Perspectives on microbial cell surface display in bioremediation

Available online at www.sciencedirect.com

Biotechnology Advances 26 (2008) 151 – 161 www.elsevier.com/locate/biotechadv

Research review paper

Perspectives on microbial cell surface display in bioremediation M. Saleem a,c,⁎, H. Brim b,⁎, S. Hussain c , M. Arshad c , M.B. Leigh d , Zia-ul-hassan c a

UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoser Str. 15, D-04318 Leipzig, Germany b Department of Microbiology and Cancer Center, Howard University, 2041 Georgia Avenue N.W., Washington, DC 20060, USA c Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad-38040, Pakistan d Institute of Arctic Biology, University of Alaska, Fairbanks, Fairbanks, AK 99775, USA Received 13 July 2007; received in revised form 4 October 2007; accepted 18 October 2007 Available online 7 November 2007

Abstract The display of heterologous proteins on the microbial cell surface by means of recombinant DNA biotechnologies has emerged as a novel approach for bioremediation of contaminated sites. Both bacteria and yeasts have been investigated for this purpose. Cell surface expression of specific proteins allows the engineered microorganisms to transport, bio-accumulate and/or detoxify heavy metals as well as to degrade xenobiotics. These otherwise would not be taken up and transformed by the microbial cell. This review focuses on the application of cell surface displays for the enhanced bio-accumulation of heavy metals by metal binding proteins. It also reviews the biodegradation of xenobiotics by enzymes/proteins expressed on microbial cell surfaces. © 2007 Elsevier Inc. All rights reserved. Keywords: Heterologous proteins; Cell surface display; Bacteria; Yeast; Bioremediation

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell surface display in Gram negative bacteria . . . . . . . . . . . . . . . . . 2.1. Enhanced bio-accumulation of heavy metals in Gram negative bacteria 2.2. Enhanced biodegradation of xenobiotics in Gram negative bacteria. . . 2.3. Rhizoremediation of heavy metals and xenobiotics . . . . . . . . . . . 3. Cell surface display in Gram positive bacteria . . . . . . . . . . . . . . . . . 3.1. Environmental applications of bioengineered Gram positive bacteria . . 4. Cell surface display in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Enhanced bio-accumulation of heavy metals in yeast . . . . . . . . . . 4.2. Enhanced biodegradation of xenobiotics in yeast . . . . . . . . . . . . 5. Biosensors based on microbial cell surface display. . . . . . . . . . . . . . . 6. Conclusions and future thrusts . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ⁎ Corresponding authors. Saleem is to be contacted at UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoser Str. 15, D-04318 Leipzig, Germany. Brim, Department of Microbiology and Cancer Center, Howard University, 2041 Georgia Avenue N.W., Washington, DC 20060, USA. Tel.: +1 202 806 4198 (Office), +1 202 806 7025 (Lab). E-mail addresses: [email protected] (M. Saleem), [email protected] (H. Brim). 0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.10.002

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1. Introduction Environmental pollution caused by inorganic and organic contaminants has steadily increased in parallel with world

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population, industrialization, and urbanization, especially in developing countries (Hettige et al., 1996). The overall damage to biodiversity, public health and the ecosystem caused by organic and inorganic contaminants has motivated scientists to develop strategies for their sequestration and removal from the contaminated bio-spheres. The application of microbial technology for the removal of xenobiotics from contaminated environments and biotransformation of heavy metals has received much attention (Siddique et al., 2005; Hussain et al., 2007). Over the last few decades, a tremendous change in bioremediation strategies has occurred due to the advent of novel biotechnological approaches for enhanced removal of these contaminants from contaminated sites. The genetic engineering of microbes expressing specific proteins for biosorption of inorganic contaminants and for biodegradation of xenobiotics, has emerged as a novel area in bioremediation research. Expression of metal binding peptides in genetically modified microbes has however, encountered some problems, such as minimal recycling of bio-adsorbents due to the slow release of accumulated metals, and the interference of cysteine rich proteins with the redox pathways in microbial cells (Bardwell, 1994; Gadd and White, 1993). Similarly, the low uptake of persistent xenobiotics (e.g. persistent organophosphate compounds) by microbial cells has led to decreased contact between enzymes and these compounds, thus preventing their removal from the contaminated sites (Dumas et al., 1989; Chen and Georgiou, 2002). Such compounds are primarily degraded by microbial organophosphorus hydrolase (OPH), which cleaves the P–O, P–F and/or P–S bonds of these pesticides (Ang et al., 2005). Unfortunately, OPH effectiveness varies greatly depending on the particular organophosphate substrate. For instance, paraoxon is the preferred substrate for hydrolase but other organophosphate pesticides like methyl parathion; chlorpyrifos and diazinon are hydrolyzed 30–1000 times slower than paraoxon (Dumas et al., 1989). The main reason for this inefficiency of degradation is low uptake of substrates by the microbial cells themselves (Dumas et al., 1989; Chen and Georgiou, 2002; Ang et al., 2005). Cell surface display permits the expression of functional proteins on the cell surface of microbes which endows intact cells with new functionalities that have a vast sphere of new applications (Ueda and Tanaka, 2000a,b). Therefore, cell surface engineering has now emerged as a novel biotechnology with potential applications in the development of live-vaccines (Zhu et al., 2006), in the biological control of pathogens (Wu et al., 2006a), in biocatalyst development (Narita et al., 2006) and for peptide library screening (Dong et al., 2006). Also recently the cell surface display of heterologous proteins responsible for enhanced bio-accumulation of inorganic contaminants and biodegradation of xenobiotics has received much attention (Dong et al., 2006; Narita et al., 2006). In this review, we describe the cell surface display of heterologous proteins in different microbial groups such as Gram negative and Gram positive bacteria as well as in yeast. We then review the ability of such proteins to act as bioadsorbents and biocatalysts. Finally, recent trends in this novel area of bioremediation research are discussed.

