Journal of Biotechnology 161 (2012) 92–103
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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Autodisplay of enzymes—Molecular basis and perspectives
Joachim Jose a,∗ , Ruth Maria Maas b , Mark George Teese a
a
b
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany
Autodisplay Biotech GmbH, Merowingerplatz 1a, D-40225 Düsseldorf, Germany
a r t i c l e
i n f o
Article history:
Received 8 October 2011
Received in revised form 14 February 2012
Accepted 4 April 2012
Available online 30 April 2012
Keywords:
Autodisplay
Biocatalysis
Synthesis
Enzymes
Whole cells
a b s t r a c t
To display an enzyme on the surface of a living cell is an important step forward towards a broader use of
biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It
is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the
enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells
can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has
some extra features that could overcome limitations in the industrial applications of enzymes. One major
advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell
surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay
enzymes with prosthetic groups can be displayed, expanding the application of surface display to the
industrial important P450 enzymes. Finally, up to 105 –106 enzyme molecules can be displayed on a
single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes
with particular attention to industrial needs and process development. Applications that will provide
sustainable solutions towards a bio-based industry are discussed.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Enzymes as biocatalysts show some outstanding advantages for
the synthesis of chemicals (Choi, 2009; Luetz et al., 2008), pharmaceutical and agrochemical intermediates (Fischer and Pietruszka,
2010; Tufvesson et al., 2011) as well as active pharmaceutical and
agrochemical compounds (Ran et al., 2009). Unlike conventional
organic chemistry, enzymes can be used under mild conditions concerning temperature, pressure and pH and usually they convert a
substrate with high regio- and enantioselectivity without protecting and de-protecting steps as necessary in conventional organic
chemistry. In many cases, the use of enzymes in chemical synthesis requires less substrate, less energy and reduces waste. Moreover
enzyme discovery and improvement could lead to completely new
processes, such as the use of cellulose for fuel production, the bioremediation of contaminated water and soil, or the production of
polymers from non–petroleum sources, which under current conditions are not feasible.
To date enzymes are already used in a distinct number of industrial processes (Busch et al., 2006). They are either applied as
preparations of purified proteins (Goldberg et al., 2007a), or as
microorganisms that produce the desired enzyme within the cell
∗ Corresponding author at: Institute for Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität, Münster, Hittorfstraße 58-62, D-48149
Münster, Germany. Tel.: +49 251 83 32210, fax: +49 251 83 32211.
E-mail address: [email protected] (J. Jose).
0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.04.001
(Goldberg et al., 2007b). Despite the advantages, there are some
drawbacks that prevent a broader application of enzymes. The
purification of enzymes is often a complex and costly production
process. In most cases purified enzymes cannot be used in repeated
reactions, they turn to waste after a single processing step. The
use of microorganisms as whole cell biocatalysts avoids the costs
associated with enzyme purification and ensures that the enzyme
is working in an optimal environment, where all co-factors and
regeneration networks are provided. Moreover, the enzyme as a
biocatalyst is largely protected from destabilizing and degrading
effects. However the intracellular location of the enzymes means
that this method will only be successful if both the substrate and
product can cross the membrane barrier. In addition, there is a
tremendous consortium of other enzymes present within the cell.
To obtain the product in a pure and unaltered form, whole cell biocatalysis is also limited to substrates and products that cannot be
converted by these native enzymes.
For many applications, the display of the enzyme at the cell
surface of the microorganism is an advancement of the whole
cell biocatalyst approach. Neither substrate nor product needs
to be membrane permeable, and both could be excluded from
any unwanted attack by other enzymes. Among the systems for
the display of recombinant proteins on microorganisms, which
include yeast (Kuroda and Ueda, 2011), gram positive (Kronqvist
et al., 2010) and gram negative bacteria (van Bloois et al., 2011),
the autodisplay system is a particularly elegant and efficient tool
with some advantageous features for biotechnological and – if
scaled up – industrial applications.
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
93
domains remain anchored on the cell surface (Linke et al., 2006).
For type Vc autotransporters, the passenger and the translocator domains are provided by separate genes (St Geme and Yeo,
2009). Both domains are transported across the inner membrane
by the Sec machinery, and interact in the periplasm via a so-called
POTRA (“polypeptide transport associated domain”) domain of the
translocator, which initiates transport of the passenger across the
outer membrane. This makes type Vc resembling similar transport
systems existing in chloroplasts and mitochondria, which are supposed to be able to transport very complex and extended folded
protein structures (Tommassen, 2007). Although these particular
features of type Vb and Vc autotransporters make them interesting candidates for the surface display of enzymes, they have not
been used for this purpose yet. Therefore this review focuses on
the application of classical autotransporters in biotechnology.
3. Display of enzymes by classical autotransporters
Fig. 1. Model of the classical autotransporter secretion mechanism. (A) Autotransporters are synthesized as precursor protein containing all domains needed to
transport the passenger to the cell surface. (B) By the aid of a classical signal peptide
the precursor is transported across the inner membrane, which is cleaved off. Subsequently, the C terminal part folds into the outer membrane as a porin-like structure,
a so-called ␤-barrel. The passenger is translocated to the cell surface by the aid of
the ␤-barrel maintains an unfolded conformation during transport. According to
this model surface translocation requires the formation of an interim hairpin structure, which was recently experimentally verified (Ieva and Bernstein, 2009). Surface
translocation is supported by folding of the passenger at the cell surface.
2. Autodisplay
Autodisplay is defined as the recombinant surface display of
proteins or peptides by means of an autotransporter protein in
any gram negative bacterium (Jose and Meyer, 2007). The autotransporter proteins are a large family of secreted proteins in
gram negative bacteria and are divided into three subgroups, the
classical autotransporters (secretion type Va), the trimeric autotransporter adhesins (Vb), and the two partner secretion systems
(Vc) (Henderson et al., 2004). All classical autotransporters are
thought to share a common general structure (Jose et al., 1995).
They are produced as precursor proteins with a standard signal
peptide at the very N terminus, which enables the transport of
the precursor protein across the cytoplasmic membrane, most frequently by the Sec machinery (Fig. 1). The signal peptide, which is
cleaved off as the protein crosses the inner membrane, is followed
by the actual passenger, which will be transported to the cell surface. Outer membrane translocation is facilitated by the C–terminal
part of the precursor, which forms a porin–like structure, a so-called
␤-barrel within the outer membrane that is frequently named
translocator domain. Because the ␤-strand that closes the barrel
is directed towards the periplasm, an additional “linker” domain
is required in between the passenger and the translocator in order
to enable full surface access of the passenger. There are over thirty
examples where the coding sequence of the natural passenger in
the autotransporter precursor protein has been replaced by the
coding sequence of a recombinant protein, resulting in the transport of the recombinant protein to the cell surface. Maurer et al.
