Engineering bacteria for efficient NP degradation and their construction method and application

Abstract

This application discloses an engineered bacterium capable of efficiently degrading NP, its construction method and application, and belongs to the field of wastewater treatment technology. kill The construction method is as follows: primers are designed based on the genomic DNA of Pseudomonas montereyii EBTHRC-PM, and PCR is performed to obtain kill Gene fragments, kill The gene fragment was homologously linked to the plasmid, and the T7 promoter of the reconstructed plasmid was replaced with the strong promoter P13. Finally, it was transferred into Pseudomonas montersii EBTHRC-PM, resulting in an engineered bacterium that can effectively inhibit the toxicity in the process of degrading NPEO. The ability to degrade NP is greatly improved, and it has good application prospects in the field of wastewater treatment.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering and wastewater treatment, and in particular relates to an engineering bacterium capable of efficiently degrading NP, a construction method thereof and an application thereof. Background Art

[0002] Nonylphenol polyoxyethylene ether (NPEO) is an important nonionic surfactant with stable properties. It is commonly used in industrial, agricultural, and commercial fields, and is therefore present in large quantities in wastewater. Biological treatment is widely used in wastewater treatment due to its low cost and wide applicability. However, during the biochemical treatment of wastewater, NPEO deethoxylates to form nonylphenol (NP), a persistent environmental pollutant with extremely strong endocrine-disrupting properties. This pollutant is extremely harmful to the normal development and reproductive behavior of humans and animals, and cannot be effectively removed within the limited hydraulic retention time (HRT). This increases the endocrine-disrupting toxicity of biochemically treated wastewater, posing an ecological risk when discharged into the environment.

[0003] In order to solve the above technical problems, in the prior art, for example, the applicant's prior application for a Chinese invention patent with publication number CN116574626A, entitled "A composite microbial agent and its preparation method and application", records the use of Priesteria gigantea ( Priestia megaterium )EBTHRC-BP and Pseudomonas moniliformis ( Pseudomonas monteilii ) EBTHRC-PM constitutes a composite bacterial agent, among which: Pseudomonas moniliformis EBTHRC-PM shows the ability to degrade both NPEO and NP, but the degradation rate of NP is low, and it cannot effectively inhibit the toxicity in the process of degrading wastewater containing NPEO; Priesteria gigantea EBTHRC-BP has a strong ability to degrade the downstream products of nonylphenol polyoxyethylene ether; the combination of the two forms a metabolic division of labor and a positive degradation feedback mechanism, achieving a synergistic effect, with a degradation effect higher than that of a single bacterium and a reduction in the endocrine disrupting toxicity of wastewater.

[0004] In this application, Priesteria gigantea EBTHRC-BP and Pseudomonas moniliformis EBTHRC-PM need to be cultured separately and mixed with equal biomass to create a composite microbial inoculum. If a microorganism with high toxicity can be obtained for the wastewater degradation of NPEO, the microbial culture process would be reduced, saving costs.

[0005] kill The gene mainly exists in bacteria and is a key gene involved in the biosynthesis of ubiquinone (also known as coenzyme Q). The current NPEO degradation process mainly focuses on screening strains with high degradation efficiency, and has not yet been tested on killResearch on gene modification to improve bacterial degradation efficiency. Summary of the Invention

[0006] 1. Problem to be solved

[0007] In order to solve the problem of increased endocrine toxicity of the existing NPEO-degrading bacteria during the degradation of NPEO, the present invention provides an engineered bacterium for efficient NP degradation and a construction method and application thereof. The engineered bacterium is an overexpression kill The construction method is as follows: primers are designed based on the genomic DNA of Pseudomonas montereyii EBTHRC-PM, and PCR is performed to obtain kill Gene fragments, kill The gene fragment was homologously connected to the plasmid, and the T7 promoter of the reconstructed plasmid was replaced with the strong promoter P13. Finally, it was transferred into Pseudomonas montersii EBTHRC-PM to obtain an engineered bacterium that can effectively inhibit the toxicity in the process of degrading NPEO, and was named engineered bacterium GC01.

