Catalytic synthesis icariside D2 glycosyltransferase mutant with improved regioselectivity and activity

Abstract

According to the invention, glycosyl transferase UGTBL1 derived from Bacillus licheniformis ZSP01 is taken as a research object, AlphaFold 2 is used for modeling, molecular docking, molecular dynamics simulation and other technologies, amino acid in an active pocket region of glycosyl transferase is analyzed, amino acid in a substrate channel is analyzed by software CAVER3.0, site-directed mutagenesis is carried out, and the glycosyl transferase UGTBL1 is obtained. The regioselectivity and the activity of the mutant for catalytically synthesizing the icariside D2 glycosyltransferase are improved.

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 region and tunneling engineering and their applications. Background Technology

[0002] Epimedium glycoside D2 is a major active ingredient extracted from the traditional Chinese medicine Epimedium, belonging to the flavonoid class of compounds. Epimedium is a traditional Chinese medicine widely used for its effects of tonifying the kidneys and strengthening yang, enhancing immunity, and improving osteoporosis. Epimedium glycoside D2 is the main pharmacologically active substance in Epimedium, possessing various biological activities, including increasing cardiovascular and cerebrovascular blood flow, promoting hematopoiesis, immune function and bone metabolism, anti-cancer, anti-aging, antioxidant, anti-inflammatory, and anti-tumor effects. Currently, the production of epimedium glycoside D2 mainly relies on extraction from the plant, but this method faces many challenges, such as endangered plant resources, complex extraction processes, long cultivation cycles, serious environmental pollution, and difficulties in separation and purification. In recent years, enzymatic synthesis has received widespread attention due to its advantages of mild conditions and environmental friendliness, providing a new direction for the efficient synthesis of epimedium glycoside D2.

[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% (SCieNtifiC 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 its application prospects in the large-scale production of icariin D2. Therefore, it is essential to obtain highly active and selective glycosyltransferases through enzyme molecular modification technology for the targeted synthesis of icariin D2. Summary of the Invention

[0004] This invention utilizes the glycosyltransferase UGT derived from Bacillus licheniformis ZSP01. BLUsing AlphaFold 2 as the research object, we used modeling, molecular docking and molecular dynamics simulation techniques to analyze the amino acids in the active pocket region of the glycosyltransferase. We also used CAVER 3.0 software to analyze the amino acids in the substrate channel and performed site-directed mutagenesis to obtain a series of glycosyltransferase mutants that can be used to synthesize icariin D2 in a targeted and efficient manner.

[0005] The specific technical solution of this invention is as follows:

[0006] A glycosyltransferase mutant comprising one or more mutations in amino acids at positions 81, 111, 112, and / or 321 of the glycosyltransferase amino acid sequence, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] The mutation at position 81 is as follows: proline is mutated to alanine, arginine, valine, phenylalanine, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.

[0008] The mutation at position 111 is: phenylalanine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.

[0009] The mutation at position 112 is as follows: methionine is mutated to alanine, arginine, valine, proline, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, aspartic acid, tyrosine, aspartic acid, or cysteine.

[0010] The mutation at position 321 is: proline is mutated to alanine, arginine, valine, phenylalanine, asparagine, glutamine, leucine, lysine, isoleucine, histidine, glycine, serine, glutamic acid, threonine, tryptophan, methionine, tyrosine, aspartic acid, or cysteine.

[0011] Preferably, the glycosyltransferase mutant of the present invention is a single point mutation, wherein the proline at position 81 is mutated to asparagine (P81N), or the phenylalanine at position 111 is mutated to glutamic acid (F111E), or the methionine at position 112 is mutated to aspartic acid (M112D), or the proline at position 321 is mutated to valine (P321V).

[0012] Preferably, the glycosyltransferase mutant of the present invention is a two-site mutation, in which phenylalanine at position 111 is mutated to glutamic acid and methionine at position 112 is mutated to aspartic acid.

