Cellulolytic Bacterium Clostridium Thermocellum Research Articles

The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.

Enzymatic synthesis of cellulose in space: gravity is a crucial factor for building cellulose II gel structure

We previously reported in vitro synthesis of highly ordered crystalline cellulose II by reverse reaction of cellodextrin phosphorylase from the cellulolytic bacterium Clostridium (Hungateiclostridium) thermocellum (CtCDP), but the formation mechanism of the cellulose crystals and highly ordered structure has long been unclear. Considering the specific density of cellulose versus water, the formation of crystalline and highly ordered structure in an aqueous solution should be affected by gravity. Thus, we synthesized cellulose with CtCDP stable variant at the International Space Station, where sedimentation and convection due to gravity are negligible. Optical microscopic observation suggested that cellulose in space has a gel-like appearance without apparent aggregation, in contrast to cellulose synthesized on the ground. Small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) indicated that cellulose synthesized in space has a more uniform particle distribution in the ~ 100 nm scale region than cellulose synthesized on the ground. Scanning electron microscopy (SEM) showed that both celluloses have a micrometer scale network structure, whereas a fine fiber network was constructed only under microgravity. These results indicate that gravity plays a role in cellulose II crystal sedimentation and the building of network structure, and synthesis in space could play a role in designing unique materials.Graphical abstract

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50–85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

Comparisons of Catalytic Efficiency during the Reduction of Lignocellulosic Substrates between Free Enzyme and Enzyme bound to Mobile Enzyme Sequestration Platforms (MESP)

The cellulolytic bacterium Clostridium thermocellum employs a flexible cellulosome that can bind a variety of enzymes to breakdown cellulosic biomass. A key feature of the cellulosome is a cohesin (Type 1) – dockerin (Type 1) interaction that binds dockerin-endowed enzymes to the cohesin-ladened scaffoldin backbone. Multiple enzymes of various enzyme classes (e.g., exocellulases, endocellulases, xylanases) can bind to the cellulosome structure for synergistic deconstruction of cellulosic substrates. Several “artificial cellulosomes” have been engineered in an attempt to mimic this system for abiotic decomposition of lignocellulosic substrates. One such engineered system is called a mobile enzyme sequestration platform (MESP). The prototype of this platform was called a “rosettasome”. MESPs are chimera of an archaeal group II chaperonin permutant fused to C. thermocellum cohesin via a 9-12 amino acid linker. The archaeal chaperonin complex is thermotolerant. In prior work our lab demonstrated that binding cellulolytic enzymes to MESPs enhances lignocellulose breakdown efficiency over free enzyme in solution (sans platform). Interestingly, our prior data indicated that MESP systems may also be acidotolerant since substrate was treated with mild sulfuric acid. In this study, we tested the acidotolerance of MESP technology as well as the efficiency of carbohydrate reduction on other feedstock (e.g., rice straw). We also tested our MESPs against the prototype rosettasome to determine if this next-generation platform outperforms the original system. Here we present the results of our comparisons. Understanding the limits of MESP enhancement will direct future engineering of this system for industrial and/or agricultural applications.

Bench‐scale fermentation for second generation ethanol and hydrogen production by Clostridium thermocellum DSMZ 1313 from sugarcane bagasse

AbstractExploration of eco‐friendly energy resources substituting conventional fossil fuels is the real challenge globally. Prospectively, current investigation accentuates the fermentative conversion of low‐cost lignocellulosic biomass waste, sugarcane bagasse (SCB) into bioethanol and biohydrogen exploiting thermophilic cellulolytic bacterium Clostridium thermocellum DSMZ 1313. Initially, the optimization of some key fermentation factors for bioethanol and biohydrogen productions was done in 150 ml serum bottles employing Taguchi orthogonal array L27 (3̂13) experimental design. Results elucidated that the most suitable factors for ethanologenesis were 70 g/L cellulose, 10 g/L corn‐steep liquor (CSL), 15 mg/L ferrous sulfate (FeSO4·6H2O), 1 g/L magnesium chloride (MgCl2·6H2O), pH 7, and 5 days incubation whereas for hydrogen fermentation were 60 g/L cellulose, 30 g/L CSL, 5 mg/L FeSO4·6H2O, 2 g/L MgCl2·6H2O, pH 7, and 3 days incubation. Quantitatively, 7.422 g/L ethanol and 56.891/50 ml hydrogen were produced from cellulose while 6.352 g/L ethanol and 51.685/50 ml hydrogen with H2SO4‐pretreated SCB (substituted with cellulose). Scaled‐up suspended‐cell fermentations in bench‐scale stirred‐tank bioreactor resulted in 11.77% increased ethanol yield (0.239 g ethanol/g of glucose) and twofold higher hydrogen volumes (106.88 ml hydrogen/g of glucose). The employment of SCB directly as substrate for biofuels production has appeared sustainable to meet the high global energy demands.

