Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 156
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Appl Microbiol Biotechnol ; 105(14-15): 5739-5749, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34283269

RESUMO

Recently, thermophilic Thermoanaerobacterium species have attracted increasing attentions in consolidated bioprocessing (CBP), which can directly utilize lignocellulosic materials for biofuels production. Compared to the mesophilic process, thermophilic process shows greater prospects in CBP due to its relatively highly efficiency of lignocellulose degradation. In addition, thermophilic conditions can avoid microbial contamination, reduce the cooling costs, and further facilitate the downstream product recovery. However, only few reviews specifically focused on the microbial applications of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery. Accordingly, this review will comprehensively summarize the recent advances of Thermoanaerobacterium species in lignocellulosic biorefinery, including their secreted xylanases and bioenergy production. Furthermore, the co-culture can significantly reduce the metabolic burden and achieve the more complex work, which will be discussed as the further perspectives. KEY POINTS: • Thermoanaerobacterium species, promising chassis for lignocellulosic biorefinery. • Potential capability of hemicellulose degradation for Thermoanaerobacterium species. • Efficient bioenergy production by Thermoanaerobacterium species through metabolic engineering.


Assuntos
Thermoanaerobacterium , Biocombustíveis , Lignina , Engenharia Metabólica , Thermoanaerobacterium/genética
2.
Arch Biochem Biophys ; 706: 108924, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34019851

RESUMO

Glycosynthases are glycoside hydrolase mutants that can synthesize oligosaccharides or glycosides from an inverted donor without hydrolysis of the products. Although glycosynthases have been characterized from a variety of glycoside hydrolase (GH) families, family GH116 glycosynthases have yet to be reported. We produced the Thermoanaerobacterium xylanolyticum TxGH116 nucleophile mutants E441D, E441G, E441Q and E441S and compared their glycosynthase activities to the previously generated E441A mutant. The TxGH116 E441G and E441S mutants exhibited highest glycosynthase activity to transfer glucose from α-fluoroglucoside (α-GlcF) to cellobiose acceptor, while E441D had low but significant activity as well. The E441G, E441S and E441A variants showed broad specificity for α-glycosyl fluoride donors and p-nitrophenyl glycoside acceptors. The structure of the TxGH116 E441A mutant with α-GlcF provided the donor substrate complex, while soaking of the TxGH116 E441G mutant with α-GlcF resulted in cellooligosaccharides extending from the +1 subsite out of the active site, with glycerol in the -1 subsite. Soaking of E441A or E441G with cellobiose or cellotriose gave similar acceptor substrate complexes with the nonreducing glucosyl residue in the +1 subsite. Combining structures with the ligands from the TxGH116 E441A with α-GlcF crystals with that of E441A or E441G with cellobiose provides a plausible structure of the catalytic ternary complex, which places the nonreducing glucosyl residue O4 2.5 Å from the anomeric carbon of α-GlcF, thereby explaining its apparent preference for production of ß-1,4-linked oligosaccharides. This functional and structural characterization provides the background for development of GH116 glycosynthases for synthesis of oligosaccharides and glycosides of interest.


Assuntos
Glicosídeo Hidrolases/metabolismo , Glicosídeos/biossíntese , Ligases/metabolismo , Oligossacarídeos/biossíntese , Thermoanaerobacterium/enzimologia , Substituição de Aminoácidos , Domínio Catalítico , Celobiose/química , Celobiose/metabolismo , Cristalografia por Raios X , Glucose/química , Glucose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeos/química , Ligases/química , Modelos Moleculares , Mutação , Nitrofenóis/química , Nitrofenóis/metabolismo , Oligossacarídeos/química , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Thermoanaerobacterium/química , Termodinâmica
3.
Bioresour Technol ; 321: 124406, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33272823

