Abstract During hydrolysis of lignocellulosic biomass, a broad range of inhibitors are generated, which interfere with yeast growth and bioethanol production. In order to improve the strain tolerance to multiple inhibitors-acetic acid, furfural, and phenol (three representative lignocellulose-derived inhibitors) and uncover the underlying tolerant mechanism, an adaptation experiment was performed in which the industrial Saccharomyces cerevisiae was cultivated repeatedly in a medium containing multiple inhibitors. The adaptation occurred quickly, accompanied with distinct increase in growth rate, glucose utilization rate, furfural metabolism rate, and ethanol yield, only after the first transfer. A similar rapid adaptation was also observed for the lab strains of BY4742 and BY4743. The metabolomic analysis was employed to investigate the responses of the industrial S. cereviaise to three inhibitors during the adaptation. The results showed that higher levels of 2-furoic acid, 2, 3-butanediol, intermediates in glycolytic pathway, and amino acids derived from glycolysis, were discovered in the adapted strains, suggesting that enhanced metabolic activity in these pathways may relate to resistance against inhibitors. Additionally, through single-gene knockouts, several genes related to alanine metabolism, GABA shunt, and glycerol metabolism were verified to be crucial for the resistance to multiple inhibitors. This study provides new insights into the tolerance mechanism against multiple inhibitors, and guides for the improvement of tolerant ethanologenic yeast strains for lignocellulose-bioethanol fermentation.
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