New Biotechnology Volume 00, Number 00 June 2015
REVIEW
Review
Going beyond E. coli: autotransporter based surface display on alternative host organisms Iasson E.P. Tozakidis1,2, Shanna Sichwart1 and Joachim Jose1,2 1 2
Institut fu¨r Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstr. 48, 48149 Mu¨nster, Germany The NRW Graduate School of Chemistry, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Wilhelm-Klemm-Str. 10, 48149 Mu¨nster, Germany
Autotransporters represent one of the most popular anchoring motifs used to display peptides, proteins or enzymes on the cell surface of a Gram-negative bacterium. Applications range from vaccine delivery to library screenings to biocatalysis and bioremediation. Although the underlying secretion mechanism is supposed to be available in most, if not all, Gram-negative bacteria, autotransporters have to date almost exclusively been used for surface display on Escherichia coli. However, for their utilisation beyond a laboratory scale, in particular for biocatalysis, host bacteria with specific features and industrial applicability are required. A few groups have addressed this issue and demonstrated that bacteria other than E. coli can also be used for autotransporter based surface display. We summarise these studies and discuss opportunities and challenges that arise from surface display of recombinant proteins using the autotransporter pathway in alternative hosts. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Going beyond E. coli: use of autotransporters in alternative Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancement of display efficiency . . . . . . . . . . . . . . Higher robustness for industrial applications . . . . . . Metabolic features . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Availability of suitable expression strains . . . . . . . . . Autotransporter compatibility . . . . . . . . . . . . . . . . . Proof of surface display . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..... hosts. ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... .....
Introduction The implementation of enzymes into chemical conversion processes has been an aspiration for a very long time and significantly Corresponding author: Jose, J. (
[email protected]) http://dx.doi.org/10.1016/j.nbt.2014.12.008 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
000 000 000 000 000 000 000 000 000 000 000 000 000 000
promoted by the advent of recombinant DNA techniques, has become an indispensable advance for the chemical and pharmaceutical industries. In terms of substrate specificity, regio- and stereoselectivity, enzymes are often superior to conventional chemical synthesis. Furthermore, enzyme reactions can in most www.elsevier.com/locate/nbt
Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
1
NBT-740; No of Pages 7 REVIEW
Review
cases be performed at low temperatures and pressures, moderate pH values, and without the extensive use of organic solvents [1]. Nevertheless, in many processes the employment of enzymes is impeded by economic considerations: the production and especially the preparation of enzymes on an industrial scale is laborious and costly. Additionally, purified enzymes are mostly applied for only one reaction and are subsequently disposed of along with the product purification procedure [2]. Therefore, strategies have been developed which aim at reducing enzyme production costs and/or enhancing enzyme stability and reusability. One way to achieve this is simply to omit all enzyme purification steps and instead use the producing cells as a whole, accordingly referred to as whole cell biocatalysis. Besides saving enzyme purification costs, such an approach allows an easy removal of the catalytic system from the reaction mixture, for example by centrifugation, and hence offers the possibility to recycle it for further reactions. Moreover, the expressed enzymes stay in a natural cellular environment, which can have beneficial effects on their stability and activity [3,4]. However, problems arise when either native enzymatic activity of the host cells interferes with the intended reaction, or when cellimpermeable substrates and/or products are involved in the conversion. In such cases, cell surface display can be of particular value, meaning not expressing the enzymes within the cell, but instead fusing them to an appropriate anchoring motif which enables their transport and linkage to the surface of the host cell.
