| Summary: | Abstract Saccharomyces boulardii (Sb) has gained significant attention for its potential therapeutic application as a probiotic yeast strain. Current approaches often leverage its secretion and display capabilities to deliver therapeutic agents aimed at alleviating intestinal disorders. However, relatively few studies have focused on optimizing its display efficiency. In this study, we evaluated two surface display systems, Aga2- and Sed1-based, for use in Sb by systematically modifying display cassette components and the host strain. Initially, both systems were tested in Saccharomyces cerevisiae (Sc) and Sb to validate their design. Sc consistently outperformed Sb in both display expression and efficiency, highlighting the need for further optimization in Sb. To enhance the display efficiency in Sb, we investigated specific modifications to the display cassette, including the use of linker sequences for Aga2 and variations in anchor length for Sed1. These experiments identified key factors influencing display performance. Subsequently, we engineered a modified Sb strain, LIP02, by overexpressing AGA1 and deleting cell wall-related genes (CCW12, CCW14, and FYV5). These modifications were expected to expand the available docking sites for the protein of interest (POI) and improve overall protein secretion and display efficiency. As a result, the modified strain exhibited a significant enhancement in display capacity compared to the wild-type Sb strain. Furthermore, genome integration of the display cassette in LIP02 enhanced both stability and expression compared to plasmid-based systems. Importantly, the functionality of β-glucosidase displayed on LIP02 was preserved, as demonstrated by improved enzymatic activity and robust growth on cellobiose as the sole carbon source. These findings establish LIP02 as a superior host for surface display applications in Sb, offering a more stable and efficient platform for the expression of therapeutic proteins and other functional biomolecules.
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