Glycosyltransferase UGTSL2 mutant and method for synthesizing rebaudioside M2

Abstract

The invention discloses a glycosyl transferase UGTSL2 mutant. The UGTSL2 mutant is subjected to any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 23rd amino acid is mutated from N to E; the 41st amino acid is mutated from R to P; the 91st amino acid is mutated from H to K; the 95th amino acid is mutated from K to D; the 123 amino acid is mutated from E to P; the 136th amino acid is mutated from L to F; the 151st amino acid is mutated from R to F; the 124 amino acid is mutated from H to E; the 168th amino acid is mutated from V to Y; the 198 amino acid is mutated from C to K; the 202nd amino acid is mutated from T to E; the 217th amino acid is mutated from W to K; the 225th amino acid is mutated from P to L; the 226 amino acid is mutated from F to V; the 285th amino acid is mutated from A to V; and the 333rd amino acid is mutated from I to V and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biocatalytic synthesis, and in particular to a glycosyltransferase UGTSL2 mutant and a one-pot biocatalytic method for synthesizing rebaudioside A into rebaudioside M2. Background Art

[0002] There is a growing demand among people, especially diabetics, for natural, calorie-free sugar substitutes. Stevioside is a natural, calorie-free ingredient extracted from stevia leaves.

[0003] Rebaudioside D is a stevioside with great application prospects. Its sweetness is 200-300 times higher than that of sucrose, but its short-term bitter taste affects its application and promotion in the market. Rebaudioside M2 ​​is a product that adds a glucose group to the C-6' position of the first glucose group connected to the C19 position of rebaudioside D. Compared with rebaudioside D, its sweetness is increased and its bitterness is eliminated. Therefore, rebaudioside M2 ​​has better sugar properties and more ideal taste than rebaudioside D.

[0004] At present, rebaudioside M2 ​​is a monoglycosylated derivative of rebaudioside D, which is obtained by bioconversion. NtUGT from tobacco (Nicotiana tomentosiformis) can catalyze the C-6' glycosylation reaction of the first glucose group attached to the C19 position of stevioside compounds, so the high-value sweetener rebaudioside M2 ​​can be obtained by catalyzing rebaudioside D with NtUGT.

[0005] NtUGT is a "Leloir" type glycosyltransferase, belonging to the GT1 family. It usually uses UDPG as a glycosyl donor to synthesize glycoside compounds. It can be obtained in large quantities by heterologous expression in Escherichia coli or Saccharomyces cerevisiae, and is used to catalyze the synthesis of rebaudioside M2 ​​from rebaudioside D. With the continuous improvement of UDPG production methods, it can be synthesized in large quantities by enzymatic methods such as SuSy. On this basis, the SuSy-UGT cascade reaction system is used, and SuSy is used to provide a glycosyl donor for NtUGT to achieve the recycling of UDPG, which greatly reduces the application cost. For the NtUGT-SuSy cascade system, SuSy provides the required glycosyl donor for the smooth progress of the glycosylation reaction. When the UDPG regeneration rate is not a limitation (for example, by increasing the sucrose concentration), the key to the efficiency of synthesizing rebaudioside M2 ​​lies in NtUGT. NtUGT from tobacco has poor thermal stability, and the enzyme is generally inactivated after 10 hours of catalysis, which limits the efficient application of glycosyltransferases. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art glycosyltransferase UGTSL2, namely, low enzyme activity and short half-life, thereby providing a glycosyltransferase UGTSL2 enzyme mutant with high enzyme activity and long half-life.

[0007] Glycosyltransferase UGTSL2 mutant, wherein the UGTSL2 mutant is any one of the following (A)-(C):

[0008] (A) A protein obtained by causing any one or more of the following mutations based on the amino acid sequence shown in SEQ ID NO.1:

[0009] The amino acid at position 23 mutated from N to E;

[0010] The amino acid at position 41 mutated from R to P;

[0011] The amino acid at position 91 mutated from H to K;

[0012] The amino acid at position 95 mutated from K to D;

[0013] The amino acid at position 123 mutated from E to P;

[0014] The amino acid at position 136 mutated from L to F;

[0015] The amino acid at position 151 mutated from R to F;

[0016] The amino acid at position 124 mutated from H to E;

[0017] The amino acid at position 168 mutated from V to Y;

[0018] The amino acid at position 198 mutated from C to K;

[0019] The amino acid at position 202 mutated from T to E;

[0020] The amino acid at position 217 mutated from W to K;

[0021] The amino acid at position 225 mutated from P to L;

[0022] The amino acid at position 226 mutated from F to V;

[0023] The amino acid at position 285 mutated from A to V;

[0024] The amino acid at position 333 mutated from I to V;

[0025] The amino acid at position 358 mutated from N to F;

[0026] The amino acid at position 392 mutated from T to V;

[0027] The amino acid at position 419 mutated from I to K;

[0028] (B) a protein having an amino acid sequence with a sequence identity of more than 95% with that of (A) and having the same function;

[0029] (C) Fusion protein obtained by ligating a tag to the end of the protein defined in (A) or (B).

[0030] Preferably, the amino acid sequence of the UGTSL2 mutant is shown in SEQ ID NO.3.

[0031] Any of the following biological materials:

[0032] (A) An expressed gene encoding the glycosyltransferase mutant according to claim 1 or 2.

[0033] (B) A recombinant plasmid, wherein the expression gene described in (A) is connected to the recombinant plasmid.

[0034] (C) A recombinant cell comprising the recombinant expression plasmid or the expression gene of the glycosyltransferase mutant.

