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1.
Int J Mol Sci ; 20(16)2019 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-31405215

RESUMO

In family GH13 of the carbohydrate-active enzyme database, subfamily 18 contains glycoside phosphorylases that act on α-sugars and glucosides. Because their phosphorolysis reactions are effectively reversible, these enzymes are of interest for the biocatalytic synthesis of various glycosidic compounds. Sucrose 6F-phosphate phosphorylases (SPPs) constitute one of the known substrate specificities. Here, we report the characterization of an SPP from Ilumatobacter coccineus with a far stricter specificity than the previously described promiscuous SPP from Thermoanaerobacterium thermosaccharolyticum. Crystal structures of both SPPs were determined to provide insight into their similarities and differences. The residues responsible for binding the fructose 6-phosphate group in subsite +1 were found to differ considerably between the two enzymes. Furthermore, several variants that introduce a higher degree of substrate promiscuity in the strict SPP from I. coccineus were designed. These results contribute to an expanded structural knowledge of enzymes in subfamily GH13_18 and facilitate their rational engineering.


Assuntos
Actinobacteria/enzimologia , Fosforilases/metabolismo , Sacarose/metabolismo , Thermoanaerobacterium/enzimologia , Actinobacteria/química , Actinobacteria/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Fosforilases/química , Conformação Proteica , Especificidade por Substrato , Thermoanaerobacterium/química , Thermoanaerobacterium/metabolismo
2.
Metab Eng ; 51: 32-42, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30218716

RESUMO

The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Clostridium thermocellum/metabolismo , Ferredoxinas/metabolismo , NADH NADPH Oxirredutases/metabolismo , Piruvato Sintase/metabolismo , Thermoanaerobacterium/metabolismo , Álcool Desidrogenase/genética , Aldeído Desidrogenase/genética , Clostridium thermocellum/genética , Fermentação , Ferredoxinas/genética , Engenharia Metabólica , NADH NADPH Oxirredutases/genética , Plasmídeos/genética
3.
PLoS One ; 13(4): e0195143, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29621294

RESUMO

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.


Assuntos
Etanol/metabolismo , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo , Adaptação Biológica , Álcool Desidrogenase/metabolismo , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Mutação , Oxirredução , Sequenciamento Completo do Genoma
4.
Curr Microbiol ; 75(5): 620-623, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29279978

RESUMO

A novel thermophilic and butanogenic Thermoanaerobacterium thermosaccharolyticum M5 was successfully isolated and characterized, which could produce butanol from hemicellulose via a unique ethanol-butanol (EB) pathway through consolidated bioprocessing (CBP). This represents the first wild-type bacterium which could produce butanol from hemicellulose via CBP under thermophilic conditions. The assembled draft genome of strain M5 is 2.64 Mp, which contains 2638 genes and 2465 protein-coding sequences with 33.90% G + C content. Among these annotated proteins, xylanases, xylosidases, and bifunctional alcohol/aldehyde dehydrogenase (AdhE) play key roles in the achievement of EB production from hemicellulose through CBP.


Assuntos
Butanóis/metabolismo , Genoma Arqueal , Polissacarídeos/metabolismo , Thermoanaerobacterium/genética , Proteínas Arqueais/genética , Composição de Bases , Etanol , Filogenia , Thermoanaerobacterium/classificação , Thermoanaerobacterium/isolamento & purificação , Thermoanaerobacterium/metabolismo
5.
Microb Cell Fact ; 16(1): 171, 2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28978312

RESUMO

BACKGROUND: Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. RESULT: Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. CONCLUSION: The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield.


Assuntos
Clostridium thermocellum/enzimologia , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Piruvato Descarboxilase/metabolismo , Acetobacteraceae/enzimologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Celulose/metabolismo , Clostridium thermocellum/genética , Fermentação , Engenharia Metabólica , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/isolamento & purificação , Temperatura , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo , Zymomonas/genética , Zymomonas/metabolismo
6.
Sci Rep ; 7(1): 10088, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855699

RESUMO

As a nongenetic engineering technique, adaptive evolution is an effective and easy-to-operate approach to strain improvement. In this work, a commercial Thermoanaerobacterium aotearoense SCUT27/Δldh-G58 was successfully isolated via sequential batch fermentation with step-increased carbon concentrations. Mutants were isolated under selective high osmotic pressures for 58 passages. The evolved isolate rapidly catabolized sugars at high concentrations and subsequently produced ethanol with good yield. A 1.6-fold improvement of ethanol production was achieved in a medium containing 120 g/L of carbon substrate using the evolved strain, compared to the start strain. The analysis of transcriptome and intracellular solute pools suggested that the adaptive evolution altered the synthesis of some compatible solutes and activated the DNA repair system in the two Thermoanaerobacterium sp. evolved strains. Overall, the results indicated the potential of adaptive evolution as a simple and effective tool for the modification and optimization of industrial microorganisms.


