APPLIEDGENETICSANDMOLECULARBIOTECHNOLOGY
GeneticengineeringofthecompletecarotenoidpathwaytowardsenhancedastaxanthinformationinXanthophyllomycesdendrorhousstartingfromahigh-yieldmutant
SörenGassel&JürgenBreitenbach&GerhardSandmann
Received:15August2013/Revised:23October2013/Accepted:25October2013/Publishedonline:17November2013#Springer-VerlagBerlinHeidelberg2013
AbstractTheyeastXanthophyllomycesdendrorhousisoneoftherareorganismswhichcansynthesizethecommerciallyinterestingcarotenoidastaxanthin.However,astaxanthinyieldinwild-typeandalsoinclassicalmutantsisstilltoolowforanattractivebioprocess.Therefore,wecombinedclassicalmuta-genesiswithgeneticengineeringofthecompletepathwaycoveringimprovedprecursorsupplyforcarotenogenesis,en-hancedmetaboliteflowintothepathway,andefficientcon-versionofintermediatesintothedesiredendproductastaxanthin.Wealsoconstructednewtransformationplasmidsforthestepwiseexpressionofthegenesof3-hydroxymethyl-3-glutarylcoenzymeAreductase,geranylgeranylpyrophos-phatesynthase,phytoenesynthase/lycopenecyclase,andastaxanthinsynthase.Startingfromtwomutantswitha15-foldhigherastaxanthin,weobtainedtransformantswithanadditional6-foldincreaseinthefinalstepofpathwayengi-neering.Thus,amaximumastaxanthincontentofalmost9mgpergdryweightwasreachedinshakingcultures.Underoptimizedfermenterconditions,astaxanthinproductionwiththeseengineeredtransformantsshouldbecomparabletoHaematococcuspluvialis,theleadingcommercialproducerofnaturalastaxanthin.
KeywordsAstaxanthin.Carotenoidpathway.Pathwayengineering.Xanthophyllomycesdendrorhous
Introduction
Thecarotenoidastaxanthin(3,3′-dihydroxy-4,4′-diketo-β-car-otene)isacommerciallyimportantfeedingredientinsalmonandtroutfarming.Itshealth-promotingactivitiestohumansareduetoitshighantioxidativepotential—areasonwhyastaxanthinisgainingimportanceinthehealthfoodsector.Todate,themarketisdominatedbychemicallysynthesizedastaxanthin.However,biologicalsystemsarecurrentlydevel-opedandimproved.Thereareonlyveryfeworganismsknownwhichareabletosynthesizeastaxanthinoritsfattyacidandglycosylatedderivatives.TheyincludestrainsfromthebacteriumParacoccus(Misawaetal.1995),thealgaHaematococcuspluvialis(Margalith1999),onlyonefungusXanthophyllomycesdendrorhous(=sexualstateofPhaffiarhodozyma)(Andrewsetal.1976),andamongplantsonlythoseofthegenusAdonis(SeyboldandGoodwin1959).Amongthem,H.pluvialisistheprimarysourcefornaturalastaxanthinwhichisapprovedasfoodsupplement(LorenzandCysewski2000).Commerciallygrowncellsofthisalgacontainastaxanthininabout1.5to3%oftheirdrymatterwhereasastaxanthininwild-typestrainsofX.dendrorhousisbelow0.1%(JohnsonandSchroeder1995).However,chemicalmutantshavebeengeneratedwithsubstantiallyincreasedastaxanthincontent(seeRodriguez-Saizetal.2010andSchmidtetal.2011forreview).Forfurtherimprovementofastaxanthinforma-tion,itisanadvantageofX.dendrorhousthatitscarot-enoidbiosynthesispathwaycanbegeneticallymanipulat-ed.Allnecessarypathwaygeneshavebeencloned.Theyincludethoseencodingaphytoenesynthase/lycopenecy-clase(Verdoesetal.1999a),aphytoenedesaturase(Verdoesetal.1999b),andanastaxanthinsynthase(Ojimaetal.2006).Aftertransformationandplasmids
S.Gassel:J.Breitenbach:G.Sandmann(*)
InstituteofMolecularBioscience,J.W.GoetheUniversität,MaxvonLaueStr.9,60438Frankfurt,Germanye-mail:sandmann@bio.uni-frankfurt.de
346hadbeendeveloped(Weryetal.1998),firstattemptshavebeenmadetoevaluateandtoovercomelimitingstepsinthepathway.Itcouldbedemonstratedthatoverexpressionofthegatewayenzymephytoenesynthase,CrtYB,stimu-latedcarotenogenesis(Verdoesetal.2003)asdidtheoverexpressionoftheenzymegeranylgeranylpyrophos-phatesynthaseCrtEwhichisespeciallylimitedunderhighoxygenconditions(Breitenbachetal.2011).
