Engineered biosynthesis of novel polyenes: a pimaricin derivative producedby targeted gene disruption in Streptomyces natalensis

Background: The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxy genases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities towar d alternative substrates, particularly epoxidases, is a Fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P4 50s belonging to the biosynthetic gene cluster of an archetype polyene, pim aricin. Results: A recombinant mutant of the pimaricin-producing actinomycete Strep tomyces natalensis produced a novel pimaricin derivative, 4,5 -deep oxypima ricin, as a major product. This biologically active product resulted from t he phage-mediated targeted disruption of the gene pimD, which encodes the c ytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following highperformance liquid chromatography purifica tion. Conclusions: We have demonstrated that PimD is the epoxidase responsible fo r the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase h as been knocked out, constitutes the first designer polyene obtained by tar geted manipulation of a polyene biosynthetic gene cluster. This novel epoxi dase could prove to be valuable for the introduction of epoxy substituents into designer macrolides. (C) 2001 Elsevier Science Ltd. All rights reserve d.