Genetically engineered bacterium for producing rhamnolipid and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing rhamnolipid and application of the genetically engineered bacterium, and belongs to the technical field of microorganisms and genetic engineering. According to the invention, a Psl polysaccharide coding gene cluster and a Pel polysaccharide coding gene cluster of Pseudomonas aeruginosa AB93066 (starting strain) are knocked out, a coding gene FleQ of a cyclic diguanylate receptor is further knocked out, and a response regulation factor BfmS is over-expressed, so that the genetically engineered bacterium for producing rhamnolipid is obtained. The strain has the advantages of short fermentation period, strong rhamnolipid synthesis capability and weak biofilm formation capability, can effectively improve the production efficiency of rhamnolipid and reduce the biosafety risk, and is suitable for industrial production and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and genetic engineering, and more specifically, relates to a genetically engineered bacterium for producing rhamnolipid and an application thereof. Background Art

[0002] Rhamnolipids are surface-active glycolipid metabolites produced by Pseudomonas microorganisms under certain culture conditions. They are water-soluble anionic surfactants. They are amphiphilic and can reduce the surface tension of water. Their CMC value, approximately 33.3 mg / L, is lower than that of common surfactants, making them useful as wetting agents, emulsifiers, and foaming agents. Furthermore, rhamnolipids possess a range of excellent properties, including mildness, temperature and salt tolerance, antibacterial properties, biodegradability, and safety. Research has shown that rhamnolipids can improve crude oil recovery, promote crop root growth, accelerate the degradation of long-chain alkanes and polycyclic aromatic hydrocarbons in soil, inhibit crop pathogens, and remove oil and stains. They hold broad application prospects in the petrochemical industry, agricultural environments, and household cleaning.

[0003] The main rhamnolipid production strains currently used in industry are natural high-yield rhamnolipid or genetically engineered Pseudomonas aeruginosa. aeruginosa), such as the invention patent with patent number CN119662485A, which discloses a strain of Pseudomonas aeruginosa that can tolerate high concentrations of ethanol and produce a high amount of rhamnolipid, with the rhamnolipid production reaching over 100 g / L after 96 hours of fermentation; the invention patent with patent number CN113481140B, which discloses a genetically engineered strain of Pseudomonas aeruginosa with the global transcription factor encoding gene MexT knocked out, whose rhamnolipid synthesis ability is significantly improved compared with the starting strain; the invention patent with application number 201410778468.3, which discloses a method for producing rhamnolipid by semi-solid fermentation of Pseudomonas aeruginosa AB93066 (purchased from CCTCC) using agricultural waste rapeseed meal and bran as raw materials, adding nutrients such as glycerol and phosphate, and fermenting for 12 days, with the final rhamnolipid production reaching 30 g / kg. However, in actual production, the fermentation cycle required for Pseudomonas aeruginosa to synthesize rhamnolipids is typically more than 5 days, which increases the energy consumption and labor costs of the fermentation process. Moreover, Pseudomonas aeruginosa is a conditionally pathogenic bacterium and is prone to forming biofilms, which poses certain safety risks. Therefore, improving the production efficiency of rhamnolipids, reducing production costs, and improving the safety of production strains remain several major issues that need to be addressed in industrial production. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provide a genetically engineered bacterium for producing rhamnolipid and its application. The technical solution adopted by the present invention is as follows:

[0005] The present invention first provides a genetically engineered bacterium that produces rhamnolipids. The genetically engineered bacterium is constructed by the following method: the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene cluster of Pseudomonas aeruginosa AB93066 (purchased from the China Center for Type Culture Collection (CCTCC), with the collection number CCTCC AB93066) are knocked out, and the cyclic diguanylate receptor encoding gene FleQ is further knocked out, and the response regulatory factor BfmS is overexpressed to obtain the genetically engineered bacterium that produces rhamnolipids.

[0006] In the technical solution of the present invention, the Psl polysaccharide encoding gene cluster includes pslA, pslB, pslC, pslD, pslE, pslF, pslG, pslH, pslI, pslJ, pslK, pslL, pslM, and pslN, and the gene sequences are shown in SEQ ID No. 1 to SEQ ID No. 14; the Pel polysaccharide encoding gene cluster includes pelA, pelB, pelC, pelD, pelE, pelF, and pelG, and the gene sequences are shown in SEQ ID No. 15 to SEQ ID No. 21. Furthermore, the gene sequence of FleQ is shown in SEQ ID No. 22; the gene sequence of bfmS is shown in SEQ ID No. 23.

[0007] According to a preferred embodiment of the present invention, the method for overexpressing the response regulatory factor BfmS is as follows: The genomic DNA of AB93066 is used as a template to amplify and obtain a bfmS gene expression fragment, which is double-digested with EcoRI and HindIII, and the obtained fragment is ligated with the shuttle vector pHERD20T double-digested with EcoRI and HindIII to obtain a recombinant plasmid pHERD20T-bfmS; the recombinant plasmid pHERD20T-bfmS is transformed into Escherichia coli β2163 competent cells to obtain the donor bacteria β2163 / pHERD20T-bfmS, and the engineered bacteria AB93066ΔpslΔpelΔfleQ in which the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene and the cyclic diguanylate receptor encoding gene FleQ are knocked out is used as the recipient bacteria for conjugation, and the bacteria are cultured until a single clone is formed to obtain the genetically engineered bacteria that produces rhamnolipid.

