An engineered bacterium for degrading the herbicide 2,4-D, its construction method and application

Abstract

The present invention belongs to the technical field of construction of genetically engineered strains, and particularly relates to an engineered bacterium for degrading the herbicide 2,4-D, and a construction method and application thereof. The present invention provides an engineered bacterium for degrading the herbicide 2,4-D, and the engineered bacterium is based on Halomonas cupida J9; genes tfdA, tfdB, tfdC, tfdD, tfdE and tfdF are inserted into non-functional sites of the genome of the Halomonas cupida J9. The engineered bacterium (J9U 2,4‑D ) using H. cupida J9 as the base bacterium can survive and maintain its activity in an environment with a high salt concentration, rapidly and efficiently degrade 2,4-D, and completely mineralize it, can effectively convert harmful pollutants into harmless substances, reduce persistent pollution in the environment, reduce treatment costs, and reduce the negative impact on the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of construction of genetically engineered strains, and particularly relates to an engineered bacterium for degrading the herbicide 2,4-D, a construction method thereof, and an application thereof. Background Art

[0002] 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide commonly used to control broad-leaved weeds in crops. This organochlorine pesticide persists in the environment and can accumulate in organisms through the food chain. There is evidence that their accumulation can have adverse effects on non-target organisms, affect the abundance and distribution of soil microorganisms, and even induce human diseases and endanger human health.

[0003] The degradation of pesticides by natural microorganisms is affected by factors such as the chemical structure of pesticides, concentration, and soil composition. Its degradation of 2,4-D is relatively slow, and relying solely on naturally isolated microorganisms is not sufficient to achieve rapid and effective biodegradation of 2,4-D. Moreover, many industrial activities discharge high-salt and high-nutrient wastewater. If not properly treated, these wastewater flows may cause significant environmental pollution. However, the use of microorganisms for wastewater treatment may be hindered by high salinity. Therefore, there is an urgent need for an engineered bacterium that can tolerate salt and efficiently degrade the herbicide 2,4-D. Summary of the Invention

[0004] The purpose of the present invention is to provide an engineered bacterium for degrading the herbicide 2,4-D, a construction method thereof, and an application thereof. The engineered bacterium of the present invention can efficiently degrade the herbicide 2,4-D, can adapt to a high-salt environment and has the ability of mineralization, can reduce the treatment cost, and reduce the negative impact on the environment.

[0005] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D. The engineered bacterium uses Halomonas cupida J9 as the base bacterium; the non-functional sites of the genome of the Halomonas cupida J9 are inserted with tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes;

[0006] The nucleotide sequence of the tfdA is as shown in SEQ ID NO.1; the nucleotide sequence of the tfdB is as shown in SEQ ID NO.2; the nucleotide sequence of the tfdC is as shown in SEQ ID NO.3; the nucleotide sequence of the tfdD is as shown in SEQ ID NO.4; the nucleotide sequence of the tfdE is as shown in SEQ ID NO.5; the nucleotide sequence of the tfdF is as shown in SEQ ID NO.6.

[0007] As a preferred embodiment, the upp gene in the genome of Halomonas desiderata J9 is deleted, and the nucleotide sequence of the upp gene is as shown in SEQ ID NO.11.

[0008] As a preferred embodiment, promoters are respectively inserted at the 5′-ends of each of the genes tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF.

[0009] As a preferred embodiment, the promoter includes the P15 promoter; the nucleotide sequence of the P15 promoter is as shown in SEQ ID NO.9.

[0010] As a preferred embodiment, the insertion site of the tfdA gene is located at positions 3150569 - 3150961 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdB gene is located at positions 2822684 - 2823050 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdC gene is located at positions 2825177 - 2825719 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdD gene is located at positions 7980206 - 789550 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdE gene is located at positions 2827638 - 2828669 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdF gene is located at positions 2834359 - 2834775 bp of the genome of Halomonas desiderata J9; the reference genome of Halomonas desiderata J9 is GenBank accession no.CP094345.

[0011] As a preferred embodiment, the gfp gene and the vgb gene are also inserted into the genome of Halomonas desiderata J9; the nucleotide sequence of the gfp gene is as shown in SEQ ID NO.7; the nucleotide sequence of the vgb gene is as shown in SEQ ID NO.8.

[0012] The present invention also provides a method for constructing the above-mentioned engineered bacterium, comprising the following steps:

[0013] Knock out the complete counter-selection marker gene upp in Halomonas desiderata J9 to obtain H. cupida J9U;

[0014] Connect the complete counter-selection marker gene upp in Halomonas desiderata J9 to pK18mobSacB to obtain the pKJU plasmid vector;

[0015] The tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes and the upstream and downstream homologous arms of each gene insertion site are respectively ligated with the pKJU plasmid vector to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, and pKJU-F;

[0016] The gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, and pKJU-F are transformed into the H. cupida J9U to obtain an engineered strain J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE, and ftdF genes.

[0017] As a preferred embodiment, transforming the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F, and pKJU-GV into the H. cupida J9U includes: transforming the gene insertion vector pKJU-A into the H. cupida J9U to obtain a strain J9U-A containing the tfdA gene; transforming the gene insertion vector pKJU-B into the strain J9U-A to obtain a strain J9U-AB containing the tfdA and tfdB genes; transforming the gene insertion vector pKJU-C into the strain J9U-AB to obtain a strain J9U-ABC containing the tfdA, tfdB, and tfdC genes; transforming the gene insertion vector pKJU-D into the strain J9U-ABC to obtain a strain J9U-ABCD containing the tfdA, tfdB, tfdC, and tfdD genes; transforming the gene insertion vector pKJU-E into the strain J9U-ABCD to obtain a strain J9U-ABCDE containing the tfdA, tfdB, tfdC, tfdD, and tfdE genes; transforming the gene insertion vector pKJU-F into the strain J9U-ABCDE to obtain a strain J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes.

[0018] As a preferred embodiment, after obtaining the strain J9U-ABCDEF, it further includes: ligating the gfp and vgb genes and the upstream and downstream homologous arms of the insertion site with the pKJU plasmid vector to obtain a gene insertion vector pKJU-GV; transforming the gene insertion vector pKJU-GV into the strain J9U-ABCDEF to obtain a strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb genes 2,4-D 。

[0019] The present invention also provides an application of the engineered bacterium described in the above solution or the engineered bacterium obtained by the construction method described in the above solution in degrading the herbicide 2,4-D.

