Metabolic Engineering

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 /leta o
PRINCIPLES AND
METHODOLOGIES

Gregory N. Stephanopoulos
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts

Aristos A. Aristidou
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts

Jens Nielsen
Department of Biotechnology
Technical University of Denmark
Lyngby, Denmark

ACADEMIC PRESS
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 CONTENTS

Preface xiii
List of Symbols xvii

1 The Essence of Metabolic Engineering

1.1 Importance of Metabolic Engineering 9
1.2 General Overview of the Book 15
References 20

2 Review of Cellular M e t a b o l i s m
2.1 An Overview of Cellular Metabolism 22
2.2 Transport Processes 25
2.2.1 Passive Transport 28
2.2.2 Facilitated Diffusion 31
2.2.3 Active Transport 34
2.3 Fueling Reactions 38
2.3.1 Glycolysis 38
2.3.2 Fermentative Pathways 45
2.3.3 TCA Cycle and Oxidative Phosphorylation 48
2.3.4 Anaplerotic Pathways 53
2.3.5 Catabolism of Fats, Organic Acids, and Amino Acids 55
2.4 Biosynthetic Reactions 57
2.4.1 Biosynthesis of Amino Acids 58
2.4.2 Biosynthesis of Nucleic Acids, Fatty Acids, and Other
Building Blocks 62
2.5 Polymerization 66

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2.6 Growth Energetics 71

References 76

3 C o m p r e h e n s i v e Models for Cellular Reactions

3.1 Stoichiometry of Cellular Reactions 82

3.2 Reaction Rates 90
3.3 Dynamic Mass Balances 93
3.4 Yield Coefficients and Linear Rate Equations 102
References 114

4 Material Balances and Data Consistency

4.1 The Black Box Model 117
4.2 Elemental Balances 119
4.3 Heat Balance 126
4.4 Analysis of Overdetermined SystemsmIdentification of
Gross Measurement Errors 130
References 146

5 Regulation of Metabolic Pathways

5.1 Regulation of Enzymatic Activity 150
5.1.1 Overview of Enzyme Kinetics 152
5.1.2 Simple Reversible Inhibition Systems 153
5.1.3 Irreversible Inhibition 163
5.1.4 Allosteric Enzymes: Cooperativity 168
5.2 Regulation of Enzyme Concentration 173
5.2.1 Control of Transcription Initiation 173
5.2.2 Control of Translation 178
5.3 Global Control: Regulation at the Whole Cell Level 180
5.4 Regulation of Metabolic Networks 188
5.4.1 Branch Point Classification 193
5.4.2 Coupled Reactions and the Role of Global
Currency Metabolites 196
References 2OO

6 Examples of P a t h w a y Manipulations: Metabolic

Engineering in Practice

6.1 Enhancement of Product Yield and Productivity 205

6.1.1 Ethanol 205
Contents ix

6.1.2 Amino Acids 212

6.1.3 Solvents 220
6.2 Extension of Substrate Range 224
6.2.1 Metabolic Engineering of Pentose Metabolism for
Ethanol Production 224
6.2.2 Cellulose-Hemicellulose Depolymerization 229
6.2.3 Lactose and Whey Utilization 230
6.2.4 Sucrose Utilization 233
6.2.5 Starch Degrading Microorganisms 233
6.3 Extension of Product Spectrum and Novel Products 235
6.3.1 Antibiotics 235
6.3.2 Polyketides 237
6.3.3 Vitamins 241
6.3.4 Biopolymers 243
6.3.5 Biological Pigments 25O
6.3.6 Hydrogen 253
6.3.7 Pentoses: Xylitol 256
6.4 Improvement of Cellular Properties 256
6.4.1 Alteration of Nitrogen Metabolism 257
6.4.2 Enhanced Oxygen Utilization 257
6.4.3 Prevention of Overflow Metabolism 259
6.4.4 Alteration of Substrate Uptake 262
6.4.5 Maintenance of Genetic Stability 263
6.5 Xenobiotic Degradation 266
6.5.1 Polychlorinated Biphenyls (PCBs) 266
6.5.2 Benzene, Toluene, p-Xylene Mixtures (BTX) 267
References 273

7 Metabolic Pathway Synthesis

7.1 Metabolic Pathway Synthesis Algorithm 288
7.2 Overview of the Algorithm 295
7.3 A Case Study: Lysine Biosynthesis 300
7.3.1 The Role of Oxaloacetate 303
7.3.2 Other Alternatives 304
7.3.3 Restrictions on the Maximum Yield 306
7.4 Discussion of the Algorithm 307
References 308

