CN112375750A

CN112375750A - Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

Info

Publication number: CN112375750A
Application number: CN202011388873.6A
Authority: CN
Inventors: 李艳; 贾红华; 余杰; 孙萍; 齐雪莲; 严明
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-02-19
Anticipated expiration: 2040-12-02
Also published as: CN112375750B

Abstract

The invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside-A thereof, wherein the amino acid sequence of the primary glycosyltransferase is as follows: 1, the experiment starts from a surface residue Q, N, T of UGT76G1, and combines the analysis results of solvent accessible surface area, B-factor and other data, single-point or multi-point iterative mutation in Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160, Thr319 and Thr 81 is screened out on the basis of the obtained prediction, and finally a better UGT-SuSy system of a mutant strain 76G1_ Q72E-N196D-T319E is screened out, so that high-efficiency catalytic synthesis of rebaudioside A is realized. And (3) connecting the glycosyltransferase UGT76G1 or mutant genes thereof with a sucrose synthase gene to obtain a recombinant plasmid, and constructing a recombinant strain with double-enzyme co-expression. By constructing a double-enzyme system, the regeneration of the UDPG in vivo is realized, the problem of the source of an expensive glycosyl donor is effectively solved, the cost is reduced, and the application of the biotechnology industry is promoted. The mutant is simple to prepare, realizes the catalytic synthesis of rebaudioside A in a short time and high efficiency, is green, environment-friendly and pollution-free in a biological enzyme catalysis method, and is more suitable for the current green industrial processing production.

Description

Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by the glycosyltransferase mutant.

Background

Steviol glycosides are tetracyclic diterpenoids, readily soluble in water and dioxane, soluble in part of the organic reagent. Good heat resistance and is not easy to decompose in the presence of light. The basic skeleton of stevioside is tetracyclic diterpene glycoside, and the variety of stevioside compounds is formed due to the variety of glycosyl groups, the addition positions (C13 and C19) and the number of the glycosyl groups. Mainly comprises steviol, stevioside, rebaudioside A, B, C, D, E, M and the like. Among them, Stevioside (St glycoside) is the most abundant in stevia rebaudiana and Rebaudioside A (RA glycoside) is the second, and accounts for about 3.8% of the dry weight of the leaves.

Rebaudioside a (RA glycoside) is a natural sweetener derived from stevia rebaudiana, has high sweetness and low calorie, and can well meet the sweet taste requirement of modern people on healthy diet. At present, RA glycoside is mainly obtained by extraction method, and can also be obtained by catalyzing Stevioside (St glycoside) by glycosyl transferase UGT76G1 from stevia rebaudiana through enzymatic conversion. Stevia-derived glycosyltransferase UGT76G1 (UDP-glycosyltransferase 76G 1) is a "Leloir" type glycosyltransferase, belongs to the GT1 family, and is usually synthesized into glycoside compounds by using UDPG as a glycosyl donor. UGT76G1 was first reported by HUMPHREY T V and RICHMAN A et al. Glycosyltransferase UGT76G1 is extracted from stevia rebaudiana Bertoni originally, and then can be obtained in large quantities by heterologous expression of escherichia coli or saccharomyces cerevisiae and the like, and is used for catalyzing St glycoside to synthesize RA glycoside in one step. The higher cost of the pre-glycosyl donor UDPG material limits the large-scale application of UGT76G 1. With the continuous improvement of the production method of UDPG, it can be synthesized in large quantities by enzyme method such as SuSy. Researchers use a SuSy-UGT cascade reaction system on the basis, and provide glycosyl donors for UGT76G1 by using SuSy, so that cyclic utilization of UDPG is realized, and application cost is greatly reduced. In the UGT76G1-SuSy cascade system, SuSy provides the necessary glycosyl donor for glycosylation reaction, and when UDPG regeneration speed is not limited (for example, by increasing sucrose concentration), the key to high and low RA glycoside synthesis efficiency is UGT76G 1. The soluble expression quantity of stevia-derived UGT76G1 in escherichia coli is very low, and the high-efficiency application of glycosyltransferase is limited.

