Bacterial surface display of metal binding peptides as whole-cell biocatalysts for 4-nitroaniline reduction

Abstract

This study employed cells that express metal binding peptides on the surface for “green” synthesis of gold and platinum nanoparticles (NPs). We began by testing several surface display systems to investigate the expression of heterologous protein/peptides in Escherichia coli and Ralstonia eutropha. Cells with gold-binding-peptides on their surfaces were used to perform a quantitative comparison of labeling efficiency using gold NPs. Next, cells expressing metal binding peptides were used to simultaneously synthesize and display the metal NPs on the cell surfaces. Our results demonstrated the possibility of using microorganisms as whole-cell biocatalysts in the production and displaying of gold and platinum NPs for the reduction of 4-nitroaniline.

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Metallic nanomaterials are widely used in nanotechnology and biotechnology applications due to their favorable catalytic, electronic, and optical properties.¹ Various chemical strategies have been developed to prepare nanomaterials within a narrow size distribution;² however, developing an efficient green chemistry approach for the synthesis of nanomaterials remains a daunting challenge.^3,4

Biomolecules such as DNA,⁵ peptides,⁶ and proteins^7,8 have often been utilized as building blocks/templates in the synthesis of nanomaterials. For example, metallothioneins, cysteine-rich metal-binding proteins, have been employed to synthesize metal NPs, wherein the size of the NPs was regulated by adjusting the concentration of the metal ions in the medium.⁷

Microbial cell-surface display systems allow target protein/peptides to be displayed on the surface of microbes through fusion with a carrier protein.^9–11 Various carrier systems have been developed to anchor foreign proteins/peptides onto cell surfaces for bioremediation, biocatalysis, and biosensing.^12–14 For example, synthetic phytochelatin and metallothionein was previously displayed for metal ion absorption.^12,15 Several enzymes were used for the whole-cell biocatalysts, including lipase, glucosidase, and endoglucanase through cell-surface engineering.^16,17 Surface-anchoring proteins can be divided into two major types: outer membrane proteins (OmpA/T/C, LamB, and FhuA)^18,19 and autotransporter secretion proteins (IgA, EstA, and EhaA).^15,20,21 Here, cells expressing metal binding peptides were used to selectively and simultaneously recover and display the metal nanoparticles from metal salt solutions as whole-cell biocatalysts.

The Gram-negative bacterium Escherichia coli is most frequently employed as a host organism in the study of microbial cell-surface display. However, the performance of aforementioned anchoring proteins in other bacteria has yet to be characterized. In this study, we evaluated display efficiency for a variety of surface-anchoring proteins in Ralstonia eutropha H16, a biotechnologically well-defined soil bacterium.

A number of proteins have been shown previously in the ability to translocate fusion protein/peptides to their outer membrane, including FhuA (an outer membrane receptor for ferrichrome-iron),¹⁹ OmpA (a truncated outer membrane protein A, OmpA_1–159),²² Lpp-OmpA (chimeric protein containing signal sequence of lipoprotein (Lpp), Lpp_1–9, and OmpA_46–159 from E. coli),^23,24 and IgA (the β domain of immunoglobulin A protease from Neisseria gonorrhoeaen).^12,15 In order to compare various surface expression systems in microorganisms, we constructed and transformed these four types of display carriers by fusing them to RFP (monomeric red fluorescence protein) to the N-terminus of IgA and C-terminus of FhuA, OmpA, and ss-Lpp-OmpA_46–159 (Fig. S1, ESI†) in E. coli and R. eutropha. The RFP fusions were driven by the control of the arabinose-inducible araBAD promoter. We then used fluorescence microscopy to examine if these molecules are exported to the outer membrane of E. coli and R. eutropha (Fig. 1) for four types of surface-display-carrier fluorescent fusion compared with RFP only control. E. coli and R. eutropha cells expressing surface-display-carrier-RFP fusion proteins present fluorescent haloes, as an indication of membrane localization (Fig. 1). In contrast, the expression of unfused cytoplasmic RFP was distributed diffusely throughout the cell (Fig. 1).

Fig. 1 Fluorescence microscopy images of E. coli or R. eutropha cells expressing RFP only (signals diffusely throughout the cell), FhuA-RFP, OmpA-RFP, ss-Lpp-OmpA-RFP, and ss′-RFP-IgA (present fluorescent haloes). Scale bar: 2 μm.

