JP2013252085A - Recombinant yeast, and method for recovering tungsten using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant yeast transferred with a gene encoding a specific protein so as to cause the presence of the specific protein on cell surface layer; and to provide a method for efficiently recovering tungsten utilizing the recombinant yeast.SOLUTION: Tungsten is efficiently recovered utilizing recombinant microorganisms expressing Pyrococcus furiosus-derived WtpA gene on cell surfaces.

Description

  The present invention relates to a recombinant yeast into which a gene encoding the protein is introduced so that a predetermined protein is present on the cell surface and a method for recovering tungsten using the recombinant yeast.

  Tungsten is used in cemented carbide tools such as special steel and cutting tools, and its usage has been increasing in recent years. In countries such as Japan where the supply amount of tungsten is low, the country depends on countries such as China where the supply amount of tungsten is large. Therefore, there is a possibility that supply instability may occur with respect to tungsten. Under such circumstances, there is a demand for simple and inexpensive recycling from so-called urban mines.

  Physical and chemical methods are known as methods for recycling rare metals from urban mines. This physicochemical method is a method capable of recovering rare metals at low cost. However, this method has a problem that the efficiency is poor when the content of the target metal in the urban mine is low. Biological methods are also known as methods for recycling rare metals from urban mines. This biological technique can supplement the physicochemical technique when the content of the target metal in an urban mine is low, and is characterized in that it can be recovered at normal temperature and normal pressure.

  As a biological method, there is a method of adsorbing a metal to a microorganism called bioadsorption. Microorganisms used for bioadsorption vary depending on the metal to be recovered. In addition, the amount adsorbed on the microorganism may vary depending on the type of metal to be collected. For example, Patent Document 1 and Non-Patent Document 1 introduce a fusion gene encoding a secretory signal peptide, a polypeptide having a metal capturing ability, and a cell surface localized protein into a host, and a metal capturing function on the cell surface of the host. A technique for imparting is disclosed. Patent Document 1 and Non-Patent Document 1 disclose that copper, nickel, and cadmium ions such as copper nitrate, copper sulfate, and copper chloride can be supplemented. More specifically, Patent Document 1 and Non-Patent Document 1 disclose that arming yeast in which (His) 6 is expressed in the cell surface layer was used, and the result of cell adsorption of copper nitrate was 1.21 μmol / g dry cell. Has been.

Non-Patent Document 2 discloses a technique for recovering molybdenum using a recombinant microorganism in which a molybdenum-adsorbing protein ModE is expressed on the cell surface. According to Non-Patent Document 2, it can be understood that the result of experiments on the amount of molybdenum adsorbed using this recombinant microorganism was about 0.9 (Number of adsorbed molybdate × 10 ⁸ molybdate / cell).

JP 2003-284556 A

Ueda, Koichi Kuroda, Bio-technology for high-efficiency metal recovery by cell surface design, construction materials, vol55 (No.8), pp. 66-70 (2007) Kouichi Kuroda ・ Mitsuyoshi Ueda, et al. Molecular design of yeast cell surface for adsorption and recovery of molybdenum one of rare metals, Appl Microbiol Biotechnol, 86, 641-648 (2010)

  However, there is no known biological method with excellent tungsten recovery efficiency, and there is a technique that can efficiently recover tungsten, in particular, a recombinant microorganism having tungsten adsorption ability, and a tungsten recovery method using the recombinant microorganism. It was sought after. Accordingly, an object of the present invention is to provide a recombinant microorganism having tungsten adsorption ability and a method for recovering tungsten using the recombinant microorganism.

  As a result of intensive studies by the present inventors to achieve the above-mentioned object, it was found that the tungsten adsorption ability in a recombinant microorganism expressing a WtpA gene derived from Pyrococcus furiosus on the cell surface is remarkably high, The present invention has been completed. That is, the present invention includes the following.

(1) A recombinant yeast having cell surface display type tungsten transport protein A (WtpA).
(2) The recombinant yeast according to (1), wherein the cell surface display type tungsten transport protein A is a fusion protein of tungsten transport protein A having a capability of capturing tungsten and a cell surface localized protein. .
(3) The recombinant yeast according to (1), wherein a fusion gene encoding a secretory signal peptide, tungsten transport protein A having tungsten capturing ability and a cell surface localized protein is introduced into a host yeast.
(4) The recombinant yeast according to (1), wherein the tungsten transport protein A is the following protein (a) or (b):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a tungsten adsorption ability. A tungsten recovery agent containing yeast.
(6) A method for recovering tungsten, comprising a step of bringing the recombinant yeast according to any one of (1) to (4) above into contact with a solution containing tungsten, and a step of separating and recovering tungsten from the recombinant yeast.

  The recombinant yeast according to the present invention has an excellent ability to adsorb tungsten. Therefore, by using the recombinant yeast according to the present invention, tungsten can be efficiently recovered from a solution containing tungsten.

It is a flowchart which builds pTOPO-TDH3P-ss.glu-wtpA. It is a flowchart which builds pTOPO-agglutinin-PGK1T. It is a flowchart which constructs pTOPO-TDH3P-ss.glu-wtpA-agglutinin-PGK1T. It is a flowchart which builds pYES2-TRP1. It is a flowchart which builds P189@pYES2-TDH3-P_ATF1-ss.gla-wtpA-agglutinin-T_PGK1-TRP1.

  Hereinafter, the recombinant yeast according to the present invention and the tungsten recovery method using the recombinant yeast will be described in detail.

<Recombinant yeast>
The recombinant yeast according to the present invention is a yeast having a cell surface display type tungsten transport protein A (WtpA). In other words, the recombinant yeast according to the present invention is characterized in that WtpA is present on the cell surface. The recombinant yeast according to the present invention can adsorb tungsten through WtpA present on the cell surface.

  The recombinant yeast according to the present invention can be prepared by introducing a cell surface display type tungsten transport protein A gene (hereinafter referred to as cell surface display type WtpA gene). Here, the cell surface presentation type WtpA gene is a gene obtained by fusing the WtpA gene and the cell surface localized protein gene so that the expressed WtpA is displayed on the surface of the yeast cell.

  The cell surface localized protein refers to a protein that is fixed on the cell surface layer of yeast and is present on the cell surface layer. For example, α- or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned. In general, a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal on the C-terminal side. Although it has the same secretory protein in that it has a secretion signal, the cell surface localized protein is different from the secreted protein in that it is transported by being immobilized on the cell membrane via a GPI anchor. When the cell surface localized protein passes through the cell membrane, the GPI anchor attachment recognition signal sequence is selectively cleaved, and is bound to the GPI anchor at the newly protruding C-terminal portion and fixed to the cell membrane. Thereafter, the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Next, the protein separated from the cell membrane is incorporated into the cell wall, fixed to the cell surface layer, and localized on the cell surface layer (see, for example, JP-A-2006-174767). Moreover, what is indicated by Unexamined-Japanese-Patent No. 2003-284556 can also be used for cell surface localized protein.

  The WtpA gene is a gene encoding tungsten transport protein A constituting a novel ABC transporter system found in Pyrococcus furiosus (J. Bacteriol., Vol. 188, No. 18, Sep. 2006, p. 6489-6505). The nucleotide sequence of the WtpA gene and the amino acid sequence of the protein encoded by the WtpA gene are shown in SEQ ID NOs: 1 and 2, respectively.

  The WtpA gene expressed as a cell surface display type in the recombinant yeast according to the present invention is not limited to the WtpA gene defined by these SEQ ID NOs: 1 and 2.

  The WtpA gene may be a gene that consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, and encodes a protein having tungsten adsorption ability. In other words, in the recombinant yeast according to the present invention, a protein having a tungsten adsorbing ability, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 2, It may be expressed as a presentation type. Here, a plurality of amino acids means, for example, 2 to 100, preferably 2 to 80, more preferably 2 to 50, and still more preferably 2 to 15 amino acids.

  Further, the WtpA gene has an identity of 70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and most preferably 98% or more with respect to the amino acid sequence of SEQ ID NO: 2. It may be a gene encoding a protein consisting of an amino acid sequence having a tungsten adsorbing ability. In other words, in the recombinant yeast according to the present invention, the amino acid sequence of SEQ ID NO: 2 is 70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, most preferably A protein having an amino acid sequence having an identity of 98% or more and having a tungsten adsorption ability may be expressed as a cell surface display type. The identity between sequences means a value (ratio) indicating the degree of coincidence between two amino acid sequences using sequence similarity search software such as BLAST, PSI-BLAST, and HMMER with default settings.

  Furthermore, the WtpA gene is a protein encoded by a polynucleotide that hybridizes under stringent conditions to at least a part of a polynucleotide consisting of a complementary strand of the nucleotide sequence of SEQ ID NO: 1, and has a tungsten adsorption capacity. It may be a gene encoding a protein having In other words, in the recombinant yeast according to the present invention, a protein encoded by a polynucleotide that hybridizes under stringent conditions to at least a part of a polynucleotide comprising a complementary strand of the base sequence of SEQ ID NO: 1. In addition, a protein having tungsten adsorption ability may be expressed as a cell surface display type. The term “stringent conditions” as used herein means the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is determined appropriately with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition). can do. Specifically, the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 × SSC (83 mM NaCl, 83 mM sodium citrate), temperature 42 ° C. The term “at least a part of the polynucleotide” means to include both the entire polynucleotide composed of a complementary strand of a predetermined base sequence and the entire continuous portion of the polynucleotide composed of the complementary strand.

  In addition, the introduction | transduction of the variation | mutation with respect to a predetermined amino acid sequence can be performed by modifying the base sequence of a WtpA gene by a method well-known in the said technical field. Mutation can be introduced into a base sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit (for example, Mutant-) using site-directed mutagenesis. Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA Bio) or the like, or using LA PCR in vitro Mutagenesis series kits (trade name, manufactured by TAKARA Bio). In addition, as a method for introducing mutations, EMS (ethyl methanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N'-nitro-N nitrosoguanidine, and other carcinogenic compounds are representative. A method using a chemical mutagen such as that described above may be used, or a method using radiation treatment or ultraviolet treatment represented by X-rays, alpha rays, beta rays, gamma rays and ion beams may be used.

  Whether a gene consisting of a predetermined base sequence encodes a protein capable of adsorbing tungsten can be prepared by preparing an expression vector in which the gene is inserted between an appropriate promoter and terminator, and using this expression vector. What is necessary is just to measure the tungsten adsorption ability of the protein which transforms a host and expresses it. Here, the tungsten adsorption ability of protein can be evaluated by, for example, mixing the protein in a sodium tungstate dihydrate solution, separating the protein component after a predetermined time, and measuring the tungsten concentration in the solution. The tungsten concentration in the solution can be measured by an emission spectroscopic analysis method using high frequency inductively coupled plasma (ICP) as a light source.

  In addition, the WtpA gene in Pyrococcus furiosus, that is, the WtpA gene defined by SEQ ID NOs: 1 and 2, has been known to study selective adsorption to tungsten by site-directed mutagenesis. (J. Bacteriol., Vol. 193, No. 18, Sep. 2011, p. 4999-5001). Specifically, genes encoding mutant proteins such as E218A, E218Q, D160A, and D160N are prepared as mutant WtpA genes, and the tungsten adsorption ability of these mutant proteins is evaluated. In the recombinant yeast according to the present invention, these known mutant WtpA genes may be used to express the mutant protein as a cell surface display type.

  As described above, the WtpA gene can be expressed as a cell surface display type by being incorporated into an expression vector and introduced into yeast serving as a host. At this time, the WtpA gene encodes a so-called secretory signal peptide (also referred to as secretory signal or secretory signal sequence) upstream of the 3 ′ end, and encodes the aforementioned cell surface localized protein downstream of the 5 ′ end. Into the expression vector so that it can be expressed as a fused gene. Here, the secretory signal peptide is a peptide containing a lot of amino acids rich in hydrophobicity, which is generally bound to the N-terminal of a protein (secretory protein) secreted extracellularly (including periplasm), Normally, secreted proteins are removed when they are secreted from inside the cell through the cell membrane. In the present invention, the secretory signal peptide is not limited at all, and conventionally known ones can be used. In particular, a secretory signal peptide known to function in yeast as a host can be used as the secretory signal peptide. As the secretion signal peptide, a pre-pro leader sequence of an α-factor precursor, a yeast α- or a-agglutinin signal sequence, a glucoamylase secretion signal, and the like can be used.

  A gene encoding a fusion protein having a secretory signal peptide, WtpA protein and cell surface localized protein in this order from the N-terminal side is PCR using primers designed based on nucleotide sequence information encoding each region, It can be prepared by a cloning method using a restriction enzyme treatment using a restriction enzyme. In addition, a gene encoding the fusion protein may be prepared by using a so-called in-fusion method. Furthermore, all or part of the gene encoding the fusion protein may be prepared by chemical synthesis. When a gene is synthesized by chemical synthesis, for example, it is preferable to synthesize the WtpA gene in a form optimized in view of yeast codon usage.

  The gene encoding the fusion protein is placed downstream of a suitable promoter so that it can be expressed in the yeast of the host. As the promoter, for example, a promoter of glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), a promoter of 3-phosphoglycerate kinase gene (PGK1), a promoter of hyperosmotic response 7 gene (HOR7), and the like can be used. Of these, the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene. In addition, downstream genes can be strongly expressed by using gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, GAP promoter, ADH promoter, AOX1 promoter and the like.

  The yeast that can be used as a host is not particularly limited, but Candida genus yeast such as Candida Shehatae, Pichia genus yeast such as Pichia stipitis, Pachysolen genus yeast such as Pachysolen tannophilus, and Saccharomyces cerevisiae genus yeast such as Saccharomyces cerevisiae. And yeast belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe, and Saccharomyces cerevisiae is particularly preferred.

  In addition, as a method for introducing the above-described gene, any conventionally known technique known as a yeast transformation method can be applied. Specifically, for example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”, lithium acetate method “J. Bacteriology, 153, p163 (1983)”, Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc. Although it can be implemented, it is not limited to this.

  As described above, the recombinant yeast according to the present invention has a cell surface display type WtpA protein. Thus, the recombinant yeast according to the present invention displays the WtpA protein on the cell surface, and therefore has a tungsten adsorption ability. That is, the ability of adsorbing tungsten in the recombinant yeast according to the present invention is different from the cell surface display type WtpA protein, and the higher the amount of the protein in the cell surface layer, the better. The amount of the cell surface display-type WtpA protein can be adjusted by the copy number of the gene encoding the protein and the strength of the promoter of the gene. In the recombinant yeast according to the present invention, the WtpA protein presented on the cell surface layer may be in a state in which all of the secretory signal peptide is cleaved or in a state in which a part or all of the secretory signal peptide remains. Also good.

  Since the recombinant yeast according to the present invention has the ability to adsorb tungsten as described above, it can be used as a tungsten recovery agent. In other words, the recombinant yeast according to the present invention can be used in a method for recovering tungsten.

  When the recombinant yeast according to the present invention is used as a tungsten recovery agent, it is not particularly limited, but the recombinant yeast can be used by being immobilized on an appropriate carrier. The carrier means a substance that can immobilize the recombinant yeast of the present invention, and preferably means a substance that is insoluble in water or a specific solvent. Examples thereof include foams or resins such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, silicon foam, and cellulose porous body. The carrier is preferably a porous carrier because it is advantageous for maintaining the growth and activity of the recombinant yeast, and for rapidly detaching dead cells. The size of the opening of the porous body varies depending on the cell, but the size capable of entering and proliferating into the porous is appropriate, and is preferably 50 μm to 1,000 μm. However, it is not limited to these sizes. Moreover, the shape of a support | carrier is not ask | required. Considering the strength of the carrier, the culture efficiency, etc., a spherical shape or a cubic shape is preferable, and the size is preferably 2 mm to 50 mm in the case of a spherical shape and 2 mm to 50 mm square in the case of a cubic shape.

  Immobilization refers to a state in which the recombinant yeast is not in a free state but is bound to or attached to a carrier, or taken into the carrier. For immobilization of the recombinant yeast, for example, known methods such as a carrier binding method, a crosslinking method and an inclusion method can be applied. The carrier binding method is optimal for immobilizing aggregating yeast. Examples of the carrier binding method include a chemical adsorption method or a physical adsorption method in which adsorption is performed on an ion exchange resin. The aggregating yeast is capable of growing despite being immobilized on a carrier, and has a property of dropping off spontaneously when the activity decreases. For this reason, the yeast bound to the carrier is characterized in that the number of viable bacteria is kept almost constant and the activity is high. Therefore, physical adsorption is most preferred for binding of the recombinant yeast to the carrier. By mixing and culturing the aggregated or adherent cells and the porous carrier, the cells enter the opening of the porous body and adhere to the carrier.

