Download High-throughput screens and selections of enzyme

High-throughput screens and selections of enzyme-encoding
genes
Amir Aharoni1, Andrew D Griffiths2 and Dan S Tawfik1
The availability of vast gene repertoires from both natural
sources (genomic and cDNA libraries) and artificial sources
(gene libraries) demands the development and application
of novel technologies that enable the screening or selection
of large libraries for a variety of enzymatic activities. We
describe recent developments in the selection of enzymecoding genes for directed evolution and functional
genomics. We focus on HTS approaches that enable selection
from large libraries (>106 gene variants) with relatively humble
means (i.e. non-robotic systems), and on in vitro
compartmentalization in particular.
Addresses
1
Department of Biological Chemistry, The Weizmann Institute of
Science, Rehovot 76100, Israel
2
The MRC Laboratory of Molecular Biology, MRC Centre, Hills Road,
Cambridge CB2 2QH, UK
Corresponding author: Tawfik, Dan S ([email protected])
Current Opinion in Chemical Biology 2005, 9:210–216
This review comes from a themed issue on
Biocatalysis and biotransformation
Edited by Dan E Robertson and Uwe T Bornscheuer
1367-5931/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2005.02.002
Introduction
Directed enzyme evolution has been used in the past two
decades as a powerful approach for generating enzymes
with desired properties. Enzyme variants have been
evolved for catalytic activity under extreme conditions
such as high temperatures, acidic and alkaline environments, and organic solvents [1,2] and with improved
catalytic activities and new substrate specificities [3,4].
Directed evolution experiments consist of two major
steps: first, the creation of genetic diversity in the target
gene in the form of gene-libraries; and second, an effective selection of the library for the desired catalytic
activity. A large variety of means for creation of genetic
diversity is currently available (for recent reviews see
[5,6]). However, the typical library size is still many orders
of magnitude larger than the number of protein variants
that can be screened. The same restriction applies to
cDNA and genomic libraries derived from natural sources
(e.g. environmental libraries), the diversity of which is
almost unlimited. Further, while methods for creating
Current Opinion in Chemical Biology 2005, 9:210–216
gene diversity are generic, the screens for activity need to
be tailored for each enzyme and reaction. The bottleneck
for most directed enzyme evolution endeavors is therefore the availability of a genuinely high-throughput (HT)
screen or selection for the target activity. This review
addresses recent enzyme screening and selection technologies with a particular focus on in vitro compartmentalization.
The basics of HT enzyme screens and
selections
The basis of all screening and selection methodologies is
a linkage between the gene, the enzyme it encodes and
the product of the activity of that enzyme (Figure 1). The
difference between screening and selection is that screening is performed on individual genes or clones and
requires some spatial organization of the screened variants on agar plates, microtiter plates, arrays, or chips,
whereas selections act simultaneously on the entire pool
of genes. Herein, we mostly use the term selection,
although formally speaking, several of the techniques
described are in fact screening methods.
Screening and selection methodologies should meet the
following demands. First, they should be, if possible,
directly for the property of interest — ‘you get what
you select for’ is the first rule of directed evolution [7].
Thus, the substrate should be identical, or as close as
possible to the target substrate, and product detection
should be under multiple turnover conditions to ensure
the selection of effective catalysts. Second, the assay
should be sensitive over the desired dynamic range.
The first rounds of any evolution experiments demand
isolation with high recovery — all improved variants,
including those that exhibit only several-fold improvement over the starting gene, should be recovered. The
more advanced rounds must be performed at higher
stringency to ensure the isolation of the best variants.
A limited dynamic range seems to be the drawback of
most selection approaches. Finally, the procedure should
be applicable in a HT format.
Numerous assays enable the detection of enzymatic
activities in agar colonies or crude cell lysates by the
production of a fluorophore or chromophore [8–10], as
well as resonance Raman scattering [11]. Assays on agarplated colonies typically enable the screening of >104
variants in a matter of days, but they are often limited in
sensitivity: soluble products diffuse away from the colony
and hence only very active variants are detected. Assays
based on insoluble products have higher sensitivity, but
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High-throughput screens and selections of enzyme-encoding genes Aharoni, Griffiths and Tawfik 211
Figure 1
(a)
Substrate
Gene
Enzyme
Product
(b)
Current Opinion in Chemical Biology
The basis of all selection and screens for enzyme-coding genes, be it in
a directed evolution or functional genomics context, is the linkage
between the gene, the enzyme it encodes, and the products of the
activity of that enzyme. The first link, between the enzyme and its
coding gene (a) can be achieved by a variety of ways, including the
cloning and expression of the library in living cells, phage-display,
ribosome and mRNA–peptide display, and by cell-free translation in
emulsion droplets (IVC). The second link, between the product of the
enzymatic activity and the linking gene (b), is usually harder to obtain.
