Recovery of anthropogenic precious metals and toxic metals using synthetic biological leaching methods.

Abstract

This invention provides a method for using enzymes to cyanide a metal and reduce the metal-cyanide complex, as well as a method for using biological techniques to hydrolyze cyanide. [Solution] The present invention relates to the use of genetically modified bacteria in a process comprising the generation of cyanide, the reduction of leached metal ions, the decomposition of cyanide, and the subsequent reuse of cyanide. Furthermore, it provides a transcriptional regulatory tool in Chromobacterium violaseum. In one embodiment, the present invention provides a genetically modified bacterium that is isolated and transformed by at least one polynucleotide molecule, wherein the polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter, and comprises at least one mutation in which a metal ion is reduced by the gene product of the MerA gene to produce a metal element as metal nanoparticles.

Description

[Technical Field]

[0001] The present invention relates to a method for degrading cyanide using enzymes, and to a method for performing a synthetic biological leaching method by recovering metals using synthetic biotechnology. More specifically, the present invention relates to the use of genetically modified bacteria used in a process comprising cyanide generation, reduction of leached metal ions, degradation of cyanide, and subsequent reuse of cyanide. Furthermore, it provides a transcriptional regulatory tool in Chromobacterium violaseum. [Background technology]

[0002] Cyanides readily combine with most major and trace metals to form cyanide complexes, and this property makes them useful in extracting metals from ore. Sodium cyanide is the most commonly used in mining sites and readily dissolves in water, forming sodium ions and cyanide ions (CN). - ) results in CN - A portion of it is converted to hydrogen cyanide (HCN), and its relative amount is determined by the pH of the water. At pH levels above 9.0, most of it is stable CN. - It exists in this form. As the pH decreases, it is converted to HCN. - As the amount of cyanide increases, HCN readily generates gas and is released into the air. Therefore, most mining solutions are maintained at a pH above 10.0, which prevents the generation of HCN gas and poisoning accidents among miners due to its inhalation. Since cyanide is a carbon-based compound, it readily reacts with other carbon-based substances and becomes toxic to many organisms. Therefore, waste containing cyanide must be detoxified before disposal. The conventional detoxification method is the alkaline chlorination method, but this method is dangerous and costly. Furthermore, problems arise if the cyanide used in mining does not decompose rapidly into harmless substances. In addition, less toxic substances may remain in the environment for long periods, causing problems in aquatic ecosystems.

[0003] The electronic waste recycling industry employs chemical methods that pose significant environmental risks. Current methods used for recovering precious metals such as gold and removing toxic metals such as lead and mercury include dry smelting (such as open combustion) and wet smelting (acid leaching and industrial cyanide treatment or cyanide baths). These methods consume a great deal of energy, require further electrolytic processes for metal separation, and are extremely environmentally 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 made to replace industrial chemical leaching methods with biotechnological leaching methods to make metal recovery and purification easier, more cost-effective, and less environmentally harmful. Many scientists and engineers, including 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 conventional techniques that recover precious metals by dissolving them in acid, current attempts to recover precious metals such as gold through bio-remediation of electronic waste utilize microorganisms that produce leaching agents (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 such microorganisms, the leaching agent used for bio-remediation and recovery of metals is typically hydrogen cyanide. While hydrogen cyanide leaks pose a significant environmental threat, the microorganisms used in the biomining industry not only produce cyanide (or its equivalents) but also decompose cyanide (detoxifying its equivalents). Therefore, concerns about hydrogen cyanide leaks are limited or minimal. Consequently, large-scale releases of cyanide into the environment rarely occur with bioleaching.

[0004] Bioleaching, which uses natural microorganisms under mild operating conditions, allows for the recycling of metals through a process that closely resembles biogeochemical cycles in nature, unlike existing methods, and as a result, can reduce the demand for resources such as ore, energy, and landfill (Brandl, H., Lehmann, S., Faramarzi, MA, and Martinelli, D. (2008), Hydrometallurgy 94, 14-17). Bioleaching is attracting attention as a "clean technology." Hydrogen cyanide is produced as an extractant by various bacteria (e.g., Chromobacterium violaceum, Pseudomonas fluorescein, 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 for a short period during the lifespan of these microorganisms. Although the production of cyanide by microorganisms has been known for many years, there has been no quantitative data on cyanide production in various species (Liang, G., Mo, Y., and Zhou, Q. (2010), Enzyme and Microbial Technology 47, 322-326). However, current attempts at biorecovery and bioremediation have failed to meet 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, rapid bioleaching, and metal separation without reliance on conventional electrolysis). As a result, metal remediation is still being carried out using conventional wet and dry smelting methods.The discrepancies between these expectations and reality stem from the insufficient metabolism of the unique leaching agents produced by microorganisms, and the lack of appropriate biological reduction pathways for recovering specific metals after bioleaching.

[0005] To protect the environment and conserve natural resources, there is a strong need to develop sustainable technologies for the reuse of electronic waste. This invention focuses on the recovery of precious metals and the removal of toxic metals from electronic waste. Conventional electronic waste treatment technologies currently used, which involve strong acids or cyanides, are highly environmentally polluting. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention generally relates to a method for cyanidating a metal and reducing a metal-cyanide complex using biological techniques, and to a method for hydrolyzing cyanides using biological techniques.

[0007] The present invention enables the construction of an integrated synthetic biosystem for metal leaching that can be introduced into a synthetic host (such as Chromobacterium violaseum, which produces cyanide), allowing for the efficient recovery of precious metals and purification of toxic metals from electronic waste. The design and construction of this synthetic host may consist of up to four main components / modules, namely, 1) cyanide production using synthetic biotechnology; 2) metal recovery using synthetic biotechnology; 3) cyanide decomposition using synthetic biotechnology; and 4) a synthetic circuit for the biological leaching method. The present invention enables the construction of a synthetic circuit for the biological leaching method.

[0008] The inventors have been able to provide industry with a sustainable method for gold cyanidation by designing and constructing a tool for removing excess cyanide from the environment. Furthermore, the inventors have designed and constructed a genome editing tool for Chromobacterium violaceum for the purpose of recovering precious and toxic metals from electronic waste, and have constructed synthetic C. violaceum. In addition, the inventors have developed a method for synthesizing gold ions (Au 3+ ) is reduced to the state of gold element (Au) as gold nanoparticles, and / or silver ions (Ag + We designed and built a tool to reduce ) back to the state of elemental silver (Ag) as silver nanoparticles, providing the industry with an alternative to conventional recovery processes that utilize electrolysis. [Means for solving the problem]

[0009] According to a first aspect, the present invention relates to an isolated genetically modified bacterium, It is transformed by at least one polynucleotide molecule, The present invention provides a genetically modified bacterium in which the at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter, and comprises at least one mutation such that the gene product of the MerA gene reduces metal ions to produce metal elements as metal nanoparticles.

[0010] Another embodiment is an isolated genetically modified bacterium, It is transformed by at least one polynucleotide molecule, The present invention provides a genetically modified bacterium comprising a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene, wherein at least one polynucleotide molecule is operably linked to at least one promoter.

[0011] In some embodiments, the isolated genetically modified bacteria are arranged from the N-terminus to the C-terminus. (i) A golS transcriptional activator gene operably linked to a constitutive promoter, and P golTS a promoter or P golB a ph1F repressor gene operably linked to a promoter; (ii) A promoter activated by CviR, an operator of PhlF, and (iii) One or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase variant gene operably linked to a promoter activated by CviR and further comprising at least one recombinant polynucleotide DNA molecule.

[0012] Another aspect is a method for recovering gold element from gold ions (Au 3+ ) as gold nanoparticles or recovering silver element from silver ions (Ag + ) as silver nanoparticles, comprising: a) contacting an isolated genetically recombinant bacterium according to any one of claims 1 to 11 with a leaching solution containing gold ions (Au 3+ ) and / or silver ions (Ag + ); and b) recovering nanoparticles of gold element and / or silver element from the leaching solution and providing a method comprising.

[0013] According to another preferred embodiment, the present invention is a method for producing an isolated genetically recombinant bacterium, comprising: a) performing error-prone PCR on a gene encoding mercury (II) reductase (MerA); i) transforming at least one bacterium with the product of the PCR; and ii) selecting a transformant that grows in a medium containing Au 3+ and / or Ag + ; or b) performing overlapping extension PCR on a gene encoding mercury (II) reductase (MerA) to introduce saturated mutations at multiple sites; i) a step of transforming at least one bacterium with the PCR product; and ii) Au 3+ and / or Ag + A step of selecting transformants that grow in a culture medium containing Includes, The genetically modified bacteria are 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 the gene product of the MerA gene contains gold ions (Au 3+ ) is reduced to produce gold elements as gold nanoparticles, or silver ions (Ag + The present invention provides a method comprising one or more mutations that cause the silver element to be reduced to produce silver nanoparticles. It is easy to understand that the gold used in the aforementioned growth medium may be in another form (e.g., AuCl).

