Copyright by Kelly Ann Markham 2018 The Dissertation Committee for Kelly Ann Markham Certifies that this is the approved version of the following dissertation: Expanding the Product Portfolio of Yarrowia lipolytica through Metabolic Engineering and Synthetic Biology Tool Development Committee: Hal Alper, Supervisor Lydia Contreras George Georgiou Adrian Keatinge-Clay Expanding the Product Portfolio of Yarrowia lipolytica through Metabolic Engineering and Synthetic Biology Tool Development by Kelly Ann Markham Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August 2018 Dedication For those who have been made to feel small. You are strong. You are important. You deserve to take up space. Acknowledgements First and foremost, I want to thank Dr. Hal Alper for being a supportive advisor and helping me grow in all aspects of being a scientist. From helpful tricks for personal development like working with Microsoft (that alignment function will always haunt me) to high expectations requiring proper experimental design, I have learned an amazing amount from Hal in the last five years. Hal has proven willing to take time to make sure that students are taken care of and meeting their full potential. Thanks for taking that time to make us all better researchers and giving us room to explore different projects and ideas. I am forever grateful for the opportunity to work with Hal and the Alper lab. Next, I would like to thank Dr. Lydia Contreras, Dr. George Georgiou, and Dr. Adrian Keatinge-Clay for serving as my dissertation committee. Each of them took time out of their busy schedules to provide helpful advice throughout my thesis work. Their questions and recommendations from my preliminary proposal led to a strengthened set of aims and challenged me to become a better scientist. Special thanks to Dr. Contreras for always being willing to help with fellowship applications and the job process, especially for writing letters of recommendation on my behalf. I am thankful for having the opportunity to work on the same floor as her group. An extra thank you to Dr. Keatinge- Clay for taking time to meet with Claire and me to help us learn more about the chemistry of polyketides and troubleshoot when we were making products we could not identify. Thank you to my committee members for all your suggestions and feedback. v Next, it would not have been the same graduate experience without the awesome members of the Alper Lab. Thanks to everyone in the lab for all the collaboration, suggestions, questions, and willingness to show up for presentations. I am especially grateful for Team Yarrowia including Claire Palmer, James Wagner, Lauren Cordova, Haibo Li, Leqian Liu, and Andrew Hill. Working with this nonconventional system is hard, and you all made it better by struggling side by side with me, sharing in successes and failures, and working to develop and share best practices for the lab. Everyone else who I had the opportunity to work with in the lab have also been instrumental to my success- Kate Curran, Nathan Crook, Sun-mi Lee, Jie Sun, John Leavitt, Joseph Cheng, Joe Abatemarco, Aaron Lin, Nick Morse, Matt Deaner, Joseph Yuan, Kevin Reed, Kristin Presnell, and Xiunan Yi. Thank you for all your collaborations, life chats, TV suggestions, food adventures, and genuinely making the Alper lab a great place to work. In addition to all the graduate students I have had the opportunity to work with in the lab, undergraduates and high school students deserve a major shout out. Thank you to Clare Murray, Arvind Swaminathan, Sofia Vazquez, Ishani Chakravarty, Sarah Coleman, Valerie Vines, Mallika Maheshwary, Cedric Ginestra, Cecilia Barnhill, and Grace Lawler. Your energy kept me on my toes and made lab a fun place to work. Each of you made me a better teacher, and this work would have been impossible without your help. Collaborations in the lab have been abundant, but I also had the opportunity to work with Dr. Nate Lynd and Gosia Chwatko. Thank you for taking the chance on working with bio-sourced products and making the TAL paper a truly great story. It was fun to see a process all the way from development to application, and that would have been impossible vi without your help along with the collaboration with Claire Palmer and the hard work of Clare, Arvind, Sofia, and Ishani who will likely hold the lab record for number of simultaneous genomic DNA preps for life. Labs do not work in isolation, and the support of staff members has not gone unnoticed. Thank you to Shallaco McDonald, Tony Le, and Jim Smitherman for keeping CPE and our lab equipment safe, functioning, running an amazing shop, and always having the best candy. Thank you to Kevin Haynes, Ben