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

Abstract

Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as "nitrate-stimulating effect (NSE)". Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn't occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.

The aminoshikimic acid pathway in bacteria as source of precursors for the synthesis of antibacterial and antiviral compounds

“…The cluster includes an operon comprising five types of modular polyketide synthases ( rifABCDE ), associated with the genes involved in the synthesis of AHBA ( rifGHIKLMN ) and rifJ (in a separated operon). Additionally, it comprises genes controlling the post-polyketide backbone modifications and conversion of rifamycin and its export (Floss et al., 2011 ; Li et al., 2017 ; Liu et al., 2020c ). Furthermore, two regulator genes are located in the rif cluster, RifZ ( rifZ ), a LuxR family transcriptional regulator activating the transcription of all the genes of the cluster; and RifQ ( rifQ ), proposed as involved in the control of the reduction of the intracellular toxicity of rifamycin by regulating the expression of the rifamycin exporter RifP ( rifP ) (Fig.…”

“…Furthermore, two regulator genes are located in the rif cluster, RifZ ( rifZ ), a LuxR family transcriptional regulator activating the transcription of all the genes of the cluster; and RifQ ( rifQ ), proposed as involved in the control of the reduction of the intracellular toxicity of rifamycin by regulating the expression of the rifamycin exporter RifP ( rifP ) (Fig. 4A ) (Li et al., 2017 ; Liu et al., 2020c ). Remarkably, besides RifZ, GlnR, a global transcriptional regulator in actinomycetes involved in the modulation of assimilation and utilization of diverse carbon sources; nitrogen sources (ammonium, urea, or nitrate), as in antibiotic biosynthesis, is also involved in the activation of the positive regulator of RifZ, promoting the indirect positive regulation of the entire rif cluster.…”

“…Remarkably, besides RifZ, GlnR, a global transcriptional regulator in actinomycetes involved in the modulation of assimilation and utilization of diverse carbon sources; nitrogen sources (ammonium, urea, or nitrate), as in antibiotic biosynthesis, is also involved in the activation of the positive regulator of RifZ, promoting the indirect positive regulation of the entire rif cluster. Additionally, GlnR was proposed to promote the biosynthesis of AHBA directly by activating the transcription of rifK coding for AHBA synthase (Liu et al., 2020c ) (Fig. 4B ).…”

“…Ansamycins produced by A. mediterranei include the rifamycins, from which derive a large number of chemically synthesized compounds, including rifampicin, rifabutin, rifapentine, and rifaximin (Kang et al., 2012 ; Liu et al., 2020c ). These compounds are still used as first-line antimycobacterial drugs against Mycobacterium tuberculosis and Mycobacterium leprae by inhibiting their RNA polymerase activity (Liu et al., 2020c ).…”

“…The elucidation of the transcriptional control mechanisms of the rif cluster for the synthesis of rifamycin in A. mediterranei by GlnR, the global nitrogen regulator in this bacterium, showed that GlnR binds specifically to the upstream region of rifZ , encoding for the RifZ specific activator of the rif cluster, acting as an indirect regulator of the entire biosynthetic cluster. Furthermore, GlnR was also determined to bind to the upstream region of rifK , coding for the AHBA synthase, acting as a direct activator of the supply of AHBA for the synthesis of rifamycin (Liu et al., 2020c ) (Fig. 4B ).…”