Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper

Heavy metal sequestration from industrial wastes and agricultural soils is a long-standing challenge. This is more critical for copper since copper pollution is hazardous both for the environment and for human health. In this study, we applied an integrated approach of Darwin’s theory of natural selection with bacterial genetic engineering to generate a biological system with an application for the accumulation of Cu2+ ions. A library of recombinant non-pathogenic Escherichia coli strains was engineered to express seven potential Cu2+ binding peptides encoded by a ‘synthetic degenerate’ DNA motif and fused to Maltose Binding Protein (MBP). Most of these peptide-MBP chimeras conferred tolerance to high concentrations of copper sulphate, and in certain cases in the order of 160-fold higher than the recognised EC50 toxic levels of copper in soils. UV–Vis spectroscopic analysis indicated a molar ratio of peptide-copper complexes, while a combination of bioinformatics-based structure modelling, Cu2+ ion docking, and MD simulations of peptide-MBP chimeras corroborated the extent of Cu2+ binding among the peptides. Further, in silico analysis predicted the peptides possessed binding affinity toward a broad range of divalent metal ions. Thus, we report on an efficient, cost-effective, and environment-friendly prototype biological system that is potentially capable of copper bioaccumulation, and which could easily be adapted for the removal of other hazardous heavy metals or the bio-mining of rare metals.

It positively effects numerous cellular enzymes involved in energy metabolism. For examples, oxidoreductase and transferase enzymes utilise copper (in nM quantity) as a cofactor to facilitate electron transfer in redox reactions involved in metabolism and bioenergetics 3 . Therefore, organisms have evolved sophisticated resistance mechanisms to maintain copper homeostasis and counteract exposure to higher copper concentrations 4,5 .
There is now an urgent need to develop a system for the efficient, cost effective and environment friendly remediation of copper. Detoxification of heavy metal pollution can occur with physio-chemical and biological methods [14][15][16][17] . Unlike physio-chemical methods that are uneconomic and generate large amounts of chemical waste, biological methods are eco-friendly and offer high specificity in the elimination and counteraction of desired heavy metals [17][18][19][20] . Moreover, counteraction of heavy metals using microorganisms, known as bioremediation, is cost-effective and can even provide a more permanent solution compared to other known methods [17][18][19] .
Bacteria expressing metal binding proteins have a range of applications that include bioremediation of toxic heavy metals, the bio-adsorption and recovery of rare metals, and in energy generation 21 . Recombinant DNA technology offers methods for anchoring metal binding peptide(s) to fusion partners such as bacterial outer membrane proteins 22 , lipopolysaccharide 23 , and the Maltose Binding Protein (MBP) 24 . For such fusions, size and amino acid composition of the cargo (poly)peptide can significantly affect the final topology of the generated chimera. For this reason, use of MBP as a fusion partner is attractive because it has solubilising properties that can promote proper folding of the fused (poly)peptide into its biologically active form 25 .

Scientific Reports
| (2020) 10:20327 | https://doi.org/10.1038/s41598-020-76178-z www.nature.com/scientificreports/ In this study, we designed a 'synthetic degenerate' DNA fragment of 30 base pairs, and which was expressed as a fusion at the C-terminal of MBP by harmless laboratory E. coli to generate a bank of semi-random peptides all of 9 amino acids length. Those predicted to contain a potential U-shaped Cu 2+ ion coordination pocket conferred to bacteria resistance to copper that was about 160-fold higher than the recognised EC 50 toxic levels of copper in soils 11 . Hence, this study demonstrates a simple, robust and innovative biological system for the efficient and effective binding of Cu 2+ ions. We also suggest that this is a generally applicable approach for the bioremediation of various toxic heavy metals and for the bio-mining of rare metals.

