Method for producing single-component rhamnolipid with high yield

Abstract

The invention provides a method for producing single-component rhamnolipid at high yield, belongs to the technical field of biology, and can produce single-component rhamnolipid with stable properties at high yield so as to improve the application effect of rhamnolipid. According to the method, wild type rhamnosyl transferase rhlB or rhlA of pseudomonas aeruginosa is modified, and the amino acid sequence of the modified rhamnosyl transferase rhlB is as shown in SEQ ID NO: 1; the amino acid sequence of the modified rhamnosyl transferase rh1A is as shown in SEQ ID NO: 2.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for high-yield single-component rhamnolipid. Background Art

[0002] Rhamnolipids (RLs) are a class of surfactants produced by certain microorganisms, the main sources of which include Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and other bacteria. Rhamnolipids are mainly formed by rhamnose and fatty acid molecules connected by glycosidic bonds. They have strong interfacial activity and can reduce the surface tension of water. They are widely used in various industrial fields, especially in environmental protection, detergents, cosmetics and biomedicine.

[0003] However, due to the complexity and heterogeneity of the components of rhamnolipids and the variability of microorganisms during the synthesis process, rhamnolipids from the same source may have different components (i.e., different combinations of fatty acid chain lengths and different rhamnose contents), resulting in unstable properties of rhamnolipids from the same source (e.g., physicochemical properties, interfacial activity, and biological activity), which in turn makes the application effect of rhamnolipids less than satisfactory. Summary of the invention

[0004] The present invention provides a method for high-yield single-component rhamnolipid, which can high-yield single-component rhamnolipid with stable properties, thereby improving the application effect of the rhamnolipid.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: The invention provides a method for high-yield single-component rhamnolipid, wherein wild-type rhamnosyltransferase rhlB or rhlA of Pseudomonas aeruginosa is modified, and the amino acid sequence of the modified rhamnosyltransferase rhlB is shown in SEQ ID NO:1.

[0006] The modified rhamnosyltransferase rhlB is obtained based on the mutation of the wild-type rhamnosyltransferase rhlB. The amino acid sequence of the wild-type rhamnosyltransferase rhlB is shown in SEQ ID NO: 3. Specifically, the 49th amino acid alanine of the wild-type rhamnosyltransferase rhlB is mutated to glutamic acid, the 94th amino acid serine is mutated to cysteine, the 144th amino acid lysine is mutated to arginine, the 151st amino acid methionine is mutated to valine, the 234th amino acid isoleucine is mutated to valine, and the 315th amino acid serine is mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlB.

[0007] The amino acid sequence of the modified wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO:2.

[0008] The modified rhamnosyltransferase rhlA is obtained based on the mutation of rhamnosyltransferase rhlA. The amino acid sequence of wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO: 4. Specifically, the 28th amino acid serine of the wild-type rhamnosyltransferase rhlA is mutated to aspartic acid, the 94th amino acid glutamic acid is mutated to alanine, the 168th amino acid glutamine is mutated to proline, the 206th amino acid alanine is mutated to serine, and the 262nd amino acid valine is mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlA.

[0009] As another aspect of the present invention, in the above method, the wild-type rhamnosyltransferase rhlB of Pseudomonas aeruginosa is modified to obtain a mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NJUXR-CC-1, deposited in China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025100.

[0010] As another aspect of the present invention, in the above method, the wild-type rhamnosyltransferase rhlA of Pseudomonas aeruginosa is modified to obtain a mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NJUXR-CC-2, deposited in China Center for Type Culture Collection on January 13, 2025, and its deposit number is CCTCC NO: M 2025102.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. The single-component rhamnolipid produced by Pseudomonas aeruginosa provided by the present invention has a high yield.

[0012] 2. The present invention screened out the modified rhamnosyltransferase rhlB and the modified rhamnosyltransferase rhlA that assist Pseudomonas aeruginosa in high production of single-component rhamnolipid through site-directed mutagenesis of the key enzymes involved in the synthesis of rhamnolipid by Pseudomonas aeruginosa. Since the single-component rhamnolipid is stable in nature, the application effect of rhamnolipid can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a schematic diagram of the protein structure and mutation site of the modified rhamnosyltransferase rhlB in Example 1; Figure 2 is a schematic diagram of the protein structure and mutation site of the modified rhamnosyltransferase rhlA in Example 2; Figure 3This is a mass spectrometry detection diagram of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 after modification in Example 2. DETAILED DESCRIPTION

[0014] The method provided in the embodiments of the present application relates to the production of rhamnolipids, and can be used to produce high-yield single-component rhamnolipids.

[0015] Understandably, the molecular structure of rhamnolipids is relatively complex, usually composed of multiple rhamnose units and fatty acids of different chain lengths. The physiological functions of rhamnolipids are mainly reflected in the following aspects: First, as a surfactant, rhamnolipids can improve the interaction between liquids and solid surfaces, and play functions such as emulsification, foam generation, and wetting. Secondly, rhamnolipids play an important role in the growth and metabolism of microorganisms, especially in the formation of biofilms, flora regulation, and interaction with the host, and have the ability to enhance the tolerance of bacteria to environmental stress (such as high salt, high temperature, and oxidative stress). In addition, rhamnolipids can also be used as potential anti-infective agents in the medical field through their antibacterial, antifungal and immunomodulatory effects, especially in the treatment of drug-resistant bacterial infections. It shows good prospects.

[0016] The main uses of rhamnolipids include: as industrial cleaners, emulsifiers, drug carriers and active ingredients in cosmetics. In environmental pollution control, rhamnolipids can effectively degrade petroleum pollutants and other organic pollutants, and therefore have significant bioremediation potential. In the pharmaceutical field, rhamnolipids are widely studied as candidate drugs for the treatment of drug-resistant bacterial infections due to their antibacterial and anti-biofilm properties. In addition, rhamnolipids also have certain applications in industries such as agriculture, cosmetics and food additives.

[0017] In order to solve the problem in the background technology that rhamnolipids from the same source may have different components, resulting in unstable properties of rhamnolipids from the same source, and then making the application effect of rhamnolipids not good enough, the embodiment of the present application provides a method for high-yield single-component rhamnolipid, which can high-yield single-component rhamnolipids with stable properties, thereby improving the application effect of rhamnolipids.

[0018] Example 1: This example describes a method for high-yield single-component rhamnolipid, wherein the wild-type rhamnosyltransferase rhlB of Pseudomonas aeruginosa is modified, and the amino acid sequence of the modified rhamnosyltransferase rhlB is shown in SEQ ID NO:1.

[0019] SEQ ID NO: 1 is as follows: MHAILIAIGSAGDVFPFIGLARTLKLRGHRVSLCTIPVFRDAVEQHGI EFVPLSDELTYRRTMGDPRLWDPKTSFGVLWQAIAGMIEPVYEYV C AQRHDDIVVVGSLWALGARIAHEKYGIPYLSAQVSPSTLLSAHLPPVHP R FWF V PLAMRKLLWRCIERFKLDRTCAPEINAVRRKVGLETPVKRIFTQWMHSPQGVVCLFPAWFAPPQQDWPQPLHMTGFPLFDGS V PGTPLDDELQRFLDQGSRPLVFTQGSTEHLQGDFYAMALRALERLGARGIFLTGAGQEPLRGLPNHVLQRAYAPLGALLP A CAGLVHPGGIGAMSLALAAGVPQVLLPCAHDQFDNAERLVRLGCGMRLGVPLREQELRGALWRLLEDPAMAAACRRFMELSQPHSIACGKAAQVVERCHREGDARWLKAAS It should be noted that the underlined content in the above SEQ ID NO: 1 is the mutated amino acid site.

[0020] The modified rhamnosyltransferase rhlB is obtained based on the mutation of the wild-type rhamnosyltransferase rhlB. The amino acid sequence of the wild-type rhamnosyltransferase rhlB is shown in SEQ ID NO: 3. Specifically, the 49th amino acid alanine of the wild-type rhamnosyltransferase rhlB is mutated to glutamic acid, the 94th amino acid serine is mutated to cysteine, the 144th amino acid lysine is mutated to arginine, the 151st amino acid methionine is mutated to valine, the 234th amino acid isoleucine is mutated to valine, and the 315th amino acid serine is mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlB.

[0021] SEQ ID NO: 3 is as follows: MHAILIAIGSAGDVFPFIGLARTLKLRGHRVSLCTIPVFRDAVEQHGIAFVPLSDELTYRRTMGDPRLWDPKTSFGVLWQAIAGMIEPVYEYVSAQRHDDIVVVGS LWALGARIAHEKYGIPYLSAQVSPSTLLSAHLPPVHPKFNVPEQMPLAMRKLLWRCIERFKLDRTCAPEINAVRRKVGLETPVKRIFTQWMHSPQGVVCLFPAWFAP PQQDWPQPLHMTGFPLFDGSIPGTPLDDELQRFLDQGSRPLVFTQGSTEHLQGDFYAMALRALERLGARGIFLTGAGQEPLRGLPNHVLQRAYAPLGALLPSCAGL VHPGGIGAMSLALAAGVPQVLLPCAHDQFDNAERLVRLGCGMRLGVPLREQELRGALWRLLEDPAMAAACRRFMELSQPHSIACGKAAQVVERCHREGDARWLKAAS The specific steps of the above method are described in detail below: Step 1, preparing Pseudomonas aeruginosa competent cells; The wild-type Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 9027, purchased from Beijing Biobo Biotechnology Co., Ltd.) was spread on LB solid medium and cultured at 37°C overnight; a single colony was picked and inoculated into 4 mL LB liquid medium, cultured at 37°C and 140 rpm for 6 h, the cells were washed 3 times with a sterile 10% glycerol solution, and finally the cells were resuspended with 0.2 mL 10% glycerol to obtain Pseudomonas aeruginosa competent cells, which were then aliquoted and stored in a -80°C refrigerator for later use; Step 2, preparing Pseudomonas aeruginosa competent cells carrying pCasPA plasmid; About 10 ng of pCasPA plasmid was added to 0.1 mL of Pseudomonas aeruginosa competent cells, and the cells were placed in a pre-cooled electroporation cup in an ice bath for 5 min, and electroporated once at 2.0 kV. The Pseudomonas aeruginosa competent cells were revived and cultured with 1 ml of LB liquid culture medium for 2 h. The revived Pseudomonas aeruginosa competent cells were spread on LB solid culture medium containing 50 ug / mL tetracycline, and cultured at 37°C overnight to obtain Pseudomonas aeruginosa competent cells carrying the pCasPA plasmid, which were stored in a -80°C refrigerator for later use. The formula of the LB liquid medium includes: 10 g / L tryptone, 5 g / L yeast powder and 10 g / L sodium chloride; the formula of the LB solid medium includes: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride and 15 g / L agar; Step 3, construct pACRISPR plasmid; The pACRISPR plasmid contains the mutation site information of rhamnosyltransferase rhlB and is inserted between the DR2 elements. The sgRNA sequences with targeting function in the pACRISPR plasmid are shown in Table 1; Table 1: sgRNA sequences for editing the rhamnosyltransferase rhlB gene ; Step 4, construct homologous fragments; The homologous fragments were constructed using a complementary primer annealing PCR procedure, and the primer sequences used are shown in Table 2 ; Table 2: Primer sequences for constructing homologous fragments of rhamnosyltransferase rhlB gene editing ; Among them, "F" represents the upstream primer, "R" represents the downstream primer, and the underlined sequence is the mutation site; In the primer sequence for constructing the homologous fragment of the rhamnosyltransferase rhlB gene editing, A represents adenine, T represents thymine, C represents cytosine, and G represents guanine; N represents any one of A, T, C, and G, K (keto) represents T or G, and M (amino) represents A or C; The PCR system of the complementary primer annealing PCR program is shown in Table 3, and the PCR program process is shown in Table 4; the PCR product obtained by the complementary primer annealing PCR program is the homologous fragment; Table 3: PCR system table ; Table 4: PCR program table ; Step 5, transforming pACRISPR plasmid and homologous fragments; Referring to the operation in step 2 above, the pACRISPR plasmid and homologous fragments were transformed into Pseudomonas aeruginosa competent cells carrying the plasmid pCasPA, and spread on LB solid medium supplemented with 50 ug / mL tetracycline and 50 ug / mL carbenicillin, and cultured overnight at 37°C; the grown single colonies can be used for liquid culture and fermentation verification; Step 6, culturing the Pseudomonas aeruginosa obtained in step 5 and fermenting to produce rhamnolipid; 48 single colonies of Pseudomonas aeruginosa (also called 48 strains of Pseudomonas aeruginosa) were picked from the plate (i.e., LB solid culture medium), and inoculated into 10 mL test tubes containing 3 ml of LB liquid culture medium, respectively, and cultured overnight at 37°C and 200 rpm in a shaking incubator as seed liquid; 1 ml of the seed liquid was added to 30 ml of fermentation medium, and cultured at 37°C and 200 rpm in a shaking incubator for 48 hours to obtain the fermentation liquid of each of the 48 strains of Pseudomonas aeruginosa; The formula of the fermentation medium is: 60 g / L rapeseed oil, 3 g / L yeast extract, 6 g / L NaNO3, 1 g / L Na2HPO4, 1 g / L KH2PO4, 0.1 g / L MgSO4, and 0.1 g / L CaCl2·2H2O; the pH value is adjusted to 7.0 using 1 M NaOH solution; According to the above operation, wild-type Pseudomonas aeruginosa is cultured and fermented to produce rhamnolipid to obtain a fermentation liquid of wild-type Pseudomonas aeruginosa; Step 7, determining the content of rhamnolipid produced by Pseudomonas aeruginosa fermentation; The following operations were performed on the fermentation broth of each of the 48 strains of Pseudomonas aeruginosa and the fermentation broth of the wild-type Pseudomonas aeruginosa; Centrifuge the Pseudomonas aeruginosa fermentation broth, take 1 ml of the supernatant, and centrifuge at 12,000 rpm for 3 min; take 200 uL of the supernatant, dilute it 3-10 times with methanol, shake it thoroughly, and centrifuge it at 12,000 rpm for 10 min. The resulting supernatant is passed through a membrane to be the sample to be tested for rhamnolipid production by Pseudomonas aeruginosa; Agilent high-throughput liquid chromatography-mass spectrometry system RapidFire 400 and Agilent triple quadrupole mass spectrometer were used as detection tools (the chromatographic column used was C18) to detect samples of 48 strains of Pseudomonas aeruginosa and wild-type Pseudomonas aeruginosa to obtain the contents of rhamnolipid components in the fermentation broth of the 48 strains of Pseudomonas aeruginosa and the contents of rhamnolipid components in the fermentation broth of the wild-type Pseudomonas aeruginosa; Specifically, the mobile phase A used in the above detection process is methanol with a flow rate of 0.8 mL / min, and the mobile phase B is ammonia water (0.05%, v / v) with a flow rate of 1.2 mL / min. The analysis procedure is: State 1 (Aspirate): 600 ms; State 2 (Wash 1): 4000 ms; State 3 (Wash 2): 0 ms; State 4 (Elute): 4000 ms; State 5 (Reequilibrate): 1000 ms. The mass spectrometer is in negative ion mode with a scanning range of 300-900 m / z. Among them, the rhamnolipid standard is prepared as a gradient concentration and a standard curve is prepared to calculate the rhamnolipid content in the fermentation broth sample.

