RESUMO
Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency.
Assuntos
Clostridium acetobutylicum , Clostridium cellulovorans , Clostridium acetobutylicum/genética , Clostridium cellulovorans/genética , Reação em Cadeia da Polimerase Multiplex , RNA Ribossômico 16S/genética , Clostridium/genética , Butanóis , 1-Butanol , FermentaçãoRESUMO
The degradation of recalcitrant polysaccharides such as cellulose and chitin requires the synergistic functionality of processive glycosidase (GH) cocktails. Understanding the fundamental phenomenon of processivity is of biological and economic importance for the conversion of biomass into biofuel. In this work, cellulase family 9 from Clostridium cellulovorans (Cel9G), which is a processive endoglucanase, was used to elucidate the processive binding mechanism with respect to polysaccharides, since it exhibits a multimodular crystallographic structure. Metadynamics and molecular dynamics simulations were performed to explore the dynamics of cellulose chain binding to Cel9G via processive motion. The processive movement of the cellulose chain towards the catalytic domain may exhibit several local minima, which are related to strong CH/π interactions between the sugar rings and the aromatic residues distributed at the active site. For the binding of the G6 and G12 molecules, the energy barriers were determined to be 4.8 and 7.4 kcal mol-1, respectively. Based on the site-directed mutagenesis simulations of Y520A, it was found that the existence of Y520 is critical for processive binding. It is likely that Y520 and H125/Y416 form two anchor points to facilitate processive binding to polysaccharides. More importantly, the straight-line morphology of the substrate could be observed after the formation of the so-called slide mode, which is different from the V-shaped Michaelis complex structure revealed by quantum mechanics/molecular mechanics simulations. This indicates that an additional step, namely, catalytic activation, probably exists between processive binding and the hydrolysis reaction. Finally, a four-step catalytic cycle was proposed for Cel9G. Our work provides novel molecular-level insights into the structure-function relationship for the processive enzyme Cel9G and should aid the development of improved GH cocktails for the efficient cleavage of glycosidic linkages.
Assuntos
Celulase , Clostridium cellulovorans , Simulação de Dinâmica Molecular , Celulose/química , Domínio Catalítico , Celulase/químicaRESUMO
Carbohydrate degradation catalyzed by glucoside hydrolases (GHs) is a major mechanism in biomass conversion. GH family 9 endoglucanase (Cel9G) from Clostridium cellulovorans, a typical multimodular enzyme, contains a catalytic domain closely linked to a family 3c carbohydrate-binding module (CBM3c). Unlike the conventional behavior proposed for other carbohydrate-binding modules, CBM3c has a direct impact on catalytic activity. In this work, extensive molecular dynamics (MD) simulations were employed to clarify the functional role of CBM3c. Furthermore, the detailed catalytic mechanism of Cel9G was investigated at the atomistic level using the combined quantum mechanical and molecular mechanical (QM/MM) method. Based on these simulations, owing to the rigidity of the peptide linker, CBM3c may affect the enzymatic activity via direct interactions with alpha helix 4 of GH9, especially with the K123 and H125 residues. In addition, using cellohexaose as a substrate, the QM/MM MD simulations confirmed that this enzyme can cleave the ß-1,4-glycosidic linkage via an inverting mechanism. An oxocarbenium ion-like transition state was located with a barrier height of 19.6 kcal mol-1. Furthermore, the G(-1) pyranose unit preferentially adopted a distorted 1S5/4H5 conformer in the enzyme-substrate complex. For the cleavage of the glycosidic bond, we were able to identify a plausible route (1S5/4H5 â [4H5/4E]# â 4C1) from the reactant to the product at the G(-1) site.
Assuntos
Celulase , Clostridium cellulovorans , Catálise , Domínio Catalítico , Celulase/química , Celulose/químicaRESUMO
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.
Assuntos
Clostridium cellulovorans , Xilanos , Clostridium cellulovorans/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.
Assuntos
Celulossomas , Clostridium cellulovorans , Lactococcus lactis , Proteínas de Bactérias/genética , Membrana Celular , Parede Celular , Clostridium cellulovorans/genética , Lactococcus lactis/genéticaRESUMO
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.
Assuntos
1-Butanol/metabolismo , Celulose/metabolismo , Clostridium cellulovorans , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismoRESUMO
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.
Assuntos
Clostridium cellulovorans , 1-Butanol , Butanóis , Celulose , Clostridium , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Fermentação , Engenharia Metabólica , ProteômicaRESUMO
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.
