ganccdp

APPLIEDANDENVIRONMENTALMICROBIOLOGY,June2009,p.
3765–3776Vol.
75,No.
110099-2240/09/$08.
000doi:10.
1128/AEM.
02594-08Copyright2009,AmericanSocietyforMicrobiology.
AllRightsReserved.
ComparativeProteomicAnalysisofToleranceandAdaptationofEthanologenicSaccharomycescerevisiaetoFurfural,aLignocellulosicInhibitoryCompoundFeng-MingLin,BinQiao,andYing-JinYuan*KeyLaboratoryofSystemsBioengineering,MinistryofEducationandDepartmentofPharmaceuticalEngineering,SchoolofChemicalEngineering&Technology,TianjinUniversity,P.
O.
Box6888,Tianjin300072,People'sRepublicofChinaReceived13November2008/Accepted1April2009ThemolecularmechanisminvolvedintoleranceandadaptationofethanologenicSaccharomycescerevisiaetoinhibitors(suchasfurfural,aceticacid,andphenol)representedinlignocellulosichydrolysateisstillunclear.
Here,18O-labeling-aidedshotguncomparativeproteomeanalysiswasappliedtostudytheglobalproteinexpressionprolesofS.
cerevisiaeunderconditionsoftreatmentoffurfuralcomparedwithfurfural-freefermentationproles.
Proteinsinvolvedinglucosefermentationand/orthetricarboxylicacidcyclewereupregulatedincellstreatedwithfurfuralcomparedwiththecontrolcells,whileproteinsinvolvedinglycerolbiosynthesisweredownregulated.
Differentiallevelsofexpressionofalcoholdehydrogenaseswereobserved.
Ontheotherhand,thelevelsofNADH,NAD,andNADH/NADwerereducedwhereasthelevelsofATPandADPwereincreased.
Theseobservationsindicatethatcentralcarbonmetabolism,levelsofalcoholdehydrogenases,andtheredoxbalancemayberelatedtotoleranceofethanologenicyeastforandadaptationtofurfural.
Furthermore,proteinsinvolvedinstressresponse,includingtheunfoldedproteinresponse,oxidativestress,osmoticandsaltstress,DNAdamageandnutrientstarvation,weredifferentiallyexpressed,andingthatwasvalidatedbyquantitativereal-timereversetranscription-PCRtofurtherconrmthatthegeneralstressresponsesareessentialforcellulardefenseagainstfurfural.
Theseinsightsintotheresponseofyeasttothepresenceoffurfuralwillbenetthedesignanddevelopmentofinhibitor-tolerantethanologenicyeastbymetabolicengineeringorsyntheticbiology.
Bioethanolproducedfromrenewableresourcessuchaslignocellulosesisconsideredtobeanattractivealternativetofossilfuels,foritisrenewable,canmakeuseoffast-rotationplants,producesfeweremissions,andgeneratesnonetcarbondioxide.
Nevertheless,therearesomebarriersinthelignocel-lulosic-to-ethanolconversionprocess,includinginhibitortol-erance,ethanoltolerance,andutilizationofxylose(62).
Inhib-itorsformedbyacid-catalyzedhydrolysisoflignocelluloses,whichincludefuranderivatives,weakacids,andphenoliccom-pounds,reduceboththegrowthrateandfermentationofeth-anologenicSaccharomycescerevisiae(2).
Themechanismsofinhibitionactinguponyeastduringfermentationoflignocel-lulosichydrolysatehavebeenstudiedintensively,butmainlywithtraditionalmethodssuchasmetaboliteanalysis,enzymeactivityanalysis,metabolicuxanalysis,andkineticanalysis(50).
Furfuralisoneofthemajorinhibitorsforlignocellulosichydrolysates.
Previousstudieshaveshownthatinmostcases,furfuralcanbeconvertedbyyeasttofurfuralalcohol(12,30).
Sometimesfuroicacid(64),furoinandfuril(47),andacyloinproducts(28,61)canalsobedetectedinthemediumunderdifferentsetsofcultivationconditions.
Thegeneticmecha-nismsinvolvedinfurfuraltolerancehavebeeninvestigatedbyscreeninganS.
cerevisiaedisruptionlibrarytondpotentialrelativegenes(18).
Throughgenecloningandenzymeactivitystudy,Liuetal.
foundthattheconversionoffurfuraliscata-lyzedbymultiplealdehydereductases(40).
Thetraditionalmethodsdescribedabovecananalyzeonlyoneorafewmetabolites,proteins,orgenesandareunabletogloballyassesstheinhibitionissue,whichiscomplexandsys-tematic.
Moreover,previousworkmainlyfocusedonextracel-lularmetabolitesandtheactivityofsomekeyenzymes,whereaswhathappensinsideyeastcellsinresponsetoinhib-itorsremainsa"blackbox"tous.
Integrationofdifferent"om-ics"tools,includingthoseoftranscriptomics,proteomics,andmetabolomics,intothestudyofsystemsbiologyisapotentiallypowerfulapproachtoaddressthesechallenges(61,68).
Manyproteomic,transcriptomic,and/ormetabolomicstudiesofS.
cerevisiaehaveprovideduswithanincreasinglyrichunder-standingoftheresponseofthisorganismtovariousenviron-mentalperturbations.
InvestigationofgenomicexpressionprolesoftheethanologenicyeastS.
cerevisiaetoHMF(5-hydroxymethylfurfural)stressconditionsshowedthatuptoseveralhundredgenesweredifferentiallyexpressedsigni-cantlyinresponsetoHMFtreatment(41,42,58).
Comparativelipidomicsanalysishasbeenappliedtostudytheethanologenicyeastresponsetodifferentinhibitors,suchasfurfural,aceticacid,andphenol(69).
Theresultsofcomparativeproteomeanalysis(8,23)andsmall-moleculemetaboliteprolingofeth-anologenicyeastduringindustrialfermentation(13)havebeenpreviouslyreported,enhancingthemolecularunderstandingofphysiologicaladaptationofindustrialstrainsforoptimizingthe*Correspondingauthor.
Mailingaddress:KeyLaboratoryofSys-temsBioengineering,MinistryofEducationandDepartmentofPhar-maceuticalEngineering,SchoolofChemicalEngineering&Technol-ogy,TianjinUniversity,P.
O.
Box6888,Tianjin300072,People'sRepublicofChina.
Phone:86-22-87401546.
Fax:86-22-27403888.
E-mail:yjyuan@tju.
edu.
cn.
Supplementalmaterialforthisarticlemaybefoundathttp://aem.
asm.
org/.
Publishedaheadofprinton10April2009.
3765onFebruary2,2021byguesthttp://aem.
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However,thespecicsofglobalproteinexpressioninresponsetothepresenceofbiomassconversioninhibitorshavenotyetbeenquantitativelymeasuredforethanologenicyeast.
Quantitativeproteomics,i.
e.
,quantifyingproteinexpressionlevelsindifferentsetsofcomplexbiologicalsamplesonalargescale,iscriticalforourunderstandingofbiologicalsystemsandpathwaysasawhole.
Itisconsideredlikelytobeapotentialcornerstoneofsystemsbiologyinthenearfuture(56).
Currentquantitativeproteomicmethodsfallintothreecategories:thetraditionaltwo-dimensional(2D)electrophoresis,stableiso-topelabeling,andnonlabelingmethods(45).
Eachofthedif-ferentlabelingmethodsundergoingdevelopmenthasitsad-vantagesanddisadvantages(seereference45forreviews).
