A method for constructing an engineered Shewanella bacterium for degrading nicotine wastewater and its application

Abstract

The invention discloses a construction method and application of an engineered Shewanella for degrading nicotine wastewater, and belongs to the technical field of wastewater treatment. Gene synthesis technology is used to obtain the flavoenzyme NicA2 gene, cytochrome c protein gene and arabinose-inducible promoter gene of Pseudomonas, and the three gene fragments are connected to the vector plasmid pYYDT backbone by Gibson connection technology to construct the pYYDT-NC plasmid. The pYYDT-NC plasmid is electrotransferred into Shewanella to obtain the engineered Shewanella MR-1 / pYYDT-NC, and the strain is used to treat nicotine in tobacco wastewater, showing the performance of efficient oxidation and degradation of nicotine. The strain has good adaptability in wastewater with different nicotine concentrations, and the actual application effect of degrading nicotine in tobacco wastewater is significant.

Description

Technical Field

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

[0002] China is the world's largest tobacco producer. A large amount of solid and liquid waste is generated during tobacco processing. Among them, nicotine is the main alkaloid in tobacco leaves and will seep into the environment with the waste. Due to its special structure, nicotine is highly addictive, toxic and carcinogenic. Long-term intake of nicotine will increase the incidence of cancer. Therefore, effectively removing nicotine from water bodies is of great significance to protecting public health and the environment.

[0003] Biodegradation is considered to be a more effective way to degrade nicotine because it is green, mild, efficient and simple. However, nicotine has significant biological toxicity, which means that most microorganisms do not have the ability to degrade nicotine, which brings many challenges to the microbial treatment system. In order to deal with nicotine and other difficult-to-degrade toxic pollutants, it is necessary to adopt advanced bio-enhancement technology, obtain strains that can efficiently degrade nicotine through genetic engineering, and improve the efficiency and stability of the treatment system.

[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 achieves electron transfer through the extracellular electron transfer system 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] Through bioinformatics analysis, it was found that Pseudomonas has a flavin enzyme NicA2, which can oxidize nicotine and transfer the oxidized electrons to cytochrome c protein. The design regulates the metabolic pathway of Shewanella and gives Shewanella the ability to oxidize nicotine, which can not only achieve the degradation of nicotine toxic pollutants, but also couple the electron transfer system to generate electricity, which is of great value in the field of biological detoxification 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 by the following technical solutions:

[0008] The present invention provides a method for constructing an engineered Shewanella for degrading nicotine wastewater, comprising the following steps:

[0009] 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;

[0010] 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;

[0011] Step 3: Using the cytochrome c protein gene fragment as a template, F-4 and R-4 as primers; using the pYYDT-NicA2 plasmid as a template, F-5 and R-5 as primers, PCR amplification was performed to obtain two gene fragments;

[0012] Step 4: The two target gene fragments in step 3 were ligated by Gibson to obtain pYYDT-NC plasmid, and positive clones were screened after transformation. PCR amplification was performed with CK-F-1 and CK-R-1 as primers to verify, and positive clone strains were cultured to extract positive pYYDT-NC plasmids;

[0013] 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.

[0014] Furthermore, 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.

[0015] Further, 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; the sequence of the R-5 is shown as SEQ ID NO.16.

[0016] Furthermore, 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.

[0017] Furthermore, the steps of the electrical transfer are as follows:

[0018] 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.

[0019] Furthermore, the preparation of the Shewanella competent cells is as follows:

[0020] 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.

[0021] Further, the activation steps of Shewanella are as follows:

[0022] 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.

[0023] 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.

[0024] 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 the strains are cultured in LB liquid culture medium containing 10 μM arabinose solution at 30°C for 24 hours, and the cultured cells are collected to extract total RNA and perform RT-qPCR reaction.

[0025] 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 Company.

[0026] 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;

[0027] 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.

[0028] 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.

