Escherichia coli engineering bacteria with high efficiency of producing plastic depolymerase and application thereof

Abstract

The present application relates to the field of microorganisms and genetic engineering, and specifically discloses an Escherichia coli engineering bacterium for efficiently producing plastic depolymerase and application thereof. The present application uses Escherichia coli as a host, and through synergistic optimization of multiple key dimensions such as signal peptide, promoter, solubility-promoting tag and co-expression of molecular chaperones, a multi-dimensional engineering system for efficiently expressing plastic depolymerase is constructed. The construction method provided by the present application includes a rational design and a screening strategy for combination and assembly of the multiple elements. The engineering bacterium can realize extracellular high-level expression of plastic depolymerase during induced fermentation, and the enzyme preparation prepared can be directly used for catalyzing efficient depolymerization of polyester plastics. The present application provides an Escherichia coli cell factory with stable high yield for industrialization and low-cost production of plastic depolymerase, and has important industrial application prospects for promoting large-scale biological degradation and resource recycling of waste plastics.

Description

An engineered Escherichia coli strain that efficiently produces plastic depolymerase and its applications Technical Field

[0001] This invention relates to the field of microorganisms and genetic engineering, specifically to an engineered strain of Escherichia coli that produces plastic depolymerase and its applications. Background Technology

[0002] Among plastic pollutants, polyester plastics, represented by polyethylene terephthalate (PET), are chemically stable and degrade extremely slowly naturally. Bioenzymatic hydrolysis, as an environmentally friendly and mild plastic recycling strategy, has received widespread attention in recent years. However, one of the key aspects of realizing the industrial application of this technology lies in how to produce highly active plastic depolymerases on a large scale at low cost and high efficiency. Currently reported microbial methods for producing PET depolymerase yields a maximum of less than 4 g / L, which is insufficient to meet the stringent requirements of large-scale biorecycling processes regarding enzyme cost and supply. Therefore, a more efficient method for producing PET depolymerase is urgently needed.

[0003] The core advantage of using *E. coli* (E. coli) to produce PET depolymerase lies in the efficient combination of a mature, classic expression platform, strong engineering capabilities, and the potential to construct an integrated bioprocess. As one of the microbial hosts with the clearest genetic background and the easiest operation, *E. coli* possesses a vast array of optimized vectors, strains, and fermentation protocols, enabling rapid production of target enzymes at extremely low cost, making it the most commonly used expression system in this field. Despite the significant advantages of the *E. coli* system, the expression of exogenous proteins also faces numerous bottlenecks, such as protein misfolding and low secretion efficiency, resulting in actual yields and activities that often fail to meet the economic requirements of industrial production, severely restricting the economic feasibility of enzyme production.

[0004] Currently, research on increasing recombinant protein yield largely focuses on single-dimensional optimization, such as screening or modifying signal peptides to enhance secretion induction efficiency, or using stronger or more tunable promoters to increase transcription levels. However, these optimization strategies often have limitations. For example, high transcription driven by strong promoters may exceed the folding and transport capabilities of the host cell; overexpression of a single molecular chaperone may disrupt the intracellular protein homeostasis network. Therefore, single-dimensional modifications lack universality and are insufficient to systematically increase protein yield to levels that meet industrial-scale cost requirements. Summary of the Invention

[0005] Objective: This invention addresses the shortcomings of existing technologies by providing a highly efficient engineered *E. coli* strain for producing plastic depolymerase and its applications. The invention focuses on the multi-dimensional and synergistic engineering modification of the *E. coli* expression system. Through rational design and synergistic optimization of multiple key dimensions, including transcriptional regulation (promoter), secretion guidance (signal peptide), solubilization tag, and folding assistance (molecular chaperone), an *E. coli* strain capable of stably and efficiently producing plastic depolymerase is constructed. This invention is of paramount importance for overcoming the bottleneck in plastic depolymerase production and promoting the practical application of plastic biorecycling technology.

[0006] To address the aforementioned technical problems, this invention discloses an engineered *E. coli* strain that efficiently produces plastic depolymerase and its applications. The specific technical solution is as follows:

[0007] In a first aspect, the present invention provides an engineered Escherichia coli strain that efficiently produces plastic depolymerase, wherein the engineered Escherichia coli co-expresses the coding gene for plastic depolymerase and a molecular chaperone; wherein the plastic depolymerase includes an enzyme with polyethylene terephthalate (PET) degradation activity; the molecular chaperone is a combination of danK, danJ, and grpE; in some embodiments of the present invention, the molecular chaperone is expressed in the form of plasmid pKJE7.

