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Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants

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Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants
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   Journalof    Biotechnology 162 (2012) 134–147 ContentslistsavailableatSciVerseScienceDirect  Journal   of    Biotechnology  j   ournal   home   page:www.elsevier.com/locate/jbiotec Bioengineering   of    carbon   fixation,   biofuels,   and   biochemicalsin   cyanobacteria   and   plants Lisa   Rosgaard a , b ,   Alice    Jara   de   Porcellinis a , b ,Jacob   H.    Jacobsen c ,Niels-Ulrik   Frigaard c ,Yumiko   Sakuragi a , b , ∗ a DepartmentofPlantBiologyandBiotechnology,FacultyofSciences,UniversityofCopenhagen,Thorvaldsensvej40,Frederiksberg1871,Denmark b VillumKannRasmussenResearchCenter-   ProactivePlants,Thorvaldsensvej40,Frederiksberg1871,Denmark c SectionforMarineBiology,DepartmentofBiology,UniversityofCopenhagen,Strandpromenaden5,   Helsingør3000,Denmark a   r   t   i   c   le   inf   o  Articlehistory: Received13February2012Receivedinrevisedform15May2012Accepted21May   2012 Available online 5 June 2012 Keywords: CyanobacteriaCarbonfixationMetabolicengineeringBiofuelSyntheticpathways a   b   s   t   ra   ct Development   of    sustainable   energy   isapivotal   steptowards   solutionsfor   today’s   global   challenges,including   mitigating   the   progression   of    climatechange   andreducing   dependence   on   fossil   fuels.Biofuelsderivedfrom   agricultural   cropshave   already   beencommercialized.   Howevertheimpactsonenvironmen-talsustainability   and   foodsupplyhave   raised   ethicalquestionsabout   the   currentpractices.   Cyanobacteriahave   attracted   interest   asan   alternativemeansfor   sustainableenergyproductions.Beingaquatic   pho-toautotrophs   they   canbe   cultivatedinnon-arable   lands   and   do   not   compete   for   landfor   food   production.Their   richgeneticresourcesoffer   meansto   engineer   metabolic   pathwaysfor   synthesis   of    valuable   bio-based   products.   Currently   themajor   obstacle   in   industrial-scale   exploitationof    cyanobacteria   astheeconomically   sustainableproduction   hosts   islow   yields.   Much   effort   has   beenmade   toimprove   thecarbon   fixation   and   manipulating   the   carbon   allocation   incyanobacteria   and   their   evolutionary   photo-synthetic   relatives,algae   andplants.   This   review   aims   atproviding   anoverviewof    the   recentprogressinthe   bioengineering   of    carbonfixation   and   allocation   incyanobacteria;wherever   relevant,the   progressmade   inplants   andalgaeis   also   discussedas   aninspiration   for   future   applicationincyanobacteria. © 2012 Elsevier B.V. All rights reserved. 1.Introduction Cyanobacteriahaveintriguedscientistsfordecadesformanyreasons.Theythriveindiverseandsometimesseeminglyharshenvironments( e.g. oceans,hotsprings,deserts,andsymbioticrelationshipswithmulticellulareukaryotes)andperformoxygen-evolvingphotosynthesisusinglight,water,andCO 2  asthebuildingmaterials.Oxygenicphotosynthesisbycyanobacteriaandchloro-plasts(theevolutionarydescendantsof    cyanobacteriain   algaeandplants)hastransformedtheEarth’satmosphereandcarboncyclesoverbillionsofyearsandtodaycyanobacteriaare   thoughtto   