分子文献介绍

AlperH,StephanopoulosG.Engineeringyeasttranscriptionmachineryforimprovedethanoltoleranceandproduction,Science,2006,314:1565-1568:Globaltranscriptionmachineryengineering(gTME)isanapproachforreprogramminggenetranscriptiontoelicitcellularphenotypesimportantfortechnologicalapplications.HereweshowtheapplicationofgTMEtoSaccharomycescerevisiaeforimprovedglucose/ethanoltolerance,akeytraitformanybiofuelsprograms.MutagenesisofthetranscriptionfactorSpt15pandselectionledtodominantmutationsthatconferredincreasedtoleranceandmoreefficientglucoseconversiontoethanol.ThedesiredphenotyperesultsfromthecombinedeffectofthreeseparatemutationsintheSPT15gene[serinesubstitutedforphenylalanine(Phe177Ser)and,similarly,Tyr195His,andLys218Arg].Thus,gTMEcanprovidearoutetocomplexphenotypesthatarenotreadilyaccessiblebytraditionalmethods.同时调控这些一直是困扰发酵工程菌构建的重题传统策略通常超表达或敲除单个或少数，以及对特定转录因子或DNA结合motif进行重组修饰。由于代谢网络的复杂性，这些“局部手术”的作法不能显著提高目的产物的产量。全局转录机器工程（global种功能启动子的广泛识别及其偏，可以大范围的转录重排(transcriptionalreprogramming)。因此建立σ因子的突变文库，然后以产量为目标进行菌种筛选，可望获得麻省理工学院建立大肠杆菌sigma的定点突变文库，筛选获得了番茄红素高产菌RNAPolⅠ、PolPol3PolAlper等研究发现Sigma的全局转录调控同样适合于酿酒酵母，从而提高了对乙醇的耐受能力，为燃提取组DNA或总RNA,以此为模板PCR法克隆sigma有可能造成转录重排（transcriptionreprogramming）,其中酶转录强度的改变，最直接MaoYB,CaiWJ,WangJW,HongGJ,TaoXY,WangLJ,HuangYP,ChenXY*.SilencingacottonbollwormP450monooxygenasegenebyplant-mediatedRNAiimpairslarvaltoleranceofgossypol.NatureBiotechnology,2007,25,1307-1313因P450。该研究基于RNA原理，首先分离了棉铃虫体内的棉酚解毒P450，经过转培育成转棉花。用表达P450双链RNA的转棉花喂食棉铃虫，发现棉铃虫的P450表达量降低，对棉酚的耐受性明显减弱，棉铃虫生长缓慢甚至。该研究利用RNA技术为农业害虫的防治提供了一种思路。Xiao-XiaXia,Zhi-GangQian,ChangSeokKi,YoungHwanPark,DavidL.Kaplan,andSangYupLee.Native-sized binantspidersilkproteinproducedinmetabolicallyengineeredEscherichiacoliresultsinastrongfiber.ProcNatlAcadSciUSA,2010,107(32):14059-14063.Spiderdraglinesilkisaremarkablystrongfiberthatmakesitattractivefornumerousapplications.Muchhasthusbeendonetomakesimilarfibersbybiomimicspinningof binantdraglinesilkproteins.However,successislimitedinpartduetotheinabilitytosuccessfullyexpress binantsilkproteins(250–320kDa).Hereweshowthata284.9binantproteinofthespiderNephilaclavipesisproducedandspunintoafiberdisplayingmechanicalpropertiescomparabletothoseofthenativesilk.Thenative-sizedprotein,predominantlyrichinglycine(44.9%),wasfavorablyexpressedinmetabolicallyengineeredEscherichiacoliwithinwhichtheglycyl-tRNApoolwaselevated.Wealsofoundthatthebinantproteinsoflowermolecularweightversionsyieldedinferiorfiberproperties.Theresultsprovideinsightintoevolutionofsilkproteinsizerelatedtomechanicalperformance,andalsoclarifywhyspinninglowermolecularweightproteinsdoesnotrecapitulatethepropertiesofnativefibers.Furthermore,thesilkexpression,purification,andspinningplatformestablishedhereshouldbeusefulforsustainableproductionofnaturalqualitydraglinesilk,potentiallyenablingbroaderapplications.