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Table 4.3 Enzyme Activities and Glycerol Utilization and Ethanol Synthesis Fluxes for Wild-Type MG1655 and Strains Overexpressing Glycerol Utilization Enzymes

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Table 4.3 Enzyme Activities and Glycerol Utilization and Ethanol Synthesis Fluxes for Wild-Type MG1655 and Strains Overexpressing Glycerol Utilization Enzymes Abstract Understanding Fermentative Glycerol Metabolism and its Application for the Production of Fuels and Chemicals by James M. Clomburg Due to its availability, low-price, and higher degree of reduction than lignocellulosic sugars, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. However, this high degree of reduction of carbon atoms in glycerol also results in significant challenges in regard to its utilization under fermentative conditions. Therefore, in order to unlock the full potential of microorganisms for the fermentative conversion of glycerol into fuels and chemicals, a detailed understanding of the anaerobic fermentation of glycerol is required. The work presented here highlights a comprehensive experimental investigation into fermentative glycerol metabolism in Escherichia coli, which has elucidated several key pathways and mechanisms. The activity of both the fermentative and respiratory glycerol dissimilation pathways was found to be important for maximum glycerol utilization, a consequence of the metabolic cycle and downstream effects created by the essential involvement of PEP-dependent dihydroxyacetone kinase (DHAK) in the fermentative glycerol dissimilation pathway. The decoupling of this cycle is of central importance during fermentative glycerol metabolism, and while multiple decoupling mechanisms were identified, their relative inefficiencies dictated not only their level of involvement, but also iii implicated the activity of other pathways/enzymes, including fumarate reductase and pyruvate kinase. The central role of the PEP-dependent DHAK, an enzyme whose transcription was found to be regulated by the cyclic adenosine monophosphate (cAMP) receptor protein (CRP)-cAMP complex, was also tied to the importance of multiple fructose 1,6-bisphosphotases (FBPases) encoded by fbp, glpX, and yggF. The activity of these FBPases, and as a result the levels of fructose 1,6-bisphosphate, a key regulatory compound, appear to also play a role in the involvement of several other enzymes during fermentative glycerol metabolism including PEP carboxykinase. Using this improved understanding of fermentative glycerol metabolism as a platform, E. coli has been engineered to produce high yields and titers of ethanol (19.8 g/L, 0.46 g/g), co-produced along with hydrogen, and 1,2-propanediol (5.6 g/L, 0.21 g/g) from glycerol, demonstrating its potential as a carbon source for the production of fuels and reduced chemicals. Acknowledgments First and foremost, I would like to express my deepest gratitude and thanks to my Ph.D. advisor Professor Ramon Gonzalez. I do not think I can overstate his contribution to both my research and continued development as a scientist and engineer. His enthusiasm and enjoyment for science and teaching is contagious and made my work enjoyable even during most difficult and frustrating periods of my research. I am truly indebted to him for all of his support and cannot thank him enough for offering me the chance to join his research group when I started at Rice. I would also like to thank all of the other members of the Gonzalez group over the years for their support and guidance, with special thanks to Dr. Syed Shams Yazdani for teaching me the majority of laboratory techniques required for my research when I first started. In addition, the teaching and guidance I received from other professors and members of the Rice community has been invaluable. My friends and family also deserve special recognition for their support during the pursuit of my degree. Lastly and most importantly, I would like to thank my parents for their love and support over the years, as without them none of this would have been possible. Contents Acknowledgments ..................................................................................................... iv Contents .................................................................................................................... v List of Figures ........................................................................................................... vii List of Tables .............................................................................................................. x Introduction ............................................................................................................... 1 Advantages and Challenges of the Use of Glycerol as a Carbon Source for the Fermentative Production of Fuels and Chemicals ...................................................... 13 2.1. Efficient production of reduced chemicals and fuels ............................................. 14 2.2. Fermentative utilization of glycerol by microorganisms ........................................ 16 2.3. Metabolic models for the fermentative utilization of glycerol in microorganisms 19 2.3.1. 1,3-Propanediol dependent model ................................................................. 20 2.3.2. 1,2-Propanediol-ethanol dependent model .................................................... 22 2.3.3. Other models for the fermentative utilization of glycerol .............................. 24 2.4. Glycerol metabolism in Escherichia coli ................................................................. 26 2.4.1. Respiratory metabolism of glycerol ................................................................. 26 2.4.2. Fermentative metabolism of glycerol ............................................................. 28 2.4.2.1. Environmental factors influencing glycerol fermentation ........................ 29 2.4.2.2. Proposed model for glycerol fermentation by Escherichia coli ................ 32 Materials and Methods ............................................................................................ 40 3.1. Strains, plasmids, and genetic methods................................................................. 40 3.2. Culture medium and cultivation conditions ........................................................... 44 3.3. Analytical methods ................................................................................................. 47 3.4. Enzyme assays ........................................................................................................ 48 3.5. Calculation of fermentation parameters ............................................................... 52 3.6. in silico Metabolic Flux Analysis ............................................................................. 52 On the Pathways and Regulation of Fermentative Glycerol Metabolism in Escherichia coli ........................................................................................................................... 54 4.1. in silico Metabolic Flux Analysis of fermentative glycerol metabolism ................. 57 vi 4.2. Experimental investigation into fermentative glycerol metabolism in Escherichia coli ................................................................................................................................. 61 4.2.1. Glycerol transport and dissimilation ............................................................... 63 4.2.2. Conversion of 3-carbon intermediates into 6-carbon intermediates ............. 71 4.2.3. PEP/Pyruvate nodes and carboxylating enzymes ............................................ 80 4.2.4. Pathways and mechanisms ensuring the availability of 3-carbon intermediates ................................................................................................................................... 86 4.2.5. Regulation of fermentative glycerol metabolism ............................................ 93 4.3. Kinetics of fermentative glycerol utilization ........................................................ 100 4.4. Improved model for fermentative glycerol metabolism in Escherichia coli ........ 106 Metabolic Engineering of Escherichia coli for the Efficient Production of Reduced Chemicals and Fuels from Glycerol ......................................................................... 113 5.1. Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol ............................................................................................................... 115 5.1.1. Proposed metabolic engineering strategies for the conversion of glycerol into 1,2-PDO .................................................................................................................... 117 5.1.2. Engineering of 1,2-propanediol synthesis pathways .................................... 120 5.1.3. Manipulation of glycerol utilization pathways .............................................. 124 5.1.4. Engineering of major fermentation pathways .............................................. 127 5.1.5. Kinetics of glycerol consumptions and product formation in E. coli strain engineered for 1,2-PDO production ........................................................................ 132 5.2. Efficient co-production of ethanol and hydrogen from glycerol waste streams . 139 Conclusions and Future Work ................................................................................. 153 References ............................................................................................................. 162 Appendix................................................................................................................ 178 List of Figures Figure 1.1 Oils, fats, biodiesel, and glycerol ................................................................... 4 Figure 1.2 Ethanol
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