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Engineering Zymomonas Mobilis for the Production of Biofuels and Other Value-Added Products

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© 2015 Kori Lynn Dunn ENGINEERING ZYMOMONAS MOBILIS FOR THE PRODUCTION OF BIOFUELS AND OTHER VALUE-ADDED PRODUCTS BY KORI LYNN DUNN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering in the Graduate College of the University of Illinois at Urbana-Champaign, 2015 Urbana, Illinois Doctoral Committee: Associate Professor Christopher V. Rao, Chair Associate Professor Yong-Su Jin Associate Professor Hyunjoon Kong Associate Professor Mary Kraft Abstract Zymomonas mobilis is a promising organism for lignocellulosic biofuel production as it can efficiently produce ethanol from simple sugars using unique metabolic pathways. Z. mobilis displays what is known as the “uncoupled growth” phenomenon, meaning cells will rapidly convert sugars to ethanol regardless of their energy requirements for growth. This makes Z. mobilis attractive not only for ethanol production, but for alternative product synthesis as well. One limitation of Z. mobilis for cellulosic ethanol production is that this organism cannot natively ferment the pentose sugars, like xylose and arabinose, which are present in lignocellulosic hydrolysates. While it has been engineered to do so, the fermentation rates of these sugars are still extremely low. In this work, we have investigated sugar transport as a possible bottleneck in the fermentation of xylose by Z. mobilis. We showed that transport limits xylose fermentations in this organism, but only when the starting sugar concentration is high. To discern additional bottlenecks in pentose fermentations by Z. mobilis, we then used adaptation and high-throughput sequencing to pinpoint genetic mutations responsible for improved growth phenotypes on these sugars. We found that the transport of both xylose and arabinose through the native sugar transporter, Glf, limits pentose fermentations in Z. mobilis, thereby confirming our previous results. We also found that mutations in the AddB protein increase plasmid stability and can reduce cellular aggregation in these strains. Consistent with previous research, we found that reduced xylitol production improves xylose fermentations in Z. mobilis. We also found that increased transketolase activity and reduced glyceraldehyde-3-phosphate dehydrogenase activity improve arabinose fermentations in Z. mobilis. ii In order for Z. mobilis to prosper as an industrial host for alternative product synthesis, the genetic techniques utilized in this organism must be improved. Toward this goal, we have adapted the λ Red recombinase system for use in Z. mobilis. We have shown that this system increases the frequency of double crossover events for the purpose of constructing gene knockouts in the strain. We have also constructed an expression system for the type II CRISPR/CRISPR-associated (Cas) bacterial adaptive immunity system in Z. mobilis. We have shown that the Cas9 nuclease can be directed by small RNAs to target the Z. mobilis genome for the purpose of genome editing, and that this system can also likely be used to facilitate gene knockouts in this organism. Finally, toward improving heterologous protein production in Z. mobilis, we have constructed a constitutive promoter library that leads to a range of gene expression levels in the strain. Collectively, our results provide the framework for the development of an industrial production process utilizing Z. mobilis as the microbial host. iii Acknowledgements I am forever indebted to the many people who have helped me throughout my years in graduate school. First and foremost, I am especially grateful for my advisor, Dr. Christopher Rao. Thank you for being patient with me as I figured out Zymomonas, for affording me the independence that I prefer, and for always trusting me to solve problems in my own way. I am leaving your lab today with a wealth of knowledge that I couldn’t have gotten under the direction of anyone else. It has been a privilege working with you. Thank you also to Dr. Yong-Su Jin, Dr. Mary Kraft, and Dr. Hyunjoon Kong for being on my committee and for providing me with valuable advice on my projects, and the Energy Biosciences Institute for funding my research. My days in lab were enjoyable thanks to the members of the Rao lab, past and present. Ahmet Badur, Santosh Koirala, and Shuyan Zhang, I couldn’t have asked for better people with whom to share all 6 years of my PhD. Thank you for always being on my side and for sharing my sense of humor. Thank you for appreciating the real me, and for making me feel like that person was someone special. To the many other members of the Rao lab, thank you for the frequent fun times and the acceptance. To my dearest friends outside of lab, Ritika Mohan and Dawn Eriksen, thank you for making the days less daunting. My years in graduate school will be remembered as some of the best of my life, thanks to all of you. To my sister, Briana, thank you for always being my biggest fan. Thank you for being by my side through it all. We made it this far together, and we will make it even further together, too. Nothing in my life means more to me than you. I don’t know what I would do without you. iv To my counterpart, Justin Bradley, thank you for moving to Illinois for me. Thank you for giving up your life to join mine. Thank you for making the most of the situation, for taking the opportunity to better yourself, and of course for never complaining when I had to work late. Thank you also to my family for making the drive to Illinois and for understanding that education is important. Thank you to my grandparents for the encouragement and for making me feel loved. Finally, thank you to my brothers, Evan and Nathan, for bringing me so much happiness in my life. I hope you will both read this document and aim to follow in my footsteps. v Table of Contents Chapter 1: Introduction ................................................................................................... 1 1.1 Motivation ................................................................................................................. 1 1.2 The physiology of Zymomonas mobilis .................................................................... 2 1.3 Genetically modifying Z. mobilis .............................................................................. 9 1.4 Z. mobilis for ethanol production from lignocellulosic biomass ............................. 12 1.5 Z. mobilis for alternative product synthesis ............................................................ 17 1.6 Conclusions ............................................................................................................. 21 1.7 Figures ..................................................................................................................... 23 Chapter 2: Expression of a xylose-specific transporter improves ethanol production in metabolically engineered Zymomonas mobilis ......................................................... 27 2.1 Introduction ............................................................................................................. 27 2.2 Materials and methods ............................................................................................ 28 2.3 Results ..................................................................................................................... 33 2.4 Discussion ............................................................................................................... 36 2.5 Tables and figures ................................................................................................... 39 Chapter 3: High-throughput sequencing reveals adaptation-induced mutations in pentose-fermenting strains of Zymomonas mobilis ...................................................... 44 3.1 Introduction ............................................................................................................. 44 3.2 Materials and methods ............................................................................................ 45 3.3 Results ..................................................................................................................... 56 3.4 Discussion ............................................................................................................... 63 3.5 Tables and figures ................................................................................................... 68 Chapter 4: Genetically modifying Z. mobilis using the λ Red recombinase system and a CRISPR-associated nuclease ............................................................................... 76 4.1 Introduction ............................................................................................................. 76 4.2 Materials and methods ............................................................................................ 80 4.3 Results ..................................................................................................................... 88 4.4 Discussion ............................................................................................................... 94 4.5 Tables and figures ................................................................................................. 100 Chapter 5: Development of a promoter library for use in Z. mobilis....................... 111 vi 5.1 Introduction ........................................................................................................... 111 5.2 Materials and methods .........................................................................................
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