Amongquantitativeproteomicmethods,18Ostableisotopelabelingisconvenienttouse,lowincost,highlyspecicintermsofspecic18OC-terminalmodications,andcapableintheoryoflabelingproteinsglobally.
The18O-labelingmethodhasdemonstrateditsapplicabilityindifferentialcomparativeproteomicswithbiologicalapplicationsperformedusingPor-phyromonasgingivalisstrainW50(4),thehumanplasmapro-teome(55),breastcancercells(5),andthelow-molecular-weightserumproteome(26).
18Olabelingisbecomingapowerfullabelingstrategyforquantitativeproteomicsap-plication.
Togiveinsightsintothetoleranceandadaptationofeth-anologenicyeasttobiomassconversioninhibitorsatthepro-teinlevel,comparativeshotgunproteomicinvestigationscom-bining18Olabelingwith2Dliquidchromatography-tandemmassspectrometry(2D-LC-MS/MS)wereperformedheretosystematicallyidentifyproteinsbytheuseofanindustrialstrainofS.
cerevisiaeandtoquantifycellstreatedwithfurfuralcomparedwithcontrolcellsunderaerobicbatchculturecon-ditions.
Quantitativereal-timereversetranscription-PCR(RT-PCR)andmetaboliteanalysiswereutilizedtoprovideorthog-onalevidenceforthecomparativeproteomeresults.
MATERIALSANDMETHODSYeaststrain.
AnindustrialstrainofS.
cerevisiae,purchasedfromAngelYeastCo.
,Ltd.
(Hubei,People'sRepublicofChina),intheformofalcoholinstantactivedryyeast,wasutilizedinthisstudy.
Thisindustrialstrainhastheadvan-tagesofthermalresistance(38to42°C),low-acidtolerance(pH2.
5),andhighglucosetolerance(60%)andcantolerate13%(vol/vol)ethanol.
Cultivationconditions.
Afterrecoveryfromalyophilizedform,S.
cerevisiaewasmaintainedonagarslantscontainingYEPDmedium(2%glucose,2%yeastextract,1%peptone,and2%agar).
S.
cerevisiaewasinitiallygrownin250-mlconicalaskscontaining50mlofYEPDmedium(2%glucose,2%yeastextract,and1%peptone)onarotaryshakerat30°Cand160rpmfor12h.
Subsequently,the50-mlseedculturesweretransferredinto2-literconicalaskscontaining450mlofYEPDmediumonarotaryshakerat30°Cand90rpmforapproximately12handgrowntoanopticaldensity(OD)ofabout3.
Cellsforthecontrolexperimentandthefurfuraltreatmentexperimentwereharvestedfromthesameinoculationculture.
AninitialODof0.
35wasusedforaerobicbathculturesperformedat30°Cin2-literconicalaskscontaining450mlofmedium,withastirrerspeedof90rpm.
Theaerobicbathculturemediumwascomposedof10%glucose,2%yeastextract,and1%peptone.
Duringexponentialgrowthintherespiratory-fermentativephase,whentheODwasapproximately3to4,50-mlvolumesofaerobicbathculturemediacontaining0and7.
33mloffurfuralwereintroducedintothemediumforthecontrolexperimentandthefurfuraltreat-mentexperiment,respectively.
Samplesforsubsequentproteinextractionwerecollectedbycentrifugationat5,000gfor10minat4°Cfromfurfural-treatedandcontrolculturesat20minand2haftertheadditionoffurfural,respectively.
Theconcentrationoffurfuralinlignocellulosichydrolysatescanrangefrom0.
5to11g/liter,andthereareabroadrangeofothercompoundsthathaveinhibitoryeffectsonmicrobialfermentation(2).
Usually,theinhibitorconcen-trationsusedtotesttheireffectsonfermentationwere10to100timeslargerthantheconcentrationfoundinthehydrolysates(31).
Therefore,basedonourpri-maryfermentationexperimentsandthepertinentliterature,thenalfurfuralconcentrationusedwassetat17g/literforthestudyoftheresponseofyeasttothepresenceoffurfuralunderanextremesetofconditions.
Analysisoffermentationparameters.
Cellgrowthwasdeterminedbymeasur-ingtheabsorbanceofthecultureat600nmwithaspectrometer(model722gratingspectrometer;ShanghaiNo.
3AnalysisEquipmentFactory,Shanghai,China).
Theconcentrationsofglucose,ethanol,glycerol,andfurfuralweremea-suredbyhigh-performanceLC.
Samplesfromanaerobicbathculturewererstlteredthrough0.
22-m-pore-sizesterileltersandloadedontoanAminexHPX-87Hion-exchangecolumn(Bio-Rad,Hercules,CA)operatedat65°Candwerethenelutedwith5mMH2SO4at0.
6ml/min.
Arefractiveindexdetectorwasused.
Cellviabilitywasassessedbymethylene-bluestainingandauores-cencemicroscope(EclipseE800;Nikon,Japan).
Proteinextraction,16O/18Olabeling,and2D-LC-MS/MSanalysis.
Theextrac-tionofwhole-yeast-cellproteinswasconductedasdescribedbyWangandYuan(66),withminormodications.
Afterreductionandalkylation,theproteinswereprecipitatedagainbyusingcoldorganicsolvent(ethanol/acetone/aceticacid,50:50:0.
1)overnightat25°C,followedbycentrifugationandlyophilization.
Thepelletswerestoredat25°Cuntiluse.
Proteinsamplesfromthecontrolexperimentandthefurfuraltreatmentex-perimentweredissolvedindigestionbuffer(1Murea,100mMNH4HCO3)madeusing16Oand18Owater(Isotec,Miamisburg,OH)(95%),witheachsolutionmaintainedataconcentrationof1g/l,andweredigestedwithtrypsin(Promega,Madison,WI)ataratioof50:1at37°Cfor24h.
Then,additionaltrypsinwasaddedtoachieveanalratioof20:1,andtheincubationwasmaintainedat37°Cforanother18h.
Digestionswereterminatedbyaddingformicacidtothenalvolumeconcentrationof5%.
Thecorresponding16O-and18O-labeledsamplesfromthesametimepointwerecombinedimmediatelybefore2D-LC-MS/MSanalysis.
NanoowLC-MS/MSanalysiswasperformedbytheuseofaLCQDecaXPMaxamassspectrometer(ThermoFinnigan,PaloAlto,CA)underthecontroloftheXcaliburdatasystem(ThermoFinnigan,PaloAlto,CA)asdescribedbyWangandYuan(66).
Thereweresomemodicationsintermsofsaltstepsandtheelutiongradient.
A14-stepseparationfromthestrongcationexchangerfollowedbyagradientelutionfromthereverse-phasechromatographyresultswasutilizedtoseparatethepeptides.
The14saltstepsused0,25,40,50,75,100,150,200,250,300,400,500,700,and1,000mMammoniumchloride,respec-tively.
Theelutiongradientforthereverse-phasechromatographyconsistedof1minof100%bufferA(5%[vol/vol]acetonitrile–0.
1%[vol/vol]formicacidinwater);a70-mingradientto30%bufferB(0.
1%[vol/vol]formicacidinaceto-nitrile);a20-mingradientto50%bufferB;a10-mingradientto95%bufferB;5minof95%bufferB;an8-mingradientto100%bufferA;and7minofre-equilibrationat100%bufferA.
Identicationandrelativequanticationofproteins.
