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UNIVERSITY of CALIFORNIA, IRVINE Metabolic Engineering Of UNIVERSITY OF CALIFORNIA, IRVINE Metabolic Engineering of Saccharomyces cerevisiae for the Enhanced Production of Biorenewable Fuels and Platform Chemicals DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemical and Biochemical Engineering By Christopher Tyler Leber Dissertation Committee: Professor Nancy A. Da Silva, Chair Professor Suzanne B. Sandmeyer Professor Szu-Wen Wang 2014 Chapter 2 © 2014 International Metabolic Engineering Society Chapter 3 © 2013 Wiley Periodicals, Inc. All other materials © 2014 Christopher Tyler Leber DEDICATION To My unbelievably supportive, caring, loving parents and wife. You guys are awesome. ii TABLE OF CONTENTS Page LIST OF FIGURES vi LIST OF TABLES xi LIST OF ABBREVIATIONS xii ACKNOWLEDGMENTS xiv CURRICULUM VITAE xv ABSTRACT OF THE DISSERTATION xvii CHAPTER 1: Introduction 1 Motivation 2 Objectives 10 Literature Overview 13 References 22 CHAPTER 2: Overproduction and Secretion of Free Fatty Acids 26 through Disrupted Neutral Lipid Recycle in Saccharomyces cerevisiae Abstract 27 Introduction 28 Materials and Methods 34 Results and Discussion 45 iii TABLE OF CONTENTS, CONT. Page Conclusions 66 Acknowledgements 67 References 68 CHAPTER 3: Engineering of Saccharomyces cerevisiae for the 72 Synthesis of Short Chain Fatty Acids Abstract 73 Introduction 74 Materials and Methods 79 Results and Discussion 91 Conclusions 107 Acknowledgements 108 References 109 CHAPTER 4: Metabolic Pathway and Enzyme Engineering for the 114 Improved Biosynthesis of Short Chain Fatty Acids in Saccharomyces cerevisiae Abstract 115 Introduction 117 Materials and Methods 121 Results and Discussion 137 Conclusions 171 Acknowledgements 173 References 174 iv TABLE OF CONTENTS, CONT. Page APPENDIX: 179 Select Gene and gBlock Sequences 179 Example FAME Standard Curves 189 Strain R1158-Tet07FAS2 Growth Curves 191 Escherichia coli FAS Background 192 Escherichia coli FAS Studies 195 v LIST OF FIGURES Page Figure 1.1 Flow-chart of manufacturing consumer goods from petroleum 3 Figure 1.2 Hydrocarbon biosynthesis pathways for fuels and chemicals 5 Figure 1.3 Chemical structures of biologically derived fuels and chemicals 6 Figure 1.4 Outline of chemical production utilizing renewable carbon feedstock 9 Figure 1.5 Structural organization of yeast fatty acid synthase 14 Figure 1.6 Structural organization of the mammalian fatty acid synthase 16 Figure 1.7 Overview of metabolic pathways involved in β-oxidation 19 Figure 1.8 Schematic overview of lipid droplet formation and degradation 21 Figure 2.1 Engineered pathways for the overproduction of free fatty acids 33 Figure 2.2 Schematic representation of vector pBTEF1 41 Figure 2.3 Effect of acyl-CoA synthetase disruption on fatty acid titers 47 Figure 2.4 Characteristic growth curve of acyl-CoA synthetase mutants 47 Figure 2.5 Effect of β-oxidation disruption on intracellular fatty acid titers 50 Figure 2.6 Effect of β-oxidation single knockouts on intracellular fatty acids 51 Figure 2.7 Effect of β-oxidation disruption on specific intracellular fatty acid titers 52 vi LIST OF FIGURES, CONT. Page Figure 2.8 Effect of interrupting acyl-CoA synthetase and β-oxidation activities 54 Figure 2.9 Characteristic growth curve of free fatty acid producing strains 55 Figure 2.10 Effect of overexpressing and disrupting the neutral lipid pathway 59 Figure 2.11 Free fatty acid levels and final cell densities of engineered strains 60 Figure 2.12 Extracellular free fatty acid chain length distribution profile 62 Figure 2.13 Visualization of extracellular free fatty acids 63 Figure 2.14 Confirmation of free fatty acid production using negative ion LC-MS 65 Figure 3.1 Confirmation of holo-hFAS formation by AcpS and Sfp using DTNB assay 93 Figure 3.2 Confirmation of hFAS activity by fatty acid synthesis using ADIFAB assay 94 Figure 3.3 Confirmation of holo-hFAS formation by growth complementation 96 Figure 3.4 Successful removal and inactivity of the TE domain 98 Figure 3.5 Confirmation of active short chain thioesterases expressed in yeast 99 Figure 3.6 Confirmation of fatty acid production using hFASΔTE mutant w/ ADIFAB 101 Figure 3.7 Intracellular and extracellular location of short chain fatty acids 102 Figure 3.8 Short chain fatty acids produced using thioesterases TEII and CpFatB1 104 vii LIST OF FIGURES, CONT. Page Figure 3.9 Short chain fatty acids produced using hFAS-TEII and hFAS-CpFatB1 106 Figure 4.1 Schematic representation of vector pXP842U-Bi 125 Figure 4.2 Full codon optimized holding vector assembly outline 128 Figure 4.3 PCR amplification and gene construction outline 129 Figure 4.4 Initial expression system evaluation 140 Figure 4.5 H. sapiens short chain TEII compared to the R. norvegicus TEII 142 Figure 4.6 H. sapiens PPT compared to the B. subtilis Sfp 144 Figure 4.7 Extracellular short chain fatty