con-tributetoaquarterofglobalcarbonfixation(Fieldetal.,   1998).ItisthisabilitytosequesterandfixCO 2  intoorganicmatterusingwaterandlightthathasdrawnrenewedattentionto   cyanobac-teriaascellfactoriesforrenewableandsustainableproductionof biofuelandbioactivecompounds.Witha   geneticplasticity,matchingthatof  Escherichiacoli andyeast,andwiththewealthof  ∗ Correspondingauthorat:   Departmentof    PlantBiologyandBiotechnology,Fac-ulty   of    Sciences,Universityof    Copenhagen,Thorvaldsensvej40,Frederiksberg1871,Denmark.Tel.:+4535333317. E-mailaddress: ysa@life.ku.dk(Y.Sakuragi). genomicinformationcomprisingmorethan170genomesequencesof    cyanobacteriathathavebeen,orisin   theprocessofbeing,determined(http://www.genomesonline.org/),   itis   nolongeraremoteideatointroducemultipleenzymesfromdifferentorgan-ismsintocyanobacteriatoconstructbiosyntheticpathwaysfortargetedproductionof    fuelsandcompoundswithhealthbene-fitsforcommercialization.Anumberof    value-addedcompoundshavealreadybeenproducedin   cyanobacteriabymeansof    geneticengineering( e.g. ethanol,isobutyraldehyde,isobutanol,1-butanol,isoprene,ethylene,hexoses,cellulose,mannitol,lacticacid,fattyacids;seeSection6)   (Fig.1).Duringthepasttwo   centuriestheatmosphericconcentrationof greenhousegasses( e.g. carbondioxide,methane)increasedsignif-icantly(Solomonetal.,2007).As   a   result,theglobaltemperatureis   risinganditisprojectedthata   totalCO 2  reductionof50–85%is   requiredby2050in   orderto   stabilizetheemissionwithinthe“safezone”of    450ppm(Schenketal.,   2008).Bio-basedsolutionstothisproblemhavebeenimplementedwithsuccess,butalsowithchallenges.Firstgenerationbiofuel( i.e. ethanol)derivedfromsugarcaneandcornhasbeencommerciallyviable;howeveritsnega-tiveimpactsonfoodsupplyhaveraisedethicalconcernsaboutitslong-termconsequences.Secondgenerationbiofuelsbasedontrees,agriculturalresidues,anddedicatedbiofuelcropshavenot 0168-1656/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2012.05.006  L.   Rosgaardetal./JournalofBiotechnology 162 (2012) 134–147  135 Fig.1. Centralmetabolicpathwaysof    cyanobacteria.Heterologouspathwaysandproductsthathavebeengeneticallyengineeredincyanobacteriaanddiscussedinthetext   areindicatedinmagenta.  Abbreviations :Enzymes:1,   ribulose-1,5-bisphosphatecarboxylase/oxygenase(RuBisCO);2,fructose-1,6-bisphosphatase;3,phosphofructok-inase;   4,sedoheptulose-1,7-bisphosphatase;5,phosphoenolpyruvatecarboxylase.Metabolites:2OG,2-oxoglutarate;2OIV,2-oxoisovalerate;2PG,2-phosphoglycerate;3PG,   3-phosphoglycerate;AcCoA,acetyl-CoA;BPG,   1,3-bisphosphoglycerate;Cit,citrate;DHAP,dihydroxyacetone-phosphate;DMAPP,dimethylallyl-pyrophosphate;DXP,   1-deoxyxylulose-5-phosphate;E4P,erythrose-4-phosphate;F6P,fructose-6-phosphate;FACoA,fattyacyl-CoA;FBP,fructose-1,6-bisphosphate;Fum,fumarate;G1P,   glucose-1-phosphate;G6P,glucose-6-phosphate;GAP,glyceraldehyde-3-phosphate;HMBPP,1-hydroxy-2-methyl-2-butenyl-4-pyrophosphate;ICit,isocitrate;IPP,isopentenyl-pyrophosphate;Mal,malate;OA,oxaloacetate;PEP,phosphoenolpyruvate;Pyr,pyruvate;R5P,ribose-5-phosphate;Ru5P,ribulose-5-phosphate;RuBP,ribulose-1,5-bisphosphate;S7P,sedoheptulose-7-phosphate;SBP,sedoheptulose-1,7-bisphosphate;SSA,succinicsemialdehyde;Suc,succinate;X5P,   xylulose-5-phosphate.Steueret   al.