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1、化学生物学与合成生物学王江云,化学生物学与合成生物学,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,人工合成生命的重大科学意义,Fossils present but rareEvolut,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,人工生命,人工合成生命的重大科学意义,改造生命，造福人类 环境健康新医药新材料新能源化工绿色农业人,化学生
2、物学与合成生物学课件,化学生物学与合成生物学课件,我们应该做什么？,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。,新人工生命体系,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,化学生物学与合成生物学课件,化学生物学与合成生物学课件,化学生物学与合成生物学课件,化学生物学与合成生物学课件,化学生物学与合成生物学课件,二氧化碳还原酶在电极上的定点偶联,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,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
3、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,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，,Metalloenzymes play important roles in alternative energies,Most important enzymes for sustainable energy are metalloenzymes.,(cytochrome c oxidasehydrogenase),(photosystem I),(photosystem
4、II),(lignin peroxidaseManganese peroxidase),Fuel cells,However, metalloenzymes are too expensive.,Fossil fuels?,Metalloenzymes play important,Why designing artificial biocatalysts?,Approaches to novel biocatalysts:Top down: reprogramming native enzymes Bottom up: design and engineering artificial bi
5、ocatalysts,Why designing artificial bioca,Why designing artificial enzymes?,Y. Lu, et al. Nature 460, 855-862 (2009).,Approaches to novel biocatalysts:Top down: reprogramming native enzymes Bottom up: design and engineering artificial biocatalysts,Why designing artificial enzym,Challenges and opport
6、unities in designing metalloenzymes as artificial biocatalysts,A wide number of metal ions and difference oxidation states of the same metal ions; Structural features (bond distance, angle and geometry) vary widely and ill-defined; Most metal ions have beautiful colors and strong magnetic properties
7、, serving as in situ probe of the design process.,J. B., Siegel et al. Science 329, 309-13 (2010).,Y. Lu, et al. Nature 460, 855-862 (2009).,Challenges and opportunities i,Advantages of Biocatalysts in fuel cells,Cytochrome c oxidase (CcO) is the best fuel cell catalysts:Has much lower overpotential
8、 (190-370 mV)uses earth abundant metal ions (iron and copper),C. H. Kjaergaard, J. Rossmeisl, and J. K. Nrskov, Inorg. Chem. 49, 35673572 (2010).,Advantages of Biocatalysts in,Bovine heart CcO (PDB ID:1OCC): membrane protein 13 subunits, MW = 200 kDa (1850 amino acids),T. Tsukihara, et al., Science
9、269, 1069-1074 (1995)S. Iwata et. al., Nature 376, 660-669 (1995),Cytochrome c Oxidase (CcO),Heme-CuB center,CuA center,CcO is a large membrane protein and unstable for fuel cell applications.,His291His290His240Tyr244CuB Bo,Using a small protein azurin to model CuA in CcO,Iwata, S.; Ostermeier, C.;
10、Ludwig, B.; Michel, H. Nature 376, 660-669 (1995).Hay, M. T.; Richard, J. H.; and Lu, Y. Proc. Natl. Acad. Sci. U. S. A. 93, 461-464 (1996).,The designed CuA looks almost identical to the native CuA.,B. CuA in Azurin (P. aeruginosa)(designed CuA, 1.65 ),A. CuA in CcO (P. denitrificans)(native CuA, 2
11、.7 ),Y. Lu, et al. Nature 460, 855-862 (2009).,Using a small protein azurin t,Overcoming over-potential problem: rationally tuning redox potentials without affecting the active site,+ 140 mV,+ 140 mV, 110 mV,Nicholas M. Marshall, Dewain K. Garner, Tiffany D. Wilson, Yi-Gui Gao, Howard Robinson, Mark
