Modified Monoterpene Indole Alkaloid Production in the Yeast Saccharomyces Cerevisiae Copyright © 2017 by Amy M. Ehrenworth
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MODIFIED MONOTERPENE INDOLE ALKALOID PRODUCTION IN THE YEAST SACCHAROMYCES CEREVISIAE A Dissertation Presented to The Academic Faculty by Amy M. Ehrenworth In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry Georgia Institute of Technology December, 2017 COPYRIGHT © 2017 BY AMY M. EHRENWORTH MODIFIED MONOTERPENE INDOLE ALKALOID PRODUCTION IN THE YEAST SACCHAROMYCES CEREVISIAE Approved by: Dr. Pamela Peralta-Yahya, Advisor Dr. Francesca Storici School of Chemistry and Biochemistry School of Biological Studies Georgia Institute of Technology Georgia Institute of Technology Dr. M.G. Finn Dr. Loren Williams School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Wendy L. Kelly School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: August 14, 2017 ACKNOWLEDGEMENTS I would like to thank all those who’ve guided me on my scientific and personal journey. I was blessed by the circumstances that have led me to this point, and I hope to make those who have influenced and inspired me proud. I want to express gratitude to my advisor Pamela Peralta-Yahya for all she has done for me in my time at Georgia Tech- from introducing me to the incredible field of synthetic biology and sharing her knowledge to helping make me a better scientist. I admire her dedication and ambition, and I am honored to have helped establish the Peralta-Yayha lab. I also appreciate the guidance I’ve received from all of my committee members throughout the years, be it via scientific discussions, mentorship, or personal inspiration. I value the friendships and experiences I’ve gained with all of the past and current members of the Peralta-Yahya lab. I want to express special thanks to Stephen Sarria, who has been through this journey with me from day one, and Emily Yasi for their companionship through years of joys and frustrations and to Dr. Kuntal Mukherjee for sharing his insights and knowledge. I’d also like to recognize the undergraduates and research technicians that have worked with me. I’ve grown as a person through my interactions with all of you and I am eager to see what you all accomplish. In addition to labmates, I’ve had a number of other friends that helped accompany me through this time. While relationships change over time, I cherish all of my interactions and memories of the last five years. I want to express special thanks to Udita iii Brahmachari. I treasure your unwavering confidence, support, and friendship. I couldn’t have done it without you. I also appreciate the continuing friendships from earlier chapters in my life that have helped support me throughout my time at Georgia Tech. I can’t express in words my thanks to my parents and grandparents for their constant support every day of my life. I am blessed with your presence and love in my life. Finally, I express my deepest gratitude to my fiancé Donovan. You are my rock in every way. You’ve stood by me through my good days and bad and always drive me to be a better person. I can’t wait to take the next steps in my career and in life together with you. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xii SUMMARY xv CHAPTER 1. Accelerating the Semisynthesis of Alkaloid-based Drugs through Metabolic Engineering 1 1.1 Abstract 1 1.2 Introduction 2 1.3 Phenylalanine- and Tyrosine-derived Alkaloid-based Pharmaceuticals 23 1.3.1 Phenylethylamines 23 1.3.2 Tetrahydroisoquinoline Alkaloids 23 1.3.3 Amaryllidaceae Alkaloids 25 1.3.4 Phenylethylisoquinoline Alkaloids 26 1.3.5 Benzylisoquinoline Alkaloids 27 1.3.6 Aporphines and Morphinans 28 1.4 Tryptophan-derived Alkaloid-based Pharmaceuticals 32 1.4.1 Tryptamines and β-Carbolines 32 1.4.2 Monoterpene Indole Alkaloids 32 1.4.3 Quinoline Alkaloids 34 1.4.4 Pyrroloquinoline Alkaloids 34 1.4.5 Ergot Alkaloids 36 1.5 Enzymatic Production of Modified Alkaloids 38 1.6 Conclusion 40 1.7 References 42 CHAPTER 2. Pterin-dependent Mono-oxidation for the Microbial Synthesis of a Modified Monoterpene Indole Alkaloid 50 2.1 Abstract 50 2.2 Introduction 51 2.3 Results 56 2.3.1 Target Choice: Hydroxystrictosidine 56 2.3.2 Microbial Synthesis of Tetrahydrobiopterin in S. cerevisiae 58 2.3.3 Optimization of Tetrahydrobiopterin Biosynthesis 64 2.3.4 Tetrahydrobiopterin Recycling for Pterin-dependent Amino Acid Mono- oxidation 67 2.3.5 Microbial Synthesis of Biogenic Amines via Pterin-dependent Mono-oxidation 70 2.3.6 Microbial Synthesis of the Modified MIA 10-Hydroxystrictosidine 72 v 2.3.7 Determining Strictosidine Synthase Functionality 77 2.4 