MODERN BIOTECHNOLOGY - Hörbücher · Modern biotechnology : ... Growth of the New Biotechnology Industry Depends on ... Biocatalysis and the Growth of Industrial Enzymes 49

MODERN BIOTECHNOLOGYConnecting Innovations in Microbiology and Biochemistry to Engineering Fundamentals

Nathan S. MosierMichael R. Ladisch

A JOHN WILEY & SONS, INC., PUBLICATION

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MODERN BIOTECHNOLOGY

MODERN BIOTECHNOLOGYConnecting Innovations in Microbiology and Biochemistry to Engineering Fundamentals

Nathan S. MosierMichael R. Ladisch

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright 2009 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Mosier, Nathan S., 1974- Modern biotechnology : connecting innovations in microbiology and biochemistry to engineering fundamentals / Nathan S. Mosier, Michael R. Ladisch. p. cm. Includes index. ISBN 978-0-470-11485-8 (cloth) 1. Biotechnology. I. Ladisch, Michael R., 1950- II. Title. TP248.2.M675 2009 660.6dc22 2009001779

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com

CONTENTS

Preface xvAcknowledgments xviiList of Illustrations xix

1 Biotechnology 1

Introduction 1The Directed Manipulation of Genes Distinguishes the New

Biotechnology from Prior Biotechnology 2Growth of the New Biotechnology Industry Depends on

Venture Capital 3Submerged Fermentations Are the Industrys Bioprocessing

Cornerstone 10Oil Prices Affect Parts of the Fermentation Industry 10

Growth of the Antibiotic/Pharmaceutical Industry 11The Existence of Antibiotics Was Recognized in 1877 11Penicillin Was the First Antibiotic Suitable for Human

Systemic Use 12Genesis of the Antibiotic Industry 12Other Antibiotics Were Quickly Discovered after the

Introduction of Penicillin 13Discovery and Scaleup Are Synergistic in the Development of

Pharmaceutical Products 15Success of the Pharmaceutical Industry in Research, Development,

and Engineering Contributed to Rapid Growth but Also Resulted in Challenges 15

Growth of the Amino Acid/Acidulant Fermentation Industry 16Production of Monosodium Glutamate (MSG) via Fermentation 17The Impact of Glutamic Acid Bacteria on Monosodium

Glutamate Cost Was Dramatic 17Auxotrophic and Regulatory Mutants Enabled Production of

Other Amino Acids 17Prices and Volumes Are Inversely Related 19Biochemical Engineers Have a Key Function in All Aspects of

the Development Process for Microbial Fermentation 21

v

vi CONTENTS

References 22

Homework Problems 24

2 New Biotechnology 27

Introduction 27

Growth of the Biopharmaceutical Industry 28The Biopharmaceutical Industry Is in the Early Part of Its

Life Cycle 31Discovery of Type II Restriction Endonucleases Opened a New

Era in Biotechnology 33The Polymerase Chain Reaction (PCR) Is an Enzyme-Mediated,

In Vitro Amplifi cation of DNA 33

Impacts of the New Biotechnology on Biopharmaceuticals, Genomics, Plant Biotechnology, and Bioproducts 34

Biotechnology Developments Have Accelerated Biological Research 35

Drug Discovery Has Benefi ted from Biotechnology Research Tools 36

The Fusing of Mouse Spleen Cells with T Cells Facilitated Production of Antibodies 36

Regulatory Issues Add to the Time Required to Bring a New Product to Market 36

New Biotechnology Methods Enable Rapid Identifi cation of Genes and Their Protein Products 39

Genomics Is the Scientifi c Discipline of Mapping, Sequencing, and Analyzing Genomes 39

Products from the New Plant Biotechnology Are Changing the Structure of Large Companies that Sell Agricultural Chemicals 42

Bioproducts from Genetically Engineered Microorganisms Will Become Economically Important to the Fermentation Industry 43

References 45


3 Bioproducts and Biofuels 49

Introduction 49

Biocatalysis and the Growth of Industrial Enzymes 49Glucose Isomerase Catalyzed the Birth of a New Process for

Sugar Production from Corn 51Identifi cation of a Thermally Stable Glucose Isomerase and an

Inexpensive Inducer Was Needed for an Industrial Process 53The Demand for High-Fructose Corn Syrup (HFCS) Resulted

in Large-Scale Use of Immobilized Enzymes and Liquid Chromatography 53

CONTENTS vii

Rapid Growth of HFCS Market Share Was Enabled by Large-Scale Liquid Chromatography and Propelled by Record-High Sugar Prices 55

