Highly-reducing iterative polyketide synthases are large multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of LovB (the Lovastatin Nonaketide Synthase) from an engineered strain of Saccharomyces cerevisiae, and complete reconstitution of its catalytic function in the presence and absence of cofactors (NADPH, SAM) and its partner enzyme, the enoyl reductase LovC. The results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding the functions and structures of this family of enzymes.Nature uses an amazing array of enzymes to make natural products (1). Among these metabolites, polyketides represent a class of over 7000 known structures of which more than 20 are commercial drugs (2). Among the most interesting but least understood enzymes making these compounds are the highly-reducing iterative polyketide synthases (HR-IPKSs) found in filamentous fungi (3). In contrast to the well-studied bacterial type I PKSs that operate in an assembly-line fashion (4), HR-IPKSs are megasynthases that function iteratively by using a set of catalytic domains repeatedly in different combinations to produce structurally diverse fungal metabolites (5). One such metabolite is lovastatin, a cholesterol-lowering drug from Aspergillus terreus (6). This compound is a precursor to simvastatin (Zocor™), a semisynthetic drug that had annual sales of over $4.3 billion prior to loss of patent protection in 2006 (7).Biosynthesis of lovastatin proceeds via dihydromonacolin L (acid form 1; lactone form 2), a product made by the HR-IPKS, LovB (Lovastatin Nonaketide Synthase), with assistance of a separate enoyl reductase, LovC (8) (Fig. 1). LovB is a 335 kDa protein that contains single copies of ketosynthase (KS), malonyl-CoA:ACP acyltransferase (MAT), dehydratase (DH), § To whom correspondence should be addressed. yitang@ucla.edu (Y.T.); john.vederas@ualberta.ca (J.C.V.).Reconstitution of catalytic function provides insight into how multifunctional enzymes synthesize important natural products. This enzyme also catalyzes a biological Diels Alder reaction during the assembly process to form the decalin ring system (10). In vitro studies of LovB (11) have been hampered by inability to obtain sufficient amounts of the functional purified megasynthase from either A. terreus or heterologous Aspergillus hosts. As a result, the programming that governs metabolite assembly by LovB or other HR-IPKSs is not understood. Key aspects that remain to be elucidated include: 1) the catalytic and structural roles of each domain in the megasynthase; 2) substrate specificities of the catalytic domains and their tolerance to...

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature.

Hypothemycin is a macrolide protein kinase inhibitor from the fungus Hypomyces subiculosus. During biosynthesis, its carbon framework is assembled by two iterative polyketide synthases (PKSs), Hpm8 (highly reducing) and Hpm3 (non-reducing). These were heterologously expressed in Saccharomyces cerevisiae BJ5464-NpgA, purified to near homogeneity and reconstituted in vitro to produce (6′S, 10′S)-trans-7′,8′-dehydrozearalenol (1) from malonyl-CoA and NADPH. The structure of 1 was determined by x-ray crystallographic analysis. In the absence of functional Hpm3, the reducing PKS Hpm8 produces and offloads truncated pyrone products instead of the expected hexaketide. The non-reducing Hpm3 is able to accept an N-acetylcysteamine thioester of a correctly functionalized hexaketide to form 1, but it is unable to initiate polyketide formation from malonyl-CoA. We show that the starter unit acyltransferase (SAT) of Hpm3 is critical for crosstalk between the two enzymes and that the rate of biosynthesis of 1 is determined by the rate of hexaketide formation by Hpm8.