• Applied Biological Chemistry
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• 제34권2호
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• pp.174-179
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• 1991

Brevibacterium flavum 과 Corynebacterium glutamicum 균주간의 원형질체 융합을 실시하였다. 원항질체 융합을 위하여, Brevibacterium flavum ATCC 21493과 Corynebacterium glutamicum ATCC 21831로부터 여러 변이주를 분리하고 융합의 최적조건을 검토하였다. 본 연구에서 저자 등은 Brevibacterium flavum 108-125와 Corynebacterium glutamicum 41-214A 균주 간의 이속간 원형질체 융합주 MWF 9031의 L-arginine발효능이 우수함을 확인하였다. 융합주 MWF 9031은 10% 포도당이 함유된 배지에서 32.5 mg/ml의 L-arginine을 생산하였다. 융합주의 생리적 성질은 두 모균주의 중간정도를 나타내고, 안정성이 60일 이상 유지되고 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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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.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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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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• 제20권1호
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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.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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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.

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.31-36
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• 1991

Tn5의 kanamycin 저항성 유전자를 가진 pBEL1 plasmid와 C.glutamicum cryptic plasmid인 pSR1으로 7.5kb의 새로운 plasmid를 만든 후, 이를 pCE1301이라 명명하였다. 이 pCE1301은 PEG1301은 PEG-protoplast법으로 C.glutamicum을 형질전환하였을 때 효율이 약 $3.0\times 10^3$형질전환제/$\mu g$이었으며 SalI과 EcoRI 제한효소 절단부위가 1개씩이었다. 또 Km이 없는 배지에서 25세대까지 안정하게 유지되었으며 B.flavum, E.coli에서 복제되었다. 이 pCE1301을 이용하여 C.glutamicum 의 homoserine dehydrogenase 유전자를 cloning하였다.

• 미생물학회지
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• 제41권4호
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• pp.300-305
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• 2005

Corynebacterium glutamicum에서 promoter-probe vector인 pSK1Cat을 이용해 분리된 프로모터를 함유하는 단편들 중 가장 높은 활성을 나타낸 $P_{19}$ 단편에 대한 심도 있는 분석을 수행하였다. Subcloning을 실시하여 프로모터 활성을 지닌 DNA 영역을 180 bp로 압축할 수 있었고 $(P_{180})$, 이를 C. glutamicum의 균주개량 측면에서 그 활용성을 분석하였다. C. glutamicum에서 메치오닌 생합성에 관여하는metX유전자의 메치오닌에 의한 repression을 해제시키기 위하여 metX유전자의 promoter를 $P_{180}$ promoter로 교체하였고 $(P_{180}-metX)$, $P_{180}-metX$를 C. glutamicum에 도입하여 발현되는 homoserine acetyltransferase 활성을 다양한 성장조건에서 측정하였다. MB 영양배지에서 배양하는 경 우 $P_{180}-metX$를 함유는 균주는 wild type보다 약 24배 높은 homoserine acetyltransferase 활성을 나타내었다. Tac 프로모터에 연계하는 경우 $(P_{tac}-metX)$, 약 13배의 활성 증가만이 관찰되었다. 최소배지에서 배양한 후 분석한 결과, $P_{180}-metX$에서의 발현양상은 배지에 첨가된 methionine에 의해 영향받지 앓음을 확인하였는데, 이는 $P_{180}$ 단편이 생합성 유전자의 derepression에 의한 아미노산 생산균의 개량에 효율적으로 이용될 수 있음을 의미한다. $P_{180}-metA$를 라이신 생산균에 도입하는 경우 최대 약 0.8g/l의 메치오닌이 생산됨을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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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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• 제4권4호
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• pp.256-263
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• 1994

The aceB gene, encoding for malate synthase, one of the key enzymes of glyoxylate bypass, was isolated from a pMT1-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli aceB mutant on an acetate minimal medium. The aceB gene was closely linked to aceA, separated by 598 base pairs, and transcribed in divergent direction. The aceB expressed a protein product of Mr 83, 000 in Corynebacterium glutamicum which was unusually large compared with those of other malate synthases. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 2, 217 base pairs which encodes a protein with the molecular weight of 82, 311 comprising 739 aminoo acids. The putative protein product showed only limited amino acid-sequence homology to its counteliparts in other organisms. The N-terminal region of the protein, which shows no apparent homology with the known sequences of other malate synthases, appeared to be responsible for the protein s unusually large size. A potential calciumbinding domain of EF-hand structure found among eukaryotes was detected in the N-terminal region of the deduced protein.

• 한국유기농업학회지
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• 제7권1호
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• pp.105-113
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• 1998

수종의 근권세균이 양액재배 오이의 생장에 미치는 영향을 구명하기 위하여 Azospirillum sp., Rhodopseudomonas sp., Pseudomonas sp., Bacillus sp.와 Corynebacterium glutamicum간의 융합균주 등을 근권처리하였다. 융합균주의 근권처리후 4일에 세균의 밀도변화르르 보면 코코피트 > 암면 > 배양액 등의 순으로 나타났다. 균주의 근권처리에 따른 오이의 생장촉진효과는 암면경보다 코코피트경에서 더 높았으며, 균주의 종류별 오이의 생장촉진효과를 보면 Azospirillum sp. > 광합성세균 $\ge$ 융합균주 Pseudomonas sp. > 대조구 등의 순으로 나타났다.