[0020] Beneficial effects:

[0021] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D. The engineered bacterium is based on Halomonas cupida J9; genes tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF are inserted into non-functional sites of the genome of Halomonas cupida J9; the nucleotide sequence of tfdA is as shown in SEQ ID NO.1; the nucleotide sequence of tfdB is as shown in SEQ ID NO.2; the nucleotide sequence of tfdC is as shown in SEQ ID NO.3; the nucleotide sequence of tfdD is as shown in SEQ ID NO.4; the nucleotide sequence of tfdE is as shown in SEQ ID NO.5; the nucleotide sequence of tfdF is as shown in SEQ ID NO.6. The engineered bacterium (J9U 2,4-D ) uses the salt-tolerant cell Halomonas cupida J9 (H.cupida J9) as the base bacterium, contains the degradation gene for degrading the herbicide 2,4-D, can survive and maintain its activity in a high-salt concentration environment, rapidly and efficiently degrade 2,4-D, and completely mineralize it, and can effectively convert harmful pollutants into harmless substances, reducing persistent pollution in the environment. Using the engineered bacterium of the present invention improves the degradation efficiency, can also reduce the treatment cost, and reduces the negative impact on the environment. The results of the examples show that J9U 2,4-D can completely degrade 25 mg / L of 2,4-D within 6 h. Description of the drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 is the fitting curve of the growth inhibition rate of H.cupida J9 cells by different concentrations of 2,4-D;

[0024] Figure 2 is the schematic diagram of the integration of the foreign gene into the genome of H.cupida J9U; in the figure, Kan is kanamycin; Upp is uracil phosphoribosyltransferase; X is the foreign gene; UP is the upstream homologous arm; DN is the downstream homologous arm;

[0025] Figure 3 is the gene metabolism schematic diagram of the engineered bacterium J9U 2,4-D ;

[0026] Figure 4 PCR detection diagram in Example 2; wherein, in the lanes: M is MarkerⅢ; 1 is tfdA, 864bp; 2 is tfdB, 1797bp; 3 is tfdC, 768bp; 4 is tfdD, 1113bp; 5 is tfdE, 705bp; 6 is tfdF, 1065bp; 7 is gfp, 720bp; 8 is vgb, 411bp;

[0027] Figure 5 Rt-PCR detection diagram in Example 3; wherein A is tfdA; B is tfdB; C is tfdC; D is tfdD; E is tfdE; F is tfdF; G is gfp; H is vgb; in the lanes: M is MarkerⅢ; 1 is PCR with cDNA as the template; 2 is PCR with genomic DNA as the template; 3 is PCR with mRNA as the template; 4 is PCR with ddH2O as the template;

[0028] Figure 6 For J9U in Example 4 2,4-D Diagram of 2,4-D degradation in MM60; wherein A is 25mg / L 2,4-D; B is 50mg / L 2,4-D; C is 100mg / L 2,4-D;

[0029] Figure 7 HPLC analysis diagram of 25mg / L 2,4-D at different sampling times in Example 4;

[0030] Figure 8 HPLC analysis diagram of 50mg / L 2,4-D at different sampling times in Example 4;

[0031] Figure 9 HPLC analysis diagram of 100mg / L 2,4-D at different sampling times in Example 4;

[0032] Figure 10 For J9U under high-concentration NaCl conditions in Example 5 2,4-D Schematic diagram of 2,4-D degradation by J9U; wherein A is 80g / L NaCl; B is 100g / L NaCl; C is 120g / L NaCl;

[0033] Figure 11 For J9U in Example 6 2,4-D Mineralization diagram of 2,4-D by J9U; wherein A is J9U for 2,4-D mineralization in MM60 added with 25mg / L 2,4-D 2,4-D For 2,4-D mineralization by J9U; B is J9U for 2,4-D mineralization in MM100 added with 25mg / L 2,4-D 2,4-D For 2,4-D mineralization;

[0034] Figure 12 Degradation diagram of 2,4-D under hypoxic conditions for J9U in Example 7 2,4-D ;

[0035] Figure 13 Growth performance diagram of J9U in Example 8 2,4-D ; where A is the growth curve diagram; B is the maximum specific growth rate (μ max );

[0036] Figure 14 Degradation schematic diagram of 2,4-D by J9U in the river water of Example 9 2,4-D ; where A is the mineralization diagram of 2,4-D by J9U in the river water added with 25 mg / L 2,4-D and 60 g / L NaCl 2,4-D ; B is the mineralization diagram of 2,4-D by J9U in the river water added with 100 mg / L 2,4-D and 100 g / L NaCl 2,4-D ; Detailed implementation mode

[0037] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D, and the engineered bacterium is based on Halomonas cupida J9; genes tfdA, tfdB, tfdC, tfdD, tfdE and tfdF are inserted into non-functional sites of the genome of Halomonas cupida J9;

[0038] The nucleotide sequence of tfdA is shown as SEQ ID NO.1; the nucleotide sequence of tfdB is shown as SEQ ID NO.2; the nucleotide sequence of tfdC is shown as SEQ ID NO.3; the nucleotide sequence of tfdD is shown as SEQ ID NO.4; the nucleotide sequence of tfdE is shown as SEQ ID NO.5; the nucleotide sequence of tfdF is shown as SEQ ID NO.6.

[0039] The Halomonas cupida J9 (H. cupida J9) described in the present invention is a halophilic microorganism that can survive and maintain its activity in an environment with a high salt concentration. Using it as the base bacterium can overcome the problem of reduced activity of traditional microorganisms in high-salt wastewater and has higher adaptability in the treatment of high-salt wastewater. The present invention has no special limitation on the source of the H. cupida J9 strain. In a specific embodiment of the present invention, the H. cupida J9 strain is derived from the Halomonas cupida J9 strain with the preservation number of CGMCC No. 24707. In the present invention, tfdA encodes 2,4-dichlorophenoxyacetic acid dioxygenase; tfdB encodes 2,4-dichlorophenol hydroxylase; tfdC encodes chlorocatechol-1,2-dioxygenase; tfdD encodes chloromuconate cycloisomerase; tfdE encodes chlorodienelactone hydrolase; tfdF encodes maleylacetate reductase. The above genes are involved in the main pathway of microbial degradation of 2,4-D. The complete metabolic pathway of the engineered bacterium of the present invention is as Figure 3 shown. The six degradation genes inserted into the engineered bacterium can convert 2,4-D into 3-oxoadipic acid, which is then converted into succinyl coenzyme A and acetyl coenzyme A by the endogenous genes PcaI, Pca J, and FadA in H. cupida J9. These products then enter the tricarboxylic acid cycle, enabling the engineered bacterium to utilize them as carbon sources. The present invention has no particular limitation on the sites where the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes are inserted into the H. cupida J9 genome, and it is sufficient to insert them into non-functional gene sites of the genome. In the present invention, the schematic diagram of the integration of foreign genes into the H. cupida J9 genome is as Figure 2 shown.

