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Lima, Serena and Webb, Conner L. and Deery, Evelyne and Robinson, Colin and Zedler, JulieA. Z. (2018) Human Intrinsic Factor Expression for Bioavailable Vitamin B12 Enrichment inMicroalgae. Biology, 7 (1). p. 19. ISSN 2079-7737.

DOI

https://doi.org/10.3390/biology7010019

Link to record in KAR

http://kar.kent.ac.uk/66137/

Document Version

Publisher pdf

ȱ

Biologyȱ2018,や7,や19;やdoi:10.3390/biology7010019や www.mdpi.com/journal/biologyや

Articleȱ

HumanȱIntrinsicȱFactorȱExpressionȱforȱBioavailableȱVitaminȱB12ȱEnrichmentȱinȱMicroalgaeȱSerenaȱLimaȱ1,2,ȱConnerȱL.ȱWebbȱ1,ȱEvelyneȱDeeryȱ1,ȱColinȱRobinsonȱ1ȱandȱJulieȱA.ȱZ.ȱZedlerȱ1,3,*ȱ1や IndustrialやBiotechnologyやCentre,やSchoolやofやBiosciences,やUniversityやofやKent,やGilesやLane,やCanterbury,や や

KentやCT2や7やNK,やUK;やserena.lima@unipa.itや(S.L.);やC.l.webb@kent.ac.ukや(C.L.W.);や やE.Deery@kent.ac.ukや(E.D.);やC.Robinsonま504@kent.ac.ukや(C.R.)や

2や Dipartimentoやdell’InnovazioneやIndustrialeやeやDigitale,やUniversitàやdegliやStudiやdiやPalermo,や やVialeやdelleやScienzeやEd.や6,や90128やPalermo,やItalyや

3や CopenhagenやPlantやScienceやCentre,やDepartmentやofやPlantやandやEnvironmentalやSciences,や やUniversityやofやCopenhagen,やThorvaldsensvejや40,や1871やFrederiksbergやC,やDenmarkや

*や Correspondence:やjz@plen.ku.dk;やTel.:や+45ま353ま301ま68や

Received:や28やNovemberや2017;やAccepted:や13やFebruaryや2018;やPublished:やdateや

Abstract:や Dietaryや supplementsや andや functionalや foodsや areや becomingや increasinglyや popularやcomplementsや toや regularやdiets.やAや recurringや ingredientや isや theや essentialや cofactorや vitaminやB12や (B12).やMicroalgaeやareやmakingやtheirやwayやintoやtheやdietaryやsupplementやandやfunctionalやfoodやmarketやbutやdoやnotやproduceやB12,やandや theirやB12やcontentや isやveryやvariable.や Inや thisやstudy,や theやsuitabilityやofやusingや theやhumanや B12まbindingやproteinや intrinsicや factorや (IF)や toや enrichや bioavailableやB12や usingやmicroalgaeやwasやtested.やTheやIFやproteinやwasやsuccessfullyやexpressedやfromやtheやnuclearやgenomeやofやtheやmodelやmicroalgaやChlamydomonasȱ reinhardtiiȱ andや theや additionや ofや anやNまterminalや ARS2や signalや peptideや resultedや inやefficientや IFやsecretionや toや theやmedium.やCoまabundanceやofやB12やandや theやsecretedや IFやsuggestsや theやalgalやproducedやIFやproteinやisやfunctionalやandやB12まbinding.やUtilizingやIFやexpressionやcouldやbeやanやefficientやtoolやtoやgenerateやB12まenrichedやmicroalgaeや inやaや controlledやmannerや thatや isや suitableや forやvegetariansやand,やpotentially,やmoreやbioavailableやforやhumans.や

Keywords:ȱ Chlamydomonas;や vitaminや B12;や cobalamin;や intrinsicや factor;や microalgae;や nuclearやtransformation;やrecombinantやprotein;やdietaryやsupplements;やfunctionalやfoodsや

や

1.ȱIntroductionȱ

Thereやisやanやincreasingやawarenessやofやtheやeffectやofやdietやonやhumanやhealthやand,やinやparticular,やinやtheやdevelopmentやofやillnessやandやchronicやdisease.やThisやhasやledやtoやanやupwardやtrendやinやconsumerやdemandやforや naturallyや occurringや bioactivesや thatや canや beや deliveredや inや theや formや ofや dietaryや supplementsや orやincorporatedやintoやfunctionalやfoods.やOneやofやtheやcommonやbioactivesやisやvitaminやB12.や

VitaminやB12やorやcobalaminや(B12),やisやanやessentialやcofactorやproducedやbyやcertainやprokaryotesやviaやtwoやmainやcomplexやbiosyntheticやpathwaysや[1].やInやhumans,やB12やisやanやessentialやcofactorやrequiredやforやaやrangeやofやcellularやmetabolismやfunctionsや(e.g.,やDNAやsynthesisやandやmethylation,やmitochondrialやmetabolism)や[2].やB12やuptakeやandやtransportやfromやingestionやtoやtheやbloodやstreamやisやaやmultiまstepやcomplexやpathwayや[3].やOneやofやtheやproteinsやplayingやaやkeyやroleやinやthisやprocessやisやtheやhumanやproteinやintrinsicやfactorや(IF).やTheやglycoproteinやbindsや freeやB12や inや theや ileumやandや theや IFまB12やcomplexや isや thenや recognizedやbyや theやcubamやreceptorやcomplexやfacilitatingやreceptorまmediatedやendocytosisや[4].やTheやlackやofやaやfunctionalやIFやproteinやleadsやtoやtheやsevereやautoimmuneやdiseaseやperniciousやanaemiaや[3].やLessやsevereやbutやratherやcommonやcasesやofやB12やdeficiencyやareやtypicallyやfoundやinやchildren,やpregnantやwomenやandやtheやelderly.やVegetariansやandやvegansやareやalsoやatやaやhigherやriskやforやB12やdeficiency,やasやhigherやplantsやdoやnotやproduceやorやuptakeやB12やandやare,やtherefore,やnotやaやdietaryやsourceやofやB12や[5].やToやcounteractやtheやlackやofやdietaryやB12やintake,やaやwideやrangeや

Biologyȱ2018,や7,や19や 2やofや13や

ofや supplementsや areや commerciallyや available.や Forや instance,やmicroalgalやChlorellaȱ supplementsやwereやfoundやtoやcontainやamountsやofやB12やvaryingやfromやtraceやamountsやupやtoや415やょgやperや100やgやdryやweightや[6].や

Microalgaeや areや versatile,やunicellularやphotosyntheticや organismsや thatや areや attractingや increasingやattention.やDespiteやedibleやmicroalgaeやhavingやaやlongやtraditionやasやaやfoodやsource,やlargeやscaleやcultivationやhasや onlyや becomeや widespreadや sinceや theや middleや ofや lastや centuryや [7,8].や Potentialや exploitationsや ofやmicroalgaeやareやasやdiverseやasやtheやpolyphyleticやgroupやitself.やInや theやcontextやofやthisやstudy,やthreeやmainやapplicationsや shouldやbeやmentioned:や (I)やmicroalgaeや forや functionalや foodsや (dueや toや theirやhighやproteinやcontent,やantiまoxidativeやpropertiesやandやotherやpotentialやhealthやbenefits)やandやasやaやvegetarianやsourceやofやbioactiveやsupplementsや[9–11];や(II)やmicroalgaeやasやaやnovelやrecombinantやbiotechnologicalやhostや[12–15];やandや(III)やmicroalgaeやasやaやsourceやofやnaturalやhighまvalueやcompoundsや(recentlyやreviewedやinや[12,16]).や

TheseやthemesやofやmicroalgalやapplicationsやareやbroughtやtogetherやinやthisやproofまofまconceptやstudyやbyやtestingやtheやpossibilityやofやenrichingやB12やinやmicroalgaeやusingやtheやhumanやIFやprotein.やToやthisやend,やnuclearやtransformantsやofやtheやgreenやmodelやalgaやChlamydomonasȱreinhardtiiȱexpressingやIFやwereやgenerated.やTheやIFや proteinやwasや successfullyや expressedや andや theや additionや ofや aや signalや peptideや fromや arsylsulfataseやresultedやinやtheやefficientやsecretionやofやIFやtoやtheやmedium.やTheやsupplementationやofやtheやcultureやmediumやwithやB12やledやtoやaやhigherやIFやabundanceやinやtheやgeneratedやmicroalgalやstrains.やThisやproofまofまconceptやshowsやmicroalgaeやareやaやviableやhostや forや theやproductionやofやaやvegetarianやIFやsourceや forやB12やenrichment.やThisやcouldやpaveやtheやwayやforやdevelopingやaやvegetarianやsourceやofやbioavailableやB12やforやdietaryやsupplementsやandやtheやenrichmentやofやfunctionalやfoods.や

2.ȱMaterialsȱandȱMethodsȱ

2.1.ȱConstructȱDesignȱandȱPlasmidȱConstructionȱ

Theや aminoや acidや sequenceや ofや theや humanや intrinsicや factorや (Uniprotや KB:や P27352)やwasや codonまoptimizedやforやnuclearやexpressionやinやtheやgreenやalgaやC.ȱreinhardtiiȱusingやtheやsoftwareやGeneDesignerや2.0や(Atum,やNewark,やCA,やUSA)や[17].やTheやNまterminalやnativeやsignalやpeptideや(aminoやacidやsequenceや(AA)や1–18)やwasやremovedやandやaやCまterminalやhumanやinfluenzaやhemagglutininや(HA)やepitopeやtagや(AAやsequence:やYPYDVPDYA)やwasやaddedやforやdetectionやofやtheやprotein.やAdditionally,やaやsecondやconstructやwasやmadeやwithやanやNまterminalやsecretionやsignalやpeptideやofやtheやC.ȱreinhardtiiȱnativeやarsylsulfataseやARS2ȱ(AAや1–21,やUniprotやKB:やQ9ATG5)や[18].やTheやsyntheticやgenesやwereやcustomやsynthesizedやbyやGenscriptや(Piscataway,やNJ,やUSA)やandやclonedやintoやtheやpCrEX1やnuclearやexpressionやvectorや(basedやonやpSR108や[19])やusingやtheやrestrictionやsitesやSapIや (LguI)やandやBglII.やFigureや1やgivesやanやoverviewやofや theやexpressionやcassettes.やTheやcorrectや constructや assemblyや hasや beenや verifiedや byや sequencing.や Allや plasmidsや wereや amplifiedや inやEscherichiaȱcoliやDH5ぼやandやpurifiedやforやtransformationやofやC.ȱreinhardtii.や

やFigureȱ 1.や Overviewや ofや theや intrinsicや factorや (IF)や expressionや cassettesや usedや forや C.ȱ reinhardtiiやtransformation.やTheやstrainsやexpressingやtheやconstructsやhaveやbeenやnamedや“IF”やandや“arsまIF”やrespectively.やTheや3たやandや5たやuntranslatedやregionsやofやtheやC.ȱreinhardtiiやPSADやgeneやareやusedやforやgeneやexpression.や(a)やTheやNまterminalやnativeやsignalやpeptideやofやtheやhumanやIFやproteinやwasやremovedやandやtheやfullやlengthやproteinやwasや codonまoptimizedや forや C.ȱ reinhardtiiや nuclearや expression.や Aや Cまterminalや humanや influenzaやhemagglutininや(HA)やepitopeやtagやwasやaddedやtoやtheやproteinやforやdetectionやpurposes.や(b)やAdditionally,やaやsecondやversionやofやIFやwasやconstructedやwithやtheやsecretionやsignalやpeptideやofやarsylsulfataseやARS2.や

Intrinsic Factor (AA19-417)

HA-tag

PSAD

5�UTR PSAD 3�UTR

PSAD

5�UTR

Intrinsic Factor (AA19-417)

HA-tagARS2 (AA1-21)

PSAD 3�UTR

(a)

(b)

�IF� strain

�ars-IF� strain

Biologyȱ2018,や7,や19や 3やofや13や

2.2.ȱStrainsȱandȱCultivationȱConditionsȱ

TheやC.ȱreinhardtiiやstrainやCCま849や(cw10,やmt┑)やwasやobtainedやfromやtheやChlamydomonasやResourceやCenterや(UniversityやofやMinnesota,やMinneapolis,やMN,やUSA).やAllやstrainsやwereやmaintainedやatや25や°Cやandやapproximatelyや50やょEやcontinuousやilluminationやonや2%やTrisまAcetateまPhosphateや(TAP)やmediumや[20]やagarやplatesやwithやaやmodifiedやtraceやelementやrecipeや[21].やLiquidやculturesやwereやgrownやinやTAPやmediumやatや25や°C,や110やrpmやshakingやandやapproximatelyや100やょEやcontinuousやillumination.やWhenやTAPやmediumやwasやsupplementedやwithやvitaminやB12や(20–100やょgぉL┑1),やcommerciallyやobtainedやcobalaminや(SigmaまAldrich,やDorset,やUnitedやKingdom)やwasやaddedやfromやaや0.1やgぉL┑1やstockやsolution.や や

Initialや screensや forやpositiveや transformantsやwereやperformedや inや 24やwellやplates.やTheやwellsやwereやinoculatedやwithやsingleやcoloniesやandやgrownやforや5やdaysやbeforeやanalysis.やForやproteinやexpressionやanalysis,や100やmLやpreまculturesやwereやinoculatedやfromやaやplate.やAfterや6やdays,や100やmLやculturesやwereやreまinoculatedやtoや anや OD750や ofや 0.01.や Cellsや wereや subsequentlyや harvestedや atや midや toや lateや logや phase.や Forや proteinやconcentrationやandやB12やassays,や1やLやculturesやwereやinoculatedやtoやanやOD750や=や0.02やfromやaや1やweekまoldや100やmLやpreまcultureやandやgrownやtoやaやmidまlogやphaseやstageやbeforeやprocessing.や