  The immobilized recombinant yeast thus obtained can be cultured in a suspended state while attached to a carrier, or packed in a column and used as a so-called bioreactor. As a column, the thing similar to what is used for normal gel filtration, ion exchange chromatography, etc. can be used, for example. In addition, the column can be used as a metal recovery device in combination with a pump, a recovery container, a metal detector, and the like.

  The yeast which concerns on this invention can adsorb | suck tungsten in the said solution by making it contact with the solution containing tungsten, for example. The solution containing tungsten is not particularly limited, and may be any of seawater, fresh water (rivers, ponds, lakes), or wastewater (domestic wastewater, kitchen wastewater, factory wastewater, etc.).

  In order to recover tungsten from recombinant yeast adsorbed with tungsten, for example, by treating the recombinant yeast with a chelating agent such as EDTA (ethylenediaminetetraacetic acid), tungsten is removed from the surface of the recombinant yeast. The tungsten that has been desorbed can be recovered. Alternatively, even when the recombinant yeast is treated with a protease such as papain instead of a chelating agent, tungsten can be detached from the surface of the recombinant yeast and recovered. Here, treating recombinant yeast with a chelating agent or protease means a method of adding the chelating agent or protease into a solution containing the recombinant yeast and stirring as necessary.

  As described above, by using the recombinant yeast according to the present invention, tungsten can be more easily recovered from a solution containing tungsten. Although it is conceivable to purify WtpA protein and recover tungsten using this according to a conventional method, purification of a sufficient amount of WtpA protein for the recovery of tungsten takes time and is not a simple method. In contrast, when the recombinant yeast according to the present invention is used, a sufficient amount of WtpA protein for tungsten recovery can be prepared simply by culturing the recombinant yeast. Further, when the recombinant yeast according to the present invention is used, tungsten can be recovered using a chelating agent or a protease as described above, and the operation of crushing the recombinant yeast and recovering tungsten therefrom is unnecessary. It becomes. Also in this point, when using the recombinant yeast according to the present invention, it is possible to more easily recover tungsten from a solution containing tungsten. Furthermore, the recombinant yeast according to the present invention can be reused after recovering tungsten using a chelating agent or protease as described above. Therefore, when the recombinant yeast according to the present invention is used, tungsten recovery from a solution containing tungsten can be performed more easily and at low cost.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

<Vector production method>
Using the genome of the S. cerevisiae OC2 strain as a template, gene amplification was performed using the primer set of primer names A1 and A2 described in Table 1 to be described later to amplify the TDH3 promoter region derived from S. cerevisiae. The obtained TDH3 promoter region was subcloned using a zero blunt TOPO PCR cloning kit (manufactured by Invitrogen). The obtained vector was named pTOPO-TDH3P.

  Next, a signal sequence derived from R. oryzae and a WtpA gene derived from Pyrococcus.furiosus (PF0080) [Reference 1: Loes E. Bevers, Peter-Leon Hagedoorn, Gerard C. Krijger, and Wilfred R. Hagen J. Bacteriol September 15, 2006 188: 6498-6505 and Reference 2: Loes E. Bevers, Guenter Schwarz, and Wilfred R. Hagen J. Bacteriol. September 2011 193: 4999-5001] in view of yeast codon usage. Optimized and synthesized artificial genes.

  Next, using pTOPO-TDH3P as a template, gene amplification was performed using the primer names of B1 and B2 listed in Table 1, and a sequence homologous to the signal sequence derived from R.oryzae at the 3 ′ end was 20 bp. The added TDH3 promoter site was amplified. In addition, gene amplification was performed using a primer sequence of the primer names C1 and C2 described in Table 1, using a signal sequence derived from artificially synthesized R. oryzae as a template, and homologous to the TDH3 promoter site at the 5 ′ end. A signal sequence derived from R. orzae having 20 bp of sequence and 20 bp of sequence homologous to the WtpA gene at the 3 ′ end was amplified. Finally, using the artificially synthesized wtpA gene as a template, gene amplification was performed using the primer sets listed in Table 1 with primer names D1 and D2, and homologous to the signal sequence derived from R.oryzae at the 5 'end. The wtpA gene with 20 bp sequence was amplified.

