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architecture.Thus,todigestlignocelluloses,manyfungi secreteacellulolyticenzymecocktail.Forexample,the brown-rotfungus Trichodermareesei simultaneously expressesthreekindsofendo- β -1,4-glucanases(EG)and twokindsofcellobiohydrolases(CBH)whentreatedwith abarleystrawsubstrate[7].Therumenfungus Neocalli- mastixpatriciarum W5isabletogrowonricestrawpow- der,byexpressinganenzymecocktailwithhighlyefficient β -glucosidases(BGL)[8,9]. Aspergillusniger canexpress EGandBGLenzymes,anditsBGLhasbeenpurified andmanufacturedasthecommercialNovozyme188[10,11]. Moreover,themodelcellulolytic Neurosporacrassa has sevenmajorfacilitatorsuperfamily(MFS)sugartranspor- ters,andthecellodextrintransportors(CDT-1andCDT-2) havebeenshowntofacilitatetransportofcellobiose,cello- triose,andcellotetraoseintothecytoplasm[12,13].But, thesecellulolyticconsumersarenotsuitableforthefer- mentationindustry,becauseofalowgrowthrate,there- quirementofaspecificculturemedium,therequirementof aspecialinducingcondition,ortheinabilitytoproducea serviceablebiofuelproduct[14].However,thesecellulolytic enzymesystemsmaybeusedtoengineerayeasthostfor thecellulosicethanolindustry. Inprinciple,cellulosicpolysaccharidescanbehydro- lyzedwhenthreetypesofcellulase(EG,CBH,andBGL) areused[15-17].However,acellulolyticfungususually possessesdifferentcellulasesofthesametype,suggest- ingthatmultipleenzymesaremoreefficientthansingle enzyme.Indeed,thecommercialCelluclast1.5L,which containsmainlyEGandCBHactivities,achievesasyner- gismwiththemixtureofCBHI,CBHII,EGI,andEGII of T.reesei [7].Thesynergisticeffectofdifferentcellulases onlignocellulosicsubstratehydrolysis,suchascellulases from Penicilliumechinulatum and T.reesei, isknownin SSFapplications[18,19].ThecommercialNovozyme188 isoneofthemostcommonlyusedenzymeforpromotion ofasynergisticeffectwith Trichoderma cellulases,anda cocktailformulationsupplementedwiththisBGLcaneffi- cientlyproducefermentableglucose[11,20,21].Although aconceptofdesigningahighlyefficientcellulasemixture forhydrolysisofcellulosehasbeenreported[21],ade- signerhostforhighlyefficientcellulasecocktailproduc- tionhasnotyetbeenachieved. ToachieveCBP,researchhasbeenpursuedinanum- berofhostorganismsincluding Escherichiacoli , Bacillus subtilis ,andespecially S.cerevisiae [2].Severaldifferent approacheshavebeenappliedtoselectorengineerfer- mentableyeastsforcellulosicmaterialutilization,such asscreeningayeastforutilizingcellobiose[22],orfor transformationofcellulasegenes[23,24],highlevelsecre- tionofcellobiohydrolases[25],andtransformationofpoly- saccharidetransportergenes[13].However,agoodCBP hostremainstobefoundorconstructed[2].Recently , K.marxianus wasproposedasapotentialyeasthostto developaCBPforconvertingcellulosicmaterialsinto bioethanol[4,24,26].Wehaverecentlyisolatedakefiryeast, K.marxianus KY3,whichisefficientinfermentingethanol fromhexoseandpentosesugarsandhasmanyother advantages,includingthermo-tolerance,ahighgrowth rate,broadgrowthtemperatureandpHranges,efficient secretionofheterologouslyexpressedproteinsanda broadsubstratespectrum[23].Moreover,asynthetic biologytool,calledthepromoter-basedgeneassembly andsimultaneousoverexpression(PGASO)technique, wasdevelopedandusedtoinsertafive-genecassette, includingthreecellulasegenes(CBH,EGandBGL),ina predesignedorderintothegenomeofKY3,resultingin arecombinantKR5strain[24].KR5coulddirectlycon- vertbeta-glycantoethanol,butcouldnotutilizecrystal avicel,whichhasamorecompactcellulosestructure thanbeta-glycan. SinceaCBPhostshouldpossessthecapabilityforsim- ultaneoussaccharificationandfermentation,KR5needsto beimproved.Forthispurpose,weneedtoknowwhich cellulasegenestotransformintothehostgenomebecause itisdifficulttotransformmanygenesintoagenome.In thisstudy,wehavedevelopedanapproachtoselectgenes toproduceanefficientcellulasecocktail.Usingthisstrat- egy,wehavechosenfivecellulasegenesfromprevious studiestoformanefficientcocktail.Inaddition,wealso choseacellodextrintransportorgene.Wethentrans- formedthesesixgenesandaselectionmarkergeneinto theKY3genomeusingPGASO.Theresultingstrain,KR7, wastestedforethanolconversionfromthecrystallinesub- strate,avicel,andthecelluloseconsumptionpathwayis showninFigure1A.Ourstudyshowsthatexpressing enzymesbyourmethodin K.marxianus KY3hasthe potentialtocreateCBPstrains. Results Anenzymecocktailformulationassayforselecting synergisticenzymes Inourpreviousstudy,wehaveengineeredarecombinant K.marxianus KY3strain,calledKR5,thatpossessesthe cbhI , egIII ,and npabgs genesandcansecretetheCBH,EG andBGLenzymessimultaneously[24].AlthoughKR5can growonmediawithcellodextrins,suchascellobioseand beta-glycan,itdoesnothavetheabilitytoutilizemore complexcellulosesubstrates,suchasfilterpaperandavi- cel.AsFPA(thefilterpaperactivity)isawidelyaccepted assayfortheoverallcellulaseactivity,weconductedan FPAassay,usingthesupernatantoftheKR5cultureasthe sourceofcrudeenzymes.TheFPAdataindicatedonlya 110U/goverallcellulaseactivityinKR5(Figure1B). ToimprovetheenzymeactivityofKR5,wedeveloped anenzymecocktailstrategytoinvestigatethesynergistic actionswiththecrudeenzymesofKR5.Manydifferent glycosylhydrolase(GH)familycellulases,includingEglA Chang etal.BiotechnologyforBiofuels 2013, 6 :19Page2of13 http://www.biotechnologyforbiofuels.com/content/6/1/19
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