construction of recombinant vectors containing clavulanic acid antibiotic regulatory gene, clar, isolated of streptomyces clavuligerus strains

the clar of streptomyces clavuligerus in the clavulanic acid gene cluster encodes a transcriptional regulator that controls clavulanic acid biosynthesis. the main goal of this study was isolation and molecular detection of the clar gene and its cloning in the streptomyces specific vector (pma:: hyg). by cinsideration of the clar gene’s start codon, the specific primers were designed. after genomic dna extraction from s. clavuligerus, the clar gene was amplified by polymerase chain reaction (pcr). the structure of the amplified clar was confirmed by nested-pcr, pcr-restriction fragment length polymorphism (pcr-rflp), and sequencing. a ligation mixture was prepared with the isolated clar gene and cut pma::hyg vector. escherichia coli competent cells were finally transformed with the ligation mixture. presence of the recombinant vector in the transformed colonies was then confirmed by the colony-pcr procedure. the clar gene was also isolated from s. clavuligerus dsm41826, cloned and sequenced in the same manner. the pma::hyg vector is a shuttle vector, which exists as a multicopy plasmid in e. coli, and as an integrative plasmid in streptomyces. therefore, the newly constructed vectors of this study can be regarded as an appropriate tool for site-directed mutagenesis and gene replacement strategies in s. clavuligerus.