Directed Enzyme Evolution: Climbing fitness Peaks One Amino .
Available online at www.sciencedirect.comDirected enzyme evolution: climbing fitness peaks one amino acidat a timeCara A Tracewell1 and Frances H Arnold1,2Directed evolution can generate a remarkable range of newenzyme properties. Alternate substrate specificities andreaction selectivities are readily accessible in enzymes fromfamilies that are naturally functionally diverse. Activities on newsubstrates can be obtained by improving variants withbroadened specificities or by step-wise evolution through asequence of more and more challenging substrates. Evolutionof highly specific enzymes has been demonstrated, even withpositive selection alone. It is apparent that many solutions existfor any given problem, and there are often many paths that leaduphill, one step at a time.Addresses1Division of Chemistry & Chemical Engineering, California Institute ofTechnology, Pasadena, CA 91125, USA2Biochemistry & Molecular Biophysics, California Institute ofTechnology, Pasadena, CA 91125, USACorresponding author: Arnold, Frances H (frances@cheme.caltech.edu)Current Opinion in Chemical Biology 2009, 13:3–9This review comes from a themed issue onBiocatalysis and BiotransformationEdited by Romas Kazlauskas and Stefan LutzAvailable online 25th February 20091367-5931/ – see front matter# 2009 Elsevier Ltd. All rights reserved.DOI 10.1016/j.cbpa.2009.01.017IntroductionDirected evolution is now well established as highlyeffective for protein engineering and optimization.Directed evolution entails accumulation of beneficialmutations in iterations of mutagenesis and screening orselection; it can be thought of as an uphill climb on a‘fitness landscape’, a multidimensional plot of fitnessversus sequence. Fitness in a directed evolution experiment – how the protein performs the target functionunder the desired conditions – is defined by the experimenter, who also controls the relationship between fitness and reproduction. There are an enormous number ofways to mutate any given protein: for a 300-amino acidprotein there are 5700 possible single amino acid substitutions and already 32,381,700 ways to make just twosubstitutions. The number of ways to make four substitutions is bigger than the US national debt, a very largenumber indeed. Because most mutational paths leaddownhill and eventually to unfolded, useless proteins(there are far many more ways to make a useless proteinwww.sciencedirect.comthan a useful one), the challenge lies in identifying anefficient path to the desired function.With accumulating evolutionary enzyme engineeringexperience and particularly owing to studies in whichthe results of evolution, both natural and directed, havebeen dissected to identify the adaptive mutations andpossible pathways of their accumulation [1–3,4 ], it isbecoming clear that (1) multiple solutions are oftenaccessible for any given functional problem and (2) thereis usually a pathway whereby the target property can beacquired in a series of single, individually beneficialamino acid mutations. Whereas negative epistatic effects(in which a combination of mutations is beneficialalthough at least one individual mutation is not) arepervasive in natural evolution [5,6], there is little evidence that such effects have played a major role infacilitating directed evolution. The vast majority of evolutionary engineering studies over the past ten yearsinvolve simple uphill walks, one step at a time. Recentwork reviewed here shows that the simple uphill walk cango to quite interesting places!Functional characterization of intermediates along evolutionary pathways has also highlighted how the acquisition of activity on new substrates often proceedsthrough enzymes that accept a much broader range ofsubstrates [7]. Studies also continue to show that subsequent re-specialization of these ‘generalists’ is possible.Finally, it is increasingly clear that the natural history ofan enzyme is a good indicator of its evolvability [8]:enzymes from large families exhibiting diverse functionsor broad substrate ranges are easy to evolve in the laboratory. The same mechanisms leading to their naturalfunctional diversity facilitate the acquisition of new functions in the laboratory.There are many ways to create sequence diversity, andthis is an important part of the search strategy formolecular optimization. The goal in choosing a mutagenesis strategy is to minimize the screening requirement and increase the chances of finding beneficialmutations [9 ]. There is no single ‘best’ mutagenesismethod. Because there are many paths to a given goal,multiple methods will work (although some are farmore efficient than others). Methods for generatingdiversity and advances in selection and screeningmethods for enzymes have been reviewed recently[10–12] and are also covered elsewhere in this issue.Here we will limit our discussion to a few selectedtopics where recent literature has made significantCurrent Opinion in Chemical Biology 2009, 13:3–9