[0035] The expressed gene is obtained by one or more of the following mutations in SEQ ID NO.2: positions 69-71 of SEQ ID NO.2 are replaced by AAC to GAA;

[0036] Positions 121-123 of SEQ ID NO.2 are replaced by CGT to CCG;

[0037] Positions 271-273 of SEQ ID NO.2 are replaced by CAC to AAA;

[0038] Positions 283-285 of SEQ ID NO.2 are replaced by AAA to GAT;

[0039] Positions 367-369 of SEQ ID NO.2 are replaced by GAG to CCG;

[0040] Positions 406-408 of SEQ ID NO.2 are replaced by CTG to TTT;

[0041] Positions 451-453 of SEQ ID NO.2 are replaced by CGT with TTT;

[0042] Positions 370-372 of SEQ ID NO.2 are replaced by CAC to GAA;

[0043] Positions 502-504 of SEQ ID NO.2 are replaced by GTG to TAT;

[0044] Positions 592-594 of SEQ ID NO.2 are replaced by TGC to AAA;

[0045] Positions 604-606 of SEQ ID NO.2 are replaced by ACC and GAA;

[0046] Positions 649-651 of SEQ ID NO.2 are replaced by TGG to AAA;

[0047] Positions 673-675 of SEQ ID NO.2 are replaced by CCG to CTG;

[0048] Positions 676-678 of SEQ ID NO.2 are replaced by TTC with GTT;

[0049] Positions 853-855 of SEQ ID NO.2 are replaced by GCG to GTT;

[0050] Positions 997-999 of SEQ ID NO.2 are replaced by ATT and GTT;

[0051] Positions 1072-1074 of SEQ ID NO.2 are replaced by AAC with TTC;

[0052] Positions 1174-1176 of SEQ ID NO.2 are replaced by ACC and GTT;

[0053] Positions 1255-1257 of SEQ ID NO.2 are replaced by ATT to AAA;

[0054] Preferably, the nucleotide sequence of the expressed gene is as shown in SEQ ID NO.4.

[0055] An enzyme composition, comprising: a glycosyltransferase UGTSL2 mutant, a glycosyltransferase NtUGT_M and a sucrose synthase AtSuSy; the glycosyltransferase UGTSL2 mutant is as shown in claim 1 or 2;

[0056] The glycosyltransferase NtUGT_M is as follows (A1) or (A2): (A1) the amino acid sequence is shown in SEQ ID NO.5;

[0057] (A2) a protein having an amino acid sequence identity of 95% or more to that of (A1) and having the same function;

[0058] The sucrose synthase AtSuSy is as follows (B1) or (B2): the amino acid sequence of (B1) is shown in SEQ ID NO.7;

[0059] (B2) A protein having an amino acid sequence identity of 95% or more to that of (B1) and having the same function.

[0060] Preferably, the enzyme activity ratio of the glycosyltransferase UGTSL2 mutant to NtUGT_M is 3-7:7-3.

[0061] A set of recombinant strains expressing the above-mentioned enzyme composition includes a recombinant strain A and a recombinant strain B: the recombinant strain A contains a recombinant plasmid A, which is obtained by jointly constructing a nucleic acid molecule encoding a mutant of the glycosyltransferase UGTSL2 of claim 1 or 2 and a nucleic acid molecule encoding a sucrose synthase AtSuSy into an expression vector; the recombinant strain B contains a recombinant plasmid B, which is obtained by jointly constructing a nucleic acid molecule encoding a glycosyltransferase NtUGT_M and a nucleic acid molecule encoding a sucrose synthase AtSuSy into a plasmid; the amino acid sequence of the glycosyltransferase NtUGT_M is shown in SEQ ID NO.5.

[0062] The host bacteria include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or Corynebacterium glutamicum.

[0063] The one-pot biocatalytic method for synthesizing rebaudioside A into rebaudioside M2 ​​comprises the following steps:

[0064] Add rebaudioside A, sucrose and the above enzyme composition or the induced expression enzyme product of the complete set of recombinant strains in claim 5 into the catalytic reaction system, react, inactivate the enzyme, and centrifuge to obtain the supernatant, which is rebaudioside M2; M2 is shown in the following formula (I).

[0065]

[0066] Preferably, the method for obtaining the induced expression enzyme product of the above-mentioned recombinant strain comprises the following steps: 1) activating the above-mentioned set of recombinant strains, transferring them to an induction medium, adding an inducer for induction culture, centrifuging and collecting the bacteria, resuspending the bacteria in an appropriate amount of buffer and breaking them, and centrifuging and collecting the supernatant, which is the induced expression enzyme product; the reaction time is 5-100 hours.

[0067] Preferably, the final concentration of the inducer is 0.02-1 g / L, and the induction time is 4-50 h.

[0068] Preferably, the concentration of rebaudioside A in the catalytic reaction system is 1-1000 g / L; the concentration of sucrose is 1-9000 g / L; the amount of induced expression enzyme product added is 1-100 MU / L; the pH of the catalytic reaction system is 7.0-8.5; preferably 7.0; the reaction temperature is 37-40°C; the enzyme activity ratio of the mutant of glycosyltransferase UGTSL2 to NtUGT_M is 3-7:7-3.

[0069] The catalytic reaction system also includes 1-4 mM UDP; preferably, 3 mM UDP.

[0070] The present invention has the following advantages:

[0071] 1. The mutant has high enzyme activity and long half-life.

[0072] 2. The one-pot biocatalytic method for synthesizing rebaudioside M2 ​​from rebaudioside A in the present application can achieve the efficient catalytic synthesis of rebaudioside M2 ​​from rebaudioside A in a short time, and the bioenzyme catalysis method is green, environmentally friendly and pollution-free, and is more suitable for current green industrial processing and production.

[0073] 3. The one-pot biocatalytic method for synthesizing rebaudioside M2 ​​from rebaudioside A realizes the regeneration of UDPG in vivo by constructing a dual enzyme system, effectively solving the problem of the source of expensive glycosyl donors, reducing costs, and promoting the application of biotechnology industry.

[0074] In summary, this application uses consensus strategy, protein molecular dynamics simulation and computer simulation screening technology to establish a site-directed mutation library of glycosyltransferase UGTSL2 based on the structure, and performs protein modification on UGTSL2. By coupling sucrose synthase with glycosyltransferase, the recycling of glycosyl donor substrate UDPG with cheap sucrose is achieved. And a "one-pot three-enzyme" cascade reaction system is established to synthesize rebaudioside M2 ​​from the cheap raw material rebaudioside A. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0076] Figure 1 It is the product yield of the one-pot reaction system temperature optimization of Example 4 of the present invention; RM21G is a self-named by-product.

[0077] Figure 2 It is the product yield of the one-pot reaction system pH optimized in Example 5 of the present invention; RM21G is a self-named by-product.

[0078] Figure 3 It is the product yield of the one-pot reaction system UGTSL2_M and NtUGT in Example 6 of the present invention with optimized enzyme activity ratio; RM21G is a self-named byproduct.

[0079] Figure 4 It is the product yield of the one-pot reaction system of Example 7 of the present invention with the substrate sucrose addition ratio optimized; RM21G is a self-named by-product.

