Trichoderma reesei engineering bacterium for secreting and expressing plastic degrading enzyme as well as construction method and application of trichoderma reesei engineering bacterium

Abstract

The invention discloses a Trichoderma reesei engineering bacterium for secreting and expressing plastic degrading enzyme as well as a construction method and application of the Trichoderma reesei engineering bacterium, and belongs to the technical field of gene engineering. An expression plasmid of the plastic degrading enzyme is constructed, trichoderma reesei is converted, and the trichoderma reesei engineering bacterium for secreting and expressing the plastic degrading enzyme is constructed. In the Trichoderma reesei engineering bacteria, an endogenous protein coding gene cbh1 with the highest secretory expression quantity in Trichoderma reesei is replaced with a gene for coding the plastic degrading enzyme. The Trichoderma reesei engineering strain for secreting and expressing the plastic degrading enzyme is successfully constructed for the first time, and the Trichoderma reesei engineering strain constructed by the invention can significantly increase the productivity of the plastic degrading enzyme, and has potential to support future large-scale application.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an engineered bacterium of Trichoderma reesei capable of secreting and expressing a plastic-degrading enzyme, and a construction method and application thereof. Background Art

[0002] Plastic-degrading enzymes are a class of enzymes produced by microorganisms or artificially modified. They can decompose chemical bonds (such as ester bonds, amide bonds, etc.) in plastics through catalytic reactions, degrading high-molecular-weight polymers into small-molecule monomers or low-harm substances. For example: PETase (polyethylene terephthalate degrading enzyme) can efficiently decompose PET plastics, reversing them into terephthalic acid (TPA) and ethylene glycol (EG) for recycling. The discovery of plastic-degrading enzymes originated from the exploration of microorganisms in nature. In the early days, scientists screened out microorganisms that can decompose plastics in environments with serious plastic pollution, such as landfills and oceans, such as Ideonella sakaiensis (Osaka bacteria, which can secrete PETase). In 2017, Guo Ruiting's team first resolved the crystal structure of the PET-degrading enzyme IsPETase, revealing its catalytic mechanism.

[0003] Currently, the industrial application of plastic-degrading enzyme PETase faces several key bottlenecks.

[0004] 1. Limitations of enzyme activity and stability

[0005] Inefficient natural enzymes: Natural PETase has low activity at room temperature and is limited in its efficiency in degrading highly crystalline PET (such as plastic bottles). For example, unmodified PETase takes several days to degrade low-crystalline PET, while highly crystalline PET requires additional pretreatment (such as high-temperature softening), which increases energy consumption.

[0006] Poor thermal stability: Most natural enzymes are easily inactivated at high temperatures, and the ideal temperature for PET degradation (e.g., 70°C) is inconsistent with its natural stability. Although the engineered FAST-PETase can work efficiently at 50°C, further expansion of its application still requires improving its heat resistance.

[0007] 2. High production costs

[0008] Investment in AI and synthetic biology: Although AI-assisted design (such as AlphaFold3) can shorten the R&D cycle, the initial investment in technologies such as gene synthesis and high-throughput screening is huge, which is difficult for small and medium-sized enterprises to afford.

[0009] Enzyme production and purification costs: Large-scale production of industrial-grade enzyme preparations relies on fermentation technology, but current enzyme production efficiency is low (traditional fermentation yields are approximately 0.5-2g / L), resulting in high costs. For example, pharmaceutical-grade enzymes can cost $2,000-8,000 per kg, far exceeding the affordable range for industrial applications.

[0010] In short, the bottlenecks to PETase's industrialization lie in enzyme performance and cost. While AI and synthetic biology have accelerated technological breakthroughs (such as the low-temperature, efficient degradation of FAST-PETase), large-scale application still requires interdisciplinary collaboration and industry chain integration. Future efforts will focus on breakthroughs in polyolefin degradation technology, reducing enzyme production costs, and building a circular economy through policy guidance.