[0040] As an implementation manner, the upp gene in the genome of the Halomonas cupida J9 is deleted, and the nucleotide sequence of the upp gene is as shown in SEQ ID NO. 11. When the upp gene in the own genome of H. cupida J9 is successfully knocked out, a mutant strain constructed with a vector carrying the upp gene as the vector for inserting genes has 5-FU resistance, and this reverse marker can be used to screen the mutant strain.

[0041] As an implementation manner, the gfp gene and the vgb gene are also inserted into the genome of the Halomonas desiderans J9; the sequence of the GFP gene is as shown in SEQ ID NO.7. In the present invention, the gfp gene can express green fluorescent protein (GFP). Introducing the GFP gene into the engineered bacteria can enable the engineered bacteria to emit green fluorescence, facilitating real-time monitoring of their activity and location through a confocal microscope. This visualization method is of great significance in the actual environmental remediation process and helps to evaluate the effect of bioremediation. The vgb gene encodes Vitreoscilla hemoglobin and can enhance the oxygen absorption ability of the engineered bacteria.

[0042] As an implementation manner, promoters are respectively inserted at the 5′-ends of each of the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb genes. In a specific implementation manner of the present invention, the promoter includes the P15 promoter; the nucleotide sequence of the P15 promoter is as shown in SEQ ID NO.9. In a specific embodiment of the present invention, an RBS sequence is also connected between the P15 promoter and the gene, and the nucleotide sequence of the RBS is as shown in SEQ ID NO.10. In the present invention, the P15 promoter is directly connected to the gene through the RBS. As an implementation manner, taking the tfdA gene as an example for illustration, the P15-RBS-tfdA will be inserted as a whole into the corresponding non-functional site.

[0043] As an implementation manner, the insertion site of the tfdA gene is located at positions 3,150,569 - 3,150,961 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdB gene is located at positions 2,822,684 - 2,823,050 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdC gene is located at positions 2,825,177 - 2,825,719 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdD gene is located at positions 7,980,206 - 7,895,50 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdE gene is located at positions 2,827,638 - 2,828,669 bp of the genome of Halomonas desiderata J9; the insertion site of the tfdF gene is located at positions 2,834,359 - 2,834,775 bp of the genome of Halomonas desiderata J9; the insertion sites of the gfp and vgb genes are located at positions 3,148,104 - 3,149,096 bp of the genome of Halomonas desiderata J9; the reference genome of Halomonas desiderata J9 is GenBank accession no. CP094345. Taking the insertion site of the tfdA gene as an example, the insertion site of the tfdA gene of the present invention can be any position within positions 3,150,569 - 3,150,961 bp of the genome of Halomonas desiderata J9; in a specific embodiment of the present invention, the insertion site of the tfdA gene is position 3,150,799 bp of the genome of Halomonas desiderata J9, that is, the tfdA gene is inserted between positions 3,150,799 - 3,150,800 bp of the genome of Halomonas desiderata J9.

[0044] The present invention also provides a method for constructing the above-mentioned engineered bacterium, including the following steps: knocking out the complete counter-selection marker gene upp in Halomonas desiderata J9 to obtain H. cupida J9U; ligating the complete counter-selection marker gene upp in Halomonas desiderata J9 to pK18mobSacB to obtain the plasmid vector pKJU; ligating the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes and the upstream and downstream homologous arms of each gene insertion site to the plasmid vector pKJU respectively to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F; transforming the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F into the H. cupida J9U to obtain the engineered bacterium J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

[0045] In the present invention, the complete reverse selection marker gene upp in H. cupida J9 was knocked out to obtain H. cupida J9U. There are no special limitations on the method for knocking out the upp gene, and conventional gene knockout methods well-known to those skilled in the art can be used for knocking out. In the present invention, the knockout of the upp gene refers to Chinese Patent 202310723544.X.

[0046] In the present invention, the complete reverse selection marker gene upp in H. cupida J9 was ligated to pK18mobSacB to obtain the plasmid vector pKJU. There are no special limitations on the ligation method, and the ligation method of genes and vectors well-known to those skilled in the art can be used. In the present invention, the preparation of the pKJU plasmid vector refers to Chinese Patent 202310723544.X. The pK18mobSacB in the present invention is a shuttle-type suicide plasmid with kanamycin resistance. This plasmid can replicate freely in Escherichia coli, but when the host bacterium is Pseudomonas, it does not have the ability to replicate independently and can only be site-specifically integrated into the chromosome of the host bacterium, otherwise it will be cut and degraded by the host strain as an exogenous fragment. The pK18mobSacB in the present invention has kanamycin (Kan) resistance, and the plasmid vector pKJU obtained by ligating the reverse selection marker gene upp to pK18mobSacB has Kan resistance and 5-FU resistance. The mutant strain constructed by transforming the H. cupida J9U with the pKJU plasmid vector has Kan resistance and 5-FU resistance, and the mutant strain can be obtained by double crossover screening.

[0047] As an embodiment, in the present invention, the P15 promoter and the RBS sequence were respectively fused with the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes to obtain the P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, and P15-tfdF genes. Taking the P15-tfdA gene as an example for illustration, the nucleotide sequence of the obtained P15-tfdA gene is the P15 promoter + RBS sequence + tfdA gene. As an embodiment, the present invention also includes: fusing the P15 promoter and the RBS sequence with the gfp and vgb genes to obtain the P15-gfp-vgb gene. The nucleotide sequence of the P15-gfp-vgb gene in the present invention is the P15 promoter + RBS sequence + gfp gene + RBS sequence + vgb gene. In the present invention, there are no special limitations on the fusion method, and the gene fusion method well-known to those skilled in the art can be used for fusion.

[0048] The present invention ligates the P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, P15-tfdF, and P15-gfp-vgb genes and the upstream and downstream homologous arms of each gene insertion site to the pKJU plasmid vector respectively to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F, and pKJU-GV.

