Improving Cellular Protein Content of Based on Adaptive Evolution and Flow Cytometry-Aided High Throughput Screening.

Enhancing the protein content and production efficiency of is crucial as an alternative protein source. This study screened nongenetically modified yeast strains with high protein content for food ingredient production and explored the underlying mechanisms. Yeast protein levels were found to correlate with RNA, leading to a high-throughput screening method using RNA fluorescence and flow cytometry. Four mutant libraries (∼200,000 cells) were generated through adaptive laboratory evolution in protein synthesis inhibitors, resulting in the high protein mutant content B1, with a protein content of 65.8 g/100 g dry cell weight in shake flasks. In a 45 L bioreactor using fed-batch fermentation with ethanol below 1.5 g/L, B1's protein content increased to 70.3 g/100 g dry cell weight, an 18.5% rise. Mannan and β-glucan levels in the cell wall decreased by 21.7 and 30.5%, potentially enhancing protein extraction for food production. Transcriptome analysis revealed that increased protein content results from down-regulating the cell cycle and meiosis-related genes. Validation of differentially expressed genes demonstrated that up-regulating and down-regulating are key for enhancing protein synthesis and accumulation. This study proposes a nongenetic screening method for high protein content strains, achieving the highest reported protein content.