[0008] The aforementioned molecular chaperones have functions such as promoting protein folding, processing, transport, or secretion. By co-expressing molecular chaperones that assist in protein folding, processing, or secretion, misfolding and inclusion body formation of plastic depolymerases within cells can be reduced, thereby achieving efficient expression and secretion of plastic depolymerases.

[0009] The gene encoding the plastic depolymerase is expressed under the regulation of a regulatory element, which includes any one or more combinations of the following (1) to (3):

[0010] (1) Signal peptide;

[0011] (2) Solubilizing tag; In some embodiments of the present invention, the solubilizing tag is directly integrated into the N-terminus of the gene encoding plastic depolymerase;

[0012] (3) Strong promoter.

[0013] The signal peptide includes any one of SEQ ID NO.13, SEQ ID NO.15 or SEQ ID NO.16, which are pectin lyase B (PelB), outer membrane porin from Escherichia coli (LamB), and outer membrane protein from Aeromonas hydrophila (TolB), respectively.

[0014] The solubilizing tag includes small ubiquitin-like modified protein (SUMO) or N-utilization substance A (NusA), the amino acid sequence of which is shown in SEQ ID NO.22 and the amino acid sequence of which is shown in SEQ ID NO.23.

[0015] The strong promoters mentioned include P T7 or P lac The P mentioned T7 The nucleotide sequence is shown in SEQ ID NO.25, and the P... lac The nucleotide sequence is shown in SEQ ID NO.26.

[0016] In some embodiments of the present invention, the regulatory element comprises a combination of a signal peptide, a lysis-promoting tag, and a strong promoter; wherein the signal peptide is the sequence TolB shown in SEQ ID NO. 16, the lysis-promoting tag is NusA shown in SEQ ID NO. 23, and the strong promoter is P shown in SEQ ID NO. 26. lac .

[0017] The host bacteria of the engineered Escherichia coli include Escherichia coli (E. coli) BL21.

[0018] In some embodiments of the present invention, the amino acid sequence of the plastic depolymerase is shown in any one of SEQ ID NO. 1 to 6, and is respectively encoded by the nucleotide sequences shown in SEQ ID NO. 7 to 12.

[0019] The present invention also provides a method for constructing the engineered *Escherichia coli* strain described in the first aspect, comprising the following steps: constructing a recombinant expression vector containing a gene encoding a plastic depolymerase and a molecular chaperone, and introducing, as needed, any one or more combinations of a promoter, a signal peptide, and a solubilizing tag into a host bacterium to obtain the engineered *Escherichia coli* strain. In other embodiments of the present invention, at least one recombinant expression vector is integrated or transformed into the engineered *Escherichia coli* strain, the recombinant expression vector comprising an expression cassette driven by a promoter, the expression cassette comprising, in sequence: a promoter, a signal peptide encoding sequence (optional), a solubilizing tag sequence (optional), a gene encoding a plastic depolymerase, and a terminator.

[0020] Secondly, this invention provides the application of the engineered *E. coli* strain described in the first aspect in the production of plastic depolymerization enzymes. This invention provides low-cost, high-efficiency enzyme preparation production technology support for the biological recycling of plastics.

[0021] Thirdly, the present invention provides a method for producing plastic depolymerase using engineered Escherichia coli as described in the first aspect. The engineered Escherichia coli is inoculated into a fermentation medium and fermented at 16-37°C for 26-38 hours to obtain the plastic depolymerase. In some embodiments of the present invention, the fermentation is first carried out at 25-37°C until the OD reaches [value missing]. 600 The concentration is 20-40 g / L, followed by the addition of the inducing agent IPTG and continued culturing at 16-25 °C for 12-18 h. In some embodiments of the present invention, the final concentration of the inducing agent IPTG is 0.1 mM. This IPTG induction concentration, induction temperature, and conditions are beneficial for maintaining cell viability while promoting the correct folding and efficient secretion of the target protein. In other embodiments of the present invention, the fermentation medium is TB medium containing 20-50 g / L glycerol.

[0022] Beneficial effects:

[0023] This invention utilizes molecular chaperone co-expression to target key limiting factors such as protein folding during the expression of plastic depolymerase, significantly improving the correct folding ratio and effective extracellular accumulation of the plastic depolymerase. This effectively reduces the ineffective consumption of the plastic depolymerase during expression, increases enzyme yield per unit fermentation volume, and exhibits more stable and reproducible expression results. Based on the molecular chaperone engineering strategy, this application combines signal peptide optimization, promoter regulation, and the addition of solubilizing tags to further enhance the expression efficiency and industrial adaptability of the plastic depolymerase, making it suitable for scale-up production. The engineered E. coli constructed in this invention has a clear genetic background, mild culture conditions, and an easily controllable fermentation process, significantly reducing the production cost of the plastic depolymerase. The obtained enzyme preparation can be directly applied to the biodegradation of polyester plastics, demonstrating promising industrial application prospects. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 shows the expression effects of different PET depolymerases in Escherichia coli.

[0026] Figure 2 shows the effect of signal peptide screening on enhancing the secretion activity of plastic depolymerase.

[0027] Figure 3 shows the effect of solubilization label screening on the enzyme activity of plastic depolymerase.

[0028] Figure 4 shows the effect of promoter screening on the activity of plastic depolymerase.

[0029] Figure 5 shows the expression of different plastic depolymerases in Escherichia coli after synergistic implementation by molecular chaperones and regulatory elements. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0031] In the following examples, the TB culture medium was formulated as follows: 12 g / L tryptone, 24 g / L yeast extract, 2.31 g / L potassium dihydrogen phosphate, 12.54 g / L dipotassium hydrogen phosphate, and 40 g / L glycerol. It was autoclaved at 121°C for 20 min, and the pH was adjusted to 5.5 with ammonia.

[0032] Example 1: Validation of the expression of exogenous plastic depolymerase in Escherichia coli

[0033] First, the codons of the PET depolymerase gene were optimized and an expression vector was constructed. This embodiment includes the following PET depolymerases: PET depolymerase 1 (PETase 1, amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 7); PET depolymerase 2 (FAST-PETase, amino acid sequence as shown in SEQ ID NO. 2, nucleotide sequence as shown in SEQ ID NO. 8); PET depolymerase 3 (MuPETase, amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 9); PET depolymerase 4 (PES-H1, amino acid sequence as shown in SEQ ID NO. 4, nucleotide sequence as shown in SEQ ID NO. 10); PET depolymerase 5 (PES-H1 mutant 1, amino acid sequence as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 11); PET depolymerase 6 (PES-H1 mutant 2, amino acid sequence as shown in SEQ ID NO. 6, nucleotide sequence as shown in SEQ ID NO. 12).

[0034] The PET depolymerase gene sequences were inserted into the multiple cloning site of the E. coli expression vector pET22b, using EcoRI and BamHI restriction enzymes, to obtain recombinant plasmids carrying the target gene: pET22b-PETase 1, pET22b-FAST-PETase, pET22b-MuPETase, pET22b-PES-H1, pET22b-PES-H1mut1, and pET22b-PES-H1mut2. These recombinant plasmids were transformed into the expression host bacterium Escherichia coli BL21, and the strains were screened using ampicillin-resistant plates to obtain stably expressing genetically engineered strains, which were named BL21 PETase 1, BL21 FAST-PETase, BL21MuPETase, BL21 PES-H1, BL21 PES-H1mut1, and BL21 PES-H1mut2, respectively.

[0035] Each of the above-mentioned monoclonal engineered bacteria was picked and inoculated into LB medium, and cultured at 37 °C with shaking until OD reached. 600 The concentration was 0.8, and expression was induced for 24 hours at 25 °C by adding 0.1 mM IPTG. After induction, the fermentation supernatant was collected by centrifugation and used as crude enzyme solution for determining enzyme activity and protein concentration.

[0036] Enzyme activity was determined using the pNPO method. The reaction was carried out at 37 °C and pH 8 for 3 minutes, and the amount of p-nitrophenol generated was measured to determine enzyme activity. The results are shown in Figure 1. Based on the fact that *E. coli* BL21 itself does not produce depolymerases, various plastic depolymerases were effectively expressed in *E. coli* and exhibited detectable enzyme activity. Protein concentration was detected using a protein assay kit.

[0037] Example 2: Co-expression of molecular chaperones

[0038] Based on the basic expression system determined in Example 1, the effect of molecular chaperone co-expression on the expression of plastic depolymerase 1 was further investigated using PET depolymerase 1 as an example. The molecular chaperones described in this example were expressed in the form of molecular chaperone plasmids, all of which were commercially available plasmids, as detailed below:

[0039] pG-KJE8: contains molecular chaperones dank, danJ, grpE, groES, and groEL;

[0040] pGro7: Contains molecular chaperones groES and groEL;

[0041] pKJE7: Contains molecular chaperones danK, danJ, and grpE;

[0042] pG-Tf2: contains molecular chaperones groES, groEL, and tig;

[0043] pTf16: Contains the molecular chaperone TIG.

[0044] The plasmids containing different molecular chaperone genes were transformed and introduced into the strain BL21 PETase 1 (i.e., the WT strain) constructed in Example 1. Stable expression strains were obtained by screening with plates containing the corresponding resistance. The genetically engineered strains were induced to express PETase 1 under the induction conditions described in Example 1, and enzyme activity was measured. The results are shown in Table 1. The co-expression control system generally showed a significant promoting effect. The extracellular enzyme activity of the three molecular chaperones, danK, danJ, and grpE, synergistically expressed with the target gene increased by 3.2-fold. The engineered strain co-expressing PETase 1 with danK, danK, and grpE chaperone proteins was named BL21 PETase 1-1.

[0045] Table 1. Enzymatic activity enhancement of co-expressed molecular chaperones on plastic depolymerization enzymes

[0046]

[0047] Example 3 Systematic screening and optimization of plastic depolymerase expression regulatory elements

[0048] Based on the engineered bacterium BL21 PETase1-1 described in Example 2, the secretory expression of plastic depolymerase was further systematically optimized from multiple expression regulatory elements such as signal peptide, solubilization tag and promoter regulation.

[0049] (1) Screening of different signal peptides

[0050] Secretory expression vectors for plastic depolymerases guided by different secretory signal peptides were constructed. The selected signal peptides included PelB, OmpA, LamB, TolB, HylA, MalE, PhoA, and DsbA, with their amino acid sequences shown in SEQ ID NO. 13-20, respectively. The plasmid vector pET22b contained the signal peptide pelB. The method for constructing the signal peptide-guided secretory expression vectors in this embodiment involved replacing the pelB signal peptide of the pET22b-PETase1 plasmid vector constructed in Example 1 with the different signal peptide sequences described above, thus obtaining secretory expression vectors containing different signal peptides. Both the above secretory expression vectors and the pKJE7 plasmid (containing danK, danJ, and grpE molecular chaperones) described in Example 2 were transformed into the BL21 strain to obtain a series of differentially expressed signal peptide strains. Each engineered strain was fermented under the same culture and induction conditions as in Example 1, and its extracellular plastic depolymerase activity was measured. The results, as shown in Figure 2, indicate that the engineered strain (named BL21 PETase 1-2) that expressed the plastic depolymerase PETase 1 guided by the TolB signal peptide (amino acid sequence shown in SEQ ID NO. 16) and simultaneously introduced into the pKJE7 plasmid exhibited the highest efficiency in secreting PETase 1. The recombinant plasmid obtained by replacing the pelB signal peptide in the pET22b-PETase 1 plasmid vector with the TolB signal peptide was named pET22b-SP. TolB -PETase 1.