2. Cell surface display in Gram negative bacteria Among Gram negative bacteria, Escherichia coli has been extensively investigated in cell surface engineering. E. coli is considered an attractive Gram negative bacterium because of the availability of various genetic tools and mutant strains and the high transformation efficiency for screening of a large peptide or protein library after surface display (Lee et al., 2003). Different strategies have been described to display heterologous proteins onto the surface of E. coli. These include the insertion of the target sequences into the surface exposed loops of outer membrane proteins; insertion of target sequences into a protein forming part of a cell surface structure such as a flagellum; or the fusion of target sequences to the N-terminus of lipoproteins (Hofnung, 1991; Little et al., 1993). In general, proteins need to cross two bacterial membranes to reach the extracellular milieu, and host bacteria resort to different secretion systems to target proteins to their surface. Bacterial proteins can be exported across the inner membrane utilizing either the twin arginine translocation (tat) pathway, or the general secretion (sec) pathway (de Keyzer et al., 2003; Palmer et al., 2005). Afterwards, the integral outer membrane proteins (OMP) are inserted from periplasm by the specialized machinery which consists of some envelope proteins (Genevrois et al., 2003; Wu et al., 2005). Since the use of LamB, OmpA and PhoE in peptide engineering, many novel proteins like FhuA, intimin, OmpC, TraT etc. have also been introduced for surface display in Gram negative bacteria (for review, Lee et al., 2003; Li et al., 2004; Rutherford and Mourez, 2006). The further detail regarding the cell surface display of contaminant-specific proteins is provided below (Section 3: Gram positive bacteria; Section 4: Yeast). 2.1. Enhanced bio-accumulation of heavy metals in Gram negative bacteria The technology for cell surface display and expression of metal binding peptides for the purpose of enhanced bioaccumulation of heavy metals came into existence to address some shortcomings in pre-existing approaches as described in the introductory section. To date, various kinds of metal binding proteins such as Glutathione (GSH), GSH-related phytochelatins (PCs), cysteine-rich metallothioneins (MTs) and synthetic phytochelatins (ECn) have been used to enhance the bio-accumulation of heavy metals. Kotrba et al. (1999) studied the metal binding properties of E. coli strains displaying short peptides as a fusion to the LamB protein. Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (namely HP) and Gly-Cys-Gly-CysPro-Cys-Gly-Cys-Gly (namely CP) were genetically engineered into LamB protein and expressed in E. coli. The potential of an E. coli expressing CP to bind cadmium (Cd2+ ) from the growth medium was increased fourfold compared to wild-type cells. However, HP display did not contribute to the accumulation of Cu2+ and Zn2+ from the growth medium. Bae et al. (2000) used synthetic phytochelatins for enhanced bio-accumulation of Cd2+ from growth medium. For this purpose, synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)n Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20))