Results
Crafting a small bank of MBP-fused semi-random peptides. The aim of this study was to engineer harmless non-pathogenic bacteria with potential to accumulate copper. An earlier study with peptides rich in either His or Cys residues and fused to LamB showed variable and modest bioaccumulation capacity for Cu 2+ , and with little specificity 26 . We capitalised on this experience by creating a small bank of semi-random peptides in which every peptide encompassed a potential Cu 2+ binding pocket formed by a random distribution of Cys, His, Arg and Tyr residues, and with an invariant Pro hinge and intermittent Gly spacer to facilitate Cu 2+ coordination. To accommodate these new design features aimed at enhancing Cu 2+ binding affinity, a minimum peptide length of 9 amino acids was required. To achieve this, nucleotide degeneracy was inbuilt into two complementary DNA oligonucleotides of 30 bases (Fig. 1A). With a unique restriction enzyme recognition site at their termini, the annealed duplex DNA motif was cloned in-frame into EcoRI and BamHI sites at the 3′ end of malE (encoding MBP) within the pMAL-p2x plasmid. The recombinant expression vector carried the chimeric malE-synthetic degenerate-dsDNA motif fusion under the control of the strong IPTG-inducible P tac promoter. We used fusion to MBP to anchor the metal binding peptides, promote their extracytoplasmic targeting, and to facilitate their purification from the bacterial periplasm for eventual biochemical studies. Critically, MBP is a fusion of choice for this role because it rarely interferes with the proper folding of the cargo (poly)peptide 25,27 .
To demonstrate proof-of-concept, we sequenced seven randomly selected plasmids purified from well-isolated colonies of newly transformed E. coli. In all cases the sequence confirmed malE-fused to degenerate DNA motifs (Fig. 1B), with the latter having a small open reading frame encoding a putative metal binding peptide of 9-amino acids in length (Fig. 1C). As expected, predicted peptide sequence positioned a Pro amino acid in the centre, with randomly distributed Cys, His, Arg and Tyr amino acids throughout that were deliberately separated by a Gly spacer placed at every alternative position (Fig. 1C). From this analysis we inferred a consensus Gly-X-Gly-X-Pro-X-Gly-Arg/Cys-Gly signature for the identified peptides (Fig. 1D).
Genetically engineered E. coli acquired resistance to copper. By expressing the malE-tagged synthetic degenerate DNA duplex proposed to encode for a bank of metal binding peptides in a harmless nonpathogenic E. coli, we aimed to select for the amino acid combinations that possess more significant metal (Cu 2+ ) binding coordination site(s). We argued that the ability of bacteria to thrive in the presence of higher copper concentrations could be an indirect measurement of their ability to produce peptides that bind Cu 2+ ions. Hence, to assess qualitatively the inhibitory copper concentrations resisted by E. coli expressing the peptide fusions, we performed initial growth analysis using a copper sulphate gradient agar plate ( Fig. 2A). In comparison to non-pathogenic E. coli expressing only MBP, bacteria expressing chimeric MBP (cMBP) with peptide, P1 to P7, conferred significantly more protection to toxic levels of copper sulphate (Fig. 2B). Semiquantification of the bacterial growth appearing on the agar plates revealed that E. coli expressing cMBP2 and cMBP3 resisted up to ~ 4 mM copper sulphate (Fig. 2C), whereas cells expressing cMBP4 to cMBP7 resisted up to ~ 2 mM copper sulphate (Fig. 2C). These growth profiles were significantly different from E. coli expressing only MBP (Fig. 2B,C). Additionally, cells expressing cMBP1 showed quite low protection with the extent of growth being only marginally better than the MBP control ( Fig. 2B,C). Consistent with growth on a copper sulphate gradient plate, cells expressing cMBP1-7 also maintained growth in liquid broth culture in the presence of 4 mM of copper sulphate compared with control bacteria (Fig. 2D). Moreover, growth of cells expressing cMBP3 was noticeably better at this concentration (4 mM). This suggested better counteraction of the toxic effect of copper by these recombinant cells. Further, exposure to higher concentrations of copper sulphate, i.e.: 6 and 8 mM, resulted in cessation of growth for all recombinant bacteria (Fig. 2E,F). Taken altogether, cells expressing cMBP2 to cMBP7 were more resistant to higher concentration of copper sulphate in comparison to the control cells and cells expressing cMBP1. Hence, the peptides P2 to P7 may incorporate copper with appreciable amount, with the most effective being the peptides P2 and P3. Protection of some recombinant bacteria from normally inhibitory copper concentrations could indicate that individual peptides confer to host bacteria the ability to assimilate copper.
