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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 transportedmore » 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. Finally, 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.« less

Authors:
 [1];  [1];  [2];  [2];  [1];  [2];  [1]
  1. Univ. of Georgia, Athens, GA (United States). Dept. of Genetics; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). The BioEnergy Science Center
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). The BioEnergy Science Center
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1220729
Report Number(s):
NREL/JA-2700-64060
Journal ID: ISSN 1932-6203
Grant/Contract Number:  
10-21-RR093-367
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 3; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; glycosylation; signal peptides; vector construction; cellulases; glycoproteins; plasmid construction; protein expression; uracils

Citation Formats

Chung, Daehwan, Young, Jenna, Bomble, Yannick J., Vander Wall, Todd A., Groom, Joseph, Himmel, Michael E., and Westpheling, Janet. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0119508.
Chung, Daehwan, Young, Jenna, Bomble, Yannick J., Vander Wall, Todd A., Groom, Joseph, Himmel, Michael E., & Westpheling, Janet. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated. United States. doi:10.1371/journal.pone.0119508.
Chung, Daehwan, Young, Jenna, Bomble, Yannick J., Vander Wall, Todd A., Groom, Joseph, Himmel, Michael E., and Westpheling, Janet. Mon . "Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated". United States. doi:10.1371/journal.pone.0119508. https://www.osti.gov/servlets/purl/1220729.
@article{osti_1220729,
title = {Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated},
author = {Chung, Daehwan and Young, Jenna and Bomble, Yannick J. and Vander Wall, Todd A. and Groom, Joseph and Himmel, Michael E. and Westpheling, Janet},
abstractNote = {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. Finally, 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.},
doi = {10.1371/journal.pone.0119508},
journal = {PLoS ONE},
number = 3,
volume = 10,
place = {United States},
year = {2015},
month = {3}
}

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    High activity CAZyme cassette for improving biomass degradation in thermophiles
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