[0013] Preferably, the glycosyltransferase mutant of the present invention is a three-point mutation, with proline at position 81 mutated to asparagine, phenylalanine at position 111 mutated to glutamic acid, and methionine at position 112 mutated to aspartic acid.

[0014] Preferably, the glycosyltransferase mutant of the present invention is a four-site mutation, in which proline at position 81 is mutated to asparagine, phenylalanine at position 111 is mutated to glutamic acid, methionine at position 112 is mutated to aspartic acid, and proline at position 321 is mutated to valine.

[0015] Another object of the present invention is to provide a DNA molecule that encodes the glycosyltransferase mutant described in the present invention.

[0016] 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.

[0017] The expression vector is a plasmid, bacteriophage, virus, or host cell.

[0018] 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.

[0019] 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 synthesis of icariin D2.

[0020] Advantages of this invention:

[0021] This invention utilizes the glycosyltransferase UGT of Bacillus licheniformis ZSP01 obtained through prior screening. BLUsing 1 as the research object, the amino acids in the active pocket region of glycosyltransferase were analyzed using techniques such as molecular docking and molecular dynamics. Saturation mutations were performed on candidate key amino acids F111 and M112. It was found that single-point mutations of F111 and M112 both resulted in mutants with significantly increased selectivity for icariin D2 synthesis. The selectivity of F111E and M112D increased to 80% and 98%, respectively. Combination mutations of F111E and M112D were performed, and the double mutant F111E / M112D was found to increase the selectivity for icariin D2 to 99.1%. To further improve its catalytic efficiency, the amino acid composition of the channel was analyzed using CAVER 3.0 software. Saturation mutations were performed on F111E / M112D using it as a template. The activities of P81N and P321V were increased by approximately 2.6-fold and 2.2-fold, respectively, compared to the template. Finally, a combination mutation was performed using F111E / M112D / P81N as a template and P321V to obtain the quadruplicate F111E / M112D / P81N / P321V, with an activity increased by 2.7-fold and a selectivity of 99.2%. This result indicates that the glycosyltransferase mutant described in this invention has higher regioselectivity and higher catalytic efficiency than the original enzyme, which is of great significance for the synthesis of icariin D2. Attached Figure Description

[0022] 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.

[0023] Figure 1 Tyrosol docking with glycosyltransferase UGT BL 1. Active pocket diagram

[0024] Figure 2 For the protein UGT during the 100ns simulation BL 1. Free energy landscape diagram between tyrosol and ligand. (a) Free energy landscape diagram of tyrosol in cavity A. (b) Free energy landscape diagram of tyrosol in cavity B.

[0025] Figure 3 UGT is a glycosyltransferase BL Candidate amino acids in cavity A of 1.

[0026] Figure 4 Selectivity plot for F111 and M112 saturation mutations.

[0027] Figure 5 The diagram shows the relative activities of the F111 and M112 saturation mutants compared to the original enzymes.

[0028] Figure 6 UGT is a glycosyltransferase BL The channels and key amino acids of 1.

[0029] Figure 7 This is a diagram showing the amino acid saturation mutation activity within the product release channel.

[0030] Figure 8 This is a diagram showing the activity of amino acid saturation mutations as the substrate enters the channel.

[0031] Figure 9 UGT is a glycosyltransferase BL 1. SDS-PAGE electrophoresis images of mutants.

[0032] Figure 10 The bar chart shows the activity of the original enzyme and each mutant.

[0033] Figure 11 This is a liquid phase diagram of the original enzyme and the mutant.

[0034] Figure 12 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

[0035] 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.

