Available online at www.sciencedirect.com
Advances in laboratory evolution of enzymes
Shimon Bershtein and Dan S Tawfik
We address recent developments in the area of laboratory, or
directed evolution, with a focus on enzymes and on new
methodologies of generic potential. We survey three main
areas: (i) library making techniques, including the application of
computational and rational methods for library design; (ii)
screening and selection techniques, including recent
applications of enzyme screening by FACS (fluorescence
activated cell sorter); (iii) new approaches for performing
directed evolution, and in particular, the application of ‘neutral
drifts’ (libraries generated by rounds of mutation and selection
for the enzyme’s original function) and of consensus mutations
to generate highly evolvable starting points for directed
evolution.
Address
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot 76100, Israel
Corresponding author: Tawfik, Dan S (tawfik@weizmann.ac.il)
Current Opinion in Chemical Biology 2008, 12:151–158
This review comes from a themed issue on
Biocatalysis and Biotransformation
Edited by Stephen G. Withers and Lindsay Eltis
Available online 7th March 2008
1367-5931/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2008.01.027
Introduction
Directed evolution techniques and applications have
widely expanded in the past few years. The literature
relevant to this area includes several hundred articles for
the period of 2005–2007 that cannot be covered by this
review. We therefore focus on a few major topics. Firstly,
although directed evolution has now been applied to a
wide range of protein functions, including ligand and
protein binding, and regulatory functions, this review’s
main focus is enzymes. Secondly, we primarily address
new methodologies and strategies for directed evolution,
rather than examples, as interesting as they may be, for
the evolution of individual enzymes. The review comprises three sections that discuss recent developments in
library making methodologies, screening and selection
methodologies, and new approaches for performing
directed evolution.
The reader is referred to other recent reviews, including
an extensive review on directed evolution of enzymes [1]
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and a review on selection strategies [2]. The application
of directed evolution to enzyme pathways has also been
reviewed [3,4]. Finally, research at the interface of fundamental and applied enzyme evolution has also been
discussed [5,6].
Library making techniques
Refining existing protocols
Several works describe the refinement of existing library
making techniques. These include variations on Stemmer’s DNaseI protocol for DNA shuffling [7,8,9] (for a
comprehensive description of various in vitro recombination strategies consult the recent review [10]) and a
method that aims at mixing wild-type, and improved
mutant genes, in certain combinations [11]. New applications for the useful incremental truncation techniques,
such as ITCHY [12], were also described [13,14]. Other
works address the fact that random libraries generated by
various methods, such as error-prone PCR, are clearly
non-random. Each method gives a different bias of base
substitutions that result in a different range of amino acid
replacements, the latter also depending on the mutated
gene itself. Statistical approaches that facilitate an educated choice of technique(s) have become available.
These assess the sequence diversity available by each
mutagenesis technique [15] and also analyze sequence
and structure indicators of the resulting libraries at the
protein level [16,17].
Experimental approaches aimed at increasing library sizes
by ‘in situ’ mutagenesis have also been described. These
are based on generating genetic diversity within the
Escherichia coli, or yeast, cells in which protein expression
and screening take place. Such in situ mutagenesis
approaches (e.g. the use of E. coli carrying a mutator
polymerase [18]) afford very large library sizes independent of transformation efficiency. Following earlier developments of in situ homologous recombination in yeast [19]
and in situ mutagenesis in RNA bacteriophage [20], new
methods for in situ recombination has now been described
for both E. coli [21] and yeast [22].
Indels
Whereas techniques for creating genetic diversity by
recombination and point mutations are well developed
and widely applied, methods for randomly incorporating
insertions and deletions (indels) are still in their infancy.
The divergence of new functions in natural enzymes
often involves such changes, and their laboratory reproduction is therefore of importance. Two new methods for
random incorporation of indels [23] and in-frame
deletions [24] were recently described, as well as a
Current Opinion in Chemical Biology 2008, 12:151–158
152 Biocatalysis and Biotransformation
method for incorporation of indels in pre-defined positions in a combinatorial manner [25]. However, the wide
applicability of these techniques, and in particular, a
convincing demonstration that indels can yield enzymatic
functions that are not accessible by point mutations and
recombination, is still pending.