[0008] According to a preferred embodiment of the present invention, the methods for knocking out the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene and the cyclic diguanylate receptor encoding gene FleQ are all homologous recombination methods mediated by the suicide plasmid pLP12.

[0009] Furthermore, the method for knocking out the Psl polysaccharide encoding gene cluster is: extracting the genomic DNA of Pseudomonas aeruginosa AB93066, using the genomic DNA as a template, PCR amplifying to obtain the fusion fragment psl; recombining the fusion fragment with the suicide vector pLP12 to construct a recombinant plasmid pLP12-psl; transforming the recombinant plasmid pLP12-psl into Escherichia coli β2163 as a donor bacterium, using Pseudomonas aeruginosa AB93066 as a recipient bacterium, performing conjugation, and culturing until a single clone is formed by secondary recombination, thereby obtaining the engineered bacterium AB93066Δpsl with the Psl polysaccharide encoding gene cluster knocked out.

[0010] Furthermore, the method for knocking out the pel polysaccharide encoding gene cluster is as follows: using the genomic DNA of Pseudomonas aeruginosa AB93066 as a template, PCR amplification is performed to obtain the fusion fragment pel; the fusion fragment is recombined with the suicide vector pLP12 to construct the recombinant plasmid pLP12-pel; the recombinant plasmid pLP12-pel is transformed into Escherichia coli β2163 as a donor bacterium, and the engineered bacterium AB93066Δpsl with the Psl polysaccharide encoding gene cluster knocked out is used as a recipient bacterium for conjugation, and cultured until a single clone is formed by secondary recombination, thereby obtaining the engineered bacterium AB93066ΔpslΔpel with the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene knocked out.

[0011] According to a preferred embodiment of the present invention, the method for knocking out the cyclic diguanylate receptor encoding gene FleQ is: obtaining a fusion fragment of the upstream and downstream homologous arms of the cyclic diguanylate receptor encoding gene FleQ; recombining the fusion fragment with the suicide vector pLP12 to construct a recombinant suicide plasmid pLP12-fleQ; transforming the recombinant plasmid pLP12-fleQ into Escherichia coli β2163 as a donor bacterium, and using the engineered bacterium AB93066ΔpslΔpel in which the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene are knocked out as a recipient bacterium for conjugation, and culturing until a single clone is formed to obtain the engineered bacterium AB93066ΔpslΔpelΔfleQ in which the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene and the cyclic diguanylate receptor FleQ are knocked out.

[0012] The present invention also provides a use of the aforementioned rhamnolipid-producing genetically engineered bacteria in the fermentation production of rhamnolipid.

[0013] The present invention further provides a method for producing rhamnolipids by fermentation based on the genetically engineered bacteria: seed liquid containing the genetically engineered bacteria is inoculated into a Pseudomonas aeruginosa fermentation medium containing ampicillin and L-arabinose, and cultured at 37° C. under ventilation conditions.

[0014] Biofilm is a group structure formed by bacterial cells attached to a solid surface, which increases the resistance of microorganisms to the external environment. In the process of fermenting rhamnolipids by Pseudomonas aeruginosa, the formation of biofilm not only increases the difficulty of equipment cleaning, but also increases the risk of the strain spreading to the outside world and wastes the cell's own energy. Psl polysaccharide and Pel polysaccharide are both skeleton components of biofilm. Psl polysaccharide mainly plays a role in the formation and structural stability of biofilm in the early stage, while Pel polysaccharide plays a role in increasing biofilm thickness and promoting biofilm diffusion in the later stage of biofilm development. The present invention first knocks out the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene cluster of Pseudomonas aeruginosa AB93066, further knocks out the encoding gene of the cyclic diguanylate receptor FleQ, overexpresses the response regulatory factor BfmS, and the obtained engineered bacteria have a shortened fermentation cycle, improved production efficiency, and reduced biofilm formation compared to the starting strain. The rhamnolipid-producing genetically engineered bacteria constructed in the present invention have the advantages of a short fermentation cycle, strong rhamnolipid synthesis ability, and weak biofilm formation ability, effectively improving the production efficiency of rhamnolipids, reducing biosafety risks, and being suitable for industrial production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the electrophoretogram of Example 1, Figure 1 In (A), (B), (C), and (D), M represents DNA Marker, and 2 represents negative control; the top "1" in (A) represents the correct colony in which the psl polysaccharide encoding gene cluster was knocked out, the top "1" in (B) represents the correct colony in which the Pel polysaccharide encoding gene cluster was knocked out, the top "1" in (C) represents the correct colony in which the FleQ encoding gene was knocked out, and the top "1" in (D) represents the correct colony in which pHERD20T-bfmS was introduced.

[0016] Figure 2 This is a graph showing changes in rhamnolipid production in Example 2.

[0017] Figure 3 These are photos of the biofilm formation detection experiment in Example 3.

[0018] Figure 4 This is a graph showing the detection results of the biofilm formation amount in Example 3.

[0019] Figure 5 This is a curve diagram of rhamnolipid production change in comparative example 1.

[0020] Figure 6 This is a curve diagram of rhamnolipid production change in comparative example 2.

