A rhamnosyltransferase mutant and its application

Abstract

This invention discloses a rhamnosyltransferase mutant and its applications, belonging to the field of genetic engineering technology. This invention improves the catalytic activity of rhamnosyltransferase through protein engineering, thereby increasing the rhamnolipid yield of *Pseudomonas aeruginosa* to 62.87 g·L⁻¹. ‑1 It yields 1.89 times that of the wild type, and has good prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for increasing rhamnolipin production based on gene fusion technology. Background Technology

[0002] Rhamnolipids (RL) are anionic biosurfactants composed of rhamnosyl groups and β-hydroxy fatty acids, forming their hydrophilic and hydrophobic groups. RL can reduce surface / interfacial tension, exhibits excellent emulsifying properties, and also possesses antibacterial and anti-biofilm activities, helping to remove biofilms and promote wound healing. Most importantly, it is biodegradable, non-toxic, and non-irritating, making it a viable alternative to chemical surfactants in fields such as petroleum extraction, pharmaceuticals, daily chemicals, and environmental protection.

[0003] The synthetic pathway of rhamnolipids consists of a cascade reaction composed of three key enzymes: RhlA, RhlB, and RhlC. RhlA (3-hydroxyacyl-ACP O-3-hydroxyacyl-transferase) catalyzes the precursor β-hydroxyacyl-ACP to yield β-hydroxy fatty acid (HAA), which reacts with one molecule of dTDP-L-rhamnose under the catalysis of RhlB (rhamnosyltransferase I) to yield monorhamnolipid. Monorhamnolipid reacts with one molecule of dTDP-L-rhamnose under the catalysis of RhlC (rhamnosyltransferase II) to generate dirhamnolipid. Among them, rhamnosyltransferase I (RhlB) is encoded by the rhlB gene (GenBank accession number 878954), which is responsible for catalyzing the conversion of the substrate dTDP-L-rhamnose and β-hydroxy fatty acids into rhamnolipids. In previous experiments, we found that RhlB is a key enzyme affecting rhamnolipid production, and patent 202210938753.1 demonstrated the importance of the rhlB gene alone in improving rhamnolipid production. However, overexpression of the rhlB gene alone has a limited effect on improving rhamnolipid production.

[0004] As is well known, the structure and function of a protein are determined by its primary amino acid sequence. Therefore, altering the amino acid sequence at specific positions can affect the catalytic performance of a protein, such as enzyme activity, stability, or selectivity. In recent years, there have been numerous reports of improving enzyme catalytic performance through protein engineering, which has become a very effective method for enzyme evolution.

[0005] For RhlB, its substrates dTDP-L-rhamnose and β-hydroxy fatty acids have large steric hindrances, making it difficult for them to enter the enzyme catalytic center. In order to further improve the yield of rhamnose lipids, this invention proposes to use protein engineering to change the size of the PaRhlB substrate binding pocket space to improve its catalytic activity. Summary of the Invention

[0006] The present invention aims to provide a method for increasing the rhamnolipid production of Pseudomonas aeruginosa by using a rhamnosyltransferase mutant.

[0007] One aspect of the present invention provides a rhamnosyltransferase mutant, which is obtained by modifying RhlB, wherein the modification includes at least one of the following schemes: mutating any one or more amino acids at sites 16, 229, 230, 306, 328, 330, and 346 of amino acids to one of glycine, alanine, or serine.

[0008] The amino acid sequence of RhlB is shown in SEQ ID NO.7, and the modification described in this article is based on this amino acid sequence.

[0009] The modifications described in this article include at least one of the above-mentioned sites, and may also include modifications at two, three, four, five, six or seven sites, wherein each site may be mutated to any one of the amino acids glycine, alanine or serine.

[0010] Another aspect of the present invention provides a gene that encodes the above-mentioned rhamnosyltransferase mutant.

[0011] Another aspect of the present invention provides a plasmid containing the aforementioned gene. The plasmid may be pBBR1MCS5, or other plasmids commonly used in the art.

