(125b) Development of a Gut-Inducible Expression Toolkit for Engineering in Probiotic Yeast Saccharomyces Boulardii

Saccharomyces boulardii is a widely used yeast probiotic, demonstrating effectiveness against various gastrointestinal disorders [1]. Thus, Sb is a model for commensal gut fungi and a promising chassis for development of personalized engineered probiotic medicines. Previous work demonstrated that constitutive promoters developed for S. cerevisiae are functional in Sb, allowing them to be used for in situ biomolecule production in the mammalian gut. However, promoters that activate in the gastrointestinal environment are necessary for programming Sb with stimuli-responsive functions, thereby enhancing the on-demand activity and fitness of the engineered Sb and limiting the off-target activity. In this work, we established a toolkit of native gut-inducible promoters in Sb. To do so, we measured the transcriptomic responses of Sb under various environmental conditions via RNA sequencing. We employed a stepwise complexity introduction model in our investigations, where in vitro cultures of single species (Sb) were followed by co-culture with commensal strains, and lastly animal models, including animal model of disease. We identified the genes that were differentially expressed under in vitro conditions mimicking the gut such as anaerobic, colon-like media. We also identified the genes that are differentially expressed when another commensal was present (Clostridioides difficile) with Sb in culture. Lastly, we investigated differentially expressed genes in Sb using several mouse models including germ-free, antibiotic-treated, and Clostridioides difficile infection. Overall, this work illuminates the physiology of Sb in vivo and enables the development of Sb with therapeutic functions that execute under defined in vivo conditions.