Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Clostridium cellulolyticum ATCC 35319 is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass. As with most truly cellulolytic clostridia, C. cellulolyticum possesses an extracellular multi-enzymatic complex, the cellulosome. The catalytic components of the cellulosome release soluble cello-oligosaccharides from cellulose providing the primary carbon substrates to support bacterial growth. As most cellulolytic bacteria, C. cellulolyticum was initially characterised by limited carbon consumption and subsequent limited growth in comparison to other saccharolytic clostridia. The first metabolic studies performed in batch cultures suggested nutrient(s) limitation and/or by-product(s) inhibition as the reasons for this limited growth. In most recent investigations using chemostat cultures, metabolic flux analysis suggests a self-intoxication of bacterial metabolism resulting from an inefficiently regulated carbon flow. The investigation of C. cellulolyticum physiology with cellobiose, as a model of soluble cellodextrin, and with pure cellulose, as a carbon source more closely related to lignocellulosic compounds, strengthen the idea of a bacterium particularly well adapted, and even restricted, to a cellulolytic lifestyle. The metabolic flux analysis from continuous cultures revealed that (i) in comparison to cellobiose, the cellulose hydrolysis by the cellulosome introduces an extra regulation of entering carbon flow resulting in globally lower metabolic fluxes on cellulose than on cellobiose, (ii) the glucose 1-phosphate/glucose 6-phosphate branch point controls the carbon flow directed towards glycolysis and dissipates carbon excess towards the formation of cellodextrins, glycogen and exopolysaccharides, (iii) the pyruvate/acetyl-CoA metabolic node is essential to the regulation of electronic and energetic fluxes. This in-depth analysis of C. cellulolyticum metabolism has permitted the first attempt to engineer metabolically a cellulolytic microorganism.     

梭热杆菌纤维小体研究进展

梭热杆菌(Clostridium thermocellum)是一种嗜热厌氧细菌,通过分泌大量纤维素酶高效降解纤维素.根据作用纤维素的不同部位,梭热杆菌分泌的纤维素酶分为内切纤维素酶和外切纤维素酶.纤维小体是由支架蛋白、锚定元件、黏合蛋白、纤维素结合域和催化单位组成的复合体,其独特的结构,使得它可以比真菌纤维素酶更紧密地结合到纤维素表面,这个复合结构结合着多种催化单位,而此特殊的结构是梭热杆菌高效降解纤维素的必要条件.近年来,为更深入透彻地了解纤维小体的结构与功能进行了大量的研究工作,现对相关研究进展进行综述,并给出了未来可能的发展方向.     

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.     

Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692-1785 from the C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L. The two-dimensional 1H-15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant beta-structure, which is in agreement with the circular dichroism data.     

Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate‐active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin‐containing proteins bear catalytic modules from numerous carbohydrate‐active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip‐cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32‐kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate‐binding module 6 or carbohydrate‐binding module 22 xylan‐binding modules along dockerins. This newly identified xyl‐doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip‐cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.     

Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria

A stable cellulose-degrading microflora enriched from composting materials has been analyzed in our laboratory. Cellulose-degrading efficiency of an anaerobic cellulolytic isolate, Clostridium straminisolvens CSK1, was remarkably lower than that of the original microflora. We successfully constructed bacterial communities with effective cellulose degradation by mixing C. straminisolvens CSK1 with aerobic non-cellulolytic bacteria isolated from the original microflora. Comparison of the cellulose degradation processes of the pure culture of C. straminisolvens CSK1 and the mixed-culture indicated that non-cellulolytic bacteria essentially contribute to cellulose degradation by supplying anaerobic environment, consuming metabolites, which otherwise deteriorate the cellulolytic activity, and by neutralizing pH.     

丝状真菌降解转化纤维素的机制与遗传改良前景简

丝状真菌可以分泌大量纤维素酶及辅助酶来降解纤维素底物,也是目前工业上纤维素酶的主要生产者。回顾并综述了丝状真菌降解转化纤维素的酶系和机制进展,详细总结了组学研究在纤维素酶研究上的新成果,并探讨了提高丝状真菌酶系效率和产量的遗传改良策略。     

Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1). Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)(-1)] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)(-1)]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1). Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.     

