Angewandte Essays Chemie
International Edition:DOI:10.1002/anie.201708408 Biocatalysis German Edition:DOI:10.1002/ange.201708408 Directed Evolution:Bringing NewChemistry to Life Frances H. Arnold* biocatalysis ·enzymes ·heme proteins · protein engineering ·synthetic methods
Survival of the Fittest Expanding Nature’s Catalytic Repertoire for aSustainable Chemical Industry In this competitive age,when new industries sprout and decay in the span of adecade,weshould reflect on how Nature,the best chemist of all time,solves the difficult acompany survives to celebrate its 350th anniversary.A problem of being alive and enduring for billions of years, prerequisite for survival in business is the ability to adapt to under an astonishing range of conditions.Most of the changing environments and tastes,and to sense,anticipate, marvelous chemistry that makes life possible is the work of and meet needs faster and better than the competition. This nature smacromolecular protein catalysts,the enzymes.By requires constant innovation as well as focused attention to using enzymes,nature can extract materials and energy from execution. Acompany that continues to provide meaningful the environment and convert them into self-replicating,self- and profitable solutions to human problems has achance to repairing,mobile,adaptable,and sometimes even thinking survive,even thrive,inarapidly changing and highly biochemical systems.These systems are good models for competitive world. asustainable chemical industry that uses renewable resources Biology has abrilliant algorithm for solving the problem and recycles agood fraction of its products.And biology is not of survival over time:evolution. Those who adapt and just amodel from which to draw inspiration:living organisms (re)produce outcompete the less agile and less fertile.Over or their components can be efficient production platforms.In the last 30 years—which seems along time but is less than fact, Ipredict that DNA-programmable microorganisms will one-tenth the time Merck KGaA, Darmstadt, Germany has be producing many of our chemicals in the not-so-distant been in business—I have tried to adapt biology smechanisms future. for innovation and optimization to solving problems in That most chemicals are made using synthetic processes chemistry and engineering.Itturns out that evolution is starting from petroleum-based feedstocks reflects the re- apowerful forward-engineering process,whose widespread markable creativity of synthetic chemists in developing adoption in enzyme engineering and synthetic biology has reaction schemes and catalysts that nature never discovered. been made possible through advances in molecular biology Synthetic chemistry has given us an explosion of products, and high-throughput screening. which feed, clothe,house,entertain, and cure us.Synthetic chemistry,however, struggles to match the efficiencyand selectivity that biology achieves with enzymes.Inmany cases, synthetic processes rely on precious metals,toxic reagents and solvents,and extreme conditions,and they generate substan- tial amounts of unwanted byproducts.DNA-programmable chemical synthesis using enzymes promises to improve on synthetic chemistry,particularly if we are able to expand biology scatalytic repertoire to include some of the most [*] Prof. F. H. Arnold Division of Chemistry and Chemical Engineering synthetically useful reactions,under physiological conditions California Institute of Technology 210-41 and with earth-abundant resources.Such clean, green chemis- 1200 E. California Blvd.,Pasadena, CA 91125 (USA) try might sound like pie in the sky,but enzymes already show E-mail:[email protected] how aprotein can orient substrates for reaction, exclude Homepage: http://fhalab.caltech.edu water from an active site,activate ametal or simple organic The ORCID identification number for the author of this article can be cofactor, or suppress competing reactions to draw out new found under https://doi.org/10.1002/anie.201708408. and admirable synthetic capabilities.Synthetic chemists have 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. been drawing inspiration from biology for decades,and now is KGaA. This is an open access article under the terms of the Creative the time for protein engineers to use inspiration from Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original synthetic chemistry to generate new enzymes that will work is properly cited, and is not used for commercial purposes. improve on and replace synthetic catalysts and reaction [1] This article is part of the Special Issue to commemorate the 350th pathways. anniversaryofMerck KGaA, Darmstadt, Germany.More articles can Unfortunately,our understanding of the link between be found at http://doi.wiley.com/10.1002/anie.v57.16. sequence and function lags well behind our desire for new
Angew.Chem. Int.Ed. 2018, 57,4143 –4148 2018 The Authors. Published by Wiley-VCH VerlagGmbH &Co. KGaA,Weinheim 4143 Angewandte Essays Chemie
