Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli

Yi-Jung Tsai, Chun-Yu Ouyang, Shi-Yuan Ma, Dong-Yu Tsai, Hsueh-Wei Tseng and Yi-Chun Yeh*
Department of Chemistry, National Taiwan Normal University, 88, Section 4, Tingzhou Road, Taipei 11677, Taiwan. E-mail: yichuny@ntnu.edu.tw

Received 21st October 2014 , Accepted 31st October 2014

First published on 31st October 2014


Abstract

This study used the biomolecule eumelanin as an agent for the reduction of metal ions. Our results demonstrate the effectiveness of synthesizing diverse metal nanoparticles through the use of recombinant E. coli expressing Rhizobium etli tyrosinase, MelA. Gold nanoparticles were recovered using cells with gold binding peptides on the surface. This study illustrates the possibility of using E. coli to produce and display diverse metal nanoparticles in a green chemistry synthetic route.


Nanomaterials are excellent platforms for applications involving diagnostic imaging, sensing and labeling.1,2 Functionalized metal nanomaterials have been studied extensively due to their unique photophysical, electronic, and magnetic properties with a wide range of potential applications in biomedicine and other industries.3–5 The preparation of metal nanomaterials generally involves the chemical reduction of metal salts using strong reducing agents.6,7 In nature, many microorganisms respond to metal stress through the precipitation of toxic metals from solutions. For example, Cupriavidus metallidurans is able to detoxify toxic gold ions through a reduction of Au(I/III) to metallic gold.8 Numerous researchers have been attracted by the robustness of biosynthesizing nanoscale materials via the green chemistry synthetic route at ambient temperature in aqueous solutions.9–12 Apte et al. recently explored the synthesis of cell-associated gold in yeast Yarrowia lipolytica by inducing the production of melanin.13

Eumelanin (EuMel), a type of melanin pigment, has been identified in prokaryotic as well as eukaryotic cells.14 The function of melanin in microorganisms remains unclear; however, metals have been shown to bind the structure of melanin.15 Tyrosinase is a key enzyme in EuMel production, catalyzing the oxidation of 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) and further to dopaquinone and melanin. Studies of EuMel indicated that this pigment contains carboxyl, amine, hydroxyl groups, quinone and semiquinone groups.16 The conversion of the phenolic groups to the quinone groups could serve as reducing agents for the transformation of metal ions into elemental forms. Recombinant E. coli cells have been shown to produce dark melanin pigment when expressing melA gene from Rhizobium etli CFN42.17,18 Herein, this study reports a new approach to the tunable bioreduction of diverse metal ions through the genetic incorporation of Rhizobium tyrosinase gene (melA) in recombinant E. coli cells with exogenous metal ions.

We began by testing the biosynthesis of gold nanoparticles in vitro. R. etli cells were incubated with 1 mM of L-DOPA for 12 h, resulting in a dark brown color. The UV-visible spectra of the solution was shown in Fig. S1A, ESI. The typical absorbance spectra of EuMel was observed.19 Next, the supernatant was collected through centrifugation at 12[thin space (1/6-em)]225g for 15 min. In the presence of L-DOPA and gold ions, a reddish color change was observed in the solution (Fig. 1A and S1B). The absence of either L-DOPA or gold ions failed to result in any change in color, indicating that the formation of gold nanoparticles depends on the presence of EuMel as well as gold ions. Similar result was observed from chemically produced EuMel (Fig. S1B). We then conducted transmission electron microscopy (TEM) analysis of gold nanoparticles (Fig. 1B). Fig. 1B presents a TEM image of the gold nanoparticles produced through the incubation of EuMel with 2 mM Au3+ salt.


image file: c4ra12805b-f1.tif
Fig. 1 EuMel from R. etli mediates the synthesis of gold nanoparticles. (A) Photograph of solutions in the presence or absence of 1 mM L-DOPA and Au3+, respectively. (B) TEM image of gold nanoparticles synthesized by EuMel. Scale bar: 20 nm. (C) TEM images of R. etli cells. Cells were incubated with or without 1 mM L-DOPA for 12 h. Subsequently, 2 mM of Au3+ or water was added. Scale bar: 200 nm.

The TEM analysis showed that the particle size was distributed with an average particle diameter of 11.7 ± 5.8 nm (n = 50). To further validate the formation of gold nanoparticles in R. etli cells, we performed TEM analysis for R. etli cells. Notably, extracellular gold nanoparticles appeared when incubating cells with L-DOPA and gold ions for 12 h (Fig. 1C, arrows). The formation of gold nanoparticles was not observed in the absence of either L-DOPA or gold ions.

