1、鏈霉菌UDP-葡萄糖脫氫酸編碼基因Ste6的克隆表達(dá)和性質(zhì)研究遺傳學(xué)報ActaGeneticaSinica,November2005,32(11):12131220SSN03794172Cloning,ExpressionandCharacterizationoftheSte6GeneEncodingaUDP-glucoseDehydrogenaSeinLIHao,WANGLingYah,XUGui-Yun,CHENYang,JIANGRong,LIYuan(1.InstituteofMedicinalBiotechnology,ChmeseAcademyofMedicalSciences,B

2、e?枷9100050,China2.1nst|tuteofChemistry.CheeseAcademyofSciences,Bejlng,00080chInaAbstract:Streptornycessp.139wasidentifiedtoproduceanewexopoIysaccharideEbosin(139A)withantirheumaticarthritisactivityvivo.TheEbosinbiosynthesisgenecluster(31.3kb:GenBankAccessionNumber:AY131229)containing22ORFs(stelste22

3、)ofStreptomycessp.139hadbeenreportedpreviously.Inthispa-per,wepresentexperimentalevidencefortheidentityoftheste6geneproductasaUDPglucosedehydrogenase(UDPGDH).WithpET-30aasvector,thegenewasclonedandexpressedinEscherichcoilBL21(DE3).TheexpressedproteinwaspurifiedtohomogeneibyHisBindresinaffinitychroma

4、tograpyanditwasabletocatalyzeUDPglucosetoUDPglucuronicacid.Toevaluatethefunctionofste6,thegenewasdisruptedbyasingle-crossoverhomologousrecombinationeventandtheresultshowedthatste6isrequiredinEbosinbiosyn?thesis.Keywords:ste6;UDP-glucosedehydrOgenase;genedisruption;Ebosinbiosynthesis鏈霉菌UDP-葡萄糖脫氫酸編碼基因

5、Ste6的克隆表達(dá)和性質(zhì)研究李顥,王玲燕,徐桂云,陳陽,(1中國醫(yī)學(xué)科學(xué)院醫(yī)藥牛物技術(shù)研究所2.中國科學(xué)院化學(xué)研究所,北京姜蓉,李,北京100050;100080)元,摘要:鏈霉菌139能夠產(chǎn)生一種全新的胞外多糖依博素(139A),該多糖體內(nèi)具有顯著抗類風(fēng)濕性關(guān)節(jié)炎活性.其生物合成基因簇(GenBankAccessionNumber:AY131229)已被鑒定約31.3kb,包含22個開放閱讀框(stelste22).以pET一30a為載體,克隆并在大腸桿菌BL21(DE3)中進(jìn)行了ste6基因的表達(dá),對該基因的克隆,表達(dá)與性質(zhì)進(jìn)行了研究.親和層析法證實,純化后重組蛋白具有催化UDP-葡萄糖脫

6、氫變成UDP-葡萄糖醛酸的活性.這表明ste6編碼產(chǎn)物是葡萄糖脫氫酶.為了證實ste6基因與依博素生物合成的關(guān)系,采用單交換基因破壞策略構(gòu)建了ste6基因阻斷突變株.結(jié)果初步顯示ste6和依博素生物合成相關(guān).關(guān)鍵詞:ste6;UDP-葡萄糖脫氫酶;基因阻斷;依博素生物合成中圖分類號:Q786文獻(xiàn)標(biāo)識碼:A文章編號:0379-4172(2005)11-1213-06收稿日期:20050107;修回日期:20050309基金項目:國家高科技項目基金(編號:2001AA一21409)SuppodedbytheChineseNationalHighTechnologyProject(No.2001AA

