Hymexazol degradation gene cluster, expression vector thereof, hymexazol degradation recombinant strain and application thereof

Abstract

The invention discloses a hymexazol degrading gene cluster, an expression vector of the hymexazol degrading gene cluster, hymexazol degrading bacteria and application of the hymexazol degrading bacteria. The gene cluster comprises an orf2179 gene with a DNA (Deoxyribose Nucleic Acid) sequence as shown in SEQ ID No. 1, an orf2181 gene with a DNA sequence as shown in SEQ ID No. 3 and an orf2182 gene with a DNA sequence as shown in SEQ ID No. 4. An expression vector and recombinant bacteria containing the gene cluster are further constructed, the degradation function of the gene cluster on the hymexazol is verified, and the result shows that the orf2180 gene does not participate in the degradation effect, but the orf2179 gene, the orf2181 gene and the orf2182 gene participate in the preliminary degradation of the hymexazol, and the results are indispensable. The Cupriavidus recombinant bacterium for expressing the orf2174-2183 gene cluster, which is constructed in the invention, has better hymexazol degradation performance and can be used for efficiently repairing a hymexazol polluted environment.

Description

Technical Field

[0001] This invention relates to a pesticide microbial degradation technology, and more particularly to a hymexazol degradation gene cluster and its expression vector, a hymexazol degradation recombinant strain and its application. Background Technology

[0002] Hymexazol (HML), chemically known as 3-hydroxy-5-methylisoxazole, is a highly effective broad-spectrum fungicide and plant growth regulator. Its long-term abuse has led to its accumulation in soil and water bodies, posing a potential threat to ecosystems and human health.

[0003] Microbial degradation technology has become an important strategy for controlling pesticide pollution due to its high efficiency and environmental friendliness. This technology transforms pesticides into non-toxic or low-toxic metabolites through specific metabolic pathways, offering advantages such as environmental friendliness, economy, and no secondary pollution. Utilizing microorganisms for the biotransformation of hymexazol has become the best way to solve the environmental pollution problem caused by hymexazol.

[0004] The core of microbial degradation technology lies in elucidating degradation gene clusters, key enzymes, and their regulatory mechanisms, providing theoretical support for the development of bioremediation technologies. Since the process of microbial degradation of pesticides relies on a series of enzymatic reactions encoded by gene clusters, upregulated genes related to degradation can be screened using multi-omics technologies such as genomics and transcriptomics. Currently, although microbial degradation pathways have been elucidated for various fungicides, the specific degradation pathways and mechanisms of hymexazol in microorganisms have not yet been reported. How to obtain hymexazol-specific degradation gene clusters and efficiently degrade hymexazol using microorganisms containing these gene clusters has become an urgent technical problem to be solved. Summary of the Invention

[0005] Objectives of this invention: First, this invention aims to provide a hymexazol-degrading gene cluster, addressing the problem of obtaining gene sequences expressing key enzymes involved in hymexazol degradation. Second, this invention aims to provide an expression vector containing the hymexazol-degrading gene cluster, addressing the problem of recombinantly expressing key enzymes involved in hymexazol degradation. Third, this invention aims to provide a recombinant bacterium containing the hymexazol-degrading gene cluster, addressing the problem of obtaining microorganisms that degrade hymexazol. Fourth, this invention aims to provide the application of a hymexazol-degrading recombinant bacterium in the remediation of hymexazol-contaminated soil and / or water, addressing the problem of utilizing microorganisms to remediate hymexazol-contaminated soil and / or water.

[0006] Technical solution: The present invention provides a gene cluster for the degradation of hymexazol, comprising the orf2179 gene with the DNA sequence shown in SEQ ID No. 1, the orf2181 gene with the DNA sequence shown in SEQ ID No. 3, and the orf2182 gene with the DNA sequence shown in SEQ ID No. 4.

[0007] The hymexazol described in this invention can be 3-hydroxy-5-methylisoxazole and / or its structural analogue 3-amino-5-methylisoxazole.

[0008] Preferably, it also includes the orf2180 gene with the DNA sequence shown in SEQ ID No.2.

[0009] A second aspect of the present invention discloses an expression vector containing the above-mentioned hymexazol degradation gene cluster.

[0010] Preferably, the expression vector is a plasmid containing a molecular chaperone gene.

[0011] In some embodiments, the molecular chaperone may be a soluble tag, SpyCatcher, which enables the target protein to maintain its correct three-dimensional structure. The plasmid may be a prokaryotic expression plasmid capable of expressing the target protein in Gram-negative bacteria.

[0012] A third aspect of the present invention discloses a recombinant bacterium of the above-described expression vector.

[0013] Preferably, the recombinant bacteria are bacteria of the genus *Codonopsis* or *Escherichia coli*.

[0014] Heterologous expression of the hymexazol degradation gene cluster in bacteria of the genus Copperalum can endow bacteria of the genus Copperalum with the ability to degrade hymexazol, and can be used to rapidly modify and obtain a variety of hymexazol-degrading microorganisms.

[0015] The fourth aspect of this invention discloses the application of the above-mentioned recombinant bacteria in the remediation of soil and / or water bodies contaminated with hymexazol.

[0016] Specifically, the application of recombinant bacteria to remediate hymexazol-contaminated soil and / or water includes the following steps: (1) Add the recombinant bacteria to the soil and / or water contaminated with hymexazol and mix well to obtain the material to be remediated; (2) Cultivate the materials to be remediated and complete the remediation of soil and / or water bodies.