Discussion 80 2.5 Materials and Methods 84 2.5.1 Reagents 84 2.5.2 Construction of W303-Ade2+ Strain 84 2.5.3 Plasmid Construction 84 2.5.4 Yeast Transformation 87 2.5.5 Microbial Synthesis of Tetrahydrobiopterin 87 2.5.6 Microbial Synthesis of L-DOPA, Dopamine, Serotonin, and Hydroxystrictosidine 88 2.5.7 Biopterin Quantification 89 2.5.8 Quantification of L-DOPA, Dopamine, and Serotonin 90 2.5.9 Mass Spectral Analysis of Hydroxystrictosidine 90 2.5.10 Hydroxystrictosidine Isomer Ratios 91 2.5.11 Statistical Analysis 93 2.5.12 Yeast Cell Lysis for Intracellular Biopterin Determination 93 2.5.13 Determination of SR Open Reading Frame from T. pseudonana 93 2.5.14 Determiniation of GTPCH, PTPS, and SR mRNA Levels 93 2.5.15 Amino Acid Limiting Experiments 94 2.6 References 95 CHAPTER 3. Medium-throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-based Sensor 102 3.1 Abstract 102 3.2 Introduction 103 3.3 Results 106 3.3.1 Serotonin Sensor 106 3.3.2 Detection of Exogenously Added Serotonin in Plain Microbial Spent Medium 110 3.3.3 Effect of Nutrient Composition on Serotonin Sensing 111 3.3.4 Detecting Microbially Produced Serotonin in a Medium-throughput Assay 114 3.4 Discussion 116 3.5 Materials and Methods 117 3.5.1 Reagents 117 3.5.2 Exogenous Serotonin Detection in Fresh Media 117 3.5.3 Serotonin Detection Controls 118 3.5.4 Colony Variation Using GFP vs. ZsGreen as Reporter Genes 118 3.5.5 Exogenous Serotonin Detection in Adjusted Fresh Media 119 3.5.6 Spent Media Preparation 120 3.5.7 Exogenous Serotonin Detection in Spent Media 120 3.5.8 Medium-Throughput Serotonin Screening Validation 121 3.5.9 Flow Cytometry Data Analysis 121 3.5.10 Microbially Produced Serotonin Identification 122 3.5.11 Plasmid Construction 122 3.6 References 123 vi CHAPTER 4. Quantifying the Efficiency of Saccharomyces cerevisiae Translocation Tags 125 4.1 Abstract 125 4.2 Introduction 126 4.3 Results 127 4.3.1 Localization to the Mitochondria 128 4.3.2 Localization to the Vacuole 130 4.3.3 Localization to the Peroxisome 133 4.3.4 Analysis of mRNA and GFP expression 135 4.4 Discussion 137 4.5 Materials and Methods 140 4.5.1 Confocal Microscopy for GFP 140 4.5.2 Confocal Microscopy for mKate2 142 4.5.3 Quantification of GFP Localization 143 4.5.4 mRNA Quantification 143 4.5.5 Protein Expression Analysis Using Flow Cytometry 144 4.5.6 Plasmid Construction 145 4.6 References 146 CHAPTER 5. Conclusions and Future Direction 151 5.1 General Conclusions 151 5.2 Future Directions 151 5.2.1 Bioproduction of Other Alkaloid Families 151 5.2.2 Bioproduction of Advanced MIAs 152 5.2.3 Screening of Serotonin-Producer Libraries 153 5.2.4 Expansion of Signal Tag Studies 153 5.3 References 153 APPENDIX A. Tables of Plasmids 155 APPENDIX B. Tables of Strains 166 APPENDIX C. Table of Primers 178 APPENDIX D. DNA Sequences 185 vii LIST OF TABLES Table 1-1 FDA approved drugs mentioned in this chapter with chemical 7 structures and selected uses, listed by class. Table 2-1 Overview of alkaloid and precursor production. 75 Table 4-1 Translocation tag origin 139 Table 4-2 Translocation tag descriptors 140 Table A-1 Plasmids used in chapter 2 155 Table A-2 Plasmids used in chapter 3 156 Table A-3 Plasmids used in chapter 4 161 Table B-1 Yeast strains used in chapter 2 166 Table B-2 Yeast strains used in chapter 3. 173 Table B-3 Yeast strains used in chapter 4 174 Table C-1 Table of primers used in this work 178 viii LIST OF FIGURES Figure 1.1 Microbial production of advanced intermediates for plant- 5 alkaloid-based pharmaceuticals. Figure 1.2 Phenylalanine- and tyrosine-derived pharmaceuticals. 22 Figure 1.3 Tryptophan-derived pharmaceuticals. 22 Figure 1.4 Proposed semisyntheses of benzylisoquinoline alkaloid- 25 derived FDA-approved pharmaceuticals. Figure 1.5 Proposed semisyntheses of morphinan- and 31 pyrroloquinoline-derived FDA-approved pharmaceuticals. Figure 1.6 Proposed microbial production of lysergic acid for the 38 semisynthesis of ergot alkaloids. Figure 2.1 Stereochemistry of pterin co-factors. 54 Figure 2.2 Pterin-dependent microbial synthesis of monoterpene indole 56 alkaloids (MIAs) in Saccharomyces cerevisiae. Figure 2.3 Modified MIAs for the semisynthesis of pharmaceuticals. 58 Figure 2.4 Microbial synthesis of tetrahydrobiopterin (BH4) in S. 60 cerevisiae. Figure 2.5 Combinatorial production of biopterin. 61 Figure 2.6 Structural alignment of Salinibacter ruber and Salmo salar 63 pyruvoyl tetrahydropterin synthase (PTPS). Figure 2.7 Structural alignment of Mortierella alpina and Thalassiosira 63 pseudonana sepiapterin reductase (SR). Figure 2.8 Purine biosynthetic pathway from the Kyoto Encyclopedia 66 of Genes and Genomes (KEGG).