Construction of nitroreductase delivery plasmid and application of nitroreductase delivery plasmid in environmental restoration

Abstract

The invention belongs to the technical field of genetic engineering and environmental remediation, and particularly discloses construction of nitroreductase delivery plasmids and application of the nitroreductase delivery plasmids in environmental remediation. The nucleotide and amino acid sequences of the nitroreductase gene derived from the actinomycetes S16 functional metagenome are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The method comprises the following steps: designing a primer by taking pQ-SacB-araC plasmid as a template, and carrying out PCR (Polymerase Chain Reaction); the method comprises the following steps: cloning an actinomycete S16 functional metagenome as a template, and designing a primer according to a target nitroreductase gene sequence to carry out PCR (Polymerase Chain Reaction) amplification; and purifying and recovering the two amplification products, and carrying out fragment recombination to obtain the nitroreductase delivery plasmid pQ-NR. The delivery plasmid pQ-NR and recombinant bacteria capable of stably and efficiently expressing nitroreductase are constructed, efficient delivery of nitroreductase genes in various microorganisms is achieved, and the application effect of the nitroreductase genes in actual environmental remediation related to nitro compound pollution is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and environmental remediation, and in particular relates to the construction of a nitroreductase delivery plasmid and its application in environmental remediation. Background Art

[0002] In the modern industrial process, nitro compounds are widely used in many fields such as chemical industry, medicine, pesticides, etc. However, these compounds enter the environment in large quantities during the production, use and discharge process, posing a serious threat to the ecosystem and human health. Nitro compounds are highly toxic, potentially carcinogenic and difficult to biodegrade. Their long-term accumulation in the environment not only destroys the ecological balance, but may also be transmitted through the food chain and ultimately endanger human health. Traditional environmental remediation methods, such as physical adsorption and chemical oxidation, can deal with nitro compound pollution to a certain extent, but they have disadvantages such as high cost, complex operation, and easy to produce secondary pollution. Bioremediation technology has become a research hotspot for solving the problem of nitro compound pollution because of its environmentally friendly, efficient and economical characteristics.

[0003] Nitroreductase (NR), a key biocatalyst that reduces nitro compounds to less toxic or more easily degradable products, plays a central role in the bioremediation of nitro-compound contamination. However, the expression levels of endogenous nitroreductase in natural microorganisms are generally low, making it difficult to meet the needs of actual environmental remediation. Therefore, constructing a delivery plasmid that efficiently expresses nitroreductase and introducing it into a suitable host bacteria to increase the expression and activity of nitroreductase is of vital importance for enhancing bioremediation effectiveness.

[0004] Currently, there have been some studies on the use of genetic engineering technology to construct engineered bacteria for the degradation of environmental pollutants, but there are still many deficiencies in the construction of nitroreductase delivery plasmids. For example, some existing plasmids have problems such as structural instability, low copy number in the host bacteria, and imprecise regulation of enzyme expression, resulting in unsatisfactory results in actual environmental remediation applications. In addition, how to optimize the combination of plasmid elements to achieve efficient and stable expression of nitroreductase, and how to ensure that the plasmid is effectively delivered and maintains its function under different environmental conditions, are still technical challenges that need to be overcome. Therefore, the construction of a delivery plasmid that can stably express nitroreductase has important application value. Summary of the Invention

[0005] In response to the shortcomings of the current existing technology, the present invention provides a nitroreductase delivery plasmid for construction and its application in environmental remediation, which realizes the efficient delivery of nitroreductase genes in various microorganisms and ensures the efficient and stable expression of nitroreductase, thereby improving its effect in actual environmental remediation applications.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] A first object of the present application is to provide a nitroreductase, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0008] A second object of the present application is to provide a gene encoding the above-mentioned nitroreductase, the nucleotide sequence of which is shown as SEQ ID NO. 1.

[0009] A third object of the present application is to provide a nitroreductase delivery plasmid pQ-NR, which carries the nucleotide sequence of the above-mentioned nitroreductase gene.

