Contents Preface vii List of Abbreviations xv Introduction1 Introductory Outlines 1 Metabolic and Cellular Engineering in the Context of Bioprocess Engineering 2 Tools for Metabolic and Cellular Engineering 3 Engineering Cells for Specific Biotransformations 5 Metabolic Areas that Have Been Subjected to MCE 8 From DNA Sequence to Biological Function 17 Temporal and Spatial Scaling in Cellular Processes 21 Scaling in Microbial and Biochemical Systems 22 Views of the Cell 24 Black and Grey Boxes: Levels of Description of Metabolic Behavior in Microorganisms 24 Transduction and Intracellular Signalling 29 Self-organized Emergent Phenomena 30 Homeodynamics and Coherence 34 Matter and Energy Balances 39 Mass Balance 39 General Formulation of Mass Balance 40 Integral and Differential Mass Balances 41 Growth Stoichiometry and Product Formation 42 Biomass and Product Yields 46 Electron Balance 47 Theoretical Oxygen Demand 48 Opening the"Black Box".Mass Balance as the Basis of Metabolic Flux Analysis 56 Energy Balance 63 Forms of Energy and Enthalpy 64 An Introduction to Metabolic and Cellular Engineering Calorimetric Studies of Energy Metabolism 67 Heat of Combustion 68 An Energetic View of Microbial Metabolism 73 Cell Growth and Metabolite Production.Basic Concepts 77 Microbial Growth under Steady and Balanced Conditions 77 Microbial Energetics under Steady State Conditions 84 Growth Kinetics under Steady State Conditions 85 The Dilution Rate 86 The Dilution Rate and Biomass Concentration 86 The Dilution Rate and the Growth-limiting Substrate Concentration 87 Biomass and Growth-limiting Substrate Concentration at the Steady State 89 Growth as a Balance of Fluxes 91 The Flux Coordination Hypothesis 93 Toward a Rational Design of Cells 96 Redirecting Central Metabolic Pathways under Kinetic or Thermodynamic Control 97 Thermodynamic or Kinetic Control of Flux under Steady State Conditions 100 Kinetic and Thermodynamic Limitations in Microbial Systems Case Studies 102 Saccharomyces cerevisiae102 Escherichia coli 105 Increasing Carbon Flow to Aromatic Biosynthesis in Escherichia coli 106 Methods of Quantitation of Cellular "Processes Performance” 111 Stoichiometry of Growth: The Equivalence between Biochemical Stoichiometries and Physiological Parameters 111 A General Formalism for Metabolic Flux Analysis 114 A Comparison between Different Methods of MFA 115 MFA Applied to Prokaryotic and Lower Eukaryotic Organisms 115 MFA as Applied to Studying the Performance of Mammalian Cells in Culture 118 Metabolic Fluxes during Balanced and Steady State Growth 119 Bioenergetic and Physiological Studies in Batch and Continuous Cultures.Genetic or Epigenetic Redirection of Metabolic Flux 121 Introduction of Heterologous Metabolic Pathways 121 Metabolic Engineering of Lactic Acid Bacteria for Optimising Essential Flavour Compounds Production 123 Metabolic Control Analysis 126 Summation and connectivity theorems 131 Control and Regulation 133 The Control of Metabolites Concentration 134 A Numerical Approach for Control Analysis of Metabolic Networks and Nonlinear Dynamics 135 The TDA Approach as Applied to the Rational Design of Microorganisms: Increase of Ethanol Production in Yeast 135 Phase I: Physiological, Metabolic and Bioenergetic Studies of Different Strains of S.cerevisiae 136 Phase II: Metabolic Control Analysis and Metabolic Flux Analysis of the Strain under the Conditions Defined in Phase I 137 Phases III and IV: To Obtain a Recombinant Yeast Strain with an Increased Dose of PFK, and to Assay the Engineered Strain in Chemostat Cultures under the Conditions Specified in Phase I 141 Appendix A 142 A Simplified Mathematical Model to Illustrate the Matrix Method of MCA 142 Appendix B 144 Conditions for Parameter Optimisation and Simulation of the Mathematical Model of Glycolysis 144 Dynamic Aspects of Bioprocess Behavior 145 Transient and Oscillatory States of Continuous Culture 145 Mathematical Model Building 145 Transfer-Function Analysis and Transient-Response Techniques 152 Theoretical Transient Response and Approach to Steady State 152 Transient Responses of Microbial Cultures to Perturbations of the Steady State 155 Dilution Rate 155 Feed Substrate Concentration 156 Growth with Two Substrates 156 Temperature 156 Dissolved Oxygen 157 The Meaning of Steady State Performance in Chemostat Culture 157 Oscillatory Phenomena in Continuous Cultures 158 1.Oscillations as a Consequence of Equipment Artifacts 158 2.Oscillations Derived from Feedback Between Cells and Environmental Parameters 159 3.Oscillations Derived from Intracellular Feedback Regulation 159 4.Oscillations Derived from Interactions between Different Species in Continuous Culture 165 5.Oscillations Due to Synchronous Growth and Division 165 An Introduction to Metabolic and Cellular Engineering Bioprocess Development with Plant Cells 171 MCE in Plants: Realities and Potentialities 172 Plant Transformation for Studies on Metabolism and Physiology 172 Improving Plants through Genetic Engineering 173 Improving Plant Resistance to Chemicals, Pathogens and Stresses 173 Improving Quality and Quantity of Plant Products 176 Using Plant Genetic Engineering to Produce Heterologous Proteins 179 Tools for the Manipulation and Transformation of Plants 180 Plant Metabolism: Matter and Energy Flows and the Prospects of MCA 183 Metabolic Compartmentation in Plant Cells 184 Carbon Assimilation, Partitioning and Allocation 186 Carbon fixation in higher plants 188 MCA Studies in Plants 194 Regulation and Control: Starch Synthesis, a Case Study 196 Concluding Remarks 199 Cellular Engineering 201 Outline201 The Global Functioning of Metabolic Networks 202 The Nature of the Carbon Source Determines the Activation of Whole Blocks of Metabolic Pathways with Global Impact on Cellular Energetics 203 Carbon Sources that Share Most Enzymes Required to Transform the Substrates into Key Intermediary Metabolites under Similar Growth Rates, Bring About Similar Fluxes through the Main Amphibolic Pathways 203 Interaction between Carbon and Nitrogen Regulatory Pathways in S.cerevisiae 204 Flux Redirection toward Catabolic (Fermentation) or Anabolic (Carbohydrates) Products May Be Generated as a Result of Alteration in Redox and Phosphorylation Potentials 206 Temperature-Dependent Expression of Certain Mutations Depend upon the Carbon Source 207 There Seems to Exist a General Pattern of Control of the Intracellular Concentration of Metabolites 207 Dependence of the Control of Glycolysis on the Genetic Background and the Physiological Status of Yeast in Chemostat Cultures 211 Cellular Engineering 212 Growth Rate, G1 Phase of the Cell Cycle, Production of Metabolites and Macromolecules as Targets for Cellular Engineering 213 Catabolite Repression and Cell Cycle Regulation in Yeast 215 Protein Production as a Function of Growth Rate 217 The Selective Functioning of Whole Metabolic Pathways is Permissive for Differentiation 220 Bibliography 223 Index 243
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