A recombinant myxobacterium displaying PET depolymerase on its surface and its construction method and application

Abstract

The present invention provides a recombinant myxobacterium displaying PET depolymerase on its surface, and a construction method and application thereof, wherein the recombinant expression plasmid of the gene sequence of the signal peptide, PET depolymerase and anchor peptide is introduced into the chassis cell using the yellow myxococcus M.xanthus DK1622 as a chassis cell; the PET depolymerase is a codon-optimized PET depolymerase DuraPETase, and its nucleotide sequence is shown in SEQ ID NO: 1; the nucleotide sequence encoding the signal peptide SP is shown in SEQ ID NO: 2; and the nucleotide sequence encoding the anchor peptide is shown in SEQ ID NO: 3. The recombinant myxococcus xanthus provided by the present invention M.xanthus DK1622 can achieve the expression of polyethylene terephthalate hydrolase (DuraPETase) and the hydrolysis of the polyester model substrate Impranil DLN, which is expected to achieve in situ remediation of plastic pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant myxobacterium displaying PET depolymerase on its surface, and a construction method and application thereof. Background Art

[0002] In today's society, the widespread use of plastics has brought convenience to our daily lives, but it has also caused a large amount of plastic waste to be discharged into the environment, causing serious environmental pollution problems, especially the environmental problem of microplastics, which has become a global concern.

[0003] Compared with the physical and chemical recycling technologies for waste plastic disposal, biodegradation and conversion technology has gradually become an important way to solve plastic pollution. Biodegradation and conversion technology is a biorefining process that uses microorganisms / enzymes to selectively degrade waste plastics into different polymer monomers under mild conditions, as well as to increase the value of mixed plastic degradation products. With the breakthrough progress made in the field of plastic biodegradation in recent years, especially in the field of enzymatic degradation of PET plastics, people have a new understanding of plastic biodegradation. Since Muller et al. identified and defined the first PET plastic hydrolase TfH in 2005, efficient PET plastic hydrolases such as cutinase HiC, Tfcut2, LCC and PETase have been identified, characterized and analyzed one after another. Through enzyme molecular design and modification, enzymes such as LCC have also been obtained. ICCG , DuraPETase, FastPETase and other PET plastic efficient depolymerases or variant enzymes. In the existing technology, it is rare to construct engineered strains using environmental microbial surface display technology, which provides a basis for the biological treatment of plastics in the environment.

[0004] Myxobacteria are a type of Gram-negative bacteria with multicellular behavior and complex life cycle. They are widely found in soil environments and can prey on other microorganisms. In addition, they have the following advantages: 1) They can reproduce by preying on other microorganisms in oligotrophic soil environments, and the abundance of the bacterial community in the soil environment is stable; 2) They have good plant rhizosphere colonization and film-forming abilities. The construction of plastic-degrading strains based on myxobacteria (dominant bacteria in the soil) is expected to achieve in-situ remediation of plastic pollution. Summary of the invention

[0005] The first object of the present invention is to provide a recombinant myxobacterium displaying PET depolymerase on its surface.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A recombinant myxobacterium displaying PET depolymerase on its surface, Myxococcus xanthus M. xanthusDK1622 is used as a chassis cell, and a recombinant expression plasmid of a gene sequence of a signal peptide, a PET depolymerase, and an anchor peptide is introduced into the chassis cell to obtain the result;

[0008] The PET depolymerase is a codon-optimized PET depolymerase DuraPETase, and its nucleotide sequence is shown in SEQ ID NO: 1; the nucleotide sequence encoding the signal peptide SP is shown in SEQ ID NO: 2; and the nucleotide sequence encoding the anchor peptide is shown in SEQ ID NO: 3.

[0009] As a preferred embodiment, the promoter for expressing PET depolymerase in the recombinant expression plasmid is a constitutive promoter PilA, and its nucleotide sequence is shown in SEQ ID NO:4.

[0010] As a preferred embodiment, the recombinant expression plasmid is constructed using the pSWU19 plasmid containing the PilA promoter as a basic expression plasmid.

