Palou, R.; van der Sloot, A. M.; Fiebig, A. A.; Zangara, M. T.; Sangwan, N.; Sanchez-Osuna, M.; Ilyas, B.; Zubyk, H.; Cook, M.; Wright, G. D.; Coombes, B. K.; Tyers, M.

Protein-based pharmaceuticals, such as engineered antibodies, form a major drug class of steadily increasing market share. However, these biologic medicines are costly to manufacture, are subject to strict supply chain and storage constraints and often require invasive administration routes. Engineered microbes that secrete bioactive products directly within the microbiome milieu may mitigate these challenges. Here, we describe a cell microfactory platform based on the probiotic yeast Saccharomyces boulardii for the production of nanobody biologics in the gastrointestinal (GI) tract. High-level secretion of nanobodies by S. boulardii was achieved by optimizing promoters, secretion signals, and antibody formats. In mice, oral gavage of S. boulardii allowed efficient and transient colonization of the colonic compartment and in situ production of a therapeutic nanobody directed against tumor necrosis factor (TNF). In a mouse model of chemical-induced colitis, GI-delivery of anti-mTNF nanobody via live S. boulardii improved both survival and disease severity without causing overt perturbation of microbiome composition. These results position S. boulardii as a synthetic biotic platform for the in situ production and delivery of protein-based therapeutics to the GI tract.