A method for improving the tolerance of gluoxydans oxydans to phenylethylene stress by overexpressing the gene marR

Abstract

The application discloses a method for improving the tolerance of Gluconobacter oxydans to phenylethylene stress by overexpressing a gene marR and belongs to the technical field of genetic engineering and microbial engineering. The application significantly improves the tolerance of Gluconobacter oxydans to phenylethylene stress by overexpressing a MarR family transcriptional regulator marR (GOX_RS11840) in Gluconobacter oxydans. The survival rate of the recombinant Gluconobacter oxydans prepared by the method of the application after stress culture for 10 hours in an environment with 15 g / L phenylethylene is 1.6 times that of a control strain of Gluconobacter oxydans.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for improving the tolerance of Gluconobacter oxydans to styrene stress by overexpressing the gene marR, belonging to the fields of genetic engineering and microbial engineering. BACKGROUND

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

[0003] However, in the industrial biological catalysis of styrene by Gluconobacter oxydans, the catalytic substrate is insoluble in water, which seriously affects the catalytic conversion rate and production performance of Gluconobacter oxydans. In order to maintain the substrate solubility of Gluconobacter oxydans, it is common in industry to add DMSO, alkanes and other organic cosolvents to improve the substrate solubility during the catalysis of Gluconobacter oxydans.

[0004] However, the addition of some organic cosolvents often leads to the accumulation of toxic stress in the catalysis of Gluconobacter oxydans. The increased substrate solubility exposes the cells to more stress, and the by-products formed by the growth and metabolism of Gluconobacter oxydans from these organic substrates will again cause the cells to be in a high stress environment, affecting the growth and metabolism of Gluconobacter oxydans again.

[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. cultured Gluconobacter oxydans in a medium containing toxic inhibitor compounds to detect the effect of the toxic inhibitor on Gluconobacter oxydans, but this method only identifies the strength of the inhibition and does not practically solve how Gluconobacter oxydans develops resistance to toxic substances and detoxification (see reference for details 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, there is an urgent need to find a better method to improve the resistance of Gluconobacter oxydans to styrene stress. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application 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 in the catalytic process of Gluconobacter oxydans, affecting the growth and metabolism of Gluconobacter oxydans.

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

[0008] The present application also provides a method for adaptive evolution of Gluconobacter oxydans to high-concentration styrene. The adaptive evolution method for tolerance to high-concentration styrene is to acclimate Gluconobacter oxydans by gradient addition of styrene in the culture medium.

[0009] In some embodiments, the starting strain for the adaptive evolution of styrene is Gluconobacter oxydans 621H.

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

[0011] In some embodiments, the initial styrene concentration in the adaptive evolution of styrene is 0.2 g / L, and the styrene concentration is increased by 0.2 g / L in sequence, and finally the highest concentration of styrene that the Gluconobacter oxydans can tolerate is 15 g / L.

[0012] The first technical solution provided by the present application is the application of transcriptional regulator MarR in improving the anti-styrene stress ability of Gluconobacter oxydans, which is overexpressing the transcriptional regulator MarR in Gluconobacter oxydans, and the amino acid sequence of the transcriptional regulator MarR is shown in SEQ ID NO. 1.

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

[0014] The second technical solution provided by the present application is a recombinant Gluconobacter oxydans, which is a host of Gluconobacter oxydans ST and overexpresses the transcriptional regulator MarR.

[0015] In some embodiments, the amino acid sequence of the transcriptional regulator MarR is shown as SEQ ID NO. 1.

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

[0017] In some embodiments, the Gluconobacter oxydans ST has been deposited in the China Center for Type Culture Collection on November 5, 2024, and the deposit number is CCTCC NO: M 20242435.

[0018] In some embodiments, the overexpression is to integrate the marR gene into a vector to obtain a recombinant plasmid containing a MarR family transcriptional regulator, and then to transfer the recombinant plasmid into the Gluconobacter oxydans ST to obtain a recombinant strain.

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

[0020] The third technical solution provided by the present application is a method for improving the stress tolerance of Gluconobacter oxydans to styrene, which is to overexpress the transcriptional regulator MarR in Gluconobacter oxydans.

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

[0022] The present application also provides a method for detecting the tolerance of Gluconobacter oxydans to styrene and other organic solvents, which is to add different concentrations of styrene to the above-mentioned Gluconobacter oxydans recombinant bacteria after they are cultured to the logarithmic growth phase, to perform stress, to obtain bacterial liquid after stress, and then to dilute and spot the bacterial liquid.

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

[0024] In some embodiments, the styrene stress culture condition is 30℃, 220 rpm for 10 h.

