During high-concentration ethanol fermentation, Saccharomyces cerevisiae often faces multiple stresses, such as high osmotic pressure, ethanol toxicity, and nutrient limitation. These factors collectively limited the production of ethanol. To identify novel targets related to fermentation performance, we employed SHPERM- bCGHR strategy (a marker free allele replacement strategy based on comparative genomics and homologous recombination). We replaced the endogenous PHO4 of the high-producing strain MF01 with the PHO4 allele from MC15, thereby constructing a novel engineered strain MF01-PHO4. Under low-phosphate conditions, compared with the wildtype strain, the PHO5/11/12 genes and ribosomal protein genes showed significant upregulation in MF01-PHO4. These changes were associated with enhanced phosphorus uptake and protein synthesis. Under high phosphate conditions, the PHO4 expression and glycolytic enzyme gene expression in MF01-PHO4 were both lower than MF01, indicating that the substitution of the PHO4 allele may be associated with the coordinated changes in phosphate signal-mediated carbon phosphorus metabolism. This study identifies PHO4 as a promising candidate target for improving high concentration ethanol fermentation efficiency. These findings provide a framework to understand the phosphate-dependent regulatory effects of PHO4 allelic variation and offer a transferable strategy for strain improvement.

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Journal Article

Authors

Shan X, Bin Q, Lyu R, Liu Z, Zou H, Lin S, Zhou J, Zhang L, Wu R, Liao Y

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