[0014] Another embodiment is an isolated genetically modified bacterium, It is transformed by at least one polynucleotide molecule, The present invention provides a genetically modified bacterium in which at least one polynucleotide molecule comprises a heterologous nitrilase gene operably linked to at least one promoter, and hydrogen cyanide is degraded by the gene product of the nitrilase gene.

[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, all operably linked to at least one promoter.

[0016] Another embodiment is a method for producing a cyanide-based leaching agent using synthetic biotechnology, The process includes the step of contacting at least one recombinant cyanide-producing bacterium with glycine, The present invention provides a method in which at least one recombinant cyanide-producing bacterium is recombinant to express heterologous hydrogen cyanide synthase (hcnABC) genes and heterologous 3-phosphoglycerate dehydrogenase mutant (serA) genes by ligating at least one promoter to these genes. An example of this is shown in Figure 2A.

[0017] In some embodiments, the recombinant cyanide-producing bacteria are arranged in the direction from the N-terminus to the C-terminus. (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB A ph1F repressor gene operably linked to a promoter; (ii) Promoter activated by CviR and operator of PhlF, and (iii) Either 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. It further comprises at least one recombinant polynucleotide DNA molecule containing [the specified compound].

[0018] In another aspect, the present invention provides at least one isolated recombinant bacterium capable of producing cyanide-based leaching agents using synthetic biotechnology as described herein.

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

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

[0021] In another embodiment, the present invention relates to an isolated recombinant DNA molecule, wherein the direction from the N-terminus to the C-terminus, (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB A ph1F repressor gene operably linked to a promoter; (ii) The promoter activated by CviR, the operator of PhlF, and (iii) One or more cyanide-producing genes operably linked to a promoter activated by CviR This provides recombinant DNA molecules containing [the specified substance].

[0022] In another embodiment, the present invention provides the use of inactive Cas9 and sgRNA for repressing the transcription of one or more genes by targeting the promoter regions of the genes in the genome of Chromobacterium violaseum, wherein the inactive Cas9 contains an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain.

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

[0024] Another embodiment provides an isolated recombinant DNA molecule comprising a golTSB operon, the golTSB operon comprising golT, golS, golB, operably ligated to a j23119 promoter, and a reporter gene (such as GFP), from the N-terminus to the C-terminus. This operon can be used for general screening of recombinant bacteria that reduce gold by MerA. [Brief explanation of the drawing]

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

[0026] [Figure 2]This paper outlines the processes of cyanide degradation and coupling. Four nitrilase variants were selected from various bacteria. These sequences were synthesized and cloned into host cells. More specifically, the four nitrilase variants used were: nitrilase from Pseudomonas pseudoalcaligenes (nit), nitrilase from the chromosome of Synechocystis genus PCC 6803 (SC-nit), cyanide dihydratase from Bacillus pumilus (BP-cynD), and cyanide dihydratase from Pseudomonas stutzeri (PS-cynD). nit contains two distinct subunits, nitB and nitC. These two subunits were cloned into the pRSF-Duet vector and expressed in Escherichia coli (DE3) BL21 strain as the host cell. The remaining three variants were cloned into pGM vectors and incorporated into the genome of Chromobacterium violaceum using the Tn7 transposition system.

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

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

[0029] [Figure 5] This shows the suppression of violacein production in C. violaceum using dCas9. (A) A schematic diagram of the dCas9 circuit is shown. (B) Target regions in the violacein operon are shown as three dCas9-sgRNA complexes, the vioA promoter, the vioB promoter, and the 5' region of vioC. (C) The amount of dCas9 induction required by gRNA targeting the vioA promoter is the smallest (0.01% arabinose), and the amount of dCas9 induction required by gRNA targeting the vioB promoter is slightly higher (0.1% arabinose). On the other hand, gRNA targeting vioC, a transcription start site further downstream, has the same effect as the two non-target-directed gRNA-negative controls shown. (D) OD600 measurements corresponding to each well shown in (C) are shown.

[0030] [Figure 6] This shows the optimization of the gold sensor circuit. (A) The dose-response to gold ions using the original GolTSB operon or GolS transcription activator and (B) PgolTS or (C) PgolB, along with schematic diagrams of these circuits.

[0031] [Figure 7] A schematic diagram of the gold sensor variant circuit is shown.

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

[0033] [Figure 9] This shows the site of the A38I mutation in GolSmt1. It is a single mutant in which the 38th residue is replaced from alanine to isoleucine (circled area). Both alanine and isoleucine are hydrophobic, but isoleucine has three more carbon atoms in its hydrophobic side chain than alanine. This is thought to increase hydrophobicity, increase aggregation to the hydrophobic center, and result in stronger binding to DNA.

[0034] [Figure 10] The A38Q and N97D mutations in GolSmt2 are shown. These are double mutants in which residue 38 is substituted from alanine to glutamine (circled in the DNA-binding domain) and residue 97 is substituted from asparagine to aspartic acid (circled in the ion-binding domain). In the first mutation, alanine is a nonpolar neutral amino acid, and glutamine is a polar neutral amino acid with an amide side chain, but the long polar side chain of glutamine is thought to improve 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 is thought to increase affinity for positively charged gold ions or promote dimerization.

[0035] [Figure 11] The A38K and V60L mutations in GolSmt3 are shown. These are double mutants in which residue 38 is replaced from alanine to lysine (outer circle) and residue 60 is replaced from valine to leucine (inner circle). In the first mutation, alanine is a nonpolar neutral amino acid and lysine is a polar basic amino acid, but it is thought that lysine improves binding to the phosphate group of the DNA backbone. In the second mutation, both valine and leucine are nonpolar neutral amino acids, and since leucine has one more carbon atom in its side chain than valine, it is thought that the overall hydrophobicity of the DNA-binding domain increases.

[0036] [Figure 12] This document describes an experimental workflow for mutagenesis and selection of gold sensor mutants in C. violaceum. A template for a mutant library of golS transcription activator was created by substituting each amino acid with 19 other amino acids using deep scanning mutagenesis. Next, the mutant library was cloned into C. violaceum for transformation. Mutants exhibiting high fluorescence output and high sensitivity to gold ions were selected.

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

[0038] [Figure 14] A schematic diagram of a synthetic circuit is shown, illustrating a method for incorporating dynamic regulation into a new system. The circuit autonomously turns on via quorum molecules, and when gold ions leach from electronic waste, a gold sensor notifies the circuit, turning it off.

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

[0040] [Figure 16] A schematic diagram of a batch culture of Chromobacterium violaseum to investigate the on / off output in response to gold ions is shown. To investigate the on / off output of the circuit of the present invention, small batch cultures of C. violaseum were cultured in 2 mL Eppendorf tubes in 300 μL of Tris minimal medium supplemented with 30 μg / mL kanamycin at 37°C for 24 hours. Each passage was diluted 1:600 ​​and cultured in a quiescent state, with 2 μM AuCl3 added every other passage. Using a BD Accuri C6 flow cytometer (BD Bioscience, Singapore), the fluorescence output of single cells at the endpoint was measured under conditions of a flow rate of 14 μL / min and a core size of 10 μm, and 10,000 events were collected for each sample. The excitation wavelength for fluorescence was 561 nm, and the detection wavelength was 610 nm or 620 nm. Cells were gated with forward and side scattering light. The background fluorescence of control cells that do not express RFP was subtracted from the average fluorescence value.

[0041] [Figure 17]The graph shows that when cells were successively passaged in the presence or absence of gold ions, and a circuit containing a mutant gold sensor was compared with a circuit containing a wild-type gold sensor, basal-level leakage expression was reduced in the circuit containing the mutant gold sensor. Each passage was cultured in minimal medium after diluting the previous passage culture to 1:600, and the circuit was either turned on without adding Au3+ or turned off with the addition of 2 μM Au3+.

[0042] [Figure 18] The graph shows that when circuits containing each GolS variant (B: GolSmt1, C: GolSmt2, D: GolS mt3) were subjected to three on / off cycles, distinguishable on and off populations were observed. However, in the circuit containing the wild-type gold sensor (A), the on and off populations were mixed.

