JP7128527B2 - Metal recovery method, metal recovery carrier, and metal recovery bioreactor using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a metal recovery method, a metal recovery carrier used in the same, and a metal recovery bioreactor using the metal recovery carrier.

Single metals such as transition metals, precious metals, rare earths, Oxides or sulfides of these metals are widely used. Some of these metals are of high rarity value.
Industrial wastewater and domestic wastewater sometimes contain high concentrations of heavy metals or heavy metal compounds that are toxic to the human body. When soil or water quality is polluted by such wastewater, it is necessary to remove elemental metals or metal compounds. On the other hand, rare earths with high scarcity value may be contained in the sludge generated in the treatment process of sewage treatment plants, the waste liquid treatment process of factories, etc., and the sludge of metal magnets.
For these reasons, there is a demand for a method for efficiently recovering elemental metals or metal compounds (these may be collectively referred to simply as "metals" hereinafter) or their ions.

Conventionally, as a method of recovering metals, a method of chemically recovering metals has been common. However, chemical recovery methods for metals require a lot of energy and have a large impact on the environment. Therefore, there is a demand for a metal recovery method that consumes less energy and has less impact on the environment.

BACKGROUND ART In recent years, attention has been focused on a recovery method using microorganisms (biosorption) as a method for recovering metals or their ions that consumes less energy and has less impact on the environment.
For example, Patent Documents 1 and 2 describe a method of introducing a metal uptake gene or metal binding gene into Escherichia coli and incorporating transition metals and heavy metals into the resulting transformant cells. Furthermore, Non-Patent Document 1 describes a method for imparting cadmium-binding ability to E. coli by modifying the E. coli transmembrane protein OmpA.

JP 2004-357566 A JP 2014-239678 A

Protein Engineering, 1998, vol.11(6), p.489-494

However, the methods described in Patent Documents 1 and 2 and Non-Patent Document 1 use genetically modified microorganisms. Such recombinant microorganisms are greatly restricted in their applicability from the viewpoint of safety.
Furthermore, in the methods described in Patent Documents 1 and 2, metals are recovered by accumulating the target metals in the cells of recombinant microorganisms. In this case, it is necessary to keep the microorganisms alive in order to take up the metal ions into the cells, but the toxicity of the taken up metals and the amount of metals exceeding the limit of accumulation can reduce the growth and viability of the recombinant microorganisms. Sometimes.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a metal recovery method that can be used under a wide range of conditions without being subject to restrictions in the applicable range.
Another object of the present invention is to provide a metal recovery carrier that can be suitably used in the metal recovery method described above.

Until now, technology has been developed to concentrate metals by biosorption of microorganisms isolated from the environment. Metal recovery technology by biosorption is expected to be more energy efficient and more efficient than chemical recovery methods. However, since biosorption is a method using microorganisms themselves, the scope of application is limited from the viewpoint of safety.

In view of such circumstances, the present inventors have conducted extensive studies.
First, the present inventors introduced a metal ion-binding peptide motif into a region that does not impair the porin function in the amino acid sequence that constitutes the porin that penetrates the cell membrane of Gram-negative bacteria, and expressed the metal ion-binding peptide motif on the surface layer. A recombinant microorganism was produced. The resulting recombinant microorganisms had binding ability and resistance to metal ions.
Non-living activities such as dead cells of microorganisms that have lost viability produced from such recombinant microorganisms and vesicles that have the same surface structure as the cell membrane (phospholipid bilayer membrane) of the original recombinant microorganisms. It has been found that the body can be suitably used in a metal recovery process without being restricted in its applicability.
The present invention has been completed based on these findings.

The above problems of the present invention have been solved by the following means.
(1) contacting a metal recovery carrier with a medium containing metal ions,
binding the metal ions contained in the medium to the surface of the outer layer of the metal recovery carrier;
separating the metal recovery carrier from the medium;
A metal recovery method comprising:
The metal recovery carrier is an inactive body whose outer layer is composed of a phospholipid bilayer membrane,
Porins derived from Gram-negative bacteria penetrate the phospholipid bilayer membrane,
a metal ion-binding peptide motif is introduced into a region of the amino acid sequence of the porin that does not impair porin function in the Gram-negative bacterium;
wherein the metal ion-binding peptide motif is displayed on the surface of the phospholipid bilayer membrane;
Metal recovery method.
(2) The method for recovering a metal according to item (1), wherein the porin is OmpA, OmpC or OmpF.
(3) the introduction site of the metal ion-binding peptide motif into the porin is any site in the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8 or a region corresponding thereto, and SEQ ID NO: 8; At least one site selected from the group consisting of the region from positions 79 to 95 of the amino acid sequence represented by or any site corresponding to the region, according to (1) or (2) above Metal recovery method.
(4) the introduction site of the metal ion-binding peptide motif into the porin is any site in the region from positions 43 to 53 of the amino acid sequence represented by SEQ ID NO: 9 or a region corresponding thereto, and SEQ ID NO: 9; At least one site selected from the group consisting of the region from positions 339 to 358 of the amino acid sequence represented by or any site corresponding to the region, according to (1) or (2) above Metal recovery method.
(5) The metal recovery method according to any one of (1) to (4) above, wherein the metal recovery carrier is a vesicle.
(6) The metal recovery method according to any one of (1) to (4) above, wherein the carrier for metal recovery is dead cells of Gram-negative bacteria.

(7) an inactive body whose outer layer is composed of a phospholipid bilayer membrane;
Porins derived from Gram-negative bacteria penetrate the phospholipid bilayer membrane,
a metal ion-binding peptide motif is introduced into a region of the amino acid sequence of the porin that does not impair porin function in the Gram-negative bacterium;
wherein the metal ion-binding peptide motif is displayed on the surface of the phospholipid bilayer membrane;
Carrier for metal recovery.
(8) The metal recovery carrier according to item (7), wherein the porin is OmpA, OmpC or OmpF.
(9) the introduction site of the metal ion-binding peptide motif into the porin is any site in the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8 or a region corresponding thereto, and SEQ ID NO: 8; At least one site selected from the group consisting of the region from positions 79 to 95 of the amino acid sequence represented by or any site corresponding to the region, according to (7) or (8) above Carrier for metal recovery.
(10) the introduction site of the metal ion-binding peptide motif into the porin is any site in the region from positions 43 to 53 of the amino acid sequence represented by SEQ ID NO: 9 or a region corresponding thereto, and SEQ ID NO: 9; At least one site selected from the group consisting of the region from positions 339 to 358 of the amino acid sequence represented by or any site corresponding to the region, according to (7) or (8) above Carrier for metal recovery.
(11) The metal recovery carrier according to any one of (7) to (10), which is a vesicle.
(12) The metal recovery carrier according to any one of (7) to (10) above, which is a killed Gram-negative bacterium.
(13) A bioreactor for metal recovery, wherein the carrier for metal recovery according to any one of (7) to (12) is immobilized.

INDUSTRIAL APPLICABILITY The metal recovery method of the present invention can be used under a wide range of conditions without being restricted in its applicable range.
In addition, the carrier for metal recovery of the present invention is not limited in its applicable range, and can be suitably used in the metal recovery method described above.
The above and other features and advantages of the present invention will become more apparent from the following description, with reference where appropriate to the accompanying drawings.

FIG. 1(A) is a schematic diagram showing a preferred embodiment of the metal recovery carrier or its precursor of the present invention, and FIG. 1(B) is a schematic diagram showing another preferred embodiment of the metal recovery carrier of the present invention. It is a diagram. FIG. 2 shows the results of alignment analysis of the amino acid sequences of OmpA, OmpC and OmpF between bacterial strains and between various porins. FIG. 2 is a diagram showing an operational flow of genome editing using the CRISPR-Cas9 system. Fig. 2 is a plasmid map of pEX-A2-sgRNA prepared as a general-purpose vector for guide RNA cloning. 1 is a diagram showing insertion positions (▴ marks) of metal ion-binding peptide motif genes into the ompA gene in transformants prepared in Test Example 1. FIG. Schematic diagram showing a method for producing a DNA fragment encoding a Cu ion-binding peptide motif. Schematic diagram showing a method for generating a DNA fragment encoding a Tb ion-binding peptide motif. Schematic diagram showing a method for generating a DNA fragment encoding a Tb ion-binding peptide motif. FIG. 2 is a schematic diagram showing the structures of OmpA of transformants YM0501, YM0901 and YM1001 prepared in Examples. FIG. 2 is a schematic diagram showing the structures of OmpA of transformants YM0701, YM1101 and YM1201 produced in Examples. 1 is a graph showing the growth ability and growth ability of transformants YM0501, YM0901 and YM1001 produced in Examples. Fig. 2 is a graph showing the growth ability and growth ability of transformants YM0701, YM1101 and YM1201 produced in Examples. 1 is a graph showing the ability of the transformant YM0501 produced in Examples and its parent strain to proliferate in the presence of metal ions. FIG. 14(A) is a drawing-substituting photograph showing how the transformant YM0501 prepared in Example was stained with FM4-64, and FIG. 14(B) is a vesicle prepared from the transformant YM0501 with FM4-64. It is a drawing substitute photograph which shows the state dyed. FIG. 2 shows the results of alignment analysis of OmpA amino acid sequences between bacterial strains and various porins. FIG. 10 is a diagram showing the results of alignment analysis of OmpC amino acid sequences between strains and various porins. 1 is a plasmid map of the ompC gene expression plasmid prepared in Examples. FIG. 18 is a diagram showing the insertion position (▴ mark) of the metal ion-binding peptide motif gene into the ompC gene in the transformant prepared in Test Example 4. FIG. FIG. 19(A) is a plasmid map of the plasmid pBADompC1-TBpep2 prepared in Test Example 4. FIG. 19(B) is a plasmid map of the plasmid pBADompC18-TBpep2 prepared in Test Example 4. FIG. FIG. 20(A) is a schematic diagram showing the structure of OmpC of a transformant containing the plasmid pBADompC1-TBpep2 produced in Test Example 4. FIG. FIG. 20(B) is a schematic diagram showing the structure of OmpC of the transformant containing the plasmid pBADompC18-TBpep2 produced in Test Example 4. FIG. FIG. 21(A) is a drawing-substituting photograph showing the results of SDS-PAGE performed using the outer membrane fraction of the transformant produced in Test Example 4. FIG. 21(B) is a drawing-substituting photograph showing the results of Western blotting performed using the outer membrane fraction of the transformant produced in Test Example 4. FIG.

As used herein, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, vol.227, p.1435-1441). Specifically, it is calculated by performing analysis using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win with a Unit size to compare (ktup) of 2.

The metal recovery carrier of the present invention (hereinafter also simply referred to as "carrier") is an inactive body having a phospholipid bilayer membrane, and the outer layer of the carrier is composed of the phospholipid bilayer membrane. Gram-negative bacterium-derived porins (also referred to as “transmembrane proteins”) penetrate the phospholipid bilayer membrane. Furthermore, via this porin, a metal ion-binding peptide motif is presented on the surface of the phospholipid bilayer membrane. In the carrier of the present invention, the metal ion-binding peptide motif is introduced into a region of the porin amino acid sequence that does not impair the porin function in Gram-negative bacteria and has low amino acid sequence conservation among bacterial species.
As used herein, the term "non-vital body" refers to a substance that does not have proliferative and metabolic capacity. Specific examples include dead cells of microorganisms that have lost viability, and vesicles (liposomes) that have a closed structure with a lipid bilayer membrane and do not contain DNA inside.

Conventional microorganisms, especially recombinant microorganisms, used for biosorption are greatly restricted in their applicability from the viewpoint of safety. On the other hand, since the metal recovery carrier of the present invention is an inanimate object, the scope of application is not limited, and it is easy to use industrially.
Furthermore, conventional microorganisms used for biosorption sometimes have reduced proliferative ability and growth ability due to the accumulation of metals in their cells. On the other hand, in the microorganism that serves as the precursor of the metal recovery carrier of the present invention, a metal ion-binding peptide motif is introduced into a site that does not affect the function of the microorganism (porin function). Therefore, the function as a microorganism is not impaired, and the ability to proliferate and grow can be maintained. Furthermore, since it is not necessary to place the microorganism, which is a precursor of the carrier for metal recovery of the present invention, in an environment in which metal ions are present, the microorganism can be grown in an environment favorable for growth.

As used herein, the term "metal" includes elemental metals and metal compounds.
The single metal is not particularly limited, but includes transition metals, noble metals, alkali metals, alkaline earth metals, and semimetals. Specifically, Sc (scandium), Ti (titanium), V (vanadium), Cr (chromium), Mn (manganese), Fe (iron), Co (cobalt), Ni (nickel), Cu (copper), Zn (zinc), Y (yttrium), Zr (zirconium), Nb (niobium), Mo (molybdenum), Tc (technetium), Ru (ruthenium), Rh (rhodium), Pd (palladium), Ag (silver), Cd (cadmium), La (lanthanum), Ce (cerium), Pr (praseodymium), Nd (neodymium), Pm (promethium), Sm (samarium), Eu (europium), Gd (gadolinium), Tb (terbium), Dy (dysprosium), Ho (holmium), Er (erbium), Tm (thulium), Yb (ytterbium), Lu (lutetium), Hf (hafnium), Ta (tantalum), W (tungsten), Re (rhenium), Os (osmium), Ir (iridium), Pt (platinum), Au (gold), Hg (mercury), Na (sodium), K (potassium), Mg (magnesium), As (arsenic).
The metal compounds include oxides, sulfides, oxoanions, and salts thereof of the single metals.

Proteins that have specific binding properties for various metal ions are usually functionally classified into metalloenzymes, metal chaperones, and metal sensors (Nature, 2009, vol. 460(7257), p. 823 -830). Of these, metal chaperones and metal sensors have peptide motifs that are widely conserved in living organisms and specifically bind to metal ions (Chem. Biol., 2002, vol. 9(6), p. 673-677; Science, 2003, vol. 301(5638), p. 1383-1387; Science, 2003, vol. 301(5638), p. 33391; Proc. Natl. Acad. Sci. USA, 2011, vol. 108, p. The carrier of the present invention appropriately selects and utilizes such metal ion-binding peptide motifs according to the target metal. The metal ion-binding peptide motif used in the present invention preferably consists of 20-30 residues.
Specific examples of metal ion-binding peptide motifs that can be used in the present invention are shown in Table 1 below. However, the present invention is not limited to these.

"Porin" in the present invention is a general term for proteins that form permeable pores (diffusion channels) present in the outer membrane of Gram-negative bacteria. Porins are rich in β-sheet structures and have a cytoplasmic β-sheet and a loop connecting the β-sheets. The β-sheet is antiparallel and has a cylindrical β-barrel structure with nonpolar groups on the outer surface and polar groups on the inside. Such structured porins penetrate phospholipid bilayer membranes such as the outer membrane of Gram-negative bacteria.
Porins that can be used in the present invention can be appropriately selected from various porins derived from Gram-negative bacteria. Specific examples include OmpA, OmpC, OmpF, PhoE, LamB, OprD2, OprE1. Among these, OmpA, OmpC, or OmpF is preferred, and OmpA, which is constantly expressed on the surface of the cell membrane regardless of osmotic conditions, is more preferred.

The aforementioned metal ion-binding peptide motif is introduced into a predetermined site of the porin by a conventional method.
In the present invention, the metal ion-binding peptide motif may be introduced into the porin by inserting or adding the metal ion-binding peptide motif to a predetermined site of the porin. Alternatively, all or part of the amino acid residues in a given region of porin may be replaced with a metal ion-binding peptide motif.

In the present invention, a metal ion-binding peptide motif is introduced into a specific site of porin that does not impair porin functions. Here, "does not impair the function of porin" means that the three-dimensional structure of porin is maintained and the function as a diffusion channel is maintained. Whether or not the three-dimensional structure of porin is maintained and the function as a diffusion channel is maintained can be determined by amino acid sequence alignment and measurement of proliferative ability and growth ability.
In the present invention, the site into which the metal ion-binding peptide motif is introduced is preferably the site corresponding to the extracellular domain in Gram-negative bacteria in the porin amino acid sequence. Furthermore, in the present invention, it is preferable to introduce a metal ion-binding peptide motif into a region with low amino acid sequence conservation among bacterial species and among various porins. Regions with low amino acid sequence conservation among bacterial strains and among various porins can be determined by alignment according to a conventional method.

A method for determining the introduction site of the metal ion-binding peptide motif in the porin amino acid sequence will be specifically described based on preferred embodiments. However, the invention is not so limited.
First, the amino acid sequence of the extracellular domain of the porin used in the present invention is specified from various databases containing information on the amino acid sequences of various porins, and a continuous region of about 10 to 20 residues is selected from the specified amino acid sequence. do. Next, the amino acid sequences of the selected regions are aligned between different strains of bacteria and between various porins. Then, in the aligned region, the number of identical or similar amino acid residues is 3 or less, preferably 2 or less, more preferably 1 or less, more preferably 0 between different bacterial strains or between various porins. A region can be determined as the introduction site for the metallopeptide motif. Alternatively, the amino acid sequence homology of the region selected as the extracellular domain (including substitution with similar amino acid residues) is 25% or less, preferably 17% or less, between different bacterial strains or between various porins. , more preferably 9% or less, more preferably 0%, can be determined as the introduction site for the metallopeptide motif.
In this specification, the similarity of amino acid residues is judged from the polarity (non-polarity, positive charge, negative charge) of the side chains of the amino acid residues. Then, residues having the same side chain polarity are classified as "similar amino acid residues".

In the present invention, the introduction site of the metal ion-binding peptide motif is the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8 or any site in the region corresponding to this, the amino acid represented by SEQ ID NO: 8 any part of the region from positions 79 to 95 of the sequence or the region corresponding thereto, any part of the region from positions 125 to 137 of the amino acid sequence represented by SEQ ID NO: 8 or the region corresponding to this, and the sequence It is preferably at least one site selected from the group consisting of the region from position 164 to 181 of the amino acid sequence represented by number 8 or any site of the region corresponding to this, represented by SEQ ID NO: 8 any part of the region from positions 38 to 54 of the amino acid sequence or the region corresponding thereto, and any part of the region from positions 79 to 95 of the amino acid sequence represented by SEQ ID NO: 8 or the region corresponding to this, At least one site selected from the group consisting of is more preferable. Here, “the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8” is the extracellular region 1 of OmpA derived from E. coli, which will be described later. In addition, “the region from positions 79 to 95 of the amino acid sequence represented by SEQ ID NO: 8” is the extracellular domain 2 of OmpA derived from E. coli to be described later. In addition, the “region from position 125 to position 137 of the amino acid sequence represented by SEQ ID NO: 8” is the extracellular region 3 of OmpA derived from E. coli to be described later. Further, “the region from positions 164 to 181 of the amino acid sequence represented by SEQ ID NO: 8” is the extracellular region 4 of OmpA derived from E. coli to be described later. (See FIGS. 2, 9, 10 and 15 above.)
Alternatively, the introduction site of the metal ion-binding peptide motif is any site in the region from positions 43 to 53 of the amino acid sequence represented by SEQ ID NO: 9 or a region corresponding to this, or the amino acid sequence represented by SEQ ID NO: 9. Any portion of the region from position 85 to 91 or the region corresponding thereto, any region from position 116 to 141 of the amino acid sequence represented by SEQ ID NO: 9 or the region corresponding to this, SEQ ID NO: 9 Any part of the region from position 172 to 200 of the amino acid sequence represented or the region corresponding thereto, any region from position 220 to 241 of the amino acid sequence represented by SEQ ID NO: 9 or the region corresponding to this site, the region from position 258 to 269 of the amino acid sequence represented by SEQ ID NO: 9 or any region corresponding to this, the region from position 300 to 318 of the amino acid sequence represented by SEQ ID NO: 9 or this At least one site selected from the group consisting of any site of the corresponding region, and any site of the region from positions 339 to 358 of the amino acid sequence represented by SEQ ID NO: 9 or the region corresponding thereto, good too. In the present invention, the region from positions 43 to 53 of the amino acid sequence represented by SEQ ID NO: 9 or any region corresponding to this, and positions 339 to 358 of the amino acid sequence represented by SEQ ID NO: 9 More preferably, it is at least one site selected from the group consisting of the region or any site corresponding to the region. Here, “the region from positions 43 to 53 of the amino acid sequence represented by SEQ ID NO: 9” is extracellular domain 1 of E. coli-derived OmpC described later. In addition, “the region from positions 85 to 91 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 2 of OmpC derived from E. coli, which will be described later. In addition, “the region from positions 116 to 141 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 3 of E. coli-derived OmpC described later. In addition, “the region from positions 172 to 200 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 4 of E. coli-derived OmpC described later. In addition, “the region from positions 220 to 241 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 5 of OmpC derived from Escherichia coli to be described later. In addition, “the region from positions 258 to 269 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 6 of OmpC derived from E. coli, which will be described later. In addition, “the region from positions 300 to 318 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 7 of E. coli-derived OmpC described later. Further, “the region from positions 339 to 358 of the amino acid sequence represented by SEQ ID NO: 9” is the extracellular region 8 of E. coli-derived OmpC described later. (Refer to FIGS. 2, 16, 20 (A) and (B) above.)
In the present invention, the introduction site of the metal ion-binding peptide motif may be one site or two or more sites.
As shown in the examples below, each of the above regions has low amino acid sequence conservation among bacterial species and among various porins. Moreover, introduction of metal ion-binding peptide motifs into these regions does not impair porin function. Metal ion-binding peptide motifs introduced into these regions are then displayed on the surface of the phospholipid bilayer membrane.
As used herein, the term "corresponding region" refers to comparing the target amino acid sequence with a reference sequence and aligning the sequences so as to give maximum homology to the conserved amino acid residues present in each amino acid sequence. can be determined by Alignments can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, the Clustal W multiple alignment program (Nucleic Acids Res., 1994, Vol. 22, p. 4673-4680) defaults, although alignments can be performed manually, such as by the Lippmann-Person method described above. This can be done by using Clustal W, for example, European Bioinformatics Institute (EBI, [www.ebi.ac.uk/index.html]) and the Japan DNA Data Bank (DDBJ, [ www.ddbj.nig.ac.jp/Welcome-j.html]).

A microorganism that serves as a precursor of the carrier of the present invention can be produced by genetic engineering techniques. For example, a host microorganism is transformed so that a gene encoding a metal ion-binding peptide motif (hereinafter referred to as "metal ion-binding peptide motif gene") is introduced into a predetermined site of porin-encoding DNA. Then, the target precursor is obtained by expressing the porin into which the metal ion-binding peptide motif has been introduced in the resulting transformant. In the present specification, precursors obtained by such methods are also referred to as recombinant microorganisms. There are no particular restrictions on the method of producing recombinant microorganisms by introducing metal ion-binding peptide motifs into porins by genetic engineering techniques, see Appl. Environ. Microbiol., 2015, vol. 81(7), p. 2506- 2514; Protein Eng., 1998, vol. 11(6), p. 489-494; Appl. Biochem. Biotechnol., 2013, vol. 169(4), p. 2011, vol. 165(7-8), p. 1674-1681; Biotechnol. Appl. Biochem., 2004, vol. 40, p. 209-228.

Host microorganisms for producing the recombinant microorganisms are not particularly limited, but Gram-negative bacteria are preferable, and bacteria described in Microbiology, 2014, vol. 160, p. 2109-2121 are more preferable.
Gram-negative bacteria that can be preferably used in the present invention include Escherichia bacteria, Shigella bacteria, Salmonella bacteria, Neisseria bacteria, Legionella bacteria, Acinetobacter , Helicobacter , Francisella , Edwardsiella , Pseudomonas , Staphylococcus , Klebsiella , bacteria of the genus Porphyromonas , bacteria of the genus Campylobacter , bacteria of the genus Bacteroides , and bacteria of the genus Vibrio . Specific examples include Escherichia coli, Shigella flexneri , Salmonella enterica , Neisseria meningitidis , Legionella pneumophila , Acinetobacter baumannii , and Helicobacter pylori. Helicobacter pylori , Myxococcus xanthus , Francisella philomiragia , Francisella tularensis , Edwardsiella tarda , Pseudomonas aeruginosa , Staphylococcus aureus ), Klebsiella pneumoniae , Pseudomonas syringae , Francisella novicida , Neisseria gonorrhoeae , Porphyromonas gingivalis , Campylobacter jejuni , Bacteroides fragilis , Bacteroides thetaiotaomicron , and Vibrio cholerae . Among these, bacteria belonging to the genus Escherichia, bacteria belonging to the genus Shigella, and bacteria belonging to the genus Salmonella are preferred, Escherichia coli, Shigella flexneri and Salmonella are more preferred, and Escherichia coli is even more preferred.
As shown in the examples below, the recombinant microorganism has excellent specificity and binding ability to specific metals, and maintains cell homeostasis.

The precursor produced as described above can be suitably used for production of the carrier of the present invention. Furthermore, if the genetically modified microorganism that is the precursor maintains the ability to proliferate, it can continue to proliferate, and the carrier of the present invention can be produced in large amounts.

Preferable forms of the carrier of the present invention include genomic DNA prepared from the above-mentioned precursors by conventional methods, and vesicles that have lost the characteristics of microorganisms such as proliferation ability and metabolic ability. As used herein, the term "vesicle" refers to a vesicle having a structure closed by a phospholipid bilayer membrane. Vesicles do not carry genetic information and are not genetically modified microorganisms, which is a major limitation in industrial applications. Therefore, even in environments and conditions where the above-mentioned recombinant microorganisms cannot be applied from the viewpoint of safety, outer membrane vesicles, which are non-biological materials, can be used for metal recovery and removal, etc. is easy to use.
1(A) and (B), the vesicle 20 is produced from the outer membrane 1 of the recombinant microorganism 10 as shown in FIG. 1(A) and contains no DNA inside. It is a vesicle (vesicle) with a membrane structure. Then, on the surface of the outer layer of the vesicle 20 (outer membrane 1 composed of a phospholipid bilayer membrane), metal ions introduced into the amino acid sequence of the porin 3 are present on the surface of the outer membrane 1 of the recombinant microorganism 10 . Binding peptide motif 4 is presented.

The vesicle of the present invention can be produced from a recombinant microorganism having a metal ion-binding peptide motif produced by the above-described method displayed on the surface of the cell membrane (phospholipid bilayer membrane). Specifically, the step of producing a recombinant microorganism 10 in which the metal ion-binding peptide motif 4 is displayed on the surface of the cell membrane (outer membrane 1 composed of a phospholipid bilayer) by the above-described method; to produce vesicles 20, centrifugation of the culture solution, removal of recombinant microorganisms by filter filtration, separation of vesicles in the culture solution by ultracentrifugation, and concentration steps. As a method for producing the vesicles of the present invention, PLos ONE, 2016, vol. 11(12), e0169186; J. Bacteriol., 1995, vol. 177(14), p. 3998-4008; Annu. Rev. Microbiol ., 2010, vol. 64, p. 163-184; J. Bacteriol., 1998, vol. 180(18), p. 4872-4878; Proteomics., 2014, vol. 222-229, etc. can be referred to.

Another preferred form of the carrier of the present invention is a killed cell produced by killing the precursor (recombinant microorganism 10) described above with reference to FIG. 1(A). From the viewpoint of safety, there are few restrictions on the use of killed cells, and they are easy to use industrially. Killed cells can be prepared by autoclave sterilization of precursor microorganisms according to a conventional method.

The metal can be recovered by contacting the carrier of the present invention with a medium containing the metal or its ions, binding the metal ions contained in the medium to the surface of the carrier of the present invention, and separating the carrier of the present invention.
There is no particular limitation on the method for extracting the metal or its ions bound to the surface of the carrier of the present invention. For example, denaturation of the secondary structure of metal ion-binding peptide motifs by high temperature (approximately 100°C) treatment, metal ion dissociation from metal ion-binding peptide motifs by high salt concentration (several M) solution treatment, irreversible metal ion-binding peptides. It can be carried out by decomposing the motif, or the like. Metals recovered from media such as natural water environments such as rivers and seas, urban mines, aqueous solutions of metal magnet sludge dissolved in acid, and dispersions of metal-contaminated soil, etc. can be reused as catalysts used in petroleum refining processes, active ingredients in preservatives, industrial oil additives, electrical and electronic equipment, and antibacterial agents used in the medical field. The carrier after separation from the metal can be reused if the structure of the metal ion-binding peptide motif is not completely destroyed.
Moreover, by the above method, metals contained in environmental water and soil contaminated with metals can be removed, making it possible to purify water and soil.

By immobilizing the carrier of the present invention on a suitable support or the like, it is possible to provide a bioreactor derived from microbial materials (for example, a column or film for recovering or removing metals, a metal recovering or removing agent). can. The support that can be used in the present invention is not particularly limited, and includes substances that are insoluble in water or specific solvents. Examples thereof include foams or resins such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, silicone foam, and cellulose porous body. As used herein, the term "immobilized" refers to a state in which the carrier of the present invention is bound or attached to a support, or incorporated in a support, rather than in a free state.

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

Test Example 1 Production of Recombinant Escherichia coli Presenting Metal Ion-Binding Peptide Motifs on the Surface of the Outer Cell (1) Examination of Target Regions in Escherichia coli Outer Cells Showing Metal Ion-Binding Peptide Motifs To present the motifs, we focused on proteins localized in the outermost layer of cells.
The phospholipid biomembranes surrounding Gram-negative bacteria such as E. coli are double-layered, the outer membrane being called the outer membrane and the inner membrane being called the inner membrane. Three types of porins (OmpA (SEQ ID NO: 8), OmpC (SEQ ID NO: 9), and OmpF (SEQ ID NO: 10)) are mainly localized in the outer membrane of E. coli. Among them, OmpA is constitutively expressed in Gram-negative bacteria and is a major outer membrane protein. Escherichia coli-derived OmpA has eight transmembrane domains, resulting in four extracellular domains that are displayed outside the cell. The four extracellular regions are hereinafter referred to as "Extracellular-1", "Extracellular-2", "Extracellular-3", and "Extracellular region", respectively. (Extracellular-4)” (see Figures 2 and 15).

OmpA, OmpC and OmpF from E. coli, OmpA (SEQ ID NO: 32), OmpC (SEQ ID NO: 33) and OmpF (SEQ ID NO: 34) from Shigella flexneri, and OmpA from Salmonella (SEQ ID NO: 35) made by ClustalW , OmpC (SEQ ID NO: 36) and OmpF (SEQ ID NO: 37), it was found that there are no identical or similar amino acid residues in extracellular domain 1 (see FIG. 2). ). Moreover, there were no identical amino acid residues in extracellular domain 2, and only one similar amino acid residue was present (see FIG. 2).
On the other hand, there were two identical amino acid residues and one similar amino acid residue in each of the extracellular regions 3 and 4 (see FIG. 2).

Based on the above results, it was considered that extracellular domain 1 and extracellular domain 2, which have extremely low amino acid sequence conservation among fungal species and among various porins, are not important for porin function, preferably OmpA function. This region was then targeted for insertion of metal ion-binding peptide motifs.

(2) General-purpose vector construction for guide RNA cloning in CRISPR-Cas system First, we applied the genome editing method using the CRISPR-Cas9 system constructed by Jiang et al. (Appl. Environ. Microbiol., 2015, vol. 81 ( 7), p. 2506-2514) (see Figure 3).
In this system, first, the temperature-sensitive plasmid pCas, which encodes Cas9 endonuclease from Streptococcus pyogenes , λ-Red recombinase (arabinose induction), and a guide RNA targeting the replication origin pMB1 (IPTG induction), was introduced into E. coli. Introduce. Next, by introducing a plasmid that expresses a target gene-specific guide RNA into E. coli into which pCas has been introduced, Cas9 expressed in the cell and the plasmid that expresses the target gene-specific guide RNA form a complex. and cleaves DNA double-strands at target locations on the genome, causing cell death. However, when a guide RNA expression plasmid is introduced, a template DNA fragment for which recombination is to be introduced, and cultured in the presence of arabinose, the action of λ-Red recombinase whose expression is induced by arabinose results in Homologous recombination occurs between the DNA cleaved at the target position and the template DNA fragment. As a result, an E. coli cell whose genome has been repaired and the template DNA fragment has been integrated onto the genome can be obtained.
In addition, pCas encodes a pMB1 replication origin-specific sgRNA that is induced by IPTG. A typical guide RNA expression plasmid is cleaved and removed by Cas9.
Furthermore, since pCas has a temperature-sensitive replication origin, the system can be easily removed by raising the culture temperature to 37°C or 42°C and culturing transformants.

First, for the construction of a guide RNA expression plasmid, a vector incorporating the designed crRNA sequence was constructed. With reference to the tracrRNA sequence and guide RNA constitutive expression promoter sequence (Cell, 2013, vol. 152(5), p. 1173-1183 ) by Qi et al. pEX-A2-sgRNA was constructed by inserting the sequence into which the recognition site of was inserted into the pEX-A2 vector using the artificial gene synthesis service of Eurofins Genomics (SEQ ID NO: 11, see FIG. 4).
In the nucleotide sequence represented by SEQ ID NO: 11, the nucleotide sequence at positions 86-147 is "tracrRNA", the nucleotide sequence at positions 155-227 is "Pj23119 promoter", and the nucleotide sequence at positions 255-370 is The nucleotide sequence at positions 587-1260 is "pMB1 ori", and the nucleotide sequence at positions 1405-2266 is "bla".

