 in 2 of the 3 strains (LMU-H1 strain and LMU-H9 strain, respectively) (MDE1, 161C → T, 54Pro → Leu; MRI1, 575G → A, 192Gly → Asp).

得られた3株の小仕込試験の結果、LMU-H9株は発酵が大きく遅れたが、LMU-H1株は良好な発酵経過を示し、実用株として有望と考えられた。 As a result of the small preparation test of the obtained 3 strains, the fermentation of the LMU-H9 strain was significantly delayed, but the LMU-H1 strain showed a good fermentation progress and was considered to be a promising practical strain.

(３) LMU-H1株の2倍体化
LMU-H1株（MATα）は1倍体であるので、2倍体化を行った。LMU-H1株とK701の野生型1倍体であるK701-a4（MATa）とを交雑し、得られた交雑株を胞子形成させた。胞子形成させたプレートから、ランダムスポア法により約400のコロニーを得た。これらのうち8株においてMTA資化能の低下がみられ、MDE1遺伝子のシークエンスの結果、MATaの1倍体で1株、MATαの1倍体で2株、MATa/αの2倍体で2株が、mde1変異株であることが確認された。上記MATa/αの2株はランダムスポア法の過程で2倍体のmde1変異株が偶発的に得られたものである（mde-D1、D2）。また、LMU-H1を含め接合型の異なる1倍体のmde1変異株どうしを交雑させ、2倍体のmde1変異株を3株得た（mde-D3～D5）。 (3) Diploidization of LMU-H1 strain
Since the LMU-H1 strain (MATα) is haploid, diploidization was performed. The LMU-H1 strain was crossed with K701-a4 (MATa), which is a wild-type monoploid of K701, and the obtained cross was sporulated. Approximately 400 colonies were obtained from the sporulated plates by the random spor method. Eight of these strains showed a decrease in MTA assimilation ability, and as a result of the sequence of the MDE1 gene, 1 strain of MATa monoploid, 2 strains of MATα monoploid, and 2 strains of MATa / α diploid. It was confirmed that the strain was an mde1 mutant strain. The above two strains of MATa / α were obtained by accidentally obtaining a diploid mde1 mutant strain in the process of the random spor method (mde-D1, D2). In addition, we crossed monoploid mde1 mutants with different mating types including LMU-H1 to obtain 3 diploid mde1 mutants (mde-D3 to D5).

(４) 2倍体mde1変異株を用いた清酒小仕込試験
mde-D1～D5の5株を用いてα化米による清酒小仕込試験を行った。精米歩合77%のα化米と精米歩合75%の麹を用い、表2の仕込配合に従い三段仕込みとし、発酵温度は13℃一定とした。mde-D2は仕込20日目に上槽し、それ以外は15日目に上槽した。生成酒は国税庁所定分析法に従い、アルコール度数、日本酒度、総酸度及びアミノ酸度を分析した。DMTS-P1はLC-MSで測定した。DMTS生成ポテンシャル（DMTS-pp、強制劣化によるDMTS生成量）は、生成酒を70℃で１週間貯蔵後、GC-MSにより測定した。 (4) Sake small preparation test using diploid mde1 mutant strain
A small sake preparation test using pregelatinized rice was conducted using 5 strains of mde-D1 to D5. Using pregelatinized rice with a rice polishing ratio of 77% and koji with a rice polishing ratio of 75%, the rice was prepared in three stages according to the preparation composition shown in Table 2, and the fermentation temperature was kept constant at 13 ° C. mde-D2 was topped on the 20th day of preparation, and on the 15th day of the rest. The alcohol content, sake content, total acidity and amino acid content of the produced sake were analyzed according to the analysis method prescribed by the National Tax Agency. DMTS-P1 was measured by LC-MS. The DMTS production potential (DMTS-pp, the amount of DMTS produced by forced deterioration) was measured by GC-MS after storing the produced liquor at 70 ° C. for 1 week.

その結果、いずれの株もDMTS-P1濃度は対照株に比べて大きく減少した（表3）。DMTS生成ポテンシャル（DMTS-pp）は5株中4株で1/9～1/3に低下した。mde-D2株ではDMTS-ppの低下がみられなかったが、アミノ酸度が高いことなどが原因と考えられる。また、5株のうちmde-D2株以外はメチオニン要求性が消失していた。 As a result, the DMTS-P1 concentration was significantly reduced in all the strains as compared with the control strain (Table 3). The DMTS generation potential (DMTS-pp) decreased to 1/9 to 1/3 in 4 out of 5 strains. No decrease in DMTS-pp was observed in the mde-D2 strain, which is considered to be due to the high amino acid content. In addition, the methionine requirement disappeared except for the mde-D2 strain among the 5 strains.

