Designed divergent evolution of enzyme function

© 2006 Nature Publishing Group

Designed divergent evolution of enzyme functionYasuo Yoshikuni1,4, Thomas E. Ferrin1,5 & Jay D. Keasling1,2,3,4

It is generally believed that proteins with promiscuous functionsdivergently evolved to acquire higher specificity and activity1–5,and that this process was highly dependent on the ability ofproteins to alter their functions with a small number of aminoacid substitutions (plasticity)6. The application of this theory ofdivergentmolecular evolution to promiscuous enzymesmay allowus to design enzymes with more specificity and higher activity.Many structural and biochemical analyses have identified theactive or binding site residues important for functional plasticity(plasticity residues)6–10. To understand how these residues con-tribute to molecular evolution, and thereby formulate a designmethodology, plasticity residues were probed in the active siteof the promiscuous sesquiterpene synthase g-humulenesynthase11,12. Identified plasticity residues were systematicallyrecombined based on a mathematical model in order to constructnovel terpene synthases, each catalysing the synthesis of one or afew very different sesquiterpenes. Here we present the construc-tion of seven specific and active synthases that use differentreaction pathways to produce the specific and very differentproducts. Creation of these enzymes demonstrates the feasibilityof exploiting the underlying evolvability of this scaffold, andprovides evidence that rational approaches based on these ideasare useful for enzyme design.Promiscuous enzyme activities have long been believed to be an

important determinant for molecular evolution of more specific andactive enzyme functions1–5. It is thought that primordial enzymesmay have been promiscuous to render primitive organisms adaptableto their environment; enzymes with higher specificity are the result ofdivergent evolution (gene duplications and subsequent mutations),driven by selective pressure, from promiscuous precursor enzymes.Numerous biochemical analyses have suggested that proteins have anability to improvise novel or altered functions with a small number ofamino acid substitutions (plasticity)5–10. We refer to those residuesthat primarily govern enzyme specificity as plasticity residues. Theunderlying evolvability of promiscuous enzymes is also thought to bevery important in molecular evolution in order to allow organismsto adapt rapidly in response to environmental changes6. Becausenatural evolution is known to be a highly accomplished designer forprotein function, understanding how proteins acquire novel oraltered functions and how plasticity residues contribute to thenatural evolution process may help to formulate an efficient designmethodology for new enzymes.To investigate how promiscuous proteins might evolve to acquire

more active and specific functions, we chose as a model enzymeg-humulene synthase, a sesquiterpene synthase from Abiesgrandis11,12 that is known to produce 52 different sesquiterpenesfrom a sole substrate, farnesyl diphosphate, through a wide variety ofcyclization mechanisms (Fig. 1 and Supplementary Fig. 1). Allterpene synthases share a similar active site scaffold13–16. The reactionis initiated by cleavage of the diphosphate group to yield a carbo-cation intermediate, which is then cyclized into many different

structures. In general, this reaction generates a large number ofterpene structures with different regio- and stereochemistries17. Innone of the previous work was it shown that one could successfullycontrol this reaction pathway of a highly reactive carbocation species,or improve the product selectivity for chemically more complexreactions.Owing to the extreme promiscuity of g-humulene synthase, the

product distribution could be very sensitive to changes in specificamino acid residues; hence, the enzyme should be an excellent modelto study plasticity residues. However, the significantly greater prom-iscuity of this enzyme compared to other enzymes makes functionaldesign more challenging, and the lack of a selection or a high-throughput screen for evolved terpene synthases makes directedevolution nearly impossible. In addition, it is extremely difficult topredict the relationships between primary sequence and enzymefunction in this class of enzymes, because enzymes are closely relatedwithin or near species regardless of their functional disparity18–20.This lack of relatedness among the limited number of knownsesquiterpene synthases with a similar function makes functionaldesign based on phylogenetic analysis nearly impossible21. Therefore,a method that would allow one to predict the effect of changes inamino acid residues in terpene synthases on product selectivitywould alleviate the need for a high-throughput screen or genomicanalysis in designing synthases useful for mass production of singleterpenes that have found use as drugs, flavours, fragrances, nutra-ceuticals and in many other applications17,22.Although plasticity residues can be found anywhere in proteins,

