A recombinant pichia pastoris indirectly displaying a petase protein on the surface

Abstract

The application discloses a kind of recombinant Pichia pastoris for indirectly surface display PETase protein, comprising: (1) 3×IM7-SED1 fusion protein is expressed heterologously in Pichia pastoris GS115, and constructs IM7 yeast.(2)CL7-PETase fusion protein is expressed heterologously in Escherichia coli BL21 (DE3), and constructs CL7-PETase protein.(3)IM7 yeast and CL7-PETase protein are combined, and the surface display of PETase protein on Pichia pastoris is realized.The construction method of indirect surface display used in the application overcomes the disadvantage of insufficient expression of direct surface display system, and compared with free enzyme, has the characteristics of high stability, easy separation and recovery, controllable continuous operation, simple process, and the surface display enzyme is different from traditional physical or chemical immobilization method.For PET rigid substrate, surface display enzyme can more fully and effectively contact with substrate, maximum catalysis substrate, and can be used as whole-cell catalyst in the application of PET plastic degradation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to a recombinant Pichia pastoris that indirectly displays PETase protein on its surface. Background Technology

[0002] PET is a widely used high-molecular-weight aromatic polyester composed of terephthalic acid (TPA) and ethylene glycol (EG) linked by ester bonds. In 2015, global PET production reached approximately 27.8 million tons, primarily used to manufacture packaging materials and beverage bottles. PET possesses excellent comprehensive properties, such as light weight, high strength, resistance to high and low temperatures, chemical corrosion resistance, good insulation, and high transparency. However, while the strong demand for PET brings convenience, it also creates a global problem: how to effectively dispose of post-consumer PET, or PET waste that, due to its durability, can accumulate in the environment and remain there for hundreds of years? Multiple studies have shown that over 690 marine organisms have accumulated PET fragments and microplastics in their bodies, meaning that PET has already spread widely in human food.

[0003] Currently, various strategies have been developed for recycling used PET, including chemical, physical, and biological treatments. In recent years, bio-recycling of plastic waste has attracted considerable attention due to its relatively mild, environmentally friendly, and safe operating conditions, and is expected to become one of the methods for plastic degradation. Biodepolymerization and conversion technology involves the selective depolymerization of waste plastics into different polymer monomers using microorganisms / enzymes under mild conditions, as well as the high-value-added biorefining process of mixed plastic degradation products. Plastics are man-made polymer materials, designed from the outset with characteristics such as dense structure, strong hydrophobicity, high molecular crystallinity, and stable bond energy. Therefore, it was traditionally believed that neither microorganisms nor enzymes could efficiently degrade plastic polymers. However, recent breakthroughs in the field of plastic biodegradation, especially in the enzymatic degradation of PET plastics, have led to a new understanding of plastic biodegradation.

[0004] Polyethylene terephthalate degrading enzyme (PETase) can degrade PET into mono(2-hydroxyethyl) terephthalic acid or terephthalic acid. Its biggest advantage is that, compared with other enzymes in fungi that can degrade PET, such as LCC, TfH, and FsC, its enzyme activity in the mesophilic range (30-40℃) is significantly higher than that of other enzymes. This means that it is easier to achieve suitable reaction conditions in practical applications.

[0005] Yeast surface-display enzymes retain their highly efficient, specific, and mild enzymatic catalytic properties while overcoming the shortcomings of free enzymes. They exhibit advantages such as high storage stability, easy separation and recovery, controllable continuous operation, and simple process. Furthermore, using indirect surface display systems can increase the protein display amount of PETase on the yeast surface. However, there are currently no reports on the indirect display of PETase on cell surfaces. Therefore, constructing indirect surface-display strains of PETase for PET transformation has significant practical implications. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant Pichia pastoris in response to the shortcomings of the prior art.

[0007] Another technical problem that this invention aims to solve is to provide a method for constructing the above-mentioned recombinant Pichia pastoris.

[0008] The final technical problem to be solved by the present invention is to provide the application of the above-mentioned recombinant Pichia pastoris in the indirect surface display of PETase protein.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] A recombinant Pichia pastoris strain that indirectly displays PETase protein was constructed by combining IM7 yeast and CL7-PETase protein.

[0011] The IM7 yeast is obtained by heterologously expressing the 3×IM7-SED1 fusion protein in Pichia pastoris as the host.

[0012] The CL7-PETase protein was obtained by heterologously expressing the CL7-PETase fusion protein in Escherichia coli BL21(DE3) and then culturing it.

[0013] The Pichia pastoris mentioned is Pichia pastoris GS115 (purchased from Beyotime Biotechnology Co., Ltd.).

[0014] The above-mentioned method for constructing recombinant Pichia pastoris includes the following steps:

[0015] (1) Construction of IM7 yeast:

[0016] (1a) Construction and transformation of recombinant plasmid: The constructed recombinant plasmid pPICZαA-HA-3×IM7-SED1 (synthesized by Nanjing Genscript Biotech Co., Ltd.) was transformed into Pichia pastoris GS115 by electroporation to obtain a recombinant strain displaying the IM7 protein surface.

[0017] (1b) The recombinant bacterial IM7 protein surface display strain obtained in step (1a) is inoculated and cultured and induced to produce enzyme. The anchoring protein SED1 will fix the linked IM7 protein on the yeast surface, resulting in IM7 yeast with IM7 fixed on the surface.

[0018] (2) Construction of CL7-PETase protein:

[0019] (2a) Construction and transformation of recombinant plasmid: The recombinant plasmid pET-22b-CL7-PETase (synthesized by Nanjing Genscript Biotech Co., Ltd.) was transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterial CL7-PETase protein expression strain;

[0020] (2b) The recombinant bacterial strain CL7-PETase protein expression obtained in step (2a) was inoculated and cultured and induced to produce enzyme to obtain CL7-PETase protein.

[0021] (3) Combine the IM7 yeast obtained in step (1b) and the CL7-PETase protein obtained in step (2b) to construct recombinant Pichia pastoris.

[0022] In step (1a), the IM7 protein has the nucleotide sequence shown in SEQ ID NO:1, and the SED1 protein has the nucleotide sequence shown in SEQ ID NO:2.

[0023] In step (1a), the electroporation method is as follows: the plasmid pPICZαA-HA-3×IM7-SED1 is cut into a linearized plasmid using sacⅠ enzyme and recovered. Prepared Pichia pastoris GS115 competent cells are taken out, 1-3 μg of the linearized plasmid is added, gently mixed, and placed on ice for 5 min before being transferred to a 0.2 cm electroporation cuvette. The electroporation instrument parameters are set as follows: voltage 1.5 kV, resistance 250 Ω, capacitance 25 μF. Electroporation is then performed. Immediately after electroporation, 1 mL of pre-chilled sorbitol is added to the electroporation cuvette, then transferred to a 1.5 mL centrifuge tube and incubated at 30℃ for 1 h. After incubation, the cells are centrifuged at 4000 rpm for 3 min at room temperature, the supernatant is discarded, the cells are resuspended in YPDS medium, and then plated on YPD plates containing 100 μg / mL bleomycin and incubated at 30℃ for 48 h.

[0024] In step (1b), the recombinant bacterial IM7 protein surface display strain is inoculated and cultured under the following conditions: the recombinant bacterial IM7 protein surface display strain is inoculated into YPD medium, 100 μg / mL of bleomycin is added, and the culture is carried out at 30℃ for 24 h. Then, the bacterial solution is added to BMGY liquid medium at an inoculation rate of 1% v / v and cultured at 30℃ for 24 h. After that, the bacterial solution is transferred to a sterile centrifuge tube and centrifuged at 6000 rpm for 10 min. The supernatant is discarded, and the bacterial cells are resuspended in an equal volume of BMMY liquid medium and then transferred to a shake flask and placed in a shaker at 30℃ for culture. 1% v / v methanol is added every 24 h. After five days of fermentation, the bacterial cells are collected by centrifugation to obtain IM7 yeast.

[0025] In step (2a), the CL7 protein has the nucleotide sequence shown in SEQ ID NO:3, and the PETase protein has the nucleotide sequence shown in SEQ ID NO:4.

[0026] In step (2a), the transformation involves adding 1 μg of plasmid pET-22b-CL7-PETase to *E. coli* BL21(DE3) competent cells (purchased from Zhuangmeng Biotechnology Co., Ltd.), placing them on ice for 30 min, then heat-shocking the competent cells in a 42°C water bath for 90 s, and immediately placing them on ice for 2 min. Subsequently, 900 μL of liquid LB medium is added, and the cells are cultured at 37°C and 200 rpm for 45 min. Finally, the cells are collected by centrifugation at 4000 rpm for 5 min and plated on LB agar plates containing 50 μg / mL ampicillin, then incubated at 37°C for 12 h.

[0027] In step (2b), the recombinant CL7-PETase protein expression strain is inoculated and cultured under the following conditions: the recombinant CL7-PETase protein expression strain is inoculated into LB medium, 50 μg / mL of ampicillin is added, and the culture is carried out at 37°C for 12-16 h. Then, the bacterial solution is added to liquid LB medium at an inoculation rate of 1% v / v and cultured at 37°C until the bacterial concentration reaches OD200. 600 When the concentration reaches 0.6-0.8, remove the cells, add 0.1mM IPTG, and culture in a shaker at 18℃ for 24h. After centrifuging at 8000rpm for 10min to obtain bacterial cells, use an ultrasonic disruptor to disrupt the cells, centrifuge at 8000rpm for 10min, and collect the supernatant to obtain CL7-PETase protein.

[0028] In step (3), the IM7 yeast and CL7-PETase protein are bound together under the following conditions: the IM7 yeast and CL7-PETase protein are incubated at 4°C for 8 hours in a 100mM Tris-HCl buffer containing 10mM CaCl2 at pH=8.

[0029] Specifically, samples were taken every 1 hour, and the supernatant and bacterial cells were separated by centrifugation at 8000 rpm for 2 min. The bacterial cells were washed 3 times with Tris-HCl buffer, and then the enzyme activity of recombinant Pichia pastoris and supernatant was determined by p-NPB method.

[0030] Specifically, enzyme activity is calculated as follows: Protease activity = A / (B*C), where A is the amount of p-nitrophenol produced (μmol) calculated from the standard curve, B is the reaction time (min), and C is the amount of enzyme added in the reaction (mL). One unit of protease activity is defined as 1 μmol of p-nitrophenol produced per minute through hydrolysis of p-NPB.

[0031] The application of the aforementioned recombinant Pichia pastoris as a whole-cell catalyst in the degradation of PET plastics is also within the scope of protection of this invention.

[0032] Beneficial Effects: The CL7 / IM7 system used in this invention enables the indirect display of PETase on the surface of Pichia pastoris, avoiding complex purification steps, improving separation and purification efficiency, and saving costs. Yeast surface-displayed enzymes maintain the specificity of free enzymes while also possessing the following advantages: (1) Surface-displayed enzymes are easier to recycle and have higher stability than free enzymes. (2) Surface-displayed enzymes are reusable. (3) Unlike traditional physical or chemical immobilization methods, surface-displayed enzymes can more effectively and fully contact the substrate, such as PET, maximizing substrate catalysis. (4) Indirect surface display systems have a higher protein display capacity compared to direct surface display systems. The PETase protein used in this invention can catalyze the biodegradation of PET plastic, which is of great significance for the biodegradation of PET and other plastics. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 A schematic diagram of the PETase protein displayed on the indirect surface of the CL7 / IM7 system.

[0035] Figure 2 The image shows the recombinant plasmid pPICZαA-HA-3×IM7-SED1.

[0036] Figure 3 The image shows the pET-22b-CL7-PETase recombinant plasmid.

[0037] Figure 4 This is a schematic diagram of IM7 yeast after immunofluorescence staining under a laser confocal microscope.

[0038] Figure 5 This is a schematic diagram of IM7 yeast binding to CL7-mCherry fluorescent protein under a fluorescence microscope.

[0039] Figure 6 This is the standard curve for p-nitrophenol.

[0040] Figure 7 The p-NPB method was used to detect changes in enzyme activity in recombinant Pichia pastoris and its supernatant during the binding of IM7 yeast with CL7-PETase. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0042] The culture media used in the following examples are as follows:

[0043] The LB medium formula is: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0044] The YPD medium formula is: 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast extract.

[0045] The formula for the yeast competent cell mother liquor is: 100mM lithium acetate, 10mM dithiothreitol, 0.6M sorbitol, and 10mM Tris-HCl.

[0046] The YPDS medium formula is: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, and 1 M sorbitol.

[0047] The formula for BMGY liquid culture medium is: 20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB (containing ammonium sulfate), 3.01 g / L K2HPO4·3H2O, 11.81 g / L KH2PO4, 0.4 mg / L biotin, and 1% glycerol.

[0048] The BMMY liquid culture medium formula is: 20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB (containing ammonium sulfate), 3.01 g / L K2HPO4·3H2O, 11.81 g / L KH2PO4, 0.4 mg / L biotin, and 1% methanol.

[0049] like Figure 1 The diagram shown is a schematic of the indirect surface display of PETase on the CL7 / IM7 system.

[0050] Example 1: Surface display of IM7 protein on Pichia pastoris and heterologous expression of CL7-PETase in Escherichia coli

[0051] (1) Construction and transformation of recombinant plasmids

[0052] The recombinant plasmid pPICZαA-HA-3×IM7-SED1 synthesized by Nanjing Genscript Biotech Co., Ltd. (plasmid map shown) Figure 2 As shown, the nucleotide sequence of the IM7 protein is shown in SEQ ID NO:1, and the nucleotide sequence of the SED1 protein is shown in SEQ ID NO:2. The IM7 protein surface display strain was obtained by transforming the strain into Pichia pastoris GS115.

[0053] The Pichia pastoris conversion method is as follows:

[0054] Preparation of competent cells for Pichia pastoris GS115: Pichia pastoris GS115 strain was inoculated into YPD test tube medium and cultured overnight at 30°C and 200 rpm. The cultured bacterial solution was then added to 50 mL of liquid YPD medium at a 5% v / v inoculation rate and cultured at 30°C and 200 rpm until OD reached [value missing]. 600 The bacterial cells were collected by centrifugation at 4500 rpm for 5 min, with a pH between 0.3 and 0.5. The cells were gently mixed with freshly prepared competent yeast cell stock solution and incubated at room temperature for 30 min, gently shaken every 10 min. The cells were then collected by centrifugation at 4500 rpm for 5 min. The cells were resuspended in 2 mL of pre-chilled 1M sorbitol and centrifuged at 4000 rpm for 5 min at 4 °C. This process was repeated three times. The cells were then resuspended in 1 mL of pre-chilled 1M sorbitol and aliquoted into 1.5 mL centrifuge tubes (80 μL per tube) and stored at -80 °C.

[0055] Pichia pastoris electroporation method: The plasmid pPICZαA-HA-3×IM7-SED1 was digested into a linearized plasmid using sacI enzyme and recovered. Prepared Pichia pastoris GS115 competent cells were taken, and 1-3 μg of the linearized plasmid was added. After gentle mixing, the cells were placed on ice for 5 min and then transferred to a 0.2 cm electroporation cuvette. The electroporator parameters were set as follows: voltage 1.5 kV, resistance 250 Ω, capacitance 25 μF. Immediately after electroporation, 1 mL of pre-chilled sorbitol was added to the cuvette, and then transferred to a 1.5 mL centrifuge tube. The cuvette was incubated at 30℃ for 1 h. After incubation, the cells were centrifuged at 4000 rpm for 3 min at room temperature, the supernatant was discarded, and the cells were resuspended in YPDS medium. The cells were then plated on YPD plates containing 100 μg / mL bleomycin and incubated at 30℃ for 48 h.

[0056] The recombinant plasmid pET-22b-CL7-PETase synthesized by Nanjing Genscript Biotech Co., Ltd. (plasmid map shown) Figure 3 As shown in SEQ ID NO:3, the nucleotide sequence of CL7 protein is shown in SEQ ID NO:4. The nucleotide sequence of PETase protein is shown in SEQ ID NO:4. The strain expressing CL7-PETase protein was obtained by transforming CL7-PETase protein into Escherichia coli BL21(DE3).

[0057] The transformation method for E. coli is as follows:

[0058] 1 μg of plasmid pET-22b-CL7-PETase was added to E. coli BL21(DE3) competent cells (purchased from Zhuangmeng Biotechnology Co., Ltd.). The cells were placed on ice for 30 min, then heat-shocked in a 42°C water bath for 90 s, and immediately placed on ice for 2 min. 900 μL of liquid LB medium was then added, and the cells were incubated at 37°C and 200 rpm for 45 min. Finally, the cells were collected by centrifugation at 4000 rpm for 5 min and plated on LB agar plates containing 50 μg / mL ampicillin. The plates were then incubated at 37°C for 12 h.

[0059] (2) Inoculation and culture of IM7 protein surface-displaying strains and CL7-PETase protein-expressing strains

[0060] The IM7 protein surface display strain was inoculated into YPD medium, and 100 μg / mL of bleomycin was added. After culturing at 30°C for 24 h, the bacterial solution was added to BMGY liquid medium at a 1% v / v inoculation rate and cultured at 30°C for 24 h. The bacterial solution was then transferred to a sterile centrifuge tube and centrifuged at 6000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in an equal volume of BMMY liquid medium and transferred to a shake flask. The flask was then placed in a shaker at 30°C and cultured. 1% v / v methanol was added every 24 h. After five days of fermentation, the cells were collected by centrifugation to obtain IM7 yeast.

[0061] The CL7-PETase protein-expressing strain was inoculated into LB medium, and 50 μg / mL ampicillin was added. After culturing at 37°C for 12-16 h, the bacterial culture was added to liquid LB medium at a 1% v / v inoculation rate and cultured at 37°C. When the bacterial concentration reached OD600... 600 When the concentration reaches 0.6-0.8, remove the cells, add 0.1mM IPTG, and culture in a shaker at 18℃ for 24h. After centrifuging at 8000rpm for 10min to obtain bacterial cells, use an ultrasonic disruptor to disrupt the cells, centrifuge at 8000rpm for 10min, and collect the supernatant to obtain CL7-PETase protein.

[0062] Example 2: Identification of IM7 protein surface display effect

[0063] (1) Immunofluorescence staining preliminarily identified the display of IM7 protein on the cell surface.

[0064] The IM7 yeast cultured in Example 1 was resuspended in PBS solution and diluted to OD. 600 =1. Take 1 mL of diluted bacterial suspension, add 1 mg / mL BSA to the 1 mL bacterial suspension and incubate at 4°C for 1 h. Then, add 1 μL of mouse anti-HA tag monoclonal antibody (purchased from Beyotime Biotechnology Co., Ltd.) to 1000 μL of cell suspension and incubate at room temperature for 2 h. Next, wash the cells 3 times with PBS solution, resuspend in 200 μL of PBS solution, add 1 μL of FITC-conjugated goat anti-mouse IgG (H+L) antibody (purchased from Beyotime Biotechnology Co., Ltd.), and incubate at room temperature for 1 h. Finally, wash the cells 3 times with PBS solution, resuspend in 1 mL of PBS solution and observe with a laser confocal microscope. The results are as follows. Figure 4 As shown, obvious green fluorescence was observed on the surface of Pichia pastoris cells, proving that the HA tag of the IM7 protein on the cell surface successfully bound to the primary antibody and to the secondary antibody containing FITC, thereby fixing the green fluorescent FITC on the cell surface. After excitation by laser confocal microscopy, obvious green fluorescence could be observed, indicating that the IM7 protein was successfully expressed on the surface of Pichia pastoris.

[0065] (2) Identification of the binding ability of CL7 to IM7 by IM7 yeast and CL7-mCherry fluorescent protein

[0066] The IM7 yeast cultured in Example 1 was resuspended in 100 mM Tris-HCl buffer and diluted to OD. 600 =1. Take 1 mL of diluted bacterial suspension, add 10 mM CaCl2 and 1 mg CL7-mCherry protein. After incubation at 4℃ for 4 h, wash the cells 3 times with Tris-HCl buffer, resuspend in 1 mL Tris-HCl buffer, and observe under a fluorescence microscope. The results are as follows. Figure 5 As shown, after the CL7 protein, which is linked to the red fluorescent protein of mCherry, binds to the IM7 protein on the cell surface, the CL7 protein interacts with the IM7 protein, thereby fixing the mCherry protein along with the CL7 protein to the cell surface. After being excited by fluorescence microscopy, obvious red fluorescence was observed on the surface of Pichia pastoris, indicating that the surface IM7 protein and CL7 protein successfully bound together.

[0067] Example 3: Identification of the indirect surface display effect of PETase protein

[0068] The IM7 yeast and CL7-PETase protein obtained in Example 1 were incubated at 4°C for 8 hours in 100 mM Tris-HCl buffer containing 10 mM CaCl2 at pH 8. Samples were taken every 1 hour, and the supernatant and bacterial cells were separated by centrifugation at 8000 rpm for 2 min. The bacterial cells were washed three times with Tris-HCl buffer, and the enzyme activity of the supernatant and recombinant Pichia pastoris was determined by the p-NPB method. The results are as follows: Figure 7 As shown, the enzyme activity in the supernatant gradually decreased, while the enzyme activity in the recombinant Pichia pastoris gradually increased, indicating that the CL7-PETase protein gradually bound to IM7 yeast, successfully displaying active PETase on the surface of Pichia pastoris.

[0069] The method for determining enzyme activity using p-NPB is as follows:

[0070] Preparation of p-nitrophenol standard curve: A 2 mM p-nitrophenol stock solution was prepared with isopropanol, and then diluted with PBS to concentrations of 10 μM, 20 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, and 200 μM, respectively. The OD values ​​of p-nitrophenol at different concentrations were measured using a microplate reader. 410 The absorbance was measured, and a standard curve was constructed (e.g., ...). Figure 6 ).

