Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase 11 and cellobiohydrolase 11 and Aspergillus aculeatus beta-glucosidase I were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase H showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase 11, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase 11, and cellobiohydrolase 11 enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase I and endoglucanase 11 could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.