We then tested whether it was possible to synthesize gold nanoparticles with EuMel based on the fact that recombinant E. coli cells express the plasmid carrying the R. etli tyrosinase (melA) gene under the control of promoter PTrc on a pBbE1k plasmid.20 EuMel was observed by inducing the exponential-phase of E. coli cells through the addition of 100 nM IPTG and 1 mM L-DOPA. The absorbance spectrum was similar to chemically produced melanin (Fig. S1C). The concentration of EuMel was calculated from standard curves (Fig. S2). After 12 h, the supernatant containing EuMel was collected and incubated with 2 mM of Au3+ solution at room temperature for 3 min. We again observed a color change similar to that previously described. The spectrum of the solution revealed a maximum absorption peak at 535 nm, which is attributed to the surface plasmon band of gold nanoparticles (Fig. S3). We next prepared a variety of metal nanoparticles such as Ag, Cu, Mn, Pt, and Ni by mixing EuMel solution with 2 mM metal salts. TEM and energy-dispersive X-ray spectroscopy (EDS) analyses were conducted to provide further confirmation of the metal particles. Representative TEM images of nanoparticles are shown in Fig. 2A–F. The spherical particles presented a size distribution of 12 to 20 nm as calculated from TEM images (Table S4). Histograms of particle size distribution are presented in Fig. S4A–F, inset. The elemental composition of the representative field was according to EDS spectra (Fig. S4A–F). We then examined the possibility of tuning the size of the nanoparticles by altering the concentration of the reactants. Gold nanoparticles were used as an example with which to compare the diameter of nanoparticles formed from various metal ions and EuMel concentrations. As shown in Table S5A, the diameter of the gold nanoparticles increased with the concentrations of Au3+ up to 2 mM. No further increase in size was observed at higher concentrations (Fig. S5A). In addition, we also evaluated the effect of EuMel concentration on particle size. The average diameter of Au nanoparticles increased slightly under higher concentrations of EuMel (Table S5B and Fig. S5B). These results suggest that the size of the gold nanoparticles is tunable between approximately 7 and 13 nm, by altering the concentration of the reactants.


image file: c4ra12805b-f2.tif
Fig. 2 TEM images of diverse metal nanoparticles synthesized by recombinant E. coli. 100 nM IPTG and 1 mM L-DOPA were used to induce the expression of EuMel for 12 h. EuMel solution was incubated with the corresponding 2 mM of metal ions. (A) Au, (B) Ag, (C) Cu, (D) Mn, (E) Pt, and (F) Ni. Scale bar: 50 nm.

We then tested the application of the EuMel-mediated metal nanoparticles as effective antibacterial agents. We compared the toxicity of Au, Ag, and Pt nanoparticles against E. coli DH10B by evaluating the number of colony forming unit (CFU) following the treatment of cells supplemented with 150 μg mL−1 of nanoparticles for 8 h. Bacterial growth was examined by spotting of cells onto agar plate. Fig. 3 shows that Ag nanoparticles exhibited better antibacterial property than do Au/Pt nanoparticles. Furthermore, we compared the antibacterial activity of Ag nanoparticles prepared by EuMel and NaBH4 (Fig. S6). Interestingly, EuMel-mediated Ag nanoparticles showed better antibacterial property.


image file: c4ra12805b-f3.tif
Fig. 3 Growth and comparison of various metal nanoparticles treated cells. Quantitative survival measurements of E. coli DH10B in the presence or absence of 150 μg mL−1 of Au, Ag, or Pt nanoparticles.

We then sought to synthesize and display the metal nanoparticles on cell surface simultaneously. The metal particles were displayed by fusing metal binding peptides with FhuA, an outer membrane iron transporter protein.3 We selected a gold binding peptide21 to examine the display of gold nanoparticles by fusing it with FhuA (FhuA-GBP). A strain containing pTrc-melA and pBAD-fhuA-gbp plasmids was grown in the presence of inducers, 1 mM L-DOPA, and 2 mM Au3+ followed by TEM analysis of the gold deposition on the cell surface (Fig. 4). As shown in Fig. 4A (arrows), the binding of gold nanoparticles occurred in cells expressing FhuA-GBP. Binding of gold nanoparticles to cells without arabinose showed the minimal background, suggesting that the observed binding was not due to nonspecific gold nanoparticle aggregation (Fig. 4B). These findings demonstrate the effectiveness of the proposed method in the synthesis and display of metal nanoparticles.


image file: c4ra12805b-f4.tif
Fig. 4 Displaying gold nanoparticles on cell surface via FhuA-GBP (A) Following incubation with 1 mM L-DOPA, 100 nM IPTG, 0.2% arabinose, and 2 mM of Au3+, the cells were washed and analysed. (B) Similar procedure was performed as described in (A), but arabinose was omitted in the medium. Scale bar: 200 nm.

In summary, this paper presents a simple, room-temperature, and the green chemistry synthetic method for the biosynthesis of nanoparticles. We illustrated how the EuMel biosynthesis pathway in R. etli can be exploited for the synthesis of metal nanoparticles through the use of recombinant E. coli with L-DOPA. While we demonstrate that the facile recovery of gold nanoparticles by gold binding peptide-tagged cells via surface display, the efficiency of gold nanoparticle recovery is low. A comprehensive screening of surface display carrier or metal binding peptide should allow us to enhance the applicability of the method in the conjugation of bacterial surfaces with diverse metal nanoparticles synthesized from EuMel.

Acknowledgements

This work was funded by the Ministry of Science and Technology of Taiwan under the project number 101-2113-M-003-010-MY2 and 103-2113-M-003-002-MY2. We thank Dr Guillermo Gosset for providing the plasmid (pTrc99A carrying R. etli melA gene). We thank Ms C.-Y. Chien and S.-J. Ji of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM, SEM and EDS experiments.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: ESI Tables S1–5 and ESI Fig. S1–6. See DOI: 10.1039/c4ra12805b

This journal is © The Royal Society of Chemistry 2014
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