The importance of pyroglutamate in cellulase Cel7A.

The commercialization of lignocellulosic biofuels relies in part on the ability to engineer cellulase enzymes to have properties compatible with practical processing conditions. The cellulase Cel7A has been a common engineering target because it is present in very high concentrations in commercial cellulase cocktails. Significant effort has thus been focused on its recombinant expression. In particular, the yeast Saccharomyces cerevisiae has often been used both in the engineering and basic study of Cel7A. However, the expression titer and extent of glycosylation of Cel7A expressed in S. cerevisiae vary widely for Cel7A genes from different organisms, and the recombinant enzymes tend to be less active and less stable than their native counterparts. These observations motivate further study of recombinant expression of Cel7A in S. cerevisiae. Here, we compare the properties of Cel7A from Talaromyces emersonii expressed in both the budding yeast S. cerevisiae and the filamentous fungus Neurospora crassa. The Cel7A expressed in N. crassa had a higher melting temperature (by 10°C) and higher specific activity (twofold at 65°C) than the Cel7A expressed in S. cerevisiae. We examined several post-translational modifications and found that the underlying cause of this disparity was the lack of N-terminal glutamine cyclization in the Cel7A expressed in S. cerevisiae. Treating the enzyme in vitro with glutaminyl cyclase improved the properties of Cel7A expressed in S. cerevisiae to match those of Cel7A expressed in N. crassa.

Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A.

Renewable fuels produced from biomass-derived sugars are receiving increasing attention. Lignocellulose-degrading enzymes derived from fungi are attractive for saccharification of biomass because they can be produced at higher titers and at significantly less cost than those produced by bacteria or archaea. However, their properties can be suboptimal; for example, they are subject to product inhibition and are sensitive to small changes in pH. Furthermore, increased thermostability would be advantageous for saccharification as increased temperature may reduce the opportunity for microbial contamination. We have developed a mutagenesis platform to improve these properties and applied it to increase the operating temperature and thermostability of the fungal glycosyl hydrolase Cel7A. Secretion of Cel7A at titers of 26 mg/L with limited hyperglycosylation was achieved using a Saccharomyces cerevisiae strain with upregulated protein disulfide isomerase, an engineered α-factor prepro leader, and deletion of a plasma membrane ATPase. Using biased clique shuffling (BCS) of 11 Cel7A genes, we generated a small library (469) rich in activity (86% of the chimeras were active) and identified 51 chimeras with improved thermostability, many of which contained mutations in the loop networks that extend over the enzyme's active site. This BCS library was far superior as a source of active and stable chimeras compared to an equimolar library prepared from the same 11 genes.