[0036] Example 1 Glycosyltransferase UGT BL 1. Identification of active pockets and channel amino acids

[0037] 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 and channel amino acids. BL 1. Regioselectivity and catalytic efficiency of UGT using Alphafold 2. BL 1. Modeling was performed using Discovery Studio with default parameters to dock the substrates tyrosol and UDPG with the enzyme. It was found that tyrosol can be present in UGT. BL 1. Inside the two cavities (cavity A and cavity B) Figure 1The docked complex was then subjected to 100 ns molecular dynamics simulations. Throughout the simulation, the hydroxyl group of tyrosol was closer to UDPG than the phenolic hydroxyl group in cavity A, which may indicate that cavity A is favorable for the synthesis of rhodioloside. To improve the selectivity of icariin D2, the amino acids in cavity A 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 the color changes represent the changes in free energy. The optimal binding conformation was identified through analysis, including the most stable conformation with a global minimum (blue) and the possible metastable and transition states represented by local minima (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 F111 and M112 were obtained. Figure 3 Channels connect the active site to the surrounding environment and play a crucial role in enzyme catalysis. The enzyme UGT was analyzed using CAVER 3.0 software. BL Channel 1 was analyzed, and principal component analysis was used again to obtain key amino acids within the channel, resulting in candidate key amino acid sites: T59, L60, N61, I62, L70, A80, P81, M83, F84, E148, E152, and P321. Figure 3 )

[0038] Example 2 Glycosyltransferase UGT BL 1. Construction of mutant libraries

[0039] The glycosyltransferase UGT obtained in Example 1 BL Candidate sites F111 and M112 were constructed using single-point saturation mutations with degenerate codons NNK and MNN, as shown in the table below.

[0040] With glycosyltransferase UGT BL Using the pET28a-(+) plasmid (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, incubated overnight at 37°C. The mutation results were verified by sequencing performed by Anhui General Biotechnology Co., Ltd. Conventional PCR techniques were used, with glycosyltransferase UGT... BL1. Using the expression vector as a template, full plasmid amplification was performed to introduce mutations. The resulting F111 and M112 mutant libraries were successfully constructed after sequencing verification.

[0041] Example 3 Recombinant Glycosyltransferase UGT BL 1. Fermentation expression of the mutant in Escherichia coli

[0042] 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.

[0043] 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.

[0044] SDS-PAGE electrophoresis of the mutant purified enzyme solution showed that the target protein (molecular weight 44.6 kDa) had a clear band at 44.6 kDa, indicating that the target protein was successfully expressed in E. coli.

[0045] Example 4: Screening Procedure for Enzyme Mutants

[0046] The enzyme activity changes of each mutant constructed in Example 3 were measured using tyrosol and UDPG as substrates, and the measurement methods are as follows:

[0047] Enzyme activity unit definition: One enzyme activity unit is the amount of enzyme required to produce 1 μmol of product per minute at 30℃ and pH 8.0. The reaction conditions for 1 mL are as follows: Dissolve 2 mM tyrosol and 5 mM UDPG in 50 mM PBS (pH 8.0), add appropriately diluted enzyme solution, react at 30℃ for 1 hour, then add an equal volume of methanol to terminate the reaction. Use an inactivated enzyme reaction solution as a control. After the reaction, filter through a 0.22 μm filter membrane for liquid chromatography detection. 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 icariin D2 were calculated based on the peak area of ​​the product.

[0048] Depend on Figure 4 and Figure 5 It can be seen that in the active pocket mutants, the selectivity for icariin D2 was significantly improved when M112 aspartic acid and F111 were mutated to glutamic acid, with M112D showing the best selectivity at 98.2%, but the activity was only 25.1% of the original enzyme. The mutant F111E had a selectivity of 80%, but the activity was only 17% of the original enzyme. Figure 10 ).