enzymes.Given that our ability to predict protein sequences, dom mutagenesis and screening,Iquickly realized that such or even just changes to asequence,which reliably give rise to mutations were easy to find and accumulate with the right whole new,finely tuned catalytic activities is rudimentary at evolutionary optimization strategy.Mystudents and Iob- best, creating new enzymes capable of improving on current served that proteins,the products of evolution, are themselves synthetic processes is apretty tall order.Wealso dream of readily evolvable.Properties we and others targeted in the going beyond known chemistry to create enzymes that early days of directed evolution (the mid-1990s) included catalyze reactions or make products that are simply not recovering activity in unusual environments (e.g.organic possible with any known method, synthetic or otherwise. solvents), improving activity on non-native substrates,en- Requiring that these new enzymes assemble and function in hancing thermostability,and changing enantioselectivity.We cells,where they can be made at low cost and incorporated learned the then-surprising fact that beneficial mutations into synthetic metabolic pathways to generate abroader array could be far from an active site,and often appeared on the of products,represents an even greater set of engineering protein surface (which in those days was generally deemed constraints and challenges. insensitive to mutation and functionally neutral). To this day, Nature senzymes are the products of evolution, not no one can explain satisfactorily how such mutations exert design. By using generations of mutation and selection for their effects,much less predict them. fitness advantages,evolution allows organisms to continu- ously update and optimize their enzyme repertoires.New enzymes even appear in real time in response to challenges Evolution of Novelty:Enzymes that Catalyze (e.g. the need to resist antibiotics or pesticides) or oppor- Reactions Invented by Synthetic Chemists tunities (e.g. the chance to occupy anew food niche by degrading recently introduced, manmade substances). Iargue Although we could enhance activity (and many other that the process that gave rise to all the remarkable biological properties) by accumulating beneficial mutations over gen- catalysts in nature should be able to produce yet more.Inthe erations of random mutagenesis and screening, evolving laboratory.Quickly.Advances in molecular biology over the awhole new catalytic activity seemed amuch more difficult past few decades—the ability to write,cut, and paste DNA problem. After all, evolution is not good for problems that and to have that DNAread and translated into proteins in require multiple,simultaneous,low-probability events,[2] and recombinant organisms—have given us the ability to breed the active sites of enzymes are so beautifully and precisely enzymes much like we breed sheep or sake yeast. We can configured that it was hard to imagine how the stepwise direct the evolution of enzymes in the laboratory by requiring accumulation of beneficial mutations could create anew one. them to perform in ways that may not be useful to abacterium Evolution sinnovation mechanisms,however, are more but are useful to us.Directed evolution achieves these simple than they might appear:evolution works best when it desirable functional outcomes while circumventing our deep does not need to generate awhole new active site from ignorance of how sequence encodes them. scratch. Instead, evolution can generate anew enzyme from Directed evolution mimics evolution by artificial selec- one that is “close”, that is,shares elements of mechanism or tion, and is accelerated in the laboratory setting by focusing machinery from which the new activity can be built. Nature on individual genes expressed in fast-growing microorgan- co-opts old machinery to do new jobs.And sometimes the isms.Westart with existing proteins (sourced from nature or ability to do the new job is already there,atleast at alow level. engineered), introduce mutations,and then screen for the Thebiological world is replete with proteins whose capabil- progeny proteins with enhanced activity (or another desirable ities extend well beyond what may be used at any given time. trait). We use the improved enzymes as parents for the next Thus new enzymes are built from promiscuous or side round of mutation and screening, recombining beneficial activities that become advantageous in anew biological mutations as needed, and continuing until we reach the target context, such as when anew food source becomes available.