A selectively improved glycosyltransferase mutant and uses thereof
By modifying the 132nd amino acid position of the Bacillus licheniformis glycosyltransferase UTGBL1, mutants S132V and S132G were obtained, solving the problems of low efficiency of glycosyltransferase and cumbersome chemical synthesis steps in the existing technology, and achieving the effect of highly selective synthesis of rhodioloside.
Patent Information
- Application Number
- CN202510078338.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2045-01-17
AI Technical Summary
In existing technologies, plant-derived glycosyltransferases have low efficiency in Escherichia coli, resulting in low selectivity in the synthesis of rhodiolosides and difficulty in efficiently separating the product. Furthermore, the chemical synthesis of rhodiolosides involves cumbersome procedures and significant environmental impact.
Using homology modeling and molecular docking methods, single-point saturation mutagenesis was performed on the glycosyltransferase UTGBL1 derived from Bacillus licheniformis, especially the modification of amino acid 132, to obtain mutants S132V and S132G, which improved their selectivity in the synthesis of rhodioloside.
The selectivity of mutants S132V and S132G was significantly improved to 89%, enabling highly efficient catalytic synthesis of rhodioloside from tyrosol and uridine diphosphate glucose. This simplified the synthesis steps and reduced the environmental impact, showing promising prospects for industrial applications.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of genetic engineering, and particularly relates to a mutant of glycosyltransferase with improved selectivity and application thereof. BACKGROUND
[0002] Rhodioloside (p-hydroxyphenethyl alcohol-beta-glucoside) is a key effective component in traditional Chinese medicine Rhodiolae, which is a product of tyrosol hydroxyl glycosylation, has anti-aging, anti-nerve cell aging, anti-tumor, anti-depression, cardiovascular protection, sedation and other activities, and is widely used in medical, health care products and skin care industry. Therefore, the demand for rhodioloside is increasing, but the content of rhodioloside in Rhodiolae plant is low (mainly exists in the blocky rhizome of Rhodiolae), which increases the complexity of the extraction process. Wild Rhodiolae resources are close to extinction due to overexploitation. In order to efficiently obtain rhodioloside, people take the way of synthesis to prepare rhodioloside. The chemical synthesis of rhodioloside cannot be mass-produced due to the more steps, protection / deprotection, and use of noble metal catalysts and bromide which have a greater impact on the environment. The enzymatic synthesis of rhodioloside has the advantages of mild conditions and no need to protect tyrosol, and can efficiently synthesize glycosylation products.
[0003] Glycosyltransferase plays a very important role in the synthesis of glycosides due to its high glycosylation efficiency, multiple sugar donors, and high stereoselectivity. At present, the plant-derived glycosyltransferase is mostly used in the enzymatic synthesis of rhodioloside, but the efficiency of the plant-derived glycosyltransferase in Escherichia coli is not very high. Microbial-derived glycosyltransferase UGT BL 1 can synthesize rhodioloside and rhodioloside isomers with uridine diphosphate glucose (UDPG) and tyrosol as substrates, and the selectivity is 51%. In the face of the difficulty of product separation, it is necessary to obtain glycosyltransferase with high selectivity through enzyme molecular modification technology. At present, directed evolution technology is one of the most effective strategies to improve the properties of proteins, which is a simulation of the natural evolution mechanism (random mutation, gene recombination, natural selection) in the laboratory, and is an effective method to obtain mutant enzymes with excellent properties in a short period of time by in vitro modification of enzyme genes and directional selection. SUMMARY
[0004] The present application provides a glycosyltransferase mutant and application thereof to overcome the deficiencies of the prior art, and takes glycosyltransferase UTG BL 1 derived from Bacillus licheniformis as the research object, adopts homology modeling and molecular docking methods, and uses single-point saturation mutation technology on the amino acids around the glycosylation site to obtain two glycosyltransferase mutants with significantly improved selectivity, which have good application potential in the synthesis of rhodioloside.
[0005] The specific technical scheme of the present application is as follows:
[0006] A glycosyltransferase mutant, wherein the mutant contains amino acid 132 of the amino acid sequence, the glycosyltransferase UTG derived from Bacillus licheniformis. BL The amino acid sequence of 1 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2.
[0007] This invention is based on the glycosyltransferase UTG derived from Bacillus licheniformis. BL 1. Optimization and modification performed. The glycosyltransferase UTG derived from Bacillus licheniformis. BL The amino acid sequence of 1 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2.
[0008] The glycosyltransferase mutant of the present invention is a mutation of serine at position 132 of the amino acid sequence shown in SEQ ID NO:1 into alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, phenylalanine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.