2.3.ȱNuclearȱTransformationȱ

Forやnuclearやtransformation,や1やょgやofやtheやrespectiveやplasmidやwasやlinearizedやbyやcuttingやwithやEcoRI.やAやnuclearやglassまbeadや transformationやprotocolやwasやusedやbasedやonやaやpreviouslyやdescribedやmethodや[22,23].やInやbrief,やaや200やmLやcultureやofやCCま849やwasやgrownやtoやearlyやlogまphaseやandやresuspendedやinや2やmLやfreshやTAPやmedium.やPerやtransformation,や300やょLやconcentratedやcellsやandや1やょgやofや linearizedやplasmidやwereやused.やTheやcellまDNAやmixtureやwasやaddedや toやapproximatelyや300やmgや400–600やょmやacidまwashedやglassやbeadsや(SigmaまAldrich,やDorset,やUnitedやKingdom)やandやagitatedやonやaやvortexやforや15やsやonやmaximumやspeed.や Theや cellsや wereや dilutedや withや TAPやmediumや toや aや finalや volumeや ofや 10やmLや andや grownや forやapproximatelyや18やh.やAfterやresuspensionやofやtheやcellやpelletsやinや500やょLやTAPやmedium,やsoftやagarや(0.5%)やwasや addedや andや theや mixtureや spreadや onや TAPや platesや containingや 10や ょgぉmL┑1や Zeocinや (InvivoGen,やToulouse,やFrance)やforやselectionやofや transformantsや thatや incorporatedや theやconstructやcontainingや theやbleやmarkerや[19].やAfterやapproximatelyや8やdays,やcoloniesやwereやreまstreakedやandやsubsequentlyやanalyzedやforやtheやpresenceやofやtheやgeneやofやinterest.や

2.4.ȱPCRȱAnalysisȱ

Transformantやcoloniesやwereやscreenedやforやtheやpresenceやofやtheやgeneやofやinterestやusingやpolymeraseやchainやreactionや(PCR).やAやgeneやfragmentやwasやamplifiedやfromやgenomicやDNAやusingやaやstandardやprotocolやwithやPhireやPlantやDirectやMasterやMixや(ThermoやFisherやScientific,やLoughborough,やUnitedやKingdom).やTheやfollowingやprimersやwereやusedやtoやamplifyやaや600やbaseやpairやfragmentやconfirmingやtheやpresenceやofやtheやIF/arsまIFや expressionや cassette:や IFまfragFや (5たま3た)や CAGCATGAAGATTAAGGACAや andや IFまfragRや (5たま3た)やGTAGTACTGCGTGAAGTTG.や

2.5.ȱPreparationȱofȱCellularȱLysatesȱandȱMediumȱSamplesȱforȱProteinȱExpressionȱAnalysisȱ

Forやproteinやexpressionやanalysis,やculturesやwereやgrownやasやindicatedやinやSectionや2.2.やCellularやlysatesやwereやpreparedやbyやresuspendingやanやequivalentやofやOD750や=や1やcellsやinやmidやtoやlateやlogやphaseやinや100やょLや10やmMやTrisまHClや(pHや8.0).やToやanalyzeやtheやmedium,や1やmLやofやcultureやwasやspunやdownやatや20,000×やgやforや5やminやandやtheやclearedやsupernatantやtransferredやtoやaやseparateやtube.や

2.6.ȱProteinȱConcentrationȱandȱIonȱExchangeȱChromatographyȱ

1やLやmidまlogやphaseやculturesやwereやharvestedやatや4000×やgやforや20やminやatや4や°C.やForやproteinやconcentrationやfromやtheやmedium,やtheやcultureやsupernatantやwasやfilteredやthroughやWhatmanやfilterやpaperやtoやremoveやanyやdebrisやandやsubsequentlyやthroughやaや0.2やょmやmembraneやtoやremoveやanyやresidualやcells.やTheやpreまpurifiedやmediumやfractionやwasやsubsequentlyやloadedやonやtheやcolumn.や

Forやconcentrationやofやcellularやproteins,やtheやharvestedやcellsやwereやresuspendedやinやlysisやbufferや(20やmMやTrisまHClや(pHや7.2),や5%やglycerol,や20やょgぉmL┑1やDNaseIや(Roche,やMannheim,やGermany),やEDTAまfreeやproteaseやinhibitorや(Roche))やandやsonicatedや(3やcyclesやofや30やsやsonicationやandや30やsやonやice).やTheやlysateやwasや

Biologyȱ2018,や7,や19や 4やofや13や

clearedやbyやultracentrifugationや (BeckmannやTLAま100.3や rotorや (Beckman,やLismeehan,や Ireland),や70,000やrpm,や30やmin)やandやtheやsolubleやsupernatantやloadedやonやtheやcolumn.や

Forや ionやexchangeや chromatography,や10やmLや columnsや (QやSepharoseやFastやFlow,やGEやHealthcare,やLittleやChalfont,やUnitedやKingdom)やwereやfreshlyやpreparedやforやeachやpurification.やAやvacuumやpumpやwasやusedや toやpassや theやmediumやsamplesや throughや theやcolumn.やAfterや loadingやofや theや sampleや (mediumやorやcellularや lysate),や theや columnやwasやwashedやwithやaやTrisやbasedやbufferや (20やmMやTrisまHClや (pHや7.2),や5%やglycerol,や1やmMやMgCl2,や5やmMやdithiothreitol)やcontainingや50やmMやNaCl.やProteinや fractionsやwereや thenやelutedやwithやbufferやcontainingやrisingやconcentrationsやofやNaClや(100やmM,や150やmM,や200やmM,や300やmM,や400やmMやandや1やMやNaCl).や

2.7.ȱVitaminȱB12ȱAssayȱ

Theや bioassayや forや theや detectionや ofや B12や contentや inや theや proteinや fractionsや fromや ionや exchangeやchromatographyやisやbasedやonやtheやSalmonellaȱentericaやserovarやTyphimuriumやLT2やstrainやAR2680や(metE�,やcbiB–).やDetailsやofやtheやassayやareやdescribedやelsewhereや[24].やInやveryやbrief,やthisやSalmonellaȱstrainやcannotやsynthesizeや B12や deや novoや (CbiB–)や andや hasや noや B12まindependentやmethionineや synthaseや (MetE–).や Thus,やgrowthやofやthisやstrainやisやdependentやonやanやexternalやsourceやofやvitaminやB12やasやaやcoまfactorやforやtheやvitaminやB12まdependentやmethionineやsynthaseやMetH.やTheやplaqueやsizeや(growth)やisやdependentやonやtheやamountやofやexternallyやsupplementedやvitaminやB12,やandやcanやthusやbeやusedやtoやsemiまquantifyやVitaminやB12やlevelsやinやtheやsample.やAやcalibrationやcurveやusingや10やょLやofや0.001やょM,や0.01やょM,や0.1やょMやandや1やょMやB12やsolutionsやledやtoやtheやequationやusedやtoやcalculateやtheやB12やcontentやinやtheやanalyzedやsamples:や

vitaminやB12やconcentrationや(ょM)や=や0.0029や×や(plaqueやdiameterやinやcm)5.4616や (1)や

UnpairedやStudent’sや tまtestsやwereやperformedやusingや theやsoftwareやPrismや (versionや7.0,やGraphPadやSoftware,やLaやJolla,やCA,やUSA).やValuesやwereやconsideredやstatisticallyやsignificantやforやpや<や0.05.や

