14-06: Mutagenesis of Trichoderma reesei Endoglucanase I for Improved Function at Elevated Temperatures

The ability of Trichoderma reesei to secrete large quantities of cellulases is key in economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (50°C) could provide significant advantages, including reduced viscosity at high solid loadings, lower risk of microbial contamination during saccharification, greater compatibility with high temperature biomass pretreatment, and potentially faster rates of hydrolysis. The instability of Trichoderma reesei cellulases at temperatures above 50°C, combined with severely limited expression and production of existing thermophilic cellulases from non-fungal sources, motivates the development of thermostable Trichoderma reesei cellulases that can hydrolyze lignocellulose at temperatures in the range of 60-70°C. A B-factor guided approach for increasing T. reesei cellulase operating temperature and improving its thermostability was used to engineer variants of Trichoderma reesei endoglucanase I (TrEGI) with 2-fold improved activity at 65°C on solid cellulosic substrates as well as a ~3°C increase in its melting temperature (T_m). TrEGI mutants expressed in N. crassa were engineered to be as efficient at hydrolyzing solid substrates at 60°C as the recombinant (N. crassa) wild-type TrEGI is at 50°C. Production of TrEGI enzymes in different hosts (cell free, S. cerevisiae and N. crassa) highlighted the significant impact of the expression host (glycosylation and/or folding) on activity and stability of TrEGI enzymes. Choosing an appropriate host for cellulase expression and evolution has a considerable impact on the properties of the resulting enzymes.