[0010] A fourth object of the present application is to provide a construction method of the nitroreductase delivery plasmid pQ-NR, which specifically comprises the following steps:

[0011] S1, obtaining an IncQ plasmid backbone: designing primers for PCR amplification with pQ-SacB-araC plasmid as a template to obtain an amplification product;

[0012] S2, obtaining a nitroreductase encoding gene: cloning from actinomycete S16 functional metagenome as a template for amplifying the nitroreductase gene, and designing specific primers according to the sequence of the target nitroreductase gene for PCR amplification to obtain an amplification product, ensuring that the encoded enzyme has good catalytic activity and substrate specificity;

[0013] S3, fragment recombination: electrophoretically purifying and recovering the above two amplification products to obtain the nitroreductase delivery plasmid pQ-NR through fragment recombination.

[0014] Further, the primer sequence of step S1 is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

[0015] Further, the primer sequence of step S2 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

[0016] Further, the electrophoresis of step S3 is agarose gel electrophoresis.

[0017] Further, the fragment recombination of step S3 is performed using a homologous recombination kit, preferably a homologous recombination kit One Step Cloning Kit from Nanjing Novizen Biological Technology Co., Ltd.

[0018] Further, the nucleotide sequence of the nitroreductase delivery plasmid pQ-NR is shown as SEQ ID NO. 7.

[0019] The fifth object of the present invention is to provide a nitroreductase recombinant bacterium, which is produced by transforming the delivery plasmid pQ-NR into Escherichia coli Dh5α competent cells, resuscitating them, and plating them on a culture medium containing ampicillin; picking single colonies from the culture medium for PCR sequencing verification, and screening positive transformants, which are recombinant bacteria containing the nitroreductase encoding gene.

[0020] The sixth object of the present invention is to provide a use of the above-mentioned delivery plasmid or recombinant bacteria in improving the activity of nitroreductase.

[0021] The seventh object of the present invention is to provide the use of the above-mentioned recombinant bacteria in the remediation of environments contaminated by nitro compounds.

[0022] The present invention also provides a method for preparing nitroreductase, which comprises culturing the above-mentioned nitroreductase recombinant bacteria containing pQ-NR and separating and purifying the nitroreductase from the culture.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Significantly increase the expression of nitroreductase: The delivery plasmid constructed by the present invention can effectively enhance the expression level of nitroreductase in microorganisms by optimizing elements such as promoters and gene sequences, thereby improving the degradation efficiency of nitro compounds and accelerating the environmental remediation process.

[0025] (2) Broad host range: The vector of the present invention is based on an IncQ-type plasmid and has a broad host range, encompassing not only different types of Gram-negative bacteria but also some Gram-positive bacteria such as Actinobacteria and Firmicutes. The rep region-related genes on the vector plasmid can perform replication functions in these strains, enabling gene delivery to a variety of host bacteria, from Gram-negative to Gram-positive bacteria, and is suitable for microorganisms in different environments.

[0026] (3) Environmentally friendly and cost-effective: Compared with traditional environmental remediation methods, the genetically engineered bacteria constructed based on the plasmid of the present invention have the advantages of low cost and no secondary pollution in the process of nitro compound pollution remediation, providing a sustainable solution for large-scale environmental remediation.

[0027] (4) Wide range of applications: This plasmid construction method has certain versatility. According to different nitro compound pollution types and environmental conditions, appropriate nitroreductase genes can be selected for construction. It is suitable for the remediation of various nitro compound-contaminated environments, such as soil, water, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Electrophoresis diagrams for the construction verification of the delivery plasmid pQ-NR of the present invention: 1: pQ-NR; 2: pQ-vector. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0031] Example 1

[0032] The construction of a nitroreductase delivery plasmid pQ-NR based on an IncQ-type plasmid comprises the following steps:

[0033] (1) Using the purchased pQ-SacB-araC plasmid as a template, primers were designed for PCR amplification. The primer sequences were rigorously screened and optimized to determine primers F1 and R1, in order to meet the needs of subsequent experiments and ensure the accuracy and efficiency of amplification. The amplification system and conditions are shown in Tables 1 and 2. After this precise process, an amplified product of 10,490 bp was successfully obtained. The mob region and rep region retained their natural functional characteristics, providing a solid structural support for the autonomous replication and transferability of the plasmid in the host bacteria.