さらに、mde-D1株、mde-D5株を用いて、総米1kgの清酒小仕込試験を行った。精米歩合75%の蒸米と麹を用い、表4の仕込配合に従い三段仕込みとし、発酵温度は13℃一定とした。K701とmde-D5は仕込14日目に上槽し、mde-D1は15日目に上槽した。生成酒をアルコール15%に調整後（表5）、40℃で貯蔵し官能評価を行った。官能評価は11名のパネルにより行った。 Furthermore, using the mde-D1 strain and the mde-D5 strain, a small sake preparation test of 1 kg of rice was conducted. Using steamed rice with a rice polishing ratio of 75% and jiuqu, the fermentation temperature was kept constant at 13 ° C. The K701 and mde-D5 were in the upper tank on the 14th day of preparation, and the mde-D1 was in the upper tank on the 15th day. After adjusting the produced liquor to 15% alcohol (Table 5), it was stored at 40 ° C and subjected to sensory evaluation. Sensory evaluation was performed by a panel of 11 people.

その結果、mde-D1株、mde-D5株による小仕込酒は対照株に比べて長期保存後の老香が少なく、総合評価において評価がよい傾向がみられた（表6）。以上の結果から、上記方法によりDMTS-P1低生産酵母を育種することができ、また、得られた株で仕込んだ清酒は貯蔵によるDMTSの生成が抑制されることが明らかとなった。 As a result, the mde-D1 strain and the mde-D5 strain had less old scent after long-term storage than the control strain, and the overall evaluation tended to be good (Table 6). From the above results, it was clarified that DMTS-P1 low-producing yeast can be bred by the above method, and that the sake brewed with the obtained strain suppresses the production of DMTS by storage.

実施例２：セルフクローニング法による変異導入株の作出
(１) LMU-H9株の変異MRI1配列を導入したセルフクローニング株の作出
親株を清酒きょうかい901号（K901株）とし、LMU-H9株の変異MRI1配列を用いて変異導入株を作成した。LMU-H9株の点変異575 G→Aを含む領域を、LMU-H9株のゲノムからPCRで増幅後、組込み型の配列除去型プラスミドpAUR135 (Takara)にクローニングした。本プラスミドは、形質転換体選択マーカーとしてオーレオバシジン耐性を付与するAUR1-C遺伝子に加え、ガラクトース誘導の自殺遺伝子を持つため、配列が除去された株だけがガラクトース培地で生き残れることを利用して、プラスミド除去を行う（Aritomi K, et al. Biosci Biotechnol Biochem. 68, 206-214 (2004); 特開2003-144164など）。具体的な工程を以下に記載する。 Example 2: Creation of a mutation-introduced strain by the self-cloning method
(1) Creation of a self-cloning strain into which the mutant MRI1 sequence of the LMU-H9 strain was introduced The parent strain was Sake Kyokai No. 901 (K901 strain), and a mutant-introduced strain was prepared using the mutant MRI1 sequence of the LMU-H9 strain. The region containing the point mutation 575 G → A of the LMU-H9 strain was amplified by PCR from the genome of the LMU-H9 strain and then cloned into the integrated sequence-removing plasmid pAUR135 (Takara). Since this plasmid has a galactose-induced suicide gene in addition to the AUR1-C gene that imparts aureobasidin resistance as a transformant selection marker, it utilizes the fact that only the strain from which the sequence has been removed can survive in the galactose medium. , Perform plasmid removal (Aritomi K, et al. Biosci Biotechnol Biochem. 68, 206-214 (2004); JP 2003-144164, etc.). The specific process is described below.

