title: Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis.
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 actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. 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 of T. bispora hydrolytic 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.
  
  
  
  Cellulose 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.
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hiras
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Wu
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biological and Materials Science Center, Sandia National Laboratories, Livermore, California, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Deng
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Aldrich
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA Faculty of Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany.
authors.forename: Dario
authors.initials: D
authors.lastname: Frey
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kolinko
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Errol W
authors.initials: EW
authors.lastname: Robinson
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Jacobs
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA Biosciences Directorate, Lawrence Berkeley National Laboratory, Berkeley, California, USA SWSinger@lbl.gov.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Singer
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000032:analysis*; D006868:Hydrolysis; D059013:Microbial Consortia; D020543:Proteome / Q000032:analysis*; D040901:Proteomics
chemical_list: D020543:Proteome; D002482:Cellulose; D006026:Glycoside Hydrolases