[0008] 2. Technical Solution

[0009] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0010] The present application provides an engineering bacterium that efficiently degrades NP. The engineering bacterium is an overexpression kill Pseudomonas monterinautica EBTHRC-PM gene, described kill The nucleotide sequence of the gene is shown in SEQ ID NO.1; the deposit number of the Pseudomonas montenosii EBTHRC-PM is CCTCC NO: M20211525, and it was deposited in the China Center for Type Culture Collection on December 3, 2021, and the deposit address is Wuhan University, Wuhan, China; Pseudomonas montenosii EBTHRC-PM is recorded in the Chinese invention patent with publication number CN116574626A, and the invention name is a composite microbial agent, its preparation method and application.

[0011] Furthermore, the above-mentioned engineered bacteria contains a recombinant plasmid, and the recombinant plasmid contains kill Gene.

[0012] Furthermore, the above recombinant plasmid is a recombinant plasmid PBBR1MCS-2 (△T7)-P13-ubiH, which contains kill The T7 promoter of the PBBR1MCS-2 plasmid was replaced by the P13 promoter.

[0013] The present application also provides a method for constructing an engineered bacterium that efficiently degrades NP, comprising the following steps:

[0014] Will kill Gene fragments were introduced into expression vectors to obtain expression kill The recombinant expression vector of the gene kill The nucleotide sequence of the gene is shown in SEQ ID NO. 1;

[0015] will express kill The recombinant expression vector of the gene was introduced into Pseudomonas montereyii EBTHRC-PM to obtain an engineered bacterium that can efficiently degrade NP. The preservation number of the Pseudomonas montereyii EBTHRC-PM is CCTCC NO: M20211525, and it was deposited in the China Center for Type Culture Collection on December 3, 2021, and the preservation address is Wuhan University, Wuhan, China.

[0016] Furthermore, the above expression vector is plasmid PBBR1MCS-2, and the above recombinant expression vector is recombinant plasmid PBBR1MCS-2-ubiH; kill The introduction of gene fragments into expression vectors includes: kill The gene fragment was introduced into plasmid PBBR1MCS-2 to obtain the recombinant plasmid PBBR1MCS-2-ubiH.

[0017] Furthermore, the above-mentioned recombinant expression vector is a recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH; the construction of the recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH includes: removing the promoter T7 in the recombinant plasmid PBBR1MCS-2-ubiH and connecting the P13 promoter.

[0018] Furthermore, the above-mentioned removal of promoter T7 in the recombinant plasmid PBBR1MCS-2-ubiH includes:

[0019] Using the recombinant plasmid PBBR1MCS-2-ubiH as a template, PCR amplification was performed with upstream and downstream primers PBBR-F2 and PBBR-R2 to obtain a linearized fragment of PBBR1MCS-2-ubiH without promoter T7. The nucleotide sequences of the upstream and downstream primers PBBR-F2 and PBBR-R2 are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0020] Furthermore, the above kill The gene fragment was amplified by PCR using the genomic DNA of Pseudomonas monteridium EBTHRC-PM as a template and the upstream and downstream primers ubiH-F and ubiH-R to obtain the fragment containing killgene fragment; the sequences of the ubiH-F and ubiH-R primers are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0021] Furthermore, the P13 promoter was amplified by PCR using the upstream and downstream primers P13-F and P13-R using the genomic DNA of Pseudomonas putida as a template to obtain the gene fragment of the P13 promoter; the sequences of the P13-F and P13-R primers are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0022] The present application also provides the use of the above-mentioned engineered bacteria that efficiently degrade NP in degrading NP and / or NPEO.

[0023] Furthermore, the above application includes adding engineered bacteria that can efficiently degrade NP into phenol-containing wastewater.

[0024] Furthermore, the above-mentioned phenol-containing wastewater is wastewater containing NP and / or NPEO.

[0025] 3. Beneficial effects

[0026] Compared with the prior art, the present application has the following advantages:

[0027] (1) The wild-type Pseudomonas monteri was modified to construct an engineered bacterium GC01 that can efficiently degrade NP. It has a high efficiency in degrading both NPEO and NP, thereby effectively inhibiting the increase of endocrine toxicity during the degradation of NPEO.