(3) Specific cleavage of E. coli outer membrane protein non-conserved region using genome editing (SEQ ID NO: 12, 5'- CCGCAGCGGTTTCAG TGTTGATGAAACCAGTGTCA GTTTTAGAGCTAGAA -3', underlined is the sequence homologous to pEX-A2-sgRNA) and its complementary oligonucleotide ompA1_sgRNA_com (SEQ ID NO: 13, 5'- TTCTAGCTCTAAAAC TGACACTGGTTTCATCAACA CTGAAACCGCTGCGG -3', underlined) is a sequence homologous to pEX-A2-sgRNA) was annealed to form a double-stranded DNA fragment, and psgRNA -ompA1 -14 was constructed (see Figure 5).
Similarly, to construct a guide RNA expression plasmid with a crRNA sequence targeting the extracellular region 2 of OmpA, oligonucleotide ompA2_sgRNA_N20 (SEQ ID NO: 14, 5′- CCGCAGCGGTTTCAG TGTTGATGAAACCAGTGTCA GTTTTAGAGCTAGAA -3′, underlined pEX-A2- sgRNA) and its complementary oligonucleotide ompA2_sgRNA_com (SEQ ID NO: 15, 5'- TTCTAGCTCTAAAAC TTGTACGGCATACGACCTAA CTGAAACCGCTGCGG -3', underlined is the homologous sequence to pEX-A2-sgRNA) to form a double-stranded DNA fragment. , Not I-cleaved pEX-A2-sgRNA was used to construct psgRNA-ompA2-29 using the In-Fusion HD Cloning kit (manufactured by Takara bio) (see Fig. 5).

psgRNA-ompA1-14 (ampicillin resistance) or psgRNA-ompA2-29 (ampicillin resistance) was introduced into E. coli W3110 typeA into which pCas (kanamycin resistance) was introduced. However, E. coli transformants with ampicillin resistance could not be obtained.
These results confirmed that the psgRNA-ompA1-14 and psgRNA-ompA2-29 cleave their respective target ompA gene sites on the genome in a Cas9-dependent manner.

(4) Introduction of Cu ion-binding peptide motif into non-conserved region of Escherichia coli outer membrane protein We focused on the Cu-binding motif (SEQ ID NO: 4) of the Escherichia coli transcription factor CueR as a Cu ion-binding peptide motif displayed on the surface of the outer membrane.
The Cu ion-binding peptide motif of CueR is composed of 21 amino acid residues ( ₂ HN-CPGDDSADCPILENLSGCCHH-COOH, SEQ ID NO: 4) (Science, 2003, vol. 301(5638), p. 1383-1387), A sequence of 27 amino acid residues with 3 glycine residues added to each end was introduced into OmpA. The details are shown below.

ompA1_cueRpep_F (SEQ ID NO: 17, 5′- TAAACTGGGCTGGTCCCAGTACCATGACACTGGTTTCATC GGCGGCGGCTGCCCTGGCGATGAC-3′, underlined corresponds to the extracellular region 1 of the ompA gene on the genome, using the pBAD33_cueR plasmid (SEQ ID NO: 16, see FIG. 6) into which the CueR gene is cloned as a template. KOD-Plus-Neo with primers of ompA1_cueRpep3_R (SEQ ID NO: 18, 5'- CAGCGCCCAGTTGGTTTTCATGGGTCGGGCCATTGTTGTT GCCGCCGCCATGATGACAGCCAGCC-3', the underline is homologous to the sequence corresponding to the extracellular region 1 of the ompA gene on the genome) as primers. Toyobo) was performed. By this operation, a DNA fragment encoding a 161 bp Cu ion-binding peptide motif (hereinafter referred to as "ompA1cueRpep3") that can be recombined into the OmpA extracellular region 1 was amplified (see FIG. 6). Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
In the nucleotide sequence represented by SEQ ID NO: 16, the nucleotide sequence at positions 96-974 is "araC", the nucleotide sequence at positions 1004-1019 is "araO2", and the nucleotide sequence at positions 1161-1172 is The nucleotide sequence at positions 1203-1216 is " CAP_BS ", the nucleotide sequence at positions 1213-1251 is "AraI1I2", the nucleotide sequence at positions 1248-1276 is " ARA_promoter ", and the nucleotide sequence at positions 1248-1276 is "ARA_promoter". The nucleotide sequence at position 1760 is " cueR ", the nucleotide sequence at positions 1761-1784 is "FLAG tag", the nucleotide sequence at positions 1878-2035 is "rrnB_terminator", and the nucleotide sequence at positions 2001-2044 is " rrnB_T1_terminator”, the nucleotide sequence at positions 2176-2203 is “rrnB_T2_terminator”, the nucleotide sequence at positions 2244-2272 is “ bla_promoter ”, and the nucleotide sequence at positions 2441-2725 is “ bla (576-860)”. The nucleotide sequence at positions 2784-3083 is "f1_origin", the nucleotide sequence at positions 3777-4436 is "CAT/CamR", and the nucleotide sequence at positions 4798-5637 is "p15A_origin".

Similarly, ompA2_cueRpep_F (SEQ ID NO: 19, 5'- AATGGGTTACGACTGGTTAGGTCGTATGCCGTACAAAGGC GGCGGCGGCTGCCCTGGCGATGAC-3', underlined is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome) and ompA2_cueRpep3_R (SEQ ID NO: 20, 5'-GTTGAACGCCCTGAGCTTTGTATGCACCGTTTCGTCAAGCCCGACTGCACCGTTTCGTCAAGCCATGACGCT 3', the underlined sequence is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome), and a 161 bp Cu ion-binding peptide motif DNA fragment that can be recombined into the extracellular region 2 of OmpA ( hereinafter referred to as "ompA2cueRpep3") was amplified (see FIG. 6). Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).

Next, electroporation competent cells of E. coli W3110 type A having pCas (J. Bacteriol., 1997, vol. 179(3), p. 959-963) were prepared. Transformation was then performed using a guide RNA expression plasmid psgRNA-ompA1-14 for specific cleavage of the extracellular region 1 of the ompA gene and a DNA fragment for recombination ompA1cueRpep3. It was applied to an LB agar medium (10 g/L tryptone (Difco), 5 g/L yeast extract (Difco), 171 mM NaCl, 15 g/L agar) and cultured overnight at 30°C to obtain transformant YM0501.
YM0501 showed ampicillin sensitivity. A primer set ompA_F (SEQ ID NO: 21, 5'-GCAGATCCCCCGGTGAAGGA-3') and ompA1_cueRpep3_R (SEQ ID NO: 18) that specifically amplifies only the recombinant ompA gene using genomic DNA extracted from this ampicillin-sensitive strain as a template. PCR was performed using As a result, while no amplification was observed in W3110 typeA, amplification of the target fragment was observed in YM0501.

Similarly, transformation was performed using a guide RNA expression plasmid psgRNA-ompA2-29 for extracellular region 2-specific cleavage of the ompA gene and a DNA fragment for recombination ompA2cueRpep3. It was plated on an LB agar medium and cultured overnight at 30°C to obtain transformant YM0701.

(5) Introduction of Tb ion-binding peptide motif into E. coli outer membrane protein non-conserved region Ca ion-binding EF-hand motif as a Tb ion-binding peptide motif displayed on the outer membrane surface (Trends Biochem. Sci., 1996, vol. 21, p. 14-17)-derived mutant (SEQ ID NO: 6) (Chembiochem., 2003, vol. 4(4), p. 272-276).
on both sides of a mutated Tb ion-binding peptide motif ( ₂ HN-YIDTNNDGWYEGDELLA-COOH, SEQ ID NO: 6) consisting of 17 amino acid residues (Chembiochem., 2003, vol. 4(4), p. 272-276) A sequence of 23 amino acid residues, each with 3 additional glycine residues, was introduced into OmpA. The details are shown below.

ompA1_TbBpep1_F (SEQ ID NO: 23, 5′- TAAAACTGGGCTGGTCCCAGTACCATGACACTGGTTTCATCGGCGGCGGC TATATTGATACCAAC-3′, underlined using the pGEX-4T-1-TbB-peptide1 plasmid (SEQ ID NO: 22, see FIG. 7) into which the Tb ion-binding peptide motif gene is cloned as a template. homologous to the sequence corresponding to the extracellular region 1 of the ompA gene on the genome) and ompA1_TbBpep1_R (SEQ ID NO: 24, 5'- CAGCGCCCAGTTGGTTTTCATGGGTCGGGCCATTGTTGTTGCCGCCGCCCGCCAACAATTCATC -3', underlined with the sequence corresponding to the extracellular region 1 of the ompA gene on the genome) (homologous) as primers and KOD-Plus-Neo (Toyobo) was used for PCR. By this operation, a DNA fragment encoding a 149 bp Tb ion-binding peptide motif (hereinafter referred to as "ompA1TbBpep1") that can be recombined into the extracellular region 1 of OmpA was amplified (see FIG. 7). Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
In the nucleotide sequence represented by SEQ ID NO: 22, the nucleotide sequence at positions 184-212 is " tac promoter ", the nucleotide sequence at positions 258-944 is "GST-a", and the nucleotide sequence at positions 945-953 is "GST-a". The sequence is " Glycine ", the nucleotide sequence at positions 954-1004 is "TbB-peptide1", the nucleotide sequence at positions 1005-1007 is "Glycine", and the nucleotide sequence at positions 1008-1026 is "GST-b , the nucleotide sequence at positions 1422-2282 is "bla", the nucleotide sequence at positions 2438-3056 is "pBR322 ori", and the nucleotide sequence at positions 3354-4445 is "lacI".

