Recovery of anthropogenic noble metal and toxic metal by utilizing synthetic biological leaching method

Abstract

To provide a method for constructing an integrated synthetic biological system for metal leaching which can be introduced into a synthetic host, efficiently recovering a noble metal from a waste electronic apparatus, and performing purification of a toxic metal.SOLUTION: The present invention generally relates to a method for cyaniding a metal and reducing a metal-cyanide composite by utilizing a biological technique and a method for hydrolyzing cyanide by utilizing a biological technique. More specifically, the present invention makes it possible to construct an integrated synthetic biological system for metal leaching which can be introduced into a synthetic host (such as Chromobacterium violaceum creating cyanide), and perform efficient recovery of a noble metal from a waste electronic apparatus and purification of a toxic metal. Design and construction of the synthetic host consists of maximum of four main constituents / modules: 1) utilizing a synthetic biological technique creation of cyanide; 2) recovery of a metal utilizing the synthetic biological technique; 3) decomposition of cyanide utilizing the synthetic biological technique; and 4) a synthesis circuit for a biological leaching method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for the decomposition of cyanide using enzymes and a synthetic biology leaching method using synthetic biology techniques to recover metals. More specifically, the present invention relates to the use of genetically engineered bacteria for use in a process involving the production of cyanide, reduction of leached metal ions, decomposition of cyanide, and subsequent recycling of the cyanide. Additionally, tools for transcriptional regulation in Chromobacterium violaceum are provided. [Background technology]

[0002] Cyanide readily bonds with most major and trace metals to form cyanide complexes, a property that makes it useful in extracting metals from their ores. Sodium cyanide, the most commonly used compound in mining, dissolves readily in water and gives rise to the sodium and cyanide ions (CN - ) is produced. - A portion of the CN is converted to hydrogen cyanide (HCN), the relative amount of which is determined by the pH of the water. At pH levels above 9.0, most of the CN is stable. - As the pH decreases, CN is converted to HCN. - However, the amount of HCN increases, but HCN easily evolves into gas and is released into the air. Therefore, most of the mining solutions are maintained at a pH level above 10.0 to prevent the generation of HCN gas and the poisoning of miners due to inhalation. Because cyanide is a carbon-based compound, it easily reacts with other carbon-based materials and becomes toxic to many living organisms. Therefore, waste containing cyanide must be detoxified before disposal. The traditional method of detoxification is the alkali chlorine method, but this method is dangerous and expensive. Furthermore, problems arise when the cyanide used in mining does not quickly decompose into harmless substances. Also, less toxic substances may remain in the environment for a long time, causing problems for aquatic ecosystems.

[0003] The electronic waste recycling industry uses chemical processes that pose considerable environmental risks. Current methods used to recover precious metals such as gold and remove toxic metals such as lead and mercury include pyrometallurgy (e.g. open combustion) and hydrometallurgy (acid leaching and industrial cyanidation or cyanide baths). These methods are energy intensive, require additional electrolytic steps for metal separation, and are highly polluting (Korte, F., Spiteller, M. & Coulston, F. (2000) Ecotoxicology and Environmental Safety 46, 241-245; Fields, S. (2001) Environ Health Perspect 109, A474-481). Research efforts are being expended to replace industrial chemical leaching methods with biotechnological leaching methods to make metal recovery and remediation easier, more cost-effective and less environmentally hazardous. Many scientists and engineers, including, for example, Brandl (Brandl, H., Lehmann, S., Faramarzi, MA, and Martinelli, D. (2008), Hydrometallurgy 94, 14-17), Watling (Watling, HR (2006), Hydrometallurgy 84, 81-108) and Rawlings (Rawlings, DE (2002), Annual Review of Microbiology 56, 65-91), have made significant contributions to the field of biotechnological leaching.Compared to traditional techniques that use acid dissolution to recover precious metals, current attempts to recover precious metals such as gold through bioremediation of e-waste utilize microorganisms that produce leachants (Korte, F., Spiteller, M. & Coulston, F. (2000) Ecotoxicology and Environmental Safety 46, 241-245; Pham, V., and Ting, YP (2009), Advanced Materials Research 71, 661-664; Liang, G., Mo, Y., and Zhou, Q. (2010), Enzyme and Microbial Technology 47, 322-326; Chi, TD, Lee, JC, Pandey, BD, Yoo, K., and Jeong, J. (2011), Miner Eng 24, 1219-1222). In these microorganisms, the leachant used for bioremediation and recovery of metals is typically hydrogen cyanide. Hydrogen cyanide leakage poses a significant threat to the environment, but the microorganisms used in the biomining industry are capable of both producing cyanide (capable of producing cyanide equivalents) and decomposing cyanide (capable of rendering the cyanide equivalents harmless), limiting or minimizing the risk of leakage. Therefore, bioleaching is unlikely to result in significant release of cyanide into the environment.

[0004] Unlike existing methods, bioleaching, which uses naturally occurring microorganisms under mild operating conditions, allows for the reuse of metals in a process that closely resembles the biogeochemical cycles found in nature, thereby reducing the demand for resources such as ores, energy, and landfill space (Brandl, H., Lehmann, S., Faramarzi, MA, and Martinelli, D. (2008), Hydrometallurgy 94, 14-17). Bioleaching has attracted attention because it is a "clean technology." Hydrogen cyanide is produced as a leaching agent by various bacteria (e.g., Chromobacterium violaceum, Pseudomonas fluorescens, and Pseudomonas aeruginosa) and various fungi (e.g., Marasmius oreades, Clitocybe sp., Polyporus sp.) (Pham, V., and Ting, YP (2009), Advanced Materials Research 71, 661-664). Cyanide is produced as a secondary metabolite during the short life span of these microorganisms. Although it has been known for many years that cyanide is produced by microorganisms, quantitative data on cyanide production in various species was lacking (Liang, G., Mo, Y., and Zhou, Q. (2010), Enzyme and Microbial Technology 47, 322-326). However, current attempts at biorecovery and bioremediation have not met industry expectations for cost-effective leaching (Faramarzi, MA, et al., (2004), Journal of Biotechnology 113, 321-326; Krebs, W., et al., (1997), FEMS Microbiology Reviews 20, 605-617) (efficient metal recovery, short bioleaching times, and metal separation without relying on traditional electrolysis), and as a result, metal remediation is performed using traditional hydrometallurgical and pyrometallurgical methods.This deviation from expectations is due to suboptimal metabolism of the intrinsic leachants produced by microorganisms and the lack of suitable biological reduction pathways to recover certain metals after bioleaching.

[0005] In order to protect the environment and conserve natural resources, there is a strong need to develop sustainable technologies for recycling electronic waste. The present invention focuses on the recovery of precious metals and the removal of toxic metals from electronic waste. Current conventional electronic waste treatment technologies using strong acids or cyanides are highly polluting to the environment. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates generally to methods for the cyanation of metals and the reduction of metal-cyanide complexes using biological techniques, and methods for the hydrolysis of cyanide using biological techniques.

[0007] The present invention allows the construction of an integrated synthetic biology system for metal leaching that can be introduced into a synthetic host (such as cyanide-producing Chromobacterium violaceum) for efficient precious metal recovery and toxic metal remediation from e-waste. The design and construction of this synthetic host may consist of up to four main components / modules: 1) synthetic biology-based cyanide production; 2) synthetic biology-based metal recovery; 3) synthetic biology-based cyanide degradation; and 4) a synthetic circuit for biological leaching. The present invention allows the construction of a synthetic circuit for biological leaching.

[0008] In this invention, we were able to provide a sustainable gold cyanidation method to the industry by designing and constructing a tool for removing excess cyanide from the environment. We also designed and constructed a genome editing tool for Chromobacterium violaceum and constructed a synthetic C. violaceum with the aim of recovering precious and toxic metals from electronic waste. Furthermore, we were able to obtain a method for the production of gold ions (Au 3+ ) to elemental gold (Au) as gold nanoparticles and / or silver ions (Ag + We have designed and constructed a tool to reduce silver (Ag) back to its elemental silver (Ag) state as silver nanoparticles, providing industry with an alternative to traditional recovery processes using electrolysis. [Means for solving the problem]

[0009] According to a first aspect, the present invention provides an isolated genetically modified bacterium comprising: transformed with at least one polynucleotide molecule, The at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter and at least one mutation that enables a gene product of the MerA gene to reduce metal ions to produce elemental metal as metal nanoparticles.

[0010] Another aspect is an isolated genetically modified bacterium comprising: transformed with at least one polynucleotide molecule, The at least one polynucleotide molecule comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter, and a genetically modified bacterium is provided.

[0011] In some embodiments, the isolated genetically modified bacterium comprises, in N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to a promoter activated by CviR; The present invention further comprises at least one recombinant polynucleotide DNA molecule comprising:

[0012] Another embodiment is a gold ion (Au 3+ ) to recover elemental gold as gold nanoparticles or silver ions (Ag + ) from elemental silver as silver nanoparticles, comprising: a) The isolated recombinant bacterium according to any one of claims 1 to 11 is treated with gold ions (Au 3+ ) and / or silver ions (Ag + with a leachate containing b) recovering elemental gold nanoparticles and / or elemental silver nanoparticles from the leachate; The present invention provides a method comprising:

[0013] According to another preferred embodiment, the present invention provides a method for producing an isolated genetically modified bacterium, comprising the steps of: a) performing error-prone PCR on a gene encoding mercury(II) reductase (MerA); i) transforming at least one bacterium with the PCR product; and ii) Au 3+ and / or Ag + selecting transformants which grow in a medium comprising b) performing multi-site saturation mutagenesis by overlap extension PCR on the gene encoding mercuric reductase (MerA); i) transforming at least one bacterium with the PCR product; and ii) Au 3+ and / or Ag + selecting a transformant that grows in a medium containing Including, the genetically modified bacterium has been transformed with at least one polynucleotide molecule; The at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter, and a gene product of the MerA gene produces gold ions (Au 3+ ) is reduced to produce elemental gold as gold nanoparticles, or silver ions (Ag + ) is reduced to produce elemental silver as silver nanoparticles. It will be readily appreciated that the gold used in the growth medium may be in other forms, such as AuCl.

[0014] Another aspect is an isolated genetically modified bacterium comprising: transformed with at least one polynucleotide molecule, The at least one polynucleotide molecule comprises a heterologous nitrilase gene operably linked to at least one promoter, and the gene product of the nitrilase gene degrades hydrogen cyanide.

