AlperH,StephanopoulosG.Engineeringyeasttranscriptionmachineryforimprovedethanoltoleranceandproduction,Science,2006,314:1565-1568:Globaltranscriptionmachineryengineering(gTME)isanapproachforreprogramminggenetranscriptiontoelicitcellularphenotypesimportantfortechnologicalapplications.HereweshowtheapplicationofgTMEtoSaccharomycescerevisiaeforimprovedglucose/ethanoltolerance,akeytraitformanybiofuelsprograms.MutagenesisofthetranscriptionfactorSpt15pandselectionledtodominantmutationsthatconferredincreasedtoleranceandmoreefficientglucoseconversiontoethanol.ThedesiredphenotyperesultsfromthecombinedeffectofthreeseparatemutationsintheSPT15gene[serinesubstitutedforphenylalanine(Phe177Ser)and,similarly,Tyr195His,andLys218Arg].Thus,gTMEcanprovidearoutetocomplexphenotypesthatarenotreadilyaccessiblebytraditionalmethods.同時調控這些一直是困擾發酵工程菌構建的重題傳統策略通常超表達或敲除單個或少數，以及對特定轉錄因子或DNA結合motif進行重組修飾。由于代謝網絡的復雜性，這些“局部手術”的作法不能顯著提高目的產物的產量。全局轉錄機器工程（global種功能啟動子的廣泛識別及其偏，可以大范圍的轉錄重排(transcriptionalreprogramming)。因此建立σ因子的突變文庫，然后以產量為目標進行菌種篩選，可望獲得麻省理工學院建立大腸桿菌sigma的定點突變文庫，篩選獲得了番茄紅素高產菌RNAPolⅠ、PolPol3PolAlper等研究發現Sigma的全局轉錄調控同樣適合于釀酒酵母，從而提高了對乙醇的耐受能力，為燃提取組DNA或總RNA,以此為模板PCR法克隆sigma有可能造成轉錄重排（transcriptionreprogramming）,其中酶轉錄強度的改變，最直接MaoYB,CaiWJ,WangJW,HongGJ,TaoXY,WangLJ,HuangYP,ChenXY*.SilencingacottonbollwormP450monooxygenasegenebyplant-mediatedRNAiimpairslarvaltoleranceofgossypol.NatureBiotechnology,2007,25,1307-1313因P450。該研究基于RNA原理，首先分離了棉鈴蟲體內的棉酚解毒P450，經過轉培育成轉棉花。用表達P450雙鏈RNA的轉棉花喂食棉鈴蟲，發現棉鈴蟲的P450表達量降低，對棉酚的耐受性明顯減弱，棉鈴蟲生長緩慢甚至。該研究利用RNA技術為農業害蟲的防治提供了一種思路。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.點過去人們試圖用工程法表達此蛋白但表達的蛋白分子量太小基本上均未獲成功。韓國SangYupLee課題組實現了技術突破，用代謝工程表達了金絲蜘蛛的蛋白大小為284.9kDa，紡織成絲后與天然蛛絲蛋白無異。由于天然蛛絲蛋白中的甘氨酸較多，所以設立的表達、蛋白純化和織造平臺技術為進一步研發奠定了基礎。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.物發酵生產青蒿素。青蒿酸是青蒿素的前體物，加州大學伯克利分校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