Investigation of Highly-Reducing Polyketide Synthase Enzymes That Produce the Fungal Polyketides Lovastatin, Fumonisin Bj, and Hypothemycin
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University of Alberta Investigation of Highly-Reducing Polyketide Synthase Enzymes that Produce the Fungal Polyketides Lovastatin, Fumonisin Bj, and Hypothemycin by Jesse W.-H Li A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry © Li, W.-H. Jesse Spring, 2011 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. 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Canada Abstract This thesis focuses on the highly-reducing polyketide synthase enzymes that biosynthesize three polyketides: lovastatin, fumonisin Bi, and hypothemycin. Lovastatin (1), a cholesterol-lowering drug, is made by the polyketide synthase enzymes LovB and LovC in the fungus Aspergillus terreus. LovB and LovC were cloned and expressed in Saccharomyces cerevisiae (strain BJ5464-NpgA). The catalytic activity of each domain of LovB and its role during biosynthesis was probed in vitro by incubation with a series of cofactors (malonyl-CoA, NADPH, SAM). Full reconstitution of the biosynthetic pathway was achieved when LovB was incubated with enoyl reductase (ER) LovC, malonyl-CoA, NADPH, and SAM to furnish dihydromonacolin L 2, an intermediate in the biosynthesis of 1. Exogenous thioesterase (TE) domain (PKS13 or PKS4) or KOH was required to liberate 2 from LovB. Standards were synthesized to confirm the structures of proposed shunt metabolites from the in vitro assays. The post-PKS enzyme Fum6p is a cytochrome P450 that hydroxylates the fumonisin carbon chain. Fum6p was expressed and purified from yeast, allowing in vitro experiments to probe its substrate specificity. Incubation of Fum6p with cofactors and the substrates stearic acid or sphinganine did not yield any detectable hydroxylated products. However, synthesis of fumonisin Bi mimic (2/?,6/?)-2,6-dimethyldecanoic acid (64), and incubation with Fum6p microsomes furnished four monohydroxylated isomeric products. Synthetic standards were prepared to allow identification of the structures from the enzyme assay. Expression and purification of the hypothemycin (105) polyketide synthases Hpm8 and Hpm3 allowed for analysis of the catalytic activity of each enzyme during the biosynthesis of 105. Hpm8 was tested in a series of in vitro assays to furnish three pyrones. The structures of these pyrones were confirmed through comparison with synthetic standards. Full reconstitution of the biosynthetic pathway was achieved by incubation of Hpm3 with Hpm8 and all cofactors to produce dehydrozearalenol (107a). Acknowledgem ents I would like to acknowledge and express my deepest gratitude to my research supervisor Professor John C. Vederas. His support and encouragement throughout my PhD has allowed me to flourish and mature as a scientist. I am thankful for the bright minds and vibrant personalities in the Vederas group, which has made my time working in the laboratory unforgettable. The rich intellectual environment they have created has prepared me well for the future. I am especially grateful to Dr. David Dietrich and Dr. Jennifer Chaytor for their assistance in proofreading my thesis. I would like to thank the dedicated support staff in the mass spectral services, NMR services, and analytical services for their invaluable assistance with my research projects. I would like to thank all my collaborators and their gifted graduate students for their assistance and expertise with my various research projects. I would like to thank