New inducible promoter for gene expression and synthetic ...

... metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in ... Skiptomaincontent Advertisement SearchallBMCarticles Search NewinduciblepromoterforgeneexpressionandsyntheticbiologyinYarrowialipolytica DownloadPDF DownloadPDF Research OpenAccess Published:15August2017 NewinduciblepromoterforgeneexpressionandsyntheticbiologyinYarrowialipolytica MarionTrassaert1 na1,MarieVandermies2 na1,FrédericCarly1,3,OliviaDenies2,StéphaneThomas1,PatrickFickers2&Jean-MarcNicaud  ORCID:orcid.org/0000-0002-6679-972X1,4  MicrobialCellFactories volume 16,Article number: 141(2017) Citethisarticle 7281Accesses 55Citations 8Altmetric Metricsdetails AbstractBackgroundTheoleaginousyeastYarrowialipolyticaisincreasinglyusedasalternativecellfactoryfortheproductionofrecombinantproteins.Atpresent,severalpromoterswithdifferentstrengthshavebeendevelopedbasedeitherontheconstitutivepTEFpromoteroronoleicacidinduciblepromoterssuchaspPOX2andpLIP2.Althoughthesepromotersarehighlyefficient,thereisstillalackofversatileinduciblepromotersforgeneexpressioninY.lipolytica. ResultsWehaveisolatedandcharacterizedthepromoteroftheEYK1genecodingforanerythrulosekinase.pEYK1inductionwasfoundtobeimpairedinmediasupplementedwithglucoseandglycerol,whilethepresenceoferythritolanderythrulosestronglyincreasedthepromoterinductionlevel.PromotercharacterizationandmutagenesisallowedtheidentificationoftheupstreamactivatingsequenceUAS1EYK1.NewhybridpromoterscontainingtandemrepeatsofeitherUAS1XPR2orUAS1EYK1weredevelopedshowinghigherexpressionlevelsthanthenativepEYK1promoter.Furthermore,promoterstrengthwasimprovedinastraincarryingadeletionintheEYK1gene,allowingthustheutilizationoferythritolanderythruloseasfreeinducer.ConclusionsNoveltunableandregulatedpromoterswithapplicationsinthefieldofheterologousproteinproduction,metabolicengineering,andsyntheticbiologyhavebeendeveloped,thusfillingthegapoftheabsenceofversatileinduciblepromoterintheyeastY.lipolytica. BackgroundInterestinnon-conventionalyeastssuchasPichiapastoris,Hansenulapolymorpha(Pichiaangusta),andYarrowialipolyticaascellfactoriesfortheproductionofrecombinantproteinsorbiomoleculeswithbiotechnologicalorpharmaceuticalapplicationshasincreasedovertheyears[1].InY.lipolytica,morethan100heterologousproteinshavebeensuccessfullyproducedathighyield,underscoringitsproductionpotential[1,2].Y.lipolyticaisamodelyeastspecies,well-knownforitsunusualmetabolicpropertiessuchastheabilitytogrowonfattyacidsoralkanesassolecarbonsourceandtoaccumulateintracellularlipidsathighyield[3,4].Thisfeaturehasenabledthedevelopmentofmetabolicengineeringstrategiestoconstructmutantstrainstoproducelipidforbiodieselandbiojetfuel[5,6,7,8,9,10,11,12],ortosynthetizeunusualfattyacids[13],suchasω−3[14],ricinoleicacid[15],conjugatedfattyacids[16,17],andfattyacidderivatives(e.g.,fattyalcoholanddicarboxilicacid)[18].Basedonitsabilitytosecretelargeamountsofproteinsandmetabolites,Y.lipolyticahasbeenusedforseveralindustrialapplications,includingheterologousproteinsynthesis,citricacidanderythritolproduction[19,20].Besides,Y.lipolyticahasbeenaccordedaGRAS(generallyrecognizedassafe)status[19].Whendevelopinganefficientcellfactory,thechoiceofthepromoterdrivingrecombinantgeneexpressioniscrucial,andthereforerepresentsoneofthekeyparameterstobeoptimized.Atpresent,fewpromotershavebeenidentifiedandtheirregulationisnotfullyunderstoodyet.Historically,thepromoterfromtheXPR2gene,whichencodesanalkalineextracellularprotease,wasthefirsttobecharacterized[21].Althoughthispromoterhasbeenusedsuccessfully,itsfullinductionrequireshighpeptidesconcentrationsandapHabovesix,conditionsthatareoftenunfeasibleatindustrialscale.Comparisonofstrengthandregulationofpromotersfromtheglycerol-3-phosphatedehydrogenase(G3P),theisocitratelyase(ICL1)andofgenesinvolvedinbeta-oxidationpathwaysuchasthe3-oxo-acyl-CoAthiolase(POT1)andtheacyl-CoAoxidases(POX2,POX1andPOX5)wasreported[22].Thisprovidedthefirststrongpromotersinduciblebyglycerol(G3P),ethanol(ICL)andoleicacid(POT1andPOX2).Otherregulatedpromoters,suchastheonefromLIP2andPOX2geneencodinganextracellularlipaseandacyl-CoAoxidase2,respectively,havebeendevelopedandcharacterized[23,24,25].UsingexpressionvectorsbasedonpLIP2,higherproteinproductivitiessuchasforLip2plipasehavebeenobtainedinY.lipolyticathaninothercellfactoriessuchasP.pastoris.UsingtheGAPconstitutivepromoter,Wangandcolleagues[26]obtainedlipaseactivitylevelsof13,500 U/mLfromaglucosefed-batchprocessina10-Lbioreactor.Incontrast,activitylevelsof150,000 U/mLwereobtainedusingLIP2promoterandatryptone-oliveoilfed-batchprocess[3].However,theutilizationofpLIP2andpPOX2isdifficultinpractice,especiallyinlarge-scalebioreactor,duetothehydrophobicnature(waterinsoluble)oftheinducer(i.e.fattyacidsortriglycerides).OtherinduciblepromotersavailableinY.lipolyticaarethosefromgenesencodingisocitratelyase(pICL1,[22]),fructose-bisphosphatealdolase(pFBA1,[27]),phosphoglyceratemutase(pGPM)orglycerol-3-phosphateO-acyltransferase(pGPAT).Theyhavebeenusedforheterologousproteinproductionwithvarioussuccesses(forareviewsee[1,28]).Constitutivepromotershavealsobeenconsidered.ThefunctionaldissectionofpXPR2allowedtheidentificationofoneofitsupstreamactivatingsequence(UAS1XPR2)thatispoorlyaffectedbycultivationconditions[29].Hybridpromoters,containinguptofourdirectrepeatsofUAS1XPR2upstreamoftheminimalLEU2promoter(mLEU2),werefirstconstructed[30].Amongthese,hp4dwaswidelyusedforheterologousproteinproduction(forreviewsee[2]).ThislatterhasbeenatthebasisoftheY.lipolyticaYLEXexpressionkitcommercialisedbyYeasternBiotechCo.