[0011] Another object of the present invention is to provide a method for constructing the above-mentioned recombinant myxobacterium, comprising:

[0012] (1) Wild-type Myxococcus xanthus M. xanthus The whole genome of DK1622 was used as a template to obtain the outer membrane protein TraA fragment by PCR amplification;

[0013] (2) Using the outer membrane protein TraA fragment obtained in step (1) as a template, PCR amplification is performed to obtain the signal peptide and anchor peptide fragments.

[0014] (3) Using the gene sequence of PET depolymerase as a template, PCR amplification was performed to obtain the DuraPETase fragment;

[0015] (4) Synthesizing homologous recombination fragments: Using the fragments obtained in steps (2) and (3) as templates, PCR amplification was performed to obtain signal peptide / PET depolymerase / anchor peptide fragments;

[0016] (5) Enzymatically ligating the fragment obtained in step (4) with the linear basic expression plasmid, transforming the fragment into Escherichia coli, and screening transformants on a solid plate containing kanamycin to obtain a recombinant expression plasmid;

[0017] (6) The recombinant expression plasmid obtained in step (5) is introduced into the wild-type Myxococcus aureus by electroporation. M. xanthus The recombinant myxobacterium was obtained by screening in DK1622 competent cells on a solid plate containing kanamycin.

[0018] Another object of the present invention is to provide the use of the above-mentioned recombinant myxobacteria in degrading polyester plastics, including: using the crude enzyme solution obtained by crushing the whole cells of the recombinant myxobacteria for degrading polyester plastics.

[0019] As a preferred embodiment, after the recombinant myxobacterium is fermented and cultured, the bacteria are collected and ultrasonically disrupted to obtain a crude enzyme solution after whole-cell disruption.

[0020] As a preferred embodiment, the culture medium for the fermentation culture is CYE liquid culture medium.

[0021] As a preferred embodiment, in the degradation system, the amount of crude enzyme solution added is 2-4 g / L substrate

[0022] As a preferred embodiment, the substrate concentration in the degradation system is 0.1-0.3% v / v.

[0023] The present invention has the following beneficial effects:

[0024] (1) The present invention obtains a method for expressing polyethylene terephthalate depolymerase (DuraPETase) M. xanthus DK1622 is a recombinant myxobacterium, and the enzyme protein obtained after fermentation culture has ester bond hydrolase activity.

[0025] (2) The present invention uses wild-type model strains M. xanthus DK1622 was used as a chassis cell to construct a recombinant Myxococcus aureus that can degrade the polyester model substrate Impranil DLN, laying the foundation for further bioremediation of soil plastic pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0027] Figure 1 Schematic diagram of recombinant plasmid construction.

[0028] Figure 2 A is the gel electrophoresis diagram for verification of recombinant single fragment and double enzyme digestion plasmid; B is M.xanthus Gel electrophoresis of PCR amplification and verification of DK1622 recombinant myxobacterium.

[0029] Figure 3 for M.xanthus Hydrolysis cycle diagram of crude enzyme solution obtained from DK1622 recombinant myxobacterium on Impranil DLN solid plate. DETAILED DESCRIPTION

[0030] Myxococcus xanthus used in the examples M. xanthusDK1622 is a model strain of Myxococcus xanthus and can be purchased from commercial channels.

[0031] The formula of CYE liquid culture medium is: magnesium sulfate 1 g / L, yeast powder 5 g / L, casein peptone 10 g / L, solvent is water, and pH is 7.6.

[0032] The solid plate containing Impranil DLN is MSM solid medium, with the formula: 2g / L (NH4)2SO4, 0.2 g / LMgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O, 0.6g / L Na2HPO4, 1.5g / LKH2PO4, 2%v / v Impranil DLN, 15 g / L agar powder, the solvent is water, and the pH is neutral. The temperature of the degradation stage is 37°C, the catalytic time is 24-48 h, and the pH is neutral.

[0033] Example 1 This example illustrates the preparation of SP / DuraPETase / MYXO-CTREM fragment.