[0012] Another aspect of the present invention provides a bioengineered bacterium containing or integrating the above-mentioned plasmids.

[0013] Furthermore, the bioengineered bacteria are Escherichia coli or Pseudomonas aeruginosa.

[0014] Another aspect of the present invention provides the application of the above-mentioned rhamnosyltransferase mutants, genes, plasmids or bioengineered bacteria in the preparation of rhamnolipids.

[0015] Another aspect of the present invention provides a method for preparing rhamnolipids, wherein the method involves modifying the RhlB gene in a microbial organism, culturing and fermenting the bioengineered bacteria to obtain a fermentation product; the modification involves site-directed mutagenesis of the RhlB gene such that its expression product contains at least one of the following: mutating any one or more amino acid positions at positions 16, 229, 230, 306, 328, 330, and 346 to one of glycine, alanine, or serine. Preferably, the mutation involves replacing position 328 or 229 of the RhlB amino acid sequence with glycine.

[0016] The bioengineered bacteria mentioned in this article are Escherichia coli or Pseudomonas aeruginosa.

[0017] Furthermore, the method for obtaining the bioengineered bacteria involves introducing a recombinant plasmid containing a mutant into the bioengineered bacteria. The plasmid can be a commonly used plasmid in the art, such as the plasmid provided in the embodiments of this specification.

[0018] Furthermore, the PCR amplification primers for the mutant are shown in Table 2.

[0019] Furthermore, the cultivation and fermentation method employs cultivation and fermentation methods commonly used in the art. Specifically, it includes: inoculating the microbial strain into a culture medium to obtain a seed culture solution, and then inoculating the seed culture solution into a fermentation culture medium for fermentation. Alternatively, it may be the method described in the embodiments of this specification.

[0020] As described above, the rhamnosyltransferase mutant of the present invention and its application have the following beneficial effects:

[0021] By modifying the RhlB gene, we obtained a large number of rhamnosyltransferase mutants, with the preferred mutant M328G producing 62.87 g·L⁻¹ rhamnolipid. -1 It is 1.89 times the yield of the wild type. Attached Figure Description

[0022] Figure 1 PCR amplification of gene P rpsJ and rhlB

[0023] Figure 2 Colony PCR verification of recombinant plasmid P rpsJ -rhlB-pBBR1MCS5

[0024] Figure 3 P rpsJ -rhlB-pBBR1MCS5 plasmid map

[0025] Figure 4 Mutant plasmids obtained by whole plasmid PCR

[0026] Figure 5 Comparison of rhamnolipid concentrations in recombinant Pseudomonas aeruginosa containing different RhlB mutant plasmids Detailed Implementation

[0027] Currently, there is no theoretical basis for the reported catalytic mechanism and key catalytic residues of RhlB. In order to obtain the catalytic center of PaRhlB more accurately, the experiment will compare the crystal structures of Q9HYD1 predicted by AlphaFold with those of vancomycin glycosyltransferase GtfD (PDB accession number: 1rrv.1, resolution 10 ... Secondary structure superposition was performed using Discovery Studio 4.5 software. Comparison revealed a high degree of consistency between the secondary structure of Q9HYD1 and GtfD, with only a partial shift in the carbon skeleton of the loop region, which did not affect the determination of the catalytic center. Since the GtfD crystal structure contains both a glycosyl donor (TDP) and a glycosyl acceptor, spatial overlap of the structures allowed us to identify the glycosyl donor binding pocket and the glycosyl acceptor binding pocket of PaRhlB. The glycosyl acceptor binding pocket consists of several α-helices at the N-terminus and is located in the outer layer of the structure, while the glycosyl donor binding pocket is located in the inner layer and is a smaller binding pocket. For sterically hindered glycosyl donors, it may be difficult to enter the catalytic center and participate in the reaction, which may explain its low catalytic activity.

