Paul Hudson is an Associate Professor (2018) of Metabolic Engineering in the School of Engineering Sciences in Chemistry, Biotechnology, and Health at the Royal Institute of Technology (KTH) in Stockholm Sweden. He has a Ph.D. degree in Chemical Engineering from U.C. Berkeley (2009). He has published 26 research papers in the fields of protein science, microbial metabolic engineering, and systems biology. The main focus of his research is on systems and synthetic biology of cyanobacteria.

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• Foreword: Cyanobacteria Biotechnology xvAcknowledgments xviiiPart I Core Cyanobacteria Processes 11 Inorganic Carbon Assimilation in Cyanobacteria: Mechanisms, Regulation, and Engineering 3Martin Hagemann, Shanshan Song, and Eva-Maria Brouwer1.1 Introduction – The Need for a Carbon-Concentrating Mechanism 31.2 The Carbon-Concentrating Mechanism (CCM) Among Cyanobacteria 41.2.1 Ci Uptake Proteins/Mechanisms 51.2.2 Carboxysome and RubisCO 81.3 Regulation of Ci Assimilation 101.3.1 Regulation of the CCM 101.3.2 Further Regulation of Carbon Assimilation 131.3.3 Metabolic Changes and Regulation During Ci Acclimation 141.3.4 Redox Regulation of Ci Assimilation 151.4 Engineering the Cyanobacterial CCM 161.5 Photorespiration 171.5.1 Cyanobacterial Photorespiration 171.5.2 Attempts to Engineer Photorespiration 191.6 Concluding Remarks 20Acknowledgments 21References 212 Electron Transport in Cyanobacteria and Its Potential in Bioproduction 33David J. Lea-Smith and Guy T. Hanke2.1 Introduction 332.2 Electron Transport in a Bioenergetic Membrane 342.2.1 Linear Electron Transport 342.2.2 Cyclic Electron Transport 372.2.3 ATP Production from Linear and Cyclic Electron Transport 372.3 Respiratory Electron Transport 382.4 Role of Electron Sinks in Photoprotection 412.4.1 Terminal Oxidases 412.4.2 Hydrogenase and Flavodiiron Complexes 412.4.3 Carbon Fixation and Photorespiration 432.4.4 Extracellular Electron Export 442.5 Regulating Electron Flux into Different Pathways 452.5.1 Electron Flux Through the Plastoquinone Pool 452.5.2 Electron Flux Through Fdx 462.6 Spatial Organization of Electron Transport Complexes 472.7 Manipulating Electron Transport for Synthetic Biology Applications 482.7.1 Improving Growth of Cyanobacteria 492.7.2 Production of Electrical Power in BPVs 492.7.3 Hydrogen Production 502.7.4 Production of Industrial Compounds 502.8 Future Challenges in Cyanobacterial Electron Transport 51References 523 Optimizing the Spectral Fit Between Cyanobacteria and Solar Radiation in the Light of Sustainability Applications 65Klaas J. Hellingwerf, Que Chen, and Filipe Branco dos Santos3.1 Introduction 653.2 Molecular Basis and Efficiency of Oxygenic Photosynthesis 673.3 Fit Between the Spectrum of Solar Radiation and the Action Spectrum of Photosynthesis 723.4 Expansion of the PAR Region of Oxygenic Photosynthesis 743.5 Modulation and Optimization of the Transparency of Photobioreactors 793.6 Full Control of the Light Regime: LEDs Inside the PBR 813.7 Conclusions and Prospects 82References 83Part II Concepts in Metabolic Engineering 894 What We Can Learn from Measuring Metabolic Fluxes in Cyanobacteria 91Xiang Gao, Chao Wu, Michael Cantrell, Melissa Cano, Jianping Yu, and Wei Xiong4.1 Central Carbon Metabolism in Cyanobacteria: An Overview and Renewed Pathway Knowledge 914.1.1 Glycolytic Routes Interwoven with the Calvin Cycle 914.1.2 Tricarboxylic Acid Cycling 944.2 Methodologies for Predicting and Quantifying Metabolic Fluxes in Cyanobacteria 954.2.1 Flux Balance Analysis and Genome-Scale Reconstruction of Metabolic Network 954.2.2 13C-Metabolic Flux Analysis 964.2.3 Thermodynamic Analysis and Kinetics Analysis 994.3 Cyanobacteria Fluxome in Response to Altered Nutrient Modes and Environmental Conditions 1014.3.1 Autotrophic Fluxome 1014.3.2 Photomixotrophic Fluxome 1044.3.3 Heterotrophic Fluxome 1054.3.4 Photoheterotrophic Fluxome 1054.3.5 Diurnal Metabolite Oscillations 1064.3.6 Nutrient States’ Impact on Metabolic Flux 1074.4 Metabolic Fluxes Redirected in Cyanobacteria for Biomanufacturing Purposes 1084.4.1 Restructuring the TCA Cycle for Ethylene Production 1084.4.2 Maximizing Flux in the Isoprenoid Pathway 1094.4.2.1 Measuring Precursor Pool Size to Evaluate Potential Driving Forces for Isoprenoid Production 1094.4.2.2 Balancing Intermediates for Increased Pathway Activity 1104.4.2.3 Kinetic Flux Profiling to Detect Bottlenecks in the Pathway 1114.5 Synopsis and Future Directions 112Acknowledgments 112References 1125 Synthetic Biology in Cyanobacteria and Applications for Biotechnology 123Elton P. Hudson5.1 Introduction 1235.2 Getting Genes into Cyanobacteria 1235.2.1 Transformation 1235.2.2 Expression from Episomal Plasmids 1255.2.3 Delivery of Genes to the Chromosome 1275.3 Basic Synthetic Control of Gene Expression in Cyanobacteria 1295.3.1 Quantifying Transcription and Translation in Cyanobacteria 1305.3.2 Controlling Transcription with Synthetic Promoters 1345.3.2.1 Constitutive Promoters 1365.3.2.2 Regulated Promoters that Are Sensitive to Added Compounds (Inducible) 1375.3.2.3 CRISPR Interference for Transcriptional Repression 1395.3.3 Controlling Translation 1415.3.3.1 Ribosome Binding Sites (Cis-Acting) 1415.3.3.2 Riboswitches (Cis-Acting) 1425.3.3.3 Small RNAs (Trans-Acting) 1435.4 Exotic Signals for Controlling Expression 1435.4.1 Oxygen 1445.4.2 Light Color 1445.4.3 Cell Density or Growth Phase 1455.4.4 Engineering Regulators for Altered Sensing Properties: State of the Art 1475.5 Advanced Regulation: The Near Future 1485.5.1 Logic Gates and Timing Circuits 1485.5.2 Orthogonal Transcription Systems 1515.5.3 Synthetic Biology Solutions to Increase Stability 1525.5.4 Synthetic Biology Solutions for Cell Separation and Product Recovery 1545.6 Conclusions 157Acknowledgments 158References 1586 Sink Engineering in Photosynthetic Microbes 171María Santos-Merino, Amit K. Singh, and Daniel C. Ducat6.1 Introduction 1716.2 Source and Sink 1726.3 Regulation of Sink Energy in Plants 1776.3.1 Sucrose and Other Signaling Carbohydrates 1786.3.2 Hexokinases 1796.3.3 Sucrose Non-fermenting Related Kinases 1806.3.4 TOR Kinase 1816.3.5 Engineered Pathways as Sinks in Photosynthetic Microbes 1826.3.6 Sucrose 1836.3.7 2,3-Butanediol 1876.3.8 Ethylene 1876.3.9 Glycerol 1886.3.10 Isobutanol 1886.3.11 Isoprene 1896.3.12 Limonene 1896.3.13 P450, an Electron Sink 1906.4 What Are Key Source/Sink Regulatory Hubs in Photosynthetic Microbes? 