Glycosyl transferase SrUGT76G1 mutant and application thereof in synthesis of rebaudioside M

Abstract

The invention discloses a glycosyl transferase SrUGT76G1 mutant and application of the glycosyl transferase SrUGT76G1 mutant in synthesis of rebaudioside M, and relates to the technical field of biological enzyme engineering. The amino acid sequence of the glycosyl transferase SrUGT76G1 mutant is as shown in SEQ IE NO. 2. According to the mutant disclosed by the invention, the enzyme activity of the glycosyl transferase SrUGT76G1 is remarkably improved, and compared with wild type glycosyl transferase SrUGT76G1, the enzyme activity of the optimal mutant is improved by 6.6 times, so that the use amount of enzyme can be remarkably reduced. And moreover, the catalytic process is simple, no by-product is generated in the reaction, and the subsequent separation is simple, so that the method has the advantages of high economical efficiency, mild reaction conditions, environment friendliness, simple product post-treatment and the like, and shows a good industrial application prospect in the synthesis of rebaudioside M.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme engineering technology, and more specifically to a glycosyltransferase SrUGT76G1 mutant and its application in the synthesis of rebaudioside M. Background Technology

[0002] RebM, a glycoside M, is used in stevia plants ( Stevia rebaudiana Bertoni RebM is one of the many sweet compounds produced by stevioside, with a sweetness approximately 200-350 times that of sucrose. It is one of the sweetest members of the steviol glycoside family, with a taste closest to sucrose, and almost none of the bitterness or metallic aftertaste of traditional steviol glycosides, offering a purer sweetness. Therefore, RebM, with its superior sensory properties and safety, is becoming the "gold standard" in the high-end sugar substitute market, especially suitable for food innovations seeking natural, zero-calorie, and pure-tasting products. Currently, RebM has obtained GRAS (Generally Recognized as Safe) certification from the US FDA, and the EU EFSA has also approved it as a food additive (E960). Furthermore, China included it in its food additive usage standards (GB 2760) in 2021. Therefore, with its three major advantages—naturalness, high sweetness, and optimized taste—RebM steviol glycosides are poised to become a star product in the sugar substitute market.

[0003] However, RebM is present in extremely low concentrations (<1%) in stevia, and plant extraction methods cannot meet market demand. Therefore, many studies have focused on synthesizing RebM through chemical and biological methods. Chemical synthesis primarily uses cyclic diterpenoids as precursors, generating steviol through multiple oxidation-reduction reactions. Glucose groups are then gradually added to the steviol skeleton with precise control over the glycosidic bond positions (e.g., C13 and C19 positions), and finally, the target product is separated by column chromatography. However, chemical synthesis still faces several challenges, including: complex reaction steps and low overall yield; multiple chiral centers, making stereoselectivity control difficult; and the use of toxic reagents (such as halogenated hydrocarbons), resulting in poor environmental friendliness. Biosynthesis, on the other hand, is the mainstream method due to its mild reaction conditions, high selectivity, yields of 60-80%, and its environmental friendliness with no toxic byproducts. SrUGT76G1 is the core modifying enzyme in RebM biosynthesis, and its main current challenge is low enzyme activity.

[0004] Therefore, how to improve the activity of SrUGT76G1 enzyme is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a glycosyltransferase SrUGT76G1 mutant and its application in the synthesis of rebaudioside M.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A glycosyltransferase SrUGT76G1 mutant, which is based on the amino acid sequence shown in SEQ IE NO.2, with mutations of A and / or G at positions 84 and 91, respectively, yielding P84A / 91A, P84G / P91G, P84A, P91A, P84G, and P91G.

[0007] Another object of the present invention is to provide a coding gene encoding the above-mentioned glycosyltransferase SrUGT76G1 mutant.

[0008] Another object of the present invention is to provide a biomaterial, wherein the biomaterial is any one of the following: A. A recombinant vector containing the gene sequence encoding the SrUGT76G1 mutant glycosyltransferase described above; B. Recombinant strains containing the recombinant vector described in A.

