BIOSYNTHESIS OF INDOLE-DITERPENOIDS AND OTHER ISOPRENOIDS BY THE RYEGRASS ENDOPHYTE, Acremonium loliae A thesis presented in fulfilment of the requirements for the degree of Ph.D. of the University of London and the Diploma of Imperial College. CHRISTOPHER MICHAEL VEEDON 1987 Department of Biochemistry Imperial College of Science and -Technology London SV7 2AY 2 ABSTRACT Acremanium loliae, the ryegrass endophyte, is implicated in causing ryegrass staggers (RGS) - a neurological syndrome of ruminants grazing ryegrass-dominant pastures, particularly in New Zealand. Tremorgenic substituted indole-diterpenoids, lolitrems A-D, isolated from A. loliae-infected perennial ryegrass iLolium perenne), are incriminated as the toxins responsible for RGS. However, the precise biosynthetic contribution to lolitrems made by the fungus and the grass is unknown, and the principal objective of this study has been to explore this, with the long-term aim of divising a strategy to avoid RGS. Although lalitrems were never detected in culture, A. loliae was shown to biasynthesise the closely related indole-diterpenoid tremorgen, paxilline. Paxilline was produced in certain culture conditions, long after growth was complete, and was detectable in culture only for a few hours on one day. Paxilline was also found, for the first time, in endophyte-infected ryegrass seed containing lolitrem B (the most abundant lolitrem). Direct transformation of 1^C-paxilline to lolitrem B could not be demonstrated, either in A. loliae culture or in endophyte-infected ryegrass seedlings. Nevertheless, paxilline remains a potential biosynthetic intermediate of lolitrems, implying a major role for A. loliae in the biosynthesis of lolitrems. A. loliae> as well as the main paxilline-producing fungus, Penicillium paxillir was shown to biosynthesise several indole- diterpenoids, closely related in structure to paxilline. These include one, that could be a biosynthetic precursor of paxilline, and another that is a paxilline isomer. 14Qf-hydroxypaxilline was also isolated and 3 was shown to arise directly from 1 'C-paxilline given to A. loliae cultures. It is postulated that lolitrem A, which has not formerly been characterized further than oxo-lolitrero B, may be 1 AO£r hydroxylolitrera B. 14G*-hydroxylatian of paxilline gave much reduced tremorgenic activity and is the first recorded 19G*-hydroxy indole- diterpenoid that is not a \ patent tremorgen. Furthermore, the new compounds may allow some revision of the indole-diterpenoid biosynthetic pathway as applied to paspalicine and the paspalinines of Claviceps paspali. Mutagenic treatment of A. loliae led to the isolation of a variant strain, that reproducibly yielded paxilline, whether or not in the special culture conditions found necessary for production by the native endophyte. Detailed characterization of the mutant remains to be carried out. Nevertheless, the feature of consistent paxilline biosynthesis might greatly facilitate the screening for a strain incapable of paxilline biosynthesis, following mutagenic treatment. When re-introduced into ryegrass, such a non-tremorgenic endophyte would theoretically be incapable of causing RGS but might still confer the benefits of native A . loliae. The principal sterol of A. loliae was 5ctf-ergosta-7,22E-dien- 3/3-ol, which is sufficiently uncommon that it can,not only provide a biochemical means of differentiating from the closely related tall fescue endophyte A . coenophialum} but may also enable detection of A. loliae in ryegrass. The indolic metabolite tryptophol was found to be a major constituent of A. loliae culture filtrate and, as a growth promoter of certain plants, may account for the enhanced vigour conferred d h ryegrass by A, loliae. 4 CONTENTS PAGE ABSTRACT .................................................. 2 CONTESTS ................................................... 4 INDEX OF FIGURES........................................... 6 INDEX OF T A B L E S ........................................... 10 ACKNOWLEDGEMENTS . '...................................... 12 1. INTRODUCTION........................................... 13 2. MATERIALS AND METHODS 2.1. Fungal Culture 2.1.1. Source of Fungi .............................. 29 2.1.2. Media ....................................... 31 2.1.3. Inoculation and Incubation ................... 33 2.1.4. Microscopy .................................. 34 2.1.5. Culture Growth Rate Assessment ................ 35 2.2. Sample Component Analysis 2.2.1. Sample Preparation ........................... 35 2.2.2. Chromatography ............................... 36 2.2.3. Spectroscopy ................................ 40 2.3. Use of Metabolic Precursors 2.3.1. Addition and Incubation of 1AC-Radiolabelled Mevalonic Acid and Amino A c i d s ................. 