Characterization of the xylose-transporting

Characterizationofthexylose-transportingpropertiesofyeasthexosetransportersandtheirinfluenceonxyloseutilization

rkGardonyi,2TanjaHamacher,1JessicaBecker,1Ma

BarbelHahn-Hagerdal2andEckhardBoles1

Authorforcorrespondence:EckhardBoles.Tel:j492118112778.Fax:j492118115370.e-mail:boles!uni-duesseldorf.de

1

InstitutfurMikrobiologie,Heinrich-Heine-Universitat,Universitatsstr.1,

Geb.26.12.01,D-40225Dusseldorf,GermanyDepartmentofAppliedMicrobiology,LundUniversity,

22100Lund,Sweden

2

Foraneconomicallyfeasibleproductionofethanolfromplantbiomassbymicrobialcells,thefermentationofxyloseisimportant.Asxyloseuptakemightbealimitingstepforxylosefermentationbyrecombinantxylose-utilizingSaccharomycescerevisiaecellsastudyofxyloseuptakewas

performed.Afterdeletionofallofthe18hexose-transportergenes,theabilityofthecellstotakeupandtogrowonxylosewaslost.Reintroductionof

individualhexose-transportergenesinthisstrainrevealedthatatintermediatexyloseconcentrationstheyeasthigh-andintermediate-affinitytransportersHxt4,Hxt5,Hxt7andGal2areimportantxylose-transportingproteins.Severalheterologousmonosaccharidetransportersfrombacteriaandplantcellsdidnotconfersufficientuptakeactivitytorestoregrowthonxylose.

Overexpressionofthexylose-transportingproteinsinaxylose-utilizingPUAyeaststraindidnotresultinfastergrowthonxyloseunderaerobicconditionsnordiditenhancethexylosefermentationrateunderanaerobicconditions.Theresultsofthisstudysuggestthatxyloseuptakedoesnotdeterminethexylosefluxundertheconditionsandintheyeaststrainsinvestigated.

Keywords:Saccharomycescerevisiae,xyloseuptake,glucoseuptake,xylose

fermentation,heterologousexpression

INTRODUCTION

Incommonfermentationprocesses,theyeastSaccharo-mycescerevisiaeiswidelyusedforbiotechnologicalethanolproduction.However,S.cerevisiaeisonlyabletofermenthexosesugars,andnotpentosesugarslikexylose.Alongwithglucose,xyloseisoneofthemajormonosaccharidecomponentsoflignocellolosicbiomass(Haynetal.,1993).Foraneconomicallyfeasibleindustrialprocessforethanolproductionfromplantbiomassinlignocellulosehydrolysates,itisnecessarytofermentallsugarspresent(vonSivers&Zacchi,1995).Effortstoestablishaxylose-utilizingpathwayinS.cerevisiaebyinsertionofthegenesencodingxylosereductaseandxylitoldehydrogenasefromPichiastipitisorotherorganismshaveresultedinonlypoorethanolproductionfromxylose(Kotter&Ciriacy,1993;Tantirungkijetal.,1993;Walfridssonetal.,1995).Varioussteps,includingtheuptakeofxylose,havebeensuggestedtolimitthemetabolismofxyloseinmetabolicallyengineeredS.cerevisiae(Kotter&Ciriacy,1993;Eliassonetal.,2000).UptakeofxylosebyS.

0002-5705#2002SGM

cerevisiaehasbeenproposedtobemediatedmoreorlessunspecificallybyitshexose-transportsystem.Thisiscomposedofalargefamilyof18relatedtransporterproteinscalledHxtsandadditionalsugartransporterswithbroadersubstratespecificity(Boles&Hollenberg,1997;Wieczorkeetal.,1999).Inxylose-uptakeexperi-mentswithS.cerevisiaecells,twokineticallydistinctuptakecomponentswithKmvaluesofabout0n19Mand1n5Mweredetermined,demonstratingthatthemono-saccharidetransportsysteminS.cerevisiaehasnearlya200-foldloweraffinityforxylosethanforglucose(Kotyk,1967;Lagunasetal.,1982;Kotter&Ciriacy,1993).

