Regulating Glycerol Metabolism to Investigate the Effects of Engineered Saccharomyces cerevisiae on Simulated Wine Flavor Compounds

This study aimed to modify metabolite synthesis in <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) under simulated wine fermentation conditions by regulating the glycerol metabolic pathway. We systematically analyzed the effects of overexpressing the aquaporin gene <i>AQY1</i> and co-expressing <i>AQY1</i> with the glycerol-3-phosphate dehydrogenase gene <i>GPD1</i> on the metabolism of ethanol, higher alcohols, and esters. Our results indicate that <i>AQY1</i> overexpression increased glycerol yield by 6.58%, reduced higher alcohol content by 14.60%, and elevated ester content by 7.15%. The downregulation of related amino acid metabolism genes correlated with the observed decrease in higher alcohol levels. Notably, co-expression of <i>AQY1</i> and <i>GPD1</i> further enhanced glycerol yield by 10.66% while decreasing ethanol content by 6.32%. By analyzing changes in gene expression alongside metabolic mechanisms, we hypothesize that the redistribution of carbon flux and NADH toward the glycerol pathway not only decreases the precursors for ethanol synthesis but also directly inhibits the activity of aldehyde dehydrogenase (<i>ALD2</i>/<i>3</i>/<i>4</i>/<i>6</i>), thereby constraining ethanol production. In comparison to <i>AQY1</i> overexpression alone, the co-expression strategy did not significantly alter glycerol accumulation; however, it reduced both ethanol and ester content by 8.38% and 8.40%, respectively, while markedly increasing higher alcohol content by 22.30%. This increase may result from enhanced glycolytic flux and pyruvate accumulation, which promote metabolic flow toward amino acid synthesis pathways. In summary, this study effectively remodeled the central carbon metabolism network by targeting glycerol metabolism, achieving diverse metabolic product synthesis and providing important references for the selection and breeding of industrial <i>S. cerevisiae</i> strains.