AsastrategyfortheincreaseofastaxanthinformationinX.dendrorhous,acombinationofclassicalmutagenesisandgeneticpathwayengineeringwassuggested(Schmidtetal.2011).Thefeasibilityofthisapproachhasbeenrecentlydemonstratedwithpromisingresultsbyfurtherincreasingcarotenoidsynthesisintwohigh-yieldastaxanthin-producingmutantsbyengineeringthemetaboliteflowintothecaroten-oidpathway(Gasseletal.2013).Therefore,inthepresentinvestigation,westartedfromtheseX.dendrorhousmutantsandoverexpressedsystematicallythelimitingenzymesstepbystepthusincreasingthemetaboliteprovisionforthepath-wayandimprovingtheintermediateconversiontotheendproductastaxanthin.Theresultingastaxanthinyieldswerethehighestreachedtodateinlaboratoryculturesandmaybefurtherincreaseunderoptimizedfermentationconditions(Gasseletal.2013).
MaterialsandmethodsStrainsandcultivation
X.dendrorhous(=sexualstateP.rhodozyma)strainATCC96594(=CBS6938)wasgrowninshakingcultures(50mlin500mlbaffledErlenmeyerflasks,180rpm,over8days)at20°C.AmodifiedGSMmedium(deBoeretal.1993)witha10-foldincreasedCuSO4anda4.8-foldin-creasedbiotincontentwasused.ThismediumandthereddishpigmentastaxanthinmutantsAXG-13andAXJ-20with15-foldimprovedastaxanthinaccumulationrelativetothewildtypeobtainedbymutagenesiswithnitrosoguanidinehavebeendescribedrecently(Gasseletal.2013).TransformationofX.dendrorhouswasasdescribed(Visseretal.2005).Selectionoftransformantswaswithgeneticin(G418sulfate,100μ/ml),hygromycin(60μg/ml),ornourseothricin(30μg/ml)onagarplates.
EscherichiacolistrainsDH5αandJM110usedforgeneticmanipulationsandplasmidamplificationweregrowninLBmediumwiththeantibioticampicillin(100μg/ml).Geneticmanipulations
PlasmidpUC8ΔEcoRI-HNNHwasconstructedstartingwiththeeliminationoftheEcoRIsitefrompUC8(Hannaetal.1984)bylinearizationwithEcoRI,blunting
ApplMicrobiolBiotechnol(2014)98:345–350
andreligationyieldingpUCΔEcoRI.Thepromoter–ter-minatorcassettewasconstructedbyfusionPCRwithpPR2TNastemplateandprimersusedbefore(Ojimaetal.2006)withsomemodifications:PGPD1shortenedforATATT,PGPD2,andTGPD1withoutmodificationandTGPD2inwhichtheAGCTAAGCTTendwasre-placedbyGCGGCCGCcreatingaNotIrestrictionsite.Thispromoter–terminatorfusionproductwasthenligatedintotheT-overhangvectorpMON38201(BorovkovandRivkin1997),cutoutwithHindIII,andligatedintotheHindIIIsiteofpUCΔEcoRI.PlasmidspPR2TNHwithhygromycinasresistancemarkerandpPR2TNNwithnourseothricinresistancewereobtainedbymodificationofpPR2TN(Visseretal.2005).Inthisplasmid,theG418(geneticin)resistancecassettecontainingthenptIIgenefromMicromonosporarhodorangeawascutout(PstI/PstI)andreplacedbythehphgenefromE.coliencodinghygromycinphosphotransferase(GritzandDavies1983)orthenatgenefromStreptomycesnourseiencodingnourseothricinacyltransferase(Krügeletal.1988),re-spectively.AftertransformationofX.dendrorhous,geneintegrationwascontrolledbyPCR.