[0028] (2) This application provides an engineered bacterium that can efficiently degrade NPs and effectively treat wastewater containing NPs.

[0029] (3) In the biochemical treatment process of wastewater, the existing technology focuses on screening out strains with high degradation efficiency. kill Gene modification can improve the degradation efficiency of bacteria, providing a new method and example for the transformation of biodegradable and detoxifying bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Electrophoresis diagram verifying that the engineered bacteria GC01 obtained the target gene (band 2 represents the positive recombinant bacteria that were successfully constructed).

[0031] Figure 2 The difference between engineered bacteria GC01 and wild bacteria kill Transcription levels of genes when degrading 10 mg / L NPEO or 10 mg / L NP.

[0032] Figure 3Strain degradation and degradation kinetics simulation results of engineered bacteria GC01 and wild bacteria under 10 mg / L NPEO or 10 mg / L NP conditions.

[0033] Figure 4 Changes in endocrine disrupting toxicity of engineered bacteria GC01 and wild bacteria during the degradation of NPEO in a simulated wastewater system. DETAILED DESCRIPTION

[0034] The present invention relates to an engineering bacterium capable of efficiently degrading NP, a construction method thereof and an application thereof, by overexpressing kill By genetically modifying wild-type Pseudomonas monteri, an engineered bacterium, named GC01, was obtained that is highly efficient in degrading NP. Compared to wild-type Pseudomonas monteri, the engineered bacterium exhibits significantly improved degradation of NP, a toxic downstream product of NPEO. GC01 can completely degrade 10 mg / L of NP, with a degradation kinetic coefficient approximately sevenfold higher. The resulting engineered bacterium can be used to treat phenolic wastewater and efficiently degrade toxic pollutants in detoxified wastewater, possessing significant application value for environmental pollution control.

[0035] The present invention is described below with reference to the accompanying drawings. Experimental methods without specific operating steps are carried out in accordance with the corresponding product specifications. Unless otherwise specified, the instruments, reagents, and consumables used in the examples can be purchased from commercial companies.

[0036] Example 1: Preparation of highly efficient NP-degrading engineered bacteria GC01

[0037] Overexpression kill Gene construction of the engineered bacteria GC01 that efficiently degrades NPs is as follows:

[0038] 1.1 kill Amplification of gene fragments

[0039] Extract Pseudomonas monteri Pseudomonas monteilli ) EBTHRC-PM genomic DNA, which was screened in the laboratory earlier, is currently deposited at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M20211525. Using this DNA as a template, upstream and downstream primers ubiH-F and ubiH-R were designed for PCR amplification to obtain a gene containing kill gene fragments, kill The nucleotide sequence of the gene fragment is shown in SEQ ID NO. 1, and the sequences of the ubiH-F and ubiH-R primers are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0040] SEQ ID NO.1:

[0041]

[0042] SEQ ID NO.2:

[0043] ubiH-F: 5'-aattctaaggaggttttctaATGAACCGGGTGAACCTGG-3';

[0044] SEQ ID NO.3:

[0045] ubiH-R: 5'-tcgacggtatcgataagcttTCATGCCTGGCCCCGCGG-3';

[0046] The PCR amplification system was as follows: 1 μL of EBTHRC-PM genomic DNA template, 0.5 μL of upstream primer ubiH-F, 0.5 μL of downstream primer ubiH-R, 10 μL of 2× Phanta Max Master Mix, 8 μL of ddH O, and a total volume of 20 μL;

[0047] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 66°C for 30 s, extension at 72°C for 40 s, 35 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0048] 1.2 Amplification of the P13 promoter fragment

[0049] Extraction of Pseudomonas putida ( Pseudomonas putida KT2440 genomic DNA was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Cat. No. B84217). Using this DNA as a template, upstream and downstream primers P13-F and P13-R were designed for PCR amplification, yielding a gene fragment containing the P13 promoter. The sequences of primers P13-F and P13-R are shown in SEQ ID NOs. 4 and 5, respectively.

