Yasuya Fujita, Junji Ito, Mitsuyoshi Ueda, Hideki Fukudaand Akihiko Kondo
Appl. Environ. Microbiol. 10.1128/AEM.70.2.1207-1212.2004.
2004, 70(2):1207. DOI:
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SynergisticAmorphousSaccharification,CodisplayingCelluloseandDirectFermentationtoEthanol,of
ThreebyUseTypesofofanCellulolyticEngineeredEnzyme
YeastStrain
YasuyaFujita,1JunjiIto,2MitsuyoshiUeda,3HidekiFukuda,1andAkihikoKondo2*
DivisionEngineering,ofMolecularScience,GraduateSchoolofScienceandTechnology,1andDepartmentFacultyofSyntheticofEngineering,2KyotoUniversity,ChemistryKobeYoshida,andUniversity,Biological1-1Sakyo-ku,Chemistry,Rokkodaicho,DepartmentofChemicalScienceand
Kyoto606-8501,GraduateNada-ku,3SchoolKobeJapan
ofEngineering,
657-8501,and
Received2September2003/Accepted3November2003
wasAwhole-cellbiocatalystwiththeabilitytoinducesynergisticandsequentialcellulose-degradationcharomycesconstructedcerevisiaethrough.Whencodisplayacellsurfaceofthreedisplaytypessystemofcellulolyticbasedonenzyme␣-agglutininonthewascellused,surfaceTrichodermaoftheyeastreactionreeseiSac-en-doglucanaseplayedenzymesasindividualIIandcellobiohydrolasefusionproteinsIIandAspergillusaculeatus-glucosidase1weresimultaneouslycodis-cencehighermicroscope.onthecellsurfacewasconfirmedwiththebyobservationC-terminal-halfofimmunofluorescence-labeledregionof␣-agglutinin.Codisplaycellswithofathefluores-threeonlycellobiohydrolaseendoglucanasehydrolyticactivityAyeaststraincodisplayingendoglucanaseIIandcellobiohydrolaseIIshowedsignificantlyaIIII,enabledandwithitsamorphouscellulose(phosphoricacid-swollencellulose)thanonedisplayingthemainyeastproductstrainwastodirectlycellobiose;producecodisplayethanoloffrom-glucosidasetheamorphous1,endoglucanasecelluloseII,(whichandperyeastconsumed)literstrainfromcodisplayingandwas100.45gperg/g,liter-glucosidasewhichwithincorresponds40h.1TheandendoglucanaseIIcouldnot),withayieldofapproximately3gtoyield88.5%(inofgramsthetheoreticalofethanolyield.producedThisindicatespergramthatofcarbohydratesimultaneousplishedsynergisticusingasaccharificationyeaststraincodisplayingandfermentationthethreeofcellulolyticamorphousenzymes.
cellulosetoethanolcanbeefficientlyaccom-Biomassistheearth’smostattractivealternativeamongfuelextracellularlysecretesthreetypesofcellulolyticenzyme,in-sourcesandmostsustainableenergyresourceandisrepro-cludingfiveendoglucanases(EG[EC3.2.1.4])(17,19–22),twoducedbythebioconversionofcarbondioxide.Ethanolpro-cellobiohydrolases(CBH[EC3.2.1.91])(9,30),andtwo-glu-ducedfrombiomassistodaythemostwidelyusedbiofuelwhencosidases(BGL[EC3.2.1.21])(3).Endoglucanasesactran-blendedwithgasoline(e.g.,E10[gasolinecontaining10%eth-domlyagainsttheamorphousregionofthecellulosechaintoanol]).Asthecarbondioxidereleasedbycombustionisrecy-producereducingandnonreducingendsforcellobiohydro-cledintobiomass,theuseofbiofuelscansignificantlyreducelases,whichproducecellobiosefromreducingornonreducingtheaccumulationofgreenhousegas.Ofthebiomassmaterials,endsofcrystallinecellulose.Cellulosechainsarethuseffi-cellulose,amajorcomponentofthecellwallofplants,isthecientlydegradedtosolublecellobioseandcellooligosacchar-mostabundantandrenewablecarbohydrate.Inrecentyears,itidesbytheendo-exosynergismofEGandCBH(9,13,29,36).hasbeenproposedthatwastecellulosicbiomasscouldbeusedInthelaststepofenzymaticcellulosedegradation,cellooligo-asacheapandreadilyavailablesugartoreplacestarchyma-saccharidesarehydrolyzedtoglucoseby-glucosidase.Inad-terialsinfermentation.Manyresearchershavepreviouslytriedditiontoendo-exosynergism,exo-exosynergismbetweentwotodevelopanefficientandinexpensiveprocessforethanolcellobiohydrolaseshasalsobeenreported(9,14,28,29).productionfromsuchwastebyusingrecombinantbacteriaandSuchcellulolyticenzymeshavebeenexpressedinbacteriayeast(e.g.,Saccharomycescerevisiae)(1,2,10,12,37),butso(34,38)andyeast(4,7,15,31,32)asawayofreducingthecostfarwithoutsuccess.Aprocessofthiskindisneededtosolveofcellulaseproductionandotherpretreatmentsintheprocessenvironmentalproblemssuchasglobalwarmingandtocon-ofethanolproductionfromcellulosicmaterials(12,26,37).structasocietyindependentoffossilfuels.
Recentlysomeresearchershavedevelopedethanologenicbac-TheanaerobicbacteriaClostridiumthermocellumandClos-teria(8,34,38)andyeast(4,7)thatcanproduceethanolfromtridiumcellulovoransandthefilamentousfungusTrichodermacellulosicmaterials.TherecombinantKlebsiellaoxytocaSZ21reeseiarewellknownasstronglycellulolyticandxylanolyticdevelopedbyZhouetal.wasabletodirectlyproduceethanolmicroorganisms.C.thermocellumandC.cellulovoransproducefromamorphouscellulose,althoughwithinsufficientethanolacellulosomecomplexconsistingofcellulaseandhemicellu-yield(38).Whenusingotherrecombinantethanologenicbac-laseorganizedonthecellsurface(5,25);T.reesei,meanwhile,
teriaoryeasttofermentcellulose,additionofcommercialcellulaseisnecessaryforethanolproduction.
Previously,wereporteddirectandefficientethanolproduc-Science*Correspondingauthor.Mailingaddress:Departmentoftionfromthesolublecellulosicpolysaccharidebarley-glucanRokkodaicho,andEngineering,withayeaststraincodisplayingonthecellsurfaceT.reesei6196.Fax:81-78-803-6196.Nada-ku,FacultyKobeE-mail:657-8501,ofEngineering,KobeUniversity,Chemical1-1kondo@cx.kobe-u.ac.jp.
Japan.Phone:81-78-803-EGII(glycosylhydrolasefamily5)andAspergillusaculeatus
1207
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APPL.ENVIRON.MICROBIOL.
TABLE1.Characteristicsofstrainsandplasmidsusedinthisstudy
Strainorplasmid
Relevantfeature(s)aReferenceorsource
Strains
S.MT8-1
cerevisiaeyeaststrainsMT8-1/pCAS1MAT27MT8-1/pFCBH2w3NoDisplaydisplayaadehis3(controlleu2strain)trp1ura324
MT8-1/pEG23u31H6PresentMT8-1/pBG211
DisplayofCBHII7studyMT8-1/pEG23u31H6/pFCBH2w3Displayof15
MT8-1/pBG211/pEG23u31H6
DisplayofEGIIDisplayofBGL1
Present7
studyBacterialMT8-1/pBG211/pEG23u31H6/pFCBH2w3DisplayofEGIIofBGL1andCBHIIBGL1,andEGII,EGII
andCBHII
PresentstudyE.coliNovaBluestrainendA1FЈ{proABhsdR17ϩ(lacIrK12ϪqZm⌬K12ϩM15::Tn)supE4410(Tetthi-1r)}gyrA96relA1lacrecA1/Novagen
PlasmidspCAS1pFCBH2w3TRP124
pEG23u31H6TRP1NoURA3Surfaceexpressionexpression(controlofT.plasmid)
reeseicellobiohydrolasegene(CBHII)PresentpBG211
7studyHIS3SurfaceSurfaceexpressionexpressionofofA.T.aculeatusreeseiendoglucanase-glucosidasegenegene(EGII)(BGL1)
15
ageneEachundergenethewascontrolexpressedoftheasglyceraldehydeafusionprotein3-phosphatewiththesecretiondehydrogenasesignalsequence(GAPDH)ofR.promoter.
