Autodisplay of Enzymes—Molecular Basis and Perspectives

Journal of Biotechnology 161 (2012) 92–103

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Journal of Biotechnology

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Autodisplay of enzymes—Molecular basis and perspectives

a,∗ b a

Joachim Jose , Ruth Maria Maas , Mark George Teese

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 a b s t r a c t

Article history: To display an enzyme on the surface of a living cell is an important step forward towards a broader use of

Received 8 October 2011

biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It

Received in revised form 14 February 2012

is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the

Accepted 4 April 2012

enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells

Available online 30 April 2012

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

Keywords:

advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell

Autodisplay

Biocatalysis surface having afﬁnity to each other will spontaneously form dimers or multimers. Using autodisplay

Synthesis enzymes with prosthetic groups can be displayed, expanding the application of surface display to the

5 6

Enzymes industrial important P450 enzymes. Finally, up to 10 –10 enzyme molecules can be displayed on a

Whole cells 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.

1. Introduction (Goldberg et al., 2007b). Despite the advantages, there are some

drawbacks that prevent a broader application of enzymes. The

Enzymes as biocatalysts show some outstanding advantages for puriﬁcation of enzymes is often a complex and costly production

the synthesis of chemicals (Choi, 2009; Luetz et al., 2008), pharma- process. In most cases puriﬁed enzymes cannot be used in repeated

ceutical and agrochemical intermediates (Fischer and Pietruszka, reactions, they turn to waste after a single processing step. The

2010; Tufvesson et al., 2011) as well as active pharmaceutical and use of microorganisms as whole cell biocatalysts avoids the costs

agrochemical compounds (Ran et al., 2009). Unlike conventional associated with enzyme puriﬁcation and ensures that the enzyme

organic chemistry, enzymes can be used under mild conditions con- is working in an optimal environment, where all co-factors and

cerning temperature, pressure and pH and usually they convert a regeneration networks are provided. Moreover, the enzyme as a

substrate with high regio- and enantioselectivity without protect- biocatalyst is largely protected from destabilizing and degrading

ing and de-protecting steps as necessary in conventional organic effects. However the intracellular location of the enzymes means

chemistry. In many cases, the use of enzymes in chemical synthe- that this method will only be successful if both the substrate and

sis requires less substrate, less energy and reduces waste. Moreover product can cross the membrane barrier. In addition, there is a

enzyme discovery and improvement could lead to completely new tremendous consortium of other enzymes present within the cell.

processes, such as the use of cellulose for fuel production, the biore- To obtain the product in a pure and unaltered form, whole cell bio-

mediation of contaminated water and soil, or the production of catalysis is also limited to substrates and products that cannot be

polymers from non–petroleum sources, which under current con- converted by these native enzymes.

ditions are not feasible. For many applications, the display of the enzyme at the cell

To date enzymes are already used in a distinct number of indus- surface of the microorganism is an advancement of the whole

trial processes (Busch et al., 2006). They are either applied as cell biocatalyst approach. Neither substrate nor product needs

preparations of puriﬁed proteins (Goldberg et al., 2007a), or as to be membrane permeable, and both could be excluded from

microorganisms that produce the desired enzyme within the cell 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),

∗

Corresponding author at: Institute for Pharmaceutical and Medicinal Chem-

the autodisplay system is a particularly elegant and efﬁcient tool

istry, Westfälische Wilhelms-Universität, Münster, Hittorfstraße 58-62, D-48149

with some advantageous features for biotechnological and – if

Münster, Germany. Tel.: +49 251 83 32210, fax: +49 251 83 32211.

E-mail address: [email protected] (J. Jose). 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 transloca-

tor 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 sup-

posed 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 interest-

ing 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

Before we come to the transport and the display of recombi-

Fig. 1. Model of the classical autotransporter secretion mechanism. (A) Autotrans- nant enzymes by the aid of an autotransporter, it appears worth

porters are synthesized as precursor protein containing all domains needed to

to have a look on their natural passengers, which are enzymes

transport the passenger to the cell surface. (B) By the aid of a classical signal peptide

as well. The prototype of an autotransporter protein and the ﬁrst

the precursor is transported across the inner membrane, which is cleaved off. Subse-

family member to be discovered middle of the eighties – although

quently, 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 not named an autotransporter at that time – was IgA1 protease

␤

the -barrel maintains an unfolded conformation during transport. According to from Neisseria gonorrhoeae (Halter et al., 1984). Together with its

this model surface translocation requires the formation of an interim hairpin struc-

structural description, the very elegant model for outer membrane

ture, which was recently experimentally veriﬁed (Ieva and Bernstein, 2009). Surface

translocation was proposed, without the requirement of energy or

translocation is supported by folding of the passenger at the cell surface.

accessory factors (Pohlner et al., 1987), which is still valid as a con-

cept today. Almost a decade later, the ﬁrst publication to mention

2. Autodisplay the term “autotransporter” listed ten ﬁrst examples of this protein

family (or eleven when IgA1 proteases form N. gonorrhoeae and

Autodisplay is deﬁned as the recombinant surface display of N. meningitidis are considered to be different examples), among

proteins or peptides by means of an autotransporter protein in which ﬁve enzymes can be found (Jose et al., 1995). Nowadays

any gram negative bacterium (Jose and Meyer, 2007). The auto- the autotransporter family of proteins comprises more than 1000

transporter proteins are a large family of secreted proteins in members, among which a considerable number bear proteases or

gram negative bacteria and are divided into three subgroups, the other hydrolases, in particular lipases as natural passengers (Benz

classical autotransporters (secretion type Va), the trimeric auto- and Schmidt, 2011; Wells et al., 2010; Wilhelm et al., 2011). At this

transporter adhesins (Vb), and the two partner secretion systems point the question arises, for what reason natural autotransporters

(Vc) (Henderson et al., 2004). All classical autotransporters are are not used more frequently for catalytic purposes (Wilhelm

thought to share a common general structure (Jose et al., 1995). et al., 2011). Autotransporter proteins have been discovered ﬁrst in

They are produced as precursor proteins with a standard signal pathogenic gram negative bacteria and are supposed to represent

peptide at the very N terminus, which enables the transport of the largest protein family in this group of microorganisms (Kajava

the precursor protein across the cytoplasmic membrane, most fre- and Steven, 2006). This reﬂects ﬁrst that pathogenic bacteria are a

quently by the Sec machinery (Fig. 1). The signal peptide, which is far more prominent subject in research as harmless commensals

cleaved off as the protein crosses the inner membrane, is followed are. Secondly, pathogenicity or the degree of pathogenicity i.e. vir-

by the actual passenger, which will be transported to the cell sur- ulence of a gram negative bacterium often appears to be associated

face. Outer membrane translocation is facilitated by the C–terminal with its proteins displayed at the cell surface, in many cases as a

part of the precursor, which forms a porin–like structure, a so-called part of an autotransporter. The application of pathogenic bacteria