[0015] In some embodiments, the at least one polynucleotide molecule further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

[0016] Another aspect is a method for producing a cyanide-based leachant using synthetic biology techniques, comprising the steps of: contacting at least one recombinant cyanide-producing bacterium with glycine; wherein the at least one recombinant cyanogenic bacterium is engineered to express a heterologous hydrogen cyanide synthase (hcnABC) gene and a heterologous 3-phosphoglycerate dehydrogenase mutant (serA) gene by linking these genes to at least one promoter. An example of this is shown in Figure 2A.

[0017] In some embodiments, the recombinant cyanogenic bacterium comprises, in N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to a promoter activated by CviR; The present invention further comprises at least one recombinant polynucleotide DNA molecule comprising:

[0018] According to another aspect, the present invention provides at least one isolated recombinant bacterium capable of carrying out the production of a cyanide-based leaching agent using synthetic biology techniques, as described herein.

[0019] In another aspect, the present invention provides a method for degrading cyanide using synthetic biology technology, comprising contacting at least one recombinant cyanide-degrading bacterium engineered to express at least one nitrilase enzyme with cyanide-containing nitriles remaining after bioleaching of e-waste.

[0020] In some embodiments, the at least one recombinant cyanide-degrading bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase.

[0021] According to another aspect, the present invention provides an isolated recombinant DNA molecule comprising, in N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or more cyanogenic genes operably linked to a promoter that is activated by CviR; The present invention provides a recombinant DNA molecule comprising:

[0022] According to another aspect, the present invention provides use of an inactive Cas9 and an sgRNA to suppress transcription of one or more genes in the genome of Chromobacterium violaceum by targeting the promoter regions of the genes, wherein the inactive Cas9 comprises an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain.

[0023] In some embodiments, the one or more genes encode the formation of the purple violacein pigment.

[0024] Another embodiment provides an isolated recombinant DNA molecule comprising the golTSB operon, the golTSB operon comprising, in an N-terminal to C-terminal direction, golT operably linked to the j23119 promoter, golS, golB operably linked to the golB promoter, and a reporter gene (such as GFP), which can be used for general screening of recombinant bacteria that perform gold reduction via MerA. [Brief description of the drawings]

[0025] [Figure 1A-1B] Figure 1A shows that the genetically engineered cyanogen-producing strain (SynLix 3.1) produces up to 80 mg / L of cyanide. The SynLix 3.1 strain was engineered to express hydrogen cyanide synthase (hcnABC) and a 3-phosphoglycerate dehydrogenase mutant (serA). Figure 1B shows an overview of the cyanide degradation and coupling process. The genetically engineered cyanogen-producing strain (SynLix 3.1) is tolerant to pH 10.0 (alkalophilic).

[0026] [Diagram 2]The process of cyanide decomposition and coupling is outlined below. Four nitrilase variants from various bacteria were selected. Their sequences were synthesized and cloned into host cells, respectively. More specifically, the four nitrilase variants used were nitrilase from Pseudomonas pseudoalcaligenes (nit), nitrilase from the chromosome of Synechocystis sp. PCC 6803 (SC-nit), cyanogen dihydratase from Bacillus pumilus (BP-cynD), and cyanogen dihydratase from Pseudomonas stutzeri (PS-cynD). nit contains two different subunits, nitB and nitC, and these two subunits were cloned into the pRSF-Duet vector and expressed in Escherichia coli (DE3) BL21 strain as host cells. The remaining three variants were cloned into the pGM vector and integrated into the genome of Chromobacterium violaceum using the Tn7 transposition system.

[0027] [Diagram 3] A schematic diagram of recombinant Chromobacterium violaceum as a host cell is shown. Formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase were used as coupling enzymes. These enzymes were cloned into pBbB8k-RFP vector (broad-host-range vector) and expressed in Chromobacterium violaceum. By linking the cyanide degradation process to downstream enzymes, the by-products carbon and nitrogen can be recycled, making this system self-sustaining.

[0028] [Figure 4]A schematic diagram of the mechanism of transcriptional repression using inactive Cas9 is shown. Cas9, whose catalytic activity is inactivated, is guided to the promoter sequence by sgRNA (blue) and physically suppresses the initiation of transcription by RNAP. The 20-base protospacer (purple) targeted in the promoter region is adjacent to the protospacer adjacent motif (PAM) sequence NGG (red), which is required for Cas9 to bind to DNA.

[0029] [Diagram 5] Repression of violacein production in C. violaceum using dCas9. (A) Schematic of the dCas9 circuit. (B) Regions targeted in the violacein operon are shown for three dCas9-sgRNA complexes, the vioA promoter, the vioB promoter, and the 5' region of vioC. (C) gRNA targeting the vioA promoter requires the least amount of dCas9 induction (0.01% arabinose), whereas gRNA targeting the vioB promoter requires slightly more (0.1% arabinose). On the other hand, gRNA targeting the further downstream transcription start site, vioC, has the same effect as the two non-targeting gRNA negative controls shown. (D) OD600 readings corresponding to each well shown in (C) are shown.

[0030] [Figure 6] Optimization of the gold sensor circuit. Dose responses to gold ions using (A) the original GolTSB operon or minimal gold sensors composed of the GolS transcriptional activator and (B) PgolTS or (C) PgolB are shown, along with schematic diagrams of these circuits.

[0031] [Figure 7] Schematic diagram of the circuit of the gold sensor mutant.

[0032] [Figure 8]The structure of GolS is shown. (A) The N-terminal domain of GolS is composed of a DNA-binding domain with a helix-turn-helix (HTH) structure, and the C-terminal domain is composed of a gold ion-binding domain. (B) The structure of the GolS homodimer bound to DNA is shown, predicted using Phyre2 (Kelley, Mezulis, Yates, Wass, & Sternberg, 2015). GolSmt1, GolSmt2, and GolSmt3 have mutations in the DNA-binding domain (right arrows). GolSmt2 has an additional mutation in the gold ion-binding domain (upper residue, upward arrow). GolSmt3 has an additional mutation in the DNA-binding domain (residue indicated by left arrow).

[0033] [Figure 9] The site of the A38I mutation in GolSmt1 is shown. This is a single mutant in which residue 38 has been substituted from alanine to isoleucine (circled). Although both alanine and isoleucine are hydrophobic, isoleucine has three more carbons in its hydrophobic side chain than alanine. This is thought to increase its hydrophobicity, leading to increased aggregation towards the hydrophobic center and therefore tighter binding to DNA.

[0034] [Figure 10] The A38Q and N97D mutations in GolSmt2 are shown. This is a double mutant with an alanine to glutamine substitution at residue 38 (circled in the DNA-binding domain) and an asparagine to aspartic acid substitution at residue 97 (circled in the ion-binding domain). In the first mutation, alanine is a non-polar neutral amino acid and glutamine is a polar neutral amino acid with an amide side chain, but the long polar side chain of glutamine may allow better binding to DNA. In the second mutation, asparagine is a polar neutral amino acid and aspartic acid is a polar acidic amino acid, but the negatively charged side chain of aspartic acid may increase affinity for positively charged gold ions or promote dimerization.

[0035] [Figure 11] The A38K and V60L mutations in GolSmt3 are shown. This is a double mutant with an alanine to lysine substitution at residue 38 (outer circle) and a valine to leucine substitution at residue 60 (inner circle). In the first mutation, alanine is a non-polar neutral amino acid and lysine is a polar basic amino acid, which would allow lysine to bind better to the phosphate groups of the DNA backbone. In the second mutation, valine and leucine are both non-polar neutral amino acids, and leucine has one more carbon in its side chain compared to valine, which would increase the overall hydrophobicity of the DNA-binding domain.

[0036] [Figure 12] The experimental workflow for mutagenesis and selection of gold sensor mutants in C. violaceum is shown. A template for a mutant library of the golS transcription activator was generated by replacing each amino acid with 19 other amino acids using deep scanning mutagenesis. The mutant library was then cloned and transformed into C. violaceum. Mutants with high fluorescence output and high sensitivity to gold ions were selected.

[0037] [Figure 13] Responses of wild-type and top four gold sensor mutants to gold ions are shown. Response curves were plotted and fitted to the Hill equation: Y=(BmaxXn) / (Kn+Xn)+C. K: [Au3+] threshold for activation to half the maximum RFU value, n: Hill coefficient, C: baseline RFU value, Bmax: maximum RFU value.

[0038] [Figure 14] A schematic of the synthetic circuit shows how dynamic regulation can be built into the new system: quorum molecules autonomously turn the circuit on, and a gold sensor signals the circuit to turn off when gold ions are leached from e-waste.

[0039] [Figure 15] A schematic diagram of a synchronized circuit that reversibly switches on and off in response to the absence and presence of gold ions is shown. The circuit is switched on by the quorum sensor (middle box) and switched off by the gold ion sensor (left box). When the repressor is diluted, the reversible circuit can be switched on again in the absence of gold ions. The gold ion sensor (left box) contains the GolS transcriptional activator, which is activated by gold ions to express the PhlF repressor. The quorum sensor (middle box) utilizes the host's endogenous CviR activator, which is induced by AHL at high cell density. In this way, cyanide production as an output indicated by RFP (right box) is synchronized at high cell density and repressed in the presence of gold ions.

[0040] [Figure 16] Schematic diagram of batch culture of Chromobacterium violaceum to investigate the on / off output in response to gold ions. To investigate the on / off output of the circuit of the present invention, small batch cultures of C. violaceum were grown in 300 μL of Tris minimal medium supplemented with 30 μg / mL kanamycin in 2 mL Eppendorf tubes at 37 °C for 24 h. Stationary phase cultures were diluted 1:600 ​​at each passage and 2 μM AuCl3 was added every other passage. Endpoint single cell fluorescence output was measured using a BD Accuri C6 flow cytometer (BD Biosciences, Singapore) at a flow rate of 14 μL / min and a core size of 10 μm, collecting 10,000 events for each sample. The fluorescence excitation wavelength was 561 nm and the detection wavelength was 610 nm or 620 nm. Cells were gated on forward and side scatter light. Background fluorescence from control cells not expressing RFP was subtracted from the mean fluorescence values.

[0041] [Figure 17]Graph showing that cells were serially passaged in the presence or absence of gold ions and circuits containing mutant and wild-type gold sensors showed reduced basal leaky expression in the mutant gold sensor circuit. Each passage was performed by diluting the previous subculture 1:600 ​​and culturing in minimal medium, with the circuit turned on without the addition of Au3+ or with the circuit turned off with the addition of 2 μM Au3+.

[0042] [Figure 18] The graphs show that circuits containing each GolS mutant (B: GolSmt1, C: GolSmt2, D: GolS mt3) were cycled three times for on / off, and distinct on and off populations were observed, whereas the circuit containing the wild-type gold sensor (A) had a mixture of on and off populations.

