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Dortmund 2018 Evaluation of C-prenylating enzymes for the heterologous biosynthesis of cannabigerolic acid Zur Erlangung des akademischen Grades eines Dr. rer. nat. von der Fakultät Bio- und Chemieingenieurwesen der Technischen Universität Dortmund genehmigte Dissertation vorgelegt von M.Sc. Sara Friederike Degenhardt aus Mainz Tag der mündlichen Prüfung: 09.10.2018 1. Gutachter: Prof. Dr. Dr. h.c. Oliver Kayser 2. Gutachter: Prof. Dr. Heribert Warzecha Dortmund 2018 i Acknowledgements Acknowledgements I would like to express my appreciation and gratitude to all the people who supported, encouraged and motivated me over the last years. Firstly, I would like to express my gratitude to my supervisor Prof. Dr. Dr. h.c. Oliver Kayser (Chair of Technical Biochemistry, TU Dortmund, Germany) for his support and guidance over the last years. He kept me going when times were though and offered me the opportunity of a Short-Term Scientific Mission in the group of Prof. Poul Nissen (Aarhus University, Denmark). Thanks to Dr. Felix Stehle (Chair of Technical Biochemistry, TU Dortmund, Germany) for helpful suggestions as well as his constant support and advice. I am also thankful for the prediction of NphB mutants. I thank PD Dr. Wolfgang Brandt (Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany) for generating the structural model of NphB complexed with GSPP and OA. Furthermore, I would like to thank the Ministry of Innovation, Science and Research of the German Federal State of North Rhine-Westphalia and TU Dortmund for awarding me with a scholarship from the CLIB-Graduate Cluster Industrial Biotechnology (CLIB2021). I am thankful to the European Cooperation in Science and Technology (COST) for financing my Short-Term Scientific Mission (STSM) in the Nissen group at the University of Aarhus (Denmark) (PlantEngine, COST Action FA1006). My sincere thank goes to Prof. Poul Nissen for offering me the opportunity to work in his lab at the University of Aarhus (DANDRITE, University of Aarhus, Denmark) and to learn a lot from him in the field of membrane-bound enzymes. Further, I am thankful to Sigrid Thirup Larsen and Joseph Lyons for guidance and support. I thank the Koch group (Medicinal Chemistry, CCB, TU Dortmund, Germany), especially Jette Pretzel and Christiane Ehrt, for proposing NphB variants. The CEN.PK-1C strain was a kind gift from Dr. Peter Kötter (Goethe University Frankfurt, Germany) and the strain S. cerevisiae Δpep4 Δgal4 was kindly provided by Prof. Poul Nissen (DANDRITE, University of Aarhus, Denmark). Thank you to Prof. David Drew (Imperial College London, United Kingdom) for making the expression vector pDDGFP-2 available for us. I am thankful to the Bachelor and Master Students for their excellent help during the laboratory work: Juliana Estrela, Christine Finger, Julia Grühn, Ramona Hansmeyer, Martin Kares, Dirk Münker, Martin Obholzer, Daniel Stalinski, Christian Staubach and Marlon Wach. A special thanks goes to Chantale Zammarelli for GPP synthesis, fun times in the lab and after work as well as for her friendship. ii Acknowledgements Thank you to Jan Schonert (CCB, TU Dortmund, Germany) for performing NMR measurements and Nico Zammarelli for help with GPP synthesis. I am grateful to my colleagues from the Biozentrum. I enjoyed fruitful discussions, mental support and the great time we spent together in the lab and after work, especially with Bastian Zirpel, Laura Kohnen, Evamaria Gruchattka, Nouman Almofti, Angela Sester, Jenny Schwarz, Anna Tippelt, Friederike Ullrich, Julia Schachtsiek, Kathleen Pamplanyil and Torsten Arndt. Last but not least I want to especially thank my family, friends and ”Sniffles“ for motivation, encouragement and the best support I could wish for. Studies were conducted with the permission of No. 458 64 16 issued by the Federal Institute for Drugs and Medical Devices (BfArM), Germany. iii Table of contents Table of contents 1. INTRODUCTION ............................................................................................................ 1 1.1. PREFACE .................................................................................................................. 2 1.2. THE BIOSYNTHESIS OF CANNABINOIDS ........................................................................ 3 1.2.1. Polyketide pathway towards olivetolic acid ..................................................................... 5 1.2.2. Biosynthesis of geranyl diphosphate ............................................................................... 7 1.2.3. Biosynthesis of cannabinoids from GPP and OA ............................................................ 8 1.3. AROMATIC PRENYLTRANSFERASES ...........................................................................14 1.3.1. Membrane-bound aromatic prenyltransferases ............................................................. 