University of Groningen
Evolution of enantioselectivity
Boersma, Ykelien
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Boersma, Y. L. (2007). Evolution of enantioselectivity: selection of improved hydrolase variants s.n.
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2
SELECTION STRATEGIES FOR
IMPROVED BIOCATALYSTS
YKELIEN L. BOERSMA, MELLONEY J. DRÖGE & WIM J. QUAX
FEBS J 2007; 274: 2181-2191
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
Selection Strategies for Improved Biocatalysts
Enzymes have become an attractive alternative to conventional catalysts in numerous
industrial processes. However, their properties do not always meet the criteria of the
application of interest. Directed evolution is a powerful tool to adapt the enzyme’s
characteristics; nevertheless, to select evolved variants remains a critical step. As a
consequence, new selection strategies have been developed during the past decades
enabling selection for the desired enzymatic activity. This review focuses on these
novel strategies for the selection of enzymes from large libraries, in particular of those
enzymes that are employed in the synthesis of pharmaceutical intermediates and
pharmaceuticals.
Introduction
Directed evolution
Over the past decades, it has become clear that enzymes hold a great potential for industry.
Enzymes are among the most remarkable biomolecules known because of their
extraordinary specificity and catalytic power 58. The specificity and (enantio- and regio-)
selectivity of certain enzymatic transformations makes them appealing for the production of
fine chemicals and pharmaceutical intermediates. To date, more than 500 products over a
wide spectrum of applications are manufactured by enzymes. Well-known examples are
ephedrine, aspartame and amoxicillin 14,64. However, in many cases naturally occurring
enzymes lack features necessary for the application of interest, since after all they have
evolved in nature to serve a different purpose than the acceleration of industrial processes.
To overcome these limitations, directed evolution has emerged as a powerful and versatile
means to tailor enzymes in order to adapt their properties to process requirements. It
mimics the process of Darwinian evolution in the test tube, combining mutagenesis and
recombination with selection or screening for improved variants with the desired
characteristics 46,47. The main advantage is that the enzyme’s properties and functions can
easily be engineered even without any required knowledge of the structure.
Figure 1: Schematic overview of a directed evolution experiment.
The technique is essentially comprised of two steps: first, mutagenesis of the gene(s)
encoding the enzyme(s) 46. Enough diversity should be created in the starting gene, such
that an improvement in the desired characteristic of the protein will be found in a library of
variants. Second, the variants are analysed on the basis of the properties of interest by either
23
CHAPTER 2
screening or selection. The gene(s) encoding the improved variants are identified and then
used to parent the next round of directed evolution 65. Thus, the ultimate goal of directed
evolution is to accumulate improvements through repetitive rounds of mutagenesis and
identification (figure 1). Directed evolution has been successfully applied to several distinct
enzymes to alter their characteristics 55,56,58,61.
To date, a broad range of strategies to introduce mutations into the starting gene(s) are
available. Moreover, methods for constructing diverse molecular libraries continue to
accumulate. These can be roughly divided into two categories: those based on point
mutations and those making use of recombination. Belonging to the first category are
methods such as oligonucleotide-directed randomisation and error-prone polymerase chain
reaction (epPCR). In principle, these methods lead to a mutant library possibly containing
any of the 20 different amino acids at a defined position (for a review, see 56,66-68). The
second method, based on recombination, is based on the breaking and rejoining of DNA in
new combinations. Many different versions of recombination exist, DNA shuffling being
the most prominent one 60. In general, DNA shuffling allows beneficial mutations from
multiple genes to be recombined. A number of methods, based on this principle, have been
developed 61,67.
The second step in directed evolution, identification of the improved variants by either
screening or selection, remains the most critical one. This review will focus on some of the
strategies to be applied in this step. The choice to apply either screening or selection is
discussed in the next paragraph.
The second step in directed evolution
The advantage of selection over screening
Assays capable of rapidly isolating rare valuable variants from a large mutant library are of
key importance to the task of evolving proteins in the laboratory, and need to be tailored for
every enzyme and reaction. Naturally, when more variants are analysed, the odds of finding
a valuable variant within the library are increased. However, the typical library size is still
many orders of magnitude larger than the number of protein variants that can be screened.
Directed evolution experiments are therefore often limited by the availability of a suitable
high-throughput screening or selection system 69,70.
At the basis of all screening and selection strategies is a linkage between the gene, the
enzyme it encodes and the product of the activity of the enzyme. The main difference
between screening and selection is that screening is performed on individual genes or
clones, whereas selections are performed simultaneously on the entire pool of variants 69,71.
Screening and selection methods should meet certain criteria. First, if possible they should
be directly associated with the property of interest. After all, you get what you select for is
the first rule of directed evolution. The substrate should be identical or at least as close as
possible to the target substrate, and product detection should be under multiple turnover
conditions to ensure selection of effective catalysts. Second, the assay should be sensitive
over the desired dynamic range. In the first rounds of directed evolution experiments, all
improved mutants should be recovered, while in more advanced rounds more stringent
24
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
conditions need to be applied to isolate only the best variants. Finally, the screening or
selection procedure should be applicable in a high-throughput format 69.
