A method for constructing a metallothionein yeast surface display engineering strain with high copper ion enrichment

Abstract

A method for constructing a metallothionein yeast surface display engineering strain with high copper ion enrichment efficiency, which relies on the construction of a metallothionein yeast surface display engineering strain based on the yeast cell surface display technology. The method connects the codon-optimized metallothionein gene CUP1 to the yeast expression vector pYD1 through double enzyme digestion, ligation, transformation and other steps, successfully constructs the recombinant display plasmid pYD1-CUP1. The recombinant display plasmid pYD1-CUP1 is transformed into the tryptophan auxotrophic Saccharomyces cerevisiae EBY100 by lithium acetate method, and several recombinant transformants are obtained through MD plate screening and are screened and verified. The yeast engineering strain constructed by the method can improve the display efficiency of metallothionein on the surface of Saccharomyces cerevisiae cells, and enhance the enrichment capacity of the engineering strain to copper ions, so as to replace inorganic copper with copper-rich yeast, reduce the side effects of copper, and improve the absorption and utilization rate of copper by livestock and poultry.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for constructing a metallothionein yeast surface display engineered strain that efficiently enriches copper ions. Background Technology

[0002] Copper is an essential trace element for animals, playing a vital role in their health and growth. Currently, most copper supplements are in inorganic form, but this type of copper presents numerous problems during use, such as low absorption rates, poor compatibility with other dietary components, high excretion rates, environmental pollution, and occasional poisoning in animals due to improper use. In recent years, the use of microorganisms to enrich trace elements needed by humans and animals has become a research hotspot both domestically and internationally.

[0003] Yeast, as a common microorganism, has shown in some wild yeast strains the ability to enrich and adsorb metals, making it possible to efficiently enrich copper ions using yeast. Metallothionein (MT), rich in cysteine ​​residues, can efficiently bind copper ions and is widely used for copper ion enrichment. However, traditional microbial adsorption methods require cell wall disruption to recover copper ions, and the adsorption efficiency is limited by the cell membrane barrier.

[0004] With the continuous advancement of biotechnology, techniques such as genetic engineering and metabolic engineering have become increasingly mature, providing technical support for constructing yeast strains with highly efficient metal accumulation capabilities. Genetic engineering can be used to modify yeast strains, enhancing their ability to absorb, transport, and accumulate metals. Metabolic engineering methods can optimize yeast metabolic pathways, enhancing their tolerance to metals and accumulation efficiency. By selecting metallothionein genes and highly expressing genes with binding and transport effects on various trace metals, engineered yeast strains capable of efficiently accumulating metal ions can be constructed.

[0005] In the prior art, the surface display technology of Saccharomyces cerevisiae cells is used to express exogenous proteins, but its application in the display of metallothionein still has the following problems: (1) the expression level of metallothionein is low, which affects the adsorption efficiency; (2) the stability of the display system is insufficient; (3) it lacks the ability to efficiently enrich copper ions. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a metallothionein yeast surface display engineered strain that efficiently enriches copper ions. This method can improve the display efficiency of metallothionein on the surface of Saccharomyces cerevisiae cells, enhance the enrichment capacity of engineered strains for copper ions, and reduce the cost of copper ion enrichment.

[0007] To achieve the purpose of this invention, the technical solution provided by this invention is: a method for constructing an engineered strain for displaying metallothionein surface with efficient enrichment of copper ions, comprising the following steps: a. Obtaining the plasmid for CUP1 gene synthesis: Based on the gene sequence of metallothionein gene CUP1-1 shown in SEQ ID NO. 1 in the NCBI database, codon optimization was performed with reference to the codon preference of Saccharomyces cerevisiae. Histidine tag and stop codon were added to the C-terminus of the gene, and KpnI restriction site sequence GGTACC and EcoRI restriction site sequence GAATTC were added to both ends of the gene, respectively, to synthesize the CUP1 gene and further obtain the synthetic plasmid pUC-CUP1 shown in SEQ ID NO. 2; b. Double digestion: The surface display vector pYD1 of Saccharomyces cerevisiae and the synthetic plasmid pUC-CUP1 obtained in step a were double digested with restriction endonucleases KpnⅠ and EcoRI. The CUP1 gene fragment and linearized plasmid vector were obtained by water bath and then detected and verified by electrophoresis. c. Construction of recombinant display plasmid: The linearized vector fragment recovered in step b was ligated with the CUP1 gene fragment using solution I to obtain the ligation product; the ligation product was transformed into Escherichia coli competent cells DH5α and verified by PCR amplification to construct a strain containing the recombinant display plasmid pYD1-CUP1 as shown in SEQ ID NO 3; d. Saccharomyces cerevisiae transformation: The recombinant display plasmid pYD1-CUP1 was extracted from the strain verified in step c. The recombinant display plasmid pYD1-CUP1 was transformed into tryptophan auxotrophic Saccharomyces cerevisiae EBY100 using the lithium acetate method. Transformants were screened by MD plates, and yeast genomic DNA was extracted and used as a template for PCR amplification. PCR amplification was performed using CUP1-F as shown in SEQ ID NO. 4 and CUP1-RV as shown in SEQ ID NO. 5 as primers for verification. e. Validation of engineered bacteria: The ability of recombinant whole cells to adsorb copper ions was determined, and strains with high adsorption capacity were screened out.

[0008] Furthermore, in step b, the reaction was carried out in a water bath at 37°C for 2 hours; the enzyme digestion system for the Saccharomyces cerevisiae surface display vector pYD1 was: vector pYD1 32.8 μL, Kpn I 1.6 μL, EcoRI 1.6 μL, 10 × buffer 4 μL; the enzyme digestion system for the synthetic plasmid pUC-CUP1 was: synthetic plasmid pUC-CUP1 32.8 μL, Kpn I 1.6 μL, EcoRI 1.6 μL, 10 × buffer 4 μL.

[0009] Furthermore, in step c, ligation is performed overnight at 16°C using ligase solution I; the ligation system consists of: 10 μL of ligase solution I, 5 μL of the CUP1 gene fragment, and 5 μL of the recovered linearized plasmid vector fragment.

[0010] Furthermore, in step c, the competent E. coli cells are DH5α; the verification process is as follows: after screening on LB plates, single colonies are picked and inoculated for culture, and bacterial culture is used as a template, with CUP1-F as shown in SEQ ID NO 4 and CUP1-RV as shown in SEQ ID NO 5 as primers for bacterial culture PCR verification.

[0011] Preferably, in steps c and d, the PCR amplification system is: 10 μL of 2 × Premix Taq, 0.4 μL of CUP1-F, 0.4 μL of CUP1-RV, 0.4 μL of bacterial culture, and 8.8 μL of deionized water; the PCR amplification program is: pre-denaturation, 94 ℃, 4 min; denaturation, 94 ℃, 30 s; annealing, 55 ℃, 30 s; extension, 72 ℃, 30 s; final extension, 72 ℃, 10 min.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High efficiency enrichment: Metallothionein is displayed on the surface of Saccharomyces cerevisiae cells, which directly enriches copper ions, avoiding membrane barrier problems and significantly improving enrichment efficiency; (2) High stability: Saccharomyces cerevisiae is a host strain that is non-toxic and easy to culture. The engineered strain has high stability in actual environments. (3) Environmental protection and economy: Engineered bacteria can be cultivated on a large scale, with low cost and no secondary pollution. Attached Figure Description

[0013] Figure 1 Electrophoresis results of double-enzyme digestion of Saccharomyces cerevisiae surface display vector pYD1 (lane 1) and pUC-CUP1 plasmid (lane 2); Figure 2 This is a gel electrophoresis image of the CUP1 gene fragment. Figure 3 This is a schematic diagram of an Escherichia coli DH5α competent cell transformation plate. Figure 4 This is a diagram showing the results of bacterial culture PCR electrophoresis. Figure 5 The image shows the PCR electrophoresis results of yeast genomic DNA. Figure 6 This is a standard curve for copper ions. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to specific embodiments.

[0015] The materials used in the following embodiments are as follows: 1. Strains and plasmids (1) Escherichia coli competent cells DH5α were purchased from Shanghai Kangwei Century Co., Ltd.

[0016] (2) The surface display vector pYD1 of Saccharomyces cerevisiae and tryptophan auxotrophic Saccharomyces cerevisiae EBY100 were purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0017] (3) The synthetic plasmid pUC-CUP1 was synthesized and constructed by Shanghai Sangon Biotech Co., Ltd.