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were linked to an lpp-ompA fusion gene and displayed onto the surface of E. coli. They compared it by displaying EC20 periplasmically as a fusion with the maltose-binding protein (MBP-EC20). The results revealed 18 mol of Cd 2+ mg− 1 dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of up to 60 mol of Cd2+ mg− 1 dry cells. Moreover, E. coli displaying EC20 onto cell surface accumulated twice the amount of Cd2+ as compared to cells expressing EC20 periplasmically. Later, Valls et al. (2000) provided the first evaluation of the potential for Ralstonia eutropha displaying metallothionein (MT) to accumulate heavy metals from the Cd2+ -polluted soil. In this study, DNA sequences encoding MT were fused to the autotransporter beta-domain of the IgA protease of Neisseria gonorrhoeae, which led the hybrid protein toward the bacterial outer membrane. The genetically engineered strain R. eutropha MTB accumulated increased amounts of Cd2+ from liquid media. Interestingly, the inoculation of R. eutropha MTB to Cd2+ contaminated soil significantly decreased the toxicity of this metal to the test organism, tobacco plants (Nicotiana bentamiana). Similarly Bae et al. (2001) described the bio-accumulation of Hg2+ from liquid medium by genetically modified Escherichia coli co-expressing a Hg2+ transport system with (Glu-Cys)20Gly (EC20) or by directly expressing EC20 on the cell surface. Kjaergaard et al. (2001) reported the identification of novel Zn2+binding peptides selected from a FimH-displayed random peptide library on the basis of some previous evidence (Schembri and Klemm, 1998; Schembri et al., 1999). In all these studies, which mostly involve cysteine-rich peptides, a major problem was the lack of specificity, which created a great deal of difficulty in the specific recovery of heavy metals like mercury (Bontidean et al., 1998). Interestingly some bacteria that are mercury resistant possess a mercury operon that is composed of a regulatory gene: MerR and other genes involved in the transport and detoxification of mercury ions within the cell. The MerR proteins have three domains: (1) an N-terminal DNA binding domain, (2) a C-terminal Hg2+ binding domain and (3) an intervening region of undefined function. Its specific affinity for Hg2+ is due to the unique trigonal thiolate coordination of Hg2+ in its metal binding centres (Wright et al., 1998). Bae et al. (2003) introduced this strategy for selective removal of mercury from liquid culture by genetically modified E. coli displaying MerR. The cell surface display of MerR enabled E. coli to accumulate Hg2+ at levels six-fold-higher than wild-type JM109 cells. The binding of Hg2+ by MerR was highly specific, with no decline even in the presence of 100-fold excess of Cd2+ and Zn2+. Interestingly, this binding was also insensitive to pH and metal chelators. Recently, Qin et al. (2006) evaluated the physiological and biochemical properties of a single polypeptide metal-binding domain (MBD) expressed either onto the cell surface or in the cytosol. This allowed better understanding of the environments in which specific metal binding occurs. They fused MBD into the AraC-controlled Lpp-OmpA fusion protein for cell surface expression and examined its ability to bind metals and protect cells from Hg2+ exposure even when secreted into the oxidized environment of the outer cell surface. More than 20,000 surface copies of MBD were expressed per E. coli cell, with metal stoichiometries of about 1.0 Hg2+ per MBD monomer. The E. coli

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cell displaying MBD on their surface accumulated not only more than 6-fold more Hg2+ from liquid culture but also recovered Hg2+ more quickly than those cells which did not express MBD (Fig. 1). Interestingly, a high specificity of the displayed protein (MBD) for Hg2+ was observed even in the presence of higher concentrations of Zn2+ and Cd2+ (a 22-fold molar excess). More recently, metal-binding peptides/proteins have been selected from random libraries constructed through cell surface display systems and are being applied to the detoxification of contaminants (Wernerus et al., 2001; Kjaergaard et al., 2001). Dong et al. (2006) constructed and screened two secondary libraries of previously isolated sequences against nickel to derive a list of peptides with the greatest affinity for nickel, (Lu et al., 1995). Through screening, additional Ni-chelating peptides were identified. They suggested that not only histidine, but also arginine is involved in Ni-binding. They also screened two particular clones (1035 and 2022) whose binding affinity was approximately nine times that of the original library derived clones. The free nickel ions completely inhibited the binding of the clones 1035 and 2022 to immobilized nickel, suggesting that the peptides were able to chelate nickel ions. On the basis of these studies, it appears that existing peptide libraries could lead to very promising results in terms of the development of metal bioadsorbents. From these diverse approaches to the general problem of bioengineering the expression of metal binding proteins, it may be concluded that further research is crucial for development of efficient bioadsorbents for sequestration of inorganic contaminants from contaminated environments. However, most of the studies lack information regarding the function of these bioadsorbents under the influence of various real-world environmental conditions, the setting which is most crucial for the development of successful bioremediation strategies. 2.2. Enhanced biodegradation of xenobiotics in Gram negative bacteria The most persistent and toxic group of pesticides, the organophosphate (OP) pesticides, is widely employed in contemporary agriculture. The most widely used organophosphates are chlorpyrifos, paraoxon, parathion, disulfoton, dimeton and carbophenothion. The microbial cell surface-display of peptides/enzymes (e.g., hydrolases) has proven to be a useful approach for in vitro detoxification of these compounds. Richins et al. (1997) successfully developed a method to display the enzyme organophosphorus hydrolase (OPH) onto the surface of E. coli. In this system, more than 80% of the hydrolase activity was displayed onto the bacterial cell surface. The cells displaying OPH removed parathion and paraoxon at seven fold higher rates than cells with similar levels of intracellular OPH. These authors suggested that immobilization of these cells on solid supports could be a useful strategy for detoxification of pesticides in the place of immobilized enzymes. Immobilization would also afford a reduced diffusional barrier. Further, Kaneva et al. (1998) investigated different factors influencing organophosphate biodegradation in liquid culture by the cell surface expressed enzyme, hydrolase. They reported that the display of active OPH onto the cell surface is highly host-specific and dependent on growth conditions.

