Method for improving styrene stress resistance of gluconobacter oxydans

Abstract

The invention discloses a method for improving the styrene stress resistance of gluconobacter oxydans, and belongs to the technical field of genetic engineering and microbial engineering. According to the method, firstly, styrene is added into a culture medium in a gradient mode, adaptive evolution of wild type gluconobacter oxydans to styrene is carried out, and the finally obtained evolved strain ST remarkably improves the tolerance of the gluconobacter oxydans to styrene. Secondly, flagellin FlgE is knocked out from the gluconobacter oxydans, so that the stress ability of the gluconobacter oxydans to styrene is also improved; the growth performance of the gene-deleted gluconobacter oxydans ST-delta flgE prepared by the method disclosed by the invention is far higher than that of wild type gluconobacter oxydans after the gene-deleted gluconobacter oxydans ST-delta flgE is cultured for 10 hours under 15g / L styrene.

Description

Technical Field

[0001] The invention relates to a method for improving the resistance of gluconobacter oxidans to styrene stress, and belongs to the technical field of genetic engineering and microbial engineering. Background Art

[0002] Gluconobacter oxydans is one of the most commonly used microorganisms in industrial biotechnology. It is rich in alcohol and aldehyde dehydrogenases on its cell outer membrane and oxidoreductases in its cytoplasm, which can quickly and incompletely oxidize alcohols, aldehydes and carbohydrates. The substrates do not need to be transported into the cell and can react in the periplasmic space. Currently, Gluconobacter oxydans has been widely used in the synthesis of compounds such as gluconic acid, miglucitol, vitamin C, etc., and has a broad market in food additives, animal feed, cosmetics, pharmaceuticals and other industries.

[0003] At present, in the catalytic production process of Gluconobacter oxidans, it is inevitable to face a variety of stresses from the external environment. Since most of the catalytic substrates are insoluble in water, an organic cosolvent is often added to improve the substrate solubility. Gluconobacter oxidans is a Gram-negative bacterium with poor tolerance to solvents, which is not conducive to the normal transformation. The environmental stress caused by the added solvent or organic substrate seriously affects the physiological activity and survival ability of Gluconobacter oxidans, thereby seriously limiting the efficiency of the biocatalytic reaction. In addition, solvent stress exposes Gluconobacter oxidans to a lethal or sublethal environment, which has a great impact on cell survival.

[0004] In order to achieve a win-win situation of production and efficiency, it is extremely important to improve the tolerance of Gluconobacter oxydans to organic substrates and solvents such as styrene. Therefore, through adaptive evolution combined with genetic modification, it is hoped that the tolerance of Gluconobacter oxydans to styrene can be improved, thus achieving a qualitative leap in the tolerance of Gluconobacter oxydans. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for improving the resistance of Gluconobacter oxydans to styrene stress, aiming to solve the technical problem that Gluconobacter oxydans has poor tolerance to solvents, which is not conducive to the normal conversion, and the environmental stress caused by the added solvent or organic substrate seriously affects the physiological activity and survival ability of Gluconobacter oxydans, thereby seriously limiting the efficiency of the biocatalytic reaction.

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

[0007] The present invention also provides a method for the adaptive evolution of Gluconobacter oxydans that tolerates high concentrations of styrene. The method for the adaptive evolution of Gluconobacter oxydans that tolerates high concentrations of styrene is to add styrene in a gradient in the culture medium to acclimate Gluconobacter oxydans.

[0008] In certain embodiments, the starting strain for the styrene adaptive evolution is Gluconacetobacter oxydans 621H.

[0009] 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.

[0010] In certain embodiments, the initial styrene concentration in the styrene adaptability is 0.2 g / L, and the styrene concentration increases in a gradient of 0.2 g / L, and finally the highest styrene concentration tolerated by Acetobacter oxydans is 15 g / L.

[0011] The second technical solution provided by the present invention is a microbial preparation containing the Gluconobacter oxidans ST described in the first technical solution.

