Glycosyltransferase mutant with improved selectivity and application thereof

Abstract

The invention relates to a glycosyl transferase UGTBL1 mutant with improved selectivity and application of the glycosyl transferase UGTBL1 mutant in catalytic synthesis of salidroside, and belongs to the field of gene engineering and enzyme engineering. A single-point mutation technology is adopted, the obtained mutant comprises 132-site amino mutation of an amino acid sequence, and the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO: 1. Research results show that the glycosyl transferase mutants S132V and S132G have good selectivity, tyrosol and uridine diphosphate glucose can be efficiently catalyzed to synthesize the salidroside, the selectivity is 89%, and the glycosyl transferase mutants S132V and S132G are suitable for enzymatic preparation of the salidroside and have wide application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a glycosyltransferase mutant with improved selectivity and application thereof. Background Art

[0002] Salidroside (p-hydroxyphenylethanol-β-glucoside) is the key active ingredient in the traditional Chinese medicine Rhodiola rosea. It is the product of glycosylation of tyrosol hydroxyl group. It has multiple activities such as anti-aging, anti-neuronal cell aging, anti-tumor, anti-depression, cardiovascular protection, sedation and sleep, and is widely used in the medical, health care and skin care industries. Therefore, people's demand for salidroside is growing, but the content of salidroside in Rhodiola rosea plants is low (mainly in the tuberous rhizomes of Rhodiola rosea), which increases the complexity of the extraction process. Wild Rhodiola rosea resources are on the verge of extinction due to over-exploitation. In order to obtain salidroside efficiently, people adopt a synthetic method to prepare salidroside. The chemical synthesis of salidroside cannot be mass-produced because it has many steps, requires protection / deprotection, and uses precious metal catalysts and bromides that have a greater impact on the environment. The enzymatic synthesis of salidroside has mild conditions, does not require the protection of tyrosol, and can efficiently synthesize glycosylated products.

[0003] Glycosyltransferase plays a very important role in the synthesis of glycoside compounds due to its advantages such as high glycosylation efficiency, a wide variety of sugar donors, and high stereoselectivity. Currently, the enzymatic synthesis of salidroside mostly uses plant-derived glycosyltransferases, but the efficiency of plant-derived glycosyltransferases in Escherichia coli is not very high. Microbial-derived glycosyltransferases UGT BL 1 can synthesize salidroside and salidroside isomers with uridine diphosphate glucose (UDPG) and tyrosol as substrates, with a selectivity of 51%. In the face of the difficulty of product separation, it is very necessary to obtain highly selective glycosyltransferases through enzyme molecular modification technology. At present, directed evolution technology is one of the most effective strategies to improve protein properties. It is an effective method to simulate natural evolution mechanisms (random mutations, gene recombination, natural selection) in the laboratory, modify enzyme genes in vitro and select them in a directed manner, and obtain mutant enzymes with excellent properties in a short period of time. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a glycosyltransferase mutant and its application, using the glycosyltransferase UTG derived from Bacillus licheniformis BL 1 was used as the research object. Homology modeling and molecular docking methods were used, and single-point saturation mutagenesis technology was used for the amino acids around the glycosyl site to obtain two glycosyltransferase mutants with significantly improved selectivity, which have good application potential in the synthesis of salidroside.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A glycosyltransferase mutant, wherein the mutant comprises the 132nd amino acid of the amino acid sequence, and 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] The present invention is based on glycosyltransferase UTG from Bacillus licheniformis BL 1. The glycosyltransferase UTG 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 mutant in which the serine at position 132 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to 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 mutant in which the serine at position 132 of the amino acid sequence shown in SEQ ID NO:1 is mutated 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 the glycosyltransferase mutant of the present invention, a recombinant vector comprising 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, preparing the glycosyltransferase mutant gene of the present invention by artificial synthesis or gene cloning, constructing an expression vector to obtain a recombinant plasmid, and transforming it into a host cell.

