Glycosyl transferase mutant for high-selectivity catalytic synthesis of salidroside

Abstract

According to the present invention, glycosyl transferase UGTBL1 derived from Bacillus licheniformis ZSP01 is adopted as a research object, AlphaFold 2 is adopted to carry out modeling, molecular docking, molecular dynamics simulation and other technologies, amino acids in an active pocket region of glycosyl transferase are analyzed, site-directed mutagenesis is performed on the amino acids to obtain a series of glycosyl transferase mutants, and salidroside can be highly selectively catalyzed and synthesized.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a class of glycosyltransferase mutants based on active pocket regions and their applications. Background Technology

[0002] Rhodioloside (p-hydroxyphenylethanol-β-glucoside) is the core active ingredient of Rhodiola rosea plants and the main pharmacodynamic material basis of the traditional Chinese medicine Rhodiola rosea. It possesses various physiological activities, including anti-aging, anti-neuronal cell aging, anti-tumor, anti-depression, hypoglycemic, anti-radiation, anti-fatigue, cardiovascular protection, and sedative-hypnotic effects, showing broad application prospects in the pharmaceutical, health product, and functional food fields. Currently, the synthesis methods of rhodioloside include plant extraction, chemical synthesis, and biological synthesis. Plant extraction is limited by the slow growth of Rhodiola rosea plants and the endangered wild resources, making it difficult to meet market demand. Chemical synthesis involves the large-scale use of organic solvents, leading to difficulties in separation and purification, low yield, and environmental pollution. The endangered plant resources also present challenges, including complex extraction processes, long cultivation cycles, severe environmental pollution, and difficulties in separation and purification. Enzymatic synthesis, due to its mild reaction conditions, high selectivity, and environmental friendliness, is considered a potential alternative for the green manufacturing of rhodioloside.

[0003] Bo Fan et al. disclosed the glycosyltransferase UGT derived from Bacillus licheniformis ZSP01. BL The selectivity for synthesizing rhodioloside using uridine diphosphate glucose (UDP-glucose) and tyrosol as substrates was 51%, and the selectivity for synthesizing icariin D2 was 49% (Scieetific Reports|7:463|DOI:10.1038 / s41598-017-00568-z). Wild-type glycosyltransferase UGT BL 1. The enzyme has significant limitations in practical applications: its catalytic efficiency is low, leading to insufficient product yield; its regioselectivity is poor, easily generating byproducts; and its enzyme stability is poor under industrial production conditions. These defects severely restrict the application prospects of this enzyme in the large-scale production of rhodioloside. Chinese patent document CN120025997A discloses a glycosyltransferase mutant, using the glycosyltransferase UGT derived from Bacillus licheniformis. BL Using 1 as the research object, single-point mutagenesis technology was used to obtain mutants S132V and S132G, which can efficiently catalyze the synthesis of rhodioloside from tyrosol and uridine diphosphate glucose, with a selectivity of 89% for both. However, their enzyme activity was reduced compared to the wild type.

[0004] UGT through protein engineering technology BL1. Targeted modification to develop glycosyltransferase mutants with high catalytic activity, excellent regioselectivity, and good stability is of great significance and application value for achieving efficient and green biosynthesis of rhodioloside. Summary of the Invention

[0005] This invention utilizes the glycosyltransferase UGT derived from Bacillus licheniformis ZSP01. BL Using AlphaFold 2 as the research object, we analyzed the amino acids in the active pocket region of glycosyltransferases through modeling, molecular docking, and molecular dynamics simulation. We then performed site-directed mutagenesis on these amino acids to obtain a series of glycosyltransferase mutants that can selectively catalyze the synthesis of rhodioloside.

[0006] The specific technical solution of this invention is as follows:

[0007] A glycosyltransferase mutant comprising one or more mutations in amino acids 61, 62, and 70 of the glycosyltransferase amino acid sequence, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] The mutation at position 61 is as follows: asparagine is mutated to alanine, arginine, valine, phenylalanine, proline, glutamine, leucine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.

[0009] The mutation at position 62 is: isoleucine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, phenylalanine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.

[0010] The 70-position mutation is as follows: leucine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, methionine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, aspartic acid, tyrosine, aspartic acid, or cysteine.

[0011] Preferably, the glycosyltransferase mutant of the present invention is a single point mutation, wherein the asparagine at position 61 is mutated to isoleucine (N61I), or the isoleucine at position 62 is mutated to threonine (I62T), or the leucine at position 70 is mutated to tyrosine (L70W).

[0012] Preferably, the glycosyltransferase mutant of the present invention is a two-site mutation, in which asparagine at position 61 is mutated to isoleucine, and isoleucine at position 62 is mutated to threonine.

