Isolation of transcription initiation signals from Corynebacterium ammoniagenes and comparison of their gene expression levels in C. ammoniagenes and Escherichia coli

With the aim of isolating transcription initiation signals, random Sau3AI fragments of Corynebacterium ammoniagenes ATCC 6872 were cloned into the promoter-probe plasmid, pEKplCm, and screened for promoter activity by chloramphenicol resistance of transformed Escherichia coli cells. Representative 22 promoter clones were analysed in C. ammoniagenes by its nucleotide sequences and promoter activities were measured by chloramphenicol acetyltransferase (CAT) assay. Activities of CAT were between 0.04 U and 2.85 U and several strong promoters, such as IJ59 clone and IJ73 clone, were found. The E. coli tac promoter was a strong promoter in C. ammoniagenes. IJ59 clone was part of the ferredoxin 3 gene and the promoter region contained a putative UP element, less conserved sequence at the -35 region and perfectly conserved sequence at the -10 region. The IJ73 clone contained a strong promoter that produced CAT at more than 10% of total cellular proteins in C. ammoniagenes and is therefore useful for expressing various genes in this coryneform bacteria.