Engineering stress tolerance in Saccharomyces cerevisiae by overexpressing PIR3 and SPI1 for efficient ethanol production from high-concentration sugarcane molasses.

The coexistence of potassium and calcium ions has been identified as a major factor limiting high-yield ethanol fermentation by Saccharomyces cerevisiae in high-concentration sugarcane molasses. To identify key genes conferring tolerance to this stress, we employed an integrated strategy combining multi-omics analysis, CRISPR-mediated gene activation/repression, and targeted overexpression. This approach pinpointed four critical genes: PIR3, SPI1, AQR1, and GUT2. Functional analysis showed that while AQR1 and GUT2 enhance ethanol biosynthesis primarily by redirecting metabolic flux, PIR3 and SPI1 are crucial for maintaining cellular integrity and viability under stress. Overexpression of PIR3 and SPI1 in a wild-type strain increased ethanol production by 24.6%, achieving a final titer of 113.3 g/L in a 5-L fermenter, a performance comparable to robust industrial strains. Furthermore, this engineering strategy boosted the synthesis of other valuable compounds, exemplified by a 12.5% increase in cinnamic acid production. Our work thus identifies precise genetic targets for engineering stress-tolerant yeast and establishes a foundation for efficient bioconversion of high-concentration sugarcane molasses.