微生物药物研究中新技术和新方法的应用

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3,,200040,RNA;;RNA;TQ465A167223651(2004)0420193206,,,B(1),Fig1Thestructuresofseveralrepresentativeantibiotics20042012073,,,,Tel:021262479808,Fax:021262791715,E2mail:baoquanzhu@sipi.com.cn,,2,3[1]2004724CJNMChinJNatMedJuly12004Vol12No.4193©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(Combinatorialbiosynthesistechnology),,,Fig3Theprocedureofdevelopingnewmicrobialmedicines,(polyketidesynthesispath2ways),aver2mectin,4,,Fig4Erythromycinbiosynthesisgeneclustersandtheirencodingenzymes,,,III(actinorhodin)ILovastatin(1),,(Kosan),,194ChinJNatMedJuly12004Vol12No.4CJNM2004724©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(microarray),1998,,Table1Microbialmedicinesandclassificationofenzymesystemsofpolyketidebiosyntheses,DNA,,,,,,,,3RNARNA,(2),,RNADNARNA,,RNA,,,RNA,RNA,RNA3.1[3]RNA,RNA,RNA2004724CJNMChinJNatMedJuly12004Vol12No.4195©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(Met)oftheopenreadingframe.Themutationpositionsareindicatedbyar2rows.Strainnumbers,nucleotidechanges,andaminoacidchangesaredenotedinthisorderinboxesFig7ActinorhodinandundecylprodigiosinproductionbyS.lividans66(parentalstrain)andtherifmutants.IncubationwascarriedoutusingGYMliquidmediumat30for6days,andActandRedweredeterminedasdescribedinmaterialsandmethods196ChinJNatMedJuly12004Vol12No.4CJNM2004724©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(InR4medium,9mutantsRC21,RH21,CT21,DE21,DV21,EN21,HC21,HL21HN21areredwhileotherarewhiteorgreyincludingtheparent;inGYMmedium,5mutantsEN21,RH21,RC21,CT21,LP21arewhitewhileotheraregreyandyellowincludingtheparent.),RNA,910Fig9S1mappinganalysisoftheactII2ORF4,redZandredDgenemRNAlevelsFig10ActinorhodinandUndecylprodigisionproductionandWesternblottinganalysisoftheActII2ORF4,ActIV2ORF1,RedZ,andRedDproteins(A)MycelialgrowthandantibioticproductionbyS.lividans66andtherifmutantsinGYMliquidcultures.Symbols:,S.lividans66(wild2typestrain);EN21(rif29);SL21(rif214);RC21(rif217).(B)WesternanalysisoftheActII2ORF4,ActIV2ORF1,andribosomalS12proteins.CellsgrowninGYMliquidculturesat30wereharvestedattheindicatedtime,followedbythepreparationofcellextractsforWesternanalysis.TheexpressionoftheribosomalS12proteinwasanalyzedinparallelasaninternalcontrol.Eachlanecontained20goftotalproteins.(C)WesternanalysisoftheRedZandRedDproteins.CellsusedforthisanalysisweresameasthoseusedinpanelB.2004724CJNMChinJNatMedJuly12004Vol12No.4197©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(1112)[4,5]Fig11Actinorhodin(blue)andundecylprodigiosin(red)produc2tion,aerialmyceliaformation,andsporulationbyStreptomyceslivi2dans66(Parent)anditsgentamicin2resistantmutants(InR4agar,mutantsHU21andHU22producemoreactinorhodin(Act),mutantsHU23andHU25producemoderateAct,mutantHU24producelessbutparentproducenoAct;inGYMagar,mutantsHU23,HU24andHU25producemoreActwhilemutantsHU21,HU22andtheparentproducenoAct.)Fig12Actinorhodin(blue)andundecylprodigiosin(red)produc2tion,aerialmyceliaformation,andsporulationbyStreptomycesceoli2colorM600anditsmutantsinR4agarandGYMagar.M6002relA,amutantofrelAgenedeletionofS.coelicolorM600;HU26andHU27,gentamicin2resistantmutantsofM6002relA.(InR4agar,M600produceblueandredantibiotics,mutantsHU26andHU27pro2duceonlyredantibioticbuttheM6002relAproducesnoredandblueantibiotics;inGYMagar,mutantsHU26andHU27producemoreblueantibioticswhileM600producesmoderatebuleandM6002relAproduceslessblue.)1066RNA,RNA,,,11,5,,,,,[1](HuHF),(ZhuBQ),(GongBY).[J].(Worldnotesonantibiotics),1998,19(6):40124081[2]XueQ,AshleyG,HutchinsonCR,etal.Amultiplasmidapproachtopreparinglargelibrariesofpolyketides[J].Biochemistry,1999,96(21):117402117451[3]HuH,ZhangQ,OchiK.ActivationofantibioticbiosynthesisbyspecifiedmutationsintherpoBgene(encodingtheRNApolymerase(subunit)ofStreptomyceslividans[J].JBacteriol,2002,184(14):3984239911[4]HuH,OchiK.Novelapproachforimprovingtheproductivityofan2tibiotic2producingstrainsbyinducingcombinedresistantmutations[J].ApplEnvironMicrobiol,2001,67:1885218921[5]TamehiroN,HosakaT,XuJ,etal.Innovativeapproachforimprove2mentofanantibiotic2overproducingindustrialstrainofStreptomycesalbus[J].ApplEnvironMicrobiol,2003,69:6412264171NovelTechnologysandMethodsAppliedinMicrobialMedicineResearchZHUBao2Quan,HUHai2Feng(ShanghaiInstituteofPharmaceuticalIndustry,Shanghai200040,China)ABSTRACTMicrobialmedicineresearchconsistsofnovelmicrobialme

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