4 Biocatalysis and Biotransformationadvances in our understanding and practice of directedenzyme evolution.Promiscuous intermediates and theimportance of natural historyEvolution has created numerous specialized enzymesthat function in living cells to catalyze the chemicalreactions of life. Their specificity is tuned so that theygenerally do not tread on each other’s toes. But that doesnot mean their specificity is absolute: a recurring observation has been that many enzymes have weak activity onnon-native substrates and that directed evolution canamplify these weak activities. When the desired activityis not measurable in the wild-type enzyme, it may bepossible to find it in close variants that have been evolvedfor activity on other substrates [13] or even in enzymesthat have been evolved neutrally, accumulatingmutations that do not significantly damage the nativeactivity [14,15]. These mutated enzymes tend to exhibitbroader functional ranges, possibly through degradationof specific interactions with the natural substrate andconferring the ability to bind multiple substrates [8].Experience indicates that changing the activities ofenzymes for which there already exists functional diversity in nature is easier than for enzymes that tend to havevery specific functions across many species. Diversifyingfunction is ‘easy’ if there are multiple single-amino acidsubstitutions that do it. Enzymes involved in secondarymetabolite formation, for example, are often quite promiscuous in their substrate specificities and reactionselectivities [16]. They are also highly evolvable: singleamino acid substitutions in carotenoid biosyntheticenzymes – synthases, desaturases, cyclases, and oxygenases – alter both substrate specificity and reaction selectivity to produce a variety of novel carotenoids [17].O’Maille et al. [18 ] analyzed the catalytic landscapebetween a sesquiterpene synthase and its functionallyorthogonal homolog (75% identity) by investigating alarge fraction of the 512 possible variants having differentsubsets of the nine amino acid changes known to interconvert reaction selectivity (one synthase produces 5-epiaristolochene from farnesyl diphosphate, while the otherproduces premnaspirodiene). About half of the sesquiterpene synthase variants catalyzed the formation of bothparental synthase products as well as several other terpenes, some produced naturally by related synthases.Alternate selectivities were accessible from these intermediate enzymes with as little as a single amino acidchange.Although amino acid residues that alter substrate selectivity or specificity are often located in the active site/substrate binding pocket, it is also often observed thatmutations conferring changes in these properties are not.Even distant mutations can significantly affect catalysisby slightly altering the geometry, electrostatic properties,Current Opinion in Chemical Biology 2009, 13:3–9or dynamics of amino acids in the active site, whichinfluence the course of a reaction, particularly after ahigh-energy intermediate is formed. For example, onlytwo of the nine synthase amino acid residues investigatedin the sesquiterpene synthases were in the active site,with the remainder scattered throughout the enzyme.Changing just the two in the active site resulted in theformation of 4-epi-eremophilene, a terpene not normallyproduced in nature, while changing two non-active siteresidues incrementally converted the synthase from amainly 5-epi-aristolochene producer to one producingmainly premnaspirodiene. Umeno et al. [17] called thistype of evolvable chemistry ‘pachinko chemistry’, referring to the popular Japanese game in which the fate of araised metal ball depends on the precise interactions ithas with small metal pins as it falls down a board.Other examples of evolvable enzymes include cytochrome P450s [19,20], glutathione transferases [21],enzymes having the common (b/a)8 barrel scaffold[22,23], and members of a/b hydrolase-fold families suchas esterases and lipases [4 ,24,25]. All these familiesexhibit wide functional diversity in nature. We haveobserved anecdotally that obtaining functional diversity– for example, activity on many new substrates – tends tobe more difficult when the targeted enzyme does not havefunctionally diverse natural homologs. It is possible thatsuch enzymes have more specific contacts with theirsubstrates that preclude functional evolution throughsingle beneficial mutations.Evolving novel activityNot long ago engineering a novel activity was consideredto be a major challenge for directed evolution. Whereasengineering catalysts for new reactions is still extremelychallenging, obtaining activity on a new substrate is farless so. If an enzyme does not already exhibit a desiredactivity, the difficulty of engineering that activitydepends on how many amino acid substitutions arerequired to reach it. If even two simultaneous amino acidsubstitutions are needed to generate measurable activity,and those mutations are made randomly, the screeningrequirement is already very high. The complexity growsexponentially with the