[0050] SEQ ID NO.4:

[0051] P13-F: 5'-cgacggccagtgagcgcgcgTCCGGATGTGGTACGGAACG-3';

[0052] SEQ ID NO.5:

[0053] P13-R: 5'-cgcggtggagctccaattcgATGCTTCATCCTTCCCGCTTATG-3';

[0054] The PCR amplification system was as follows: KT2440 genomic DNA template 1 μL, upstream primer P13-F 0.5 μL, downstream primer P13-R 0.5 μL, 2× Phanta Max Master Mix 10 μL, ddH O 8 μL, total volume 20 μL;

[0055] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 66°C for 13 s, extension at 72°C for 40 s, 35 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0056] 1.3 Preparation of linearized PBBR1MCS-2

[0057] The PBBR1MCS-2 plasmid was introduced into the Escherichia coli DH5α strain via a heat shock method. Heat shock experiments were performed in a 42°C water bath for 90 seconds, followed by a 2-minute ice bath. DH5α strains containing the PBBR1MCS-2 plasmid were cultured and amplified at 37°C and 150 rpm. The plasmids were extracted. PBBRMCS-2 was obtained from Core Biotechnology Co., Ltd. (Cat. No. VT8173), and competent DH5α strains were obtained from Sangon Biotech (Shanghai) Co., Ltd. (Cat. No. B528413). Using plasmid DNA as a template, upstream and downstream primers PBBR-F1 and PBBR-R1 were designed for inverse PCR amplification, resulting in a linearized fragment containing PBBR1MCS-2. The sequences of the PBBR-F1 and PBBR-R1 primers are shown in SEQ ID NOs. 6 and 7, respectively.

[0058] SEQ ID NO.6:

[0059] PBBR-F1: 5'-AAGCTTATCGATACCGTCGACC-3';

[0060] SEQ ID NO.7:

[0061] PBBR-R1: 5'-GAATTCCTGCAGCCCGGG-3';

[0062] The PCR amplification system was as follows: PBBR1MCS-2 plasmid DNA template 1 μL, upstream primer PBBR-F1 0.5 μL, downstream primer PBBR-R1 0.5 μL, 2× Phanta Max Master Mix 10 μL, ddH O 8 μL, total volume 20 μL;

[0063] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 56°C for 2 min 30 s, extension at 72°C for 40 s, 35 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0064] 1.4 Preparation of PBBR1MCS-2-ubiH-containing plasmid

[0065] In 1.1 kill The gene fragment and the linearized plasmid fragment in 1.3 were homologously ligated according to the instructions of the homologous ligation kit (Nanjing Novozyme Biotechnology Co., Ltd., Cat. No. C116-01). The homologous ligation product was introduced into DH5α competent cells by heat shock method. The heat shock experiment was carried out in a 42℃ water bath, and then ice-bathed for 2 minutes. After recovery, the product was spread on 50μg / mL kanamycin solid medium, and positive recombinant bacteria with kanamycin resistance were selected, cultured and amplified, and the product containing kill The PBBR1MCS-2-ubiH plasmid was used to express the gene.

[0066] 1.5 Linearize the recombinant plasmid to remove the T7 promoter

[0067] Using the recombinant plasmid PBBR1MCS-2-ubiH obtained in 1.4 as a template, upstream and downstream primers PBBR-F2 and PBBR-R2 were designed for PCR amplification to obtain a linearized fragment of PBBR1MCS-2-ubiH without promoter T7. The sequences of the primers PBBR-F2 and PBBR-R2 are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0068] SEQ ID NO.8:

[0069] PBBR-F2: 5'-CGAATTGGAGCTCCACCGC-3';

[0070] SEQ ID NO.9:

[0071] PBBR-R2: 5'-CGCGCGCTCACTGGCCGT-3';

[0072] The PCR amplification system was as follows: 1 μL of PBBR1MCS-2-ubiH plasmid DNA template, 0.5 μL of upstream primer PBBR-F2, 0.5 μL of downstream primer PBBR-R2, 10 μL of 2× Phanta Max Master Mix, 8 μL of ddH O, and a total volume of 20 μL;