[0012] The expression vector is a plasmid, a phage, a virus or a host cell.

[0013] The host cell is a prokaryotic cell or a eukaryotic cell, and may be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma, preferably Escherichia coli.

[0014] Another object of the present invention is to provide the use of the glycosyltransferase mutant, DNA molecule or expression vector of the glycosyltransferase mutant in the catalytic synthesis of salidroside, specifically, catalyzing the synthesis of salidroside from tyrosol and uridine diphosphate glucose.

[0015] Beneficial effect: The present invention targets glycosyltransferase UGT BL 1. Rational design was performed to improve the enzymatic properties, and the single-site mutants S132V and S132G were obtained, which had good selectivity for the synthesis of salidroside, significantly higher than the glycosyltransferase UGT. BL 1. The selectivity of S132V and S132G is 89%, which can efficiently catalyze tyrosol and uridine diphosphate glucose (UDPG) to synthesize salidroside. Compared with the existing direct extraction method and chemical synthesis method, the salidroside enzymatic synthesis technology established in this application has the advantages of high conversion rate and fewer catalytic synthesis steps, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 UGT BL 1Structure diagram.

[0017] Figure 2 This is the amino acid map of the sugar site.

[0018] Figure 3 The SDS-PAGE electrophoresis diagram of glycosyltransferase UGTBL1 and its alanine mutant.

[0019] Figure 4 The selectivity diagram of the synthesis of salidroside catalyzed by glycosyltransferase UGTBL1 and its alanine mutant.

[0020] Figure 5 This is the liquid phase spectrum of the reaction products of glycosyltransferase UGTBL1 (A), S132V (B) and S132G (C). The retention time of the by-product is 6.4 min, and the retention time of salidroside is 10.3 min.

[0021] Figure 6 Effects of temperature and pH on glycosyltransferase UT BL 1 and the effects of mutants S132V and S132G on the activity and stability, (A) optimum temperature, (B) temperature stability, (C) optimum pH, (D) pH stability. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only for illustrating the present invention, and are not intended to limit the scope of the invention in any way. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without paying creative work are within the scope of protection of the present invention.

[0023] Example 1 Determination of key amino acid residues in the glycosyl site of glycosyltransferase UGTBL1

[0024] (1) Homology modeling: BLAST analysis was performed based on the NCBI and PDB databases, using the Bacillus UDP-glycosyltransferase (PDB ID: 7VLB) as a template, and its sequence was similar to the original enzyme UGT BL The amino acid sequence of SEQ ID NO: 1 is 56.85% similar. The MODELER program of Discovery Studio 3.5 (DS 3.5) was used to construct the UGT BL The three-dimensional structure of 1 is Figure 1 The final quality of the constructed model was analyzed using PROCHECK and Profile-3D programs. The visualization and analysis of the model were performed using Discovery Studio 2018Client and MOE2019.01 software.

[0025] (2) Use Autodock 4.2 software to dock the substrate to the constructed UGT model BL The catalytic center of 1 was selected by computer-predicted free binding energy to optimize the docking conformation of the ligand and receptor.

[0026] (3) Determination of key amino acids: 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 acids determined in Example 1 were mutated to alanine to carry the glycosyltransferase UGT BL 1 gene sequence (SEQ ID NO: 2) as a template, with reference to the Vazyme biological products and operation manual, using mutation primer pairs, the whole plasmid amplified the site-directed mutation sequence. The PCR product was treated with Dpn I. After the template digestion, it was transformed into Escherichia coli BL21 (DE3) cells using the heat shock method, and spread on LB agar plates containing 100 μg / ml kanamycin sulfate, and inverted at 37°C overnight. The mutation results were verified by sequence determination by Anhui General Biological Company. Taking the S132 site as an example, the construction of the mutant was carried out using conventional PCR technology and the glycosyltransferase UGT BL1 expression vector was used as a template for full plasmid amplification to introduce mutations, and the resulting S132 mutant library was successfully constructed by sequencing verification.

