• Journal of Microbiology and Biotechnology
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• v.24 no.1
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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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• v.15 no.1
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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.

• 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.