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 tuberculosisandMycobacterium lepraeAlthough the biosynthetic pathway of rifamycin has been extensively studied inAmycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, anin vivotransposon system was employed to identify genes involved in the regulation of rifamycin production inA. mediterraneiU32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted inAMED_0655(rifZ, encoding a LuxR family regulator). TherifZgene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rifcluster) was remarkably reduced in therifZnull mutant. Based on the cotranscription assay results, genes within therifcluster 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 wholerifcluster. 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 bothrifKandorf18(AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within therifcluster, we propose that RifZ is a pathway-specific regulator for therifcluster.IMPORTANCETo 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 (rifcluster), 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 therifcluster. As RifZ directly regulates the transcription of the entirerifcluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation ofrifcluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.
State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Chen
C
Li
University of Chinese Academy of Sciences, Beijing, China.|CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Xinqiang
X
Liu
CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Chao
C
Lei
CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Han
H
Yan
CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Zhihui
Z
Shao
State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.
Ying
Y
Wang
State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China.|CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.|Department of Microbiology and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.
Guoping
G
Zhao
CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China wangj01@hotmail.com xmding74@fudan.edu.cn.
Jin
J
Wang
State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, China wangj01@hotmail.com xmding74@fudan.edu.cn.
Xiaoming
X
Ding
D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D059765:Homologous Recombination; D005810:Multigene Family; D009154:Mutation; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D012294:Rifamycins / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D012294:Rifamycins; D015534:Trans-Activators
Amycolatopsis mediterranei; RifZ; pathway-specific regulator; rif; rifamycin