Microorganisms that accumulate platinum group metals

Abstract

Provided is a microorganism in which at least one gene selected from the group consisting of the nikA gene or a gene corresponding thereto, the nikR gene or a gene corresponding thereto, the rcnA gene or a gene corresponding thereto, the rcnR gene or a gene corresponding thereto, and the zntA gene or a gene corresponding thereto is deleted or inactivated.

Description

[Technical Field]

[0001] The present invention relates to microorganisms that accumulate platinum group metals. [Background technology]

[0002] The combined annual consumption of palladium and platinum in catalysts for purifying automobile exhaust gases is approximately 400 tons, accounting for 90% of the platinum group metals. Although electric vehicles (EVs) are becoming more popular, the ratio of hybrid vehicles (HVs) to gasoline-powered vehicles is expected to remain at a certain level in the future, meaning the need for exhaust gas purification will not disappear. Palladium, an important metal for the economy and industry, has a domestic supply of approximately 90 tons in Japan, of which over 70% is imported from countries such as Russia and South Africa. Of Japan's domestic demand of approximately 80 tons, just under 40% is used in exhaust catalysts for fuel-powered vehicles, and recycling from these exhaust catalysts is progressing. Many countries have put forward policies to achieve an EV penetration rate of over 30% by 2030, and considering the current global vehicle population of 1.3 billion, a large amount of exhaust catalyst waste will be generated in the future when fuel-powered vehicles are replaced by EVs. On the other hand, platinum group metals, which exhibit diverse catalytic activity, are expected to continue to have promising industrial applications in the future. Although platinum group metals are useful industrially, their supply source is dependent solely on ores, which have uneven deposit locations, and it has become clear that there is a need for highly selective recovery and concentration technologies that target sources other than ores. Therefore, the development of new platinum group metal resources that will realize a recycling-oriented society and circular economy is an urgent issue.

[0003] Current recycling technologies for platinum group metals, which are used in exhaust gas purification catalysts, etc., have inefficient concentration processes due to the low solubility of platinum group metals, making it difficult to separate specific platinum group metals. Therefore, there is a need to improve the recycling rate.

[0004] Bioprocesses, which are comprehensively understood biological functions, are utilized in a variety of fields due to their industrial benefits of low environmental impact, energy conservation, and low cost. Metal biology, which has emerged due to advances in microanalysis and genome biology, has deepened our understanding of the in vivo functions of metals, providing an opportunity to convert biotechnology techniques currently used in metal refining and recovery into bioprocessing technologies that utilize comprehensive biological functions. Based on an understanding of metal homeostasis in the model bacterium Escherichia coli as part of basic academic research, we designed a metal-accumulating Escherichia coli strain using two types of genes as part of applied exploratory research, and demonstrated the improvement of its metal accumulation ability using molybdenum (see Patent Document 1).

[0005] In recent years, highly sensitive elemental analysis methods such as atomic absorption spectrophotometry, ICP-MS, and ICP-AES have been used to measure elements in living organisms, leading to a better understanding of the metallic elements that make up microorganisms. It has been discovered that in addition to the nonmetallic elements hydrogen, carbon, nitrogen, oxygen, phosphorus, and sulfur, 26 metallic elements are also found to make up microorganisms. However, there have been no reports of palladium being utilized by living organisms. Meanwhile, microorganisms that adsorb palladium have been isolated (see Non-Patent Documents 1 to 4), and attempts have been made to utilize them for resource recovery, but they have not yet been put to practical use due to the low selectivity of adsorption. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-239678 [Non-patent literature]

[0007] [Non-Patent Document 1] Steven, MY. et al. : Current opinion in biotechnology: analytical biotech. Curr Opin Biotechnol., 23, 89-95, (2007) [Non-patent document 2] Lin, Z. et al. : Adsorption and reduction of palladium (Pd2+) by Bacillus licheniformis R08. Chinese Science Bulletin, 4, 357-360, (2002) [Non-patent document 3] Beata, GZ. et al. : Removal of platinum and palladium from wastewater by means of biosorption on fungi Aspergillus sp. and yeast Saccharomyces sp. Water, 11, 1522, (2019) [Non-patent document 4] Kim, S. et al. : Selective biosorption behavior of Escherichia coli biomass toward Pd(II) in Pt(IV)-Pd(II) binary solution. Journal of Hazardous Materials, 283, 657-662, (2015) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a microorganism capable of accumulating platinum group metals such as palladium within its cells. Another object of the present invention is to provide a microbial bioreactor for recovering or removing metals, which is capable of efficiently recovering or removing metals. Another object of the present invention is to provide a method for recovering or removing metals that has a low environmental impact and enables efficient recovery or removal of metals. Another object of the present invention is to provide a method for treating a metal-containing solution that has a low environmental impact and is capable of efficiently recovering or removing metals. [Means for solving the problem]

[0009] The present inventors conducted research and development using Escherichia coli, which is easily genetically modified and genome-edited, to achieve high selectivity and recovery rates by using bioaccumulation technology to accumulate palladium intracellularly. As a result, they discovered that microorganisms in which specific genes were deleted or inactivated were able to accumulate platinum group metals such as palladium intracellularly. The present invention has been completed based on these findings.

[0010] The object of the present invention has been achieved by the following means. (1) nikA a gene or a gene corresponding to said gene, nikR a gene or a gene corresponding to said gene, rcnA a gene or a gene corresponding to said gene, rcnR a gene or a gene corresponding to said gene, and zntA A microorganism in which at least one gene selected from the group consisting of a gene or a gene corresponding to said gene has been deleted or inactivated.

[0011] (2) The above nikA a gene or a gene corresponding to said gene, and nikR a gene or a gene corresponding to said gene, rcnR a gene or a gene corresponding to said gene, and zntA The microorganism according to (1) above, wherein one kind of gene selected from the group consisting of a gene, a gene corresponding to said gene, and a gene selected from the group consisting of a gene, a gene corresponding to said gene, is deleted or inactivated. (3) The above nikA The microorganism according to (1) or (2), wherein the gene is a gene consisting of the following DNA (a) or (b): (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA in (a) and encodes NikA. (4) The above nikR The microorganism according to (1) or (2), wherein the gene is a gene consisting of the following DNA (c) or (d): (c) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 (d) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (c) and encodes NikR. (5) The above rcnA The microorganism according to (1) or (2), wherein the gene is a gene consisting of the following DNA (e) or (f): (e) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 (f) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (e) and encodes RcnA. (6) The above rcnR The microorganism according to (1) or (2), wherein the gene is a gene consisting of the following DNA (g) or (h): (g) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 (h) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA in (g) and encodes RcnR. (7) The above zntA The microorganism according to (1) or (2), wherein the gene is a gene consisting of the following DNA (i) or (j): (i) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 (j) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (i) above and encodes ZntA. (8) The microorganism according to any one of (1) to (7), wherein the microorganism is Escherichia coli.

[0012] (9) A microbial bioreactor for recovering or removing metals, in which the microorganism according to any one of (1) to (8) above is immobilized. (10) The microbial bioreactor according to (9) above, wherein the metal is at least one metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (11) The microbial bioreactor according to (9) or (10), wherein the metal is at least one elemental metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum, or a metal compound containing at least one metal element selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (12) A method for recovering metals, comprising contacting the microorganism according to any one of (1) to (8) with a metal-containing medium, incorporating the metal contained in the medium into the cells of the microorganism, and recovering the metal incorporated into the cells. (13) The method for recovering a metal according to (12) above, wherein the metal is at least one metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (14) The method for recovering a metal according to (12) or (13), wherein the metal is at least one elemental metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum, or a metal compound containing at least one metal element selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (15) A method for removing metals, comprising contacting the microorganism according to any one of (1) to (8) above with a metal-containing medium to remove the metal from the medium. (16) The method for removing metals according to (15) above, wherein the metal is at least one metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (17) The method for removing metals according to (15) or (16), wherein the metal is at least one elemental metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum, or a metal compound containing at least one metal element selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (18) A method for treating a metal-containing solution, comprising contacting the microorganism according to any one of (1) to (8) above with a metal-containing solution, and removing or recovering the metal from the solution. (19) The method for treating a metal-containing solution according to (18), wherein the metal is at least one metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. (20) The method for treating a metal-containing solution according to (18) or (19), wherein the metal is at least one elemental metal selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum, or a metal compound containing at least one metal element selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum. [Effects of the Invention]

[0013] The microorganism of the present invention is capable of intracellularly accumulating platinum group metals such as palladium. Furthermore, the microbial bioreactor of the present invention is capable of efficiently recovering or removing metals. Furthermore, the method for recovering or removing metals of the present invention places a low burden on the environment and enables efficient recovery or removal of metals. Furthermore, the method for treating a metal-containing solution of the present invention places a low burden on the environment and enables efficient recovery or removal of metals. The above and other features and advantages of the present invention will become more apparent from the following description, taken in conjunction with the accompanying drawings where appropriate. [Brief explanation of the drawings]

[0014]

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[0015] The microorganism according to the present invention is a microorganism in which a specific gene described below has been deleted or inactivated. By deleting or inactivating the specific gene described below, the microorganism according to the present invention can accumulate platinum group metals such as palladium within the bacterial cell. In this specification, the term "metal" encompasses both elemental metals and metal compounds. Examples of the elemental metals include nickel (Ni) and platinum group elements such as ruthenium (Ru), rhodium (Rh), palladium (Pd), osmium (Os), iridium (Ir), and platinum (Pt). The term "metal compound" refers to a metal compound containing at least one metal element selected from the group consisting of nickel, ruthenium, rhodium, palladium, osmium, iridium, and platinum, and examples thereof include oxides, chlorides, sulfides, and oxoanions of the elemental metals, or salts thereof.

[0016] In the present invention, the genes to be deleted or inactivated are: nikA gene, nikR gene, rcnA gene, rcnR Genes, and zntAThe gene is selected from a gene and a group of genes corresponding to the gene. In this specification, the names of the genes are written based on the E. coli genome database published by the National Center for Biotechnology Information (see URL: http: / / www.ncbi.nlm.nih.gov / nucleotide / ), and the name, number, function, etc. of each gene can be referenced. The genes to be deleted or inactivated in the present invention will be described below.

[0017] In this specification, nikA The "nik gene" encodes the protein NikA (see C Navarro, LF Wu, MA Mandland-Berthelot, The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel, Mol Microbiol. 1993;9(6):1181-91. doi: 10.1111 / j.1365-2958.1993.tb01247.x). NikA is known to cooperate with other proteins (subunits) to form a multimeric protein with nickel transport function (nickel uptake function) (see Jonathan Heddle, David J Scott, Satoru Unzai, Sam-Yong Park, Jeremy RH Tame, Crystal structures of the liganded and unliganded nickel-binding protein NikA from Escherichia coli, J Biol Chem. 2003;278(50):50322-9. doi: 10.1074 / jbc.M307941200). nikR "Gene" is nikA Binds to the upstream gene nikAIt is a transcription factor that represses gene expression (see Jeffrey S Iwig, Jessica L Rowe, Peter T Chivers, Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function, Mol Microbiol. 2006 62(1):252-62. doi: 10.1111 / j.1365-2958.2006.05369.x.). In this specification, zntA The "zntA gene" encodes the protein ZntA, which transports (excretes) zinc from the body (see C Rensing, B Mitra, BP Rosen, The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase, Proc Natl Acad Sci U S A. 1997;94(26):14326-31. doi: 10.1073 / pnas.94.26.14326). In addition, in this specification, rcnA The "gene" encodes the protein RcnA, which functions to excrete nickel from the body (see Agnes Rodrigue, Geraldine Effantin, Marie-Andree Mandlund-Berthelot, "Identification of rcnA (yohM), a nickel and cobalt resistance gene in Escherichia coli," J Bacteriol. 2005;187(8):2912-6. doi: 10.1128 / JB.187.8.2912-2916.2005). rcnR "Gene" is rcnAThis gene encodes the protein RcnR, which functions to regulate gene expression (see Jeffrey S Iwig, Jessica L Rowe, Peter T Chivers, Nickel, "Homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function," Mol Microbiol. 2006 62(1):252-62. doi: 10.1111 / j.1365-2958.2006.05369.x). The complete base sequences of these genes, as well as the amino acids of the proteins encoded by these genes, are described in the Escherichia coli genome database published by the National Center for Biotechnology Information (URL: http: / / www.ncbi.nlm.nih.gov / nucleotide), and the present invention can use these as references.

[0018] Genes to be deleted or inactivated in the present invention include, for example, genes that have a base sequence in which one or several base sequences have been deleted, substituted, or added to the base sequence of each gene published in the aforementioned databases, etc., and that have the same function as the gene. Furthermore, genes that have 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more identity to the base sequence of each gene published in the aforementioned databases, etc., and that have the same function as the gene are also included.

[0019] The aforementioned nikA The gene is preferably a gene consisting of the following DNA (a) or (b): The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of NikA of Escherichia coli strain MG1655. (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA in (a) and encodes NikA. The DNA (b) is preferably a DNA consisting of an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes a protein having NikA activity. Furthermore, the DNA (b) is also preferably a DNA in which one or more amino acids (for example, 1 to 209, preferably 1 to 183, more preferably 1 to 157, more preferably 1 to 131, more preferably 1 to 104, more preferably 1 to 78, more preferably 1 to 52, more preferably 1 to 36, more preferably 1 to 26, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5) have been deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NO: 1, and which encodes a protein having NikA activity.

[0020] The aforementioned nikA Specific examples of the gene include a gene consisting of the following DNA (A) or (B): The base sequence represented by SEQ ID NO: 26 is a sequence corresponding to the base sequence of the E. coli MG1655 strain genome. nikA This is the base sequence of a gene. (A) DNA consisting of the base sequence shown in SEQ ID NO: 26. (B) A DNA having a base sequence that is 60% or more identical to the base sequence of DNA (A) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes NikA. Furthermore, DNA (B) is also preferred that has one or more bases (for example, 1 to 630, preferably 1 to 551, more preferably 1 to 472, more preferably 1 to 393, more preferably 1 to 315, more preferably 1 to 236, more preferably 1 to 157, more preferably 1 to 110, more preferably 1 to 78, more preferably 1 to 47, more preferably 1 to 31, more preferably 1 to 15) deleted, substituted, inserted, or added in the base sequence of DNA (A), and encodes NikA. Furthermore, DNA (B) that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA (A) and that encodes NikA is also preferred.

[0021] The aforementioned nikR The gene is preferably a gene consisting of the following DNA (c) or (d): The amino acid sequence of SEQ ID NO: 2 is the amino acid sequence of NikR of the Escherichia coli MG1655 strain. (c) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 (d) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (c) and encodes NikR. The DNA (d) is preferably a DNA consisting of an amino acid sequence that is 60% or more identical to the amino acid sequence set forth in SEQ ID NO: 2 (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes a protein having NikR activity. Furthermore, the DNA (d) is also preferably a DNA in which one or more amino acids (for example, 1 to 53, preferably 1 to 46, more preferably 1 to 39, more preferably 1 to 33, more preferably 1 to 26, more preferably 1 to 19, more preferably 1 to 13, more preferably 1 to 9, more preferably 1 to 6, more preferably 1 to 3, more preferably 1 to 2, more preferably 1 or less) have been deleted, substituted, inserted, or added to the amino acid sequence shown in SEQ ID NO: 2, and which encodes a protein having NikR activity.

[0022] The aforementioned nikR Specific examples of the gene include a gene consisting of the following DNA (C) or (D). The base sequence shown in SEQ ID NO: 27 is a sequence corresponding to the base sequence of the E. coli MG1655 strain genome. nikR This is the base sequence of a gene. (C) DNA consisting of the base sequence represented by SEQ ID NO: 27. (D) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (C) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes NikR. Furthermore, DNA (D) is also preferred that has one or more bases (for example, 1 to 160, preferably 1 to 140, more preferably 1 to 120, more preferably 1 to 100, more preferably 1 to 80, more preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 28, more preferably 1 to 20, more preferably 1 to 12, more preferably 1 to 8, more preferably 1 to 4) deleted, substituted, inserted, or added in the base sequence of DNA (C), and encodes NikR. Furthermore, DNA (D) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (C) and encodes NikR.

[0023] The aforementioned rcnA The gene is preferably a gene consisting of the following DNA (e) or (f): The amino acid sequence of SEQ ID NO: 3 is the amino acid sequence of RcnA of the Escherichia coli MG1655 strain. (e) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 (f) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (e) and encodes RcnA. The DNA (f) is preferably a DNA consisting of an amino acid sequence that is 60% or more identical to the amino acid sequence shown in SEQ ID NO: 3 (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes a protein having RcnA activity. Furthermore, DNA (f) is also preferred that encodes a protein having RcnA activity, in which one or more amino acids (for example, 1 to 109, preferably 1 to 95, more preferably 1 to 82, more preferably 1 to 68, more preferably 1 to 54, more preferably 1 to 41, more preferably 1 to 27, more preferably 1 to 19, more preferably 1 to 13, more preferably 1 to 8, more preferably 1 to 5, more preferably 1 to 2) have been deleted, substituted, inserted, or added to the amino acid sequence shown in SEQ ID NO: 3.

[0024] The aforementioned rcnA Specific examples of the gene include a gene consisting of the following DNA (E) or (F). The base sequence shown in SEQ ID NO: 28 is a sequence corresponding to the DNA (E) or (F) on the genome of the Escherichia coli MG1655 strain. rcnA This is the base sequence of a gene. (E) DNA consisting of the base sequence represented by SEQ ID NO: 28. (F) A DNA having a base sequence that is 60% or more identical to the base sequence of the DNA (E) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and encoding RcnA. Furthermore, DNA (F) is also preferred that has one or more bases (for example, 1 to 330, preferably 1 to 288, more preferably 1 to 247, more preferably 1 to 206, more preferably 1 to 165, more preferably 1 to 123, more preferably 1 to 82, more preferably 1 to 57, more preferably 1 to 41, more preferably 1 to 24, more preferably 1 to 16, more preferably 1 to 8) deleted, substituted, inserted, or added in the base sequence of DNA (E), and encodes RcnA. Furthermore, DNA (F) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (E) and encodes RcnA.

[0025] The aforementioned rcnR The gene is preferably a gene consisting of the following DNA (g) or (h): The amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of RcnR of the Escherichia coli strain MG1655. (g) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 (h) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA in (g) and encodes RcnR. The DNA (h) is preferably a DNA consisting of an amino acid sequence that has 60% or more identity (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 4, and that encodes a protein having RcnR activity. Furthermore, the DNA (h) is also preferably a DNA in which one or more amino acids (for example, 1 to 36, preferably 1 to 31, more preferably 1 to 27, more preferably 1 to 22, more preferably 1 to 18, more preferably 1 to 13, more preferably 1 to 9, more preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 2, more preferably 1) have been deleted, substituted, inserted, or added to the amino acid sequence shown in SEQ ID NO: 4, and which encodes a protein having RcnR activity.

[0026] The aforementioned rcnR Specific examples of the gene include a gene consisting of the following DNA (G) or (H). The base sequence shown in SEQ ID NO: 29 is a sequence corresponding to the base sequence of the E. coli MG1655 strain genome. rcnR This is the base sequence of a gene. (G) DNA consisting of the base sequence represented by SEQ ID NO: 29. (H) DNA having a base sequence that is 60% or more identical to the base sequence of DNA (G) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more). The base sequence of DNA (G) is 60% or more identical to the base sequence of DNA (G) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and encoding RcnR. Furthermore, DNA (H) is also preferred that has one or more bases (for example, 1 to 109, preferably 1 to 95, more preferably 1 to 81, more preferably 1 to 68, more preferably 1 to 54, more preferably 1 to 40, more preferably 1 to 27, more preferably 1 to 19, more preferably 1 to 13, more preferably 1 to 8, more preferably 1 to 5, more preferably 1 to 2) deleted, substituted, inserted, or added in the base sequence of DNA (G), and encodes RcnR. Furthermore, DNA (H) is also preferably a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA (G) and encodes RcnR.

