Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Appl Microbiol Biotechnol. 2020 Jun;104(11):4971-4983. doi: 10.1007/s00253-020-10573-4. Epub 2020 Apr 4.
During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex-mediated degradation. Next, we investigated the effects of GID protein-coding genes (GID1-GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle-related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation. KEY POINTS: An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production. The protein expression profiles in the GID disruptants differed from one another.
在酒精发酵过程中，酿酒酵母会产生包括琥珀酸、乙酸和苹果酸在内的有机酸。由于苹果酸有助于清酒（一种日本酒精饮料）的口感醇厚，因此开发了各种生产高产苹果酸酵母的方法。我们之前分离到一株高产苹果酸的酵母菌株，发现 GID4 的一个错义突变是导致高产苹果酸表型的原因。Gid4 是 GID（葡萄糖诱导降解缺陷）复合物的一个组成部分，可刺激糖异生酶的分解代谢降解。在这项研究中，研究了该突变导致酵母细胞中苹果酸产量增加的机制。对糖异生酶的破坏体和突变体的评估表明，细胞质苹果酸脱氢酶（Mdh2）参与了苹果酸的生成。此外，靶标蛋白质组分析表明，由于 GID 复合物介导的降解丧失，gid4 破坏体中 Mdh2 的积累导致苹果酸产量增加。接下来，我们研究了 GID 蛋白编码基因（GID1-GID9）对酵母中有机酸产生和酶表达谱的影响。GID1、2、3、4、5、8 和 9 的破坏体表现出高产苹果酸。GID 破坏体之间的蛋白质丰度比较揭示了蛋白质表达谱的变化，包括糖酵解和三羧酸循环相关酶。高产苹果酸的破坏体表现出几种糖酵解酶的激活和参与丙酮酸转化为乙醇的酶的减少。我们的结果表明，高产苹果酸的破坏体通过调节葡萄糖同化和乙醇发酵中蛋白质表达来适应代谢，以过量产生苹果酸。关键点：GID4 突变体中苹果酸水平的增加是由于 Mdh2 的积累。GID1、2、3、4、5、8 和 9 的破坏体表现出高产苹果酸。GID 破坏体之间的蛋白质表达谱彼此不同。
