A method and strain for improving the yield of pet depolymerase based on cell membrane protection strategy

Abstract

This invention relates to the fields of microbial fermentation and genetic engineering, specifically to a method and strain for increasing the yield of PET depolymerase based on a cell membrane protection strategy. This method, by simultaneously adding exogenous phosphatidylethanolamine and enhancing the endogenous phosphatidylethanolamine synthesis pathway in the host bacteria through genetic engineering, synergistically enhances cell membrane stability, achieving protection and repair of cell membrane integrity, and significantly improving the expression level and extracellular secretion efficiency of PET depolymerase in *E. coli*. This method is applicable to various PET depolymerases such as PETase and FastPETase, demonstrating strong versatility. High-density fermentation in a 5 L fermenter yielded an enzyme production of 5.1 g / L, the highest reported level to date. This invention's method is simple to operate, cost-effective, and the resulting fermentation broth can be directly used for the enzymatic degradation of PET plastics, showing broad application prospects in the field of PET waste biorecycling.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and genetic engineering, and specifically to a method and strain for increasing the yield of PET depolymerase based on a cell membrane protection strategy. Background Technology

[0002] Polyethylene terephthalate (PET) is one of the most widely used polyester plastics globally, and its difficulty in natural degradation has caused serious white pollution. Utilizing PET hydrolase to degrade PET plastic into monomers or oligomers is an important way to achieve green recycling of PET waste.

[0003] Escherichia coli is widely used as a host bacterium for the recombinant expression of PET depolymerase due to its advantages such as simple genetic manipulation, rapid growth, and low culture cost. However, the following technical challenges exist in the fermentation production of PET depolymerase from E. coli:

[0004] First, overexpression of recombinant proteins often leads to cell membrane damage. Studies have shown that phospholipase activity can disrupt cell membrane structure and reduce the production of PET depolymerase. The integrity of the cell membrane is crucial for the proper folding, transmembrane transport, and extracellular secretion of recombinant proteins.

[0005] Second, phosphatidylethanolamine (PE) is the most abundant phospholipid component in the *E. coli* cell membrane, accounting for approximately 70%–80% of the total membrane phospholipids. It not only forms the structural basis of the cell membrane but also participates in the proper folding and functional maintenance of membrane proteins. Existing research indicates that the absence of PE significantly reduces the secretion efficiency of secretory proteins such as alkaline phosphatase. However, there are currently no reports of applying PE supplementation and synthesis strategies to improve the fermentation yield of PET depolymerase.

[0006] Third, existing E. coli fermentation processes using PET depolymerase generally face problems such as low yield and low extracellular secretion efficiency. Although multi-dimensional engineering strategies have been attempted, a systematic strategy for cell membrane protection remains lacking.

[0007] Therefore, developing a method to increase the yield of PET depolymerase through cell membrane protection, particularly through the supplementation and synthetic enhancement of phosphatidylethanolamine, has significant industrial application value. Summary of the Invention

[0008] The technical problem this invention aims to solve is the low yield and low extracellular secretion efficiency of PET depolymerase in E. coli fermentation processes in existing technologies. This invention provides a method for improving PET depolymerase yield based on a cell membrane protection strategy. This method involves adding exogenous phosphatidylethanolamine to the fermentation medium during fermentation, and / or enhancing the host bacteria's endogenous phosphatidylethanolamine synthesis pathway through genetic engineering. This achieves the protection and repair of cell membrane integrity, thereby increasing the expression level and extracellular secretion efficiency of PET depolymerase.

[0009] Another technical problem to be solved by the present invention is to provide a strain that improves the yield of PET depolymerase based on a cell membrane protection strategy.

[0010] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned method or strain in the degradation of PET plastic.

[0011] The final technical problem to be solved by this invention is to provide a method for degrading PET plastic.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a method for increasing the yield of PET depolymerase based on a cell membrane protection strategy, comprising the following steps:

[0014] (1) Construct a recombinant Escherichia coli that co-expresses PET depolymerase and phosphatidylserine decarboxylase, wherein the recombinant Escherichia coli carries a first recombinant vector containing a gene encoding PET depolymerase and a second recombinant vector containing a gene encoding phosphatidylserine decarboxylase psd.

[0015] (2) The seed culture of recombinant Escherichia coli in step (1) is inoculated into the fermentation medium for fermentation culture and induction expression of PET depolymerase. During the induction period, exogenous phosphatidylethanolamine is added to the fermentation medium.

