(175ae) Establishing Probiotic Saccharomyces Boulardii As a Model Organism for Synthesis and Delivery of Biomolecules to the Mammalian Gut

Saccharomyces boulardii is a widely used yeast probiotic, demonstrating effectiveness against various gastrointestinal disorders. Thus, S. boulardii is a promising chassis for development of personalized engineered probiotic medicines. In this work, we measured S. boulardii’s residence time in the gut of antibiotic-treated and germfree mice. We found that S. boulardii can colonize antibiotic-treated mice for at least 5 days and can stably colonize germfree mice for longer than 30 days. We then enabled tunable metabolic engineering of S. boulardii by measuring the activities of a previously-established molecular toolkit for Saccharomyces cerevisiae in this strain. We found that during growth as a pure culture, gene expression levels and their cell-to-cell variability varied substantially based on the selective marker, origin of replication, promoter, or terminator which was used. We additionally performed CRISPR/Cas9-mediated genome editing in S. boulardii which enabled us to compare genomic versus plasmid-based gene expression. Finally, we performed combinatorial assembly of pathways for drug and vitamin synthesis, which allowed us to probe the effects of pathway architecture on product titers in pure culture and in the mammalian gut. This work establishes S. boulardii as a genetically tractable commensal fungus and provides a set of procedures for engineering S. boulardii to synthesize and deliver therapeutic biomolecules during colonization.