Use of synthetic leaching agent biology for recovery of valuable and toxic metals from human sources

Abstract

The invention relates to a method for biologically recovering valuable and toxic metals from human sources using synthetic leaching agents, and generally relates to a method for biologically reducing metal cyanide complexes after cyanidation of metals and a method for biologically hydrolyzing cyanides. More specifically, the present invention allows for biosystematic engineering of an integrated synthetic leaching agent to be contained within a synthetic host, such as cyanogenic Chromobacterium violaceum, for efficient precious metal recovery and toxic metal repair of electronic waste. Also disclosed are bacteria capable of reducing ionic metals to ionic metals in nanoparticles, such as gold or silver. Also disclosed are methods of synthetic cyanide leaching agent production using genetically engineered bacteria transformed with a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene. Synthetic cyanolysis using genetically engineered bacteria transformed with heterologous nitrilase genes is also disclosed.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of April 8, 2020, application number 202080040580.0 (international application number PCT / SG2020 / 050216), and titled “Use of synthetic leaching agents for biological recovery of precious and toxic metals from anthropogenic sources”. Technical Field

[0002] The present invention relates to a method for decomposing cyanide using enzymes, and a synthetic metal recovery method for synthetic lixiviant biology. More specifically, the present invention relates to the use of engineered bacteria in processes involving cyanogenesis, reduction of leached metal ions, cyanolysis, and downstream recycling of cyanide. Tools for transcriptional control in C. violaceum are also provided. Background Art

[0003] Cyanide readily combines with most major and trace metals to form cyanide complexes, a property that makes it useful for extracting metals from ores. Sodium cyanide is most commonly used in mining areas. It is readily soluble in water to produce sodium ions and cyanide ions CN. - . Some CN - will be converted to hydrogen cyanide HCN, and the relative amount depends on the pH of the water. At a pH above 9.0, CN - It is the main stable form. As the pH decreases, CN converted to HCN - The amount of HCN will increase, and HCN tends to form gas and be released into the air. Therefore, most mining solutions are maintained at a pH above 10.0 to prevent the formation of HCN gas and accidental poisoning of miners due to inhalation. Because cyanide is carbon-based, it tends to react with other carbon-based substances, making it toxic to many living organisms. Therefore, waste containing cyanide must be detoxified before disposal. Conventional methods include alkaline chlorination, which is both dangerous and expensive. In addition, problems arise when cyanide used in mining does not decompose quickly into harmless substances. Even these less toxic substances will persist in the environment for a long time, which may cause problems for aquatic ecosystems.

[0004] The e-waste recycling industry uses chemical processes that pose serious environmental risks: current processes for recovering precious metals (such as gold) and removing toxic metals (such as lead and mercury) include pyrometallurgy (open-air burning, etc.) and hydrometallurgy (acid leaching and industrial cyanidation or cyanide baths); these methods are energy intensive, require an additional electrolysis step for metal separation, and are extremely polluting (Korte, F., Spiteller, M. and Coulston, F. (2000) Ecotoxicology and Environmental Safety 46, 241-245; Fields, S. (2001) Environ Health Perspect 109, A474-481). Efforts have been made to use biotechnological leaching processes to replace industrial chemical processes to make metal recovery and remediation simpler, more cost-effective and environmentally friendly, and scientists and engineers such as 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 for precious metal recovery by acid dissolution, current efforts in bioremediation of electronic waste to recover precious metals such as gold involve the use of microorganisms that produce lixiviants (Korte, F., Spiteller, M., and 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 lixiviant involved in the bioremediation and recovery of metals is usually hydrogen cyanide.Although possible hydrogen cyanide leaks pose a serious threat to the environment, the use of microorganisms in the biomining industry limits and minimizes such problems because the microorganisms in this context are both cyanogenic (capable of generating cyanide equivalents) and cyanolytic (capable of detoxifying cyanide equivalents), thereby virtually ensuring that no significant amounts of cyanide are released into the environment during the bioleaching process.

[0005] In contrast to existing processes, bioleaching by naturally occurring microorganisms operating under mild operating conditions can allow metal recycling in a process similar to natural biogeochemical cycles and thus reduce the demand for resources such as ores, energy or landfill space (Brandl, H., Lehmann, S., Faramarzi, MA and Martinelli, D. (2008), Hydrometallurgy 94, 14-17). Bioleaching has received attention because it represents a "clean technology". As a leaching agent, hydrogen cyanide is formed by a variety of bacteria (e.g., Chromobacterium violaceum, Pseudomonas fluorescens, and Pseudomonas aeruginosa) and fungi (e.g., Marasmius oreades, Clitocybe species, Polysporus species) (Pham, V. and Ting, YP (2009), Advanced Materials Research 71, 661-664). Cyanide is formed as a secondary metabolite and lasts for a short duration in the life of the microorganism. Although cyanide production by microorganisms was known many years ago, quantitative data on cyanide production by many species are still lacking (Liang, G., Mo, Y. and Zhou, Q. (2010), Enzyme and Microbial Technology 47, 322-326). However, current biorecovery and bioremediation efforts cannot match the industry expectations of cost-efficiency of leaching (effective metal recovery, short bioleaching time, and metal separation independent of conventional electrolysis) (Faramarzi, MA et al., (2004), Journal of Biotechnology 113, 321-326; Krebs, W. et al., (1997), FEMS Microbiology Reviews 20, 605-617), resulting in the continued use of conventional hydrometallurgical and pyrometallurgical methods for metal remediation. This difference in expectations is due to the inherent suboptimal lixiviant metabolism of microorganisms and the lack of suitable bioreduction pathways for specific metal recovery after bioleaching.

[0006] There is an urgent need to develop sustainable technologies for recycling electronic waste to protect our environment and conserve natural resources. The present invention focuses on the recovery of precious metals and removal of toxic metals from electronic waste. Current conventional electronic waste treatment technologies using strong acids or cyanides are polluting. Summary of the invention

[0007] The present invention generally relates to methods for the bioreduction of metal cyanide complexes following metal cyanidation and to methods for the biohydrolysis of cyanide.

[0008] The present invention allows the engineering of an integrated synthetic lixiviant biological system to be housed within a synthetic host, such as the cyanogenic Chromobacterium violaceum, for efficient precious metal recovery and toxic metal remediation of electronic waste. There may be up to four major components / modules in the design and engineering of the synthetic host: 1) synthetic cyanogenesis; 2) synthetic metal recovery; 3) synthetic cyanolysis; and 4) a synthetic loop for lixiviant biology. The present invention enables the construction of a synthetic loop for lixiviant biology.

[0009] In the present invention, the inventors have designed and constructed tools to remove excess cyanide from the environment, thereby providing a sustainable gold cyanidation process for industry. In addition, the inventors have designed and constructed genome editing tools for Chromobacterium violaceum for the construction of synthetic Chromobacterium violaceum to recover precious toxic metals from electronic waste. In addition, the inventors have designed and constructed tools to convert ionic gold (Au 3+ ) and / or ionic silver (Ag + ) are reduced back to their elemental states (Au) and (Ag) as gold and silver nanoparticles, respectively, offering industry an alternative to the conventional recovery step involving electrolysis.

[0010] According to a first aspect, the present invention provides an isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprises at least one mutation that enables the gene product to reduce ionic metals to elemental metals in the form of metal nanoparticles.

[0011] Another aspect provides an isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

[0012] In some embodiments, the isolated genetically engineered bacteria further comprises at least one polynucleotide molecule, the at least one polynucleotide molecule comprising in sequence from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0013] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a ph1F repressor gene operably linked to the promoter;

[0014] (ii) a promoter activated by CviR and an operator of PhlF, and

[0015] (iii) one or more of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

[0016] On the other hand, there is provided a process for recovering elemental gold in the form of gold nanoparticles from ionic gold (Au3+) or recovering elemental silver in the form of silver nanoparticles from ionic silver (Ag+), respectively, the process comprising the following steps:

[0017] a) contacting the isolated genetically engineered bacterium according to any one of claims 1 to 11 with a leachate containing ionic gold (Au3+) and / or ionic silver (Ag+); and

[0018] b) recovering the elemental gold nanoparticles and / or elemental silver nanoparticles from the leachate.

[0019] According to another preferred embodiment, the present invention provides a method for producing an isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprises one or more mutations that enable the gene product to convert ionic gold (Au) to 3+ ) to elemental gold in the form of gold nanoparticles or to ionic silver (Ag + ) is reduced to elemental silver in the form of silver nanoparticles, the method comprising the steps of:

[0020] a) error-prone PCR for the gene encoding mercury(II) reductase (MerA);

[0021] i) transforming at least one bacterium with the product of the PCR;

[0022] ii) Select the 3+ and / or Ag+ medium; or

[0023] b) multi-site saturation mutagenesis of the gene encoding mercury(II) reductase (MerA) by overlap extension PCR;

[0024] i) transforming at least one bacterium with the product of the PCR;

[0025] ii) Select the 3+ and / or Ag + Transformants grown on medium.

[0026] It will be appreciated that other forms of gold may be used in the growth medium, such as AuCl.

[0027] In another aspect, an isolated genetically engineered bacterium is provided, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous nitrilase gene operably linked to at least one promoter, the product of which gene causes cyanolysis of hydrogen cyanide.

[0028] In some embodiments, the at least one polynucleotide molecule further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

[0029] Another aspect provides a process for producing a synthetic cyanide lixiviant, the process comprising: contacting at least one recombinant cyanogenic bacterium with glycine, wherein the at least one bacterium is engineered to express a heterologous hydrogen cyanide synthase (hcnABC) gene and a heterologous 3-phosphoglycerate dehydrogenase mutant (serA) gene linked to at least one promoter. Examples are shown in Figure 1A middle.

[0030] In some embodiments, the recombinant cyanogenic bacteria further comprises at least one polynucleotide molecule, wherein the at least one polynucleotide molecule comprises in sequence from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0031] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a phlF repressor gene operably linked to the promoter;

[0032] (ii) a promoter activated by CviR and an operator of PhlF, and

[0033] (iii) one or more of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

[0034] According to another aspect, the present invention provides at least one isolated recombinant bacterium as defined herein capable of synthetic cyanide lixiviant production.

[0035] According to another aspect, the present invention provides a process for synthetic cyanolysis, the process comprising: contacting at least one recombinant cyanolytic bacterium with nitriles including cyanide present after bioleaching of electronic waste, wherein the at least one bacterium is engineered to express at least one nitrilase.

[0036] In some embodiments, the at least one recombinant cyanolytic bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase.

[0037] According to another aspect, the present invention provides an isolated recombinant DNA molecule comprising, in order from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0038] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a ph1F repressor gene operably linked to a PgolTS or PgolB promoter;

[0039] (ii) a promoter activated by CviR and an operator of PhlF, and

[0040] (iii) one or more cyanogenic genes operably linked to the CviR-activated promoter.