UV-Vis spectroscopy analysis in the presence of Cu 2+ ions identified peptide-Cu 2+ complexes only for cMBP2 and cMBP3 fusions, as indicated by absorption peaks at around 800 nm ( Fig. 3B-E). Both cMBP2 and cMBP3 in the presence of Cu 2+ showed a shift in the absorption peak at 280 nm (Fig. 3D,E). This is reflective of efficient copper incorporation by these two peptides. In contrast, cMBP1 (least active peptide) generated UV-Vis spectra similar to the MBP control, regardless of the presence or absence of Cu 2+ ions (Fig. 3B,C). This indicates negligible incorporation of Cu 2+ by the cMBP1 fusion. Overall, purified cMBP2 and cMBP3 showed significant copper binding in vitro, and this corroborates protection to toxic Cu 2+ levels by our recombinant non-pathogenic E. coli expressing either cMBP2 or cMBP3.
Employing UV-Vis spectroscopy, we further examined relative copper incorporation by cMBP2 and cMBP3 fusions via a titration experiment (Fig. 3F-I). Successive increments in Cu 2+ concentration enhanced the intensity of absorption peak at both 280 nm and 800 nm, indicating absorption maxima at our highest tested copper were designed with nucleotides degeneracy (highlighted in grey) in order to code for potential metal binding residues, Cys, His, Arg and Tyr, distributed randomally, at the site of degeneracy, with an invariant Gly spacer and Pro at the centre. Restriction enzyme sites, EcoRI and BamHI (underlined) were incorporated at respective termini to facilitate the cloning of the anneled dsDNA motif into the pMAL-p2x plasmid to express malEmetal binding peptide fusions (chimeric MBP, cMBP). (B. Sequenced plasmids from 7 random recombinant E. coli strains encoded a cMBP fusion (cMBP1-7), containing putative Cu 2+ binding motifs (yellow) flanked by BamHI and EcoRI restriction sites. (C) Translated putative Cu 2+ binding motifs (yellow), encoding peptides 1-7 (P1-P7). As expected all encoded peptides contain a central Pro amino acid (P), Gly (G) spacer at alternative positions of the intended potential metal binding residues, Cys (C), His (H), Arg (R) and Tyr (Y) amino acids, and all are terminated by stop codon (−).(D) The consensus signature, Gly-X-Gly-X-Pro-X-Gly-Arg/Cys-Gly for P1-P7 peptides.  www.nature.com/scientificreports/ concentration (10 mM) for both cMBP. We used 10 mM copper sulphate as the highest concentration of Cu 2+ simply because this value is considerably more than the recognised EC 50 toxic levels of copper in soils. Michaelis-Menten and Allosteric Sigmoid kinetics calculations indicated the cooperativity of cMBP2 with Cu 2+ is slightly lower than that of cMBP3, in the order of a 1:3 cMBP: Cu 2+ molar ratio compared to 1:2, respectively (Fig. 3G,H). Altogether, these in vitro assays suggest that cMBP2 and cMBP3 can bind substantial amounts of copper (i.e.: > 4.0 mM).  www.nature.com/scientificreports/ Structure modelling and molecular dynamic simulation of the cMBP-Cu 2+ interaction. We observed that MBP-fused P2 and P3 possess an appreciable amount of copper binding propensity. With the idea to explain favorable Cu 2+ binding, and whether this binding correlated to peptides possessing a proposed U-shape metal-binding pocket, we used in silico analysis to compare and contrast the potential molecular structure of peptides P2 and P3. We hypothesized a U-shape binding pocket from several known illustrations of active Cu 2+ binding pockets encased in various different protein families 29 . For comparison, we included the poorest copper binder, MBP-fused P1 peptide. Possible structural features of these peptides were suggested using the structure-modelling predictions and Molecular dynamics (MD) simulation after removing from the respective full-length cMBP model in the absence of Cu 2+ ion. Peptide P1 appears