title: Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species.
abstract: The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensisencodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptorspecies genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique toC. kronotskyensisand contains SLH domains. Calkro_0111 was produced recombinantly inEscherichia colias two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo- and exoglucanase activity on the β-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs inC. kronotskyensis, when produced inE. coli, was active on a variety of xylans and β-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptorand among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii As such, the gene encoding Calkro_0402 was inserted into the C. besciigenome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-typeC. bescii The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptorspecies.
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Conway
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: William S
authors.initials: WS
authors.lastname: Pierce
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Jaycee H
authors.initials: JH
authors.lastname: Le
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: George W
authors.initials: GW
authors.lastname: Harper
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: John H
authors.initials: JH
authors.lastname: Wright
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Allyson L
authors.initials: AL
authors.lastname: Tucker
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Jeffrey V
authors.initials: JV
authors.lastname: Zurawski
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Laura L
authors.initials: LL
authors.lastname: Lee
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Blumer-Schuette
authors.affiliation: From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695 rmkelly@ncsu.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D000068016:Eubacteriales / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016680:Genome, Bacterial; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014934:Wood / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D011134:Polysaccharides; D011994:Recombinant Proteins; D014990:Xylans; C007916:hemicellulose; C008247:laminaran; D006026:Glycoside Hydrolases
keywords: Caldicellulosiruptor; S-layer; biofuel; cell surface; cell surface enzyme; enzyme; glycoside hydrolase; lignocellulose; plant cell wall
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