Table Of ContentCyanobacteriaBiotechnology
Cyanobacteria Biotechnology
Edited by
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v
Contents
Foreword:CyanobacteriaBiotechnology xv
Acknowledgments xviii
PartI CoreCyanobacteriaProcesses 1
1 InorganicCarbonAssimilationinCyanobacteria:Mechanisms,
Regulation,andEngineering 3
MartinHagemann,ShanshanSong,andEva-MariaBrouwer
1.1 Introduction–TheNeedforaCarbon-ConcentratingMechanism 3
1.2 TheCarbon-ConcentratingMechanism(CCM)Among
Cyanobacteria 4
1.2.1 C UptakeProteins/Mechanisms 5
i
1.2.2 CarboxysomeandRubisCO 8
1.3 RegulationofC Assimilation 10
i
1.3.1 RegulationoftheCCM 10
1.3.2 FurtherRegulationofCarbonAssimilation 13
1.3.3 MetabolicChangesandRegulationDuringC Acclimation 14
i
1.3.4 RedoxRegulationofC Assimilation 15
i
1.4 EngineeringtheCyanobacterialCCM 16
1.5 Photorespiration 17
1.5.1 CyanobacterialPhotorespiration 17
1.5.2 AttemptstoEngineerPhotorespiration 19
1.6 ConcludingRemarks 20
Acknowledgments 21
References 21
2 ElectronTransportinCyanobacteriaandItsPotentialin
Bioproduction 33
DavidJ.Lea-SmithandGuyT.Hanke
2.1 Introduction 33
2.2 ElectronTransportinaBioenergeticMembrane 34
2.2.1 LinearElectronTransport 34
vi Contents
2.2.2 CyclicElectronTransport 37
2.2.3 ATPProductionfromLinearandCyclicElectronTransport 37
2.3 RespiratoryElectronTransport 38
2.4 RoleofElectronSinksinPhotoprotection 41
2.4.1 TerminalOxidases 41
2.4.2 HydrogenaseandFlavodiironComplexes 41
2.4.3 CarbonFixationandPhotorespiration 43
2.4.4 ExtracellularElectronExport 44
2.5 RegulatingElectronFluxintoDifferentPathways 45
2.5.1 ElectronFluxThroughthePlastoquinonePool 45
2.5.2 ElectronFluxThroughFdx 46
2.6 SpatialOrganizationofElectronTransportComplexes 47
2.7 ManipulatingElectronTransportforSyntheticBiologyApplications 48
2.7.1 ImprovingGrowthofCyanobacteria 49
2.7.2 ProductionofElectricalPowerinBPVs 49
2.7.3 HydrogenProduction 50
2.7.4 ProductionofIndustrialCompounds 50
2.8 FutureChallengesinCyanobacterialElectronTransport 51
References 52
3 OptimizingtheSpectralFitBetweenCyanobacteriaandSolar
RadiationintheLightofSustainabilityApplications 65
KlaasJ.Hellingwerf,QueChen,andFilipeBrancodosSantos
3.1 Introduction 65
3.2 MolecularBasisandEfficiencyofOxygenicPhotosynthesis 67
3.3 FitBetweentheSpectrumofSolarRadiationandtheActionSpectrumof
Photosynthesis 72
3.4 ExpansionofthePARRegionofOxygenicPhotosynthesis 74
3.5 ModulationandOptimizationoftheTransparencyof
Photobioreactors 79
3.6 FullControloftheLightRegime:LEDsInsidethePBR 81
3.7 ConclusionsandProspects 82
References 83
PartII ConceptsinMetabolicEngineering 89
4 WhatWeCanLearnfromMeasuringMetabolicFluxesin
Cyanobacteria 91
XiangGao,ChaoWu,MichaelCantrell,MelissaCano,JianpingYu,and
WeiXiong
4.1 CentralCarbonMetabolisminCyanobacteria:AnOverviewand
RenewedPathwayKnowledge 91
