具有细胞内积累的异亮氨酸(Ile)的各种酿酒酵母将是开发独特清酒的有前途的菌株,一种传统的日本酒精饮料,因为Ile衍生的挥发性化合物对发酵食品的风味和味道有很大影响。在这项研究中,我们通过常规诱变从酿酒酵母的二倍体清酒酵母中分离出Ile积累突变体(菌株K9-I48)。菌株K9-I48在编码苏氨酸脱氨酶(TD)的His480Tyr变体的ILV1基因中携带新的突变。有趣的是,His480Tyr变体的TD活性对Ile的反馈抑制明显不敏感,但没有被缬氨酸上调,导致Ile的细胞内积累和2-甲基-1-丁醇的细胞外过量生产,一种来源于Ile的杂醇,在酵母细胞中。本研究首次证明,位于两个调节域之间的接头区域中的保守组氨酸残基参与了TD的变构调节。此外,用菌株K9-I48酿造的清酒所含的2-甲基-1-丁醇和乙酸2-甲基丁酯比用亲本菌株酿造的清酒多2至3倍。这些发现对于TD工程以提高Ile及其衍生的杂醇的生产率很有价值。重要性异亮氨酸衍生的挥发性化合物的果样风味,2-甲基-1-丁醇(2MB)及其乙酸酯,有助于酒精饮料的各种口味和口味。除了它作为食品和化妆品中的香气成分的价值外,2MB作为第二代生物燃料引起了极大的关注。苏氨酸脱氨酶(TD)催化异亮氨酸生物合成的第一步,其活性受到异亮氨酸的反馈抑制。这里,我们分离了一个积累异亮氨酸的清酒酵母突变体,并鉴定了一个编码新型TD变体的突变基因。变体TD对异亮氨酸的敏感性低得多,导致2MB和异亮氨酸的产量高于野生型TD。此外,用表达变体TD的突变酵母酿造的清酒比用亲本菌株酿造的清酒含有更多的2MB及其乙酸酯。这些发现将有助于开发用于高水平生产异亮氨酸及其相关杂醇的优良工业酵母菌株。
A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.