Pseudomonas aeruginosa with high yield of di-rhamnolipid, and construction method and application thereof

Abstract

The application provides a high-yield double-rhamnolipid Pseudomonas aeruginosa DM01 and a construction method and application thereof, and belongs to the technical field of genetic engineering; the Pseudomonas aeruginosa YM4 is used as a starting strain, a homologous recombination technology is used to knock out the key gene phaC of a by-product PHA metabolic pathway, meanwhile, an endogenous promoter P rhlA The rhlC gene is overexpressed in situ, the metabolic flow is directionally regulated, and a high-yield double-rhamnolipid strain DM01 is obtained. The strain realizes high-purity Rha-Rha-C 10 -C 10 component directional synthesis. In the oil reservoir engineering application such as enhanced oil recovery, the application has outstanding performance advantages and broad application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic engineering and relates to a Pseudomonas aeruginosa DM01 with high production of dirhamnolipid, a construction method and an application thereof. Background Art

[0002] Rhamnolipid is a biosurfactant mainly produced by Pseudomonas aeruginosa. With its excellent emulsifying, foaming and surface activity, as well as excellent biodegradability and low toxicity, it has shown great application potential in agriculture, medicine, daily chemicals and oil extraction. Currently, there are more than 60 known rhamnolipid homologues, among which the disrhamnolipid Rha-Rha-C 10 -C 10 and monorhamnolipid Rha-C 10 -C 10 There are two main structures. Influenced by the characteristics of the strain, fermentation raw materials and process, the proportion of the disrhamnolipid component in the total product can range from 0-90%. The structure of rhamnolipids has a significant impact on their physical and chemical properties and application effects. Studies have shown that in the oil production field, the higher the proportion of disrhamnolipids, the better the viscosity reduction effect on crude oil and the oil sand cleaning effect. In the agricultural field, disrhamnolipids have stronger antifungal activity than monorhamnolipids, and can more effectively inhibit the growth of pathogenic fungal hyphae and lyse spores. Therefore, increasing the proportion of disrhamnolipids in the product can help achieve better application performance.

[0003] In recent years, increasing the proportion of rhamnolipids by enhancing rhlC gene expression has been proven to be an effective strategy, but existing research still faces many technical bottlenecks, such as low yield, insufficient conversion efficiency, and diverse structures of rhamnolipid products. For example, in Chinese patent CN112941001B, although a rhamnolipid yield of 65.0 g / L was obtained, the substrate conversion rate was low and the rhamnolipids were a mixture of various structures. 10 -C 10 This accounts for only 73.91%. The bottlenecks of existing technologies stem primarily from two key factors: first, the stability of the exogenous expression system. Plasmid vectors are easily lost during the scale-up industrial fermentation process, resulting in unstable yields. Second, differences in the genetic background of the strains. Different chassis strains have varying abilities to synthesize dirhamnolipids and differ in their responses to rhlC gene expression. Therefore, it is crucial to select chassis strains with superior genetic characteristics and optimize genetic manipulation strategies to achieve efficient dirhamnolipid synthesis.

[0004] This study proposes the following strategy: A production strain capable of efficiently synthesizing dirhamnolipids was selected as a base strain. The key gene phaC, which is involved in the synthesis of the competing product PHA, was knocked out, blocking the PHA synthesis pathway. Simultaneously, in situ expression of the key gene for dirhamnolipid synthesis (rhlC) was enhanced within phaC. The goal was to construct a genetically engineered strain capable of high-yield dirhamnolipid production using soybean oil as a carbon source. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to obtain a high-purity Rha-Rha-C 10 -C 10 The strain is used to ferment and produce high-concentration and high-purity rhamnolipid.

[0006] The first object of the present invention is to overcome the defects of the above-mentioned prior art and provide a high-yield rhamnolipid production strain, which is classified and named Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) DM01, deposit number: CCTCC NO: M2025560. It was deposited with the China Center for Type Culture Collection (CCTCC) on March 24, 2025. The depository address is Wuhan University.

