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ABSTRACT FROCK, ANDREW DAVID. Functional Genomics

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ABSTRACT FROCK, ANDREW DAVID. Functional Genomics ABSTRACT FROCK, ANDREW DAVID. Functional Genomics Analysis of the Microbial Physiology and Ecology of Hyperthermophilic Thermotoga Species. (Under the direction of Dr. Robert Kelly.) Generation of renewable fuels through biological conversion of inexpensive carbohydrate feedstocks has been the focus of intense research, which has mostly involved metabolic engineering of model mesophilic microorganisms. However, hyperthermophilic (Topt ≥ 80°C) bacteria of the genus Thermotoga exhibit several desirable traits for a potential biofuel-producing host. They ferment a variety of carbohydrates to hydrogen with high yields approaching the theoretical limit for anaerobic metabolism of 4 mol H2/mol glucose. High temperature growth facilitates biomass feedstock degradation, decreases risk of contamination, and eliminates the reactor cooling costs associated with growth of mesophiles. To further understand the glycoside hydrolase and carbohydrate transporter inventory of these bacteria, four closely-related Thermotoga species were compared. T. maritima, T. neapolitana, T. petrophila, and T. sp. strain RQ2 share approximately 75% of their genomes, yet each species exhibited distinguishing features during growth on simple and complex carbohydrates that correlated with their genotypes. T. maritima and T. neapolitana displayed similar monosaccharide utilization profiles, with a strong preference for glucose and xylose. Only T. sp. strain RQ2 has a fructose specific phosphotransferase system (PTS), and it was the only species found to utilize fructose. T. petrophila consumes glucose to a lesser extent and up-regulates a transporter normally used for xylose (XylE1F1K1) to compensate for the absence of the primary glucose transporter in these species (XylE2F2K2). In some cases, the use of mixed cultures has been proposed to combine the complementary strengths of different microbes for the development of improved bioprocesses. For this reason, bioprocessing would benefit from better insight into the factors that trigger stationary phase and affect cell viability in both pure and mixed cultures. Analysis of population dynamics of mixed cultures of these Thermotoga species showed that they do not form stable co-cultures under the conditions tested. This illustrated that establishment of stable, synthetic, mixed cultures requires an existing (or engineered) dependence of one species upon another. To cast light on signaling and survival mechanisms, especially the likely involvement of poorly characterized small ORFs (< 100 AA), T. maritima was cultivated in pure culture and nutrient levels were varied by changing the yeast extract concentration. The transition from exponential phase to stationary phase on richer medium resulted in differential transcription of 359 genes involved in cell division, carbon metabolism, and biosynthesis of flagella, amino acids, and vitamins. Growth on leaner medium triggered a comparable number of genes (254) with similar functional roles, but also a ~3-fold lower cell density, with many cells becoming bloated and coccoidal upon entry into stationary phase. An ORF corresponding to a putative 31-amino acid peptide (TM1316) was dramatically up-regulated in stationary phase on the lean medium, such that it was the most highly transcribed gene in the transcriptome. TM1316 is associated with a unique region of the genome characterized by repetitive organization of toxin/antitoxin pairs, small hypothetical ORFs, radical SAM proteins, and ABC-2 transporters. The only functionally characterized loci with similar organization were found to be responsible for bacteriocin production in Bacillus species. TM1316, consistent with transcriptomic data, was produced at high levels during stationary phase and localized to the cell envelope. Taken together, this study highlights the fact that subtle genomic variations can have important consequences for industrially relevant phenotypes such as carbohydrate utilization. This insight supports the value of studies targeted at such variations and the value of high-throughput screening of even closely related strains. Furthermore, the high- level transcription of TM1316 and associated phenotype contributes to the body of literature on physiology outside of central metabolic processes, the understanding of which is paramount for the development of robust bioprocesses. Functional Genomics Analysis of the Microbial Physiology and Ecology of Hyperthermophilic Thermotoga Species by Andrew David Frock 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 2013 APPROVED BY: ___________________________ ___________________________ David F. Ollis Balaji M. Rao ___________________________ ___________________________ Amy M. Grunden Robert M. Kelly Chair of Advisory Committee BIOGRAPHY Andrew David Frock was born in Fletcher, NC on April 20, 1985. He grew up living on-site at Lutheridge Conference Center and Camp in Arden, NC. He graduated from T.C. Roberson High School in 2003. As a high school senior, he went on his first date with his future wife, Brittany Nicole Watson, on February 14, 2003. In 2007, he received a Bachelor of Science degree from Virginia Polytechnic Institute and State University (Blacksburg, VA) with a major in Biological Systems Engineering, a concentration in Bioprocess Engineering, and a minor in Chemistry. He then entered the graduate program in Chemical and Biomolecular Engineering at North Carolina State University. During this time, he happily married Brittany Watson Frock on October 3, 2009. Following completion of his doctoral degree in early 2013, he began work as a process engineer for the production of enzymes at Novozymes in Franklinton, NC. ii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ v LIST OF FIGURES ........................................................................................................ vii Chapter 1: The genus Thermotoga: Recent developments.............................................. 1 ABSTRACT ......................................................................................................... 2 INTRODUCTION ................................................................................................. 2 DIVERSITY WITHIN THE THERMOTOGA GENUS ............................................ 4 HYDROGEN PRODUCTION AND REDUCTANT DISPOSAL DURING CARBOHYDRATE FERMENTATION .................................................................. 5 RESPONSE TO OXYGEN EXPOSURE .............................................................. 8 MICROBIAL ECOLOGY OF THERMOTOGA SPECIES ...................................... 9 BIOCATALYSIS USING THERMOTOGA ENZYMES ........................................ 14 CONCLUSIONS ................................................................................................ 17 ACKNOWLEDGEMENTS .................................................................................. 17 REFERENCES .................................................................................................. 18 Chapter 2: Extreme thermophiles: Moving beyond single-enzyme biocatalysis ............. 36 ABSTRACT ....................................................................................................... 37 INTRODUCTION ............................................................................................... 37 ISOLATION OF NOVEL EXTREME THERMOPHILES ...................................... 39 GENOMICS OF EXTREME THERMOPHILES .................................................. 40 MOLECULAR GENETICS TOOLS FOR EXTREME THERMOPHILES ............. 42 BIOCATALYSIS AT ELEVATED TEMPERATURES .......................................... 44 METABOLIC ENGINEERING APPLICATIONS AND OPPORTUNITIES AT HIGH TEMPERATURES ................................................................................... 47 CONCLUSIONS ................................................................................................ 49 ACKNOWLEDGEMENTS .................................................................................. 50 REFERENCES .................................................................................................. 50 Chapter 3: Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases ..................................................... 67 ABSTRACT ....................................................................................................... 68 INTRODUCTION ............................................................................................... 69 MATERIALS AND METHODS ........................................................................... 72 RESULTS AND DISCUSSION........................................................................... 74 Genomic comparison of Thermotoga species .................................................... 74 Utilization of a mixture of monosaccharides ....................................................... 76 Design of the Thermotoga multispecies cDNA microarray ................................. 77 Monosaccharide and polysaccharide transcriptomes for Thermotoga species .......................................................................................... 77 Glucose transport .............................................................................................. 79 Regulation
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