title: 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.
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, 201 Calvin Laboratory, Berkeley, California, 94720; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, 491 Tan Hall, Berkeley, California, 94720.
authors.forename: Craig M
authors.initials: CM
authors.lastname: Dana
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dotson-Fagerstrom
authors.forename: Christine M
authors.initials: CM
authors.lastname: Roche
authors.forename: Sarala M
authors.initials: SM
authors.lastname: Kal
authors.forename: Harshal A
authors.initials: HA
authors.lastname: Chokhawala
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Blanch
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism; D002482:Cellulose / Q000032:analysis / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D002482:Cellulose; D002480:Cellulase; D011761:Pyrrolidonecarboxylic Acid
keywords: Cel7A; N-terminal glutamine cyclization; Saccharomyces cerevisiae; glutaminyl cyclase; protein engineering; pyroglutamate; recombinant expression