[0028] To increase the substrate binding pocket of the glycosyl donor, we [discussed the distance from TDP]. We analyzed 30 amino acid sites within the range. Based on literature reports, we inferred that Ser11 and Asp13 are the amino acid residues involved in catalysis (Biochemistry, 2004, 43, 5170-5180). The remaining 28 amino acids can be roughly divided into amino acid residues with relatively small steric hindrance (Ala11, Gly12, Gly232, Ser233, Gly259, Ala303, Ala307, Gly11, Gly232, Ser233, Gly259, Ala303, Ala307, Gly233, Gly259, Ala303, Ala307, Gly259 ... The substrate-binding pocket is divided into two groups: y310, Gly324, Gly326, Ala327, Ser329, and sterically hindered amino acid residues (Phe15, Pro16, Val128, Pro228, Leu229, Phe230, Asp231, Leu286, Tyr306, Pro308, Leu309, ILe325, Met328, Leu330, Asp346, Gln347). Among these, the sterically hindered amino acids occupy 42.8% of the substrate-binding pocket, and the majority are Gly and Ala. This suggests that the natural evolution of PaRhlB has also progressed towards increasing the substrate-binding pocket size.

[0029] Therefore, for these 16 sterically hindered amino acid residues, this invention proposes to construct mutant libraries using glycine scanning, alanine scanning, or serine scanning strategies, and screen out sites with significant changes in enzyme activity. Since the construction methods of mutant libraries are similar, this invention patent uses the construction and screening of glycine scanning libraries as an example to demonstrate and illustrate the embodiments.

[0030] Terminology Definition

[0031] In this document, the term "bioengineered bacteria" refers to cells used for transformation, i.e., cells used to express the target gene. Bioengineered bacteria can be isolated cells or cell lines cultured in cultures, or cells present in living tissues or organisms. In the context of this invention, the host cell is preferably a cell capable of growing in a culture. The cells described in this invention are bacteria or fungi, and examples of *Escherichia coli* and *Pseudomonas aeruginosa* are used for illustration and explanation.

[0032] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0033] Example 1: Construction of recombinant plasmid P rpsJ -rhlB-pBBR1MCS5

[0034] Using the genome of Pseudomonas aeruginosa ATCC27853 as a template, P was amplified by PCR. rpsJ -rhlB fragment, the reaction system was 50μL: 1μL genome, 1μL upstream primer (10μM), 1μL downstream primer (10μM), 25μL 2×Phanta Master Mix high-fidelity DNA polymerase, 22μL ultrapure water, primers are shown in Table 1. The PCR program was: (1) 94℃, 10min; (2) 30 cycles: 95℃, 30s; 55℃, 30s; 72℃, 1min or 1min 30s; (3) 72℃, 10min; (4) 16℃, +∞.

[0035] Table 1 Primer List

[0036]

[0037] Among them, P rpsJ The nucleotide sequence is as follows (SEQ ID NO.5):

[0038]

[0039]

[0040] The nucleotide sequence of rhlB is as follows (SEQ ID NO.6):

[0041]

[0042]

[0043] The amino acid sequence of rhlB is as follows (SEQ ID NO.7):

[0044]

[0045] The results of gene retrieval are as follows Figure 1 As shown, P rpsJ The gene sizes of rhlB were 500bp and 1281bp, respectively. As shown in the figure, bands of the corresponding sizes were successfully obtained. The PCR products were then purified using a gel extraction purification kit (Shanghai Jierui Biotechnology Co., Ltd.) for subsequent use.

[0046] The purified PCR product fragments and linearized pBBR1MCS5 vector (digested with BamHI and HindIII) were ligated using a one-step cloning multi-fragment ligation kit (Nanjing Novizan Biotechnology Co., Ltd.). The ligation process was as follows: 10 μL: 60–100 ng of vector. Fragment P was added at a molar ratio of gene fragment to vector fragment of 5:1. rpsJ Add rhlB, 5 μL of 2×CloneExpress, and ultrapure water to a final volume of 10 μL. Incubate at 50 °C for 15 min. After the reaction, transform the ligation product into E. coli BL21(DE3) competent cells: heat shock at 42 °C for 90 s, culture at 37 °C for 1–1.5 h, then plate the transformation product onto LB agar plates containing gentamicin resistance and incubate overnight at 37 °C.

