skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction

Abstract

We present that the ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivoroles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergisticallyin vivoand accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on themore » secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. Lastly, these results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [2]; ORCiD logo [2]; ORCiD logo [2];  [3];  [3];  [3];  [1]
  1. North Carolina State Univ., Raleigh, NC (United States). Department of Chemical and Biomolecular Engineering
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  3. Univ. of Georgia, Athens, GA (United States). Department of Biochemistry and Molecular Biology
Publication Date:
Research Org.:
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:
1474620
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 24; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES

Citation Formats

Conway, Jonathan M., McKinley, Bennett S., Seals, Nathaniel L., Hernandez, Diana H., Khatibi, Piyum A., Poudel, Suresh, Giannone, Richard J., Hettich, Robert L., Williams-Rhaesa, Amanda M., Lipscomb, Gina L., Adams, Michael W. W., and Kelly, Robert M. Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction. United States: N. p., 2017. Web. doi:10.1128/AEM.01828-17.
Conway, Jonathan M., McKinley, Bennett S., Seals, Nathaniel L., Hernandez, Diana H., Khatibi, Piyum A., Poudel, Suresh, Giannone, Richard J., Hettich, Robert L., Williams-Rhaesa, Amanda M., Lipscomb, Gina L., Adams, Michael W. W., & Kelly, Robert M. Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction. United States. doi:10.1128/AEM.01828-17.
Conway, Jonathan M., McKinley, Bennett S., Seals, Nathaniel L., Hernandez, Diana H., Khatibi, Piyum A., Poudel, Suresh, Giannone, Richard J., Hettich, Robert L., Williams-Rhaesa, Amanda M., Lipscomb, Gina L., Adams, Michael W. W., and Kelly, Robert M. Fri . "Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction". United States. doi:10.1128/AEM.01828-17. https://www.osti.gov/servlets/purl/1474620.
@article{osti_1474620,
title = {Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction},
author = {Conway, Jonathan M. and McKinley, Bennett S. and Seals, Nathaniel L. and Hernandez, Diana H. and Khatibi, Piyum A. and Poudel, Suresh and Giannone, Richard J. and Hettich, Robert L. and Williams-Rhaesa, Amanda M. and Lipscomb, Gina L. and Adams, Michael W. W. and Kelly, Robert M.},
abstractNote = {We present that the ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivoroles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergisticallyin vivoand accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. Lastly, these results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.},
doi = {10.1128/AEM.01828-17},
journal = {Applied and Environmental Microbiology},
number = 24,
volume = 83,
place = {United States},
year = {2017},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 9 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

The catalytic mechanism and unique low pH optimum of Caldicellulosiruptor bescii family 3 pectate lyase
journal, August 2015

  • Alahuhta, Markus; Taylor, Larry E.; Brunecky, Roman
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 9
  • DOI: 10.1107/S1399004715013760

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii
journal, August 2014

  • Yokoyama, H.; Yamashita, T.; Morioka, R.
  • Journal of Bacteriology, Vol. 196, Issue 21
  • DOI: 10.1128/JB.01897-14

S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus
journal, December 2011

  • Ozdemir, Inci; Blumer-Schuette, Sara E.; Kelly, Robert M.
  • Applied and Environmental Microbiology, Vol. 78, Issue 3
  • DOI: 10.1128/AEM.07031-11

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii
journal, May 2016

  • Lipscomb, Gina L.; Conway, Jonathan M.; Blumer-Schuette, Sara E.
  • Applied and Environmental Microbiology, Vol. 82, Issue 14
  • DOI: 10.1128/AEM.00570-16

Classification of 'Anaerocellum thermophilum' strain DSM 6725 as Caldicellulosiruptor bescii sp. nov.
journal, October 2009

  • Yang, S. -J.; Kataeva, I.; Wiegel, J.
  • INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, Vol. 60, Issue 9
  • DOI: 10.1099/ijs.0.017731-0

Natural genetic variability reduces recalcitrance in poplar
journal, May 2016

  • Bhagia, Samarthya; Muchero, Wellington; Kumar, Rajeev
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-016-0521-2

The cellulolytic system of Clostridium cellulolyticum
journal, September 1997


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
  • DOI: 10.1016/j.jbiosc.2012.07.019

Protein O-mannosylation: Conserved from bacteria to humans
journal, May 2009


PAV1, the First Virus-Like Particle Isolated from a Hyperthermophilic Euryarchaeote, "Pyrococcus abyssi"
journal, July 2003


Real-Time DNA Sequencing from Single Polymerase Molecules
journal, January 2009


The structure and mode of action of Caldicellulosiruptor bescii family 3 pectate lyase in biomass deconstruction
journal, March 2013

