ROS signaling mediates aeration-dependent thermotolerance in Kluyveromyces marxianus for high-temperature xylitol production.

Abstract

This study uncovers that moderate reactive oxygen species (ROS) signaling mediates aeration-dependent thermotolerance in Kluyveromyces marxianus, challenging the long-held paradigm that ROS function solely as toxic metabolic byproducts. Through integrated transcriptomic profiling and RT-qPCR validation, 19 key transcription factors regulating this adaptive response were identified, and functional assays demonstrated that targeted knockout of GSF2 and RIM101 coupled with MED15 overexpression significantly enhances hypoxic thermotolerance. The ERG6-overexpressing strain YZB559 yields 72.45 g/L xylitol at 45 °C under medium-high oxygen (MHO) condition, marking a 20% improvement over the parental strain, while the combinatorially engineered YZB639 (ΔGSF2::MED15) achieves 74.13 g/L xylitol with complete xylose consumption at 46 °C and 30.64 g/L ethanol under micro-oxygen conditions, representing a 23% increase in ethanol production. Notably, at 47 °C, the highest temperature reported for xylitol fermentation, YZB639 accumulates 51.68 g/L xylitol under constant MHO condition, and an optimized two-stage oxygen supply strategy further elevates the titer to 67.78 g/L, with robust performance also observed when using industrial xylose mother liquor as feedstock to produce 57.34 g/L xylitol. This work resolves the fundamental oxygen contradiction between thermotolerance enhancement and product biosynthesis in high-temperature fermentation, providing a transformative strategy for cost-effective and sustainable industrial biomanufacturing.