[0073] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 56°C for 2 min 30 s, extension at 72°C for 40 s, 35 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0074] 1.6 Preparation of plasmid PBBR1MCS-2(△T7)-P13-ubiH

[0075] Homologously ligate the P13 fragment from 1.2 with the linearized fragment from 1.5, following the instructions for the homologous ligation kit (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. C116-01). Heat-shock the ligated product into a DH5α competent culture. After recovery, plate the product onto solid medium supplemented with 50 μg / mL kanamycin. Select for kanamycin-resistant recombinant strains, expand the culture, and extract the plasmid to obtain the plasmid PBBR1MCS-2(△T7)-P13-ubiH.

[0076] 1.7 Construction of highly efficient NPEO degradation and detoxification engineered bacteria GC01

[0077] The recombinant plasmid PBBR1MCS-2(ΔT7)-P13-ubiH described in 1.6 was electroporated into the wild-type Pseudomonas monteroides strain EBTHRC-PM at 600V for 12ms. After recovery, the cells were plated onto solid medium supplemented with 30μg / mL kanamycin and screened for kanamycin-resistant recombinant strains. The resulting strain, which efficiently detoxified NPEO, was named GC01.

[0078] The target gene (P13-ubiH) obtained by engineering bacteria GC01 was verified by PCR using primers M13F and M13R. The electrophoresis pattern is shown in the figure. Figure 1 As shown, band 1 is wild Pseudomonas monteri, and band 2 is the successfully constructed positive recombinant bacteria. The sequences of primers M13F and M13R are shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

[0079] SEQ ID NO.10:

[0080] M13F: 5'-GTTGTAAAACGACGGCCAG-3';

[0081] SEQ ID NO.11:

[0082] M13R: 5'-CAGGAAACAGCTATGAC-3'.

[0083] Example 2: Detection of engineered bacteria GC01 kill Gene expression

[0084] The wild-type strain EBTHRC-PM and the engineered strain GC01 were inoculated into LB medium overnight and cultured to an OD600 of 1.0. The cells were centrifuged at 7000 rpm for 3 minutes, the supernatant discarded, and the pellet collected. Washing with MSM was repeated three times, and the pellets were resuspended in an equal volume of inorganic salt medium. The pellets were then inoculated into inorganic salt medium containing 10 mg / L NPEO and NP, respectively, at a 5% volume ratio. Inoculation with inorganic salt medium alone served as a blank control. The cells were harvested at 0, 12, 24, and 48 hours, and then centrifuged at 8000 rpm for 5 minutes. Total RNA was extracted using an RNA extraction kit (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. RC113) and immediately reverse-transcribed into cDNA using a reverse transcription kit (Nanjing Novozymes Biotechnology Co., Ltd., Cat. No. R223) for real-time fluorescence quantitative polymerase chain reaction. Mineral salts medium (MSM) contained the following components: 3000 mg / L Na2HPO4, 200 mg / L MgSO4·7H2O, 10 mg / L CaCl2, 2650 mg / L KH2PO4and 1500 mg / L (NH4)2SO4.

[0085] The fluorescent quantitative kit (Nanjing Novozyme Biotechnology Co., Ltd., product number: Q331-02) was used to detect the expression of the engineered bacteria GC01. kill The expression of genes at the transcriptional level, detailed operation steps are shown in the kit instructions. Figure 2 As shown in the figure, during the degradation of NPEO, the engineered bacteria GC01 kill The gene was upregulated 2-3 times compared with the wild strain EBTHRC-PM. During the degradation of NP, the engineered strain GC01 kill The gene was upregulated 3-5 times compared with the wild-type EBTHRC-PM.

[0086] Example 3: Detection of the degradation of engineered bacteria GC01 in NPEO or NP environment

[0087] The engineered bacteria GC01 prepared in Example 1 was inoculated into inorganic salt culture media containing 10 mg / L NPEO and 10 mg / L NP, respectively, to detect the degradation of NPEO and NP by the strain.