Targeted and designed libraries
The scrutiny of various library making strategies continues. One school surmises that incorporating mutations
at random is the most effective approach, primarily
because many function altering mutations occur at unexpected positions away from the active site. The other
school focuses on the randomization of a given set of
residues that are within the active site and in direct
contact with the substrate [26,80]. Indeed, in certain
cases, targeted active-site libraries yield preferable results
[27–29,69], but in others, the results of random and
targeted libraries were comparable [30]. It therefore
appears that mutations in both direct contact, and distal,
mutations can improve activity, and the former can be
particularly successful in cases where a small number of
active-site residues that trigger the required change in
substrate specificity can be identified [27]. The snag is
that in most cases, the choice of active-site residues for
diversification is far from obvious. Active-site walls comprise numerous residues, and the libraries in which these
are simultaneously randomized are far too large and
comprise almost only inactive variants. This difficulty
can be addressed by iteratively exploring the active-site
residues, namely by libraries that randomize one or two
residues at a time and then combine the advantageous
mutations [31,32]. This strategy might be limited, however, by the non-additivity (epistasis) of evolutionary
pathways, namely, in many cases where the order by
which mutations accumulate is crucial [33]. Alternatively,
combinatorial methods that generate random subsets of
the entire set of randomized active-site residues have
been explored [25,30].
Finally, recent works indicate that a successful choice of
which positions should be diversified, and how (e.g. into
which amino acids) could also be made using a variety of
statistical and computational methods [34,35–37]. While
space limitations do not enable a detailed discussion of
these methodologies, it appears that computationally
designed libraries will play a growingly important role
in protein engineering. The facile making of such ‘smart’
libraries can be greatly facilitated by sophisticated, automated DNA synthesis methods [38].
Screening/selection techniques
FACS based enzyme screens
Modern FACS machines can easily sort >104 events/s,
accurately and reproducibly, using multiple parameters.
FACS therefore holds much potential in the area of
enzyme evolution. What remains a main challenge,
Current Opinion in Chemical Biology 2008, 12:151–158
however, is maintaining the linkage between the
enzyme, a diffusable product, and the enzyme coding
gene. In some cases, where the target reaction involves
the modification of a hydrophobic fluorescent substrate
with a charged group, the unmodified substrate can be
washed out of the cells, while the product remains
within (Figure 1a). For example, modification of the
fluorophore 7-amino-4-chloromethyl coumarin with
glutathione resulted in its entrapment, allowing large
libraries of E. coli cells carrying active glutathione-Stransferases (GSTs) variants to be selected [39,40]. In
another example, fluoroscein di-b-galactopyranoside
with a lipohilic tail that retains the fluorescent product
upon cleavage by b-galactosidase was applied [7]. An
alternative scheme for the entrapment of a fluorescent
product by coupled enzymatic reactions that occur
within the sorted E. coli cells was devised for the
selection of glycosyltransferases [41].
Another type of application involved the display of
enzyme on cell surfaces and the entrapment of the
product onto the surface (Figure 1b). The protease
OmpA was displayed on the surface of E. coli, and a
fluorescence resonance energy transfer (FRET) substrate was added that adheres to the surface of the
bacteria. Enzymatic cleavage of the substrate releases
a quencher group, and the resulting fluorescent cells were
isolated by FACS. This system was used for a dual
selection (a selection for a cleavage of a given peptide
substrate against the cleavage of others), and a variant of
OmpT proteinase with a change in selectivity of >106
relative to wild-type was obtained [[42], see also note
added in proof]. A complementary approach was developed whereby the substrates of surface-displayed
enzymes (esterases or lipases) release a tyramide–biotin
moiety that is then covalently linked to the cell surface
using peroxidase-activated tyramide conjugation. This
labeling enables the isolation of E. coli cells presenting
active enzyme variants by either FACS or magnetic-bead
selections [43,69].
