Engineering bacterium capable of efficiently degrading NP as well as construction method and application of engineering bacterium

Abstract

The invention discloses an engineering bacterium capable of efficiently degrading NP as well as a construction method and application of the engineering bacterium, and belongs to the technical field of wastewater treatment. The engineering bacterium is pseudomonas monteilii EBTRC-PM with an overexpressed ubiH gene, and the engineering bacterium is a pseudomonas monteilii EBTRC-PM The construction method comprises the following steps: designing a primer according to the genomic DNA of the pseudomonas monteilii EBTRC-PM, carrying out PCR to obtain a ubiH gene segment, carrying out homologous connection on the ubiH gene segment and a plasmid, replacing a T7 promoter of a reconstructed plasmid with a strong promoter P13, and finally transferring into the pseudomonas monteilii EBTRC-PM to obtain the engineering bacterium capable of efficiently inhibiting the toxicity in the NPEO degradation process, so that the NP degradation capability is greatly improved, and the engineering bacterium has the advantages that the construction is simple, and the construction cost is low. Good application prospects are realized in the field of wastewater treatment.

Description

Technical Field

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

[0002] Nonylphenol polyoxyethylene ether (NPEO) is an important non-ionic surfactant with stable properties and is commonly used in industries, agriculture, commerce and other fields. Therefore, it exists 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 will generate a persistent environmental pollutant with extremely strong endocrine disrupting properties - nonylphenol (NP) through de-ethoxylation. It has great harm to the normal development and reproductive behavior of humans and animals, and cannot be effectively removed within a limited hydraulic retention time (HRT), which increases the endocrine disrupting toxicity of the biochemical treated wastewater and poses an ecological risk when discharged into the environment.

[0003] To solve the above technical problems, in the prior art, for example, in a Chinese invention patent with the publication number of CN116574626A and the invention name of a composite microbial agent and its preparation method and application applied by the applicant, it is recorded that Priestia megaterium ( Priestia megaterium ), EBTHRC-BP and Pseudomonas monteilii ( Pseudomonas monteilii ), EBTHRC-PM form a composite microbial agent, wherein: Pseudomonas monteilii EBTHRC-PM shows the degradation ability to both NPEO and NP, but the degradation rate of NP is relatively low, and it cannot efficiently inhibit toxicity during the degradation of wastewater containing NPEO; Priestia megaterium EBTHRC-BP has a strong ability to degrade the downstream products of nonylphenol polyoxyethylene ether; combining the two forms a metabolic division of labor and a positive degradation feedback mechanism to achieve a synergistic effect, and has an effect of degrading and reducing the endocrine disrupting toxicity of wastewater higher than that of single bacteria.

[0004] In the above application, it is necessary to separately culture Priestia megaterium EBTHRC-BP and Pseudomonas monteilii EBTHRC-PM, and take the same biomass and mix them evenly to prepare a composite microbial agent. If microorganisms capable of efficiently inhibiting toxicity during the degradation of NPEO-containing wastewater can be obtained, the culture process of microorganisms will be reduced and the cost will be saved.

[0005] ubiH Genes mainly exist in bacteria and are a key gene involved in the biosynthesis pathway of ubiquinone (also known as coenzyme Q). The current NPEO degradation process mainly focuses on screening strains with high degradation efficiency, and has not passed ubiHResearch on modifying genes to improve the degradation efficiency of bacteria Summary of the Invention

[0006] 1. Problems to be Solved To solve the problem of increased endocrine toxicity during the degradation of NPEO by existing discovered NPEO-degrading bacteria, the present invention provides an engineered bacterium for highly efficient degradation of NP, its construction method and application. This engineered bacterium is Pseudomonas monteilii EBTHRC-PM overexpressing the ubiH gene; the construction method is as follows: primers are designed based on the genomic DNA of Pseudomonas monteilii EBTHRC-PM, and PCR is performed to obtain the ubiH gene fragment. The ubiH gene fragment is homologously ligated to the plasmid, and then the T7 promoter of the reconstructed plasmid is replaced with the strong promoter P13. Finally, it is transferred into Pseudomonas monteilii EBTHRC-PM to obtain an engineered bacterium that can highly efficiently inhibit toxicity during the degradation of NPEO, named engineered bacterium GC01.