Disclosure of Invention

The invention aims to provide a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by using the glycosyltransferase mutant; the mutant gene is utilized to realize glycosylation modification of stevioside, rebaudioside A is prepared by high-efficiency catalysis, and a certain reference is provided for industrial production.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

a glycosyltransferase mutant suitable for rebaudioside A synthesis, the glycosyltransferase amino acid sequence is shown in SEQ ID NO. 1, and the glycosyltransferase mutant is obtained by single-point or multi-point iterative mutation of Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160, Thr319 and Thr 81 through at least one of the following mutations:

N196D is the mutation of amino acid 196 in the sequence of SEQ ID NO. 1 from aspartyl (N) to aspartic acid (D);

N78D is the mutation of 78 th amino acid from aspartyl (N) to aspartic acid (D) in the sequence of SEQ ID NO. 1;

N400D is SEQ ID NO. 1, wherein the 400 th amino acid of the sequence is mutated from aspartyl (N) to aspartic acid (D);

N69D is SEQ ID NO. 1, wherein the 69 th amino acid is mutated from aspartyl (N) to aspartic acid (D);

Q72E is the 72 th amino acid in the SEQ ID NO. 1 sequence, and glutamine (Q) is mutated into glutamic acid (E);

Q198E is the mutation of amino acid 198 in the sequence of SEQ ID NO. 1 from glutamine (Q) to glutamic acid (E);

Q178E is the amino acid 478 of the SEQ ID NO:1 sequence, and glutamine (Q) is mutated into glutamic acid (E);

Q160E is the amino acid at position 160 in the sequence of SEQ ID NO. 1, which is mutated from glutamine (Q) to glutamic acid (E);

T319E is SEQ ID NO. 1 in which amino acid 319 is mutated from threonine (T) to glutamic acid (E);

T81E is SEQ ID NO. 1, wherein the 81 st amino acid is mutated from threonine (T) to glutamic acid (E);

Q72E-N196D is the amino acid 72 of the SEQ ID NO. 1 sequence mutated from glutamine (Q) to glutamic acid (E) and from aspartyl (N) to aspartic acid (D);

N196D-T319E is SEQ ID NO 1 sequence with aspartyl (N) at amino acid position 196 mutated to aspartic acid (D) and threonine (T) at amino acid position 319 mutated to glutamic acid (E);

Q72E-N196D-T319E is the mutation of amino acid 72 from glutamine (Q) to glutamic acid (E) and amino acid 196 from aspartyl (N) to aspartic acid (D) and amino acid 319 from threonine (T) to glutamic acid (E) in the sequence of SEQ ID NO: 1.

Single-point mutants Q72E, N196D, T319E and multi-point mutants Q72E-N196D, N196D-T319E, Q72E-N196D-T319E of glycosyltransferase UGT76G1 (Unit prot ID: Q6VAB 4; Access: AY 345974).

An expressed gene encoding any of the glycosyltransferase mutants described above.

The nucleotide sequence of the 76G1_ Q72E mutant is shown as SEQ ID NO. 5, the nucleotide sequence of the 76G1_ N196D mutant is shown as SEQ ID NO. 6, the nucleotide sequence of the 76G1_ T319E mutant is shown as SEQ ID NO. 7, the nucleotide sequence of the 76G1_ Q72E-N196D mutant is shown as SEQ ID NO. 8, the nucleotide sequence of the 76G1_ N196D-T319E mutant is shown as SEQ ID NO. 9, and the nucleotide sequence of the 76G1_ Q72E-N196D-T319E mutant is shown as SEQ ID NO. 10.

A recombinant plasmid to which the above-described expression gene and sucrose synthase gene SUS are ligated.

A recombinant cell comprising an expression gene of the recombinant expression vector or the glycosyltransferase mutant.

The glycosyltransferase mutant is applied to preparing rebaudioside A.

A method for catalytically synthesizing rebaudioside a comprising the steps of:

1) constructing a recombinant strain containing a double-enzyme co-expression system: coexpression of the nucleotide sequence of the glycosyltransferase mutant and the nucleotide sequence of sucrose synthase to obtain recombinant plasmid, and transformation of the recombinant plasmid into competent cells of escherichia coli BL21(DE3) to obtain a recombinant strain containing a double-enzyme coexpression system;

2) induction of recombinant strains to produce enzymes: activating the recombinant strain, transferring the activated recombinant strain into a TB induction culture medium according to the proportion of 1-3%, culturing for about 2 hours at 37 ℃, reducing the temperature to 25 ℃ with the addition of lactose of 0.02g/L, continuing induction culture for 20 hours at 200rpm, centrifuging at low temperature and collecting thalli. The thalli is resuspended in a proper amount of buffer solution and crushed, and the supernatant fluid is collected by centrifugation to obtain crude enzyme solution;

3) catalytic synthesis of rebaudioside a: adding stevioside, cane sugar and crude enzyme liquid into a catalytic reaction system, reacting for 30 hours, inactivating the enzyme at high temperature, and centrifuging to obtain supernatant, namely rebaudioside-A.

