1-naphthoic acid and 2-naphthoic acid degradation gene cluster npa and use thereof

Abstract

The application discloses a 1-naphthalene carboxylic acid and 2-naphthalene carboxylic acid degradation gene cluster npa and application thereof. The application identifies a gene cluster related to 1-naphthalene carboxylic acid and 2-naphthalene carboxylic acid degradation from a strain M3 based on a random insertion mutation technology npa , and the gene cluster can realize effective degradation of 1-naphthalene carboxylic acid and 2-naphthalene carboxylic acid after heterologous expression, which indicates that the gene cluster plays a key role in the naphthalene carboxylic acid degradation process, and provides a theoretical basis and technical support for analyzing a naphthalene carboxylic acid degradation pathway and constructing an engineering bacterium for pollution remediation.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to 1-naphthoic acid and 2-naphthoic acid degradation gene clusters. npa And its applications. Background Technology

[0002] Naphthoic acids are an important class of naphthalene-based aromatic derivatives, mainly including 1-naphthoic acid (1-NPA) and 2-naphthoic acid (2-NPA). These compounds have wide industrial applications, serving as intermediates or raw materials in pharmaceuticals, pesticides, dyes, and fine chemicals. However, with the expansion of related industrial production and use, 1-naphthoic acid and 2-naphthoic acid can enter the environment through industrial wastewater discharge, production process leaks, and improper disposal, accumulating in water bodies, soil, and sediments, posing potential risks to the ecological environment. Existing studies have shown that 1-naphthoic acid and 2-naphthoic acid possess certain environmental persistence and ecotoxicity, potentially adversely affecting microbial community structure, aquatic organisms, and soil ecosystems. Furthermore, these compounds may undergo further transformations in the environment, generating derivatives or metabolites with even higher toxicity, thus exacerbating their environmental harm. Therefore, research on pollution control and remediation technologies for 1-naphthoic acid and 2-naphthoic acid is of great significance.

[0003] Currently, the main remediation methods for 1-naphthoic acid and 2-naphthoic acid include physical, chemical, and biological methods. While physical and chemical methods can remove these pollutants to some extent, they often suffer from high costs, low efficiency, and the potential for secondary pollution. In contrast, biological methods, due to their high efficiency, environmental friendliness, and sustainability, are considered an important research direction for the remediation of aromatic organic pollutants.

[0004] However, current research on the degrading strains, functional genes, and molecular mechanisms of 1-naphthoic acid and 2-naphthoic acid remains limited, especially regarding the scarcity of functional gene clusters related to key steps in their degradation initiation. Therefore, screening microbial resources capable of efficiently degrading 1-naphthoic acid and 2-naphthoic acid, identifying their degradation-related functional genes, and elucidating their degradation mechanisms are of significant value for improving the theoretical framework of naphthoic acid-related pollutant biodegradation and developing efficient environmental remediation technologies. Summary of the Invention

[0005] The purpose of this invention is to provide gene clusters for the degradation of 1-naphthoic acid and 2-naphthoic acid. npa And its applications, to address the shortcomings of existing technologies.

[0006] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a 1-naphthoic acid and 2-naphthoic acid degradation gene cluster. npa Its nucleotide sequence is shown in SEQ ID NO.6.

[0007] A second aspect of the present invention provides a recombinant vector containing the aforementioned 1-naphthoic acid and 2-naphthoic acid degradation gene clusters. npa .

[0008] A third aspect of the present invention provides a recombinant microorganism containing the above-described recombinant vector.

[0009] The fourth aspect of this invention provides the above-mentioned 1-naphthoic acid and 2-naphthoic acid degradation gene clusters. npa Application in the degradation of 1-naphthoic acid and 2-naphthoic acid.

[0010] The fifth aspect of the present invention provides the application of the above-mentioned recombinant carrier in the degradation of 1-naphthoic acid and 2-naphthoic acid.

[0011] The sixth aspect of the present invention provides the application of the above-mentioned recombinant microorganisms in the degradation of 1-naphthoic acid and 2-naphthoic acid.

[0012] The beneficial effects of this invention are: This invention, based on random insertion mutation technology, identifies gene clusters in strain M3 associated with the degradation of 1-naphthoic acid and 2-naphthoic acid. npa After heterologous expression, the gene cluster can effectively degrade 1-naphthoic acid and 2-naphthoic acid, indicating that the gene cluster plays a key role in the degradation of naphthoic acid. This provides a theoretical basis and technical support for elucidating the degradation pathway of naphthoic acid and constructing engineered bacteria for pollution remediation. Attached Figure Description

[0013] Figure 1 The degradation and growth curves of 1-naphthoic acid by strain M3 are shown.

[0014] Figure 2 This is a liquid chromatogram of strain M3 degrading 1-naphthoic acid.

