Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity
Abstract
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:
-
- Univ. of Georgia, Athens, GA (United States). Dept. of Genetics; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
- 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:
- Research Org.:
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1409498
- Report Number(s):
- NREL/JA-2700-70537
Journal ID: ISSN 0099-2240
- Grant/Contract Number:
- AC36-08GO28308; AC05-00OR22725
- Resource 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
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; consolidated bioprocessing; biomass deconstruction; cellobiose phosphorylase; Caldicellulosiruptor
Citation Formats
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., 2017.
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. https://doi.org/10.1128/AEM.02348-17
Kim, Sun-Ki, Himmel, Michael E., Bomble, Yannick J., and Westpheling, Janet. Fri .
"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. https://doi.org/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 = {Fri Nov 03 00:00:00 EDT 2017},
month = {Fri Nov 03 00:00:00 EDT 2017}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 8 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus
journal, November 2011
- Kado, Yuji; Inoue, Tsuyoshi; Ishikawa, Kazuhiko
- Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 67, Issue 12
The Major Autolysin Acm2 from Lactobacillus plantarum Undergoes Cytoplasmic O-Glycosylation
journal, November 2011
- Fredriksen, L.; Mathiesen, G.; Moen, A.
- Journal of Bacteriology, Vol. 194, Issue 2
Engineering the N -terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose : Engineering the
journal, March 2017
- Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E.
- Biotechnology and Bioengineering, Vol. 114, Issue 5
Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes
journal, May 2011
- Chung, Dae-Hwan; Huddleston, Jennifer R.; Farkas, Joel
- Journal of Industrial Microbiology & Biotechnology, Vol. 38, Issue 11
Isolation of Recombinant Secretory Proteins by Limited Induction and Quantitative Harvest
journal, February 1998
- Rosenberg, Helene F.
- BioTechniques, Vol. 24, Issue 2
Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose
journal, August 2015
- Chung, Daehwan; Young, Jenna; Cha, Minseok
- Biotechnology for Biofuels, Vol. 8, Issue 1
Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates
journal, August 2016
- Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E.
- Biotechnology for Biofuels, Vol. 9, Issue 1
Product inhibition of cellulases studied with 14C-labeled cellulose substrates
journal, January 2013
- Teugjas, Hele; Väljamäe, Priit
- Biotechnology for Biofuels, Vol. 6, Issue 1
Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii
journal, January 2013
- Kanafusa-Shinkai, Sumiyo; Wakayama, Jun'ichi; Tsukamoto, Kazumi
- Journal of Bioscience and Bioengineering, Vol. 115, Issue 1
Microbial Cellulose Utilization: Fundamentals and Biotechnology
journal, September 2002
- Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.
- Microbiology and Molecular Biology Reviews, Vol. 66, Issue 3, p. 506-577
Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar
journal, March 1959
- Miller, G. L.
- Analytical Chemistry, Vol. 31, Issue 3
Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium, Thermotoga maritima MSB8
journal, January 2002
- Rajashekhara, Eranna; Kitaoka, Motomitsu; Kim, Yeon-Kye
- Bioscience, Biotechnology, and Biochemistry, Vol. 66, Issue 12
Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii
journal, June 2014
- Chung, D.; Cha, M.; Guss, A. M.
- Proceedings of the National Academy of Sciences, Vol. 111, Issue 24, p. 8931-8936
Heterologous expression of a β-d-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose
journal, September 2017
- Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E.
- Journal of Industrial Microbiology & Biotechnology, Vol. 44, Issue 12
Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725
journal, January 2011
- Dam, Phuongan; Kataeva, Irina; Yang, Sung-Jae
- Nucleic Acids Research, Vol. 39, Issue 8
Revealing Nature's Cellulase Diversity The Digestion Mechanism of Caldicellulosiruptor bescii CelA
journal, December 2013
- Brunecky, R.; Alahuhta, M.; Xu, Q.
- Science, Vol. 342, Issue 6165, p. 1513-1516
Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes
journal, October 2002
- Fujita, Y.; Takahashi, S.; Ueda, M.
- Applied and Environmental Microbiology, Vol. 68, Issue 10
Isolation and Characterization of Acidothermus cellulolyticus gen. nov., sp. nov., a New Genus of Thermophilic, Acidophilic, Cellulolytic Bacteria
journal, July 1986
- MOHAGHEGHI, A.; GROHMANN, K.; HIMMEL, M.
- International Journal of Systematic Bacteriology, Vol. 36, Issue 3, p. 435-443
Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii
journal, October 2015
- Chung, Daehwan; Cha, Minseok; Snyder, Elise N.
- Biotechnology for Biofuels, Vol. 8, Issue 1
Molecular and Biochemical Analyses of CbCel9A/Cel48A, a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose
journal, December 2013
- Yi, Zhuolin; Su, Xiaoyun; Revindran, Vanessa
- PLoS ONE, Vol. 8, Issue 12
Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated
journal, March 2015
- Chung, Daehwan; Young, Jenna; Bomble, Yannick J.
- PLOS ONE, Vol. 10, Issue 3
Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola.
journal, January 1996
- Lou, J.; Dawson, K. A.; Strobel, H. J.
- Applied and environmental microbiology, Vol. 62, Issue 5
Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe "Anaerocellum thermophilum" DSM 6725
journal, May 2009
- Yang, Sung-Jae; Kataeva, Irina; Hamilton-Brehm, Scott D.
- Applied and Environmental Microbiology, Vol. 75, Issue 14, p. 4762-4769
Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis
journal, April 2011
- Lochner, Adriane; Giannone, Richard J.; Rodriguez, Miguel
- Applied and Environmental Microbiology, Vol. 77, Issue 12
Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters
journal, January 2013
- Ha, Suk-Jin; Galazka, Jonathan M.; Joong Oh, Eun
- Metabolic Engineering, Vol. 15
Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation
journal, May 2005
- Zhang, Y. -H. P.; Lynd, L. R.
- Proceedings of the National Academy of Sciences, Vol. 102, Issue 20
Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalisgenerates a new host for the analysis of biomass deconstruction
journal, September 2014
- Groom, Joseph; Chung, Daehwan; Young, Jenna
- Biotechnology for Biofuels, Vol. 7, Issue 1
Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement
journal, January 2013
- Chung, Daehwan; Farkas, Joel; Westpheling, Janet
- Biotechnology for Biofuels, Vol. 6, Issue 1
Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum
journal, March 2004
- Zhang, Y. -H. P.; Lynd, L. R.
- Applied and Environmental Microbiology, Vol. 70, Issue 3
Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii
journal, November 2012
- Farkas, Joel; Chung, Daehwan; Cha, Minseok
- Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 1, p. 41-49
Works referencing / citing this record:
Genomic and physiological analyses reveal that extremely thermophilic Caldicellulosiruptor changbaiensis deploys uncommon cellulose attachment mechanisms
journal, August 2019
- Khan, Asma M. A. M.; Mendoza, Carl; Hauk, Valerie J.
- Journal of Industrial Microbiology & Biotechnology, Vol. 46, Issue 9-10