  • Alahuhta, Markus; Brunecky, Roman; Chandrayan, Puja
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 4
  • DOI: 10.1107/S0907444912050512

Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance
journal, October 2014

  • Chung, Daehwan; Pattathil, Sivakumar; Biswal, Ajaya K.
  • Biotechnology for Biofuels, Vol. 7, Issue 1
  • DOI: 10.1186/s13068-014-0147-1

Pour some sugar on it: the expanding world of bacterial protein O -linked glycosylation : Protein
journal, June 2013

  • Iwashkiw, Jeremy A.; Vozza, Nicolas F.; Kinsella, Rachel L.
  • Molecular Microbiology, Vol. 89, Issue 1
  • DOI: 10.1111/mmi.12265

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose
journal, February 2015

  • Blumer-Schuette, Sara E.; Alahuhta, Markus; Conway, Jonathan M.
  • Journal of Biological Chemistry, Vol. 290, Issue 17
  • DOI: 10.1074/jbc.M115.641480

Hemicelluloses for fuel ethanol: A review
journal, July 2010


Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species
journal, January 2016

  • Conway, Jonathan M.; Pierce, William S.; Le, Jaycee H.
  • Journal of Biological Chemistry, Vol. 291, Issue 13
  • DOI: 10.1074/jbc.M115.707810

Thermophilic lignocellulose deconstruction
journal, May 2014

  • Blumer-Schuette, Sara E.; Brown, Steven D.; Sander, Kyle B.
  • FEMS Microbiology Reviews, Vol. 38, Issue 3
  • DOI: 10.1111/1574-6976.12044

Clostridium cellulolyticum : model organism of mesophilic cellulolytic clostridia
journal, September 2005


Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases
journal, February 2011

  • Lipscomb, Gina L.; Stirrett, Karen; Schut, Gerrit J.
  • Applied and Environmental Microbiology, Vol. 77, Issue 7, p. 2232-2238
  • DOI: 10.1128/AEM.02624-10

Population genomics of Populus trichocarpa identifies signatures of selection and adaptive trait associations
journal, August 2014

  • Evans, Luke M.; Slavov, Gancho T.; Rodgers-Melnick, Eli
  • Nature Genetics, Vol. 46, Issue 10
  • DOI: 10.1038/ng.3075

moFF: a robust and automated approach to extract peptide ion intensities
journal, November 2016

  • Argentini, Andrea; Goeminne, Ludger J. E.; Verheggen, Kenneth
  • Nature Methods, Vol. 13, Issue 12
  • DOI: 10.1038/nmeth.4075

Complete Genome Sequences of Caldicellulosiruptor sp. Strain Rt8.B8, Caldicellulosiruptor sp. Strain Wai35.B1, and “ Thermoanaerobacter cellulolyticus
journal, May 2015

  • Lee, Laura L.; Izquierdo, Javier A.; Blumer-Schuette, Sara E.
  • Genome Announcements, Vol. 3, Issue 3
  • DOI: 10.1128/genomeA.00440-15

Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT
journal, August 2002

  • McDonald, W. Hayes; Ohi, Ryoma; Miyamoto, David T.
  • International Journal of Mass Spectrometry, Vol. 219, Issue 1
  • DOI: 10.1016/S1387-3806(02)00563-8

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
  • DOI: 10.1128/AEM.00236-09

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
  • DOI: 10.1128/AEM.02811-10

Phylogenetic, Microbiological, and Glycoside Hydrolase Diversities within the Extremely Thermophilic, Plant Biomass-Degrading Genus Caldicellulosiruptor
journal, October 2010

  • Blumer-Schuette, S. E.; Lewis, D. L.; Kelly, R. M.
  • Applied and Environmental Microbiology, Vol. 76, Issue 24, p. 8084-8092
  • DOI: 10.1128/AEM.01400-10

Molecular diversity of thermophilic cellulolytic and hemicellulolytic bacteria
journal, February 1999


Faster SEQUEST Searching for Peptide Identification from Tandem Mass Spectra
journal, September 2011

  • Diament, Benjamin J.; Noble, William Stafford
  • Journal of Proteome Research, Vol. 10, Issue 9
  • DOI: 10.1021/pr101196n

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725
journal, August 2012


Genome Sequence of the Anaerobic, Thermophilic, and Cellulolytic Bacterium "Anaerocellum thermophilum" DSM 6725
journal, April 2009

  • Kataeva, I. A.; Yang, S. -J.; Dam, P.
  • Journal of Bacteriology, Vol. 191, Issue 11
  • DOI: 10.1128/JB.00256-09

Semi-supervised learning for peptide identification from shotgun proteomics datasets
journal, October 2007