Carotenoidextractionandanalysis
CellsofX.dendrorhouswereharvestedandfreeze-dried.Carotenoidsweresubsequentlyextractedfromthesecellswithdimethylsulfoxidebyheatingfor15minat60°C.Aftercentrifugation,theextractwastransferredintoaseparatorfunnel,diethyletherwasadded,andthemixturewaskeptonicefor3minbeforewaterwasaddedforphaseseparation.Theupperphasewascollectedtowhichstepbystepacetone,10%(v/v)diethyletherinpetro-leumether(bp60–80°C),andwaterwereadded.Again,theupperphasewascollected,washedwithwater,driedinastreamofN2,andresuspendedinacetone.Detailsoftheextractionprocedurearegivenelsewhere(Visseretal.2005).High-performanceliquidchromatography(HPLC)separationofcarotenoidswasperformedinanisocraticwayona15-cmNucleosil100C18,5μcolumnwithacetonitrile/methanol/2-propanol(85:10:5,byvolume)asthemobilephaseat20°C.Spectrawererecordedonlinewithaphotodiodearraydetector440(Kontron,Straubenhard,Germany).Carotenoididentificationandquantificationwascarriedoutwithauthenticstandards.TheyweregeneratedbygeneticengineeringofE.colileadingtothereconstructionofpathwaytothedesiredcarotenoidsastaxanthin,HO-canthaxanthin,keto-hydroxy-torulene,HO-echinenone,echinenone,torulene,andβ-carotene(Sandmann2002).Carotenoidswerequantifiedfromfiveindependentlygrownculturesandaregiventogetherwiththeirstandarddeviations.
ApplMicrobiolBiotechnol(2014)98:345–350Results
Transformationvectors
Integrationintothegenomeisgenerallypreferredforengi-neeringoforganismstoincreasestability,reducetheeffectofplasmidburden,andtoavoidtheneedforselectiveagentsinfinalindustrialprocesses.However,thereisalimitationofintegrationvectorsavailableforX.dendrorhous.Previously,theintegrationplasmidpPR2TNwithG418asselectionmark-erhasbeenused(Visseretal.2005).Toexpandtheavailabil-ityofselectablemarkersandallowintegrationofseveralinsertsintoonestrain,weconstructedanewsetofplasmidsbyexchangingtheG418resistanceforhygromycinandnourseothricinresistanceobtainingpPR2TNHandpPR2TNN,respectively(Fig.1).Thethreeresistancegenesareunderthecontroloftheglyceraldehydephosphatedehy-drogenase(GAPDH)promoterandterminator,andtherestoftheplasmidareidentical.Multicopyintegrationisachievedthroughthe18S-ribosomalDNAsequencefromX.dendrorhous.ThemultiplecloningregionsoftheseplasmidscomprisetheEcoRI/XhoI/NotIrestrictionsite.ForinsertionofanygeneorcDNAofinterest,wealsoconstructedapUC8-basedmodificationplasmidpUC8ΔEcoRI-HNNH(Fig.1).ByinsertionintoitsEcoRI/XhoIsites,theGAPDHpromoterandterminatorarefusedtothereadingframe.TheflankingBamHI,NotI,andHindIIIsitesallowtheexcisionofthewholecassetteandtheinsertionofuptotwogenecassettesintoanyofthethreeintegrationplasmids.Thus,tandemgenesinthreeplasmidsallowtheinsertionofsixgenesforpathwaymanipulationofX.dendrorhous.Allthreeplasmidswereusedforgeneticpathwayengineeringbyinsertionofgenesoftheearlystepofprenylpyrophosphatesynthesisandlatestepsofthecarotenoidbiosynthesispathway.
InordertoovercomethelimitationofcarotenoidbiosynthesisinX.dendrorhouswhichinitiallyisphytoenesynthesiscatalyzedbyCrtYBandthenastaxanthinsynthesisfromβ-carotenecatalyzedbyAsyaftercrtYBoverexpression(Verdoesetal.2003),weconstructedpPR2TN-YB-ASYwithgeneticinasselec-tionmarkerforthesimultaneousintegrationofthecrtYBandtheasygeneasafirststep.StartingfrompPR2TN(Fig.1),theentirepromoter–terminatorcassetteswiththeasycDNAfrompPR2TN2BPAT(Ojimaetal.2006)andthecrtYBgenecassettefrompPR13F(Verdoesetal.2003)wereinserted.Bothgeneproducts,crtYBandasy,shouldenhancethemetaboliteflowintothecarot-enoidpathwaybyhigherphytoenesynthaseactivity,amoreactivecyclizationandabetterconversionofβ-caroteneandallketolatedintermediatesintoastaxanthin(Fig.2).