Toimprovethexylose-uptakeactivityofS.cerevisiaeitisnecessarytoidentifythespecifictransportersmediat-inguptakeofxylose.Additionally,heterologousxylosetransportersfromotherorganismsmaybeexpressedinS.cerevisiaetoimprovexyloseuptake.UsingS.cerevi-siaeTMB3201,expressingafunctionalxyloseutilizationpathwaybutlackingthecompletemonosaccharide-transportsystem,wehavecharacterizedthexylose-transportingpropertiesofmonosaccharidetransporters

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fromyeast,bacteriaandplants,bygrowthand[\"%C]xyloseuptake.Especiallythehigh-affinityglucosetransportersofS.cerevisiaecanmediateuptakeofxylose.Nevertheless,overexpressionofthesetrans-portersinthexylose-fermentingyeaststrainPUA-X1,2didnotincreaseitsgrowthrateonxylosenoritsxylosefermentationrate.METHODS

CEN.PK2-1C(MATaleu2-3,112ura3-52trp1-289his3-∆1MAL2-8cSUC2),EBY.VW4000(MATa∆hxt1-17∆gal2∆stl1∆agt1∆mph2∆mph3leu2-3,112ura3-52trp1-289his3-∆1MAL2-8cSUC2)(Wieczorkeetal.,1999),TMB3001(MATahis3-∆1::YIpXR\\XDH\\XKMAL2-8cSUC2)(Eliassonetal.,2000),TMB3201(MATa∆hxt1-17∆gal2∆stl1∆agt1∆mph2∆mph3leu2-3,112ura3-52trp1-289his3-∆1::YIpXR\\XDH\\XKMAL2-8cSUC2)(thiswork),PUA-X1,2[his3-11ura3-52leu2::(LEU2:PDC1pr-XYL1,ADH1pr-XYL2)](kindlypro-videdbyT.Weierstall,Dusseldorf).Syntheticmediaconsistedof6n7gl−\"Difcoyeastnitrogenbase(YNB)supplementedwithaminoacidsandadenine,withorwithouturacilasaplasmid-selectionmarker,andwithvariouscarbonsources.Yeastcellsweregrownaerobicallyat30mConarotaryshakeroronagarplates.Xylosefermentationwasinvestigatedbyincubating50mlyeastcultureswithahighcelldensityinsealed50mlbottlesundergentlestirring.

Constructionofplasmids.The0n4kbSacI\\SpeIMET25promoterfragmentofmulticopyplasmidp426MET25(Mumbergetal.,1994)wasreplacedbya0n4kbDNAfragmentcontaininganHXT7promoterfragmentfromk392bptok1bpthatwasamplifiedbyPCRwithprimersP426H7-1(5h-CTAGAGCTCGTAGGAACAATTTCGG-3h)andP426H7-2(5h-CGACTAGTGTGATGGTGATGGTGATG-CATGTTAACTTTTTGATTAAAATTAAAAAAACTT-3h),andplasmidYEpkHXT7(Krampeetal.,1998)asthetemplate,resultinginplasmidp4H7.Allmonosaccharide-transportergeneswereclonedbyrecombination-cloningintop4H7usingthestrategiesdescribedbyWieczorkeetal.(1999).Theyeasthexose-transportergeneswereamplifiedbywhole-cellPCRfromstrainFY1679withprimersobtainedfromResearchGenetics.TheArabidopsisthalianaSTP2andSTP3ORFswereamplifiedfromplasmidspUC19STP2andpUC19STP3usingtheprimersF-ATSTP2(5h-GGAATTC-CAGCTGACCACCATGGCTGTTGGTTCGATGA-3h),R-ATSTP2(5h-GATCCCCGGGAATTGCCATGCTAGT-CTTTGAAATATTTCTTCC-3h),F-ATSTP3(5h-GGAAT-TCCAGCTGACCACCATGGTAGCAGAAGAAGCAAG-3h)andR-ATSTP3(5h-GATCCCCGGGAATTGCCATGTCAATGGCTAAGAATGGTG-3h),respectively.ThexylEORFwasamplifiedfromEscherichiacolistrainJM109withprimersF-XYLE(5h-GGAATTCCAGCTGACCACCATG-AATACCCAGTATAATTCC-3h)andR-XYLE(5h-GATCC-CCGGGAATTGCCATGTTACAGCGTAGCAGTTTG-TTG-3h).PCRproductswerefurtheramplifiedwithprimersT3(5h-GAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAAGGAATTCCAGCTGACCACC-3h)andT2-ORFs(5h-GGGGGAGGGCGTGAATGTAAGCGTGACA-TAACTAATTACATGACTCGAGGATCCCCGGGAATTGCCATG-3h).ThepredictedA.thalianaxylose-protonsymporterORFwasamplifiedfromaseedlingcDNAbank(Minetetal.,1992)withprimersF7-AT5G(5h-TAATTTT-AATCAAAAAATGTGTTTTAAATCGAGGCCTGAC-3h)andR-AT5G(5h-CCCCGGGAATTGCCATGTCACTT-CAAGATTTTTGATTC-3h),andthePCRproductwas