[0049] The present invention transforms the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F, and pKJU-GV into H. cupida J9U to obtain the engineered strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, GFP, and Vgb genes. 2,4-D 。

[0050] The transformation of the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F, and pKJU-GV into the H. cupida J9U in the present invention includes: transforming the gene insertion vector pKJU-A into the H. cupida J9U to obtain the strain J9U-A containing the tfdA gene; transforming the gene insertion vector pKJU-B into the strain J9U-A to obtain the strain J9U-AB containing the tfdA and tfdB genes; transforming the gene insertion vector pKJU-C into the strain J9U-AB to obtain the strain J9U-ABC containing the tfdA, tfdB, and tfdC genes; transforming the gene insertion vector pKJU-D into the strain J9U-ABC to obtain the strain J9U-ABCD containing the tfdA, tfdB, tfdC, and tfdD genes; transforming the gene insertion vector pKJU-E into the strain J9U-ABCD to obtain the strain J9U-ABCDE containing the tfdA, tfdB, tfdC, tfdD, and tfdE genes; transforming the gene insertion vector pKJU-F into the strain J9U-ABCDE to obtain the strain J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes. As an embodiment, the present invention transforms the gene insertion vector pKJU-GV into the strain J9U-ABCDEF to obtain the strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, GFP, and Vgb genes. 2,4-DIn the specific embodiments of the present invention, a method of single and double exchange screening is used to screen strains. First, strains with a first single exchange are obtained by kanamycin resistance screening. The screened strains with single exchange are screened again in a medium containing 5-Fu to obtain target gene-inserted strains after the second exchange, and finally the target strains are obtained.

[0051] The present invention also provides the application of the engineered bacteria described in the above solution or the engineered bacteria obtained by the construction method described in the above solution in degrading the herbicide 2,4-D.

[0052] To further illustrate the present invention, the following describes in detail an engineered bacterium for degrading the herbicide 2,4-D, its construction method and application provided by the present invention in conjunction with the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0053] Main experimental materials:

[0054] LB60 medium (1L): 70 g NaCl, 10 g tryptone, 5 g yeast extract.

[0055] MM60 medium (1L): 60 g NaCl, 30 g glucose, 0.5 g yeast extract, 2 g NH4Cl, 0.2 g MgSO4, 9.65 g Na2HPO4·12H2O, 1.5 g KH2PO4, 10 mL trace element I, 1 mL trace element II.

[0056] Trace element solution I: 5 g / L ammonium ferric citrate, 2 g / L CaCl2, prepared with 1M HCl.

[0057] Trace element solution II: 100 mg zinc sulfate heptahydrate, 30 mg manganese chloride tetrahydrate, 300 mg boric acid, 200 mg cobalt chloride hexahydrate, 10 mg copper sulfate pentahydrate, 20 mg nickel chloride hexahydrate, 30 mg sodium aluminate dihydrate, dissolved in 1M HCl.

[0058] 2,4-dichlorophenoxyacetic acid (2,4-D) stock solution (10 g / L): Weigh 0.1 g of 2,4-D and dissolve it in 10 mL of methanol solution, and store it in the dark.

[0059] Example 1 Toxicity analysis

[0060] The inhibitory activity of 2,4-D against H. cupida J9 was evaluated using the median effective concentration (EC50): H. cupida J9 was inoculated into MM60 medium and cultured at 37°C for 24 h, and transferred 3 times repeatedly; then the strain was transferred to fresh MM60 medium to make the OD 600When nm was 0.1 and the culture was continued for 6 h to reach the logarithmic growth phase of the strain, different concentrations of 2,4-D (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L) were added respectively; after culturing and incubating at 37 °C for 12 h, OD 600 values were measured.

[0061] The inhibition data were fitted to obtain a fitted curve, as Figure 1 shown. According to the fitted curve, the EC50 value was 1554 mg / L. According to previous studies (W. Wilms, M. -Karczewska, M. Niemczak, A. Parus, R. Frankowski, Wolko, J. Czarny, A. Piotrowska-Cyplik, A. H. J. Heipieper, Chrzanowski, 2,4-D versus 2,4-D based ionic liquids: Effect of cation on herbicide biodegradation, tfdA genes abundance and microbiome changes during soil bioaugmentation, J. Hazard. Mater. 452 (2023) 131209.), an EC50 value greater than 1000 mg / L was considered harmless. It was shown that H. cupida J9 was a powerful chassis cell that could survive in an environment with high concentrations of 2,4-D, making it a promising candidate for treating 2,4-D contaminated wastewater.

[0062] Example 2 Construction of engineered bacteria

[0063] 1. Amplify genes: A. Entrust Genscript Biotech Co., Ltd. in Nanjing to synthesize the genes responsible for the 2,4-D degradation pathway (tfdA, tfdB, tfdC, tfdD, tfdE and tfdF), as well as the genes encoding GFP and VHB proteins (gfp and vgb), and optimize according to the codons of the genus Halomonas, and the sequence information is as SEQ ID NO.1~SEQ ID NO.6. B. Using the genes obtained in step A as templates, design primers and use PCR to amplify the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes.

[0064] 2. Genomic DNA extraction: Extract the genomic DNA of Halomonas cupida J9 strain according to the kit instructions of Vazyme Co., Ltd.; the reference genome of the Halomonas cupida J9 strain is GenBank accession no. CP094345. The upstream homologous arm UP of the tfdA gene is 679 bp upstream of the 3,150,799th bp of the genome, and the downstream homologous arm DN is 679 bp downstream of the 3,150,799th bp of the genome; the upstream homologous arm UP of the tfdB gene is 800 bp upstream of the 2,823,040th bp of the genome, and the downstream homologous arm DN is 800 bp downstream of the 2,823,040th bp of the genome; the upstream homologous arm UP of the tfdC gene is 666 bp upstream of the 2,825,294th bp of the genome, and the downstream homologous arm DN is 666 bp downstream of the 2,825,294th bp of the genome; the upstream homologous arm UP of the tfdD gene is 666 bp upstream of the 7,980,210th bp of the genome, and the downstream homologous arm DN is 665 bp downstream of the 7,980,210th bp of the genome; the upstream homologous arm UP of the tfdE gene is 680 bp upstream of the 2,828,468th bp of the genome, and the downstream homologous arm DN is 680 bp downstream of the 2,828,468th bp of the genome; the upstream homologous arm UP of the tfdF gene is 666 bp upstream of the 2,834,728th bp of the genome, and the downstream homologous arm DN is 666 bp downstream of the 2,834,728th bp of the genome; the upstream homologous arm UP of the GFP and Vgb genes is 700 bp upstream of the 3,148,803rd bp of the genome, and the downstream homologous arm DN is 700 bp downstream of the 3,148,803rd bp of the genome. Taking the upstream and downstream homologous arms of the tfdA gene as an example, the 3,150,798th bp of the H. cupida J9 strain genome is the 679th bp at the 3′ end of the upstream homologous arm UP of the tfdA gene; the 3,150,800th bp of the genome is the 1st bp at the 5′ end of the downstream homologous arm DN of the tfdA gene. Use PCR to amplify the upstream and downstream homologous arm sequences UP and DN of the insertion sites of the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb genes. The gene insertion sites are shown in Table 1.

