Precursor supply and translational machinery engineering of Saccharomyces cerevisiae for improving cellular protein...

The sustainable production of biomass-based microbial protein (MP) requires efficient microbial cell factories for accumulating cellular protein with high content, which is beneficial both for improving protein production and downstream cellular protein isolation and purification. To overcome the limited protein content of the Saccharomyces cerevisiae, we designed a systematic multilevel metabolic engineering strategy. Initially, single-gene edits based on predictions using the genome-scale model Yeast 9.0.2 and the OptForce algorithm failed to increase protein content due to precursor supply limitations. Enhancing genes in nitrogen metabolic (GDH1, GDH2, GLN1, GLT1) and central carbon (CIT1, IDH1) pathways were implemented to synergistically enhance ammonium assimilation. Subsequently, overexpression of valyl-tRNA synthetase (VAS1) alleviated the translational bottleneck, increasing cellular protein content to 52.3 g/100 g dry cell weight (DCW). The ribosomal synthesis pathway was further enhanced via ribosomal regulator IFH1 and ribosomal protein gene overexpression, with cellular protein content reaching 57.3 g/100 g DCW. Finally, diploidization and global transcriptional regulator SUT1 integration in strain D3 achieved a protein content of 66.5 g/100 g DCW in shake flask culture. Under controlled 5 L bioreactor conditions, its protein content further increased to a peak of 75.2 g/100 g DCW, representing a 50.3% increase over the parental strain Y1. This study developed a multilevel engineering strategy to enhance yeast protein production by optimizing precursor supply, translation machinery, and diploid construction. Using marker-free editing and endogenous gene regulation, it provides both improved protein content and key targets for breeding high-protein microbial strains.