A method for improving the ability of gluoxydibacter oxidans to resist phenylethene stress

Abstract

The application discloses a method for improving the anti-styrene stress ability of Gluconobacter oxydans, and belongs to the technical field of genetic engineering and microbial engineering. Firstly, the adaptability evolution of wild-type Gluconobacter oxydans to styrene is carried out by gradient addition of styrene in a culture medium, and finally, the evolved strain ST significantly improves the tolerance of Gluconobacter oxydans to styrene. Secondly, the knocking out of flagellin FlgE in Gluconobacter oxydans also improves the stress ability of Gluconobacter oxydans to styrene; the growth performance of the gene deletion Gluconobacter oxydans ST-ΔflgE prepared by the method of the application after 10h culture under 15g / L styrene is far better than that of the wild-type Gluconobacter oxydans.

Description

Technical Field

[0001] This invention relates to a method for improving the resistance of *Glucosamine oxidans* to styrene stress, belonging to the fields of genetic engineering and microbial engineering technology. Background Technology

[0002] Gluconobacter oxydans is one of the most commonly used microorganisms in industrial biotechnology. Its outer membrane is rich in alcohol and aldehyde dehydrogenases, and its cytoplasm contains oxidoreductases that can rapidly and incompletely oxidize alcohols, aldehydes, and carbohydrates. Moreover, the substrates do not need to be transported into the cell; the reaction can take place in the periplasmic space. Currently, Gluconobacter oxydans is widely used in the synthesis of compounds such as gluconic acid, migritol, and vitamin C, and has a broad market in the food additive, animal feed, cosmetic, and pharmaceutical industries.

[0003] Currently, the catalytic production process of *Glucobacterium oxidans* inevitably faces various stresses from the external environment. Since most catalytic substrates are poorly soluble in water, organic co-solvents are often added to improve substrate solubility. However, *Glucobacterium oxidans* is a Gram-negative bacterium with poor solvent tolerance, which hinders the normal transformation process. The environmental stress caused by added solvents or organic substrates severely affects the physiological activity and survival ability of *Glucobacterium oxidans*, thus significantly limiting the efficiency of the biocatalytic reaction. Furthermore, solvent stress exposes *Glucobacterium oxidans* to lethal or sublethal environments, which have a significant impact on cell survival.

[0004] To achieve a balance between yield and efficiency, improving the tolerance of *Glucosamine oxidans* to organic substrates and solvents such as styrene is crucial. Therefore, this study aims to enhance the styrene tolerance of *Glucosamine oxidans* through a combination of adaptive evolution and genetic modification, thereby achieving a qualitative leap in its tolerance. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a method for improving the resistance of *Glucobacterium oxysporum* to styrene stress. The aim is to solve the technical problem that *Glucobacterium oxysporum* has poor solvent tolerance, which is not conducive to the normal progress of transformation. The environmental stress caused by added solvents or organic substrates seriously affects the physiological activity and survival ability of *Glucobacterium oxysporum*, and thus seriously limits the efficiency of biocatalytic reactions.

[0006] The first technical solution provided by this invention is a strain of Gluconobacteroxydans ST, which was deposited at the China Center for Type Culture Collection on November 5, 2024, with accession number CCTCC NO:M20242435.

[0007] This invention also provides a method for the adaptive evolution of *Gluconobacterium oxyacetylene* to tolerate high concentrations of styrene. The method involves acclimating *Gluconobacterium oxyacetylene* to high concentrations of styrene by gradually adding styrene to a culture medium.

[0008] In some embodiments, the starting strain for the adaptive evolution of styrene is *Glucosamine oxyacetobacter* 621H.

[0009] In some embodiments, the culture conditions for 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.

[0010] In some embodiments, the initial styrene concentration in the styrene adaptation is 0.2 g / L, and the styrene concentration increases sequentially in a gradient of 0.2 g / L, with the highest final styrene concentration tolerated by *Glucosamine oxyacetylene* being 15 g / L.