[0029] Beneficial effects of the present invention:

[0030] Gene synthesis of flavoenzyme NicA2, cytochrome c protein ( cy ) gene and arabinose-inducible promoter gene, which are connected with the vector plasmid pYYDT to construct plasmid pYYDT-NC, and then electrotransformed into Shewanella competent cells to construct engineered Shewanella MR-1 / pYYDT-NC. The engineered strain exhibits excellent performance in nicotine oxidation and actual nicotine wastewater treatment, and can efficiently oxidize and degrade nicotine. The present invention confirms that cytochrome c protein CycN is an efficient and specialized electron acceptor of NicA2, which can accelerate the oxidative degradation of nicotine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings.

[0032] Figure 1 The pYYDT-NC plasmid and the pYYDT-NicA2 plasmid maps of the present invention;

[0033] in, Figure 1 (a) is the plasmid map of pYYDT-NC. Figure 1 (b) is the plasmid map of pYYDT-NicA2;

[0034] Figure 2 It is the RT-qPCR graph of the transcription of the exogenous gene of the engineering strain of the present invention;

[0035] 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;

[0036] Figure 4 The figure shows the results of the degradation experiment of different concentrations of nicotine by strain MR-1 / pYYDT-NC;

[0037] Figure 5 It is the growth curve of strain MR-1 / pYYDT-NC under different initial nicotine concentrations;

[0038] Figure 6 To add OD 600 =0.3 strains MR-1 / pYYDT and MR-1 / pYYDT-NC and a blank group without added strains to treat tobacco wastewater nicotine concentration results;

[0039] Figure 7 To add OD 600 =0.5 and the blank group without added strains to treat nicotine concentration in tobacco wastewater. DETAILED DESCRIPTION

[0040] 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0041] In order to achieve the nicotine degradation of Shewanella, 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 (see the plasmid map for details). Figure 1 ), forming plasmid pYYDT-NicA2 (plasmid map see Figure 3 In order to 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 transferred into Shewanella by electroporation, and positive engineering strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were obtained by antibiotic screening.

[0042] Example 1

[0043] This embodiment provides a method for constructing an engineered Shewanella for degrading nicotine wastewater, comprising the following steps:

[0044] 1) Strain culture

[0045] Turbo Escherichia coli competent cells and Shewanella Shewanella oneidensis MR-1) strain was taken out from the -80℃ refrigerator, and after thawing, it was streaked and inoculated into LB solid medium (LB liquid medium formula: 15g / L agar, 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl) for overnight culture. The culture temperatures of Turbo Escherichia coli competent cells and Shewanella were 37℃ and 30℃, respectively. Single colonies were picked and inoculated into 5mL LB liquid medium (LB liquid medium formula: 10g / L tryptone, 5g / L yeast extract, 10g / L NaCl) and cultured overnight with shaking at 200 rpm. 40μL of bacterial solution was taken out and put into 4mL LB liquid medium respectively, and the activated Turbo Escherichia coli competent cells and Shewanella strains were obtained by shaking and culture at 200 rpm for 12h.

[0046] 2) Plasmid construction

[0047] The flavoenzyme NicA2 gene fragment (as shown in SEQ ID NO.1), the arabinose-inducible promoter gene fragment (as shown in SEQ ID NO.2), the vector plasmid pYYDT gene fragment (as shown in SEQ ID NO.3, containing kanamycin resistance) and the cytochrome c protein gene fragment (as shown in SEQ ID NO.4) were gene synthesized, and the flavoenzyme NicA2 gene fragment was used as a template, and F-1 (as shown in SEQ ID NO.5) and R-1 (as shown in SEQ ID NO.6) were used as primers; the arabinose-inducible promoter gene fragment was used as a template, and F-2 (as shown in SEQ ID NO.7) and R-2 (as shown in SEQ ID NO.8) were used as primers; the vector plasmid pYYDT gene fragment was used as a template, and F-3 (as shown in SEQ ID NO.11) and R-3 (as shown in SEQ ID NO.12) were used as primers; PCR amplification was performed, and the target gene fragments were recovered by gel electrophoresis, and three purified gene fragments were obtained using a gel recovery kit. The three gene fragments were connected by Gibson ligation to obtain the pYYDT-NicA2 plasmid, which was then transferred into Turbo E. coli competent cells and positive clones were screened using LB plates containing kanamycin. Using CK-F-1 (as shown in SEQ ID NO.9) and CK-R-1 (as shown in SEQ ID NO.10) as primers, PCR amplification was performed to obtain the positive plasmid pYYDT-NicA2. See the schematic diagram for details. Figure 1 (b). The cytochrome c protein gene fragment was used as a template, and F-4 (as shown in SEQ ID NO.13) and R-4 (as shown in SEQ ID NO.14) were used as primers; pYYDT-NicA2 was used as a template, and F-5 (as shown in SEQ ID NO.15) and R-5 (as shown in SEQ ID NO.16) were used as primers; PCR amplification was used to obtain two gene fragments. The two gene fragments were connected by Gibson and transferred into Turbo Escherichia coli competent cells. Positive clones were screened using LB plates containing kanamycin. CK-F-1 and CK-R-1 were used as primers, and PCR amplification was performed to verify that the positive plasmid pYYDT-NC was obtained. The schematic diagram is shown in Figure 1 (a). Use a plasmid extraction kit to extract the target plasmids from the bacterial solution containing pYYDT-NicA2 and pYYDT-NC for later use.

