Combination mutant of glycosyl transferase UGT91C1-F208M

Abstract

The invention belongs to the technical field of genetic engineering and protein modification, and particularly relates to a novel combined mutant of glycosyl transferase UGT91C1-F208M. The gene sequence of the starting protein UGT91C1-F208M is Seq ID (Identity) NO. 1, and the primary sequence of the protein is Seq ID NO. 2. The mutant is obtained through a site-directed mutagenesis technology. According to the invention, combined mutation of an H93 site is further carried out on the basis of the UGT91C1-F208M, and compared with the UGT91C1-F208M, the activity of the optimal mutant UGT91C1-F208M-H93F is improved by 1.3 times, and the activity of the optimal mutant UGT91C1-F208M-H93F is improved by 1.3 times; and compared with the wild type UGT91C1, the activity is improved by 2.6 times. The activity of the mutant is remarkably improved, and the thermal stability of the mutant is kept unchanged, so that the mutant has remarkable advantages in large-scale industrial production of rebaudioside D (RD). According to the mutant disclosed by the invention, the catalytic efficiency of enzyme is remarkably improved, good thermal stability is also maintained, and a more efficient and economical solution is provided for related industrial production.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and protein engineering, and molecularly modifies an enzyme through site-directed mutagenesis technology. Specifically, it relates to a combinatorial mutant of glycosyltransferase UGT91C1-F208M. Background Art

[0002] In people's daily diet patterns, if sugars are ingested in the long term and in excessive amounts, it will bring many potential hazards to human health and is extremely likely to induce a series of health problems such as obesity, diabetes, cardiovascular diseases, etc. Therefore, sugar substitutes with high sweetness and low calories have emerged as the times require. With its remarkable characteristics of high sweetness and low calories, it has quickly become an ideal substitute for traditional sugars, attracting much attention from all walks of life and gradually entering the public's field of vision.

[0003] There are significant differences among different types of sugar substitutes. In the case of artificial sweeteners, their production processes may involve relatively complex chemical processes, which not only increase production costs but may also introduce some impurities that are difficult to remove. The potential health risks of long-term consumption of artificial sweeteners containing trace impurities are not yet clear. Therefore, natural sweeteners such as steviol glycosides are increasingly favored by people. Steviol glycosides are extracted from the natural stevia plant, which ensures their natural and pure characteristics. Moreover, steviol glycosides have extremely high sweetness, being 200 - 300 times that of sucrose. At the same time, a large number of scientific studies and practices have shown that steviol glycosides are relatively stable during human metabolism and do not cause large fluctuations in blood sugar. For diabetic patients and those who pay attention to health and pursue a low-sugar diet, it is undoubtedly an ideal sweetening choice. Steviol glycosides include stevioside (ST), rebaudioside A (RA), RD, and rebaudioside M (RM). The latter two are better ideal components, with a taste similar to sucrose and no bitter aftertaste, but their natural contents are very low and it is difficult to extract them in large quantities.

[0004] With the continuous progress of technology and in-depth exploration, the synthetic pathway of RD has been revealed. It has been found that the glycosyltransferase UGT91C1 (UDP-glycosyltransferase 91C1; EC: 2.4.1.-) can be used as a key "catalytic key" to promote the conversion of RA with a relatively high natural content, thereby synthesizing RD. However, the natural UGT91C1 enzyme has an inherent defect of low activity, which means that its conversion efficiency is not satisfactory during the catalytic reaction, severely restricting the application of this synthesis method in large-scale industrial production. Therefore, it is necessary to improve the enzyme activity.

[0005] In the prior art, mutants of UGT91C1 have been developed (such as F208M, L204A, etc.) to improve catalytic performance. For example, Patent CN119265152A discloses the application of UGT91C1 mutants in the synthesis of steviol glycoside M, and its catalytic activity is 1.1 - 2 times that of the wild type. However, the existing mutants still have problems of insufficient catalytic efficiency and limited site selection. For example, the existing mutations are concentrated at sites such as 204, 208, 380, etc., the functional exploration of site H93 has not been involved, and combinatorial mutations can be carried out on the basis of mutants with improved activity to further enhance the activity. Therefore, in the present invention, by systematically screening mutations at site H93 and performing combinatorial mutations with UGT91C1 - F208M, it is found that the activity of the F208M - H93A / F / M mutant is further improved, filling the blank in this field and can be applied to the production process of RD. Summary of the Invention

[0006] The purpose of the present invention is to provide a combinatorial mutant of a novel glycosyltransferase UGT91C1 - F208M. The activity of the mutant is significantly improved, which is beneficial to its large - scale application in the synthesis of RD.