7、一21409)作者簡介:李顥(1981一)男,江蘇南京人,在讀碩士生,研究方向:微生物與生物藥學(xué)通訊作者.E-mail:yuanwli;Tel214遺傳學(xué)報ActaGeneticaSinicaVo1.32No.112005CellsurfacepOIysaccharidescancompletelysecreteintoenvironmentasexOpOIysaccharides(EPSs)inawidevarietyofbacteria.ManyEPSshaveuniquephysicalandrheologicalpropertiesforfoodapplic

8、ationsandareusedasnaturalbiOthickenersandstabilizerstoimprovethetex-tureandviscosityoffermentedproducts.Moreover,ithasbeensuggestedthatsomeEPSsproducedbylacticacidbacteriamayconferhealthbenefitstotheconsumerandmousemodeIstudieshaveindicatedthatEPSmayhaveimmu-nostimulatory.antitumoralorcholestero1.1o

9、w.eringactivity.ThebiosynthesisofEPSsconsis-tingofrepeatingunitshasasimliarmechanism.Therepeatingunitsareassembledatmembranebyglycosyltransfer-rasesthatlinksugarsfrOmnucleotidesugartoalipidcarrier,subsequentlypolymerizedandexportedtothecellsurface.Ingram.positivebacteria,theinvestigationofEPSsgenecl

10、usterhasadvancedrecently.Strep-tomyces,aGram-positivebacterium,iswellknownasanimportantindustrialmicroorganismfortheirproductionofnaturallyderivedantibiotics.Howev-er,littleisknownofEPSproductioninStreptomy-ces.Recently,inourlaboratory,Streptomycessp.139wasidentifiedtoproduceanewEPSdesig?natedEbosin

11、(139A)thatshowedantirheumaticarthritisactivitynvivo.Qualitativesugaranaly-sisindicatedthatEbosincontainsgalactose,arabi-nose,mannose,fucose,xylose,rhamnose,galac-turonicacidandglucose.Wehavealsorepor-tedtheidentificatiOnandcharacterizatiOnofano.velgeneclusterinvolvedinthebiosynthesisofEbosin(139A)fr

12、omStreptomycessp.139.Itcon-sistsof22ORFs,namedstelthroughste22,Ioca-tedona32kbchromosomalregionofStreptomy-cessp.139.Amongthem.sixhypotheticalproteins(ste6,ste10,stel1,stel6,stel7andstel9)maybeinvolvedinnucleotidesugarprecursorsynthe-sisandEPSmodification.ste6,byhomologycomparison.appearedtocodefora

13、putativeUDP-glucose/GDP.mannosefamilydehydrOgenase.Theobjectiveofthisstudywastoexperimentallyi-dentifytheproteinencodedbyste6andinvesti.gateitsfunctioninthebiosynthesisofEbosin.MaterialsandMethods1.1Strains,media,cultivationandplasmidsStreptomycessp.139.whichtaxonomicspe-cieswasidentifiedbutunpublis

14、hedfChinaGener-aIMicrobiologyCultureCollectionCenter.No.0405)wasgrownat28oc.eitherinTSBmediumsupplementedwith5mmol/LMgCI2and0.5%glycine,orinfermentationmedium(1%glucose,2%starch,2%soybeanextract,0.2%tryptone,0.2%beefextract,0.4%yeastextract,0.05%K2HPO4,0.3%CaCO3,pH7.3).EscherichcoliBL21(DE3)wasgrown

15、inLBmediumat37C.PlasmidpKC1139wasprovidedbyDr.WenLiu,plasmidpET-30awaspresentedbyProf.ChenandplasmidpLY5015containingEbosinbiosyn-thesisgeneswasconstructedpreviously.1.2Cloningandexpressionoftheste6geneTheste6genewasPCRamplifiedfrOmplas-midpLY5015.Theprimersusedwere5GCGATCCATGCGTGTCAGCGTGTTCG3and5GC

16、AAGCTTTTACCAGGCAAGGCCCACG3(theun-derlinedIetterscorrespondtoBamHlandHindrestrictionsitesrespectively).ThePCRamplificationwasperformedunderthefollowingconditions:aninitiaIdenaturationat94ocfor3minthen30cyclesof1minat94.1minat66and2minat72oc.andfinally10minat72oc.Theamplified1.3kbDNAfragmentwasclonedi