[0011] Trichoderma reesei is a mesophilic, saprophytic, filamentous fungus. The isolated strain was identified as T. reesei QM6a. Starting from the original QM6a strain, a series of mutagenesis breeding programs led to the identification of several high-producing cellulase strains, such as RUT-C30. T. reesei is widely used due to its ability to naturally secrete large amounts of endogenous proteins into the extracellular space. After fermentation, endogenous cellulases can produce up to 100 g / L. As a result, T. reesei has become a commonly used strain for industrial enzyme production. Unmodified T. reesei strains primarily produce cellulases, with the gene encoding the most highly secreted protein being cellulase cbh1.

[0012] To reduce the production cost of plastic-degrading enzymes, there have been reports of using Escherichia coli and Pichia pastoris to ferment and produce plastic-degrading enzymes. However, neither E. coli nor Pichia pastoris are food-safe strains, requiring IPTG or methanol to induce gene expression, respectively. Furthermore, the expression levels of PETase are low, making them unsuitable for future large-scale environmental applications. Trichoderma reesei is a food-safe strain that, through sugar-induced enzyme gene expression, is suitable for large-scale industrial production in 100-ton fermentation tanks. Therefore, engineering Trichoderma reesei to produce high-yield plastic-degrading enzymes is crucial for addressing these technical challenges. Summary of the Invention

[0013] The purpose of the present invention is to provide an engineered Trichoderma reesei that secretes and expresses a plastic-degrading enzyme, as well as a construction method and application thereof, to solve the problems existing in the above-mentioned prior art. By replacing the endogenous cellulase cbh1 of Trichoderma reesei with a heterologous plastic-degrading enzyme gene, an engineered Trichoderma reesei that secretes and expresses a plastic-degrading enzyme is successfully constructed, providing a new strain and data support for the large-scale production of plastic-degrading enzymes.

[0014] To achieve the above object, the present invention provides the following solutions:

[0015] The present invention provides a plastic degrading enzyme variant, whose amino acid sequence is any one of the sequences shown in SEQ ID NO.4-8; or a functionally identical sequence after 1 to 2 amino acids are substituted and / or deleted and / or added to any one of the sequences shown in SEQ ID NO.4-8.

[0016] In the art, substitutions with amino acids of similar or similar properties generally do not alter protein function. Adding several amino acids to the N-terminus can form a secretory peptide with secretory function, which helps secrete the protein into the fermentation broth outside the cell, and generally does not alter protein function. In the present invention, the disclosed plastic degrading enzyme variant undergoes mutations at one to two amino acid sites compared to the original plastic degrading enzyme sequence (GenBank: WCL40211.1): the R at positions 72 and 73 mutates to Q, A, K, or S. This mutation does not negatively affect the hydrolytic activity of the plastic degrading enzyme and may even significantly enhance its activity. In the present invention, the secretory peptide selected is up to 270 bases long, encoding a polypeptide of 90 amino acids in length. In the present invention, a 90-amino acid polypeptide is added to the N-terminus of the plastic degrading enzyme to form the plastic degrading enzyme proenzyme, the amino acid sequence of which is shown in SEQ ID NO. 9, and the encoding gene sequence is shown in SEQ ID NO. 16. This operation does not affect the expression and activity of the plastic degrading enzyme. Therefore, in the present invention, the amino acid sequence of the plastic degrading enzyme expressed by Trichoderma reesei also includes variant forms of the sequences of SEQ ID NO. 4 to 8 having the function of plastic degrading enzyme, as well as further variant forms based on this, which include: 1 to 2 amino acid substitutions, and adding one or several (1 to 90) amino acids to the N-terminus of the sequences of SEQ ID NO. 4 to 8.

[0017] The present invention also provides a plastic degrading enzyme proenzyme, the amino acid sequence of which is the N-terminus of the plastic degrading enzyme variant with a Trichoderma reesei secretory peptide containing 1 to 90 amino acids added thereto.

[0018] In the embodiment of the present invention, the amino acid sequence of the peptide secreted by Trichoderma reesei is shown as SEQ ID NO.2 or SEQ ID NO.3.