1916.5 Concluding Remarks 194Acknowledgment 195References 1957 Design Principles for Engineering Metabolic Pathways in Cyanobacteria 211Jason T. Ku and Ethan I. Lan7.1 Introduction 2117.2 Cofactor Optimization 2127.2.1 Recruiting NADPH-Dependent Enzymes Wherever Possible 2157.2.2 Engineering NADH-Specific Enzymes to Utilize NADPH 2177.2.3 Increasing NADH Pool in Cyanobacteria Through Expression of Transhydrogenase 2187.3 Incorporation of Thermodynamic Driving Force into Metabolic Pathway Design 2197.3.1 ATP Driving Force in Metabolic Pathways 2207.3.2 Increasing Substrate Pool Supports the Carbon Flux Toward Products 2227.3.3 Product Removal Unblocks the Limitations of Product Titer 2237.4 Development of Synthetic Pathways for Carbon Conserving Photorespiration and Enhanced Carbon Fixation 2257.5 Summary and Future Perspective on Cyanobacterial Metabolic Engineering 229References 2298 Engineering Cyanobacteria for Efficient Photosynthetic Production: Ethanol Case Study 237Guodong Luan and Xuefeng Lu8.1 Introduction 2378.2 Pathway for Ethanol Synthesis in Cyanobacteria 2388.2.1 Pyruvate Decarboxylase and Type II Alcohol Dehydrogenase 2388.2.2 Selection of Better Enzymes in the Pdc–AdhII Pathway 2408.2.3 Systematic Characterization of the PdcZM–Slr1192 Pathway 2418.3 Selection of Optimal Cyanobacteria “Chassis,” Strain for Ethanol Production 2428.3.1 Synechococcus PCC 6803 and Synechococcus PCC 7942 2438.3.2 Synechococcus PCC 7002 2458.3.3 Anabaena PCC 7120 2458.3.4 Nonconventional Cyanobacteria Species 2468.4 Metabolic Engineering Strategies Toward More Efficient and Stable Ethanol Production 2468.4.1 Enhancing the Carbon Flux via Overexpression of Calvin Cycle Enzymes 2488.4.2 Blocking Pathways that Are Competitive to Ethanol 2488.4.3 Arresting Biomass Formation 2498.4.4 Engineering Cofactor Supply 2498.4.5 Engineering Strategies Guided by In Silico Simulation 2508.4.6 Stabilizing Ethanol Synthesis Capacity in Cyanobacterial Cell Factories 2518.5 Exploring the Response in Cyanobacteria to Ethanol 2538.5.1 Response of Cyanobacterial Cells Toward Exogenous Added Ethanol 2548.5.2 Response of Cyanobacteria to Endogenous Synthesized Ethanol 2558.6 Metabolic Engineering Strategies to Facilitate Robust Cultivation Against Biocontaminants 2568.6.1 Engineering Cyanobacteria Cell Factories to Adapt for Selective Environmental Stresses 2568.6.2 Engineering Cyanobacteria Cell Factories to Utilize Uncommon Nutrients 2588.7 Conclusions and Perspectives 258References 2599 Engineering Cyanobacteria as Host Organisms for Production of Terpenes and Terpenoids 267João S. Rodrigues and Pia Lindberg9.1 Terpenoids and Industrial Applications 2679.2 Terpenoid Biosynthesis in Cyanobacteria 2709.2.1 Methylerythritol-4-Phosphate Pathway 2709.2.2 Formation of Terpene Backbones 2729.3 Natural Occurrence and Physiological Roles of Terpenes and Terpenoids in Cyanobacteria 2749.4 Engineering Cyanobacteria for Terpenoid Production 2759.4.1 Metabolic Engineering 2779.4.1.1 Terpene Synthases 2779.4.1.2 Increasing Supply of Terpene Backbones 2859.4.1.3 Engineering the Native MEP Pathway 2869.4.1.4 Implementing the MVA Pathway 2879.4.1.5 Enhancing Precursor Supply 2889.4.2 Optimizing Growth Conditions for Production 2899.4.3 Product Capture and Harvesting 2919.5 Summary and Outlook 292Acknowledgments 293References 29310 Cyanobacterial Biopolymers 301Moritz Koch and Karl Forchhammer10.1 Polyhydroxybutryate 30110.1.1 Introduction 30110.1.2 PHB Metabolism in Cyanobacteria 30210.1.3 Industrial Applications of PHB 30510.1.3.1 Physical Properties of PHB and Its Derivatives 30510.1.3.2 Biodegradability 30610.1.3.3 Application of PHB as a Plastic 30610.1.3.4 Reactor Types 30610.1.3.5 Production Process 30710.1.3.6 Downstream Processing 30810.1.4 Metabolic Engineering of PHB Biosynthesis 30810.1.5 Limitations and Potential of PHB Production in Cyanobacteria 31010.2 Cyanophycin Granules in Cyanobacteria 31110.2.1 Biology of Cyanophycin 31110.2.2 Genes and Enzymes of CGP Metabolism 31510.2.2.1 Cyanophycin Synthetase 31510.2.2.2 Cyanophycin Degrading Enzymes 31610.2.3 Regulation of Cyanophycin Metabolism 31710.2.4 Cyanophycin Overproduction and Potential Industrial Applications 318Acknowledgement 319References 31911 Biosynthesis of Fatty Acid Derivatives by Cyanobacteria: From Basics to Biofuel Production 331Akihito Kawahara and Yukako Hihara11.1 Introduction 33111.2 Overview of Fatty Acid Metabolism 33211.2.1 Fatty Acid Biosynthesis 33211.2.2 Fatty Acid Degradation and Turnover 33511.2.3 Accumulation of Storage Lipids 33611.3 Basic Technologies for Production of Free Fatty Acids 33711.3.1 Production of Free Fatty Acids in E. coli 33711.3.2 Production of Free Fatty Acids in Cyanobacteria 33811.4 Advanced Technologies for Enhancement of Free Fatty Acid Production 33911.4.1 