Crystallization and preliminary X-ray crystallographic analysis of a novel ?-L-arabinofuranosidase (CtGH43) from Clostridium thermocellum ATCC 27405.
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SUBMITTER: Goyal A
PROVIDER: S-EPMC4014331 | biostudies-literature | 2014 May
REPOSITORIES: biostudies-literature
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Crystallization and preliminary X-ray crystallographic analysis of a novel α-L-arabinofuranosidase (CtGH43) from Clostridium thermocellum ATCC 27405.
Goyal Arun A Ahmed Shadab S Fontes Carlos M G A CM Najmudin Shabir S
Acta crystallographica. Section F, Structural biology communications 20140415 Pt 5
The truncated carbohydrate-active enzyme belonging to family 43 glycoside hydrolase from Clostridium thermocellum (CtGH43) is an α-L-arabinofuranosidase that in combination with endoxylanase leads to complete breakdown of L-arabinosyl-substituted xylans. The recombinant enzyme CtGH43 from C. thermocellum was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. The recombinant CtGH43 has a molecular mass of 35.86 kDa. Preliminary structural characteriza ...[more]
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Project description:BackgroundThe ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations.ResultsA time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase.ConclusionsOverall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cells' movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered.
| S-EPMC3130646 | biostudies-literature
Project description:The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.
| S-EPMC3855739 | biostudies-literature
Project description:Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate isomerases. Recently, Clostridium thermocellum d-ribose-5-phosphate isomerase (CTRPI), an aldose-ketose isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 69.5, c = 154.4 angstrom, and diffracted to 1.9 angstrom resolution. According to Matthews coefficient calculations, the crystallographic structure consists of a dimer in the asymmetric unit, with a V(M) of 3.2 angstrom(3) Da(-1) and a solvent content of 61.7%.
| S-EPMC2777044 | biostudies-literature
Project description:Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results: A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions: Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligo-saccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cells’ movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered.
2011-05-27 | GSE29554 | GEO
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production. However, C. thermocellum is relatively sensitive to ethanol compared to yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains, but relatively little is known about the genome changes associated with the ethanol tolerant C. thermocellum strain. In this study, C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the supplementation of ethanol to a final concentration of 3.95 g/L (equal to 0.5% [v/v]) or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically reduced the C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption in the treated cultures. The response of C. thermocellum to ethanol was dynamic and involved more than six hundred genes that were significantly and differentially expressed between the different conditions over time and every functional category was represented. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and cellobiose-6-P. The comparison and correlation among intracellular metabolites, proteomic and transcriptomics profiles as well as the ethanol effects on cellulosome, hydrogenase glycolysis and nitrogen metabolism are discussed, which led us to propose that C. thermocellum may utilize the nitrogen metabolism to bypass the arrested carbon metabolism in responding to ethanol stress shock, and the nitrogen metabolic pathway and redox balance may be the key target for improving ethanol tolerance and production in C. thermocellum.
2012-08-16 | GSE25236 | GEO
Project description:Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results: A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions: Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligo-saccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cellsM-bM-^@M-^Y movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered. Total RNA was extracted from the cell pellets and the reverse transcribed cDNA was hybridized to oligo-arrays containing duplicated probes representing ~90% of the annotated ORFs in C. thermocellum ATCC27405 genome.Dual-channel dye swap experimental design was used to analyze the time-course of gene expression during cellulose fermentation using two biological replicate fermentation. The 6hr sample as the reference, to which all other time-point samples (8, 10, 12, 14, 16hr) were compared.
2011-05-27 | E-GEOD-29554 | biostudies-arrayexpress
Project description:The cellulosome, a highly elaborate extracellular multi-enzyme complex of cellulases and hemicellulases, is responsible for the degradation of plant cell walls. The xylanase CtXyl5A (Cthe_2193) is a multimodular arabinoxylanase which is one of the largest components of the Clostridium thermocellum cellulosome. The N-terminal catalytic domain of CtXyl5A, which is a member of glycoside hydrolase family 5 (GH5), is responsible for the hydrolysis of arabinoxylans. Appended after it are three noncatalytic carbohydrate-binding modules (CBMs), which belong to families 6 (CBM6), 13 (CBM13) and 62 (CBM62). In addition, CtXyl5A has a fibronectin type III-like (Fn3) module preceding the CBM62 and a type I dockerin (DOK) module following it which allows the enzyme to be integrated into the cellulosome through binding to a cohesin module of the protein scaffold CipA. Crystals of the pentamodular enzyme without the DOK module at the C-terminus, with the domain architecture CtGH5-CBM6-CBM13-Fn3-CBM62, have been obtained. The structure of this pentamodular xylanase has been determined by molecular replacement to a resolution of 2.64?Å using coordinates of CtGH5-CBM6, Fn3 and CBM62 from the PDB as search models.
| S-EPMC3144809 | biostudies-literature
Project description:BACKGROUND:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms. RESULTS:C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was prepared for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNA-seq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5% false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared. CONCLUSIONS:Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.
| S-EPMC3880215 | biostudies-literature
Project description:Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a approximately 10-fold range of dilution rates (0.013 to 0.16 h(-1)). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h(-1): cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.
| S-EPMC1183361 | biostudies-literature
Project description:BackgroundEconomic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.Methodology/principal findingsIn this study, we used quantitative proteomics (multidimensional LC-MS/MS and (15)N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to (15)N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.Conclusions/significanceOverall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.
| S-EPMC2668762 | biostudies-literature