␤-barrel within the outer membrane that is frequently named for catalytic purposes bears a safety problem and appears not rec-

translocator domain. Because the ␤-strand that closes the barrel ommendable for industrial purposes. Therefore it was necessary

is directed towards the periplasm, an additional “linker” domain to express the autotransporter genes from a pathogenic bacterium

is required in between the passenger and the translocator in order in laboratory strains of E. coli. However this led in many cases to

to enable full surface access of the passenger. There are over thirty incompatibility problems and affected also the early work with

examples where the coding sequence of the natural passenger in IgA1 protease from N. gonorrhoeae expressed in E. coli. Although the

the autotransporter precursor protein has been replaced by the potential of this enzyme for proteolytic purposes, in particular site

coding sequence of a recombinant protein, resulting in the trans- speciﬁc cleavage of fusion proteins, and for the transport of recom-

port of the recombinant protein to the cell surface. Maurer et al. binant proteins was anticipated from the beginning (Pohlner et al.,

(1997) convincingly showed this using the AIDA-I autotransporter, 1992) and the pioneering work based thereon delivered the proof of

and it was the ﬁrst report that used the term “autodisplay” for such principle for many following studies (Jose et al., 1996; Klauser et al.,

purpose. 1990, 1992), all these early experiments were performed with E. coli

The trimeric autotransporters (type Vb) show a similar organi- cells grown on agar plates, which is acceptable for basic research

zation as the classical autotransporters, with the difference that the but cumbersome for most biotechnical applications. Incompatibil-

␤

-domain is rather truncated and cannot function as a monomer. ity problems due to phylogenetic distant relationship could also

␤

The translocator within the outer membrane is formed by the - account for the incomplete transport of a lipase and an esterase

domains of three precursors and as a consequence, three passenger from Bacillus subtilis and Serratia marcescens by the aid of a lipase

94 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 prob-

lems, 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 bac-

terium 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 autotrans-

porters 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 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

et al., 2010). Recently more than 215 autotransporter genes could

of the passenger domain across the outer membrane and ␤-barrel integration is

be located within the 28 complete genome sequences of E. coli,

facilitated by the Bam complex, thus enabling the surface translocation of folded

and their basic structure could be analyzed and phylogenetically

passenger proteins. This model is according to the model initially developed for

compared (Wells et al., 2010). This leads to the assumption that Omp85 (Tommassen, 2007). Beside the Bam complex consisting of BamA-E, several

most probably all gram negative bacteria contain autotransporter chaperones are involved, which appeared also to play a role in the classical secretion

model (Fig. 1) (Benz and Schmidt, 2011).

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 in order to adapt the enzymatic activity of the autotransporter to a

gram negative bacterium than that where it is originated from, synthetic reaction that is in need (Wilhelm et al., 2007). There have

it can take use of this machinery being present as a biological been no comprehensive studies on native autotransporter func-

principle (Walther et al., 2009). On one hand this would mimic tions, from strains which are not associated with pathogenicity. At

a kind of self-facilitated process which initially made these pro- this point it would be interesting to know whether there are other

teins appointed to be “auto”-transporters, and which now requires enzymatic activities manifested within autotransporter passenger

some sort of reassessment. On the other hand, this would account domains beyond the proteolytic and hydrolytic activities identi-

also for the observed incompatibility problems in case an auto- ﬁed so far (Henderson et al., 2004). From the thousands of putative

transporter gene is expressed in a foreign host background. There autotransporter sequences in public databases to which no known

is a well documented example for this kind of interspecies incom- function has been ascribed, we think it is highly likely that many

patibility given by Robert et al. (2006), who could show that the of these will be ascribed enzymatic functions completely unre-

restriction in recombinant expression of the neisserial ␤-barrel pro- lated to pathogenicity (Wells et al., 2010). To solve incompatibility

tein PorA could be overcome by exchanging the sequence of the problems, one could consider identifying autotransporter–speciﬁc

last C terminal membrane ␤-strand with that of the corresponding chaperones from the original organism, and co–expressing these in

E. coli sequence. They called this a C terminal signature sequence by the non-pathogenic lab strain in order to optimize autotransporter

virtue an outer membrane protein assembly machinery recognizes mediated surface display of enzymes. Although there is experimen-

outer membrane proteins including autotransporters (Robert et al., tal evidence for positive effects of such co-expression (Binder et al.,

2006). 2010; Schlapschy and Skerra, 2011), this would be a case to case

Outer membrane translocation within the autotransporter solution which might be too cumbersome for biocatalytic purposes

secretion pathway is thought to require the assistance of chaperone directed towards industrial applications.

proteins such as BamA, a component of a hetero-oligomeric com-

plex (Bam complex) with several lipoproteins (BamB-E) (Knowles 4. Autodisplay of recombinant enzymes

et al., 2009; Tommassen, 2007), SurA, Skp, DegP and DnaK (Benz and

Schmidt, 2011) (Fig. 2). Moreover, intramolecular domains have The breakthrough in the autodisplay of recombinant enzymes

been identiﬁed with chaperone–like function for the support of appeared when a homologous autotransporter was used for expres-

correct folding of the passenger (Kajava and Steven, 2006; Peterson sion in a homologous host, namely an E. coli autotransporter in

et al., 2010; Renn and Clark, 2008), but we are still far away from an E. coli host strain. Although other E. coli autotransporters were

understanding by what molecular mechanism the passenger of an known at that time, such as Tsh, (Jose et al., 1995) and EspP,

autotransporter protein traverse the outer membrane. Recent stud- (Brunder et al., 1997), the adhesin involved in diffuse adherence

ies on the autotransporter Esp (Ieva and Bernstein, 2009; Ieva et al., (AIDA-I) was chosen, most probably because it was the most tho-