[0007] The second object of the present invention is to provide a method for constructing a high-yield rhamnolipid production strain, using Pseudomonas aeruginosa YM4 as the host strain, adopting a "knockout + overexpression" strategy, using homologous recombination technology to knock out the key gene phaC in the metabolic pathway of the byproduct PHA, and at the same time using the endogenous promoter P rhlA The rhlC gene was overexpressed in situ to construct the Pseudomonas aeruginosa DM01, and the phaC nucleotide sequence after overexpression was shown in SEQ ID NO. 4. The engineered bacteria can efficiently synthesize rhamnolipids and block the synthesis pathway of the metabolic byproduct polyhydroxyalkanoate (PHA).

[0008] Preferably, the P rhlA The nucleotide sequence is shown as SEQ ID NO.1.

[0009] Preferably, the nucleotide sequence of rhlC is shown as SEQ ID NO.2.

[0010] Preferably, the nucleotide sequence of phaC is shown as SEQ ID NO.3.

[0011] Preferably, the fermentation of Pseudomonas aeruginosa DM01 using 40 g / L soybean oil as a carbon source produces disrhamnolipids at a yield of 24.06 g / L. The product structure is highly homogeneous, with the disrhamnolipid Rha-Rha-C10-C10 accounting for up to 95%.

[0012] The third object of the present application is the use of the high-yield biosurfactant-producing strain or fermentation broth in enhancing oil recovery.

[0013] The present application has the following advantages: the high-yield biosurfactant-producing Pseudomonas aeruginosa DM01, the construction method and the application thereof are provided, the Pseudomonas aeruginosa YM4 is used as the starting strain, the homologous recombination technology is used to knock out the key gene phaC of the metabolic pathway of the by-product PHA, the endogenous strong promoter P rhlA The rhlC gene is overexpressed in situ, the metabolic flow is directionally regulated, and the high-yield biosurfactant-producing strain DM01 is obtained. When the DM01 strain uses 40 g / L soybean oil as the carbon source, the yield of the biosurfactant reaches 24.06 g / L; and the product structure presents high singleness, in which the Rha-Rha-C 10 -C 10 The proportion reaches 95%. Compared with the original strain YM4 (47.88%), the proportion of Rha-Rha-C 10 -C 10 is increased by 47.12%, and the high-purity Rha-Rha-C 10 -C 10 component is directionally synthesized. In particular, compared with the conventional mixed-type mono / di-rhamnolipid product, the high-purity di-rhamnolipid exhibits significant advantages in physical and chemical properties and application effects, has stronger wetting property, higher tolerance to salinity, and more prominent oil sand cleaning capacity, and these characteristics make it have outstanding performance advantages and broad application prospects in oil reservoir engineering applications such as enhancing oil recovery. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a schematic diagram of the principle of constructing the Pseudomonas aeruginosa DM01 in the embodiment 1 of the present application;

[0015] Figure 2 It is a colony PCR gel map result in the embodiment 1 of the present application; wherein, (a) is a colony PCR result gel map of DH5α, (b) is a colony PCR result gel map of S17-1λpir;

[0016] Figure 3 It is a colony photograph map at different culture stages in the embodiment 1 of the present application; wherein, (a) is a LBNS plate double parent combination map, (b) is a sucrose streak plate map, (c) is a LB plate and a Qingda plate live bacteria map;

[0017] Figure 4 It is a PCR verification gel map result of the Pseudomonas aeruginosa DM01 in the embodiment 1 of the present application;

[0018] Figure 5This is a TEM electron micrograph of the YM4 strain in the control group after fermentation in Example 3 of the present invention;

[0019] Figure 6 This is a TEM electron micrograph of the Pseudomonas aeruginosa DM01 strain after fermentation in Example 3 of the invention;

[0020] Figure 7 This is a diagram showing the structure of rhamnolipid identified by HPLC-ELSD in Example 3 of the present invention;

[0021] Figure 8 This is a diagram showing the precipitation of rhamnolipid in mineralized water in Example 4 of the present invention;

[0022] Figure 9 This is a graph showing the supernatant retention rate of rhamnolipid at various temperatures and salinities in Example 4 of the present invention;

[0023] Figure 10 This is a graph showing the contact angle of rhamnolipid on oil film at different salinities in Example 4 of the present invention.

[0024] The deposit information of Pseudomonas aeruginosa in this application is as follows

[0025] Pseudomonas aeruginosa, classified as Pseudomonas aeruginosa DM01 was deposited in the China Center for Type Culture Collection on March 24, 2025, with the deposit number CCTCCNO: M2025560. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0027] The present invention is further described below with reference to the accompanying drawings and embodiments.

