• Korean Journal of Microbiology
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• v.29 no.4
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• pp.203-208
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• 1991

The dapA-complementing gene (L-2, 3-dihydrodipicolinate synthetase: DHDP synthetase, dapA) has been cloned by using a cosmid genomic bank of Corynebacterium glutamicum JS231 that is a lysine overproducer, AEC (s-(2-aminoethyl)-L-cysteine) resistant mutant. By enzymatic deletion analysis, the DNA region complementing the escherichia coli dapA host could be confined to 4.5kb SalI-generated DNA fragment. This DNA fragment was inserted into the C. glutamicum/E. coli shuttle vector pECCG117 to construct pDHDP5812. The specific activity of DHDP synthetase detected in C. glutamicum JS231/pDHDP5812 was increased about 10 fold above that of C. glutamicum JS231. The addition of leucine during growth did not repress the expressin of dapA, and the enzyme activity was not inhibited by lysine.

• Korean Journal of Microbiology
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• v.29 no.3
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• pp.149-154
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• 1991

Escherichia coli/Corynebacterium glutamicum shuttle vectors, pECCG1 and pECCG2 were constructed by joining a 3.00 kb cryptic plasmid pCB 1 from C. glutamicum and a 3.94 kb plasmid pACYC 177 from E. coli. By trimming unessential parts and introducing mulitiple cloning site into the plasmid pECCG 1, a plasmid pECCG122(5.1kb) was constructed. All the shuttle vectors were stably maintained in C. glutamicum up to about 40 generations irrespective of kanamycin addition in the medium. Threonine operon (homoserine dehydrogenase/homoserine kinase) and dapA gene (dihydrodipicolinate synthetase) of C. glutamicum were cloned into the plasmid pECCG122, and the resultant plasmids were designated pTN31 and pDHDP19, respectively. They were used to study the effect of cloned foreign gene on the stability of the plasmid pECCG122. Plasmids pTN31 and pDHDP19 were segregated rapidly from C. glutamicum when cultured in the medium without kanamycin. In medium with $50\mu${\g/ml} of kanamycin, their segregation rates were much slower than those in medium without kanamycin, but the danamycin addition didn't guarantee the complete maintenance of the plasmids in C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.999-1009
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• 2006

Physiological, biochemical and genetic studies of Corynebacterium glutamicum, a workhorse of white biotechnology used for amino acid production, led to a waste knowledge mainly about amino acid biosynthetic pathways and the central carbon metabolism of this bacterium. Spurred by the availability of the genome sequence and of genome-based experimental methods such as DNA microarray analysis, research on genetic regulation came into focus. Recent progress on mechanisms of genetic regulation of the carbon, nitrogen, sulfur and phosphorus metabolism in C. glutamicum will be discussed.

• Journal of Microbiology and Biotechnology
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• v.7 no.5
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• pp.287-292
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• 1997

The role of glyoxylate bypass in lysine production by Corynebacterium glutamicum ssp. lactofermentum ATCC21799 was analyzed by using cloned aceA and aceB genes which encode enzymes catalyzing the bypass. Introduction of a plasmid carrying aceA and aceB to the strain increased enzyme activities of the bypass to approximately 5 fold on acetate minimal medium. The strain with amplified glyoxylate bypass excreted 25% more lysine to the growth medium than the parental strain, apparently due to the increased availability of intracellular oxaloacetate. The final cell yield was lower in the strain with amplified glyoxylate bypass. These changes were specific to the lysine-producing C. glutamicum ssp. lactofermentum ATCC21799, since the lysine-nonproducing wild type Corynebacterium glutamicum strain grew faster and achieved higher cell yield when the glyoxylate bypass was amplified. These findings suggest that the lysine producing C. glutamicum ssp. lactofermentum ATCC21799 has the ability to efficiently channel oxaloacetate, the TCA cycle intermediate, to the lysine biosynthesis pathway whereas lysine-nonproducing strains do not. Our results show that amplification of the glyoxylate bypass efficiently increases the intracellular oxaloacetate in lysine producing Corynebacterium species and thus results in increased lysine production.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.128-134
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• 2008