[0016] In step (1), the PET depolymerase is selected from at least one of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase.

[0017] In some embodiments of the present invention, the PET depolymerase is PETase, or FastPETase, or PES-H1, or TurboPETase, or SsPETase.

[0018] The amino acid sequences of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase are shown in SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.11, respectively.

[0019] In step (1), the amino acid sequence of the phosphatidylserine decarboxylase is shown in SEQ ID NO.3.

[0020] The vectors include, but are not limited to, pET28a and pACYC Duet-1. Any existing vector tools that can carry the PET depolymerase gene and the phosphatidylserine decarboxylase gene psd can be used as vectors in this invention.

[0021] In some embodiments of the present invention, the first recombinant vector is pET28a and the second recombinant vector is pACYC Duet-1.

[0022] In step (2), the induction is performed by inducing with IPTG at a final concentration of 0.1-0.5 mM for 4-24 h; preferably, the induction is performed by inducing with IPTG at a final concentration of 0.5 mM for 20 h.

[0023] In step (2), the final concentration of the exogenous phosphatidylethanolamine is 0.1~10 mM; preferably, the final concentration of the exogenous phosphatidylethanolamine is 5 mM. As a key component of the cell membrane, an adequate supply of phosphatidylethanolamine contributes to the normal function of membrane proteins and the transmembrane transport of recombinant proteins.

[0024] In step (2), the fermentation culture is either shake flask fermentation or high-density fermentation.

[0025] In some embodiments of the present invention, the high-density fermentation is a scale-up culture in a 5 L fermenter, including a cell growth stage, a fed-batch fermentation stage, and an induction stage.

[0026] Specifically, the culture conditions for the cell growth stage are: temperature 30~37℃, pH 7.0~8.0, initial stirring speed 200~500 rpm, aeration rate 2~4 L / min, dissolved oxygen (DO) controlled at 30%~35%, and culture time approximately 10 hours.

[0027] Specifically, the fed-batch fermentation stage begins from the point where dissolved oxygen (DO) rises sharply until the OD of the fermentation broth cells increases. 600 To reach 30, the flow rate of the fed culture medium was 6-12 mL / L / h, and the dissolved oxygen (DO) was maintained at 30%-35% during this period.

[0028] Specifically, the culture conditions for the induction phase are: temperature 25℃, dissolved oxygen controlled at 20%~30%, and total induction time 16~30 h.

[0029] Secondly, the present invention provides a recombinant Escherichia coli that produces PET depolymerase based on a cell membrane protection strategy, which is obtained by overexpressing the PET depolymerase gene and the phosphatidylserine decarboxylase gene using Escherichia coli as the chassis strain.

[0030] The PET depolymerase is selected from at least one of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase.

[0031] In some embodiments of the present invention, the PET depolymerase is PETase, or FastPETase, or PES-H1, or TurboPETase, or SsPETase.

[0032] The nucleotide sequences of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase are shown in SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, and SEQ ID NO.12, respectively.

[0033] The nucleotide sequence of the phosphatidylserine decarboxylase is shown in SEQ ID NO.4.

[0034] Thirdly, the present invention provides the application of the method described in the first aspect or the recombinant Escherichia coli described in the second aspect in the degradation of PET plastic.

[0035] Fourthly, the present invention provides a method for degrading PET plastic, wherein the crude enzyme solution of PET depolymerase prepared by the method described in the first aspect, or the crude enzyme solution of PET depolymerase obtained by fermentation and induced expression of recombinant Escherichia coli described in the second aspect, is directly reacted with PET plastic to carry out a depolymerization reaction.

[0036] The depolymerization reaction is carried out under the following conditions: pH 7.0~9.0, 100~500 rpm, and 55~70℃ for 12~36 h.

[0037] In some embodiments of the present invention, the PET plastic is an amorphous PET film and / or waste PET products.

[0038] Beneficial effects:

[0039] (1) This invention, through a combined strategy of supplementing exogenous phosphatidylethanolamine and enhancing the endogenous phosphatidylethanolamine synthesis pathway in the host bacteria (overexpression of the psd gene) using genetic engineering, effectively protects cell membrane integrity, reduces yield decline caused by membrane damage, and achieves a synergistic increase in PET depolymerase yield. In a high-density fermentation scale-up experiment in a 5 L fermenter, the yield reached 5.1 g / L, which is the highest reported yield level for PET hydrolase to date, demonstrating potential for industrial scale-up and enabling large-scale production.

