7-68: Biased clique shuffling reveals stabilizing mutations in Cel7A

 

Lignocellulose-degrading enzymes from fungi are attractive for use in commodity scale biofuels production because they can be expressed at high titers relative to bacterial or archael cellulases. Still, their native properties can be suboptimal.  In addition to being subject to product inhibition and sensitive to small changes in pH, these enzymes typically exhibit low activity and poor thermostability. We have developed a mutagenesis platform to improve these properties and applied it to increase the thermostability of Cel7A. Secretion of Cel7A at high titers with limited hyperglycosylation was achieved using a Saccharomyces cerevisiae strain with upregulated protein disulfide isomerase, an engineered α-factor prepro leader, and a gene deletion of a plasma membrane ATPase. Using biased clique shuffling (BCS) of eleven Cel7A genes, we generated a small library (469) rich in activity (86%) and identified 51 chimeras with improved thermostability, many of which contained mutations in the loop networks that extend over the enzyme's active site.  The BCS library was far superior as a source of active and stable mutants compared to an equimolar library prepared from the same eleven genes.