[0043] [Figure 19] A schematic diagram of the biosensor's structure is shown. The gol operon of the Salmonella genus consists of golT, which encodes a P-type ATPase; golS, which encodes an Au sensor; and golB, which encodes a metal-binding chaperone. The constitutive promoter J23119 in E. coli expresses the Au sensor, which is used for binding Au3+ ions. This Au sensor then binds to the Au-sensing promoter golB, inducing GFP expression.

[0044] [Figure 20] This graph shows the biosensitivity of golGFP as the Au3+ concentration (μM) increases.

[0045] [Figure 21] A schematic diagram of the screening process for identifying gold-reducing mutants is shown.

[0046] [Figure 22] This shows the target sites of MerA proteins whose sequences have been altered to change their binding affinity and / or specificity to metals.

[0047] [Figure 23] (A) The effect of Au3+ on the proliferation of Escherichia coli Rosetta(DE3)pLysS cells in liquid medium and (B) agar medium is shown.

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

[0049] [Figure 25] TEM images of gold nanoparticles synthesized using the MerA mutant are shown.

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

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

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

[0053] [Figure 29]This shows a comparison of the Au content before and after Au recovery using DM11, as well as at downstream processing stages.

[0054] [Figure 30] The following are TEM images of gold nanoparticles (AuNPs) recovered by DM11 from AuCl3 solution or leachate from electronic waste. (A) Shows truncated tetrahedral gold nanoparticles recovered from AuCl3 solution. (B) Shows gold nanoparticles in the form of two truncated tetrahedrons joined together, recovered from AuCl3 solution. (C) Shows truncated tetrahedral gold nanoparticles recovered from leachate.

[0055] [Figure 31] (A) A schematic diagram of gold and silver biosensors in C. violaceum is shown. (B) A graph showing the sensitivity of the variant gold sensor to various metal ions in minimal culture medium, namely 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] [Figure 32] The dose-response curves of wild-type and mutant biosensors for various metal ions in minimal culture media are shown.

[0057] [Figure 33] This document outlines the biosensing process of C. violaceum against gold and silver using metals leached from scrap metals of electronic devices.

[0058] [Figure 34] This graph shows the noble metal ion sensing ability of wild-type and mutant biosensors in a mixture of metal ions leached from electronic scrap metal (ESM). [Modes for carrying out the invention]

[0059] For easy reference, a list of references cited herein is appended after the examples. While the entire contents of each of these references are incorporated herein by reference, the references made herein do not imply that these references constitute common technical knowledge.

[0060] To use genetically modified bacteria in a process involving cyanide generation, reduction of leached metal ions, degradation of cyanide, and subsequent reuse of cyanide, it is assumed that separate bacterial strains with distinct mutant enzymes are necessary. Some bacterial strains selectively reduce gold, and others selectively reduce silver. A feasible workflow would involve oxidizing the metal with a bacterial strain that produces a biological leachate, then selectively reducing the metal to recover it with another bacterial strain utilizing gold-reducing and silver-reducing mutants, and finally biologically purifying the biological leachate by biodegrading the cyanide using yet another bacterial strain.

[0061] Before disclosing and describing the compounds, compositions, articles, apparatus and / or methods of the present invention, it should be noted that the compounds, compositions, articles, apparatus and / or methods of the present invention are not limited to specific synthesis methods or specific recombinant biotechnology methods unless otherwise specified, nor are they limited to specific reagents unless otherwise specified, and therefore, needless to say, may be modified in various ways. It should also be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not limited to any particular embodiment.

[0062] Definition of Terms For convenience, the specific terms used in this specification, the examples, and the appended claims are summarized below.

[0063] In this specification, the terms "a" and "an" are used to refer to one or more (i.e., at least one) things that are the grammatical objects of these articles.

[0064] In this specification, the terms “comprising” or “including” are interpreted as identifying the presence of features, components, processes, or ingredients described herein as indicated by those terms, but do not exclude the presence or addition of one or more other features, components, processes, or ingredients or groups thereof. Furthermore, 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” and “comprises,” and variations of the term “including,” such as “include” and “includes,” have similarly broad meanings.

[0065] In this specification, numerical ranges may be indicated as an "approximate" specific number and / or a range from an "approximate" specific number to another "approximate" specific number. Where such ranges are described, another embodiment includes the non-approximate specific number and / or the non-approximate range from the non-approximate specific number to the other specific number. Similarly, where a specific number is described as an approximate number by using the antecedent "about," it is understood that another embodiment is comprised of the non-approximate specific number. The upper and lower limits of each numerical range may or may not be related to the limit of one limit. Furthermore, while various numerical values ​​are disclosed in this specification, these numbers include not only the specific number itself but also "approximate" values ​​of that number. For example, where the number "10" is disclosed, the number "about 10" is also disclosed. Also, as will be readily apparent to those skilled in the art, where a specific number is disclosed, numerical ranges that can be set between "less than or equal to that number," "greater than or equal to that number," and those numbers are also disclosed. For example, if the number "10" is disclosed, then numbers such as "10 or less" and "10 or more" are also disclosed. Throughout this application, data is provided in various formats, and this data indicates upper and lower limits, as well as ranges representing any combination of data points. For example, if a specific data point "10" and another specific data point "15" are disclosed, then numbers greater than 10, numbers greater than or equal to 10, numbers less than 10, numbers less than or equal to 10, numbers equal to 10, numbers greater than 15, numbers greater than or equal to 15, numbers less than 15, numbers less than or equal to 15, numbers equal to 15, and numbers between 10 and 15 are also disclosed. In addition, each unit between two specific units is also disclosed. For example, if the numbers "10" and "15" are disclosed, then the numbers 11, 12, 13, and 14 are also disclosed.

[0066] In a first embodiment of the present invention, an isolated genetically modified bacterium, It is transformed by 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 such that the gene product of the MerA gene reduces metal ions to produce metal elements as metal nanoparticles.

[0067] In some embodiments, the MerA gene contains one or more mutations encoding an amino acid substitution, the amino acid substitution located at a position selected from the group including V317, Y441, and C464. In another embodiment, the mutation in the MerA gene is located 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 ion is reduced to produce gold elements as gold nanoparticles, or the metal ion is a silver ion (Ag + ) and the silver ions are reduced to produce silver elements as silver nanoparticles.

[0069] In some embodiments, the isolated bacteria exhibit reduced reducing ability to the substrate mercury compared to bacteria containing an unmutated MerA gene.

[0070] In some embodiments, isolated genetically modified bacteria are 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 hydrogen cyanide production, thereby leaching precious metals from metal sources such as electronic waste.

[0071] In some embodiments, hydrogen cyanide generation is controlled by an on / off switch including a gold ion sensor and a quorum sensor (an example is shown in Figure 15). More specifically, the gold sensor turns off the leaching process when the leached gold reaches a critical value. This off circuit responds to the gold ions produced by cyanide and includes a “weak” constitutive promoter that induces the golS gene and another promoter that induces a downstream repressor gene. An example of a minimal gold sensor is a weak constitutive promoter (e.g., P Con6 The golS transcription activator gene under the control of (SEQ ID NO: 37) and the Ph1F repressor which blocks the operator of PhlF in the quorum sensor, golTS Promoter or P golB One example is a gold sensor containing a promoter-induced ph1F repressor gene. The quorum sensor's ON circuit is responsive to high cell density and includes a promoter activated by endogenous CviR (induced by AHL at high cell density) and a PhlF operator downstream of this quorum sensor promoter. Downstream and under its regulation are one or more cyanide-producing genes, 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. This off circuit is upstream of the on circuit, which 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 P golTS Promoter and P golB The circuit includes a downstream heterologous ph1F repressor gene operably linked to a promoter selected from the promoters. The ON circuit includes a promoter activated by endogenous CviR, and includes a PhlF operator between this 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 mutation (an example is shown in Figure 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 includes the golTSB operon shown in Figure 19.

[0075] In another embodiment, an isolated recombinant DNA molecule is provided comprising a golTSB operon, wherein the golTSB operon comprises golT, golS, golB, operably ligated to a j23119 promoter from N-terminus to C-terminus, and a reporter gene (such as GFP). This operon can be used for general screening of recombinant bacteria that reduce gold by MerA.

[0076] Nitriles are a group of enzymes that hydrolyze nitriles into ammonia and their corresponding carboxylic acids. There are two types of such cyanide-degrading enzymes. [ka]

[0077] The first type of cyanide dihydratase includes bacterial enzymes. These enzymes behave as true nitrilases, directly converting cyanide to formic acid and ammonia (upper scheme). On the other hand, fungal cyanide hydratases hydrolyze cyanide to formamide (lower scheme). These hydrolases are promising candidates for bio-remediation of cyanide because they do not require other cofactors or substrates and exhibit catalytic activity over a wide range of substrate concentrations.