New Biotechnology Volume 00, Number 00 June 2015
A plethora of such anchoring motifs has been described and applied for diverse applications besides biocatalysis, among them high throughput library screening, biosensors, vaccine delivery and bioremediation [5]. For surface display on Gram-negative bacteria, monomeric autotransporters (classified as secretion type Va) represent the most prominent anchoring motif, probably because of their apparent simplicity and modular structure. These proteins, which are very divers in their native function, consist of an N-terminal signal peptide, followed by a so-called passenger domain, a linker and a bbarrel domain (Fig. 1). The term ‘autotransporter’ has inspired lively discussion on the question of whether or not these proteins do transport themselves autonomously to the cell surface [6]. It is now known that autotransporters comprise all the mechanistic elements for a self-sufficient folding in vitro [7]. In vivo, however, autotransporter biogenesis is connected to a complex and not fully understood interplay with the cellular machinery of the host organism. After translocation of the autotransporter protein into the periplasm via the Sec pathway [8–10], the unfolded protein interacts with several periplasmic chaperones, among them SurA, DegP and Skp, that are presumed to keep the autotransporter in a suitable form for outer membrane incorporation [11–13]. The bbarrel assembly machinery (BAM complex), in particular BamA (formerly Omp85) and BamD, play an essential role in the integration of the autotransporter into the outer membrane and display of the passenger domain on the cell surface [14,15], for
FIGURE 1
Above: Modular protein structure of a classical autotransporter as used for surface display. SP, signal peptide. Below: Illustration of important factors to be considered when establishing a new autotransporter host. Basic requirements include the availability of genetic engineering and protein expression tools. After expression in the cytoplasm, the autotransporter protein is transported to the periplasm via the Sec machinery (SEC) and the signal peptide is cleaved. Both protein expression and translocation to the periplasm can influence the amount of displayed protein. The incorporation of the b-barrel and display of the passenger domain on the cell surface is facilitated by the b-barrel assembly machinery (BAM). Here, the compatibility of the autotransporter used with the host’s BAM, as well as the efficiency of this machinery, are crucial for the overall catalytic performance of the cell. For industrial applications, metabolic pathways and robustness of the host cell are crucial parameters. 2
www.elsevier.com/locate/nbt Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
NBT-740; No of Pages 7 New Biotechnology Volume 00, Number 00 June 2015
Acetobacter, Gluconacetobacter and Gluconobacter are also interesting candidates due to their oxidising metabolism, not only for the food and beverage industry, but also for the development of new production routes for industrial chemicals [32]. Although only a few reports have been published in this context, we want to emphasise the benefits, but also the difficulties associated with the use of alternative hosts for autotransportermediated surface display. These points will be discussed in the light of our own experiences made so far with surface display on alternative hosts.
Going beyond E. coli: use of autotransporters in alternative hosts Opportunities Enhancement of display efficiency The primary criterion for the evaluation of a catalytic system is its efficiency. In the case of enzyme-displaying cells, this parameter strongly correlates with the number of enzymes displayed on their surface. In this context it must be considered that an enzyme, when fused to an autotransporter, first has to traverse the inner membrane and subsequently needs to be integrated into the outer membrane. This process is naturally limited by the efficiency of the Sec pathway, and, more importantly, the capacity of the host’s outer membrane to incorporate b-barrel proteins. Although E. coli is a bacterium with the natural ability to secrete autotransporter proteins, it has to be explored whether there might be other bacteria that could possibly perform better in this regard. We recently displayed an esterase from Burkholderia gladioli on the surface of the ethanologenic bacteria Zymobacter palmae and Z. mobilis and observed that Z. palmae cells displaying the esterase exhibited higher activity than E. coli cells displaying the same enzyme [33]. By contrast, Z. mobilis cells exhibited a comparably low enzymatic activity. These results have to be interpreted with regard to the fact that the plasmid used for all three bacteria was codon-optimised for expression in E. coli (approx. 50% GC-content), so that even higher expression levels could possibly have been achieved in Z. palmae (approx. 55.8% GC-content) when optimising the codon usage accordingly. Furthermore, the EhaA autotransporter used [34] was originally derived from E. coli, suggesting that this autotransporter would be expected to be recognised better by the E. coli than by the Z. palmae and Z. mobilis folding machineries. Further investigations are needed to elucidate if Z. palmae cells can actually transport larger amounts of autotransporter proteins into their outer membrane than E. coli and, if so, for what reason. As the secretion of autotransporters is based on a general machinery of the host bacterium, one would expect that differences in display efficiency would be caused by differences within this machinery–for instance, the performance of the Sec translocon or the Bam complex could vary in different host bacteria, or expression levels of these machineries could be different. Furthermore, the host’s outer membrane composition could play a role in the folding kinetics and the membranes capacity for b-barrel proteins. In any case, what can be noted from this example is that there might be bacteria whose physiological properties allow a better exploitation of the autotransporter secretion pathway and thus could, when optimised, reach higher numbers of enzymes on their surface than E. coli. Therefore
www.elsevier.com/locate/nbt 3 Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
Review
