Downloaded from orbit.dtu.dk on: Oct 09, 2021 Genomics-driven discovery, characterization, and engineering of fungal secondary metabolites Wolff, Peter Betz Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Wolff, P. B. (2020). Genomics-driven discovery, characterization, and engineering of fungal secondary metabolites. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. GENOMICS-DRIVEN DISCOVERY, CHARACTERIZATION, AND ENGINEERING OF FUNGAL SECONDARY METABOLITES Peter Betz Wolff A thesis submitted for the degree of Doctor of Philosophy February 2020 Genomics-driven discovery, characterization, and engineering of fungal secondary metabolites Peter Betz Wolff, 27th of February 2020 Address: Technical University of Denmark Department of Biotechnology and Biomedicine Sølftofts Plads, Building 223 2800 Kgs. Lyngby Denmark Supervisors: Professor Uffe Hasbro Mortensen Professor Thomas Ostenfeld Larsen Preface Preface This Ph.D. thesis describes the results of my study initiated at the Technical University of Denmark (DTU) in September 2016 and finished in February 2020. The study was conducted at DTU Bioengineering, department of Biotechnology and Biomedicine under the supervi- sion of professor Uffe Hasbro Mortensen and Professor Thomas Ostenfeld Larsen. The Novo Nordisk foundation supported the work, grand NNF15OC0016610. During my Ph.D. project, I have had the opportunity to learn new techniques for molecular engineering within fungal secondary metabolism, gain a broader knowledge for project management, and participate in inspiring conferences worldwide. Kgs. Lyngby, February 2020 Peter Betz Wolff Page i Contents Contents Preface i Acknowledgements v Summary vi Sammenfatning ix Publications xii Conference contributions xiii Abbreviations xiv 1 Project motivation and objective 1 Project motivation . .1 Project objective . .1 References . .3 2 Background 4 Biology and chemistry . .4 The fifth kingdom . .4 From genes to natural products . .5 The biosynthesis of natural products . .8 Pathways . 14 Tetracyclines . 18 Mode of action . 19 Structure activity relationship . 20 Bacteria resistance mechanisms . 22 Tools......................................... 23 Bioinformatics . 23 Quantitative real-time PCR . 23 Chemical analysis and dereplication . 24 Molecular networking for biosynthetic pathway elucidation . 24 References . 26 3 Fungal Synthetic Biology Tools to Facilitate Utilization of Secondary Metabo- lites: Discovery; Heterologous Production; and Biosynthesis of Biocombinato- rial Compounds 38 Abstract . 38 Introduction . 38 Page ii Contents Discovery of secondary metabolite pathways in natural hosts . 39 CRISPR Cas9 in filamentous fungi . 40 Gene-expression cassettes . 41 Activation of silent BGCs in the native host . 43 Global regulation on and beyond chromatin level . 44 Linking fungal biosynthetic gene clusters to secondary metabolite products 46 Discovery and characterization using heterologous expression . 47 Fungal cell factories . 49 Optimization of fungal cell factories . 49 Expanding the chemical space via biocombinatorial synthetic biology . 51 Synthetic Pathways for Production of Natural and Non-natural Products . 51 Synthetic Enzymes for Production of Non-Native Products . 54 Perspective . 56 References . 57 4 Acurin A; a novel hybrid compound; biosynthesized by individually translated PKS- and NRPS-encoding genes in Aspergillus aculeatus 69 Abstract . 69 Introduction . 70 Materials and Methods . 71 Results and Discussion . 76 Acurin A – a novel PK-NRP hybrid compound from Aspergillus aculeatus . 76 Identification of putative PKS and NRPS genes for the production of acurin 78 Separate PKS- and NRPS enzymes synthesize the acurin A core structure . 80 Uncoupled PKS and NRPS activity is conserved in a clade in section Nigri . 81 The acurin BGC contains genes encoding tailoring enzymes and a TF . 85 Gene deletions identify genes in the acr biosynthetic gene cluster . 87 Proposed biosynthesis of acurin A . 89 Conclusion . 91 Acknowledgements . 92 References . 92 5 Genetic Manipulation and Molecular Networking for Biosynthetic Pathway Characterization of Tetracycline-like Compounds in Aspergillus sydowii 97 Abstract . 97 Introduction . 97 Results . 98 Screening the genus Aspergillus for a potential tetracycline-likeBGC . 98 Homologous expression of a putative pathway-specific transcription factor . 