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Fig. 1. Recovery of E. coli cells expressing MBD after Hg2+ exposure. U (open symbols), uninduced; I (filled symbols), induced for MBD expression; the numbers following U- and I- indicate the concentration of Hg2+ (mM) used for challenge, with permission (Qin et al., 2006).

Supplementing growth medium with cobalt chloride enhanced OPH activity due to the active role of cobalt chloride in the formation of the metal active center, and the timing of cobalt addition influenced parathion biodegradation. There are several reports which depict the potential of transformed microbial species that display degradative genes/enzymes on their cell surfaces to accelerate the biodegradation of organophosphate and other pesticides (Table 1). The strategy is not only highly conducive to the biodegradation of organophosphate pesticides, but also could be employed for biodegradation of other classes of xenobiotics in soil and water environments. Recently, the P450 proteins responsible for xenobiotic metabolism in pigs (Sus scrofa), humans (Homo sapiens) and other organisms have been characterized, and their metabolism of pesticides has been studied in different genetically engineered plants species (Kawahigashi et al., 2005; Bode et al., 2006). A novel study was reported by Yim et al. (2006) where diflavin-containing mammalian NADPH-cytochrome P450 oxidoreductase was displayed onto the cell surface of E. coli by using ice-nucleation protein from P. syringae. This study has the potential to allow the selection and development of oxidoreductases encompassing bulky and complex prosthetic groups of FAD and FMN. This may permit their development into whole-cell biocatalysts useful for the removal of other classes of xenobiotics such as polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) etc. However, in most situa-

tions, it is typically the case that a variety of contaminants exist at a single site requiring decontamination. This requires the bioremediating organism to be multipotent and able to deal with a variety of different contaminants at the same time. Recently, Lan et al. (2006) described a novel strategy using a co-expression vector (pETDuet) for construction of bacteria expressing the organophosphate hydrolase gene (opd) from Flavobacterium sp. and the carboxylesterase B1 gene (b1) from Culex pipiens. The genetically modified bacterium was capable of producing both enzymes for degradation of organophosphorus, carbamate and pyrethroid classes of pesticides. This finding strongly supports the evidence that bacteria displaying more than one enzyme could be useful in detoxification of a variety of xenobiotics that may occur simultaneously in a contaminated environment. 2.3. Rhizoremediation of heavy metals and xenobiotics In the previous sections, the bio-accumulation of heavy metals and biodegradation of organophosphorus compounds by genetically engineered Gram negative bacteria have been described. But there is a potential problem in the practical application of this approach as the survival and functioning of such recombinant Gram negative bacterial strains may be limited under natural environmental conditions. Under these circumstances, the use of rhizobacteria exhibiting engineered cell surface displays might be

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Table 1 Cell surface display of enzymes in recombinant strains for the detoxification of xenobiotics Strain

Fusion system

Verification of fusion system

Comments

Reference

Escherichia coli

Truncated ice nucleation protein

–

Cho et al., 2002

Escherichia coli

Ice nucleation protein (INPNC)

Immunoblotting

Escherichia coli

Ice nucleation protein (INP).

SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy, proteinase accessibility and cell fractionation.