Similarly, ompA2_TbBpep1_F (SEQ ID NO: 25, 5'- AATGGGTTACGACTGGTTAGGTCGTATGCCGTACAAAGGCGGCGGCGGC TATATTGATACCAAC-3', underlined is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome) and ompA2_TbBpep1_R (SEQ ID NO: 26, 5'- GTTTGAACGCCCTGAGCTTTGTATGCACCGTTTTCAACGCTGCACCGTTTTCAACGCTGCACCGTTTTCAACGCTGCCAT3 ', the underlined sequence is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome), and a 149 bp Tb ion-binding peptide motif DNA fragment that can be recombined into the extracellular region 2 of OmpA (hereinafter , termed “ompA2TbBpep1”) was amplified (see FIG. 7). Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).

Next, in the same manner as described above, E. coli W3110 typeA carrying pCas (J. Bacteriol., 1997, vol. 179(3), p. 959-963) was electroporated to extract the extracellular region of the ompA gene. Transformation was performed using the guide RNA expression plasmid psgRNA-ompA1-14 for specific cleavage of 1 and the DNA fragment ompA1TbBpep1 for recombination to obtain transformant YM0901.
Similarly, transformation was performed using a guide RNA expression plasmid psgRNA-ompA2-29 for specific cleavage of the extracellular region 2 of the ompA gene and a DNA fragment for recombination ompA2TbBpep1 to obtain transformant YM1101.

Furthermore, as a Tb ion-binding peptide motif different from the Tb ion-binding peptide motif, a Tb ion-binding peptide motif with a different mutation consisting of 17 amino acid residues ( ₂ HN-ACVDWNNDGWYEGDECA-COOH, SEQ ID NO: 7) (Chembiochem., 2003, vol. 4(4), p. 272-276), a sequence of 23 amino acid residues with three glycine residues added to each end, was introduced into OmpA.

ompA1_TbBpep2_F (SEQ ID NO: 28, 5'- TAAACTGGGCTGGTCCCAGTACCATGACACTGGTTTCATCGGCGGCGGCGCGTGCGTGGATTGG -3', underlined) using the pGEX-4T-1-TbB-peptide2 plasmid (SEQ ID NO: 27, see FIG. 8) into which this Tb ion-binding peptide motif gene is cloned as a template. is homologous to the sequence corresponding to the extracellular region 1 of the ompA gene on the genome) and ompA1_TbBpep2_R (SEQ ID NO: 29, 5'- CAGCGCCCAGTTGGTTTTCATGGGTCGGGCCATTGTTGTTGCCGCCGCC CGCGCATTCATCGCC-3', underlined is the sequence corresponding to the extracellular region 1 of the ompA gene on the genome) PCR was performed using KOD-Plus-Neo (Toyobo) using KOD-Plus-Neo (homologous to ) as primers. By this operation, a DNA fragment (hereinafter referred to as "ompA1TbBpep2") encoding a 149 bp Tb ion-binding peptide motif that can be recombined into the extracellular domain 1 of OmpA was amplified. Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
In the nucleotide sequence represented by SEQ ID NO: 27, the nucleotide sequence at positions 184-212 is " tac promoter ", the nucleotide sequence at positions 258-944 is "GST-a", and the nucleotide sequence at positions 945-953 is The sequence is " Glycine ", the nucleotide sequence at positions 954-1004 is "TbB-peptide2", the nucleotide sequence at positions 1005-1007 is "Glycine", and the nucleotide sequence at positions 1008-1026 is "GST-b , the nucleotide sequence at positions 1422-2282 is " bla ", the nucleotide sequence at positions 2438-3056 is "pBR322 ori", and the nucleotide sequence at positions 3354-4445 is "lacI".

Similarly, ompA2_TbBpep2_F (SEQ ID NO: 30, 5- AATGGGTTACGACTGGTTAGGTCGTATGCCGTACAAAGGCGGCGGCGGCGCGTGCGTGGATTGG -3', underlined is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome) and ompA2_TbBpep2_R (SEQ ID NO: 31, 5'-GTTGAACGCCCTGAGCTTTGTATGCACCGTTTTCCGACGCGCGCCATCGCCCGCGCTGCC3). , the underlined sequence is homologous to the sequence corresponding to the extracellular region 2 of the ompA gene on the genome), and a 149 bp Tb ion-binding peptide motif DNA fragment that can be recombined into the extracellular region 2 of OmpA (hereinafter referred to as "ompA2TbBpep2") was amplified (see Figure 8). Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
Next, in the same manner as described above, E. coli W3110 typeA carrying pCas (J. Bacteriol., 1997, vol. 179(3), p. 959-963) was electroporated to extract the extracellular region of the ompA gene. Transformation was performed using the guide RNA expression plasmid psgRNA-ompA1-14 for specific cleavage of 1 and the DNA fragment ompA1TbBpep2 for recombination to obtain transformant YM1001.
Similarly, transformation was performed using a guide RNA expression plasmid psgRNA-ompA2-29 for specific cleavage of the extracellular region 2 of the ompA gene and a DNA fragment for recombination ompA2TbBpep2 to obtain transformant YM1201.

FIG. 9 shows the outline of the structures of transformants YM0501, YM0901 and YM1001 prepared as described above. Schematic structures of transformants YM0701, YM1101 and YM1201 are shown in FIG.

Test Example ₂ Growth of Recombinants Displaying Metal Ion _- Binding Motifs _on the Surface of the Outer Layer of E. coli Each transformant prepared in Test Example 1 was placed in M9 glucose medium (0.4 mM _2PO4 , 0.8 mM NaCl, 0.1 mM _CaCl2.2H2O , ₂₀ mM NH4Cl, ₁ mM _MgCl2.6H2O , 0.25 mM _K2SO4 , ₁₀ mM D- _{( +} )-glucose) at 37°C. and measured OD _{600 nm} over time. The results are shown in FIGS. 11 and 12. FIG.
As shown in FIGS. 11 and 12, all transformants exhibited growth curves similar to those of the parent strain W3110 typeA. These results demonstrate that the transformants of the present invention can display metal ion-binding peptide motifs on the surface of the cell outer layer without affecting their proliferation.

The E. coli transformant YM0501 in which the Cu ion-binding motif was inserted into the extracellular region 1 of the ompA gene presents the Cu ion-binding motif on the surface of the extracellular layer.
This transformant YM0501 and its parent strain (W3110 typeA) were each added to 5 mM CuCl ₂ in an LB liquid medium (10 g/L tryptone (Difco), 5 g/L yeast extract (Difco), 171 mM NaCl) at 37°C. was cultured overnight and OD _{600 nm} was measured.
As a result, the parent strain W3110 typeA had an OD 600 _nm value of 0.410, whereas the transformant YM0501 had an OD _{600 nm} value of 1.299, which was higher than that of the parent strain (see FIG. 13).
From this, it was proved that Cu ion tolerance of transformant YM0501 was improved by binding Cu ion binding motifs presented on the surface of the outer cell layer to Cu ions in the environment.

Test Example 3 Preparation of Vesicles from Recombinant Escherichia coli The transformant YM0501 was cultured overnight at 37° C. in 1 L of LB liquid medium (10 g/L tryptone (Difco), 5 g/L yeast extract (Difco), 171 mM NaCl). Cells were removed from the culture solution by centrifugation and filtration using a φ0.45 μm filter. The filtrate was then ultracentrifuged (150,000×g, 1 hour) to separate and concentrate naturally occurring vesicles present in the solution. The precipitate containing vesicles was suspended in a phosphate buffer (pH 7.0) to prepare a vesicle solution.

