Genome Sequence of Klebsiella oxytoca M5al, a Promising Strain for Nitrogen Fixation and Chemical Production.

Klebsiella oxytoca is an important microorganism for nitrogen fixation and chemical production. Here, we report an annotated draft genome of K. oxytoca strain M5al that contains 5,256 protein-coding genes and 95 structural RNAs, which provides a genetic basis for a better understanding of the physiology of this species.

Characterization of glycerol dehydratase expressed by fusing its alpha- and beta-subunits.

The gdh and gdhr genes, encoding B(12)-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the beta-subunit was lost during GDH purification when co-expressing alpha, beta and gamma subunit.

Recombinant glutamine synthetase (GS) from C. glutamicum existed as both hexamers & dedocamers and C-terminal His-tag enhanced inclusion bodies formation in E. coli.

In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.

Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol.

Redox cofactors play crucial roles in the metabolic and regulatory network of living organisms. We reported here the effect of introducing a heterogeneous NADH regeneration system into Klebsiella oxytoca on cell growth and glycerol metabolism. Expression of fdh gene from Candida boidinii in K.

Novel redox potential-based screening strategy for rapid isolation of Klebsiella pneumoniae mutants with enhanced 1,3-propanediol-producing capability.

This report describes a novel redox potential (oxidoreduction potential [ORP])-based screening strategy for the isolation of mutants of Klebsiella pneumoniae which have an increased ability to produce 1,3-propanediol (1,3-PD). This method can be described as follows: first, to determine an ORP range which is preferred for the wild-type strain to grow and to produce 1,3-PD; second, to subject a chemically mutagenized culture to a reduced ORP level which is deleterious for the wild-type strain. Colonies that showed high specific growth rates at deleterious ORP levels were selected, and their abilities to produce 1,3-PD were investigated.

[Expression and characterization of formate dehydrogenase gene in Klebisella pneumoniae].

Glycerol can be converted to 1,3-propanediol by the anaerobic fermentation of Klebsiella pneumoniae, during which reducing equivalent NADH was consumed. Therefore, the availability of NADH would be critical for the yield of 1, 3-propanediol. In this paper, formate/formate dehydrogenase system was used for the regeneration of in vivo NADH and the improvement of 1, 3-propanediol production.

Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae.

Production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is restrained by ethanol formation. The first step in the formation of ethanol from acetyl-CoA is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme that competes with 1,3-PD oxidoreductase for the cofactor NADH. This study aimed to improve the production of 1,3-PD by engineering the ethanol formation pathway.

Intracellular pH and metabolic activity of long-chain dicarylic acid-producing yeast Candida tropicalis.

The intracellular pH (pH(i) of alpha-omega-dicarylic acid producing Candida tropicalis was determined by a fluorescence technique using the pH-sensitive fluorescent probe 5(6)-carboxyfluorescein diacetate. Fermentations with n-tridecane substrate to produce alpha,omega-tridecanedioic acid were carried out in a 5-l bioreactor in which growth and production were separated. During the growth phase, the measured pH(i) values were varied from 5.

[Measurement of intracellular pH in long-chain dicarboxylic acid-producing yeast Candida tropicalis and its growth activity].

Intracellular pH (pHi) has an important influence on the metabolic activity of cells or cellular processes. The intracellular pH (pHi) of long-chain alpha,omega-dicarboxylic acid-producing Candida tropicalis was determined by fluorescence technique using a pH-sensitive fluorescent probe 5(6)-carboxyfluorescein diacetate. Optimal loading conditions of the fluorescent probe into the cells were experimentally determined.

[Effects of carnitine acetyltransferase gene knockout on long chain dicarboxylic acid production and metabolism of Candida tropicalis].

Candida tropicalis can assimilate n-alkane as a sole carbon source and produce dicarboxylic acids (DCAs). The synthesis of DCAs is thought to be reduced by beta-oxidation. Carnitine acetyltransferase (CAT) is the major enzyme to transfer DCAs into beta-oxidative pathway, then DCAs would be catalyzed to generate ATP to supply cells with energy.

[Effects of H2O2 addition on cell growth and product formation in long-chain dicarboxylic acid fermentation].

When Candida tropicalis was cultivated in shakig-flask with the H2O2 addition, DCA (Dicarboxylic Acid, DCA) concentration was increased, especially at 2 mmol/L H2O2 concentration. The cytochrome P450 activity assays indicated that H2O2 addition significantly increased the activities of cytochrome P450 and DCA production positively correlated with the activities of cytochrome P450. The study on the cell growth demonstrated that the H2O2 addition inhibited the cell growth rate.

Isolation and enzyme determination of Candida tropicalis mutants for DCA production.

Techniques, named two-step enrichment and double-time replica-plating method (TEDR), are described that allow a mutated population of Candida tropicalis to be enriched efficiently for mutants deficient in the alkane degradation pathway (Alk(-)) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutated with ethyl methane sulphonate and ultraviolet, the Alk(-) mutants were enriched (the first step enrichment, up to eightfold in one round of enrichment) by treatment with nystatin in medium SEL1-1.