Biocatalysts Are Used in Fine-Chemical Manufacture 56

Growth of Renewable Resources as a Source of Specialty Products and Industrial Chemicals 58

A Wide Range of Technologies Are Needed to Reduce Costs for Converting Cellulosic Substrates to Value-Added Bioproducts and Biofuels 59

Renewable Resources Are a Source of Natural Plant Chemicals 63Bioseparations Are Important to the Extraction, Recovery, and

Purifi cation of Plant-Derived Products 64

Bioprocess Engineering and Economics 65

Bioseparations and Bioprocess Engineering 66

References 67


4 Microbial Fermentations 73

Introduction 73

Fermentation Methods 75Fermentations Are Carried Out in Flasks, Glass Vessels,

and Specially Designed Stainless-Steel Tanks 75

Microbial Culture Composition and Classifi cation 78Microbial Cells: Prokaryotes versus Eukaryotes 78

Classifi cation of Microorganisms Are Based on Kingdoms 81Prokaryotes Are Important Industrial Microorganisms 81Eukaryotes Are Used Industrially to Produce Ethanol,

Antibiotics, and Biotherapeutic Proteins 82Wild-Type Organisms and Growth Requirements in

Microbial Culture 83Wild-Type Organisms Find Broad Industrial Use 83Microbial Culture Requires that Energy and All Components

Needed for Cell Growth Be Provided 86

Media Components and Their Functions (Complex and Defi ned Media) 86

Carbon Sources Provide Energy, and Sometimes Provide Oxygen 86

Complex Media Have a Known Basic Composition but a Chemical Composition that Is Not Completely Defi ned 89

Industrial Fermentation Broths May Have a High Initial Carbon (Sugar) Content (Ethanol Fermentation Example) 91

The Accumulation of Fermentation Products Is Proportional to Cell Mass in the Bioreactor 92

viii CONTENTS

A Microbial Fermentation Is Characterized by Distinct Phases of Growth 93

Expressions for Cell Growth Rate Are Based on Doubling Time 94

Products of Microbial Culture Are Classifi ed According to Their Energy Metabolism (Types I, II, and III Fermentations) 96

Product Yields Are Calculated from the Stoichiometry of Biological Reactions (Yield Coeffi cients) 102

The EmbdenMeyerhof Glycolysis and Citric Acid Cycles Are Regulated by the Relative Balance of ATP, ADP, and AMP in the Cell 104

References 105


5 Modeling and Simulation 111

Introduction 111

The RungeKutta Method 112Simpsons Rule 112Fourth-Order RungeKutta Method 113Ordinary Differential Equations (ODEs) 115

RungeKutta Technique Requires that Higher-Order Equations Be Reduced to First-Order ODEs to Obtain Their Solution 115

Systems of First-Order ODEs Are Represented in Vector Form 116

Kinetics of Cell Growth 117Ks Represents Substrate Concentration at Which the Specifi c

Growth Rate Is Half Its Maximum 120

Simulation of a Batch Ethanol Fermentation 122Ethanol Case Study 123

LuedekingPiret Model 127

Continuous Stirred-Tank Bioreactor 128

Batch Fermentor versus Chemostat 132

References 133


6 Aerobic Bioreactors 141

Introduction 141

Fermentation Process 144Fermentation of Xylose to 2,3-Butanediol by Klebsiella

oxytoca Is Aerated but Oxygen-Limited 144Oxygen Transfer from Air Bubble to Liquid Is Controlled by

Liquid-Side Mass Transfer 153

CONTENTS ix

Chapter 6 Appendix: Excel Program for Integration of Simultaneous Differential Equations 159

References 161


7 Enzymes 165

Introduction 165

Enzymes and Systems Biology 165

Industrial Enzymes 166

Enzymes: In Vivo and In Vitro 167

Fundamental Properties of Enzymes 169

Classifi cation of Enzymes 170

Sales and Applications of Immobilized Enzymes 172

Assaying Enzymatic Activity 173Enzyme Assays 181

Batch Reactions 187

Thermal Enzyme Deactivation 187

References 192


8 Enzyme Kinetics 199

Introduction 199

Initial Rate versus Integrated Rate Equations 200Obtaining Constants from Initial Rate Data Is an Iterative

Process 204

Batch Enzyme Reactions: Irreversible Product Formation (No Inhibition) 207

Rapid Equilibrium Approach Enables Rapid Formulation of an Enzyme Kinetic Equation 207