[0027] The aforementioned zntA The gene is preferably a gene consisting of the following DNA (i) or (j): The amino acid sequence of SEQ ID NO: 5 is the amino acid sequence of ZntA of the Escherichia coli MG1655 strain. (i) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 (j) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of the DNA of (i) above and encodes ZntA. The DNA (j) is preferably a DNA consisting of an amino acid sequence that is 60% or more identical to the amino acid sequence shown in SEQ ID NO: 5 (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more), and that encodes a protein having ZntA activity. Furthermore, DNA (j) is also preferred that encodes a protein having ZntA activity, in which one or more amino acids (for example, 1 to 292, preferably 1 to 256, more preferably 1 to 219, more preferably 1 to 183, more preferably 1 to 146, more preferably 1 to 109, more preferably 1 to 73, more preferably 1 to 51, more preferably 1 to 36, more preferably 1 to 21, more preferably 1 to 14, more preferably 1 to 7) have been deleted, substituted, inserted, or added to the amino acid sequence shown in SEQ ID NO: 5.

[0028] The aforementioned zntASpecific examples of the gene include a gene consisting of the following DNA (I) or (J). The base sequence shown in SEQ ID NO: 30 is a sequence corresponding to the base sequence of the E. coli MG1655 strain genome. zntA This is the base sequence of a gene. (I) DNA consisting of the base sequence represented by SEQ ID NO: 30. (J) A DNA having a base sequence that is 60% or more identical to the base sequence of DNA (I) (preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, and even more preferably 99% or more). The DNA encodes ZntA. Furthermore, DNA (J) is also preferred that has one or more bases (for example, 1 to 879, preferably 1 to 769, more preferably 1 to 659, more preferably 1 to 549, more preferably 1 to 439, more preferably 1 to 329, more preferably 1 to 219, more preferably 1 to 153, more preferably 1 to 109, more preferably 1 to 65, more preferably 1 to 43, more preferably 1 to 21) deleted, substituted, inserted, or added in the base sequence of DNA (I), and encodes ZntA. Furthermore, the DNA (J) is preferably a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA (I) and encodes ZntA.

[0029] As used herein, "stringent conditions" include, for example, the method described in Molecular Cloning—A Laboratory Manual, Third Edition [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press]. For example, hybridization conditions include incubation of a solution containing 6×SSC (1×SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% sodium dodecyl sulfate (hereinafter also referred to as "SDS"), 5×Denhardt's, and 100 mg / mL herring sperm DNA together with a probe at 65°C for 8 to 16 hours. In this specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227, p. 1435) or the like. Specifically, the identity can be calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win (manufactured by Software Development Co., Ltd.) with the parameter Unit size to compare (ktup) set to 2.

[0030] The species of the microorganism of the present invention is not particularly limited, and includes Escherichia coli ( Escherichia ) bacteria, Methanococcus ( Methanococcus ) bacteria, Rhodococcus ( [[ID= ) bacteria, Synechococcus ( ​ ) bacteria, Bacillus ( ​ Among these, bacteria of the genus Escherichia are preferred, with Escherichia coli being more preferred. Furthermore, in addition to wild-type bacterial species, mutant strains in which specific mutations have been introduced into wild-type bacterial species can also be used.

[0031] The microorganism of the present invention can be prepared according to conventional methods, such as by inserting another DNA fragment (e.g., a stop codon or a marker gene) into the target gene, physically deleting the target gene, or mutating the transcription / translation initiation region of the target gene. Methods for deleting or inactivating a target gene include methods for deleting or inactivating a target gene using genetic recombination and methods for deleting or inactivating a target gene using genome editing. Methods for deleting or inactivating a target gene using genetic recombination include methods for inserting a marker gene such as a drug resistance gene or an auxotrophic gene into the target gene using information. Methods for deleting or inactivating a target gene using genome editing include methods using the CRISPR-Cas system (e.g., Miyake, Y. and Yamamoto, K.: Epistatistics effect of regulators to the adaptive growth of ​ ​ . Sci. Rep. 10:3661, (2020) etc.

[0032] In the microorganism of the present invention, the gene that has been deleted or inactivated may be one of the above-mentioned genes, or two or more of the above-mentioned genes may be deleted or inactivated. ​ a gene or a gene corresponding to said gene, and ​ a gene or a gene corresponding to said gene, ​ a gene or a gene corresponding to said gene, ​ a gene or a gene corresponding to said gene, and ​ and one gene selected from the group consisting of a gene or a gene corresponding to said gene, are preferably deleted or inactivated, ​ a gene or a gene corresponding to said gene, and ​ a gene or a gene corresponding to said gene, ​ a gene or a gene corresponding to said gene, and ​It is more preferable that the gene or one gene selected from the group consisting of the gene or a gene corresponding to the gene is deleted or inactivated, ​ a gene or a gene corresponding to said gene, and ​ a gene or a gene corresponding to said gene, and ​ It is more preferable that the gene or one gene selected from the group consisting of the gene or a gene corresponding to the gene is deleted or inactivated, ​ a gene or a gene corresponding to said gene, and ​ It is more preferable that the gene or a gene corresponding to the gene is deleted or inactivated, respectively. Furthermore, in addition to the above-mentioned genes, other genes may be modified within the scope that does not impair the effects of the present invention.

[0033] As will be shown in the Examples below, the ability of a microorganism to accumulate a metal within its cells can be evaluated by drying and calcining the cells of the microorganism to prepare a powder, dissolving the powder in a liquid, and then measuring the metal concentration using an atomic absorption spectrophotometer.

[0034] By immobilizing the microorganisms of the present invention on a suitable carrier, the microbial bioreactor of the present invention (e.g., a column for recovering or removing metals, a metal recovery or removal agent) can be provided. The material of the carrier used in the present invention is not particularly limited, but examples include substances insoluble in water or specific solvents. Examples include polyvinyl alcohol, polyurethane, polystyrene, polyacrylamide, silicone, cellulose, polyaluminum chloride, ferric chloride, aluminum sulfate, and polyferric sulfate. Furthermore, the shape of the carrier used in the present invention is not particularly limited, and any of the usual shapes used for immobilizing microorganisms can be used, such as a sponge structure, wall structure, pellet structure, foam, porous body, foam, resin molded body, and aggregate structure. As used herein, "immobilized" refers to a state in which the microorganisms are bound or attached to a carrier, or are incorporated into the carrier, rather than being free.

[0035] The microorganism according to the present invention can be contacted with a metal-containing medium in which a platinum group metal is dissolved, the metal contained in the medium can be taken up into the cells of the microorganism, and the metal taken up into the cells can be recovered.Furthermore, the microorganism according to the present invention can be contacted with a metal-containing medium in which a platinum group metal is dissolved, and the metal can be removed from the medium. Furthermore, by contacting the microorganism according to the present invention with a solution containing a metal dissolved in a solvent, it becomes possible to remove or recover the metal from the solution. The microorganism according to the present invention, which has accumulated platinum group metals within its cells, can be obtained by a conventional method, such as centrifuging a culture solution of the microorganism and recovering the precipitate.

[0036] Bioprocesses, which are comprehensively understood biological functions, are utilized in a variety of fields due to their industrial advantages of low environmental impact, energy conservation, and low cost. Metal biology, which has emerged due to advances in microanalysis and genome biology, has deepened our understanding of the in vivo functions of metals, providing an opportunity to convert biotechnology techniques used in metal refining and recovery into bioprocessing technologies that utilize comprehensive biological functions. Until now, valuable rare metals have been sourced from ores with high metal content, which are unevenly distributed around the globe. Due to a global policy shift toward building a recycling-oriented society, waste containing valuable rare metals is being considered a new resource and refined into raw materials. However, both processes require large-scale facilities, entailing enormous costs and making it difficult to replace existing facilities and technologies. Meanwhile, in addition to urban mines, there are also unused metal environments. These include ores with low metal content and dilute dissolved metals in seawater, rivers, and lakes. These unused environments require an active "concentration" step. In response, the inventors' proposed metal-accumulating bacteria, utilizing a metal homeostatic bioprocess, have the potential to actively concentrate metals from these unused metal resources. [Example]

[0037] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0038] (1) Bioaccumulation of palladium by Escherichia coli To evaluate the bioaccumulation of palladium by E. coli, it is necessary to accurately measure the amount of platinum group metals, such as palladium, in the cells. To this end, we established a method for quantifying palladium using an atomic absorption spectrophotometer with a sensitivity 10 to 100 times greater than that of spectrophotometric determination. Using this measurement, we investigated whether E. coli cells contained palladium and, if so, what its concentration was.

[0039] First, we used BW25113, the parent strain of a comprehensive gene deletion strain set used as a genome research resource, as the E. coli K strain (see Baba, T., et al.: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2:2006.0008, (2006)). We confirmed that nickel, ruthenium, rhodium, palladium, iridium, and platinum are each soluble in LB liquid medium. Therefore, we used LB liquid medium containing palladium, one of the platinum group metals, and cultured BW25113 overnight in LB liquid medium with a palladium concentration of 25 μM. To count the cell number in the culture medium, a portion was diluted 100-fold, placed on a cell counter plate, and counted under an optical microscope to calculate the cell concentration. In the palladium-free culture medium, an average of 1.77 × 10 cells / mL was obtained. 9 cells, an average of 2.10 × 10 per mL in palladium-added culture medium 9 As a result of multiple experiments, the culture medium prepared under these conditions contained 1.94 × 10 cells per mL. 9 The cells were found to contain E. coli cells.

[0040] One liter of this culture was centrifuged to recover the E. coli cells, which were then washed with EDTA solution, then washed again with sterile water, and dried at 120°C for four hours. The dried E. coli cell sample was then calcined at 1,000°C for two hours to produce a metal powder containing palladium. The metal mixture was dissolved in hydrochloric acid, and its concentration was measured using an atomic absorption spectrophotometer along with a solution of known palladium concentration.

[0041] The amount of palladium measured was calculated using the number of cells contained in the E. coli cell preparation. In the culture medium without palladium, the amount was 0.12 × 10 per cell. -18 g (1.10 μM) of palladium was present (see Table 1). Furthermore, in the culture medium supplemented with 25 μM palladium, 0.29 × 10 -18 g (2.76 μM) of palladium was detected, and the accumulation amount was increased by approximately three times compared to when palladium was not added (see Table 1). Furthermore, the recovery rate of palladium from 1 L of 25 μM palladium-added medium in E. coli bioaccumulation was 0.02%.

[0042] (2) Impact of the nickel homeostasis system in Escherichia coli on palladium accumulation After confirming the bioaccumulation of palladium by Escherichia coli, we investigated the effect of the E. coli NikABCDE system on the intracellular accumulation of nickel. The NikABCDE system is a nickel transport system protein, and we expected to see an effect on its homologous palladium. First, we investigated the effect of the NikABCDE system on nickel sensitivity.

[0043] Based on the literature (Monica Riley, Takashi Abe, Martha B Arnaud, Mary KB Berlyn, Frederick R Blattner, Roy R Chaudhuri, Jeremy D Glasner, Takashi Horiuchi, Ingrid M Keseler, Takehide Kosuge, Hirotada Mori, Nicole T Perna, Guy Plunkett 3rd, Kenneth E Rudd, Margrethe H Serres, Gavin H Thomas, Nicholas R Thomson, David Wishart, Barry L Wanner, Escherichia coli K-12: a cooperatively developed annotation snapshot--2005, Nucleic Acids Res. 2006 Jan 5;34(1):1-9. doi: 10.1093 / nar / gkj405), any codon in the middle of each gene of BW25113 was replaced with a stop codon, ​ gene deletion strains, ​ gene deletion strains, ​ gene deletion strains, ​ gene deletion strains, ​ Gene deletion strains were constructed for each gene.

[0044] When BW25113 was cultured in M9 glucose minimal medium supplemented with various concentrations of nickel, the initiation of growth was delayed according to the nickel concentration (see Figure 2(a)). ​ gene deletion strains, ​ gene deletion strains, ​ gene deletion strains, ​ gene deletion strains, ​ When the gene deletion strains were grown in media containing various concentrations of nickel, all strains showed the same nickel-dependent growth retardation as the parent strain (see Figure 1(b)-(f)). These results were unexpected, as growth in minimal medium did not confirm the effect of the NikABCDE system on nickel sensitivity of E. coli.

[0045] Next, we investigated the effect of the NikABCDE system on palladium sensitivity. When BW25113 was cultured in a medium prepared by adding various concentrations of palladium to the M9 glucose minimal medium, growth of BW25113 was inhibited in the presence of 2.5 μM or more palladium, but growth was confirmed in the presence of 2.5 μM or less palladium (see Figure 2(a)). ​ Although the gene deletion strain showed a delayed growth initiation, growth was confirmed in the presence of 2.5 μM palladium (Figure 2(b)). Furthermore, BW25113 and BW25113 were grown in LB medium supplemented with various concentrations of nickel. ​ When the gene deletion strain was cultured, growth was suppressed in the presence of 25 μM or more palladium, but growth was confirmed in the presence of 25 μM or less palladium (see Figures 3(a) and (b)). These results suggest that the NikABCDE system is involved in palladium resistance in E. coli.

[0046] So, ​ The amount of palladium accumulated in the gene deletion strain was measured. ​ The gene deletion strains were cultured in LB liquid medium containing 25 μM palladium, and each E. coli cell was harvested, washed, and dried to prepare a cell sample. The resulting metal powder was then dissolved in hydrochloric acid and the amount of palladium was measured using an atomic absorption spectrophotometer. As a result, in the culture medium without palladium, 0.31 × 10 -18 g (2.96 μM) of palladium was present (see Table 1). Furthermore, in the 25 μM palladium-supplemented culture medium, 1.04 × 10 -18 g (9.74 μM) of palladium was detected, and the accumulation increased approximately threefold compared to when palladium was not added, as with the parent strain (see Table 1). Furthermore, the recovery rate of this palladium in E. coli was 0.07% from 1 L of medium supplemented with 25 μM palladium, confirming a three-fold increase compared to the parent strain.

[0047] [Table 1]

[0048] (3) Palladium-binding protein NikR NikR binds nickel ​ Binds to the upstream gene ​ It is a transcription factor that suppresses gene expression. Therefore, we investigated the binding of the NikR protein with palladium. First, we constructed a system for overexpressing recombinant NikR protein in E. coli cells and succeeded in overexpressing the recombinant NikR protein in E. coli cells (see Watanabe Hiroki and Yamamoto Kaneyoshi, "Genome Editing Using the HoSeI Method for Rare Metal Resource Development Using E. coli Bioprocesses," Chemical Industry, 2020 71 (12), in press). The E. coli cells were disrupted, and the crude extract was fractionated by affinity chromatography to purify the recombinant NikR protein. Next, we measured the binding of NikR to nickel and palladium using isothermal titration calorimetry (ITC). We evaluated the specific binding of NikR to nickel and palladium by gradually increasing the concentration of nickel or palladium added to 10 μM recombinant NikR protein and measuring the heat release upon each addition.

[0049] First, as a control, measurements were taken using cobalt, which does not bind to NikR. As expected, no increase in the amount of heat generated was observed even when the cobalt concentration was increased (see Figure 4(C)). A similar experiment was conducted with nickel. The results showed that the amount of heat generated increased as the nickel concentration increased (see Figure 4(A)). A similar experiment was also conducted with palladium. The results showed that the amount of heat generated increased as the palladium concentration increased (see Figure 4(B)). These results demonstrate that NikR specifically binds not only to nickel but also to palladium.

[0050] (4) Search for E. coli genes whose expression is induced by palladium The above results suggest that the NikABCDE system is involved in palladium excretion. ​ We decided to examine whether gene expression is suppressed by palladium. Therefore, using a luciferase reporter system, ​ - ​ Fusion genes were created. These fusion genes were introduced into E. coli cells, and gene expression was evaluated by measuring the luciferase activity of the E. coli cells as luminescence. E. coli cells into which the fusion genes had been introduced were cultured in LB medium until the logarithmic growth phase. The culture was then divided into two portions, and one portion was cultured in LB medium with a nickel concentration of 1 mM for an additional 8 hours while measuring the luciferase activity in the culture medium. ​ The results showed that the presence of nickel ​ It was confirmed that gene expression was significantly suppressed (see FIG. 5(A)). A similar experiment was carried out using LB medium with a palladium concentration of 25 μM, and the results showed that the presence of palladium, like nickel, ​ Gene expression was suppressed (see Figure 5(A)).

[0051] Therefore, we decided to comprehensively search for genes in the E. coli transport system similar to NikABCDE whose expression is regulated by palladium. Based on their structural characteristics, the metal transport systems of Escherichia coli were divided into three groups: Group A, which consists of one polypeptide; Group B, which consists of three polypeptides; and Group C, which is an ABC transporter consisting of multiple polypeptides. In group A, we identified Kch, Kup, TrkA, TrkG, and TrkH, which transport potassium, and CorA and MgtA, which transport magnesium. In addition, we also investigated KefB, KefC, CvrA, YbjL, YfbS, YidE, CopA, and ZntA, which have similar domains to these proteins. KdpABC, which is involved in potassium transport, and TonB / ExbBD, which is involved in iron transport, were identified in group B. In addition, MgtA, CopA, ZntA, MotA, TolQ, and TolR, which have domains similar to these proteins, were also investigated. In addition to NikABCDE, group C also contained FecBCDEE, FepBDGC, and FhuDBC, which are involved in iron transport, ZnuABC, which is involved in zinc transport, and ModABC, which is involved in molybdenum transport. Additionally, DdpA, DppA, GslB, MppA, OppA, SapA, YejA, YgiS, SgrR, YbaE, BtuF, CysP, and Sbp, which share similar domains to these proteins, were also investigated. In addition, we also included RcnA, a recently discovered novel nickel efflux pump with a structure different from those of these groups.

[0052] Of the 41 gene promoters investigated, we successfully constructed luciferase reporter systems for 21 types ( ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ , ​ - ​ ,​ - ​ , ​ - ​ E. coli cells carrying these fusion genes were cultured in LB medium until they reached the logarithmic growth phase. The culture was then divided into two portions, and one portion was cultured in LB medium containing 25 μM palladium for an additional 8 hours, during which the luciferase activity in the culture medium was measured. the result, ​ It was confirmed that gene expression was induced by the presence of palladium (see Figure 5(B)). The expression of the other 20 genes was not affected by the presence of palladium. Similar experiments were performed using ruthenium instead of palladium. As a result, it was confirmed that the expression of the exbD gene was induced by the presence of ruthenium, while the expression of the trkG, trkH, and copA genes was repressed by ruthenium (see Figure 5(C)). The expression of the other 20 genes was not affected by the presence of palladium.

[0053] (5) Identification of genes involved in palladium accumulation in Escherichia coli They were the first in the world to discover that "E. coli cells accumulate palladium and ruthenium," and further confirmed that "the amount of palladium and ruthenium accumulated in E. coli cells increases when high concentrations of palladium or ruthenium are present in the culture medium." Furthermore, bioprocess modifications to improve these capabilities include: ​ We confirmed that the deletion of gene function was effective. ​ Furthermore, we found that genes may be candidates for this. ​ Genes were also expected to affect the bioaccumulation of elements such as palladium and ruthenium. So, ​ genes and their regulatory genes ​ gene, ​ genes and their regulatory genes ​Four gene deletion strains were prepared (see Tomoya Baba, Takeshi Ara, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Kirill A Datsenko, Masaru Tomita, Barry L Wanner, Hirotada Mori, Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection, Mol Syst Biol. 2006;2:2006.0008. doi: 10.1038 / msb4100050.), and their ability to accumulate palladium was measured.

[0054] ​ Deletion strains, ​ Deletion strains, ​ Deletion strains, ​ The deletion strains were cultured in LB liquid medium containing 25 μM palladium, and each E. coli cell was harvested, washed, and dried to prepare a cell sample. The resulting metal powder was then dissolved in hydrochloric acid and the amount of palladium was measured using an atomic absorption spectrophotometer. As a result, in the culture medium without palladium, ​ The amount of palladium per cell in the deletion strain was 0.07 × 10 -18 g (0.61 μM), ​ The deletion strain was 0.28 × 10 -18 g (2.66 μM), ​ The deletion strain was 0.02 × 10 -18 g (0.16 μM), ​ The deletion strain was 0.17 × 10 -18 g (1.64 μM). Furthermore, in the culture medium containing 25 μM palladium, ​ The amount of palladium per cell in the deletion strain was 0.18 × 10 -18 g (1.68 μM), ​ The deletion strain was 0.99 × 10 -18 g (9.33 μM), ​ The deletion strain was 0.32 × 10 -18 g (2.98 μM), ​ The deletion strain was 0.35 × 10 -18g (3.33 μM), ​ Deletion strains, ​ Deletion strains, ​ Similar to the parent strain, the deletion strain showed an approximately three-fold increase in the amount of accumulated palladium compared to when no palladium was added (see Figure 6). ​ The deletion strain showed an approximately 10-fold increase in accumulated amount compared to when palladium was not added (see Figure 6). ​ In the deletion strain, the palladium recovery rate from 1 L of culture medium supplemented with 25 μM palladium was 0.07%, which was confirmed to be three times higher than that of the parent strain (see FIG. 6).

[0055] (6) Development of the HoSeI genome editing method without the use of exogenous genes Recombinant Escherichia coli, in which the genes involved in palladium homeostasis in Escherichia coli have been modified, is expected to be applied as a low-environmental-impact technology to replace the energy-intensive dry-wet rare metal purification method. However, existing gene recombination and genome editing technologies require the introduction of foreign genes, which makes it difficult to eliminate the impact of the resulting recombinant organisms on the ecosystem, a major factor hindering their practical application. Therefore, we developed a genome editing technique called HoSeI (Homologous Sequence Integration) that does not use exogenous genes (Miyake, Y. and Yamamoto, K.: Epistatistic effect of regulators on the adaptive growth of ​ ​ Sci. Rep. 10:3661, (2020)). The HoSeI method uses the CRISPR-Cas system and does not use markers that are exogenous genes, making it easy to modify many genes in the genome. This artificially creates mutations that occur naturally in the target E. coli, solving the problem of genetically modified organisms that poses a risk in industrial applications. Using this method, we modified the gene functions that contribute to palladium bioaccumulation in Escherichia coli and created genome-edited strains.

[0056] (7) Application of the HoSeI method to palladium bioaccumulation in Escherichia coli The HoSeI method has been shown to be somewhat versatile in terms of hosts, so we began by selecting a parent strain suitable for genome editing that contributes to palladium accumulation. In addition to the BW25113 strain used in the above experiments, three strains widely used in experiments, MG1655 strain (Blattner, FR et al.: The complete genome sequence of ​ ​ K-12. Science 277(5331), 1453-1462, (1997)), W3110 strain (Hayashi, K. et al.: Highly accurate genome sequences of ​ ​ K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2:2006.0007, (2006)), W3110 type-A strain (Jishage, M. and Ishihama, A.: Variation in RNA polymerase sigma subunit composition within different stocks of ​ ​ W3110. J. Bacteriol. 179, 959-963, (1997)) were cultured in LB liquid medium containing 25 μM palladium, and each E. coli cell was harvested, washed, and dried to prepare a sample of E. coli cells. The resulting metal powder was then dissolved in hydrochloric acid solution and the amount of palladium was measured using an atomic absorption spectrophotometer. As a result, the amount of palladium per cell of BW25113 strain in the palladium-free culture medium was 0.12 × 10 -18 g (1.10 μM), whereas the MG1655 strain had a 0.26 × 10 -18 The presence of palladium was not confirmed in the W3110 and W3110 type-A strains (see Figure 7(A)). On the other hand, in the culture medium supplemented with 25 μM palladium, the amount of palladium per cell of the BW25113 strain was 0.29 × 10-18 g (2.76 μM), whereas the MG1655 strain had a concentration of 5.16 × 10 -18 g (48.50 μM), and the W3110 strain was 5.89 × 10 -18 g (55.36 μM), and the W3110 type-A strain was 3.89 × 10 -18 g (36.57 μM) (see Figure 7(A)). These results suggest that the MG1655, W3110, and W3110 type-A strains accumulate palladium more efficiently than the BW25113 strain. Therefore, the MG1655 strain, which accumulated palladium in the absence of palladium like the BW25113 strain and accumulated approximately 20-fold more palladium in the presence of palladium, was selected as the parent strain for genome editing using the HoSeI method.

[0057] on the genome of strain MG1655 ​ gene, ​ gene, ​ gene, ​ gene, ​ We created strains in which a stop codon was introduced upstream of the protein-coding region of each gene using the HoSeI method (see Figures 8 to 12). These genome-edited strains were cultured in LB liquid medium containing 25 μM palladium, and the amount of palladium in the resulting E. coli cells was measured. The oligonucleotides listed in Table 2 were used to create the various gene deletion strains. Details of the various gene deletion strains are shown in Table 3. Furthermore, the plasmids used for deleting the various genes are shown in Table 4. on the genome of strain MG1655 ​ The base sequence of the gene is shown in SEQ ID NO: 26. ​ The base sequence of the gene is shown in SEQ ID NO: 27. ​ The base sequence of the gene is shown in SEQ ID NO: 28. ​ The base sequence of the gene is shown in SEQ ID NO: 29. ​ The base sequence of the gene is shown in SEQ ID NO:30.

[0058] [Table 2]

[0059] [Table 3]

[0060] [Table 4]

[0061] where: ​ Using gene deletion strains as examples, the following will be described with reference to oligonucleotides consisting of the nucleotide sequences shown in any of SEQ ID NOS: 6 to 9, pCas plasmids, psgRNA plasmids, and psgRNA_nikA02_08 plasmids. The pCas plasmid was obtained from Addgene (https: / / www.addgene.org / 62225 / ), and the psgRNA plasmid was obtained from Miyake, Y. and Yamamoto, K., “Epistatistic effect of regulators to the adaptive growth of ​ ​ ", Sci. Rep., 10:3661, (2020). The genome editing technology HoSeI (Homologous Sequence Integration) method (Miyake, Y. and Yamamoto, K., "Epistatistic effect of regulators to the adaptive growth of ​ ​ ”, Sci. Rep., 10:3661, (2020)), a nikA gene deletion strain was created. ​The guide RNA expression plasmid psgRNA_nikA02_08 targeting the upstream region of a gene was constructed by inserting a DNA fragment annealed with the oligonucleotides SEQ ID NOs: 6 and 7 into the psgRNA plasmid. The plasmid was introduced into MG1655 containing pCas9, along with a DNA fragment annealed with the oligonucleotides SEQ ID NOs: 8 and 9. Growth of the plasmid on agar medium containing ampicillin was confirmed. ​ The gene deletion strain was screened. ​ The gene deletion strain has ​ The gene sequence was confirmed and the desired genome editing was confirmed. ​ In a double mutant strain in which two genes, including the ​ Gene deletion was performed in a similar manner.

