Taxol and Taxane productionTaxomyces andreanae, an Endophytic Fungus of Pacific Yew.docx

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1、A.Scicr1.candG.Strobe1.DepartmentofP1.antPaiho1.ogy,MonunaSuieUniversity.Bozeman,MT59717.D.Sticr1.c.DepanmcniofCbcmistAandGcchemistry.MontanaCo1.kgCofMiDeridScienceandTechno1.ogytBune4MT59701.Taxo1.andTaxaneproductionhTaxomyctsandreanaeanEndoph)1.ieFUngUSfPaC1.fkYevAndgaSiierk,GaryS11be1.,Donak1.Si

2、ierkM.C.Wani.H.1.Tav1.or.M.E.Wa1.1.P.Coggon.A.T.MCPmAm.Ciwm.93、2325(1971)Taxomyccsandrearwe.funga1.endophyte,wniso1.atedfnthehkcminnerbark)ofthePacificyew.Taxusbrevifi)1.u.Thefungusishyphomycctcosand.whengrowninascmi-Synthctic1.iquid11dium.produced(axo1.andre1.atedcompounds.Taxo1.wasidentifiedbymass

3、ecircneuychmwgraphy,andreac(iviywithmonoc1.ona1.aniibodicsspecificfortaxo!.Both1.-uC1.aceticacidand1.-IU-uCphcny1.a1.anin?WrViCedasprecursorsof,4CJtaxoIinfunga1.cu1.tures.Notaxo!wasdetectedinzero-timecu1.turesorinthesma1.1.agarp1.ugsusedtoirxu1.atcthecu1.turef1.asks.Taxo1.ahigh1.yderivatizedditerpen

4、oid11)hasshownPrUmisCasanan(i(umuragentinbrcasandovariancancers(Fig.1)(2.3)Theprimarysourceofaxo1.hasbeen(heharvestedanddriedinnerbarkIph1.oem-cambia1.(ies)orTaxbreifo1.ia(Pacifkyew)(4,5).Pacificyewisas1.ow-growingtreetbundinn)istsoi1.snearstreamsand1.akesincertainregionsof(hePacificNorthWe$t(6).On1

5、y0.01(o0.03%ofthedryPhIOCmweightisIaXO1.yetasmuchas2gofpurifiedtaxo1.isrequiredforafu1.1.regimenofanti(umorIreaimein(5).Thesupp1.yissueisfurthercomp1.icatedbytheSCarCiIyoftheycu,tree.A1.thougha1.1.11speciesofTaxusmaketaxo1.natura1.Maiuisofthesetreesarvofenna1.1.andIocutedinremoteareas.HereWCreport(

6、hePrOduai(Inoftaxo1.byTaxomycesatidreaiiae,anendophyticfungusassociatedwith7vbrevifo1.ia(7).Wcconfirmedthepresenceof(axo1.andre1.atedIaxancsin3wxko1.dcu1.turesofthefungusbymassSPCCtrOmetry.imnwnochcmistry.chromatographicmethods,andradihemca1.techniques.Thesearchforyew-associatedmicrobesthatProdUcCUi

7、xo1.isjustifiedbypreviousexamp1.esofp1.ant-associatedmicrobesproducingfc,1.anf,compounds,suchasgibbere1.1.ins(8).Thepathwaysofgibbere1.1.inbosyn1.hcsisinIiKfungusandthehigherp1.antareidentica1.upoGA11(9),Weexaminedmicroorganismsiso1.atedfrommorethan25T.brevifo1.iatreesfrom201.ocations.OFthe200microb

8、esscreenedtodate,on1.yT.utdreiuuehasdemonstratedtheabi1.itytoproducetaxo1.Thisfungus,wasiso1.atedf11nIheurface-disinfec(ed(80%ethano1.)innerbarkofonetreeinano1.d-growthcedarforestinnorthernMonimui.T1.nycesatuireMcwascuhuredbyt11mscrringhypha1.tipsfromwateragar,onwhichbarkpieceshadbeencu1.tured,ontoa