2.8.ȱReducingȱSDSȬPAGE,ȱNativeȱPAGEȱandȱWesternȱBlottingȱ

Samplesやwereやseparatedやbyやelectrophoresisやonや12%やpolyacrylamideやgelsやusingやaやBioまRadやMiniやProteanやGelや systemや (BioまRadやLaboratories,やWatford,やUnitedやKingdom).やForや reducing,やdenaturingやsodiumやdodecylやsulfateやpolyacrylamideやgelやelectrophoresisや(SDSまPAGE),やsamplesやwereやboiledやwithやLaemmliやbufferやcontainingやぽまmercaptoethanolやatや95や°Cやforや5やminや(crudeやcellularやlysatesやandやmediumやsamples)やorや10やminやatや50や°Cや(fractionsやfromやionやexchangeやchromatography).やForやnativeや や や analysis,やsamplesやwereやnotやboiledやandやnoやsodiumやdodecylやsulfateやnorやぽまmercaptoethanolやwasやusedやinやsamples,やgelsやorやrunningやbuffer.やTheやpolyacrylamideやgelsやwereやsubsequentlyやimmunoblottedやusingやantibodiesやagainstやtheやCまterminalやHAまtagや(SigmaまAldrich,やDorset,やUnitedやKingdom)やandやagainstやtheやCまterminusやofやtheやIFやproteinや(abcam)やtoやdetectやtheやproteinやofやinterest.や

2.9.ȱDensitometricȱAnalysisȱ

Densitometryやanalysisやofや immunoblotsやwasやperformedやusingや ImageLabやSoftwareやVersionや4.1や(BioまRadやLaboratories,やWatford,やUnitedやKingdom).やTheや relativeやabundanceやofや theや IFやproteinやwasやcalculatedやinやrelationやtoやtheやproteinやbandやfoundやwithoutやsupplementationやofやB12やtoやtheやmedium.やForやtheやarsまIFやstrains,やtheやcalculatedやabundanceやwasやnormalizedやtoやtheやOD750やofやtheやculturesやmeasuredやatやharvestingやtoやaccountやforやgrowthやdifferences.やThisやcorrectionやwasやnotやperformedやforやtheやIFやstrainsやasやequalやamountsやofやcellularやlysateやstandardizedやtoやOD750やwereやloadedやonやtheやgel.や

3.ȱResultsȱ

3.1.ȱGenerationȱofȱC.やreinhardtiiȱNuclearȱTransformantsȱ�IF�ȱandȱ�arsȬIF�ȱ

Forやthisやstudy,やconstructsやforやnuclearやtransformationやofやaやwallまdeficientやC.ȱreinhardtiiȱstrainや(CCま849)やwereやassembledやwithやtheやhumanやgeneやencodingやtheやmatureやsequenceやofやtheやB12まbindingやproteinやIFや flankedや byや 5たや andや 3たや untranslatedや regionsや (UTRs)や ofや theや PSADや gene.や Twoや constructsや wereやgenerated—oneやforやcytoplasmicやexpressionや(Figureや1a)やencodingやIFやandやaやsecondやversionやwithやtheや

Biologyȱ2018,や7,や19や 5やofや13や

additionやofやanやNまterminalやsignalやpeptideやfromやARS2や(Figureや1b).やThisやsignalやpeptideやhasやpreviouslyやbeenや shownや toや enableや secretionや toや theや mediumや inや C.ȱ reinhardtiiや ofや recombinantや proteinsや [18].やAdditionally,やaやCまterminalやHAやepitopeやtagやwasやaddedやtoやbothやproteinsやforやdetectionやpurposes.やTheやbleやmarkerやdownstreamやofやtheやgeneやofやinterestやonやtheやpCrEX1やplasmidやwasやusedやtoやselectやforやpositiveやtransformantsやafterやtransformationやbyやagitationやofやaやDNAまcellやmixtureやwithやglassやbeadsや(modifiedやfromやpreviouslyやdescribedやmethodsや [22,25],や seeやSectionや 2.3).やTheやpresenceやofや theや IFや cassetteやwasやverifiedやbyやPCRや(FigureやS1)やinやcoloniesやthatやgrewやonやZeocinやafterやtransformation.やPositiveやcandidatesやbasedやonやPCRやresultsやwereやthenやsubjectedやtoやimmunoblotやanalysisやtoやscreenやforやexpressionやofやtheやIFやprotein.やTwoや strainsやwithや theや respectiveや constructsやwereや foundや toや expressやdetectableや levelsやofや IFや(Figureや2).やTheやstrainsやwereやnamedや“IF”やandや“arsまIF”や(Figureや1)やandやsubjectedやtoやfurtherやanalysis.や

3.2.ȱExpressionȱofȱHumanȱIntrinsicȱFactorȱinȱC.やreinhardtiiȱ

TheやexpectedやsizeやofやtheやmatureやsizeやIFやbasedやonや itsやaminoやacidやsequenceやisや44.6やkDa.やTheやIFやproteinやexpressedやinやtheやIFやstrainやhasやaやsimilarやsizeやmigratingやonやanやSDSまPAGEやslightlyやfasterやthanやtheや46やkDaやmolecularやweightやstandardや(Figureや2a).やAdditionally,やaやsecondやnonまspecificやbandやcrossまreactingやwithやtheやHAやantiserumやwasやdetectedやinやcellularやlysatesやofやboth,やtheやIFやandやtheやcontrolやstrainやaroundや32やkDaやthatやweやpreviouslyやobservedやinやC.ȱreinhardtiiȱcellularやlysatesや[26].やTheやIFやproteinやwasやexpressedやwithやanやNまterminalやARS2やsignalやpeptideやinやtheやarsまIFやstrain.やTheやsignalやpeptideやandやtheやmatureやIFやproteinやhaveやaやcombinedやpredictedやmolecularやweightやofや46.3やkDa,やhowever,やtheやexpectedやsizeやofやtheやmatureやproteinやafterやcleavageやofやtheやARS2やsignalやpeptideやisやtheやsameやmolecularやweightやasやtheやcytoplasmicやIFやproteinやwithや44.6やkDaや(thisやobservationやisやdiscussedやinやmoreやdetailやinやSectionや4.2).やImmunoblotやanalysisやofやtheやarsまIFやcultureやmediumやshowsやtheやproteinやmigratingやatやaやsizeやaroundや50やkDaやwhichやisやslightlyやbiggerやthanやexpected.やToやfurtherやverifyやtheやband,やwhichやisやnotやpresentやinやtheやCCま849やcontrolやstrainや(Figureや2b),やtheやproteinやwasやalsoやimmunoblottedやusingやanやIFまspecificやantibody.やWithやbothやantibodies,やtheやsameやbandやwasやdetected.や

Toやdetermineやtheや localizationやofやtheやprotein,やcultureやsupernatantや(medium)やandやpelletedやcellsや(cellularやlysate)やwereやimmunoblottedやseparately.やInやtheやIFやstrain,やtheやIFやproteinやwasやonlyやdetectedやinやcellularやlysatesや(Figureや2a,d).やInやcontrastやtoやthis,やtheやIFやproteinやexpressedやinやtheやarsまIFやstrainやwithやtheやARS2やsignalやpeptideやwasやonlyやfoundやinやtheやcultureやsupernatantや(Figureや2b),やnoやproteinやwasやdetectedやinやcellularやlysatesや(Figureや2c).や や

Biologyȱ2018,や7,や19や 6やofや13や

やFigureȱ2.やExpressionやofやhumanやintrinsicやfactorや(IF)やproteinやandやarsまIFやinやC.ȱreinhardtiiȱCCま849やdetectedやbyや immunoblotやanalysisやusingやHAや(ぼHA)やandや intrinsicやfactorや(ぼIF)やantibodies,やrespectively,や inやtheやcultureやmediumや andや cellularや lysates.や (a)やCellularや lysatesや ofや IFや strainや expressingや theやprotein.やTheやproteinやbandやisやindicatedやwithやanやarrowや(IF).やAやsecond,やnonまspecificやbandやcrossまreactingやwithやtheやHAやantiserumや isや indicatedや (N.S.).や (b)や Cultureや supernatantや (medium)や ofや transformedや CCま849や strainやexpressingや arsまIF.や ‘C’や indicatesや aや negativeや controlや (cellularや lysateや andや supernatantや ofや theやuntransformedやCCま849やstrain).や(c)やCellularやlysatesやofやarsまIFやstrainやandやCCま849.や(d)やCultureやmediumやofやarsまIFやandやIFやstrain.や