Enhancement of Fatty Acid Biosynthesis 33911.4.2 Enhancement of Carbon Fixation Activity 34511.4.3 Engineering of Carbon Flow: Modification of Key Regulatory Factors 34511.4.4 Engineering of Carbon Flow: Deletion of Competitive Pathways 34611.4.5 Mitigation of the Toxicity of FFAs 34711.4.6 Enhancement of FFA Secretion 34811.4.7 Induction of Cell Lysis 34911.4.8 Recovery of Produced FFAs from Medium 35011.4.9 Identification of Cyanobacterial Strains Suitable for FFA Production 35011.5 Hydrocarbon Production in Cyanobacteria 35111.6 Advanced Technologies for Enhancement of Hydrocarbon Production 35311.6.1 Enhancement of Alk(a/e)ne Biosynthesis 35311.6.2 Improvement of the Performance of Alkane Biosynthetic Enzymes 35411.7 Basic Technologies for Production of Fatty Alcohols 35511.8 Advanced Technologies for Enhancement of Fatty Alcohol Production 35511.9 Basic Technologies for Production of Fatty Acid Alkyl Esters 35611.10 Perspectives 357References 35812 Product Export in Cyanobacteria 369Cátia F. Gonçalves, Steeve Lima, and Paulo Oliveira12.1 Introduction 36912.2 Secretion Mediated by Membrane-Embedded Systems 37312.2.1 Proteins 37312.2.2 Extracellular Polymeric Substances (EPS) 37712.2.3 Soluble Sugars and Organic Acids 37912.2.4 Fatty Acids 38112.2.5 Alcohols 38212.2.6 Terpenes 38412.3 MV-Mediated Secretion 38612.3.1 Structure and Biogenesis of Bacterial MVs 38612.3.1.1 Cyanobacterial MVs 38812.3.2 MVs as Novel Biotechnological Tools 38912.4 Concluding Remarks 391Acknowledgments 392References 392Part III Frontiers of Cyanobacteria Biotechnology 40713 Harnessing Solar-Powered Oxic N2-fixing Cyanobacteria for the BioNitrogen Economy 409James Young, Liping Gu, William Gibbons, and Ruanbao Zhou13.1 Introduction 40913.2 Physiology and Implications of Oxic Nitrogen Fixation 41013.2.1 Ecological Range 41113.2.2 Balancing Photosynthesis and Nitrogen Fixation 41213.2.3 Energetic Demands and How the Cells Adapt 41213.2.4 Impacts of Continuous Light vs Dark–Light Cycles 41613.3 Major Biotechnology Applications for Diazotrophic Cyanobacteria 41713.3.1 General Economic and Environmental Considerations of Diazotrophic Cyanobacteria 41713.3.2 Metabolic Engineering of N2-Fixing Cyanobacteria for Carbon Compound Production 42013.3.2.1 Direct Production of Biofuels 42013.3.2.2 Cyanobacteria as a Fermentable Substrate 42013.3.3 Metabolic Engineering of Nitrogen Fixing Cyanobacteria for Nitrogen-Rich Compound Production 42213.3.3.1 Ammonia 42213.3.3.2 Guanidine 42313.3.3.3 Cyanophycin 42313.3.3.4 Amino Acids and Proteins 42313.3.4 Application of Diazotrophic Cyanobacteria in Agriculture 42513.4 Conclusions 428References 42814 Traits of Fast-Growing Cyanobacteria 441Meghna Srivastava, Elton P. Hudson, and Pramod P. Wangikar14.1 Introduction 44114.2 Why is Growth Rate Significant? 44214.3 An Overview of Factors Affecting the Growth Rates of Cyanobacteria 44614.3.1 Light Intensity and Quality 44814.3.2 Mixotrophic Growth 45114.3.3 Circadian Rhythm 45114.3.4 Additional Factors Relating to Growth Rates in Cyanobacteria 45214.3.4.1 Cell Morphology 45314.3.4.2 Genome Size 45314.3.4.3 Saltwater Tolerance 45414.3.4.4 Nutrient Supplementation 45414.3.5 Carbon Storage 45514.4 Overview of the Fast-Growing Model Cyanobacteria 45514.4.1 Synechococcus elongatus UTEX 2973 45514.4.2 Synechococcus elongatus PCC 11801 45614.4.3 Synechococcus sp. PCC 11901 45614.4.4 Synechococcus sp. PCC 7002 45714.5 Relationship Between Light Usage and Growth Rate in Model Strains 45814.5.1 Case Study: The pmgA Mutant of Synechocystis 45814.5.2 Case Study: The S. elongatus 7942 and S. elongatus 2973 Strains 46014.6 Molecular Determinants of Fast Growth of S. elongatus UTEX 2973 46014.7 Carbon Fluxes in Fast-Growing Strains Determined Using Metabolic Flux Analysis 46314.8 Engineering Cyanobacteria for Fast Growth 46514.8.1 Calvin Cycle Enzymes 46514.8.2 PEP Carboxylase 46614.8.3 Carbon and Light Uptake Proteins 46714.9 Conclusion 468References 46815 Cyanobacterial Biofilms in Natural and Synthetic Environments 477Christian David, Rohan Karande, and Katja Bühler15.1 Motivation 47715.2 Introduction to Biofilms: Biology and Applications 47815.3 Cyanobacteria in Natural Biofilms and Microbial Mats 48315.4 Introduction to (Photo-)biotechnology 48415.5 Benefits of Microscale Systems for (Photo-)biofilm Cultivation 48715.6 Oxygen Accumulation and Its Impacts 48815.7 Resource Management in Biofilms 49115.8 Applications of Photosynthetic Biofilms 49315.8.1 Biofilms Enable High Cell Densities 49715.8.2 Biofilms Enable Continuous Production 49815.9 Outlook 499References 49916 Growth of Photosynthetic Microorganisms in Different Photobioreactors Operated Outdoors 505Eleftherios Touloupakis and Pietro Carlozzi16.1 Background 50516.1.1 Photobiological Hydrogen Production 50616.1.2 Polyhydroxyalkanoate Production by Photosynthetic Microbes 50816.1.3 Photobioreactors 50916.2 Case Studies of Outdoor Cultivations of Photosynthetic Microorganisms 51316.2.1 Outdoor Cultures of Purple Non-Sulfur Bacteria for H2 and PHB Production 51316.2.2 Outdoor Cultures of Cyanobacteria 51616.3 Conclusion 517Acknowledgments 519References 519Index 531

Paul Hudson is an Associate Professor (2018) of Metabolic Engineering in the School of Engineering Sciences in Chemistry, Biotechnology, and Health at the Royal Institute of Technology (KTH) in Stockholm Sweden. He has a Ph.D. degree in Chemical Engineering from U.C. Berkeley (2009). He has published 26 research papers in the fields of protein science, microbial metabolic engineering, and systems biology. The main focus of his research is on systems and synthetic biology of cyanobacteria.