9、nodifkd-Inyco1.ogica1.agar(10).T1.iemyce1.iumthensuccessive1.ytransferredtoe1.iminatetracesof(axo1.orother(ax;incsCarriCdoverfimtheorigina1.treeM)urcc.Transfersofsma1.1.agarp1.ugstobrothcu1.turesweremadefrommycerixi.nyce1.iaawfvdgoinaquiescentstate.TuxoMycesandreataeWaJistoredinwaterat4cVandgrownona

10、semi-definedcu1.turemedium.TheconidiaofT.uu1.reanuedonogcrmimte.IhCBfUrCWCImnMZrrodpieces(0.5by0.5cm)ofagarb1.ockconfiningIhemyce1.ia1.n11sosteri1.izedS-7medium(10).OptimumconditionswereasaS1.i1.1.cu1.uirctat25*C.withe11t100f1.3(cm2:m1.).After2)daysofincubation,thecu1.turewasfi1.teredthroughcheesec1

11、oth.TheresiduewasgroundinaSorva1.1.OmnimixeradG1.tcredagain.ThecombinedI1.uidswereextractedwithanequa1.vo1.umenfdich1.oromeIhaneOrch1.o11fnaograhy(T1.C)ofIheorganicextractdemonstratedthepresenceofacompound(hatmimickedUiAC1.inthreeMMVCntsystems(11).Theorganicextractwasdisso1.vedin2m1.ofCh1.orQforman

12、dp1.acedOnasiIiaige1.co1.umn.Vhcco1.umnwasrinsedWiIhch1.oroformatie1.uedwith20m1.ofacetonitri1.e.Thee1.uentwasdried.ChrOma1.UgRIPhCdonapreparativeT1.Cp1.ate(0.5mmsi1.icage1.),anddeve1.opedinCh1.orofbmvacctoni(ri1.c7:3v.v.De1.ectab1.eUxo1.ainezero.OdiMiKhoIineChk1.ride(atImg1.,m1.)inthemedium(iabo1.i

13、shedtaxo1.riHucti(Hi.Thiscompoundisa1.soaneffectiveinhibitorofgibbere1.1.inproductiontnG.Jigihtmta1.thoughitstimu1.atedpctaso1.(Scsquitcrpcnoid)ProdUCtiOuntsOfmXO1.andaxanesproducedbyT.andeafwearc1.ow.asref1.ectedbythe1.imitedincorrahonofuC-IabeIedprecursorsi11o(axo1.(Tab1.eI).QuantitationbyCkcirosp

14、raymassspectrometryandCIE1.Aindicated(hat24to50ngofUxo1.Wereptn1.ucaiper1.iter.However,manyp1.an-associatcdmicrobesrequireoneormorep1.antmetabo1.itestoactivatethesynthesisofsccx)ndaryIKiiumIproducts(19).Iinproveiicu1.turingIechniquexandtheapp1.icationofgcnc(kengineeringmayimproveU1.Xc1.PnXhICIiOnby7

15、andrcanae(20).REFERENCESANDNOTESd.dich1.o11Mne1.une(erahydrofuran6:2;ande.bexane-acetonc1:1.12. T.D.McC1.ure.K.H.Schnim.M.1.J.Rci11r.V,S”r112.3,672(1992).13. J.H.CaR1.c1.Iina.J.1.q.Gtroniatogr.14.659(1991).14. P.G.GnHhau*.ea1.,secoxiNaiiomdCancerInstituteworkshopotaxo1.andTaxtts.Nationa1.Cancer1.ns

16、tiu(e;P.G.GnXhiIsTJGRaytxw1.d;G.S.Bignami.C.B.1.az).J.B.Byrnes,unpub1.ishedresu1.ts.15. CycoioxicPn1.peH沁*offunga1.uo1.WefSeS1.edasdescribedT.Mossman.J.InununoI.Meihodt65.55(1983).KBce1.1.sIcpidcmioidcarcinoma)wereincuhedin96we1.1.p1.atesa37Xfor24hours.Testsamp1.esoftaxo1.weredisso1.vedindimethy1.M1