acid production using RTEII or HTEII 145 Figure 4.8 Short chain free fatty acid production using engineered strains 151 Figure 4.9 Schematic representation of CAI and CFD of gene hFAS-TEII 153 Figure 4.10 Schematic representation of CAI and CFD of gene PChFAS-TEII 154 Figure 4.11 Schematic representation of CAI and CFD of gene FChFAS-TEII 155 Figure 4.12 Extracellular short chain free fatty acid production using PChFAS-TEII 157 Figure 4.13 Specific intracellular fatty acid levels of Strain R1158-Tet07FAS2 160 viii LIST OF FIGURES, CONT. Page Figure 4.14 Extra- and intracellular fatty acid production using pADH1-FAS2 162 Figure 4.15 Extra- and intracellular fatty acid production using pPGK1-ACC1M 165 Figure 4.16 Extra- and intracellular fatty acid production using pADH2-ACC1M 167 Figure 4.17 Extra- and intracellular fatty acid levels from pathway interventions 170 Figure A.1 Native hFAS-TEII Gene Sequence 179 Figure A.2 Native hFAS-HTEII Gene Sequence 180 Figure A.3 First 834 bp codon optimized hFAS-TEII gene sequence 182 Figure A.4 Full ORF codon optimized hFAS-HTEII gene sequence 182 Figure A.5 Full ORF codon optimized hFAS-TEII gene sequence 184 Figure A.6 IDT DNA gBlock gene sequences 186 Figure A.7 Example of octanoic acid fatty acid methyl ester standard curve 189 Figure A.8 Example of decanoic acid fatty acid methyl ester standard curve 189 Figure A.9 Example of palmitic acid fatty acid methyl ester standard curve 190 Figure A.10 Example of stearic acid fatty acid methyl ester standard curve 190 Figure A.11 Characteristic growth curve of strain R1158-Tet07FAS2 – Dox 8 h 191 ix LIST OF FIGURES, CONT. Page Figure A.12 Characteristic growth curve of strain R1158-Tet07FAS2 – Dox 15 h 191 Figure A.13 Outline of the E. coli type II FAS pathway 193 Figure A.14 List of genes used in the E. coli type II fatty acid pathway 195 Figure A.15 List of cloned E. coli genes and vector descriptions 197 Figure A.16 Western blot and NADPH assay using E. coli proteins 198 x LIST OF TABLES Page Table 1.1 The six known acyl-CoA synthetases found in S. cerevisiae 17 Table 2.1 List of strains (constructed and used in Chapter 2) 37 Table 2.2 List of plasmids (constructed and used in Chapter 2) 38 Table 2.3 List of primer sequences (used in Chapter 2) 39 Table 2.4 List of gBlock sequences (used in Chapter 2) 40 Table 2.5 Intracellular, extracellular and total specific fatty acid levels 61 Table 3.1 List of strains (constructed and used in Chapter 3) 79 Table 3.2 List of plasmids (constructed and used in Chapter 3) 84 Table 3.3 List of primer sequences (used in Chapter 3) 85 Table 4.1 List of plasmids (constructed and used in Chapter 4) 130 Table 4.2 List of strains (constructed and used in Chapter 4) 133 Table 4.3 List of integrating primers (used in Chapter 4) 134 Table 4.4 List of cloning primers (used in Chapter 4) 135 Table 4.5 Select plasmid stability values as percent 140 xi LIST OF ABBREVIATIONS ACC American Chemistry Council ACP Acyl carrier protein ADIFAB Acrylodan-labeled Intestinal fatty acid binding AT Acetyl transferase CAI Codon adaptation index CFD Codon frequency distribution CN Copy number CoA Coenzyme-A DTNB 5,5’-Dithio-bis-2-nitrobenzoic acid FA Fatty acid FAB Fatty acid biosynthesis FAEE Fatty acid ethyl ester FAME Fatty acid methyl ester FAS Fatty acid synthase FFA Free fatty acid GC-GC-MS Gas-chromatography gas-chromatography mass spectrometry GC-MS Gas-chromatography mass spectrometry hFAS H. sapiens fatty acid synthase hPPT H. sapiens phosphopantetheine transferase xii LIST OF ABBREVIATIONS, CONT. hTEII H. sapiens thioesterase II IDT DNA Integrated DNA Technologies LB Luria-Bertani LCFA Long chain fatty acid LC-MS Liquid-chromatography mass spectrometry LD Lipid droplet MCFA Medium chain fatty acid MPT Malonyl/palmitoyl transacylase NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate OD Optical density PPT Phosphopantetheine transferase RTEII R. norvegicus TEII SCFA Short chain fatty acid SE Steryl ester TAG Triacylglyceride TE Thioesterase yFAS Yeast fatty acid synthase xiii ACKNOWLEDGEMENTS I am deeply thankful and grateful for the constant guidance and support my advisor, Professor Nancy Da Silva, has given me throughout my stay at UCI. She has always been extremely approachable, resourceful and accommodating and I’m appreciative to her for that. I’m also thankful to all my past and present committee members including Professor Suzanne Sandmeyer, Professor Szu-Wen Wang, Professor Hung Nguyen and Professor Shiou-Chuan Tsai for their constructive comments, insights and guidance during my Ph.D. tenure. I would also like to acknowledge Dr. John Greaves and Dr. Beniam Berhane at the Mass Spectrometry Facility at UCI for their guidance and troubleshooting with mass spectrometry. I have been extremely fortunate to have unbelievable hands-on research support from Brian Polson and Andres Aguirre.
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