(Taiwan).Morerecently,anextendedseriesofhybridpromoters,carryingvariouscopynumbers(upto32)ofUAS1XPR2upstreamofmLEU2,wereconstructed[31].SomeofthesehybridpromoterswereshowntopossessanefficiencyeightfoldhigherthananyknownendogenouspromoterfromY.lipolytica[31].ThepromoterfromtheTEF1geneencodingthetranslationelongationfactor-1α[32]isalsowidelyusedtodriveconstitutivegeneexpressioninY.lipolytica.Hybridpromoterswithvariablestrengthsderivedfromthelatterwererecentlytestedfortheproductionofsecretedproteinsofindustrialinterestsuchasxylanaseandglucoamylase[33].Thisstudyhighlightedthathigherproteinproductivitydoesnotnecessarilyrelyonthestrengthofthepromoterusedfortheexpressionofthecorrespondinggene.Insyntheticbiology,geneexpressionmustbefine-tunedinordertoensureoptimalfluxesinthecorrespondingpathwayortoavoidametabolicburden.Hussainandcolleagues[34]investigatedpromoterstrengthbyshufflingpromoterconstitutiveelements(UAS,proximalpromoter,TATAboxandcorepromoter)ofvariousfungalgenepromoters(TEF,POX2,LEU2,PAT1)inY.lipolytica.Theyfoundoutthatengineeringpromoterarchitectureallowstomodulateandtofine-tunegeneexpressionlevel.However,toexpendtherangeofthisregulation,novelregulatoryelements(UAS)andthusnovelregulatedpromotersremaintobediscovered.Inthisstudy,wereportontheidentificationoftheinduciblepromoterfromtheEYK1geneencodinganerythrulosekinaseinY.lipolytica,thecharacterisationofitsregulatoryelementsandthedevelopmentofhybridderivativespromotersshowingdifferentinductionstrengthsandregulatorypatternsdependingonthegeneticbackgroundoftherecipientstrain(WTor∆eyk1).Thissetofnovelpromotershasdirectapplicationsforheterologousproteinproduction,metabolicengineeringandsyntheticbiology.MethodsGrowthandcultureconditionsTheY.lipolyticastrainsusedinthisstudywerederivedfromthewild-typeY.lipolyticaW29strain(ATCC20460).TheauxotrophicderivativePo1d(Leu−Ura−)waspreviouslydescribedbyBarthandGaillardin[35].EscherichiacolistrainDH5αwasusedforhostingandamplificationofrecombinantplasmidDNA.AllthestrainsusedinthisstudyarelistedinTable 1.ThemediaandgrowthconditionsusedforE.coliweredescribedbySambrookandcolleagues[36].YPDandYNBmediumtogetherwithgrowthconditionsforY.lipolyticahavebeenpreviouslydescribedbyBarthandGaillardin[35].Tomeetauxotrophicrequirements,uracil(0.1 g/L)and/orleucine(0.1 g/L)wereaddedtotheculturemediumwhennecessary.Casaminoacids(0.2%BactoCasaminoAcids,Difco,Paris,France),wereaddedtobioreactorsforfastergrowthrate.GrowthofY.lipolyticawasperformedinbaffled250 mLflaskincubatedat28 °Cat160 rpm.YNBmediumwassupplementedwithcarbonsource(10 g/L)asfollows:glucose(YNBD),glycerol(YNBG),erythritol(YNBOL)orerythrulose(YNBOSE).Growthof∆eyk1strainswereperformedinYNBmediumwith0.25%glucoseorglycerolascarbonsourceand0.25%erythritolorerythruloseasinducer.Table 1ListofstrainsandplasmidsusedinthisstudyFullsizetable Growthinmicroplateandfluorescenceanalysis YarrowialipolyticapreculturesweregrownovernightinYNBD,beforebeingcentrifuged,washedwithanequalvolumeofYNBmediumwithoutcarbonsourceandresuspendedin1 mLofthesamemedium.96-wellmicroplatescontaining200 μLoftheappropriatedmedium(finalvolume)wereinoculatedwithwashedcellsatanOD600nmof0.1.GrowthwasperformedinamicrotiterplatereaderSynergyMx(Biotek,Colmar,France)followingthemanufacturer’sinstructionsat28 °Cand110 rpm.OD600nmandfluorescenceweremeasuredevery20 minfor72 h.YFPfluorescencewasanalyzedwiththewavelengthsettingsex:505 nm/em:530 nm.Fluorescencewasexpressedasspecificfluorescenceunit(SFU,normalizedtobiomassvalue)ormeanspecificfluorescencevalue(mSFU,meanvalueofSFUforthedifferentsamplingtimes).TheSFUvalueofthewild-typestrainJMY2900(i.e.cellintrinsicfluorescence)wassystematicallydeducedfromthatoftheYFPreporterstraininthesameexperimentalconditions(samplingtimeandmedium).Cultureswereperformedinduplicates.Growthinbioreactorandmonitoringofpromoterinductionbyflowcytometry YarrowialipolyticapreculturesweregrownovernightinYPD,beforebeingcentrifuged,washedwithanequalvolumeofYNBmediumwithoutcarbonsourceandresuspendedin5 mLoftheculturemedium.ThewashedcellswereusedforbioreactorinoculationatanOD600nmof0.5.Chemostatwereperformedin200 mL(150 mLworkingvolume)DASGIP®DASboxMiniBioreactorsSR0250ODLS(Eppendorf,Hamburg,Germany).Arunof7 hinbatchmodewasperformedbeforebeingshiftedincontinuousmodewithdilutionratesasstipulatedinthetext.FeedingoffreshmediumwasensuredbyaWatsonMarlow323Speristalticpump(WatsonMarlow,FalmouthCornwall,UK),andremovalofspentmediumwasensuredbyaWatsonMarlow120U/DM3peristalticpump.Cultureparametersweresetasfollows:temperature,30 °C;agitationrate,800 rpm;aerationrateat1 vvm.Carbonsourcepulses(CSP)inthereactorswereatfixedvolume(4.2 mL),regardlessofthepulseconcentration.AftereachCSP,biomass,YFPfluorescenceandcarbonsourceconcentrationsweremonitoredfor8 hwithasamplingfrequencyof1 h.CSPwereperformedatsteadystate.Chemostatcultureswereperformedinduplicates.YFPfluorescencewasmonitoredusingaBDAccuri™C6FlowCytometer(BDBiosciences,NJ,USA).Flowratewasfixedat14 µL/min,andsamplesweredilutedwithphosphatesalinebuffer(PBS)toreachacelldensityrangingbetween500and2500 