Dataset Information

Conjugation of ?BT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32.

ABSTRACT: The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 × 10-5 CFU per recipient cell. This integrative vector, based on the ?BT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.

SUBMITTER: Li C

PROVIDER: S-EPMC5639847 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Conjugation of ϕBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32.

Li Chen C Zhou Li L Wang Ying Y Zhao Guoping G Ding Xiaoming X

Bioengineered 20170103 5

The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack ...[more]

PMID: 28045587

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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.

Dataset Information

Conjugation of ?BT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32.

Publications

Conjugation of ϕBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32.

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