  After purifying these gene-amplified fragments, PCR is performed again using the three fragments simultaneously as a template, so that the TDH3 promoter region, the signal sequence derived from R. oryzae and the wtpA gene derived from Pyrococcus.furiosus are in this order. Nucleic acid fragments connected by Amplified nucleic acid fragments were subcloned using the Zerobrand TOPO PCR cloning kit. The obtained vector was named pTOPO-TDH3P-ss.glu-wtpA (see FIG. 1).

  On the other hand, using the genome of S. cerevisiae OC2 strain as a template, gene amplification was carried out using the primer names listed in Table 1: primer set of E1 and E2, and primer set of F1 and F2, respectively. The side α-agglutinin and PGK1 terminator regions were each amplified. The obtained S. cerevisiae-derived 3 ′ α-agglutinin and PGK1 terminator regions were each subcloned using a zero blunt TOPO PCR cloning kit. The obtained vectors were named pTOPO-agglutinin and pTOO-PGK1T, respectively.

  Next, using pTOPO-agglutinin as a template, gene amplification was performed using the primer names listed in Table 1 with primer sets G1 and G2, and α-agglutinin with a 20-bp sequence homologous to the PGK1 terminator region at the 3 ′ end was added. Was amplified. In addition, using pTOPO-PGK1T as a template, gene amplification was performed using the primer set of H1 and H2 listed in Table 1, and a PGK1 terminator region with a 20 bp sequence homologous to α-agglutinin at the 5 ′ end was added. Amplified. After purifying these gene-amplified fragments, PCR was carried out again using the two fragments simultaneously as a template, thereby linking the 3'-side α-agglutinin derived from S. cerevisiae and the PGK1 terminator region to produce nucleic acid fragments. The resulting nucleic acid fragment was subcloned using the Zerobrand TOPO PCR cloning kit. The obtained vector was named pTOPO-agglutinin-PGK1T (see FIG. 2).

  On the other hand, pTOPO-TDH3P-ss.glu-wtpA was used as a template, gene amplification was performed using the primer name: I1 and I2 primer sets described in Table 1, and a sequence homologous to α-agglutinin at the 3 ′ end was 20 bp. The given gene fragment was amplified. In addition, a gene fragment obtained by performing gene amplification using pTOPO-agglutinin-PGK1T as a template and the primer names of J1 and J2 listed in Table 1 and adding a 20 bp sequence homologous to the wtpA gene at the 5 ′ end. Was amplified. After purifying these gene-amplified fragments, PCR is performed again using the two fragments simultaneously as a template, so that the TDH3 promoter region, the signal sequence derived from R.oryzae, the wtpA gene derived from Pyrococcus.furiosus, and S A nucleic acid fragment was prepared by linking cerevisiae 3'-side α-agglutinin and PGK1 terminator region in this order. The resulting nucleic acid fragment was subcloned using the Zerobrand TOPO PCR cloning kit. The obtained vector was named pTOPO-TDH3P-ss.glu-wtpA-agglutinin-PGK1T (FIG. 3).

On the other hand, using pYES2 (manufactured by Invitrogen) as a template, gene amplification was performed using the primer set of primer names L1 and L2 described in Table 1, and a terminator of S. cerevisiae-derived TRP1 was placed on the 5 ′ side of the gene amplified fragment. A gene fragment having 16 bp of sequence homologous to the region and 16 bp of sequence homologous to the TRP1 promoter region on the 3 ′ side was amplified (E. coli derived Amp ^r , PUCori, S. cerevisiae derived 2 μm gene etc. Including). On the other hand, using the genome of OC2 strain as a template, gene amplification was carried out using the primer names of K1 and K2 listed in Table 1, and the promoter region of TRP1 derived from S. cerevisiae, the TRP1 gene, and the terminator region of TRP1 The contained nucleic acid fragment was amplified. Next, each amplified fragment was purified with a PCR purification kit and reacted by an in-fusion method (manufactured by Clontech) to prepare pYES2-TRP1 (see FIG. 4).

