Method for improving styrene stress tolerance of gluconobacter oxydans through overexpression gene marR

Abstract

The invention discloses a method for improving styrene stress tolerance of gluconobacter oxydans through overexpression gene marR, and belongs to the technical field of gene engineering and microbial engineering. According to the invention, through overexpression of a MarR family transcriptional regulation factor marR (GOXRS11840) in the gluconobacter oxydans, the tolerance of the gluconobacter oxydans to styrene stress is obviously improved; the survival rate of the recombinant gluconobacter oxydans prepared by the method is 1.6 times of that of a gluconobacter oxydans control strain after the recombinant gluconobacter oxydans are subjected to stress culture for 10 hours in an environment of 15g / L styrene.

Description

Technical Field

[0001] The invention relates to a method for improving the tolerance of Gluconobacter oxydans to styrene stress by overexpressing gene marR, and belongs to the technical fields of genetic engineering and microbial engineering. Background Art

[0002] Gluconobacter oxydans is a strain widely used in industry. It is often used to produce vitamin C, dihydroxyacetone, unnatural amino acids, and other products with wide applications and broad market prospects in food, medicine, and daily chemical products.

[0003] However, in the industrial biocatalytic styrene production process using Gluconobacter oxydans, the water-insolubility of the catalytic substrate severely impacts the catalytic conversion rate and production performance of the organism. To maintain substrate solubility in the catalytic process, the industry often enhances substrate solubility by adding organic cosolvents such as DMSO and alkanes.

[0004] However, the addition of some organic cosolvents often leads to the accumulation of toxic stress during the catalytic process of Gluconobacter oxydans. The increased substrate solubility exposes the cells to more stress. The byproducts formed by the growth and metabolism of these organic substrates through Gluconobacter oxydans will again cause the cells to be in a high-stress environment, which will again affect the growth and metabolism of Gluconobacter oxydans.

[0005] The existing biological detoxification inhibition still has certain defects, among which the long-term accumulation of toxicity is the most important factor hindering the growth of Gluconobacter oxydans. For example, Jiang et al. detected the effect of the toxicity inhibitor on Gluconobacter oxydans by inoculating Gluconobacter oxydans into a culture medium containing a toxicity inhibitory compound. However, this method only identified the intensity of the inhibitory effect and did not effectively solve how Gluconobacter oxydans produces resistance to toxic substances and detoxification (specifically, see reference Jiang W, Dai L, Tan X, Zhou X, Xu Y. Screening of Gluconobacter oxydans in xylonic acid fermentation for tolerance of the inhibitors formed dilute acid pretreatment. Bioprocess Biosyst Eng. 2023 Apr; 46 (4): 589-597.). Therefore, it is urgent to find a better method for improving the ability of Gluconobacter oxydans to resist styrene stress. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for improving the tolerance of Gluconobacter oxydans to styrene stress by overexpressing the gene marR, aiming to solve the technical problem that organic solvents can cause the accumulation of toxic stress during the catalytic process of Gluconobacter oxydans, affecting the growth and metabolism of Gluconobacter oxydans.

[0007] The present invention provides a strain of Gluconobacter oxydans ST, which has been deposited in the China Center for Type Culture Collection on November 5, 2024, with a deposit number of CCTCC NO: M 20242435.

[0008] The present invention also provides a method for the adaptive evolution of Gluconobacter oxydans that tolerates high concentrations of styrene. The method comprises the step of adding styrene in a gradient to a culture medium to acclimate Gluconobacter oxydans.

[0009] In certain embodiments, the starting strain for the styrene-adaptive evolution is Gluconacetobacter oxydans 621H.

[0010] In certain embodiments, the culture conditions of the styrene-adaptive evolution strain are a culture temperature of 30° C., a rotation speed of 220 rpm, and a culture time of 36-48 h.

[0011] In certain embodiments, the initial styrene concentration in the styrene adaptability test is 0.2 g / L, and the styrene concentration increases in a gradient of 0.2 g / L, and the maximum styrene concentration that Gluconacetobacter oxidans tolerates is 15 g / L.

[0012] The first technical solution provided by the present invention is the application of the transcriptional regulatory factor MarR in improving the resistance of Gluconobacter oxydans to styrene stress. The application is to overexpress the transcriptional regulatory factor MarR in Gluconobacter oxydans, and the amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.1.

[0013] In certain embodiments, the Gluconobacter oxydans is wild-type Gluconobacter oxydans 621H and Gluconobacter oxydans ST, which is the result of styrene-adaptive evolution. The evolved strain ST has been deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number CCTCC NO: M 20242435.

[0014] The second technical solution provided by the present invention is a recombinant bacterium of Gluconobacter oxydans, wherein the recombinant bacterium of Gluconobacter oxydans uses Gluconobacter oxydans ST as a host and overexpresses the transcriptional regulatory factor MarR.

[0015] In certain embodiments, the amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.1.

[0016] In certain embodiments, the nucleotide sequence of the gene encoding the transcriptional regulatory factor MarR is shown as SEQ ID NO.2.

[0017] In certain embodiments, the Gluconobacter oxydans ST was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number being CCTCC NO: M 20242435.

[0018] In certain embodiments, the overexpression is achieved by integrating the marR gene into a vector to obtain a recombinant plasmid encoding a MarR family transcriptional regulatory factor, and then transferring the recombinant plasmid into Gluconobacter oxydans ST to obtain a recombinant strain.

[0019] In certain embodiments, the pBBR1MCS-2 plasmid is used as the expression vector.

[0020] The third technical solution provided by the present invention is a method for improving the ability of Gluconobacter oxydans to resist styrene stress, wherein the method is to overexpress the transcriptional regulatory factor MarR in Gluconobacter oxydans.

[0021] The present invention also provides a microbial preparation containing the marR-overexpressing Gluconobacter oxydans ST-pBBR1MCS-2-marR.

[0022] The present invention also provides a method for detecting the tolerance of Gluconobacter oxydans to styrene and other organic solvents. The method comprises culturing the above-mentioned recombinant Gluconobacter oxydans to the logarithmic growth phase, adding different concentrations of styrene thereto for stress, obtaining a stressed bacterial solution, and then diluting the bacterial solution for spot plating.

[0023] In certain embodiments, the styrene concentration is 0, 7, or 15 g / L.

[0024] In certain embodiments, the styrene stress culture condition is culturing at 30° C. and 220 rpm for 10 h.

[0025] In certain embodiments, the forced bacterial solution dilution is to dilute the bacterial solution to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0026] The fourth technical solution provided by the present invention is the use of the recombinant Gluconobacter oxydans described in the second technical solution or the method described in the third technical solution in the microbial treatment of wastewater containing styrene or in biocatalysis using styrene as a substrate.

[0027] The fifth technical solution provided by the present invention is the application of the recombinant Gluconobacter oxydans described in the second technical solution or the method described in the third technical solution in the fields of food, medicine and daily chemical products.

[0028] The technical effects of the present invention are as follows:

[0029] The present invention provides a recombinant Gluconobacter oxydans strain ST-pBBR1MCS-2-marR that is highly resistant to styrene. The recombinant Gluconobacter oxydans strain ST-pBBR1MCS-2-marR is obtained by overexpressing a MarR family transcriptional regulatory factor GOX_RS111840 (marR). After the recombinant Gluconobacter oxydans strain is cultured to a logarithmic phase, it is subjected to styrene stress for 10 hours and then diluted and plated to obtain a recombinant Gluconobacter oxydans strain with significantly improved styrene stress resistance. The survival rate of the overexpressed Gluconobacter oxydans strain after being cultured in a 15 g / L styrene environment for 10 hours is increased by 1.6 times compared with a control strain.

[0030] Biomaterial Deposit

[0031] A strain of Gluconobacter oxydans ST, with the taxonomic name Gluconobacter oxydans, was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO:M 20242435. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effects of styrene and cosolvents DMSO and n-hexadecane on the growth of Gluconobacter oxydans.

[0033] Figure 2 This is the plasmid map of the recombinant plasmid pBBR1MCS-2-marR.

[0034] Figure 3 Shown are the growth curves of the control strain and the overexpression strain.

[0035] Figure 4 Dot plate diagram showing the tolerance of the control strain and the overexpression strain to styrene. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are intended to better explain the present invention and are not intended to limit the present invention. The Gluconobacter oxydans 621H involved in the following examples was purchased from the DSM Culture Collection (DSMZ) in Germany; the pBBR1MCS-2 plasmid involved in the following examples was purchased from the BioVector Plasmid Vector Bacteria Cell Gene Collection.

[0037] The culture medium involved in the following examples is as follows:

[0038] Sorbitol liquid medium: 20 g / L sorbitol, 20 g / L yeast extract, 5 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.25 g / L anhydrous magnesium sulfate, and 0.1 g / L glutamine.

[0039] Sorbitol solid medium: Add 2% agar to the sorbitol liquid medium.

[0040] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast powder.

[0041] Styrene liquid medium: Add corresponding concentration of styrene to sorbitol liquid medium.