[0018] 2. Reagents are listed in Table 1 below: Table 1. Reagent Names and Manufacturers Kpn I, EcoRI, and ligase solution I Baori Biotechnology (Beijing) Co., Ltd. Agarose, agar powder, glucose Shanghai Lanji Technology Development Co., Ltd. Yeast extract, trypsin, peptone Beijing Aoboxing Biotechnology Co., Ltd. Proteinase K, 50 × TAE buffer Sangon Biotech (Shanghai) Co., Ltd. Ampicillin Hebei Yuancheng Pharmaceutical Co., Ltd. Lithium acetate dihydrate Shanghai Zhanyun Chemical Co., Ltd. Fish sperm DNA Thermo Fisher Scientific YNB, PEG-4000, DMSO Nanjing Aoduofuni Biotechnology Co., Ltd. Plasmid mini extraction kit Corning Life Sciences (Wujiang) Co., Ltd. DNA Gel Recovery Kit Corning Life Sciences (Wujiang) Co., Ltd. Yeast Genome Extraction Kit Corning Life Sciences (Wujiang) Co., Ltd. 3. Preparation of culture medium and solution (1) LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl, deionized water. If solid medium is required, add 2% agar powder.

[0019] (2) LB-Amp liquid medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride (NaCl), 0.1% Amp, deionized water. If solid medium is required, add 2% agar powder.

[0020] (3) YPD medium: 2% peptone, 1% yeast extract, 2% glucose. If solid medium is required, add 2% agar powder.

[0021] (4) MD solid culture medium: amino-free yeast nitrogen source (containing ammonium sulfate) / YNB 0.67%, glucose 2%, leucine 0.01%, agar powder 2%.

[0022] (5) 10 × TE (pH 7.5): 1 M Tris-HCl 10 ml, 0.5 M EDTA 2 ml, add distilled water to make up to 100 ml, and adjust pH to 7.5.

[0023] (6) 1 × TE (pH 7.5): Add 10 ml of 10 × TE to distilled water to a final volume of 100 ml and adjust the pH to 7.5.

[0024] (7) 0.1 mol / L LiAc (pH 6.0): Add 1.02 g LiAC to 10 ml 10 × TE, distilled water to a final volume of 100 ml, and adjust pH to 6.0.

[0025] (8) 40% PEG-4000 (prepared and used immediately): Dissolve 0.4 g PEG-4000 in 1 ml LiAc. Example

[0026] A method for constructing a highly efficient copper-enriched metallothionein yeast surface-displaying engineered strain includes the following steps: a. Obtaining the plasmid for CUP1 gene synthesis Based on the gene sequence of the metallothionein gene CUP1 shown in SEQ ID NO. 1 in the NCBI database, codon optimization was performed with reference to the codon preference of Saccharomyces cerevisiae. Histidine tag (sequence CATCACCATCACCATCAC) and stop codon (sequence TAA) were added to the C-terminus of the gene. KpnI restriction site sequence (GGTACC) and EcoRI restriction site sequence (GAATTC) were added to both ends of the gene, respectively. The gene was then synthesized by a biotechnology company, and the synthetic plasmid pUC-CUP1 shown in SEQ ID NO. 2 was obtained.

[0027] b. Double digestion of the Saccharomyces cerevisiae surface display vector pYD1 and the synthetic plasmid pUC-CUP1 and ligation The *Saccharomyces cerevisiae* surface display vector pYD1 and the synthetic plasmid pUC-CUP1 were double-digested with restriction endonucleases Kpn I and EcoRI under 37 °C water bath conditions for 2 h to obtain the CUP1 gene fragment and the linearized plasmid vector. The restriction enzyme digestion system of *Saccharomyces cerevisiae* surface display vector pYD1 is shown in Table 2, and the restriction enzyme digestion system of synthetic plasmid pUC-CUP1 is shown in Table 3.

[0028] Table 2. Double enzyme digestion system of pYD1 surface display vector for Saccharomyces cerevisiae carrier pYD1 32.8 EcoR I 1.6 Kpn I 1.6 10 × buffer 4 Table 3. Double enzyme digestion system of the synthesized plasmid pUC-CUP1 Synthetic plasmid pUC-CUP1 32.8 EcoR I 1.6 Kpn I 1.6 10 × buffer 4 The enzyme digestion products were subjected to electrophoresis on a 1% (m / v) agarose gel and observed under UV light. The electrophoresis detection steps are as follows: (1) Select a suitable comb, glue-making board, and empty slot. Place the glue-making board into the appropriate empty slot and insert the comb into the glue-making board for use in pouring glue.