[0088] 200 mL of inorganic salt culture medium was added to a 250 mL conical flask, and NPEO was added at a final concentration of 10 mg / L. The engineered bacteria G01 and wild bacteria EBTHRC-PM were inoculated into the inorganic salt culture medium, respectively, with an initial inoculation size of 5%. The degradation experimental conditions were 28°C and 150 rpm. Samples were taken at different time points, and the residual amount of NPEO was detected by high-performance liquid chromatography.

[0089] 50 mL of inorganic salt medium was added to 100 mL of the medium, followed by NP at a final concentration of 10 mg / L. The engineered strain GC01 and the wild strain EBTHRC-PM were inoculated into the inorganic salt medium, cultured at 28°C for 14 hours, and then harvested by centrifugation. The initial inoculum size was 5%. Degradation experiments were performed at 28°C and 150 rpm. Samples were taken at different time points, and the residual NP content was determined by high-performance liquid chromatography.

[0090] Figure 3 The results show the degradation performance and degradation kinetics of the two strains inoculated into MSM containing 10 mg / L NPEO or NP, respectively. There was no significant difference in the degradation ability of the two strains for NPEO, and the degradation kinetic constants were similar. The engineered strain GC01 exhibited significantly higher NP degradation than the wild strain. While the wild strain only achieved a 40% degradation rate of NP in 72 hours, the engineered strain GC01 achieved 100% complete degradation, with a degradation kinetic constant seven times that of the wild strain.

[0091] Example 4: Detoxification of NPEO by Engineered Bacteria in a Simulated Wastewater Environment

[0092] The engineered bacteria GC01 prepared in Example 1 was inoculated into 10 mg / L NPEO simulated wastewater. 100 mL of samples were taken at different time points, filtered, and subjected to solid-phase extraction with a 3cc HLB column. The concentration was enriched 100-fold to 1 mL, and changes in endocrine disrupting toxicity during the entire process were detected.

[0093] Endocrine-disrupting toxicity was determined using luminescent yeast bioassays, according to the method described in the article "Xu T, Young A, Narula J, Sayler G, Ripp S. High-Throughput Analysis of Endocrine-Disrupting Compounds Using BLYES and BLYAS Bioluminescent Yeast Bioassays. Methods Mol Biol. 2020;2081:29-41. doi: 10.1007 / 978-1-4939-9940-8_3. PMID: 31721116." Estrogen E2 was used as a positive control, and methanol was used as a negative control. The assay was performed in a 96-well white plate. 20 μL of the positive control was added to each well, and the actual sample concentration gradient was 20 μL, 10 μL, 5 μL, 2 μL, and 1 μL, for a total of 5 concentrations. After evaporation of the methanol, 200 μL of the yeast suspension was added, and the cells were incubated at 28°C for 8-12 hours. Subsequently, the luminescence intensity of each well was measured using a multifunctional plate reader (Tecan, Austria). The EC50 value was used to characterize the estrogenic toxicity equivalence. Each sample was tested in triplicate to ensure reliability. The effect value of each sample was normalized to the percentage of the maximum effect value of the positive control E2. The dose-effect curve of the sample was fitted using logistic regression analysis. The EC50 of the sample was calculated from the dose-effect curve, and the estradiol equivalent quality (EEQ) of the sample was calculated using the following formula:

[0094]

[0095] EEQ is the estradiol equivalent of the sample (ng / L), EC50(E2) is the EC50 of the positive control estradiol (E2) (ng / L), EC50(sample) is the EC50 of the sample, and sampleEF is the enrichment factor of the sample.

[0096] Figure 4 The results showed that the engineered bacteria GC01 significantly weakened the toxicity during the degradation of NPEO, which was mainly reflected in the reduction of the peak value and the shortening of the detoxification time.

[0097] It is proved that the engineered bacteria prepared for efficient NP degradation can achieve efficient toxicity inhibition in the process of NPEO degradation. Under the condition of an initial biological inoculation rate of 5%, the toxicity peak of the engineered bacteria GC01 in the detoxification process of 10 mg / L NPEO in a simulated wastewater environment decreased by about 80% compared with wild bacteria.

