Rhamnolipid-producing genetically engineered bacteria and application thereof

Abstract

The application discloses a rhamnolipid-producing genetically engineered bacterium and application thereof, and belongs to the technical field of microorganisms and genetic engineering. A Psl polysaccharide coding gene cluster and a Pel polysaccharide coding gene cluster of Pseudomonas aeruginosa AB93066 (a starting strain) are knocked out, a coding gene FleQ of a cyclic diguanylate receptor is further knocked out, and a response regulator BfmS is overexpressed to obtain the rhamnolipid-producing genetically engineered bacterium. The strain has the advantages of a short fermentation cycle, strong rhamnolipid synthesis capacity and weak biofilm formation capacity, can effectively improve the production efficiency of rhamnolipid, reduces the biological safety risk, and is suitable for industrial production and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, and more specifically, relates to a genetically engineered bacterium that produces rhamnolipin and its applications. Background Technology

[0002] Rhamnolipids are surface-active glycolipid metabolites produced by Pseudomonas microorganisms under certain culture conditions. They belong to the category of water-soluble anionic surfactants. They are amphiphilic, reducing the surface tension of water. With a CMC value of approximately 33.3 mg / L, lower than that of commonly used surfactants, they can be used as wetting agents, emulsifiers, and foaming agents. Furthermore, rhamnolipids possess a range of excellent properties, including mildness, temperature and salt tolerance, antibacterial activity, biodegradability, and safety. Studies have shown that rhamnolipids can improve 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 have broad application prospects in petrochemicals, agricultural environments, and daily cleaning products.

[0003] Currently, the main industrial strains used for rhamnolipin production are naturally occurring high-yield rhamnolipin strains or genetically engineered Pseudomonas aeruginosa strains. For example, patent CN119662485A discloses a strain of *Pseudomonas aeruginosa* that can tolerate high concentrations of ethanol and produce high levels of rhamnolipin, with a rhamnolipin yield exceeding 100 g / L after 96 hours of fermentation; patent CN113481140B discloses a genetically engineered *Pseudomonas aeruginosa* strain with the global transcription factor coding gene MexT knocked out, whose rhamnolipin synthesis capacity is significantly improved compared to the original strain; and patent application 201410778468.3 discloses a method for producing rhamnolipin using agricultural waste such as rapeseed meal and wheat bran as raw materials, with added nutrients such as glycerol and phosphate, and using *Pseudomonas aeruginosa* AB93066 (purchased from CCTCC) for semi-solid fermentation, with a final rhamnolipin yield of 30 g / kg after 12 days of fermentation. However, in actual production, the fermentation cycle required for Pseudomonas aeruginosa to synthesize rhamnolipids typically exceeds 5 days, increasing energy consumption and labor costs. Furthermore, Pseudomonas aeruginosa, as an opportunistic pathogen, is prone to biofilm formation, posing certain safety risks. Therefore, improving the production efficiency of rhamnolipids, reducing production costs, and enhancing the safety of the production strains remain key challenges for industrial-scale production. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a genetically engineered bacterium that produces rhamnolipids and its applications. The technical solution adopted by this invention is as follows:

[0005] This invention first provides a genetically engineered bacterium that produces rhamnolipids, which is constructed using the following method: the Psl polysaccharide-coding gene cluster and the Pel polysaccharide-coding gene cluster of Pseudomonas aeruginosa AB93066 (purchased from the China Center for Type Culture Collection, accession number CCTCC AB93066) are knocked out, and the gene encoding the cyclic diguanosine monophosphate receptor FleQ is further knocked out, and the response regulator BfmS is overexpressed to obtain the genetically engineered bacterium that produces rhamnolipids.

[0006] In 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, with gene sequences 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, with gene sequences shown in SEQ ID No. 15 to SEQ ID No. 21. Further, the gene sequence of FleQ is shown in SEQ ID No. 22; and 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 regulator BfmS is as follows: using Pseudomonas aeruginosa Using the genomic DNA of AB93066 as a template, the bfmS gene expression fragment was amplified. The bfmS gene expression fragment was double-digested with EcoRI and HindIII. The resulting fragment was ligated with the shuttle vector pHERD20T, which had been 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 strain β2163 / pHERD20T-bfmS. The engineered bacterium AB93066ΔpslΔpelΔfleQ, which had the Psl polysaccharide coding gene cluster, the Pel polysaccharide coding gene, and the cyclic diguanylate receptor coding gene FleQ knocked out, was used as the recipient strain for conjugation. The strain was cultured until monoclonal formation was achieved to obtain the genetically engineered bacterium that produces rhamnolipids.

