Rhamnosyltransferase mutant and application thereof

Abstract

The invention discloses a rhamnosyltransferase mutant and application thereof, and belongs to the technical field of genetic engineering. The catalytic activity of the rhamnosyltransferase is improved through a protein engineering modification method, so that the rhamnolipid yield of the pseudomonas aeruginosa is improved, reaches 62.87 g.L <-1 > and is 1.89 times that of a wild type, and the pseudomonas aeruginosa has a good industrial application prospect.

Description

[0001] Parent application number: 202310149228.6; filing date: 2023-02-22; invention name: A rhamnosyltransferase mutant and its application TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to a method for improving rhamnolipid production based on gene fusion technology. BACKGROUND

[0003] Rhamnolipids (RL) is a kind of anionic biosurfactant of glycolipid, which is composed of rhamnose and β-hydroxy fatty acid as its hydrophilic group and hydrophobic group. RL can reduce surface / interface tension, has good emulsifying performance, and also has antibacterial activity and antibiofilm activity, which can help to remove biofilm and promote wound healing; most importantly, it is biodegradable, non-toxic and non-irritating, and can replace chemical surfactants to be applied in the fields of oil exploitation, medicine, daily chemicals and environmental protection.

[0004] The synthesis pathway of rhamnolipid is 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 obtain β-hydroxy fatty acid (HAA), and the latter reacts with one molecule of dTDP-L-rhamnose under the catalysis of RhlB (rhamnosyltransferase I) to obtain monorhamnolipid, and 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 rhlB gene (GenBank accession number 878954), which is responsible for catalyzing the substrate dTDP-L-rhamnose and β-hydroxy fatty acid to generate rhamnolipid. In the previous experiments, we found that RhlB is a key enzyme affecting the yield of rhamnolipid, and it is proved in patent 202210938753.1 that the independent rhlB gene is important for improving the yield of rhamnolipid, but overexpression of rhlB gene alone has limited effect on the improvement of rhamnolipid yield.

[0005] It is well known that the structure and function of a protein are determined by its primary structure amino acid sequence, therefore, changing the amino acid sequence at a specific position can affect the catalytic performance of the protein, such as enzyme activity, stability or selectivity, etc. In recent years, there have been many reports on improving enzyme catalytic performance through protein engineering, which has become a very effective means of enzyme evolution.

[0006] For RhlB, its substrates dTDP-L-rhamnose and β-hydroxy fatty acid have a huge steric hindrance, which is difficult to enter the catalytic center of the enzyme. In order to further improve the rhamnolipid yield, the present application intends to change the size of the substrate binding pocket of PaRhlB by protein engineering to improve its catalytic activity. SUMMARY

[0007] The present application aims to provide a method for improving the rhamnolipid yield of Pseudomonas aeruginosa by rhamnosyltransferase mutants.

[0008] One aspect of the present application provides a rhamnosyltransferase mutant, which is obtained by modifying RhlB, and the modification comprises at least any one of the following schemes: mutating the amino acid at any one or several of positions 16, 229, 230, 306, 328, 330 and 346 to one of glycine, alanine or serine.

[0009] The amino acid sequence of RhlB is shown in SEQ ID NO. 7, and the modification described herein is also based on the modification of this amino acid sequence.

[0010] The modification described herein at least comprises the mutation of one of the above positions, and can also comprise the modification of two, three, four, five, six or seven positions, wherein each position can be mutated to any one of glycine, alanine or serine.

[0011] Another aspect of the present application provides a gene encoding the above-mentioned rhamnosyltransferase mutant.

[0012] Another aspect of the present application provides a plasmid containing the above-mentioned gene. The plasmid can be pBBR1MCS5, or other plasmids commonly used in the art.

[0013] Another aspect of the present application provides a bioengineering bacteria containing or integrated with the above-mentioned plasmid.

[0014] Further, the bioengineering bacteria is Escherichia coli or Pseudomonas aeruginosa.

[0015] Another aspect of the present application provides the use of the above-mentioned rhamnosyltransferase mutant, gene, plasmid or bioengineering bacteria in the preparation of rhamnolipid.