Hydrolysis of methyl parathion occurred 25-fold faster than does the wild type in liquid culture. Biodegradation of paraoxon in liquid culture occurred at rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. More than 90% of the malathion was degraded in batch degradation experiments after duration of about 4 h.

Moraxella sp.

Ice nucleation protein (INPNC)

Shimazu et al., 2001a

Pseudomonas putida KT2440

Ice-nucleation protein anchor

Pseudomonas putida JS444

Ice-nucleation protein (INP) from Pseudomonas syringae

Western blotting,cell fractionation, and immunofluorescence microscopy

Pseudomonas syringae

Ice nucleation protein from Pseudomonas syringae INA5 Ice-nucleation protein (INP) anchoring motif from Pseudomonas syrinage. Truncated OprF, an outer membrane protein of Pseudomonas aeruginosa

Protease accessibility, immunofluorescence microscopy and cell fractionation Flow cytometry, protease accessibility and whole-cell enzyme activity

Hydrolysis of methyl parathion, parathion, and paraoxon in liquid culture occurred at rate of 0.6 μmol/h/mg dry weights, 1.5 μmol/h/mg dry weights, and 9.0 μmol/h/mg dry weights respectively. The whole cell activity increased up to 10 times higher which could be helpful in accelerated biodegradation. Hydrolysis of paraoxon, parathion, and methyl parathion in liquid culture occurred at rate of 7.90, 3.54, and /h/mg dry weights. Most of the OPH activity was located onto the cell surface and retained almost 100% over a period of 3 weeks. A whole cell presenting lipase reactions appeared to be useful for bioconversions of organic solvents and thus could also be used for detoxification of xenobiotics. The whole-cell lipase activity was greater than 90% and could be used for detoxification of xenobiotics.

Pseudomonas putida GM730

Pseudomonas putida KT2442

–

–

Western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity

effective when inoculated with hyperaccumulator plants for phytoremediation purposes. Lee et al. (2006) displayed EC20 onto the cell surface of Pseudomonas strain Pb2-1 and Rhizobium strain 10320D. The cells expressing EC20 demonstrated sixfold higher cadmium bio-accumulation than native bacteria in the presence of 16 mM CdCl2. The biodegradation of trichloroethylene (TCE) was decreased in the presence of cadmium for cells without EC20 expression. Interestingly, however, the expression of EC20 restored the biodegradation rate of TCE. The results revealed that EC20 expression not only enhanced the bioaccumulation of cadmium but also decreased the negative effect of cadmium on TCE degradation. They concluded that it is very likely that these engineered rhizobacteria could be used for inoculation of plant roots. Wu et al. (2006b) expressed a metalbinding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, which not only enhanced cadmium binding but also eliminated the cellular toxicity of cadmium. Interestingly, the inoculation of sunflower roots with the engineered rhizobacterium caused a marked decrease in cadmium phytotoxicity and increased the cadmium accumulation in the plant root up to 40%. They concluded that the use of EC20-expressing P. putida endowed with organic-degrading capabilities could be a promising strategy for bioremediation of mixed organic-metal-contaminated sites. Moreover, the rhizobacterial enzyme ACC deaminase is known to

Wang et al., 2002

Zhang et al., 2004

Shimazu et al., 2003 Lei et al., 2005a

Shimazu et al., 2001b Jung et al., 2006

Lee et al., 2005

enhance the rhizoremediation/phytoremediation of both inorganic and organic contaminants (Arshad et al., 2007). The genetic engineering of ACC deaminase rhizobacteria displaying contaminant-specific heterologous proteins onto their cell surfaces may therefore yield promising results in terms of enhanced phytoremediation/rhizoremediation of contaminated sites. 3. Cell surface display in Gram positive bacteria The cell surface display in Gram-positive bacteria enjoys some advantages over Gram-negative bacteria. In particular, the translocation of peptides involves only a single membrane, and the thick and rigid cell wall is relatively resistant to shear forces. Such factors make these bacteria suitable for a variety of biotechnological applications (Lee et al., 2003; Narita et al., 2006). Like E. coli among Gram negative bacteria, Staphylococcus xylosus and Staphylococcus carnosus have been investigated in this regard. The peptide engineering in Gram positive bacteria has been pursued by taking advantage of the anchoring mechanism of S. auresus protein A (SpA) (Schneewind et al., 1995). Recently, Narita et al. (2006) introduced a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. Here, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion

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protein was expressed on the cell surface. In another study, Bosma et al. (2006) developed a novel surface display system for proteins on non-genetically modified Gram-positive bacteria. This system is comprised of non-genetically modified and non-living Gram-positive bacterial cells which are designated Gram-positive enhancer matrix (GEM) particles. These particles act as substrates for binding externally added heterologous proteins by means of a high-affinity binding domain derived from the Lactococcus lactis peptidoglycan hydrolase AcmA (Lee et al., 2003; Desvaux et al., 2006; Marraffini et al., 2006). There are, however, relatively few existing studies of the cell display of contaminant-specific proteins onto Gram-positive bacteria. These are described in next section. 3.1. Environmental applications of bioengineered Gram positive bacteria To test the possibility of displaying different metal binding proteins on Gram positive bacteria for the purpose of the bioaccumulation of heavy metals, Samuelson et al. (2000) displayed two different polyhistidyl peptides, His3-Glu-His3 and His6 on bacterial cells for the first time. They constructed two novel S. xylosus and S. carnosus strains with surface-exposed chimeric proteins, for example polyhistidyl peptides for binding of the divalent metal ions. The chimeric surface proteins were found to be functional in binding of metals like Ni2+ and Cd2+. For the last few years, the fungal cellulose-binding domain (CBD), derived from T. reesei cellulase Cel6A, has been expressed in its non-engineered form onto the surface of S. carnosus (Lehtio et al., 2001). The cell surface expression of the CBD scaffold could prove helpful for enhanced bio-accumulation of heavy metals. Wernerus et al. (2001) constructed Ni2+-binding staphylococci expressing CBD derived from Trichoderma reesei cellulase. The engineered strains expressing CBD were shown to

be highly efficient in bio-accumulation of Ni2+ (Fig. 2). Currently, however, there are relatively few published studies examining the application of Gram positive bacteria in bioremediation but in future this strategy may receive more attention due to greater suitability of these bacteria for cell surface engineering. 4. Cell surface display in yeast Yeast, being a eukaryote, is generally regarded as safe (GRAS) in its applications in different fields. It bears a number of attractive features as a protein engineering platform (Boder and Wittrup, 2000). The cell surface engineering in yeast has been demonstrated in bakers' yeast Saccharomyces cerevisiae. The S. cerevisiae is highly advantageous host for cell surface display as it may allow the folding and glycosylation of expressed heterologous eukaryotic proteins. Moreover it may also be subjected to many other genetic manipulations. In addition to all these, its rigid structure is highly conducive for surface engineering of peptides (Kondo and Ueda, 2004). That is why novel strains are being constructed equipped with a variety of functional proteins like antibodies, enzymes and combinatorial protein libraries (Boder and Wittrup, 1997; Breinig et al., 2006; Parthasarathy et al., 2006; Furukawa et al., 2006). For cell surface display, the cell wall of S. cerevisiae has two kinds of mannoproteins: sodium dodecyl sulfate (SDS)-extractable and glucanase-extractable mannoproteins. These proteins are mostly rich in serine and/or threonine, and contain a putative glycosyl phosphatidylinositol (GPI) attachment signal at the Ctermini (Van Der Vaart et al., 1997). An addition of a GPI anchor to C-termini is needed for the covalent association of these proteins with the cell wall. The foreign peptide to be expressed is fused to a mannoprotein and is mostly carried and anchored covalently onto the cell surface. Mostly the cell surface display in yeast is GPI anchor-dependent (Lee et al., 2003). Currently the

Fig. 2. Histogram representation of results from the whole-cell Ni2+-binding assay. Wild-type (wt) and recombinant staphylococci were incubated with a nickelchelated alkaline phosphatase conjugate. After addition of substrate, the color shift was monitored at 405 nm. Sc:wt (bar 1), Sc:ABP (bar 2), Sc:CBD1 (bar 3), Sc:CBD2 (bar 4), Sc:CBD3 (bar 5), Sc:CBD4 (bar 6), Sc:CBD5 (bar 7), Sc:CBD6 (bar 8), Sc:CBD7 (bar 9), Sc:CBD8 (bar 10). Error bars show standard deviation, with permission (Wernerus et al., 2001).

