In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 degrees C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (beta-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled beta-carotene synthesis (194 mu g total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional beta-carotene recovered in feces was 56-fold higher than the beta-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.