[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 FleQ gene encoding the cyclic diguanosine receptor 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 as follows: genomic DNA of *Pseudomonas aeruginosa* AB93066 is extracted, and the fusion fragment psl is amplified by PCR using the genomic DNA as a template; the fusion fragment is recombined with the suicide vector pLP12 to construct the recombinant plasmid pLP12-psl; the recombinant plasmid pLP12-psl is transformed into *Escherichia coli* β2163 as the donor bacterium and *Pseudomonas aeruginosa* AB93066 as the recipient bacterium for conjugation, and cultured until a single clone is formed through secondary recombination to obtain 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, the fusion fragment PEL is amplified by PCR; 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 the donor bacterium, and the engineered bacterium AB93066Δpsl with the Psl polysaccharide-encoding gene cluster knocked out is used as the recipient bacterium for conjugation, and cultured until a single clone is formed that undergoes secondary recombination, thus 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 (cDR) receptor encoding gene FleQ is as follows: obtaining a fusion fragment of the upstream and downstream homologous arms of the cDR receptor encoding gene FleQ; recombining the fusion fragment with the suicide vector pLP12 to construct the recombinant suicide plasmid pLP12-fleQ; transforming the recombinant plasmid pLP12-fleQ into Escherichia coli β2163 as the donor bacterium, and using the engineered bacterium AB93066ΔpslΔpel, which knocks out the Psl polysaccharide encoding gene cluster and the Pel polysaccharide encoding gene, as the recipient bacterium for conjugation, culturing until single-clone formation, to obtain the engineered bacterium AB93066ΔpslΔpelΔfleQ, which knocks out the Psl polysaccharide encoding gene cluster, the Pel polysaccharide encoding gene, and the cDR receptor FleQ.

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

[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 fermentation medium of Pseudomonas aeruginosa containing ampicillin and L-arabinose, and cultured at 37°C under aeration.

[0014] Biofilms are community structures formed by bacterial cells adhering to solid surfaces, enhancing the resistance of microorganisms to the external environment. In the fermentation process of *Pseudomonas aeruginosa* to produce rhamnolipids, biofilm formation not only increases the difficulty of equipment cleaning but also raises the risk of the strain spreading to the outside world and wastes cellular energy. Both Psl polysaccharide and Pel polysaccharide are skeletal components of biofilms. Psl polysaccharide mainly plays a role in the early formation and structural stability of biofilms, while Pel polysaccharide increases biofilm thickness and promotes biofilm diffusion in the later stages of development. This invention first knocks out the Psl polysaccharide and Pel polysaccharide coding gene clusters of *Pseudomonas aeruginosa* AB93066, further knocking out the coding gene for the cyclic diguanylate receptor FleQ, and overexpressing the response regulator BfmS. Compared to the starting strain, the resulting engineered strain has a shorter fermentation cycle, higher production efficiency, and lower biofilm formation. The genetically engineered bacterium for producing rhamnolipin constructed in this invention has the advantages of short fermentation cycle, strong rhamnolipin synthesis ability and weak biofilm formation ability, which effectively improves the production efficiency of rhamnolipin, reduces biosafety risks, and is suitable for industrial production and application. Attached Figure Description

[0015] Figure 1 This is an electrophoresis image of Example 1. Figure 1 In (A), (B), (C), and (D), M represents DNA Marker, and 2 represents negative control. The "1" at the top of (A) represents the correct colony with the psl polysaccharide-encoding gene cluster knocked out. The "1" at the top of (B) represents the correct colony with the Pel polysaccharide-encoding gene cluster knocked out. The "1" at the top of (C) represents the correct colony with the FleQ-encoding gene knocked out. The "1" at the top of (D) represents the correct colony with pHERD20T-bfmS introduced.