Assuntos
Adaptação Biológica/genética , Biotecnologia/métodos , Pressão Osmótica/fisiologia , Thermoanaerobacterium/metabolismo , Evolução Biológica , Metabolismo dos Carboidratos/genética , Etanol/metabolismo , Fermentação , Mutação , Thermoanaerobacterium/genética
7.
Appl Environ Microbiol ; 83(18)2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28710263

RESUMO

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 ß-l-arabinopyranosidase (CpAbp27A), and two GH127 ß-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved ß-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusIMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as ß-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.


Assuntos
Proteínas de Bactérias/metabolismo , Polissacarídeos/metabolismo , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Família Multigênica , Polissacarídeos/química , Especificidade por Substrato , Thermoanaerobacterium/química , Thermoanaerobacterium/genética
8.
Microb Cell Fact ; 16(1): 77, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468624

RESUMO

BACKGROUND: Energy shortage and environmental pollution are two severe global problems, and biological hydrogen production from lignocellulose shows great potential as a promising alternative biofuel to replace the fossil fuels. Currently, most studies on hydrogen production from lignocellulose concentrate on cellulolytic microbe, pretreatment method, process optimization and development of new raw materials. Due to no effective approaches to relieve the inhibiting effect of inhibitors, the acid pretreated lignocellulose hydrolysate was directly discarded and caused environmental problems, suggesting that isolation of inhibitor-tolerant strains may facilitate the utilization of acid pretreated lignocellulose hydrolysate. RESULTS: Thermophilic bacteria for producing hydrogen from various kinds of sugars were screened, and the new strain named MJ1 was isolated from paper sludge, with 99% identity to Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. The hydrogen yields of 11.18, 4.25 and 2.15 mol-H2/mol sugar can be reached at an initial concentration of 5 g/L cellobiose, glucose and xylose, respectively. The main metabolites were acetate and butyrate. More important, MJ1 had an excellent tolerance to inhibitors of dilute-acid (1%, g/v) pretreated sugarcane bagasse hydrolysate (DAPSBH) and could efficiently utilize DAPSBH for hydrogen production without detoxication, with a production higher than that of pure sugars. The hydrogen could be quickly produced with the maximum hydrogen production reached at 24 h. The hydrogen production reached 39.64, 105.42, 111.75 and 110.44 mM at 20, 40, 60 and 80% of DAPSBH, respectively. Supplementation of CaCO3 enhanced the hydrogen production by 21.32% versus the control. CONCLUSIONS: These results demonstrate that MJ1 could directly utilize DAPSBH for biohydrogen production without detoxication and can serve as an excellent candidate for industrialization of hydrogen production from DAPSBH. The results also suggest that isolating unique strains from a particular environment offers an ideal way to conquer the related problems.


Assuntos
Celulose/metabolismo , Hidrogênio/metabolismo , Saccharum , Thermoanaerobacterium/isolamento & purificação , Thermoanaerobacterium/metabolismo , Ácidos , Biocombustíveis , Celobiose/metabolismo , Fermentação , Glucose/metabolismo , Lignina/metabolismo , RNA Ribossômico 16S , Thermoanaerobacterium/genética , Xilose/metabolismo
9.
Appl Microbiol Biotechnol ; 101(6): 2619-2627, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28110397

RESUMO

Conversion of organic wastes to syngas is an attractive way to utilize wastes. The produced syngas can be further used to produce a variety of chemicals. In this study, a hollow-fiber membrane biofilm reactor with mix cultures was operated at 55 °C to convert syngas (H2/CO2) into acetate. A high concentration of acetate (42.4 g/L) was reached in batch experiment while a maximum acetate production rate of 10.5 g/L/day was achieved in the continuous-flow mode at hydraulic retention time (HRT) of 1 day. Acetate was the main product in both batch and continuous-flow experiments. n-Butyrate was the other byproduct in the reactor. Acetate accounted for more than 98.5 and 99.1% of total volatile fatty acids in batch and continuous modes, respectively. Illumina Miseq high-throughput sequencing results showed that microorganisms were highly purified and enriched in the reactor. The main genus was Thermoanaerobacterium (66% of relative abundance), which was usually considered as H2 producer in the literature, however, likely played a role as a H2 consumer in this study. This study provides a new method to generate the high producing rate and purity of acetate from syngas.