[0048] 3) Preparation of Shewanella competent cells

[0049] 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.

[0050] 4) Construction of engineered Shewanella

[0051] 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, electroporated at 2.5 kV for 5 ms, and then immediately added to 1 mL of LB medium and incubated at 30°C for 1 h. Then, it was 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 and positive clones were verified using PCR amplification technology, and positive clone strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were selected.

[0052] Example 2

[0053] Molecular validation of engineered Shewanella transcript levels:

[0054] The strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were cultured in LB liquid medium containing 10 μM arabinose solution at 30°C for 24 hours, and the cultured bacteria were collected by centrifugation. Total RNA was extracted using Takara's RNAisoPlus Kit reagent, and residual DNA contaminants in the total RNA samples were removed using RNase-free deoxyribonuclease (Takara Co., China). cDNA synthesis was performed using Takara's PrimeScript II first Strand cDNA Synthesis Kit, and the expression of the target gene was detected and analyzed using Takara's SYBR Premix Ex Taq Kit and StepOne Real-Time PCR System (Applied Biosystems Inc., USA). The RT-PCR reactions of all samples were accompanied by the amplification of the housekeeping gene RecA, and the negative control was used to exclude nonspecific amplification interference from DNA contamination. All test groups had three replicates, and the cycle threshold (Ct) value ranged from 20 to 30 to ensure the 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 Figure 4 .

[0055] 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.

[0056] Example 3

[0057] Nicotine degradation experiment in the laboratory using engineered Shewanella:

[0058] A control group was set up: the vector plasmid pYYDT was electroporated into Shewanella competent cells, and the specific operation steps were the same as those for constructing engineered Shewanella in Example 1, to obtain strain MR-1 / pYYDT, and strain MR-1 / pYYDT was used as the control group.

[0059] Set up experimental groups: strains MR-1 / pYYDT-NicA2 and MR-1 / pYYDT-NC were used as experimental groups.

[0060] 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, and the cells were collected and washed, and injected into 10 mL of mineral salt medium at OD 600 =0.3 strain MR-1 / pYYDT, strain MR-1 / pYYDT-NicA2 or strain MR-1 / pYYDT-NC, add 10μL 10mM arabinose solution respectively, and then add nicotine at the same time, wherein the nicotine content in each system is 200mg / L. This moment is defined as degradation time 0, and then 200μL of culture is taken at the specified time point, centrifuged at 15000g for 5min, the supernatant is collected, 100μL of supernatant is taken and mixed with 100μL water to prepare HPLC samples. Prepare nicotine standard samples of 2.5mg / L, 6.25mg / L, 12.5mg / L, 25mg / L, 50mg / L, 75mg / L, and 150 mg / L, use the standard samples and samples for liquid phase detection, and calculate the sample concentration by the standard curve.

