A strain for efficiently degrading 2,4,6-trichlorophenol and a construction method thereof

Abstract

The application discloses a strain for efficiently degrading 2,4,6-trichlorophenol and a construction method thereof. The strain is obtained by integrating exogenous TcpA gene, TcpC gene and TcpD gene before phaC gene of Cupriavidus necator JMP134 as a starting strain to obtain JMP134-1 strain, and overexpressing PcaI gene, PcaJ gene and PcaF gene derived from the strain itself in the JMP134-1 strain to obtain JMP134-2 strain. Compared with the starting strain JMP134, the modified engineering strain JMP134-2 can reach 0.63 in OD 600 , which is 1.625 times higher than that of the starting strain, and the degradation capacity of 2,4,6-TCP is 2.19 times that of the starting strain.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a strain that efficiently degrades 2,4,6-trichlorophenol and its construction method. Background Technology

[0002] 2,4,6-Trichlorophenol (2,4,6-TCP) is a chlorinated organic pollutant that is poorly soluble in water but readily soluble in organic solvents. It is commonly used as a preservative in wood, textiles, and leather. TCP is listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA). Because TCP has been used as a preservative in wood, leather, and adhesives since the 1950s, it is widely present in the environment. TCP can be absorbed by the human body through drinking water, food, or polluted air, causing toxic effects such as coughing, chronic bronchitis, chest tightness, altered lung function, and lung disease. Animal studies have shown that long-term exposure to 10,000 μg / L TCP in mice induced lymphoma and leukemia. Therefore, identifying microorganisms capable of degrading TCP is crucial for addressing the environmental pollution caused by TCP.

[0003] *Cupriavidus necator* JMP134 is a Gram-negative bacterium whose genome has been sequenced. Isolated from Australian soil, it is capable of consuming copper-dependent copper (TCP) as its sole energy and carbon source, and the TCP degradation pathway within *C. necator* JMP134 has been elucidated. However, its TCP-degrading ability requires further enhancement. Therefore, modifying TCP-degrading strains through metabolic engineering will further effectively address the pollution problem caused by TCP. Summary of the Invention

[0004] In view of this, the present invention provides a strain that efficiently degrades 2,4,6-trichlorophenol.

[0005] In a first aspect, the present invention provides a method for constructing a strain that efficiently degrades 2,4,6-trichlorophenol. The method uses *Cupriavidus necator* JMP134 as the starting strain, integrating TCP degradation-related genes (i.e., the TcpA gene from *Caballeronia glathei*, the TcpC gene from *Rhodococcus jostii*, and the TcpD gene from *Lachnellula obcidentalis*) immediately before the phaC gene to obtain strain JMP134-1. Subsequently, overexpression of the PcaI, PcaJ, and PcaF genes derived from JMP134-1 further enhances the strain's ability to degrade 2,4,6-trichlorophenol, resulting in strain JMP134-2.

[0006] The degradation pathway of TCP in hookworm basidiomycete JMP134 is as follows: Figure 1 As shown, this pathway ultimately degrades 2,4,6-TCP into acetyl-CoA and succinyl-CoA.

[0007] In some embodiments, the TcpA gene sequence in the above method is shown as positions 1 to 1554 of SEQ ID NO:7, and the amino acid sequence of the 3,5,6-trichloro-2-pyridinol monooxygenase it encodes is shown as SEQ ID NO:8.

[0008] In some embodiments, in any of the methods described above, the sequence of the TcpC gene is shown as positions 1568 to 2416 of SEQ ID NO:7, and the amino acid sequence of the 6-chlorohydroxyquinoline-1,2-dioxygenase it encodes is shown as SEQ ID NO:9.

[0009] In some embodiments, in any of the methods described above, the sequence of the TcpD gene is shown as positions 2430 to 3029 of SEQ ID NO:7, and the amino acid sequence of the maleic acid reductase it encodes is shown as in SEQ ID NO:10.

