Construction method and application of engineering shewanella for degrading nicotine wastewater

Abstract

The invention discloses a construction method and application of engineering shewanella for degrading nicotine wastewater, and belongs to the technical field of wastewater treatment. The method comprises the following steps: acquiring a flavinase NicA2 gene, a cytochrome c protein gene and an arabinose inducible promoter gene of pseudomonas by using a gene synthesis technology, and connecting the three gene segments to a vector plasmid pYYDT skeleton by using a Gibson connection technology to construct a pYYDT-NC plasmid; the engineering shewanella MR-1 / pYYDT-NC is obtained by electrically transferring the pYYDT-NC plasmid into shewanella, the strain is used for treating nicotine in tobacco wastewater, the performance of efficient oxidative degradation of nicotine is shown, the strain has good adaptability to wastewater with different concentrations of nicotine, and the practical application effect of degrading nicotine in tobacco wastewater is remarkable.

Description

Technical Field

[0001] The invention belongs to the technical field of wastewater treatment, and particularly relates to a construction method of an engineered Shewanella bacterium for degrading nicotine wastewater and its application. Background Art

[0002] China is the world's largest tobacco producer. Tobacco processing generates significant amounts of solid and liquid waste. Nicotine, the primary alkaloid in tobacco leaves, can leach into the environment along with this waste. Due to its unique structure, nicotine is highly addictive, toxic, and carcinogenic. Long-term nicotine consumption increases the incidence of cancer. Therefore, effectively removing nicotine from water bodies is crucial for public health and environmental protection.

[0003] Biodegradation is considered a relatively effective method for nicotine degradation due to its environmentally friendly, gentle, efficient, and simple nature. However, nicotine's significant biotoxicity makes it incapable of degradation by most microorganisms, posing numerous challenges to microbial treatment systems. To address these refractory toxic pollutants, advanced bioaugmentation techniques are required. Genetically engineering strains that can efficiently degrade nicotine is crucial for improving the efficiency and stability of treatment systems.

[0004] Shewanella is an electroactive bacterium widely distributed in aquatic environments such as lake sediments and marine sediments. As an electroactive microorganism, this type of strain uses an extracellular electron transfer system to achieve electron transfer for the reduction of toxic and harmful difficult-to-degrade pollutants. It has strong environmental tolerance and is adapted to the biodegradation of wastewater containing a variety of complex toxic and difficult-to-degrade pollutants. However, no research has shown that this strain has the ability to oxidize toxic pollutants.

[0005] Bioinformatics analysis revealed that Pseudomonas contains the flavoenzyme NicA2, which can oxidize nicotine and transfer the oxidized electrons to the cytochrome c protein. By designing and manipulating the metabolic pathway of Shewanella, and endowing it with the ability to oxidize nicotine, this method not only degrades the toxic pollutant nicotine but also generates electricity through coupling with the electron transfer system, which has important value in the field of biodetoxification of pollutants. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing an engineered Shewanella for degrading nicotine wastewater and its application, so as to achieve efficient degradation of nicotine in wastewater.

[0007] The purpose of the present invention can be achieved through the following technical solutions: The present invention provides a method for constructing an engineered Shewanella bacterium for degrading nicotine wastewater, comprising the following steps: Step 1: PCR amplification was performed using the flavoenzyme NicA2 gene fragment as a template, F-1 and R-1 as primers, the arabinose-inducible promoter gene fragment as a template, F-2 and R-2 as primers, the vector plasmid pYYDT gene fragment as a template, and F-3 and R-3 as primers to obtain three target gene fragments; Step 2: The three target gene fragments were ligated to the vector plasmid pYYDT via Gibson to obtain the pYYDT-NicA2 plasmid. After transformation, positive clones were screened and verified by PCR amplification using CK-F-1 and CK-R-1 as primers. The positive clones were cultured and the pYYDT-NicA2 plasmid was extracted. Step 3: Using the cytochrome c protein gene fragment as a template and F-4 and R-4 as primers, and using the pYYDT-NicA2 plasmid as a template and F-5 and R-5 as primers, PCR amplification was performed to obtain two gene fragments. Step 4: The two target gene fragments from step 3 were ligated by Gibson ligation to obtain the pYYDT-NC plasmid. After transformation, positive clones were screened and verified by PCR amplification using CK-F-1 and CK-R-1 as primers. The positive clone strains were cultured and the positive pYYDT-NC plasmids were extracted. Step 5: The positive pYYDT-NicA2 plasmid and pYYDT-NC plasmid were electroporated into Shewanella competent cells to construct engineered Shewanella MR-1 / pYYDT-NC and engineered Shewanella MR-1 / pYYDT-NicA2, and gene expression was verified.

