CN115725491A - Recombinant myxobacteria with PET (polyethylene terephthalate) depolymerase displayed on surface as well as construction method and application thereof - Google Patents
Recombinant myxobacteria with PET (polyethylene terephthalate) depolymerase displayed on surface as well as construction method and application thereof Download PDFInfo
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Images
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a recombinant myxobacteria with a surface displaying PET depolymerase and a construction method and application thereof, which are obtained by taking M.xanthorrhus DK1622 of myxococcus xanthus as a chassis cell and introducing recombinant expression plasmids of gene sequences of signal peptide, PET depolymerase and anchoring peptide into the chassis cell; the PET depolymerizing enzyme is codon-optimized PET depolymerizing enzyme DuraPETase, and the nucleotide sequence of the PET depolymerizing enzyme is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence for coding the signal peptide SP is shown as SEQID NO:2 is shown in the specification; the nucleotide sequence encoding the anchoring peptide is shown in SEQ ID NO:3, respectively. The invention provides a recombinant myxococcus xanthusM.xanthusDK1622 can realize the expression of polyethylene terephthalate hydrolase (DuraPETase), and the hydrolysis of polyester mode substrate Impranil DLN, and is expected to realize the in-situ remediation of plastic pollution.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant myxobacteria with a PET (polyethylene terephthalate) depolymerase displayed on the surface, and a construction method and application thereof.
Background
In the modern society, the wide application of plastics brings convenience to daily life, but causes a great amount of plastic waste to be discharged into the environment, so that the problem of serious environmental pollution is caused, and particularly the environmental problem of micro plastics becomes a global concern.
Compared with the physical and chemical recovery technology of waste plastic disposal, the biodegradation and conversion technology is gradually becoming an important way to solve the plastic pollution. The biodegradation and conversion technology is a biorefinery process for selectively degrading waste plastics into different polymer monomers by using microorganisms/enzymes under mild conditions and performing high-valued biorefinery on mixed plastic degradation products. With the breakthrough development in the field of plastic biodegradation, particularly the field of enzymatic degradation of PET plastics in recent years, people have brand new knowledge on the plastic biodegradation. Starting from the first PET plastic hydrolase TfH identified and defined by Muller et al in 2005, PET plastic high efficiency hydrolases such as cutinases HiC, tfcut2, LCC and PETase were identified, characterized and analyzed in succession. LCC is also obtained by enzyme molecule design and modification ICCG PET plastics such as DuraPETase, fastPETase and the like are efficient depolymerase or variant enzyme, and in the prior art, an engineering strain is rarely constructed and obtained by utilizing an environmental microorganism surface display technology, so that a foundation is provided for biological treatment of plastics in the environment.
Slime bacteria are a group of gram-negative bacteria that have a multicellular behaviour and a complex life cycle, are widely present in soil environments, are able to prey on other microorganisms, and have the following advantages: 1) The microbial inoculum can be bred by predating other microorganisms in the oligotrophic soil environment, and the abundance of the flora in the soil environment is stable; 2) Has good plant rhizosphere colonization ability and film forming ability. The plastic degradation strain taking slime bacteria (dominant bacteria in soil) as a chassis is constructed, and the in-situ remediation of plastic pollution is hopeful to be realized.
Disclosure of Invention
The first object of the present invention is to provide a recombinant myxobacterium having a PET depolymerase surface-displayed thereon.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a recombinant myxobacteria with surface display of PET depolymerase is prepared from Myxococcus xanthusM. xanthusDK1622 is used as a chassis cell, and is obtained by introducing recombinant expression plasmids of gene sequences of signal peptide, PET depolymerase and anchoring peptide into the chassis cell;
the PET depolymerase is codon-optimized PET depolymerase DuraPEtase, and the nucleotide sequence of the PET depolymerase is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence encoding the signal peptide SP is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the anchoring peptide is shown as SEQ ID NO:3, respectively.
As a preferred embodiment, the promoter for expressing PET depolymerase in the recombinant expression plasmid is a constitutive promoter PilA, and the nucleotide sequence thereof is as shown in SEQ ID NO:4, respectively.
In a preferred embodiment, the pSWU19 plasmid containing the PilA promoter is used as a basic expression plasmid to construct the recombinant expression plasmid.
