title: Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.
abstract: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which 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. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure and function of this important enzyme in biomass deconstruction.
authors: Department of Genetics, University of Georgia, Athens, Georgia, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Daehwan
authors: D
authors: Chung
authors: Department of Genetics, University of Georgia, Athens, Georgia, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Jenna
authors: J
authors: Young
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Yannick J
authors: YJ
authors: Bomble
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Todd A
authors: TA
authors: Vander Wall
authors: Department of Genetics, University of Georgia, Athens, Georgia, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Joseph
authors: J
authors: Groom
authors: Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Michael E
authors: ME
authors: Himmel
authors: Department of Genetics, University of Georgia, Athens, Georgia, United States of America; The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.
authors: Janet
authors: J
authors: Westpheling
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018533:Biomass; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology*; D006868:Hydrolysis; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins; D002482:Cellulose; D006026:Glycoside Hydrolases; C074775:CelA endoglucanase; D002480:Cellulase