[0016] Figure 2 This is a graph showing the change in rhamnolipin production in Example 2.

[0017] Figure 3 This is a photograph of the biofilm formation detection experiment in Example 3.

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

[0019] Figure 5 The graph shows the change in rhamnolipin production as a comparative example 1.

[0020] Figure 6 The graph shows the change in rhamnolipin production as a comparative example 2.

[0021] Figure 7The graph shows the change in rhamnolipin production as a comparative example 2. Detailed Implementation

[0022] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0023] I. Construction Method of Engineered Bacteria Producing Rhamnolipids

[0024] The rhamnolipid-producing genetically engineered bacterium of the present invention was constructed using the following approach: the Psl polysaccharide-coding gene cluster and the Pel polysaccharide-coding gene cluster of the starting strain Pseudomonas aeruginosa AB93066 were knocked out, and the cyclic diguanylate receptor-coding gene FleQ was further knocked out, and the response regulator BfmS was overexpressed to obtain the rhamnolipid-producing genetically engineered bacterium.

[0025] Typically, but not limited to, in one alternative implementation, the engineered bacteria are further constructed using the following method:

[0026] The Psl polysaccharide-encoding gene cluster (pslA, pslB, pslC, pslD, pslE, pslF, pslG, pslH, pslI, pslJ, pslK, pslL, pslM, pslN, gene sequences shown in SEQ ID No. 1~SEQ ID No. 14) of *Pseudomonas aeruginosa* AB93066 was knocked out using homologous recombination mediated by the suicide plasmid pLP12, resulting in an engineered bacterium lacking the Psl polysaccharide-encoding gene cluster, named AB93066Δpsl.

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

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

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

[0030] II. Description of the culture medium formulation and fermentation method used in this 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] Solid culture medium for Pseudomonas aeruginosa: 5 g / L tryptone, 5 g / L beef extract, 3 g / L sodium chloride, 20 g / L agar powder, pH=7.0.

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

[0035] Fermentation medium for Pseudomonas aeruginosa: 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] Supplemental culture medium ①: corn oil.

[0037] Feeding medium ②: Sodium nitrate 100g / L.

[0038] The strain described in this invention was activated by streaking on Pseudomonas aeruginosa solid culture medium and then incubated at 37°C for 12-14 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 primary seed culture.

[0040] At an inoculation rate of 3% (V / V), the primary seed culture was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured on a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed culture.

[0041] At an inoculum rate of 3% (V / V), the secondary seed culture was transferred to a 5L fermenter containing 2.5L of Pseudomonas aeruginosa fermentation medium. The culture temperature was 37℃, the rotation speed was 400rpm, and the aeration rate was 1vvm. At 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 rhamnolipin yield. Fermentation was terminated when the rhamnolipin yield no longer changed or decreased.

[0042] When culturing the engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS using Pseudomonas aeruginosa solid medium or Pseudomonas aeruginosa seed medium, 50 μg / mL of ampicillin needs to be added. When culturing it using Pseudomonas aeruginosa fermentation medium, 50 μg / mL of ampicillin and 0.2 wt% L-arabinose need to be added.

[0043] III. Methods for Detecting Rhamnose Lipid Production

[0044] Preparation of L-rhamnose standard working solution: Weigh 0.25 g of dried L-rhamnose standard to a 250 mL volumetric flask, dissolve and dilute to volume with ultrapure water to prepare a standard stock solution with a concentration of 1.0 g / L. Transfer 1 mL, 3 mL, 5 mL, 7 mL, and 9 mL of the standard L-rhamnose solution to five 10 mL volumetric flasks, 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, respectively.

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

[0046] Establishment of the standard curve: Transfer 0.2 mL of each concentration of L-rhamnose standard working solution into clean test tubes, place the test tubes in an ice-water bath, and then add 0.8 mL of anthrone-sulfuric acid reagent to each tube, shaking well after addition. Then immerse the tubes in a 100℃ water bath, boil for 10 min, remove, and allow to cool naturally to room temperature. Transfer 200 μL of each solution to an ELISA plate and measure the absorbance at 620 nm. Plot the standard curve with L-rhamnose concentration on the x-axis and absorbance on the y-axis, and fit the curve to obtain the regression equation (R²). 2 ≥0.99).

