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University of Florida Thesis Or Dissertation Formatting Template

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University of Florida Thesis Or Dissertation Formatting Template BIOSYNTHESIS AND MECHANISM OF NATURAL PRODUCTS IN NEMATODES By LIKUI FENG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Likui Feng To my family ACKNOWLEDGMENTS I would like to dedicate my success in abtaining a PhD at the University of Florida to the many people who have given me support since I came to Gainesville, because without their considerate help, I do not believe that I would have obtained it. First of all, I would like to give the special thanks to my research advisor Dr. Rebecca Butcher. It was her who gave me the chance to transfer to this great university and program when I tried to transfer from Indiana University Bloomington. Most importantly, her knowledge, passion and confidence have helped me to become a successful scientist over the past five years. To be a great mentor, she also inspired me with new ideas in multiple perspectives. I would also like to thank my kind committee members, Dr. Steven Bruner, Dr. Nicole Horenstein, Dr. Keith Choe and Dr. Robert McKenna. These professors have provided me with valuable suggestions in many aspects of my work. They are also very nice to get along with and good friends. In addition, I would like to thank Dr. Ben Smith and Ms. Lori Clark, for their endless help in processing documents and my program transfer. Furthermore, I would like to thank Dr. Kari Basso and others in the mass spec facility for their help in running and analyzing samples. I am very glad and lucky to work with so many fantastic people in the Butcher lab. It is all of you who make our big lab family full of happiness. In particular, I would like to thank Dr. Qingyao Shou, who inspired me a lot in the nemamide project and has continued help me to figure out experimental problems even after he left our lab. Dr. Xinxing Zhang, full of academic thoughts, is a very good friend both in research and life, and provided me with a lot of fresh ideas and new techniques. I would like to thank Dr. Yue Zhou and Dr. Yuting Wang for their help in the lab and life. I would like to thank Dr. 4 Rouf Dar for synthesizing intermediates and Ying Liu for her collaborative work on metabolomics. In my last year of PhD, I am so lucky to work with Matthew Gordon, who is very helpful, funny and talented as a promising scientist. I thank all other current Butcher lab members, including Dr. Guohui Li, Prashant Singh, Subhradeep Bhar, Nasser Faghih, David Perez and previous group members Dr. Satya Chinta, Dr. Rachel Jones, Dr. Jungsoo Han, Asyegul Ozdogan, Mayra Tuiche, Jasmine Gonzalez, Priyanka Raichoudhury, and Lauren Suarez. I am also grateful to all Bruner lab members, including Dr. Kunhua Li, Dr. Wei- Hung Chen, Dr. Matthew Burg, Aleksandra Zagulyaeva, Brian Mctavish, Dr. Naga Sandhya Guntaka, Qiang Li, Prabhanshu Tripathi and Gengnan Li. I would also like to thank my dear friends in the Department of Microbiology and Cell Sciences, because they made my PhD life colorful and amazing. Finally, I would like to give my deepest gratitude to my family. My parents gave me the the life and continue supporting me to decorate my world in many aspects. Their endless love makes me go through lots of difficulties to further improve myself to be a better person and a successful scientist. I am very thankful to my younger brother for his effort to take care of our parents. Most importantly, I would like to thank my dear wife Yuanyuan Leng, who cheers me up and motivates me all along to make me succeed. She is always patient no matter how late and how long I work and she tries to bring me out of the lab to make me feel relaxed. I will do my best to make her proud and love her forever. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 17 ABSTRACT ................................................................................................................... 20 CHAPTER 1 INTRODUCTION .................................................................................................... 22 1.1 Nematodes and Life Cycle of C. elegans .......................................................... 22 1.2 Polyketide and Nonribosomal Peptides ............................................................ 25 1.2.1 Initiation Module of PKSs and NRPSs ..................................................... 27 1.2.2 Mechanism of Extension in PKSs and NRPSs ........................................ 29 1.2.3 Accessory Enzymes Acting in trans or in cis ........................................... 31 1.3 Signaling Molecules in C. elegans .................................................................... 32 1.3.1 Dauer Pheromone Ascarosides ............................................................... 32 1.3.2 Discovery of the Nemamides ................................................................... 34 1.3.3 Other Signaling Molecules in Worms ....................................................... 39 1.4 Significance and Summary ............................................................................... 39 2 MECHANISM OF THE NEMAMIDES IN PROMOTING LARVAL SURVIVAL AND PATHOGEN RESISTANCE ........................................................................... 42 2.1 Insulin Signaling Pathway in Worm Development and Arrest ........................... 42 2.2 Experimental Methods ...................................................................................... 43 2.2.1 Methods for Roles of Nemamides in Larval Survival ............................... 43 2.2.2 Screen for Pathogen Resistance ............................................................. 49 2.2.3 Methods for PKS-1b Verification and Gene Cloning ................................ 51 2.3 Results .............................................................................................................. 52 2.3.1 Site of nrps-1 and pks-1 Expression in C. elegans .................................. 53 2.3.2 Nemamides Promoting Arrested Larval Survival ..................................... 54 2.3.3 Screening by Pathogen Avoidance Assay and Killing Assay ................... 68 2.3.4 Verification of the Presence of pks-1b ..................................................... 71 2.3.5 Gene Cloning of pks-1b ........................................................................... 72 2.4 Discussion and Future Work ............................................................................. 73 3 NONCANONICAL FEATURES IN BIOSYNTHESIS OF THE NEMAMIDES .......... 75 3.1 Proposed Nemamide Biosynthetic Pathway ..................................................... 75 6 3.2 Domain Properties of PKS-1 and NRPS-1 ........................................................ 76 3.2.1 Ketosynthase (KS) Domains ................................................................... 76 3.2.2 Carrier Protein (CP) Domain ................................................................... 77 3.2.3 Acyltransferase (AT) Domains ................................................................. 78 3.2.4 Dehydratase (DH) Domains .................................................................... 79 3.2.5 Ketoreductase (KR) Domains .................................................................. 80 3.2.6 Adenylation domain (A) Domains ............................................................ 82 3.2.7 Condensation (C) Domains ..................................................................... 86 3.2.8 Thioesterase (TE) Domains ..................................................................... 86 3.3 Experimental Methods ...................................................................................... 88 3.3.1 Strains and Transgenic Lines .................................................................. 88 3.3.2 Single Worm PCR and CRISPR-Cas9 .................................................... 93 3.3.3 Transgenic Line Construction .................................................................. 98 3.3.4 Plasmid Construction, Protein Overexpression and Purification .............. 99 3.3.5 Small Scale Worm Extraction and Intermediate Extractions .................. 102 3.3.6 LC-MS-based ACS and ACOX Activity Assay ....................................... 104 3.3.7 Profiling Ascaroside Production ............................................................. 105 3.4 Functional Analysis of Enzymatic Domains .................................................... 105 3.4.1 Ketosynthase (KS) Domain ................................................................... 106 3.4.2 Carrier Protein (CP) Domains ................................................................ 108 3.4.3 Condensation (C) Domains ................................................................... 111 3.4.4 Adenylation (A) Domains ....................................................................... 115 3.4.5 Thioesterase (TE) Domains ................................................................... 117 3.5 Genome Mining of Accessory Enzymes ......................................................... 122 3.6 Functional Analysis of Accessory Enzymes
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