Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase

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 the lovastatin nonaketide synthase (LovB) from an engineered strain...

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Bibliographic Details
Published in:	Science (American Association for the Advancement of Science) 2009-10, Vol.326 (5952), p.589-592
Main Authors:	Ma, Suzanne M, Li, Jesse W.-H, Choi, Jin W, Zhou, Hui, Lee, K.K. Michael, Moorthie, Vijayalakshmi A, Xie, Xinkai, Kealey, James T, Da Silva, Nancy A, Vederas, John C, Tang, Yi
Format:	Article
Language:	eng
Subjects:	Adenosylmethionine Aspergillus Aspergillus - enzymology Aspergillus - genetics Aspergillus - metabolism Aspergillus terreus Biocatalysis Biological and medical sciences Biosynthesis Biotechnology Catalytic Domain Cellular biology Chemical engineering Cloning, Molecular Condensation Enzymes Fundamental and applied biological sciences. Psychology Fungal Proteins - metabolism Fungi Gene expression Ketones - metabolism Lactones Lactones - metabolism Lovastatin - biosynthesis Malonyl Coenzyme A - metabolism Mathematical functions Methods. Procedures. Technologies Molecular Structure Multienzyme Complexes - metabolism NAD - metabolism Naphthalenes - metabolism Polyketide Synthases - chemistry Polyketide Synthases - genetics Polyketide Synthases - isolation & purification Polyketide Synthases - metabolism Polyketides Protein engineering Pyrones Pyrones - metabolism Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism S-Adenosylmethionine - metabolism Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Substrate Specificity Tailoring Yeast
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Summary:	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 the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our 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 of the functions and structures of this family of enzymes.
ISSN:	0036-8075 1095-9203