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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.<\/jats:p>","DOI":"10.3390\/life16081374","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T10:55:18Z","timestamp":1787223318000},"page":"1374","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Transcriptomic Analysis Reveals How PHO4 Gene Modulates Growth and Ethanol Fermentation in Saccharomyces cerevisiae"],"prefix":"10.3390","volume":"16","author":[{"given":"Xinran","family":"Shan","sequence":"first","affiliation":[{"name":"Guangxi Key Laboratory of Polysaccharide Materials and Modification, Guangxi-Indonesia Joint Laboratory for Microbial Resources and Artificial Intelligence, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning 530008, 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Joint Laboratory for Microbial Resources and Artificial Intelligence, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning 530008, China"},{"name":"National Key Laboratory of Non-Food Biomass Technology, National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0088-7011","authenticated-orcid":false,"given":"Yanjuan","family":"Liao","sequence":"additional","affiliation":[{"name":"Guangxi Key Laboratory of Polysaccharide Materials and Modification, Guangxi-Indonesia Joint Laboratory for Microbial Resources and Artificial Intelligence, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning 530008, 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