[0009] Preferably, the mutant of the present invention is a mutation of serine at position 132 of the amino acid sequence shown in SEQ ID NO:1 to valine or glycine, and the amino acid sequence is shown in SEQ ID NO:3 and SEQ ID NO:4.
[0010] Another object of the present invention is to provide a gene encoding a glycosyltransferase mutant as described in the present invention, a recombinant vector containing the glycosyltransferase mutant gene, and a transformant of the recombinant vector.
[0011] Furthermore, the present invention also provides a method for preparing the recombinant vector, wherein the glycosyltransferase mutant gene of the present invention is prepared by artificial synthesis or gene cloning, an expression vector is constructed to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a host cell.
[0012] The expression vector is a plasmid, bacteriophage, virus, or host cell.
[0013] The host cell can be a prokaryotic or eukaryotic cell, and can be Escherichia coli, yeast, Bacillus, lactobacillus, Aspergillus or Trichoderma, with Escherichia coli being preferred.
[0014] Another objective of this invention is to provide the application of the glycosyltransferase mutant, DNA molecule, or expression vector of the glycosyltransferase mutant described herein in the catalytic synthesis of rhodioloside. Specifically, it is used to catalyze the synthesis of rhodioloside from tyrosol and uridine diphosphate glucose.
[0015] Beneficial effects: This invention targets the glycosyltransferase UGT BL 1. Through rational design and improvement of enzymatic properties, single-point mutants S132V and S132G were obtained, exhibiting good selectivity for the synthesis of rhodioloside, significantly higher than that for glycosyltransferase UGT. BL 1. Both S132V and S132G exhibit a selectivity of 89%, enabling efficient catalysis of the synthesis of rhodioloside from tyrosol and uridine diphosphate glucose (UDPG). Compared with existing direct extraction and chemical synthesis methods, the enzymatic synthesis technology for rhodioloside established in this application has advantages such as high conversion rate and fewer catalytic synthesis steps, and has good prospects for industrial application. Attached Figure Description
[0016] Figure 1 UGT is a glycosyltransferase BL 1. Structural diagram.
[0017] Figure 2 This is a diagram of the amino acid sites at the glycosyl sites.
[0018] Figure 3 This is an SDS-PAGE electrophoresis image of glycosyltransferase UGTBL1 and its alanine mutant.
[0019] Figure 4 This diagram illustrates the selectivity of rhodioloside synthesis catalyzed by the glycosyltransferase UGTBL1 and its alanine mutant.
[0020] Figure 5 The liquid phase chromatograms of the reaction products of glycosyltransferases UGTBL1(A), S132V(B) and S132G(C) are shown. The retention time of the byproducts is 6.4 min, and the retention time of rhodioloside is 10.3 min.
[0021] Figure 6 Temperature and pH effects on glycosyltransferase UT BL 1. Effects of the mutants S132V and S132G on activity and stability, (A) optimal temperature, (B) temperature stability, (C) optimal pH, (D) pH stability. Detailed Implementation
[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Example 1: Determination of key amino acid residues at the glycosyl site of glycosyltransferase UGTBL1
[0024] (1) Homology modeling: BLAST analysis was performed based on the NCBI and PDB databases. Using the Bacillus-derived UDP-glycosyltransferase (PDB ID: 7VLB) as a template, its sequence was similar to that of the original enzyme UGT. BL The amino acid sequence SEQ ID NO: 1 has a similarity of 56.85%. The UGT was constructed using the MODELER program in Discovery Studio 3.5 (DS 3.5). BL The three-dimensional structure of 1 is as follows Figure 1 The final quality analysis of the constructed model was performed using the PROCHECK and Profile-3D programs. Model visualization and analysis were conducted using Discovery Studio 2018 Client and MOE2019.01 software.
[0025] (2) Use Autodock 4.2 software to dock the substrate to the already constructed UGT model. BL The catalytic center of 1 selects the optimal ligand-receptor docking conformation based on the free binding energy predicted by computer.
[0026] (3) Identification of key amino acids: such as Figure 2 As shown, by analyzing the interaction between UDPG and tyrosol and the enzyme, the amino acids G15, S132, A235, F236, G300, M301, E322, and Q323 at the glycosyl site were identified as key amino acids for mutation.