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• Foreword: Cyanobacteria Biotechnology xvAcknowledgments xviiiPart I Core Cyanobacteria Processes 11 Inorganic Carbon Assimilation in Cyanobacteria: Mechanisms, Regulation, and Engineering 3Martin Hagemann, Shanshan Song, and Eva-Maria Brouwer1.1 Introduction – The Need for a Carbon-Concentrating Mechanism 31.2 The Carbon-Concentrating Mechanism (CCM) Among Cyanobacteria 41.2.1 Ci Uptake Proteins/Mechanisms 51.2.2 Carboxysome and RubisCO 81.3 Regulation of Ci Assimilation 101.3.1 Regulation of the CCM 101.3.2 Further Regulation of Carbon Assimilation 131.3.3 Metabolic Changes and Regulation During Ci Acclimation 141.3.4 Redox Regulation of Ci Assimilation 151.4 Engineering the Cyanobacterial CCM 161.5 Photorespiration 171.5.1 Cyanobacterial Photorespiration 171.5.2 Attempts to Engineer Photorespiration 191.6 Concluding Remarks 20Acknowledgments 21References 212 Electron Transport in Cyanobacteria and Its Potential in Bioproduction 33David J. Lea-Smith and Guy T. Hanke2.1 Introduction 332.2 Electron Transport in a Bioenergetic Membrane 342.2.1 Linear Electron Transport 342.2.2 Cyclic Electron Transport 372.2.3 ATP Production from Linear and Cyclic Electron Transport 372.3 Respiratory Electron Transport 382.4 Role of Electron Sinks in Photoprotection 412.4.1 Terminal Oxidases 412.4.2 Hydrogenase and Flavodiiron Complexes 412.4.3 Carbon Fixation and Photorespiration 432.4.4 Extracellular Electron Export 442.5 Regulating Electron Flux into Different Pathways 452.5.1 Electron Flux Through the Plastoquinone Pool 452.5.2 Electron Flux Through Fdx 462.6 Spatial Organization of Electron Transport Complexes 472.7 Manipulating Electron Transport for Synthetic Biology Applications 482.7.1 Improving Growth of Cyanobacteria 492.7.2 Production of Electrical Power in BPVs 492.7.3 Hydrogen Production 502.7.4 Production of Industrial Compounds 502.8 Future Challenges in Cyanobacterial Electron Transport 51References 523 Optimizing the Spectral Fit Between Cyanobacteria and Solar Radiation in the Light of Sustainability Applications 65Klaas J. Hellingwerf, Que Chen, and Filipe Branco dos Santos3.1 Introduction 653.2 Molecular Basis and Efficiency of Oxygenic Photosynthesis 673.3 Fit Between the Spectrum of Solar Radiation and the Action Spectrum of Photosynthesis 723.4 Expansion of the PAR Region of Oxygenic Photosynthesis 743.5 Modulation and Optimization of the Transparency of Photobioreactors 793.6 Full Control of the Light Regime: LEDs Inside the PBR 813.7 Conclusions and Prospects 82References 83Part II Concepts in Metabolic Engineering 894 What We Can Learn from Measuring Metabolic Fluxes in Cyanobacteria 91Xiang Gao, Chao Wu, Michael Cantrell, Melissa Cano, Jianping Yu, and Wei Xiong4.1 Central Carbon Metabolism in Cyanobacteria: An Overview and Renewed Pathway Knowledge 914.1.1 Glycolytic Routes Interwoven with the Calvin Cycle 914.1.2 Tricarboxylic Acid Cycling 944.2 Methodologies for Predicting and Quantifying Metabolic Fluxes in Cyanobacteria 954.2.1 Flux Balance Analysis and Genome-Scale Reconstruction of Metabolic Network 954.2.2 13C-Metabolic Flux Analysis 964.2.3 Thermodynamic Analysis and Kinetics Analysis 994.3 Cyanobacteria Fluxome in Response to Altered Nutrient Modes and Environmental Conditions 1014.3.1 Autotrophic Fluxome 1014.3.2 Photomixotrophic Fluxome 1044.3.3 Heterotrophic Fluxome 1054.3.4 Photoheterotrophic Fluxome 1054.3.5 Diurnal Metabolite Oscillations 1064.3.6 Nutrient States’ Impact on Metabolic Flux 1074.4 Metabolic Fluxes Redirected in Cyanobacteria for Biomanufacturing Purposes 1084.4.1 Restructuring the TCA Cycle for Ethylene Production 1084.4.2 Maximizing Flux in the Isoprenoid Pathway 1094.4.2.1 Measuring Precursor Pool Size to Evaluate Potential Driving Forces for Isoprenoid Production 1094.4.2.2 Balancing Intermediates for Increased Pathway Activity 1104.4.2.3 Kinetic Flux Profiling to Detect Bottlenecks in the Pathway 1114.5 Synopsis and Future Directions 112Acknowledgments 112References 1125 Synthetic Biology in Cyanobacteria and Applications for Biotechnology 123Elton P. Hudson5.1 Introduction 1235.2 Getting Genes into Cyanobacteria 1235.2.1 Transformation 1235.2.2 Expression from Episomal Plasmids 1255.2.3 Delivery of Genes to the Chromosome 1275.3 Basic Synthetic Control of Gene Expression in Cyanobacteria 1295.3.1 Quantifying Transcription and Translation in Cyanobacteria 1305.3.2 Controlling Transcription with Synthetic Promoters 1345.3.2.1 Constitutive Promoters 1365.3.2.2 Regulated Promoters that Are Sensitive to Added Compounds (Inducible) 1375.3.2.3 CRISPR Interference for Transcriptional Repression 1395.3.3 Controlling Translation 1415.3.3.1 Ribosome Binding Sites (Cis-Acting) 1415.3.3.2 Riboswitches (Cis-Acting) 1425.3.3.3 Small RNAs (Trans-Acting) 1435.4 Exotic Signals for Controlling Expression 1435.4.1 Oxygen 1445.4.2 Light Color 1445.4.3 Cell Density or Growth Phase 1455.4.4 Engineering Regulators for Altered Sensing Properties: State of the Art 1475.5 Advanced Regulation: The Near Future 1485.5.1 Logic Gates and Timing Circuits 1485.5.2 Orthogonal Transcription Systems 1515.5.3 Synthetic Biology Solutions to Increase Stability 1525.5.4 Synthetic Biology Solutions for Cell Separation and Product Recovery 1545.6 Conclusions 157Acknowledgments 158References 1586 Sink Engineering in Photosynthetic Microbes 171María Santos-Merino, Amit K. Singh, and Daniel C. Ducat6.1 Introduction 1716.2 Source and Sink 1726.3 Regulation of Sink Energy in Plants 1776.3.1 Sucrose and Other Signaling Carbohydrates 1786.3.2 Hexokinases 1796.3.3 Sucrose Non-fermenting Related Kinases 1806.3.4 TOR Kinase 1816.3.5 Engineered Pathways as Sinks in Photosynthetic Microbes 1826.3.6 Sucrose 1836.3.7 2,3-Butanediol 1876.3.8 Ethylene 1876.3.9 Glycerol 1886.3.10 Isobutanol 1886.3.11 Isoprene 1896.3.12 Limonene 1896.3.13 P450, an Electron Sink 1906.4 What Are Key Source/Sink Regulatory Hubs in Photosynthetic Microbes? 