Beta-1,3-glycosyltransferase mutants and their use in catalyzing the synthesis of rebaudioside m

Abstract

The application belongs to the technical field of enzyme engineering, and particularly relates to a beta-1,3-glycosyltransferase mutant and application thereof in catalyzing synthesis of rebaudioside M. The beta-1,3-glycosyltransferase mutant is obtained by mutating the beta-1,3-glycosyltransferase with an amino acid sequence shown in SEQ ID NO. 1 by at least one mutation, i.e. mutating isoleucine at the 14th position into alanine; and / or mutating asparagine at the 50th position into alanine; and / or mutating isoleucine at the 92nd position into leucine; and / or mutating leucine at the 141st position into cysteine or isoleucine. The mutant is applied to catalyzing synthesis of rebaudioside M, and is significantly superior to the wild-type enzyme in catalytic efficiency and thermal stability.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to β-1,3-glycosyltransferase mutants and their application in the catalytic synthesis of rebaudioside M. Background Technology

[0002] Stevia rebaudiana Bertoni belongs to the Asteraceae family and is native to South America, Paraguay, and Brazil. Steviosides are zero-calorie sweeteners extracted from stevia leaves and are considered one of the "world's three major sugar sources," along with sucrose and beet sugar. Steviosides in stevia leaves are a mixture of molecules with different structures, mainly including stevioside (St), rebaudioside A (Reb A), rebaudioside D (Reb D), and rebaudioside M (Reb M). Among them, Reb M is 250-300 times sweeter than sucrose, possessing the advantages of high sweetness, zero calories, and a taste similar to sucrose, and is considered an ideal substitute for high-calorie sugars with broad application prospects. However, the content of Reb M in dried stevia leaves is very low (0.4%-0.5%), traditional plant extraction methods are costly, and existing enzyme catalysis methods have problems such as low glycosyltransferase catalytic efficiency and poor thermal stability, which make it difficult to meet the needs of industrial production. Summary of the Invention

[0003] The purpose of this invention is to provide a β-1,3-glycosyltransferase mutant for the catalytic synthesis of Reb M, which has a significantly improved catalytic efficiency compared to the wild type.

[0004] The technical solution of the present invention is as follows:

[0005] A β-1,3-glycosyltransferase mutant is obtained by mutating the β-1,3-glycosyltransferase with the amino acid sequence shown in SEQ ID NO.1 through at least one of the following mutations:

[0006] The isoleucine (I) at position 14 is mutated to alanine (A);

[0007] And / or mutate the 50th position of asparagine (N) to alanine (A);

[0008] And / or mutate isoleucine (I) at position 92 to leucine (L);

[0009] And / or mutate leucine (L) at position 141 to cysteine ​​(C) or isoleucine (I).

[0010] A gene encoding the β-1,3-glycosyltransferase mutant, the nucleotide sequence of which is obtained by the above mutation based on the sequence shown in SEQ ID NO:2.

[0011] A recombinant expression vector comprising a gene encoding the β-1,3-glycosyltransferase mutant, wherein the vector is preferably a pET-28a(+) vector.

[0012] A recombinant cell comprising the recombinant expression vector, or with a gene encoding the β-1,3-glycosyltransferase mutant integrated into its genome. The cell is a prokaryotic cell, preferably *Escherichia coli* (E. coli), more preferably *E. coli* BL21(DE3).

[0013] The application of the β-1,3-glycosyltransferase mutant, encoding gene, recombinant expression vector, or recombinant cell in the catalytic synthesis of Reb M.

[0014] A method for catalytically synthesizing Reb M using Reb D as a substrate involves contacting the β-1,3-glycosyltransferase mutant, or recombinant cells expressing the mutant, or a crude enzyme solution containing the mutant, with the substrate Reb D and a glycosyl donor to catalyze the synthesis of Reb M. The glycosyl donor is either UDP-glucose (UDPG) or ADP-glucose (ADPG).