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Yeaststrainsandgrowthconditions.Yeaststrainswere:

furtheramplifiedwithprimersT71-ORFs(5h-AACACAA-AAACAAAAAGTTTTTTTAATTTTAATCAAAAA-3h)andT2-ORFs.p4H7waslinearizedwithBamHIandHindIIIandtransformedintoyeaststrainEBY.VW4000,togetherwiththePCR-amplifiedsugar-transportergenes,selectingforuracilprototrophyonamediumcontaining2%maltoseafterhomologousrecombinationinyeast.Plasmidswerereisolated,amplifiedinE.coliSURE(Stratagene),andanalysedbyrestrictionenzymemappingorsequencing(Seqlab).TheresultingplasmidswerenamedpTH[nameofgene](e.g.pTHHXT1,pTHHXT3).ConstructionofplasmidsYEpkHXT7,p426MET25-HXT11andpHL125-GAL2isde-scribedelsewhere(Krampeetal.,1998;Wieczorkeetal.,1999;Liang&Gaber,1996).

formedusingpublishedprocedures(Sambrooketal.,1989).Xyloseuptakewasassayedasdescribedpreviously(Weierstalletal.,1999).Forthedeterminationoftheethanolproductionrates,ethanolconcentrationsinyeastculturesupernatantsweremeasuredusinganenzymictestkit(Roche).

Othertechniques.Molecularbiologytechniqueswereper-

RESULTS

Yeasthexosetransportersmediatinguptakeofxylose

TheyeaststrainTMB3001(Eliassonetal.,2000),constructedbyintegratingavectorexpressingP.stipitisXYL1andXYL2(encodingxylosereductaseandxylitoldehydrogenase,respectively)andS.cerevisiaeXKS1(encodingxylulosekinase)intothegenomeofanS.cerevisiaeCEN.PKstrain,grewslowlyinshaken-flaskcultureswithsyntheticmediumand2%xyloseasthecarbonsource(Fig.1).Incontrast,strainTMB3201,constructedbyintegratingthesameplasmidintotheCEN.PK-derivedstrainEBY.VW4000(Wieczorkeetal.,1999)lackingallitshexose-transportergenes,didnotgrowatallinthesamemedium.Thisdemonstratesthatoneormoreoftheyeasthexosetransportersisre-sponsiblefortheuptakeofxyloseinS.cerevisiae.Inordertofindthetransporter(s)responsible,theS.cerevisiaehexose-transportergenesHXT1,3,4,5,8,9,10,13,14and15wereclonedbyrecombination-cloningintothemulticopyexpressionvectorp4H7,drivingexpressionfromtheverystrongandconstitutiveHXT7\"–$*#bppromoterfragment.Theresultingplas-mids,theemptyvector,andplasmidsYEpkHXT7,p426MET25-HXT11andpHL125-GAL2(expressingtheS.cerevisiaeHxt7,Hxt11andGal2transporters,respectively),weretransformedintotheyeaststrainTMB3201.Thecellswereplatedontoagarplatesofsyntheticmediumwithouturacil,containing2%malt-oseasthecarbonsource.Afterreplica-plating,allthetransformantsexceptthosewiththeemptyvectorcouldgrowonsyntheticmediumwithglucoseasthesolecarbonsource,indicatingthatalltransporterswerefunctionallyexpressedandabletomediateuptakeofsugarsintothecells.

Toidentifywhichhexosetransportersareabletotakeupxylose,thegrowthratesofthetransformantsinliquidsyntheticmediumwithouturacilandwith2%xyloseasthesolecarbonsourceweredetermined.Only

XyloseuptakeinS.cerevisiae

1·5

(a)

CEN.PK2-1C

1·0

TMB3201 (Dhxt)(Dhxt) HXT4

+

0·5

(Dhxt) HXT5+(Dhxt) HXT7+

OD6000·75

50

(b)

100150

(Dhxt) GAL2+

0·50

1020304050

–1

Xylose uptake [nmol min (mg dry wt)–1]

.................................................................................................................................................