[0021] Figure 7This is a curve diagram of rhamnolipid production change in comparative example 2. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0023] 1. Construction method of engineering bacteria producing rhamnolipids

[0024] The rhamnolipid-producing genetically engineered bacteria of the present invention are constructed using the following approach: the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene cluster of the starting strain Pseudomonas aeruginosa AB93066 are knocked out, and the cyclic diguanylate receptor encoding gene FleQ is further knocked out, and the response regulatory factor BfmS is overexpressed to obtain the rhamnolipid-producing genetically engineered bacteria.

[0025] Typically but not limiting, in an optional specific embodiment, the following method is further used to achieve the construction of the engineered bacteria:

[0026] The Psl polysaccharide encoding gene cluster (pslA, pslB, pslC, pslD, pslE, pslF, pslG, pslH, pslI, pslJ, pslK, pslL, pslM, and pslN, gene sequences shown in SEQ ID No. 1 to SEQ ID No. 14) of Pseudomonas aeruginosa AB93066 was deleted by homologous recombination mediated by the suicide plasmid pLP12. An engineered strain lacking the Psl polysaccharide encoding gene cluster was obtained and named AB93066Δpsl.

[0027] The Pel polysaccharide encoding gene cluster (pelA, pelB, pelC, pelD, pelE, pelF, pelG, gene sequences shown in SEQ ID No. 15 to SEQ ID No. 21) of AB93066Δpsl was knocked out by homologous recombination mediated by the suicide plasmid pLP12, resulting in an engineered bacterium lacking the Pel polysaccharide encoding gene, named AB93066ΔpslΔpel.

[0028] The cyclic diguanylate receptor FleQ encoding gene (fleQ, gene sequence shown in SEQ ID No. 22) of AB93066ΔpslΔpel was knocked out by homologous recombination mediated by suicide plasmid pLP12 to obtain an engineered bacterium lacking the FleQ encoding gene, named AB93066ΔpslΔpelΔfleQ.

[0029] By constructing the shuttle vector pHERD20T-bfmS (bfmS, gene sequence is shown in SEQ ID No. 23) and introducing it into AB93066ΔpslΔpelΔfleQ, the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS overexpressing the response regulatory factor BfmS was obtained.

[0030] II. Description of the culture medium formulation and fermentation method used in the present invention

[0031] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L, pH = 7.0.

[0032] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, pH = 7.0.

[0033] Pseudomonas aeruginosa solid culture medium: tryptone 5 g / L, beef extract 5 g / L, sodium chloride 3 g / L, agar powder 20 g / L, pH=7.0.

[0034] Pseudomonas aeruginosa seed culture medium: tryptone 5 g / L, beef extract 5 g / L, sodium chloride 3 g / L, pH = 7.0.

[0035] Pseudomonas aeruginosa fermentation medium: corn oil 40 g / L, sodium nitrate 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate trihydrate 2 g / L, magnesium sulfate heptahydrate 4 g / L, yeast extract 1.5 g / L, manganese sulfate 0.05 g / L, ferrous sulfate 0.04 g / L, pH = 7.0.

[0036] Feed medium ①: corn oil.

[0037] Feed medium ②: sodium nitrate 100g / L.

[0038] The strain of the present invention was activated by streaking on a solid culture medium of Pseudomonas aeruginosa, and then cultured in a 37° C. incubator for 12 to 14 hours.

[0039] A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium, and cultured in a shaker at 37°C and 220 rpm for 12 h to obtain a first-level seed solution.

[0040] According to the inoculation volume of 3% (V / V), the first-level seed liquid was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured in a shaker at 37°C and 220 rpm for 8 h to obtain the second-level seed liquid.

[0041] At an inoculum rate of 3% (V / V), the secondary seed liquid was transferred to a 5-L fermentor containing 2.5 L of Pseudomonas aeruginosa fermentation medium. The culture temperature was 37°C, the rotation speed was 400 rpm, and the ventilation volume was 1 vvm. After culturing for 12 h, 24 h, 36 h, and 48 h, 50 g of feed medium ① and 50 mL of feed medium ② were added, respectively. During the fermentation process, samples were taken regularly to detect rhamnolipid production. Fermentation was terminated when the rhamnolipid production no longer changed or decreased.

[0042] Among them, the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS needs to be cultured using Pseudomonas aeruginosa solid culture medium and Pseudomonas aeruginosa seed culture medium, and needs to be cultured using Pseudomonas aeruginosa fermentation culture medium. 50 μg / mL of ampicillin and 0.2 wt% L-arabinose need to be added.

[0043] 3. Rhamnolipid production detection method

[0044] Preparation of L-rhamnose standard working solution: Weigh 0.25 g of dried L-rhamnose standard into a 250 mL volumetric flask and dissolve it with ultrapure water to a constant volume to prepare a standard stock solution with a concentration of 1.0 g / L. Pipette 1 mL, 3 mL, 5 mL, 7 mL, and 9 mL of the standard L-rhamnose solution into five 10 mL volumetric flasks, respectively. Add ultrapure water to the mark and shake well to obtain L-rhamnose standard working solutions with concentrations of 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, and 0.9 g / L.