[0012] In certain embodiments, the concentration of the Gluconobacter oxydans ST in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0013] The third technical solution provided by the present invention is an engineered bacterium of Gluconobacter oxydans, wherein the engineered bacterium of Gluconobacter oxydans ST described in the first technical solution has the flagellin gene flgE knocked out; the flagellin FlgE is a protein related to regulating cell movement and biofilm formation.

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

[0015] In certain embodiments, the nucleotide sequence of the gene encoding the flagellin FlgE is shown as SEQ ID NO.2.

[0016] In certain embodiments, the gene deletion is achieved by first synthesizing the 1000 bp upstream and downstream homologous arms of the flagellin FlgE gene into a 2000 bp fragment through fusion PCR, and then connecting it to the knockout expression vector to construct a recombinant plasmid containing the gene deletion, and then introducing the recombinant plasmid into Bacillus oxydans.

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

[0018] In certain embodiments, the gene deletion is performed by first fusing the upstream and downstream homology arms of the flgE gene, and then connecting the digestion products obtained by double digestion of the pK18mobSacB plasmid with EcoR I and Sal I to construct a recombinant plasmid containing the flgE gene deletion, and finally introducing the recombinant plasmid into Gluconobacter oxydans.

[0019] The fourth technical solution provided by the present invention is a method for improving the ability of Gluconobacter oxydans to resist styrene stress, wherein the method comprises inhibiting or weakening the expression of flagellin FlgE in the Gluconobacter oxydans.

[0020] In certain embodiments, the Gluconobacter oxydans comprises Gluconobacter oxydans 621H or Gluconobacter oxydans ST.

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

[0022] In certain embodiments, the nucleotide sequence of the gene encoding the flagellin FlgE is shown as SEQ ID NO.2.

[0023] The fifth technical solution provided by the present invention is the application of flagellin FlgE in improving the ability of Gluconobacter oxydans to resist styrene stress. The application is to knock out the flagellin FlgE gene of Gluconobacter oxydans, 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 use of the Gluconobacter oxidans 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 wastewater containing styrene or in biocatalysis using styrene as a substrate.

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

[0026] (1) The present invention obtains a highly styrene-tolerant Gluconacetobacter oxidans ST through adaptive evolution to styrene. The Gluconacetobacter oxidans ST can tolerate 15 g / L styrene, and its ability to tolerate styrene is increased by 2.56 times compared with the wild-type Gluconacetobacter oxidans.

[0027] (2) The present invention knocks out the flagellin gene flgE in the evolved strain ST to obtain a gene-deficient strain ST-ΔflgE with improved resistance to styrene stress; the survival ability of the gene-deficient strain ST-ΔflgE of the present invention after being cultured in a 15 g / L styrene environment for 10 hours is much stronger than that of the wild type.

[0028] (3) The method of the present invention is effective in improving the ability of Gluconobacter oxydans to resist styrene stress, and is suitable for the microbial treatment of wastewater containing styrene and biocatalysis using styrene as a substrate.

[0029] Biomaterial Deposit

[0030] A strain of Gluconobacter oxydans ST, with the taxonomic name of Gluconobacter oxydans, was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCCNO:M 20242435. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The effects of styrene and co-solvents DMSO and n-hexadecane on the growth of Gluconobacter oxydans.

[0032] Figure 2 The growth of wild strain and styrene-evolved strain under different concentrations of styrene stress.

[0033] Figure 3 This shows the tolerance of the wild strain and the styrene-evolved strain to other organic solvents.

[0034] Figure 4 The wild strain 621H, the evolved strain ST and the gene-deficient strain ST-ΔflgE were plated under different concentrations of styrene.

[0035] Figure 5 Cell motility of wild strain 621H, evolved strain ST and gene-deficient strain ST-ΔflgE. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. 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 Gluconobacter oxydans 621H involved in the following embodiments was purchased from the German DSM Culture Collection (DSMZ); the pK18mobSacB plasmid involved in the following embodiments was purchased from the BioVector Plasmid Vector Culture Cell Gene Collection.