[0008] Furthermore, the sequence of the flavoenzyme NicA2 gene fragment is shown as SEQ ID NO.1, the sequence of the arabinose-inducible promoter gene fragment is shown as SEQ ID NO.2, the sequence of the vector plasmid pYYDT gene fragment is shown as SEQ ID NO.3, and the sequence of the cytochrome c protein gene fragment is shown as SEQ ID NO.4.

[0009] Furthermore, the sequence of the F-1 is shown as SEQ ID NO.5; the sequence of the R-1 is shown as SEQ ID NO.6; the sequence of the F-2 is shown as SEQ ID NO.7; the sequence of the R-2 is shown as SEQ ID NO.8; the sequence of the CK-F-1 is shown as SEQ ID NO.9; the sequence of the CK-R-1 is shown as SEQ ID NO.10; the sequence of the F-3 is shown as SEQ ID NO.11; the sequence of the R-3 is shown as SEQ ID NO.12; the sequence of the F-4 is shown as SEQ ID NO.13; the sequence of the R-4 is shown as SEQ ID NO.14; the sequence of the F-5 is shown as SEQ ID NO.15; and the sequence of the R-5 is shown as SEQ ID NO.16.

[0010] Furthermore, in step 2 and step 4, the specific steps for screening positive clones after transformation are as follows: the pYYDT-NicA2 plasmid and the pYYDT-NC plasmid obtained by Gibson ligation are respectively transformed into Turbo Escherichia coli competent cells, and the plasmids are extracted after positive verification by PCR amplification to obtain the pYYDT-NicA2 plasmid and the pYYDT-NC plasmid.

[0011] Furthermore, the steps of the electrical transfer are as follows: 1 μg each of the positive pYYDT-NicA2 plasmid and pYYDT-NC plasmid were mixed with 100 μL of Shewanella competent cells and transferred to a 1 mm sterile electroporation cuvette. After electroporation at 2.5 kV for 5 ms, the mixture was immediately added to 1 mL of LB liquid medium and incubated at 30°C for 1 h. The mixture was spread on an LB agar plate containing 50 μg / mL kanamycin and cultured at 30°C for 1 day. Single clones were picked for positive clone verification to obtain positive clone engineered Shewanella strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC.

[0012] Furthermore, the preparation of the Shewanella competent cells is as follows: 4 mL of activated Shewanella was cultured in 50 mL of LB liquid medium for 16 h. The strain was collected after centrifugation, washed twice with 300 mM sucrose solution at 4°C, and then resuspended in 100 μL of 300 mM sucrose solution to obtain Shewanella competent cells.

[0013] Further, the activation steps of Shewanella are as follows: The Shewanella strain was taken out of the -80°C refrigerator, 40 μL of the bacterial solution was placed in 4 mL of LB medium and cultured with shaking for 12 h to obtain the activated Shewanella strain.

[0014] Furthermore, the shaking culture temperature was set to 37° C. for Escherichia coli and 30° C. for Shewanella, the rotation speed was set to 200 rpm, and the LB medium formula was 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0015] Furthermore, the gene expression verification is to perform molecular verification of the transcription level of the engineered Shewanella MR-1 / pYYDT-NicA2 and the engineered Shewanella MR-1 / pYYDT-NC, and culture the strains in LB liquid culture medium containing 10 μM arabinose solution at 30°C for 24 hours, collect the cultured cells, extract total RNA, and perform RT-qPCR reaction.