[0025] In some embodiments, the dilution of the stress bacterial liquid is to dilute the bacterial liquid to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0026] The fourth technical solution provided by the present application is the application of the recombinant Gluconobacter oxydans bacteria of the second technical solution or the method of the third technical solution in microbial treatment of styrene-containing wastewater or biological catalysis with styrene as a substrate.

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

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

[0029] The present application provides a recombinant Gluconobacter oxydans bacteria ST-pBBR1MCS-2-marR which can tolerate styrene at a high level. The recombinant Gluconobacter oxydans bacteria ST-pBBR1MCS-2-marR is obtained by overexpressing the MarR family transcription regulator GOX_RS111840 (marR). After the bacteria are cultured to the logarithmic phase, they are stressed with styrene for 10 hours, and then diluted and plated. As a result, the recombinant Gluconobacter oxydans bacteria with significantly improved resistance to styrene stress are obtained. The survival rate of the overexpressing Gluconobacter oxydans bacteria of the present application after being cultured in a 15g / L styrene environment for 10 hours is 1.6 times higher than that of the control strain.

[0030] Biological material preservation

[0031] A Gluconobacter oxydans strain ST, the taxonomic name of which is Gluconobacter oxydans, has been preserved in the China Center for Type Culture Collection on November 5, 2024, at the address of Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20242435. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Effects of styrene, DMSO and n-hexadecane on the growth of Gluconobacter oxydans.

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

[0034] Figure 3 Growth curves of the control strain and the overexpression strain.

[0035] Figure 4 Tolerance point plate of the control strain and the overexpression strain to styrene. DETAILED DESCRIPTION

[0036] The following describes preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not intended to limit the present application. The Gluconobacter oxydans 621H involved in the following examples was purchased from the German DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH); the pBBR1MCS-2 plasmid involved in the following examples was purchased from the BioVector plasmid vector strain cell gene preservation center.

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

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

[0039] The sorbitol solid culture medium: 2% agar was added to the sorbitol liquid culture medium.

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

[0041] The styrene liquid culture medium: styrene was added to the sorbitol liquid culture medium at a corresponding concentration.

[0042] The preparation method involved in the following examples is as follows:

[0043] Preparation of Gluconobacter oxydans competence:

[0044] A single colony of Gluconobacter oxydans was inoculated into 10 mL of sorbitol culture medium, and cultured at 30°C and 220 rpm until the logarithmic growth phase; 1% (v / v) was inoculated into 100 mL of sorbitol culture medium, and cultured at 30°C and 220 rpm for 6-8 h, and then centrifuged at 4°C to remove the supernatant, the bacterial cells were collected, resuspended with 10% (v / v) glycerol and washed three times, finally 1 mL of 10% (v / v) glycerol was added for resuspension, 100 μL of the resuspension was aliquoted in a sterile 1.5 mL centrifuge tube, and stored at -80°C for standby.

[0045] Electrotransformation method of Gluconobacter oxydans:

[0046] 1000 ng of the constructed recombinant plasmid was added to the pre-cooled electrotransformation cup together with the competence, and electroshocked at 1.8 kv for 4.8 ms, and then 800 μL of fresh sorbitol culture medium was immediately added, and cultured at 30°C and 220 rpm for 5 h, and then plated on a sorbitol solid plate containing 50 μg / mL kanamycin, and cultured at 30°C for 48-60 h until the colonies grew.

[0047] Example 1 Preparation of Gluconobacter oxydans ST

[0048] The specific steps are as follows:

[0049] (1) First, the wild-type Gluconobacter oxydans was cultured in a sorbitol medium to the logarithmic growth phase as a seed liquid, and then 1.5 mL of fresh sorbitol medium was added to a 24-well plate, 100 μL of the 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 commonly used cosolvents (n-hexadecane and DMSO) and styrene were added, respectively. After 24 h of culture at 30°C and 220 rpm, the absorbance value of each well of the bacterial liquid at 600 nm was detected to reflect the growth condition, and the optimal initial concentration of styrene for the adaptive evolution of the wild-type Gluconobacter oxydans was determined.

[0050] The results are shown in Table 1. Figure 1 As shown in Table 1, the dose-limiting growth test of Gluconobacter oxydans with two cosolvents (n-hexadecane and DMSO) found that the cosolvents n-hexadecane and DMSO had no significant effect on the growth of the wild-type Gluconobacter oxydans. In contrast, 9 g / L of styrene caused the bacteria to die completely. In the initial stage of evolution, high cell density is essential, and the survival rate of Gluconobacter oxydans under the condition of 3 g / L of styrene is similar to that under the condition of 0 g / L, which is also sufficient to provide a selection pressure, so 3 g / L of styrene is selected as the initial concentration for evolution.

