Directed evolution of a beta-glucosidase for ionic liquid tolerance

Towards the conversion of renewable plant biomass to fuels, ionic liquids (ILs) have emerged as a powerful method for overcoming the recalcitrance of lignocellulosic biomass. However, after the removal of lignin and hemicellulose, approximately 20% residual IL remains with the resulting cellulose used for enzymatic saccharification; certain ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), inhibit enzyme activity. Therefore, one bottleneck in establishing a cellulase cocktail is identifying enzymes that are compatible with IL pretreated biomass. Currently, IL tolerant enzymes are identified by screening hundreds of enzymes from microbial communities, a costly and labor-intensive process. We hypothesized that cellulases and other enzymes could be engineered for IL tolerance. Therefore, we chose a highly active, thermostable (T50 = 80°C) beta-glucosidase (BG) that is only moderately IL tolerant ([C2mim][OAc] IL50  = 7.5%) for directed evolution. Error prone PCR (EpPCR) was used to create mutant libraries of the BG and a high throughput robotic screen was established to identify mutants with increased activity in the presence of IL. Selected mutants were then characterized for IL50, T50, melting temperature (Tm), and half-life, and the most stable mutants were subjected to subsequent rounds of EpPCR until an IL tolerant BG was evolved. The engineered BG also served as a comparison between the wild type and homologous BGs to gain insight into sequence and structural characteristics that could be used for the rational design of IL tolerant enzymes.