MS/MSdataweresearchedusingtheSEQUESTalgorithm,andadatabaseofS.
cerevisiaeopenreadingframeswasdownloadedfromtheSaccharomycesGenomeDatabaseon9March2007.
TheparametersusedfortheanalysisofMS/MSspectraweredetailedinapreviousstudy(66).
Inthisstudy,carboxyl-terminaldouble18Osweredesignatedforuseinvariablemodication.
Then,SEQUESToutputlesweresubmittedtothePeptideProphetandProteinProphetwebsitesoftheSe-attleProteomicsCenter(http://tools.
proteomecenter.
org)forstatisticalassess-mentofpeptideandproteinsequencematches,respectively(65).
INTERACTsoftwarewasutilizedtoorganizeanddisplaytheresults.
Theacceptederrorrateforpeptideandproteinidenticationwascontrolledtoremainbelow10%.
Theabundanceratios(18O/16O)forlabeledpeptidepairswerecalculatedusingreconstructedionchromatogramsandthefollowingequation,whichissimilartoonepreviouslyreported(70)butwithslightmodications:Ratio18O16OI0M4M0I21M2M01M2M0M2M0I0I0(1)whereI0,I2,andI4representthemeasuredrelativeintensitiesforthersttwoisotopicvariantsofunlabeledpeptides,monolabeledpeptides,anddilabeledpeptides,respectively,andM0,M2,andM4representthesumsofthetheoreticalrelativeintensitiesforthersttwoisotopicvariantsofunlabeledpeptides,mono-labeledpeptides,anddilabeledpeptides,respectively.
Thenaturalisotopicdis-tributionwascalculatedusingthepeptidesequenceandtheMS-isotopeprogram(http://prospector.
ucsf.
edu/).
Theisotopedistributionpatternforthe18O-la-beledpeptidewasassumedtobethesameasfortheunlabeledpeptide.
The3766LINETAL.
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Theremainingpeptideratioswereusedtocalculatetheproteinmeansandstandarddeviations.
RT-PCR.
TotalRNAsfromthreebiologicalreplicateswereisolatedusingTrizolreagent(Invitrogen)forRT-PCR.
Eachbiologicalreplicatewasanalyzedthrice.
Quantitativereal-timePCRassayswereperformedusingSYBRgreenPCRmastermix(ABI)andanABI7300real-timePCRsystem(ABI).
ThesequencesofprimersusedforquantitativePCRaredescribedinTableS1inthesupplementalmaterial.
Thecyclingprogramwasasfollows:aninitialcycleof2minat50°Cand10minat95°C,followedby40cyclesof10sat95°Cand30sat60°C.
Thedisassociationanalysiswascarriedoutinaroutinefashionbyacquiringuorescentreadingsfor1°Cincreasesfrom55to95°C.
Datawereanalyzedusingsystem7300SDSsoftwaretocalculatethresholdcycle(CT)values.
Subsequently,therelativeexpressionratiosforthegenesweredeter-minedaccordingtothefollowingequations:SampleCTCTSampleCTTDH2(2)CTsampleCTcontrolCT(3)Severalfoldincrease(sampleversuscontrol)2CT(4)NAD/NADHandATP/ADPassays.
Rapidsampling,quenching,andmetab-oliteextractionofbiomass(inapproximately10mlofculturebroth)wereperformedaccordingtothemethodofLuoetal.
(44).
AllLC-MSexperimentswerecarriedoutusinganLCQDecaXPMaxmassspectrometer(ThermoFinni-gan,PaloAlto,CA)underthecontroloftheXcaliburdatasystem(ThermoFinnigan,PaloAlto,CA).
Asampleof20lwasloadedontoanAtlantisT34.
6-by250-mmcolumn(Waters,Ireland)(5mporesize),whichwasequilibratedfor30minbeforeloadingwith98%solutionA(5mMNH4HCO3inwater)and2%solutionB(80%methanol–5mMNH4HCO3inwater).
A20-mingradientto60%solutionAanda5-mingradientto60%solutionAwereused.
Thecolumneluentwaselectrosprayeddirectlyintoamassspectrometer.
Themassspectrom-eterwasoperatedinthenegative-ionandselected-reaction-monitoringmode.
Theoptimizedparameterswereasfollows:ion-sprayvoltage,4.
5kV;sheathgasandauxiliarygas,33and5(arbitraryunits),respectively.
Thecapillarytemperaturewas300°C,andthescanrangewas140to850m/z.
Thestandardcurvewasobtainedbyanalyzingstandardsolutions(NADHandNAD;Sigma,St.
Louis,MO)atveconcentrations(50,10,1,0.
5,and0.
1g)selectedforNADHandNADconcentrationcalculations.
ThelevelsofNADH/NADandATP/ADPwerecalculatedaccordingtotheratiosoftheintegratedioncurrents.
Threebiologicalreplicateexperimentswereperformed,withtwoinjectionsforeachsample.
Dataanalysis.
Thefunctionalcategoryandsubcellularlocalizationdetermi-nationsforproteinswerecarriedoutwithFunCatDBsoftware(http://mips.
gsf.
de/projects/funcat),andbiochemicalpathwayswereclassiedandreconstructedbyreferencetotheKEGGdatabase(http://www.
genome.
jp/kegg/)andtheSac-charomycesGenomeDatabase(http://www.
yeastgenome.
org/).
TheSaccharo-mycesGenomeDatabasewasalsoutilizedtoobtaininformationonproteins.
Proteinsquantiedwithrespecttotwoormorepeptideswereconsideredtobesignicantlychangedwhenoneofthefollowingthreecriteriawassatised:(i)expressionchangedbynolessthan1.
5-foldandwithrelativestandarddevia-tions(RSDs)below40%;(ii)determinationofan18O/16Oratiohigherthan3orlowerthan0.
33andanRSDbelow50%;or(3)allcorrespondingpeptideabundanceschangedbynolessthan1.
5-foldwithoutregardtoRSDvalues.
RESULTSANDDISCUSSIONPhysiologicalproleofS.
cerevisiaetreatedwithfurfuralorleftuntreated.
Thephysiologicaleffectoffurfuralonthein-dustrialS.
cerevisiaestrainwasstudiedbycomparisonofcul-tivationwiththeadditionoffurfural(17g/liter)intoaerobicbatchcultivationsduringexponentialgrowthintherespiratory-fermentativephasetothecontrolcultivationsgrownunderthesameconditionswithouttheintroductionoffurfural.
There-sultsarepresentedinFig.
1.
Theadditionoffurfuralalmostcompletelysuppressedcellgrowth,leadingtoveryslowcellgrowthintherst2hafteradditionoffurfuralandahaltinbiomassformationafterthat.
Uponextendedincubationfor9h,noincreaseingrowthwasobserved.
Bothethanolforma-tionandglucoseconsumptionwereinhibited,showingslowratesintherst4handthenstoppingat4haftertheadditionoffurfural.
Glycerolformationwashaltedimmediately,andtherewasnochangeintheglycerolconcentrationaftertheadditionoffurfural.
Incontrast,thefurfuralconcentrationinthemediumdecreasedsharplyduringtherst4handslowlyinthenext4handthenshowednochangewitharesidualcon-centrationofabout6g/liter,indicatingthatfurfuralhadbeenconvertedbyyeastcellstocompoundsoflessertoxicity.
Usingthemethylenebluetechniqueforcellviabilitydeterminationsduringtheexperiment,weobservedthatyeastcellswerenotabletorecoverfromtheinhibitioncausedbyfurfuralandthatallthecellsdiedat8haftertheadditionoffurfural(seeFig.