[0040] (2) Regardless of the source, amino acid sequence and structural characteristics of PET depolymerase, this combination strategy can significantly improve its expression level in recombinant Escherichia coli and simultaneously increase the fermentation density of the host strain, which has strong universality.

[0041] (3) The crude enzyme solution produced by the method of this application (the fermentation broth can be used directly after centrifugation and membrane filtration) can efficiently degrade PET plastics of different forms without complicated purification steps. The depolymerization rates for amorphous PET films and waste PET products are 72.65% and 83.25%, respectively.

[0042] (4) The technical solution of this application does not involve complex genetic engineering modification. The strategy of genetic engineering modification of the phosphatidylethanolamine synthesis pathway has sustainability and cost advantages. At the same time, the addition of exogenous phosphatidylethanolamine is simple to operate and easy to implement in industrial fermentation. The overall operation is simple and the cost is controllable. Attached Figure Description

[0043] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0044] Figure 1 The results show the optimized conditions for exogenous phosphatidylethanolamine addition. In this table, A represents cell growth under different induction durations with phosphatidylethanolamine addition, B represents cell enzyme production under different induction durations with phosphatidylethanolamine addition, C represents cell growth under different concentrations of phosphatidylethanolamine addition, and D represents cell enzyme production under different concentrations of phosphatidylethanolamine addition.

[0045] Figure 2 The images show cell growth and enzyme production data for cells overexpressing key enzyme genes in the phosphatidylethanolamine synthesis pathway. In the images, A represents cell growth and B represents enzyme production.

[0046] Figure 3 This diagram illustrates cell growth and enzyme production under a strategy combining exogenous addition of phosphatidylethanolamine and overexpression of key enzyme genes in the phosphatidylethanolamine biosynthesis pathway. In the diagram, A represents cell growth, and B represents cell enzyme production.

[0047] Figure 4 This section demonstrates the applicability of the combined strategy across different PET depolymerases. In this diagram, A represents cell growth, and B represents cell enzyme production. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0050] The culture media involved in the following examples are as follows:

[0051] (1) LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0052] (2) Fermentation medium: 24 g / L yeast extract, 12 g / L peptone, 5 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, initial pH 7.

[0053] The detection methods involved in the following embodiments are as follows:

[0054] (1) Biomass determination method

[0055] The wet cell weight (WCW) of the cells during fermentation was determined using a specific gravity method. A 2 mL capped centrifuge tube was dried to constant weight, weighed, and then 1 mL of fermentation broth was added. The tube was centrifuged at 12000 rpm for 5 min. After removing the supernatant, the cell pellet was collected and washed twice with 1 mL of physiological saline. After centrifugation, the supernatant was discarded, and the cells were weighed. The wet cell weight was calculated as: Wet cell weight (g / L) = (Weight of centrifuge tube containing wet cells (g) - Net weight of centrifuge tube (g)) / Sampling volume of fermentation broth (L).

[0056] The turbidimetric method was used to determine the cellular optical density (OD) during fermentation. 1 mL of fermentation broth was added to a capped centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was discarded, and 1 mL of physiological saline was added and mixed thoroughly. After appropriate dilution, the absorbance of physiological saline was used as a blank control. The appropriate dilution factor (n) was considered when the absorbance at 600 nm was between 0.2 and 0.8. Cellular optical density (OD) = n × OD 600 .

[0057] (2) Depolymerase activity detection method

[0058] Add 10 μL of 10 mM p-nitrophenol octanoate (pNPO), 10 μL of enzyme solution, and 980 μL of PBS buffer (50 mM, pH 8.0) to a 5 mL centrifuge tube to a final concentration of 1 mL. Heat the assay system at 37 °C for 10 min, then immediately cool it on ice. Measure the absorbance at 410 nm using a microplate reader. The absorbance is determined by p-nitrophenol and OD... 410 The standard curve was plotted to calculate the molar amount of p-nitrophenol. Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute at 37°C.