[0071] Prepare 10 mM p-NPB with isopropanol. Add 980 μL PBS, 10 μL p-NPB, and 10 μL enzyme solution to a 2 mL centrifuge tube. Incubate at 37°C for 5 min, then measure the OD value using a microplate reader. 410 The absorbance was measured, and the concentration of p-nitrophenol produced was determined by comparing it with the standard curve.

[0072] Enzyme activity calculation: Protease activity = A / (B*C), where A is the amount of p-nitrophenol produced (μmol) calculated from the standard curve, B is the reaction time (min), and C is the amount of enzyme added in the reaction (mL). One unit of protease activity is defined as 1 μmol of p-nitrophenol produced per minute by hydrolysis of p-NPB.

[0073] This invention provides a method and approach for indirectly displaying PETase protein on the surface of recombinant Pichia pastoris. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of a recombinant Pichia pastoris containing indirect surface-displayed PETase protein as a whole-cell catalyst in the degradation of PET plastics, wherein the recombinant Pichia pastoris is constructed by combining IM7 yeast and CL7-PETase protein. in, The IM7 yeast was obtained by heterologously expressing the 3×IM7-SED1 fusion protein in Pichia pastoris as a host, and the Pichia pastoris was *Pichia pastoris*. Pichia pastoris GS115; The CL7-PETase protein was obtained by heterologously expressing the CL7-PETase fusion protein in Escherichia coli BL21(DE3) and then culturing it. The binding conditions for IM7 yeast and CL7-PETase protein are as follows: incubation at 4°C for 8 h in 100 mM Tris-HCl buffer containing 10 mM CaCl2 at pH 8.

0. The nucleotide sequence of the IM7 protein is shown in SEQ ID NO:1, the nucleotide sequence of the SED1 protein is shown in SEQ ID NO:2, the nucleotide sequence of the CL7 protein is shown in SEQ ID NO:3, and the nucleotide sequence of the PETase protein is shown in SEQ ID NO:

4.

2. The application according to claim 1, characterized in that, The method for constructing the recombinant Pichia pastoris includes the following steps: (1) Construction of IM7 yeast: (1a) Construction and transformation of recombinant plasmid: The constructed recombinant plasmid pPICZαA-HA-3×IM7-SED1 was transformed into Pichia pastoris GS115 by electroporation to obtain a recombinant strain displaying the IM7 protein surface. (1b) The recombinant bacterial IM7 protein surface display strain obtained in step (1a) is inoculated and cultured and induced to produce enzyme. The anchoring protein SED1 will fix the linked IM7 protein on the yeast surface to obtain IM7 yeast with IM7 fixed on the surface. (2) Construction of CL7-PETase protein: (2a) Construction and transformation of recombinant plasmid: The recombinant plasmid pET-22b-CL7-PETase was transformed into Escherichia coli BL21(DE3) to obtain a recombinant bacterial strain expressing CL7-PETase protein; (2b) The recombinant bacterial strain expressing CL7-PETase protein obtained in step (2a) was inoculated and cultured and induced to produce enzyme to obtain CL7-PETase protein; (3) Combine the IM7 yeast obtained in step (1b) and the CL7-PETase protein obtained in step (2b) to construct recombinant Pichia pastoris.

3. The application according to claim 2, characterized in that, In step (2b), the recombinant CL7-PETase protein-expressing strain is inoculated and cultured under the following conditions: 50 μg / mL ampicillin is added to LB medium and cultured at 37°C for 12-16 h. Then, the bacterial solution is added to liquid LB medium at an inoculation rate of 1% v / v and cultured at 37°C until the bacterial concentration reaches OD200. 600 When the pH reaches 0.6-0.8, remove the sample and add 0.1 mM IPTG, then incubate at 18℃ for 24 h.

4. The application according to claim 2, characterized in that, In step (1b), the recombinant bacterial IM7 protein surface display strain is inoculated and cultured under the following conditions: 100 μg / mL of bleomycin is added to YPD medium and cultured at 30°C for 24 h. The bacterial solution is then added to BMGY liquid medium at an inoculation rate of 1% v / v and cultured at 30°C for 24 h. After centrifugation, the culture is resuspended in an equal volume of BMMY liquid medium and cultured at 30°C again. 1% v / v methanol is added every 24 h, and fermentation is carried out for five days.

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