0·25

25

50Time (h)

75100

Fig.2.XyloseuptakeinHXTmutantstrains.YeasttransformantsCEN.PK2-1C(p4H7),TMB3201(p4H7),TMB3201(pTHHXT4),TMB3201(pTHHXT5),TMB3201(YEpkHXT7)andTMB3201(pHL125-GAL2)weregrownintotheexponentialgrowthphaseinsyntheticmediumwithouturacilandwith2%maltosebeforeharvesting.Xyloseuptakewasmeasuredfor1minwith50mMD-[U-14C]xylose.TheresultsaremeansofthreedeterminationspSEM.

.................................................................................................................................................

Fig.1.GrowthofHXTmutantstrainsinxylosemedium.Yeasttransformantswerepregrowninsyntheticmediumwithouturacilandwith2%maltose,washedtwiceandinoculatedinsyntheticmediumwithouturacilandwith2%xyloseataninitialOD600ofabout0n35(a)or0n25(b).Cellsweregrownaerobicallyat30mConarotaryshaker.GrowthwasmonitoredbymeasuringtheOD600ofthecultures.Strainsare:(aandb)TMB3001(p4H7)(󰀫),TMB3201(p4H7)($);(a)TMB3201(pTHHXT1)(7),TMB3201(YEpkHXT7)(=),TMB3201(pHL125-GAL2)(#);(b)TMB3201(pTHHXT4)(#),TMB3201(pTHHXT5)(=),TMB3201(pTHHXT9)(7).Representa-tiveresultsofatleasttwoindependentexperimentsforeachstrainareshown.

xylose(Fig.2).Incontrast,expressionofHxt4,5and7,andGal2,conferredxyloseuptakeactivitiesuponthestrain.Xyloseuptakeactivitywith50mMradiolabelledxylosewashighestforthestrainexpressingHxt7[48nmolmin−\"(mgdryweight)−\"]andlowestforthestrainexpressingHxt5(11nmolmin−\"(mgdryweight)−\"](Fig.2).TheparentalstrainCEN.PK2-1Ctransformedwiththeemptyvectorp4H7exhibitedanuptakeactivityof15nmolmin−\"(mgdryweight)−\".

ExpressionofheterologousmonosaccharidetransportersinS.cerevisiae

transformantsexpressingtheHxt4,Hxt5,Hxt7orGal2transporterswereabletogrowinthexylosemedium(Fig.1).Incontrast,transformantsexpressingthetransportersHxt1,3,8,9,10,11,13,14and15didnotgrowwith2%xylose.OurresultsindicatedthatHxt4,5and7,andGal2,areabletomediatetheuptakeofxyloseintoS.cerevisiaecells.WedidnotanalyseHxt2asitwasnotpossibletocloneitbehindtheHXT7promoterfragmentonvectorp4H7.Hxt6,Hxt16andHxt17werenotincludedbecausetheyarealmostidenticaltoHxt7,Hxt15andHxt13,respectively.HXT12isapseudogene(Wieczorkeetal.,1999).

Determinationofxylose-uptakeactivities

Toconfirmtheresultsobtainedinthegrowthtests,short-term(60s)xyloseuptakeassayswereperformedwithradiolabelledxylose.Xyloseuptakewaslinearforatleast60sundertheconditionsused(datanotshown).TransformantsofstrainTMB3201expressingHxt4,Hxt5,Hxt7orGal2,orcontainingtheemptyvectorp4H7,weregrownintotheexponentialgrowthphaseinsyntheticmediumwithouturacilandwith2%maltose.Asexpected,thehexose-transport-deficientstrainTMB3201withtheemptyvectorwasnotabletotakeup

TheS.cerevisiaehexosetransporterssolelymediatefacilitateddiffusionoftheirsubstratesdownacon-centrationgradient(Lagunas,1993).Incontrast,severalotheryeasts,plantcellsandbacterialcellscanactivelytransporthexosesandpentosesbyproton-symportmechanismsagainstconcentrationgradients(Boles&Hollenberg,1997;Buttner&Sauer,2000).Moreover,theseproton-symportsystemsnormallyexhibitmuchhigheraffinitiesfortheirsubstrates.Wethereforeinvestigatedwhethersuchheterologoustransporterscouldrestoregrowthonxylosetothemonosaccharide-transport-deficientyeaststrainTMB3201.