The vesicles were stained with FM4-64 (trade name: SynapseRed C2 (equivalent to FM4-64), available from Takara Bio) and confirmed using a confocal laser microscope Zeiss LSM510 META (Carl Zeiss). The results are shown in FIG. 14(B). A micrograph of transformant YM0501 stained with FM4-64 is also shown in FIG. 14(A). FM4-64 is a hydrophobic red fluorescent substance that stains lipid bilayer membranes. In this test example, FM4-64 was used to stain the phospholipid bilayer membrane of E. coli and vesicles.
In FIG. 14(B), E. coli cells as seen in FIG. 14(A) were not confirmed. In fact, in FIG. 14(B), a large number of vesicles with a diameter of about several hundred nm were observed, which agreed with the fact that the diameter of vesicles produced from E. coli is generally several tens to several hundred nm. Furthermore, the presence of protein in the vesicle solution was confirmed by the Bradford method.
The above results demonstrate that vesicles were successfully prepared from transformants by the above-described procedure.

Test Example 4 Recovery of metals using dead cells of recombinant E. coli that displays a metal ion-binding peptide motif on the surface of the cell outer layer It has eight extracellular domains. Hereinafter, the eight extracellular regions are respectively referred to as "Extracellular-1", "Extracellular-2", "Extracellular-3", and "Extracellular region." 4 (Extracellular-4)”, “Extracellular-5”, “Extracellular-6”, “Extracellular-7”, “Extracellular-8 ( Extracellular-8)” (see Figure 16).
Of these, extracellular domain 1 and extracellular domain 8 were targeted for insertion of metal ion-binding peptide motifs (see FIG. 18).

First, a plasmid for ompC gene expression was constructed.
Primers were designed to insert the ompC region into pBAD33 (J. Bacteriol. 1995, vol. 177(14), p. 4121-4130). A primer OmpC_BAD_F (SEQ ID NO: 38) with a total of 54 bases including 15 bases homologous to pBAD33, 14 bases including an SD sequence, and 25 bases from the initiation codon of the target gene ompC was designed. A total of 70 bases of primer OmpC_BAD_R (SEQ ID NO: 39) was designed, including 25 bases excluding the termination codon of the ompC gene from the upstream, 30 bases including the FLAG sequence and the termination codon, and 15 bases of the homologous sequence to pBAD33.

Using the E. coli W3110 Type A genome (J. Bacteriol. 1997, vol. 179(3), p. 959-963) as a template DNA, PCR was performed using the primers OmpC_BAD_F and OmpC_BAD_R to amplify the ompC gene region. . PCR was performed using ExTaq polymerase (Takara bio), and the PCR product was confirmed by 2% agarose gel electrophoresis. As a result, amplification of the target DNA fragment (1177 bp) was confirmed. Therefore, the DNA fragment was purified using NucleoSpin Gel and PCR Clean-up Kit (Cherey-Nagel). The obtained ompC fragment containing the sequence homologous to pBAD33 was designated as OmpC-BAD fragment.
Thereafter, pBAD33 treated with restriction enzymes HindIII and SacI and the OmpC-BAD fragment were ligated by homologous recombination using the In-Fusion HD Cloning Kit (Takara bio), and introduced into Escherichia coli DH5α by transformation using the _CaCl2 method. Colonies were obtained on LB agar medium containing chloramphenicol. Plasmids were extracted from the colonies that appeared using FavorPrep Plasmid DNA Extraction Mini Kit (Favorgen), and the extracted plasmids were subjected to DNA nucleotide sequencing to obtain plasmid pBADompC-FLAG (see FIG. 17).

Next, in order to introduce the Tb-binding peptide motif 2 (H ₂ N-ACVDWNNDGWYEGDECA-COOH, SEQ ID NO: 7) into the coding region of the extracellular region 1 of pBADompC-FLAG, the linear DNA of the pBADompC-FLAG plasmid was analyzed by Inverse PCR. was prepared.
By PCR using Pfu Ultra High Fidelity DNA Polymerase AD (Agilent) using primer ompC1a_inv_F (5'-ATCTTTGTTGTCAGAGAAATAGTGC-3', SEQ ID NO: 40) and primer ompC1a_inv_R (5'-GATGGCGACCAGACCTACATGCGTC-3', SEQ ID NO: 41) The DNA of interest (6452 base pairs) was amplified. After that, the PCR product was digested with Dpn I, and DNA purification was performed using NucleoSpin Gel and PCR Clean-up Kit.

Using the pGEX-4T-1-TbB-peptide2 plasmid in which the Tb ion-binding peptide motif 2 gene is cloned as a template, primer ompC1a_TbBpep2_F-2 (5'-TCTGACAACAAAGATGGCGGCGGCGCGTGCGTGGATTGG-3', SEQ ID NO: 42) and primer ompC1a_TbBpep2_R-2 (5') -GGTCTGGTCGCCATCGCCGCCGCCCGCGCATTCATCGCC-3', SEQ ID NO: 43) was used to perform PCR with KOD-Plus-Neo (Toyobo). By this operation, a 99 base pair DNA fragment encoding Tb ion-binding peptide motif 2 (hereinafter referred to as "ompC1TbBpep2") that can be introduced into the extracellular domain 1 of OmpC was amplified. Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
The pBADompC-FLAG linear vector and ompC1TbBpep2 were ligated by homologous recombination using In-Fusion HD Enzyme, introduced into Escherichia coli DH5α by transformation using the CaCl ₂ method, and colonies were obtained on LB agar medium containing chloramphenicol. Plasmids were extracted from the colonies that appeared using FavorPrep Plasmid DNA Extraction Mini Kit (Favorgen), and the extracted plasmids were subjected to DNA nucleotide sequencing to obtain plasmid pBADompC1-TBpep2 (see FIG. 19(A)).

Furthermore, a Tb-binding peptide motif 2 (H ₂ N-ACVDWNNDGWYEGDECA-COOH, SEQ ID NO: 7) was additionally introduced into the OmpC extracellular domain 8 coding region of pBADompC1-TBpep2. Linear DNA of the pBADompC1-TBpep2 was prepared by inverse PCR.
PCR using Pfu Ultra High Fidelity DNA Polymerase AD (Agilent) using primer ompC8f_inv_F (5'-CTGGTTGTCGTCCAGCAGGTTGATT-3', SEQ ID NO: 44) and ompC8f_inv_R (5'-ACTCGTGACGCTGGCATCAACACTG-3', SEQ ID NO: 45) The DNA of interest was amplified. The PCR product was then digested with DpnI and DNA purified using NucleoSpin Gel and PCR Clean-up Kit.
Using the pGEX-4T-1-TbB-peptide2 plasmid in which the Tb ion-binding peptide motif 2 gene is cloned as a template, primer ompC8f_TbBpep2_F-2 (5'-CTGGACGACAACCAGGGCGGCGGCCGCGTGCGTGGATTGG-3', SEQ ID NO: 46) and primer ompC8f_TbBpep2_R-2 (5') were used. -GCCAGCGCTCACGAGTGCCGCCGCCCGCGCATTCATCGCC-3', SEQ ID NO: 47) was used to perform PCR with KOD-Plus-Neo (Toyobo). By this operation, a 99 base pair DNA fragment encoding Tb ion-binding peptide motif 2 (hereinafter referred to as "ompC8TbBpep2") that can be introduced into the extracellular region 8 of OmpC was amplified. Amplified DNA fragments were purified using the QIA quick PCR purification kit (Qiagen).
The pBADompC-FLAG linear vector and ompC8TbBpep2 were ligated by homologous recombination with In-Fusion HD Enzyme, introduced into Escherichia coli DH5α by transformation by the CaCl ₂ method, and colonies were obtained on LB agar medium containing chloramphenicol. Plasmids were extracted from the colonies that appeared using FavorPrep Plasmid DNA Extraction Mini Kit (Favorgen), and the extracted plasmids were subjected to DNA nucleotide sequencing to obtain plasmid pBADompC18-TBpep2 (see FIG. 19(B)).

The constructed plasmids pBADompC1-TBpep2, pBADompC18-TBpep2, pBADompC-FLAG and plasmid pBAD33 were introduced into E. coli JW2203 (ompC::Kmr) (Mol. Syst. Biol. 2006, Vol. 2, 2006.0008.).
Create E. coli JW2203 competent cells for CaCl2, add pBADompC1-TBpep2, pBADompC18-TBpep2, pBADompC _- FLAG, pBAD33 plasmid solutions, perform transformation by CaCl2 method, and place on LB agar medium containing chloramphenicol. acquired a colony. The resulting transformants (see FIGS. 20(A) and (B)) were cultured in LB medium containing chloramphenicol and arabinose (final concentration 0.002%). -HCl pH 8.0/20% sucrose). Lysozyme (200 μg/mL) and EDTA (final concentration 0.1 M) were added to the cell suspension and allowed to stand on ice for 40 minutes. After that, MgCl ₂ (0.5 M final concentration) was added, and the spheroplast precipitate obtained by centrifugation (10,000 rpm, 20 min, 4°C) was separated into a spheroplast suspension solution (10 mM Tris-HCl pH 8.0). After being suspended in , ultrasonication (Amplitude 15%, Pulse ON/OFF 10 seconds, total 90 seconds) was performed.
Centrifuge the solution (10,000 rpm, 15 minutes, 4oC), then ultracentrifuge the supernatant twice (30,000 rpm, 60 minutes), freeze-thaw, and divide the obtained membrane fraction into 1% Suspended with N-lauroylsarcinosinate (0.5% final concentration). After incubating at 25°C for 20 minutes, ultracentrifugation was performed again (30,000 rpm, 90 minutes), and the resulting precipitate was used as the outer membrane fraction. The outer membrane fraction was dissolved in an outer membrane soluble solution (20 mM Tris-HCl pH 8.0/20% sucrose/2% SDS), and the amount of protein was quantified by the Bradford method.