[0045] Preparation of anthrone-sulfuric acid reagent: Weigh 0.1 g of anthrone into a volumetric flask, add 50 mL of 85 wt% sulfuric acid solution to dissolve and dilute to volume.

[0046] Establishment of the standard curve: Pipette 0.2mL of each concentration of L-rhamnose standard working solution into a clean test tube, place the test tube in an ice water bath, and then add 0.8mL of anthrone-sulfuric acid reagent to each test tube. Shake well after addition. Then immerse in a 100℃ water bath, boil for 10 minutes, remove, and cool naturally to room temperature. Pipette 200μL of each solution into the ELISA plate and measure the absorbance at a wavelength of 620nm. Plot the standard curve with L-rhamnose concentration as the horizontal axis and absorbance as the vertical axis, and fit to obtain the regression equation (R 2 ≥0.99).

[0047] Take the fermentation broth of the strain described in the present invention, dilute it with ultrapure water by an appropriate multiple, take 0.2mL of the diluted fermentation broth into a clean test tube, place the test tube in an ice-water bath, add 0.8mL of anthrone-sulfuric acid reagent, and shake well after addition. Then immerse it in a 100°C water bath, boil for 10 minutes, remove it, and cool it naturally to room temperature. Pipette 200μL of the reaction solution into an ELISA plate and measure the absorbance at a wavelength of 620nm. Substitute the absorbance value into the standard curve equation to calculate the L-rhamnose concentration, and then multiply it by the dilution multiple and the coefficient 3.4 to obtain the concentration of rhamnolipid in the fermentation broth.

[0048] 4. Biofilm Formation Quantity Detection Method

[0049] The strain of the present invention was streaked and activated on LB solid medium and cultured in a 37°C incubator for 12-14 hours. A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL LB liquid medium and cultured in a shaker at 37°C and 220 rpm for 12 hours to obtain a seed solution. The OD value of the seed solution was measured. 600 , press the initial OD 600 =0.1 was connected to a test tube containing 1.5mL LB liquid culture medium, and an uninoculated test tube containing 1.5mL LB liquid culture medium was set as a blank control, and they were placed at 37℃ for static culture for 3d. After the culture is completed, the bacterial liquid or culture medium in the test tube was aspirated out, washed three times with deionized water and dried. Add 2mL of 0.1wt% crystal violet solution to the dried test tube, stain at room temperature for 20min, then aspirate the dye, wash three times with deionized water and dry. Compare the color depth of the purple circle on the wall of the test tube, and take pictures and record. Add 2mL of 80wt% ethanol to the test tube, use an ultrasonic cleaner to sonicate until the purple circle is completely dissolved, and measure the OD 595 , which is the amount of biofilm formation.

[0050] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0051] Example 1 Specific construction of engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS:

[0052] The reaction system and reaction conditions used in this example are as follows:

[0053] PCR reaction system (DNA fragment amplification or fusion PCR): 10 μL of 5× PrimeSTAR buffer, 4 μL of dNTPs, 1 μL each of upstream and downstream primers, 0.5 μL of DNA template, 0.5 μL of HS PrimeStar DNA polymerase, and make up to a total volume of 50 μL with ddH2O.

[0054] PCR reaction conditions (DNA fragment amplification or fusion PCR): pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; extension at 72°C (extension time calculated at 1 kb / min) for 32 cycles; extension at 72°C for 10 min; storage at 4°C.

[0055] PCR reaction system (colony PCR verification): 2× Rapid Taq Master Mix 10 μL, upstream primer and downstream primer 0.4 μL each, ddH2O 9.2 μL, appropriate amount of DNA template, total volume 20 μL.

[0056] PCR reaction conditions (colony PCR verification): pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec; annealing at 58°C for 15 sec; extension at 72°C (extension time calculated at 4 kb / min) for 28 cycles; extension at 72°C for 2 min; storage at 4°C.

[0057] Enzyme digestion system: restriction endonuclease I 1 μL, restriction endonuclease II 1 μL, 10× QuickCut Buffer 5 μL, plasmid DNA or PCR product 500-1000 ng, and fill with ddH2O to a total volume of 50 μL.

[0058] Enzyme digestion conditions: incubate at 37°C for 5-15 min, incubate plasmid DNA for 15 min, and incubate PCR product for 5 min.

[0059] Ligation system: 10× Ligation Buffer 2μL, vector DNA 50ng, DNA fragment (molar ratio to vector DNA is about 3), T4 DNA Ligase 1μL, and fill with ddH2O to a total volume of 20μL.

[0060] Connection conditions: Keep warm at 16℃ for 1~5h.

[0061] Construction of engineered bacteria AB93066Δpsl:

[0062] Genomic DNA from Pseudomonas aeruginosa AB93066 was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). Using this genomic DNA as a template, PCR amplification was performed using primers psl-UF and psl-UR, and psl-DF and psl-DR, respectively, to generate the upstream homology arm psl-U and the downstream homology arm psl-D, which contain complementary sequences. The fusion fragment psl was amplified using primers psl-UF and psl-DR, respectively, using the upstream homology arm psl-U and the downstream homology arm psl-D as templates. The fusion fragment psl was then double-digested with XbaI and EcoRI, and the resulting fragment was ligated into the suicide vector pLP12, which had been double-digested with XbaI and EcoRI. The ligation product was chemically transformed into competent Escherichia coli DH5αλpir cells. Positive transformants were screened by adding 20 μg / mL chloramphenicol and 0.3 wt% D-glucose to LB solid medium. Positive transformants were further verified by colony PCR using primers psl-UF and psl-DR. Plasmids from confirmed transformants were extracted using a plasmid mini kit (Jiangsu Kangwei Century Biotechnology Co., Ltd.) and confirmed by sequencing to obtain the recombinant plasmid pLP12-psl.