to lose the helical structure propensity during the simulation, probably because of the inherently unstable 3 10 -helical feature (Fig. 4A). Only the first 4 residues, Gly1 to Arg4, seem to create a Turn-like structure, and the peptide possesses an overall linear conformation (Fig. 4A). In contrast, peptide P2 modelling indicated a possible Turn-like structure from the coil feature through to 70 ns simulation (Fig. 4B). Interestingly, peptide P3 modelling predicted two turns and the absence of any extended configuration that might be a basis for better accommodation of the Cu 2+ ion (Fig. 4C). Moreover, only in P2 and P3 models was the conserved Pro residue at position 5 contributing to the putative Turn-like structure. Collectively, only in models of P2 and P3 was there suggestion of a stable loop conformation. These findings are consistent with our initial hypothesis that a U-shaped structure would favour better Cu 2+ ion coordination, as inspired from known active-site Cu 2+ binding configurations 29 . To our knowledge, this is the first report of a synthetic 'degenerate' DNA motif of 30 bp used to liberate a bank of semi-random peptides with potential to generate U-shaped conformations that coordinate copper binding. Further, the predicted full-length modelled structures upon interaction with Cu 2+ ion were similar at the N-terminal end reflecting the MBP molecule, and with some differences towards the C-terminal end reflecting contributions from the fused peptide (Fig. 4D). Critically, the peptides of cMBP2 and cMBP3 fold with a hairpin secondary structure that could help in accommodating the metal ions 30 In contrast, the peptide of cMBP1 forms a non-hairpin like secondary structure (Fig. 4D). Subsequently, simulated full length MBP and cMBP structures in the absence of Cu 2+ ions were analysed under physiological conditions. Simulation of these modelled structures indicate that in the absence of Cu 2+ ion, the cMBP3 structure is dynamically more stable given that the root mean square deviation (RMSD), a dynamic stability index, is lower for the structures of cMBP1 and cMBP2 when simulated with identical parameters (Fig. 4E). The control MBP structure shows lowest RMSD value, indicating it to be dynamically very stable (Fig. 4E). However, we caution that this low RMSD value can be partly attributed to MBP alone having a shorter sequence. On the other hand, the greater dynamic instability of cMBP1 corroborates the lower Cu 2+ binding potency measured both in vitro and in vivo (see Figs. 2 and 3). Thus, we surmise from these MD simulations in the absence of Cu 2+ ion that cMBP2 and cMBP3 structures are dynamically more stable than cMBP1, and this may partly explain their enhanced ability to bind Cu 2+ both in vitro as well as in vivo.
Intriguingly in the presence of Cu 2+ , throughout the entire simulation run the molecular interaction distance of Cu 2+ ion with the N-atom of the first Gly1 residue in all three chimeric peptides routinely indicated a lower Å distance between the ion and cMBP2 or cMBP3 compared to the ion and cMBP1 (Fig. 4F). The cMBP2-Cu 2+ interaction, and to a lesser extent the cMBP3-Cu 2+ interaction, appears to be more stable at least until 50 ns. On the other hand, the cMBP1-Cu 2+ interaction appears very unstable considering the observed higher distance of cMBP-Cu 2+ interaction and many fluctations in simulation (Fig. 4F). Remarkably, the cMBP3-Cu 2+ interaction generated very low distance until 10 ns, implying that the docked Cu 2+ ion could remain in the binding pocket. In contrast, the docked Cu 2+ ion appeared to leave the binding pocket of cMBP1 and cMBP2 within 3 ns of stimulation. Hence, MD simulations predict that a Cu 2+ ion binds preferentially to cMBP2 and cMBP3, and not to cMBP1.