[0029] (2) Weigh 0.25 g agarose and 25 mL of 1×TAE buffer into an Erlenmeyer flask and shake slowly. Heat in a microwave oven for 30 s until the solution is clear and transparent, then add 3 μL of nucleic acid dye.

[0030] (3) Pour the fully dissolved agarose into the prepared gel casting plate, pouring slowly to prevent foaming. After the gel cools, slowly pull out the comb and place the solidified gel and the gel casting plate into the electrophoresis tank of the electrophoresis apparatus.

[0031] (4) Slowly add 1×TAE buffer to the electrophoresis tank until the gel block is submerged.

[0032] (5) Add the marker and sample to the spotting wells in sequence, with each adding 5 μL. If sample recovery is required, add all the sample to the wells and add 7 μL of marker.

[0033] (6) After adding the sample, close the lid and turn on the electrophoresis apparatus, paying attention to the positions of the positive and negative electrodes. The electrophoresis conditions are 100 V for 20 minutes. The detection results are as follows: Figure 1 As shown, where, Figure 1 Lane 1 shows the results of double digestion of the pYD1 vector. Figure 1 Lane 2 shows the results of double digestion of the pUC-CUP1 plasmid, which is as expected and can be recovered by gel. The upper band was recovered.

[0034] (7) Remove the gel and observe it in a UV analyzer. Cut out the expected band and recover the target fragment from the gel according to the DNA gel recovery kit operation steps. Detect the obtained fragment by electrophoresis on a 1% (m / v) agarose gel. See the electrophoresis verification results below. Figure 2 .

[0035] c. Construction of recombination display plasmid 1. The linearized vector fragment recovered in step b was ligated with the CUP1 gene fragment using ligase solution I to obtain the ligation product. The ligation conditions were 16 °C overnight. The ligation system is shown in Table 4.

[0036] Table 4 Connection System Ligase Solution I 10 CUP1 gene fragment 5 linearized vector pYD1 fragment 5 2. Transformation of E. coli: The ligation product and E. coli DH5α cells were mixed and transformed, following the steps below: (1) Quickly remove 100 μl from the -70 °C ultra-low temperature freezer. E. coli DH5α cells were thawed in an ice bath.

[0037] (2) To E. coli Add 10 μl of the ligation product to be transformed into DH5α cell suspension, gently mix, and let stand in an ice bath for 30 min.

[0038] (3) Place the centrifuge tube in a 42 ℃ water bath for 1 min 30 s, and then quickly transfer the centrifuge tube to an ice bath to cool the cells for 3 min. Do not shake the centrifuge tube during this process.

[0039] (4) Add 900 μl of sterile LB medium (without ampicillin) to each centrifuge tube, mix well, and place in a shaker at 37°C and 150 rpm for 45 min. The purpose is to express the relevant resistance marker gene on the plasmid and revive the bacteria.

[0040] (5) Mix the contents of the centrifuge tube thoroughly, and add 100 μL of the transformed E. coli competent cells to an LB agar plate containing ampicillin. Gently spread the cells evenly with a spreader. Incubate the plate at room temperature until the liquid is absorbed. Invert the plate and incubate at 37 °C for 12–16 h. See transformation plate for details. Figure 3 The single colonies that grew were initially identified as transformants of the recombinant plasmid, but further verification is needed.

[0041] 3. Screening and verification of positive clones: Single colonies from transformation plates were picked and inoculated into 5 ml LB-Amp liquid tubes and cultured at 37 ℃ and 180 rpm / min for 12-16 hours. Using the bacterial culture as a template, and CUP1-F as shown in SEQ ID NO. 4 and CUP1-RV as shown in SEQ ID NO. 5 as primers, the CUP1 gene was amplified. The PCR reaction consisted of 30 cycles, and the amplification system and reaction program are shown in Table 5. The PCR results are as follows. Figure 4 As shown, the electrophoretic band in lane 1 is at 220 bp, consistent with the theoretical value, confirming that the recombinant plasmid has been successfully transformed into E. coli.

