Metabolic engineering of Pichia pastoris for high-efficiency production of branched-chain amino acids -enriched single-cell protein.

Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential nutrients for animals that must be obtained from the diet, as mammals cannot synthesize them. This study developed an engineered Pichia pastoris strain for efficient production of BCAAs-enriched single-cell protein (SCP) through synergistic integration of metabolic engineering and artificial intelligence. We first enhanced BCAAs biosynthesis by overexpressing the Ilv3 gene (encoding dihydroxy-acid dehydratase) via CRISPR-Cas9. Subsequently, the PMPEPE (Pichia pastoris Mutation Predictor for Enhanced Protein Expression) model screened endogenous proteins with high BCAAs content, enabling AI-driven in silico design of a BCAAs-rich variant, M0504 (35% BCAAs composition). This engineered protein was successfully expressed in P. pastoris. High-cell-density fermentation demonstrated that the engineered strain HTX33-ILV3-M0504 exhibited significantly increased crude protein content during methanol induction and superior biomass accumulation compared to controls. The BCAAs content in SCP reached 9.8 mg/100 mg (equivalent to 11.9 g/L in the fermentation broth), representing a 37.1% increase over the wild-type strain HTX-33 (7.2 mg/100 mg). Transcriptomics analysis revealed that Ilv3 overexpression upregulated key genes in the BCAAs synthesis pathway while modulating metabolic homeostasis through the TCA cycle, methanol assimilation, and carbon-nitrogen co-utilization. This work establishes a scalable strategy for industrial production of functional SCP enriched with BCAAs.