[0027] Example 2 Glycosyltransferase UGT BL 1. Construction of mutants
[0028] The key amino acid identified in Example 1 was mutated with alanine to produce a product containing the glycosyltransferase UGT. BL Using the pET-28a(+) plasmid (SEQ ID NO: 2) as a template, and referring to the Vazyme biological products and operation manual, the site-directed mutant sequence was amplified from the whole plasmid using mutant primer pairs. The PCR product was digested with Dpn I. After template digestion, it was transformed into E. coli BL21(DE3) competent cells using the heat shock method and plated on LB agar plates containing 100 μg / ml kanamycin sulfate, and incubated overnight at 37°C. The mutation results were verified by sequencing by Anhui General Biotechnology Co., Ltd. The mutant was constructed using the S132 site as an example, employing conventional PCR techniques with glycosyltransferase UGT... BL1. Using the expression vector as a template, full plasmid amplification was performed to introduce mutations, and the resulting S132 mutant library was successfully constructed after sequencing verification.
[0029] Example 3: Fermentation expression of recombinant glycosyltransferase mutant in Escherichia coli
[0030] The expression mutant constructed in Example 2 and the original enzyme UGT were respectively used. BL 1. The engineered bacteria were inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37°C and 180 rpm. 2% of the seed culture was then inoculated into 50 mL of fresh LB liquid medium and cultured at 37°C and 180 rpm until OD (dose-free survival) reached. 600 When the concentration is 0.6–0.8, the sample is removed, cooled in an ice-water bath for 5 min, and then IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) is added. Expression is induced at 20 °C and 180 rpm for 16 h.
[0031] The induced fermentation broth was centrifuged at 12,000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in a 50 mM Na2HPO4-KH2PO4 (pH 8.0) buffer to wash them. The cells were then centrifuged again at 12,000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in the buffer again. The cells were then sonicated. The lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant was used for SDS-PAGE electrophoresis. The stacking gel concentration was 4%, and the separating gel concentration was 12.5%. The sample and loading buffer were mixed at a 3:1 ratio, and the mixture was reacted in a boiling water bath for 5 min before loading for electrophoresis. The electrophoresis apparatus was set to an initial voltage of 120 V. When the sample moved to the separating gel, the voltage was increased to 230 V until the sample reached the bottom of the electrophoresis tank, at which point the electrophoresis was stopped.
[0032] The results are as follows Figure 3 As shown, glycosyltransferase UGT BL The molecular weight of 1 is 44.6 kDa. The results showed that the mutant had a clear band at 44.6 kDa after induction, indicating that the glycosyltransferase mutant was successfully induced to express.
[0033] Example 4: Screening Procedure for Optimal Glycosyltransferase Mutants
[0034] The mutant obtained in Example 2 was used to determine changes in enzyme activity using UDPG and tyrosol as substrates. The determination method is as follows:
[0035] Enzyme activity unit definition: One enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of product per minute under conditions of 30°C and pH 8.0.
[0036] The 1 mL reaction system consisted of 5 mM uridine diphosphate glucose (UDPG), 3.6 mM tyrosol, 5 mM MgCl2, 5% DMSO, and 0.5 U enzyme. After mixing the above reaction system, the mixture was placed in a 30°C shaker for 15 min, and then the reaction was terminated by boiling in a water bath for 5 min. The sample was filtered through a 0.22 μm filter and analyzed by HPLC. Detection conditions: 20% methanol as the mobile phase, flow rate 1 mL / min, C18 column, and detection wavelength 278 nm. The product concentration was calculated using the internal standard method, and the selectivity (percentage of rhodioloside yield to total product yield) and enzyme activity of the mutant-catalyzed synthesis of rhodioloside were calculated based on the product peak area.
[0037] The selectivity results of the mutant after alanine mutation are as follows: Figure 4 The results showed that the selectivity of most amino acids increased after mutation, while the selectivity of the S132 site increased the most after mutation with alanine.
[0038] The enzyme activity results of the alanine mutant are shown in the table below:
[0039] Strains WT G15 S132 A235 F236 G300 M301 E322 Q323 Enzyme activity (U) 0.027 0.034 0.026 0.016 0.009 0.004 0.002 0.021 0.020
[0040] Taking into account both the selectivity and enzyme activity of the mutant, the S132 site was selected as the hotspot residue for modification.
[0041] Following the method in Example 2, site-directed saturation mutagenesis was performed on site S132. The corresponding mutagenesis primers are shown below:
[0042]
[0043]
[0044]
[0045] Example 5: Screening Procedure for Enzyme Mutants
[0046] Following the method described in Example 4, the enzyme activity and selectivity changes of each S132 mutant obtained in Example 4 were measured using uridine diphosphate glucose (UDPG) and tyrosol as substrates. The results are shown in the table below (no activity was detected in mutants not listed in the table). The results indicate that after mutating the serine residue at position 132, only S132V, S132G, S132A, S132E, S132F, S132P, S132C, and S132T showed changes relative to the original enzyme UGT. BL 1. Selectivity was increased, while other mutant enzyme activities were almost lost. Among them, the selectivity of mutants S132V and S132G for the synthesis of rhodioloside was increased compared to the original enzyme UGT. BL The percentage of 1 increased from 51% to 89%, and the original enzyme UGT BL1. The liquid phase spectrum after the reaction of S132V and S132G is as follows: Figure 5 As shown.