In silico prediction of metal ion interaction sites. To have any relevance to environmental clean-up applications, it is important to appreciate the metal binding affinity and specificity of our engineered MBPpeptide chimeras. Experimental identification of metal binding sites within poly(peptides) can be challenging as it requires expensive and specialized techniques, such as NMR spectroscopy 31 , absorption spectroscopy 32 , metalaffinity column chromatography and electrophoretic mobility shift assay 33 . In contrast, bioinformatic analysis of metal ion binding sites capatilises on the vast information accumulated in public database to generate rapid, accurate and reliable predictions. We used the MIB server 34 to assign predictions for metal binding affinity because it has proven reliability for predicting binding sites for up to 12 metal ions. The prediction of metal ion binding is based upon a fragment transformation method 35 , where each amino acid within a given peptide was considered as an individual structural unit in order to search for potential metal ions against a reference list of ~ 40,000 metal binding poly(peptides) in the Protein Data bank 36 . Of eight tested divalent metal ions, only Cu 2+ and Ni 2+ ions were predicted to engage with peptide P1 (Fig. 5A). Residue Cys2 displayed predicated affinity for both ions, while residue Arg4 only for Cu 2+ ion. The modest metal binding prediction mirrors the in vitro results of negligible binding of cMBP1 with Cu 2+ ion (see Figs. 2 and 3). The peptide P2 displayed a predicted metal ion binding affinity toward the ions Cu 2+ , Ni 2+ , Co 2 and Hg 2+ (Fig. 5B). Affinity for Cu 2+ ion was found at residues His2, Tyr4, Cys6 and Arg8, indicating a potent Cu 2+ coordination within this peptide. The peptide P3 displayed a predicted broad metal ion binding affinity towards the ions Cu 2+ , Co 2+ , Mn 2+ , Ni 2+ and Zn 2+ (Fig. 5C). Within peptide P3, the residues Arg2, His6 and Arg8 appear to have a marked metal ion binding potential for Cu 2+ ion. The His6 residue also displayed predicted affinity for Mn 2+ , Ni 2+ and Zn 2+ ions. Collectively, the fused peptides appear to possess a broad metal ion binding affinity in the order cMBP3 > cMBP2 > cMBP1, corroborating the degree of copper binding achieved by the three peptides (see Figs. 2 and 3).

Discussion
Copper is an essential trace element for living organisms and their ecosystems, but at higher concentrations it is toxic. Anthropogenic activities have elevated copper concentrations well beyond the current environmental quality standard 1,[6][7][8][9][10][11][12][13]37 . This is concerning given that copper pollution of the environment is commonplace. Hence, there is an urgent need to develop systems for the efficient, cost-effective and environment friendly remediation of copper. With a view to develop a technology to overcome copper pollution, we constructed a biological system intended for the eventual bioaccumulation of Cu 2+ ions. A harmless non-pathogenic E. coli strain DH5ᾳ was engineered to express a small bank of semi-random peptides fused at their C-termini to MBP harboured within the IPTG inducible plasmid, pMAL-p2x. Under the laboratory conditions tested, recombinant E. coli expressing six of the seven MBP-peptide chimeras acquired tolerance to high concentrations of copper. Among these, bacteria expressing either cMBP2 or cMBP3 were particularly tolerant to higher Cu 2+ concentrations. In this study, we did not perform Cu 2+ uptake experiments to demonstrate Cu 2+ accumulation by peptide-expressing bacteria. However, we showed clearly that the extent of copper tolerance by cultured non-pathogenic E. coli bacteria expressing either cMBP2 or cMBP3 was consistent with in vitro incorporation of copper by the purified cMBP2 and cMBP3 variants.
This study was not intended to be an exhaustive screen of Cu 2+ -ion binding peptides. Of the many recovered white colony transformants from the original transformation plated on selective agar plates supplemented with X-gal and IPTG, sequencing of a selection of these revealed seven unique DNA sequences, each encoding for a subtly different peptide. Had we continued to screen, we may have recovered other unique sequences. However, after examining the positive effect of expressing this random selection of seven peptides -P1 to P7 -in E. coli grown in the presence of a toxic Cu 2+ ion gradient, we considered that these were a sufficient representation to establish this proof-of-concept study.
Noted published studies have shown enhancement of Cu 2+ absorption up to 32-fold using engineered bacteria expressing metal binding peptides. For example, Pazirandeh and colleagues engineered E. coli cells expressing a triplet of small peptides, N CGCCG C in fusion with periplasmic MBP, but reported removal of less than 40% of a modest 5 µM copper ions from a liquid medium after 1 h incubation 24 . Moreover, Ueki and co-authors reported that E. coli BL21 cells expressing two vanabins, which are small cysteine-rich proteins distantly related to metallothioneins, in fusion with periplasmic MBP, absorbed about 70% of a modest 10 µM copper (II) ions from an aqueous medium 27 . Although we did not perform Cu 2+ adsorption experiments in this study, bacteria expressing some of our peptides conferred much higher resistance to Cu 2+ ions. These observations correlated to in vitro and in silico Cu 2+ incorporation studies. In fact, measurements produced herein suggest that our biological system has the potential to assimilate copper at levels almost 160-fold higher than the recognised EC 50 toxic levels of copper in soils 11 .