which the underlying mechanism is not yet clarified. Display of the desired peptide, protein or enzyme is attained by simply exchanging the native passenger domain with a recombinant one, with only a few limitations imposed on the nature of the recombinant passenger. Excellent articles on the structural and mechanistic details [11,16–19], as well as fields of application [20–22] are available. Here, we will focus on the role of the host organism, an issue which in our opinion has been neglected in the literature so far. In contrast to other anchoring motifs, autotransporters are considered ubiquitous in Gram-negative bacteria [23]; their use should therefore not be restricted to a specific host, making them suitable to be extended to a universal surface display system. Until now, Escherichia coli has served as host organism for nearly all reported studies about autotransporter-mediated surface display. The benefits of working with this bacterium are indisputable: E. coli is one of the best-studied prokaryotic organisms, its genome is completely sequenced and the organism is easily cultivatable and genetically modifiable, with a large set of expression systems and optimised strains for specific purposes being available [24]. From the laboratory viewpoint, E. coli therefore is very well suitable as an autotransporter host, for example when screening surface displayed variants of mutagenesis libraries: A target protein, which is subject to mutation or recombination to obtain variants with specifically desired properties, is expressed as the passenger domain within an autotransporter and thus is directly accessible on the cell surface, allowing its rapid selection (e.g. by fluorescenceactivated cell sorting) without the need for preceding cell lysis or protein purification [25]. In such a procedure, each bacterial cell carries a different variant of the target protein, so that selected variants can easily be multiplied for further analysis by cultivating the respective bacterium. As the success of such approaches strongly correlates with the number of screened variants, fast cultivation and high transformation efficiencies of the host bacterium are needed to allow the generation of large libraries. From an industrial point of view, however, E. coli appears not to be necessarily the best choice for autotransporter-based surface display. Although a broad range of enzymes has meanwhile been successfully displayed on E. coli, none of these approaches have so far been further developed towards a commercial scale [4]. This has to be ascribed to diverse yet unsolved problems, of which some can be addressed to the host organism itself. The establishment of new hosts thus appears to be a rewarding objective. Several species could be taken into account, among them the biotechnologically well-defined ones from the Pseudomonas and Ralstonia genera, but also bacteria being in the exploratory or early development stage for industrial applications. For example, Klebsiella species have shown to be of value for producing the bulk chemicals 1,3-propanediol and 2,3-butanediol under anoxic conditions [26,27]. The microbial production of these compounds, which are to date produced petrochemically, is expected to gain more attention with increasing oil prices, especially when considering waste products (e.g. glycerol) or sustainable biomass (e.g. lignocellulose) as feedstock for the fermentation [28]. In the field of biofuel production, Zymomonas mobilis has for a longer time been subject to research due to its high sugar uptake and ethanol productivity [29], and fermentation with this bacterium has meanwhile reached pilot plant stage [30,31]. Acetic acid bacteria such as
REVIEW
NBT-740; No of Pages 7 REVIEW
systematic studies need to be performed to allow a quantitative comparison between different host organisms.
Higher robustness for industrial applications
Review
The requirements of bacteria used for laboratory procedures differ from those imposed on bacteria for scaled-up production. To run a large process economically, the cells have to be employed as directly as possible with a minimised number of intermediate steps. Depending on the number and nature of upstream processes, it is often unavoidable that the biocatalytic conversion step takes place under conditions which are not optimal for a bacterium. Such stress conditions include extreme temperatures and pH values, as well as the presence of toxic or growth inhibiting compounds and organic solvents. For example, the unsymmetric epoxidation of styrene by means of whole cells suffers the limitation that the product, (S)-styrene oxide, is a cell-toxic compound [35]. Other transformations in turn require the addition of organic solvents due to the participation of water-insoluble substrates or products [36]. When applying whole cells to such mixtures, it is essential to maintain their cell integrity, and for fermentation processes their viability as well. E. coli as an enterobacterium does not possess appropriate protective functions to sufficiently withstand the aforementioned toxic influences. It can therefore be helpful to have host bacteria to hand which are naturally adapted to difficult environmental conditions. Although not for biocatalytic purposes, two species with such properties have already been proven to be applicable as autotransporter hosts. The soil bacterium Ralstonia eutropha CH34 exhibits high natural resistance towards heavy metal contaminations and therefore has been used by Valls et al. [37] as host for the display of a mouse metallothionein. The authors could demonstrate that R. eutropha cells displaying the metal-complexing protein could adsorb significantly more Cd2+ ions than unmodified cells, at a cadmium concentration of 300 mM, which normally inhibits growth of E. coli. Biondo et al. [38] followed the same concept by displaying a synthetic phytochelatin on R. eutropha and showed that recombinant cells were also able to adsorb Pb2+, Zn2+, Cu2+, Mn2+ and Ni2+. In a similar approach, Pseudomonas putida has also been proven to be suitable as host organism for autotransporter-based surface display [39]. This soil bacterium is of particular interest for industrial purposes because of its capability to cope with organic solvents and various toxins, high temperatures and pH values [40]. Both of these organisms are suitable for scale up applications and therefore should also be considered as autotransporter hosts for biocatalytic applications.