TheE.colixylEgeneencodesaxylose–protonsym-porter(Davis&Henderson,1987).Thehexose–protonsymportersHup1fromChlorellakessleri,andStp2andStp3fromA.thaliana,havebeenshowntomediateuptakenotonlyofvarioushexosesbutalsoofxylose(reviewedbyButtner&Sauer,2000).Moreover,aftercompletionoftheA.thalianagenomesequencingproject,asequencehasbeenfoundonchromosome5encodingapredictedxylose–proton-symporter-likepro-teintargetedtochloroplasts(accessionnumberAt5g59250).ThexylE,STP2andSTP3ORFs,aswellas

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theputativexylose–protonsymportersequencelackingthe31aminoacidsencodingthechloroplast-targetingsequence,wereclonedintothemulticopyexpressionvectorp4H7.AftertransformationoftheresultingplasmidsandplasmidpNEV-EexpressingHUP1behindtheyeastPMA1promoter(Robletal.,2000)intostrainTMB3201,Stp2,Stp3andHup1,butnot–asexpected–XylEandtheputativeA.thalianaxylose–protonsymporter,supportedgrowthoftheyeasttransformantsonsyntheticmediumwith2%glucose(datanotshown).However,noneofthetransportersrestoredgrowthinsyntheticmediumwith2%xylose,indicatingthattheyarenotabletotakeupxyloseintotheyeastcells.Moreover,wetransformedstrainTMB3201withtheP.stipitisgenomicDNAlibraryYEpTW(Weierstalletal.,1999)andanA.thalianaseedlingcDNAbank(Minetetal.,1992),andselectedthetransformantsforgrowthinsyntheticmediumwith2%xylose.Fromatotalofmorethan18000and10000transformants,respectively,wecouldnotfindanytransformantabletoutilizeandtogrowonxylose.

XylosefermentationsandgrowthtestswithHXT-overexpressingstrains

1·5Ethanol (g l–1)1·0

0·5

25

Time (h)

5075

.................................................................................................................................................

Fig.3.EthanolproductionofPUA-X1,2cellsoverexpressingxylosetransporters.TransformantsTMB3001(p4H7)(7),TMB3201(pTHHXT5)(󰀁),TMB3201(YEpkHXT7)(#)andTMB3201(pHL125-GAL2)(=)werepregrowninsyntheticmediumwithouturacilandwith2%xylosetoanOD600of1n9,washedtwice,resuspendedin0n1vol.ofthesamemedium,andincubatedunder(semi-)anaerobicconditionsat30mC.Ethanolconcentrationsintheculturesupernatantsweredeterminedatvarioustimepoints.Representativeresultsofatleasttwoindependentexperimentsforeachstrainareshown.

TheS.cerevisiaePUAstrainswereselectedforfastergrowthonxylulosemedia(Porep,1987;Kotter&Ciriacy,1993).Subsequently,theP.stipitisXYL1andXYL2ORFsundercontroloftheS.cerevisiaePDC1andADH1promoters,respectively,werechromosomallyintegratedinthesestrains,resultinginPUA-X1,2.Inordertoseewhetheroverexpressionofxylose-trans-portingproteinscouldincreasetheethanolproductionratefromxylosebyrecombinantS.cerevisiaestrains,pTHHXT5,YEpkHXT7,pHL125-GAL2andtheemptyvectorweretransformedintothePUA-X1,2strain.Thetransformantswerepre-grownonsyntheticmediumwith2%xylosetoanODof1n9.Cellswereharvested

'!!bycentrifugationandwashedtwicewith2%xylose

medium.Theywerethenresuspendedin0n1vol.ofthevolumeofthesamemedium,incubatedat30mCandtheethanolconcentrationinthemediumdeterminedatdifferenttimepoints.AscanbeseeninFig.3,over-expressionofthemonosaccharidetransportersdidnotincreasetheethanolproductionrate.Instead,itseemedthatethanolproductionwasevenslightlydelayed,especiallyinthecaseoftheGal2-overproducingtrans-formants.