[0041] According to another aspect, the present invention provides the use of an inactivated Cas9 and sgRNA comprising mutations H840A and D10A in the HNH endonuclease domain and the RuvC endonuclease domain, respectively, for inhibiting the transcription of one or more genes in the Chromobacterium violaceum genome by targeting the promoter region of the one or more genes.

[0042] In some embodiments, the one or more genes encode violacein violacein formation.

[0043] On the other hand, a kind of isolated recombinant DNA molecule comprising golTSB operon is provided, wherein said operon comprises in order from N-terminus to C-terminus of said recombinant DNA molecule: golT, golS, golB and reporter gene such as GFP operably connected to j23119 promoter. This operon can be used for general screening of MerA gold reduction engineered bacteria.

[0044] The present invention further relates to the following technical solutions:

[0045] 1. An isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprising at least one mutation that enables the gene product to reduce ionic metals to elemental metals in the form of metal nanoparticles.

[0046] 2. The isolated bacterium of claim 1 , wherein the MerA gene comprises one or more mutations encoding an amino acid substitution, wherein the amino acid substitution is located at a position selected from the group consisting of V317, Y441, C464, A323D, A323D (delΔ324-365), A414E, G415I, E416C, L417I, I418D and A422N comprising the MerA coding sequence.

[0047] 3. The isolated bacterium according to claim 1 or 2, wherein the ionic metal is ionic gold (Au 3+ ), which is reduced to elemental gold in the form of gold nanoparticles, or ionic silver (Ag + ), which is reduced to elemental silver in the form of silver nanoparticles, and / or wherein the at least one isolated bacterium has a reduced reduction capacity for a mercury substrate when compared to a bacterium comprising an unmutated MerA gene.

[0048] 4. An isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

[0049] 5. The isolated bacterium according to item 4, wherein the hydrogen cyanide synthase gene is hcnABC and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or wherein the isolated genetically engineered bacterium further comprises at least one polynucleotide molecule, the at least one polynucleotide molecule being included in sequence from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0050] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a ph1F repressor gene operably linked to the promoter;

[0051] (ii) a promoter activated by CviR and an operator of PhlF, and

[0052] (iii) one or more of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

[0053] 6. The isolated bacterium of item 5, wherein the golS gene is codon-optimized for Chromobacterium violaceum, and / or wherein the golS gene is a mutant selected from GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

[0054] 7. The isolated bacterium according to any one of the preceding items, wherein the bacterium is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli and / or wherein the bacterium is stable at pH 10.

[0055] 8. A method for recovering elemental gold in the form of gold nanoparticles from ionic gold (Au3+) or elemental silver in the form of silver nanoparticles from ionic silver (Ag+), respectively, the method comprising the following steps:

[0056] a) contacting the isolated genetically engineered bacterium according to any one of items 1 to 7 with a leachate containing ionic gold (Au3+) and / or ionic silver (Ag+); and

[0057] b) recovering the elemental gold nanoparticles and / or elemental silver nanoparticles from the leachate.

[0058] 9. A method according to item 8, wherein the contacting is carried out under alkaline conditions.

[0059] 10. A method for producing an isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprising one or more mutations that enable the gene product to convert ionic gold (Au) to 3+ ) to elemental gold in the form of gold nanoparticles or to ionic silver (Ag + ) is reduced to elemental silver in the form of silver nanoparticles, the method comprising the steps of:

[0060] a) error-prone PCR for the gene encoding mercury(II) reductase (MerA);

[0061] i) transforming at least one bacterium with the product of the PCR;

[0062] ii) Select the 3+ and / or Ag+ medium; or

[0063] b) multi-site saturation mutagenesis of the gene encoding mercury(II) reductase (MerA) by overlap extension PCR;

[0064] i) transforming at least one bacterium with the product of the PCR;

[0065] ii) Select the 3+ and / or Ag + Transformants grown on medium.

[0066] 11. A method according to claim 10, wherein in part a), the PCR is carried out using forward and reverse primers, wherein the forward primer comprises the nucleotide sequence shown in SEQ ID NO:1 and the reverse primer comprises the sequence shown in SEQ ID NO:2, and / or wherein in part b), the PCR is carried out using primers containing NNK and / or MNN at target sites V317, Y441 and C464.

[0067] 12. A method according to item 10 or 11, wherein the selection involves the selection of at least 2 forms, one of which comprises 3+ and / or Ag + The other form consists of selecting on agar plates containing Au 3+ and / or Ag + Selection was performed in liquid culture.

[0068] 13. An isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a heterologous nitrilase gene operably linked to at least one promoter, the product of the gene causing the cyanolysis of hydrogen cyanide.

[0069] 14. The isolated genetically engineered bacterium of claim 13, wherein the heterologous nitrilase gene encodes an enzyme selected from the group comprising cyanide dehydratase and cyanide hydratase, and / or wherein the at least one polynucleotide molecule further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

[0070] 15. The isolated genetically engineered bacterium according to item 13 or 14, wherein the bacterium is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli.

[0071] 16. A method for producing a synthetic cyanide lixiviant, the method comprising:

[0072] contacting at least one recombinant cyanogenic bacterium with glycine,

[0073] The bacteria comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

[0074] 17. A method according to item 16, wherein the hydrogen cyanide synthase gene is hcnABC and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or wherein the recombinant cyanogenic bacterium further comprises at least one polynucleotide molecule, wherein the at least one polynucleotide molecule is sequentially included from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0075] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a ph1F repressor gene operably linked to the promoter;

[0076] (ii) a promoter activated by CviR and an operator of PhlF, and

[0077] (iii) one or more of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

[0078] 18. A method according to claim 17, wherein the golS gene is codon-optimized for Chromobacterium violaceum, and / or wherein the golS gene is a mutant selected from GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

[0079] 19. The method according to any one of items 16 to 18, wherein the at least one recombinant cyanogenic bacterium can tolerate about pH 10.

[0080] 20. A process according to any one of clauses 16 to 19, wherein the synthetic cyanide lixiviant production is carried out together with the metals for bioleaching in a single reactor.

[0081] 21. At least one isolated recombinant cyanogenic bacterium according to any one of items 16 to 20, which is capable of synthetic cyanide lixiviant production.

[0082] 22. At least one recombinant cyanogenic bacterium according to item 21, which is selected from the group consisting of Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Escherichia coli.

[0083] 23. A method of synthetic cyanolysis comprising contacting at least one recombinant cyanolytic bacterium with nitriles, including cyanide, present following bioleaching of electronic waste, wherein the at least one bacterium is engineered to express at least one nitrilase.

[0084] 24. A method according to claim 23, wherein the at least one nitrilase is selected from the group comprising cyanide dehydratase and cyanide hydratase, and / or wherein the at least one recombinant cyanolytic bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase and phosphoenolpyruvate carboxylase.

[0085] 25. The method according to claim 23 or 24, wherein the at least one nitrilase is derived from at least one bacterial species selected from the group comprising Pseudomonas pseudoalcaligenes (nit), Synechocystis sp. PCC 6803 chromosome (SC-nit), and the cyanide dihydratase is derived from Bacillus pumilus (BP-cynD) and Pseudomonas stutzeri (PS-cynD).

[0086] 26. An isolated recombinant DNA molecule comprising, in order from the N-terminus to the C-terminus of the recombinant DNA molecule: (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and golTS or P golB a ph1F repressor gene operably linked to the promoter;

[0087] (ii) a promoter activated by CviR and an operator of PhlF, and

[0088] (iii) one or more cyanogenic genes operably linked to the CviR-activated promoter.

[0089] 27. An isolated recombinant DNA molecule according to item 26, wherein the golS transcriptional activator gene is a mutant selected from the group comprising or consisting of: GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

[0090] 28. Use of an inactivated Cas9 and sgRNA comprising mutations H840A and D10A in the HNH endonuclease domain and the RuvC endonuclease domain, respectively, for inhibiting the transcription of one or more genes in the Chromobacterium violaceum genome by targeting the promoter region of the one or more genes.

[0091] 29. The use according to item 28, wherein the gene encoding the inactivated Cas9 is araBADThe promoter is operably linked, and the gene encoding the RNA guide (sgRNA) is operably linked to a strong constitutive promoter such as J23119.

[0092] 30. An isolated recombinant DNA molecule comprising a golTSB operon, wherein the operon comprises, in order from the N-terminus to the C-terminus of the recombinant DNA molecule: golT operably linked to a j23119 promoter, golS, golB operably linked to a golB promoter, and a reporter gene such as GFP. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1A-1B : Figure 1A ; The engineered cyanogenic strain (SynLix 3.1) was shown to produce up to 80 mg / L cyanide. SynLix 3.1 was engineered to express a mutant of hydrogen cyanide synthase (hcnABC) and 3-phosphoglycerate dehydrogenase (serA). Figure 1B ; Overview of the cyanolysis and conjugation processes. The engineered cyanogenic strain (SynLix 3.1) can tolerate pH 10.0 (alkaliphilic).

[0094] Figure 2 An overview of the cyanolysis and coupling processes is shown. Four variants of nitrilase from different bacteria were selected; their sequences were synthesized and subsequently cloned into their corresponding host cells. Nitrilases from Pseudomonas alginate (nit) and Synechocystis species PCC 6803 chromosome (SC-nit), cyanide dihydratase from Bacillus pumilus (BP-cynD) and Pseudomonas stutzeri (PS-cynD). Nit contains 2 different subunits, nitB and nitC, which were cloned into pRSF-Duet vectors and expressed in E. coli (DE3) BL21 host cells. For the remaining three variants, they were cloned into pGM vectors and subsequently integrated into the Chromobacterium violaceum genome via the Tn7 transposition system.

[0095] Figure 3 The schematic diagram of recombinant Chromobacterium violet host cell is shown. Coupling enzymes include formate dehydrogenase, glutamate dehydrogenase and phosphoenolpyruvate carboxylase. These enzymes are cloned in pBbB8k-RFP (broad-spectrum host range) carrier, and expressed in Chromobacterium violet subsequently. By coupling the cyanolysis process with downstream enzymes, byproduct carbon and nitrogen can be recycled, making it a self-sustaining system.

[0096] Figure 4Schematic representation of the inactivated Cas9 mechanism showing transcriptional repression. The catalytically inactivated Cas9 is guided to the promoter sequence by sgRNA (blue) and physically inhibits RNAP from initiating transcription. The 20-nucleotide protospacer sequence (purple) targeted in the promoter region is adjacent to the protospacer adjacent motif (PAM) sequence NGG (red) required for Cas9 to bind to DNA.

[0097] Figure 5 Shown is the repression of violacein production in Chromobacterium violaceum by dCas9. (A) Schematic diagram of the dCas9 circuit. (B) The targeted regions of the violacein operon are indicated by the three dCas9-sgRNA complexes, which are the vioA promoter, the vioB promoter, and the 5' region of vioC. (C) The gRNA targeting the vioA promoter requires the lowest amount of dCas9 induction (0.01% arabinose), the gRNA targeting the vioB promoter requires slightly higher amounts of dCas9 induction (0.1% arabinose), and the gRNA targeting vioC (further down from the transcription start site) has the same effect as the non-targeting gRNA negative control. (D) Measured OD600 of the corresponding wells in (C).

