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.

清酒是日本传统的酒精饮料，用酿酒酵母（Saccharomyces cerevisiae）酿造。清酒的味道受糖，有机酸和氨基酸的影响。我们先前分离了对源自二倍体清酒酵母菌株的脯氨酸类似物氮杂环丁烷-2-羧酸盐具有抗性的突变体。一些突变体在酿造的酒中产生了大量的脯氨酸。其中之一（菌株K-9-AZC）在编码γ-谷氨酰激酶Pro1的Gln79His变体的PRO1基因中携带了一个新突变，该基因是酿酒酵母脯氨酸生物合成中的关键酶。该突变导致对脯氨酸的反馈抑制极度脱敏，从而导致脯氨酸过度生产。有趣的是，用K-9-AZC酿造的清酒所含的脯氨酸含量比用亲本菌株酿造的清酒高3.7倍，但琥珀酸盐含量却仅降低25％。代谢组学分析表明，琥珀酸酯含量的降低归因于2-氧代戊二酸酯含量的降低，后者转化为谷氨酸盐。这里的方法可能是一种用于培养清酒口味多样性的酵母菌株的实用方法。