一种新的链霉菌表达载体启动子

一种新的链霉菌表达载体启动子【摘要】eryA基因直接控制着红霉素母环6-脱氧-红霉内酯B的合成，在红霉素生物合成过程中具有重要作用。本文克隆了eryA基因的启动子PeryA，以绿色荧光蛋白基因为报告基因，构建了大肠埃希菌-糖多孢红霉菌穿梭型质粒。PEG介导原生质体转化法将穿梭型质粒分别转入糖多孢红霉菌A226与变铅青链霉菌JT46，荧光显微镜检测发现，此启动子在两菌株中都具有功能。随后，以变铅青链霉菌JT46为宿主，对PeryA启动子区域进行了深入研究，结果发现该启动子的-35区并不是必需的，仅有-10区、长度为41bp的该启动子在链霉菌中仍具有功能。定点突变证明-10区对于该启动子是必不可少的。因此，41bp的该启动子片段可作为链霉菌的有效启动子，这是迄今为止所发现的最短的启动子之一，可用于构建新的链霉菌表达载体。

【关键词】eryA基因；启动子；糖多孢红霉菌；变铅青链霉菌；增强型绿色荧光蛋白

ABSTRACTTheeryAgenesplayaveryimportantroleinthebiosynthesisoferythromycinmacrolactonering(6-deoxy-erythronolideB,6-dEB).Inthispaper,anEscherichiacoli-SaccharopolysporaerythraeashuttlevectorcontainingtheeryApromoterregionwasconstructedusingtheenhancedgreenfluorescentprotein(EGFP)geneasareporter.TheshuttleplasmidwastransformedintoA226andStreptomyceslividansJT46byPEGmediation,respectively.FluorescencemicroscopyconfirmedthattheEGFPwasexpressedinbothstrains.Subsequently,thecharacteristicsoftheeryApromoterregionwereexclusivelystudiedinthehoststrainJT46,andtheresultsshowedthatthepredicted-35regionwasnotnecessaryforthepromoterandthe41-bppromoterDNAfragmentonlycontainingpredicted-10regionwasstillfunctionedinStreptomyces.Site-directedmutantdemonstratedthatthepredicted-10regionwasindispensableforthepromoter.Thus,the41-bppromotersegmentcanfunctionasaneffectivepromoterofStreptomycesexpressionvector,whichisoneoftheshortestpromotershithertofoundinStreptomycesandisusefulforconstructingnewStreptomycesexpressionvectors.

KEYWORDSeryAgene;Promoter;Saccharopolysporaerythraea;Streptomyceslividans;Enhancedgreenfluorescentprotein(EGFP)

Saccharopolysporaerythraeaasamycelium-formingactinomycete,isthemajorproduceroferythromycin,amacrolideantibioticthathasanimportantapplicationinclinic.Extensivegeneticstudieshaveprovidedsomeinsightsintothegenesinvolvedintheerythromycinbiosynthesis［1］.Ithasbeenproposedthatthebiosynthesisoferythromycinmightbeseparatedintotwostages［1］:(i)thesynthesisoferythromycinmacrolactonering(6-deoxy-erythrolideB,6-dEB)requiringonemoleculeofpropionyl-CoAandsixmoleculesofmethylmalonyl-CoA;and(ii)thetransformationof6-dEBintoerythromycinAinaseriesofreactionscatalyzedbystereospecifichydroxylases,glycosyltransferasesandmethyltransferases.Similartoothersecondarymetabolicpathwaygenes［2～5］,theerythromycinbiosyntheticgenesexistinclusteronachromosome.Nevertheless,thereisnoregulatorassociatedwiththeerygenesofandnoothergeneinthisorganismhasyetbeenshowntocontrolerythromycinproduction［1］,whichisobviouslydifferentfromtheantibioticbiosyntheticregulatorygenesinmostStreptomycesspp.［6,7］.Inrecentyears,theyieldsofantibioticshavebeengreatlyenhancedbyoverexpressionorknockoutofcertainkeygenesandsomenewantibioticshavealsobeenobtainedbycombinatorialbiosynthesis［8～11］,whichinspiresustobemoreinterestedintheexpressionsystemofStreptomycessp.andAsanimportantelementofgeneexpression,promoterisnodoubtakeyfactorofunderstandingthegeneexpressionsystems.

ThecentralportionoferythromycinbiosyntheticclustercontainsthethreeeryAgenes(eryAI,eryAIIanderyAIII)encoding6-deoxy-erythronolideBsynthase(DEBS)［12,13］,whichareco-transcribedunderthecontroloferyApromoter.Reeveetal.［14］havelocatedthetranscriptionalinitiationsiteoferyApromoterusingS1nucleaseprotectionassayandpredictedthe-10and-35regionsofthepromoterbythealignmentof10differentpromotersinerygenecluster.However,amoredetailedanalysisofthepromoterregionhasnotyetbeenreported.