[0016] Another aspect of the present application provides a method for preparing rhamnolipid, which is to modify RhlB gene in a biological bacteria, culture the fermentation bioengineering bacteria, and obtain the fermentation product. The modification is to perform site-directed mutation on the RhlB gene, so that the expression product thereof contains at least any one of the following schemes: the amino acid at any one or several of positions 16, 229, 230, 306, 328, 330 and 346 is mutated into one of glycine, alanine or serine. Preferably, the mutation is to replace the amino acid at position 328 or 229 of the RhlB amino acid sequence with glycine.

[0017] The bioengineering bacteria described herein is Escherichia coli or Pseudomonas aeruginosa.

[0018] Further, the bioengineering bacteria obtaining method is to introduce the recombinant plasmid containing the mutant into the bioengineering bacteria. The plasmid can be a plasmid commonly used in the art, for example, the plasmid provided in the examples of the specification herein can be used.

[0019] Further, the PCR amplification primer of the mutant is shown in Table 2.

[0020] Further, the culture fermentation method adopts a culture fermentation method commonly used in the art. Specifically, it includes inoculating the bacterial 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. It can also be the method described in the examples of the specification herein.

[0021] As described above, the rhamnosyltransferase mutant of the present application and the application thereof have the following beneficial effects:

[0022] By modifying the RhlB gene, we obtained a large number of rhamnosyltransferase mutants, and the yield of the more preferred mutant M328G rhamnolipid reached 62.87 g·L -1 , which is 1.89 times the yield of the wild type. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 PCR amplification of gene P rpsJ and rhlB

[0024] Figure 2 Colony PCR verification of recombinant plasmid P rpsJ -rhlB-pBBR1MCS5

[0025] Figure 3 P rpsJ -rhlB-pBBR1MCS5 plasmid map

[0026] Figure 4 Full plasmid PCR to obtain mutant plasmid

[0027] Figure 5 Comparison of rhamnolipid concentration of recombinant Pseudomonas aeruginosa containing different RhlB mutant plasmids DETAILED DESCRIPTION

[0028] At present, there is no theoretical basis for reporting the catalytic mechanism and key catalytic residues of RhlB. In order to more accurately obtain the catalytic center of PaRhlB, the structure Q9HYD1 predicted from AlphaFold was superimposed with the crystal structure (PDB accession number: 1rrv.1, resolution 2.2 A) of vancomycin glycosyltransferase GtfD using Discovery studio 4.5 software. ) using Discovery studio 4.5 software. Through comparison, it was found that the secondary structure of Q9HYD1 showed high consistency with GtfD, and only the carbon skeleton of part of the loop region deviated, which did not affect the determination of the catalytic center. Since the crystal structure of GtfD contains a glycosyl donor TDP and a glycosyl acceptor, we determined the binding pocket of the glycosyl donor and the binding pocket of the glycosyl acceptor of PaRhlB through spatial coincidence of the structure. The glycosyl acceptor binding pocket is composed of several alpha helices at the N terminus, located on the outer layer of the structure, while the glycosyl donor binding pocket is located on the inner layer of the structure and is a smaller binding pocket. For larger steric hindrance glycosyl donors, it may be difficult to enter the catalytic center to participate in the reaction, which may be the reason for its low catalytic activity.

[0029] In order to increase the substrate binding pocket of the glycosyl donor, we analyzed 30 amino acid sites within a distance of 5 A from TDP According to literature reports, we inferred that Ser11 and Asp13 are involved in catalysis (Biochemistry, 2004, 43, 5170-5180), and the remaining 28 amino acids can be roughly divided into two groups: amino acid residues with small steric hindrance (Ala11, Gly12, Gly232, Ser233, Gly259, Ala303, Ala307, Gly310, Gly324, Gly326, Ala327, Ser329) and amino acid residues with large steric hindrance (Phe15, Pro16, Val128, Pro228, Leu229, Phe230, Asp231, Leu286, Tyr306, Pro308, Leu309, ILe325, Met328, Leu330, Asp346, Gln347). Among them, amino acids with small steric hindrance account for 42.8% of the substrate binding pocket, and most of them are Gly and Ala, which means that the natural evolution of PaRhlB is also towards the direction of increasing the substrate binding pocket.