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

[0048] IV. Methods for detecting biofilm formation

[0049] The strain described in this invention was activated by streaking on LB solid medium and incubated at 37°C for 12-14 hours. Single colonies with good growth were inoculated into 250 mL shake flasks containing 25 mL of LB liquid medium and incubated at 37°C and 220 rpm for 12 hours to obtain the seed culture. The OD of the seed culture was measured. 600 According to the initial OD 600 =0.1 was inoculated into a test tube containing 1.5 mL of LB liquid medium, and an uninoculated test tube containing 1.5 mL of LB liquid medium was set up as a blank control. Both were placed incubated at 37℃ for 3 days. After incubation, the bacterial culture or 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 staining was performed at room temperature for 20 min. Then, the staining solution was aspirated, washed three times with deionized water, and dried. The intensity of the purple ring on the test tube wall was compared and photographed. 2 mL of 80 wt% ethanol was added to the test tube, and ultrasonic cleaning was performed until the purple ring was completely dissolved. The OD was measured. 595 This refers to the amount of biofilm formed.

[0050] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

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

[0052] The reaction system and reaction conditions used in this embodiment 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 bring the total volume to 50 μL with ddH2O.

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

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

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

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

[0058] Enzyme digestion conditions: Incubate at 37℃ for 5-15 min, incubate plasmid DNA for 15 min, and incubate PCR products for 5 min.

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

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

[0061] Construction of engineered bacteria AB93066Δpsl:

[0062] Genomic DNA was extracted from *Pseudomonas aeruginosa* AB93066 using a bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). Using the genomic DNA as a template, PCR amplification was performed with primers psil-UF and psil-UR, and psil-DF and psil-DR, respectively, to obtain upstream homologous arms psil-U and downstream homologous arms psil-D containing complementary sequences. Using upstream homologous arms psil-U and downstream homologous arms psil-D as templates, PCR amplification was performed with primers psil-UF and psil-DR to obtain the fusion fragment psil. The fusion fragment psil was 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 *E. coli* DH5α λpir competent cells. Positive transformants were screened by adding 20 μg / mL chloramphenicol and 0.3 wt% D-glucose to LB agar. Colony PCR was then performed to verify the positive transformants using pSL-UF and pSL-DR primers. For verified transformants, plasmids were extracted using a plasmid miniprep kit (Jiangsu Kangwei Century Biotechnology Co., Ltd.) and sequenced to obtain the recombinant plasmid pLP12-psl.

[0063] The recombinant plasmid pLP12-psl was chemically transformed into *E. coli* β2163 competent cells. Positive transformants were selected by adding 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose to LB solid medium, yielding 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, while the recipient strain was inoculated into LB liquid medium. The cultures were incubated at 37°C with shaking at 220 rpm for 12 h. 100 μL of each culture was then mixed and incubated at 37°C with shaking at 220 rpm for 6 h for conjugation. The cultured bacterial suspension was spread on LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. Genomic DNA was extracted from single colonies and used as templates. PCR verification was performed using psl-UF and psl-DR primers to screen for single colonies that underwent one recombination. Verified single colonies were inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37°C with shaking at 220 rpm for 12 h. The cultured suspension was then spread on LB solid medium containing 0.4 wt% L-arabinose. Colony PCR verification was performed using psl-UF and psl-DR primers (the correct colony band size was 1304 bp; the negative control showed no band; electrophoresis image as shown). Figure 1 As shown in (A) in the figure, single colonies that underwent secondary recombination were screened out. The PCR products were purified and sequenced for verification. After verification, the engineered strain AB93066Δpsl was obtained.

[0064] Construction of engineered bacteria AB93066ΔpslΔpel:

[0065] Using genomic DNA of *Pseudomonas aeruginosa* AB93066 as a template, PCR amplification was performed using pel-UF and pel-UR, and pel-DF and pel-DR primers, respectively, to obtain upstream homologous arms pel-U and pel-D containing complementary sequences. Using upstream homologous arms pel-U and downstream homologous arms pel-D as templates, PCR amplification was performed using pel-UF and pel-DR primers to obtain the fusion fragment pel. 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 transformed into *Escherichia coli* DH5α λpir competent cells by chemical transformation. Positive transformants were screened by adding 20 μg / mL chloramphenicol and 0.3 wt% D-glucose to LB solid medium. Colony PCR was then performed to verify the positive transformants using pel-UF and pel-DR primers. To verify the correct transformants, plasmids were extracted using a plasmid miniprep kit and sequenced to obtain the recombinant plasmid pLP12-pel.

[0066] The recombinant plasmid pLP12-pel was chemically transformed into *E. coli* β2163 competent cells. Positive transformants were selected 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. Using the engineered strain AB93066Δpsl 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 inoculated into LB liquid medium and cultured at 37°C with shaking at 220 rpm for 12 h. 100 μL of each culture was then mixed and cultured at 37°C with shaking at 220 rpm for 6 h for conjugation. The cultured bacterial suspension was spread on LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. Genomic DNA was extracted from single colonies and used as templates. PCR verification was performed using pel-UF and pel-DR primers to screen for single colonies that underwent one recombination. Verified single colonies were inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37°C with shaking at 220 rpm for 12 h. The cultured bacterial suspension was then spread on LB solid medium containing 0.4 wt% L-arabinose. Colony PCR verification was performed using pel-UF and pel-DR primers (the correct colony band size was 1279 bp; the negative control showed no band; electrophoresis image as shown). Figure 1As shown in (B) in the figure, single colonies that underwent secondary recombination were screened out. The PCR products were purified and sequenced for verification. After verification, the engineered bacteria AB93066ΔpslΔpel were obtained.

[0067] Construction of engineered bacteria AB93066ΔpslΔpelΔfleQ:

[0068] Using genomic DNA of *Pseudomonas aeruginosa* AB93066 as a template, PCR amplification was performed using fleQ-UF and fleQ-UR, and fleQ-DF and fleQ-DR as primers, respectively, to obtain upstream homologous arms fleQ-U and fleQ-D containing complementary sequences. Using fleQ-U and fleQ-D as templates, PCR amplification was performed using fleQ-UF and fleQ-DR as primers to obtain the fusion fragment fleQ. The fusion fragment fleQ 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 *Escherichia coli* DH5α λpir competent cells, and positive transformants were screened by adding 20 μg / mL chloramphenicol and 0.3 wt% D-glucose to LB solid medium. Further colony PCR was performed on positive transformants using fleQ-UF and fleQ-DR primers. For correctly verified transformants, plasmids were 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 *E. coli* β2163 competent cells. Positive transformants were selected by adding 20 μg / mL chloramphenicol, 0.3 mM diaminopimelic acid, and 0.3 wt% D-glucose to LB solid medium, yielding the donor strain β2163 / pLP12-fleQ. Using engineered strain AB93066ΔpslΔpel 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, while AB93066ΔpslΔpel was inoculated into LB liquid medium. The cultures were incubated at 37°C with shaking at 220 rpm for 12 h. 100 μL of each culture was then mixed and incubated at 37°C with shaking at 220 rpm for 6 h for conjugation. The cultured bacterial suspension was spread onto LB solid medium containing 20 μg / mL chloramphenicol and 0.3 wt% D-glucose. Genomic DNA was extracted from single colonies and used as templates. PCR verification was performed using fleQ-UF and fleQ-DR primers to screen for single colonies that underwent one recombination. Verified single colonies were inoculated into LB liquid medium containing 0.3 wt% D-glucose and cultured at 37°C with shaking at 220 rpm for 12 h. The cultured bacterial suspension was then spread onto LB solid medium containing 0.4 wt% L-arabinose. Colony PCR verification was performed using fleQ-UF and fleQ-DR primers (the correct colony band size was 1010 bp, and the negative control band size was 1491 bp; electrophoresis results are shown below). Figure 1 As shown in (C), single colonies that underwent secondary recombination were screened out. The PCR products were purified and sequenced for verification. After verification, 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, the bfmS gene expression fragment was amplified using primers bfmS-F and bfmS-R. The bfmS gene expression fragment was double-digested with EcoRI and HindIII, and the resulting fragment was ligated into the shuttle vector pHERD20T, which had also been double-digested with EcoRI and HindIII. The ligation product was chemically transformed into *E. coli* DH5α competent cells, and positive transformants were screened by adding 50 μg / mL ampicillin to LB agar. Colony PCR was then performed to verify the positive transformants using primers bfmS-F and bfmS-R. For verified transformants, plasmids were extracted using a plasmid miniprep kit and sequenced to obtain the recombinant plasmid pHERD20T-bfmS.

[0072] The recombinant plasmid pHERD20T-bfmS was chemically transformed into *E. coli* β2163 competent cells. Positive transformants were selected by adding 50 μg / mL ampicillin and 0.3 mM diaminopimelic acid to LB solid medium, yielding the donor strain β2163 / pHERD20T-bfmS. Using engineered strain AB93066ΔpslΔpelΔfleQ as the recipient strain, the donor strain was inoculated into LB liquid medium containing 50 μg / mL ampicillin and 0.3 mM diaminopimelic acid, and AB93066ΔpslΔpelΔfleQ was inoculated into LB liquid medium. The cultures were incubated at 37°C and 220 rpm for 12 h with shaking. 100 μL of each culture was then mixed and incubated at 37°C and 220 rpm for 6 h for conjugation. The cultured bacterial suspension was spread onto LB agar containing 50 μg / mL ampicillin. Single colonies were selected as templates, and colony PCR was performed using bfmS-test-F and bfmS-R primers for verification (correct colony band size was 1581 bp; negative control showed no band; electrophoresis image as shown). Figure 1 (As shown in (D)). A single colony that is verified correctly is 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 list) are shown in Table 1.

[0074] Table 1 - Primers used in Example 1

[0075]

[0076] Example 2: Fermentation of engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS

[0077] The engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS was streaked and activated on a Pseudomonas aeruginosa solid medium containing 50 μg / mL ampicillin and incubated at 37°C for 12–14 h.

[0078] 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℃ and 220 rpm for 12 h to obtain a primary seed culture.

[0079] At an inoculum rate of 3% (V / V), the primary seed culture was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium containing 50 μg / mL ampicillin, and cultured on a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed culture.

[0080] At an inoculum rate of 3% (v / v), the secondary seed culture was transferred to a 5L fermenter containing 2.5L of *Pseudomonas aeruginosa* fermentation medium containing 50 μg / mL ampicillin and 0.2 wt% L-arabinose. The culture temperature was 37℃, the rotation speed was 400 rpm, and the aeration rate was 1 vvm. At 12h, 24h, 36h, and 48h, 50g of feed medium ① and 50mL of feed medium ② were added, respectively. Rhamnolipid production was measured periodically during fermentation. Fermentation was terminated when rhamnolipid production no longer changed or decreased (rhamnolipid production curve shown in Figure 1). Figure 2 As shown in the figure, the fermentation cycle in this embodiment is 78 hours, the final yield of rhamnolipin is 73 g / L, and the production efficiency is 0.94 g / L / h.

[0081] Example 3 Detection of biofilm formation

[0082] Pseudomonas aeruginosa AB93066 and engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS were activated by streaking on LB solid medium and incubated at 37°C for 12–14 h. Single colonies with good growth were inoculated into 250 mL shake flasks containing 25 mL of LB liquid medium and incubated at 37°C and 220 rpm for 12 h to obtain seed culture. The OD of the seed culture was measured. 600 According to the initial OD 600=0.1 was inoculated into a test tube containing 1.5 mL of LB liquid medium, and an uninoculated test tube containing 1.5 mL of LB liquid was set up as a blank control. Both were placed in a static culture at 37℃ for 3 days. After the culture was completed, the bacterial culture or 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 staining was performed at room temperature for 20 min. Then the staining solution was aspirated, washed three times with deionized water and dried. The color intensity of the purple ring on the test tube wall was compared and photographed for recording (e.g., ...). Figure 3 (As shown). Add 2 mL of 80 wt% ethanol to the test tube, and sonicate with an ultrasonic cleaner until the purple circle is completely dissolved. Measure the OD. 595 This is the amount of biofilm formed (e.g.) Figure 4 (As shown). The results showed that the biofilm formation rate of the engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS was reduced by 82% compared with the starting strain Pseudomonas aeruginosa AB93066, indicating that the biofilm formation ability of the engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS obtained in this invention was significantly reduced.