The advantage of screening is that the difference between substrate and product of an
enzymatic reaction can be determined directly or indirectly in almost every case. Most
screening methods rely on the use of fluorogenic or chromogenic substrates, which are
converted in spectroscopically different products 12,69,72. Recently, FACS analysis has
emerged as an important high-throughput means for detection 73. However, the
disadvantage of screening can be put down to the fact that every single mutant must be
tested for the desired enzymatic reaction, even variants that might not be active or are
incorrectly folded. In a directed evolution experiment using recombination, these nonactive enzymes are typically 50-80% of the total library. Therefore, selection is preferred
over screening 12.
In general, selection techniques are less labour-intensive and more efficient than screening
techniques as they allow for the analysis of more mutants simultaneously. Screening limits
the number of individual library members to be analysed to roughly 104 variants, whereas
in selection strategies the library size is extended to 1010 up to 1013 variants 11,74. Selection
strategies exploit conditions favouring the exclusive survival of desired variants, therefore
uninteresting variants are never seen. Thus, the evolutionary character of the overall
process is stimulated, which makes the directed evolution approach rational in a different
sense. However, with every selection system the possibility always exists that viable but
unanticipated variants will surface. If these false positives become too abundant, an
efficient screening step or a redesign of the selection procedure may be necessary 62. One
drawback of selections for enzymatic activity though is the required substantial up-front
customisation or the availability of relevant auxotrophs in genetic complementation 62,70,75.
This review discusses some of the genotype-phenotype linkages possible for the selection
of interesting enzyme variants. The focus will be on selection for pharmaceutically relevant
substrates.
In vivo and in vitro selection systems
The genotype-phenotype linkage can be acquired in different ways. A cell-type linkage can
be created by compartmentalising the protein and the gene together in cells. All in vivo
selection systems have in common that the genetic library obtained in the first step of
directed evolution must be transformed into cells, either bacteria or yeast. The selection is
based on the presence of a catalytic activity which provides a growth advantage to
microorganisms possessing that specific activity. Thus, cell-type linkage methods have
been used mainly for the selection of catalytic enzymes. However, low transformation
efficiency or cells circumventing selection pressure or lack of transport of the substrate are
some of the limitations of in vivo selection 11,69,71.
These limitations can be overcome by in vitro selection techniques. With these systems, no
transformation step is necessary. Transformation efficiency is therefore no longer a limiting
factor. Thus, library sizes can be much larger, up to 1014 members, enabling the exploration
of a larger fraction of sequence space. Furthermore, these selections can be performed
under more stringent conditions, as selection is not dependent on viable cells. Thus,
nonphysiological conditions such as elevated temperatures, extreme pH or even organic
25
CHAPTER 2
media can be applied. As in vitro selection does not require living cells membrane barriers
between the enzyme and the substrate are non-existing. Examples of (partially) in vitro
selection techniques are phage display, ribosome display, cell surface display and in vitro
compartmentalisation (IVC) 76-78.
In this review, both in vivo and in vitro techniques for the selection of enzymatic activity
and some examples of their applications will be discussed.
In vivo selection for enzymatic activity
In vivo selection links cell survival to enzyme activity. This strategy is termed genetic or
growth selection. Historically, genetic selection has been widely used to identify
biosynthetic genes and pathways. The general strategy for genetic selection involves the
introduction of a metabolic requirement for the desired activity into the host cells (figure
2A). Plasmids, encoding for a mutant library of the protein of interest, are introduced into a
suitable host for selection, preferably a mutant strain of a well-characterised bacterium,
such as Escherichia coli. Selective conditions for the target function of the protein encoded
by the plasmid are imposed in such a way that only those cells expressing variants with the
desired phenotype are viable. By means of the enzymatic activity that is used for selection,
usually an essential nutrient is provided to compensate for a deficiency in the strain used,
but the product can also enable cell survival from increasing concentrations of toxic
compounds by neutralising them. These selected variants can be further characterised after
selection 79.
Figure 2: A) General strategy for genetic selection. In the bacterium, the plasmid DNA is transcribed
and translated to the enzyme, which can in its turn convert the substrate into the nutrient. B) Strategy
for chemical complementation, as performed by van Sint Fiet et al 79. After enzymatic conversion, the
product of the transcriptional activator NahR, which activates TetA, enables cell growth on selective
minimal medium.
26
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
Prokaryotic selection systems offer a number of advantages over selection in eukaryotes.
Transformation efficiencies are much higher in prokaryotes and cell division times are
much shorter than for eukaryotes; besides that, prokaryotic genomes are less complex 80.
Genetic complementation
Genetic selections have been rated extremely valuable for evolving enzymes with improved
catalytic activity, improved stability, and altered specificity. In literature, many examples of
successful genetic selections have been described 62,74,81-88. In this review, we will mainly
focus on the experiments that have been developed for the selection of enzymes by growth.
In particular, enzymes that can be used for the synthesis of pharmaceuticals and
pharmaceutical intermediates will be discussed.
DeSantis et al broadened the substrate specificity of E. coli 2-deoxyribose-5-phosphate
aldolase (DERA), which is a unique enzyme amongst the aldolase family as it catalyses the
reversible condensation of two aldehydes. DERA is an attractive biocatalyst as it accepts a
broad range of substrates, with a strong preference for phosphorylated substrates. An
important application of this enzyme is found in the synthesis of the antitumour agent
Epothilone A and the enzyme might also be used in the synthesis of precursors of statins
89,90
. To expand and improve DERA’s activity towards nonphosphorylated substrates, the
so-called E. coli SELECT was engineered auxotrophic for acetaldehyde. Afterwards, this E.
coli strain was transformed with a DERA mutant library and grown on minimal medium
supplemented with the non-phosphorylated unnatural substrate D-2-deoxyribose. This
substrate can only be converted to acetaldehyde by DERA variants. The selection and
identification of novel DERA variants using this system is currently in progress 90,91.