[0007] To achieve the above purpose, the main experimental steps involved in the present invention include:

[0008] (1) Through molecular docking simulation, it is found that site H93 is located at the edge of the substrate - binding pocket, and the steric hindrance of its side chain affects the entry of the substrate and the release of the product. Therefore, this site is selected to be mutated to the following hydrophobic amino acids: A / F / M / I / L / Y / V on the basis of UGT91C1 - F208M, in order to optimize the hydrophobic interaction of the substrate channel.

[0009] (2) Construction of the plasmid of the UGT91C1 - F208M combinatorial mutant.

[0010] (3) Protein expression and purification of the UGT91C1 - F208M combinatorial mutant.

[0011] (4) Activity characterization of the UGT91C1 - F208M combinatorial mutant.

[0012] The gene sequence of the starting mutant UGT91C1 - F208M applied in the present invention is shown as Seq ID NO.1.

[0013] The primary protein sequence of the starting mutant UGT91C1 - F208M is Seq ID NO.2.

[0014] The present invention relates to a DNA molecule encoding the above - mentioned UGT91C1 - F208M combinatorial mutant.

[0015] The present invention relates to a recombinant expression vector containing the above DNA molecule.

[0016] The present invention relates to a host cell containing the above expression vector.

[0017] The present invention relates to the application of the glycosyltransferase UGT91C1-F208M combinatorial mutant in the production of RD.

[0018] Compared with the existing mutants, on the basis of the existing mutant UGT91C1-F208M, the present invention systematically mutated the key site H93 that was excavated, and constructed the UGT91C1-F208M combinatorial mutants: UGT91C1-F208M-H93A; UGT91C1-F208M-H93F; UGT91C1-F208M-H93M. The activities were increased by 1.2, 1.3, and 1.28 times respectively compared with UGT91C1-F208M, further improving the activity and without reducing the thermal stability. Description of the Drawings

[0019] Figure 1 It is a conversion rate result diagram of the mutant protein prepared in Example 3 for catalyzing RA to generate RD;

[0020] Figure 2 It is a schematic diagram comparing the activities of the optimal mutant prepared in Example 3 with the wild-type UGT91C1 and the starting protein UGT91C1-F208M. Detailed Embodiments

[0021] The present invention discloses a combinatorial mutant of glycosyltransferase UGT91C1-F208M and its preparation method. The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0022] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products or can be prepared by conventional methods known in the technical field.

[0023] The methods used in the following examples, unless otherwise specified, are all conventional operations in the technical field or are operated according to the product instructions.

[0024] Main solutions involved in the experiment:

[0025] (1) LB liquid medium: 2.5 g of LB Broth, 100 mL of deionized water;

[0026] (2) LB solid medium: 2.5 g of LB Broth, 1.5 g of Agar, 100 mL of deionized water;

[0027] (3) TB Media medium: 12 g of tryptone, 24 g of yeast extract, 4 mL of glycerol, 900 mL of deionized water;

[0028] (4) 10×TB Salt: 23.1 g of KH2PO4, 164.32 g of K2HPO4·3H2O, 1 L of deionized water, sterilized.

[0029] (5) Electrophoresis buffer 50×TAE: 242 g of Tris, 37.2 g of Na2EDTA·2H2O, 800 mL of deionized water. After stirring well until dissolved, add 57.1 mL of acetic acid and make up the volume to 1000 mL with deionized water. Store at room temperature. Dilute 50×TAE 50 times to 1×TAE for use as nucleic acid electrophoresis buffer in experiments.

[0030] (6) 1% TAE gel: 0.25 g of agarose, 25 mL of 1×TAE, heat to melt and then cool for use.

[0031] (7) Buffer A: 50 mM of Tris, 500 mM of NaCl, 20 mM of imidazole, 10% (v / v) of glycerol, 1 mM of β-mercaptoethanol, adjust the pH to 8, filter through a 0.45 μm aqueous filter membrane for use.

