A Genomics-Led Approach to Deciphering Heterocyclic Natural Product Biosynthesis Karen Hoi-Lam Chan Churchill College Department of Biochemistry University of Cambridge October 2018 This dissertation is submitted for the degree of Doctor of Philosophy. Abstract A Genomics-Led Approach to Deciphering Heterocyclic Natural Product Biosynthesis Karen Hoi-Lam Chan Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N- formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily. Preface The dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualitification at the University of Cambridge or any other University or similar institution. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution. It does not exceed the prescribed word limit of 60,000 words. Karen Hoi-Lam Chan Churchill College Department of Biochemistry University of Cambridge October 2018 Acknowledgements First and foremost, my sincere gratitude goes to my PhD supervisor, Professor Peter Leadlay. I thank Peter for offering the opportunity for me to embark on this challenging yet fun, fruitful and memorable journey, as well as for his immense support, guidance and encouragement throughout my PhD. I could not have asked for a better research group to work with. I thank all past and present group members I have met for the warm welcome and for creating an enjoyable atmosphere at work. Special thanks to Dr. Fanglu Huang who is always so kind and generous with advices, for supervising much of my lab work, to Dr. Hui Hong for her advices and help with analytics, and to Dr. Annabel Murphy for her help with chemical synthesis. I express my gratitude to Dr. Youngjun Zhou, Dr. Qiulin Wu and Joachim Hug who started the oxazole and benzoxaozle projects. Their insights and results served as a foundation of my work. Special thanks to my good friend Vinzent Schulz for the much enjoyable time working on thiotetronates and for his constant support and encouragement. I thank Dr. Freddie Dudbridge and Dr. Marie Yurkovich for their help with microbiology and molecular biology, and Dr. Anya Luhavaya and Dr. Katharina Dornblut for proofreading parts of this thesis and their detailed feedback, and most importantly, for being great friends. I appreciate the sequencing work conducted by Shilo Dickens, Nataliya Scott, Anna Efimova and Reda Deglau of the DNA Sequencing Facility. I truly cherish the time spent and the friendships I have with all who are already mentioned, Felix Trottmann, Roel van Harten, Hannah Büttner, Rory Little, Oana Sadiq, Karen Lebe, Constance Wu, Carsten Schotte, Katsiaryna Usachova, Anna Reva, Jake Pollock, David Möller, Dr. Oksana Bilyk and Dr. Kwaku Kyeremeh. Additionally, I thank Professor Satoshi Ômura and Dr. Yuki Inahashi of the Kitasato Institute for providing the phthoxazolin producer strains and sharing their findings, Dr. Markiyan Samborskyy for his work on genome assembly and annotation, and the Mass Spectrometry Service, Department of Chemistry for performing accurate mass analyses. My thanks also go to all my other friends in Cambridge and around the world for their support. Financial support from the Henry Lester Trust and the Herchel Smith Foundation aided me to complete this PhD. Finally, I would like to thank and dedicate this work to my parents and my sister, who always believed in me and offered their support unconditionally. I hope I have made you proud. Contents Abbreviations ...........................................................................................................15 Chapter 1 Introduction.............................................................................................21 1.1 A brief history of antibiotic discovery .......................................................21 1.1.1 Antibiotics of microbial origin ...................................................................21 1.1.2 Antibiotic classification and mechanisms of action..................................22 1.1.3 Mechanisms of antibiotic self-resistance .................................................23 1.1.4 Emergence of antibiotic resistance in pathogens ....................................23 1.2 Streptomyces as a prolific source of bioactive natural products...........24 1.2.1 The genus Streptomyces.........................................................................25 1.2.2 The ecology and evolution of natural product biosynthesis.....................26 1.3 Harnessing the biosynthetic potential of microorganisms .....................28 1.3.1 Conventional methods for natural product discovery ..............................28 1.3.2 Mining for biosynthetic gene clusters in the age of genome sequencing 28 1.3.3 Heterologous expression of biosynthetic pathways.................................29 1.3.4 In vitro reconstitution of biosynthetic pathways .......................................30 1.4 The enzymes of natural product biosynthesis .........................................31 1.4.1 Terpenes and terpene synthases ............................................................31 1.4.2 Ribosomally-synthesised and post-translationally modified peptides......34 1.4.3 Polyketides and polyketide synthases.....................................................35 1.4.4 Substrate specificity and stereochemistry of PKS ...................................41 1.4.5 Trans-AT PKS: a non-canonical class of modular type I PKS.................46 1.4.6 Overall architecture of PKS .....................................................................47 1.4.7 Non-ribosomal peptides and non-ribosomal peptide synthetases...........49 1.4.8 Substrate specificity and overall architecture of NRPS domains.............50 1.4.9 Hybrid PKS-NRPS...................................................................................51 1.4.10 Heterocycles in polyketide and peptide natural products ........................52 1.5 Aims of this work.........................................................................................54 Chapter 2 Genome analysis of the phthoxazolin-producing strains Streptomyces sp. OM-5714 and Streptomyces sp. KO-7888 ...............................55 2.1 Introduction..................................................................................................55 2.2 Results and Discussion ..............................................................................58 2.2.1 Whole-genome sequencing of Streptomyces sp. OM-5714 and Streptomyces sp. KO-7888, the phthoxazolin producers ........................58 2.2.2 Phylogenetic studies and whole-genome comparisons of Streptomyces sp. OM-5714 and Streptomyces sp. KO-7888.........................................58 2.2.3 Annotation of the genomes of Streptomyces sp. OM-5714 and Streptomyces sp. KO-7888 .....................................................................63 2.2.4 Bioinformatics analysis of uncharacterised