1916.5 Concluding Remarks 194Acknowledgment 195References 1957 Design Principles for Engineering Metabolic Pathways in Cyanobacteria 211Jason T. Ku and Ethan I. Lan7.1 Introduction 2117.2 Cofactor Optimization 2127.2.1 Recruiting NADPH-Dependent Enzymes Wherever Possible 2157.2.2 Engineering NADH-Specific Enzymes to Utilize NADPH 2177.2.3 Increasing NADH Pool in Cyanobacteria Through Expression of Transhydrogenase 2187.3 Incorporation of Thermodynamic Driving Force into Metabolic Pathway Design 2197.3.1 ATP Driving Force in Metabolic Pathways 2207.3.2 Increasing Substrate Pool Supports the Carbon Flux Toward Products 2227.3.3 Product Removal Unblocks the Limitations of Product Titer 2237.4 Development of Synthetic Pathways for Carbon Conserving Photorespiration and Enhanced Carbon Fixation 2257.5 Summary and Future Perspective on Cyanobacterial Metabolic Engineering 229References 2298 Engineering Cyanobacteria for Efficient Photosynthetic Production: Ethanol Case Study 237Guodong Luan and Xuefeng Lu8.1 Introduction 2378.2 Pathway for Ethanol Synthesis in Cyanobacteria 2388.2.1 Pyruvate Decarboxylase and Type II Alcohol Dehydrogenase 2388.2.2 Selection of Better Enzymes in the Pdc–AdhII Pathway 2408.2.3 Systematic Characterization of the PdcZM–Slr1192 Pathway 2418.3 Selection of Optimal Cyanobacteria “Chassis,” Strain for Ethanol Production 2428.3.1 Synechococcus PCC 6803 and Synechococcus PCC 7942 2438.3.2 Synechococcus PCC 7002 2458.3.3 Anabaena PCC 7120 2458.3.4 Nonconventional Cyanobacteria Species 2468.4 Metabolic Engineering Strategies Toward More Efficient and Stable Ethanol Production 2468.4.1 Enhancing the Carbon Flux via Overexpression of Calvin Cycle Enzymes 2488.4.2 Blocking Pathways that Are Competitive to Ethanol 2488.4.3 Arresting Biomass Formation 2498.4.4 Engineering Cofactor Supply 2498.4.5 Engineering Strategies Guided by In Silico Simulation 2508.4.6 Stabilizing Ethanol Synthesis Capacity in Cyanobacterial Cell Factories 2518.5 Exploring the Response in Cyanobacteria to Ethanol 2538.5.1 Response of Cyanobacterial Cells Toward Exogenous Added Ethanol 2548.5.2 Response of Cyanobacteria to Endogenous Synthesized Ethanol 2558.6 Metabolic Engineering Strategies to Facilitate Robust Cultivation Against Biocontaminants 2568.6.1 Engineering Cyanobacteria Cell Factories to Adapt for Selective Environmental Stresses 2568.6.2 Engineering Cyanobacteria Cell Factories to Utilize Uncommon Nutrients 2588.7 Conclusions and Perspectives 258References 2599 Engineering Cyanobacteria as Host Organisms for Production of Terpenes and Terpenoids 267João S. Rodrigues and Pia Lindberg9.1 Terpenoids and Industrial Applications 2679.2 Terpenoid Biosynthesis in Cyanobacteria 2709.2.1 Methylerythritol-4-Phosphate Pathway 2709.2.2 Formation of Terpene Backbones 2729.3 Natural Occurrence and Physiological Roles of Terpenes and Terpenoids in Cyanobacteria 2749.4 Engineering Cyanobacteria for Terpenoid Production 2759.4.1 Metabolic Engineering 2779.4.1.1 Terpene Synthases 2779.4.1.2 Increasing Supply of Terpene Backbones 2859.4.1.3 Engineering the Native MEP Pathway 2869.4.1.4 Implementing the MVA Pathway 2879.4.1.5 Enhancing Precursor Supply 2889.4.2 Optimizing Growth Conditions for Production 2899.4.3 Product Capture and Harvesting 2919.5 Summary and Outlook 292Acknowledgments 293References 29310 Cyanobacterial Biopolymers 301Moritz Koch and Karl Forchhammer10.1 Polyhydroxybutryate 30110.1.1 Introduction 30110.1.2 PHB Metabolism in Cyanobacteria 30210.1.3 Industrial Applications of PHB 30510.1.3.1 Physical Properties of PHB and Its Derivatives 30510.1.3.2 Biodegradability 30610.1.3.3 Application of PHB as a Plastic 30610.1.3.4 Reactor Types 30610.1.3.5 Production Process 30710.1.3.6 Downstream Processing 30810.1.4 Metabolic Engineering of PHB Biosynthesis 30810.1.5 Limitations and Potential of PHB Production in Cyanobacteria 31010.2 Cyanophycin Granules in Cyanobacteria 31110.2.1 Biology of Cyanophycin 31110.2.2 Genes and Enzymes of CGP Metabolism 31510.2.2.1 Cyanophycin Synthetase 31510.2.2.2 Cyanophycin Degrading Enzymes 31610.2.3 Regulation of Cyanophycin Metabolism 31710.2.4 Cyanophycin Overproduction and Potential Industrial Applications 318Acknowledgement 319References 31911 Biosynthesis of Fatty Acid Derivatives by Cyanobacteria: From Basics to Biofuel Production 331Akihito Kawahara and Yukako Hihara11.1 Introduction 33111.2 Overview of Fatty Acid Metabolism 33211.2.1 Fatty Acid Biosynthesis 33211.2.2 Fatty Acid Degradation and Turnover 33511.2.3 Accumulation of Storage Lipids 33611.3 Basic Technologies for Production of Free Fatty Acids 33711.3.1 Production of Free Fatty Acids in E. coli 33711.3.2 Production of Free Fatty Acids in Cyanobacteria 33811.4 Advanced Technologies for Enhancement of Free Fatty Acid Production 33911.4.1 Enhancement of Fatty Acid Biosynthesis 33911.4.2 Enhancement of Carbon Fixation Activity 34511.4.3 Engineering of Carbon Flow: Modification of Key Regulatory Factors 34511.4.4 Engineering of Carbon Flow: Deletion of Competitive Pathways 34611.4.5 Mitigation of the Toxicity of FFAs 34711.4.6 Enhancement of FFA Secretion 34811.4.7 Induction of Cell Lysis 34911.4.8 Recovery of Produced FFAs from Medium 35011.4.9 Identification of Cyanobacterial Strains Suitable for FFA Production 35011.5 Hydrocarbon Production in Cyanobacteria 35111.6 Advanced Technologies for Enhancement of Hydrocarbon Production 35311.6.1 Enhancement of Alk(a/e)ne Biosynthesis 35311.6.2 Improvement of the Performance of Alkane Biosynthetic Enzymes 35411.7 Basic Technologies for Production of Fatty Alcohols 35511.8 Advanced Technologies for Enhancement of Fatty Alcohol Production 35511.9 Basic Technologies for Production of Fatty Acid Alkyl Esters 35611.10 Perspectives 357References 35812 Product Export in Cyanobacteria 369Cátia F. Gonçalves, Steeve Lima, and Paulo Oliveira12.1 Introduction 36912.2 Secretion Mediated by Membrane-Embedded Systems 37312.2.1 Proteins 37312.2.2 Extracellular Polymeric Substances (EPS) 37712.2.3 Soluble Sugars and Organic Acids 37912.2.4 Fatty Acids 38112.2.5 Alcohols 38212.2.6 Terpenes 38412.3 MV-Mediated Secretion 38612.3.1 Structure and Biogenesis of Bacterial MVs 38612.3.1.1 Cyanobacterial MVs 38812.3.2 MVs as Novel Biotechnological Tools 38912.4 Concluding Remarks 391Acknowledgments 392References 392Part III Frontiers of Cyanobacteria Biotechnology 40713 Harnessing Solar-Powered Oxic N2-fixing Cyanobacteria for the BioNitrogen Economy 409James Young, Liping Gu, William Gibbons, and Ruanbao Zhou13.1 Introduction 40913.2 Physiology and Implications of Oxic Nitrogen Fixation 41013.2.1 Ecological Range 41113.2.2 Balancing Photosynthesis and Nitrogen Fixation 41213.2.3 Energetic Demands and How the Cells Adapt 41213.2.4 Impacts of Continuous Light vs Dark–Light Cycles 41613.3 Major Biotechnology Applications for Diazotrophic Cyanobacteria 41713.3.1 General Economic and Environmental Considerations of Diazotrophic Cyanobacteria 41713.3.2 Metabolic Engineering of N2-Fixing Cyanobacteria for Carbon Compound Production 42013.3.2.1 Direct Production of Biofuels 42013.3.2.2 Cyanobacteria as a Fermentable Substrate 42013.3.3 Metabolic Engineering of Nitrogen Fixing Cyanobacteria for Nitrogen-Rich Compound Production 42213.3.3.1 Ammonia 42213.3.3.2 Guanidine 42313.3.3.3 Cyanophycin 42313.3.3.4 Amino Acids and Proteins 42313.3.4 Application of Diazotrophic Cyanobacteria in Agriculture 42513.4 Conclusions 428References 42814 Traits of Fast-Growing Cyanobacteria 441Meghna Srivastava, Elton P. Hudson, and Pramod P. Wangikar14.1 Introduction 44114.2 Why is Growth Rate Significant? 