(1997) convincingly showed this using the AIDA-I autotransporter,
and it was the first report that used the term “autodisplay” for such
purpose.
The trimeric autotransporters (type Vb) show a similar organization as the classical autotransporters, with the difference that the
␤-domain is rather truncated and cannot function as a monomer.
The translocator within the outer membrane is formed by the ␤domains of three precursors and as a consequence, three passenger
Before we come to the transport and the display of recombinant enzymes by the aid of an autotransporter, it appears worth
to have a look on their natural passengers, which are enzymes
as well. The prototype of an autotransporter protein and the first
family member to be discovered middle of the eighties – although
not named an autotransporter at that time – was IgA1 protease
from Neisseria gonorrhoeae (Halter et al., 1984). Together with its
structural description, the very elegant model for outer membrane
translocation was proposed, without the requirement of energy or
accessory factors (Pohlner et al., 1987), which is still valid as a concept today. Almost a decade later, the first publication to mention
the term “autotransporter” listed ten first examples of this protein
family (or eleven when IgA1 proteases form N. gonorrhoeae and
N. meningitidis are considered to be different examples), among
which five enzymes can be found (Jose et al., 1995). Nowadays
the autotransporter family of proteins comprises more than 1000
members, among which a considerable number bear proteases or
other hydrolases, in particular lipases as natural passengers (Benz
and Schmidt, 2011; Wells et al., 2010; Wilhelm et al., 2011). At this
point the question arises, for what reason natural autotransporters
are not used more frequently for catalytic purposes (Wilhelm
et al., 2011). Autotransporter proteins have been discovered first in
pathogenic gram negative bacteria and are supposed to represent
the largest protein family in this group of microorganisms (Kajava
and Steven, 2006). This reflects first that pathogenic bacteria are a
far more prominent subject in research as harmless commensals
are. Secondly, pathogenicity or the degree of pathogenicity i.e. virulence of a gram negative bacterium often appears to be associated
with its proteins displayed at the cell surface, in many cases as a
part of an autotransporter. The application of pathogenic bacteria
for catalytic purposes bears a safety problem and appears not recommendable for industrial purposes. Therefore it was necessary
to express the autotransporter genes from a pathogenic bacterium
in laboratory strains of E. coli. However this led in many cases to
incompatibility problems and affected also the early work with
IgA1 protease from N. gonorrhoeae expressed in E. coli. Although the
potential of this enzyme for proteolytic purposes, in particular site
specific cleavage of fusion proteins, and for the transport of recombinant proteins was anticipated from the beginning (Pohlner et al.,
1992) and the pioneering work based thereon delivered the proof of
principle for many following studies (Jose et al., 1996; Klauser et al.,
1990, 1992), all these early experiments were performed with E. coli
cells grown on agar plates, which is acceptable for basic research
but cumbersome for most biotechnical applications. Incompatibility problems due to phylogenetic distant relationship could also
account for the incomplete transport of a lipase and an esterase
from Bacillus subtilis and Serratia marcescens by the aid of a lipase
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J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
from Pseudomonas aeruginosa when expressed in E. coli (Becker
et al., 2005).
In order to understand what could account for these problems, we need to critically reflect the term autotransporter. It was
assigned to this family of proteins based on the observation that
surface translocation of the passenger was achieved by the mere
transfer of the autotransporter gene from one gram negative bacterium to the other (Jose et al., 1995; Loveless and Saier, 1997;
Pohlner et al., 1987). This led to the impression that this type of
secretion does not require further accessory factors, and hence the
assignment autotransporter. Today it is obvious that autotransporters are a common theme in gram negative bacteria (Benz and
Schmidt, 2011). They have been found by sequence comparisons
in most if not all pathogenic gram negative bacteria (Nishimura
et al., 2010). Recently more than 215 autotransporter genes could
be located within the 28 complete genome sequences of E. coli,
and their basic structure could be analyzed and phylogenetically
compared (Wells et al., 2010). This leads to the assumption that
most probably all gram negative bacteria contain autotransporter
proteins, and in case additional accessory factors are required for
transport, they are available in these bacteria. As a consequence, if
an autotransporter protein is expressed recombinantly in another
gram negative bacterium than that where it is originated from,
it can take use of this machinery being present as a biological
principle (Walther et al., 2009). On one hand this would mimic
a kind of self-facilitated process which initially made these proteins appointed to be “auto”-transporters, and which now requires
some sort of reassessment. On the other hand, this would account
also for the observed incompatibility problems in case an autotransporter gene is expressed in a foreign host background. There
is a well documented example for this kind of interspecies incompatibility given by Robert et al. (2006), who could show that the
restriction in recombinant expression of the neisserial ␤-barrel protein PorA could be overcome by exchanging the sequence of the
last C terminal membrane ␤-strand with that of the corresponding
E. coli sequence. They called this a C terminal signature sequence by
virtue an outer membrane protein assembly machinery recognizes
outer membrane proteins including autotransporters (Robert et al.,
2006).
Outer membrane translocation within the autotransporter
secretion pathway is thought to require the assistance of chaperone
proteins such as BamA, a component of a hetero-oligomeric complex (Bam complex) with several lipoproteins (BamB-E) (Knowles
et al., 2009; Tommassen, 2007), SurA, Skp, DegP and DnaK (Benz and
Schmidt, 2011) (Fig. 2). Moreover, intramolecular domains have
been identified with chaperone–like function for the support of
correct folding of the passenger (Kajava and Steven, 2006; Peterson
et al., 2010; Renn and Clark, 2008), but we are still far away from
understanding by what molecular mechanism the passenger of an
autotransporter protein traverse the outer membrane. Recent studies on the autotransporter Esp (Ieva and Bernstein, 2009; Ieva et al.,
2008) were able to experimentally verify the hairpin formation of
the linker domain during transport, and also to demonstrate chaperone interactions. It cannot be excluded at the moment that two
of the models (reflected by Fig. 1 and Fig. 2) will eventually be
absorbed in a common scheme (Benz and Schmidt, 2011). Although
these observations clearly indicate that autotransporters do not
deserve the prefix “auto” and that their suffix “transporter” is under
re-evaluation, we would prefer to keep this name to facilitate convenient discussion.