[0051] (2) Effect of solubilization tags on expression

[0052] Based on the strain BL21 PETase1-2, the effects of N-terminal fusion of a solubilizing tag on protein expression and secretion performance of plastic depolymerase were further compared. The solubilizing tag included any one of Trx, SUMO, NusA, and MBP. The amino acid sequences of the solubilizing tags Trx, SUMO, NusA, and MBP are shown in SEQ ID NO. 21-24, respectively. The Trx, SUMO, NusA, and MBP sequences were synthesized by the company and directly ligated into pET22b-SP. TolBVectors containing different solubilization tags were obtained by extracting the N-terminus of the PETase 1 gene from the PETase 1 plasmid. These vectors, along with the pKJE7 plasmid described in Example 2, were transformed into the BL21 strain to obtain a series of strains with differentially expressed solubilization tags. Each engineered strain was fermented under the same culture and induction conditions as in Example 1, and its extracellular plastic depolymerase activity was measured. The results, as shown in Figure 3, indicate that adding a solubilization tag to the N-terminus promoted the extracellular expression of PETase 1 to varying degrees. NusA (amino acid sequence shown in SEQ ID NO. 23) was identified as the optimal solubilization tag, significantly enhancing the extracellular protein expression level of PETase 1. The pET22b-SP strain containing the NusA solubilization tag was selected as the optimal solubilization tag. TolB -PETase 1 plasmid named pET22b-SP TolB -NusA-PETase 1, containing pET22b-SP TolB The engineered bacteria of the -NusA-PETase 1 plasmid and pKJE7 plasmid were named BL21PETase1-3.

[0053] (3) Comparison of the regulatory effects of different promoters

[0054] Promoter screening was performed based on the engineered bacterium BL21 PETase1-3. Specifically, P... lac、 P araBAD and P Trc The promoter (nucleotide sequences shown in SEQ ID NO. 26~28) replaces pET22b-SP. TolB -NusA-PETase 1 vector originally contained P T7 A promoter (its nucleotide sequence is shown in SEQ ID NO. 25) was used to drive the expression of plastic depolymerase. Recombinant vectors containing different promoters were constructed, and both the above vectors and the pKJE7 plasmid described in Example 2 were transformed into the BL21 strain to obtain a series of strains with differentially expressed promoters. Among them, those containing P araBAD The strain containing the promoter was induced using arabinose at a final concentration of 13 mM during the induction phase. The remaining culture methods were the same as in Example 1. Other engineered strains were fermented under the same culture and induction conditions as in Example 1. The extracellular plastic depolymerase activity of the above engineered strains was measured. The results, as shown in Figure 4, indicate that different promoters significantly affect the expression level of plastic depolymerase, with IPTG-induced P... lacThe promoter (nucleotide sequence shown in SEQ ID NO.26) exhibited high expression levels in the described co-engineering system. The resulting engineered strain BL21 PETase1-4 showed an enzyme activity of 380 U / mL and a protein concentration of 1.9 g / L, representing a 7.6-fold increase in PETase1 expression compared to the unoptimized strain. (Note: The last part, "P," is a direct translation of the last part and doesn't need a direct translation.) lac The recombinant plasmid for the promoter was named pET22b-P lac -SP TolB -NusA-PETase1 will contain pET22b-P lac -SP TolB The engineered strain containing the -NusA-PETase1 plasmid and pKJE7 plasmid was named BL21 PETase1-4.

[0055] Example 4: 5L fermenter-scale production

[0056] The above-mentioned preferred strain BL21 PETase1-4 was used for high-density fermentation in a 5 L fermenter. The specific steps are as follows:

[0057] (1) Cell growth stage: The cells were cultured in TB medium containing 40 g / L glycerol. The culture conditions were: temperature 37℃, pH 7, pressure 0.05 MPa, stirring speed adjusted according to dissolved oxygen, and dissolved oxygen (DO) controlled at 30%~35%. The culture time was about 10 h.

[0058] (2) Fed-feed fermentation stage: Start feeding with 50% v / v glycerol at a rate of 6~12 mL / L / h, and continue culturing for 4~8 h until the cell OD of the fermentation broth reaches zero. 600 Once it reaches 30, maintain DO at 30%~35% during this stage.

[0059] (3) Induction phase: IPTG with a final concentration of 0.1 mM was added for induction. The induction temperature was maintained at 25℃. During the entire induction phase, DO was maintained at 20%~30%, and the total induction time was 12~18 h.

[0060] After 16 hours of induction and fermentation, the enzyme activity of the target depolymerase in the fermentation supernatant was measured to be up to 1660 U / mL, and the protein concentration was 8.3 g / L.

[0061] The results of this embodiment show that the multidimensional optimized engineered bacteria obtained by applying the fermentation method of the present invention exhibit excellent growth stability and high efficiency in producing the target protein in 5 L scale fermentation, verifying the potential for large-scale application of the strain and the supporting fermentation process.

