RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteriaMycobacterium tuberculosis andMycobacterium leprae . Although the biosynthetic pathway of rifamycin has been extensively studied inAmycolatopsis mediterranei , little is known about the regulation in rifamycin biosynthesis. Here, anin vivo transposon system was employed to identify genes involved in the regulation of rifamycin production inA. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted inAMED_0655 (rifZ , encoding a LuxR family regulator). TherifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in therifZ null mutant. Based on the cotranscription assay results, genes within therif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the wholerif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of bothrifK andorf18 (AMED_0645 ) was further verified. As RifZ directly regulates the transcription of all operons within therif cluster, we propose that RifZ is a pathway-specific regulator for therif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for therif cluster. As RifZ directly regulates the transcription of the entirerif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation ofrif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.