Multi-omics data and model integration reveal the main mechanisms associated with respiro-fermentative metabolism and ethanol stress responses in Kluyveromyces marxianus

Kluyveromyces marxianus is a yeast capable of fermenting sugars into ethanol and growing at high temperatures (>37{o}C). However, it is less tolerant to ethanol than Saccharomyces cerevisiae, which limits its application in second-generation ethanol production. Since the mechanisms of ethanol stress response are still poorly described, especially compared to S. cerevisiae, we used an integrative multi-omics approach, combining transcriptomics, coexpression networks, gene regulation, and genome-scale metabolic modelling to gain insights about these mechanisms. Through metabolic modelling, we predicted the occurrence of a respiro-fermentative metabolism and its onset as the dilution rate increased. From gene coexpression networks, we detected that the protein quality control system is a main mechanism involved in the ethanol stress response. Further, we identified key regulators in the ethanol stress response, such as HAP3, MET4, and SNF2, and assessed how disturbances in their gene expression affect cellular metabolism. We also found that amino acid metabolism, membrane lipid metabolism, and ergosterol exhibit increased metabolic flux under the explored conditions. These findings provide useful cues to develop and implement genetic and metabolic engineering strategies to enhance ethanol tolerance.