A variety of methods that provide the above link in a genuinely HT
format are discussed in the text.
their scope is rather limited (for example, see [12]). The
range of assays that are applicable for crude cell lysates is
obviously much wider, but their throughput is rather
restricted: in the absence of sophisticated robotics, 103
variants are typically screened. These low-to-medium
throughput screens have certainly proved effective for
the isolation of enzyme variants with improved properties
as described in several recent reviews [4,13,14]. However,
a far more efficient sampling of sequence space is
required for the isolation of rare variants with dramatically
altered phenotypes.
While HT selections for binding activity have become
abundant [15–17], enzymatic selections remain a challenge. The main obstacle is that catalytic screens or
selections demand a linkage between the gene, the
encoded enzyme, and multiple product molecules
(Figure 1) [18,19]. This review focuses on methodologies
that enable the selection of large libraries, typically well
over 106 variants, for enzymatic activities. This throughput is still beyond the reach of HT technologies that are
based on 2D-arrays and robotics (micro-plates, chips, etc.)
(for an exception see [20]).
HTS of enzymes using cell- and phage-display
libraries
The display of the screened enzyme on the surface of
cells or phages has several advantages: first, the bacterium
or phage provide the link between the gene and the
protein it encodes (Figure 1a); second, although library
size is limited by transformation, 107 transformants can be
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obtained quite easily, and 1011–1012 are not beyond reach;
third, display on the surface allows unhindered accessibility of the substrate and reaction conditions of choice
(e.g. buffer, pH, metals). The main challenge with display
systems is maintaining the linkage between the enzyme
and the products of its activity (Figure 1b). Previously,
selections for enzymatic activities, of either catalytic
antibodies or enzymes displayed on phage, were performed indirectly, by binding to transition state analogues
or suicide inhibitors [21,22]. In recent years, direct selections for product formation under single [23,24] as well
multiple turnovers have been achieved. A notable example is the selection of a synthetic antibody library for
alkaline phosphatase activity [25]. Hydrolysis of a soluble substrate generated a product that acts as an electrophilic reagent and couples onto the phage particle that
displays the catalytically active antibody variant. These
phage particles were then captured by affinity chromatography to the product. Two rounds of selection from an
initial library of >109 antibody variants yielded an antibody with catalytic efficiency (kcat/KM) that is 1000-fold
higher than other alkaline phosphatase antibodies generated by immunization with a transition state analogue.
This high catalytic efficiency was mainly the result of an
increase in kcat value, demonstrating the importance of
selection for multiple turnovers. Another example regards
the selection of catalytic antibodies with peroxidase
activity. Selection was based on a biotin-linked tyramine
substrate being oxidized and coupled to the antibody’s
binding site. Phages displaying active antibody catalysts
were isolated by binding to streptavidin [26].
Another strategy for selection by phage display is based
on linking the substrate to the phage particle expressing
the target enzyme. Active enzyme variants transform the
phage-linked substrate to product, which remains
attached to the phage, and the phage can then be isolated
by affinity chromatography to the product [23,24,27,28].
The strategy was recently applied for a model selection of
phages displaying adenylate cyclase from a large excess of
phages that do not display the enzyme. The enzymatic
conversion of a chemically linked ATP into cAMP was
selected using immobilized anti-cAMP antibodies, and an
enrichment factor of about 70-fold was demonstrated
[29]. A selection for DNA polymerases was also
described [30,31]. In this case, the substrate (a DNA
primer) was also covalently attached via a flexible tether
to the phage coat protein. Active variants were selected by
virtue of elongation of the linked primer, first by incorporation of the target nucleotides, and finally by a biotinylated nucleotide that was used to capture the phage
onto avidin-coated beads. Using this strategy, a DNA
polymerase was evolved to efficiently incorporate rNTPs
and thereby function as an RNA polymerase [30]. The
same selection system was used to evolve DNA polymerases that incorporate 20 -O-methylribonucleosides
with high efficiency [31].