LMU-H9株のゲノムを鋳型とし、プライマーMRI1-SF（CTTTGATAGATCGGAGCCAGA、配列番号７）及びMRI1-SR（TTGAATTCCTCAGGGTTACGTT、配列番号８）を用いたPCRにより、点変異を含む993bpの領域（配列番号９）を増幅した。pAUR135への挿入のため、増幅断片をEcoRIで処理し、986bpのインサート断片を回収した。pAUR135をSmaI及びEcoRIで切断し、インサート断片をライゲーションして形質転換用プラスミド（配列番号１０、741～1726位がインサートの変異型MRI1部分配列）を得た。 A 993 bp region containing a point mutation (SEQ ID NO: 9) by PCR using the genome of the LMU-H9 strain as a template and the primers MRI1-SF (CTTTGATAGATCGGAGCCAGA, SEQ ID NO: 7) and MRI1-SR (TTGAATTCCTCAGGGTTACGTT, SEQ ID NO: 8). Was amplified. The amplified fragment was treated with EcoRI for insertion into pAUR135 and the 986 bp insert fragment was recovered. pAUR135 was cleaved with SmaI and EcoRI, and the insert fragment was ligated to obtain a transformation plasmid (SEQ ID NO: 10, 741-1726 positions are mutant MRI1 partial sequences of the insert).

形質転換用プラスミドをSpe Iで切断してリニア化し、K901株に導入した後、ゲノム中にプラスミドが組み込まれた株をプラスミドの薬剤耐性（オーレオバシジン耐性）で選抜した。選抜した株をガラクトース培地で培養することにより、プラスミド配列が除かれた株を得た。プラスミドの除去は、プラスミド配列部分に設定したプライマー（pAUR135-checkF3: GCCTGATGCGGTATTTTCTC（配列番号１１）及びpAUR135-checkR3: TCGTCGTTTGGTATGGCTTC（配列番号１２））を用いたPCRにより確認した。また、プラスミド配列が除かれる際、正常なMRI1配列に戻る場合と変異型MRI1配列になる場合があるため、MRI1遺伝子の塩基配列をシークエンスすることで変異型配列の組み込みを確認した。 The transformation plasmid was cleaved with Spe I, linearized, introduced into the K901 strain, and then the strain in which the plasmid was integrated into the genome was selected based on the drug resistance (aureobasidine resistance) of the plasmid. The selected strain was cultured in galactose medium to obtain a strain from which the plasmid sequence had been removed. The removal of the plasmid was confirmed by PCR using the primers set in the plasmid sequence portion (pAUR135-checkF3: GCCTGATGCGGTATTTTCTC (SEQ ID NO: 11) and pAUR135-checkR3: TCGTCGTTTGGTATGGCTTC (SEQ ID NO: 12)). In addition, when the plasmid sequence is removed, it may return to the normal MRI1 sequence or it may become a mutant MRI1 sequence, so we confirmed the integration of the mutant sequence by sequencing the base sequence of the MRI1 gene.

K901株は2倍体であるため、プラスミド配列が除かれ変異型配列の組み込みが確認された株に対して、形質転換用プラスミドの導入作業を再度行い、両アリルのMRI1遺伝子が変異型に置換された株30-5株を得た。 Since the K901 strain is a diploid strain, the plasmid sequence was removed and the mutant sequence was confirmed to be integrated, and the transformation plasmid was introduced again, and the MRI1 genes of both alleles were replaced with the mutant type. 30-5 strains were obtained.

(２) セルフクローニング株を用いた発酵試験
K901株（親株、2倍体）、LMU-H9株（1倍体）、及びLMU-H9株のMRI1変異をホモに持つセルフクローニング株30-5株（2倍体）を用いた総米300gの小仕込み試験（表7）を3連で行い、製成酒のDMTS-P1濃度、DMTS-pp測定及び一般分析を行った。なお、アルコール度数は簡易分析法（アルコメイト）、日本酒度は振動密度計、酸度・アミノ酸度はpHによる滴定法を用いて測定した。また、DMTS-P1はLC-MSで、DMTS-ppはGC-MSにより測定した。 (2) Fermentation test using self-cloning strain
Total rice 300g using K901 strain (parent strain, diploid), LMU-H9 strain (haploid), and self-cloning strain 30-5 strain (diploid) having MRI1 mutation of LMU-H9 strain homozygous The small preparation test (Table 7) was carried out in triplets, and the DMTS-P1 concentration, DMTS-pp measurement and general analysis of the brewed sake were performed. The alcohol content was measured by a simple analysis method (alcomate), the sake content was measured by a vibration densitometer, and the acidity and amino acid content were measured by a pH titration method. DMTS-P1 was measured by LC-MS, and DMTS-pp was measured by GC-MS.

