DOCSLIB.ORG

Directed Evolution: Bringing New Chemistry to Life

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Directed Evolution: Bringing New Chemistry to Life 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,
Load more

Recommended publications
  • UNIVERSITY of CALIFORNIA, IRVINE an Expanded Genetic Code
    UNIVERSITY OF CALIFORNIA, IRVINE An expanded genetic code for the characterization and directed evolution of tyrosine-sulfated proteins DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILSOPHY in Biomedical Engineering by Xiang Li Dissertation Committee: Assistant Professor Chang C. Liu, Chair Associate Professor Wendy Liu Associate Professor Jennifer A. Prescher 2018 Portion of Chapter 2 © John Wiley and Sons Portion of Chapter 3 © Springer Portion of Chapter 4 © Royal Society of Chemistry All other materials © 2018 Xiang Li i Dedication To My parents Audrey Bai and Yong Li and My brother Joshua Li ii Table of Content LIST OF FIGURES ..................................................................................................................VI LIST OF TABLES ................................................................................................................. VIII CURRICULUM VITAE ...........................................................................................................IX ACKNOWLEDGEMENTS .................................................................................................... XII ABSTRACT .......................................................................................................................... XIII CHAPTER 1. INTRODUCTION ................................................................................................ 1 1.1. INTRODUCTION .................................................................................................................
    [Show full text]
  • Machine Learning-Assisted Directed Evolution Navigates a Combinatorial Epistatic Fitness Landscape with Minimal Screening Burden
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.04.408955; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Machine Learning-Assisted Directed Evolution Navigates a Combinatorial Epistatic Fitness Landscape with Minimal Screening Burden Bruce J. Wittmann1, Yisong Yue2, & Frances H. Arnold1,3,* 1Division of Biology and Biological Engineering, 2Department of Computing and Mathematical Sciences, and 3Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA *To whom correspondence should be addressed Abstract Due to screening limitations, in directed evolution (DE) of proteins it is rarely feasible to fully evaluate combinatorial mutant libraries made by mutagenesis at multiple sites. Instead, DE often involves a single-step greedy optimization in which the mutation in the highest-fitness variant identified in each round of single-site mutagenesis is fixed. However, because the effects of a mutation can depend on the presence or absence of other mutations, the efficiency and effectiveness of a single-step greedy walk is influenced by both the starting variant and the order in which beneficial mutations are identified—the process is path-dependent. We recently demonstrated a path- independent machine learning-assisted approach to directed evolution (MLDE) that allows in silico screening of full combinatorial libraries made by simultaneous saturation mutagenesis, thus explicitly capturing the effects of cooperative mutations and bypassing the path-dependence that can limit greedy optimization.
    [Show full text]
  • 1 Engineering Lipases with an Expanded Genetic Code Alessandro De Simone, Michael Georg Hoesl, and Nediljko Budisa
    3 1 Engineering Lipases with an Expanded Genetic Code Alessandro De Simone, Michael Georg Hoesl, and Nediljko Budisa 1.1 Introduction Lipases (EC 3.1.1.3) are a class of ubiquitous enzymes that catalyze both the hydrolysis and synthesis of acylglycerols with long acyl chains (carbon atoms >10) [1]. Currently, they constitute one of the most important groups of biocatalysts applied in many fields, including foods, detergents, flavors, fine chemicals, cosmetics, biodiesel, and pharmaceuticals owing to their high specificity, regios- electivity, and enantioselectivity [2]. Lipases can be found in a broad range of organisms, including plants and animals, however, it is chiefly the microbial lipases that find immense application. This is because of their high yields and ease of genetic manipulation, as well as wide substrate specificity [3]. Although lipases’ properties (molecular weight, pH and temperature optima, stability, substrate specificity) are source dependent, they all share a common structure consisting of a compact minimal α/β hydrolase fold. The hydrolase fold is composed of a central β-sheet consisting of up to eight different β strands connected by up to six α helices [1]. The active site of the α/β hydrolase fold enzymes contains a nucleophilic residue (serine), a catalytic acid residue (aspartate/glutamate), and a histidine residue, always in this order in the amino acid sequence. These residues act cooperatively in the catalytic mechanism of ester hydrolysis [4]. The lipolytic reaction takes place at the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved. Lipases are activated by the presence of this emulsion interface, a phenomenon called interfacial activation, which differentiates them from similar esterases.