[0010] In some implementations, in any of the methods described above, the integration of the TCP-degrading genes is achieved through a suicide plasmid-mediated target gene deletion strategy.

[0011] A suicide plasmid-mediated target gene deletion strategy, with recombination primarily mediated by the endogenous RecA-dependent system. A sacB-based anti-selection method is employed. The sacB gene encodes levoglucosidase, which hydrolyzes sucrose to release glucose and synthesize fructose. Because fructose accumulates in the periplasm of Gram-negative bacteria, leading to cell lysis, it is highly toxic to many bacteria. After the first single crossover occurs during resistance selection, bacteria are placed on a reverse-selection medium containing a high concentration of sucrose. Under these conditions, mutants that have undergone one single crossover express the sacB gene; only cells reverting to wild-type or target mutants can grow. PCR can then be used to distinguish between wild-type and target mutants.

[0012] In some embodiments, in any of the methods described above, the TcpA-TcpC-TcpD fragment containing the TcpA, TcpC, and TcpD genes is integrated into the genome of *Hypophthalmia ulmoides* JMP134 using a suicide plasmid-mediated target gene deletion strategy to obtain strain JMP134-1. Specifically, this can be achieved by ligating the TcpA-TcpC-TcpD-phaCDonor fragment shown in SEQ ID NO:15 and the suicide pK19mobsacB plasmid to obtain the pK19-TcpA-TcpC-TcpD-phaCDonor plasmid, then transferring the pK19-TcpA-TcpC-TcpD-phaCDonor plasmid into *Hypophthalmia ulmoides* JMP134 via conjugation, and selecting for homologous single crossover and homologous recombination to obtain a strain in which the TcpA, TcpC, and TcpD genes are integrated into the genome of *Hypophthalmia ulmoides* JMP134 and inserted immediately before the original phaC gene, i.e., strain JMP134-1.

[0013] In some embodiments, in any of the methods described above, the sequence of the PcaI gene is shown as positions 1 to 570 of SEQ ID NO:26, and the amino acid sequence of the 3-oxoadipic acid-co-A transferase it encodes is shown as SEQ ID NO:27.

[0014] In some embodiments, in any of the methods described above, the sequence of the PacJ gene is shown as positions 584 to 1222 of SEQ ID NO:26, and the amino acid sequence of the coenzyme A transferase it encodes is shown as SEQ ID NO:28.

[0015] In some embodiments, in any of the methods described above, the sequence of the PacF gene is shown as positions 1236 to 2438 of SEQ ID NO:26, and the amino acid sequence of the 3-oxoadiacyl-CoA thiolase it encodes is shown as SEQ ID NO:29.

[0016] In some embodiments, in any of the methods described above, the PcaI, PacJ, and PacF genes are initiated by the Pj5 promoter, the sequence of which is shown in SEQ ID NO:30.

[0017] In some embodiments, in any of the methods described above, the overexpression of the PcaI, PcaJ, and PcaF genes derived from the JMP134-1 strain is achieved by transferring recombinant plasmids containing these genes into the JMP134-1 strain. The recombinant plasmid can be a pBBR1-PcaI-PcaJ-PcaF plasmid, which can be constructed using the following method:

[0018] The PcaI-PcaJ-PcaF fragment shown in SEQ ID NO:26 was inserted into the XbaⅠ site of the pBBRMSC1 plasmid.

[0019] In a second aspect, the present invention provides strains constructed by any of the methods described above.

[0020] In a third aspect, the present invention provides the use of the above-mentioned strain in the degradation of 2,4,6-trichlorophenol.

[0021] Compared to the original strain JMP134, the engineered strain JMP134-2 showed higher OD values ​​on MM medium containing 2,4,6-trichlorophenol as the sole carbon source. 600 It can reach 0.63, which is 1.625 times higher, and the degradation ability of 2,4,6-TCP is 2.19 times that of TCP. Attached Figure Description

[0022] Figure 1 The degradation pathway of TCP in hookworm copper-loving bacteria JMP134.

