A method for producing a high-yield single-component rhamnolipid

Abstract

The present invention provides a method for highly producing a single-component rhamnolipid, belonging to the field of biotechnology, which can highly produce a rhamnolipid with stable properties of a single component, thereby improving the application effect of the rhamnolipid. This method modifies the wild-type rhamnosyltransferase rhlB or rhlA of Pseudomonas aeruginosa, and the amino acid sequence of the modified rhamnosyltransferase rhlB is as shown in SEQ ID NO: 1; the amino acid sequence of the modified rhamnosyltransferase rhlA is as shown in SEQ ID NO: 2.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for highly producing single-component rhamnolipids. Background Art

[0002] Rhamnolipids (RLs) are a class of surfactants produced by certain microorganisms, and their microbial sources mainly include bacteria such as Pseudomonas aeruginosa ( Pseudomonas aeruginosa ). Rhamnolipids are mainly formed by the glycosidic bond connection of rhamnose and fatty acid molecules, have strong interfacial activity, and can reduce the surface tension of water. They are widely used in a variety of industrial fields, especially in environmental protection, detergents, cosmetics, and biomedical fields, with important application prospects.

[0003] However, due to the certain complexity and heterogeneity of the components of rhamnolipids, and the certain variability in the synthesis process of microorganisms, 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 (such as physical and chemical properties, interfacial activity, and biological activity, etc.) of rhamnolipids from the same source, and further making the application effect of rhamnolipids not good enough. Summary of the Invention

[0004] The present invention provides a method for highly producing single-component rhamnolipids, which can highly produce rhamnolipids with stable properties of a single component, thereby improving the application effect of rhamnolipids.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for highly producing single-component rhamnolipids, which modifies the wild-type rhamnosyltransferase rhlB or rhlA of Pseudomonas aeruginosa, and the amino acid sequence of the modified rhamnosyltransferase rhlB is as shown in SEQ ID NO: 1.

[0007] The above-mentioned modified rhamnosyltransferase rhlB is obtained by mutating the wild-type rhamnosyltransferase rhlB. The amino acid sequence of the wild-type rhamnosyltransferase rhlB is as 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, to obtain the modified rhamnosyltransferase rhlB.

[0008] The amino acid sequence of the modified wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO: 2.

[0009] The above-mentioned modified rhamnosyltransferase rhlA is obtained by mutating rhamnosyltransferase rhlA. The amino acid sequence of wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO: 4. Specifically, the 28th amino acid serine in 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, resulting in the modified rhamnosyltransferase rhlA.

[0010] As another aspect of the present invention, in the above method, the wild-type rhamnosyltransferase rhlB of Pseudomonas aeruginosa is modified to obtain mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), NJUXR-CC-1, which was deposited at the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025100.

[0011] As another aspect of the present invention, in the above method, the wild-type rhamnosyltransferase rhlA of Pseudomonas aeruginosa is modified to obtain mutant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), NJUXR-CC-2, which was deposited at the China Center for Type Culture Collection on January 13, 2025, and its deposit number is CCTCC NO: M 2025102.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The yield of the single-component rhamnolipid produced by the Pseudomonas aeruginosa provided by the present invention is relatively high.

[0014] 2. The present invention screens out the modified rhamnosyltransferase rhlB and the modified rhamnosyltransferase rhlA that assist Pseudomonas aeruginosa in producing high-yield single-component rhamnolipids by site-directed mutagenesis of the key enzymes involved in the synthesis of rhamnolipids by Pseudomonas aeruginosa. Since the single-component rhamnolipid has stable properties, the application effect of rhamnolipid can be improved. Description of the Drawings

[0015] Figure 1 is a schematic diagram of the protein structure and mutation sites of the modified rhamnosyltransferase rhlB in Example 1;

[0016] Figure 2 is a schematic diagram of the protein structure and mutation sites of the modified rhamnosyltransferase rhlA in Example 2;

[0017] Figure 3 It is the mass spectrometry detection chart of rhamnolipid in the fermented broth of Pseudomonas aeruginosa NJUXR-CC-2 after modification in Example 2. Detailed implementation manners

[0018] The method provided by the embodiment of the present application relates to the production of rhamnolipid and can be used for producing high-yield single-component rhamnolipid.

