Construction method and application of novel multi-enzyme display platform for efficiently degrading PET (Polyethylene Terephthalate) plastic

Abstract

The invention discloses a construction method and application of a novel multi-enzyme display platform for efficiently degrading PET (Polyethylene Terephthalate) plastic, and belongs to the technical field of plastic waste biodegradation. According to the invention, an integrated self-assembly multi-enzyme display platform (ISA-MEDP) based on a SpyTag-SpyCatcher system is constructed in candida tropicalis, Tag is used as a scaffold to be fused with an endogenous anchoring protein gene, and Catcher is respectively fused with FAST-PETase and MHETase genes. The recombinant strain DFM2 constructed by the invention can completely depolymerize a PET film within 7-13 days, can be applied to PET plastic degradation as a whole-cell catalyst, has the characteristics of easiness in separation and recovery, controllability in continuous operation and simplicity and convenience in process, and has a large-scale application prospect.

Description

Technical Field

[0001] The present invention relates to a construction method and application of a novel multi-enzyme display platform for efficiently degrading PET plastics, belonging to the technical field of biodegradation of plastic waste. Background Art

[0002] Plastics are important basic materials that bring great convenience to human production and life. More than 36 million tons of plastics are produced every year, and among them, PET plastic is one of the most widely used plastics, mainly used in food packaging and textile fibers. Due to its excellent mechanical properties and moisture resistance, it can exist in nature for hundreds of years, inevitably causing environmental pollution and an increase in CO2 emissions. And many studies have found that PET waste will enter the animal body in the form of microplastics, ultimately endangering human health.

[0003] Recycling PET waste is an important means to solve PET pollution, and how to recycle waste is currently the focus of attention of scientists. Here, some strategies have been developed. Through physical thermomechanical recycling, however, it will cause a decline in the mechanical properties of PET materials. Through chemical recycling, it often causes high energy consumption and secondary pollution. The biological method, because of its environmental protection and energy saving, conforms to the green route and is becoming a potential way to solve this problem.

[0004] The key to biological degradation is the enzyme. In recent years, researchers have identified a novel PET hydrolase called PETase. Compared with other well-known PET hydrolases, this enzyme has several different characteristics. In addition to its high substrate specificity for PET and maintaining enzyme activity at low temperatures such as 30 °C, the most valuable characteristic of PETase is that it shows enzyme activity towards PET with high crystallinity. These unique characteristics make PETase a promising candidate in the field of PET recycling. However, the enzyme activity of PETase towards PET is still very low, inhibited by the substrate, and the high purification cost and short working life of free enzymes limit its application in large-scale recycling. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a strain capable of efficiently degrading PET plastics, which contains an integrated multi-enzyme surface display platform (ISA-MEDP) constructed based on the SpyTag-SpyCatcher system and the SnoopTag-SnoopCatcher system. FAST-PETase and MHETase are co-displayed on Candida tropicalis, and then they play a synergistic role in degrading PET plastics.

[0006] The present invention provides an expression cassette, including (a) or (b):

[0007] (a) Display platform expression cassette: containing the coding genes of signal peptide S.S, carbohydrate module CBM, V5-tag, scaffold protein SpyTag (ST), scaffold protein SnoopTag (SNT) and endogenous anchor protein CtGCW65;

[0008] (b) Plastic-degrading enzyme expression cassette: containing the coding gene fragments of signal peptide S.S and plastic-degrading enzyme, capture proteins SpyCatcher and SnoopCatcher.

[0009] In one embodiment, the display platform expression cassette has the structure shown as S.S-CBM-V5-ST-SNT-CtGCW65, S.S-CBM-V5-ST-SNT-ST-CtGCW65 or S.S-CBM-V5-ST-ST-SNT-ST-CtGCW65; wherein, S.S is the signal peptide coding sequence, FAST or MHETase is the PET-degrading enzyme gene sequence, 6xHis, cMyc or V5 is the coding sequence of the protein tag; ST or STN is the coding sequence of the scaffold protein; SC or SNC is the coding sequence of the capture protein; CBM is the carbohydrate module coding sequence; CtGCW65 is the coding gene of the endogenous anchor protein CtGCW65.