[0023] Figure 2 The image shows a gel electrophoresis diagram of nucleic acid in which TcpACD has been integrated into the genome of *Hookworm Copper-Loving Bacterium* JMP134 for PCR verification; where M is the DNA Marker; lane C is the PCR product of the wild-type *Hookworm Copper-Loving Bacterium* JMP134 genome; lanes 1-4 are the PCR products of the JMP134-1 strain obtained by colony PCR screening.

[0024] Figure 3 The liquid chromatogram is for 2,4,6-TCP standard.

[0025] Figure 4 The image shows the liquid chromatogram of the fermentation broth of strain JMP134-2. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0027] The hookworm copper-degrading bacterium JMP134 was disclosed in the literature "Q, Lam M, LK, et al. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase[J]", and is available to the public from Wanhua Chemical Group Co., Ltd.

[0028] The bacterium *Caballeronia glathei* was disclosed in the literature “Sadauskas M,R Statkeviiūt,JVaitekūnas, et al. Bioconversion of Biologically Active Indole Derivatives with Indole-3-Acetic Acid-Degrading Enzymes from *Caballeronia glathei* DSM50014[J]”, and is available to the public from Wanhua Chemical Group Co., Ltd.

[0029] pET-28a(+)-RjTcpC plasmid is a product of Qingke Biotechnology.

[0030] pET-28a(+)-LoTcpD plasmid is a product of Qingke Biotechnology.

[0031] The pK19mobsacB plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P1363.

[0032] The pBBRMSC1 plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P0305.

[0033] Example 1: Test of TCP degradation ability of Cupriavidus necator JMP134 strain

[0034] Streaking of *Hookworm Copper-Loving Bacterium* JMP134 onto LB agar plates containing 10 μg / L gentamicin and incubating at 30°C for 16 h. The next day, single colonies were picked and inoculated into 5 mL of LB medium and incubated at 30°C and 220 rpm for 16 h. Then, 1% (v / v) was inoculated into 250 mL shake flasks containing 50 mL of MM liquid medium (5 mM 2,4,6-TCP, 9 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 1 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L ferric citrate, 200 mg / L NaHCO3, 1 mL / L trace elements). Initial OD... 600 The concentration was 0.03, and fermentation was carried out at 30℃ and 220rpm for 5 days.

[0035] After fermentation, the fermentation broth was centrifuged at 8000 rpm for 5 min, the supernatant was collected, filtered through a 0.45 μm aqueous filter membrane, and the filtrate was analyzed by HPLC. HPLC detection conditions were as follows: Waters... C18 column (4.6×75mm, 3.5μm); column temperature: 40℃; detector: UV detector, detection wavelength: 294nm; mobile phase: methanol / 0.01mol / L ammonium acetate (volume ratio = 75:25); flow rate: 0.6mL / min; injection volume: 10μL; run time: 6min.

[0036] Testing revealed that after fermentation, the residual 2,4,6-TCP in the fermentation broth was 3.15 mM. Five days after fermentation, the OD500 of the strain... 600 It reached 0.24.

[0037] Example 2: Integration of exogenous TCP degradation-related genes

[0038] 1. Obtaining the TcpA-TcpC-TcpD fragment

[0039] (1) Using the genomic DNA of Burkholderia glathei as a template, PCR amplification was performed using primers 1 and 2 to obtain a fragment containing the TcpA gene.

[0040] Primer 1: 5'-ggcagagagacaatcaaatcatgatccgtaccggaaagca-3' (SEQ ID NO: 1);

[0041] Primer 2: 5'-tggtcatcaaaggaggaagtttagttcgcgaaggcttcag-3' (SEQ ID NO: 2).

[0042] (2) Using pET-28a(+)-RjTcpC plasmid as a template, PCR amplification was performed using primers 3 and 4 to obtain a fragment containing the TcpC gene.

[0043] Primer 3: 5'-gaactaaacttcctcctttgatgaccacgtacttcaccga-3' (SEQ ID NO: 3);

[0044] Primer 4: 5'-cggccatcaaaggaggaagttcagctgctgccccgcgtga-3' (SEQ ID NO: 4).