100 Targeted gene deletions for tetracycline-like BGC characterization . 102 Molecular networking as a tool for intermediate characterization . 103 Page iii Contents Proposed biosynthetic pathway . 106 Discussion . 109 Screening the genus Aspergillus for potential tetracycline BGCs . 109 Overexpression of nstR leads to an unnatural behavior in A. sydowii .... 109 Targeted gene deletions verify neosartoricinB as the main BGC product . 110 Molecular networking as a tool for biosynthetic pathway characterization . 110 Enzyme order in the pathway is defined by the environmental factors . 110 Conclusion . 111 Acknowledgements . 112 Experimental . 112 References . 115 6 Expression Platform for Biocombinatorial Decoration of a Fungal-derived Tetra- cycline in Aspergillus niger 118 Abstract . 118 Introduction . 118 Results . 119 Homologous reconstitution of the ada pathway in A. niger ......... 119 Verification and analysis of tetracycline production in the platform strains . 122 AMA1-based expression of two tailoring-enzymes for further validation . 122 Identifying tailoring enzyme-encoding genes for further expression . 124 Discussion . 126 Conclusion . 127 Acknowledgements . 127 Experimental section . 127 References . 129 7 Overall discussion, conclusion, and perspectives 131 References . 131 Appendix I (Chapter 4) 137 Appendix II (Chapter 5) 183 Appendix III (Chapter 6) 214 Page iv Acknowledgements Acknowledgements Firstly, I would like to express my gratitude to my supervisors Prof. Uffe Hasbro Mortensen and Prof. Thomas Ostenfeld Larsen, for the opportunity to be part of this research project. These last three and a half years have been an exciting, rewarding, and educational experience for me, and it would not have been possible without the invaluable guidance I received from both throughout this research. They have taught me the methodology needed to carry out my research and to present the results in the most precise way possible. Their motivation and vision for the project have deeply inspired me, and I feel incredibly grateful to have had the chance to work with them. My former master supervisor Assoc. Prof. Jakob Blæsbjerg Hoof has given me valuable support and been a mentor for me throughout this project. I thank you for all the enjoyable scientific discussion we have had over the years, they have inspired me to do more. I want to thank Karolina Subko for the fantastic cooperation on this project. You are a brilliant chemist with innovative ideas, an eye for details, and a great drive. I have enjoyed working together with you, and I have gained a lot more insights into the world of chemistry through our many talks. Thank you. Working at the Technical University of Denmark (DTU) and especially in the Aspergillus laboratory at the section for synthetic biology, has been one of the best working experiences in my career. My co-workers and fellow Ph.D. candidates have always been helpful, and with a great sense of humor. I have particularly appreciated my productive discussions with Kresten Jon Korup Kromphardt and Jakob Kræmmer Haar Rendsvig, they have both contributed to improving my understanding of fungal science. Also, Zofia Dorota Jarczynska has been a great help in the laboratory with guidance and advice. And a special thanks to Andreas Møllerhøj Vestergaard for keeping us all entertained in his inexhaustible search for fungal monooxygenases. Finally, to my caring, loving, and supportive wife, Naja: you have my deepest gratitude. You have been a guardian angel throughout these years, and the project would never have been finished if it wasn’t for you. Your loving care for our toddler daughter and baby son, in my absence, while writing, has been admirable and an inspiration to me. You have helped me in my darkest hour of frustration, listened, and always reassured me that I could do this. For that, I owe you everything. Page v Summary Summary Filamentous fungi can produce many natural products (NPs) with both beneficial properties (e.g., bioactive pharmaceutical compounds), as well as harmful substances (e.g., mycotoxins). For the past decade, microbial whole-genome sequencing projects have identified a sizeable genetic potential for discovering yet more unknown natural products, which include complex derivatives of already-approved drugs. Filamentous fungi, here among the genus Aspergillus, has been a source