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Teleomorph Hypocrea Jecorina) Downloaded from orbit.dtu.dk on: Dec 20, 2017 Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei ( Teleomorph Hypocrea jecorina) Jørgensen, Mikael Skaanning; Larsen, Thomas Ostenfeld; Mortensen, Uffe Hasbro; Aubert, Dominique Publication date: 2013 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Jørgensen, M. S., Larsen, T. O., Mortensen, U. H., & Aubert, D. (2013). Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei ( Teleomorph Hypocrea jecorina). Kgs. Lyngby: Technical University of Denmark (DTU). 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Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei (Teleomorph Hypocrea jecorina) MJ-T-030 Mikael Skaanning Jørgensen Ph.D. Thesis November 2012 MJ-T-020 MJ-T-032 28 8 20 27 5 28 1 30 17 8 24 25 9 25 31 13 15 26 16 4 24 12 32 18 11 3 14 13 19 10 5 7 2 9 2521 22 23 Unraveling the Secondary Metabolism of the Biotechnological Important Filamentous Fungus Trichoderma reesei (Teleomorph Hypocrea jecorina) Mikael Skaanning Jørgensen Ph.D. Thesis November 2012 I. Preface and Funding The work regarding this thesis was primarily carried out at department 463, Fungal Gene Technology at Novozymes in Bagsvaerd. My first hands-on experience with Trichoderma reesei was acquired at the Fungal Expression I department of Novozymes, California, where I stayed for two months in the beginning of the project. All experimental work regarding Aspergillus nidulans and metabolite profiling using UHPLC-TOF-MS was carried out in building 223 and 221, respectively, at the Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark (DTU). The project was funded by Novozymes, Center for Microbial Biotechnology at DTU and Research School for Biotechnology (FOBI). Supervisors: Uffe Hasbro Mortensen Center Director, Professor DTU Systems Biology De partment of Systems Biology Center for Microbial Biotechnology Technical University of Denmark Dominique Aubert Skovlund Senior Scientist Department 463, Fungal Gene Technology Novozymes Mikael Skaanning Jørgensen II. Acknowledgements The past few years have been an incredible experience for me and include some of the most interesting times of my life and also some of the hardest. Through both I have been surrounded by magnificent people that deserve my acknowledgement. I owe my gratitude to everyone at Novozymes department 463, Fungal Gene Technology, for making me feel so welcome and for putting their interest into my project. It has truly been a pleasure to get to know everyone in the department and I know that I will miss their great company in the future. I also greatly appreciate the help I have received from the department’s laboratory technicians when time has been sparse – and I owe them for making my long days in the laboratory a joy. I have had a tremendous degree of freedom in the laboratory and for that I can only credit 463’s Department Manager Pia Francke Johannesen who has also contributed with great input regarding my project. Special thanks go to my supervisor from Novozymes, Senior Scientist Dominique Aubert Skovlund, who I have also been so fortunate to share an office with. I thank her for always taking her time to discuss important aspects of my project and for the inputs I have received through the last couple of years. Our mutual office mate, Senior Professional Jesper Henrik Rung, also deserves to be acknowledged for making the days at the office such a good experience. At DTU everyone at the Center for Microbial Biotechnology (CMB) has always made me feel welcome. The great expertise regarding fungal secondary metabolism at CMB has always resulted in great feedback after presentations, interesting discussions and good inputs for the project. In that regard PhD student Dorte Koefoed Holm deserves special attention – I truly admire her for her diligence. I would like to thank Associate Professor Kristian Fog Nielsen for introducing me to the world of analytical chemistry and for making this field of study much less frightening than it seemed before. My supervisor at DTU, Center Director and Professor Uffe Hasbro Mortensen, also deserves huge credit for his unique insight into my project. Even though his time has been sparse, our meetings have always been fruitful and I am grateful for his feedback during preparation of the manuscripts included in this thesis. I also want to thank my friends for hanging on even though I have been missing in action for several long periods during the extent of this project. I really look forward to be able to spend more time with them again. Last, but not least, I am full of gratitude for the extraordinary family I have been rewarded with. I truly appreciate the unconditionally support that I have always been given, not just through this project, but in all aspects and I will always be grateful for having them in my life. III. Abstract The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the most important industrial production organisms, owing to its highly efficient (hemi- )cellulase synthesis and secretion machineries. These enzymes, which in nature allow the fungus to utilize energy bound in cellulosic biomass, has wide application in the pulp and paper, textile and biofuel industries. The genomic sequence for T. reesei was published in 2008 and provides the basis for introducing targeted genetic alterations that could for example increase cellulase production yields. Like all other filamentous fungi, also T. reesei synthesizes a range of small bioactive metabolites which are not required for growth, but can broaden the ecological niche of the fungus. Since these bioactive metabolites are not involved in the primary metabolism, they are often referred to as secondary metabolites. These metabolites include some of the most potent toxins of natural source and can therefore have a vast implication on human health and also on the economy if crops are contaminated or livestock affected. Luckily, the high bioactivity for these metabolites also provides a range of beneficial activities and they therefore constitute one of the most important sources for pharmaceuticals; including drugs for treatment of infections, for lowering of the cholesterol level in the bloodstream and for minimizing undesirable immune responses. The primary objective of this study was to gain more knowledge about the genetic mechanisms leading to biosynthesis of two of the most prominent types of secondary metabolites produced by T. reesei, polyketides and non-ribosomal peptides. Since the molecular tools for T. reesei are not as well-developed as for many other species, the study was initiated with creation of new genetic tools that would enable pursuance of the primary objective. The developed molecular tools were assembled into an expression system for high-throughput construction of defined integrated T. reesei mutants and combined inactivation of the non-homologous end joining pathway that facilitates ectopic integration of exposed DNA fragments, and a color maker so that the mutants, in which the substrate had been integrated correct, could be identified by their phenotype. A new bidirectional selective marker system was developed based on the pyr2 gene, involved in the pyrimidine biosynthesis pathway, and was included in the expression system. The developed expression system was subsequently utilized to overexpress several transcription factor genes located in the proximity of polyketide synthase- or non-ribosomal synthetase genes in T. reesei. Overexpression of one of these transcription factor genes led to a significantly increased production of 78 different metabolites, some of which had previously been described and belong to the polyketide derived sorbicillinoids. Genetic deletions performed subsequently, demonstrated that two polyketide synthase genes, located near the overexpressed transcription factor gene, were both essential for biosynthesis of the sorbicillinoids. Hence, genes involved in biosynthesis of this group of polyketides were identified for the first time. Comparative genomics was subsequently used to identify a highly similar polyketide synthase gene cluster in another well-known sorbicillinoid producer, Penicillium chrysogenum. Twenty three T. reesei polyketide synthase- and non-ribosomal peptide synthetase genes were selected for heterologous expression in Aspergillus nidulans, using a previously developed expression system. Several of the resulting mutants exhibited altered phenotypes and could be divided into two groups; one with mutants producing a pigment and another where the mutants had changed colony
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