Metabolic features The host organism should not only be seen as a platform for the immobilisation of enzymes. Instead, a conversion process can be enriched significantly by combining the reaction catalysed by a displayed enzyme with the native (or engineered) metabolism of the host cell. For example, a polymeric structure, which is too large to enter a bacterial cell, could first be degraded on the cell surface, and the monomeric units produced could then be converted intracellularly to the desired end product. Such a concept has not been realised with autotransporter-based surface display so far, obviously because nearly no hosts with appropriate metabolic features have been reported until now. The above 4
New Biotechnology Volume 00, Number 00 June 2015
mentioned proof-of-principle study concerning the usability of autotransporters in Z. palmae and Z. mobilis represents a first step into this direction. The ethanol productivity of these species is uniquely high among bacteria and makes them interesting candidates for the production of biofuels [41,42]. Other Gram-negative species with interesting metabolic capabilities are also eligible or already applied for industrial biocatalysis, but have not been used as autotransporter hosts so far. To mention some of them, strains of the genus Klebsiella are being developed for the production of the platform chemical 2,3-butanediol [43], and also for biofuels [44]. Bacteria belonging to the Erwinia genus have found use for the production of the anti-leukaemic L-asparaginase, which in E. coli could only be produced with contamination of toxic glutaminase [45]. The acetic acid bacterium Gluconobacter oxydans has diverse applications in the enantioselective oxidation of alcohols and the production of polymers [46]. Meanwhile, a considerable number of engineered Pseudomonas strains are available and already in use for the production of diverse pharmaceuticals and platform chemicals [40], among them a butanol producing strain [47].
Challenges Availability of suitable expression strains The successful establishment of a new autotransporter host depends on some prerequisites that have to be fulfilled: first of all, the cultivation of the organism in liquid medium and under defined conditions needs to be possible. In this regard, the strength of E. coli becomes clear when considering growth rates. For example, with similar cultivation conditions using standard growth media, E. coli has a doubling time of 20–30 min, while Z. palmae needs about one hour and Z. mobilis about two hours for replication (Tozakidis et al., unpublished data). These comparably long cultivation times make laboratory experiments more timeconsuming. At the industrial scale, the focus also lies on obtaining high cell densities to increase overall product yield. In our experiments, Z. palmae and Z. mobilis reached final cell densities of about 7, which was comparable to E. coli. The industrial cultivation of E. coli has already been developed intensively towards high cell densities, although limitations of oxygen supply and heat transfer in large reactors are still challenging [48]. High cell density cultures of P. putida and R. eutropha have already been proven to be feasible and represent promising alternatives to E. coli [49,50]. However, cell density should not be the only criterion: as the feeding strategy used strongly affects cell metabolism and protein expression, it can also have an influence on display efficiency and stability. Thus, when considering a bacterium as an autotransporter host on a large scale, its response to the occurring stress conditions require particular attention. Secondly, the organism needs to be genetically modifiable, either by means of a plasmid or by stable DNA insertion into the genome. The genetic engineering of Z. mobilis has been well developed, resulting in the availability of a large set of Z. mobilis strains with extended substrate range, gene knockouts and adaptions to different cultivation conditions [29]. To date, Z. palmae has only merely been subject to such genetic engineering [42,51]. For both, it was not possible to utilise the vector backbones normally used for surface display on E. coli. In consequence, a new broad-host-range vector had to be constructed to express
www.elsevier.com/locate/nbt Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
NBT-740; No of Pages 7
recombinant autotransporters in these bacteria [33]. It turned out that a mob gene [52] was necessary for stable replication in Z. mobilis and Z. palmae. This gene was not required for replication in P. putida, but was also needed in R. eutropha (Sichwart et al., unpublished data). While we could obtain very high numbers of Z. palmae, P. putida and R. eutropha transformants using this vector backbone, the insertion of the same plasmid into Z. mobilis was very inefficient, yielding only one to five clones after electroporation. While this low transformation efficiency was acceptable for us to prove the general applicability of autotransporters in Z. mobilis, it would be unsatisfactory for screening purposes and requires improvement. Regarding the vector backbone, possible natural resistance of the host has to be tested and appropriate selection markers identified. For the organisms mentioned, kanamycin was shown to work well. Thirdly, a suitable protein expression system for the respective organism has to be available. In particular, the promoter used for expression of the autotransporter protein must be chosen carefully. Too strong expression can cause the accumulation of protein in the cytoplasm or impairment of cell integrity, while too low expression levels decrease the catalytic activity of the cell. The use of a constitutive promoter avoids the necessity of adding an inducer, but cell growth can deteriorate, cells can be genetically unstable due to the constant pressure of protein expression, or the displayed protein become untimely degraded. For surface display on E. coli, both inducible [53] and constitutive [54] promoters have been shown to be applicable. Although no reports about the use of the inducible pBAD promoter in Z. mobilis and Z. palmae had been available, we found out that this promoter is functional in both bacteria and, in the case of the displayed esterase, is suitable for the expression of autotransporter proteins [33]. Currently, we are testing a set of constitutive promoters to see whether higher display efficiencies can be obtained. Experiences based on E. coli have led to the formulation of some further demands on the host: typically, E. coli strains lacking the outer membrane protease OmpT, such as BL21 or UT5600, are applied to avoid proteolytic cleavage of the passenger [54]. When displaying an esterase on Z. palmae, enzymatic activity was also detected in the cell supernatant. Because cell lysis could be experimentally excluded as an explanation, we assume that outer membrane protease activity resulted in the partial release of the passenger domain. This is supported by the finding that the esterase-displaying E. coli strain BL21 did not show any enzyme activity in the supernatant [33]. For new host organisms, outer membrane proteases have to be identified and subsequently disabled to ensure stable surface exposure. According to the proposed
REVIEW
autotransporter secretion mechanism, the passenger domain has to be in a mainly unfolded state to be translocated to the cell surface. Consequently, early folding of the passenger domain in the periplasm has to be prevented. This applies especially when the passenger domain possesses disulphide bonds. For example, the trypsin inhibitor aprotinin could only be displayed when 2-mercaptoethanol was externally supplemented to impede disulphide bond formation. Without the addition of this reducing agent, the autotransporter protein resided in the periplasm and was degraded by proteases located therein [55]. For surface translocation of cysteine-rich passengers, it was shown that it can be helpful to disable the periplasmic oxidoreductase DsbA, which is involved in disulphide bond formation [56]. As in the case of outer membrane proteases, analogous (or also homologous) enzymes have to be identified and possibly knocked out in new host organisms to enable the surface translocation of disulphide bond containing enzymes.