4.1.1 GlycolyticRoutesInterwovenwiththeCalvinCycle 91
4.1.2 TricarboxylicAcidCycling 94
Contents vii
4.2 MethodologiesforPredictingandQuantifyingMetabolicFluxesin
Cyanobacteria 95
4.2.1 FluxBalanceAnalysisandGenome-ScaleReconstructionofMetabolic
Network 95
4.2.2 13C-MetabolicFluxAnalysis 96
4.2.3 ThermodynamicAnalysisandKineticsAnalysis 99
4.3 CyanobacteriaFluxomeinResponsetoAlteredNutrientModesand
EnvironmentalConditions 101
4.3.1 AutotrophicFluxome 101
4.3.2 PhotomixotrophicFluxome 104
4.3.3 HeterotrophicFluxome 105
4.3.4 PhotoheterotrophicFluxome 105
4.3.5 DiurnalMetaboliteOscillations 106
4.3.6 NutrientStates’ImpactonMetabolicFlux 107
4.4 MetabolicFluxesRedirectedinCyanobacteriaforBiomanufacturing
Purposes 108
4.4.1 RestructuringtheTCACycleforEthyleneProduction 108
4.4.2 MaximizingFluxintheIsoprenoidPathway 109
4.4.2.1 MeasuringPrecursorPoolSizetoEvaluatePotentialDrivingForcesfor
IsoprenoidProduction 109
4.4.2.2 BalancingIntermediatesforIncreasedPathwayActivity 110
4.4.2.3 KineticFluxProfilingtoDetectBottlenecksinthePathway 111
4.5 SynopsisandFutureDirections 112
Acknowledgments 112
References 112
5 SyntheticBiologyinCyanobacteriaandApplicationsfor
Biotechnology 123
EltonP.Hudson
5.1 Introduction 123
5.2 GettingGenesintoCyanobacteria 123
5.2.1 Transformation 123
5.2.2 ExpressionfromEpisomalPlasmids 125
5.2.3 DeliveryofGenestotheChromosome 127
5.3 BasicSyntheticControlofGeneExpressioninCyanobacteria 129
5.3.1 QuantifyingTranscriptionandTranslationinCyanobacteria 130
5.3.2 ControllingTranscriptionwithSyntheticPromoters 134
5.3.2.1 ConstitutivePromoters 136
5.3.2.2 RegulatedPromotersthatAreSensitivetoAddedCompounds
(Inducible) 137
5.3.2.3 CRISPRInterferenceforTranscriptionalRepression 139
5.3.3 ControllingTranslation 141
5.3.3.1 RibosomeBindingSites(Cis-Acting) 141
5.3.3.2 Riboswitches(Cis-Acting) 142
5.3.3.3 SmallRNAs(Trans-Acting) 143
viii Contents
5.4 ExoticSignalsforControllingExpression 143
5.4.1 Oxygen 144
5.4.2 LightColor 144
5.4.3 CellDensityorGrowthPhase 145
5.4.4 EngineeringRegulatorsforAlteredSensingProperties:Stateofthe
Art 147
5.5 AdvancedRegulation:TheNearFuture 148
5.5.1 LogicGatesandTimingCircuits 148
5.5.2 OrthogonalTranscriptionSystems 151
5.5.3 SyntheticBiologySolutionstoIncreaseStability 152
5.5.4 SyntheticBiologySolutionsforCellSeparationandProduct
Recovery 154
5.6 Conclusions 157
Acknowledgments 158
References 158
6 SinkEngineeringinPhotosyntheticMicrobes 171
MaríaSantos-Merino,AmitK.Singh,andDanielC.Ducat
6.1 Introduction 171
6.2 SourceandSink 172
6.3 RegulationofSinkEnergyinPlants 177
6.3.1 SucroseandOtherSignalingCarbohydrates 178
6.3.2 Hexokinases 179
6.3.3 SucroseNon-fermentingRelatedKinases 180
6.3.4 TORKinase 181
6.3.5 EngineeredPathwaysasSinksinPhotosyntheticMicrobes 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,anElectronSink 190