[0032] (8) Buffer B: 50 mM of Tris, 500 mM of NaCl, 250 mM of imidazole, 10% (v / v) of glycerol, 1 mM of β-mercaptoethanol, adjust the pH to 8, filter through a 0.45 μm aqueous filter membrane for use.

[0033] (9) Desalting buffer: 20 mM of Tris, 10% (v / v) of glycerol, 1 mM of β-mercaptoethanol, adjust the pH to 8, filter through a 0.45 μm aqueous filter membrane for use.

[0034] (10) Lysis buffer: 50 mM of Tris, 500 mM of NaCl, 20 mM of imidazole, 10% (v / v) of glycerol, 1 mM of β-mercaptoethanol, Tween 20, adjust the pH to 8, filter through a 0.45 μm aqueous filter membrane for use.

[0035] (11) Stripping Buffer: 18.612 g of Na2EDTA, 1000 mL of 1×PBS. After fully dissolving, filter through a 0.45 μm aqueous filter membrane for use.

[0036] (12) Protein electrophoresis staining solution: 92 mL of acetic acid, 454 mL of methanol, 1 g of Coomassie Brilliant Blue, 454 mL of deionized water.

[0037] (13) Phosphate buffer: 50 mM phosphate, 5 mM MgCl2, pH = 7.3.

[0038] The present invention will be further described below in conjunction with embodiments:

[0039] Example 1: Preparation of glycosyltransferase UGT91C1-F208M combinatorial mutant

[0040] The nucleotide sequence of glycosyltransferase UGT91C1 after codon optimization was inserted into the expression vector pET-32a to obtain the vector pET-32a-UGT91C1 containing the target gene, which was then mutated to obtain pET-32a-UGT91C1-F208M, as shown in SEQ ID NO.1, as the starting mutant.

[0041] To further enhance the activity of glycosyltransferase UGT91C1-F208M enzyme, molecular docking simulation found that the H93 site is located at the edge of the substrate binding pocket, and the steric hindrance of its side chain affects the entry of the substrate and the release of the product. Mutating this site to hydrophobic amino acids: A / F / M / I / L / Y / V can optimize the hydrophobic interaction of the substrate channel. Therefore, combinatorial mutations were carried out on the basis of the UGT91C1-F208M mutant in order to further improve the catalytic efficiency.

[0042] Using the pET-32a-UGT91C1-F208M plasmid as a template, primers for the mutation sites were designed by snap gene, and the primer sequences are shown in Table 1. The PCR reaction system was 50 μL, in which: 32 μL of ultrapure water, 10 μL of 10×GXL Buffer, 4 μL of dNTP, 1 μL of template, 1 μL of each upstream and downstream primer, and 1 μL of GXL enzyme. Site-directed mutagenesis of the gene was achieved using circular PCR technology. The PCR reaction procedure was as follows: pre-denaturation at 98°C for 10 min, then denaturation at 98°C for 30 s, annealing at 55°C for 50 s, extension at 72°C for 10 min, and cycling 30 times, and finally extension at 72°C for 10 min and storage at 4°C.

[0043] Table 1. Primers used for mutation at the H93 site

[0044]

[0045] The degraded PCR products were transformed into competent Escherichia coli cells DH5α. Single colonies were picked and cultured, and then plasmids were extracted. The correct recombinant plasmids pET-32a-UGT91C1-F208M-H93A, pET-32a-UGT91C1-F208M-H93F, pET-32a-UGT 91C1-F208M-H93M, pET-32a-UGT91C1-F208M-H93I, pET-32a-UGT91C1-F208M-H93L, pET-32a-UGT91C1-F208M-H93Y, and pET-32a-UGT91C1-F208M-H93V were obtained by sending the plasmids to Tsingke Biotechnology for sequencing. Then they were transformed into competent Escherichia coli cells C41 and spread onto LB solid plates containing ampicillin resistance. They were cultured upside down at 37°C. Single colonies were picked and the bacteria were preserved, named F208M-H93A, F208M-H93F, F208M-H93M, F208M-H93I, F208M-H93L, F208M-H93Y, and F208M-H93V respectively.