[0062] ​ nikR_sgRNA_N20-1 (SEQ ID NO: 10) and nikR_sgRNA_com-1 (SEQ ID NO: 11) were used as oligonucleotides for cloning the gene into the psgRNA plasmid. ​ The oligonucleotides used to introduce a stop codon into the gene were nikR_PAMstop-1 (SEQ ID NO: 12) and nikR_PAMstop_com-1 (SEQ ID NO: 13). ​ In the same way as the gene deletion strain, ​ A gene deletion strain was constructed. ​ In a double mutant strain in which two genes, including the ​ Gene deletion was performed in a similar manner. ​ Using rcnA_sgRNA_N20-2 (SEQ ID NO: 14) and rcnA_sgRNA_com-2 (SEQ ID NO: 15) as oligonucleotides for cloning the gene into the psgRNA plasmid, ​ Except that rcnA_PAMstop_F (SEQ ID NO: 16) and rcnA_PAMstop_R (SEQ ID NO: 17) were used as oligonucleotides to introduce a stop codon into the gene, ​The rcnA gene deletion strain was prepared in the same manner as the gene deletion strain. ​ In a double mutant strain in which two genes, including the ​ Gene deletion was performed in a similar manner. ​ Using rcnR_sgRNA_N20-4 (SEQ ID NO: 18) and rcnR_sgRNA_com-4 (SEQ ID NO: 19) as oligonucleotides for cloning the gene into the psgRNA plasmid, ​ The oligonucleotides used to introduce a stop codon into the gene were rcnR_PAMstop_F (SEQ ID NO: 20) and rcnR_PAMstop_R (SEQ ID NO: 21). ​ In the same way as the gene deletion strain, ​ A gene deletion strain was constructed. ​ In a double mutant strain in which two genes, including the ​ Gene deletion was performed in a similar manner. ​ zntA_sgRNA_N20-4 (SEQ ID NO: 22) and zntA_sgRNA_com-4 (SEQ ID NO: 23) were used as oligonucleotides for cloning the gene into the psgRNA plasmid. ​ The oligonucleotides used to introduce a stop codon into the gene were zntA_PAMstop_F (SEQ ID NO: 24) and zntA_PAMstop_R (SEQ ID NO: 25). ​ In the same way as the gene deletion strain, ​ A gene deletion strain was constructed. ​ In a double mutant strain in which two genes, including the ​ Gene deletion was performed in a similar manner.

[0063] When the amount of palladium was measured, the amount of intracellular palladium in both genome-edited strains was comparable to that of the parent strain in the palladium-free culture medium (see Figure 7(B)). However, in the 25 μM palladium-added culture medium, ​ 31.52 × 10 per cell in the gene deletion strain -18 g (296.16 μM) of palladium, ​The gene deletion strain was 6.21 × 10 -18 g (58.37 μM), ​ The gene deletion strain was 18.15 × 10 -18 g (170.54 μM), ​ The gene deletion strain was 11.62 × 10 -18 g (109.22 μM), which was a higher amount of palladium accumulated than the mutant strain derived from BW25113 (see Figure 7(B)). ​ In the gene deletion strain, the palladium recovery rate from 1 L of culture medium supplemented with 25 μM palladium was ​ The rate was 2.29% in the gene deletion strain. Among the double mutants with all combinations of the nikA gene, the nikR gene, the rcnA gene, the rcnR gene, and the zntA gene, the double mutant strains with deletions of the nikA gene and the rcnR gene (nikA·rcnR deletion strain), the double mutant strains with deletions of the nikA gene and the zntA gene (nikA·zntA deletion strain), and the double mutant strains with deletions of the nikA gene and the nikR gene (nikA·nikR deletion strain) showed particularly high palladium recovery (see Figure 7(B)).

[0064] As described above, the microorganism of the present invention in which a specific gene has been deleted or inactivated can efficiently accumulate platinum group metals such as palladium within the bacterial cell.

[0065] While the present invention has been described in connection with embodiments thereof, we do not intend to limit our invention to any of the details of the description unless otherwise specified, and believe that the claims should be construed broadly without departing from the spirit and scope of the invention as set forth in the appended claims.

[0066] This application claims priority based on Japanese Patent Application No. 2020-194565, filed on November 24, 2020, the contents of which are incorporated herein by reference as part of the present specification.

Claims

1. The nikA gene, and one gene selected from the group consisting of the rcnR gene and the zntA gene; are deleted or inactivated, respectively.

2. 2. The Escherichia coli according to claim 1, wherein the nikA gene is a gene consisting of the following DNA (a) or (b): (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 (b) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of the DNA of (a) above and encodes NikA (wherein the stringent conditions are those in which the DNA is incubated together with the probe in a solution containing 6xSSC (1xSSC has a composition of 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% sodium dodecyl sulfate, 5x Denhardt's buffer, and 100 mg / mL herring sperm DNA at 65°C for 8 to 16 hours to allow hybridization).

3. The Escherichia coli according to claim 1, wherein the rcnR gene is a gene consisting of the following DNA (g) or (h): (g) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 (h) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of the DNA of (g) above and encodes RcnR (wherein the stringent conditions are those in which the DNA is incubated together with the probe in a solution containing 6x SSC (1x SSC has a composition of 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% sodium dodecyl sulfate, 5x Denhardt's buffer, and 100 mg / mL herring sperm DNA at 65°C for 8 to 16 hours to allow hybridization).

4. The Escherichia coli according to claim 1, wherein the zntA gene is a gene consisting of the following DNA (i) or (j): (i) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 (j) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of the DNA of (i) above and encodes ZntA (wherein the stringent conditions are conditions in which the DNA is incubated together with the probe in a solution containing 6xSSC (1xSSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% sodium dodecyl sulfate, 5x Denhardt's, and 100 mg / mL herring sperm DNA at 65°C for 8 to 16 hours to allow hybridization).

5. Escherichia coli in which at least one gene selected from the group consisting of the nikA gene, the nikR gene, and the rcnR gene has been deleted or inactivated; or E. coli in which the nikA gene and one gene selected from the group consisting of the nikR gene, the rcnR gene, and the zntA gene have been deleted or inactivated, 1. An Escherichia coli bioreactor for recovering or removing palladium, wherein:

6. Escherichia coli in which at least one gene selected from the group consisting of the nikA gene, the nikR gene, and the rcnR gene has been deleted or inactivated; or E. coli in which the nikA gene and one gene selected from the group consisting of the nikR gene, the rcnR gene, and the zntA gene have been deleted or inactivated, A method for recovering palladium, comprising contacting the E. coli with a palladium-containing medium, incorporating the palladium contained in the medium into the E. coli cells, and recovering the palladium incorporated into the cells.

7. Escherichia coli in which at least one gene selected from the group consisting of the nikA gene, the nikR gene, and the rcnR gene has been deleted or inactivated; or E. coli in which the nikA gene and one gene selected from the group consisting of the nikR gene, the rcnR gene, and the zntA gene have been deleted or inactivated, A method for removing palladium, comprising contacting a palladium-containing medium with a palladium-containing solvent to remove palladium from the medium.

8. Escherichia coli in which at least one gene selected from the group consisting of the nikA gene, the nikR gene, and the rcnR gene has been deleted or inactivated; or E. coli in which the nikA gene and one gene selected from the group consisting of the nikR gene, the rcnR gene, and the zntA gene have been deleted or inactivated, A method for treating a palladium-containing solution, comprising contacting a metal oxide with a solution containing palladium and removing or recovering palladium from the solution.

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