Engineering a robust cell-surface display platform in the multi-stress-tolerant yeast Issatchenkia orientalis.

Displaying cellulolytic enzymes on the yeast cell surface via the anchoring domain of glycosylphosphatidylinositol-anchored proteins can integrate enzyme production, saccharification, and fermentation into a single step in biomass fermentation. Therefore, this technique represents a promising strategy for cost-effective and sustainable production of value-added chemicals and biofuels from lignocellulosic biomass (LCB), the most abundant renewable resource, which typically requires multiple complex processing steps. However, pretreatment of LCB, such as acidic thermochemical treatment, causes harsh cultivation conditions characterized by low pH, high temperature, and lignocellulosic fermentation inhibitors. Thus, host strains displaying cellulolytic enzymes must exhibit remarkable tolerance to these stresses. Here, we employed the non-conventional yeast Issatchenkia orientalis, which has remarkable multi-stress tolerance, and successfully established the display system in this yeast. We found that the cell-surface display cassette that functions in the model yeast Saccharomyces cerevisiae was not functional in I. orientalis; therefore, we constructed a modified display cassette based on the I. orientalis SED1 gene. Using this cassette, we successfully displayed fluorescent protein and β-glucosidase (BGL) on the cell surface. The BGL-displaying strain grew robustly on cellobiose as the sole carbon source and, notably, maintained growth even in the presence of lignocellulosic fermentation inhibitors. To the best of our knowledge, this is the first report demonstrating the successful immobilization of functional proteins on the cell surface of I. orientalis and assimilation of LCB-derived intermediates, even under stress conditions, by displaying cellulolytic enzymes, highlighting its potential as a general industrial platform for sustainable LCB bioconversion. KEY POINTS: A cell-surface display cassette for Issatchenkia orientalis was constructed. The β-glucosidase-display strain utilized cellobiose as the sole carbon source. Cellobiose assimilation was observed even in the presence of fermentation inhibitors.