[0028] The host bacteria Pseudomonas aeruginosa YM4 in the examples has been disclosed in the applicant's prior patent application CN107557324A, and the Pseudomonas aeruginosa YM4 deposit number is CCTCCNO: M2017494.

[0029] Promoter P rhlA The gene sequence and rhlC gene sequence are both derived from Pseudomonas aeruginosa YM4, and their nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. rhlA and rhlC gene sequence, the nucleotide sequence of the phaC gene sequence is shown as SEQ ID NO: 3; the nucleotide sequence of the replaced phaC gene sequence is shown as SEQ ID NO: 4.

[0030] In the following examples, the culture medium was composed as follows:

[0031] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0032] LB plate: LB liquid medium with 20 g / L agar added.

[0033] LBNS plate: peptone 10 g / L, yeast extract 5 g / L, agar 20 g / L.

[0034] Gentamicin resistance plate: Add 30 μg / mL gentamicin to LB plate.

[0035] 10% sucrose plates: LBNS plates were supplemented with 100 g / L sucrose.

[0036] Double-antibody plate: Triclosan and gentamicin were added to LBNS plates at a final concentration of 25 μg / mL and 30 μg / mL, respectively.

[0037] Fermentation medium: 50 g / L (YM4 strain) or 40 g / L (DM01 strain) of commercially available soybean oil, 8 g / L sodium nitrate, 1.9 g / L potassium dihydrogen phosphate, 7.8 g / L sodium hydrogen phosphate dodecahydrate, 0.1 g / L anhydrous calcium chloride, 0.2 g / L magnesium sulfate heptahydrate, 1 g / L potassium chloride, 1 g / L sodium chloride, 1 g / L yeast extract, 1 g / L defoamer, 2 mL / L trace elements (0.5 g / L ferrous sulfate heptahydrate, 0.3 g / L copper sulfate pentahydrate, 2.9 g / L zinc sulfate heptahydrate, 1.7 g / L manganese sulfate heptahydrate). The initial pH of the fermentation medium was adjusted to 7.0 with 2 M sodium hydroxide.

[0038] Example 1: Construction of Pseudomonas aeruginosa DM01 with high production of dirhamnolipid

[0039] (1) Construction of suicide vector pEX18GM-ΔphaC::rhlC

[0040] Schematic diagram of the principle of constructing high-yield rhamnolipid engineering bacteria. The main technical means is homologous recombination. The construction process is shown in the figure Figure 1 Using Pseudomonas aeruginosa YM4 as template, primer pairs P1-F / P1-R and P2-F / P2-R were used to amplify the upstream and downstream homology arms of the phaC gene (nucleotide sequence shown in SEQ ID NO.3), and primer pairs P3-F / P3-R and P4-F / P4-R were used to amplify the P rhlA(nucleotide sequence as shown in SEQ ID NO.1) and rhlC target gene fragment (nucleotide sequence as shown in SEQ ID NO.2). The linearized vector was obtained using the plasmid pEX18GM as a template and the primer pair P-18GM-F / P-18GM-R. The primer sequences are shown in Table 1. After gel recovery of the PCR product, the vector and fragment were connected using the overlapping PCR principle and a one-step cloning kit (Nanjing Novozyme C116-02) and transformed into DH5α competent cells. After plating and screening on Qingda resistance plates, the suicide vector pEX18GM-ΔphaC::rhlC was obtained after sequencing verification. The PCR reaction system for amplifying gene fragments is shown in Table 2. The PCR program for amplifying gene fragments is shown in Table 3. The Tm values ​​of the upstream and downstream homology arm primers of the phaC gene are both 65°C, and the reaction extension time is 19s; P rhlA The Tm value of the primers was 55°C, and the reaction extension time t was 14s; the Tm value of the rhlC primers was 55°C, and the reaction extension time t was 30s; the Tm value of the linearized vector primers was 60°C, and the reaction extension time was 174s. When performing colony PCR verification on Qingda resistance plates, the verification primer pair was Yan-F / Yan-R. The colony PCR verification reaction system is shown in Table 4, and the reaction procedure is shown in Table 3. The Tm value of the verification primer pair Yan-F / Yan-R was 63°C, and the reaction extension time t was 22s. The band pattern of DH5α of the suicide vector is shown in Figure 2 As shown in (a), the bright band with a length of 1401 bp is DH5α carrying a suicide vector, which was preserved and sent to Universal Biotech for sequencing.