[0078] In some embodiments, the isolated genetically modified bacteria may contain a heterologous nitrilase gene operably linked to at least one promoter. This 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 derived from at least one bacterial species selected from the group including Pseudoalkaligenes (nit), Synechocystis PCC 6803 chromosome (SC-nit), cyanide dihydratase from Bacillus pumilus (BP-cynD), and Pseudomonas stazzeri (PS-cynD). These cyanide-degrading bacterial strains for bioremediation may be used alone or as part of an integrated workflow, and may be used as containment in industries handling cyanide and heavy metals.

[0079] In some embodiments, the isolated genetically modified bacteria further include heterologous formate dehydrogenase genes, heterologous glutamate dehydrogenase genes, and heterologous phosphoenolpyruvate carboxylase genes, operably linked to at least one promoter. By linking the cyanide degradation process to downstream enzymes—formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase—by reusing the byproducts carbon and nitrogen, the system can be made self-sustaining (Figure 3).

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

[0081] In some embodiments, the bacteria are selected from the group including Chromobacterium violaceum, Pseudomonas fluorescein, Pseudomonas aeruginosa, and Escherichia coli.

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

[0083] In a second aspect of the present invention, gold ions (Au 3+ ) to recover the element gold as gold nanoparticles, or silver ions (Ag + A method for recovering silver element as silver nanoparticles from ) a) A genetically modified bacterium isolated according to any aspect of the present invention is subjected to gold ions (Au 3+ ) and / or silver ions (Ag + The process of contacting the leachate containing ) with the leachate; and b) A step of recovering gold element nanoparticles and / or silver element nanoparticles from the leachate. A method including this is provided.

[0084] In some embodiments, the contact is carried out under alkaline conditions.

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

[0086] A third aspect of the present invention relates to a method for producing isolated bacteria, a) A step of performing error-prone PCR on the gene encoding mercury(II) reductase (MerA); i) a step of transforming at least one bacterium with the PCR product; and ii) A step of selecting transformants that grow in a culture medium containing metal ions; or b) A process of introducing saturated mutations at multiple sites by performing overlap extension PCR on the gene encoding mercury(II) reductase (MerA); i) a step of transforming at least one bacterium with the PCR product; and ii) A step of selecting transformants that grow in a culture medium containing metal ions. Includes, The bacterium contains at least one polynucleotide molecule, A method is provided in which the at least one polynucleotide molecule comprises a heterologous mercury(II) reductase (MerA) gene operably linked to at least one promoter, and includes one or more mutations such that the gene product of the MerA gene reduces metal ions to produce metal elements as metal nanoparticles.

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

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

[0089] In some embodiments, the selection includes at least two forms, one of which is Au 3+ and / or Ag + This is a selection on agar plates containing Au, and another form is Au 3+ and / or Ag + This is a selection for liquid cultures that include this method.

[0090] A fourth aspect of the present invention provides a method for producing a cyanide-based leachate using synthetic biotechnology, comprising the step of contacting recombinant cyanide-producing bacteria with glycine, wherein the bacteria comprises 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. One example is shown in Figure 1A, where the mutant, named SynLix 3.1, was grown in LB medium and cyanide production was monitored for 48 hours. Cyanide production was detected using a cyanide-sensitive ion-selective electrode (ISE).

[0092] In some embodiments, the hcnABC and serA genes are controlled by an inducible promoter. In some embodiments, the isolated recombinant bacterium includes a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to an on / off circuit. This off circuit is upstream of the on circuit, which is upstream of the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene. The off circuit includes a heterologous golS gene operably linked to a promoter, and P golTS Promoter and P golB The circuit includes a downstream heterologous ph1F repressor gene operably linked to a promoter selected from the promoters. The ON circuit includes a promoter activated by endogenous CviR, and includes a PhlF operator between this 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 cyanide-producing bacteria are tolerant to at least about pH 10.

[0094] In some embodiments, the production of cyanide-based leaching agents using the synthetic biotechnology and the bioleaching of metals are carried out in a single reactor.

[0095] In another aspect of the present invention, at least one isolated recombinant bacterium is provided that can be used to produce a cyanide-based leachate using synthetic biotechnology as described in any aspect of the present invention.

[0096] In some embodiments, the recombinant cyanide-producing bacteria are selected from the group including Chromobacterium violaceum, Pseudomonas fluorescein, Pseudomonas aeruginosa, and Escherichia coli.

[0097] Another aspect of the present invention provides a method for degrading cyanide using synthetic biotechnology, comprising the steps of a) contacting at least one recombinant cyanide-degrading bacterium recombinant to express at least one nitrilase enzyme with nitrile containing cyanide remaining after bioleaching of electronic waste.

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

[0099] In some embodiments, the at least one recombinant cyanide-degrading bacterium is further recombinant 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 Pseudoalkaligenes (nit), Synechocystis PCC 6803 chromosome (SC-nit), Bacillus pumilus-derived cyanidilatase (BP-cynD), and Pseudomonas stazzeri (PS-cynD).

[0101] In another aspect of the present invention, an isolated recombinant DNA molecule, wherein the direction from the N-terminus to the C-terminus, (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB A ph1F repressor gene operably linked to a promoter; (ii) The promoter activated by CviR, the operator of PhlF, and (iii) One or more cyanide-producing genes operably linked to a promoter activated by CviR Recombinant DNA molecules containing [the specified substance] are provided.

[0102] In some embodiments, the golS transcription activator gene is a weak constitutive promoter (P Con6 It is subject to control by (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 Figure 12). In some embodiments, the golS gene is a mutant selected from the group comprising or consisting of GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

[0104] Another aspect of the present invention provides the use of an inactive Cas9 and an RNA guide (sgRNA) for repressing the transcription of one or more genes by targeting the promoter regions of those genes in the genome of Chromobacterium violaseum, wherein the inactive Cas9 comprises an H840A mutation in its HNH endonuclease domain and a D10A mutation in its 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 ligated to a promoter, and is operably ligated to a strong constitutive promoter such as J23119.

[0106] In some embodiments, since the violacein dye can complicate downstream process steps, inactive Cas9 is used to target the violacein operon and prevent the formation of the purple violacein dye. In some embodiments, this inactive Cas9 targets the vioA promoter, the vioB promoter, and / or the vioC promoter, preferably all three promoters. While the outline of the present invention has been described, the present invention can be more readily understood by referring to the following examples, which are provided for illustrative purposes. The following examples are not intended to limit the present invention. [Examples]

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

[0108] Example 1 Integration of nitrilase into host cells for the purpose of cyanide degradation. Four nitrilase variants were selected from various bacteria. These sequences were synthesized and cloned into host cells. More specifically, the four nitrilase variants used were: nitrilase (nit) (SEQ ID NO: 11) from Pseudomonas pseudoalcaligenes, nitrilase (SC-nit) (SEQ ID NO: 12) from the chromosome of Synechocystis genus PCC 6803, cyanidehydratase (BP-cynD) (SEQ ID NO: 13) from Bacillus pumilus, and cyanidehydratase (PS-cynD) (SEQ ID NO: 14) from Pseudomonas stutzeri. The nit gene contains two distinct subunits, nitB (sequence number 15) and nitC (sequence number 16). These two subunits were cloned into the pRSF-Duet vector and expressed in the host cell strain E. coli (DE3) BL21. The remaining three variants were cloned into the pGEM vector and incorporated into the genome of Chromobacterium violaceum using the Tn7 translocation system (Figures 2 and 3).

[0109] Formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase were used as coupling enzymes. These enzymes were cloned into the 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 reused, making this system self-sustaining (Figure 3).

[0110] The ability of cyanide-degrading Chromobacterium violaseum strains to remove cyanide from the extracellular environment was tested. The recombinant cyanide-degrading C. violaseum strains were able to completely remove exogenous cyanide within 24 hours in the presence of 100 mg / L potassium cyanide.

[0111] Example 2 Development of a dCas9-based genome transcription control tool in Chromobacterium violaseum CRISPR-Cas9 is widely used as a genome editing tool in many organisms. CRISPR-Cas9 has primarily been used in eukaryotes such as mammalian cells, insect cells, and yeast cells. The CRISPR-Cas system is derived from the bacterial adaptive immune system and stores small fragments of invading bacteriophage DNA as a memory by inserting them into the host genome, enabling monitoring of future invading bacteriophages composed of the same DNA fragment. The CRISPR-Cas9 system derived from Streptococcus pyogenes consists of a Cas9 endonuclease that, in cooperation with an RNA guide, binds to a DNA sequence homologous to the RNA guide. This bound Cas9 endonuclease then 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-base scaffold that contacts the Cas9 endonuclease. The 20-base spacer of the RNA guide guides the Cas9 endonuclease to the target DNA, causing a double-strand break in the host chromosome. The host then activates its DNA repair mechanism to repair the broken chromosome, resulting in insertion, deletion, or homologous recombination of the introduced DNA fragment at the target DNA site. Because this RNA-guided mechanism was easily available in a modular manner, CRISPR-Cas9 genome editing has rapidly become available in many organisms.

[0112] CRISPR-Cas has been applied to a variety of other uses. Because RNA can easily and specifically recognize DNA sequences, there are many applications and variations of Cas9 targeting specific genomic loci. Two functionalities have been added to the CRISPR / Cas gene tool: 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. This steric hindrance inhibits the binding of RNA polymerase to the promoter region, thereby suppressing gene transcription. In CRISPRa, gene transcription is enhanced via a protein fused with an activator that recruits RNA polymerase and dCas. CRISPRa has been shown to triple the expression of target genes [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 Escherichia, Pseudomonas, Mycobacterium, Corynebacterium, Clostridium, or Bacillus, and can suppress gene expression by up to 1 / 300th [Cho, S. et al., Int J Mol Sci, 19(4). doi:10.3390 / ijms19041089 (2018); Qi et al., 2013]. Below, we investigate whether transcription of chromosomal gene expression can be suppressed in Chromobacterium violaseum using CRISPRi tools.

[0113] While CRISPR / Cas genome editing tools are powerful tools for eukaryotic cells, their use in prokaryotes is more limited because double-strand DNA breaks are lethal in prokaryotes. Furthermore, prokaryotic DNA repair mechanisms are insufficient for cell recovery, leading to bacterial death when CRISPR / Cas is used.

[0114] Despite these circumstances, an inactive Cas9 (dCas9) with D10A and H840A mutations in its catalytic domain was adapted to block the transcription of the target gene (Figure 5). In this study, dCas9 was used to target the promoter region of the violacein operon and prevent the formation of the purple violacein pigment (Figure 5B). This repression was most effective when targeting the promoter (SEQ ID NO: 18) of the first gene (vioA), and was sufficiently repressed with 0.01% arabinose. This was followed by the vioB promoter (SEQ ID NO: 19), but to repress violacein via this vioB promoter, it was necessary to induce dCas9 expression with a higher concentration of 0.1% arabinose. Repression of the vioC promoter (SEQ ID NO: 20), further downstream in this operon, had little effect on repressing violacein transcription. The slight decrease in optical density is not thought to be due to the metabolic load caused by dCas expression. One of the main reasons for the slight decrease in optical density is OD 570 This may be due to overlap with the absorbance spectrum of violacein, which has the highest absorbance [Swem, LR et al., Mol Cell, 35(2), 143-153 (2009)]. Another possible reason for the slight decrease in optical density is that cells with insufficient suppression of the violacein dye formed aggregates (Figure 5C).

[0115] Transcriptional regulation of C. violaseum chromosomes has not been reported to date. Controlling endogenous genes is useful for metabolic engineering, particularly for controlling metabolic fluxes, as it allows for rapid and efficient knockdown of gene expression. Furthermore, because it can be easily duplicated, many genes can be efficiently and rapidly knocked down 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 other proteins, for example, to introduce single nucleotide mutations by fusing it to other proteins such as cytidine deaminase or adenine deaminase [Arazoe, T. et al., Biotechnology journal, 13(9): e1700596 (2018); Komor, Kim, Packer, Zuris, & Liu, 2016]. In this way, the gene toolbox of C. violaseum can be developed in a diverse range of ways.

[0116] Cloning and expression of dCas in Chromobacterium violaceum Mutation primers and Gibson assemblies were used to introduce mutations (D10A and H840A) into Cas9 at two catalytic sites. Next, P araBAD dCas was cloned under the control of (SEQ ID NO: 21). Using the strong constitutive promoter J23119 (SEQ ID NO: 22), expression of sgRNAs targeting the 5' end of the vioA promoter, vioB promoter, or two non-target sequences of vioC was induced (Figure 5A). C. violaceum was cultured overnight, diluted 1:100 in a 96-deep-well block (Nunc, Denmark), and dCas was induced by adding 0.01% arabinose, 0.1% arabinose, or 1% arabinose. The control was not induced. After growing the cultures overnight at 37°C and 280 rpm, they were transferred to 96-deep-well plates, and violacein production was visualized and OD was measured. 600 We measured it.

[0117] Example 3 Construction of a gold sensor from a natural gold operon in Chromobacterium violaseum A gold sensor is one of the important sensors in the gold bioleaching circuit. It can provide dynamic feedback to the bioleaching circuit depending on the amount of gold leached, and the leaching process can be stopped when the gold ion concentration reaches a critical value. The only gold biosensor demonstrated to date is the golTSB operon (SEQ ID NO: 23) derived from Salmonella enterica serovar Typhimurium str. LT2 strain. This gold biosensor contains GolS (SEQ ID NO: 24), which is the only MerR family transcription factor 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 had previously been demonstrated only in Escherichia coli and S. enterica, but in this report, it has been optimized for C. violaceum for the first time to broaden its dynamic range.

[0118] To characterize this gold sensor, first, the entire golTSB operon is cloned upstream of the production of the fluorescent protein. However, Au in the range of 0.001 μM to 10 μM 3+ At high concentrations, the dynamic range of fluorescence output is low (Figure 6A). This low dynamic range is due to Au 3+ The leakage phenomenon in the absence of [the substance] is attributed to the high value of 14842 RFU (Figure 6A). Furthermore, high concentrations of Au 3+ At concentrations of 1 μM or higher, cell density decreases significantly. This toxicity to cells is thought to be due to the overexpression of GolT, a transmembrane P-type ATPase responsible for membrane transport, and it is believed that overexpression of GolT may disrupt the integrity of the cell membrane. Since this gold sensor showed a low dynamic range and cytotoxicity in C. violaceum, the golTSB operon was optimized.

[0119] To suppress the leakage activation caused by GolS, P golTS When GolS expression was induced by replacing it with a weak constitutive promoter, the promoter strength decreased and the positive feedback loop disappeared (Figure 6B). Furthermore, by removing GolT and GolB to construct a minimal gold sensor, the harmful effects on cell proliferation were minimized. Next, P golTS (Sequence ID 25) or P golB This construct was tested using (SEQ ID NO: 26) to induce the production of a fluorescent protein (Figures 6B and 6C). Au in the range of 0–10 μM. 3+ In terms of concentration, the maximum value of each dynamic range was 151 times for pGolTS (minimum 110 RFU to maximum 16416 RFU) and 113 times for pGolB (minimum 50 RFU to maximum 1648 RFU).

[0120] No toxicity was observed to C. violaseum from 0-1 μM Au. 3+ When comparing pGolTS and pGolB in terms of concentration, pGolTS showed a larger magnification change. 0~1 μM Au 3+ At different concentrations, the dynamic range of gene expression was 38-fold for PgolB (minimum 50 RFU to maximum 1887 RFU) (Figure 6B) and 62-fold for PgolTS (minimum 110 RFU to maximum 6737 RFU) (Figure 6C). Due to its high dynamic range, the PgolTS construct was selected as the gold sensor to be used in the next experiment.

[0121] Improving the dynamic range and sensitivity of gold sensors using mutagenesis in Chromobacterium violaceum After optimizing the gold sensor circuit and regulatory mechanism, the sensitivity and dynamic range of the gold sensor are further improved by performing deep scanning mutagenesis on the GolS transcription activator. Although the structure of the GolS protein is not yet understood, GolS belongs to the MerR protein family, which has a helix-turn-helix structure with a DNA-binding domain and a gold ion-binding domain (Figure 8A) [Checa, SK et al., Mol Microbiol, 63(5), 1307-1318 (2007)]. GolS is constitutively weakly expressed, and P induces RFP. golTS An optimized circuit consisting of (Figure 7) was used to screen and select GolS mutants with a wider dynamic range and higher sensitivity than wild-type GolS (Figure 12). Four mutants were selected: GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P), and their characteristics 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 exhibit higher sensitivity and a wider dynamic range than previously reported wild-type gold sensors (Table 1). max The value increases, up to the wild type B max The value changed in multiples of 3.5 times. Since other parameters such as K, n, and C did not change by more than 2 times, it was shown that other characteristics remained relatively similar.