As a result of analyzing these outer membranes by SDS-PAGE using an acrylamide gel containing urea (final concentration 7.5%), E. coli JW2203 strain having pBAD33 at around 45 kDa could not be confirmed (Fig. 21(A)). Lane 1), and a protein band presumed to be OmpC confirmed in the E. coli strain JW2203 harboring pBADompC-FLAG was detected (see lane 2 in FIG. 21(A)). In addition, since the mobility of this protein band was slightly slow in the JW2203 strain having pBADompC1-TBpep2, it was presumed to be OmpC containing the Tb ion-binding peptide motif 2 (see lane 3 in FIG. 21(A). ).
In order to confirm the above results, Western blotting using a FLAG antibody was performed. The outer membrane fraction derived from each transformant was electrophoresed by SDS-PAGE using an acrylamide gel containing urea (final concentration 7.5%), and the proteins in the gel were transferred to iBlot Gel Transfer Stacks PVDF (Invitrogen). transcribed. The transferred PVDF membrane was blocked with TBS solution (50mM Tris/150mM NaCl/1M HCl pH7.5) containing skimmed milk (final concentration 3%), followed by blocking with TBS containing skimmed milk (final concentration 1%) to which FLAG antibody (Sigma) was added. Immunoreactivity on PVDF with primary antibody was performed by exposing to 30°C overnight with shaking. After that, the transferred PVDF membrane was washed with TBS containing skimmed milk (final concentration 1%), shaken in TBS containing skimmed milk (final concentration 1%) to which HRP-conjugated mouse IgG antibody was added for 2 hours, and then washed with secondary antibody. An immune response was performed. The immunoreacted transferred PVDF membrane was washed with TBS containing skimmed milk (final concentration 1%) and purified water, and HRP chemiluminescence on the membrane was detected with LAS-4000 (Fujifilm) using Chemilmi One Super (Nacalai Tesque, Inc.). did. As a result, a band was detected in E. coli JW2203 strain having pBADompC-FLAG at around 45 kDa, and a band with slightly slower mobility was detected in E. coli JW2203 strain having pBADompC1-TBpep2 (see lanes 2 and 3 in FIG. 21(B). ). However, no band could be confirmed with the E. coli strain JW2203 harboring pBAD33 (see lane 1 in FIG. 21(B)).
These results confirmed that pBADompC1-TBpep2 expresses OmpC containing Tb ion-binding peptide motif 2 on the outer membrane of E. coli cells.

The E. coli transformants were cultured in LB medium containing chloramphenicol and arabinose (final concentration 0.002%) and killed by autoclaving. The adsorption behavior of this dead cell was evaluated.
When the dead cells were suspended in 1 mL of pure water, the amount of liquid that gave a turbidity of 1.0 OD (Optical Density) at a wavelength of 660 nm was collected by centrifugation. The collected dead cells were suspended in 1 mL of a 50 mM MES (pH 4.5) aqueous solution containing 100 μM TbCl ₃ , stirred for 30 minutes to prevent reprecipitation, and then centrifuged to remove the cells. The supernatant was separated. This supernatant was used as the supernatant after adsorption.

Each of the metal solution undiluted solution (MES aqueous solution containing TbCl ₃ ) before suspension of dead cells and the supernatant after adsorption was diluted 10 times with 5% nitric acid, and ICP-AES (Inductively Coupled Plasma-Atomic Each Tb ³⁺ ion concentration was determined by Emission Spectroscopy), and the adsorption rate of Tb ³⁺ ions adsorbed to the dead cells was calculated. The adsorption rate was calculated from the following formula.

(adsorption rate/%) =
(1-(Tb ³⁺ ion concentration of post-adsorption supernatant)/(Tb ³⁺ ion concentration of undiluted metal solution))

Table 2 shows the results.

As shown in Table 2, the amount of Tb ³⁺ ions adsorbed by E. coli strain 1 is considered to be due to non-specific adsorption of Tb ³⁺ ions to the surface layer of the E. coli body. On the other hand, E. coli strains 2 and 3, in which the Tb ion-binding peptide motif is displayed on the outer membrane, show a higher Tb ³⁺ ion adsorption rate than E. coli strain 1.
Therefore, in dead E.coli strains 2 and 3 expressing OmpC1a-TbBpep2 or OmpC1aC8f-Tbpep2 on the outer membrane, Tb ³⁺ ion is dominant compared to dead E.coli strain 1 that is not expressing , it was confirmed that OmpC1a-TbBpep2 or OmpC1aC8f-Tbpep2 possesses the ability to adsorb Tb ³⁺ ions.

While we have described our invention in conjunction with embodiments thereof, we do not intend to limit our invention in any detail to the description unless specified otherwise, which is contrary to the spirit and scope of the invention as set forth in the appended claims. I think it should be interpreted broadly.

This application claims priority based on Japanese Patent Application No. 2017-091503 filed in Japan on May 2, 2017, the contents of which are incorporated herein by reference. taken in as a part.

1 Outer membrane 2 Inner membrane 3 Porin 4 Metal ion-binding peptide motif 10 Recombinant microorganism or dead cell thereof 20 Vesicle

Claims (5)

Bringing the metal recovery carrier into contact with a medium containing metal ions,
binding the metal ions contained in the medium to the surface of the outer layer of the metal recovery carrier;
separating the metal recovery carrier from the medium;
A metal recovery method comprising:
The metal recovery carrier is a vesicle produced from live cells of a genetically modified microorganism that maintains its ability to proliferate, and whose outer layer is composed of a phospholipid bilayer membrane,
Porins derived from Gram-negative bacteria penetrate the phospholipid bilayer membrane,
In the amino acid sequence of the OmpA porin among the porins , any one of SEQ ID NOs: 1 to 7 represents a region that maintains the three-dimensional structure of the OmpA porin in the Gram-negative bacterium and does not impair the function of the OmpA porin as a diffusion channel. substituted with a metal ion-binding peptide motif consisting of an amino acid sequence ,
wherein the metal ion-binding peptide motif is displayed on the surface of the phospholipid bilayer membrane;
Metal recovery method. The substitution site of the metal ion-binding peptide motif to the OmpA porin is any site in the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8 or the region corresponding to this, represented by SEQ ID NO: 8. Any site of the region from positions 79 to 95 of the amino acid sequence represented by SEQ ID NO. , and at least one site selected from the group consisting of the region from positions 164 to 181 of the amino acid sequence represented by SEQ ID NO: 8 or any region corresponding to this, The metal according to claim 1 collection method. A vesicle made from live cells of a genetically modified microorganism that maintains its ability to proliferate and having an outer layer composed of a phospholipid bilayer membrane,
Porins derived from Gram-negative bacteria penetrate the phospholipid bilayer membrane,
In the amino acid sequence of the OmpA porin among the porins , any one of SEQ ID NOs: 1 to 7 represents a region that maintains the three-dimensional structure of the OmpA porin in the Gram-negative bacterium and does not impair the function of the OmpA porin as a diffusion channel. substituted with a metal ion-binding peptide motif consisting of an amino acid sequence ,
wherein the metal ion-binding peptide motif is displayed on the surface of the phospholipid bilayer membrane;
Carrier for metal recovery. The substitution site of the metal ion-binding peptide motif to the OmpA porin is any site in the region from positions 38 to 54 of the amino acid sequence represented by SEQ ID NO: 8 or the region corresponding to this, represented by SEQ ID NO: 8. Any site of the region from positions 79 to 95 of the amino acid sequence represented by SEQ ID NO. , and at least one site selected from the group consisting of the region from positions 164 to 181 of the amino acid sequence represented by SEQ ID NO: 8 or any region corresponding to this, The metal according to claim 3 , Recovery carrier. A bioreactor for metal recovery, wherein the carrier for metal recovery according to claim 3 or 4 is immobilized.

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