The entrapment of fluorescent products can also be
achieved by compartmentalization in emulsion droplets
(in vitro compartmentalization, or IVC) (Figure 1c). The
technology makes use of the standard water-in-oil (w/o)
droplets to compartmentalize the gene, the encoded
enzyme, and the resulting product. Conversion of the
primary emulsion to a double water-in-oil-in-water (w/o/
w) emulsion provides an external aqueous phase that
enables sorting of the droplets by FACS. Double emulsions were recently applied for the directed evolution of
different enzymatic systems. A library of b-galactosidase
variants was translated in vitro in the primary w/o emulsion and then FACS sorted in a double emulsion [44]. In
the case of PON1, intact E. coli cells in which the library
variants are expressed were emulsified and FACS sorted
[30]. The same strategy has been recently applied to the
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Advances in laboratory evolution of enzymes Bershtein and Tawfik 153
Figure 1
Screening enzyme libraries by FACS (fluorescence activated cell sorter). A wealth of enzyme reactions can be screened using fluorogenic substrates,
provided that the enzyme, the product, and the gene encoding the enzyme can be linked during the sort. Various strategies were developed that meet
this prerequisite: (a) Entrapment of the product within the enzyme expressing cells [7,39,40,41]. The enzyme-transformed substrate is modified in a
way that prevents its diffusion out of the cells (usually by the attachment of a polar or charged group). The excess of unreacted substrate is washed
out, and cells containing the entrapped fluorescent product are isolated. (b) Entrapment of the product onto the surface of cells displaying the target
enzyme [42,43,69]. The surface-displayed (or periplasmic) enzyme converts a non-fluorescent substrate into a fluorescent product that is then
attached to the cell surface (either covalently, or non-covalently). (c) Entrapment of the fluorescent product in an emulsion droplet carrying the enzyme
and its encoding gene. In this case, the enzyme can be expressed in E. coli cells (either in the cytoplasm, or on the surface), or by cell-free translation.
Conversion of the emulsion to a double, w/o/w emulsion enables the FACS sorting of the droplets [30,44–46].
selection of lactonase activity [45], using an oxo-lactone
substrate whose enzymatic hydrolysis generates a thiol
that was subsequently detected with a fluorogenic probe.
Detailed protocols for the preparation and sorting of
double emulsions are now available [46].
Other screening/selection methodologies
The development and application of other emulsionbased selections are described in a recent review [47],
with the most recent developments being the selection of
thermostable polymerase variants for the amplification of
damaged DNA samples, such as ancient DNA [48];
selection by product driven PCR amplification of enzyme
coding genes [81]; and the selection of restriction endonucleases [82].
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In vitro techniques other than IVC have also been applied
for directed evolution. Most recently, messenger RNA
display was used to select a library of >1012 in vitro
translated protein variants derived from a novel zincfinger scaffold, and thus identify novel RNA ligases
[49]. In vitro (cell-free) transcription/translation has also
been used in conjunction with standard screens in 96-well
plates [29].
Besides the development of novel approaches, the application of traditional methodologies, such as genetic
screens and selections and screens in 96-well plates,
has produced interesting results. A book describing a
wide range of generally applicable approaches has been
recently published [50]. Screening assays for highly useful
Current Opinion in Chemical Biology 2008, 12:151–158
154 Biocatalysis and Biotransformation
enzyme types such as hydroxynitrile lyases [51] and
epoxygenases [52] have been developed and applied
for library selections. Despite their low throughput, assays
in microtiter plates (96- or 384-plates) are advantageous
because they can be applied to complex enzyme systems
such as the adenylation domains involved in combinatorial biosynthesis [53] or new glycosyl transferases [54]. The
throughput of plate assays can also be increased by
pooling up to 10 colonies in a single well [55].
Notable extensions for the application of traditional
genetic in vivo screens to enzyme library selections have
been also described [56], including the construction of a
chemosensor E. coli host strain that recognizes the product of the targeted enzymatic conversion [57] and the
application of tunable expression vectors for extending
the dynamic range of in vivo selections [58].