[0015] Figure 3 To screen mutant strains that have lost the ability to use 1-naphthoic acid as the sole carbon source using a photocopy plate method, A shows the growth on MSM solid plates containing a final concentration of 0.6 mM 1-naphthoic acid, and B shows the growth on LB double antibody plates containing 50 mg / L Gm and 30 mg / L Cm.

[0016] Figure 4 Naphthoic acid degradation gene cluster npa A diagram of heterogeneous expression strategies.

[0017] Figure 5 The recombinant strain KT2440(pBBR- npa Degradation curves of 1-naphthoic acid and 2-naphthoic acid.

[0018] Figure 6 The recombinant strain KT2440(pBBR- npa ) Liquid chromatogram of degradation of 1-naphthoic acid and 2-naphthoic acid, A is recombinant strain KT2440 (pBBR- npa ) Liquid chromatogram of 1-naphthoic acid degradation, B is recombinant strain KT2440 (pBBR- npa Liquid chromatogram of degradation of 2-naphthoic acid. Detailed Implementation

[0019] The present invention will be further explained below with reference to the embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] The culture medium formulations involved in the following examples are as follows: The inorganic salt liquid medium (MSM medium) has the following formulation for 1 L: NaCl 1.0 g, NH4Cl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, and ultrapure water to a final volume of 1 L. Sterilize at 121℃ for 20 min. The pH is 7.0. The inorganic salt solid medium (MSM solid medium) is prepared by adding 18 g of agar powder to this formulation.

[0021] LB liquid medium, 1L formulation: NaCl 5.0 g, yeast extract 5.0 g, tryptone 10.0 g, ultrapure water to 1 L, sterilized at 121℃ for 20 min. pH 7.0. LB solid medium is prepared by adding 18 g agar powder to this formulation.

[0022] Example 1: Degradation experiment of 1-naphthoic acid by strain M3 Strain M3 (strain M3 disclosed in CN 118291328 B) was inoculated into LB liquid medium and cultured with shaking at 30℃ and 180 rpm until mid-log phase. The culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected and washed twice with MSM medium. The washed bacterial cells were then resuspended in MSM medium to obtain the M3 seed culture. The M3 seed culture was then inoculated at the initial inoculum amount OD. 600Approximately 0.1 μL was inoculated into an Erlenmeyer flask containing 20 mL of MSM medium. The medium was then inoculated with 1-naphthoic acid to a final concentration of 0.2 mM, serving as the experimental group (M3 + 1-naphthoic acid group). MSM medium containing only the same final concentration of 1-naphthoic acid but without inoculation with strain M3 served as the control group (1-naphthoic acid group). Each treatment group was divided into triplicates. All treatment groups were cultured at 30°C and 180 rpm with shaking. 1 mL samples were collected at 0, 8, 16, 24, 48, and 72 h. Cell growth was measured using a UV spectrophotometer, and 1-naphthoic acid was quantitatively analyzed by HPLC. The specific procedures are as follows: The sample pretreatment method is as follows: Take 500 µL of sample, add an equal volume of methanol, mix well, centrifuge at 12000 rpm for 5 min, filter the supernatant through a 0.22 μm filter membrane, and then perform HPLC analysis. The chromatographic conditions for HPLC detection of 1-naphthoic acid concentration are as follows: Column: Agilent ZORBAX SB C18 (250 mm × 4.6 mm × 5 μm); Mobile phase: methanol: 0.5 v / v% acetic acid / water (v:v = 75:25); Flow rate: 1.0 mL / min. -1 The detection wavelength was 230 nm; the column temperature was 35°C; and the injection volume was 20 μL. 500 µL of sample was taken and detected using a UV spectrophotometer at a detection wavelength of 600 nm.

[0023] The results are as follows Figure 1 and Figure 2 As shown, strain M3 can degrade 0.2 mM 1-naphthoic acid within 72 h. At the same time, the biomass of strain M3 increased from about 0.1 to about 0.15, indicating that strain M3 can utilize 1-naphthoic acid as the sole carbon and energy source for growth.

[0024] Degradation of 2-naphthoic acid by strain M3 is described in CN 118291328 B.