[0045] (3) Using pET-28a(+)-LoTcpD plasmid as a template, PCR amplification was performed with primers 5 and 6 to obtain a fragment containing the TcpD gene.

[0046] Primer 5: 5'-cagctgaacttcctcctttgatggccgatgttacgcccat-3' (SEQ ID NO:5);

[0047] Primer 6: 5'-cgcgcctttgccggtcgccatttagcaggaggaccttcta-3' (SEQ ID NO: 6).

[0048] (4) Using the fragment containing the TcpA gene obtained in step (1), the fragment containing the TcpC gene obtained in step (2), and the fragment containing the TcpD gene obtained in step (3) as templates, fusion PCR was performed using primers 1 and 6 to obtain the TcpA-TcpC-TcpD fragment. The sequence of this fragment is shown in SEQ ID NO:7, where positions 1 to 1554 are the TcpA gene, positions 1568 to 2416 are the TcpC gene, and positions 2430 to 3029 are the TcpD gene. The TcpA gene is derived from *Caballeronia glathei*, the TcpC gene from *Rhodococcus jostii*, and the TcpD gene from *Lachnellula occidentalis*.

[0049] The amino acid sequence of 3,5,6-trichloro-2-pyridinol monooxygenase encoded by the TcpA gene is shown in SEQ ID NO:8.

[0050] The amino acid sequence of 6-chlorohydroxyquinoline-1,2-dioxygenase encoded by the TcpC gene is shown in SEQ ID NO:9.

[0051] The amino acid sequence of maleate reductase encoded by the TcpD gene is shown in SEQ ID NO:10.

[0052] 2. Obtaining the TcpA-TcpC-TcpD-phaCDonor fragment

[0053] (1) Using the genomic DNA of hookworm copper-loving bacteria JMP134 as a template, PCR amplification was performed using primers 7 and 8 to obtain the upstream fragment of the phaC gene.

[0054] Primer 7: 5'-aacgacaacaggagacaggcagaccggccgcatggccagcc-3' (SEQ ID NO: 11);

[0055] Primer 8: 5'-tgctttccggtacggatcatgatttgattgtctctctgcc-3' (SEQ ID NO: 12).

[0056] (2) Using the genomic DNA of hookworm copper-loving bacteria JMP134 as a template, PCR amplification was performed using primers 9 and 10 to obtain the downstream fragment of the phaC gene.

[0057] Primer 9: 5'-tagaaggtcctcctgctaaatggcgaccggcaaaggcgcg-3' (SEQ ID NO: 13);

[0058] Primer 10: 5'-atgcttcttcagcagcgcgttgaagcctcgggattggtggcaa-3' (SEQ ID NO: 14).

[0059] (3) Using the upstream fragment of the phaC gene obtained in step (1), the downstream fragment of the phaC gene obtained in step (2), and the TcpA-TcpC-TcpD fragment as templates, fusion PCR was performed using primers 7 and 10 to obtain the TcpA-TcpC-TcpD-phaCDonor fragment, the sequence of which is shown in SEQ ID NO:15.

[0060] 3. Construction of pK19-TcpA-TcpC-TcpD-phaCDonor plasmid

[0061] The pK19mobsacB plasmid was digested with BamHI to obtain the backbone; the TcpA-TcpC-TcpD-phaCDonor fragment and the backbone were ligated using Gibson ligation to obtain the pK19-TcpA-TcpC-TcpD-phaCDonor plasmid. The plasmid was sent for sequencing, and the results were correct.

[0062] 4. The constructed plasmid was transferred into hookworm copper-loving bacteria JMP134 via conjugation. The specific steps are as follows:

[0063] (1) The pK19-TcpA-TcpC-TcpD-phaCDonor plasmid was transformed into Escherichia coli S17 by chemical transformation to obtain a single colony of Escherichia coli S17 containing the target plasmid.