[3] level of performance. Thus aconservative process of accumulating beneficial Engineering enzymes in the 1980s and 1990s,Ilearned the mutations can innovate because the innovation is already hard way that there was no reliable method to predict there!The magnificent diversity of the biological world performance-enhancing mutations.Turning instead to ran- provides the fuel for further innovations. Fordirected evolution to be areliable approach to Frances Arnold is the Linus Pauling Profes- creating new enzymes,wethe breeders of proteins must first sor of Chemical Engineering, Bioengineering, identify potential catalytic novelty in the form of starting and Biochemistry at the California Institute proteins which have at least low levels of anew activity.We of Technology,where her research focuses therefore look for activities that are known to synthetic on enzyme engineering by directed evolu- tion, with applications in sustainable fuels chemistry,but perhaps not explored in nature.Cytochrome and chemicals. She uses evolution’s innova- P450s,whose native functions include avariety of extremely tion mechanisms to bring new chemical challenging transformations such as hydroxylation, epoxida- reactions to biology.Her honors include the tion, heteroatom oxidations,nitration and more,looked to me MillenniumTechnology Prize (2016). She like apromising place to start hunting for new activities. has been elected to the US National Nature had already exploited this evolvable heme-protein Academies of Science, Medicine, and Engi- assembly and the various reactive intermediates in the neering. catalytic cycle to create all the natural P450 functions.We
4144 www.angewandte.org 2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem. Int. Ed. 2018, 57,4143 –4148 Angewandte Essays Chemie quickly discovered that many more new,non-natural func- greatly promotes carbene-transfer and nitrene-transfer activ- tions were possible.Inthe last few years we have engineered ities. P450s and other heme proteins to carry out aplethora of After demonstrating that the P411 derived from Bacillus reactions known to synthetic chemists,but not found in megaterium cytochrome P450 could be engineered for intra- biology.[4] molecular C Hamination, and intermolecular aziridination À Forexample,olefin cyclopropanation by carbene transfer and sulfimidation activities unknown in biological systems, is areaction well known in the area of transition-metal our efforts culminated in cytochrome P411CHA,which cata- catalysis,but not known to be catalyzed by an enzyme.In lyzes intermolecular benzylic C Hamination.[9] Efficient and À 2012, inspired by much older reports of heme mimics highly enantioselective intermolecular amination of C(sp3) À performing such reactions in organic solvents,wediscovered Hbonds has long been achallenge in chemical catalysis. that heme proteins catalyze cyclopropanation when provided Despite screening many different heme proteins and protein with diazo carbene precursors and asuitable olefin, in water.[5] variants,however, we never found one with the desired This promiscuous activity is manifested when the protein activity until postdoctoral fellow Chris Prier discovered that encounters the diazo reagent, forms the reactive carbene,and the P411 variant “P4”, evolved for an intermolecular sulfimi- then transfers it to the olefin. Our lab took advantage of this dation and rearrangement reaction, had acquired promiscu- inherent ability of abacterial cytochrome P450 to evolve ous activity for benzylic C Hamination. Chris Prier and À ahighly efficient enzyme for production of the chiral cis- doctoral student Kelly Zhang then directed the evolution of cyclopropane precursor to the antidepressant medication P4 to create P411CHA,which exhibits hundreds of turnovers levomilnacipran.[6] Our group and that of Rudi Fasan have for the amination of benzylic C Hbonds with excellent À since pushed avariety of heme proteins to synthesize other enantioselectivities (> 99% ee).[9] chiral cyclopropane pharmaceutical precursors,including one Free heme does not catalyze any of these nitrene-transfer used in the synthesis of ticagrelor,amedication used to reactions,and small-molecule catalysts for direct C H À prevent the reoccurrence of heart attacks.[7] In our case,we amination rely heavily on precious metals which are not identified atruncated globin from Bacillus subtilis,which sustainable.The protein, however, can impart this new catalyzes the reaction at low levels and also showed some reactivity to earth-abundant iron in its porphyrin cofactor, selectivity for producing the single,desired diastereomer of and it is evolvable.Evolution enabled P411CHA to promote the ticagrelor cyclopropane precursor from ethyl diazoacetate nitrenoid formation and transfer to asecond substrate over and 3,4-difluorostyrene (Figure 1). Just afew generations of the competing nitrene reduction heavily favored in the parent directed evolution improved the activity and selectivity of the enzyme,[9] aproperty that would be extremely challenging, if enzyme so that, of the four possible stereoisomers,itproduces not impossible,todesign. In fact, we think of these proteins as the ticagrelor cyclopropane almost exclusively.Because the chiral, self-assembling,DNA-encoded macromolecular