Hester, Tammy McDade, and Eddie Ibarra for ensuring that resources were promptly procured. Additional support from Courtney Hazlett, Randy Rife, Jason Barborka, Kate West Baird, and Melinda Heidenreich ensured that I had all the tools to succeed. Each of these staff members have gone out of their way to help me to be a successful graduate student and always did so with kindness. Outside of the department, I would like to especially thank my family for their support. Thank you to my Mom, Kate, Kyle, Kurt, Joel, Margeaux, Esme, and my grandparents. You all helped to make sure that I was a functioning human, especially with Kate and Joel keeping me caffeinated. Though not related by blood, my girl squad has supported me like a family. Thank you Haley Streff, Rachel Casciato, Erin Christensen, Sarah Waldschmidt, Callie Larson, Laura Chemler, AK Rockwell, Melissa Donahue, Andrea DiVenere, and Claire Palmer. You challenge me to be a better person and I am grateful for your unconditional support. Thank you as well to my graduate school cohort and other Austin friends for making this city a fun place to live and explore. Finally, thanks to 7-Eleven and podcasts for fueling this graduate degree. vii Expanding the Product Portfolio of Yarrowia lipolytica through Metabolic Engineering and Synthetic Biology Tool Development Kelly Ann Markham, Ph.D. The University of Texas at Austin, 2018 Supervisor: Hal S. Alper Yarrowia lipolytica is a potent microbial cell factory for metabolic engineering that will enable the sustainable production of chemicals beyond first generation biofuels and polymer precursors. As an alternative host, there are many limitations yet to be resolved that require much more intensive research. Yet, this oleaginous yeast has already proven superior to its conventional yeast counterpart Saccharomyces cerevisiae for the production of a variety of different products most notably including lipids. The work presented here offers two metabolic engineering stories demonstrating the potential of Y. lipolytica to produce value-added chemicals at high titers. Additionally, seeking to alleviate bottlenecks in the engineering process, this work presents details of an optimized and streamlined protocol for transformation as well as a strategy for tuning gene expression via synthetic terminators. First, the focus was set on engineering a high-lipid strain for cyclopropane fatty acid production. This work leveraged a strain of Y. lipolytica that had been previously metabolically engineered to produce high levels of fatty acids. Cyclopropane fatty acids (a non-native molecule to Y. lipolytica) serve as a simple way to improve biodiesel properties and are additionally useful in lubricants. Through engineering, 3 g/L production was enabled. In a second study, the inherent flux towards lipids was hijacked and redirected towards polyketides. Specifically, extensive metabolic engineering led to production of 36 viii g/L of triacetic acid lactone, an important biorenewable precursor molecule. Third, the development and optimization of an electroporation transformation protocol alleviated a critical bottleneck in the engineering process, allowing for highly efficient incorporation of exogenous DNA into Y. lipolytica. This protocol streamlines transformations and does not require the addition of carrier DNA. Fourth, synthetic terminators originally developed for S. cerevisiae were tested in Y. lipolytica enabling improved tuning of expression. Incorporation of these synthetic terminators allowed for a 70% increase in gene expression over a commonly used terminator. Collectively, these chapters demonstrate the potential of Y. lipolytica as a host for metabolic engineering through study of tools and investigation of native pathways that can be redirected towards non-native products. ix Table of Contents List of Tables .....................................................................................................................xv List of Figures .................................................................................................................. xvi Chapter 1: Introduction ........................................................................................................1 1.1 Chapter Summary .................................................................................................1 1.2 Introduction ...........................................................................................................1 1.3 Recent advances in Y. lipolytica metabolic engineering ......................................3 1.3.1 Oleochemicals ........................................................................................5 1.3.2