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

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

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

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

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

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

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

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

[0045] Adaptive evolution of Gluconobacter oxydans to styrene

[0046] (1) Cultivate the wild type Gluconobacter oxydans to the logarithmic growth phase and use it as the starting culture for adaptive evolution.

[0047] (2) The starting bacterial liquid is transferred to the initial styrene liquid culture medium, and after culturing to the logarithmic phase, it is further transferred to the liquid culture medium with increasing styrene concentrations. In this way, the strain is continuously subcultured in the culture medium with increasing styrene concentrations until the strain stops growing completely.

[0048] (3) Take 2 mL of the final gradient-evolved bacterial solution, spread it on a sorbitol solid medium, and cover it with about 3 mm of styrene at the final screening concentration. After culturing for 72-84 h, the final highly styrene-tolerant single colony was isolated and named Gluconobacter oxydans ST.

[0049] Preparation of competent cells of Gluconobacter oxydans:

[0050] Inoculate a single colony of Gluconobacter oxydans into 10 mL of sorbitol medium and culture at 30°C and 220 rpm until the logarithmic growth phase. Transfer to 100 mL of sorbitol medium at 1% (v / v), culture at 30°C and 220 rpm for 6-8 h, then centrifuge at 4°C and 8000 rpm for 20 min to remove the supernatant, collect the cells, resuspend and wash the cells three times with 10% (v / v) glycerol, and finally add 1 mL of 10% (v / v) glycerol to fully resuspend, dispense 100 μL of the resuspension into each 1.5 mL centrifuge tube, and store at -80°C.

[0051] Oxidized gluconate rod electroporation method:

[0052] Add 1000 ng of the constructed DNA knockout fragment and the competent cell into a pre-cooled electroporation cup, electroporate at 1.8 kV for 4.8 ms, and then immediately add 600 μL of fresh sorbitol medium. After culturing at 30°C and 220 rpm for 5 h, spread it on a sorbitol solid plate containing 50 μg / mL kanamycin and culture it upside down at 30°C for 48-60 h until colonies grow.

[0053] The tolerance detection method involved in the following embodiments is as follows:

[0054] Cultivate Gluconobacter oxydans to the logarithmic growth phase, measure its absorbance at 600 nm using a spectrophotometer, and dilute its OD with fresh culture medium. 600 The concentration was 2.0, and a uniform concentration treatment was performed; then different concentrations of styrene were added to 10 mL of the bacterial suspension treated with the uniform concentration, and the OD was measured again after culturing at 30°C and 220 rpm for 10 h. 600 OD of bacterial suspension after styrene treatment 600 Characterizing tolerance.

[0055] Example 1 Preparation of Gluconacetobacter oxidans ST

[0056] The specific steps are as follows:

[0057] (1) First, the wild-type Gluconobacter oxydans was inoculated into sorbitol medium and cultured to the logarithmic growth phase as seed solution. Then, 1.5 mL of fresh sorbitol medium was added to a 24-well plate, 100 μL of seed solution 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, respectively. 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.

[0058] The results are as follows Figure 1 As shown, the dose-limiting growth test of two cosolvents (n-hexadecane and DMSO) on Gluconobacter oxydans found 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. In the initial stage of evolution, high cell density is essential. The survival rate of Gluconobacter oxydans under 3 g / L styrene conditions is similar to that under 0 g / L conditions, which is sufficient to provide screening pressure. Therefore, 3 g / L styrene was selected as the starting concentration for evolution.

[0059] (2) After the wild-type Gluconobacter oxydans was inoculated into a sorbitol medium and cultured to the logarithmic growth phase, it was inoculated into a sorbitol medium with 3 g / L styrene added for adaptive evolution. Three independent repetitions were set. After culturing to the logarithmic growth phase, it was transferred to the next adaptive evolution gradient at a 1% (v / v) inoculum amount. Each time the styrene concentration was increased by 0.2 g / L to promote adaptation. After 10 transfers, cell growth slowed down and resistant cells appeared. As the number of transfers increased, the styrene concentration also gradually increased, and the time 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 it took 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 was terminated.