[0043] [Figure 19] A schematic diagram of the biosensor configuration is shown. The Salmonella gol operon consists of golT, which encodes a P-type ATPase, golS, which encodes an Au sensor, and golB, which encodes a metal-binding chaperone. The Au sensor used to bind Au3+ ions is expressed by the constitutive promoter J23119 of E. coli, and this Au sensor binds to the Au-sensing promoter golB, inducing the expression of GFP.

[0044] [Figure 20] 1 shows a graph depicting the biosensitivity of golGFP with increasing Au3+ concentration (μM).

[0045] [Figure 21] FIG. 1 shows a schematic of screening to identify gold-reducing mutants.

[0046] [Figure 22] 1 shows target sites in the MerA protein whose sequence was altered to alter metal binding affinity and / or specificity.

[0047] [Figure 23] Figure 2 shows the effect of Au3+ on the growth of E. coli Rosetta(DE3)pLysS cells in (A) liquid medium and (B) agar medium.

[0048] [Figure 24] OD600 of wild-type E. coli cells and all mutants expressing MerA on Au3+-containing agar medium is shown.

[0049] [Diagram 25] TEM images of gold nanoparticles synthesized by MerA mutants are shown.

[0050] [Figure 26] Comparison of kinetic parameters for Au3+ reduction by mutants identified by DM selection (black circles - DM mutants, black hexagons - wild type (WT) MerA). In (A) and (B), mutants above the dotted line have improved kcat / KM or kcat compared to WT MerA. (A) Comparison of kcat / KM values ​​for DM mutants. (B) Comparison of kcat values ​​for DM mutants. (C) Comparison of KM values ​​for DM mutants. Mutants below the dotted line have improved KM compared to WT MerA. (D) Table showing the various mutants and their associated sequences.

[0051] [Figure 27] Shown are TEM images of gold nanoparticles (AuNPs) recovered by reduction of AuCl3 with (A) WT MerA or (B) DM11.

[0052] [Figure 28] 1 shows the reduction of Au3+ from AuCl3 solutions or leachates by (A) WT MerA or (B) DM11 in vivo.

[0053] [Figure 29]A comparison of Au content before and after Au recovery by DM11, as well as during downstream processing steps, is shown.

[0054] [Diagram 30] TEM images of gold nanoparticles (AuNPs) recovered by DM11 from AuCl3 solution or e-waste leachate: (A) truncated tetrahedral gold nanoparticles recovered from AuCl3 solution, (B) bipartite truncated tetrahedral gold nanoparticles recovered from AuCl3 solution, and (C) truncated tetrahedral gold nanoparticles recovered from leachate.

[0055] [Diagram 31] (A) Schematic diagram of gold and silver biosensors in C. violaceum. (B) Graph showing sensitivity of mutant gold sensors to various metal ions in minimal medium: AuCl3 (40 μM), AgNO3 (10 μM), CdCl2 (80 μM), ZnCl2 (100 μM), HgCl2 (5 μM), NiSO4 (50 μM), CoCl2 (120 μM), FeSO4 (25 μM), and CuSO4 (35 μM).

[0056] [Diagram 32] 1 shows dose-response curves of wild-type and mutant biosensors to various metal ions in minimal medium.

[0057] [Diagram 33] We outline the biosensing process of C. violaceum for gold and silver using metals leached from scrap metal of electronic devices.

[0058] [Diagram 34] 1 shows a graph illustrating the precious metal ion sensing ability of wild-type and mutant biosensors in a mixture of metal ions leached from electronics scrap metal (ESM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] For ease of reference, the references cited herein are listed at the end of the Examples, all of which are incorporated herein by reference in their entirety, but no reference to these references herein is intended to imply that they form part of the common general knowledge.

[0060] The use of genetically engineered bacteria in a process involving cyanide production, reduction of leached metal ions, decomposition of cyanide, and subsequent recycling of the cyanide is expected to require the use of separate bacterial strains with separate mutant enzymes. Some bacterial strains selectively reduce gold and some selectively reduce silver. A possible workflow would be to oxidize the metals with a bacterial strain that produces a biological leachant, then selectively reduce and recover the metals with another bacterial strain utilizing gold- and silver-reducing mutants, and bioremediate the biological leachant by biodegrading the cyanide using yet another bacterial strain.

[0061] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that the present compounds, compositions, articles, devices, and / or methods are not limited to particular synthetic methods or to particular recombinant biotechnology methods, unless otherwise specified, nor are they limited to particular reagents, unless otherwise specified, and as such may, of course, vary in many ways. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting to particular embodiments.

[0062] Definition of Terms For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0063] The words "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0064] In this specification, the terms "comprising" or "including" are to be construed as specifying the presence of the features, components, steps or ingredients described herein indicated by these terms, but not excluding the presence or addition of one or more other features, components, steps or ingredients or groups thereof. In the context of this disclosure, the terms "comprising" or "including" also encompass the meaning of "consisting of". Thus, variations of the term "comprising", such as "comprise" or "comprises", and variations of the term "including", such as "include" or "includes", are similarly broad in meaning.

[0065] In the present specification, a range of values ​​may be expressed as "approximately" a particular value and / or as a range from "approximately" a particular value to "approximately" another particular value. When such a range is stated, another embodiment includes the particular value that is not approximate, and / or the range from the particular value that is not approximate to the other particular value. Similarly, when a particular value is stated as an approximation by using the antecedent "about", it is understood that the particular value that is not approximate constitutes another embodiment. The upper and lower limits of each numerical range may be related to one limit or may not be related to the other limit. In addition, various numerical values ​​are disclosed in this specification, and these numerical values ​​are disclosed herein not only as the specific numerical value itself, but also as "approximate" numerical values ​​of the numerical value. For example, when a numerical value of "10" is disclosed, the numerical value of "about 10" is also disclosed. In addition, as can be easily understood by a person skilled in the art, when a specific numerical value is disclosed, "less than or equal to that numerical value", "more than or equal to that numerical value", and a numerical range that can be set between those numerical values ​​are also disclosed. For example, when the number "10" is disclosed, numbers such as "10 or less" and "10 or more" are also disclosed. In addition, data are provided throughout this application in various formats, and these data indicate upper and lower limits as well as ranges indicating any combination of data points. For example, when a specific data point "10" and another specific data point "15" are disclosed, numbers greater than 10, numbers greater than 10, numbers less than 10, numbers less than 10, numbers equal to 10, numbers greater than 15, numbers greater than 15, numbers less than 15, numbers less than 15, numbers equal to 15, and numbers between 10 and 15 are also disclosed. Also, each unit between two specific units is disclosed. For example, when the numbers "10" and "15" are disclosed, the numbers 11, 12, 13, and 14 are also disclosed.

[0066] In a first aspect of the present invention there is provided an isolated genetically modified bacterium comprising: transformed with at least one polynucleotide molecule, A genetically modified bacterium is provided, wherein the at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter and comprises one or more mutations that cause a gene product of the MerA gene to reduce metal ions to produce elemental metal as metal nanoparticles.

[0067] In some embodiments, the MerA gene contains one or more mutations encoding amino acid substitutions at positions selected from the group including V317, Y441, and C464. In other embodiments, the MerA gene mutations are at one or more positions selected from the group including A323D, A323D (deletion of amino acids 324-365), A414E, G415I, E416C, L417I, I418D, and A422N.

[0068] In some embodiments, the metal ion is a gold ion (Au 3+ ), and the gold ions are reduced to produce elemental gold nanoparticles, or the metal ions are silver ions (Ag + ) and the silver ions are reduced to produce elemental silver as silver nanoparticles.

[0069] In some embodiments, the isolated bacterium has a decreased ability to reduce the substrate mercury compared to a bacterium that comprises a non-mutated MerA gene.

[0070] In some embodiments, the isolated genetically modified bacteria is transformed with at least one polynucleotide molecule, the at least one polynucleotide molecule comprising a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter. In some embodiments, the hydrogen cyanide synthase gene is hcnABC (SEQ ID NO: 35) and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA (SEQ ID NO: 36). These genes increase the production of hydrogen cyanide to leach precious metals from metal sources, such as electronic waste.

[0071] In some embodiments, hydrogen cyanide production is under the control of an on / off switch that includes a gold ion sensor and a quorum sensor (an example is shown in FIG. 15). More specifically, the gold sensor turns off the leaching process when a critical amount of leached gold is reached. This off circuit is responsive to gold ions produced by cyanidation and includes a "weak" constitutive promoter that drives the golS gene and another promoter that drives a downstream repressor gene. An example of a minimal gold sensor would be one that drives a weak constitutive promoter (e.g., P Con6 37) and the PhlF repressor, which blocks the operator of PhlF in the quorum sensor. golTS Promoter or P golB The gold sensor includes a quorum sensor that includes a ph1F repressor gene induced by a promoter. The on-circuit of the quorum sensor is responsive to high cell density and includes a promoter that is activated by endogenous CviR (induced by AHL at high cell density) and an operator of PhlF downstream of the quorum sensor promoter. Downstream of and under the regulation of the quorum sensor are one or more cyanogenic genes, which are selected from heterologous hydrogen cyanide synthase genes and heterologous 3-phosphoglycerate dehydrogenase mutant genes.

[0072] In some embodiments, the isolated genetically modified bacterium comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to an on / off circuit, the off circuit being upstream of an on circuit, and the on circuit being upstream of the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene. The off circuit comprises a heterologous golS gene operably linked to a promoter and a heterologous P golTS Promoter and P golB and a downstream heterologous ph1F repressor gene operably linked to a promoter selected from the group consisting of promoters. The on-circuit comprises a promoter activated by endogenous CviR, and a PhlF operator between the CviR-activated promoter and the hydrogen cyanide synthase gene or the 3-phosphoglycerate dehydrogenase mutant gene. The hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene are operably linked to the CviR-activated promoter.

[0073] The golS gene is preferably codon-optimized for C. violaceum and its dynamic range and sensitivity are optimized by mutagenesis (an example is shown in FIG. 12). In some embodiments, the golS gene is a mutant selected from GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

[0074] A useful gold ion sensor comprises the golTSB operon shown in FIG.

[0075] In another aspect, an isolated recombinant DNA molecule is provided that comprises the golTSB operon, the golTSB operon comprising, from N-terminus to C-terminus, golT operably linked to the j23119 promoter, golS, golB operably linked to the golB promoter, and a reporter gene, such as GFP, which can be used for general screening of recombinant bacteria that perform gold reduction via MerA.

