化学生物学与合成生物学课件

化学生物学与合成生物学王江云FossilspresentbutrareEvolutionandexpansionoflifePlantsinvadethelandAgeofreptilesAgeofmammalsInsectsandamphibiansinvadetheland近40亿年的进化历程，创造了丰富多彩的生物。据估计地球物种总数多达1亿种。亚里斯多德达尔文沃森克里克人类基因组文特人类对生命本质的研究有两千多年了。基因组测序之后，我们对生物的认识更加深入，而对生命本质的理解还远远不够千人基因组不能创造的东西就不能被理解。人工生命可以更好理解生命本质。人工合成生命的重大科学意义

改造生命，造福人类快速发展的领域，生命科学的新方法工程、生物和信息科学的多学科综合交叉产生广泛的工业应用

环境健康新医药新材料新能源化工绿色农业人工生命人工合成生命的重大科学意义

1965年，第一个人工合成的蛋白质1972年，第一个人工合成的真核基因2003年，合成噬菌体基因组2006年，酵母菌中合成青蒿酸2008年，第一个人工合成原核基因组2010年，第一次人造基因组的活细胞2014年，第一次合成真核染色体

未来5-10年人造生命展现诱人前景光合作用及人造叶绿体取革命性突破合成生物体将革新工业生物制造人工生命是当前重要科学前沿我们应该做什么？衣原体基因组人工合成，只有合成，没有设计酵母基因组合成，少量设计我们应该做什么？化能自养光能自养有氧代谢能量利用水平是人类进步的标志电能不能在自然界稳定存在，自然进化生物不能高效利用电能人工合成电能、光能、生物质能高效利用的新生命体系具有重要意义。风能水能核能太阳能电能新人工生命体系二氧化碳还原酶在电极上的定点偶联利用电能进行淀粉生物合成形成电能细胞，创新生物的能量来源，引领下一代生物技术的发展，如生物计算机、生物传感器、分子马达等。电子催化酶与电子传递通道设计电能驱动人工生命体系电能驱动线粒体基因组人工合成与优化组装电能高效利用MetalloenzymesplayimportantrolesinalternativeenergiesMostimportantenzymesforsustainableenergyaremetalloenzymes.(cytochromecoxidasehydrogenase)(photosystemI)(photosystemII)(ligninperoxidaseManganeseperoxidase)FuelcellsHowever,metalloenzymesaretooexpensive.Fossilfuels?Whydesigningartificialbiocatalysts?Approachestonovelbiocatalysts:

Topdown:reprogrammingnativeenzymes Bottomup:designandengineeringartificialbiocatalystsWhydesigningartificialenzymes?UltimatetestourknowledgeRevealnewconceptsDesignnewbiocatalystswith unprecedentednewpropertiesProvidecheaperalternativesfor biotechnologicaland pharmaceuticalapplications

EngineeringartificialbiocatalystsY.Lu,etal.Nature460,855-862(2009).Approachestonovelbiocatalysts:

Topdown:reprogrammingnativeenzymes Bottomup:designandengineering

artificialbiocatalystsChallengesandopportunitiesin

designingmetalloenzymesasartificialbiocatalysts

Awidenumberofmetalionsanddifferenceoxidationstatesofthesamemetalions;Structuralfeatures(bonddistance,angleandgeometry)varywidelyandill-defined;

Mostmetalionshavebeautifulcolorsandstrongmagneticproperties,servingasinsituprobeofthedesignprocess.J.B.,Siegeletal.Science329,309-13(2010).Y.Lu,etal.Nature460,855-862(2009).AdvantagesofBiocatalystsinfuelcellsCytochromecoxidase(CcO)isthebestfuelcellcatalysts:Hasmuchloweroverpotential(190-370mV)usesearthabundantmetalions(ironandcopper)C.H.Kjaergaard,J.Rossmeisl,andJ.K.Nørskov,Inorg.Chem.49,3567–3572(2010).His291His290His240Tyr244CuBBovineheartCcO(PDBID:1OCC):membraneprotein13subunits,MW=200kDa(1850aminoacids)T.Tsukihara,etal.,Science