Underanaerobicconditions,therecombinantPUAstrainscannotgrowonxylosemedium(Kotter&Ciriacy,1993;datanotshown).Therefore,thegrowthratesofthetransformantsweredeterminedunderaerobicconditionsinshaken-flaskcultureswithsyn-theticmediumand2%xylose.GrowthonxyloseoftherecombinantPUAstrainswastwiceasfastasthatoftheCEN.PK-derivedstrains(doublingtimesabout13and29h,respectively).However,thegrowthratesofthetransformantsoverexpressingthexylose-transportingproteinsHxt4,Hxt7andGal2werethesameasthoseofthetransformantswiththeemptyvector.Moreover,noethanolcouldbedetectedintheculturesupernatantsof

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anyofthetransformants.Ourresultsindicatethatxyloseuptakedoesnotdeterminexyloseflux,atleastnotastheonlystepinxyloseutilization.DISCUSSION

Wehaveshownthatafterdeletionofallofthe18hexose-transportergenesfromarecombinantyeaststrainabletoutilizethepentosesugarxylose,itsabilitytotakeupandtogrowonxyloseislost.Thisis,forthefirsttime,ageneticalproofthattheyeasthexose-uptakesystemalsomediatesuptakeofxylose.Itconfirmsearlierresultsobtainedbykineticandphysiologicalanalysisofxyloseuptakeinyeastcells(Serrano&delaFuente,1974;Busturia&Lagunas,1986;Kotter&Ciriacy,1993;Meinander&Hahn-Hagerdal,1997).Afteroverexpressionofindividualhexose-transportergenesinthexyloseuptake-deficientyeaststrain,wecoulddemonstratethatatxyloseconcentrationsof2%onlyHxt4,Hxt5,Hxt7andGal2areabletotransportthesugarinsignificantamounts.Hxt7andGal2arehigh-affinityhexosetransporterswhereasHxt4andHxt5aremoderatelylowinaffinityforglucose(Reifenbergeretal.,1997;Diderichetal.,2001).WehavenottestedHxt2andHxt6.However,asHxt6isnearlyidenticaltoHxt7,andHxt2showstransportkineticssimilartoHxt4,weproposethatalsothesetransporterscanmediateuptakeofxylose.However,thelow-affinityhexosetransportersHxt1andHxt3,andalsothenormallyveryweaklyexpressedtransportersHxt8toHxt17werenotabletotransportxylose,atleastinamountssufficienttosupportgrowth.

Theresultswereobtainedafterconstitutiveoverexpres-sionofthehexose-transportergenes.Normally,thesegenesareregulatedbythepresenceandconcentrationofglucoseorgalactose(reviewedbyBoles&Hollenberg,

zcan&Johnston,1999).Whereasexpressionof1997;O

XyloseuptakeinS.cerevisiae

HXT1toHXT4requiresthepresenceofglucoseinthemedium,GAL2isonlyexpressedinthepresenceofgalactoseandabsenceofglucose.Incontrast,HXT5andHXT7arealsoexpressedintheabsenceofglucose(Schulteetal.,2000;Diderichetal.,1999,2001).Thismeansthatinrecombinantxylose-utilizingS.cerevisiaecellsduringgrowthonxylosemainlyHxt5andHxt7maycontributetotheuptakeofthesugar.

Noneoftheheterologoussugartransporterswasabletosupportgrowthonxylose.InthecaseofE.coliXylEandthepredictedA.thalianaxylose–protonsymporter,wecannotexcludethatthesetransportersarenotcorrectlytargetedtotheplasmamembraneinafunctionalform,ashasbeenobservedforseveralheterologousmembraneproteinsinyeast(Villalbaetal.,1992;Jahnetal.,2002;ourownunpublishedresults).However,C.kessleriHup1andA.thalianaStp2andStp3werefunctionallyexpressedintheplasmamembrane,astheysupportedgrowthofthehexose-transport-deficientyeaststrainonglucose.Growthofthesestrainsonglucosewassignificantlylowerthanparentalgrowth,indicatingthatthecapacityoftheheterologoustrans-porterswasverylow.Forallthreetransporters,xylose-uptakeactivityhasbeendemonstrated(Buttner&Sauer,2000).Therefore,weproposethatthexylose-uptakeactivitymediatedbyHup1,Stp2andStp3istoolowtosupportgrowthonxylose.