2008) were able to experimentally verify the hairpin formation of roughly investigated candidate at that time (Benz and Schmidt,

the linker domain during transport, and also to demonstrate chap- 1989, 1992a,b; Suhr et al., 1996). The term “autodisplay” was coined

erone interactions. It cannot be excluded at the moment that two initially for the use of AIDA-I in the surface display of recombinant

of the models (reﬂected by Fig. 1 and Fig. 2) will eventually be proteins in E. coli (Maurer et al., 1997). For autodisplay the ␤-barrel

absorbed in a common scheme (Benz and Schmidt, 2011). Although and the linker region of AIDA-I was employed in combination with

these observations clearly indicate that autotransporters do not the signal peptides of various origins (CTB, PelB, OmpA and also

deserve the preﬁx “auto” and that their sufﬁx “transporter” is under the original AIDA-I signal peptide) (Fig. 3). The DNA encoding the

re-evaluation, we would prefer to keep this name to facilitate con- recombinant passenger was inserted in frame between the cod-

venient discussion. ing regions for the domains indispensable for transport and this

To overcome the pathogenicity and incompatibility problems resulted in proper surface translocation of the passenger. For suc-

discussed above, a systematic approach to identify autotrans- cessful surface display the artiﬁcial construct had to be expressed in

porters with enzyme passengers in harmless commensals or soil an E. coli strain lacking the outer membrane protease (OmpT) such

bacteria would be helpful. These could serve as translocators for as UT5600 (Elish et al., 1988) or BL21, because it had been shown

the construction of whole cell biocatalysts with surface displayed that this protease cleaved a region within the autodisplay linker,

enzymes, or as the starting point for a molecular evolution approach 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). outer membrane translocation by autodisplay is thought to involve

Using this system, quite a number of enzymes have been displayed the formation of a hairpin structure (Fig. 1), which would lead to

in a functional form on E. coli (Table 1), including ␤-lactamase from a scenario, in which the C-terminus of the passenger reaches the

E. coli, adrenodoxin (Adx) from Bos taurus, sorbitol dehydrogenase surface ﬁrst followed by its N-terminus. This is exactly the oppo-

(SDH) from Rhodobacter sphaeroides, different esterases (e.g. ApeE site of the order in which proteins are released from the ribosome.

and EstA), nitrilases from Alcaligenes faecalis and Klebsiella pneumo- It would also require that during entire transport, the polypep-

niae, human hyaluronidases, isoprenyltransferase from Aspergillus tide chain needs to be kept in an unfolded conformation, a model

formigatus and ﬁnally a P450 enzyme from Bacillus megaterium which is experimentally supported by several examples (Ieva and

(CYP106A2) and the human P450 enzyme CYP3A4. Bernstein, 2009; Jose et al., 1996; Jose and Zangen, 2005). In the case

From the list of enzymes which can be displayed (Table 1) of the displayed enzymes discussed here, this is obviously working

it becomes obvious that the origin of the protein, whether it is for proteins that are not naturally secreted, independent whether

bacterial or eukaryotic, does not really matter. This is remark- they are of eukaryotic or prokaryotic origin. To answer the under-

able, because eukaryotic and prokaryotic proteins are composed lying questions, a more systematic approach would be helpful. In

of different domain structures and are assumed to exhibit differ- particular, it would be useful to understand the role of the linker

ent folding behaviour (Netzer and Hartl, 1997). As outlined above, domain, which contains the so-called autochaperones. It would be

Table 1

Autodisplay of enzymes using different autransporter proteins.

Auto-transporter Autotransporter Enzyme Passenger origin Application Reference

origin

AIDA-I E. coli ß-Lactamase (bla) Escherichia coli Translocation studies (Lattemann et al., 2000)

bovine adrenodoxin (Adx) Bos taurus Whole cell biocatalysis (Jose et al., 2001, 2002)

esterase A (EstA) Burkholderia gladioli Whole cell biocatalysis (Schultheiss et al., 2002)

sorbitol dehydrogenase Rhodobacter sphaeroides Whole-cell biocatalysis (Jose and von Schwichow, 2004a,b)

esterase (ApeE) Salmonella enterica Whole cell biocatalysis (Schultheiss et al., 2008)

nitrilase Alcaligenes faecalis Whole-cell biocatalysis (Detzel et al., 2011)

hyaluronidase (hPH-20) Homo sapiens Inhibitor screening (Kaessler et al., 2011)

prenyltransferase (FgaPT2) Aspergillus fumigatus Whole-cell biocatalysis (Kranen et al., 2011)

cytochrome P450 106A2 Bacillus megaterium Whole-cell biocatalysis (Schumacher et al., 2012)

cytochrome P450 3A4 Homo sapiens Drug metabolism studies (Schumacher and Jose, 2012)

organophosphate Flavobacterium ATCC 27551 Bioremediation (Li et al., 2008)

hydrolase (opd)

EstA P. aeruginosa lipase (LipA) cutinase Bacillus subtilis Translocation studies (Becker et al., 2005)

lipase Fusarium solani pisi

Serratia marcescens

foldase, lipase-speciﬁc Pseudomonas aeruginosa Enzyme refolding (Wilhelm et al., 2007) (lipH)

P. putida ß-Lactamase (bla) Escherichia coli Translocation studies (Yang et al., 2004)

lipase (PAL), foldase Pseudomonas aeruginosa Whole cell biocatalysis (Yang et al., 2010)

lipase (BCL), foldase Burkholderia cepacia

lipase (PFL) Pseudomonas ﬂuorescens

IcsA (VirG) S. ﬂexneri alkaline phosphatase Escherichia coli Translocation studies (Suzuki et al., 1995) (phoA)

There is 100% amino acid identity between the EstA gene of the closely related species, Pseudomonas aeruginosa and Pseudomonas putida.

96 J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103

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 auto-

transporter 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 sur-

rounding. The whole cell biocatalyst obtained by the surface display

of Adx and the addition of AdR and P450 enzyme exhibited activities

Fig. 4. Passenger driven dimerization of SDH on the surface of E. coli by autodisplay.

in the same range as they were obtained with traditional recon-

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 stituted membrane approaches (Jose and Meyer, 2007). Therefore

the example of SDH, where the crystal structure showed that the two subunits bind

autodisplay overcame all the obstacles to CYP activity, and is

in reverse, the linker has be long and ﬂexible enough to allow such conformations.

a promising tool for accessing the synthetic potential of P450

monooxygenases, an interesting but difﬁcult to handle class of

reasonable to investigate the efﬁciency of functional enzyme dis- enzymes. Finally, by setting up a calibration curve with puriﬁed

play with one or two model enzymes i.e. one of prokaryotic and Adx, the number of functional Adx dimers at the cell surface was

one of eukaryotic origin. An example of an enzyme of eukaryotic determined as 1.8 10 molecules per single cell. To put this in

origin that failed to be expressed on the surface of E. coli by autodis- perspective, the diameter of a ␤-barrel was calculated to be 1.1 nm

␣

␤

play is human steroid 5 -reductase type II (Panter et al., 2005). (a crystal structure of the AIDA-I -barrel is not yet available).