number of required changes.Strategies to overcome this exponential explosion include(1) converting the big challenge into a series of smallerones by incrementally changing the selection pressure toachieve the desired activity (analogous to increasingtemperature slightly in each generation to find highlythermostable enzymes), for example, by choosing intermediate target substrates that individually representsmall hurdles but lead to the desired activity [26], and(2) creating a ‘generalist’ enzyme, with broader specificitythat includes weak activity toward the desired substrate,and then improving the activity of that generalist. Directing multiple simultaneous mutations to a smaller set ofamino acid positions in a hybrid ‘rational’/evolutionarywww.sciencedirect.com
Directed enzyme evolution: climbing fitness peaks one amino acid at a time Tracewell and Arnold 5engineering approach is also possible [9 ,27–30], but ofcourse works only if the desired function is encoded bychanges in the targeted sites.The incremental challenge approach to obtaining a newactivity was used by Fasan et al. [31 ], who converted acytochrome P450 fatty acid hydroxylase into a highlyefficient propane hydroxylase, an activity absent in thenative enzyme. They first improved the existing weakactivity on octane until there was sufficient side activityon propane to allow screening directly on a surrogate ofthe smaller alkane. The propane hydroxylase theyobtained has sufficient side activity on ethane to allowscreening for hydroxylation of that substrate.Early steps in directed evolution for activity on a nonnative substrate often create ‘generalists’ that are activeon a much broader range of substrates. These have beenused in a more serendipitous approach to obtaining newactivities. For example, a broad-range, stereoselective Damino acid dehydrogenase was generated from mesodiaminopimelate D-hydrogenase [32] with a first roundof directed evolution to identify variants that could acceptsubstrates similar to the native substrate, meso-diaminopimelic acid. One variant had activity on D-lysine, andadditional rounds of evolution further broadened its substrate range to include straight-chain aliphatic and aromatic amino acids. In another example, galactose oxidasewas initially evolved to increase its activity on D-glucose;one variant had a broader substrate range that included 1phenylethanol. Further directed evolution improved itsactivity on 1-phenylethanol as well as other secondaryalcohols [33].New activities can also arise during ‘neutral drifts’, inwhich mutations that do not abolish the native activity areaccumulated in multiple rounds of high-error-rate mutagenesis and screening [15,34,35]. Similarly, the largenumber of mutations made by recombination of homologous glutathione transferases [21] and cytochrome P450s[36] – which can be thought of as a type of intense neutraldrift – led to the emergence of activities not observed inthe parent enzymes.Enzyme engineers also want to catalyze new reactions,but these are harder to obtain by directed evolution. Thisis where rational design can provide a crucial boost byassembling at least the rudiments of an active site. Recentenzymes designed de novo using computational methodsshow both the promise and limitations of the approach(and of our understanding of enzyme function and abilityto translate that into a design) [37 ,38]. Reactions forwhich there are no known counterparts in nature becomeaccessible when the designed protein binds the transitionstate with higher affinity than the substrates or products.Unfortunately, transition state binding is just one piece ofthe catalysis puzzle, and the resulting catalysts are notwww.sciencedirect.comparticularly impressive, at least compared to mostenzymes. Directed evolution, however, can take overwhere rational design necessarily leaves off: with thefine-tuning. Seven rounds of random mutagenesis, recombination, and screening improved the kcat/Km of adesigned Kemp elimination catalyst 200-fold [37 ].The eight accumulated amino acid substitutions occurredat positions adjacent to the designed residues as well asfarther from the active site.Evolving specific enzymesOn rare occasions an enzyme with high specificity for anew substrate can be generated with a single amino acidsubstitution [39]. Activity on a new substrate, however, isusually achieved by broadening the substrate range(Figure 1), which indicates that these ‘generalist’enzymes are the most accessible, and frequent, solutions.In fact, there are usually many ways to obtain low activityon a new substrate [9 ,34,36,40 ,41]. If a substrate-specificenzyme is required, it may be possible to eliminatevariants that maintain activity on the native or anotherundesired substrate using negative selection. Furthermutagenesis may also be required to obtain the desiredspecificity, with positive selection to improve the desiredactivity and negative selection to remove the undesiredone(s). Recent examples include highly active and selective endopeptidases generated using positive