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Title: Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Abstract

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 degrees C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essentialmore » engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.« less

Authors:
ORCiD logo [1];  [2];  [3]; ORCiD logo [3];  [3];  [1];  [3];  [1]; ORCiD logo [3]
  1. Weizmann Inst. of Science, Rehovot (Israel)
  2. Weizmann Inst. of Science, Rehovot (Israel); Univ. of the Negev (Israel)
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1602688
Report Number(s):
[NREL/JA-2700-76211]
[Journal ID: ISSN 1742-464X]
Grant/Contract Number:  
[AC36-08GO28308]
Resource Type:
Accepted Manuscript
Journal Name:
Federation of European Biochemical Societies (FEBS) Journal
Additional Journal Information:
[Journal Name: Federation of European Biochemical Societies (FEBS) Journal]; Journal ID: ISSN 1742-464X
Publisher:
Federation of European Biochemical Societies
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; cellulosome; cellulase; thermostability; scaffoldin; glycosylation; expression host; Caldicellulosiruptor bescii

Citation Formats

Kahn, Amaranta, Morais, Sarah, Chung, Daehwan, Sarai, Nicholas, Hengge, Neal, Kahn, Audrey, Himmel, Michael E., Bayer, Edward A., and Bomble, Yannick J. Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis. United States: N. p., 2020. Web. doi:10.1111/febs.15251.
Kahn, Amaranta, Morais, Sarah, Chung, Daehwan, Sarai, Nicholas, Hengge, Neal, Kahn, Audrey, Himmel, Michael E., Bayer, Edward A., & Bomble, Yannick J. Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis. United States. doi:10.1111/febs.15251.
Kahn, Amaranta, Morais, Sarah, Chung, Daehwan, Sarai, Nicholas, Hengge, Neal, Kahn, Audrey, Himmel, Michael E., Bayer, Edward A., and Bomble, Yannick J. Mon . "Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis". United States. doi:10.1111/febs.15251.
@article{osti_1602688,
title = {Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis},
author = {Kahn, Amaranta and Morais, Sarah and Chung, Daehwan and Sarai, Nicholas and Hengge, Neal and Kahn, Audrey and Himmel, Michael E. and Bayer, Edward A. and Bomble, Yannick J.},
abstractNote = {Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 degrees C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.},
doi = {10.1111/febs.15251},
journal = {Federation of European Biochemical Societies (FEBS) Journal},
number = ,
volume = ,
place = {United States},
year = {2020},
month = {2}
}

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