A strain of Comamonas capable of degrading microplastics and its construction method and application

Abstract

The present invention relates to a microplastic-degradable Comamonas strain and a construction method and application thereof. Using DuraPETase as an element and testosterone-producing Comamonas CNB-1 as a chassis cell, the expression and secretion of DuraPETase in CNB-1 are optimized to optimize its ability to degrade microplastics, and finally an artificial strain with microplastic degradation activity is obtained. The strain can degrade PET plastic film and PET microplastics in shake flask culture. A new solution is provided for the environmental pollution problem of PET microplastics.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and synthetic biology, and relates to a strain of Comamonas capable of degrading microplastics, and a construction method and application thereof. Background Art

[0002] According to statistics, nearly 80 million tons of PET plastics were produced worldwide in 2020. 91% of these plastic products were incinerated, landfilled or discarded after consumption, causing serious waste of resources and environmental pollution. Compared with these visible plastic pollution, the harm caused by the tiny microplastics (MPs) produced during the use and treatment of PET plastics is more serious. Microplastics are widely present in water bodies and sediments, acting as carriers of various pathogenic microorganisms, assisting the spread and diffusion of microbial resistance. High concentrations of microplastics can even inhibit the feeding activities of marine organisms, prolong the intestinal residence time, and affect their growth and reproduction. Nanoplastics (NPs) with smaller diameters can also enter the brain through food ingestion or maternal transfer, causing DNA oxidative damage. According to statistics, about 1,500 tons of microplastics from personal care products enter the sewage treatment system every year worldwide. In addition, the washing process of textiles and clothing in daily life will also release a large amount of fibrous microplastics that are collected in urban sewage treatment plants and eventually deposited in activated sludge. Using microorganisms to degrade microplastics in activated sludge in situ is the preferred means to solve the problem of microplastic pollution.

[0003] The latest research found that Is The mutant DuraPETase of PETase can effectively degrade PET plastics. The current research goal is to integrate the DuraPETase enzyme protein into microorganisms to achieve direct degradation in the environment. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Comamonas capable of degrading microplastics, and a construction method and application thereof.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A microplastic-degrading Comamonas strain is obtained by introducing a recombinant vector capable of secreting and expressing PET hydrolase into a host cell; wherein:

[0007] The host cell is Comamonas testosteroni CNB-1;

[0008] The recombinant vector capable of secreting and expressing PET hydrolase comprises a fusion protein expression frame, wherein the fusion protein expression frame is derived from 5 ， End to 3 ，The end is formed by the promoter, PET hydrolase and terminator in series;

[0009] The promoter is the strong endogenous promoter Pfer of Comamonas, whose nucleotide sequence is shown in SEQ ID NO: 1; the PET hydrolase is DuraPETase, whose nucleotide sequence is shown in SEQ ID NO: 3.

[0010] As a preferred embodiment, a signal peptide is further connected between the promoter and the PET hydrolase; the nucleotide sequence of the signal peptide is shown in SEQ ID NO:2.

[0011] As a preferred embodiment, the PET hydrolase 3 ， The end is also connected to green fluorescent protein GFP, whose nucleotide sequence is shown in SEQ ID NO:4.

[0012] Another object of the present invention is to provide a method for constructing the above-mentioned Comamonas, comprising: connecting the signal peptide, PET hydrolase and / or green fluorescent protein GFP and the terminator through overlapping extension PCR technology, and then connecting it with a linearized vector through recombinant cloning to construct a recombinant plasmid; introducing the recombinant plasmid into a host cell to obtain the Comamonas that can degrade microplastics.

[0013] As a preferred embodiment, the vector is pBBR1MCS-2 vector.

[0014] Another object of the present invention is to provide the use of the above-mentioned Comamonas in the degradation of PET plastics.

[0015] As a preferred embodiment, the microplastic-degradable Comamonas bacteria are inoculated into the culture medium, and then the PET plastic to be degraded is added.

[0016] As a preferred embodiment, the culture medium is MSM medium supplemented with 5% LB medium and 1% glucose.

[0017] As a preferred embodiment, the PET plastic is pretreated with alcohol and ultraviolet rays to remove surface bacteria before degradation, preferably soaked in 75% alcohol and then irradiated with ultraviolet rays.

[0018] As a preferred embodiment, the PET plastic is PET film plastic or PET microplastic.