[0034] The pQ-SacB-araC plasmid can be constructed according to conventional methods: using pQ-mini (the plasmid constructed earlier can refer to patent document CN 115109791 A) as the backbone, isolating the SacB gene from Bacillus subtilis as a negative selection marker, isolating the araC arabinose operon regulatory gene from Escherichia coli, and finally performing homologous recombination to form the pQ-SacB-araC plasmid.

[0035] (2) Obtaining and cloning the nitroreductase gene: The nitroreductase gene was cloned from the functional metagenome of actinomycete S16 as a template for amplifying the nitroreductase gene. Specific primers F2 and R2 were designed based on the sequence of the target nitroreductase gene (nucleotide sequence shown in SEQ ID NO. 1), and high-fidelity PCR amplification was performed. The amplification system and conditions are shown in Tables 1 and 2, and an amplified product was obtained. The size of the amplified product was 722 bp. The nitroreductase encoded by this gene (amino acid sequence shown in SEQ ID NO: 2) has good catalytic activity and substrate specificity.

[0036] (3) The two amplified products were subjected to agarose gel electrophoresis, purified and recovered, and then fragments were recombined using a homologous recombination kit to obtain the pQ-NR plasmid. The kit is preferably the One Step Cloning Kit of Nanjing Novozymes Biotechnology Co., Ltd., and then transformed into E. coli Dh5α competent cells. After recovery for a certain period of time, the cells were spread on LB agar plates containing ampicillin at a final concentration of 100 μg / mL;

[0037] The agarose gel electrophoresis method includes preparing a 1.0% TAE agarose gel, then mixing the PCR amplification product using 10X loading, spotting all the products for electrophoresis, and performing electrophoresis at 100V, 88mA, and 40min. The purification and recovery is performed using a gel recovery and purification kit (Takara). The homologous recombination system configuration is shown in Table 3. The homologous recombination reaction conditions are preferably: after preparation on ice, gently flicking to mix, brief centrifugation, and reaction at 50°C for 30min.

[0038] (4) Transformation and screening of recombinant plasmid pQ-NR:

[0039] Under sterile conditions, add 300–500 ng of the ligation product to 100 μL of E.coli Dh5α competent cells. Gently shake the tube and place on ice for 30 minutes to allow the plasmid DNA to fully adsorb to the competent cell surface. Then, heat shock the tube for 60 seconds in a 42°C water bath. After the heat shock, quickly cool the tube on ice for 5 minutes to allow the pores in the cell membrane to reclose and minimize cell death. Add 1 mL of LB liquid medium to the tube, pipette to mix thoroughly, and incubate the tube at 37°C, shaking at 220 rpm for 1 hour to allow the cells to recover and express the resistance gene.

[0040] An appropriate amount of the revived bacterial liquid was spread on LB solid medium containing ampicillin (final concentration of 100 μg / mL) and cultured at 37°C overnight to allow colonies containing the target plasmid to grow stably and form.

[0041] (5) Identification and verification of recombinant plasmid pQ-NR

[0042] Single colonies were picked from the LB agar plates obtained through culture and verified by colony PCR using the designed primers F3 and R3. The reaction system remained the same as the previous amplification step. Through this verification process, positive clones whose amplified bands were consistent with the expected ones were screened out and sent to a professional sequencing company for high-precision sequencing analysis. Only when the sequencing results were completely consistent with the sequence of the target nitroreductase gene could it be determined that the recombinant plasmid construction was accurate and correct, and the nitroreductase delivery vector pQ-NR (nucleotide sequence shown in SEQ ID NO.7) was successfully obtained. The electrophoresis diagram of the construction verification of the delivery plasmid pQ-NR is shown in Figure 1 , laying a solid and reliable material foundation for subsequent extensive application and in-depth research.

[0043] At this point, the nitroreductase delivery plasmid pQ-NR based on IncQ-type plasmid was successfully constructed.

[0044] Primer F1 sequence (SEQ ID NO. 3): 5`-ATGGAGAAACAGTAGAGAGTTGC-3`;

[0045] Primer R1 sequence (SEQ ID NO. 4): 5′-ATCATTGCTTTAGTAAAACGAAGAGG-3′;

[0046] Primer F2 sequence (SEQ ID NO. 5): 5′-TCGCAACTCTCTACTGTTTCTCCATCGGATGAATAAGTATGATGTAAGCTG-3′;

[0047] Primer R2 sequence (SEQ ID NO. 6): 5′-CTCTTCGTTTTACTAAAGCAATGATGAGGCCCTTATTTCTAAAATGTTTCTG-3′;

[0048] Primer F3 sequence: 5`-CGATTCATTAATGCAGCTGGC-3`;

[0049] Primer R3 sequence: 5`-GGATCATTTTGCGCTTCAGC-3`.

