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Understanding the Redox Metabolism of Pyrococcus Furiosus For UNDERSTANDING THE REDOX METABOLISM OF PYROCOCCUS FURIOSUS FOR EFFICIENT METABOLIC ENGINEERING by DIEP MINH NGOC NGUYEN (Under the Direction of Michael W. W. Adams) ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus has been a valuable source for thermostable and thermoactive enzymes for many biotechnological applications. With the availability of its genome sequence and a genetic system, P. furiosus became the subject for metabolic engineering projects to produce various alcohols and industrially relevant chemicals. Proofs of concepts have been established to show that P. furiosus could be engineered to produce ethanol, butanol and 3-hydroxypropionate. These fuel synthesis pathways were completed by insertion of one or more genes from foreign donors into the P. furiosus genome. Product synthesis was controlled in a temperature-dependent manner, in which the host was subjected to suboptimal growth temperatures to induce product formation. However, improvements in yield are necessary to advance P. furiosus as a platform relevant for industrial applications. To optimize yield in the current engineered P. furiosus strains, a deeper understanding of the electron metabolism at the suboptimal temperatures and knowledge of the host’s redox flux will be beneficial to redirect carbon and electron flow toward desired products. In this study, at temperature ranging from 70 to 80C, P. furiosus was shown to produce acetoin as a side-product of the biosynthesis of the branched chain amino acids. This work established that another industrially relevant compound could be produced from this organism. Additionally, we also were able to redirect carbon from acetoin toward ethanol production by deleting the gene encoding for an acetolactate synthase (ALS). Deletion of ALS increased the ethanol yield by 50% in the ethanol producing strain. This result implied that the glycolytic enzyme pyruvate ferredoxin oxidoreductase (POR) was most likely to be the bottleneck step in the current pathways for ethanol production. The second aspect of this study focuses on how electron flow is regulated in P. furiosus. We investigated the physiological functions of the two ferredoxin:NADP+ oxidoreductases, NfnI and Xfn. The bifurcating NfnI is responsible for NADPH generation by utilizing both reduced ferredoxin and NADH as electron donors. Xfn harbors a similar physiological function to NfnI, however, via a different mechanism. Xfn does not catalyze the same reaction as NfnI and whether it is another type of electron bifurcation is to be determined. The mechanism by which NfnI utilizes reduced ferredoxin and NADH to generate NADPH involves novel flavin and iron sulfur cluster biochemistry and understanding it in detail will not only be beneficial to control NfnI catalysis in metabolically engineered strains, but also provide insights into designing synthetic enzymes or bioinspired catalysts. INDEX WORDS: Pyrococcus furiosus, archaea, anaerobe, hyperthermophile, biotechnology, metabolic engineering, electron bifurcation, biofuels UNDERSTANDING THE REDOX METABOLISM OF PYROCOCCUS FURIOSUS FOR EFFICIENT METABOLIC ENGINEERING by DIEP MINH NGOC NGUYEN BS, Virginia Polytechnic Institute and State University, 2013 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2018 © 2018 Diep Minh Ngoc Nguyen All Rights Reserved UNDERSTANDING THE REDOX METABOLISM OF PYROCOCCUS FURIOSUS FOR EFFICIENT METABOLIC ENGINEERING by DIEP MINH NGOC NGUYEN Major Professor: Michael W. W. Adams Committee: William Lanzilotta Yajun Yan Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia August 2018 DEDICATION To my mother who loves and supports me unconditionally, who always believes in the choice I make in this life. There are things she wished she could have done differently, I hope that raising me to the person I am today is not one of those. iv ACKNOWLEDGEMENTS There are many who have helped me through my development from a mere student into a scientist. Throughout the 24 years of being students, I have encountered the right persons that helped and showed me the way. Special thanks go to Dr. Michael W. W. Adams for his support, oversight, and insight throughout my graduate school career. I also would like to thank my committee members, Dr. William Lanzilotta and Dr. Yajun Yan for their help and advice during my committee meetings. Big thanks and appreciation go to Dr. Gina Lipscomb and Dr. Gerrit Schut, my two special mentors. They have worked with me closely on my research projects, taught me the knowledge I need and above all, became friends who also support me emotionally. It was my luck to be able to work under their wings. Moreover, I have to thank the entire Adams lab, especially my manager Farris Poole, for their individual help, technical and emotional support. They have become friends that I can count on and enjoy working together. I would like to express my gratitude to Dr. Timothy Larson, my undergraduate advisor, who first taught me and showed me that I could be a scientist. For me to be who I am today, my deepest thanks and appreciation to my mother-Tuyet Thi Nguyen, my father-Hanh Minh Nguyen, my step-father-Tuan Anh Nguyen, my sister-Tram Minh Bao Nguyen and my fiancée-Arthur Boon Teck Ong for their unconditional love and support. Without them, I could have lost my way. And lastly, to my pet Daiki, he has worked very hard to keep me sane during my graduate school career. v “The only way of finding the limits of the possible is going beyond them into the impossible” -Arthur C. Clarke- vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 1.1 Introduction to archaea ......................................................................................1 1.2 Pyrococcus furiosus and its metabolism ............................................................3 1.3 Electron bifurcation .........................................................................................12 1.4 Research objectives ..........................................................................................17 1.5 Tables and figures ............................................................................................19 2 TEMPERATURE-DEPENDENT ACETOIN PRODUCTION BY PYROCOCCUS FURIOSUS IS CATALYZED BY A BIOSYNTHETIC ACETOLACTATE SYNTHASE AND ITS DELETION IMPROVES ETHANOL PRODUCTION .......37 2.1 Introduction ......................................................................................................39 2.2 Materials and methods .....................................................................................41 2.3 Results and discussion .....................................................................................46 2.4 Conclusion .......................................................................................................55 2.5 Figures..............................................................................................................56 vii 3 TWO FUNCTIONALLY DISTINCT NADP+-DEPENDENT FERREDOXIN OXIDOREDUCTASES MAINTAIN THE PRIMARY REDOX BALANCE OF PYROCOCCUS FURIOSUS ..................................................................................76 3.1 Introduction ......................................................................................................78 3.2 Experimental procedures .................................................................................81 3.3 Results and discussion .....................................................................................91 3.4 Tables and figures ..........................................................................................106 4 THE BIFURCATING NFN OF PYROCOCCUS FURIOSUS: A SIMPLE MODEL TO DETERMINE THE MECHANISM OF FLAVIN-BASED ELECTRON BIFURCATION .........................................................................................................150 4.1 The bifurcating center of P. furiosus NfnI: L-FAD .......................................151 4.2 The high potential electron transfer pathway.................................................153 4.3 The low potential electron transfer pathway ..................................................154 4.4 Protein dynamics and the effect of cluster-ligands on bifurcating activity ...155 4.5 Figures............................................................................................................160 5 DISCUSSION AND CONCLUSION........................................................................178 5.1 Optimizing P. furiosus for biofuel and chemical production ........................180 5.2 NfnI as model for an efficient bioinspired catalyst ........................................183 5.3 Conclusion and outlook .................................................................................189 5.4 Figures............................................................................................................191 REFERENCES ............................................................................................................................197
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