cells/µL.Foreachsample,40,000cellswereanalyzedusingtheFL1-AchanneltoidentifyfluorescenceassociatedwiththeYFP(excitationwasperformedwitha20-mW,488-nmsolid-statebluelaser;theemissionwavelengthwas533/30 nm).Additionally,datafromtheforwardscatterchannel(FSC-A)werecollectedtogetinformationonthesizedispersionamongthecellpopulation.Theflowcytometrydotplots(FL1-A/FSC-A)wereanalyzedusingCFlowPlussoftware(Accuri,BDBioscience).Forfurtherprocessing,therawdatawereexportedas.fcsfilesandloadedinMatLabusingthefca_readfscfunction(downloadedfromtheMatLabFileExchangefileserver;[37]).Backgroundnoise(cellintrinsicfluorescence)wasfixedat4000fluorescenceunits.Thisvalueencompassesthefluorescencelevelofatleast99.3%ofthewild-typecells(strainJMY2900)growninYNBG(glycerol),YNBOL(erythritol)andofJMY6245(pEYK300-YFP)growninYNBG(glycerol).Relativefluorescence(RFU)wasdefinedasthesamplemedianfluorescencevalueminustheintrinsicfluorescencevalue.Proportionofinducedcellsreferstothenumberofcellsshowingafluorescencesignalhigherthan4000fluorescenceunits,relativetothetotalnumberofanalyzedcellsinthesample(i.e.40,000).GateQ1-URofFSC-A/FL1-Acytogramsencompassesinducedcells.PlasmidandyeaststrainconstructionPlasmidconstructionRestrictionenzymes,DNApolymerases,andligaseswereusedinaccordancewiththemanufacturer’srecommendations.RestrictionenzymeswereobtainedfromOZYME(Saint-Quentin-en-Yvelines,France).PCRamplificationswereperformedusinganEppendorf2720thermalcyclerwithPyroBestDNApolymerase(Takara)forcloningpurposeandwithGoTaqDNApolymerase(Promega)fordeletion/overexpressionverification.PCRfragmentswerepurifiedusingaQIAgenPurificationKit(Qiagen,Hilden,Germany),andDNAfragmentswererecoveredfromagarosegelsusingaQIAquickGelExtractionKit(Qiagen,Hilden,Germany).DNAsequencingwasperformedbyGATCBiotechandprimersweresynthetizedbyEurogentec(Seraing,Belgium).TheCloneManagersoftwarepackage(Sci-EdSoftware)wasusedforgenesequenceanalysisandprimerdesign.Disruptionandexpressioncassetteswereusedtotransformyeastcellsusingthelithiumacetatemethod[38].TransformantswereselectedonYNBcasa,YNBura,orYNBdependingontheirgenotype.ThegenomicDNAfromyeasttransformantswasobtainedasdescribedbyQuerolandcolleagues[39].PrimersMT-URA3-for,MT-YFP-rev,pTEF-start,61stopwereusedtoverifysuccessfulinsertionoftheexpressioncassetteandthepromotersequences.Foreachtransformation,atleastthreeindependenttransformantscarryingthecorrectintegrationwereanalysed.Therepresentativecloneswereusedforthisstudy.ThestrainsandplasmidsusedinthisstudyaresummarizedinTable 1andprimersarelistedinTable 2.Thevectorscarryingtheyellowfluorescentprotein(YFP)underthecontrolofthepTEFandhp4dhavebeenpreviouslydescribed(Table 1).ThepEYK1promoteranditsderivatives(mutatedandhybridpromoters)wereintroducedbyexchangeoftheClaI-BamH1regionortheClaI-SpeIregionofYFP-encodingplasmidasdescribedbelow.Table 2ListofprimersusedinthisstudyFullsizetable ConstructionofpEYK300ThepromoterregionofEYK1gene(pEYK300)wasamplifiedfromgenomicDNAofY.lipolyticastrainW29withprimerpairpEYK300F/pEYKR,designedtointroduceClaIandBamHIrestrictionsites,respectively,intheamplifiedfragment.TheresultingampliconwaspurifiedandclonedintopJET1.2,toyieldplasmidFCP007.ThepEYK300fragmentwasthenreleasedfromFCP007andclonedatthecorrespondingsiteofJMP1427,yieldingtheplasmidJMP3934.ConstructionofpEYK450,pEYK300AbandpEYK300aBpromotersPlasmidcontainingpEYK450wasobtainedbyPCRamplificationoftheintergenicregionbetweengenesYALI0F01628gandYALI0F01606gwithprimerpairMT-TATAampli-F/MT-TATAampli-R.Thisresultedina252 bpfragmentcarryingT,AandBboxeswithinaClaI-SpeIfragment(sitesaddedatthe5′and3′ends,respectively).ThisfragmentwasligatedintoFCP013digestedbyClaI-SpeI,toyieldtheplasmidJMP3994(pEYK450).PlasmidscontainingpEYK300AbandpEYK300aBwereobtainedbyexchangeoftheClaI-SpeIfragmentofJMP3934(pEYK300)bytwoClaI-SpeIDNAfragmentscarryingtheA(aB)orB(Ab)mutatedregions,respectively.TheywereobtainedbyannealingoligonucleotidesST044/ST045/ST050/ST047(fragmentaB)andST043/ST046/ST049tB/ST048(fragmentAb)(Table 2).TheoligonucleotidesST044andST046containaMluIsitefortheverificationoftheinsertionofthemutation.TheresultingplasmidsweredesignatedJMP3988(pEYK300aB)andJMP3991(pEYK300Ab),respectively.ConstructionofhybridpHU4EYK300promoterThefragmentcarryingfourtandemrepeatsoftheUAS1XPR2(HU4derivingfromhp4d)wasobtainedbyClaI-BstBIdigestionfromtheJMP2027vector[33].Aftergelpurification,itwasthenligatedattheClaIsiteofJMP3934(previouslydigestedbyClaIanddephosphorylated).CorrectorientationoftheHU4regionwasverifiedbyClaI-BamHIrestrictionandDNAsequencing.TheresultingplasmidwasnamedJMP3998(pHU4EYK300).ConstructionofhybridEYKpromoterSyntheticpromoterscarryingthreerepeatedofdomainsofAboxupstreamofthewild-typeBbox(A3B,JMP4123)andthemutatedBbox(A3b,JMP4124)weresynthesisedbyGenScriptBiotechCo.