  Finally, using pTOPO-TDH3P-ss.glu-wtpA-agglutinin-PGK1T as a template, gene amplification was carried out using the primer names M1 and M2 described in Table 1, and pYES2-TRP1 was 16 bp of sequence homologous to the terminator region of PGK1 derived from S. cerevisiae is added to the 5 'side of the amplified gene fragment by performing gene amplification using the primer name: N1 and N2 primer sets described in Table 1 as a template Then, a gene fragment having 16 bp of a sequence homologous to the promoter region of TDH3 on the 3 ′ side was amplified. P189@pYES2-TDH3-P_ATF1-ss.gla-wtpA-agglutinin-T_PGK1-TRP1 was prepared by purifying each gene amplification fragment with PCR purification kit and reacting by in-fusion method (Clontech) . The obtained vector was used as the final gene transfer vector (see FIG. 5).

<Method of gene introduction into yeast>
Frozen-EZ Yeast Transformation II kit (manufactured by ZYMO RESEARCH) was used for gene introduction into yeast. As a host yeast, S. cerevisiae OC2-T strain (tryptophan requirement) was used. That is, P189@pYES2-TDH3-P_ATF1-ss.gla-wtpA-agglutinin-T_PGK1-TRP1 prepared as described above was introduced into the S. cerevisiae OC2-T strain using the above kit. The obtained transformant was SDC-Trp agar (Yeast Nitrogen base w / o amimo acids and ammonium slufate 0.34%, sodium glutamate 0.1%, potassium aspartate 0.3%, urea 0.3%, glucose 2%, CSM-Trp0. 2% and 1.5% agar) were selected and colonies with good growth were used as recombinant yeast.

<Culture method of yeast>
Recombinant yeast selected as described above was transformed into SDC-Trp (Yeast Nitrogen base w / o amimo acids and ammonium slufate 0.34%, sodium glutamate 0.1%, potassium aspartate 0.3%, urea 0.3%, glucose 2% and CSM. -Trp 0.2%) Incubation was performed at 30 ° C. for 48 hours using a flask with a 150 ml / 500 ml baffle. The cells were cultured until OD (Optical density) reached 10, and 20 ml of the culture solution was collected, and then centrifuged to collect the cells.

  On the other hand, the OC2-T strain to be compared was cultured at 30 ° C. for 24 hours in a 150 ml / 500 ml baffled flask using YPD (yeast extract 1%, peptone 2% and glucose 2%). The cells were cultured until OD was 10, and 20 ml of the culture solution was collected and centrifuged to collect the cells.

<Tungsten recovery experiment method>
To the collected cells, 10 ml of sodium tungstate dihydrate was added to a 50 ml falcon tube to a final concentration of 1 mM, and the mixture was well suspended, followed by shaking at 32 ° C. and 150 rpm for 2 hours. Centrifugation was performed at 5000 rpm for 5 minutes to separate the cells and supernatant, and the supernatant was placed in a vial and analyzed. In the tungsten analysis, the sample was diluted with ultrapure water and directly introduced into the ICP emission spectroscopic analyzer ICPS-8100 for analysis. The results are shown in Table 2.

As shown in Table 2, the amount of tungsten (mg / L) adsorbed on the cell surface of the bacterial cells was 50 mg / L for the recombinant yeast having the cell surface display-type WtpA protein, and 20 mg / L for the control OC2-T strain. Met. If this result is expressed in units of (μmol / g dry cell), the amount of tungsten adsorbed is 49.45 μmol / g dry cell (= 9.09 [mg / g dry cell] = 4.09 [Number of adsorbed tungstate × 10 ⁸ ) for recombinant yeast. tungstate / cell]). From this result, it was revealed that the amount of tungsten adsorbed can be remarkably increased by expressing WtpA protein on the surface layer of the cell.

Claims (6)

  A recombinant yeast having cell surface display type tungsten transport protein A (WtpA).   2. The recombinant yeast according to claim 1, wherein the cell surface display type tungsten transport protein A is a fusion protein of tungsten transport protein A having a capability of capturing tungsten and a cell surface localized protein.   The recombinant yeast according to claim 1, wherein a fusion gene encoding a secretory signal peptide, tungsten transport protein A having a tungsten capturing ability and a cell surface localized protein is introduced into a host yeast. The recombinant yeast according to claim 1, wherein the tungsten transport protein A is the following protein (a) or (b):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having a tungsten adsorption ability   The tungsten collection | recovery agent containing the recombinant yeast as described in any one of Claims 1 thru | or 4. Contacting the recombinant yeast according to any one of claims 1 to 4 with a solution containing tungsten;
Separating and recovering tungsten from the recombinant yeast.

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