3.3.ȱC.やreinhardtiiȬProducedȱIFȱStabilityȱIncreasesȱwithȱExternalȱVitaminȱB12ȱSupplementationȱ

ToやtestやtheやB12やbindingやcapacitiesやofやtheやmicroalgalやIFやproducedやprotein,やtheやIFやandやarsまIFやstrainsやwereやgrownやinやTAPやmediumやsupplementedやwithやvitaminやB12やatやaやconcentrationやofや0,や20,や50やandや100やょgぉL–1.やAsやshownやbyやimmunoblotting,やaやcorrelationやbetweenやhigherやB12やlevelsやandやtheやIFやproteinや(arsまIFやstrain)やinやtheやmediumやisやobservedや(Figureや3a).やTheやproteinやabundanceやincreasesやapproximatelyや2.1やfoldや(±0.50)やwithやtheやadditionやofや100やょgぉL–1やB12やinやtheやcultureやmediumや(Tableや1).やSimilarly,やbasedやonやdensitometricやanalysisやofやrelativeやproteinやabundance,やtheやcytoplasmicやIFやproteinやinやtheやIFやstrainやisやalsoやestimatedやtoやbeやmoreやabundantやwithやB12やsupplementedやinやtheやmedium.やHowever,やhereやtheやhighestやabundanceやofやIFやisやestimatedやtoやbeやatや50やょgぉL–1やB12や(Figureや3bやandやTableや1).や

やFigureȱ3.やAccumulationやofやhumanやintrinsicやfactorや(IF)やproteinやinやC.ȱreinhardtiiやarsまIFやandやIFやculturesやwithや increasingや concentrationsや ofや vitaminや B12や (cobalamin)や supplementedや TAPやmedium.や (a)やHAまimmunoblotやofやmediumやharvestedやfromやCCま849やandやarsまIFやculturesやgrownやtoやlateやlogやphaseやcontainingや0,や20,や50やandや100やょgぉL–1やVitaminやB12.やEqualやamountsやofやcultureやsupernatantや(20やょL)やloadedやperやlane.や(b)やHAまimmunoblotやofやcellularやlysatesやfromやCCま849やandやIFやculturesやgrownやtoやlateやlogやphaseやwithや0,や20,や50やandや100やょgぉL–1やVitaminやB12やsupplemented.やEqualやamountsやofやcellularやlysateやstandardizedやbyやOD750やloaded.やMobilityやofやIFやproteinやindicatedやbyやarrow,やN.S.やdenotesやaやnonまspecificやproteinやcrossまreactingや withや theや HAまserum.や Supplementaryや Figureや S2や showsや Coomassieや stainedや SDSまpolyacrylamideやgelsやwithやtotalやproteinやloadedやforやrespectiveやwesternやblotやsamples.や

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Biologyȱ2018,や7,や19や 7やofや13や

Tableȱ1.やRelativeやhumanや intrinsicや factorや (IF)やproteinやexpressionや levelsや inや IFやandやarsまIFや strainsや forやdifferentやB12やconcentrationやsupplementedやtoやgrowthやmediumや(nや=や3や±やstandardやerror).や

VitaminȱB12ȱinȱMediumȱ(ΐgȉLƺ1) arsȬIFȱStrainȱ(Medium) 1ȱ IFȱStrainȱ(Cytoplasmic)ȱ2ȱ0や 1.0や 1.0や20や 1.1や± 0.01 1.4や±や0.15や50や 1.6や±や0.17や 2.3や±や0.17や100や 2.1や±や0.50や 1.4や±や0.11や

1や Densitometricや relativeや quantityや ofや IFや wasや normalizedや toや theや OD750や ofや theや respectiveや culturesやmeasuredやwhenや samplesやwereや harvested.や 2や Equalや amountsや ofや cells,や normalizedや toやOD750,や forや allやculturesやwereやloadedやonやgelやforやimmunoblotting.や

3.4.ȱCorrelationȱofȱVitaminȱB12ȱandȱIFȱProteinȱLevelsȱ

ToやfurtherやinvestigateやifやtheやIFやproteinやwasやbindingやB12,やtheやproteinやwasやconcentratedやfromやcellularやlysatesや (IFや strain,や CCま849や control)や andや theやmediumや (arsまIFや strain,や CCま849や control).や Ionや exchangeやchromatographyやwasやusedやtoやconcentrateやandやseparateやproteinやfractionsやfromやtotalやcellularや(IFやstrain)やorやsecretedやproteinや(arsまIF).やProteinやfractionsやwereやelutedやwithやrisingやNaClやconcentrationsやfromや100やmMやtoや1やMやNaCl.やBothやIFやexpressingやstrainsやandやtheやnegativeやcontrolやstrainやCCま849やwereやgrownやwithやeitherやnoやB12やorや20やょgぉL–1やB12やsupplementedやtoやtheやmedium.やTheやmajorityやofやtheやIFやproteinやwasやelutedやwithや100–150やmMやNaClやinやbothやcases,やtheやmediumやandやtheやcytoplasmicやIFやversion,やasやshownやbyやimmunoblotやanalysisやofやtheやindividualやproteinやfractionsや(Figureや4).やImmunoblotsやofやIFやconcentratedやfromやcellularやlysatesやofやtheやIFまstrainやshowedやoneやprominentやbandやofやtheやproteinやwithやbothやreducingやSDSまPAGEやandやnativeやPAGEや(Figureや4a).やInやtheやmediumやversion,やbandsやmigratingやdifferentlyやinやnativeやPAGEやthanやwithやreducingやSDSまPAGEやwereやobservedや(Figureや4b).や や

ȱ

Figureȱ4.やProteinやfractionsやpurifiedやfromや(a)やcellularやlysatesや(humanやintrinsicやfactorや(IF)やstrain)やandや(b)やmediumや(arsまIFやstrain)やHAまImmunoblotsやofやreducingやSDSまやandやnativeまPAGEやshowやtheやpresenceやofやtheやIFやproteinやinやtheやseparateやfractions.やLまlysate;やFまT—flowまthrough;やWまwashやfractionsや(washや1やandやwashや2やasやshownやinやFigureや4やwereやpooled);や100やmM–1000やmM:やNaClやconcentrationsやofやelutedやfractions.や

