Unites a biological and a biotechnological perspective on cyanobacteria, and includes the industrial aspects and applications of cyanobacteria
Cyanobacteria Biotechnology offers a guide to the interesting and useful features of cyanobacteria metabolism that keeps true to a biotechnology vision. In one volume the book brings together both biology and biotechnology to illuminate the core acpects and principles of cyanobacteria metabolism.
Designed to offer a practical approach to the metabolic engineering of cyanobacteria, the book contains relevant examples of how this metabolic "module" is currently being engineered and how it could be engineered in the future. The author includes information on the requirements and real-world experiences of the industrial applications of cyanobacteria. This important book:
- Brings together biology and biotechnology in order to gain insight into the industrial relevant topic of cyanobacteria
- Introduces the key aspects of the metabolism of cyanobacteria
- Presents a grounded, practical approach to the metabolic engineering of cyanobacteria
- Offers an analysis of the requirements and experiences for industrial cyanobacteria
- Provides a framework for readers to design their own processes
Written for biotechnologists, microbiologists, biologists, biochemists, Cyanobacteria Biotechnology provides a systematic and clear volume that brings together the biological and biotechnological perspective on cyanobacteria.
Table of Contents
Foreword: Cyanobacteria Biotechnology xv
Acknowledgments xviii
Part I Core Cyanobacteria Processes 1
1 Inorganic Carbon Assimilation in Cyanobacteria: Mechanisms, Regulation, and Engineering 3
Martin Hagemann, Shanshan Song, and Eva-Maria Brouwer
1.1 Introduction - The Need for a Carbon-Concentrating Mechanism 3
1.2 The Carbon-Concentrating Mechanism (CCM) Among Cyanobacteria 4
1.2.1 Ci Uptake Proteins/Mechanisms 5
1.2.2 Carboxysome and RubisCO 8
1.3 Regulation of Ci Assimilation 10
1.3.1 Regulation of the CCM 10
1.3.2 Further Regulation of Carbon Assimilation 13
1.3.3 Metabolic Changes and Regulation During Ci Acclimation 14
1.3.4 Redox Regulation of Ci Assimilation 15
1.4 Engineering the Cyanobacterial CCM 16
1.5 Photorespiration 17
1.5.1 Cyanobacterial Photorespiration 17
1.5.2 Attempts to Engineer Photorespiration 19
1.6 Concluding Remarks 20
Acknowledgments 21
References 21
2 Electron Transport in Cyanobacteria and Its Potential in Bioproduction 33
David J. Lea-Smith and Guy T. Hanke
2.1 Introduction 33
2.2 Electron Transport in a Bioenergetic Membrane 34
2.2.1 Linear Electron Transport 34
2.2.2 Cyclic Electron Transport 37
2.2.3 ATP Production from Linear and Cyclic Electron Transport 37
2.3 Respiratory Electron Transport 38
2.4 Role of Electron Sinks in Photoprotection 41
2.4.1 Terminal Oxidases 41
2.4.2 Hydrogenase and Flavodiiron Complexes 41
2.4.3 Carbon Fixation and Photorespiration 43
2.4.4 Extracellular Electron Export 44
2.5 Regulating Electron Flux into Different Pathways 45
2.5.1 Electron Flux Through the Plastoquinone Pool 45
2.5.2 Electron Flux Through Fdx 46
2.6 Spatial Organization of Electron Transport Complexes 47
2.7 Manipulating Electron Transport for Synthetic Biology Applications 48
2.7.1 Improving Growth of Cyanobacteria 49
2.7.2 Production of Electrical Power in BPVs 49
2.7.3 Hydrogen Production 50
2.7.4 Production of Industrial Compounds 50
2.8 Future Challenges in Cyanobacterial Electron Transport 51
References 52
3 Optimizing the Spectral Fit Between Cyanobacteria and Solar Radiation in the Light of Sustainability Applications 65