Autotransporter compatibility Table 1 lists the reported uses of recombinant autotransporters in bacteria beyond E. coli. It is noticeable that nearly all reported studies made use of a heterologous autotransporter, meaning that b-barrel and linker domains were not derived from the genome of the host itself, but from a different bacterium. The Neisseria gonorrhoeae IgA autotransporter was successfully applied in P. putida [39] as well as in R. eutropha [37,38]. This autotransporter was the first one used to display a recombinant passenger on the E. coli surface [57]. Interestingly, the IgA autotransporter was soon rated as insufficiently functioning in E. coli, which resulted in a widespread use of E. coli autotransporters, the most frequently used being AIDA-I [58]. The poor display efficiency of the IgA autotransporter in E. coli was later ascribed to a species incompatibility [59]. A C-terminal, species-specific motif within the b-barrel domain was identified which is recognised by the core component of the BAM complex, BamA and triggers the translocation of the protein into the outer membrane [23]. Seemingly, the IgA autotransporter was not properly recognised by the E. coli BAM complex. This problem, however, did not occur when using P. putida and R. eutropha as hosts [37,39]. Surface display on Z. palmae and Z. mobilis was also carried out by means of a heterologous autotransporter, namely EhaA from E. coli [33]. Again, no incompatibility could be observed. Vice versa, the autotransporter EstA from Pseudomonas was shown to be functional for surface display in E. coli [60,61]. Marin et al. [62] performed a comparative study of various autotransporters and reported that the expression and display levels of heterologous autotransporters in E. coli are de-
TABLE 1
Surface display on alternative hosts using recombinant autotransporters Host bacterium
Recombinant passenger
Autotransporter
Reference
Pseudomonas stutzeri A15
b-Lactamase
P. stutzeri EstA
[63]
Pseudomonas putida KT2442
Mouse metallothionein
N. gonorrhoeae IgA
[39]
Ralstonia eutropha CH34
Synthetic phytochelatin
N. gonorrhoeae IgA
[38]
Ralstonia eutropha CH34
Mouse metallothionein
N. gonorrhoeae IgA
[37]
Zymobacter palmae
B. gladioli EstA
E. coli EhaA
[33]
Zymomonas mobilis
B. gladioli EstA
E. coli EhaA
[33]
www.elsevier.com/locate/nbt 5 Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
Review
New Biotechnology Volume 00, Number 00 June 2015
NBT-740; No of Pages 7 REVIEW
Review
pendent on the phylogenetic distance between the host and the bacterium from which the autotransporter is derived. By contrast, Nicolay et al. [63] evaluated the applicability of a native autotransporter in Pseudomonas stutzeri and discovered that the P. stutzeri EstA autotransporter used was only able to transport one of several tested recombinant passenger domains to the cell surface. Because these findings cannot be ascribed to a species incompatibility problem, one can only speculate whether in this particular case the autotransporter, the inserted passenger domains, or a combination of both, lead to the observed defective translocation. In any case, a general statement about the functionality of an autotransporter in a specific host does not seem possible at the moment. More details of the autotransporter secretion pathway have to be elucidated to allow predictions about the compatibilities of autotransporters and hosts. Meanwhile, autotransporters of a wide range of bacteria with industrial prospects have been identified, among them Pseudomonas, Ralstonia, Paracoccus and Zymomonas species [23]. The use of native autotransporters thus has become possible in cases where heterologous autotransporters turn out not to be optimally functional.