点过去人们试图用工程法表达此蛋白但表达的蛋白分子量太小基本上均未获成功。韩国SangYupLee课题组实现了技术突破，用代谢工程表达了金丝蜘蛛的蛋白大小为284.9kDa，纺织成丝后与天然蛛丝蛋白无异。由于天然蛛丝蛋白中的甘氨酸较多，所以设立的表达、蛋白纯化和织造平台技术为进一步研发奠定了基础。DKRo,EMParadise,MOuellet,KJFisher,KL,JMNdungu,KAHo,RAEachus,TSHam,JKirby,MCChang,STWithers,YShiba,RSarpong,andJDKeasling.Productionoftheantimalarialdrugprecursorartemisinicacidinengineeredyeast.Nature,2006,440(7086):Malariaisaglobalhealthproblemthatthreatens300-500millionpeopleandkillsmorethanonemillionpeopleannually.Diseasecontrolishamperedbytheoccurrenceofmulti-drug-resistantstrainsofthemalariaparasitePlasmodiumfalciparum.Syntheticantimalarialdrugsandmalarialvaccinesarecurrentlybeingdeveloped,buttheirefficacyagainstmalariaawaitsrigorousclinicaltesting.Artemisinin,asesquiterpenelactoneendoperoxideextractedfromArtemisiaannuaL(familyAsteraceae;commonlyknownassweetwormwood),ishighlyeffectiveagainstmulti-drug-resistantPlasmodiumspp.,butisinshortsupplyandunaffordabletomostmalariasufferers.Althoughtotalsynthesisofartemisininisdifficultandcostly,thesemi-synthesisofartemisininoranyderivativefrommicrobiallysourcedartemisinicacid,itsimmediateprecursor,couldbeacost-effective,environmentallyfriendly,high-qualityandreliablesourceofartemisinin.HerewereporttheengineeringofSaccharomycescerevisiaetoproducehightitres(upto100mgl(-1))ofartemisinicacidusinganengineeredmevalonatepathway,amorphadienesynthase,andanovelcytochromeP450monooxygenase(CYP71AV1)fromA.annuathatperformsathree-stepoxidationofamorpha-4,11-dienetoartemisinicacid.Thesynthesizedartemisinicacidistransportedoutandretainedontheoutsideoftheengineeredyeast,meaningthatasimpleandinexpensivepurificationprocesscanbeusedtoobtainthedesiredproduct.AlthoughtheengineeredyeastisalreadycapableofproducingartemisinicacidatasignificantlyhigherspecificproductivitythanA.annua,yieldoptimizationandindustrialscale-upwillberequiredtoraiseartemisinicacidproductiontoalevelhighenoughtoreduceartemisinincombinationtherapiestosignificantlybelowtheircurrentprices.物发酵生产青蒿素。青蒿酸是青蒿素的前体物，加州大学伯克利分校JDKeaslingParayilKumaranAjikumar,Wen-HaiXiao,KeithE.J.Tyo,YongWang,FritzSimeon,EffendiLeonard,OliverMucha,TooHengPhon,BlainePfeifer,GregoryStephanopoulos.IsoprenoidpathwayoptimizationfortaxolprecursoroverproductioninEscherichiacoli.Science,2010,330(6000):70-74Taxol(pa axel)isapotentanticancerdrug isolatedfromtheTaxusbrevifoliaPacificyewtree.Currently,cost-efficientproductionofTaxolandits ogsremainslimited.Here,wereportamultivariate-modularapproachtometabolic-pathwayengineeringthatsucceededinincreasingtitersoftaxadiene—the committedTaxolintermediate—approxima y1gramperliter(~15,000-fold)inanengineeredEscherichiacolistrain.Ourapproachpartitionedthetaxadienemetabolicpathwayintotwomodules:ana