[0030] Therefore, for the 16 large steric hindrance amino acid residues, the application adopts the strategy of glycine scanning or alanine scanning or serine scanning to construct a mutant library, and the sites with significant changes in enzyme activity are screened from the mutant library. Since the construction methods of the mutant library are similar, the application patent constructs and screens the glycine scanning library to demonstrate and illustrate the examples.

[0031] Definitions of terms

[0032] In this context, the term "bioengineered bacteria" refers to those cells used for transformation, i.e. cells used to express the gene of interest. The bioengineered bacteria can be isolated cells or cell lines cultured in culture, or cells present in living tissues or organisms. In the context of the present application, the host cells are preferably cells that can grow in culture. The cells described in the present application are bacteria or fungi, and the present application demonstrates and illustrates examples with Escherichia coli and Pseudomonas aeruginosa.

[0033] The embodiments of the present application are described below by way of specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in this specification based on different views and applications without departing from the spirit of the present application. It should be noted that the process equipment or devices not specifically mentioned in the following examples are conventional equipment or devices in the art. In addition, it should be understood that the one or more method steps mentioned in the present application do not exclude the presence of other method steps before and after the combination steps or the insertion of other method steps between the explicitly mentioned steps, unless otherwise stated; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present application does not exclude the presence of other devices / apparatuses before and after the combination devices / apparatuses or the insertion of other devices / apparatuses between the two explicitly mentioned devices / apparatuses, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool to identify each method step, and is not a limitation on the arrangement order of each method step or the scope of the application that can be implemented, and changes or adjustments of the relative relationship without substantial changes in technical content are also considered as the scope of the application that can be implemented.

[0034] Example 1 Construction of recombinant plasmid P rpsJ -rhlB-pBBR1MCS5

[0035] The Pseudomonas aeruginosa ATCC27853 genome was used as a template for PCR amplification of P 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℃, +∞.

[0036] Table 1 Primer List

[0037]

[0038] Among them, P rpsJ The nucleotide sequence is as follows (SEQ ID NO.5):

[0039]

[0040]

[0041] The nucleotide sequence of rhlB is as follows (SEQ ID NO.6):

[0042]

[0043]

[0044] The amino acid sequence of rhlB is as follows (SEQ ID NO.7):

[0045]

[0046] 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.

[0047] 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. rpsJAdd 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, incubate 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.

[0048] 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℃, +∞.

[0049] 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.

[0050] Example 2: Construction of glycine scanning mutant plasmid

[0051] 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.

[0052] Table 2. Glycine scanning primer design

[0053]

[0054]

[0055] PCR reaction system (50 μL): 1.0 μL KOD enzyme, 1.0 μL template (5-50 ng), 4.0 μL dNTP, 5.0 μL 10x reaction buffer, 1.0 μL of each upstream and downstream primer, and ddH2O to 50 μL.

[0056] The PCR amplification program is as follows: (1) denaturation at 94℃ for 3 min, (2) denaturation at 94℃ for 30 sec, (3) annealing at 54℃ for 30 sec, (4) extension at 72℃ for 150 sec, repeating steps (2)-(4) for 10-15 cycles, finally extending at 72℃ for 10 min, and storing the PCR amplification product at 4℃.

[0057] The PCR results are shown in Fig. 1. Figure 4 As shown in the figure, the plasmid bands of the corresponding size were successfully amplified. Then, DpnI restriction endonuclease was added to the PCR product and incubated at 37℃ for 1 h to remove the template plasmid. 10 μL of the digestion reaction solution was transferred into 100 μL of E. coli BL21 (DE3) competent cells by CaCl2 / MgCl2 heat transformation method, and cultured at 37℃ for 1-1.5 h. Then, it was uniformly coated on LB agar plates containing 50 μg·mL-1 of gentamicin and cultured at 37℃ for 12 h under inversion. -1 The LB agar plates containing 50 μg·mL-1 of gentamicin were cultured at 37℃ for 12 h under inversion.