[0011] The second technical solution provided by the present invention is a microbial preparation containing *Gluconobacterium oxysporum* ST as described in the first technical solution.

[0012] In some embodiments, the concentration of *Glucobacterium oxyphylla* ST in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0013] The third technical solution provided by the present invention is an engineered strain of *Glucobacterium oxysporum*, wherein the engineered strain of *Glucobacterium oxysporum* is obtained by knocking out the flagellin gene flgE in *Glucobacterium oxysporum* ST as described in the first technical solution; the flagellin FlgE is a protein related to regulating cell movement and biofilm formation.

[0014] In some embodiments, the amino acid sequence of the flagellin FlgE is shown in SEQ ID NO.1.

[0015] In some embodiments, the nucleotide sequence of the gene encoding the flagellate protein FlgE is shown in SEQ ID NO.2.

[0016] In some embodiments, the gene deletion is achieved by first synthesizing a 2000bp fragment by fusion PCR of the upstream and downstream homologous arms of the flagellin FlgE gene (1000bp each), then ligating it to a knockout expression vector to construct a recombinant plasmid containing the gene deletion, and finally introducing the recombinant plasmid into *Glucosobacterium oxysporum*.

[0017] In some embodiments, the gene knockout expression vector is the pK18mobSacB plasmid.

[0018] In some embodiments, the gene deletion is achieved by first fusing the upstream and downstream homologous arms of the flgE gene, then ligating it with the digestion product obtained by double digestion of pK18mobSacB plasmid with EcoRI and SalI to construct a recombinant plasmid containing the flgE gene deletion, and finally introducing the recombinant plasmid into glucosamine oxidase.

[0019] The fourth technical solution provided by the present invention is a method for improving the resistance of *Glucobacterium oxysporum* to styrene stress, wherein the method is to inhibit or weaken the expression of flagellin FlgE in *Glucobacterium oxysporum*.

[0020] In some embodiments, the glucosobacterium oxidase includes glucosobacterium oxidase 621H or glucosobacterium oxidase ST.

[0021] In some embodiments, the amino acid sequence of the flagellin FlgE is shown in SEQ ID NO.1.

[0022] In some embodiments, the nucleotide sequence of the gene encoding the flagellate protein FlgE is shown in SEQ ID NO.2.

[0023] The fifth technical solution provided by the present invention is the application of flagellin FlgE in improving the resistance of Glucosamine oxidans to styrene stress. The application is to knock out the flagellin FlgE gene of Glucosamine oxidans. The amino acid sequence of the flagellin FlgE is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the flagellin FlgE is shown in SEQ ID NO.2.

[0024] The sixth technical solution provided by the present invention is the application of the *Gluconobacterium oxysporum* ST described in the first technical solution, or the microbial preparation described in the second technical solution, or the engineered bacteria described in the third technical solution, or the method described in the fourth technical solution in the microbial treatment of styrene-containing wastewater or in biocatalysis using styrene as a substrate.

[0025] The technical effects of this invention are as follows:

[0026] (1) Through adaptive evolution to styrene, the present invention obtained a highly styrene-tolerant glucosamine oxyacetobacter ST, which can tolerate 15 g / L of styrene. Compared with wild-type glucosamine oxyacetobacter, the ability to tolerate styrene is increased by 2.56 times.

[0027] (2) By knocking out the flagellin gene flgE in the evolved strain ST, the present invention obtained a gene-deleted strain ST-ΔflgE with improved resistance to styrene stress; the survival ability of the gene-deleted strain ST-ΔflgE after being cultured in a styrene environment of 15g / L for 10h was much stronger than that of the wild type.

[0028] (3) The method of the present invention has a good effect on improving the resistance of styrene stress of Gluconobacterium oxidans and is applicable to the current microbial treatment of styrene-containing wastewater and biocatalysis with styrene as a substrate.