[0019] Comamonas testosteroni CNB-1 is a core bacterial community in soil and activated sludge environments. This strain has the ability to degrade a variety of environmental pollutants and has a mature genetic operating system. In addition, the genome of CNB-1 contains a TPA degradation gene cluster tq I C I A2 I A3 I B I A1 I (GenBank accession, AB238678) and tq II C II A2 II A3 II B II A1 II (GenBank accession, AB238679), which can further metabolize and utilize TPA, the hydrolysis product of PET.

[0020] This application selects Comamonas testosteroni CNB-1 as the chassis cell, and uses DuraPETase with high PET hydrolysis activity as the element. By optimizing the expression and secretion of DuraPETase protein, an artificial strain with microplastic degradation activity is constructed, and GFP determination further verifies that DuraPETase can be successfully secreted outside the cell. The recombinant strain has strong activity and good extracellular secretion, and has a significant degradation effect on PET plastic film and PET microplastics. It provides a new solution to the environmental pollution problem of PET microplastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of DuraPETase and the detection of its aqueous polyurethane hydrolysis activity were performed.

[0022] Figure 2 The influence of different promoters on the expression and enzyme activity of DuraPETase.

[0023] Figure 3 It is the effect of signal peptide on the extracellular enzyme activity of DuraPETase.

[0024] Figure 4 GFP was used to measure the secretion of DuraPETase.

[0025] Figure 5 This is the case of artificial strains of bacteria degrading PET microplastics. DETAILED DESCRIPTION

[0026] Unless otherwise specified, the materials in the examples can be obtained from commercial channels.

[0027] In the embodiment, Comamonas testosteroni CNB-1 is already described in the literature The Complete Genome of Comamonas testosterone Reveals Its Genetic Adaptations to Changing Environments. Applied and Environmental Microbiology 2009, 75: 6812-6819, and the public can obtain it from Nanjing University of Technology. The applicant promises to release the biological material to the public within 20 years from the date of application.

[0028] The components of the MSM medium in the embodiment are: (NH4)2SO4, 0.2g / L MgSO4·7H2O, 0.01g / L CaCl2·2H2O, 0.001g / L FeSO4·7H2O, 0.6g / L Na2HPO4, 1.5g / L KH2PO4, the rest is deionized water, and agar is added to the solid.

[0029] Example 1: This example is used to illustrate the activity of gene-synthesized DuraPETase

[0030] The DuraPETase gene sequence (SEQ ID NO: 3) used in the present invention was synthesized by GenScript Biotech Co., Ltd., and the corresponding codon optimization was performed according to the different hosts during the synthesis of the gene sequence. In order to verify that the synthesized DuraPETase has PET plastic degrading esterase activity, the pET29a(+) vector was used to express and purify DuraPETase in the Escherichia coli host and verify its degradation activity. The seed liquid was inoculated into 100 ml LB liquid culture medium at 1%, and cultured at 37°C and 200rpm until the OD was about 0.8-1.0, and then IPTG was added to a final concentration of 100 μmol / L, and the culture was induced at 18°C ​​and 200 rpm for 24 hours. After the induction, the bacterial sludge was broken by centrifugation, and DuraPETase was purified using a Ni affinity chromatography column. After purification, SDS-PAGE verification was performed, and the results are as follows. Figure 1 As shown in Figure A, a single DuraPETase band of approximately 29 kDa can be obtained when the imidazole concentration is 200 mM. 10 μL of the purified DuraPETase pure enzyme solution and PBS buffer were added to the aqueous polyurethane plate, and the polyurethane degradation activity was verified using the transparent circle. The results are shown in Figure 1. Figure 1As shown in B, DuraPETase showed obvious hydrolysis circles on the aqueous polyurethane plate, indicating that the genetically synthesized DuraPETase has polyurethane hydrolysis activity.

[0031] Example 2: This example is used to illustrate the effect of different promoters on the expression of DuraPETase in CNB-1

[0032] In order to express the target gene DuraPETase more efficiently in strain CNB-1, the present invention uses different promoters to verify their effects on the expression of DuraPETase. Endogenous promoters P0838, P3064, P0955 and Pfer (SEQ ID NO: 1) with different expression strengths in the CNB-1 strain were selected to express DuraPETase.