[0080] Figure 5 The present invention is the product yield of Example 8 one-pot reaction system with optimized UDP addition amount; RM21G is a self-named by-product. DETAILED DESCRIPTION

[0081] Example 1 Preparation of recombinant bacteria expressing glycosyltransferase UGTSL mutant and sucrose synthase AtSuSy

[0082] Select the dual gene expression vector pRSFDuet-1 (Han Guangwei. Co-expression and immunogenicity study of α, β_1, β_2, ε toxin proteins of Clostridium perfringens [D]. Chinese Academy of Agricultural Sciences, 2014. This vector was disclosed), insert the nucleotide sequence of the glycosyltransferase UGTSL or mutant encoding gene from Stevia at the NdeI / XhoI site of the vector, and insert the sucrose synthase AtSuSy nucleotide sequence from Arabidopsis at the site NcoI / EcoRI to obtain the recombinant expression plasmid pRSFDuet-1-AtSuSy-UGTSL. The recombinant plasmid was introduced into Escherichia coli BL21 (DE3) for induction and expression to obtain BL21-pRSFDuet-1-AtSuSy-UGTSL. The amino acid sequence of the glycosyltransferase UGTSL is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0083] The above mutants include the following single point mutations: UGTSL2_N23E, UGTSL2_R41P, UGTSL2_H91K, UGTSL2_K95D, UGTSL2_E123P, UGTSL2_L136F, UGTSL2_R151F, UGTSL2_H124E, UGTSL2_V168Y, UGTSL2_C198K, UGTSL2_T202E, UGTSL2_W217K, UGTSL2_P225L, UGTSL2_F226V, UGTSL2_A285V, UGTSL2_I333V, UGTSL2_N358F, UGTSL2_T392V and UGTSL2_I419K.

[0084] N23E is the amino acid at position 23 of SEQ ID NO.1 mutated from N to E; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 69-71 of SEQ ID NO.2 substituted from AAC to GAA;

[0085] R41P is the amino acid at position 41 of SEQ ID NO.1 mutated from R to P; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 121-123 of SEQ ID NO.2 substituted from CGT to CCG;

[0086] H91K is the amino acid at position 91 of SEQ ID NO.1 mutated from H to K; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 271-273 of SEQ ID NO.2 substituted from CAC to AAA;

[0087] K95D is the amino acid at position 95 of SEQ ID NO.1 mutated from K to D; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 283-285 of SEQ ID NO.2 substituted from AAA to GAT;

[0088] E123P is the amino acid at position 123 of SEQ ID NO.1 mutated from E to P; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 367-369 of SEQ ID NO.2 substituted from GAG to CCG;

[0089] L136F is the amino acid at position 136 of SEQ ID NO.1 mutated from L to F; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 406-408 of SEQ ID NO.2 substituted from CTG to TTT;

[0090] R151F is the amino acid at position 151 of SEQ ID NO.1 mutated from R to F; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 451-453 of SEQ ID NO.2 substituted from CGT to TTT;

[0091] H124E is the amino acid at position 124 of SEQ ID NO.1 mutated from H to E; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 370-372 of SEQ ID NO.2 substituted from CAC to GAA;

[0092] V168Y is the amino acid at position 168 of SEQ ID NO.1 mutated from V to Y; the nucleic acid molecule encoding the mutant protein is the position 502-504 of SEQ ID NO.2 substituted from GTG to TAT;

[0093] C198K is the amino acid at position 198 of SEQ ID NO.1 mutated from C to K; the nucleic acid molecule encoding the mutant protein is the 592-594 positions of SEQ ID NO.2 substituted from TGC to AAA;

[0094] T202E is the amino acid at position 202 of SEQ ID NO.1 mutated from T to E; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 604-606 of SEQ ID NO.2 substituted from ACC to GAA;

[0095] W217K ​​is the amino acid at position 217 of SEQ ID NO.1 mutated from W to K; the nucleic acid molecule encoding the mutant protein is the 649-651 positions of SEQ ID NO.2 substituted from TGG to AAA;

[0096] P225L is the amino acid at position 225 of SEQ ID NO.1 mutated from P to L; the nucleic acid molecule encoding the mutant protein is the position 673-675 of SEQ ID NO.2 substituted from CCG to CTG;

[0097] F226V is the amino acid at position 226 of SEQ ID NO.1 mutated from F to V; the nucleic acid molecule encoding the mutant protein is the position 676-678 of SEQ ID NO.2 substituted from TTC to GTT;

[0098] A285V is the amino acid at position 285 of SEQ ID NO.1 mutated from A to V; the nucleic acid molecule encoding the mutant protein is the amino acid at positions 853-855 of SEQ ID NO.2 substituted from GCG to GTT;

[0099] I333V is the amino acid at position 333 of SEQ ID NO.1 mutated from I to V; the nucleic acid molecule encoding the mutant protein is the position 997-999 of SEQ ID NO.2 substituted from ATT to GTT;

[0100] N358F is the amino acid at position 358 of SEQ ID NO.1 mutated from N to F; the nucleic acid molecule encoding the mutant protein is the position 1072-1074 of SEQ ID NO.2 substituted from AAC to TTC;

[0101] T392V is the amino acid at position 392 of SEQ ID NO.1 mutated from T to V; the nucleic acid molecule encoding the mutant protein is the position 1174-1176 of SEQ ID NO.2 substituted from ACC to GTT;

[0102] I419K is the amino acid at position 419 of SEQ ID NO.1 mutated from I to K; the nucleic acid molecule encoding the mutant protein is the position 1255-1257 of SEQ ID NO.2 substituted from ATT to AAA;

[0103] UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K-

[0104] The - / T202E / W217K / P225L / F226V / A285V / I333V / N358F / T392V / I419K protein mutant includes all the above mutations, named UGTSL2_M, and its amino acid sequence is shown in SEQ ID NO.3. The nucleotide sequence of the nucleic acid molecule encoding the mutant protein is shown in SEQ ID NO.4.