17、1.fbXkk(DMSO)anddi1.(odwithmedium.Thesamp1.esWereXHIdedoIhep1.atesintrip1.icateforeachconcentration,andin1.-2y1.-2,5di1.wny1.Ictrazo1.iunibromide(10ofMTTin20m1.ofphosphatc-buffcrodsa1.ine)wasadded,andthep1.ateswereshakenSUnUyforimin.todisso1.vetheIdrmazancrysta1.s.Absorbanccat535nmwasnasurwia1.EDmw

18、asCi1.cuh(edasIMdi1.utionofsamp1.ewithaneffectone-ha1.fthatofthecontro1.(nagentadded).16. D.1.KiiigMon,mute.77rr.PnnHS3,1(991.).17. P.E.1.1.cmng.U.Mck,H.G,11w.ZAfntChcm.115,805(1993).18. GJ.Bunkers.D.Kcnfic1.d.G.A.Strobe1.Myco1.w.95.347(1991)19. F.PinkcrtonandG.A.Strobe1.Proc.Nai1.Acad.Sei.U.S.A.73.

19、47(1976).20A1.Dcma11.Science214,987(1981).2i.SupportedinparibyNationa1.5kienceFoundationgrantCHE-92068()3.theMontanaScctioaoftheznehcanCancerSociety,axitheMontaiwScienceandTechno1.ogyA1.1.iance.WedunktheNationa1.CancerInstituteandBristo1.-M)x:rsSquibbCo.finassiMance1.J.Searsfor11ssSpectra1.data.J.B1

20、ackbum.B.Niedens.andR.Wi1.kinsonforung1.cu1.turingandex(hausadG.S.BignamiforCIEIAoffunga1.taxo1.andM.Nicdcr.W.Ma.AndF.MacDona1.dtorassayingcytotoxicityoffunga1.Taxo1.11Janiwry1993：玳CePtCd16Marcy19932.E.W.Rowcnsky.1.A.Cazcnave.F.C.Donc-howr.J.Nat1.Cancer1.11f,82.1247(1990).3.PB.SchiOeia1.,Nature277,

21、665(1979).4.PBSchiiVandS.B.Honowitz.Proc.NaI1.Acad.Sc1.U.S.A,77.1561(1980).5.0.M.CniggandK.M.Sniukr,CunctrCe1.1.s3.233(1991).6.G.Strobe1.A.Sticr1.c.TJG.M.VanKuijk.，储儿，“九X4.65119927.G.A.Strobe1.A.Sticr1.c.D.Sticric.Mwnaxon47,71(1993).8.B.B.StoweandT.Yanuki.Atnu.Rev.P1.uniPhvsio1.S.ISI(1957).9.A.以咚枷“2

22、1,53,1970).10.S-7mediumconsistsofIgofg1.ucose.3goff11rtojic.6gofSuCrWSc.1gofNa,-acetate.1gofsoyone.Imgofthiamine,Imgofbiod1ngofpyridoxa1.1mgofCaI.23*-antohenate.3.6IngofMgSOi,6.5mgofCaNO?,ImgofCu(NOs):.2.5rngofZ11SOper1.iter.Sugarratioisidcniica1.tothatoccurringintheInnerbarkofPacitkyew.Modifiedmyco1.ogica!Agarconsistsof10gbnct(-soytone.40gofG1.ucose.!5gofbctoagar.1gofNa*-acctatc,and50mgofsodiumbenzoateper1.iter.11.Thiiv1.ayerchromatographyso1.vtmsystems,v/v:a.chkrof(rm-3cetonitri1.e7:3;b.Ch1.oroform-mc1.ha1.7:1;c.c(hy1.acctate-iupropan1.95:5;