[0042] The preparation methods involved in the following examples are as follows:

[0043] Preparation of competent cells of Gluconobacter oxydans:

[0044] A single colony of Gluconobacter oxydans was inoculated into 10 mL of sorbitol medium and cultured at 30°C and 220 rpm until the logarithmic growth phase; a 1% (v / v) transfer volume was inoculated into 100 mL of sorbitol medium and cultured at 30°C and 220 rpm for 6-8 h, followed by centrifugation at 4°C to remove the supernatant, collect the cells, resuspend in 10% (v / v) glycerol, and wash the cells three times. Finally, 1 mL of 10% (v / v) glycerol was added to fully resuspend the cells, and 100 μL of the resuspension was dispensed into each sterile 1.5 mL centrifuge tube and stored at -80°C until use.

[0045] Oxidized gluconate rod electroporation method:

[0046] Add 1000 ng of the constructed recombinant plasmid and competent cells to a pre-cooled electroporation cup, electroporate at 1.8 kV for 4.8 ms, and then immediately add 800 μL of fresh sorbitol medium. After incubation at 30°C and 220 rpm for 5 h, spread it on a sorbitol solid plate containing 50 μg / mL kanamycin and incubate it upside down at 30°C for 48-60 h until colonies grow.

[0047] Example 1 Preparation of Gluconacetobacter ST

[0048] The specific steps are as follows:

[0049] (1) First, the wild-type Gluconobacter oxydans was cultured in sorbitol medium until the logarithmic growth phase as seed liquid. Then, 1.5 mL of fresh sorbitol medium was added to a 24-well plate, 100 μL of seed liquid was transferred to each well, and different concentrations (0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35 and 40 g / L) of common cosolvents (n-hexadecane and DMSO) and styrene were added. After culturing at 30°C and 220 rpm for 24 h, the absorbance of each well of the bacterial solution at 600 nm was detected. This absorbance value was used to reflect the growth situation and to determine the optimal starting styrene concentration for the adaptive evolution of the wild-type Gluconobacter oxydans.

[0050] The results are as follows Figure 1 As shown, a dose-limiting growth test of two cosolvents (n-hexadecane and DMSO) on Gluconobacter oxydans revealed that the cosolvents n-hexadecane and DMSO had no significant effect on the growth of wild-type Gluconobacter oxydans. In contrast, 9 g / L styrene caused complete bacterial death. High cell density is essential in the initial stages of evolution. The survival rate of Gluconobacter oxydans at 3 g / L styrene was similar to that at 0 g / L, sufficient to provide screening pressure. Therefore, 3 g / L styrene was selected as the starting concentration for evolution.

[0051] (2) After the wild-type Gluconobacter oxydans was cultured in sorbitol medium to the logarithmic growth phase, it was inoculated in sorbitol medium supplemented with 3 g / L styrene for adaptive evolution. Three independent replicates were set up. After culturing to the logarithmic growth phase, it was transferred to the next adaptive evolution gradient at a 1% (v / v) inoculum size. The styrene concentration was increased by 0.2 g / L each time to promote adaptation. After 10 transfers, cell growth slowed down and resistant cells appeared. As the number of transfers increased, the styrene concentration gradually increased, and the time it took for Gluconobacter oxydans to reach the logarithmic growth phase became longer and longer. However, as long as the adaptability improved significantly over time, the evolution continued. After multiple transfers, Gluconobacter oxydans showed limited growth and required up to 72 hours to reach the logarithmic phase. At this time, the styrene concentration increased to 15 g / L. It was believed that the adaptive evolution entered the plateau phase and the evolution of tolerance to styrene terminated.

[0052] The final adaptively evolved bacterial liquid was spread on a sorbitol solid medium and covered with approximately 3 mm of 15 g / L styrene (using ethanol as the solvent). A single colony was isolated after cultivation, which was designated as the final evolved strain and named Gluconoacetobacter oxydans ST. This Gluconoacetobacter oxydans ST was deposited with the China Center for Type Culture Collection with the accession number CCTCCNO: M 20242435.