44214.3 An Overview of Factors Affecting the Growth Rates of Cyanobacteria 44614.3.1 Light Intensity and Quality 44814.3.2 Mixotrophic Growth 45114.3.3 Circadian Rhythm 45114.3.4 Additional Factors Relating to Growth Rates in Cyanobacteria 45214.3.4.1 Cell Morphology 45314.3.4.2 Genome Size 45314.3.4.3 Saltwater Tolerance 45414.3.4.4 Nutrient Supplementation 45414.3.5 Carbon Storage 45514.4 Overview of the Fast-Growing Model Cyanobacteria 45514.4.1 Synechococcus elongatus UTEX 2973 45514.4.2 Synechococcus elongatus PCC 11801 45614.4.3 Synechococcus sp. PCC 11901 45614.4.4 Synechococcus sp. PCC 7002 45714.5 Relationship Between Light Usage and Growth Rate in Model Strains 45814.5.1 Case Study: The pmgA Mutant of Synechocystis 45814.5.2 Case Study: The S. elongatus 7942 and S. elongatus 2973 Strains 46014.6 Molecular Determinants of Fast Growth of S. elongatus UTEX 2973 46014.7 Carbon Fluxes in Fast-Growing Strains Determined Using Metabolic Flux Analysis 46314.8 Engineering Cyanobacteria for Fast Growth 46514.8.1 Calvin Cycle Enzymes 46514.8.2 PEP Carboxylase 46614.8.3 Carbon and Light Uptake Proteins 46714.9 Conclusion 468References 46815 Cyanobacterial Biofilms in Natural and Synthetic Environments 477Christian David, Rohan Karande, and Katja Bühler15.1 Motivation 47715.2 Introduction to Biofilms: Biology and Applications 47815.3 Cyanobacteria in Natural Biofilms and Microbial Mats 48315.4 Introduction to (Photo-)biotechnology 48415.5 Benefits of Microscale Systems for (Photo-)biofilm Cultivation 48715.6 Oxygen Accumulation and Its Impacts 48815.7 Resource Management in Biofilms 49115.8 Applications of Photosynthetic Biofilms 49315.8.1 Biofilms Enable High Cell Densities 49715.8.2 Biofilms Enable Continuous Production 49815.9 Outlook 499References 49916 Growth of Photosynthetic Microorganisms in Different Photobioreactors Operated Outdoors 505Eleftherios Touloupakis and Pietro Carlozzi16.1 Background 50516.1.1 Photobiological Hydrogen Production 50616.1.2 Polyhydroxyalkanoate Production by Photosynthetic Microbes 50816.1.3 Photobioreactors 50916.2 Case Studies of Outdoor Cultivations of Photosynthetic Microorganisms 51316.2.1 Outdoor Cultures of Purple Non-Sulfur Bacteria for H2 and PHB Production 51316.2.2 Outdoor Cultures of Cyanobacteria 51616.3 Conclusion 517Acknowledgments 519References 519Index 531

Paul Hudson is an Associate Professor (2018) of Metabolic Engineering in the School of Engineering Sciences in Chemistry, Biotechnology, and Health at the Royal Institute of Technology (KTH) in Stockholm Sweden. He has a Ph.D. degree in Chemical Engineering from U.C. Berkeley (2009). He has published 26 research papers in the fields of protein science, microbial metabolic engineering, and systems biology. The main focus of his research is on systems and synthetic biology of cyanobacteria.

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• Foreword: Cyanobacteria Biotechnology xvAcknowledgments xviiiPart I Core Cyanobacteria Processes 11 Inorganic Carbon Assimilation in Cyanobacteria: Mechanisms, Regulation, and Engineering 3Martin Hagemann, Shanshan Song, and Eva-Maria Brouwer1.1 Introduction – The Need for a Carbon-Concentrating Mechanism 31.2 The Carbon-Concentrating Mechanism (CCM) Among Cyanobacteria 41.2.1 Ci Uptake Proteins/Mechanisms 51.2.2 Carboxysome and RubisCO 81.3 Regulation of Ci Assimilation 101.3.1 Regulation of the CCM 101.3.2 Further Regulation of Carbon Assimilation 131.3.3 Metabolic Changes and Regulation During Ci Acclimation 141.3.4 Redox Regulation of Ci Assimilation 151.4 Engineering the Cyanobacterial CCM 161.5 Photorespiration 171.5.1 Cyanobacterial Photorespiration 171.5.2 Attempts to Engineer Photorespiration 191.6 Concluding Remarks 20Acknowledgments 21References 212 Electron Transport in Cyanobacteria and Its Potential in Bioproduction 33David J. Lea-Smith and Guy T. Hanke2.1 Introduction 332.2 Electron Transport in a Bioenergetic Membrane 342.2.1 Linear Electron Transport 342.2.2 Cyclic Electron Transport 372.2.3 ATP Production from Linear and Cyclic Electron Transport 372.3 Respiratory Electron Transport 382.4 Role of Electron Sinks in Photoprotection 412.4.1 Terminal Oxidases 412.4.2 Hydrogenase and Flavodiiron Complexes 412.4.3 Carbon Fixation and Photorespiration 432.4.4 Extracellular Electron Export 442.5 Regulating Electron Flux into Different Pathways 452.5.1 Electron Flux Through the Plastoquinone Pool 452.5.2 Electron Flux Through Fdx 462.6 Spatial Organization of Electron Transport Complexes 472.7 Manipulating Electron Transport for Synthetic Biology Applications 482.7.1 Improving Growth of Cyanobacteria 492.7.2 Production of Electrical Power in BPVs 492.7.3 Hydrogen Production 502.7.4 Production of Industrial Compounds 502.8 Future Challenges in Cyanobacterial Electron Transport 51References 523 Optimizing the Spectral Fit Between Cyanobacteria and Solar Radiation in the Light of Sustainability Applications 65Klaas J. Hellingwerf, Que Chen, and Filipe Branco dos Santos3.1 Introduction 653.2 Molecular Basis and Efficiency of Oxygenic Photosynthesis 673.3 Fit Between the Spectrum of Solar Radiation and the Action Spectrum of Photosynthesis 723.4 Expansion of the PAR Region of Oxygenic Photosynthesis 743.5 Modulation and Optimization of the Transparency of Photobioreactors 793.6 Full Control of the Light Regime: LEDs Inside the PBR 813.7 Conclusions and Prospects 82References 83Part II Concepts in Metabolic Engineering 894 What We Can Learn from Measuring Metabolic Fluxes in Cyanobacteria 91Xiang Gao, Chao Wu, Michael Cantrell, Melissa Cano, Jianping Yu, and Wei Xiong4.1 Central Carbon Metabolism in Cyanobacteria: An Overview and Renewed Pathway Knowledge 914.1.1 Glycolytic Routes Interwoven with the Calvin Cycle 914.1.2 Tricarboxylic Acid Cycling 944.2 Methodologies for Predicting