[0051] (2) The wild-type Gluconobacter oxydans was cultured in a sorbitol medium to the logarithmic growth phase, and then inoculated in a sorbitol medium added with 3 g / L of styrene for adaptive evolution, with 3 independent repeats. After being cultured to the logarithmic growth phase, 1% (v / v) of the inoculum was transferred to the next adaptive evolution gradient, and the concentration of styrene was increased by 0.2 g / L each time to promote adaptation. After 10 transfers, the cell growth slowed down, and resistant cells appeared. With the increase of the number of transfers, the concentration of styrene also gradually increased, and the time for Gluconobacter oxydans to reach the logarithmic growth phase became longer and longer, but as long as the adaptive capacity improved significantly over time, the evolution continued. After multiple transfers, Gluconobacter oxydans showed limited growth, and it took as long as 72 h to reach the logarithmic phase. At this time, the concentration of styrene increased to 15 g / L, and it was considered that the adaptive evolution entered a plateau, and the evolution of styrene tolerance ended.

[0052] The last adaptive evolution bacterial liquid was spread on a sorbitol solid medium and covered with about 3 mm of 15 g / L styrene (with ethanol as the solvent). After culture, the single colonies isolated were the final evolved strain, which was named Gluconobacter oxydans ST. The Gluconobacter oxydans ST was preserved in the China Center for Type Culture Collection, and the preservation number was CCTCC NO: M 20242435.

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

[0054] The specific steps are as follows:

[0055] The DNA fragment of the transcriptional regulator MarR gene was obtained by PCR amplification using the genome of Gluconobacter oxydans ST in Example 1 as the template and the two primers marR-F and marR-R, and the fragment had homologous arms of the two enzyme digestion sites EcoR I and Hind III in plasmid pBBR1MCS-2 at both ends; the DNA fragment with the homologous arms was ligated with the linearized plasmid pBBR1MCS-2 digested by EcoR I and Hind III 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 incubated at 37°C for 12-16 h to obtain the transformants; the transformants were randomly picked for colony PCR verification, and the positive transformants were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 220 rpm for 8-12 h, after which 2 mL of bacterial cells were collected, the plasmid was extracted, and the EcoR I and Hind III enzyme digestion and electrophoresis verification and sequencing verification were performed; after the sequencing verification was correct, the recombinant E. coli JM109 / pBBR1MCS-2-marR was obtained; the recombinant expression plasmid JM109 / pBBR1MCS-2-marR was extracted and electroporated into the evolution strain ST, and the transformation product was spread on sorbitol solid medium containing 50 μg / mL kanamycin and incubated at 30°C for 48-60 h to obtain the transformants; the transformants were randomly picked for colony PCR verification, and the positive transformants were sent to the company for genome sequencing verification; after the sequencing verification was correct, the gene overexpression strain ST-pBBR1MCS-2-marR was obtained.

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

[0057] Table 1. Primers used for constructing recombinant bacteria

[0058]

[0059] Example 3: Analysis of the growth ability of the control strain ST and the overexpression strains

[0060] The specific steps are as follows:

[0061] The strains ST-pBBR1MCS-2, ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were activated, and single colonies were inoculated into sorbitol liquid medium, and then cultured at 30°C and 220 rpm to obtain seed liquid at the early logarithmic growth phase. The absorbance values of the two bacterial liquids at 600 nm were measured, and the bacterial liquids were diluted with the medium to OD 600 values of 0.1; then the diluted seed liquid was inoculated into 100 ml of fresh sorbitol liquid medium at an inoculation amount of 1% (v / v), and G. oxydans ST-pBBR1MCS-2 was used as a control and cultured at 30°C and 220 rpm.

[0062] During the culture, the OD 600 values of the bacterial liquids were measured every 8 h, and the growth curves were plotted (see Figure 3). The results showed that the growth states of the control strain ST-pBBR1MCS-2 and the three recombinant strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were good without styrene stress. However, the growth performance of the recombinant strains ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW was not as good as that of the control strain ST-pBBR1MCS-2, and the growth ability of the recombinant strain ST-pBBR1MCS-2-marR was the strongest.