(2012),ZhangandBryant(2011)andreferencesin   thetext. yetbeencommerciallyavailableandareunlikelytobefreefromchallengesincludingenvironmentalsustainabilityanditsimpacton   thefoodsupplychain.Developmentof    a   suiteof    strategiesforgenerationofrenewableenergiesisa   pressingneedforoursoci-ety,becauseno   singlesourceof    biomassandbiofueltechnologyislikelytoreplacefossilfuels.Cyanobacteriaholda   potentialasanalternativebio-basedsystemforsustainableproductionof    biofuelandotherbioproducts.Amajorchallengein   futurecommercialexploitationof cyanobacteriais   theproductyield.Designandoperationalprac-ticesofcultivationsandharvestsoftheproductsholdthekeystoeconomicfeasibility,asdostrainoptimizationforhigherproduc-tivities.Substantialimprovementsin   carbonfixingandrecyclingpathwaysareneededfor   thecyanobacterialcellfactorytobecomecommercialrealityandto   offeraneconomicallyandenvironmen-tallysustainableproductionsystem.Theaimof    thisreviewis   toprovideanoverviewoftherecentprogressingeneticengineeringforimprovingcarbonfixationandallocationforenhancedproduc-tionofbiofuelandbioactivecompoundsincyanobacteria.Sincemuchinspiringworkinthisfieldhasbeenmadeinhigherplantsduetoagriculturalintereststhisprogressisalsodiscussedasa   lessonforfutureapplicationincyanobacteria. 2.   Geneticallymodifiablecyanobacteria Cyanobacteriaconstituteoneofthelargestgroupsofbacteriawithmanyhundredsofstrainsdepositedinculturecollectionsworldwide(CohenandGurevitz,2006).Themethodsavailableforgenetransferto   cyanobacteriaarenaturaltransformation,chemicaltransformation,electroporation,andconjugation.Amongthemanystrainsthat   aregeneticallyamenableinthismanner(KoksharovaandWolk,2002)onlyafewstrainsdominatetheresearchongeneticandmetabolicmanipulation(Floresetal.,2008;Heidornetal.,2011).Thesestrainsincludetheunicellular,freshwaterstrains Synechocystis sp.PCC6803(IkeuchiandTabata,2001)and Syne-chococcuselongatus sp.PCC7942(Holtmanetal.,   2005)that   botharenaturallytransformable. Synechocystis sp.PCC6803was   thefirstphotosyntheticorganismtohaveitsgenomesequencedandisoneofthebestcharacterizedcyanobacteria.Acommercialkitforengineeringexpressionofrecombinantproteinsspecificallyin S.elongatus sp.PCC7942is   nowavailable(LifeTechnologiesCorp.,Carlsbad,CA).Thephysiologyandgenomesequenceof anothermodelorganism, S.elongatus sp.PCC6301isnearlyiden-ticaltothatof    S.   elongatus sp.PCC7942.Theunicellular,marinestrains Synechococcus sp.PCC7002(Frigaardetal.,   2004)and  136  L.Rosgaardetal./     Journalof    Biotechnology 162 (2012) 134–147  Thermosynechococcuselongatus BP-1(Iwaietal.,   2004)arealsonaturallytransformablealthoughthelatterstrainhasahighertransformationefficiencybyelectroporation. T.   elongatus BP-1istheonlythermophilicstrain(growthoptimum55 ◦ C)com-monlyusedingeneticandphysiologicalstudiesofcyanobacteria.  Anabaenavariabilis ATCC29413(Happeetal.,2000),  Anabaena (alsocalled Nostoc  )sp.PCC7120(Kanekoetal.,   2001),and Nostocpuncti- forme ATCC29133(alsocalledPCC73102)(Meeksetal.,2001),are allfilamentous,freshwaterstrainsthat   performN 2  fixationin   spe-cializedcellscalledheterocysts.Genetransfertothesethreestrainsistypicallyperformedbyconjugation.Onesignificantadvantageofdoingmetabolicengineeringincyanobacteria,incontrastto   algaeandplants,is   thatthegenetictoolsaremuchmoredevelopedin   cyanobacteria(CohenandGurevitz,2006;Heidornetal.,   2011;Huangetal.,2010;KoksharovaandWolk,2002).Thegenomesof    allabove-mentionedmodelcyanobacteriahavebeensequencedandannotated.Thegenomesofunicellularcyanobacteriaare   