Allや fractions,や theや lysatesや andやmediumや samplesやwereや subsequentlyやusedや inや aやB12やbioassayや toやquantifyやtheirやB12やcontent.やThisやbioassayやisやbasedやonやtheやgrowthやdependencyやofやaやSalmonellaȱentericaȱ(metE�やcbiB–)やstrainやonやexternalやB12やsupplementation.やAllやexperimentsやwereやperformedやinやtriplicateやandやtheやB12やcontentやfoundやinやtheやindividualやfractionsやisやshownやinやFigureや5aやforやtheやIFまstrainやandやFigureや5bやforやtheやarsまIFやstrainやwhenやculturesやwereやsupplementedやwithや20やょgぉL┑1やB12.やInやbothやcases,やtheやhighestやB12や contentや wasや foundや inや theや lysateや (medium),や flowまthroughや andや washや fractions.や SignificantやdifferencesやinやB12やcontentやbetweenやtheやcontrolやstrainやandやtheやIFやstrainやwereやonlyやfoundやinやtheやcellularやlysateや(pや=や0.01)やhoweverやthisやobservationやwasやnotやsupportedやbyやtheやelutedやproteinやfractionsや(Figureや5a).やInやcaseやofやtheやarsまIFやstrain,やsignificantやdifferencesやwereやonlyやfoundやinやtheや100やandや150やmMやNaClやelutedやproteinや fractionsや (pや =や 0.02)や (Figureや 5b).やAllやpurificationsや fromや culturesやgrownやwithoutやB12やsupplementedや inや theやmediumやdidやnotやcontainやB12や levelsやdetectableやwithや theやbioassay.やTheやelutionやpatternやofやtheやIFやproteinやinやtheseやsamplesやwas,やhowever,やfoundやtoやbeやoverallやtheやsameやasやfromやculturesやsupplementedやwithやB12.やTheやmajorityやofやsecretedや IFやproteinやwasや foundや toやbeやcleavedやorやdegradedや

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Biologyȱ2018,や7,や19や 8やofや13や

withoutやB12やsupplementationやasやseenやbyやSDSまPAGEやanalysisやduringやtheやpurificationやprocessや(FigureやS3b).やWhereasやaやsimilarやpattern,やwithやandやwithoutやB12やsupplementationやofやtheやmedium,やwasやfoundやforやtheやcytoplasmicやIFやproteinや(FigureやS3a).や

やFigureȱ5.やVitaminやB12やcontentやofやproteinやfractionsやpurifiedやfromやIFやandやarsまIFやstrainsやgrownやwithや20やょgぉL–

1や supplementedやTAPやmediumやdeterminedやusingやaやplaqueや assayやofやSalmonellaȱ entericaȱ (metE�や cbiB–).やFractionsやwereやelutedやwithや increasingやconcentrationsやofやNaClや (100やmMや toや1やM).やDataやshowやaverageやvalues,やerrorやbars:やstandardやerrorや(nや=や3).やStatisticallyやsignificantやdifferencesやbetweenやtheやcontrolやandやtheやrespectiveやstrainやforやeachやfractionやareやindicatedやwithやanやasteriskや(Student’sやtまtest,やpや<や0.05).や(a)やB12やamountsやdetectedやinやfractionsやofや1やLやcellularやlysatesやfromやIFやandやCCま849やstrains.や(b)やB12やamountsやdetectedやinやproteinやfractionsやpurifiedやfromや1やLやmediumやofやarsまIFやorやCCま849やcultures.や

4.ȱDiscussionȱ

4.1.ȱExpressionȱandȱEfficientȱSecretionȱofȱaȱHumanȱGlycoproteinȱIntrinsicȱFactorȱinȱMicroalgaeȱ

InやthisやstudyやweやhaveやsuccessfullyやexpressedやtheやhumanやB12やreceptorやproteinやIFやinやC.ȱreinhardtii.やTheやproteinやwasやexpressedやfromやtheやnuclearやgenomeやasやthisやallowedやforやsecretionやofやtheやproteinやtoやtheやmediumやandやpotentialやglycosylationやofや theやprotein.やTwoやdifferentやstrainsやwereやgeneratedや forや thisやstudy—oneやstrainやexpressingやtheや44.6やkDaやIFやproteinやinやtheやcytoplasmや(IFまstrain)やandやaやsecondやstrainやthatやsecretesやtheやproteinやtoやtheやmediumや(arsまIF).やTheやarsまIFやexpressedやIFやproteinやhasやbeenやfoundやtoやbeやefficientlyやsecretedやtoやtheやmediumやbyやmeansやofやanやARS2やsignalやpeptideやthatやhasやpreviouslyやbeenやusedやtoやsecreteやaやrecombinantやenzymeや [18].やNoやproteinやwasやdetectedや inや theやcellularや lysateや inや theやarsまIFやstrainや(Figureや2c).やThisやsuggestsやmoreやefficientやsecretionやofやtheやarsまIFやthanやwhatやhasやbeenやreportedやforやsecretionやofやaやluciferaseやusingやtheやsameやARS2やsignalやpeptideや[18].やToやdate,やonlyやfewやstudiesやonやtheやsecretionやofやrecombinantやproteinsやinやmicroalgaeやareやavailable.やMostやreportsやareやfromやtheやgreenやalgaやC.ȱreinhardtiiや[18,27–30]ȱorやtheやdiatomやPhaeodactylumȱtricornutumȱ[31,32].や

4.2.ȱPosttranslationalȬModificationȱofȱtheȱSecretedȱIFȱProteinȱ

BasedやonやmigrationやonやSDSまPAGEやgels,やtheやsecretedやmicroalgalやIFやproteinやshowedやaやconsistentlyやlargerやmolecularやsizeやcomparedやtoやtheやcytoplasmicやproteinや(Figuresや3,や4やandや5b,c).やThereやareやseveralやreasonsやthatやcouldやexplainやthis.やOneやpossibilityやisやtheやfailureやtoやcleaveやtheやsignalやpeptide.やHowever,やtheやARS2やsignalやpeptideやonlyやaddsやapproximatelyや1.7やkDaやtoやtheやmatureやproteinやwhichやseemsやunlikelyやtoやcauseやtheやobservedやshiftやofやaroundや5やtoや10やkDa.やAやpreviousやstudyやhasやusedやanやARS1やsignalやpeptideやforやsecretionやofやaやproteinや toや theやmediumやandやobservedやaやsimilarやpattern.や Inや thisやstudy,や theやsignalやpeptideやwasやnotやfoundやwithやmassやfingerprintやanalysisやsuggestingやthatやtheやnonまcleavageやofやtheやsignalやpeptideやwasやunlikelyや[18].や

AnotherやpossibilityやthatやcouldやexplainやtheやsizeやdifferenceやisやtheやpostまtranslationalやmodificationやofやtheやIFやprotein.やThisやisやlikelyやasやtheやhumanやIFやproteinやcontainsやanやNȬglycosylationやsiteや[33]やandやisや

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Biologyȱ2018,や7,や19や 9やofや13や