Metaboliteflowintocarotenogenesiscanbeimprovedbyoverexpressionofthekeyenzymeshydroxymethylglutaryl
347
coenzymeAreductaseofthemevalonatepathwayinX.dendrorhous(Shimadaetal.1998)andbyoverexpressionofgeranylgeranylpyrophosphatesynthase(CrtE)forsubstrateprovisiontophytoenesynthase(Breitenbachetal.2011).ThecrtEgeneandthe3-hydroxymethyl-3-glutarylcoenzymeA(HMG-CoA)reductasegene(HMG)fromX.dendrorhous(Verwaaletal.2007)werefirstclonedintopUC8ΔEcoRI-HNNH(Fig.1)forfusionwiththepromoterandterminatorofglyceraldehydephosphatedehydrogenaseandthewholecas-settescutout.InsertionofbothcassettesintopPR2TNHwithhygromycinasselectionmarker(Fig.1)yieldedpPR2TNH-HMG-CrtE.ThehydroxymethylglutarylcoenzymeAreduc-tasegenewasusedasatruncatedformlackingthemembrane-bindingregion.ForadditionalincreaseofAsyactivity,plas-midpPR2TNN-asywithnourseothricinasselectionmarkerwasobtainedbyinsertionoftheasycDNAcassettewiththeasycDNAintopPR2TNN.Thisresultingplasmidwasusedinthethirdtransformationstep.
TransformantsofmutantsAGX-13andAXJ-20
Twohigh-astaxanthinmutants,AXG-13andAXJ-20,wereselectedforcarotenoidpathwayengineeringsinceithasbeenshownthatphytoenesynthaseandastaxanthinsyn-thasewerestilllimitinginbothofthem(Gasseletal.2013).Therefore,plasmidpPR2TN-YB-asywithG418selectionhadbeenconstructedandwasusedforthetransformationofbothmutantsasafirststep.Severaltransformantswereselectedandanalyzedforcarotenoidcomposition.ThebestlineswithrespecttoastaxanthincontentsareshowninFigs.3and4.ThebestcrtYB-asytransformantsfromAXG-13andAXJ-20wereAXG#5with3.1mg/gdwandAXJ#37with3.7mg/gdwastaxanthin,respectively.EnhancedprovisionofprecursorsforcarotenogenesisviathemevalonatepathwaywasattemptedbyoverexpressingtheHMG-CoAreductasegene(HMG)incombinationwiththegeranylgeranylpy-rophosphatesynthasegenecrtEasoutlinedinFig.2.Therefore,inthenextstageofpathwayengineering,weusedplasmidpPR2TNH-HMG-CrtEwithhygromycinse-lectiontoenhanceprecursorsupplyforthecarotenoidpathway.Forthispurpose,AXG#5andAXJ#37wereused.Thesetransformationsresultedinafurtherincreaseoftheastaxanthinyieldreaching5.2mg/gdwinAXG#521andinAXG#522,4.7mg/gdwinAXJ#3726and5.8mg/gdwinAXJ#3728.DetailedanalysisofthecarotenoidcompositionofthedoubletransformantsAXG#522andAXJ#3728showedthatonly52and61%oftheastaxanthinprecursorcarotenoidswerefinallymetabolizedintoastaxanthin(Table1).Inaddition,HO-canthaxanthinandHO-echinenonewhicharesubstratesofAsyaccumulateinthedoubletransformantstoahigherpercentagecomparedtothesingletransformants.This
348
Fig.1ModificationandtransformationvectorsforthegeneticengineeringofX.