A natural progression of this work is therefore to express our copper-binding peptides in E. coli strains with improved copper sensing capacity. Hence, the recent report by Ravikumar and colleagues is significant. These authors describe an E. coli system based upon a small copper binding peptide, N SPHHGGW C fused to OmpC, and with its expression controlled by the extracellular copper sensing two-component system, CusSR 38,39 . Assembly of this genetic circuit improved copper adsorption affinity up to 92.2 μM 38 . Given this, it is possible that incorporation of our hyper copper-sequestering peptides (P2 and P3) with OmpC and expressed under the control of an intact CusSR two-component system might generate a strain with improved environmental copper sensing ability in association with hyper copper bioaccumulation capacity. It is also relevant that Wang and co-authors recently engineered a copA and cueO mutant of E. coli that was capable of detecting 0.01-25 µM copper and efficiently adsorbing 125 µM copper ions from aqueous solutions 40 . Our hyper copper-sequestering peptides could easily be incorporated into the genetic system of Wang and co-authors 40 to generate an E. coli strain with capacity for simultaneous detection and removal of copper in a temperature dependent manner. Thus, options exist to refine our system to enhance copper removal capacity and avoid reliance on artificial induction by expensive chemical synthetics.
Interestingly, structural modelling by IntFOLD coupled to protein dynamics assessment by RMSD and MD simulation indicated that the peptide sequence when appended to MBP negatively influenced the protein stability in the order of cMBP3 < cMBP2 < cMBP1. Although this would need to be confirmed by biophysical experiments, such as by performing circular dichroism studies, it is a possibility that enhanced peptide P3 and P2 stability maximises their apparent Cu 2+ binding affinity as measured experimentally in both in vitro and in vivo contexts. As protein structures are unrestrained and free to move in all directions during the period of simulation, bound ion releases from the binding site. The rate of ion release is based on the flexibility of the binding site, which in turn infers information about the strength of the interaction 41 . MD simulation studies are therefore commonly employed to access this information 30 . A well-established example of this is with the assessment of Na + and Clion shuttling on the surface of the S6 ribosomal protein 42 . When applied to our peptides, 100 ns MD simulations predicted release rates of Cu 2+ ions that in turn indicated superior ion binding in the order of cMBP3 > cMBP2 > cMBP1. This makes sense considering the prediction of a distinct U-shaped turn for P2 and P3, compared to an overall linear conformation for P1. We believe that the U-shape could help P2 and P3 peptides to accommodate the Cu 2+ ion. Interestingly, the guanidine group of arginine residues within both P2 ( N GHGYP-CGRG C ) and P3 ( N GRGYPHGRG C ) appeared to anchor Cu 2+ ions in a stable metal-peptide complex. Such a role for arginine has been suggested previously 43 . Finally, proline residues are favourable for the turn formation 44 . Proline was engineered at position 5 of each peptide. However, its presence in P1 was unable to promote loop formation. Perhaps arginine residues on either side of this proline might be the reason that stable loop conformation could not be formed as previously shown by the role of the arginine in increasing the unfolding rate 45 .

Scientific Reports
| (2020) 10:20327 | https://doi.org/10.1038/s41598-020-76178-z www.nature.com/scientificreports/ Computational predication of metal ion binding affinity is a powerful recent development. Compared to traditional specialised methods that are often expensive and tedious, such as NMR, chromatography, gel electrophoresis and spectroscopy 31,32,46,47 , available in silico prediction tools now enable an accurate, fast and efficient approach to identify metal ion binding affinity and specificity 48 . Corroborrating data obtained with Cu 2+ in both in vitro and in vivo experimental contexts, the bioinformatic MIB server 34 predicted broad range metal ion affinity in the order of cMBP3 > cMBP2 > cMBP1. Moreover, the MIB server indicated affinity for the Cu 2+ ion was coordinated through histidine, tyrosine, cysteine and arginine residues, consistent with information inferred from our structural modelling and protein dynamic simulations. The histidine residue is of particular importance, not only for enabling broad binding specificity to multiple metal ions, but also for establishing the extent of the binding affinity. In this regard, the positioning of histidine within the peptide sequence seems to have consequence for both specificity and affinity given that it is positioned at residue 6 in the most potent peptide, P3, while positioned at residue 2 in the somewhat inferior performed peptide, P2. Significantly, there is precedent for a role of histdine residues in influencing copper-binding site behavior as implied by mutagenesis and catalytic activity studies of human tyrosinase 49 .
