Design and in situ biomanufacturing of targeted peptide inhibitors via engineered probiotic yeast.

Many biological processes such as cancer cell proliferation, inflammation, and infection occur in the human gut. Biologic therapies, or treatments using substances made from living organisms, are particularly useful in treating these diseases and infections. The biological molecules used in biologic therapy tightly bind to specific molecules or organisms within the gut to modulate their behavior. Unfortunately, production and distribution of biologic therapies is incredibly complex and expensive, limiting availability to the public. A possible alternative approach is to make the biologic drugs directly in the patient's gut using unused dietary material and engineered microbes. Since the human gut is much more complex than an industrial bioreactor used to produce conventional biologics, there is still much to learn about how to accomplish this feat. This project will investigate strategies for biomanufacturing short chains of amino acids (peptides) directly in the large intestine using engineered probiotic yeast. The peptides will be optimized to bind with the toxins produced by Clostridioides difficile, the hospital-acquired gastrointestinal infection commonly referred to as C. diff. A yeast strain related to baker's yeast, Saccharomyces boulardii, will be engineered to produce the peptides under the harsh conditions of the human gut. The effectiveness of this approach will be examined in model organs, or 'miniguts.' This process of delivering drug molecules directly to the gut using engineered microbes promises to substantially reduce the cost of biologic therapeutics and, subsequently, improve human health. The investigators will also engage in research-related education and outreach, including developing a weekend symposium for high school teachers on microbiome engineering. Activities toward broadening the participation of underrepresented groups in STEM will include mentoring and active recruitment efforts.

In situ biomanufacturing, wherein therapeutic molecules are synthesized directly in the human gut by engineered microbes, is an attractive alternative to the complex manufacturing and oral delivery of biologic therapies. However, the human gut is unlike any industrial bioreactor, and many outstanding questions remain before in situ biomanufacturing becomes reality. The investigators posit that manufacturing peptide-based drugs in the probiotic yeast Saccharomyces boulardii to target Clostridioides difficile infection will serve as an ideal platform and model system by which to develop design strategies enabling efficient production of biologic molecules directly in the gut. Two complementary objectives will be pursued toward testing the engineering strains and peptides. First, peptides that specifically bind to C. difficile toxin A and to its surface layer protein (SlpA) will be designed. The peptides will be designed by combining solid phase library screening and computational peptide design/optimization approaches. The efficiency of thousands of peptides will be tested using in vitro gut models. Second, the physiological state of S. boulardii in gut models will be characterized by determining which genes are expressed and which proteins are secreted while residing in the gut. This knowledge will be applied to develop engineered S. boulardii strains that can effectively secrete and display the peptides designed in the first objective. The ability of the engineered strains to counteract C. difficile pathogenesis in 3D organoids will also be determined. The project will advance understanding of in situ biomanufacturing and reveal best practices for ensuring peptide efficacy.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.