Washington University in St. Louis Washington University Open Scholarship Engineering and Applied Science Theses & McKelvey School of Engineering Dissertations Winter 12-15-2018 Investigating Cyanobacteria Metabolism and Channeling-based Regulations via Isotopic Nonstationary Labeling and Metabolomic Analyses Mary Helen Abernathy Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/eng_etds Part of the Biomedical Engineering and Bioengineering Commons, and the Chemical Engineering Commons Recommended Citation Abernathy, Mary Helen, "Investigating Cyanobacteria Metabolism and Channeling-based Regulations via Isotopic Nonstationary Labeling and Metabolomic Analyses" (2018). Engineering and Applied Science Theses & Dissertations. 396. https://openscholarship.wustl.edu/eng_etds/396 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 Tang, Chair Douglas Allen Marcus Foston Tae Seok Moon Himadri Pakrasi Investigating Cyanobacteria Metabolism and Channeling-based Regulations via Isotopic Nonstationary Labeling and Metabolomic Analyses by Mary H. Abernathy A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2018 St. Louis, Missouri © 2018, Mary H. Abernathy Table of Contents List of Figures ................................................................................................................................ vi List of Tables ............................................................................................................................... viii List of Abbreviations ….…………………………………………………………………………ix Acknowledgments........................................................................................................................... x Abstract ......................................................................................................................................... xii Chapter 1: Introduction ................................................................................................................... 1 1.1 Cyanobacteria as cell factories ......................................................................................... 2 1.1.1 Inherent challenges facing cyanobacteria ............................................................................. 2 1.1.2 Rewiring cellular metabolism is difficult .............................................................................. 4 1.1.3 Channeling has significant biotechnology impacts ............................................................... 5 1.2 INST-MFA as a tool for decoding cellular phenotypes ................................................... 7 1.2.1 Materials associated with INST-MFA ................................................................................ 11 1.2.2 Methods for performing INST-MFA .................................................................................. 13 1.2.3 Consideration of metabolite channeling in INST-MFA ...................................................... 19 1.2.4 Notes on INST-MFA .......................................................................................................... 20 Chapter 2: Deciphering cyanobacterial phenotypes for fast photoautotrophic growth via INST- MFA .............................................................................................................................................. 24 2.1 Introduction .................................................................................................................... 25 2.2 Results ............................................................................................................................ 27 2.2.1 Syn.2973 growth and biomass composition ....................................................................... 27 2.2.2 INST-MFA of photoautotrophic metabolism ..................................................................... 29 2.2.3 Synechococcus flux maps in PBR and SF cultures ............................................................. 31 2.2.4 Isotopic metabolite profiling for relative pool size estimation .......................................... 32 2.2.5 Effect of exogeneous organic acids on Syn. 2973’s growth .............................................. 33 2.3 Discussion ...................................................................................................................... 35 2.3.1 Flux comparison among cyanobacterial species ................................................................ 35 2.3.2 The optimal photoauotrophic metabolism ........................................................................ 37 2.3.3 Metabolic features of Syn. 2973 for bioproduction applications ........................................ 39 2.3.4 Subpopulations, metabolic inactive pools and substrate channeling .................................. 40 ii 2.3.5 Conclusions ....................................................................................................................... 42 2.4 Methods .......................................................................................................................... 43 2.4.1 Cultivation conditions and transient labeling experiments ................................................. 43 2.4.2 Metabolite quenching, extraction, and analysis .................................................................. 44 2.4.3 Estimation of relative pool sizes of metabolites ................................................................ 44 2.4.4 Biomass composition analysis ............................................................................................ 45 2.4.5 GC-MS analysis of proteinogenic amino acids ................................................................... 45 2.4.6 Isotopically nonstationary MFA ........................................................................................ 45 2.4.7 Construction of the zwf, pgl, and succinate dehydrogenase deletion mutants ................... 46 Chapter 3: Cyanobacteria carboxysome mutant reveals the influence of enzyme compartmentations on cellular metabolisms and metabolic network rigidity ............................. 47 3.1 Introduction .................................................................................................................... 48 3.2 Results ............................................................................................................................ 50 3.2.1 Growth comparison between the control strain and ∆ccmKLMN mutant ........................... 50 3.2.2 Biomass composition analysis ........................................................................................... 51 3.2.3 INST-MFA reveals flux topology in ∆ccmKLMN mutant by INCA .................................. 52 3.2.4 Change in pool sizes in response to carboxysome knock out ............................................ 56 3.3 Discussion ...................................................................................................................... 58 3.3.1 Cyanobacteria maintain rigid flux networks despite differeing phenotypes ....................... 58 3.3.2 Differences in the assumed metabolic netwrok of PCC 7002 compared to other cyano .... 59 3.3.3 Using carboxysome-deficient mutants to redirect carbon fluxes ........................................ 60 3.3.4 Impact of carboxysome mutations on photomixotrophic metabolism ............................... 61 3.3.5 Insights into channeling-based flux regulation ................................................................... 63 3.3.6 Conclusions ......................................................................................................................... 65 3.4 Materials and Methods ................................................................................................... 65 3.4.1 Strains and Cultivation conditions ...................................................................................... 65 3.4.2 INST-MFA and 13C Labeling experiments ......................................................................... 66 3.4.3 Metabolite extraction and analysis ..................................................................................... 67 3.4.4 Isotopically nonstationary MFA ......................................................................................... 67 3.4.5 Biomass composition analysis ............................................................................................ 68 3.4.6 GC-MS analysis of proteinogenic amino acids ................................................................... 68 iii 3.4.7 Measuring photosynthetic parameters and oxygen evolution ............................................. 68 Chapter 4: Metabolic engineering of cyanobacteria for photoautotrophic production of heparosan, a pharmacutical precursor of heparin ........................................................................ 70 4.1 Introduction ...................................................................................................................