Synbio Production of Terpenes through Computationally Guided Enzyme Engineering

Abstract

Terpenes, terpenoids, or isoprenoids, are the largest class of natural products. Finding use in many established and emerging industries, their ubiquity is matched only by their structural variety. Despite this variety, terpenoids are built from a single, acyclic, isoprenoid precursor, derived from universally distributed precursor pathways. The enzymes responsible for converting this precursor into terpenoids are terpene synthases (TSs), which carry out stereo- and regiochemistry far beyond that capable by conventional chemical synthesis. However, due to the highly reactive nature of the carbocationic intermediates handled by TSs, they have evolved to prioritise chemical control over reaction enhancement, and the means by which they promote the formation of particular products is often extremely subtle. Adding to the difficulty is the fact that TSs share very low sequence identity, even between those which make the same product. This makes sequence-based rational engineering very challenging for this family of enzymes. Instead, a combined computational and experimental approach is required to characterise and understand these enzymes, with the ultimate aim of engineering so-called “designer terpene synthases” with chosen product outcomes. The aim of this thesis was therefore to establish the catalytic motifs underpinning TS chemistry, and to use these insights to characterise and engineer TSs. Chapter 1 is a published review article that probes the recent literature to establish the important catalytic motifs in TS chemistry, and looks forward to the near future of high-throughput and data-driven characterisation of TSs. Chapter 2 is a published journal article in which a sesquiterpene synthase is characterised and its mechanism deduced using a combined experimental and computational approach. Chapters 3 and 4 are computational and experimental chapters, respectively, dedicated to characterising two TSs from a naturally thermostable bacterium. Rational mutation of one of these enzymes based on insights gained in these chapters revealed two variant enzymes with improved activity and altered mechanistic preferences. In summary, the work in this thesis has helped establish important motifs in TS chemistry, and demonstrates the power of a combined computational and experimental approach in characterising and engineering TSs. The insights and results presented here will now be taken forward into the era of high-throughput characterisation to move us closer to the ultimate goal of designer terpene synthases.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Sam Hay (Supervisor)