Klaas J. Hellingwerf, Que Chen, and Filipe Branco dos Santos
3.1 Introduction 65
3.2 Molecular Basis and Efficiency of Oxygenic Photosynthesis 67
3.3 Fit Between the Spectrum of Solar Radiation and the Action Spectrum of Photosynthesis 72
3.4 Expansion of the PAR Region of Oxygenic Photosynthesis 74
3.5 Modulation and Optimization of the Transparency of Photobioreactors 79
3.6 Full Control of the Light Regime: LEDs Inside the PBR 81
3.7 Conclusions and Prospects 82
References 83
Part II Concepts in Metabolic Engineering 89
4 What We Can Learn from Measuring Metabolic Fluxes in Cyanobacteria 91
Xiang Gao, Chao Wu, Michael Cantrell, Melissa Cano, Jianping Yu, and Wei Xiong
4.1 Central Carbon Metabolism in Cyanobacteria: An Overview and Renewed Pathway Knowledge 91
4.1.1 Glycolytic Routes Interwoven with the Calvin Cycle 91
4.1.2 Tricarboxylic Acid Cycling 94
4.2 Methodologies for Predicting and Quantifying Metabolic Fluxes in Cyanobacteria 95
4.2.1 Flux Balance Analysis and Genome-Scale Reconstruction of Metabolic Network 95
4.2.2 13C-Metabolic Flux Analysis 96
4.2.3 Thermodynamic Analysis and Kinetics Analysis 99
4.3 Cyanobacteria Fluxome in Response to Altered Nutrient Modes and Environmental Conditions 101
4.3.1 Autotrophic Fluxome 101
4.3.2 Photomixotrophic Fluxome 104
4.3.3 Heterotrophic Fluxome 105
4.3.4 Photoheterotrophic Fluxome 105
4.3.5 Diurnal Metabolite Oscillations 106
4.3.6 Nutrient States’ Impact on Metabolic Flux 107
4.4 Metabolic Fluxes Redirected in Cyanobacteria for Biomanufacturing Purposes 108
4.4.1 Restructuring the TCA Cycle for Ethylene Production 108
4.4.2 Maximizing Flux in the Isoprenoid Pathway 109
4.4.2.1 Measuring Precursor Pool Size to Evaluate Potential Driving Forces for Isoprenoid Production 109
4.4.2.2 Balancing Intermediates for Increased Pathway Activity 110
4.4.2.3 Kinetic Flux Profiling to Detect Bottlenecks in the Pathway 111
4.5 Synopsis and Future Directions 112
Acknowledgments 112
References 112
5 Synthetic Biology in Cyanobacteria and Applications for Biotechnology 123
Elton P. Hudson
5.1 Introduction 123
5.2 Getting Genes into Cyanobacteria 123
5.2.1 Transformation 123
5.2.2 Expression from Episomal Plasmids 125
5.2.3 Delivery of Genes to the Chromosome 127
5.3 Basic Synthetic Control of Gene Expression in Cyanobacteria 129
5.3.1 Quantifying Transcription and Translation in Cyanobacteria 130
5.3.2 Controlling Transcription with Synthetic Promoters 134
5.3.2.1 Constitutive Promoters 136
5.3.2.2 Regulated Promoters that Are Sensitive to Added Compounds (Inducible) 137
5.3.2.3 CRISPR Interference for Transcriptional Repression 139
5.3.3 Controlling Translation 141
5.3.3.1 Ribosome Binding Sites (Cis-Acting) 141
5.3.3.2 Riboswitches (Cis-Acting) 142
5.3.3.3 Small RNAs (Trans-Acting) 143
5.4 Exotic Signals for Controlling Expression 143
5.4.1 Oxygen 144
5.4.2 Light Color 144
5.4.3 Cell Density or Growth Phase 145
5.4.4 Engineering Regulators for Altered Sensing Properties: State of the Art 147
5.5 Advanced Regulation: The Near Future 148
5.5.1 Logic Gates and Timing Circuits 148
5.5.2 Orthogonal Transcription Systems 151
5.5.3 Synthetic Biology Solutions to Increase Stability 152
5.5.4 Synthetic Biology Solutions for Cell Separation and Product Recovery 154
5.6 Conclusions 157
Acknowledgments 158
References 158
6 Sink Engineering in Photosynthetic Microbes 171
María Santos-Merino, Amit K. Singh, and Daniel C. Ducat
6.1 Introduction 171
6.2 Source and Sink 172
6.3 Regulation of Sink Energy in Plants 177
