Biosynthesis of the Allylmalonyl-CoA Extender Unit for the FK506 Polyketide Synthase (PKS) Proceeds Through a Dedicated PKS and Facilitates the Mutasynthesis of Novel Analogs

The allyl moiety of the immunosuppressive agent FK506 is structurally unique amongst polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multi-step enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogs, namely 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogs.