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JP-2026076232-A - Recovery of anthropogenic precious metals and toxic metals using synthetic biological leaching methods.

JP2026076232AJP 2026076232 AJP2026076232 AJP 2026076232AJP-2026076232-A

Abstract

[Problem] To provide a method for cyanidating a metal using enzymes and reducing the metal-cyanide complex, and a method for hydrolyzing cyanide using biological techniques. [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 an isolated genetically modified bacterium 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. [Selection Diagram] None

Inventors

  • ユー，ウェンシャン
  • ゴー，メイベル ダーレーン コー
  • リオウ，ルーティン
  • ラジャサバーイ，ラシュミ

Assignees

  • ナショナル ユニヴァーシティー オブ シンガポール

Dates

Publication Date
20260511
Application Date
20260120
Priority Date
20190408

Claims (16)

  1. An isolated recombinant DNA molecule, in the direction from the N-terminus to the C-terminus, (i) a golS transcription activator gene operably linked to a constitutive promoter and a ph1F repressor gene operably linked to the P golTS promoter or the P golB promoter; (ii) a promoter activated by CviR, a PhlF operator, and (iii) one or more cyanide-producing genes operably linked to the promoter activated by CviR.
  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 in the genome of Chromobacterium violaseum by targeting the promoter region of said genes, wherein the inactive Cas9 contains the H840A mutation in the HNH endonuclease domain and the D10A mutation in the RuvC endonuclease domain.
  4. The use according to claim 3, wherein the gene encoding the inactive Cas9 is operably ligated to the ParaBAD promoter, and the gene encoding the RNA guide (sgRNA) is operably ligated to a strong constitutive promoter such as J23119.
  5. A recombinant DNA molecule isolated from the golTSB operon, wherein the golTSB operon comprises golT, golS, golB, 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) a golS transcription activator gene operably linked to a constitutive promoter and a ph1F repressor gene operably linked to the P golTS promoter or the P golB promoter; (ii) a promoter activated by CviR and an operator of PhlF, and (iii) at least one recombinant polynucleotide DNA molecule comprising one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the promoter activated by CviR, according to claim 6.
  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 mutant 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) a golS transcription activator gene operably linked to a constitutive promoter and a ph1F repressor gene operably linked to the P golTS promoter or the P golB promoter; The method of claim 10, further comprising (ii) a promoter activated by CviR and an operator of PhlF, and (iii) at least one recombinant polynucleotide DNA molecule comprising one or both of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.
  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 approximately 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.

Description

This invention relates to a method for degrading cyanide using enzymes, and to a method for performing synthetic biological leaching by recovering metals using synthetic biotechnology. More specifically, this invention relates to the use of genetically modified bacteria 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. 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; it readily dissolves in water, producing sodium ions and cyanide ions ( CN⁻ ). Some of the CN⁻ are converted to hydrogen cyanide (HCN), the relative amount of which is determined by the pH of the water. At pH levels above 9.0, most exist in the stable form of CN⁻ . As the pH decreases, the amount of CN⁻ converted to HCN increases, but 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 the poisoning accidents of miners due to its inhalation. Since cyanides are carbon-based compounds, they readily react with other carbon-based substances and become toxic to many organisms. Therefore, waste containing cyanides must be detoxified before disposal. A 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 quickly into harmless substances. Also, less toxic substances may persist in the environment for extended periods, potentially causing problems for aquatic ecosystems. 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 vast amounts 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, M. A., and Martinelli, D. (2008), Hydrometallurgy 94, 14-17), Watling (Watling, H. R. (2006), Hydrometallurgy 84, 81-108), and Rawlings (Rawlings, D. E. (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-purification 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, Y. P. (2009), Advanced Materials Research 71, 661-664; Liang, G., Mo, Y., and Zhou, Q. (2010), Enzyme and Microbial Technology 47, 322-326; Chi, T. D., Lee, J. C., Pandey, B. D., Yoo, K., and Jeong, J. (2011), Miner Eng 24, 1219-1222). In these microorganisms, the leaching agent used for the bio-cleaning and recovery of metals is typically hydrogen cyanide. While hydrogen cyanide leaks pose a significant environmental threat, the microorganisms used in the biomining industry both produce cyanide (or its equivalents) and decompose cyanide (detoxifying its equivalents). Therefore, concerns about hydrogen cyanide leaks are limited or minimized. Consequently, large-scale releases of cyanide into the environment rarely occur with bioleaching. 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, M. A., and Martinelli, D. (2008), Hydrometallurgy 94, 14-17). Bioleaching is attracting attention as a "clean technology." Various bacteria (e.g., Chromobacterium violaceum, Pseudomonas fluorescein, and Pseudomonas aeruginosa) and various fungi (e.g., Marasmius oreades, Clitocybe sp., Polyporus sp.) produce hydrogen cyanide as a leachate (Pham, V., and Ting, Y. P. (2009), Advanced Materials Research 71, 661-664). Cyanide is produced as a secondary metabolit