In summary, a combinatory approach of synthetic biology with bacterial physiology enabled the generation of a small bank of Cu 2+ assimilating peptides. When expressed by E. coli DH5α, the peptide fusions -cMBP2 and cMBP3 -could bind Cu 2+ ions with higher capacity than previously reported. This includes being almost 32-fold higher than an E. coli strain engineered to sequester Cu 2+ from Chinese wastewater 40 , and about 160-fold higher than the recognised EC 50 toxic levels of copper in soils 11 . Thus, we demonstrate a concept for potential development of a cost-effective and environment friendly biological system to counteract copper pollution. This system is also open for possible broad-spectrum use to sequester other hazardous heavy metal contaminants or might even be applied to the bio-mining of rare metals.

Materials and methods
Designing of synthetic degenerate DNA oligonucleotides. In order to craft a bank of metal binding peptides, a previously described approach 26 was employed with modifications. Briefly, two synthetic degenerate DNA oligonucleotides (30 nt long), 5′-AAT TCG GTYRTGGC YRCCCG YRTGGC YRCGG TTG AG-3′ (oligo 1) and 5′-GCCA RYACCG RYGGGC RYACCG RYGCC AAC TCC TAG -3′ (oligo 2) were designed and purchased from Eurofins MWG Operon, Ebersberg, Germany. Both oligos were complementary to each other and annealed (in-house) together to form a duplex 'degenerate' metal binding DNA motif that would have potential to encode for small 'degenerate' peptides of 9 amino acids in length. All peptides were to initiate with Gly and terminate with a stop codon. These peptides would have random distribution of potent metal binding amino acids Cys, His, Arg, and Tyr at position 2 nd , 4 th , 6 th and 8 th with an invariant Gly spacers and Pro hinge at the centre (5 th position) to form a hypothesised U-shaped pocket to occupy metal ion(s).
Growth of recombinant non-pathogenic E. coli on LA agar supplemented with Cu 2+ . Resistance to copper of recombinant E. coli strains expressing sequence confirmed cMBP (in the periplasm) was initially assessed in the presence of copper sulphate (CuSO 4 .5H 2 O). A gradient (0 to 10 mM) of copper sulphate was established in sterile square petri plates. Molten LB agar (25 ml), supplemented with ampicillin (50 μg/ml), IPTG (0.1 mM) and CuSO 4 .5H 2 O (10 mM) was poured diagonally into the plates under sterile conditions. Following solidification, a further 25 ml molten LB supplemented only with ampicillin (50 μg/ml) and IPTG (0.1 mM) was overlaid onto the gradient of copper sulphate. A standardised inoculum (10 µl of 0.1 OD 600nm ) of overnight grown (in selective LB liquid broth containing ampicillin (50 μg/ml) and IPTG (0.2 mM) at 37 °C with agitation) bacterial culture was aseptically streaked directionally from predictable lower to higher concentration of copper Purification of MBP and cMBP fusions from the periplasmic fractions. The MBP alone and the cMBP fusions were purified by one-step Maltose affinity chromatography according to the protocol provided by the manufacturer (New England Biolabs protocol) with some modifications. Briefly, recovered periplasmic fractions were mixed in 20 ml of amylose-column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA and 10 mM β-Mercaptoethanol; pH 7.45), supplemented with 1 × cOmplete protease inhibitor cocktail (Sigma-Aldrich, Gillingham, Dorset, UK). Columns (2.5 × 10 cm) were packed with amylose resin, equilibrated with 100 ml of column binding buffer, and then loaded with periplasmic fraction mixtures at a rate of 0.5 ml/min. Flow-through was collected and the columns washed with 100 ml of column buffer at 1 ml/min. Unbound and washed samples were collected. Amylose-resin bound with MBP or the cMBP fusions were eluted at a rate of 0.5 ml/min with 10 mM maltose containing column buffer. Ten fractions (1 ml each) were collected and analysed by SDS-PAGE. Fractions containing most of the MBP or cMBP protein were pooled and concentrations of each was measured using Protein Kit (Bradford Method) reagent solution. An equal volume (25 µl) of the purified-pooled fraction containing either MBP or a cMBP fusion was mixed with sample buffer and analysed by SDS-PAGE. To de-salt and exchange the buffer of the eluted pooled fractions of MBP and cMBP, PD-10 desalting columns prepacked with Sephadex G-25 medium were used. Briefly, columns pre-equilibrated with 25 ml of elution buffer (20 mM Tris-HCl, pH 7.45), were loaded with 2.5 ml of the respective protein fraction. Bound protein was eluted in 1 ml fractions with 4 ml of elution buffer. An equal volume from each fraction was analysed by SDS-PAGE. Those fractions containing most of the eluted MBP or cMBP fusion protein was pooled together and stored at 4 °C until further biochemical analysis.