[0049] To obtain a mutant capable of targeted synthesis of icariin D2, following the methods described in Examples 2 and 3, F111E and M112D were combined to prepare the mutant F111E / M112D, which exhibited a selectivity of 99.1% and an activity of 20% of the original enzyme. Figure 10 and Figure 11 To obtain mutants with enhanced catalytic activity, amino acids within the enzyme channel were mutated using F111E / M112D (M1-1) as a template. Free energy morphology analysis identified the key amino acids as T59, L60, N61, I62, L70, A80, P81, M83, F84, E148, E152, and P321. Since L60, N61, I62, L70, and F84 are points within the active pocket, a saturation mutation involving three mutations was performed on T59, A80, P81, M83, E148, E152, and P321. To avoid affecting selectivity, the selection criteria were set at selectivity greater than 99% and activity increased by 15%. The corresponding three-mutants were prepared according to Examples 2 and 3.

[0050]

[0051] The activity and selectivity of each mutant are as follows: Figure 7 and Figure 8 As shown. The results indicate that in the saturation mutation experiment of the three mutants of amino acids in the channel using F111E / M112D as a template, saturation mutation of T59, A80, and M83 did not produce mutants with both increased activity and selectivity. However, after the three mutations of P81 to asparagine (2.6-fold) and valine (1.25-fold), E148 to alanine (1.16-fold) and valine (1.2-fold), E152 to glycine (1.6-fold), and P321 to valine (2.2-fold), the activity increased while the selectivity remained unchanged. Figure 6 and Figure 7 Other mutants failed to maintain both high selectivity and high activity simultaneously. Combining the most significantly enhanced activities of P81N and P321V with the template F111E / M112D resulted in the optimal four-mutant F111E / M112D / P81N / P321V (M2-1), which exhibited 99.1% selectivity for the synthesis of icariin D2, with an activity 2.7 times higher than the original enzyme. Figure 10 and Figure 11 The SDS-PAGE electrophoresis results of the pure enzyme solution of mutant F111E / M112D / P81N / P321V are as follows: Figure 9 As shown.

[0052] Example 5 Enzymatic Properties Analysis of Mutants

[0053] 1. Optimal temperature and temperature stability

[0054] To determine the optimal temperatures for the mutant and the original enzyme, the mutant F111E / M112D / P81N / P321V obtained in Example 4 and the original enzyme solution were diluted to specific concentrations and reacted at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 1 hour, 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 enzyme activity changing with temperature was plotted.

[0055] To determine the temperature stability of the mutant and the original enzyme, enzyme solutions of the original enzyme and the mutant F111E / M112D / P81N / P321V were incubated in water baths at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C for 2 hours, respectively. Enzyme activity changes were then measured according to the method described in Example 4. The highest enzyme activity was taken as 100%, and relative enzyme activities were calculated sequentially to plot enzyme activity change curves under different incubation conditions.

[0056] The results are as follows Figure 12 (a) As shown in the optimal temperature chart, the optimal temperature for both the original enzyme and the mutant is 30°C. Figure 12(b) Temperature stability: It can be seen that the temperature stability of the mutant decreases sharply with increasing temperature. After incubation at 35℃ for 2 hours, the mutant F111E / M112D / P81N / P321V has only 50% activity, while the original enzyme still has 50% activity after incubation at 35℃ for 2 hours, indicating that the thermostability of the mutant is lower than that of the original enzyme.

[0057] 2. Optimal pH and pH stability

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The results are as follows Figure 12 (c) As shown in the optimal pH, both the original enzyme and the mutant exhibit the highest activity at pH 8.0. Figure 12 (d) The pH stability graph 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 a higher enzyme activity than the original enzyme, indicating that the pH stability of the mutant is improved.

[0062] 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 of the mutations P81N, F111E, M112D, and P321V.

2. The glycosyltransferase mutant as described in claim 1, characterized in that... The mutant has F111E and M112D mutations.

3. The glycosyltransferase mutant as described in claim 2, characterized in that... The mutant further has the P81N mutation and / or the P321V 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 icariin D2.

10. The application according to claim 9, characterized in that... A glycosyltransferase mutant catalyzes the synthesis of icariin D2 from tyrosol and uridine diphosphate glucose.

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