Physiological, transcriptomic, and metabolomic insights into the sugar stress tolerance mechanisms of Clavispora lusitaniae and its fermentation potential.

High-gravity fermentation (HGF) is a powerful strategy to increase volumetric productivity in beer, wine, and bioethanol production but imposes severe osmotic stress that limits yeast viability and process stability. Non-Saccharomyces yeasts such as Clavispora lusitaniae have shown promise for improving flavor complexity and stress tolerance; however, their sugar stress adaptation mechanisms remain poorly characterized. Here, we systematically investigated the physiological, transcriptional, and metabolic responses of C. lusitaniae CP022 under 60% glucose stress and evaluated its fermentation performance. Physiological assays revealed a 4.3-fold increase in glycerol production (3.5 → 15 g/L) and a significant rise in ergosterol content (p < 0.01), accompanied by a 50% reduction in membrane fluidity. Cell wall analysis showed β-1,3-glucan and chitin contents increased by 1.7-fold and 1.5-fold, respectively, indicating wall reinforcement. Transcriptomic profiling identified 1035-1438 upregulated and 1082-1536 downregulated genes at 36-60 h, with significant enrichment of glycolysis, sterol biosynthesis, and amino acid metabolism pathways. Metabolomics detected 902 differential metabolites, predominantly in carbon and nitrogen metabolism. During grape juice fermentation, co-inoculation with S. cerevisiae increased ethanol yield by 15%, boosted phenethyl alcohol accumulation by 22%, and enhanced the accumulation of multiple esters, thereby intensifying the floral flavor profile. These findings provide the important multi-omics insight into the sugar stress tolerance of C. lusitaniae and demonstrate its potential as a functional adjunct yeast for optimizing high-sugar fermentation systems and improving process robustness.