Assuntos
Butanóis/metabolismo , Clostridium cellulovorans/metabolismo , Engenharia Metabólica , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Butanóis/química , Carbono/metabolismo , Xilose/metabolismoRESUMO
Single layered two-dimensional (2D) materials such as transition metal dichalcogenides (TMDs) show great potential in many microelectronic or nanoelectronic applications. For example, because of extremely high sensitivity, TMD-based biosensors have become promising candidates for next-generation label-free detection. However, very few studies have been conducted on understanding the fundamental interactions between TMDs and other molecules including biological molecules, making the rational design of TMD-based sensors (including biosensors) difficult. This study focuses on the investigations of the fundamental interactions between proteins and two widely researched single-layered TMDs, MoS2, and WS2 using a combined study with linear vibrational spectroscopy attenuated total reflectance FTIR and nonlinear vibrational spectroscopy sum frequency generation vibrational spectroscopy, supplemented by molecular dynamics simulations. It was concluded that a large surface hydrophobic region in a relatively flat location on the protein surface is required for the protein to adsorb onto a monolayered MoS2 or WS2 surface with preferred orientation. No disulfide bond formation between cysteine groups on the protein and MoS2 or WS2 was found. The conclusions are general and can be used as guiding principles to engineer proteins to attach to TMDs. The approach adopted here is also applicable to study interactions between other 2D materials and biomolecules.
Assuntos
Proteínas de Bactérias/química , Dissulfetos/química , Glucosidases/química , Hidrolases/química , Molibdênio/química , Tungstênio/química , beta-Glucosidase/química , Adsorção , Clostridium cellulovorans/enzimologia , Interações Hidrofóbicas e Hidrofílicas , Lactococcus lactis/enzimologia , Simulação de Dinâmica Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Sphingomonas/enzimologia , Propriedades de Superfície , VibraçãoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridium cellulovorans/crescimento & desenvolvimento , Plantas/química , Polissacarídeos/química , Proteômica/métodos , Técnicas Bacteriológicas , Metabolismo dos Carboidratos , Parede Celular/química , Clostridium cellulovorans/metabolismo , Análise por Conglomerados , Regulação Bacteriana da Expressão Gênica , Anotação de Sequência MolecularRESUMO
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.
Assuntos
Clostridium cellulovorans/genética , Expressão Gênica , Engenharia Metabólica/métodos , Plasmídeos/genética , Transformação Bacteriana , Biocombustíveis , Biomassa , Butanóis/metabolismo , Celulose/metabolismo , Clostridium cellulovorans/metabolismo , Eletroporação , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Células-TroncoRESUMO
With high cellulolytic and acetic/butyric acids production abilities, Clostridium cellulovorans is promising for use to produce cellulosic n-butanol. Here, we introduced three different aldehyde/alcohol dehydrogenases encoded by bdhB, adhE1, and adhE2 from Clostridium acetobutylicum into C. cellulovorans and studied their effects on ethanol and n-butanol production. Compared to AdhE2, AdhE1 was more specific for n-butanol biosynthesis over ethanol. Co-expressing adhE1 with bdhB produced a comparable amount of butanol but significantly less ethanol, leading to a high butanol/ethanol ratio of 7.0 and 5.6â¯(g/g) in glucose and cellulose fermentation, respectively. Co-expressing adhE1 or adhE2 with bdhB did not increase butanol production because the activity of BdhB was limited by the NADPH availability in C. cellulovorans. Overall, the strain overexpressing adhE2 alone produced the most n-butanol (4.0â¯g/L, yield: 0.22⯱â¯0.01â¯g/g). Based on the insights from this study, further metabolic engineering of C. cellulovorans for cellulosic n-butanol production is suggested.
Assuntos
Clostridium acetobutylicum , Clostridium cellulovorans , 1-Butanol , Álcool Desidrogenase , Aldeídos , Butanóis , Celulose , Clostridium , Etanol , FermentaçãoRESUMO
Endoglucanase E (EngE) is a cellulosomal enzyme of the glycoside hydrolase family 5 generated by the cellulosome-producing bacterium Clostridium cellulovorans 743B. Although its basic activities and properties have been characterized, its substrate specificity, product range, and steady-state kinetics remain unclear. The current study prepared recombinant EngE (rEngE) and analyzed its substrate specificity and product range using thin layer chromatography. When carboxymethyl cellulose (CMC) or phosphoric acid swollen cellulose was used as a substrate, disaccharides and trisaccharides were the main products. However, no product was detected with microcrystalline cellulose as the substrate. This indicated that rEngE is a cellulase that hydrolyzes low-crystallinity cellulose. Furthermore, products were detected when glucomannan, lichenan, or ß-glucan was used, but no product was obtained with xylan. These results suggested that rEngE hydrolyzes the ß-1,4 glycosidic bond between glucose residues of the substrate. In the kinetic analysis, at CMC concentrations of ≥3 mg/mL, the reaction rate decreased. Application of the above data to three substrate inhibition models generated a better fit to a model that generates products not only from the enzyme-substrate complex but also from enzyme-substrate-substrate (ESS) complexes, in which two substrates are bound to the enzymes. In addition, it was found that a carbohydrate-binding module (CBM) contained in EngE binds to cellulose. Therefore, substrate inhibition likely occurred because the binding site of CBM may correspond to one of the substrate-binding sites in the ESS complex.
Assuntos
Celulase/metabolismo , Clostridium cellulovorans/enzimologia , Sítios de Ligação , Hidrólise , Cinética , Especificidade por SubstratoRESUMO
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.