6.3.1 Sucrose and Other Signaling Carbohydrates 178
6.3.2 Hexokinases 179
6.3.3 Sucrose Non-fermenting Related Kinases 180
6.3.4 TOR Kinase 181
6.3.5 Engineered Pathways as Sinks in Photosynthetic Microbes 182
6.3.6 Sucrose 183
6.3.7 2,3-Butanediol 187
6.3.8 Ethylene 187
6.3.9 Glycerol 188
6.3.10 Isobutanol 188
6.3.11 Isoprene 189
6.3.12 Limonene 189
6.3.13 P450, an Electron Sink 190
6.4 What Are Key Source/Sink Regulatory Hubs in Photosynthetic Microbes? 191
6.5 Concluding Remarks 194
Acknowledgment 195
References 195
7 Design Principles for Engineering Metabolic Pathways in Cyanobacteria 211
Jason T. Ku and Ethan I. Lan
7.1 Introduction 211
7.2 Cofactor Optimization 212
7.2.1 Recruiting NADPH-Dependent Enzymes Wherever Possible 215
7.2.2 Engineering NADH-Specific Enzymes to Utilize NADPH 217
7.2.3 Increasing NADH Pool in Cyanobacteria Through Expression of Transhydrogenase 218
7.3 Incorporation of Thermodynamic Driving Force into Metabolic Pathway Design 219
7.3.1 ATP Driving Force in Metabolic Pathways 220
7.3.2 Increasing Substrate Pool Supports the Carbon Flux Toward Products 222
7.3.3 Product Removal Unblocks the Limitations of Product Titer 223
7.4 Development of Synthetic Pathways for Carbon Conserving Photorespiration and Enhanced Carbon Fixation 225
7.5 Summary and Future Perspective on Cyanobacterial Metabolic Engineering 229
References 229
8 Engineering Cyanobacteria for Efficient Photosynthetic Production: Ethanol Case Study 237
Guodong Luan and Xuefeng Lu
8.1 Introduction 237
8.2 Pathway for Ethanol Synthesis in Cyanobacteria 238
8.2.1 Pyruvate Decarboxylase and Type II Alcohol Dehydrogenase 238
8.2.2 Selection of Better Enzymes in the Pdc-AdhII Pathway 240
8.2.3 Systematic Characterization of the PdcZM-Slr1192 Pathway 241
8.3 Selection of Optimal Cyanobacteria “Chassis,” Strain for Ethanol Production 242
8.3.1 Synechococcus PCC 6803 and Synechococcus PCC 7942 243
8.3.2 Synechococcus PCC 7002 245
8.3.3 Anabaena PCC 7120 245
8.3.4 Nonconventional Cyanobacteria Species 246
8.4 Metabolic Engineering Strategies Toward More Efficient and Stable Ethanol Production 246
8.4.1 Enhancing the Carbon Flux via Overexpression of Calvin Cycle Enzymes 248
8.4.2 Blocking Pathways that Are Competitive to Ethanol 248
8.4.3 Arresting Biomass Formation 249
8.4.4 Engineering Cofactor Supply 249
8.4.5 Engineering Strategies Guided by In Silico Simulation 250
8.4.6 Stabilizing Ethanol Synthesis Capacity in Cyanobacterial Cell Factories 251
8.5 Exploring the Response in Cyanobacteria to Ethanol 253
8.5.1 Response of Cyanobacterial Cells Toward Exogenous Added Ethanol 254
8.5.2 Response of Cyanobacteria to Endogenous Synthesized Ethanol 255
8.6 Metabolic Engineering Strategies to Facilitate Robust Cultivation Against Biocontaminants 256
8.6.1 Engineering Cyanobacteria Cell Factories to Adapt for Selective Environmental Stresses 256
8.6.2 Engineering Cyanobacteria Cell Factories to Utilize Uncommon Nutrients 258
8.7 Conclusions and Perspectives 258
References 259
9 Engineering Cyanobacteria as Host Organisms for Production of Terpenes and Terpenoids 267
João S. Rodrigues and Pia Lindberg
9.1 Terpenoids and Industrial Applications 267
9.2 Terpenoid Biosynthesis in Cyanobacteria 270
9.2.1 Methylerythritol-4-Phosphate Pathway 270
9.2.2 Formation of Terpene Backbones 272
9.3 Natural Occurrence and Physiological Roles of Terpenes and Terpenoids in Cyanobacteria 274
9.4 Engineering Cyanobacteria for Terpenoid Production 275
9.4.1 Metabolic Engineering 277
9.4.1.1 Terpene Synthases 277