Current Opinion in Chemical Biology 2005, 9:210–216
212 Biocatalysis and biotransformation
Several groups applied a phage selection strategy termed
‘catalytic elution’ for enzymes for which catalytic activity
depends on cofactor binding [23,32]. Following protein
expression on the phage surface, the cofactor (e.g. a
metal) is removed and the catalytically inactive phages
are bound to an immobilized substrate. The cofactor is
then added, and phages displaying an active enzyme are
eluted by conversion of the substrate into product.
utility, for example in the identification of promiscuous
enzyme activities [39,40]. Furthermore, the scope of
genetic selections has been significantly widened by the
application of the three-hybrid system to link the catalytic
activity of an enzyme to the transcription of a reporter
gene in yeast cells. The utility of this approach has now
been demonstrated with several different enzymes
[2,3,41,42].
The application of cell-surface display for directed evolution has gained much momentum. In particular, screening
by FACS (fluorescence-activated cell sorter) has yielded
several highly potent binding proteins such as antibodies
[15,33]. However, sorting for enzymatic activity has thus
far been achieved only in a few particular cases where the
product of the enzymatic reaction could be captured
within bacterial cells [34] or on their surface [35,36].
The protease OmpA was displayed on the surface of
Escherichia 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 approach
allowed the screening of a library of 106 OmpA variants
and the subsequent isolation of a variant exhibiting a
60-fold improvement in catalytic activity [35]. A complementary approach was developed by Kolmar and
co-workers, whereby the products of a surface-displayed
enzyme (e.g. an esterase) are covalently linked to the
cell surface using the peroxidase-activated tyramide conjugation [18].
In vitro compartmentalization
Another potentially useful screening approach is based on
the immobilization of enzyme-displaying cells on beads
[37]. The cells are adsorbed to polyacrylamide beads preequilibrated with growth medium to form a bead population containing, on average, a single cell per bead. The
beads are immobilized on a solid glass support and cells
are allowed to grow and form microcolonies while utilizing the medium retained within the hydrogel matrix. The
microbead colonies are then equilibrated with a chromogenic or fluorogenic substrate, and the beads are screened
under the microscope. This technique was recently
demonstrated by enriching E. coli cells expressing
b-lactamase on their surface from a large excess of other
E. coli cells.
Genetic screens and selections
Genetic, or in vivo, selections comprised the traditional
tool of directed evolution at its early stages [38]. As the
scope of targets for evolution widened, the utility of this
approach became limited. After all, these selections are
usually based on the evolving activity complementing an
auxotroph strain in which an enzyme was knocked-out.
Thus, broadly speaking, the target for evolution (substrate, reaction, etc.) needs to parallel an already existing
enzyme. Nevertheless, genetic screens can be of much
Current Opinion in Chemical Biology 2005, 9:210–216
In vitro compartmentalization (IVC) is based on water-inoil emulsions, where the water phase is dispersed in the
oil phase to form microscopic aqueous compartments.
Each droplet contains, on average, a single gene, and
serves as an artificial cell in enabling transcription, translation and the activity of the resulting proteins, to take
place within the compartment. The oil phase remains
largely inert and restricts the diffusion of genes and
proteins between compartments. The droplet volume
(5 femtoliters) enables a single DNA molecule to be
transcribed and translated [43], as well as the detection of
single enzyme molecules [44]. The high capacity of the
system (>1010 in 1 ml of emulsion), the ease of preparing
emulsions, and their high stability over a broad range of
temperatures, render IVC an attractive system for
enzyme HTS.
