A random evolution approach for improving fungal cellobiohydrolase activity

Hydrolysis of lignocellulosic biomass via enzymatic means involves the breakdown of crystalline cellulose by cellobiohydrolases (CBHs).  This step is considered as the rate limiting step in this whole process, which requires additional enzymes such as endo-β-1,4-endoglucanases and β -1,4-glucosidases. Among the non-complexed fungal cellulose systems, glycosyl hydrolase family 7 cellobiohydrolases are the most well studied glycosyl hydrolase enzymes and have dominated the industrial applications of cellulases. However, owing to their extremely slow rate of catalysis, industrial application of these enzymes would benefit greatly from improvements in their ability to decrease the degree of polymerization of cellulose. Here, we have used a random evolution approach to screen and identify Cel7A mutants with improved cellulase activities. Point mutations were introduced into the Cel7a gene (from Talaromyces emersonii) using error-prone mutagenesis. DNA sequencing of this mutation library revealed a mutation rate of 1-19 mutations per gene. This mutant library was cloned into a yeast expression vector and transformed into the yeast Saccharomyces cerevisiae containing a β -1,4-glucosidase expressing plasmid.  Approximately 5000 colonies that appeared on selection plates were screened for their ability to utilize Avicel as the carbon source as well as improved Cel7A activity using biochemical assays. Results of this mutant screen analysis are presented.