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An in Silico Characterization of Microbial Electrosynthesis for Metabolic Engineering of Biochemicals

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An in Silico Characterization of Microbial Electrosynthesis for Metabolic Engineering of Biochemicals An in silico Characterization of Microbial Electrosynthesis for Metabolic Engineering of Biochemicals by Aditya Pandit A thesis submitted in conformity with the requirement for the degree of Masters of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto (c) Copyright by Aditya Pandit 2012 An in silico Characterization of Microbial Electrosynthesis for Metabolic Engineering of Biochemicals Aditya Vikram Pandit Masters of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto 2012 ABSTRACT A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates. Bioelectrochemical techniques offer a promising method to alleviate redox imbalances during the synthesis of biochemicals. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell‘s redox balance. The cellular response to such redox changes and the additional reducing can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can improve product yields and/or productivity. We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation ii processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity. iii ACKNOWLEDGMENTS I would like to give my thanks to my supervisor, Prof. Mahadevan for giving me the opportunity to do my masters degree in his lab. It has been a great experience and I have learned a lot under his guidance. As an undergraduate, I never thought that I would ever work in anything related to biology. However, Prof. Mahadevan has passed on his passion and enthusiasm for metabolic engineering and microbiology on to me, and I am very glad to have done graduate work in metabolic engineering and microbiology. I would like to extend my appreciation to my parents for their continuing support, guidance and encouragement. Most importantly, they have instilled in me a passion for learning – and for that I am grateful. Finally, I would like to extend my thanks to all my colleagues in Biozone for their support, great conversations and levity during my studies. In particular, thanks to Nicholas Bourdakos for all the insightful conversations. iv TABLE OF CONTENTS Abstract .................................................................................................................................. ii Acknowledgments .............................................................................................................. iv Table of Contents................................................................................................................ v List of Tables....................................................................................................................... vi List of Figures .................................................................................................................... vii List of Appendices ........................................................................................................... viii Glossary................................................................................................................................ ix Chapter 1 ....................................................................................................................... 1 Introduction and Background ............................................................................. 1 1.1 Importance of Developing Biochemicals ............................................ 1 1.2 Cofactor Manipulation as a Means to Drive Product Synthesis ..... 2 1.3 Bioelectrochemcial Techniques to Drive Product Synthesis ........... 3 1.4 Bioelectrochemcial Systems ................................................................... 4 1.5 Extracellular Electron Transfer ............................................................. 6 1.6 Computational Strategies for Strain Design ........................................ 7 Chapter 2 ....................................................................................................................... 8 Knowledge Gaps and Statement of Objectives ............................................... 8 Chapter 3 ..................................................................................................................... 10 Methods and Materials ........................................................................................10 3.1 Modelling Bioelectrosynthesis for Chemical Production ...............10 3.2 Flux Balance Analysis ............................................................................11 3.3 Modelling Electrode Interactions ........................................................12 3.4 Augmenting the Model to Incorporate Heterologous Pathways ..14 3.5 Selection of Substrates and Products for Analysis ...........................14 Chapter 4 ..................................................................................................................... 17 Results and Discussion .......................................................................................17 4.1 Impact on ATP Yield and Biomass ....................................................17 4.2 Impact on CO2 Fixing Pathways .........................................................23 4.3 Impact of Product and Substrate Degree of Reduction on Bioelectrosynthesis .......................................................................................25 4.4 Growth Coupled Electrical Enhancement ........................................31 4.5 Bioelectrosynthesis on Substrate Mixtures ........................................39 4.6 Limitations of Modelling Results ........................................................41 Chapter 5: .................................................................................................................... 44 Conclusions and Recommendations ................................................................44 Bibliography ....................................................................................................................... 47 Appendix A ........................................................................................................................ 54 Appendix B......................................................................................................................... 57 Appendix C ........................................................................................................................ 62 Appendix D ........................................................................................................................ 66 v LIST OF TABLES Number Page 1 Milestones of Achievements in Bioelectrosynthesis ....................................... 4 2 NADH Produced or Consumed per Substrate or Product .........................15 3 SPEEQ Values for Substrate Product Couplings ........................................15 4 Effect of CO2 Fixation on Growth Rate .........................................................24 5 SPEEQ Values for Substrate Product Couplings ..........................................27 6 Predicted Fluxes Through Selected Reactions ...............................................29 7 Predicted Changes in Fluxes Through Selected Pathways ...........................37 8 Summary of Increases in Theoretical Product Yield Coupled to Biomass Growth ...................................................................................................................40 vi LIST OF FIGURES Number Page Figure 1 Milestones of Achievements in Bioelectrosynthesis ...................................... 4 Figure 2 Typical Bioelectrochemical System (BES) ....................................................... 5 Figure 3 Central Metabolism and NADH Regeneration Maps .................................13 Figure 4 Percentage Improvements in ATP Yield and Biomass Yield as a Result of Electrical Enhancement .................................................................................18 Figure 5 Map of Metabolic Flux Distributions for the Wild Type Metabolism .....22 Figure 6 Theoretical Increases in Product Yield Resulting from Electrical Enhancement ........................................................................................................27 Figure 7 Theoretical Increases in Product Yield of Growth Coupled Resulting from Electrical Enhancement............................................................................33 Figure 8 Production Envelopes for Growth Coupled and Electrically Enhanced 35 Figure 9 Production Envelopes for Three Growth Coupled Strategies for Ethanol...................................................................................................................38 Figure 10 Changes in Product Yield, Biomass Yield and Substrate Specific Productivity as a Function of Electron Exchange Rate ...............................39 vii LIST OF APPENDICES Page Appendix A Model Reactions/Deletion Strategies .....................................................54 Appendix B Modelling Results - Maximizing Biomass ...............................................57 Appendix C Changes in Metabolic Flux Distribution
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