[0025] Example 2 Identification of key naphthoic acid degradation genes in strain M3 2.1 Construction of transposon mutant libraries To screen key genes involved in naphthoic acid degradation, a transposon mutant library of strain M3 was constructed using the random insertion plasmid pSC137 (reference: Wang Y, Qian G, Li Y, et al. Biosynthetic mechanism for sunscreens of the biocontrol agent Lysobacter enzymogenes[J]. PLoS One, 2013,8(6): e66633.). The specific steps are as follows: Fresh strain M3 and helper strains were selected separately. E. coli HB101 (pRK2013) and donor bacteria E. coli Single colonies of DH5α (pSC137) were inoculated into liquid LB medium containing 50 mg / L gentamicin (Gm), 50 mg / L kanamycin (Km), and 30 mg / L chloramphenicol (Cm), respectively. After inoculation, strain M3 was cultured at 30°C and 180 rpm to the logarithmic growth phase. E. coli HB101 (pRK2013) E. coli DH5α (pSC137) was cultured to the logarithmic growth phase at 37℃ and 180 rpm. 1 mL of each of the above cultures was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with MSM medium. The washed cells from each of the three strains were resuspended in the same centrifuge tube with MSM medium, mixed thoroughly, and 100 μL was added dropwise to a sterilized filter membrane placed on the surface of antibiotic-free LB solid medium. The mixture was incubated at 30℃ for 24 h. After incubation, the cells on the filter membrane were washed off with 1 mL of MSM medium, and serial dilutions were performed. 100 μL of the appropriately diluted bacterial solution was spread onto LB agar plates containing 50 mg / L Gm and 30 mg / L Cm, and incubated at 30℃ for 36–48 h. The colonies growing on the LB agar plates were identified as the transposon mutant strain M3.

[0026] 2.2 Screening of mutants lacking 1-naphthoic acid degradation ability After single bacteria have grown on LB agar plates, mutant strains that have lost the ability to use 1-naphthoic acid as the sole carbon source are screened using a replica plate method. The specific steps are as follows: LB agar plates containing 50 mg / L Gm and 30 mg / L Cm were prepared, as well as MSM solid plates containing 0.6 mM 1-naphthoic acid (using 1-naphthoic acid as the sole carbon source). Single colonies were picked from the LB agar plates using sterile toothpicks and inoculated onto corresponding positions on the MSM solid plates and LB agar plates, respectively, and incubated at 30°C for 36-48 h. By comparing the growth on the two plates, bacteria that could grow on the LB agar plates were screened. Figure 3 (B) strains, but strains that cannot grow on MSM solid plates with 1-naphthoic acid as the sole carbon source. Figure 3 (A) was selected as a candidate 1-naphthoic acid degradation deletion mutant. The selected candidate 1-naphthoic acid degradation deletion mutants were further purified by streaking on LB agar plates, and their degradation ability for 1-naphthoic acid was further verified using the same method as in Example 1. The 1-naphthoic acid degradation deletion mutant M3-RI was successfully obtained through screening.

[0027] 2.3 Transposon insertion site analysis and gene cluster localization Genomic DNA was extracted from the 1-naphthoic acid degradation deletion mutant M3-RI, and the sequences flanking the transposon insertion site were amplified and analyzed using SEFA-PCR (Self-Formed Adaptor PCR). Primers SP1, SP2, and SP3 were designed based on the transposon sequence on the random insertion plasmid pSC137, and their sequences are as follows: SP1: GTGGCCAATATGGACAACTTCTTCGCCCCC (SEQ ID NO.1); SP2: GGCGACAAGGTGCTGATGCCGC (SEQ ID NO. 2); SP3: CCGTTTGTGATGGCTNNNNNNNNNGCAGAT (SEQ ID NO.3); The SEFA-PCR amplification process involves two rounds of consecutive amplification reactions.

[0028] The first round of SEFA-PCR amplification used SP1 and SP3 as primers. The amplification system (50 μL) consisted of: 25 μL 2×phanta master mix, 1 μL DNA template, 2 μL primer SP3, and 22 μL ddH2O. The PCR amplification program was as follows: 95℃ for 5 min, 95℃ for 30 s, 40℃ for 3 min, ramping up to 70℃ at 0.2℃ / s, 72℃ for 5 min, and 10℃ for 5 min. After amplification, 2 μL of SP1 was added to the PCR tube as primer, and PCR was continued using the following program: 95℃ for 30 s, 70℃ for 30 s, 72℃ for 5 min, repeated 25 times. The PCR products were then subjected to 8 rounds of asymmetric PCR amplification, with each round using the following program: 95℃ for 30 s, 70℃ for 30 s, 72℃ for 5 min, 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 5 min. The reaction was then stopped at 72℃ for 5 min and 10℃ for 5 min.

[0029] SEFA-PCR second round amplification: SP2 was used as the primer. The amplification system (50 μL) consisted of: 25 μL 2×phantamaster mix, 1 μL of the previous round PCR product, 2 μL of primer SP2, and 22 μL ddH2O. The PCR amplification program was as follows: Stage 1, 95℃ for 5 min; Stage 2, 95℃ for 30 s, 70℃ for 30 s, 72℃ for 5 min, repeated 20 times; Stage 3, 72℃ for 5 min, 10℃ for 5 min.