    [Show full text]
  • Methods for the Directed Evolution of Proteins
    REVIEWS STUDY DESIGNS Methods for the directed evolution of proteins Michael S. Packer and David R. Liu Abstract | Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature. Natural selection Over many generations, iterated mutation and natural In this Review, we summarize techniques that gener- A process by which individuals selection during biological evolution provide solutions ate single-gene libraries, including standard methods as with the highest reproductive for challenges that organisms face in the natural world. well as novel approaches that can generate superior diver- fitness pass on their genetic material to their offspring, thus However, the traits that result from natural selection sity containing a larger proportion of functional mutants. maintaining and enriching only occasionally overlap with features of organ- We also review screening and selection methods that heritable traits that are isms and biomolecules that are sought by humans. identify or isolate improved variants within these librar- adaptive to the natural To guide evolution to access useful phenotypes more ies. Although these strategies can be applied to multi­ environment. frequently, humans for centuries have used artificial gene pathways3,4 and gene networks5–7, the examples in selection Artificial selection , beginning with the selective breeding of this Review will focus exclusively on the laboratory evolu- 1 2 (Also known as selective crops and domestication of animals .
    [Show full text]
  • Deep Learning Applications for Computer-Aided Design of Directed Evolution Libraries
    Deep Learning Applications for Computer-Aided Design of Directed Evolution Libraries Anthony J. Agbay Department of Bioengineering Stanford University [email protected] Abstract In the past decades, the advancement in industrial enzymes, biological therapeutics, and biologic-based "green" technologies have been driven by advances in protein engineering. Specifically, the development of directed evolution techniques have enabled scientists to generate genetic diversity to iteratively test randomized li- braries of variants to generate optimized enzymes for specific functions. However, throughput of directed evolution is limited due to the potential number number of variants in a randomized library. Training neural network models that utilize the growing number of datasets from deep mutational scanning experiments that characterize entire mutational landscapes and new computational representations of protein properties provides a unique opportunity to significantly improve the efficiency and throughput of directed evolution , alleviating a major bottleneck and cost in many research and development projects. 1 Introduction From reducing manufacturing waste and developing green energy alternatives, to biocatalytic cascades, designing new therapeutics, and developing inducible protein switches, engineering proteins enable enormous opportunities across a diverse array of fields. (Huffman et al., 2019; Nimrod et al., 2018; Langan et al., 2019; Smith et al., 2012) Directed evolution, or utilizing evolutionary principles to guide iterative mutations
    [Show full text]
  • The Evolution of the Genetic Code: Impasses and Challenges
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository of the Academy's Library Special Issue on Code Biology in BioSystems The evolution of the genetic code: impasses and challenges Ádám Kun1,2,3 & Ádám Radványi4 1 Parmenides Center for the Conceptual Foundations of Science, Munich/Pullach, Germany. 2 MTA-ELTE Theoretical Biology and Evolutionary Ecology Research Group, Budapest, Hungary. 3 Evolutionary Systems Research Group, Centre for Ecological Research, Tihany, Hungary 4 Department of Plant Systematics, Ecology and Theoretical Biology, Institute of Biology, Eötvös University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary Abstract The origin of the genetic code and translation is a “notoriously difficult problem”. In this survey we present a list of questions that a full theory of the genetic code needs to answer. We assess the leading hypotheses according to these criteria. The stereochemical, the coding coenzyme handle, the coevolution, the four-column theory, the error minimization and the frozen accident hypotheses are discussed. The integration of these hypotheses can account for the origin of the genetic code. But experiments are badly needed. Thus we suggest a host of experiments that could (in)validate some of the models. We focus especially on the coding coenzyme handle hypothesis (CCH). The CCH suggests that amino acids attached to RNA handles enhanced catalytic activities of ribozymes. Alternatively, amino acids without handles or with a handle consisting of a single adenine, like in contemporary coenzymes could have been employed. All three scenarios can be tested in in vitro compartmentalized systems. Keywords Origin of Life; genetic code; RNA world; ribozyme; coding coenzyme handle; Introduction Modern cells store information in DNA and have peptide enzymes to carry out the metabolism for the cell.