  • Käll, Lukas; Canterbury, Jesse D.; Weston, Jason
  • Nature Methods, Vol. 4, Issue 11
  • DOI: 10.1038/nmeth1113

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii
journal, May 2017

  • Williams-Rhaesa, Amanda M.; Poole, Farris L.; Dinsmore, Jessica T.
  • Applied and Environmental Microbiology, Vol. 83, Issue 14
  • DOI: 10.1128/AEM.00444-17

Glycoside hydrolase inventory drives plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus
journal, March 2011

  • VanFossen, Amy L.; Ozdemir, Inci; Zelin, Samantha L.
  • Biotechnology and Bioengineering, Vol. 108, Issue 7
  • DOI: 10.1002/bit.23093

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization
journal, August 2015

  • Zurawski, Jeffrey V.; Conway, Jonathan M.; Lee, Laura L.
  • Applied and Environmental Microbiology, Vol. 81, Issue 20
  • DOI: 10.1128/AEM.01622-15

Deletion of Caldicellulosiruptor besciiCelA reveals its crucial role in the deconstruction of lignocellulosic biomass
journal, October 2014

  • Young, Jenna; Chung, Daehwan; Bomble, Yannick J.
  • Biotechnology for Biofuels, Vol. 7, Issue 1
  • DOI: 10.1186/s13068-014-0142-6

The MPB83 Antigen from Mycobacterium bovis Contains O -Linked Mannose and (1 → 3)-Mannobiose Moieties
journal, January 2003

  • Michell, Stephen L.; Whelan, Adam O.; Wheeler, Paul R.
  • Journal of Biological Chemistry, Vol. 278, Issue 18
  • DOI: 10.1074/jbc.M207959200

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease
journal, October 2016

  • Wiemels, Richard E.; Cech, Stephanie M.; Meyer, Nikki M.
  • Journal of Bacteriology, Vol. 199, Issue 1
  • DOI: 10.1128/JB.00453-16

Large-scale analysis of the yeast proteome by multidimensional protein identification technology
journal, March 2001

  • Washburn, Michael P.; Wolters, Dirk; Yates, John R.
  • Nature Biotechnology, Vol. 19, Issue 3
  • DOI: 10.1038/85686

Revealing Nature's Cellulase Diversity The Digestion Mechanism of Caldicellulosiruptor bescii CelA
journal, December 2013


Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake
journal, January 2007

  • Kingsford, Carleton L.; Ayanbule, Kunmi; Salzberg, Steven L.
  • Genome Biology, Vol. 8, Issue 2
  • DOI: 10.1186/gb-2007-8-2-r22

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus
journal, January 2015


Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii
journal, January 2012

  • Su, Xiaoyun; Mackie, Roderick I.; Cann, Isaac K. O.
  • Applied and Environmental Microbiology, Vol. 78, Issue 7
  • DOI: 10.1128/AEM.06814-11

Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated
journal, March 2015


Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.
journal, May 1996


Effect of Prolyl Isomerase on the Folding Reactions of Staphylococcal Nuclease
journal, December 1997

  • Veeraraghavan, Sudha; Nall, Barry T.; Fink, Anthony L.
  • Biochemistry, Vol. 36, Issue 49
  • DOI: 10.1021/bi971357r

Multidomain and Multifunctional Glycosyl Hydrolases from the Extreme Thermophile Caldicellulosiruptor Isolate Tok7B.1
journal, May 2000

  • Gibbs, Moreland D.; Reeves, Rosalind A.; Farrington, G. King
  • Current Microbiology, Vol. 40, Issue 5, p. 333-340
  • DOI: 10.1007/s002849910066

The genome portal of the Department of Energy Joint Genome Institute: 2014 updates
journal, November 2013

  • Nordberg, Henrik; Cantor, Michael; Dusheyko, Serge
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt1069

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
  • DOI: 10.1186/1754-6834-6-82

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
  • DOI: 10.1007/s10295-012-1202-1

Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii
journal, July 2012

  • Ye, Libin; Su, Xiaoyun; Schmitz, George E.
  • Applied and Environmental Microbiology, Vol. 78, Issue 19
  • DOI: 10.1128/AEM.02009-12

Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass
journal, January 2013

  • Cha, Minseok; Chung, Daehwan; Elkins, James G.
  • Biotechnology for Biofuels, Vol. 6, Issue 1
  • DOI: 10.1186/1754-6834-6-85

    Works referencing / citing this record:

    The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan
    journal, December 2019


    Deletion of a single glycosyltransferase in Caldicellulosiruptor bescii eliminates protein glycosylation and growth on crystalline cellulose
    journal, September 2018


    The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan
    journal, December 2019