New approaches for laboratory enzyme
evolution
Multi-parameter selections
Whereas traditional screening and selection strategies aim
at isolating variants that exhibit an increase in a single
target activity, more sophisticated schemes arise that
screen for more than one parameter. Multi-parameter
screens can include many substrates [45,59,60]. The
aim can be, for example, to maintain the enzymatic
properties by screening for multiple substrates while
increasing expression levels and stability (for example
see reference [61]), or enhancing only one enzymatic
activity without altering others [62]. Coevolving stability
and enzymatic activity is another target for multiparameter selections [63,64]. Finally, it is becoming
apparent that while improving the activity toward a weak,
promiscuous substrate is easily achieved, under selection
for a ‘new’ activity, the original, native activity of the
enzyme often persists [65,66]. Hence, a ‘dual selection’ –
a selection for the new function and against the old one –
needs often to be applied to accelerate the evolution of a
new function while erasing the original one [[42,67,69],
see also note added in proof].
Neutral drift—a novel approach for directed evolution
An emerging development in the area of laboratory evolution regards the reproductions of ‘neutral drifts’, that is,
the gradual accumulation of mutations under selection to
maintain the protein’s original function and structure
[7,45,60,68,70]. Theoreticians have predicted fascinating properties of ‘neutral networks’ or quasi-species such
as higher mutational robustness and evolvability that
develop when proteins drift and create a ‘cloud’ of
sequences around an existing sequence [71–73].
Although a neutral enzyme drift was described a while
ago, but its potential in the evolution of new function has
not been demonstrated [7]. More recently, neutral drifts
of two different enzymes PON1 and P450 were perCurrent Opinion in Chemical Biology 2008, 12:151–158
formed by applying iterative rounds of mutation and
selection for the enzymes’ native or primary activity.
These showed how the potential for new functions develops when the neutral network of a protein expands
[45,60]. The PON1 drift led to the isolation and characterization of 311 neutral variants that maintain wild-typelike activity and expression level. About half of these
neutral variants exhibited significant changes in promiscuous activities, specificities, or inhibition (the latter
simulating the emergence of drug resistance) [45].
Similar fluctuations in promiscuous activities were
observed in the P450 neutral variants [60]. A number
of the PON1 neutral variants isolated were one, or even
two, mutations closer to a new enzyme specificity (aryl
esterase, thiolactonase, phosphotriesterase, or drug resistance). Indeed, a subsequent screen of these 311 variants
with a new substrate (an analog of the nerve agent
cyclosarin) yielded two variants with up to 72-fold higher
activity relative to wild-type PON1 [45]. Additional
variants exhibiting large improvements with other promiscuous substrates were recently identified (Gupta D
et al., unpublished results).
How can a small library of only 311 members yield a range
of improved variants? The standard libraries used for
directed evolution comprise a majority of ‘dead’ variants
owing to deleterious mutations, and hence demand the
screening of much larger diversities. The neutral drift
libraries comprise enzyme variants that are all stable and
active. Nevertheless, these variants carry numerous
mutations in, and around, the active site. Some of these
mutations have the ability to mediate new specificities by
increasing or decreasing existing promiscuous activities
while exerting little effect on native activity (Figure 2).
Boosting enzyme evolvability
An oft-forgotten point is that the mutation rates practiced
in directed laboratory evolution are orders of magnitude
higher than those of natural organism (>1 mutation/gene/
generation, in contrast to 10 6 in most of the natural
organisms). The prevailing view is that proteins tolerate
most mutations with no loss of function. But in fact,
although proteins generally withstand the first one or
few mutations, with no apparent loss of activity, their
stability is severely compromised, and so is their ability to
stomach more mutations [68]. Most proteins used as
starting points for laboratory evolution never evolved to
withstand high mutational loads, but because thermodynamic stability correlates with mutational tolerance, the
application of thermodynamically stable proteins, either
natural thermophylic proteins (for example see reference
[74]), or laboratory evolved stabilized proteins, as starting
points for library making is one way of solving this
problem [75,79].
Neutral drifts provide another way of tackling the problem of sensitivity to mutations. As previously predicted
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Advances in laboratory evolution of enzymes Bershtein and Tawfik 155
Figure 2
[72,73], neutral drifts can prompt the emergence of mutational robustness. The neutral drift performed with a
P450 demonstrated that increased stability and mutational tolerance underlies large polymorphic populations,
and not in a drift taken through a bottleneck, that is, when
a single variant was passed from one generation to another
[70]. A drift we performed with TEM-1 b-lactamase by
selecting for its native penicillinase activity showed that
several mutations that act as ‘global suppressors’ were
enriched that increase TEM-1’s stability and suppress the
effect of a broad range of deleterious destabilizing
mutations. These global suppressors enabled a higher
number of mutations to accumulate without deleterious
consequences and increased the frequency of positive
variants observed in a selection for a new function (degradation of a synthetic, ‘third-generation antibiotic) (Bershtein S, et al., unpublished results).