[0022] Specifically, the above-mentioned rhamnolipid standard was purchased from Shanghai McLean, and was prepared with 50% methanol into gradient concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, and 500 mg / L rhamnolipid standard solutions, which were detected using the above-mentioned scheme respectively, and a standard curve was prepared using rhamnolipid concentration-total ionic strength.

[0023] Finally, the strain with the highest rhamnolipid production among the 48 Pseudomonas aeruginosa strains was named NJUXR-CC-1.

[0024] In addition, the above method also includes step 8.

[0025] Step 8: Gene sequencing of dominant strains.

[0026] Specifically, a bacterial genomic DNA extraction kit (Beijing Solebow) was used to extract NJUXR-CC-1 genomic DNA, and the modified rhamnosyltransferase rhlB gene on the genome was sequenced. The modified rhamnosyltransferase rhlB in the NJUXR-CC-1 genomic DNA is shown in SEQ ID NO: 1. The 49th amino acid alanine of the wild-type rhamnosyltransferase rhlB was mutated to glutamic acid, the 94th amino acid serine was mutated to cysteine, the 144th amino acid lysine was mutated to arginine, the 151st amino acid methionine was mutated to valine, the 234th amino acid isoleucine was mutated to valine, and the 315th amino acid serine was mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlB. The positions of the above mutation sites (49th, 94th, 144th, 151st, 234th, and 315th) in the protein structure of the modified rhamnosyltransferase rhlB are shown in FIG. Figure 1 shown.