[0098] The embodiments of the present invention are described in detail above. However, the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. An engineered bacterium capable of efficiently degrading NP, characterized in that: The engineered bacteria is overexpressed in Pseudomonas monteri EBTHRC-PM ubiH Genetically engineered bacteria, ubiH The nucleotide sequence of the gene is shown in SEQ ID NO.1; the preservation number of the Pseudomonas monterosoni EBTHRC-PM is CCTCC NO:M20211525, and it was deposited in the China Center for Type Culture Collection on December 3, 2021, and the preservation address is Wuhan University, Wuhan, China.

2. The engineered bacteria for efficiently degrading NP according to claim 1, characterized in that: The engineered bacteria comprises a recombinant plasmid, which comprises ubiH Gene.

3. The engineered bacteria for efficiently degrading NP according to claim 2, characterized in that: The recombinant plasmid is the recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH, which contains ubiH The T7 promoter of the PBBR1MCS-2 plasmid was replaced by the P13 promoter.

4. A method for constructing an engineered bacterium capable of efficiently degrading NP, characterized in that: The method comprises the following steps: Will ubiH Gene fragments were introduced into expression vectors to obtain expression ubiH The recombinant expression vector of the gene ubiH The nucleotide sequence of the gene is shown in SEQ ID NO.1; will express ubiH The recombinant expression vector of the gene was introduced into Pseudomonas montereyii EBTHRC-PM to obtain an engineered bacterium that can efficiently degrade NP. The preservation number of the Pseudomonas montereyii EBTHRC-PM is CCTCC NO: M20211525, and it was deposited in the China Center for Type Culture Collection on December 3, 2021, and the preservation address is Wuhan University, Wuhan, China.

5. The method for constructing an engineered bacterium capable of efficiently degrading NP according to claim 4, characterized in that: The expression vector is plasmid PBBR1MCS-2, and the recombinant expression vector is recombinant plasmid PBBR1MCS-2-ubiH; ubiH The introduction of gene fragments into expression vectors includes: ubiH The gene fragment was introduced into plasmid PBBR1MCS-2 to obtain the recombinant plasmid PBBR1MCS-2-ubiH.

6. The method for constructing an engineered bacterium capable of efficiently degrading NP according to claim 5, characterized in that: The recombinant expression vector is a recombinant plasmid PBBR1MCS-2 (△T7)-P13-ubiH; the construction of the recombinant plasmid PBBR1MCS-2 (△T7)-P13-ubiH includes: removing the promoter T7 in the recombinant plasmid PBBR1MCS-2-ubiH and connecting the P13 promoter.

7. The method for constructing an engineered bacterium capable of efficiently degrading NP according to claim 6, wherein: The removal of promoter T7 in the recombinant plasmid PBBR1MCS-2-ubiH comprises: Using the recombinant plasmid PBBR1MCS-2-ubiH as a template, PCR amplification was performed with upstream and downstream primers PBBR-F2 and PBBR-R2 to obtain a linearized fragment of PBBR1MCS-2-ubiH without promoter T7. The nucleotide sequences of the upstream and downstream primers PBBR-F2 and PBBR-R2 are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

8. The method for constructing an engineered bacterium capable of efficiently degrading NP according to claim 7, characterized in that: described ubiH The gene fragment was amplified by PCR using the genomic DNA of Pseudomonas monteridium EBTHRC-PM as a template and the upstream and downstream primers ubiH-F and ubiH-R to obtain the fragment containing ubiH gene fragments; The sequences of the ubiH-F and ubiH-R primers are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively; The P13 promoter is amplified by PCR using the genomic DNA of Pseudomonas putida as a template and the upstream and downstream primers P13-F and P13-R to obtain the gene fragment of the P13 promoter; the sequences of the primers P13-F and P13-R are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

9. Use of the engineered bacteria capable of efficiently degrading NP according to any one of claims 1 to 3 in degrading NP and / or NPEO.

10. The use according to claim 9, characterized in that The application comprises adding an engineered bacterium capable of efficiently degrading NP into phenol-containing wastewater.

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