Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production

We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.Production of bioethanol from biomass has recently attracted attention due to the mandate for a billion gallons of renewable fuel by the new Energy Policy Act (22). Current production processes using sugar cane and cornstarch are well established (19, 23). However, utilization of a cheaper substrate would render bioethanol more competitive with fossil fuel (29). Cellulosic biomass found in many low-value agricultural or wood pulping wastes is particularly well suited because of its large-scale availability, low cost, and environmentally benign production (23). The primary obstacle impeding the more widespread production of ethanol from cellulose is the absence of a low-cost technology for overcoming its recalcitrant nature (21).Recently, a new method known as consolidated bioprocessing (CBP) has been proposed that combines enzyme production, cellulose saccharification, and fermentation into a single process to dramatically reduce the cost of ethanol production (22). An ideal microorganism for CBP should possess the capability of simultaneous cellulose saccharification and ethanol fermentation. One attractive candidate is Saccharomyces cerevisiae, which is widely used for industrial ethanol production due to its high ethanol productivity and high inherent ethanol tolerance (24). Attempts have been made to engineer S. cerevisiae to hydrolyze cellulose (6, 7, 16). However, due to energetic limitations under anaerobic conditions, only a small amount of cellulases can often be secreted. An alternative is to display the cellulolytic enzymes on the yeast cell surface (13, 14). Up to three different cellulases have been displayed, permitting the hydrolysis of cellulose with concomitant ethanol production. While these results point to a potential strategy of combining ethanol-producing capability with cellulose hydrolysis, the efficiency of hydrolysis must be significantly improved before it can be employed for practical applications.Many anaerobic bacteria have developed an elaborately structured enzyme complex on the cell surface, called the cellulosome, to maximize the catalytic efficiency of cellulose hydrolysis with only a limited amount of enzymes (1, 8, 9). The major component of these cellulosome complexes is a structural scaffoldin consisting of at least one cellulose-binding domain (CBD) and repeating cohesin domains, which are docked individually with a different cellulase tagged with the corresponding dockerin domain (26). Since the interaction between dockerin and cohesin is species specific (17, 25), designer minicellulosomes composed of three different dockerin-cohesin pairs with a cellulose hydrolysis efficiency up to sixfold higher than that of similar free enzymes have been generated (11, 12). Recently, it has been shown that the specific cellulose hydrolysis rates of metabolically active cultures of C. thermocellum displaying cellulosomes are more than fourfold higher than those of purified cellulosomes (20). This significant improvement appears to be a surface phenomenon involving adhesion to cellulose for enhanced substrate capture.In the present report, we demonstrate the functional assembly of a minicellulosome composed of three different cellulases on the S. cerevisiae cell surface and the feasibility of using the engineered yeast strains for cellulosic ethanol production. The success of displaying a functional cellulosome on the surface of an organism that already produces high titers of ethanol could lay a foundation for the achievement of an industrially relevant CBP-enabling microorganism.