We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4–5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.

我们以前报道过细胞内脯氨酸 (Pro) 赋予酵母酿酒酵母对乙醇的耐受性。在这项研究中，为了提高日本传统酒精饮料清酒的乙醇生产力，我们通过常规诱变成功地分离了几个源自酿酒酵母二倍体清酒酵母的促积累突变体。有趣的是，与亲本菌株（K901）相比，其中一个（菌株 A902-4）产生的鸟氨酸 (Orn) 和 Pro 的量高出 10 倍以上。Orn 是一种非蛋白氨基酸，是精氨酸 (Arg) 和 Pro 的前体。它具有一定的生理功能，如改善倦怠等消极状态，改善睡眠质量。我们还发现了一个同源等位基因突变ARG5,6基因编码菌株 A902-4 中的 Thr340Ile 变体N-乙酰谷氨酸激酶 (NAGK)。Thr340Ile 变体的 NAGK 活性对 Arg 的反馈抑制极其不敏感，导致细胞内 Orn 积累。这是工业酵母中去除 NAGK 活性反馈抑制，导致细胞内 Orn 水平高的第一份报告。此外，用菌株 A902-4 酿造的清酒和清酒蛋糕所含的奥恩比用菌株 K901 酿造的多 4-5 倍。此处描述的方法可能是开发具有过量生产的 Orn 的工业酵母菌株的实用方法。