Autodisplay of an endo-1,4-ß-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation.

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.

Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlACA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnrCA -thlACA -hbdCT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Clostridium cellulovorans metabolism of cellulose as studied by comparative proteomic approach.

Clostridium cellulovorans is among the most promising candidates for consolidated bioprocessing (CBP) of cellulosic biomass to liquid biofuels (ethanol, butanol). C. cellulovorans metabolizes all the main plant polysaccharides and mainly produces butyrate. Since most butyrate and butanol biosynthetic reactions from acetyl-CoA are common, introduction of single heterologous alcohol/aldehyde dehydrogenase can divert the branching-point intermediate (butyryl-CoA) towards butanol production in this strain. However, engineering C. cellulovorans metabolic pathways towards industrial utilization requires better understanding of its metabolism. The present study aimed at improving comprehension of cellulose metabolism in C. cellulovorans by comparing growth kinetics, substrate consumption/product accumulation and whole-cell soluble proteome (data available via ProteomeXchange, identifier PXD015487) with those of the same strain grown on a soluble carbohydrate, glucose, as the main carbon source. Growth substrate-dependent modulations of the central metabolism were detected, including regulation of several glycolytic enzymes, fermentation pathways (e.g. hydrogenase, pyruvate formate lyase, phosphate transacetylase) and nitrogen assimilation (e.g. glutamate dehydrogenase). Overexpression of hydrogenase and increased ethanol production by glucose-grown bacteria suggest a more reduced redox state. Higher energy expenditure seems to occur in cellulose-grown C. cellulovorans (likely related to overexpression and secretion of (hemi-)cellulases), which induces up-regulation of ATP synthetic pathways, e.g. acetate production and ATP synthase. SIGNIFICANCE: C. cellulovorans can metabolize all the main plant polysaccharides (cellulose, hemicelluloses and pectins) and, unlike other well established cellulolytic microorganisms, can produce butyrate. C. cellulovorans is therefore among the most attractive candidates for direct fermentation of lignocellulose to high-value chemicals and, especially, n-butanol, i.e. one of the most promising liquid biofuels for the future. Recent studies aimed at engineering n-butanol production in C. cellulovorans represent milestones towards production of biofuels through one-step fermentation of lignocellulose but also indicated that more detailed understanding of the C. cellulovorans central carbon metabolism is essential to refine metabolic engineering strategies towards improved n-butanol production in this strain. The present study helped identifying key genes associated with specific catabolic reactions and indicated modulations of central carbon metabolism (including redox and energy balance) associated with cellulose consumption. This information will be useful to determine key enzymes and possible metabolic bottlenecks to be addressed towards improved metabolic engineering of this strain.

Metabolic Engineering of Clostridium cellulovorans to Improve Butanol Production by Consolidated Bioprocessing.

Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (ter), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides.

BACKGROUND: Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. RESULTS: We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. CONCLUSION: In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans.

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering.

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.

Transcriptomic Responses of the Interactions between Clostridium cellulovorans 743B and Rhodopseudomonas palustris CGA009 in a Cellulose-Grown Coculture for Enhanced Hydrogen Production.

UNLABELLED: Coculturing dark- and photofermentative bacteria is a promising strategy for enhanced hydrogen (H2) production. In this study, next-generation sequencing was used to query the global transcriptomic responses of an artificial coculture of Clostridium cellulovorans 743B and Rhodopseudomonas palustris CGA009. By analyzing differentially regulated gene expression, we showed that, consistent with the physiological observations of enhanced H2 production and cellulose degradation, the nitrogen fixation genes in R. palustris and the cellulosomal genes in C. cellulovorans were upregulated in cocultures. Unexpectedly, genes related to H2 production in C. cellulovorans were downregulated, suggesting that the enhanced H2 yield was contributed mainly by R. palustris A number of genes related to biosynthesis of volatile fatty acids (VFAs) in C. cellulovorans were upregulated, and correspondingly, a gene that mediates organic compound catabolism in R. palustris was also upregulated. Interestingly, a number of genes responsible for chemotaxis in R. palustris were upregulated, which might be elicited by the VFA concentration gradient created by C. cellulovorans In addition, genes responsible for sulfur and thiamine metabolism in C. cellulovorans were downregulated in cocultures, and this could be due to a response to pH changes. A conceptual model illustrating the interactions between the two organisms was constructed based on the transcriptomic results. IMPORTANCE: The findings of this study have important biotechnology applications for biohydrogen production using renewable cellulose, which is an industrially and economically important bioenergy process. Since the molecular characteristics of the interactions of a coculture when cellulose is the substrate are still unclear, this work will be of interest to microbiologists seeking to better understand and optimize hydrogen-producing coculture systems.

