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A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/81849 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Unlocking the potential of novel taxa – a study on Actinoallomurus João Carlos Santos Cruz Submitted for the degree of Doctor of Philosophy School of Life Sciences, University of Warwick March, 2016 1 TABLE OF CONTENTS Table of Contents .......................................................................................................... i Acknowledgments...................................................................................................... iv List of Figures ................................................................................................................. v List of Tables ................................................................................................................. ix Declaration .................................................................................................................. xi Summary ....................................................................................................................... xii Abbreviations .............................................................................................................. xiii 1. Introduction ............................................................................................................ 1 1.1. State of the art - antibiotics ................................................................ 1 1.2. Importance of natural products ........................................................ 7 1.3. Changing the screening paradigm ............................................................................. 16 1.3.1. The screening strategies ..................................................................................... 16 1.3.2. Introducing elements of novelty ......................................................................... 19 1.3.3. The Genus of Actinoallomurus ........................................................................... 21 1.4. Hypothesis ................................................................................................................. 24 2. Screening Actinoallomurus ........................................................................................................... 25 2.1. Introduction ............................................................................................................... 25 2.2. Experimental Section ................................................................................................ 27 2.3. The screening strategy .............................................................................................. 29 2.4. Screening Overview .................................................................................................. 31 2.4.1. The universe of the unknown ............................................................................. 36 2.4.2. Polyethers from Actinoallomurus ....................................................................... 36 2.4.3. The case of coumermycin ................................................................................... 38 i 2.5. Another Screening Tool ............................................................................................ 40 2.5.1. Rationale ............................................................................................................. 40 2.5.2. Evaluation of the tool ......................................................................................... 42 2.6. Discussion ................................................................................................................. 47 3. Contributions for the discovery of a new metabolite .................................................................... 49 3.1. Introduction ............................................................................................................... 49 3.2. Experimental Section ................................................................................................ 50 3.3. Results ....................................................................................................................... 52 3.4. Discussion ................................................................................................................. 64 4. Allocyclinones, hyperchlorinated angucyclinones from Actinoallomurus .................................... 66 4.1. Introduction ............................................................................................................... 66 4.2. Experimental Section ................................................................................................ 67 4.3. Results ....................................................................................................................... 70 4.4. Discussion ................................................................................................................. 80 5. A Brominated Variant of the Lantibiotic NAI-107 with Enhanced Antibacterial Potency ........... 82 5.1. Introduction ............................................................................................................... 82 5.2. Experimental Section ................................................................................................ 83 5.3. Results ....................................................................................................................... 86 5.4. Discussion ................................................................................................................. 95 6. Genomic analysis of Actinoallomurus .......................................................................................... 97 6.1. Introduction ............................................................................................................... 97 6.2. Experimental Section ................................................................................................ 97 6.3. Results ..................................................................................................................... 100 6.3.1. Whole-genome comparisons ............................................................................ 101 6.3.2. Biosynthetic gene clusters ................................................................................ 106 6.3.3. Creation of a genomic library from Strain ID145113 ...................................... 118 6.4. Discussion ............................................................................................................... 122 7. General Discussion ...................................................................................................................... 125 ii 7.1. Introduction ............................................................................................................. 125 7.2. Critical overview of the results ............................................................................... 126 7.3. Insights into Actinoallomurus ................................................................................. 129 7.4. The concept of low-hanging fruits .......................................................................... 130 7.5. The genomic potential of Actinoallomurus vs the screening results ....................... 132 7.6. Finding new chemical classes and finding improved antibiotics ............................ 133 7.7. Outlook ................................................................................................................... 134 Appendix 1 .......................................................................................................................................... 136 Appendix 2 .......................................................................................................................................... 157 Appendix 3 .......................................................................................................................................... 159 8. References ................................................................................................................................... 160 iii ACKNOWLEDGMENTS To those who gave me the opportunity to have this wonderful experience, who went above and beyond to make me feel welcome and ensure I was getting the most out of this project. For their advices professionally, but also personal, I thank you Dr. Stefano Donadio and Dr. Margherita Sosio. I also want to thank Professor Elizabeth Wellington, who has always made an effort to help, support and guide me on this journey, despite being based different countries. None of this work would have been accomplished without the guidance of Marianna, the support of Matteo or the comments of Alice; the advices of Paolo, Arianna, or Sonia were equally important, and last, but certainly not least, Cristina, Eleonora, and Stefania and their ever present helping hand. NAICONS Research group, I thank you all. Because these three years were spent also outside of the Lab, I have to thank Marcello, Maxime, and Thomas for welcoming me in Milan with open arms and creating a bond that I will always

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