The amino acid sequence of glycosyltransferase UGT76G1 is shown as SEQ ID NO. 1, the nucleotide sequence is shown as SEQ ID NO. 2, the amino acid sequence of sucrose synthase is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4. 76G1_ Q72E is shown as SEQ ID NO. 5, 76G1_ N196D is shown as SEQ ID NO. 6, 76G1_ T319E is shown as SEQ ID NO. 7, 76G1_ Q72E-N196D is shown as SEQ ID NO. 8, 76G1_ N196D-T319E is shown as SEQ ID NO. 9, and 76G1_ Q72E-N196D-T319E is shown as SEQ ID NO. 10.

The solubility of the protein is related to the hydrophilicity of surface residues, and the related amino acid residues on the surface of the protein are changed by utilizing a site-directed mutagenesis technology to improve the solubility of the protein. The contribution of the aspartic acid (D), the glutamic acid (E) and the serine (S) to the protein solubility is obviously higher than that of other hydrophilic amino acids, particularly under the condition of high net charge, the asparagine (N), the glutamine (Q) or the threonine (T) in the amino acid residues on the surface of the enzyme protein is replaced by D, E or S, the protein solubility expression level can be improved, and the high-efficiency catalysis of the enzyme can be promoted. According to the invention, based on glycosyltransferase UGT76G1, key amino acid site-directed mutagenesis is carried out on the surface residue of the glycosyltransferase UGT76G1, and the optimal glycosyltransferase mutant 76G1_ Q72E-N196D-T319E is obtained by comparing the relative activity and conversion rate of the mutant strain. The glycosyltransferase mutant is cascaded with sucrose synthase, stevioside is used as a substrate, a proper amount of sucrose is added, and rebaudioside A is catalytically synthesized. The mutant is simple to prepare, the high-efficiency catalytic synthesis of rebaudioside-A is realized, the activity of the mutant is improved by about 2.8 times compared with that of the original enzyme under the same condition, and the yield of rebaudioside-A is remarkably improved compared with that of the original enzyme. The catalytic effect of the reaction system based on the mutant strain 76G1_ Q72E-N196D-T319E is better than that of the non-mutant system, the concentration of RA glycoside is 20G/L when the reaction is carried out for 7 hours, and the catalytic rate is high.

According to the invention, the soluble expression of glycosyltransferase UGT76G1 and the enhancement of the hydrophobic acting force between the mutation site and the substrate are improved through mutation, the substrate conformation is drawn towards the glycosyl donor direction, the enzyme activity can be improved, the combination with the substrate stevioside is more suitable, and the rebaudioside A catalytic production efficiency is further improved.

Has the advantages that:

the invention improves the enzyme activity and soluble expression of glycosyltransferase UGT76G1 through site-directed mutagenesis, realizes high-efficiency catalytic synthesis of rebaudioside A by utilizing the mutant, and has the advantages of mild synthesis method conditions, simple operation, short time, high catalytic efficiency, high yield and better application prospect.

Drawings

FIG. 1UGT76G1 mutation site; the three stick structures are mutation points Asn196, Thr319 and Gln72 respectively.

Detailed Description

The technical solution of the present invention is further described below with reference to examples, but the scope of the present invention is not limited thereto.

The detection methods used in the following examples are as follows:

HPLC determination method:

the mobile phase was checked to be 80% acetonitrile and adjusted to pH 4.0 with glacial acetic acid. Filtering with a 0.45 mu m organic filter membrane, and removing bubbles by ultrasonic. The chromatographic column is BF-NH2, 5 μm 120A (Wuxi Jia Laike chromatography science and technology Co., Ltd.), the temperature of the chromatographic column is 40 ℃, the detection wavelength is 210 nm, and the detection time is 20 min. Filtration is required before all samples are detected (0.45 mu m organic filter membrane)

Methods for confirming mutants used in the following examples:

in the research, glycosyltransferase UGT76G1 is used for mutation, and the mutation of surface residue N, Q, T of the enzyme protein can improve the hydrophobicity and surface configuration between the enzyme structure and a substrate, improve the enzyme activity, and enhance the specific combination with the substrate, thereby greatly improving the catalytic effect. The replacement of N, Q, T in amino acid residues on the surface of the protein with D, E or S can effectively increase the soluble expression of the protein, and can make the substrate stevioside close to the sugar donor direction and increase the hydrophobic effect between the mutation site and the substrate. And the protein soluble expression is closely related to the relative enzyme activity of the protein. Therefore, the experiment starts from the surface residue Q, N, T of UGT76G1, the configuration of the binding reaction substrate is combined with the analysis results of the solvent accessible surface area, B-factor and other data (Table I), mutation sites of Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160, Thr319, Thr 81 and the like are further screened out on the basis of the obtained prediction, and then are mutated into Asp, Glu or Glu respectively, the relative activity of the mutated enzyme and the concentration of the rebaudioside A generated by conversion are compared, and better mutation sites are further screened out for iterative mutation, so that the biological method for efficiently and catalytically synthesizing rebaudioside A is realized.

EXAMPLE 1 preparation of glycosyltransferase mutants

PCR amplification of site-directed mutant coding gene: rapid mutation was carried out using PCR amplification technique with non-mutated strain pRSFDUet-UGT 76G1-AtSUS1 as template DNA:

the primers for site-directed mutagenesis of Q72E were:

a forward primer: 5' -AACGACCCGGAAGATGAACGCATCTCTAATCTGCCG-3' (the mutated base is underlined) is shown in SEQ ID NO:11

Reverse primer: 5' -GCGTTCATCTTCCGGGTCGTTATCCAGAATAAAACG-3' (the mutated base is underlined) is shown in SEQ ID NO:12

Primers for site-directed mutagenesis of N196D were:

a forward primer: 5' -GCGTACTCGGACTGGCAGATTCTGAAAGAAATCCTG-3' (the mutated base is underlined) is shown in SEQ ID NO:13

Reverse primer: 5' -AATCTGCCAGTCCGAGTACGCTGACTTAATATCCTT-3' (the mutated base is underlined) is shown in SEQ ID NO:14

Primers for site-directed mutagenesis of T319E were:

a forward primer: 5' -AAGGGCTCTGAATGGGTTGAACCGCTGCCGGACGGC-3' (the mutated base is underlined) is shown in SEQ ID NO:15

Reverse primer: 5' -TTCAACCCATTCAGAGCCCTTCACAAAACCCGGGCG-3' (the mutated base is underlined) is shown in SEQ ID NO:16

The PCR target plasmid amplification reaction system is as follows: 2. mu.L each of 10. mu.M forward and reverse primers; 1 μ L of dNTPmix; 2 × MaxBuffer 25 μ L; 1 μ L of template plasmid; 2U/50. mu.L Super-FidelityDNA polymerase 1. mu.L, sterile water dd H₂O make up to 50. mu.L.

PCR target plasmid amplification reaction conditions: pre-denaturation at 95 ℃ for 30 s; 30 cycles (95 ℃ denaturation 15 s; 65 ℃ annealing 15 s; 72 ℃ extension 7 min); completely extending for 5min at 72 ℃; finally, keeping the temperature at 16 ℃. And carrying out agarose gel nucleic acid electrophoresis detection on the PCR amplification product.

Adding DpnI1 mu L into a PCR amplification product which is determined to have mutation, then placing a reaction system at 37 ℃ for constant temperature reaction for 1-2 h, transferring the reaction system into escherichia coli BL21(DE3) competent cells, placing the cells on ice for 30min, performing heat shock at 42 ℃ for 45-90 s, placing the cells on ice for 2min, adding 600 mu L of LB culture medium (formula: NaCl 10 g/L, yeast powder 5g/L, peptone 10 g/L), shaking the bed at 37 ℃ for 200rpm for 45min, taking all bacterial liquid, uniformly spreading the bacterial liquid on an LB plate containing kanamycin resistance, and performing overnight culture at 37 ℃.2 single colonies on the plate are picked, inoculated with LB liquid culture medium (formula: NaCl 10 g/L, yeast powder 5g/L, peptone 10 g/L), after 9h, a bacterial liquid glycerol tube is preserved and sequenced, the bacterial liquid glycerol tube with the correct sequencing result is coated on an LB plate (formula: NaCl 10 g/L, yeast powder 5g/L, peptone 10 g/L, agar 20 g/L) containing kanamycin resistance, the tube is shaken to activate and extract plasmids, and the recombinant plasmids are introduced into escherichia coli BL21(DE 3).

(1) Single point mutation

The mutant was obtained by introducing the recombinant plasmid into E.coli BL21(DE3) according to the site-directed mutagenesis method described above. The nucleotide sequence of the mutant 76G1_ Q72E is shown as SEQ ID NO. 5; the mutant 76G1_ N196D has the nucleotide sequence shown in SEQ ID NO 6; the mutant 76G1_ T319E has the nucleotide sequence shown in SEQ ID NO. 7.

Wherein, the amino acid sequence of glycosyltransferase UGT76G1 is shown in SEQ ID NO. 1, and the mutated amino acid sites are respectively selected from one of Gln72, Asn196 and Thr 319. The mutant Q72E is the amino acid 72 in the sequence of SEQ ID NO. 1, which is mutated from glutamine (Q) to glutamic acid (E); the mutant N196D is the mutant of the 196 th amino acid of SEQ ID NO. 1 sequence which is mutated from aspartyl (N) to aspartic acid (D); the mutant T319E is the amino acid 319 of the SEQ ID NO. 1 sequence mutated from threonine (T) to glutamic acid (E). The experimental results show that (Table 2), the relative enzyme activities of the 3 single-point mutants Q72E, N196D and T319E are all higher than that of UGT76G 1. The enzyme activity of the single-point mutant N196D is obviously higher than that of the mutants Q72E and T319E, and the single-point mutant N196D is about 1.5 times of the relative enzyme activity of an unmutated strain due to the increase of hydrophobic interaction before and after mutation.

(2) Multiple point mutation

On the basis of single-point mutation, overlapping Q72E and T319E on an effective mutation site N196D respectively to carry out double-point mutation and 3-point overlapping mutation to form a mutant 76G1_ Q72E-N196D which is shown as SEQ ID NO: 8; the mutant 76G1_ N196D-T319E is shown as SEQ ID NO 9; the mutant 76G1_ Q72E-N196D-T319E is shown as SEQ ID NO: 10.

Wherein, the amino acid sequence of glycosyltransferase UGT76G1 is shown in SEQ ID NO. 1, and the mutated amino acid sites are respectively selected from two or three of Gln72, Asn196 and Thr 319. The double-point mutant Q72E-N196D is SEQ ID NO:1, wherein the 72 th amino acid is mutated from glutamine (Q) to glutamic acid (E), and the 196 th amino acid is mutated from aspartyl (N) to aspartic acid (D); the double-point mutant N196D-T319E is SEQ ID NO. 1, wherein the 196 th amino acid is mutated from aspartyl (N) to aspartic acid (D), and the 319 th amino acid is mutated from threonine (T) to glutamic acid (E); the three-point mutant Q72E-N196D-T319E is characterized in that in the sequence of SEQ ID NO 1, the 72 th amino acid is mutated into glutamic acid (E) from glutamine (Q), the 196 th amino acid is mutated into aspartic acid (D) from aspartyl (N), and the 319 th amino acid is mutated into glutamic acid (E) from threonine (T); the results of the experiments (Table 3) show that all the multi-point mutants have higher relative enzyme activities than the non-mutant strains. In the double-point mutation, the relative enzyme activity of the double-point mutant Q72E-N196D is obviously higher than that of the double-point mutant N196D-T319E. The effect of the three-point combination mutant Q72E-N196D-T319E is superior to the relative enzyme activity of other two-point mutants N196D-T319E and Q72E-N196D, and the relative enzyme activity of the three-point combination mutant Q72E-N196D-T319E is about 2.8 times that of an unmutated strain. In conclusion, the relative enzyme activity of the multi-point mutant Q72E-N196D-T319E is highest. The recombinant strain 76G1_ Q72E-N196D-T319E containing three-point combined mutation shows increased hydrophobic interaction between a mutated site and a substrate in a cascade reaction, when the substrate concentration is increased to 30G/L and the crude enzyme concentration is 5mg/mL, the capacity of catalyzing and synthesizing RA glycoside by the mutant strain 76G1_ Q72E-N196D-T319E is obviously superior to that of an unmutated strain (Table 4) within 0-24 h, the yield of rebaudioside A is catalytically produced after 24h of reaction, and the yield of the mutant strain 76G1_ Q72E-N196D-T319E is 2.1 times that of the unmutated strain, so that the mutant strain 76G1_ Q72E-N196D-T319E can realize a high-efficiency catalytic synthesis method within a short time.

EXAMPLE 2 construction of the two-enzyme expression System

Selection of the Dual Gene expression vector pRSFDuet-1, on whichNdeI / XhoThe nucleotide sequence of glycosyltransferase UGT76G1 (Access: AY 345974) from stevia rebaudiana Bertoni or mutant nucleotide sequence thereof is inserted into the I siteNcoI/ EcoRI was inserted into the Arabidopsis-derived sucrose synthase AtSUS3 nucleotide sequence (Access: AY 051001) to construct a corresponding recombinant expression plasmid, and constructed into pRSFDuet-1 vector. The recombinant plasmid is introduced into Escherichia coli BL21(DE3) to form a double-enzyme co-expression recombinant strain.