typicallysmaller(2.5–4Mbp)thanthatof  E.coli .Inaddition,cyanobacterialgenomeshavenointronsandhighlyefficientgenetransferprotocolsandwell-characterizedplasmidvectors,selectionmarkers,andgeneexpressionsystemsareavailable.Finally,manygenesencodingreporterproteinsandenzymesusedformetabolicmanipulationin   otherbacterialike E.   coli functionwellin   cyanobacteria(KoksharovaandWolk,2002). Withrespecttophysiology,cyanobacteriahavecertainadvan-tagesoveralgaeandplants:cyanobacteriaexhibitrapidgrowthandhavelesscomplexintracellularstructuresandcellwalls.Cyanobac-teriahaveabacterial-typepeptidoglycancellwallunlikethecomplexcarbohydratecellwallfoundinalgaeandplants.Finally,somecyanobacteriaarecapableof    usingN 2  asthesolesourceof nitrogen–somethingthatalgaeandplantscannot(unlesstheyhavesymbioticN 2 -fixingbacteria). 3.ImprovementofCO 2  fixation Improvementofcarbonfixationhasbeena   subjectoflongandextensiveresearchoverthepastfourdecades.Oxygenicphototrophs,includingcyanobacteria,algae,andplants,usethereductivepentosephosphatecycle,alsoknownastheCalvin–Benson–Basshamcycle(hereafterCalvincycle),forassimi-lationofCO 2  (Fig.1).Numerouseffortstoimprovetherateof    CO 2 fixation via theCalvincycle,PEPcarboxylaseandthroughsyntheticpathwayintroductionhavebeenattemptedwithvaryingdegreesofsuccess.Theapproachescanbelargelygroupedintothefollow-ingfourcategories:(1)engineeringofribulose-1,5-bisphosphatecarboxylase/oxygenase(RuBisCO)forimprovementsoftheratesofcatalysisofcarboxylationandreductionof    theoxygenationreaction,(2)enhancementoftheactivationstateofRuBisCO,(3)improvementsoftheregenerationphaseof    theCalvincycle,and(4)enrichmentofCO 2  concentrationaroundRuBisCOforsuppressionoftheoxygenasereaction.  3.1.RuBisCOengineering  RuBisCOhaslongbeena   targetforimprovingcarbonfixationbecauseofitspivotalrole   incarbonfixationin   photoautotrophicorganisms.Localizationof    RuBisCOin   cellsvariesbetweencyanobacteria,algae,andplants.Incyanobacteriaandalgae,RuBisCOarefoundin   proteinaceousmicrocompartmentsknownascarboxysomesin   thecytosolandpyrenoidin   chloroplaststroma,respectively,whileinplantRuBisCois   largelysolublein   thestromaofchloroplasts(MoroneyandSomanchi,1999).Thereare   fourdis-tinctformsofRuBisCOinnature;formI,II,IIIandRuBisCO-likeformIVbasedonaminoacidsequences,phylogenyandstruc-ture(AnderssonandBacklund,2008;Tabitaetal.,2008b).FormI RuBisCOis   comprisedof    8smalland8largesubunits.Itis   themostabundantformandisfoundin   plants,algaeandcyanobacteriaandsomemembersof   -,  -and  -proteobacteria.In   prokaryotesandnon-greenalgae,the rbcL and rbcS  ,encodingthelargeandsmallsubunit,respectively,areco-transcribedfroma   singlepromoterwhereasinplantsandgreenalgae rbcL   is   encodedin   thechloro-plastgenomewhilethe rbcS  is   encodedinthenucleargenome(seeareviewbyTabitaetal.,   2008aandreferencestherein).FormII   iscomposedof    onlythelargesubunitandis   presentindinoflagellatesandsomemembersof   -,  -   and  -proteobacteria.Interest-ingly,thephototrophicpurplenon-sulfurbacteria( e.g.Rhodobacter sphaeroides ,   Rhodobactercapsulatus )   andotherorganismsinclud-ing Hydrogenovibriomarinus andsome Thiobacillus speciescontainbothformIandformII   RuBisCO,andin R.capsulatus bothformsareexpressedunderphotoautotrophicconditions(AnderssonandBacklund,2008;Paolietal.,1998).FormIIIis   foundinarcheaandconsistsof    a   largesubunitina   