Synthetic biology and metabolic engineering for the production of monoterpenoids

Abstract

Synthetic biology can contribute to the development of new technologies, products and manufacturing processes in different fields. This technology is based on the rational design and standardisation of approaches applied to biology and cell metabolism. Its most promising and recognised application is in the development of microbial cell factories to produce, for example, biofuels, valuable drugs and commodity chemicals. Terpenoids are the largest class of natural compounds and have a vast application. Microbial cell factories have the potential to displace traditional processes used to produce these molecules, providing a more sustainable alternative. Terpenoids are produced in nature through canonical metabolic pathways which can be long, highly regulated and difficult to engineer in a microbial chassis. This thesis explores synthetic biology and metabolic engineering tools to engineer the alternative isopentanol utilisation (IU) pathway in Escherichia coli. This pathway requires the use of isoprenol and/or prenol alcohols as substrates which are phosphorylated in only three enzymatic steps to produce terpenoid building blocks. The IU pathway was assembled in a linalool-producing strain, and optimisation of the plasmid system, strain, culture conditions and linalool synthase allowed the production of 178 mg/Lorg linalool. Next, new enzymes and variants for each step of the pathway were investigated, together with its ribosome binding site (RBS). High strength RBSs increased linalool titres for the majority of the enzymes tested, and the improved IU pathway increased linalool titres to almost 500 mg/Lorg. These pathways were cloned into the industrially relevant halophile host, Halomonas bluephagenesis, producing 12.5 mg/Lorg linalool. Two possible precursor pathways to produce prenol from leucine were also identified and engineered, although further enzyme and cell strain investigation is necessary to achieve target production. Finally, a new analytical method was developed to detect and quantify eight phosphorylated intermediates of the terpenoid pathways. Liquid chromatography tandem mass spectrometry was used coupled with a hydrophilic interaction system. A new synthetic route to produce deuterium-labelled intermediates was also developed to produce internal standards for the analytical methodology. The alternative pathways and the analytical method developed in this study provide valuable insights and can guide future research in the terpenoid field.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Sam Hay (Supervisor)