[0034] (1) Using wild-type model bacteria M. xanthus The whole genome of DK1622 was used as a template, and the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 were used as primers to obtain the TraA fragment by PCR amplification;

[0035] (2) Using the TraA fragment obtained in step (1) as a template and the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7, and SEQ ID NO: 8 and SEQ ID NO: 6 as primers, amplify the signal peptide SP and MYXO-CTREM anchor peptide fragments through PCR reaction.

[0036] (3) Using the synthetic plasmid pET22b as a template and the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 as primers, PCR amplification was performed to obtain the codon-optimized DuraPETase fragment.

[0037] (4) Using the single fragment obtained in steps (2) and (3) as a template, the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 as primers for overlap extension ligation, and PCR amplification to obtain the SP / DuraPETase / MYXO-CTREM fragment. The results are as follows: Figure 1 .

[0038] The PCR amplification system was: 1 µL of the obtained DNA template, 2 µL of the upstream and downstream primers, 25 µL of 2 × Phanta MaxMaster Mix buffer, and 20 µL of ddH2O. The PCR reaction program was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s; then 58°C annealing for 30 s, 72°C extension, 34 cycles; 72°C extension for 5 min. The amplified bands were cut and recovered using a column-type gel recovery kit.

[0039] Example 2 This example describes M.xanthus Construction of DK1622 recombinant myxobacterium. The plasmid construction is shown in the figure below: Figure 1 .

[0040] The plasmid containing PilA promoter p For SWU19 Xba Ⅰ. Hin d Ⅲ double enzyme digestion. The enzyme digestion system is: plasmid 50μL, Xba Ⅰ5µL, Hind Ⅲ5µL, 10× buffer 10µL, ddH2O30µL, total volume 100µL. After the enzyme cutting system is cut into the gel, it is recovered using a column cutting recovery kit. The nucleotide sequence of the PilA promoter is shown in SEQ ID NO: 4.

[0041] The SP / DuraPETase / MYXO-CTREM fragment obtained in Example 1 was recovered by gel ligation and then connected to the linear plasmid after double restriction digestion. The ligation reaction system was: 2 µL of PCR product, 3 µL of restriction digestion plasmid, 2 µL of one-step cloning ligase, 3 µL of 5× ligase buffer, 5 µL of ddH2O, and a total volume of 15 µL. The recombinant plasmid was obtained by ligation. p SWU19-PilA / SP / DuraPETase / MYXO-CTREM, screen transformants on a solid plate containing kanamycin and verify by enzyme digestion, and obtain the polyethylene terephthalate depolymerase (DuraPETase) expression plasmid. The results are as follows Figure 2 As shown in A.

[0042] Wild-type M. xanthus DK1622 was prepared into competent cells and inoculated with wild type M. xanthus DK1622 was added to 30 mL CYE liquid medium and cultured overnight at 30°C and 200 rpm until the bacterial OD600 was about 0.6-1 (OD600 of 1 represents 5×10 8 cells / mL), centrifuged at 8000 rpm for 10 min at 4°C to collect the cells, washed three times with deionized water, and finally resuspended with an appropriate amount of water to make the cell density as high as possible.

[0043] The above plasmid p SWU19-PilA / SP / DuraPETase / MYXO-CTREM was transferred to a 0.1 cm electroporation cuvette in 50 µL competent cells under the following electroporation conditions: 0.65 kV, 400 Ω. Immediately add 2 mL CYE liquid medium to the electroporated cells, and resuscitate them at 30°C, 200 rpm for 8 h. Then mix them with semi-solid medium (5 g / L casein peptone, 10 mM Tris-HCl (7.6), 1mM KH2PO4, 8 mM MgSO4, 5 g / L agar powder) and spread them evenly on CYE liquid medium containing selection markers. Culture them at 30°C for 4-6 days until a single colony grows. Pick a single colony and transfer it repeatedly for verification. The recombinant myxobacterium is obtained. The results are shown in Figure 2. Figure 2 As shown in B.

[0044] Example 3 This example illustrates the recombinant myxobacterium M. xanthus DK1622 expresses polyethylene terephthalate depolymerase (DuraPETase) protein and is used in the degradation of polyester-type plastic model substrate Impranil DLN.

