• Title/Summary/Keyword: Corynebacterium glutamicum glutamicum

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Isolation and characterization of sigH from Corynebacterium glutamicum (Corynebacterium glutamicum의 sigH 유전자의 분리 및 기능분석)

  • Kim Tae-Hyun;Kim Hyung-Joon;Park Joon-Sung;Kim Younhee;Lee Heung-Shick
    • Korean Journal of Microbiology
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    • v.41 no.2
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    • pp.99-104
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    • 2005

  • Corynebacterial clones which exert regulatory effects on the expression of the glyoxylate bypass genes were isolated using a reporter plasmid carrying the enteric lacZ fused to the aceB promoter of Corynebacterium glutamicum. Some clones carried common fragments as turned out by DNA mapping technique. Subcloning analysis followed by the measurement of $\beta-galactosidase$ activity in Escherichia coli identified the region responsible for the aceB-repressing activity. Sequence analysis of the DNA fragment identified two independent ORFs of ORF1 and ORF2. Among them, ORF2 was turned out to be responsible for the aceB-repressing activity. ORF1 encoded a 23,216 Da protein composed of 206 amino acids. Sequence similarity search indicated that the ORF may encode a ECF-type $\sigma$ factor and designated sigH. To identify the function of sigH, C. glutamicum sigH mutant was constructed by gene disruption technique and the sigH mutant showed growth retardation as compared to the wild type strain. In addition, the mutant strain showed sensitivity to oxidative-stress generating agent plumbagin. This result imply that sigH is probably involved in the stress response occurring during normal cell growth.

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The protoplast formation, regeneration and fusion of coryneform bacteria (Coryneform bacteria의 原形質體 形成, 再生 및 融合에 관한 硏究)

  • Shin, Myung-Gyo;Lee, Se-Yong;Lim, Bun-Sam;Chun, Moon-Jin
    • Korean Journal of Microbiology
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    • v.22 no.3
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    • pp.175-181
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    • 1984

  • In order to develope a protoplast fusion system for industrial coryneform bacteria, the optimum conditions for the formation and regeneration of progoplast were examined for Brevibacterium flavum and Corynebacterium glutamicum and the protoplast fusion was performed. For the formation of the protoplast of B. flavum and C. glutamicum, the optimum time for penicillin G. treatment to obtain protoplast was mid-exponential growth phase ($O.D_{580}=0.6-0.8,\;8.0{\times}10^7-1.0{\times}10^8cell/ml$). At the optimum conditions (0.3units/ml penicillin G and $400{\mu}g/ml$ lysoyme for treatement), frequencies of protoplast formation and protoplast regeneration were 99% and 25%, respectively. Protoplast regeneration frequency was highest under the optimum conditions for the protoplast formation. Addition of 25mM $Mg^{2+}\;and\;50mM\;Ca^{2+}$ to the regeneration medium further increased the regeneration frequencies. The protoplast fusion frequencies of B. flavum and C. glutamicum in intraspecies fusion were $1.0{\times}10^{-8}\;and\;7.8{\times}10^{-4}$, of the regenerated protoplast respectively, when 33% of PEG (polythylene glycol) 6,000 was used as the fusing agent.

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Towards Methionine Overproduction in Corynebacterium glutamicum - Methanethiol and Dimethyldisulfide as Reduced Sulfur Sources

  • Bolten, Christoph J.;Schroder, Hartwig;Dickschat, Jeroen;Wittmann, Christoph
    • Journal of Microbiology and Biotechnology
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    • v.20 no.8
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    • pp.1196-1203
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    • 2010

  • In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.

  • https://doi.org/10.4014/jmb.1002.02018 인용 PDF KSCI