S1inthesupplementalmaterial).
Itisobviousthattheintroductionoffurfuralintoaerobicbatchyeastcultivationshadanimmediateanddrasticeffectonthephysiologicalbehaviorofyeastandthattheresponseofyeasttothepresenceoffurfuralwasacontinueddynamicprocess.
Confrontedwithfurfural,theindustrialyeastcellsshowedanotabledecreaseincellgrowth,glucoseconsump-tion,ethanolproduction,andglycerolproductionbutstilltriedtodetoxifyfurfuraltoalleviateitsinhibitoryeffects.
Theseresultsmayreectametabolicrearrangementinyeasttocopewithfurfuraldetoxicationandwithdiverseeffectscausedbythepresenceoffurfural,suchasthearrestofcellgrowthand/ortheaccumulationofacetaldehyde,underscoringtheneedforutilizationofsystemsbiologyapproachesforfurtherFIG.
1.
Comparisonofcellgrowthratesandmetaboliteconcentra-tionsduringaerobicbatchcultivationofS.
cerevisiaeinthepresence(lledsymbols)orabsence(emptysymbols)offurfural.
(A)Concen-trationoffurfural(diamond)andglucose(circle);(B)biomass(trian-gle),ethanol(square),glycerol(circle).
Allexperimentswereper-formedinduplicate.
Furfural(17g/liter)wasaddedwhenthecultureshadreachedanODof3to4(denotedbytime0).
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75,2009COMPARATIVEPROTEOMEOFYEASTINRESPONSETOFURFURAL3767onFebruary2,2021byguesthttp://aem.
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Proteinsdifferentiallyexpressedinresponsetofurfural.
TostudytheproteomicresponseoftheindustrialS.
cerevisiaestrainunderconditionsoftreatmentwithfurfural,ashotgunyeastcomparativeproteomeinvestigationhasbeencarriedout.
Twoequivalentwhole-yeastextractsacquiredfromthecontrolexperimentandthefurfuraltreatmentexperimentweretrypsinizedintopeptidesinH216OandH218O,respec-tively.
Samplesfromthesametimepoint(the20-minor2-htimepoint)werecombinedina1:1ratioandsubjectedtothreeroundsofanalysisusing2D-LC-MS/MS.
Proteinswereidenti-edthroughdatabasesearching,andrelativeproteinexpres-sionlevelsweredeterminedbycalculatingcorrected18O/16Opeptideratiosandusingpeakareas.
Atotalof2,037and3,655peptides(correspondingto205and309proteins,respectively)derivedfromthreereplicateexperimentsrunforboth20minand2hwerequantiedmanually.
Ofthoseproteins,175werequantiedforbothofthedatasets.
Accordingtothedata-miningcriteriadescribedabove(seeMaterialsandMethods)asusedforanalysisofproteinsthataredifferentiallyexpressed,70proteinswereupregulatedand6proteinsweredownregu-latedat20min,whereas31proteinswereupregulatedand35proteinsweredownregulatedat2hinresponsetothepresenceoffurfural.
Togetanoverviewofthedifferentiallyexpressedproteinsandguidesubsequentdataanalysis,determinationsofthesub-cellularlocalizationsandfunctionalcategoriesofthedifferen-tiallyexpressedproteinswerecarriedoutusingFunCatDBsoftware.
Thenumbersofdifferentiallyexpressedproteinsforeachcellularcompartmentorfunctionalcategoryfortimepoints20minand2hareshownintheformof100%stackedcolumns,asdisplayedinFig.
2.
Subcellularlocalizationdistributionsofdifferentiallyex-pressedproteinsat20minwerenotablydifferentfromthoseseenat2h.
Proteinswithanabundancechangeat20minwerenotlocalizedtothebud,golgi,orperoxisomecompartments,whereasproteinswithanabundancechangeat2hdidnotoriginatefromthecellwall.
Theothersubcellularcompart-mentswerewellrepresentedandhaddifferentproportionsofdifferentiallyexpressedproteinsineachdataset.
Thediversesubcellularlocalizationdistributionprolingresultsfor20minofcultivationversus2hdemonstratethatfurfuralhasdifferenteffectsoncellularcompartmentsovertime.
Whenintroducedintotheaerobicbatchculture,furfuralrstenteredintotheyeastcellthroughthecellwallandplasmamembraneandaffectedyeastgeneexpressioninaslittleas10min(39).
Asaresult,thenumbersofdifferentiallyexpressedproteinslocal-izedtothecellwall,plasmamembrane,andnucleusweremuchgreaterat20minthanat2h.
Ontheotherhand,morecompartments,includingthebud,golgi,andperoxisomecom-partments,wereaffectedbyfurfuralat2hthanat20min.
Thefunctiondistributionofproteinswithalteredexpressioninresponsetothepresenceoffurfuralat20minand2hispresentedinFig.
2B.
Asawhole,differentiallyexpressedpro-teinsat20minwereoverrepresentedcomparedtothoseseenat2hformostfunctiongroups,withtheexceptionoftheregulationofmetabolismandproteinfunction,unclassiedprotein,anddevelopmentfunctiongroups.
At20minofcul-tivation,furfuralmayaffectyeastmoreseverelyandresultinFIG.
2.
Subcellularlocalizationdistribution(A)andfunctionalcategories(B)of76and65proteinsdifferentiallyexpressedinresponsetothepresenceoffurfuralat20minand2h,respectively.
The100%stackedcolumnchartsareusedtocomparethenumbersofdifferentiallyexpressedproteinsineachcellularcompartmentorfunctioncategoryat20minand2h.
Thetotalnumberofdifferentiallyexpressedproteinsfromthetwotimepointsrepresents100%,whiletheindividualnumbersofdifferentiallyexpressedproteinsat20minand2hcomposethetwostackedelementsthatmakeuponecolumn.
(B)PF,proteinfate;CRDV,cellrescue,defense,andvirulence;CCDP,cellcycleandDNAprocessing;IE,interactionwiththeenvironment;BCC,biogenesisofcellularcomponents;CTD,celltypedifferentiation;CTTFTR,cellulartransport,transportfacilities,andtransportroutes;PS,proteinsynthesis;PBFCR,proteinwithbindingfunctionorcofactorrequirement;CF,cellfate;CC/STM,cellularcommu-nication/signaltransductionmechanism;UP,unclassiedproteins;RMPF,regulationofmetabolismandproteinfunction.
3768LINETAL.
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9g/literand10.
5g/literat20minand2h,respectively;seeFig.
1).
Yeastcellscanconvertfurfuraltoless-toxiccompounds,resultinginadecreasedconcentrationoffurfuralovertime.
Adose-dependentresponseofethanolo-genicyeasttothepresenceoffurfuralandHMFatconcentra-tionsfrom10to120mMhasbeencharacterizedbyLiuetal.
(43).
Proteinswithabundancechangescausedbythepresenceoffurfuralwerelocalizedtomostcompartmentsandwerefoundtobeinvolvedinalmostallthefunctionsandpathwaysinyeastcells,revealingthattheresponseofyeasttofurfuralisglobalandsystematic.
Furthermore,ourobservationsalsosug-gestthattheresponseofyeasttofurfuralisacontinueddy-namicandcomplexprocess.
Glycolysispathway.
AsimpliedbytheKEGGpathwayanal-ysisofthedifferentiallyexpressedproteins,furfuralmayhaveagreatimpactontheglycolysispathway.
