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Revelation of Yin-Yang Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering Gang Wu Washington University in St

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Revelation of Yin-Yang Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering Gang Wu Washington University in St Washington University in St. Louis Washington University Open Scholarship Engineering and Applied Science Theses & McKelvey School of Engineering Dissertations Spring 5-15-2016 Revelation of Yin-Yang Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering Gang Wu Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/eng_etds Part of the Biodiversity Commons, Computer Sciences Commons, Engineering Commons, and the Evolution Commons Recommended Citation Wu, Gang, "Revelation of Yin-Yang Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering" (2016). Engineering and Applied Science Theses & Dissertations. 163. https://openscholarship.wustl.edu/eng_etds/163 This Dissertation is brought to you for free and open access by the McKelvey School of Engineering at Washington University Open Scholarship. It has been accepted for inclusion in Engineering and Applied Science Theses & Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS School of Engineering and Applied Science Department of Energy, Environmental & Chemical Engineering Dissertation Examination Committee: Yinjie J. Tang, Chair Pratim Biswas Zi Chen Cynthia Lo Tae Seok Moon Ryan Senger Revelation of ‘Yin-Yang’ Balance in Microbial Cell Factories by Data Mining, Flux Modeling, and Metabolic Engineering by Gang Wu A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2016 Saint Louis, Missouri © 2016, Gang Wu Table of Contents List of Figures ......................................................................................................ix List of Tables ..................................................................................................... xiv Acknowledgement ............................................................................................... iii Abstract ..............................................................................................................xvi Chapter 1: Introduction of Fluxomics Studies and Metabolic Burden Modeling ................................................................................................................1 1.1. Background of fluxomics ..............................................................................................1 1.2. A brief overview of FBA...............................................................................................1 1.3. Introduction of 13C-MFA...............................................................................................1 1.4. Overview of published 13C-MFA papers on bacteria species .........................................6 1.5. Modeling work related with metabolic burden ...............................................................9 1.6. Outline of this disserttation ......................................................................................... 13 1.7. Reference .................................................................................................................... 14 Chapter 2: Evaluating Physiological State of Engineered E. coli Strains by Isotopomer Constrained Flux Balance Analysis ............................................... 22 2.1. Abstract .............................................................................................................................. 22 2.2. Introduction ........................................................................................................................ 23 2.3. Materials and Methods ..................................................................................................... 26 ii 2.3.1. Strains and plasmids ...................................................................................................... 26 2.3.2. Medium and culture conditions ...................................................................................... 27 2.3.3. Quantification of biomass and extracellular metabolites ................................................. 27 2.3.4. Mass isotopomer distribution of proteinogenic amino acids ........................................... 28 2.3.5. Central metabolic flux determined by 13C-MFA ............................................................. 29 2.3.6. Genome-scale model constrained by 13C-MFA flux ........................................................ 30 2.4. Results and discussions ..................................................................................................... 31 2.4.1. Physiological states of different Strains .......................................................................... 31 2.4.2. Central metabolic flux determined by 13C-MFA .............................................................. 32 2.4.3. Energy metabolism and evolutionary fitness analysis ...................................................... 33 2.4.4. The effects of P/O ratio, oxygen flux, and maintenance energy on isobutanol production.34 2.5. Conclusions ........................................................................................................................ 36 2.6. References .......................................................................................................................... 36 Chapter 3: Investigate Energy Metabolism of Microbial Cell Factories by Yin- Yang Theory Research ....................................................................................... 60 3.1. Abstract .............................................................................................................................. 60 3.2. Introduction ........................................................................................................................ 61 3.3. The energy losses in microbial cell factories ..................................................................... 62 3.4. The tradeoff between product yield and energy fitness........................................................ 64 3.5. Sensitivity analysis of the energy penalty on biofuel synthesis ............................................ 66 iii 3.6 Yin-Yang theory in metabolic engineering ....................................................................... 68 3.7. Conclusions ........................................................................................................................ 73 3.8. References .......................................................................................................................... 73 Chapter 4: Enhance Energy State of Fatty Acid Producing E. coli Strains With Vitreoscilla Hemoglobin ............................................................................ 87 4.1. Abstract .............................................................................................................................. 87 4.2. Introduction ........................................................................................................................ 87 4.3. Experimental and Methods ............................................................................................... 90 4.3.1. Chemicals and Strains .................................................................................................... 90 4.3.2. Plasmid construction ...................................................................................................... 91 4.3.3. Medium and culture conditions ...................................................................................... 92 4.3.4. Fatty acid measurement ................................................................................................. 92 4.3.5. Simulate cellular physiology with flux balance model ................................................... 93 4.4. Results and discussions ..................................................................................................... 94 4.4.1. Growth kinetics and fatty acid production ....................................................................... 99 4.4.2. Expression of VHb affects the degree of unsaturation of free fatty acid ........................... 95 4.4.3. Effect of oxygen and maintenance energy on fatty acid production ................................ 96 4.5. Conclusions ........................................................................................................................ 98 4.6. References .......................................................................................................................... 98 iv Chapter 5: ... Build Web-Based Platform for Fluxomics Studies: Microbesflux Rebuild and Website Development .................................................................. 112 5.1. Abstract ............................................................................................................................ 112 5.2. Introduction ...................................................................................................................... 113 5.3. Implementation .............................................................................................................. 115 5.3.1. MicrobesFlux update ................................................................................................... 115 5.3.2. New features of reloaded MicrobesFlux......................................................................... 116 5.3.3. Development
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