Another object of the present invention is to provide a method for constructing the recombinant myxobacteria, comprising:
(1) Using wild type myxococcus xanthusM. xanthus Taking the whole genome of DK1622 as a template, and carrying out PCR amplification to obtain an outer membrane protein TraA fragment;
(2) And (2) performing PCR amplification by using the outer membrane protein TraA fragment obtained in the step (1) as a template to obtain a signal peptide and an anchoring peptide fragment.
(3) Using the gene sequence of the PET depolymerase as a template, and carrying out PCR amplification to obtain a DuraPETase fragment;
(4) Synthesizing homologous recombination fragments: taking the fragment obtained in the step (2) and (3) as a template, and carrying out PCR amplification to obtain a signal peptide/PET depolymerase/anchored peptide fragment;
(5) Enzymatically connecting the fragment obtained in the step (4) with a linear basic expression plasmid, converting the fragment into escherichia coli, and screening a transformant in a solid plate containing kanamycin to obtain a recombinant expression plasmid;
(6) The recombinant expression plasmid obtained in the step (5) is introduced into wild type myxococcus xanthus through electrotransformationM. xanthus DK1622 competent cells were screened on kanamycin-containing solid plates to obtain the recombinant myxobacteria.
The invention also aims to provide the application of the recombinant myxobacteria in degrading polyester plastics. The method comprises the following steps: and (3) using the crude enzyme liquid obtained after the recombinant myxobacteria whole cell disruption to degrade polyester plastics.
In a preferred embodiment, the recombinant myxobacteria are subjected to fermentation culture, and then the cells are collected and disrupted by sonication to obtain a crude enzyme solution after disruption of whole cells.
As a preferred embodiment, the fermentation culture medium is CYE liquid medium.
As a preferred embodiment, the amount of the crude enzyme solution added in the degradation system is 2 to 4 g/L of the substrate
As a preferred embodiment, the substrate concentration in the degradation system is from 0.1 to 0.3% v/v.
The invention has the following beneficial effects:
(1) The invention obtains the expression of the polyethylene glycol terephthalate depolymerizing enzyme (DuraPETase)M. xanthus DK1622 recombines myxobacteria, and the zymoprotein obtained after fermentation culture has ester bond hydrolase activity.
(2) The invention relates to a wild type strainM. xanthusDK1622 as a chassis cell for constructing degradable polyesterThe recombinant mucococcus xanthus of the model substrate Impranil DLN lays a foundation for further bioremediation of soil plastic pollution.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram of recombinant plasmid construction.
In FIG. 2, A is a gel electrophoresis diagram of recombinant single-fragment and double-restriction enzyme plasmid validation; b isM.xanthusThe gel electrophoresis pattern was verified by PCR amplification of DK1622 recombinant myxobacteria.
FIG. 3 is a drawing showingM.xanthusHydrolysis circles of crude enzyme solutions from DK1622 recombinant myxobacteria on Impranil DLN solid plates.
Detailed Description
Examples used Myxococcus xanthusM. xanthusDK1622 is a strain of Myxococcus xanthus, and is commercially available.
The formula of the CYE liquid culture medium is as follows: 1 g/L magnesium sulfate, 5 g/L yeast powder, 10 g/L casein peptone and water as solvent, and the pH value is 7.6.
The solid plate containing Impranil DLN is MSM solid culture medium, and the formula is as follows: 2g/L (NH) 4 ) 2 SO 4 ，0.2 g/L MgSO 4 ·7H 2 O，0.01 g/L CaCl 2 ·2H 2 O，0.001 g/L FeSO 4 ·7H 2 O，0.6g/L Na 2 HPO 4 ，1.5g/L KH 2 PO 4 2% v/v Impranil DLN,15 g/L agar powder, solvent water, pH neutral. The temperature of the degradation stage is 37 ℃, the catalysis time is 24-48h, and the pH is neutral.
EXAMPLE 1 this example illustrates the acquisition of SP/DuraPEtase/MYXO-CTREM fragments.
(1) Wild type model bacteriumM. xanthusThe whole genome of DK1622 is the template, SEQ ID NO:5 and SEQ ID NO:6 is used as a primer, and a TraA segment is obtained by PCR amplification;
(2) Taking the TraA fragment obtained in the step (1) as a template, and respectively taking the TraA fragment as a template and the fragment as shown in SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:6 is a primer, and a signal peptide SP and a MYXO-CTREM anchoring peptide fragment are obtained by PCR amplification.