[0010] In one embodiment, the plastic-degrading enzyme expression cassette is S.S-FAST-6xHis-SC or S.S-MHETase-cMyc-SNC; wherein, S.S is the signal peptide coding sequence, FAST or MHETase is the PET-degrading enzyme gene sequence, 6xHis and cMyc are the coding sequences of the protein tags.

[0011] The present invention provides recombinant Candida tropicalis for expressing plastic-degrading enzymes based on the SpyTag-SpyCatcher system and the SnoopTag-SnoopCatcher system; the SpyTag-SpyCatcher system includes scaffold protein SpyTag (ST) and capture protein SpyCatcher (SC); the SnoopTag-SnoopCatcher system includes scaffold protein SnoopTag (SNT) and capture protein SnoopCatcher (SNC); the scaffold protein is fusion-expressed with the carbohydrate module CBM; the capture protein is fusion-expressed with the plastic-degrading enzyme.

[0012] In one embodiment, the plastic-degrading enzyme includes but is not limited to PET-degrading enzyme.

[0013] In one embodiment, the plastic-degrading enzyme is FAST-PETase (FAST) and MHETase.

[0014] In one embodiment, the expression of the scaffold protein and / or the plastic-degrading enzyme is regulated by a non-constitutive promoter.

[0015] In one embodiment, the recombinant Candida tropicalis integrates the expression cassette into the genome.

[0016] In one embodiment, the nucleotide sequence encoding the signal peptide S.S of the display platform is as shown in SEQ ID NO.1; the nucleotide sequence encoding the carbohydrate module CBM is as shown in SEQ ID NO.2.

[0017] In one embodiment, the nucleotide sequences encoding the scaffold proteins ST and SNT, the capture proteins SC and SNC, and the anchoring protein CtGCW65 are as shown in SEQ ID NOs.3 to 7, respectively.

[0018] In one embodiment, the genes of the PET-degrading enzyme include but are not limited to FAST and MHETase; the nucleotide sequence encoding FAST is as shown in SEQ ID NO.8; the nucleotide sequence encoding MHETase is as shown in SEQ ID NO.9.

[0019] In one embodiment, the PET-degrading enzyme tag is linked to a protein tag, and the protein tag includes but is not limited to V5-tag, 6xHis-tag, cMyc-tag; the nucleotide sequences of V5-tag, 6xHis-tag, and cMyc-tag are as shown in SEQ ID NOs.10 to 12, respectively.

[0020] In one embodiment, the promoters regulating the expression cassette include but are not limited to P CTR1 and P GAL1 / P GAL10 ; the nucleotide sequence of the promoter P CTR1 is as shown in SEQ ID NO.13; the nucleotide sequence of the promoter P GAL1 / P GAL10 is as shown in SEQ ID NO.14.

[0021] In one embodiment, the expression of the scaffold CBM-V5-ST-SNT-CtGCW65 or CBM-V5-ST-SNT-ST-CtGCW65 or CBM-V5-ST-ST-SNT-ST-CtGCW65 is controlled by the promoter P CTR1 , the expression of FAST-PETase-SC is initiated by the promoter P GAL10 , and the expression of MHETase-SNC is controlled by the promoter P GAL1 .

[0022] In one embodiment, the display platform S.S-CBM-V5-ST-SNT-ST–CtGCW65 and the PET-degrading enzyme are expressed sequentially.

[0023] In one embodiment, the expression cassette of the PET-degrading enzyme display platform is inserted into the DPP3 locus of the Candida tropicalis genome.

[0024] The present invention also provides a cell catalyst containing the recombinant Candida tropicalis.

[0025] The present invention also provides a method for degrading PET plastics, which is to contact the recombinant Candida tropicalis with PET plastics to degrade PET plastics.

[0026] In one embodiment, the cell catalyst is reacted in a reaction system including but not limited to 50 mM glycine-NaOH (pH 9.0) at 25°C to 50°C for 7 to 13 days.

[0027] In one embodiment, the PET plastics include but are not limited to PET powder or PET film.

[0028] In one embodiment, the particle size of the PET powder is 100 mesh.

[0029] In one embodiment, the diameter of the PET film is ≤ 6 mm.

[0030] In one embodiment, the crystallinity of the PET plastics includes but is not limited to 12.59% to 23.2%.

[0031] In one embodiment, the reaction pH is controlled at 8.0 to 9.0 during the process of degrading PET plastics.

[0032] In one embodiment, the pH of the reaction system is controlled at 8.5 ± 0.2.

[0033] The present invention also provides the application of the recombinant Candida tropicalis or the cell catalyst in the field of plastic degradation.

[0034] Beneficial effects:

[0035] (1) The present invention provides a novel multi-enzyme display platform for the depolymerization of PET plastics. The expression level of the enzyme is controlled by the number of scaffold proteins in one cell, and the expression order is controlled by a non-constitutive promoter. The typical process in which the target protein and the scaffold are expressed in two different cells simplifies the operation steps and realizes the integrated degradation of PET plastics.

[0036] (2) During the process of degrading PET plastics, the operation steps of the integrated self-assembled multi-enzyme display platform (ISA-MEDP) of the present invention are simple. During the reaction process, only the pH needs to be controlled, and there is no need to additionally supplement fresh bacterial liquid or enzyme liquid, avoiding the cumbersome operation of adding free enzymes multiple times to maintain enzyme activity, reducing the operation cost. Moreover, this reaction process can adapt to a lower operation temperature, reducing energy consumption and achieving depolymerization at near ambient temperature.

[0037] (3) The recombinant Candida tropicalis constructed in the present invention can be used as a cell catalyst. When reacting with PET powder at 45 °C for 48 h, the product release amount reaches more than 1.2 mM; when reacting with PET film at 45 °C for 7 to 13 days, complete degradation of the PET film can be achieved. Brief Description of the Drawings

[0038] Figure 1 Schematic diagram for constructing a multi-enzyme display platform.

[0039] Figure 2 Map of recombinant plasmid pDFM-01.

[0040] Figure 3 Immunofluorescence labeling of the display platform.

[0041] Figure 4 Degradation of PET plastics by the multi-enzyme display platform.

[0042] Figure 5 Effect of degrading PET plastics at different temperatures and cell densities (OD 600 ).

[0043] Figure 6 Degradation processes of different PET plastics.

[0044] Figure 7 Comparison of the processes of typical PETase protein degradation and engineering yeast catalyzing PET waste. Detailed Embodiments

[0045] The following combines examples to further describe in detail the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0046] Sequence Description:

[0047] The nucleotide sequence of the signal peptide S.S is shown in SEQ ID NO.1;

[0048] The coding sequence of the carbohydrate module CBM is shown in SEQ ID NO.2;

[0049] The coding sequences of the scaffold protein SpyTag (ST), the scaffold protein SnoopTag (SNT), the capture protein SpyCatcher (SC), and the capture protein SnoopCatcher (SNC) are shown in SEQ ID NO.3 to SEQ ID NO.6 respectively;

[0050] The coding gene of the anchor protein CtGCW65 is shown in SEQ ID NO.7;

[0051] The nucleotide sequence encoding the PET-degrading enzyme FAST-PETase is shown in SEQ ID NO.8; the nucleotide sequence encoding the PET-degrading enzyme MHETase is shown in SEQ ID NO.9;

[0052] The nucleotide sequences of the protein tags V5-tag, 6xHis-tag, and cMyc-tag are shown in SEQ ID NO.10 to SEQ ID NO.12 respectively;

[0053] Promoter P CTR1 The nucleotide sequence is shown in SEQ ID NO.13; Promoter P GAL1 / P GAL10 The nucleotide sequence is shown in SEQ ID NO.14.

[0054] The formula of the culture medium used:

[0055] LB medium (g / L): yeast extract 5, peptone 10, sodium chloride 10.

[0056] MM medium (g / L): YNB 6.7, ammonium sulfate 10, glucose 20.

[0057] YPD medium (g / L): glucose 20, peptone 20, yeast powder 10.

[0058] Galactose medium (g / L): YNB 6.7, ammonium sulfate 10, galactose 20.

