Structural and Biosynthetic Studies of Microbial Natural Products A thesis submitted for the degree of . Doctor of Philosophy by Maree Louise Burgess School of Biochemistry and Molecular Genetics University of New South Wales June 1998 Table of Contents ACKNOWLEDGEMENTS ............................................................................................................................... i ABSTRACT ................................................................................................................................................. ii PART A SECONDARY METABOLISM OF OMPHALOTUS NIDIFORMIS ................................ 1 CHAPTER 1 AN INTRODUCTION TO SECONDARY METABOLISM AND OMPHALOTUS NIDIFORMIS .............2 Secondary Metabolites ................................................................................................................................ 2 Sesquiterpenes ............................................................................................................................................. 3 Humulene-Derived Sesquiterpenes ............................................................................................................. 4 Sesquiterpene Cyclases ............................................................................................................................... 6 Omphalotus nidiformis ................................................................................................................................ 6 CHAPTER 2 0MPHALOTUS NIDIFORMIS METABOLITES ........................................................................... 9 Introduction ...................................................................................................................................... 9 Results and Discussion ................................................................................................................... 22 0. nidiformis Cultures ............................................................................................................................... 23 Production of Secondary Metabolites in Surface Culture ......................................................................... 25 Reproducibility of Growth in Shake Culture ............................................................................................. 25 Survey of 0. nidiformis Secondary Metabolites ........................................................................................ 30 Detailed NMR Spectroscopy of Selected Structural Types ....................................................................... 39 Experimental .................................................................................................................................. 73 Strains of 0. nidiformis ............................................................................................................................. 73 Culturing ................................................................................................................................................... 73 Thin Layer Chromatography ..................................................................................................................... 74 Extraction and Comparison of Fruiting Body Metabolites ........................................................................ 75 Production of Secondary Metabolites in Surface Culture ......................................................................... 76 Quantification ofilludin Sand Illudin M .................................................................................................. 76 Time Course of Secondary Metabolite Production ................................................................................... 77 A Survey of 0. nidiformis Secondary Metabolites .................................................................................... 77 CHAPTER 3 FEEDING STUDIES ............................................................................................................ 92 Introduction .................................................................................................................................... 92 Labelling Studies ....................................................................................................................................... 92 Illudin Biosynthesis ................................................................................................................................... 94 Results and Discussion ................................................................................................................. 100 Acetate Feeding Studies .......................................................................................................................... 100 Incorporation of Labelled Acetates into Illudins ..................................................................................... 101 Proposed Mechanism of Biosynthesis of Illudins ................................................................................... 131 Experimental ................................................................................................................................ 139 2H and 13C Labelled Acetate Feedings .................................................................................................... 139 NMR Analyses of Labelled Metabolites ................................................................................................. 140 CHAPTER4 CELL-FREE STUDIES ...................................................................................................... 143 Introduction ......................................................................................., ..... ,;.::: ................................ 143 Characterisation of Sesquiterpene Cyclases ........................................... :::·.. .':.·: ... : .................................... 143 Cell-Free Studies of Sesquiterpene Biosynthesis .................................................................................... 150 Results and Discussion ................................................................ ::.:.':'. .... :......... :........................... 155 Synthesis ofFarnesyl Diphosphate ..................................................... :............. ,, .... : ..... , ......................... 155 Preparation of a Cell-Free Extract of 0. nidiformis ............................. :... :.:: ..... :... ~ ................................ 157 Table of Contents Humulene Cold-Trap .............................................................................................................................. 158 Characterisation of the Products of the Cell-Free System ....................................................................... 159 Time-Course of ~-6-Protoilludene Production ........................................................................................ 166 Mg2+ Requirement of the Cyclase ........................................................................................................... 167 Assay for Cyclase Activity ...................................................................................................................... 169 Determination of the Isoelectric Point of the Cyclase ............................................................................. 170 Purification of the Sesquiterpene Cyclase ............................................................................................... 17 4 Experimental ................................................................................................................................ 181 Synthesis of [l-3H] Farnesyl Diphosphate .............................................................................................. 181 Synthesis of Unlabelled FPP ................................................................................................................... 185 Synthesis of(E,Z)- a-, (Z,Z)- a-, and (Z)- ~ - Farnesenes ...................................................................... 186 Preparation of a Cell-Free System from 0. nidiformis ............................................................................ I 86 Incubation of Cell-Free Preparations with [l-3H]-FPP ........................................................................... 188 Tritium-Labelled Products of the Cell-Free System ................................................................................ 188 Humulene Cold Trap ............................................................................................................................... 189 Characterisation of the Products of the Cell-Free System ....................................................................... 189 Enzyme Assays ....................................................................................................................................... 191 Requirement of Mg2+ for Cyclase Activity ............................................................................................. 192 Protein Estimation using the Bradford Dye-Binding Method ................................................................. 193 Isoelectric Point Determination ............................................................................................................... 193 Cyclase Purification ................................................................................................................................ 195 PART B BACTERIAL CAROTENOIDS ........................................................................................