Proof of surface display When displaying proteins on the surface of E. coli, a protease accessibility test is often used to verify the actual surface exposure of the passenger domain. In this procedure, whole cells expressing the recombinant autotransporter are treated with a protease, such as proteinase K or trypsin, and outer membrane proteins of the cells are subsequently isolated and separated by SDS-PAGE. The protein band assigned to the recombinant autotransporter protein should then disappear or be reduced in apparent molecular weight, indicating that the protein was accessible to the protease, which due to its size cannot penetrate an intact bacterial cell membrane. This procedure is popular because the success of the surface display approach can be assessed quickly and without special devices or reagents. However, the integrity of the bacterial outer membrane during the proteinase treatment has to be controlled – otherwise, the degradation of the autotransporter could also be a result of an intrusion of the protease into the periplasmic space, giving a false-positive result. For this reason, the outer membrane proteins OmpA, OmpC and OmpF are often used as reference proteins, giving a characteristic double band pattern at approx. 35 kDa (with OmpC and OmpF having identical sizes). The appearance of unprocessed OmpA in the outer membrane isolates provides evidence that the protease could not enter the periplasm of the cells. OmpA has a C-terminal extension in the periplasm, which would otherwise be degraded [64].
New Biotechnology Volume 00, Number 00 June 2015
Unfortunately, such reference proteins have not been identified for other Gram-negative bacteria so far, so that either other control proteins for cell integrity will have to be identified, or a completely different methodology for the verification of surface display applied. Labelling of the passenger domain with specific antibodies, combined with subsequent quantification by means of an enzyme linked immunosorbent assay (ELISA, using an enzyme-coupled second antibody) or flow cytometry (using a fluorescent second antibody) are the most obvious ones. As antibodies are too large to enter the periplasm, binding can only take place when the passenger is indeed located outside of the cell. Van Gerven et al. [65] pointed out that the expression of autotransporters can lead to cell leakiness and lysis, giving falsepositive results in the proteinase accessibility assay and ELISA. Flow cytometry offers the advantage of analysing individual cells, not only in terms of their fluorescence, but also of their outer appearance, which eliminates the risk of misinterpreting the localisation of the passenger domain.
Conclusion Although many other anchoring motifs are in principal suitable, autotransporters, due to their probably ubiquitous presence in Gram-negative bacteria, appear to be the most practical ones for displaying enzymes on alternative hosts. To enable the transfer of this technique from E. coli to other bacteria, we suggest focusing research onto two major areas. (1) Autotransporter secretion pathways have to be understood better, particularly their recognition by chaperones, and related to that, their compatibility in a heterologous background. This would help avoiding much trial and error in the establishment of new host organisms. (2) Strains have to be developed that are optimised in terms of surface display. This necessitates having detailed genetic information about the respective bacterium as well as the tools for its genetic engineering at hand. With these prerequisites fulfilled, alternative host organisms could help to bring biocatalysis with surface displayed enzymes a step further towards industrial application.
Author contributions I.T. wrote the manuscript, S.S. contributed experimental details and literature research, J.J. designed the concept of the review and wrote the manuscript. All authors have read and approved the final manuscript.
Acknowledgement We thank the NRW Graduate School of Chemistry (GSC-MS) for providing a scholarship to I.T.
References [1] Reetz MT. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J Am Chem Soc 2013;135(34):12480–96. [2] Jose J, Maas RM, Teese MG. Autodisplay of enzymes – molecular basis and perspectives. J Biotechnol 2012;161(2):92–103. [3] Ishige T, Honda K, Shimizu S. Whole organism biocatalysis. Curr Opin Chem Biol 2005;9(2):174–80. [4] Schuurmann J, Quehl P, Festel G, Jose J. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application. Appl Microbiol Biotechnol 2014;98(19):8031–46. [5] Lee SY, Choi JH, Xu Z. Microbial cell-surface display. Trends Biotechnol 2003;21(1):45–52.