[0027] On this basis, the above-mentioned Pseudomonas aeruginosa NJUXR-CC-1 was deposited. The above-mentioned Pseudomonas aeruginosa NJUXR-CC-1 was deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China, the deposit date was January 13, 2025, the deposit number was CCTCC NO: M 2025100, and the classification name was Pseudomonas aeruginosa NJUXR-CC-1 Pseudomonas aeruginosa NJUXR-CC-1.

[0028] Example 2: This example describes a method for high-yield single-component rhamnolipid, in which the wild-type rhamnosyltransferase rhlA of Pseudomonas aeruginosa is modified. The amino acid sequence of the modified rhamnosyltransferase rhlA is shown in SEQ ID NO:2.

[0029] SEQ ID NO: 2 is as follows: MRRESLLVSVCKGLRVHVERVGQDPGR D TVMLVNGAMATTASFARTCKCLAEHFNVVLFDLPFAGQSRQHNPQRGLITKDDEVEILLALIERF A VNHLVSASWGGISTLLALSRNPRGIRSSVVMAFAPGLNQAMLDYVGRAQALIELDDKSAIGHLLNETVGKYLP P RLKASNHQHMASLATGEYEQARFHIDQVLALNDRGYL S CLERIQSHVHFINGSWDEYTTAEDARQFRDYLPHCSFSRVEGTGHFLDLESKLAA A RVHRALLEHLLKQPEPQRAERAGFHEMAIGYA It should be noted that the underlined amino acid sites in the above SEQ ID NO: 2 are mutated amino acid sites.

[0030] The modified rhamnosyltransferase rhlA is obtained based on the mutation of rhamnosyltransferase rhlA. The amino acid sequence of wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO: 4. Specifically, the 28th amino acid serine of the wild-type rhamnosyltransferase rhlA is mutated to aspartic acid, the 94th amino acid glutamic acid is mutated to alanine, the 168th amino acid glutamine is mutated to proline, the 206th amino acid alanine is mutated to serine, and the 262nd amino acid valine is mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlA.

[0031] SEQ ID NO: 4 is as follows: MRRESLLVSVCKGLRVHVERVGQDPGRSTVMLVNGAMATTASFARTCKCLAEHFNVVLFDLPFAGQSRQHNPQRGLITKDDEVEILLALIERFEVNHLVSASWGGISTLLALSRNPRGIRSSVVMAFAPGLNQAMLDYVGRAQALIE LDDKSAIGHLLNETVGKYLPQRLKASNHQHMASLATGEYEQARFHIDQVLALNDRGYLACLERIQSHVHFINGSWDEYTTAEDARQFRDYLPHCSFSRVEGTGHFLDLESKLAAVRVHRALLEHLLKQPEPQRAERAAGFHEMAIGYA The difference between the above method recorded in this embodiment and the method recorded in Example 1 is that: In step 3, the pACRISPR plasmid contains the mutation site information of rhamnosyltransferase rhlA and is inserted between the DR2 elements. The sgRNA sequence with targeting function in the pACRISPR plasmid is shown in Table 5.

[0032] Table 5: sgRNA sequences for editing the rhamnosyltransferase rhlA gene ; In step 4, the primer sequences used for the homologous fragments are shown in Table 6.

[0033] Table 6: Primer sequences of homologous fragments of rhamnosyltransferase rhlA ; In the primer sequence of the homologous fragment of rhamnosyltransferase rhlA, N represents any one of A, T, C, and G; In step 7, the strain with the highest rhamnolipid production among the 48 Pseudomonas aeruginosa strains was finally recorded as NJUXR-CC-2.

