Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.
Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
J Ind Microbiol Biotechnol. 2020 Oct;47(9-10):715-723. doi: 10.1007/s10295-020-02297-1. Epub 2020 Aug 3.
Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.
清酒是一种传统的日本酒精饮料，由酿酒酵母 Saccharomyces cerevisiae 酿造而成。清酒的口感受糖、有机酸和氨基酸的影响。我们之前从一株二倍体清酒酵母菌株中分离出了对脯氨酸类似物氮杂环丁烷-2-羧酸（azetidine-2-carboxylate）具有抗性的突变体。其中一些突变体能在酿造的清酒中产生更多的脯氨酸。其中一个（菌株 K-9-AZC）在编码 γ-谷氨酰激酶 Pro1 的 PRO1 基因中发生了一个新的突变，该基因编码脯氨酸生物合成中的关键酶，Pro1 的 Gln79His 变体。这种突变导致对脯氨酸的反馈抑制极度脱敏，从而导致脯氨酸过量产生。有趣的是，用 K-9-AZC 酿造的清酒中含有 3.7 倍的脯氨酸，但与亲本菌株相比，琥珀酸含量仅低 25%。代谢组学分析表明，琥珀酸的减少归因于 2-氧戊二酸水平较低，2-氧戊二酸转化为谷氨酸。这里的方法可能是一种实用的方法，可以用于培育参与清酒口感多样性的酵母菌株。