17、ntopET-30avectortoconstructpET-LH17.Thenu.cleotidesequenceofamplifiedDNAfragmentclonedinpET-LH17wasanalyzedusinganABIPRISM377XLDNASequencerrAppliedBiosys-tems).E.coilBL21(DE3)wastransformedwithplasmidpLH17andculturedovernightinLBbrothcontainingkanamycin(20ug/mL).Theculturewasdiluted1:20withLBbrothan

18、dsubjectedtoLIHaoeta1.:Cloning,ExpressionandCharacterizationoftheSte6Gene1215furtherincubationat37oCuntiItheabsorbanceat600nmreachedto0.61.0.Isopropyl3-Dthio-gaIactopyranoside(IPTG)wasaddedtothecul-tureatafinaIcOncentratiOnOf0.3mmol/L.Afterfurtherincubationat37oCfor6h.thebacterialcellswereharvesteda

19、ndresuspendedinthebind-ingbuffer(5mmol/Limidazole,0.5mol/LNaCI,20mmol/LTris-HCIpH8.0).Bacteriaweresoni-catedandthecelIdebriswasremovedbycentrifu-gation(10000g,15min)at4oC.1.3Purificationoftherecombinantproductofsfe6AfterremovingthecelIdebris.the2.5mLsu-pernatantwasIoadedona2.5mLNi.NTAHis.Bindresinco

20、lumn(Novagen)pre-equilibratedwith7.5mLbindingbuffer.Thecolumnwasthenelutedwith2x5mLbindingbuffer.2x5mLwashbuffer(60mmol/Limidazole.0.5mol/LNaCI,20mmol/LTris-HCIpH8.0)and10mLelutebuffer(750mmol/Limidazole,0.5mol/LNaCI.20mmol/LTris-HCIpH8.0)successively.Thefrac.tionscontainingtherecombinantproteinenco

21、dedbyste6werecollectedanddialysedwithH.Oat4oC.ThepurityoftheproteinwasascertainedbySDS-polyaclamidegelelectrophoresis(PAGE).1.4EnzymeassayUDP.glucosedehydrOgenaseactivitywasmeasuredasdescribedbyLineta1.Thereactionmixture(1mL)containing0.2mol/LTris-HCIpH8.5,1pmol/LNAD.0.5pmol/LUDP-glucoseand300Ugpuri

22、fiedste6proteinwasincubatedat25oCfor30min.Afterthereaction,10uLsam.piewastakenforHPLCanalysis.ThesamplewasinjectedtoaKromasiIKR100-5NH,(4.6x250mm)column,mobilephase:55%acetonitrileand45%0.05mol/LTris-HCIinH,0,flowrate:0.4mL/min.Theeluentsweremonitoredat260nm.OneunitofUDP-glucosedehydrOgenaseisde-fin

23、edastheamountoftheenzymecatalyzingthereductionof1mol/LofNADpermin.1.5Disruptionofste6Toevaluatethefunctionoftheste6gene,knockouttransformantsweregenerated.A0.5kbinternalfragmentofste6(residues8232-8716)wasPCRamplifiedfrOmpLY5015usingprimers:5GCGGATCCCGTGTCAGCGTGTTCG3and5GCAAGC-I-TCCAGCGTGCGGGACTTCTT

24、3(HindandBamHIrestrictionsitesareunderlined).ThePCRproductwasclonedintopKC1139.tOcreateplasmidpLH1327.AfterpropagationinE.coilET12567J.pLH1327weretransducedin.toStreptomycessp.139bypOIyethyIenegIycOI(PEG)mediatedprotoplasttransformation12.Af-terincubationat28for16to20h.theplateswereoverlaidwithsoftR