40 2.3.2. Addition and Incubation of Paxilline ............ 41 2.3.3. Autoradiography .............................. 43 2.4. Mutagenic Treatment of A. l o l i a e ...................... 43 3 . RESULTS 3.1. Confirmation of Identity of Isolated Ryegrass Endophyte as Acremonium l o l i a e ....................................44 3.2. A . loliae Culture in Optimum Growth Conditions 3.2.1. Determination of Optimum Growth Conditions .... 43 3.2.2. Products of A,loliae in Optimum Growth Conditions . 51 3.3. Preparation of 1^C-Radiolabelled Paxilline 3.3.1. Penicillium paxilli Culture .................... 82 3.3.2. Additional Compounds Isolated from P. paxilli Culture...................................... 91 3.4. Pectin-Supplemented A. loliae Culture ................ 115 3.5. Incubation of Paxilline with A. loliae 3.5.1. Incubation of Paxilline with A. loliae in Optimum' Growth-Rate Conditions ....................... 125 3.5.2. Incubation of 1^C-Paxilline with A. loliae in Paxilline Production Medium ................ 131 3.6. Tremorgen Investigation in Lolium perenne 3.6.1. Examination of Ryegrass Seeds .................. 139 3.6.2. Introduction of 1^C-Radiolabelled Paxilline into A. loliae-Infected Seedlings .............. 139 3.7. Administration of Indole-Diterpenoids to M i c e .......... 144 3.8. Mutagenic Treatment of A. lo l i a e ....................... 144 4. DISCUSSIOM......................... 147 5. REFERE5CES............................................... 151 6 IflDEX OF FIGURES PAGE Figure 1. The Indole-Diterpenoids .......................... 17 Figure 2. Peramine......................................... 19 Figure 3. Phaseolin......................................... 19 Figure 4. Verruculogen, 15-Acetoxyverruculogen and Fumitremorgin A ....................................24 Figure 5. Proposed Mechanism of Cyclization of Geranyl Geranyl Pyrophosphate in Indole-Diterpenoid Biosynthesis.................. 26 Figure 6. Proposed Mechanism of Attachment of an Isoprenoid Moiety to the Indolic Benzene Ring of Indole- Diterpenoids ..................................... 27 Figure 7. Proposed Mechanism of Lolitrem B Biosynthesis via Paxilline......................................... 28 Figure 8. Emergence of Mui Endophyte from Ryegrass Seedling . 30 Figure 9. Cultures of isolated Nui endophyte and a typical strain of A. l o l i a e ................................ 44 Figure 10. Effect of Incubation Temperature on A, loliae Growth . 49 Figure 11. The Major Acetone-Soluble Products of A. loliae .... 54 Figure 12. Thin Layer Chromatography of Acetone-Soluble Products of A. l o l i a e .............................. 56 Figure 13. High Performance Liquid Chromatography of the Major Acetone-Soluble Products of A . loliae ......... 58 Figure 14. Electron Impact Mass Spectroscopy of the Major Acetone-Soluble Products of A. loliae .............. 62 7 Figure 15. Autoradiographs and Thin Layer Chromatographs of Acetone Extracts of A. loliae Cultures after Incubation with 1AC-Radiolabelled Primary Metabolites...................................... 71 Figure 16. P. paxilli C u l t u r e.............................. 85 Figure 17. Paxilline Standard Curve ......................... 86 Figure 18. Time-Course of Paxilline and Biomass Yield in P. paxilli Culture .................................. 88 Figure 19. Autoradiographs and Thin Layer Chromatographs of Acetone Extracts of P, paxilli Cultures After Incubation with 1AC-Mevalonate and 1^C-Benzene Ring Tryptophan.................................. 89 Figure 20. The Indole-Diterpenoids of A. loliae and P, p a x i l l i..........................................91 Figure 21. Thin Layer Chromatography of Acetone Extracts of A. loliae and P. paxilli Cultures.................. 92 Figure 22. High Performance Liquid Chromatography of the Indole-Diterpenoids of A. loliae and P. paxilli .... 93 Figure 23. El Mass Spectrum of Paxilline (70eV).............. 95 Figure 24. 1HMMR Spectrum of Paxilline (DMSO, 500MHz> ......... 97 Figure 25. Proton-Proton Couplings and Full Stereochemistry of Paxilline.................................... 99 Figure 26. El Mass Spectrum of Prepaxilline-16/3-ol (70eV) .... 100 Figure 27. 1HNMR Spectrum of Prepaxilline-16/3-ol CDMSO, 500MHz)........................................... 102 Figure 28. Proton-Proton Couplings and Full Stereochemistry of Prepaxilline-16j^-ol.............................101 Figure 29. El Mass Spectrum of 140Phydroxyprepaxilline (70eV)........................................... 104 8 Figure 30. 1 HNMR Spectrum of 14Qf-hydroxyprepaxi 11 ine (DMSO, 500MHz)........................................... 106 Figure 31. Proton-Proton Couplings and Full Stereochemistry of 14QJ-hydroxypre paxilline......................... 108 Figure