[0007] 2. Technical Solutions To solve the above problems, the technical solutions adopted in this application are as follows: This application provides an engineered bacterium for highly efficient degradation of NP. This engineered bacterium is Pseudomonas monteilii EBTHRC-PM overexpressing the ubiH gene, and the nucleotide sequence of the ubiH gene is shown in SEQ ID NO.1; the preservation number of Pseudomonas monteilii EBTHRC-PM is CCTCC NO:M20211525, and it was preserved at the China Center for Type Culture Collection on December 3, 2021. The preservation address is Wuhan University, Wuhan, China; Pseudomonas monteilii EBTHRC-PM is described in the Chinese invention patent with the publication number CN116574626A and the invention name "A Composite Microbial Agent and Its Preparation Method and Application".

[0008] Furthermore, the above-mentioned engineered bacterium contains a recombinant plasmid, and the recombinant plasmid contains the ubiH gene.

[0009] Furthermore, the above-mentioned recombinant plasmid is recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH. The recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH is a PBBR1MCS-2 plasmid containing the ubiH gene, and the T7 promoter of the PBBR1MCS-2 plasmid is replaced with the P13 promoter.

[0010] This application also provides a construction method for an engineered bacterium for highly efficient degradation of NP, including the following steps: The ubiHThe gene fragment is introduced into an expression vector for expression ubiH a recombinant expression vector of the gene, wherein ubiH the nucleotide sequence of the gene is as shown in SEQ ID NO.1; The recombinant expression vector expressing the ubiH gene is introduced into Pseudomonas monteilii EBTHRC-PM to obtain an engineered bacterium with high efficiency in degrading NP. The preservation number of Pseudomonas monteilii EBTHRC-PM is CCTCC NO:M20211525, and it was preserved in the China Center for Type Culture Collection on December 3, 2021, with the preservation address being Wuhan University, Wuhan, China.

[0011] Furthermore, the above-mentioned expression vector is plasmid PBBR1MCS-2, and the above-mentioned recombinant expression vector is recombinant plasmid PBBR1MCS-2-ubiH; the introduction of the ubiH gene fragment into the expression vector includes: introducing the ubiH gene fragment into plasmid PBBR1MCS-2 to obtain recombinant plasmid PBBR1MCS-2-ubiH.

[0012] Furthermore, the above-mentioned recombinant expression vector is recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH; the construction of recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH includes: removing the promoter T7 in recombinant plasmid PBBR1MCS-2-ubiH and ligating the P13 promoter.

[0013] Furthermore, the removal of the promoter T7 in recombinant plasmid PBBR1MCS-2-ubiH includes: Using recombinant plasmid PBBR1MCS-2-ubiH as a template, PCR amplification is carried out with upstream and downstream primers PBBR-F2 and PBBR-R2 to obtain a linearized fragment of PBBR1MCS-2-ubiH without the promoter T7. The nucleotide sequences of the upstream and downstream primers PBBR-F2 and PBBR-R2 are as shown in SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0014] Furthermore, the above-mentioned ubiH gene fragment is amplified by PCR using the genomic DNA of Pseudomonas monteilii EBTHRC-PM as a template with upstream and downstream primers ubiH-F and ubiH-R to obtain a gene fragment containing ubiH ; the primer sequences of ubiH-F and ubiH-R are as shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0015] Further, the above-mentioned P13 promoter uses the genomic DNA of Pseudomonas putida as a template, and PCR amplification is carried out with upstream and downstream primers P13-F and P13-R to obtain the gene fragment of the P13 promoter; the primer sequences of P13-F and P13-R are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0016] The present application also provides the application of the above-mentioned engineering bacteria capable of efficiently degrading NP in degrading NP and / or NPEO.

[0017] Further, the above-mentioned application includes adding the engineering bacteria capable of efficiently degrading NP to the phenol-containing wastewater.

[0018] Further, the above-mentioned phenol-containing wastewater is wastewater containing NP and / or NPEO.

[0019] 3. Beneficial effects Compared with the prior art, the beneficial effects of the present application are as follows: (1) A strain of engineering bacteria GC01 capable of efficiently degrading NP is constructed by transforming wild-type Pseudomonas monteilii, which has high-efficiency degradation ability for both NPEO and NP, so as to effectively inhibit the increase of endocrine toxicity during the degradation process of NPEO.

[0020] (2) The engineering bacteria capable of efficiently degrading NP provided by the present application can effectively treat the wastewater containing NP.