25、2YE(0.7%agar)con-tainingapramycin(40pg/mL).PlasmidpLH1327bearsatemperature?-sensitiveStreptomycesrepli?-cationoriginthatisunabletoreplicateattempera-turesabove34.Therefore.thetransformantswerefirstincubatedat28(3for2daysuntilpin-pointsizecoloniesbecamevisible,thenshiftedto37oCforfurtherincubation.Mu

26、tantsresultingfromsingle.crossoverhomologusrecombinationgrewoutoftheoriginalpinpoint-sizecoloniesinseveraldays.1.6lsolationofEbosinandthederivatesThefiltrateobtainedbyfiltrationofthewholebrothwasappliedtoaDiaionHP-20column.Theeluentwassubjectedto0.07x7styrenecationexchangeresintoprecipitationtwiceby

27、adding60%ethano1.TheresultantbrownishproductwasdissolvedinaminimumamountofwaterandIoad.edonaDEAE-DextranA-25column.ThecolumnwaselutedwithH2O,0.2mol/LNH4CIand0.5mol/LNH4CIsuccessfully.Ebosinandthederiva.tivespresentedinfractionselutedbyH2Owerelyophilized.1.7CompositionanalysisofEbosinandthederivatiVe

28、SThecompositionanalysiswasperformedas1216遺傳學(xué)報ActaGeneticaSinicaVo1.32No.112005described.Inshort.thesample(2mg)wasdis?solvedin2mLofTFAtohydrolysefor1hat120oC.thendryingunderastreamofnitrogen.Subsequently,theresiduewastreatedwith0.5mLof0.5mol/LNaOHandNaBHatroomtern?peratureovernight,neutralizedwithace

29、ticacid,thendischargedtheexcessNaBH4withmethanoI.TheresiduewasdriedwithP2O5invacuofor4h,thenacetylatedwith0.5mLofaceticanhydridein0.5mLofpyridineat100oCfor30min.Aftercool-ing,theexcessreagentwasdischargedunderastreamofnitrogen,thensamplewasanalyzedbyGas.Chromatography?MassSpectrum(GC?MSTR12000,Engla

30、nd).2Results2.1Cloningandexpressionoftheste6geneinE.coflTheste6geneofStreptomycessp.139wasPCR?amplifiedusingplasmidpLY5015asatern?plateandclonedintothepET?30avectortocon?structpLH17.Thenucleotidesequenceanalysisofste6bythedideoxychainterminationmethodcon?firmeditsidentity,comparedwiththepublishedse.

31、quence(GenBankaccessionnumber:AYI31229,.ThegenewasexpressedinE.coilBL21(DE3)andtheproteinencodedbyste6waspro.ducedbothinsolublefOrmandininclusionbody.Afterthebacteriawassonicated,analysisbySDS?PAGE(Fig.1)withCoomassiebluestainingindi.catedthatthesupernatantandthepelletscon-tainedtherecombinantprotei

32、nwithmolecularweightinagreementwiththeexpectedsize48.98kD,(including2.5kDofhis-tag,s?tagorigina.tedfrOmpET?30a).2.2Purificationoftheproteinencodedbyste6Theste6geneproductexpressedinthesu.pernatantofsonicatedE.coliBL21(DE3)waspurifiedtohomogeneitybytheHis?Bindresinaf.34Fig.1SDS-PAGEanalysisoftheprote

33、inencodedbyste6expressedinE.coliBL211:Molecularmassstandard;2:Thesupernatant.3:Thepellets:4:ThelysissampleofE.coil(pET一30a);Thearrowshowstheproteinband.finityChrOmatOgraphy.Eachfractionselutedbydif?ferentbufferswascheckedbySDS?PAGE.AfterthechrOmatOgraphy,HPLCanalysisdemonstratedthepurityoftheprotein

