Downloaded from orbit.dtu.dk on: Mar 30, 2019 Genus-level studies of gene dynamics for the Aspergillus genus Theobald, Sebastian Publication date: 2018 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Theobald, S. (2018). Genus-level studies of gene dynamics for the Aspergillus genus. Kgs. Lyngby: Technical University of Denmark (DTU). 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. 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Genus-level studies of gene dynamics for the Aspergillus genus PhD thesis by Sebastian Theobald Technical University of Denmark January 2018 2 Contents 1 Introduction 13 1.1 Fungi and their impact on society . 13 1.2 Non ribosomal peptide synthetases (NRPS) . 15 1.3 Polyketide synthases (PKS) . 17 1.4 Polyketide - non ribosomal peptide synthetase hybrids . 19 1.5 Genome research in Aspergilli . 20 1.6 Approaches for Comparative Genomics in Aspergillus and Peni- cillium .............................. 21 2 The genomes of Aspergillus section Nigri reveal drivers in fungal speciation 63 3 Uncovering bioactive compounds in Aspergillus section Ni- gri by genetic dereplication using secondary metabolite gene cluster networks 81 4 Genus level analysis of PKS-NRPS and NRPS-PKS hybrids reveals their origin in Aspergilli 93 5 Comparative genomics of A. nidulans and section Nidu- lantes 109 6 Conclusions and perspectives 121 A Appendix 123 A.1 Supplementary information: The genomes of Aspergillus sec- tion Nigri reveal drivers in fungal speciation . 123 A.2 Supplementary information: Uncovering bioactive compounds in Aspergillus section Nigri by genetic dereplication using sec- ondary metabolite gene cluster networks . 145 A.3 Supplementary information: Genus level analysis of PKS-NRPS and NRPS-PKS hybrids reveals their origin in Aspergilli . 151 3 A.4 Supplementary information: Comparative genomics of A. nidu- lans and section Nidulantes ................... 156 4 Contents Preface This thesis serves as a partial fulfillment of the requirements to obtain a PhD degree of the Technical University of Denmark (DTU). The work was carried out at the Department of Biotechnology and Biomedicine under the supervision of Prof. Mikael R. Andersen, Assistant Prof. Tammi C. Vesth, Prof. Thomas O. Larsen and Anders G. Pedersen. The project has been funded by the Villum foundation (grant VKR023437). Kongens Lyngby, January 2018 Sebastian Theobald 5 Acknowledgements This project would have not been possible without the many wonderful people involved who supported me professionally and personally. First, I would like to thank my supervisors Mikael Rørdam Andersen and Tammi Camilla Vesth for giving me the opportunity to pursue this project and mentoring me during the three years. You were putting me on the right path when I was getting lost in the details of the project | which in my case happens quite frequently. I learned so much during these three years. Thank you for making this possible and also teaching me that there's much more to science than just the project. Furthermore, I would like to thank the whole Network Engineering of Eukaryotic Cell Factories group. Especially Jane Nybo, the best office mate you can wish for, with constant positive and energetic attitude, Inge Kjærbølling for also being full of positive energy and curiosity, and Julian Brandl for his humor and supporting me with python. It was a great experience to work and travel together with you guys to so many different places. This mostly bioinformatic project tried to bridge the gap between com- parative genomics and molecular biology, which would have not been possible without the help of my colleagues. I would like to thank Jakob B. Hoof and Jakob K. Rendsvig for a great collaboration to elucidate the malformin gene cluster and Thomas Ostenfeld Larsen, who joined as co-supervisor later in the process, for his advice and mentoring regarding the chemical aspects of this thesis. Also I would like to thank Matthias Brock for the opportunity to visit his laboratory at University of Nottingham and Elena Geib for col- laboration. Here also a big thanks to Justyna Kuska! It was great to have a small masters reunion in the UK! DTU Bioengineering has been a great place to work and I want to thank everyone in building 223 and 221 for creating such a good atmosphere. I'm very grateful that I could work together with so many inspiring people during my PhD | I definitely learned a lot from everyone. Also, I would like to thank my family and friends for their constant sup- port they gave me during these past three years. Finally, a very special thanks goes to Angela^ de Carvalho for the love, the endless support, and for just always being there. 