[0029] Preservation of biological materials

[0030] A strain of *Gluconobacter oxydans* ST was deposited on November 5, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO:M 20242435. Attached Figure Description

[0031] Figure 1 The effects of styrene and cosolvents DMSO and n-hexadecane on the growth of Gluconobacterium oxidans.

[0032] Figure 2 The growth of wild-type strains and styrene-evolved strains under different concentrations of styrene stress is shown.

[0033] Figure 3 Tolerance of wild-type strains and styrene-evolved strains to other organic solvents.

[0034] Figure 4 The wild strain 621H, the evolved strain ST, and the gene-deleted strain ST-ΔflgE were plate-stamped at different concentrations of styrene.

[0035] Figure 5 Cell motility was assessed for wild-type strain 621H, evolved strain ST, and gene-deleted strain ST-ΔflgE. Detailed Implementation

[0036] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. The *Gluconobacterium oxysporum* 621H involved in the following embodiments was purchased from the DSM Culture Collection (DSMZ) in Germany; the pK18mobSacB plasmid involved in the following embodiments was purchased from the BioVector plasmid vector culture and gene bank.

[0037] The culture media involved in the following examples are as follows:

[0038] Sorbitol liquid culture 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, 0.1 g / L glutamine.

[0039] Sorbitol solid medium: 2% agar is added to sorbitol liquid medium.

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

[0041] Sucrose medium: Add 100 g / L sucrose to sorbitol liquid medium.

[0042] Styrene liquid medium: Add the corresponding concentration of styrene to sorbitol liquid medium.

[0043] Semi-solid culture medium: Add 0.3% and 0.4% agar to sorbitol liquid medium.

[0044] The preparation methods involved in the following examples are as follows:

[0045] The adaptive evolution of Gluconobacterium oxidans to styrene

[0046] (1) Wild-type glucosinolate bacteria were cultured to the logarithmic growth phase to prepare an adaptive evolutionary starter culture.

[0047] (2) Transfer the starting bacterial culture to the initial styrene liquid medium and culture it to the logarithmic phase. Then, transfer it to a liquid medium with increased styrene concentration. Repeat this process to culture the culture medium with increased styrene concentration until the strain stops growing completely.

[0048] (3) Take 2 mL of the final gradient evolution bacterial culture, spread it on sorbitol solid medium, and cover it with about 3 mm of styrene at the final screening concentration. After culturing for 72-84 h, isolate the final high styrene-tolerant single colony and name it Gluconobacterium oxysporum ST.

[0049] Preparation of competent cells of *Gluconobacterium oxidans*:

[0050] Inoculate a single colony of *Glucobacter oxidans* into 10 mL of sorbitol medium and incubate at 30 °C and 220 rpm until the logarithmic growth phase. Transfer the colony to 100 mL of sorbitol medium at 1% (v / v) and incubate at 30 °C and 220 rpm for 6–8 h. Then centrifuge at 4 °C and 8000 rpm for 20 min, discard the supernatant, collect the bacterial cells, resuspend in 10% (v / v) glycerol and wash the cells three times. Finally, add 1 mL of 10% (v / v) glycerol to fully resuspend the cells. Aliquot 100 μL of the resuspended solution into 1.5 mL centrifuge tubes and store at -80 °C.

[0051] Oxidized gluconate electroconversion method:

[0052] Add 1000 ng of the constructed DNA knockout fragment and competent cells to a pre-cooled electroporation cuvette, electroporate at 1.8 kV for 4.8 ms, and then immediately add 600 μL of fresh sorbitol medium. Incubate at 30°C and 220 rpm for 5 h, then spread onto sorbitol solid plates containing 50 μg / mL kanamycin and incubate upside down at 30°C for 48-60 h until colonies grow.

[0053] The tolerance testing methods involved in the following examples are as follows:

[0054] Gluconobacterium oxidans was cultured to the logarithmic growth phase, and its absorbance at 600 nm was measured using a spectrophotometer. Its OD was then diluted with fresh culture medium. 600 The concentration was set to 2.0, and a uniform concentration treatment was performed. Then, different concentrations of styrene were added to 10 mL of the uniformly concentrated bacterial suspension, and the suspension was incubated at 30℃ and 220 rpm for 10 h. The OD value was then measured again. 600 OD of the styrene-treated bacterial suspension 600 Characterize tolerance.

[0055] Example 1: Preparation of *Glucosamine oxyacetobacter* ST

[0056] The specific steps are as follows:

[0057] (1) First, wild-type Gluconobacterium oxygenase was inoculated into sorbitol medium and cultured to the logarithmic growth phase to prepare seed culture. Then, 1.5 mL of fresh sorbitol medium was added to each well of a 24-well plate, and 100 μL of seed culture was transferred to each well. Different concentrations (0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35 and 40 g / L) of commonly used cosolvents (n-hexadecane and DMSO) and styrene were added. After culturing at 30℃ and 220 rpm for 24 h, the absorbance of each well was measured at 600 nm. The absorbance was used to reflect the growth status and determine the optimal initial styrene concentration for the adaptation and evolution of wild-type Gluconobacterium oxygenase.

[0058] The results are as follows Figure 1 As shown, dose-limit growth experiments using two co-solvents (n-hexadecane and DMSO) revealed that n-hexadecane and DMSO had no significant effect on the growth of wild-type *Glucobacter oxidans*. In contrast, 9 g / L styrene led to complete bacterial death. High cell density is essential in the initial stages of evolution. The survival rate of *Glucobacter oxidans* at 3 g / L styrene was similar to that at 0 g / L, which was sufficient to provide selection pressure. Therefore, 3 g / L styrene was chosen as the starting concentration for evolution.

[0059] (2) After wild-type *Glucobacterium oxysporum* was inoculated into sorbitol medium and cultured to the logarithmic growth phase, it was inoculated into sorbitol medium supplemented with 3 g / L styrene for adaptive evolution. Three independent replicates were set up. After reaching the logarithmic growth phase, it was transferred to the next adaptive evolution gradient at an inoculation rate of 1% (v / v), with the styrene concentration increasing by 0.2 g / L with each transfer to promote adaptation. After 10 transfers, cell growth slowed down, and resistant cells appeared. With the increase of the number of transfers, the styrene concentration gradually increased, and the time for *Glucobacterium oxysporum* to reach the logarithmic growth phase also became longer. However, as long as the adaptive ability improved significantly over time, the evolution continued. After multiple transfers, *Glucobacterium oxysporum* showed limited growth, requiring up to 72 hours to reach the logarithmic phase. At this time, the styrene concentration increased to 15 g / L, and the adaptive evolution was considered to have entered a plateau phase, and the evolution of tolerance to styrene terminated.

[0060] The final adaptive evolutionary bacterial suspension was spread on sorbitol solid medium and covered with about 3 mm of 15 g / L styrene (using ethanol as solvent). The single colony isolated after cultivation was the final evolved strain, named *Glucosamine oxyacetobacter* ST. *Glucosamine oxyacetobacter* ST was deposited at the China Center for Type Culture Collection, with accession number CCTCCNO:M 20242435.

[0061] Example 2: Styrene-tolerant test of evolved strain ST

[0062] The specific steps are as follows:

[0063] Wild-type strain 621H and the evolved strain ST obtained in Example 1 were activated separately, and then cultured at 30°C and 220 rpm until the logarithmic growth phase to obtain seed culture. The absorbance of the two bacterial cultures at 600 nm was measured, and the bacterial cultures were diluted with culture medium to the initial OD value. 600 Both were 0.1; then, the two seed cultures were inoculated at a rate of 1% (v / v) into 100 ml of fresh sorbitol liquid medium and styrene medium containing 7 g / L and 15 g / L respectively, and cultured at 30 °C and 220 rpm. The OD of the bacterial culture was measured every 4 h. 600 Values, plot growth curves (see) Figure 2 ).