[0041] Table 1. Primer sequences used in the present invention

[0042]

[0043] Table 2. PCR reaction system

[0044]

[0045] Table 3. PCR program

[0046]

[0047] Table 4. Colony PCR verification reaction system

[0048]

[0049] (2) Introduce the suicide vector pEX18GM-ΔphaC::rhlC into Escherichia coli S17-1λpir

[0050] In E. coli S17-1 lambda pir competent, add 20 μL of suicide vector pEX18GM-ΔphaC::rhlC, after 2.2 kV shock in the electrotransformation cup, add 1 mL of LB liquid medium in the super-clean bench, recover at 37°C, 200 rpm for 45 min, spread on the cefotaxime-resistant plate, and culture for 12 h. Pick single colonies and perform colony PCR verification using the same primer pair Yan-F / Yan-R. The reaction system is shown in Table 4, and the reaction procedure is shown in Table 3. The Tm value of the verification primer is 63°C, the reaction extension time t is 22 s, the band is bright and the length is 1401 bp, which is the S17-1 lambda pir strain with the suicide vector, named pEX18GM-ΔphaC::rhlC-S17-1 lambda pir strain. The PCR verification picture is shown in Figure 2 (b) of FIG. 3.

[0051] (3) Introduction of the suicide vector pEX18GM-ΔphaC::rhlC into P. aeruginosa YM4

[0052] Using the double parent binding strategy, overnight culture P. aeruginosa YM4 and pEX18GM-ΔphaC::rhlC-S17-1 lambda pir strain, mix 120 μL of pEX18GM-ΔphaC::rhlC-S17-1 lambda pir fermentation broth and 10 μL of YM4 fermentation broth in a 2 mL centrifuge tube. Take 10 μL of the mixed bacterial solution and drop it on the LBNS plate. After the bacterial solution is air-dried, place it in a 37°C incubator for overnight culture. The colony picture is shown in Figure 3 (a) of FIG. 3. Take appropriate sludge and spread it on the double-antibiotic plate and culture at 37°C for 24 h. Pick single colonies and streak them on 10% sucrose plates and culture for 12 h. Obtain single colonies, and the colony picture is shown in

[0053] (4) Screening and verification of the gene scarless replacement strain

[0054] Pick single colonies from the 10% sucrose plate and culture on cefotaxime-resistant plates and LB plates at 37°C for 12 h. Strains that cannot survive on cefotaxime plates may be correct knockout strains or wild-type strains. The colony picture is shown in Figure 3 (c) of FIG. 3. The corresponding colonies grown on LB plates were subjected to PCR amplification using primer pair P1-F / Yan-R. The original strain was the negative control, and the constructed suicide vector was the positive control (band length 1152 bp). The results are shown in Figure 4 FIG. 3. The gene scarless replacement strain with the same length as the positive band. After PCR verification, further sequencing verification was performed, and the final correct P rhlA -rhlC recombinant fragment replacement phaC gene engineering strain, named DM01.

[0055] Example 2: Preparation of mono / di-rhamnolipid standard curve

[0056] Mono / di-rhamnolipid crude product was prepared by the following steps: the rhamnolipid fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was collected; the pH of the supernatant was adjusted to below 2.0 using 2M HC1, and the mixture was placed at 4°C overnight, and the precipitate was collected by centrifugation; the precipitate was dissolved with 0.5M NaOH, and the pH was adjusted to 7.0-7.3, and the supernatant was collected by centrifugation, and the water in the supernatant was removed by freeze-drying; the sample was dissolved with ethanol after freeze-drying, and the insoluble substances such as proteins were removed by centrifugation, and finally the ethanol was removed by rotary evaporation to obtain a yellow oil of rhamnolipid, which was dissolved with water, and freeze-dried again to obtain the mono / di-rhamnolipid crude product in powder form.