6 Abstract The fungal genus Aspergillus has a great impact on society. It includes species with industrial value A. niger and A. oryzae, medically relevant species As- pergillus fumigatus and A. terreus, and the model organisms A. nidulans. A. nidulans, chosen as model organism due to its sexual cycle and pos- sibility to study genetics using spore color, provided insights into key regu- lators of cell cycle control, development and secondary metabolism. Results obtained from studies on A. nidulans were found to be applicable on other Aspergilli as well. Genome sequencing of A. nidulans, A. fumigatus and A. oryzae further revealed a large phylogenetic distance between species of the genus. Hence, conclusions derived from studies on model organisms might not apply to the genus as a whole but rather on smaller taxonomical groups. Taxonomy of Aspergilli is constantly under evaluation and the information added by new methods and technologies is aiding the definition of new taxonomic groups (Chen et al., 2016; Hubka et al., 2016). In the 300 Aspergillus genome project, we are extending the set of se- quenced species to further investigate genome and gene diversity across the entire genus. This specific project deals with the diversity of secondary metabolite (SM) genes and conservation of regulators. To achieve a com- parison of several species at once, we created methods which classify genes into families. This approach highlighted that regulators like mcrA, a master regulator of secondary metabolism, is conserved throughout Aspergilli and that galR, a regulator which was thought to be unique for A. nidulans is present in many other species. SM genes needed a slightly different type of clustering since they show high similarity between each other | making them difficult to separate. The full pathway for a secondary metabolite is encoded on a spatial collective of genes, a secondary metabolite gene cluster (SMGC). Thus, to find families of SMGC for production of similar SMs we compared whole gene clusters against each other. Our analysis highlights differences of SMGC families shared on several taxonomic levels. Characterizing regulators and SMGC over several species as families has the advantage that we are able to follow their distribution throughout the genus. This has several benefits. Examination of conserved genes can give insights into the adaptations of a section of Aspergilli. Examining SMGC families can give hints to uninvestigated SMGC producing promising drug leads. Overall, analyzing Aspergillus species with this comparative approach will reveal their gene dynamics and diversity and give insights into adapta- tions of sections throughout the genus. 7 Dansk resume Den filamentøse svampeslægt Aspergillus har stor indflydelse p˚asamfundet. Slægten indeholder arter med stor industriel værdi (f.eks. A. niger og A. oryzae), medicinsk indflydelse (f.eks. A. fumigatus og A. terreus), og biol- ogisk model organismer (f.eks. A. niger og A. nidulans). A. nidulans blev en klassisk model organisme grundet sin seksuelle cyklus og muligheden for at studere genetik ved hjælp af sporefarve. Dette har givet indsigt i essen- tielle regulatorer af cellecykluskontrol, udvikling og sekundær metabolisme. Studier foretaget i A. nidulans har vist sig at være anvendelige i flere af arterne fra Aspergillus slægten. I 2005 blev A. nidulans, A. fumigatus og A. oryzae genom sekventeret og dette afslørede en langt større fylogenetisk afstand mellem arterne end før antaget. Derfor er det vigtigt ikke at antage at konklusioner fundet i studier af model organismer kan overføres direkte p˚aslægtensom helhed, men snarere p˚amindretaksonomiske grupper. Tax- onomien for Aspergillus slægten er konstant under evaluering, hvor informa- tion opn˚aetvia nye metoder og teknologier understøtter definitionen af nye taxonomiske grupper (Chen et al., 2016; Hubka et al., 2016). Vi vil, igennem Aspergillus hel-slægts sekventeringsprojektet, udvide sættet af sekventeret arter for yderligere at undersøge genom og gendiversiten over hele slægten. Denne afhandling omhandler diversiteten af sekundære metabolit (SM) gener og konserveringen af regulatorer. For at sammenligne flere arter p˚a´en gang, har vi skabt metoder der grupperer gener i familier af samme funktion eller produkt. Disse metoder har fremhævet at regulatorer som mcrA, en master- regulator af sekundær metabolisme, er bevaret i hele Aspergillus slægten og at galR, en regulator der blev anset for at være unik i A. nidulans er til stede i langt flere arter. SM gener bruger en speciel type gruppering, da de viser høj genetisk similaritet mellem hinanden | hvilket gør dem vanske- lige at adskille. Den fulde pathway af en sekundær metabolit er kodet i et spatial kollektiv af gener, et sekundært metabolit genkluster (SMGC).