AftertheadditionoffurfuralintotheaerobicbatchculturesoftheindustrialS.
cerevisiaestrain,12outof16proteinsquantiedintheglyco-lysispathwayhadbeendramaticallyupregulatedat20min,whileonly1of17proteinshadbeenupregulatedand3pro-teinsdownregulatedat2h(Fig.
3).
Evidently,theglycolysisofS.
cerevisiaehadrstbeenrapidlyinducedbytheadditionoffurfuralandhadthenreachedcontrollevelsat2hcomparedtothecontrolexperimentresults(veriedbyNADH/NADandATP/ADPassays).
Thisgivesfurtherevidencethatthere-sponseofyeasttothepresenceoffurfuralisacontinuingdynamicprocess,asdescribedabove.
ItisreasonabletobelievethattheexpressionlevelsofproteinscatalyzingthereactionsofFIG.
3.
Relativeexpressionlevelsofproteinsinvolvedincentralcarbonmetabolism,includingglycolysis,theTCAcycle,glycerolbiosynthesis,andthePPP.
The18O/16Oratiosofproteinsarepresentedforthe20-mintimepoint(therstvalueinparentheses)andthe2-htimepoint(thesecondvalueinparentheses).
Theunderlined18O/16Oratioswereobtainedonthebasisofanalysisofonepeptide.
The20-mintimepointdataaremissingbecauseproteinswerenotdetectedat20minby2D-LC-MS/MS.
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Theideaoftheactivationofglycolysisinthepresenceoffurfuralissupportedbyotherstudies.
Taherzadehetal.
(61)determinedthatglycolysisinducingchangesinabundancewasimmediatelyaffectedbyfurfuralandthatthereductionoffurfuralreliedonactiveglycolysisbyinvestigatingconversionoffurfuralinaerobicandanaerobicbathculturesofS.
cerevi-siaeCBS8066growingonglucose.
Analysisofthemetabolicuxdistributionsforaerobicsteady-statecultureswithandwithoutfurfuralinthemediumshowedthatthepresentationoffurfuralinthemediumresultedinanincreaseof30%inthespecicrateofglycolysiscomparedtotherateseenwithfurfural-freemedium(27).
Throughmetaboliteanalysis,Palmqvistetal.
(48)foundthatfurfuraldecreasedcellrepli-cationwithoutinhibitingcellactivityandhadatwofoldeffectonthekineticsofglucosemetabolisminS.
cerevisiae(i.
e.
,theglucosemetabolismratewasinhibitedbutthenalethanolyieldwasslightlyincreasedatanonlethalconcentrationoffurfural)(48).
Incontrasttotheresultsseenwithactivatedglycolysis,thegrowthandfermentationperformanceoftheindustrialS.
cer-evisiaestrainweresignicantlyretarded.
However,notablere-ductionoffurfuralwasobservedintherst4h,demonstratingthatactivatedglycolysismaycorrelatewiththeconversionoffurfural.
Presumably,thereductionoffurfuraltofurfuralal-coholmaybecatalyzedbyalcoholdehydrogenases(ADHs),withNADHasacofactor(12,46,67),whichwasevidencedherebytheupregulationofAdh5pandAdh1pasdescribedbelow.
ItispossiblethatglycolysisisactivatedbyfurfuraltoprovideasmuchNADHasisrequiredforthereductionoffurfural,whereasethanolproductionisreduced,becausethereductionoffurfuralcompeteswiththereactionofacetalde-hydetoethanolforbothNADHandADHs.
Glycerolbiosynthesis.
DownregulatedlevelsofGpd1p(at20min,notquantied;at2h,0.
46),Rhr2p(0.
58;0),andHor2p(notquantied;0.
43)catalyzingglycerolbiosynthesiswereobservedinfurfural-treatedcellscomparedtocontrolcells(Fig.
3).
Furthermore,asshowninFig.
1,theglycerolconcentrationstayedconstantaftertheintroductionoffurfuraltotheaerobicbathculturesofyeast,indicatingthatfurfuralseverelyinhibitsglycerolformation.
Ithasbeendemonstratedthatthereductionoffurfuraltofurfuralalcoholispreferredtoglycerolproductionasaredoxsink,subsequentlyresultinginthereplacementofglycerolformationbyfurfuralalcoholpro-duction(48,61).
Palmqvistetal.
(49)andTaherzadehetal.
(61)observedthatglycerolproductionwasdecreasedinthepresenceoffurfuralunderanaerobicconditions.
Glycerolbio-synthesisactsasaredoxsink,providingadditionalreoxidationofcytosolicNADH.
Atthesametime,NADHisthemajorcofactorrequiredforreductionoffurfuraltofurfuralalcohol.
Asaresult,glycerolproductionandfurfuralreductioncom-peteforasharedpoolofNADH,asisalsoobservedwiththereactionofacetaldehydetoethanolandfurfuralreduction.
Inthepresenceofahighconcentrationoffurfural,yeastreducestheproductionofglyceroltomeettheurgentrequirementofNADHfortheconversionoffurfuraltofurfuralalcohol(seeFig.
8).
TCAcycle.
Asacentralmetabolicpathway,thetricarboxylicacid(TCA)cycleprovidesprecursorsformanycompounds,includingsomeaminoacids,andgeneratesusefulamountsofATPandNADHunderaerobicconditions.
Noneoftheen-zymesintheTCAcyclewasidentiedat20min,whereasCit1p,Aco1p,Aco2p,Idh1p,andMah1pwereidentiedandquantiedat2handalldisplayedatrendtowardincreasedproduction,withAco2pandMdh1pnoticeablyupregulatedinthepresenceoffurfural.
Analysisofthemetabolicuxdistri-butionsfortheaerobicsteady-statecultureswithandwithoutfurfuralinthemediumshowedthatthepresenceoffurfuralinthemediumresultedina50%increaseinthespecicrateoftheTCAcyclecomparedtotherateseenwithfurfural-freemedium(27).
Theupregulationofenzymesat2hrevealsthattheTCAcyclecanbeactivatedinthepresenceoffurfuraltoproducemoreNADHforthereductionoffurfural(seeFig.
8).
PPP.
Thepentosephosphatepathway(PPP)isanimportantcarbohydratemetabolismpathway,oxidizingglucosetogener-ateNADPHforreductivebiosynthesisreactionswithincellsandribose-5-phosphateforthesynthesisofthenucleotidesandnucleicacid.
However,theexpressionlevelsofGnd1p(at20min,1.
46;at2h,1.
08),Tkl1p(0.
91;0.
98),andTal1p(1.
23;1.
30)involvedinthePPPwerenotaffectedbythepresenceoffurfuralateithertimepoint(Fig.
3).
Thisobservationiscon-sistentwiththeresultsofapreviousstudyshowingthatal-thoughselective-deletionmutantscodedbygenesinthePPPshowedgrowthdecienciesinthepresenceoffurfural,thesemutantswereinefcientinreducingfurfuraltofurfuralalcohol(18).
Therefore,itissuggestedthatthePPPmayhavenodirectcorrelationwithfurfuralconversionundertheconditionsstud-iedhere.
Sinceithasbeenreportedthatthecentralcarbonmetabo-lismofS.
cerevisiaeiscontrolledtoalargeextentviaposttran-scriptionalmechanismsinchemostatstudies(11,32,33),quan-titativeRT-PCRwasnotcarriedouttosubstantiateourndingsconcerningtheinvolvementofthecentralcarbonme-tabolismincellularresponsetothepresenceoffurfural.