9.4.1.2 Increasing Supply of Terpene Backbones 285
9.4.1.3 Engineering the Native MEP Pathway 286
9.4.1.4 Implementing the MVA Pathway 287
9.4.1.5 Enhancing Precursor Supply 288
9.4.2 Optimizing Growth Conditions for Production 289
9.4.3 Product Capture and Harvesting 291
9.5 Summary and Outlook 292
Acknowledgments 293
References 293
10 Cyanobacterial Biopolymers 301
Moritz Koch and Karl Forchhammer
10.1 Polyhydroxybutryate 301
10.1.1 Introduction 301
10.1.2 PHB Metabolism in Cyanobacteria 302
10.1.3 Industrial Applications of PHB 305
10.1.3.1 Physical Properties of PHB and Its Derivatives 305
10.1.3.2 Biodegradability 306
10.1.3.3 Application of PHB as a Plastic 306
10.1.3.4 Reactor Types 306
10.1.3.5 Production Process 307
10.1.3.6 Downstream Processing 308
10.1.4 Metabolic Engineering of PHB Biosynthesis 308
10.1.5 Limitations and Potential of PHB Production in Cyanobacteria 310
10.2 Cyanophycin Granules in Cyanobacteria 311
10.2.1 Biology of Cyanophycin 311
10.2.2 Genes and Enzymes of CGP Metabolism 315
10.2.2.1 Cyanophycin Synthetase 315
10.2.2.2 Cyanophycin Degrading Enzymes 316
10.2.3 Regulation of Cyanophycin Metabolism 317
10.2.4 Cyanophycin Overproduction and Potential Industrial Applications 318
Acknowledgement 319
References 319
11 Biosynthesis of Fatty Acid Derivatives by Cyanobacteria: From Basics to Biofuel Production 331
Akihito Kawahara and Yukako Hihara
11.1 Introduction 331
11.2 Overview of Fatty Acid Metabolism 332
11.2.1 Fatty Acid Biosynthesis 332
11.2.2 Fatty Acid Degradation and Turnover 335
11.2.3 Accumulation of Storage Lipids 336
11.3 Basic Technologies for Production of Free Fatty Acids 337
11.3.1 Production of Free Fatty Acids in E. coli 337
11.3.2 Production of Free Fatty Acids in Cyanobacteria 338
11.4 Advanced Technologies for Enhancement of Free Fatty Acid Production 339
11.4.1 Enhancement of Fatty Acid Biosynthesis 339
11.4.2 Enhancement of Carbon Fixation Activity 345
11.4.3 Engineering of Carbon Flow: Modification of Key Regulatory Factors 345
11.4.4 Engineering of Carbon Flow: Deletion of Competitive Pathways 346
11.4.5 Mitigation of the Toxicity of FFAs 347
11.4.6 Enhancement of FFA Secretion 348
11.4.7 Induction of Cell Lysis 349
11.4.8 Recovery of Produced FFAs from Medium 350
11.4.9 Identification of Cyanobacterial Strains Suitable for FFA Production 350
11.5 Hydrocarbon Production in Cyanobacteria 351
11.6 Advanced Technologies for Enhancement of Hydrocarbon Production 353
11.6.1 Enhancement of Alk(a/e)ne Biosynthesis 353
11.6.2 Improvement of the Performance of Alkane Biosynthetic Enzymes 354
11.7 Basic Technologies for Production of Fatty Alcohols 355
11.8 Advanced Technologies for Enhancement of Fatty Alcohol Production 355
11.9 Basic Technologies for Production of Fatty Acid Alkyl Esters 356
11.10 Perspectives 357
References 358
12 Product Export in Cyanobacteria 369
Cátia F. Gonçalves, Steeve Lima, and Paulo Oliveira
12.1 Introduction 369
12.2 Secretion Mediated by Membrane-Embedded Systems 373
12.2.1 Proteins 373
12.2.2 Extracellular Polymeric Substances (EPS) 377
12.2.3 Soluble Sugars and Organic Acids 379
12.2.4 Fatty Acids 381
12.2.5 Alcohols 382
12.2.6 Terpenes 384
12.3 MV-Mediated Secretion 386
12.3.1 Structure and Biogenesis of Bacterial MVs 386
12.3.1.1 Cyanobacterial MVs 388
12.3.2 MVs as Novel Biotechnological Tools 389
12.4 Concluding Remarks 391
Acknowledgments 392
References 392
Part III Frontiers of Cyanobacteria Biotechnology 407
13 Harnessing Solar-Powered Oxic N2-fixing Cyanobacteria for the BioNitrogen Economy 409