人工合成启动子文库研究进展

随着合成生物学的发展,人工合成功能元件在代谢工程领域显示出巨大应用潜力。启动子是调控基因转录水平的重要元件,可以利用人工合成启动子文库对代谢途径进行精细调控,使基因适量表达,实现代谢途径的组合优化。文章综述了人工合成启动子文库的研究进展,评述了不同文库的构建策略,讨论了人工合成启动子文库在代谢工程领域的应用,并展望了人工合成启动子文库的发展方向。     

Pretreatment of microcrystalline cellulose flakes with CaCl2 increases the surface area, and thus improves enzymatic saccharification

Glucose production from cellulose flakes with cellulases was improved after pretreatment with saturated CaCl2 at room temperature. When pretreated microcrystalline cellulose flakes (Funacel II, Funakoshi Co., Ltd, Tokyo, Japan) were saccharified with the cellulases, 76.8% of the substrate was converted into glucose within 5 h, whereas the corresponding conversion rate of water-pretreated cellulose flakes was 33.8%. To clarify the mechanism of the promotion, cellobiohydrolase I purified from Trichoderma longibrachiatum was used as the model cellulase, which degraded CaCl2-pretreated cellulose more quickly than the water-pretreated cellulose under tested conditions. The maximum amount of the enzyme bound to CaCl2-pretreated cellulose at 37 degrees C was estimated as 1.14 nmol/mg of cellulose, whereas that to water-pretreated cellulose was 0.527 nmol/mg of cellulose. The specific activity of the bound enzyme greatly decreased with the increase of the surface density (rho) of the bound enzyme, and no significant positive effects of the CaCl2-pretreatment on the specific activity could be observed at the same rho value, suggesting that the promotion was attributed mainly to the increase of the surface area of cellulose. The effect was also observed with dewaxed cotton or filter paper, but not with nata de coco cellulose or bagasse cellulose as the substrates. This suggests that the CaCl2-pretreatment serves to increase the surface area of cellulose flakes via liberation of cellulose particles which were artificially aggregated during harsh drying processes of the flakes.     

Pre-acclimation of a wastewater inoculum to cellulose in an aqueous-cathode MEC improves power generation in air-cathode MFCs

Cellulose has been used in two-chamber microbial fuel cells (MFCs), but power densities were low. Higher power densities can be achieved in air-cathode MFCs using an inoculum from a two-chamber, aqueous-cathode microbial electrolysis cell (MEC). Air-cathode MFCs with this inoculum produced maximum power densities of 1070 mW m⁻² (cathode surface area) in single-chamber and 880 mW m⁻² in two-chamber MFCs. Coulombic efficiencies ranged from 25% to 50%, and COD removals were 50-70% based on total cellulose removals of 60-80%. Decreasing the reactor volume from 26 to 14 mL (while maintaining constant electrode spacing) decreased power output by 66% (from 526 to 180 mW m⁻²) due to a reduction in total mass of cellulose added. These results demonstrate that air-cathode MFCs can produce high power densities with cellulose following proper acclimation of the inoculum, and that organic loading rates are important for maximizing power densities from particulate substrates.     

Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.     

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.     

Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5 and 3 flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5 upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in March 2005 with corrections to Dr. Tatarinovas name.     

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.     

Isolation and characterization of cellulose nanofibrils from wheat straw using steam explosion coupled with high shear homogenization

Cellulose nanofibrils of diameter 10–50 nm were obtained from wheat straw using alkali steam explosion coupled with high shear homogenization. High shear results in shearing of the fiber agglomerates resulting in uniformly dispersed nanofibrils. The chemical composition of fibers at different stages were analyzed according to the ASTM standards and showed increase in α-cellulose content and decrease in lignin and hemicellulose. Structural analysis of steam exploded fibers was carried out by Fourier Transform Infrared (FT-IR) spectroscopy and X-ray diffraction (XRD). Thermal stability was higher for cellulose nanofibrils as compared to wheat straw and chemically treated fibers. The fiber diameter distribution was obtained using image analysis software. Characterization of the fibers by AFM, TEM, and SEM showed that fiber diameter decreases with treatment and final nanofibril size was 10–15 nm. FT-IR, XRD, and TGA studies confirmed the removal of hemicellulose and lignin during the chemical treatment process.     

Solid acid and microwave-assisted hydrolysis of cellulose in ionic liquid

Solid acid-catalyzed hydrolysis of cellulose in ionic liquid was greatly promoted by microwave heating. H-form zeolites with a lower Si/Al molar ratio and a larger surface area showed a relatively higher catalytic activity. These solid catalysts exhibited better performance than the sulfated ion-exchanging resin NKC-9. Compared with conventional oil bath heating mode, microwave irradiation at an appropriate power significantly reduced the reaction time and increased the yields of reducing sugars. A typical hydrolysis reaction with Avicel cellulose produced glucose in around 37% yield within 8 min.