(China)withClaIandSpeIsitesatthe5′and3′ends,respectively.TheClaI-SpeIfragmentsfromJMP4123andJMP4124wereligatedintoJMP918digestedbyClaI-SpeI,yieldingtheplasmidsJMP4137(pEYK300A3B)andJMP4139(pEYK300A3b),respectively.DeletionoftheEYK1geneTheEYK1disruptioncassettewasgeneratedbyPCRamplificationaccordingtoVandermiesandcolleagues[40].First,theupstream(Up)anddownstream(Dn)regionsoftheEYK1genewereamplifiedusingY.lipolyticaW29genomicDNAasthetemplatewiththeEYK-P-F/EYK-P-R-SfiIandEYK-T-L-SfiI/EYK-T-Rasprimerpairs.URA3exmarkerwasamplifiedfromJME803withtheprimerpairLPR-L-SfiI/LPR-R-SfiI(Table 2).AmpliconsweredigestedwithSfiIbeforebeingpurifiedandligated,usingT4DNAligase.TheligationproductwasamplifiedbyPCRusingtheprimerpairEYK-P-F/EYK-T-R.Theeyk1::URA3exdisruptioncassettewasfinallyusedtotransformY.lipolyticastrainPo1d.TheresultingstrainwasdesignatedRIY147(eyk1::URA3ex,Leu−).TheauxotrophicderivativeRIY176wasisolatedaftertransformationofRIY147withthereplicativeplasmidpRRQ2,accordingtoFickersandcolleagues[41],formarkerrescue(Table 2).TheprimersEYK-V1andEYK-V2(Table 2)wereusedforgenedisruptionverification.EYK1promotersequenceanalysisMultiplealignmentsofnucleotidesequenceofEYK1genepromotersamongtheYarrowiaclade:Y.lipolytica(YALI),Yarrowiaphangngensis(YAPH),Yarrowiayakushimensis(YAYA),Yarrowiaalimentaria(YAAL),andYarrowiagalli(YAGA)wereperformedusingclustalW2software[42]accordingtoLarkinandcolleagues[43].GenomesequencesofYarrowiaspecieswereassembledandannotatedbyCécileNeuvéglise,HugoDevillersandcoworkers(tobepublished).HomologuesofYALI0F01606ginYarrowiaspecieswereidentifiedbyBlastontheprivatesiteofGRYC(GenomeResourcesforYeastChromosomes;http://gryc.inra.fr)usingYALI0F01606ggeneastemplate.PromoterregionswereretrievedusingthedownloadfunctionalitydevelopedbyH.Devillers.AnalyticalmethodsCellgrowthwasmonitoredbyopticaldensity(OD600nm).Erythritol,erythrulose,glucoseandglycerolconcentrationsintheculturesupernatantweremeasuredbyHPLC(AgilentTechnologies1200series)usinganAminexHPX-87Hionexclusioncolumn(Biorad300 × 7.8 mm).Elutionwasperformedusing15 mMtrifluoroaceticacidasthemobilephaseataflowrateof0.5 mL/minandatemperatureof65 °C.Erythritol,glucoseandglycerolweredetectedusingarefractiveindexdetector(RID,AgilentTechnologies),whileerythrulosewasmeasuredat210 nmwithaUVdetector(AgilentTechnologies).ResultsanddiscussionEYK1promoterisinducedbyerythritolanderythruloseTodate,twodifferentpathwayshavebeenreportedforerythritolcatabolism.Inafirstone,erythritolisphosphorylatedintoerythritol-phosphateandthenoxidizedinerythrulose-phosphate[44].Inasecondone,erythritolisfirstconvertedintoerythrulosebeforebeingphosphorylatedintoerythrulose-phosphate[45].WehaverecentlyidentifiedandcharacterizedEYK1gene(YALI0F1606g)[46]inY.lipolytica.Disruptionofthelatterabolishedyeastgrowthonerythritolmedium,showingthatEYK1geneisinvolvedinerythritolcatabolism.Inaddition,a∆eyk1mutantwasfoundtoaccumulatel-erythrulose.Fromthis,ithasbeenconcludedthatEYK1encodeanerythrulosekinase(Eyk)andthaterythritolcatabolisminY.lipolyticafollowsthepathwaydepictedinFig. 1.Fig. 1ErythritolcatabolismpathwayinYarrowialipolytica.Basedon[45]and[46].Erythritolisconvertedintol-erythrulosebyanerythritoldehydrogenaseencodedbyEYD1,thenl-erythruloseisphosphorylatedbythel-erythrulose-1-kinaseencodedbyEYK1(YALI0F01606g)intol-erythrulose-1-phosphateFullsizeimage WethereforeexpectthatEYK1geneexpressionisregulatedbyerythrulose,thesubstrateofEyk,and/orbyerythritol.ToassesstheregulationoftheEYK1promoter,twofragmentsof450and300 bp,(EYK450andEYK300,respectively),correspondingtotheintergenicregionofgenesYALI0F01606gandYALI0F01628gwereusedtoconstructareportergenesystembasedonayellowfluorescentreporterprotein(YFP)(Fig. 2).Indeed,theYFPfluorescencewasusedtoquantifythepromoterinductionlevel.Fig. 2Schematicrepresentationofpromotersusedinthisstudy.SchematicrepresentationofthegenomiclocuscontainingtheupstreamgeneYALI0F01628gandtheEYK1gene,YALI0F01606g.aSchematicrepresentationofthenativepromoterspEYK450(TATAbox + nativeAbox + nativeBbox)andpEYK300(nativeAbox + nativeBbox)controllingtheexpressionofYFP;bSchematicrepresentationofthemutatedpromoterspEYK300aB(mutatedAbox + nativeBbox)andpEYK300Ab(nativeAbox + mutatedBbox)controllingtheexpressionofYFP;cSchematicrepresentationofthehybridpromoterspEYK300A3B(3Aboxes + nativeBbox)andpHu4EYK300(4tandemcopiesofUAS1xpr2 + nativeAbox + nativeBbox)controllingtheexpressionofYFP.Symbolsarewhitefilledsquare:TATAbox(T),blackfilledsquare:Abox(A),greyfilledsquare:Bbox(B),blackfilledtriangle:mutatedAbox,greyfilledtriangle:mutatedBbox,greyrightwardsarrow:YFPgene,rectangle:fourtandemcopiesofUAS1xpr2Fullsizeimage FragmentsEYK450andEYK300thatspanover438and 291 bpupstreamoftheEYK1startcodon(Fig. 2a),wereclonedinJMP1427asdescribedinMaterialsandmethodstoyieldplasmidsJMP3934(pEYK300)andJMP3994(pEYK450),respectively(Fig. 3).TheywerethenusedtotransformY.lipolyticastrainJMY2101.Severalindependenttransformants(3–6)wererandomlyselectedforeachconstructandthecorrespondingYFPfluorescencemeasuredduringcellgrowthonerythritolmedium(YNBOL).SincenodifferencesinYFPfluorescencelevel,andthuspromoterinduction,couldbeobserved(datanotshown),onetransformantofeachconstructwasusedforfurtherstudies,namelystrainsJMY6245(pEYK300-YPF)andJMY6375(pEYK450-YFP),respectively(Table 1).Fig. 3Schematicrepresentationofplasmidsconstructedinthisstudy.PlasmidpEYK300containedtheyellowfluorescentproteinYFP,underthe285 bppromoterregionoftheEYK1gene(erythrulosekinase;YALI0F01606g).ThevectorscontainthezetasequencefortargetingtheexpressioncassetteobtainedafterNotIdigestion.KanRandURA3markersareforselectioninE.coliandY.lipolytica,respectively.TheURA3isflankedbyLoxP/LoxRregionformarkerrescue(excisablemarkerURA3ex).JME3934(pEYK300-YPF);JME3994(pEYK450-YFP);JMP3988(pEYK300aB-YPF);JMP3991(pEYK300Ab-YPF);JMP4137(pEYK300A3B-YPF)-YPF;JMP4139(pEYK300A3b-YPF)andJMP3998(pHU4EYK300-YPF)Fullsizeimage