␣

5 -reductase type II is a natural integral inner membrane pro- After estimating the cell surface area of an average E. coli cell,

␣

tein with ﬁve transmembrane spanning helices. When its coding the mean distance between two ␤-barrels was determined to be

sequence was inserted at the passenger site of the autotransporter around 8.6 nm in each direction (Jose and Meyer, 2007). Although

precursor gene and this construct was transferred into an E. coli this is a rough estimation, it makes clear, that such a large number

host, no expression product was detectable. This indicates that the of molecules displayed per single cell is not unreasonable. In addi-

surface display of a natural inner membrane protein by the autodis- tion a similar number was later experimentally veriﬁed for other

play strategy is not possible. It possibly interferes with the proteins passengers such as SDH (Jose and von Schwichow, 2004b).

inherent signalling or membrane targeting. Therefore autodisplay The major consequence from the results obtained with Adx was

appears to be restricted to the surface display of soluble proteins to investigate the autodisplay of a P450 enzyme. Cytochrome P450

or – at least – the soluble domains of membrane proteins, so long monooxygenases (P450s or CYPs) play essential roles in the biosyn-

as they are not involved in membrane embedding, targeting or thesis of prostaglandins, steroids or secondary metabolites of plants

signalling. and microorganisms, as well as in the detoxiﬁcation of a wide range

of foreign compounds as drugs or chemical pollutants (Bernhardt,

5. Autodisplay for P450 enzyme whole cell biocatalysis 2006). Most P450s are membrane-bound or need a membrane envi-

ronment to gain functional conformation. All require at least one

␤

The ﬁrst two enzymes to be autodisplayed, -lactamase redox partner protein in addition to be active. As mentioned above,

(Lattemann et al., 2000) and esterase EstA (Schultheiss et al., 2002), the redox partner proteins for the mammalian mitochondrial P450

provided the proof of principle, but the ﬁrst biocatalytic application enzymes (Class I) are Adx and AdR. The puriﬁcation of Adx and

of autodisplay appeared with the surface display of Adx (Jose et al., AdR is laborious, and may be a reason why the use of the synthetic

2002; Jose et al., 2001). Adx, a bovine iron-sulfur protein, delivers potential of P450s in bio-transformations or organic synthesis is not

electrons to the mitochondrial type of P450 enzymes in order to very common (Urlacher and Girhard, 2011). Nevertheless, proof of

maintain their activity (Ewen et al., 2011). These electrons are pro- principle in the autodisplay of a P450 was achieved using naturally

vided by adrenodoxin reductase (AdR). In addition to containing soluble CYP106A2 from Bacillus megaterium (Schumacher et al.,

an inorganic prosthetic group, Adx is a functional dimer (Pikuleva 2012). CYP106A2 accepts bovine Adx and AdR as redox partners,

et al., 2000). Adx was expressed as a monomeric passenger protein and is known to catalyze the 15␤-hydroxylation of various steroids

by autodisplay and subsequently Adx dimers were detectable at the or steroid like compounds such as the diterpene abietic acid, as well

cell surface. One of the most convenient features of autodisplay is as the N-demethylation of the antidepressant imipramine (Bleif

␤

the membrane anchoring domain, the -barrel, which is not cova- et al., 2011). For autodisplay the CYP106A2 encoding sequence

lently linked to the cell envelope as it is in other display systems, was inserted as described above and after expression of the cor-

but instead is motile within the plane of the outer membrane. If responding gene construct in E. coli BL21(DE3), sufﬁcient amounts

the displayed passenger domains have afﬁnity to each other, this of protein were detectable at the cell surface (Schumacher et al.,

will lead to a passenger driven or self-driven dimerization or mul- 2012). Enzyme assays included the cells displaying CYP106A2, the

timerization as it has been shown in addition for SDH (Jose and von redox partners Adx and AdR, and NADPH.

Schwichow, 2004a) (Fig. 4), nitrilase (Detzel et al., 2011), prenyl- Unexpectedly, cells displaying CYP106A2 showed enzymatic

transferase (Kranen et al., 2011), or the lacZ domain of protein A activity towards the substrate deoxycorticosterone without the

(Jose et al., 2009). By electron spin resonance experiments it was need to externally incorporate the heme prosthetic group. As

shown that when it reached the surface, the Adx dimer was devoid described above, the translocation mechanism of autodisplay

of the iron-sulfur group and hence inactive (Jose et al., 2001). By is thought to occur while the protein is in an unfolded form.

a simple titration step under mild but anaerobic conditions, the The lack of activity of autodisplayed Adx without the external

iron sulfur group could be incorporated in apo–Adx displayed at addition of the prosthetic group was consistent with this translo-

the E. coli surface and after the addition of AdR and P450 enzyme cation model (Jose et al., 2002, 2001). Experiments aiming to

(either CYP11A1 or CYP11B1) under aerobic conditions a whole cell 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 solution to cells of E. coli displaying the CYP106A2 apoprotein

be explained in two ways. Either, the poryphrin was incorporated the enzymatic activity in the TolC mutant could be completely

during transport and – as a consequence – CYP106A2 was translo- restored.

cated to the cell surface along with the heme prosthetic group For biotechnology purposes, the origin of the heme group

in a folded form, which contradicts the current theories regard- is of less importance than the clear activity towards substrates

ing translocation. Or alternatively, CYP106A2 was translocated as which mirror those previously described for the puriﬁed CYP106A2

an apoprotein without the heme and the prosthetic group was enzyme. In addition to the substrate deoxycorticosterone, the

incorporated after transport. In this case the porphyrin must whole cell biocatalyst obtained by autodisplay of CYP106A2

have been present in the supernatant, either as a component of also hydroxylated abietic acid and enabled N-demethylation of

the growth medium or released by the cells themselves. Very imipramin (Schumacher et al., 2012).

