title: Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose.
abstract: CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C. bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain-an 82% increase over wild type CelA. In addition, expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel. Biotechnol. Bioeng. 2017;114: 945-950. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Genetics, University of Georgia, Athens, Georgia, 30602.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831.
authors.forename: Sun-Ki
authors.initials: SK
authors.lastname: Kim
authors.affiliation: The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831.|Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado.
authors.forename: Daehwan
authors.initials: D
authors.lastname: Chung
authors.affiliation: The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831.|Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.affiliation: The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831.|Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado.
authors.forename: Yannick J
authors.initials: YJ
authors.lastname: Bomble
authors.affiliation: Department of Genetics, University of Georgia, Athens, Georgia, 30602.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831.
authors.forename: Janet
authors.initials: J
authors.lastname: Westpheling
mesh_terms: D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D000068536:Bacillota / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D000072417:Protein Domains / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase
keywords: Caldicellulosiruptior; CelA; biomass deconstruction; repeated aspartate residues