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

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:
Grant/Contract Number:
AC05-00OR22725
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
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)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES
OSTI Identifier:
1474620

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., 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.. 2017. "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}
}

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