A Trichoderma reesei engineered strain that secretes and expresses plastic-degrading enzymes, its construction method and application

Abstract

This invention discloses an engineered Trichoderma reesei strain that secretes and expresses a plastic-degrading enzyme, its construction method, and its applications, belonging to the field of genetic engineering technology. This invention constructs an expression plasmid for the plastic-degrading enzyme, transforms it into Trichoderma reesei, and constructs an engineered Trichoderma reesei strain that secretes and expresses the plastic-degrading enzyme. In this engineered Trichoderma reesei strain, the endogenous protein-coding gene cbh1, which has the highest secretion expression level in Trichoderma reesei, is replaced with the gene encoding the plastic-degrading enzyme. This invention is the first successful construction of an engineered Trichoderma reesei strain that secretes and expresses a plastic-degrading enzyme. The engineered Trichoderma reesei strain constructed by this invention can significantly increase the production capacity of the plastic-degrading enzyme and has the potential to support future large-scale applications.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered Trichoderma reesei strain that secretes and expresses plastic-degrading enzymes, its construction method, and its application. Background Technology

[0002] Plastic-degrading enzymes are a class of enzymes, either microorganisms or artificially modified, that can break down chemical bonds (such as ester and amide bonds) in plastics through catalytic reactions, degrading high-molecular polymers into smaller monomers or less harmful substances. For example, PETase (polyethylene terephthalate degrading enzyme) can efficiently decompose PET plastic, reversing it into terephthalic acid (TPA) and ethylene glycol (EG), enabling recycling. The discovery of plastic-degrading enzymes stemmed from the exploration of microorganisms in nature. Early on, scientists screened microorganisms capable of degrading plastics in heavily plastic-polluted environments such as landfills and oceans, such as *Ideonella sakaiensis* (which secretes 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 the plastic degrading enzyme PETase faces several key bottlenecks.

[0004] 1. Limitations on enzyme activity and stability

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

[0006] Poor thermal stability: Most natural enzymes are easily deactivated at high temperatures, and the ideal degradation temperature of PET (e.g., 70℃) contradicts its natural stability. Although the engineered FAST-PETase can work efficiently at 50℃, its heat resistance still needs to be improved for further expansion of its applications.

[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 upfront 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 yield is approximately 0.5-2 g / L), resulting in high costs. For example, pharmaceutical-grade enzymes can cost between $2,000 and $8,000 per kg, far exceeding the affordability of industrial applications.

[0010] In summary, the industrialization bottlenecks of PETase are concentrated in enzyme performance and cost. Although AI and synthetic biology have accelerated technological breakthroughs (such as the low-temperature, high-efficiency degradation of FAST-PETase), large-scale application still requires interdisciplinary collaboration and supply chain integration. Future efforts should focus on breakthroughs in polyolefin degradation technology, reducing enzyme production costs, and building a circular economy system through policy guidance.

[0011] *Trichoderma reesei* is a thermophilic, saprophytic filamentous fungus. The isolated strain was identified as *Trichoderma reesei* QM6a. Starting from the original strain QM6a, a series of mutagenesis breeding experiments were conducted, resulting in the selection of several high-yielding cellulase strains, such as RUT-C30. *Trichoderma reesei* is widely used due to its ability to naturally secrete large amounts of endogenous proteins extracellularly, and the yield of endogenous cellulase after fermentation can reach up to 100 g / L. Therefore, *Trichoderma reesei* has become a commonly used strain for the production of industrial enzyme preparations. The proteins produced by unmodified *Trichoderma reesei* strains are mainly cellulases, with the most highly expressed protein encoding the cellulase gene cbh1.

[0012] To reduce the production cost of plastic-degrading enzymes, there are research reports on obtaining these enzymes through fermentation using *E. coli* and *Pichia pastoris*. However, neither *E. coli* nor *Pichia pastoris* are food-safe strains, requiring IPTG or methanol to induce gene expression, and their PETase expression levels are low, failing to meet future large-scale applications. *Trichoderma reesei*, on the other hand, is a food-safe strain, and its sugar-induced enzyme gene expression makes it suitable for large-scale industrial production in fermenters with capacities of hundreds of tons. Therefore, modifying *Trichoderma reesei* to obtain engineered strains capable of high-yield plastic-degrading enzymes is of great significance for solving the aforementioned technical problems. Summary of the Invention

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

[0014] To achieve the above objectives, the present invention provides the following solution:

[0015] The present invention provides a plastic degrading enzyme variant having an amino acid sequence as shown in any of SEQ ID NO. 4-8; or a sequence as shown in any of SEQ ID NO. 4-8 with the same function after substitution and / or deletion and / or addition of 1-2 amino acids.

[0016] In this field, substitution with amino acids of similar or identical properties generally does not alter protein function; adding several amino acids to the N-terminus can form a secretory peptide with secretory function, helping the protein to be secreted into the extracellular fermentation broth, which generally does not change the protein's function. In this invention, the disclosed plastic degrading enzyme variant has a mutation of 1 to 2 amino acid sites compared to the original plastic degrading enzyme sequence (GenBank: WCL40211.1)—the R at positions 72 and 73 is mutated to Q, A, K, or S. This mutation does not negatively affect the hydrolytic activity of the plastic degrading enzyme and may even significantly increase its activity. In this invention, the selected secretory peptide is up to 270 bases long, encoding a polypeptide of 90 amino acids. This invention adds a 90-amino acid polypeptide to the N-terminus of the plastic degrading enzyme to form the original plastic degrading enzyme, with the amino acid sequence shown in SEQ ID NO. 9 and the encoding gene sequence shown in SEQ ID NO. 16. This operation does not affect the expression and activity of the plastic degrading enzyme. Therefore, in this invention, the amino acid sequence of the plastic degrading enzyme expressed by Trichoderma reesei also includes variations of the sequence of SEQ ID NO. 4 to 8 that have plastic degrading enzyme function, as well as further variations based thereon, including: substitution of 1 to 2 amino acids, and addition of one or more (1 to 90) amino acids to the N-terminus of the sequence 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 the addition of a Trichoderma reesei secretory peptide containing 1 to 90 amino acids.

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

[0019] The plastic degrading enzyme variants shown in SEQ ID NO.4-8 are linked with secretory peptides to form plastic degrading enzyme proenzymes, with amino acid sequences as shown in SEQ ID NO.9-15.

[0020] The present invention also provides a gene encoding the plastic degradation enzyme proenzyme, the nucleotide sequence of which is shown in SEQ ID NO.16-22, or a sequence having more than 60% homology with the sequence of SEQ ID NO.16-22.

[0021] In this field, codons are degenerate, meaning that multiple codons encode the same amino acid. Similar to the sequences shown in SEQ ID NO. 16–22, other DNA sequences with more than 60% homology that can encode the same amino acid sequence can also achieve the functions described in this invention.

[0022] The secretory peptide at the N-terminus of the plastic degrading enzyme proenzyme, as shown in SEQ ID NO. 9–15, can cleave the plastic degrading enzyme proenzyme into a plastic degrading enzyme with amino acid sequences as shown in SEQ ID NO. 1 and 4–8, and secrete the plastic degrading enzyme out of Trichoderma reesei cells.

[0023] The present invention also provides a recombinant vector containing the aforementioned gene. After the recombinant vector is introduced into *Trichoderma reesei*, it can replace the endogenous cellulase cbh1 gene in *Trichoderma reesei*. The recombinant vector sequentially contains the promoter sequence of the cbh1 cellulase gene, the encoding gene for the plastic degradation proenzyme, and the terminator sequence of the cbh1 cellulase gene.

[0024] The present invention also includes engineered Trichoderma reesei containing the recombinant vector.

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

[0026] Obtain the gene of the plastic degradation enzyme proenzyme with the nucleotide sequence 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 includes the promoter sequence of the endogenous cbh1 cellulase gene of Trichoderma reesei, the encoding gene of the plastic degradation enzyme proenzyme, and the terminator sequence of the endogenous cbh1 cellulase gene of Trichoderma reesei.

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

[0029] Specifically, this invention involves replacing the gene sequence of cellulase cbh1, the highest-expressed endogenous protein secreted by *Trichoderma reesei*, with the gene sequence encoding the proenzyme of plastic degrading enzyme using a recombinant plasmid. Further, after replacing the endogenous cellulase cbh1 gene sequence in *Trichoderma reesei*, hygromycin resistance is induced through self-recombination, and strains with the highest plastic degrading enzyme activity are selected as engineered strains. Because the engineered strains lack resistance markers, theoretically, iterative modifications can be continued to gradually increase the number of plastic degrading enzyme genes in the chromosome, thereby gradually increasing enzyme activity.

[0030] The present invention also provides the application of the engineered Trichoderma reesei strain in the production of plastic degrading enzymes.

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

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

[0033] This invention replaces the endogenous cellulase cbh1 of *Trichoderma reesei* with a heterologous plastic-degrading enzyme gene (the amino acid sequence of the replaced plastic-degrading enzyme is shown in SEQ ID NO. 9-15, and the nucleotide sequence is shown in SEQ ID NO. 16-22), thus transforming *Trichoderma reesei* into an engineered strain capable of producing plastic-degrading enzyme preparations. This invention represents the first successful construction of an engineered *Trichoderma reesei* strain that secretes and expresses plastic-degrading enzymes. This engineered strain can significantly increase the production capacity of plastic-degrading enzymes and has the potential to provide a foundation for the future large-scale industrial production of plastic-degrading enzyme preparations. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a diagram illustrating the construction of the expression plasmid for the plastic-degrading enzyme in this invention.

[0036] Figure 2 This is a flowchart illustrating the construction of the engineered strain of plastic-degrading enzyme in this invention;

[0037] Figure 3 This refers to the activity of the plastic-degrading enzyme secreted and expressed by the engineered strain of plastic-degrading enzyme in this invention. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

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

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

[0045] (2) Formula for glucose or xylose PDA medium: 6g potato extract powder, 20g glucose (or xylose) and 16g agar, bring the volume to 1L with tap water, and set the pH to natural.

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

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

[0048] (4) Agrobacterium conjugation transfer of Trichoderma reesei: The expression module was electroporated into Agrobacterium rhizogenes, and then the successfully electroporated Agrobacterium was co-cultured with the Trichoderma reesei host strain on IM plate medium (Covert et al.Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum.Mycol.Res.105(3):259-264) to carry out Agrobacterium rhizogenes-mediated conjugation transfer. After co-culturing for 2 days, the strains were screened on glucose PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grew. Then, PCR verification was performed to prove that the grown cells were the correct transformants.

[0049] (5) Trichoderma reesei shuttle plasmid and resistance marker deletion scheme: After eliminating the hygromycin resistance marker 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 gene modification can be carried out. The principle is as follows: Hygromycin resistance will be deleted when cultured and passaged in xylose PDA plates, with a deletion efficiency of close to 70-100%; the deleted strains cannot grow in PDA plates containing hygromycin, and this characteristic can be used to verify the deleted strains.

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

[0051] (6) Method for determining the activity of plastic-degrading enzymes: A 10 mM substrate solution was prepared by dissolving p-nitrophenol butyrate (pNPB) in isopropanol, sealing the solution, and storing it at -20°C. A 50 mM phosphate buffer solution (PBS buffer, pH 8.0) was prepared using sterile water. Then, 980 μL of the 50 mM phosphate buffer solution was taken, preheated to 37°C, and 10 μL of enzyme solution and 10 μL of substrate solution were added. The mixture was heated at 37°C for 10 min, and the absorbance was measured 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 defined as one enzyme activity unit (U).

[0052] The plasmid extraction kit was purchased from AXYGEN, the gel extraction kit from MAGEN, the seamless cloning kit from TransGen, and the DNA restriction endonucleases and ligases from NEB, or similar products from other companies.

[0053] Experimental methods in the following examples, unless otherwise specified, were performed 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 for the plastic degradation enzyme proenzyme

[0055] The method for obtaining the encoding gene of the plastic degradation enzyme is as follows: The amino acid sequence (SEQ ID NO. 9–15) is provided, and a conventional genetic engineering company is commissioned to synthesize the corresponding nucleotide coding sequence. The synthesis rules can be based on the host's codon preference. Due to host differences, codon preferences also vary. Based on the codon preference of *Trichoderma reesei*, the final synthesized DNA sequences are shown in SEQ ID NO. 16–22. Because codon preference is a range, other DNA sequences encoding the same amino acid sequence, similar to those shown in SEQ ID NO. 16–22, can also be used.

[0056] Example 2 Construction of expression plasmid for plastic degrading enzyme

[0057] To transcribe the gene from Example 1 into Trichoderma reesei, a promoter and a terminator are required. This invention provides the sequences of the endogenous promoter with the highest expression level in Trichoderma reesei, and its terminator (as shown in SEQ ID NO. 23-24, respectively).

[0058] The construction scheme is as follows: Using PCR, the promoter and plastic degrading enzyme gene fragments were cloned separately. Using the Vazyme One Step Clone Kit, these two fragments were ligated into 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) to construct an intermediate plasmid. Using PCR, the terminator was cloned. Using the seamless ligation kit, the terminator fragment was ligated into the SmiI single restriction site of the intermediate plasmid, thus obtaining the plastic degrading enzyme expression plasmid. The entire process is as follows: Figure 1 As shown.

[0059] The specific experimental procedures are as follows.

[0060] A. The nucleotide sequence of the CBHI promoter was amplified using the Trichoderma reesei genome as a template, using primers CBHI-F1 and CBHI-F2. 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: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers CBHI-F1 / CBHI-F2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

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

[0065] B. Using primers CBHI-R1 and CBHI-R2, the nucleotide sequence of the CBHI terminator was amplified using the *Trichoderma reesei* genome as a template. A 16-base sequence from the *Trichoderma reesei* shuttle plasmid LML2.0a was inserted at the 5' end of both primers CBHI-R1 and CBHI-R2 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: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers CBHI-R1 / CBHI-R2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

[0069] Reaction program: 94℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ 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. A 15-base sequence from the CBHI promoter was added to the 5' end of primer C2CPET-F1 for seamless ligation. A 19-base sequence from the *Trichoderma reesei* shuttle plasmid LML2.0a was added to the 5' end of primer C2CPET-F2 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: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO4, 1.5 μL each of 10 μM primers C2CPET-F1 / C2CPET-F2, 1 μL of genomic template (200 ng), and 1 μL of KOD-Plus-Neo (1 U / μL).

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

[0075] D. Using primers 74PET-F1 and 74PET-F2, the nucleotide sequences of plastic degrading enzyme proenzymes 2–7 were amplified using SEQ ID NO. 17–22 as templates. A 15-base sequence from the CBHI promoter was added to the 5' end of primer 74PET-F1 for 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℃ for 2 min; 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 30 sec, run for 30 cycles; 68℃ for 5 min.

[0080] An expression plasmid was constructed using the *Trichoderma reesei* shuttle plasmid LML2.0a as a backbone. The plasmid was double-digested with the restriction endonucleases PacI / XbaI on the existing plasmid LML2.0a, and then seamlessly ligated using the Vazyme One Step Clone Kit to insert the promoter and the proenzyme DNA fragments, respectively. The plasmid was then digested with the restriction endonuclease SwaI, and seamlessly ligated again using the Vazyme One Step Clone Kit to insert the unique terminator DNA fragment, thus completing the plastic degrading enzyme expression plasmid. Figure 1 ).

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

[0082] The seven plastic degrading enzyme expression plasmids 1-7 from Example 2 were transformed into *Trichoderma reesei* QM6a (ATCC13631) and RUT-C30 (ATCC 56765), respectively, to obtain transformants. In this invention, *Trichoderma reesei* strain Y (QM6a or RUT-C30) was transformed using the *Agrobacterium tumefaciens-mediated transformation* (Covert et al. *Agrobacterium tumefaciens-mediated transformation of *Fusarium circinatum*. *Mycol. Res.* 105(3):259-264). The plastic degrading enzyme replaced the cellulase cbh1, resulting in first-generation *Trichoderma reesei* strains Y-PET(1-7)-1. After culturing in xylose-PDA medium and deleting the hygromycin resistance marker, the final resistance-free *Trichoderma reesei* engineered strain Y-PET(1-7)-2 expressing the plastic degrading enzyme was obtained. In the engineered strains, the most important cellulase cbh1 was replaced by the plastic degrading enzyme gene. The entire construction process is as follows: Figure 2 As shown.

[0083] The final Trichoderma reesei engineered strain Y-PET(1~7)-2 was fermented in Trichoderma reesei fermentation medium to induce the secretion and expression of plastic degrading enzymes. The fermentation tank culture method was based on the literature "Chen et al. Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3. Biotechnol Biofuels. 2020, 13:62". After fermentation, the fermentation broth was centrifuged at 12500 rpm for 10 min, and the supernatant was collected, which was the crude enzyme solution of plastic degrading enzymes. The activity of plastic degrading enzymes was determined according to the above "(6) Plastic Degrading Enzyme Activity Determination Method", and the results are as follows. Figure 3 As shown.

[0084] The results showed that the Trichoderma reesei originating strains QM6a and RUT-C30 had almost no plastic degrading enzyme activity. Plastic degrading enzyme activity could only be detected in the fermentation broth after replacing the cellulase CBHI gene with the plastic degrading enzyme proenzyme gene, indicating that both secreted peptides effectively expressed and secreted the plastic degrading enzyme extracellularly (see RUTC30-PET(1)-2 and RUTC30-PET(2)-2). As a host cell for expressing plastic degrading enzymes, Trichoderma reesei RUT-C30 (ATCC 56765) was superior to QM6a (ATCC13631).

[0085] Different plastic degrading enzyme variants exhibited varying activities during shake-flask fermentation. The highest activity was achieved by RUTC30-PET(6)-2, reaching 1745 U / mL, while RUTC30-PET(5)-2 and RUTC30-PET(7)-2 showed relatively high activities of 1567 U / mL and 1538 U / mL, respectively. Even when directly expressed compared to the original, unmutated plastic degrading enzyme sequence RUTC30-PET(2)-2, RUTC30-PET(3)-2 and RUTC30-PET(4)-2 showed increases of 3.6 and 3.1 times, respectively. These data demonstrate the significant increase in expression level or activity resulting from site mutations in different variants.

[0086] In summary, this invention discloses the sequences of various plastic-degrading enzyme variants and the genetic modification process 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 for the production of plastic-degrading enzymes, providing a foundation for the future large-scale industrial production of plastic-degrading enzyme preparations.

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

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

[0089] SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDGPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELCNASHIAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALCDFRTNNRHCQ.

[0090] Functional secretory peptides in Trichoderma reesei enable the secretion of plastic-degrading enzymes from Trichoderma reesei cells. The amino acid sequences encoding the secretory peptides are 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 NO.4-8.

[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] Linking the secreted peptide sequence with the plastic-degrading enzyme sequence will form the proenzyme sequence to be expressed in Trichoderma reesei, as shown in SEQ ID NO. 9–15.

[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 plastic degradation proenzyme are shown in SEQ ID NO.16–22. These sequences are 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 24 respectively.

[0137] SEQ ID NO.23:

[0138]

[0139] SEQ ID NO.24:

[0140]

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

Claims

1. A plastic-degrading enzyme variant, characterized in that, Its amino acid sequence is any one of the sequences shown in SEQ ID NO.4-8.

2. A plastic-degrading enzyme precursor, characterized in that, Its amino acid sequence is the N-terminus of the plastic degrading enzyme variant of claim 1 with the addition of a Trichoderma reesei secretory peptide containing 1 to 90 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.

3.

3. The gene encoding the plastic-degrading enzyme variant of claim 1 or the plastic-degrading enzyme proenzyme of claim 2.

4. A recombinant vector comprising the gene of claim 3.

5. A Trichoderma reesei engineered strain comprising the recombinant vector of claim 4.

6. A method for constructing the engineered Trichoderma reesei strain according to claim 5, characterized in that, Includes the following steps: Obtain the gene of the plastic degradation enzyme proenzyme with the nucleotide sequence shown in SEQ ID NO.18-22, 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 as described in claim 6, characterized in that, The recombinant vector contains a promoter sequence, a gene encoding a plastic degradation proenzyme, and a terminator sequence.

8. The construction method as described in claim 6, characterized in that, The Trichoderma reesei includes Trichoderma reesei QM6a and RUT-C30.

9. The application of the engineered Trichoderma reesei strain as described in claim 5 in the production of plastic-degrading enzymes, characterized in that, The amino acid sequence of the plastic degrading enzyme is shown in SEQ ID NO.

1.

10. A method for producing plastic-degrading enzymes, characterized in that, The method includes the step of obtaining a plastic degrading enzyme by fermentation culture using the engineered Trichoderma reesei strain as described in claim 5, wherein the amino acid sequence of the plastic degrading enzyme is shown in SEQ ID NO.1.

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