UGT mutant and method for preparing rebaudioside M by using UGT mutant

Abstract

The invention relates to the technical field of stevioside production, and discloses a UGT mutant and a method for preparing rebaudioside M by using the UGT mutant. The UGT mutant is obtained by modifying a stevia rebaudiana wild type UGT76G1, the activity of glycosyl transferase is remarkably improved after mutation, rebaudioside A can be efficiently catalyzed by taking ADP-G as a substrate to generate rebaudioside M, the production cost is reduced, and the UGT mutant is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of steviol glycoside production technology, and in particular to a UGT mutant and a method for preparing lebodiin M. Background Technology

[0002] Steviol glycosides (SGs), extracted from the leaves of stevia rebaudiana Bertoni, are characterized by low calories and high sweetness, and have been widely studied as an excellent sucrose substitute. This sweetener is 200 to 300 times sweeter than sucrose, while its calorie content is only one-three-hundredth that of sucrose, hence it is sometimes referred to as a "third type of sugar source." The safety of steviol glycosides has been verified by international organizations including the FAO and WHO, and it has passed numerous tests for carcinogenicity, teratogenicity, and toxicity.

[0003] Rebaudioside M (RM) is a natural steviol glycoside compound widely used in the food, beverage, and health ingredient industries. As a natural, virtually calorie-free sweetener, it is highly sweet, 250-350 times sweeter than sucrose. Its taste is also similar to sucrose, making it suitable as a substitute for traditional sugars in beverages and baking. Compared to other steviol glycosides such as rebaudioside A, rebaudioside M offers a superior sweetness and is considered an important direction for the development of steviol glycoside products. Currently, the core method for producing RM using enzymatic catalysis involves the conversion of RD (reverse glycosides) catalyzed by glycosyltransferases.

[0004] UGT, also known as UDP-glucosyltransferase, is a class of key enzymes that use uridine diphosphate glucose (UDPG) as a glycosyl donor to catalyze the transfer of glycosyl groups to acceptor molecules to form glycosidic bonds. It is widely involved in the glycosylation modification of natural products.

[0005] Currently, the enzymatic synthesis of RM requires the addition of expensive UDP as one of the substrates. UDP is catalyzed by sucrose synthase to produce UDPG, and then RM and UDP are catalyzed by UGT enzyme using RD and UDPG as substrates. Due to the high price of UDP, the cost of industrial-scale RM production remains high, making it difficult to achieve market competitiveness. It is necessary to mutate the UGT enzyme so that it can couple with sucrose synthase, using ADP as a substrate, to efficiently catalyze the conversion of RA to RM. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a UGT mutant that overcomes the limitations of existing wild-type UGT, such as low catalytic activity, limited substrate range, and reliance on expensive glycosyl donors, thereby achieving efficient glycosylation of RA substrates and reducing production costs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A UGT mutant, obtained by modifying wild-type stevia UGT76G1, the amino acid sequence of which is shown in SEQ ID NO: 2; the mutant is a mutant in which at least one site of the amino acid sequence shown in SEQ ID NO: 2 is mutated; wherein the mutant has improved glycosyltransferase activity compared with the wild type and can catalyze the production of rebaudioside A to rebaudioside M using ADP-G as a substrate.

[0009] As a preferred technical solution, the mutant is a mutant in which at least one site selected from positions 23, 278, 282, or 309 of the amino acid sequence shown in SEQ ID NO: 2 is mutated.

[0010] As a preferred technical solution, the amino acid at position 23 is mutated to glutamic acid (E) or alanine (A); the amino acid at position 278 is mutated to glycine (G) or alanine (A); the amino acid at position 282 is mutated to glutamine (Q) or alanine (A); and the amino acid at position 309 is mutated to glycine (G) or alanine (A).

[0011] As a preferred technical solution, the mutant is a double mutant 23E / 309G, 278G / 309G or 282Q / 309G, or a triple mutant 23E / 282Q / 309G, or a quadruple mutant 23E / 278G / 282Q / 309G.

[0012] The present invention also discloses a nucleotide sequence that encodes a UGT mutant.

[0013] The present invention also discloses an expression vector containing a nucleotide sequence including a UGT mutant.