[0045] The recombinant myxobacterium M. xanthus DK1622 was inoculated into 5 mL of CYE liquid medium supplemented with kanamycin resistance and cultured at 30°C overnight; then, 1% of the inoculum was transferred into 100 mL of CYE liquid medium containing kanamycin (pH 7.2-7.6) and fermented at 30°C for 24-27 hours.

[0046] The bacteria were collected by centrifugation, washed three times with 0.05 M phosphate buffer (PBS), and ultrasonically disrupted to obtain a crude enzyme solution. Western Blot analysis revealed that the target protein DuraPETase was expressed in a soluble form.

[0047] Place an Oxford cup on a solid plate containing Impranil DLN (200uL DLN is evenly poured into 100mL MSM solid culture medium), drop 0.3mg of crude enzyme solution, and place it in a 37℃ incubator. After 24-48h of catalysis, an obvious transparent circle appears, indicating that the ester bond in the polyester model substrate Impranil DLN has been broken. The results are as follows: Figure 3 .

Claims

1. A recombinant myxobacterium displaying PET depolymerase on its surface, characterized in that: Myxococcus xanthus M. xanthus DK1622 is used as a chassis cell, and a recombinant expression plasmid of a gene sequence of a signal peptide, a PET depolymerase, and an anchor peptide is introduced into the chassis cell to obtain the result; The PET depolymerase is a codon-optimized PET depolymerase DuraPETase, whose nucleotide sequence is shown in SEQ ID NO: 1; the nucleotide sequence encoding the signal peptide SP is shown in SEQ ID NO: 2; the nucleotide sequence encoding the anchor peptide is shown in SEQ ID NO: 3; The promoter for expressing PET depolymerase in the recombinant expression plasmid is a constitutive promoter PilA, and its nucleotide sequence is shown in SEQ ID NO:

4.

2. The recombinant myxobacterium according to claim 1, characterized in that The recombinant expression plasmid is constructed by using the pSWU19 plasmid containing the PilA promoter as a basic expression plasmid.

3. The method for constructing the recombinant myxobacterium according to any one of claims 1 to 2, characterized in that: include: (1) Wild-type Myxococcus xanthus M. xanthus The whole genome of DK1622 was used as a template to obtain the outer membrane protein TraA fragment by PCR amplification; (2) Using the outer membrane protein TraA fragment obtained in step (1) as a template, PCR amplification was performed to obtain the signal peptide and anchor peptide fragments; (3) Using the gene sequence of PET depolymerase as a template, PCR amplification was performed to obtain the DuraPETase fragment; (4) Synthesizing homologous recombination fragments: Using the fragments obtained in steps (2) and (3) as templates, PCR amplification is performed to obtain signal peptide / PET depolymerase / anchor peptide fragments; (5) Enzymatically ligating the fragment obtained in step (4) with the linear basic expression plasmid, transforming the fragment into Escherichia coli, and screening transformants on a solid plate containing kanamycin to obtain a recombinant expression plasmid; (6) The recombinant expression plasmid obtained in step (5) is introduced into the wild-type Myxococcus aureus by electroporation. M. xanthus The recombinant myxobacterium was obtained by screening in DK1622 competent cells on a solid plate containing kanamycin.

4. Use of the recombinant myxobacterium according to any one of claims 1 to 2 in degrading polyester plastics; the polyester plastic is a polyurethane plastic Impranil DLN; the crude enzyme solution obtained by crushing the whole cells of the recombinant myxobacterium is used to degrade polyester plastics.

5. The use according to claim 4, characterized in that: After the recombinant myxobacterium is fermented and cultured, the bacterial cells are collected and ultrasonically disrupted to obtain a crude enzyme solution after whole-cell disruption.

6. The use according to claim 5, characterized in that: The culture medium for the fermentation culture is CYE liquid culture medium.

7. The use according to claim 5, characterized in that In the degradation system, the amount of crude enzyme solution added was 2-4 g / L substrate.

8. The use according to claim 5, characterized in that The substrate concentration in the degradation system was 0.1-0.3% v / v.

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