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Copyright by Rebecca Anne Knight 2014 The Dissertation Committee for Rebecca Anne Knight Certifies that this is the approved version of the following dissertation: Coordinated Response and Regulation of Carotenogenesis in Thermosynechococcus elongatus (BP-1): Implications for Commercial Application Committee: Jerry J. Brand, Supervisor Hal S. Alper, Co-Supervisor Enamul Huq Robert K. Jansen David R. Nobles Coordinated Response and Regulation of Carotenogenesis in Thermosynechococcus elongatus (BP-1): Implications for Commercial Application by Rebecca Anne Knight, B.A., B.S.E., M.S.E. 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 December 2014 Dedication Dedicated to my parents, who taught me to always work hard, and never stop learning. Acknowledgements I think there is a unique experience that people like myself undergo when working in industry then returning to academia. Very little is taken for granted on campus, so much to learn and take advantage of knowledge-wise. Then there is the flip side; long hours in the lab, the struggle to find your niche and contribution to the scientific community, and a much tighter budget both for research and personal life. I am here to say that without my family and their encouragement and sacrifices I would not have made it. My loving husband, who’s been my biggest supporter, and my wonderful step-daughters, whom I have dragged to campus numerous times and have essentially lived in my shoes for six whole years. To you three, I credit your love and support for keeping me going amidst the most trying of times. To my advisors, Dr. Brand and Dr. Alper, it takes a tremendous amount of courage to take on an independent student such as myself. I came with a busy family life, work experience, ideas, and many interests. So much that early on Dr. Brand saw the need to help me stay focused and not diverge on side- projects. He took the risk of taking me on, without dedicated funding or a defined project. For years both of my advisors met with me in the best of times and the worst of times, helping to guide me, yet always giving me the liberty to follow my desired research and ambitions. The other three committee members were also extremely supportive, allowing me to stop in and discuss my project with them individually. I particularly want to thank Dr. Nobles, whom I spoke with on an almost weekly basis. v One of the best attributes of the campus lifestyle is the diverse community of friends and colleagues that can be developed. From my sustainably-minded political, engineering, and innovation-driven friends at Purdue, to my closest lunch, coffee, and happy-hour friends in the University of Texas community, I have been so fortunate to be in an environment that allows such diverse relationships and sharing of ideas. I am thankful for the camaraderie of Tinisha Hancock, Dr. Schonna Manning, and Dr. David Nobles in Dr. Brand’s lab. Also, the friendships and training I received working in the Lambowitz lab were instrumental early on, with Dr. Georg Mohr, his wife Dr. Sabine Mohr, and Marta Mastroianni being wonderful mentors. Finally, I once again want to thank my husband and step-daughters. They have kept my life balanced, offered new perspective, and allowed me to be a kid again when I needed it most. To my dear husband, I thank you for all that you do, out of love and commitment to me and your children. You are my best of friends and my strongest of allies. I am so fortunate to share this project, and this life journey, with you. vi Coordinated Response and Regulation of Carotenogenesis in Thermosynechococcus elongatus (BP-1): Implications for Commercial Application Rebecca Anne Knight, Ph.D. The University of Texas at Austin, 2014 Supervisor: Jerry J. Brand Co-Supervisor: Hal S. Alper If small isoprenoids, the starting component of carotenoids, can be efficiently excreted from thermophilic cyanobacteria, they could help satisfy the demand for sustainably produced hydrocarbons. This is the driving force behind wanting to understand the response and regulation of isoprenoid pathways to environmental stimuli in the thermophilic cyanobacterium, Thermosynechococcus elongatus, BP-1. The portion of the isoprenoid pathway studied here is the carotenoid pathway since these products are critical to adaptation and they encompass the largest pool of isoprenoid compounds in cyanobacteria. Although synthetic biology in cyanboacteria has improved in recent years, there are many undiscovered metabolic complexities that make large-scale commercial production challenging. To address this need, I quantify and report for the first time metabolic shifts within the carotenoid pathway of BP-1 due to combined effects of temperature, pH and blue light. I show that metabolism shifts from the dicyclic into the monocyclic carotenoid pathway in response to pH, and that decreasing temperature drives flux into the end products of both pathways. Also, vii I report that the productivity of an uncommon carotenoid, 2-hydroxymyxol 2’- fucoside (HMF), approached 500 μg/L-day in cultures grown at 45 °C, high light intensity, and pH 8. In order to further elucidate these responses, I analyzed 42 RNAseq samples taken over time of BP-1 induced by cold and heat stress and compared these results to metabolomics data. I showed that crtR and crtG, two central carotenogenesis genes, are transcriptionally controlled and used weighted gene co-expression network analysis (WGCNA) to determine eight separate co-expressed modules of biological significance. Among the co- regulated heat response and cold response genes there were three and five non- coding RNA, respectively, providing targets for future investigation. Using subtractive genomics and transcriptional data I narrowed the potential missing steps of the myxol pathway in cyanobacteria to seven unknown BP-1 genes, two of which were confirmed not to be involved in the missing step(s). Finally, by generating a ΔcrtG mutant and testing it under different environmental parameters, I showed that HMF does not protect against high pH or low temperature (despite up-regulation at these conditions), and that CrtG has a higher affinity for monocyclic than dicyclic carotenoids. viii Table of Contents List of Tables ........................................................................................... xiv List of Figures ........................................................................................... xv List of Abbreviations .................................................................................. xx Chapter 1: Introduction .............................................................................. 1 1.1 Utilization of cyanobacteria as microbial cell factories ................. 1 1.1.1 Cyanobacteria structure .................................................... 1 1.1.2 Exploitation of cyanobacteria natural products ................. 3 1.1.3 Metabolic engineering of cyanobacteria ............................ 5 1.1.4 Thermosynechococcus elongatus, BP-1, and other thermophilic cyanobacteria ................................................ 7 1.2 isoprenoid products .................................................................... 10 1.2.1 Isoprenoid biosynthesis .................................................. 10 1.2.2 Carotenoid products and carotenogenesis ..................... 11 1.2.3 Isoprenoids for pharmaceutical and neutraceutical applications ...................................................................... 14 1.2.4 Isoprenoids for fuel and chemicals.................................. 16 1.2.5 Volatile isoprenoids ......................................................... 17 1.3 Motivation for isoprenoid research in BP-1 ................................ 19 1.3.1 BP-1 isoprenoid pathway ................................................ 19 1.3.2 Potential engineering points in the BP-1 isoprenoid pathway ......................................................................................... 19 1.3.3 Motivation for researching the coordinated response and regulation of carotenogenesis in BP-1 ............................. 21 1.3.3.1 Probing the effects of cultural triggers .................. 21 1.3.3.2 Probing the effects of environmental triggers ....... 23 1.4 Conclusions ............................................................................... 24 ix Chapter 2: Development of Equipment and Protocols for Growth, Screening, Isoprenoid Analysis, and RNA Extraction of BP-1 ........................... 27 2.1 Introduction ................................................................................ 27 2.2 Materials and Methods ............................................................... 27 2.2.1 Strain and growth media ................................................. 27 2.2.2 Gradient Heat Block ........................................................ 28 2.2.3 Custom photobioreactor .................................................. 28 2.2.4 Volatile Isoprenoid Analysis ............................................ 29 2.2.5 Carotenoid Analysis ........................................................ 30 2.2.6 RNA extraction ................................................................ 31 2.3 Results ....................................................................................... 32 2.3.1 BP-1 temperature growth characteristics ........................ 32 2.3.2 Photobioreactor construction