(3) Using synthetic plasmid pET22b as a template, SEQ ID NO:9 and SEQ ID NO:10 as primer, and obtaining DuraPETase fragment after codon optimization by PCR amplification.
(4) Taking the single fragment obtained in the steps (2) and (3) as a template, and obtaining the fragment shown in SEQ ID NO:5 and SEQ ID NO:6, overlapping extension connection is carried out on the primer, and the SP/DuraPEtase/MYXO-CTREM fragment is obtained by PCR amplification, and the result is shown in figure 1.
The PCR amplification system is as follows: the obtained DNA template is 1 muL, the upstream and downstream of the primer are respectively 2 muL, 2 XPhanta Max Master Mix buffer solution is 25 muL, ddH 2 O20 muL. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min, and denaturation at 95 ℃ for 30 s; then annealing at 58 ℃ for 30 s, extending at 72 ℃ and circulating for 34 times; extension at 72 ℃ for 5 min. And cutting the amplified strips, and then recovering by using a column type rubber tapping recovery kit.
EXAMPLE 2 this example illustratesM.xanthusConstruction of DK1622 recombinant Myxobacteria. The plasmid construction is schematically shown in FIG. 1.
Plasmid containing PilA promoterpFor SWU19XbaⅠ、HinAnd d III double enzyme digestion. The enzyme cutting system is as follows: the plasmid was made to be 50 μ L,XbaⅠ5µL， Hind III 5 muL, 10 Xbuffer solution 10 muL, ddH 2 O30 muL and the total volume is 100 muL. And (4) cutting the gel of the enzyme digestion system, and then recovering the gel by using a column-type gel cutting recovery kit. The nucleotide sequence of the PilA promoter is shown as SEQ ID NO:4, respectively.
The SP/DuraPEtase/MYXO-CTREM fragment obtained in example 1 was recovered by gel and ligated to the double digested linearized plasmid. The connection reaction system is as follows: 2 muL of PCR product, 3 muL of enzyme digestion plasmid, 2 muL of one-step cloning ligase, 3 muL of 5 Xligase buffer solution, ddH 2 O5 muL and the total volume is 15 muL. Obtaining recombinant plasmid by connectionpSWU19-PilA/SP/DuraPETase/MYXO-CTREM, screening transformants in a solid plate containing kanamycin and carrying out enzyme digestion verification to obtain an expression plasmid of polyethylene glycol terephthalate depolymerizing enzyme (DuraPETase), wherein the result is shown as A in figure 2.
The wild type is addedM. xanthusDK1622 was prepared as competent cell, inoculated with wild typeM. xanthusDK1622 to 30 mL CYE liquid medium, cultured overnight at 30 ℃ and 200rpm until the OD600 of the cells is about 0.6-1 (OD 600 is 1 represents 5X 10) 8 cell/mL), centrifuging at 4 deg.C and 8000rpm for 10min to collect thallus, washing thallus with deionized water three times, and finally resuspending with appropriate amount of water to make thallus density as high as possible.
Mixing the above plasmidspTransferring the SWU19-PilA/SP/DuraPETAse/MYXO-CTREM in 50 mu L competent cells to a 0.1 cm electric shock cup, wherein the electric transfer conditions are as follows: 0.65 kV,400 omega. The electroporated cells were immediately added to 2 mL CYE broth, thawed at 200rpm at 30 ℃ for 8h, and then incubated with semi-solid medium (5 g/L casein peptone, 10 mM Tris-HCl (7.6), 1 mM KH 2 PO 4 ，8 mM MgSO 4 5 g/L agar powder) are uniformly mixed and spread on a CYE liquid culture medium containing a screening marker, the mixture is cultured at 30 ℃ for 4-6 days until a single colony grows out, and the single bacterium is selected and repeatedly transferred and then verified to obtain the recombinant sticky bacteria, wherein the result is shown as B in figure 2.
Example 3 this example illustrates recombinant myxobacteriaM. xanthusDK1622 expresses polyethylene terephthalate depolymerizing enzyme (DuraPETase) protein and its use for degradation in polyester-type plastic model substrate Impranil DLN.
Recombinant myxobacteria to be constructedM. xanthusDK1622 was inoculated into 5 mL of CYE liquid medium supplemented with kanamycin resistance and cultured overnight at 30 ℃; then, 100mL of CYE liquid medium (pH 7.2 to 7.6) containing kanamycin was inoculated at an inoculation amount of 1%, and fermentation culture was carried out at 30 ℃ for 24 to 27 hours.
