Enhanced cellulosic ethanol production by increasing p-benzoquinone tolerance of Saccharomyces cerevisiae in adaptive evolution.

p-Benzoquinone, a lignin-derived inhibitor generated during lignocellulosic biomass pretreatment, severely restricts microbial fermentation and remains a major obstacle to efficient cellulosic ethanol production. In this study, p-benzoquinone concentrations in different lignocellulosic hydrolysates ranged from 130 to 210 mg/L under high-solids conditions. Growth analysis showed that p-benzoquinone strongly inhibited Saccharomyces cerevisiae DQ1 in a concentration-dependent manner, with 100 mg/L p-benzoquinone causing 68.5% growth inhibition. Adaptive evolution under gradually increasing p-benzoquinone stress generated an evolved strain, S. cerevisiae ZX100. Compared with the parental strain, ZX100 exhibited markedly enhanced tolerance and produced approximately 2.7 times more ethanol in biodetoxified corn stover hydrolysate. In biodetoxified hydrolysate supplemented with 100 mg/L p-benzoquinone, ZX100 achieved an ethanol yield of 0.43 g ethanol/g sugar consumed, a glucose consumption rate of 2.3 g/L/h, and a volumetric ethanol productivity of 1.0 g/L/h. Phenotypic analyses further demonstrated that ZX100 possessed improved membrane integrity and reduced intracellular ROS accumulation under p-benzoquinone stress. Transcriptomic analysis indicated that the improved p-benzoquinone tolerance of the evolved strain may be associated with enhanced antioxidant defense, membrane stabilization, energy metabolism, and sugar transport capacity. Overall, this work deepens our understanding of the physiological and molecular mechanisms underlying p-benzoquinone tolerance in S. cerevisiae and provides potential candidate genes for engineering robust biorefinery strains with enhanced p-benzoquinone tolerance.