[0019] It can be understood that the molecular structure of rhamnolipid is relatively complex and is usually composed of multiple rhamnose units and fatty acids with different chain lengths. The physiological functions of rhamnolipid are mainly reflected in the following aspects: First, as a surfactant, rhamnolipid can improve the interaction between liquid and solid surfaces and play functions such as emulsification, foam generation, and wetting. Second, rhamnolipid plays an important role in the growth and metabolism of microorganisms, especially in the processes of biofilm formation, flora regulation, and interaction with hosts, and has the ability to enhance the tolerance of bacteria to environmental stresses (such as high salt, high temperature, and oxidative stress). In addition, rhamnolipid can also be used as a potential anti-infective agent in the medical field through its antibacterial, antifungal, and immunomodulatory effects, especially showing good prospects in the treatment of drug-resistant bacterial infections.

[0020] The main uses of rhamnolipid include: as an industrial cleaner, emulsifier, drug carrier, and active ingredient in cosmetics. In environmental pollution treatment, rhamnolipid can effectively degrade petroleum pollutants and other organic pollutants, so it has significant potential for bioremediation. In the medical field, rhamnolipid has been widely studied as a candidate drug for the treatment of drug-resistant bacterial infections due to its antibacterial and anti-biofilm formation properties. In addition, rhamnolipid also has certain applications in industries such as agriculture, cosmetics, and food additives.

[0021] 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 thus poor application effects of rhamnolipids, the embodiment of the present application provides a method for producing high-yield single-component rhamnolipid, which can produce high-yield single-component rhamnolipid with stable properties, thereby improving the application effects of rhamnolipid.

[0022] Example 1: This example describes a method for producing high-yield single-component rhamnolipid, which modifies the wild-type rhamnosyltransferase rhlB of Pseudomonas aeruginosa. The amino acid sequence of the modified rhamnosyltransferase rhlB is shown in SEQ ID NO: 1.

[0023] SEQ ID NO: 1 is as follows:

[0024] MHAILIAIGSAGDVFPFIGLARTLKLRGHRVSLCTIPVFRDAVEQHGI E FVPLSDELTYRRTMGDPRLWDPKTSFGVLWQAIAGMIEPVYEYV C AQRHDDIVVVGSLWALGARIAHEKYGIPYLSAQVSPSTLLSAHLPPVHP R FNVPEQ V PLAMRKLLWRCIERFKLDRTCAPEINAVRRKVGLETPVKRIFTQWMHSPQGVVCLFPAWFAPPQQDWPQPLHMTGFPLFDGS V PGTPLDDELQRFLDQGSRPLVFTQGSTEHLQGDFYAMALRALERLGARGIFLTGAGQEPLRGLPNHVLQRAYAPLGALLP A CAGLVHPGGIGAMSLALAAGVPQVLLPCAHDQFDNAERLVRLGCGMRLGVPLREQELRGALWRLLEDPAMAAACRRFMELSQPHSIACGKAAQVVERCHREGDARWLKAAS

[0025] It should be noted that the underlined content in SEQ ID NO: 1 above represents the mutated amino acid sites.

[0026] The above-mentioned modified rhamnosyltransferase rhlB is obtained by mutating 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 glutamate, 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, resulting in the modified rhamnosyltransferase rhlB.

