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Signature Redacted Signature of Author Biosensor-based Strategies for Improving Pathway Production in Escherichia coli MASSACHUSETTS INSTITUTE OF TECHNOLOGY By MAY 23 2019 Stephanie J. Doong LIBRARIES ARCHIVES B.S. Chemical Engineering Stanford University, 2014 Submitted to the Department of Chemical Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2019 2019 Massachusetts Institute of Technology. All rights reserved Signature redacted Signature of Author ............................................................ (1/ Stephanie J. Doong Department of Chemical Engineering May 7, 2019 Certified by ................................................................. Signature redacted 61 Kristala L. J. Prather Professor of Chemical Engineering Thesis Supervisor Signature redacted Accepted by.......................................................... Patrick S. Doyle Professor of Chemical Engineering Chairman, Committee for Graduate Students 1 2 Biosensor-based Strategies for Improving Pathway Production in Escherichia coli By Stephanie J. Doong Submitted to the Department of Chemical Engineering on May 7, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemical Engineering Abstract Microbial production of chemicals and fuels is an attractive renewable alternative to petroleum- based processes. D-glucaric acid, a Department of Energy top value-added chemical from biomass, is a precursor to polymers such as nylons and used in detergents. An engineered metabolic pathway requiring three heterologous enzymes to convert glucose into glucaric acid in Escherichia coli was previously demonstrated by the Prather lab. Glucaric acid production has been shown to be limited by the two downstream enzymes myo-inositol-1-phosphate synthase (MIPS) and myo-inositol oxygenase (MIOX). This work develops and deploys a biosensor that recognizes a pathway intermediate in order to overcome both limitations. A biosensor for myo-inositol (MI) was developed using the transcriptional regulator IpsA from the organism Corynebacterium glutamicum. A hybrid promoter was designed to enable function in the desired host organism E. coli. The modular design of the biosensor permitted the behavior and Ml dose response to be adjusted for the pathway applications. The myo-inositol biosensor was used to regulate expression of Miox, the enzyme that consumes myo-inositol, such that Miox was transcribed only in the presence of its substrate. Controlled expression of Miox led to a 2.5-fold increase in glucaric acid titer compared to the static case where Miox was constitutively expressed. This dynamic regulation scheme was then paired with a system that dynamically knocked down glycolysis, which independently improved glucaric acid production by relieving competition of glycolysis with MIPS, the first pathway enzyme. The layered dynamic regulation scheme improved glucaric acid production by up to 9-fold. Next, the biosensor was used as a high-throughput screen for mutants of MIPS generated by directed evolution. The biosensor enabled a large library of MIPS to be screened by fluorescence- activated cell sorting (FACS). The screen identified MIPS mutants with up to 20% improvement in myo-inositol production. This work used a biosensor to tackle two pathway limitations and improve glucaric acid production, showcasing the biosensor as a powerful metabolic engineering tool. 3 4 Acknowledgements Many thanks to Kris for your guidance, mentorship, and excitement about science over these past few years. I left every meeting feeling motivated, no matter how unprepared I felt before or how awful the results looked. Thank you for maintaining that positive attitude! I would also like to thank my committee members Professors Hadley Sikes and Tim Lu for their support. To all the members of the Prather lab past and present - thank you for making the lab a friendly and fun place to be. Thank you to Kat and Dity for welcoming me to the group and training me. Many thanks to Sue Zanne for all of your pro tips and assistance even after you went on to do bigger and better things. To Jason, a huge thank you for your expertise, experience, and the time you took to troubleshoot many experiments with me. Thank you to Lisa for teaching me about glucaric acid, going on adventures with me, and commiserating about being an adult. Many thanks to Kevin and Jennifer, who brought new energy and enthusiasm to the lab when my motivation was at an all-time low and for organizing team Phage Attack. Thank you to my undergraduate student Pep, who stuck around despite your disgust at the smell of autoclaved LB and multiple experiments gone awry. Many thanks to all the musicians and groups I have had the pleasure to play with. MITSO, Mercury, and Du Bois Orchestras, thank you for giving me the opportunity to play and grow musically. Thank you to the "Fish Heads" quintet (Anne, Isabelle, Eva, and Alan) for very entertaining rehearsals. To Carina, Roger, and Hoon, thank you for welcoming me to the group. Thank you to my teacher, Sarita, for believing in me, helping developing my sound, and improving my violin playing. To those of you who went to practice school with me, it is not a small achievement that we are still friends! Thanks for being team players as colleagues, great travel companions, and very cool people. Special thanks to Sarah for being a wonderful roommate. Thank you to Team Celery - I would not have survived first semester without you. I am so lucky to have an amazing group of friends. Thank you to my roommate, Christy, for listening to me complain about my day, fabulous wine nights, and for always telling me to use a larger pot when cooking. To Shuting, thank you for your sense of humor and always staying calm through any situation. Thank you to Miao, yeah buddy, for making everything seem like it "ain't nothing but a peanut". Thank you to Mike, my first friend at MIT, for enjoying horror movies and climbing with me. German, thank you for singing with me and encouraging me to get out, see concerts, and have fun. Max, thank you for eating food with me, coming to my concerts (my biggest fan!), and for always listening. To my family - thank you for all your support. Many thanks to my brother Justin for being my adviser on all things tech and for very interesting conversations. Thank you to my parents for living the "graduate student lifestyle" so we could attend college. And I very much appreciate you taking us on amazing trips all around the world. It has been a challenging journey, but I have truly enjoyed it, and I've also laughed quite a bit along the way. Stephanie Doong 5 6 Contents 1. Introduction ....................................................................................................................... 15 1.1 Engineering Organism s............................................................................................... 16 1.2 Biosensors as Tools for M etabolic Engineering ......................................................... 19 1.3 Dynam ic Regulation Strategies for Pathw ay Im provem ent....................................... 22 1.4 High-Throughput Screening of Directed Evolution Libraries ...................................... 26 1.5 D-Glucaric Acid Pathway ............................................................................................ 30 1.6 Thesis Objectives ............................................................................................................ 33 1.7 Thesis Organization ........................................................................................................ 33 2. m yo-Inositol Biosensor Developm ent .............................................................................. 35 2.1 Introduction.................................................................................................................... 37 2.2 M aterials and M ethods ............................................................................................. 39 2.2.1 M l Sensor Plasm id Construction ......................................................................... 39 2.2.2 Fluorescence M easurem ents............................................................................... 41 2.2.3 M I Ferm entation ................................................................................................ 42 2.2.4 M I Quantification ................................................................................................ 42 2.3 Results and Discussion ................................................................................................ 45 2.3.1 Construction and Characterization of the myo-Inositol Biosensor ..................... 45 7 2.4 Conclusions..................................................................................................................... 52 3. Dynam ic Regulation of M IOX............................................................................................ 53 3.1 Introduction.................................................................................................................... 55 3.2 M aterials and M ethods ............................................................................................... 59 3.2.1 Glucaric Acid Pathw ay Plasm id Construction ...................................................... 59 3.2.2 Strain Construction .............................................................................................. 61 3.2.3 Glucaric acid Ferm entation...............................................................................
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