[0098] Figure 6 Gold sensor circuit optimization. Gold ion dose response and circuit schematic, where (A) the original GolTSB operon or the minimal gold sensor, which consists of the GolS transcriptional activator and (B) P golTS or (C)P golB composition.

[0099] Figure 7 Schematic diagram showing the circuit of the gold mutant sensor.

[0100] Figure 8 The structure of GolS is shown. (A) The N-terminal and C-terminal domains of GolS consist of a helix-turn-helix (HTH) DNA binding domain and a gold ion binding domain, respectively. (B) The structure of a GolS homodimer bound to DNA predicted using Phyre2 (Kelley, Mezulis, Yates, Wass, & Sternberg, 2015). In GolSmt1, GolSmt2, and GolSmt3, the residues at the DNA binding domain (right arrows) are mutated. GolSmt2 has an additional mutation at the gold ion binding domain (top residues, up arrows). GolSmt3 has an additional mutation at the DNA binding domain (left arrow residues).

[0101] Fig. 9The mutation site of GolSmt1A38I is shown. A single mutant from alanine to isoleucine (circled) at residue 38. Both residues are hydrophobic, and isoleucine has 3 more carbons in the hydrophobic side chain than alanine. This may increase hydrophobicity and aggregation of the hydrophobic core and allow for stronger binding to DNA.

[0102] Fig.10 Shown is the mutation of GolSmt2 A38Q N97D. Double mutant from alanine to glutamine at residue 38 (circle at DNA binding domain) and from asparagine to aspartic acid at residue 97 (circle at ion binding domain). For the first mutation, alanine is a non-polar neutral amino acid, while glutamine is polar neutral with an amide side chain, and the longer polar side chain of glutamine may have better binding to DNA. For the second mutation, asparagine is polar neutral, while aspartic acid is polar acidic, and the negatively charged side chain of aspartic acid may have a higher affinity with positively charged gold ions or improve dimerization.

[0103] Fig.11 Shown is the mutation of GolSmt3 A38K V60L. Double mutant from alanine to lysine at residue 38 (outer circle) and from valine to leucine at residue 60 (inner circle). For the first mutation, alanine is non-polar neutral, while lysine is polar basic, which can allow better binding to the backbone phosphate groups. For the second mutation, both valine and leucine are non-polar neutral, and leucine has one more carbon atom in its side chain, which can increase the overall hydrophobicity of the DNA binding domain.

[0104] Fig.12 Experimental workflow showing mutagenesis and selection of gold sensor mutants in Chromobacterium violaceum. A template for a mutant library of the golS transcriptional activator was created by deep scanning mutagenesis, in which each amino acid was replaced with 19 other amino acids. The mutant library was then cloned and transformed into Chromobacterium violaceum. Mutants with higher fluorescence output and sensitivity to gold ions were selected.

[0105] Fig.13 The responses of the wild type and the top four gold sensor mutants to gold ions are shown. The response curves were plotted and fitted to the Hill equation: Y = (B max X n ) / (K n +X n )+C. K: Activation at half-maximum RFU [Au 3+ ] threshold, n: Hill coefficient, C: baseline RFU, B max : Maximum RFU.

[0106] Fig.14 A schematic diagram of a synthetic circuit is shown, which provides a way to incorporate dynamic regulation into new systems. A quorum of molecules allows the circuit to be turned on autonomously, while a gold sensor signals the circuit to close when gold ions have been leached from the electronic waste.

[0107] Fig.15 Schematic diagram showing a reversible synchronized on-off circuit in response to the absence and presence of gold ions. The circuit is turned on by a population sensor (middle frame) and closed by a gold ion sensor (left frame). The reversible circuit can be turned on again by dilution of the repressor in the absence of gold ions. The gold ion sensor (left frame) consists of a GolS transcriptional activator that is activated by gold ions for expressing the PhlF repressor. The population sensor (middle frame) utilizes the host's endogenous CviR activator that is induced by AHLs at high cell density. The cyanogenic output (right frame) represented by RFP is thus synchronized at high cell density and inhibited in the presence of gold ions.

[0108] Fig.16 Schematic diagram showing batch culture of Chromobacterium violaceum for on-off output in response to gold ions. To study the on-off output of the circuit, small-scale batch cultures of Chromobacterium violaceum were grown in 2 mL eppendorf tubes in 300 μL of Tris minimal medium supplemented with 30 μg / mL kanamycin at 37°C for 24 hours. A 1:600 ​​dilution of the stationary phase culture was performed at each passage, and 2 μM AuCl was added at alternate passages. 3 Endpoint single-cell fluorescence output was measured using a BD Accuri C6 flow cytometer (BD Biosciences, Singapore) with a flow rate of 14 μL / min and a core size of 10 μm, collecting 10 000 events per sample. Fluorescence excitation was performed at 561 nm and detection was performed at 610 / 620 nm. Cells were gated using forward and side scatter. Background fluorescence of control cells without RFP was subtracted from the mean fluorescence values.

[0109] Fig.17 Graph showing the reduction in basal leak expression of a circuit using a mutant gold sensor compared to a wild-type gold sensor by successive cell passaging in the presence or absence of gold ions. Each passaging is a step-wise increase from the previous passaging in the absence of Au. 3+ To open the circuit or contain 2μM Au 3+ Dilute 1:600 ​​in closed loop minimal medium.

[0110] Fig.18Shown are graphs representing the separation of the on and off populations during three on-off cycles using GolS mutants (B-GolSmt1, C-GolSmt2, D-GolSmt3), while the on-off populations were fused in a circuit containing a wild-type gold sensor (A).

[0111] Fig.19 Schematic diagram showing the construction of the biosensor. The Salmonella gol operon consists of golT, golS, and golB, which encode a P-type ATPase, an Au sensor, and a metal-binding chaperone, respectively. The constitutive E. coli promoter J23119 ensures the use of Au 3+ The ion-bound Au sensor is expressed, and the Au sensor then binds to the Au sensing promoter golB and drives the expression of GFP.

[0112] Fig. 20 Display of golGFP in response to increasing concentrations of Au 3+ (μM) of biological sensitivity.

[0113] Fig.21 Schematic showing the screen used to identify gold-reducing mutants.

[0114] Fig. 22 Target sites in the MerA protein for sequence modification to alter metal binding affinity and / or specificity are shown.

[0115] Fig.23 Au is shown in (A) liquid broth and (B) agar plates. 3+ Effect on the growth of Escherichia coli Rosetta (DE3) pLysS cells.

[0116] Fig.24 Shown from Au 3+ OD of E. coli cells expressing wild-type MerA and all mutants on agar plates 600 .

[0117] Fig.25 TEM images of gold nanoparticles synthesized by the MerA mutants are shown.

[0118] Fig.26 Au of mutants identified by DM selection is shown 3+ Comparison of reduction kinetic parameters. ●-DM mutant, ●-WT MerA. For (A) and (B), the mutants above the dashed line show k cat / K M or k cat Improvements compared to WT MerA. (A) k of DM mutants cat / KM Comparison of (A) kcat values ​​of DM mutants, (C) K M Comparison of K values. Mutants below the dashed line show K M Improvements relative to WT MerA. (D) - Table of mutants and related sequences.

[0119] Fig. 27 Shown is the reduction of AuCl by (A) WT MerA (B) DM11 3 TEM images of recovered AuNPs.

[0120] Fig.28 Shown from AuCl 3 and Au in the leaching solution 3+ In vivo reduction by (A) WT MerA (B) DM11.

[0121] Fig.29 Shown is a comparison of Au content before and after recovery through DM11 and at downstream processing stages.

[0122] Fig.30 Shows that AuCl 3 TEM images of AuNPs recovered from AuCl 3 Recycled truncated tetrahedral AuNP (B) from AuCl 3 Recovered truncated bi-tetrahedral AuNPs (C) Truncated tetrahedral AuNPs recovered from the leaching solution.

[0123] Fig.31 Figure 2 shows (A) a schematic diagram of the gold and silver biosensors in Chromobacterium violaceum and (B) a graph showing the sensitivity of the mutant gold sensors to the following different metal ions in minimal medium: AuCl 3 (40μM), AgNO 3 (10 μM), CdCl 2 (80μM), ZnCl 2 (100μM), HgCl 2 (5μM), NiSO 4 (50μM), CoCl 2 (120μM), FeSO 4 (25 μM) and CuSO 4 (35μM).

[0124] Fig.32 Shown are the dose-response curves of the wild-type and mutant biosensors to different metal ions in minimal medium.

[0125] Fig.33An outline of the method for gold and silver biosensing in Chromobacterium violaceum using metals leached from electronic waste metals is shown.

[0126] Fig.34 Graph showing the ability of wild-type and mutant biosensors to sense noble metal ions in a mixture of metal ions leached from electronic waste metals (ESM).

[0127] For convenience, the references mentioned in this specification are listed in the form of a reference list and attached at the end of the examples. The entire contents of such references are incorporated herein by reference, but the references mentioned in the specification do not mean that they constitute part of the common general knowledge. DETAILED DESCRIPTION

[0128] It is envisioned that the use of engineered bacteria in processes involving cyanogenesis, reduction of leached metal ions, cyanolysis, and downstream recycling of cyanide will require the use of separate strains of bacteria with separate mutant enzymes. Some strains will selectively reduce gold, while others will selectively reduce silver. Possible workflows would include: strains that generate biolixiviants to oxidize metals; strains that would then selectively reduce metals using gold-reducing and silver-reducing mutants to recover metals; and strains that would be used to bioremediate biolixiviants by biodegrading cyanide.

[0129] Before the compounds, compositions, articles, devices and / or methods of the present invention are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to specific reagents unless otherwise specified, as such are, of course, variable. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0130] definition

[0131] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0132] The terms "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article.

[0133] As used herein, the terms "comprising" or "including" should be interpreted as specifying the presence of the stated features, integers, steps or components mentioned, but not excluding the presence or addition of one or more features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the terms "comprising" or "including" also include "consisting of". Variations of the word "comprising", such as "comprise" and "comprises", and variations of "including", such as "include" and "includes", have correspondingly varying meanings.

[0134] In this article, the range can be expressed as from "about" a specific value and / or to "about" another specific value. When such a range is expressed, another embodiment includes from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the specific value forms another embodiment. It should be further understood that the endpoints of each range are important relative to the other endpoints and are independent of the other endpoints. It should also be understood that multiple values ​​are disclosed herein, and in addition to the value itself, each value is also disclosed as "about" this specific value in this article. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to" the value, and the possible range between the values ​​are also disclosed, as the technician appropriately understands. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and this data represents the endpoints and starting points and ranges of any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15 are considered disclosed. It is also understood that every unit between the two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0135] In a first aspect of the present invention, an isolated genetically engineered bacterium is provided, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprises one or more mutations that enable the gene product to reduce ionic metals to elemental metals in the form of metal nanoparticles.

[0136] In some embodiments, the MerA gene comprises one or more mutations encoding amino acid substitutions, wherein the amino acid substitution is located at a position selected from the group consisting of V317, Y441, and C464. In other embodiments, the MerA gene mutation is located at one or more sites selected from the group consisting of: A323D, A323D (delΔ324-365), A414E, G415I, E416C, L417I, I418D, and A422N.

[0137] In some embodiments, the ionic metal is ionic gold (Au 3+ ), which is reduced to elemental gold in the form of gold nanoparticles, or ionic silver (Ag + ), which is reduced to elemental silver in the form of silver nanoparticles.

[0138] In some embodiments, the isolated bacterium has a decreased reducing ability for a mercury substrate when compared to a bacterium comprising a non-mutated MerA gene.

[0139] In some embodiments, an isolated genetically engineered bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter. 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). The presence of these genes increases hydrogen cyanide production to leach precious metals from sources such as electronic waste.

[0140] In some embodiments, the production of hydrogen cyanide is under the control of an on / off switch comprising a gold ion sensor and a quorum sensor (e.g. Fig.15 More specifically, the gold sensor shuts down the leaching process once the leached gold reaches a critical level. The shut-off circuit is responsive to gold ions generated by cyanidation and comprises a "low" constitutive promoter driving the golS gene and a promoter driving a downstream repressor gene. An example of a minimal gold sensor comprises a low constitutive promoter (e.g., P Con6 ; SEQ ID NO:37) under the control of golS transcriptional activator gene, and by P golTS or P golBThe quorum sensor comprises a promoter driven to produce a Ph1F repressor to block the Ph1F operator in the quorum sensor. The on-loop of the quorum sensor is responsive to high cell density and comprises a promoter activated by endogenous CviR (induced by AHL at high cell density) and an operator of Ph1F downstream of the quorum sensor promoter. Downstream of the quorum sensor and under its regulation are one or more cyanogenic genes selected from a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene as described above.

[0141] In some embodiments, the isolated genetically engineered bacteria comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to an on / off loop, wherein the off loop is located upstream of the on loop, the on loop being located upstream of the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene, wherein the off loop comprises a heterologous golS gene operably linked to a promoter, and a promoter selected from the group consisting of golTS and P golB A downstream heterologous ph1F repressor gene is operably connected to a promoter of CviR, wherein the open loop comprises a promoter activated by endogenous CviR and an operator gene of PhlF between the CviR-activated promoter, and wherein the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene are operably connected to the CviR-activated promoter.

[0142] Preferably, the golS gene is codon optimized for Chromobacterium violaceum and the dynamic range and sensitivity are optimized by mutagenesis (e.g. Fig.12 In some embodiments, the golS gene is a mutant selected from the group consisting of: GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L, and GolSmt4_D33P.

[0143] Useful gold ion sensors include the golTSB operon, shown in Fig.19 middle.

[0144] On the other hand, a kind of isolated recombinant DNA molecule comprising golTSB operon is provided, wherein said operon comprises in order from N-terminus to C-terminus of said recombinant DNA molecule: golT, golS, golB and reporter gene such as GFP operably connected to j23119 promoter. This operon can be used for general screening of MerA gold reduction engineered bacteria.

[0145] Nitrilases are a group of enzymes that hydrolyze nitriles into ammonia and the corresponding carboxylic acid. There are two types of such cyanide-degrading enzymes.

[0146]

[0147] First, cyanide dihydratases, comprise a group of bacterial enzymes. These enzymes behave like true nitrilases, converting cyanide directly into formic acid and ammonia (top). On the other hand, cyanide hydratases of fungal origin hydrolyze cyanide to formamide (bottom). These hydrolases do not require additional cofactors or substrates and catalyze a wide range of substrate concentrations, making them good candidates for bioremediation of cyanide.

[0148] In some embodiments, a kind of isolated genetically engineered bacteria can include a heterologous nitrilase gene operably connected to at least one promoter. The nitrilase gene causes the cyanolysis of hydrogen cyanide. In some embodiments, the heterologous nitrilase gene encodes an enzyme selected from the group comprising cyanide dehydratase and cyanide hydratase. In some embodiments, at least one nitrilase is derived from at least one bacterial species selected from the group comprising pseudoalcaligenes Pseudomonas (nit), Synechocystis species PCC 6803 chromosome (SC-nit), and cyanide dihydratase is derived from Bacillus pumilus (BP-cynD) and Pseudomonas stutzeri (PS-cynD). In addition to being part of an integrated workflow, the cyanolysis strain for bioremediation can be used by itself, or as a restriction to the cyanide heavy industry.

[0149] In some embodiments, the isolated genetically engineered bacteria further comprises a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter. By coupling the cyanolysis process with downstream enzymes (i.e., formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase), byproduct carbon and nitrogen can be recycled, making it a self-sustaining system ( Figure 3 ).

[0150] In some embodiments, the isolated genetically engineered bacteria comprises a heterologous nitrilase gene, a heterologous formate dehydrogenase gene, a heterologous glutamate dehydrogenase gene, and a heterologous phosphoenolpyruvate carboxylase gene operably linked to at least one promoter.

[0151] In some embodiments, the bacteria is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Eschericia coli.

[0152] In some embodiments, the bacteria are stable at pH 10.

[0153] The second aspect of the present invention provides a method of obtaining a 3+ ) to recover elemental gold as gold nanoparticles or from ionic silver (Ag +) A process for recovering elemental silver in the form of silver nanoparticles, the process comprising the following steps:

[0154] a) contacting the isolated genetically engineered bacteria according to any aspect of the present invention with a nucleic acid comprising ionic gold (Au) 3+ ) and / or ionic silver (Ag + ); and

[0155] b) recovering the elemental gold nanoparticles and / or elemental silver nanoparticles from the leachate.

[0156] In some embodiments, the contacting is performed under basic conditions.

[0157] In some embodiments, the contacting is performed at a pH of at least about 10.

[0158] A third aspect of the invention provides a method of producing an isolated bacterium comprising at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a heterologous mercury (II) reductase (MerA) gene operably linked to at least one promoter and comprising one or more mutations, wherein the one or more mutations enable the gene product to reduce an ionic metal to an elemental metal in the form of metal nanoparticles, the method comprising the steps of:

[0159] a) error-prone PCR for the gene encoding mercury(II) reductase (MerA);

[0160] i) transforming at least one bacterium with the product of the PCR;

[0161] ii) selecting transformants that grow on a medium containing the ionic metal; or

[0162] b) multi-site saturation mutagenesis of the gene encoding mercury(II) reductase (MerA) by overlap extension PCR;

[0163] i) transforming at least one bacterium with the product of the PCR;

[0164] ii) selecting transformants that grow on the medium containing the ionic metal.

[0165] In some embodiments, the gene product is capable of converting ionic gold (Au 3+ ) and / or ionic silver (Ag + ) are reduced to elemental gold in the form of gold nanoparticles and / or elemental silver in the form of silver nanoparticles, respectively. In some embodiments, in part ii), the ionic metal is selected from AuCl 3 and / or AgNO 3In some embodiments, in part a), the PCR is performed with forward and reverse primers, wherein the forward primer comprises the nucleotide sequence 5'-GTGGTGGTGGTGGTGCTCGAGTTA-3' (SEQ ID NO: 1), and the reverse primer comprises the nucleotide sequence 5'-GATATACATATGCACCACCATCACCATCAT-3' (SEQ ID NO: 2).

[0166] In some embodiments, in part b), the PCR is performed using primers containing NNK and / or MNN at target sites V317, Y441, and C464 of the MerA protein.

[0167] In some embodiments, the selection involves selection of at least 2 forms, one of which comprises 3+ and / or Ag + The other form consists of selecting on agar plates containing Au 3+ and / or Ag + Selection was performed in liquid culture.

[0168] A fourth aspect of the present invention provides a process for producing a synthetic cyanide lixiviant, the process comprising:

[0169] contacting the recombinant cyanogenic bacteria with glycine,

[0170] The bacteria comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

[0171] In some embodiments, the hydrogen cyanide synthase gene is hcnABC and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA. Examples are shown in Figure 1A In the experiment, a mutant called SynLix 3.1 was grown in LB and cyanide production was monitored over 48 hours. Cyanide production was detected using a cyanide-sensitive ion selective electrode (ISE).

[0172] In some embodiments, the hcnABC and serA genes are under the control of an inducible promoter. In some embodiments, the isolated genetically engineered bacteria comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to an on / off loop, wherein the closed loop is located upstream of the open loop, and the open loop is located upstream of the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene, wherein the closed loop comprises a heterologous golS gene operably linked to a promoter, and a heterologous 3-phosphoglycerate dehydrogenase mutant gene selected from P golTS and P golBA downstream heterologous ph1F repressor gene is operably connected to a promoter of CviR, wherein the open loop comprises a promoter activated by endogenous CviR and an operator gene of PhlF between the CviR-activated promoter, and wherein the hydrogen cyanide synthase gene and the 3-phosphoglycerate dehydrogenase mutant gene are operably connected to the CviR-activated promoter.

[0173] In some embodiments, the recombinant cyanogenic bacteria can tolerate a pH of at least about 10.

[0174] In some embodiments, the synthetic cyanide lixiviant generation is performed with the metals in a single reactor for bioleaching.

[0175] Another aspect of the present invention provides at least one isolated recombinant bacterium as defined in any aspect of the present invention capable of synthetic cyanide lixiviant production.

[0176] In some embodiments, the recombinant cyanogenic bacteria is selected from the group consisting of Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Escherichia coli.

[0177] Another aspect of the present invention provides a process for synthetic cyanolysis, the process comprising:

[0178] a) contacting at least one recombinant cyanolytic bacterium with nitriles including cyanide present after bioleaching of electronic waste,

[0179] wherein the at least one bacterium is engineered to express at least one nitrilase.

[0180] In some embodiments, the at least one nitrilase is selected from the group consisting of cyanide dehydratases and cyanide hydratases.

[0181] In some embodiments, the at least one recombinant cyanolytic bacterium is further engineered to express formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase.

[0182] In some embodiments, the at least one nitrilase is derived from at least one bacterial species selected from the group comprising Pseudomonas pseudoalcaligenes (nit), Synechocystis sp. PCC 6803 chromosome (SC-nit) and the cyanide dihydratase is derived from Bacillus pumilus (BP-cynD) and Pseudomonas stutzeri (PS-cynD).

[0183] Another aspect of the present invention provides an isolated recombinant DNA molecule comprising, in order from the N-terminus to the C-terminus of the recombinant DNA molecule;

[0184] (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a ph1F repressor gene operably linked to a PgolTS or PgolB promoter;

[0185] (ii) a promoter activated by CviR and an operator of PhlF, and

[0186] (iii) one or more cyanogenic genes operably linked to the CviR-activated promoter.

[0187] In some embodiments, the golS transcriptional activator gene is under a low constitutive promoter (such as P Con6 ) is under the control of.

[0188] In some embodiments, the golS gene is codon optimized for Chromobacterium violaceum and the dynamic range and sensitivity are optimized by mutagenesis (e.g. Fig.12 In some embodiments, the golS gene is a mutant selected from the group consisting of: GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L, and GolSmt4_D33P.

[0189] Another aspect of the present invention provides the use of an inactivated Cas9 and sgRNA comprising mutations H840A and D10A in the HNH endonuclease domain and the RuvC endonuclease domain, respectively, for inhibiting the transcription of one or more genes in the Chromobacterium violaceum genome by targeting the promoter region of the one or more genes.

[0190] In some embodiments, the gene encoding the inactivated Cas9 is fused to the P araBAD The promoter is operably linked, and the gene encoding the RNA guide (sgRNA) is operably linked to a strong constitutive promoter (such as J23119).

[0191] In some embodiments, the inactivated Cas9 targets the violacein operon to prevent the formation of violacein violacein, as the pigment may complicate downstream processing steps. In some embodiments, the inactivated Cas9 targets the vioA, vioB and / or vioC promoters, preferably all three promoters. Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention.

[0192] Example

[0193] Standard molecular biology techniques known in the art and not specifically described were generally followed, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0194] Example 1

[0195] Integrating nitrilase into host cells for cyanolysis

[0196] Four variants of nitrilase from different bacteria were selected; their sequences were synthesized and subsequently cloned into their corresponding host cells. Nitrilases from Pseudomonas pseudoalcaligenes (nit) (SEQ ID NO: 11) and Synechocystis sp. PCC 6803 chromosome (SC-nit) (SEQ ID NO: 12), cyanide dihydratase from Bacillus pumilus (BP-cynD) (SEQ ID NO: 13) and Pseudomonas stutzeri PS-cynD) (SEQ ID NO: 14). Nit contains two different subunits, nitB (SEQ ID NO: 15) and nitC (SEQ ID NO: 16), which were cloned into pRSF-Duet vector and expressed in E. coli (DE3) BL21 host cells. For the remaining three variants, they were cloned into pGEM vector and subsequently integrated into the Chromobacterium violaceum genome via the Tn7 transposition system ( Figure 2 and Figure 3 ).

[0197] The coupled enzymes include formate dehydrogenase, glutamate dehydrogenase, and phosphoenolpyruvate carboxylase. These enzymes were cloned into the pBbB8k-RFP (broad host range) vector and subsequently expressed in Chromobacterium violaceum. By coupling the cyanolysis process with downstream enzymes, the byproduct carbon and nitrogen can be recycled, making it a self-sustaining system ( Figure 3 ).

[0198] The cyanolytic strain of Chromobacterium violaceum was tested for its ability to remove cyanide from the extracellular environment. In the presence of 100 mg / L potassium cyanide, the engineered cyanolytic strain of Chromobacterium violaceum was able to completely remove exogenous cyanide within 24 hours.

[0199] Example 2

[0200] Development of genomic transcriptional control tools using dCas9 in Chromobacterium violaceum.

[0201] CRISPR-Cas9 is widely used as a genome editing tool in many organisms. It is mainly used in eukaryotes, including mammals, insects and yeast cells. The CRISPR-Cas system is derived from the adaptive immune system of bacteria, in which a small fragment of the phage DNA of the invasion is inserted into its host genome as a memory, to supervise the possible invasion of phages composed of the same DNA fragments in the future. The CRISPR-Cas9 system from Streptococcus pyogenes (Streptomyces pyogenes) is composed of Cas9 endonucleases, which are combined with RNA guides to bind to a DNA sequence homologous to the RNA guide, and the Cas9 endonucleases are cut into the double-stranded DNA of the invasion phage. When used as a genome editing tool, Cas9 endonucleases are expressed together with RNA guides, and the RNA guides are composed of a spacer of 20 nucleotides complementary to the target DNA and a 76bp support of the Cas9 endonucleases contacted thereafter. After being guided to the target DNA by the 20bp spacer of the RNA guide, the Cas9 endonucleases cause double-strand breaks in the host chromosome. The host activates DNA repair mechanisms against the cut chromosome, resulting in insertion, deletion or homologous recombination of the introduced DNA fragment in the DNA target site. Due to the ease and modularity of the RNA-guided mechanism, the CRISPR-Cas9 genome editing method has been rapidly adopted in many organisms.

[0202] CRISPR-Cas has been adapted for many other applications. Due to the ease and specificity of RNA recognition of DNA sequences, many applications and variations of Cas9 have been applied to target specific genomic loci. CRISPRi (CRISPR-interference) and CRISPRa (CRISPR-activation) that target gene transcription are two additions to the CRISPR / Cas genetic toolkit. CRISPRi ( Figure 4) is mediated by an inactivated Cas9 (dCas) (SEQ ID NO: 17) having mutations H840A and D10A in the HNH endonuclease domain and the RuvC endonuclease domain, respectively, which inhibits gene transcription via steric hindrance of RNA polymerase binding to the promoter region. CRISPRa is achieved via a fusion protein between dCas and an activator that recruits RNA polymerase, thereby enhancing gene transcription. Although CRISPRa has been shown to increase the expression of target genes by 3-fold [Bikard, D. et al., Nucleic Acids Res, 41(15), 7429-7437 (2013)], CRISPRi has been more widely applied to species belonging to the genera Escherichia, Pseudomonas, Mycobacterium, Corynebacterium, Clostridium, and Bacillus, thereby inhibiting gene expression by up to 300-fold [Cho, S. et al., Int J Mol Sci, 19(4). doi: 10.3390 / ijms19041089 (2018); Qi et al., 2013]. The CRISPRi tool will be explored in Chromobacterium violaceum for transcriptional inhibition of chromosomal gene expression.

[0203] CRISPR / Cas genome editing tools are already powerful tools for use in eukaryotic cells, however, the use of CRISPR / Cas is more limited in prokaryotes because of the lethality of double-stranded DNA cuts in prokaryotes. The DNA repair mechanisms in prokaryotes are not sufficient to rescue the cells, resulting in bacterial death when CRISPR / Cas is employed.

[0204] However, the use of inactivated Cas9 (dCas9) with D10A mutation and H840A mutation in the catalytic domain has been adapted to block the transcription of target genes ( Figure 5 In this study, dCas9 was used to target the promoter region of the violacein operon, thereby preventing the formation of violacein violacein ( Figure 5B). The repression was most effective when targeting the first gene (vioA) promoter (SEQ ID NO: 18), which only required 0.01% arabinose. This was followed by the vioB promoter (SEQ ID NO: 19), which required a higher amount of 0.1% arabinose to induce dCas9 expression for violacein repression. Inhibition of vioC (SEQ ID NO: 20) further downstream of the operon had little effect on violacein transcriptional repression. The slight drop in OD should not be due to the metabolic load of dCas expression. One of the main reasons may be due to the overlap of the absorption spectra of violacein, which has a maximum absorbance at OD 570 [Swem, LR et al., Mol Cell, 35(2), 143-153 (2009)]. Another reason for the slight decrease in optical density may be due to the clumping of cells when the violacein pigment is not completely repressed ( Figure 5 C).

[0205] Chromosomal transcriptional control of Chromobacterium violaceum has not been reported so far. Control of endogenous genes will be useful for metabolic engineering, especially for the control of metabolic flux, so as to quickly and effectively knock down gene expression. Because multiplexing is simple and direct, multiple genes can be effectively and quickly knocked down at one time [Cobb, RE et al., ACS Synth Biol, 4 (6), 723-728 (2014); Cress et al., 2015]. In addition, dCas9 can be fused with other proteins (including cytidine deaminase or adenine deaminase for single nucleotide mutation) [Arazoe, T. et al., Biotechnology journal, 13 (9): e1700596 (2018); Komor, Kim, Packer, Zuris and Liu, 2016], thereby providing an excellent opportunity to expand the genetic toolbox of Chromobacterium violaceum.

[0206] Cloning and expression of dCas for Chromobacterium violaceum

[0207] Two catalytic mutations, D10A and H840A, were introduced into Cas9 via mutagenesis primers and Gibson assembly. araBAD The dCas was cloned under the control of (SEQ ID NO: 21). The strong constitutive promoter J23119 (SEQ ID NO: 22) was used to drive the expression of sgRNAs targeting the vioA promoter, vioB promoter, the 5' end of vioC and two non-targeting sequences ( Figure 5A). Overnight cultures of Chromobacterium violaceum were diluted 1:100 into 96 deep-well blocks (Nunc, Denmark) and dCas was induced by adding 0.01%, 0.1% or 1% arabinose, or not induced as a control. The cultures were grown overnight at 37°C, 280 rpm, after which they were transferred to 96 deep-well plates for visualization of violaceum production and OD600 measurement.

[0208] Example 3

[0209] Construction of a gold sensor from the native gold operon in Chromobacterium violaceum

[0210] An important sensor for a gold bioleaching circuit is a gold sensor. The amount of gold leached will be able to provide dynamic feedback to the circuit and stop the leaching process whenever the gold ion concentration reaches a critical level. The only gold biosensor shown so far is the goltSB operon (SEQ ID NO: 23) from the Salmonella enterica Typhimurium serotype LT2 strain. It contains GolS (SEQ ID NO: 24), which is shown to be the only MerR family transcriptional regulator reported to be able to distinguish gold ions from copper and silver ions [Cerminati, S. et al., Biotechnol Bioeng, 108 (11), 2553-2560 (2011)]. Although this gold biosensor has only been shown previously in Escherichia coli and Salmonella enterica, it is optimized in Chromobacterium violaceum for the first time in this report to achieve a large dynamic range.

[0211] The entire goltSB operon was first cloned upstream of the fluorescent protein output for characterization of the gold sensor. However, the fluorescence output was 3+ Low output dynamic range at concentrations ( Figure 6 A). This low dynamic range is due to the absence of Au 3+ In the case of high leakage expression of 14842RFU ( Figure 6 A). In addition, at high Au concentrations from 1 μM upwards 3+ The cell density decreased significantly at 2.5 ng / mL concentration. The toxicity to the cells may be due to the overexpression of GolT, a transmembrane efflux P-type ATPase that may disrupt the integrity of the cell membrane if overexpressed. This low dynamic range and cytotoxicity of the gold sensor in Chromobacterium violaceum led to subsequent optimization in the goltSB operon.

[0212] In order to reduce the leakage activation caused by GolS, P golTSInstead, a weak constitutive promoter was used to drive GolS expression, thereby reducing promoter strength and eliminating the positive feedback loop ( Figure 6 B) In addition, GolT and GolB were removed to construct a minimal gold sensor and minimize possible harmful effects on cell growth. golTS (SEQ ID NO: 25) or P golB (SEQ ID NO:26) Test construct ( Figure 6 B and Figure 6 C) 0 and 10 μM Au 3+ The maximum dynamic range was 151-fold change for pGolTS (min 110 RFU max 16416 RFU) and 113-fold change for pGolB (min 50 RFU max 1648 RFU).

[0213] In the presence of 0 and 1 μM Au 3+ There was also a higher fold change for pGolTS compared to pGolB, where no toxicity to Chromobacterium violaceum was observed. 3+ , there was a 38-fold change in the dynamic range of gene expression for PgolB (min50max 1887RFU) ( Figure 6 B), and for PgolTS there was a 62-fold change in the dynamic range of gene expression (min110max 6737RFU) ( Figure 6 C) The PgolTS construct was subsequently chosen as the gold sensor due to its higher dynamic range.

[0214] Improving the dynamic range and sensitivity of gold sensors in Chromobacterium violaceum by mutagenesis

[0215] After optimizing the circuit and regulatory mechanism of the gold sensor, the next step is to perform deep scanning mutagenesis on the GolS transcription activator to further increase the sensitivity and dynamic range of the gold sensor. Although the structure of the GolS protein has not yet been resolved, GolS belongs to the MerR protein family with a helix-turn-helix DNA binding domain and a gold ion binding domain ( Figure 8 A) [Checa, SK et al., Mol Microbiol, 63(5), 1307-1318 (2007)]. Using a low constitutively expressed GolS and a P driving RFP golTS The optimization loop composed of Figure 7 ), screen and select GolS mutants with wide dynamic range and high sensitivity compared with wild-type GolS ( Fig.12). Four mutants were selected and characterized; GolSmt1 (A38I); GolSmt2 (A38Q and N97D); GolSmt3 (A38K and V60L); and GolSmt4 (D33P). The structures of wild-type GolS and mutants GolSmt1, GolSmt2, and GolSmt3 are shown in Figures 8 to 11 middle.

[0216] Compared with the wild-type gold sensors reported to date, the gold sensor mutants have increased sensitivity and dynamic range (Table 1). max Wild-type B max The maximum fold change of 3.5-fold increase in the value of 1.5-fold was observed for the other parameters including K, n and C, which did not change more than 2-fold, indicating that the other characteristics remained relatively similar.

[0217] Table 1: Parameter values ​​from the fitted response curves of gold sensor mutants.

[0218] K(μM) N <![CDATA[B max ]]> C <![CDATA[R 2 ]]> SEQ ID GolS-mt1 2.25±0.19 1.07±0.07 45088±1672 51±225 0.998 27 GolS-mt2 1.36±0.15 1.12±0.10 36074±1611 54±370 0.996 28 GolS-mt3 1.48±0.14 1.23±0.11 41388±1646 97±392 0.996 29 GolS-mt4 4.05±0.87 1.15±0.13 43451±4356 78±234 0.998 30 GolS-WT 5.51±3.33 1.15±0.29 12811±3825 47±111 0.994 24

[0219] Sensitivity is a key parameter of the sensor, determining its functionality in the system. The results obtained show that the sensitivity of the gold sensor mutants to gold ions is at least two times that of the wild-type gold sensor. For the gold sensor mutants, there was at least a three-fold induced detectable change fold at 8 nM gold ions, while there was no detectable change fold for the wild-type gold sensor (Table 2).

[0220] Table 2: For the wild type and the top three gold sensor mutants, as Au 3+ The output changes between the on and off states as the concentration increases.

[0221]

[0222] This value is more sensitive than the reported 33 nM gold ions for detection in E. coli biosensors, with a 2.3-fold induction as the threshold [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)]. 3+ The wild-type gold sensor achieved a more than 10-fold output change at this gold ion concentration, while the wild-type gold sensor had only a 3-fold induction at this gold ion concentration, with a sensitivity to gold ions of 4.5 RFU nM. -1. There was also low leaky expression of the off state in both the wild-type and mutant gold sensors. The off state was lower, with all outputs below 100 RFU. (GolSmt1-51 RFU, GolSmt2-54 RFU, GolSmt3-97 RFU, WT-47 RFU). Before reaching toxic levels of gold ions, the gold sensor mutants were also able to produce a higher maximum output than the wild-type gold sensor, with more than 100-fold changes (GolSmt1-45 088 RFU, GolSmt2-36 074 RFU, GolSmt3-41 388 RFU, WT-12 811 RFU).

[0223] All mutants had mutations in the helix-turn-helix DNA binding domain (left and right arrows, Figure 8 B), indicating that the increased output of the transcriptional activator may be due to better binding to and subsequent activation of the promoter region, resulting in a higher maximum output of the gold sensor mutant ( Fig.13 ). The activator mutant also has an increased affinity for gold ion binding, supported by a leftward shift in the gold response function ( Fig.13 ). At the same time, no leaky basal expression was found, indicating that the mutation did not increase the activation of the non-induced activator. In conclusion, the increased transcriptional output, increased affinity for gold ions, and tight expression of the gold-sensing transcriptional activator mutants can serve as useful tools for future gold ion sensing applications.

[0224] Mutagenesis of gold sensors

[0225] The golS mutant library was created by QuikScan-19 and established using QuikChange-HT kit (Agilent Technologies, Santa Clara). Each amino acid was repeatedly replaced with 19 other amino acids, thereby obtaining 19 mutagenesis custom oligomers for each amino acid in the GolS used in the QuikChange reaction. For 154 aa of GolS, in addition to the first amino acid methionine, a total of 153 x 19 = 2907 possible single amino acid mutants will be generated. A total of 6 libraries were generated in the protein according to the manufacturer's scheme, and each library spanned approximately 25 amino acid mutation regions. Each library was subsequently converted into competent violet chromobacterium and plated in a culture medium containing 30 μg / mL kanamycin and 2 μM AuCl 3 The colonies were selected by observation under blue light for higher RFP output compared to wild-type GolS and plated into 96-well plates for growth and subsequent fluorescence quantification using a BioTek Synergy H1 microplate reader.

[0226] Fluorescence measurements for population and gold sensor characterization

[0227] For gold sensor characterization, cells were grown overnight from frozen stocks in LB medium in 96 deep-well blocks (Nunc, Denmark) at 37° C. Cells were diluted 1:200 in Tris minimal medium (pH 7.5) containing 80 mM NaCl, 50 mM Tris, 22 mM glucose, 20 mM KCl, 20 mM NH 4 Cl, 3 mM Na 2 SO 4 , 1mM thiamine hydrochloride, 0.5g / L yeast extract, 1mM MgCl 2 , 0.65 mM Na 2 HPO 4 and 0.1 mM CaCl 2 .

[0228] The diluted cells were then grown overnight at 37°C in a BioTek Synergy H1 microplate reader. Red fluorescence was measured every 10 minutes with excitation at 530 nm and emission at 600 nm (gain 50) for 14-20 hours. Optical density was measured by absorbance at 600 nm. The OD of the medium alone without cells was 0.0447 W / v. 600 and fluorescence values ​​as blank.

[0229] Example 4

[0230] Incorporating dynamic regulation and biosensors via synthetic circuits for robust microbial cell factories

[0231] In a gold bioleaching microbial cell factory, the two main components are the target element gold and cyanide, which is the leaching agent required to oxidize solid gold to aqueous gold ions. This can pose a challenge to live microbial cell factories because both gold ions and cyanide are highly toxic to microorganisms. Gold ion toxicity originates from the accumulation of Au(I)-S complexes that induce oxidative stress [Reith, F. et al., Proc Natl Acad Sci USA, 106 (42), 17757-17762 (2009)], while cyanide inhibits the respiratory process by binding to the metal in cytochrome oxidase [Knowles, CJ Bacteriol Rev, 40 (3), 652-680 (1976); Knowles, CJ and Bunch, AWA Adv Microb Physiol, 27, 73-111 (1986)]. Gold ions have been shown to be toxic to bacteria at concentrations as low as 0.35 μM [Shareena Dasari, TP et al., Biochem Pharmacol (Los Angel), 4(6), 199 (2015)], while cyanide is toxic to bacteria as low as 0.4 μM [Liu, W. et al., Chinese Journal of Chemistry, 25(2), 203-207 (2007)]. Although there are many cyanide detoxification mechanisms in Chromobacterium violaceum [Brysk, MM et al., J Bacteriol, 97(1), 322-327 (1969); Brysk, MM and Ressler, Journal of Biological Chemistry, 245(5), 1156-1160 (1970); Ressler, C. et al., Biochemistry, 12(26), 5369-5377 (1973)] to protect itself from its own cyanide production, gold ion toxicity will be a new and urgent challenge for the inexperienced Chromobacterium violaceum during the gold bioleaching process. Therefore, a dynamic on-off circuit was constructed to turn on cyanide production only at high cell density and turn off the circuit when toxic gold ions are sensed ( Fig.14 ).

[0232] Adding an endogenous quorum sensing system to the circuit will ensure that the output is activated only when the cell density is high enough ( Fig.15). Because the gold sensor that initiates the negative feedback loop of the synthesis circuit is an activator, a repressor must be introduced downstream to mediate repression. A strong repressor of the TetR family that has been characterized was added downstream of the gold sensor, which mediates up to a 193-fold change in gene expression of PhlF (SEQ ID NO: 31) [Stanton, BC et al., Nat Chem Biol, 10(2), 99-105(2014)] for transcriptional repression of the output. An operator gene of PhlF (SEQ ID NO: 32) was added downstream of the quorum sensing promoter for physical inhibition of RNAP during transcription ( Fig.15 ). The negative feedback loop provided by the gold sensor will provide real-time feedback to the bioleaching process and inhibit the process when the cyanogenic process has produced high concentrations of gold ions through cyanidation. Once the repressor is expressed, it will then bind to the promoter region of the cyanogenic gene. This means that when high concentrations of gold ions are detected, no further expression of the bioleaching enzyme will occur.

[0233] The loop was tested in serial batch cultures with a 1:600 ​​dilution from the previous passage. The cultures were maintained at 2 μM Au 3+ to close the circuit, or in the absence of gold ions to open the circuit ( Fig.16 ). In our study, 2 μM gold ions were used to induce the closed state because it already resulted in the highest repression before gold ion toxicity was observed at 10 μM. However, closing the circuit means that there should be low leaky expression or no leaky expression at all. The basal expression level of the circuit containing the wild-type gold sensor in the closed state increased with each cycle. The closed output increased from 245 RFU in the first cycle to 765 RFU and 1047 RFU in the second and third cycles, respectively ( Fig.17 This is in contrast to the circuit with the gold sensor mutant, whose off-state output remained consistently below 250 RFU in all three cycles ( Fig.17 ).

[0234] The reduced basal expression of the GolS mutant resulted in an increased dynamic range of expression through continuous cell culture. The dynamic range of the WT gold sensor dropped from 15-fold in the first cycle to only 5-fold and 4-fold dynamic range in the second and third cycles, respectively, while the circuit containing the GolS mutant maintained a dynamic range above 10-fold in all three cycles ( Fig.17 ). This maintenance of high dynamic range in the circuit containing the GolS mutant will confer robustness and increased functionality to the gold-leaching microbial cytokines to undergo multiple on-off cycles.