dendrorhous.PGAPDHpromoterofglyceraldehydephosphatedehydrogenase,TGAPDHterminatorofglyceraldehydephosphatedehydrogenase,G418RG418resistancegene,HygRhygromycinresistancegene,NurRnourseothricin
resistancegene,AmpRampicillinresistancegene,rDNAribosomalDNA
ApplMicrobiolBiotechnol(2014)98:345–350
resultindicatedthatalthoughAsywasoverexpressedinthefirsttransformationround,therewasstillalimitationintheactivityofthisenzyme.Inordertostrengthenthemetabolizationofβ-carotene,3-HO-canthaxanthin,echinenone,and3-HO-echinenoneintoastaxanthin,plas-midpPR2NN-asycontainingtheasycDNAwithnourseothricinselectionwasusedforadditionalintegrationandexpressioninAXG#522andAXJ#3728.Afterthisthirdtransformationround,theselectedtransformantsshowedahigherastaxanthincontent.TheywereAXG#5221with8.0,AXG#5223with6.5mg/gdw,AXJ#37284with8.7mg/gdwandAXJ#37287with7.8mg/gdwastaxanthin(Figs3and4).Inallofthem,thehigheradditionalexpressionofAsydecreasedtheintermediatelevelsresultinginahigherpercentageofastaxanthinpertotalcarotenoidsandahigherabsoluteamountinthecells.DataforAXG#5221andAXJ#37287areshowninTable1.Integrationofaddition-alcopiesofasyinthethirdtransformationroundwasdemonstratedbySouthernhybridizationwithaprobeagainstthenourseothricinresistancegeneasintegrationmarker.
Fig.2StrategiesforgeneticmodificationofcarotenoidbiosynthesisforenhancedastaxanthinformationinX.dendrorhousbyexpressionoflimitinggenesinthepathway.Thespecificcarotenoidpathwayisboxedthereactionscatalyzedbyastaxanthinsynthaseencodedbytheasygeneiscircled.Fromtheearlymevalonatepathwayandformationandinterconversionofprenylpyrophosphates,theoverexpressedgeneswerethosefor3-hydroxymethyl-3-glutarylcoenzymeAreductase(HMG)andgeranylgeranylpyrophosphatesynthase(crtE).GenecrtYBencodesabifunctionalphytoenesynthase/lycopenecyclase.KHTketo-hydroxy-torulene,Astastaxanthin
ApplMicrobiolBiotechnol(2014)98:345–350Fig.3AstaxanthinformationafterseveralmodificationstepsofmutantAXG-13resultingfromchemicalmutagenesisandselectionforhighastaxanthincontent.Concentrationsaremeansofdeterminations±stan-darddeviationfromfiveindividualcultures.ArrowsindicatewhichofthestrainsandlineswasusedforstepwisetransformationstartingwithAXG-13;genecombinationsontoprepresentthetransgenicbackgroundoftherecipientstrain.Mumutant,crtYBphytoenesynthase/lycopenecyclasegene,asyastaxanthinsynthasegene,crtEgeranylgeranylpyrophosphatesynthasegene,HMG3-hydroxymethyl-3-glutarylcoenzymeAreductasegene
Discussion
Overdecades,attemptshavebeenmadetogeneratehigh-yieldastaxanthinmutantsbychemicalmutagenesis(Rodriguez-Saizetal.2010;Schmidtetal.2011).Althoughsignificantprogresshasbeenmade,thelevelsreachedwerenotsufficient
Fig.4AstaxanthinformationafterseveralmodificationstepsofmutantAXJ-20resultingfromchemicalmutagenesisandselectionforhighastaxanthincontent.Concentrationsaremeansofdeterminations±SDfromfiveindividualcultures.ArrowsindicatewhichofthestrainsandlineswasusedforstepwisetransformationstartingwithAXJ-20;genecombinationontoprepresentsthetransgenicbackgroundoftherecipientstrain.Mumutant,crtYBphytoenesynthase/lycopenecyclasegene,asyastaxanthinsynthasegene,crtEgeranylgeranylpyrophosphatesynthasegene,HMG3-hydroxymethyl-3-glutarylcoenzymeAreductasegene
349
Table1CarotenoiddistributionintransformantsofX.dendrorhousmutantsAXG-13andAXJ-20
AXG-13AXJ-20#522
#5221
#3728
#37284
Astaxanthincontent(mg/gdw)5.2±0.89.0±0.75.8±0.68.6±1.4%DistributionAstaxanthin
52646169HO-canthaxanthina14090Keto-hydroxy-torulene5766HO-echinenonea14131210Echinenonea5644Torulene0107β-Carotenea9
