METABOLIC ENGINEERING OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS FOR THE RENEWABLE PRODUCTION OF BIOFUELS AND COMMODITY CHEMICALS by MATTHEW WILLIAM KELLER (Under the Direction of MICHAEL W. W. ADAMS) ABSTRACT The human race depends primarily on fossil fuels for the production of carbon based commodity chemicals and transportation fuels. Plant biomass is the leading feedstock in efforts to renewably produce liquid transportation fuels, but its use at large scales is inefficient and results in similar carbon emissions as traditional gasoline on an energy basis. Microorganisms, with their extremely diverse metabolic abilities, offer a wide range of alternative strategies for producing renewable fuels. One such strategy is to use a metabolically-engineered microbe to direct carbon dioxide into a carbon fixation cycle and directly into a fuel synthesis pathway. Our strategy is to engineer the hyperthermophile Pyrococcus furiosus (Topt 100°C) to express the 3- hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation, drive that pathway with molecular hydrogen via the native soluble hydrogenase, and direct the resulting acetyl-CoA to a bacterial butanol or ethanol fermentation pathway. The work here focuses on three parallel goals of the larger objective. The first is to demonstrate the first sub-pathway of the carbon fixation cycle, the second is to assemble a hybrid pathway for butanol production from acetyl-CoA, and the third is to explore multiple gene donors for ethanol production from acetyl-CoA. The first sub-pathway of the carbon fixation cycle generates the key intermediate 3- HP, which is a valuable plastics precursor that is currently produced from petroleum. The insertion of the five genes encoding this three enzyme pathway into P. furiosus resulted in the accumulation of 50 mg/L 3-HP in the medium at 75°C. Under the controlled conditions of an in vitro assay, 3-HP production was demonstrated to be dependent upon H2 and CO2. Since no single butanol pathway sufficiently thermophilic for expression in P. furiosus was known, an artificial pathway was assembled from multiple gene donors. Concentrated cell suspensions were incubated at 60°C and butanol (70 mg/L) was produced from maltose. Unlike 3-HP and butanol, a small amount of ethanol (40 mg/L) is natively produced by P. furiosus at low growth temperatures. The bifunctional AdhE is capable of the sequential reduction of acetyl-CoA to ethanol. Since acetyl-CoA is the intended link between carbon fixation and fuel synthesis, AdhE was inserted into P. furiosus to demonstrate ethanol production from acetyl-CoA. Eight thermophilic bacteria were used as gene donors and a maximum of 200 mg/L ethanol was produced by recombinant P. furiosus. This demonstrates a new functional route of ethanol production from acetyl-CoA that is directly compatible with the overall strategy. INDEX WORDS: Pyrococcus furiosus, electrofuels, biofuels, Metallosphaera sedula, carbon fixation, anaerobe, archaea, biotechnology, metabolic engineering, thermophile, Thermoanaerobacter METABOLIC ENGINEERING OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS FOR THE RENEWABLE PRODUCTION OF BIOFUELS AND COMMODITY CHEMICALS by MATTHEW WILLIAM KELLER B.S. College of Charleston, 2010 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 2016 © 2016 Matthew William Keller All Rights Reserved METABOLIC ENGINEERING OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS FOR THE RENEWABLE PRODUCTION OF BIOFUELS AND COMMODITY CHEMICALS by MATTHEW WILLIAM KELLER Major Professor: Michael W. W. Adams Committee: Claiborne V. Glover III Joy Doran-Peterson William N. Lanzilotta Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia May 2016 DEDICATION I dedicate this to my wife Jessica, and I thank her for her wonderful loving support. iv ACKNOWLEDGMENTS First I would like to thank my advisor Dr. Michael Adams for his support and guidance. I would also like to thank the Adams lab members and the ARPA-E team, especially Dr. Gerrit Schut and Dr. Gina Lipscomb, for helping to foster a fun, positive, and productive research experience. This work was supported by the US Department of Energy as part of the Electrofuels Project of ARPA-E (grant number DE-AR0000081) and the National Science Foundation (grant number 1021RR166824). We thank Brian Vaccaro, Dennis Phillips and Zhirui Wang of the University of Georgia for running the NMR, ESI-MS and GC-MS analyses of 3- hydroxypropionate, respectively. The GC-MS facility is supported in part by the Center for Plant and Microbial Complex Carbohydrates funded by the US Department of Energy (DE-FG02-93ER- 20097). v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .................................................................................................................... v LIST OF TABLES ................................................................................................................................. x LIST OF FIGURES ............................................................................................................................. xii CHAPTER 1 INTRODUCTION ................................................................................................................... 1 GOAL OF THIS WORK .............................................................................................. 1 The current need and status of renewable liquid transportation fuels ................. 2 Thermophilic Microorganisms .............................................................................. 13 Archaea ................................................................................................................. 16 Pyrococcus furiosus ............................................................................................... 17 Metallosphaera sedula and a thermophilic pathway for carbon fixation ............ 26 Thermoanaerobacter ethanolicus and a thermophilic pathway for fuel synthesis ............................................................................................................................... 29 vi Tables and Figures ................................................................................................ 31 2 EXPLOITING MICROBIAL HYPTERTHERMOPHILICITY TO PRODUCE AN INDUSTRIAL CHEMICAL, USING HYDROGEN AND CARBON DIOXIDE ................................................................ 59 Abstract ................................................................................................................. 60 Introduction .......................................................................................................... 61 Results and Discussion .......................................................................................... 63 Materials and Methods ......................................................................................... 69 Tables and Figures ................................................................................................ 75 3 A HYBRID SYNTHETIC PATHWAY FOR BUTANOL PRODUCTION BY A HYPERTHERMOPHILIC MICROBE ................................................................................................ 105 Abstract ............................................................................................................... 106 Introduction ........................................................................................................ 107 Materials and Methods ....................................................................................... 108 Results and Discussion ........................................................................................ 112 Conclusion ........................................................................................................... 117 Tables and Figures .............................................................................................. 119 vii 4 ETHANOL PRODUCTION FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS FURIOSUS BY EXPRESSION OF BACTERIAL BIFUNCTIONAL ALCOHOL DEHYDROGENASES ........ 135 Summary ............................................................................................................. 136 Introduction ........................................................................................................ 137 Results ................................................................................................................. 141 Discussion............................................................................................................ 146 Experimental Procedures .................................................................................... 154 Tables and Figures .............................................................................................. 159 5 EXPRESSION OF THE COMPLETE 3-HP/4-HB CYCLE FOR CARBON FIXATION IN PYROCOCCUS FURIOSUS ............................................................................................................. 173 Abstract ............................................................................................................... 174 Introduction ........................................................................................................ 175 Results ................................................................................................................. 176 Discussion...........................................................................................................