[0013] Preferably, the glycosyltransferase mutant of the present invention is a three-point mutation, wherein the asparagine at position 61 is mutated to isoleucine, the isoleucine at position 62 is mutated to threonine, and the leucine at position 70 is mutated to tyrosine.

[0014] Another object of the present invention is to provide a DNA molecule that encodes the glycosyltransferase mutant described in the present invention.

[0015] Another object of the present invention is to provide an expression vector for a glycosyltransferase mutant, expressing the carbonyltransferase mutant as described in the present invention. The expression vector contains a DNA molecule encoding the glycosyltransferase mutant described in the present invention.

[0016] The expression vector is a plasmid, bacteriophage, virus, or host cell.

[0017] 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.

[0018] Another objective of this invention is to provide the application of the glycosyltransferase mutant, DNA molecule, or expression vector of the glycosyltransferase mutant described in this invention in the synthesis of rhodioloside.

[0019] Advantages of this invention:

[0020] This invention utilizes the glycosyltransferase UGT of Bacillus licheniformis ZSP01 obtained through prior screening. BL Using 1 as the research object, the amino acids in the active pocket region of the glycosyltransferase were analyzed using techniques such as molecular docking and molecular dynamics. Saturation mutations were performed on candidate key amino acid sites L60, N61, I62, L70, M77, and F84. The results showed that single-point mutations at N61, I62, and L70 resulted in mutants with significantly increased selectivity for rhodioloside synthesis. Specifically, the selectivity of N61I, I62T, and L70W increased to 85%, 80%, and 84%, respectively. Combination mutations of N61I and I62T yielded a double mutant N61I / I62T with 96% selectivity for rhodioloside. Subsequent combination mutations of L70W with these mutants resulted in a triple mutant N61I / I62T / L70W with 99.2% selectivity for rhodioloside, representing a 6.9-fold increase in activity compared to the original enzyme. These results indicate that the glycosyltransferase mutants described in this invention exhibit higher regioselectivity and higher catalytic efficiency than the original enzyme, which is of significant importance for the synthesis of rhodioloside. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 Tyrosol docking with glycosyltransferase UGT BL 1. Active pocket diagram

[0023] Figure 2 This is a landscape diagram showing the free energy of the ligand in cavities A and B during a 100 ns simulation.

[0024] Figure 3 UGT is a glycosyltransferase BL Candidate amino acids in cavity B of 1.

[0025] Figure 4 Selectivity plot for saturation mutations of L60, N61, I62, L70, M77, and F84.

[0026] Figure 5 The relative activity plots for saturation mutants L60, N61, I62, L70, M77, and F84 are shown.

[0027] Figure 6 UGT is a glycosyltransferase BL 1. SDS-PAGE electrophoresis images of mutants.

[0028] Figure 7 Bar chart showing the relative activities of the mutant and the original enzyme.

[0029] Figure 8 This is a liquid phase diagram of the mutant and the original enzyme.

[0030] Figure 9 The image shows the enzymatic properties of the mutant N61I / I62T / L70W. (a) Optimal temperature, (b) Temperature stability, (c) Optimal pH, (d) pH stability. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that these embodiments are only for explaining the invention and not for limiting its scope. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0032] Example 1 Glycosyltransferase UGT BL 1. Determination of active pocket amino acids

[0033] The microbial glycosyltransferase UGT selected in this invention BL 1. The soluble protein content in *E. coli* is relatively high, enabling the synthesis of icariin D2 and rhodioloside from tyrosol and uridine diphosphate glucose (UDPG) as substrates, with selectivity of 49% and 51%, respectively. Therefore, this invention improves UGT by modifying the active pocket amino acids. BL 1. Regioselectivity and catalytic efficiency of UGT using Alphafold 2. BL 1. Modeling was performed using Discovery Studio to dock the substrate tyrosol and UDPG with the enzyme using default parameters. It was found that tyrosol can be present in UGT. BL 1. Inside the two cavities (cavity A and cavity B) Figure 1 The docked complex was then subjected to 100 ns molecular dynamics simulations. Throughout the simulation, the phenolic hydroxyl group of tyrosol was closer to UDPG in cavity B than the alcoholic hydroxyl group, which may indicate that cavity B is favorable for the synthesis of icariin D2. To improve the selectivity of rhodioloside, the amino acids in cavity B were modified. The optimal conformation and key binding sites during protein-ligand binding were analyzed using a free energy landscape diagram. The conformation after equilibrium was calculated, and RMSD and Rg were selected as reaction coordinates to generate a free energy heatmap. The horizontal and vertical axes represent the reaction coordinates, and color changes represent free energy changes. The optimal binding conformation was identified through analysis, including the most stable conformation (global minimum, i.e., blue) and the possible metastable and transition states (local minimum, i.e., green). Figure 2 By analyzing the amino acids with strong interactions between tyrosine and the enzyme in the conformation represented by the energy potential well and local minima, candidate key amino acid sites L60, N61, I62, L70, M77, and F84 were obtained. Figure 3 ).