[0042] Table 5. Bacterial PCR System and Conditions

[0043] d. Transformation of brewer's yeast The recombinant display plasmid pYD1-CUP1 was extracted from the strain verified in step c using a plasmid mini-extraction kit. The recombinant display plasmid pYD1-CUP1 was then transformed into the tryptophan-auxotrophic Saccharomyces cerevisiae strain EBY100 using the lithium acetate method, as follows: (1) Preparation of competent yeast cells ① Inoculate the activated EBY100 strain into 50 mL of YPD liquid medium and culture overnight at 28 ℃ and 200 rpm / min with shaking until the logarithmic growth phase.

[0044] ② Centrifuge in a 50 mL sterile centrifuge tube at 2500 rpm for 5 min, and discard the supernatant.

[0045] ③ Place the suspended cells in 1 mL of 1×TE, centrifuge at 3000 rpm for 4 min, and discard the supernatant.

[0046] ④ Suspend cells in 600 μL of 0.1M pH6.0 LiAc.

[0047] ⑤ Incubate in a 28°C water bath for 1 hour without shaking. Centrifuge at 3000 rpm for 4 minutes and discard the supernatant.

[0048] ⑥ Gently suspend the cells in 120 μL of 0.1 M pH 6.0 LiAc.

[0049] (2) Transformation ① Take 40 μL of competent cells, 2 μL of fish sperm DNA, and 3 μL of transformed DNA.

[0050] ② Water bath at 28 ℃ for 15 min.

[0051] ③ Add 350 μL of 40% PEG-4000 and 16 μL of 1mol / L DTT.

[0052] ④ Bathe in a 28°C water bath for 1 hour, without shaking.

[0053] ⑤ Add 40 μL DMSO (final concentration 10%).

[0054] ⑥ Heat shock at 39 ℃ for 10 min.

[0055] ⑦ Add 400 μL of 0.1 M pH 6.0 LiAc, spread it on an MD plate for screening, and incubate at 28 ℃ for 2-3 days until transformants appear on single copies.

[0056] ⑧ Select a single colony of the well-grown yeast and inoculate it into 5 mL of liquid YPD medium. After culturing for 12-16 hours, preserve the culture and name it CUP-16 strain.

[0057] Activate the recombinant yeast strain and extract yeast genomic DNA using a yeast genomic DNA extraction kit, following the instructions.

[0058] After extraction, recombinant yeast genomic DNA was used as the template for PCR amplification. Primers CUP1-F (SEQ ID NO. 4) and CUP1-RV (SEQ ID NO. 5) were used to amplify and verify the fragment size. Detailed PCR amplification systems and reaction procedures are shown in Table 5 above. After the PCR reaction, electrophoresis was performed on a 1% agarose gel, and the results are shown below. Figure 5 .from Figure 5 As can be seen, the selected yeast strain CUP-16 was verified as positive.

[0059] The adsorption performance of the engineered strains displaying metallothionein on the surface constructed in this embodiment was tested, and the specific process is as follows: Pretreatment of bacterial cells: Take a 10 mL centrifuge tube, add 3 mL of engineered CUP-16 bacterial suspension, centrifuge at 12,000 rpm for 5 min, and discard the supernatant; add 2 mL of deionized water to resuspend the bacterial cells, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and repeat the washing twice to remove culture medium residue.

[0060] Enrichment reaction: Add 5 mL of 10 mg / L CuSO4 solution to a 10 mL centrifuge tube, resuspend the washed bacterial cells in it, and shake gently; transfer the mixture to a 50 mL centrifuge tube and place it in a constant temperature shaker (28℃, 180 rpm) for 12 h.

[0061] Sample detection: After the reaction was completed, the sample was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected. The Cu content in the supernatant was determined using an atomic absorption spectrophotometer (AAS). 2+ Concentration. The absorbance of a 10 mg / L CuSO4 solution was measured separately.

[0062] Preparation of standard curve: Select a 0-5 mg / L CuSO4 solution and measure its absorbance to prepare a standard curve, such as... Figure 6 As shown in Table 6, the experimental results demonstrate that the engineered strain CUP-16 achieved a copper enrichment rate of 89.95%, significantly higher than that of strain EBY100. This indicates that the present invention successfully constructed an engineered strain for displaying metallothionein surface and efficiently enriching copper ions.

