THE POLYKETIDE ORIGINS OF CANNABINOIDS IN CANNABIS SATIVA A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Biology University of Saskatchewan Saskatoon By STEVE JOSEPH GAGNE Copyright Steve J. Gagne, October, 2013. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Biology University of Saskatchewan Saskatoon, Saskatchewan, S7N 5E2 i ABSTRACT Phytocannabinoids are the active substances responsible for the medicinal and psychotropic effects of Cannabis sativa. Although the bioactivity of cannabis and its preparations have been known for millennia, several steps in the biosynthetic pathway leading to phytocannabinoids remain unclear. Phytocannabinoids are prenylated resorcylic acids which are formed in specialized plant organs called glandular trichomes. Following the analysis of a pre-generated cannabis trichome cDNA library, a type III polyketide synthase (tetraketide synthase; TKS) was identified and assayed, yielding three major compounds, hexanoyl triacetic acid lactone (HTAL), pentyl diacetic acid lactone (PDAL), and olivetol, yet no resorcylic acid was detected. This lack of resorcylic acid in enzyme assays has instigated the characterization of TKS and a search for putative cyclases in the cannabis trichome cDNA library, and involved protein pulldown, co- immunoprecipitation, and co-assay experiments. These experiments led to the discovery of a novel polyketide cyclase protein named olivetolic acid cyclase (OAC) responsible for the proper cyclization of a polyketide intermediate produced by TKS. This thesis shows that TKS assays conducted with OAC produce olivetolic acid (OA), an intermediate required during the biosynthesis of cannabinoids. The TKS/OAC spatial relationship was also investigated following the creation of fluorescent fusion proteins which show that the enzymes co-localized in vivo when viewed with confocal microscopy. Furthermore, yeast two-hybrid assays using TKS and OAC were performed to establish whether the enzymes physically interact. Finally, an attempt to determine the responsible amino acids involved in OAC’s mechanism was conducted by comparing the activity of single point OAC mutants with the wild-type OAC. Based on the available data, mechanisms for the production of HTAL, PDAL, olivetol, and OA are proposed. ii ACKNOWLEDGEMENTS Although the research presented in this dissertation is my work, this project would not have been possible without the involvement of many people who have ceaselessly provided their valuable support. I would primarily like to thank my supervisor, Dr. Jonathan Page, who offered me the opportunity to tackle the cannabinoid conundrum, and continued to provide encouragement and guidance throughout each process of this work. I’d like to also thank Jon for the important bits of advice he has passed on to me with regard to professionalism and work flow, which will continue to assist me in my future career, whether it is in industry, academia, or government settings. I also warmly thank each member of my committee who have provided important comments that helped to shape this thesis to its current state; each have generously provided flexibility with their time and personally contributed important input during the course of this project; thank you Dr. Gordon Gray, Dr. Chris Todd, and Dr. David Palmer. I also thank the external examiner, Dr. Reinhart Jetter, who has also contributed a lot of significant input during the final stages of my dissertation preparation. I would like to express my sincere gratitude to each lab member, whether past or present, which I had the pleasure to work with during these last few years. This includes Dr. Jake Stout, Dr. Shawn Clark, Enwu Liu, David Konkin, Sandra Polvi, Dr. Zakia Boubakir, Shawn Whitfield, and Nikki Theaker. Aside from providing their invaluable technical expertise, exemplary teaching and guidance, and stellar participation and inspiration, I am deeply appreciative for their friendship, openness, and for being the best lab mates one could ask for. I would also like to thank each and every individual that I had the opportunity to meet at the University of Saskatchewan and the Plant Biotechnology Institute. The environment that iii these people have collectively created is one of highest integrity and good cheer. A special thanks to Dr. Patrick Covello, Darwin Reed, Stephen Ambrose, Dr. Shawn Gibson, Sharla Lozinsky, Dr. Julien Cotelesage, Deidre Wasyliw, Brenda Haug, Dr. Leonid Akhov, Ioannis Mavraganis, and Aaron Karapinka; your precious words have coaxed me to the finish line. I acknowledge the financial support provided to me by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Department of Biology, and the University of Saskatchewan. Lastly, but most importantly, I would like to thank my best friend and wife, Christine, as well as my children, Spring and Sethia Om, for I know well enough that without their enduring love, kindness, and patience, I would not have completed one word of this dissertation. iv TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES ....................................................................................................................... xiii LIST OF FIGURES ..................................................................................................................... xiv LIST OF ABBREVIATIONS .................................................................................................... xviii 1. INTRODUCTION ...................................................................................................................... 1 1.1 Research Goals and Objectives .............................................................................................. 2 2. LITERATURE REVIEW ........................................................................................................... 3 2.1 Overview of Cannabis sativa and Cannabinoids ................................................................... 3 2.1.1 The Biology of Cannabis .................................................................................................. 5 2.1.2 Bioactivity of Cannabinoids ............................................................................................ 11 2.1.3 The Current Understanding of Cannabinoid Biosynthesis.............................................. 11 2.2 Olivetolic Acid Biosynthesis ................................................................................................ 14 2.2.1 Type III PKSs: Diversity, Structure and Mechanism ..................................................... 14 2.2.2 Instances of Olivetolic Acid Biosynthesis ...................................................................... 21 2.2.3 The Biosynthesis of Olivetolic Acid Requires a Polyketide Synthase ........................... 21 2.2.4 Type III PKSs from Cannabis ......................................................................................... 22 2.3 Polyketide Cyclization and DABB Proteins ......................................................................... 25 2.3.1 Polyketide Cyclases ........................................................................................................ 25 v 2.3.1.1 Secondary Polyketide Cyclases ................................................................................. 28 2.3.1.2 A Possible Role for Polyketide Cyclases in Cannabinoid Biosynthesis.................... 29 2.3.2 DABB Proteins ............................................................................................................... 30 2.3.2.1 The Structure and Occurrence of DABB Proteins ..................................................... 31 2.3.2.2 The Function of DABB Proteins ............................................................................... 33 2.3.3 Aldolases ......................................................................................................................... 36 3. THE TKS REACTION