[0076] Nitrilases are a class of enzymes that hydrolyze nitriles to ammonia and the corresponding carboxylic acids. There are two types of such cyanide-decomposing enzymes: [ka]

[0077] The first group, cyanide dihydratases, includes a group of bacterial enzymes. These enzymes behave as true nitrilases and directly convert cyanide to formic acid and ammonia (upper scheme), whereas fungal cyanide hydratases hydrolyze cyanide to formamide (lower scheme). These hydrolases do not require additional cofactors or substrates and exhibit catalytic activity over a wide range of substrate concentrations, making them promising candidates for cyanide bioremediation.

[0078] In some embodiments, the isolated genetically modified bacteria may include a heterologous nitrilase gene operably linked to at least one promoter. The heterologous nitrilase gene degrades hydrogen cyanide. In some embodiments, the heterologous nitrilase gene encodes an enzyme selected from the group including cyanide dehydratase and cyanide hydratase. In some embodiments, the at least one nitrilase enzyme is from at least one bacterial species selected from the group including Pseudomonas pseudoalcaligenes (nit), Synechocystis sp. PCC 6803 chromosome (SC-nit), Bacillus pumilus cyanide dihydratase (BP-cynD) and Pseudomonas stutzeri (PS-cynD). These cyanide degrading bacterial strains for bioremediation may be used alone, rather than as part of an integrated workflow, or as industrial containment for cyanide and heavy metals.

[0079] In some embodiments, the isolated genetically modified bacterium further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter. The cyanide decomposition process can be coupled to downstream enzymes formate dehydrogenase, glutamate dehydrogenase and phosphoenolpyruvate carboxylase, which can recycle the carbon and nitrogen by-products, making the system self-sustaining (Figure 3).

[0080] In some embodiments, the isolated genetically modified bacterium comprises a heterologous nitrilase gene, a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

[0081] In some embodiments, the bacterium is selected from the group including Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Escherichia coli.

[0082] In some embodiments, the bacteria is stable at pH 10.

[0083] In a second aspect of the present invention, gold ions (Au 3+ ) to recover elemental gold as gold nanoparticles or silver ions (Ag + ) from elemental silver as silver nanoparticles, comprising: a) The isolated genetically modified bacterium according to any of the aspects of the present invention is treated with gold ions (Au 3+ ) and / or silver ions (Ag + with a leachate containing b) recovering elemental gold nanoparticles and / or elemental silver nanoparticles from the leachate; A method is provided that includes:

[0084] In some embodiments, the contacting is performed under alkaline conditions.

[0085] In some embodiments, the contacting is performed at a pH of at least about 10.

[0086] In a third aspect of the invention there is provided a method for producing an isolated bacterium comprising the steps of: a) performing error-prone PCR on a gene encoding mercury(II) reductase (MerA); i) transforming at least one bacterium with the PCR product; and ii) selecting transformants that grow in a medium containing metal ions; or b) performing multi-site saturation mutagenesis by overlap extension PCR on the gene encoding mercuric reductase (MerA); i) transforming at least one bacterium with the PCR product; and ii) selecting transformants that grow in a medium containing metal ions; Including, the bacterium comprises at least one polynucleotide molecule; Methods are provided, wherein the at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter and comprises one or more mutations that cause a gene product of the MerA gene to reduce metal ions to produce elemental metal as metal nanoparticles.

[0087] In some embodiments, the gene product is a gold ion (Au 3+ ) can be reduced to produce elemental gold as gold nanoparticles and / or silver ions (Ag + ) to produce elemental silver as silver nanoparticles. In some embodiments, ii) a portion of the metal ions is selected from AuCl3 and / or AgNO3. In some embodiments, a) the portion of the PCR is performed using a forward primer and a reverse primer, the forward primer comprises the nucleotide sequence of 5'-GTGGTGGTGGTGGTGCTCGAGTTA-3' (SEQ ID NO: 1) and the reverse primer comprises the nucleotide sequence of 5'-GATATACATATGCACCACCATCACCATCAT-3' (SEQ ID NO: 2).

[0088] In some embodiments, b) the PCR portion is performed using primers containing NNK and / or MNN at target sites V317, Y441 and C464 of the MerA protein.

[0089] In some embodiments, the selection includes at least two forms, one of which is Au 3+ and / or Ag + The other form is selection on agar plates containing Au 3+ and / or Ag + The selection is carried out in liquid culture containing

[0090] In a fourth aspect of the present invention there is provided a method for producing a cyanide-based leaching agent utilising synthetic biology techniques, comprising the step of contacting a recombinant cyanide-producing bacterium with glycine, said bacterium comprising a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

[0091] In some embodiments, the hydrogen cyanide synthase gene is hcnABC and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA. An example of this is shown in Figure 1A, where the mutant, designated SynLix 3.1, was grown in LB medium and cyanide production was monitored over a 48 hour period. Cyanide production was detected using a cyanide-sensitive ion selective electrode (ISE).

[0092] In some embodiments, the hcnABC and serA genes are under the control of an inducible promoter. In some embodiments, the isolated genetically modified bacterium comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to an on / off circuit. The off circuit is upstream of an on circuit, and the on circuit is upstream of the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene. The off circuit comprises a heterologous golS gene operably linked to a promoter and a heterologous P golTS Promoter and P golB and a downstream heterologous ph1F repressor gene operably linked to a promoter selected from the group consisting of promoters. The on-circuit comprises a promoter activated by endogenous CviR, and a PhlF operator between the CviR-activated promoter and the hydrogen cyanide synthase gene or the 3-phosphoglycerate dehydrogenase mutant gene. The hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene are operably linked to the CviR-activated promoter.

[0093] In some embodiments, the recombinant cyanogenic bacterium is tolerant to a pH of at least about 10.

[0094] In some embodiments, the synthetic biology production of cyanide leachant and metal bioleaching are carried out in a single reactor.

[0095] In another aspect of the present invention, there is provided at least one isolated recombinant bacterium capable of carrying out the synthetic biology production of a cyanide-based leaching agent according to any of the aspects of the present invention.

[0096] In some embodiments, the recombinant cyanogenic bacterium is selected from the group including Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Escherichia coli.

[0097] In another aspect of the present invention, there is provided a method for degrading cyanide using synthetic biology techniques, comprising the steps of: a) contacting at least one recombinant cyanide-degrading bacterium engineered to express at least one nitrilase enzyme with cyanide-containing nitriles remaining after bioleaching of e-waste.

[0098] In some embodiments, the at least one nitrilase enzyme is selected from the group comprising cyanogen dehydratase and cyanogen hydratase.

[0099] In some embodiments, the at least one recombinant cyanide-degrading bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase.

[0100] In some embodiments, the at least one nitrilase enzyme is derived from at least one bacterial species selected from the group including Pseudomonas pseudoalcaligenes (nit), Synechocystis sp. PCC 6803 chromosome (SC-nit), Bacillus pumilus cyanide hydratase (BP-cynD), and Pseudomonas stutzeri (PS-cynD).

[0101] In another aspect of the invention, there is provided an isolated recombinant DNA molecule comprising, in N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or more cyanogenic genes operably linked to a promoter that is activated by CviR; A recombinant DNA molecule comprising:

[0102] In some embodiments, the golS transcriptional activator gene is driven by a weak constitutive promoter (P Con6 etc.)

[0103] In some embodiments, the golS gene is codon-optimized for C. violaceum and its dynamic range and sensitivity are optimized by mutagenesis (an example is shown in FIG. 12). In some embodiments, the golS gene is a mutant selected from the group including or consisting of GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

[0104] In another aspect of the present invention, there is provided a use of an inactive Cas9 and an RNA guide (sgRNA) for suppressing transcription of one or more genes in the genome of Chromobacterium violaceum by targeting the promoter regions of the one or more genes, wherein the inactive Cas9 comprises an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain.

[0105] In some embodiments, the gene encoding the inactive Cas9 is P araBAD The gene encoding the RNA guide (sgRNA) is operably linked to a promoter, and the gene encoding the RNA guide (sgRNA) is operably linked to a strong constitutive promoter, such as J23119.

[0106] In some embodiments, the inactive Cas9 targets the violacein operon to prevent the formation of purple violacein pigment, which may complicate downstream process steps. In some embodiments, the inactive Cas9 targets the vioA promoter, the vioB promoter, and / or the vioC promoter, preferably all three promoters. Having described the general outline of the invention, the invention may be more readily understood by reference to the following examples, which are provided for illustrative purposes. However, the following examples are not intended to limit the invention. EXAMPLES

[0107] Standard molecular biology techniques known in the art that are not specifically described herein were generally performed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0108] Example 1 Incorporation of nitrilase into host cells for the degradation of cyanide Four nitrilase variants from various bacteria were selected. Their sequences were synthesized and cloned into host cells, respectively. More specifically, the following four nitrilase variants were used: nitrilase (nit) from Pseudomonas pseudoalcaligenes (SEQ ID NO: 11), nitrilase (SC-nit) from the chromosome of Synechocystis sp. PCC 6803 (SEQ ID NO: 12), cyandihydratase (BP-cynD) from Bacillus pumilus (SEQ ID NO: 13), and cyandihydratase (PS-cynD) from Pseudomonas stutzeri (SEQ ID NO: 14). Nit contains two distinct subunits, nitB (SEQ ID NO: 15) and nitC (SEQ ID NO: 16), and these two subunits were cloned into the pRSF-Duet vector and expressed in E. coli (DE3) BL21 strain as host cells. The remaining three variants were cloned into the pGEM vector and integrated into the genome of Chromobacterium violaceum using the Tn7 transposition system (Figures 2 and 3).

[0109] The coupling enzymes used were formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase. These enzymes were cloned into the pBbB8k-RFP vector (a broad-host range vector) and expressed in Chromobacterium violaceum. By linking the cyanide degradation process to downstream enzymes, the by-products carbon and nitrogen could be recycled, making the system self-sustaining (Figure 3).

[0110] A cyanide-degrading Chromobacterium violaceum strain was tested for its ability to remove cyanide from the extracellular environment. The engineered cyanide-degrading C. violaceum strain was able to completely remove exogenous cyanide within 24 h in the presence of 100 mg / L potassium cyanide.