dimericorpentamericarrange-ment.IthasbeensuggestedthatphotosyntheticRuBisCOevolvedfromtheformIIIRuBisCO(Ashidaetal.,2008;Tabitaetal.,2008b). Thecatalyticcoreofthethreeformsof    RuBisCOconsistsofnine-teenconservedaminoacidresidues.Incontrast,theRuBisCO-likeform(formIV)asfoundin   Chlorobaculumtepidum and Bacillussub-tilis lacksnineof    theseresiduesrenderingitunableto   catalyzeneithertheoxygenasenorcarboxylasereaction(AnderssonandBacklund,2008;Tabitaetal.,2007).TheRuBisCO-likeformhasbeenshowntoplaya   roleinthemethioninesalvagepathwaywhereitcatalyzestheenolizationof    2,3-dioxo-5-methylthiopentyl-1-P,ananalogof    ribulose-1,5-bisphophate(RuBP)in   B.subtilis (Ashidaetal.,   2008).WithintheRuBisCO rbcL superfamilytheaverageaminoacidsequenceidentityis   31%(Tabitaetal.,   2007),yetthesecondarystructureiswellconservedandthefouraminoacidsrequiredforthecatalysisarealsostrictlyconserved(Tabitaetal.,2008a).ThecarboxylasereactionofRuBisCOresultsin   productionof twomoleculesof3-phosphoglycerate,oneof    whichis   subse-quentlyrecycledto   regenerateribulose-1,5-bisphosphate(RuBP)whereastheotherisconvertedtobiomassandgrowth(Fig.1).Theoxygenasereactionontheotherhandresultsinproductionofonemoleculeof    3-phosphoglycerateandonemoleculeof    2-phosphoglycolate.2-Phosphoglycolateismetabolizedtoglycolateandsecretedorfurthermetabolizedtoaminoacidsandothercom-pounds(seeSection4).Boththecarboxylaseandtheoxygenasereactionsrequireactivationof    RuBisCO via carbamylationof    theconservedlysineresidueonthelargesubunitanditsstabilizationbyMg 2+ .RuBPboundattheactivesiteisconvertedto   anenediolformwhichreactswitheitherCO 2  orO 2  (RoyandAndrews,2000).Oneof    themajorobstaclesinimprovingRuBisCOis   theinverserelationshipbetweentheCO 2 /O 2  specificityandcatalyticeffi-ciency(Tcherkezetal.,   2006;Whitneyetal.,2011).Comparisonof thekineticpropertiesof    RuBisCOfromdifferentphotoautotrophicorganismshasshownthattheMichaelis–MentenconstantsforCO 2 ( K  C )varyfrom3.3  M   in Galdierasulfuraria to246  Min Syne-chococcus sp.PCC7002(Tcherkezetal.,   2006;Whitneyetal.,   2011).TheRuBisCOfrom G.sulfuraria hasthehighestCO 2 /O 2  specificity(166)andoneof    thelowestratesof    catalyticturnoverof1.2s − 1 .Incontrast,theRuBisCOfrom Synechococcus sp.PCC7002hastheCO 2 /O 2  specificityof    52witha   catalyticturnoverof13.4s − 1 .Thefactthatthevaluesof    catalyticturnoverforRuBisCOseemtocor-relatenegativelywiththeCO 2 /O 2  specificityvalueshassuggestedthatimprovementoftheCO 2  fixationratecomesattheexpenseofCO 2  affinityandthattheenzymemightalreadybeoptimizedduringevolution(KapralovandFilatov,2007;Raines,2006;Saviretal.,   2010;Tcherkezetal.,2006).Yet,therestillseemstoberoomforsomeimprovementsinthespecificityof    RuBisCOparticularlyin   plantsandcyanobacteria.WhencomparingdifferentRuBisCOs,itisevidentthatRuBisCOfromredalgaesuchas Griffithsiamonilis  138  L.Rosgaardetal./     Journalof    Biotechnology 162 (2012) 134–147  inincreasedRuBisCOactivityby1.4folds(Atsumietal.,2009).Theoverexpressionconstructsweresubsequentlyintroducedtothe S.elongatus sp.PCC7942strainharboringthe alsS  genefrom B.sub-tilis ,the ilvC  and ilvD genesfrom E.coli forthesynthetic-biologicalproductionofisobutyraldehyde.Thisresultedina2-foldincreaseintheproductionrateandthetotalyieldofisobutyraldehyde.Theseresultsemphasizetheimportanceof    