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Title: Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.
Authors:
 [1] ;  [2] ;  [2] ;  [1]
  1. Univ. of Georgia, Athens, GA (United States). Dept. of Genetics; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). 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). BioEnergy Science Center
Publication Date:
Report Number(s):
NREL/JA-2700-70537
Journal ID: ISSN 0099-2240
Grant/Contract Number:
AC36-08GO28308; AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 84; Journal Issue: 3; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
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)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; consolidated bioprocessing; biomass deconstruction; cellobiose phosphorylase; Caldicellulosiruptor
OSTI Identifier:
1409498

Kim, Sun-Ki, Himmel, Michael E., Bomble, Yannick J., and Westpheling, Janet. Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity. United States: N. p., Web. doi:10.1128/AEM.02348-17.
Kim, Sun-Ki, Himmel, Michael E., Bomble, Yannick J., & Westpheling, Janet. Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity. United States. doi:10.1128/AEM.02348-17.
Kim, Sun-Ki, Himmel, Michael E., Bomble, Yannick J., and Westpheling, Janet. 2017. "Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity". United States. doi:10.1128/AEM.02348-17. https://www.osti.gov/servlets/purl/1409498.
@article{osti_1409498,
title = {Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity},
author = {Kim, Sun-Ki and Himmel, Michael E. and Bomble, Yannick J. and Westpheling, Janet},
abstractNote = {Members of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose. CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. Here, this work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.},
doi = {10.1128/AEM.02348-17},
journal = {Applied and Environmental Microbiology},
number = 3,
volume = 84,
place = {United States},
year = {2017},
month = {11}
}

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