[0105] The specific preparation method of the recombinant expression strain of the glycosyltransferase UGTSL mutant comprises the following steps:

[0106] 1. PCR amplification of site-directed mutant encoding genes: Using PCR amplification technology, the plasmid of the non-mutated strain BL21-pRSFDuet-1-AtSuSy-UGTSL was used as a template DNA for rapid mutation (underlined mutant bases)

[0107] The primers for N23E site-directed mutagenesis are:

[0108] Forward primer: 5'-CCGTTTCTG GAA ATTGCGAAACAGCTGGCGGAT-3'(SEQ ID NO.9)

[0109] Reverse primer: 5'-CGCAAT TTC CAGAAACGGGCTGATGTGACCGTA-3'(SEQ ID NO.10)

[0110] The primers for R41P site-directed mutagenesis are:

[0111] Forward primer: 5'-AGCACC CCG ATTAACCTGGAGAGCATCATTAAGAAAATC-3'(SEQ ID NO.11)

[0112] Reverse primer: 5'-GTTAAT CGG GGTGCTGCACAGGTAGATCAGGAA-3'(SEQ ID NO.12)

[0113] The primers for H91K site-directed mutagenesis are:

[0114] Forward primer: 5'-ACCCTG AAA AAGGCGCTGAAAATGAGCAAGCCG-3'(SEQ ID NO.13)

[0115] Reverse primer: 5'-CGCCTT TTT CAGGGTCGGGTTCAGGTGCGGCGG-3'(SEQ ID NO.14)

[0116] The primers for K95D site-directed mutagenesis are:

[0117] Forward primer: 5'-GCGCTG GAT ATGAGCAAGCCGAACTTTAGCCGT-3'(SEQ ID NO.15)

[0118] Reverse primer: 5'-GCTCAT ATC CAGCGCCTTGTGCAGGGTCGGGTT-3'(SEQ ID NO.16)

[0119] The primers for E123P site-directed mutagenesis are:

[0120] Forward primer: 5'-TGGGCG CCG CACGTTGCGAACGAACAAAACATT-3'(SEQ ID NO.17)

[0121] Reverse primer: 5'-AACGTG CGG CGCCCACGGCTGCAGCACGTCGTA-3'(SEQ ID NO.18)

[0122] The primers for L136F site-directed mutagenesis are:

[0123] Forward primer: 5'-GCGGGTAAA TTT CTGACCAGCTGCGCGGCGGTG-3'(SEQ ID NO.19)

[0124] Reverse primer: 5'-GGTCAG AAA TTTACCCGCCGGAATGTTTTGTTC-3'(SEQ ID NO.20)

[0125] The primers for R151F site-directed mutagenesis are:

[0126] Forward primer: 5'-TTT TTT AAGAACCCGGGCGTTGAGTTCCCGTTT-3'(SEQ ID NO.21)

[0127] Reverse primer: 5'-GCCCGGGTTCTT AAA AAAGCTAAAGAAATAGCTGAACACCGC-3'(SEQ IDNO.22)

[0128] The primers for H124E site-directed mutagenesis are:

[0129] Forward primer: 5'-GCGGAG GAA GTTGCGAACGAACAAAACATTCCGGCG-3'(SEQ ID NO.23)

[0130] Reverse primer: 5'-TTCGTTCGCAAC TTC CTCCGCCCACGGCTGCAGCAC-3'(SEQ ID NO.24)

[0131] The primers for V168Y site-directed mutagenesis are:

[0132] Forward primer: 5'-CCGGAA TAT GAAAAGGTTAAAATCCGTGAGATTCTGGCG-3'(SEQ ID NO.25)

[0133] Reverse primer: 5'-CTTTTC ATA TTCCGGCAGGTGGATCGCCGGAAA-3'(SEQ ID NO.26)

[0134] The primers for C198K site-directed mutagenesis are:

[0135] Forward primer: 5'-CTGATG AAA ACCAGCCGTACCCATCGAGGCGAAA-3'(SEQ ID NO.27)

[0136] Reverse primer: 5'-GCTGGT TTT CATCAGCATCATCTGCTTGTTACC-3'(SEQ ID NO.28)

[0137] The primers for T202E site-directed mutagenesis are:

[0138] Forward primer: 5'-AGCCGT GAA ATCGAGGCGAAATACATTGACTATTGC-3'(SEQ ID NO.29)

[0139] Reverse primer: 5'-CTCGAT TTC ACGGCTGGTGCACATCAGCAT-3'(SEQ ID NO.30)

[0140] The primers for W217K ​​site-directed mutagenesis are:

[0141] Forward primer: 5'-CTGTGCAAC AAA AAGGTGGTTCCGGTGGGTCCG-3'(SEQ ID NO.31)

[0142] Reverse primer: 5'-CACCTT TTT GTTGCACAGTTCGGTGCAATAGTC-3'(SEQ ID NO.32)

[0143] The primers for P225L site-directed mutagenesis are:

[0144] Forward primer: 5'-GGTCCG CTG TTCCAAGATCTGATCACCAACGAT-3'(SEQ ID NO.33)

[0145] Reverse primer: 5'-TTGGAA CAG CGGACCCACCGGAACCACCTTCCA-3'(SEQ ID NO.34)

[0146] The primers for F226V site-directed mutagenesis are:

[0147] Forward primer: 5'-CCGCCG GTT CAAGATCTGATCACCAACGATGCG-3'(SEQ ID NO.35)

[0148] Reverse primer: 5'-ATCTTG AAC CGGCGGACCCACCGGAACCAC-3'(SEQ ID NO.36)

[0149] The primers for site-directed mutagenesis of A285V are:

[0150] Forward primer: 5'-TGGGTT GTT CGTTTTCCGAAAGGCGAGGAACGT-3'(SEQ ID NO.37)

[0151] Reverse primer: 5'-AAAACG AAC AACCCAGATGAAGTTCACGTTGCT-3'(SEQ ID NO.38)

[0152] The primers for I333V site-directed mutagenesis are:

[0153] Forward primer: 5'-GGCTTC GTT AGCCACTGCGGTTGGAACAGCGCG-3'(SEQ ID NO.39)

[0154] Reverse primer: 5'-GTGGCT AAC GAAGCCACCGGTGCTCGGGTGGTT-3'(SEQ ID NO.40)

[0155] The primers for N358F site-directed mutagenesis are:

[0156] Forward primer: 5'-CCGATCCAC TTC GACCAACCGATTAACGCGAAACTGATGGT-3'(SEQ IDNO.41)

[0157] Reverse primer: 5'-CGGTTGGTC GAA GTGGATCGGCATCGCAATGATCGGAACGC-3'(SEQ IDNO.42)

[0158] The primers for T392V site-directed mutagenesis are:

[0159] Forward primer: 5'-GCGGAA GTT CTGAAGAGCGTGGTTACCGGC-3' (SEQ ID NO. 43) Reverse primer: 5'-CTTCAG AAC TTCCGCAATCTCGCCACGGTG-3'(SEQ ID NO.44)

[0160] The primers for I419K site-directed mutagenesis are:

[0161] Forward primer: 5'-AAGAGC AAA CGTGATGAGGAAATGGACGCGGTT-3'(SEQ ID NO.45)

[0162] Reverse primer: 5'-CTCATCACG TTT GCTCTTCAGGTTTTTGCTGATTTCACG-3'(SEQ ID NO.46)

[0163] 2. The PCR target plasmid amplification reaction system is: 2 μL each of 10 μM forward primer and reverse primer; 1 μL of dNTP Mix; 25 μL of 2×Max Buffer; 1 μL of template plasmid; 1 μL of 2U / 50 μL Super-Fidelity DNA polymerase, add sterile water ddH2O to make up to 50 μL.

[0164] The reaction conditions for PCR target plasmid amplification were as follows: 95°C pre-denaturation for 30 s; 30 cycles (95°C denaturation for 15 s; 65°C annealing for 15 s; 72°C extension for 7 min); 72°C complete extension for 5 min; and finally, insulation at 16°C. The PCR amplification products were detected by agarose gel nucleic acid electrophoresis.

[0165] 3. Add 1 μL of DpnI endonuclease to the PCR amplification product that has been confirmed to have mutated, then place the reaction system at 37°C for 1-2 hours, transfer to E. coli BL21 (DE3) competent cells, place on ice for 30 minutes, heat shock at 42°C for 45-90 seconds, place on ice for 2 minutes, add 600 μL of LB medium, shake at 37°C at 200rpm for 45 minutes, take all the bacterial solution and evenly spread it on the LB plate containing kanamycin resistance, and culture at 37°C overnight. Pick 2 single colonies on the plate, inoculate them into LB liquid culture medium, and store the bacterial solution in glycerol tubes after 9 hours and sequence. Spread the bacterial solution glycerol tube with the correct sequencing result on the LB plate containing kanamycin resistance, shake the tube to activate the extracted plasmid, and introduce the recombinant plasmid into E. coli BL21 (DE3).

[0166] 4. The above method was used to obtain the following recombinant expression strains of mutants: UGTSL2_N23E, UGTSL2_R41P, UGTSL2_H91K, UGTSL2_K95D, UGTSL2_E123P, UGTSL2_L136F, UGTSL2_R151F, UGTSL2_H124E, UGTSL2_V168Y, UGTSL2_C198K, UGTSL2_T202E, UGTSL2_W217K, UGTSL2_P225L, UGTSL2_F226V, UGTSL2_A285V, UGTSL2_I333V, UGTSL2_N358F, UGTSL2_T392V and UGTSL2_I419K.

[0167] 5. UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K-

[0168] The method for obtaining the recombinant expression strain of - / T202E / W217K / P225L / F226V / A285V / I333V / N358F / T392V / I419K protein mutant is as follows:

[0169] (1) Using the plasmid of the BL21-pRSFDuet-1-AtSuSy-UGTSL strain as the template plasmid for the target plasmid amplification reaction system in step 2 above, and the primers for site-directed mutagenesis of N23E, repeat steps 2 and 3 above to obtain the N23E strain.

[0170] (2) To determine that the mutated N23E plasmid is used as the template for the target plasmid amplification reaction system in step 2 above, the primers are primers for site-directed mutation of R41P, and the above steps 2 and 3 are repeated to obtain the N23E / R41P strain.

[0171] (3) To determine that the mutated N23E / R41P plasmid is the template for the target plasmid amplification reaction system in step 2 above, the primers are primers for site-directed mutagenesis of H91K, and the above steps 2 and 3 are repeated to obtain the N23E / R41P / H91K strain.

[0172] By analogy, the recombinant expression strain of UGTSL2_N23E / R41P / H91K / K95D protein mutant, the recombinant expression strain of N23E / R41P / H91K / K95D / E123P protein mutant, the recombinant expression strain of UGTSL2_N23E / R41P / H91K / K95DN23E / R41P / H91K / K95D / E123P / L136F protein mutant, and the recombinant expression strain of UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F protein mutant recombinant expression strain, UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E protein mutant recombinant expression strain, UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y protein mutant recombinant expression strain,

[0173] Recombinant expression strain of UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K protein mutant,

[0174] Recombinant expression strains of UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E protein mutants, Recombinant expression strains of UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E protein mutants, / W217K / P225L protein mutant recombinant expression strain, UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V protein mutant recombinant expression strain, UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V / A285V protein mutant recombinant expression strain, UGTSL2_ Recombinant expression strains of N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V / A285V / I333V protein mutants, UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V / A285V / I333V protein mutants. The recombinant expression strain of P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V / A285V / I333V / N358F / T392V protein mutant, the recombinant expression strain of UGTSL2_N23E / R41P / H91K / K95D / E123P / L136F / R151F / H124E / V168Y / C198K / T202E / W217K / P225L / F226V / A285V / I333V / N358F / T392V / I419K protein mutant.

[0175] Example 2 Obtaining crude enzyme solution of UGTSL2 and mutants

[0176] The recombinant expression strain containing UGTSL2 and its mutants prepared in Example 1 was spread on LB solid plates (NaCl 10g / L, yeast powder 5g / L, peptone 10g / L, agar 20g / L) containing 50μg / L kanamycin, and placed in a 37°C incubator for constant temperature culture for 12h. The next day, a single colony was selected from the plate and placed in a shaking tube containing 5mL LB liquid culture medium (containing 50μg / L kanamycin), and cultured in a shaking table at 37°C, 200rpm for 12h as seed liquid. The seed liquid was transferred to 100mL TB culture medium (yeast powder 25g / L, peptone 15g / L, NaCl 10g / L, glucose 2g / L, lactose 0.5g / L, containing 50μg / L kanamycin) according to an inoculation amount of 1% (v:v). Placed in a shaking table at 37°C, 200rpm, shaken culture. After 2 h, the temperature was adjusted to 25 °C and the culture was continued for 20-22 h.