James Young, Liping Gu, William Gibbons, and Ruanbao Zhou
13.1 Introduction 409
13.2 Physiology and Implications of Oxic Nitrogen Fixation 410
13.2.1 Ecological Range 411
13.2.2 Balancing Photosynthesis and Nitrogen Fixation 412
13.2.3 Energetic Demands and How the Cells Adapt 412
13.2.4 Impacts of Continuous Light vs Dark-Light Cycles 416
13.3 Major Biotechnology Applications for Diazotrophic Cyanobacteria 417
13.3.1 General Economic and Environmental Considerations of Diazotrophic Cyanobacteria 417
13.3.2 Metabolic Engineering of N2-Fixing Cyanobacteria for Carbon Compound Production 420
13.3.2.1 Direct Production of Biofuels 420
13.3.2.2 Cyanobacteria as a Fermentable Substrate 420
13.3.3 Metabolic Engineering of Nitrogen Fixing Cyanobacteria for Nitrogen-Rich Compound Production 422
13.3.3.1 Ammonia 422
13.3.3.2 Guanidine 423
13.3.3.3 Cyanophycin 423
13.3.3.4 Amino Acids and Proteins 423
13.3.4 Application of Diazotrophic Cyanobacteria in Agriculture 425
13.4 Conclusions 428
References 428
14 Traits of Fast-Growing Cyanobacteria 441
Meghna Srivastava, Elton P. Hudson, and Pramod P. Wangikar
14.1 Introduction 441
14.2 Why is Growth Rate Significant? 442
14.3 An Overview of Factors Affecting the Growth Rates of Cyanobacteria 446
14.3.1 Light Intensity and Quality 448
14.3.2 Mixotrophic Growth 451
14.3.3 Circadian Rhythm 451
14.3.4 Additional Factors Relating to Growth Rates in Cyanobacteria 452
14.3.4.1 Cell Morphology 453
14.3.4.2 Genome Size 453
14.3.4.3 Saltwater Tolerance 454
14.3.4.4 Nutrient Supplementation 454
14.3.5 Carbon Storage 455
14.4 Overview of the Fast-Growing Model Cyanobacteria 455
14.4.1 Synechococcus elongatus UTEX 2973 455
14.4.2 Synechococcus elongatus PCC 11801 456
14.4.3 Synechococcus sp. PCC 11901 456
14.4.4 Synechococcus sp. PCC 7002 457
14.5 Relationship Between Light Usage and Growth Rate in Model Strains 458
14.5.1 Case Study: The pmgA Mutant of Synechocystis 458
14.5.2 Case Study: The S. elongatus 7942 and S. elongatus 2973 Strains 460
14.6 Molecular Determinants of Fast Growth of S. elongatus UTEX 2973 460
14.7 Carbon Fluxes in Fast-Growing Strains Determined Using Metabolic Flux Analysis 463
14.8 Engineering Cyanobacteria for Fast Growth 465
14.8.1 Calvin Cycle Enzymes 465
14.8.2 PEP Carboxylase 466
14.8.3 Carbon and Light Uptake Proteins 467
14.9 Conclusion 468
References 468
15 Cyanobacterial Biofilms in Natural and Synthetic Environments 477
Christian David, Rohan Karande, and Katja Bühler
15.1 Motivation 477
15.2 Introduction to Biofilms: Biology and Applications 478
15.3 Cyanobacteria in Natural Biofilms and Microbial Mats 483
15.4 Introduction to (Photo-)biotechnology 484
15.5 Benefits of Microscale Systems for (Photo-)biofilm Cultivation 487
15.6 Oxygen Accumulation and Its Impacts 488
15.7 Resource Management in Biofilms 491
15.8 Applications of Photosynthetic Biofilms 493
15.8.1 Biofilms Enable High Cell Densities 497
15.8.2 Biofilms Enable Continuous Production 498
15.9 Outlook 499
References 499
16 Growth of Photosynthetic Microorganisms in Different Photobioreactors Operated Outdoors 505
Eleftherios Touloupakis and Pietro Carlozzi
16.1 Background 505
16.1.1 Photobiological Hydrogen Production 506
16.1.2 Polyhydroxyalkanoate Production by Photosynthetic Microbes 508
16.1.3 Photobioreactors 509
16.2 Case Studies of Outdoor Cultivations of Photosynthetic Microorganisms 513
16.2.1 Outdoor Cultures of Purple Non-Sulfur Bacteria for H2 and PHB Production 513
16.2.2 Outdoor Cultures of Cyanobacteria 516
16.3 Conclusion 517
Acknowledgments 519
References 519
Index 531