[0009] Another object of the present invention is to provide the application of the above-mentioned mutant or the coding gene of the above-mentioned mutant P84A / P91A or the above-mentioned biological material in the catalytic synthesis of rebaudioside D into rebaudioside M.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned mutant, comprising the following steps: (1) The gene sequence of glycosyltransferase SrUGT76G1 was obtained according to the Uniport ID: Q6VAB4, and the codons were optimized to obtain SEQ ID NO.1; (2) The whole gene was synthesized and ligated to the NdeI / XhoI position of the vector pETDuet-1 to obtain the recombinant plasmid pETDuet-1-SrUGT76G1; (3) Using recombinant plasmid pETDuet-1-SrUGT76G1 as a template, perform PCR, and carry out one or two rounds of site-directed mutagenesis to construct the corresponding recombinant plasmid carrying the mutant. (4) Transform the recombinant plasmid carrying the mutant into the host bacteria to obtain transformants, and then sequence them for verification; (5) Ferment the correctly sequenced transformants to obtain the SrUGT76G1 mutant of glycosyltransferase.

[0011] Preferably, the primer sequences for the site-directed mutation in step (3) are shown in SEQ ID NO.3-SEQ ID NO.8.

[0012] Preferably, the host bacterium in step (4) is Escherichia coli.

[0013] Another objective of this invention is to provide a method for synthesizing rebaudioside M, which uses the above-mentioned SrUGT76G1 mutant as a catalyst and rebaudioside D as a substrate to carry out a catalytic reaction to obtain rebaudioside M.

[0014] As can be seen from the above technical solution, compared with the prior art, this invention discloses a glycosyltransferase SrUGT76G1 mutant. This invention significantly improves the enzyme activity of glycosyltransferase SrUGT76G1 through site-directed mutagenesis. Compared with wild-type glycosyltransferase SrUGT76G1, the optimal mutant enzyme activity is increased by 6.6 times, thus significantly reducing the amount of enzyme used. The glycosyltransferase mutant provided by this invention has the advantages of high catalytic activity and no other byproducts. Furthermore, the method for catalyzing the production of rebaudioside D from rebaudioside M using the glycosyltransferase mutant provided by this invention has a simpler catalytic process compared with other preparation methods, with no byproducts generated and simple subsequent separation. Therefore, it has advantages such as high economy, mild reaction conditions, environmental friendliness, and simple product post-processing, showing good industrial application prospects in the synthesis of rebaudioside M. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The reaction mechanism by which glycosyltransferase SrUGT76G1 catalyzes the conversion of rebaudioside D to rebaudioside M; Figure 2 Rational site design for the catalytic pocket of glycosyltransferase SrUGT76G1; Figure 3 The relative activities of glycosyltransferase SrUGT76G1 and its various mutants; Figure 4 The reaction curve for the crude enzyme-catalyzed synthesis of RebM is shown. Figure 5 The results are HPLC results for RebD and RebM. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1 1. Construction of engineered strains The gene encoding the amino acid sequence (Uniport ID: Q6VAB4) of the glycosyltransferase SrUGT76G1 derived from Stevia rebaudiana was codon-optimized in E. coli to obtain SEQ ID NO. 1. This gene was then synthesized and inserted into the NdeI / XhoI position of the expression plasmid pETDuet-1 to obtain pETDuet-1-SrUGT76G1, which was then transformed into the expression host E. coli. E. coli BL21(DE3) is used for subsequent expression of recombinase.

[0019]

[0020] 2. Sr Rational modification of the active pocket of UGT76G1 protein In proteins, the cyclic side chain of proline (Pro) has a rigid conformation, limiting the flexibility of local structures. Proline often appears at turns or in rigid regions, hindering the formation of α-helices or β-sheets. (See attached image) Figure 2 As shown, P84 and P91 are located at the substrate / product inlet and outlet. The rigidity of P84 / P91 may "lock" the local conformation around the channel, hindering the closure of the active site after substrate binding. This rigidity may slow down the conformational change rate, leading to a decrease in catalytic efficiency.

[0021] Based on this, the present invention mutates P84 and / or P91 to alanine (A) and / or glycine (G), respectively, thereby increasing the conformational freedom of the local region and making it easier for the active center to change from an open state to a closed state. This shortens the time for the substrate to enter the active center, enhances the "wrapping" of the substrate by the catalytic center, and improves the reaction efficiency (such as the glycosyl transfer rate).

