Replacing Glycerol-3-Phosphate Dehydrogenase with NADH Oxidase: Effects on Glucose Fermentation and Product Formation in Saccharomyces cerevisiae

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.