IVC provides a facile means for co-compartmentalizing
genes and the proteins they encode, but the selection of
an enzymatic activity requires a link between the desired
reaction product and the gene (Figure 1). One possible
selection format is to have the substrate, and subsequently the product, of the desired enzymatic activity
physically linked to the gene. Enzyme-encoding genes
can then be isolated by virtue of their attachment to the
product while other genes, that encode an inactive
protein, carry the unmodified substrate. The simplest
applications of this strategy lie in the selection of
DNA-modifying enzymes where the gene and substrate
comprise the same molecule. Indeed, IVC was first
applied for the selection of DNA-methyltransferases
(MTases) [43]. Selection was performed by extracting
the genes from the emulsion and subjecting them to
digestion by a cognate restriction enzyme that cleaves
the non-methylated DNA [43,45,46]. Other applications
include the selection of restriction endonucleases [47]
and DNA polymerases [48,49]. The selection of DNA
polymerases was based on the fact that inactive variants
failed to amplify their own genes and therefore disappeared from the library pool. These modes of selection
are obviously restricted, not only in the scope of the
selected enzymatic activities, but also by the stochiometry — typically, one gene, and hence one substrate
molecule (or several substrate sites) is present per droplet,
together with 10–100 enzyme molecules. Despite these
restrictions, several new and interesting enzyme variants
have been evolved by IVC. A variant of HaeIII MTase
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High-throughput screens and selections of enzyme-encoding genes Aharoni, Griffiths and Tawfik 213
was evolved with up to 670-fold improvement in catalytic
efficiency for a non-palindromic target sequence (AGCC)
and ninefold improvement for the original recognition
site (GGCC) [46]. Active FokI restriction nuclease variants were also selected by IVC from a large library of
mutants with low residual activity [47]. Additionally,
DNA polymerase variants with increased thermal stability [48], and the ability to incorporate a diverse range of
bases including fluorescent dye-labeled nucleotides were
evolved [49].
The first application of IVC beyond DNA-modifying
enzymes was demonstrated by a selection of bacterial
phosphotriesterase variants [44]. The selection strategy
was based on two emulsification steps: in the first step,
microbeads, each displaying a single gene and multiple
copies of the encoded protein variant, were formed by
translating genes immobilized to microbeads in emulsion
droplets and capturing the resulting protein via an affinity
tag. The microbeads were isolated and re-emulsified in
the presence of a modified phosphotriester substrate. The
product and any unreacted substrate were subsequently
coupled to the beads. Product-coated beads, displaying
active enzymes and the genes that encode them, were
detected with fluorescently labeled anti-product antibodies and selected by FACS. Selection from a library of
>107 different variants led to the isolation of a variant
with a very high kcat value (>105 sec1). Microbead display libraries formed by IVC can also be selected for
binding activity [50] (for other binding selection by IVC
see [51,52]).
Some of the IVC selection modes take advantage of the
fact that this system is purely in vitro, and can allow
selection for substrates, products and reaction conditions
that are incompatible with in vivo systems. However, the
cell-free translation must be performed under defined
pH, buffer, ionic strength and metal ion composition. In
the selection for phosphotriesterase using IVC described
above [44], translation is completely separated from
catalytic selection by using two sequential emulsification
steps, allowing selection for catalysis under conditions
that are incompatible with translation. However, it is also
possible to use a single emulsification step, and to modify
the content of the droplets without breaking the emulsion
once translation is completed. There are currently several
ways of modulating the emulsion content without affecting its integrity. These include the delivery of hydrophobic substrates through the oil phase, reduction of the
droplet’s pH by delivery of acid, and photoactivation of a
substrate contained within the aqueous droplets [19].
More recently, a nano-droplet delivery system was developed that allows the transport of various solutes, including
metal ions, into the emulsion droplets. This transport
mechanism was applied for the selection of DNA-nuclease inhibitors. Inactive DNA-nucleases were co-compartmentalized with a gene-library, and once translation has
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been completed, the nuclease was activated by delivery
of nickel or cobalt ions. Genes encoding nuclease inhibitors survived the digestion and were subsequently
amplified and isolated. Selection was therefore performed
directly for inhibition, and not for binding of the nuclease
[53].