recently, the TolC channel protein was identiﬁed to be respon- The successful display of CYP106A2 as a P450 model enzyme

sible for the active transport of porphyrins into the supernatant was not an isolated case, and it is likely that surface display

of E. coli (Tatsumi and Wachi, 2008). Based on these ﬁndings can be applied to other P450s of interest for chemical synthesis

autodisplay of CYP106A2 was analyzed in the TolC negative and toxicology. The strategy described above was subsequently

mutant JW55301-1 of E. coli (Baba et al., 2006). The amount of applied to the human P450 enzyme CYP3A4, a membrane asso-

protein displayed at the surface of the TolC mutant remained ciated liver enzyme of the human ﬁrst-pass metabolism (Kato,

unaltered in comparison to the TolC positive host background, 2008). The enzyme was displayed at the cell surface and activ-

however, enzymatic activity was substantially reduced. This was ity was tested in a 2-component system, requiring the external

an indirect but strong indication that CYP106A2 was translo- addition of NADPH–P450–reductase (Schumacher and Jose, 2012).

cated to the cell surface without poryphyrin, most probably in Whole cells displaying the enzyme converted the substrate testos-

an unfolded form, consistent with the mechanism recently sup- terone to yield 6␤-OH-deoxycorticosterone (Schumacher and Jose,

ported by data obtained with natural autotransporter passengers 2012). We would like to clearly state that the rate of CYP expres-

(Ieva and Bernstein, 2009). Finally by adding heme as a salt sion 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 cycle, 55% of the intial activity remained after 120 h of enzymatic

number of CYP enzymes in general and that of CYP3A4 in particular, conversion (Detzel et al., 2011). In these experiments, the cell se-

needs improvement. diment harvested from one cycle was put into the next production

Nevertheless these experiments open the door for a broader cycle of R-mandelic acid without normalization on the cell number.

application of P450 whole cell biocatalysis for the conversion of Therefore, this loss in activity also includes the loss in cell material

substrates and products that are not able to pass membrane bar- during harvesting and transfer. Similar results were obtained with a

riers. For purposes of chemical synthesis the native form of the whole cell biocatalyst displaying prenyltransferase from Aspergillus

enzyme does not need to be retained, and it is likely that the appli- niger for the efﬁcient prenylation of indole derivatives, which was

cation of sophisticated enzyme improvement techniques can select shown to convert the substrates indole-3-propionic acid and L-

␤ enzyme variants with increased rates of turnover. The next step -homotryptophan (Kranen et al., 2011). It could be stored one

◦

will be the co-expression of the other proteins required for P450 month at 8 C without loss in activity and could also be reused in

activity on a single cell of E. coli, (e.g. Adx and AdR for class I, and cyclic synthesis protocols. For nitrilase from A. faecalis the whole

NADPH-P450-reductase and cytochrome b5 for class II) in order to cell biocatalyst obtained by autodisplay showed a KM value for

obtain a self-assembling functioning electron delivery system on mandelonitrile (3.6 mM), which was in accordance with the pub-

the cell surface (Fig. 5B). Cells of these types could subsequently lished KM value for the free enzyme (5.75 mM) (Yamamoto et al.,

be combined with cells displaying redox co-factor (NADP/NADPH) 1992) as well as an identical pH optimum of 7.5. It also converted

regenerating enzymes to enable a continued enzymatic conversion phenylacetonitrile ﬁve times faster than mandelonitrile (9.3 mM in

of the substrates. Finally after this conﬁguration has been success- 16 h) indicating the same substrate speciﬁcity as the free enzyme

fully assembled and shown to operate for synthesis purposes in (Yamamoto et al., 1992). This would imply that autodisplay of the

the lab scale, conditions need to be identiﬁed and optimized for nitrilase of A. faecalis does not affect its substrate speciﬁcity. Such

the scale up of this process and ﬁnally would lead to products in unaltered substrate speciﬁcity was also found when nitrilase from

the gram scale. Klebsiella aerogenes was displayed on the cell surface of E. coli (Det-

zel and Jose, unpublished). However, this is not a general rule,

because it was observed before in the autodisplay of sorbitol dehy-

6. Enzyme autodisplay and product preparation drogenase from R. sphaeroides that the preferences for different

substrates, polyols and sugars, was altered in comparison to the

The ﬁrst chemical compound prepared and puriﬁed in the sub- free enzyme (Jose and von Schwichow, 2004a). An altered substrate

gram range using the autodisplay technology was achieved with preference was also found for esterase ApeE when displayed at the

a whole cell biocatalyst displaying nitrilase from Alcaligenes fae- cell surface of E. coli by autodisplay (Schultheiss et al., 2008). In

calis (Detzel et al., 2011). Nitrilases (EC 3.5.5.1) are enzymes that principle for autodisplay, the enzyme remains connected to the cell

convert nitriles to the corresponding carboxylic acid and ammo- surface via its C-terminus by fusion to the linker domain. This fusion

nia in a single step, a reaction of substantial industrial interest. can limit the ﬂexibility of the enzyme resulting in an altered activ-

The carboxylic acids produced are used as intermediates in a great ity or substrate speciﬁcity. There are also a small number of extra

variety of chemical production processes. In most cases they are amino acids at the N-terminus which remain after cleavage of the

enantiomerically pure and can be produced under mild conditions. signal peptide. And ﬁnally, one cannot discount the possibility that

However, nitrilases are known to be rather labile and immobi- the environment the enzyme molecule is facing at the cell surface is

lization and aggregation has been attempted in order to adapt different from that of the cell interior, resulting in an altered activ-

these enzymes to biocatalytic purposes (Martinkova and Mylerova, ity or substrate speciﬁcity. In summary it can be concluded that the

2003). Using the whole cell biocatalyst displaying nitrilase from activity of an autodisplayed enzyme does not need to be altered a

Alcaligenes faecalis, 0.4 g of R-mandelic acid with an ee value >99% priori in comparison to the free enzyme, but it appears to depend on

could be produced with 120 h of a 1 l batch culture. Nevertheless, it the enzyme displayed and must be investigated on a case by case

was about 22-fold less than a similar culture of A. faecalis cells could basis.

have produced in the same time (Kaul et al., 2007). This could have At this point it appears worth discussing how the activity of

been due to a lower number of enzyme molecules displayed on the whole cell biocatalysts displaying an enzyme can be measured

surface (75,000) in comparison to those which were expressed in and compared to pure enzymes, or other whole cell biocatalyst,

A. faecalis (not determined). It could also reﬂect that the conditions in particular which dimension should be used. The activity in most

−1

within the cell are more convenient for this enzyme reaction than puriﬁed enzyme preparations is given as U mg protein, which

on the cell surface and e.g. would allow higher degrees of multimer- means ␮mol substrate converted per min per mg of enzyme. Also

ization than on the cell surface. Nitrilase has been found to increase in case kcat would be used instead, an estimation of the amount

its enzymatic activity with increasing states multimerization and of protein would be required. Different as in the case of a puriﬁed

nonamers up to dodecamers have been reported (Yamamoto et al., enzyme, where all proteins are supposed to be enzyme molecules,