many biochemical and genomic analyses have indicated that theytend to bemore focused inside or near active sites6–10. Consistent withthis observation, results from directed evolution23, which can searchfor such residues over entire proteins, also indicate that these residuesmost often occur in the active site6,9. To determine the active-siteresidues important for g-humulene synthase function, a homologystructure for g-humulene synthase was first built using the crystalstructure of 5-epi-aristolochene synthase (Protein Data Bank entry5eat14) as a guide24,25. Although mutations to residues in the con-served aspartate-rich motif in the active site are known to alter thereaction mechanisms of terpene synthases12,26, these residues werenot considered further because mutations in this motif are usuallyaccompanied by significant losses of activity12,26. As a result, the 19residues composing the active-site contour were selected for satur-ation mutagenesis to investigate how each residue contributes to aparticular reaction mechanism (Fig. 2). Saturation mutagenesis ofresidue S484 and subsequent screening by gas chromatography-massspectrometry (GC-MS) suggested that 80 mutants were sufficientto obtain almost all possible amino acid changes (SupplementaryTable 1). The altered product distribution from each mutant wasnormalized to that of the wild-type enzyme and profiled. Althoughmany of these residues were identified to be plastic, four residuessignificantly affected catalysis: W315, M447, S484 and Y566 (Sup-plementary Figs 2–5). Mutations to these residues shifted the relative

LETTERS

1UCSF/UCB Joint Graduate Group in Bioengineering, 2Department of Chemical Engineering, and 3California Institute for Quantitative Biomedical Research (QB3), University ofCalifornia at Berkeley, Berkeley, California 94720, USA. 4Synthetic Biology Department, Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California94710, USA. 5Department of Pharmaceutical Chemistry and Biopharmaceutical Sciences, University of California at San Francisco, San Francisco, California 94143, USA.

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selectivity (the amount of one product relative to another product)by 100- to 1,000-fold.To investigate further how these plasticity residues contribute to

molecular evolution and to formulate a design methodology forproduct selectivity, the mutations were systematically recombinedbased on the profiles obtained from saturation mutagenesis. Therecombination was carried out using an algorithm (detailed in theMethods) based on the assumption that each plasticity residue isindependent—the effect of a particular mutation on the reactionmechanism should be the same for the wild-type enzyme and anymutants. Thus, a set of mutations to achieve a desired enzymefunction was predicted based on howmuch the product distributionmoved towards the desired product distribution, as measured by qfor a particular combination of mutations. For example, in the

construction of a b-bisabolene synthase (BBA; product 7 in Fig. 1),two mutants (M447H/A336V/I562T (q ¼ 6.7) and M447H/A336V/I562V (q ¼ 5.4)) were predicted to be the best combinations ofmutations to these three amino acids to maximize b-bisaboleneselectivity (q ¼ 24.4 for wild type). M447 was important in specify-ing the 10,1 or 6,1 closure from the trans- or cis-farnesyl cation(q ¼ 16.3, Fig. 3b, c); A336 was important in specifying the 11,1closure from the cis-farnesyl cation (q ¼ 10.8, Fig. 3d, e); and I562was important in specifying acyclic terpene formation (Fig. 3f).M447H/A336V/I562T reduced production of product 1 and showed2.5-fold better selectivity for production of 7 over 1 compared to thatof M447H/A336V/I562V (Fig. 3f; see also Supplementary Table 2).Thus, a b-bisabolene synthase was successfully constructed whilemaintaining its activity (Table 1).

Figure 1 | g-Humulene synthase cyclization reaction mechanisms. Whenthe substrate, farnesyl diphosphate, binds to the enzyme active site viadivalent magnesium cations, the diphosphate group is released to yieldeither trans- or cis-farnesyl cation. From the trans-farnesyl cation, sibirene(2) is produced by the 10,1 cyclization reaction. From the cis-farnesyl cation,g-humulene (3), longifolene (4) and a-longipinene (5) are produced

through an 11,1 cyclization reaction; a-ylangene (6) through a 10,1cyclization reaction; and b-bisabolene (7) through a 6,1 cyclization reaction.E-b-farnesene (1) and Z,E-a-farnesene (8) can be produced by directeddeprotonation from either farnesyl cation (see Supplementary Fig. 1 formore details).

Figure 2 | The homology structural model for the g-humulene synthaseactive site. a, The residues that were not considered are shown in yellow. Sixaspartate residues in two different aspartate-rich motifs and two arginineresidues, which are generally conserved in all sesquiterpene cyclases, werenot considered, because these residues are thought to be catalyticallyimportant, and mutations to these residues would have decreased enzyme

activity significantly. b, The 19 residues in the active site are shown, andthese residues were targeted for saturation mutagenesis and systematicremodelling. (See Supplementary Fig. 11 for the primary sequencealignment and Supplementary Data 1 for the three-dimensional coordinatesfor the homology model of g-humulene synthase.)