[0058] After the transformants grew, a toothpick was used to pick a single colony and inoculate into LB liquid medium, which was cultured at 37℃ for 12 h. Then, the plasmid was extracted and sent to Suzhou Hongxin Biotechnology Co., Ltd. for sequence determination. The sequencing results showed that 15 RhlB mutants were successfully obtained: F15G, P16G, V128G, P228G, L229G, F230G, D231G, L286G, Y306G, P308G, L309G, I325G, M328G, L330G, and D346G. Multiple PCR at Q347 site failed to obtain the correct mutant sequence, so the construction of this mutant was abandoned.

[0059] Example 3 Construction of recombinant Pseudomonas aeruginosa containing RhlB mutants

[0060] 3 μL of the above-mentioned 15 RhlB mutant plasmids and RhlB WTThe plasmid was added to the Pseudomonas aeruginosa ATCC27853 competent cells in turn and mixed by blowing and sucking, and then incubated on ice for 30 min. The BIO-RAD Micro Pulser electroporator was used for transformation, and a 0.1 cm electroporation cup was selected. The transformation voltage was 2.5 kV, the time was 3.0 ms, and after the end of the electric shock, the sample was placed on ice, 800 μL of LB medium was quickly added, and after resuspension by blowing and sucking, the bacterial solution was aspirated and placed in a centrifuge tube. The bacterial solution was cultured at 37°C on a shaker for 2 h, and then the bacterial solution was inoculated on an LB plate containing gentamicin. The transformants obtained were the Pseudomonas aeruginosa containing the recombinant plasmid. Then, a single colony was picked and inoculated into LB liquid medium, and the next day, an appropriate amount of bacterial solution was taken for preservation.

[0061] Example 4 Comparison of the production of recombinant Pseudomonas aeruginosa rhamnolipids containing RhlB mutants

[0062] The recombinant Pseudomonas aeruginosa mutants obtained in Example 3 were sequentially subjected to fermentation culture to compare the production of rhamnolipids. Different recombinant Pseudomonas aeruginosa mutants and wild bacteria were inoculated into LB medium, and the seed culture liquid was cultured overnight at 37°C on a shaker. Then, the seed culture liquid was inoculated into a 500 mL conical flask containing 100 mL of fermentation medium (80 g·L -1 Soybean oil, 0.3 g·L -1 K2HPO4, 15 g·L -1 NaNO3, 1.0 g·L -1 KCl, 0.5 g·L -1 MgSO4·7H2O) at a 10% inoculation amount, and 0.1% trace elements (2 g·L -1 Trisodium citrate·2H2O, 0.28 mg·L -1 FeCl3·6H2O, 1.4 g·L -1 ZnSO4·7H2O, 1 g·L -1 CoCl2·7H2O, 3 g·L -1 CuSO4·7H2O) and gentamicin (50 mg·mL -1 ) were added, and the mixture was incubated at 30°C, 180 rpm·min -1 for 7 days. After the fermentation was completed, the rhamnolipid content was determined by sampling.

[0063] Rhamnolipid concentration determination

[0064] Take 100 μL of the above fermentation broth, adjust the pH to 2.0 with hydrochloric acid; add 300 μL of ethyl acetate and shake for 10 min, then centrifuge at 12000 rpm for 5 min after the extraction is completed; aspirate the organic phase, then add 300 μL of ethyl acetate and extract for 10 min, then centrifuge at 12000 rpm for 5 min after the extraction is completed; 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 is completely volatilized, add 100 μL of H2O to the centrifuge tube, shake to mix uniformly, and use as the sample to be tested.

[0065] After the above 100 μL of treated sample is appropriately diluted, take 100 μL to a 1.5 mL centrifuge tube, add 100 μL of orcinol reagent (1.6% w / v), add 800 μL of 60% concentrated sulfuric acid (v / v), and react at 80°C, 1000 r / min for 30 min. After cooling to room temperature, measure the absorbance value at 421 nm, and bring the reading into the standard curve to calculate the rhamnose concentration. The rhamnolipid concentration = rhamnose concentration x 3.4 x dilution factor.