Interestingly, all the global suppressors we identified
emerged in positions where the sequence of TEM-1
deviates from its family consensus, and thereby bring
TEM-1 closer to its consensus. Such changes, which can
be readily predicted from sequence alignments, have
been shown to stabilize numerous proteins [76–78].
Neutral drifts and the evolution of new enzyme variants. (a) The neutral
drift of serum paraoxonase (PON1) introduced a wide range of
apparently neutral mutations in the periphery and within PON1’s active
site [45]. The active site pocket of PON1 comprises 32 residues and is
presented as a blue mesh. The highly conserved calcium ion (magenta
sphere), and histidine dyad H115 and H134 (green spheres) that mediate
PON1’s native activity as a lactonase are shown. Active site residues
found to be mutated in variants isolated during three rounds of neutral
drift are colored in either red, or yellow. The red colored residues were
identified in variants with native lactonase activity and expression levels
that are essentially identical to wild-type PON1, whereas yellow residues
refer to variants in which a significant lactonase activity is maintained
although not at wild-type levels [45]. Note that the active site wall in the
vicinity of the 115–134 dyad remained intact in contrast to extensive
changes seen in other parts. (b) A schematic representation of PON1’s
putative neutral network based on the experimentally characterized
neutral variants and variants identified in previous directed evolution
experiments PON1 (adopted from reference [45]). The neutral variants
comprise a ‘cloud’ of sequences scattered around the wild-type
sequence that is primarily a lipo-lactonase. The expansion of PON1’s
neutral network, through the mutations highlighted in panel a enables
gradual (by single mutation steps) and smooth (without loss of protein
stability or function) transitions to a whole range of new enzyme variants,
each possessing a new and different substrate specificity.
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These experiments point out an alternative approach that
can yield interesting and useful results despite the use of
relatively small libraries. The latter is particularly important in a wide range of emerging applications for which
screening can only be performed with limited throughput,
for example, under industrially relevant conditions, or for
substrate/reaction that is not amenable to high throughput screens. The neutral drift (i.e. repetitive rounds of
mutation and selection for the original function of the
starting gene) requires relatively small libraries because,
unlike a selection for a new function where active variants
are rare, most library-mutated variants are active. The
resulting library contains large, yet non-deleterious, variation. As such, neutral drift libraries – although relatively
small in size – can possess a vastly increased potential to
evolve new functions. Another useful approach that
emerges concerns libraries enriched with consensus
mutations. These mutations could be spiked into libraries
using synthetic oligonucleotides and DNA shuffling
[25,34]). The presence of these mutations can boost
the protein’s tolerance to mutations, and compensate for
the deleterious effects of function-altering mutation,
thereby increasing the frequency of variants conferring
new functions (Bershtein S, et al., unpublished results).
Note added in proof
Varadarajan et al. report the application of enzyme display
and FACS (see Ref. [42]; Figure 1b) and the engineering
of a remarkable series of site-specific endopeptidases
capable of cleaving a wide range of peptide sequences
with high selectivity and catalytic efficiency (Varadarajan
N, Rodriguez S, Hwang B-Y, Georgiou G, Iverson BL: A
Current Opinion in Chemical Biology 2008, 12:151–158
156 Biocatalysis and Biotransformation
family of engineered endopeptidases. Nature Chem Biol;
in press). These enzyme variants were obtained by iterative rounds using different diversification techniques, and
by applying both positive selection for variants cleaving
the desired peptide substrate, and negative selection to
purge variants cleaving other peptides.
Acknowledgements
Research grants from the Minerva Foundation, the EU MiFEM consortium,
and the Estate of Fannie Sherr are gratefully acknowledged.
References and recommended reading
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developed for enzyme optimization. The method is based on the statistical identification of beneficial mutations in early rounds variants, including variants with reduced activity, and combining these mutations by the
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