[0059] (3) Protein content detection methods

[0060] This invention uses a protein assay kit (BSA protein assay kit, Genstar, Beijing, China) to detect protein concentration and SDS-PAGE analysis to determine the purity of each protein. First, a 1 mg / mL bovine serum albumin (BSA) stock solution is prepared. Then, PBS (50 mM, pH 8.0) is added to the stock solution to prepare a series of BSA stock solutions with concentrations ranging from 0.2 to 1 mg / L. This series of solutions is then diluted 10-fold to obtain a series of diluted BSA solutions with concentrations ranging from 0.02 to 0.1 mg / L. 20 μL of each of the different concentration gradients of BSA standard solutions is added to a 96-well plate, followed by 200 μL of Coomassie Brilliant Blue solution. After thorough mixing, the mixture is allowed to stand for 3 min. The absorbance of the different concentrations of BSA standard solutions at 595 nm is measured using a microplate reader, and a standard curve is plotted with protein concentration on the x-axis and absorbance on the y-axis. The absorbance of the samples can be measured using the same method, and the protein concentration can be calculated from the standard curve.

[0061] In this invention, the protein expression level refers to the concentration of active protein in the protein solution, and the calculation formula is: protein expression level (mg / L) = protein solution enzyme activity (U / mL) / protein specific enzyme activity (U / mg).

[0062] (4) Liquid chromatography method for detecting the depolymerization rate of PET and BHET

[0063] PET degradation enzymatic hydrolysates were filtered using a 0.22 μm filter, diluted appropriately according to product concentration, and then analyzed using an Agilent 1260 Infinity II high-performance liquid chromatography system via a C18 column (Thermo Fisher Scientific, 150 × 4.6 mm, 5 μm). A mobile phase of 18% acetonitrile, 1% formic acid, and 81% water (pH 2.5) was used. The detection time was 25 min, the flow rate was 0.8 mL / min, and the detection wavelength was 254 nm. Standard curves were constructed using TPA and BHET gradient standard solutions at concentrations of 1 mM, 0.1 mM, 0.01 mM, 0.001 mM, and 0.0001 mM, respectively. The product concentration of the enzymatic hydrolysate could be calculated from the standard curves.

[0064] Example 1: Addition of exogenous phosphatidylethanolamine to increase PET depolymerase yield

[0065] 1. Construction of recombinant vector pET28a-PETase and expression strain

[0066] The gene encoding PET depolymerase was codon-optimized (optimized sequence shown in SEQ ID NO.2), and the target gene fragment was obtained through chemical synthesis. The target gene fragment and the pET28a expression vector (carrying kanamycin resistance) were double-digested with restriction endonucleases NcoI and XhoI, purified, and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Positive clones were picked for sequencing verification, and the results showed that the pET28a-PETase recombinant vector was constructed correctly.

[0067] The recombinant vector pET28a-PETase, which was verified to be correct by sequencing, was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Positive clones were picked, and PCR verification confirmed the successful introduction of the recombinant vector, thus obtaining the expression strain.

[0068] 2. Fermentation Culture and Induction Expression: Select verified single colonies and inoculate them into 5 mL of LB medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 220 rpm. Transfer the inoculum to a 250 mL shake flask containing 50 mL of fermentation medium at a 2% v / v inoculation rate and incubate at 37°C and 220 rpm until OD500 reaches the target value. 600After the temperature reached approximately 0.6~0.8, IPTG was added to a final concentration of 0.5 mM for induction. Simultaneously, phosphatidylethanolamine (PE, dissolved in an appropriate amount of Triton X-100) was added to a final concentration of 1 mM for induction. The mixture was then incubated at 25°C and 200 rpm for 24 hours. A control group was used without the addition of phosphatidylethanolamine.

[0069] Samples were taken at 0, 4, 8, 12, 16, 20, and 24 hours after induction to measure OD. 600 value( Figure 1 The expression level of PET depolymerase was detected by enzyme activity assay (A in the text). Figure 1 (B in the text). The results showed that the production of PET depolymerase reached its peak after 20 h of induction, increasing by approximately 66% compared to the control group, and OD... 600 The value was 32% higher than that of the control group.

[0070] Furthermore, with a fixed induction time of 20 h, the concentration of phosphatidylethanolamine was optimized. Phosphatidylethanolamine at concentrations of 0, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM was added during induction. After 20 h of induction, samples were taken to measure OD. 600 ( Figure 1 The C value and the enzyme activity of PET depolymerase (in the text) Figure 1 (D in the text). The results showed that the production of PET depolymerase was highest with the addition of 5 mM phosphatidylethanolamine, increasing by approximately 123% compared to the control group, OD... 600 The value was approximately 78% higher than that of the control group.