To overcome the pathogenicity and incompatibility problems
discussed above, a systematic approach to identify autotransporters with enzyme passengers in harmless commensals or soil
bacteria would be helpful. These could serve as translocators for
the construction of whole cell biocatalysts with surface displayed
enzymes, or as the starting point for a molecular evolution approach
Fig. 2. Bam complex model of autotransporter secretion. In this model the precursor
including the passenger folds at least partially already in the periplasm. Transport
of the passenger domain across the outer membrane and ␤-barrel integration is
facilitated by the Bam complex, thus enabling the surface translocation of folded
passenger proteins. This model is according to the model initially developed for
Omp85 (Tommassen, 2007). Beside the Bam complex consisting of BamA-E, several
chaperones are involved, which appeared also to play a role in the classical secretion
model (Fig. 1) (Benz and Schmidt, 2011).
in order to adapt the enzymatic activity of the autotransporter to a
synthetic reaction that is in need (Wilhelm et al., 2007). There have
been no comprehensive studies on native autotransporter functions, from strains which are not associated with pathogenicity. At
this point it would be interesting to know whether there are other
enzymatic activities manifested within autotransporter passenger
domains beyond the proteolytic and hydrolytic activities identified so far (Henderson et al., 2004). From the thousands of putative
autotransporter sequences in public databases to which no known
function has been ascribed, we think it is highly likely that many
of these will be ascribed enzymatic functions completely unrelated to pathogenicity (Wells et al., 2010). To solve incompatibility
problems, one could consider identifying autotransporter–specific
chaperones from the original organism, and co–expressing these in
the non-pathogenic lab strain in order to optimize autotransporter
mediated surface display of enzymes. Although there is experimental evidence for positive effects of such co-expression (Binder et al.,
2010; Schlapschy and Skerra, 2011), this would be a case to case
solution which might be too cumbersome for biocatalytic purposes
directed towards industrial applications.
4. Autodisplay of recombinant enzymes
The breakthrough in the autodisplay of recombinant enzymes
appeared when a homologous autotransporter was used for expression in a homologous host, namely an E. coli autotransporter in
an E. coli host strain. Although other E. coli autotransporters were
known at that time, such as Tsh, (Jose et al., 1995) and EspP,
(Brunder et al., 1997), the adhesin involved in diffuse adherence
(AIDA-I) was chosen, most probably because it was the most thoroughly investigated candidate at that time (Benz and Schmidt,
1989, 1992a,b; Suhr et al., 1996). The term “autodisplay” was coined
initially for the use of AIDA-I in the surface display of recombinant
proteins in E. coli (Maurer et al., 1997). For autodisplay the ␤-barrel
and the linker region of AIDA-I was employed in combination with
the signal peptides of various origins (CTB, PelB, OmpA and also
the original AIDA-I signal peptide) (Fig. 3). The DNA encoding the
recombinant passenger was inserted in frame between the coding regions for the domains indispensable for transport and this
resulted in proper surface translocation of the passenger. For successful surface display the artificial construct had to be expressed in
an E. coli strain lacking the outer membrane protease (OmpT) such
as UT5600 (Elish et al., 1988) or BL21, because it had been shown
that this protease cleaved a region within the autodisplay linker,
efficiently releasing recombinant passenger proteins from the cell
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
95
Fig. 3. Typical structure of a fusion protein precursor for autodisplay by the example of CYP3A4. It consists of a signal peptide at the very N terminus followed by the passenger
domain. The signal peptides in use were most frequently derived from CtxB, PelB or OmpA, as well as the natural signal peptide of AIDA-I (Jose and Meyer, 2007). Due to the
cloning procedure several amino acids are added at the C terminus and at the N terminus of the passenger. The passenger domain is followed by the so-called linker, which
optionally can contain protease cleavage site and tags for antibody detection or specific protein labeling (Jose and Handel, 2003).
surface into the supernatant (Jose et al., 2002; Maurer et al., 1997).
Using this system, quite a number of enzymes have been displayed
in a functional form on E. coli (Table 1), including ␤-lactamase from
E. coli, adrenodoxin (Adx) from Bos taurus, sorbitol dehydrogenase
(SDH) from Rhodobacter sphaeroides, different esterases (e.g. ApeE
and EstA), nitrilases from Alcaligenes faecalis and Klebsiella pneumoniae, human hyaluronidases, isoprenyltransferase from Aspergillus
formigatus and finally a P450 enzyme from Bacillus megaterium
(CYP106A2) and the human P450 enzyme CYP3A4.
From the list of enzymes which can be displayed (Table 1)
it becomes obvious that the origin of the protein, whether it is
bacterial or eukaryotic, does not really matter. This is remarkable, because eukaryotic and prokaryotic proteins are composed
of different domain structures and are assumed to exhibit different folding behaviour (Netzer and Hartl, 1997). As outlined above,
outer membrane translocation by autodisplay is thought to involve
the formation of a hairpin structure (Fig. 1), which would lead to
a scenario, in which the C-terminus of the passenger reaches the
surface first followed by its N-terminus. This is exactly the opposite of the order in which proteins are released from the ribosome.
It would also require that during entire transport, the polypeptide chain needs to be kept in an unfolded conformation, a model
which is experimentally supported by several examples (Ieva and
Bernstein, 2009; Jose et al., 1996; Jose and Zangen, 2005). In the case
of the displayed enzymes discussed here, this is obviously working
for proteins that are not naturally secreted, independent whether
they are of eukaryotic or prokaryotic origin. To answer the underlying questions, a more systematic approach would be helpful. In
particular, it would be useful to understand the role of the linker
domain, which contains the so-called autochaperones. It would be
Table 1
Autodisplay of enzymes using different autransporter proteins.
Auto-transporter
Autotransporter
origin
Enzyme
Passenger origin
Application
Reference
AIDA-I
E. coli
Escherichia coli
Bos taurus
Burkholderia gladioli
Rhodobacter sphaeroides
Salmonella enterica
Alcaligenes faecalis
Homo sapiens
Aspergillus fumigatus
Bacillus megaterium
Homo sapiens
Flavobacterium ATCC 27551
Translocation studies
Whole cell biocatalysis
Whole cell biocatalysis
Whole-cell biocatalysis
Whole cell biocatalysis
Whole-cell biocatalysis
Inhibitor screening
Whole-cell biocatalysis
Whole-cell biocatalysis
Drug metabolism studies
Bioremediation
(Lattemann et al., 2000)
(Jose et al., 2001, 2002)
(Schultheiss et al., 2002)
(Jose and von Schwichow, 2004a,b)
(Schultheiss et al., 2008)
(Detzel et al., 2011)
(Kaessler et al., 2011)
(Kranen et al., 2011)
(Schumacher et al., 2012)
(Schumacher and Jose, 2012)
(Li et al., 2008)
EstA
P. aeruginosa*
ß-Lactamase (bla)
bovine adrenodoxin (Adx)
esterase A (EstA)
sorbitol dehydrogenase
esterase (ApeE)
nitrilase
hyaluronidase (hPH-20)
prenyltransferase (FgaPT2)
cytochrome P450 106A2
cytochrome P450 3A4
organophosphate
hydrolase (opd)
lipase (LipA) cutinase
lipase
Bacillus subtilis
Fusarium solani pisi
Serratia marcescens
Pseudomonas aeruginosa
Translocation studies
(Becker et al., 2005)
Enzyme refolding
(Wilhelm et al., 2007)
Escherichia coli
Pseudomonas aeruginosa
Burkholderia cepacia
Pseudomonas fluorescens
Translocation studies
Whole cell biocatalysis
(Yang et al., 2004)
(Yang et al., 2010)
Escherichia coli
Translocation studies
(Suzuki et al., 1995)
P. putida*
IcsA (VirG)
*
S. flexneri
foldase, lipase-specific
(lipH)
ß-Lactamase (bla)
lipase (PAL), foldase
lipase (BCL), foldase
lipase (PFL)
alkaline phosphatase
(phoA)
There is 100% amino acid identity between the EstA gene of the closely related species, Pseudomonas aeruginosa and Pseudomonas putida.
