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Cloning, Expression and Characterisation of the Starter Module from Indanomycin Biosynthesis

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Cloning, Expression and Characterisation of the Starter Module from Indanomycin Biosynthesis Cloning, expression and characterisation of the starter module from indanomycin biosynthesis Sasha Rebecca Derrington Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Astbury Centre for Structural Molecular Biology September 2014 The candidate confirms that the submitted work is her own and that the appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from this thesis may be published without proper acknowledgement. © 2014 The University of Leeds and Sasha Rebecca Derrington Acknowledgements Firstly, I would like to thank Professor Alan Berry for giving me the opportunity to work in this lab, allowing me to start something completely new, and for the support that he has provided me throughout the project, especially during the thesis dark days. I would also like to thank my second supervisor, Professor Adam Nelson for his supervision, guidance and encouragement. I don’t want to spoil the ending, but a big part of the results includes all of the work I have completed in the NMR spectroscopy facility. None of this would have been possible without the help and support of Dr Gary Thompson and also Dr Arnout Kalverda as well. I would like to thank my friend Dr Theo Karamanos, who has kept me calm and also helped me in NMR spectroscopy. Next I would like to thank my friend Dr James Ault, who not only carried out the awesome mass spectrometry work but for our chats and being supportive, and I would like to thank Dr Chi Trinh for all his help with the crystallography. I would also like to thank Prof. Arwen Pearson and Prof. Sheena Radford for all their support and advice. I have thoroughly enjoyed my four years working in the Astbury centre and have an amazing group of friends who have supported me, made me laugh and above everything made me happy. To Oli, Claire, Rachel, Lydia, Sophia, Jan, Hazel, Rob, Chris, Laura, Tom, Patrick, Paul and everyone else on level 10 (and mass spec), thanks for an amazing four years and putting up with me! And let’s not forget the past Berry members and friends, whom I miss, Lucy Woods, Nicole Timms, Adam Daniels and Sophie Forrest. Finally, I would like to thank my family, my Mum, Dad, Sister and Brother. To my Sister and Brother, thanks for putting up with me and the late phone calls because “I’m scared” and for supporting me. To my Mum and Dad, thank you for everything you have done and continue to do for me, you really are amazing parents and this would have been impossible without you. I really hope I’ve made you proud! i ii I would like to dedicate this thesis work to my Bubbe, Zayde, Grandma, Grandpa and the rest of my wonderful family for all of the love, support and encouragement they have always given me. I am so thankful for everything you have done for me and I hope I have made you proud. iii iv Abstract Nonribosomal peptides and polyketides form important classes of pharmaceutical agents. Several architectures of biosynthetic machinery exist for the construction of these structurally diverse molecules. Many attempts to engineer these proteins have concentrated on the multimodular type I polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). Here, work was carried out to express the NRPS module responsible for starter unit formation from indanomycin biosynthesis in E. coli. Three synthetic genes, for IdmI, IdmJ and IdmK, were cloned and subsequently used in expression trials. Despite multiple attempts, IdmI was always expressed as an insoluble protein. IdmJ was expressed as a soluble protein, and an unexpected post-translational modification was found and investigated. Mutagenesis studies suggested that the unknown post-translational modification was occurring at a cysteine 127. IdmK, the carrier protein, was expressed in a soluble form with good yield. Analysis by mass spectrometry showed that, surprisingly, E. coli was able to phosphopantetheinylate IdmK, which is required for a functional module. Preliminary structure determination was carried out by X-ray crystallography. A complete 3D structure was obtained using NMR spectroscopy of the [15N] and [13C, 15N] labelled protein. Structure determination was performed using CS-ROSETTA, which only uses chemical shift data, and ARIA, which assigns ambiguous NOE distance restraints. Both structure calculations produced comparable structures for IdmK. The structure showed a 3 α-helix bundle, with the same topology, lengths and locations of helices as other carrier proteins. Initial investigations into holo-IdmK suggest that the phosphopantetheine co-factor is directed into the hydrophobic core of the protein. This research has set up the system for future studies to engineer the pathway for novel product biosynthesis. v vi Contents Acknowledgments…………………………………………………..……i Abstract………………………………………………………………..…v Table of contents…………………………………………………...…….vii List of figures…………………………………………………………….xiii Abbreviations…………………………………………………………….xvii 1. Introduction ........................................................................................................ 1 1.1 The Golden Age of Antibiotics ..................................................................... 1 1.2 Natural products as antibiotics ...................................................................... 3 1.2.1 Polyketides ............................................................................................. 4 1.2.2 Nonribosomal peptides........................................................................... 6 1.2.3 Polyketide-nonribosomal peptide hybrid products ................................ 8 1.3 Polyketide synthases ...................................................................................... 9 1.3.1 PKS biosynthetic gene clusters ............................................................ 10 1.3.2 PKS module organisation ..................................................................... 11 1.3.2.1 Acyltransferase (AT) domain ....................................................... 12 1.3.2.2 Ketosynthase (KS) domain ........................................................... 13 1.3.2.3 Acyl carrier protein ....................................................................... 13 1.3.2.4 Chain initiation and termination ................................................... 14 1.3.2.5 Linker regions ............................................................................... 15 1.3.2.6 Accessory domains: KR, DH and ER ........................................... 15 1.3.2.7 Biosynthetic pathways of polyketide synthases and fatty acid synthases 15 1.3.2.8 A model system ............................................................................ 17 1.3.3 Structure determination of PKSs .......................................................... 19 1.3.4 Engineering of PKSs ............................................................................ 24 1.4 Nonribosomal peptide synthetases .............................................................. 28 1.4.1 NRPS biosynthetic gene clusters ......................................................... 28 1.4.2 Biosynthesis of nonribosomal peptides and module organisation ....... 29 1.4.2.1 Adenylation domain ...................................................................... 30 1.4.2.2 Condensation domain ................................................................... 32 1.4.2.3 Peptidyl carrier protein ................................................................. 32 1.4.2.4 Chain termination and macrocyclisation ...................................... 33 1.4.2.5 Epimerase domain......................................................................... 33 vii 1.4.2.6 Cyclisation domain ....................................................................... 34 1.4.2.7 Additional NRP chain modifications ............................................ 36 1.4.2.8 Linking modules in NRPS ............................................................ 36 1.4.2.9 A model system ............................................................................ 36 1.4.3 Structural and mechanistic studies of NRPSs ...................................... 38 1.4.4 Engineering NRPSs ............................................................................. 41 1.5 NRPS-PKS hybrid enzymes ........................................................................ 42 1.5.1 The hybrid NRP/PK indanomycin ....................................................... 43 1.5.1.1 Biosynthetic gene cluster and structure of indanomycin .............. 43 1.5.1.2 Starter unit biosynthesis................................................................ 44 1.6 Project outlines ............................................................................................ 46 2 Materials and methods ..................................................................................... 47 2.1 Materials ...................................................................................................... 47 2.1.1 Chemicals ............................................................................................. 47 2.1.2 Bacterial strains and plasmids .............................................................. 47 2.1.3 Enzymes ............................................................................................... 48 2.1.4 Antibodies ...........................................................................................
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