[0177] Collect the fermentation liquid and centrifuge it at 4°C, 7000rpm, 6min, discard the supernatant to get the bacterial mud, and rinse it twice with potassium phosphate buffer. Then add an appropriate amount of potassium phosphate buffer, place it in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacteria, with the parameters set to Ф6, 300W, 30min. Then centrifuge it in a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, 30min, and take the supernatant as the crude enzyme solution, which is stored in a 4°C refrigerator for later use.

[0178] The method for determining the activity of glycosyltransferase NtUGT_M is as follows: add 1mM substrate rebaudioside D, 2mM UDPG, 0.5mg crude enzyme to 3mL of enzyme catalytic reaction system, and then supplement with 100mM, pH 7.2 potassium phosphate buffer. The reaction conditions are 30℃, 200rpm; the sampling time is 0min, 20min, and 30min. Sample treatment: take 500μL, inactivate in a water bath at 95℃ for 5min, and wait for HPLC detection. Enzyme activity definition (U): The amount of enzyme required to convert 1μmol of product within 1 minute is 1 enzyme activity unit. Taking the wild enzyme activity as 100%, the relative enzyme activity of other mutant enzymes is obtained.

[0179] The method for determining the activity of glycosyltransferase UGTSL2 is as follows: add 1mM substrate rebaudioside A, 2mM UDPG, 0.5mg crude enzyme to 3mL of enzyme catalytic reaction system, and then supplement with 100mM, pH 7.2 potassium phosphate buffer. The reaction conditions are 30℃, 200rpm; the sampling time is 0min, 20min, and 30min. Sample treatment: take 500μL, inactivate in a water bath at 95℃ for 5min, and wait for HPLC detection. Enzyme activity definition (U): The amount of enzyme required to convert 1μmol of product within 1 minute is 1 enzyme activity unit. Taking the wild enzyme activity as 100%, the relative enzyme activity of other mutant enzymes is obtained.

[0180] The half-life detection method is: incubate the wild enzyme and the mutant enzyme for different time periods in a 37°C water bath, measure the enzyme activity, draw the half-life curve and calculate the half-life time. The enzyme activity detection scheme is the same as above. The enzyme activity detection shows that the multi-point mutant UGTSL2_M not only has an enzyme activity of 690% of the wild enzyme, but also has a half-life extended by 9.4h, and its ability to catalyze rebaudioside D is far superior to that of the wild enzyme. The specific data are shown in Table 1.

[0181] Table 1. Relative enzyme activities and half-lives of UGTSL2 and its mutants

[0182]

[0183] Note: The enzyme activities of other mutants are lower than that of the UGTSL2 strain and are not listed here.

[0184] Example 3 Construction of a three-enzyme coupling system

[0185] The glycosyltransferase UGTSL2_M (nucleotide sequence as shown in SEQ ID NO.4) derived from Stevia was inserted into the NdeI / XhoI site of the pRSFDuet-1 vector obtained in Example 1, and the sucrose synthase AtSuSy nucleotide sequence (SEQ ID NO.8) derived from Arabidopsis was inserted into the site NcoI / EcoRI to obtain the recombinant expression plasmid pRSFDuet-1-AtSuSy-UGTSL. It was introduced into Escherichia coli BL21 (DE3) for induction and expression to obtain BL21 (DE3)-pRSFDuet-1-AtSuSy-UGTSL. The amino acid sequence of the sucrose synthase AtSuSy derived from Arabidopsis is shown in SEQ ID NO.7.

[0186] The double gene expression vector pRSFDuet-1 was selected, and the glycosyltransferase NtUGT_M nucleotide sequence (SEQ ID NO.6) from Stevia was inserted into the NdeI / XhoI site of the vector, and the sucrose synthase AtSuSy nucleotide sequence (SEQ ID NO.8) from Arabidopsis was inserted into the site NcoI / EcoRI to obtain pRSFDuet-1-NtUGT_M-AtSuSy. The recombinant plasmid was introduced into Escherichia coli BL21 (DE3) for induction and expression to obtain BL21 (DE3)-pRSFDuet-1-NtUGT_M-AtSuSy. The amino acid sequence of glycosyltransferase NtUGT_M is shown in SEQ ID NO.5.

[0187] Fermentation induction of three enzymes: BL21(DE3)-pRSFDuet-1-AtSuSy-UGTSL and BL21(DE3)-pRSFDuet-1-NtUGT_M-AtSuSy strains were spread on LB solid plates (Na Cl 10g / L, yeast powder 5g / L, peptone 10g / L, agar 20g / L) containing 50μg / L kanamycin, and placed in a 37°C incubator for constant temperature culture for 12h. The next day, a single colony was selected from the plate and placed in a shaking tube containing 5mL LB liquid culture medium (containing 50μg / L kanamycin), and cultured in a shaking incubator at 37°C, 200rpm for 12h as seed liquid. The seed liquid was transferred to 100mL TB culture medium (containing 50μg / L kanamycin) at a 1% (v:v) inoculation amount. Place in a shaking incubator at 37°C, 200rpm for shaking culture. After 2 h, the temperature was adjusted to 25 °C and the culture was continued for 20-22 h.

[0188] The fermentation broth was collected and centrifuged at 4°C, 7000rpm, 6min, and the supernatant was discarded to obtain bacterial mud, which was rinsed twice with potassium phosphate buffer. An appropriate amount of potassium phosphate buffer was added, placed in an ice-water mixture, and the bacterial cells were ultrasonically disrupted with an ultrasonic disruptor, with the parameters set to Ф6, 300W, and 30min. Centrifuge with a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, and 30min. The supernatant was taken as the crude enzyme solution, which was stored in a refrigerator at 4°C for later use. The crude enzyme solution containing UGTSL2_M and the crude enzyme solution containing NtUGT_M and were obtained, which were the crude enzyme solutions used in the following Examples 4-8.

[0189] Example 4 Optimizing the reaction temperature of the one-pot reaction system

[0190] For UGTSL2_M, NtUGT_M and AtSuSy, although increasing the reaction temperature can obtain higher enzyme activity, it is often accompanied by the inactivation of enzyme proteins. Therefore, selecting a suitable reaction temperature to balance the effectiveness between enzyme activity and protein inactivation is also an important part of optimizing the catalytic reaction system. The reaction system was incubated at 30°C, 33°C, 37°C and 40°C, respectively, and other conditions remained the same, including 50g / L rebaudioside A, 450g / L sucrose, pH 8.0 100mM sodium phosphate buffer, additional addition of 2mM UDP (human uridine diphosphate, Shanghai Yuanye Biological Co., Ltd.) and 1:1 UGTSL2_M (25mU / mL) and NtUGT_M (25mU / mL) enzyme activity ratio. After 48h of reaction, the reaction was terminated by heating, and the reaction solution was analyzed by HPLC. UGTSL2_M and NtUGT_M enzymes are both crude enzyme solutions prepared in Example 3.