    [Show full text]
  • Correction for Wu Et Al., Machine Learning-Assisted Directed Protein Evolution with Combinatorial Libraries
    Correction APPLIED BIOLOGICAL SCIENCES, COMPUTER SCIENCES Correction for “Machine learning-assisted directed protein The authors note that Fig. 4 appeared incorrectly. The cor- evolution with combinatorial libraries,” by Zachary Wu, rected figure and its legend appear below. S. B. Jennifer Kan, Russell D. Lewis, Bruce J. Wittmann, and The authors also note that Table 2 appeared incorrectly. The Frances H. Arnold, which was first published April 12, 2019; corrected table appears below. 10.1073/pnas.1901979116 (Proc.Natl.Acad.Sci.U.S.A.116, Lastly, the authors also note that Table S4 in the SI Appendix 8852–8858). appeared incorrectly. The SI Appendix has been corrected online. Fig. 4. (A) Structural homology model of Rma NOD and positions of mutated residues made by SWISS-MODEL (47). Set I positions 32, 46, 56, and 97 are shown in red, and set II positions 49, 51, and 53 are shown in blue. (B) Evolutionary lineage of the two rounds of evolution. (C) Summary statistics for each round, including the number of sequences obtained to train each model, the fraction of the total library represented in the input variants, each model’s leave- one-out cross-validation (CV) Pearson correlation, and the number of predicted sequences tested. 788–789 | PNAS | January 7, 2020 | vol. 117 | no. 1 www.pnas.org Downloaded by guest on September 23, 2021 Table 2. Summary of the most (S)- and (R)-selective variants in the input and predicted libraries in position set II (P49, R51, I53) Residue Selectivity, % ee Cellular activity Variant 49 51 53 (enantiomer) increase over KFLL Input variants _ From VCHV P* R* I* 86 (S)_ YVF 86(S)_ NDV 75(S)_ † † † From GSSG P R I 62 (R)_ YFF 57(R)_ CVN 52(R)_ Predicted variants From VCHV Y V V 93 (S) 2.8-fold PV I 93(S) 3.2-fold PVV 92(S) 3.1-fold From GSSG P R L 79 (R) 2.2-fold PGL 75(R) 2.1-fold PFF 70(R) 2.2-fold Mutations that improve selectivity for the (S)-enantiomer appear in the background of [32V, 46C, 56H, 97V (VCHV)] and for the (R)-enantiomer are in [32G, 46S, 56S, 97G (GSSG)].
    [Show full text]
  • A Novel Framework for Engineering Protein Loops Exploring Length and Compositional Variation Pedro A
    www.nature.com/scientificreports OPEN A novel framework for engineering protein loops exploring length and compositional variation Pedro A. G. Tizei1, Emma Harris2, Shamal Withanage3, Marleen Renders3 & Vitor B. Pinheiro1,2,3* Insertions and deletions (indels) are known to afect function, biophysical properties and substrate specifcity of enzymes, and they play a central role in evolution. Despite such clear signifcance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specifcity, identifying multiple novel extended spectrum β-lactamases with loops of modifed length and composition—areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efcient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters. Directed evolution is a powerful tool for optimizing, altering or isolating novel function in proteins and nucleic acids1, 2. Cycles of sequence diversifcation to generate libraries followed by partitioning of those populations through selection, enable a desired function to be isolated and systematically optimised.
