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Mining of Cryptic Secondary Metabolism in Aspergillus O O O N R HN O N N N O O O OH OH O O OH OH OH O OH O OH OH OH OH HO O OH OH O OH OH OH O Yaojie Guo PhD Thesis Department of Biotechnology and Biomedicine Technical University of Denmark September 2020 Mining of Cryptic Secondary Metabolism in Aspergillus Yaojie Guo PhD Thesis Department of Biotechnology and Biomedicine Technical University of Denmark September 2020 Supervisors: Professor Thomas Ostenfeld Larsen Professor Uffe Hasbro Mortensen Preface This thesis is submitted to the Technical University of Denmark (Danmarks Tekniske Universitet, DTU) in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. This work was carried out from September 2017 to September 2020 under the supervision of the main supervisor Professor Thomas Ostenfeld Larsen and co-supervisor Professor Uffe Hasbro Mortensen at the Department of Biotechnology and Biomedicine (DTU Bioengineering). Throughout the three years´ research, I have received tons of help without which I wouldn´t be able to complete this thesis. First of all, I would like to give my sincere and deep gratitude to my supervisor Thomas, who is leading the Natural Product Discovery Group at Section for Microbial and Chemical Ecology, DTU Bioengineering. I was always feeling lucky to have such an experienced, open-minded, and easy-going supervisor. During the whole PhD study, Thomas not only gave me numerous scientific guidance, valuable discussions, and cheerful encouragements which kept me focused and motivated in the research but also supported me to learn new things beyond traditional natural product chemistry to expand my expertise. Secondly, I would like to thank my co-supervisor Uffe Hasbro Mortensen and Dr. Fabiano Jares Contesini. Uffe offered me an opportunity to perform molecular engineering research in B223 which was an amazing environment to study fungi. The professional suggestions by him always helped me out when I met trouble in the Aspergillus engineering world. Fabiano taught me a lot of methodologies and practical operations during the genetic manipulation process. I also want to express my thanks to my collaborator Malgorzata Ewa Futyma for the amazing cooperation on several projects during which l got to learn molecular biology. A huge thanks go to Assoc. Prof. Charlotte Held Gotfredsen and Dr. Kasper Enemark-Rasmussen for all the NMR related stuff. Special thanks go to Professor Jens Christian Frisvad for his experienced advice on all the fungal stuff. I am also very thankful to people in natural product discovery lab Ling Ding, Thomas Isbrandt Petersen, Mario Wibowo, Karolina Subko, Xinhui Wang, Aaron Andersen, Johan Vormsborg Christiansen, Lisette Knoth-Nielsen, Mette Amfelt, Elisabeth Varga, Andreas Heidemann, Christopher Phippen, as well as people in Aspergillus lab Jakob Blæsbjerg Hoof, i Andreas Møllerhøj Vestergaard and Jakob Kræmmer Haar Rendsvig. Besides, I feel like to extend my gratitude to all the people in B221 and B223 who offered a fantastic and collaborative atmosphere for research. Financial support for this study from the China Scholarship Council and DTU Bioengineering is highly acknowledged. Finally, I want to thank my family for their continuous and boundless support thousand miles away all over the past three years. Yaojie Guo Kgs. Lyngby, Denmark September 2020 ii Table of contents Contents Preface ........................................................................................................................................................... i Table of contents ......................................................................................................................................... iii Summary ....................................................................................................................................................... v Sammenfatning .......................................................................................................................................... viii List of publications and conference contributions ...................................................................................... xi List of abbreviations ....................................................................................................................................xiii 1. Introduction .............................................................................................................................................. 1 1.1 Fungi .................................................................................................................................................... 1 1.2 Aspergillus ........................................................................................................................................... 1 1.3 Natural products ................................................................................................................................. 4 1.4 Natural products from a biosynthetic perspective ............................................................................. 6 1.4.1 Polyketides ................................................................................................................................... 6 1.4.2 Nonribosomal peptides ................................................................................................................ 9 1.4.3 Terpenoids ................................................................................................................................. 11 1.4.4 Others......................................................................................................................................... 12 1.3 Natural product discovery techniques .............................................................................................. 13 1.3.1 High-performance liquid chromatography (HPLC) .................................................................... 13 1.3.2 Mass spectrometer (MS) ............................................................................................................ 15 1.3.3 Nuclear magnetic resonance (NMR) spectroscopy .................................................................... 17 1.3.4 Chiroptical spectroscopy and electronic circular dichroism (ECD) ............................................ 19 1.4 Genome editing technologies ........................................................................................................... 21 1.5 Establishment of the link between secondary metabolites and biosynthetic gene clusters ........... 23 1.5.1 From secondary metabolites to biosynthetic gene clusters ...................................................... 24 1.5.2 From biosynthetic gene clusters to secondary metabolites ...................................................... 25 2. Overall results and discussions ............................................................................................................... 27 2.1. Natural products discovery from Aspergillus californicus ................................................................... 28 2.1.1 Characterization of octaketides from Aspergillus californicus ...................................................... 28 2.1.2 Two new naphthyl derivatives from Aspergillus californicus ........................................................ 31 2.1.3 Characterization of two new OPK NRPs from Aspergillus californicus .......................................... 33 2.2. Investigation of the biosynthetic pathways of secondary metabolites in Aspergilli ........................... 34 iii 2.2.1 Investigation of the biosynthesis of calipyridone A in Aspergillus californicus ............................. 34 2.2.2 Genetic origin of homopyrones, a rare type of phenylpropanoid and polyketide derived yellow pigments from Aspergillus homomorphus .............................................................................................. 38 2.2.3 Linking of oxepinamide L to its biosynthetic gene cluster and linking of five genes to the cryptic metabolites in Aspergillus californicus ................................................................................................... 40 3. Conclusions and perspectives ................................................................................................................. 45 References .................................................................................................................................................