improvingcarbonfixationforincreasingproductivityincyanobacteria.TheroleofmolecularchaperoneinRuBisCOfoldingis   begin-ningtobeunderstood.Insomecyanobacteria, rbcX  co-localizeswith rbcL and rbcS  in   thechromosome.IthasbeenshownthatRbcXgreatlyimprovestheassemblyof    RuBisCOfrom S.   elonga-tus sp.PCC7942, Synechococcus sp.PCC7002and  Anabaena sp.CAintofunctionalcomplexeswhenexpressedin E.coli (Emlyn-Jonesetal.,2006;LiandTabita,1997;Liuetal.,2010;Onizukaetal.,2004;Saschenbreckeretal.,2007).The rbcX    deletionmutantof  S.elongatus sp.PCC7942displayednormalgrowthphenotypeandthecellularlevelandcarboxysome-partitioningofRuBisCOweresimilartothosein   wildtype(Emlyn-Jonesetal.,2006).In   astarkcontrast,introductionof    aframeshiftmutationinthe rbcX  genein Synechococcus sp.PCC7002resultedin   a   lethalphenotype(Onizukaetal.,2004). rbcX  formsageneclusterwith rbcL   and rbcS  in Syne-chococcus sp.PCC7002whereasin S.elongatus sp.PCC7942 rbcX  islocatedmorethana100kilo-baseawayfromthe rbcL – rbcS  clus-ter.Thedifferencesinmutantphenotypesandgeneorganizationmightreflectthedifferencesinimportanceof    theRbcXinthesetwospecies.Theroleof    RbcXinhigherplantsis   currentlyunknown.RbcXof  Synechococcus sp.PCC7002isfoundto   specificallybindto   asevenaminoacidstretchontheC-terminalofRbcL(Liuetal.,   2010;Saschenbreckeretal.,2007).ThesesevenaminoacidsarepresentintheRbcLsequencesoforganismscontainingFormIRuBisCObutabsentinformII   containingspecies.It   is   thereforepossiblethatthesimilarRbcX-RuBisCOassemblymechanismisfunctionalin   higherplantsaswell.  3.2.RuBisCOactivase Inplants,activationof    RuBisCOisessentialforcatalysisandismediatedbyRuBisCOactivase.RuBisCOactivaseremovestightlyboundsugarphosphate( i.e. ribulose-1,5-bisphosphate(RuBP))fromtheactivesitebyATPhydrolysistopromotecarbamylationandtheproductivebindingofRuBP(SalvucciandOgren,1996). RuBisCOactivaseisessentialforgrowthofArabidopsisatambi-entCO 2  sincethedeletionmutantsdisplayedseverelystuntedgrowthphenotypeascomparedtothewildtype(Kureketal.,2007).ItiswellknownthathightemperatureinhibitstheactivityofRuBisCOandreducescarbonfixationinC3plants,whichispar-tiallyattributedtothethermostabilityof    RuBisCOactivase(SalvucciandCrafts-Brandner,2004a).It   hasbeenknownthatRuBisCOacti-vasefromspeciesfromdiverseclimates(drytropicaltopolar)differwithrespecttotheirheatstabilities(SalvucciandCrafts-Brandner,2004b)andthatspeciesselectivityexistssuchthatRuBisCOacti-vasesin e.g. solanaceaearepooractivatorsof    RuBisCOascomparedtothoseinnon-solanaceae(Portisetal.,   2008).AchimericRuBisCOactivasewithhigherthermostabilityhasbeenconstructedwheretheRuBisCOrecognitiondomainofthemorethermostableactivasefromtobaccowasreplacedbythelessthermostableactivasefromArabidopsis.TransgenicArabidopsislinesexpressingthechimeradisplayedhigherratesof    photosyn-thesiswhenexposedtohightemperatures(38 ◦ C)(Kumaretal.,2009).Inaddition,theactivationstateofRuBisCOwasalsohigheratamoderatelyelevatedtemperature(30 ◦ C)resultingin   improvedgrowthwhiletherewasnodifferencebetweenwildtypeandthetransgeniclinesatnormalgrowthtemperature(22 ◦ C).Enhancedthermostabilityof    ArabidopsisRuBisCOactivasewasalsoobtainedbygeneratinga   seriesofvariantsof    theRuBisCOactivaseusingageneshufflingtechnology(Kureketal.,   2007).ThetransgeniclinescontainingtheRCAvariantsexhibitedincreasedactivationofRuBisCO,photosyntheticrates,aswellasin   biomassandseedyieldsascomparedtothewildtypewhengrownundermoderateheatstress.Incontrastto   