[0064] The results showed that both wild-type strain 621H and the evolved strain ST exhibited good growth in the absence of styrene stress. Wild-type strain 621H could barely survive under styrene stress above 7 g / L, while the evolved strain ST could still grow under styrene stress of 15 g / L, indicating that the adaptive evolution of strain ST significantly improved its tolerance to styrene. However, styrene inhibits bacterial growth; even without the addition of styrene stress, the growth of the evolved strain was partially inhibited.

[0065] Example 3: Tolerance of the evolved strain ST to other organic solvents

[0066] The specific steps are as follows:

[0067] (1) Wild-type strain 621H and evolved strain ST were activated separately, and then cultured at 30℃ and 220 rpm until the logarithmic growth phase to obtain seed culture. The absorbance of the seed culture was measured at 600 nm. The culture medium was diluted to the initial OD value. 600 Both were 2.0, resulting in bacterial suspensions of the evolved strain ST and the original strain 621H.

[0068] (2) Organic solvents exhibit a logP dependence. o / w The toxicity, at low logP o / w The toxicity was even higher at certain times. The final evolved strain could tolerate 15 g / L styrene (logP). o / w =3), which also shows its tolerance to other different logP values. o / w The potential of the solvent. Therefore, five different logP values ​​were selected. o / w Growth experiments were conducted at different levels to assess the tolerance of the evolved strains, including phenol (logP). o / w =1.5), benzene (logP) o / w =2), Toluene (logP) o / w =2.5), p-xylene (logP) o / w =3) and propylbenzene (logP) o / w =3.8). Add 15 g / L of the selected logP to the above bacterial suspension respectively. o / w The OD of the bacterial suspension was measured after culturing in an organic solvent at 30°C and 220 rpm for 10 h. 600 The result is as follows Figure 3 As shown in Table 1.

[0069] The results showed that, compared with the wild type, the evolved strain ST exhibited higher tolerance to solvents other than toluene, with tolerance to phenol, benzene, toluene, p-xylene, and propylbenzene increasing by 1.77, 1.81, 1.18, 1.66, and 2.65 times, respectively. This indicates that adaptive evolution enhanced the solvent tolerance of *Gluconobacterium oxidans* and successfully produced a highly tolerant mutant. Therefore, the evolved strain ST possesses broad-spectrum tolerance to benzene-based solvents.

[0070] Table 1. Enhanced ability of evolutionary strains to withstand different organic solvent stresses

[0071]

[0072] Example 4: Construction of gene-deleted strains

[0073] The specific steps are as follows:

[0074] Using the ST genome of *Glucosamine oxyphylla* as a template, 1000 bp fragments of the upstream and downstream homologous arms of the flagellin flgE gene were amplified by PCR. These two homologous arms were then fused into a 2000 bp knockout fragment using fusion PCR. The knockout fragment was then ligated into the linearized plasmid pK18mobSacB, which had been digested with EcoRI and SalI, and transformed into *Escherichia coli* JM109. The transformation product was plated on LB solid medium (containing 50 μg / mL kanamycin) and incubated at 37°C for 12–16 h to obtain transformants. Transformants were randomly selected for colony PCR verification. Positive transformants were then inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 220 rpm for 8–12 h. Two mL of bacterial cells were then collected, plasmids were extracted, and EcoRI and SalI digested. After enzyme digestion, electrophoresis and sequencing verification were performed. Once sequencing verification was confirmed, the recombinant *E. coli* strain JM109 / pK18mobSacB-HA was obtained. The recombinant expression plasmid JM109 / pK18mobSacBmobSacB-HA was extracted and electroporated into the evolved strain *Glucobacterium oxysporum* ST. The transformation product was plated on solid medium containing 50 μg / mL kanamycin and sorbitol, and incubated at 30°C inverted for 48–60 h to obtain transformants. Transformants were randomly selected for colony PCR verification. Positive transformants were then inoculated into sorbitol medium containing 50 μg / mL kanamycin. The bacterial culture was inoculated in sorbitol liquid medium at 30°C and 220 rpm for 16–24 h until the logarithmic growth phase. Then, it was transferred to sucrose medium at an inoculation rate of 1% (v / v) and incubated at 30°C and 220 rpm for 16–24 h. 100 μL of the bacterial culture was then spread on sorbitol solid plates and incubated upside down at 30°C for 48 h. After colonies grew on the plates, colony PCR was performed for verification. The verified positive transformants were inoculated into sorbitol liquid medium and allowed to grow until the logarithmic growth phase. The genome was then extracted and sent to the company for genome sequencing verification, finally obtaining the gene-deleted strain ST-ΔflgE.