Assuntos
1-Butanol/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Evolução Molecular , Engenharia Metabólica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Clostridium cellulovorans/crescimento & desenvolvimento , Coenzima A/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Fermentação , Regulação Bacteriana da Expressão Gênica , Lignina/metabolismo , Microrganismos Geneticamente Modificados/genética , Oxirredutases/genéticaRESUMO
The processive mechanism of cellulases against cellulose represents one of the key mechanisms in the conversion of biomass. A reliable model of substrate binding in a multidomain cellulase is a prerequisite for fully understanding this mechanism. In this study, the specificity of the recognition of the polysaccharide by the multidomain endoglucanase Cel9G from Clostridium cellulovorans was investigated by molecular dynamics simulations. Aromatic ring-containing residues were found to be critical for stabilizing the substrate. The calculated subtotal contributions of polar residues close to the active site, e.g., D58, E244, R315 and D420, also have some critical functions in substrate binding. Unlike other members of the carbohydrate-binding module family, CBM3c alone is shown not to bind cellulose very well, which is also consistent with experimental conclusions.
Assuntos
Proteínas de Bactérias/química , Celulase/química , Celulose/química , Clostridium cellulovorans/química , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Sequência de Aminoácidos , Domínio Catalítico , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
Clostridium cellulovorans produces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2â M sodium acetate trihydrate, 0.1â M Tris-HCl pH 8.5, 32%(w/v) PEG 4000 at 293â K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95â Å resolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20â Å, α = ß = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans.
Assuntos
Clostridium cellulovorans/enzimologia , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Endo-1,4-beta-Xilanases/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Difração de Raios X/métodosRESUMO
Plant-virus-based expression vectors have been used as an alternative to the creation of transgenic plants. Using a virus-based vector, we investigated the feasibility of producing the endoglucanase D (EngD) from Clostridium cellulovorans in Nicotiana benthamiana. This protein has endoglucanase, xylanase, and exoglucanase activities and may be of value for cellulose digestion in the generation of biofuels from plant biomass. The EngD gene was cloned between the nuclear inclusion b (NIb)- and coat protein (CP)-encoding sequences of pSP6PepMoV-Vb1. In vitro transcripts derived from the clone (pSP6PepMoV-Vb1/EngD) were infectious in N. benthamiana but caused milder symptoms than wild-type PepMoV-Vb1. RT-PCR amplification of total RNA from non-inoculated upper leaves infected with PepMoV-Vb1/EngD produced the target band for the CP, partial NIb and EngD-CP regions of PepMoV-V1/EngD, in addition to nonspecific bands. Western blot analysis showed the CP target bands of PepMoV-Vb1/EngD as well as non-target bands. EngD enzymatic activity in infected plants was detected using a glucose assay. The plant leaves showed increased senescence compared with healthy and PepMoV-Vb1-infected plants. Our study suggests the feasibility of using a viral vector for systemic infection of plants for expression of heterologous engD for the purpose of digesting a cellulose substrate in plant cells for biomass production.
Assuntos
Celulase/biossíntese , Clostridium cellulovorans/enzimologia , Expressão Gênica , Vetores Genéticos , Nicotiana/enzimologia , Potyvirus/genética , Proteínas Recombinantes/biossíntese , Western Blotting , Celulase/genética , Clonagem Molecular , Clostridium cellulovorans/genética , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/genéticaRESUMO
Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 µM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome.
Assuntos
Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Clostridium cellulovorans/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Proteínas de Bactérias/genética , Biomassa , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clostridium cellulovorans/genética , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Complexos Multienzimáticos/genética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , CoesinasRESUMO
The presence of the family of 3c cellulose binding module (CBM3c) is important for the catalytic activity of family 9 endoglucanases such as the EngZ from Clostridium cellulovorans. To determine the role of CBM3c in catalytic activity, we made a tryptophan to alanine substitution because tryptophan can bind strongly to both substrates and other amino acids. The conserved tryptophan substitution (W483A) did not influence substrate binding, but it reduced enzyme activity to 10-14% on both amorphous and crystalline cellulose. CBM3c is directly involved in the endoglucanase reaction independent of substrate binding. EngZ W483A was also inactivated independent of substrate concentrations. Specially, EngZ W483A restored its catalytic base activity (31.6±1.2U/nM) which is similar to the wild-type (29.4±0.3U/nM) on Avicel in the presence of 50mM sodium azide which is instead of catalytic base reaction. These results suggest that CBM3c is deeply involved in the cellulolytic reaction, specifically at the catalytic base region. Moreover, EngZ W483A was also easily denatured by DTT, an outer disulfide bond breaker, compared to the wild-type. CBM3c could influence the surface stability. These features of CBM3c result from the hydrophobic interaction of tryptophan with the catalytic domain that is unrelated to substrate binding.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias , Celulase , Clostridium cellulovorans , Mutação de Sentido Incorreto , Triptofano , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Celulase/química , Celulase/genética , Clostridium cellulovorans/enzimologia , Clostridium cellulovorans/genética , Estabilidade Enzimática/genética , Estrutura Secundária de Proteína , Triptofano/química , Triptofano/genéticaRESUMO
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.