RT Journal Article SR Electronic T1 Establishing Probiotic Saccharomyces boulardii as a Model Organism for Synthesis and Delivery of Biomolecules JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.01.22.915389 DO 10.1101/2020.01.22.915389 A1 Deniz Durmusoglu A1 Ibrahim Al’Abri A1 Scott P. Collins A1 Chase Beisel A1 Nathan Crook YR 2020 UL http://biorxiv.org/content/early/2020/01/23/2020.01.22.915389.abstract AB Saccharomyces boulardii is a widely used yeast probiotic which can counteract various gastrointestinal disorders1. As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid growth and is easy to transform2 and thus represents a promising chassis for the engineered secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to the mammalian gut, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements in this organism. These genetic elements were characterized in plasmidic and genomic contexts, revealing strategies for tunable control of gene expression and CRISPR-mediated genome editing in this strain. We then leveraged this set of genetic parts to combinatorially assemble pathways enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardii’s residence time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the relationships between dosing strategy and colonization level. This work establishes S. boulardii as a genetically tractable commensal fungus and provides a set of strategies for engineering S. boulardii to synthesize and deliver biomolecules during gut colonization.