[0016] Furthermore, an RNA extraction kit is used in the extraction of total RNA; the RNA extraction kit is the RNAiso Plus Kit extraction reagent of Takara.

[0017] Furthermore, the RT-qPCR reaction is performed using a single-strand cDNA synthesis reagent and a probe-based qPCR reagent using a real-time fluorescence quantitative PCR system; The first-strand cDNA synthesis reagent is the PrimeScript II first Strand cDNA Synthesis Kit reagent of Takara Company, and the probe method qPCR reagent is the SYBR Premix Ex Taq reagent of Takara Company.

[0018] The present invention also provides an application of an engineered Shewanella for degrading nicotine wastewater, wherein the engineered Shewanella obtained by the above construction method is used to degrade nicotine in tobacco wastewater.

[0019] Beneficial effects of the present invention: Gene synthesis of flavoenzyme NicA2, cytochrome c protein ( cycN ) gene and an arabinose-inducible promoter gene were ligated with the vector plasmid pYYDT to construct the plasmid pYYDT-NC. This was then electroporated into competent Shewanella cells to create the engineered Shewanella MR-1 / pYYDT-NC strain. This engineered strain demonstrated excellent performance in nicotine oxidation and in actual nicotine wastewater treatment, efficiently oxidizing and degrading nicotine. Furthermore, the present invention demonstrates that the cytochrome c protein CycN is a highly efficient and specialized electron acceptor for NicA2, accelerating the oxidative degradation of nicotine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below with reference to the accompanying drawings.

[0021] Figure 1The pYYDT-NC plasmid and the pYYDT-NicA2 plasmid maps of the present invention are shown; in, Figure 1 (a) is the map of pYYDT-NC plasmid, Figure 1 (b) is the plasmid map of pYYDT-NicA2; Figure 2 This is an RT-qPCR graph of exogenous gene transcription in the engineered strain of the present invention; Figure 3 The graph shows the laboratory degradation results of 200 mg / L nicotine by strains MR-1 / pYYDT, MR-1 / pYYDT-NicA2, and MR-1 / pYYDT-NC; Figure 4 The graph shows the experimental results of the degradation of nicotine at different concentrations by strain MR-1 / pYYDT-NC; Figure 5 is the growth curve of strain MR-1 / pYYDT-NC at different initial nicotine concentrations; Figure 6 To add OD 600 =0.3 strains MR-1 / pYYDT and MR-1 / pYYDT-NC and the blank group without strain addition treated tobacco wastewater nicotine concentration results diagram; Figure 7 To add OD 600 =0.5 and the blank group without added strains to treat tobacco wastewater with nicotine concentration results. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] In order to enable Shewanella to degrade nicotine, the flavoenzyme NicA2 gene sequence was obtained by gene synthesis and assembled together with the arabinose-inducible promoter gene on the vector plasmid pYYDT backbone (plasmid map see Figure 1 ), forming plasmid pYYDT-NicA2 (plasmid map see Figure 3To further explore the effects of Shewanella endogenous electron acceptors and Pseudomonas nicotine oxidation electron acceptor cytochrome c protein (CycN protein) on nicotine oxidation, we added the CycN protein gene to the plasmid pYYDT-NicA2 to form the plasmid pYYDT-NC (see the plasmid map for details). Figure 2 The plasmid was then transformed into Shewanella by electroporation, and the positive engineered strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were obtained by antibiotic screening.

[0024] Example 1

[0025] This embodiment provides a method for constructing an engineered Shewanella bacterium for degrading nicotine wastewater, comprising the following steps: 1) Strain culture Turbo Escherichia coli competent cells and Shewanella ( Shewanella oneidensis The MR-1) strain was taken out of the -80°C freezer and, after thawing, streaked onto LB solid medium (LB liquid medium formula: 15 g / L agar, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) for overnight culture. The culture temperatures for Turbo E. coli competent cells and Shewanella were 37°C and 30°C, respectively. Single colonies were picked and inoculated into 5 mL of LB liquid medium (LB liquid medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured with shaking at 200 rpm overnight. 40 μL of the bacterial solution was then pipetted into 4 mL of LB liquid medium and cultured with shaking at 200 rpm for 12 h to obtain activated Turbo E. coli competent cells and Shewanella strains.

