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Optimization of Isopropanol Production by Engineered Escherichia Coli

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Optimization of Isopropanol Production by Engineered Escherichia Coli Optimization of Isopropanol Production by Engineered Escherichia coli DISSERTATION In Partial Fulfillment of the Requirements For the Academic Degree of Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Faculty of Sciences I – Life Sciences Martin Luther University Halle-Wittenberg By Ramona Engelhardt, née Konrad, MSc Born on January 15, 1982 in Buende, Germany Halle (Saale) 2020 Examiners: 1. Prof. Dr. Markus Pietzsch, Martin Luther University Halle-Wittenberg 2. Prof. Dr. Bruno Bühler, Martin Luther University Halle-Wittenberg 3. Prof. Dr. Udo Rau, Technical University Braunschweig Oral Defense: 03.11.2020 2 Declaration of Academic Honesty I hereby declare and confirm with my signature that the present doctoral dissertation is solely the result of my own work, based on my research and without using any other resources than the ones indicated. All thoughts taken directly or indirectly from external sources are properly denoted as such. I further declare that all persons and institutions that have assisted in the preparation of the thesis have been acknowledged and that this thesis has not been submitted, in part or wholly, as an examination document to any other authority. Halle (Saale), 08.06.2020 (Signature) 3 Acknowledgement I would like to express my sincere gratitude to my doctoral advisor Prof. Dr. Markus Pietzsch for his continuous support during my PhD studies, for his guidance, motivation and immense knowledge. Thank you for being a great teacher in every aspect of research. I would particularly like to thank the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) for the financial support and funding of the Leading-Edge Cluster “BioEconomy” and the Cluster project “TG 2 – Chemistry, VP 2.5, Energy-efficient synthesis of olefins from their corresponding alcohols”. I greatly appreciate the fruitful collaboration and constructive meetings with the project cooperation partners: Linde Engineering Dresden GmbH, CRI Catalyst Leuna GmbH, University of Leipzig (Institute of Technical Chemistry) and Fraunhofer Society (Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.) - Center for Chemical-Biotechnological Processes (CBP) - Institute for Interfacial Engineering and Biotechnology (IGB) - Institute for Chemical Technology (ICT). I am indebted to the Fraunhofer CBP for provision and analysis of the beech wood hydrolysate (BWH). I had the pleasure of meeting and working with many great Fraunhofer people: Dr. Daniela Pufky- Heinrich, Gerd Unkelbach, Tino Elter, Dr.-Ing. Katja Patzsch, Sandra Torkler, Anita May, Anja Hauffe, Holger Becker. Thank you also for giving me the opportunity to participate in the 35 L bioreactor scale cultivations of one of my isopropanol-producing strains. I am very grateful to the examiners of my dissertation: Prof. Dr. Markus Pietzsch, Prof. Dr. Bruno Bühler and Prof. Dr. Udo Rau. Special thanks to the people who read parts of my thesis before submission and who provided me with valuable feedback: Anne Kind, Dr. Christian Marx and especially Dr. Bodo Moritz. Profound thanks to Martina Anwand who taught me almost everything I know about bioreactor cultivation. Thank you for your advice and your friendship. I also thank Dr. Norbert Volk for his support in bioreactor cultivation planning. I would like to acknowledge the technical assistance of Martina Schreiber and Bal Mukund Sharma. Many thanks to Dr. Angelika Schierhorn who performed the peptide mass fingerprint analyses. I also wish to thank my former master students Joyshree Ganguly and Benjamin Schrank (now Böhme) for their dedicated work. I cannot begin to express my thanks to my co-workers of the Department of Downstream Processing (AG Aufarbeitung biotechnischer Produkte). Thank you, not only for helpful advice, practical suggestions and insightful discussions, but also for making the lab my “second home”. I would like to thank my family and friends for their unwavering support and patience. I owe my deepest gratitude to my parents Gabi and Erwin Konrad for their love and encouragement. Thanks to my friend Carsten Struessmann for believing in me from the beginning. Heartfelt thanks to my husband Matthias Engelhardt. Thank you for your love, help, and your priceless computer tools. Special thanks to my cat Julie who was always by my side, while I was writing this thesis. Last, but not least, I would like to thank my daughter Isabelle Aurélie for being the light of my life. 4 The scientific man does not aim at an immediate result. He does not expect that his advanced ideas will be readily taken up. His work is like that of the planter - for the future. His duty is to lay the foundation for those who are to come, and point the way. He lives and labors and hopes. Nikola Tesla The Problem of Increasing Human Energy - (The Century Magazine, June 1900) ~ This thesis is dedicated to my parents. ~ In loving memory of my grandmother Alma and my grandfather Edmund. Table of Contents 5 Table of Contents Declaration of Academic Honesty ....................................................................................................... 2 Acknowledgement ............................................................................................................................. 3 Table of Contents ............................................................................................................................... 5 List of Abbreviations .......................................................................................................................... 8 List of Figures ................................................................................................................................... 12 List of Tables .................................................................................................................................... 15 1 Introduction ......................................................................................................................... 18 2 State of the Art .................................................................................................................... 21 2.1 Isopropanol - Basic Facts .................................................................................................. 21 2.2 Isopropanol Production by Chemical Processes ................................................................. 22 2.3 Isopropanol Production by Microorganisms ...................................................................... 22 2.3.1 Natural Isopropanol Producers.............................................................................................. 22 2.3.2 Recombinant Isopropanol Producers .................................................................................... 25 2.3.3 Enzymes of the Isopropanol Pathway ................................................................................... 29 2.4 Lignocellulose Hydrolysates as Feedstock for Microorganisms ........................................... 31 3 Aims and Objectives ............................................................................................................. 36 4 Material and Methods .......................................................................................................... 37 4.1 Material ........................................................................................................................... 37 4.1.1 Chemicals ............................................................................................................................... 37 4.1.2 Buffers and Solutions ............................................................................................................. 39 4.1.3 Lignocellulose Hydrolysate .................................................................................................... 40 4.1.4 Bacterial Strains ..................................................................................................................... 41 4.1.5 Bacterial Growth Media, Antibiotics and Supplements ........................................................ 41 4.1.6 Plasmid DNA .......................................................................................................................... 42 4.1.7 Primer Oligonucleotides ........................................................................................................ 43 4.1.8 Enzymes and Molecular Biology Reagents ............................................................................ 44 4.1.9 Antibodies .............................................................................................................................. 45 4.1.10 Markers .................................................................................................................................. 45 4.1.11 Kits ......................................................................................................................................... 46 4.1.12 Instruments, Devices, Laboratory Equipment and Consumables.......................................... 46 4.1.13 Computer Programs and Online Tools .................................................................................. 49 4.2 Methods .......................................................................................................................... 49 4.2.1 Microbiological Methods ....................................................................................................... 49 4.2.1.1 Cultivation of Bacterial Cells in Shake Flask Scale .........................................................
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