[0019] The plastic degrading enzyme variants shown in SEQ ID NOs. 4-8 are connected to the secretory peptide to form the plastic degrading enzyme proenzyme, and the amino acid sequence is shown in SEQ ID NOs. 9-15.

[0020] The present invention also provides a gene encoding the plastic degrading enzyme proenzyme, the nucleotide sequence of which is shown in SEQ ID NOs. 16 to 22, or a sequence having more than 60% homology with the sequences of SEQ ID NOs. 16 to 22.

[0021] In the art, codons are degenerate, meaning multiple codons encode the same amino acid. Other DNA sequences similar to those shown in SEQ ID NOs. 16-22, with at least 60% homology, that encode the same amino acid sequence can also achieve the functions described in the present invention.

[0022] The secretory peptide at the N-terminus of the plastic degrading enzyme proenzyme with amino acid sequences such as SEQ ID NOs. 9 to 15 can cleave the plastic degrading enzyme proenzyme into plastic degrading enzymes with amino acid sequences such as SEQ ID NOs. 1 and 4 to 8, and secrete the plastic degrading enzyme out of the Trichoderma reesei cells.

[0023] The present invention also provides a recombinant vector containing the gene. When introduced into Trichoderma reesei, the recombinant vector can replace the endogenous cellulase cbh1 gene of Trichoderma reesei. The recombinant vector sequentially contains the promoter sequence of the cbh1 cellulase gene, the gene encoding the proenzyme of the plastic-degrading enzyme, and the terminator sequence of the cbh1 cellulase gene.

[0024] The present invention also includes an engineered Trichoderma reesei bacterium comprising the recombinant vector.

[0025] The present invention also provides a method for constructing the engineered Trichoderma reesei, comprising the following steps:

[0026] Obtain the gene of the plastic degrading enzyme proenzyme with nucleotide sequence as shown in SEQ ID NO.16-22, connect the gene with the expression plasmid to construct a recombinant vector, and then introduce the recombinant vector into Trichoderma reesei to obtain the engineered Trichoderma reesei.

[0027] Preferably, the recombinant vector comprises a promoter sequence of the cbh1 cellulase gene endogenous to Trichoderma reesei, a gene encoding a proenzyme of the plastic degrading enzyme, and a terminator sequence of the cbh1 cellulase gene endogenous to Trichoderma reesei;

[0028] And / or the Trichoderma reesei includes QM6a and RUT-C30.

[0029] Specifically, the present invention uses a recombinant plasmid to replace the gene sequence encoding the proenzyme of the plastic-degrading enzyme with the gene sequence of the cellulase cbh1, the most highly expressed endogenous protein secreted in Trichoderma reesei. Furthermore, after replacing the endogenous cellulase cbh1 gene sequence in Trichoderma reesei, hygromycin resistance is deleted through induced self-recombination, and strains with the highest plastic-degrading enzyme activity are selected as engineered strains. Because engineered strains lack resistance markers, theoretically, iterative modification can be performed to gradually increase the number of plastic-degrading enzyme genes on the chromosome, thereby gradually improving enzyme activity.

[0030] The present invention also provides the use of the engineered Trichoderma reesei in producing plastic-degrading enzymes.

[0031] The present invention also provides a method for producing plastic-degrading enzyme, comprising the step of obtaining the plastic-degrading enzyme by fermentation and culture using the engineered Trichoderma reesei bacteria.

[0032] The present invention discloses the following technical effects:

[0033] The present invention replaces the endogenous cellulase cbh1 in Trichoderma reesei with a heterologous plastic-degrading enzyme gene (the amino acid sequences of the replaced plastic-degrading enzymes are shown in SEQ ID NOs. 9-15, and the nucleotide sequences are shown in SEQ ID NOs. 16-22), transforming Trichoderma reesei into an engineered strain capable of producing plastic-degrading enzyme preparations. This invention successfully constructs an engineered strain of Trichoderma reesei that secretes and expresses a plastic-degrading enzyme. This engineered strain significantly increases plastic-degrading enzyme production capacity and has the potential to provide a foundation for future large-scale industrial production of plastic-degrading enzyme preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a diagram of the construction of the plastic degrading enzyme expression plasmid in the present invention;

[0036] Figure 2 This is a flow chart for the construction of the plastic-degrading enzyme engineering strain of the present invention;

[0037] Figure 3 It is the activity of the plastic-degrading enzyme secreted and expressed by the plastic-degrading enzyme engineering strain of the present invention. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] In the following examples of the present invention, the formulas of culture medium and reagents are as follows:

[0044] (1) Luria Bertani (LB) medium formula: 5 g yeast powder, 10 g peptone, and 10 g sodium chloride, dilute to 1 L with tap water, natural pH.

[0045] (2) Glucose or xylose PDA culture medium formula: 6 g potato extract, 20 g glucose (or xylose) and 16 g agar, dilute to 1 L with tap water, natural pH.

[0046] (3) Formula of Trichoderma reesei fermentation medium (1 L): 5 g glucose, 37 g lactose, 6 g ammonium sulfate, 10 g cellulose powder, 5 g bran, 20 g corn steep liquor, 2 g peptone, 1 g yeast powder, 10 g KH2PO4, 0.6 g MgSO4·7H2O, 0.5 g CaCl2, 1 mL Mandels trace element solution, and 1 mL Tween 80.

[0047] The formula of Mandels trace element solution (1000×) is as follows: FeSO4·7H2O 5g, CoCl·6H2O 2g, ZnSO4·7H2O 1.4g, MnSO4·H2O 1.6g, and the volume is adjusted to 1L with purified water.

[0048] (4) Agrobacterium conjugative transfer scheme for Trichoderma reesei: The expression module is electroporated into Agrobacterium tumefaciens, and then the successfully electroporated Agrobacterium is co-cultured with the Trichoderma reesei host strain on IM plate culture medium (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264) for Agrobacterium tumefaciens-mediated conjugative transfer. After 2 days of co-cultivation, the cells are screened on glucose PDA plates supplemented with cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grow. PCR verification is then performed to confirm that the grown bacteria are correct transformants.

[0049] (5) Trichoderma reesei shuttle plasmid and resistance marker deletion scheme: After the hygromycin resistance marker is eliminated in each round of engineered strains according to the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN1.0. Scientific Reports. 2016, 6: 20761), the next round of genetic modification can be carried out. The principle is as follows: by culturing and passage on xylose PDA plates, hygromycin resistance will be lost, and the deletion efficiency is close to 70-100%. After the deletion, the strain cannot grow on PDA plates containing hygromycin, and this characteristic can be used to verify the deletion strain.

[0050] The starting strains of Trichoderma reesei used in the examples of the present invention are Trichoderma reesei QM6a (ATCC 13631) and RUT-C30 (ATCC 56765). This protocol is also applicable to other Trichoderma reesei strains, such as QM9414 (ATCC 26921), RL-P37 (NRRL15709) and NG14 (ATCC 56767) and their derivatives.

[0051] (6) Plastic degrading enzyme activity assay: Dissolve p-nitrophenol butyrate (pNPB) in isopropanol to prepare a 10 mM substrate solution, seal the container, and store at -20°C. Prepare a 50 mM phosphate buffered saline solution (PBS buffer, pH 8.0) with sterile water. Then, take 980 μL of the 50 mM phosphate buffered saline solution, preheat it at 37°C, add 10 μL of enzyme solution and 10 μL of substrate solution, heat it at 37°C for 10 min, and measure the absorbance at 410 nm using a spectrophotometer. Enzyme activity definition: The amount of enzyme required to catalyze the hydrolysis of pNPB to produce 1 μmol of p-nitrophenol per minute at 37°C is one unit of enzyme activity (U).

[0052] The plasmid extraction kit was purchased from AXYGEN, the gel recovery kit was purchased from MAGEN, the seamless cloning kit was purchased from Quanshijin, and the DNA restriction endonuclease and ligase were purchased from NEB. Similar products from other companies can also be used instead.

[0053] The experimental methods in the following examples where specific conditions are not specified were generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0054] Example 1 Obtaining the Gene of Plastic Degrading Enzyme Proenzyme

[0055] The method for obtaining the gene encoding the plastic degrading enzyme proenzyme is: provide the amino acid sequence (SEQ ID NO.9-15) and entrust a conventional gene company to synthesize the corresponding nucleotide coding sequence. The synthesis rules can be based on the codon preference of the host. Because of the differences in hosts, the codon preference is also different. According to the codon preference of Trichoderma reesei, the final synthesized DNA sequence is shown in SEQ ID NO.16-22. Because the codon preference is a range. Similar to the sequences shown in SEQ ID NO.16-22, other DNA sequences that can encode the same amino acid sequence can also be used.

[0056] Example 2 Construction of Plastic Degrading Enzyme Expression Plasmid

[0057] In order to transcribe and express the gene in Example 1 in Trichoderma reesei, a promoter and a terminator are required. The present invention provides the endogenous promoter with the highest expression level in Trichoderma reesei and its terminator sequence (respectively shown in SEQ ID NOs. 23-24).

[0058] The construction scheme is as follows: using the PCR scheme, the promoter and plastic degrading enzyme proenzyme gene fragments are cloned separately, and the two fragments are connected to the PacI / XbaI site of the Trichoderma reesei shuttle plasmid LML2.0a (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761) using the Vazyme One Step Clone Kit seamless ligation kit to construct an intermediate plasmid; using the PCR scheme, the terminator is cloned, and using the seamless ligation kit, the terminator fragment is connected to the SmiI single enzyme cutting site of the above intermediate plasmid, which is the plastic degrading enzyme expression plasmid. The whole process is as follows. Figure 1 shown.

[0059] The specific experimental operations are as follows.

[0060] A. Using primers CBHI-F1 and CBHI-F2, the nucleotide sequence of the CBHI promoter was amplified using the Trichoderma reesei genome as a template. A 14-base sequence from the Trichoderma reesei shuttle plasmid LML2.0a was added to the 5' end of primer CBHI-F1 for seamless ligation.

[0061] CBHI-F1: 5'-TTACGAATTCTTAATTCTGGAGACGGCTTGTTGAATCAT-3' (SEQ ID NO. 25);

[0062] CBHI-F2: 5'-CATGATGCGCAGTCCGCGGTTGA-3' (SEQ ID NO. 26).

[0063] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL, 2mM dNTPs 5μL, 25mM MgSO4 3μL, 10μM primers CBHI-F1 / CBHI-F2 1.5μL each, genomic template (200ng) 1μL and KOD-Plus-Neo (1U / μL) 1μL.

[0064] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0065] B. Using primers CBHI-R1 and CBHI-R2, the nucleotide sequence of the CBHI terminator was amplified using the T. reesei genome as a template. A 16-base sequence from the T. reesei shuttle plasmid LML2.0a was added to the 5' end of both primers for seamless ligation.

[0066] CBHI-R1: 5'-ACTAGTGAGCTCATTTAGCTCCGTGGCGAAAGCCT-3' (SEQ ID NO. 27);

[0067] CBHI-R2: 5'-AGTGCCAAGCTTATTTCATCGTAACCGAGAATCCAGAGCTG-3' (SEQ ID NO. 28).

[0068] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL, 2mM dNTPs 5μL, 25mM MgSO4 3μL, 10μM primers CBHI-R1 / CBHI-R2 1.5μL each, genomic template (200ng) 1μL and KOD-Plus-Neo (1U / μL) 1μL.

[0069] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0070] C. Using primers C2CPET-F1 and C2CPET-F2, the nucleotide sequence of proenzyme 1 was amplified using SEQ ID NO. 16 as a template. Primer C2CPET-F1 had a 15-base sequence from the CBHI promoter added to its 5' end for seamless ligation. Primer C2CPET-F2 had a 19-base sequence from the Trichoderma reesei shuttle plasmid LML2.0a added to its 5' end for seamless ligation.

[0071] C2CPET-F1: 5'-GGACTGCGCATCATGATCGTCGGCATCCTCACCA-3' (SEQ ID NO. 29);

[0072] C2CPET-F2: 5'-CATACATTATACGAAGTTATTACTGGCAGTGGCGGTTGTTG-3' (SEQ ID NO. 30).

[0073] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL, 2mM dNTPs 5μL, 25mM MgSO4 3μL, 10μM primers C2CPET-F1 / C2CPET-F2 1.5μL each, genomic template (200ng) 1μL and KOD-Plus-Neo (1U / μL) 1μL.

[0074] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0075] D. Using primers 74PET-F1 and 74PET-F2, the nucleotide sequences of proplasticases 2 to 7 were amplified using SEQ ID NOs. 17 to 22 as templates, respectively. A 15-base sequence from the CBHI promoter was added to the 5' end of primer 74PET-F1 to ensure seamless ligation. Primer 74PET-F2 is shown in SEQ ID NO. 30 above.

[0076] 74PET-F1: 5'-GGACTGCGCATCATGAAGGTCAGCCGTGCT-3' (SEQ ID NO. 31);

[0077] 74PET-F2: 5'-CATACATTATACGAAGTTATTACTGGCAGTGGGCGGTTGTTG-3' (ie, SEQ ID NO. 30 above).

[0078] Amplification reaction system: 10× PCR Buffer for KOD-Plus-Neo 5μL; 2mM dNTPs 5μL; 25mM MgSO4 3μL; 10μM primers HEWL-F1 / HEWL-F2 1.5μL each; genomic template (200ng) 1μL; KOD-Plus-Neo (1U / μL) 1μL.

[0079] Reaction program: 94°C for 2 min; 98°C for 10 sec, 58°C for 30 sec, 68°C for 30 sec, for 30 cycles; 68°C for 5 min.

[0080] The expression plasmid was constructed using the Trichoderma reesei shuttle plasmid LML2.0a as the backbone. The restriction endonucleases PacI / XbaI on the existing plasmid LML2.0a were double-digested, and the Vazyme One Step Clone Kit was used for seamless ligation, and the two DNA fragments of the promoter and the plastic degrading enzyme proenzyme were respectively connected; then the restriction endonuclease SwaI was used for single digestion, and the Vazyme One Step Clone Kit was used for seamless ligation, and the only DNA fragment of the terminator was connected, which is the plastic degrading enzyme expression plasmid ( Figure 1 ).

[0081] Example 3 Construction of an engineered strain of Trichoderma reesei expressing plastic-degrading enzymes

[0082] The seven plastic degrading enzyme expression plasmids No. 1 to 7 in Example 2 were transformed into Trichoderma reesei QM6a (ATCC13631) and RUT-C30 (ATCC 56765) respectively to obtain transformants. The present invention uses the Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264) to transform Trichoderma reesei strain Y (QM6a or RUT-C30), and the plastic degrading enzyme will replace the cellulase cbh1, and the first generation of Trichoderma reesei process strain Y-PET (1 to 7) -1 is obtained respectively. After culturing in xylose PDA medium and deleting the hygromycin resistance marker, the final Trichoderma reesei engineered strain Y-PET (1 to 7) -2 expressing plastic degrading enzyme without resistance marker is obtained. In the engineered strain, the most important cellulase cbh1 is replaced by the plastic degrading enzyme gene. The entire construction process is as follows. Figure 2 shown.

[0083] The final engineered strain of Trichoderma reesei Y-PET (1-7) -2 was fermented in the fermentation medium of Trichoderma reesei to induce the secretory expression of plastic degrading enzyme. The fermentation tank culture method refers to the literature "Chen et al. Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass bytruncation of the cellulase activator ACE3. Biotechnol Biofuels. 2020, 13: 62". After the fermentation is completed, the fermentation liquid is taken, centrifuged at 12500rpm for 10min, and the supernatant is taken, which is the crude enzyme solution of plastic degrading enzyme. The activity of plastic degrading enzyme is measured according to the above "(6) Plastic degrading enzyme activity determination method", and the results are as follows. Figure 3 shown.