[0123] [Table 1]

[0124] In this system, sensitivity is a crucial parameter determining 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. These gold sensor mutants showed a magnification change of at least three times for 8 nM gold ions, while no magnification change was detected in the wild-type gold sensor (Table 2).

[0125] [Table 2]

[0126] These values ​​demonstrate higher sensitivity than the previously reported 2.3-fold induction by 33 nM gold ions as the detection threshold for biosensors introduced into E. coli [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)]. These gold sensor mutants require only 80 nM Au 3+ While the output multiplier change exceeded 10 times at higher concentrations, 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. Furthermore, slight leakage was observed in both the wild-type and mutant gold sensors in the off state. Output in the off state was low, all below 100 RFU (GolSmt1: 51 RFU, GolSmt2: 54 RFU, GolSmt3: 97 RFU, WT: 47 RFU). Additionally, the gold sensor mutants were able to generate higher maximum output than the wild-type gold sensor before gold ions reached toxic levels, with a multiplier change exceeding 100 times (GolSmt1: 45,088 RFU, GolSmt2: 36,074 RFU, GolSmt3: 41,388 RFU, WT: 12,811 RFU).

[0127] Since all of these mutants have mutations in the DNA-binding domain with a helix-turn-helix structure (left and right arrows, Figure 8B), the increase in the output of the transcription activator is thought to be due to the activation of the promoter region by improved binding to it, which suggests that the maximum output was higher in the gold-sensing mutant (Figure 13). Furthermore, these mutants of the transcription activator showed increased affinity for binding to gold ions, which was supported by a leftward shift in the response function to gold (Figure 13). In addition, no leaky expression was observed at the basal level, suggesting that these mutations did not enhance activation when the transcription activator was not induced. Therefore, such enhancement of transcription output, increased affinity for gold ions, and robust expression of gold-sensing transcription activator mutants could be useful tools for future applications utilizing gold ion sensing.

[0128] Introduction of mutations in gold sensors A library of golS mutants was prepared using QuikScan-19 and constructed using the QuikChange-HT kit (Agilent Technologies, Santa Clara). By repeatedly substituting each amino acid with 19 other amino acids, custom mutant oligos were obtained in which each amino acid of GolS was substituted with 19 other amino acids, and these were used for the QuikChange reaction. In the 154-amino acid length of GolS, excluding the first amino acid residue, methionine, a total of 153 × 19 = 2907 single-amino acid mutants could be created. Following the manufacturer's protocol, a total of six libraries were prepared, each containing a mutant region approximately 25 amino acids long relative to the full length of the GolS protein. Next, each library was introduced into competent C. violaceum cells, transformed, and plated in Tris minimal medium containing 1.5% bacterial agar supplemented with 30 μg / mL kanamycin and 2 μM AuCl3. Colonies were observed under blue light, and colonies with high RFP production compared to wild-type GolS were selected. These were inoculated into 96-well plates for growth, and fluorescence was quantified using a BioTek Synergy H1 microplate reader.

[0129] Fluorescence measurement for characterization of quorum sensors and gold sensors To evaluate the properties of the gold sensor, cells were seeded from frozen stocks in LB medium into 96 deep-well blocks (Nunc, Denmark) and grown overnight at 37°C. Next, the cells were diluted 1:200 in 96-well plates (Costar, Kennebank) 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 to 10 μM gold(III) chloride (Sigma-Aldrich) was added.

[0130] Next, diluted cells were grown overnight at 37°C using a BioTek Synergy H1 microplate reader. The excitation wavelength was set to 530 nm and the emission wavelength to 600 nm (gain set to 50). Red fluorescence was measured every 10 minutes, and measurements continued for 14-20 hours. Optical density was also measured by measuring the absorbance at 600 nm. OD 600 Furthermore, only culture media without cells were used as a blank for fluorescence values.

[0131] Example 4 Dynamic regulation and biosensor integration using synthetic circuits for robust microbial cell factories Two main components used in microbial cell factories for gold bioleaching are gold, the element of interest, and cyanide, a leaching agent necessary for oxidizing solid gold to obtain an aqueous solution of gold ions. Both gold ions and cyanide are highly toxic to microorganisms, making their use in living microbial cell factories difficult. The toxicity of gold ions arises from the induction of oxidative stress due to the accumulation of Au(I)-S complexes [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 metals contained in cytochrome oxidase [Knowles, CJ Bacteriol Rev, 40(3), 652-680 (1976); Knowles, CJ & Bunch, AW Adv Microb Physiol, 27, 73-111 (1986)]. Furthermore, gold ions have been shown to be toxic to bacteria at a low concentration of 0.35 μM [Shareena Dasari, TP et al., Biochem Pharmacol (Los Angel), 4(6), 199 (2015)]. In contrast, cyanide is toxic to bacteria at a low concentration of 0.4 μM [Liu, W. et al., Chinese Journal of Chemistry, 25(2), 203-207 (2007)].C. violaceum possesses 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)], which protect C. violaceum itself from the cyanide it generates. 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, a dynamic on / off circuit was constructed in which cyanide generation is turned on only when the cell density is high, and the circuit is turned off when it senses toxic gold ions (Figure 14).

[0132] By adding an endogenous quorum sensing system to the circuit, the output can be activated only when the cell density is sufficiently high (Figure 15). Since the gold sensor that initiates the negative feedback loop of the synthesis circuit is an activator, it is necessary to introduce a repressor downstream to suppress it. PhlF (SEQ ID NO: 31) [Stanton, BC et al., Nat Chem Biol, 10(2), 99-105 (2014)], a potent repressor of the TetR family that has been previously characterized and can reduce gene expression by up to 1 / 193, is added downstream of the gold sensor to suppress the transcription of the output. Furthermore, to physically suppress RNAP in the transcription process, an operator of PhlF (SEQ ID NO: 32) is added downstream of the quorum sensing promoter (Figure 15). The negative feedback loop by the gold sensor allows for real-time feedback on the bioleaching process, and when cyanide is generated and the concentration of gold ions becomes high due to cyanide, the bioleaching process is suppressed. When the repressor is expressed, it binds to the promoter region of the cyanide-producing gene. This means that if high concentrations of gold ions are detected, the expression of this bioleaching enzyme will not occur any further.

[0133] This circuit will be tested in a continuous batch culture where the culture is performed after diluting the previously subcultured culture to 1:600. 2 μM Au 3+The circuit is either 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 research, 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 we use 2 μM gold ions to induce the off state. On the other hand, turning off the circuit means that low leakiness expression is observed, or no leakiness expression is observed at all. When the circuit containing the wild-type gold sensor is turned off, the expression level at the basal level increases with each cycle. The output in the off state of the circuit containing the wild-type gold sensor 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, in the circuit with the gold sensor mutant, the output in the off state was maintained at less than 250 RFU in all three cycles (Figure 17).

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

[0135] Analysis of each cell using a flow cytometer further supported the improved robustness of the GolS mutant circuit, as shown in the representative histogram (Figure 18), by maintaining distinguishable on- and off-populations throughout multiple consecutive cycles. (A) represents wild-type, (B) represents GolSmt1, (C) represents GolSmt2, and (D) represents GolSmt3. The extent of the off-cell population observed in the wild-type gold sensor (Figure 18A) suggests a decrease in the circuit's dynamic range, mainly due to increased heterogeneity of expression in this cell population. The strong maintenance of the off state in circuits containing gold sensor mutants may be due to increased sensitivity to gold ions and an increased dynamic range of the gold sensor. 3+ Under the same induction conditions, the higher the expression level of PhlF repressor, the better the quorum sensing promoter may be suppressed.

[0136] Another observation revealed that in two of the GolS mutants (GolSmt2 (Figure 18C) and GolSmt3 (Figure 18D)), the population that remained in the off state throughout the three cycles increased, and the dynamic range was slightly reduced. This may be due to insufficient dilution of the PhlF repressor throughout the cycle, resulting in a small population of cells remaining in the off state. In these GolS mutants, increased PhlF repressor expression maintained the off state output well, but this effect may have been too strong, resulting in insufficient dilution of the PhlF repressor in the cells and preventing the circuit from returning to the on state. This suggests that GolSmt2 and GolSmt3 may not be suitable as gold sensors for robust and continuous on / off cycling in the gold bioleaching synthesis circuit.

[0137] In the gold sensor mutant, the robustness of the circuit increased over three on / off cycles consisting of six cell subculturing cycles (Figures 16-18). Since the toxicity of gold ions to cells means that gold leaching requires many cycles, this characteristic greatly contributes to the development of robust microbial cell factories for gold bioleaching.

