Efficient surface display of single-chain variable fragments against tumor necrosis factor α on engineered probiotic Saccharomyces boulardii and its application in alleviating intestinal inflammation in vivo

Saccharomyces boulardii is a probiotic that can alleviate inflammation in the gut. In this study, a novel surface display system was developed by employing two different single-chain variable fragments (scFvs) against human tumor necrosis factor alpha as model anti-inflammatory proteins. We first optimized the expression levels of the scFvs by selecting strong constitutive promoters, of which expression cassettes were integrated into the S. boulardii chromosome using the CRISPR/Cas9-based genome editing system. As the signal peptide and anchoring motif are key factors affecting the display efficiency of target proteins, we next sought to find their optimal combination for the development of an efficient surface display system in S. boulardii. Among various combinations of signal peptide and anchoring motif, the engineered S. boulardii, which displayed scFvs using the SED1 signal peptide and DAN4 glycophosphatidylinositol domain (derived from Saccharomyces cerevisiae) as the signal peptide and anchoring motif, respectively, exhibited the highest display efficiency. Finally, the anti-inflammatory effects of the engineered S. boulardii strains displaying a high yield of scFvs, including a low disease activity index score, prevention of colon shortening, and reduction in pro-inflammatory cytokines, were validated using a colitis mouse model. Therefore, we believe that our approach has potential applications in the development of engineered S. boulardii displaying other valuable proteins.