Improving inhibitor tolerance of xylitol-producing Saccharomyces cerevisiae by overexpressing key target genes mined...

Xylitol, a valuable five-carbon sugar alcohol widely used in the food and pharmaceutical industries, can be biosynthesized through the reduction of xylose by engineered Saccharomyces cerevisiae. A major challenge in producing xylitol from lignocellulosic feedstocks is the sensitivity of yeast to multiple inhibitors generated during biomass pretreatment. Developing robust microbial cell factories with enhanced tolerance to these inhibitors is therefore essential for efficient and sustainable xylitol production. In this study, we employed comparative transcriptomic analysis to investigate the response mechanisms of two xylitol-producing S. cerevisiae strains, CXAU and TX2022, to vanillin and PCS-L (liquid hydrolysate from pretreated corn stover).

Under vanillin stress, CXAU exhibited downregulation of glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, accompanied by upregulation of amino acid and ergosterol biosynthesis. In contrast, TX2022 showed repression of central carbon metabolism, oxidative phosphorylation, and heme and thiamine synthesis, while enhancing amino acid synthesis and glutathione (GSH) regeneration. Under PCS-L exposure, CXAU experienced severe metabolic disruption but prioritized improving the fidelity of protein translation. Meanwhile, TX2022 upregulated amino acid and ergosterol synthesis, purine metabolism, and ribosome biogenesis, while downregulating oxidative phosphorylation and peroxisomal functions. Based on transcriptomic insights, 11 candidate genes potentially involved in stress tolerance were identified and individually overexpressed. Overexpression of SIP18 or CTT1 significantly enhanced tolerance to both vanillin and complex inhibitors. Additionally, overexpression of AAD4 or AAD6 improved vanillin tolerance, whereas SPI1 or GRE1 overexpression conferred increased resistance to the complex inhibitors. Notably, the engineered strain TX2022-SIP18 achieved high-level xylitol production of 43.50 g/L (yield: 0.961 g/g xylose) in concentrated hydrolysate from pretreated corn cob containing high concentrations of inhibitors.

This study provides the first experimental evidence that SIP18, AAD4, AAD6, SPI1, CTT1, and GRE1 contribute to inhibitor tolerance of S. cerevisiae, highlighting their potential as targets for engineering robust industrial strains for sustainable lignocellulosic xylitol production.