Calorimetric studies of the growth of anaerobic microbes.

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method.

Metabolic and process engineering of Clostridium cellulovorans for biofuel production from cellulose.

Production of cellulosic biofuels has drawn increasing attention. However, currently no microorganism can produce biofuels, particularly butanol, directly from cellulosic biomass efficiently. Here we engineered a cellulolytic bacterium, Clostridium cellulovorans, for n-butanol and ethanol production directly from cellulose by introducing an aldehyde/alcohol dehydrogenase (adhE2), which converts butyryl-CoA to n-butanol and acetyl-CoA to ethanol. The engineered strain was able to produce 1.42 g/L n-butanol and 1.60 g/L ethanol directly from cellulose. Moreover, the addition of methyl viologen as an artificial electron carrier shifted the metabolic flux from acid production to alcohol production, resulting in a high biofuel yield of 0.39 g/g from cellulose, comparable to ethanol yield from corn dextrose by yeast fermentation. This study is the first metabolic engineering of C. cellulovorans for n-butanol and ethanol production directly from cellulose with significant titers and yields, providing a promising consolidated bioprocessing (CBP) platform for biofuel production from cellulosic biomass.

Hydrolytic effects of scaffolding proteins CbpB and CbpC on crystalline cellulose mediated by the major cellulolytic complex from Clostridium cellulovorans.

The role of the scaffolding proteins, cellulose binding protein B and C (CbpB and CbpC, respectively) were identified in cellulolytic complex (cellulosome) of Clostridium cellulovorans for efficient degradation of cellulose. Recombinant CbpB and CbpC directly anchored to the cell surface of C. cellulovorans. In addition, CbpB and CbpC showed increased hydrolytic activity on crystalline cellulose incubated with exoglucanase S (ExgS) and endoglucanase Z (EngZ) compared with the activity of free enzymes. Moreover, the results showed synergistic effects of crystalline cellulose hydrolytic activity (1.8- to 2.2-fold) when CbpB and CbpC complex with ExgS and EngZ are incubated with cellulolytic complex containing mini-CbpA. The results suggest C. cellulovorans critically uses CbpB and CbpC, which can directly anchor cells for the hydrolysis of cellulosic material with the major cellulosome complex.

Artificial symbiosis for acetone-butanol-ethanol (ABE) fermentation from alkali extracted deshelled corn cobs by co-culture of Clostridium beijerinckii and Clostridium cellulovorans.

BACKGROUND: Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to solvents (acetone, butanol and ethanol) production from the renewable and inexpensive substrates, for example, lignocellulose, on account of the depletion of oil resources, increasing gasoline prices and deteriorating environment. Limited to current tools for genetic manipulation, it is difficult to develop a genetically engineered microorganism with combined ability of lignocellulose utilization and solvents production. Mixed culture of cellulolytic microorganisms and solventogenic bacteria provides a more convenient and feasible approach for ABE fermentation due to the potential for synergistic utilization of the metabolic pathways of two organisms. But few bacteria pairs succeeded in producing biobutanol of high titer or high productivity without adding butyrate. The aim of this work was to use Clostridium cellulovorans 743B to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be subsequently utilized by Clostridium beijerinckii NCIMB 8052 to produce butanol in one pot reaction. RESULTS: A stable artificial symbiotic system was constructed by co-culturing a celluloytic, anaerobic, butyrate-producing mesophile (C. cellulovorans 743B) and a non-celluloytic, solventogenic bacterium (C. beijerinckii NCIMB 8052) to produce solvents by consolidated bioprocessing (CBP) with alkali extracted deshelled corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. Under optimized conditions, the co-culture degraded 68.6 g/L AECC and produced 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and 0.87 g/L ethanol) in less than 80 h. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain during the co-culturing process, which figured out the roles of each strain at different periods in the symbiosis. CONCLUSION: Our work illustrated the great potential of artificial symbiosis in biofuel production from lignocellulosic biomass by CBP. The dynamics of the abundance of C. beijerinckii and C. cellulovorans revealed mechanisms of cooperation and competition between the two strains during the co-culture process.

Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum ß-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose.

BACKGROUND: The complete degradation of the cellulose requires the synergistic action of endo-ß-glucanase, exo-ß-glucanase, and ß-glucosidase. But endo-ß-glucanase and exo-ß-glucanase can be recovered by solid-liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the ß-glucosidases cannot be recovered because of most ß-glucosidases without the CBD, so additional ß-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation. RESULTS: The glucose-tolerant ß-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum ß-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in Vmax/Km in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. CONCLUSIONS: The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

Exoproteome profiles of Clostridium cellulovorans grown on various carbon sources.

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.

Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans.

The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the ß-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.

Putative role of cellulosomal protease inhibitors in Clostridium cellulovorans based on gene expression and measurement of activities.

This study is the first to demonstrate the activity of putative cellulosomal protease/peptidase inhibitors (named cyspins) of Clostridium cellulovorans, using the Saccharomyces cerevisiae display system. Cyspins exhibited inhibitory activities against several representative plant proteases. This suggests that these inhibitors protect their microbe and cellulosome from external attack by plant proteases.

Comparative genomics of the mesophilic cellulosome-producing Clostridium cellulovorans and its application to biofuel production via consolidated bioprocessing.

Clostridium cellulovorans is an anaerobic, mesophilic bacterium that efficiently degrades native substrates in soft biomass such as corn fibre and rice straw by producing an extracellular enzyme complex called the cellulosomes. By examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Recently, we reported the whole genome sequence of C. cellulovorans. A total of 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipase, peptidase and proteinase inhibitors, in addition to two novel genes encoding scaffolding proteins CbpB and CbpC. Interestingly, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia: mesophilic C. cellulolyticum and thermophilic C. thermocellum. Since the C. cellulovorans genome included not only cellulosomal genes but also a large number of genes encoding non-cellulosomal enzymes, the genome expansion of C. cellulovorans included genes more related to degradation of polysaccharides, such as hemicelluloses and pectins, than to cellulose. In this review, we propose a strategy for industrial applications such as biofuel production using enhanced mesophilic cellulosome- and solvent-producing clostridia.

The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers.

The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology.

Cellulosomic profiling produced by Clostridium cellulovorans during growth on different carbon sources explored by the cohesin marker.

Clostridium cellulovorans produces large extracellular enzyme complex, called cellulosomes. The diversity of the cellulosomal enzymes, which are secreted by C. cellulovorans that has been cultured on different carbon sources, such as Avicel, xylan, AXP (Avicel-xylan-pectin, 3:1:1) and cellobiose, was explored by two-dimensional gel electrophoresis. To identify the cellulosomal enzymes, we constructed a biomarker using cohesin 6, one of the CbpA cohesins, that was labeled with fluorescence. The major apparent spots were isolated and identified by ESI MS/MS protein sequencing. Fluorescently labeled cohesin clearly showed that the amount of the cellulosomal enzymes was influenced by the available carbon source. EngE, ExgS, EngK, XynB and ManA were most frequently expressed under all conditions. However, EngY was only observed on the AXP culture. We found two novel putative cellulosomal proteins, NC1[GH9] and NC2[GH26], and five unknown proteins, NU1, NU2, NU3, NU4 and NU5. The cohesin biomarker clearly showed different production patterns of the cellulosomal subunits under different culture conditions and revealed novel cellulosomal subunits.