Downloaded from orbit.dtu.dk on: Sep 26, 2021 The Transcriptional Landscape of the Production Organism Pseudomonas putida D'Arrigo, Isotta Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): D'Arrigo, I. (2016). The Transcriptional Landscape of the Production Organism Pseudomonas putida. Novo Nordisk Foundation Center for Biosustainability. 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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The Transcriptional Landscape of the Production Organism Pseudomonas putida Ph.D. Thesis Isotta D’Arrigo The Novo Nordisk Foundation Center for Biosustainability The Technical University of Denmark April 2016 The Transcriptional Landscape of the Production Organism Pseudomonas putida Ph.D. Thesis 2016 © Isotta D’Arrigo Novo Nordisk Center for Biosustainability Technical University of Denmark Kogle Alle 6, DK - 2970 Hørsholm Denmark Cover art: image adapted by Se Hyeuk Kim. “It’s not what you look at that matters, it’s what you see” (Henry David Thoreau) To my grandfather, I did not know why I am here Preface This thesis is written as a partial fulfillment of the requirements to obtain a Ph.D. degree at the Technical University of Denmark. The work presented here was performed between May 2013 and April 2016 at the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark. The work was supervised by Katherine S. Long, Associate Professor from the Technical University of Denmark. Funding was provided by The Novo Nordisk Foundation and a Ph.D. grant from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme, [FP7-People-2012-ITN], under grant agreement no. 317058, “BACTORY”. The thesis was evaluated by Professor Juan Luis Ramos Martin from Abengoa Research, by Jeppe Vinther, Associate Professor at the University of Copenhagen, and by Alex Toftgaard Nielsen, Professor at Technical University of Denmark. Isotta D’Arrigo Hørsholm, April 2016 i Acknowledgements First, I would like to thank my supervisor Katherine S. Long for her continuous support and motivation during my entire Ph.D. You have been an encouraging guide, who let me choose the direction but also drive me on the proper way. Thanks to all the members (and ex-) of the RNA group, I cannot imagine how it would be without you! Special thanks to Holger, for inspiring me so many times and in so many occasions even without being at the center. You have been my first teacher, colleague and friend in the lab. I have learnt how to “bend the rules” thanks to you! Martin, for your generous patience and passion in making my things work. For all those days spent in front of a black screen with weird codes running…people watching me were thinking I was doing difficult programming work…I let them think of it, they did not know I was just trying to learn the “cd” command. A big thanks to Klara; even if you talk a lot, really a lot, now I can honestly say “it has been a big pleasure to work with you!” Together we worked in the RNA lab, together we crashed our “head” with bioinformatics in Český Krumlov, together we crashed my head in Cancun, and together we had also a lot of fun! Beside at work, I am so glad to have a friend like you…thanks a lot for everything! A huge thanks to Michelino, because you are not just a helpful and always happy co-worker but mainly a great boyfriend. Thank you for listening, for understanding me, for your continuous reinforcement. Thanks Miche, for always taking care of me... and for teaching me the proper use of “by chance”. Xiaochen, for all our discussions about work problems and scientific doubts, but at the end let’s order pizza vah! ii I would like to express my thanks to the Bactory people. Thanks to Søren and Mette, who built an amazing program and gave me the opportunity to take part in it. Thanks to all the Bactory Ph.D. students, it was a great pleasure starting with you that 1st of May three years ago, and since then we have always helped and strengthen each other. In particular many thanks to Mimi, for all your microbiology lessons, for your continuous support at work and in daily life, for all the laughs we had from Stockholm to China and for the pleasure of having you “endless” and singing Chandelier. Thanks to papiciulo Henrique, for your incredible help in every situation. You know “Hey brother…if the sky comes falling down for you, There’s nothing in this world I wouldn’t do.” Thanks to Giuggiolina for all our conversations, all our outbursts, and all our relaxing “cenette”. I will miss everything! I would like to thank the Cfb collaborators João, Niko, Markus, Maja, thanks a lot for this final rush, I really appreciated all of your efforts. Thanks to the Cool Office and all the people who make it cool, it was awesome to spend these three years with you (Sofie, Patri, Dario)! I also would like to thank Carlotta who has always stood on my side. Thank you for our long talks, funny moments, and Elvira breaks! Big big thanks to Mr. Kim for all the help in the lab, with the figures, and with any kind of doubts. Always available to help, you are really awesome! Thanks to Laura, Virginia, Roberto, Alicia, Anna, Gigi, Nabin, Christian, Ida, Rosa, Math because all of you guys have helped me to enjoy these three years at Cfb. I would like to thank Allan and Anne from Novozymes, for hosting me in your department. It has been a wonderful experience with you! I also would to thank the Italian group here in Copenhagen. Thanks to my brother Niccolò, who bravely took a decision and started a new adventure here. My historical friends Giulia and Isacco; I am closing iii another circle and I will bring you in the next one. Thanks to Marchino for all the funny moments and Eugene. My flatmate Nicola; it has been great to know you, even if you support Djokovic. I would like to thank my mum, dad, and grandmother Erichina, because even with the distance I have felt your strong support. You have always believed in me and push me to follow my dreams. Thanks a lot for that! Finally, I would like to thank all my friends in Italy and abroad that never give up on me. I cannot mention you all but you are amazing! iv Abstract Bacterial cell factories represent a valid alternative to fossil fuel-based production. A promising bacterium that can be optimized as cell factory is Pseudomonas putida. However, its development in bioproduction applications poses some challenges including a clear understanding of the bacterial system biology. This thesis has the aim of facilitating the development of P. putida KT2440 as a bacterial cell factory by investigating the transcriptome of the bacterium under different conditions (e.g. growth and stress). The main goals are the identification of differentially expressed genes, which provide information on bacterial adaptation to different environments, and the identification of non-coding RNAs, which regulate gene expression. This work focuses on several aspects of P. putida highlighting genomic features such as transcription start sites (TSSs), RNA regulatory elements such as riboswitches and small RNAs (sRNAs), metabolic pathways and transporter systems. The results reported here significantly increase knowledge of the P. putida transcriptome, adaptation mechanisms, and reveal novel bacterial features that will aid the design and optimization of the bacterium as a cell factory. v Dansk resumé Bakterielle cellefabrikker udgør et reelt alternativ til produktion baseret på fossile brændstoffer. En lovende kandidatbakterie, der kan optimeres som cellefabrik, er Pseudomonas putida. Brugen af denne bakterie til bioproduktion har dog nogle udfordringer, hvilket inkluderer forståelsen af det bakterielle system. Denne afhandling har som mål fremme udviklingen af P. putida KT2440 som en bakteriel celle fabrik ved at undersøge transkriptomet af bakterien under forskellige forhold (fx vækst og stress). De overordnende mål er identifikation af differentielt udtrykte gener, hvilket giver information om den bakterielle adaptation til forskellige miljøer, og identifikation af ikke-kodende RNA’er, der regulerer genekspression. Arbejdet fokuserer på adskillige aspekter af P. putida og fremhæver genomiske egenskaber som transkriptionelle startsteder (TSSs), regulatoriske RNA elementer som ’riboswithes’ og små RNA’er (sRNAs), metaboliske netværk og transportsystemer. Resultaterne, der her rapporteres, øger markant forståelsen af P. putidas transkriptom, tilpasningsmekanismer og beskriver bakterielle egenskaber, der vil hjælpe med designet og optimeringen af denne bakterielle cellefabrik. vi Publications Included in this thesis I. D’Arrigo, K. Bojanovič, X. Yang, M. H. Rau, K. S. Long. (2016). Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida. Environmental Microbiology. Manuscript in press. K. Bojanovič, I. D’Arrigo, K. S. Long. (2016). Global transcriptional responses to oxidative, osmotic, and imipenem stress conditions in Pseudomonas putida. Manuscript in preparation.