CellgrowthandYFPfluorescencewerequantifiedovertimeduringcultureofstrainJMY6245inYNBminimalmediasupplementedwithglucose(YNBD),glycerol(YNBG),erythritol(YNBOL)anderythrulose(YNBOSE).Inmediumcontainingerythritol(YNBOL)anderythrulose(YNBOSE),YFPfluorescence,andthereforepEYK300inductionlevels,weresignificantlyhigherthaninthepresenceofglucose(YNBD)andglycerol(YNBG)(3157and4844 mSFUascomparedto344and357 mSFU,respectively)(Fig. 4a).ThisclearlyhighlightsthaterythruloseanderythritolpositivelyregulatepEYK300inductionbycontrasttoglucoseandglycerol.However,thelowfluorescencelevelsobservedinYNBDandYNBGmediumsuggestthatpEYK300isslightlyinducedbyglucoseandglycerol.After60 hofculture,thefluorescencelevelinmediumsupplementedwitherythrulosewas1.5-foldhigherthaninthepresenceoferythritol(3536and5904 SFU,respectively).Thissuggeststhaterythrulosecouldbeabetterinducerthanerythritol.ExperimentsperformedwithstrainJMY6375(pEYK450-YFP)inthesameexperimentalconditionsyieldedsimilarresults(datanotshown).Therefore,thepEYK300promoterseemstoencompassthedifferentregulatoryelementsrequestedforgeneexpression(UASandURS).Consequently,pEYK450promoterwasnotfurtheranalysedandwefocusedonlyonpEYK300promoterinfurtherexperiments.Fig. 4TimecourseofYFPfluorescencedependingonculturemediumfornativeEYK1andpTEFpromoters.Specificfluorescence(SFU)correspondingtotheexpressionofYFPunder:apEYK300(JMY6245)andbpTEF(JMY2878).GrowthinminimummediaYNBcontaining1%ofspecifiedcarbonsource(OLerythritol,OSEerythrulose,Ddextrose,Gglycerol)Fullsizeimage InordertoassessthestrengthofpEYK300inductionbyerythritolanderythrulose,itwascomparedtothestrengthofthestrongconstitutivepTEFpromoter.YFPfluorescenceofstrainJMY2876(pTEF-YFP)wasmeasuredinthesameexperimentalconditionsandcomparedtothatofstrainJMY6245.AsshowninFig. 4b,pTEFexpressionwassimilarinthefourmediatested,withfluorescencevaluesbeing1192,1369,1485and1016mSFUinYNBOL,YNBOSE,YNBDDandYNBG,respectively.ExpressionlevelsforpEYK300inYNBOLandYNBOSEwereinaverage2.6-and3.5-foldhigherthantheexpressionlevelofpTEF,respectively.IdentificationofEYK1regulatoryelementsInordertoidentifytheregulatoryelement(i.e.UAS)ofpEYK1,weanalysedthenucleotidesequenceoftheEYK1promoterregionusingtheintergenicregionbetweenYALI0F01628gandYALI0F01606g(Fig. 5;Additionalfile1:TableS1).BlastanalysisoftheEYK1promoterdidnotevidencedanyconservedmotifwithinYarrowialipolyticagenome(datanotshown).Therefore,wecomparedthepromoterregionoftheEYK1genetothosepresentinotherspeciesoftheYarrowiaclade(namely,Yarrowiaphangngensis,Yarrowiayakushimensis,YarrowiaalimentariaandYarrowiagallithathavebeenrecentlysequencedandannotatedinourlaboratory[47]andNeuvegliseN.,DevillersH.etcollaborator(unpublished).AlignmentoftheEYK1promotersequences(Fig. 5;Additionalfile1:TableS1)highlightedthreeputativeconservedelements;aputativeTATAbox(BoxTATA)andaconservedAmotif(BoxA)withthemainsignature[GGAAAGCCGCY]andaconservedBmotif(BoxB)withthemainsignature[CNTGCATWATCCGAYGAC].Fig. 5MultiplealignmentofEYKpromoter.AlignmentoftheintergenicregionbetweenYALI0F01628gandYALI0F01606ginYarrowialipolyticaandstrainsfromtheYarrowiacladehighlightingconservedblocsthatrepresentsputativeregulatoryelementsfortheexpressionandregulationoftheYALI0F01606ggenebyerythritolanderythrulose.BoxedCATandATGcorrespondtotheStopandstartcodonoftheYALI0F01628gandYALI0F01606g,respectively.ClaIandSpeIrestrictionsitesareunderlined.Localizationofp300primercontainingtheClaIsiteisindicatedabovethegenomicsequence.GenomicsequencesarefromY.lipolyticaW29(YALI;YALI0F01606g),Yarrowiaphangngensis(YAPH),Yarrowiayakushimensis(YAYA),Yarrowiaalimentaria(YAAL),andYarrowiagalli(YAGA).SequencesareinAdditionalfile1:Table S1.TheClaIsiteupstreamofthep300sequenceandtheSpeIsitesareunderlined.TheregioncontainingUAS1eykusedfortandemrepeatsconstructionisboxedFullsizeimage ThecomparisonofYFPfluorescenceunderpEYK450andpEYK300indicatesthattheTATAboxmaybeinvolvedintheexpressionofgeneYALIPF01628gratherthangeneYALI0F01606g.Thus,todeterminetheroleofBoxAandBoxBinpEYKregulation,twomutatedpromoters,namelypEYK300aBandpEYL300Ab,wereconstructedasdescribedinmaterialandmethodbyexchangeoftheClaI-SpeIfragment.MutationoftheconservedBoxAandBoxBwereperformedbyintroducingaMulIsite.ThemotifA[GGAAAGCCGCC]wasreplacedby[GGAACGCGTCC]andnamedmotifa.ThemotifB[CTTGCATAATCCGATGAC]wasreplacedby[CTTGT ACGCGT AGATGAC]andnamedmotifb.ThisyieldedtopEYK300aBandpEYK300Ab,respectively(Fig. 2b).ThemutatedpEYK300aBandpEYK300AbwereintroducedintostrainJMY2101(Po1dLeu+)togiverisetorepresentativestrainsJMY6369andJMY6372,respectively(Table 1).ForstrainJMY6369carryingthepEYK300aBmutantpromoter,YFPfluorescencewasremarkablyreducedinthepresenceoferythritol(YNBOL)anderythrulose(YNBOSE)(683and1481mSFU,respectively)(Fig. 6a).ThisobservationsuggestedthattheBoxAcorrespondstotheupstreamactivatingsequence(UAS1EYK1)requiredforthepromoterinductionbybotherythritolanderythrulose.Fig. 6TimecourseofYFPfluorescencedependingonculturemediumformutatedpEYK300promoter.Specificfluorescence(SFU)correspondingtotheexpressionofYFPunder:apromoterpEYK300withamutatedAbox;pEYK300aB(JMY6369)andbpromoterEYK300withamutatedBbox;pEYK300Ba(JMY6372).GrowthinminimummediaYNBcontaining1%ofspecifiedcarbonsource(OLerythritol,EOSE:erythruloseDdextrose,Gglycerol)Fullsizeimage