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Cloning and Expression of Zymobacter Palmae Pyruvate Decarboxylase for Enhanced Ethanol Production in the Cyanobacterium Synechocystis Sp

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Cloning and Expression of Zymobacter palmae pyruvate decarboxylase for enhanced ethanol production in the cyanobacterium Synechocystis sp. PCC 6803 Lorraine Quinn B.Sc. Project supervised by Prof. J. Tony Pembroke, Dr. Patricia Armshaw, Prof. Tewfik Soulimane and Dr. Con Sheahan. A thesis submitted to the University of Limerick in candidature for the degree of Masters of Science. Submitted to the University of Limerick. ii Abstract Cloning and Expression of Zymobacter palmae pyruvate decarboxylase for enhanced ethanol production in the cyanobacterium Synechocystis sp. PCC 6803. Lorraine Quinn Recently, photoautotrophic cyanobacteria have gained much attention due to their ability to convert carbon dioxide using light energy into carbon metabolites, some with potential biofuel applications. This is significant due to the sustainable nature of production and the global limitation of fossil fuels in the future. Model organisms such as Synechocystis sp. PCC 6803 have been described as “cell factories” for this reason and can be genetically modified and manipulated to produce compounds such as ethanol. It was decided to construct a recombinant strain of Synechocystis 6803 expressing a Zymomonas mobilis pyruvate decarboxylase (pdc) and an alcohol dehydrogenase (adh) native to Synechocystis 6803 which is capable of autotrophic conversion of pyruvate to ethanol. In an attempt to improve ethanol yield it was hypothesised that utilising a PDC with a reported lower Km might improve flux to ethanol and have identified and utilised a novel pdc gene from Zymobacter palmae in this respect. A pET22b (+) expression vector with a strong T7 promoter was primarily used to overexpress the ZpPDC for enzymatic analysis and purification purposes for potential structural studies. The pdc gene from Zymobacter palmae was amplified and a codon optimised version was also synthesised. Two constructs were created which contained the native/codon optimised Zppdc and the adh native to Synechocystis 6803 under the control of the strong light driven pPSBAII promoter. These cassette constructs were transformed into wildtype Synechocystis 6803 and integration of the cassettes into the polyploidy chromosome at the PSBAII neutral site was confirmed via DNA sequencing and PCR screening. Strains confirmed as verified recombinants were then characterised for growth, biomass and ethanol productivity relative to UL004 (expressing the Zymomonas mobilis pdc). Unfortunately and to our surprise, no increase in ethanol production was observed in the fully segregated strains in comparison to UL004, the original production strain expressing the ZmPDC despite testing a number of different segregants and utilising repeated assays. Although there may in fact be several reasons for this it was hypothesised that the ZpPDC may be less compatible than the ZmPDC in terms of its pH optimum, functional linkage to the native ADH or just sensitivity to ethanol. Going forward, it may be worthwhile focusing on other PDC enzymes from organisms such as Gluconacetobacter diazotrophicus or Gluconobacter oxydans which have recently been reported to display even lower Km values than the PDC from Zymobacter palmae. iii Declaration I hereby declare that the work detailed in this thesis is the results of my own investigations. No part of this work has been or is being submitted in candidature for any other degree. ______________________________ Lorraine Quinn ______________________________ Date iv List of Publications Armshaw, P., Carey, D., Quinn, L., Sheahan, C. and Pembroke, J.T. (2015) 'Optimisation of ethanol production in Synechocystis PCC 6803, the DEMA approach.', in 1st International Solar Fuels Conference (ISF-1), Uppsala, Sweden, April 26th-May 1st 2015, University of Limerick. Carey, D., Armshaw, P., Quinn, L., Sheahan, C. and Pembroke, J.T. (2015) 'Phenotypical analysis of different strains of Synechocystis sp. PCC 6803 for determination of the optimal strain for ethanol production’, in Society for General Microbiology: Irish Division Meeting: Microbial Interfaces, NUIG, Galway, 17th-19th June 2015, University of Limerick. Manuscript in preparation for Biotechnology Letters, ‘The pyruvate decarboxylase from Zymomonas mobilis out performs that of Zymobacter palmae for ethanol production in metabolic engineered Synechocystis sp. PCC 6803’, Quinn, L., Armshaw, P., Soulimane, T., Sheahan, C. and Pembroke, J.T. Pembroke, J.T., Quinn, L., O' Riordan, H. and Armshaw, P. (2017) 'Ethanol Production in Cyanobacteria: Impact of Omics of the Model Organism Synechocystis on Yield Enhancement' in Los, D. A., ed., Cyanobacteria: Omics and Manipulation, Norfolk, UK: Caister Academic Press, 