Assuntos
Ácido Acético/metabolismo , Dióxido de Carbono/metabolismo , Hidrogênio/metabolismo , Consórcios Microbianos/genética , Esgotos/microbiologia , Thermoanaerobacterium/genética , Técnicas de Cultura Celular por Lotes , Biofilmes/crescimento & desenvolvimento , Reatores Biológicos , Ácido Butírico/metabolismo , Fermentação , Sequenciamento de Nucleotídeos em Larga Escala , Esgotos/química , Thermoanaerobacterium/metabolismo
10.
World J Microbiol Biotechnol ; 33(1): 7, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27858340

RESUMO

Biohydrogen is considered as one of the most promising energy alternatives considering the climate and energy crisis. The dark fermentative hydrogen production from xylose at extreme thermophilic condition (70 °C) using mixed culture was conducted in this study. The effects of initial pH values (ranged from 5.0 to 10.0) and substrate concentrations (ranged from 2.5 to 15.0 g/L) on the hydrogen production, substrate degradation and metabolite distributions were investigated using batch-mode operations. Results showed that initial substrate pH values in the neutral region (6.0-7.0) were beneficial for hydrogen production. The fermentation at initial pH 7.0 and 7.5 g/L xylose reached an optimal hydrogen yield of 1.29 mol-H2/mol-xyloseconsumed. Ethanol, butyrate, and propionate were the major liquid metabolites. The xylose biodegradation efficiency of the mixed culture decreased sharply at high initial culture pH values. The increase of xylose concentration resulted in the accumulation of propionate and an obvious decrease in the final pH value, as well as a low hydrogen yield. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis indicated that hydrogen producing bacteria were enriched by repeated culture under extreme thermophilic conditions. Also, the mixed culture was dominated with bacterial species related to Clostridium and Thermoanaerobacterium.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Hidrogênio/metabolismo , Xilose/metabolismo , Bactérias/genética , Técnicas de Cultura Celular por Lotes , Butiratos/metabolismo , Clostridium/genética , Clostridium/isolamento & purificação , Clostridium/metabolismo , DNA Bacteriano/genética , Eletroforese em Gel de Gradiente Desnaturante , Etanol/metabolismo , Fermentação , Temperatura Alta , Concentração de Íons de Hidrogênio , Reação em Cadeia da Polimerase , Thermoanaerobacterium/genética , Thermoanaerobacterium/isolamento & purificação , Thermoanaerobacterium/metabolismo
11.
Appl Environ Microbiol ; 82(24): 7134-7141, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27694237

RESUMO

Ferredoxin:NAD+ oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+ This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol. IMPORTANCE: Redox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD+, a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.


Assuntos
Proteínas de Bactérias/metabolismo , Etanol/metabolismo , Ferredoxinas/metabolismo , NAD/metabolismo , Oxirredutases/metabolismo , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Oxirredução , Oxirredutases/genética , Thermoanaerobacterium/genética
12.
Appl Microbiol Biotechnol ; 100(19): 8607-20, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27538932

RESUMO

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.


Assuntos
Celulose/metabolismo , Clostridium thermocellum/crescimento & desenvolvimento , Clostridium thermocellum/metabolismo , Hidrogênio/metabolismo , Thermoanaerobacterium/crescimento & desenvolvimento , Thermoanaerobacterium/metabolismo , Clostridium thermocellum/enzimologia , Clostridium thermocellum/genética , Técnicas de Cocultura , Evolução Molecular , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hidrogenase/genética , Hidrogenase/metabolismo , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/genética
13.
FEMS Microbiol Lett ; 363(11)2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27190292

RESUMO

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass. Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at high yield (>90% of theoretical) and titer (>70 g/l). Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors. These cofactors (NAD(+), NADH, NADP(+) and NADPH) and their oxidation state are important in the context of microbial redox metabolism. In this study we directly measured the concentrations and reduced oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays. We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered. We have found that the ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum.