1992). Although the motile ␤-barrel allows a multimerization of the enzyme displayed at the cell surface makes up only a tiny

passenger domains as seen with various examples, this multimer- proportion of the whole cell protein, to which it remains intrin-

ization will be hindered by the extension of the barrel itself and sically tied to. Therefore, to determine the activity of a surface

will be limited by the operating distance that is provided by the displayed enzyme and set it into relation of the whole cell pro-

linker region. Therefore it is at least doubtful whether dodecamers tein appears to be not reasonable and will give a wrong impression

indeed can be formed on the cell surface after autodisplay, and up to about the enzyme’s real activity. An enzyme displayed at the cell

now such have not been experimentally detected yet. Surface dis- surface is excluded from the vast majority of cell protein which

play of nitrilase from A. faecalis nevertheless proved the evidence is expressed intracellular and as a consequence, to set the activ-

obtained before that autodisplay allows multimerization, and gave ity at the cell surface into relation to the entire cell protein would

an excellent indication of the suitability of autodisplay for industrial rather cover the real circumstances than being useful informa-

processes. tion. Another possibility is to calculate the amount of substrate

The whole cell biocatalyst displaying nitrilase could be stored that is converted by a single cell of E. coli displaying the enzyme

◦

for 180 days at −70 C without any signiﬁcant loss in enzymatic of interest, as it has been done for sorbitol dehydrogenase (Jose

activity. Cells were reused in subsequent cycles of R-mandelic acid and von Schwichow, 2004a) and prenyltransferase (Kranen et al.,

production in batch culture and it turned out that within the last 2011). This allows to compare the activities of different whole cell

J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103 99

biocatalysts, which is in the range of pU per single cell of E. coli, possesses restricted LPS at the cell surface, sufﬁcient enzymatic

but does not allow to compare these activities with those of activity was obtained. This was not surprising and could have

the corresponding free enzyme. For better comparison we sug- been due to a competitive inhibition of hPH20, because the sub-

gest to indicate the enzymatic activity of a whole cell biocatalyst strate of hPH20, HA and LPS share sufﬁcient structural similarity.

−1

in U (␮mol min ) per volume of a bacterial suspension with a A similar competitive inhibition of LPS was observed with the sur-

deﬁned optical density. This can then be compared with a solu- face display of sorbitol dehydrogenase (Jose and von Schwichow,

tion of the puriﬁed enzyme preparation. For the autodisplay of 2004a). We cannot at this point exclude the possibility that LPS is

−1

enzymes, whole cell activities of 0.1 until up to 50 mU ml of a a general problem when sugar modifying enzymes are displayed

cell culture with an OD578 of 1 were observed for various whole at the cell surface of E. coli. For hPH20 the enzymatic activity was

cell biocatalyst displaying enzymes including SDH (Jose and von signiﬁcantly reduced in comparison to the free enzyme produced

Schwichow, 2004a), esterase (Schultheiss et al., 2008), lipase (Det- in eukaryotic cells. A similar reduction was found with surface

zel, Kranen, Jose unpublished) and CYP106A2 (Schumacher et al., display of nitrilase from A. faecalis in comparison to intracellu-

2012). lar prokaryotic expression. But unlike the nitrilase, which is of

Preliminary experiments to increase the OD values by a contin- bacterial origin, the human hPH20 is predicted to be glycosy-

uous culture system and subsequent substrate fermentation have lated, and the reduced activity could have been due to the lack

been performed with the whole cell biocatalyst displaying nitrilase of eukaryotic-like glycosylation machinery in the E. coli host cell.

from A. faecalis. The optical density could indeed be increased but It was demonstrated by the example of human hyaluronidase

the overall enzymatic activity was not higher than that obtained Hyal-1 that incubation with N-glycosidase (PNGase F) resulted in

in the batch culture (Detzel and Jose, unpublished). This means, a reduction of enzyme activity to 60% (Hoﬁnger et al., 2007a).

that transferring the substrate conversion with whole cell biocat- Three N-glycosylation sites within hHyal-1 were identiﬁed and

alysts displaying enzymes at the cell surface from the lab scale proposed to support correct protein folding (Chao et al., 2007).

to production scale or even industrial scale is an investigation Within the hPH-20 amino acid sequence four possible glycosy-

area of its own and needs further efforts. It can also indicate lation sites were found at positions 31, 96, 260 and 369 and it

that the observed product inhibition for nitrilase from A. faecalis is supposed that like hHyal–1, the lack of glycosylation of hPH-

accounts for this observation (Yamamoto et al., 1992) and sys- 20 in E. coli is a reason for the lower enzyme activity. Despite

tematic process development will be required to overcome this some progress in understanding bacterial protein glycosylation

limitation. (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

7. Autodisplay of challenging enzymes exception. Nevertheless the experience with human hyaluronidase

hPH20 shows that autodisplay is a viable alternative expression

Human hyaluronidases are interesting pharmaceutical tar- system for challenging enzymes, especially where intracellular

gets particularly to address cancer diseases (Kovar et al., 2006; expression results in inclusion bodies. Whole cells displaying

Lokeshwar et al., 2006). Speciﬁc inhibitors of this group of enzymes hyaluronidases can be used to test for novel inhibitors, but also

could allow new therapeutic options to treat such diseases. More- for the production of hyaluronic acids of deﬁned chain length

over, hyaluronidases could be used for the production of selected (Fig. 6), necessary for research of physiological function or for use

fragments of the biopolymer hyaluronic acid (HA) for research in cosmetic or therapeutic preparations (Bogdan Allemann and

and cosmetic purposes. HA consists of ␤-1,3 linked d-glucuronic Baumann, 2008; Burdick and Prestwich, 2011), currently restricted

acid and N-acetyl-d-glucosamine disaccharide units. Disaccharide to crude hyaluronic acid preparations with a broad variation in

␤ units are -1,4 linked and joined up to 25,000 times, reaching chain length.

a molecular mass up to 4 × 10 Da. HA is the main component

of the extracellular matrix and belongs to the glycosaminogly-

cans, but in contrast to heparin or chondroitin it is not sulfated. 8. Autodisplay of recombinant enzymes by other

Its concentration depends on the balance between synthesis via autotransporters

hyaluronate synthases and degradation via human hyaluronidases,

mainly hyaluronidase 1 (hHyal-1), hyaluronidase 2 (hHyal-2) and Beside AIDA-I, surface display of enzymes has been tested with