IVC in double emulsions
The need to link the product to the enzyme-coding gene
complicates and restricts the scope of selection especially
for non-DNA modifying enzymes. Recently, an alternative strategy has been developed based on compartmentalizing, and sorting, single genes, together with the
fluorescent product molecules generated by their
encoded enzymes. The technology makes use of double,
water-in-oil-in-water (w/o/w) emulsions that are amenable to sorting by FACS (Figure 2). It circumvents the
need to tailor the selection for each substrate and reaction,
and allows the use of a wide variety of existing fluorogenic
substrates. Recently it has been shown that the making
and sorting of w/o/w emulsion droplets does not disrupt
the content of the aqueous droplets of the primary w/o
emulsions. Further, sorting by FACS of w/o/w emulsion
droplets containing a fluorescent marker and parallel gene
enrichments have been demonstrated [54]. More
recently, w/o/w emulsions were applied for the directed
evolution of two different enzymatic systems: new variants of serum paraoxonase (PON1) with thiolactonase
activity (Aharoni et al., unpublished data), and new
enzyme variants with b-galactosidase activity (Mastrobattista et al., unpublished data) were selected from libraries
of >107 mutants. The b-galactosidase variants were translated in vitro as with previously described IVC selections
[19,43]. In the case of PON1, intact E. coli cells in which
the library variants were expressed, were emulsified and
FACS sorted, thus demonstrating the applicability of
double emulsions for single-cell phenotyping and directed enzyme evolution.
Concluding remarks
The past few years have seen significant progress in the
development and application of HT screens for enzymatic activities. Most significantly, a variety of methods
have become available for the direct selection of the
target enzymatic activity under multiple turnover conditions. The maturity of this area is also reflected in the shift
from model selections (i.e. demonstrating the isolation of
an existing enzyme-coding gene from a large excess of
irrelevant genes) to library selections, and the subsequent
isolation of a variety of new enzyme variants [6]. Cytoplasmic, periplasmic (and periplasmic-anchored [55])
expression, phage- and cell-display, cell-free translation
in emulsion droplets (IVC) and other in vitro display
technologies such as ribosome- or mRNA-peptide display
[16], were all shown to provide an effective means of
linking genes to their encoded proteins (Figure 1a). However, linking the gene to the products of the desired
Current Opinion in Chemical Biology 2005, 9:210–216
214 Biocatalysis and biotransformation
Figure 2
(b)
Oil
(a)
Enzyme
Gene library
RNA
Gene
Water-in-oil
emulsion
(c)
Hydrodynamically
focused stream
Sheath Fluid
Enzyme
Excitation laser(s)
Substrate
Product
(f)
Microdroplets
Fluorescence
(up to 16 colours)
Gene
Oil
Water
(d)
Breakoff point
+
+
-
+
Charged
deflection plates
Genes encoding
active enzymes
−
Waste
+
(e)
Current Opinion in Chemical Biology
Selections by flow sorting of double emulsion microdroplets using a fluorescence activated cell sorter (FACS). (a) A library of genes, each
encoding a different enzyme variant, is dispersed to form a water-in-oil (w/o) emulsion with typically one gene per aqueous microdroplet.
(b) The genes are transcribed and translated within their microdroplets using either in vitro (cell-free) transcription/translation, or by
compartmentalizing single cells (e.g. bacteria into which the gene library is cloned) in the microdroplets. (c) The w/o emulsion is converted into
a water-in-oil-in-water (w/o/w) emulsion and proteins with enzymatic activity convert the non-fluorescent substrate into a fluorescent product.
(d) Fluorescent microdroplets are separated from non-fluorescent microdroplets (or microdroplets containing differently coloured fluorochromes)
using FACS. (e) Genes from fluorescent microdroplets, which encode active enzymes, are recovered and amplified. (f) These genes can be
re-compartmentalised for further rounds of selection.
enzymatic activity (Figure 1b) still needs to be specifically tailored for each enzyme, reaction and substrate.
The use of fluorogenic substrates and sorting by FACS
appears to be the most promising avenue. Modern FACS
machines routinely analyze and sort >107 events per
hour. Fluorescence is a sensitive, versatile and general
signal that can detect a huge range of enzymatic reactions.
And, unlike selections performed in bulk, FACS allows
the stringency and recovery of the selection to be fineCurrent Opinion in Chemical Biology 2005, 9:210–216
tuned [18,36]. Thus, the combination of FACS, display
technologies and compartmentalization in double emulsions holds much potential in the areas of directed
enzyme evolution and functional genomics.
Acknowledgements
We acknowledge financial support to DST by the Israel Science
Foundation through its Bikura program, and to both ADG and DST by
the European Union through the ENDIRPRO training network. DST
is the incumbent of the Elaine Blond Career Development Chair.
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High-throughput screens and selections of enzyme-encoding genes Aharoni, Griffiths and Tawfik 215
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