Example 3 fermentation Induction of mutant enzymes

Recombinant bacteria constructed by single-point mutants Q72E, N196D and T319E of glycosyltransferase UGT76G1 (UniProt ID: Q6VAB 4) and multi-point mutants Q72E-N196D, N196D-T319E and Q72E-N196D-T319E are respectively coated on LB solid plates (NaCl 10G/L, yeast powder 5G/L, peptone 10G/L and agar 20G/L) containing 50 mu G/L kanamycin and are placed in a 37 ℃ incubator for overnight constant-temperature culture. The next day, single colonies were picked from the plates into shake tubes containing 5 mL of LB liquid medium (containing 50. mu.g/L kanamycin) and cultured overnight as seed liquid in a shaker at 37 ℃ and 200 rpm. The seed solution was inoculated into 100 mL TB medium (containing 50. mu.g/L kanamycin) at 1% (v: v). The mixture was placed in a shaker at 37 ℃ and 200rpm and cultured with shaking. After 2h, the temperature was adjusted to 25 ℃ and the culture was continued for 20-22 h.

The fermentation broth was collected and subjected to refrigerated centrifugation (4 ℃, 7000 rpm, 6 min), and the supernatant was discarded to obtain a sludge, which was washed twice with potassium phosphate buffer. Adding a proper amount of potassium phosphate buffer solution, placing in an ice water mixture, and ultrasonically crushing thalli by using an ultrasonic crusher, wherein the parameters are phi 6, 300W and 30 min. Centrifuging with refrigerated centrifuge at 4 deg.C and 8000rpm for 30min, collecting supernatant as crude enzyme solution, and storing in 4 deg.C refrigerator.

Example 4 method for determining the yield of a glycosyltransferase mutant coupled to a sucrose synthase

The method for measuring the enzyme activity of the glycosyltransferase comprises the following steps: enzyme catalyzed reaction at 3 mLAdding 1 mM substrate stevioside, 2 mM UDPG and 3 mM MgCl into the reaction system₂0.5 mg of crude enzyme, supplemented with 100 mM, pH 7.2 potassium phosphate buffer. The reaction conditions were 30 ℃ and the sampling time at 200rpm was 0min, 20 min, 30 min. Sample treatment: taking 500 mu L, boiling for 5min, centrifuging, taking the supernatant and n-butanol, extracting at a ratio of 1:1, filtering with an organic filter membrane (0.45 mu m), and placing in a sample bottle for HPLC detection. Definition of enzyme activity (U): the enzyme amount required for generating 1 mu mol of product by conversion within 1 minute is 1 enzyme activity unit.

The method for measuring the enzyme activity of the sucrose synthase comprises the following steps: 500 mM of sucrose as a substrate, 10 mM of UDP xNa and 3 mM of MgCl were added to 3 mL of the enzyme-catalyzed reaction system₂6 mg of crude enzyme, supplemented with 100 mM, pH 7.2 potassium phosphate buffer. The reaction conditions were 30 ℃, 200rpm, and the sampling time was 0min, 30min, 60 min. Sample treatment: boiling the sample for 5min, centrifuging to obtain 500 muL, adding 500 muL water, adding 2 mL DNS, boiling for 2min, cooling, adding 7 mL distilled water, and measuring A₅₄₀Fructose content was calculated against DNS standard. Definition of enzyme activity (U): the enzyme amount required for generating 1 mu mol of product by conversion within 1 minute is 1 enzyme activity unit.

Catalytic reaction system (20 mL): substrate St glycoside 20 g/L, sucrose 60 g/L, crude enzyme 5mg/mL, supplemented with 100 mM, pH 7.2 potassium phosphate buffer. The catalytic reaction conditions were 30 ℃ and 200 rpm. Sampling according to a certain time, processing the sample, and detecting and analyzing by using High Performance Liquid Chromatography (HPLC). Sample treatment: diluting the sample to 20 times, boiling for 5min, centrifuging to obtain 500 muL, and adding n-butyl alcohol 1:1, extracting, filtering with an organic filter membrane (0.45 mu m), putting into a sample bottle, and waiting for HPLC detection and analysis.

EXAMPLE 5 comparison of the non-mutated strains with the optimal mutant for rebaudioside A Synthesis

And catalyzing a substrate stevioside to synthesize RA glycoside by using the unmutated strain and the optimal mutated strain 76G1_ Q72E-N196D-T319E under the same catalytic reaction condition, sampling within a certain time, and carrying out sample treatment and HPLC detection and analysis. Substrate St glycoside: the sucrose is 1: 3, the amount of crude enzyme added was 5mg/mL, supplemented with 100 mM, pH 7.2 potassium phosphate buffer, and the catalytic reaction conditions were 30 ℃ and 200 rpm. In the catalytic reaction in Table four, the addition amount of the substrate was 30g/L of St glycoside and 90 g/L of sucrose.

