Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures.
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ABSTRACT:
SUBMITTER: Seo H
PROVIDER: S-EPMC6792240 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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:Specific cellulose hydrolysis rates (g of cellulose/g of cellulase per h) were shown to be substantially higher (2.7- to 4.7-fold) for growing cultures of Clostridium thermocellum as compared with purified cellulase preparations from this organism in controlled experiments involving both batch and continuous cultures. This "enzyme-microbe synergy" requires the presence of metabolically active cellulolytic microbes, is not explained by removal of hydrolysis products from the bulk fermentation broth, and appears due to surface phenomena involving adherent cellulolytic microorganisms. Results support the desirability of biotechnological processes featuring microbial conversion of cellulosic biomass to ethanol (or other products) in the absence of added saccharolytic enzymes.
| S-EPMC1637554 | biostudies-literature
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 bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 +/- 0.46. Determination of intracellular beta-glucan radioactivity resulting from 14C-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For 14C-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was approximately 2. For 14C-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was approximately 4. These results indicate that: (i) C. thermocellum hydrolyzes cellulose by a different mode of action from the classical mechanism involving solubilization by cellobiohydrolase; (ii) bioenergetic benefits specific to growth on cellulose are realized, resulting from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of beta-glucosidic bonds; and (iii) these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic biotechnological processing of cellulosic biomass without added saccharolytic enzymes.
| S-EPMC1129095 | biostudies-literature
Project description:Background:Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results:We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase-hydroxybutyryl-CoA dehydrogenase-crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions:Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h.
| S-EPMC6652007 | 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 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:BackgroundCost-efficient saccharification is one of the main bottlenecks for industrial lignocellulose conversion. Clostridium thermocellum naturally degrades lignocellulose efficiently using the cellulosome, a multiprotein supermolecular complex, and thus can be potentially used as a low-cost catalyst for lignocellulose saccharification. The industrial use of C. thermocellum is restrained due largely to the inhibition of the hydrolysate cellobiose to its cellulosome. Although the supplementation of beta-glucosidase may solve the problem, the production of the enzymes greatly complicates the process and may also increase the cost of saccharification.ResultsTo conquer the feedback inhibition and establish an efficient whole-cell catalyst for highly efficient cellulose saccharification, we constructed a recombinant strain of C. thermocellum ∆pyrF::CaBglA which produced a secretory exoglucanase CelS-bearing heterologous BGL using a newly developed seamless genome editing system. Without the extra addition of enzymes, the relative saccharification level of ∆pyrF::CaBglA was stimulated by over twofolds compared to its parent strain ∆pyrF through a two-stage saccharification process with 100 g/L Avicel as the carbon source. The production of reducing sugars and the relative saccharification level were further enhanced to 490 mM and 79.4%, respectively, with increased cell density.ConclusionsThe high cellulose-degrading ability and sugar productivity suggested that the whole-cell-catalysis strategy for cellulose saccharification is promising, and the C. thermocellum strain ∆pyrF::CaBglA could be potentially used as an efficient whole-cell catalyst for industrial cellulose saccharification.
| S-EPMC5429504 | biostudies-literature
Project description:Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a C. thermocellum cipA mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The in vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.
| S-EPMC3370548 | biostudies-literature
Project description:Clostridium thermocellum is a potent cellulolytic bacterium. C. thermocellum strain PAL5, was derived from strain S14 that was isolated from bagasse paper sludge, possesses higher cellulose-degradation ability than representative strains ATCC27405 and DSM1313. In this work, we determined the draft genome sequence of C. thermocellum PAL5. Genomic DNA was used for whole-genome sequencing using the Illumina HiSeq 2500. We obtained 215 contigs of >200 bp (N50, 78,366 bp; mean length, 17,378 bp). The assembled data were subjected to the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline, and 3198 protein-coding sequences, 53 tRNA genes, and 4 rRNA genes were identified. The data are accessible at NCBI (the accession number SBHL00000000). Our data resource will facilitate further studies of efficient cellulose-degradation using C. thermocellum.
| S-EPMC6685675 | biostudies-literature
Project description:The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively. The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD(CelK) contains 1 mol of calcium per mol. The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N(CelK). The CBD-N(CelK) and D192A retained binding parameters close to that of the intact CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded.
| S-EPMC95039 | biostudies-literature