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 naturesmacromolecular 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 biologysmechanisms 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 biologyscatalytic 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, Naturesenzymes 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 Evolutionsinnovation 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,