Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

Abstract

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 ␣-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus ␤-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of ␣-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 II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of ␤-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying ␤-glucosidase 1 and endoglucanase II 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.

Ethanol production from cellulosic materials using cellulase‐expressing yeast

“…However, S. cerevisiae cannot utilize cellulosic materials, and a cellulose saccharification process to produce glucose is necessary before fermentation [5].…”

Recombinant Cellulase and Cellulosome Systems

“…However, levels of cellulase expression were deemed low, and did not enable growth and ethanol production using cellulose as the sole carbon source. A later study similarly expressed the three types of cellulases required for cellulose degradation in S. cerevisiae [13]. The EGL and CBH, from Trichoderma reesei, and the BGL, from Aspergillus aculeatus, were co-displayed as -agglutinin fusions on the surface of yeast cells, enabling the liberation of glucose from phosphoric acid swollen cellulose (PASC), and fermentation to ethanol when the cells were pre-grown in rich media.…”

Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa

“…Cho et al [35] showed that, for SSF experiments with a strain producing both a b-glucosidase and enzymes with exo-and endocellulase activity, loadings of externally added cellulase could be reduced. Fujita et al [36,37] reported co-expression and surface display of cellulases in S. cerevisiae. High-cell-density suspensions of a recombinant strain displaying the Trichoderma reesei endoglucanase II, cellobiohydrolase II and the Aspergillus aculeatus b-glucosidase were able to directly convert 10 g l 21 phosphoric acid swollen cellulose (PASC) to approximately 3 g l 21 ethanol.…”