[0061] Mineral salt medium formula: 0.46g / L NaCl, 0.117g / L MgSO4·7H2O, 0.225g / L (NH4) 2SO4, 0.225g / L KH2PO4, 0.225g / L K2HPO4, 50mM HEPES, 20mM sodium lactate, 10mL trace elements, pH = 7.1-7.2. Place the mineral salt medium in an anaerobic bottle, pass nitrogen for 20 minutes to remove oxygen, and sterilize it with high-pressure steam at 121℃ for later use.

[0062] The high performance liquid chromatography detection method is as follows: the samples and standards are detected using a high performance liquid chromatograph (HPLC, LC-16, Shimadzu Co., Japan), using a ZORBAX Eclipse Plus C18 (4.6×250 mm column, 5μm) as the chromatographic column and acetonitrile / 0.1% methanolic acid (volume ratio 1:9) as the mobile phase. The samples are filtered using a 0.22μm membrane and added to a liquid phase vial. The vial is placed in an autosampler. The samples and standards are within the linear detection range, and the absorbance peaks are integrated and quantified.

[0063] 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 receptor for NicA2.

[0064] 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.

[0065] Figure 5 The graph shows 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.

[0066] Example 4

[0067] Actual wastewater treatment experiment of engineered Shewanella:

[0068] In order to verify the practical application potential of strain MR-1 / pYYDT-NC, its performance in treating real tobacco wastewater containing nicotine was tested. The activated sludge strengthened by strain MR-1 / pYYDT-NC was used to treat the actual tobacco wastewater. The activated sludge strengthened by strain MR-1 / pYYDT-NC (experimental group) and the activated sludge strengthened by strain MR-1 / pYYDT (control group) were heated to different OD values. 600The results of the degradation experiment are shown in Figure 6 and Figure 7 , the specific experimental steps are as follows:

[0069] The activated control strain MR-1 / pYYDT and the engineered strain MR-1 / pYYDT-NC were cultured overnight in LB liquid medium at 30°C, and the bacteria were collected by centrifugation the next day and washed twice with 1×PBS buffer for later use. The tobacco wastewater was centrifuged to remove incompatible impurities, 200 mg / L glucose and 20 mM sodium lactate were added to 100 mL of tobacco wastewater for activation and stabilization, and 100 μL of 10 mM arabinose solution was added to the wastewater as an inducer; according to the principle of wastewater treatment by activated sludge method, activated sludge was added to 100 mL of tobacco wastewater according to MLSS=3000 mg / L; then the collected strain MR-1 / pYYDT and the engineered strain MR-1 / pYYDT-NC were added to the tobacco wastewater, so that the cell biomass was OD at the initial reaction time. 600 =0.3 or OD 600 =0.5. No strain was added to the tobacco wastewater in the activated sludge treatment group without bacteria, and the other steps and conditions were the same as above. The reaction system was cultured in an incubator at 30°C. After that, 1 mL of the reaction sample was taken at regular intervals, the sample was centrifuged to obtain the supernatant, and part of the supernatant was filtered through a 0.22μm membrane and then tested by HPLC.

[0070] Figure 6 The degradation experiment results showed that strain MR-1 / pYYDT-NC showed high nicotine degradation ability in actual tobacco wastewater treatment. When the biomass of strain MR-1 / pYYDT-NC added 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 can be degraded within 2 h. However, the nicotine degradation effect of the MR-1 / pYYDT group and the blank group was not obvious. Therefore, in the actual wastewater biological treatment process, the engineered strain MR-1 / pYYDT-NC can completely oxidize nicotine in a very short hydraulic retention time and eliminate the toxicity of nicotine.

[0071] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0072] Although 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 the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an engineered Shewanella for degrading nicotine wastewater, 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, and verify gene expression; 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; 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; The strain of the Shewanella competent cells is Shewanella oneidensis MR-1.

2. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, 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.

3. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, 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.

4. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 3, 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.

5. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 4, 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.

6. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 5, 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.

7. The method for constructing an engineered Shewanella for degrading nicotine wastewater according to claim 1, 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.

8. An application of engineered Shewanella for degrading nicotine wastewater, characterized in that: The engineered Shewanella obtained by the construction method according to any one of claims 1 to 7 is used to degrade nicotine in tobacco wastewater.

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