7
6
4
Astaxanthincontentasmeansofdeterminations±SDfromfiveindividualcultures
aSubstrateorintermediateswhichcanbeconvertedbyastaxanthinsyn-thaseintoastaxanthin
forcommercialastaxanthinproduction.Twoofthosemutants,AXG-13andAXJ-20,havebeenrecentlyanalyzed.Inaddi-tiontotheirincreasedastaxanthincontent,itwasshownthatphytoenesynthaseandastaxanthinsynthasewerestilllimitinginbothofthem(Gasseletal.2013).Therefore,theywereusedforgeneticpathwayengineeringaimingforanadditionalincreaseoftheastaxanthinproduction.AsoutlinedinFig.2,thesemetaboliclimitationsofthecarotenoidpathwayinX.dendrorhouswereovercomebyintegrationofadditionalcop-iesofthephytoenesynthase/lycopenecyclasegenecrtYBandtheastaxanthinsynthasecDNAasyviaasingletransforma-tionvector.Thisleftthetwoothertandemintegrationvectorswithotherselectionmarkers(Fig.1)openforfurthergeneticmodifications.OneofthemwasusedtoprovideprecursormetabolitesofthemevalonatepathwaysinceanenhancedcarotenoidpathwayneedsanefficientmetabolitesupplybyoverexpressinggeranylgeranylpyrophosphatesynthaseCrtE(Breitenbachetal.2011)incombinationwiththeHMG-CoAreductasegene(HMG).Thelatteristhekeyenzymeofthemevalonatepathway(Shimadaetal.1998)providingprecur-sorstotheterpenoidpathwaysincludingcarotenoidandsterolbiosynthesisinX.dendrorhous.Thisadditionaltransforma-tionstepincreasedtotalcarotenoidsynthesisinbothX.dendrorhousmutants.However,theenzymeAsyalthoughalreadyoverexpressedhasbecomeabottleneckofthecarot-enoidpathwayintheresultingdoubletransformant.Theen-dogenousastaxanthinsynthaseAsyandtheproductofthecopiesfromthefirsttransformationroundwerenotsufficientforefficientmetabolizationofβ-caroteneandintermediatesoftheAsycatalyzedreactions(Table1).ThefinaltransformationwithanotherasycontainingplasmidpPR2TNN-asyassuredaconstantmetabolicflowfromthemevalonatepathwayvia
350prenylpyrophosphatesintocarotenogenesisandtothedesiredendproductastaxanthin.
Inconclusion,thecombinationofclassicalmutagenesiswithgeneticpathwayengineeringwashighlyeffectivetoobtainanextremelyhighastaxanthinaccumulationinX.dendrorhous.Thisproductionlevelisregardedtobeattractiveforcommercialexploitation(JohnsonandSchroeder1995)especiallywhentheamountofastaxanthinofengineeredtransformantscanbeincreasedadditionallyupto3-foldbyoptimizedfermentercultivationcomparedtoshakingculturesaspreviouslyshown(Gasseletal.2013).Startingfromtwomutantswitha15-foldhigherastaxanthincontentthanthewild-type,thisproductioncouldbetoppedbyanother6-foldincreaseduetopathwayengineering.Itwaspossibletoge-neticallymodifytheentirepathwayofbothX.dendrorhousmutants,theprovisionofmetabolitesforcarotenoidsynthesisandthecarotenoidtowardstheendproductastaxanthin.ThisindicatesthatthemutationsinAXG-13andAXJ-20mostlikelyaffectedupregulationofastaxanthinsynthesisbyaprocesswhichisnotunderstoodyet.Nevertheless,wedem-onstratedherethatthecombinedstrategyofrandommutagen-esishittinganunknownregulatorysystemfollowedbysys-tematicgeneticpathwayimprovementwashighlysuccessfulforthedevelopmentofastaxanthinproductionstrains.ThepotentialforcommercialuseasacellfactoryforastaxanthinofthistypeofgeneticallyengineeredX.dendrorhousstrainshasnowtobeproveninoptimizedfermenterculturesbydevelopmentofaroutineandcost-competitivebioprocess.Thisshouldalsoincludeadetailedinvestigationonthemet-abolicfluxeswithinthecarotenoidpathway.
AcknowledgmentsThisworkwasfundedbytheGermanFederalMinistryofEducationandResearch(BMBF)(FKZ0315327).
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