Besides selection for altered substrate specificity, genetic selection can also be applied for
the selection of enzymes with improved catalytic activity. The ultimate goal of Otten et al
was to convert the glutaryl acylase from Pseudomonas SY-77 into an adipyl acylase by
directed evolution. This enzyme could be applied in the one-step bioconversion of adipyl-7aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, which is of key
importance for the synthesis of semi-synthetic cephalosporins. After constructing an epPCR
mutant library, genetic selection was applied by using adipyl-leucine as a mimic substrate
and a leucine auxotrophic E. coli strain as selection host. Consequently, only enzymatic
hydrolysis of adipyl-leucine would allow for growth. Variants with an improved growth
capability on the mimic substrate also showed an improved activity towards the real βlactam substrate. Selected mutants demonstrated a nearly 10-fold improved ratio of adipyl7-ADCA over glutaryl-7-ACA hydrolysis. Expansion of the acyl binding pocket of the
enzymes formed the explanation for this improvement 92.
Improvement of enantioselectivity is difficult to select for. Hwang et al proposed a
selection system for enantioselectivity based on differential cell growth. The principle is
based on toxic product formation using chiral antibiotic esters. Upon enantioselective
hydrolysis by commercially available esterases and lipases, the antibiotic was released and
cell growth was inhibited. Thus, the difference in cell density is directly correlated with the
enantioselectivity 93.
Recently, we have developed a genetic selection system using an aspartate auxotroph E.
coli strain to select for both enantioselectivity and enzymatic activity. The system was
applied to the selection of hydrolase variants such as Bacillus subtilis lipase A. Using this
27
CHAPTER 2
system, we were able to select mutants with inverted and improved enantioselectivities
towards the chiral synthon 1,2-O-isopropylidene-sn-glycerol (IPG), an important precursor
in the synthesis of β-adrenergic receptor antagonists (unpublished data).
Chemical complementation
Genetic complementation, as described in the previous section, has proven to be a powerful
approach to enzyme evolution. However, these complementations are often limited by the
(natural) reactions that have to be used for selection. Therefore, a chemical
complementation strategy was developed, in which complementation is solely dependent on
the product that is formed and not on the reaction pathway itself 94. In this approach,
enzymatic activity is linked to the transcription of an essential gene using a yeast threehybrid system. The enzyme substrate acts as an inducer for dimerisation to reconstitute a
DNA-binding and transcriptional activation domain of an artificial transcription factor.
Thus, enzymatic activity can result in the activation or repression of expression of either an
essential or a toxic reporter gene. The system enables selection for biocatalytically active
cells from a background of inactive cells. Baker et al explored the cephalosporin hydrolysis
by a β-lactam hydrolase from Enterobacter cloacae P99 as a model reaction. They linked
the enzymatic activity of this enzyme to transcription of a lacZ reporter gene in vivo 94,95.
The utility of this system has also been demonstrated for glycosynthase from Humicola
insolens, yielding a variant with a five-fold increase in glycosynthase activity 96.
Witholt et al developed a selection system using an engineered E. coli strain which detects
the production of benzoate and 2-hydroxybenzoate from their corresponding aldehydes
catalysed by benzaldehyde dehydrogenase (XylC) of Pseudomonas putida. Detection was
carried out by a mutant of the transcriptional activator protein NahR from P. putida. This
mutant specifically recognises the products and not the aldehyde substrates. Furthermore, it
activates transcription from its cognate salicylate promoter. Genes encoding either a
fragment of β-galactosidase or the tetracycline antiporter TetA were cloned behind this
promoter to enable the selection of biocatalytically active cells producing XylC. On
selective minimal medium plates containing tetracycline E. coli could only grow after
addition of the aldehyde substrates (figure 2B). As this system depends merely on the
synthesis of the product and not on its reaction pathway, several different enzymatic
reactions can be monitored. This system was successfully used to detect nitrilase, amidase,
aldehyde oxidase, and aldehyde dehydrogenase activities yielding differently substituted
benzoates 79,97.
In vitro selection for enzymatic activity
Similar to in vivo selection systems, in vitro selection systems also link genotype and
phenotype. Examples of these selection strategies are systems which display variants on the
surface, such as phage display, cell surface display and ribosome display. Surface display
allows unhindered accessibility of the substrate as well as reaction conditions of choice.
These systems will be discussed in the next sections, together with their applications.
28
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
Phage display
Phage display technology provides a versatile tool for exploring interactions between
proteins, peptides, and small molecule ligands. Derivatives of M13 filamentous phages are
most commonly used for display of proteins on the surface of E. coli. This viral particle
replicates and assembles without killing the host cell. The proteins and peptides to be
displayed can be expressed on the surface of a phage by inserting the gene of interest into
the gene of one of the phage coat proteins, such as g3p and g8p. During phage assembly in
the periplasmic space, the fusion proteins are incorporated into the nascent phage particle 98100
.