[0021] (3) In the biochemical treatment process of wastewater, the prior art focuses on screening strains with high degradation efficiency. The present application modifies genes to improve the bacterial degradation efficiency, providing a new method and example for the transformation of biodegradable detoxifying bacteria. ubiH For the transformation of biodegradable detoxifying bacteria. Description of the drawings

[0022] Figure 1 Electrophoresis diagram for verifying that the engineering bacteria GC01 obtain the target gene (band 2 is the successfully constructed positive recombinant bacteria).

[0023] Figure 2 The ubiH Transcription levels of genes of engineering bacteria GC01 and wild bacteria when degrading 10 mg / L NPEO or 10 mg / L NP.

[0024] Figure 3 Degradation and degradation kinetic simulation results of engineering bacteria GC01 and wild bacteria under the conditions of 10 mg / L NPEO or 10 mg / L NP.

[0025] Figure 4 Changes in endocrine disrupting toxicity during the degradation of NPEO by engineering bacteria GC01 and wild bacteria in a simulated wastewater system. Detailed implementation manners

[0026] The present invention relates to an engineering bacterium for efficiently degrading NP, a construction method and an application thereof, by overexpressing ubiH The wild-type Pseudomonas montenosii was genetically modified to obtain an engineered bacterium that can efficiently degrade NP, named the engineered bacterium GC01. Compared with the wild-type Pseudomonas montenosii, the engineered bacterium obtained after the modification has significantly improved its ability to degrade NP, the toxic product downstream of NPEO. GC01 can completely degrade 10mg / L NP, and the degradation kinetic coefficient is increased by about 7 times. The prepared engineered bacteria can be used to treat phenol-containing wastewater and efficiently degrade toxic pollutants in detoxified wastewater, which has important significance and application value for the treatment of environmental pollutants.

[0027] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0028] Example 1: Preparation of highly efficient NP-degrading engineered bacteria GC01 Overexpression ubiH Gene construction of the engineered bacteria GC01 that efficiently degrades NPs, the specific steps are as follows: 1.1 ubiH Amplification of gene fragments Extract Pseudomonas monteri Pseudomonas monteilli ) EBTHRC-PM genomic DNA, which was screened out by our laboratory in the early stage, is now stored at Wuhan University, Wuhan, China, with the deposit 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 ubiH The gene fragments ubiH 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.

[0029] SEQ ID NO.1: SEQ ID NO.2: ubiH-F: 5’-aattctaaggaggttttctaATGAACCGGGTGAACCTGG-3’; SEQ ID NO.3: ubiH-R: 5’-tcgacggtatcgataagcttTCATGCCTGGCCCCGCGG-3’; The PCR amplification system is 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 ddH2O, with a total volume of 20 μL; The PCR amplification reaction procedure is 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, for 35 cycles; final extension at 72°C for 5 min, and hold at 4°C.

[0030] 1.2 Amplification of P13 promoter fragment Extract the genomic DNA of Pseudomonas putida ( Pseudomonas putida ), KT2440, which was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (product number: B84217). Using the DNA as a template, design upstream and downstream primers P13-F and P13-R for PCR amplification to obtain a gene fragment containing the P13 promoter. The primer sequences of P13-F and P13-R are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0031] SEQ ID NO.4: P13-F: 5’-cgacggccagtgagcgcgcgTCCGGATGTGGTACGGAACG-3’; SEQ ID NO.5: P13-R: 5’-cgcggtggagctccaattcgATGCTTCATCCTTCCGCTTATG-3’; The PCR amplification system is as follows: 1 μL of KT2440 genomic DNA template, 0.5 μL of upstream primer P13-F, 0.5 μL of downstream primer P13-R, 10 μL of 2×Phanta Max Master Mix, 8 μL of ddH2O, with a total volume of 20 μL; The PCR amplification reaction procedure is 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, for 35 cycles; final extension at 72°C for 5 min, and hold at 4°C.

[0032] 1.3 Preparation of Linearized PBBR1MCS-2 The PBBR1MCS-2 plasmid was introduced into Escherichia coli DH5α strain by heat shock method. The heat shock experiment was carried out in a 42 °C water bath, and after heat shock for 90 s, it was ice-bathed for 2 minutes. DH5α containing the PBBR1MCS-2 plasmid was cultured and amplified at 37 °C and 150 rpm, and the plasmid was extracted. PBBRMCS-2 was from Kele Biotechnology Co., Ltd. (product number: VT8173), and DH5α competent cells were from Sangon Biotech (Shanghai) Co., Ltd. (product number: B528413). Using plasmid DNA as a template, upstream and downstream primers PBBR-F1 and PBBR-R1 were designed for inverse PCR amplification to obtain a linearized fragment containing PBBR1MCS-2. The primer sequences of PBBR-F1 and PBBR-R1 are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively.

