Using synthetic lixiviant biology for the recovery of precious and toxic metals from anthropogenic sources

Patent metadata

Field	Value
Publication number	US-2022205062-A1
Application number	US-202017601802-A
Country	US
Kind code	A1
Filing date	Apr 8, 2020
Priority date	Apr 8, 2019
Publication date	Jun 30, 2022
Grant date	—

Abstract

The present invention generally relates to methods of biological reduction of metal-cyanide complexes after metal-cyanidation and methods of biologically hydrolysing cyanide. More particularly, 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; with up to four main components/modules in the design and engineering of the synthetic host: 1) synthetic cyanogenesis; 2) synthetic metal recovery; 3) synthetic cyanolysis; and 4) synthetic circuits for lixiviant biology. Bacteria capable of reducing ionic metal to ionic metal (such as gold or silver) as nanoparticles, comprising mercury(ll) reductase (MerA) comprising a substitution mutation at position V317, Y441, C464, A323D, A414E, G415I, E416C, L417I, I418D, or A422N, are also disclosed. Processes of synthetic cyanide lixiviant production using genetically engineered bacterium transformed with a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene are also disclosed. Processes of synthetic cyanolysis using a genetically engineered bacterium transformed with a heterologous nitrilase gene are also disclosed.

First claim

1 . An isolated genetically engineered bacterium, wherein the bacterium has been transformed by 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 which renders the gene product capable of reducing ionic metal to elemental metal as metal nanoparticles.

2 . The isolated bacterium of claim 1 , wherein the MerA gene comprises one or more mutations which encode amino acid substitutions, wherein the amino acid substitutions are at positions selected from the group comprising V317, Y441, C464, A323D, A323D (delΔ324-365), A414E, G415I, E416C, L417I, I418D and A422N of the MerA coding sequence.

3 . The isolated bacterium of claim 1 or 2 , wherein said ionic metal is ionic gold (Au 3+ ) which is reduced to elemental gold as gold nanoparticles, or ionic silver (Ag + ) which is reduced to elemental silver as silver nanoparticles and/or wherein said at least one isolated bacterium has reduced reductive capacity for mercury substrate when compared to a bacterium comprising a non-mutated MerA gene.

4 . An isolated genetically engineered bacterium, wherein the bacterium has been transformed by 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.

5 . The isolated bacterium of claim 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 comprising, in order from N-terminus to C-terminus of the recombinant DNA molecule;
(i) a go/S transcriptional activator gene operably linked to a constitutive promoter, and a ph1F repressor gene operably linked to a P golTS or P golB promoter; (ii) a promoter activated by CviR and an operator of PhIF, and (iii) one or more of said heterologous hydrogen cyanide synthase gene and said heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

6 . The isolated bacterium of claim 5 , wherein the go/S gene is codon optimized for C. violaceum and/or wherein the go/S gene is a mutant selected from GolSmt1_A38I, GolSmt2_A38Q&N97D, GolSmt3_A38K&V60L and GolSmt4_D33P.

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

8 . A process for recovering elemental gold or silver, as gold nanoparticles from ionic gold (Au3+) or as silver nanoparticles from ionic silver (Ag+), respectively, said process comprising the steps of:
a) contacting the isolated genetically engineered bacterium according to any one of claims 1 to 7 with a leachate comprising ionic gold (Au3+) and/or ionic silver (Ag+); and b) recovering the elemental gold and/or silver nanoparticles from the leachate.

9 . The process according to claim 8 , wherein the said contact is performed in alkaline conditions.

10 . A method for producing an isolated genetically engineered bacterium, wherein the bacterium has been transformed by 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 which renders the gene product capable of reducing ionic gold (Au 3+ ) to elemental gold as gold nanoparticles, or ionic silver (Ag + ) which is reduced to elemental silver as silver nanoparticles, said method comprising the steps:
a) performing error-prone PCR on a gene encoding mercury(II) reductase (MerA);
i) transforming at least one bacterium with the products of said PCR;
ii) selecting transformants that grow on a media comprising Au 3+ and/or Ag+; or
b) performing multiple site-saturated mutagenesis by overlap-extension PCR on a gene encoding mercury(II) reductase (MerA);
i) transforming at least one bacterium with the products of said PCR;
ii) selecting transformants that grow on a media comprising Au 3+ and/or Ag + .

11 . The method of claim 10 , wherein in part a) the PCR is performed with forward and reverse primers, wherein the forward primer comprises the nucleotide sequence set forth in SEQ ID NO: 1 and the reverse primer comprises the sequence set forth in SEQ ID NO: 2 and/or wherein in part b) the PCR is performed with primers containing NNK and/or MNN at the target sites V317, Y441 and C464.

12 . The method of claim 10 or 11 , wherein said selection involves at least 2 forms of selection, wherein one form comprises selection on agar plates comprising Au 3+ and/or Ag + and another form comprises selection in liquid culture comprising Au 3+ and/or Ag + .

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

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.

15 . The isolated genetically engineered bacterium of claim 13 or 14 , wherein the bacterium is selected from the group comprising Chromobacterium violaceum, Pseudomonas fluorescens, P. aeruginosa and Escherichia coli.

16 . A process of synthetic cyanide lixiviant production, said process comprising:
contacting at least one recombinant cyanogenic bacterium with glycine, wherein the bacterium comprises a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to at least one promoter.

17 . The process of 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 comprising, in order from N-terminus to C-terminus of the recombinant DNA molecule;
(i) a golS transcriptional activator gene operably linked to a constitutive promoter, and a ph1F repressor gene operably linked to a P golTS or P golB promoter; (ii) a promoter activated by CviR and an operator of PhIF, and (iii) one or more of said heterologous hydrogen cyanide synthase gene and said heterologous 3-phosphoglycerate dehydrogenase mutant gene operably linked to the CviR-activated promoter.

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

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

20 . The process of

Assignees

• Nat Univ Singapore

Inventors

• Yew Wen Shan
• Go Maybelle Darlene Kho
• Liow Lu Ting
• Rajasabhai Rashmi

Classifications

• C12R2001/19

  Escherichia coli · CPC title

• C12R2001/385

  Pseudomonas aeruginosa · CPC title

• C12R2001/39

  Pseudomonas fluorescens · CPC title

• C12R2001/01

  Bacteria or Actinomycetales {; using bacteria or Actinomycetales} · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title