[0060] The final adaptively evolved bacterial liquid was spread on a sorbitol solid culture medium and covered with about 3 mm of 15 g / L styrene (with ethanol as the solvent). The single colony isolated after cultivation was the final evolved strain, which was named Gluconoacetobacter oxidans ST; the Gluconoacetobacter oxidans ST was deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20242435.

[0061] Example 2: Experiment on the growth of evolved strain ST tolerant to styrene

[0062] The specific steps are as follows:

[0063] The wild strain 621H and the evolved strain ST obtained in Example 1 were activated respectively, and then cultured at 30°C and 220 rpm until the logarithmic growth phase to obtain seed solution. The absorbance values ​​of the two bacterial solutions at 600 nm were measured respectively, and the bacterial solutions were diluted with culture medium to an initial OD 600 The two seed solutions were inoculated at 1% (v / v) into 100 ml of fresh sorbitol liquid medium and 7 g / L and 15 g / L styrene medium, respectively, and cultured at 30 ° C and 220 rpm. The OD of the bacterial solution was measured every 4 h. 600 Values, draw growth curves (see Figure 2 ).

[0064] The results showed that both the wild strain 621H and the evolved strain ST grew well without styrene stress. The wild strain 621H could hardly survive under styrene stress above 7g / L, while the evolved strain ST could still grow under styrene stress of 15g / L, indicating that the strain ST's ability to tolerate styrene was significantly improved after adaptive evolution. However, styrene inhibits bacterial growth, and the growth of the evolved strain will be partially inhibited even without the addition of styrene stress.

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

[0066] The specific steps are as follows:

[0067] (1) The wild strain 621H and the evolved strain ST were activated separately, and then cultured at 30°C and 220 rpm until the logarithmic growth phase to obtain seed solution, and its absorbance at 600 nm was measured. The culture medium was used to dilute the bacterial solution to the initial OD 600 Both were 2.0, and bacterial suspensions of the evolved strain ST and the original strain 621H were obtained.

[0068] (2) Organic solvents show a dependence on logP o / w Toxicity, at low logP o / w The final evolved strain was able to tolerate 15 g / L styrene (logP o / w =3), which also shows that it is tolerant to other o / w Therefore, five different logP o / w Growth experiments were performed at 1:1 level 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). 15 g / L of different selected logP o / w The OD of the bacterial suspension was measured after culturing at 30°C and 220 rpm for 10 h. 600 , the results are as follows Figure 3 And as shown in Table 1.

[0069] The results showed that compared with the wild type, the evolved strain ST showed higher tolerance to solvents except toluene, and its tolerance to phenol, benzene, toluene, p-xylene, and propylbenzene increased by 1.77, 1.81, 1.18, 1.66, and 2.65 times, respectively; this indicates that adaptive evolution has enhanced the solvent tolerance of Gluconobacter oxydans and successfully produced highly tolerant mutants. Therefore, the evolved strain ST has a broad spectrum of tolerance to benzene-based solvents.

[0070] Table 1 The ability of evolved strains to improve stress under different organic solvents

[0071]

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

[0073] The specific steps are as follows:

[0074] Using the ST genome of Evolved Gluconobacter oxydans as a template, 1000 bp fragments of the upstream and downstream homologous arms of the flagellar protein flgE gene were obtained by PCR amplification, and then the two homologous arms were fused into a 2000 bp knockout fragment by fusion PCR, and then the knockout fragment was connected to the linearized plasmid pK18mobSacB after EcoR I and Sal I digestion, and transformed into Escherichia coli (Escherichia coli) JM109; the transformation product was spread on LB solid culture medium (containing 50 μg / mL kanamycin), and inverted cultured at 37°C for 12 to 16 hours to obtain transformants; after randomly picking transformants for colony PCR verification, the positive transformants were picked and inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220rpm for 8 to 12 hours, and then 2 mL of bacteria were collected, the plasmid was extracted, and EcoR I and Sal I were performed. I enzyme digestion was followed by electrophoresis verification and sequencing verification. After sequencing verification was correct, the recombinant E. coli JM109 / pK18mobSacB-HA was obtained; the recombinant expression plasmid JM109 / pK18mobSacBmobSacB-HA was extracted and electrotransformed into the evolved strain Gluconobacter oxydans ST, and the transformation product was spread on a solid medium containing 50 μg / mL kanamycin and sorbitol, and inverted at 30°C for 48 to 60 hours to obtain transformants; transformants were randomly selected for colony PCR verification, and positive transformants were selected and inoculated into a sorbitol solid medium containing 50 μg / mL kanamycin. The culture medium was incubated at 30°C and 220rpm for 16 to 24h until the logarithmic growth phase, then transferred to a sucrose medium at a 1% (v / v) inoculation amount, and cultured at 30°C and 220rpm for 16 to 24h. 100 μL of the bacterial solution was spread on a sorbitol solid plate, and incubated at 30°C for 48h. After the colonies on the plate grew, colony PCR verification was performed. The verified positive transformants were inoculated into sorbitol liquid medium until they grew to the logarithmic growth phase, and then their genomes were extracted and sent to the company for genome sequencing verification, and finally the gene-deficient strain ST-ΔflgE was obtained.

[0075] Table 2 Primers used for flgE gene deletion

[0076]

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

[0078] The specific steps are as follows:

[0079] (1) Gluconobacter oxydans (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, and bacterial suspensions of the gene-deficient strain ST-ΔflgE evolved strain ST and the wild strain 621H were prepared respectively;

[0080] (2) Add styrene to the bacterial suspension prepared in step (1) at final concentrations of 0, 7, and 15 g / L, respectively, and culture at 30°C and 220 rpm for 10 h. Dilute to 10% by gradient concentration. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 times, then take 3 μL of the diluted bacterial suspension of the four strains, spot it on a sorbitol solid plate, incubate it upside down at 30°C for 48 hours, and monitor the growth status of the colonies on the plate. The results are as follows Figure 5 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 .

[0081] The results are as follows Figure 4 The results showed that under organic solvent stress, the growth performance of the gene-deficient Gluconobacter oxydans ST-ΔflgE was better than that of strains in other groups. When 7 g / L or 15 g / L styrene was added, the gene-deficient strain ST-ΔflgE showed superior growth performance, confirming that knocking out the flagellar protein FlgE in Gluconobacter oxydans can effectively improve its stress tolerance to styrene.

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

[0083] The specific steps are as follows: after streaking activation of the wild-type strain 621H, the strain ST obtained in Example 1, and the strain ST-ΔflgE obtained in Example 4 on a sorbitol solid medium, a single colony was picked and inoculated into a sorbitol liquid medium, and cultured 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 bacterial solution was diluted with a sorbitol liquid medium to an OD of 0. 600All were treated with a uniform concentration of 2.0; then 15 g / L of styrene was added to the bacterial solution after the uniform concentration, and cultured at 30°C and 220 rpm for 10 h; a sterile inoculation needle was used to dip the diluted bacterial solution and vertically inserted into the center of the semi-solid culture medium. The agar concentration of the semi-solid culture medium was 0.3% and 0.4% (w / v), respectively, to determine the swimming and surging ability of the cells. Then the semi-solid culture medium was placed upright at 30°C for static culture for about 72 h and photographed and recorded ( Figure 5 ).

[0084] Results from Figure 5 As shown in Table 3, under styrene stress, the diameter and area of ​​the movement circle formed on the surface of the culture medium plate decreased, and the motility of the evolved strain was significantly reduced compared with the wild type of Gluconobacter oxydans, including cluster swimming ability and surging ability. After knocking out the flagellin FlgE, the motility and tolerance to styrene were improved, indicating that flagellin FlgE is one of the methods to improve the tolerance of Gluconobacter oxydans to styrene.