[0034] Example 2 Glycosyltransferase UGT BL 1. Construction of mutant libraries

[0035] The glycosyltransferase UGT obtained in Example 1 BL Candidate sites L60, N61, I62, L70, M77, and F84 were subjected to single-point saturation mutations using the degenerate codons NNK and MNN to obtain mutants with higher regioselectivity and catalytic efficiency. The construction method is as follows, and the primers used are listed in the table below:

[0036]

[0037] With glycosyltransferase UGT BLUsing the pET28a-(+) plasmid of gene sequence 1 (SEQ ID NO:2) as a template, the site-directed mutant sequence was amplified from the whole plasmid using mutation primer pairs, following the Vazyme biological products and operation manual. 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 performed by Anhui General Biotechnology Co., Ltd. Taking the N61 site as an example, conventional PCR techniques were used, with glycosyltransferase UGT... BL 1. Mutations were introduced by amplifying the whole plasmid using the expression vector as a template. The resulting L60, N61, I62, L70, M77 and F84 mutant libraries were successfully constructed by sequencing.

[0038] Example 3 Recombinant Glycosyltransferase UGT BL 1. Fermentation expression of the mutant in Escherichia coli

[0039] The mutant recombinant strains obtained in Example 2 were inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate and cultured overnight at 37°C and 180 rpm. Seed culture was inoculated into 50 mL of fresh LB liquid medium at a 2% inoculation rate and cultured at 37°C and 180 rpm until OD (dose retardation). 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 20 h.

[0040] The induced fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 50 mM PBS (pH 8.0) to wash them. The cells were then sonicated. The lysate was centrifuged at 12000 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 incubated in a boiling water bath for 5 min before loading and 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 terminated.

[0041] SDS-PAGE electrophoresis of the mutant purified enzyme solution showed a clear band at 44.6 kDa for the target protein (molecular weight 44.6 kDa), indicating successful expression of the target protein in E. coli. Figure 6 ).

[0042] Example 4 Screening of enzyme mutants

[0043] The enzyme activity changes of each mutant constructed in Example 3 were measured using tyrosol and UDPG as substrates. The measurement method is as follows: Enzyme activity unit definition: One enzyme activity unit is the amount of enzyme required to generate 1 μmol of product per minute at 30℃ and pH 8.0. The reaction conditions for 1 mL were as follows: 2 mM tyrosol and 5 mM UDPG were dissolved in 50 mM PBS (pH 8.0), and an appropriately diluted enzyme solution was added. After reacting at 30℃ for 1 hour, an equal volume of methanol was added to terminate the reaction. An inactivated enzyme reaction solution was used as a control. After the reaction, the solution was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography. Detection conditions: Column: C18 column (Kromasil, 4.6*250 mm, 5 μm, Bohus, Sweden); Mobile phase: methanol:water = 20:80; Flow rate: 1.0 mL / min; UV detector (278 nm); Column temperature: 30℃. The concentration of the product was calculated using the internal standard method, and the selectivity (percentage of rhodioloside yield to total product yield) and enzyme activity of the mutant catalytic synthesis of rhodioloside were calculated based on the product peak area.

[0044] Figure 4 The diagram shows the selectivity for the synthesis of rhodioloside catalyzed by saturation mutations of L60, N61, I62, L70, M77, and F84. The results show that mutations of L60 to arginine (40%) and aspartic acid (45%), and mutations of F84 to arginine (37%) and proline (39%), decreased the selectivity for rhodioloside. Mutations of N61 to isoleucine, leucine, methionine, proline, and valine increased the selectivity for rhodioloside, ranging from 78% to 85%. Mutations of I62 to proline and threonine increased the selectivity to 78% and 80%, respectively. Mutations of L70 to arginine, cysteine, histidine, phenylalanine, and tyrosine increased the selectivity by 78% to 86%. The selectivity for rhodioloside was significantly improved when N61 was mutated to isoleucine, I62 to threonine, and L70 to tyrosine, reaching 85%, 80%, and 84%, respectively.

[0045] Figure 5 The diagram shows the relative activities of the L60, N61, I62, L70, M77, and F84 saturation mutants compared to the original enzymes. The results indicate that mutations of N61 to isoleucine, methionine, and proline; I62 to proline and threonine; and L70 to cysteine ​​and tyrosine increased the activity by 1.5 to 2.0 times compared to the original enzyme. Mutations of L60 to arginine, aspartic acid, and lysine, and F84 to lysine and valine, significantly reduced the activity, all below 30% of the original enzyme.

[0046] Referring to the methods in Examples 2 and 3, the mutants N61I and I62T, which show the most significant improvement in selectivity and activity after single mutation of N61, I62 and L70 sites, were combined with L70W to prepare double mutants and triple mutants.