The successful display of many enzymes, such as amylases 101, ß-lactamases 102, lipases103,
subtiligases 104, endoxylanases 105, and transferases 106 has been reported previously. As
phage particles are assembled in the cell envelope of E. coli, translocation of the fusion
protein across the inner membrane of E. coli to the periplasm of the cell is a prerequisite for
proper phage display. Thus, mostly periplasmic instead of intracellular proteins are
employed in phage display. However, even periplasmic proteins that fold well in the
periplasmic space frequently show a poor display on filamentous phages. Recently,
presentation of poorly displayed enzymes was improved by exchanging the Sec signal
peptide for translocation for SRP-dependent signal sequences. resulting in a >1,000-fold
enrichment per selection round 107. The display of enzymes that fold in the cytoplasm was
recently demonstrated by exchanging the signal peptide, either by using a Sec-dependent
signal peptide 108, or by making use of the Tat translocation pathway followed by
association with the g3p coat protein in the periplasm 109. These developments open doors
to new possibilities for the selection of enzymes.
Indirect selection based upon affinity
One of the first selection strategies for phage-displayed enzymes was based on binding to
substrates, products or transition state analogues (figure 3). Phage-enzymes are added to an
immobilised target: only those that do bind are isolated. These phages are amplified and
undergo subsequent rounds of selection. In this way, enzymes have been selected on the
basis of their stability since only stably folded phage-enzymes will have bound to the target
110
. As the catalytic power of enzymes stems from their higher affinity for transition state
analogues than for substrates, selection by transition state analogues should be more
efficient. This has been shown for several catalytic antibodies 111,112.
Selection of enzymes using suicide inhibitors is based on this same affinity principle.
Suicide substrates irreversibly bind and thus inhibit the selected enzyme 113,114. This
principle has been demonstrated by Beliën et al using endoxylanases. Endoxylanases are
important enzymes for industrial processes such as bread making and beer production.
Endoxylanase inhibitors considerably affect the functionality of endoxylanases in
biotechnological processes. Endoxylanase I from Aspergillus niger and endoxylanase A
from B. subtilis were functionally displayed on the coat of M13 filamentous phages and
incubated with immobilised endoxylanase inhibitors. The phage-enzymes bound with high
specificity to the immobilised inhibitors. However, for industrial processes, low specificity
binding of endoxylanases to the inhibitor is required. Thus, these results will find their
application in the selection of inhibitor-insensitive endoxylanase variants 105.
29
CHAPTER 2
Figure 3: Affinity-capture of phage-displayed enzyme variants on an immobilised substrate. The
enzyme is fused to the g3p coat protein. Some enzymes will bind to the immobilised substrate (♦),
others will not (○).
The affinity selection strategy was also employed in the selection of B. subtilis 168 lipase A
variants with improved and inverted enantioselectivity towards the chiral substrate IPG.
Dröge et al used a phosphonate ester of enantiopure IPG, coupled to SIRAN beads to
facilitate recovery of selected variants. A library of variants was constructed on a region
near the active site, and displayed on filamentous phages. Their selection strategy was dual:
first, undesired mutants were removed by incubating phage-enzymes with the phosphonate
inhibitor coupled to the unwanted enantiomer of IPG. Second, phage-enzymes that did not
bind to the first inhibitor were incubated with a second inhibitor, coupled to the desired
enantiomer of IPG, thus selecting for variants with desired enantioselectivity. After four
rounds of selection, a variant with an inverted enantioselectivity towards the desired wanted
enantiomer of IPG was found and characterised. The increase in enantioselectivity
however, was modest, which can be explained by the fact that in the 3D model of the
structure both enantiomers fit equally well 103,115.
To select B. cereus metallo-β-lactamase variants with improved catalytic activity towards
benzylpenicillin, Ponsard et al used a slightly different affinity selection technique. An
epPCR library of metallo-β-lactamase was constructed and displayed on phages. The
phage-enzymes were inactivated by complexing the enzyme’s cofactor zinc(II) with EDTA.
Then inactivated variants with affinity for the substrate benzylpenicillin were absorbed onto
immobilised benzylpenicillin. Finally, the inactivated variants were catalytically eluted by
adding a zinc(II) salt, thereby reactivating the selected variants. After two rounds of
selection, the catalytic activity of the selected mutants was increased 60-fold. In general,
this method is applicable for all enzymes necessitating the presence of a cofactor.
Nevertheless, the apoenzyme still needs to possess affinity for the substrate 116.
However, affinity selections do have some limitations. The immobilisation of the target
(substrate) can have a great influence on the outcome of the selection process 99. Besides
that, a disadvantage of the use of suicide inhibitors is that the selection process is based on
binding and not on product release and catalytic turnover. Consequently, binding does not
necessarily correlate with the catalytic activity of the enzyme. Thus, affinity selection
30
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
should only be used if a change of substrate specificity is desired, not if an enhancement of
rate acceleration or turnover is required.
Direct selection for catalytic activity
Direct selection of enzymes on the basis of catalytic activity is more difficult, as reaction
products readily diffuse from the reaction site. In order to inhibit this diffusion, a physical
link between the phage-enzyme and the substrate should be established 111. Several methods
are available to achieve this link, a number of them will be discussed here.