[0111] Example 2 Development of a genome transcriptional control tool using dCas9 in Chromobacterium violaceum CRISPR-Cas9 has been widely used as a genome editing tool in many organisms. It has been mainly used in eukaryotic organisms, such as mammalian cells, insect cells, and yeast cells. The CRISPR-Cas system is derived from the bacterial adaptive immune system and inserts small fragments of invading bacteriophage DNA into the host genome to store them as memory and monitor future invading bacteriophages of the same DNA fragment. The CRISPR-Cas9 system from Streptococcus pyogenes consists of the Cas9 endonuclease, which works in conjunction with an RNA guide to bind to DNA sequences homologous to the RNA guide, and the bound Cas9 endonuclease cleaves the double-stranded DNA of the invading bacteriophage. When Cas9 endonuclease is used as a genome editing tool, it is expressed with an RNA guide consisting of a 20-base spacer complementary to the target DNA followed by a 76-bp scaffold that contacts the Cas9 endonuclease. The RNA guide guides the Cas9 endonuclease to the target DNA via the 20-bp spacer, where it creates a double-strand break in the host chromosome. The host activates DNA repair machinery to repair the broken chromosome, which results in insertion, deletion, or homologous recombination of the introduced DNA fragment into the DNA target site. The modular and easy accessibility of this RNA-guided machinery has led to the rapid adoption of CRISPR-Cas9 genome editing in many organisms.

[0112] CRISPR-Cas has been applied to a variety of other uses. There are many applications and variations of Cas9 that target specific genomic loci, as DNA sequences can be easily and specifically recognized by RNA. There are two CRISPR / Cas gene tools with added functions: CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation), which target gene transcription. CRISPRi (Figure 4) is performed using inactive Cas9 (dCas) (SEQ ID NO: 17), which has an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain, which inhibits the binding of RNA polymerase to the promoter region due to steric hindrance, thereby suppressing gene transcription. In CRISPRa, gene transcription is enhanced through a protein in which dCas is fused to an activator that recruits RNA polymerase. CRISPRa has been shown to increase the expression of target genes by 3-fold [Bikard, D. et al., Nucleic Acids Res, 41(15), 7429-7437 (2013)]. On the other hand, CRISPRi is more widely used in bacterial genera belonging to the genera Escherichia, Pseudomonas, Mycobacterium, Corynebacterium, Clostridium or Bacillus, and can suppress gene expression up to 300-fold [Cho, S. et al., Int J Mol Sci, 19(4). doi:10.3390 / ijms19041089 (2018); Qi et al., 2013]. Hereinafter, we investigate whether the expression of chromosomal genes can be transcriptionally silenced in Chromobacterium violaceum by using CRISPRi tools.

[0113] Although the CRISPR / Cas genome editing tool is a powerful tool for eukaryotic cells, its use in prokaryotes is more limited because double-stranded DNA breaks are lethal in prokaryotes, and CRISPR / Cas use kills bacteria because prokaryotic DNA repair mechanisms are insufficient to repair the cell.

[0114] Nevertheless, we have adapted an inactive Cas9 (dCas9) with D10A and H840A mutations in the catalytic domain to block transcription of target genes (Figure 5). In this study, we use dCas9 to target the promoter region of the violacein operon and block the formation of the purple violacein pigment (Figure 5B). This repression is most effective when targeting the promoter (sequence number 18) of the first gene (vioA), which is sufficiently repressed with 0.01% arabinose. This is followed by the vioB promoter (sequence number 19), but to repress violacein via this promoter, dCas9 expression must be induced with a higher concentration of 0.1% arabinose. Repression further downstream in this operon, at the vioC promoter (sequence number 20), has little effect on the repression of violacein transcription. There is a slight decrease in optical density, but this is not likely due to the metabolic burden of dCas expression. One of the main reasons for the slight decrease in optical density is the OD 570 This may be due to the overlap of the absorbance spectrum with that of violacein, which has an absorbance maximum at 100 nm [Swem, LR et al., Mol Cell, 35(2), 143-153 (2009)]. Another reason for the slight decrease in optical density may be due to the formation of clumps of cells with insufficient suppression of violacein pigment (Figure 5C).

[0115] Transcriptional control of chromosomes in C. violaceum has not been reported so far. Control of endogenous genes is useful for metabolic engineering, especially for controlling metabolic fluxes, allowing rapid and efficient knockdown of gene expression. It can also be easily multiplexed, allowing efficient and rapid knockdown of many genes at once [Cobb, RE et al., ACS Synth Biol, 4(6), 723-728 (2014); Cress et al., 2015]. In addition, dCas9 can be fused to another protein, for example, cytidine deaminase or adenine deaminase, to introduce single-base mutations [Arazoe, T. et al., Biotechnology journal, 13(9): e1700596 (2018); Komor, Kim, Packer, Zuris, & Liu, 2016]. In this way, the genetic toolbox of C. violaceum can be developed in a versatile way.

[0116] Cloning and expression of dCas in Chromobacterium violaceum Using mutagenesis primers and Gibson assembly, two catalytic site mutations (D10A and H840A) were introduced into Cas9. araBAD dCas was cloned under the control of (SEQ ID NO: 21). A strong constitutive promoter, J23119 (SEQ ID NO: 22), was used to induce expression of sgRNAs targeting the vioA promoter, the vioB promoter, the 5' end of vioC or two non-targeting sequences (Figure 5A). Overnight cultures of C. violaceum were diluted 1:100 in 96 deep-well blocks (Nunc, Denmark) and dCas was induced by adding 0.01% arabinose, 0.1% arabinose, or 1% arabinose. Controls were not induced. Cultures were grown overnight at 37 °C and 280 rpm, then transferred to 96 deep-well plates and violacein production was visualized by OD 600 was measured.

[0117] Example 3 Construction of a gold sensor from the native gold operon in Chromobacterium violaceum One of the important sensors in the gold bioleaching cycle is the gold sensor. Depending on the amount of leached gold, it can provide dynamic feedback to the bioleaching cycle and stop the leaching process when the gold ion concentration reaches a critical value. The only gold biosensor that has been demonstrated so far is the golTSB operon (SEQ ID NO: 23) from Salmonella enterica serovar Typhimurium str. LT2 strain. This gold biosensor contains GolS (SEQ ID NO: 24), which is the only MerR family transcriptional regulator that has been reported to be able to distinguish gold ions from copper and silver ions [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)]. This gold biosensor has previously been demonstrated only in E. coli and S. enterica, but in this report, we optimized it for the first time in C. violaceum to expand its dynamic range.

[0118] To characterize this gold sensor, the entire golTSB operon was first cloned upstream of the production of a fluorescent protein. 3+ The dynamic range of the fluorescence output is low at various concentrations (Figure 6A). 3+ This is due to the leaky expression in the absence of Au, which showed a high value of 14,842 RFU (Figure 6A). 3+ In C. violaceum, the gold sensor showed a low dynamic range and cytotoxicity, leading to a significant decrease in cell density at concentrations above 1 μM. This toxicity to cells may be due to the overexpression of GolT, a transmembrane P-type ATPase involved in membrane transport, which may disrupt the integrity of the cell membrane. The poor dynamic range and cytotoxicity of this gold sensor in C. violaceum led to the optimization of the golTSB operon.

[0119] To suppress leaky activation caused by GolS, P golTS When we induced the expression of GolS by replacing GolT with a weak constitutive promoter, the strength of the promoter was reduced and the positive feedback loop was abolished (Figure 6B). Furthermore, we constructed a minimal gold sensor by removing GolT and GolB, thereby minimizing the detrimental effects on cell growth. Next, P golTS (SEQ ID NO: 25) or P golB This construct was tested using (SEQ ID NO: 26) to induce the production of fluorescent protein (Figure 6B and Figure 6C). 3+ The maximum dynamic range of each concentration was 151-fold (minimum 110 RFU to maximum 16,416 RFU) for pGolTS and 113-fold (minimum 50 RFU to maximum 1,648 RFU) for pGolB.

[0120] No toxicity was observed for C. violaceum at 0–1 μM Au. 3+ Comparing pGolTS and pGolB in terms of concentration, pGolTS showed a larger fold change. 3+ In terms of concentration, the dynamic range of gene expression was 38-fold change (minimum 50 RFU to maximum 1887 RFU) for PgolB (Figure 6B) and 62-fold change (minimum 110 RFU to maximum 6737 RFU) for PgolTS (Figure 6C). The PgolTS construct was selected as the gold sensor to be used in the following experiments due to its high dynamic range.

[0121] Improving the dynamic range and sensitivity of gold sensors by mutagenesis in Chromobacterium violaceum After optimizing the circuit and regulatory mechanism of the gold sensor, we will further improve the sensitivity and dynamic range of the gold sensor by performing deep scanning mutagenesis on the GolS transcription activator. Although the structure of the GolS protein has not been elucidated, GolS belongs to the MerR protein family, which has a helix-turn-helix DNA-binding domain and a gold ion-binding domain (Figure 8A) [Checa, SK et al., Mol Microbiol, 63(5), 1307-1318 (2007)]. GolS, which is constitutively and weakly expressed, and P, which induces RFP, are expressed in the cytoplasm of the gold sensor. golTS Using the optimized circuit consisting of the above (Figure 7), we screened and selected GolS mutants with a wider dynamic range and higher sensitivity than wild-type GolS (Figure 12). Four mutants, GolSmt1 (A38I); GolSmt2 (A38Q and N97D); GolSmt3 (A38K and V60L); and GolSmt4 (D33P), were selected and their properties were evaluated. The structures of wild-type GolS and the GolSmt1, GolSmt2, and GolSmt3 mutants are shown in Figures 8 to 11, respectively.

[0122] These gold sensor mutants have higher sensitivity and a wider dynamic range than the wild-type gold sensor reported so far (Table 1). max The value increased to a maximum of B max The fold change was 3.5 times the value of the original value. Other parameters such as K, n, and C did not change by more than 2-fold, indicating that other properties remained relatively similar.

[0123] [Table 1]

[0124] In this system, sensitivity is an important parameter that determines the functionality of the gold sensor. The results showed that the gold sensor mutants were at least twice as sensitive to gold ions as the wild-type gold sensor. Fold changes indicating at least 3-fold induction were detected for these gold sensor mutants at 8 nM gold ions, whereas no fold change was detected for the wild-type gold sensor (Table 2).

[0125] [Table 2]

[0126] These values ​​indicate a higher sensitivity than the previously reported 2.3-fold induction of detection threshold by 33 nM gold ions in E. coli biosensors [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)]. These gold sensor mutants were able to detect only 80 nM Au. 3+ The fold change in output was more than 10-fold at the same gold ion concentration, whereas the wild-type gold sensor showed only a 3-fold induction at the same gold ion concentration, with a gold ion sensitivity of 4.5 RFU / nM. Both the wild-type and mutant gold sensors also showed slight leaky expression in the off state. The output in the off state was low, less than 100 RFU in both cases (GolSmt1: 51 RFU, GolSmt2: 54 RFU, GolSmt3: 97 RFU, WT: 47 RFU). Furthermore, the gold sensor mutants were able to generate a higher maximum output than the wild-type gold sensor before the gold ions reached toxic levels, with a fold change of more than 100-fold (GolSmt1: 45,088 RFU, GolSmt2: 36,074 RFU, GolSmt3: 41,388 RFU, WT: 12,811 RFU).

