University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2015-03-16 Characterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum) Beaudoin, Guillaume Arthur Welch Beaudoin, G. A. (2015). Characterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum) (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25284 http://hdl.handle.net/11023/2115 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Characterization of Oxidative Enzymes Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in Opium Poppy (Papaver somniferum) by Guillaume Arthur Welch Beaudoin A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA MARCH, 2015 © Guillaume Arthur Welch Beaudoin 2015 Abstract Benzylisoquinoline alkaloids (BIAs) are a large group of nitrogen-containing specialized metabolites. Opium poppy (Papaver somniferum) is an important pharmaceutical plant and has been cultivated for thousands of years for its analgesic constituents: the morphinan BIAs codeine and morphine. In addition, opium poppy produces other BIAs with biological activities, such as the vasodilator papaverine, the potential anti-cancer drug noscapine and the antimicrobial agent sanguinarine. The objective of this work was to identify and characterize oxidative enzymes involved in BIA biosynthesis, with a specific focus on cytochromes P450 (P450s). The biosynthesis of sanguinarine from (S)-reticuline requires four P450s. Our first objective was to identify these unidentified P450s in opium poppy. Our approach was based on the coordinated induction of all sanguinarine biosynthetic enzyme transcripts and proteins in elicited opium poppy cell suspension cultures. Using data from a previous microarray study, we identified 12 uncharacterized inducible P450s. Recombinant expression in Saccharomyces cerevisiae revealed that 4 of these enzymes (CYP719A20, CYP719A25, CYP82N3 and CYP82N4) were involved in sanguinarine biosynthesis. Unexpectedly, in planta silencing using virus-induced gene silencing showed that plants suppressed in these P450s transcripts have significantly increased levels of sanguinarine and dihydrosanguinarine. Many changes in the accumulation of sanguinarine biosynthetic intermediates were also detectable. In CYP82N3- and CYP82N4-silenced plants, we detected a significant accumulation of some alkaloids in roots and a significant reduction of some of these in latex. Previous silencing of the penultimate step in morphine biosynthesis, codeinone reductase (COR), showed a dramatic decrease in morphinan alkaloids, as well as a significant ii increase in reticuline, the central BIA branch point intermediate. A search for similar enzymes that may be co-silenced revealed a P450 reductase fusion. Further in vitro and in planta characterization showed that this enzyme is responsible for the final unidentified and first committed steps in morphine biosynthesis, the epimerization of (S)-reticuline. Papaver rhoeas, which does not accumulate morphinan alkaloids, possesses this enzyme as two polypeptides. The significance of this difference remains to be studied. We were also able to identify a paralog of COR which can catalyze the isomerization of many morphinans, potentially opening new routes to manufacture novel opiates. iii Acknowledgements I would first like to thank the people that kept me on track to finish this work. If it wasn’t for mental health breaks with my parents, siblings, Mylène, Theo, Fred, my grandmother and others, my time at the University of Calgary might have gone very differently. Not to mention the invaluable contributions friends in no particular order such as Glen, Ola, Donald, Alexis, Scott, Crystal and Mat in Calgary, as well as Keenan, Lauren and Natalie in Montreal and many other friendly people there and elsewhere! I would also like to thank the people that gave me the technical skills that helped me complete the work in this thesis. Dre. Johanne Tremblay and Suzanne Cossette at the CHUM gave me the background to all the molecular biology skills I needed. The chemistry skills I gained working with Dr. Oswy Pereira and the rest of the group at ViroChem Pharma as well as the protein work I did with Dr. Joanne Turnbull at Concordia also have me an enormous head start when I began this work. I would also like to thank Dr. Isabel Desgagné-Penix and Dr. Eun-Jeong Lee for their general technical help, as well as Scott Farrow with anything to do with analytical chemistry. Donald Dinsmore and Crystal Bross were also excellent people to discuss all aspects of on-going work and gave many helpful comments throughout my time in Calgary. Without them, much of this work would have taken much longer. Although each chapter has its own acknowledgements, some of the most discoveries in this work would likely not have occurred if it wasn’t for discussions with Scott Farrow. It’s also worth noting that without scholarships from the University of Calgary, the National Sciences and Engineering Research Council of Canada, Alberta Ingenuity – Technology Futures and the fonds Québécois de la recherché sur la nature et les technologies, there would not have been nearly as much time invested in this work. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... xi List of Figures .................................................................................................................. xiii List of Symbols, Abbreviations and Nomenclature ....................................................... xviii Epigraph ........................................................................................................................... xxi CHAPTER 1: INTRODUCTION ........................................................................................1 1.1 Plant specialized metabolism .....................................................................................1 1.2 Benzylisoquinoline alkaloids in opium poppy .....................................................3 1.3 Biosynthesis of the major alkaloids in opium poppy .................................................4 1.3.1 Many reactions from a limited number of enzyme families ..............................4 1.3.2 (S)‑Reticuline ....................................................................................................5 1.3.3 Papaverine .........................................................................................................9 1.3.4 Morphine .........................................................................................................10 1.3.5 Protoberberines, protopines and sanguinarine .................................................13 1.3.6 Noscapine ........................................................................................................15 1.3.7 Other alkaloids .................................................................................................16 1.4 Regulation of alkaloid metabolism ..........................................................................17 1.4.1 Cellular localization and transport ...................................................................17 1.4.2 Subcellular compartmentalization and trafficking ..........................................18 1.4.3 Hormonal regulation ........................................................................................22 1.4.4 Transcriptional regulation ...............................................................................23 1.5 Metabolic engineering .............................................................................................24 1.5.1 Plants ...............................................................................................................24 1.5.2 Microbes ..........................................................................................................25 1.6 Cytochromes P450 ...................................................................................................26 1.7 Strategies for triage and discovery of novel genes ..................................................28 1.7.1 Traditional approach ........................................................................................28 1.7.2 Homology-based cloning .................................................................................29 1.7.3 “Guilt by association” ......................................................................................30