[0059] Detection method:

[0060] Flow cytometry detection method:

[0061] First, collect the bacterial cells with a sterile centrifuge tube, and then centrifuge at 6000×g for 5 minutes; then discard the supernatant, add PBS buffer to wash and resuspend the bacterial cells, and centrifuge at 6000×g for 5 minutes again. This washing step is repeated 3 times; after washing, resuspend the bacterial cells with an appropriate amount of PBS containing 1% BSA (bovine serum albumin), and adjust the OD of the sample 600Adjust to 10; then take 100 μL of the cell suspension, add 1 μL of the primary antibody (mouse monoclonal antibody), and let it stand at room temperature for 2 hours, gently shaking continuously during this period to keep the cells in suspension; then centrifuge at 6000×g for 5 minutes; then wash the cells 3 times with 1 mL of PBS solution, each time centrifuging at 6000×g for 5 minutes; then wash the cells once with 1 mL of PBS containing 1% BSA (bovine serum albumin), also centrifuging at 6000g for 5 minutes; then resuspend the cells with 100 μL of PBS containing 1% BSA (bovine serum albumin), add 1 μL of the secondary antibody (FITC or Cy3-labeled goat anti-mouse IgG antibody) to each tube, and let it stand at room temperature in the dark for 1 hour, gently mixing to keep the cells in suspension during this period; then wash the cells 3 times with 1 mL of PBS solution, centrifuging at 6000×g for 5 minutes; finally, suspend the cells with an appropriate amount of PBS solution, take an appropriate amount of the cells and place them in a flow cytometer for detection and record the results.

[0062] Enzyme activity assay of ISA-MEDP whole-cell catalysis:

[0063] Soak 10 mg of PET powder together with the strain in 1000 μL of buffer containing 50 mM glycine-NaOH (pH 9.0). The reaction mixture is incubated at 30 °C for 48 hours. After the reaction is completed, add 3M HCl to adjust the pH to 2, heat at 100 °C for 5 minutes, and then adjust the pH to 8 with 3M NaOH. Then, the supernatant obtained by centrifugation (18000×g, 5 minutes) is subjected to HPLC analysis. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 nmol of product (TPA + MHET) per minute. The enzyme activity on the cell surface was normalized by the dry cell weight of each strain.

[0064] Optimize the temperature and cell optical density (OD 600 ) of the ISA-MEDP whole-cell catalysis reaction:

[0065] To optimize the reaction temperature, soak 10 mg of PET powder with the cells (OD 600 = 10) in 1000 μL of buffer containing 50 mM glycine-NaOH (pH 9.0), and then set different temperatures (25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C) in a shaker (ThermoFisher Scientific, USA) at 800 rpm for the reaction. Similarly, to optimize the reaction OD 600 , react with cells with different OD 600 (2, 5, 10, 15, and 20) at 45 °C.

[0066] Preparation method of the degradation enzyme FAST-PETase:

[0067] The pET28a-FAST plasmid carrying the FAST-PETase encoding gene was transformed into Escherichia coli BL21(DE3) and cultured in LB medium at 37 °C until the OD 600 reached 0.8 - 1.0. Induction was carried out with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 16 °C for 16 h. Cells were collected by centrifugation at 4000×g for 10 min, resuspended in buffer A (25 mM Tris-HCl, 150 mM NaCl, pH 7.5), and lysed by high-pressure homogenization. Cell debris was precipitated by centrifugation at 16000×g for 1 h, and the supernatant was loaded onto a Ni-NTA agarose resin column. After washing with buffer A containing 20 mM imidazole, the protein was eluted with buffer A containing 300 mM imidazole. The purified protein was concentrated to 10 mg / mL in buffer B (50 mM Na2HPO4-HCl, 100 mM NaCl, pH 7.0) for subsequent experiments.

[0068] Degradation of PET film by ISA-MEDP whole cells:

[0069] The PET film and DFM2 cells (OD 600 = 15) were soaked in 1000 μL of buffer containing 50 mM glycine-NaOH pH 9.0. The reaction mixture was incubated at 45 °C. After the reaction, 3 M HCl was added to adjust the pH to 2, heated at 100 °C for 5 min, and then the pH was adjusted to 8 with 3 M NaOH. The supernatant after centrifugation (18000×g, 5 min) was subjected to HPLC analysis.

[0070] Method for detecting products by HPLC:

[0071] The instrument was Agilent1260, and the column was C18; the mobile phase consisted of 80% buffer A (0.1% formic acid in ultrapure water) and 20% buffer B (acetonitrile). The flow rate was 1 mL / min. Separation was carried out at 28 °C, and the detection wavelength was 260 nm.

[0072] Example 1: Construction of gene expression cassette plasmid of ISA-MEDP

[0073] Using the pDFM-01 plasmid (DPP3-gda324-URA3-T PGK1 -SCH-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-CSNC-T 7syn -P CTR1-S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 ) Construction is taken as an example. The specific construction method is as follows:

[0074] (1) Download the signal peptide S.S, CBM, ST, SNT, SC, SNC gene sequences and the sequences of the plastic-degrading enzymes FAST and MHETase from NCBI and send them to the company for codon-optimized synthesis to obtain the nucleotide sequences shown in SEQ ID NO.1-6 and SEQ ID NO.8-9 respectively. Amplify the Candida tropicalis genome with the corresponding primers to obtain the CtGCW65 gene (shown in SEQ ID NO.7) and the promoters P CTR1 (shown in SEQ ID NO.13), P GAL1 and P GAL10 (shown in SEQ ID NO.14). The plasmid Ts-CAT2-gda324-URA3-P-yeGFP-T (published in the paper "Establishment of the Genetic Operating System of Candida tropicalis and Its Application in the Synthesis of Dicarboxylic Acids") linearized by XbaⅠ and SalⅠ and the fragment S.S-FAST-6 × His with a histidine tag linked to ST and FAST are ligated by a one-step ligation kit to obtain the recombinant plasmid Ts-CAT2-gda324-URA3-P-S.S-FAST-6×His-SC-T.

[0075] Similarly, construct the recombinant plasmid Ts-CAT2-gda324-URA3-P-S.S-MHETase-cMyc-SNC-T, and further amplify the S.S-MHETase-cMyc-SNC fragment for standby. The plasmid PBRP01 (Ts-T PGK1 -carB-P FBA1 -P GAPDH -carRP-T ENO1 (published in the paper "Systematic Metabolic Engineering of Candida tropicalis for the Production of Terpenoid Natural Products") and the synthetic fragment S.S-CBM-V5-ST-SNT-CtGCW65, the promoter fragments P CTR1 and P GAL1-10 and the terminator fragment T 7syn (T 7syn published in the paper "Systematic Metabolic Engineering of Candida tropicalis for the Production of Terpenoid Natural Products") are connected by a one-step ligation kit to obtain the recombinant plasmid pGC65-01 (Ts-T PGK1 -P GAL10-GAL1 -T 7syn -P CTR1-S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 )。The linearized pGC65-01 by XhoⅠ was ligated with the fragment S.S-FAST-6×His one-step kit to obtain the recombinant plasmid pGC65-02 (Ts-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -P CTR1 -S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 );The linearized PGC65-02 by NotⅠ was ligated with the fragment S.S-MHETase-cMyc-SNC one-step kit to obtain the recombinant plasmid pGC65-03 (Ts-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-cMyc-SNC-T 7syn -P CTR1 -S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 )。

[0076] (2) The plasmid Ts-DPP3-gda324-URA3 was disclosed in the paper "Metabolic engineering of Candida tropicalis for the production of β-ionone" and the recombinant plasmid Ts-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-cMyc-SNC-T 7syn -P CTR1 -S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 was digested with MluⅠ and ligated by one-step method to obtain the recombinant plasmid pDFM-01 (Ts-DDP3-gda324-URA3-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-cMyc-SNC-T 7syn -P CTR1 -S.S-CBM-V5-ST-SNT-CtGCW65-T ENO1 )( Figure 1 )。

[0077] (3) Referring to the methods of steps (1) to (2), the plasmid pDFM-02 (DPP3-gda324-URA3-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-cMyc-SNC-T 7syn-P CTR1 -S.S-CBM-V5-ST-SNT-ST-CtGCW65

[0078] -T ENO1 ) and pDFM-03 (DPP3-gda324-URA3-T PGK1 -SC-6×His-FAST-S.S-P GAL10-GAL1 -S.S-MHETase-cMyc-SNC-T 7syn -P CTR1 -S.S-CBM-V5-ST-ST-SNT-ST-CtGCW65

[0079] -T ENO1 )。

[0080] (4) Digest the above recombinant plasmids pDFM-01, pDFM-02 and pDFM-03 with MluⅠ to obtain purified fragments for standby for the next transformation.