Ontheopposite,themeanrelativeYFPfluorescencemeasuredforstrainJMY6372carryingthepEYK300Abmutatedpromoter(Fig. 6b),was2.4-foldhigherinthepresenceoferythritol(YNBOLmedium)thanforthenon-mutatedpEYK300promoterinthesameconditions(8389and3536SFUafter60 h,respectively).Incontrast,YFPfluorescenceinthepresenceoferythrulose(YNBOSEmedium)wasinthesamerangeasYFPfluorescenceofthenon-mutatedpromoter.Furthermore,pEYK300AbwaslessrepressedonglucosemediaascomparedtopEYK300(withameanspecificfluorescenceof718versus279 mSFU),suggestingthattheBboxmaybeinvolvedinglucoserepression.ThisclearlydemonstratesthatdomainAisinvolvedinerythritolanderythruloseinductionandthatdomainBmaybeinvolvedinglucoserepressionsinceexpressionofthepEYK300Abincreasedattheendofthecultureinglucosemedia,whichisnotthecaseinglycerolmedia.TamdemrepeatsofUAS1EYK1increasepromoterstrengthMulticopyrepeatsofUASelementsupstreamofapromoterhavebeenshowntoincreasepromoterstrength[30,31,34,48].Therefore,weconstructedpromoterpEYK300A3Bcomposedofthreerepeatsofthe48 bpUAS1EYK1fragmentencompassingtheBoxA(GGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGC)upstreamofthewild-typepEYK300promoter(Fig. 2c).TheresultingconstructwasintroducedintostrainJMY2101togiverisetostrainJMY6681.Promoterstrengthwasmonitoredinthepresenceofglucose(YNBD),glycerol(YNBG),erythritol(YNBOL)anderythrulose(YNBOSE)andcomparedtothatofpEYK300(strainJMY6245).AsshowninFig. 7a,YFPfluorescencemeasuredforpEYK300A3Bwas3.4-foldhigherinaverageinthepresenceoferythritolascomparedtopEYK300(10,538and3157 mSFU,respectively).Incontrast,inductionofpEYK300A3BwasfoundsimilarinaverageinthepresenceoferythruloseascomparedtopEYK300(5034and4844 mSFU,respectively).BycontrasttopreviousobservationwithpEYK300(Fig. 4),pEYK300A3Binductionlevelwas2.1-foldhigherinaverageinthepresenceoferythritolthanforerythrulose(10,538and5034 mSFU,respectively).SimilarexperimentsperformedwithstrainJMY6684(pEYK300A3b),showedthattheinductionprofileonYNBOLwasnotsignificantlydifferentfromtheoneofJMY6681(pEYK300A3B),exceptthatinductionwassignificantlylessrepressedbyglucoseandglycerol,confirmingthepreviousobservations(datanotshown).Fig. 7TimecourseofYFPexpressiondependingonmediaforEYKhybridpromoters.Specificfluorescence(SFU)correspondingtotheexpressionofYFPunder:apEYK300A3B(JMY6681)andbpHU4EYK300(JMY6380).GrowthinminimummediaYNBcontaining1%ofspecifiedcarbonsource(OLerythritol,OSEerythrulose,Ddextrose,Gglycerol)Fullsizeimage Sincetheinsertionofseveralcopiesofthe48 bpregionencompassingtheBoxAmotifresultedinastrongerpromoterinductionlevel,itcouldbeassumedthatincreasingthecopynumberofUAS1EYK1wouldallowtofinetunethestrengthofpromoterinduction.Indeed,severalstrongsynthetichybridpromotershavebeencreatedbyfusingtandemrepeatsofupstreamactivationsequence(UAS)upstreamtoacorepromoterregion.Thefirstone(hp4d)wasbasedonfourtandemrepeatsofthe108 bpUAS1XPR2oftheXPR2geneupstreamontheminimalLEU2corepromoter[30].LaterBlazekandcoworker’sconstructedhybridpromoterscontainingupto32copiesofUAS1XPR2oftheXPR2geneupstreamontheminimalLEU2corepromoterand16copiesofUAS1XPR2oftheXPR2geneupstreamofTEFcorepromotersofdifferentlength[31].PromoterstrengthincreasedwithcopynumberoftheUAS,andthebestoneshowedatenfoldincreaseexpressioncomparedtothepTEFpromoter.Similarexpressionlevelswereobtainedbyinsertingthreetandemcopiesofthe230 bpUAS1TEFupstreamofthepTEFpromoter[48]anditsexpressiondidnotvarysignificantlywithcarbonsource(glucose,sucrose,glycerolandoleicacid).TheonlystronginduciblepromoteristhePOX2one[22].OleicacidinduciblehybridsyntheticpromoterswereobtainedcomprisingeightcopiesofUAS1xpr2upstreamofthe100 bpproximalcorePOX2promoter.ThisUAS-corepromoterchimerashoweda4.2-foldhigherexpressionlevelinoleicacidmediathaninglucoseincontrasttoatwofoldhigherexpressionlevelforthe8copiesofUAS1xpr2upstreamofthe136 bpproximalcoreTEFpromoter[34].Hereweshowedthatahybridpromotercontainingtwoadditionaltandemcopiesoftheshort48 bpUAS1EYK1upstreamoftheEYK1promoterresultsina3.3-foldstrongerpromoter,thuswecouldexpecttobeabletoconstructstrongererythritol/erythruloseinduciblepromotersbyintroducingadditionaltandemrepeatsoftheUAS1EYK1.UAS1BfromXPR2enhancedpromoterstrengthwithoutaffectingerythritolanderythruloseinductionMadzakandcolleaguesreportedthatthefusionoffourtandemsrepeatsofUAS1BofXPR2geneupstreamofaminimalpromoteroftheLEU2gene(yieldingtheso-calledhp4dhybridpromoter)allowedasignificanttranscriptionalactivity[30].Inthesameline,wecombinedfourcopiesofUAS1XPR2(UAS1B)withthepEYK300promoterleadingtopromoterHU4EYK300(JME3998)(Fig. 2c).ThelatterwasintroducedintoJMY2101,givingrisetostrainJMY6380.TheregulationofthepHU4EYK300wasinvestigatedbymonitoringcellgrowthandYFPfluorescencelevelsduringcultureofstrainJMY6380inYNBmediumsupplementedwitherythritol(YNBOL),erythrulose(YNBOSE),glucose(YNBD)andglycerol(YNBG).AsshowninFig. 