8 Metabolic Flux Analysis

8.1 Theory 313
8 . 2 0 v e r d e t e r m i n e d Systems 333
8.3 Underdetermined SystemsuLinear Programming 341
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8.4 Sensitivity Analysis 347

References 350

9 M e t h o d s for the E x p e r i m e n t a l D e t e r m i n a t i o n of M e t a b o l i c
Fluxes by Isotope Labeling
9.1 Direct Flux Determination from Fractional Label Enrichment 356
9.1.1 Flux Determination from Transient Intensity Measurements 356
9.1.2 Metabolic and Isotopic Steady State Experiments 357
9.2 Applications Involving Complete Enumeration of
Metabolite Isotopomers 367
9.2.1 Distribution of TCA Cycle Metabolite Isotopomers
from Labeled Pyruvate 371
9.2.2 Distributions of T CA Cycle Metabolite Isotopomers
from Labeled Acetate 377
9.2.3 Interpretation of Experimental Data 382
9.3 Carbon Metabolite Balances 397
9.3.1 Direct Metabolite Carbon Balances 397
9.3.2 Use of Atom Mapping Matrices 405
References 409

10 A p p l i c a t i o n s of M e t a b o l i c F l u x A n a l y s i s
10.1 Amino Acid Production by Glutamic Acid Bacteria 413
10.1.1 Biochemistry and Regulation of Glutamic Acid Bacteria 414
10.1.2 Calculation of Theoretical Yields 419
10.1.3 Metabolic Flux Analysis of Lysine Biosynthetic
Network in C. glutamicum 426
10.1.4 Metabolic Flux Analysis of Specific Deletion
Mutants of C. glutamicum 439
10.2 Metabolic Fluxes in Mammalian Cell Cultures 445
10.2.1 Determination of Intracellular Fluxes 446
10.2.2 Validation of Flux Estimates by 13C Labeling Studies 452
10.2.3 Application of Flux Analysis to the Design of
Cell Culture Media 457
References 458

11 M e t a b o l i c C o n t r o l A n a l y s i s
11.1 Fundamentals of Metabolic Control Analysis 464
11.1.1 Control Coefficients and the Summation Theorems 465
11.1.2 Elasticity Coefficients and the
Connectivity Theorems 470
11.1.3 Generalization of MCA Theorems 473
Contents xi

11.2 Determination of Flux Control Coefficients 475

11.2.1 Direct Methods for FCC Determination 478
11.2.2 Indirect Methods for FCC Determination 484
11.2.3 Use of Transient Metabolite Measurements 488
11.2.4 Kinetic Models 491
11.3 MCA of Linear Pathways 492
11.4 MCA of Branched Pathways 499
11.5 Theory of Large Deviations 513
11.5.1 Unbranched Networks 514
11.5.2 Branched Networks 522
11.5.3 Response to Changes in Nutrient Concentrations
and External Effectors 527
11.5.4 Discussion 529
References 53O

12 Analysis of Structure of Metabolic Networks

12.1 Control of Flux Distribution at a Single Branch Point 539
12.2 Grouping of Reactions 545
12.2.1 Group Flux Control Coefficients 546
12.2.2 Identification of Independent Pathways 547
12.3 Case Study: Aromatic Amino Acid Biosynthetic Pathway 554
12.3.1 Model of Aromatic Amino Acid Biosynthesis in S. cerevisiae 554
12.3.2 Identification of Independent Pathways 557
12.3.3 Identification of Link Metabolites and Group
Flux Determination 563
References 580

13 F l u x A n a l y s i s of Metabolic Networks
13.1 Relationships among Group and Individual Control
Coefficients (Bottom-Up Approach) 582
13.2 Determination of Group Control Coefficients from
Flux Measurements (Top-Down Approach) 585
13.2.1 Determination of gFCCs from Three Perturbations 586
13.2.2 Determination of gFCCs from a
Characterized Perturbation 590
13.2.3 Determination of gCCCs 591
13.2.40bservability of Perturbations 592
13.3 Case Study 592
13.3.1 Analytical Determination of Group Control
Coefficients (Bottom-Up Approach) 593
13.3.2 Simulation of Experimental Determination of
gFCCs (Top-Down Approach) 601
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13.4 Extension of Control Analysis to Intermetabolite

Reaction Groups 603
13.4.1 The Perturbation Constant 603
13.4.2 Analysis of Overlapping Reaction Groups in
Multiple Branch Points 605
13.4.3 Case Study 607
13.5 Optimization of Flux Amplification 609
13.5.1 Derivation of Optimization Algorithm 610
13.5.2 Case Study 613
13.6 Consistency Tests and Experimental Validation 616
13.6.1 Development of Consistency Tests Using
Multiple Perturbations 618
13.6.2 Application to the Prephenate Branch Point 623
13.6.3 Effects of Measurement Error 625
References 626