[0085] Table 3 Average diameter of bacterial rings formed by cell movement

[0086]

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

[0088] The specific steps are as follows:

[0089] The wild-type strain 621H, the strain ST obtained in Example 1, and the strain ST-ΔflgE obtained in Example 4 were streaked on a sorbitol solid culture medium, and single colonies grew after inverted culture at 30°C for 48 hours; single colonies were picked and inoculated into sorbitol liquid culture medium, and cultured at 30°C and 220rpm for 24 hours to obtain seed liquid; the seed liquid was transferred to fresh sorbitol liquid culture medium at an inoculation amount of 1% (v / v), and cultured at 30°C and 220rpm for 12 hours to obtain culture liquid; the OD of the culture liquid was measured. 600 Dilute with culture medium to OD 600The concentrations of styrene and 15 g / L styrene were added to the bacterial solution with the same concentration, and the solution was cultured at 30°C and 220 rpm for 10 h. The culture solution after styrene stress culture was diluted one thousand times, and 700 μL of the diluted bacterial solution was taken into a 1.5 mL EP tube. After culturing at 30°C for 48 h, all the bacterial solution was poured out, and 700 μL of 0.1% (w / v) crystal violet dye solution was added and dyed for 30 minutes. Then 800 μL of physiological saline was added several times to rinse the crystal violet dye solution and free bacteria in the tube until the physiological saline in the tube became clear and transparent. Finally, 200 μL of anhydrous ethanol was added to each tube to dissolve the crystal violet dye in the tube. After standing for 10 minutes, the wavelength A of each sample was measured. 595 Lower absorbance value.

[0090] As shown in Table 4, the biofilm formation ability of the evolved strain ST is slightly higher than that of the wild type, which is 1.06 times that of the wild type. The gene-deficient strain ST-ΔflgE has the strongest biofilm formation ability, which is 1.34 times that of the wild type 621H and 1.25 times that of the evolved strain ST, indicating that the gene-deficient strain ST-ΔflgE has significantly improved tolerance to styrene stress. This proves that flagellar protein FlgE can regulate biofilm formation to regulate the tolerance of cells to styrene stress.

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

[0092]

[0093] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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. A strain of Gluconobacter oxydans ST was deposited in the China Center for Type Culture Collection on November 5, 2024, with the deposit number CCTCC NO:M 20242435.

2. A microbial preparation containing the Gluconobacter oxydans ST according to claim 1.

3. An engineered bacterium of Gluconobacter oxidans, characterized in that: The engineered bacterium of Gluconobacter oxydans is obtained by knocking out the flagellin gene flgE in the Gluconobacter oxydans ST described in claim 1.

4. The engineered bacteria according to claim 3, characterized in that The amino acid sequence of the flagellin FlgE is shown in SEQ ID NO.

1.

5. A method for improving the ability of Gluconobacter oxydans to resist styrene stress, characterized in that: The method is to inhibit or weaken the expression of flagellin FlgE in the Gluconobacter oxydans.

6. The method according to claim 5, characterized in that The method is to knock out the flagellin gene flgE in Gluconobacter oxydans.

7. The method according to claim 5 or 6, characterized in that The Gluconobacter oxydans includes Gluconobacter oxydans 621H or Gluconobacter oxydans ST.

8. The method according to any one of claims 5 to 7, characterized in that The amino acid sequence of the flagellin FlgE is shown in SEQ ID NO.

1.

9. The use of flagellin FlgE in improving the ability of Gluconobacter oxydans to resist styrene stress, characterized in that: The application is to knock out the flagellin FlgE gene of Gluconobacter oxydans. 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.

10. Use of the Gluconobacter oxydans ST according to claim 1, or the microbial preparation according to claim 2, or the engineered bacteria according to claim 3 or 4, or the method according to any one of claims 5 to 8 in the microbial treatment of wastewater containing styrene or in biocatalysis using styrene as a substrate.

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