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We used this same method to create an E-b-farnesene (1)/Z,E-a-farnesene (8) synthase (BFN: W315P), a sibirene (2) synthase (SIB:F312Q/M339A/M447F), a longifolene (4) synthase (LFN: A336S/S484C/I562V), an a-longipinene (5) synthase (ALP: A336C/T445C/S484C/I562L/M565L) and two new g-humulene (3) synthases(HUM: M339N/S484C/M565I and AYG: S484A/Y566F), the latterhaving significantly improved a-ylangene (6) production (Fig. 4,Table 1, Supplementary Figs 6–10 and Supplementary Tables 3–7).SIB, HUM, ALP and AYG are new synthases that have not yet beendiscovered in nature. Although the construction of a-ylangenesynthase was not achieved (AYG), the algorithm predicted that itwould not be possible to create such an enzyme from the current setof plasticity residues.Almost all mutations were added by saturation mutagenesis so as

not to miss any better substitutions that were not predicted by thealgorithm. However, in almost all cases, the predicted substitutions

gave the desired product distribution (Fig. 3; see also SupplementaryTables 2–7 and Supplementary Figs 6–10). In general, all of thedesigned enzymes maintained a level of specific activity comparableto the wild-type ancestor (Table 1). These results indicate that inorder to generate a specific enzyme from another specific enzyme, theenzyme must first acquire promiscuous function, supporting thetheory that specific enzymes could evolve from promiscuous pre-cursor enzymes with surprisingly few mutations6. In addition, weobserved convergent evolution, as the product profiles of HUM andAYG are very similar to each other despite the significant differencesin mutations that gave rise to these specific functions.Althoughwe assumed that the plasticity residues behaved as if they

were independent and that changes to these residues were additive,the effects could be partially additive, synergistic, antagonistic, oreven absent altogether27. If two residues to be mutated do notinteract, then the effect of mutating the residues is likely to be

Figure 3 | Systematic remodelling of plasticity residues to designb-bisabolene synthase. Chromatograms in a, c, e and g show the GC-MSanalysis of terpene production for wild type, M447H, A336V/M447H andA336V/M447H/I562T, respectively. The numbers for each peak correspondto those in Fig. 1. b, d and f show the free energy change for formation of 7

over that for formation of 1–6 for each successive change to residues M447,A336 and I562 compared to that of wild type, respectively. Quantitativeanalyses are shown in Supplementary Table 2. Mutations were sequentiallyadded, so as to reduceq. b-Bisabolene synthase was successfully constructedfrom g-humulene synthase through A336V/M447H/M562T.

Table 1 | Summary for wild-type g-humulene synthase and its derivatives

Clones* Mutations Product distributions (%)† Yield(times) ‡

kcat(s21)

Km

(mM)kcat/Km

(M21 s21)1 2 3 4 5 6 7 8

Wild type Wild type 3.0 23.1 45.1 13.4 4.7 3.8 6.9 ND 1 2.36 ^ 0.16 ( £ 1022) 4.66 ^ 0.97 5.07 £ 103

