The importance of pyroglutamate in cellulase Cel7A

Abstract

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.

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

“…3A). These results recapitulated previous data demonstrating a difference in activity in pGlu-modified versus unmodified TeCBH-1 at different temperatures (13). Furthermore, these data suggested that the N. crassa QC enzymes are functionally redundant.…”

“…The T. emersonii cbh - 1 coding sequence regulated by the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter (13) was introduced into the wild type (WT) and the Δqc - 1 , Δqc-2 , and Δqc - 1 Δqc - 2 strains. Activity of TeCBH-1 culture supernatants was assayed from 35°C to 60°C using a 4-methylumbelliferyl β- d -lactopyranoside (MuLac) assay for enzyme activity (Fig.…”

“…However, two recent studies showed that heterologous production of a CBH-1 and an endoglucanase 1 (EG1) from a thermotolerant filamentous fungus Talaromyces emersonii in Saccharomyces cerevisiae showed decreased thermal stability (12, 13) (Fig. 1B).…”

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

“…In addition, it is worth noting that the differences in glycosylation between Sc_TrEGI and Nc_TrEGI (Additional file 1: Figure S8) are irrelevant to the activity and stability differences between these enzymes in comparison to the cyclization of the N-terminal glutamine. Recently our group also demonstrated a similar increase in stability and activity upon cyclization of N-terminal glutamine to pyroglutamate in S. cerevisiae -expressed Talaromyces emersonii Cel7A [33]. The ubiquitous presence of the N-terminal pyroglutamate in GH7 enzymes (Cel7A and Cel7B), along with its role in imparting high stability/activity for these enzymes, highlights the importance of N-terminal glutamine cyclization for achieving high-efficiency cellulases.…”

“…T. reesei EGI and engineered mutants were expressed in N. crassa as described previously [33]. The wild-type TrEGI gene was amplified from T. reesei gDNA.…”