[0017] Preferably, in step (1), the content of recombinant bacteria in the material to be repaired is at least 1 × 10⁻⁶. 6 CFU / g; Preferably, in step (2), when the material to be remediated is a mixture of recombinant bacteria and contaminated soil, the culture conditions are as follows: adjust the pH of the material to be remediated to neutral, control the temperature at 25-30℃, maintain the humidity at 30-40%, and culture for at least 7 days; When the material to be remediated is a mixture of recombinant bacteria and polluted water, the culture conditions are as follows: adjust the pH of the material to be remediated to neutral, control the temperature of the material to be remediated to 29-30℃, and aerate for at least 5 days.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: In this invention orf2179-2182 Functional validation of the gene cluster revealed the multi-enzyme synergistic mechanism of hymexazol degradation: by orf2181 FAD gene expression is catalyzed by oxidoreductase-dependent hydroxylation or aldehyde grouping of the isoxazole ring, by... orf2179 Fe-S protein expressed by the gene and orf2182 The ferricyanide expressed by the gene is responsible for electron transport. orf2181 The inactivity of expression alone may indicate the need for cofactors (such as NADH) or transmembrane transporters, while the coenzyme supply system present in the DL-D2 strain (host bacterium), which is also a member of the Coccidioides genus, may compensate for this deficiency.

[0019] Gene knockout experiments further confirmed that orf2179 Gene, orf2181 Gene, orf2182 The gene is essential for degradation, and orf2180 The degradation activity of the recombinant bacteria (expressing small proteins) was almost unaffected after knockout. RT-qPCR validation showed that... orf2174-2183 The expression levels of the gene cluster were significantly increased under the induction of hymexazol, consistent with the transcriptome data, indicating a multi-gene synergistic mechanism in this gene cluster.

[0020] The expression constructed in this invention orf2174-2183 The recombinant strains of the genus *Copper-loving Bacteria* exhibit good degradation performance against hymexazol, making them suitable for efficient remediation of environments contaminated with hymexazol and showing promising prospects for industrial applications. Attached Figure Description

[0021] Figure 1 A phylogenetic tree of EML bacteria constructed based on 16S rDNA; Figure 2 Results of agarose gel analysis of EML bacterial genomic DNA; Figure 3 Volcano diagram of differentially expressed genes; Figure 4 for orf2174-2183 Gene cluster size and function annotation diagram; Figure 5 pET-28a(+)-SpyCatcher linearized vector and corresponding orf2181 Gene fragment amplification results; Figure 6 Electrophoresis diagram of SpyCatcher-2181 protein induced by recombinant bacteria; Figure 7 for orf2179 gene fragments orf2182Gene fragments and corresponding pET-28a linearized vector amplification results; Figure 8 Electrophoresis images of recombinant bacteria induced to express 2179 and 2182 proteins; Figure 9 for orf2179-2182 Amplification results of gene cluster fragments and their corresponding pBBR1MCS-2 linearized vectors; Figure 10 The degradation of hymexazol under the catalysis of DL-pBBR-EML recombinant bacteria; Figure 11 Single knockout of pBBR-EML plasmid orf2179, orf2180, orf2181, orf2182 Results of gene linearization vector amplification; Figure 12 Validation results for single clones of different single-gene knockout expression strains; Figure 13 HPLC detection results were used to verify the hymexazol degradation function of different bacteria after different single gene knockouts. Figure 14 The results validate the degradation function of hymexazol after knockout of different single genes. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1: Functional verification and cloning of key genes for hymexazol degradation: (1) A dominant strain capable of growth using hymexazol as the sole carbon source was isolated from paddy soil (named EML strain). Based on morphological observation and 16S rRNA gene sequence analysis, EML strain was preliminarily identified as copper-loving bacterium. Cupriavidus ) , such as Figure 1 As shown in the figure. Tests showed that this strain, in 20 mL MSM liquid medium, could completely degrade 200 mg / L of hymexazol within five days.

[0024] (2) Whole genome and transcriptome sequencing of EML bacteria Genomic DNA extraction: A single EML bacterial culture was picked from a plate and transferred to 3 mL of sterile LB liquid. The culture was incubated overnight at 30 °C and 150 rpm. The next day, a loopful of EML bacterial culture was streaked onto a plate to check for contamination. The remaining bacterial culture was centrifuged at 10,000 rpm to collect the bacterial cells. Genomic DNA was extracted using the bacterial genomic DNA extraction kit (DP302) provided by Tiangen Biotech (Beijing). 3 μL of the extracted DNA was used for agarose gel electrophoresis to assess its quality. The results are shown below. Figure 2 As shown, M is the DNA Marker, and lane 1 is the genomic DNA of EML bacteria.

[0025] Total RNA extraction: EML cells were cultured in 100 mL of liquid MSM with 300 mg / L hymexazol and succinic acid as the sole carbon sources, respectively. After 5 days, the cells were collected by high-speed centrifugation. Total RNA was then extracted from the EML cells using the SteadyPure rapid RNA extraction kit provided by Aikerui (Hunan). 1 μL of RNA was measured on a NanoDrop micro-UV spectrophotometer at absorbance A260 and A260 / A280 to determine its purity and concentration. Each experiment was performed in triplicate.

[0026] Genome and transcriptome sequencing and analysis: Quality-tested EML genomic DNA samples were identified and sequenced using a scaffold diagram. Genome assembly analysis, gene prediction, and functional annotation were then performed. The following results were obtained: The strain's EML genome scaffold diagram contained 140 scaffolds, with a total base count of 7,873,078 bp, 7,311 predicted genes, and a G+C content of 64.06%.