[0081] Example 2: Construction of recombinant strains

[0082] (1) Using the uracil auxotrophic strain Candida tropicalis CU-208 (disclosed in the paper "Systematic metabolic engineering of Candida tropicalis for the production of terpene natural products") as the starting strain, the knockout cassette pDFM-01, pDFM-02 or pDFM-03 was respectively transferred into Candida tropicalis CU-208 by the LiCl transformation method, and then spread on the MM solid medium to pick transformants. Genomic DNA was extracted and correct transformants were identified by PCR verification (the specific construction method and process refer to the paper "Establishment of the genetic operating system of Candida tropicalis and its application in the synthesis of dibasic acids").

[0083] (2) Send the PCR products to the company for sequencing. After the sequence alignment is correct, the recombinant Candida tropicalis strains are respectively named DFM1, DFM2 and DFM3.

[0084] (3) CU-208, DFM1, DFM2 and DFM3 were labeled with primary antibody and secondary antibody in sequence and then analyzed by flow cytometry. The results showed that compared with the control strain, the recombinant strains all had obvious fluorescence signals ( Figure 3 ), proving that the strain can successfully display FAST and MHETase, indicating that the multi-enzyme display platform is successfully constructed.

[0085] Example 3: Enzyme activity assay of recombinant strains

[0086] The DFM1, DFM2, and DFM3 constructed in Example 2 were streaked on a plate, and single colonies were picked. After culturing in YPD at 30 °C for 18 hours, the cells were collected by centrifugation, washed 3 times with sterile water, and the cells were divided into two equal parts. One part of the cells was cultured in MM liquid medium for 30 hours; the other part of the cells was cultured in MM liquid medium with the addition of a CuSO4 solution with a final concentration of 10 μM for 6 hours, then the cells were collected by centrifugation, transferred to galactose medium for induction for 24 hours, and then washed 3 times with 50 mM glycine-NaOH buffer, and the OD 600 was controlled at 10, and 10 mg of PET powder (100 mesh, crystallinity 23.2%) was used as the substrate to react at 30 °C for 48 hours.

[0087] The results showed that enzyme activities were detected in the strains DFM1, DFM2, and DFM3 after induced expression, while no obvious enzyme activity was detected in the non-induced control group. Among them, the enzyme activity shown by the DFM2 strain reached 220.1 U / (g dry cells), and their release products were almost all TPA, and MHET was almost absent ( Figure 4 ), proving that the FAST and MHETase genes were successfully expressed and active in Candida tropicalis.

[0088] Example 4: Optimization of conditions for the multi-enzyme display platform

[0089] The PET powder used in this example had a particle size of 100 mesh and a crystallinity of 23.2%.

[0090] The strain DFM2 constructed in Example 2 was cultured according to the method of Example 3, and the bacterial liquid was collected. After washing 3 times with 50 mM glycine-NaOH buffer, the OD 600 was controlled at 10, and it was reacted with PET powder at different temperatures. After 48 hours, the degradation products were detected. The results showed ( Figure 5 ) that products were released at 25 - 50 °C, indicating that the DFM2 strain had enzyme activity in this range, and 45 °C was the optimal temperature for the reaction, and the TPA concentration was 3.8 mM.

[0091] Similarly, by changing the OD 600 of the DFM2 strain and reacting with PET powder at 45 °C for 48 hours, the results showed that when the OD 600 was 1 - 30, the product release amount reached more than 1.2 mM, and the highest product release amount was shown when the OD 600 was 15, and the TPA concentration was 4.3 mM.

[0092] Example 5: Determination of products from the degradation of PET film by the recombinant strain

[0093] The crystallinities of the PET films (#1 - #4) used in this example are 13.84%, 12.59%, 16.83% and 19.82% respectively.