[0065] 3. Connect the P15 promoter: Using the J9 genome in step 2 as a template, design primers according to the gene insertion site to amplify the promoters linked to different genes, and introduce the same RBS sequence (SEQ ID NO.10) at the end of each promoter sequence; use Vazyme high-fidelity polymerase to fuse the amplified P15 promoters with the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes respectively to obtain P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, and P15-tfdF fragments; use Vazyme high-fidelity polymerase to fuse the P15 strong promoter with the gfp and vgb genes to obtain the P15-gfp-vgb fragment.

[0066] Table 1 Gene Sources and Their Integration Sites

[0067]

[0068] 4. Use Vazyme high-fidelity polymerase to fuse the upstream and downstream homologous arms amplified in step 2 with the P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, P15-tfdF, and P15-gfp-vgb genes respectively (refer to step 3) to obtain the UP-P15-tfdA-DN, UP-P15-tfdB-DN, UP-P15-tfdC-DN, UP-P15-tfdD-DN, UP-P15-tfdE-DN, UP-P15-tfdF-DN, and UP-P15-gfp-vgb-DN fragments. Taking the UP-P15-tfdA-DN fragment as an example, the UP-P15-tfdA-DN is obtained by sequentially connecting the upstream homologous arm UP of the tfdA gene, the P15 promoter, the RBS sequence, the tfdA gene, and the downstream homologous arm DN of the tfdA gene, without a linker. The UP-P15-gfp-vgb-DN fragment is obtained by sequentially connecting the upstream homologous arm UP of the gfp-vgb gene, the P15 promoter, the RBS sequence, the gfp gene, the RBS sequence, the vgb gene, and the downstream homologous arm DN of the gfp-vgb gene, without a linker.

[0069] 5. Insertion vector construction: Knock out the reverse selection marker gene upp in the H. cupida J9 strain to obtain H. cupida J9U; connect the reverse selection marker gene upp in H. cupida J9 to pK18mobSacB to obtain the pKJU plasmid vector (see Chinese Patent 202310723544.X).

[0070] 6. Construction of strain J9U-A: (1) The plasmid pKJU was digested with the restriction endonuclease EcoRⅠ to obtain a linearized plasmid. Subsequently, the UP-P15-tfdA-DN sequence in step 4 was ligated to the linearized plasmid using Vazyme single-fragment ligase to obtain pKJU-A, which was then transferred into DH5α competent cells.

[0071] (2) Plasmid extraction: The plasmid was extracted using the FastPure Plasmid Mini Kit from Vazyme. Primers were designed based on the pKJU plasmid vector, and colony PCR was used to verify whether the inserted gene was inserted. The verified plasmid was sequenced, and after sequencing was completed, the target plasmid was transferred into Escherichia coli E.coli S17-1λpir.

[0072] (3) Conjugative transformation: A. The E.coli S17-1λpir containing the inserted vector was streaked and activated on an LB plate, and the H.cupida J9 strain was streaked and activated on an LB60 plate. B. The E.coli S17-1λpir was inoculated into an LB liquid medium, and the H.cupida J9 strain was inoculated into an LB60 liquid medium. Both were cultured at 37°C and 180 rpm for 16 h. C. An aliquot of the cultured bacterial solution from step 2 was transferred to fresh LB and LB60 liquid media at a ratio of 1% (v / v) respectively, and cultured at 37°C and 180 rpm for 6 h. D. The cultured E.coli S17-1λpir and H.cupida J9 bacterial solutions containing the plasmid were centrifuged at 5000 rpm for 5 min, and the supernatant was discarded completely. E. The E.coli S17-1λpir and H.cupida J9 cells were washed and resuspended three times with 10 mmol / L MgSO4 and 10 mmol / L MgSO4 containing 60 g / L NaCl respectively. F. The cells were resuspended in 100 μL of LB liquid respectively, and the two bacterial solutions were mixed and dropped onto the cellulose membrane on an LB60 solid plate.

[0073] (4) Screening for single and double exchanges: A. Scrape the bacterial lawn obtained after the combined transformation in step (3), dissolve it in 1 mL of LB60 liquid medium, and spread it on an LB60 plate containing kanamycin sulfate resistance. Incubate it upside down at 37 °C for 24 h until colonies grow on the plate. B. Pick a single colony into an LB60 liquid medium containing 100 μg / mL Kan, incubate at 37 °C and 180 rpm for about 6 h. Design primers based on the homologous arms upstream and downstream of the inserted gene, and perform colony PCR verification to screen for single exchanges. C. Transfer the verified correct strain to a non-resistant LB60 test tube, incubate at 37 °C and 180 rpm for 24 h to complete double exchange and the suicide plasmid is lost. D. Take 100 μL of the cultured bacterial liquid, dilute it 1000 times with LB60 liquid medium, spread it on an LB60 liquid medium containing 5-Fu resistance for culture. Design primers based on the inserted gene, verify double exchange by colony PCR, and sequence the PCR product of the double-exchanged strain. F. Insert the correctly sequenced gene into the strain, and the strain containing the tfdA gene is stored in a glycerol tube at -80 °C, denoted as J9U-A strain.

[0074] 7. Construction of J9U-AB strain: Construct the pKJU-B plasmid with UP-P15-tfdB-DN obtained in step 5 according to the method described in step 6(1), and then transfer it into the J9U-A strain by conjugation transformation according to the methods described in steps 6(2) - (3). Screen the strains with kanamycin resistance for the exchanged strains according to the method described in step 6(4). Screen the verified correct strains again in a 5-Fu medium to obtain the target gene-inserted strains after the second exchange, and obtain the J9U-AB strain containing the tfdA and tfdB genes.