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principle technique for the cell surface engineering of yeasts mainly includes agglutinin and flocculin systems (Kondo and Ueda, 2004). Further detail about display of heterologous proteins on yeast cells with respect to their potential in biodegradation of contaminants is discussed in Section 4.2. 4.1. Enhanced bio-accumulation of heavy metals in yeast For accumulation of heavy metals, peptide engineering in S. cerevisiae involves the surface display of histidine oligopeptide, hexa-His. This is a known chelator of divalent heavy metal ions like Ni, Cu, Cd and Zn. Kuroda et al. (2001) expressed histidine oligopeptide (hexa-His) on the yeast cell surface for the enhanced bio-accumulation of heavy metal ions. The genetically modified yeast accumulated three to eight times more copper ions than the parent strain and was more resistant to copper (4 mM) than the parent (b1 mM at pH 7.8). They recovered about a half of the copper ions adsorbed by whole cells with EDTA treatment without disintegrating the cells. However there were some problems with utilizing this approach: for example difficulties in separation of bioadsorbents from contaminated media and relatively little recycling of heavy metals. Moreover, in the remediation of these issues, the centrifugation of the cultures may be problematic in terms of cost of instruments (Kondo and Ueda, 2004). On the basis of knowledge about the GTS1 gene responsible for aggregation of yeast cells, the aggregation of cells has been considered as a possible alternative, since it is an expensive and straightforward procedure (Yaguchi et al., 2000), Kuroda et al. (2002) engineered a novel S. cerevisiae strain expressing hexa-His with the ability to self-aggregate in response to heavy metals with the help of a fusion gene for the expression of GTS1, which encodes a putative zinc-finger transcription factor responsible for cell-aggregation, under the control of the copper ioninducible CUP1 promoter from the yeast metallothionein gene. The genetically modified strain aggregated in the medium only in the presence of copper ion without interfering with the copper ion-adsorbing function of the engineered yeast, indicating twin features of engineered strains. Like histidine oligopeptides, metallothioneins (cysteine-rich proteins) may play a major role in bio-accumulation and detoxification of heavy-metals like copper, cadmium, zinc, silver, and mercury (Amiard et al., 2006). The S. cerevisiae metallothionein (YMT) contains 12 cysteines which are highly likely to bind up to eight Cu1+ ions and four Zn2+ ions (Winge et al., 1985). Kuroda and Ueda (2003) expressed cysteine-rich and an active YMT on the yeastcell surface for bioremediation of Cd2+ from contaminated medium. The genetically engineered cells with YMT cell surface expression demonstrated superior cell-surface adsorption and recovery of Cd2+ under EDTA treatment than the yeast cells expressing hexa-His-displaying cells. Interestingly, in this study they reported that unlike hexa-His-displaying cells, cells displaying YMT and hexa-His were resistant to Cd2+ exposure, mainly due to active and enhanced adsorption of toxic Cd2+. The genetically modified strain showed a greater potential for the bio-accumulation of Cd2+ than E. coli cells displaying these molecules (Sousa et al., 1998). In another study, Kuroda and

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Ueda (2006) attempted to increase the metal-binding capacity of a displayed protein by adding tandem repeats of a metal-binding protein. The adsorption and recovery of Cd2+ on the cell surface was enhanced substantially with increasing the number of tandem repeats. They reported that cells displaying four repeats of YMT demonstrated a 5.9-fold higher adsorption and recovery while the cells displaying eight repeats of YMT demonstrated a 8.7-fold higher adsorption and recovery than the control cells displaying only one repeat. But they did not determine the maximum number of repeats where the adsorption and recovery efficiency is maximized. All the Cd2+-binding sites in the YMT tandem repeats were thought to be completely occupied. Moreover, the growth rate of yeast in Cd2+-containing liquid medium was found to be dependent on the number of displayed tandem repeats of YMT. They suggested that the characteristics of surface engineered yeasts as a bioadsorbent were dependent on the ability of the displayed proteins to bind metal ions while the adsorption of heavy metal ions on the cell surface plays a central role in the ability of the cells to resist the toxic effects of metal ions. 4.2. Enhanced biodegradation of xenobiotics in yeast As described in a previous section, both Lpp-OmpA and INPNC anchors have been used to express the OPH gene on surface of E. coli for enhanced biodegradation of OPC. Keeping in mind the potential of S. cerevisiae, Takayama et al. (2006) recently reported the construction of S. cerevisiae cells expressing the OPH gene using the glycosylphosphatidylinositol (GPI) anchor system, in which the secretion signal sequence of α-agglutinin and a GPI anchor attachment signal sequence have been genetically fused, respectively, to the N- and C-terminal regions of the target protein (Ueda and Tanaka, 2000a). To accomplish this task, the gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed onto the cell surface of S. cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH (Fig. 3). The genetically modified strain S. cerevisiae MT8-1 exhibited a greater potential for the OPHdisplaying system than E. coli (Takayama et al., 2006). Recently, Breinig et al. (2006) demonstrated bacterial esterase cell surface display in yeast, which was remarkably more effective than esterase surface display in E. coli. These findings strongly highlight the applications of yeast cell surface display for enhanced removal of xenobiotics. Likewise in another study, Matsui et al. (2006) characterized several genes of S. cerevisiae responsible for inducing higher tolerance to the hydrophobic organic-solvents. From this study, it is very likely that these genes could be expressed on cell surfaces of yeast, enabling them to thrive at higher concentrations of xenobiotics. This could potentially be helpful for the accelerated removal of organic contaminants. 5. Biosensors based on microbial cell surface display The use of microbial biosensors has emerged as a novel approach for rapid as well as accurate determination of