[0027] SEQ ID NO: 3 is as follows:

[0028] MHAILIAIGSAGDVFPFIGLARTLKLRGHRVSLCTIPVFRDAVEQHGIAFVPLSDELTYRRTMGDPRLWDPKTSFGVLWQAIAGMIEPVYEYVSAQRHDDIVVVGSLWALGARIAHEKYGIPYLSAQVSPSTLLSAHLPPVHPKFNVPEQMPLAMRKLLWRCIERFKLDRTCAPEINAVRRKVGLETPVKRIFTQWMHSPQGVVCLFPAWFAPPQQDWPQPLHMTGFPLFDGSIPGTPLDDELQRFLDQGSRPLVFTQGSTEHLQGDFYAMALRALERLGARGIFLTGAGQEPLRGLPNHVLQRAYAPLGALLPSCAGLVHPGGIGAMSLALAAGVPQVLLPCAHDQFDNAERLVRLGCGMRLGVPLREQELRGALWRLLEDPAMAAACRRFMELSQPHSIACGKAAQVVERCHREGDARWLKAAS

[0029] The following is a detailed description of the specific steps of the above method:

[0030] Step 1: Prepare Pseudomonas aeruginosa competent cells;

[0031] Spread wild-type Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 9027, purchased from Beijing Bio-win Biotechnology Co., Ltd.) on LB solid medium and culture it overnight at 37°C. Pick a single colony and inoculate it into 4 mL of LB liquid medium, culture it at 37°C and 140 rpm for 6 h, wash the cells 3 times with sterile 10% glycerol solution, and finally resuspend the cells with 0.2 mL of 10% glycerol to obtain Pseudomonas aeruginosa competent cells. Aliquot and store the Pseudomonas aeruginosa competent cells in an -80°C refrigerator for later use;

[0032] Step 2: Prepare Pseudomonas aeruginosa competent cells carrying the pCasPA plasmid;

[0033] Add approximately 10 ng of the pCasPA plasmid to 0.1 mL of Pseudomonas aeruginosa competent cells, ice-bath in a pre-chilled electroporation cuvette for 5 min, perform electroporation once at 2.0 kV, and resuscitate and culture the Pseudomonas aeruginosa competent cells with 1 ml of LB liquid medium for 2 h. Spread the resuscitated and cultured Pseudomonas aeruginosa competent cells on LB solid medium containing 50 μg / mL tetracycline and culture it overnight at 37°C to obtain Pseudomonas aeruginosa competent cells carrying the pCasPA plasmid, and store them in an -80°C refrigerator for later use;

[0034] The formula of the above LB liquid medium includes: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride; the formula of the above LB solid medium includes: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and 15 g / L of agar;

[0035] Step 3: Construct the pACRISPR plasmid;

[0036] The pACRISPR plasmid contains the mutation site information of rhamnosyltransferase rhlB and is inserted between the DR2 elements. The sgRNA sequence with targeting function in the pACRISPR plasmid is shown in Table 1;

[0037] Table 1: sgRNA sequences for gene editing of rhamnosyltransferase rhlB

[0038] ;

[0039] Step 4: Construct the homologous fragment;

[0040] The homologous fragment is constructed using the complementary primer annealing PCR procedure, and the primer sequences used are shown in Table 2;

[0041] Table 2: Primer sequences for constructing homologous fragments for gene editing of rhamnosyltransferase rhlB

[0042] ;

[0043] Among them, "F" represents the upstream primer, "R" represents the downstream primer, and the underlined sequence is the mutation site;

[0044] In the above primer sequences for constructing homologous fragments for gene editing of rhamnosyltransferase rhlB, A represents adenine, T represents thymine, C represents cytosine, G represents guanine; N represents any one of A, T, C, G, K (keto) represents T or G, and M (amino) represents A or C;

[0045] The PCR system of the above complementary primer annealing PCR procedure is shown in Table 3, and the PCR procedure process is shown in Table 4; the PCR product obtained by the above complementary primer annealing PCR procedure is the homologous fragment;

[0046] Table 3: PCR system table

[0047] ;

[0048] Table 4: PCR procedure table

[0049] ;