[0083] Comparative Example 1: Fermentation of Pseudomonas aeruginosa AB93066

[0084] Pseudomonas aeruginosa AB93066 was activated by streaking on Pseudomonas aeruginosa solid medium and incubated at 37°C for 12-14 hours.

[0085] 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 primary seed culture.

[0086] At an inoculation rate of 3% (V / V), the primary seed culture was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured on a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed culture.

[0087] At an inoculum rate of 3% (v / v), the secondary seed culture was transferred to a 5L fermenter containing 2.5L of Pseudomonas aeruginosa fermentation medium. The culture temperature was 37℃, the rotation speed was 400 rpm, and the aeration rate (air) was 1 vvm. At 12h, 24h, 36h, and 48h, 50g of feed medium ① and 50mL of feed medium ② were added, respectively. Rhamnolipid production was measured periodically during fermentation. Fermentation was terminated when rhamnolipid production no longer changed or decreased (rhamnolipid production change curve is shown in Figure 1). Figure 5 As shown in the figure, the fermentation cycle of this comparative example was 142 hours, the final yield of rhamnolipin was 87 g / L, and the production efficiency was 0.61 g / L / h.

[0088] Comparative Example 2: Fermentation of engineered bacteria AB93066ΔpslΔpelΔfleQ

[0089] The engineered strain AB93066ΔpslΔpelΔfleQ was activated by streaking on Pseudomonas aeruginosa solid medium and incubated at 37°C for 12-14 hours.

[0090] 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 primary seed culture.

[0091] At an inoculation rate of 3% (V / V), the primary seed culture was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured on a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed culture.

[0092] At an inoculum rate of 3% (v / v), the secondary seed culture was transferred to a 5L fermenter containing 2.5L of Pseudomonas aeruginosa fermentation medium. The culture temperature was 37℃, the rotation speed was 400rpm, and the aeration rate was 1vvm. At 12h, 24h, 36h, and 48h, 50g of feed medium ① and 50mL of feed medium ② were added, respectively. Rhamnolipin production was measured periodically during fermentation. Fermentation was terminated when rhamnolipin production no longer changed or decreased (rhamnolipin production change curve is shown in Figure 1). Figure 6 As shown in the figure, the fermentation cycle of this comparative example was 118 hours, the final yield of rhamnolipin was 79.5 g / L, and the production efficiency was 0.67 g / L / h.

[0093] Comparative Example 3

[0094] The engineered bacteria AB93066ΔpslΔpel were streaked and activated on Pseudomonas aeruginosa solid medium and incubated at 37°C for 12-14 hours.

[0095] 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 primary seed culture.

[0096] At an inoculation rate of 3% (V / V), the primary seed culture was transferred to a 250 mL shake flask containing 25 mL of Pseudomonas aeruginosa seed culture medium and cultured on a shaker at 37°C and 220 rpm for 8 h to obtain the secondary seed culture.

[0097] At an inoculum rate of 3% (v / v), the secondary seed culture was transferred to a 5L fermenter containing 2.5L of Pseudomonas aeruginosa fermentation medium. The culture temperature was 37℃, the rotation speed was 400rpm, and the aeration rate was 1vvm. At 12h, 24h, 36h, and 48h, 50g of feed medium ① and 50mL of feed medium ② were added, respectively. Rhamnolipin production was measured periodically during fermentation. Fermentation was terminated when rhamnolipin production no longer changed or decreased (rhamnolipin production change curve is shown in Figure 1). Figure 7 As shown in the figure, the fermentation cycle of this comparative example was 132 hours, the final yield of rhamnolipin was 82.7 g / L, and the production efficiency was 0.63 g / L / h.