oryzaeglucoamylasegeneandtheC-terminalhalfregionofthe␣-agglutininBGL1(family3)(7).Inthepresentstudy,weattemptedsi-Enzymeassay.Endoglucanaseandcellobiohydrolaseactivitiesweredeter-multaneousandsynergisticsaccharificationandfermentationminedbyhydrolysisof1gofamorphouscelluloseperliterin50mMsodiumofamorphouscellulosetoethanolwiththeuseofonlyare-acetatebuffer(pH5.0)at30°C.Phosphoricacid-swollencellulosewaspreparedcombinantyeaststraincodisplayingthreetypesofcellulolyticfromAvicelPH-101(FlukaChemieGmbH,Buchs,Switzerland)asamorphousenzyme,namely,T.reeseiEGIIandCBHII(family6)andcellulose(33).AfterprecultivationinSDmediumfor24handaerobiccultiva-tioninSDCmediumfor72hat30°C,cellswerecollectedbycentrifugationatA.aculeatusBGL1.
6,000ϫgfor10minat4°C,washedwithdistilledwatertwice,andresuspendedinareactionmixturewiththeopticaldensityat600nm(ODsupernatantwasseparatedby600)adjustedto10.Afterahydrolysisreaction,thecentrifugationforMATERIALSANDMETHODS
3minat20,000ϫgand4°CandusedformeasurementoftheamountofStrainsandmedia.Thebacterialandyeaststrainsusedaresummarizedinreducingsugar,totalsolublesugar,andinsolublesugarandforhigh-performanceTable1.EscherichiacoliwasgrowninLuria-Bertanimedium(10goftryptoneliquidchromatography(HPLC)analysisofhydrolysisproductsasdescribedperliter,5gofyeastextractperliter,5gofsodiumchlorideperliter)containingbelow.Theamountofreducingsugarandtotalsolublesugarreleasedfrom100gofampicillinperml.Followingprecultivationinsyntheticmedium(SDinsolublesubstrateandinsolublesugarwasmeasuredusingaSomogyi-Nelsonmedium;6.7gofyeastnitrogenbasewithoutaminoacid[DifcoLaboratories,method(35)andaphenol-sulfuricacidmethod(6)todeterminethenumberofDetroit,Mich.]perliterwithappropriatesupplementscontaining20gofglucoseglucoseequivalents.
perliter),yeastcellswereaerobicallycultivatedat30°CinSDmediumcontain--Glucosidaseactivitywasmeasuredusingp-nitrophenyl--D-glucopyranosideing20gofCasaminoAcids(Difco)perliter(SDCmedium).
(NacalaiTesque,Inc.,Kyoto,Japan)asthesubstrateaccordingtoapreviouslyConstructionofplasmids.TheplasmidpFCBH2w3forcell-surfacedisplayofdescribedmethod(7)butwithanOD600of0.1inthereactionmixture.
theT.reeseiCBHIIgenewasconstructedasfollows:withfirst-strandcDNADeterminationofhydrolysisproducts.TheendproductsreleasedfromthepreparedfromT.reeseiQM9414asthetemplate,a1.39-kbpSacII-BglIIDNAphosphoric-acidswollencellulosewereanalyzedaccordingtoapreviouslyde-fragmentofthematureregionoftheT.reeseiCBHIIgenefusedwiththegenescribedmethod(7).
