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 alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 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 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 beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-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.

FIG. 1.

Expression plasmid for display of T. reesei CBHII on the yeast cell surface (pFCBH2w3). s.s., secretion signal sequence of R. oryzae glucoamylase gene.

FIG. 2.

Immunofluorescence labeling of transformants: Nomarski differential interference micrographs (columns 1 and 4) and immunofluorescence micrographs (columns 2, 3, and 5) of S. cerevisiae MT8-1/pCAS1 (control) (A), MT8-1/pFCBH2w3 (B), MT8-1/pEG23u31H6 (C), MT8-1/pBG211 (D), MT8-1/pEG23u31H6/pFCBH2w3 (E), MT8-1/pBG211/pEG23u31H6 (F), and MT8-1/pBG211/pEG23u31H6/pFCBH2w3 (G). Cells were labeled with rabbit anti-FLAG IgG antibody and goat anti-rabbit IgG conjugated with Alexa Fluor 546 (column 2), with mouse anti-RGS(His)₄ antibody and goat anti-mouse IgG conjugated with Alexa Fluor 488 (column 3), and with rabbit anti-A. aculeatus BGL1 antibody and goat anti-rabbit IgG conjugated with Alexa Fluor 546 (column 5).

FIG. 3.

Time course of synergistic hydrolysis of amorphous cellulose by S. cerevisiae MT8-1/pCAS1 (control) (open square), MT8-1/pFCBH2w3 (open triangle), MT8-1/pEG23u31H6 (closed triangle), MT8-1/pEG23u31H6/pFCBH2w3 (closed circle), and MT8-1/pBG211/pEG23u31H6/pFCBH2w3 (open circle). The data points represent the averages of five independent experiments.

FIG. 4.

Graph (a) and photograph (b) representing the residual amount of cellulose in hydrolysis reaction mixture after 72 h of reaction with S. cerevisiae MT8-1/pCAS1 (control) (A), MT8-1/pFCBH2w3 (B), MT8-1/pEG23u31H6 (C), MT8-1/pEG23u31H6/pFCBH2w3 (D), and MT8-1/pBG211/pEG23u31H6/pFCBH2w3 (E). The data represent the averages of three independent experiments.

FIG. 4.

Graph (a) and photograph (b) representing the residual amount of cellulose in hydrolysis reaction mixture after 72 h of reaction with S. cerevisiae MT8-1/pCAS1 (control) (A), MT8-1/pFCBH2w3 (B), MT8-1/pEG23u31H6 (C), MT8-1/pEG23u31H6/pFCBH2w3 (D), and MT8-1/pBG211/pEG23u31H6/pFCBH2w3 (E). The data represent the averages of three independent experiments.

FIG. 5.

Time course of production of ethanol from amorphous cellulose as the sole carbon source with strain MT8-1/pBG211/pEG23u31H6/pFCBH2w3. Symbols: triangle, ethanol; circle, total sugar; square, glucose in culture broth. The data points represent the averages of seven independent experiments.