[0015] A method for catalytic synthesis of Reb M using Reb A as a substrate employs a multi-enzyme cascade reaction system, which includes:

[0016] 1) The β-1,3-glycosyltransferase mutant;

[0017] 2) β-1,2-glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO:3 and the nucleotide sequence of which is shown in SEQ ID NO:5;

[0018] 3) Sucrose synthase, the amino acid sequence of which is shown in SEQ ID NO:4 and the nucleotide sequence of which is shown in SEQ ID NO:6;

[0019] The method uses Reb A as the starting substrate and sucrose and adenosine diphosphate (ADP) or uridine diphosphate (UDP) as raw materials to generate Reb M through the multi-enzyme cascade reaction.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Dual enhancement of core enzyme performance: high efficiency and stability

[0022] This invention, through the rational design of β-1,3-glycosyltransferase, yielded a mutant that significantly outperforms the wild-type in both catalytic efficiency and thermal stability. In particular, the preferred mutant GTM0-L141C exhibits a significantly enhanced ability to catalyze the synthesis of Reb M, implying shorter reaction times and higher yields in production. Furthermore, thermal stability tests showed that the mutant maintained a high catalytic yield even after heat treatment, with GTM0-L141C still achieving a Reb M yield of 7.95 g / L, approximately 33% higher than the wild-type (5.98 g / L). This provides strong evidence and demonstrates significant potential for adapting to higher temperature conditions and extending service life in industrial production.

[0023] 2. Integrated innovation of technical solutions: comprehensive optimization from enzymes to processes

[0024] This invention integrates high-performance mutants into an innovative multi-enzyme cascade reaction system, achieving comprehensive optimization from enzymes to processes. By constructing an efficient cascade reaction, starting with Reb A, this invention efficiently synthesizes Reb M, achieving a Reb A conversion rate of up to 99% within 10 hours. This system utilizes sucrose synthase to regenerate expensive glycosyl donors in situ with inexpensive sucrose, reducing production costs. Attached Figure Description

[0025] Figure 1 A standard curve showing the relationship between protein content and absorbance;

[0026] Figure 2 HPLC detection of peak elution times for Reb A, Reb D, and Reb M;

[0027] Figure 3 The HPLC chromatogram is shown for a multi-enzyme cascade system catalyzing a reaction for 1 hour.

[0028] Figure 4 This is an HPLC chromatogram of the multi-enzyme cascade system catalyzing the reaction for 10 hours. Detailed Implementation

[0029] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; where specific techniques or conditions are not specified in the experiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials mentioned are all commercially available.

[0030] Example 1

[0031] In this embodiment, β-1,3-glycosyltransferase was rationally designed and site-directedly mutated using techniques such as molecular docking, molecular dynamics, AlphaFold, and EvolvePro to obtain several mutants. The mutation sites are shown in Table 1.

[0032] The β-1,3-glycosyltransferase gene (SEQ ID NO.2) was cloned into the pET28a(+) vector to construct the recombinant plasmid pET28a-GTM0 (synthesized by Beijing Qingke Biotechnology Co., Ltd.). Using this plasmid as a template, PCR amplification was performed using the primers listed in Table 1 to obtain the mutant gene fragment. The PCR product was digested with DpnI and purified by gel extraction. The purified product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, incubated at 37°C for 12–16 hours. After single colonies appeared on the plates, colony PCR verification and sequencing confirmation were performed.

[0033] Table 1. Mutation sites and corresponding primers

[0034]

[0035] Example 2

[0036] The recombinant mutant plasmid with correct sequencing from Example 1 was transformed into... E. coli BL21(DE3) competent cells were used to obtain a genetically engineered strain containing a point mutation. A single colony was picked and inoculated into 3 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 200 rpm for 8 h. Then, a 1% (v / v) inoculum was transferred to 50 mL of fresh liquid TB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 200 rpm until OD (out of control) was reached. 600 When the concentration reaches 0.6-0.8, add IPTG (isopropyl) β D Isopropyl β-thiogalactoside D Thiogalactoside was added to a final concentration of 0.5 mM and incubated at 25°C and 200 rpm for 20 h.

[0037] After culturing, the culture medium was centrifuged at 4°C and 4000 rpm for 15 min, the supernatant was discarded, and the bacteria were collected. The bacterial cells were resuspended in 1×PBS, with the amount of 1×PBS being 5 times the mass of the bacterial culture. Then, the cells were sonicated to disrupt the cytotoxicity using an ultrasonic homogenizer at 40% power (total power 560W) for 2 seconds, followed by a 4-second pause, for a total of 15 min. The resulting cell lysis buffer was centrifuged at 4°C and 4000 rpm for 15 min, and the supernatant was collected to obtain the crude enzyme solution of the mutant.