[0075] 8. Construction of J9U-ABC strain: Construct the pKJU-C plasmid with UP-P15-tfdC-DN in step 5 according to the method described in step 6(1), and then transfer it into J9U-AB by conjugation transformation according to the methods described in steps 6(2) - (3). Screen the strains with kanamycin resistance for the exchanged strains according to the method described in step 6(4). Screen the verified correct strains again in a 5-Fu medium to obtain the target gene-inserted strains after the second exchange, and obtain the J9U-ABC strain containing the tfdA, tfdB and tfdC genes.

[0076] 9. Construction of strain J9U-ABCD: Construct the plasmid pKJU-D with UP-P15-tfdD-DN in step 5 according to the method described in step 6(1). Then, transfer it into strain J9U-ABC by conjugation transformation according to the method described in steps 6(2)-(3). Screen the strains with kanamycin resistance according to the method described in step 6(4) to obtain the strains with exchange. Screen the strains with correct verification again in the medium containing 5-Fu to obtain the target gene-inserted strains after the second exchange, and obtain strain J9U-ABCD containing tfdA, tfdB, tfdC, and tfdD genes.

[0077] 10. Construction of strain J9U-ABCDE: Construct the plasmid pKJU-E with UP-P15-tfdE-DN in step 5 according to the method described in step 6(1). Then, transfer it into strain J9U-ABCD by conjugation transformation according to the method described in steps 6(2)-(3). Screen the strains with kanamycin resistance according to the method described in step 6(4) to obtain the strains with exchange. Screen the strains with correct verification again in the medium containing 5-Fu to obtain the target gene-inserted strains after the second exchange, and obtain strain J9U-ABCDE containing tfdA, tfdB, tfdC, tfdD, and tfdE genes.

[0078] 11. Construction of strain J9U-ABCDEF: Construct the plasmid pKJU-F with UP-P15-tfdF-DN in step 5 according to the method described in step 6(1). Then, transfer it into strain J9U-ABCDE by conjugation transformation according to the method described in steps 6(2)-(3). Screen the strains with kanamycin resistance according to the method described in step 6(4) to obtain the strains with exchange. Screen the strains with correct verification again in the medium containing 5-Fu to obtain the target gene-inserted strains after the second exchange, and obtain strain J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes.

[0079] 12. Construction of strain J9U2,4-D: Construct the plasmid pKJU-GV with UP-P15-gfp-vgb-DN in step 5 according to the method described in step 6(1). Then, transfer it into strain J9U-ABCDEF by conjugation transformation according to the method described in steps 6(2)-(3). Screen the strains with kanamycin resistance to obtain the strains with exchange. Screen the strains with correct verification again in the medium containing 5-Fu to obtain the target gene-inserted strains after the second exchange, and obtain strain J9U2,4-D containing tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb genes.

[0080] After the foreign gene is integrated into the genome of H. cupida J9U, J9U 2,4-D The complete metabolic pathway is asFigure 3 As shown in the figure, 2,4-D is converted into 2,4-dichlorophenol (2,4-DCP) by the action of 2,4-dichlorophenoxyacetic acid dioxygenase encoded by the tfdA gene; then, 2,4-DCP is degraded into 3,5-dichlorocatechol (3,5-DCC) by the action of 2,4-dichlorophenol hydroxylase encoded by the tfdB gene; 3,5-DCC is converted into 2,4 -dichloro-cis, cis-muconate; then it is converted into 2,4-chlorodienolide by the action of dichloromuconate cycloisomerase (encoded by tfdD gene); 2,4-chlorodienolide generates 2,4-chloromaleacetic acid by the action of dienolide hydrolase encoded by tfdE gene; then it is further converted into maleoacetic acid by the action of maleoacetate reductase encoded by tfdF, and then becomes β-ketoadipate; β-ketoadipate generates β-ketoadipate-CoA by the action of succinyl-CoA:3-oxooxalate-CoA transferase α subunit and β subunit encoded by J9 endogenous genes Pca I and Pca J; then it generates acetyl-CoA and succinyl-CoA by the action of β-ketoadipate-CoA thiolase encoded by J9 endogenous FadA gene, and finally enters the tricarboxylic acid cycle to realize the mineralization of 2,4-D. It can be seen that the six degradation genes can convert 2,4-D into 3-oxoadipate, but the subsequent conversion into succinyl-CoA and acetyl-CoA is completed by the endogenous genes PcaI, PcaJ and FadA in H. cupida J9, and these products then enter the tricarboxylic acid cycle, making J9U 2,4-D They can be used as a carbon source.

[0081] 13. Strain verification: A. Use SparkJade Bacterial RNA Rapid Extraction Kit to extract RNA, see the instructions for details; use Vazyme IIQ RT SuperMix for qPCR kit removes residual DNA in the solution more thoroughly, followed by reverse transcription. Primers are designed based on the inserted gene, and PCR is used to detect J9U 2,4-D Whether the strain is successfully constructed. Figure 4 shown.

[0082] The strain was subjected to Sanger sequencing, and the eight exogenous genes were successfully inserted into the H. cupida J9 genome, which was verified by PCR and Sanger sequencing. Finally, the engineered strain J9U was constructed. 2,4-D .

[0083] Example 3 RT-PCR detection:

[0084] J9U in Example 2 2,4-DThe strain was activated; the activated strain was incubated in LB60 overnight for 12 h; the cells were collected after centrifugation, and total RNA was extracted from J9U using the SPARKeasy improved bacterial RNA kit (Sparkjade). 2,4-D Genomic DNA residues in the RNA were removed using the Vazyme reverse transcription kit to obtain mRNA; the RNA was reverse transcribed into cDNA after removing the residual DNA; the genomic DNA of the J9U 2,4-D strain was extracted using the Vazyme bacterial DNA rapid extraction and isolation kit to obtain genomic DNA.

[0085] Subsequently, primers were designed according to the inserted genes, and PCR amplification was performed using cDNA, genomic DNA, mRNA, and ddH2O as templates to analyze the transcription of tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb. The results are as Figure 5 shown.

[0086] The results showed that the bands corresponding to cDNA and genomic DNA showed bands matching the expected sizes of the target genes, while no bands were observed in the negative control. These results confirmed the successful transcription of all eight foreign genes in J9U 2,4-D strain.

