Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Metabolic engineering of microorganisms for the overproduction of fatty acids Ting Wei Tee Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Chemical Engineering Commons Recommended Citation Tee, Ting Wei, "Metabolic engineering of microorganisms for the overproduction of fatty acids" (2013). Graduate Theses and Dissertations. 13516. https://lib.dr.iastate.edu/etd/13516 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Metabolic engineering of microorganisms for the overproduction of fatty acids by Ting Wei Tee A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy Major: Chemical Engineering Program of Study Committee: Jacqueline V. Shanks, Major Professor Laura R. Jarboe R. Dennis Vigil David J. Oliver Marna D. Nelson Iowa State University Ames, Iowa 2013 Copyright © Ting Wei Tee, 2013. All rights reserved. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................ v ABSTRACT ........................................................................................................................ vi CHAPTER 1 INTRODUCTION ................................................................................... 1 Introduction of chemical industry ................................................................................. 1 Carboxylic acids and pyrones….…. ............................................................................. 2 Metabolic engineering cycle ......................................................................................... 3 Transcriptomics....................................................................................................... 5 Preteomics ............................................................................................................... 5 Metabolomics .......................................................................................................... 7 Fluxomics ............................................................................................................... 8 Optimization-based approaches .................................................................................... 9 Organization of dissertation .......................................................................................... 10 References… ................................................................................................................. 11 CHAPTER 2 Literature Review..................................................................................... 16 Metabolic flux analysis (MFA) ..................................................................................... 16 Conventional metabolic flux analysis (c-MFA) ........................................................... 16 13C metabolic flux analysis ........................................................................................... 17 Isotopomer….. .............................................................................................................. 19 Nuclear magnetic resonance (NMR) and mass spectroscopy (MS) techniques ……. 21 Enumerating flux .......................................................................................................... 24 Non-Stationary flux analysis......................................................................................... 26 References………. ........................................................................................................ 27 CHAPTER 3 Systems Metabolic Enginering Design: Fatty Acid Production as Emerging Case Study .......................................................................................................... 30 Abstract …… ................................................................................................................ 30 Introduction .................................................................................................................. 31 Classical metabolic enginering ..................................................................................... 32 Integrated computational and experimental Approach ................................................. 34 Synthetic biology .......................................................................................................... 35 Conclusion and future challenges ................................................................................. 37 Acknowledgement ....................................................................................................... 38 References .................................................................................................................. 38 List of figures ................................................................................................................ 42 iii CHAPTER 4 An Integrated Computational and Experimental Study for Overproducing Fatty Acids in Escherichia Coli ................................................................. 47 Abstract…………… ..................................................................................................... 47 1. Background and introduction .................................................................................. 48 2. Materials and methods ............................................................................................ 51 2.1. Strains and plasmids ...................................................................................... 51 2.2. Metabolic flux analysis experiments ............................................................. 51 2.3. Using OptForce for fatty acid overproduction ............................................... 54 2.4. Metabolic interventions and fatty acid titer determinations .......................... 57 3. Results….. ............................................................................................................... 58 3.1. Flux measurements ........................................................................................ 58 3.2. OptForce results ............................................................................................. 59 3.3. Experimental characterization of metabolic interventions ............................ 65 4. Summary and discussion......................................................................................... 67 Authors’ contributions .................................................................................................. 71 Acknowledgement ........................................................................................................ 71 References… ................................................................................................................. 71 List of figures ................................................................................................................ 77 Supporting information ................................................................................................. 89 CHAPTER 5 Closing Metabolic Engineering Cycle by Integrated Approach of Computational, Experimental and Omics Tools for Overproduction of Fatty Acids ......... 103 Abstract…………… ..................................................................................................... 103 1. Background and introduction .................................................................................. 104 2. Materials and methods ............................................................................................ 107 2.1. Strains and plasmids ........................................................................................ 107 2.2. Metabolic flux analysis experiments ............................................................... 108 2.3. Using OptForce for fatty acid overproduction ................................................. 109 2.4. Intracellular metabolite profilings.................................................................... 111 2.5. RNAseq transcriptome profilings .................................................................... 112 2.6. Metabolic interventions and fatty acid titer determinations ............................ 113 3. Results….. ............................................................................................................... 113 3.1. Flux measurements .......................................................................................... 113 3.2. OptForce results ............................................................................................... 115 3.3. Intracellular metabolite profiling results .......................................................... 117 3.4. Experimental characterizarion of metabolic interventions .............................. 118 3.5. RNAseq analysis .............................................................................................. 119 4. Summary and discussion......................................................................................... 121 Authors’ contributions .................................................................................................