In order to examine efficient L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS) synthesis process using whole cell biocatalyst, a thermostable L-threonine aldolase (L-TA), which cloned from Streptomyces coelicolor A3(2) and improved for stability, was expressed in a Corynebacterium glutamicum R strain. The constructed Corynebacterium expression vector, pCG-H44(1) successfully expressed L-TA in C. glutamicum R strain, but showed very low expression level. In order to improve the expression level, the expression vector named pCG-H44(2) was reconstructed by eliminating 1 nucleotide between SD sequence and start codon of L-TA. The pCG-H44(2) vector plasmid was able to overexpress L-TA approximately 3.2 times higher than pCG-H44(1) in C. glutamicum R strain (CGH-2). When the whole cell of CGH-2 was examined in a repeated batch system, L-threo-DOPS was successfully synthesized with a yield of 4.0 mg/ml and maintain synthesis rate constantly after 30 repeated batch reactions for 130 h.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.279-283
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• 1985

As a method of breeding L-lysine producing strains, the intergeneric protoplast fusion between Brevibacterium flavum and Corynebacterium glutamicum was performed. As a results, Brevibacterium flavum ATCC 21528 R showed 99％ of protoplast formation and 10％ of regeneration frequencies when treated with 400$\mu\textrm{g}$/$m\ell$ of lysozyme for 12hrs. In Corynebacterium glutamicum ATCC 21514 S, 99％ and 12％ were obtained by treatment of 300$\mu\textrm{g}$/$m\ell$ lysozyme for 12 hrs. In intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528 R and Corynebacterium glutamicum ATCC 21831 S, 1.0$\times$10$^{-6}$ of recombinant frequency per regenerable cells was observed by use of PEG 6000, 30％(w/v). Among the strains obtained KR$_{43}$ strain showed 12％ higher productivity of L-lysine than the parental cell. Then, the activity of aspartokinase of KR$_{43}$ was about 13％ higher than the parental cell.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.807-822
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• 2016

Starting as a glutamate producer, Corynebacterium glutamicum has played a variety of roles in the industrial production of amino acids, one of the most important areas of white biotechnology. From shortly after its genome information became available, C. glutamicum has been applied in various production processes for value-added chemicals, fuels, and polymers, as a key organism in industrial biotechnology alongside the surprising progress in systems biology and metabolic engineering. In addition, recent studies have suggested another potential for C. glutamicum as a synthetic biology platform chassis that could move the new era of industrial microbial biotechnology beyond the classical field. Here, we review the recent progress and perspectives in relation to C. glutamicum, which demonstrate it as one of the most promising and valuable workhorses in the field of industrial biotechnology.

• 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.25 no.4
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• pp.452-458
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• 2015

The biocatalytic efficiency of recombinant Corynebacterium glutamicum ATCC 13032 expressing the secondary alcohol dehydrogenase of Micrococcus luteus NCTC2665 was studied. Recombinant C. glutamicum converts ricinoleic acid to a product, identified by gas chromatography/mass spectrometry as 12-ketooleic acid (12-oxo-cis-9-octadecenoic acid). The effects of pH, reaction temperature, and non-ionic detergent on recombinant C. glutamiucm whole cell bioconversion were examined. The determined optimal conditions for production of 12-ketooleic acid are pH 8.0, 35℃, and 0.05 g/l Tween80. Under these conditions, recombinant C. glutamicum produces 3.3 mM 12-ketooleic acid, with a 72% (mol/mol) maximum conversion yield, and 1.1 g/l/h volumetric productivity in 2 h; and 3.9 mM 12-ketooleic acid, with a 74% (mol/mol) maximum conversion yield, and 0.69 g/l/h maximum volumetric productivity in 4 h of fermentation. This study constitutes the first report of significant production of 12-ketooleic acid using a recombinant Corynebacterium glutamicum-based biocatalyst.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.45-49
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• 1998

Two kinds of Mutants of Corynebacterium glutamicum, which were resistant to branched chain amino acid analogues, were obtained for L-leucine production; C. glutamicum LT26 resistant to 4-azaleucine and $\alpha$-amino-$eta$-hydroxyvaleric acid, and from which C. glutamicum LT3811-70 resistant to DL-4-thiaisoleucine were derived. Accumulation of L-leucine in the culture broths of these mutant strains, C. glutamicum LT26 and LT3811-70, were much higher than those of their parent strains even though they were non-auxotrophic mutants. Enzymatic analyses were performed to measure the activities of $\alpha$-acetohydroxy acid synthase (AHAS) and $\alpha$-isopropylmalate synthase (IPMS), which were the key enzymes for the L-isoleucine, L-valine and L-leucine biosynthetic pathways branching from a common precursor. In C. glutamicum LT26 and LT3811-70, AHAS and IPMS were found to be derepressed and desensitized to L-leucine. In addition, in C. glutamicum LT3811-70, IPMS was further more derepressed by L-leucine and AHAS was more desensitized by L-isoleucine and L-valine compared to its parent strain, C. gIEitamicum LT26.