Overexpressionofthexylose-transportinghexosetrans-portersinaxylose-utilizingS.cerevisiaePUAstraindidnotresultinfastergrowthon2%xylosemediumunderaerobicconditions,nordiditresultinanincreaseoftheethanolproductionrateunder(semi-)anaerobiccondi-tions.Thisisincontrasttotherecentobservationthatunderanaerobicconditionsinchemostatcultureswithmineralmediumincreasingthexyloseconcentrationinthefeedenhancedthexyloseflux,suggestingthattransportlimitsflux(Eliassonetal.,2000).However,inthatstudyadifferent,CEN.PK-derivedyeaststrainwasused.Indeed,thePUAstrainthatwehaveusedhasbeenselectedforfastergrowthonxylulosemedium(Porep,1987;Kotter&Ciriacy,1993).Oneofitsnewpropertiesisahigherendogenouslevelofxylulosekinaseactivity.ThegrowthrateonxylosewasmorethantwofoldhigherforthePUA-derivedrecombinantstrainthanfortheCEN.PK-derivedstrain.Interestingly,thexylose-uptakeactivitywasalsoabouttwofoldhigherinPUAcells(datanotshown).ThiscouldmeanthatinthePUAstrainduringselectionforfastergrowthonxylulosethesugar-uptakesystemwasoneofthestepsthatwereoptimized.Unfortunately,theparentalstrainisnolongeravailabletoconfirmthisassumption.Never-theless,itseemsthatindifferentyeaststrainsdifferentfactorsmightcontributetolimitedxylosefermentation,withxyloseuptakebeingoneofthem.ACKNOWLEDGEMENTS

WethankProfessorC.P.Hollenbergforhiskindsupport.WealsothankT.Weierstall,I.Robl,W.Tanner,R.Gaber,M.ButtnerandN.Sauerfortheirkindgiftsofyeaststrainsand

plasmids.Thisworkwassupportedbyagrantfromthe

EuropeanCommission(QLK3-CT-1999-00080)toE.B.andB.H.-H.

REFERENCES

Boles,E.&Hollenberg,C.P.(1997).Themoleculargeneticsof

hexosetransportinyeasts.FEMSMicrobiolRev21,85–111.Busturia,A.&Lagunas,R.(1986).CataboliteinactivationoftheglucosetransportsysteminSaccharomycescerevisiae.JGenMicrobiol132,152–160.Buttner,M.&Sauer,N.(2000).Monosaccharidetransportersinplants:structure,functionandphysiology.BiochimBiophysActa1465,263–274.

Davis,E.O.&Henderson,P.J.(1987).ThecloningandDNAsequenceofthegenexylEforxylose-protonsymportinEscher-ichiacoliK12.JBiolChem262,13928–13932.

Diderich,J.A.,Schepper,M.,vanHoek,P.&8otherauthors(1999).GlucoseuptakekineticsandtranscriptionofHXTgenes

inchemostatculturesofSaccharomycescerevisiae.JBiolChem274,15350–15359.

Diderich,J.A.,MerijnSchuurmans,J.,VanGaalen,M.C.,Kruckeberg,A.L.&VanDam,K.(2001).Functionalanalysisofthe

hexosetransporterhomologueHXT5inSaccharomycescerevi-siae.Yeast18,1515–1524.

Eliasson,A.,Christensson,C.,Wahlbom,C.F.&Hahn-Hagerdal,B.(2000).AnaerobicxylosefermentationbyrecombinantSac-

charomycescerevisiaecarryingXYL1,XYL2,andXKS1inmineralmediumchemostatcultures.ApplEnvironMicrobiol66,3381–3386.

Hayn,M.,Steiner,W.,Klinger,R.,Steinmuller,H.,Sinner,M.&Esterbauer,H.(1993).Basicresearchandpilotplantstudiesonthe

enzymaticconversionoflignocellulosics.InBioconversionofForestandAgriculturalPlantResidues,pp.33–72.EditedbyJ.N.Saddler.Wallingford,UK:CABInternational.

Jahn,T.P.,Schulz,A.,Taipalensuu,J.&Palmgren,M.G.(2002).

Post-translationalmodificationofplantplasmamembraneH+-ATPaseasarequirementforfunctionalcomplementationofayeasttransportmutant.JBiolChem277,6353–6358.Kotter,P.&Ciriacy,M.(1993).XylosefermentationbySac-charomycescerevisiae.ApplMicrobiolBiotechnol38,776–783.Kotyk,A.(1967).Mobilityofthefreeandtheloadedmono-saccharidecarrierinSaccharomycescerevisiae.BiochimBiophysActa135,112–119.

Krampe,S.,Stamm,O.,Hollenberg,C.P.&Boles,E.(1998).

Cataboliteinactivationofthehigh-affinityhexosetransportersHxt6andHxt7ofSaccharomycescerevisiaeoccursinthevacuoleafterinternalizationbyendocytosis.FEBSLett441,343–347.Lagunas,R.(1993).SugartransportinSaccharomycescerevisiae.FEMSMicrobiolRev104,229–242.

Lagunas,R.,Dominguez,C.,Busturia,A.&Saez,M.J.(1982).