[0057] Mono / di-rhamnolipid pure product was prepared by further purifying the rhamnolipid crude product by column chromatography gradient elution. The eluent was dichloromethane, methanol and 1% acetic acid, and the dichloromethane and methanol were changed from 20:1 to 7:1. The chromogenic agent (a-naphthol, sulfuric acid, ethanol, water, 10.5 / 6.5 / 40.5 / 4.5, m / v / v / v) was heated to develop color or the alkaline potassium permanganate solution was used to develop color, and the alkaline potassium permanganate solution was composed of potassium permanganate, potassium carbonate, sodium hydroxide and water in a ratio of 2.5:20:0.25:40 (m / m / m / v). The eluate was collected, concentrated by rotary evaporation, and then the acetic acid was removed by vacuum pump. The product was dissolved with pure water, and the pH was adjusted to 7.0-7.3 with 0.2M sodium hydroxide. The aqueous solution was freeze-dried to obtain the mono / di-rhamnolipid pure product.

[0058] The structure of rhamnolipid was qualitatively analyzed by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) according to the following procedure: the mono / di-rhamnolipid pure product was diluted with pure water to an appropriate multiple, filtered through a 0.22μm water filter membrane, and then transferred to a liquid phase vial for standby. The analysis was performed on an Agilent HPLC-ELSD system (USA, Palo Alto, California) equipped with a C18 chromatographic column (4.6x150mm, 5μm; Sepax Technology Company, Suzhou, China). The detection conditions were set as follows: ELSD drift tube temperature 103°C, atomizing gas flow rate 2.8L / min. The mobile phase was acetonitrile (A phase) and water containing 0.05% formic acid (B phase), and eluted with a linear gradient of 30%-100% in 26 minutes, and the injection volume was 20μL. Under the above conditions, the mono-rhamnolipid Rha-C 10 -C 10 , the di-rhamnolipid Rha-Rha-C 10 -C 10 and the di-rhamnolipid Rha-Rha-C 10 -C 12The peak times were 10.6min, 13.4min and 14.5min respectively.

[0059] High-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) was used to quantitatively analyze the structure of rhamnolipids. The following steps were performed: Using pure mono- and di-rhamnolipids obtained by column chromatography as standards, calibration curves were established for the concentration and peak area of ​​each rhamnolipid using the HPLC-ELSD instrument. XY scatter plots were plotted with concentration (logarithm) as the abscissa and peak area (logarithm) as the ordinate, and linear fits were performed. Results showed that within the instrument's detection limit, the calibration curve equation for di-rhamnolipid within the concentration range of 0.2-0.7 mM was y=2.1252x+2.5146 (R²=0.9997). Good linearity was observed for mono-rhamnolipid within the concentration range of 0.2-1.0 mM, with the calibration curve equation being y=1.9796x+2.3015 (R²=0.9992).

[0060] Example 3: Fermentation of rhamnolipids by Pseudomonas aeruginosa YM4 and DM01 using oil as a carbon source

[0061] Pseudomonas aeruginosa strains YM4 and DM01 were fermented in 5L tanks. The fermentation process was as follows: a single colony was inoculated into 50mL of LB liquid medium and incubated at 37°C, 2000rpm for 12 hours to obtain a seed solution. A 2% inoculum of the seed solution was transferred to 300mL of LB medium and incubated for 5 hours. Finally, a 10% inoculum was inoculated into the fermentation medium in a 5L tank. Fermentation was maintained at 37°C, 200rpm, and oxygen was supplied at 0.02vvm for 64 hours. The rhamnolipid fermentation broth was obtained by centrifugation to remove the bacterial cells. Pseudomonas aeruginosa strain YM4 served as the control.

[0062] Morphological testing of rhamnolipid-producing strains: 5 mL of fermentation broth from a 48-hour fermentation was transferred to a 50 mL centrifuge tube. The cells were collected by low-speed centrifugation (5000 g / 10 min) at 4°C. The cells were washed twice with 10 mL of sterile water, transferred to a 2 mL centrifuge tube, and centrifuged at 1500 rpm and 4°C for 10 min. The supernatant was removed and the precipitated cells were retained. The cells were quickly frozen in liquid nitrogen and sent to the scientific dog testing platform for transmission electron microscopy (TEM) analysis of cell morphology. The cell morphology of the YM4 strain is shown in Figure 1. Figure 5 As shown in the figure, it can be seen that there are a lot of white particles in the cells, which are polyhydroxyalkanoates (PHA); and the cell morphology of the DM01 strain is as follows Figure 6 As shown, there is no PHA accumulation in the genetically recombinant DM01 strain.