6.4 WhatAreKeySource/SinkRegulatoryHubsinPhotosynthetic
Microbes? 191
6.5 ConcludingRemarks 194
Acknowledgment 195
References 195
7 DesignPrinciplesforEngineeringMetabolicPathwaysin
Cyanobacteria 211
JasonT.KuandEthanI.Lan
7.1 Introduction 211
7.2 CofactorOptimization 212
Contents ix
7.2.1 RecruitingNADPH-DependentEnzymesWhereverPossible 215
7.2.2 EngineeringNADH-SpecificEnzymestoUtilizeNADPH 217
7.2.3 IncreasingNADHPoolinCyanobacteriaThroughExpressionof
Transhydrogenase 218
7.3 IncorporationofThermodynamicDrivingForceintoMetabolicPathway
Design 219
7.3.1 ATPDrivingForceinMetabolicPathways 220
7.3.2 IncreasingSubstratePoolSupportstheCarbonFluxToward
Products 222
7.3.3 ProductRemovalUnblockstheLimitationsofProductTiter 223
7.4 DevelopmentofSyntheticPathwaysforCarbonConserving
PhotorespirationandEnhancedCarbonFixation 225
7.5 SummaryandFuturePerspectiveonCyanobacterialMetabolic
Engineering 229
References 229
8 EngineeringCyanobacteriaforEfficientPhotosynthetic
Production:EthanolCaseStudy 237
GuodongLuanandXuefengLu
8.1 Introduction 237
8.2 PathwayforEthanolSynthesisinCyanobacteria 238
8.2.1 PyruvateDecarboxylaseandTypeIIAlcoholDehydrogenase 238
8.2.2 SelectionofBetterEnzymesinthePdc–AdhIIPathway 240
8.2.3 SystematicCharacterizationofthePdc –Slr1192Pathway 241
ZM
8.3 SelectionofOptimalCyanobacteria“Chassis,”StrainforEthanol
Production 242
8.3.1 SynechococcusPCC6803andSynechococcusPCC7942 243
8.3.2 SynechococcusPCC7002 245
8.3.3 AnabaenaPCC7120 245
8.3.4 NonconventionalCyanobacteriaSpecies 246
8.4 MetabolicEngineeringStrategiesTowardMoreEfficientandStable
EthanolProduction 246
8.4.1 EnhancingtheCarbonFluxviaOverexpressionofCalvinCycle
Enzymes 248
8.4.2 BlockingPathwaysthatAreCompetitivetoEthanol 248
8.4.3 ArrestingBiomassFormation 249
8.4.4 EngineeringCofactorSupply 249
8.4.5 EngineeringStrategiesGuidedbyInSilicoSimulation 250
8.4.6 StabilizingEthanolSynthesisCapacityinCyanobacterialCell
Factories 251
8.5 ExploringtheResponseinCyanobacteriatoEthanol 253
8.5.1 ResponseofCyanobacterialCellsTowardExogenousAdded
Ethanol 254
8.5.2 ResponseofCyanobacteriatoEndogenousSynthesizedEthanol 255
x Contents
8.6 MetabolicEngineeringStrategiestoFacilitateRobustCultivationAgainst
Biocontaminants 256
8.6.1 EngineeringCyanobacteriaCellFactoriestoAdaptforSelective
EnvironmentalStresses 256
8.6.2 EngineeringCyanobacteriaCellFactoriestoUtilizeUncommon
Nutrients 258
8.7 ConclusionsandPerspectives 258
References 259
9 EngineeringCyanobacteriaasHostOrganismsforProduction
ofTerpenesandTerpenoids 267
JoãoS.RodriguesandPiaLindberg
9.1 TerpenoidsandIndustrialApplications 267
9.2 TerpenoidBiosynthesisinCyanobacteria 270
9.2.1 Methylerythritol-4-PhosphatePathway 270
9.2.2 FormationofTerpeneBackbones 272
9.3 NaturalOccurrenceandPhysiologicalRolesofTerpenesandTerpenoids
