AbstractThe assembly of functional cellulolytic enzymes was displayed using a synthetic, cell-surface engineered diploid yeast consortium. Trichoderma reesei endoglucanase II (EGII), cellobiohydrolase II (CBHII), and Aspergillus aculeatus β-glucosidase I (BGLI) were displayed as fusion proteins with the AGA2p C-terminus of a-agglutinin on the cell surface of the diploid yeast strain Saccharomyces cerevisiae Y5. The cell-surface immobilization of each enzyme was confirmed by immunofluorescence microscopy. This type of yeast consortium allowed convenient optimization of ethanol production by adjusting the combination ratios of each cell type for inducing synergy in cellulose hydrolysis. Next, the direct ethanol fermentation from steam-exploded corn stover was investigated. The optimized cellulase-displaying consortium produced 20.4 g/L ethanol from 48.4 g cellulose per liter after 72 h in the presence of a small amount of cellulase reagent (0.9 FPU/mL). These results suggested the feasibility of the cellulase-displaying yeast consortium for simultaneous saccharification and fermentation from insoluble cellulosic materials.