encodingtheFLAGpeptidetagattheNterminuswaspreparedbyPCRasFermentation.AfterprecultivationinSDmediumfor24h,yeastcellsweredescribedpreviously(18)withthetwoprimers5Ј-GAGCCGCGGGAGACTAaerobicallycultivatedfor72hat30°CinSDCmedium,collectedbycentrifuga-CAAGGATGACGATGACAAGCAAGCTTGCTCAAGCGTCTGGGGCC-3Јtionfor10minat6,000ϫgand4°C,andwashedwithdistilledwatertwice.Theand5Ј-CGAACGAGATCTAGGAACGATGGGTTTGCGTTTGTGAGAAGcellpelletsweretheninoculatedintofermentationmedium(6.7gofyeastC-3Ј.TheDNAfragmentwasdigestedbySacIIandBglIIandintroducedintothenitrogenbasewithoutaminoacid[Difco]perliterwithappropriatesupplements,SacII-BglIIsiteofthecell-surfaceexpressionplasmidpCAS1(24)containingthe20gofCasaminoAcidsperliter,10gofnonsterilizedphosphoricacid-swollengenesencodingthesecretion-signalsequenceoftheglucoamylasegenefromcelluloseperliterasthesolecarbonsource),andethanolfermentationwasRhizopusoryzaeandthe3Ј-halfregionofthe␣-agglutiningene(thegeneencod-anaerobicallyperformedat30°CwiththeOD600ofthefermentationmedium
ingtheC-terminal320aminoacidresiduesand446bpofthe3Ј-flankingregion)(11).TheresultingplasmidwasnamedpFCBH2w3(Fig.1).TheplasmidsusedaresummarizedinTable1.
Yeasttransformation.TransformationoftheexpressionplasmidsintoS.cere-visiaeMT8-1wascarriedoutbyalithiumacetatemethodusingaYEASTMAKERyeast-transformationsystem(ClontechLaboratories,Inc.,PaloAlto,Calif.).ThetransformantsconstructedandusedinthestudyaresummarizedinTable1.Immunofluorescencelabelingofcells.Immunofluorescencelabelingofcellswascarriedoutaccordingtoamethoddescribedpreviously(7).Astheprimaryantibody,mouseanti-RGS(His)Calif.),rabbitanti-FLAGIgG(Sigma4immunoglobulinG(IgG)(Qiagen,Valencia,ChemicalCo.,St.Louis,Mo.)andrabbitanti-A.aculeatusBGL1IgGwereusedatdilutionratesof1:100,1:100,and1:500withtheRGS(His)BGL1–␣-agglutininfusion6–EGII–␣-agglutinin,FLAGϪCBHII–␣-agglutinin,andproteins,respectively.Assecondaryantibody,goatanti-mouseIgGconjugatedwithAlexaFluor488andgoatanti-rabbitIgGcon-jugatedwithAlexaFluor546(MolecularProbes,Inc.,Eugene,Oreg.)wereusedyeastFIG.1.Expressionplasmidfordisplayofatadilutionrateof1:250.
oryzaecellglucoamylasesurface(pFCBH2w3).T.reeseiCBHIIonthegene.
s.s.,secretionsignalsequenceofR.Downloaded from http://aem.asm.org/ on November 21, 2011 by guestVOL.70,2004DIRECTFERMENTATIONTOETHANOLOFAMORPHOUSCELLULOSE1209
rescenceFIG.2.1/pBG211micrographsImmunofluorescence(columns2,labeling3,and5)ofoftransformants:S.cerevisiaeNomarskidifferentialinterferencemicrographs(columns1and4)andimmunofluo-Cellsanti-RGS(His)were(D),labeledMT8-1/pEG23u31H6/pFCBH2w3MT8-1/pCAS1(control)(A),MT8-1/pFCBH2w3(B),MT8-1/pEG23u31H6(C),MT8-withrabbitanti-FLAGIgGantibody(E),MT8-1/pBG211/pEG23u31H6andgoatanti-rabbitIgGconjugated(F),andMT8-1/pBG211/pEG23u31H6/pFCBH2w3withAlexaFluor546(column2),withmouse(G).andgoatanti-rabbit4antibodyIgGandconjugatedgoatanti-mousewithAlexaIgGFluorconjugated546(columnwithAlexa5).