Assuntos
Clostridium thermocellum/metabolismo , Niacinamida/metabolismo , Thermoanaerobacterium/metabolismo , Biocombustíveis , Biomassa , Clostridium thermocellum/genética , Etanol/metabolismo , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas , NAD/metabolismo , NADP/metabolismo , Oxirredução , Thermoanaerobacterium/genética
14.
J Microbiol ; 54(6): 440-4, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27225461

RESUMO

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Chaperoninas/química , Chaperoninas/isolamento & purificação , Thermoanaerobacterium/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Thermoanaerobacterium/química , Thermoanaerobacterium/genética
15.
Appl Microbiol Biotechnol ; 100(11): 5165-76, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27052381

RESUMO

Bio-hydrogen production from mixed culture fermentation (MCF) of glucose was studied by conducting a comprehensive product measurement and detailed mass balance analysis of their contributions to the final H2 yield. The culture used in this study was enriched on glucose at 60 °C through a sequential batch operation consisting of daily glucose feeds, headspace purging and medium replacement every third day in serum bottles for over 2 years. 2-Bromoethanesulfonate (BES) was only required during the first three 3-day cycles to permanently eliminate methanogenic activity. Daily glucose feeds were fully consumed within 24 h, with a persistent H2 yield of 2.7 ± 0.1 mol H2/mol glucose, even when H2 was allowed to accumulate over the 3-day cycle. The measured H2 production exceeded by 14 % the theoretical production of H2 associated with the fermentation products, dominated by acetate and butyrate. Follow-up experiments using acetate with a (13)C-labelled methyl group showed that the excess H2 production was not due to acetate oxidation. Chemical formula analysis of the biomass showed a more reduced form of C5H11.8O2.1N1.1 suggesting that the biomass formation may even consume produced H2 from fermentation.


Assuntos
Meios de Cultura/química , Fermentação , Temperatura Alta , Hidrogênio/metabolismo , Acetatos/metabolismo , Ácidos Alcanossulfônicos/química , Anaerobiose , Técnicas de Cultura Celular por Lotes , Biomassa , Reatores Biológicos/microbiologia , Butiratos/metabolismo , Dióxido de Carbono/metabolismo , DNA Bacteriano/isolamento & purificação , Glucose/metabolismo , Microbiologia Industrial , Modelos Teóricos , Análise de Sequência de DNA , Thermoanaerobacterium/metabolismo
16.
Bioresour Technol ; 209: 80-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26954308

RESUMO

A series of standardized activity experiments were performed to characterize three different temperature ranges of hydrogen fermentation from different carbon sources. 16S rRNA sequences analysis showed that the bacteria were close to Enterobacter genus in the mesophilic mixed culture (MMC) and Thermoanaerobacterium genus in the thermophilic and hyper-thermophilic mixed cultures (TMC and HMC). The MMC was able to utilize the glucose and cellulose to produce methane gas within a temperature range between 25 and 45 °C and hydrogen gas from 35 to 60°C. While, the TMC and HMC produced only hydrogen gas at all temperature ranges and the highest activity of 521.4mlH2/gVSSd was obtained by TMC. The thermodynamic analysis showed that more energy is consumed by hydrogen production from cellulose than from glucose. The experimental results could help to improve the economic feasibility of cellulosic biomass energy using three-phase technology to produce hythane.


Assuntos
Biotecnologia/métodos , Celulose/metabolismo , Hidrogênio/metabolismo , Consórcios Microbianos/fisiologia , Biocombustíveis , Carbono/metabolismo , Enterobacter/genética , Enterobacter/metabolismo , Fermentação , Glucose/metabolismo , Metano/biossíntese , Consórcios Microbianos/genética , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Temperatura , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
17.
Bioresour Technol ; 198: 198-206, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26386423

RESUMO

Anaerobic digestion by two-stage process, containing hydrogen-producing (acidogenic) first stage and methanogenic second stage, has been proposed to degrade substrates which are difficult to be treated by single stage anaerobic digestion process. This research was aimed to evaluate the bio-hydrogen and the bio-methane potentials (BHP and BMP) of skim latex serum (SLS) by using sequential batch hydrogen and methane cultivations at thermophilic conditions (55°C) and with initial SLS concentrations of 37.5-75.0% (v/v). The maximal 1.57 L H2/L SLS for BHP and 12.2L CH4/L SLS for BMP were both achieved with 60% (v/v) SLS. The dominant hydrogen-producing bacteria in the H2 batch reactor were Thermoanaerobacterium sp. and Clostrdium sp. Meanwhile, the CH4 batch reactor was dominated by the methanogens Methanosarcina mazei and Methanothermobacter defluvii. The results demonstrate that SLS can be degraded by conversion to form hydrogen and methane, waste treatment and bioenergy production are thus combined.


