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-AL_80-4, 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.

• PMID: 40905401
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Journal Article

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Chen G, Shan Y, Wang J, Zhang Q, Yao L, Chen X

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