15 1.3.2. Soluble, aromatic prenyltransferases ............................................................................ 18 1.4. SCOPE OF THIS THESIS .............................................................................................23 2. MATERIAL AND METHODS ........................................................................................24 2.1. MATERIALS ..............................................................................................................25 2.2. METHODS ................................................................................................................25 2.2.1. Expression of membrane-bound prenyltransferases in S. cerevisiae ........................... 25 2.2.2. Expression of nphB in Escherichia coli ......................................................................... 33 2.2.3. Expression of nphB and thcas in Saccharomyces cerevisiae ....................................... 40 3. RESULTS AND DISCUSSION ......................................................................................48 3.1. EXPRESSION OF MEMBRANE-BOUND PRENYLTRANSFERASES IN S. CEREVISIAE ............48 3.1.1. Comparison of CsPT1, CsPT2, CsPT3 and CsPT1P .................................................... 49 3.1.2. Sequence analysis ........................................................................................................ 49 3.1.3. Expression strategy ....................................................................................................... 55 3.1.4. Expression of CsPT1, CsPT2 and CsPT3 ..................................................................... 58 3.1.5. Discussion ..................................................................................................................... 67 3.2. EXPRESSION OF NPHB IN ESCHERICHIA COLI .............................................................74 3.2.1. Screening for an E. coli strain with the highest nphB expression level ......................... 74 3.2.2. NphB activity in the presence of 1,6-DHN ..................................................................... 76 3.2.3. NphB activity in the presence of olivetol and olivetolic acid .......................................... 78 3.2.4. Influence of the N-terminal TrxA tag .............................................................................. 81 iv Table of contents 3.2.5. Protein engineering of NphB ......................................................................................... 83 3.2.6. Screening of different NphB mutants for high CBGA formation .................................... 85 3.2.7. Discussion ..................................................................................................................... 87 3.3. EXPRESSION OF NPHB AND THCAS IN SACCHAROMYCES CEREVISIAE ..........................95 3.3.1. Test of different NphB coding sequences ..................................................................... 95 3.3.2. Whole cell assay ............................................................................................................ 98 3.3.3. Fusion of nphB and thcas .............................................................................................. 99 3.3.4. Discussion ................................................................................................................... 105 4. CONCLUSION AND OUTLOOK ................................................................................. 113 4.1. EXPRESSION OF MEMBRANE-BOUND PRENYLTRANSFERASES IN S. CEREVISIAE .......... 113 4.2. EXPRESSION OF NPHB IN E. COLI ............................................................................ 114 4.3. EXPRESSION OF NPHB AND THCAS IN S. CEREVISIAE ................................................ 115 ERKLÄRUNG ZUR REPRODUKTION VORAB VERÖFFENTLICHTER INHALTE ........... VIII REFERENCES ..................................................................................................................... IX SUPPLEMENTARY INFORMATION .............................................................................. XXIX LIST OF ABBREVIATIONS .................................................................................................... XXX EXPRESSION OF MEMBRANE-BOUND PRENYLTRANSFERASES IN S. CEREVISIAE ................... XXXV EXPRESSION OF NPHB IN ESCHERICHIA COLI ......................................................................XLIII EXPRESSION OF NPHB AND THCAS IN S. CEREVISIAE .............................................................. LI v Abstract Abstract Aromatic prenyltransferases catalyze the prenylation of an electron-rich aromatic compound by a prenyl diphosphate
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