One approach to procure the enzyme-substrate link is to couple the substrate to a reactive
thiol or amine of the phage coat using a maleimide-based linker (figure 4A). This strategy
was used in the directed evolution of Bordetella pertussis adenylate cyclase variants. The
wild type enzyme catalyses the conversion of ATP into cAMP. Active phage-cyclases were
able to convert a substrate analogue which was coupled to the phage into cAMP. The
phage-enzymes were recovered by incubation on beads derivatised with a single-chain
antibody fragment against cAMP. With these tools, they devised a selection scheme that
permits the selection of active phage-cyclases with an enrichment factor of approximately
70-fold for each round of selection 117. However, the disadvantage of this system is that
cross-reactivity is not excluded, as the phage-enzyme and the substrate are at some distance
from each other.
Figure 4: A) Direct selection of phage-displayed enzyme variants by linking the substrate to the
phage coat. B) Selection of polymerases; both an acidic peptide and the enzyme variants are fused to
g3p. By incorporating a biotinylated nucleotide, the phage enzyme is captured using streptavidin. C)
In vivo selection combined with in vitro display. The substrate, fused to g3p, is biotinylated in the
cytoplasm, and the whole complex is then displayed on a phage and affinity-captured with avidin.
31
CHAPTER 2
In an alternative approach, both the enzyme and the substrate can be specifically colocalised on the g3p coat protein, resulting in a display of both the enzyme and the substrate
(figure 4B). This would limit cross-reactivity, as the phage-enzyme and the substrate are in
each other’s vicinity. To expand the substrate specificity of a DNA polymerase towards the
synthesis of unnatural polymers from 2’-O-methyl ribonucleoside triphosphates, a
combined display was achieved by reengineering the helper phage genome: it now
contained a gIII gene fused to DNA encoding an ‘acidic’ peptide. This peptide is able to
form a leucine zipper and a disulfide bond with a ‘basic’ peptide. The latter is conjugated to
an oligonucleotide, the primer in the polymerisation reaction. In the selection, extension of
the primer that was still bound to the polymerase ultimately resulted in the incorporation of
a biotinylated nucleotide, which in turn could be used for affinity–capture of the complex
on streptavidin. The method led to one mutant with modified substrate specificity, though it
had the same fidelity with unnatural substrates as the wild type enzyme with natural
substrates 118,119. This principle was applied to the evolution of thermostable reverse
transcriptases as well 120, but an application apart from nucleic acid polymerases is unlikely.
Routenberg Love et al developed a substrate attachment-strategy for the phage display of
glycosyltransferase. They focused on E. coli glycosyltransferase MurG, an important
enzyme for antibiotic synthesis. Cultures containing a MurG encoding plasmid were
infected with selenocysteine (Sec)-expressing helper phages. The outcome was a phage
with both the enzyme and a Sec handle on the same end of the phage particle. The Sec
handle could be used for binding to biotin and subsequent capture on streptavidin. The
activity of the phage-enzyme was established by incubation with UDP-[14C]-GlcNAc and a
biotin-labelled lipid I analogue. Phage-enzymes were found to be active, thereby opening
doors for the phage-display evolution of related glycosyltransferases 121.
A relatively novel approach is the combination of in vitro selection with in vivo enzymatic
activity. Here, substrate- and enzyme-encoding DNA are both introduced in E. coli and
expressed in the cytoplasm (figure 4C). The substrate however, is expressed as a fusion
protein to one of the coat proteins of M13. After a catalytic reaction in the cytoplasm, the
product, likewise fused to the coat protein, is incorporated in the phage coat and displayed
on its surface. Upon affinity-capture of the product in vitro, the gene encoding for the
selective enzyme is automatically selected for as well. The power of this system was
demonstrated with E. coli biotin protein ligase (BPL) as a model enzyme. BPL catalyses the
highly specific formation of biotinyl-5’-adenylate from biotin and ADP, and transfers biotin
to a specific lysine residue on the biotin carboxyl carrier protein, a subunit of acetyl-CoA
carboxylase. The researchers used the biotin-tag-peptide (Btag) as the substrate to be fused
to g3p and subsequently to be displayed on the phage coat. Phages displaying the reaction
product could be captured with avidin coated beads. An advantage of this method is that the
selected enzymes are stable in vivo, since the enzymatic reaction takes place in the (natural)
cellular environment. This makes the system extremely suitable for the selection of
enzymes that catalyse modifications of peptides or proteins, such as protein ligases,
acetylases, kinases, phosphatases, ubiquitinases, and proteases. Furthermore, the enzyme
does not need to be secreted to the periplasmic space; consequently, size, nature and folding
problems do not propose a limiting factor to display 122. The researchers also demonstrated
the utility of the g8p coat protein for substrate fusion. Due to the higher copy number of
g8p, more product could be displayed. If the catalytic power of the enzyme of interest is
32
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
moderate or the affinity of the product of the enzymatic reaction for the capture molecule is
relatively low, this method could be very effective 123.
Bacterial cell surface display
Cell surface display is a technique to present peptides or proteins on the surface of Gramnegative or Gram-positive bacteria by fusing them to surface anchoring motifs or otherwise
named carrier proteins. This technique has had its impact on a wide range of
biotechnological and industrial applications. It has led to substantial progress in whole cell
biocatalysis, live-vaccine development, biosorbent and biosensor development, epitope
mapping, antigen delivery, inhibitor design and protein library screening. In contrast to
phage display, the size of the displayed protein is not a limiting factor in cell surface
display. An additional advantage of this system is that bacterial cells are used: they are selfreplicative and are sufficiently large to be examined by optical methods, including
fluorescence microscopy or FACS analysis. The latter allows for high-throughput screening
124-127
.