tran- reaction proceeds in whole Escherichia coli cells which sition-metal complexes whose steric and electronic properties express the evolved enzyme,producing the catalyst is as are readily tuned by directed evolution to achieve desired simple as growing bacteria. activities and selectivities. Recently we have been exploring enzymes that open yet more chemical space for biocatalysis,including enzymes that form chemical bonds unknown in biology.Inthe last year we described heme enzymes that catalyze carbene insertion into Si Hand B Hbonds,thus giving living systems their first À À carbon–silicon[10] and carbon–boron[11] bond-forming activi- ties.C Si bonds are useful in medicinal chemistry,imaging Figure 1. A B. subtilis globin variant, engineered by directed evolution, À catalyzes the cyclopropanation of 3,4-difluorostyrene to make the agents,elastomers,and awide variety of consumer products, desired stereoisomer of aticagrelor precursor with high selectivity and but they have never been found in biological systems.Until yield.[7a] now,the only methods to create these bonds enantioselec- tively involved multistep syntheses just to prepare chiral While we were investigating carbene-transfer reactions reagents or chiral transition-metal complexes.The resulting catalyzed by heme proteins,wealso looked into the possibility catalysts are often only poorly active,and an iron-based of evolving enzymes for nitrene-transfer reactions.Inspired catalyst had never been reported for this carbene-insertion by ahint in the literature from the 1980’stoattempt reaction. Upon screening acollection of heme proteins, intramolecular C Hamination, we were delighted to find postdoctoral fellow Jennifer Kan and her team discovered À that acytochrome “P411” exhibited alow level of promiscu- that asmall (124 aa), highly stable cytochrome c from ous activity with an aryl sulfonyl azide nitrene precursor,and Rhodothermus marinus (Rma cyt c)could catalyze the that activity could be improved by directed evolution.[8] We reaction between ethyl 2-diazopropanoate and phenyldime- purposefully engineered the P411 by replacing the completely thylsilane to form the chiral organosilicon product with high conserved cytochrome P450 cysteine ligand, which is bound to enantioselectivity (Figure 2A). Directed evolution discov- the iron center,with serine,aligand not found in any known ered three mutations that enable the enzyme to form C Si À natural heme protein. This change shifts the characteristic bonds with up to 8200 total turnovers (based on Rma cyt c peak in the CO difference spectrum from l = 450 to 411 nm concentration) and enantioselectivities with greater than and abolishes the native monooxygenase activity.Italso 99% ee for awide range of silicon-containing substrates.
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Figure 3. Production of chiral organoboranes by E. coli expressing Rhodothermus marinus cytochrome c.[11] The bacterial catalyst uses borane-Lewis base complexesand diazo reagents to construct boron- containing carbon stereocenters efficiently and selectivelyincells by carbene B Hinsertion. The bioconversion can be conducted readily À on gram scale, and the enantio-preference of borylation was switched to give either enantiomer of the organoborane products. Figure 2. A) Chiral Si Cbond formation catalyzed by alaboratory- À evolved variant of Rhodothermus marinus cytochrome c.[10] The three amino acid residues that were mutated to increase this abiological even create chemical bonds not known in biology (C Si, C À À activity include the methionine axial ligand (M100).B)The enzyme B). These new reactivities were made possible by 1) use of catalyzes formation of different organosilane products with high enantiomeric excess from silane and diazo substrates. reagents not found naturally (carbene and nitrene precur- sors), 2) the promiscuous reactivities of proteins in the presence of these synthetic reagents,3)directed evolution Doctoral student Kai Chen used the engineered enzyme to to enhance and tune the new activities,and 4) chemical make 20 organosilicon products,most of which were obtained intuition and know-how to identify the right conditions and as single enantiomers.The evolved enzyme also became reactions to test with the right enzymes. highly chemoselective for Si Hinsertion using substrates À having other potentially reactive functional groups (alkene, alkyne,N H, O H; Figure 2B). Enzymes to Conquer New Chemistry À À We also asked whether an enzyme could catalyze carbene insertion into B Hbonds.NoC Bbonds are known in the Thenext big challenge is to create enzymes for reactions À À biological world, and the few natural products that include that neither biology nor synthetic chemistry has conquered. boron in different forms are thought to incorporate boric acid, As macromolecular catalysts,for example,enzymes can available from the environment, non-enzymatically.Postdoc- stabilize transition