and Quantifying Metabolic Fluxes in Cyanobacteria 954.2.1 Flux Balance Analysis and Genome-Scale Reconstruction of Metabolic Network 954.2.2 13C-Metabolic Flux Analysis 964.2.3 Thermodynamic Analysis and Kinetics Analysis 994.3 Cyanobacteria Fluxome in Response to Altered Nutrient Modes and Environmental Conditions 1014.3.1 Autotrophic Fluxome 1014.3.2 Photomixotrophic Fluxome 1044.3.3 Heterotrophic Fluxome 1054.3.4 Photoheterotrophic Fluxome 1054.3.5 Diurnal Metabolite Oscillations 1064.3.6 Nutrient States’ Impact on Metabolic Flux 1074.4 Metabolic Fluxes Redirected in Cyanobacteria for Biomanufacturing Purposes 1084.4.1 Restructuring the TCA Cycle for Ethylene Production 1084.4.2 Maximizing Flux in the Isoprenoid Pathway 1094.4.2.1 Measuring Precursor Pool Size to Evaluate Potential Driving Forces for Isoprenoid Production 1094.4.2.2 Balancing Intermediates for Increased Pathway Activity 1104.4.2.3 Kinetic Flux Profiling to Detect Bottlenecks in the Pathway 1114.5 Synopsis and Future Directions 112Acknowledgments 112References 1125 Synthetic Biology in Cyanobacteria and Applications for Biotechnology 123Elton P. Hudson5.1 Introduction 1235.2 Getting Genes into Cyanobacteria 1235.2.1 Transformation 1235.2.2 Expression from Episomal Plasmids 1255.2.3 Delivery of Genes to the Chromosome 1275.3 Basic Synthetic Control of Gene Expression in Cyanobacteria 1295.3.1 Quantifying Transcription and Translation in Cyanobacteria 1305.3.2 Controlling Transcription with Synthetic Promoters 1345.3.2.1 Constitutive Promoters 1365.3.2.2 Regulated Promoters that Are Sensitive to Added Compounds (Inducible) 1375.3.2.3 CRISPR Interference for Transcriptional Repression 1395.3.3 Controlling Translation 1415.3.3.1 Ribosome Binding Sites (Cis-Acting) 1415.3.3.2 Riboswitches (Cis-Acting) 1425.3.3.3 Small RNAs (Trans-Acting) 1435.4 Exotic Signals for Controlling Expression 1435.4.1 Oxygen 1445.4.2 Light Color 1445.4.3 Cell Density or Growth Phase 1455.4.4 Engineering Regulators for Altered Sensing Properties: State of the Art 1475.5 Advanced Regulation: The Near Future 1485.5.1 Logic Gates and Timing Circuits 1485.5.2 Orthogonal Transcription Systems 1515.5.3 Synthetic Biology Solutions to Increase Stability 1525.5.4 Synthetic Biology Solutions for Cell Separation and Product Recovery 1545.6 Conclusions 157Acknowledgments 158References 1586 Sink Engineering in Photosynthetic Microbes 171María Santos-Merino, Amit K. Singh, and Daniel C. Ducat6.1 Introduction 1716.2 Source and Sink 1726.3 Regulation of Sink Energy in Plants 1776.3.1 Sucrose and Other Signaling Carbohydrates 1786.3.2 Hexokinases 1796.3.3 Sucrose Non-fermenting Related Kinases 1806.3.4 TOR Kinase 1816.3.5 Engineered Pathways as Sinks in Photosynthetic Microbes 1826.3.6 Sucrose 1836.3.7 2,3-Butanediol 1876.3.8 Ethylene 1876.3.9 Glycerol 1886.3.10 Isobutanol 1886.3.11 Isoprene 1896.3.12 Limonene 1896.3.13 P450, an Electron Sink 1906.4 What Are Key Source/Sink Regulatory Hubs in Photosynthetic Microbes? 1916.5 Concluding Remarks 194Acknowledgment 195References 1957 Design Principles for Engineering Metabolic Pathways in Cyanobacteria 211Jason T. Ku and Ethan I. Lan7.1 Introduction 2117.2 Cofactor Optimization 2127.2.1 Recruiting NADPH-Dependent Enzymes Wherever Possible 2157.2.2 Engineering NADH-Specific Enzymes to Utilize NADPH 2177.2.3 Increasing NADH Pool in Cyanobacteria Through Expression of Transhydrogenase 2187.3 Incorporation of Thermodynamic Driving Force into Metabolic Pathway Design 2197.3.1 ATP Driving Force in Metabolic Pathways 2207.3.2 Increasing Substrate Pool Supports the Carbon Flux Toward Products 2227.3.3 Product Removal Unblocks the Limitations of Product Titer 2237.4 Development of Synthetic Pathways for Carbon Conserving Photorespiration and Enhanced Carbon Fixation 2257.5 Summary and Future Perspective on Cyanobacterial Metabolic Engineering 229References 2298 Engineering Cyanobacteria for Efficient Photosynthetic Production: Ethanol Case Study 237Guodong Luan and Xuefeng Lu8.1 Introduction 2378.2 Pathway for Ethanol Synthesis in Cyanobacteria 2388.2.1 Pyruvate Decarboxylase and Type II Alcohol Dehydrogenase 2388.2.2 Selection of Better Enzymes in the Pdc–AdhII Pathway 2408.2.3 Systematic Characterization of the PdcZM–Slr1192 Pathway 2418.3 Selection of Optimal Cyanobacteria “Chassis,” Strain for Ethanol Production 2428.3.1 Synechococcus PCC 6803 and Synechococcus PCC 7942 2438.3.2 Synechococcus PCC 7002 2458.3.3 Anabaena PCC 7120 2458.3.4 Nonconventional Cyanobacteria Species 2468.4 Metabolic Engineering Strategies Toward More Efficient and Stable Ethanol Production 2468.4.1 Enhancing the Carbon Flux via Overexpression of Calvin Cycle Enzymes 2488.4.2 Blocking Pathways that Are Competitive to Ethanol 2488.4.3 Arresting Biomass Formation 2498.4.4 Engineering Cofactor Supply 2498.4.5 Engineering Strategies Guided by In Silico Simulation 2508.4.6 Stabilizing Ethanol Synthesis Capacity in Cyanobacterial Cell Factories 2518.5 Exploring the Response in Cyanobacteria to Ethanol 2538.5.1 Response of Cyanobacterial Cells Toward Exogenous Added Ethanol 2548.5.2 Response of Cyanobacteria to Endogenous Synthesized Ethanol 2558.6 Metabolic Engineering Strategies to Facilitate Robust Cultivation Against Biocontaminants 2568.6.1 Engineering Cyanobacteria Cell Factories to Adapt for Selective Environmental Stresses 2568.6.2 Engineering Cyanobacteria Cell Factories to Utilize Uncommon Nutrients 2588.7 Conclusions and Perspectives 258References 2599 Engineering Cyanobacteria as Host Organisms for Production of Terpenes and Terpenoids 267João S. Rodrigues and Pia Lindberg9.1 Terpenoids and Industrial Applications 2679.2 Terpenoid Biosynthesis in Cyanobacteria 2709.2.1 Methylerythritol-4-Phosphate Pathway 2709.2.2 Formation of Terpene Backbones 2729.3 Natural Occurrence and Physiological Roles of Terpenes and Terpenoids in Cyanobacteria 2749.4 Engineering Cyanobacteria for Terpenoid Production 2759.4.1 Metabolic Engineering 2779.4.1.1 Terpene Synthases 2779.4.1.2 Increasing Supply of Terpene Backbones 2859.4.1.3 Engineering the Native MEP Pathway 2869.4.1.4 Implementing the MVA Pathway 2879.4.1.5 Enhancing Precursor Supply 2889.4.2 Optimizing Growth Conditions for Production 2899.4.3 Product Capture and Harvesting 2919.5 Summary and Outlook 292Acknowledgments 293References 29310 Cyanobacterial Biopolymers 301Moritz Koch and Karl Forchhammer10.1 Polyhydroxybutryate 30110.1.1 Introduction 30110.1.2 PHB Metabolism in