    [Show full text]
  • 5. Directed Evolution of Artificial Metalloenzymes: Bridging Synthetic Chemistry and Biology Ruijie K
    1 5. Directed evolution of artificial metalloenzymes: bridging synthetic chemistry and biology Ruijie K. Zhang1, David K. Romney1, S. B. Jennifer Kan1, Frances H. Arnold1 1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Abstract: Directed evolution is a powerful algorithm for engineering proteins to have novel and useful properties. However, we do not yet fully understand the characteristics of an evolvable system. In this chapter, we present examples where directed evolution has been used to enhance the performance of metalloenzymes, focusing first on ‘classical’ cases such as improving enzyme stability or expanding the scope of natural reactivity. We then discuss how directed evolution has been extended to artificial systems, in which a metalloprotein catalyzes reactions using abiological reagents or in which the protein utilizes a non-natural cofactor for catalysis. These examples demonstrate that directed evolution can also be applied to artificial systems to improve catalytic properties, such as activity and enantioselectivity, and to favor a different product than that favored by small-molecule catalysts. Future work will help define the extent to which artificial metalloenzymes can be altered and optimized by directed evolution and the best approaches for doing so. Keywords: directed evolution, fitness landscape, non-natural reactivity, artificial metalloenzyme 5.1. Evolution enables chemical innovation Nature is expert at taking one enzyme framework and repurposing it to perform a multitude of chemical transformations. For example, the P450 superfamily consists of structurally similar heme-containing proteins that catalyze C–H oxygenation, alkene epoxidation, oxidative cyclization (1), aryl-aryl coupling (2), and nitration (3), among others (4).
    [Show full text]
  • Machine Learning-Assisted Directed Protein Evolution with Combinatorial Libraries Zachary Wua, S. B. Jennifer Kana, Russell D. L
    Machine Learning-Assisted Directed Protein Evolution with Combinatorial Libraries Zachary Wua, S. B. Jennifer Kana, Russell D. Lewisb, Bruce J. Wittmannb, Frances H. Arnolda,b,1 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and bDivision of Biology and Bioengineering, California Institute of Technology, Pasadena, CA 91125 1To whom correspondence may be addressed. Email: [email protected] Author contributions: Z.W., S.B.J.K., R.D.L. and F.H.A. designed research; Z.W. and B.J.W. performed research; Z.W. contributed new analytic tools; Z.W., S.B.J.K., R.D.L. and B.J.W. analyzed data; and Z.W., S.B.J.K, R.D.L., B.J.W. and F.H.A. wrote the paper. Abstract To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning into the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine learning models trained on tested variants provide a fast method for testing sequence space computationally. We validated this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (stereodivergence) of a new-to- nature carbene Si–H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee.
    [Show full text]
  • A High-Throughput Platform for Feedback-Controlled Directed Evolution
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.01.021022; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A high-throughput platform for feedback-controlled directed evolution Erika A. DeBenedictis1,2, Emma J. Chory1,2*, Dana Gretton2*, Brian Wang2, Kevin Esvelt2 1Department of Biological Engineering, Massachusetts Institute of Technology. 2Department of Media Arts and Sciences, Massachusetts Institute of Technology. *Designates equal-contribution Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activity- dependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system
    [Show full text]
  • Machine-Learning-Guided Directed Evolution for Protein Engineering
    REVIEW ARTICLE https://doi.org/10.1038/s41592-019-0496-6 Machine-learning-guided directed evolution for protein engineering Kevin K. Yang, Zachary Wu and Frances H. Arnold* Protein engineering through machine-learning-guided directed evolution enables the optimization of protein functions. Machine-learning approaches predict how sequence maps to function in a data-driven manner without requiring a detailed model of the underlying physics or biological pathways. Such methods accelerate directed evolution by learning from the prop- erties of characterized variants and using that information to select sequences that are likely to exhibit improved properties. Here we introduce the steps required to build machine-learning sequence–function models and to use those models to guide engineering, making recommendations at each stage. This review covers basic concepts relevant to the use of machine learn- ing for protein engineering, as well as the current literature and applications of this engineering paradigm. We illustrate the process with two case studies. Finally, we look to future opportunities for machine learning to enable the discovery of unknown protein functions and uncover the relationship between protein sequence and function. rotein engineering seeks to design or discover proteins with landscape8. Recombination methods may allow for bigger jumps properties useful for technological, scientific, or medical appli- in sequence space while retaining function9, but these methods are Pcations. Properties related to a protein’s function, such as its restricted to combinations of previously explored mutations. expression level and catalytic activity, are determined by its amino Whereas directed evolution discards information from unim- acid sequence. Protein engineering inverts this relationship in order proved sequences, machine-learning methods can use this informa- to find a sequence that performs a specified function.
    [Show full text]