[0087] Example 4 Degradation experiment of 2,4-D at different concentrations

[0088] Degradation experiments were carried out in MM60 medium supplemented with different concentrations of 2,4-D. A. The J9U 2,4-D strain cultured overnight in Example 2 was inoculated into 100 mL of LB60 medium at an inoculation amount of 1% (v / v) and cultured for 9 - 12 h until the logarithmic growth phase. The cells were washed 3 times with 10 mM phosphate buffer solution (PBS60) containing 60 g / L NaCl and inoculated into 20 mL of MM60 medium, and the initial OD 600 nm value was adjusted to 0.4. B. 25, 50, and 100 mg / L of 2,4-D were added to the MM60 medium respectively, and J9U 2,4-DDegradation of 2,4-D. The control group transferred the H. cupida J9U strain without any transferred genes. C. Incubate the above cultures on a shaker at 37 °C and 180 rpm, and collect samples at different time points for analysis. D. Use HPLC to determine the residual concentration of 2,4-D: Perform liquid chromatography analysis using Agilent 1260 Infinity II, equipped with a ZORBAX StableBond C18 column and a variable wavelength detector (VWD). The detection wavelength of 2,4-D is set at 230 nm, and the column temperature is maintained at 30 °C. The mobile phase consists of 70% methanol and 30% water (containing 0.1% phosphoric acid, v / v), the flow rate is 1 mL / min, and the sample volume is 5 μL.

[0089] In the HPLC chromatogram, the retention time of the characteristic peak of 2,4-D is 3.5 min, and the results are as Figures 6 to 9 shown, indicating that J9U 2,4-D can degrade a large amount of herbicide within 2 h and completely degrade 25 mg / L of 2,4-D within 6 h. This strain also shows good degradation ability at higher concentrations of 2,4-D, with a degradation rate of 95% for 50 mg / L of 2,4-D and 90% for 100 mg / L of 2,4-D within 6 h. The control group did not show significant degradation of 2,4-D.

[0090] Example 5 Degradation experiment with different concentrations of NaCl:

[0091] Conduct degradation experiments in MM medium supplemented with different concentrations of NaCl. A. Operate according to step A of Example 4. B. Use MM medium containing 25 mg / L of 2,4-D and 80 g / L, 100 g / L, or 120 g / L of NaCl (MM80, MM100, and MM120) to evaluate the performance of the engineered bacteria under high-salt conditions; the control group transferred the H. cupida J9U strain without any transferred genes. C. Incubate the cultures on a shaker at 37 °C and 180 rpm, and collect samples at different time points for analysis. D. Use HPLC to determine the residual concentration of 2,4-D, and the conditions are set the same as in step D of Example 4.

[0092] The results are as Figure 10 shown, indicating that J9U 2,4-D can rapidly degrade 25 mg / L of 2,4-D within 8 h in MM80 medium containing 80 g / L of NaCl. This strain also maintained excellent degradation performance in MM100 and MM120 media, indicating its potential for treating pollutants in high-salt wastewater environments.

[0093] Example 6 Mineralization experiment (isotope experiment)

[0094] By monitoring J9U 2,4-D During the degradation 13 of C6-2,4-D, the 13 released CO2 is used to evaluate its ability to mineralize pollutants. The J9U 2,4-D from Example 2 was inoculated into MM60 and MM100 media containing 25 mg / L 13 C6-2,4-D, and the initial OD 600 nm value was adjusted to 0.4, and it was incubated at 37 °C for 3 d under sealed culture conditions. At the end of the incubation, the culture was acidified to a pH below 2 with 2 M HCl; then it was shaken at 180 rpm for 30 min, and the gas in the sealed vial was analyzed using an isotope mass spectrometer (CAAS ESI lab). The carbon isotope ratio was defined using the δ13C value (in ‰) relative to the V-PDB standard. The control group was transferred to the H. cupida J9U strain without any gene transfer.

[0095] The results are as Figure 11 shown, indicating that after incubating J9U 2,4-D for 3 d in MM60 medium containing 25 mg / L 2,4-D, the 2,4-D C value (2712‰) in the CO2 produced by J9U 13 was much higher than that of the control group (128‰). At the same time, in the incubation gas of J9U 2,4-D , the value of 13 13C / (12C + 12 13C) % increased by 319.87% compared with the control group. J9U 13 also maintained the ability to mineralize pollutants under high-salt conditions. In MM100 medium, the mineralization ability of J9U 2,4-D was also confirmed by stable isotope experiments, showing that it could perfectly mineralize pollutants under high-salt conditions. 2,4-D Example 7 Degradation experiment under hypoxic conditions

[0096] To evaluate the degradation ability of J9U

[0097] under hypoxic conditions, the J9U 2,4-D strain was inoculated into MM60 medium containing 25 mg / L 2,4-D, and the initial OD 2,4-D nm was adjusted to 0.4, and it was cultured in a sealed bottle at 37 °C and 80 rpm to maintain hypoxic conditions. The degradation of 2,4-D was analyzed at different time points, and HPLC was used for detection. The detection and analysis were the same as in Example 4. 600 nm

[0098] The results are as Figure 12 shown, indicating that the engineered strain J9U 2,4-DExhibits better growth ability under hypoxic conditions and can completely degrade 25 mg / L of 2,4-D within 8 h.

[0099] Example 8 Gene stability and growth kinetics study:

[0100] J9U 2,4-D Passaged 20 times in 5 mL of MM60 and LB60 media. The presence of eight foreign genes in the twentieth-generation strains was verified using PCR. The overnight cultures of H. cupida J9 and J9U 2,4-D were separately inoculated into 100 mL of LB60 and MM60 media. After adjusting the initial OD 600 at nm to 0.1, the cultures were incubated at 37 °C and 180 rpm for 48 h, and the OD 600 at nm was measured every 3 h to construct a growth curve.

[0101] The results are as Figure 13 shown: The eight foreign genes of the twentieth-generation strains were stably expressed in both MM60 and LB60. The degradation experiment also demonstrated that J9U still had a very prominent ability to degrade 2,4-D after multiple passages. By measuring the growth curves of H. cupida J9 and J9U 2,4-D under the same conditions, it was found that there was no significant difference in the growth trends and maximum specific growth rates of the two strains, indicating that the insertion of foreign genes did not affect the growth of J9U.

