IDENTIFICATION, ANALYSIS AND MANIPULATION OF THE TORRUBIELLONE A GENE CLUSTER By GUILLERMO CARLOS FERNANDEZ BUNSTER School of Biological Sciences University of Bristol United Kingdom A dissertation submitted to the UNIVERSITY OF BRISTOL in accordance with the requirements of the DOCTOR OF PHILOSOPHY in the FACULTY OF SCIENCE June / December 2016 Word count = 43.800 i Abstract Torrubiellones A-D, extracted from Torrubiella sp. BCC2165, are structurally similar to 2- pyridone compounds. Torrubiellone A is particularly interesting because it has antimalarial activity. Combining knowledge of the gene clusters responsible for the biosynthesis of the structurally similar compounds with in-silico analysis of the Torrubiella genome sequence lead to the identification of the torrubiellone A biosynthetic gene cluster. Torrubiella sp. BCC2165 DNA was extracted, sequenced and analysed to reveal a putative torrubiellone A gene cluster, comprising torS encoding a hybrid polyketide synthase- nonribosomal peptide synthetase, torA and torB encoding two P450 cytochromes and torC encoding an enoyl reductase. Comparison to the tenellin and desmethylbassianin gene clusters identified two additional genes, torD and torE, which could be responsible for structural differences between torrubiellone A and desmethylbassianin. torS was assembled without introns by homologous recombination in yeast and combined with other biosynthetic genes from the putative torrubiellone cluster, on a multigene expression vector. Assembled plasmids were used to transform the filamentous fungus Aspergillus oryzae NSAR1, yielding strongly yellow-pigmented transformants. Analysis of organic extracts from transformants by liquid chromatography-mass spectroscopy indicated that the production of torrubiellone- related compounds has been achieved. torD and torE gene functions were investigated by co- expressing these genes in a tenellin-producing A. oryzae, resulting in the addition of a hydroxyl group to the tenellin compound. A Torrubiella transformation system was developed, initially for promoter analysis of the torD and torE genes because the start codon of torE lies only 17 bp downstream of the torD stop codon in the Torrubiella genome, yet the two genes are transcribed independently, and that torE promoter is encoded within the torD coding region. The potential regulatory function of two transcription factors, ZnTF and C6TF, whose genes flank the torrubiellone biosynthetic genes, was investigated by over-expression in the native host. Overexpression of ZnTF led to the production of novel compounds, but no role, either positive or negative, was found for C6TF. The potential for genetic analysis by gene knockout in Torrubiella was tested with the torS gene. Transformants generated by homologous recombination were detected, as well as ectopic events, but the system requires improvement particularly in the isolation of homokaryons post-transformation. ii Dedication and acknowledgements I would like to thank to my supervisor for giving me the opportunity to study a PhD in England and accepting me as his student. I have learnt lots by listening, talking and watching him work, such as the first day, with joy and excitement of every new finding he could get. I will always have him as example in how a researcher should be, always curious, with constantly one theory to test, tweaking protocols to enhance them and teaching me how to express and write properly. Those are lessons that I will never forget. Also thanks to Elisabeth, who always was present for us, and especially for my daughter, who adores her. Also thanks to Jeroen Maertens, Luis Pablo and Sandra Alvarez, the best friends I could have got in UK, who offered me a word of support, discussion and even constructive criticisms, and always available for a cup of coffee at any time of the day, and to Magdalena Koziol for teaching me (almost) all the techniques required to perform my research. To my parents, for their continuous support while I was away. Amelia and Angela, I really appreciate what you did for me, you decided to come with me for this four-year adventure, in a foreign country, almost not speaking the language. You are the bravest people I know, and I am very lucky to have you as my beloved wife and my adorable daughter. I am sure I wouldn’t have been able to do this without you. You are my pillars and my motivation, the reason why I am here, and I hope to be the best for you. iii Author’s declaration declare that the work in this dissertation was carried out in accordance with the requirements of the University’s Regulations and Code of IPractice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ................................................. DATE: ............................................ iv ABBREVIATIONS: PK: Polyketide PKS: Polyketide synthase NRP: Non-Ribosomal peptide NRPS: Non-Ribosomal Peptide Synthase PCR: Polymerase Chain Reaction LC-MS: Liquid Chromatography – Mass Spectrometry TLC: Thin Layer Chromatography ZnTF: Zn Finger Domain- Transcription Factor C6TF: C6-Transcription Factor TF: Transcription Factor FAS: Fatty Acid Synthase FA: Fatty Acid KS: Keto synthase domain of PKS ACP: Acyl-carrier protein domain of PKS AT: Acyl transferase domain of PKS KR: Keto-reductase domain of PKS DH: Dehydratase domain of PKS ER: Active enoyl reductase domain of PKS. ER°: Inactive enoyl reductase domain of PKS. TE: Thio-esterease domain of NRPS SAT: Starter-Unit ACP transacylase domain of PKS PT: product template domain of PKS C-Met: Methylation domain of PKS A: Adenylation domain of NRPS PCP: Thiolation and Peptide Carrier Protein domain of NRPS C: Condensation domain of NRPS NR-PKS: Non-reducing polyketide synthase v PR-PKS: Partially-reducing polyketide synthase HR-PKS: Highly-reducing polyketide synthase MO: 3-methylorcinaldehyde OSA: Orsenillic acid NSAS: Norsolorinic acid synthase MSAS: 6-methylsalicylate synthase ten, dmb, mil pathway: tenellin, desmethylbassianin, militarinone ACT: Artemisinin-based combination therapies WHO: World Health Organization GMAP: Global Malaria Action Plan SAM: S-adenosyl methionine SM: Secondary metabolism NMR: Nuclear Magnetic Resonance antiSMASH: antibiotics & Secondary Metabolite Analysis Shell ORF: Open Reading Frame MEA: Malt Extract Agar. A. oryzae production media. CDA or Cdox: Czapek-Dox agar. A. oryzae selection media. ES+: Positive electrospray ionisation mass spectrometry ES-: Negative electrospray ionisation mass spectrometry Standard genomic nomenclature: torS: gene TorS: protein sequence TORS: Enzyme vi INDICE Abbreviations: .......................................................................................................................... v Indice ...................................................................................................................................... vii List of tables ............................................................................................................................. x List of Figures ........................................................................................................................ xi 1. Introduction ...................................................................................................... 1 1.1 Specialized metabolism ..................................................................................................... 1 1.2 Fungal secondary metabolites .......................................................................................... 1 1.3 Biosynthesis ................................................................................................................ 3 1.3.1 Fatty acid biosynthesis ........................................................................................ 4 1.3.2 Polyketide biosynthesis ....................................................................................... 6 1.3.3 PKS- NRPS hybrid systems ................................................................................ 9 1.4 Torrubiella spp: Characteristics and compounds .................................................... 13 1.5 Malaria and antimalarial activities .......................................................................... 16 1.6 Gene mining approach for discovering new natural products ................................ 21 1.7 Heterologous expression of fungal secondary metabolites ...................................... 24 1.8 Molecular approaches to the study of torrubiellone A biosynthesis ....................... 26 1.8.1 Yeast Recombination ............................................................................................. 27 1.8.2 Gateway Recombination System........................................................................... 28 1.9 Aims ............................................................................................................................ 29 2. Materials and Methods .................................................................................. 30 2.1 Microbial strains and growth media ........................................................................ 30 2.1.1 Routine Chemicals ................................................................................................. 30 2.1.2 Escherichia