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J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
Fig. 4. Passenger driven dimerization of SDH on the surface of E. coli by autodisplay.
Due to the motility of the ␤ barrel within the plane of the outer membrane, passenger
domains that have affinity to each other will form stable dimers or multimers. As in
the example of SDH, where the crystal structure showed that the two subunits bind
in reverse, the linker has be long and flexible enough to allow such conformations.
reasonable to investigate the efficiency of functional enzyme display with one or two model enzymes i.e. one of prokaryotic and
one of eukaryotic origin. An example of an enzyme of eukaryotic
origin that failed to be expressed on the surface of E. coli by autodisplay is human steroid 5␣-reductase type II (Panter et al., 2005).
5␣-reductase type II is a natural integral inner membrane protein with five transmembrane spanning ␣ helices. When its coding
sequence was inserted at the passenger site of the autotransporter
precursor gene and this construct was transferred into an E. coli
host, no expression product was detectable. This indicates that the
surface display of a natural inner membrane protein by the autodisplay strategy is not possible. It possibly interferes with the proteins
inherent signalling or membrane targeting. Therefore autodisplay
appears to be restricted to the surface display of soluble proteins
or – at least – the soluble domains of membrane proteins, so long
as they are not involved in membrane embedding, targeting or
signalling.
5. Autodisplay for P450 enzyme whole cell biocatalysis
The first two enzymes to be autodisplayed, ␤-lactamase
(Lattemann et al., 2000) and esterase EstA (Schultheiss et al., 2002),
provided the proof of principle, but the first biocatalytic application
of autodisplay appeared with the surface display of Adx (Jose et al.,
2002; Jose et al., 2001). Adx, a bovine iron-sulfur protein, delivers
electrons to the mitochondrial type of P450 enzymes in order to
maintain their activity (Ewen et al., 2011). These electrons are provided by adrenodoxin reductase (AdR). In addition to containing
an inorganic prosthetic group, Adx is a functional dimer (Pikuleva
et al., 2000). Adx was expressed as a monomeric passenger protein
by autodisplay and subsequently Adx dimers were detectable at the
cell surface. One of the most convenient features of autodisplay is
the membrane anchoring domain, the ␤-barrel, which is not covalently linked to the cell envelope as it is in other display systems,
but instead is motile within the plane of the outer membrane. If
the displayed passenger domains have affinity to each other, this
will lead to a passenger driven or self-driven dimerization or multimerization as it has been shown in addition for SDH (Jose and von
Schwichow, 2004a) (Fig. 4), nitrilase (Detzel et al., 2011), prenyltransferase (Kranen et al., 2011), or the lacZ domain of protein A
(Jose et al., 2009). By electron spin resonance experiments it was
shown that when it reached the surface, the Adx dimer was devoid
of the iron-sulfur group and hence inactive (Jose et al., 2001). By
a simple titration step under mild but anaerobic conditions, the
iron sulfur group could be incorporated in apo–Adx displayed at
the E. coli surface and after the addition of AdR and P450 enzyme
(either CYP11A1 or CYP11B1) under aerobic conditions a whole cell
biocatalyst for the efficient conversion of different steroids was
obtained (Fig. 5a) (Jose et al., 2002). Three important conclusions
could be drawn from this investigation. First, cells displaying a
recombinant protein on E. coli by the use of a homologous autotransporter were robust and cell viability was not disturbed by
incorporation of an anorganic prosthetic group under unaerobic
conditions followed by long time use under aerobic conditions
(Jose et al., 2001). Second, the outer membrane of E. coli provides
sufficient membrane environment to the investigated membrane
associated P450 enzymes in order to be enzymatically active. The
activity assay for Adx involved the external addition of a P450
enzyme, whose activity is thought to depend on membrane surrounding. The whole cell biocatalyst obtained by the surface display
of Adx and the addition of AdR and P450 enzyme exhibited activities
in the same range as they were obtained with traditional reconstituted membrane approaches (Jose and Meyer, 2007). Therefore
autodisplay overcame all the obstacles to CYP activity, and is
a promising tool for accessing the synthetic potential of P450
monooxygenases, an interesting but difficult to handle class of
enzymes. Finally, by setting up a calibration curve with purified
Adx, the number of functional Adx dimers at the cell surface was
determined as 1.8 × 105 molecules per single cell. To put this in
perspective, the diameter of a ␤-barrel was calculated to be 1.1 nm
(a crystal structure of the AIDA-I ␤-barrel is not yet available).
After estimating the cell surface area of an average E. coli cell,
the mean distance between two ␤-barrels was determined to be
around 8.6 nm in each direction (Jose and Meyer, 2007). Although
this is a rough estimation, it makes clear, that such a large number
of molecules displayed per single cell is not unreasonable. In addition a similar number was later experimentally verified for other
passengers such as SDH (Jose and von Schwichow, 2004b).
The major consequence from the results obtained with Adx was
to investigate the autodisplay of a P450 enzyme. Cytochrome P450
monooxygenases (P450s or CYPs) play essential roles in the biosynthesis of prostaglandins, steroids or secondary metabolites of plants
and microorganisms, as well as in the detoxification of a wide range
of foreign compounds as drugs or chemical pollutants (Bernhardt,
2006). Most P450s are membrane-bound or need a membrane environment to gain functional conformation. All require at least one
redox partner protein in addition to be active. As mentioned above,
the redox partner proteins for the mammalian mitochondrial P450
enzymes (Class I) are Adx and AdR. The purification of Adx and
AdR is laborious, and may be a reason why the use of the synthetic
potential of P450s in bio-transformations or organic synthesis is not
very common (Urlacher and Girhard, 2011). Nevertheless, proof of
principle in the autodisplay of a P450 was achieved using naturally
soluble CYP106A2 from Bacillus megaterium (Schumacher et al.,
2012). CYP106A2 accepts bovine Adx and AdR as redox partners,
and is known to catalyze the 15␤-hydroxylation of various steroids
or steroid like compounds such as the diterpene abietic acid, as well
as the N-demethylation of the antidepressant imipramine (Bleif
et al., 2011). For autodisplay the CYP106A2 encoding sequence
was inserted as described above and after expression of the corresponding gene construct in E. coli BL21(DE3), sufficient amounts
of protein were detectable at the cell surface (Schumacher et al.,
2012). Enzyme assays included the cells displaying CYP106A2, the
redox partners Adx and AdR, and NADPH.
