A Branched Extender Unit Shared between Two Orthogonal Polyketide Pathways in an Endophyte

The large polyketide family of natural products impressively demonstrates how nature generates a wealth of diverse and complex compounds from simple building blocks. During the past two decades, a large body of knowledge on the various types of polyketide synthases (PKS) and their modes of action has been gained. In almost all cases, the polyketide backbone is assembled exclusively from malonyl(MCoA) and methylmalonyl-CoA (mMCoA) units, and structural diversity is primarily governed through variations in chain length, processing of the b-keto group, versatile cyclizations, and numerous post-PKS tailoring reactions. While extender units other than MCoA and mMCoA are scarce, several complex polyketides in part composed of substituted malonyl building blocks have recently been discovered. Furthermore, it is known that modular polyketide synthases employ acyltransferase (AT) domains for the selection of the correct extender unit, and elegant studies have demonstrated that it is possible to graft alternative building blocks into complex polyketide backbones through domain swapping, mutagenesis, and mutasynthesis. Thus, with the aim to increase natural polyketide diversity, much effort is currently devoted to enlarging the repertoire of possible biosynthetic building blocks. Apart from ethylmalonyl-CoA and the recently discovered methoxymalonyl and hydroxyand aminomalonyl building blocks, there is evidence for the utilization of longer alkyl residues that account for the propenyl and hexyl side chains of FK506 and thuggacin, respectively. Although isotope labeling experiments have pointed to an a-methylbutyryl-derived extender in the polyoxypeptin pathway, to date the molecular basis for the formation of branched-chain malonyl units has remained fully unknown. Herein we report the discovery and biosynthetic origin of an unprecedented isobutylmalonyl (ibMCoA) extender unit, which is surprisingly employed in two unrelated polyketide pathways in an endophytic Streptomyces species. In the course of evaluating the metabolic capabilities of mangrove endophytes, we have discovered a set of unusual ansa macrolides, named divergolides, which differ markedly in their overall architecture but share an unprecedented branched side chain. This unusual residue suggested that the polyketide backbone has incorporated a novel type of branched extender unit that is likely derived from isobutyrate. Initial attempts to support this hypothesis through stableisotope labeling failed because of the minute amounts of divergolides produced. We eventually succeeded in feeding experiments with [D7]isobutyrate using a highly sensitive HPLC–HRMS (orbitrap) setup. In this way, we identified the expected mass shifts for the major components of the divergolide complex (e.g., divergolide C, 1), thus providing evidence that the extender unit originates from the branched isobutyrate precursor (Figure 1). Surprisingly, a closer inspection of the endophytes metabolome in the presence or absence of [D7]isobutyrate