[0047]

[0048] The double mutant N61I / I62T exhibited 96% selectivity for rhodioloside, with a 2.3-fold increase in activity compared to N61I. Subsequently, the mutant N61I / I62T was combined with L70W to obtain the triple mutant N61I / I62T / L70W (M1), which showed 99.2% selectivity for rhodioloside, a 3.0-fold increase in activity compared to N61I / I62T, and a 6.9-fold increase in activity compared to the original enzyme. Figure 7 and Figure 8 The SDS-PAGE electrophoresis results of the pure enzyme solution of mutant N61I / I62T / L70W are as follows: Figure 6 As shown.

[0049] Example 5: Enzymatic Properties Analysis of Mutants N61I / I62T / L70W

[0050] 1. Optimal temperature and temperature stability

[0051] To determine the optimal temperatures for the mutant and the original enzyme, the mutant N61I / I62T / L70W(M1) obtained in Example 4 and the original enzyme solution were diluted to specific concentrations and reacted at 20℃, 30℃, 40℃, 50℃, and 60℃ for 1 h, respectively. An equal volume of methanol was added to terminate the reaction, and the enzyme activity was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activities were calculated sequentially. A curve showing the change in enzyme activity with temperature was plotted.

[0052] To determine the temperature stability of the mutant and the original enzyme, the enzyme solutions of the original enzyme and the mutant N61I / I62T / L70W were incubated in water baths at 20℃, 30℃, 40℃, 50℃, and 60℃ for 2 hours, respectively. Then, the enzyme activity changes were measured according to the method described 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.

[0053] The results are as follows Figure 9 (a) As shown in the optimal temperature chart, the optimal temperature for both the original enzyme and the mutant is 30°C. Figure 9 (b) Temperature stability shows that the temperature stability of the mutant decreases sharply with increasing temperature. The mutant N61I / I62T / L70W has more than 80% activity after being incubated at 40℃ for 2 hours, while the original enzyme has only about 60% activity after being incubated at 40℃ for 2 hours, indicating that the thermostability of the mutant is higher than that of the original enzyme.

[0054] 2. Optimal pH and pH stability

[0055] Buffers and substrates 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 8.0-10.0) to prepare UDPG and tyrosol substrates under different pH conditions.

[0056] The mutant and the original enzyme solution obtained in Example 4 were diluted at a certain concentration and added to UDPG and tyrosol substrate reaction systems prepared with different pH buffers. The reaction was carried out at 30°C for 1 hour. After the reaction was completed, an equal amount of methanol was added to terminate the reaction, 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 of enzyme activity with substrate pH was plotted.

[0057] The mutant and original enzyme solutions were diluted with buffers of different pH values, incubated on ice at 4°C for 2 hours, and then enzyme activity was measured. The highest enzyme activity was taken as 100%, and relative enzyme activities were calculated sequentially to plot the enzyme activity change curves under different incubation conditions.

[0058] The results are as follows Figure 9 (c) As shown in the optimal pH, both the original enzyme and the mutant exhibit the highest activity at pH 8.0. Figure 9 (b) pH stability shows that the enzyme activity decreases with increasing or decreasing pH. Both the mutant and the original enzyme are relatively stable at pH 7.0–10.0, retaining more than 60% of their activity. Under all pH conditions, the mutant retains higher enzyme activity than the original enzyme, indicating that the pH stability of the mutant is improved.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that... The mutant is an amino acid sequence shown in SEQ ID NO:1 with one or more mutations of N61I, I62T, and L70W.

2. The glycosyltransferase mutant as described in claim 1, characterized in that... The mutant has N61I and I62T mutations.

3. The glycosyltransferase mutant as described in claim 2, characterized in that... The mutant further exhibits the L70W mutation.

4. A DNA molecule, characterized in that, The DNA molecule encodes the glycosyltransferase mutant according to any one of claims 1-3.

5. An expression vector for a glycosyltransferase mutant, characterized in that... Express the glycosyltransferase mutants as described in claims 1-3.

6. The expression vector as described in claim 5, characterized in that... The expression vector is a plasmid, bacteriophage, virus, or host cell.

7. The expression vector according to claim 6, characterized in that... The host cell is a prokaryotic cell or a eukaryotic cell.

8. The expression carrier according to claim 7, characterized in that... The host cells are selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.

9. The use of the glycosyltransferase mutant according to any one of claims 1-3, the DNA molecule according to claim 4, or the expression vector of the glycosyltransferase mutant according to any one of claims 5-8 in the catalytic synthesis of rhodioloside.

10. The application according to claim 9, characterized in that... Glycosyltransferase mutants catalyze the synthesis of rhodioloside from tyrosol and uridine diphosphate glucose.

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