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Fig. 3. Plasmid pMWOPH constructed for display of OPH on yeast cell surface. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. The FLAG-encoding gene was fused for the detection of successful display of OPH on the yeast cell surface, with permission (Takayama et al., 2006).

xenobiotics in environmental samples. Microbial hydrolases are involved in the hydrolysis of a wide range of organophosphate compounds. This hydrolysis produces an acid and an alcohol that can be detected directly. This approach has been employed for the development of biosensors based on microbial cell surface display technology, for example, for determination of organophosphate nerve agents and other xenobiotics. To date, numerous examples of applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors exist in the literature (Lei et al., 2004, 2005b). Recently Lei et al. (2006) developed the first microbial biosensor for

rapid and cost-effective determination of organophosphorus pesticides fenitrothion and ethyl p-nitrophenyl thionobenzenephosphonate (EPN). This biosensor was composed of a transformed p-nitrophenol (PNP)-degrading P. putida JS444 displaying the OPH biological sensing element and a dissolved oxygen electrode as the transducer. The detection limits for this biosensor were 277 ppb of fenitrothion and 1.6 ppm of EPN under optimum operating conditions even in the presence of other pesticides like carbamate, triazine herbicides organophosphate and phenolic compounds. Mulchandani et al. (2006) developed a microbial biosensor consisting of a dissolved

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oxygen electrode modified with recombinant PNP-degrader Moraxella sp. displaying OPH for sensitive, selective, rapid and direct determination of PNP-substituted compounds. The biosensor was capable of measuring paraoxon at levels as low as 0.1 M (27.5 ppb) and under optimum conditions, demonstrated excellent selectivity against triazines, carbamates and organophosphates without PNP-substitutents. The biosensor showed stability for a week when stored at 4 °C. In another study, Lei et al. (2007) developed a whole cell-based amperometric biosensor comprised of P. putida JS444 displaying OPH on its cell surface as biological sensing element and a carbon paste electrode as the amperometric transducer for highly selective, sensitive, rapid, and cost-effective determination of fenitrothion and EPN. They reported that operating at optimum conditions, 0.086 mg dry weight of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citratephosphate buffer, pH 7.5, with 50 μM CoCl2 at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN without interfering from phenolic compounds, carbamate pesticides, triazine. The development of biosensors capable of working under a wide range of environmental conditions is imperative and there exists a critical need for further research in this area of great potential biotechnological applications. 6. Conclusions and future thrusts Although the biotechnology for the display of heterologous proteins for novel biocatalyst and bioadsorbents on microbial cell surfaces is far better developed than conventional approaches, the majority of studies to date are in-vitro laboratory-based ones. Also, even under laboratory conditions, their efficacy of the developed systems has not been evaluated under a variety of environmental (biotic and abiotic) parameters. This is a necessary step in bringing this biotechnology to real-world environmental applications. To date, the available studies have addressed some important specific group of organic contaminants (organophosphate pesticides) and selected heavy metals but systems to address many other groups of key organic and inorganic environmental contaminants are yet to be developed. In addition, the expression of such proteins onto cell surface is limited to some specific bacteria and yeast. However, the expression of these proteins onto the surface of environmentally relevant bacteria such as Fe (III)- and sulfate-reducing bacteria and methanogens for completion of carbon cycling from the contaminant could potentially be a useful strategy for the cleaning up of contaminated terrestrial sites. Apart from all these considerations, a number of regulatory barriers exist that can block the release of genetically engineered microorganisms (GEM) in the environment. Based on these facts, it is clear that further intensive laboratory and field research are needed to address these shortcomings and to explore the unexploited aspects of microbial cell display for remediation of contaminated terrestrial sites. Acknowledgements We thank Dr. Charles F. Dillon and an anonymous reviewer for the many useful comments, technical input and editing of the manuscript.

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