• 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.

• Archives of Pharmacal Research
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• 제15권1호
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• pp.30-34
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• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.78-86
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• 2019

대사공학을 이용하여 N-아세틸글루코사민(GlcNAc)을 생산하는 재조합 Corynebacterium glutamicum을 개발하였다. 먼저 GlcNAc를 생산하는 기반균주를 제작하기 위하여, N-acetylglucosamine-6-phosphate deacetylase와 glucosamine-6-phosphate deaminase를 암호화하는 nagAB와 N-acetylmannosamine-6-phosphate epimerase를 암호화하는 nanE를 C. glutamicum ATCC 13032에서 순차적으로 결손하여, 최종적으로 KG208 균주를 제작하였다. 또한, glucosamine-6-phosphate synthase를 암호화하는 C. glutamicum 유래의 glmS와 glucosamine-6-phosphate N-acetyltransferase를 암호화하는 Saccharomyces cerevisiae 유래의 gna1을 각각 여러 발현벡터에 클로닝하였다. 여러 발현 조합의 플라스미드들 중에서 pCXI40-glmS와 pCEI40-gna1을 함유한 제조합균주 KG440은 삼각플라스크 발효에서 1.77 g/l의 GlcNAc와 0.63 g/l의 글루코사민을 생산하였다.