MechanismsoftheappearanceofthePasteureffectinSac-charomycescerevisiae:inactivationofsugartransportsystems.JBacteriol152,19–25.

Liang,H.&Gaber,R.F.(1996).AnovelsignaltransductionpathwayinSaccharomycescerevisiaedefinedbySnf3-regulatedexpressionofHXT6.MolBiolCell7,1953–1966.Meinander,N.Q.&Hahn-Hagerdal,B.(1997).Influenceofcosubstrateconcentrationonxyloseconversionbyrecombinant,XYL1-expressingSaccharomycescerevisiae:acomparisonofdifferentsugarsandethanolascosubstrates.ApplEnvironMicrobiol63,1959–1964.

2787

T.Hamacherandothers

Minet,M.,Dufour,M.-E.&Lacroute,F.(1992).ComplementationofSaccharomycescerevisiaeauxotrophicmutantsbyArabidopsisthalianacDNAs.PlantJ2,417–422.Mumberg,D.,Muller,R.&Funk,M.(1994).Regulatablepromo-torsofSaccharomycescerevisiae:comparisonoftranscriptionalactivityandtheiruseforheterologousexpression.NucleicAcidsRes22,5767–5768.Ozcan,S.&Johnston,M.(1999).Functionandregulationofyeasthexosetransporters.MicrobiolMolBiolRev63,554–569.

Porep,H.J.(1987).XyluloseverwertungbeiSaccharomycescer-evisiae.PhDthesis,UniversityofDusseldorf.

Reifenberger,E.,Boles,E.&Ciriacy,M.(1997).Kineticcharac-terizationofindividualhexosetransportersofSaccharomycescerevisiaeandtheirrelationtothetriggeringmechanismsofglucoserepression.EurJBiochem245,324–333.

Robl,I.,Grassl,R.,Tanner,W.&Opekarova,M.(2000).Propertiesofareconstitutedeukaryotichexose\\protonsymportersolubi-lizedbystructurallyrelatednon-ionicdetergents:specificre-quirementofphosphatidylcholineforpermeasestability.BiochimBiophysActa1463,407–418.

Sambrook,J.,Fritsch,E.F.&Maniatis,T.(1989).MolecularCloning:aLaboratoryManual,2ndedn.ColdSpringHarbor,NY:ColdSpringHarborLaboratory.

Schulte,F.,Wieczorke,R.,Hollenberg,C.P.&Boles,E.(2000).

theconstitutivehexosetransportinSaccharomycescerevisiae.MolCellBiochem5,161–171.

Tantirungkij,M.,Nakashima,N.,Seki,T.&Yoshida,T.(1993).

Constructionofxylose-assimilatingSaccharomycescerevisiae.JFermBioeng75,83–88.

Villalba,J.M.,Palmgren,M.G.,Berberian,G.E.,Ferguson,C.&Serrano,R.(1992).Functionalexpressionofplantplasma

membraneH+-ATPaseinyeastendoplasmicreticulum.JBiolChem267,12341–12349.

vonSivers,M.&Zacchi,G.(1995).Atechno-economicalcomparisonofthreeprocessesfortheproductionofethanolfrompine.BioresourTechnol51,43–52.

Walfridsson,M.,Hallborn,J.,Penttila,M.,Keranen,S.&Hahn-gerdal,B.(1995).Xylose-metabolizingSaccharomycescerevi-HasiaestrainsoverexpressingTKL1andTAL1genesencodingthepentosephosphatepathwayenzymestransketolaseandtransaldo-lase.ApplEnvironMicrobiol61,4184–4190.

Weierstall,T.,Hollenberg,C.P.&Boles,E.(1999).Cloningandcharacterizationofthreegenes(SUT1–3)encodingglucosetransportersoftheyeastPichiastipitis.MolMicrobiol31,871–883.

Wieczorke,R.,Krampe,S.,Weierstall,T.,Freidel,K.,Hollenberg,C.P.&Boles,E(1999).Concurrentknock-outofatleast20

TheHTR1geneisadominantnegativemutantalleleofMTH1andblocksSnf3-andRgt2-dependentglucosesignalinginyeast.JBacteriol182,540–542.

Serrano,R.&delaFuente,G.(1974).Regulatorypropertiesof

transportergenesisrequiredtoblockuptakeofhexosesinSaccharomycescerevisiae.FEBSLett464,123–128.

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Received29April2002;revised7June2002;accepted13June2002.

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