ABSTRACT STRAUB, CHRISTOPHER THOMAS. Metabolic Engineering of Extreme Thermophiles for Conversion of Renewable Feedstocks to Industrial Chemicals (Under the direction of Dr. Robert M. Kelly.) Extremely thermophilic microorganisms (Topt 70°C) challenge assumptions about the origins and limits of life. The diverse and specialized biological machinery, mechanisms, and systems utilized by these microorganisms ensure that these bacteria and archaea thrive in extreme thermal environments. These facets can be exploited for contributions to biotechnology in the pursuit of renewable, sustainable, and environmentally compatible solutions to needs for energy and materials. Caldicellulosiruptor bescii is an anaerobic bacterium that was isolated from thermal hot springs. It grows optimally at 78°C (172°F) on plant matter that has washed into the hot springs in which it resides. C. bescii natively deconstructs and ferments the cellulose and hemi-cellulose primarily to acetate, CO2 and H2. C. bescii has previously been shown to utilize approximately 30% of the available carbohydrate content in biomass, limited by the complex network of lignocellulosic biomass. However, by implementation of a mild sodium hydroxide pretreatment, C. bescii metabolized 70% of the carbohydrate content of pretreated switchgrass. Wild-type and transgenic black cottonwood (Populus trichocarpa) were also evaluated as feedstocks for C. bescii conversion. With milled wild-type poplar, C. bescii converted only 25% of the carbohydrate content, in contrast to nearly 90% of the carbohydrate content of a low lignin transgenic line created by down-regulation of the coumarate-3-hydroxylase (C3H3) gene. Furthermore, when entire stem samples of the transgenic line were utilized without milling, C. bescii solubilized over 50% of the feedstock, which has important biotechnological and economic consequences. The native C. bescii fermentation products have little economic value. By re-engineering the organism’s metabolism, the fermentation can be directed toward chemicals of value. Thermophilic enzymes capable of producing acetone at 70°C in vitro were identified, expressed, and characterized. These enzymes were expressed in a metabolically engineered strain of C. bescii, producing acetone at titers up to 4.5 mM (0.26 g/L), thereby providing a platform strain for further optimization. An engineering analysis illustrated the process benefits of producing volatile compounds, such as acetone or ethanol in extreme thermophiles, exploiting product volatility to facilitate separation. Pyrococcus furious is found in ocean volcanic vents, rich in sulfur, hydrogen gas, carbon monoxide, and carbon dioxide. Optimally growing at 100°C (212°F), P. furiosus metabolizes the most abundant organic compounds found in the ocean - laminarin from seaweed, chitin from the exoskeletons of arthropods, and peptides from deceased animals to name a few. As with C. bescii, P. furiosus has adapted to thrive by producing primarily acetate, H2 and CO2. Its central glycolytic metabolism differs somewhat from the canonical Embden-Meyerhof pathway which affects its suitability as a metabolic engineering platform for production of bio-based chemicals. Engineered strains were generated that utilized either ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase (Gapor) or non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (Gapn) for a key step in central glycolysis to examine how this effects the resulting fermentation profile. In addition, an NADPH-dependent primary alcohol dehydrogenase was identified in Caldanaerobacter subterraneus and further characterized. Insertion of this gene alone allowed P. furiosus to produce ethanol at high yield at 80°C. Its insertion into various strains demonstrated the effects of modification of central glycolysis on a heterologous pathway. Insights into the glycolytic pathway in engineered strains further elucidated the potential for P. furiosus as a metabolic engineering platform. These two extreme thermophiles provide a strategic set of native capabilities to degrade and metabolize nature’s renewable resources. Metabolic engineering to significantly alter their fermentation profiles has resulted in both fundamental understandings of key factors affecting their performance and demonstrated improvements toward this goal. Further work to optimize pathways to improve yield and titers and to maintain metabolic activity are the two major challenges remaining for these two extreme thermophiles to reach their biotechnological potential. © Copyright 2019 by Christopher T. Straub All Rights Reserved Metabolic Engineering of Extreme Thermophiles for Conversion of Renewable Feedstocks to Industrial Chemicals By Christopher Thomas Straub A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical Engineering Raleigh, North Carolina 2019 APPROVED BY: Dr. Robert M. Kelly Dr. Rodolphe Barrangou Committee Chair Dr. Carlos Goller Dr. Nathan Crook DEDICATION To my high school chemistry teacher, Mrs. Eleanor Herbstritt, and my high school physics teacher, Mr. James Lallman. ii BIOGRAPHY Christopher Thomas Straub was born and raised in Saint Marys, Pennsylvania, as were both of his parents, all four of his grandparents, and all eight of his great-grandparents. He attended Catholic schools in Saint Marys and graduated from Elk County Catholic High School in 2003. A child of two Penn State graduates and a lifelong fan of the Nittany Lions, Chris pursued his undergraduate studies at The Pennsylvania State University – University Park where he obtained his degree (BS) in Chemical Engineering. Following graduation, he moved to Delaware where he worked for the E.I. du Pont de Nemours Company in manufacturing and technical roles in the fluorine chemicals and polymers division. After a seven year career at Dupont, Chris decided to pursue a PhD and began his studies at the North Carolina State University in August 2014. He spent the following years engineering extreme thermophiles in order to advance the field of renewable biofuels and biochemicals under the advisement of Dr. Robert M. Kelly. Following receipt of his PhD, Chris plans to return to industry with a focus on process development to bring innovative ideas to industrial scale. iii ACKNOWLEDGEMENTS There are many people to thank for their help along the way. I would like to thank my family and friends for their support and encouragement. I would like to extend my sincere graditute to all of the undergraduates who worked with me during my time here. Your assistance was incredibly value and the mentorship experience was rewarding. I owe a great deal of gratitude to my graduate advisor, Dr. Robert M. Kelly, and all of the members of the Kelly Lab at NC State. For all those who helped me along the way, no one put more trust in me than the taxpayers of the United States. More than $500,000 of taxpayer funds went toward my tuition, stipend, student health insurance, lab supplies, travel, and other expenses. I will work each and every day to demonstrate that not only was I a worthwhile venture, but also that the public funding of science is a high-return investment for our citizens, industries, markets, and the entirety of our economy. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x CHAPTER 1: Extremely Thermophilic Energy Metabolisms: Biotechnological Prospects . 1 Abstract ..................................................................................................................................... 2 Introduction ............................................................................................................................... 2 Genetics..................................................................................................................................... 3 Carbon Dioxide Fixation........................................................................................................... 3 Carbon Monoxide ..................................................................................................................... 4 Alcohols .................................................................................................................................... 5 Impact of environmental factors on host metabolism ............................................................... 6 Chemolithotrophy ..................................................................................................................... 7 Hydrogen/Hydrogenases/Bifurcation ....................................................................................... 8 Lignocellullose & Biomass Degradation ................................................................................ 10 Conclusions ............................................................................................................................. 10 Acknowledgments................................................................................................................... 11 References ............................................................................................................................... 12 CHAPTER 2: Biotechnology of Extremely Thermophilic Archaea......................................