[0191] The results are as follows Figure 1 As shown in the figure, the experimental results show that when the reaction is carried out at 30℃ and 33℃, the yield of RM2 (a byproduct of unknown structure) is low, 15.5mM and 24.0mM respectively, and both will accumulate more rebaudioside D and rebaudioside D2. Due to the lower temperature, the catalytic efficiency is reduced, and the two intermediate products of rebaudioside D and rebaudioside D2 (RD2) cannot be converted into the target product rebaudioside M2 ​​in time, and even the substrate rebaudioside A is not completely consumed. When the reaction temperature is increased to 37℃ and 40℃, the substrate rebaudioside A is completely converted, and the synthesized rebaudioside M2 ​​is 30.9mM and 31.1mM respectively. The reaction temperature is set to 40℃ to obtain the highest yield of rebaudioside M2.

[0192] Example 5 Optimizing the reaction pH of the one-pot reaction system

[0193] In the UGT-SuSy coupling reaction system, the catalysis was performed under buffer conditions of pH 7, 7.5, 8, 8.5, 9 and 9.5, respectively. Other conditions remained the same, including 50 g / L rebaudioside A, 450 g / L sucrose, additional 2 mM UDP and 1:1 enzyme activity ratio of UGTSL2_M (25 mU / mL) and NtUGT_M (25 mU / mL), and the reaction was incubated at 35 °C. After 48 h of catalysis, the reaction was terminated by heating and the reaction solution was analyzed by HPLC. The experimental results are shown in Figure 2. Figure 2As shown in the figure, it can be intuitively found that alkaline buffer conditions inhibit the efficiency of UGT-SuSy coupling reaction, especially in the buffer environment of pH 9 and pH 9.5, a large amount of substrate rebaudioside A was not converted, and the yield of rebaudioside M2 ​​was 3.36mM and 0.58mM, respectively. In the slightly alkaline buffer environment (pH 7.5, 8.0, 8.5), the productivity of rebaudioside M2 ​​was still insufficient, with yields of 25.47mM, 21.31mM and 9.36mM, respectively. Considering the buffer requirements of AtSuS y, UGTSL2_M and NtUGT_M, the buffer condition was set to pH 7.0 100mM sodium phosphate buffer to synthesize the highest content of rebaudioside M2, which reached 28.46mM.

[0194] Example 6 Optimization of the enzyme activity ratio of UGTSL2_M and NtUGT_M in the one-pot reaction system

[0195] UGTSL2_M and NtUGT_M are key enzymes for two-step glycosylation, and their addition amount and addition ratio are closely related to the synthesis efficiency of the target and intermediate products. In the UGTSL2_M and NtUGT_M addition amount optimization experiment, the UGTSL2_M and NtUGT_M enzyme activity addition ratios were set to 3:7, 4:6, 5:5, 6:4 and 7:3 (total enzyme activity was 50mU / mL), and other conditions remained the same, including 50g / L rebaudioside A, 450g / L sucrose, and additional 2mM UDP, catalyzing the reaction in pH 8.0 100mM sodium phosphate buffer and 35°C. The reaction was terminated by heating after 48h of catalysis, and the reaction solution was analyzed by HPLC.

[0196] The results are as follows Figure 3 As shown in the figure, when the enzyme activity addition ratio of UGTSL2_M and NtUGT_M is 3:7, 4:6, 5:5, 6:4 and 7:3, 23.72mM, 26.1mM, 23.08mM, 21.96mM and 19.19mM are synthesized in sequence, and the enzyme activity addition ratio of 4:6 is the highest in the adjustment of enzyme amount ratio. Adding UGTSL2_M or NtUGT_M on the basis of the enzyme activity addition ratio of 4:6 will lead to excessive accumulation of intermediates.

[0197] Example 7 Optimization of the substrate sucrose addition ratio in the one-pot reaction system

[0198] In the experiment of optimizing the added sucrose content, 6, 9, 12, 15 and 18 times the amount of sucrose as the acceptor substrate were added to the reaction system, and other conditions remained the same, including 50 g / L of rebaudioside A, additional 2 mM UDP and 1:1 enzyme activity ratio of UGTSL2_M (25 mU / mL) and NtUGT_M (25 mU / mL), and the reaction was catalyzed in pH 8.0 100 mM sodium phosphate buffer and 35 ° C. After catalysis for 48 hours, the reaction was terminated by heating and the reaction solution was analyzed by HPLC. The results are shown in Figure 4 As shown in the figure, the experimental results show that excessive addition of sucrose in the three-enzyme coupling catalytic system will reduce the yield of rebaudioside M2, excessive accumulation of the intermediate product rebaudioside D, and a decrease in the conversion efficiency of rebaudioside A. It is speculated that excessive sucrose may strengthen the pathway of UGTSL2_M to convert rebaudioside A to rebaudioside D, causing excessive precipitation of rebaudioside D and reducing the catalytic efficiency of NtUGT_M to convert rebaudioside D to rebaudioside M2. 6, 9, 12, 15 and 18 times the amount of sucrose synthesized 27.90, 29.69, 24.02, 17.51 ​​and 8.04 mM, respectively, among which the substrate and sucrose addition ratio of 1:9 can catalyze the synthesis of the highest rebaudioside M2 ​​yield.

[0199] Example 8 Optimizing the addition ratio of substrate UDP in the one-pot reaction system

[0200] The UDP contained in the catalytic system comes from the host cell lysate. If the UDP content in the lysate is insufficient, it will limit the regeneration rate of UDPG. Adding a small amount of additional UDP can solve this problem. In the experiment to explore the amount of UDP added, 0, 1, 2, 3 and 4 mM UDP were added to the three-enzyme coupling catalytic reaction system, and other reaction conditions remained consistent, including 50 g / L rebaudioside A (RA), 9 times the sucrose / substrate ratio and 1:1 enzyme activity ratio of UG TSL2_M (25 mU / mL) to NtUGT_M (25 mU / mL). The reaction was catalyzed in pH 8.0 100 mM sodium phosphate buffer and 35°C. The reaction was terminated by heating after 48 hours of catalysis, and the reaction solution was analyzed by HPLC. The results are shown in Figure 5 As shown, the results showed that compared with the blank control group with zero addition of UDP, the yield of RM2 in the coupling reaction with the additional addition of 1-4 mM UDP was significantly increased, generating 19.38, 21.98, 23.98, 24.99 and 24.74 mM, respectively, among which the addition of 3 mM UDP could catalyze the synthesis of the highest yield of rebaudioside M2.

