Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2015 The deI ntification of Alkaloid Pathway Genes from Non-Model Plant Species in the Amaryllidaceae Matthew .B Kilgore Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Recommended Citation Kilgore, Matthew B., "The deI ntification of Alkaloid Pathway Genes from Non-Model Plant Species in the Amaryllidaceae" (2015). Arts & Sciences Electronic Theses and Dissertations. 657. https://openscholarship.wustl.edu/art_sci_etds/657 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Plant Biology Dissertation Examination Committee: Toni Kutchan, Chair Elizabeth Haswell Jeffrey Henderson Joseph Jez Barbara Kunkel Todd Mockler The Identification of Alkaloid Pathway Genes from Non-Model Plant Species in the Amaryllidaceae by Matthew Benjamin Kilgore 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 December 2015 St. Louis, Missouri © 2015, Matthew Benjamin Kilgore Table of Contents List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................ vii Acknowledgments........................................................................................................................ viii Abstract ........................................................................................................................................... x Chapter 1: Introduction: The Amaryllidaceae Alkaloids: Biosynthesis and Methods for Enzyme Discovery .................................................................................................... 1 1.1 Amaryllidaceae Alkaloids ................................................................................................ 2 1.1.1 Amaryllidaceae Description with a Narcissus spp. Emphasis ................................... 2 1.1.2 Amaryllidaceae Alkaloid Introduction ..................................................................... 3 1.1.3 Prevalent Biosynthetic Gene Superfamilies.............................................................. 6 1.1.4 Core Biosynthetic Pathway ....................................................................................... 8 1.1.5 Galanthamine-Type Alkaloid Biosynthesis ............................................................ 16 1.1.6 Haemanthamine, Pancratistatin, Gracilamine, Cripowellin, Tazettine, Plicamine, Hostasinine, Graciline, Augustamine, and Montanine-Type Alkaloid Biosynthesis ........................................................................... 18 1.1.7 Lycorine and Homolycorine-Type Alkaloid Biosynthesis ..................................... 22 1.1.8 Other Amaryllidaceae Alkaloid Biosynthesis: Cherylline, Galanthindole, Galasine, and Buflavine ........................................................................... 24 1.2 Methods of Interest to Pathway Elucidation ................................................................... 26 1.2.1 Introduction ............................................................................................................. 26 1.2.2 Gene Clusters and Co-Regulation of Biosynthetic Pathways ................................. 27 1.2.3 Sequencing Technologies ....................................................................................... 30 1.2.4 Nuclear Magnetic Resonance Spectroscopy ........................................................... 33 1.2.5 Mass Spectrometry.................................................................................................. 34 1.2.6 Substrate Considerations ..................................................................................... 35 1.3 Conclusion ...................................................................................................................... 36 1.4 Chapter Summary ........................................................................................................... 37 1.4.1 Chapter 2 ................................................................................................................. 37 1.4.2 Chapter 3 ................................................................................................................. 38 1.4.3 Chapter 4 ................................................................................................................. 38 1.4.4 Chapter 5 ................................................................................................................. 39 Chapter 2: Cloning and Characterization of a Norbelladine 4’-O-Methyltransferase Involved in the Biosynthesis of the Alzheimer’s Drug Galanthamine in Narcissus sp. aff. pseudonarcissus ................................................................................................................ 40 2.1 Introduction .................................................................................................................... 41 2.2 Materials and Methods ................................................................................................... 46 2.2.1 Plant Tissue and Chemicals ................................................................................... 46 2.2.2 Alkaloid Extraction and Euantification ................................................................. 46 ii 2.2.3 Illumina Sequencing and Transcriptome Assembly .............................................. 47 2.2.4 Candidate Gene Identification ............................................................................... 48 2.2.5 Phylogenetic Tree .................................................................................................. 49 2.2.6 PCR and Cloning ................................................................................................... 49 2.2.7 Protein Purification ................................................................................................ 50 2.2.8 Screening Enzyme Assays ..................................................................................... 51 2.2.9 Kinetic Characterization ........................................................................................ 51 2.2.10 NMR ..................................................................................................................... 53 2.2.11 Quantitative Real Time-PCR (qRT-PCR) ............................................................ 53 2.3 Results ............................................................................................................................. 54 2.4 Discussion ........................................................................................................................ 62 2.5 Acknowledgments ........................................................................................................... 66 Chapter 3: CYP96T1 of Narcissus sp. aff. pseudonarcissus Catalyzes Formation of the Para-Para’ C-C Phenol Couple in the Amaryllidaceae Alkaloids .................................... 67 3.1 Introduction .................................................................................................................... 68 3.2 Materials and Methods ................................................................................................... 73 3.2.1 Plant Tissue and Chemicals .................................................................................... 73 3.2.2 Transcriptome Assembly and Transcript Abundance Estimation .......................... 74 3.2.3 Candidate Gene Identification ................................................................................ 75 3.2.4 Polymerase Chain Reaction (PCR) and Cloning .................................................... 76 3.2.5 Protein Expression .................................................................................................. 78 3.2.6 3’-O-Methylnorbelladine and 3’,4’-O-Dimethylnorbelladine Synthesis ................ 79 3.2.7 Enzyme Assays ....................................................................................................... 79 3.2.8 LC-MS/MS ............................................................................................................. 80 3.3 Results ............................................................................................................................ 82 3.3.1 Transcriptome Assembly and Transcript Abundance Estimation .......................... 82 3.3.2 Candidate Gene Identification and Cloning ............................................................ 83 3.3.3 Enzyme Assays and Analysis by LC-MS/MS ........................................................ 85 3.3.4 Sodium Borohydride Assays and Analysis by LC-MS/MS .................................... 91 3.4 Discussion ....................................................................................................................... 93 3.5 Acknowledgments