Biosynthesis and Mode of Action of the Β-Lactone Antibiotic Obafluorin
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Biosynthesis and mode of action of the β-lactone antibiotic obafluorin Thomas Alexander Scott John Innes Centre Department of Molecular Microbiology Norwich Research Park, Colney Lane, Norwich, NR4 7UH, UK A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy September 2017 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Biosynthesis and mode of action of the β-lactone antibiotic obafluorin Thomas Alexander Scott, John Innes Centre, September 2017 Abstract DNA sequencing technologies have advanced rapidly in the 21st century and there are now an abundance of microbial genomes available to mine in search of novel biosynthetic gene clusters and the bioactive natural products they encode. Whilst an enormously exciting prospect, this abundance of genomic data presents a challenge in determining how to select clusters for further study. To address this issue, this project focusses on the biosynthesis of the β-lactone antibiotic obafluorin produced by the soil bacterium Pseudomonas fluorescens. β- Lactones occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from β-hydroxy-α-amino acids, which are themselves privileged chiral building blocks in a variety of pharmacologically and agriculturally important natural products and medicines. I report the delineation of the entire obafluorin biosynthetic pathway using complementary mutagenesis and biochemical assay-based approaches. As part of this work I have been able to characterise ObaG, a novel PLP-dependent L-threonine transaldolase responsible for the biosynthesis of an unusual nonproteinogenic β- hydroxy-α-amino acid precursor from which the β-lactone ring of obafluorin is derived. Phylogenetic analysis has shed light on the evolutionary origin of this rare enzyme family and has identified further gene clusters encoding putative L-threonine transaldolases. Furthermore, I have been able to biochemically assay an entire intact nonribosomal peptide synthetase that displays a noncanonical domain architecture and is responsible for obafluorin assembly and β-lactone ring formation. These studies allowed both mechanism- and redundancy-guided genome mining strategies to be developed that might allow the specific targeting of novel chemistry in the uncharted reaches of the natural product world. i Acknowledgements I would immediately like to express my enormous gratitude to Prof. Barrie Wilkinson for his invaluable guidance and support throughout this project. I have enjoyed countless discussions with him, both scientific and otherwise, and have benefitted immensely from his help and encouragement in conversations about my future. It has been both a tremendous honour and pleasure to be his first PhD student. I would also like to thank my supervisory committee members Dr. Jacob Malone and Prof. Mervyn Bibb FRS for their advice and careful scrutiny of my thesis work. I also owe special thanks to Dr. Neil Holmes, who invested a great deal of his time in helping me learn molecular methods in my first year, and to Dr. Daniel Heine for his many contributions in the more chemistry-based elements of my research. I thoroughly enjoyed my time working with them both. Thanks also to Dr. Zhiwei Qin and Dr. Gerhard Saalbach for help in purifying and characterising obafluorin, and with proteomics analyses respectively. I am extremely grateful to Dr. Govind Chandra for helping me explore my ideas about new genome mining strategies and for his bioinformatics support on this project, and to Ms. Eposi Enjema Carine Solange for all her work investigating obafluorin bioactivity. I feel extremely proud to have belonged to the Molecular Microbiology department at the John Innes Centre. It cultivates a unique research environment in which members readily share their experiences and support one another in their research in whatever way they can. My thesis would not have been possible without the help and support of numerous members of the department, both past and present: I think particularly of Dr. Silke Alt, Mr. Thomas Booth, Dr. Matt Bush, Dr. Juan-Pablo Gomez-Escribano, Dr. Richard Little and Dr. Javier Santos-Aberturas. I have really enjoyed my time in Mol. Micro. and have made many great friends. It will be with a very heavy heart that one day I move on to something new. Finally, I would like to thank my family who have always been a source of unremitting support and my partner, Dhana Thomy, who has been constantly at my shoulder through both good times and more challenging times. ii Author’s declaration The research described in this thesis was conducted entirely at the John Innes Centre between October 2013 and September 2017. All the data described are original and were obtained by the author, except where specific acknowledgement has been made. No part of this thesis has previously been submitted as for a degree at this or any other academic institution. A significant proportion of this work has been the subject of the following publication, which is included in the appendices of this thesis: Scott, T. A., Heine, D., Qin, Z. & Wilkinson, B. An L-threonine transaldolase is required for L-threo-β-hydroxy-α-amino acid assembly during obafluorin biosynthesis. Nat. Commun. 