[0022] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEE DEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESE LETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEV LAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL* , SEQ ID: NO.2.

[0023] 3. Glycosyltransferase Sr Construction of UGT76G1 single / double point mutants Based on the concept in step 2, this invention performs site-directed mutagenesis on two sites, 84 and 91, of SrUGT76G1 to determine their potential to enhance the catalytic activity of SrUGT76G1.

[0024] Using the recombinant plasmid pETDuet-1-SrUGT76G1 obtained in step 1 as a template, primers were designed for the proposed mutation sites (as shown in Table 1), and one or two rounds of site-directed mutagenesis were performed to construct the corresponding recombinant plasmids carrying the mutants. PCR system: 10 μL of 2x KOD mix, 1 μL each of forward and reverse primers, 1 μL of template, 7 μL of double-distilled water, for a total volume of 20 μL. PCR program: 98℃ pre-denaturation for 5 min; then cycled 30 times according to the following program: 98℃ denaturation for 10 s, 58℃ annealing for 5 s, 68℃ extension for 1 min 40 s; 68℃ final extension for 5 min, and storage at 4℃.

[0025] Table 1 Primers used for the SrUGT76G1 glycosyltransferase mutant

[0026] Add 2 μL of DpnI enzyme to the PCR product and incubate overnight at 37°C to digest the template plasmid. Transform 5 μL of the digested PCR product into *E. coli* BL21(DE3) competent cells, incubate on ice for 30 min, then heat shock at 42°C for 90 s, incubate on ice for 2 min, add 900 μL of LB medium, incubate at 37°C with a shaker at 200 rpm for 45 min, centrifuge to resuspend the cells, and evenly spread them on LB agar plates containing kanamycin resistance. Incubate overnight at 37°C. Once single colonies have grown on the plates, send them to a third-party sequencing platform. After successful sequencing, preserve the bacteria and name the strains accordingly. Sr UGT76G1P84A (P at position 84 is mutated to A), Sr UGT76G1P84G (P at position 84 is mutated to G) Sr UGT76G1P91A (P at position 91 is mutated to A) Sr UGT76G1P91G (P at position 91 is mutated to G) Sr UGT76G1P84A / P91A (P at positions 84 and 91 both mutate to A), Sr UGT76G1P84G / P91G (P at positions 84 and 91 are both mutated to G) was used for subsequent experiments.

[0027] 4. Strains culture The wild-type *Escherichia coli* BL21(DE3) / pETDuet-1-SrUGT76G1 obtained in step 1 and the mutant strain obtained in step 3 were inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin. The cultures were incubated at 37°C and 200 rpm for 16 hours. Then, 1% of the cultures were inoculated into 100 mL of fresh LB medium containing ampicillin and incubated at 37°C and 200 rpm until OD (dose retardation). 600 When the β-thiogalactoside ratio is approximately 0.6-0.8, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) is added at 20℃ and 150 rpm to induce fermentation for 16 hours to obtain the fermentation broth.

[0028] 5. Determination of the enzyme activity of glycosyltransferase SrUGT76G1 and its mutants The fermentation broth of the recombinant bacteria was centrifuged at 8,000 rpm for 10 min at 4 °C. After discarding the supernatant, the bacteria were washed twice with 100 mM sodium phosphate buffer (pH 7.2), then resuspended and sonicated (300 W, 5 s on, 5 s off, 99 cycles). Afterward, the mixture was centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was collected as the crude enzyme extract. The protein concentration of the crude enzyme solution was determined using the Bradford method.

[0029] The enzyme activity of the mutant was determined. Glycosyltransferase (U) activity was defined as the amount of enzyme producing 1 μmol of Reb M from Reb D per minute. The crude enzyme reaction system consisted of 3 mL of 100 mM NaH₂PO₄-Na₂HPO₄ buffer (PBS, pH 7.2), containing 1 mg of crude enzyme extract (cell lysate supernatant), 1 mM Reb D substrate, 2 mM UDPG, and 3 mM MgCl₂. After incubation at 30 °C for 10 min, the reaction was quenched by heating at 95 °C for 15 min. The mixture was then centrifuged, and the supernatant was filtered through a 0.22 μm organic filter for HPLC analysis.