[0071] Example 2: Overexpression of phosphatidylethanolamine synthesis pathway gene increases PET depolymerase yield

[0072] 1. Construction of the recombinant vector pACYC Duet-1-psd

[0073] The phosphatidylserine decarboxylase gene psd (amino acid sequence shown in SEQ ID. NO. 3, nucleotide sequence shown in SEQ ID. NO. 4), a key enzyme gene in the phosphatidylethanolamine synthesis pathway of *E. coli*, was amplified by PCR and cloned into the pACYC Duet-1 vector (carrying chloramphenicol resistance). The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 20 μg / mL chloramphenicol, and incubated overnight at 37°C. Positive clones were selected for sequencing verification, yielding the recombinant vector pACYC Duet-1-psd with the correct sequence.

[0074] 2. Co-transformation to prepare recombinant strains

[0075] The obtained pACYC Duet-1-psd recombinant vector was co-transformed with the PET depolymerase recombinant vector pET28a-PETase (carrying kanamycin resistance) from Example 1 into BL21(DE3) competent cells. The cells were plated on LB agar plates containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol and incubated overnight at 37°C. Positive clones were picked and verified by PCR to obtain the recombinant strain. A strain containing only the pET28a-PETase vector served as a control. Fermentation and IPTG induction were performed according to the method in Example 1 for 20 h, and OD was measured. 600 value( Figure 2 The expression level of PET depolymerase was detected by enzyme activity assay (A in the text). Figure 2 (B in the middle).

[0076] The results showed that the recombinant strain overexpressing the phosphatidylserine decarboxylase gene psd exhibited approximately 162% higher PET depolymerase production and OD... 600 The value was approximately 93% higher than that of the control group.

[0077] Example 3: Combination Strategy (Exogenous Addition + Synthetic Enhancement)

[0078] Simultaneously, fermentation experiments were conducted using the exogenous phosphatidylethanolamine addition strategy from Example 1 and the phosphatidylethanolamine synthesis pathway overexpression strategy from Example 2. Fermentation culture and IPTG induction for 20 h (with a final concentration of 5 mM phosphatidylethanolamine added) were performed according to the method in Example 1 (replacing the LB medium containing 50 μg / mL kanamycin with LB medium containing 50 μg / mL kanamycin + 20 μg / mL chloramphenicol). OD was measured. 600 value( Figure 3 The expression level of PET depolymerase was detected by enzyme activity assay (A in the text). Figure 3 (B in the middle).

[0079] The results showed that the combined strategy of exogenous addition and synthetic enhancement had a synergistic effect, further increasing the yield of PET depolymerase compared to the single strategy, and increasing the total yield of PET depolymerase by approximately 313%, OD 600 The value increased by approximately 243%.

[0080] Example 4: Scale-up verification of high-density fermentation in a 5 L fermenter

[0081] 1. Preparation of seed liquid

[0082] Single colonies of the recombinant strain prepared in Example 2 were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin + 20 μg / mL chloramphenicol. The culture was carried out at 37°C and 220 rpm for 8 h with shaking to obtain the primary seed culture.

[0083] The primary seed culture was transferred at a 2% v / v inoculation rate to a 500 mL shake flask containing 100 mL of LB liquid medium containing 50 μg / mL kanamycin + 20 μg / mL chloramphenicol, and cultured at 37°C with shaking at 220 rpm until OD was reached. 600 ≈2.0, used as secondary seed liquid.

[0084] 2. Preparation of culture medium

[0085] (1) Prepare TB medium containing 20 g / L glucose as fermentation medium, specifically including: 24 g / L yeast extract, 12 g / L peptone, 5 g / L glycerol, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, 20 g / L glucose, pH 7.0; sterilize at 121℃ for 20 min, and after cooling, add filtered sterilized kanamycin and chloramphenicol to a final concentration of 50 μg / mL and 20 μg / mL, respectively.

[0086] (2) Feeding medium: 500 g / L glucose, sterilized at 121℃ for 20 min for later use.

[0087] 3. Cell growth stage

[0088] The secondary seed culture was inoculated into a 5 L fermenter containing 2 L of sterile fermentation medium at an inoculation rate of 5% v / v. The culture conditions were: temperature 37℃, pH 7 (adjusted with ammonia), stirring speed adjusted according to dissolved oxygen, initial stirring speed of 200 rpm, aeration rate of 2 L / min, dissolved oxygen (DO) controlled at 30%~35%, and culture time of about 10 hours.

[0089] 4. Feeding and Fermentation Stage

[0090] When the dissolved oxygen (DO) value rises sharply, it indicates that the glucose in the fermentation medium has been depleted. Initiate feeding: add 500 g / L glucose using a peristaltic pump at a rate of 6–12 mL / L / h, and continue culturing until the cell OD in the fermentation broth reaches zero. 600 Once it reaches 30, maintain DO at 30%~35% during this stage.

[0091] 5. Induction Phase

[0092] When OD 600 When the temperature reaches 30°C, 0.1 mM IPTG and 5 mM phosphatidylethanolamine are added to the fermenter simultaneously for induction. The induction temperature is maintained at 25°C. During the entire induction phase, the DO is kept at 20%~30%, and the total induction time is 18 hours.

[0093] After induced fermentation was completed, samples were taken and the expression level of PET depolymerase was detected by enzyme activity assay. The results showed that the enzyme activity of the target depolymerase in the fermentation supernatant increased by approximately 382% compared to before optimization, reaching 5.1 g / L, and the OD of the fermentation broth... 600 The score reached 93, an improvement of approximately 258% compared to before optimization.

[0094] The above results demonstrate that, using the strategy of this invention, the recombinant strain exhibits excellent growth stability and high efficiency in producing the target protein during 5 L-scale fermentation, verifying the potential of this strain and its supporting fermentation process for large-scale application.

[0095] Example 5: Application of crude enzyme solution in PET degradation

[0096] 1. Preparation of crude enzyme solution

[0097] The fermentation broth from Example 4 was centrifuged at 8000×g for 20 min at 4°C. The supernatant was collected and filtered through a 0.45 μm membrane to obtain the crude enzyme solution. The crude enzyme solution was stored at 4°C for later use.

[0098] 2. Degradation reaction system

[0099] Commercially available amorphous PET film was used as PET substrate 1, and waste PET pillow cores (crushed and washed) were used as PET substrate 2. The commercially available amorphous PET film was cut into small pieces of approximately 1 cm × 1 cm, washed with deionized water before use, and dried at 60°C to constant weight. The waste PET pillow cores were crushed to a particle size of approximately 2-5 mm, washed with a 1% (w / v) SDS solution at 60°C for 30 min to remove surface impurities, repeatedly rinsed with deionized water until neutral, and dried at 60°C to constant weight.

[0100] Degradation reaction system: 200 g / L PET substrate 1, 200 g / L PET substrate 2, 0.06 g enzyme added based on enzyme protein content (the protein concentration of the crude enzyme solution was determined in advance using the Bradford method), pure water added to a total volume of 100 mL, pH controlled at 8.0 using 2M sodium hydroxide, reaction at 65℃ and 200 rpm for 24 hours. After the reaction, the remaining solid was collected by filtration, washed three times with deionized water, dried at 60℃ to constant weight, and the mass loss rate, i.e., depolymerization rate, was calculated by weighing. Depolymerization rate (%) = (initial substrate dry weight - dry weight of remaining solid after reaction) / initial substrate dry weight × 100%. Simultaneously, the reaction supernatant was filtered through a 0.22 μm filter membrane, and the concentrations of degradation products terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected by high performance liquid chromatography (HPLC).

[0101] The results showed that the depolymerization rate of amorphous PET film was 72.65%, and the depolymerization rate of waste PET pillow core was 83.25%. This embodiment demonstrates that the crude enzyme solution produced by the recombinant strain of the present invention can efficiently degrade PET plastics of different forms without complex purification.

[0102] Example 6: Verification of the universality of the synergistic implementation of the combination strategy

[0103] This embodiment aims to verify the applicability and universal promoting effect of the synergistic engineering strategy of exogenous addition of phosphatidylethanolamine and overexpression of the psd gene described in Example 3 in different plastic depolymerization enzymes.

[0104] 1. Construction of expression vectors for different PET depolymerases

[0105] The target gene of plasmid PETase in Example 3 was replaced with FastPETase (SEQ ID NO. 6), PES-H1 (SEQ ID NO. 8), TurboPETase (SEQ ID NO. 10) and SsPETase (SEQ ID NO. 12), respectively.

