Multiple Lignocellulosic Inhibitor-Tolerant Saccharomyces cerevisiae Strains Developed by Evolutionary Engineering and CRISPR/Cas9 Gene Editing Technology.

Through evolutionary engineering strategies, scientists have successfully cultivated multiple strains of Saccharomyces cerevisiae with enhanced tolerance, demonstrating significant potential in improving S. cerevisiae resistance. In this study, S. cerevisiae CEN.PK113-7D was continuously cultured for 80 days in a medium containing lignocellulosic inhibitors (furfural, acetic acid, and vanillin). The evolved strain, S. cerevisiae CEN.PK113-AL80-4, exhibited 12 h reduction in lag phase under multiple stress conditions and 17% increase in the ethanol conversion rate. The double mutant strain RG was constructed by mutating genes such as Rad18 and Gcn1 using CRISPR/Cas9 gene editing technology. Under the stress of 2 g/L furfural, 3 g/L acetic acid, and 1.5 g/L vanillin, ethanol yield reached 5.88 ± 0.28 g/L (the conversion rate was 0.29 ± 0.01 g/g). However, the original strain cannot grow. Mechanism studies have shown that Rad18 and Gcn1 significantly enhance stress tolerance by increasing the activities of catalase (CAT) (75%) and superoxide dismutase (SOD) (27.6%), increasing intracellular glycerol content, and strengthening carbon metabolism and oxidative stress responses. This study lays a solid theoretical foundation for developing more robust strains and advancing efficient utilization of lignocellulosic biomass.