6
[6] Bernstein HD. Are bacterial ‘autotransporters’ really transporters? Trends Microbiol 2007;15(10):441–7. [7] Leyton DL, Johnson MD, Thapa R, Huysmans GH, Dunstan RA, Celik N, et al. A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes. Nat Commun 2014;5:4239. [8] Desvaux M, Scott-Tucker A, Turner SM, Cooper LM, Huber D, Nataro JP, et al. A conserved extended signal peptide region directs posttranslational protein translocation via a novel mechanism. Microbiology 2007;153(Pt 1): 59–70. [9] Peterson JH, Szabady RL, Bernstein HD. An unusual signal peptide extension inhibits the binding of bacterial presecretory proteins to the signal recognition
www.elsevier.com/locate/nbt Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
NBT-740; No of Pages 7
[10] [11]
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
[20] [21]
[22]
[23]
[24] [25]
[26]
[27] [28]
[29] [30] [31]
[32] [33]
[34]
[35]
[36] [37]
[38]
particle, trigger factor, and the SecYEG complex. J Biol Chem 2006;281(14): 9038–48. Xie K, Dalbey RE. Inserting proteins into the bacterial cytoplasmic membrane using the Sec and YidC translocases. Nat Rev Microbiol 2008;6(3):234–44. Leyton DL, Rossiter AE, Henderson IR. From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis. Nat Rev Microbiol 2012;10(3):213–25. Purdy GE, Fisher CR, Payne SM. IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA. J Bacteriol 2007; 189(15):5566–73. Ruiz-Perez F, Henderson IR, Leyton DL, Rossiter AE, Zhang Y, Nataro JP. Roles of periplasmic chaperone proteins in the biogenesis of serine protease autotransporters of Enterobacteriaceae. J Bacteriol 2009;191(21):6571–83. Knowles TJ, Scott-Tucker A, Overduin M, Henderson IR. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat Rev Microbiol 2009;7(3):206–14. Rossiter AE, Leyton DL, Tveen-Jensen K, Browning DF, Sevastsyanovich Y, Knowles TJ, et al. The essential beta-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis. J Bacteriol 2011;193(16):4250–3. Benz I, Schmidt MA. Structures and functions of autotransporter proteins in microbial pathogens. Int J Med Microbiol 2011;301(6):461–8. Gawarzewski I, DiMaio F, Winterer E, Tschapek B, Smits SH, Jose J, et al. Crystal structure of the transport unit of the autotransporter adhesin involved in diffuse adherence from Escherichia coli. J Struct Biol 2014;187(1):20–9. Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala’Aldeen D. Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 2004;68(4):692–744. Pavlova O, Peterson JH, Ieva R, Bernstein HD. Mechanistic link between beta barrel assembly and the initiation of autotransporter secretion. Proc Natl Acad Sci U S A 2013;110(10):E938–47. Jong WS, Sauri A, Luirink J. Extracellular production of recombinant proteins using bacterial autotransporters. Curr Opin Biotechnol 2010;21(5):646–52. Nicolay T, Vanderleyden J, Spaepen S. Autotransporter-based cell surface display in Gram-negative bacteria. Crit Rev Microbiol 2013. http://dx.doi.org/10.3109/ 1040841X.2013.804032. van Ulsen P, Rahman S, Jong WS, Daleke-Schermerhorn MH, Luirink J. Type V secretion: from biogenesis to biotechnology. Biochim Biophys Acta 2014; 1843(8):1592–611. Celik N, Webb CT, Leyton DL, Holt KE, Heinz E, Gorrell R, et al. A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters. PLoS ONE 2012;7(8):432–45. Makino T, Skretas G, Georgiou G. Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 2011;10:32. Becker S, Schmoldt HU, Adams TM, Wilhelm S, Kolmar H. Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Curr Opin Biotechnol 2004;15(4):323–9. Cheng KK, Zhang JA, Liu DH, Sun Y, Liu HJ, Yang MD, et al. Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae. Process Biochem 2007;42(4):740–4. Ji XJ, Huang H, Ouyang PK. Microbial 2,3-butanediol production: a state-of-theart review. Biotechnol Adv 2011;29(3):351–64. Hermann BG, Patel M. Today’s and tomorrow’s bio-based bulk chemicals from white biotechnology – a techno-economic analysis. Appl Biochem Biotechnol 2007;136(3):361–88. He MX, Wu B, Qin H, Ruan ZY, Tan FR, Wang JL, et al. Zymomonas mobilis: a novel platform for future biorefineries. Biotechnol Biofuels 2014;7:101. Reisch MS. Fuels of the future. Chem Eng News 2006;84(47):30–2. chemicals-technology.com. DuPont Cellulosic Ethanol Biorefinery Project. United States of America. http://www.chemicals-technology.com/projects/ dupont-cellulosic-ethanol-biorefinery-project/ [accessed 15.10.14]. Raspor P, Goranovic D. Biotechnological applications of acetic acid bacteria. Crit Rev Biotechnol 2008;28(2):101–24. Tozakidis IE, Sichwart S, Teese MG, Jose J. Autotransporter mediated esterase display on Zymomonas mobilis and Zymobacter palmae. J Biotechnol 2014. http:// dx.doi.org/10.1016/j.jbiotec.2014.07.009. Wells TJ, Sherlock O, Rivas L, Mahajan A, Beatson SA, Torpdahl M, et al. EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation. Environ Microbiol 2008; 10(3):589–604. Kuhn D, Buhler B, Schmid A. Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas. J Ind Microbiol Biotechnol 2012;39(8):1125–33. Leon R, Fernandes P, Pinheiro HM, Cabral JMS. Whole-cell biocatalysis in organic media. Enzyme Microb Technol 1998;23(7/8):483–500. Valls M, Atrian S, de Lorenzo V, Fernandez LA. Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil. Nat Biotechnol 2000;18(6):661–5. Biondo R, da Silva FA, Vicente EJ, Souza Sarkis JE, Schenberg AC. Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for