[0033] SEQ ID NO.6: PBBR-F1: 5’-AAGCTTATCGATACCGTCGACC-3’; SEQ ID NO.7: PBBR-R1: 5’-GAATTCCTGCAGCCCGGG-3’; The PCR amplification system was: 1 μL of PBBR1MCS-2 plasmid DNA template, 0.5 μL of upstream primer PBBR-F1, 0.5 μL of downstream primer PBBR-R1, 10 μL of 2×Phanta Max Master Mix, 8 μL of ddH2O, with a total volume of 20 μL; The PCR amplification reaction program was: 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, for 35 cycles; final extension at 72 °C for 5 min, and placed at 4 °C.

[0034] 1.4 Preparation of PBBR1MCS-2-ubiH Plasmid The ubiH gene fragment in 1.1 and the linearized plasmid fragment in 1.3 were ligated homologously. The homologous ligation was carried out referring to the instruction manual of the kit (Nanjing Novoprotein Scientific Co., Ltd., product number: C116-01). The product after homologous ligation was introduced into DH5α competent cells by heat shock method. The heat shock experiment was carried out in a 42 °C water bath, and after heat shock for 90 s, it was ice-bathed for 2 minutes. After recovery, it was spread on a solid medium containing 50 μg / mL kanamycin to screen for positive recombinant bacteria with kanamycin resistance, cultured and amplified, and the PBBR1MCS-2-ubiH plasmid containing the ubiH gene was extracted.

[0035] 1.5 Linearize the recombinant plasmid to remove the promoter T7 Using the recombinant plasmid PBBR1MCS-2-ubiH obtained in 1.4 as a template, design upstream and downstream primers PBBR-F2 and PBBR-R2 for PCR amplification to obtain a linearized fragment of PBBR1MCS-2-ubiH without the promoter T7. The primer sequences of PBBR-F2 and PBBR-R2 are shown in SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0036] SEQ ID NO.8: PBBR-F2: 5’-CGAATTGGAGCTCCACCGC-3’; SEQ ID NO.9: PBBR-R2: 5’-CGCGCGCTCACTGGCCGT-3’; The PCR amplification system is 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 ddH2O, with a total volume of 20 μL; The PCR amplification reaction procedure is 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, for 35 cycles; final extension at 72°C for 5 min, hold at 4°C.

[0037] 1.6 Preparation of plasmid PBBR1MCS-2(△T7)-P13-ubiH Homologously ligate the P13 fragment in 1.2 and the linearized fragment in 1.5. The homologous ligation is carried out according to the instructions of the homologous ligation kit (Nanjing Novoprotein Scientific Inc., product number: C116-01). The product after homologous ligation is introduced into DH5α competent cells by heat shock method. After recovery, it is spread on a solid medium containing 50 μg / mL kanamycin to screen for positive recombinant bacteria with kanamycin resistance. Expand the positive strains and extract the plasmid to obtain plasmid PBBR1MCS-2(△T7)-P13-ubiH.

[0038] 1.7 Construction of highly efficient NPEO-degrading and detoxifying engineering bacterium GC01 The recombinant plasmid PBBR1MCS-2(△T7)-P13-ubiH in 1.6 was electrotransformed into the wild strain EBTHRC-PM of Pseudomonas monteilii under the electrotransformation conditions of 600V and 12ms. After recovery, it was spread on a solid medium containing 30 μg / mL kanamycin to screen for positive recombinant bacteria with kanamycin resistance, and the engineered bacteria with high-efficiency degradation and detoxification of NPEO were obtained and named GC01.

[0039] The target gene (P13-ubiH) obtained by the engineered bacteria GC01 was verified by PCR using primers M13F and M13R, and the electrophoresis pattern was as Figure 1 shown. Band 1 was the wild Pseudomonas monteilii, and band 2 was the successfully constructed positive recombinant bacteria. The primer sequences of M13F and M13R are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively.

