Metabolic Engineering and Genome-Wide Adaptive Evolution for Efficient Reduction of Glycerol in Industrial Saccharomyces cerevisiae

The production of glycerol as a major by-product during yeast-based bioethanol fermentation arises directly from the need to re-oxidize excess NADH, which reduces conversion efficiency. In this study, an optimized Cas9-based genome editing method was performed to develop a mixotrophic CO2-fixing industrial Saccharomyces cerevisiae by heterologous expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO form Pseudomonas sp.) and phosphoribulokinase (PRK form Spinach). Additionally, the gene encoding alcohol dehydrogenase (ADH2) responsible for converting ethanol to acetaldehyde was deleted, while the great wall-family protein kinase Rim15 gene was overexpressed to facilitate the reduction in glycerol content. The resulting CO2-fixing yeast M-2 led to a 21.5% reduction of the by-product glycerol in corn mash fermentation cultures at 39 ℃. Moreover, we established a novel gene mutators mediated genome-wide mutations system that accumulates distinct mutations in the industrial S. cerevisiae strains under the stress conditions to improve the robustness in the S. cerevisiae strains efficiently.