Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
Faculty of Science, Al-Azhar University, Assiut, 71524, Egypt.
Arch Microbiol. 2024 Nov 25;207(1):3. doi: 10.1007/s00203-024-04187-x.
The NADH/NAD balance plays a critical role in regulating cellular and metabolic pathways. In Saccharomyces cerevisiae, glycerol-3-phosphate dehydrogenase (ScGPD) enzymes are essential for NADH homeostasis, glycerol biosynthesis, and osmotic stress adaptation. This study investigates the replacement of ScGPD isoforms with the water-forming NADH oxidase from Lactococcus lactis (LlnoxE) and its effects on 10% glucose fermentation dynamics in minimal medium under microaerobic conditions. We engineered S. cerevisiae strains by individually or sequentially deleting or substituting ScGPD isoforms with LlnoxE, generating strains with varying NADH oxidation levels, fermentation rates, and byproduct formation. The engineered strains exhibited three distinct fermentation profiles: faster strains (∆GPD2 and ∆GPD1,2), five medium-speed strains (native, ∆GPD1, LlnoxE/∆GPD1, LlnoxE/∆GPD2, and LlnoxE with GPD), and three slower strains (LlnoxE/∆GPD1,2, LlnoxE/∆GPD1-∆GPD2, and LlnoxE/∆GPD2-∆GPD1). Increased NADH oxidation correlated strongly with higher acetic acid production, which inhibited cell growth and reduced fermentation speed, especially when glycerol biosynthesis was abolished. For instance, LlnoxE/ΔGPD1 reduced glycerol production by 88% and increased ethanol yield by 6.2%, despite a 9% increase in acetic acid production. This study underscores the importance of NADH oxidation in optimizing fermentation efficiency and metabolic balance in S. cerevisiae strains lacking GPD during glucose fermentation.
NADH/NAD 平衡在调节细胞和代谢途径方面起着关键作用。在酿酒酵母中，甘油-3-磷酸脱氢酶（ScGPD）酶对于 NADH 稳态、甘油生物合成和渗透胁迫适应至关重要。本研究探讨了用来自乳球菌的形成水的 NADH 氧化酶（LlnoxE）替代 ScGPD 同工酶及其对微需氧条件下最小培养基中 10%葡萄糖发酵动力学的影响。我们通过单独或顺序缺失或用 LlnoxE 替代 ScGPD 同工酶来工程化酿酒酵母菌株，生成具有不同 NADH 氧化水平、发酵速率和副产物形成的菌株。工程菌株表现出三种不同的发酵谱：更快的菌株（∆GPD2 和 ∆GPD1,2）、五个中等速度的菌株（天然、∆GPD1、LlnoxE/∆GPD1、LlnoxE/∆GPD2 和 LlnoxE 与 GPD）和三个较慢的菌株（LlnoxE/∆GPD1,2、LlnoxE/∆GPD1-∆GPD2 和 LlnoxE/∆GPD2-∆GPD1）。增加的 NADH 氧化与更高的乙酸产量密切相关，乙酸产量抑制细胞生长并降低发酵速度，特别是当甘油生物合成被废除时。例如，LlnoxE/ΔGPD1 减少了 88%的甘油产量，并增加了 6.2%的乙醇产量，尽管乙酸产量增加了 9%。这项研究强调了在缺乏 GPD 的酿酒酵母菌株葡萄糖发酵过程中，NADH 氧化对于优化发酵效率和代谢平衡的重要性。