[0235] The increased robustness of the GolS mutant circuit is further supported by the maintenance of separate on-off populations during continuous cycling, as can be seen from representative flow cytometry histograms ( Fig.18 ), where (A) is WT; (B) GolSmt1; (C) GolSmt2; and (D) GolS mt3. Spreading of the shut-off cell population observed in the WT gold sensor ( Fig.18 A) shows that the increased heterogeneity of cell population expression is the main reason for the reduced dynamic range of the circuit. The stronger maintenance of the closed state of the circuit containing the gold sensor mutant can be attributed to the increased sensitivity of the gold sensor to gold ions and the increased dynamic range. Even with increasing cell passages, the dynamic range of the circuit decreased at 2 μM Au. 3+ Higher expression of the PhlF repressor under the same induction of may lead to better repression of the quorum sensing promoter.

[0236] Another observation is that two GolS mutants ( Fig.18 GolSmt2 and Fig.18 D's GolSmt3) had an increased population that remained in the closed state during three cycles, resulting in a slightly reduced dynamic range. This may be due to insufficient dilution of the PhlF repressor in the cycle, which caused a small population of cells to remain in the closed state. Although the GolS mutant allowed increased PhlF repressor expression to better maintain the closed state output, it may be too strong that the cellular dilution of the PhlF repressor was insufficient to switch the loop back to the open state. This suggests that GolSmt2 and GolSmt3 may not be as suitable as gold sensors for robust continuous on-off cycling in a gold bioleaching synthesis loop.

[0237] The gold sensor mutants increased the robustness of the circuit over three on-off cycles consisting of six cell passages ( Figures 16 to 18 ). This has made a significant contribution to the development of gold bioleaching microbial cell factories, as the toxicity of gold ions to cells would mean that many gold leaching cycles would be required.

[0238] Example 5

[0239] Directed evolution of mercury(II) reductase for gold reduction

[0240] Determination of Minimum Inhibitory Concentration of Au(III)

[0241] Au(III) is known to be toxic to many bacterial cells due to its high affinity for thiol groups (-SH) and may therefore affect many metabolically important enzymes and membrane-bound proteins. E. coli cells expressing a MerA mutant enzyme with improved gold-reducing activity can potentially exhibit higher resistance to toxic levels of Au(III) supplemented in the culture medium.

[0242] For the broth method, the ligation mixture was transformed into E. coli Rosetta (DE3) pLysS competent cells, which were plated on normal LB agar plates. For broth medium, 5 mL of medium supplemented with the same antibiotics and different concentrations of AuCl 3 Tris-buffered low phosphate medium was inoculated with a single colony at 37 °C for 24 h. The OD 600 The lowest concentration of heavy metal that completely blocked the growth was defined as MIC. Fig.23 A shows that at a concentration greater than 140 μM Au 3+ At this concentration, cells cannot grow.

[0243] For the plate method, E. coli cells expressing wild-type MerA were cultured, diluted, and plated in a flask supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG, and various concentrations of AuCl 3 Tris-buffered low phosphate agar medium (Tris 6.06 g / L, NaCl 4.68 g / L, KCl 1.49 g / L, NH 4 Cl 1.07g / L, Na 2 SO 4 0.43g / L, MgCl 2 .6H 2 O 0.2g / L, CaCl 2 .2H 2 O 0.03g / L, Na 2 HPO 4 .12H 2 O 0.23g / L, glucose 5.0g / L, yeast extract 0.5g / L and agar 15g / L) and grown at 37°C for 24h. 3+ As the concentration increased, fewer colonies were observed on the plate. 3+ When the concentration reached 160 μM, cell growth was completely inhibited ( Fig.23 B).

[0244] The toxicity of gold may be due to its high affinity for thiol groups (-SH) present in many metabolically important enzymes and membrane-bound proteins. Upon binding to gold ions, these proteins are no longer accessible to their native biologically relevant metal ions. The broth method exhibited a slightly lower minimum inhibitory concentration compared to the plate method. This difference may be due to the Au 3+ Different distribution patterns of ions between the plate and the liquid broth.

[0245] Directed evolution library construction

[0246] A synthetic, codon-optimized gene encoding mercury (II) reductase (MerA) was cloned in frame with an N-terminal 6x His tag into the expression vector pET20b (Novagen) using restriction endonucleases NdeI and XhoI. To evolve MerA with improved gold-reducing activity, a mutant library was constructed using error-prone PCR and site-saturation mutagenesis.

[0247] 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 used in the PCR reaction to achieve a moderate mutation frequency (4.5-9 mutations / kb) and confirmed by sequencing several randomly picked colonies. The following primers were used: epPCR-fw: 5'-GTGGTGGTGGTGGTGGTGCTCGAGTTA-3' (SEQ ID NO: 1) and epPCR-rv: 5'-GATATACATATGCACCACCATCACCATCAT-3' (SEQ ID NO: 2). The thermal cycling program consisted of first denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1.5 min, and extension at 72°C for 10 min after the last cycle. The PCR product was then treated with DpnI for template degradation and purified using a QIAquick PCR purification kit (Qiagen). The purified DNA fragment was double digested with NdeI and XhoI, purified and ligated into a pET20b vector.

[0248] Multisite saturation mutagenesis was performed by overlap extension PCR using degenerate primers containing NNK at target sites V317, Y441, C464, and C465'. These positions are in close contact with potential gold binding sites within the active site of MerA ( Fig. 22). The redox active cysteines C136 and C141 were kept unchanged to prevent possible loss of redox activity. The primers used are listed in Table 3.

[0249] Table 3: Sequences of primers used for site-saturation mutagenesis

[0250]

[0251] a Degenerate alphabet: N = (A, T, C, G); K = (T, G); M = (A, C)

[0252] PCR reactions were performed with the following primer pairs: T7-fw / V317NNK-rv, V317NNK-fw / Y441NNK-rv, Y441NNK-fw / C464NNK-rv, and C464NNK-fw / T7-rv to generate partially overlapping DNA fragments. A total volume of 50 μL of the reaction mixture containing 50 ng of pET20bMerA plasmid, 500 nM of each primer, and 1 x PrimeSTAR Max premix (Clontech) was incubated for 30 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 10 s. The PCR products were purified and equimolar amounts of each fragment were mixed in 1 x PrimeSTAR Max premix for short overlap extension reactions (5 cycles of 98°C for 10 s, 55°C for 5 s, 72°C for 10 s), and 1 μL of the reaction mixture was used as a template to amplify the full-length MerA mutant gene using primers epPCR-fw and epPCR-rv. The PCR products were purified, digested with NdeI and XhoI, and ligated into the pET20b vector.

[0253] Selection of gold-tolerant mutants

[0254] Ligation mixtures from error-prone PCR or site-saturation mutagenesis were used to transform E. coli Rosetta (DE3) pLysS competent cells, and transformants were plated on Tris-buffered low phosphate agar medium (Tris 6.06 g / L, NaCl 4.68 g / L, KCl 1.49 g / L, NH 4 Cl 1.07 g / L, Na 2 SO 4 0.43 g / L, MgCl 2 .6H 2 O 0.2 g / L, CaCl 2 .2H2 O0.03 g / L, Na 2 HPO 4 .12H 2 O 0.23 g / L, glucose 5.0 g / L, yeast extract 0.5 g / L and agar 15 g / L) and grown at 37°C for 24 h. Colonies were then picked and cultured in medium supplemented with 100 μg / mL ampicillin, 34 μg / mL chloramphenicol, 0.1 mM IPTG and 300 μM AuCl 3 After several rounds of selection, toxic levels of Au 3+ Ten mutants showed significantly better cell growth at 300 μM, at the toxic level of Au 3+ Under these conditions, cells expressing wild-type MerA may barely grow ( Fig.24 ). It is worth mentioning that all colonies that survived two selections were from the site saturation library, indicating that this semi-rational designed library rather than a library generated by random mutagenesis may have a higher chance of containing functionally improved variants. The improved tolerance compared to the wild type may come from two sources: 1) enzymatic reduction of Au 3+ ions, and 2) detoxification of MerA by reacting with Au 3+ 2. MerA Binding to Isolate Au 3+ .

[0255] Generated from AuCl 3 The MerA mutants synthesized gold nanoparticles, as shown in the TEM images ( Fig.25 ). Through our directed evolution approach, an enhanced gold reductase, MerA mutant V317S, has been identified. This mutant has the kinetic parameters listed in Table 5. The enhanced gold reductase 3+ Reduced to elemental gold (Au 0 ) has a 67-fold increase in catalytic efficiency.

[0256] Table 5: Kinetic parameters of enhanced gold reductase

[0257]

[0258] Further improved mutants

[0259] Previously, 9.1±3.2×10 1 M -1 s -1 The catalytic efficiency of MerA established the gold reduction ability of MerA. A combination of directed evolution and rational design methods was used to generate improved Au 3+A library of MerA mutants was restored. More than 50% of the isolated mutants showed improved activity, with the most improved mutants displaying up to 15-fold improvement in catalytic efficiency ( Fig.26 ). Due to the update rate (k cat ) and binding affinity (K M ) both, mutant DM11 (G415I) (SEQ ID NO: 48) showed the greatest improvement in catalytic efficiency (15-fold increase). The kinetic parameters of all mutants can be found in Table 6.

[0260] This improvement in catalytic efficiency is also reflected in the complexity of the gold nanoparticles (AuNPs) formed after reduction. For example, DM11 produced AuNPs with larger size and higher complexity ( Fig. 27 This improvement in catalytic efficiency is also reflected in the improved reduction and recovery of gold from electronic waste leachate by DM11 as compared to WT MerA ( Fig.28 DM11 from AuCl 3 Au recovery from solution (67%) and electronic waste leaching solution (67%) 3+ The same is true for Fig.29 ).

[0261] Table 6: Kinetic parameters of mutants identified by DM selection. Mutants in the dimerization region (DM10-15) and residue position 323 (DM4 and DM5) showed the greatest increase in catalytic efficiency. *(delΔ324-365)

[0262]

[0263]

[0264] However, for AuCl 3 For the reduction from the leaching solution, AuNPs with increased complexity are observed, while for the reduction from the leaching solution, AuNPs with limited complexity are observed ( Fig.30 This may be due to the Au 3+ The amount of gold reduction is generally low. It is believed that if this limitation can be overcome, greater improvements in gold recovery can be observed. MerA is an essential enzyme in bacterial mercury resistance systems. Based on its reaction mechanism and available crystal structures, it is envisioned that it can also be engineered for effective gold reduction. To this end, a high-throughput selection program was established, which involved toxic agar plate selection and subsequent more stringent liquid culture selection. A mutant library was constructed by both error-prone PCR and multi-site saturation mutagenesis, and the library was subjected to this two-step selection. As a result, MerA mutants with enhanced gold reduction / gold recovery properties have been identified. This constitutes the synthetic metal recovery part of synthetic lixiviant biology.