[0094] The bacterial liquid was collected after culturing the strain DFM2 constructed in Example 2 according to the method of Example 3. After washing 3 times with 50 mM glycine - NaOH buffer, the OD 600 was controlled at 15. Four kinds of plastics with different crystallinities were cut into films (#1 - #4) with a diameter of 6 mm and placed together with DFM2 cells in a 2 mL centrifuge tube with a liquid volume of 1 mL. Degradation was carried out in a shaker at 45 °C and 900 rpm. The pH was adjusted to 8.5 every 6 hours, and observations and photographs were taken every 12 hours or 24 hours until complete degradation (duration: 7 - 13 days). The results showed that the film first changed from transparent to white, and then as time increased, the white color on the surface deepened and corrosion gradually appeared, followed by rupture, and finally complete degradation. The gradual increase in the product also reflected this process ( Figure 6 a). In addition, the amount of enzyme shown by controlling the purified FAST and DFM2 (1 mL, OD 600 = 15) was the same. It was placed with PET plastic (#1) in a 2 mL centrifuge tube with a liquid volume of 1 mL. Degradation was carried out in a shaker at 50 °C and 900 rpm. The enzyme solution was refreshed every 24 hours, and observations and photographs were taken every 12 hours or 24 hours until complete degradation, with a duration of 8 days ( Figure 6 b). The results showed that under the optimal catalytic conditions of DFM2 and FAST, the complete depolymerization time of DFM2 whole - cell catalysis was shorter, and almost all the released products were TPA, while obvious MHET was released in the products of FAST depolymerization ( Figure 6 c). In addition, only the pH during the reaction process needs to be controlled, and there is no need to replace the enzyme solution ( Figure 7 ).

[0095] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Expression cassette, characterized in that, Including (a) or (b): (a) Display platform expression cassette: containing the gene fragments encoding the signal peptide SS, carbohydrate module CBM, V5-tag, scaffold protein SpyTag, scaffold protein SnoopTag, and anchor protein CtGCW65; (b) Plastic degrading enzyme expression cassette: contains the coding gene fragments of signal peptide SS, plastic degrading enzyme, capture proteins SpyCather and SnoopCather.

2. Recombinant Candida tropicalis, characterized in that Expression of plastic degrading enzymes based on the SpyTag-SpyCatcher system and the SnoopTag-SnoopCatcher system; the SpyTag-SpyCatcher system includes a scaffold protein SpyTag and a capture protein SpyCatcher; the SnoopTag-SnoopCatcher system includes a scaffold protein SnoopTag and a capture protein SnoopCatcher; The scaffold protein is expressed in fusion with the carbohydrate module CBM; The capture protein is fused with the plastic degrading enzyme for expression.

3. The recombinant Candida tropicalis according to claim 2, characterized in that, The plastic degrading enzyme includes but is not limited to PET degrading enzyme FAST-PETase and / or MHETase.

4. The recombinant Candida tropicalis according to claim 2 or 3, characterized in that, The scaffold protein and / or plastic degrading enzyme are expressed by a non-constitutive promoter.

5. The recombinant Candida tropicalis according to any one of claims 2 to 4, characterized in that The recombinant Candida tropicalis has the expression cassette of claim 1 integrated into its genome.

6. The method according to claims 2 to 5, characterized in that, regulated by promoter P CTR1 regulates the expression of the scaffold protein, and P GAL1 and P GAL10 promoter regulates the expression of the plastic-degrading enzyme.

7. A cell catalyst comprising the recombinant Candida tropicalis according to any one of claims 2 to 6.

8. A method for degrading PET plastic, characterized in that: The recombinant Candida tropicalis according to any one of claims 2 to 6 or the cell catalyst according to claim 7 is brought into contact with PET plastic to degrade the PET plastic.

9. The method according to claim 8, characterized in that, The method comprises reacting at 25° C. to 50° C. for 7 to 13 days.

10. Use of the recombinant Candida tropicalis according to any one of claims 2 to 6, or the cell catalyst according to claim 7, or the method according to any one of claims 8 to 9 in the field of plastic degradation.

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