14 T h e r m o d y n a m i c s of Cellular P r o c e s s e s
14.1 Thermodynamic Principles: A Review 630
14.2 Thermodynamic Feasibility 639
14.2.1 The Algorithm 641
14.2.2 Determination of AGo' from Group Contributions 649
14.3 Nonequilibrium Thermodynamics 663
14.4 Application of Thermokinetics to MCA 686
References 692

Glossary 695

Index 707
 PREFACE

Metabolic engineering is about the analysis and modification of metabolic

pathways. The field emerged during the past decade, and powered by
techniques from applied molecular biology and reaction engineering, it is
becoming a focal point of research activity in biological and biochemical
engineering, cell physiology, and applied microbiology. Although the notion
of pathway manipulation had been discussed before, the vision of metabolic
engineering as defining a discipline in its own right was first suggested by
Bailey in 1991. It was embraced soon thereafter by both engineers and life
scientists who saw in it the opportunity to capture the potential of sequence
and other information generated from genomics research.
We first attempted to convey the excitement and basic concepts of
metabolic engineering to our students in a course that was taught at MIT in
1993. The experiment was repeated again in 1995 and 1997, at which time a
definite syllabus and tentative set of notes had emerged as a result of these
offerings. A similar development occurred at the Technical University of
Denmark (DTU), where metabolic engineering has been a central topic in
biochemical engineering courses at both the undergraduate and the graduate
level. In 1996, a standard one-semester course on metabolic engineering was
offered for the first time. The growing interest in metabolic engineering and
requests to share the course material led us to the decision to write this
book. In so doing, we have tried to formulate a framework of quantitative
biochemistry for the analysis of pathways of enzymatic reactions. In this
sense, the book reflects a shift of focus from equipment toward single cells,
as it concentrates on the elucidation and manipulation of their biochemical

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xiv Preface

functions. As such, this text can support a graduate or advanced undergradu-

ate course on metabolic engineering to complement current offerings in
biochemical engineering.
The book manuscript was used to teach courses on metabolic engineering
at MIT and DTU, as well as a summer course at MIT. The material can be
covered in a single semester with no prerequisites, although some prior
exposure to an introductory biochemistry course is helpful. Assigned read-
ings from biochemistry texts during the first quarter of the semester can
complement the first part of the book. Problem sets aiding the understanding
of basic concepts will be posted periodically at the web site listed below.
Although the focus of this book is on metabolism, the concepts of pathway
analysis are broad and as such generally applicable to other types of reaction
sequences, including those involved in protein expression and post-transla-
tional modification or in signal transduction pathways.
Writing a book on a subject that is still in its formative stage is a challenge
that carries with it increased responsibility. For this reason, we set as our
goal to define core principles central to pathway design and analysis,
complemented with specific methods derived from recent research. We
expect these methods to evolve further and hope that this book will play
a role in catalyzing such activity. Software implementing the various meth-
ods can be found at the book web site, h t t p : / / w w w . c p b . d t u . d k / c p b /
metabol.htm, with hyperlinks to other sites where public domain software is
available. To facilitate broad interdisciplinary participation, only codes with a
minimum of service and user-friendliness have been selected. Furthermore,
the mathematical complexity of the book has been kept to an absolute
minimum, and background material has been provided wherever possible to
assist the less mathematically inclined. We are aware of the challenges of this
task and the difficulties in satisfying all segments of the readership spectrum.
We encourage readers to continue their review of the book undeterred by
any temporary difficulties.
We are indebted to many individuals for their direct contributions or
indirect input in planning and executing this project. First, we thank our
students for their boundless energy and refreshing creativity, in particular
Mafia Klapa, for a thorough review of metabolic flux analysis, and Troy
Simpson, whose research provided the basis for complex pathway analysis.
Also, we thank Martin Bastian Pedersen for drafting many of the figures, and
Christian Mfiller, Susanne Sloth Larsen, Birgitte Karsbol, and Kristen Nielsen
for their help in finalizing the manuscript. We thank our colleagues, in
particular Tony Sinskey, for his enthusiasm about the unlimited possibilities
of metabolic engineering, and Sue Harrison and Eduardo Agosin for their
most constructive comments. Finally, we thank our collaborators and friends,
Preface XV

in particular, Barry Buckland, Bernhard Palsson, John Villadsen, Maish

Yarmush, and D. Ramkrishna. Their vision and unwavering support when it
mattered meant a lot.

Gregory N. Stephanopoulos
Aristos A. Aristidou
Jens Nielsen
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