states and promote reactions through toral fellows Jennifer Kan and Xiongyi Huang proposed that pathways that would be difficult, if not impossible,toaccess B Hinsertion could be genetically encoded, thus giving living with small-molecule catalysts because of competition with À cells the ability to make chiral organoborane products from other, lower-energy reaction pathways.Inapowerful example appropriate,biocompatible carbene precursors and borylat- of how an enzyme active site can be engineered to promote ing agents.Starting with the Rma cytochrome c, they evolved one reaction pathway over another, postdoctoral fellow avery efficient biocatalyst:bacteria expressing evolved Rma Stephan Hammer directed the evolution of an alkene anti- cytcprovided access to 16 chiral organoboranes,which had Markovnikov oxygenase (aMOx), which catalyzes the con- never been made previously,from borane-Lewis base com- version of alkenes into the anti-Markovnikov carbonyl plexes and various carbene precursors.[11] Suitable for gram- compounds.[12] Intrigued by areport that the cytochrome scale biosynthesis,the catalyst offered up to 15300 turnovers P450 from Labrenzia aggregata made some phenyacetalde- (based on cytochrome c concentration), a99:1enantiomeric hyde as aside product when it oxidized styrene to the epoxide, ratio (e.r.), and 100%chemoselectivity (Figure 3). These Hammer looked more deeply and discovered that this catalyst turnovers are more than 400 times greater than those promiscuous reactivity did not involve epoxidation followed for known chiral catalysts for the same class of transforma- by isomerization to the aldehyde,ashad been proposed.[13] He tion. Furthermore,the enzyme senantio-preference could be correctly surmised that it instead went through acompeting, switched to make either product enantiomer.Fully genet- stepwise mechanism involving radical/cation intermediates ically encoded and functional for hours,these new enzymes and a1,2-hydride migration (Figure 4A).[14] He then exploit- open anew world of silicon and boron chemistry in living ed this side activity to direct the evolution of by far the most systems. active,and the first enantioselective,direct aMOx catalyst.[12] It has been fascinating to see that at least some of nature s Using earth-abundant iron, dioxgen, and arecyclable cofactor vast catalogue of proteins can be evolved in the laboratory, (NADPH), the laboratory-evolved P450 enzyme catalyzes often with only afew mutations,tocatalyze abiological thousands of turnovers for anti-Markovnikov oxidation of reactions (cyclopropanation, N Hinsertion, S Hinsertion, different substituted styrenes,including hindered substrates À À amination, aziridination, and more). Thenew enzymes can such as internal and 1,1-disubstituted alkenes.
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principle approachable through the combination of an evolved aMOx with other enzymes or chemical catalysts (Figure 4C). Knowing that such adirect aMOx catalyst is possible may inspire creation of even better enzymes or even invention of smaller-molecule mimics.
Closing Thoughts
Ihave focused this essay on the efforts of my own group to re-purpose heme proteins and to use them to demonstrate that directed evolution (and alittle design) can readily create new genetically encoded enzyme catalysts for reactions first invented by synthetic chemists and even for reactions which have eluded synthetic approaches.The active sites of the new enzymes can confer high activities and chemo-, regio-, and enantioselectivies,aswell as product selectivities,which are difficult or impossible to achieve with small-molecule cata- lysts.Furthermore,laboratory-evolved enzymes can stabilize and direct the fates of highly reactive intermediates to promote reactions that are disfavored without the precise Figure 4. Acytochrome P450 anti-Markovnikov oxygenase.[12] A) Com- control of the enzyme.Wehope that these demonstrations peting reaction pathways for P450-catalyzed oxo transfer to alkenes. will soon be accompanied by deeper insights into the The concerted epoxidation pathway is favored over the stepwise anti- mechanisms of the new enzyme-catalyzed reactions gained Markovnikov oxidation consisting of oxo transfer followed by an from ongoing structural, spectroscopic,and computational (enantioselective) 1,2-hydridemigration. B) Tenrounds of directed studies. evolution accumulated 12 amino acid mutations,many of which are distant from the active site. C) aMOx can be combined with estab- Other excellent examples of directed evolution for non- lished (bio)catalystsfor various challenging anti-Markovnikov alkene natural chemistry have been produced by other groups, functionalization reactions. including designed enzymes and enzymes with artificial cofactors,some of which are described in our recent review.