• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.517.2-517
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• 1986

The regulation of 3 ammonia assimilatory enzymes GDH(glutamate dehydrogenase), GS(glutamine synthetase) and GOGAT (glutamate synthase), have been examined in C. glutamicum for the biosynthesis of glutamate and glutmine. The cell free extracts of 3 kinds of arg, his and trp auxotrophs were investigated the activities of -ketoglutarate dehydrogenase, GDH, GS, and GOGAT on the media cultured with nitrogen excess and limiting conditions. Trp and his howed higher level of glutamate and glutamine than that of parental strain. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with his, trp, glyc, ala, ser, and GMP implied that a system of feedback inhibition were effective. Three enzyme biosynthesis is repressed by nitrogen sources such as trp, pro, glyc, ala, ser and tyrosine.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1353-1360
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• 2007

Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of $30^{\circ}C$ but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to $15^{\circ}C$, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at $20^{\circ}C$, but not at $30^{\circ}C$. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.337-342
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• 2019

We published that bacterial heme was over-produced in a recombinant Corynebacterium glutamicum expressing 5-aminolevulinic acid synthase ($hemA^+$) under control of a constitutive promoter ($P_{180}$) and the heme-producing C. glutamicum had commercial potentials; as an iron feed additive for swine and as a preservative for lactic acid bacteria. To enhance the heme production, the $hemA^+$ gene was expressed under controls of various promoters in the recombinant C. glutamicum. The $hemA^+$ expression by $P_{gapA}$ (a constitutive glycolytic promoter of glyceraldehyde-3-phosphate dehydrogenase) led 75% increase of heme production while the expression by $P_{H36}$ (a constitutive, very strong synthetic promoter) resulted in 50% decrease compared with the control ($hemA^+$ expression by $P_{180}$ constitutive promoter). The $hemA^+$ expression by a late log-phase activating $P_{sod}$ (an oxidative-stress responding promoter of superoxide dismutase) led 50% greater heme production than the control. The $hemA^+$ expression led by a heat-shock responding chaperone promoter ($P_{dnaK}$) resulted in 121% increase of heme production at the optimized heat-shock conditions. The promoter strength and induction phase are discussed based on the results for the heme production at an industrial scale.

• KSBB Journal
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• 제6권1호
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• pp.63-68
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• 1991

본 연구에서는 L-phcn ylalaninc을 생산하는 조절기작을 상실한 영양요구성 변이주인 Corynebacterium glulamicum ATCC 21674를 이용하여 플라스크내에서의 회분식 배양시의 특성을 조사하였다. 이 균주는 회분반효시 2.1-3.4 g/I 의 phcnllalanine파 2.9-4.4 g/I 의 tyrosine을 생산하였고, 당농도가 높을 경우 생산성이 저하됨을 알 수 있었다. 또한 온도의 변화는 이들 아미노산 생산에 큰 영향을 미침이 관찰되었다. $30^{\circ}C$에사 배양하는 경우, $37^{\circ}C$에서 배양하는 것보다 훨씬 많은 아미노산이 생산되었다. 배양도즙 pH는 급격한 변화를 보였다, 이 균주는 tyrosine이 없는 최소배지에서도 자라는 것이 확인되었고, tyrosine를 과량 생성까시 함으로 보아 영양 요구 성질을 상실한 revertant로서 조전기작 상실성을 ­유지한 것으로 판단된다.

• 한국식품과학회지
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• 제23권5호
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• pp.561-567
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• 1991

본 연구는 Brevibacterium flavum ATCC 14067 및 Corynebacterium glutamicum ATCC 13032간의 protoplast fusion을 행하여 L-methionine의 생산성을 검토하고 발효조건을 개선하기 위하여 연속배양을 행하였다. N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) $500{\mu}g/ml$로 처리하였으며 B. flavum K-104($Thr\;Met\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}$)와 C. glutamicum B-70($Thr\;Hos\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}$)의 변이주를 분리하였다. 이들 변이주에 $500{\mu}g/ml$의 lysozyme을 처리하였을 때 원형질체 형성을 및 재생율은 각각 99% 및 $64{\sim}66%$를 나타내었으며 융합 빈도는 3% PVP를 함유한 35% PEG 용액에서 $3.5{\times}10^{5}$을 나타내었다. Sodium alginate로 고정화시킨 융합주 BFCG 37은 72시간 회분배양에서 0.89g/l의 methionine을 생산하였고 연속배양에서는 $18.75mg/^{1}hr\;^{1}$의 L-methionine를 안정적으로 생산할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1174-1179
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• 2006