[0066] Plotting of standard curve

[0067] According to Table 3, prepare 0, 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.2 mg·mL -1 L-rhamnose solution.

[0068] Table 3 Preparation of rhamnose standard curve

[0069]

[0070]

[0071] Take 100 μL of the above fermentation broth, adjust the pH to 2.0 with hydrochloric acid; add 300 μL of ethyl acetate and shake for 10 min, then centrifuge at 12000 rpm for 5 min after the extraction is completed; aspirate the organic phase, then add 300 μL of ethyl acetate and extract for 10 min, then centrifuge at 12000 rpm for 5 min after the extraction is completed; 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 is completely volatilized, add 100 μL of H2O to the centrifuge tube, shake to mix uniformly, and use as the sample to be tested. -1 After cooling to room temperature, measure the absorbance value at 421 nm, and draw the standard curve.

[0072] After the above sample is measured, the corresponding rhamnolipid concentration is calculated, and the determination results are as shown in Figure 5 From the figure, it can be seen that the rhamnolipid production of P16G, L229G, F230G, Y306G, M328G, L330G, and D346G is higher than that of WT, which is 1.41, 1.75, 1.26, 1.62, 1.89, 1.26, and 1.22 times that of WT, respectively. The optimal mutant is M328G, and the rhamnolipid production reaches 62.87 g·L-1 Secondly, mutant L229G, the rhamnolipid production reached 58.21 g·L -1 , their product yield Y p / s reached 0.79 and 0.73 g·g –1 , far more than the Y p / s level: 0.278 g·g –1 and 0.392 g·g –1 (Appl. Microbiol. Biotechnol. 2011, 89, 585-592; Biore source. Technol. 2012, 117, 208-213), indicating that the rhamnolipid production can be significantly improved by protein modification, and the rhamnolipid engineering bacteria with high product yield are obtained, and the method is also suitable for modification of other engineering bacteria.

[0073] According to the above construction method, we constructed alanine scanning mutation library and serine scanning mutation library for 16 mutation sites, and obtained some better mutants such as P16A, L229A, M328A, P16S, L229S, M328S through screening. The rhamnolipid production of these mutants is increased to 1.21, 1.36, 1.43, 1.29, 1.26 and 1.52 times of WT, respectively, which indicates that the enzyme activity of RhlB can be effectively improved by reducing the steric hindrance, and then the rhamnolipid production is improved.

[0074] In summary, the application provides a method for improving the rhamnolipid production of Pseudomonas aeruginosa by protein engineering, and a high-yield rhamnolipid RhlB mutant is obtained by glycine scanning or alanine scanning or serine scanning mutation technology, which further reduces the production cost and has good industrial application prospect.

[0075] The above examples are intended to illustrate the embodiments disclosed in the present application, and should not be understood as limiting the present application. In addition, various modifications listed herein and changes in the method and composition of the application are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the application which are obvious to those skilled in the art should be included in the scope of the present application.

Claims

1. A rhamnosyltransferase mutant, characterized in that, The rhamnose transferase mutant is obtained by modifying the amino acid sequence of RhlB, i.e. mutating the amino acid at position 230 to glycine.

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 bioengineered bacteria contain the plasmid of claim 3.

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

6. A method for increasing the yield of rhamnolipid, the method comprising modifying the amino acid sequence of RhlB in bioengineered bacteria, culturing and fermenting the bioengineered bacteria to obtain a fermentation product; the modification comprising site-directed mutagenesis of the rhlB gene so that the expressed product has the amino acid at position 230 mutated to glycine.

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

8. The method of claim 7, wherein, The bioengineered bacteria are obtained by introducing the recombinant plasmid containing the mutant into the bioengineered bacteria.

9. The method of claim 6, wherein, The bioengineered bacteria are Pseudomonas aeruginosa.

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