Fluor488(column3),andwithrabbitanti-A.aculeatusBGL1antibodyadjustedto50.Ethanol,totalsugar,andglucoseconcentrationsweremeasuredusinggaschromatography,aphenol-sulfuricacidmethod(6)asaglucoseequiv-constructedayeaststraincodisplayingthreetypesofcellulo-alent,andaGlucoseCIItest(WakoPureChemicalIndustries,Ltd.,Osaka,lyticenzymeonthecellsurfacesimultaneously.TheexpressionJapan),respectively.Thegaschromatograph(modelGC-8A,Shimadzu,Kyoto,plasmidspBG211,pEG23u31H6,andpFCBH2w3(Fig.1)(forJapan)(fittedwithaflameionizationdetector)wasoperatedunderthefollowingdisplayoftheBGL1–␣-agglutinin,RGS(His)6–EGII–␣-agglu-conditions:glasscolumn(2.0mby3.2mm)packedwithThermon-3000(Shi-madzu);temperatureofcolumn,injector,anddetector,180°C;nitrogencarriertinin,andFLAG–CBHII–␣-agglutininfusiongenes)weregasflowrate,25ml/min.Totalsugarconcentrationsweredeterminedbysub-transformedorcotransformedintotheyeastS.cerevisiaeMT8-tractingtheyeastcell-derivedsugarfromtheculturemediumcontainingthe1strainsimultaneously,andtheresultanttransformantswereyeastcellsandcellulose.
designatedstrainsMT8-1/pFCBH2w3,MT8-1/pEG23u31H6,MT8-1/pBG211,MT8-1/pEG23u31H6/pFCBH2w3,MT8-1/RESULTS
pBG211/pEG23u31H6,andMT8-1/pBG211/pEG23u31H6/pFCBH2w3(Table1).
CodisplayofthreetypesofcellulolyticenzymeontheyeastToconfirmcodisplayofBGL1,EGII,andCBHIIonthecellsurface.Tofermentamorphouscellulosetoethanol,we
yeastcellsurface,immunofluorescencelabelingofthecells
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aerobiccultivationofcellsinSDCmediumfor72hat30°C.IntheyeaststraindisplayingCBHII(MT8-1/pFCBH2w3),reduc-ingsugarwasnotdetectedbyaSomogyi-Nelsonmethod(Fig.3)andonlyalittlesugarwasdetectedbyaphenol-sulfuricacidmethod(datanotshown).However,theyeaststrainco-displayingEGIIandCBHII(MT8-1/pEG23u31H6/pFCBH2w3)showedmuchhigheractivitythantheyeaststraindisplayingEGIIalone(MT8-1/pEG23u31H6)(Fig.3),andasignificantreductionintheinsolublecelluloseinthereactionmixturewasobserved(Fig.4).ThisresultsuggeststhatCBHIIisactiveontheyeastcellsurfaceandplaysaveryimportantroleinamor-phouscellulosedegradation.TheactivitiesofEGIIandCBHIItaggedwithRGS(His)loseFIG.3.Timecourseofsynergistichydrolysis6andFLAGwerenearlyequaltothosepFCBH2w3byS.cerevisiaeMT8-1/pCAS1(control)(openofamorphouscellu-ofnontaggedEGIIandCBHII(datanotshown).