RESUMO

The current study aimed to develop an anaerobic wastewater treatment and reuse module enabled by thermophilic bioprocessing, a microbial fuel cell (MFC) and ultrafiltration (UF) treatment. A previously unexplored consortium based on Thermoanaerobacterium thermosaccharolyticum and Arcobacter sp. was used to remove ~73% of chemical oxygen demand (COD) from wastewater under anaerobic conditions (CODi = 200 mg/L). The subsequent MFC and UF treatment removed the COD remnants to meet the secondary treatment standards and reuse criteria. The energy efficiency of polyethersulfone UF membranes was improved by modifying their surfaces with coatings based on self-polymerized dopamine, mixtures of dopamine and poly(2-dimethylamino) ethyl methacrylate methyl, and dopamine analog norepinephrine. The resulting hydrophilic, anti-fouling layers were found to reduce interactions between rejected species and the membrane surface. Finally, this study presents a comparative treatment performance and energy efficiency of the wastewater treatment and reuse modules arranged in six different configurations.


Assuntos
Ultrafiltração , Purificação da Água , Anaerobiose , Membranas Artificiais , Thermoanaerobacterium , Águas Residuárias
4.
RNA ; 27(2): 133-150, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33184227

RESUMO

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.


Assuntos
Genoma Arqueal , Genoma Bacteriano , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Alteromonadaceae/classificação , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Pareamento de Bases , Sequência de Bases , Clostridiales/classificação , Clostridiales/genética , Clostridiales/metabolismo , Firmicutes/classificação , Firmicutes/genética , Firmicutes/metabolismo , Halobacteriales/classificação , Halobacteriales/genética , Halobacteriales/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 5S/química , RNA Ribossômico 5S/metabolismo , Thermoanaerobacterium/classificação , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
5.
Biotechnol Bioeng ; 117(10): 2985-2995, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32946127

RESUMO

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.


Assuntos
Biomassa , Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Lignina/metabolismo , Thermoanaerobacterium/metabolismo , Bioengenharia , Biotecnologia , Consórcios Microbianos , Xilanos/metabolismo
6.
Appl Environ Microbiol ; 86(23)2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32978139

RESUMO

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids.IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.


Assuntos
Clostridium thermocellum/metabolismo , Difosfatos/metabolismo , Nitrogênio/metabolismo , Thermoanaerobacterium/metabolismo , Reatores Biológicos , Análise do Fluxo Metabólico
7.
Methods Mol Biol ; 2096: 21-43, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32720144

RESUMO

In this work, we describe genetic tools and techniques for engineering Thermoanaerobacterium saccharolyticum. In particular, the T. saccharolyticum transformation protocol and the methods for selecting for transformants are described. Methods for determining strain phenotypes are also presented.


Assuntos
Engenharia Metabólica/métodos , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Ensaios Enzimáticos , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Ribossômico 16S/genética , Thermoanaerobacterium/genética , Transformação Genética
8.
PLoS One ; 15(7): e0236518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702033

RESUMO

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.


Assuntos
Celulose/metabolismo , Polissacarídeos/metabolismo , Microbiologia do Solo , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Biomassa , Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Filogenia , Especificidade por Substrato , Thermoanaerobacterium/classificação , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/isolamento & purificação
9.
Bioresour Technol ; 310: 123435, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32361198

RESUMO

Although Thermoanaerobacterium aotearoense SCUT27 (SCUT27) could co-utilize glucose and xylose, the presence of glucose still repressed xylose catabolism. Arginine repressors (ArgRs) were involved in several key metabolic pathways and might be the global regulator. In SCUT27, three genes (V518_0585; V518_1870; V518_1864) were annotated as argR and only the deficiency of argR1864 could greatly improve the co-utilization of glucose and xylose, due to the enhanced activity of xylose isomerase, xylulokinase and the higher energy level. The metabolic flux of SCUT27/ΔargR1864 indicated that new carbon distribution had been re-established and the ethanol yield had increased by 82.95%, strains growth and acetate yield improved by ~35.91% without detectable lactate for the poor activity of lactate dehydrogenase. The improved concentration of ATP and NAD(H) in SCUT27/ΔargR1864 provided more energy to respond the stress, which enabled the mutant the better cell viability to utilize lignocellulosic hydrolysates for enhanced ethanol formation.


