Kinetic Analysis of the Catabolism of Tabersonine in Catharanthus roseus Leaf Protein Extracts A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Matthew Dale Evans IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE CHEMICAL ENGINEERING Dr. Guy W. Sander August 2018 © Matthew Evans 2018 Acknowledgments I would like to express gratitude toward my adviser Dr. Guy Sander for teaching me the principles of metabolic engineering, and for providing great guidance and support throughout the entire process of this work. I also would like to thank Dr. Michael Rother and Dr. Victor Lai for serving on my thesis committee. Thanks also goes out to the faculty of the Chemical Engineering Department for teaching me the principles of chemical engineering in your courses. I would also like to thank my fellow Chemical Engineering graduate students for their support and conversations, both on- and off-campus. And finally, I would like to thank my family and friends for their continued support throughout my graduate studies. Funding for this project was provided by the Chancellor’s Faculty Small Grant and Teaching & Research assistantships through the University of Minnesota Duluth. i Abstract Vinblastine and vincristine are valuable chemotherapy compounds produced by Catharanthus roseus. However, they are produced in small quantities in the plant because of their cytotoxicity and spatial separation of the metabolic pathways of the two monomeric molecules, vindoline and catharanthine, that condense to form them. There have been extensive attempts to metabolically engineer greater production of these molecules, but the specialized cell types needed for the terminal steps of vindoline biosynthesis from tabersonine and the high regulation of the pathway in the plant have made use of a homologous host difficult. Recently, all metabolic steps of the pathway have been elucidated, but there remains difficulty in culturing a heterologous host to express all metabolic steps. There is almost no knowledge of the catabolism of these alkaloids in the plant, and this knowledge could be used in future metabolic engineering attempts. Leaf protein extracts were incubated with tabersonine to attempt to find possible catabolic products, and initial rate experiments were used to calculate kinetic parameters of a possible catabolic enzyme. Results showed no formation of previously known or unknown metabolites. After performing kinetic studies, evidence was found of -1 " an allosteric enzyme acting on tabersonine with a vm of 5.4 ± 1.9 μM min , a Km of 16,000 ± 14,000 μM, and a Hill coefficient of 2.0 ± 0.2. ii Table of Contents Abstract ......................................................................................................................................................... ii List of Tables.................................................................................................................................................iv List of Figures ................................................................................................................................................ v Background .................................................................................................................................................... 1 Introduction ................................................................................................................................................. 1 Terpenoid Indole Alkaloid Enzymatic Pathway ......................................................................................... 2 Metabolic Engineering for Increased Alkaloid Production ......................................................................... 3 Tabersonine Metabolism ............................................................................................................................. 6 Enzyme Kinetics ......................................................................................................................................... 7 Materials and Methods ................................................................................................................................. 8 Chemicals.................................................................................................................................................... 8 Protein Extraction ....................................................................................................................................... 8 Search for Unknown Metabolites ............................................................................................................... 8 Enzymatic Reactions ............................................................................................................................... 8 Ethyl Acetate Extraction of Alkaloids ..................................................................................................... 9 Kinetic Studies ............................................................................................................................................ 9 Initial Rate Experiments ......................................................................................................................... 9 Acetone Protein Precipitation Protocol for Alkaloid Extraction ............................................................ 9 HPLC-UV Analysis .................................................................................................................................. 10 Theory ....................................................................................................................................................... 10 Determination of Experimental Reaction Rates ................................................................................... 10 Initial Kinetic Model to Determine Time Scale .................................................................................... 11 Results and Discussion ................................................................................................................................ 12 Search for Unknown Metabolites ............................................................................................................. 12 Initial Kinetic Model to Determine Time Scale .................................................................................... 12 Initial Enzymatic Reactions .................................................................................................................. 13 Kinetic Studies .......................................................................................................................................... 13 Error in Ethyl Acetate Extraction ......................................................................................................... 13 Kinetic Studies ...................................................................................................................................... 14 Kinetic Model ....................................................................................................................................... 16 Conclusion .................................................................................................................................................... 18 Future Works............................................................................................................................................... 19 Illustrations .................................................................................................................................................. 20 References .................................................................................................................................................... 28 Appendices ................................................................................................................................................... 31 Appendix 1: Calculating Rate Constants from Initial Rate Experiments .................................................. 31 Appendix 2: Polymath ODE Solver Input ................................................................................................ 36 iii List of Tables Table 1: Kinetic constants used in model .. .................................................................................................... 21 iv List of Figures Figure 1: Terpenoid Indole Alkaloid Metabolic Pathway ............................................................................ 20 Figure 2: Metabolic pathway from tabersonine to vindoline. ....................................................................... 21 Figure 3: Results of metabolic model ........................................................................................................... 22 Figure 4: 254 nm chromatogram of extracted reaction medium.. ................................................................ 22 Figure 5: 329 nm chromatogram of extracted reaction medium. ................................................................. 23 Figure 6: Tabersonine consumption in samples incubated at 30°C for 2 hours with a starting tabersonine concentration of 30 μM and: A: 300 μM of NADPH and s-adenosyl-L-methionine; B: 300 μM s- adenosyl-L-methionine; and C: 300 μM of NADPH ............................................................................. 23 Figure 7: Specific activity of samples without (control) and with clotrimazole. .......................................... 24 Figure 8: Results of testing both extraction protocols