[0040] SEQ ID NO.10: M13F: 5’-GTTGTAAAACGACGGCCAG-3’; SEQ ID NO.11: M13R: 5’-CAGGAAACAGCTATGAC-3’.

[0041] Example 2: Detection of ubiH gene expression in the engineered bacteria GC01 The wild-type strain EBTHRC-PM and the engineered bacteria GC01 were respectively inoculated in LB medium and cultured overnight until the OD600 reached 1.0. After centrifugation at 7000 rpm for 3 min, the supernatant was discarded, and the bacterial precipitate was collected. It was washed with MSM three times and resuspended with an equal volume of inorganic salt medium. Then, they were inoculated into the inorganic salt medium containing 10 mg / L NPEO and NP at a volume ratio of 5% respectively, with only inoculation in the inorganic salt medium as the blank control. The bacteria were collected at 0, 12, 24, and 48 h, and then the bacteria were collected by centrifugation at 8000 rpm for 5 min. Total RNA was extracted using an RNA extraction kit (Vazyme Biotech Co., Ltd., product number: RC113), and immediately reverse transcribed into cDNA using a reverse transcription kit (Vazyme Biotech Co., Ltd., product number: R223) for real-time fluorescence quantitative polymerase chain reaction. The inorganic salt medium (MSM) contained the following components: 3000 mg / L Na2HPO4, 200 mg / L MgSO4·7H2O, 10 mg / L CaCl2, 2650 mg / L KH2PO4, and 1500 mg / L (NH4)2SO4.

[0042] The engineered bacteria GC01 were detected using a fluorescence quantitative kit (Vazyme Biotech Co., Ltd., product number: Q331-02)ubiH The expression of the gene at the transcriptional level. For the detailed operation steps, please refer to the kit instruction manual. The results are as Figure 2 shown. During the degradation of NPEO, the ubiH gene of the engineered strain GC01 was up-regulated by 2-3 folds compared with the wild strain EBTHRC-PM. During the degradation of NP, the ubiH gene of the engineered strain GC01 was up-regulated by 3-5 folds compared with the wild strain EBTHRC-PM.

[0043] Example 3: Detection of the degradation of the engineered strain GC01 in an NPEO or NP environment The engineered strain GC01 prepared in Example 1 was inoculated into the inorganic salt medium containing 10 mg / L NPEO and 10 mg / L NP respectively, and the degradation of NPEO and NP by the strain was detected.

[0044] Add 200 mL of inorganic salt medium into a 250 mL conical flask, add NPEO with a final concentration of 10 mg / L. The engineered strain G01 and the wild strain EBTHRC-PM were inoculated into the inorganic salt medium respectively, with an initial inoculation amount of 5%. The degradation experiment 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.

[0045] Add 50 mL of inorganic salt medium into 100 mL, add NP with a final concentration of 10 mg / L. The engineered strain GC01 and the wild strain EBTHRC-PM were inoculated into the inorganic salt medium respectively, and cultured at 28 °C for 14 h. The cells were collected by centrifugation, with an initial inoculation amount of 5%. The degradation experiment conditions were 28 °C and 150 rpm. Samples were taken at different time points, and the residual amount of NP was detected by high performance liquid chromatography.

[0046] Figure 3 The results were the degradation of the two strains inoculated into the MSM containing 10 mg / L NPEO or NP respectively and the degradation kinetic simulation curve. There was no significant difference in the degradation ability of the two strains to NPEO, and the degradation kinetic constants were similar. The degradation ability of the engineered strain GC01 to NP was significantly higher than that of the wild strain. The degradation rate of the wild strain to NP could only reach 40% at 72 h, while the engineered strain GC01 could reach 100% and achieve complete degradation, and its degradation kinetic constant was 7 times that of the wild strain.

[0047] Example 4: Detoxification of NPEO by the engineered strain in a simulated wastewater environment The engineered strain GC01 prepared in Example 1 was inoculated into the simulated wastewater containing 10 mg / L NPEO. 100 mL of samples were taken at different time points, filtered, and solid phase extracted with a 3 cc HLB small column, enriched 100 times to 1 mL, and the change of endocrine disrupting toxicity during the whole process was detected.