[0027] After passing quality control, total RNA samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for transcriptome sequencing. Libraries were constructed using the Illumina NovaSeq 6000 platform. Raw data underwent FastQC quality control, HISAT2 alignment with the reference genome, and DESeq2 analysis was used to analyze differentially expressed genes. The results are as follows: Three biological replicates were used for each group during succinic acid culture and hymexazol induction, resulting in six samples submitted for transcriptome analysis. A total of 21.83 Gb of clean data was obtained, with each sample achieving a clean data of over 3.31 Gb and a Q30 base percentage of over 93.57%. A total of 9634 expressed genes were detected in this analysis, including 7299 known genes, 2038 novel genes, and 297 sRNAs.

[0028] Based on the quantitative expression results, differentially expressed genes were analyzed between groups using DESeq2. The screening threshold was |log2FC|≥1 and padjust<0.05. A total of 1807 differentially expressed genes were identified, of which 270 were upregulated genes and 1537 were downregulated genes. To further screen genes involved in the initial degradation function in EML bacteria, a volcano plot of differentially expressed genes was drawn, as shown below. Figure 3 As shown. By Figure 3 The data shows that four consecutive genes among the upregulated genes exhibited significant differences, namely... orf2179 , orf2180 , orf2181 and orf2182 Because several genes were upregulated simultaneously, it is speculated that... orf2179-2182 It may participate in the initial degradation of hymexazol.

[0029] Further analysis of the volcano map revealed that orf2174-2183 All genes showed upregulation, suggesting that this gene cluster may be involved in the complete degradation process of hymexazol, up to the generation of metabolic waste. See below for size and functional annotations of the ten genes. Figure 4 The white arrows represent genes that have not yet been reported.

[0030] (3) Contains orf2179 Gene, orf2180 Gene, orf2181 Genes and orf2182 Construction of gene expression vectors and verification of gene functions: Gene clusters were discovered through genome and transcriptome sequencing analysis. orf2174-2183 The expression of these substances was significantly upregulated in the induced group, and they were selected from among them. orf2179 Gene, orf2180 Gene, orf2181 Genes and orf2182 Genetically designed heterologous expression strains were used to verify their function.

[0031] (3.1) orf2181 Construction and functional validation of gene expression vectors The genome sequencing results revealed orf2181 The gene expression product is an oxidase. orf2181 The gene is annotated in the NR database as FAD-dependent oxidoreductase, with a DNA sequence shown in SEQ ID No. 3. It is 1221 bp in length and encodes a protein consisting of 406 amino acids with a molecular weight of 43.9 kDa, the amino acid sequence of which is shown in SEQ ID No. 8. Based on previous experience, oxidases expressed heterologously in vitro tend to form inclusion bodies. Therefore, the soluble tag SpyCatcher was used to assist expression; this molecular chaperone helps the protein maintain its correct three-dimensional structure.

[0032] Based on the principle of seamless cloning primer design, specific amplification primers were designed using pET-28a(+)-SpyCatcher-5958 plasmid (an empty vector containing SpyCatcher expression elements) as a template: Upstream primer: 5'-ACCATGGCCGATTACGATATTCCGACCA-3'; Downstream primer: 5'-GTCGCATACTGCCACTACTGCCGGTATGG-3'; The PCR amplification system is as follows: Table 1 PCR reaction system

[0033] The PCR reaction conditions are as follows: Table 2 PCR reaction procedures

[0034] Using a high-fidelity PCR kit ApexHF HS DNA Polymerase-FL was used to amplify the pET-28a-SpyCatcher linearized vector via PCR. The extension time of the pET-28a(+)-SpyCatcher vector was 5.5 min. After amplification, 50 μL of PCR product was mixed with 5 μL of 10× loading buffer, and TAE electrophoresis was performed on a 0.9% agarose gel. After EB staining, the PCR amplification quality was observed using a gel imaging system.

[0035] PCR amplification orf2181 Gene fragments: Based on the principle of seamless cloning primer design, design orf2181 Gene-specific amplification primers: Upstream primer: 5'-GCAGTAGTGGCAGTATGCGACATGGTCTAGTCATC-3'; Downstream primer: 5'-ATCGTAATCGGCCATGGTATATCTCCTTC-3'; use orf2181 Gene-specific amplification primers were used, with genomic DNA from strain EML as a template, and amplification was performed using the Apex HF HS DNA polymerase-FL high-fidelity PCR kit. orf2181 The gene fragment (using pET-28a(+)-SpyCatcher as the vector) was processed in the same reaction system as in Table 1. The PCR amplification program was identical to that in Table 2, except for the extension time, which was 1 min 10 sec. After amplification, 50 μL of PCR product was mixed with 5 μL of 10× loading buffer and subjected to agarose gel electrophoresis. The amplification quality was assessed using a gel imaging system after electrophoresis.

[0036] The linearized vector of pET-28a(+)-SpyCatcher and its corresponding orf2181 Gene fragment PCR amplification results are as follows Figure 5 As shown, Figure 5 In the diagram, lanes 5 and 6 represent the linearized pET-28a(+)-SpyCatcher vector; lanes 7 and 8 represent the corresponding linearized pET-28a(+)-SpyCatcher vectors. orf2181 Gene fragment, M stands for DNA Marker; Depend on Figure 5As can be seen, the length of the pET-28a(+)-SpyCatcher linearized vector is 5655 bp, corresponding to orf2181 All gene fragments were approximately 1200 bp in size. After gel extraction and recovery, homologous arms were ligated using a seamless cloning enzyme.