269,1069-1074(1995)S.Iwataet.al.,Nature376,660-669(1995)CytochromecOxidase(CcO)Heme-CuBcenterCuAcenterCcOisalargemembraneproteinandunstableforfuelcellapplications.UsingasmallproteinazurintomodelCuAinCcOIwata,S.;Ostermeier,C.;Ludwig,B.;Michel,H.Nature376,660-669(1995).Hay,M.T.;Richard,J.H.;andLu,Y.Proc.Natl.Acad.Sci.U.S.A.93,461-464(1996).ThedesignedCuAlooksalmostidenticaltothenativeCuA.B.CuAinAzurin(P.aeruginosa)(designedCuA,1.65Å)A.CuAinCcO(P.denitrificans)(nativeCuA,2.7Å)Y.Lu,etal.Nature460,855-862(2009).Overcomingover-potentialproblem:rationallytuningredoxpotentialswithoutaffectingtheactivesite+~140mV+~140mV–~110mVNicholasM.Marshall,DewainK.Garner,TiffanyD.Wilson,Yi-GuiGao,HowardRobinson,MarkJ.Nilges,YiLuNature462,113-116(2009).NativeazurinY.Lu,etal.Nature460,855-862(2009).Howgoodisit?SohiniMukherjee,SabyasachiBandyopadhyay,ArnabMukherjee,YiLu,AbhishekDeyHowgoodisit?SohiniMukherjee,SabyasachiBandyopadhyay,ArnabMukherjee,YiLu,AbhishekDeyCatalystkcatTurnoverNo.NativeCcO105M-1s-1-Syntheticmodel1.2x105M-1s-11.2x104Designedprotein4.6

x107

M-1s-11.0x105J.P.Collman,etal.Science315,1565(2007).IntroducingunnaturalaminoacidintoartificialbiocatalystsXiaohongLiu,YangYu,ChengHu,WeiZhang,YiLu,JiangyunWangAngew.Chem.,Int.Ed.51,4312-4316(2012).Thecross-linkedHis-TyrincreasedthereactivityandturnoversdramaticallyKcat

=120S-1LigninbiodegradationLigninisthesecondmostabundantbiopolymeronearthnexttocelluloseAcriticalbarriertobiomassconversionWhilerotfungiisknowndegradeligninnaturally,butisverydifficultfor large-scaleindustrialapplicationsWhiterotfungiBakers’yeastvs.whiterotfungi

Bakers’yeast

WhiterotfungiBakers’yeastismuchcheaperandwidelyavailableforindustrialapplicationsCytochromecperoxidase(CcP)Manganeseperoxidase(MnP)

CcP

MnP(baker’syeast) (whiterotfungi)Oxidationofcytc Degradationofligninandmany aromaticpollutantsincludingPCBs1.B.C.Finzel,T.L.Poulos,andJ.Kraut,J.Biol.Chem.259,13027(1984).2.M.Sundaramoorthy,K.Kishi,M.H.Gold,andT.L.Poulos,J.Biol.Chem.269,32759(1994).ActiveSite

CcP(Baker’syeast)MnP(whiterotfungi)CcPandMnPareverysimilar.MajorDifference

Trp51Trp191Mn(II)Phe45Phe190B.KSYeung,X.Wang,J.A.Sigman,P.A.Petillo,andY.LuChem.&Biol.4,215(1996).X.WangandY.Lu,Biochemistry,38,9146-9157(1999).A.Gengenbach,S.Syn,X.Wang,andY.Lu,Biochemistry,38,11425-11432(1999).CcP(Baker’syeast)MnP(whiterotfungi)TransformingCcPintofunctionalMnPMnCcPW191FMnCcPMnCcPW191F/W51FMnCcPW51FWTCcPNoenzymeB.KSYeung,X.Wang,J.A.Sigman,P.A.Petillo,andY.LuChem.&Biol.4,215(1996).X.WangandY.Lu,Biochemistry,38,9146-9157(1999).A.Gengenbach,S.Syn,X.Wang,andY.Lu,Biochemistry,38,11425-11432(1999).ImprovingKMthroughengineeringnon-covalentinteractionsY36FK179RI40GH-bondingMobilityofligandStericclashesFlexibilitySaltbridgeH-bondingMnCcPActivityassaysoftripletmutantonligninmodelsTestedactivityassayson:Dimericligninmodelcompound(HPLCanalysis)Guaiacylglycerol-β-guaiacoleth