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[Thesis Title Goes Here] METABOLIC ENGINEERING AND OMICS ANALYSIS OF AGROBACTERIUM SP. ATCC 31749 FOR OLIGOSACCHARIDE SYNTHESIS A Dissertation Presented to The Academic Faculty by Anne M. Ruffing In Partial Fulfillment of the Requirements for the Degree Ph.D. in Chemical Engineering in the School of Chemical & Biomolecular Engineering Georgia Institute of Technology May 2010 METABOLIC ENGINEERING AND OMICS ANALYSIS OF AGROBACTERIUM SP. ATCC 31749 FOR OLIGOSACCHARIDE SYNTHESIS Approved by: Dr. Rachel Ruizhen Chen Advisor Dr. Jim Spain School of Chemical & Biomolecular School of Civil & Environmental Engineering Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Martha Grover Dr. Donald Doyle School of Chemical & Biomolecular School of Chemistry & Biochemistry Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Mark Prausnitz School of Chemical & Biomolecular Engineering Georgia Institute of Technology Date Approved: January 12, 2010 ACKNOWLEDGEMENTS I am deeply indebted to those who have supported me throughout my time here at Georgia Tech. First, I wish to thank my advisor, Dr. Rachel Chen, for her support and guidance. Her ideas and insight were essential in the progression of this thesis project. I would like to thank my other committee members Dr. Martha Grover, Dr. Mark Prausnitz, Dr. Jim Spain, and Dr. Donald Doyle for their time and suggestions. Several others have directly contributed to the work presented in this dissertation, deserving both my thanks and acknowledgment for their contribution. Dr. Zichao Mao, a former postdoctoral fellow of the Chen group, contributed to the development of the LacNAc- producing biocatalyst by constructing the Agrobacterium expression vector, pBQ, and the recombinant plasmid, pKEL. We collaborated with Dr. Wei-Shou Hu and Marlene Castro at the University of Minnesota for the genome sequencing of ATCC 31749. Marlene ran the program for sequence assembly and assisted with gene prediction. I am grateful to Dr. Hyun-Dong Shin for sharing his knowledge of molecular biology and for his helpful suggestions regarding this thesis work. I also wish to thank all members of the Chen group throughout my years at Georgia Tech, particularly Shara McClendon, my officemate for the last 5 years, and Manoj Agrawal. I am fortunate to have an amazing group of people who have supported me throughout the many tribulations, setbacks, and triumphs of my graduate career. Thanks to my parents, Bob and Margie, who have driven to Atlanta numerous times over the years to help me move apartments, to celebrate birthdays, or just to make sure I take a break from the lab. I would also like to thank my siblings, Mark, Nick, and Kristen, for iv their support. And last, but certainly not least, I wish to thank my fiancé, Carter Dietz, for his friendship and encouragement, for making me laugh, and for the many lunch-time discussions trouble-shooting my research problems. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES xiii LIST OF FIGURES xv LIST OF ABBREVIATIONS xviii LIST OF GENE AND ENZYME NOMENCLATURE xxiii SUMMARY xxvi PREFACE xxviii CHAPTER 1 INTRODUCTION 1 1.1 Oligosaccharides 1 1.1.1 Oligosaccharide structure 1 1.1.2 Biological function 3 1.1.3 Medical applications 4 1.1.3.1 Oligosaccharide-based treatments for bacterial, 5 parasitic, and viral infections 1.1.3.2 Oligosaccharide-based treatments for cancer 6 1.2 Oligosaccharide Synthesis 8 1.2.1 Chemical synthesis 8 1.2.2 Biological synthesis 10 1.2.2.1 Enzymatic synthesis 10 1.2.2.1.1 Glycosidases 11 1.2.2.1.2 Glcosyltransferases 13 vi 1.2.2.2 Whole-cell synthesis 17 1.2.2.2.1 Metabolic engineering 18 1.2.2.2.2 Single-cell synthesis 20 1.2.2.2.3 Bacterial coupling 21 1.3 Agrobacterium sp. ATCC 31749 24 1.3.1 Phylogeny of the Agrobacterium genus 24 1.3.2 History of ATCC 31749 26 1.3.2.1 Curdlan polysaccharide 27 1.3.2.2 Curdlan production in ATCC 31749 29 1.4 Project Objectives 31 1.4.1 Metabolic engineering of ATCC 31749 for oligosaccharide 32 synthesis 1.4.2 What factors effect production of the curdlan polysaccharide in 32 ATCC 31749? 1.5 References 33 2 METABOLIC ENGINEERING OF AGROBACTERIUM SP. FOR 41 UDP-GALACTOSE REGENERATION AND LACNAC SYNTHESIS 2.1 Abstract 41 2.2 Introduction 42 2.3 Results 45 2.3.1 Engineering a UDP-galactose regeneration system for 45 disaccharide synthesis 2.3.2 Rifampicin effect and intracellular nucleotide concentrations 48 2.3.3 Product selectivity 51 2.3.4 Identification of oligosaccharide byproducts 54 2.4 Discussion and Conclusions 56 2.5 Materials and Experimental Methods 58 vii 2.5.1 Materials 58 2.5.2 Bacterial strains and plasmids 58 2.5.3 Construction of Agrobacterium sp. expression vector, pKEL 59 and analysis of GalE-LgtB fusion enzyme activity 2.5.4 LacNAc and lactose synthesis with LTU265 transformants 62 2.5.5 Analytical methods 62 2.5.5.1 Carbohydrate analysis 62 2.5.5.2 Nucleotide extraction and assay 63 2.5.5.3 β-Galactosidase assay 63 2.6 References 64 3 METABOLIC ENGINEERING OF AGROBACTERIUM SP. STRAIN 67 ATCC 31749 FOR PRODUCTION OF AN α-GAL EPITOPE 3.1 Abstract 67 3.2 Introduction 68 3.3 Results 73 3.3.1 α-Gal epitope synthesis in ATCC 31749/pBQET 73 3.3.2 Increased uptake of the acceptor, lactose 75 3.3.3 Curdlan synthase knockout for improved UDP-glucose 79 availability 3.4 Discussion and Conclusions 84 3.5 Materials and Experimental Methods 88 3.5.1 Materials 88 3.5.2 Bacterial strains and plasmids 88 3.5.3 Construction of pBQET and pBQETY for α-Gal epitope 90 synthesis 3.5.4 Gene knockout of curdlan synthase ( crdS ) in ATCC 31749 92 3.5.5 Cell growth and GalE:α1,3GalT enzyme activity assay 94 viii 3.5.6 Cell viability 94 3.5.7 α-Gal epitope synthesis 95 3.5.8 Analytical techniques 96 3.5.8.1 SDS-PAGE 96 3.5.8.2 Carbohydrate analysis 96 3.5.8.3 Intracellular lactose measurement 97 3.5.8.4 Curdlan detection 98 3.6 References 98 4 CITRATE STIMULATION OF OLIGOSACCHARIDE SYNTHESIS 102 IN METABOLICALLY ENGINEERED AGROBACTERIUM SP. 4.1 Abstract 102 4.2 Introduction 103 4.3 Results 105 4.3.1 Citrate stimulation of oligosaccharide synthesis 105 4.3.2 Mechanisms of citrate stimulation 106 4.3.2.1 Citrate as carbon source for energy production 106 4.3.2.2 Citrate as chelating agent 108 4.3.2.3 Citrate as buffer 110 4.3.3 Simultaneous uptake of multiple carbon sources 112 4.3.4 Metabolic flux analysis 113 4.3.5 Citrate effect unique to Agrobacterium sp. 119 4.4 Discussion and Conclusions 