[0046] Example 2: Expression and purification of glycosyltransferase UGT91C1 mutant protein

[0047] The preserved bacterial strains were cultured overnight in 100 mL LB medium to activate the strains. In a laminar flow hood, they were inoculated into 1 L TB medium (1 mL ampicillin and 100 mL 10×TB salt should be added to 900 mL medium in sequence) at an inoculation amount of 1% and cultured in a shaker at 37°C and 170 rpm for 3 h. When the OD600nm of the bacterial liquid reached 0.6 - 0.8, 0.1 mM IPTG was added for induction. The induction temperature was 18°C, and after culturing at 170 rpm for 16 h, the bacterial liquid was collected by centrifugation at 4°C and 6000 rpm for 15 min. After discarding the supernatant, the bacterial pellet was resuspended in lysis buffer.

[0048] 0.05 g lysozyme and 1% protease inhibitor PMSF were added to every 100 mL resuspended bacterial liquid to improve the lysis effect and protect the target enzyme from being degraded by proteases. After stirring evenly, the bacterial suspension was lysed by high-pressure homogenization (pressure at 1000 - 1500 bar) 5 - 6 times at 4°C. After lysis, the bacterial liquid was centrifuged (4°C, 9000 rpm, 20 min) and the supernatant was collected. The mutant protein was purified by nickel ion affinity chromatography. The specific operation method is as follows:

[0049] (1) The crude enzyme solution was centrifuged at 4°C and 8000 rpm for 25 min to remove cell debris, and the supernatant was filtered through a 0.45 μm filter membrane for standby. In a chromatography cabinet at 4°C, the sample was pumped into a nickel ion chromatography column using a peristaltic pump.

[0050] (2) Pretreat the AKTA primer plus protein liquid purification system with water and Buffer A / Buffer B. Equilibrate the system with Buffer A. Connect the nickel ion chromatography column after sample loading to the protein liquid purification system. Set the system flow rate at 2 mL / min. Wash away the enzymes not bound to the nickel ion chromatography column and weakly bound miscellaneous proteins with Buffer A. After the baseline stabilizes, adjust the ratio of Buffer B to Buffer A. Elute the proteins weakly bound to the nickel ion chromatography column successively with 10% (25 mM imidazole), 20% (50 mM imidazole), and 30% (75 mM imidazole) Buffer B. Finally, adjust to 100% (250 mM imidazole) Buffer B to elute the target enzyme, and collect the eluate.

[0051] (3) Since the salt ion concentration of the enzyme solution eluted from the nickel ion chromatography column is too high and the presence of imidazole ions may affect the subsequent determination of enzyme activity and stability, it is necessary to further separate the ions and the enzyme through a gel filtration chromatography column to desalt the enzyme. After washing the system pipeline 3 times with water and desalting buffer respectively, connect the processed gel filtration chromatography column. After equilibrating the chromatography column with desalting buffer and waiting for the baseline to stabilize, slowly inject the enzyme sample into the gel filtration chromatography column with a syringe at an injection volume of 1 / 5 column volume. Elute with desalting buffer at a flow rate of 1 mL / min, and collect the eluted enzyme sample according to the elution peaks of the ultraviolet absorption peak at OD280 nm and the ion strength peak in the chromatogram.

[0052] The purified protein sample was detected by SDS-PAGE as a single band, and the protein molecular weight was shown to be 69 kDa.

[0053] Example 3: Activity Characterization of Glycosyltransferase UGT91C1 Mutant

[0054] Prepare the mother liquors of UDPG, RA, and mutant protein with a pH 7.0, 5 mM magnesium chloride, potassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[0055] In a 2.0 mL round-bottom centrifuge tube, successively add 100 μL of uridine diphosphate glucose (UDPG), 50 μL of rebaudioside A (RA), and 50 μL of the mutant protein. Place the centrifugation tube after sample addition in a shaker with a temperature set at 37 °C and a rotation speed of 170 rpm to allow it to react for a certain time. When the timing ends, immediately add 200 μL of methanol to the reaction system to terminate the reaction. After completing the termination operation, place the reaction mixture in a centrifuge and centrifuge at a centrifugal force of 12000×g for 10 min. Subsequently, take the supernatant and filter it through an organic phase filter membrane with a pore size of 0.22 μm.

[0056] The filtered supernatant was used for high performance liquid chromatography (HPLC) analysis. The specific experimental conditions are as follows:

[0057] (1) The chromatographic column used was a Silgreen ODS C18 column ( ).

[0058] (2) The mobile phase was 70% ultrapure water and 30% acetonitrile, and an isocratic elution mode was adopted.

[0059] (3) The injection volume was set at 20 μL.

[0060] (4) The detection wavelength was determined to be 210 nm.

