• Korean Journal of Food Science and Technology
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• v.23 no.5
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• pp.561-567
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• 1991

This study was designed to investigate the productivity of L-methionine by the method of protoplast fusion between Brevibacterium flavum ATCC 14067 and Corynebacterium glutamicm ATCC 13032, and then L-methionine production was performed to continuous fermentation using the immobilized fusant cells. Mutants B. flavum K 104($thr\;met\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}\;as\;genetic\;marker$) and C. glutamicum B 70($thr\;Hos\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}as\;genetic\;marker$) were isolated by MNNG treatment. On the other hand, protoplast of mutants were formed to treat with lysis solution containing $500{\mu}g/ml$ of lysozyme. The ratios of protoplast formation and regeneration were 99% and $64{\sim}66%$ respectively. Fusion frequency between B. flavum K 104 and C. glutamicum B 70 showed the $3.5{\times}10^{5}$ in the 35% polyethylene glycol(PEG6000) containing 3% PVP solution. The productivity of L-methionine by fusant BFCG 37 immobilized with sodium alginate was 0.89 g/l the batch fermentation and was $18.75mg/^{1}hr\;^{1}$ on the continuous fermentation at $30^{\circ}C$ for 72 hr.

• 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.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.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.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1683-1690
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• 2013

The cyclic AMP receptor protein (CRP) homolog, GlxR, controls the expression of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. In silico analysis has revealed the presence of glxR binding sites upstream of genes ptsG, adhA, and ald, encoding glucose-specific phosphotransferase system protein, alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH), respectively. However, the involvement of the GlxR-cAMP complex on the expression of these genes has been explored only in vitro. In this study, the expressions of ptsG, adhA, and ald were analyzed in detail using an adenylate cyclase gene (cyaB) deletion mutant and glxR deletion mutant. The specific activities of ADH and ALDH were increased in both the mutants in glucose and glucose plus ethanol media, in contrast to the wild type. In accordance, the promoter activities of adhA and ald were derepressed in the cyaB mutant, indicating that glxR acts as a repressor of adhA. Similarly, both the mutants exhibited derepression of ptsG regardless of the carbon source. These results confirm the involvement of GlxR on the expression of important carbon metabolic genes; adhA, ald, and ptsG.

• KSBB Journal
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• v.6 no.3
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• pp.223-230
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• 1991

Using the shuttle vector pECCGl between Escherichia coli and Corynebacterium glutamicum and C. glutamicum strain JS231 grown in the medium supplemented with penicillin-G, which inhibits the formation of cross-links in the peptidoglycan of bacterial cell wall, various parameters involved in electroporation system including resistance, electric field strength, capacitance, DNA concentration, and cell density were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Using cells grown with 0.3U/ml of penicillin-G and harvested at A600 of 0.7-0.8, transformation efficiencies of 107-l08 transformants/$\mu\textrm{g}$ of DNA with Corynebcctertum glutamicum strain JS231 and wild type ATCC13032 were achieved under conditions of 12.5kV/cm of electric field strength, 400 ohms of resistance, $25\mu$F of capacitance, 3$\times$108 cells per transformation(1.2$\times$1010 cells/ml) and 100ng of plasmid DNA per transformation.

• 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.

• Journal of Microbiology and Biotechnology
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• v.33 no.12
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• pp.1595-1605
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• 2023

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQD^WT with the citrate at a resolution of 1.80Å and CgDHQD^R19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQD^S103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQD^S103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1916-1927
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• 2018

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.