[0063] Rhamnolipid structure and yield assay: 1 mL of fermentation broth from a 64-hour fermentation was centrifuged at 8000 rpm for 15 minutes in a centrifuge tube. The supernatant was diluted with purified water at an appropriate multiple and analyzed by HPLC-ELSD. The molar concentrations of the disrhamnolipids and monorhamnolipids were calculated using the disrhamnolipid and monorhamnolipid standard curves in Example 2, respectively. The molar concentrations were then multiplied by the corresponding molar masses (504 for monorhamnolipid and 650 for disrhamnolipid) to obtain the mass concentrations.

[0064] The results are as follows Figure 7 As shown: The fermentation product of the control group is a mixture of mono- / di-rhamnolipids, the main structure of which is Rha-Rha-C 10 -C 10 (peak area accounted for 47.88%) and Rha-C 10 -C 10 (peak area accounts for 40.05%), and the fermentation product is named mixed rhamnolipid (Mixture). Pseudomonas aeruginosa DM01 strain group does not synthesize single rhamnolipid Rha-C 10 -C 10 The main product is rhamnolipid Rha-Rha-C 10 -C 10 and Rha-Rha-C 10 -C 12 ; Among them, Rha-Rha-C 10 -C 10 The peak area accounts for as much as 95%, and the fermentation product is named as dirhamnolipid (Rha-Rha-C 10 -C 10 After 64 hours of fermentation in a 50 g / L soybean oil medium, the control strain YM4 produced a total rhamnolipid yield of 28.55 g / L (13.67 g / L disrhamnolipid and 14.88 g / L monorhamnolipid). The Pseudomonas aeruginosa strain DM01, fermented in a 40 g / L soybean oil medium for 64 hours, produced only disrhamnolipid at a yield of 24.06 g / L, significantly improving product purity.

[0065] Therefore, Pseudomonas aeruginosa YM4 was selected as the starting strain, which retained its efficient rhamnolipid synthesis pathway; through genome modification strategy (knockout of phaC competitive pathway and use of endogenous promoter P rhlA Overexpression of rhlC not only avoided the problem of plasmid instability, but also optimized the carbon metabolic flow without the addition of exogenous inducers; it achieved efficient production with a short fermentation cycle (64 hours) and high yield (24.06g / L); most importantly, the Pseudomonas aeruginosa DM01 strain was able to specifically produce rhamnolipids, of which Rha-Rha-C 10 -C 10The structure accounts for as much as 95%. It has demonstrated significant technical advantages in terms of fermentation efficiency and product specificity, providing a better solution for the industrial production of rhamnolipids.

[0066] The carbon source utilization efficiency of rhamnolipid fermentation by the strains was calculated: using trilinoleylglycerol (molar mass 878 g / mol) as a proxy for soybean oil, the carbon molar recovery of strain YM4 was 44.38%, while that of strain DM01 increased to 45.61%. In summary, these genetic modifications not only optimized the product composition but also improved carbon source utilization efficiency, providing important technical support for the industrial production of high-purity dirhamnolipids.

[0067] Example 4: Effect of dirhamnolipid on crude oil recovery

[0068] Currently, surfactant products used for enhanced oil recovery (EOR) include anionic surfactants such as petroleum sulfonates, alkylbenzene sulfonates, and olefin sulfonates. They are effective and widely used in conventional oil reservoirs with temperatures of 80°C and salinity below 30,000 mg / L. However, for harsh oil reservoirs, the above surfactants are inefficient or even ineffective due to their low activity and poor salt tolerance.

[0069] Test of heat and salt resistance of rhamnolipids: Example 3 Rhamnolipids (Rha-Rha-C 10 -C 10 ) was used as the experimental group to examine the tolerance of dirhamnolipids to salinity. The experiment used simulated formation water as the test medium. The formula is shown in Table 5. Its salt ion composition is highly similar to that of real reservoir water. Dirhamnolipid solutions were prepared in formation water with different salinities and their turbidity was observed after standing to determine the stability of rhamnolipids in high-salinity environments. Mixed rhamnolipids (Mixture) served as the control group. The results are shown in Figure 5. Figure 8 As shown in the figure: 2mM rhamnolipid solution was prepared in formation water with a mineralization of 50000mg / L. The control solution immediately became turbid and formed obvious precipitation after standing for 2h. The experimental group of rhamnolipid (Rha-Rha-C 10 -C 10 ) The solution always remains clear and transparent, showing excellent salt tolerance.