[0102] Example 9 River water degradation experiment

[0103] River water was collected from an unpolluted stream on the campus of Nankai University in Tianjin, China. J9U 2,4-D was inoculated into river water media containing 25 mg / L of 2,4-D and 60 g / L of NaCl or 100 mg / L of 2,4-D and 100 g / L of NaCl to achieve a cell density of 10 6 . The cultures were incubated in a shaker at 37 °C and 180 rpm, and samples were taken regularly for HPLC analysis. The mineralization ability of J9U 2,4-D in river water was further evaluated using 13 C6-2,4-D, using 25 mg / L of 2,4-D and 100 mg / L of NaCl.

[0104] The results are as Figure 14 shown, indicating that J9U 2,4-D effectively degraded the pollutants in river water containing 25 mg / L of 2,4-D and 60 g / L of NaCl and completed the degradation within 8 h. Even in river water with a high salt concentration (100 mg / L of 2,4-D and 100 g / L of NaCl), J9U 2,4-DStill maintains its high degradation ability, degrading 90% of 2,4-D within 15 h, while showing good growth performance. And J9U 2,4-D The 13 C value in the CO2 produced is significantly higher than that of the control group, indicating the successful mineralization of the herbicide in the natural water environment.

[0105] In summary, in the present invention, the halophilic microorganism H. cupida J9 is used as a chassis cell, and a herbicide-degrading bacterium J9U with a complete metabolic pathway containing six degradation genes (tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF) is successfully constructed through synthetic biology 2,4-D , which can convert 2,4-D into 3-oxoadipic acid, and then be converted into succinyl coenzyme A and acetyl coenzyme A by the endogenous genes PcaI, PcaJ, and FadA in H. cupida J9 and enter the tricarboxylic acid cycle, enabling J9U 2,4-D to utilize them as carbon sources, ultimately achieving efficient degradation of the herbicide 2,4-D. And it can effectively convert harmful pollutants into harmless substances, reducing persistent pollution in the environment. The engineered bacterium described in the present invention improves the degradation efficiency of the herbicide 2,4-D, reduces the treatment cost, and reduces the negative impact on the environment.

[0106] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An engineered bacterium for degrading the herbicide 2,4-D, characterized in that, The engineered bacterium is based on Halomonas cupida J9; the non-functional sites of the genome of Halomonas cupida J9 are inserted with tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes; The nucleotide sequence of tfdA is shown in SEQ ID NO.1; the nucleotide sequence of tfdB is shown in SEQ ID NO.2; the nucleotide sequence of tfdC is shown in SEQ ID NO.3; the nucleotide sequence of tfdD is shown in SEQ ID NO.4; the nucleotide sequence of tfdE is shown in SEQ ID NO.5; the nucleotide sequence of tfdF is shown in SEQ ID NO.6; The preservation number of Halomonas cupida J9 is CGMCC No.24707; The insertion site of the tfdA gene is at the 3,150,799th bp of the genome of Halomonas cupida J9; the insertion site of the tfdB gene is at the 2,823,040th bp of the genome of Halomonas cupida J9; the insertion site of the tfdC gene is at the 2,825,294th bp of the genome of Halomonas cupida J9; the insertion site of the tfdD gene is at the 7,980,210th bp of the genome of Halomonas cupida J9; the insertion site of the tfdE gene is at the 2,828,468th bp of the genome of Halomonas cupida J9; the insertion site of the tfdF gene is at the 2,834,728th bp of the genome of Halomonas cupida J9; the reference genome of Halomonas cupida J9 is GenBank accession no.CP094345.

2. The engineered bacterium according to claim 1, wherein The upp gene in the genome of Halomonas cupida J9 is deleted, and the nucleotide sequence of the upp gene is shown in SEQ ID NO.

11.

3. The engineered bacterium according to claim 1, wherein Promoters are respectively inserted at the 5′ ends of each of the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

4. The engineered bacterium according to claim 3, wherein The promoter includes the P15 promoter; the nucleotide sequence of the P15 promoter is shown in SEQ ID NO.

9.

5. The engineered bacterium according to claim 1, wherein, The gfp gene and the vgb gene are also inserted into the genome of Halomonas cupida J9; the nucleotide sequence of the gfp gene is shown in SEQ ID NO.7; the nucleotide sequence of the vgb gene is shown in SEQ ID NO.

8.

6. The method for constructing the engineering bacteria according to any one of claims 1 to 5, characterized in that, It includes the following steps: Knock out the complete counter-selection marker gene upp in Halomonas cupida J9 to obtain H.cupida J9U; Connect the complete counter-selection marker gene upp in Halomonas cupida J9 to pK18mobSacB to obtain the pKJU plasmid vector; The tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes and the upstream and downstream homologous arms of each gene insertion site are respectively ligated to the pKJU plasmid vector to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, and pKJU-F; The gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, and pKJU-F are transformed into the H. cupida J9U to obtain an engineered bacterium J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE, and ftdF genes.

7. The construction method according to claim 6, characterized in that Transforming the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F, and pKJU-GV into the H. cupida J9U includes: transforming the gene insertion vector pKJU-A into the H. cupida J9U to obtain a strain J9U-A containing the tfdA gene; transforming the gene insertion vector pKJU-B into the strain J9U-A to obtain a strain J9U-AB containing the tfdA and tfdB genes; transforming the gene insertion vector pKJU-C into the strain J9U-AB to obtain a strain J9U-ABC containing the tfdA, tfdB, and tfdC genes; transforming the gene insertion vector pKJU-D into the strain J9U-ABC to obtain a strain J9U-ABCD containing the tfdA, tfdB, tfdC, and tfdD genes; transforming the gene insertion vector pKJU-E into the strain J9U-ABCD to obtain a strain J9U-ABCDE containing the tfdA, tfdB, tfdC, tfdD, and tfdE genes; transforming the gene insertion vector pKJU-F into the strain J9U-ABCDE to obtain a strain J9U-ABCDEF containing the tfdA, tfdB, tfdC, tfdD, tfdE, and tfdF genes.

8. The construction method according to claim 7, characterized in that, After obtaining the strain J9U-ABCDEF, it further includes: ligating the gfp and vgb genes and the upstream and downstream homologous arms of the insertion site with the pKJU plasmid vector to obtain the gene insertion vector pKJU-GV; transforming the gene insertion vector pKJU-GV into the strain J9U-ABCDEF to obtain the strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes 2,4-D .

9. Application of the engineered bacterium according to any one of claims 1 to 5 or the engineered bacterium obtained by the construction method according to any one of claims 6 to 8 in degrading the herbicide 2,4-D.

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