[0050] Step 5: Transform the pACRISPR plasmid and the homologous fragment;

[0051] Referring to the operation in Step 2 above, transform the pACRISPR plasmid and the homologous fragment into the competent cells of Pseudomonas aeruginosa carrying the plasmid pCasPA, and spread them on the LB solid medium supplemented with 50 ug / mL tetracycline and 50 ug / mL carbenicillin, and culture them overnight at 37°C; the grown single colonies can be used for liquid culture and fermentation verification;

[0052] Step 6: Culture the Pseudomonas aeruginosa obtained in Step 5 and ferment to produce rhamnolipid;

[0053] Pick 48 single colonies of Pseudomonas aeruginosa (also known as 48 strains of Pseudomonas aeruginosa) from the plate (i.e., the LB solid medium), and inoculate them into 10 mL test tubes containing 3 ml of LB liquid medium respectively, and culture them overnight at 37°C and 200 rpm on a shaker as the seed liquid; add 1 ml of the seed liquid to 30 ml of the fermentation medium, and culture it at 37°C and 200 rpm on a shaker for 48 h to obtain the fermentation broth of each strain of Pseudomonas aeruginosa among the 48 strains of Pseudomonas aeruginosa;

[0054] The formula of the above 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; adjust the pH value to 7.0 with 1 M NaOH solution;

[0055] According to the above operation, culture the wild-type Pseudomonas aeruginosa and ferment to produce rhamnolipid to obtain the fermentation broth of the wild-type Pseudomonas aeruginosa;

[0056] Step 7: Determine the content of rhamnolipid produced by the fermentation of Pseudomonas aeruginosa;

[0057] Perform the following operations on the fermentation broth of each strain of Pseudomonas aeruginosa among the 48 strains of Pseudomonas aeruginosa and the fermentation broth of the wild-type Pseudomonas aeruginosa respectively;

[0058] Centrifuge the fermentation broth of Pseudomonas aeruginosa, take 1 ml of the supernatant, and centrifuge it at 12000 rpm for 3 min; take 200 uL of the supernatant, appropriately dilute it 3 - 10 times with methanol, fully shake it and then centrifuge it at 12000 rpm for 10 min, and the obtained supernatant passing through the membrane is the sample to be detected for rhamnolipid produced by Pseudomonas aeruginosa;

[0059] Using the Agilent high-throughput liquid chromatography-mass spectrometry system RapidFire 400 and the Agilent triple quadrupole mass spectrometer as detection tools (the chromatographic column used was C18), the samples to be detected of 48 Pseudomonas aeruginosa strains and wild-type Pseudomonas aeruginosa were detected to obtain the contents of each component of rhamnolipid in the fermentation broth of 48 Pseudomonas aeruginosa strains and the contents of each component of rhamnolipid in the fermentation broth of wild-type Pseudomonas aeruginosa;

[0060] Specifically, the mobile phase A used in the above detection process was methanol with a flow rate of 0.8 mL / min, and the mobile phase B was ammonia water (0.05%, v / v) with a flow rate of 1.2 mL / min. The analysis program was: 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 spectrometry was in negative ion mode, and the scanning range was 300 - 900 m / z. Among them, the rhamnolipid standard was prepared into gradient concentrations and a standard curve was made to calculate the rhamnolipid content in the fermentation broth sample.

[0061] Specifically, the above rhamnolipid standard was purchased from Shanghai Macklin and prepared into rhamnolipid standard solutions with gradient concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, and 500 mg / L with 50% methanol, and were detected respectively using the above-mentioned scheme. A standard curve was made with rhamnolipid concentration - total ion intensity.

[0062] Finally, the strain with the highest rhamnolipid production among the 48 Pseudomonas aeruginosa strains was designated as NJUXR-CC-1.

[0063] In addition, the above method further includes step 8.

[0064] Step 8: Gene sequencing of the dominant strain.