[0053] Example 2: Construction of Gluconobacter oxydans overexpression strain

[0054] The specific steps are as follows:

[0055] The ST genome of Gluconacetobacter oxydans in Example 1 was used as a template, and two primers, marR-F and marR-R, were used to amplify the DNA fragment of the transcriptional regulatory factor MarR gene by PCR. The fragment had homology arms of the two restriction sites EcoR I and Hind III in the plasmid pBBR1MCS-2 at both ends. The DNA fragment with homology arms was ligated with the linearized plasmid pBBR1MCS-2 after EcoR I and HindIII digestion to construct the recombinant plasmid pBBR1MCS-2-marR (see Figure 2 ) and transformed into Escherichia coli JM109; the transformation product was spread on LB solid medium (containing 50 μg / mL kanamycin) and inverted at 37°C for 12-16 hours to obtain transformants; transformants were randomly picked for colony PCR verification, and positive transformants were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 220 rpm for 8-12 hours. Then, 2 mL of bacteria were collected, plasmids were extracted, and EcoRI and Hind After enzyme digestion with III, electrophoresis verification and sequencing verification were performed. After sequencing verification was correct, the recombinant Escherichia coli JM109 / pBBR1MCS-2-marR was obtained; the recombinant expression plasmid JM109 / pBBR1MCS-2-marR was extracted and electroporated into the evolved strain ST, and the transformation product was spread on a solid culture medium containing 50 μg / mL kanamycin sorbitol, and inverted cultured at 30°C for 48 to 60 hours to obtain transformants; transformants were randomly picked for colony PCR verification, and the positive transformants were picked and sent to the company for genome sequencing verification. After sequencing verification was correct, the gene overexpression strain ST-pBBR1MCS-2-marR was obtained.

[0056] Referring to the same method as that for constructing the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-marR, the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-GOX_RS15020, ST-pBBR1MCS-2-ompW and the control strain ST-pBBR1MCS-2 were constructed; wherein, the primers used to construct the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-GOX_RS15020 were GOX_RS15020-F and GOX_RS15020-R, and the DNA fragment of GOX_RS15020 used was shown in SEQ ID NO: 3; the primers used to construct the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-ompW were ompW-F and ompW-R, and the DNA fragment of ompW used was shown in SEQ ID NO: 4.

[0057] Table 1 Primers used to construct recombinant bacteria

[0058]

[0059] Example 3: Growth ability analysis of control strain ST and overexpression strains

[0060] The specific steps are as follows:

[0061] The strain ST-pBBR1MCS-2 and three overexpression strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were activated respectively, and single colonies were picked and inoculated into sorbitol liquid medium. Then, they were cultured at 30°C and 220 rpm until the early logarithmic growth phase to obtain seed liquid. The absorbance values ​​of the two bacterial liquids at 600 nm were measured respectively, and the bacterial liquids were diluted with culture medium to OD 600 The diluted seed solution was then transferred to 100 ml of fresh sorbitol liquid culture medium at a 1% (v / v) inoculation volume, and cultured at 30°C and 220 rpm using Gluconobacter oxydans ST-pBBR1MCS-2 as a control.

[0062] During the culture process, the OD of the bacterial solution was measured every 8 h. 600 Value, draw the growth curve (see Figure 3The results showed that the control strain ST-pBBR1MCS-2 and the three recombinant strains overexpressing the gene, ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020, and ST-pBBR1MCS-2-ompW, all grew well in the absence of styrene stress. However, the growth performance of the recombinant strains ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW was inferior to that of the control strain ST-pBBR1MCS-2, while the recombinant strain ST-pBBR1MCS-2-marR showed the strongest growth ability.

[0063] Example 4: Tolerance test of control strain ST and overexpression strain under styrene stress conditions

[0064] The specific steps are as follows:

[0065] The control strain ST-pBBR1MCS-2 and three overexpression strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020, and ST-pBBR1MCS-2-ompW were activated and cultured to the logarithmic growth phase. The absorbance at 600 nm was measured and then diluted with sorbitol liquid medium to control the initial OD 600 The control strain ST-pBBR1MCS-2 and three recombinant strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW bacterial suspensions were prepared respectively; styrene at final concentrations of 0 and 15 g / L was added to the bacterial suspensions prepared above, and the OD values ​​were measured after culturing at 30°C and 220 rpm for 10 h. 600 and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Then take 3 μL of the diluted bacterial suspension and spot it on a sorbitol solid plate. Incubate it upside down at 30℃ for 48 hours until colonies grow on the plate. The results are as follows: Figure 4 As shown, from left to right are the gradient dilutions of bacterial suspension 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0066] The results are as follows Figure 4 The results showed that under the stress of styrene, the growth performance of overexpressing Gluconobacter oxydans ST-pBBR1MCS-2-marR was better than that of the control strain ST-pBBR1MCS-2. When 15 g / L styrene was added, overexpressing Gluconobacter oxydans ST-pBBR1MCS-2-marR showed superior growth performance, confirming that overexpressing the MarR family transcriptional regulatory factor GOX_RS11840 (marR) in Gluconobacter oxydans can effectively improve its tolerance to styrene stress.

