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

Through evolutionary engineering strategies, scientists have successfully cultivated multiple strains of <i>Saccharomyces cerevisiae</i> with enhanced tolerance, demonstrating significant potential in improving <i>S. cerevisiae</i> resistance. In this study, <i>S. cerevisiae</i> <i>CEN.PK113-7D</i> was continuously cultured for 80 days in a medium containing lignocellulosic inhibitors (furfural, acetic acid, and vanillin). The evolved strain, <i>S. cerevisiae</i> <i>CEN.PK113-AL_80-4</i>, 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.