[0047] Strains WT S132V S132G S132A S132E S132F S132P S132C S132T Enzyme activity (U) 0.027 0.012 0.011 0.026 0.021 0.019 0.011 0.014 0.026 Selectivity (%) 51 89 89 77 63 72 63 59 66
[0048] Example 6 Enzymatic Properties Analysis of Enzyme Mutants
[0049] 1. Optimal temperature and temperature stability
[0050] To determine the optimal temperature for the original enzyme and the mutants, the mutants S132V and S132G obtained in Example 5 were added to the reaction system and reacted at 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 15 min, respectively. After the reaction, the mixture was boiled for 5 min, and the enzyme activity was measured. Using the highest enzyme activity as 100%, the relative enzyme activity was calculated sequentially, and a curve showing the change in enzyme activity with temperature was plotted. The results are shown below. Figure 6 As shown in Figure A.
[0051] To determine the temperature stability of the original enzyme and the mutants, the enzyme solutions of mutants S132V and S132G were incubated in water baths at 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 2 hours, respectively. Then, the enzyme activity changes were measured according to the method in Example 4. Using the highest enzyme activity as 100%, the relative enzyme activity was calculated sequentially, and enzyme activity change curves under different incubation conditions were plotted. The results are shown below. Figure 6 As shown in B.
[0052] 2. Optimal pH and pH stability
[0053] To determine the optimal pH for the original enzyme and the mutant, buffers with different pH values were prepared: citric acid-sodium citrate (pH 3.0-6.0), Na₂HPO₄-KH₂PO₄ (pH 6.0-8.0), and glycine-sodium hydroxide (pH 8.0-9.0). The substrate was dissolved in each buffer, and the reaction was carried out at 30°C for 15 min. After the reaction, the mixture was boiled for 5 min, and the enzyme activity was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated sequentially. A curve showing the change in enzyme activity with substrate pH was plotted.
[0054] To determine the pH stability of the original enzyme and the mutant, the enzyme was suspended in different buffers, incubated on ice at 4°C for 2 hours, reacted at 30°C for 15 minutes, and then boiled for 5 minutes after the reaction. The enzyme activity was then measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated sequentially to plot the enzyme activity change curves under different incubation conditions.
[0055] The results are as follows Figure 6 As shown in C and D, the original enzyme UGT BLThe optimal reaction pH for both mutants S132V and S132G is 8.0. At pH 8.0, both the mutants and the original enzyme exhibit the highest activity; enzyme activity decreases with increasing or decreasing pH. The original enzyme UGT... BL The enzyme activity of the mutants S132V and S132G was relatively stable between pH 6.0 and 9.0, retaining more than 70% of its activity after incubation for 2 hours under these conditions. However, the stability of both the original enzyme and its mutants decreased between pH 3.0 and 6.0. Under different pH conditions, the activity of the original enzyme UGT... BL The enzyme activity retained was lower than that of mutants S132V and S132G, indicating that the pH stability of the mutant was improved.
Claims
1. A glycosyltransferase mutant, characterized in that... The mutant is an amino acid sequence shown in SEQ ID NO:1 where the 132nd amino acid is mutated from Ser to Val or Gly.
2. A DNA molecule, characterized in that, The DNA molecule encodes the glycosyltransferase mutant of claim 1.
3. An expression vector for a glycosyltransferase mutant, characterized in that... Express the glycosyltransferase mutant of claim 1.
4. The expression vector according to claim 3, characterized in that... Express the DNA molecule as described in claim 2.
5. The expression vector according to claim 3, characterized in that... The expression vector is a plasmid, a virus, or a host cell.
6. The expression vector according to claim 5, characterized in that... The host cell is a prokaryotic cell or a eukaryotic cell.
7. The expression carrier according to claim 6, characterized in that... The host cells are selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.
8. The use of the glycosyltransferase mutant according to claim 1, the DNA molecule according to claim 2, or the expression vector of the glycosyltransferase mutant according to any one of claims 3-7 in the catalytic synthesis of rhodioloside.
9. The application according to claim 8, characterized in that... Glycosyltransferase mutants catalyze the synthesis of rhodioloside from tyrosol and uridine diphosphate glucose.