[0067] Example 5: Survival rate experiment of overexpression strain under styrene stress conditions

[0068] The specific steps are as follows:

[0069] The control strain ST-pBBR1MCS-2 and three overexpression strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were streaked onto sorbitol solid medium for activation and cultured at 30°C until colonies grew. Single colonies were picked and inoculated into sorbitol liquid medium and cultured at 30°C and 220 rpm until the logarithmic growth phase to obtain seed solution. The absorbance at 600 nm was measured and the solution was diluted with sorbitol liquid medium to control the initial OD 600 The seed solution was inoculated into sorbitol liquid medium at an inoculum rate of 1% (v / v), and cultured at 30°C and 220 rpm for 18 h to obtain the control strain ST-pBBR1MCS-2 and the culture solutions of the three recombinant strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020, and ST-pBBR1MCS-2-ompW, respectively; the absorbance of the culture solution at 600 nm was measured again and then diluted with sorbitol liquid medium, and the initial OD was also controlled. 600 =2.0; 10 mL of culture medium was taken from each sample and the cells were collected by centrifugation at 12,000 rpm. The collected cells were washed twice with PBS and resuspended in 10 mL of sorbitol liquid medium supplemented with 15 g / L styrene. The cells were cultured at 30°C and 220 rpm for 10 h to obtain bacterial suspensions. 200 μL of the bacterial suspensions were spread on a sorbitol solid medium containing 50 μg / mL kanamycin to determine the number of viable bacteria and the survival rate (the results are shown in Table 2); wherein, the survival rate = (A / A0) × 100%; A represents the number of viable colonies grown on a sorbitol solid medium containing 50 μg / mL kanamycin after cultured with styrene for 10 h; and A0 is the number of viable colonies grown on a sorbitol solid medium containing 50 μg / mL kanamycin of a bacterial suspension not subjected to styrene stress.

[0070] As can be seen from Table 2, according to the survival rate experiment analysis, after 10 hours of stress in sorbitol liquid medium containing 15 g / L styrene, the survival rate of the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-marR was 1.6 times that of the control, indicating that the tolerance of the recombinant Serratia marcescens ST-pBBR1MCS-2-marR to styrene stress was significantly improved; after 10 hours of stress in sorbitol liquid medium containing 15 g / L styrene, the survival rates of the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were only 0.9 and 0.8 times that of the control, respectively, indicating that the tolerance of the recombinant Gluconobacter oxydans ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW to styrene stress was not improved. This demonstrates that the transcriptional regulatory factor MarR can regulate the cell tolerance to styrene stress, while proteins GOX_RS15020 and OmpW cannot.

[0071] Table 2 Effects of styrene stress on the survival rates of different overexpression strains

[0072]

[0073] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The application of the transcriptional regulatory factor MarR in improving the ability of Gluconobacter oxydans to resist styrene stress, characterized in that: The application is to overexpress the transcriptional regulatory factor MarR in Gluconobacter oxydans, and the amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the transcriptional regulatory factor MarR is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The Gluconobacter oxydans is a wild-type Gluconobacter oxydans 621H or Gluconobacter oxydans ST. The Gluconobacter oxydans ST has been deposited in the China Center for Type Culture Collection on November 5, 2024, with a deposit number of CCTCC NO: M 20242435.

4. A recombinant bacterium of Gluconobacter oxydans, characterized in that: The recombinant bacterium Gluconobacter oxydans uses Gluconobacter oxydans ST as a host and overexpresses the transcriptional regulatory factor MarR.

5. The recombinant bacterium according to claim 4, characterized in that The Gluconobacter oxydans ST was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number being CCTCC NO:M 20242435.

6. The recombinant bacterium according to claim 4, characterized in that The amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.

1.

7. A method for improving the ability of Gluconobacter oxydans to resist styrene stress, characterized in that: The method is to over-express the transcriptional regulatory factor MarR in Gluconobacter oxydans, wherein the amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The Gluconobacter oxydans is a wild-type Gluconobacter oxydans 621H or Gluconobacter oxydans ST. The Gluconobacter oxydans ST has been deposited in the China Center for Type Culture Collection on November 5, 2024, with a deposit number of CCTCC NO: M 20242435.

9. Use of the recombinant Gluconobacter oxydans according to any one of claims 4 to 6 or the method according to claim 7 or 8 in the microbial treatment of wastewater containing styrene or in biocatalysis using styrene as a substrate.

10. Use of the recombinant Gluconobacter oxydans according to any one of claims 4 to 6 or the method according to claim 7 or 8 in the fields of food, medicine and daily chemical products.

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