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Robert M Kelly - One of the best experts on this subject based on the ideXlab platform.
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multidomain surface layer associated glycoside hydrolases contribute to Plant Polysaccharide degradation by caldicellulosiruptor species
Journal of Biological Chemistry, 2016Co-Authors: Jonathan M Conway, William S Pierce, George W Harper, John H Wright, Allyson L Tucker, Jeffrey V Zurawski, Laura L Lee, Sara E Blumerschuette, Robert M KellyAbstract: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.
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s layer homology domain proteins csac_0678 and csac_2722 are implicated in Plant Polysaccharide deconstruction by the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Applied and Environmental Microbiology, 2012Co-Authors: Inci Ozdemir, Sara E Blumerschuette, Robert M KellyAbstract:The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on Plant Polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex Polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to Plant biomass utilization.
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glycoside hydrolase inventory drives Plant Polysaccharide deconstruction by the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Biotechnology and Bioengineering, 2011Co-Authors: Amy L Vanfossen, Inci Ozdemir, Samantha L Zelin, Robert M KellyAbstract:The genome of Caldicellulosiruptor saccharoly- ticus encodes a range of glycoside hydrolases (GHs) that mediate Plant biomass deconstruction by this bacterium. Two GH-based genomic loci that appear to be central to the hydrolysis of hemicellulosic and cellulosic substrates were examined. XynB-XynF (Csac_2404-Csac_2411) encodes intracellular and extracellular GHs that are active towards xylan and xylan side-chains, as well as carboxymethyl cellu- lose (CMC). XynD (Csac_2409) and XynE (Csac_2410) were produced recombinantly and confirmed to be xyla- nases. XynF (Csac_2411) was produced in two separate polypeptides, each with one GH43 catalytic domain displaying a-L-arabinofuranosidase activity. CelA-ManB (Csac_1076-Csac_1080) encodes four multi-domain, extra- cellular GHs, including CelB (Csac_1078), a 118 kDa extra- cellular enzyme not present in the other genome-sequenced member of this genus, Caldicellulosiruptor bescii (formerly Anaerocellum thermophilum). CelB contains both GH10 and GH5 domains, separated by a family 3 carbohydrate-binding module (CBM3). CelB encoded in Csac_1078 differed from the version originally reported (Saul et al., 1990, Appl Environ Microbiol 56:3117-3124) with respect to linker regions. CelB hydrolyzed xylan and CMC, as well as barley b-glucan, glucomannan, and arabinoxylan. For all substrates tested, intact CelB was significantly more active than either the individual GH5 and GH10 domains or the two discrete domains together, indicating that the multi-domain archi- tecture is essential for complex carbohydrate hydrolysis. Transcriptomes for C. saccharolyticus grown at 708 Co n glucose, xylose, xyloglucan, switchgrass, and poplar revealed that certain GHs were particularly responsive to growth on switchgrass and poplar and that CelB was in the top decile of all transcripts during growth on the Plant biomass. Biotechnol. Bioeng. 2011;9999: xxx-xxx.
Henri Pierre Fierobe - One of the best experts on this subject based on the ideXlab platform.
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mechanisms involved in xyloglucan catabolism by the cellulosome producing bacterium ruminiclostridium cellulolyticum
Scientific Reports, 2016Co-Authors: Julie Ravachol, Romain Borne, Pascale De Philip, Maria Mate, Pascal Mansuelle, Stephanie Perret, Henri Pierre FierobeAbstract:Xyloglucan, a ubiquitous highly branched Plant Polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This Polysaccharide also triggers the synthesis of cytoplasmic β-galactosidase, α-xylosidase, and β-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic Polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.
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Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum
Scientific Reports, 2016Co-Authors: Julie Ravachol, Romain Borne, Pascale De Philip, Pascal Mansuelle, Stephanie Perret, Maria J. Mate, Henri Pierre FierobeAbstract:Xyloglucan, a ubiquitous highly branched Plant Polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This Polysaccharide also triggers the synthesis of cytoplasmic beta-galactosidase, alpha-xylosidase, and beta-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic Polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.
Inci Ozdemir - One of the best experts on this subject based on the ideXlab platform.
