HARNESSING SYNTHETIC BIOLOGY FOR THE BIOPROSPECTING AND ENGINEERING OF AROMATIC POLYKETIDE SYNTHASES A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering (2017) MATTHEW J. CUMMINGS SCHOOL OF CHEMISTRY 1 THIS IS A BLANK PAGE 2 List of contents List of contents .............................................................................................................................. 3 List of figures ................................................................................................................................. 8 List of supplementary figures ...................................................................................................... 10 List of tables ................................................................................................................................ 11 List of supplementary tables ....................................................................................................... 11 List of boxes ................................................................................................................................ 11 List of abbreviations .................................................................................................................... 12 Abstract ....................................................................................................................................... 14 Declaration .................................................................................................................................. 15 Copyright statement .................................................................................................................... 16 Acknowledgements ..................................................................................................................... 17 Thesis preface ............................................................................................................................. 18 1 Introduction.......................................................................................................................... 19 1.1 Specialised metabolites .............................................................................................. 19 1.1.1 Antimicrobial discovery ....................................................................................... 19 1.2 Biosynthetic genes cluster .......................................................................................... 24 1.2.1 Operons and evolution ........................................................................................ 24 1.2.2 BGC prediction .................................................................................................... 28 1.3 Synthetic biology for specialised metabolites ............................................................. 32 1.3.1 Rebuilding biosynthetic gene clusters ................................................................. 32 1.3.2 Semantics in synthetic biology ............................................................................ 34 1.4 Polyketides .................................................................................................................. 35 1.4.1 Type I polyketide synthases ................................................................................ 37 1.4.2 Type III polyketide synthases .............................................................................. 38 1.4.3 Type II polyketide synthases ............................................................................... 39 1.5 Aims of the thesis ........................................................................................................ 49 1.6 References .................................................................................................................. 52 2 Steps towards the synthetic biology of polyketide biosynthesis ......................................... 66 2.1 Preface ........................................................................................................................ 67 2.2 Abstract ....................................................................................................................... 68 2.3 Steps towards the synthetic biology of polyketide biosynthesis ................................. 69 2.3.1 Polyketides: magnificently modular ..................................................................... 69 2.3.2 Modularity of scaffold biosynthesis ..................................................................... 72 2.3.3 Modularity of tailoring reactions .......................................................................... 75 2.4 Synthetic biology: the future of combinatorial biosynthesis ........................................ 76 2.5 Acknowledgements ..................................................................................................... 78 3 2.6 References................................................................................................................... 79 3 The Minimum Information about a Biosynthetic Gene cluster (MIBiG) specification .......... 83 3.1 Preface ......................................................................................................................... 86 3.2 Abstract ........................................................................................................................ 87 3.3 Introduction .................................................................................................................. 88 3.4 Results and discussions .............................................................................................. 90 3.4.1 Design of the MIBiG standard .............................................................................. 90 3.4.2 MIBiG: designed to address key research needs ................................................ 92 3.4.3 Community annotation effort ................................................................................ 94 3.4.4 Planned implementation in databases and web services .................................... 95 3.5 Future perspectives ..................................................................................................... 95 3.6 Acknowledgements ...................................................................................................... 96 3.7 Competing Financial Interests Statement .................................................................... 96 3.8 References................................................................................................................... 97 4 An Output Ordering and Prioritisation System (OOPS) for ranking biosynthetic gene clusters to enhance bioactive metabolite discovery .................................................................... 99 4.1 Preface ....................................................................................................................... 100 4.2 Abstract ...................................................................................................................... 101 4.3 Introduction ................................................................................................................ 102 4.4 Methods and materials .............................................................................................. 103 4.4.1 Prioritization protocol ......................................................................................... 104 4.4.2 Guanine and cytosine content ........................................................................... 104 4.4.3 Codon bias ......................................................................................................... 104 4.4.4 Similarity to known cluster ................................................................................. 105 4.4.5 Self-similarity...................................................................................................... 105 4.4.6 Phylogenetic diversity ........................................................................................ 105 4.4.7 Availability .......................................................................................................... 106 4.4.8 Precomputation of Actinobacterial BGCs .......................................................... 106 4.4.9 Processing commercially sensitive data ............................................................ 106 4.5 Results and Discussion ............................................................................................. 107 4.5.1 Computational pipeline for OOPS ..................................................................... 107 4.5.2 Prioritisation Parameters ................................................................................... 107 4.5.3 Exploring Actinobacterial BGCs......................................................................... 112 4.6 Conclusion ................................................................................................................. 112 4.7 References................................................................................................................. 114 5 Assembling a modular production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli ................................................................................................... 118 5.1 Preface ....................................................................................................................... 119 5.2 Abstract .....................................................................................................................