Directed Evolution of a Thermophilic Cellulase Using GFP as a Reporter

Cellulases, composed of endo-β-1,4-glucanases (EC. 3.2.1.4), cellobiohydrolases (3.2.1.91) and β-1,4-glucosidases (3.2.1.21), are important biocatalysts for many industrial processes, including the resurgent area of cellulosic biofuels production where cellulases may be exposed to extremes of temperature, pH and harsh pretreatment protocols. To date little progress has been made in improving the catalytic efficiency of cellulases in these environments, which is important for reducing production costs and making biofuels economically viable.

Here we report on engineering of a thermoplilic Cel5A for enhanced activity in at 70 °C in an ionic liquid environment. We describe the use of GFP fused to Cel5A, which allows us to use flow cytometry to eliminate mutants that were not fully transcribed because of truncations or frameshifts and potentially mutants that are misfolded. This in turn allows us to screen larger libraries generated using higher error prone PCR rates. Additionally, the fluorescent intensity represents the protein expression level, allowing the mutant library to be screened for both total activity and specific activity. By using error-prone PCR with a high error rate (~6 amino acid mutations on average), highly diverse mutant libraries of Cel5A were constructed and screened for improved activity. One novel mutant was characterized to have ~4 folds increase in specific activity compared to wild-type enzyme.