Characterization of the post-PKS modification steps of FK506 biosynthesis

FK506 is a macrocyclic polyketide produced by Streptomyces and widely used to prevent the rejection of organ transplants. The hybrid polyketide synthase/nonribosomal peptide synthetase system biosynthesizes FK506. Although it has been known that the post-PKS tailoring steps of FK506 included C9-oxidation catalyzed by cytochrome P450 hydroxylase (FkbD) and 31-O-methylation by S-adenosylmethionine (SAM)-dependent methyltransferase (FkbM), the detailed biosynthetic intermediate has remained unresolved. In this study, we report a comprehensive characterization of all the FK506 biosynthetic intermediates involved in post-PKS modification based on in-depth NMR, HPLC−ESI-MS/MS, and high-resolution MS (HR-MS) data. Also we have discovered the parallel pathways responsible for the post-modification step in the biosynthesis of FK506. Our results clearly demonstrate that there are two independent biosynthetic routes to FK506, and it has been shown that 9-deoxoFK506 derivatives, 9-deoxo-31-O-demethylFK506 and 9-deoxoFK506, can be used as substrates for FkbD, whereas FkbM can utilize 31-O-demethyl derivatives, 31-O-demethylFK506 and 9-deoxo-31-O-demethylFK506, as substrates. These substrate-flexible post-PKS modification enzymes, FkbD and FkbM, can provide a potential tool for the combinatorial biosynthesis of novel macrolide derivatives.