[0037] Construction of recombinant plasmid pET-28a(+)-spy2181: The DNA obtained from the above PCR amplification was processed using the ordinary agarose gel DNA recovery kit (DP209) provided by Tiangen Biotech (Beijing). orf2181 Gene fragments and linearized vectors were purified. The concentration of recovered DNA was then determined by 0.9% agarose gel electrophoresis. The purified DNA was then... orf2181 The gene fragment and the linearized vector fragment were ligated using a seamless cloning kit (MC40201, Mona Biosciences). The reaction mixture consisted of approximately 2.5 μL each of the target gene and the linearized vector (adjusted according to the brightness of the electrophoresis bands), and 5 μL of MonClone™ Single Assembly Cloning Mix. After gently mixing the reaction mixture, it was incubated in a 50 °C water bath for 15 min. The reaction solution was then briefly centrifuged to collect the residue at the bottom of the tube, yielding the seamless cloning product, which was then stored in an ice box for later use.

[0038] Construction of BL21 recombinant bacteria: Add 5 μL of seamless cloning product to each 50 μL tube of BL21(DE3) competent cells, mix gently, and incubate on ice for 30 minutes. Then, heat shock in a 42 °C water bath for 1 min 30 s, followed immediately by an ice bath for 5 minutes. In a clean bench, add 500 μL of sterile LB medium to each tube and mix well. Place the centrifuge tubes on a 37 °C shaker and incubate at 150 rpm for 1 h to promote cell recovery. After recovery, centrifuge the tubes at 5000 rpm for 5 minutes, discard approximately 400 μL of supernatant, and resuspend the cells in the remaining culture medium. Then, take 100 μL of the resuspended solution and spread it evenly on an LB agar plate containing 50 mg / L kanamycin. Invert the plate and incubate at 37 °C for 12–16 h to obtain visible single colonies. Finally, pick positive clones.

[0039] Induction and gene function verification of recombinant bacteria: Two positive clone expression strains were streaked onto LB agar plates containing 50 mg / L kanamycin. Freshly grown single colonies were picked and inoculated into 3 mL LB tubes (containing 50 mg / L kanamycin). The tubes were incubated at 37 °C with shaking at 150 rpm for 12 h. Then, 1% of the colony was inoculated into 100 mL LB liquid medium containing kanamycin and incubated at 37 °C with shaking at 150 rpm for 2-3 h until the OD600 reached approximately 0.6. One mL of this inoculum was stored at 4 °C for later use. After the bacterial culture cooled to 20 °C, IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 20 °C with shaking at 150 rpm for 16-20 h. One mL of the pre-induction bacterial culture and one mL of the same amount of induced bacterial culture (estimated based on the OD values ​​before and after induction) were centrifuged at 12000 rpm for 1 min. After discarding the supernatant, add 25 μL of 1× protein loading buffer and mix thoroughly. Incubate at 100 °C for 10 min, then centrifuge at 12000 rpm for 5 min and cool to room temperature. The induction effect was detected by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Results Figure 6 As shown, Figure 6 Lane 3 represents the recombinant bacteria before induction; lane 4 represents the recombinant bacteria after induction; M represents the DNA marker.

[0040] Whole-cell catalysis: 100 mL each of the induced expression strain and the empty vector strain were centrifuged at 5000 rpm for 5 min. The cells were collected and resuspended in 20 mL of sterile MSM containing 80 mg / L hymexazol. The mixture was then incubated at 30 ℃ with shaking at 150 rpm for 10 h. 500 μL of the culture solution was taken, mixed thoroughly with 500 μL of methanol, centrifuged at 12000 rpm for 1 min, and the supernatant was collected. The sample was filtered through a 0.22 μm organic filter, and the degradation of hymexazol was detected by HPLC. Even with the assistance of the soluble tag SpyCatcher during induction... orf2181 Gene expression can also lead to the formation of inclusion bodies; therefore, high-performance liquid chromatography (HPLC) analysis of substrate degradation showed no changes in the liquid chromatography chromatogram. orf2181 The recombinant gene expression product has no hymexazol degradation activity.

[0041] (3.2) orf2179 and orf2182 Construction and functional validation of gene expression vectors orf2179The gene is annotated in the NR database as (Fe-S)-binding protein. Its DNA sequence is shown in SEQ ID No. 1, with a length of 1332 bp, encoding a protein composed of 443 amino acids with a molecular weight of 48.3 kDa. Its amino acid sequence is shown in SEQ ID No. 6. orf2182 The gene is annotated as ferredoxin in the NR database. Its DNA sequence is shown in SEQ ID No. 4, with a length of 774 bp, encoding a protein consisting of 257 amino acids with a molecular weight of 29.3 kDa. Its amino acid sequence is shown in SEQ ID No. 9. 。

[0042] orf2179 Genes and orf2182 The primers for seamless gene cloning are as follows: Table 3 orf2179 and orf2182 Gene primer sequence

[0043] Using the primers in Table 3 and the same seamless cloning method as in step (3.2), recombinant plasmids pET-28a-2179 and pET-28a-2182 were constructed. These two recombinant expression vectors were then transformed into engineered bacterium BL21 to obtain expression vectors. orf2179 The gene-producing strain BL21-pET28a-2179 and its expression orf2182 Gene BL21-pET28a-2182.

[0044] orf2179 and orf2182 The PCR amplification results of the gene fragments and the corresponding pET-28a(+) linearized vectors are as follows: Figure 7 As shown. Figure 7 In the middle, lanes 1 and 2 are orf2179 The pET-28a linearized vector corresponding to the gene fragment; lanes 3 and 4 are... orf2179 Gene fragment; M is DNA marker; lanes 5 and 6 are... orf2182 Gene fragments; lanes 7 and 8 are orf2182 The pET-28a linearized vector corresponding to the gene fragment.