The wild-type Corynebacterium glutamicum ATIC 13032 and Corynebacterium glutamicum ATTC 13032 lysC S301Y, exhibiting a deregulated aspartokinase, were compared concerning growth, lysine production, and intracellular carbon fluxes. Both strains differ by only one single nucleotide over the whole genome. In comparison to the wild-type, the mutant showed significant production of lysine with a molar yield of 0.087 mol (mol glucose$^{-1}$) whereas the biomass yield was reduced. The deregulation of aspartokinase further led to a global rearrangement of carbon flux throughout the whole central metabolism. This involved an increased flux through the pentose phosphate pathway (PPP) and an increased flux through anaplerosis. Because of this, the mutant revealed an enhanced supply of NADPH and oxaloacetate required for lysine biosynthesis. Additionally, the lumped flux through phosphoenolpyruvate carboxykinase and malic enzyme, withdrawing oxaloacetate back to the glycolysis and therefore detrimental for lysine production, was increased. The reason for this might be a contribution of malic enzyme to NADPH supply in the mutant in the mutant. The observed complex changes are remarkable, because they are due to the minimum genetic modification possible, the exchange of only one single nucleotide.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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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.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1420-1429
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• 2020

Corynebacterium glutamicum, an important industrial strain, has a relatively slower reproduction rate. To acquire a growth-boosted C. glutamicum, a descendant strain was isolated from a continuous culture after 600 generations. The isolated descendant C. glutamicum, JH41 strain, was able to double 58% faster (t_d=1.15 h) than the parental type strain (PT, t_d=1.82 h). To understand the factors boosting reproduction, the transcriptomes of JH41 and PT strains were compared. The mRNAs involved in respiration and TCA cycle were upregulated. The intracellular ATP of the JH41 strain was 50% greater than the PT strain. The upregulation of NCgl1610 operon (a putative dyp-type heme peroxidase, a putative copper chaperone, and a putative copper importer) that presumed to role in the assembly and redox control of cytochrome c oxidase was found in the JH41 transcriptome. Plasmid-driven expression of the operon enabled the PT strain to double 19% faster (t_d=1.82 h) than its control (t_d=2.17 h) with 14% greater activity of cytochrome c oxidase and 27% greater intracellular ATP under the oxidative stress conditions. Upregulations of genes those might enhance translation fitness were also found in the JH41 transcriptome. Plasmid-driven expressions of NCgl0171 (encoding a cold-shock protein) and NCgl2435 (encoding a putative peptidyl-tRNA hydrolase) enabled the PT to double 22% and 32% faster than its control, respectively (empty vector: t_d=1.93 h, CspA: t_d=1.58 h, and Pth: t_d=1.44 h). Based on the results, the factors boosting growth rate in C. gluctamicum were further discussed in the viewpoints of cellular energy state, oxidative stress management, and translation.

• KSBB Journal
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• 제6권3호
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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.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.520.3-521
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• 1986

L-Lysine 생성균인 Corynebacterium glutamicum의 동종간 융합주 RS 99를 담체인 Alginate와 여기에 TiCl$_4$로부터 제조된 Titanium hydroxide를 혼합하여 각각 고정하고 이들의 Gel strength, 활성도 및 회분식 발효조건을 비교하였다. 그 결과 Alginate-Titanium hydroxide를 담체로 선정하여 고정화 C, glutamicum 융합주의 재사용성 및 안정성을 검토하였으며, Continuous-Flow Stirred-Tank Reactor를 구성하여 L-Lysine 의 연속발효를 시도하였다.