MT8-1/pEG23u31H6/pFCBH2w3(opentriangle),MT8-1/pEG23u31H6square),MT8-1/HPLCanalysiswascarriedouttoexaminethehydrolysispEG23u31H6/pFCBH2w3(closedcircle),(closedtriangle),productsreleasedfromamorphouscellulosebystrainsMT8-averagesoffiveindependent(openexperiments.
circle).ThedataandpointsMT8-1/pBG211/representthe1/pEG23u31H6andMT8-1/pEG23u31H6/pFCBH2w3.Sam-plessubjectedtoahydrolysisreactionfor72hwereusedforHPLCanalysis.Cellobioseandcellotrioseweredetectedasthewascarriedoutusingrabbitanti-FLAGantibody,mouseanti-mainproductsofstrainMT8-1/pEG23u31H6,whilealargeRGS(His)4antibody,andrabbitanti-A.aculeatusBGL1anti-amountofcellobiosewasdetectedasthemainproductofbodyastheprimaryantibody.AsshowninFig.2,theredstrainMT8-1/pEG23u31H6/pFCBH2w3(datanotshown).fluorescenceofAlexaFluor546-conjugatedgoatanti-rabbitOnlyasmallamountofcellobiosewasdetectedasthemainIgGwasobservedforstrainsMT8-1/pFCBH2w3,MT8-1/productofstrainMT8-1/pFCBH2w3,andnohydrolysisprod-pEG23u31H6/pFCBH2w3,andMT8-1/pBG211/pEG23u31H6/uctwasobservedinthereactionmixtureofthecontrolstrainpFCBH2w3(Fig.2,column2);thatofAlexaFluor546-con-MT8-1/pCAS1(datanotshown).
jugatedanti-rabbitIgGwasobservedforstrainsMT8-1/Degradationofamorphouscellulosebyayeaststraincodis-pBG211,MT8-1/pBG211/pEG23u31H6,andMT8-1/pBG211/playingthreetypesofcellulolyticenzyme.TheabilityofthepEG23u31H6/pFCBH2w3(Fig.2,column5);andthegreenyeaststrainMT8-1/pBG211/pEG23u31H6/pFCBH2w3tohy-fluorescenceofAlexaFluor488-conjugatedgoatanti-mousedrolyzeamorphouscellulosewasexaminedusingthesameIgGwasobservedforstrainsMT8-1/pEG23u31H6,MT8-1/methodasdescribedabove.InspiteofcodisplayofthreetypespEG23u31H6/pFCBH2w3,MT8-1/pBG211/pEG23u31H6,ofenzymeonthecellsurface,noreducingsugarwasdetectedandMT8-1/pBG211/pEG23u31H6/pFCBH2w3(Fig.2,col-inthereactionmixtureofthestrain(Fig.3).However,theumn3).TheseresultsconfirmsingledisplayandcodisplayofresidualamountofinsolublesugarinthereactionmixtureofBGL1,EGII,andCBHII.Specifically,threetypesofcellu-thisstrainwassmallerthanthatseenwithyeaststrainMT8-1/lase(namely,BGL1,EGII,andCBHII)weresuccessfullypEG23u31H6/pFCBH2w3(Fig.4).Thereducingsugarpro-codisplayedonthecellsurfaceofstrainMT8-1/pBG211/ducedbythehydrolysisofamorphouscellulosewasthusim-pEG23u31H6/pFCBH2w3.
mediatelytakenupbytheyeastcells.
Degradationofamorphouscellulosebyyeaststraincodis-Directfermentationofamorphouscellulosetoethanol.Di-playingEGIIandCBHII.Toexaminetheeffectofcodisplayofrectproductionofethanolfromamorphouscellulosewasper-EGIIandCBHIIonhydrolysisactivity,ahydrolysisexperi-formedusingtheyeaststraincodisplayingBGL1,EGII,andmentwasperformedusingstrainsMT8-1/pFCBH2w3,MT8-1/CBHII(MT8-1/pBG211/pEG23u31H6/pFCBH2w3).Fermen-pEG23u31H6,andMT8-1/pEG23u31H6/pFCBH2w3after
tationwasanaerobicallyperformedat30°Cinfermentation
S.FIG.4.Graph(a)andpBG211/pEG23u31H6/pFCBH2w3cerevisiaeMT8-1/pCAS1photograph(control)(b)representingtheresidualamountofcelluloseinhydrolysisreactionmixtureafter72hofreactionwith(E).(A),TheMT8-1/pFCBH2w3datarepresentthe(B),averagesMT8-1/pEG23u31H6ofthreeindependent(C),MT8-1/pEG23u31H6/pFCBH2w3experiments.