[0050] Table 1 High-fidelity PCR reaction system

[0051]

[0052] Table 2 Reaction conditions for high-fidelity enzyme PCR

[0053]

[0054] Table 3 Homologous recombination system

[0055]

[0056] Example 2 Nitroreductase activity determination comprises the following steps:

[0057] (1) Cultivation of engineered bacteria and preparation of crude enzyme solution

[0058] The engineered E. coli Dh5α strain containing pQ-NR was inoculated into LB liquid medium (containing ampicillin) and cultured at 37°C, 200 rpm, and shaken until the logarithmic growth phase. 10 mL of the bacterial suspension was collected by centrifugation at 8000 rpm for 5 minutes at 4°C. The cells were resuspended in PBS buffer and disrupted by ultrasonication in an ice bath (ultrasonication power of 250 W, 3 seconds on-time, 3 seconds off-time, total ultrasonication time of 10 minutes). The disrupted cell suspension was centrifuged at 12000 rpm for 3 minutes at 4°C. The supernatant was used as the crude enzyme solution for enzyme activity determination.

[0059] (2) Enzyme activity determination method: The activity of nitroreductase in the crude enzyme solution was determined by spectrophotometry. An appropriate amount of p-nitrobenzoic acid and the crude enzyme solution were added to the reaction system. The absorbance change was monitored in real time at a wavelength of 340 nm. The absorbance values ​​at different time points were recorded. A curve of the absorbance change over time was plotted to calculate the nitroreductase activity.

[0060] Obviously, the specific implementation scheme described above is only a further detailed description of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nitroreductase, characterized in that The amino acid sequence of the nitroreductase is shown in SEQ ID NO.

2.

2. The gene encoding the nitroreductase according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A nitroreductase delivery plasmid pQ-NR, characterized in that The plasmid carries the nucleotide sequence of the nitroreductase gene according to claim 2.

4. The method for constructing a nitroreductase delivery plasmid pQ-NR according to claim 3, characterized in that: The steps include: S1. Using the pQ-SacB-araC plasmid as a template, primers were designed and PCR was performed to obtain the amplified product. S2. Clone the functional metagenome of actinomycete S16 as a template for amplifying the nitroreductase gene, design specific primers based on the target nitroreductase gene sequence, and perform PCR amplification to obtain the amplified product; S3. Purify and recover the two amplification products described in steps S1 and S2 by electrophoresis, and reassemble the fragments to obtain the nitroreductase delivery plasmid pQ-NR.

5. The method for constructing a nitroreductase delivery plasmid pQ-NR according to claim 4, characterized in that: The primer sequences in step S1 are shown as SEQ ID NO.3 and SEQ ID NO.

4.

6. The method for constructing a nitroreductase delivery plasmid pQ-NR according to claim 4, characterized in that: The primer sequences in step S2 are shown as SEQ ID NO.5 and SEQ ID NO.

6.

7. The method for constructing a nitroreductase delivery plasmid pQ-NR according to claim 4, characterized in that: The nucleotide sequence of the delivery plasmid pQ-NR is shown in SEQ ID NO.

7.

8. A nitroreductase recombinant bacterium, characterized in that: The recombinant bacteria are prepared by transforming the delivery plasmid pQ-NR according to claim 3 into Escherichia coli Dh5α competent cells, resuscitating the cells, and plating the cells on a culture medium containing ampicillin; a single colony is picked from the culture medium for PCR sequencing verification, and the positive transformants are screened as nitroreductase recombinant bacteria.

9. Use of the delivery plasmid pQ-NR according to claim 3 or the recombinant bacterium according to claim 8 in improving nitroreductase activity.

10. A method for preparing nitroreductase, characterized in that: The nitroreductase recombinant bacteria according to claim 8 are cultured and separated and purified from the culture.

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