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Transient Expression for Engineering Triterpenoid Diversity in Plants

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Transient Expression for Engineering Triterpenoid Diversity in Plants Transient expression for engineering triterpenoid diversity in plants James Ashley Reed A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy © September 2016 John Innes Centre Norwich Research Park Norwich Norfolk United Kingdom © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract The triterpenes are one of the largest and most structurally diverse classes of plant specialised metabolites, with important commercial, agronomical and medical potential. However the structural complexity and low abundance of these compounds in nature presents significant challenges for exploiting this diversity. In recent years, advances in our understanding of triterpene biosynthesis in various plant species have greatly facilitated the ability to produce these compounds in other hosts. Plant-based host systems hold great promise because they may provide substrates, cofactors and subcellular compartmentalisation mechanisms facilitating engineering production of complex triterpenoids. Agroinfiltration is a rapid and scalable process that enables transient expression of biosynthetic genes in amenable plants such as Nicotiana benthamiana. This technique holds great promise for selective production and modification of triterpenes, but studies in this area have been limited. In this PhD thesis, transient expression is used to produce and oxidise triterpenes in N. benthamiana. Triterpene yields are improved through expression of rate-limiting genes in the mevalonate pathway (Chapter 3). An enzymatic ‘toolkit’ is established from a collection of previously characterised triterpene biosynthetic genes, allowing production of milligram quantities of a diverse array of simple and oxidised triterpenes in N. benthamiana. Furthermore, combinatorial biosynthesis is utilised to engineer production of novel oxidised triterpenes in planta (Chapter 4). A set of oxidised derivatives of β-amyrin are purified and evaluated for antiproliferative and anti-inflammatory activity in human cell lines, yielding insights into structural features underlying these activities in humans (Chapter 5). Finally, N. benthamiana is used for functional screening of cytochrome P450s implicated in the biosynthesis of agronomically important antifungal triterpene glycosides (avenacins) in oat, leading to identification of new P450s required for disease resistance (Chapter 6). This work highlights the utility of N. benthamiana as triterpene engineering platform and provides a basis for future studies exploring triterpene structure-activity relationships. I Acknowledgements I would like to express a huge thanks to my supervisors, Prof Anne Osbourn at JIC and Prof Maria O’Connell in the School of Pharmacy at UEA both for giving me the opportunity to take on such a unique and fulfilling project, as well as for always giving me fantastic support and advice needed throughout. I would also like to extend additional thanks to Anne for giving me so many opportunities over the last few years to embrace my creativity during this PhD and to learn and develop in areas outside this project. I would also like to say an additional thank you to the members of the Osbourn lab, both current and former for making the last four years such an enjoyable experience. Without your help and talents many aspects of this project would not have been possible. In particular I would like to say a huge thank you to Aymeric Leveau, Tessa Moses, Thomas Louveau and Michael Stephenson both for help in practical and scientific matters but also your support through the tougher times, particularly in the last year. I am also hugely grateful to Lionel Hill and Paul Brett within the metabolite services team at JIC for always taking the time to provide excellent help and advice with analytical chemistry. Particularly, without Paul’s insight into the many complexities of GC-MS this thesis would have turned out very differently. Finally I would like to thank my family, without whose encouragement and good humour I would not be here. In addition, my partner Rhiannon’s family, for all the support they’ve provided during the last four years and the years before. Lastly and most importantly thanks to Rhiannon for her always unwavering support at home and constant encouragement throughout this PhD. II List of common abbreviations AsbAS1 - Avena strigosa β-amyrin synthase CA - Cycloartenol CP - Chloroplast CPMV - Cowpea mosaic virus DD-II - Dammarenediol-II DMAPP - Dimethylallyl diphosphate DOS - Dioxidosqualene EI - Electron-impact ELISA - Enzyme-Linked ImmunoSorbent Assay EpDM - Epoxydammarane EpHβA - 12,13β-epoxy,16β-hydroxy-β-amyrin ER - Endoplasmic Reticulum ESI - ElectroSpray Ionisation FPP - Farnesyl diphosphate FPS - Farnesyl diphosphate synthase GC-MS - Gas Chromatography-Mass Spectrometry GFP - Green Fluorescent Protein GGPP - Geranylgeranyl diphosphate GPP - Geranyl diphosphate GPPS - Geranyl diphosphate synthase HMGR - 3-hydroxy,3-methylglutaryl-CoA reductase HT - Hypertranslatable IPP - Isopentenyl diphosphate III IS - Internal Standard Keap1 - Kelch-like ECH-associated protein 1 LC - Liquid Chromatography LPS - Lipopolysaccharide MEP - 2-C-methyl-D-erythritol 4-phosphate MVA - Mevalonate NFκB - Nuclear Factor Kappa B NMR - Nuclear Magnetic Resonance Nrf2 - Nuclear Factor Erythroid 2 Related Factor 2 OS - Oxidosqualene OSC - Oxidosqualene cyclase P450 - Cytochrome P450 rDA - Retro Diels-Alder RT-PCR - Reverse Transcriptase PCR SAD1 - Avena strigosa β-amyrin synthase SAD2 - Avena strigosa CYP51H10 SQE - Squalene epoxidase SQS - Squalene synthase T-DNA - Transfer DNA tHMGR - truncated HMGR TIC - Total Ion Chromatogram TLC - Thin Layer Chromatography TMS - Trimethylsilyl TNFα - Tumour Necrosis Factor alpha IV Contents Abstract .................................................................................................................... I Acknowledgements .................................................................................................. II List of common abbreviations ................................................................................. III Contents .................................................................................................................. V List of Figures .......................................................................................................... X List of Tables ........................................................................................................ XIII Chapter 1 – General Introduction ............................................................................. 1 1.1 – Plant specialised metabolism ...................................................................... 2 1.2 – Terpenoids: functions in nature and human uses ........................................ 3 1.3 – Triterpenes: Importance and bioactivity. ...................................................... 5 1.4 – Triterpenoid biosynthesis in plants............................................................... 8 1.4.1 – Oxidosqualene cyclisation generates diversity in triterpene scaffolds.. 11 1.4.2 – Oxidation allows further diversification of triterpenes ........................... 13 1.4.3 – Clustering aids identification of new triterpene oxidases ..................... 16 1.5 – Engineering diversity in triterpene biosynthesis ......................................... 22 1.5.1 – Combinatorial biosynthesis ................................................................. 22 1.5.2 – Protein engineering augments combinatorial biosynthesis .................. 25 1.6 – Triterpene production in heterologous hosts .............................................. 26 1.6.1 – Nicotiana benthamiana is an emerging host for producing plant specialised metabolites ................................................................................... 28 1.6.2 – Realising N. benthamiana as a (tri)terpene production platform .......... 29 1.7 – PhD overview: ........................................................................................... 31 Chapter 2 – General materials and methods .......................................................... 32 2.1 – General methods ....................................................................................... 33 2.1.1 – Gateway cloning ................................................................................. 33 2.1.2 – Microbiological techniques .................................................................. 37 2.1.3 – Agroinfiltration ..................................................................................... 38 2.1.4 – General considerations for purification of compounds ......................... 42 V 2.1.5 – GC-MS programs: ............................................................................... 44 2.1.6 – Common reagents and solutions ......................................................... 44 Chapter 3 – Improving triterpene production in N. benthamiana ............................ 46 3.1 – Introduction ............................................................................................... 47 3.1.1 – Engineering early pathway steps in triterpene production ................... 48 3.1.2 – The importance of compartmentalisation in terpene biosynthesis ....... 51 3.1.3 – Aims...................................................................................................
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