REVIEW
[39]
[40]
[41] [42]
[43]
[44] [45] [46]
[47]
[48] [49]
[50]
[51]
[52]
[53]
[54]
[55] [56]
[57]
[58] [59]
[60]
[61]
[62]
[63]
[64]
[65]
enhanced metals bioremediation. Environ Sci Technol 2012;46(15): 8325–32. Valls M, de Lorenzo V, Gonzalez-Duarte R, Atrian S. Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption. J Inorg Biochem 2000;79(1–4):219–23. Poblete-Castro I, Becker J, Dohnt K, dos Santos VM, Wittmann C. Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol 2012;93(6):2279–90. Rogers PL, Jeon YJ, Lee KJ, Lawford HG. Zymomonas mobilis for fuel ethanol and higher value products. Adv Biochem Eng Biotechnol 2007;108:263–88. Yanase H, Sato D, Yamamoto K, Matsuda S, Yamamoto S, Okamoto K. Genetic engineering of Zymobacter palmae for production of ethanol from xylose. Appl Environ Microbiol 2007;73(8):2592–9. Kim B, Lee S, Park J, Lu M, Oh M, Kim Y, et al. Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes. J Microbiol Biotechnol 2012;22(9):1258–63. Dien BS, Cotta MA, Jeffries TW. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 2003;63(3):258–66. Robertbaudouy J. Molecular biology of Erwinia – from soft-rot to antileukemics. Trends Biotechnol 1991;9(9):325–9. De Muynck C, Pereira CSS, Naessens M, Parmentier S, Soetaert W, Vandamme EJ. The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications. Crit Rev Biotechnol 2007;27(3):147–71. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL. Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 2009;11(4/5):262–73. Choi JH, Keum KC, Lee SY. Production of recombinant proteins by high cell density culture of Escherichia coli. Chem Eng Sci 2006;61(3):876–85. Diniz SC, Taciro MK, Gomez JG, da Cruz Pradella JG. High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates. Appl Biochem Biotechnol 2004;119(1):51–70. Srinivasan S, Barnard GC, Gerngross TU. A novel high-cell-density protein expression system based on Ralstonia eutropha. Appl Environ Microbiol 2002; 68(12):5925–32. Yanase H, Yamamoto K, Sato D, Okamoto K. Ethanol production from cellobiose by Zymobacter palmae carrying the Ruminocuccus albus beta-glucosidase gene. J Biotechnol 2005;118(1):35–43. Szpirer CY, Faelen M, Couturier M. Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1. J Bacteriol 2001;183(6): 2101–10. Kranen E, Detzel C, Weber T, Jose J. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs. Microb Cell Fact 2014;13(1):19. Maurer J, Jose J, Meyer TF. Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli. J Bacteriol 1997;179(3):794–804. Jose J, Zangen D. Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 2005;333(4):1218–26. Jose J, Kramer J, Klauser T, Pohlner J, Meyer TF. Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway. Gene 1996;178(1/2):107–10. Klauser T, Pohlner J, Meyer TF. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J 1990;9(6):1991–9. Jose J, Meyer TF. The autodisplay story, from discovery to biotechnical and biomedical applications. Microbiol Mol Biol Rev 2007;71(4):600–19. Robert V, Volokhina EB, Senf F, Bos MP, Van Gelder P, Tommassen J. Assembly factor Omp85 recognizes its outer membrane protein substrates by a speciesspecific C-terminal motif. PLoS Biol 2006;4(11):e377. Becker S, Theile S, Heppeler N, Michalczyk A, Wentzel A, Wilhelm S, et al. A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS Lett 2005;579(5):1177–82. Yang TH, Pan JG, Seo YS, Rhee JS. Use of Pseudomonas putida EstA as an anchoring motif for display of a periplasmic enzyme on the surface of Escherichia coli. Appl Environ Microbiol 2004;70(12):6968–76. Marin E, Bodelon G, Fernandez LA. Comparative analysis of the biochemical and functional properties of C-terminal domains of autotransporters. J Bacteriol 2010;192(21):5588–602. Nicolay T, Lemoine L, Lievens E, Balzarini S, Vanderleyden J, Spaepen S. Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15. Microb Cell Fact 2012; 11:158. Jose J, Bernhardt R, Hannemann F. Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli. J Biotechnol 2002;95(3): 257–68. Van Gerven N, Sleutel M, Deboeck F, De Greve H, Hernalsteens JP. Surface display of the receptor-binding domain of the F17a-G fimbrial adhesin through the autotransporter AIDA-I leads to permeability of bacterial cells. Microbiology 2009;155(Pt 2):468–76.
www.elsevier.com/locate/nbt 7 Please cite this article in press as: Tozakidis, I.EP. et al., Going beyond E. coli: autotransporter based surface display on alternative host organisms, New Biotechnol. (2015), http://dx.doi.org/ 10.1016/j.nbt.2014.12.008
Review
New Biotechnology Volume 00, Number 00 June 2015