[15] Thedesigned enzymes don tyet have the sophistication of Remarkably,the aMOx enzyme reacts with 1,1-disubsti- nature sproducts,and design struggles with the metals and tuted alkenes and generates chiral centers by controlling the other cofactors that drive so much interesting chemistry.But enantioselectivity of the 1,2-hydride migration. It is difficult when that changes,directed evolution will be there to draw to envision how this could be done outside of amacro- out the new capabilities and fine-tune the designs,just as it molecular active site.The 12 mutations that confer this can do with the promiscuous activities of natural proteins. impressive reactivity and control over substrate orientation With the power of evolution realized for engineering,we during the 1,2-hydride migration are distributed all over the now have awhole new way to look at the diverse products of protein (Figure 4B). Their specific effects on the enzyme s natural evolution. Instead of simply asking what enzymes do structure and mechanism that led to this remarkable activity in nature,wecan now ask the question, “what CANthey do?” are difficult to rationalize,much less predict. It will turn out that they can do alot more than we ever Synthetic catalysts for anti-Markovnikov oxidation have imagined, especially when we use evolution to unleash their not been able to exploit the aMOx mechanism because of latent potentials.Whatisclear is that evolution is an competition from the highly favored concerted epoxidation innovation machine,and nature sproducts are ready to be pathway.Instead, synthetic approaches use alternative strat- let loose to take on new functions,under the discerning eye of egies that require precious metals and/or stoichiometric the breeder of molecules.Atreasure trove of new enzymes is oxidants such as iodosobenzene.Their very limited activities just waiting to be discovered and used for chemistry that we cannot compete with the laboratory-evolved enzyme.No could only have dreamed of just afew years ago. catalytic,enantioselective method to convert prochiral al- kenes into their chiral anti-Markovnikov carbonyl com- pounds was known before this enzyme was engineered. Acknowledgements To enhance utility for production of fine chemicals,aMOx can be coupled with other catalysts,such as an alcohol Ithank Sabine Brinkmann-Chen, Kai Chen, Stephan Ham- dehydrogenase,toenable anti-Markovnikov redox hydration mer, Kari Hernandez, Xiongyi Huang, Jennifer Kan, Rusty of alkenes.[12] This coupled enzyme system yields valuable Lewis,Chris Prier and Ruijie Zhang for the ideas,inspiration, chiral alcohols with high enantiomeric excess and has the data, and hard work that went into the examples presented added benefit of recyclingthe NADPH cofactor. Theanti- here.Ithank Kim Mayer for excellent editorial assistance. Markovnikov alkene functionalization is an important step in This work was supported by the National Science Foundation, the production of many valuable chemicals,which are now in Division of Molecular and Cellular Biosciences (grant MCB-
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1513007), the Resnick Sustainability Institute,and the Cal- Catal. 2016, 6,7810;b)P.Bajaj, G. Sreenilayam, V. Tyagi, R. tech CI2 innovation program. Fasan, Angew.Chem. Int. Ed. 2016, 55,16110; Angew.Chem. 2016, 128,16344. [8] J. A. McIntosh, P. S. Coelho,C.C.Farwell, Z. J. Wang,J.C. Lewis,T.R.Brown, F. H. Arnold, Angew.Chem. Int. Ed. 2013, Conflict of interest 52,9309; Angew.Chem. 2013, 125,9479;see also R. Singh, M. Bordeaux,R.Fasan, ACSCatal. 2014, 4,546. Theauthor declares no conflict of interest. [9] C. K. Prier,R.K.Zhang,F.H.Arnold, Nat. Chem. 2017, 9,629. [10] S. B. J. Kan, R. D. Lewis,K.Chen, F. H. Arnold, Science 2016, Howtocite: Angew.Chem. Int. Ed. 2018, 57,4143–4148 354,1048. Angew.Chem. 2018, 130,4212–4218 [11] S. B. J. Kan, X. Huang,Y.Gumulya, K. Chen, F. H. Arnold, Nature 2017,DOI:10.1038/nature24996. [1] C. K. Prier,F.H.Arnold, J. Am. Chem. Soc. 2015, 137,13992. [12] S. C. Hammer,G.Kubik, E. Watkins,S.Huang,H.Minges,F.H. [2] P. Romero,F.H.Arnold, Nat. Rev.Mol. Cell Biol. 2009, 10,866. Arnold, Science 2017, 358,215. [3] P. J. O Brien, D. Herschlag, Chem. Biol. 1999, 6,R91. [13] Y. C. Yin, H. L. Yu,Z.J.Luan, R. J. Li, P. F. Ouyang,J.Liu, J. H. [4] O. Brandenberg, R. Fasan, F. H. Arnold, Curr.Opin. Biotechnol. Xu, ChemBioChem 2014, 15,2443. 2017, 47,102. [14] J. T. Groves,R.S.Myers, J. Am. Chem. Soc. 1983, 105,5791. [5] P. S. Coelho,E.M.Brustad, A. Kannan, F. H. Arnold, Science [15] S. C. Hammer,A.M.Knight, F. H. Arnold, Curr.Opin. Green 2013, 339,307. Sustainable Chem. 2017, 7,23. [6] Z. J. Wang,H.Renata, N. E. Peck, C. C. Farwell, P. S. Coelho, F. H. Arnold, Angew.Chem. Int. Ed. 2014, 53,6810; Angew. Chem. 2014, 126,6928. [7] a) K. E. Hernandez, H. Renata, R. D. Lewis,S.B.J.Kan, C. Manuscript received:August 15, 2017 Zhang,J.Forte,D.Rozzell, J. A. McIntosh,F.H.Arnold, ACS Version of record online: November 28, 2017
4148 www.angewandte.org 2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem. Int. Ed. 2018, 57,4143 –4148