[0064] (2) Take out the preserved hookworm copper-loving bacterium JMP134 strain from the -80℃ freezer, use a sterilized pipette tip to draw 10μL of bacterial solution, streak it on LB solid medium containing 10μg / L gentamicin, and incubate it overnight in a 30℃ incubator. The next day, pick a single colony and incubate it on LB liquid medium.

[0065] (3) Pick a single colony of Escherichia coli S17 containing the target plasmid with a pipette tip and culture it in liquid LB containing 50 μg / L kanamycin at 37°C and 250 rpm for 12 h with shaking.

[0066] (4) Take 1 mL of each of the bacterial suspensions of Escherichia coli S17 containing the target plasmid and Hookworm Copper-Loving Bacteria JMP134 and mix them. Centrifuge at 12000 rpm for 1 min. Remove the supernatant, wash the cells with 1 mL LB, and then resuspend the cells in 100 μL LB. Spread the cells on LB plates without antibiotics and incubate at 30°C for 24 h.

[0067] (5) Genotype verification after homologous single crossover: Pick 3-5 single colonies from the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and culture for 24 h; then extract 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0068] Primers A1 and B1 are primers located on the pK19-TcpA-TcpC-TcpD-phaCDonor plasmid:

[0069] Primer A1: 5'-tcccctgtacacctgcgctgtg-3' (SEQ ID NO:16);

[0070] Primer B1: 5'-cgcgcgcggcatcagccgcc-3' (SEQ ID NO:17);

[0071] Primers C1 and D1 are located upstream and downstream of the phaC gene in the JMP134 genome of hookworm copper-eating bacteria:

[0072] Primer C1: 5'-tggctggcggactgagcccgcc-3' (SEQ ID NO: 18);

[0073] Primer D1: 5'-gaagttggcgggcgacatcgcat-3' (SEQ ID NO:19);

[0074] To verify whether homologous single exchange was successful, colony PCR was performed using primers C1 and B1 or primers A1 and D1. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 min, only one pair of primers can produce a band for the same clone.

[0075] Finally, a single clone that could amplify the band was obtained. During the homologous single exchange process, the entire plasmid pK19-TcpA-TcpC-TcpD-phaCDonor was integrated into the genome of Hookworm Copper-Loving Bacteria JMP134 through a single exchange of one homologous arm.

[0076] (6) Homologous recombination: The single clones obtained in step (5) were induced twice with sucrose (i.e., once with liquid induction and once with plate induction), and then resistance screening was performed to obtain clones that grew on LB plates containing 10 μg / L gentamicin but did not grow on LB plates containing 50 μg / L kanamycin. Colony PCR verification was performed using primers C1 and D1 to obtain positive clones that could amplify the target band. In this positive clone, homologous recombination was performed between homologous regions within the integrated fragment, and the TcpA-TcpC-TcpD fragment was integrated into the genome of Hookworm Copper-Loving Bacteria JMP134 and inserted immediately before the original phaC gene. This positive clone was named strain JMP134-1. In the colony PCR verification, Hookworm Copper-Loving Bacteria JMP134 was used as a control.

[0077] The specific steps for the two sucrose inductions are as follows:

[0078] The single clone was liquid-induced for 72 h in LB agar plates without NaCl and with 100 g / L sucrose. After liquid induction, 1 μL of bacterial culture was diluted 1000 times, and 100 μL was spread on LB agar plates without NaCl and with 50 g / L sucrose. The plates were incubated for 24 h and then induced again.

[0079] Generally, the gene knockout efficiency using the suicide-type pK19mobsacB plasmid is up to 50%, so 16-24 single colonies are usually selected for PCR verification. In order to obtain a stable monoclonal strain, after PCR verification, it is generally necessary to perform subculture and PCR verification again.

[0080] Colony PCR validation results are as follows Figure 2 As shown, the results indicate that the engineered strain JMP134-1, which integrates the exogenous TcpA-TcpC-TcpD gene before the original phaC gene locus, was successfully screened.