[0014] The present invention also discloses a host cell containing a nucleotide sequence of a UGT mutant or an expression vector containing a nucleotide sequence including a UGT mutant.

[0015] This invention also discloses a method for preparing rebaudioside M using a UGT mutant. The method uses the UGT mutant as a catalyst, rebaudioside A as a substrate, and ADP as a glycosyl donor precursor. Sucrose, AtSUS sucrose synthase, OsUGT β-1,2-glycosidase, and a buffer system are added to carry out the reaction. As a preferred technical solution,

[0016] As a preferred technical solution, the reaction system further contains disodium EDTA, the buffer system includes potassium dihydrogen phosphate, and the reaction is carried out in a solution containing the following components, wherein:

[0017] The initial reaction concentration of rebaudioside A was 75-125 g / L.

[0018] The initial reaction concentration of sucrose was 175-225 g / L.

[0019] The initial reaction concentration of ADP was 0.38-0.62 g / L.

[0020] The initial reaction concentration of disodium EDTA is 3.3-4.1 g / L.

[0021] The initial reaction concentration of potassium dihydrogen phosphate is 6.4-7.2 g / L.

[0022] The initial reaction concentration of AtSUS sucrose synthase is 0.8-1.2 g / L.

[0023] The initial reaction concentration of OsUGT β-1,2-glucosidase is 2.4-3.7 g / L.

[0024] The initial reaction concentration of the crude enzyme solution of the UGT mutant was 2.4-3.7 g / L.

[0025] The reaction time is 1-6 hours.

[0026] This invention also discloses the application of a UGT mutant in the preparation of sweeteners in the food and beverage industry, wherein the sweetener uses rebaudioside M as the active ingredient and the addition amount is 0.001-1g / L.

[0027] Beneficial effects

[0028] The UGT mutant provided by this invention significantly improves the glycosylation activity of RA through site-directed mutagenesis, enabling the use of ADP as a substrate to replace expensive UDP and reduce the production cost of RM. Meanwhile, the mutant C5 (23E / 282Q / 309G) achieved a conversion rate of 88% in scale-up experiments, far exceeding the conversion efficiency of the wild type, verifying that this mutant has the potential for industrial application and is suitable for large-scale production. Detailed Implementation

[0029] To facilitate understanding of the present invention, it will be described in detail below with reference to specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0030] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice of this invention, preferred methods and materials are now described.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] LB medium: peptone 10.0 g / L, sodium chloride 10.0 g / L, yeast extract 5.0 g / L.

[0034] High performance liquid chromatography analysis: The instrument was an Agilent HPLC 1200, the chromatographic column was a C18 column, the mobile phase was water + 0.1% formic acid and acetonitrile + 0.1% formic acid, the flow rate was 1 mL / min, and the sample loading volume was 20 μL.

[0035] PCR reaction system: 5 μL of 10x high-fidelity enzyme buffer containing MgSO4, 1 μL of 10 mM dNTP mixture, 1 μL of upstream primer solution, 1 μL of downstream primer solution, 1 μL (5 ng) of template plasmid solution, 1.2 μL of high-fidelity DNA polymerase (2.5 U / µl), 39.8 μL of ddH2O, total system 50 μL.

[0036] PCR setup: initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 12 min, 18 cycles, final extension at 68°C for 10 min, and hold at 12°C.

[0037] I. Evaluation of UGT Expression and Activity

[0038] The wild-type UGT76G1 gene from Stevia rebaudiana (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) was ligated into the pET28a vector and transformed into Escherichia coli expression strain BL21(DE3). Heterologous protein expression was induced in LB medium under the following conditions: 0.5 mM IPTG added, induction overnight at 25°C. The bacterial cells were collected by centrifugation, resuspended in Tris hydrochloric acid buffer, and then sonicated. The supernatant was then centrifuged to obtain the crude enzyme solution.

[0039] The glycosyltransferase activity assay system is as follows: Rebaudioside A 1.0 g was used as the substrate, along with ADP 5 mg, sucrose 2 g, disodium EDTA 37 mg, potassium dihydrogen phosphate 68 mg, AtSUS sucrose synthase 10.0 mg, OsUGT β-1,2-glucosidase 30.0 mg, and then 30.0 mg of crude UGT76G1 or mutant enzyme solution was added. Each sample had a total volume of 10.0 ml. The reaction was carried out at pH 7.2 and 30℃ for 30 minutes. After the glycosylation reaction was complete, an equal volume of methanol was added to terminate the reaction, and the activity was determined by liquid chromatography.