Selection of a host strain for surface display is an important consideration. A good host has
to meet certain criteria, such as compatibility with the protein to be displayed and easy
cultivation without cell lysis. Furthermore, the host cell should exhibit low activities of cell
wall- and extracellular proteases. For Gram-negative bacteria, the fragility of the outer cell
membrane can be a problem; nevertheless, E. coli remains an attractive host for cell surface
display, because of its high transformation efficiency. Gram-positive bacteria seem more
suitable for surface display purposes, as they have only one cell membrane and their cell
walls are thicker and consequently more rigid. Bacillus and Staphylococcus strains are most
commonly used for this purpose 125-127.
Not only is the host strain of importance, the anchoring motif to which the protein of
interest is fused needs to meet some criteria as well. First, it should have an efficient signal
sequence to transport a premature fusion protein across the inner membrane. Naturally, the
characteristics of passenger proteins to be displayed on the surface have an influence on the
transport as well. Second, it should have a strong anchoring structure to insert into the outer
membrane and keep fusion proteins attached to the cellular surface. Again, this attachment
is influenced by the passenger protein. Third, the anchoring motif should be compatible
with the foreign sequences to be inserted or fused. Last, it should be resistant to any attack
of proteases in the periplasm or in the medium. Each type of anchoring motif has its own
characteristics and can therefore be useful for specific applications 126. A number of these
applications will be discussed on the basis of their anchoring motif in the following
paragraphs.
Outer membrane proteins as an anchoring motif
Outer membrane proteins (Omp) span the membrane several times, and are therefore
attractive as an anchoring motif for cell surface display. They are mainly built up of antiparallel β-strands, resulting in β-barrel structures. Sequences of the protein of interest are
inserted into a permissive site of the anchoring motif. Examples of studied membrane
proteins used as anchoring motifs are OmpA, OmpS, LamB, PhoE, and OmpC. The
Lpp’OmpA system allows for C-terminal fusions, thus being more suitable as a carrier of
larger inserts 125,128. Cell surface display on the basis of Omp has been applied previously
33
CHAPTER 2
for whole cell biocatalysis 129, in particular for the enzymatic resolution of chiral
compounds. Displayed enzymes act as whole cell biocatalysts. Lee et al applied the cell
surface display strategy in the enantioselective resolution of racemic compounds by
Pseudomonas fluorescens lipase while anchoring the gene of interest to Salmonella
typhimurium OmpC. 130. A second lipase, the thermostable Bacillus sp. strain TG43 lipase,
was also applied in whole cell biocatalysis while displayed on the cellular surface. The
lipase was fused to the ninth loop of FadL by C-terminal deletion-fusion. FadL, an Omp
involved in long-chain fatty acid transport in E. coli, was shown to be useful as an
anchoring motif for the display of lipases on the surface of E. coli 131.
Autotransporter proteins as anchoring motif
The so-called autodisplay system has been developed on the basis of the secretion
mechanism of the autotransporter family of proteins. Autotransporters are synthesised as
precursor proteins and the passenger is an integral part of the protein. Beside the passenger,
the precursor protein contains a signal peptide necessary for transport across the inner
membrane, a C-terminal β-barrel for transport across the outer membrane and a connecting
peptide. The latter, a so-called linker, will ensure full surface access. Hydrolases, foldases,
β-lactamases and oxidoreductases have been autodisplayed on the surface of E. coli using
autodisplay 124,132. Some examples and their applications will be discussed below.
For the molecular evolution of Burkholderia gladioli esterase A, a system autodisplaying
the enzyme was developed. The esterase was genetically fused to the autotransporter
domains of the adhesin (AIDA-I) involved in diffuse adherence to HeLa cells. After
transformation of E. coli with the plasmid, the fusion protein was transported to and
expressed on the cell surface. The enzyme was displayed in its active form, as was
demonstrated by the conversion of p-nitrophenyl acetate to p-nitrophenol. These results
provide a basic selection strategy for the evolution of a biocatalyst such as esterase A 133.
An antibody-independent detection method was developed for future selections.
Apart from the autotransporter AIDA-I, a membrane-anchored esterase (EstA) from
Pseudomonas aeruginosa was also used in autodisplay. EstA is enzymatically active when
anchored to the cell surface of P. aeruginosa, but also on the surface of the heterologous
host E. coli. As a consequence, EstA can only be used as an autotransporter for hydrolytic
enzymes when in an inactive form. Three hydrolytic enzymes, B. subtilis lipase A,
Fusarium solani cutinase, and the large Serratia marcescens lipase, were fused to an
inactive variant of EstA. All three enzymes were displayed as fusion proteins on the surface
of E. coli, as was revealed by FACS analysis and more importantly, the three lipases
retained enzymatic activity 134. EstA was also employed in the autotransportation of a lipase
specific foldase (Lif), the protein LipH from P. aeruginosa, required in the folding of
extracellular lipases from Pseudomonads and related strains to convert lipases in their
active form. Surface displayed LipH was analysed by FACS, and proved to be functional
by efficiently refolding chemically denatured lipase. This system can therefore be used in
the selection of large libraries of foldase variants 135.
