CRISPR-Cas9 engineering for high-level β-galactosidase production in Saccharomyces boulardii

Saccharomyces boulardii is a clinically validated probiotic yeast widely used to treat gastrointestinal disorders. Its natural probiotic effects, combined with the capacity of surviving the harsh gastrointestinal conditions, makes it an attractive chassis for therapeutic protein delivery. However, its engineering has relied on a limited set of genetic tools and lacks systematic validation, which has constrained its development as a next generation engineered probiotic. In this study, the compatibility of the EasyClone-MarkerFree vector system with CRISPR-Cas9 editing in S. boulardii was systematically assessed. Eight genomic loci were identified in silico as suitable for integration, and four were used to construct strains with the Aspergillus niger lacA gene integrated at one or multiple genomic loci, encoding an extracellular β-galactosidase capable of hydrolysing lactose into glucose and galactose, an activity of potential therapeutic relevance for lactose intolerance. Engineered strains efficiently hydrolysed lactose, with the strain with the lacA cassette integrated in three genomic loci (Sb-Triple) displaying up to an 11.76-fold increase in specific β-galactosidase activity relative to single-integration strains, without compromising growth. Importantly, Sb-Triple retained the native pH tolerance characteristic of S. boulardii and was able to grow on, and hydrolyse lactose from, cheese whey powder, a food-grade substrate. These results demonstrate a validated CRISPR-Cas9 strategy for high extracellular β-galactosidase secretion in the clinically established S. boulardii CNCM I-745, while evaluating the compatibility of EasyClone-MarkerFree vector set with this strain. Overall, this work provides a foundation for future studies where the potential of the developed strains is evaluated in clinically relevant settings.