[0081] Example 3: Overexpression of autologous PcaI, PcaJ, and PcaF genes

[0082] 1. Obtaining the PcaI-PcaJ-PcaF gene fragment

[0083] (1) Using the genomic DNA of hookworm copper-loving bacteria JMP134 as a template, PCR amplification was performed using primers 11 and 12 to obtain a fragment containing the PcaI gene.

[0084] Primer 11: 5'-atgccatcaaaggaggaagttcaggcgaccttgacgacgc-3' (SEQ ID NO: 20);

[0085] Primer 12: 5'-aaacctaatggatcgaccttatgcccgcggaactgatcga-3' (SEQ ID NO: 21).

[0086] (2) Using the genomic DNA of hookworm copper-loving bacteria JMP134 as a template, PCR amplification was performed using primers 13 and 14 to obtain a fragment containing the PcaJ gene.

[0087] Primer 13: 5'-cggtcatacttcctcctttgttacagcagcggagcgccag-3' (SEQ ID NO: 22);

[0088] Primer 14: 5'-cgcctgaacttcctcctttgatggcatggacacgtgacga-3' (SEQ ID NO: 23).

[0089] (3) Using the genomic DNA of hookworm copper-loving bacteria JMP134 as a template, PCR amplification was performed using primers 15 and 16 to obtain a fragment containing the PcaF gene.

[0090] Primer 15: 5'-tgagcctttcgttttatttgcttagacccgctcgatcacca-3' (SEQ ID NO: 24);

[0091] Primer 16: 5'-gctgtaacaaaggaggaagtatgaccgaagcctttatctgc-3' (SEQ ID NO: 25).

[0092] (4) Using the fragment containing the PcaI gene obtained in step (1), the fragment containing the PcaJ gene obtained in step (2), and the fragment containing the PcaF gene obtained in step (3) as templates, fusion PCR was performed using primers 11 and 16 to obtain the PcaI-PcaJ-PcaF fragment. The sequence of the fragment is shown in SEQ ID NO:26, where positions 1 to 570 are the PcaI gene, positions 584 to 1222 are the PcaJ gene, and positions 1236 to 2438 are the PcaF gene.

[0093] The amino acid sequence of the 3-oxoadipic acid-co-A transferase encoded by the PcaI gene is shown in SEQ ID NO:27.

[0094] The amino acid sequence of the coenzyme A transferase encoded by the PcaJ gene is shown in SEQ ID NO:28.

[0095] The amino acid sequence of 3-oxoadiacyl-CoA thiolytic enzyme encoded by the PcaF gene is shown in SEQ ID NO:29.

[0096] 2. Construction of pBBR1-PcaI-PcaJ-PcaF plasmid

[0097] The pBBRMSC1 plasmid was digested with XbaI to obtain the backbone. The PcaI-PcaJ-PcaF fragment and the backbone were ligated using the Gibson method to obtain the pBBR1-PcaI-PcaJ-PcaF plasmid. This plasmid was sequenced, and the results were correct. The PcaI, PcaJ, and PcaF genes are promoted by the Pj5 promoter, the sequence of which is shown in SEQ ID NO:30.

[0098] 3. The pBBR1-PcaI-PcaJ-PcaF plasmid was transferred into hookworm copper-loving bacterium JMP134-1 by electroporation. The recombinant engineered bacterium was obtained by kanamycin screening and named JMP134-2 strain.

[0099] Example 4: Test of TCP degradation ability of JMP134-2 strain

[0100] The TCP degradation ability of strain JMP134-2 was tested according to the method in Example 1, except that the strain to be tested was replaced with JMP134-2.

[0101] The liquid chromatogram of 2,4,6-TCP standard is shown below. Figure 3 As shown, the TCP retention time for 2, 4, and 6 is 3.412 minutes.

[0102] The liquid chromatogram of the fermentation broth sample of strain JMP134-2 is shown below. Figure 4 As shown, the retention time for TCP 2, 4, and 6 is 3.412 min.