(D),andMT8-1/Downloaded from http://aem.asm.org/ on November 21, 2011 by guestVOL.70,2004DIRECTFERMENTATIONTOETHANOLOFAMORPHOUSCELLULOSE1211
celluloseFIG.5.pEG23u31H6/pFCBH2w3.asTimethecoursesolecarbonofproductionofethanolfromamorphousar;Symbols:sourcetriangle,withethanol;strainMT8-1/pBG211/circle,averagessquare,ofglucosesevenindependentinculturebroth.experiments.
Thedatapointsrepresenttotalsug-themediumcontaining10gofphosphoricacid-swollencelluloseperliterasthesolecarbonsourceandusingyeastcells(ODϭat50)30°C.subjectedEthanoltowasaerobicnotproducedcultivationfrominSDCamorphousmediumcellulosefor72600hwhentheyeaststraincodisplayingEGIIandBGL1(MT8-1/pBG211/pEG23u31H6)wasused(datanotshown),butwithstrainMT8-1/pBG211/pEG23u31H6/pFCBH2w3,ethanolwasefficientlyproducedandthemaximumconcentrationofaround2.9g/literwasreachedwithin40hofcommencingfermentation(Fig.5).Whenfermentationwasstarted,theethanolconcentrationincreasedandthetotalsugarconcen-trationdecreasedwithoutatimelag.Glucosewasnotdetectedintheculturebrothduringfermentation.Theyield(ingramsofethanolproducedpergramofsugarconsumed)was0.45g/g,whichcorrespondsto88.5%ofthetheoreticalyieldfor40hoffermentation.
DISCUSSION
Toreducethecostofethanolproductionfromcellulosicbiomass,recombinantmicroorganismswiththeabilitytofer-mentcellulosehavebeendevelopedbymanyresearchers(4,8,34,38).Thesewhole-cellbiocatalystswiththeabilitytode-gradecellulosehaveseveraladvantages:conversionofcello-bioseandglucose,whichinhibitcellulaseand-glucosidaseactivities;lowersterilizationrequirements,asglucoseisimme-diatelytakenupbycellsandethanolisproduced;andasinglereactor.Inthefermentationofcellulosewithyeastcells,cel-lulosemustfirstbedegradedtoglucose,asyeastcellsarenotabletoutilizecelluloseorcellooligosaccharides.Enzymaticdegradationofcelluloserequiresthreetypesofcellulolyticenzyme(endoglucanase,cellobiohydrolase,and-glucosidase),andasynergisticeffectbetweenendoglucanaseandcellobio-hydrolaseisessentialforefficienthydrolysisofcellulose(9,13,29,36).Wethereforeconstructedayeaststraininwhichthethreetypesofcellulasenecessarytoefficientlydegradecellu-losearecodisplayedonthecellsurface,with␣-agglutininasananchor(Fig.2).Whilecodisplayoftwoproteinsthroughtheuseofacellsurfacedisplaysystembasedon␣-agglutininhasbeenreported(7,15,16,23),thereisnoreportofcodisplayof
threeormoreproteins.AsdemonstratedbytheresultshowninFig.5,wesucceededindirectlyproducingethanolfromamorphouscellulosewithouttheadditionofcellulaseenzymesbydevelopingayeaststraincodisplayingT.reeseiEGIIandCBHIIandA.aculeatusBGL1onthecellsurface.Thisisthefirstreportofcodisplayontheyeastcellsurfaceofthreefunctionalproteins.
IthaspreviouslybeenreportedthatEGandCBHactsyn-ergisticallyoncellulosechainstoproducesolublecellooligo-saccharidesandthatCBHisthekeycellulaseincellulosehydrolysis(9,13,29,36).Althoughsurface-displayedCBHIIhadonlyalittleactivitywithrespecttoamorphouscellulose,theyeaststraincodisplayingEGIIandCBHIIshowedsignifi-cantlyhigheractivitythantheyeaststraindisplayingonlyEGIIandproducedcellobioseasthemainproduct(Fig.3).ThisresultindicatesthatCBHIIplaysaveryimportantroleincellulosedegradationandthatsynergismbetweenEGIIandCBHIIissuccessfullyinducedontheyeastcellsurface.