andnegative selection with a fluorescent activated cell sorting(FACS) screen [29,30] and a D-xylose-specific xylosereductase engineered using positive and negative selection for growth or inhibition on the substrates [42].Specificity can also come as a side result of continuedpressure for higher activity on the new substrate wheninteractions with the new substrate interfere with recognition of the old. Fasan et al. evolved their highly activepropane monooxygenase with positive selection aloneover multiple rounds of mutagenesis and screening foractivity on propane [31 ]. The substrate range of thisenzyme turned out to be very narrow and no longerincluded the native C12–C20 fatty acid substrates, or evenoctane [43 ]. When new activities are obtained in earlygenerations of directed evolution via a generalist enzyme,these new activities are usually well below that of thenative enzyme on its preferred substrates. Further evolution to enhance one activity comes at the cost of theothers when the substrates differ structurally and chemically and therefore interact with the enzyme in mutuallyincompatible ways. The ease of re-specializationobviously depends on how easily the enzyme can recognize those differences.Changing or increasing enzyme enantioselectivity isimportant for creating biocatalysts for synthetic organicchemistry [44]. Because the solution to this problem isreconfiguring the active site to accept (or produce) onlyone enantiomer, it was anticipated to be difficult toCurrent Opinion in Chemical Biology 2009, 13:3–9
6 Biocatalysis and BiotransformationFigure 1Directed evolution of enzyme activity on a new substrate often proceeds via a ‘generalist’ that shows weak activity on multiple substrates. Evolution ofa specific enzyme from a generalist can be done with positive selection for the new activity and negative selection to remove those variants having theundesired activity. Specificity can also be achieved by positive selection alone, if the solution to high activity on one substrate precludes high activityon others.engineer in enzymes, probably requiring multiple simultaneous mutations. To evolve an enantioselective lipase,Reetz et al. [4 ] initially used random mutagenesis with ahigh error rate and then saturation mutagenesis of residues in the active site. The best enantioselective lipasefrom that set contained six amino acid mutations. Computational analysis predicted that only two of these werenecessary, and the double variant in fact had greaterenantioselectivity than the variant with all six mutations.Because both mutations contributed positively to the newphenotype (no negative epistasis), an uphill walk couldhave found the double variant, particularly if beneficialmutations were recombined (e.g. by shuffling) or if several improved mutants were evolved in parallel after thefirst round.Directed evolution usually goes through singleamino acid improvementsAnalysis of the directed evolution literature shows that awide range of problems can be solved by uphill walksinvolving single amino acid changes. Often, singlemutations are responsible for the functional change, evenwhen multiple mutations are made [45]. Or, whenmultiple beneficial mutations are found, they all contribute and could have been found separately, as in theenantioselectivity example discussed above. Analysis ofreconstructed evolutionary intermediates supports theseobservations by demonstrating that multiple pathways ofsmall incremental improvements exist [1,2].Not surprisingly, then, a highly effective and efficientdirected evolution strategy is to gradually accumulateCurrent Opinion in Chemical Biology 2009, 13:3–9single beneficial mutations, either sequentially or byrecombination, while applying (often increasing) selection pressure. Low error-rate random mutagenesis byerror-prone PCR is very simple to implement, but onlyaccesses a limited set of (mostly conservative) amino acidchanges. Other mutagenesis methods, including saturation mutagenesis, can effectively generate additionalamino acid possibilities in targeted residues. Such a walkdoes not require construction and screening of very largelibraries: a few thousand clones can cover much of thesingle-mutant possibilities in each generation. With thereduced screening load comes the possibility of usingscreens that are higher in quality and more likely toaccurately interrogate the desired properties.An uphill walk via single beneficial mutations works onlyif (1) intermediates exist along the path from the startingpoint to the desired enzyme that incrementally improvethe desired properties, and (2) the path chosen in eachgeneration does not lead to a dead end (Figure 2). Thereare effective ways to circumvent apparent dead ends,including incorporation of stabilizing mutations that allowthe accumulation of new functional, but
Directed enzyme evolution: climbing fitness peaks one amino acid at a time Cara A Tracewell1 and Frances H Arnold1,2 Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzym
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