Biocontainment strategies forin vivoapplications ofSaccharomyces boulardii

Abstract

AbstractThe human gastrointestinal tract is a complex and dynamic environment, playing a crucial role in human health. Microorganisms engineered to express a therapeutic activity have emerged as a novel modality to manage numerous diseases. Such advanced microbiome therapeutics (AMTs) must be contained within the treated individual. Hence safe and robust biocontainment strategies are required to prevent the proliferation of microbes outside the treated individual. Here we present the first biocontainment strategy for a probiotic yeast, demonstrating a multilayered strategy combining an auxotrophic and environmental-sensitive strategy. We knocked out the genesTHI6andBTS1, causing thiamine auxotrophy and increased sensitivity to cold, respectively. The biocontainedSaccharomyces boulardiiwas unable to grow in the absence of thiamine above 1 ng/mL and exhibited a severe growth defect at temperatures below 20°C. The biocontained strain was well tolerated and viable in mice and demonstrated equal efficiency in peptide production as the ancestral non-biocontained strain. In combination, the data support thatthi6Δ andbts1Δ enable biocontainment ofS. boulardii, which could be a relevant chassis for future yeast-based AMTs.