[0038] Example 3

[0039] This embodiment tested the protein concentration of the mutant crude enzyme solution prepared in Example 2, using the following method:

[0040] (1) Constructing a standard curve:

[0041] Take 7 test tubes and number them (each numbered tube should be tested in triplicate). Add the reagents according to Table 2 and mix well. After standing for 2 minutes, perform colorimetric determination at a wavelength of 595 nm (colorimetric determination should be completed within 1 hour). Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis. Figure 1 ).

[0042] Table 2. Data on the amount of reagents added

[0043]

[0044] (2) Take another test tube, accurately add 100 μL of sample, then add 0.9 mL of distilled water and 5 mL of Coomassie Brilliant Blue G-250 reagent, mix thoroughly, let stand for 2 min, use the standard curve tube No. 1 as a reference, measure the color at a wavelength of 595 nm, record the absorbance value, calculate the protein concentration, and the protein concentration of the mutant crude enzyme solution is shown in Table 3.

[0045] Table 3 Protein concentrations of crude enzyme solutions from wild-type and mutant strains

[0046]

[0047] Example 4

[0048] In this example, a 1 mL reaction system was prepared with a final substrate concentration of Reb D 10 g / L, UDPG 10 mM, crude enzyme solution 0.5 g / L (based on protein concentration), and 1×PBS to a final volume of 1 mL. The reaction was carried out at 60 °C and 800 rpm for 20 min. After the reaction, 100 μL of the solution was taken and 900 μL of methanol was added. The mixture was shaken to terminate the reaction. The solution was centrifuged at 12000 rpm for 2 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The Reb M content was detected by HPLC to screen the catalytic efficiency of the mutant. The results are shown in Table 4.

[0049] Table 4 Results of crude enzyme solution catalysis of wild type and mutant

[0050]

[0051] Table 4 shows the initial screening results: the mutants GTM0-I14A, GTM0-N50A, GTM0-I92L, GTM0-L141C, and GTM0-L141I all produced higher amounts of Reb M than the wild-type glycosyltransferase GTM0, indicating that the activities of these mutants were all improved compared to the wild type. Among them, the mutant GTM0-L141C had the highest Reb M production.

[0052] Example 5

[0053] In this embodiment, the thermal stability of the mutants was tested. 1.5 mL of crude enzyme solution from wild-type GTM0 and mutants GTM0-I14A, GTM0-N50A, GTM0-I92L, GTM0-L141C, and GTM0-L141I was taken and placed in a water bath at 65°C for 30 min. After heat treatment, it was used to catalyze the production of Reb M. The method was the same as in Example 4, except that the reaction temperature in this embodiment was a metal bath at 65°C. After the reaction, the Reb M content was measured, and the results are shown in Table 5.

[0054] Table 5. Results of catalytic reaction of crude enzyme solutions from wild type and mutant strains at 65°C in a metal bath.

[0055]

[0056] As can be seen from Table 5, after heat treatment in a 65℃ water bath for 30 min, the Reb M yield produced by the preferred mutant GTM0-L141C of this invention reached 7.95 g / L, which is 33% higher than that of wild-type GTM0 (5.98 g / L). This proves that the mutant GTM0-L141C has significantly enhanced catalytic ability and thermal stability.

[0057] Example 6

[0058] In this embodiment, crude enzyme solutions of β-1,2-glycosyltransferase (β-1,2-GT) and sucrose synthase (SUS) were first prepared, following the preparation method described in Example 2. The amino acid sequence of β-1,2-glycosyltransferase is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.5; the amino acid sequence of sucrose synthase is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.6. The β-1,2-GT and SUS genes were inserted into the pET28a(+) plasmid vector, respectively, to obtain the recombinant plasmids pET28a-β12GT and pET28a-SUS, which were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0059] Furthermore, this embodiment constructed a multi-enzyme cascade catalytic reaction system to catalyze the synthesis of Reb M. Using Reb A97 (Reb A content 97 wt.%) as the substrate, in a 100 mL reaction system, the final concentrations of Reb A97 were 100 g / L, ADP 0.4 g / L, sucrose 150 g / L, sucrose synthase 0.3 g / L, β-1,2-glycosyltransferase 0.5 g / L, and the β-1,3-glycosyltransferase mutant GTM0-L141C 1 g / L. Finally, 1×PBS was added to 100 mL. The reaction was carried out at 60 °C and 800 rpm for 10 h. After the reaction, 100 μL was taken, 900 μL of methanol was added, the mixture was shaken to terminate the reaction, and the mixture was centrifuged at 12000 rpm for 2 min. The supernatant was filtered through a 0.22 μm filter membrane and detected by HPLC. The elution times of Reb A, Reb D, and Reb M as determined by HPLC are as follows: Figure 2 The HPLC results of the Reb A reaction catalyzed by the multi-enzyme cascade system for 1 h are as follows: Figure 3 The HPLC detection results after 10 hours of reaction are as follows: Figure 4 The results showed that after 10 hours of reaction, Reb A was almost completely reacted, with a conversion rate of 99%. The content of intermediate product Reb D was 1.9 g / L, and the content of product Reb M was 113.5 g / L.