8, 15935 (2017). iii Abbreviations α-OH-β-AA α-Hydroxy-β-amino acid β-OH-α-AA β-Hydroxy-α-amino acid 2,3-DHBA 2,3-Dihydroxybenzoic acid 2,4-DAPG 2,4-Diacetylphloroglucinol 3-HBA 3-Hydroxybenzoic acid 4-ABA 4-Aminobenzoic acid 4-APA 4-Aminophenylalanine 4-APP 4-Aminophenylpyruvate 4-HBA 4-Hydroxybenzoic acid 4-NPA 4-Nitrophenylacetaldehyde 4-NPAA 4-Nitrophenylacetic acid 4-NPE 4-Nitrophenylethanol 4-NPP 4-Nitrophenylpyruvate AaRS Aminoacyl-tRNA synthetase ADC 4-Amino-4-deoxychorismate ADH Aldehyde dehydrogenase AHAB (2S,3R)-2-Amino-3-hydroxy-4-(4’-aminophenyl)butanoate AHBAS 3-Amino-5-hydroxybenzoic acid synthase AHNB (2S,3R)-2-Amino-3-hydroxy-4-(4’-nitrophenyl)butanoate AMP Adenosine monophosphate ANA Anthranilic acid ATP Adenosine triphosphate BA Benzoic acid BGC Biosynthetic gene cluster CLP Cyclic lipopeptide CoA Coenzyme A DAHP Deoxy-D-arabino-heptulosonate-7-phosphate DNA Deoxyribonucleic acid DTT DL-Dithiothreitol EDTA Ethylenediaminetetraacetic acid FAS Fatty acid synthase GCS Glycine cleavage system GHMT Glycine hydroxymethyltransferase GlyU 5′-C-glycyluridine HCN Hydrogen cyanide HGT Horizontal gene transfer HPLC High-performance liquid chromatography HTS High-throughput screening IAA Indole-3-acetic acid IPM Isopropylmalate IPMS Isopropylmalate synthase IPP Isopentenyl diphosphate LCMS Liquid chromatography-mass spectrometry MGE Mobile genetic element MIC Minimum inhibitory concentration MS Mass spectrometry iv NAC N-Acetylcysteamine NADH Nicotinamide adenine dinucleotide (reduced) NMR Nuclear magnetic resonance NP Natural product NRP Nonribosomal peptide NRPS Nonribosomal peptide synthetase O/N Overnight PCS Protein coding sequence PK Polyketide PKS Polyketide synthase PLP Pyridoxal-5’-phosphate PPant Phosphopantetheine pPDC Phenylpyruvate decarboxylase PPi Inorganic pyrophosphate PQQ Pyrroloquinolone quinone PQS Pseudomonas quinolone signal PTM Post-translational modification QS Quorum sensing RiPP Ribosomally synthesised and post-translationally modified peptide RNA Ribonucleic acid SAL Salicylic acid SAM S-adenosyl methionine SHMT Serine hydroxymethyltransferase TA Threonine aldolase TEAB Tetraethylammonium bicarbonate ThDP Thiamine diphosphate THF Tetrahydrofolate ThrRS Threonyl-tRNA synthetase TTA Threonine transaldolase NRPS/PKS domains A Adenylation ACP Acyl carrier protein ArCP Aryl carrier protein C Condensation Cy Cyclisation DH Dehydratase E Epimerisation ER Enoyl reductase KR Ketoreductase KS Ketosynthase PCP Peptidyl carrier protein TE Thioesterase v Compound Key vi Table of contents Abstract ................................................................................................................... i Acknowledgments .................................................................................................. ii Author’s declaration .............................................................................................. iii Abbreviations ......................................................................................................... iv Compound Key ...................................................................................................... vi Table of contents .................................................................................................. vii Index of Figures and Tables ................................................................................ xiii Chapter 1: Introduction ........................................................................................ 1 1.1 Natural product discovery .......................................................................... 2 1.1.1 What are natural products? .................................................................... 2 1.1.2 The rise of NP drug discovery ................................................................ 2 1.1.3 The fall of NP drug discovery ................................................................. 4 1.1.4 The second coming of NP drug discovery – the genome mining era ...... 4 1.2 Pseudomonas spp. as producers