[0075] Table 2 Primers used for flgE gene deletion

[0076]

[0077] Example 5: Tolerance test of gene-deleted strains under styrene stress

[0078] The specific steps are as follows:

[0079] (1) Glucose oxidase (strain ST-ΔflgE, strain ST and strain 621H) were cultured to the logarithmic growth phase, diluted with sorbitol liquid medium, and the initial OD600 was controlled to be 2.0 to prepare bacterial suspensions of gene-deleted strain ST-ΔflgE evolved strain ST and wild strain 621H respectively.

[0080] (2) Add styrene to the bacterial suspension prepared in step (1) at final concentrations of 0, 7, and 15 g / L respectively, incubate at 30°C and 220 rpm for 10 h, and then dilute to 10 g / L according to the concentration gradient. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Then, 3 μL of the diluted bacterial suspension of the four strains was taken and spotted onto a sorbitol solid plate. The plate was incubated upside down at 30°C for 48 hours, and the growth status of the colonies was monitored. The results are as follows: Figure 5 As shown, from left to right, the bacterial suspension is serially diluted 10-10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 .

[0081] The results are as follows Figure 4 The results showed that under organic solvent stress, the gene-deleted *Gluconobacterium oxysporum* ST-ΔflgE exhibited superior growth performance compared to other strains. Under conditions of 7 g / L or 15 g / L styrene addition, the gene-deleted strain ST-ΔflgE showed excellent growth performance, confirming that knocking out the flagellin FlgE in *Gluconobacterium oxysporum* can effectively improve its tolerance to styrene stress.

[0082] Example 6: Cell motility of gene-deleted strain ST-ΔflgE under styrene stress

[0083] The specific steps are as follows: After activating the wild-type strain 621H, the strain ST obtained in Example 1, and the strain ST-ΔflgE obtained in Example 4 by streaking on sorbitol solid medium, a single colony was picked and inoculated into sorbitol liquid medium. The culture was carried out at 30°C and 220 rpm until the logarithmic growth phase. The absorbance of the diluted bacterial solution at 600 nm was measured, and the solution was diluted with sorbitol liquid medium to an OD value of [missing value]. 600All cultures were treated to a uniform concentration of 2.0. Then, 15 g / L styrene was added to the uniformly concentrated bacterial culture, and the culture was incubated at 30°C and 220 rpm for 10 hours. A sterile inoculation needle was used to pick up the diluted bacterial culture and vertically insert it into the center of the semi-solid culture medium. The agar concentrations of the semi-solid culture medium were 0.3% and 0.4% (w / v), respectively, to measure cell migration and pulsation. The semi-solid culture medium was then placed upright at 30°C and incubated statically for approximately 72 hours before photographing and recording the results. Figure 5 ).

[0084] The result is Figure 5 As shown in Table 3, under styrene stress, the diameter and area of ​​the motility rings formed on the surface of the culture medium plates decreased, and the motility of the evolved strains was significantly reduced compared to the wild type of *Glucobacter oxidans*, including clustering and surging abilities. However, after knocking out the flagellin FlgE, the motility and tolerance to styrene were improved, indicating that the flagellin FlgE is one of the methods to improve the styrene tolerance of *Glucobacter oxidans*.

[0085] Table 3. Average diameter of the microbial spheres formed by cell movement.

