Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from<i>Amycolatopsis mediterranei</i>

Schupp, Thomas; Toupet, Christiane; Engel, Nathalie; Goff, Stephen A.

doi:10.1111/j.1574-6968.1998.tb12861.x

Cited by 96 publications

(61 citation statements)

References 16 publications

(16 reference statements)

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“…The formation of the polyketide backbone of the ansamitocins is catalyzed by four genes of 38 kbp encoding a type I PKS with an aromatic loading domain closely resembling that of the rif cluster (27,58) and seven chain-extension modules. Each module contains the predicted functional domains for chain extension and chain modification, in an arrangement matching that in other type I PKSs analyzed (59), and the modules are arranged colinear with their function in the biosynthetic assembly process (Fig.…”

Section: Discussionmentioning

confidence: 99%

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

279

256

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Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual ''methoxymalonate'' polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

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Section: Discussionmentioning

confidence: 99%

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

279

256

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“…1). The cluster includes genes involved in 3-amino-5-hydroxybenzoic acid (AHBA) biosynthesis (rifG-rifN operon and rifJ), polyketide biosynthesis (rifA-rifE operon), and rifamycin modification and export (6,(10)(11)(12)(13)(14). Although two rif genes are annotated as regulatory genes ( Fig.…”

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confidence: 99%

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

Liu

Shao

et al. 2017

Appl Environ Microbiol

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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.

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“…The rifamycin biosynthetic gene cluster recently has been cloned from A. mediterranei and sequenced (12,13,15). On one side of the cluster of about 90 kb are located about 36 kb of DNA-containing genes required for AHBA synthesis, genes involved in export and resistance, regulatory genes, and a substantial number of downstream-processing genes.…”

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confidence: 99%

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

Shen²,

Doi-Katayama³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

133

130

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The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra-to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta-through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with 18 O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7,8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.

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Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster fromAmycolatopsis mediterranei

Cited by 96 publications

References 16 publications

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

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

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

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