[0098] As shown in Examples 2, 1, 2, and 3, the engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS, compared to Pseudomonas aeruginosa AB93066 (the starting strain), shortened the fermentation cycle by 45.1% and increased the production efficiency by 54.1%; compared to engineered strain AB93066ΔpslΔpelΔfleQ, the engineered strain AB93066ΔpslΔpelΔfleQ / pHERD20T-bfmS, shortened the fermentation cycle by 33.9% and increased the production efficiency by 40.3%; and compared to engineered strain AB93066ΔpslΔpel, the engineered strain AB93066ΔpslΔpel, shortened the fermentation cycle by 40.9% and increased the production efficiency by 49.2%.

[0099] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they 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 rhamnolipids, characterized in that, It is constructed using the following method: against Pseudomonas aeruginosa Pseudomonas aeruginosa AB93066 Psl Polysaccharide-encoding gene clusters and Pel The polysaccharide-encoding gene cluster was knocked out, and the gene encoding the cyclic diguanylate receptor was further knocked out. FleQ Overexpression of response regulators BfmS The genetically engineered bacterium that produces rhamnolipid was obtained. The overexpression response regulator BfmS The method is as follows: With Pseudomonas aeruginosa Pseudomonas aeruginosa Using AB93066's genomic DNA as a template, amplification was performed to obtain... bfmS Gene expression fragments, bfmS The gene expression fragment was double-digested with EcoRI and HindIII. The resulting fragment was then ligated with the shuttle vector pHERD20T, which had been double-digested with EcoRI and HindIII, to obtain the recombinant plasmid pHERD20T-. bfmS , The recombinant plasmid pHERD20T- bfmS Transformed into E. coli β2163 competent cells to obtain donor bacteria β2163 / pHERD20T- bfmS To knock out Psl Polysaccharide-encoding gene clusters, Pel Polysaccharide-coding genes and cyclic diguanylate receptor-coding genes FleQ engineered bacteria AB93066Δ psl Δ pel Δ fleQ The recipient bacteria were conjugated and cultured until monoclonal formation was achieved, thus obtaining the genetically engineered bacterium that produces rhamnolipid.

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

21.

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

23.

4. The genetically engineered bacterium producing rhamnolipids according to claim 1, characterized in that, Knockout Psl Polysaccharide-encoding gene clusters, Pel Polysaccharide-coding genes and cyclic diguanylate receptor-coding genes FleQ All methods used were homologous recombination mediated by the suicide plasmid pLP12.

5. The genetically engineered bacterium producing rhamnolipids according to claim 4, characterized in that, Knockout of the gene encoding the cyclic diguanylate receptor FleQ The method is as follows: Obtain the gene encoding the cyclic diguanylate receptor. FleQ Fusion fragments of upstream and downstream homologous arms; The fusion fragment was recombinated with the suicide vector pLP12 to construct the recombinant suicide plasmid pLP12- fleQ ; Recombinant plasmid pLP12- fleQ Transformed into Escherichia coli β2163 as the donor bacterium to knock out Psl Polysaccharide-encoding gene clusters, Pel Engineered bacterium AB93066Δ encoding polysaccharide gene psl Δ pel The recipient bacteria are conjugated and cultured until a single colony is formed, thus obtaining the knockout strain. Psl Polysaccharide-encoding gene clusters, Pel Polysaccharide-encoding genes and cyclic diguanylate receptors FleQ engineered bacteria AB93066Δ psl Δ pel Δ fleQ .

6. The application of genetically engineered bacteria in the fermentation production of rhamnolipids, characterized in that, The genetically engineered bacteria is the rhamnolipin-producing genetically engineered bacteria according to any one of claims 1-5.

7. A method for producing rhamnolipids through fermentation using genetically engineered bacteria according to any one of claims 1-5, characterized in that, include: The seed culture containing the genetically engineered bacteria was inoculated into a fermentation medium of Pseudomonas aeruginosa containing ampicillin and L-arabinose, and cultured at 37°C under aeration.

8. The method according to claim 7, characterized in that, During fermentation, corn oil and sodium nitrate are added regularly. Rhamnolipid production is measured regularly during fermentation, and fermentation is stopped when the rhamnolipid production no longer changes or decreases.

9. The method according to claim 7, characterized in that, The formula for the Pseudomonas aeruginosa fermentation medium is as follows: 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; In the fermentation medium of Pseudomonas aeruginosa, the concentration of ampicillin was 50 μg / mL, and the mass percentage of L-arabinose was 0.2%.

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