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Title: Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

Abstract

ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCECellulose is the most abundant organic polymermore » on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1340800
Report Number(s):
PNNL-SA-120275
Journal ID: ISSN 2150-7511; 40022; KP1704020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Volume: 7; Journal Issue: 4; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Hiras, Jennifer, Wu, Yu-Wei, Deng, Kai, Nicora, Carrie D., Aldrich, Joshua T., Frey, Dario, Kolinko, Sebastian, Robinson, Errol W., Jacobs, Jon M., Adams, Paul D., Northen, Trent R., Simmons, Blake A., and Singer, Steven W.. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis. United States: N. p., 2016. Web. doi:10.1128/mBio.01106-16.
Hiras, Jennifer, Wu, Yu-Wei, Deng, Kai, Nicora, Carrie D., Aldrich, Joshua T., Frey, Dario, Kolinko, Sebastian, Robinson, Errol W., Jacobs, Jon M., Adams, Paul D., Northen, Trent R., Simmons, Blake A., & Singer, Steven W.. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis. United States. https://doi.org/10.1128/mBio.01106-16
Hiras, Jennifer, Wu, Yu-Wei, Deng, Kai, Nicora, Carrie D., Aldrich, Joshua T., Frey, Dario, Kolinko, Sebastian, Robinson, Errol W., Jacobs, Jon M., Adams, Paul D., Northen, Trent R., Simmons, Blake A., and Singer, Steven W.. Tue . "Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis". United States. https://doi.org/10.1128/mBio.01106-16.
@article{osti_1340800,
title = {Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis},
author = {Hiras, Jennifer and Wu, Yu-Wei and Deng, Kai and Nicora, Carrie D. and Aldrich, Joshua T. and Frey, Dario and Kolinko, Sebastian and Robinson, Errol W. and Jacobs, Jon M. and Adams, Paul D. and Northen, Trent R. and Simmons, Blake A. and Singer, Steven W.},
abstractNote = {ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.},
doi = {10.1128/mBio.01106-16},
url = {https://www.osti.gov/biblio/1340800}, journal = {mBio (Online)},
issn = {2150-7511},
number = 4,
volume = 7,
place = {United States},
year = {2016},
month = {8}
}

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    Works referencing / citing this record:

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    Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger
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