InadditiontocodisplayofEGIIandCBHII,BGL1wassimultaneouslycodisplayedtoproduceethanolfromamor-phouscellulose.The-glucosidaseactivityoftheyeaststraincodisplayingEGII,CBHII,andBGL1(164.5U/g[dryweight]ofcells)wasapproximately2.0and1.6timeshigher,respec-tively,thanthatofthestraindisplayingBGL1andthestraincodisplayingBGL1andEGII(80.8and129.8U/g[dryweight]ofcells).Interestingly,flowcytometricanalysisoffluorescence-labeledyeastcellsconfirmedthatthetotalproteinofyeaststrainsdisplayingtwoorthreeenzymeswasgreaterthanthatofsingle-displaystrains,asmeasuredbymeanfluorescenceintensity(datanotshown).ThisresultindicatedthatthesumofthenumberofBGL1,EGII,andCBHIImoleculesinacodisplaystrainislargerthanthatseeninasingle-displaystrain.
Inthehydrolysisexperiment,althoughthereducingsugarproducedbytheyeaststraincodisplayingEGII,CBHII,andBGL1wasnotdetectedinthesupernatantofthereactionmixture(Fig.3),adecreaseininsolublecellulosewasobserved(Fig.4).Thisisbecausethecellobioseproducedbythesyner-gisticreactionofEGIIandCBHIIisfurtherconvertedtoglucosebyBGL1andtheglucoseisimmediatelytakenupbycells.TheyeaststrainMT8-1/pBG211/pEG23u31H6/pFCBH2w3wasthusabletodirectlyproduceethanolfromamorphouscellulose(Fig.5).Ontheotherhand,theyeaststraincodis-playingBGL1andEGIIcouldnotfermentamorphouscellu-losetoethanol.Throughcodisplayofthethreetypesofcellu-lolyticenzyme,amorphouscellulosewassynergisticallyandsequentiallyhydrolyzedtoglucoseontheyeastcellsurfaceandimmediatelyconvertedtoethanolbyintracellularmetabolicenzymes.TheseresultsindicatethatcodisplayofCBHIIisveryeffectiveininducinghydrolysisanddirectfermentationofamorphouscellulose.Theethanolproductionwasalsoeffi-cient,astherewasnotimelaginthedecreaseininsolublecelluloseandtheethanolyieldwashigh(88.5%oftheoreticalyield).Moreover,glucosewasnotdetectedinthefermentationmedium,whichisadvantageoustopreventorminimizecon-tamination.
Asdescribedabove,efficientdirectfermentationofamor-phouscellulosetoethanolwasachievedbydevelopingayeaststraincodisplayingthreetypesofcellulolyticenzyme.Codis-playofCBHIIsignificantlyaffectedcellulosehydrolysis,sug-
Downloaded from http://aem.asm.org/ on November 21, 2011 by guest1212FUJITAETAL.gestingthatcombinationofcellulaseswithvariousfunctionsiseffectiveinproducingefficientdegradation.Furtherworkisneededtoanalyzethesynergisticreactionofthecellulasescodisplayedonthecellsurfaceandtoconstructayeastwhole-cellbiocatalystwithanimprovedabilitytocatalyzecellulosedegradationandfermentation.
ACKNOWLEDGMENTS
gaokaWethankYasushiMorikawa,CBHIIUniversitylegeandMotooofArai,Technology,DepartmentDepartmentforprovidingoftheBioengineering,cDNAofT.reeseiNa-rabbitofAgriculture,UniversityofOsakaofAgriculturalPrefecture,forChemistry,providingCol-theTechnologyThisanti-BGL1workDevelopmentwasantiserum.
alsofinancedOrganizationbythe(NEDO),NewEnergyTokyo,andJapan.
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