[0030] From the appendix Figure 3 It can be seen that the mutant enzyme activity is significantly increased compared to the wild type, with the following being particularly high. Sr The UGT76G1P84A / P91A showed the most significant improvement, being 6.6 times that of the wild type. Sr UGT76G1P84G / P91G is 5.2 times that of the wild type. Sr UGT76G1P91A is 4.8 times more potent than the wild type. SrUGT76G1P84A is 4.5 times more potent than the wild type. Sr UGT76G1P84G is 4.2 times more potent than the wild type. Sr UGT76G1P91G is 3.6 times that of the wild type.

[0031] 6. Crude enzyme-catalyzed synthesis of RebM After fermentation, the bacterial cells were collected by centrifugation, washed twice with 100 mM pH 7.2 PBS, and then resuspended in 100 mL PBS. Sr UGT76G1P84A / P91A bacterial suspension and sucrose synthase At SUS3 bacterial suspensions were all at a concentration of 5 g / 100 mL, sonicated on ice, and centrifuged at 12000 rpm for 30 min. The supernatant was collected. Subsequently, 3 g / L Reb D, 15 g / L sucrose, 3 mM MgCl2, and 5% (v / v) dimethyl sulfoxide (DMSO) solution were added to this supernatant to catalyze the reaction. o C, reaction for 24 h. Samples were taken during the reaction process, and the samples were subjected to 95°C. o Heating was stopped at C for 10 min. After centrifugation at 12,000 rpm for 10 min and filtration through a 0.22 μm filter membrane, the Reb M yield was determined by HPLC. The results are attached. Figure 4 As shown, the final system contained 0.207 g / L Reb D and 3.08 g / L Reb M. Calculations showed that the conversion rate of steviol glycoside D was greater than 93.1%, and the yield of Reb M was 89.8%. Liquid chromatography results indicated that no byproduct RebM2 was generated during this process (see Appendix). Figure 5 ).

[0032] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0033] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase SrUGT76G1 mutant, characterized in that, The mutants are based on the amino acid sequence shown in SEQ IE NO.2, with mutations at positions 84 and 91 to A and / or G, respectively, to obtain P84A / 91A, P84G / P91G, P84A, P91A, P84G, and P91G.

2. The gene encoding the SrUGT76G1 mutant glycosyltransferase as described in claim 1.

3. A biomaterial, characterized in that, The biomaterial is any one of the following: A. A recombinant vector having a gene sequence encoding the SrUGT76G1 mutant glycosyltransferase of claim 1; B. Recombinant strains containing the recombinant vector described in A.

4. The application of the mutant of claim 1 or the gene encoding the mutant of claim 2 or the biomaterial of claim 3 in the catalytic synthesis of rebaudioside D into rebaudioside M.

5. The method for preparing the mutant according to claim 1, characterized in that, Includes the following steps: (1) The gene sequence of glycosyltransferase SrUGT76G1 was obtained according to the Uniport ID: Q6VAB4, and the codons were optimized to obtain SEQ ID NO.1; (2) The whole gene was synthesized and ligated to the NdeI / XhoI position of the vector pETDuet-1 to obtain the recombinant plasmid pETDuet-1-SrUGT76G1; (3) Using recombinant plasmid pETDuet-1-SrUGT76G1 as a template, perform PCR, and carry out one or two rounds of site-directed mutagenesis to construct the corresponding recombinant plasmid carrying the mutant. (4) Transform the recombinant plasmid carrying the mutant into the host bacteria to obtain transformants, and then sequence them for verification; (5) Ferment the correctly sequenced transformants to obtain the SrUGT76G1 mutant of glycosyltransferase.

6. The method for preparing the mutant according to claim 5, characterized in that, The primer sequences for step (3) site-directed mutagenesis are shown in SEQ ID NO.3-SEQ ID NO.

8.

7. The method for preparing the mutant according to claim 5, characterized in that, Step (4) The host bacterium is Escherichia coli.

8. A method for synthesizing rebaudioside M, characterized in that, Rebadinoside M was obtained by using the SrUGT76G1 mutant of claim 1 as a catalyst and rebadinoside D as a substrate for catalytic reaction.

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