• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.704-710
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• 2008

Glutamate availability in the argF-argR-proB${\Delta}$ strain of Corynebacterium glutamicum was increased by addition of glutamate to the cell or inactivation of the phosphoenolpyruvate carboxykinase activity and simultaneous overexpression of the pyruvate carboxylase activity to assess its effect on L-ornithine production. When glutamate was increased in an L-ornithine-producing strain, the production of L-ornithine was not changed. This unexpected result indicated that the intracellular concentration and supply of glutamate is not a rate-limiting step for the L-ornithine production in an L-ornithine-producing strain of C. glutamicum. In contrast, overexpression of the L-ornithine biosynthesis genes (argCJBD) resulted in approximately 30% increase of L-ornithine production, from 12.73 to 16.49 mg/g (dry cell weight). These results implied that downstream reactions converting glutamate to L-ornithine, but not the availability of glutamate, is the rate-limiting step for elevating L-ornithine production in the argF-argR-proB${\Delta}$ strain of C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.127-131
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• 2010

In this study, Corynebacterium glutamicum and its derived mutants were used to demonstrate the relationship between proline, glutamate, and ornithine. The maximum ornithine production was shown in the culture medium (3,295.0 mg/l) when the cells were cultured with 20 mM proline, and was 15.5 times higher than in the presence of 1 mM proline. However, glutamate, which is known as an intermediate in the process of converting proline to ornithine, did not have any positive effect on ornithine production. This suggests that the conversion of proline to ornithine through glutamate, is not possible in C. glutamicum. Comparative analysis between the wild-type strain, SJC 8043 ($argF^-$, $argR^-$), and SJC 8064 ($argF^-$, $argR^-$, and $ocd^-$), showed that C glutamicum could regulate ornithine production by ornithine cyclodeaminase (Ocd) under proline-supplemented conditions. Therefore, proline directly caused an increase in the endogenous level of ornithine by Ocd, which would be a primary metabolite in the ornithine biosynthesis pathway.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.552-555
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• 1989

A 150-fold purified preparation of NADPH-specific glutamate dehydrogenase of Corynebacterium glutamicum (1) was used for the determination of kinetic parameters of the substrates, NADPH, NH$_4$Cl, and $\alpha$-ketoglutarate in the direction of glutamate synthesis. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme, Based on the analysis of the result derived from initial velocity, the reaction mechanism was postulated to be ordered addition with NADPH as a first substrate to bind in the forward direction. Of the several metabolites tested for a possible function in the regulation of glutamate dehydrogenase activity, only malate and citrate were appeared to have an appreciable influence on the enzyme, Potassium chloride showed to be the most effective for the enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1361-1369
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• 2023

Corynebacterium glutamicum (C. glutamicum) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from C. glutamicum ATCC 13032 (Cg6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of Cg6PGD, which are crucial for understanding this enzyme. Based on the findings of our research, Cg6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.

• Proceedings of the Polymer Society of Korea Conference
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• 2006.10a
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• pp.352-352
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• 2006

Corynebacterium glutamicum, which is well known as an amino acid fermentation bacterium, has been used as a producer of poly(3-hydroxybutyrate) [P(3HB)]. P(3HB) was synthesized in recombinant C. glutamicum harboring the expression plasmid vector with a strong promoter for cell surface protein gene derived from C. glutamicum and P(3HB) biosynthetic gene operon derived from Ralstonia eutropha. The expression of P(3HB) synthase gene was detected by enzyme activity assay. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5 % (w/w) with molecular weight of $2.1{\times}10^{5}$ and polydispersity of 1.63.

• KSBB Journal
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• v.5 no.3
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• pp.299-306
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• 1990

Escherkchla coli/Cownebacterium glutamicum shuttle vectors, pECCGl and pECCG2 were constructed by joining a 3.0 kb C. glutamicum cryptic plasmid pCBl and a 3.94 kb E. coli plasmid pACYC177. Using the plasmid pECCGl, various parameters involved in electroporation system including electric field strength, resistance, DNA concentration, cell concentration and growth stage were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Transformation efficiencies of 106 transformants/$\mu\textrm{g}$ of plasmid DNA were achieved with Corynebacterium glutamicum.

• KSBB Journal
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• v.5 no.2
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• pp.125-131
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• 1990

Microbial production of L-phenylalanine using Corynebacterium glutamicum ATCC 21674, a tyrosine auxotroph resistant to aromatic amino acid analogues, has been studied and kinetic analysis was performed. Even though the strain was reported as a tyrosine auxotroph, it produced tyrosine and was able to grow on the minimal medium where no tyrosine was present. The average specific growth rate at the exponential growth phase was 0.087 hr-1. There was a dissociation of growth from the formation of the product. Linear correlation between biomass production and total CO2 production was obtained. The relationship between CO2 evolution rate and sugar consumption rate was also found to be linear.

• Journal of Microbiology and Biotechnology
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• v.7 no.6
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• pp.435-437
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• 1997

The role of $Ca^{2+}$ in making functional malate synthase in Corynebacterium glutamicum was investigated using the cloned DNA coding for the enzyme. Introduction of cloned aceB into C. glutamicum overexpressed malate synthase as judged by SDS-PAGE. However, the increase in enzyme activity of the expressed malate synthase did not match the level of overexpression observed in SDS-PAGE. Addition of $Ca^{2+}$ to the growth medium specifically increased the activity. The malate synthase could be stained with ruthenium red in a $Ca^{2+}$-specific manner. This agrees with the previous observation which reported a potential $Ca^{2+}$-binding domain in the N-terminal region of the protein.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.187-194
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• 2007

In order to utilize different nitrogen sources and to survive in a situation of nitrogen limitation, microorganisms have developed sophisticated mechanisms to adapt their metabolism to a changing nitrogen supply. In this communication, the recent knowledge of nitrogen regulation in the amino acid producer Corynebacterium glutamicum is summarized. The core adaptations of C. glutamicum to nitrogen limitation on the level of transcription are controlled by the global regulator AmtR. Further components of the signal pathway are GlnK, a $P_{II}-type$ signal transduction protein, and GlnD. Mechanisms involved in nitrogen control in C. glutamicum regulating gene expression and protein activity are repression of transcription, protein-complex formation, protein modification by adenylylation, change of intracellular localization, and proteolysis.