[0062] Example 5: Application of crude enzyme solution in PET degradation

[0063] Based on the fermentation results described in Example 4, the degradation activity was verified in a 5 L fermenter. Specifically, the fermentation broth from Example 4 was centrifuged and filtered through a 0.45 μm membrane to obtain a crude enzyme solution. Commercially available amorphous PET film and waste PET pillow cores (crushed and washed) were used as substrates. The degradation reaction system consisted of 400 g of PET substrate, 1.2 g of enzyme, and reacted in 2 L of pure water. The pH was controlled at 8.0 using 4 M sodium hydroxide, and the reaction was carried out at 65°C and 200 rpm for 24 hours. After the reaction, the remaining solids were collected by filtration, dried, weighed, and the mass loss rate was calculated. Simultaneously, the supernatant was taken, and the concentrations of the degradation products terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were determined by HPLC.

[0064] The results showed that the depolymerization rate of amorphous PET film was 76.65%, and the depolymerization rate of waste PET pillow core was 85.25%. This embodiment demonstrates that the crude enzyme solution produced by the engineered bacteria of this invention can efficiently degrade PET plastics of different forms without complex purification.

[0065] Example 6: Universality Verification of Synergistic Implementation of Molecular Chaperones and Regulatory Elements

[0066] This embodiment aims to verify the applicability and universal promoting effect of the gene editing strategy optimized in the above embodiments on different plastic depolymerases.

[0067] The plasmid pET22b-P from Example 3 was used. lac -SP TolB After replacing the target gene PETase1 in the -NusA-PETase1 plasmid with any one of FAST-PETase, MuPETase, PES-H1, PES-H1 mutant 1, and PES-H1 mutant 2, the resulting vector and the pKJE7 plasmid described in Example 2 were transformed into the expression host bacterium BL21, and the strains were screened using the corresponding resistance plates to obtain engineered strains that stably integrate different depolymerase genes.

[0068] Single clones of the co-engineered bacteria corresponding to the different plastic depolymerization enzymes were selected and fermented in a 5 L fermenter according to the method described in Example 4. The fermentation supernatant of the different plastic depolymerization enzyme engineered bacteria was analyzed for enzyme activity and expression level. The results are shown in Figure 5. Under the same molecular chaperone and expression element conditions, the plastic depolymerization enzymes from different sources, with different sequences and different structural characteristics can all achieve stable secretory expression.

[0069] The above results demonstrate that the synergistic engineering strategy designed in this invention, which combines molecular chaperone co-expression with regulatory elements, is not only applicable to the expression optimization of a single plastic depolymerase, but also has a good universal promoting effect on a variety of different types of plastic depolymerases, thus verifying the universal applicability of this strategy in constructing engineered bacteria for the production of highly efficient plastic depolymerases.

[0070] This invention provides an engineered Escherichia coli strain that efficiently produces plastic depolymerization enzymes, along with its application and related ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A highly efficient engineered Escherichia coli strain for producing plastic depolymerization enzyme, characterized in that, The engineered *E. coli* strain co-expresses a gene encoding a plastic depolymerase and a molecular chaperone; wherein the plastic depolymerase is a polyethylene terephthalate (PET) degradative enzyme; the amino acid sequence of the plastic depolymerase is shown in SEQ ID NO. 1; the molecular chaperone is a combination of danK, danJ, and grpE; the expression of the gene encoding the plastic depolymerase is regulated by a regulatory element, which includes a combination of a signal peptide, a solubilizing tag, and a strong promoter; the signal peptide is the sequence shown in SEQ ID NO. 16, the solubilizing tag is NusA as shown in SEQ ID NO. 23, and the strong promoter is P as shown in SEQ ID NO.

26. lac .

2. The engineered Escherichia coli strain according to claim 1, characterized in that, Its host bacteria include Escherichia coli BL21.

3. The application of the engineered Escherichia coli strain according to any one of claims 1 to 2 in the production of plastic depolymerase.

4. The method for producing plastic depolymerase using engineered Escherichia coli according to any one of claims 1 to 2, characterized in that, The engineered Escherichia coli strain was inoculated into a fermentation medium and fermented at 16-37 °C for 26-38 h to obtain the plastic depolymerization enzyme.

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