Surface-displayed whole-cell glucose oxidase from engineered Pichia pastoris exhibits enhanced intestinal repair and barrier restoration in a polymicrobial-infected mouse model.

Pathogenic infections pose significant threats to livestock production, necessitating antibiotic-free alternatives. This study engineered a high-yield whole-cell glucose oxidase (GOD) in Pichia pastoris via yeast cell surface display technology by optimizing anchor proteins (Gcw21), gene copy numbers (3 copies), and auxiliary proteins (HAC1) co-expression. The engineered strain yielded a GOD activity of 9322 U/g dry cell weight. The whole-cell GOD exhibited superior thermal stability, acid resistance, and tolerance to proteases and metal ions compared to free GOD. In vitro, its catalytic products, gluconic acid (GA) and hydrogen peroxide (H₂O₂), demonstrated potent antibacterial activity against enterotoxigenic Escherichia coli (ETEC) and Staphylococcus aureus. In a murine co-infection model (ETEC and S. aureus), whole-cell GOD outperformed free GOD in mitigating intestinal damage and reducing oxidative stress; it also better restored barrier integrity, reflected by increased tight junction proteins and villus-to-crypt ratio. The gastrointestinal stability of whole-cell GOD enabled sustained GA and H₂O₂ production, lowering luminal pH while inhibiting pathogens and promoting probiotic growth. These findings establish whole-cell GOD as a promising antibiotic-free feed additive for maintaining intestinal homeostasis, with engineered cell surface display enhancing its catalytic efficiency and therapeutic potential in animal husbandry.