all the friends that I have made in the Department of Chemistry, which has made the time during my PhD truly memorable. I am thankful for the financial support by the University of Alberta. I am indebted to my family, who have supported and encouraged me throughout my life. Finally, I would like to thank Anita who has provided endless love and support throughout this process. I am eternally grateful for her presence in my life. Table of Contents 1 Chapter 1: Introduction to Polyketides Biosynthesis, and Biosynthetic Studies on Lovastatin 1 1.1 Introduction 1 1.2 Types of PKS enzymes 8 1.2.1 Type 1 9 1.2.2 Type II 11 1.2.3 Type III 13 1.3 The fungal polyketide lovastatin and its biological properties 15 1.3.1 Statin drugs and inhibition of cholesterol biosynthesis 15 1.3.2 Lovastatin biosynthesis 17 1.4 Results and discussion 26 1.4.1 Expression and purification of LovB and LovC 26 1.4.2 Investigation of the catalytic activity of pure LovB with malonyl- CoA in the absence of NADPH and SAM 29 1.4.3 In vitro enzyme assay of LovB with malonyl-CoA and NADPH .... 33 1.4.4 In vitro enzyme assay of LovB with malonyl-CoA, NADPH, and SAM 37 1.4.5 Complete reconstitution of the biosynthetic pathway to dihydromonacolin L 39 1.4.6 Substrate specificity of LovC 42 1.4.7 Investigation of interactions between LovB and ER enzymes 43 1.4.8 Synthesis of standards to confirm structures of pyrones 4,5, and 7 produced from the various enzyme assays 47 1.4.9 Synthesis of standards to confirm tetra- and pentaketide ketones 9 and 12 generated from the enzyme assay 49 1.4.10 Synthesis of desmethyl-dihydromonacolin L (32) standard to confirm its structure from the enzyme assay 50 1.4.11 Role of truncated NRPS section in lovastatin biosynthesis 57 1.4.12 In vitro studies with purified LovC 63 1.4.13 In vitro fluorometric enzyme assay 70 2 Chapter 2: Biosynthetic Studies on Fumonisin Bi 76 2.1 Fumonisin Biosynthesis 76 2.2 Cytochrome P450 and Fum6p 81 2.3 Results and discussion 84 2.3.1 In vitro experiments with Fum6p 84 3 Chapter 3: Biosynthetic Studies on Hypothemycin 99 3.1 Hypothemycin Biosynthesis 99 3.2 Results and discussion 103 3.2.1 In vitro experiments with Hpm8 103 3.2.2 In vitro experiments with Hpm8 and Hpm3 107 4 Experimental procedures 114 4.1 General experimental methods 114 4.1.1 Reagents, solvents, and solutions 114 4.1.2 Purification Techniques 114 4.1.3 Instrumentation for compound characterization 115 4.2 Synthesis and characterization of compounds 116 (3E, 5E, 7£)-Nona-3, 5, 7-trien-2-one (9) 116 (2E, 4E, 6£)-Ar-Methoxy-iV-methylocta-2,4, 6-trienamide (10) 117 {IE, 4E, 6E)-Octa-2,4, 6-trienal (11) 118 (3E, 5E, IE, 9£)-Undeca-3, 5, 7,9-tetraen-2-one (12) 119 2-(3-Bromopropyl)-l,3-dioxolane (14) 120 2-[(5E, 7£)-Nona-5, 7-dienyl]-l, 3-dioxolane (16) 121 (6E, 8£)-Deca-6, 8-dienal (17) 122 (2E, 8E, 10£)-Dodeca-2, 8,10-trienal (19) 123 (15, 25,4a/?, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-octahydronaphthalene-l- carbaldehyde (21) 124 Ethyl 3-[( 15,25,4a/?, 8a5)-2-methyl-l, 2, 4a, 5, 6, 7, 8, 8a- octahydronapthalen-l-yl]acrylate (24) 126 Methyl 3-[(15, 25, 4a/?, 8a5)-2-methyl-l, 2,4a, 5, 6, 7, 8, 8a- octahydronapthalen-l-yl]propanoate (25) 127 3-[(15,25,4a/?, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a-octahydronapthalen-l- yl]propanal (27) 128 (/?)-l-[(15,25,4aR, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a- octahydronapthalen-l-yl]hex-5-en-3-ol (28) 130 (/?)-l-[(15,25,4aR, 8a5)-2-Methyl-l, 2,4a, 5, 6, 7, 8, 8a- octahydronapthalen-l-yl]hex-5-en-3-yl aery late (29) 131 (/?)-6-[2-Ethyl]-5, 6-dihydro-2//-pyran-2-one