The Pseudo-Steady-State Method Requires More Effort to Obtain the Hart Equation but Is Necessary for Reversible Reactions 209

Irreversible Product Formation in the Presence of Inhibitors and Activators 210

Inhibition 212Competitive Inhibition 213Uncompetitive Inhibition 214(Classical) Noncompetitive Inhibition 216Substrate Inhibition 217

x CONTENTS

Example of Reversible Reactions 220Coenzymes and Cofactors Interact in a Reversible Manner 223

KingAltman Method 225

Immobilized Enzyme 234Online Databases of Enzyme Kinetic Constants 236

References 237


9 Metabolism 243

Introduction 243

Aerobic and Anaerobic Metabolism 245Glycolysis Is the Oxidation of Glucose in the Absence of Oxygen 245Oxidation Is Catalyzed by Oxidases in the Presence of O2,

and by Dehydrogenases in the Absence of O2 246A Membrane Bioreactor Couples Reduction and Oxidation

Reactions (R-Mandelic Acid Example) 247Three Stages of Catabolism Generate Energy, Intermediate

Molecules, and Waste Products 248The Glycolysis Pathway Utilizes Glucose in Both Presence

(Aerobic) and Absence (Anaerobic) of O2 to Produce Pyruvate 249Glycolysis Is Initiated by Transfer of a High-Energy Phosphate

Group to Glucose 250Products of Anaerobic Metabolism Are Secreted or Processed

by Cells to Allow Continuous Metabolism of Glucose by Glycolysis 253

Other Metabolic Pathways Utilize Glucose Under Anaerobic Conditions (Pentose Phosphate, EntnerDoudoroff, and Hexose Monophosphate Shunt Pathways) 255

Knowledge of Anaerobic Metabolism Enables Calculation of Theoretical Yields of Products Derived from Glucose 257

Economics Favor the Glycolytic Pathway for Obtaining Oxygenated Chemicals from Renewable Resources 258

Citric Acid Cycle and Aerobic Metabolism 259Respiration Is the Aerobic Oxidation of Glucose and Other

Carbon-Based Food Sources (Citric Acid Cycle) 260The Availability of Oxygen, under Aerobic Conditions,

Enables Microorganisms to Utilize Pyruvate via the Citric Acid Cycle 260

The Citric Acid Cycle Generates Precursors for Biosynthesis of Amino Acids and Commercially Important Fermentation Products 264

Glucose Is Transformed to Commercially Valuable Products via Fermentation Processes: A Summary 264

Essential Amino Acids Not Synthesized by Microorganisms Must Be Provided as Nutrients (Auxotrophs) 267

CONTENTS xi

The Utilization of Fats in Animals Occurs by a NonTricarboxylic Acid (TCA) Cycle Mechanism 267

Some Bacteria and Molds Can Grow on Hydrocarbons or Methanol in Aerated Fermentations (Single-Cell Protein Case Study) 269

Extremophiles: Microorganisms that Do Not Require Glucose, Utilize H2, and Grow at 80100 C and 200 atm Have Industrial Uses 270

The Terminology for Microbial Culture Is Inexact: Fermentation Refers to Both Aerobic and Anaerobic Conditions While Respiration Can Denote Anaerobic Metabolism 271

Metabolism and Biological Energetics 272

References 272


10 Biological Energetics 277

Introduction 277

Redox Potential and Gibbs Free Energy in Biochemical Reactions 277

Heat: Byproduct of Metabolism 286

References 292


11 Metabolic Pathways 295

Introduction 295Living Organisms Control Metabolic Pathways at Strategic and

Operational Levels 296Auxotrophs Are Nutritionally Defi cient Microorganisms that

Enhance Product Yields in Controlled Fermentations (Relief of Feedback Inhibition and Depression) 296

Both Branched and Unbranched Pathways Cause Feedback Inhibition and Repression (Purine Nucleotide Example) 299

The Accumulation of an End Metabolite in a Branched Pathway Requires a Strategy Different from that for the Accumulation of an Intermediate Metabolite 301

Amino Acids 305The Formulation of Animal Feed Rations with Exogeneous

Amino Acids Is a Major Market for Amino Acids 306Microbial Strain Discovery, Mutation, Screening, and

Development Facilitated Introduction of Industrial, Aerated Fermentations for Amino Acid Production by Corynbacteriumglutamicum 308