[0030] The obtained PCR products were sequenced and compared with the whole genome sequence of wild-type strain M3 to identify the gene region inactivated by transposon insertion. The results showed that the insertion site was located in a potential functional gene cluster related to naphthoic acid degradation, and this gene cluster was named... npa ( Figure 4 ), npa The nucleotide sequence is shown in SEQ ID NO. 6.

[0031] Example 3 Naphthoic acid degradation gene cluster npa Cloning and functional verification 3.1 Naphthoic acid degradation gene cluster npa Cloning Primers were designed based on the M3 genomic sequence of strain M3 to target the naphthoic acid degradation gene cluster. npa Perform PCR amplification. Use primers pBBR1- npa _F and pBBR1- npa _R amplification of naphthoic acid degradation gene cluster npa Fragment (SEQ ID NO. 6).

[0032] Forward primer pBBR- npa _F: AAAAGCTGGGTACCGGGCCCGAACCGTCCTCCAGAGTTCTTAATGTT (SEQ ID NO. 4); Reverse primer pBBR- npa _R: TATAGGGCGAATTGGAGCTCTCAGACGCTGTAAATGTC (SEQ ID NO. 5).

[0033] PCR amplification program: Stage 1, pre-denaturation at 95℃ for 3 min; Stage 2, denaturation at 95℃ for 15 s, annealing at 57℃ for 15 s, extension at 72℃ for 3 min, 30 cycles; Stage 3, final extension at 72℃ for 5 min.

[0034] The pBBR1MCS-2 plasmid was digested with restriction endonucleases Apa I and Sac I at 30℃ for 2 h. Then, the PCR-amplified gene fragment was ligated into the linearized plasmid pBBR1MCS-2 using homologous recombination to obtain the recombinant plasmid pBBR- npa The recombinant plasmid pBBR- npa Transform to E. coli In DH5α, positive clones (recombinant expression strains) were selected. E. coli DH5α(pBBR- npa Then with helper strains E. coli HB101(pRK600) was recombined with the plasmid pBBR- using a triparental conjugation method. npa Import to Pseudomonas putida KT2440 (i.e. strain KT2440) was used to obtain a heterologous expression strain, namely the recombinant strain KT2440(pBBR- npa (See process) Figure 4 ).

[0035] 3.2 Naphthoic acid degradation gene cluster npa Functional verification To verify the naphtholic acid degradation gene cluster npa The recombinant strain KT2440 (pBBR-) exhibits enhanced degradation capabilities for 1-naphthoic acid and 2-naphthoic acid. npa Inoculate into LB liquid medium and culture with shaking at 30°C and 180 rpm until the logarithmic growth phase, then inoculate according to the initial inoculum size OD. 600Approximately 1.0 μL was inoculated into an Erlenmeyer flask containing 20 mL of MSM medium. 1-Naphthoic acid or 2-naphthoic acid was added to the medium to a final concentration of 0.2 mM as a substrate (denoted as KT2440(pBBR-...). npa )+1-naphthoic acid group, KT2440(pBBR- npa The KT2440+1-naphthoic acid group and the KT2440+2-naphthoic acid group were used as control groups, respectively, and strain KT2440 was inoculated under the same conditions. Each treatment group was replicated in triplicate. Each treatment group was cultured at 30℃ and 180 rpm with shaking, and samples were taken at 0, 1, 2, 3, 4, 6, 8 and 10 h. The substrate residue concentration of the samples was determined by HPLC, and the detection method was the same as in Example 1.

[0036] The results are as follows Figure 5 and Figure 6 As shown, the retention time for 1-naphthoic acid was 6.1 min, and the retention time for 2-naphthoic acid was 6.8 min. Recombinant strain KT2440 (pBBR- npa It can degrade approximately 0.16 mM of 1-naphthoic acid within 8 hours, approximately 0.12 mM of 2-naphthoic acid within 3 hours, and approximately 0.2 mM of 2-naphthoic acid within 6 hours. These results indicate that the gene cluster... npa Conferring host bacteria the ability to degrade 1-naphthoic acid and 2-naphthoic acid is a key gene involved in the metabolism of these compounds.

Claims

1. A gene cluster for the degradation of 1-naphthoic acid and 2-naphthoic acid npa Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

6.

2. A recombinant vector, characterized in that, It contains the 1-naphthoic acid and 2-naphthoic acid degradation gene clusters as described in claim 1. npa .

3. A recombinant microorganism, characterized in that, It contains the recombinant vector as described in claim 2.

4. The 1-naphthoic acid and 2-naphthoic acid degradation gene clusters as described in claim 1 npa Application in the degradation of 1-naphthoic acid and 2-naphthoic acid.

5. The application of the recombinant carrier according to claim 2 in the degradation of 1-naphthoic acid and 2-naphthoic acid.

6. The application of the recombinant microorganisms according to claim 3 in the degradation of 1-naphthoic acid and 2-naphthoic acid.

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