Factors affecting ethanol tolerance in Kazachstania unispora Mkaz: membrane characteristics and antioxidative stress.

Kazachstania unispora is extensively utilized as a food-grade fermentation agent due to its strong stress-resistance and superior fermentation properties. However, ethanol stress during fermentation negatively impacts its performance. Therefore, selecting ethanol-tolerant K. unispora and understanding its tolerance mechanisms are critical for enhancing fermentation efficiency and product quality. In this study, a mutant strain K. unispora Mkaz, capable of tolerating up to 20 % (vol/vol) ethanol and exhibiting favorable fermentation characteristics, was developed through mutagenesis. Comprehensive phenotypic and multi-omics analyses were conducted to elucidate the mechanisms underlying its ethanol tolerance. The results indicated that increased levels of total fatty acid (TFA), particularly long-chain fatty acid (LCFA) and very long-chain fatty acid (VLCFA), as well as trehalose and ergosterol under ethanol stress, improved membrane characteristics, thereby enhancing ethanol tolerance. Furthermore, elevated activities of adenosine triphosphatase (ATPase), superoxide dismutase (SOD), and catalase (CAT) contributed to ethanol tolerance by reducing oxidative stress. Genomic and transcriptomic analyses revealed key mutations and differentially expressed genes (DEGs) associated with the biosynthesis of fatty acid (FA), trehalose, and ergosterol, as well as the regulation of oxidative stress. This study provides novel insights into the ethanol tolerance mechanisms in K. unispora, laying a foundation for its potential application in food fermentation.