[0034] In step 8, a bacterial genomic DNA extraction kit (Beijing Solebow) is used to extract NJUXR-CC-2 genomic DNA, and the rhamnosyltransferase rhlA gene on the genome is sequenced. The modified rhamnosyltransferase rhlA in NJUXR-CC-2 is shown in SEQ ID NO: 2, and the amino acid serine at position 28 of the rhamnosyltransferase rhlA is mutated to aspartic acid, the amino acid glutamic acid at position 94 is mutated to alanine, the amino acid glutamine at position 168 is mutated to proline, the amino acid alanine at position 206 is mutated to serine, and the amino acid valine at position 262 is mutated to alanine, thereby obtaining the modified rhamnosyltransferase rhlA. The positions of the above mutation sites (positions 28, 94, 168, 206, and 262) in the protein 3D structure of the modified rhamnosyltransferase rhlA are shown in FIG. Figure 2 shown.

[0035] On this basis, the above-mentioned Pseudomonas aeruginosa NJUXR-CC-2 was deposited. The above-mentioned Pseudomonas aeruginosa NJUXR-CC-2 was deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China, the deposit date was January 13, 2025, the deposit number was CCTCC NO: M 2025102, and the classification name was Pseudomonas aeruginosa NJUXR-CC-2 Pseudomonas aeruginosa NJUXR-CC-2.

[0036] Experimental Example 1: This experimental example is based on a method for high-yield single-component rhamnolipid described in Example 1, and verifies whether the Pseudomonas aeruginosa strain NJUXR-CC-1 obtained in Example 1 has the ability to high-yield single-component rhamnolipid.

[0037] Specifically, referring to step 6 of Example 1, the Pseudomonas aeruginosa obtained in step 5 was cultured and fermented to produce rhamnolipids, and referring to step 7 of Example 1, the contents of the various components of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and NJUXR-CC-1 were measured. The contents of the various components of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and NJUXR-CC-1 are shown in Table 7.

[0038] Table 7: Ratio of rhamnolipid components in the fermentation broth of wild-type Pseudomonas aeruginosa and NJUXR-CC-1 ; Note: Rha-C10-C10 contains 1 rhamnose and 2 fatty acid chains of 10 carbon atoms; Rha-Rha-C10-C12 contains 2 rhamnose, 1 fatty acid chain of 10 carbon atoms, and 1 fatty acid chain of 12 carbon atoms; Rha-C12:1-C10 contains 1 rhamnose, 1 fatty acid chain of 10 carbon atoms, 1 fatty acid chain of 12 carbon atoms and 1 unsaturated bond; Rha-C8-C10 contains 1 rhamnose, 1 fatty acid chain of 8 carbon atoms, and 1 fatty acid chain of 10 carbon atoms.

[0039] As can be seen from Table 7, the rhamnolipid components produced by the wild-type Pseudomonas aeruginosa include 56.3% Rha-C10-C10, and the remaining 43.7% are other types of components (24.0% Rha-Rha-C10-C12, 8.3% Rha-C12:1-C10, 7.2% Rha-C8-C10, and 4.2% other components); the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-1 include 84.1% Rha-C10-C10, and the remaining 15.9% are other types of components (4.1% Rha-Rha-C10-C12, 2.1% Rha-C12:1-C10, 4.2% Rha-C8-C10, and 1.5% other components). By comparison, among the rhamnolipid components produced by wild-type Pseudomonas aeruginosa, there are four components (Rha-C10-C10, Rha-Rha-C10-C12, Rha-C12:1-C10, and Rha-C8-C10) that account for more than 5%; while among the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-1, only Rha-C10-C10 accounts for more than 5%. Therefore, it can be considered that compared with wild-type Pseudomonas aeruginosa that cannot produce single-component rhamnolipid, Pseudomonas aeruginosa NJUXR-CC-1 can produce single-component rhamnolipid.

[0040] Continuing to refer to Table 7, it can be seen that the total production of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa is 4.7 g / L; the total production of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-1 is 12.7 g / L, which is almost three times the total production of rhamnolipids in wild-type Pseudomonas aeruginosa. Compared with wild-type Pseudomonas aeruginosa, Pseudomonas aeruginosa NJUXR-CC-1 can produce high rhamnolipids.

[0041] In summary, it can be seen from Table 7 that the Pseudomonas aeruginosa NJUXR-CC-1 obtained by the above method can produce a high yield of single-component rhamnolipid compared to the wild-type Pseudomonas aeruginosa.