In-stead,thelevelsofintracellularNADH,NAD,NADH/NAD,andATP/ADPweremeasuredbyLC/MS(Fig.
4).
TheintracellularconcentrationsofNADandNADHwerede-creasedatleasttwofoldandfourfold,respectively,leadingtoalowerNADH/NADratioinfurfural-treatedcellscomparedwithcontrolcellsatbothtimepoints.
Incontrast,theATP/ADPratiowasincreasedsignicantlyat20minbutnotat2h.
TheurgentrequirementoffurfuralreductioncausedasevereshortageofNADH,leadingtotheupregulationofsomeen-zymesinthecentralcarbonmetabolismtoprovideasmuchNADHandATPaspossibleincellstreatedwithfurfuralcomparedwithcontrolcellsat20minand2h(althoughtheeffectwaslesssignicantat2h).
TheneedforATPmaybesmallerthanthatforNADHinfurfural-treatedcells.
Thus,thelevelsofintracellularNAD,NADH,andNADH/NADweredecreasedwhereastheATP/ADPratiowasincreasedaftertheadditionoffurfural.
Theseresultscorroboratethecomparativeproteomeresultsdescribedabove.
NADandNADHareinvolvedinvariousbiologicalprocesses,includingaging,apoptosis,celldeath,energymetabolism,mitochondrialfunctions,calciumhomeostasis,antioxidation-generationofoxidativestress,geneexpression,andsoon(71).
IthasbeensuggestedthatNADdepletionmediatespoly(ADP-ribose)3770LINETAL.
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Previousworkhassug-gestedthatNADandNADHmaybeinvolvedinapoptosis:itwasreportedthatselectiveinhibitorsofNADsynthesiscaninduceapoptosis(24)andthatNADH/NADPHdepletionisanearlyeventinapoptosis(52).
Thus,thedecreaseinbothNADandNADHlevelsinyeastcellsupontreatmentwithfurfural,resultingfromaninhibitionofsynthesisofthesecom-poundsorfromaccelerateddegradation,maybeultimatelyresponsibleforthecelldeathobserved.
Furthermore,ourdatasuggestthatacetaldehydelikelyaccumulatesinthecultureduringfurfuralreductionduetoadecreasedNADHconcen-trationinthecell.
Inapreviousstudy,theaccumulationofacetaldehydeaftertheadditionoffurfuralwasobservedandtheeffectoffurfuraloncellreplicationwasshowntoberelatedtoacetaldehydeformation(48).
Acetaldehydehasbeenfoundtoexertinhibitioneffectsonyeastgrowth(59),whichiscon-sistentwiththearrestofgrowthobservedinthisstudy.
Obvi-ously,thepresenceoffurfuralcausedseveralsecondaryeffects,includingthedropinbothNADandNADHlevels,theac-cumulationofacetaldehyde,andacontributiontothegeneralstressenvironment,whichinturnmayaffectthecellularme-tabolisminyeastcellstreatedwithfurfural.
Thealteredexpressionlevelsofmostproteinscatalyzingthereactionsofcentralcarbonmetabolism(withtheexceptionofthoseinvolvedinthePPP)revealedthattheadditionoffur-furalledtoacentralcarbonmetabolismrearrangementinyeastcellsinordertoinducetolerationoffurfural.
Glycolysisand/ortheTCAcyclewerestimulatedtoprovidesufcientNADHforefcientconversionoffurfural.
Also,NADHwasdivertedfromglycerolsynthesisandethanolproductionintofurfuralalcoholformation.
Thus,theformationofethanolandglycerolwasinhibitedduringtheadaptationofyeasttofurfural(seeFig.
8).
ADHs.
AmongthesevenADHsinyeastcells,threewerequantiedat20minandsixat2h(Fig.
5).
When17goffurfural/literofmediumwasused,theexpressionofAdh5pwasmarkedlyupregulated(i.
e.
,itwasmorethan4timeshigherthanthatseeninfurfural-freemediumatbothtimepoints)whereastranslationallevelsshowednosignicantchanges.
ThisdiscrepancyforAdh5pattheproteinlevelandthetran-scriptlevelhasbeenreportedbyothergroups(6,7,9).
Adh5pcancatalyzetheconversionofacetaldehydetoethanol,withactivityapparentonlyinanadh1andadh3double-deletionstrain(57),andthisconversioncanbeinducedbythepresenceofdimethylsulfoxide(72).
Adh6pwassignicantlyupregulatedat20min(withonepeptidequantied)andshowednochangeat2h.
Larroyetal.
reportedthatAdh6pisanNADPH-dependentADHofbroadsubstratespecicitythatisabletoreducealdehydes,includingcinnamaldehyde,veratraldehyde,andfurfural(34).
Inaddition,thereductionof5-hydroxy-methylfurfuralandfurfuralwithNADPHasacofactorwasincreasedincell-freecrudeextractsfromAdh6p-overexpress-ingstrains(51).
Adh1p,themajorenzymecatalyzingthereac-tionofacetaldehydetoethanol,displayedmediateincreasesatbothtimepoints,whichwereveriedbyquantitativeRT-PCR(seeFig.
7).
Adh1phasbeensuggestedinpreviousstudiestobeanenzymepossiblycatalyzingthereductionoffurfuraltofurfuralalcohol(21).
TheoverexpressionofAdh1pcanin-creasetheformaldehyderesistanceofS.
cerevisiae(20).
Sinceethanolformationwasseverelyinhibitedatbothtimepoints,theenhancementofADHswasnotrelatedtothereactionofFIG.
4.
IntracellularNADHandNADlevels(A),ATP/ADPra-tios(B),andNAD/NADHratios(C)infurfural(fur)-treatedcells(graybars)andcontrol(con)cells(blackbars)at20minand2h.
Threebiologicalreplicateexperimentswereperformedalongwithtwoinjec-tionsforeachsample.
ThePvaluesindicatethestatisticalsignicanceoftheobserveddifferences,withP0.
05consideredtobestatisticallysignicantandP0.
05tobenotstatisticallysignicant,asdeterminedbyatwo-tailedStudentttest.
CDW,celldryweight.
FIG.
5.
HistogramshowingsixmembersoftheADHgenefamilyandtheirrespectivechangeratiosat20minand2h.
Thelledcirclesindicatethatproteinswerenotdetectedatthattimepoint.
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Furthermore,ithasbeenreportedthatthereductionoffurfuraltofurfuralalcoholislikelycat-alyzedbyADHs(12,46,67).
Thus,inthecontextoftheliteratureandourexperiments,itispossiblethatAdh5p,Adh1p,andAdh6pmaycatalyzethereductionoffurfuraltofurfuralalcohol.
However,furthervalidationstudiesarenec-essarytoconrmthishypothesis,sincetheinductionofAdh5p,Adh1p,andAdh6pbyfurfuralmaybeassociatedwithotherbiologicalprocesses.
Adh2p,Adh4p,andSfa1pweredetectedandquantiedonlyat2h.
Adh2pandSfa1phaddecreasedexpressionlevels,whereastheexpressionabundanceofAdh4pwasnotaffectedbythepresenceoffurfural.
Adh2p,unlikeotherADHs,cata-lyzesthereactionofethanoltoacetaldehydeandisrepressedinthepresenceofglucose(9).
Additionoffurfuralinhibitedtheglucoseconsumptionandledtohigherglucoseconcentra-tionsinthefurfuraltreatmentexperiment(Fig.