higherplantsandalgae,evidenceof    involve-mentofRuBisCOactivasein   activationofRuBisCOislackingincyanobacteria.AsidefromRuBisCOactivase,RuBisCOindiversespeciesareknowntobeactivatedbyeffectormoleculesincludingphosphorylatedintermediates,NADPH,andorthophosphate(Pi)(AnwaruzzamanSawadaetal.,   1995;BuchananandSchurmann,1972;CholletandAnderson,1976;ChuandBassham,1975;Marcusetal.,2011;MarcusandGurevitz,2000).IthasbeenshownthatPiincreasestheaffinityofRuBisCOfortheactivatorCO 2  byseveralfolds(AnwaruzzamanSawadaetal.,   1995).Thecrystalstructureof RuBisCOfromtobaccoincomplexwithphosphaterevealedthreePi-bindingssitesatthelatchandbindingsitesofthetwo   phosphategroupsofRuBP(Duffetal.,2000).Site-directedmutagenesisofthephosphate-bindingglycine(Gly404)andthreonine(Thre65)toala-nineorserinediminishedthePi-stimulatedRuBisCOactivationin Synechocystis sp.PCC6803,underpinningtheimportanceof    PiinRuBisCOactivationinthisspecie(Marcusetal.,   2011).ThemutantRuBisCOexhibitedreduced K  cat  bybetween6and20foldsascom-paredtothewild-typeRuBisCOwhile K  M  forRuBPalsodecreasedfrom146  Minthewildtypetoaslowas46  MinthemutantRuBisCOs.Interestingly,thereductionof  K  cat  wascompensatedbyincreaseofthecontentsof    themutantRuBisCOsby3fold,result-ingin   thecompensationofthereducedcarboxylaserateof    themutantuptoone-thirdofthewild-typelevel.Intriguingly,boththewildtypeandthestrainscarryingthemutantRuBisCOsgrewsimilarlyandexhibitedthesimilarrateof    oxygenevolutionundersaturatedlightintensityandCO 2  supply.TheauthorssuggestedthatRuBisCOactivationandactivitiesarenot   limitingphotosyn-thesisandgrowthandthattheregenerationofRuBP,ratherthanRubiscoactivity,maybelimitingphotosynthesisin   cyanobacteria(Marcusetal.,2011).  3.3.Sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase Sedoheptulose-1,7-bisphosphatase(SBPase)isauniqueenzymetotheCalvincycleandfunctionsin   theregenerativepartof    theCalvincycle(Fig.1)(Raines,2003).Incyanobacteria,two   distinctformsof    SBPasehave   beenfound(Tamoietal.,   1996).Oneformcon-fersSBPaseactivitywhiletheotherisbi-functionalconferringbothSBPaseandfructose-1,6-bisphosphatase(FBPase)activities.FBPaseispositionedasabranchpointwherecarboneithercontinuesin   thecycleforregenerationof    RuBPorleavestheCalvincycletoenterthecentralcarbonmetabolism.Thebi-functionalSBPase/FBPasehassofarbeenfoundinfourcyanobacterialspecies: Synechocystis sp.PCC6803,  Anabaena sp.PCC7120, Plectonemaboryanum and S.elonga-tus PCC7942andhasbeenshownto   bemoreresistantto   hydrogenperoxidethanthespinachcounterparts(Tamoietal.,   1996,1998a).SeveralstudieshavedescribedtheimportanceofSBPaseinplantsbasedonanalysisoftransgenictobaccoandricein   whichtheexpressionofSBPasewassuppressedbyRNAinterference(Fengetal.,   2009;Lawsonetal.,   2006;Rainesetal.,   2000).In   thesestudies,mutantswithreducedSBPaseactivitiesdisplayedlowerCO 2 assim-ilationandlowerstarchaccumulationthanthewild-type.In   otherstudiesSBPaseswereoverexpressedinplants,whichhasresultedinfastergrowth,a   higherdryweightcontentandhigherstarch,sucroseandhexosecontentinleaves(Fengetal.,   2007;Lefebvreetal.,2005;Miyagawaetal.,2001).Ithasbeenestimatedthatintobaccoincreaseof    activitiesof    FBPaseandSBPaseby1.7foldand1.3fold,respectively,wouldhaveprofoundimpactsonphotosyn-thesis(Tamoietal.,   2006).
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