PH–20 (hPH-20) (Stern, 2005; Stern et al., 2006). Until today access only a few other autotransporters, none of which are of E. coli

to these enzymes and hence to speciﬁc inhibitors is limited because origin (Table 1). EstA, an esterase from Pseudomas spp., was the

human hyaluronidases form inclusion bodies (IBs) when expressed most often used autotransporter for such purpose. Autodisplay has

in E. coli and need to be puriﬁed and refolded, as observed in been performed in several cases with the EstA of Pseudomonas

expression studies using hHyal-1 (Hoﬁnger et al., 2007b) and aeruginosa origin (Becker et al., 2008, 2007, 2005; Wilhelm et al.,

bee venom hyaluronidase (Soldatova et al., 1998). Expression in 2007, 1999), and performed once with the EstA from Pseudomanas

eukaryotic cells is slow, expensive and inefﬁcient in comparison putida (Yang et al., 2004), but both EstA proteins share the iden-

to E. coli. In the best case, several days are needed to obtain only tical amino acid sequence and can be considered as being the

low amounts of enzyme for activity determinations (Bookbinder same autotransporter. EstA has been used for the surface display

et al., 2006; Hoﬁnger et al., 2007b; Soldatova et al., 1998). Human of lipases (Becker et al., 2005), a foldase (Wilhelm et al., 2007), and

hyaluronidase PH20 was displayed on the surface of E. coli by ␤-lactamase (Yang et al., 2004). The aim of most of these studies was

autodisplay and considerable enzymatic activity could be mea- to demonstrate the use of this autotransporter for surface display of

sured with whole cells (Kaessler et al., 2011). Autodisplay of hPH20 recombinant proteins and also to investigate the translocation and

yielded a simple, reproducible and reliable source for this inter- the folding of the passenger protein. It was also used as a platform

esting enzyme and the ﬁrst three inhibitors could be identiﬁed. for clever screening approaches, either to identify cells displaying

However, when the enzyme was expressed on the surface of E. coli esterases with catalytic activity (Becker et al., 2007, 2004, 2005) or

hosts strains with standard lipopolysaccharide (LPS) like BL21or to identify variants with improved enantioselectivity (Becker et al.,

UT5600, only marginal or no enzymatic activity was detectable. 2008). Therefore EstA can be considered alongside AIDA-I as the

Only when autodisplay of hPH20 was performed in E. coli host most commonly used autotransporter protein for biotechnological

strain F470 (Schop et al., 2000; Vinogradov et al., 1999), which applications (Wilhelm et al., 2011). Also Yang et al. (2004) reported

100 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 NaH2PO4 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 mitochondrial type of P450 enzyme and AdR on the surface of a

cell surface of E. coli by the aid of EstA. single cell (Fig. 5b). AdR is an FAD containing enzyme and CYP

A very early example concerning autodisplay was a report of P450 reductase to be co-expressed with type II P450s contains FMN

an alkaline phosphatase displayed on the E. coli cell surface by the in addition to FAD. It has already been shown that ﬂavin contain-

aid of VirG autotransporter from Shigella ﬂexneri, but its enzymatic ing enzymes can be expressed in an active form at the cell surface

activity remained obscure (Suzuki et al., 1995). by autodisplay (Kranen and Jose, unpublished). As with the heme

group of P450 enzymes, the ﬂavin component was delivered by

9. Perspectives the host cell, most probably released via Tolc and incorporated

into apoprotein displayed at the surface. In case it will be possi-

The examples of autodisplay based whole cell biocatalysts pre- ble to create cells displaying different functional P450s including

sented here are promising, but have only been tested in the lab co-factors and partner proteins, modular synthetic systems are

scale. Preliminary experiments with nitrilase displaying cells in a technically feasible combining different enzymatic activities. For

2 l bioreactor were successsful, however, more systematic studies example, cells displaying CYP3A4, cells displaying CYP2D6 and

with whole cell biocatalysts displaying enzymes will be needed cells displaying CYP2C9, the major enzymes of human ﬁrst pass

to identify conditions for up-scaling and for process development metabolism in the liver could be used in concert in order to simu-

in order to bring the applications from the lab scale to scales of late human metabolism of drugs or drug like compounds. Moreover

industrial interest. they could be used to prepare metabolic intermediates of new

For whole cell biocatalysts displaying P450 enzymes, it was drugs to be provided to analytical purposes, i.e. as reference com-

shown by several examples (CYP11A1, CYP11B1, CYP3A4) that the pounds. It would be most suitable to combine these modules with

surface of E. coli provides sufﬁcient membrane environment to surface displayed P450 enzymes with another whole cell biocat-

these enzymes in order to be active and in the case of CYP3A4 alyst displaying another enzyme which is able to regenerate the

and CYP106A2 it could be demonstrated that the heme group is redox equivalents NADP or NADPH in order to assure continues

provided by the E. coli host, most probably via TolC, and incor- reactions.

porated at the cell surface into the P450 apoprotein (Schumacher Another interesting approach, which is not restricted to P450

et al., 2012; Schumacher and Jose, 2012). This makes the autodis- enzymes, is to use whole cell biocatalyst with autodisplayed

play of P450 enzymes a convenient system to access the synthetic enzymes as modules in a sequential row for synthesis (Fig. 7). One

potential of these enzymes and it provides a new expression plat- could imagine a start with an easy accessible compound, perhaps a

form for these and other difﬁcult to handle biocatalysts. The next plant ingredient or waste material, whether aromatic, heterocyclic,

steps will be the co-expression of those proteins that are needed cyclic or non-cyclic, and lead it along a series of whole cell biocata-

to deliver the electrons to the P450 enzyme, such as Adx and AdR lysts in order to produce a new valuable or bioactive compound (i.e.

for the 3-component system, and CYP P450 reductase for the 2- a drug). In a similar fashion, a series of whole cell biocatalysts could

component system (Bernhardt, 2006; Urlacher and Girhard, 2011). be used in bioremediation, to degrade persistent pollutants or to-

Functional surface display of Adx has already been reported (Jose xins (Li et al., 2008; Scott et al., 2009). Many pollutants (e.g. isomers

et al., 2002, 2001). It is suggested to be co-expressed with the 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) 92–103 101

requiring several steps before detoxiﬁcation is achieved, and in approach has been successfully used in combination with plate

some cases, multiple enzymes (Lal et al., 2010). Most of the whole assays and the yeast model (Luthi et al., 2003; Murai et al., 1997).