[0047] The next day, single colonies were picked for colony PCR verification. The colony PCR system (10 μL) consisted of: picking one colony with a toothpick, 5 μL of 2×Taq DNA polymerase, 0.5 μL of M13F, 0.5 μL of M13R, and adding ultrapure water to a final volume of 10 μL. The M13F sequence was: GTAAAACGACGGCCAGT (SEQ ID NO.8); the M13R sequence was: CAGGAAACAGCTATGAC (SEQ ID NO.9). The colony PCR program was as follows: (1) 94℃, 10 min; (2) 30 cycles: 95℃, 30 s; 55℃, 30 s; 72℃, 1 min; (3) 72℃, 10 min; (4) 16℃, +∞.

[0048] Colony PCR results as follows Figure 2 As shown in the figure, P rpsJ The -rhlB gene has been successfully ligated into the vector pBBR1MCS5. The gene is approximately 1781 bp in length and has been named P. rpsJ -rhlB-MCS5 (image as shown) Figure 3 (As shown in the figure), then the corresponding positive transformants were picked and inoculated into LB medium containing gentamicin resistance, and cultured overnight at 37°C and 220 rpm. The next day, plasmids were extracted using a plasmid mini-extraction kit (Shanghai Jierui Biotechnology Co., Ltd.) and sequenced and verified by Suzhou Hongxun Technology Co., Ltd.

[0049] Example 2: Construction of glycine scanning mutant plasmid

[0050] Site-directed mutagenesis was performed on Phe15, Pro16, Val128, Pro228, Leu229, Phe230, Asp231, Leu286, Tyr306, Pro308, Leu309, ILe325, Met328, Leu330, Asp346, and Gln347 using whole plasmid PCR. The primer designs are shown in Table 2 (all described in 5-3' direction), with underlined sites representing mutation sites.

[0051] Table 2. Glycine scanning primer design

[0052]

[0053]

[0054] PCR reaction system (50μL): 1.0μL KOD enzyme, 1.0μL template (5-50ng), 4.0μL dNTP, 5.0μL 10×reaction buffer, 1.0μL each of forward and reverse primers, and ddH2O to bring the total to 50μL.

[0055] The PCR amplification program is as follows: (1) denature at 94℃ for 3 min, (2) denature at 94℃ for 30 sec, (3) anneal at 54℃ for 30 sec, (4) extend at 72℃ for 150 sec. Repeat steps (2) to (4) for 10 to 15 cycles. Finally, extend at 72℃ for 10 min and store the PCR amplification product at 4℃.

[0056] PCR results as follows Figure 4 As shown in the figure, the plasmid band of the corresponding size was successfully amplified. DpnI restriction endonuclease was then added to the PCR product, and the mixture was incubated at 37℃ for 1 h to remove the template plasmid. 10 μL of the digestion solution was then transferred into 100 μL of E. coli BL21(DE3) competent cells using the CaCl2 / MgCl2 thermal conversion method. After culturing at 37℃ for 1–1.5 h, the cells were evenly spread onto a substrate containing 50 μg / mL of DpnI restriction endonuclease. -1 Gentamicin was incubated on LB agar plates at 37°C upside down for 12 hours.

[0057] After transformants grew, single colonies were picked with toothpicks and inoculated into LB liquid medium. After culturing at 37°C for 12 hours, plasmids were extracted and sent to Suzhou Hongxun Biotechnology Co., Ltd. for sequencing. The sequencing results showed that 15 mutants of RhlB were successfully obtained: F15G, P16G, V128G, P228G, L229G, F230G, D231G, L286G, Y306G, P308G, L309G, I325G, M328G, L330G, and D346G. Multiple PCRs at the Q347 site failed to obtain the correct mutant sequence, so the construction of this mutant was abandoned.