[0070] To comprehensively evaluate the effects of high temperature and high mineralization on the solubility of rhamnolipids, 2mM rhamnolipid solutions were prepared in a mineralization gradient range of 5000-90000 mg / L and stability tested at different temperature conditions for 4 hours (121°C, autoclaved for 30 minutes). The retention rate of rhamnolipids in the supernatant was calculated after centrifugation of the turbid solution. The results are shown in Figure 2. Figure 9The results show that: 1) temperature has little effect on the stability of rhamnolipids; 2) the retention rate of mixed rhamnolipids in the supernatant decreases sharply with increasing mineralization. At a mineralization of 90,000 mg / L, the retention rate in the supernatant is less than 20%, indicating that a large amount of precipitation has occurred; 3) disrhamnolipids show excellent tolerance to high mineralization. Even at high mineralization (90,000 mg / L), the retention rate in the supernatant remains above 90%.

[0071] The above examples have significant industrial application value: Disrhamnolipid's excellent tolerance to high salt and high temperatures makes it particularly suitable for operations such as enhanced oil recovery (EOR) and reservoir plugging removal under harsh reservoir conditions. This invention not only reveals the structure-property relationship of disrhamnolipid but also provides reliable technical support for its practical application in the field of oil recovery.

[0072] Table 5. Formulations of simulated formation water with different salinities

[0073]

[0074] Effect of rhamnolipids on the wettability of oil films: Contact angle, a key parameter characterizing the spreading ability of a liquid on a solid surface, effectively reflects surface wetting properties. This study systematically evaluated the wetting properties of rhamnolipids with different structures on crude oil films by measuring the contact angle changes of rhamnolipid solutions. The contact angle value is negatively correlated with the wetting effect, with lower contact angles indicating superior wetting properties, which directly impacts oil stripping efficiency and oil recovery enhancement.

[0075] The oil film preparation method is as follows: a standard glass slide is used as a carrier and ultrasonically cleaned (30 minutes each time) using 10% dilute hydrochloric acid, anhydrous ethanol, and pure water to ensure the surface is clean. An oil solution is prepared with a volume ratio of petroleum ether to crude oil of 10:1, and the treated glass slide is completely immersed in it. The oil-loaded glass slide is removed and placed in an incubator at 80°C for 7 days to obtain an oil film model with a stable hydrophobic surface.

[0076] The contact angle was measured as follows: the mixed rhamnolipid (Mixture) produced by the YM4 strain and the dirhamnolipid (Rha-Rha-C 10 -C 10 ) The crude product was prepared into 1mM rhamnolipid test solution with pure water and mineral water respectively. A contact angle meter (DropMeterA100P Ningbo Haishu Maishi Testing Technology Co., Ltd.) was used to absorb a certain amount of test solution and drop it onto the oil film surface. The wetting performance was evaluated by analyzing the droplet contact angle. The experimental results are shown in Figure 2. Figure 10 As shown in the figure, under the same test conditions, the fermentation product of DM01 strain rhamnolipid (Rha-Rha-C 10-C 10 ) exhibited a smaller contact angle, confirming its superior wetting properties. The mineralized water environment significantly affected the wetting properties of rhamnolipids: as the mineralization increased, the contact angle increased, indicating a corresponding decrease in wetting effect. Specifically, Figure 10 In the study, the contact angle of mixed rhamnolipids reached 62.17° at a salinity of 50,000 mg / L, a significant increase of 66.10% compared to its contact angle in pure water (37.43°). In contrast, the contact angle of disrhamnolipids under the same conditions only increased from 32.59° to 39.34°, a mere 20.99% increase. This comparative data strongly demonstrates that disrhamnolipids maintain excellent wettability in high-salinity environments, being significantly less affected by salinity than mixed rhamnolipids. This finding provides important theoretical support for the application of disrhamnolipids in oil recovery in complex formations, particularly demonstrating significant advantages in enhancing oil recovery in high-salinity reservoirs.