[0061] (5) The detection temperature was maintained at 30 °C.

[0062] The obtained activity characterization results are shown in the appendix Figure 1 as follows. Compared with the starting mutant UGT91C1-F208M, the conversion rates of the combinatorial mutants UGT91C1-F208M-H93A; UGT91C1-F208M-H93F; UGT91C1-F208M-H93M were all improved, and their relative activities were calculated to be increased by 1.2, 1.3, and 1.28 times respectively.

[0063] Example 4: Reaction kinetic parameters and stability characterization of activity-improved mutants

[0064] To further analyze the performance of the activity-improved mutants, the mutants with improved activity were characterized for reaction kinetic parameters using HPLC and for thermal stability using CD.

[0065] The specific experimental conditions for HPLC detection were the same as those in Example 3. In this experiment, an enzyme concentration of 0.5 μM was selected for the experiment, and the reaction times were 5, 10, 15, 20, and 25 min. First, the concentration of one substrate RA was controlled at 250 μM unchanged, and the concentration of the other substrate UDPG was gradually increased from 0 to 500 μM. Then, the concentration of UDPG was controlled at 250 μM, and the concentration of RA was gradually increased from 0 to 500 μM. The initial rate of the enzymatic reaction and the substrate concentration were fitted into a Michaelis curve equation using Origin, and the reaction kinetic parameters calculated therefrom are shown in Table 2. The catalytic efficiency of the activity-improved mutants was further improved.

[0066] Table 2. Reaction kinetic parameters of UGT91C1-F208M and its combinatorial mutants

[0067]

[0068] When performing thermal stability characterization using CD, a sample cell with an optical path length of 1 cm was selected, and the enzyme concentration used was 0.5 μM. First, the blank value of PB Buffer was subtracted, and the CD spectrum of the UGT91C1 enzyme sample was measured at 20 °C, with a wavelength range of 190 - 250 nm, a step size of 1 nm, and 0.5 s of acquisition time for each point. The instrument was changed to the kinetic detection mode, the wavelength was fixed at 222 nm, the voltage was changed to the optimized voltage value of the instrument, the signal acquisition time was 1 s, and continuous monitoring was carried out for 7200 s. The temperature measurement range was 20 - 95 °C, the temperature change amplitude was set to 1 °C, and the temperature fluctuation range was 2 °C, with the temperature automatically controlled by the instrument. According to the experimental data, a thermal denaturation curve was plotted, and the experimental data was fitted with formula (1 - 1). The results are shown in Table 3. The thermal stability of the activity - enhanced mutants was not affected, and the Tm of UGT91C1 - F208M - H93A increased by 14 K.

[0069]

[0070] where y - CD signal, deg·cm 2 ·dmol -1 ;

[0071] x - temperature, K;

[0072] aN / aD - CD signal of the native / denatured state of the protein, deg·cm 2 ·dmol -1 ;

[0073] bN / bD - slope of the baseline of the native / denatured state of the protein, fraction;

[0074] △HTm - enthalpy change of melting, kJ / mol;

[0075] △Cp - specific heat capacity at constant pressure, J / (kg·K);

[0076] Tm - melting temperature, K;

[0077] R - gas constant, J / (mol·K).

[0078] Table 3. Thermal stability parameters of UGT91C1 - F208M and its combined mutants

[0079] Variants Tm / K F208M 317.014±0.55 H93A 331.54±1.75 H93F 316.95±0.27 H93M 318.23±0.66

Claims

1. A combined mutant of glycosyltransferase UGT91C1-F208M, characterized in that: The mutant is based on the UGT91C1-F208M enzyme with amino acid sequence SeqID NO.1, wherein the 93rd amino acid is mutated from His to any one of Ala, Val, Ile, Tyr, Leu, Phe or Met, wherein the enzyme activity is improved after the mutation of Ala, Phe and Met.

2. A DNA molecule encoding the UGT91C1-F208M combined mutant of claim 1.

3. A recombinant expression plasmid, characterized in that: The plasmid comprises the DNA molecule of claim 2.

4. A host cell, characterized in that The host cell comprises the recombinant expression plasmid according to claim 3.

5. The host cell according to claim 4, characterized in that The host cell is Escherichia coli.

6. Use of the UGT91C1 mutant described in claim 1 in the production of Rebaudigan D (RD).

Images