[0042] Experimental Example 2: This experimental example is based on a method for high-yield single-component rhamnolipid described in Example 2, and verifies whether the Pseudomonas aeruginosa strain NJUXR-CC-2 obtained in Example 2 has the ability to high-yield single-component rhamnolipid.

[0043] Specifically, referring to step 6 of Example 2, the Pseudomonas aeruginosa obtained in step 5 was cultured and fermented to produce rhamnolipids, and referring to step 7 of Example 2, the contents of various components of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and Pseudomonas aeruginosa NJUXR-CC-2 were measured. The mass spectrometry signal of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 is shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the types of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 are complex, but the signal intensity mainly comes from rhamnolipids with a mass-to-heterotype ratio of 503.2, indicating that the main rhamnolipid component produced by Pseudomonas aeruginosa NJUXR-CC-2 is Rha-C10-C10 type rhamnolipid. In addition, the figure also shows signals with mass-to-heterotype ratios of 531.3, 529.3, and 475.2, indicating that the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 also contains Rha-Rha-C10-C12 type rhamnolipids, Rha-C12:1-C10 type rhamnolipids, and Rha-C8-C10 type rhamnolipids. However, the contents of Rha-Rha-C10-C12 rhamnolipids, Rha-C12:1-C10 rhamnolipids, and Rha-C8-C10 rhamnolipids are much lower than those of Rha-C10-C10 rhamnolipids.

[0044] The contents of rhamnolipid components in the fermentation broth of wild-type Pseudomonas aeruginosa and Pseudomonas aeruginosa NJUXR-CC-2 are shown in Table 8.

[0045] Table 8: Ratio of rhamnolipid components in the fermentation broth of wild-type Pseudomonas aeruginosa and Pseudomonas aeruginosa NJUXR-CC-2 ; As can be seen from Table 8, the rhamnolipid components produced by the wild-type Pseudomonas aeruginosa include 56.3% Rha-C10-C10, and the remaining 43.7% are other types of components (24.0% Rha-Rha-C10-C12, 8.3% Rha-C12:1-C10, 7.2% Rha-C8-C10, and 4.2% other components); the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-2 include 95.1% Rha-C10-C10, and the remaining 4.9% are other types of components (1.6% Rha-Rha-C10-C12, 1.3% Rha-C12:1-C10, 1.2% Rha-C8-C10, and 0.8% other components). By comparison, among the rhamnolipid components produced by wild-type Pseudomonas aeruginosa, there are four components (Rha-C10-C10, Rha-Rha-C10-C12, Rha-C12:1-C10, and Rha-C8-C10) that account for more than 5%; while among the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-2, only Rha-C10-C10 accounts for more than 5%. Therefore, it can be considered that compared with wild-type Pseudomonas aeruginosa that cannot produce single-component rhamnolipid, Pseudomonas aeruginosa NJUXR-CC-2 can produce single-component rhamnolipid.

[0046] Continuing to refer to Table 8, it can be seen that the total production of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa is 4.7 g / L; the total production of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 is 16.5 g / L, which is 3.5 times the total production of rhamnolipids in wild-type Pseudomonas aeruginosa. Compared with wild-type Pseudomonas aeruginosa, Pseudomonas aeruginosa NJUXR-CC-2 can produce high rhamnolipids.

[0047] In summary, it can be seen from Table 8 that the Pseudomonas aeruginosa NJUXR-CC-2 obtained by the above method can produce a high yield of single-component rhamnolipid compared to the wild-type Pseudomonas aeruginosa.

Claims

1. A method for high-yield single-component rhamnolipid, characterized in that: The wild-type rhamnosyltransferase rhlB or rhlA of Pseudomonas aeruginosa is modified. The amino acid sequence of the modified rhamnosyltransferase rhlB is shown in SEQ ID NO: 1, and the amino acid sequence of the modified rhamnosyltransferase rhlA is shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that The wild-type rhamnosyltransferase rhlB of Pseudomonas aeruginosa was modified to obtain a mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NJUXR-CC-1, deposited in China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025100.

3. The method according to claim 1, characterized in that The wild-type rhamnosyltransferase rhlA of Pseudomonas aeruginosa was modified to obtain a mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa )NJUXR-CC-2, deposited in China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025102.

Images