[0058] Example 3: Construction of recombinant Pseudomonas aeruginosa containing the RhlB mutant

[0059] 3 μL of the 15 successfully validated RhlB mutant plasmids and RhlB WT The plasmid was added sequentially to competent *Pseudomonas aeruginosa* cells ATCC27853 and mixed by pipetting and aspiration. The cells were incubated on ice for 30 min. Transformation was performed using a BIO-RAD Micro Pulser electroporator with a 0.1 cm electroporation cuvette. The electroporation voltage was 2.5 kV for 3.0 ms. After electroporation, the cells were placed on ice, and 800 μL of LB medium was quickly added. The cells were resuspended by pipetting and aspiration, and the bacterial culture was transferred to a centrifuge tube and incubated at 37°C with shaking for 2 h. The inoculated culture was then plated on LB agar plates containing gentamicin resistance. The transformed cells were *Pseudomonas aeruginosa* containing the recombinant plasmid. Single colonies were then picked and inoculated into LB liquid medium. A suitable amount of the bacterial culture was collected the following day for preservation.

[0060] Example 4: Comparison of rhamnolipid production in recombinant Pseudomonas aeruginosa containing the RhlB mutant

[0061] The recombinant *Pseudomonas aeruginosa* mutants obtained in Example 3 were sequentially fermented to compare rhamnolipid production. Different recombinant *Pseudomonas aeruginosa* mutants and wild-type bacteria were inoculated into LB medium and cultured overnight at 37°C using a shaker as the seed culture. The seed culture was then inoculated at a 10% inoculation rate into 100 mL of fermentation medium (80 g·L⁻¹). -1 Soybean oil, 0.3 g / L -1 K2HPO4, 15g·L -1 NaNO3, 1.0 g·L -1 KCl, 0.5 g·L -1 Add MgSO4·7H2O to a 500mL Erlenmeyer flask, and add 0.1% trace element (2g·L⁻¹). -1 Trisodium citrate·2H₂O, 0.28 mg·L⁻¹ -1 FeCl3·6H2O, 1.4 g·L -1 ZnSO4·7H2O, 1g·L -1 CoCl2·7H2O, 3g·L -1 CuSO4·7H2O) and gentamicin (50 mg·mL) -1 At 30℃ and 180 rpm·min -1 Fermentation lasted 7 days, and samples were taken after fermentation to determine the content of rhamnolipin.

[0062] Determination of rhamnolipid concentration

[0063] Take 100 μL of the above fermentation broth and adjust the pH to 2.0 with hydrochloric acid; add 300 μL of ethyl acetate and extract with shaking for 10 min; after extraction, centrifuge at 12000 rpm for 5 min; aspirate the organic phase, add another 300 μL of ethyl acetate and extract for 10 min; after extraction, centrifuge at 12000 rpm for 5 min; aspirate the organic phase again, combine the two aspirated organic phases and place them under a nitrogen blower to volatilize the organic phase. After the ethyl acetate has completely volatilized, add 100 μL of H2O to the centrifuge tube, shake and mix well, and use it as the sample to be tested.

[0064] After appropriately diluting the 100 μL of the treated sample, take 100 μL into a 1.5 mL centrifuge tube, add 100 μL of phenol reagent (1.6% w / v), add 800 μL of 60% concentrated sulfuric acid (v / v), react at 80℃ and 1000 r / min for 30 min, cool to room temperature, and measure the absorbance value at 421 nm. Substitute the reading into the standard curve to calculate the rhamnolipid concentration. Rhamnolipid concentration = rhamnolipid concentration × 3.4 × dilution factor.

[0065] Plotting the standard curve

[0066] Prepare solutions of 0, 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, and 0.2 mg / mL as shown in Table 3. -1 L-rhamnose solution.

[0067] Table 3. Preparation of the rhamnose standard curve

[0068]

[0069] Take 100 μL of standard solutions of different concentrations into 1.5 mL centrifuge tubes, add 100 μL of phenol reagent (1.6% w / v), add 800 μL of 60% concentrated sulfuric acid, and incubate at 80 °C and 1000 rpm. -1 After reacting for 30 min and cooling to room temperature, the absorbance was measured at 421 nm, and a standard curve was plotted.

