Biosynthetic regulation of the major opiates in Papaver somniferum Fergus Meade Doctor of Philosophy University of York Biology September 2015 Abstract Opium poppy, Papaver somniferum, is the sole source of the analgesic alkaloids morphine and codeine as well as thebaine, a precursor for semi-synthetic opiates. T6ODM (thebaine 6- O-demethylase) and CODM (codeine O-demethylase) are dioxygenases involved in morphine biosynthesis and represent promising targets for metabolic engineering of the morphinan alkaloid pathway through reverse genetic screening. An EMS (ethyl methanesulfonate)- mutagenised population of a morphine accumulating cultivar (>4000 plants) was screened for mutations in CODM and T6ODM. Although nonsense mutations were found in both, complete metabolic blocks and codeine and thebaine were not observed owing to the presence of multiple copies of these genes in the genome. Crosses and further mutagenesis were attempted to produce new cultivars of opium poppy with increased yields of codeine and thebaine. 2 Table of Contents ABSTRACT .............................................................................................................................................. 2 TABLE OF CONTENTS ......................................................................................................................... 3 LIST OF FIGURES ............................................................................................................................... 11 LIST OF TABLES ................................................................................................................................ 15 ACKNOWLEDGEMENTS .................................................................................................................. 17 AUTHOR’S DECLARATION ............................................................................................................. 18 CHAPTER 1- INTRODUCTION ....................................................................................................... 19 1.1 THE OPIUM POPPY, PAPAVER SOMNIFERUM L. .................................................................................... 19 1.2 HISTORICAL IMPORTANCE OF OPIUM POPPY ........................................................................................ 20 1.3 BENZYLISOQUINOLINE ALKALOIDS ......................................................................................................... 23 1.3.1 Alkaloids .................................................................................................................................................... 24 1.3.2 Society’s use of BIAs ............................................................................................................................. 25 1.3.3 Roles of BIAs in the plant ................................................................................................................... 26 1.4 BIOSYNTHESIS OF BIAS IN OPIUM POPPY .............................................................................................. 27 1.4.1 Structural diversity from a common origin .............................................................................. 28 1.4.2 Benzophenanthridine and protoberberine biosynthesis .................................................... 29 1.4.3 Phthalideisoquinoline biosynthesis .............................................................................................. 30 1.4.4 Promorphinan and morphinan biosynthesis ........................................................................... 30 1.4.5 Enzymes involved in BIA metabolism .......................................................................................... 32 1.4.6 Sites of BIA biosynthesis and storage .......................................................................................... 33 1.5 CODM AND T6ODM ARE 2-ODDS INVOLVED IN O-DEMETHYLATION IN ALKALOID METABOLISM ..................................................................................................................................................................................... 35 1.5.1 O-demethylation in plant metabolism ........................................................................................ 36 1.5.2 2-ODDs are involved in both primary and secondary metabolism in plants ............ 36 1.5.3 Broader roles of T6ODM, CODM and PODA in opium poppy ............................................ 40 3 1.5.4 Regulation of CODM and T6ODM .................................................................................................. 42 1.6 SUPPLY OF OPIATES .................................................................................................................................. 42 1.6.1 Controlled substances ......................................................................................................................... 43 1.6.2 Tasmania .................................................................................................................................................. 43 1.6.3 Other producers ..................................................................................................................................... 44 1.6.4 Potential alternative BIA production systems ......................................................................... 44 1.7 POPPY BREEDING ...................................................................................................................................... 46 1.7.1 Requirement for varieties with high yields of codeine ........................................................ 47 1.7.2 Requirement for varieties with high yields of thebaine and oripavine ........................ 49 1.7.3 GSK Australia’s opiates division ..................................................................................................... 50 1.7.4 Chemical sprays used by the poppy industry ........................................................................... 51 1.8 SOURCES OF GENETIC VARIATION FOR BREEDING ............................................................................... 51 1.8.1 Genetically engineered opium poppy........................................................................................... 52 1.8.2 Chemical mutagenesis ........................................................................................................................ 52 1.9 TARGETING INDUCED LOCAL LESIONS IN GENOMES (TILLING) ..................................................... 56 1.9.1 Choice of TILLING platforms ........................................................................................................... 56 1.9.2 Applications of TILLING outside of model plants ................................................................... 58 1.10 SUMMARY AND AIMS .............................................................................................................................. 58 CHAPTER 2- METHODS................................................................................................................... 62 2.1 CHARACTERISATION OF GENE TARGETS ................................................................................................ 62 2.1.1 Cloning of full length PCR products and sequencing ............................................................ 63 2.1.2 Plasmid extraction ............................................................................................................................... 64 2.1.3 PCR product purification ................................................................................................................... 64 2.1.4 Preparation of samples for sequencing ...................................................................................... 65 2.1.5 BAC isolation ........................................................................................................................................... 65 2.1.6 LongAmp PCR ......................................................................................................................................... 66 2.1.7 RNA extraction ....................................................................................................................................... 66 2.1.8 cDNA preparation ................................................................................................................................. 68 4 2.2 SAMPLE TRACKING .................................................................................................................................... 68 2.3 REVERSE GENETIC SCREEN ..................................................................................................................... 69 2.3.1 Ethyl methanesulfonate (EMS) mutagenesis ........................................................................... 69 2.3.2 TILLING populations ........................................................................................................................... 70 2.3.3 Plant growth conditions .................................................................................................................... 71 2.3.4 DNA extraction ....................................................................................................................................... 71 2.3.5 Normalisation of DNA plates ........................................................................................................... 72 2.3.6 Primer design for TILLING ..............................................................................................................