[0026] 2) Plasmid construction A flavoenzyme NicA2 gene fragment (shown in SEQ ID NO.1), an arabinose-inducible promoter gene fragment (shown in SEQ ID NO.2), a vector plasmid pYYDT gene fragment (shown in SEQ ID NO.3, containing kanamycin resistance), and a cytochrome c protein gene fragment (shown in SEQ ID NO.4) were gene synthesized using the flavoenzyme NicA2 gene fragment as a template and F-1 (shown in SEQ ID NO.5) and R-1 (shown in SEQ ID NO.6) as primers; the arabinose-inducible promoter gene fragment as a template and F-2 (shown in SEQ ID NO.7) and R-2 (shown in SEQ ID NO.8) as primers; and the vector plasmid pYYDT gene fragment as a template and F-3 (shown in SEQ ID NO.11) and R-3 (shown in SEQ ID NO.12) as primers. PCR amplification was performed, and the target gene fragments were recovered by gel electrophoresis. Three purified gene fragments were obtained using a gel recovery kit. The three gene fragments were ligated via Gibson ligation to obtain the pYYDT-NicA2 plasmid, which was then transformed into Turbo E. coli competent cells. Positive clones were screened using LB plates containing kanamycin. PCR amplification was performed using primers CK-F-1 (shown in SEQ ID NO. 9) and CK-R-1 (shown in SEQ ID NO. 10) to obtain the positive plasmid pYYDT-NicA2. See the schematic diagram for details. Figure 1 (b). Using the cytochrome c protein gene fragment as a template, F-4 (as shown in SEQ ID NO.13) and R-4 (as shown in SEQ ID NO.14) as primers; using pYYDT-NicA2 as a template, F-5 (as shown in SEQ ID NO.15) and R-5 (as shown in SEQ ID NO.16) as primers, PCR amplification was performed to obtain two gene fragments. These two gene fragments were Gibson ligated and transformed into Turbo Escherichia coli competent cells. Positive clones were screened using LB plates containing kanamycin. Positive clones were verified by PCR amplification using primers CK-F-1 and CK-R-1 to obtain the positive plasmid pYYDT-NC. See the schematic diagram. Figure 1 (a) Use a plasmid extraction kit to extract the target plasmids from the bacterial suspension containing pYYDT-NicA2 and pYYDT-NC, respectively.

[0027] 3) Preparation of Shewanella competent cells 4 mL of activated Shewanella was cultured in 40 mL of LB liquid medium for 16 h, and then the bacteria were collected and washed twice with a 300 mM sucrose aqueous solution at 4°C, and then resuspended with 100 μL of a 300 mM sucrose aqueous solution to obtain competent cells.

[0028] 4) Construction of engineered Shewanella 1 μg of each pYYDT-NicA2 and pYYDT-NC plasmid was mixed with 100 μL of Shewanella competent cells, transferred to a 1 mm sterile electroporation cuvette, and electroporated at 2.5 kV for 5 ms. The mixture was then immediately added to 1 mL of LB medium and incubated at 30°C for 1 hour. The cells were then plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 30°C for 1 day. Single colonies were picked and verified by PCR amplification. Positive clones were selected, including strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC.

[0029] Example 2

[0030] Molecular validation of engineered Shewanella transcriptome at the transcript level: Strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were cultured in LB liquid medium containing 10 μM arabinose at 30°C for 24 hours. Bacteria were harvested by centrifugation, and total RNA was extracted using the Takara RNAisoPlus Kit. Residual DNA contaminants were removed using RNase-free deoxyribonuclease (Takara Co., China). cDNA synthesis was performed using the Takara PrimeScript II First Strand cDNA Synthesis Kit, and target gene expression was detected and analyzed using the Takara SYBR Premix Ex Taq Kit and the StepOne Real-Time PCR System (Applied Biosystems Inc., USA). RT-PCR reactions for all samples were accompanied by amplification of the housekeeping gene RecA, and negative controls were used to exclude nonspecific amplification interference from DNA contamination. All assays were performed in triplicate, with threshold cycle (Ct) values ​​ranging from 20 to 30 to ensure reproducibility and reliability of the results. The sample cDNA was normalized using RecA cDNA abundance to calculate the relative expression of the target gene. The analysis of the test results is shown in the figure. Figure 4 .

[0031] according to Figure 2 The results showed that NicA2 of strain MR-1 / pYYDT-NicA2 was successfully transcribed, and both NicA2 and CycN genes of strain MR-1 / pYYDT-NC were transcribed at a high level, proving that the engineering strain was successfully constructed.

[0032] Example 3

[0033] Nicotine degradation experiment in the laboratory using engineered Shewanella: A control group was set up: the vector plasmid pYYDT was electroporated into Shewanella competent cells. The specific operation steps were the same as those for constructing engineered Shewanella in Example 1 to obtain strain MR-1 / pYYDT. Strain MR-1 / pYYDT was used as the control group.

[0034] Set up experimental groups: strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were used as experimental groups.

[0035] The strains MR-1 / pYYDT (control group), MR-1 / pYYDT-NicA2, and MR-1 / pYYDT-NC were cultured in LB liquid medium at 30°C and 200 rpm overnight. The cells were collected and washed, and the OD values ​​were injected into 10 mL of mineral salt medium. 600 = 0.3, 10 μL of 10 mM arabinose solution was added to strains MR-1 / pYYDT, MR-1 / pYYDT-NicA2, or MR-1 / pYYDT-NC, respectively. Nicotine was then added simultaneously, with the nicotine content in each system being 200 mg / L. This time was designated as degradation time zero. At the specified time point, 200 μL of the culture was collected and centrifuged at 15,000 g for 5 minutes. The supernatant was collected and 100 μL of the supernatant was mixed with 100 μL of water to prepare HPLC samples. Nicotine standards at concentrations of 2.5 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, and 150 mg / L were prepared. The standards and samples were used for liquid chromatography-mass spectrometry, and sample concentrations were calculated using a standard curve.

[0036] Mineral salts medium formulation: 0.46 g / L NaCl, 0.117 g / L MgSO₄·7H₂O, 0.225 g / L (NH₄)₂SO₄, 0.225 g / L KH₂PO₄, 0.225 g / L K₂HPO₄, 50 mM HEPES, 20 mM sodium lactate, 10 mL trace elements, pH = 7.1-7.2. Place the mineral salts medium in an anaerobic bottle, aerate with nitrogen for 20 minutes to remove oxygen, and autoclave at 121°C until ready for use.

[0037] High-performance liquid chromatography (HPLC) analysis was performed using a high-performance liquid chromatograph (LC-16, Shimadzu Co., Japan) using a ZORBAX Eclipse Plus C18 column (4.6 × 250 mm, 5 μm) and a mobile phase of acetonitrile / 0.1% methanolic acid (volume ratio 1:9). Samples were filtered through a 0.22 μm membrane and transferred to liquid chromatography vials. The vials were placed in an autosampler. Samples and standards were within the linear detection range, and absorbance peaks were integrated and quantified.

[0038] according to Figure 3 The results showed that the control strain MR-1 / pYYDT had no ability to oxidize nicotine, but exogenously introduced NicA2 could achieve nicotine oxidation; the nicotine oxidation efficiency of strain MR-1 / pYYDT-NC was much higher than that of strain MR-1 / pYYDT-NicA2, that is, the addition of CycN protein could rapidly accelerate the oxidation of nicotine, and the presence of only endogenous cytochrome protein of Shewanella could not achieve rapid nicotine oxidation. Therefore, it was analyzed that CycN protein was a special electron acceptor for NicA2.

[0039] The engineered strain MR-1 / pYYDT-NC was put into different nicotine concentrations for degradation experiments. The highest nicotine concentration reached 1g / L. Figure 4 The results showed that the engineered strain could completely degrade 200 mg / L of nicotine within 7 hours, 500 mg / L of nicotine within 10 hours, and 1 g / L of nicotine within 20 hours. The engineered strain MR-1 / pYYDT-NC has the ability to efficiently oxidize nicotine.