[0065] Specifically, the genomic DNA of NJUXR-CC-1 was extracted using a bacterial genomic DNA extraction kit (Solarbio, Beijing). The modified rhamnosyltransferase rhlB gene on the genome was sequenced. The modified rhamnosyltransferase rhlB in the genomic DNA of NJUXR-CC-1 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, resulting in the modified rhamnosyltransferase rhlB. The positions of the above mutation sites (the 49th, 94th, 144th, 151st, 234th, and 315th positions) in the protein structure of the modified rhamnosyltransferase rhlB are as Figure 1 shown.

[0066] On this basis, the above-mentioned Pseudomonas aeruginosa NJUXR-CC-1 was preserved. The above-mentioned Pseudomonas aeruginosa NJUXR-CC-1 was preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China, the preservation date being January 13, 2025, and the preservation number being CCTCC NO: M 2025100, and the taxonomic name being Pseudomonas aeruginosa NJUXR-CC-1 Pseudomonas aeruginosa NJUXR-CC-1.

[0067] Example 2: This example describes a method for producing a high-yield single-component rhamnolipid. The wild-type rhamnosyltransferase rhlA of Pseudomonas aeruginosa was modified, and the amino acid sequence of the modified rhamnosyltransferase rhlA is shown in SEQ ID NO: 2.

[0068] SEQ ID NO: 2 is as follows:

[0069] MRRESLLVSVCKGLRVHVERVGQDPGR D TVMLVNGAMATTASFARTCKCLAEHFNVVLFDLPFAGQSRQHNPQRGLITKDDEVEILLALIERF A VNHLVSASWGGISTLLALSRNPRGIRSSVVMAFAPGLNQAMLDYVGRAQALIELDDKSAIGHLLNETVGKYLP P RLKASNHQHMASLATGEYEQARFHIDQVLALNDRGYL SCLERIQSHVHFINGSWDEYTTAEDARQFRDYLPHCSFSRVEGTGHFLDLESKLAA A RVHRALLEHLLKQPEPQRAERAAGFHEMAIGYA

[0070] It should be noted that the underlined amino acid sites in the above SEQ ID NO: 2 are the mutated amino acid sites.

[0071] The above-mentioned modified rhamnosyltransferase rhlA is obtained by mutating the rhamnosyltransferase rhlA. The amino acid sequence of the wild-type rhamnosyltransferase rhlA is shown in SEQ ID NO: 4. Specifically, the 28th amino acid serine in 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, resulting in the modified rhamnosyltransferase rhlA.

[0072] SEQ ID NO: 4 is as follows:

[0073] MRRESLLVSVCKGLRVHVERVGQDPGRSTVMLVNGAMATTASFARTCKCLAEHFNVVLFDLPFAGQSRQHNPQRGLITKDDEVEILLALIERFEVNHLVSASWGGISTLLALSRNPRGIRSSVVMAFAPGLNQAMLDYVGRAQALIELDDKSAIGHLLNETVGKYLPQRLKASNHQHMASLATGEYEQARFHIDQVLALNDRGYLACLERIQSHVHFINGSWDEYTTAEDARQFRDYLPHCSFSRVEGTGHFLDLESKLAAVRVHRALLEHLLKQPEPQRAERAAGFHEMAIGYA

[0074] The difference between the method described in this example and the method described in Example 1 lies in:

[0075] In step 3, the pACRISPR plasmid contains the mutation site information of the rhamnosyltransferase rhlA and is inserted between the DR2 elements. The sgRNA sequence with targeting function in the pACRISPR plasmid is shown in Table 5.

[0076] Table 5: sgRNA sequences for gene editing of rhamnosyltransferase rhlA

[0077] ;

[0078] In step 4, the primer sequences of the homologous fragments are shown in Table 6.

[0079] Table 6: Primer sequences of the homologous fragments of rhamnosyltransferase rhlA

[0080] ;

[0081] In the above primer sequences of the homologous fragments of rhamnosyltransferase rhlA, N represents any one of A, T, C, and G;

[0082] In step 7, finally, the strain with the highest rhamnolipid production among the 48 Pseudomonas aeruginosa strains was designated as NJUXR-CC-2.

[0083] In step 8, the genomic DNA of NJUXR-CC-2 was extracted using a bacterial genomic DNA extraction kit (Beijing Solarbio), and the rhamnosyltransferase rhlA gene on the genome was sequenced. The modified rhamnosyltransferase rhlA in NJUXR-CC-2 is shown in SEQ ID NO: 2. The 28th amino acid serine of rhamnosyltransferase rhlA was mutated to aspartic acid, the 94th amino acid glutamic acid was mutated to alanine, the 168th amino acid glutamine was mutated to proline, the 206th amino acid alanine was mutated to serine, and the 262nd amino acid valine was mutated to alanine, resulting in the modified rhamnosyltransferase rhlA. The positions of the above mutation sites (the 28th, 94th, 168th, 206th, and 262nd positions) in the protein 3D structure of the modified rhamnosyltransferase rhlA are as Figure 2 shown.

[0084] On this basis, the above-mentioned Pseudomonas aeruginosa NJUXR-CC-2 was preserved. The above-mentioned Pseudomonas aeruginosa NJUXR-CC-2 was preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, China, the preservation date is January 13, 2025, and the preservation number is CCTCC NO: M 2025102, and the taxonomic name is Pseudomonas aeruginosa NJUXR-CC-2 Pseudomonas aeruginosa NJUXR-CC-2.

[0085] Experimental Example 1: Based on the method for producing a high-yield single-component rhamnolipid described in Example 1, this experimental example verified whether the Pseudomonas aeruginosa strain NJUXR-CC-1 obtained in Example 1 has the ability to produce a high-yield single-component rhamnolipid.

[0086] Specifically, referring to step 6 of Example 1, the Pseudomonas aeruginosa obtained in step 5 was cultured and fermented to produce rhamnolipids. Referring to step 7 of Example 1, the contents of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of NJUXR-CC-1 were determined. The contents of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of NJUXR-CC-1 are shown in Table 7.

[0087] Table 7: Proportion table of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of NJUXR-CC-1

[0088] ;

[0089] Note: Rha-C10-C10 contains 1 rhamnose and 2 fatty acid chains with 10 carbon atoms; Rha-Rha-C10-C12 contains 2 rhamnoses, 1 fatty acid chain with 10 carbon atoms, and 1 fatty acid chain with 12 carbon atoms; Rha-C12:1-C10 contains 1 rhamnose, 1 fatty acid chain with 10 carbon atoms, 1 fatty acid chain with 12 carbon atoms and 1 unsaturated bond; Rha-C8-C10 contains 1 rhamnose, 1 fatty acid chain with 8 carbon atoms, and 1 fatty acid chain with 10 carbon atoms.

[0090] As can be seen from Table 7, the components of rhamnolipids produced by wild-type Pseudomonas aeruginosa include 56.3% of Rha-C10-C10 and the remaining 43.7% of other types of components (24.0% of Rha-Rha-C10-C12, 8.3% of Rha-C12:1-C10, 7.2% of Rha-C8-C10, 4.2% of other components); the components of rhamnolipids produced by Pseudomonas aeruginosa NJUXR-CC-1 include 84.1% of Rha-C10-C10 and the remaining 15.9% of other types of components (4.1% of Rha-Rha-C10-C12, 2.1% of Rha-C12:1-C10, 4.2% of Rha-C8-C10, 1.5% of other components). By comparison, among the components of rhamnolipids produced by wild-type Pseudomonas aeruginosa, there are four components with a proportion exceeding 5% (Rha-C10-C10, Rha-Rha-C10-C12, Rha-C12:1-C10, Rha-C8-C10); while among the components of rhamnolipids produced by Pseudomonas aeruginosa NJUXR-CC-1, there is only one component with a proportion exceeding 5%, namely Rha-C10-C10. Therefore, it can be considered that compared with wild-type Pseudomonas aeruginosa that cannot produce single-component rhamnolipids, Pseudomonas aeruginosa NJUXR-CC-1 can produce single-component rhamnolipids.

