Harnessing a germ‑free mouse gut bioreactor for directed evolution of probiotics to combat non-alcoholic fatty liver disease.

Directed evolution has transformed biomolecular engineering but remains largely untapped for probiotic optimization, despite its immense promise for human health maintenance and disease therapy. Here, we present an in vivo, host-mediated directed-evolution platform that harnesses the gut's endogenous selective pressures to drive functional enhancement of probiotics. Using Bifidobacterium animalis subsp. lactis as a model, we expose germ-free male mice to stepwise increases in bile-acid stress via a high-fat, high-cholesterol diet. Compared to in vitro evolution, which fails to produce any functional gains, our host-mediated approach yields a variant exhibiting a 77% increase in bile acid metabolism. Multi-omics analysis identifies two critical single-nucleotide variants (SNVs) simultaneously: one in the upstream region of cbh, encoding bile salt hydrolase, and a non-synonymous mutation in mdr, a bile-acid efflux transporter. Functional validation assays confirm that these mutations drive increased corresponding gene expression and enhance substrate binding efficiency. Moreover, to demonstrate its translational potential, we administer the parental and adapted strains daily in a male diet-induced mouse model of non-alcoholic fatty liver disease (NAFLD). We find that the adapted strain significantly improves bile-acid homeostasis, reduces hepatic steatosis, lowers inflammatory and lipid biomarkers, and enhances histological liver health compared to the parental strain. Our work establishes the host gut as a living evolutionary bioreactor for precision engineering of probiotics, enabling targeted phenotypic enhancement in vivo through natural selection. This framework paves the way for personalized, functionally tailored microbiome therapeutics and sets a foundation for next-generation live biotherapeutic agents.