[0045] Depend on Figure 7 It can be seen that the length of the pET-28a(+) linearized vector is approximately 5200 bp. orf2179 and orf2182 The gene fragments are approximately 1300 bp and 800 bp, respectively.

[0046] After induction, strains BL21-pET28a-2179 and BL21-pET28a-2182 were subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 8 As shown, Figure 8 M: marker; Lane 1: after BL21-pET28a-2179 induction; Lane 2: before BL21-pET28a-2179 induction; Lane 3: after BL21-pET28a-2182 induction; Lane 4: before BL21-pET28a-2182 induction. According to... Figure 8 The comparison of protein bands before and after induction shows that both proteins were successfully induced to express. orf2179 and orf2182 The molecular weights of the expressed proteins 2179 and 2182 are approximately 49 kDa and 30 kDa, respectively.

[0047] After whole-cell catalysis, the substrate degradation was detected by high-performance liquid chromatography. The liquid chromatogram showed no change, indicating that neither of the two expression strains was active.

[0048] (3.3) orf2179 - 2182 Construction and functional validation of expression vectors for gene clusters because orf2180 The gene is 300 bp in size, and its DNA sequence is shown in SEQ ID No. 2. It is a small protein consisting of only 99 amino acids, and its amino acid sequence is shown in SEQ ID No. 7. Its structural characteristics determine that it cannot independently complete the initial degradation process of the hymexazol molecule. Furthermore... orf2179 , orf2181 and orf2182 No degradation activity was detected when expressed alone. Figure 4 It is evident that gene clusters orf2179-2182 The consistent transcriptional direction across the genome suggests that these four genes may participate in the initial degradation process through synergistic action. Based on these experimental results, this invention employs a multi-gene co-expression strategy to... orf2179-2182 Gene clusters are cloned and heterologously expressed as complete transcriptional units to maintain the integrity of their degradation function. orf2179-2182 The DNA sequence of the gene cluster is shown in SEQ ID No. 5.

[0049] This invention will orf2179-2182 The four genes were inserted as a whole into the pBBR1MCS-2 plasmid, and then transformed into [the target gene] by chemical transformation. E. coli Within DH5α. A large number of plasmid copies were obtained by culturing DH5α-pBBR-EML. The plasmid was extracted using a kit and then transformed into the existing copper-degrading bacterium DL-D2 strain (genus *Copper-degrading bacterium*) without hymexazol degradation ability using electroporation. Cupriavidus The DL-D2 strain, disclosed in Chinese patent CN118703349A, is a strain capable of efficiently degrading sodium 4-chlorophenoxyacetate and growing solely using it as a carbon source. This strain has been identified as lacking the ability to degrade hymexazol. This was verified within the *Copper-Gnawing Bacterium* DL-D2 strain. orf2179 - 2182 The function of gene clusters is explained in the following ways: pBBR1MCS-2 is a commonly used plasmid in molecular biology. Its pBBR1 origin is a broad-host-range replication origin, which allows the plasmid to replicate in a variety of Gram-negative bacteria.

[0050] Following the principle of seamless cloning primer design, specific amplification primers were designed using pBBR1MCS-2 plasmid as a template. The sequences are as follows: Upstream primer pBBR-BanII-F: 5'-CACCAGAGCTCCAATTCGCCCTA-3'; Downstream primer pBBR-BanI-R: 5'-GAGAGGTACCCAGCTTTTGTTCCCTT-3'; The pBBR1MCS-2 linearized vector was obtained by amplification using the ApexHF HS DNA polymerase-FL high-fidelity PCR kit. The amplification system is shown in Table 1, and the PCR program is shown in Table 2. The extension time of the pBBR1MCS-2 linearized vector was 4 min 30 sec. After amplification, 50 μL of PCR product was mixed with 5 μL of 10× loading buffer and subjected to TAE electrophoresis on a 0.9% agarose gel. After EB staining, the PCR amplification quality was observed using a gel imaging system.

[0051] To ensure orf2179-2182 It can be successfully expressed in this plasmid, so a design was made. orf2179-2182 The cloning start point of the gene fragment is 500 bp before the start codon of gene 2179, and the cloning end is 450 bp after the stop codon of gene 2182 (approximately...). orf2179-2182 The ribosome binding sites or promoters of the gene clusters were also cloned to prevent false negative results due to non-transcriptional translation. Using the genomic DNA of strain EML as a template, a design was developed. orf2179 - 2182 The specific amplification primers for the gene cluster are as follows: Upstream primer WF-EML-500F: 5'-CAAAAGCTGGGTACCTCTCAGTGATTGTCCTAGTGTTG-3'; Downstream primer WF-EML-R: 5'-CGAATTGGAGCTCTGGTGATAGCCGCCGTATG-3'; The linearized vector for pBBR1MCS-2 is approximately 5000 bp in length. orf2179-2182 Because approximately 500 bp bases were added before and after (to ensure successful cloning of the promoter and ribosome binding site), the actual length is approximately 5300 bp. orf2179-2182 and pBBR1MCS-2 linearized vector PCR amplification as follows Figure 9 As shown, Figure 9 M: marker; Lanes 1 and 2: orf2179-2182 Amplification products; lanes 3 and 4: orf2179-2182 The corresponding linearized amplification product of the pBBR1MCS-2 vector. Figure 9 The amplified product bands were correctly positioned. After gel extraction and recovery, homologous arms were ligated using a seamless cloning enzyme.