[0048] Endocrine disruption toxicity was determined using luminescent yeast. This method referred to the literature "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". Using estrogen E2 as the positive control and methanol as the negative control, detection was carried out using a 96-well white plate. 20 μL was added to each well of the positive control, and the actual sample gradients were 20 μL, 10 μL, 5 μL, 2 μL, and 1 μL, with a total of 5 concentrations. After the methanol had evaporated, 200 μL of yeast suspension was added and incubated at 28 °C for 8 - 12 hours. Subsequently, a multifunctional plate reader (Tecan, Austria) was used to measure the luminescence intensity of each well. The estrogen toxicity equivalence was characterized by the EC50 value. 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. A 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:

[0049] 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.

[0050] Figure 4 The results showed that the engineered bacterium GC01 significantly weakened the toxicity during the degradation of NPEO, mainly reflected in the reduction of the peak value and the shortening of the detoxification time.

[0051] It was proved that the prepared engineered bacterium with high efficiency in degrading NP could achieve high-efficiency toxicity inhibition during the degradation of NPEO. Under the condition of an initial biological inoculation amount of 5%, during the detoxification of 10 mg / L NPEO by the engineered bacterium GC01, compared with the wild bacterium, the toxicity peak value in the simulated wastewater environment decreased by approximately 80%.

[0052] The above has described the embodiments of the present invention in detail, but the above content is only the preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention shall still fall within the scope covered by the patent of the present invention.

Claims

1. An engineered bacterium for highly efficient degradation of NP, characterized in that, The engineered bacterium is Pseudomonas monteilii EBTHRC-PM overexpressing ubiH the ubiH gene, and the nucleotide sequence of the ubiH gene is shown in SEQ ID NO.1; the preservation number of Pseudomonas monteilii EBTHRC-PM is CCTCC NO:M20211525, and it was preserved in the China Center for Type Culture Collection on December 3, 2021, with the preservation address being Wuhan University, Wuhan, China.

2. The engineered bacteria for highly efficient degradation of NP according to claim 1, characterized in that, The engineered bacteria contain a recombinant plasmid, and the recombinant plasmid contains ubiH gene.

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

4. Method for constructing engineered bacteria for efficiently degrading NP, characterized in that, The method includes the following steps: Introduce ubiH the gene fragment into an expression vector for expression ubiH to obtain a recombinant expression vector of the gene, wherein the ubiH nucleotide sequence of the gene is as shown in SEQ ID NO.1; Introduce the recombinant expression vector expressing ubiH the gene into Pseudomonas monteilii EBTHRC-PM to obtain an engineered bacterium with high efficiency in degrading NP. The preservation number of Pseudomonas monteilii EBTHRC-PM is CCTCC NO: M20211525, and it was preserved at the China Center for Type Culture Collection on December 3, 2021, with the preservation address being Wuhan University, Wuhan, China.

5. The method for constructing an engineered bacterium for 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; the ubiH introduction of the gene fragment into the expression vector includes: ubiH introducing the gene fragment into plasmid PBBR1MCS-2 to obtain recombinant plasmid PBBR1MCS-2-ubiH.

6. The method for constructing an engineered bacterium for highly efficient degradation of NP according to claim 5, characterized in that, The recombinant expression vector is 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 ligating the P13 promoter.

7. The method for constructing an engineered bacterium for highly efficient degradation of NP according to any one of claims 4-6, characterized in that, The removing of the promoter T7 in the recombinant plasmid PBBR1MCS-2-ubiH includes: Using the recombinant plasmid PBBR1MCS-2-ubiH as a template, performing PCR amplification with upstream and downstream primers PBBR-F2 and PBBR-R2 to obtain a linearized PBBR1MCS-2-ubiH fragment without the 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 with high efficiency in degrading NP according to claim 7, characterized in that The said ubiH gene fragment was amplified by PCR using the genomic DNA of Pseudomonas monteilii EBTHRC-PM as a template and the upstream and downstream primers ubiH-F and ubiH-R to obtain a gene fragment containing ubiH ; The primer sequences of ubiH-F and ubiH-R are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; The P13 promoter is obtained by performing PCR amplification with upstream and downstream primers P13-F and P13-R using the genomic DNA of Pseudomonas putida as a template to obtain a gene fragment of the P13 promoter; the primer sequences of P13-F and P13-R are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

9. The application of the engineered bacterium with high efficiency in degrading NP according to any one of claims 1-3 in degrading NP and / or NPEO.

10. The application according to claim 9, characterized in that, The application includes adding the engineered bacterium with high efficiency in degrading NP into the phenol-containing wastewater.

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