Parallel Pathways and Alternative Macrocyclizations in the Biosynthesis of Largimycins by Cytochrome P450 Minus Mutants of Streptomyces argillaceus.

Largimycins are a recently discovered group of hybrid nonribosomal peptide-polyketide compounds belonging to the leinamycin family of natural products. They are produced in Streptomyces argillaceus by the lrg biosynthetic gene cluster, which includes three cytochrome P450 encoding genes. By characterizing the compounds accumulated by mutants lacking these three genes, we have determined their essential roles in largimycin biosynthesis. Specifically, LrgC2 is involved in converting an olefinic exomethylene group into an oxo (ketone) group at position C-9, while LrgC1 is responsible for forming an epoxide on the C-3 side chain. Also, it is proposed that LrgC3 acts at an early stage of the biosynthetic pathway, probably hydroxylating an alanine residue during peptide assembly. The chemical structure of the compounds produced by lrgC1- and lrgC2-minus mutants suggests that a hydrolytic dehalogenation step proceeds before the peptide-polyketide chain is off-loaded from the polyketide synthase LrgJ. Remarkably, some compounds generated by these mutants bear a C-3 side chain that is cyclized into a unique β-thiolactone ring, a structural feature not seen before in natural products. Furthermore, this work has revealed alternative ways of peptide-polyketide chain off-loading by macrocyclization, unveiling the unprecedented flexibility of the thioesterase domain of LrgJ for selecting different intramolecular nucleophiles.