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Sustained in situ protein production and release in the mammalian gut by an engineered bacteriophage

Nature Biotechnology (2025)Cite this article

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Abstract

Oral administration of biologic drugs is challenging because of the degradative activity of the upper gastrointestinal tract. Strategies that use engineered microbes to produce biologics in the lower gastrointestinal tract are limited by competition with resident commensal bacteria. Here we demonstrate the engineering of bacteriophage (phage) that infect resident commensals to express heterologous proteins released during cell lysis. Working with the virulent T4 phage, which targets resident, nonpathogenic Escherichia coli, we first identify T4-specific promoters with maximal protein expression and minimal impact on T4 phage titers. We engineer T4 phage to express a serine protease inhibitor of a pro-inflammatory enzyme with increased activity in ulcerative colitis and observe reduced enzyme activity in a mouse model of colitis. We also apply the approach to reduce weight gain and inflammation in mouse models of diet-induced obesity. This work highlights an application of virulent phages in the mammalian gut as engineerable vectors to release therapeutics from resident gut bacteria.

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Fig. 1: Phage-induced lysis of bacteria releases intracellular proteins.
Fig. 2: Survey of T4 phage promoters for in vitro sfGFP production.
Fig. 3: In vivo sfGFP production by engineered phage.
Fig. 4: Reduction of DSS-induced colitis via Serpin production.
Fig. 5: The efficacy of T4::clpB to reduce food consumption and weight gain.

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Data availability

Source data for figures in the main text and Supplementary Information are provided with this paper. 16S amplicon reads are available on Mendeley Data85. Source data are provided with this paper.

Code availability

No code was generated in this study.

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Acknowledgements

We thank T. Thorn, S. DiLorenzo and J. Cartwright at Virginia Tech for technical assistance, as well as E. Ng at the FLSI Light Microscopy Facility at Virginia Tech for fluorescence microscopy support. Research reported in this publication was supported in part by the National Institutes of Health (R35 GM147484 (to B.B.H.) and R01 AI172133 (to L.L.)) and Virginia Tech Dean’s Discovery Fund (fund 446728 to B.B.H. and. L.L.).

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Authors and Affiliations

  1. Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA

    Zachary R. Baker, Yao Zhang, Hollyn C. Franklin, Liwu Li & Bryan B. Hsu

  2. Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA, USA

    Zachary R. Baker, Hollyn C. Franklin & Bryan B. Hsu

  3. Center for Emerging, Zoonotic, and Arthropod-Borne Pathogens, Virginia Tech, Blacksburg, VA, USA

    Zachary R. Baker, Hollyn C. Franklin & Bryan B. Hsu

  4. Metabolism Core, Department of Human Nutrition, Foods, and Exercise, Virginia Tech, Blacksburg, VA, USA

    Haiyan Zhang

  5. Department of Biomedical Sciences and Pathobiology, VA–MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

    Priscila B. S. Serpa, Teresa Southard & Bryan B. Hsu

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Contributions

B.B.H. and L.L. conceptualized the project. Z.B. and B.B.H. developed the methodology. Z.B., Y.Z., H.Z., H.F., P.B.S.S. and T.S. conducted the investigation. Z.B. and B.B.H. wrote the original draft of the paper. L.L. and B.B.H. reviewed and edited the paper, provided supervision and secured funding.

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Correspondence to Liwu Li or Bryan B. Hsu.

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Competing interests

Z.B., L.L. and B.B.H. are inventors on a pending patent application related to the phage-based heterologous gene expression system described in this paper. The other authors declare no competing interests.

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Nature Biotechnology thanks Jeremy Barr, Laurent Debarbieux and Tae Seok Moon for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figs. 1–7 and Supplementary Tables 1 and 2.

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Supplementary Data 1

Supporting data for Supplementary Figs. 1, 3–5 and 7.

Supplementary Data 2

Oligonucleotide sequences used in this study.

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Baker, Z.R., Zhang, Y., Zhang, H. et al. Sustained in situ protein production and release in the mammalian gut by an engineered bacteriophage. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02570-7

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