Research Article: The biosynthetic pathway to ossamycin, a macrocyclic polyketide bearing a spiroacetal moiety

Date Published: April 30, 2019

Publisher: Public Library of Science

Author(s): Oksana Bilyk, Markiyan Samborskyy, Peter F. Leadlay, Marie-Joelle Virolle.


Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster. The presence in the cluster of a specific crotonyl-CoA carboxylase/reductase homologue suggests that the 5-methylhexanoate extension unit used in construction of the macrocyclic core is incorporated intact from the unusual precursor isobutyrylmalonyl-CoA. Surprisingly, the modular polyketide synthase uses only 14 extension modules to accomplish 15 cycles of polyketide chain extension, a rare example of programmed iteration on a modular polyketide synthase. Specific deletion of genes encoding cytochrome P450 enzymes has given insight into the late-stage tailoring of the ossamycin macrocycle required for the attachment of the unusual 2,3,4,6-deoxyaminohexose sugar l-ossamine to C-8 of the ossamycin macrocycle. The ossamycin cluster also encodes a putative spirocyclase enzyme, OssO, which may play a role in establishing the characteristic spiroketal moiety of the natural product.

Partial Text

Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is a cytotoxic polyketide first reported in 1965 [1]. It is one of a family of 22- to 26-membered macrocyclic polyketides whose structural hallmark is a 6,6-spiroacetal (1,7-dioxaspiro[5,5]-undecanyl) moiety welded to one side of the macrocycle (Fig 1). The best-known and most widely-studied of these compounds are the 26-membered oligomycins/rutamycins [2], potent inhibitors of the mitochondrial F1F0-ATPase [3]. Various other macrocyclic polyketides related to the oligomycins/rutamycins have been isolated (Fig 1) by screening for antifungal, immunosuppressive or cytotoxic bioactivity, including for example the 24-membered dunaimycins [4], and the 22-membered cytovaricin [5], A82548A (also known as yokonolide B) [6], yokonolide A [7], phthoramycin [8], ushikulides A and B [9] and kaimonolide A [10], all produced by Streptomyces spp. and many of them by different strains of S. diastatochromogenes. The chemical structures of these compounds have been established by detailed NMR analysis. Also, assignment of relative and absolute stereochemistry at the multiple stereocentres has been achieved for representative compounds through single-crystal X-ray crystallography and elegant total synthesis [6, 11–14]. This has disclosed the striking structural consistency between the compounds of Fig 1. Even more remarkably, comparison [11,13] of the X-ray crystal structures of cytovaricin and rutamycin A has shown that, despite the differences in ring size and in substituents, their aglycones are superimposable. Consistent with this, cytovaricin and ossamycin act as selective cytotoxic agents through inhibition of the Fo component of F1F0-ATPases, the known target of oligomycin [15].

Apart from the oligomycin biosynthetic gene cluster [62] the genes and enzymes for biosynthesis of 22- to 26-membered macrocyclic polyketides bearing a spiroacetal moiety have remained unexplored. We have now identified the biosynthetic gene cluster for ossamycin biosynthesis in the whole-genome sequence of S. hygroscopicus var. ossamyceticus Bioinformatic analysis of the cluster strongly suggests that programmed iteration of a specific extension module on the modular PKSs is required to furnish the 24-membered macrocycle of ossamycin. Such exceptions to the co-linearity rule are rare and offer insight into a possible mechanism for evolution of modular polyketide megasynthases. It is tempting to suggest that the ossamycin PKS evolved from an assembly-line catalysing production of a 22-membered macrocycle (Fig 1) by local mutation within the PKS, allowing a freshly-elongated intermediate to reacylate the KS domain and undergo a second identical cycle of chain extension. It will be of considerable interest to sequence and compare the PKS genes for a naturally-occurring 22-membered macrocyclic polyketide.




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