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ICHIKAWA-DISSERTATION-2019.Pdf BIOSYNTHESIS OF THE THIOPEPTINS AND IDENTIFICATION OF AN F420H2-DEPENDENT DEHYDROPIPERIDINE REDUCTASE A Thesis Presented to The Academic Faculty By Hiroyuki Ichikawa In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Chemistry Georgia Institute of Technology August 2019 Copyright © 2019 by Hiroyuki Ichikawa Biosynthesis of the thiopeptins and identification of an F420H2-dependent dehydropiperidine reductase Approved by: Dr. Wendy L. Kelly, Advisor Dr. Kirill S. Lobachev School of Chemistry and Biochemistry School of Biological Sciences Georgia Institute of Technology Georgia Institute of Technology Dr. Andreas S. Bommarius Dr. Adegboyega K. Oyelere School of Chemical and Biomolecular School of Chemistry and Biochemistry Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Julia M. Kubanek Date Approved: May 9, 2019 School of Biological Sciences School of Chemistry and Biochemistry Georgia Institute of Technology ACKNOWLEDGEMENTS I would like to express my deepest gratitude for my Ph.D. advisor, Dr. Wendy Kelly, for her expertise, guidance, and patience throughout my graduate studies. Her rigor and dedication to science has taught me to apply myself more effectively to research and inspired me to complete my doctorate work. I am extremely grateful for her generosity and support in and outside the lab, especially during the last year of my tenure. I would like to thank my committee members, Dr. Andreas Bommarius, Dr. Julia Kubanek, Dr. Kirill Lobachev, and Dr. Adegboyega Oyerele, for their valuable time, understanding, and expert advice. My appreciation goes to Dr. Christy O’Mahony, Dr. Mary Peek, and Dr. Hui Zhu who have been supportive and immensely accommodating with my teaching schedule. I am grateful for my friends and colleagues who have made my time at Georgia Institute of Technology a wonderful and memorable experience. It has been a great pleasure to work with former and present members of the Agarwal, Kelly, Oyelere, and Reddi groups. In particular, I would like to thank Dr. Bradley Carpenter, Mr. Iramofu Dominic, Ms. Verjine Khodaverdian, Ms. Osiris Martinez-Guzman, Ms. Ipsita Mohanty, Dr. Marietou Paye, Dr. Idris Raji, Mr. Daniel Sircar, Dr. Hem Thapa, Dr. Arren Washington, and Dr. Feifei Zhang for their support and encouragement. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ iii LIST OF TABLES .......................................................................................................... vii LIST OF FIGURES ....................................................................................................... viii LIST OF SCHEMES ..................................................................................................... xiv LIST OF SYMBOLS AND ABBREVIATIONS .......................................................... xv SUMMARY .................................................................................................................... xix CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1 Thiopeptides ........................................................................................................... 1 1.2 Structural classification of thiopeptides .............................................................. 6 1.3 Biological activities of thiopeptides ...................................................................... 7 1.4 Thiostrepton A ....................................................................................................... 9 1.5 Thiopeptins ........................................................................................................... 10 1.6 Thiopeptide biosynthesis ..................................................................................... 11 1.7 Biosynthesis of thiostrepton A ............................................................................ 15 1.8 Dehydropiperidine and piperidine biosyntheses .............................................. 22 1.9 Reductive coenzymes ........................................................................................... 23 1.10 Biosynthesis of cofactor F420 ............................................................................... 27 1.11 Scope of this work ................................................................................................ 29 1.12 References ............................................................................................................. 30 CHAPTER 2: IDENTIFICATION OF THE THIOPEPTIN BIOSYNTHETIC GENE CLUSTER AND CHARACTERIZATION OF TPNW, A 2-METHYL-L-TRYPTOPHAN AMINOTRANSFERASE ............... 42 2.1 Introduction ......................................................................................................... 42 2.2 Materials and methods ........................................................................................ 47 2.2.1 General ........................................................................................................... 47 2.2.2 Bacterial strains, plasmids, and growth medium............................................ 48 2.2.3 Growth of S. tateyamensis and analysis of thiopeptin production ................. 48 2.2.4 Construction of the S. tateyamensis fosmid library and screening for selected sequences ....................................................................................................... 49 iv 2.2.5 Bioinformatics analyses of the tpn gene cluster ............................................. 50 2.2.6 Sporulation of S. tateyamensis ....................................................................... 50 2.2.7 Intergeneric conjugation in S. tateyamensis ................................................... 50 2.2.8 Disruption of tpnA .......................................................................................... 51 2.2.9 Additional conditions for S. tateyamensis protoplast preparation.................. 53 2.2.10 Towards disruption of tpnL ............................................................................ 54 2.2.11 Towards disruption of tpnM ........................................................................... 55 2.2.12 Towards disruption of tpnN ........................................................................... 56 2.2.13 Towards disruption of tpnR ............................................................................ 56 2.2.14 Towards the genetic complementation of S. tateyamensis HI1 ..................... 57 2.2.15 Expression and purification of TpnW ............................................................ 58 2.2.16 In vitro reconstitution of TpnW activity ........................................................ 59 2.3 Results and discussion ......................................................................................... 60 2.3.1 Production of thiopeptins by S. tateyamensis ................................................ 60 2.3.2 Identification of the thiopeptin biosynthetic gene cluster .............................. 65 2.3.3 The deduced functions of the tpn cluster ....................................................... 68 2.3.4 Disruption of open reading frames of the tpn cluster ..................................... 73 2.3.5 Transaminase activity of TpnW ..................................................................... 84 2.4 Conclusions........................................................................................................... 88 2.5 References ............................................................................................................. 89 CHAPTER 3: HETEROLOGOUS EXPRESSION OF TPNLMNR IN STREPTOMYCES LARUENTII ........................................................... 96 3.1 Introduction ......................................................................................................... 96 3.2 Materials and methods ........................................................................................ 97 3.2.1 General ........................................................................................................... 97 3.2.2 Bacterial strains, plasmids, and growth medium............................................ 98 3.2.3 Growth of S. laurentii and analysis of thiostrepton production ..................... 98 3.2.4 Transformation of S. laurentii with pSET1520 and cultivation of S. laurentii HI0 ............................................................................................. 100 3.2.5 Expression of TpnL in S. laurentii ............................................................... 101 3.2.6 Expression of TpnM in S. laurentii .............................................................. 102 3.2.7 Expression of TpnN in S. laurentii............................................................... 103 3.2.8 Expression of TpnR in S. laurentii ............................................................... 104 3.2.9 Expression of TpnLM in S. laurentii ........................................................... 105 3.2.10 Expression of TpnMN in S. laurentii ........................................................... 106 3.2.11 Expression of TpnLMN in S. laurentii......................................................... 107 3.2.12 Purification and characterization of thiostrepton Aa (31) ............................ 108 3.2.13 Determination of thiostrepton Aa (31) solubility ......................................... 108 3.2.14 Antimicrobial activity of thiostrepton Aa
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