Assuntos
Thermoanaerobacterium , Etanol , Fermentação , Lignina , Xilose
10.
Appl Microbiol Biotechnol ; 104(12): 5605-5617, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32248440

RESUMO

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD+ concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol production.


Assuntos
Etanol/metabolismo , Regulação Bacteriana da Expressão Gênica , Thermoanaerobacterium/genética , Fatores de Transcrição/genética , Ácido Acético/metabolismo , Álcool Desidrogenase/metabolismo , Fermentação , Deleção de Genes , Ácido Láctico/metabolismo , Oxirredução , Thermoanaerobacterium/metabolismo , Fatores de Transcrição/metabolismo , Xilose/metabolismo
11.
Nat Commun ; 11(1): 1937, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321909

RESUMO

The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp. strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum. Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.


Assuntos
Clostridiales/metabolismo , Técnicas de Cocultura/métodos , Etanol/metabolismo , Microbiologia Industrial/métodos , Thermoanaerobacterium/metabolismo , Zea mays/microbiologia , Celulose/metabolismo , Clostridiales/genética , Fermentação , Temperatura Alta , Thermoanaerobacterium/genética , Xilanos/metabolismo , Zea mays/metabolismo
12.
Bioresour Technol ; 310: 123426, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32344241

RESUMO

In this work, accelerated start-up of biological hydrogen production system fed with glucose and molasses at 55 °C by regulating pH and COD concentration was investigated in two groups. Then three reactors in each group were compared: controlling pH, controlling pH with COD, and controlling the COD. The reactors in group A presented best hydrogen yield of 1.84 mol H2/mol glucose·day and worked stably at the 8th day. The highest hydrogen yield in group B was 2.13 mol H2/mol molasses·day and steadily at the 11th day. It proved that controlling two key parameters of the inflow pH (8.0) and substrate concentration (4000 mg COD/L) could realize fast start-up of hydrogen production reactor. This study demonstrated that Thermoanaerobacterium sp. strain RBIITD could produce hydrogen and provide a new avenue for biological hydrogen production by dark fermentation using cheap substrate towards a more sustainable and feasible technology.


Assuntos
Thermoanaerobacterium , Reatores Biológicos , Fermentação , Hidrogênio , Concentração de Íons de Hidrogênio , Melaço
13.
Protein Expr Purif ; 169: 105571, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31978533

RESUMO

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.


Assuntos
Glucosiltransferases/biossíntese , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Engenharia Genética/métodos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Chaperonas Moleculares/metabolismo , Plasmídeos , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
14.
J Agric Food Chem ; 68(3): 818-825, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31845578

RESUMO

ß-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 ß-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for ß-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 °C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 °C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Thermoanaerobacterium/enzimologia , beta-Manosidase/química , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , Thermoanaerobacterium/química , Thermoanaerobacterium/genética , beta-Manosidase/genética , beta-Manosidase/metabolismo
15.
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
16.
J Ind Microbiol Biotechnol ; 46(5): 687-695, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30783893

RESUMO

The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of plant lignocellulosic biomass to biofuels and bioproducts. To investigate the synergy of enzymes involved and to further improve the ability of C. bescii to degrade cellulose, we introduced CAZymes that act synergistically with the C. bescii exoproteome in vivo and in vitro. We recently demonstrated that the Acidothermus cellulolyticus E1 endo-1,4-ß-D-glucanase (GH5) with a family 2 carbohydrate-binding module (CBM) increased the activity of C. bescii exoproteome on biomass, presumably acting in concert with CelA. The ß-glucanase, GuxA, from A. cellulolyticus is a multi-domain enzyme with strong processive exoglucanase activity, and the cellobiose phosphorylase from Thermotoga maritima catalyzes cellulose degradation acting synergistically with cellobiohydrolases and endoglucanases. We identified new chromosomal insertion sites to co-express these enzymes and the resulting strain showed a significant increase in the enzymatic activity of the exoproteome.