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s layer homology domain proteins csac_0678 and csac_2722 are implicated in Plant Polysaccharide deconstruction by the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Applied and Environmental Microbiology, 2012Co-Authors: Inci Ozdemir, Sara E Blumerschuette, Robert M KellyAbstract:The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on Plant Polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex Polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to Plant biomass utilization.
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glycoside hydrolase inventory drives Plant Polysaccharide deconstruction by the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Biotechnology and Bioengineering, 2011Co-Authors: Amy L Vanfossen, Inci Ozdemir, Samantha L Zelin, Robert M KellyAbstract:The genome of Caldicellulosiruptor saccharoly- ticus encodes a range of glycoside hydrolases (GHs) that mediate Plant biomass deconstruction by this bacterium. Two GH-based genomic loci that appear to be central to the hydrolysis of hemicellulosic and cellulosic substrates were examined. XynB-XynF (Csac_2404-Csac_2411) encodes intracellular and extracellular GHs that are active towards xylan and xylan side-chains, as well as carboxymethyl cellu- lose (CMC). XynD (Csac_2409) and XynE (Csac_2410) were produced recombinantly and confirmed to be xyla- nases. XynF (Csac_2411) was produced in two separate polypeptides, each with one GH43 catalytic domain displaying a-L-arabinofuranosidase activity. CelA-ManB (Csac_1076-Csac_1080) encodes four multi-domain, extra- cellular GHs, including CelB (Csac_1078), a 118 kDa extra- cellular enzyme not present in the other genome-sequenced member of this genus, Caldicellulosiruptor bescii (formerly Anaerocellum thermophilum). CelB contains both GH10 and GH5 domains, separated by a family 3 carbohydrate-binding module (CBM3). CelB encoded in Csac_1078 differed from the version originally reported (Saul et al., 1990, Appl Environ Microbiol 56:3117-3124) with respect to linker regions. CelB hydrolyzed xylan and CMC, as well as barley b-glucan, glucomannan, and arabinoxylan. For all substrates tested, intact CelB was significantly more active than either the individual GH5 and GH10 domains or the two discrete domains together, indicating that the multi-domain archi- tecture is essential for complex carbohydrate hydrolysis. Transcriptomes for C. saccharolyticus grown at 708 Co n glucose, xylose, xyloglucan, switchgrass, and poplar revealed that certain GHs were particularly responsive to growth on switchgrass and poplar and that CelB was in the top decile of all transcripts during growth on the Plant biomass. Biotechnol. Bioeng. 2011;9999: xxx-xxx.
Julie Ravachol - One of the best experts on this subject based on the ideXlab platform.
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mechanisms involved in xyloglucan catabolism by the cellulosome producing bacterium ruminiclostridium cellulolyticum
Scientific Reports, 2016Co-Authors: Julie Ravachol, Romain Borne, Pascale De Philip, Maria Mate, Pascal Mansuelle, Stephanie Perret, Henri Pierre FierobeAbstract:Xyloglucan, a ubiquitous highly branched Plant Polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This Polysaccharide also triggers the synthesis of cytoplasmic β-galactosidase, α-xylosidase, and β-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic Polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.
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Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum
Scientific Reports, 2016Co-Authors: Julie Ravachol, Romain Borne, Pascale De Philip, Pascal Mansuelle, Stephanie Perret, Maria J. Mate, Henri Pierre FierobeAbstract:Xyloglucan, a ubiquitous highly branched Plant Polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This Polysaccharide also triggers the synthesis of cytoplasmic beta-galactosidase, alpha-xylosidase, and beta-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic Polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.
Sara E Blumerschuette - One of the best experts on this subject based on the ideXlab platform.
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multidomain surface layer associated glycoside hydrolases contribute to Plant Polysaccharide degradation by caldicellulosiruptor species
Journal of Biological Chemistry, 2016Co-Authors: Jonathan M Conway, William S Pierce, George W Harper, John H Wright, Allyson L Tucker, Jeffrey V Zurawski, Laura L Lee, Sara E Blumerschuette, Robert M KellyAbstract: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.
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s layer homology domain proteins csac_0678 and csac_2722 are implicated in Plant Polysaccharide deconstruction by the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Applied and Environmental Microbiology, 2012Co-Authors: Inci Ozdemir, Sara E Blumerschuette, Robert M KellyAbstract:The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on Plant Polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex Polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to Plant biomass utilization.