In vitro quantification of Cu 2+ incorporation by cMBP fusions. A JASCO-V560 UV-Vis Spectro-
photometer was used to quantitate Cu 2+ ions incorporation into the MBP and cMBP1 to cMBP3 fusions. Briefly, an equal volume of purified-desalted periplasmic samples containing 1 × cOmplete protease inhibitor cocktail (Sigma-Aldrich, Gillingham, Dorset, UK) was mixed with a final concentration of 4 mM CuSO 4 .5H 2 O in a total sample volume of 1 ml. Samples were incubated overnight at room temperature and UV-Vis spectra of each was recorded at 190 to 900 nm wavelength. Additionally, cMBP2 and cMBP3 samples (also contain 1 × cOmplete protease inhibitor cocktail) were incubated overnight at room temperature with 0.1 to 10 mM CuSO 4 .5H 2 O and on the following day their UV-Vis spectra recorded. We used 10 mM copper sulphate as the highest concentration of Cu 2+ because this is considerably more than the recognised EC 50 toxic levels of copper in soils.
Peak absorption values at 800 nm peaks (representing Cu 2+ bound cMBP) were further analysed by well-known Michaelis-Menten and Allosteric Sigmoid kinetics in order to demonstrate Cu 2+ ions cooperativity to cMBP2 and cMBP3 fusions.

Structure modelling and MD simulation.
A homology structure-modelling server, IntFOLD 52 was used to generate the model structures of MBP alone, cMBP1, cMBP2 and cMBP3. Best quality modelled structure of each was selected and were subjected to MD simulation without bound ions. The modelled proteins were solvated with a water cubic box of appropriate dimensions as per the size of protein. Counter ions were added to compensate the charges in the system. Solvated protein structurers were minimised and equilibrated for 500 picoseconds 52 each. Further, these structures were subjected to production run of 100 nano seconds (ns) of Scientific Reports | (2020) 10:20327 | https://doi.org/10.1038/s41598-020-76178-z www.nature.com/scientificreports/ molecular dynamics (MD) simulations using GROMACS-2019 program by utilising GROMOS96 54a7 force field 53 for protein, water and ions. The protein dynamic was evaluated by root mean square deviation (RMSD) method. Furthermore, each modelled structures was brought into the AutoDock 54 environment and one Cu 2+ ion was docked into the fused peptide portion of each cMBP. The docked poses were subjected to run for 100 ns MD simulation by applying similar parameters as described above. Interatomic distances were calculated for the N-atom of Gly417 (G1 in the peptide). Secondary structures of the fused peptides were calculated (in absence of Cu 2+ ) from the reterieved MD simulations, using the Timeline plugin in VMD-1.9.2 software 55 .
Predictions of metal ions binding affinity. Metal ions binding affinity of each structure was predicted by MIB server 34 . This program uses fragment transformation method 35 and combines the structural and sequence information to search for local metal interaction sites in the protein. Metal binding affinity for MBP and cMBPs structures was calculated for 8 different types of metal ions with default input parameters.
Statistical analysis. Statistical analyses were performed using GraphPad Prism statistical software (V8.2.0; San Diego, CA, USA). One-way ANOVA followed by Bonferroni post-test was performed to examine for differences among the various strains and growth conditions. Differences with a value of p ≤ 0.05 were considered statistically significant. The densitometric analysis of MBP, MBP-peptide fusion proteins and copper sulphate linear gradient plate was performed using ImageJ 1.48v (NIH).