Although EstA itself is an excellent anchoring motif for autodisplay, the protein itself can
also be used for surface display using its hydrolytic characteristics. To select for this
property, the product should not diffuse from the reaction environment. Thus, Becker et al
developed a cell surface display system termed ESCAPED (Enzyme Screening by Covalent
34
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
Attachment of Products via Enzyme Display) in which the product is linked to the outer
membrane of E. coli by tyramide conjugation. In a model experiment, the researchers used
an octanoic ester of biotin-tyramide to be hydrolysed by EstA. The resulting tyramideconjugate was used as a substrate for horse radish peroxidase (HRP), the latter being
surface-displayed as well. HRP reacts with hydrogen peroxide and biotin-tyramide to
produce a quinone structure bearing a radical. This radical interacted with tyrosine residues
in close vicinity to HRP on the cellular surface, resulting in biotin deposition and
subsequent affinity-capture using streptavidin-coated magnetic beads. Thus, only cells
displaying an active hydrolase were selected. After only two rounds of cell sorting, the
enriched population of bacterial cells showed an increased esterase activity. This method
can be applied in the selection of proteases and phosphatases (figure 5) 73,136.
Figure 5: The ESCAPED system. The substrate, a biotin-tyramide ester, is converted by the cell
surface-displayed enzyme EstA (◊); then released tyramide (▲) reacts with cell surface-displayed
HRP (○) to form radicals that will bind to the cell surface of E. coli. Detection is achieved with
tagged streptavidin.
Cell surface display of Gram positive bacteria
In some applications, the use of genetically modified bacteria is less desirable, e.g. in food
processing and vaccine development. Recently, a novel surface display system using nongenetically modified Gram-positive bacteria was developed. The system is based on the
peptidoglycan-binding domain of the major autolysin AcmA of Lactococcus lactis and
enables functional display of heterologous proteins on the surface of genetically unmodified
Gram-positive bacteria. The cell wall-binding domain is designated the protein anchor
(PA), which directs the protein to the cell membrane. Hybrid PA fusion proteins exhibit the
same properties. To prove the principle of the display system, two biocatalysts, B.
licheniformis α-amylase and E. coli β-lactamase, were functionally displayed on the
surface of L. lactis by coupling them to PA. Activity assays showed that the enzymes were
still active. This system can be used for the immobilisation of enzymes to be used in
industrial processes. In conclusion, this surface display system provides a cheap, flexible
and easy-to-handle alternative to display proteins on non-genetically modified bacteria 137.
This may prove to be an advantage in developing vaccines.
35
CHAPTER 2
Ribosome display
The previously discussed selection strategies, phage- and cell surface display, only work
‘partially’ in vitro. In the last years, cell-free selection strategies such as ribosome display
and in vitro compartmentalisation have gained momentum, as a transformation step is
circumvented. In ribosome display, the physical link between genotype and phenotype is
achieved by mRNA-ribosome-protein complexes formed in the translation step which can
be directly used for selection. In vitro translation occurs in a cell-free system, such as E.
coli S30 or rabbit reticulocytes (figure 6). The system can either be coupled or uncoupled.
Coupled systems make use of DNA; they are simpler, more efficient and avoid problems of
mRNA degradation. In contrast, uncoupled systems require mRNA 138.
Figure 6: Schematic overview of selection by ribosomal display. In a cell-free system, dsDNA is
transcribed to mRNA, which forms a complex with the riboxome. mRNA lacks a stop codon, thereby
stalling translation. Selection is based on affinity-capture of the complex. After disassembly of the
complex, mRNA is amplified by RT-PCR.
During translation, ribosomal complexes are formed containing a functionally folded
protein from the ribosomal tunnel. For the protein to fold, the fusion protein is constructed
in such a way that the domain of interest is fused to a C-terminal spacer. This enables the
protein to fold while the spacer is still in the ribosomal tunnel. The fusion lacks a stop
codon at the mRNA level; as a consequence, the release of mRNA and the polypeptide is
stalled. The complex is further stabilised by high concentrations of magnesium(II) and low
temperatures. Following selection in an RNase-free environment, mRNA is released from
the ternary complex by removing magnesium(II) and cDNA is prepared by RT-PCR. This
step allows for additional mutations to be incorporated 76.
36
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
Ribosome display has been mostly applied to the selection of single-chain antibody
fragments and peptides from designed libraries. Some enzymatic selections have been
reported as well, which will be discussed in the next section.
The first selection of an enzyme based on catalytic activity was reported by Amstutz et al.
As a model system, the researchers displayed RTEM β-lactamase on the surface of the
ribosome in complex with its mRNA. The gene was fused to the C-terminus of E. coli
TolA, which served as a spacer. The enzyme was correctly folded and displayed in an
active form on the ribosome. Selection was based on affinity by making use of a
mechanism based suicide inhibitor, biotinylated ampicillin sulfone. After translation, the
complexes were incubated with the inhibitor and rescued with streptavidin-coated beads.
Per round of selection, active β-lactamase could be enriched over an inactive mutant >100fold 139. This same affinity principle was also employed in the selection of dehydrofolate
reductase (DHFR) variants. Mutants were selected using a substrate analogue, methotrexate
immobilised on agarose beads. Only a ribosome complex containing an active DHFR could
bind to the methotrexate beads. Four mutants showing the same activity as the wild type
enzyme were selected and further characterised 140.