[0070] After substituting the above sample measurements into the standard curve, the corresponding rhamnolipid concentrations were calculated, and the results are as follows: Figure 5 As shown in the figure, the rhamnolipin production of mutants P16G, L229G, F230G, Y306G, M328G, L330G, and D346G is higher than that of WT, being 1.41, 1.75, 1.26, 1.62, 1.89, 1.26, and 1.22 times that of WT, respectively. Among them, the best mutant is M328G, with a rhamnolipin production of 62.87 g·L⁻¹. -1 The second highest yield was from the mutant L229G, which produced 58.21 g·L⁻¹ of rhamnolipin. -1 Their product yield Y p / s They reached 0.79 and 0.73 g·g respectively. –1 It far exceeds the Y reported in the literature. p / s Level: 0.278 g·g –1 and 0.392 g·g –1 (Appl. Microbiol. Biotechnol. 2011, 89, 585–592; Bioresour. Technol. 2012, 117, 208–213) This study shows that protein modification can significantly increase rhamnolipid production and obtain rhamnolipid engineered bacteria with high product yield. This method is also applicable to the modification of other engineered bacteria.

[0071] Following the above construction method, we constructed alanine-scanning mutant libraries and serine-scanning mutant libraries for 16 mutation sites. Through screening, we obtained some superior mutants, such as P16A, L229A, M328A, P16S, L229S, and M328S. The rhamnolipin yield of these mutants was increased to a certain extent compared with WT, by 1.21, 1.36, 1.43, 1.29, 1.26, and 1.52 times that of WT, respectively. This indicates that reducing steric hindrance can indeed effectively improve the enzyme activity of RhlB, thereby increasing the yield of rhamnolipin.

[0072] In summary, this invention provides a method for increasing the rhamnolipin yield of Pseudomonas aeruginosa through protein engineering, and obtains a high-yield RhlB mutant of rhamnolipin through glycine scanning, alanine scanning, or serine scanning mutation technology, which further reduces production costs and has good prospects for industrial application.

[0073] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A rhamnosyltransferase mutant, characterized in that, The rhamnose transferase mutant is obtained by modifying the amino acid sequence of RhlB, wherein the amino acid sequence of RhlB is shown as SEQ ID NO. 7, and the modification is mutating the amino acid sequence at position 16 into one of glycine, alanine or serine.

2. A gene, characterized in that, The gene encodes the rhamnose transferase mutant of claim 1.

3. A plasmid, characterized in that, The plasmid contains the gene of claim 2.

4. A bioengineered bacterium, characterized in that, The bioengineering bacteria contain the plasmid of claim 3.

5. The bioengineered bacteria of claim 4, wherein, The bioengineering bacteria are Pseudomonas aeruginosa.

6. A method for improving the yield of rhamnolipid, the method being to modify the amino acid sequence of RhlB in a bioengineered bacterium, culturing the bioengineered bacterium, and obtaining the fermentation product; wherein the amino acid sequence of RhlB is shown as SEQ ID NO. 7, and the modification is to perform site-directed mutagenesis on the B gene so that the expression product thereof is to mutate the amino acid at the 16th position to one of glycine, alanine or serine. rhl B gene so that the expression product thereof is to mutate the amino acid at the 16th position to one of glycine, alanine or serine.

7. The method of claim 6, wherein, The method comprises the following steps: inoculating the bacteria into a culture medium to obtain a seed culture solution, and inoculating the seed culture solution into a fermentation culture medium to obtain a fermentation product.

8. The method of claim 7, wherein, The method for obtaining the bioengineering bacteria is to introduce the recombinant plasmid containing the mutant into the bioengineering bacteria.

9. The method of claim 7, wherein, The bioengineering bacteria are Pseudomonas aeruginosa. The bioengineering bacteria are Pseudomonas aeruginosa.

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