Abstract
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.
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Data availability
All the data that support the findings of this study are available within the paper and its Supplementary Data. All third-generation long-read, whole-genome resequencing, transcriptomic (RNA-seq), and metagenomic sequencing data generated in this study have been deposited in the NCBI database under the following BioProject accession numbers: PRJNA1281804, PRJNA1281807, PRJNA1380636, PRJNA1281849, and PRJNA1283298. Metagenomic data for sample H22B656-6 are not obtained due to DNA extraction failure. The predicted structures of the wild-type protein and the N192D mutant have been deposited in Figshare (https://doi.org/10.6084/m9.figshare.31272826). Targeted metabolomics data for bile acids are available in the MetaboLights database under identifier MTBLS13718. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 32525049) awarded to J.Z.
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J.Z. and Z.S. conceptualized the research framework and formulated the experimental design. The experimental work and sample preparation were conducted by Z.H., X.L., D.Z., Q.G., L.Z., and S.S. Data analysis was performed by Z.H. in collaboration with Z.S. Z.H., Z.S., S.J., and Z.Z. contributed to figure preparation. The manuscript was drafted by Z.S. and Z.H., and J.Z. and Z.S. provided critical review and substantially refined the final version. All authors read and approved the final manuscript.
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Han, Z., Sun, Z., Liu, X. et al. Harnessing a germ‑free mouse gut bioreactor for directed evolution of probiotics to combat non-alcoholic fatty liver disease. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69823-0
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DOI: https://doi.org/10.1038/s41467-026-69823-0
