Nishimura Akira, Takasaki Yurie, Isogai Shota, Toyokawa Yoichi, Tanahashi Ryoya, Takagi Hiroshi
Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Nara 630-0192, Japan.
Microorganisms. 2021 Sep 7;9(9):1902. doi: 10.3390/microorganisms9091902.
Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast . During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues.
泡盛是日本冲绳的传统蒸馏酒精饮料，由酵母酿造而成。在发酵后的蒸馏过程中，必须处理大量含有酵母细胞的蒸馏残渣，最近这在环境和经济方面都被视为一个主要问题。脯氨酸是一种多功能氨基酸，在氨基酸中具有最高的保水能力。因此，含有大量脯氨酸的蒸馏残渣可能在化妆品中有用。在这里，我们分离出了一种细胞内脯氨酸水平较高的酵母突变体，并在编码γ-谷氨酰激酶Pro1（脯氨酸生物合成中的一种限速酶）的基因上发现了一个错义突变（Gln79His）。Pro1中Gln79突变为His导致对脯氨酸介导的GK活性反馈抑制脱敏，从而导致脯氨酸在细胞中积累。生化和计算机模拟分析表明，79位的氨基酸残基通过氢键网络参与Pro1中脯氨酸结合口袋的稳定，这在反馈抑制中起重要作用。因此，我们目前的研究提出了γ-谷氨酰激酶活性反馈抑制的一种可能机制。该机制可用于构建脯氨酸积累酵母菌株，以有效利用蒸馏残渣。
