SUNY College of Environmental Science and Forestry Digital Commons @ ESF Dissertations and Theses Spring 4-15-2020 Enhancing polyhydroxyalkanoate biosynthesis in Escherichia coli: A genetic engineering and process optimization approach towards functionalized polymeric nanomedicine Ryan Scheel SUNY College of Environmental Science and Forestry, [email protected] Follow this and additional works at: https://digitalcommons.esf.edu/etds Part of the Environmental Engineering Commons Recommended Citation Scheel, Ryan, "Enhancing polyhydroxyalkanoate biosynthesis in Escherichia coli: A genetic engineering and process optimization approach towards functionalized polymeric nanomedicine" (2020). Dissertations and Theses. 161. https://digitalcommons.esf.edu/etds/161 This Open Access Dissertation is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. ENHANCING POLYHYDROXYALKANOATE BIOSYNTHESIS IN ESCHERICHIA COLI: A GENETIC ENGINEERING AND PROCESS OPTIMIZATION APPROACH TOWARDS FUNCTIONALIZED POLYMERIC NANOMEDICINE By Ryan A. Scheel A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree State University of New York College of Environmental Science and Forestry Syracuse, New York April 2020 Department of Chemistry Approved by: Christopher T. Nomura, Major Professor John C. Stella, Chair, Examining Committee Avik P. Chatterjee, Department Chair S. Scott Shannon, Dean, The Graduate School Acknowledgements The ESF community has played a pivotal role in shaping me into the person I am today; I have discovered new passions, formed many incredible friendships, and learned so much about the environment from many different angles. There are too many people here who have touched my life to properly thank you all, so to the ESF community at large: thank you for helping me become the person I want to be. May you continue to inspire future generations! The completion of this dissertation would not have been possible without the support of my committee members, Dr. Chris Nomura, Dr. Art Stipanovic, and Dr. Lee Newman. Thank you for your insight and guidance, as well as your patience throughout the research process. I am especially grateful to my advisor Dr. Chris Nomura. You have taught me what it means to be a good scientist. I would also like to extend my sincere thanks to Dr. Erica Majumder. Although our time together at ESF was brief, I truly value the scientific, professional, and personal insight you offered me in the final year of my research. I look forward to seeing the great things you will accomplish here at ESF. To my colleagues in the Nomura Research Group, thank you for both your friendship and your endless assistance in the lab. I’m extremely grateful to Dr. Ben Lundgren, Dr. Zaara Sarwar, and Dr. Ata Pinto; thank you for devoting your time to training me in all manner of lab techniques, and for the research opportunities within your own projects that you offered me. I would also like to thank all the undergraduates who worked with me on my research. You allowed me to become a better mentor, and your work in the lab was instrumental to this research. I am particularly indebted to Sarah Alam el Din and Seamus McKenney, whose work laid the foundations for the research on nanoparticles and high-density fermentation, respectively. And finally, to my family. To my mother, Carla: for always teaching and supporting me, especially when I didn’t appreciate it as you deserved. To my grandmother, Marcia: for instilling scientific curiosity in me for as long as I can remember. To my partner, Megan Gallagher: for your encouragement and your inspiring work ethic, which impresses me to this day. And to my best friend, Ryan Crandall: for your enduring friendship, your endless enthusiasm, and for teaching me nearly everything I know about bugs. ii Table of Contents List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ......................................................................................................................... ix Abstract ......................................................................................................................................... xii Chapter 1: Introduction and literature review for polyhydroxyalkanoates ..................................... 1 1.1 Introduction to polyhydroxyalkanoates ............................................................................ 1 1.2 PHA composition and physical properties ....................................................................... 2 1.3 PHA biosynthesis in native producers ............................................................................. 7 1.4 PHA biosynthesis in Escherichia coli LSBJ .................................................................... 9 1.4.1 Engineering of E. coli LS5218.................................................................................. 9 1.4.2 SCL and MCL biosynthesis pathways .................................................................... 10 1.4.3 Transcriptional regulation of MCL PHA biosynthesis pathway............................. 13 1.5 Recent developments in the biosynthesis of chemically modifiable PHAs ................... 28 1.5.1 Fluorination of the PHA α-carbon .......................................................................... 29 1.5.2 Development and biosynthesis of azido-PHAs....................................................... 35 1.5.3 Biosynthesis and modifications of unsaturated PHAs ............................................ 40 1.6 Objectives of this dissertation ........................................................................................ 42 1.7 References ...................................................................................................................... 44 Chapter 2: Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA........................................................................................................... 70 2.1 Abstract ............................................................................................................................... 70 2.2 Introduction ......................................................................................................................... 71 2.3 Methods ............................................................................................................................... 75 2.3.1 Materials ....................................................................................................................... 75 2.3.2 Gene deletions .............................................................................................................. 76 2.3.3 PHA production ............................................................................................................ 77 2.3.4 GC analysis ................................................................................................................... 78 2.3.5 Polymer purification and molecular weight determination .......................................... 78 2.3.6 Growth profiles of LSBJ and RSC02 ........................................................................... 79 2.3.7 RNA extraction and qPCR analysis .............................................................................. 80 2.4 Results ................................................................................................................................. 81 2.4.1 PHA homopolymer production .................................................................................... 81 iii 2.4.2 Molecular weight comparison ...................................................................................... 84 2.4.3 Growth profile of LSBJ and RSC02 ............................................................................. 85 2.4.4 PHA yield at 24 vs 48 hours ......................................................................................... 86 2.4.5 Relative gene expression of RSC02 ............................................................................. 87 2.5 Discussion ........................................................................................................................... 88 2.6 References ........................................................................................................................... 94 Chapter 3: Increased production of the value-added biopolymers poly(R-3-hydroxyalkanoate) and poly(γ-glutamic acid) from hydrolyzed paper recycling waste fines ................................... 104 3.1 Abstract ............................................................................................................................. 104 3.2 Introduction ....................................................................................................................... 105 3.3 Methods ............................................................................................................................. 106 3.3.1 Hydrolysate from waste fines ....................................................................................