[0138] Example 5 Conferring gold reduction activity to mercury(II) reductase using 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), and therefore can affect many metabolically important enzymes and membrane-bound proteins. E. coli cells expressing a MerA mutant enzyme with enhanced gold-reducing activity may exhibit high resistance 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 a ligation mixture and cultured on standard LB agar. As the liquid medium, single colonies were inoculated into 5 mL of Tris-buffered hypophosphatemia supplemented with the same antibiotic and various concentrations of AuCl3, and cultured at 37°C for 24 hours. OD 600 The levels were measured and cell proliferation was compared. The lowest concentration of heavy metal that completely inhibited cell growth was defined as the minimum inhibitory concentration (MIC). Figure 23A shows that E. coli cells were inhibited by Au above 140 μM. 3+ This indicates that the organism could not grow at that concentration.

[0140] To perform the agar plate dilution method for E. coli cells expressing wild-type MerA, E. coli cells expressing wild-type MerA were cultured, diluted, and spread on Tris-buffered hypophosphate 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. The results showed that Au... 3+ As the concentration increased, it was shown that fewer colonies were observed on the agar plate. Au 3+ Cell proliferation was completely suppressed when the concentration reached 160 μM (Figure 23B).

[0141] The toxicity of gold is likely due to its high affinity for thiol groups (-SH) present in many metabolically important enzymes and membrane-bound proteins. When metabolically important enzymes and membrane-bound proteins bind to gold ions, these enzymes and proteins become unable to bind to the metal ions that are normally biologically relevant to them. The liquid dilution method showed a slightly lower minimum inhibitory concentration than the agar plate dilution method. This difference is due to the difference in Au between plate medium and liquid medium. 3+ This can be due to differences in ion distribution patterns.

[0142] Building a directional evolution library The codon-optimized synthesis gene encoding mercury(II) reductase (MerA) was cloned in-frame into the pET20b expression vector (Novagen) with a 6×His tag at the N-terminus using restriction enzymes NdeI and XhoI. To obtain MerA with enhanced gold-reducing activity, a mutant library was constructed using error-prone PCR and site-saturated mutagenesis.

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

[0144] Saturated mutagenesis was performed at multiple target sites, V317, Y441, C464, and C465', using overlap extension PCR with degenerate primers containing NNK. These locations are extremely close to the gold binding sites within the MerA active site (Figure 22). The redox-active cysteine ​​at positions C136 and C141 were maintained without modification 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 such as T7-fw / V317NNK-rv, V317NNK-fw / Y441NNK-rv, Y441NNK-fw / C464NNK-rv, or C464NNK-fw / T7-rv to produce partially overlapping DNA fragments. A total volume of 50 μL of reaction mixture containing 50 ng of pET20bMerA plasmid, 500 nM of each primer, and 1×PrimeSTAR Max Premix (Clontec) was incubated for 30 cycles of 10 seconds at 98°C, 5 seconds at 55°C, and 10 seconds at 72°C. The PCR products were purified, and equimolar amounts of each fragment were mixed with 1×PrimeSTAR Max Premix. A short overlap extension reaction (5 cycles of 10 seconds at 98°C, 5 seconds at 55°C, and 10 seconds at 72°C) was performed. Using 1 μL of the resulting reaction mixture as a template, the full-length MerA mutant gene was amplified using epPCR-fw primers 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 Rosetta(DE3)pLysS competent cells of Escherichia coli were transformed using ligation mixtures obtained by error-prone PCR or site-saturated mutagenesis. The resulting transformants were seeded 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 hours. Next, colonies were picked and grown in liquid Tris-buffered hypophosphatemia medium supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG, and 300 μM AuCl3. By repeating this selection several times, cells expressing wild-type MerA were found to have toxic levels of Au that could barely grow. 3+ Ten mutants were obtained that showed substantially good cell proliferation in the presence of (300 μM) (Figure 24). All colonies that survived the second selection were obtained from the site-saturated mutagenesis library, and it is noteworthy that this semi-rationally designed library has the potential to yield variants with improved functionality at a higher rate than the random mutagenesis library. The improved resistance compared to the wild type was due to 1) Au by MerA. 3+ Detoxification via enzymatic reduction of ions, and 2) Au 3+ Au by binding MerA to 3+ This is thought to be due to two factors: the capture of the insect and the capture of the insect.

[0148] As shown in the TEM image (Figure 25), a MerA mutant that synthesizes gold nanoparticles from AuCl3 was obtained. Directed evolution identified the MerA mutant V317S as an enhanced gold reductase. This mutant has the reaction kinetic parameters summarized in Table 5. This enhanced gold reductase is Au 3+Gold element (Au 0 The catalytic efficiency for reducing it to ) has increased 67 times.

[0149] [Table 4]

[0150] Further improved mutants The catalytic efficiency of MerA for gold reduction is 9.1 ± 3.2 × 10⁻⁶. 1 It has been previously established that it is / M / sec. By combining directional evolution and rational design methods, improved Au 3+ A library of MerA mutants with reducing ability was constructed. More than 50% of the isolated mutants showed improved activity, and the best-improved mutant showed up to a 15-fold improvement in catalytic efficiency (Figure 26). The DM11 mutant (G415I) (SEQ ID NO: 48) showed a high turnover rate (k cat ) and binding affinity (K M Since all parameters improved, catalytic efficiency was the most improved (15-fold increase). The kinetic parameters of all mutants are shown in Table 6.

[0151] This improvement in catalytic efficiency is also reflected in the complexity of the gold nanoparticles (AuNPs) formed by reduction. DM11 generates larger and more complex gold nanoparticles compared to MerA (Figure 27). Furthermore, this improvement in catalytic efficiency is reflected in the fact that DM11 is more effective than wild-type MerA in reducing and recovering gold from leachate of electronic waste (Figure 28). Au by DM11 3+ The recovery is from AuCl3 solution. 3+ Even with the recovery rate (67%), Au was found in the leachate from electronic waste. 3+ A recovery rate of 67% is equally effective (Figure 29).

[0152] [Table 5]

[0153] However, while the complexity of gold nanoparticles (AuNPs) increased upon reduction from the AuCl3 solution, the complexity of gold nanoparticles remained limited upon reduction from the leachate (Figure 30). This is because the Au in the leachate... 3+ This is thought to be due to the overall low amount of [the substance]. If this constraint can be overcome, it is believed that gold recovery can be greatly improved. MerA is an essential enzyme in bacterial mercury-resistant systems. Based on the reaction mechanism of MerA and the available crystal structures, it was hypothesized that genetic manipulation of MerA for efficient gold reduction was possible. To achieve this, a high-throughput selection procedure was established, which involves selection using toxic agar medium followed by selection using a more stringent liquid culture. A mutant library was constructed using error-prone PCR and saturated mutagenesis at multiple sites and subjected to this two-step selection. As a result, a MerA mutant with enhanced gold reduction / gold recovery properties was identified. This result plays a part in synthetic metal recovery technology in synthetic biological leaching.

[0154] Protein expression and purification Recombinant proteins were expressed using a T7 expression system. Rosetta(DE3)pLysS cells were transformed with this plasmid and selected on LB agar containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. Single colonies were picked and inoculated into 5 mL of LB medium containing the two antibiotics and grown overnight at 37°C. Diluted 100-fold, OD 600The cultures were grown at 37°C until the pH reached 0.6. Next, protein expression was induced by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM. After induction of protein expression, the cells were grown for a further 18 hours at 16°C. The cells were lysed by sonication, the lysate was clarified, and the His-tagged protein was purified by passing it through a nickel chelate column (Qiagen). The protein sample in the eluate (500 mM imidazole, 50 mM Tris-Cl (pH 7.5), 300 mM NaCl) was concentrated and dialyzed against 20 mM sodium phosphate (pH 7.4) using an Amicon Ultracentrifugal filter (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. NADPH oxidation was tracked at 340 nm by spectrophotometric analysis. Enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1.0 μmol of NADPH per minute in an Au-dependent manner.

[0156] Example 6 Kinetic parameters of gold reduction by MerA The kinetic parameters of purified mercury reductase (MerA) against an AuCl3 substrate were measured using a continuous spectroscopic assay (Scheme 1).

[0157] Scheme 1: Assay to monitor MerA activity by measuring changes in absorbance at 340 nm. M is Hg 2+ or Au 3+ X represents GSH - Alternatively, it corresponds to Cl. [ka]

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

[0159] The kinetic parameters of MerA when using Hg(GSH)2 and AuCl3 were measured (Table 4).

