University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 3-24-2017 A Novel Approach to the Discovery of Natural Products From Actinobacteria Rahmy Tawfik University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Microbiology Commons Scholar Commons Citation Tawfik, Rahmy, "A Novel Approach to the Discovery of Natural Products From Actinobacteria" (2017). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/6766 This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. A Novel Approach to the Discovery of Natural Products From Actinobacteria by Rahmy Tawfik A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Cell Biology, Microbiology & Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Lindsey N. Shaw, Ph.D. Edward Turos, Ph.D. Bill J. Baker, Ph.D. Date of Approval: March 22, 2017 Keywords: Secondary Metabolism, Soil, HPLC, Mass Spectrometry, Antibiotic Copyright © 2017, Rahmy Tawfik Acknowledgements I would like to express my gratitude to the people who have helped and supported me throughout this degree for both scientific and personal. First, I would like to thank my mentor and advisor, Dr. Lindsey Shaw. Although my academics were lacking prior to entering graduate school, you were willing to look beyond my shortcomings and focus on my strengths. In doing so, I was able to overcome what I was lacking and although I still have much to learn, I am better for being your graduate student. Thank you for pushing me beyond what I thought possible and all the continuous support throughout my graduate career. Without your knowledge and guidance, this thesis would not be possible. I would also like to thank my committee members, Dr. Bill Baker and Dr. Edward Turos for all of the assistance and knowledge provided throughout this degree. Dr. Baker, thank you for allowing the collaborations with your laboratories so we could push this research further than I previously perceived and sharing your knowledge and mastery of natural products chemistry. I would also like to acknowledge all members of the Shaw and Baker labs who have helped me along the way and provided me with immeasurable aid. Renee Fleeman, thank you for all your help with laboratory techniques as well as for introducing me to the lab. Andrew Shilling, I cannot express the countless ways you have bettered this research. Your extensive knowledge of natural products has been crucial to the success of this thesis and I am thankful that we were able to work together throughout this endeavor. Haider Manzer and Sarah Kennedy, thank you both for all your help through the years. Without your consistent aid, we would not have been able to push this project any further, with your help we were able to look beyond the petri dish. To my family, thank you for your support throughout all my years as an undergraduate and graduate. Your love and guidance has provided me so much stability and allowed me to focus on my passions, which I cannot express enough gratitude for. Mom, you have always been an excellent source for clear thinking, and I would not be where I am today if you did not insist on explaining the roads ahead, but allowing me to make the appropriate choice. Dad, although we do not see everything the same way, you have always loved, supported, and provided for me and for this I am forever grateful. To Arielle Sharp, I truly could not have made it through the toughest parts of my graduate career without you. Thank you for making sure I was fed, slept appropriate hours, had activities to clear my mind, and ensure I didn’t go crazy, you are sincerely my foundation. Table of Contents List of Tables ................................................................................................................................ iii List of Figures ............................................................................................................................... iv Abstract ......................................................................................................................................... v Introduction ................................................................................................................................... 1 Antibiotics: Costs and Resistance. .................................................................................... 1 ESKAPE Pathogens ......................................................................................................... 2 Antibiotic Resistance Mechanisms ................................................................................... 6 Actinobacteria ................................................................................................................. 11 Natural Products ............................................................................................................. 14 DNA Methyltransferase ................................................................................................... 16 Epigenetic Modification ................................................................................................... 18 Anthracyclines ................................................................................................................. 21 Project Aim ...................................................................................................................... 25 Materials and Methods ............................................................................................................... 27 Initial Soil Collection and Processing Optimization ......................................................... 27 Media .................................................................................................................. 27 Inoculation Techniques ....................................................................................... 28 Isolation and Purification of species .................................................................... 29 Current Actinobacteria Culture Techniques .................................................................... 30 Isolate Identification ............................................................................................ 30 Isolates Used for Optimization Techniques ......................................................... 31 Secondary Metabolite Extraction Optimization ............................................................... 32 Epigenetic Modification Optimization .............................................................................. 33 Screening Extracts Against the ESKAPE Pathogens ..................................................... 34 Small-Scale Growth ........................................................................................................ 34 Large-Scale Growth ........................................................................................................ 35 HPLC Purification and Bioassay Guided Fractionation ................................................... 36 Modified HPLC Purification and Bioassay Guided Fractionation .................................... 37 Results ........................................................................................................................................ 38 Investigating Cultivation Strategies Necessary for Actinobacteria Growth ..................... 38 Model for Rapid Identification of Actinobacterial Cultures and Strain Collection Development ............................................................................................................. 41 Secondary Metabolite Extraction Optimization ............................................................... 49 Epigenetic Modification Optimization .............................................................................. 49 i Quantitative Analysis of Secondary Metabolite Production of Actinobacteria Strain Collection ........................................................................................................ 56 Evaluating the Effects of Crude Secondary Metabolites Against the ESKAPE Pathogens ................................................................................................................. 56 Initial High Performance Liquid Chromatography Coupled with Bioassay Guided Fractionation ............................................................................................................. 67 High Performance Liquid Chromatography Coupled with Bioassay Guided Fractionation ............................................................................................................. 70 Discussion .................................................................................................................................. 78 Actinobacteria Diversity .................................................................................................. 78 Drug Discovery ............................................................................................................... 81 Epigenetics ..................................................................................................................... 86 Future Directions .......................................................................................................................