BFN W315P 54.1 2.9 2.2 ND ND ND 6.7 34.0 2.1 1.94 ^ 0.04 ( £ 1023) 0.179 ^ 0.024 1.08 £ 104

SIB F312Q, M339A, M447F 6.4 78.1 11.4 2.6 0.1 1.0 0.4 ND 1.8 4.63 ^ 0.19 ( £ 1024) 3.01 ^ 0.54 1.54 £ 102

HUM M339N, S484C, M565I 2.7 0.4 85.7 3.4 3.5 4.3 ND ND 1.2 1.81 ^ 0.09 ( £ 1023) 2.08 ^ 0.53 8.70 £ 102

LFN A317N§, A336S, S484C,I562V

1.7 1.4 12.6 63.0 12.6 3.2 5.5 ND 4.4 6.96 ^ 0.50 ( £ 1022) 3.83 ^ 0.95 1.81 £ 104

ALP A336C, T445C, S484C,I562L, M565L

7.7 ND 11.7 13.3 61.5 2.2 3.6 ND 13 3.81 ^ 0.18 ( £ 1023) 4.59 ^ 0.84 8.31 £ 102

AYG S484A, Y566F 2.6 0.4 54.6 3.5 15.8 14.7 8.4 ND 3.3 1.21 ^ 0.06 ( £ 1022) 6.05 ^ 1.04 1.99 £ 103

BBA A336V, M447H, I562T 6.4 0.4 3.8 4.7 1.1 0.1 83.6 ND 4.2 2.24 ^ 0.10 ( £ 1022) 2.88 ^ 0.40 7.77 £ 103

BFN, E-b-farnesene synthase; SIB, sibirene synthase; HUM, g-humulene synthase; LFN, longifolene synthase; ALP, a-longipinene synthase; AYG, a-ylangene over-producer (another g-humulene synthase); BBA, b-bisabolene synthase; ND, production not detected; Km, Michaelis constant.*All constructs are made based on a soluble variant (data not shown).†All product distributions were represented for 1–8 as 100%; these products generally correspond to more than 85–95% and to 75% of total products in mutants and wild type, respectively.All product distributions were determined from triplicates, and standard deviations were lower than 2%.‡ In vivo productivity over wild type for each desired product.§A317N occurred during recombination, and improved in vivo terpene production without a change in product distribution.

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additive. As direct or indirect interactions between two residuesincrease, the impact of multiple mutations may be far fromadditive27. All effects other than antagonistic, however, would stillbe predicted to some extent using the methodology outlinedhere. Interestingly, with the exception of the double mutantM339N/S484C, all mutations introduced into the enzyme wereeffectively additive. Hence, in practice, the assumption thatmutations will have an additive effect is rational, and the resultingenzyme design methodology is simple, yet powerful.Construction of the large number of sesquiterpene synthases

required fewer than 2,500 mutants to be screened. However, indirected evolution—currently touted as an effective tool to alterprotein function6,9,10,23,28—tens of thousands to a million or moremutants must be screened to find a few critical mutations; hence itsapplication is limited by the availability of an efficient screeningmethod. The systematic recombination approach described hereinenabled us to design enzyme specificity rapidly and efficiently with-out a screen for the desired activity. Because plasticity residues havevery important roles in proteins29, this approach, with some modi-fications, may be useful for designing novel functions for many otherproteins, including enzymes, protein ligands/receptors, transcriptionfactors and antibodies.On the basis of the theories of molecular evolution and experi-

mental observations, we formulated an approach for systematicrecombination of the promiscuous g-humulene synthase. We suc-cessfully constructed a large number of novel specific sesquiterpenesynthases, each producing one or a few products derived from apredominant reaction pathway while largely maintaining the specificactivity of the original enzyme. These results suggest that: (1)divergent evolution by rational design may be feasible on a signifi-cantly larger scale than currently possible; (2) plasticity residuescould significantly drive molecular evolution; and (3) most of the

substitutions in plasticity residues additively affect protein functions.Although we demonstrated systematic recombination using thesubset of plasticity residues located in the active site, other residuescan also be considered in order to construct other specific enzymes oreven enzymes that produce unnatural products.

METHODSGC-FID and GC-MS analysis for sesquiterpenes. A single colony harbouringpTrcHUM and pBBRMBIS (kanamycin; antibiotic-resistance gene wasreplaced)22 was inoculated into Luria Bertani (LB) medium containing50 mgml21 carbenicillin and 50mgml21 kanamycin and grown overnight at30 8C. An aliquot (50 ml) of this seed culture was inoculated into fresh LBmedium containing 10mMmevalonate, 0.1mM isopropyl-1-thio-b-D-galacto-pyranoside (IPTG), 50 mgml21 carbenicillin and 50 mgml21 kanamycin (5ml),overlaid with 500ml dodecane, and grown for 24 h at 30 8C. To screen the libraryresulting from site-directed saturation mutagenesis, a single colony harbouringonly pTrcHUM was inoculated into LB medium containing 0.1mM IPTG and50 mgml21 carbenicillin overlaid with 500ml dodecane, and grown for 24 h at30 8C. An aliquot of dodecane (50ml) was diluted into 200ml of ethyl acetate, andthe mixture was analysed by GC-MS using a GC oven temperature programmeof 80 8C for 1min, then steps of 30 8Cmin21 to 110 8C, 5 8Cmin21 to 160 8C and130 8Cmin21 to 250 8C for Cyclosil-B capillary column analysis, or at 80 8Cfor 3min, then steps of 5 8Cmin21 to 160 8C and 120 8Cmin21 to 300 8C forDB-5MS capillary column analysis. Gas chromatography-flame ionizationdetector (GC-FID) analysis was also carried out to quantify each sesquiterpeneproduct using the method described herein. The proportion of each productwas determined based on the ratio of the relative peak abundance for eachproduct. Sesquiterpenes were identified from their mass spectra and GCretention times by comparison to available authentic standards (g-humulene,longifolene, a-longipinene and E-b-farnesene) and spectra in libraries pre-viously reported in the literature (sibirene11, a-ylangene30, b-bisabolene30 andZ,E-a-farnesene30).Homology structural modelling of g-humulene synthase. The homologystructural model for g-humulene synthase (Supplementary Data 1) was built

