A genetic engineering toolkit for the probiotic yeast Saccharomyces boulardii

Saccharomyces boulardii ( S. boulardii ) is the only yeast probiotic widely used in clinical practice and consumer products. It combines gastrointestinal robustness, intrinsic antibiotic tolerance and eukaryotic protein-processing capacity, making it an attractive chassis for engineered probiotics, in situ biomanufacturing and live biotherapeutic delivery. However, host-specific characterization of regulatory elements, candidate genomic integration sites, and multicopy integration strategies remains limited in the clinically used S. boulardii CNCM I-745 background. Here, using S. boulardii CNCM I-745 as the host, we developed and characterized a genetic engineering toolkit that integrates transcriptional control elements with genome-integration strategies. We first quantified the activities of 20 endogenous constitutive promoters, two inducible promoters and six terminators. We then established a markerless CRISPR-Cas9 workflow and evaluated 10 chromosomal integration loci; six of which showed Editing efficiency above 80% while supporting comparable transgene expression without detectable growth penalties. Finally, we constructed an rDNA-repeat-based multicopy integration system that increased transgene expression by 9.8- to 22.6-fold relative to single-copy integration and revealed a significant positive but non-proportional association between estimated copy number and reporter output. Together, this toolkit provides S. boulardii with a set of tunable promoters and terminators, candidate chromosomal integration sites compatible with a markerless CRISPR-Cas9 workflow. These resources expand the genetic accessibility of this probiotic yeast and provide a foundation for developing engineered S. boulardii strains with enhanced probiotic, therapeutic and biomanufacturing functions.