Abstract

DNA fragments showing promoter activity were obtained from the chromosomal DNA of Lactococcus lactis ssp. cremoris LM0230 by using a promoter-screening vector pBV5030, which contains a promoterless chloramphenicol acetyltransferase gene. Ten fragments were identified based on their ability to confer resistance against chloramphenicol in Escherichia coli. DNA sequencing revealed that all the fragments had a consensus region recognized by the sigma factor and only the nucleotide sequence of fragment 15C had the identical consensus region with the promoter P2 from L. lactis ssp. lactis MG1614. To compare their promoter strengths, an E. coli-lactococcal shuttle vector pWL1 containing a luciferase gene as the reporter gene was constructed based on lactococcal plasmid pMG36e. The putative promoter regions of 10 fragments exhibiting promoter activity were characterized in E. coli and L. lactis by measuring the luciferase activity, among which the putative promoter P6C exhibited the highest promoter activity both in E. coli JM109 and L. lactis ssp. cremoris MG1363. The luciferase system endowed significantly different expression levels enough to compare promoter strengths in E. coli and lactococcal host. The transcription-initiation sites of P6C and P13C were mapped by primer extension, which showed that they corresponded to a purine residue. The characterized promoters could be useful for the industrial production of heterologous proteins in L. lactis in case the proteins require a high safety level.