[0084] The results showed that the starting strains of Trichoderma reesei, QM6a and RUT-C30, had almost no plastic degrading enzyme activity. Only after the cellulase CBHI gene was replaced with the plastic degrading enzyme proenzyme gene, could the plastic degrading enzyme activity be detected in the fermentation broth. This indicates that both secretory peptides can well express and secrete the plastic degrading enzyme to the extracellular space, as shown in RUTC30-PET(1)-2 and RUTC30-PET(2)-2. As a host cell for expressing plastic degrading enzymes, Trichoderma reesei RUT-C30 (ATCC 56765) is superior to QM6a (ATCC13631).

[0085] Different plastic degrading enzyme variants have different levels of plastic degrading enzyme activity in shake flask fermentation. The highest enzyme activity of RUTC30-PET(6)-2 reaches 1745U / mL, while the higher ones of RUTC30-PET(5)-2 and RUTC30-PET(7)-2 reach 1567U / mL and 1538U / mL respectively. Even when RUTC30-PET(3)-2 and RUTC30-PET(4)-2 are directly expressed compared with the original non-mutated plastic degrading enzyme sequence RUTC30-PET(2)-2, they are increased by 3.6 and 3.1 times respectively. The above data show that the expression level or activity of different variants is significantly improved by site mutation.

[0086] In summary, this invention discloses the sequences of multiple plastic-degrading enzyme variants and the genetic modification of Trichoderma reesei, successfully constructing, for the first time, an engineered strain that secretes and expresses plastic-degrading enzymes. This engineered strain can be used to produce plastic-degrading enzymes, providing a foundation for future large-scale industrial production of plastic-degrading enzyme preparations.

[0087] The sequence involved in the present invention is as follows:

[0088] The original plastic degrading enzyme sequence (GenBank: WCL40211.1) is SEQ ID NO.1:

[0089] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0090] A functional secretory peptide in Trichoderma reesei can cause the plastic-degrading enzyme to be secreted out of the Trichoderma reesei cell. The amino acid sequence encoding the secretory peptide is shown in SEQ ID NO. 2-3.

[0091] SEQ ID NO.2:

[0092] MIVGILTTLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYY SQCLPGAASSSSSTRAASTTSRVSPTTSRDKR.

[0093] SEQ ID NO.3:

[0094] MKVSRVLALVLGAVIPAHA.

[0095] The amino acid sequences of the plastic degrading enzyme variants in the following examples are shown in SEQ ID NOs. 4 to 8.

[0096] SEQ ID NO.4:

[0097] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGARLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0098] SEQ ID NO.5:

[0099] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRALASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0100] SEQ ID NO.6:

[0101] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGQRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0102] SEQ ID NO.7:

[0103] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGKKLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0104] SEQ ID NO.8:

[0105] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGKSLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0106] By connecting the secretory peptide sequence and the plastic-degrading enzyme sequence, the original enzyme sequence to be expressed in Trichoderma reesei will be formed, as shown in SEQ ID NOs. 9 to 15.

[0107] SEQ ID NO.9:

[0108] MIVGILTTLATLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSSSSTRAASTTSRVSPTTSRDKRSNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0109] SEQ ID NO.10:

[0110] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0111] SEQ ID NO.11:

[0112] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGARLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0113] SEQ ID NO.12:

[0114] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRALASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0115] SEQ ID NO.13:

[0116] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGQRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ。

[0117] SEQ ID NO.14:

[0118] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGKKLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLD ANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0119] SEQ ID NO.15:

[0120] MKVSRVLALVLGAVIPAHASNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGKSLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLD ANRLAVAGHSMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0121] The nucleotide sequences encoding the proenzyme of the plastic degrading enzyme are shown in SEQ ID NOs. 16 to 22, which are nucleotide sequences optimized according to the codon preference of Trichoderma reesei.

