Improving Cellular\nProtein Content of Saccharomyces cerevisiae Based on Adaptive Evolution\nand Flow Cytometry-Aided High Throughput Screening

Enhancing the protein content and production efficiency\nof Saccharomyces cerevisiae is crucial\nas an alternative\nprotein source. This study screened nongenetically modified yeast\nstrains with high protein content for food ingredient production and\nexplored the underlying mechanisms. Yeast protein levels were found\nto correlate with RNA, leading to a high-throughput screening method\nusing RNA fluorescence and flow cytometry. Four mutant libraries (∼200,000\ncells) were generated through adaptive laboratory evolution in protein\nsynthesis inhibitors, resulting in the high protein mutant content\nB1, with a protein content of 65.8 g/100 g dry cell weight in shake\nflasks. In a 45 L bioreactor using fed-batch fermentation with ethanol\nbelow 1.5 g/L, B1′s protein content increased to 70.3 g/100\ng dry cell weight, an 18.5% rise. Mannan and β-glucan levels\nin the cell wall decreased by 21.7 and 30.5%, potentially enhancing\nprotein extraction for food production. Transcriptome analysis revealed\nthat increased protein content results from down-regulating the cell\ncycle and meiosis-related genes. Validation of differentially expressed\ngenes demonstrated that up-regulating <i>SUT1</i> and down-regulating <i>CNM67</i> are key for enhancing protein synthesis and accumulation.\nThis study proposes a nongenetic screening method for high protein\ncontent S. cerevisiae strains, achieving\nthe highest reported protein content.