[0063] Table 6. Results of copper enrichment performance tests for strains CUP-16 and S21 Copper enrichment performance test experimental group (engineered bacteria CUP-16 strain): 0.134 89.9% Control group (EBY100 strain): 0.856 36.0%

Claims

1. A method for constructing a highly efficient copper-ion-enriched metallothionein yeast surface-displaying engineered strain, characterized in that, Includes the following steps: a. Obtaining the plasmid for CUP1 gene synthesis: Based on the gene sequence of the metallothionein gene CUP1 shown in SEQ ID NO. 1 in the NCBI database, codon optimization was performed with reference to the codon preference of Saccharomyces cerevisiae. Histidine tag and stop codon were added to the C-terminus of the gene, and Kpn I restriction site sequence GGTACC and EcoR I restriction site sequence GAATTC were added to both ends of the gene, respectively, to further obtain the synthetic plasmid pUC-CUP1 shown in SEQ ID NO. 2; b. Double digestion: The surface display vector pYD1 of Saccharomyces cerevisiae and the synthetic plasmid pUC-CUP1 obtained in step a were double digested with restriction endonucleases Kpn I and EcoRI. The CUP1 gene fragment and linearized plasmid vector were obtained by water bath and then detected and verified by electrophoresis. c. Construction of recombinant display plasmid: The linearized vector fragment recovered in step b was ligated with the CUP1 gene fragment using solution I to obtain the ligation product; the ligation product was transformed into Escherichia coli competent cells DH5α and verified by PCR amplification to construct a strain containing the recombinant display plasmid pYD1-CUP1 as shown in SEQ ID NO 3; d. Saccharomyces cerevisiae transformation: The recombinant display plasmid pYD1-CUP1 was extracted from the strain verified in step c. The recombinant display plasmid pYD1-CUP1 was transformed into tryptophan auxotrophic Saccharomyces cerevisiae EBY100 using the lithium acetate method. Transformants were screened by MD plates, and yeast genomic DNA was extracted and used as a template for PCR amplification. PCR amplification was performed using CUP1-F as shown in SEQ ID NO. 4 and CUP1-RV as shown in SEQ ID NO. 5 as primers for verification. e. Validation of engineered bacteria: Determine the adsorption performance of recombinant strains and screen out strains with high enrichment capacity.

2. The method for constructing a highly efficient copper-ion-enriched metallothionein yeast surface-displaying engineered strain according to claim 1, characterized in that, In step b, the reaction was carried out in a water bath at 37℃ for 2 h. The enzyme digestion system for the Saccharomyces cerevisiae surface display vector pYD1 was: 32.8 μL of vector pYD1, 1.6 μL of Kpn I, 1.6 μL of EcoRI, and 4 μL of 10 × buffer. The enzyme digestion system for the synthetic plasmid pUC-CUP1 was: 32.8 μL of synthetic plasmid pUC-CUP1, 1.6 μL of Kpn I, 1.6 μL of EcoRI, and 4 μL of 10 × buffer.

3. The method for constructing a highly efficient copper-ion-enriched metallothionein yeast surface-displaying engineered strain according to claim 1, characterized in that, In step c, ligation was performed overnight at 16°C using ligase solution I. The ligation system consisted of 10 μL of ligase solution I, 5 μL of the CUP1 gene fragment, and 5 μL of the recovered linearized plasmid vector fragment.

4. The method for constructing a highly efficient copper-ion-enriched metallothionein yeast surface-displaying engineered strain according to claim 1, characterized in that, In step c, the verification process is as follows: after screening on LB plates, single colonies are picked and inoculated for culture. Using the bacterial culture as a template, and using CUP1-F as shown in SEQ ID NO 4 and CUP1-RV as shown in SEQ ID NO 5 as primers, bacterial culture PCR verification is performed.

5. The method for constructing a highly efficient copper-ion-enriched metallothionein yeast surface-displaying engineered strain according to claim 5, characterized in that, In steps c and d, the PCR amplification system was: 10 μL of 2 × Premix Taq, 0.4 μL of CUP1-F, 0.4 μL of CUP1-RV, 0.4 μL of bacterial culture, and 8.8 μL of deionized water; the PCR amplification program was: pre-denaturation, 94 ℃, 4 min; denaturation, 94 ℃, 30 s; annealing, 55 ℃, 30 s; extension, 72 ℃, 30 s; final extension, 72 ℃, 10 min.

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