格尔德霉素基因工程高产菌株的构建和培养

生物工程学报ChinJBiotech2008,January25;24(1):15-[e-mail]©2008InstituteofMicrobiology,CAS&CSM,Allrightsreserved.Received:April6,2007;Accepted:June4,2007Supportedby:theNationalDepartmentofSciencesandTechnologyunderPreliminaryBasicResearch973project(No.2001CCA00500).Correspondingauthor:YiguangWang.Tel:[phone];Fax:[phone];E-mail:[e-mail]973(No.2001CCA00500)研究报告赫卫清,周红霞,王红远,高群杰,王以光/,,100050:在格尔德霉素产生菌吸水链霉菌17997(Streptomyceshygroscopicus17997)中存在两种3-氨基-5-羟基苯甲酸(3-amino-5-hydroxybenzoicacid,AHBA)的生物合成基因簇,根据同源性可分为苯醌类和萘醌类。已证明其中苯醌类的AHBA生物合成基因簇负责格尔德霉素(geldanamycin,Gdm)起始单位的合成,而萘醌类的AHBA基因簇可能参与未知安莎化合物的生物合成。为提高吸水链霉菌17997菌种的Gdm发酵产量,并研究高产菌种在固体培养基上孢子的生长周期。采用基因阻断技术,将吸水链霉菌17997中的萘醌类AHBA生物合成基因簇(shnSOP)进行破坏,以获得ΔSOP菌株,从而减少对合成所需共同底物AHBA的争夺。HPLC分析结果表明ΔSOP菌株Gdm的发酵产量比原株提高185%。同时,通过孢子计数发现该菌株在固体培养基上的孢子生长经历2个周期,第2代孢子菌种的Gdm产量较高。:吸水链霉菌17997,格尔德霉素,基因阻断,孢子形成周期ConstructionandCultivationofGenetically-engineeredStraintoImproveGeldanamycinProductionWeiqingHe,HongxiaZhou,HongyuanWang,QunjieGao,andYiguangWangInstituteofMedicinalBiotechnology,CAMS&PUMC,KeyLaboratoryofBiotechnologyofAntibiotics,MinistryofHealth,Beijing100050,ChinaAbstract:ToimprovetheproductionofgeldanamycininStreptomyceshygroscopicus17997,genedisruptionwasdonetodeletethenaphthalenicAHBAgenes(shnSOP),encodingtheproductsthatsharethecommonbiosyntheticsubstrateswithgeldanamycin.Theresultingmutantstrain(ΔSOP)wascultivatedonasolidmediumandtheamountofsporescollectedfromtheplateswascalculatedfrom5to14daysandtheyieldofgeldanamycinwasmeasuredbyHPLC.ThegeldanamycinproductionoftheΔSOPstrainincreasedby185%comparingwiththatoftheparentstrain.Onsolidmedium,theΔSOPstrainunderwent2cyclesofsporulationandthegrowthofthesecondsporulationhadthehighestgeldanamycinproduction.Keywords:Streptomyceshygroscopicus17997,geldanamycin,genedisruption,sporeformation(Streptomyceshygroscopicus)Gdm(1),(),AHBA16ISSN1000-3061CN11-1998/QChinJBiotechJanuary25,2008Vol.24No.1Journals.im.ac.cn图1格尔德霉素的化学结构Fig.1ChemicalstructureofgeldanamycinSasakiGdm[1],Gdm[2]Gdm90(Hsp90)ATP/ADP[3,4],Hsp90GdmGdmGdm(17)GdmC1717-AAG17-DMAGIII[5,6]Gdm,GdmGdm[7]GdmAHBA[8,9],GdmAHBAPKS,Gdm[10],AHBA(AHBA-shn),[11]AHBA,AHBA,(N-AHBA),Gdm(B-AHBA),Gdm,,1材料和方法1.1菌种和质粒Gdm17997/pGH112(tsrR)[12]/[13],DH5α,(Apramycin,AmR)pUC18,1.2培养基17997(MY)(Yeastex-tract4g,Maltextract10g,Glucose4g,15g,1000mL)Gdm(starch2g,cottonmeal0.5g,glucose0.5g,cornstarchliquor0.5g,yeastpowder0.5g,CaCO30.2g,100mL)/(soybeanpowder20g,mannitol20g,agar15g,1000mL)1.3药品和试剂LA-Taq(CIAP)TaKaRaDNADNA/Squibb&Sons,INC,(Am)1.4仪器ShimadzuLC-10Avp,SPD-M10AvP,CLASS-VPPCRTechgeneThermalCycler-PROGENE1.5DNA操作[14],DNA[15]TaKaRaPCR:PCRTaKaRaLA-Taq,GC-I(20μL):(ddH2O4.6μL,GC-Ibuffer(2×)10.0μL,Primer:17Journals.im.ac.cn(25μmol/L)0.5μL,Template1μL,dNTP(2.5mmol/L)3.2μL,Taq0.2μL):401min,962min,96×40s,60×40s,72×1.5min,30,725min,P5:5′GGCCTCTAGAAGCGCATAGGTTACGA3′(XbaI)P6:5′CTAGTCTAGATGATGTCGGGAAAGTAGCG3′(XbaI)1.10孢子生长周期的观察和计数,28℃7d,3mL,0.1mL20mL,28℃567891011121314d,,5mL,,0.1mL,28℃7d,1.6大肠杆菌ET1567/pUZ8002与链霉菌进行接合转移[15]1.7Gdm基因阻断工程菌发酵培养及产物HPLC分析[7]1.8基因阻断载体的构建shnSOP,17997PCR2结果:P15′-CCGGAATTCACACTCGCACGTCCAGCC-3′EcoRI2.1萘醌型AHBA基因阻断重组质粒的构建AHBA,17997AHBA[AHBA-gdnAHBA-shn],AHBA-shn(2),Gdm,17997AHBA-shn,pGH112AHBA-shnShnSN(P1)ShnOC(P2),ShnOC(P3)ShnPN(P4),17997DNAPCR,S1S2S1S2Am,pGH112pGEX-shn3:P25′-GCGGGTACCGCAGCCAGATGCAGTCGG-3′KpnI1257bp1:P35′-AAAACTGCAGCTGTGGCTCAGACTGCTGC-3′PstI:P45′-CTAGTCTAGATGATGTCGGAAAGTAGCG-3′XbaI1265bp2PCR12,PstIKpnIAm(1.5kb),EcoRI-XbaIpGH112,DH5α,1.9基因阻断株的获得及鉴定2.2基因阻断株的获得和鉴定[16]PCR,12,PCRpGEX-shnET12567/pUZ800217997,MY3~4,图2AHBA-shn基因簇的组成Fig.2TheorganizationofAHBA-shngeneclustershnQ:aminodehydroquinatesynthasegene,shnS:AHBAsynthasegeneshnO:oxidoreductasegene,shnP:phosphataseshnK:kinasegene,shnJ:aminodehydroquinatedehydratasegene18ISSN1000-3061CN11-1998/QChinJBiotechJanuary25,2008Vol.24No.1Journals.im.ac.cn图3pGEX-shn基因阻断载体的构建Fig.3ConstructionofthegenedisruptionvectorpGEX-shn,TsrAm,AmRTsrSΔSOPΔSOPPCRΔSOP,12(P5图4基因阻断变株PCR产物电泳图Fig.4ElectrophoresisofPCRproductsofgenedisruptionmutantgenome1:S.hygroscopicus17997;2:ΔSOPmutant;M1:λDNA/HindⅢmarker;M2:DNAmarkerⅢP6)PCR,44PCR4kb,ΔSOP6kb,1.5kbAm,ΔSOPAmR,DNAAmR,shnSOP2.3ΔSOP变株发酵产量的HPLC检测17997ΔSOP7d,,5μL,:=40%~100%30min,304nmHPLC,5,ΔSOPGdm17997185%shnSOPGdm2.4ΔSOP变株在固体培养基上的生长周期与发酵产量的关系17997:19Journals.im.ac.cnΔSOP,ΔSOPMY,7d,,10d17997RTPeakareaA:Gdm27.4331076622B:1799724.470285489C:ΔSOP24.480529614图5HPLC检测ΔSOP变株发酵液中Gdm的含量Fig.5HPLCanalysisofGdmcontentinΔSOPmutantbroth图6ΔSOP变株在MY固体培养基上不同培养天数形成孢子数的变化Fig.6SporeamountofΔSOPmutantculturingontheMYmediumatdifferentdaysYaxisindicatingthesporeamountperPetridish(unit:tenthousands)IncubationdaysRTΔSOPmutantparentstrainPeakareaA:7day27.43352961476616B:10day24.470747856139125图7HPLC检测ΔSOP变株固体培养基上不同培养时间发酵液效价Fig.7HPLCanalysisofGdmfermentationtitleofΔSOPmutantculturingonsolidmediumatdifferentdays,ΔSOP6(7d)(10d)ΔSOP,17997,HPLC,7,7d,ΔSOP10dGDM3讨论,,AHBAAHBA,,,AHBA[17]17997AHBA,GdmAHBA-BAHBA-NAHBA4-20ISSN1000-3061CN11-1998/QChinJBiotechJanuary25,200