Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Abstract

Ni2؉ -binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni 2؉ -binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni 2؉ -binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.Bacterial surface display of heterologous proteins has in recent years become an increasingly active research area with a wide range of applications in immunology, vaccinology, and biotechnology (11, 49). An interesting application area is the display of metal-binding proteins on bacteria to create wholecell tools for improved sequestration of toxic metals in wastewater (3), a field in which naturally occurring bacteria have been evaluated previously (10, 31). The heterologous expression of peptides and proteins with inherent metal-binding capacity have been employed to create bacteria with improved metalloadsorption properties (39), and surface display has been used as an attractive strategy in this context (22,38,47,51).Short metal-binding peptides, such as hexahistidyl peptides, have been introduced into bacterial surface proteins in order to create bacteria with improved metal-binding capacity (21,46). Using this strategy, both Escherichia coli (46) and Staphylococcus carnosus (43) strains with increased ability to bind Ni 2ϩ and Cd 2ϩ ions have been generated. The introduction of combinatorial protein engineering has also made it possible to select peptides, from large peptide libraries with increased selectivity for certain metals (4,30,37,44), and such peptides might become interesting for surface display applications (44).Gram-positive surface display systems have been suggested to exhibit some advantages compared to gram-negative bacteria (28, 49): (i) the translocation involves only a single membrane, and (ii) gram-positive bacteria have been shown to be more rigid and thus less sensitive to shear forces (19, 36) due to the thick cell wall surrounding the cells, and thus potentially more suitable for field applications such as bioadsorption. For metal adsorption applications, gram-positive bacteria have the additional advantage of having inherent metal-binding capacity due to the thick pe...

Advances in molecular engineering of carbohydrate-binding modules

“…This strategy has been applied to three families of carbohydrate binding domains, specifically CBM1, CBM4, and CBM21 . In every case, the authors used assembly PCR and the introduction of mutations with degenerate codons, such as NNN or NNK, which allow incorporation of any of the four bases or partly randomized bases by restricting the mixture in some positions, for example, NNK ( N = A / C / C / G / T , K = G / T ).…”

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

“…Furthermore, single-chain Fv antibody fragments, as well as IgE-and IgA-specific affibody ligands, have been displayed, creating potential whole-cell diagnostic devices (12,13). In addition, staphylococci with increased metal-binding capacity have been created through display of polyhistidyl peptides (35), and novel metal-binding proteins have been generated through combinatorial protein engineering approaches (19,44).…”

“…Furthermore, single-chain Fv antibody fragments, as well as IgE-and IgA-specific affibody ligands, have been displayed, creating potential whole-cell diagnostic devices (12, 13). In addition, staphylococci with increased metal-binding capacity have been created through display of polyhistidyl peptides (35), and novel metal-binding proteins have been generated through combinatorial protein engineering approaches (19,44 (45). Here, we report the use of a model system employing surface-displayed engineered SpA domains to test the functionality of this S. carnosus display system for the specific enrichment of target cells from a large excess of background cells by flow cytometry.…”

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid‐binding peptide

“…Finally, one could apply the strategies used in this paper to target other typically smaller molecular weight compounds such as mediators, substrates, inhibitors, or pro‐drugs to active molecules, such as enzymes or receptors, of choice. In effect, several investigators have successfully applied conventional phage display technologies to other small molecules such as fluorophores or rhodopsin (36,37) or even metals such as Ni 2+ (38). Applications of these technologies and use of peptides to other areas such as health care or personal care are currently being addressed (39–42).…”