Dataset Information

A preliminary study of the mechanism of nitrate-stimulated remarkable increase of rifamycin production in Amycolatopsis mediterranei U32 by RNA-seq.

ABSTRACT:

Background

Rifamycin is an important antibiotic for the treatment of infectious disease caused by Mycobacteria tuberculosis. It was found that in Amycolatopsis mediterranei U32, an industrial producer for rifamycin SV, supplementation of nitrate into the medium remarkably stimulated the yield of rifamycin SV. However, the molecular mechanism of this nitrate-mediated stimulation remains unknown.

Results

In this study, RNA-sequencing (RNA-seq) technology was employed for investigation of the genome-wide differential gene expression in U32 cultured with or without nitrate supplementation. In the presence of nitrate, U32 maintained a high transcriptional level of genes both located in the rifamycin biosynthetic cluster and involved in the biosynthesis of rifamycin precursors, including 3-amino-5-dihydroxybenzoic acid, malonyl-CoA and (S)-methylmalonyl-CoA. However, when nitrate was omitted from the medium, the transcription of these genes declined sharply during the transition from the mid-logarithmic phase to the early stationary phase. With these understandings, one may easily propose that nitrate stimulates the rifamycin SV production through increasing both the precursors supply and the enzymes for rifamycin biosynthesis.

Conclusion

It is the first time to thoroughly illustrate the mechanism of the nitrate-mediated stimulation of rifamycin production at the transcriptional level, which may facilitate improvement of the industrial production of rifamycin SV, e.g. through optimizing the global rifamycin biosynthetic pathways on the basis of RNA-seq data.

SUBMITTER: Shao ZH

PROVIDER: S-EPMC4453227 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Publications

A preliminary study of the mechanism of nitrate-stimulated remarkable increase of rifamycin production in Amycolatopsis mediterranei U32 by RNA-seq.

Shao Zhi Hui ZH Ren Shuang Xi SX Liu Xin Qiang XQ Xu Jian J Yan Han H Zhao Guo Ping GP Wang Jin J

Microbial cell factories 20150604

<h4>Background</h4>Rifamycin is an important antibiotic for the treatment of infectious disease caused by Mycobacteria tuberculosis. It was found that in Amycolatopsis mediterranei U32, an industrial producer for rifamycin SV, supplementation of nitrate into the medium remarkably stimulated the yield of rifamycin SV. However, the molecular mechanism of this nitrate-mediated stimulation remains unknown.<h4>Results</h4>In this study, RNA-sequencing (RNA-seq) technology was employed for investigati ...[more]

PMID: 26041361

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Project description: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 pathway-specific 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 high-yield industrial strains.

Project description: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.

Dataset Information

A preliminary study of the mechanism of nitrate-stimulated remarkable increase of rifamycin production in Amycolatopsis mediterranei U32 by RNA-seq.

Background

Results

Conclusion

Publications

A preliminary study of the mechanism of nitrate-stimulated remarkable increase of rifamycin production in Amycolatopsis mediterranei U32 by RNA-seq.

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