[0052] Following the same seamless cloning method in step (3.2), the recombinant plasmid pBBR1MCS-2-EML was constructed and transformed into DH5α engineered bacteria to obtain the cloned strain DH5α-pBBR-EML.

[0053] Amplification and extraction of pBBR1MCS-2-EML plasmid: DH5α-pBBR-EML was inoculated into 3 mL LB and cultured overnight at 150 rpm and 30 °C. The cells were then collected by centrifugation at 12000 rpm for 5 min, and plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit (DP105).

[0054] The method for preparing DL-D2 bacterial electrocompetent cells is as follows: (a) Inoculate DL-D2 bacteria at a 1% inoculum in 50 mL LB and incubate at 37 °C with shaking at 150 rpm for 3-5 h until OD. 600 It is approximately 0.8.

[0055] (b) Incubate the culture medium in ice water for 30 min, then centrifuge at 5000 rpm for 10 min at low temperature, discard the supernatant and collect the bacterial cells.

[0056] (c) Add 3 mL of pre-sterilized and pre-cooled ultrapure water to the tip of a pipette and slowly and gently resuspend the bacterial cells. Then bring the volume up to about 20 mL and wash the bacterial cells again by low-temperature centrifugation.

[0057] (d) Repeat (c) twice.

[0058] (e) Add pre-sterilized and pre-cooled 10% glycerol to resuspend the bacterial cells. Centrifuge at low temperature, discard the supernatant, and finally add 2 mL of 10% glycerol to resuspend the bacterial cells. Aliquot the bacterial cells into 90 μL vials of electrocompetent DL-D2 bacteria and store them in a -80 ℃ freezer for later use.

[0059] Construction of DL-pBBR-EML expression strain: One vial of DL-D2 electroporation competent cells was slowly thawed on ice. After complete thawing, 10 μL of pBBR1MCS-2-EML plasmid was added to the DL-D2 electroporation competent cells, gently mixed to avoid generating air bubbles, and then transferred to a pre-chilled 1 mm gap electroporation cuvette. Electroporation was performed using a Gene Pluser Xcell (BIO-RAD) electroporator with the following parameters set: voltage 1.8 kV, resistance 200 Ω, and capacitance 25 μF. Electroporation efficiency was best when the electroporation time was maintained between 4 ms and 5 ms. Immediately after electroporation, 900 μL of liquid LB medium was added, and the cells were incubated at 150 rpm for 1 hour at 37 ℃. The cells were then collected by centrifugation at 5000 rpm for 5 min. Approximately 900 μL of supernatant was discarded, and the remaining supernatant was used to remix the cells. 100 μL of the remixed supernatant was plated on a plate containing 50 μg / mL kanamycin. After incubating upside down at 37 ℃ for 1 day, single clones were selected for colony PCR verification. After overnight incubation, 750 μL of the culture medium was mixed with 250 μL of 80% glycerol and stored at -80 ℃ for later use; this is the DL-pBBR-EML expression strain.

[0060] Functional validation of DL-pBBR-EML expression strains: pBBR1MCS-2 is constitutively expressed, and a large amount of the target protein cannot be obtained through induction; only prolonged culture time can yield sufficient protein. The DL-pBBR-EML recombinant strain was first inoculated at a 1% inoculum in 100 mL LB broth and cultured at 30℃ and 150 rpm for 12–16 h. Then, the cells were centrifuged at 5000 rpm for 5 min, collected, and resuspended in 20 mL of sterile MSM containing 80 mg / L hymexazol. The cells were then cultured at 30℃ with shaking at 150 rpm for 10 h. 500 μL of the culture medium was taken, mixed thoroughly with 500 μL of methanol, centrifuged at 12000 rpm for 1 min, and the supernatant was collected. The sample was filtered through a 0.22 μm organic filter, and the degradation of hymexazol was detected by HPLC (same as the whole-cell catalysis method).

[0061] The results of liquid phase detection are as follows Figure 10As shown, the substrate peak that originally appeared at 7.5 min disappeared completely after 10 h of incubation, but a new peak appeared at 4.2 min, which is speculated to be the initial product of hymexazol degradation.

[0062] Example 2: orf2179-2182 On the recombinant expression vector of gene clusters orf2179 Gene, orf2180 Gene, orf2181 Genes and orf2182 The methods for gene knockout and functional verification are as follows: Add pBBR1MCS-2-EML plasmid orf2179-2182 In gene clusters orf2179 Gene, orf2180 Gene, orf2181 Genes and orf2182 The genes were knocked out individually and then re-electrotransfused into competent DL-D2 bacteria to verify whether the remaining genes could still function together.

[0063] After culturing the DH5α-pBBR-EML strain from Example 1, the pBBR1MCS-2-EML plasmid was extracted. Using this plasmid as a template and with the start and end points of the gene to be knocked out as forward and reverse starting points, seamless cloning primers were designed. A large number of linearized plasmids were obtained through PCR amplification. Finally, the homologous arms of the linearized plasmids were ligated using a seamless cloning enzyme, thus successfully knocking out the gene. The newly constructed plasmids were then electroporated into competent DL-D2 bacteria to obtain the gene knockout strains.