To select for catalytic activity of ligases, T4 DNA ligase was used as a model enzyme.
mRNA encoding for both T4 DNA ligase and a spacer was hybridised with double stranded
DNA (dsDNA), a substrate of T4 DNA ligase. The resulting hybrid was translated in vitro,
up to the point where the ribosome reached the site of RNA-DNA hybridisation. The
enzyme was functionally displayed on the ribosome, which was shown by ligation of the
dsDNA in the complex with biotinylated dsDNA probe. Thus, the complex displaying
active T4 DNA ligase was labelled with biotin and selected by binding to streptavidincoated beads. A 40-fold enrichment over an inactive mutant could be achieved. Using this
method, it is possible to evolve new functions of T4 DNA ligase as well as of other ligases
141
. Ribosome display has not been widely used, however.
Ribozymes, enzymes made of RNA, have been shown to be capable of biocatalysis as well
(for a review, see 142). However, the focus is on their proteinaceous counterparts, thus
ribozymes are beyond the scope of this review.
In vitro compartmentalisation
In vitro compartmentalisation (IVC) simulates cellular compartments in which only the
reaction to be selected for is performed. The technique is based on water-in-oil emulsions,
where the water phase is dispersed in the oil phase with the aid of surfactants to form
microscopic aqueous compartments. Thus, artificial cells of approximately 5 fL are created.
These droplets contain on average a single gene; here, transcription, translation and
expression of the resulting proteins can all take place from in vitro active components. The
oil phase remains mostly inert and limits the diffusion of DNA and proteins between
compartments (figure 7) 71,143.
37
CHAPTER 2
Figure 7: General strategy for selection by IVC.
Active enzyme-encoding genes can be isolated on the basis of the presence of the product,
while inactive enzyme-encoding genes can be discarded on the basis of an unmodified
substrate. This strategy was employed in the selection of the methyltransferases HaeIII
(M.HaeIII) 144 and HhaI (M.HhaI). Selection was performed by extracting the genes from
the emulsion and subjecting them to digestion using a restriction enzyme that cleaves the
non-methylated DNA 145,146. By biotinylation, the active enzyme-encoding genes could be
affinity-captured and used in subsequent rounds of selection. This strategy resulted in
catalytically improved enzyme variants.
IVC was also used as the selection strategy of choice in the evolution of a bacterial
phosphotriesterase (PTE), which is applied in the degradation of pesticides. Here, a socalled microbead-display library technique was used. Streptavidin-coated microbeads
displaying a library of the gene and the enzyme PTE it encodes were compartmentalised in
a first emulsion. Then, after breaking the first emulsion, the microbeads were
recompartmentalised together with a soluble substrate coupled to caged-biotin. After the
enzymatic reaction, the biotinylated product was captured on the streptavidin microbeads,
allowing for selection with a fluorescent labelled anti-product antibody. The second
emulsion was broken and product-coated beads could then be enriched and analysed by
FACS. This method resulted in an enrichment of PTE activity, with one variant exhibiting a
63 times higher activity than the wild type enzyme 147.
As seen previously, the direct sorting by FACS may allow for versatile and powerful highthroughput systems. Using fluorogenic substrates, artificial cells compartmentalising genes
encoding active enzymes would become fluorescent, making them applicable for selection
by FACS. However, the continuous oil phase currently employed in IVC is not compatible
with FACS analysis. Thus, double emulsions (water-oil-water) were developed. As a proof
of principle, M.HaeIII encoding for DNA methyltransferase, and FolA encoding for E. coli
dehydrofolate reductase were compartmentalised, FolA together with FITC-BSA. Both
genes were tagged with biotin to allow affinity-capture on streptavidin after breakage of the
emulsions. The genes isolated from the droplets sorted by FACS were amplified by PCR
38
SELECTION STRATEGIES FOR IMPROVED BIOCATALYSTS
and appeared at a 1:3 FolA:M.HaeIII ratio, indicating a 30-fold enrichment. This result
demonstrated that there was no mixing of either DNA or FITC-BSA, thus the genotypephenotype link is conserved in this system 148. The applicability of this system was
demonstrated in the selection of novel β-galactosidase variants 149 and serum paraoxonase
(PON1) with thiolactonase activity 143. Mastrobattista et al evolved Ebg, a protein with at
present an unknown function, into an enzyme with considerable β-galactosidase activity,
while Aharoni et al achieved a 100-fold improvement in thiolactonase activity.
Concluding remarks
Directed evolution has become an important means to improve an enzyme or alter its
substrate specificity. To be able to analyse libraries of at least 1010 variants at a time,
several selection strategies have been set up, always linking phenotype to genotype.
Nevertheless, selection for catalysis remains a difficult task: enzymatic activity still needs
to be specifically tailored for each enzyme, reaction and substrate. Each system has its own
advantages and disadvantages. In vivo methods, though elegant, can be limited in their use
because of the narrow range of reactions that can be used in selection. Partial in vitro
techniques have been developed to overcome these limitations, although they still involve a
transformation step. Cell–free systems combined with FACS analysis hold enormous
potential, allowing a rapid analysis of enzyme variants. Thus, to our opinion these systems
will prove the most promising for the future of directed enzyme evolution.
39