Following this, we constructed gold-binding-peptides (GBP containing VSGSSPDS)²⁵ in conjunction with surface-display-carrier-RFP, yielding the constructs shown in Fig. S2.† Localization of RFP–GBP fusions remained unchanged within this pattern, thereby verifying that the GBP was correctly displayed on cell surface of both E. coli and R. eutropha cells (Fig. S3†). Gold NP labeling was then used to (i) verify whether the GBP was properly localized on the external cell surface and (ii) validate the function of GBP to compare the display efficiencies of these systems. For this, citrate-stabilized gold NPs were synthesized²⁶ to test the binding efficiency of various E. coli and R. eutropha strains. The diameter of gold NPs was distributed with an average 13 nm (Fig. S4A†) determined by transmission electron microscopy (TEM) analysis. The UV-visible spectrum of the gold NP solutions was confirmed and shown in Fig. S4B.† The display of gold NPs on strains expressing the GBP was examined. Binding efficiency was evaluated using the exponential-phase of cells and 11.54 nM of gold NPs. The concentration of the gold nanoparticles was determined using an extinction coefficient.²⁷ The number of NPs deposited on the cell surface was visually examined using TEM. Representative TEM images of the cells are presented in Fig. 2A–D. Minimal background labeling was observed in cases without GBP expression (Fig. 2E–H), which suggests that the nonspecific aggregation of gold NPs did not occur under the labeling conditions in this study. The labeling of gold NPs was observed in all of the display systems, and its efficiency increased with arabinose concentrations up to 0.2%. However, we did not observe further increase of labeling at higher concentration of arabinose. Notably, the efficiency mainly depends on the anchor domain. The quantitative analysis of number of gold NPs observed on cells is listed in Table S4.† Our findings revealed that GBP-IgA was the most efficient at binding gold NPs in R. eutropha. Consist with previous studies, autotransporter type of proteins, which have distinct functions in native hosts, act most prominent as anchoring motifs in other Gram-negative bacteria.²⁸ Thus, IgA was used throughout the rest of the study.

Fig. 2 Gold nanoparticle labeling of R. eutropha cells expressing (A) FhuA-GBP, (B) OmpA-GBP, (C) ss-Lpp-OmpA-GBP, (D) ss′-GBP-IgA, (E) FhuA-RFP, (F) OmpA-RFP, (G) ss-Lpp-OmpA-RFP, and (H) ss′-RFP-IgA. Following incubation with 11.54 nM of gold nanoparticles, the cells were washed and analysed by TEM. Scale bar: 1 μm.

Next, we tested the surface display platform with regard to its effects on the recovery of metal ion and display NPs simultaneously. Previous study indicated that E. coli biosynthesized silver NPs via c-type cytochromes efficiently under anaerobic conditions.³ We therefore examined if it was possible to synthesize gold NPs by inducing the expression of GBP-IgA in E. coli or R. eutropha cells followed by adding a final 1 mM Au³⁺ solution for 1 h without other reducing agents under anaerobic condition. Cells were washed with PBS buffer and examined by TEM. The formation of gold NPs was observed (Fig. 3A and B). Fig. S5† illustrates the same field but at a lower magnification of the cells. TEM images revealed that the Au³⁺-treated cells had spherical gold particles deposited predominantly on the surface of the bacterial cells, suggesting that the strong affinity between metal binding peptides and NPs. The UV-visible spectra of the gold NP solutions were examined to confirm the surface plasma resonance peak and shown in Fig. S6.† Size distribution histograms of gold NPs are presented in Fig. S7A and B.† The average diameter of gold NPs was between 11 and 14 nm. To further evaluate the elemental composition of NPs, energy-dispersive X-ray spectroscopy (EDS) was performed (Fig. S8A and B†).

Fig. 3 Cells expressing gold/platinum binding peptides mediated recovery of (A) and (B) gold or (C) and (D) platinum NPs under anaerobic conditions. TEM images of E. coli or R. eutropha cells expressing ss′-GBP-IgA, and ss′-S7-IgA. Scale bar: 200 nm.

We next constructed and transformed platinum facet-specific peptides S7 (SSFPQPN)⁶ – IgA in E. coli or R. eutropha cells in the presence of a final 1 mM of Pt⁴⁺ solution. We again observed platinum NPs. TEM images of typical platinum NPs on cells were shown in Fig. 3C and D. Particle size histograms and EDS analyses are presented in Fig. S7C, D and S8C, D.†

We sought to test the catalytic property of whole-cell biocatalyst for the reduction of 4-nitroaniline to 4-aminoaniline with NaBH₄ as reducing agent at room temperature. Using procedures similar to those outlined above, E. coli or R. eutropha cells (0.78 mg) with display of gold and platinum NPs were washed and resuspended in 4-nitroaniline solution. The reaction was monitored using the UV-vis spectroscopy. The absorption spectra for the reduction of 4-nitroaniline using E. coli-gold NP-biocatalysts at different time points were shown in Fig. 4A. The band at 385 nm of 4-nitroaniline decreases and disappears after 3 min. In addition, new bands at 242 and 307 nm gradually increased, indicating the formation of 4-aminoaniline.

