Declaration of Originality I confirm that all materials in this thesis are my own work, and any sources of information used therein have been appropriately cited. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Acknowledgements First of all, I am grateful to my supervisor, Dr Patrik Jones for providing the opportunity to work on this exciting project in an inspiring environment in the heart of London. He was always open to new ideas, guiding me through my path to becoming an independent scientist. I also would like to thank the European Union’s FP7 People Programme (Marie Curie Actions, 317184) and the BBSRC sLoLa grant (BB/N003608/1) for providing funding for this project. I thank the entire PHOTO.COMM consortium for the excellent training events and the great community, especially Dr Kristine Groth Kirkensgaard and Ms Karin Norris for arranging the project transfer from Turku to London. I am thankful to Professor Enrique Flores at University of Seville for kindly providing the Anabaena sp. PCC 7120 base strains and Mr Anthony Riseley, a fellow PhD student at the University of Cambridge for his essential contribution to investigating the potential of the nitrogen excretion strains. I am also grateful to Dr Dennis Nürnberg at Imperial College London for all his help and endless patience in teaching me the conjugative transformation of cyanobacteria. I would like to thank the members of the MME group at Imperial College for the friendly atmosphere and their contribution to my work in one way or another. I especially thank Ms Phoebe Tickell for her great support in the laboratory by making my everyday life so much easier. I am grateful to Dr András Pásztor and Zsu Horváth for supporting us in many different ways while in Finland, and also afterwards in spite of the time difference. Finally, I would like to thank my lab buddies, Ms Marine Valton, Dr Paulina Bartasun and Dr John Rowland for their presence; the amazing conversations, the laughs, as well as the invaluable discussions on many different matters of my thesis. It would not have been the same without them. Lastly, I am thankful to the SCR at Imperial College London for the unmatched fish and chips. Some might even say it is the best one in London. To Lilla, Luca and Laura for supporting me all the way on this great endeavour; and to Máté for patiently waiting until it has been completed. Modelling and Engineering Anabaena sp. PCC 7120 for Nitrogen Excretion David Malatinszky Imperial College London Department of Life Sciences Submitted for the Degree of Doctor of Philosophy 2017 Abstract Nitrogen is an essential element for every organism on Earth. Modern agricultural activity depletes soil in nitrogen much faster than it is naturally replenished. Therefore, fertilization is key for feeding a fast-growing population. However, the use efficiency of fertilizer nitrogen is only about 60%. The rest of reactive nitrogen leaches to the environment, and the pollution caused demands modern societies tens of billions of euros annually as remediation costs. Rationalisation of current agricultural practices is essential, including a more targeted application of fertilizers, to tackle the nitrogen crisis. One way is the use of nitrogen-fixing organisms as biofertilizer in close association to agricultural crops. In this thesis, a stoichiometric model was reconstructed for the heterocystous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 to understand the nature of metabolite exchange between its photosynthetic and diazotrophic cell types, and design metabolic engineering strategies for nitrogen excretion. Using flux balance analysis of diazotrophically grown filaments, excretion of ammonia followed by urea achieved the highest molar nitrogen flux. To achieve a similar effect experimentally, glutamine synthetase (GS) was inhibited using L-methionine sulfoximine. It was possible to accumulate about 760 μM ammonia in a 7-day assay with stagnating growth. For stable excretion, GS has been replaced for an active-site mutant exhibiting decreased specific activity for ammonia. The metabolic changes have been implemented in both wild type and an ammonium uptake transporter mutant (Δamt). The resulting strains displayed increased ammonia excretion up to about 8-fold compared to the wild type. Furthermore, IF7A, a small oligopeptide controlling the activity of GS, has been overexpressed under four different promoters. The constructs driven by two of these promoters, PnifHDK and PpetE enabled the growth of the non-diazotrophic alga Chlorella vulgaris at 68% of that of the algal monoculture on combined nitrogen. Overall, the ammonia-excreting strains provided an important proof-of-principle for the development of more efficient biofertilizers in future agriculture. 9 Table of Contents Abstract ................................................................................................................................................... 9 List of Figures ........................................................................................................................................ 14 List of Tables ......................................................................................................................................... 17 Abbreviations ........................................................................................................................................ 18 1 Introduction .................................................................................................................................. 22 1.1 Synopsis................................................................................................................................. 22 1.2 Essential nitrogen .................................................................................................................. 24 1.2.1 Nitrogen for life ............................................................................................................. 24 1.2.2 The nitrogen cycle ......................................................................................................... 25 1.2.3 Haber–Bosch ammonia ................................................................................................. 28 1.3 The Nitrogen Crisis ................................................................................................................ 30 1.3.1 Environmental and health effects ................................................................................. 30 1.3.2 Costs and measures ...................................................................................................... 31 1.4 Metabolic modelling of entire organisms ............................................................................. 32 1.4.1 Systems biology ............................................................................................................. 32 1.4.2 Flux Balance Analysis .................................................................................................... 33 1.5 Cyanobacteria ....................................................................................................................... 36 1.5.1 Nitrogen assimilation .................................................................................................... 38 1.5.1.1 Heterocysts and nitrogen fixation ............................................................................ 38 1.5.1.2 Ammonia assimilation and excretion ....................................................................... 41 1.5.1.3 Nitrogenase enzyme complex ................................................................................... 44 1.5.2 Importance of iron ........................................................................................................ 46 1.5.3 Natural and synthetic communities .............................................................................. 47 1.6 Challenges in metabolic engineering of Anabaena sp. PCC 7120 ......................................... 49 1.6.1 Developing synthetic biology ........................................................................................ 49 10 1.6.2 Homologous recombination favours single recombination ......................................... 51 1.6.3 Oligoploid cyanobacteria .............................................................................................. 51 1.6.4 Multicellularity in filamentous cyanobacteria .............................................................. 52 1.7 Aims and objectives .............................................................................................................. 53 2 Materials and methods ................................................................................................................. 56 2.1 Chemicals and reagents ........................................................................................................ 56 2.2 General protocols.................................................................................................................. 56 2.2.1 Incubators and sterile work .........................................................................................