34、elutedbytheelutebuffert0beabout90%(Fig.2).10534931q)1416449Fig.2HPLCanalysisofthepurifiedproteinencodedbyste6ThesamplewasinjectedtoareversedphaseSUPELCSILLC一308column(C8):mobilephase.(A)acetontrile(20%一80%),(B)0.05%TFA(80%一20%),flowrate:0.5mL/min.Theeluatesweremonitoredat280nm.2.3UDP-glucosedehydrog

35、enaseactivityoftheste6geneproductTheHPLCKromasilKR100?5NH2column藤LIHaoeta1.:Cloning.ExpressionandCharacterizatiOnoftheSte6Genel2l7(4.6x250mm),mobilephase:55%acetonitrile45%0.05mol/LTrisHCIinH,O,flowrate:0.4mL/minanalysisresultsshowedthatthereten.tiontimeofstandardUDPglucoseis18.192minandUDPglucuroni

36、cacidis19.036min(Fig.3.AandC).Aftertheenzymaticreactioncatalyzedby5623O89TimefmIn1A3theste6protein,theretentiontimeoftheproductis18.94min(Fig.3.B),whichissimilartothatofUDPglucuronicacid.Suchresultsdemonstratedthattheste6geneproductinStreptomycessp.139isaUDPglucosedehydrOgenase,whichisabletocatalyze

37、UDPglucosetoUDPglucuronicacid.昌45938蚤Ir一一號627.TImefmIn1BFig3HPLCanalysisforUDPglucosedehydrogenaseactivityoftheste6geneproductA:HPLCanalysisofthestandardUDP.glucose;B:HPLCanalysisfortheproductoftheenzymaticreactiOn:C:HPLCanalysisofthestandardUDPglucuronicacid.2.4Disruptionofste6Toinvestigatethefunct

38、ionrelatedtoEbosinbiosynthesisfortheste6gene,ste6wasinser.tionallydisruptedbyasinglecrossoverhomolo.gousrecombinationevent.A0.5kbinternaIfrag.mentofste6wasclonedintovectorpKC1139tOyieldpLH1327,whichwastransductedintoStrep.tomycessp.139(Fig.4,A).CorrectintegrationinStreptomycesLH9827ste6.mutantstrain

39、wasidentifiedbySouthernhybridization(Fig.4,B).AsshowninFig.4B,adistinctivehybridizationbandofthepredictedsizeof8.15kbwasdetectedwiththevectorpKC1139asaprobeinthemutantstrainStreptomycesLH9827(Lane2),butthisbandwasabsentinthewildtypestrain(Lane1).Complimentarily,whenthe0.5kbinternalfrag.mentofste6was

40、usedasaprobe(Fig.4,C),the3.85kbbandinthewildtypestrain(Lane1)wassplitandchangedintotwofragmentsof8.15kband3.0kbinthemutantstrain(Lane2).Suchresultswereconsistentwiththeexpectancyfordisruptionofste6byasingle.crossoverho.mologousrecombinationevent.AfterisolationoftheEbosinderivativespro.ducedbythemuta

41、ntstrainStreptomycesLH9827,compositionanalysiswascarriedoutu.singthemethodmentionedbefore.Gas.Chroma.tographyMassSpectrumresultsshowedthatquantityofgalactosewasabout47.5%inthede.rivatives(Fig.5),whichisremarkablyhigherthan36.9%inEbosin.Cheneta1.reportedthatEb.oslniscomposedofrhamnose,xylose,glucose,

42、mannose,arabinose,fucose,galactoseandgalac.turonicacid.Afterdisruptionofste6,thepercent.ageofgalactosewaschangeableinthederiva.tivesproducedbythemutant.Fromthisresult.geneste6appearedtoberequiredforthebiosyn.thesisofEbosininStrepmycessp.139.l2l8遺傳學(xué)報ActaGeneticaSinicaVo1.32No.112005A139CeSLH9827128.15kbB12385kb+C謦+一8.15kb搴+一30kbFig.4Disruptionofgeneste