(392d) Expression and Reconstitution of Fungal Polyketide Synthases

Hypercholesterolemia is the primary risk factor for coronary heart diseases. By inhibiting 3-hydroxy-3-methylglytaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for catalyzing the committed step in cholesterol biosynthesis, serum levels of cholesterol can be effectively lowered, thus lowering plaque accumulation and decreasing the risk for coronary heart disease. The cholesterol lowering statin natural products, such as the prototypical lovastatin, are the drug therapy of choice in effectively treating hypercholesterolemia. Lovastatin is biosynthesized in its natural host, Aspergillus terreus by an iterative type I fungal polyketide (IPKS). Using the genetically amendable host Saccharomyces cerevisiae, we have cloned and expressed two central enzymes involved in lovastatin biosynthesis: the lovastatin nonaketide synthase LovB (LNKS, 335kDa) and the accessory enoylreductase LovC (40kDa). We demonstrate for the first time successful verification of the activities of LovB and LovC in vitro, essentially reconstituting the upstream biosynthetic pathway in lovastatin biosynthesis. We verified 1) the recombinant LovB retained complete biosynthetic capabilities of all their catalytic domains; 2) LovB megasynthase alone afforded a series of truncated, elongated polyketide products derived from malonyl-CoA; and 3) LovB and LovC can interface in vitro towards the coordinated catalysis of the lovastatin precursor dihydramonacolin L. Our work paves the way for detailed mechanistic analysis of the programming rules associated with this large, yet cryptic family of fungal megasynthases. The successful reconstitution study also enables combinatorial biosynthesis of the next generation of therapeutically important statin molecules.