結果を図２及び表８、９に示す。1倍体のLMU-H9株の炭酸ガス減量は低く、30-5株の炭酸ガス減量は親株のK901株とほぼ同じであった（図2、表8）。親株のK901株に比べ、LMU-H9株・30-5株ではDMTS-P1生成能が大幅に低減した（表8）。一般成分分析の結果によると、1倍体のLMU-H9株はアルコールが低い等の特徴があったが、30-5株はK901株とほぼ同程度であった（表9）。なお、セルフクローニング株の作出過程で得られた、変異をヘテロで持つ2倍体株についてもDMTS-P1生成能を調べたところ、DMTS-P1生成能の低下は認められなかった。 The results are shown in FIGS. 2 and 8 and 9. The carbon dioxide weight loss of the monoploid LMU-H9 strain was low, and the carbon dioxide weight loss of the 30-5 strain was almost the same as that of the parent strain K901 strain (Fig. 2, Table 8). Compared with the parent strain K901 strain, the DMTS-P1 production ability was significantly reduced in the LMU-H9 strain and 30-5 strain (Table 8). According to the results of general component analysis, the monoploid LMU-H9 strain was characterized by low alcohol content, but the 30-5 strain was almost the same as the K901 strain (Table 9). When the DMTS-P1 producing ability was examined for the diploid strain having the mutation heterozygous obtained in the process of producing the self-cloning strain, no decrease in the DMTS-P1 producing ability was observed.

実施例３：MTA培地での増殖能低下を指標としたDMTS-P1低生産変異酵母株のスクリーニング（その２）
(１) メチオニン要求性変異株のスクリーニング
清酒酵母きょうかい7号（K7、2倍体）より、Pb添加培地上でのコロニー色を指標としてMet要求性変異株のスクリーニングを行なった。 Example 3: Screening of a DMTS-P1 low-producing mutant yeast strain using a decrease in proliferation ability in an MTA medium as an index (Part 2)
(1) Screening of methionine-requiring mutant strains Met-requiring mutant strains were screened from sake yeast Kyokai No. 7 (K7, diploid) using the colony color on the Pb-added medium as an index.

K7株をUV照射（10分間、生存率80％）したもの、およびUV照射しないものをPb添加培地（Pb²⁺濃度3mM）上に播種し、30℃で４～５日間培養後、暗褐色～黒色に発色したコロニーを回収した。合計約30万コロニーからのスクリーニングの結果、UV照射ありで2株、UV照射なしで1株のMet要求性変異株を取得した。UV処理ありで取得した変異株をU5およびU6株、UV処理なしで取得した自然突然変異株をS1-2株と命名した。 The K7 strain was inoculated on a Pb-added medium (Pb 2 ⁺ concentration 3 mM) with UV irradiation (10 minutes, survival rate 80%) and without UV irradiation, cultured at 30 ° C for 4 to 5 days, and then dark brown. -The colonies that developed black color were collected. As a result of screening from a total of about 300,000 colonies, 2 strains with UV irradiation and 1 strain without UV irradiation were obtained as Met-requiring mutant strains. The mutant strains obtained with UV treatment were named U5 and U6 strains, and the spontaneous mutant strains obtained without UV treatment were named S1-2 strains.

(２) MTA培地及びMet培地での増殖差を指標としたDMTS-P1低生産変異株のスクリーニング
得られたMet要求性変異株S1-2株を培養して増殖させた後、UV照射（30分間、生存率10～20％）して変異を誘発させた。変異処理後の酵母細胞をYPDプレートに播種して培養後、出てきたコロニーをMTA培地およびMet培地にレプリカし、Met培地に比べてMTA培地での増殖が劣る株を候補株とした。MTA培地及びMet培地の組成は、Sodium glutamate 7.1g/LをUrea 2.3g/Lに変更し、Biotin及びThiamineを除いたほかは表１と同じ組成とした（MTA及びMet以外の硫黄分は含有しない）。 (2) Screening of DMTS-P1 low-production mutant strain using the growth difference between MTA medium and Met medium as an index After culturing and growing the obtained Met-requiring mutant S1-2 strain, UV irradiation (30). The mutation was induced with a survival rate of 10 to 20% for a minute. After seeding the yeast cells after the mutation treatment on a YPD plate and culturing, the colonies that emerged were replicated in MTA medium and Met medium, and the strain that grew poorly in MTA medium compared to Met medium was selected as a candidate strain. The composition of MTA medium and Met medium was the same as Table 1 except that Sodium glutamate 7.1 g / L was changed to Urea 2.3 g / L and Biotin and Thiamine were excluded (sulfur content other than MTA and Met was contained). do not do).