Figure 4 | Divergent evolution of novel sesquiterpene synthases fromg-humulene synthase. Chromatograms show the GC-MS analysis forsesquiterpene production from both wild type (centre) and variants ofg-humulene synthase. The numbers for each peak and the colours for eachchromatogram correspond to those in Fig. 1. All g-humulene synthasevariants were designed based on the systematic remodelling and constructed

by site-directed saturation mutagenesis and site-directed mutagenesis.Primary sequence and reaction mechanism relationships were clearlyobserved. For example, enzymes that produce 3, 4 and 5, all of which areproduced from the 11,1 cyclization pathway, are more closely related(sharing S484C) to each other than to any of the other enzymes.

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using MODELLER25 (http://salilab.org/modeller/). The alignment (Supplemen-tary Fig. 11) and the structure of 5-epi-aristolochene synthase14 (PDB entry 5eat)were used as guides. The resulting homology structure was visualized usingChimera (http://www.cgl.ucsf.edu/chimera).Algorithm for systematic remodelling of plasticity residues. To design thespecificity for novel sesquiterpene synthases, combinations of mutations wereselected based on the results from the previous screening. Assuming that there isno interaction between plasticity residues, the effect of a certain mutation is thesame for both wild type and other mutants. Therefore, the product distributionprofile upon another round of mutagenesis can easily be calculated using thefollowing equation:

Di ¼dixiPnj¼1 djxj

£ 100 ð%Þ

where Di is the predicted percentage of product distribution of compound i forall compounds 1 to n (in this study n ¼ 17), di is percentage of parent productdistribution of compound i (which can be predicted) and x i is the effect of aparticular mutation on compound i productivity relative to the wild-typeenzyme.

The predicted percentage of product distribution of compound i by additionofm distinctivemutations (mth generation),Di,m, is then represented as follows:

Di;m ¼di;0

Qmg¼1 xi;gPn

j¼1 dj;0Qm

g¼1 xi;g£ 100 ð%Þ

where di,0 represents the parent (0th generation) product distribution ofcompound i (which can be predicted) and x i,m represents the effects by themth mutation. To select the mutations that probably introduce the desiredfunction, the root mean square deviation of the predicted product distributionfrom the desired product distribution was calculated using the followingequation:

qm ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPni¼1 Di;m 2 d

0

i

� �2

n

vuutð%Þ

where qm represents the relative closeness of product profiles of mutants withmth generation (m mutations) to the one specified, and d

0

i is the percentage ofdesired product distribution for compound i (for example, for b-bisabolenesynthase d 0

7 ¼ 100% and d 0

i–7 ¼ 0%). To improve the accuracy of the designmethodology, we included 17 different product profiles, which correspond to95% of total products.

To maintain specific activity and productivity, the overall productivity wascalculated using the following equation:

P¼Xni¼1

Pi ¼Xni¼1

pixi

where P is total productivity, Pi is predicted productivity for compound i, and p iis parent productivity for compound i. Similarly, the overall productivity at themth generation was calculated using the following equation:

Pm ¼Xni¼1

Pi;m ¼Xni¼1

piYmg¼1

xi;g

The combinations of mutations were selected so as to decrease the qm value andtomaintain Pm. The selectedmutations were added to g-humulene synthase andthe results were inspected. See Supplementary Methods for additional methods.

Received 25 October 2005; accepted 26 January 2006.Published online 22 February; corrected 27 February 2006.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We would like to thank P. C. Babbitt, J. D. Newman,M. C. Chang and S. C.-H. Pegg for discussions and critical reading of themanuscript. We are also grateful for D. Herschlag for critical comments. Thisresearch was funded by the Bill & Melinda Gates Foundation, the USDepartment of Agriculture, and the National Science Foundation.

Author Contributions Y.Y. and J.D.K. conceived the project; Y.Y., J.D.K. andT.E.F. designed the experiments; and Y.Y. and J.D.K. wrote the paper.

Author Information Reprints and permissions information is available atnpg.nature.com/reprintsandpermissions. The authors declare competingfinancial interests: details accompany the paper at www.nature.com/nature.Correspondence and requests for materials should be addressed to J.D.K.([email protected]).

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Designed divergent evolution of enzyme function