Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Mais filtros

Intervalo de ano de publicação
Appl Microbiol Biotechnol ; 100(3): 1523-1529, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26572521


In typical acetone-butanol-ethanol (ABE) fermentation, acetone is the main by-product (50 % of butanol mass) of butanol production, resulting in a low yield of butanol. It is known that some Clostridium tetanomorphum strains are able to produce butanol without acetone in nature. Here, we described that C. tetanomorphum strain DSM665 can produce 4.16 g/L butanol and 4.98 g/L ethanol at pH 6.0, and 9.81 g/L butanol and 1.01 g/L ethanol when adding 1 mM methyl viologen. Butyrate and acetate could be reassimilated and no acetone was produced. Further analysis indicated that the activity of the acetate/butyrate:acetoacetyl-CoA transferase responsible for acetone production is lost in C. tetanomorphum DSM665. The genome of C. tetanomorphum DSM665 was sequenced and deposited in DDBJ, EMBL, and GenBank under the accession no. APJS00000000. Sequence analysis indicated that there are no typical genes (ctfA/B and adc) that are typically parts of an acetone synthesis pathway in C. tetanomorphum DSM665. This work provides new insights in the mechanism of clostridial butanol production and should prove useful for the design of a high-butanol-producing strain.

1-Butanol/metabolismo , Acetona/metabolismo , Proteínas de Bactérias/genética , Clostridium tetanomorphum/genética , Clostridium tetanomorphum/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium tetanomorphum/crescimento & desenvolvimento , Fermentação , Genoma Bacteriano , Genômica , Dados de Sequência Molecular
Metab Eng ; 30: 190-196, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26070834


Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.

Proteínas de Bactérias/biossíntese , Clostridium tetanomorphum/genética , Escherichia coli , Fumaratos/metabolismo , Transferases Intramoleculares/biossíntese , Maleatos/metabolismo , Proteínas de Bactérias/genética , Clostridium tetanomorphum/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Transferases Intramoleculares/genética
Syst Appl Microbiol ; 37(1): 1-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24331236


A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474(T). GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v). The solventogenesis genes crt, bcd, etfA/B and hbd composing the bcs (butyryl-CoA synthesis) operon of C. tetanomorphum GT6 were sequenced. They occur in a genomic arrangement identical to those in other solventogenic clostridia. Furthermore, the sequence of a potential regulator gene highly similar to that of the NADH-sensing Rex family of regulatory genes was found upstream of the bcs operon. Potential binding sites for Rex have been identified in the promoter region of the bcs operon of solvent producing clostridia as well as upstream of other genes involved in NADH oxidation. This indicates a fundamental role of Rex in the regulation of fermentation products in anaerobic, and especially in solventogenic bacteria.

1-Butanol/metabolismo , Clostridium tetanomorphum/isolamento & purificação , Clostridium tetanomorphum/metabolismo , Glicerol/metabolismo , Redes e Vias Metabólicas , Óperon , Metabolismo dos Carboidratos , Clostridium tetanomorphum/classificação , Clostridium tetanomorphum/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fermentação , Sedimentos Geológicos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Temperatura
Protein Pept Lett ; 17(6): 759-64, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20397969


The coupling of an aspartate residue with an active site histidine plays a pivotal role in enzyme catalysis. The His-Asp pair in glutamate mutase and other B(12)-dependent mutases is not only responsible for coenzyme-binding, but is also involved in fine-tuning the enzymatic activities. Our modeling results show that the His-Asp pair is arranged in a highly organized manner. Except for carboxymethylated Cys or Glu, a less hindered or non-charged amino acid residue is preferred between the conserved histidine and aspartate residue.

Proteínas de Bactérias/química , Clostridium tetanomorphum/enzimologia , Transferases Intramoleculares/química , Vitamina B 12/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Sequência Conservada , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Simulação de Dinâmica Molecular , Vitamina B 12/química
Chembiochem ; 10(13): 2236-45, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19670200


3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site Mg(II) ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the Mg(II) ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and (1)H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of k(cat) and k(cat)/K(M); this implicates Lys331, His194 and Gln329 as mechanistically important. The (1)H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.

Amônia-Liases/química , Amônia-Liases/genética , Amônia-Liases/metabolismo , Domínio Catalítico , Clostridium tetanomorphum/enzimologia , Cinética , Magnésio/química , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/metabolismo , Estereoisomerismo
FEBS J ; 275(23): 5960-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19021770


Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostridium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme. Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (AdoCbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale. Equilibrium dialysis was used to measure the binding of coenzyme B(12) analogs to glutamate mutase. Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio k(cat)/K(m) decreased approximately 10(4)-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor. The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme's active site cannot correctly accommodate the coenzyme analog AdoCbi. The results presented here shed some light on the cobalt-carbon cleavage mechanism of B(12).

Proteínas de Bactérias/química , Clostridium tetanomorphum/enzimologia , Cobamidas/química , Transferases Intramoleculares/química , Proteínas de Bactérias/metabolismo , Catálise , Cobamidas/síntese química , Cobamidas/metabolismo , Diálise , Transferases Intramoleculares/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação Proteica , Espectrofotometria , Espectrofotometria Ultravioleta , Termodinâmica