[0029] Example 3 Fermentation expression of recombinant glycosyltransferase mutants in Escherichia coli

[0030] The expression mutant constructed in Example 2 and the original enzyme UGT BL 1. Inoculate the engineered bacteria into 50 mL of LB liquid medium containing 100 μg / mL kanamycin sulfate, and culture at 37°C, 180 rpm overnight; inoculate the seed liquid into 50 mL of fresh LB liquid medium at a 2% inoculum amount, and culture at 37°C, 180 rpm until OD 600 When the pH value was 0.6-0.8, the cells were taken out and cooled in an ice-water bath for 5 min, and the inducer IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) was added, and the expression was induced at 20°C and 180 rpm for 16 h.

[0031] Take the fermentation broth after induced expression, centrifuge at 12000rpm for 20min, discard the supernatant, and then add 50mM Na 2 HPO 4 -KH 2 PO 4 Resuspend and wash the cells with buffer (pH 8.0), centrifuge at 12000rpm for 20min, discard the supernatant, resuspend with buffer, and then ultrasonically disrupt. Centrifuge the disrupted liquid at 12000rpm for 20min, take the supernatant for SDS-PAGE electrophoresis, the concentration of concentrated gel is 4%, the concentration of separation gel is 12.5%, the sample and loading buffer are mixed in a ratio of 3:1, react in a boiling water bath for 5min, and then load the sample for electrophoresis. Set the initial voltage of the electrophoresis instrument to 120V, increase the voltage to 230V when the sample moves to the separation gel, and end the electrophoresis when the sample moves to the bottom of the electrophoresis tank.

[0032] The results are as follows Figure 3 As shown, glycosyltransferase UGT BL The molecular weight of 1 is 44.6 kDa, and the results showed that after induction, the mutants all had obvious bands at 44.6 kDa, indicating that the glycosyltransferase mutants were successfully induced to express.

[0033] Example 4 Screening procedure for optimal mutants of glycosyltransferases

[0034] The mutant obtained in Example 2 was used to measure the change in enzyme activity using UDPG and tyrosol as substrates. The measurement method is as follows:

[0035] Definition of enzyme activity unit: One unit of enzyme activity is the amount of enzyme required to generate 1 μmol of product per minute at 30°C and pH 8.0.

[0036] 1 ml reaction system is as follows: 5mM uridine diphosphate glucose (UDPG), 3.6mM tyrosol, 5mM MgCl2, 5% DMSO, 0.5U enzyme. After mixing the above reaction system, place it in a 30 degree oscillator for 15 minutes, and then terminate the reaction in a boiling water bath for 5 minutes. After filtering through a 0.22 micron filter membrane, HPLC detection is performed under the following detection conditions: 20% methanol as the mobile phase, flow rate 1ml / min, C18 chromatographic column, detection wavelength 278nm. The concentration of the product is calculated according to the internal standard method, and the selectivity (the percentage of salidroside production in the total product production) and enzyme activity of the mutant catalytic synthesis of salidroside are calculated according to the product peak area.

[0037] The selectivity results of the mutants after alanine mutation are as follows Figure 4 The results showed that the selectivity of most amino acids increased after mutation, and the selectivity of the S132 site increased the most after mutation to alanine.

[0038] The enzyme activity results of the mutant after alanine mutation are shown in the following table:

[0039] strain 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 the selectivity and enzyme activity of the mutant, the S132 site was selected as the hotspot residue for modification.

[0041] Referring to the method of Example 2, site-directed saturation mutagenesis was performed on the S132 site. The corresponding mutation primers designed are as follows:

[0042]

[0043]

[0044]

[0045] Example 5 Screening Procedure for Enzyme Mutants

[0046] Referring to the method shown in Example 4, the S132 mutants obtained in Example 4 were tested for enzyme activity and selectivity changes using uridine diphosphate glucose (UDPG) and tyrosol as substrates. The results are shown in the following table (no activity was detected for mutants not included in the table). The results show that after the serine at position 132 was mutated, only S132V, S132G, S132A, S132E, S132F, S132P, S132C, and S132T were significantly different from the original enzyme UGT. BL 1 had a slight increase in selectivity, while the other mutants almost lost their enzyme activity. The selectivity of mutants S132V and S132G for synthesizing salidroside was higher than that of the original enzyme UGT BL 1 increased from 51% to 89%, the original enzyme UGTBL 1. The liquid phase spectrum after the reaction of S132V and S132G is as follows Figure 5 shown.

[0047] strain 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 Analysis of Enzymatic Properties of Enzyme Mutants

[0049] 1. Optimum temperature and temperature stability

[0050] In order to determine the optimal temperature of the original enzyme and the mutants, the mutants S132V and S132G screened in Example 5 were added to the reaction system and reacted at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 15 min, respectively. After the reaction, the mixture was boiled for 5 min and the enzyme activity was determined. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in turn. The curve of enzyme activity changing with temperature was plotted. The results are shown in Figure 2. Figure 6 As shown in A.

[0051] In order to determine the temperature stability of the original enzyme and the mutant, the mutant S132V and S132G enzyme solutions were placed in a water bath at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 2 h, and then the enzyme activity changes were determined according to the method in Example 4. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in turn. The change curves of enzyme activity under different incubation conditions were plotted. The results are shown in Figure 4. Figure 6 As shown in B.

[0052] 2. Optimum pH and pH stability

[0053] In order to determine the optimal pH of the original enzyme and the mutant, buffer solutions of different pH were prepared: citric acid-sodium citrate (pH 3.0-6.0), Na 2 HPO 4 -KH 2 PO 4 (pH 6.0-8.0), glycine-sodium hydroxide (pH 8.0-9.0), dissolve the substrate with buffers of different pH, react at 30℃ for 15min, boil for 5min after the reaction, and measure the enzyme activity. The highest enzyme activity is taken as 100%, and the relative enzyme activity is calculated in turn, and a curve of enzyme activity changing with substrate pH is drawn.

[0054] In order to determine the pH stability of the original enzyme and mutants, the enzyme was suspended in different buffers, incubated on ice at 4°C for 2 hours, reacted at 30°C for 15 minutes, and boiled for 5 minutes after the reaction to determine its enzyme activity. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in turn, and the change curve of enzyme activity under different incubation conditions was drawn.

[0055] The results are as follows Figure 6As shown in C and D, the original enzyme UGT BL The optimal reaction pH of 1 and mutants S132V and S132G is 8.0. Under the condition of pH 8.0, the mutant and the original enzyme have the highest activity. As the pH increases or decreases, their enzyme activities decrease. BL The enzyme activities of 1 and mutants S132V and S132G were relatively stable between pH 6.0 and 9.0, and they could retain more than 70% of their activities after incubation for 2 h under this condition. However, the stability of the original enzyme and its mutants decreased between pH 3.0 and 6.0. Under different pH conditions, the original enzyme UGT BL 1 The retained enzyme activity 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 a mutant in which the amino acid at position 132 of the amino acid sequence shown in SEQ ID NO: 1 is mutated from Ser to Val or Gly.

2. A DNA molecule, characterized in that The DNA molecule encodes the glycosyltransferase mutant according to claim 1.

3. An expression vector for a glycosyltransferase mutant, characterized in that Expressing the glycosyltransferase mutant according to claim 1.

4. The expression vector according to claim 3, characterized in that Express the DNA molecule of claim 2.

5. The expression vector according to claim 3, characterized in that The expression vector is a plasmid, a phage, 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 vector according to claim 6, characterized in that The host cell is selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

8. 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 to 7 in catalyzing the synthesis of salidroside.

9. The use according to claim 8, characterized in that Glycosyltransferase mutants catalyze the synthesis of salidroside from tyrosol and uridine diphosphate glucose.

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