200-217. Quinn, L., Armshaw, P., Carey, D., Sheahan, C. and Pembroke, J.T. (2015) 'Kinetic optimisation of the pyruvate decarboxylase from Zymomonas mobilis to maximise ethanol productivity in the cyanobacterium Synechocystis sp. PCC 6803', in Society for General Microbiology: Irish Division Meeting: Microbial Interfaces, NUIG, Galway, 17-19th of June 2015, University of Limerick. v Acknowledgements First of all I would like to sincerely thank Dr. Patricia Armshaw for her continued support and guidance over the last two years as both a supervisor and friend. Without her help and patience the completion of this work would not have been possible. I am eternally grateful. To Professor Tony Pembroke for his words of wisdom and supervision throughout this project and my undergraduate degree. Words cannot express how thankful I am and I will always remember the anecdotes as well as the advice. To Professor Tewfik Soulimane for his advice and help as I progressed from strength to strength (I think) in my knowledge of proteins and also in my undergraduate years. For his persistence in convincing me to continue in research and for the belief that I could achieve something great. For that I thank you. To Dr. Michael Ryan for all his help and guidance at the final hurdle. The finishing touches made all the difference and I can be truly proud of what I’ve written thanks to your knowledge and expertise. To all of the staff in the CES department who assisted me with my work throughout the years, notably Sinéad, Brian, Ciara, Miriam and Maria. Your continued support will never be forgotten. Also a huge thank you to Dr. Fintan Bracken in the UL Glucksman Library for all his help in the final stages. I too would like to gratefully thank Dr. Con Sheahan and all in the DEMA consortium with a special word of thanks to Davide Montesarchio for all his help and advice. Also to DEMA for funding my research. To all in L2-007. Not all of us may have worked together but I still looked on you as my science family. Wishing you all the best in the future wherever it may take you and I hope we can stay in contact and continue to socialise as the years move on. vi To my fellow lab friends Kevin and Helen. Where would I be without your friendship and support? I truly wish you both the very best of luck as you continue with your studies and I know that you will do great! I’ll never forget the craic and banter of A2-005. Memories forever. To my close Industrial Biochemistry friend Michael. For all your help throughout the past six years, I cannot thank you enough. Never were you too busy to lend a hand when I was in need of advice or a miracle. Wishing you luck and success in the years ahead. To my parents Margaret and Liam and my sister Rachel. Thank you for all the love and support over the past six years, for putting up with the mood swings and tantrums. I don’t think I was as bad for the second degree but then again I could be wrong. All that you have done will never be forgotten. vii Table of Contents Abstract ................................................................................................................. iii Declaration............................................................................................................ iv List of Publications ............................................................................................... v Acknowledgements .............................................................................................. vi Table of Contents ............................................................................................... viii List of Figures ..................................................................................................... xiii List of Tables ..................................................................................................... xvii List of Abbreviations ......................................................................................... xix Units of Measurement....................................................................................... xxii Chapter 1: Introduction ....................................................................................... 2 1.1 Biofuel necessity ............................................................................................... 3 1.1.1 First, second and third generation biofuels .................................................... 3 1.2 Cyanobacteria: Model organisms ..................................................................... 5 1.2.1 Synechocystis sp. PCC 6803 .......................................................................... 7 1.2.2 Ploidy levels ................................................................................................... 8 1.2.3 Cultivation.....................................................................................................
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