[0063] The recombinant plasmid pLP12-psl was chemically transformed into competent Escherichia coli β2163 cells. Positive transformants were screened by adding 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose to LB solid medium to obtain the donor strain β2163 / pLP12-psl. Pseudomonas aeruginosa AB93066 was used as the recipient strain. The donor strain was inoculated into LB liquid medium containing 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose. The donor strain was then inoculated into LB liquid medium containing 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose. The cells were shaken at 37°C and 220 rpm for 12 h. 100 μL of each culture was mixed and shaken at 37°C and 220 rpm for 6 h for conjugation. The cultured bacterial liquid was spread on LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. A single colony was selected and genomic DNA was extracted as a template. PCR verification was performed using psl-UF and psl-DR as primers to screen out single colonies that underwent a single recombination. The correct single colony was inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37 ° C and 220 rpm for 12 h. The cultured bacterial liquid was spread on LB solid medium containing 0.4 wt% L-arabinose. A single colony was selected as a template and colony PCR verification was performed using psl-UF and psl-DR as primers (the correct colony band size was 1304 bp, the negative control had no band, and the electrophoresis pattern was as shown in the figure). Figure 1 (A) in Figure 1) , single colonies that underwent secondary recombination were screened. The PCR product was purified and sequenced for verification. Once verified, the engineered strain AB93066Δps1 was obtained.

[0064] Construction of engineered bacteria AB93066ΔpslΔpel:

[0065] Using genomic DNA from Pseudomonas aeruginosa AB93066 as a template, PCR amplification was performed using primers pel-UF and pel-UR, and pel-DF and pel-DR, respectively, to generate the upstream homology arm pel-U and the downstream homology arm pel-D, which contain complementary sequences. The fusion fragment pel was amplified using primers pel-UF and pel-DR, respectively, using the upstream homology arm pel-U and the downstream homology arm pel-D as templates. The fusion fragment pel was double-digested with XbaI and EcoRI, and the resulting fragment was ligated with the suicide vector pLP12, which had been double-digested with XbaI and EcoRI. The ligation product was chemically transformed into competent Escherichia coli DH5αλpir cells, and positive transformants were selected by LB solid medium supplemented with 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. Positive transformants were further verified by colony PCR using primers pel-UF and pel-DR. The correct transformants were verified by extracting the plasmid using a plasmid extraction kit and sequencing to obtain the recombinant plasmid pLP12-pel.

[0066] The recombinant plasmid pLP12-pel was chemically transformed into competent Escherichia coli β2163 cells. Positive transformants were screened by adding 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose to LB solid medium to obtain the donor strain β2163 / pLP12-pel. The engineered strain AB93066Δpsl was used as the recipient strain. The donor strain was inoculated into LB liquid medium containing 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose. The engineered strain AB93066Δpsl was then inoculated into LB liquid medium and cultured with shaking at 37°C and 220 rpm for 12 h. 100 μL of each culture was mixed and cultured with shaking at 37°C and 220 rpm for 6 h for conjugation. The cultured bacterial liquid was spread on LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. A single colony was selected and genomic DNA was extracted as a template. PCR verification was performed using pel-UF and pel-DR as primers to screen out single colonies that underwent a single recombination. The correct single colony was inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37 ° C and 220 rpm for 12 hours. The cultured bacterial liquid was spread on LB solid medium containing 0.4 wt% L-arabinose, and a single colony was selected as a template. pel-UF and pel-DR were used as primers to perform colony PCR verification (the correct colony band size was 1279 bp, and the negative control had no band. The electrophoresis pattern is as shown below). Figure 1(B) in Figure 1) , single colonies that underwent secondary recombination were screened. The PCR product was purified and sequenced for verification. Once verified, the engineered strain AB93066ΔpslΔpel was obtained.

[0067] Construction of engineered bacteria AB93066ΔpslΔpelΔfleQ:

[0068] Using genomic DNA from Pseudomonas aeruginosa AB93066 as a template, PCR amplification was performed using primers fleQ-UF and fleQ-UR, and fleQ-DF and fleQ-DR, respectively, to generate the upstream homology arm fleQ-U and the downstream homology arm fleQ-D, which contain complementary sequences. The fusion fragment fleQ was amplified using primers fleQ-UF and fleQ-DR, respectively, using the upstream homology arm fleQ-U and the downstream homology arm fleQ-D as templates. The fleQ fusion fragment was double-digested with XbaI and EcoRI, and the resulting fragment was ligated with the suicide vector pLP12, which had been double-digested with XbaI and EcoRI. The ligation product was chemically transformed into competent Escherichia coli DH5αλpir cells, and positive transformants were selected by incubating in LB solid medium supplemented with 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. Positive transformants were further verified by colony PCR using flEQ-UF and flEQ-DR primers. Correct transformants were then extracted using a plasmid miniprep kit and sequenced to obtain the recombinant plasmid pLP12-fleQ.

[0069] The recombinant plasmid pLP12-fleQ was chemically transformed into competent Escherichia coli β2163 cells. Positive transformants were screened by adding 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose to LB solid medium to obtain the donor strain β2163 / pLP12-fleQ. The engineered strain AB93066ΔpslΔpel was used as the recipient strain. The donor strain was inoculated into LB liquid medium containing 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose. AB93066ΔpslΔpel was then inoculated into LB liquid medium and cultured with shaking at 37°C and 220 rpm for 12 h. 100 μL of each culture was mixed and cultured with shaking at 37°C and 220 rpm for 6 h for conjugation. The cultured bacterial liquid was spread on LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. A single colony was selected and genomic DNA was extracted as a template. PCR verification was performed using flEQ-UF and flEQ-DR as primers to screen out single colonies that underwent a single recombination. The correct single colony was inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37 ° C and 220 rpm for 12 hours. The cultured bacterial liquid was spread on LB solid medium containing 0.4 wt% L-arabinose, and a single colony was selected as a template. Using flEQ-UF and flEQ-DR as primers, colony PCR verification was performed (the correct colony band size was 1010 bp, and the negative control band size was 1491 bp. The electrophoresis pattern is shown in Figure 2). Figure 1 (C) in Figure 1) Single colonies that underwent secondary recombination were screened. The PCR product was purified and sequenced for verification. Once verified, the engineered strain AB93066ΔpslΔpelΔfleQ was obtained.

[0070] Construction of engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS:

[0071] Using genomic DNA from Pseudomonas aeruginosa AB93066 as a template, primers bfmS-F and bfmS-R were used to amplify the bfmS gene expression fragment. The bfmS gene expression fragment was double-digested with EcoRI and HindIII, and the resulting fragment was ligated with the shuttle vector pHERD20T, which had been double-digested with EcoRI and HindIII. The ligation product was chemically transformed into competent Escherichia coli DH5α cells, and positive transformants were selected by LB solid medium supplemented with 50 μg / mL ampicillin. Positive transformants were further verified by colony PCR using primers bfmS-F and bfmS-R. Plasmids from confirmed transformants were extracted using a plasmid miniprep kit and confirmed by sequencing to obtain the recombinant plasmid pHERD20T-bfmS.

[0072] The recombinant plasmid pHERD20T-bfmS was chemically transformed into competent Escherichia coli β2163 cells. Positive transformants were screened in LB solid medium supplemented with 50 μg / mL ampicillin and 0.3 mM diaminopimelic acid to obtain the donor strain β2163 / pHERD20T-bfmS. The engineered strain AB93066ΔpslΔpelΔfleQ was used as the recipient strain. The donor strain was inoculated into LB liquid medium supplemented with 50 μg / mL ampicillin and 0.3 mM diaminopimelic acid, and AB93066ΔpslΔpelΔfleQ was inoculated into LB liquid medium. The cells were shaken at 37°C and 220 rpm for 12 h. 100 μL of each culture was mixed and shaken at 37°C and 220 rpm for 6 h for conjugation. The cultured bacterial liquid was spread on LB solid medium containing 50 μg / mL ampicillin, and a single colony was selected as a template. BfmS-test-F and bfmS-R were used as primers for colony PCR verification (the correct colony band size was 1581 bp, and the negative control had no band. The electrophoresis pattern was as shown in the figure). Figure 1 (D) in the figure). The correct single colony was verified to be the engineered bacterium AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS.

[0073] The primer sequences used in Example 1 (corresponding to SEQ ID No. 24 to SEQ ID No. 38 in the sequence listing) are shown in Table 1.

[0074] Table 1 - Primers used in Example 1

[0075] Example 2 Fermentation of engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS

[0076] The engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS were streaked and activated on a solid culture medium of Pseudomonas aeruginosa containing 50 μg / mL ampicillin and cultured in a 37°C incubator for 12-14 h.

[0077] A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium containing 50 μg / mL ampicillin, and cultured in a shaker at 37° C. and 220 rpm for 12 h to obtain a primary seed solution.

[0078] According to the inoculum volume of 3% (V / V), the primary seed solution was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium containing 50 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed solution.

[0079] According to the inoculum size of 3% (V / V), the secondary seed liquid was transferred to a 5L fermentor containing 2.5L of Pseudomonas aeruginosa fermentation medium containing 50μg / mL ampicillin and 0.2wt% L-arabinose. The culture temperature was 37℃, the rotation speed was 400rpm, and the ventilation volume was 1vvm. At the 12th, 24th, 36th, and 48th hours of culture, 50g of feed medium ① and 50mL of feed medium ② were added respectively. During the fermentation process, samples were taken regularly to detect the rhamnolipid production. The fermentation was terminated when the rhamnolipid production stopped changing or decreased (the rhamnolipid production change curve is shown in Figure 2). Figure 2 As shown), the fermentation cycle of this embodiment is 78h, the final rhamnolipid yield is 73g / L, and the production efficiency is 0.94g / L / h.

[0080] Example 3 Detection of biofilm formation

[0081] Pseudomonas aeruginosa AB93066 and engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS were streaked onto LB solid medium and incubated at 37°C for 12-14 hours. A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of LB liquid medium and incubated at 37°C, 220 rpm, for 12 hours to obtain a seed solution. The seed solution OD was measured. 600 , press the initial OD 600=0.1 was transferred to a test tube containing 1.5 mL of LB liquid culture medium. An uninoculated test tube containing 1.5 mL of LB liquid culture medium was set up as a blank control and incubated at 37°C for 3 days. After the incubation period, the bacterial liquid or culture medium in the test tube was aspirated, washed three times with deionized water, and dried. 2 mL of 0.1 wt% crystal violet solution was added to the dried test tube and dyed at room temperature for 20 minutes. The dye was then aspirated, washed three times with deionized water, and dried. The color depth of the purple ring on the test tube wall was compared and photographed (e.g. Figure 3 Add 2 mL of 80 wt% ethanol to the test tube and use an ultrasonic cleaner to sonicate until the purple circle is completely dissolved. Measure the OD 595 , which is the amount of biofilm formation (e.g. Figure 4 The results showed that the biofilm formation amount of the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS was reduced by 82% compared with the starting strain Pseudomonas aeruginosa AB93066, indicating that the biofilm formation ability of the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS obtained by the present invention was significantly reduced.

[0082] Comparative Example 1 Fermentation of Pseudomonas aeruginosa AB93066

[0083] Pseudomonas aeruginosa AB93066 was activated by streaking on Pseudomonas aeruginosa solid culture medium and cultured in a 37°C incubator for 12 to 14 hours.

[0084] A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium, and cultured in a shaker at 37°C and 220 rpm for 12 h to obtain a first-level seed solution.

[0085] According to the inoculation volume of 3% (V / V), the first-level seed liquid was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured in a shaker at 37°C and 220 rpm for 8 h to obtain the second-level seed liquid.

[0086] According to the inoculum volume of 3% (V / V), the secondary seed liquid was transferred to a 5L fermenter containing 2.5L Pseudomonas aeruginosa fermentation medium. The culture temperature was 37°C, the rotation speed was 400 rpm, and the ventilation (air) volume was 1 vvm. After culturing for 12h, 24h, 36h, and 48h, 50g of feed medium ① and 50mL of feed medium ② were added respectively. During the fermentation process, samples were taken regularly to detect the rhamnolipid production. The fermentation was terminated when the rhamnolipid production stopped changing or decreased (the rhamnolipid production change curve is shown in Figure 2). Figure 5 As shown), the fermentation period of this comparative example was 142 h, the final rhamnolipid yield was 87 g / L, and the production efficiency was 0.61 g / L / h.

[0087] Comparative Example 2 Fermentation of engineered bacteria AB93066ΔpslΔpelΔfleQ

[0088] The engineered bacteria AB93066ΔpslΔpelΔfleQ were streaked and activated on Pseudomonas aeruginosa solid culture medium and cultured in a 37°C incubator for 12-14 hours.

[0089] A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium, and cultured in a shaker at 37°C and 220 rpm for 12 h to obtain a first-level seed solution.

[0090] According to the inoculation volume of 3% (V / V), the first-level seed liquid was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured in a shaker at 37°C and 220 rpm for 8 h to obtain the second-level seed liquid.

[0091] According to the inoculum volume of 3% (V / V), the secondary seed liquid was transferred to a 5L fermenter containing 2.5L Pseudomonas aeruginosa fermentation medium. The culture temperature was 37°C, the rotation speed was 400 rpm, and the ventilation volume was 1 vvm. At the 12th, 24th, 36th, and 48th hours of culture, 50 g of feed medium ① and 50 mL of feed medium ② were added respectively. During the fermentation process, samples were taken regularly to detect the rhamnolipid production. The fermentation was terminated when the rhamnolipid production stopped changing or decreased (the rhamnolipid production change curve is shown in FIG). Figure 6 As shown), the fermentation period of this comparative example was 118 h, the final rhamnolipid yield was 79.5 g / L, and the production efficiency was 0.67 g / L / h.

[0092] Comparative Example 3

[0093] The engineered bacteria AB93066ΔpslΔpel were activated by streaking on Pseudomonas aeruginosa solid culture medium and cultured in a 37°C incubator for 12-14 hours.

[0094] A single colony with good growth was inoculated into a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium, and cultured in a shaker at 37°C and 220 rpm for 12 h to obtain a first-level seed solution.

[0095] According to the inoculation volume of 3% (V / V), the first-level seed liquid was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured in a shaker at 37°C and 220 rpm for 8 h to obtain the second-level seed liquid.

[0096] According to the inoculum volume of 3% (V / V), the secondary seed liquid was transferred to a 5L fermenter containing 2.5L Pseudomonas aeruginosa fermentation medium. The culture temperature was 37°C, the rotation speed was 400 rpm, and the ventilation volume was 1 vvm. At the 12th, 24th, 36th, and 48th hours of culture, 50 g of feed medium ① and 50 mL of feed medium ② were added respectively. During the fermentation process, samples were taken regularly to detect the rhamnolipid production. The fermentation was terminated when the rhamnolipid production stopped changing or decreased (the rhamnolipid production change curve is shown in FIG). Figure 7 As shown), the fermentation period of this comparative example was 132 h, the final rhamnolipid yield was 82.7 g / L, and the production efficiency was 0.63 g / L / h.

[0097] It can be seen from Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 that the fermentation period of the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS is shortened by 45.1% and the production efficiency is increased by 54.1% compared with Pseudomonas aeruginosa AB93066 (starting strain); the fermentation period of the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS is shortened by 33.9% and the production efficiency is increased by 40.3% compared with the engineered bacteria AB93066ΔpslΔpelΔfleQ; the fermentation period of the engineered bacteria AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS is shortened by 40.9% and the production efficiency is increased by 49.2% compared with the engineered bacteria AB93066ΔpslΔpel.

[0098] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A genetically engineered bacterium producing rhamnolipid, characterized in that: It is constructed using the following method: The Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene cluster of Pseudomonas aeruginosa AB93066 are knocked out, and the cyclic diguanylate receptor encoding gene FleQ is further knocked out, and the response regulatory factor BfmS is overexpressed to obtain the rhamnolipid-producing genetically engineered bacteria.

2. The rhamnolipid-producing genetically engineered bacterium according to claim 1, characterized in that The Psl polysaccharide encoding gene cluster includes pslA, pslB, pslC, pslD, pslE, pslF, pslG, pslH, pslI, pslJ, pslK, pslL, pslM and pslN, and the gene sequences are shown in SEQ ID No. 1 to SEQ ID No. 14; The Pel polysaccharide encoding gene cluster includes pelA, pelB, pelC, pelD, pelE, pelF and pelG, and the gene sequences are shown in SEQ ID No. 15 to SEQ ID No.

21.

3. The rhamnolipid-producing genetically engineered bacterium according to claim 1, characterized in that The gene sequence of FleQ is shown in SEQ ID No. 22; the gene sequence of bfmS is shown in SEQ ID No.

23.

4. The rhamnolipid-producing genetically engineered bacterium according to claim 1, characterized in that The method for overexpressing the response regulatory factor BfmS is: The genomic DNA of Pseudomonas aeruginosa AB93066 was used as a template to amplify the bfmS gene expression fragment. The bfmS gene expression fragment was double-digested with EcoRI and HindIII, and the obtained fragment was ligated with the shuttle vector pHERD20T double-digested with EcoRI and HindIII to obtain the recombinant plasmid pHERD20T-bfmS. The recombinant plasmid pHERD20T-bfmS was transformed into Escherichia coli β2163 competent cells to obtain the donor bacteria β2163 / pHERD20T-bfmS, and the engineered bacteria AB93066ΔpslΔpelΔfleQ in which the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene and the cyclic diguanylate receptor encoding gene FleQ were knocked out were used as the recipient bacteria for conjugation, and the cells were cultured until a single clone was formed to obtain the genetically engineered bacteria producing rhamnolipid.

5. The rhamnolipid-producing genetically engineered bacterium according to claim 1, characterized in that The methods for knocking out the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene, and the cyclic diguanylate receptor encoding gene FleQ are all homologous recombination methods mediated by the suicide plasmid pLP12.

6. The rhamnolipid-producing genetically engineered bacterium according to claim 5, characterized in that: The method for knocking out the gene encoding cyclic diguanylate receptor FleQ is: Obtaining a fusion fragment of the upstream and downstream homology arms of the gene encoding the cyclic diguanylate receptor FleQ; The fusion fragment was recombined with the suicide vector pLP12 to construct the recombinant suicide plasmid pLP12-fleQ; The recombinant plasmid pLP12-fleQ was transformed into Escherichia coli β2163 as the donor bacterium, and the engineered bacterium AB93066ΔpslΔpel in which the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene were knocked out was used as the recipient bacterium for conjugation. The cells were cultured until a single clone was formed, and the engineered bacterium AB93066ΔpslΔpelΔfleQ in which the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene and the cyclic diguanylate receptor FleQ were knocked out was obtained.

7. The use of genetically engineered bacteria in the fermentation production of rhamnolipids, characterized in that: The genetically engineered bacteria is the rhamnolipid-producing genetically engineered bacteria according to any one of claims 1 to 6.

8. A method for producing rhamnolipids by fermentation using the genetically engineered bacteria according to any one of claims 1 to 6, characterized in that: include: The seed liquid containing the genetically engineered bacteria is inoculated into a Pseudomonas aeruginosa fermentation medium containing ampicillin and L-arabinose, and cultured at 37° C. under ventilation conditions.

9. The method according to claim 8, characterized in that During the fermentation process, corn oil and sodium nitrate are added regularly; during the fermentation process, samples are taken regularly to detect the rhamnolipid production, and the fermentation is terminated when the rhamnolipid production no longer changes or decreases.

10. The method according to claim 8, characterized in that The formula of the Pseudomonas aeruginosa fermentation medium is: 40 g / L corn oil, 5 g / L sodium nitrate, 1 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate trihydrate, 4 g / L magnesium sulfate heptahydrate, 1.5 g / L yeast extract, 0.05 g / L manganese sulfate, 0.04 g / L ferrous sulfate, pH=7.0; In the Pseudomonas aeruginosa fermentation medium, the concentration of ampicillin is 50 μg / mL, and the mass percentage content of L-arabinose is 0.2%.

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