cell biocatalysts obtained by the autodisplay of enzymes were With autodisplay, the location of the enzyme on the cell envelope

using E. coli as a host organism. The advantages of E. coli as a host might also confer an advantage to the organism in liquid cultures.

include that genetic engineering protocols and tools are at hand, This allows the system to be self-selective, as the cells containing

detailed knowledge on physiology and protein function, and avail- the enzyme variant with the most improved catalysis will grow to

able expertise in industrial fermentation. But the expression of dominate the culture, reducing the number of variants which need

recombinant proteins in E. coli as a host organism also has dis- to be investigated.

advantages. E. coli contains LPS and the contamination of pro- Finally, cells with autodisplayed redox enzymes could be used

ducts, of peptides or proteins used for therapeutic or pharma- to create novel microbial fuel cells (Fishilevich et al., 2009). Low

ceutical purposes is inacceptable because it causes an impetuous value compounds such as waste products could be used as substrate

immune response (Freudenberg et al., 2008). Although an increased and the electrons produced can be bled off by carbon electrodes in

rate of cell lysis in the case of autodisplay in E. coli using homol- order to produce current. Similar systems could also be applied

ogous autotransporters has not yet been observed (Kranen et al., for analytical purposes such as a biosensor in which the current

2011; Schumacher and Jose, 2012), it is unrealistic to expect an produced is a function of the concentration of substrate analyte.

E. coli supernatant to remain completely free of LPS. In addition In conclusion, what can be learned from the past experience

E. coli does not grow to such high densities as it has been reported is that autodisplay can achieve much more than initially realized.

for other gram negative bacteria and it appears to be not as robust Therefore it seems worthwhile to take the next step towards a

as natural soil bacteria in remediation experiments in soil or other large scale application of whole cell biocatalysts with autodisplayed

mechanically crude processes. Therefore it could be beneﬁcial to enzymes and to adapt it to industrial needs.

establish autodisplay in other gram negative bacteria more suit-

able for such purposes, e.g. Rhodobacter capsulatus (Katzke et al., Acknowledgements

2010) or Ralstonia eutropha (Valls et al., 2000). However, E. coli

autotransporter constructs used for autodisplay in these organisms The authors would like to thank all colleagues, former and cur-

may encounter incompatibility problems in the host organism and rent co-workers and collaboration partners for their contributions

it remains to be seen whether this is as efﬁcient as in E. coli. An in the autodisplay of enzymes. We would like to apologize if not all

alternative would be to isolate and test naturally occurring auto- was considered within the narrow focus of this review.

transporters in these organisms, which could be used as a transport

vehicle for recombinant enzymes. References

Autodisplay has the advantage, that neither substrate nor pro-

duct need to cross a membrane barrier. This makes it most suit- Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A.,

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Reetz, M.T., Kolmar, H., 2008. Single-cell high-throughput screening to identify

in the sequence coding the enzyme, and single cells can be selected

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replicate and ﬁnally load themselves with the new enzyme iden- Becker, S., Michalczyk, A., Wilhelm, S., Jaeger, K.E., Kolmar, H., 2007. Ultrahigh-

throughput screening to identify E. coli cells expressing functionally active

tiﬁed. Autodisplay is also compatible with some high throughput

enzymes on their surface. Chembiochem 8, 943–949.

screening approaches, for example agar plate clearing assays or ﬂu-

Becker, S., Schmoldt, H.U., Adams, T.M., Wilhelm, S., Kolmar, H., 2004. Ultra-high-

orescence activated cell sorting (FACS). So long as the cells can be throughput screening based on cell-surface display and ﬂuorescence-activated

cell sorting for the identiﬁcation of novel biocatalysts. Current Opinion in

labelled based on their activity, FACS in particular will allow single

Biotechnology 15, 323–329.

cell high throughput screening (Becker et al., 2008; Jose et al., 2005).

Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.E.,

Such approach towards enzymes with new synthetic features could Kolmar, H., 2005. A generic system for the Escherichia coli cell-surface display of

start with the surface display of a recombinant enzyme that is sub- lipolytic enzymes. FEBS Letters 579, 1177–1182.

Benz, I., Schmidt, M.A., 1989. Cloning and expression of an adhesin (AIDA-I) involved

sequently varied by random in order to get libraries of bacteria

in diffuse adherence of enteropathogenic Escherichia coli. Infection and Immu-

which bear a single variant in high numbers at the cell surface. It

nity 57, 1506–1511.

would also be possible to start with a natural enzyme passenger of Benz, I., Schmidt, M.A., 1992a. AIDA-I, the adhesin involved in diffuse adherence of

the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a

an autotransporter as the template to introduce random variation

precursor molecule. Molecular Microbiology 6, 1539–1546.

in order to identify improve enzyme variant (Becker et al., 2008,

Benz, I., Schmidt, M.A., 1992b. Isolation and serologic characterization of AIDA-I, the

2005). For the latter case it would be interesting to start a more adhesin mediating the diffuse adherence phenotype of the diarrhea-associated

Escherichia coli strain 2787 (O126:H27). Infection and Immunity 60, 13–18.

systematic approach in order to identify autotransporter proteins

Benz, I., Schmidt, M.A., 2002. Never say never again: protein glycosylation in

with enzymes as passengers in soil bacteria or harmless gram nega-

pathogenic bacteria. Molecular Microbiology 45, 267–276.

tive commensals. At least 53 putative autotransporters from gram Benz, I., Schmidt, M.A., 2011. Structures and functions of autotransporter pro-

teins in microbial pathogens. International Journal of Medical Microbiology 301,

negative commensals have already been identiﬁed (Wells et al., 461–468.

2010).

Bernhardt, R., 2006. Cytochromes P450 as versatile biocatalysts. Journal of Biotech-

Autodisplay could also allow screening for novel catalytic acti- nology 124, 128–145.

Binder, U., Matschiner, G., Theobald, I., Skerra, A., 2010. High-throughput sorting of

vity, where improved variants of the enzyme confer a growth

an anticalin library via EspP-mediated functional display on the Escherichia coli

advantage on the organism. This can be achieved by nutrient

cell surface. Journal of Molecular Biology 400, 783–802.

limitation, where the product of the catalysis is the sole source Bleif, S., Hannemann, F., Lisurek, M., von Kries, J.P., Zapp, J., Dietzen, M., Antes, I., Bern-

of the particular nutrient (e.g. carbon, nitrogen or phosphate). hardt, R., 2011. Identiﬁcation of CYP106A2 as a regioselective allylic bacterial

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