• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.789-795
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• 2004

A promoter-probe shuttle vector pSK1Cat was constructed for the isolation of transcriptional signal sequences from Corynebacterium glutamicum. Besides conferring resistance to kanamycin in Escherichia coli and C. glutamicum, the vector carried a promoterless cat gene to confer resistance to chloramphenicol upon insertion of the appropriate transcriptional signals in the multiple cloning site. By utilizing the vector, a series of transcriptionally active fragments were isolated from the genome of C. glutamicum. The clones, ranging from 200 bp to 1 kb in size, were grouped into 3 classes of strong, medium, and weak, based on the chloramphenicol acetyltransferase (CAT) activity and sensitivity to the chloramphenicol of the clone-carrying C. glutamicum cells. C. glutamicum cells carrying the $P_{19}$ clone, a representative in the strong class, were able to grow on minimal agar plates containing over $40 mg/mell$ chloramphenicol, and showed CAT activity of 10 m㏖/mgㆍmin, performing slightly better than the cells carrying $P_{tac}$ , a strong E. coli promoter. Subcloning analysis of the $P_{19}$ clone identified a 180 bp intergenic fragment ($P_{180}$), which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by $P_{180}$ was not affected by either the kinds of carbon sources or changes in temperature. These properties make the $P_{180}$ clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.78-86
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• 2019

Recombinant Corynebacterium glutamicum producing N-acetylglucosamine (GlcNAc) was constructed by metabolic engineering. To construct a basal strain producing GlcNAc, the genes nagA, nagB, and nanE encoding N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, and N-acetylmannosamine-6-phosphate epimerase, respectively, were sequentially deleted from C. glutamicum ATCC 13032, yielding strain KG208. In addition, the genes glmS and gna1 encoding glucosamine-6-phosphate synthase and glucosamine-6-phosphate N-acetyltransferase, which originated from C. glutamicum and Saccharomyces cerevisiae, respectively, were expressed in several expression vectors. Among several combinations of glmS and gna1 expression, recombinant cells expressing glmS and gna1 under control of the ilvC promoter produced 1.77 g/l of GlcNAc and 0.63 g/l of glucosamine in flask cultures.

• 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.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.294-301
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• 2001

The effects of including glycine and isonicotinic acid hydrazide (INH) in the growth medium (Luria broth, LBG) on the subsequent lysozyme-imduced protoplast formation and transformation efficiency of Corynebacterium glutamicum were studied. The transformation efficiency of C. glutamicum AS019 increased up to 100-fold as the ocncentrationof glycine in the media increased from 0% to 5% (w/v), relative to cells grown in the absence of glycine. The presence of 5 mg/ml INH in the growth medium led to a further 10-fold increase in transformation efficiency. In addition, this transformation protocol was successfully applied to other strains of C. glutamicum. Both chemicals affected the mycolic acid attachment to the cell surface of C. glutamicum, when INH, the relative percentage of fatty acids of AS019 to the total lipids (mycolic acid plus fatty acids) decreased from 76.9% (in LBG) to 72.9% (in LBG-2% glycine) and 66.4% (in LBG-8 mg InG/ml), thereby suggeting that these chemicals also inhibit fatty acid synthesis.

• 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.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1404-1408
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• 2016

In recent years, foot-and-mouth disease has occurred in all parts of the world. The animals with the disease are buried in the ground; therefore, their concentration could affect ground or groundwater. Moreover, the complete degradation of carcasses is not a certainty, and their disposal is important to prevent humans, livestock, and the environment from being affected with the disease. The treatment of Corynebacterium glutamicum is a feasible method to reduce the risk of carcass decomposition affecting humans or the environment. Therefore, this study aimed to investigate the effect of C. glutamicum on the soil environment with a carcass. The composition of amino acids in the soil treated with C. glutamicum was generally higher than those in the untreated soil. Moreover, the plant root in the soil samples treated with C. glutamicum had 84.0% amino acids relative to the standard value and was similar to that of the control. The results of this study suggest the possibility to reduce the toxicity of a grave land containing animals with this disease.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2005

A transcriptionally active fragment $(P_{19})$ isolated by utilizing the promoter-probe shuttle vector pSK1Cat was analyzed. By subcloning analysis, the 180 bp region $(P_{180})$ responsible for the activity was determined. Transcriptional fusion of the C. glutamicum metX gene to $P_{180}\;(P_{180}-metX)$ resulted in a 24-fold increase in MetX activity in a complex medium, while a 13-fold increase was observed with the $P_{tac}$ promoter. Additionally, the expression conferred by $P_{180}$ was not affected by methionine added to the growth medium, suggesting that the $P_{180}$ clone is useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation. Introduction of $P_{180}-metX$ into a lysine-producing C. glutamicum resulted in the production of methionine to 0.8 g/l.

• 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.

• Korean Journal of Veterinary Service
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• v.40 no.1
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• pp.53-59
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• 2017

Foot and mouth disease (FMD) is highly infectious disease of cloven-hoofed animals, particularly problematic in cattle, sheep, pigs and goats for economic reasons. Last FMD outbreak in February, 2017 caused tremendous social and economical impacts. The Korean FMD policy aims to vaccinate intact animals and euthanize and bury infected animals to prevent the disease spread. However, there was a problem that the buried livestock did not decompose after several years. Therefore, the study was purposed to investigate the effect of Corynebacterium glutamicum and Bacillus licheniformis on the degradation of buried cow carcasses and on the soil condition; such as temperature, decomposition course of carcasses, composition of amino acids in the soil around carcasses, and plant root elongation to measure soil conditions. As a result, the composition of amino acids in the soil treated with C. glutamicum and B. licheniformis was generally higher than those in the untreated soil. Plant roots in soil treated with C. glutamicum and B. licheniformis grew longer than in non-treated soil. The results suggested that the toxic effect on a grave land buried with FMD infected livestock is reduced when treated with C. glutamicum and B. licheniformis in regard of odor reduction, promoted decaying process, and soil fertilization.

• 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.