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Thesis Submitted for the Degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2017

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Thesis Submitted for the Degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2017 University of Bath PHD Esterification as a strategy for the detoxification of value compounds in Escherichia coli Chacon, Micaela Award date: 2018 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: 07. Oct. 2021 Esterification as a strategy for the detoxification of value compounds in Escherichia coli Micaela Chacón A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2017 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 the author 1 Abstract The use of microbial platforms for the production of commercially valuable compounds has grown over the last few decades as concern over the sustainability of other production methods, such as extraction and chemical synthesis, has been come into question. Advancements in the understanding of microbial metabolism and regulation with the aid of -omics research, and a steady increase in the number of available genetic tools, has allowed model organisms such as the bacteria Escherichia coli and the yeast Saccharomyces cerevisiae to be engineered for high titre production of a number of value products. The economical production of these compounds on an industrial scale can, however, be hampered by the toxicity of a given end-product upon accumulation in culture. The mode of action by which these products interfere with cellular homeostasis is often multifaceted, making it difficult to address. As such, considerable research has gone into developing strategies to minimize this microbial toxicity in platform hosts; including engineering strains for increased tolerance, and the development of fermentation strategies for the in situ removal of toxic end-products. These efforts have had mixed success, with the microbial titres of a number of value compounds continuing to be limited due low tolerance thresholds of the host. In this work, an alternative strategy for minimizing the toxicity of value alcohols in E. coli was investigated. This strategy involved the in vivo sequestration of an endogenously produced alcohol into a more neutral short/medium chain ester via enzyme mediated esterification by an alcohol acyl transferase (AAT). The rationale behind this strategy being that the incorporation of a toxic alcohol into a less toxic ester molecule may facilitate higher product accumulation in culture, as the ester end-product would be minimally inhibitory. Further, the alcohol component of the ester could be easily recovered downstream through hydrolysis, while the recovered acid component could be recycled as substrate for this strategy in future cultures. In this work, the feasibility of this detoxification strategy was first validated and then applied to two commercially valuable alcohols in E. coli. The first alcohol to which this strategy was applied was the proposed gasoline alternative short chain alcohol, butanol, which is detrimental to culture health when present at concentrations above 1.5% (v/v); and the second alcohol trialled was the common flavour and fragrance constituent monoterpene alcohol, geraniol, which is inhibitory when present above 0.05% (v/v) in culture. 2 Acknowledgements I would like to thank my thesis supervisors, Professor David Leak and Dr. Albert Bolhuis, for giving me the opportunity to pursue a stream of research that I truly enjoy. Thank you both for your great insight and guidance throughout this project, and for the flexibility in letting me design my own experiments – some ending successfully, and some ending as horrible failures. I’m a better scientist for having worked with you both. Thank you for taking a chance on me. I would also like to thank all the member of labs 1.28 and 1.33, past and present, for always being generous with their time and good spirits, and for making my time at the University of Bath and absolute joy. Especially Alex, for being the best friend I could ask for, and for shining some of her charisma, uniqueness, nerve and talent down on me; and Emanuele for being so generous with his optimism, patience (Gibson primers), and time – and for having a drawer full of premium snacks. Also to Jonathan, thank you for somehow simultaneously knocking me off my feet and holding me steady, I love you. I would like to dedicate this thesis to my family, who have always offered endless support and encouragement. Especially when it involved long nights helping me with homework that I left to the last minute. I have loved every moment watching Stargate SG-1 with you dad, every girly weekend eating almost nothing but candy with you mom, and every time you have accidentally called me Janelle, Benj. Thank you all for everything. In particular, I would especially like to dedicate this to my grandad, who was the kindest person I’ve ever known. 3 Contents Abstract ......................................................................................................................................... 2 Acknowledgements ...................................................................................................................... 3 Contents ........................................................................................................................................ 4 Figures list..................................................................................................................................... 8 Tables list .................................................................................................................................... 12 Abbreviations ............................................................................................................................. 13 1. General Introduction ............................................................................................................. 16 1.1 Natural products and their biological origin ................................................................ 16 1.2 Methods for the commercial production of natural products: extraction, chemical synthesis and microbial fermentation ...................................................................................... 18 1.3 Microbial expression platforms for value product synthesis ............................................. 20 1.4 E. coli as an expression platform ....................................................................................... 22 1.5 Metabolic engineering of microbes for improved product formation ................................ 24 1.6 Production of value compounds in E. coli ......................................................................... 26 1.6.1 Butanol production in E. coli ...................................................................................... 27 1.6.2 Monoterpene production in E. coli.............................................................................. 32 1.7 Obstacles to high titre product formation in microbial platforms – a focus on toxicity ... 36 1.8 Strategies for increasing microbial solvent tolerance ........................................................ 38 1.8.1 Strain engineering ....................................................................................................... 39 1.8.2 Product sequestration .................................................................................................. 41 1.9 General aims and objectives .............................................................................................. 43 2. Materials and Methods .......................................................................................................... 44 2.1 Microbial Growth Media ................................................................................................... 44 2.1.1 Luria Broth (LB) ......................................................................................................... 44 2.1.2. 2X Tryptone Yeast Broth (2TY) ................................................................................ 44 2.1.3 Terrific Broth (TB)...................................................................................................... 44 2.1.4 Modified Terrific Broth (MTB) .................................................................................
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