Inthisstudy,anSac.erythraeashuttlevectorwasconstructedusingenhancedgreenfluorescentprotein(EGFP)asareporterinordertoconvenientlyfacilitatetheinvestigationoftheeryApromoter.Asiswellknown,StreptomyceslividanshasclosekindredtoandthespecieshasbeenthegeneralhostofStreptomycesingeneticmanipulationbecauseofitscleargeneticbackground.Therefore,wechoseasthehoststraintoexaminewhethertheeryApromoterwasactiveinthestrain.Inaddition,forfurthercharacterizationoftheeryApromoterregionasapromoterofStreptomycesexpressionsystem,aseriesofnovelreportshuttleplasmidswereconstructedcontainingdifferenteryApromoterDNAfragmentsandtransformedintoThroughthefluorescencedetectionofthetransformants,weconfirmedapromoterthatcontainsonly-10regionoferyApromoter,from-41to-1relativetothestartpointoftranslation,wasfunctionalintheStreptomyces.ThepromoterhaspotentialforconstructingnewStreptomycesexpressionvectorsbecauseofitsveryshortlength.

1Materialsandmethods

Strains,plasmidsandcultureconditions

A226andJT46weregiftsfromProf.WangYi-guang,InstituteofMedicinalBiotechnology,ChineseAcademyofMedicalSciences.A226wasgrownintrypticsoybroth(TSB;Difco,)［15］forpreparationoftotalDNA,andonR3Magarplate［16］fortheregenerationofprotoplastsandtheselectionoftransformants.EscherichiacoliDH5αwasusedasageneralcloninghostandwasgrownandmaintainedaccordingtothestandardmethod［17］.ThedetailsofandculturewerereferredtoKieseretal［15］.ThevectorpUC18wasusedforgeneticmanipulationandcloning.PlasmidspEGFP-N1(Clontech,),pUEKML［18］andpWOR120-N［18］wereusedforconstructingreportershuttleplasmids.

CloningoftheeryApromoterregions

ThegenomicDNAofwasisolatedasdescribed［15］.BasedontheeryAgenesequences(GenBankaccessionno.M63676),fourpairsofprimers()weredesignedtoacquirethepromoterregions.Polymerasechainreaction(PCR)wasusedtoamplifythePeryAandPeryA1fragmentsandperformedat96℃for5min,andthencycled30timesat94℃for1min,65℃for1min,72℃for2min,followedbyincubationat72℃for5min.Theamplifiedproductswererecoveredfromanagarosegel,digestedwithAatIIandNdeI,andligatedintothepUC18togeneratepUPandpUP1.However,throughonlydenaturalizationandannealingofforwardandreverseprimers,PeryA2andPeryA3fragmentswereobtainedwithAatIIandNdeIadhesiveendsandclonedintothepUC18tocreatepUP2andpUP3.

Constructionofreportershuttleplasmids

TocharacterizetheeryApromoter,pUPW-EGFP,areportershuttleplasmid,wasconstructed.TheDNAfragmentcarryingmultiplycloningsites(MCS)andfdtranscriptionterminatorwasremovedfrompUEKMLwithNdeIandEcoRIandclonedintothesamesitesofpUP,yieldingpUPKML.PCRwasperformedusingplasmidpEGFP-N1asatemplate,andthePCRproductwasclonedintothepUC18toyieldpUC-EGFP.TheEGFPgenefrompUC-EGFPwasthensubclonedintoNdeI/HindIII-digestedpUPKMLtogeneratepUP-EGFP.Finally,aKpnI-digestedandCIAP(calfintestinalalkalinephosphatase)-dephosphorylatedpUP-EGFPwasrelocatedtothesamesiteofpWOR120-N,creatingpUPW-EGFP.OtherthreereportershuttleplasmidscontainingdifferenteryApromoterDNAfragmentswereconstructedasdescribedabove.

TransformationofandStreptomycesandmicroscopicanalysisoftransformants

Thepreparationandtransformationofandprotoplastsweredescribedinliterature［19］and［15］,respectively.TheplasmidpUPW-EGFPwasintroducedintoA226andJT46protoplastsbypolyethyleneglycol(PEG)mediation,thuscreatingA226(pUPW-EGFP)andJT46(pUPW-EGFP).ThemicroscopywascarriedoutonaZeissAxiophotphotomicroscopeequippedwithafluorescencefiltersetfordetectingEGFPofthesetransformants.ImageswereobtainedwithanAxiocam.TheAxioVisionsoftware(AxioVisionVersion)wasusedtoacquireimages.