[0122] SEQ ID NO.16:

[0123]

[0124] SEQ ID NO.17:

[0125] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCCGACGCCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0126] SEQ ID NO.18:

[0127] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCGCCCGCCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0128] SEQ ID NO.19:

[0129] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCCGCGCCCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0130] SEQ ID NO.20:

[0131] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCCAGCGCCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0132] SEQ ID NO.21:

[0133] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCAAGAAGCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0134] SEQ ID NO.22:

[0135] ATGAAGGTCAGCCGCGTGCTCGCCCTCGTCCTCGGCGCCGTCATTCCCGCTCACGCTAGCAACCCCTACCAGCGAGGCCCCAACCCTACGCGCAGCGCCCTCACCGCTGACGGCCCCTTCAGCGTCGCCACCTACACCGTCAGCCGCCTCAGCGTCAGCGGCTTCGGCGGCGGCGTCATCTACTACCCCACCGGCACCAGCCTCACGTTCGGCGGCATTGCCATGTCGCCCGGCTACACCGCCGACGCCAGCAGCCTCGCCTGGCTCGGCAAGAGCCTCGCCAGCCACGGCTTCGTCGTCCTCGTCATCAACACCAACAGCCGCTTCGACGGCCCCGACAGCCGCGCCTCGCAGCTCAGCGCCGCTCTCAACTACCTCCGCACCAGCTCGCCCAGCGCCGTCCGCGCTCGCCTCGACGCCAACCGCCTGGCCGTCGCCGGCCACAGCATGGGCGGCGGCGGCACCCTCCGCATTGCCGAGCAGAACCCCAGCCTCAAGGCCGCCGTGCCTCTCACGCCCTGGCACACCGACAAGACCTTCAACACGAGCGTCCCCGTCCTCATCGTCGGCGCGGAGGCCGACACCGTCGCTCCCGTCAGCCAGCACGCCATTCCGTTCTACCAGAACCTGCCTAGCACGACGCCCAAGGTCTACGTCGAGCTGTGCAACGCCTCGCACATTGCCCCTAACAGCAACAACGCCGCCATCTCCGTCTACACCATCAGCTGGATGAAGCTCTGGGTCGACAACGACACCCGCTACCGCCAGTTCCTCTGCAACGTCAACGACCCCGCGCTCTGCGACTTCCGCACCAACAACCGCCACTGCCAGTAA。

[0136] The promoter and terminator sequences of the cellulase gene cbh1 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively.

[0137] SEQ ID NO.23:

[0138]

[0139] SEQ ID NO.24:

[0140]

[0141] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A plastic degrading enzyme variant, characterized in that The amino acid sequence is any one of the sequences shown in SEQ ID NOs. 4-8; or a functionally identical sequence of any one of the sequences shown in SEQ ID NOs. 4-8 with 1 to 2 amino acids substituted and / or deleted and / or added.

2. A plastic degrading enzyme, characterized in that The amino acid sequence is the plastic degrading enzyme variant according to claim 1 with a Trichoderma reesei secretory peptide containing 1 to 90 amino acids added to the N-terminus.

3. A gene encoding the proenzyme of the plastic degrading enzyme according to claim 1 or 2.

4. A recombinant vector comprising the gene according to claim 3.

5. An engineered strain of Trichoderma reesei comprising the recombinant vector according to claim 4.

6. A method for constructing the engineered Trichoderma reesei according to claim 5, characterized in that: The following steps are involved: Obtain the gene of the plastic degrading enzyme proenzyme with nucleotide sequence as shown in SEQ ID NO.16-22, connect the gene with the expression plasmid to construct a recombinant vector, and then introduce the recombinant vector into Trichoderma reesei to obtain the engineered Trichoderma reesei.

7. The construction method according to claim 6, wherein: The recombinant vector comprises a promoter sequence, a gene encoding a plastic degrading enzyme proenzyme and a terminator sequence.

8. The construction method according to claim 6, wherein: The Trichoderma reesei includes Trichoderma reesei QM6a and RUT-C30 and derivative strains thereof.

9. Use of the engineered Trichoderma reesei according to claim 5 in producing plastic-degrading enzymes.

10. A method for producing a plastic degrading enzyme, characterized in that: The method comprises the step of obtaining plastic degrading enzyme by fermentation culture using the engineered bacteria of Trichoderma reesei as claimed in claim 5.

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