And (3) centrifugally collecting thalli, washing the thalli for three times by using 0.05M Phosphate Buffer Solution (PBS), carrying out ultrasonic disruption to obtain a crude enzyme solution, and detecting by Western Blot to find that the target protein polyethylene glycol terephthalate depolymerizing enzyme (DuraPETase) is expressed and is soluble expression.
An oxford cup is placed on a solid plate containing Impranil DLN (200 uL DLN is uniformly poured into 100mL MSM solid culture medium), 0.3mg of crude enzyme solution is dropped into the oxford cup, the oxford cup is kept still in an incubator at 37 ℃, and an obvious transparent ring appears after catalysis is carried out for 24-48h, which indicates that the cleavage of ester bonds in the polyester mode substrate Impranil DLN is realized, and the result is shown in figure 3.
Claims (10)
1. A recombinant myxobacteria with PET depolymerase displayed on surface is characterized in that the recombinant myxobacteria is prepared from Myxococcus xanthusM. xanthusDK1622 is used as a chassis cell, and is obtained by introducing recombinant expression plasmids of gene sequences of signal peptide, PET depolymerase and anchoring peptide into the chassis cell;
the PET depolymerase is codon-optimized PET depolymerase DuraPEtase, and the nucleotide sequence of the PET depolymerase is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence for coding the signal peptide SP is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the anchoring peptide is shown as SEQ ID NO:3, respectively.
2. The recombinant myxobacterium according to claim 1, wherein the promoter for expressing the PET depolymerase in the recombinant expression plasmid is a constitutive promoter PilA, and the nucleotide sequence of the promoter is shown in SEQ ID NO:4, respectively.
3. The recombinant myxobacterium of claim 2, wherein the recombinant expression plasmid is constructed using a pSWU19 plasmid containing a PilA promoter as a base expression plasmid.
4. The method for constructing a recombinant myxobacterium according to any one of claims 1 to 3, comprising:
(1) Using wild type myxococcus xanthusM. xanthus Taking the whole genome of DK1622 as a template, and carrying out PCR amplification to obtain an outer membrane protein TraA fragment;
(2) And (2) performing PCR amplification by using the outer membrane protein TraA fragment obtained in the step (1) as a template to obtain a signal peptide and an anchoring peptide fragment.
(3) Using the gene sequence of the PET depolymerase as a template, and carrying out PCR amplification to obtain a DuraPETase fragment;
(4) Synthesizing a homologous recombination fragment: taking the fragment obtained in the step (2) and (3) as a template, and carrying out PCR amplification to obtain a signal peptide/PET depolymerase/anchor peptide fragment;
(5) Enzymatically connecting the fragment obtained in the step (4) with a linear basic expression plasmid, converting the fragment into escherichia coli, and screening a transformant in a solid plate containing kanamycin to obtain a recombinant expression plasmid;
(6) The recombinant expression plasmid obtained in the step (5) is introduced into wild type myxococcus xanthus through electrotransformationM. xanthus DK1622 competent cells were screened on kanamycin-containing solid plates to obtain the recombinant myxobacteria.
5. Use of the recombinant myxobacteria of any one of claims 1 to 3 for degrading polyester-based plastics.
6. The use according to claim 5, wherein the crude enzyme solution obtained by disrupting whole cells of the recombinant myxobacteria is used for degrading polyester-based plastics.
7. The use according to claim 6, wherein the recombinant myxobacteria are subjected to fermentation culture, and then the cells are harvested and disrupted by sonication to obtain a crude enzyme solution after disruption of whole cells.
8. Use according to claim 7, wherein the culture medium of the fermentation culture is CYE liquid medium.
9. The use of claim 7, wherein the crude enzyme solution is added in an amount of 2-4 g/L substrate in the degradation system.
10. Use according to claim 7, wherein the substrate concentration in the degradation system is 0.1-0.3% v/v.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116555091A (en) * | 2023-04-20 | 2023-08-08 | 南京农业大学 | Salt-tolerant myxobacteria and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116555091A (en) * | 2023-04-20 | 2023-08-08 | 南京农业大学 | Salt-tolerant myxobacteria and application thereof |
| CN116555091B (en) * | 2023-04-20 | 2024-01-26 | 南京农业大学 | Salt-tolerant myxobacteria and application thereof |
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