[0103] Testing revealed that after fermentation, the residual 2,4,6-TCP in the fermentation broth was only 0.95 mM. Five days after fermentation, the OD of the strain... 600 The value reached 0.63. Compared to hookworm copper-degrading bacterium JMP134, JMP134-2's 2,4,6-TCP degradation ability is 2.19 times that of JMP134-2.

[0104] sequence

[0105] SEQ ID NO:7

[0106]

[0107] SEQ ID NO:8

[0108] MIRTGKQYLESLNDGRDVWVGNEKIDNIATHPKTRDYAQRHADFYDLHHRPDLQDVLTYVDEDGERRSMQWFSHRDKEQLRRKRKYHETIMRELAGASFPRTPDVNNYVLATYIDDPQPWETQTIGAEGRVRASNIVDFVKFARKHDFNCAPQFVDPQMDRSNPDAQQRSPGLRVVEKNEKGIVVDGVKAIGTGVAFADWIHIGVFFRPGIPGEQVIFAATPVNTKGVTIVCRESVVKEDATEHPLASQGDELDGMTVFDKVFIPWTHVFHIGNSEHAKLYPQRVFDWLHYHALIRQMVRAELMAGLAVLITEHIGTNKIPAVQTRVAKLVGFHQAMLAHVIASEELGFHTPGGHYKPNILIYDFGRALYLERFSEMIYELIDLSGRSALIFASEKQWNDGKLNEWFERMNNGPIGKPHDRVKIGRVIRDLFLTDWGNRLFVFENFNGTPLQAIRTLTMQRAEFSGAGPYAKLARQVCGIELEEAEQTDYKATADYAKALDSARHQEQRALTEAFAN*

[0109] SEQ ID NO:9MTTYFTEDQSVDTVNGRMGIDAGDRAAVVMESLVRHLHSFVKDVGITQAEWGLAIDFLTRTGQICGPERQEFILLSDTLGVSMLVDAINHRRPTGATENTVFGPFHVEGAPIRQMGDDISLDGKGESCLFAGQVRDLDGHPIEGACVDVWSDNADGYYDVQQPDIQPQWNNRGRFLTGADGRYLFRGIKPTAYPIPDDGPVGQLLDRLGRHPYRPAHMHFLVTAEGCERLVTHTFVEGDSYLESDAVFGVKEALIATYDRNSDDPATAWSSQYDFVLTRGSS*

[0110] SEQ ID NO:10

[0111] MADVTPIDPFVYNVSPSRVIFGSGSIKKLAAELQRQGLSSPLLLSTPQQVSQVEELSKQLNGKIAGTFSEATMHTPSHITEKALSYTDEVKADSVVSIGGGSTIGLGKAISIRTGMPHICIPTTYAGSEMTPILGETENGLKRTRSDPKILPGVVIYDVDLTMTLPTIMSATSGVNAIAHSGKSALPLGKKLSSRRSSC*

[0112] SEQ ID NO:15

[0113]

[0114] SEQ ID NO:26

[0115]

[0116] SEQ ID NO:27

[0117] MPAELIDALIEQGARDLTIVNNNAGNGDTGLAALLKTKRVRKIICSFPRQADSYVFDSLYHAGEIELELVPQGNLAERIRAAGAGIGGFFTRTAYGTPLAEGKETRIIDGQGYVFETPIHADFALIKADTADRWGNLTYRKTARNFGPIMAMAAKCAIVQVSHVVELGEMDPEHIVTPGLFVKRVVKVA*

[0118] SEQ ID NO:28

[0119] MAWTRDEMAARAATELQDGFYVNLGIGLPTLVANWVPEGMEVWLQSENGLLGIGPFPTEDEVDADMINAGKQTVTTLPGSSIFSSADSFAMIRGGHINLAILGAMQVSEKGDLANWMIPGKMVKGMGGAMDLVAGVGRVVVLMEHTAKKKDGTEDIKILKDCNLPLTGVGVVNRIITDLGVIDVTDEGLKLVETAPGVSREEIQAKTGAPLL*