Fig. 4 (A) Representative UV-vis absorption spectra for the 4-nitroaniline reduction in the presence of E. coli-gold NP-biocatalysts at the 0 and 3 min. (B) Kinetics plots of the 4-nitroaniline reduction using different biocatalysts generated under anaerobic condition, wild type cells, and distilled deionized water (uncatalyzed) in the presence of NaBH₄ at 385 nm. E. c: E. coli; R. e: R. eutropha. (C) Kinetics plots using different biocatalysts generated under aerobic condition. (D) The comparison of the rate constants for the 4-nitroaniline reduction using different biocatalysts under anaerobic or aerobic conditions.

Previous studies have demonstrated that microorganisms are capable of reducing metal ions to metal species, which might involve in utilizing metals as final electron acceptors in anaerobic respiration.³ We therefore performed the kinetic studies of biocatalysts generated under anaerobic or aerobic conditions (Fig. 4B and C). To further evaluate the relative reaction rates of different biocatalysts, the kinetics plots of biocatalysts including E. coli or R. eutropha gold/platinum NPs were generated. The reduction reaction in the presence of NaBH₄ using wild type cells without metal NP display shows similar pattern as in the case of uncatalyzed reaction which confirms that the NPs on the cell surface act as catalysts but not the cells in this reaction (Fig. 4B). Under aerobic conditions, the reaction rates of biocatalysts showed similar but significantly slower patterns compared with that grown under anaerobic conditions (Fig. 4B and C). Fig. 4D shows the comparison of the rate constants which were determined from the slope of the linear relationship of the natural logarithm of the absorbance versus time at 385 nm. These results indicate that E. coli-gold NP-biocatalyst was the best catalyst compared with others. Since the size distribution analyses of metal NP between two hosts are relatively similar, it suggests that a greater number of metal NPs were deposited on the surface of E. coli might attribute to higher density of metal binding peptides on the surface. In addition, we observed a consistent enhancement of catalytic activity of gold/platinum NPs in both E. coli and R. eutropha under anaerobic condition (Fig. 4C and D) conditions, suggesting that anaerobically-induced enzymes might play an important role for bioreduction during anaerobic respiration.^3,29 Bioreduction of metal ion was observed in wild type cells in the presence of high concentration of metal salts.³⁰ We therefore examine the catalytic activity of wild type E. coli cells in the presence of Au³⁺/Pt⁴⁺. The catalytic activity of wild type is significantly lower than metal binding expressing cells (Fig. S9†). Furthermore, the catalytic activity of the whole-cell biocatalyst did not show significant decrease after five rounds of reuse (Fig. S10†).

Organisms that are naturally adapted to extreme environmental conditions are required for the practical recovery of metal ions. While extensive research has been conducted on the topic of bacterial cell-surface display, most of these studies have used E. coli as a tool to understand the underlying mechanism of membrane transport.^10,31 Surface display carriers in other Gram-negative bacteria have not been systematically examined. R. eutropha is a hydrogen-oxidizing chemolithoautotroph, which is attracting increasing biotechnological attention as a producer of bioplastics and biofuels.^32,33 Therefore, platinum NPs on R. eutropha could potentially serve as catalysts for generation of hydrogen gas which acts as energy donor for final electron acceptor for cells.

In summary, this paper examined the display efficacy of a variety of carrier proteins in both E. coli and R. eutropha. We present a cell-surface display system expressing metal binding peptides for the recovery and display of metal NPs simultaneously. Furthermore, nanoparticle-coated cells serve as whole-cell biocatalysts with excellent catalytic activity and reusability for the reduction of 4-nitroaniline. This system could potentially be applied for environmental remediation.

Acknowledgements

This work was funded by the Ministry of Science and Technology of Taiwan under the project number 103-2113-M-003-002-MY2. We thank Ms C.-Y. Chien and S.-J. Ji of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM and EDS experiments.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Supporting Tables S1–S4 and supporting Fig. S1–S8. See DOI: 10.1039/c5ra18561k

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