119 4.5 Materials and Experimental Methods 122 4.5.1 Materials 122 4.5.2 Bacterial strains 122 4.5.3 Oligosaccharide synthesis 122 ix 4.5.4 Analytical methods 123 4.5.4.1 Carbohydrate analysis 123 4.5.4.2 Citrate measurement 124 4.5.4.3 Acetate and glycerol measurement 124 4.5.4.4 Curdlan measurement 124 4.5.5 Metabolic flux analysis 125 4.6 References 125 5 GENOME SEQUENCING OF AGROBACTERIUM SP. ATCC 31749 129 5.1 Abstract 129 5.2 Introduction 129 5.3 Results 132 5.3.1 ATCC 31749 genome sequence and annotation 132 5.3.2 Phylogeny and comparative genomics of ATCC 31749 138 5.3.3 Genes relevant to curdlan synthesis and regulation 141 5.4 Discussion and Conclusions 145 5.5 Methods 145 5.5.1 16S rRNA sequencing and phylogentic tree construction 145 5.5.2 Genomic DNA isolation, sequencing, and annotation 146 5.5.3 Operon and metabolic pathway prediction 147 5.5.4 Crd and cel operon analysis 147 5.5.5 Comparative genomics 148 5.6 References 148 6 TRANSCRIPTOME ANALYSIS OF AGROBACTERIUM SP. 151 ATCC 31749 REVEALS GENES IMPORTANT FOR CURDLAN SYNTHESIS AND REGULATION 6.1 Abstract 151 x 6.2 Introduction 152 6.3 Results 153 6.3.1 Microarray analysis of ATCC 31749 at pH 7 vs. pH 5.5 153 6.3.2 Nitrogen-dependent microarray analysis of ATCC 31749 159 6.3.3 Gene knockouts identify genes influencing curdlan 164 production 6.3.3.1 Nitrogen-limited regulatory genes 164 6.3.3.2 Energy-related genes 168 6.3.3.3 GTP-derived second messengers 172 6.3.4 Analysis of possible mechanisms for the regulation of 175 curdlan synthesis 6.4 Discussion and Conclusions 185 6.5 Materials and Experimental Methods 187 6.5.1 Materials 187 6.5.2 Bacterial strains and plasmids 187 6.5.3 Fermentation for curdlan synthesis 188 6.5.4 RNA isolation and DNA microarray processing 190 6.5.5 Microarray data analysis 191 6.5.6 Gene knockouts 193 6.5.6.1 ATCC 31749 electrocompetent cell preparation 196 6.5.7 Growth and cell viability studies of gene KO mutants 196 6.5.8 Shake-flask curdlan synthesis for analysis of KO mutants 197 6.6 References 198 7 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK 202 7.1 Conclusions 202 7.1.1 Biocatalyst construction for oligosaccharide production 202 xi 7.1.2 Multiple factors influence curdlan and oligosaccharide 204 synthesis in ATCC 31749 7.2 Significant Contributions 208 7.3 Recommendations for Future Work 209 7.3.1 Biocatalyst development for improved oligosaccharide 210 production 7.3.1.1 Large-scale oligosaccharide synthesis 210 7.3.1.2 Targets for metabolic engineering 211 7.3.2 Determination of the mechanisms regulating curdlan 212 synthesis in ATCC 31749 7.4 References 214 APPENDIX A: Metabolic Flux Analysis 217 A.1 Reactions included in the metabolic network of 217 ATCC 31749/pKEL A.2 MATLAB code for metabolic flux analysis 218 APPENDIX B: Perl Scripts 222 B.1 blast_XML_extraction.pl 222 B.2 Pathologic_format.pl 228 B.3 matchandsort.pl 230 xii LIST OF TABLES Page Table 1.1: Changes in oligosaccharide expression in malignant tissues 7 Table 1.2: Commercially available carbohydrate-based treatments 8 Table 1.3: Bacterial glycosyltransferases 16 Table 2.1: Activities of β-1,4-galactosyltransferase and fusion enzyme in E.
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