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KMID : 0545120040140040789
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 4 p.789 ~ p.795
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Isolation and Characterization of Transcriptional Elements from Corynebacterium glutamicum
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Park SD
Lee SN/Park IH/Choi JS/Jeong WK/Kim YH/Lee HS
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Abstract
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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 P19 clone a representative in the strong class were able to grow on minimal agar plates containing over 40 mg/ml chloramphenicol and showed CAT activity of 10 mmol/mg·min performing slightly better than the cells carrying Ptac a strong E. coli promoter. Subcloning analysis of the P19 clone identified a 180 bp intergenic fragment (P180) which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by P180 was not affected by either the kinds of carbon sources or changes in temperature. These properties make the P180 clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.
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