Expression of Melanocarpus albomyces cellobiohydrolase Cel7B in Saccharomyces cerevisiae and directed evolution to improve its thermostability

Cellulose is an important industrial raw material and a source of renewable energy. Cellulases catalyse the hydrolysis of cellulose to smaller oligosaccharides. This catalysis is exploited in many biotechnological processes. In the present study, a one-domain cellobiohydrolase (Cel7B) from the thermophilic fungus Melanocarpus albomyces was expressed in Saccharomyces cerevisiae. The expression plasmid pSV7 contains Cel7B cDNA under a constitutive triose phosphate isomerase (TPI) promoter, and URA3 was used as the selection marker. This yeast expression system makes the Cel7B available for directed evolution methods to improve its characteristics, i.e. thermostability. The cDNA sequence coding for the M. albomyces Cel7B was subjected to random mutagenesis by an error-prone PCR method resulting into a mutation rate on average of 1.7 amino acid changes per gene. After one round of random mutagenesis, a robotic screen of 15,000 clones was performed. Mutant libraries were screened in a 96-well microtiter plate format for higher temperature stability in an enzymatic activity assay based on a soluble chromophoric substrate, methylumbelliferyl lactoside (MUL). The yeast supernatants were pre-incubated at 76°C, and the activity after the heat treatment was compared to the activity of the untreated control samples. Mutants with improved thermostability were chosen for replica assays, after which a manual secondary screen was performed. The most interesting mutant cellulases from the secondary screen are now being purified for more detailed enzymatic characterisation.