Metabolic engineering of the yeast Yarrowia lipolytica for the production of even- and odd-chain fatty acids Young Kyoung Park To cite this version: Young Kyoung Park. Metabolic engineering of the yeast Yarrowia lipolytica for the production of even- and odd-chain fatty acids. Biotechnology. Université Paris-Saclay, 2020. English. NNT : 2020UPASB010. tel-03191617 HAL Id: tel-03191617 https://pastel.archives-ouvertes.fr/tel-03191617 Submitted on 7 Apr 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Metabolic engineering of the yeast Yarrowia lipolytica for the production of even- and odd-chain fatty acids Thèse de doctorat de l'université Paris-Saclay École doctorale n° 581 : agriculture, alimentation, biologie, environnement et santé (ABIES) Spécialité de doctorat : Biotechnologies Unité de recherche : Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas 78352 France Référent : AgroParisTech Thèse présentée et soutenue à Paris-Saclay, le 1er Octobre 2020, par Young-Kyoung PARK Composition du Jury Marie-Joëlle VIROLLE Presidente Directrice de recherche, CNRS, Gif-sur-Yvette Fayza DABOUSSI Rapporteur & Examinatrice Directrice de recherche, INRAE, Toulouse Frédéric DOMERGUE Rapporteur & Examinateur Chargé de recherche (HDR), CNRS, Bordeaux Sébastien BAUD Examinateur Directeur de recherche, CNRS, Versailles Ioana POPESCU Examinatrice Maître de conférences, Université d'Evry-Val-Essonne B010 Michael SAUER Examinateur Professeur, BOKU University Vasiliki TSAKRAKLIDES Examinatrice Responsable, Novogy Inc. : 2020UPAS Jean-Marc NICAUD Directeur de thèse Directeur de recherche, INRAE, Jouy-en-Josas NNT Thèse de doctorat Acknowledgements Last four years at INRA(E) in Jouy-en-Josas were great experiences for not only doing my PhD but also working with wonderful, admirable, and magnificent people. I am very grateful for everyone who welcome me with opened arms and support my PhD life in many ways. First and foremost, I want to give many thanks to the director of thesis, Jean-Marc Nicaud. I really appreciate to give me the opportunity for doing my PhD in BIMLip group. I am very grateful to all his contribution to this PhD project with unlimited and fruitful discussion, encouragements, and trust, which were great driving forces to progress myself and the PhD works. I learned a lot from his passion on the scientific research and enthusiasm towards Yarrowia lipolytica, which helped me to develop myself as a researcher. I greatly appreciate to all jury members of my thesis. I would like to thank two reporters, Fayza Daboussi and Frédéric Domergue, for their evaluation and kind reports. I also appreciate Marie-Joëlle Virolle, Sébastien Baud, Ioana Popescu, Michael Sauer, and Vasiliki Tsakraklides for the evaluation and many valuable comments for improving my thesis and establishing many ideas for future work. I express my gratitude to all my thesis committee members, Gilles Truan and Vincent Sauveplane, for not only their scientific suggestions and discussions but also kind encouragements and advices for future career. I extend my thanks to our collaborators, Ewelina Celińska, Polina Korpys, and Monika Kubiak (Poznan University of Life Sciences), Patrick Fickers and Marie Vandermies (University of Liege-Gembloux Agro-Bio Tech), Fabien Letisse and Florence Bordes (INSA Toulouse). I have no doubt that my PhD would be very difficult without these great collaborations. I greatly express my appreciation for their excellent scientific discussions, advices, and supports. I deeply appreciate the kind help of the graduate school ABIES in AgroParisTech, especially to Alexandre Pery, Irina Vassileva, and Christine Duvaux-Ponter. Their kind and interactive supports were superb and hugely helpful for continuing and finalizing PhD works without problems. I also would like to appreciate to Kwanjeong Education Foundation for the scholarship and generous supports of this adventure for PhD study in France. Many thanks to all BIMLip members for their kindness and supports since the beginning of the work. Especially, I appreciate to Rodrigo Ledesma-Amaro for helping me to settle down and initiate this PhD project. I learned many things from him even after his leaving, thanks for all his contributions. Great thanks to Stephane Thomas for all his kind support, especially managing analysis equipment which was one of important parts in my thesis. Many thanks to Tristan Rossignol, Anne-Marie Le Coq, and Heber Gamboa-Melendez for their kind scientific discussion and supports. Special thanks to Macarena Larroude and Pauline Trebulle who spent more than three years together in BIMLip. I was really lucky to do PhD together with you, thanks for all our conversations and encouragements each other which gave me a strength during this PhD. Many thanks to Lea Vidal for her pleasant supports, kind information, and experimental assistance for many times. I am very grateful for people in our building 526, the group COMBAC, PAPPSO, and FME. I cannot thank you enough for their kindness and encouragement. Many thanks to Vincent Juillard for his kindness and time to help solving HPLC problems and to Abarna Lingeswaran for her splendid organizing for several events in the building and transport system for RER C users. Last but not the least, I would like to express my great thanks to all my family for their endless support and encouragement. Thanks to my husband for being with me and giving huge supports in this hard time. Table of Contents CHAPTER 1. INTRODUCTION ........................................................................................... 1 1.1. CONTEXT OF THE STUDY ................................................................................................................................................ 1 1.2. BACKGROUND ............................................................................................................................................................. 3 1.2.1. Yarrowia lipolytica ........................................................................................................................................... 3 1.2.2. Metabolic engineering tools for Y. lipolytica ................................................................................................... 6 1.2.3. The biotechnological applications of Y. lipolytica .......................................................................................... 11 1.2.4. Lipid production in Y. lipolytica ...................................................................................................................... 16 1.2.5. Odd-chain fatty acids (OCFAs) ....................................................................................................................... 29 1.3. OBJECTIVES .............................................................................................................................................................. 39 CHAPTER 2. MATERIALS AND METHODS ...................................................................... 40 2.1. STRAINS, MEDIA, AND GROWTH CONDITIONS ................................................................................................................... 40 2.2. CLONING .................................................................................................................................................................. 40 2.2.1. General molecular biology ............................................................................................................................ 40 2.2.2. Gene synthesis ............................................................................................................................................... 41 2.2.3. Plasmid construction ..................................................................................................................................... 41 2.3. CONSTRUCTION OF Y. lipolytica STRAIN ........................................................................................................................ 44 2.3.1. Transformation ............................................................................................................................................. 44 2.3.2. Verification of construction in Y. lipolytica .................................................................................................... 44 2.3.3. Re-use of marker ........................................................................................................................................... 45 2.4. ANALYSIS ................................................................................................................................................................. 45 2.4.1. Growth .......................................................................................................................................................... 45 2.4.2. Fluorescence .................................................................................................................................................. 46 2.4.3. Metabolites ..................................................................................................................................................