[0086]

[0087] Example 7: Biofilm formation experiment of gene-deleted strain ST-ΔflgE under styrene stress

[0088] The specific steps are as follows:

[0089] Wild-type strain 621H, strain ST obtained in Example 1, and strain ST-ΔflgE obtained in Example 4 were streaked onto sorbitol solid medium. After incubation at 30°C for 48 hours, single colonies grew. Single colonies were picked and inoculated into sorbitol liquid medium and cultured at 30°C and 220 rpm for 24 hours to obtain seed culture. The seed culture was then transferred to fresh sorbitol liquid medium at an inoculation rate of 1% (v / v) and cultured at 30°C and 220 rpm for 12 hours to obtain culture medium. The OD of the culture medium was measured. 600 Dilute with culture medium to OD 600All values ​​were 2.0. Then, 15 g / L styrene was added to the bacterial culture at the same concentration, and the culture was incubated at 30℃ and 220 rpm for 10 h. The culture culture after styrene stress was diluted 1000 times, and 700 μL of the diluted bacterial culture was placed in a 1.5 mL EP tube. After incubation at 30℃ for 48 h, all the bacterial culture was poured out, and 700 μL of 0.1% (w / v) crystal violet staining solution was added, and staining was performed for 30 minutes. Then, 800 μL of physiological saline was added repeatedly to rinse the remaining crystal violet staining solution and free bacterial cells in the tube until the physiological saline solution in the tube became clear and transparent. Finally, 200 μL of anhydrous ethanol was added to each tube to dissolve the crystal violet dye, and after standing for 10 min, the wavelength of each sample was measured at wavelength A. 595 Lower absorbance value.

[0090] Table 4 shows that, based on biofilm formation experiments, the biofilm formation ability of the evolved strain ST was slightly higher than that of the wild type, being 1.06 times that of the wild type. The gene-deleted strain ST-ΔflgE exhibited the strongest biofilm formation ability, being 1.34 times that of the wild type 621H and 1.25 times that of the evolved strain ST. This indicates that the gene-deleted strain ST-ΔflgE significantly improved tolerance to styrene stress. This demonstrates that flagellin FlgE can regulate cell tolerance to styrene stress by modulating biofilm formation.

[0091] Table 4 Effects of styrene stress on the biofilm formation ability of bacterial strains

[0092]

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An engineered strain of *Glucobacterium oxysporum*, characterized in that, The engineered strain of *Glucosamine oxidase* is derived from the *Glucosamine oxidase* (… Gluconobacter oxydans Knockout flagellin gene in ST flgE The *Gluconobacterium oxysporum* ST strain was deposited at the China Center for Type Culture Collection on November 5, 2024, with accession number CCTCCNO:M 20242435. (Flagellin gene...) flgE The nucleotide sequence is shown in SEQ ID NO.

2.

2. A method for improving the resistance of *Glucobacterium oxysporum* to styrene stress, characterized in that, The method involves inhibiting or reducing the expression of flagellin FlgE in *Glucosamine oxidans*, and the flagellin gene... flgE The nucleotide sequence is shown in SEQ ID NO.

2. The glucosamine oxidase includes glucosamine oxidase 621H or glucosamine oxidase ST. The glucosamine oxidase ST was deposited at the China Center for Type Culture Collection on November 5, 2024, with accession number CCTCC NO:M 20242435.

3. The method according to claim 2, characterized in that, The method involves knocking out the flagellin gene in *Glucobacter oxytetracycline*. flgE .

4. The application of flagellin FlgE in enhancing the resistance of *Glucobacterium oxidans* to styrene stress, characterized in that... The application involves knocking out the flagellin FlgE gene of *Glucosamine oxidans*, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding which is shown in SEQ ID NO.

2.

5. The engineered bacteria of claim 1, or the method of any one of claims 2 to 3, in the microbial treatment of styrene-containing wastewater or in styrene-based biocatalysis.

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