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

[0064] The specific steps are as follows:

[0065] The control strain ST-pBBR1MCS-2 and the 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, and then their absorbance at 600 nm was measured before being diluted with a sorbitol liquid medium, and the initial OD 600 was controlled to be 2.0. The control strain ST-pBBR1MCS-2 and the three recombinant strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were prepared. 0 and 15 g / L styrene was added to the above prepared bacterial suspensions, respectively, and the OD 600 was measured after 10 h of stress culture at 30°C and 220 rpm, and then the bacterial suspensions were diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 , respectively. Then 3 μL of the diluted bacterial suspensions were spotted on sorbitol solid plates, and the plates were cultured at 30°C for 48 h until the colonies grew out. The results are shown in Figure 4 , wherein, from left to right, the gradient dilutions of the bacterial suspensions are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0066] The results are shown inFigure 4 It is shown that the overexpression of GOX_RS11840 (marR) in O. oleoxydans can effectively improve its stress tolerance to styrene. The overexpression strain ST-pBBR1MCS-2-marR shows superior growth performance under the condition of adding 15 g / L styrene, which proves that overexpression of MarR family transcriptional regulator GOX_RS11840 (marR) in O. oleoxydans can effectively improve its stress tolerance to styrene.

[0067] Example 5: Survival rate experiment of overexpression strains under the condition of styrene stress

[0068] The specific steps are as follows:

[0069] The control strain ST-pBBR1MCS-2 and the three overexpression strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW were streaked on sorbitol solid medium for activation, and cultured at 30°C until colonies grew. Single colonies were picked and inoculated in sorbitol liquid medium, and cultured at 30°C, 220 rpm to the logarithmic growth phase to obtain seed liquid. After measuring the absorbance at 600 nm, the seed liquid was diluted with sorbitol liquid medium to control the initial OD 600 at 2.0. The seed liquid was inoculated in sorbitol liquid medium at a 1% (v / v) inoculation amount, and cultured at 30°C, 220 rpm for 18 h to obtain the culture liquid of the control strain ST-pBBR1MCS-2 and the three recombinant strains ST-pBBR1MCS-2-marR, ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW. After measuring the absorbance at 600 nm again, the culture liquid was diluted with sorbitol liquid medium to control the initial OD 600 at 2.0. 10 mL of the culture liquid was collected and centrifuged at 12000 rpm to collect the cells. The collected cells were washed twice with PBS and resuspended in 10 mL of sorbitol liquid medium containing 15 g / L styrene. After being cultured at 30°C, 220 rpm for 10 h, bacterial suspension was obtained. 200 μL of bacterial suspension was spread on 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). The survival rate = (A / A0) x 100%; A represents the number of viable bacteria grown on sorbitol solid medium containing 50 μg / mL kanamycin after being cultured with styrene for 10 h; A0 is the number of viable bacteria of the bacterial suspension without styrene stress treatment on sorbitol solid medium containing 50 μg / mL kanamycin.

[0070] As shown in Table 2, after 10 hours of stress in the 15 g / L styrene sorbitol liquid medium, the survival rate of the recombinant S. oxidans ST-pBBR1MCS-2-marR was 1.6 times that of the control, indicating that the tolerance of the recombinant S. oxidans ST-pBBR1MCS-2-marR to styrene stress was significantly improved; after 10 hours of stress in the 15 g / L styrene sorbitol liquid medium, the survival rates of the recombinant S. oxidans 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 S. oxidans ST-pBBR1MCS-2-GOX_RS15020 and ST-pBBR1MCS-2-ompW to styrene stress was not improved. This proves that the transcriptional regulator MarR can regulate the tolerance of cells to styrene stress, while the proteins GOX_RS15020 and OmpW cannot.

[0071] Table 2 Influence of styrene stress on the survival rate of different overexpression strains

[0072]

[0073] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. The application of transcriptional regulator MarR in enhancing the resistance of *Glucobacterium oxidans* to styrene stress, characterized in that, The application involves overexpressing the transcriptional regulatory factor MarR in *Glucosamine oxyphylla*, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The application 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 application according to claim 1, characterized in that, The *Glucosamine oxidans* is a wild-type *Glucosamine oxidans* (… Gluconobacter oxydans )621H or Glucosamine oxidase ( Gluconobacter oxydans The *Gluconobacterium oxysporum* ST was deposited at the China Center for Type Culture Collection on November 5, 2024, with accession number CCTCC NO:M 20242435.

4. A recombinant strain of *Glucobacterium oxysporum*, characterized in that, The recombinant glucosamine oxidase strain uses glucosamine oxidase ST as the host and overexpresses the transcriptional regulatory factor MarR. 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. The amino acid sequence of the transcriptional regulatory factor MarR is shown in SEQ ID NO.

1.

5. A method for improving the resistance of *Glucobacterium oxidans* to styrene stress, characterized in that, The method involves overexpressing the transcriptional regulatory factor MarR in *Glucosamine oxyphylla*, the amino acid sequence of which is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, The glucosobacterium oxidans is wild-type glucosobacterium oxidans 621H or glucosobacterium oxidans ST. The glucosobacterium oxidans ST was deposited at the China Center for Type Culture Collection on November 5, 2024, with accession number CCTCC NO:M 20242435.

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