Cyanobacteria 30210.1.3 Industrial Applications of PHB 30510.1.3.1 Physical Properties of PHB and Its Derivatives 30510.1.3.2 Biodegradability 30610.1.3.3 Application of PHB as a Plastic 30610.1.3.4 Reactor Types 30610.1.3.5 Production Process 30710.1.3.6 Downstream Processing 30810.1.4 Metabolic Engineering of PHB Biosynthesis 30810.1.5 Limitations and Potential of PHB Production in Cyanobacteria 31010.2 Cyanophycin Granules in Cyanobacteria 31110.2.1 Biology of Cyanophycin 31110.2.2 Genes and Enzymes of CGP Metabolism 31510.2.2.1 Cyanophycin Synthetase 31510.2.2.2 Cyanophycin Degrading Enzymes 31610.2.3 Regulation of Cyanophycin Metabolism 31710.2.4 Cyanophycin Overproduction and Potential Industrial Applications 318Acknowledgement 319References 31911 Biosynthesis of Fatty Acid Derivatives by Cyanobacteria: From Basics to Biofuel Production 331Akihito Kawahara and Yukako Hihara11.1 Introduction 33111.2 Overview of Fatty Acid Metabolism 33211.2.1 Fatty Acid Biosynthesis 33211.2.2 Fatty Acid Degradation and Turnover 33511.2.3 Accumulation of Storage Lipids 33611.3 Basic Technologies for Production of Free Fatty Acids 33711.3.1 Production of Free Fatty Acids in E. coli 33711.3.2 Production of Free Fatty Acids in Cyanobacteria 33811.4 Advanced Technologies for Enhancement of Free Fatty Acid Production 33911.4.1 Enhancement of Fatty Acid Biosynthesis 33911.4.2 Enhancement of Carbon Fixation Activity 34511.4.3 Engineering of Carbon Flow: Modification of Key Regulatory Factors 34511.4.4 Engineering of Carbon Flow: Deletion of Competitive Pathways 34611.4.5 Mitigation of the Toxicity of FFAs 34711.4.6 Enhancement of FFA Secretion 34811.4.7 Induction of Cell Lysis 34911.4.8 Recovery of Produced FFAs from Medium 35011.4.9 Identification of Cyanobacterial Strains Suitable for FFA Production 35011.5 Hydrocarbon Production in Cyanobacteria 35111.6 Advanced Technologies for Enhancement of Hydrocarbon Production 35311.6.1 Enhancement of Alk(a/e)ne Biosynthesis 35311.6.2 Improvement of the Performance of Alkane Biosynthetic Enzymes 35411.7 Basic Technologies for Production of Fatty Alcohols 35511.8 Advanced Technologies for Enhancement of Fatty Alcohol Production 35511.9 Basic Technologies for Production of Fatty Acid Alkyl Esters 35611.10 Perspectives 357References 35812 Product Export in Cyanobacteria 369Cátia F. Gonçalves, Steeve Lima, and Paulo Oliveira12.1 Introduction 36912.2 Secretion Mediated by Membrane-Embedded Systems 37312.2.1 Proteins 37312.2.2 Extracellular Polymeric Substances (EPS) 37712.2.3 Soluble Sugars and Organic Acids 37912.2.4 Fatty Acids 38112.2.5 Alcohols 38212.2.6 Terpenes 38412.3 MV-Mediated Secretion 38612.3.1 Structure and Biogenesis of Bacterial MVs 38612.3.1.1 Cyanobacterial MVs 38812.3.2 MVs as Novel Biotechnological Tools 38912.4 Concluding Remarks 391Acknowledgments 392References 392Part III Frontiers of Cyanobacteria Biotechnology 40713 Harnessing Solar-Powered Oxic N2-fixing Cyanobacteria for the BioNitrogen Economy 409James Young, Liping Gu, William Gibbons, and Ruanbao Zhou13.1 Introduction 40913.2 Physiology and Implications of Oxic Nitrogen Fixation 41013.2.1 Ecological Range 41113.2.2 Balancing Photosynthesis and Nitrogen Fixation 41213.2.3 Energetic Demands and How the Cells Adapt 41213.2.4 Impacts of Continuous Light vs Dark–Light Cycles 41613.3 Major Biotechnology Applications for Diazotrophic Cyanobacteria 41713.3.1 General Economic and Environmental Considerations of Diazotrophic Cyanobacteria 41713.3.2 Metabolic Engineering of N2-Fixing Cyanobacteria for Carbon Compound Production 42013.3.2.1 Direct Production of Biofuels 42013.3.2.2 Cyanobacteria as a Fermentable Substrate 42013.3.3 Metabolic Engineering of Nitrogen Fixing Cyanobacteria for Nitrogen-Rich Compound Production 42213.3.3.1 Ammonia 42213.3.3.2 Guanidine 42313.3.3.3 Cyanophycin 42313.3.3.4 Amino Acids and Proteins 42313.3.4 Application of Diazotrophic Cyanobacteria in Agriculture 42513.4 Conclusions 428References 42814 Traits of Fast-Growing Cyanobacteria 441Meghna Srivastava, Elton P. Hudson, and Pramod P. Wangikar14.1 Introduction 44114.2 Why is Growth Rate Significant? 44214.3 An Overview of Factors Affecting the Growth Rates of Cyanobacteria 44614.3.1 Light Intensity and Quality 44814.3.2 Mixotrophic Growth 45114.3.3 Circadian Rhythm 45114.3.4 Additional Factors Relating to Growth Rates in Cyanobacteria 45214.3.4.1 Cell Morphology 45314.3.4.2 Genome Size 45314.3.4.3 Saltwater Tolerance 45414.3.4.4 Nutrient Supplementation 45414.3.5 Carbon Storage 45514.4 Overview of the Fast-Growing Model Cyanobacteria 45514.4.1 Synechococcus elongatus UTEX 2973 45514.4.2 Synechococcus elongatus PCC 11801 45614.4.3 Synechococcus sp. PCC 11901 45614.4.4 Synechococcus sp. PCC 7002 45714.5 Relationship Between Light Usage and Growth Rate in Model Strains 45814.5.1 Case Study: The pmgA Mutant of Synechocystis 45814.5.2 Case Study: The S. elongatus 7942 and S. elongatus 2973 Strains 46014.6 Molecular Determinants of Fast Growth of S. elongatus UTEX 2973 46014.7 Carbon Fluxes in Fast-Growing Strains Determined Using Metabolic Flux Analysis 46314.8 Engineering Cyanobacteria for Fast Growth 46514.8.1 Calvin Cycle Enzymes 46514.8.2 PEP Carboxylase 46614.8.3 Carbon and Light Uptake Proteins 46714.9 Conclusion 468References 46815 Cyanobacterial Biofilms in Natural and Synthetic Environments 477Christian David, Rohan Karande, and Katja Bühler15.1 Motivation 47715.2 Introduction to Biofilms: Biology and Applications 47815.3 Cyanobacteria in Natural Biofilms and Microbial Mats 48315.4 Introduction to (Photo-)biotechnology 48415.5 Benefits of Microscale Systems for (Photo-)biofilm Cultivation 48715.6 Oxygen Accumulation and Its Impacts 48815.7 Resource Management in Biofilms 49115.8 Applications of Photosynthetic Biofilms 49315.8.1 Biofilms Enable High Cell Densities 49715.8.2 Biofilms Enable Continuous Production 49815.9 Outlook 499References 49916 Growth of Photosynthetic Microorganisms in Different Photobioreactors Operated Outdoors 505Eleftherios Touloupakis and Pietro Carlozzi16.1 Background 50516.1.1 Photobiological Hydrogen Production 50616.1.2 Polyhydroxyalkanoate Production by Photosynthetic Microbes 50816.1.3 Photobioreactors 50916.2 Case Studies of Outdoor Cultivations of Photosynthetic Microorganisms 51316.2.1 Outdoor Cultures of Purple Non-Sulfur Bacteria for H2 and PHB Production 51316.2.2 Outdoor Cultures of Cyanobacteria 51616.3 Conclusion 517Acknowledgments 519References 519Index 531