[0120] SEQ ID NO:29

[0121] MTEAFICDAIRTPIGRYGGSLSAVRPDDLGAVPLKALMARNPNLDWKAIDDVIYGNANQAGEDNRNVARMSALLAGLPQEVPGATINRLCGSGMDATGTAARAIKAGEATLMIAGGVESMSRAPFVMGKATSAFSRDAQIFDTTIGWRFINPAMRAAYGVDSMPETAENVATDYKISREDQDLMALRSQEKASRAQADGTLAQEITPVTIAQKKGDPIVVERDEHPRATSMEALAKLRGVVRPDGTVTAGNASGVNDGACALLLASEAGLKQHGLTPRARIVGMATAGVAPRVMGIGPAPASQKLLRQLGMTMDQIDVIELNEAFAAQGLAVLRELGVADDDKRVNPNGGAIALGHPLGMSGARLVTTAMYQLHRTGGHFALCTMCIGVGQGIAMVIERV*

[0122] SEQ ID NO:30

[0123] AAGGTCGATCCATTAGGTTTACTAACAGTATATTCTAAATTTCCACCTGTGTCAATAACGGTTTTTATATCCGCT

Claims

1. A method for constructing a strain that degrades 2,4,6-trichlorophenol, wherein the strain is *Hookworm Copper-Degrading Bacterium* JMP134 ( Cupriavidus necator JMP134 was used as the starting strain, and the strain originated from Burkholderia glyphus (JMP134). Caballeronia glathei The TcpA gene, derived from Rhodococcus johnsonii ( Rhodococcus jostii The TcpC gene of *Cyclophorus rubrum* and *Cyclophorus rubrum* ( Lachnellula occidentalis The TcpD gene of JMP134 was integrated before the phaC gene to obtain strain JMP134-1; strain JMP134-2 was obtained by overexpressing the PcaI, PcaJ and PcaF genes derived from itself in strain JMP134-1.

2. The method according to claim 1, characterized in that: The sequence of the TcpA gene is shown in positions 1 to 1554 of SEQ ID NO: 7, and the amino acid sequence of the 3,5,6-trichloro-2-pyridinol monooxygenase it encodes is shown in SEQ ID NO:

8.

3. The method according to claim 1, characterized in that: The sequence of the TcpC gene is shown from position 1568 to position 2416 in SEQ ID NO: 7, and the amino acid sequence of the 6-chlorohydroxyquinoline-1,2-dioxygenase it encodes is shown in SEQ ID NO:

9.

4. The method according to claim 1, characterized in that: The sequence of the TcpD gene is shown from position 2430 to position 3029 in SEQ ID NO: 7, and the amino acid sequence of the maleate reductase it encodes is shown in SEQ ID NO:

10.

5. The method according to claim 1, characterized in that: The sequence of the PcaI gene is shown from position 1 to position 570 in SEQ ID NO: 26, and the amino acid sequence of the 3-oxoadipic acid coenzyme A transferase it encodes is shown in SEQ ID NO:

27.

6. The method according to claim 1, characterized in that: The sequence of the PcaJ gene is shown from position 584 to position 1222 in SEQ ID NO: 26, and the amino acid sequence of the coenzyme A transferase it encodes is shown in SEQ ID NO:

28.

7. The method according to claim 1, characterized in that: The sequence of the PcaF gene is shown from position 1236 to position 2438 in SEQ ID NO: 26, and the amino acid sequence of the 3-oxoadiacyl-CoA thiolase it encodes is shown in SEQ ID NO:

29.

8. The method according to any one of claims 1-7, characterized in that: The integration of the TcpA, TcpC, and TcpD genes is achieved through a suicide plasmid-mediated target gene deletion strategy.

9. The strain constructed by the method according to any one of claims 1-8.

10. The use of the strain according to claim 9 in the degradation of 2,4,6-trichlorophenol.

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