Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan.
Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto 612-8385, Japan.
FEMS Yeast Res. 2023 Jan 4;23. doi: 10.1093/femsyr/foad012.
The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.
两种缬氨酸衍生挥发物——异丁醇和异丁酸乙酯的果香对酒精饮料的风味和口感有很大影响，包括日本传统的酒精饮料——清酒。随着人们对清酒的兴趣日益浓厚，通过增加缬氨酸衍生的香气，培育具有细胞内缬氨酸积累能力的酵母菌株是满足人们对具有各种风味和口感的清酒需求的一种有前途的方法。在这里，我们分离到一株缬氨酸积累型清酒酵母突变株（K7-V7），并在 Ilv6 上鉴定到一个新的氨基酸取代，即 Ala31Thr，Ilv6 是乙酰羟酸合酶的调节亚基。Ala31Thr 变体 Ilv6 的表达赋予了实验室酵母细胞缬氨酸积累能力，导致异丁醇产量增加。此外，酶分析表明 Ilv6 中的 Ala31Thr 取代降低了对缬氨酸的反馈抑制的敏感性。这项研究首次证明，真菌乙酰羟酸合酶调节亚基的 N 端臂参与了缬氨酸的变构调节。此外，用 K7-V7 菌株酿造的清酒中异丁醇和异丁酸乙酯的含量比用亲本菌株酿造的清酒高 1.5 倍。我们的发现将有助于酿造具有独特风味的清酒和开发具有增加缬氨酸衍生化合物产量的酵母菌株。