12、 J. Nilges, Yi Lu Nature 462, 113-116 (2009).,Native azurin,Y. Lu, et al. Nature 460, 855-862 (2009).,Overcoming over-potential prob,How good is it?,Sohini Mukherjee, Sabyasachi Bandyopadhyay, Arnab Mukherjee, Yi Lu, Abhishek Dey,How good is it?Sohini Mukherje,How good is it?,Sohini Mukherjee, Sabya
13、sachi Bandyopadhyay, Arnab Mukherjee, Yi Lu, Abhishek Dey,J. P. Collman, et al. Science 315, 1565 (2007).,How good is it?Sohini Mukherje,Introducing unnatural amino acid into artificial biocatalysts,Xiaohong Liu, Yang Yu, Cheng Hu, Wei Zhang, Yi Lu, Jiangyun Wang Angew. Chem., Int. Ed. 51, 4312-4316
14、 (2012).,The cross-linked His-Tyr increased the reactivity and turnovers dramatically,Kcat = 120 S-1,Introducing unnatural amino ac,Lignin biodegradation,Lignin is the second most abundant biopolymer on earth next to celluloseA critical barrier to biomass conversionWhile rot fungi is known degrade l
15、ignin naturally, but is very difficult forlarge-scale industrial applications,White rot fungi,Lignin biodegradationLignin is,Bakers yeast vs. white rot fungi,Bakers yeastWhite rot fungi,Bakers yeast is much cheaper and widely available for industrial applications,Bakers yeast vs. white rot fu,Cytoch
16、rome c peroxidase (CcP) Manganese peroxidase (MnP),CcP MnP(bakers yeast)(white rot fungi)Oxidation of cyt c Degradation of lignin and many aromatic pollutants including PCBs,1. B. C. Finzel, T. L. Poulos, and J. Kraut, J. Biol. Chem. 259, 13027 (1984).2. M. Sundaramoorthy, K. Kishi, M. H. Gold, and
17、T. L. Poulos, J. Biol. Chem.269, 32759 (1994).,Cytochrome c peroxidase (CcP),Active Site,CcP(Bakers yeast),MnP(white rot fungi),CcP and MnP are very similar.,Active SiteCcPMnPCcP and MnP a,Major Difference,Trp 51,Trp 191,Mn(II),Phe 45,Phe 190,B. KS Yeung, X. Wang, J. A. Sigman, P. A. Petillo, and Y.
18、 Lu Chem. & Biol. 4, 215 (1996).X. Wang and Y. Lu, Biochemistry, 38, 9146-9157 (1999).A. Gengenbach, S. Syn, X. Wang, and Y. Lu, Biochemistry, 38, 11425-11432 (1999).,CcP(Bakers yeast),MnP(white rot fungi),Major DifferenceTrp 51Trp 191M,Transforming CcP into functional MnP,MnCcPW191FMnCcP,MnCcPW191F
19、/W51FMnCcPW51F,WTCcPNo enzyme,B. KS Yeung, X. Wang, J. A. Sigman, P. A. Petillo, and Y. Lu Chem. & Biol. 4, 215 (1996).X. Wang and Y. Lu, Biochemistry, 38, 9146-9157 (1999).A. Gengenbach, S. Syn, X. Wang, and Y. Lu, Biochemistry, 38, 11425-11432 (1999).,Transforming CcP into function,Improving KM th
20、rough engineering non-covalent interactions,H-bondingMobility of ligand,Steric clashesFlexibility,Salt bridgeH-bonding,MnCcP,Improving KM through engineeri,Activity assays of triplet mutant on lignin models,Tested activity assays on:Dimeric lignin model compound (HPLC analysis)Guaiacylglycerol-guaia
21、col ether*Alkali-treated lignin (HPLC),*Lignin model is courtesy of Dr. Michelle Chang from UC Berkeley.See: Brown M E et al, ACS Chem. Biol.2012,7, 2074-2081,Activity assays of triplet mut,Fluorescent protein design for Mn(III) sensing,Liu, X.; Wang, J.* J. Am. Chem. Soc. 2014, 136, 1309413097.,Flu
22、orescent protein design for,Liu, X.; Wang, J.* J. Am. Chem. Soc. 2014, 136, 1309413097.,Fluorescent protein design for Mn(III) sensing,Liu, X.; Wang, J.* J. Am. C,CO2+ H2O,(CH2O)n + O2,2H2O,hv,O2 + 4H+ + 4e-,NAPH,CO2+ H2O (CH2O)n + O22H2OhvO2,37,Green fluorescent protein: Efficient photo trigged electron transfer,LV, X.; Wang, J.* Angew. Chem. Intl. Ed. 2014,37Green fluorescent protein: L,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。,新人工生命体系,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风能水能,