7b,YFPfluorescence,andthereforepromoterinductionwere17.1-and9.8-foldhigherinthepresenceoferythritol(YNBOLmedium)anderythrulose(YNBOSE)thanforpEYK300promoter(54,063and47,487 mSFUascomparedto3157and4844 mSFU,respectively).pHU4EYKwasinducedinstationaryphase(i.e.after60 hofculture,63,380 SFU)onglucosemedia(YNBD)incontrasttopEYK300(344 SFU).Nevertheless,pHU4EYKwasnotfoundhighlyexpressedonglycerolmedia.SincepHU4EYKpromoteryieldedmuchstrongerinductionandthusgeneexpression,itsregulationwasfurthercharacterisedinregardtothecellgrowthrateofstrainJMY6380andtotheinductioneffectoferythritolanderythruloseonpHU4EYK.HybridpromoterHU4EYK300isinduciblebyerythritolanderythruloseInordertofurthercharacterisetheregulationofthehybridpromoterpHU4EYK300,itsregulationwasanalysedatsteadystateinchemostatculture.Thisensuresthatoncethesteadystateisestablished,theeffectofanyperturbations—e.g.,theadditionofaknownamountofaspecificcompound(aninducerorarepressor)inthemedium—onpHU4EYK300inductioncanbespecificallyassessedovertime.TheregulationofpHU4EYK300wasinvestigatedinregardtothegrowthrateofstrainJMY6380andthecompositionoftheculturemedium,morespecificallyinthepresenceofamixtureofglycerol/erythritolorglycerol/erythrulose. GrowthratehasnoeffectonpHU4EYK300induction Yeastcellphysiologyisdirectlyinfluencedbythegrowthrate.WiththeaimtoevaluatetheinfluenceofcellgrowthrateonpHU4EYK300inductionbyerythritol,chemostatcultureswereperformedinYNBOLmediumattwodistinctdilutionrates(i.e.0.16and0.08 h−1).ThefluorescencelevelsofYFPweremonitoredbyflowcytometrytoassesstheinductionlevelatthesinglecelllevel.Nosignificantdifferenceinthepromoterinductionlevelscouldbeobservedforthetwodilutionratestested(datanotshown).Indeed,themeanrelativefluorescenceofthecellpopulationwasequalto8.86 ± 0.62 × 104RFUatD = 0.16 h−1,andto9.47 ± 0.31 × 104RFUatD = 0.08 h−1.Moreover,cytogramsshowedthatthecellpopulationishomogenouslyinducedinpresenceoferythritol(Additionalfile2:FigureS1).ErythritolanderythruloseconcentrationmodulatethestrengthpHU4EYK300inductioninthepresenceofglycerolToassesstheinfluenceofinducerconcentrationontheregulationofpHU4EYK300,chemostatculturesofJMY6380wereperformedonYNBGmediumatadilutionrateof0.2 h−1.Atsteadystate,differentamountsoferythritolorerythrulosewereinjectedinthebioreactortoreachafinalconcentrationof0.2and0.6%(hereafter0.2CSPand0.6CSP),respectively.Glycerol,erythritol,erythruloseandYFPfluorescenceweremonitoredfor8 hafterinduceraddition.Inallexperimentalconditionstested,glycerolconcentrationremainedalmostconstant(i.e.3 g/L)inthebioreactor,confirmingthatasteadystatewasmaintainedinthoseexperimentalconditions.AsshowninFig. 8,pHU4EYK300inductionlevelseemstobemodulatedbytheinducerconcentrationinthoseexperimentalconditions(i.e.inthepresenceofglycerol).For0.2CSP,inductionincreasedduringthethreefirsthoursafterinducer(erythritolanderythrulose)addition(Fig. 8a,c).After,whentheinducerconcentrationwasbelow1 g/L,itremainedalmostconstantforthenext6 h.Bycontrast,for0.6CSP,inductionincreasedalmostlinearlyduring8 hafterinduceraddition(Fig. 8b,d).Itisworthmentioningthattheamplitudeofinductionalsoseemstobecorrelatedtotheinducerconcentration.ThemaximalYFPfluorescenceandthuspHU4EYK300induction,obtainedafter8 hoferythritoladditionwashigherforthe0.6CSPthanforthe0.2CSP(1.4 × 103and1.1 × 103RFU,respectively).Similarobservationsweremadeforerythrulose.ThemaximalYFPfluorescenceobtained8 haftererythruloseadditionwashigherforthe0.6CSPthanforthe0.2CSP(2.5 × 103and1.1 × 103RFU,respectively).ItcouldalsobededucedfromFig. 8,thaterythruloseyieldstohigherinductionlevelthanerythritol,eveninthepresenceof3 g/Lofglycerol.TheseresultsobtainedfromachemostatexperimentconfirmtheobservationsmadeinFig. 7b,i.e.pHU4EYK300isastronginduciblepromoter,respondingtoerythritolandevenmoretoerythruloseasaninducer.Fig. 8InductionofhybridEYK1promoterpHU4EYK300incontinuousculturebyerythritol(a,b)andbyerythrulose(c,d).Erythritolorerythruloseandglycerolconcentrationsintheculturemedium,andrelativefluorescenceofthecellsduringchemostatofJMY6380(pHU4EYK300)onYNB-glycerolmedium(1%glycerol).aInductionwithapulseof0.2%oferythritol.bInductionwithapulseof0.6%oferythritol.cInductionwithapulseof0.2%oferythrulose.dInductionwithapulseof0.6%oferythrulose.Time0correspondstothetimeofthepulse.Symbolsare:blackfilledsquare:erythritolorerythrulose;opencircle:glycerol;blackfilledtriangle:relativefluorescence(×103).FigureillustratesrepresentativeexperimentsFullsizeimage DeletionofEYKenhancedpEYKexpressionErythritolanderythrulosecouldbeusedbyY.lipolyticaasmaincarbonsource(Fig. 1).AlthoughglycerolwasfoundtorepressEYK1promoter,experimentsinchemostatdemonstratedthatamixtureofglycerol/erythritolorglycerol/erythrulosecouldbeusedforinduction.Moreover,botherythritolanderythruloseinducedpHU4EYK300promoterinadose-dependentmanner(Fig. 8).Therefore,wehypothesisethatpEYKexpressioncouldbeenhancedbydeletionoftheEYK1gene,thuserythritolanderythrulosecouldserveasinducerswhileavoidingtheiruseascarbonsourcesforgrowth.Therefore,theauxotrophiceyk1∆strainRIY176(Table 2)wasconstructedasdescribedin“Methods”.TheexpressioncassettecarryingpEYK300-YFP-LEU2exwasthenintroducedintoRIY176,givingrisetostrainRIY180(JMY6637).Sinceeyk1∆couldnotgrowonerythritolanderythruloseassolecarbonsource,strainRIY180wasgrowninthepresenceofglucoseorglycerol,usedasenergysource.Therefore,JMY6245(pEYK300-WT)andRIY180(pEYK300-eyk1∆)weregrowninYNBDOL(glucose,erythritol),YNBGOL(glycerol,erythritol),YNBDOSE(glucose,erythrulose),orYNBGOSE(glycerol,erythrulose).Inductionofthepromoterswasfollowedduringtimeinmicroplateswithglucoseorglycerolforgrowth(0.25%)andwitherythritolorerythruloseforinduction(0.25%).AsshowninFig. 