Overproduction of Glutamate by C. glutamicum Depends on an Increase in Bacterial Membrane Permeability (Biotin-Defi cient Mutant) 309

xii CONTENTS

A Threonine and Methionine Auxotroph of C. glutamicum Avoids Concerted Feedback Inhibition and Enables Industrial Lysine Fermentations 310

Cell (Protoplast) Fusion Is a Method for Breeding Amino Acid Producers that Incorporate Superior Characteristics of Each Parent (Lysine Fermentation) 312

Amino Acid Fermentations Represent Mature Technologies 313

Antibiotics 314Secondary Metabolites Formed During Idiophase Are Subject

to Catabolite Repression and Feedback Regulation (Penicillin and Streptomycin) 314

The Production of Antibiotics Was Viewed as a Mature Field Until Antibiotic-Resistant Bacteria Began to Appear 317

Bacteria Retain Antibiotic Resistance Even When Use of the Antibiotic Has Ceased for Thousands of Generations 318

Antibiotic Resistance Involves Many Genes (Vancomycin Example) 318

References 320


12 Genetic Engineering: DNA, RNA, and Genes 331

Introduction 331

DNA and RNA 332DNA Is a Double-Stranded Polymer of the Nucleotides:

Thymine, Adenine, Cytosine, and Guanine 332The Information Contained in DNA Is Huge 332Genes Are Nucleotide Sequences that Contain the Information

Required for the Cell to Make Proteins 333Transcription Is a Process Whereby Specifi c Regions of the

DNA (Genes) Serve as a Template to Synthesize Another Nucleotide, Ribonucleic Acid (RNA) 333

Chromosomal DNA in a Prokaryote (Bacterium) Is Anchored to the Cells Membrane While Plasmids Are in the Cytoplasm 333

Chromosomal DNA in a Eukaryote (Yeast, Animal or Plant Cells) Is Contained in the Nucleus 334

Microorganisms Carry Genes in Plasmids Consisting of Shorter Lengths of Circular, Extrachromosomal DNA 334

Restriction Enzymes Enable Directed In Vitro Cleavage of DNA 337

Different Type II Restriction Enzymes Give Different Patterns of Cleavage and Different Single-Stranded Terminal Sequences 339

DNA Ligase Covalently Joins the Ends of DNA Fragments 341

CONTENTS xiii

DNA Fragments and Genes of 150 Nucleotides Can Be Chemically Synthesized if the Nucleotide Sequence Has Been Predetermined 342

Protein Sequences Can Be Deduced and Genes Synthesized on the Basis of Complementary DNA Obtained from Messenger RNA 343

Genes and Proteins 344Selectable Markers Are Genes that Facilitate Identifi cation of

Transformed Cells that Contain Recombinant DNA 344A Second Protein Fused to the Protein Product Is Needed to

Protect the Product from Proteolysis (-Gal-SomatostatinFusion Protein Example) 346

Recovery of Protein Product from Fusion Protein Requires Correct Selection of Amino Acid that Links the Two Proteins (Met Linker) 347

Chemical Modifi cation and Enzyme Hydrolysis Recover an Active Molecule Containing Met Residues from a Fusion Protein (-Endorphin Example) 347

Metabolic Engineering Differs from Genetic Engineering by the Nature of the End Product 348

References 349


13 Metabolic Engineering 355

Introduction 355

Building Blocks 359

l-Threonine-Overproducing Strains of E. coli K-12 359Genetically Altered Brevibacterium lactoferrin Has Yielded

Improved Amino AcidProducing Strains 360Metabolic Engineering May Catalyze Development of New

Processes for Manufacture of Oxygenated Chemicals 362Gene Chips Enable Examination of Glycolytic and Citric

Acid Cycle Pathways in Yeast at a Genomic Level (Yeast Genome Microarray Case Study) 362

The Fermentation of Pentoses to Ethanol Is a Goal of Metabolic Engineering (Recombinant Bacteria and Yeast Examples) 364

Metabolic Engineering for a 1,3-Propanediol-Producing Organism to Obtain Monomer for Polyester Manufacture 370

Redirection of Cellular Metabolism to Overproduce an Enzyme Catalyst Results in an Industrial Process for Acrylamide Production (YamadaNitto Process) 373

References 377


MODERN BIOTECHNOLOGY - Hörbücher · Modern biotechnology : ... Growth of the New Biotechnology Industry Depends on ... Biocatalysis and the Growth of Industrial Enzymes 49

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