1),andthisinturnrepressedtheexpressionofAdh2p.
ThedownregulationofSfa1pisconsistentwiththelowerethanolconcentrationinthemediumwithfurfuralcomparedtothefurfural-freeme-diumat2h,asithadbeenreportedthatSfa1pisinducedbythepresenceofethanol(17).
Thedecreasedlevelsofexpres-sionofAdh2pandSfa1psuggestthattherippleeffectimposedbythepresenceoffurfuralexistsandbecomesmoresignicantwiththepassageoftimeaftertheadditionoffurfural.
Thus,ADHsmayplayaroleinthetoleranceandadaptationofethanologenicyeasttofurfural.
Proteinsrelatedtostressresponse.
Althoughithasbeenmentionedbeforethatfurfuralmayresultintheaccumulationofreactiveoxygenspecies,vacuoleandmitochondrialmem-branedamage,andchromatinandactindamageinS.
cerevisiae(39),therehasbeennopreviousstudyfocusingonthestresseffectscausedinS.
cerevisiaebythepresenceoffurfural.
Thelevelsofabundanceof23proteinsrelatedtostressresponsedisplayedsignicantchangeseitheratonetimepointoratbothtimepoints(Fig.
6A).
Analysiswithrespecttofunctionalcategoryshowedthattheseproteinsareinvolvedinthere-sponseofyeasttounfoldedproteins,oxidativestress,osmoticandsaltstress,DNAdamage,andnutrientstarvation.
Thereareeightproteinsrelatedtounfoldedproteinre-sponse(UPR),includingveHSP70proteins:Ssb1p,Ssb2p,Ssc1p,Ssz1p,andKar2p.
At20minaftertheadditionoffurfural,proteinsrelatedtothefolding,sorting,andtranslo-cationofnewlysynthesizedpolypeptidechainssuchasEgd2p(16),Hsp10p(25),andSsb1pwereupregulated.
At2h,nochangeintheexpressionofEgd2pandSsb1pwasseen,whereasHsp10pwasslightlydownregulated,asshownbytheresultsofquanticationofonepeptide.
Instead,anothergroupFIG.
6.
Proteinsrelatedtostressresponsethatexhibitedsignicantchangesinabundanceinthisstudy(A)andribosomalproteinsquantiedinthisstudy(B).
Thelledcirclesindicatethatproteinswerenotdetectedatthattimepoint.
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ItisworthnotingthatKar2pwasupregu-latedat2h,sinceKar2pnotonlyisinducedbyUPRbutisalsoinvolvedintheregulationofUPRthroughinteractionwithIre1p.
TheinductionoftheseproteinsrelatedtotheUPRimpliesthattheadditionoffurfuralmayleadtotheaccumu-lationofunfoldedproteins,subsequentlyresultingintriggeringoftheUPR.
Furtherevidencewasobservedwhentheresultsofstimulationofribosomeproteinswererecorded(Fig.
6B).
At20min,allofthe32ribosomalproteinsquantiedinthisstudyexhibitedatrendtoincreasedregulationchangesasagroup;17ofthoseproteinsweresignicantlyupregulatedinresponsetofurfural.
At2h,16of34ribosomalproteinsquantiedshoweddistinctlyincreasedexpressionlevels,while1proteinwasslightlydownregulated.
Atbothtimepoints,27proteinswerequantied,with11proteinsupregulated.
Themainfunc-tionofribosomeistoorganizeproteinsynthesis.
Theupregu-lationofribosomalproteinssuggeststhatproteinsynthesisintheaerobicbatchculturecontainingfurfuralmayhavebeenacceleratedcomparedtothatseenwithfurfural-freeculturesunderthesameconditions.
Theaccelerationofproteinsyn-thesismayleadtotheaccumulationofunfoldedproteinsintheendoplasmicreticulum(ER)lumen,inturnactivatingtheUPRtorestoreprotein-foldingcapacityandadapttonewconditionscausedbythepresenceoffurfural.
TheUPRcanprotectcellsagainstERstress,butwhenthisobjectivecannotbeachievedwithinacertaintimeperiodorwhenERstressisprolonged,theUPRcaninitiatecelldeathorapoptosis.
Theconcentrationoffurfuralappliedinthisstudywassohighthathigh-intensityandlong-termERstressexisted,andwashoutofculturesoccurredat8h.
Proteinsthatrespondtooxidativestressrepresentthesec-ond-largestgroupamongthestress-response-relatedproteinsthatshowedabundancechangesinthepresenceoffurfural.
ThethioredoxinsTrx1pandTrx2p,heatshockproteinHsp12p,andsuperoxidedismutaseSod1pweresignicantlyupregu-latedat20minbutshowednoabundancechangeat2hinresponsetothepresenceoffurfural.
ExpressionofTsa1p,ahousekeepingthioredoxinperoxidase,wasrstnotablyup-regulatedat20minandthenslightlydownregulatedat2h.
TheexpressionlevelofAhp1wasdownregulatedatbothtimepointsaftertheadditionoffurfuralintoaerobicbatchculturesofS.
cerevisiae.
Ahp1pisathiol-specicperoxiredoxinprotect-ingcellsfromoxidativedamagebyreducinghydroperoxides(35).
Yhb1p,anitricoxideoxidoreductasedetoxifyingnitricoxide(38),wasquantiedonlyat2handshowedanincreasedexpressionlevel.
TheoxidativestressmayberelatedtothedecreaseoftheNAD,NADH,andNADH/NADlevels,sinceithasbeensuggestedthatNADandNADHinuenceantioxidationandthegenerationofoxidativestress(71).
Atbothtimepoints,furfuralinducedtheexpressionlevelsofRps3p(at20min,1.
70;at2h,1.
42)andStm1p(3.
25;1.
87),whichrespondtoDNAdamage.
Geneticanalyseshavesug-gestedthatStm1p,aG4quadruplexandpurinemotiftriplexnucleicacid-bindingprotein,participatesinseveralbiologicalprocesses,includinginteractionwithribosomeandsubtelo-mericYDNA(63),telomeremaintenancebyinteractionwithCdc13p,andapoptosis(25,36).
Furthermore,itsaccumulationinducescelldeath(36).
LiketheUPR,theupregulationofStm1patbothtimepointsmaybeinvolvedinthewashoutofculturesat8h.
Incontrast,Rps3pisessentialforviability(15)andisinvolvedinDNAdamageprocessingandwithapurinic-apyrimidinicendonucleaseactivity(29).
Thus,theupregula-tionofexpressionofthesetwoproteinsrevealsthatfurfuralcausesDNAdamageinS.
cerevisiae.
ThePkc1p–mitogen-activatedproteinkinase(Pkc1p-MAPkinase)pathwayregulatescellwallmaintenanceandintegrity,whichareessentialforthegrowthandtheintegrityofprolif-eratingcells(60).
ThePkc1p-MAPkinasepathwayisreportedtobenegativelyregulatedbyZeo1p(at20min,2.
3;at2h,1.
3)(19)andLsp1p(at20min,notquantied;at2h,2.
83)(73),sotheupregulationofexpressionofZeo1pat20minandofLsp1pat2hrevealedthatthispathwayisinactivatedinthepresenceoffurfural,leadingtolackofcellwallintegrityanddefectivecellgrowth.
ThishypothesisisfurthersupportedbythedownregulationofRho1p(at20min,notquantied;at2h,0.
