BIOSYNTHESIS OF THE ANSAMYCIN ANTIBIOTIC RIFAMYCIN - DEDUCTIONS FROM THE MOLECULAR ANALYSIS OF THE RIF BIOSYNTHETIC GENE-CLUSTER OF AMYCOLATOPSIS-MEDITERRANEI S699

Background: The ansamycin class of antibiotics are produced by various Actinomycetes. Their carbon framework arises from the polyketide path way via a polyketide synthase (PKS) that uses an unusual starter unit. Rifamycin (rif), produced by Amycolatopsis mediterranei, is the arche type ansamycin and it is medically important. Although its basic precu rsors (3-amino-5-hydroxy benzoic acid AHBA, and acetic and propionic a cids) had been established, and several biosynthetic intermediates had been identified, very little was known about the origin of AHBA nor h ad the PKS and the various genes and enzymes that modify the initial i ntermediate been characterized. Results: A set of 34 genes clustered a round the rifK gene encoding AHBA synthase were defined by sequencing all but 5 kilobases (kb) of a 95 kb contiguous region of DNA from A. m editerranei. The involvement of some of the genes in the biosynthesis of rifamycin B was examined, At least five genes were shown to be esse ntial for the synthesis of AHBA, five genes were determined to encode the modular type I PKS that uses AHBA as the starter unit, and 20 or m ore genes appear to govern modification of the polyketide-derived fram ework, and rifamycin resistance and export. Putative regulatory genes were also identified. Disruption of the PKS genes at the end of rifA a bolished rifamycin B production and resulted in the formation of P8/1- OG, a known shunt product of rifamycin biosynthesis, whereas disruptio n of the orf6 and orf9 genes, which may encode deoxysugar biosynthesis enzymes, had no apparent effect. Conclusions: Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of st ructural, resistance and export, and regulatory genes. The genes chara cterized here could be modified to produce novel forms of the rifamyci ns that may be effective against rifamycin-resistant microorganisms.