候補株について、上記１．(２)と同様にスポット法により増殖差を確認し、差がみられた株を選択した。これらをYPD培地で一晩前培養後、菌体数が1×10⁶cells/mLとなるようにYPD培地に植菌し、30℃で1週間静置培養して、培養液の上清のDMTS-P1濃度をLC-MSにより測定した。 Regarding the candidate strains, the above 1. In the same manner as in (2), the growth difference was confirmed by the spot method, and the strain in which the difference was observed was selected. After culturing these in YPD medium overnight, inoculate them into YPD medium so that the number of cells is 1 × 10 ⁶ cells / mL, and incubate at 30 ° C for 1 week to obtain the supernatant of the culture solution. DMTS-P1 concentration was measured by LC-MS.

S1-2株由来の約60,000コロニーからのスクリーニングの結果、MTA培地での増殖能が低い株が51株得られた。そのうちの5株で親株と比較して顕著なDMTS-P1濃度の減少が認められた。図３にはDMTS-P1測定結果を示す。 As a result of screening from about 60,000 colonies derived from the S1-2 strain, 51 strains having low growth ability in MTA medium were obtained. Five of these strains showed a marked decrease in DMTS-P1 concentration compared to the parent strain. FIG. 3 shows the measurement results of DMTS-P1.

Claims

A step of obtaining a methionine-requiring strain from a yeast parent strain, and after mutating a methionine-requiring strain, a methionine-containing medium and a methionine-free MTA-containing medium containing 5'-methylthioadenosine (MTA). A step of replicating to both and culturing and selecting a strain having a lower growth ability than a methionine-containing medium in an MTA-containing medium.
It is a method for producing yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one, and the methionine-containing medium and the MTA-containing medium contain sulfur sources other than methionine and MTA. A method of limiting medium, the medium having a sulfur concentration other than methionine and MTA of less than 0.005 mM.

Strains selected as low-growing strains in MTA-containing medium and parent strains were liquid-cultured, and the concentration of 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one in the culture supernatant was measured. The method according to claim 1, further comprising a step of selecting a strain having a lower production amount of 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one as compared with the parent strain.

The second aspect of claim 2, further comprising acquiring a strain having lost methionine requirement from a yeast strain having a low productivity of 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one obtained. Method.

Using monoploid yeast as the yeast parent strain, further including the step of diploidizing the obtained yeast strain having low productivity of 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one. The method according to claim 2.

The method according to claim 4, further comprising a step of selecting a strain having lost methionine requirement from a diploid strain.

Select a strain having a lower growth ability than a methionine-containing medium in an MTA-containing medium, or select a strain having a lower production amount of 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one as compared with the parent strain. The method according to any one of claims 1 to 5, which comprises the step of sequence of the MDE1 gene and the MRI1 gene of the selected strain and identifying the mutation site.

The method according to any one of claims 1 to 6, wherein the yeast is brewed yeast.

The method according to claim 7, wherein the yeast is sake yeast.

1,2-Dihydroxy-5- (methylsulfinyl) having a mutation in which the MDE1 gene-encoded protein No. 54 proline becomes leucine or the MRI1 gene-encoded protein No. 192 glycine becomes aspartic acid. Pentan-3-one low-protein yeast.

The 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one low-productivity yeast according to claim 9, which is a brewing yeast.

The 1,2-dihydroxy-5- (methylsulfinyl) pentane-3-one low-productivity yeast according to claim 10, which is a sake yeast.

A brewed yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one is produced by the method according to claim 7, and alcoholic fermentation is carried out using the brewed yeast, or claimed. Item 10. Production of an alcoholic beverage having reduced sulfide-like off-flavor, which comprises performing alcoholic fermentation using 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one low-producing yeast according to Item 10. Method.

The method according to claim 8 is used to produce a sake yeast having a low ability to produce 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one, and to perform alcoholic fermentation using the sake yeast, or claim. Item 12. A method for producing sake in which the generation of old aroma is suppressed, which comprises performing alcoholic fermentation using 1,2-dihydroxy-5- (methylsulfinyl) pentan-3-one low-producing yeast according to Item 11.