Chapter 1 : Introduction

A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/89468 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 Antibiotic Biosynthesis and its Transcriptional Regulation in Streptomyces Bacteria Shanshan Zhou Supervisor: Prof. Gregory L. Challis A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Department of Chemistry, University of Warwick September 2016 Table of Contents List of Figures ............................................................................................................................. v List of Schemes ........................................................................................................................ xiii List of Tables ........................................................................................................................... xvi Acknowledgements ................................................................................................................ xvii Declaration ............................................................................................................................ xviii Abstract .................................................................................................................................... xix Abbreviations ........................................................................................................................... xx 1. Introduction ........................................................................................................................ 1 1.1 Streptomyces ........................................................................................................................ 2 1.1.1 Streptomyces coelicolor A3(2) ................................................................................. 5 1.2 Streptomyces signalling molecules ...................................................................................... 7 1.2.1 Structural types of Streptomyces signalling molecules ............................................ 7 1.2.2 Biosynthesis of γ-butyrolactones ........................................................................... 10 1.2.3 Biosynthesis of AHFCAs ....................................................................................... 14 1.3 Transcriptional regulation of specialised metabolites in Streptomyces ............................. 17 1.3.1 TetR transcriptional repressors .............................................................................. 17 1.3.2 ArpA-like transcriptional repressors ...................................................................... 18 1.3.3 Regulation of methylenomycin biosynthesis in S. coelicolor ................................ 20 1.4 Siderophores ...................................................................................................................... 24 1.4.1 Bioactivity of siderophores .................................................................................... 26 1.4.2 Biosynthesis of siderophores ................................................................................. 26 1.5 2-Hydroxyphenothiazolines ............................................................................................... 27 1.5.1 Pyochelin and enantiopyochelin ............................................................................ 29 1.5.2 Biosynthesis of pyochelin and enantio-pyochelin .................................................. 31 1.5.3 Pulicatins, thiazostatin and watasemycin ............................................................... 35 1.6 S-Adenosylmethionine (SAM)-dependent methylation ..................................................... 36 1.6.1 Class B radical SAM methylases ........................................................................... 38 1.6.1.1 Stereochemical course of C-methylation by class B radical SAM methylases .. 40 1.7 Aims of project .................................................................................................................. 42 2. Investigation of the role of MmfL in AHFCA biosynthesis .......................................... 43 2.1 Introduction to previous studies ......................................................................................... 44 2.2 Synthesis of the N-acetylcysteamine (NAC) β-ketothioester substrate analogue .............. 46 2.3 Synthesis of the butenolide authentic standard .................................................................. 47 2.4 Overproduction, purification and characterisation of recombinant MmfL ........................ 51 i 2.5 Characterisation of dephosphorylated product of MmfL-catalysed reaction ..................... 52 2.6 Summary ............................................................................................................................ 56 3. Interactions between DNA, ligand and ArpA-like repressors ..................................... 57 3.1 Introduction ........................................................................................................................ 58 3.2 Interactions of MmfR with three MARE sequences .......................................................... 60 3.2.1. Confirmation of MmfR binding to MARE sequences ........................................... 60 3.2.2. Comparison of the binding affinity of MmfR to three MAREs ............................. 64 3.3 Interactions of MmfR with natural AHFCAs .................................................................... 67 3.3.1 Synthesis of AHFCA1 and AHFCA3 .................................................................... 67 3.3.2 Comparison of the binding affinity of the different intergenic regions to MmfR with natural AHFCAs 1-5 ...................................................................................................... 70 3.4 Key residues in the ligand binding of AHFCAs by MmfR ................................................ 72 3.4.1 Analysis of the crystal structure ............................................................................. 72 3.4.2 Preparation of MmfR mutants ................................................................................ 73 3.4.3 Attempts to mutate Leu 110 ................................................................................... 75 3.4.4 Effect of mutations on ligand binding .................................................................... 76 3.4.4.1. Tyr 85 ............................................................................................................. 76 3.4.4.2. Gln 130........................................................................................................... 77 3.4.4.3. Tyr 144 ........................................................................................................... 78 3.5 Ligand binding specificity of MmfR ................................................................................. 78 3.5.1 Synthesis of SCB1 ................................................................................................. 79 3.5.2 Interaction of MmfR with SCB1 ............................................................................ 83 3.6 Investigation of a MmfR homologue, SgnR ...................................................................... 84 3.6.1 Cloning of sgnR ..................................................................................................... 85 3.6.2 Overproduction and purification of recombinant His6-SgnR protein .................... 86 3.6.3 Synthesis of AHFCA6 and AHFCA7 .................................................................... 87 3.6.4 EMSAs with SgnR ................................................................................................. 88 3.7 Key notes for studying in vitro DNA-protein-ligand interaction using EMSAs ................ 90 3.8 Summary ............................................................................................................................ 92 4. Watasemycin biosynthesis in Streptomyces venezuelae ATCC10712 ........................... 94 4.1 Introduction to previous work ............................................................................................ 95 4.1.1 Upregulation of ‘pyochelin-like’ gene cluster ....................................................... 95 4.1.2 Isolation and characterisation of watasemycin and thiazostatin from S. venezuelae . ............................................................................................................................... 97 4.2 Identification and characterisation of isopyochelin ......................................................... 100 4.2.1 Synthesis of pyochelin ......................................................................................... 100 4.2.2 LC-MS analysis of synthetic pyochelin ............................................................... 104 4.2.3 Synthesis and characterisation of isopyochelin .................................................... 105 ii 4.3 Roles of Sven0508 and Sven0516 in the biosynthetic pathway ...................................... 108 4.3.1 Sven0516 is required for 2-hydroxyphenylthiazoline biosynthesis

Load more