[0040] II. Site-specific alanine scan

[0041] Twenty-three amino acid sites closely related to activity were selected from SEQ ID NO: 2: Q23, G24, H25, N27, Y278, S278, G282, S283, T285, V309, K337, W338, V339, P340, Q341, Q342, H356, G358, W359, N360, S361, E364, and Q381. Site-directed alanine mutation was performed. The specific procedure was as follows: Using the recombinant plasmid pET28a-UGT76G1 as a template, a pair of primers containing the mutation site was used for full-plasmid PCR amplification with a high-fidelity enzyme to obtain a linearized plasmid with the specified mutation site. The amplification product was digested with DpnI enzyme at 37℃ for 2 hours to degrade the initial template. The digestion products were converted to E. coli DH5α, spread onto LB agar plates containing 50 μg / mL Kana, and incubated overnight at 37°C.

[0042] A mutant screening method was established to analyze and determine the catalytic activity of the enzyme. The mutation site information and relative activity of the mutants with enhanced activity are shown in Table 1. Relative activity refers to the multiple of the catalytic activity of the mutant on the same substrate under the same conditions relative to the wild type, when the relative activity of wild-type UGT76G1 is defined as 1.

[0043] Table 1

[0044]

[0045] III. Site-directed saturation mutations

[0046] Four sites closely related to activity were selected from positions 23, 278, 282, and 309 of SEQ ID NO: 2 for site-directed saturation mutagenesis. The specific procedure was as follows: Using recombinant plasmid pET28a-UGT76G1 as a template, a pair of primers containing the mutation sites were used for full-plasmid PCR amplification with a high-fidelity enzyme to obtain the recombinant plasmid with the specified mutation sites. The amplification product was digested with DpnI enzyme at 37°C for 2 hours to degrade the initial template. The digestion product was transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / mL Kana, and incubated overnight at 37°C.

[0047] Table 2 lists the relative activities and mutation details of mutants with significantly increased relative activity, while the relative activity of wild type is 1.

[0048] Table 2

[0049]

[0050] IV. Combination mutations at loci

[0051] Based on the above site-directed mutagenesis, mutations at amino acid residues 23 and 309 significantly enhanced enzyme activity. Therefore, two sites were selected for combination to construct double mutants 23E / 309G, 278G / 309G, and 282Q / 309G. Activity measurements of these double mutants revealed that the activity of 23E / 309G was significantly higher than that of the single mutant. Therefore, a further round of combined mutagenesis was conducted based on the double mutant 23E / 309G to construct triple mutants 23E / 278G / 309G, 23E / 282Q / 309G, and quadruple mutants 23E / 278G / 282Q / 309G.

[0052] Table 3 lists the relative activity and mutation details of the combined mutants, with the wild-type relative activity being 1.

[0053] Table 3

[0054]

[0055] Example 1: Construction and transformation rate determination of the double mutant C1 (23E / 309G)

[0056] 1. Site-directed mutagenesis construction

[0057] Using the recombinant plasmid pET28a-UGT76G1 as a template, corresponding primers were designed to introduce a 23E mutation. The PCR product was digested and transformed into DH5α. After sequencing confirmation, the circularized pET28a-UGT76G1-23E plasmid was used as a template to introduce a 309G mutation via PCR using corresponding primer pairs (the mutated nucleic acid sequence is shown in SEQ ID NO: 3, and the mutated amino acid sequence is shown in SEQ ID NO: 4). The PCR amplification product was digested with DpnI enzyme at 37°C for 2 hours to degrade the initial template. The digestion product was transformed into E. coli DH5α and plated onto LB agar plates containing 50 μg / mL Kana, and incubated overnight at 37°C. Single colonies were picked, plasmids were extracted, and sequencing verification was performed to obtain the correct double mutant plasmid pET28a-UGT76G1-C1 (23E / 309G).