Unexpectedly, cells displaying CYP106A2 showed enzymatic
activity towards the substrate deoxycorticosterone without the
need to externally incorporate the heme prosthetic group. As
described above, the translocation mechanism of autodisplay
is thought to occur while the protein is in an unfolded form.
The lack of activity of autodisplayed Adx without the external
addition of the prosthetic group was consistent with this translocation model (Jose et al., 2002, 2001). Experiments aiming to
add the prosthetic group from the exterior did no yield a higher
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
97
Fig. 5. Whole biocatalyst for steroid synthesis obtained by autodisplay of Adx and the addition of AdR and CYP enzyme for steroid biosynthesis (A, Jose et al., 2002). The next
step is to display Adx, AdR and CYP enzyme as CYP106A2 on the surface of a single cell in order to have a complete and stable P450 biocatalyst without the need of adding
components from exterior (B).
enzymatic activity (Schumacher et al., 2012). In principle this could
be explained in two ways. Either, the poryphrin was incorporated
during transport and – as a consequence – CYP106A2 was translocated to the cell surface along with the heme prosthetic group
in a folded form, which contradicts the current theories regarding translocation. Or alternatively, CYP106A2 was translocated as
an apoprotein without the heme and the prosthetic group was
incorporated after transport. In this case the porphyrin must
have been present in the supernatant, either as a component of
the growth medium or released by the cells themselves. Very
recently, the TolC channel protein was identified to be responsible for the active transport of porphyrins into the supernatant
of E. coli (Tatsumi and Wachi, 2008). Based on these findings
autodisplay of CYP106A2 was analyzed in the TolC negative
mutant JW55301-1 of E. coli (Baba et al., 2006). The amount of
protein displayed at the surface of the TolC mutant remained
unaltered in comparison to the TolC positive host background,
however, enzymatic activity was substantially reduced. This was
an indirect but strong indication that CYP106A2 was translocated to the cell surface without poryphyrin, most probably in
an unfolded form, consistent with the mechanism recently supported by data obtained with natural autotransporter passengers
(Ieva and Bernstein, 2009). Finally by adding heme as a salt
solution to cells of E. coli displaying the CYP106A2 apoprotein
the enzymatic activity in the TolC mutant could be completely
restored.
For biotechnology purposes, the origin of the heme group
is of less importance than the clear activity towards substrates
which mirror those previously described for the purified CYP106A2
enzyme. In addition to the substrate deoxycorticosterone, the
whole cell biocatalyst obtained by autodisplay of CYP106A2
also hydroxylated abietic acid and enabled N-demethylation of
imipramin (Schumacher et al., 2012).
The successful display of CYP106A2 as a P450 model enzyme
was not an isolated case, and it is likely that surface display
can be applied to other P450s of interest for chemical synthesis
and toxicology. The strategy described above was subsequently
applied to the human P450 enzyme CYP3A4, a membrane associated liver enzyme of the human first-pass metabolism (Kato,
2008). The enzyme was displayed at the cell surface and activity was tested in a 2-component system, requiring the external
addition of NADPH–P450–reductase (Schumacher and Jose, 2012).
Whole cells displaying the enzyme converted the substrate testosterone to yield 6␤-OH-deoxycorticosterone (Schumacher and Jose,
2012). We would like to clearly state that the rate of CYP expression and the quite low enzymatic activity of the CYP3A4 whole cell
98
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
biocatalyst, which is at least partly owed to the very low turnover
number of CYP enzymes in general and that of CYP3A4 in particular,
needs improvement.
Nevertheless these experiments open the door for a broader
application of P450 whole cell biocatalysis for the conversion of
substrates and products that are not able to pass membrane barriers. For purposes of chemical synthesis the native form of the
enzyme does not need to be retained, and it is likely that the application of sophisticated enzyme improvement techniques can select
enzyme variants with increased rates of turnover. The next step
will be the co-expression of the other proteins required for P450
activity on a single cell of E. coli, (e.g. Adx and AdR for class I, and
NADPH-P450-reductase and cytochrome b5 for class II) in order to
obtain a self-assembling functioning electron delivery system on
the cell surface (Fig. 5B). Cells of these types could subsequently
be combined with cells displaying redox co-factor (NADP/NADPH)
regenerating enzymes to enable a continued enzymatic conversion
of the substrates. Finally after this configuration has been successfully assembled and shown to operate for synthesis purposes in
the lab scale, conditions need to be identified and optimized for
the scale up of this process and finally would lead to products in
the gram scale.
6. Enzyme autodisplay and product preparation
The first chemical compound prepared and purified in the subgram range using the autodisplay technology was achieved with
a whole cell biocatalyst displaying nitrilase from Alcaligenes faecalis (Detzel et al., 2011). Nitrilases (EC 3.5.5.1) are enzymes that
convert nitriles to the corresponding carboxylic acid and ammonia in a single step, a reaction of substantial industrial interest.
The carboxylic acids produced are used as intermediates in a great
variety of chemical production processes. In most cases they are
enantiomerically pure and can be produced under mild conditions.
However, nitrilases are known to be rather labile and immobilization and aggregation has been attempted in order to adapt
these enzymes to biocatalytic purposes (Martinkova and Mylerova,
2003). Using the whole cell biocatalyst displaying nitrilase from
Alcaligenes faecalis, 0.4 g of R-mandelic acid with an ee value >99%
could be produced with 120 h of a 1 l batch culture. Nevertheless, it
was about 22-fold less than a similar culture of A. faecalis cells could
have produced in the same time (Kaul et al., 2007). This could have
been due to a lower number of enzyme molecules displayed on the
surface (75,000) in comparison to those which were expressed in
A. faecalis (not determined). It could also reflect that the conditions
within the cell are more convenient for this enzyme reaction than
on the cell surface and e.g. would allow higher degrees of multimerization than on the cell surface. Nitrilase has been found to increase
its enzymatic activity with increasing states multimerization and
nonamers up to dodecamers have been reported (Yamamoto et al.,
1992). Although the motile ␤-barrel allows a multimerization of
passenger domains as seen with various examples, this multimerization will be hindered by the extension of the barrel itself and
will be limited by the operating distance that is provided by the
linker region. Therefore it is at least doubtful whether dodecamers
indeed can be formed on the cell surface after autodisplay, and up to
now such have not been experimentally detected yet. Surface display of nitrilase from A. faecalis nevertheless proved the evidence
obtained before that autodisplay allows multimerization, and gave
an excellent indication of the suitability of autodisplay for industrial
processes.
