A Novel Mycolactone Toxin Obtained by Biosynthetic Engineering

Mycolactones are polyketide macrolide toxins produced by the emerging human pathogen Mycobacterium ulcerans, the causative agent of the destructive skin disease Buruli ulcer. 2] Mycolactone appears to be the primary virulence determinant for the infection, and purified mycolactone has potent cytotoxic, apoptotic and immunomodulatory properties. The structures of mycolactones A/B ([M+Na] at m/z 765) have been determined, and their configuration has been confirmed by chemical synthesis. 5–7] The molecules are Z and E isomers of a 12-membered macrolactone linked via an ester bond with a highly unsaturated polyketide chain (Scheme 1). Further work has revealed the heterogeneity of the toxin from different geographical sources, with structural variations in the side chain that could be traced to specific changes in the biosynthetic pathway. More recently, distinctive mycolactones with characteristically different side chains have also been isolated from the frog pathogen Mycobacterium liflandii and from several mycobacterial fish pathogens (see below); this raises the question of which structural features are crucial for the several effects of mycolactone on human cells. The genetic basis for mycolactone biosynthesis has been determined. M. ulcerans has a 174 kb megaplasmid harbouring three genes encoding type I modular polyketide synthases (PKS). Genes mlsA1 and mlsA2 together encode the PKS for production of the 12-membered core lactone, while mlsB encodes the PKS for the side chain. Three additional genes are present, MUP_045, MUP_038 and MUP_053. MUP_045 encodes an enzyme resembling FabH-like type III ketosynthases (KS), MUP_038 encodes a discrete thioesterase, and MUP_053 encodes a cytochrome P450 hydroxylase Cyp140A7 that is strongly implicated in the catalysis of oxidation at C-12’ of the side chain. For example, Australian strains of M. ulcerans, whose virulence plasmid lacks MUP_053, typically produce mycolactone C, in which the C12’ hydroxy group is missing. We wished to test whether Cyp140A7 might oxidise an alternative