[0064] The primer sequences used to knock out each gene are as follows: orf2179 Gene knockout upstream primer pBBR-EMLΔ79F: 5'-GAGGAGACTTGACGTAAGAGTATTTCCTC-3' orf2179 Gene knockout downstream primer pBBR-EMLΔ79R: 5'-TCTTACGTCAAGTCTCCTCCATGTCACT-3' orf2180 Gene knockout upstream primer pBBR-EMLΔ80F: 5'-GGATCTAATCACGAGAAGGAACGTCTCC-3' orf2180 Gene knockout downstream primer pBBR-EMLΔ80R: 5'-CCTTCTCGTGATTAGATCCTCTTTCAAAAG-3' orf2181 Gene knockout upstream primer pBBR-EMLΔ81F: 5'-GACATGGTTTTCCTTATGCGCAATTGAA-3' orf2181 Gene knockout downstream primer pBBR-EMLΔ81R: 5'-CATAAGGAAAACCATGTCGCATCAGACA-3' orf2182 Gene knockout upstream primer pBBR-EMLΔ82F: 5'-CGGAACATGCTTTCGTAGAAAATTAGAAGC-3' orf2182 Gene knockout downstream primer pBBR-EMLΔ82R: 5'-TCTACGAAAGCATGTTCCGCCTGTCTCTC-3' PCR amplification was performed using the knockout primers described above to obtain linearized plasmid products. The amplification system and program were the same as those in Tables 1 and 2. The extension times for PCR amplification of the linearized plasmids pBBR-EMLΔ79, pBBR-EMLΔ80, pBBR-EMLΔ81, and pBBR-EMLΔ82 were 8 min, 9 min, 8 min 30 sec, and 9 min, respectively. After amplification, 50 μL of PCR product was mixed with 5 μL of 10× loading buffer and subjected to TAE electrophoresis on a 0.9% agarose gel. After EB staining, the PCR amplification quality was observed using a gel imaging system. The results are as follows: Figure 11 As shown, Figure 11 In the diagram, M stands for DNA marker; lanes 1 and 2 are... orf2179 Gene knockout linearized vector amplification products; lanes 3 and 4 are orf2180 Gene knockout linearized vector amplification products; lanes 5 and 6 are orf2181 Gene knockout linearized vector amplification products; lanes 7 and 8 are orf2182 Gene knockout linearized vector amplification product. Figure 11 The results showed that the band sizes of all PCR products were as expected. Subsequently, the four linearized vectors were recovered and homologous arms were ligated using a seamless cloning enzyme to restore their circular structures.

[0065] The PCR products were successfully recovered from the gel, and homologous arms of the DNA fragments were ligated using a seamless cloning kit (method as in Example 1). This yielded pBBR-EMLΔ79 (knockout). orf2179 (gene), pBBR-EMLΔ80 (knocked out) orf2180 (gene), pBBR-EMLΔ81 (knocked out) orf2181 (gene), pBBR-EMLΔ82 (knocked out) orf2182 (Gene) Circular plasmid.

[0066] Four circular plasmids were electroporated into DL-D2 bacteria to obtain strains DL-pBBR-EMLΔ79, DL-pBBR-EMLΔ80, DL-pBBR-EMLΔ81, and DL-pBBR-EMLΔ82 (method as in Example 1). The results of single-clone verification of the gene knockout expression strains are as follows... Figure 12 As shown, Figure 12 M: DNA marker; Lanes 1-4 show the PCR verification results for DL-pBBR-EMLΔ79 colonies; lanes 5-8 show the PCR verification results for DL-pBBR-EMLΔ80 colonies; lanes 9-12 show the PCR verification results for DL-pBBR-EMLΔ81 colonies; lanes 13-16 show the PCR verification results for DL-pBBR-EMLΔ82 colonies. Because there are non-coding regions among the four genes, the PCR amplification lengths are the lengths of the non-coding regions, which are 560 bp, 611 bp, 380 bp, and 527 bp, respectively. Figure 12 As can be seen, the size of each band is as expected, proving that four gene knockout expression strains have been successfully constructed.

[0067] Inoculate 1% of the culture medium into 100 mL LB medium and incubate at 30 °C and 150 rpm for 12–16 h. Then, centrifuge at 5000 rpm for 5 min, collect the cells, and resuspend them in 20 mL of sterile MSM containing 80 mg / L hymexazol. Incubate at 30 °C and 150 rpm for 10 h. Take 500 μL of the culture medium, add 500 μL of methanol, mix thoroughly, centrifuge at 12000 rpm for 1 min, and collect the supernatant. Filter the sample through a 0.22 μm organic filter and use HPLC to detect the degradation of hymexazol (same as the whole-cell catalysis method).

[0068] Liquid phase detection results as follows Figure 13 As shown, Figure 13 The strains corresponding to Figures (a), (b), (c), and (d) are DL-pBBR-EMLΔ79, DL-pBBR-EMLΔ80, DL-pBBR-EMLΔ81, and DL-pBBR-EMLΔ82, respectively. Figure 13 The display shows that when orf2179, orf2181, orf2182 When the gene was knocked out, the whole-cell post-catalytic liquid chromatography spectrum showed no change compared to the control. However, orf After the 2180 gene was knocked out, its liquid chromatography results were compared with those of DL-pBBR-EML whole-cell catalysis. Figure 10 Almost identical. To gain a more intuitive understanding of the results of functional verification after gene knockout, [further details are needed]. Figure 14 . Figure 13 and Figure 14Joint Explanation orf2180 The gene did not participate in the initial degradation process of hymexazol. On the other hand, it can be proven that... orf2179、 orf2181, orf2182 Genes are involved in the initial degradation of hymexazol, and none of them can be omitted.

