University of Bath PHD Discovering and Engineering Novel Thermostable Terpene Synthases Nesbitt, Edward Award date: 2020 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Discovering and Engineering Novel Thermostable Terpene Synthases Edward Andrew Nesbitt A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2019 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with the author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that they must not copy it or use material from it except as permitted by law or with the consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. Signed by Author 1 Abstract Terpenes 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. 2 Acknowledgements I would like to thank my supervisor, Professor David Leak, for the opportunity to work on this project and in Lab 1.28. You gave me the freedom to make this project my own from the outset but gave me support when it was required as well as pushing me to take on my fear of presenting. I would also like to thank my co-supervisor, Dr Susan Crennell, for the regular support and for always being available at short notice to talk to about experiments. I express my gratitude to BBSRC for funding my project. I am so thankful to everyone in the Leak and Pudney labs, past and present, who have helped me from when I was a shy, anxious final year undergraduate project student to what I have become today. I am extremely appreciative of all the help from Dr Styles and Dr Chacon with all of my terpene work as well as the undergraduate and master’s students I have supervised. I would also like to thank Rory, Hannah, Dragana and Alex, for their much-needed help with everything protein related. To Emanuele, Rory and Helen, I will miss our spontaneous Chinese dinners on our walk home. To Mavi, it has been my pleasure to work alongside you from our first day of sharing a lab bench to our graduation together. To Liz, you have been a great friend throughout our PhDs and I can’t thank you enough for all of the support throughout. I hope I provided just as much support in return. To Abby, thank you for all the time you put into helping me find strategies to deal with my anxiety. All of you have made my PhD immensely enjoyable and provided many great memories that I will take away with me. To my parents, this is entirely your fault. For my whole life, you made science unavoidable and piqued my interest at a young age. Since then you have supported me every step of the way and given me a wealth of opportunities to help me get this far. To my brother, Haza, you continue to motivate me to be a better person even though we still bicker in our mid-20s. Lastly to my partner in crime, Rebecca. I couldn’t have done this without you. We have been on a rollercoaster of a journey over the past six years where you have supported me through the darkest times and celebrated with me at the best of times. You gave me the confidence me to take opportunities and try new things that I would not have been able to do on my own. Thank you for making me a better person. You are incredible, you are my champion and I am truly “greatful” that you were the one I got to share the PhD experience with. I am excited to find out what the future holds for us. 3 Contents Abstract ................................................................................................................... 2 Acknowledgements .................................................................................................. 3 Contents .................................................................................................................. 4 Figures List .............................................................................................................. 8 Tables List ............................................................................................................. 14 Abbreviations ......................................................................................................... 16 1 Introduction ..................................................................................................... 19 1.1 Natural products ...................................................................................... 19 1.2 Terpenes ................................................................................................. 23 1.2.1 Terpene pathways ............................................................................ 24 1.2.2 Hemiterpenes ................................................................................... 26 1.2.3 Monoterpenes................................................................................... 26 1.2.4 Sesquiterpenes ................................................................................. 27 1.2.5 Diterpenes ........................................................................................ 29 1.2.6 Commercial terpene production ........................................................ 30 1.3 Terpene synthase .................................................................................... 34 1.4 The Parageobacillus terpene platform ..................................................... 40 1.5 Aims and Objectives ................................................................................ 46 2 Materials and Methods ................................................................................... 47 2.1 DNA Manipulation .................................................................................... 47 2.1.1 Plasmid DNA .................................................................................... 47 2.1.2 Polymerase Chain Reaction (PCR) ................................................... 48 2.1.3 Golden Gate Cloning ........................................................................ 49 2.1.4 Colony PCR (cPCR) ........................................................................