The whole cell biocatalyst displaying nitrilase could be stored
for 180 days at −70 ◦ C without any significant loss in enzymatic
activity. Cells were reused in subsequent cycles of R-mandelic acid
production in batch culture and it turned out that within the last
cycle, 55% of the intial activity remained after 120 h of enzymatic
conversion (Detzel et al., 2011). In these experiments, the cell sediment harvested from one cycle was put into the next production
cycle of R-mandelic acid without normalization on the cell number.
Therefore, this loss in activity also includes the loss in cell material
during harvesting and transfer. Similar results were obtained with a
whole cell biocatalyst displaying prenyltransferase from Aspergillus
niger for the efficient prenylation of indole derivatives, which was
shown to convert the substrates indole-3-propionic acid and L␤-homotryptophan (Kranen et al., 2011). It could be stored one
month at 8 ◦ C without loss in activity and could also be reused in
cyclic synthesis protocols. For nitrilase from A. faecalis the whole
cell biocatalyst obtained by autodisplay showed a KM value for
mandelonitrile (3.6 mM), which was in accordance with the published KM value for the free enzyme (5.75 mM) (Yamamoto et al.,
1992) as well as an identical pH optimum of 7.5. It also converted
phenylacetonitrile five times faster than mandelonitrile (9.3 mM in
16 h) indicating the same substrate specificity as the free enzyme
(Yamamoto et al., 1992). This would imply that autodisplay of the
nitrilase of A. faecalis does not affect its substrate specificity. Such
unaltered substrate specificity was also found when nitrilase from
Klebsiella aerogenes was displayed on the cell surface of E. coli (Detzel and Jose, unpublished). However, this is not a general rule,
because it was observed before in the autodisplay of sorbitol dehydrogenase from R. sphaeroides that the preferences for different
substrates, polyols and sugars, was altered in comparison to the
free enzyme (Jose and von Schwichow, 2004a). An altered substrate
preference was also found for esterase ApeE when displayed at the
cell surface of E. coli by autodisplay (Schultheiss et al., 2008). In
principle for autodisplay, the enzyme remains connected to the cell
surface via its C-terminus by fusion to the linker domain. This fusion
can limit the flexibility of the enzyme resulting in an altered activity or substrate specificity. There are also a small number of extra
amino acids at the N-terminus which remain after cleavage of the
signal peptide. And finally, one cannot discount the possibility that
the environment the enzyme molecule is facing at the cell surface is
different from that of the cell interior, resulting in an altered activity or substrate specificity. In summary it can be concluded that the
activity of an autodisplayed enzyme does not need to be altered a
priori in comparison to the free enzyme, but it appears to depend on
the enzyme displayed and must be investigated on a case by case
basis.
At this point it appears worth discussing how the activity of
whole cell biocatalysts displaying an enzyme can be measured
and compared to pure enzymes, or other whole cell biocatalyst,
in particular which dimension should be used. The activity in most
purified enzyme preparations is given as U mg−1 protein, which
means ␮mol substrate converted per min per mg of enzyme. Also
in case kcat would be used instead, an estimation of the amount
of protein would be required. Different as in the case of a purified
enzyme, where all proteins are supposed to be enzyme molecules,
the enzyme displayed at the cell surface makes up only a tiny
proportion of the whole cell protein, to which it remains intrinsically tied to. Therefore, to determine the activity of a surface
displayed enzyme and set it into relation of the whole cell protein appears to be not reasonable and will give a wrong impression
about the enzyme’s real activity. An enzyme displayed at the cell
surface is excluded from the vast majority of cell protein which
is expressed intracellular and as a consequence, to set the activity at the cell surface into relation to the entire cell protein would
rather cover the real circumstances than being useful information. Another possibility is to calculate the amount of substrate
that is converted by a single cell of E. coli displaying the enzyme
of interest, as it has been done for sorbitol dehydrogenase (Jose
and von Schwichow, 2004a) and prenyltransferase (Kranen et al.,
2011). This allows to compare the activities of different whole cell
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
biocatalysts, which is in the range of pU per single cell of E. coli,
but does not allow to compare these activities with those of
the corresponding free enzyme. For better comparison we suggest to indicate the enzymatic activity of a whole cell biocatalyst
in U (␮mol min−1 ) per volume of a bacterial suspension with a
defined optical density. This can then be compared with a solution of the purified enzyme preparation. For the autodisplay of
enzymes, whole cell activities of 0.1 until up to 50 mU ml−1 of a
cell culture with an OD578 of 1 were observed for various whole
cell biocatalyst displaying enzymes including SDH (Jose and von
Schwichow, 2004a), esterase (Schultheiss et al., 2008), lipase (Detzel, Kranen, Jose unpublished) and CYP106A2 (Schumacher et al.,
2012).
Preliminary experiments to increase the OD values by a continuous culture system and subsequent substrate fermentation have
been performed with the whole cell biocatalyst displaying nitrilase
from A. faecalis. The optical density could indeed be increased but
the overall enzymatic activity was not higher than that obtained
in the batch culture (Detzel and Jose, unpublished). This means,
that transferring the substrate conversion with whole cell biocatalysts displaying enzymes at the cell surface from the lab scale
to production scale or even industrial scale is an investigation
area of its own and needs further efforts. It can also indicate
that the observed product inhibition for nitrilase from A. faecalis
accounts for this observation (Yamamoto et al., 1992) and systematic process development will be required to overcome this
limitation.
7. Autodisplay of challenging enzymes
Human hyaluronidases are interesting pharmaceutical targets particularly to address cancer diseases (Kovar et al., 2006;
Lokeshwar et al., 2006). Specific inhibitors of this group of enzymes
could allow new therapeutic options to treat such diseases. Moreover, hyaluronidases could be used for the production of selected
fragments of the biopolymer hyaluronic acid (HA) for research
and cosmetic purposes. HA consists of ␤-1,3 linked d-glucuronic
acid and N-acetyl-d-glucosamine disaccharide units. Disaccharide
units are ␤-1,4 linked and joined up to 25,000 times, reaching
a molecular mass up to 4 × 109 Da. HA is the main component
of the extracellular matrix and belongs to the glycosaminoglycans, but in contrast to heparin or chondroitin it is not sulfated.
Its concentration depends on the balance between synthesis via
hyaluronate synthases and degradation via human hyaluronidases,
mainly hyaluronidase 1 (hHyal-1), hyaluronidase 2 (hHyal-2) and
PH–20 (hPH-20) (Stern, 2005; Stern et al., 2006). Until today access
to these enzymes and hence to specific inhibitors is limited because
human hyaluronidases form inclusion bodies (IBs) when expressed
in E. coli and need to be purified and refolded, as observed in
expression studies using hHyal-1 (Hofinger et al., 2007b) and
bee venom hyaluronidase (Soldatova et al., 1998). Expression in
eukaryotic cells is slow, expensive and inefficient in comparison
to E. coli. In the best case, several days are needed to obtain only
low amounts of enzyme for activity determinations (Bookbinder
et al., 2006; Hofinger et al., 2007b; Soldatova et al., 1998). Human
hyaluronidase PH20 was displayed on the surface of E. coli by
autodisplay and considerable enzymatic activity could be measured with whole cells (Kaessler et al., 2011). Autodisplay of hPH20
yielded a simple, reproducible and reliable source for this interesting enzyme and the first three inhibitors could be identified.
