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

Abstract

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ 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 the rifZ null mutant. Based on the cotranscription assay results, genes within the rif 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 whole rif 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 both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathwayspecific regulator for the rif 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 the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of highyield industrial strains.

GlnR Dominates Rifamycin Biosynthesis by Activating the rif Cluster Genes Transcription Both Directly and Indirectly in Amycolatopsis mediterranei

“…In addition, the genome annotation results of A. mediterranei U32 indicate that the rif cluster contains two canonical transcriptional regulators: the LuxR-family transcriptional regulator RifZ and the TetR-family transcriptional regulator RifQ (Zhao et al, 2010). Recently, our studies have revealed that RifZ is a pathway-specific regulator of the rif cluster and directly activates the transcription of all rif cluster genes (Li et al, 2017), and RifQ could reduce the intracellular toxicity of rifamycin via directly regulating the expression of the rifamycin exporter-encoding gene rifP (Lei et al, 2018).…”

“…The glnR null mutant ( glnR) was constructed using double crossover recombination (Yu et al, 2007;Lin et al, 2014). To obtain the glnR complemented strain (glnR+), the glnR gene (with its native promoter) was integrated into the chromosome of glnR, employing the integrative plasmid pDZL803 as the vector (Lin et al, 2014;Li et al, 2017). As a control, glnR was integrated with the vector to obtain glnR/803 (Lin et al, 2014), which was used to investigate the influence of the empty plasmid on both bacterial growth and rifamycin production.…”

“…Later, to better understand rifamycin biosynthesis, the rifamycin biosynthetic gene cluster (rif cluster) and the complete genome of A. mediterranei were sequenced (August et al, 1998;Zhao et al, 2010). The rif cluster, consisting of 43 genes (i.e., from rifS to rifZ, Supplementary Figure S1) (Floss and Yu, 1999;Li et al, 2017), includes genes involved in the synthesis of the starter unit AHBA (3-amino-5-hydroxybenzoic acid), assembly and modification of the polyketide backbone, downstream processing and conversion of rifamycin, and resistance Xu et al, 2003Xu et al, , 2005Floss and Yu, 2005;Floss et al, 2011;Yuan et al, 2011). In addition, the genome annotation results of A. mediterranei U32 indicate that the rif cluster contains two canonical transcriptional regulators: the LuxR-family transcriptional regulator RifZ and the TetR-family transcriptional regulator RifQ (Zhao et al, 2010).…”

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

“…Previous studies in rare Actinomycete species have largely focused on the use of the well-characterised ϕC31-based integration vectors, and have mostly overlooked tools based on other phage integrases(43-45). Additionally, the conjugation methods used widely in Streptomyces gene transfer because of their ease have shown little success in rare Actinomycetes, including species in the genus Amycolatopsis , so electroporation has been the long-preferred method of gene transfer for species in this genus(5, 9, 46, 47). However, the growing interest in the use of serine integrases for synthetic biology applications(11) has led to further research into expanding the pool of available enzymes and their potentials as genetic tools(48-50).…”

“…Amongst rare Actinomycetes, the Amycolatopsis genus is of particular interest for its production of important antibiotics such as vancomycin(4) and rifamycin(5), as well as a diverse range of active natural products(6-8). The publicly available NCBI database contains nearly 70 genomes of Amycolatopsis strains, covering more than 40 species from this genus.…”