[0069] In summary, genome sequencing revealed that the G+C content of EML bacteria was 64.06%, and it carried 7311 predicted genes, including multiple efflux pumps (such as MexAB-OprM) and antioxidant genes (such as...). sodA This may be related to its environmental adaptability. Transcriptome analysis revealed that in EML bacteria induced by hymexazol... orf2179-2183 Gene clusters were significantly upregulated (log2FC≥3), among which orf2181 (FAD-dependent oxidoreductase) orf2179 (Fe-S binding protein) and orf2182 (Ferredoxin) is presumed to be involved in the initial degradation. Heterologous expression experiments showed that expressing gene 2181 alone... E. coli No degradation activity was detected in BL21, and the protein existed in the form of inclusion bodies. However, the four genes ( orf2179-2182 Gene clusters) co-expressed in Cupriavidus sp. Following DL-D2, hymexazol was completely degraded, generating an intermediate product (HPLC detection of a new peak at 4.2 min). Gene knockout experiments confirmed that... orf2179 , orf2181 , orf2182 Genes essential for degradation, and orf2180 The activity was not affected after gene knockout.

[0070] The high G+C content (64.09%) of the genome may enhance gene expression under extreme conditions by stabilizing mRNA secondary structure, a characteristic widely found in actinomycetes.

[0071] orf2179-2182 Functional validation of the gene cluster revealed a multi-enzyme synergistic mechanism for the degradation of hymexazol: FAD-dependent oxidoreductase ( orf2181 ) may catalyze the hydroxylation or aldehyde formation of the isoxazole ring, Fe-S protein ( orf2179 ) and ferrolin ( orf2182 (Then) is responsible for electronic transmission. orf2181 The inactivity of isolated expression may indicate a need for cofactors (such as NADH) or transmembrane transporters, while the coenzyme supply system present in DL-D2, also belonging to the genus *Codonopsis*, may compensate for this deficiency. Gene knockout experiments confirmed this. orf2180 The gene is not essential for the degradation of hymexazol, but its conservation in the gene cluster and upregulation suggest that it may be involved in regulation or bind to cofactors.

[0072] Example 3: The DL-pBBR-EML recombinant bacteria constructed in Example 1 were used for the remediation of farmland soil contaminated with 0.2 wt‰ hymexazol. The method is as follows: After routine culture, the DL-pBBR-EML recombinant bacteria were obtained with a concentration of 10. 8 A liquid microbial agent (CFU / g) was applied evenly to soil samples contaminated with hymexazol at a concentration of 5% of the soil mass. The soil pH was adjusted to 7.0-7.5, the temperature controlled at 25-30℃, and the soil moisture maintained at 30-40%. After 7 days of incubation, the hymexazol content in the soil was measured. The hymexazol degradation rate was calculated using the following formula: Hymexazol degradation rate (%) = (Initial hymexazol content in soil - Hymexazol content in soil after remediation by recombinant bacteria) / Initial hymexazol content in soil × 100%; Tests showed that the recombinant bacteria could degrade hymexazol in the soil by up to 92.4%.

[0073] Example 4: The DL-pBBR-EML recombinant bacteria constructed in Example 1 were used for the remediation of water bodies contaminated with 200 mg / L hymexazol. The method is as follows: After routine culture, the DL-pBBR-EML recombinant bacteria were obtained with a concentration of 10. 9 A liquid bacterial agent at CFU / g was added to a water sample contaminated with hymexazol to control the initial bacterial concentration (OD) in the water sample. 600 =0.1, adjust the pH of the water to 7.0-7.5, control the temperature at 29-30 ℃, and incubate with aeration for 5 days. Then, measure the concentration of hymexazol in the water sample. Calculate the hymexazol degradation rate using the following formula: Hymexazol degradation rate (%) = (initial hymexazol concentration in water sample - hymexazol concentration in water sample after recombinant bacterial remedy) / initial hymexazol concentration in water sample × 100%; Tests showed that the recombinant bacteria could degrade hymexazol in water samples contaminated with hymexazol up to 95.7%.

Claims

1. A gene cluster for degrading hymexazol, characterized in that, The gene comprising the orf2179 gene with the DNA sequence shown in SEQ ID No. 1, the orf2181 gene with the DNA sequence shown in SEQ ID No. 3, and the orf2182 gene with the DNA sequence shown in SEQ ID No.

4.

2. The hymexazol degradation gene cluster according to claim 1, characterized in that, It also includes the orf2180 gene with the DNA sequence shown in SEQ ID No.

2.

3. An expression vector comprising the hymexazol degradation gene cluster of claim 1 or 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is a plasmid containing a molecular chaperone gene.

5. A recombinant bacterium comprising the expression vector of claim 3.

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria are bacteria of the genus *Codonopsis* or *Escherichia coli*.

7. The application of the recombinant bacteria according to claim 5 in the remediation of soil and / or water bodies contaminated with hymexazol.

8. The application according to claim 7, characterized in that, Includes the following steps: (1) Add the recombinant bacteria to the soil and / or water contaminated with hymexazol and mix well to obtain the material to be remediated; (2) Cultivate the materials to be remediated and complete the remediation of soil and / or water bodies.

9. The application according to claim 8, characterized in that, In step (1), the content of recombinant bacteria in the material to be repaired is at least 1 × 10⁻⁶. 6 CFU / g.

10. The application according to claim 8, characterized in that, In step (2), when the material to be remediated is a mixture of recombinant bacteria and contaminated soil, the culture conditions are as follows: adjust the pH of the material to be remediated to neutral, control the temperature at 25-30℃, maintain the humidity at 30-40%, and culture for at least 7 days; When the material to be remediated is a mixture of recombinant bacteria and polluted water, the culture conditions are as follows: adjust the pH of the material to be remediated to neutral, control the temperature of the material to be remediated to 29-30℃, and aerate for at least 5 days.

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