However, when the enzyme was expressed on the surface of E. coli
hosts strains with standard lipopolysaccharide (LPS) like BL21or
UT5600, only marginal or no enzymatic activity was detectable.
Only when autodisplay of hPH20 was performed in E. coli host
strain F470 (Schop et al., 2000; Vinogradov et al., 1999), which
99
possesses restricted LPS at the cell surface, sufficient enzymatic
activity was obtained. This was not surprising and could have
been due to a competitive inhibition of hPH20, because the substrate of hPH20, HA and LPS share sufficient structural similarity.
A similar competitive inhibition of LPS was observed with the surface display of sorbitol dehydrogenase (Jose and von Schwichow,
2004a). We cannot at this point exclude the possibility that LPS is
a general problem when sugar modifying enzymes are displayed
at the cell surface of E. coli. For hPH20 the enzymatic activity was
significantly reduced in comparison to the free enzyme produced
in eukaryotic cells. A similar reduction was found with surface
display of nitrilase from A. faecalis in comparison to intracellular prokaryotic expression. But unlike the nitrilase, which is of
bacterial origin, the human hPH20 is predicted to be glycosylated, and the reduced activity could have been due to the lack
of eukaryotic-like glycosylation machinery in the E. coli host cell.
It was demonstrated by the example of human hyaluronidase
Hyal-1 that incubation with N-glycosidase (PNGase F) resulted in
a reduction of enzyme activity to 60% (Hofinger et al., 2007a).
Three N-glycosylation sites within hHyal-1 were identified and
proposed to support correct protein folding (Chao et al., 2007).
Within the hPH-20 amino acid sequence four possible glycosylation sites were found at positions 31, 96, 260 and 369 and it
is supposed that like hHyal–1, the lack of glycosylation of hPH20 in E. coli is a reason for the lower enzyme activity. Despite
some progress in understanding bacterial protein glycosylation
(Benz and Schmidt, 2002; Nothaft and Szymanski, 2010), a major
drawback of all mechanisms of expression in E. coli is the lack
of mammalian-like protein glycosylation, and autodisplay is no
exception. Nevertheless the experience with human hyaluronidase
hPH20 shows that autodisplay is a viable alternative expression
system for challenging enzymes, especially where intracellular
expression results in inclusion bodies. Whole cells displaying
hyaluronidases can be used to test for novel inhibitors, but also
for the production of hyaluronic acids of defined chain length
(Fig. 6), necessary for research of physiological function or for use
in cosmetic or therapeutic preparations (Bogdan Allemann and
Baumann, 2008; Burdick and Prestwich, 2011), currently restricted
to crude hyaluronic acid preparations with a broad variation in
chain length.
8. Autodisplay of recombinant enzymes by other
autotransporters
Beside AIDA-I, surface display of enzymes has been tested with
only a few other autotransporters, none of which are of E. coli
origin (Table 1). EstA, an esterase from Pseudomas spp., was the
most often used autotransporter for such purpose. Autodisplay has
been performed in several cases with the EstA of Pseudomonas
aeruginosa origin (Becker et al., 2008, 2007, 2005; Wilhelm et al.,
2007, 1999), and performed once with the EstA from Pseudomanas
putida (Yang et al., 2004), but both EstA proteins share the identical amino acid sequence and can be considered as being the
same autotransporter. EstA has been used for the surface display
of lipases (Becker et al., 2005), a foldase (Wilhelm et al., 2007), and
␤-lactamase (Yang et al., 2004). The aim of most of these studies was
to demonstrate the use of this autotransporter for surface display of
recombinant proteins and also to investigate the translocation and
the folding of the passenger protein. It was also used as a platform
for clever screening approaches, either to identify cells displaying
esterases with catalytic activity (Becker et al., 2007, 2004, 2005) or
to identify variants with improved enantioselectivity (Becker et al.,
2008). Therefore EstA can be considered alongside AIDA-I as the
most commonly used autotransporter protein for biotechnological
applications (Wilhelm et al., 2011). Also Yang et al. (2004) reported
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J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
Fig. 6. HPLC monitoring of the production of hyaluronic acid (HA) with defined chain length by hyaluronidase. Crude HA extracts are incubated with hyaluronidase and
samples were taken at 24 h and 48 h and analyzed by HPLC using a NH2 -column with a linear gradient of 16 mM to 800 mM NaH2 PO4 as solute. The smallest fragment that
could be identified was a tetrasaccharide at a retention time of 10 min. The elution was stopped at 33 min with the appearance of a 22mer. Whether the fragment appearing
at around 3 min was a monomer or a dimer was not possible to determine. The crude extract contained a 11mer (retention time around 18 min), which was not degradable.
on whole cell biocatalysis with different lipases displayed at the
cell surface of E. coli by the aid of EstA.
A very early example concerning autodisplay was a report of
an alkaline phosphatase displayed on the E. coli cell surface by the
aid of VirG autotransporter from Shigella flexneri, but its enzymatic
activity remained obscure (Suzuki et al., 1995).
9. Perspectives
The examples of autodisplay based whole cell biocatalysts presented here are promising, but have only been tested in the lab
scale. Preliminary experiments with nitrilase displaying cells in a
2 l bioreactor were successsful, however, more systematic studies
with whole cell biocatalysts displaying enzymes will be needed
to identify conditions for up-scaling and for process development
in order to bring the applications from the lab scale to scales of
industrial interest.
For whole cell biocatalysts displaying P450 enzymes, it was
shown by several examples (CYP11A1, CYP11B1, CYP3A4) that the
surface of E. coli provides sufficient membrane environment to
these enzymes in order to be active and in the case of CYP3A4
and CYP106A2 it could be demonstrated that the heme group is
provided by the E. coli host, most probably via TolC, and incorporated at the cell surface into the P450 apoprotein (Schumacher
et al., 2012; Schumacher and Jose, 2012). This makes the autodisplay of P450 enzymes a convenient system to access the synthetic
potential of these enzymes and it provides a new expression platform for these and other difficult to handle biocatalysts. The next
steps will be the co-expression of those proteins that are needed
to deliver the electrons to the P450 enzyme, such as Adx and AdR
for the 3-component system, and CYP P450 reductase for the 2component system (Bernhardt, 2006; Urlacher and Girhard, 2011).