2Resultsanddiscussion

Constructionofreportershuttleplasmid

Inordertoinvestigatethepromoteroferythromycinbiosyntheticgenes,theprimers()weredesignedtoamplifyaneryApromoterregion(PeryA)andtheDNAfragmentwasclonedtoyieldpUPasshownin“Materialsandmethods”.SequencingconfirmedthatthePeryAwasentirelyconsistentwiththecorrespondingsequenceinreference［13］.Inmanyorganisms,thegreenfluorescentprotein(GFP)ofthejellyfishAequoreavictoriahasprovedtobeaparticularlyusefulandsensitivereporter［20,21］.TheEGFPgenewasamodifiedversionoftheGFPgeneandwasobtainedfromtheeukaryoticexpressionplasmidpEGFP-N1.PreviousworkshavedemonstratedthattheEGFPcouldbeexpressedin［18］.SowefusedthePeryAtothepromoterlessEGFPgenetohandilystudythepromoteractivity.Thus,thereportershuttleplasmidpUPW-EGFPwasconstructedbyaseriesofsubcloningasdescribedin

CharacteristicoftheeryApromoter

PermE(promoteroferythromycinresistancegene)isusedwidelyfortheexpressionofheterologousgenesinStreptomycesandhascomparativelystrong,constitutiveactivityandrelativelywellcharacterized［22］.Moreover,theEGFPexpressioncouldbeinducedinunderthecontrolofthePermEfromconstructed

ConstructionofreportshuttleplasmidpUPW-EGFPplasmidpZM-EGFP［18］.Therefore,wetooktheA226(pZM-EGFP)andJT46(pZM-EGFP)transformantsaspositivecontrols.TheplasmidpUPW-EGFPwastransformedintoA226andJT46byPEGmediation,respectively.Then,theA226(pUPW-EGFP)andJT46(pUPW-EGFP)transformantswerescreenedunderthepresenceofthiostrepton.Myceliumsofthefourtransformantsallemittedgreenfluorescence.TheseresultsrevealedthattheEGFPwasnotonlyexpressedinbutalsoinStreptomyces,showingthattheeryApromoterisactiveinbothstrains,namely,itseemednottobegenus-specific,andthatthepromoterwasnotcontrolledbypathway-specificregulators,whichapparentlydifferedfromthepromotersofmostStreptomycesantibioticbiosyntheticgenes［23］.Thus,itwasimperativeforustofurtherstudyitscharacteristicsasapromoterofStreptomycesexpressionvector.

DeterminationoferyApromoteractivityregioninStreptomyces

EvidencesabovehaveconfirmedthatthePeryAdidworkinStreptomyces.SoitwasnecessarytocharacterizetheminimalfunctionalregionoftheeryApromoterinthehoststrainThePeryA1fragmentwasclonedcarryingthepredicted-10and-35regions(),andtheplasmidpUP1W-EGFPwasconstructed.ThetransformantJT46(pUP1W-EGFP)emittedgreenfluorescence.Furthermore,theplasmidpUP2W-EGFPwasbuiltcontainingonlythepredicted-10regionofthepromoter.Tooursurprise,theEGFPexpressioninStreptomyceswasstillvisible.Tofurthertestwhetherthepredicted-10regionservesasanindispensableelementofStreptomycespromoter,wemutatedinitialATTofthisregiontoCGGasshowninHowever,therewasnoEGFPexpressioninStreptomyces,meaningthatthepredicted-10regionwasessentialforthepromoter.TheseresultsshowedthatthePeryAwithonly-10regioncouldfunctionasaneffectivepromoterofStreptomyces.

Bibbetal［22］pointedoutthatsequencescorrespondingtotheconsensus-35regionoftheermEpromoterhadgreatervariabilitythanits“-10”sequence.BydeletionmutationofStreptomycescoelicolorsapAgenepromotersequences,Hana［24］foundthateven18-bppromoterthatextendedfrom-8to+10relativetothetranscriptionalstartpointretainedbasalactivityofthewild-typesapApromoter,anditwasdeducedthattheadjoiningvectorDNAupstreamofthe18-bppromoterwasprovidingcis-actingsitesforexpressionofthesapAgene.SowemanagedtoaligntheupstreampromotersequencesofthePeryA2fragmentanditsadjoiningpUC18vectorDNA.Asshownin,therewaslittlesimilarity,whichsuggestedthatinaccordwiththeresultsofBibb［22］andHana［24］,theupstreampromotersequencesofthepredicted-10regionwasalsonotconserved,predicatingthatitseemednottoberequiredfortheeryApromoterinStreptomyces.

Streptomycetegeneshavesofarshownawidediversityofpromotersequencesandtranscriptionalpatterns.Thepromotersfallintothreebasiccategoriesatleast［25,26］:(i)thepromotersdesignatedas,likeStreptomycespromotersonthebasisofsimilaritytothepromotersrecognizedbytheσ70;(ii)promotersjustsimilartoprokaryoticclassicalpromoter10region;and(iii)promoterspossessingnoanalogoussequencesto-10and-35regionsofprokaryotictypicalpromoters.Inthisstudy,wefoundthattheeryApromotermaybelongtothesecondtypeofStreptomycespromoters.Toourbestknowledge,thisisthefirstreportindicatingthatthepredicted-35regiondoesnotappeartobeindispensableforthepromoterinStreptomyces;moreover,theonly41-bppromoterDNAsegmentcanworkasaneffectivepromoterofStreptomyces,whichisveryshort.Thus,itwillbeveryusefulforbuildingnewStreptomycesexpressionvectors.

AlignmentoftheupstreamDNAsequencesfromtranslatingsiteinpUP1W-EGFPandpUP2W-EGFP

(TheclonedDNAfragmentscarryingdifferenteryApromoterregionsareunderlined

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