Dual-phase Hog1 activation and transporter gene reprogramming enable extreme sugar tolerance in food osmophilic yeasts.

The protein kinase Hog1 plays a central role in cellular responses, including cell volume and gene expression regulation during osmoregulation in the model yeast Saccharomyces cerevisiae. Despite sharing the conserved kinase Hog1 for osmotic response, Zygosaccharomyces rouxii and S. cerevisiae exhibit markedly different sugar resistance. Here, we systematically compared the phenotypes, Hog1 phosphorylation kinetics, and transcriptomic profiles of both yeasts under 60 % (w/v) extremely high-glucose stress. Under 60 % (w/v) extremely high-glucose stress, Z. rouxii exhibits prolonged survival with volume recovery post-shrinkage, contrasting S. cerevisiae's irreversible collapse. Additionally, we found that the important Hog1 kinase shows transient activation with Hsp70-coupled recovery in Z. rouxii versus sustained activation in S. cerevisiae. Correspondingly, transcriptome data showed different expression patterns of transmembrane transport differentially expressed genes (DEGs): S. cerevisiae upregulated high-affinity transporter genes (HXT3: 5.2-fold; HXT4: 4.7-fold), whereas Z. rouxii induced low-affinity transporter genes (ZYRO0E10054 (FFZ1): 1.6-fold; ZYRO0F02090 (FFZ2): 25.8-fold) under 60 % (w/v) extremely high-glucose stress. Most transmembrane transport gene expression patterns persist in 60 °brix apple juice stress (complex sugar), except for stress-type-specific induction of ZYRO0F02090 (FFZ2) and ZYRO0E09988 (FLR1). Our work deciphers the evolutionary divergence of sugar osmoadaptation strategies in yeasts, providing actionable targets for engineering microbial sugar tolerance.