[0060] The amino acid sequence of β-1,3-glycosyltransferase, SEQ ID NO.1:

[0061] MPNKTETTVRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

[0062] Nucleotide sequence of β-1,3-glycosyltransferase SEQ ID NO.2:

[0063]

[0064] Amino acid sequence of β-1,2-glycosyltransferase SEQ ID NO.3:

[0065] MHHHHEGVSDQTLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTLINLESIIKKIPEKYSESIRFVELHLPELPELPPHYHTTNGLPPHLNHTLHKALKMSKPNFSKILQNLKPDLVIYDILQPWAEHVVNEQNIPAVKILTSGAALFSYFFNFLKNPGVEFPFPAIYLPKVEQVKMREMFEKEPNEEDRLAEGNMQIMLMCTSRTIEAKYLDYCTELSNWKVVPVGPPFQDPITNDVDDMELIDWLGTKDENSTVFVCFGSEYFLSREDMEEVAFGLELSNVNFIWVARFPKGEEQNLEDVLPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESLDFGVPIIAMPMHNDQPINAKLIVELGVAMEIVRDDDGNIHRGEITETLKDVITGETGEILRGKVRDISKNLKSIREEEMNAAAEELIQLCRNSNKYK

[0066] Nucleotide sequence of β-1,2-glycosyltransferase SEQ ID NO.5:

[0067]

[0068] Amino acid sequence of sucrose synthase SEQ ID NO.4:

[0069] MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQEGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME

[0070] Nucleotide sequence of sucrose synthase SEQ ID NO.6:

[0071]

Claims

1. A β-1,3-glycosyltransferase mutant, characterized in that: It is obtained by mutating β-1,3-glycosyltransferase with the amino acid sequence shown in SEQ ID NO.1 by the following mutation: mutating leucine at position 141 to cysteine.

2. A gene encoding the β-1,3-glycosyltransferase mutant of claim 1.

3. The gene as described in claim 2, characterized in that, Its nucleotide sequence is obtained by the mutation described in claim 1 based on the sequence shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, The vector contains the gene as described in claim 2 or 3.

5. A recombinant cell, characterized in that, The cell contains the recombinant expression vector of claim 4, or has the gene of claim 2 or 3 integrated into its genome.

6. The recombinant cell as described in claim 5, characterized in that, The cells in question are prokaryotic cells.

7. The use of the β-1,3-glycosyltransferase mutant of claim 1, the gene of claim 2 or 3, the recombinant expression vector of claim 4, or the recombinant cell of claim 5 or 6 in the catalytic synthesis of rebaudioside M.

8. A method for catalytic synthesis of rebaudine M using rebaudine D as a substrate, characterized in that, The method involves contacting the β-1,3-glycosyltransferase mutant of claim 1, or recombinant cells expressing the mutant, or crude enzyme solution containing the mutant, with the substrate rebaudioside D and a glycosyl donor to catalyze the synthesis of rebaudioside M.

9. The method as described in claim 8, characterized in that, The glycosyl donor is either UDP-glucose or ADP-glucose.

10. A method for catalytic synthesis of rebaudine M using rebaudine A as a substrate, characterized in that, The method employs a multi-enzyme cascade reaction system, which includes: 1) The β-1,3-glycosyltransferase mutant according to claim 1; 2) β-1,2-glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO:3; 3) Sucrose synthase, the amino acid sequence of which is shown in SEQ ID NO:4; The method uses rebaudioside A as the starting substrate and sucrose and adenosine diphosphate or uridine diphosphate as raw materials to generate rebaudioside M through the multi-enzyme cascade reaction.

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