[0033] The recombinant strain was constructed as follows: using the CNB-1 full genome sequence as a template, the promoter sequence was PCR amplified by designing homologous primers, and using the pET29a-DuraPETase plasmid as a template, homologous primers were designed to amplify the DuraPETase sequence, and then the PBBR1MCS-2 vector was linearized using XbaI and KpnI. After the PCR product was recovered, the promoter and DuraPETase sequences were connected to the vector using recombinant cloning technology. After the recombination reaction product was co-incubated with the competent Escherichia coli DH5α, a 42°C heat shock reaction was performed, and then positive clones were screened. The screened positive clones were extracted with plasmids and sequenced, and the correctly sequenced plasmids were transformed into CNB-1 competent cells again. After resistance screening, the recombinant strains were obtained. The construction of the recombinant strains in the subsequent examples (connection of signal peptides, GFP) was also obtained by the aforementioned recombinant cloning method. After the recombinant strains were cultured overnight in LB medium, 10 μL of each was added to the MSM inorganic salt solid plate containing aqueous polyurethane and cultured at 30°C for 2 days. The results are shown in Figure 2 As shown, only DuraPETase expressed by the Pfer promoter showed an obvious transparent circle. The Pfer promoter will be selected in the future to construct an artificial strain for microplastic degradation.

[0034] Example 3: This example is used to illustrate the effect of signal peptide on the extracellular enzyme activity of DuraPETase

[0035] Since microplastic particles are large and cannot enter the microbial cells, it is necessary to construct a DuraPETase secretion system to secrete the expressed DuraPETase outside the cells. Therefore, the present invention selects the conservative membrane pore protein signal peptide OrpC (the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2) as the secretion signal peptide of DuraPETase. In order to verify whether the signal peptide plays a secretion function, the strains CNB-1 / pBBR1MCS-2-Pfer, CNB-1 / OrpC-DuraPETase, and CNB-1 / Pfer-DuraPETase were cultured overnight in MSM medium, respectively, and the supernatant of the bacterial liquid was collected by centrifugation, and the esterase activity of DuraPETase in the supernatant was detected. pNPB was used as the substrate for the esterase activity detection, and the results are as follows. Figure 3 As shown in A, compared with the blank control CNB-1 / Pfer, the extracellular enzyme activity of strains CNB-1 / Pfer-DuraPETase and strain CNB-1 / OrpC-DuraPETase was higher, and the extracellular enzyme activity of strain CNB-1 / OrpC-DuraPETase was the highest.

[0036] In addition, 10 μL of each of the three bacterial strains was dropped onto a MSM inorganic salt solid plate containing aqueous polyurethane. After culturing at 30 °C for 2 days, the transparent zone diameter / colony diameter (D / d) was measured. The results are as follows: Figure 3 As shown in B, strain CNB-1 / Pfer has almost no transparent circle on the plate, while strain CNB-1 / OrpC-DuraPETase has the largest D / d value of 1.48±0.08. The results of enzyme activity detection and the results of aqueous polyurethane hydrolysis circle experiment both prove that the signal peptide OrpC used in the present invention has a secretion function and can secrete DuraPETase outside the cell.

[0037] Example 4: This example is used to illustrate the effect of signal peptide on DuraPETase secretion

[0038] In order to further explore the effect of the signal peptide on the secretion effect of DuraPETase, the present invention uses green fluorescent protein (GFP) to associate the expression of DuraPETase with the extracellular secretion and the distribution of GFP fluorescence signals, and judges the secretion of DuraPETase by detecting the strength of the GFP fluorescence signal in the intracellular and extracellular supernatants. The GFP protein (the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 4) is connected in series to the downstream of DuraPETase to construct the plasmid PBBR1MCS2-Pfer-OrpC-DuraPETase-GFP (the nucleotide sequence of the fusion protein expression frame is shown in SEQ ID NO: 5, wherein positions 1-251 are Pfer promoters; positions 252-280 are RBS sequences and surrounding base nucleotide sequences, i.e., ribosome binding sites; positions 281-350 are signal peptides OrpC; positions 351-1110 are DuraPETase; positions 1111-1825 are GFP; positions 1826-1828 are stop codons). The constructed plasmid was transferred into CNB-1 cells and cultured overnight in MSM medium. Cells with the same OD were selected to measure the GFP fluorescence signal of the total cells. Subsequently, the supernatant was centrifuged to measure the GFP level in the supernatant. The bacterial sludge was resuspended and crushed and centrifuged again to measure the intracellular GFP content. The results are shown in Figure 3 As shown, the secretion efficiency of the negative control without a signal peptide was about 19.9%, while the secretion efficiency of DuraPETase containing the OrpC signal peptide was about 35.5%.