Table 1:

TABLE 1 analysis of parameters of the mutation sites

Amino acids	Site of the body	Two-stage structure	Solvent accessible surface area (ASA, A)²)	Pocket	Channel	Average B-factor (A)²)	Hot spot
								Asn(N)	196	extend strand(E)	80.66	1	1.2.4	62.99	ok
Asn(N)	78	3-10 helix(G)	154.36	3	-	69.73	ok
								Asn(N)	400	bend(S)	107.42	-	-	49.41	ok
Asn(N)	69	extend strand(E)	55.01	-	-	35.83	ok
								Gln(Q)	72	extend strand(E)	87.2	3	-	61.62	ok
Gln(Q)	198	alpha helix(H)	163.53	-	4	70.95	ok
								Gln(Q)	178	extend strand(E)	121.26	-	-	53.62	ok
Gln(Q)	160	alpha helix(H)	92.31	-	-	53.56	ok
								Thr(T）	319	extend strand(E)	109.25	24	-	52.57	ok
Thr(T）	81	extend strand(E)	92.43	3.4	3.5	47.7	ok

TABLE 2 relative enzyme Activity of Single Point mutations

Bacterial strains	Relative enzyme activity (%)
		Not mutated	100±9.0
76G1_Q72E	113.4±8.3
		76G1_T319E	114.5±13.3
76G1_N196D	147.2±3.8

TABLE 3 relative enzyme Activity of multiple mutations

Bacterial strains	Relative enzyme activity (%)
		Not mutated	100±6.1
76G1_N196D-T319E	169.5±31.2
		76G1_Q72E-N196D	264.4±61.4
76G1_Q72E-N196D-T319E	276.4±40.3

TABLE 4 comparison of the concentrations of RA glycosides catalytically synthesized by the mutant and the control group (stevioside substrate 30g/L, crude enzyme amount 5 mg/L)

Time (h)	Control	76G1_Q72E-N196D-T319E
			0	0.4	0.8
7	10.9	20.5
			17	16.6	28.4
24	21.9	45.3

In the reaction of 24h, compared with the non-mutant strain, the mutant strain has the advantages that the catalytic synthesis rate of RA glycoside is obviously high, the final rebaudioside A concentration reaches 45g/L, and the rebaudioside A is efficiently catalytically synthesized.

Sequence listing

<110> Nanjing university of industry

<120> glycosyltransferase mutant and method for catalytically synthesizing rebaudioside-A by using same

