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Quantitative Analysis of Metabolic Pathways in Catharanthus Roseus

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Quantitative Analysis of Metabolic Pathways in Catharanthus Roseus Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots metabolically engineered for terpenoid indole alkaloid overproduction Guy William Sander Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biological Engineering Commons, and the Chemical Engineering Commons Recommended Citation Sander, Guy William, "Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots metabolically engineered for terpenoid indole alkaloid overproduction" (2009). Graduate Theses and Dissertations. 10820. https://lib.dr.iastate.edu/etd/10820 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]. Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots metabolically engineered for terpenoid indole alkaloid overproduction by Guy William Sander II 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 Brent H. Shanks Peter J. Reilly Eve Syrkin Wurtele David J. Hannapel Iowa State University Ames, Iowa 2009 Copyright © Guy William Sander II, 2009. All rights reserved. ii Dedicated to My Parents iii Table of Contents Table of Contents ................................................................................................................. iii List of Tables .................................................................................................................... xvii Acknowledgements .......................................................................................................... xviii Abstract .............................................................................................................................. xxi Chapter 1 - Introduction .............................................................................................................1 Thesis Organization ...............................................................................................................4 Figures ...................................................................................................................................7 References ..............................................................................................................................9 Chapter 2 – Literature Review .................................................................................................11 Metabolic Engineering of Secondary Metabolism ..............................................................11 Measurement of Metabolic Fluxes ......................................................................................15 Figures .................................................................................................................................17 References ............................................................................................................................19 Chapter 3 - Materials and Methods ..........................................................................................22 Alkaloid Extraction from Fresh Biomass ............................................................................22 HPLC Analysis of Metabolites ............................................................................................22 Alkaloid Extraction from Growth Media .............................................................................23 References ............................................................................................................................24 Chapter 4 – Metabolite and Gene Expression Analysis of Terpenoid Indole Alkaloid Pathways in Catharanthus roseus Seedlings Treated with Methyl Jasmonic Acid .................25 Abstract ................................................................................................................................25 Abbreviations .......................................................................................................................26 Introduction ..........................................................................................................................27 Materials and Methods .........................................................................................................29 iv Plant growth .....................................................................................................................29 Treatment with MeJA ......................................................................................................30 Isolation of housekeeping genes ......................................................................................30 Analysis of reference mRNA levels ................................................................................31 qRT-PCR analysis of TIA genes......................................................................................32 Extraction of alkaloids and HPLC analysis .....................................................................33 Results ..................................................................................................................................34 Isolation of C. roseus reference genes .............................................................................34 Analysis of the expression stability of C. roseus reference genes ...................................35 Time course of TIA gene expression in response to MeJA .............................................36 Metabolite analysis of C. roseus seedlings treated with MeJA .......................................37 Discussion ............................................................................................................................38 Isolation and characterization of C. roseus reference genes ............................................38 Timing of MeJA induction of TIA mRNA and metabolite levels ...................................39 Acknowledgements ..............................................................................................................42 Tables ...................................................................................................................................43 Figures .................................................................................................................................45 References ............................................................................................................................49 Chapter 5 - Transient Effects of Overexpressing Anthranilate Synthase α and β Subunits in Catharanthus roseus Hairy Roots in Light and Dark Conditions ........................52 Abstract ................................................................................................................................52 Introduction ..........................................................................................................................53 Materials and Methods .........................................................................................................55 Chemicals .........................................................................................................................55 Culture Conditions ...........................................................................................................56 Induction of the Catharanthus roseus Hairy root Lines ..................................................56 Harvesting Hairy Root Cultures.......................................................................................57 Alkaloid Extraction ..........................................................................................................57 HPLC Analysis of Metabolites ........................................................................................58 LC-MS Analysis of Metabolites ......................................................................................59 Flux Modeling Methodology ...........................................................................................59 Statistical Analysis ...........................................................................................................63 v Results ..................................................................................................................................63 Trends in Metabolite Levels ............................................................................................64 Minimum Metabolic Flux Model.....................................................................................70 Discussion ............................................................................................................................71 Effect of Light Adaptation ...............................................................................................72 Effect of Induced Overexpression of AS α with Constitutive Overexpression of AS β ..................................................................................................................................73 Effect of Light Adaptation with Induced Overexpression of AS α and
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