[0058] 2. Protein expression and preparation of crude enzyme solution

[0059] The validated plasmid was transformed into E. coli BL21(DE3), and heterologous protein expression was induced using LB medium. The bacterial cells were collected by centrifugation, resuspended in Tris hydrochloric acid buffer, sonicated, and then centrifuged again to obtain the crude enzyme solution from the supernatant.

[0060] 3. Conversion rate determination

[0061] 1. Take the prepared C1 mutant crude enzyme solution and add 1.0g of rebaudioside A, 5mg of ADP, 2g of sucrose, 37mg of disodium EDTA, and 68mg of potassium dihydrogen phosphate to a sterile centrifuge tube in sequence. Then add 10.0mg of AtSUS sucrose synthase and 30.0mg of OsUGT β-1,2-glucosidase. Finally, add 30.0mg of C1 mutant crude enzyme solution and bring the total volume to 10.0mL with Tris hydrochloric acid buffer at pH 7.2. Mix well.

[0062] 2. Place the above reaction system in a 65℃ constant temperature water bath and keep it at this temperature for 30 minutes. Gently shake the system once every 10 minutes during this period to ensure that the system is mixed evenly.

[0063] 3. After the reaction is complete, immediately add 10 mL of methanol to the system, shake for 30 seconds to terminate the reaction, and at the same time precipitate the protein in the system.

[0064] 4. Place the mixture after the reaction is terminated in a high-speed centrifuge and centrifuge at 10,000 rpm for 5 minutes at room temperature. Take the supernatant and filter it through a 0.22 μm organic phase filter membrane to remove residual impurities to obtain the HPLC detection sample. The detection conversion rate is 78.9%.

[0065] Example 2: Construction and activity determination of the double mutant C3 (282Q / 309G)

[0066] 1. Site-directed mutagenesis construction

[0067] Using the recombinant plasmid pET28a-UGT76G1 as a template, corresponding primers were designed to introduce a 282Q mutation. The PCR product was digested and transformed into DH5α. After sequencing confirmation, the circularized pET28a-UGT76G1-282Q plasmid was used as a template to introduce a 309G mutation via PCR using the corresponding primer pair (the mutated nucleic acid sequence is shown in SEQ ID NO: 5, and the mutated amino acid sequence is shown in SEQ ID NO: 6). The PCR reaction system and procedure were the same as in Example 1. After digestion, transformation, and culture with DpnI, single colonies were picked for sequencing verification to obtain the double mutant plasmid pET28a-UGT76G1-C3 (282Q / 309G).

[0068] 2. Protein expression and preparation of crude enzyme solution

[0069] The plasmid was transformed into BL21 (DE3), and expression was induced in LB medium. The crude enzyme solution was obtained by sonication and centrifugation, following the same steps as in Example 1.

[0070] 3. Conversion rate determination

[0071] Add 30.0 mg of crude C3 enzyme solution to carry out the reaction, and perform the remaining operations as in Example 1. The conversion rate of C3 was determined by HPLC and calculated to be 65.2%.

[0072] Example 3: Construction and activity determination of the three mutant C5 (23E / 282Q / 309G)

[0073] 1. Site-directed mutagenesis construction

[0074] Using the double mutant plasmid pET28a-UGT76G1-C1 (23E / 309G) as a template, primers were designed to introduce a mutation at position 282 Q (the mutated nucleic acid sequence is shown in SEQ ID NO: 7, and the mutated amino acid sequence is shown in SEQ ID NO: 7). The double mutant plasmid pET28a-UGT76G1-C3 (23E / 282Q / 309G) was obtained.

[0075] 2. Protein expression and preparation of crude enzyme solution

[0076] The plasmid was transformed into BL21 (DE3), and expression was induced in LB medium. The crude enzyme solution was obtained by sonication and centrifugation, following the same steps as in Example 1.

[0077] 3. Conversion rate determination

[0078] Add 30.0 mg of crude C5 enzyme solution to carry out the reaction, and perform the remaining operations as in Example 1. The conversion rate of C5 was determined by HPLC and calculated to be 92.1%.

[0079] 4. 1L scale-up experiment of preparing RM from triple mutant C5

[0080] Using 100.0 g of rebaudioside A as a substrate, 0.5 g of ADP, 200 g of sucrose, 3.72 g of disodium EDTA, and 6.8 g of potassium dihydrogen phosphate, 1 g of AtSUS sucrose synthase and 3 g of OsUGT β-1,2-glucosidase were added, followed by 3 g of crude enzyme solution of UGT76G1 mutant C5, for a total volume of 1 L. The reaction was carried out at pH 7.2 and 65 °C for 3 h. After the reaction, 50 μL of the reaction solution was filtered through a 0.22 μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. 88% conversion was achieved after 3 h of reaction.

[0081] Comparative preparation of wild-type UGT RM

[0082] Using 1.0 g of rebaudioside A as a substrate, 5 mg of ADP, 2 g of sucrose, 37 mg of disodium EDTA, and 68 mg of potassium dihydrogen phosphate, 10.0 mg of AtSUS sucrose synthase and 30.0 mg of OsUGT β-1,2-glucosidase were added, followed by 30.0 mg of UGT76G1 wild-type crude enzyme solution, for a total volume of 10.0 ml. The reaction was carried out at pH 7.2 and 65 °C for 3 hours, with a conversion rate of 3%.

[0083] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A UGT mutant, characterized in that, The mutant was obtained by modifying wild-type stevia UGT76G1, the amino acid sequence of which is shown in SEQ ID NO: 2; the mutant is a mutant in which at least one site of the amino acid sequence shown in SEQ ID NO: 2 is mutated; the mutant has improved glycosyltransferase activity compared with the wild type and can catalyze the production of rebaudioside A to rebaudioside M using ADP as a glycosyl donor precursor.

2. The UGT mutant according to claim 1, characterized in that, The mutant is a mutant in which at least one site selected from positions 309, 23, 278, or 282 in the amino acid sequence shown in SEQ ID NO: 2 is mutated.

3. The UGT mutant according to claim 2, characterized in that, The amino acid at position 23 is mutated to glutamic acid (E) or alanine (A); the amino acid at position 278 is mutated to glycine (G) or alanine (A); the amino acid at position 282 is mutated to glutamine (Q) or alanine (A); and the amino acid at position 309 is mutated to glycine (G) or alanine (A).

4. The UGT mutant according to claim 3, characterized in that, The mutants are double mutants 23E / 309G, 278G / 309G or 282Q / 309G, or triple mutants 23E / 282Q / 309G, or quadruple mutants 23E / 278G / 282Q / 309G.

5. A nucleotide sequence, characterized in that, Its encoding is the UGT mutant as described in claims 1-4.

6. An expression carrier, characterized in that, It comprises the nucleotide sequence of claim 5.

7. A host cell, characterized in that, It comprises the nucleotide sequence of claim 5 or the expression vector of claim 6.

8. A method for preparing rebaudioside M from a UGT mutant, characterized in that, Using the UGT mutant described in any one of claims 1-4 as a catalyst, rebaudioside A as a substrate, ADP as a glycosyl donor precursor, sucrose, AtSUS sucrose synthase, OsUGT β-1,2-glycosidase and a buffer system are added to carry out the reaction.

9. The method for preparing rebaudioside M from the UGT mutant according to claim 8, characterized in that, The reaction system also contains disodium EDTA, the buffer system includes potassium dihydrogen phosphate, and the reaction is carried out in a solution containing the following components, wherein: The initial reaction concentration of rebaudioside A was 75-125 g / L. The initial reaction concentration of sucrose was 175-225 g / L. The initial reaction concentration of ADP was 0.38-0.62 g / L. The initial reaction concentration of disodium EDTA is 3.3-4.1 g / L. The initial reaction concentration of potassium dihydrogen phosphate is 6.4-7.2 g / L. The initial reaction concentration of AtSUS sucrose synthase is 0.8-1.2 g / L. The initial reaction concentration of OsUGT β-1,2-glucosidase is 2.4-3.7 g / L. The initial reaction concentration of the crude enzyme solution of the UGT mutant was 2.4-3.7 g / L. The reaction time is 1-6 hours.

10. The application of the UGT mutant according to any one of claims 1-4 in the preparation of sweeteners in the food and beverage industry, wherein the sweetener uses rebaudioside M as the active ingredient and is added at an amount of 0.001-1 g / L.

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