Functional surface display of Adx has already been reported (Jose
et al., 2002, 2001). It is suggested to be co-expressed with the
mitochondrial type of P450 enzyme and AdR on the surface of a
single cell (Fig. 5b). AdR is an FAD containing enzyme and CYP
P450 reductase to be co-expressed with type II P450s contains FMN
in addition to FAD. It has already been shown that flavin containing enzymes can be expressed in an active form at the cell surface
by autodisplay (Kranen and Jose, unpublished). As with the heme
group of P450 enzymes, the flavin component was delivered by
the host cell, most probably released via Tolc and incorporated
into apoprotein displayed at the surface. In case it will be possible to create cells displaying different functional P450s including
co-factors and partner proteins, modular synthetic systems are
technically feasible combining different enzymatic activities. For
example, cells displaying CYP3A4, cells displaying CYP2D6 and
cells displaying CYP2C9, the major enzymes of human first pass
metabolism in the liver could be used in concert in order to simulate human metabolism of drugs or drug like compounds. Moreover
they could be used to prepare metabolic intermediates of new
drugs to be provided to analytical purposes, i.e. as reference compounds. It would be most suitable to combine these modules with
surface displayed P450 enzymes with another whole cell biocatalyst displaying another enzyme which is able to regenerate the
redox equivalents NADP or NADPH in order to assure continues
reactions.
Another interesting approach, which is not restricted to P450
enzymes, is to use whole cell biocatalyst with autodisplayed
enzymes as modules in a sequential row for synthesis (Fig. 7). One
could imagine a start with an easy accessible compound, perhaps a
plant ingredient or waste material, whether aromatic, heterocyclic,
cyclic or non-cyclic, and lead it along a series of whole cell biocatalysts in order to produce a new valuable or bioactive compound (i.e.
a drug). In a similar fashion, a series of whole cell biocatalysts could
be used in bioremediation, to degrade persistent pollutants or toxins (Li et al., 2008; Scott et al., 2009). Many pollutants (e.g. isomers
of hexachlorocyclohexane) are extremely resistant to degradation,
Fig. 7. Modular system of whole cell biocatalysts with autodisplayed enzymes for the synthesis of drugs or building blocks.
J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103
requiring several steps before detoxification is achieved, and in
some cases, multiple enzymes (Lal et al., 2010). Most of the whole
cell biocatalysts obtained by the autodisplay of enzymes were
using E. coli as a host organism. The advantages of E. coli as a host
include that genetic engineering protocols and tools are at hand,
detailed knowledge on physiology and protein function, and available expertise in industrial fermentation. But the expression of
recombinant proteins in E. coli as a host organism also has disadvantages. E. coli contains LPS and the contamination of products, of peptides or proteins used for therapeutic or pharmaceutical purposes is inacceptable because it causes an impetuous
immune response (Freudenberg et al., 2008). Although an increased
rate of cell lysis in the case of autodisplay in E. coli using homologous autotransporters has not yet been observed (Kranen et al.,
2011; Schumacher and Jose, 2012), it is unrealistic to expect an
E. coli supernatant to remain completely free of LPS. In addition
E. coli does not grow to such high densities as it has been reported
for other gram negative bacteria and it appears to be not as robust
as natural soil bacteria in remediation experiments in soil or other
mechanically crude processes. Therefore it could be beneficial to
establish autodisplay in other gram negative bacteria more suitable for such purposes, e.g. Rhodobacter capsulatus (Katzke et al.,
2010) or Ralstonia eutropha (Valls et al., 2000). However, E. coli
autotransporter constructs used for autodisplay in these organisms
may encounter incompatibility problems in the host organism and
it remains to be seen whether this is as efficient as in E. coli. An
alternative would be to isolate and test naturally occurring autotransporters in these organisms, which could be used as a transport
vehicle for recombinant enzymes.
Autodisplay has the advantage, that neither substrate nor product need to cross a membrane barrier. This makes it most suitable for screening purposes in order to obtain taylor-made enzymes
for pre-given reactions (Becker et al., 2008, 2007; Gratz and Jose,
2008, 2011). Molecular biology tools are used to create variations
in the sequence coding the enzyme, and single cells can be selected
by the enzyme variant displayed at the cell surface which will
replicate and finally load themselves with the new enzyme identified. Autodisplay is also compatible with some high throughput
screening approaches, for example agar plate clearing assays or fluorescence activated cell sorting (FACS). So long as the cells can be
labelled based on their activity, FACS in particular will allow single
cell high throughput screening (Becker et al., 2008; Jose et al., 2005).
Such approach towards enzymes with new synthetic features could
start with the surface display of a recombinant enzyme that is subsequently varied by random in order to get libraries of bacteria
which bear a single variant in high numbers at the cell surface. It
would also be possible to start with a natural enzyme passenger of
an autotransporter as the template to introduce random variation
in order to identify improve enzyme variant (Becker et al., 2008,
2005). For the latter case it would be interesting to start a more
systematic approach in order to identify autotransporter proteins
with enzymes as passengers in soil bacteria or harmless gram negative commensals. At least 53 putative autotransporters from gram
negative commensals have already been identified (Wells et al.,
2010).
Autodisplay could also allow screening for novel catalytic activity, where improved variants of the enzyme confer a growth
advantage on the organism. This can be achieved by nutrient
limitation, where the product of the catalysis is the sole source
of the particular nutrient (e.g. carbon, nitrogen or phosphate).
Alternatively, the substrate of the reaction can be toxic to the
cells (e.g. an antibiotic). The autodisplay of antibiotic-degrading
enzymes such as Bla has been shown to convey resistance to ampicillin (Lattemann et al., 2000). Cells displaying enzymes with high
turnover efficiency would have a selective advantage over cells
expressing enzymes with poor efficiency. This “growth selection”
101
approach has been successfully used in combination with plate
assays and the yeast model (Luthi et al., 2003; Murai et al., 1997).
With autodisplay, the location of the enzyme on the cell envelope
might also confer an advantage to the organism in liquid cultures.
This allows the system to be self-selective, as the cells containing
the enzyme variant with the most improved catalysis will grow to
dominate the culture, reducing the number of variants which need
to be investigated.
Finally, cells with autodisplayed redox enzymes could be used
to create novel microbial fuel cells (Fishilevich et al., 2009). Low
value compounds such as waste products could be used as substrate
and the electrons produced can be bled off by carbon electrodes in
order to produce current. Similar systems could also be applied
for analytical purposes such as a biosensor in which the current
produced is a function of the concentration of substrate analyte.
In conclusion, what can be learned from the past experience
is that autodisplay can achieve much more than initially realized.
Therefore it seems worthwhile to take the next step towards a
large scale application of whole cell biocatalysts with autodisplayed
enzymes and to adapt it to industrial needs.
Acknowledgements
The authors would like to thank all colleagues, former and current co-workers and collaboration partners for their contributions
in the autodisplay of enzymes. We would like to apologize if not all
was considered within the narrow focus of this review.
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