31),whichisrequiredforthePkc1plocalizationtositesofpolarizedgrowththroughoutthecellcycleandalsotoregionsofcellwalldamage(3,54).
Rho1palsoregulatescellwallsynthesizingenzyme1,alsocalled3-beta-glucansynthase(Fks1pandGsc2p)(14,54).
ExpressionofAct1p,astructuralconstituentofthecytoskeletonthatisinvolvedincellpolar-ization,endocytosis,andmanyothercytoskeletalfunctions(53),wassignicantlyupregulatedat20minandunchangedat2h.
Thealteredexpressionofproteinsthatareinvolvedincellintegrity,growth,andsurvivalstronglyimpliesthatthepres-enceoffurfuralhadcausedarearrangementofcellstructureanddamagetoyeastcellintegrity,growth,andsurvival.
More-over,Pho2p,whichparticipatesinnutrientstarvation,wassig-nicantlyupregulatedat20min.
PHO2isknownasatran-scriptionalactivatorofPHO5andPHO81(phosphateutilization),HIS4(histidinebiosynthesis),CYC1,TRP4,andHO;italsoactivatesexpressionoftheADE1,ADE2,ADE5,ADE7,andADE8genes,whichareinvolvedinthemetabolicpathwayofpurinenucleotidebiosynthesis(10,37).
Toconrmtheproteinexpressiondataobtainedby18Olabeling,thetranscriptlevelsofthe10selectedstress-relatedproteinsdescribedaboveweremeasuredbyquantitativeRT-PCRat20minand2h(Fig.
7).
ThequantitativeRT-PCRresultsforAct1p,Hsp10p,Hsp12p,Pho2p,Tsa1p,andZeo1pat20minandforLsp1pandStm1pat2hareconsistentwiththerelativequantitativeproteinexpressionresultsobtainedusing18Olabeling.
Furthermore,thelevelsofexpressionofZeo1p,Hsp10p,andHsp12pchangedinthesamedirectionatboththeproteinlevelandthetranscriptionlevel,althoughthechangesatthetranscriptlevelwerestatisticallysignicantwhereasthoseattheproteinlevelwerenot.
Generally,therewerediscrepanciesbetweenquantitativeRT-PCRresultsandtherelativequantitativeproteinexpressionresultssuchasthoseseenwithAhp1patbothtimepoints.
Thesediscrepan-ciesmayhavebeenduetothefactthat,apartfromtheeffectdeterminedbytheamountofmRNApresent,theproteinexpressionlevelisinuencedbyproteinturnoverandpost-translationalmodications.
Whatismore,themRNAmole-culesmayberelativelyunstablecomparedtoproteinsingen-eral,contributingtothedifferenceinturnoverratesbetweenmRNAandprotein,asreportedinapreviousstudyusingS.
cerevisiae(22).
ThequantitativeRT-PCRresultsprovideor-VOL.
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Clearly,furfuralnotonlyinuencesyeastwithrespecttoprimarycarbonatemetabolicpathwaysandproteinsynthesisbutalsocausestheformationofacomplexstressenvironmentinyeastcells.
Theexpressionlevelsofproteinsinvolvedincommonstressresponses,includingtheUPR,oxidativestress,osmoticandsaltstress,DNAdamage,andnutrientstarvation,werealteredduetothecomplexstressenvironmentformedbytheadditionoffurfuraltotheaerobicbatchculture.
There-directionofresourcestowardstressdefensemayleadtodi-minishingamountsoffreeavailableenergysuppliedbycatab-olismforcellgrowthandinsufcientATPforphosphorylationofglucosetoformglucose-6-phosphate,whichiscriticalfortheutilizationofglucose.
Thus,cellgrowthandglucoseconsump-tionareinhibitedbyfurfural.
Withthepassageoftime,yeastcellsrstdisplayalagphaseandeventuallyadapttofurfuralstress,butwhentheconcentrationoffurfuralistoohighandrequirestoomuchNADH,theyeastcellisseverelydamagedandwashoutoccurs(Fig.
8).
Conclusion.
Theadditionofhighconcentrationsoffurfuraltotheaerobicbatchculturesofanindustrialstrainseverelyinhibitedbiomassgrowth,glucoseconsumption,ethanolpro-duction,andglycerolproduction.
Relativequantitativepro-teomicdatahereprovideadeeperunderstandingofthemo-lecularmechanisminvolvedintheresponseofS.
cerevisiaetofurfuralunderaerobicbatchconditions.
Togetherwithupregu-lationofAdh5pandAdh1p,activationofglycolysisand/ortheTCAcycleandrepressionofglycerolbiosynthesiswereob-served,suggestingthatthereductionoffurfuraltofurfuralalcoholcatalyzedbyAdh5pandAdh1pwithNADHasaco-factormaybeapotentialpathwayfortheconversionoffur-FIG.
7.
Asetof13transcriptlevelswasmeasuredbyquantitativeRT-PCR.
Ratiosbetweenfurfural-treatedandcontrolcelllevelsat20minand2hareshown.
Threebiologicalreplicateexperimentswereperformedalongwiththreetechnicalanalysesforeachbiologicalreplicate.
Astatisticalanalysisofindependentculturereplicateswasperformedusingatwo-tailedStudentttest.
Theproteinscodedbygenesthatweresignicantlytranscriptionallyregulatedaredepicted(*,P0.
05).
FIG.
8.
Modeldepictingtheeffectsofthepresenceoffurfuralonethanologenicyeastunderaerobicbatchconditions.
Underconditionsthatincludedtreatmentwithfurfural,glycolysisand/ortheTCAcyclewasactivated(green)whereasglycerolandethanolproduction(red)wererepressedtoprovideasmuchNADHasrequiredforfurfuralreductiontofurfuralalcohol.
Ontheotherhand,ageneralstressresponse(purple)wascausedbyfurfural.
Asaresult,yeastcellsdisplayedalagphasetotolerateandadapttofurfural,butwhentheconcentrationoffurfuralwastoohigh,leadingtoarequirementfortoomuchNADHandresultinginseveredamagetoyeastcells,washoutoccurred.
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Whatismore,proteinsinvolvedinstressresponsewerealsodifferentiallyexpressedduetothecomplexstressenvironmentwhoseformationwascausedbythepresenceoffurfural.
Theredirectionofre-sourcestowardfurfuralconversionandstressdefensemayleadtotheinhibitionofyeastcellgrowthandfermentation.
Quan-titativeRT-PCRandmetaboliteanalysis(ofthelevelsofNADH,NAD,NADH/NAD,andATP/ADP)wereutilizedtoprovideorthogonalevidencesupportingthecomparativeproteomicsresults.
Secondaryeffectsduetothepresenceoffurfuralwereobservedandmayhavebeenrelatedtothein-hibitoryeffectsoffurfural.
Theseinsightsintotheresponseofyeasttofurfuralwillbenetthedesignanddevelopmentofinhibitor-tolerantethanologenicyeaststrainsforlignocellu-lose-bioethanolfermentation,whichisoneofthesignicantchallengesforcost-competitivebioethanolproduction.
ACKNOWLEDGMENTSWeareverygratefulforthenancialsupportfromtheNationalScienceFundofChinaforDistinguishedYoungScholars(project20425620),theKeyProgram(project20736006),NationalBasicRe-searchProgram973ofChina(grant2007CB714301),theInternationalCollaborationProjectofMOST(grant2006DFA62400),andKeyProjectsintheNationalScience&TechnologyPillarProgram(grant2007BAD42B02).
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