[0127] Since both mutants have mutations in the DNA-binding domain with a helix-turn-helix structure (left and right arrows, Figure 8B), the increased output of the transcription activator may be due to improved binding to the promoter region, which activates the promoter region, and this resulted in a higher maximum output in the gold sensor mutant (Figure 13). These transcription activator mutants also had increased affinity for binding to gold ions, which was supported by a left-shift in the response function to gold (Figure 13). Furthermore, there was no leaky expression at basal levels, suggesting that these mutations did not enhance activation in the uninduced state of the transcription activator. Thus, the enhanced transcription output, enhanced affinity for gold ions, and robust expression of the gold-sensing transcription activator mutants may be useful tools in future applications of gold ion sensing.

[0128] Mutagenesis of gold sensors Libraries of golS mutants were generated using QuikScan-19 and constructed using the QuikChange-HT kit (Agilent Technologies, Santa Clara, CA). By repeatedly replacing each amino acid with 19 other amino acids, custom mutagenesis oligos were obtained in which each amino acid in GolS was replaced with another 19 amino acid, and these were used in the QuikChange reaction. In the 154 amino acid long GolS, excluding the first amino acid residue, methionine, a total of 153 × 19 = 2907 single amino acid mutations can be generated. A total of six libraries with mutation regions of approximately 25 amino acids long relative to the full length of the GolS protein were generated according to the manufacturer's protocol. Competent cells of C. violaceum were then transformed with each library and plated on 1.5% bacterial agar-containing Tris minimal medium supplemented with 30 μg / mL kanamycin and 2 μM AuCl3. Colonies were observed under blue light, and colonies with higher RFP production compared to wild-type GolS were selected, inoculated into 96-well plates, grown, and fluorescence quantified using a BioTek Synergy H1 microplate reader.

[0129] Fluorescence measurements for characterization of quorum and gold sensors To characterize the gold sensor, cells were seeded into 96-deep-well blocks (Nunc, Denmark) from frozen stocks in LB medium and grown overnight at 37 °C. Cells were then diluted 1:200 in 96-well plates (Costar, Kennebunk) containing Tris minimal medium (pH 7.5) containing 80 mM NaCl, 50 mM Tris, 22 mM glucose, 20 mM KCl, 20 mM NH4Cl, 3 mM Na2SO4, 1 mM thiamine hydrochloride, 0.5 g / L yeast extract, 1 mM MgCl2, 0.65 mM Na2HPO4, and 0.1 mM CaCl2, and 0.001 μM–10 μM gold(III) chloride (Sigma-Aldrich) was added.

[0130] The diluted cells were then grown overnight at 37°C in a BioTek Synergy H1 microplate reader. Red fluorescence was measured every 10 minutes with an excitation wavelength of 530 nm and an emission wavelength of 600 nm (gain set to 50) over a period of 14-20 hours. Optical density was also measured by measuring absorbance at 600 nm. OD 600 And as a blank for the fluorescence values, only medium without cells was used.

[0131] Example 4 Dynamic regulation and biosensor integration using synthetic circuits for robust microbial cell factories The two main components used in microbial cell factories for gold bioleaching are the target element gold and cyanide, a leaching agent required to oxidize solid gold to obtain an aqueous solution of gold ions. Both gold ions and cyanide are highly toxic to microorganisms, making it difficult to use these components in living microbial cell factories. The toxicity of gold ions is due to the accumulation of Au(I)-S complexes, which induce oxidative stress [Reith, F. et al., Proc Natl Acad Sci USA, 106(42), 17757-17762 (2009)]. On the other hand, cyanide inhibits the respiratory system by binding to the metal in cytochrome oxidase [Knowles, CJ Bacteriol Rev, 40(3), 652-680 (1976); Knowles, CJ & Bunch, AW Adv Microb Physiol, 27, 73-111 (1986)]. Gold ions have also been shown to be toxic to bacteria at concentrations as low as 0.35 μM [Shareena Dasari, TP et al., Biochem Pharmacol (Los Angel), 4(6), 199 (2015)], whereas cyanide is toxic to bacteria at concentrations as low as 0.4 μM [Liu, W. et al., Chinese Journal of Chemistry, 25(2), 203-207 (2007)].C. violaceum has various cyanide detoxification mechanisms [Brysk, MM et al., J Bacteriol, 97(1), 322-327 (1969); Brysk, MM & Ressler, Journal of Biological Chemistry, 245(5), 1156-1160 (1970); Ressler, C. et al., Biochemistry, 12(26), 5369-5377 (1973)] that protect C. violaceum from the cyanide produced by C. violaceum. However, in the gold bioleaching method, the toxicity of gold ions becomes an urgent new problem for C. violaceum, which has never experienced the toxicity of gold ions. Therefore, we constructed a dynamic on / off circuit that turns on the production of cyanide only when the cell density is high and turns off the circuit when toxic gold ions are detected (Figure 14).

[0132] By adding an endogenous quorum sensing system to the circuit, the output can be activated only when the cell density is high enough (Figure 15). The gold sensor, which initiates the negative feedback loop of the synthetic circuit, is an activator, so a repressor must be introduced downstream to suppress it. PhlF (SEQ ID NO: 31), a strong repressor of the TetR family that has been previously characterized and reduces gene expression up to 193-fold [Stanton, BC et al., Nat Chem Biol, 10(2), 99-105 (2014)] is added downstream of the gold sensor to suppress the transcription of the output. In addition, an operator for PhlF (SEQ ID NO: 32) is added downstream of the quorum sensing promoter to physically suppress RNAP in the transcription process (Figure 15). The negative feedback loop of the gold sensor provides real-time feedback to the bioleaching process, and the bioleaching process is suppressed when cyanide is produced and gold ions are highly concentrated due to cyanide. When the repressor is expressed, it binds to the promoter region of the cyanogenic gene, which means that no further expression of the bioleaching enzyme occurs when high concentrations of gold ions are detected.

[0133] The circuit is tested in continuous batch cultures in which a 1:600 ​​dilution from the previous subculture is performed. 3+The circuit is turned off by culturing in the presence of gold ions or turned on by culturing in the absence of gold ions (Figure 16). In our study, the inhibitory effect was highest at a gold ion concentration of 2 μM, and toxicity of gold ions was observed at a concentration of 10 μM, so 2 μM gold ions are used to induce the off state. On the other hand, turning off the circuit means that low or no leaky expression is observed. When the circuit containing the wild-type gold sensor is turned off, the basal expression level increases with each cycle. The output of the circuit containing the wild-type gold sensor in the off state increased from 245 RFU in the first cycle to 765 RFU in the second cycle and 1047 RFU in the third cycle (Figure 17). In contrast, the output of the circuit containing the gold sensor mutant remained below 250 RFU in the off state in all three cycles (Figure 17).

[0134] The reduced basal expression level of the GolS mutants indicates an increased dynamic range of expression in continuous cell culture. The dynamic range of the wild-type gold sensor decreased from 15-fold in the first cycle to only 5-fold in the second cycle and 4-fold in the third cycle, whereas the circuit containing the GolS mutant maintained a dynamic range of more than 10-fold in all three cycles (Figure 17). The high dynamic range maintained in the circuit containing the GolS mutants suggests that this microbial cell factory for gold leaching has robustness and improved functionality capable of multiple on / off cycles.

[0135] Analysis of individual cells by flow cytometry further supports the improved robustness of the GolS mutant circuit by maintaining distinct ON and OFF populations through multiple consecutive cycles, as shown in representative histograms (Figure 18). (A) shows wild type, (B) shows GolSmt1, (C) shows GolSmt2, and (D) shows GolSmt3. The broadening of the OFF cell population observed in the wild type gold sensor (Figure 18A) suggests that the dynamic range of the circuit is reduced, mainly due to increased expression heterogeneity in this cell population. The robust maintenance of the OFF state in the circuit containing the gold sensor mutants may be due to the increased sensitivity to gold ions and thus the increased dynamic range of the gold sensor. Even with increasing cell passages, the 2 μM Au 3+ The higher the expression level of the PhlF repressor under the same induction condition by PhlF, the better the quorum sensing promoter can be repressed.

[0136] Another observation is that two of the GolS mutants (GolSmt2 (Figure 18C) and GolSmt3 (Figure 18D)) showed an increased population that remained in the off state throughout the three cycles, resulting in a slightly reduced dynamic range. This may be due to insufficient dilution of the PhlF repressor throughout the cycles, which resulted in a small population of cells being maintained in the off state. In these GolS mutants, the expression of the PhlF repressor was increased to maintain a good off-state output, but this effect may have been too strong, leading to insufficient dilution of the PhlF repressor in the cells to return the circuit to the on state. This suggests that GolSmt2 and GolSmt3 may not be suitable gold sensors to perform robust continuous on / off cycles in the gold bioleaching synthesis circuit.

[0137] The gold sensor mutants showed increased robustness of the circuit over three on / off cycles consisting of six cell passages (Figures 16-18). Since the toxicity of gold ions to cells means that more cycles are required for gold leaching, these properties will contribute greatly to the development of microbial cell factories for robust gold bioleaching.

[0138] Example 5 Conferring gold-reducing activity to mercury(II) reductase by directed evolution Determination of the minimum inhibitory concentration of Au(III) Au(III) is known to be toxic to many bacterial cells due to its high affinity for thiol groups (-SH), which can affect many metabolically important enzymes and membrane-bound proteins. Escherichia coli cells expressing a mutant MerA enzyme with improved gold reduction activity can be highly resistant to toxic levels of Au(III) added to the culture medium.

[0139] To perform the liquid dilution method, E. coli Rosetta(DE3)pLysS competent cells were transformed with the ligation mixture and plated on regular LB agar medium. A single colony was inoculated into 5 mL of Tris-buffered low-phosphate medium supplemented with the same antibiotic and various concentrations of AuCl3 for the liquid medium and grown at 37°C for 24 h. OD 600 The minimum inhibitory concentration (MIC) was determined as the lowest concentration of heavy metal that completely inhibited growth. Figure 23A shows that E. coli cells were inhibited by over 140 μM Au. 3+ This indicates that the bacteria were unable to grow at the above concentrations.

