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

Abstract

Background: The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (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 toward 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 P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin.

Results: A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification.

Conclusions: We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.