knownやtoやbeやhighlyやglycosylated.やWhenやpreviouslyやexpressedやinやhigherやplantsや(Arabidopsisȱthaliana)やglycosylationやwasや observedや andや theや proteinや detectedや hadや aや similarや sizeや ofや 50や kDaや toや theや algalやexpressedやIFや[34].やTheやglycosylationやofやIFやseemsやtoやbeやnonまessentialやforやB12やandやreceptorやbindingや[35],やhowever,やitやmayやstabilizeやandやprotectやtheやproteinやfromやdegradationやinやtheやlowやpHやandやproteolyticやconditionsやinやtheやintestine.やNotやmuchやisやknownやaboutやNȬglycosylationやinやChlamydomonasȱ[27,36,37]やandやeffortsやtoやcharacterizeやtheやsecretedやIFやproteinやinやmoreやdetailやwereやpreventedやbyやlowやexpressionやlevels.や Treatmentや ofや theや proteinや withや aや commercialや deglycosylationや enzymeや cocktailや (ProteinやDeglycosylationやMixやII,やNewやEnglandやBiolabs,やHitchin,やUnitedやKingdom)やwasやinconclusiveやandやweやdidやnotやobtainやsufficientやamountsやofやpureやproteinやforやmassやspectrometryや(dataやnotやshown)やfromや1やLやculturesや(OD750nmやbetweenや0.6やandや0.8).やLowやexpressionやlevelsやofやrecombinantやproteinsやareやtoやdateやaやcommonやproblemや seenや inやChlamydomonasや (previousや studiesやhaveや reportedや 0.25%やofや totalや cellularやproteinや[38]やorや0.25%やofやtotalやsolubleやproteinや[30]やforやexample).やThisやisやlikelyやdueやtoやvariousやfactorsやsuchやasやrandomやinsertionやofやtheやconstructやinやtheやnuclearやgenomeやandやefficientやsilencingやmechanismsや[39].やRecentやeffortsや toやestablishや targetedやDNAやapproachesや (seeや [14,40]や forや recentや reviews)やsuchやasやCRISPR/Cas9やtechnologyや[41–43]やforやnuclearやtransformationやinやC.ȱreinhardtiiやcouldやbeやpromisingやtoolsやtoや increaseやrecombinantやproteinやyieldsやallowingや forやaやmoreやdetailedやcharacterizationやofや theやalgaeまproducedやIFやprotein.や

4.3.ȱVitaminȱB12ȱEnrichmentȱinȱIFȱExpressingȱC.やreinhardtiiやStrainsȱ

EukaryoticやmicroalgaeやdoやnotやproduceやB12やandやmoreやthanやhalfやofやmicroalgalやspeciesやareやthoughtやtoやbeやB12やauxotrophsやrelyingやonやB12やobtainedやfromやsymbioticやrelationshipsやwithやbacteriaや[44,45].やTheやmodelやalgaやC.ȱreinhardtiiやusedやinやthisやstudyやhasやbeenやshownやtoやbeやB12やindependentやasやitやcontainsやMETEや(B12まindependentやmethionineやsynthaseやgene).やHowever,やtheやalgaやalsoやcontainsやtheやB12まdependentやB12やmethionineやsynthaseやMetHやwhichやisやpreferablyやusedやinやtheやpresenceやofやB12や[44].や や

NoやB12やwasやdetectedや inや anyやproteinや fractionsや concentratedや fromや culturesやgrownやwithoutやB12やsupplementationや (Tableや S1)や whichや isや expectedや asや theや algaeや wereや grownや axenically.や Whenやsupplementingや theやmediumやwithやB12,や increasedやamountsやofや theや IFやproteinやwereやdetectedや inやbothやstrains,やIFやandやarsまIFや(Figureや3やandやTableや1).やThisやindicatesやthatやtheやIFやproteinやisやstabilizedやdueやtoやB12やbinding.やIFやisやaやknownやB12やbindingやproteinやandやformationやofやtheやholoまIFやformやstabilizesやtheやproteinや[33].やBindingやofやB12やtoやtheやcytoplasmicやIFやproteinやpresumesやtheやcellularやuptakeやofやB12やfromやtheやmediumやintoやtheやcell.やB12やtransportやintoやtheやcellやinやChlamydomonasやisやexistent,やevidentやbyやtheirやabilityやtoやutilizeやB12や[44,46]やbutやmolecularやmechanismsやofやB12やuptakeやinやmicroalgaeやhaveやhardlyやbeenやdescribedや[47].やTheやsemiまquantitativeやB12やassayやshowsやaやsignificantやdifferenceやofやtheやtotalやB12やamountやaccumulatedやinやtheやIFやstrainやlysatesやcomparedやtoやaやnegativeやcontrolやstrainや(Figureや5a).やHowever,やthisやobservationやwasやnotやsupportedやbyやtheやindividualやelutionやfractions.やIfやtheやincreasedやamountやofやB12やfoundやinやtheやIFやlysatesやwasやdirectlyやdueやtoやbindingやtoやtheやIFやprotein,やoneやwouldやexpectやtoやseeやaやsignificantやdifferenceやinやtheやelutionsやcontainingやtheやmajorityやofやtheやIFやproteinや(100やmM,や150やmMやNaCl,やFigureや4).やHowever,やnoやsignificantやdifferencesやofやanyやofやtheやelutionやfractionsやwereやfoundやforやtheやIFやandやCCま849やcontrolやstrain.やConsequently,や itや isやpossibleや thatや theや expressionやofや IFや inや theや algaeやdoesや leadや toや anや enrichmentやofやintracellularやvitaminやB12,やbutやfurtherやworkやisやneededやtoやsupportやthisやhypothesis.や や

WithやtheやarsまIFやstrain,やtheやIFやproteinやwasやpurifiedやfromやtheやmediumやwhereやtheやbackgroundやfromやotherやcoまpurifiedやproteinsやisやsignificantlyやlowerやthanやinやtheやcellularやlysates.やHere,やtheやfractionsやthatやcontainやtheやhighestやamountやofやIFやproteinやshowやaやsignificantlyやhigherやB12やcontentやwhenやcomparedやwithやtheやcontrolやstrainやCCま849や(Figureや5b).や や

Weやalsoやobservedやthat,やinやtheやflowまthroughやandやtheやwashやfractionsや(Figureや5b),やnoやsignificantやdifferencesやbetweenやtheやarsまIFやandやtheやcontrolやstrainやisやseen.やThisやisやlikelyやtoやbeやdueやtoやtheやunboundやB12やwashingやoffや andや theやB12やdetectedや inや subsequentや elutionや fractionsやmostや likelyやbeingやboundや toやprotein.やHowever,やtheやB12やbioassayやusedやinやthisやstudyやdoesやnotやallowやdistinguishingやbetweenやproteinまboundやB12やandやfreeやB12.やTherefore,やweやcannotやconcludeやifやtheやB12やwasやboundやtoやtheやsecretedやIFやprotein.や

InやtheやcaseやofやtheやcytoplasmicやIFやprotein,やthereやisやaやstatisticallyやsignificantやdifferenceやbetweenやB12やconcentrationsやmeasuredやinやcellularやlysatesやexpressingやIFやandやtheやcontrolやstrainや(Figureや5a).やHowever,やthereやwasやnoやsignificantやdifferenceやobservedやinやtheやelutionやfractions.やOneやreasonやforやthisやmightやbeやthatや

Biologyȱ2018,や7,や19や 10やofや13や

theやassayやdoesやnotやdistinguishやbetweenやB12やboundやtoやtheやIFやproteinやorやotherやcellularやproteins.やOtherやenzymesやalreadyやknownやtoやbeやassociatedやwithやintracellularやB12やmetabolismやinやalgaeやwillやmostやlikelyやinteractやwithや internalizedやB12や [45].やThisや couldや explainや theやhighやbackgroundや foundや inや theやproteinやelutionsやfromやbothやcellularやlysates,やCCま849やandやIFやstrainや(Figureや5a).やAdditionally,やaやrelativeやdecreaseやofや cytoplasmicや IFや wasや observedや fromや culturesや supplementedや withや 50や ょgぉL–1や B12や toや culturesやsupplementedやwithや100やょgぉL–1やB12や(Figureや3bやandやTableや1).やAlthoughやweやcanやonlyやhypothesize,やthisやdecreaseやcouldやbeやdueやtoやconcentrationまdependentやtoxicityやofやtheやproteinやandやtherewithやassociatedやcellularやregulatoryやprocesses.やAdditionally,やdueやtoやtheやrandomやinsertionやofやtheやexpressionやcassetteやinやtheやnuclearやgenome,やthereやisやtheやpossibilityやthatやotherやcellularやfunctionsやmayやbeやaffected.や