[0160]

Table 6

[0161] When mercury was used as a substrate, the K M value of MerA was 96.3 ± 57.6 μM, which was higher than the previously reported 10.7 μM in the literature. Also, the kcat value of MerA was 14.6 ± 5.1 / second, which was slightly faster than the previously reported 9.43 / second, but there was no significant difference. The overall catalytic efficiency (k cat / K M ) value was 1.5 ± 0.7 × 10 5 / M / second, which was lower than expected compared to the previously reported 8.8 × 10 5 / M / second in the literature and was one-sixth (Moore, M.J., Miller, S.M., Walsh, C.T. C-Terminal Cysteines of Tn501 Mecuric Ion Reductase (1992) Biochemistry 31(6):1677-85). For AuCl3, there is no reported data. Compared with 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 to different substrates.

[0162] Screening using a gold-sensing biosensor identified a MerA mutant with improved gold-reducing ability. The golTSB operon isolated from Salmonella enterica serovar typhimurium functions as a biosensor together with green fluorescent protein (GFP) as a reporter (Zammit et al., 2013) (Figure 19). This biosensor is Au + Complex or Au 3+ It is under the control of GolS regulators induced in the presence of the complex. Au + Ion or Au 3+ When ions interact with the GolS regulator, the GolS regulator binds to the target promoter sequence, golB. This induces a structural change in the GolS / golB complex, promoting the transcription of the GFP reporter. In this specification, we propose incorporating this circuit into the chromosome of E. coli according to a previously reported method [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)] and using this E. coli strain as an endogenous reporter for gold reduction ability.

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

number

[0164] Au up to 100 μM 3+ Fluorescence increased with increasing concentration (Figure 20). At higher concentrations, the fluorescence decreased sharply, likely because E. coli became sensitive to metal toxicity. Au in this sensor 3+ The detection limit, i.e., the minimum and maximum concentrations of gold at which an increase in fluorescence compared to the background can be detected, was found to be 100 nm to 100 μM. Therefore, this sensor can detect various concentrations of Au 3+ Since it has been proven that these can be distinguished, it can be used for screening for optimal gold-reducing MerA mutants.

[0165] Screening for the optimal gold-reducing MerA mutant can be performed based on the observation of a decrease in fluorescence, which indicates the presence of Au in the cell. 3+ This corresponds to a decrease in ions and therefore functions as an indirect reporter of gold reduction activity (Figure 21). In the presence of gold ions and a control plasmid, the biosensor is induced and a strong fluorescence signal is produced, but 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 violaseum The biosensor shown in Figure 31A was tested for sensitivity to various metal ions in minimal culture medium. This biosensor sensed gold and silver ions, but did not show high responsiveness to other metals such as cadmium, zinc, mercury, nickel, cobalt, iron, and copper ions (Figure 31B). This dose-response suggests that this 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 Detection of precious metal ions leached from electronic waste by C. violaceum. C. violaseum 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. violaseum cells were centrifuged, and the resulting spent culture medium was added to C. violaseum containing the biosensor used in Example 7 (Figure 33). This transcription factor biosensor was activated in the spent culture medium containing scrap metal (ESM) leached from electronic equipment, but not in the control culture medium (Figure 34). The fluorescence output of the mutant biosensor in response to the spent culture on day 6 containing scrap metal (ESM) leached from electronic equipment was 2515 RFU, compared to 1101 RFU for the wild-type biosensor. ICP analysis of cultures used for 6 days revealed the presence of 1.1 ppm (5.6 μM) of gold ions, 0.34 ppm (3.2 μM) of silver ions, and 12.3 ppm (193.2 μM) of copper ions in the used medium. This result indicates that the C. violaceum biosensor was activated in the presence of gold and silver ions leached from a medium containing electronic waste. This property is useful for activating further synthetic circuit modules in C. violaceum.

[0168] summary 1. In the present invention, by using a mercury reductase (Mer) system, it was possible to cyanide the metal by biological technique and reduce the metal-cyanide complex. 2. In the present invention, the tool of the present invention makes it possible to construct a synthetic circuit for biological leaching and can be applied to the continuous and appropriate expression and activation of individual biological systems with the aim of efficiently recovering metals. The tool of the present invention has the following features: a. By adding electronic waste to a bioreactor, the expression of a synthetic module for cyanide production can be controlled. This allows for the timely production of cyanide-based leaching agents for metal bioleaching. b. Upon detection of individual metal-cyanide complexes, a metal recovery synthesis module is specifically and transiently expressed. Because electronic waste is heterogeneous, the concentrations of each metal (and the resulting metal ion concentrations) vary considerably, making it necessary to temporarily remove each metal. This removal can be achieved by selectively expressing a recombinant Mer system specifically according to the valence and type of metal ion. c. After the metal is recovered, excess cyanide-based leaching agents can be metabolized and the carbon and nitrogen atoms of cyanide returned to the central metabolic pathway by regulated induction of the expression of the cyanide degradation synthetic module.

[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. An isolated recombinant DNA molecule, in the direction from the N-terminus to the C-terminus, (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB The ph1F repressor gene is operablely linked to the promoter; (ii) The promoter activated by CviR and the operator of PhlF, and (iii) One or more cyanide-producing genes operably linked to a promoter activated by CviR Recombinant DNA molecules containing this molecule.

2. The isolated recombinant DNA molecule according to claim 1, wherein the golS transcription activator gene is a mutant selected from the group comprising or consisting of GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

3. The use of an inactive Cas9 and an RNA guide (sgRNA) to repress the transcription of one or more genes by targeting the promoter region of said genes in the genome of Chromobacterium violaseum, wherein the inactive Cas9 contains an H840A mutation in the HNH endonuclease domain and a D10A mutation in the RuvC endonuclease domain.

4. The gene encoding the inactive Cas9 is P araBAD The use according to claim 3, wherein the gene encoding the RNA guide (sgRNA) is operably ligated to a promoter, and the gene is operably ligated to a strong constitutive promoter such as J23119.

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

6. Isolated genetically modified bacteria, It is transformed by at least one polynucleotide molecule, A genetically modified bacterium comprising a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene, wherein at least one polynucleotide molecule is operably linked to at least one promoter.

7. The hydrogen cyanide synthase gene is hcnABC, the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or the isolated genetically modified bacterium is arranged in the direction from the N-terminus to the C-terminus. (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB The ph1F repressor gene is operablely linked to the promoter; (ii) The promoter activated by CviR and the operator of 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 isolated bacterium according to claim 6, further comprising at least one recombinant polynucleotide DNA molecule containing the above.

8. The isolated bacterium according to claim 7, wherein the golS gene is codon-optimized for Chromobacterium violaseum and / or the golS gene is a variant selected from GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

9. An isolated bacterium according to any one of the claims, selected from the group including Chromobacterium violaseum, Pseudomonas fluorescein, Pseudomonas aeruginosa and Escherichia coli, and / or stable at pH 10.

10. A method for producing cyanide-based leaching agents using synthetic biotechnology, The process includes the step of contacting at least one recombinant cyanide-producing bacterium with glycine, A method comprising the bacterium comprising a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene, both operably linked to at least one promoter.

11. The hydrogen cyanide synthase gene is hcnABC, the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or the recombinant cyanide-producing bacterium is such that, from the N-terminus to the C-terminus, (i) golS transcription activator gene operably linked to a constitutive promoter, and P golTS Promoter or P golB The ph1F repressor gene is operablely linked to the promoter; (ii) The promoter activated by CviR and the operator of 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 method according to claim 10, further comprising at least one recombinant polynucleotide DNA molecule containing the

12. The method according to claim 11, wherein the golS gene is codon-optimized for Chromobacterium violaseum and / or the golS gene is a mutant selected from GolSmt1 (A38I), GolSmt2 (A38Q and N97D), GolSmt3 (A38K and V60L), and GolSmt4 (D33P).

13. The method according to any one of claims 10 to 12, wherein the at least one recombinant cyanide-producing bacterium is tolerant to about pH 10.

14. The method according to any one of claims 10 to 13, wherein the production of a cyanide-based leaching agent using the aforementioned synthetic biotechnology and the bioleaching of a metal are carried out in a single reactor.

15. At least one isolated recombinant cyanide-producing bacterium capable of producing a cyanide-based leachate using synthetic biotechnology as described in any one of claims 10 to 14.

16. At least one recombinant cyanide-producing bacterium according to claim 15, selected from the group including Chromobacterium violaseum, Pseudomonas fluorescein, Pseudomonas aeruginosa, and Escherichia coli.

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