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1.
BMC Microbiol ; 19(1): 118, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159733

RESUMO

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 Molecular
2.
Appl Microbiol Biotechnol ; 103(13): 5391-5400, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115632

RESUMO

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-Tronco
3.
J Biosci Bioeng ; 128(4): 398-404, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30981600

RESUMO

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 Substrato
4.
Bioresour Technol ; 285: 121316, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30959389

RESUMO

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 5.6 (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 (4.0 g/L, yield: 0.22 ±â€¯0.01 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 , Fermentação
5.
Appl Environ Microbiol ; 85(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658972

RESUMO

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/genética
6.
Phys Chem Chem Phys ; 20(7): 5235-5245, 2018 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29399685

RESUMO

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 , Termodinâmica
7.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 2): 113-116, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29400321

RESUMO

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 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Šresolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 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/métodos
8.
Arch Virol ; 162(12): 3717-3726, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28864903

RESUMO

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 , Potyvirus/genética , Proteínas Recombinantes/biossíntese , Tabaco/enzimologia , Western Blotting , Celulase/genética , Clonagem Molecular , Clostridium cellulovorans/genética , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tabaco/genética
9.
J Biosci Bioeng ; 124(4): 376-380, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28533157

RESUMO

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
10.
Int J Biol Macromol ; 92: 159-166, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27375055

RESUMO

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/genética
11.
Sci Rep ; 6: 26128, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27184298

RESUMO

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-ß-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg).


Assuntos
Celulase/genética , Celulase/metabolismo , Evolução Molecular Direcionada/métodos , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala , Sistema Livre de Células , Clostridium cellulovorans/enzimologia , Clostridium cellulovorans/genética , Escherichia coli/genética
12.
Appl Environ Microbiol ; 82(15): 4546-4559, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27208134

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Celulose/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Hidrogênio/metabolismo , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Transcriptoma , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura
13.
J Biosci Bioeng ; 122(3): 364-9, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27012376

RESUMO

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.


Assuntos
Bactérias Anaeróbias/crescimento & desenvolvimento , Bactérias Anaeróbias/metabolismo , Calorimetria/métodos , Temperatura Alta , Trifosfato de Adenosina/metabolismo , Clostridium acetobutylicum/crescimento & desenvolvimento , Clostridium acetobutylicum/metabolismo , Clostridium beijerinckii/crescimento & desenvolvimento , Clostridium beijerinckii/metabolismo , Clostridium cellulovorans/crescimento & desenvolvimento , Clostridium cellulovorans/metabolismo
14.
Int J Biol Macromol ; 86: 269-76, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26808019

RESUMO

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30-60 °C). In addition, the optimal temperature of K94R/S365P was increased by 7.5 °C. K94R/S365P retained 78.3% relative activity at 70 °C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70 °C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5 °C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl2 and delayed aggregation temperature in the presence of CaCl2 compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12h reaction; however, the yields consistently increased until 72h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50 °C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.


Assuntos
Substituição de Aminoácidos , Biocatálise , Celulase/química , Celulase/metabolismo , Temperatura , Sequência de Aminoácidos , Cálcio/farmacologia , Celulase/genética , Clostridium cellulovorans/enzimologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Mutação , Agregados Proteicos
15.
Appl Microbiol Biotechnol ; 100(5): 2289-99, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26590584

RESUMO

Clostridium cellulovorans, a cellulolytic bacterium producing butyric and acetic acids as main fermentation products, is a promising host for biofuel production from cellulose. However, the transformation method of C. cellulovorans was not available, hindering its genetic engineering. To overcome this problem, its restriction modification (RM) systems were analyzed and a novel in vivo methylation was established for its successful transformation in the present study. Specifically, two RM systems, Cce743I and Cce743II, were determined. R. Cce743I has the same specificity as LlaJI, recognizing 5'-GACGC-3' and 5'-GCGTC-3', while M. Cce743I methylates the external cytosine in the strand (5'-GACG(m)C-3'). R. Cce743II, has the same specificity as LlaI, recognizing 5'-CCAGG-3' and 5'-CCTGG-3', while M. Cce743II methylates the external cytosine of both strands. An in vivo methylation system, expressing M. Cce743I and M. Cce743II from C. cellulovorans in Escherichia coli, was then established to protect plasmids used in electrotransformation. Transformants expressing an aldehyde/alcohol dehydrogenase (adhE2), which converted butyryl-CoA to n-butanol and acetyl-CoA to ethanol, were obtained. For the first time, an effective transformation method was developed for metabolic engineering of C. cellulovorans for biofuel production directly from cellulose.


Assuntos
Clostridium cellulovorans/enzimologia , Clostridium cellulovorans/genética , Metilação de DNA , Enzimas de Restrição-Modificação do DNA , Técnicas de Transferência de Genes , Transformação Bacteriana , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Butanóis/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Engenharia Metabólica
16.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 10): 1264-72, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26457517

RESUMO

Exoglucanase/cellobiohydrolase (EC 3.2.1.176) hydrolyzes a ß-1,4-glycosidic bond from the reducing end of cellulose and releases cellobiose as the major product. Three complex crystal structures of the glycosyl hydrolase 48 (GH48) cellobiohydrolase S (ExgS) from Clostridium cellulovorans with cellobiose, cellotetraose and triethylene glycol molecules were solved. The product cellobiose occupies subsites +1 and +2 in the open active-site cleft of the enzyme-cellotetraose complex structure, indicating an enzymatic hydrolysis function. Moreover, three triethylene glycol molecules and one pentaethylene glycol molecule are located at active-site subsites -2 to -6 in the structure of the ExgS-triethylene glycol complex shown here. Modelling of glucose into subsite -1 in the active site of the ExgS-cellobiose structure revealed that Glu50 acts as a proton donor and Asp222 plays a nucleophilic role.


Assuntos
Celulases/química , Celulases/metabolismo , Celulose/análogos & derivados , Clostridium cellulovorans/enzimologia , Tetroses/metabolismo , Biocatálise , Cálcio/metabolismo , Domínio Catalítico , Celulose/química , Celulose/metabolismo , Cristalização , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Tetroses/química
17.
Metab Eng ; 32: 39-48, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26365585

RESUMO

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.


Assuntos
Biocombustíveis , Celulose/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Engenharia Metabólica/métodos , Álcool Desidrogenase/genética , Aldeído Desidrogenase/genética , Biomassa , Butanóis/metabolismo , Coenzima A/metabolismo , Meios de Cultura , Etanol/metabolismo , Paraquat/farmacologia , Plasmídeos
18.
J Struct Biol ; 191(3): 352-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26160737

RESUMO

The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs.


Assuntos
beta-Glucosidase/metabolismo , Catálise , Domínio Catalítico , Celulose/metabolismo , Clostridium cellulovorans/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Trichoderma/enzimologia
19.
Bioresour Technol ; 191: 505-11, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25748018

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridium cellulovorans/metabolismo , Celulose/metabolismo , Hidrólise
20.
Enzyme Microb Technol ; 70: 1-8, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25659626

RESUMO

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75-25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.


Assuntos
Mananas/metabolismo , alfa-Galactosidase/metabolismo , beta-Manosidase/metabolismo , Aspergillus niger/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotecnologia , Sequência de Carboidratos , Clostridium cellulovorans/enzimologia , Cyamopsis/enzimologia , Proteínas Fúngicas/metabolismo , Galactanos/química , Galactanos/metabolismo , Hidrólise , Mananas/química , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Gomas Vegetais/química , Gomas Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Especificidade por Substrato
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