[0077] Testing the Oil Sand Cleaning Performance of Rhamnolipids: The cleaning ability of rhamnolipids in oil sands is a microscopic manifestation of their application in enhanced oil recovery (EOR). Their surface activity effectively reduces oil-sand interfacial tension, promoting the stripping of crude oil from the sand surface. This study simulated a laboratory reservoir environment and used an artificial standard oil sand model to evaluate the oil cleaning efficiency of rhamnolipids with different structures, revealing differences in their oil recovery performance under real reservoir conditions.

[0078] Standard oil sand samples for measuring oil washing efficiency were prepared according to the Shengli Oilfield standard method. 170 g of quartz sand was mixed with 4 g of artificial oil (Shengli Oilfield, China) and 10 mL of petroleum ether with a boiling range of 60°C–90°C. The mixture was heated at 80°C for 1 hour to remove the solvent, and then aged at 60°C for 7 days to prepare the standard oil sand sample. 0.5 g of the artificial oil was transferred to a 100 mL volumetric flask, dissolved in 60°C–90°C petroleum ether, and diluted to volume. This solution contained 5.0 mg / mL of oil. This stock solution was then diluted appropriately, and a standard curve was plotted against the absorbance at 225 nm and the corresponding oil content. For the test, 2 g of aged oil sand was incubated with 20 mL of rhamnolipid cleaning solution of varying concentrations (0.5–3 mM) at 70°C and 90 rpm for 12 hours. After the reaction, 50 mL of petroleum ether was used to extract the crude oil from the cleaning solution. The absorbance at 225 nm was read to determine the oil content in the cleaning solution. The absorbance of 2 g of oil sand directly extracted was used as the total oil content of the oil sand. The oil washing efficiency was calculated using formula (1).

[0079] Oil sand cleaning efficiency = oil content in cleaning fluid / total oil content in oil sand × 100%........... (1)

[0080] The experimental data are shown in Table 6: in a pure water system, both of the rhamnolipid samples exhibit good oil washing capacity, and the oil washing efficiency is more than 85% at 0.5 mM; and in a 50000 mg / L mineralized water system, the oil washing performance of the double rhamnolipid is improved by 20-40% than that of the mixed rhamnolipid under the same concentration, indicating that the double rhamnolipid has more excellent crude oil stripping performance in a high-salt oil reservoir, and exhibits a broader application prospect.

[0081] Table 6. Oil sand cleaning efficiency of rhamnolipid in a pure water system and a mineralized water system

[0082]

[0083] The above has disclosed the present application with preferred embodiments, but it is not used to limit the present application, and the technical solutions obtained by taking equivalent replacement or equivalent transformation all fall within the protection scope of the present application.

Claims

1. A Pseudomonas aeruginosa DM01 that produces high amounts of dirhamnolipid, characterized by: The Pseudomonas aeruginosa DM01 was deposited in the China Center for Type Culture Collection on March 24, 2025, with the deposit number being CCTCCNO: M2025560.

2. The Pseudomonas aeruginosa DM01 having a high production of dirhamnolipid according to claim 1, characterized in that: The construction method is as follows: using Pseudomonas aeruginosa YM4 as the starting strain, using homologous recombination technology to knock out the key gene phaC in the metabolic pathway of the byproduct PHA, and using the endogenous promoter P rhlA The rhlC gene was overexpressed in situ to construct the Pseudomonas aeruginosa DM01. The nucleotide sequence after in situ overexpression is shown in SEQ ID NO.

4.

3. The Pseudomonas aeruginosa DM01 with high production of dirhamnolipid according to claim 2, characterized in that: The endogenous promoter P rhlA The nucleotide sequence of the rhlC gene fragment is shown as SEQ ID NO.1; the nucleotide sequence of the rhlC gene fragment is shown as SEQ ID NO.2; and the nucleotide sequence of the phaC gene fragment is shown as SEQ ID NO.

3.

4. Use of the high-yield disrhamnolipid-producing Pseudomonas aeruginosa DM01 according to claim 1 in the fermentation production of disrhamnolipid.

5. The use according to claim 4, characterized in that The Pseudomonas aeruginosa DM01 fermentation uses soybean oil as a carbon source, and the Rha-Rha-C10-C10 component in the fermented synthesized disrhamnolipid accounts for more than 95%.

6. Use of the high-di-rhamnolipid-producing Pseudomonas aeruginosa DM01 or its fermentation liquid according to claim 1 in improving crude oil recovery.

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