[0140] To perform the agar dilution assay of E. coli cells expressing wild-type MerA, cells expressing wild-type MerA were cultured, diluted, plated, and grown for 24 h at 37 °C in Tris-buffered low-phosphate agar medium (6.06 g / L Tris, 4.68 g / L NaCl, 1.49 g / L KCl, 1.07 g / L NH4Cl, 0.43 g / L Na2SO4, 0.2 g / L MgCl2·6H2O, 0.03 g / L CaCl2·2H2O, 0.23 g / L Na2HPO4·12H2O, 5.0 g / L glucose, 0.5 g / L yeast extract, and 15 g / L agar) supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG, and various concentrations of AuCl3. 3+ As the concentration increased, fewer colonies were observed on the agar plates. 3+ When the concentration reached 160 μM, cell proliferation was completely inhibited (FIG. 23B).

[0141] The toxicity of gold is probably due to its high affinity for thiol groups (-SH) present in many metabolically important enzymes and membrane-bound proteins. The binding of gold ions to metabolically important enzymes and membrane-bound proteins prevents these enzymes and proteins from binding to otherwise biologically relevant metal ions. The broth dilution method showed a slightly lower minimum inhibitory concentration than the agar plate dilution method. This difference was due to the higher Au concentration in the plate and broth cultures. 3+ This may be due to different distribution patterns of ions.

[0142] Construction of directed evolution libraries A codon-optimized synthetic gene encoding mercury(II) reductase (MerA) was cloned in frame into the pET20b expression vector (Novagen) with an N-terminal 6×His tag using the restriction enzymes NdeI and XhoI. To obtain MerA with improved reduction activity toward gold, a mutant library was constructed using error-prone PCR and site-saturation mutagenesis.

[0143] Error-prone PCR of the MerA gene (SEQ ID NO: 33; SEQ ID NO: 34) was performed using 50-100 ng of target DNA using the GeneMorph II Random Mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. Several colonies were randomly picked up and sequenced, confirming that a moderate mutation frequency (4.5-9 mutations / kb) was obtained. The primers used in this PCR reaction were epPCR-fw (5'-GTGGTGGTGGTGGTGCTCGAGTTA-3' (SEQ ID NO: 1)) and epPCR-rv (5'-GATATACATATGCACCACCATCACCATCAT-3' (SEQ ID NO: 2)). The thermal cycle was programmed as follows: initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1.5 min, followed by extension at 72°C for 10 min after the final cycle. The PCR product was then treated with DpnI to degrade the template and purified with a QIAquick PCR Purification Kit (Qiagen). The purified DNA fragment was double-digested with NdeI and XhoI, purified, and ligated into the pET20b vector.

[0144] Multi-site saturation mutagenesis was performed by overlap extension PCR using degenerate primers containing NNK at the target sites V317, Y441, C464 and C465'. These positions are in close proximity to the putative gold binding site within the active site of MerA (Figure 22). The redox-active cysteines at C136 and C141 were kept unchanged to prevent loss of redox activity. The primers used are summarized in Table 3.

[0145] [Table 3]

[0146] PCR reactions were performed using primer pairs T7-fw / V317NNK-rv, V317NNK-fw / Y441NNK-rv, Y441NNK-fw / C464NNK-rv, or C464NNK-fw / T7-rv to generate partially overlapping DNA fragments. A total reaction mixture of 50 μL containing 50 ng of pET20bMerA plasmid, 500 nM of each primer, and 1× PrimeSTAR Max Premix (Clontech) was incubated at 98°C for 10 s, 55°C for 5 s, and 72°C for 10 s for 30 cycles. The PCR products were purified, and equimolar amounts of each fragment were mixed with 1× PrimeSTAR Max Premix to carry out short overlap extension reactions (5 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 10 s), and 1 μL of the resulting reaction mixture was used as a template to amplify the full length of the MerA mutant gene with epPCR-fw and epPCR-rv primers. The PCR products were purified, digested with NdeI and XhoI, and ligated into the pET20b vector.

[0147] Selection of gold-resistant mutants Ligation mixtures obtained from error-prone PCR or site-saturation mutagenesis were used to transform E. coli Rosetta(DE3)pLysS competent cells, and the resulting transformants were plated on Tris-buffered low-phosphate agar medium (6.06 g / L Tris, 4.68 g / L NaCl, 1.49 g / L KCl, 1.07 g / L NH4Cl, 0.43 g / L Na2SO4, 0.2 g / L MgCl2·6H2O, 0.03 g / L CaCl2·2H2O, 0.23 g / L Na2HPO4·12H2O, 5.0 g / L glucose, 0.5 g / L yeast extract, and 15 g / L agar) supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG, and 250 μM AuCl3 and grown at 37 °C for 24 h. Colonies were then picked and grown in liquid Tris-buffered low-phosphate medium supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG, and 300 μM AuCl3. By repeating this selection process several times, toxic levels of AuCl3 were obtained that barely allowed cells expressing wild-type MerA to grow. 3+ We obtained 10 mutants that showed substantially better cell growth in the presence of MerA (300 μM) (Figure 24). It is noteworthy that all colonies that survived the second round of selection were from the site-saturation mutagenesis library, and this semi-rationally designed library may have a higher probability of yielding variants with improved functionality than a random mutagenesis library. The improved resistance over the wild type was due to: 1) MerA-mediated Au 3+ ion via enzymatic reduction and 2) Au 3+ MerA binding to Au 3+ This is thought to be due to two factors:

[0148] As shown in the TEM images (Figure 25), a MerA mutant was obtained that synthesizes gold nanoparticles from AuCl3. Directed evolution identified the MerA mutant V317S as an enhanced gold reductase. This mutant has the kinetic parameters summarized in Table 5. This enhanced gold reductase synthesizes gold nanoparticles from AuCl3. 3+Gold element (Au 0 ) is reduced by 67-fold.

[0149] [Table 4]

[0150] Further improved mutants The catalytic efficiency of MerA for gold reduction was 9.1 ± 3.2 × 10 1 It has been previously established that the Au 3+ A library of reducing MerA mutants was generated. Over 50% of the isolated mutants showed improved activity, with the best-improved mutants exhibiting up to 15-fold increased catalytic efficiency (Figure 26). The DM11 mutant (G415I) (SEQ ID NO: 48) reduced the turnover number (k cat ) and binding affinity (K M ) was improved, resulting in the greatest improvement in catalytic efficiency (15-fold increase). The kinetic parameters of all mutants are shown in Table 6.

[0151] This improved catalytic efficiency is also reflected in the complexity of the gold nanoparticles (AuNPs) formed upon reduction. DM11 produces larger sized and more complex gold nanoparticles compared to MerA (Figure 27). This improved catalytic efficiency is also reflected in the improved reduction and recovery of gold from e-waste leachates by DM11 compared to wild-type MerA (Figure 28). 3+ The recovery of Au from AuCl3 solution 3+ Recovery of Au from electronic waste leachate (67%) was also poor. 3+ It is equally effective in recovering (67%) the

[0152] [Table 5]

[0153] However, while an increased complexity of gold nanoparticles (AuNPs) is observed in the reduction from the AuCl3 solution, the complexity of gold nanoparticles is limited in the reduction from the leachate (Figure 30). This is because the AuNPs in the leachate 3+ This is likely due to the overall low amount of mercury in the mercury-resistant bacteria. If this limitation could be overcome, gold recovery could be greatly improved. MerA is an essential enzyme in bacterial mercury resistance systems. Based on the reaction mechanism of MerA and the available crystal structure, it was envisioned that genetic engineering of MerA for efficient gold reduction was possible. To achieve this, a high-throughput selection procedure was established that included a toxic agar medium selection followed by a more stringent liquid culture selection. A mutant library was constructed using error-prone PCR and multi-site saturation mutagenesis and subjected to this two-step selection. As a result, MerA mutants with enhanced gold reduction / gold recovery properties were identified. This result represents a part of the synthetic metal recovery technology in synthetic biology leaching.

[0154] Protein expression and purification The recombinant protein was expressed using the T7 expression system. The plasmid was transformed into Rosetta(DE3)pLysS cells and selected on LB agar medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. A single colony was picked and inoculated into 5 mL of LB medium containing the two antibiotics and grown overnight at 37°C. After 100-fold dilution, the OD 600Cultures were grown at 37°C until a Kα reached 0.6. Protein expression was then induced by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM. After induction of protein expression, cells were grown for an additional 18 h at 16°C. Cells were lysed by sonication, and the lysate was clarified and applied to a nickel chelate column (Qiagen) to purify the His-tagged protein. Protein samples in the eluate (500 mM imidazole, 50 mM Tris-Cl (pH 7.5), 300 mM NaCl) were concentrated and dialyzed against 20 mM sodium phosphate (pH 7.4) using Amicon Ultra centrifugal filters (Millipore).

[0155] Gold reduction assay Enzyme assays were performed at 25 °C in 20 mM sodium phosphate (pH 7.4), 200 μM NADPH and 100 μM AuCl3. The oxidation of NADPH was followed spectrophotometrically at 340 nm. A unit of enzyme activity is defined as the amount of enzyme that catalyzes the Au-dependent oxidation of 1.0 μmol of NADPH per minute.

[0156] Example 6 Kinetic parameters of gold reduction by MerA A continuous spectrophotometric assay (Scheme 1) was used to measure the kinetic parameters of purified mercuric reductase (MerA) towards AuCl3 substrate.

[0157] Scheme 1: Assay to monitor MerA activity by change in absorbance at 340 nm. M is Hg 2+ or Au 3+ and X is GSH - or Cl. [ka]

[0158] Natural substrate Au 3+ FromAu 0 The reduction of NADPH to NADP+ The oxidation of NADPH was monitored by measuring the change in absorbance at 340 nm. The reaction mixture (50 μL) contained 100 mM PIPES (pH 7.0), 400 μM NADPH, 17.9 μM MerA, and various amounts of Au. 3+ In this assay, Hg(GSH)2 was used as a substrate to detect Hg, the natural substrate of MerA. 2+ from Hg 0 A reduction to was observed.

[0159] The kinetic parameters of MerA with Hg(GSH)2 and AuCl3 were determined (Table 4).

[0160] [Table 6]

[0161] When mercury was used as a substrate, the K M The kcat value for MerA was 96.3 ± 57.6 μM, which is higher than the 10.7 μM reported previously in the literature. The kcat value for MerA was 14.6 ± 5.1 / s, which is slightly faster than the previously reported 9.43 / s, but not significantly different. The overall catalytic efficiency (k cat / K M ) value is 1.5±0.7×10 5 / M / sec, which is 8.8 × 10 5 / M / sec, which is lower than expected, 6-fold lower (Moore, MJ, Miller, SM, Walsh, CT C-Terminal Cysteines of Tn501 Mecuric Ion Reductase (1992) Biochemistry 31(6):1677-85). No data are reported for AuCl3. Compared to the natural substrate Hg(GSH)2, the k cat / K MThe value is four orders of magnitude smaller. Thus, this assay can be used to measure the reducing ability of MerA towards different substrates.