4.4.ȱIntrinsicȱFactorȱforȱDevelopingȱB12ȬEnrichedȱMicroalgaeȱ

Inや thisや studyや weや expressedや IFや inや theや eukaryoticや microalgaeや Chlamydomonasȱ exploringや theやpotentialやofやenrichingやbioavailableやB12やusingやaやmicroalgalやhost.やWeやproposeやthatやthisやmodelやalgaやcouldやbeやaやgoodやchassisや forや futureやproductionやofやmicroalgalや functionalや foodsやandやdietaryやB12やcontainingやsupplements.や Forや example,や recentlyや itや hasや beenや shownや thatや cyanobacteriaや produceや aや differentやchemicalやvariantやofやB12や[48].やThisやchemicalやvariantやofやB12や(alsoやknownやasやpseudocobalamin)やbindsやwithやaやmuchやlowerやaffinityやtoやB12やreceptorやproteinsやsuchやasやIFやmakingやitやlessやbioavailableやforやhumansや[49].やEukaryoticやmicroalgae,やonやtheやotherやhand,やutilizeやtheやsameやB12やvariantsやasやhumans,やtherefore,やmakingやthemやaやbetterやsourceやofやB12やsupplementation.や や

Asや shownや recentlyや forや Chlorellaや supplementsや [6],や currentlyや availableや eukaryoticやmicroalgalやsupplementsや canや containやvaryingや amountsやofやB12.やThisや isやmostや likelyやdueや toやdifferentや cultivationやconditionsやalteringやtheやcompositionやofやtheやassociatedやbacterialやbiofilmsやthatやareやtheやoriginalやsourceやofやB12や foundや inや theや supplements.や Inや thisや respect,や theや establishmentや ofや closed,や standardizedや growthやsystems,や asや previouslyや shownや forや Chlamydomonasや [50,51],や allowsや forや muchや moreや controlや overやcontaminationやcomparedやtoやopenやsystemsやandやherebyやalsoやofやtheやB12やcontentやinやtheやfinalやproduct.やInやaddition,やeffortsやtoやengineerやmicroalgalやcommunitiesや(reviewedやrecentlyやinや[12,52])やwithやsymbioticやrelationships,やasやfoundやbetweenやMesorhizobiumȱlotiやandやLobomonasȱrostrataや[53,54],やcouldやallowやforやaやmoreやcontrolledやinteraction,やandやthusやfluxやofやB12,やofやtheやmicroalgaやandやassociatedやbacteria.やFinally,やlookingやatやtheやcurrentやmarket,やIF,やofやanimalや(porcine)やorigin,やisやcommerciallyやavailableやinやsomeやB12やsupplements.やThisやproofまofまconceptやstudyやshowsやthatやmicroalgaeやareやaやpotentialやvegetarianまfriendlyやalternativeやtoやIFまB12やcoまformulationやinやfunctionalやfoodsやandやdietaryやsupplements.や

5.ȱConclusionsȱ

Hereや weや haveや shownや thatや theや humanや glycoproteinや Intrinsicや Factorや canや beや expressedや inやChlamydomonasȱreinhardtiiやfromやtheやnuclearやgenome.やTheやadditionやofやanやARS2ȱsignalやpeptideやleadsやtoやefficientやsecretionやtoやtheやmedium.やOurやdataやsuggestや thatやtheやsecretedやIFやproteinやdirectlyやbindsやB12,やhowever,や itや isや lessや clearや ifや theや cytoplasmicや IFや bindsや theや cofactorや orや hasや anや indirectや effectや onやB12やenrichmentやinやtheやIFやstrain.やOurやdataやalsoやshowやaやcorrelationやofやhigherやproteinやlevelsやwithやhigherやB12やconcentrationsやsuggestingやaやpotentialやstabilizingやeffectやonやtheやprotein.やThisやstudyやisやaやfirstやproofまofまconceptやutilizingや aやhumanやB12やbindingやproteinや asや aや toolや forや enrichingやB12や inやmicroalgaeや andや canやcontributeやtoやtheやfutureやdevelopmentやofやmicroalgalやfunctionalやfoods.や

SupplementaryȱMaterials:やTheやfollowingやareやavailableやonlineやatやwww.mdpi.com/2079ま7737/7/1/19/s1,やFigureやS1:やPCRやanalysisやofやChlamydomonasȱreinhardtiiやstrainsやIFやandやarsまIF.やFigureやS2:やCoomassieまstainedやpolyacrylamideやgelsやofやsamplesやshownや inやFigureや3.やFigureやS3:やIFやpurifiedやfromやculturesやgrownやwithoutやB12やsupplementation.やTableやS1:やVitaminやB12やassayやplaqueやdiametersやmeasuredやinやdifferentやfractionsやfromやionやexchangeやchromatographyやandやcalculatedやVitaminやB12やcontentやofやsamplesや(nM).や

Acknowledgments:やTheやauthorsやthankやSaulやPurtonやforやadviceやonやnuclearやtransformationやandやforやprovidingやtheやpCrEX1やplasmid,やRachelやBoneやforやpreliminaryやworkやonやtheやprojectやleadingやtoやthisやmanuscript,やMartinやJ.やWarrenやforやinputやtoやtheやexperimentalやdesignやofやthisやstudyやandやPoulやErikやJensenやandやDavidやA.やRussoやforやcriticalやreadingやofやtheやmanuscript.やTheやresearchやleadingやtoやtheseやresultsやhasやreceivedやfundingやfromやtheやPeopleやProgrammeや(MarieやCurieやActions)やofや theやEuropeanやUnion’sや SeventhやFrameworkやProgrammeやFP7/2007�2013/やunderやREAやgrantや

Biologyȱ2018,や7,や19や 11やofや13や

agreementやno.や317184,やProofやofやConceptやFundingやfromやPHYCONETやandやfromやtheやBiotechnologyやandやBiologicalやSciencesやResearchやCouncilやunderやgrantやagreementやnumberやBB/L010208/1.や

AuthorȱContributions:ȱ J.A.Z.Z.やandやC.R.やconceivedやandやdesignedや theやexperiments;やS.L.,やC.L.W.やandや J.A.Z.Z.やperformedや theやexperiments;やS.L.やandや J.A.Z.Z.やanalyzedや theやdata;やE.D.やprovidedや theやvitaminやB12やassayやplates,やJ.A.Z.Z.やwroteやtheやmanuscript.や

ConflictsȱofȱInterest:やTheやauthorsやdeclareやnoやconflictやofやinterest.やTheやfundingやsponsorsやhadやnoやroleやinやtheやdesignやofやtheやstudy;やinやtheやcollection,やanalyses,やorやinterpretationやofやdata;やinやtheやwritingやofやtheやmanuscript,やandやinやtheやdecisionやtoやpublishやtheやresults.や

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©や2018やbyやtheやauthors.やLicenseeやMDPI,やBasel,やSwitzerland.やThisやarticleやisやanやopenやaccessやarticleやdistributedやunderやtheやtermsやandやconditionsやofやtheやCreativeやCommonsやAttributionや(CCやBY)やlicenseや(http://creativecommons.org/licenses/by/4.0/).や