AbstractTerpenes are the most diverse group of natural products with more than 80000 structures and have a wide range of applications in industry ranging from pharmaceuticals to flavours and fragrances. Most commercial terpene are extracted from plants, but this method typically results in low yields. Microbial platforms such as the Saccharomyces cerevisiae and Rhodobacter sphaeroides platforms can provide a cheaper, more sustainable alternative. Recently, the first thermostable terpene platform was developed in the thermophile, Parageobacillus thermoglucosidasius. As well as consuming the breakdown products of waste lignocellulosic biomass as a feedstock rather than sugar like the other microbial terpene platforms, this platform is believed to have other advantages including lower risk of contamination, increased substrate solubility and lower running costs due to running at high temperatures. One of the final steps in the terpene pathways, catalysed by the terpene synthase (TPS), is mainly responsible for the structural diversity of terpenes. Before this work, the Parageobacillus platform could only synthesise the sesquiterpene, τ-muurolol, as no other thermostable TPSs had been identified other than the two τ-muurolol synthases from Roseiflexus species. For this system to be industrially viable, valuable terpenes need to be produced in high yields.
To increase the number of terpenes produced by the Parageobacillus platform, this work aimed to characterise more thermostable TPSs. Hidden Markov Models (HMMs) were used to search and identify novel thermostable TPSs from thermophiles which were then characterised using in vitro assays. This strategy identified the first naturally thermostable germacrene D-4-ol synthase as well as the first (+)-sativene synthase. In order to identify methods of increasing the thermostability and robustness of mesostable TPSs without screening large numbers of mutants, structural comparisons between mesostable and thermostable τ-muurolol synthases were conducted. Computational methods were then used to identify thermostabilising mutations in mesostable τ-muurolol and selinadiene synthases which resulted in small increases in thermostability. Finally, the active site of the thermostable τ-muurolol synthase, RoseRS_3509, was mutated to create a TPS that synthesised the prospective biofuel, β-farnesene. This work also led to increased understanding about which active site amino acids are involved in the cyclisation mechanism.
|Date of Award||1 Apr 2020|
|Supervisor||David Leak (Supervisor) & Susan Crennell (Supervisor)|
Discovering and Engineering Novel Thermostable Terpene Synthases
Nesbitt, E. (Author). 1 Apr 2020
Student thesis: Doctoral Thesis › PhD