[0201] Example 9 One-pot catalytic synthesis of rebaudioside A into rebaudioside M2

[0202] The three-enzyme coupling reaction system uses 70 g / L rebaudioside A, 420 g / L sucrose, 3 mM UDP, and an enzyme activity ratio of 4:6 between UGTSL2_M (20 mU / mL) and NtUGT_M (30 mU / mL). Under the reaction conditions of pH 7.0 100 mM sodium phosphate buffer and 37°C, the substrate rebaudioside A is completely converted to generate 61.0 mM rebaudioside M2 ​​(78.8 g / L), and the product yield is over 80%.

Claims

1. A glycosyltransferase UGTSL2 mutant, characterized in that: The UGTSL2 mutant is any one of the following (A)-(C): (A) A protein obtained by causing any one or more of the following mutations based on the amino acid sequence shown in SEQ ID NO.1: The amino acid at position 23 mutated from N to E; The amino acid at position 41 mutated from R to P; The amino acid at position 91 mutated from H to K; The amino acid at position 95 mutated from K to D; The amino acid at position 123 mutated from E to P; The amino acid at position 136 mutated from L to F; The amino acid at position 151 mutated from R to F; The amino acid at position 124 mutated from H to E; The amino acid at position 168 mutated from V to Y; The amino acid at position 198 mutated from C to K; The amino acid at position 202 mutated from T to E; The amino acid at position 217 mutated from W to K; The amino acid at position 225 mutated from P to L; The amino acid at position 226 mutated from F to V; The amino acid at position 285 mutated from A to V; The amino acid at position 333 mutated from I to V; The amino acid at position 358 mutated from N to F; The amino acid at position 392 mutated from T to V; The amino acid at position 419 mutated from I to K; (B) a protein having an amino acid sequence with a sequence identity of more than 95% with that of (A) and having the same function; (C) Fusion protein obtained by ligating a tag to the end of the protein defined in (A) or (B).

2. The glycosyltransferase UGTSL2 mutant according to claim 1, characterized in that The amino acid sequence of the UGTSL2 mutant is shown in SEQ ID NO.

3.

3. Any of the following biological materials: (A) an expressed gene encoding the glycosyltransferase mutant according to claim 1 or 2; (B) a recombinant plasmid, the recombinant plasmid being connected to the expression gene described in (A); (C) A recombinant cell comprising the recombinant expression plasmid or the expression gene of the glycosyltransferase mutant.

4. An enzyme composition, characterized in that The enzyme composition comprises: a glycosyltransferase UGTSL2 mutant, a glycosyltransferase NtUGT_M and a sucrose synthase AtSuSy; the glycosyltransferase UGTSL2 mutant is as shown in claim 1 or 2; The glycosyltransferase NtUGT_M is as follows (A1) or (A2): (A1) the amino acid sequence is shown in SEQ ID NO.5; (A2) a protein having an amino acid sequence identity of 95% or more to that of (A1) and having the same function; The sucrose synthase AtSuSy is as follows (B1) or (B2): the amino acid sequence of (B1) is shown in SEQ ID NO.7; (B2) A protein having an amino acid sequence identity of 95% or more to that of (B1) and having the same function.

5. The composition according to claim 4, characterized in that The enzyme activity ratio of glycosyltransferase UGTSL2 mutant and NtUGT_M is 3-7:7-3.

6. A set of recombinant strains expressing the enzyme composition according to claim 4 or 5, characterized in that: It comprises a recombinant strain A and a recombinant strain B: the recombinant strain A contains a recombinant plasmid A, which is obtained by co-constructing a nucleic acid molecule encoding a mutant of the glycosyltransferase UGTSL2 according to claim 1 or 2 and a nucleic acid molecule encoding a sucrose synthase AtSuSy into an expression vector; the recombinant strain B contains a recombinant plasmid B, which is obtained by co-constructing a nucleic acid molecule encoding a glycosyltransferase NtUGT_M and a nucleic acid molecule encoding a sucrose synthase AtSuSy into a plasmid; the amino acid sequence of the glycosyltransferase NtUGT_M is shown in SEQ ID NO.

5.

7. The set of recombinant strains according to claim 6, characterized in that: The host bacteria include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or Corynebacterium glutamicum.

8. A one-pot biocatalytic method for synthesizing rebaudioside M2 ​​from rebaudioside A, characterized in that: The steps include: Add rebaudioside A, sucrose and the enzyme composition of claim 4 or the induced expression enzyme product of the complete set of recombinant strains of claim 5 into the catalytic reaction system, react, inactivate the enzyme, and centrifuge to obtain the supernatant, which is rebaudioside M2; M2 is shown in the following formula (I); 9. The method according to claim 8: The method for obtaining the induced expression enzyme product of the recombinant strain in claim 5 or 6 comprises the following steps: 1) activating the set of recombinant strains according to claim 5 or 6, transferring them to an induction medium, adding an inducer for induction culture, centrifuging and collecting the bacteria, resuspending the bacteria in an appropriate amount of buffer and breaking them, and centrifuging and collecting the supernatant, which is the induced expression enzyme product; the reaction time is 5-100 hours.

10. The method according to claim 9, characterized in that The final concentration of the inducer is 0.02-1 g / L, and the induction time is 4-50 h.

11. The method according to claim 9 or 10, characterized in that: The concentration of rebaudioside A in the catalytic reaction system is 1-1000 g / L; the concentration of sucrose is 1-9000 g / L; the amount of the induced expression enzyme product added is 1-100 MU / L; The pH of the catalytic reaction system is 7.0-8.5, preferably 7.0; the reaction temperature is 37-40°C; the enzyme activity ratio of the mutant of glycosyltransferase UGTSL2 to NtUGT_M is 3-7:7-3.

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