[0162] A gold-sensing biosensor screen identified MerA mutants with improved gold reduction activity. The golTSB operon isolated from Salmonella enterica serovar typhimurium functions as a biosensor with green fluorescent protein (GFP) as a reporter (Zammit et al., 2013) (Figure 19). This biosensor detects Au + Complex or Au 3+ It is under the control of the GolS regulator, which is induced in the presence of the Au complex. + ion or Au 3+ When ions interact with the GolS regulator, it binds to the target promoter sequence golB. This induces a conformational change in the GolS / golB complex, which promotes transcription of the GFP reporter. Here, we propose to integrate this circuit into the chromosome of E. coli as previously reported [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)] and use this E. coli strain as an endogenous reporter for gold reduction.

[0163] The biosensor was cloned into the pRSFDuet-1 vector and tested for its responsiveness to gold as a substrate. Fluorescence was measured using a filter at 485.20 nm as the excitation wavelength and a filter at 528.20 nm as the emission wavelength. The final optical density (OD) of each sample at 600 nm was 600 ) was also measured. Fluorescence measurements (F s ) was normalized using the following formula (Equation 1):

number

[0164] Up to 100 μM Au 3+ Fluorescence increased with increasing concentration (Figure 20). At higher concentrations, the fluorescence decreased rapidly, probably due to the sensitivity of E. coli to metal toxicity. 3+ The detection limits, i.e., the lowest and highest concentrations of gold at which an increase in fluorescence compared to the background can be detected, were found to be between 100 nm and 100 μM. Thus, the sensor was able to detect a wide range of concentrations of Au. 3+ The ability to distinguish between these two mutants can be used to screen for optimal gold-reducing MerA mutants.

[0165] Screening for optimal gold-reducing MerA mutants can be performed based on the observation of a decrease in fluorescence, which is in turn related to the presence of Au in the cells. 3+ This corresponds to a decrease in the gold ions and therefore serves as an indirect reporter of gold-reducing activity (Figure 21). In the presence of gold ions and a control plasmid, the biosensor is induced to produce a strong fluorescent signal, whereas in the presence of the gold-reducing MerA mutant, the gold ions are reduced and no fluorescence is produced.

[0166] Example 7 High sensitivity of gold biosensor to silver ions in Chromobacterium violaceum The biosensor shown in Figure 31A was tested for its sensitivity to various metal ions in minimal medium. The biosensor senses gold and silver ions, but does not show high response to other metals such as cadmium, zinc, mercury, nickel, cobalt, iron, and copper ions (Figure 31B). The dose response indicates that the mutant biosensor has a wider dynamic range and higher sensitivity to gold and silver ions than the wild-type biosensor (Figure 32).

[0167] Example 8 Sensing of precious metal ions leached from electronic waste by C. violaceum C. violaceum was incubated with electronic waste and cultured for 6 days to leach precious metals from the electronic waste into the aqueous culture. After 6 days of culture, C. violaceum cells were centrifuged and the resulting spent medium was added to C. violaceum containing the biosensor used in Example 7 (Figure 33). The transcription factor biosensor was activated in the spent culture medium containing leached scrap metal (ESM) from electronics, but not in the control culture medium (Figure 34). The mutant biosensor had a fluorescence output of 2515 RFU in response to the 6-day spent culture containing leached scrap metal (ESM) from electronics, whereas the wild-type biosensor had a fluorescence output of 1101 RFU. ICP analysis of the 6-day culture showed the presence of 1.1 ppm (5.6 μM) gold ions, 0.34 ppm (3.2 μM) silver ions, and 12.3 ppm (193.2 μM) copper ions in the spent medium. This result indicates that the C. violaceum biosensor was activated in the presence of gold and silver ions leached from the medium containing e-waste. This property is useful for activating further synthetic circuit modules in C. violaceum.

[0168] summary 1. In the present invention, by using the mercuric reductase (Mer) system, it is possible to cyanate metals and reduce metal-cyanide complexes by biological techniques. 2. In the present invention, the tool of the present invention allows the construction of a synthetic circuit for biological leaching, which can be applied to the continuous and appropriate expression and activation of individual biological systems for the purpose of efficient metal recovery. The tool of the present invention has the following characteristics: a. The expression of the cyanide-producing synthetic module can be regulated and induced by adding e-waste to the bioreactor, allowing for the timely production of cyanide-based leachants for metal bioleaching. b. Detection of individual metal-cyanide complexes results in the specific and transient expression of synthesis modules for metal recovery. Due to the heterogeneity of e-waste, the concentrations of each metal (and therefore the metal ion concentrations) vary widely, making it necessary to temporarily remove each metal. This can be achieved by selective expression of the genetically engineered Mer system specific for the valence and type of metal ion. c. After metals are recovered, excess cyanide leaching agents can be metabolized by regulated induction of the expression of a synthetic module for cyanide degradation, returning the carbon and nitrogen atoms of cyanide to the central metabolic pathway.

[0169] References Arazoe, T., Kondo, A., & Nishida, K. (2018). Targeted nucleotide editing technologies for microbial metabolic engineering. Biotechnology journal, 1700596. Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res, 41(15), 7429-7437. doi:10.1093 / nar / gkt520. Brandl, H., Lehmann, S., Faramarzi, M. A., and Martinelli, D. (2008), Biomobilization of silver, gold, and platinum from solid waste materials by HCN forming microorganisms, Hydrometallurgy 94, 14-17. Brysk, M. M., Corpe, W. A., & Hankes, L. V. (1969). Beta-cyanoalanine formation by Chromobacterium violaceum. J Bacteriol, 97(1), 322-327. Brysk, M. M., & Ressler, C. (1970). γ-Cyano-α-l-aminobutyric acid a new product of cyanide fixation in Chromobacterium violaceum. Journal of Biological Chemistry, 245(5), 1156-1160. Cerminati, S., Soncini, F. C., & Checa, S. 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Claims

1. 1. An isolated genetically modified bacterium, comprising: transformed with at least one polynucleotide molecule, A genetically modified bacterium, wherein said at least one polynucleotide molecule comprises a heterologous nitrilase gene operably linked to at least one promoter, and wherein hydrogen cyanide is decomposed by a gene product of said nitrilase gene.

2. 2. The isolated genetically modified bacterium of claim 1, wherein the heterologous nitrilase gene encodes an enzyme selected from the group consisting of cyanogen dehydratase and cyanogen hydratase, and / or the at least one polynucleotide molecule further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

3. 3. The isolated genetically modified bacterium of claim 1 or 2, which is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli.

4. 1. A method for degrading cyanide utilizing synthetic biology technology, comprising contacting at least one recombinant cyanide-degrading bacterium engineered to express at least one nitrilase enzyme with cyanide-containing nitriles remaining after bioleaching of electronic waste.

5. 5. The method of claim 4, wherein the at least one nitrilase enzyme is selected from the group comprising cyanogen dehydratase and cyanogen hydratase, and / or the at least one recombinant cyanide degrading bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase and phosphoenolpyruvate carboxylase.

6. 6. The method of claim 4 or 5, wherein the at least one nitrilase enzyme is derived from at least one bacterial species selected from the group comprising Pseudomonas pseudoalcaligenes (nit), Synechocystis sp. PCC 6803 chromosome (SC-nit), Bacillus pumilus cyandihydratase (BP-cynD) and Pseudomonas stutzeri (PS-cynD).

7. 1. An isolated recombinant DNA molecule comprising, in N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or more cyanogenic genes operably linked to a promoter that is activated by CviR; A recombinant DNA molecule comprising:

8. The isolated recombinant DNA molecule of claim 7, wherein the golS transcriptional activator gene is a mutant selected from the group including or consisting of GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L) and GolSmt4 (D33P).

9. 1. Use of an inactive Cas9 and an RNA guide (sgRNA) to suppress transcription of one or more genes by targeting the promoter regions of the one or more genes in the genome of Chromobacterium violaceum, wherein the inactive Cas9 comprises an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain.

10. The gene encoding the inactive Cas9 is araBAD 10. The use of claim 9, wherein the gene encoding the RNA guide (sgRNA) is operably linked to a promoter, and the gene encoding the RNA guide (sgRNA) is operably linked to a strong constitutive promoter, such as J23119.

11. An isolated recombinant DNA molecule comprising a golTSB operon, the golTSB operon comprising, from N-terminus to C-terminus, golT operably linked to the j23119 promoter, golS, golB operably linked to the golB promoter, and a reporter gene (such as GFP).

12. 1. An isolated genetically modified bacterium, comprising: transformed with at least one polynucleotide molecule, A genetically modified bacterium, wherein said at least one polynucleotide molecule comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

13. the hydrogen cyanide synthase gene is hcnABC, the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or the isolated genetically modified bacterium comprises, in the N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to a promoter activated by CviR; 13. The isolated bacterium of claim 12, further comprising at least one recombinant polynucleotide DNA molecule comprising:

14. The isolated bacterium of claim 13, wherein the golS gene is codon-optimized for Chromobacterium violaceum and / or the golS gene is a mutant selected from GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L) and GolSmt4 (D33P).

15. 2. The isolated bacterium according to any one of the preceding claims, which is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli and / or is stable at pH 10.

16. A method for producing a cyanide-based leachant utilizing synthetic biology techniques, comprising: contacting at least one recombinant cyanide-producing bacterium with glycine; The method, wherein the bacterium comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

17. the hydrogen cyanide synthase gene is hcnABC, the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or the recombinant cyanide-producing bacterium contains, in the N-terminal to C-terminal direction: (i) a golS transcriptional activator gene operably linked to a constitutive promoter; and golTS Promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) a promoter activated by CviR and an operator for PhlF; and (iii) one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to a promoter activated by CviR; 17. The method of claim 16, further comprising at least one recombinant polynucleotide DNA molecule comprising:

18. The method of claim 17, wherein the golS gene is codon-optimized for Chromobacterium violaceum and / or the golS gene is a mutant selected from GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L) and GolSmt4 (D33P).

19. 19. The method of any one of claims 16 to 18, wherein said at least one recombinant cyanogenic bacterium is tolerant to a pH of about 10.

20. 20. The method of any one of claims 16 to 19, wherein the production of a cyanide leachant using synthetic biology techniques and the bioleaching of metals are carried out in a single reactor.

21. At least one isolated recombinant cyanogenic bacterium capable of carrying out the synthetic biology based production of a cyanide based leaching agent according to any one of claims 16 to 20.

22. 22. At least one recombinant cyanogenic bacterium according to claim 21, selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli.

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