Transient expression for engineering triterpenoid diversity in plants

Reed, James (2016) Transient expression for engineering triterpenoid diversity in plants. Doctoral thesis, University of East Anglia.

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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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Megan Ruddock
Date Deposited: 15 May 2018 10:34
Last Modified: 31 Dec 2019 01:38


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