dhaT Gene Research Articles

Optimization of polymerase chain reaction‐amplified conditions using the uniform design method

AbstractPolymerase chain reaction (PCR) is a key procedure in modern molecular biology but may be adversely affected by many factors and it is difficult to optimize reaction conditions; a technically simple and effective method to set up an optimal PCR‐amplified system is required. Uniform design (UD) is a method that enables many factors to be investigated. For example, the dhaT gene from Klebsiella pneumoniae was amplified and the optimization of the amplified parameters including concentration of Mg2+, annealing temperature, annealing time and number of cycles based on UD was reported. After only 12 experimental trials, an optimal PCR‐amplified result was obtained, demonstrating the value of UD for optimization of PCR. Copyright © 2004 Society of Chemical Industry

Cloning and sequence analysis of the dhaT gene of the 1,3-propanediol regulon from Klebsiella pneumoniae.

1,3-Propanediol oxidoreductase encoded by dhaT gene, a gene of 1,3-propanediol regulon, is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. DhaT gene was amplified from the genome of K. pneumoniae, sequenced and its amino acid sequence deduced. A predicted secondary structure and 3D-structural model was constructed by homology modelling. Based on these results, we infer that 1,3-propanediol oxidoreductase belongs to NAD(P)-dependent alcohol dehydrogenase group III of iron-activated dehydrogenases.

Cargill and Codexis to collaborate on bio-processes

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD+-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B12, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD+ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD+ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD+ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.

Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii.

The coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E. coli strain producing both His6-tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably alpha2beta2. The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. The DhaF-DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF-DhaG complex of C. freundii efficiently cross-activated the enzyme-cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.

Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase

When grown on glycerol as sole carbon and energy source, cell extracts of Clostridium pasteurianum exhibited activities of glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1,3-propanediol dehydrogenase. The genes encoding the latter two enzymes were cloned by colony hybridization using the dhaT gene of Citrobacter freundii as a heterologous DNA probe and expressed in Escherichia coli. The native molecular mass of 1,3-propanediol dehydrogenase (DhaT) is 440 000 Da. The dhaT gene of C. pasteurianum was subcloned and its nucleotide sequence (1158 bp) was determined. The deduced gene product (41 776 Da) revealed high similarity to DhaT of C. freundii (80.5% identity; 89.8% similarity).

Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli

1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.