Proline protects Wickerhamomyces anomalus against ethanol‑induced damage during logarithmic growth: integrated physiological, transcriptomic, and metabolomic analyses.

BACKGROUND

Proline plays a critical regulatory role in microbial stress responses; however, its specific mechanism of action under ethanol stress in Wickerhamomyces anomalus during logarithmic growth remains poorly understood.

RESULTS

This study investigated the protective effects of proline against ethanol-induced injury and elucidated the mechanism by which it enhances resistance to 9% (v/v) ethanol stress. The results indicated that proline supplementation significantly increased the viability of ethanol-stressed yeast and stimulated cellular proline transport and catabolism. Furthermore, proline mitigated the impairment in morphology and ultrastructure of yeast cells by maintaining cell wall, cell membrane, and mitochondrial integrity, ameliorating oxidative stress, and restoring cellular homeostasis under ethanol stress. Further analysis revealed that the protective effect of proline on cell membrane homeostasis was related to decreased membrane permeability, increased membrane fluidity, and elevated intracellular K⁺ levels. The alleviation of oxidative stress by proline supplementation was associated with enhanced antioxidant enzyme activities and increased glutathione levels. Transcriptomic and metabolomic analyses indicated that proline regulated ribosome synthesis, the cell wall integrity pathway, ABC transporters, and arginine and proline metabolism.

CONCLUSIONS

These findings indicate that proline effectively protects yeast from ethanol stress, supporting the development of ethanol-tolerant W. anomalus strains for industrial fermentation.