inCyanobacteria 274
9.4 EngineeringCyanobacteriaforTerpenoidProduction 275
9.4.1 MetabolicEngineering 277
9.4.1.1 TerpeneSynthases 277
9.4.1.2 IncreasingSupplyofTerpeneBackbones 285
9.4.1.3 EngineeringtheNativeMEPPathway 286
9.4.1.4 ImplementingtheMVAPathway 287
9.4.1.5 EnhancingPrecursorSupply 288
9.4.2 OptimizingGrowthConditionsforProduction 289
9.4.3 ProductCaptureandHarvesting 291
9.5 SummaryandOutlook 292
Acknowledgments 293
References 293
10 CyanobacterialBiopolymers 301
MoritzKochandKarlForchhammer
10.1 Polyhydroxybutryate 301
10.1.1 Introduction 301
10.1.2 PHBMetabolisminCyanobacteria 302
10.1.3 IndustrialApplicationsofPHB 305
10.1.3.1 PhysicalPropertiesofPHBandItsDerivatives 305
10.1.3.2 Biodegradability 306
10.1.3.3 ApplicationofPHBasaPlastic 306
10.1.3.4 ReactorTypes 306
10.1.3.5 ProductionProcess 307
10.1.3.6 DownstreamProcessing 308
10.1.4 MetabolicEngineeringofPHBBiosynthesis 308
10.1.5 LimitationsandPotentialofPHBProductioninCyanobacteria 310
Contents xi
10.2 CyanophycinGranulesinCyanobacteria 311
10.2.1 BiologyofCyanophycin 311
10.2.2 GenesandEnzymesofCGPMetabolism 315
10.2.2.1 CyanophycinSynthetase 315
10.2.2.2 CyanophycinDegradingEnzymes 316
10.2.3 RegulationofCyanophycinMetabolism 317
10.2.4 CyanophycinOverproductionandPotentialIndustrial
Applications 318
Acknowledgement 319
References 319
11 BiosynthesisofFattyAcidDerivativesbyCyanobacteria:From
BasicstoBiofuelProduction 331
AkihitoKawaharaandYukakoHihara
11.1 Introduction 331
11.2 OverviewofFattyAcidMetabolism 332
11.2.1 FattyAcidBiosynthesis 332
11.2.2 FattyAcidDegradationandTurnover 335
11.2.3 AccumulationofStorageLipids 336
11.3 BasicTechnologiesforProductionofFreeFattyAcids 337
11.3.1 ProductionofFreeFattyAcidsinE.coli 337
11.3.2 ProductionofFreeFattyAcidsinCyanobacteria 338
11.4 AdvancedTechnologiesforEnhancementofFreeFattyAcid
Production 339
11.4.1 EnhancementofFattyAcidBiosynthesis 339
11.4.2 EnhancementofCarbonFixationActivity 345
11.4.3 EngineeringofCarbonFlow:ModificationofKeyRegulatory
Factors 345
11.4.4 EngineeringofCarbonFlow:DeletionofCompetitivePathways 346
11.4.5 MitigationoftheToxicityofFFAs 347
11.4.6 EnhancementofFFASecretion 348
11.4.7 InductionofCellLysis 349
11.4.8 RecoveryofProducedFFAsfromMedium 350
11.4.9 IdentificationofCyanobacterialStrainsSuitableforFFA
Production 350
11.5 HydrocarbonProductioninCyanobacteria 351
11.6 AdvancedTechnologiesforEnhancementofHydrocarbon
Production 353
11.6.1 EnhancementofAlk(a/e)neBiosynthesis 353
11.6.2 ImprovementofthePerformanceofAlkaneBiosyntheticEnzymes 354
11.7 BasicTechnologiesforProductionofFattyAlcohols 355
11.8 AdvancedTechnologiesforEnhancementofFattyAlcohol
Production 355
11.9 BasicTechnologiesforProductionofFattyAcidAlkylEsters 356
11.10 Perspectives 357
References 358