Assuntos
Celulose/química , Glucosiltransferases/biossíntese , Glicosídeo Hidrolases/biossíntese , Thermoanaerobacterium/enzimologia , beta-Glucanas/química , Actinomycetales/metabolismo , Biomassa , Celobiose , Celulase/metabolismo , Clostridiales/metabolismo , Engenharia Genética , Técnicas Genéticas , Hidrólise , Microbiologia Industrial , Plantas/microbiologia , Proteoma , Proteômica , Açúcares/química
17.
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
18.
Biotechnol Adv ; 36(8): 2077-2100, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30266344

RESUMO

Novel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.


Assuntos
Proteínas de Bactérias , Hidrolases , Thermoanaerobacterium/enzimologia , DNA Arqueal/genética , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Temperatura Alta , Metagenoma/genética , Metagenômica , Thermoanaerobacterium/genética
19.
J Biol Inorg Chem ; 23(5): 809-817, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29882029

RESUMO

Carboxydothermus hydrogenoformans is a model microorganism for the study of [NiFe]-CODH, a key enzyme of carbon cycle in anaerobic microorganisms. The enzyme possesses a unique active site (C-cluster), constituted of a distorted [NiFe3S4] cubane linked to a mononuclear Fe(II) center. Both the biogenesis of the C-cluster and the activation of CODH by nickel insertion remain unclear. Among the three accessory proteins thought to play a role in this latter step (CooC, CooJ, and CooT), CooT is identified as a nickel chaperone involved in CODH maturation in Rhodospirillum rubrum. Here, we structurally and biophysically characterized a putative CooT protein present in C. hydrogenoformans (pChCooT). Despite the low sequence homologies between CooT from R. rubrum (RrCooT) and pChCooT (19% sequence identity), the two proteins share several similarities, such as their overall structure and a solvent-exposed Ni(II)-binding site at the dimer interface. Moreover, the X-ray structure of pChCooT reveals the proximity between the histidine 55, a potential nickel-coordinating residue, and the cysteine 2, a highly conserved key residue in Ni(II)-binding.


Assuntos
Proteínas de Bactérias/química , Chaperonas Moleculares/química , Níquel/química , Thermoanaerobacterium/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Fenômenos Biofísicos , Cristalografia por Raios X , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Mutagênese Sítio-Dirigida , Conformação Proteica , Homologia de Sequência de Aminoácidos
20.
Bioresour Technol ; 263: 120-127, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29738974

RESUMO

The feasibility and performance of hydrogen production from paper sludge without inoculation was investigated under thermophilic conditions. The maximum hydrogen production reached 64.32 mM with 7.4% PS. The dynamic changes in bacterial community structures during hydrogen production were investigated by analyzing 16S rDNA gene sequences using high throughput sequencing technology. The results showed that microbial community was dominated by order Clostridiales and Thermoanaerobacterales. Genus Thermoanaerobacterium and Ruminiclostridium played a leading role in the fermentation process, which was responsible for the hydrolysis of PS and hydrogen production. Effect of inoculation with Clostridium thermocellum on hydrogen production from PS was also studied. The results showed that C. thermocellum supplement significantly increased hydrogen yield and holocellulose degradation rate by 96.80% and 32.95%, respectively. In addition, inoculation of C. thermocellum enhanced VFA generation and shortened the lag phase of hydrogen production. The present study lays the foundation on the valorization of waste lignocellulose.


Assuntos
Clostridium thermocellum , Hidrogênio , Celulose , Fermentação , Lignina , Esgotos , Thermoanaerobacterium
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...