[0265] Protein expression and purification

[0266] Recombinant protein was expressed using the T7 expression system. Rosetta (DE3) pLysS was transformed with the plasmid and selected on LB agar plates containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. Single colonies were picked into 5 mL of LB medium containing both antibiotics and grown overnight at 37°C. A 100-fold dilution was performed and the culture was grown at 37°C until OD 600 The protein expression was then induced by adding isopropyl β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM. After induction, the cells were grown for another 18 h at 16 ° C. The cells were lysed by ultrasonic treatment and the His-tagged protein was purified from the clarified lysate on a nickel chelate column (Qiagen). The protein sample in the eluent (500 mM imidazole, 50 mM Tris-Cl pH 7.5, 300 mM NaCl) was concentrated by an Amicon Ultra centrifugal filter (Millipore) and dialyzed against 20 mM sodium phosphate (pH 7.4).

[0267] Gold reduction assay

[0268] The precipitate was stirred at 25 °C in 20 mM sodium phosphate, pH 7.4, 200 μM NADPH, 100 μM AuCl 3 The enzyme assay was performed in . The oxidation of NADPH was followed spectrophotometrically at 340 nm. A unit of enzyme activity was defined as the amount of enzyme that catalyzes the Au-dependent oxidation of 1.0 μmol NADPH per minute.

[0269] Example 6

[0270] Kinetic parameters for gold reduction by MerA

[0271] For substrate AuCl 3 Continuous spectrophotometric assays were used to determine the kinetic parameters of purified mercury reductase (MerA) (Scheme 1).

[0272] Protocol 1: Assay for monitoring MerA activity via absorbance changes at 340 nm. M refers to Hg 2+ or Au 3+ , then X corresponds to GSH - or Cl.

[0273]

[0274] Natural substrate Au 3+ Reduction to Au 0Oxidized with NADPH to NADP + 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 varying amounts of Au. 3+ The assay was also performed using the substrate Hg(GSH) 2 To observe the natural substrate of MerA, Hg 2+ Reduction to Hg 0 .

[0275] Use of Hg(GSH) 2 and AuCl 3 The kinetic parameters of MerA were determined (Table 4).

[0276] Table 4: Kinetic parameters of purified MerA

[0277]

[0278] Regarding the mercury substrate, MerA showed a higher KM of 96.3 ± 57.6 μM compared to 10.7 μM reported in the literature. -1 Compared with the findings of -1 The kcat value of 8.8x10 5 M -1 s -1 Compared with the literature findings, 1.5±0.7 x 10 5 M -1 s -1 The overall catalytic efficiency (k cat / K M ) value is six times lower than expected (Moore, MJ, Miller, SM, Walsh, C-Terminal Cysteines of Tn501 Mecuric Ion Reductase (1992) Biochemistry 31(6):1677-85). 3 , no public data was available. 2 Compared with AuCl 3 K cat / K M Therefore, the assay can be used to determine the reducing capacity of MerA on different substrates.

[0279] Improved gold-reducing MerA mutants were identified by screening with a gold sensing biosensor. The goltSB operon isolated from Salmonella enterica serovar Typhimurium functioned as a biosensor with green fluorescent protein (GFP) as a reporter (Zammit et al., 2013) ( Fig.19 ). This biosensor is under the control of the GolS regulator, which is a + / Au 3+ The presence of the complex induced + / Au 3+ When the ion interacts with the GolS regulator, the GolS regulator binds to its target promoter sequence golB. This induces a conformational change in the GolS / golB complex, thereby promoting transcription of the GFP reporter. We propose to integrate this circuit into the E. coli chromosome as previously shown [Cerminati, S. et al., Biotechnol Bioeng, 108(11), 2553-2560 (2011)] and use this E. coli strain for endogenous reporting of gold reducing ability.

[0280] The biosensor was cloned into the pRSFDuet-1 vector and tested for its reactivity to the gold substrate. Fluorescence measurements were performed using 485-20 and 528-20 nm filters for excitation and emission wavelengths, respectively. The final optical density (OD) at 600 nm was also measured for each sample. 600 ). According to the following formula (Formula 1), the fluorescence measurement (F s )Normalization.

[0281]

[0282] RFU 样品 is fluorescence (measured in arbitrary relative fluorescence units of the instrument), and is the final optical density determined for each sample obtained from the sensor bacteria, and RFU PRSF and The induction coefficient (IC) was calculated as follows: is the normalized fluorescence value of the sensor bacteria exposed to the metal, and is the normalized fluorescence (background fluorescence) of the biosensor incubated without added metal.

[0283] Fluorescence with Au 3+ The concentration increased with increasing concentration until 100 μM ( Fig. 20). At higher concentrations, the fluorescence decreases dramatically, probably due to the sensitivity of E. coli to metal toxicity. 3+ The limits of detection (i.e., the lowest and highest gold concentrations that produced a detectable increase in fluorescence compared to background) were 100 nm to 100 μM. Thus, it has been demonstrated that the sensor is able to distinguish Au 3+ Different concentrations of , and will be used to screen for the best gold-reducing MerA mutant.

[0284] The screening for the best gold-reducing MerA mutants was based on the observation of a decrease in fluorescence corresponding to the presence of Au in the cell. 3+ ions, thus acting as an indirect reporter of gold reduction activity ( Fig.21 ). In the presence of gold ions and a control plasmid, the biosensor will be induced, resulting in a strong fluorescence signal, but in the presence of the gold-reducing MerA mutant, the gold ions will be reduced, resulting in a lack of fluorescence.

[0285] Example 7

[0286] The gold biosensor in Chromobacterium violaceum is sensitive to silver ions.

[0287] test Fig.31 The sensitivity of the biosensor shown in A to different metal ions in the minimal medium. The biosensor senses gold and silver ions, but does not respond highly to other metals including calcium, zinc, mercury, nickel, cobalt, iron, and copper ions ( Fig.31 B). Dose response shows that the mutant biosensor is more sensitive to gold and silver ions than the wild-type biosensor and has a larger dynamic range ( Fig.32 ).

[0288] Example 8

[0289] Sensing of noble metal ions leached from electronic waste by Chromobacterium violaceum

[0290] The purple Chromobacterium was incubated with the electronic waste and cultured for 6 days to leach the precious metals from the electronic waste into the aqueous culture. After 6 days of culture, the purple Chromobacterium cells were centrifuged and the spent culture medium was added to the purple Chromobacterium containing the biosensor used in Example 7 ( Fig.33 ). The transcription factor biosensor was activated in spent medium containing leached ESM but not in control medium ( Fig.34). In response to the 6-day spent culture medium containing leached ESM, the mutant biosensor fluorescence output was 2515 RFU, while the wild-type biosensor was 1101 RFU. ICP analysis showed that in the 6-day spent culture, there were 1.1 ppm (5.6 μM) gold ions, 0.34 ppm (3.2 μM) silver ions, and 12.3 ppm (193.2 μM) copper ions in the spent culture medium. This shows that the biosensor in Chromobacterium violaceum is activated in the presence of gold and silver ions from the leached e-waste culture medium. This will be useful in activating other synthetic circuit modules in Chromobacterium violaceum.

[0291] Summarize

[0292] 1. For the present invention, the bioreduction of metal cyanide complexes after metal cyanidation has been achieved using the mercury reductase (Mer) system.

[0293] 2. For the present invention, tools will allow the construction of synthetic circuits for lixiviant biology and involve the continuous and appropriate expression and activation of the corresponding biological systems for efficient metal recovery and include the following features:

[0294] a. Regulated inducible expression of the synthetic cyanogenetic module after adding electronic waste to the bioreactor. This enables the timely production of cyanide lixiviants for metal bioleaching.

[0295] b. Specific transient expression of synthetic metal recovery modules upon detection of the corresponding metal cyanide complex. Due to the heterogeneity of electronic waste, different concentrations of metals (and associated metal ions) necessitate the temporary removal of the corresponding metals; this will be achieved through the selective expression of engineered Mer systems that are specific for the valence and identity of the metal ions.

[0296] c. Regulated inducible expression of the synthetic cyanolysis module after metal recovery to metabolize excess cyanide lixiviant, thereby transporting the carbon and nitrogen atoms of cyanide back into the central metabolic pathway.

[0297] References

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Claims

1. An isolated genetically engineered bacterium, wherein the bacterium has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising a heterologous nitrilase gene operably linked to at least one promoter, the product of the gene causing the cyanolysis of hydrogen cyanide.

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

3. The isolated genetically engineered bacterium according to claim 1 or 2, wherein the bacterium is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, Pseudomonas aeruginosa and Eschericia coli.

4. A method for synthetic cyanolysis, the method comprising contacting at least one recombinant cyanolytic bacterium with nitriles, including cyanide, present following bioleaching of electronic waste, wherein the at least one bacterium is engineered to express at least one nitrilase.

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

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

7. An isolated recombinant DNA molecule comprising, in order from the N-terminus to the C-terminus of the recombinant DNA molecule; (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a ph1F repressor gene operably linked to the promoter; (ii) a promoter activated by CviR and an operator of PhlF, and (iii) one or more cyanogenic genes operably linked to the CviR-activated promoter.

8. The isolated recombinant DNA molecule of claim 7, wherein the golS transcriptional activator gene is a mutant selected from the group consisting of GolSmt1 A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

9. Use of an inactivated Cas9 and sgRNA comprising mutations H840A and D10A in the HNH endonuclease domain and the RuvC endonuclease domain, respectively, for inhibiting the transcription of one or more genes in the Chromobacterium violaceum genome by targeting the promoter region of the one or more genes.

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

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

12. An isolated genetically engineered bacterium, wherein the bacterium has been 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.

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

14. The isolated bacterium of claim 13, wherein the golS gene is codon optimized for Chromobacterium violaceum, and / or wherein the golS gene is a mutant selected from the group consisting of GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L, and GolSmt4_D33P.

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

16. A method for producing a synthetic cyanide lixiviant, the method comprising: contacting at least one recombinant cyanogenic bacterium with glycine, The bacteria comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

17. The method according to claim 16, wherein the hydrogen cyanide synthase gene is hcnABC and / or the 3-phosphoglycerate dehydrogenase mutant gene is serA, and / or wherein the recombinant cyanogenic bacterium further comprises at least one polynucleotide molecule, wherein the at least one polynucleotide molecule is sequentially included from the N-terminus to the C-terminus of the recombinant DNA molecule; (i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a golTS or P golB a ph1F repressor gene operably linked to the promoter; (ii) a promoter activated by CviR and an operator of PhlF, and (iii) one or more of the heterologous hydrogen cyanide synthase gene and the heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

18. The method of claim 17, wherein the golS gene is codon optimized for Chromobacterium violaceum, and / or wherein the golS gene is a mutant selected from the group consisting of GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

19. The method according to any one of claims 16 to 18, wherein the at least one recombinant cyanogenic bacterium can tolerate about pH 10.

20. A process according to any one of claims 16 to 19, wherein the synthetic cyanide lixiviant production is carried out together with the metals for bioleaching in a single reactor.

21. At least one isolated recombinant cyanogenic bacterium as defined in any one of claims 16 to 20, capable of synthetic cyanide lixiviant production.

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

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