[0091] Continuing to refer to Table 7, 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, 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 yields of rhamnolipids.

[0092] In summary, as can be seen from Table 7, Pseudomonas aeruginosa NJUXR-CC-1 obtained by the above method can produce high yields of single-component rhamnolipids compared with wild-type Pseudomonas aeruginosa.

[0093] Experimental Example 2: Based on the method for producing high yields of single-component rhamnolipids described in Example 2, this experimental example verified whether the Pseudomonas aeruginosa strain NJUXR-CC-2 obtained in Example 2 has the ability to produce high yields of single-component rhamnolipids.

[0094] Specifically, referring to Step 6 of Example 2, the Pseudomonas aeruginosa obtained in Step 5 was cultured and fermented to produce rhamnolipids. Referring to Step 7 of Example 2, the contents of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 were determined. The mass spectrometry signal pattern of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 is as Figure 3 shown. As can be seen from Figure 3 , 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-charge ratio of 503.2, indicating that the main component of rhamnolipids produced by Pseudomonas aeruginosa NJUXR-CC-2 is Rha-C10-C10 type rhamnolipids. In addition, signals with mass-to-charge ratios of 531.3, 529.3, and 475.2 are also shown in the figure, 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 type rhamnolipids, Rha-C12:1-C10 type rhamnolipids, and Rha-C8-C10 type rhamnolipids are much less than those of Rha-C10-C10 type rhamnolipids.

[0095] The contents of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 are shown in Table 8.

[0096] Table 8: Proportion table of each component of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa and the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2

[0097] ;

[0098] As can be seen from Table 8, the rhamnolipid components produced by wild-type Pseudomonas aeruginosa include 56.3% of Rha-C10-C10 and the remaining 43.7% of other types of components (24.0% of Rha-Rha-C10-C12, 8.3% of Rha-C12:1-C10, 7.2% of Rha-C8-C10, 4.2% of other components); the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-2 include 95.1% of Rha-C10-C10 and the remaining 4.9% of other types of components (1.6% of Rha-Rha-C10-C12, 1.3% of Rha-C12:1-C10, 1.2% of Rha-C8-C10, 0.8% of other components). By comparison, among the rhamnolipid components produced by wild-type Pseudomonas aeruginosa, there are four components with a proportion exceeding 5% (Rha-C10-C10, Rha-Rha-C10-C12, Rha-C12:1-C10, Rha-C8-C10); while among the rhamnolipid components produced by Pseudomonas aeruginosa NJUXR-CC-2, there is only one component with a proportion exceeding 5%, namely Rha-C10-C10. Therefore, it can be considered that compared with wild-type Pseudomonas aeruginosa that cannot produce single-component rhamnolipids, Pseudomonas aeruginosa NJUXR-CC-2 can produce single-component rhamnolipids.

[0099] Continuing to refer to Table 8, the total yield of rhamnolipids in the fermentation broth of wild-type Pseudomonas aeruginosa is 4.7 g / L; the total yield of rhamnolipids in the fermentation broth of Pseudomonas aeruginosa NJUXR-CC-2 is 16.5 g / L, which is 3.5 times the total yield of rhamnolipids in wild-type Pseudomonas aeruginosa. Compared with wild-type Pseudomonas aeruginosa, Pseudomonas aeruginosa NJUXR-CC-2 can produce rhamnolipids with high yield.

[0100] In summary, as can be seen from Table 8, Pseudomonas aeruginosa NJUXR-CC-2 obtained by the above method can produce rhamnolipids with single components and high yield compared with 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.

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