Assuntos
Anaerobiose/fisiologia , Hidrogênio/metabolismo , Látex/metabolismo , Metano/biossíntese , Clostridium/metabolismo , Euryarchaeota/metabolismo , Thermoanaerobacterium/metabolismo
18.
Bioresour Technol ; 198: 47-54, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26363501

RESUMO

In this study, an advanced biorefinery technology that uses mixed bakery waste has been developed to produce l-lactic acid using an adaptively evolved Thermoanaerobacterium aotearoense LA1002-G40 in a non-sterilized system. Under these conditions, mixed bakery waste was directly hydrolysed by Aspergillus awamori and Aspergillus oryzae, resulting in a nutrient-rich hydrolysate containing 83.6g/L glucose, 9.5 g/L fructose and 612 mg/L free amino nitrogen. T. aotearoense LA1002-G40 was evaluated and then adaptively evolved to grow in this nutrient-rich hydrolysate. Using a 5-L fermenter, the overall lactic acid production from mixed bakery waste was 0.18 g/g with a titer, productivity and yield of 78.5 g/L, 1.63 g/L/h and 0.85 g/g, respectively. This is an innovative procedure involving a complete bioconversion process for l-lactic acid produced from mixed bakery waste under non-sterilized conditions. The proposed process could be potentially applied to turn food waste into l-lactic acid in an economically feasible way.


Assuntos
Fermentação , Hidrólise , Ácido Láctico/biossíntese , Thermoanaerobacterium/metabolismo , Resíduos , Aspergillus , Bactérias Aeróbias , Reatores Biológicos , Alimentos , Glucose , Nitrogênio
19.
J Bacteriol ; 197(18): 2920-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26124241

RESUMO

UNLABELLED: NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+). The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity. IMPORTANCE: Thermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)(+). The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+) and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes.


Assuntos
Proteínas de Bactérias/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/genética , Metabolismo dos Carboidratos , Transporte de Elétrons , Etanol/metabolismo , Ferredoxinas/metabolismo , NAD , NADP , Oxirredução , Thermoanaerobacterium/genética
20.
PLoS One ; 10(7): e0133183, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26196387

RESUMO

Caldicellulosiruptor saccharolyticus has proven itself to be an excellent candidate for biological hydrogen (H2) production, but still it has major drawbacks like sensitivity to high osmotic pressure and low volumetric H2 productivity, which should be considered before it can be used industrially. A whole genome re-annotation work has been carried out as an attempt to update the incomplete genome information that causes gap in the knowledge especially in the area of metabolic engineering, to improve the H2 producing capabilities of C. saccharolyticus. Whole genome re-annotation was performed through manual means for 2,682 Coding Sequences (CDSs). Bioinformatics tools based on sequence similarity, motif search, phylogenetic analysis and fold recognition were employed for re-annotation. Our methodology could successfully add functions for 409 hypothetical proteins (HPs), 46 proteins previously annotated as putative and assigned more accurate functions for the known protein sequences. Homology based gene annotation has been used as a standard method for assigning function to novel proteins, but over the past few years many non-homology based methods such as genomic context approaches for protein function prediction have been developed. Using non-homology based functional prediction methods, we were able to assign cellular processes or physical complexes for 249 hypothetical sequences. Our re-annotation pipeline highlights the addition of 231 new CDSs generated from MicroScope Platform, to the original genome with functional prediction for 49 of them. The re-annotation of HPs and new CDSs is stored in the relational database that is available on the MicroScope web-based platform. In parallel, a comparative genome analyses were performed among the members of genus Caldicellulosiruptor to understand the function and evolutionary processes. Further, with results from integrated re-annotation studies (homology and genomic context approach), we strongly suggest that Csac_0437 and Csac_0424 encode for glycoside hydrolases (GH) and are proposed to be involved in the decomposition of recalcitrant plant polysaccharides. Similarly, HPs: Csac_0732, Csac_1862, Csac_1294 and Csac_0668 are suggested to play a significant role in biohydrogen production. Function prediction of these HPs by using our integrated approach will considerably enhance the interpretation of large-scale experiments targeting this industrially important organism.


Assuntos
Proteínas de Bactérias/genética , Biomassa , Genoma Bacteriano , Hidrogênio/metabolismo , Thermoanaerobacterium/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Evolução Molecular , Anotação de Sequência Molecular , Dados de Sequência Molecular , Filogenia , Thermoanaerobacterium/crescimento & desenvolvimento , Thermoanaerobacterium/metabolismo
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