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...

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A convergent, nine‐step (LLS), enantioselective synthesis of α‐cyclopiazonic acid and related natural products is reported. The route features a) an enantioselective aziridination of an imine with a chiral sulfur ylide; b) a bioinspired (3+2)‐cycloaddition of the aziridine onto an alkene; and c) installation of the acetyltetramic acid by an unprecedented tandem carbonylative lactamization/N−O cleavage of a bromoisoxazole.

An efficient, high yielding synthesis of ethyl 5-hydroxymethyl-3-methylisoxazole-4-carboxylate has been developed, based on a procedure by Gelin which involves reaction of ethyl acetoacetate with chloroacetyl chloride followed by treatment with hydroxylamine hydrochloride. The product of this reaction was then converted into the bromide and reacted with tetrahydrothiophene to give sulfonium salts in up to 71% overall yield (from ethyl acetoacetate). The synthesis is suitable for use with a chiral sulfide and for large-scale use. The synthesis of ethyl 5-formyl-3-methyl-4-isoxazolecarboxylate and the corresponding tosylhydrazone are also reported. These isoxazoles are starting materials for a proposed convergent, biomimetic synthesis of α-cyclopiazonic acid.

Ac onvergent, nine-step (LLS), enantioselective synthesis of a-cyclopiazonic acid and related natural products is reported. The route features a) an enantioselective aziridination of an imine with ac hiral sulfur ylide;b )abioinspired (3+ +2)-cycloaddition of the aziridine onto an alkene;a nd c) installation of the acetyltetramic acid by an unprecedented tandem carbonylative lactamization/NÀOc leavage of ab romoisoxazole.