[0039] Example 5: This example is used for the degradation of microplastics by artificial strains

[0040] In order to test the effect of artificial strains on the degradation of microplastics, the present invention first cuts the PET film into small pieces of 0.5×0.5 cm, then adds the small pieces to the pulverizer and pulverizes them for about 5 min, and passes the pulverized PET through a 40-mesh sieve to obtain PET microplastic particles less than 425 μm. Further, the engineered strain CNB-1 / Pfer-OrpC-DuraPETase is inoculated at a 5% rate in an MSM medium supplemented with 5% LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) and 1% glucose. Before the degradation experiment, the PET microplastics are soaked in 75% alcohol for 1 h, the supernatant is removed after centrifugation, and the microplastics are irradiated with ultraviolet light for 30 min. This step is to remove the bacteria on the surface of the microplastics. The microplastics are then placed in a 37 °C oven for drying for 24 h. About 0.1 g of PET microplastics are added to the degradation system, and the mass change of the microplastics is uniformly weighed using an electronic balance with an accuracy of one ten-thousandth, and 3 parallels are made for each of the experimental group, the control group, and the blank group. After 20 days of degradation, the weight loss of PET microplastics was measured. The results are as follows: Figure 5 As shown, the blank group was used to calculate the recovery rate of microplastics when collected, and the recovery rate of microplastics using the above method was 96.406%. There was almost no change in the control group before and after degradation, and the average weight loss of microplastics after degradation by the engineered strain constructed by the present invention was about 0.0112 g.

[0041] The present invention preliminarily characterized the plastic degradation ability of the artificial strain by measuring the extracellular esterase activity of DuraPETase and the size of the hydrolysis circle of the waterborne polyurethane plate, and measured the expression and secretion of DuraPETase in Comamonas testosteroni CNB-1 with GFP signal. The PET microplastic degradation ability of the artificial strain CNB-1 / OrpC-DuraPETase-GFP was further determined by the PET microplastic degradation experiment, providing a new idea for the biodegradation of PET microplastics.

Claims

1. A strain of Comamonas that can degrade microplastics, characterized in that: The method is obtained by introducing a recombinant vector capable of secreting and expressing PET hydrolase into a host cell; wherein: The host cell is Comamonas testosteroni CNB-1; The recombinant vector capable of secreting and expressing PET hydrolase comprises a fusion protein expression frame, wherein the fusion protein expression frame is derived from 5 ， End to 3 ， The end is formed by the promoter, PET hydrolase and terminator in series; The promoter is the strong endogenous promoter Pfer of Comamonas, and its nucleotide sequence is shown in SEQ ID NO: 1; the PET hydrolase is DuraPETase, and its nucleotide sequence is shown in SEQ ID NO: 3; a signal peptide is also connected between the promoter and the PET hydrolase; the nucleotide sequence of the signal peptide is shown in SEQ ID NO:

2.

2. The Comamonas according to claim 1, characterized in that The PET hydrolase 3 ， The end is also connected to green fluorescent protein GFP, whose nucleotide sequence is shown in SEQ ID NO:

4.

3. The method for constructing Comamonas according to any one of claims 1 to 2, characterized in that: include: The signal peptide, PET hydrolase and / or green fluorescent protein GFP and terminator are connected by overlapping extension PCR technology, and then connected to the linearized vector by recombinant cloning to construct a recombinant plasmid; the recombinant plasmid is introduced into the host cell to obtain the Comamonas bacteria that can degrade microplastics.

4. The construction method according to claim 3, characterized in that: The vector used is pBBR1MCS-2pfer vector.

5. Use of the Comamonas described in any one of claims 1 to 2 in the degradation of PET plastics.

6. The use according to claim 5, characterized in that: The microplastic-degradable Comamonas bacteria are inoculated into the culture medium, and then the PET plastic to be degraded is added.

7. The use according to claim 6, characterized in that The culture medium is MSM medium supplemented with 5% LB medium and 1% glucose.

8. The use according to claim 6, characterized in that: Before PET plastic is degraded, it is pre-treated with alcohol and ultraviolet light to remove surface bacteria.

9. The use according to claim 5, characterized in that: The PET plastic is PET film plastic or PET microplastic.

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