Rifamycin B biosynthesis by Amycolatopsis mediterranei S699 involves a number of unusual modification reactions in the formation of the unique polyketide backbone and decoration of the molecule. A number of genes believed to be involved in the tailoring of rifamycin B were investigated and the results confirmed that the formation of the naphthalene ring moiety of rifamycin takes place during the polyketide chain extension and is catalysed by Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded by rif-orf5 is required for the conversion of the D12, 29 olefinic bond in the polyketide backbone of rifamycin W into the ketal moiety of rifamycin B. Furthermore, Rif-Orf3 may be involved in the regulation of rifamycin B production, as its knock-out mutant produced about 40 % more rifamycin B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in rifamycin biosynthesis, but might be evolutionary remnants carried over from an ancestral lineage.
INTRODUCTIONRifamycin continues to play a significant role in clinical medicine. Synthetically modified derivatives, such as rifampicin, rifabutin and rifapentine, remain the principal chemotherapeutic agents used for combating tuberculosis, leprosy and AIDS-related mycobacterial infections (Maggi et al., 1966;Ramos-e-Silva & Rebello, 2001;Sepkowitz et al., 1995). The potent antibacterial activity of this class of antibiotics is due to their specific inhibition of bacterial DNA-dependent RNA polymerases (Campbell et al., 2001;Wehrli & Staehelin, 1969). At the same time, they have relatively low if any activity against eukaryotic RNA polymerases. Due to their high selectivity for their molecular target, the rifamycins have become a safe and effective medication. Unfortunately, as with many other antibiotics, the incidence of resistance of Mycobacterium tuberculosis, the causative agent of tuberculosis, to rifamycins is continuing to increase over time, due largely to mutational alterations of the target molecule, the b subunit of RNA polymerase (Kirschbaum & Gotte, 1993;Suzuki et al., 1995). This high-level resistance has contributed to the recent reemergence of tuberculosis as a major health problem and the consequent increase in the death toll among world populations (Dye et al., 2002). Therefore, new drug discovery and continued development of the existing drugs to combat tuberculosis is indispensable.Despite the preparation of a large number of rifamycin derivatives by semi-synthetic approaches, the structural modifications have been limited primarily to one region of the molecule, the C-3 or C-4 positions of the aromatic core unit (Cricchio et al., 1974(Cricchio et al., , 1975Maggi et al., 1965; Wehrli et al., 1987). Alterations at other locations are difficult to accomplish chemically due to the complexity of the molecule, and require the implementation of alternative methodology, including combinatorial biosynthesis or mutasynthesis, to achieve additional...