[0106] Specifically, gene fragments encoding FastPETase, PES-H1, TurboPETase, and SsPETase were synthesized and cloned into the pET28a expression vector according to the method in Example 1 to obtain the corresponding recombinant plasmids. The recombinant plasmids were verified to be correct by sequencing.

[0107] The four recombinant plasmids described above were co-transformed with the pACYC Duet-1-psd recombinant vector (carrying chloramphenicol resistance) constructed in Example 2 into Escherichia coli BL21(DE3) competent cells. The cells were then plated on LB agar plates containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol and incubated overnight at 37°C. Positive clones were picked, and PCR verification confirmed successful introduction of both plasmids, resulting in four co-engineered bacterial strains.

[0108] Single colonies of the co-engineered strains corresponding to the different PET depolymerases were picked and fermented and induced according to the method in Example 1 (IPTG 0.5 mM, 25℃, 200~800 rpm, induced for 20 h, with the addition of 5 mM phosphatidylethanolamine).

[0109] Samples were taken 20 h after induction, and the OD values ​​of the fermentation broth in each group were analyzed. 600 value( Figure 4 The expression levels of A in the sample and the co-engineered strains corresponding to different PET depolymerases (A) and (B) Figure 4The results showed that by using a combination strategy of exogenous addition of phosphatidylethanolamine and overexpression of the psd gene, the expression levels of the four PET depolymerases from different sources, with different sequences and structural characteristics were significantly increased, and the fermentation OD of the host strain was improved. 600 The values ​​also showed a significant increase, indicating that the collaborative engineering strategy has good universality.

[0110] This invention provides a method, strain, and approach for improving PET depolymerase yield based on a cell membrane protection strategy. 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 method for increasing PET depolymerase yield based on a cell membrane protection strategy, characterized in that, Includes the following steps: (1) Construct a recombinant Escherichia coli that co-expresses PET depolymerase and phosphatidylserine decarboxylase, wherein the recombinant Escherichia coli carries a first recombinant vector containing a gene encoding PET depolymerase and a second recombinant vector containing a gene encoding phosphatidylserine decarboxylase psd. (2) The seed culture of recombinant Escherichia coli in step (1) was inoculated into the fermentation medium for fermentation culture and induced expression of PET depolymerase. During the induction period, exogenous phosphatidylethanolamine was added to the fermentation medium. In step (1), the PET depolymerase is selected from at least one of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase, and the amino acid sequences of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase are shown in SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.11, respectively. In step (1), the amino acid sequence of the phosphatidylserine decarboxylase is shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that, In step (2), the induction is performed by inducing with IPTG at a final concentration of 0.1-0.5 mM for 4-24 h.

3. The method according to claim 1, characterized in that, The final concentration of the exogenous phosphatidylethanolamine is 0.1~10 mM.

4. The method according to claim 3, characterized in that, The final concentration of the exogenous phosphatidylethanolamine is 5 mM.

5. A recombinant *E. coli* strain for producing PET depolymerase based on a cell membrane protection strategy, characterized in that, It was obtained by overexpressing the PET depolymerase gene and the phosphatidylserine decarboxylase gene using Escherichia coli as the chassis strain; The PET depolymerase is selected from at least one of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The nucleotide sequences of PETase, FastPETase, PES-H1, TurboPETase, and SsPETase are shown in SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, and SEQ ID NO.12, respectively.

7. The recombinant Escherichia coli according to claim 5, characterized in that, The nucleotide sequence of the phosphatidylserine decarboxylase is shown in SEQ ID NO.

4.

8. The method according to any one of claims 1 to 4 or the recombinant Escherichia coli according to any one of claims 5 to 7 in the degradation of PET plastic.

9. A method for degrading PET plastic, characterized in that, The crude enzyme solution of PET depolymerase prepared by the method described in any one of claims 1 to 4, or the crude enzyme solution of PET depolymerase obtained by fermentation and induced expression of recombinant Escherichia coli as described in any one of claims 5 to 7, is directly reacted with PET plastic to carry out a depolymerization reaction.

10. The method according to claim 9, characterized in that, The depolymerization reaction is carried out under the following conditions: pH 7.0~9.0, 100~500 rpm, 55~70℃ for 12~36 h.

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