9,YFPexpressioninwild-typeandeyk1∆strainsinthepresenceoferythritoloccurredduringthegrowthphaseinmediacontaining0.25%ofglucoseor0.25%ofglycerol(Fig. 9a,b).YFPfluorescenceat34 hofgrowthwas8.3-and7.8-foldhigherintheeyk1∆straincomparedtothewild-typestraininglucoseandglycerol,respectively(25,672SFUversus3078SFUwithglucoseand19,478SFUversus2500SFUwithglycerol).Incontrast,inthepresenceoferythrulose,YFPexpressioninwild-typeandeyk1∆strainswassomewhatdelayedfromthegrowthphaseinmediacontaining0.25%ofglucoseor0.25%ofglycerol(Fig. 9c,d).However,YFPfluorescencewas4.9-and2.6-foldhigherintheeyk1∆straincomparedtothewild-typeinglucoseandglycerol,respectively(9106SFUversus2993SFUwithglucoseand7934SFUversus3564SFUwithglycerol).Fig. 9TimecourseofYFPfluorescenceinwild-typeandeyk1∆strainunderpEYK300.YFPfluorescenceunderpEYK300inwild-typeandeyk1∆mutant,JMY6245andJMY6638,respectively.GrowthinminimummediaYNBcontaining0.25%ofcarbonsourceand0.25%ofinducer.aGlucoseanderythritol.bGlycerolanderythritol.cGlucoseanderythrulose.dGlycerolanderythrulose.Symbolsarewild-type(square)andmutant(triangle).Growth(fullsymbols)andfluorescence(emptysymbols)Fullsizeimage ForstrainJMY6245(pEYK300-WT),theratesofincreaseofYFPfluorescenceinthepresenceoferythritolwere97and83 FU/hinglucoseandglycerol,respectively.Incomparison,inthemutanteyk1∆,theratesofincreaseofYFPfluorescencewere10.5-foldhigher(1034and875 FU/hinglucoseandglycerol,respectively).Similarly,inthepresenceoferythrulose,higherinductionlevelswereobtainedfortheeyk1∆mutant(pEYK300-eyk1∆)ascomparedtothenon-disruptedmutant(pEYK300-EYK1).TherateofYFPproductioninthemutantstrainwas6.1-foldhigheringlucoseascomparedtothewild-typestrain(4000and347 FU/h,respectively).Inthepresenceofglycerol,thisincreasewas7.3-fold(2527and875 FU/h,respectively).TheseresultsdemonstratethatexpressionlevelscouldbefurtherimprovedbyusingastraindeletedfortheEYK1gene.Insuchastrain,erythritolorerythrulosecouldbeusedasafreeinducerandindependentlyfromgrowth,forhavinginductioneitherduringthegrowthphaseordelayedfromthislatter.ConclusionsSeveralgroupshaveconstructedhybridpromotersbasedoncombinationofrepeatsofupstreamactivatingsequence(UAS),TATAboxandcorepromoterforgeneexpressioninY.lipolytica[30,31,34,48].Thisgaverisetohybridpromoterswithvariousstrengths,uptotenfoldhigherexpressionthantheconstitutivepTEFpromoter[32],thislateronebeingaconstitutivestrongpromotercommonlyusedforgeneexpressionandforpromoterstrengthcomparison.AmongthemarefewstronginduciblepromoterssuchaspICL1,pLIP2,pPOX2[23,24,25,49].TheLIP2andPOX2promotersareinduciblebyoleicacidwhichhasthedrawbacktorequireoilemulsionforinduction.Thisstudyhasidentifiedanewshortpromoter(136 bp)induciblebyerythritolorbyerythrulose.Furthermore,promotercomparisonallowedtoidentifyaveryshort(43 bp)upstreamactivatingsequence(UAS1EYK1)andapotentialupstreamregulatorysequence(URSEYK).ThisstudyhasgeneratednewhybridpromoterscombiningcoreEYKpromoterwitheitherUAS1EYK1orUAS1XPR2upstreamactivatingsequences,allowingatleastatenfoldhigherexpressionthanthepTEFpromoter.ThisopensthepathtothedesignofnewsyntheticpromoterscontainingUASEYKand/orURSEYKwithhighertandemrepeatsnumberorwithvariouscorepromoterstofurtherwidentheexpressionrangeandtheinductionprofiles.Thesepromotersarepoorlyexpressedinglucoseorglycerolandcouldbeinducedbyerythritolorbyerythrulosewithatremendousadvantageofbeingdosedependantthusallowingfinetuningofinductionwhichwillpermittovarythedegreesofexpressionthatcouldbeobtained.OnewouldbeeasilyabletoregulatetheexpressionleveldependingonUAS1EYK1copynumber,theinductiontimedependingontheinducerused(erythritolorbyerythrulose),theinductionleveldependingonthemediaandtheinducerconcentration.Onewillhavealsotochoiceusingerythritolorerythruloseasinducerandsourceofcarbonforgrowthoruseonlyasinducerina∆eyk1geneticbackground.TheseinduciblepromotersandUAS1EYK1expandthepartsavailableforproteinexpression[33]andforthedevelopmentoftoolsforgeneticengineeringsuchasadditionalmarkerforgenedeletionormarkerrescue[40,41]andforinducibleexpressionofgenesuchasCAS9forgenomeediting[50,51].ThesenewpromoterscouldbealsoapowerfultoolforfundamentalresearchaswasthedevelopmentoftheGAL1promoterinSaccharomycescerevisiae[52]. AbbreviationsCSP: carbonsourcepulse D: dilutionrate EYK1: erythrulosekinase FU: fluorescenceunits(flowcytometry) GRAS: generallyrecognizedassafe Leu/LEU2: 3-isopropylmalatedehydrogenasegene PCR: polymerasechainreaction SFU: specificfluorescenceunits(Biolector) mSFU: meanspecificfluorescence UAS: upstreamactivatingsequence Ura/URA3: orotidine-5′-phosphatedecarboxylase Vvm: volume(ofgas)pervolume(ofculturemedium)perminute YFP: yellowfluorescentprotein YNBD: YNB + glucose YNBG: YNB + glycerol YNBOL: YNB + erythritol YNBOSE: YNB + erythrulose YNBDOL: YNB + glucose + erythritol YNBGOL: YNB + glycerol + erythritol YNBDOSE: YNB + glucose + erythrulose YNBGOSE: YNB + glycerol + erythrulose ReferencesMadzakC.Yarrowialipolytica:recentachievementsinheterologousproteinexpressionandpathwayengineering.ApplMicrobiolBiotechnol.2015;99:4559–77.doi:10.1007/s00253-015-6624-z.CAS  Article  GoogleScholar  MadzakC,BeckerichJM.HeterologousproteinexpressionandsecretioninYarrowialipolytica.In:BarthG,editor.Yarrowialipolytica.Springer:Berlin;2013.p.1–76.doi:10.1007/978-3-642-38583-4_1. 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