Construction of lactic acid overproducing Clostridium thermocellum through enhancement of lactate dehydrogenase expression

Rapid expansion of global market of lactic acid (LA) has prompted research towards cheaper and more eco-friendly strategies for its production. Nowadays, LA is produced mainly through fermentation of simple sugars or starchy biomass (e.g. corn) and its price is relatively high. Lignocellulose could be an advantageous alternative feedstock for LA production owing to its high abundance and low cost. However, the most effective natural producers of LA cannot directly ferment lignocellulose. So far, metabolic engineering aimed at developing microorganisms combining efficient LA production and cellulose hydrolysis has been generally based on introducing designer cellulase systems in natural LA producers. In the present study, the approach consisted in improving LA production in the natural cellulolytic bacterium Clostridium thermocellum DSM1313. The expression of the native lactate dehydrogenase was enhanced by functional replacement of its original promoter with stronger ones resulting in a 10-fold increase in specific activity, which resulted in a 2-fold increase of LA yield. It is known that eliminating allosteric regulation can also increase lactic acid production in C. thermocellum, however we were unable to insert strong promoters upstream of the de-regulated ldh gene. A strategy combining these regulations and inactivation of parasitic pathways appears essential for developing a homolactic C. thermocellum.

Developing a Cell-Free Extract Reaction (CFER) System in Clostridium thermocellum to Identify Metabolic Limitations to Ethanol Production

The cellulolytic bacterium Clostridium thermocellum is a promising candidate for lignocellulosic biofuel production, however ethanol titer needs to be improved for commercialization. To understand the factors limiting ethanol titer in C. thermocellum, we developed a cell-free extract reaction (CFER) system. We demonstrated that 15 mM cellobiose could be converted, in vitro, to 25 mM ethanol, and that this reaction functions both at thermophilic (55C) and mesophilic (37C) temperatures. Although the yield was similar to that produced by whole cells, the rate was much slower (~0.5 vs 12 mM/h). In order to reliably quantify metabolites, rapid CFER quenching is necessary. Among the methods tested, filtration with a 3 kDa molecular weight cutoff filter proved to be the most effective. Metabolomic analysis revealed high levels of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) in the CFER, identifying potential rate limiting enzymes downstream of F6P. NADH was also found to accumulate in the CFER, suggesting NADH recycling rate-limiting. We used two complementary strategies to identify enzymes that limit metabolic flux, including feeding different substrates and supplementing with exogenous enzymes. In the enzyme addition experiment, the largest improvement was observed with the addition of yeast alcohol dehydrogenase (ADH), indicating a limitation at that reaction. The development of a CFER system for C. thermocellum, combined with detailed measurements of intermediate metabolites, allowed us to directly observe the metabolism of this organism, and suggested several potential metabolic engineering interventions for increasing ethanol titer. This demonstrates a technique that may be of general use for metabolic engineering in non-model organisms.

Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I–B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I–B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I–B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I–B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum

Membrane vesicles released from bacteria contribute to cell-cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50-300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Enhancement of methane production by addition of Clostridium thermocellum in the anaerobic digestion of switchgrass

The low efficiency of hydrolysis is usually the rate-limiting step in the anaerobic digestion of lignocellulosic substrates. In this investigation, bioaugmentation and pretreatment using the cellulolytic anaerobic bacterium Clostridium thermocellum have been found to be beneficial for the improvement of the hydrolysis of switchgrass and enhancing methane production. By adopting these two methods, the methane production of switchgrass reached 289.309 and 278.34 ml/g volatile solids, with an increase of 24.2% and 19.5%, respectively, compared to the blank group. The outcome of this study clearly indicated that the fermentation efficiency could be improved by the use of microorganisms in the above two methods. The methane yield of bioaugmentation not only was slightly higher than that of biopretreatment but also had a higher efficiency in terms of total time and was more adaptive to the industrial implementation. Moreover, this study proved that Clostridium thermocellum required about one week to acclimatize the anaerobic condition and the optimum adding time was at the beginning of anaerobic digestion.

Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization

BackgroundThe development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium Clostridium thermocellum is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass.ResultsIn this study, 60 cellulosomal components were recombinantly produced in E. coli and systematically permuted in synthetic complexes to study the function–activity relationship of all available enzymes on Kraft pulp from pine wood as the substrate. Starting from a basic exo/endoglucanase complex, we were able to identify additional functional classes such as mannanase and xylanase for optimal activity on the substrate. Based on these results, we predicted a synthetic cellulosome complex consisting of seven single components (including the scaffoldin protein and a β-glucosidase) and characterized it biochemically. We obtained a highly thermostable complex with optimal activity around 60–65 °C and an optimal pH in agreement with the optimum of the native cellulosome (pH 5.8). Remarkably, a fully synthetic complex containing 47 single cellulosomal components showed comparable activity with a commercially available fungal enzyme cocktail on the softwood pulp substrate.ConclusionsOur results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of C. thermocellum can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

Background Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate of ΔhydG decreased substantially compared to wild type.ResultsAddition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ∆hydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ΔhydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG. Clostridium thermocellum ΔhydGΔech had a higher growth rate than ΔhydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate.ConclusionsSulfur metabolism is perturbed in C. thermocellum ΔhydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.

Single-step bioconversion of lignocellulose to hydrogen using novel moderately thermophilic bacteria.

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to hydrogen offers great potential for lower cost and higher efficiency compared to processes featuring dedicated cellulase production. Current studies on CBP-based hydrogen production mainly focus on using the thermophilic cellulolytic bacterium Clostridium thermocellum and the extremely thermophilic cellulolytic bacterium Caldicellulosiruptor saccharolyticus. However, no studies have demonstrated that the strains in the genus Thermoanaerobacterium could be used as the sole microorganism to accomplish both cellulose degradation and H2 generation.ResultsWe have specifically screened for moderately thermophilic cellulolytic bacteria enabling to produce hydrogen directly from conversion of lignocellulosic materials. Three new strains of thermophilic cellulolytic bacteria in the genus Thermoanaerobacterium growing at a temperature of 60°C were isolated. All of them grew well on various plant polymers including microcrystalline cellulose, filter paper, xylan, glucose, and xylose. In particular, the isolated bacterium, designated as Thermoanaerobacterium thermosaccharolyticum M18, showed high cellulolytic activity and a high yield of H2. When it was grown in 0.5% microcrystalline cellulose, approximately 82% cellulose was consumed, and the H2 yield and maximum production rate reached 10.86 mmol/g Avicel and 2.05 mmol/L/h, respectively. Natural lignocellulosic materials without any physicochemical or biological pretreatment also supported appreciable growth of strain M18, which resulted in 56.07% to 62.71% of insoluble cellulose and hemicellulose polymer degradation in corn cob, corn stalk, and wheat straw with a yield of 3.23 to 3.48 mmol H2/g substrate and an average production rate of 0.10 to 0.13 mmol H2/L/h.ConclusionsThe newly isolated strain T. thermosaccharolyticum M18 displayed effective degradation of lignocellulose and produced large amounts of hydrogen. This is the first report of a Thermoanaerobacterium species presenting cellulolytic characteristics, and this species thus represents a novel cellulolytic bacterium distinguished from all other known cellulolytic bacteria. In comparison, the extraordinary yield and specific rate of hydrogen for strain M18 obtained from lignocellulose make it more attractive in monoculture fermentation. T. thermosaccharolyticum M18 is thus a potential candidate for rapid conversion of lignocellulose to biohydrogen in a single step.

Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

BackgroundAn industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.Methodology/Principal FindingsIn this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).Conclusion/SignificanceThe findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.

Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure.

The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca(2+) ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.

Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

The unique set of putative membrane-associated anti-σ factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation

Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative sigma factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-sigma(I) factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-sigma factors form predicted bicistronic operons, in which the first gene encodes a putative alternative sigma factor, similar to B. subtilissigma(I), but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and sigma(I)-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.

Interplay between Clostridium thermocellum family 48 and family 9 cellulases in cellulosomal versus noncellulosomal states.

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability

Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high-yield production of enzymatic hydrogen. The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64.9 kDa. This enzyme was found to be a dual-specificity enzyme - PGM/phosphomannomutase (PMM). Mg(2+) and Mn(2+) were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The k(cat) and K(m) of PGM were 190 s(-1) and 0.41 mmol l(-1) on glucose-1-phosphate and 59 s(-1) and 0.44 mmol l(-1) on mannose-1-phosphate, respectively, at 60 degrees C. Thermostability of PGM at a low concentration (2 nmol l(-1), 100 U l(-1)) was enhanced by 12-fold (i.e. t(1/2) = 72 h) at 60 degrees C with addition of bovine serum albumin, Triton X-100, Mg(2+)and Mn(2+). The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).