[0040] Figure 5 The results show that there is no significant difference in the growth curves of the engineered strain MR-1 / pYYDT-NC under different nicotine concentrations, indicating that the engineered strain has good adaptability to nicotine.

[0041] Example 4

[0042] Actual wastewater treatment experiment using engineered Shewanella: To verify the practical application potential of strain MR-1 / pYYDT-NC, its performance in treating real tobacco wastewater containing nicotine was tested. Activated sludge enhanced with strain MR-1 / pYYDT-NC was used to treat real tobacco wastewater. Activated sludge enhanced with strain MR-1 / pYYDT-NC (experimental group) and activated sludge enhanced with strain MR-1 / pYYDT (control group) were subjected to different OD values. 600The results of the degradation experiment are shown in Table 2. Figure 6 and Figure 7 , the specific experimental steps are as follows: The activated control strain MR-1 / pYYDT and engineered strain MR-1 / pYYDT-NC were cultured in LB liquid medium at 30°C overnight, and the bacteria were collected by centrifugation the next day and washed twice with 1×PBS buffer for use. The tobacco wastewater was centrifuged to remove incompatible impurities, and 200 mg / L glucose and 20 mM sodium lactate were added to 100 mL of tobacco wastewater for activation and stabilization. 100 μL of 10 mM arabinose solution was added to the wastewater as an inducer. The wastewater was treated according to the principle of activated sludge method, and activated sludge was added to 100 mL of tobacco wastewater according to MLSS = 3000 mg / L. The collected strain MR-1 / pYYDT and engineered strain MR-1 / pYYDT-NC were then added to the tobacco wastewater, so that the cell biomass at the initial reaction time was OD 600 =0.3 or OD 600 = 0.5. In the activated sludge treatment group without bacterial addition, no strains were added to the tobacco wastewater. Other steps and conditions were the same as above. The reaction system was incubated in a 30°C incubator. Afterwards, 1 mL of the reaction sample was periodically taken, centrifuged, and the supernatant was filtered through a 0.22 μm membrane and analyzed by HPLC.

[0043] Figure 6 The degradation experiment results showed that the strain MR-1 / pYYDT-NC showed high nicotine degradation ability in actual tobacco wastewater treatment. When the biomass of the added strain MR-1 / pYYDT-NC reached OD 600 =0.3, the treatment system can complete the degradation of nicotine within 4 hours; Figure 7 The results of the degradation experiment showed that when the biomass reached OD 600 = 0.5, nicotine was degraded within 2 hours. However, neither the MR-1 / pYYDT group nor the blank group showed significant nicotine degradation. Therefore, in actual wastewater biological treatment, the engineered strain MR-1 / pYYDT-NC can completely oxidize nicotine within a very short hydraulic retention time, eliminating its toxicity.

[0044] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0045] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an engineered Shewanella bacterium for degrading nicotine wastewater, It is characterized in that The steps include: Step 1, using the flavin enzyme NicA2 gene fragment as a template, F-1 and R-1 as primers, the arabinose-inducible promoter gene fragment as a template, F-2 and R-2 as primers, the vector plasmid pYYDT gene fragment as a template, and F-3 and R-3 as primers, PCR amplification was performed to obtain three target gene fragments; Step 2: Connect the three target gene fragments to the vector plasmid pYYDT through Gibson to obtain the pYYDT-NicA2 plasmid, screen the positive clones after transformation, perform PCR amplification verification with CK-F-1 and CK-R-1 as primers, culture the positive clone strains, and extract the pYYDT-NicA2 plasmid; Step 3, using the cytochrome c protein gene fragment as a template and F-4 and R-4 as primers; Using pYYDT-NicA2 plasmid as template and F-5 and R-5 as primers, PCR amplification was used to obtain two gene fragments; Step 4: The two target gene fragments in step 3 were ligated by Gibson to obtain the pYYDT-NC plasmid, and positive clones were screened after transformation. PCR amplification was performed using CK-F-1 and CK-R-1 as primers to verify the positive clones, and the positive clone strains were cultured to extract the pYYDT-NC plasmid; Step 5: Electroporate the positive pYYDT-NicA2 plasmid and pYYDT-NC plasmid into Shewanella competent cells to construct engineered Shewanella MR-1 / pYYDT-NC and engineered Shewanella MR-1 / pYYDT-NicA2 for gene expression verification.

2. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, It is characterized in that The sequence of the flavin enzyme NicA2 gene fragment is shown in SEQ ID NO.1, the sequence of the arabinose inducible promoter gene fragment is shown in SEQ ID NO.2, the sequence of the vector plasmid pYYDT gene fragment is shown in SEQ ID NO.3, and the sequence of the cytochrome c protein gene fragment is shown in SEQ ID NO.

4.

3. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, It is characterized in that The sequence of F-1 is shown in SEQ ID NO.5; the sequence of R-1 is shown in SEQ ID NO.6; the sequence of F-2 is shown in SEQ ID NO.7; the sequence of R-2 is shown in SEQ ID NO.8; the sequence of CK-F-1 is shown in SEQ ID NO.9; the sequence of CK-R-1 is shown in SEQ ID NO.10; the sequence of F-3 is shown in SEQ ID NO.11; the sequence of R-3 is shown in SEQ ID NO.12; the sequence of F-4 is shown in SEQ ID NO.13; the sequence of R-4 is shown in SEQ ID NO.14; the sequence of F-5 is shown in SEQ ID NO.15; the sequence of R-5 is shown in SEQ ID NO.

16.

4. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, It is characterized in that In step 2 and step 4, the specific steps of screening positive clones after transformation are as follows: the pYYDT-NicA2 plasmid and the pYYDT-NC plasmid obtained by Gibson ligation are respectively transferred into Turbo Escherichia coli competent cells, and the plasmids are extracted after positive verification by PCR amplification to obtain pYYDT-NicA2 plasmid and pYYDT-NC plasmid.

5. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, It is characterized in that The specific steps of the electrical transfer are as follows: 1 μg of each positive pYYDT-NicA2 plasmid and pYYDT-NC plasmid were mixed with 100 μL of Shewanella competent cells, transferred to a 1 mm sterile electroporation cuvette, and immediately added to 1 mL of LB liquid culture medium after electroporation at 2.5 kV for 5 ms. The mixture was incubated at 30°C for 1 h, spread on an LB agar plate containing 50 μg / mL kanamycin, and after incubation at 30°C for 1 day, a single clone was picked for positive clone verification to obtain the positive clone engineered Shewanella strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC.

6. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 5, It is characterized in that The preparation of the Shewanella competent cells is as follows: 4 mL of activated Shewanella was cultured in 50 mL of LB liquid culture medium for 16 h, the strain was collected after centrifugation, washed twice with 300 mM sucrose solution at 4° C., and then resuspended in 100 μL of 300 mM sucrose solution to obtain Shewanella competent cells.

7. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 6, It is characterized in that The activation steps of Shewanella are as follows: The Shewanella strain was taken out from the -80°C refrigerator, and 40 μL of the bacterial solution was put into 4 mL of LB medium for shaking culture for 12 h to obtain the activated Shewanella strain.

8. A method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 7, It is characterized in that The temperature of the shaking culture was set at 30° C., the rotation speed was set at 200 rpm, and the LB medium formula was: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

9. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, It is characterized in that The gene expression verification is to perform molecular verification of the transcription level of the engineered Shewanella MR-1 / pYYDT-NicA2 and the engineered Shewanella MR-1 / pYYDT-NC, and culture the strains in LB liquid culture medium containing 10 μM arabinose solution at 30° C. for 24 hours, collect the cultured cells, extract total RNA, and perform RT-qPCR reaction.

10. Application of an engineered Shewanella bacterium for degrading nicotine wastewater, It is characterized in that The engineered Shewanella obtained by the construction method according to any one of claims 1 to 9 is used to degrade nicotine in tobacco wastewater.

Images