<141> 2020-12-02

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 458

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 1

Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu

20 25 30

Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr

35 40 45

Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg

50 55 60

Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro

65 70 75 80

Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His

85 90 95

Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser

100 105 110

Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr

115 120 125

Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu

130 135 140

Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln

145 150 155 160

Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu

165 170 175

Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser

180 185 190

Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile

195 200 205

Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu

210 215 220

Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu

450 455

<210> 2

<211> 1616

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 2

cttgcgtgta aacgtcagtc aaacccaatg gaaaataaaa cggagaccac cgttcgccgg 60

cgccggagaa taatattatt cccggtacca tttcaaggcc acattaaccc aattcttcag 120

ctagccaatg tgttgtactc taaaggattc agtatcacca tctttcacac caacttcaac 180

aaacccaaaa catctaatta ccctcacttc actttcagat tcatcctcga caacgaccca 240

caagacgaac gcatttccaa tctaccgact catggtccgc tcgctggtat gcggattccg 300

attatcaacg aacacggagc tgacgaatta cgacgcgaac tggaactgtt gatgttagct 360

tctgaagaag atgaagaggt atcgtgttta atcacggatg ctctttggta cttcgcgcaa 420

tctgttgctg acagtcttaa cctccgacgg cttgttttga tgacaagcag cttgtttaat 480

tttcatgcac atgtttcact tcctcagttt gatgagcttg gttacctcga tcctgatgac 540

aaaacccgtt tggaagaaca agcgagtggg tttcctatgc taaaagtgaa agacatcaag 600

tctgcgtatt cgaactggca aatactcaaa gagatattag ggaagatgat aaaacaaaca 660

aaagcatctt caggagtcat ctggaactca tttaaggaac tcgaagagtc tgagctcgaa 720

actgttatcc gtgagatccc ggctccaagt ttcttgatac cactccccaa gcatttgaca 780

gcctcttcca gcagcttact agaccacgat cgaaccgttt ttcaatggtt agaccaacaa 840

ccgccaagtt cggtactgta tgttagtttt ggtagtacta gtgaagtgga tgagaaagat 900

ttcttggaaa tagctcgtgg gttggttgat agcaagcagt cgtttttatg ggtggttcga 960

cctgggtttg tcaagggttc gacgtgggtc gaaccgttgc cagatgggtt cttgggtgaa 1020

agaggacgta ttgtgaaatg ggttccacag caagaagtgc tagctcatgg agcaataggc 1080

gcattctgga ctcatagcgg atggaactct acgttggaaa gcgtttgtga aggtgttcct 1140

atgattttct cggattttgg gctcgatcaa ccgttgaatg ctagatacat gagtgatgtt 1200

ttgaaggtag gggtgtattt ggaaaatggg tgggaaagag gagagatagc aaatgcaata 1260

agaagagtta tggtggatga agaaggagaa tacattagac agaatgcaag agttttgaaa 1320

caaaaggcag atgtttcttt gatgaagggt ggttcgtctt acgaatcatt agagtctcta 1380

gtttcttaca tttcatcgtt gtaaataaca cgatgattaa tcaagcactt ggattgcatg 1440

ctagctgagt agctggtaat ttgagttatt agaagcaaag actacttggt ttaaattaaa 1500

taaaggatgg ttgttggtta tgtgagctag tttatgttat gttttgtagg ctataaaagc 1560

cttcatatgt ttcttattgt ttctgtttct aaggtgaaaa aaatgctcgt ttttat 1616

<210> 3

<211> 809

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 3

Met Ala Asn Pro Lys Leu Thr Arg Val Leu Ser Thr Arg Asp Arg Val

1 5 10 15

Gln Asp Thr Leu Ser Ala His Arg Asn Glu Leu Val Ala Leu Leu Ser

20 25 30

Arg Tyr Val Asp Gln Gly Lys Gly Ile Leu Gln Pro His Asn Leu Ile

35 40 45

Asp Glu Leu Glu Ser Val Ile Gly Asp Asp Glu Thr Lys Lys Ser Leu

50 55 60

Ser Asp Gly Pro Phe Gly Glu Ile Leu Lys Ser Ala Met Glu Ala Ile

65 70 75 80

Val Val Pro Pro Phe Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Val Arg Val Asn Val Phe Glu Leu Ser Val Glu Gln Leu

100 105 110

Thr Val Ser Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Pro

115 120 125

Asn Ser Asp Pro Phe Cys Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Asn Val Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn Gly Val Gln

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ser Val Met Phe Arg Asn Lys Asp Cys

165 170 175

Leu Glu Pro Leu Leu Asp Phe Leu Arg Val His Lys Tyr Lys Gly His

180 185 190

Pro Leu Met Leu Asn Asp Arg Ile Gln Ser Ile Ser Arg Leu Gln Ile

195 200 205

Gln Leu Ser Lys Ala Glu Asp His Ile Ser Lys Leu Ser Gln Glu Thr

210 215 220

Pro Phe Ser Glu Phe Glu Tyr Ala Leu Gln Gly Met Gly Phe Glu Lys

225 230 235 240

Gly Trp Gly Asp Thr Ala Gly Arg Val Leu Glu Met Met His Leu Leu

245 250 255

Ser Asp Ile Leu Gln Ala Pro Asp Pro Ser Ser Leu Glu Lys Phe Leu

260 265 270

Gly Met Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Thr Glu Met Leu

305 310 315 320

Leu Arg Ile Lys Arg Gln Gly Leu Asp Ile Ser Pro Ser Ile Leu Ile

325 330 335

Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Thr Cys Asn Gln Arg

340 345 350

Leu Glu Arg Val Ser Gly Thr Glu His Thr His Ile Leu Arg Val Pro

355 360 365

Phe Arg Ser Glu Lys Gly Ile Leu Arg Lys Trp Ile Ser Arg Phe Asp

370 375 380

Val Trp Pro Tyr Leu Glu Asn Tyr Ala Gln Asp Ala Ala Ser Glu Ile

385 390 395 400

Val Gly Glu Leu Gln Gly Val Pro Asp Phe Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Met Ala His Arg Met Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Asp Phe Asp Asn Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Leu Ile Ala Met Asn Asn Ala Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Gly Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Thr Ile Tyr Phe Pro Tyr Ser Glu Glu Thr Arg Arg Leu Thr

530 535 540

Ala Leu His Gly Ser Ile Glu Glu Met Leu Tyr Ser Pro Asp Gln Thr

545 550 555 560

Asp Glu His Val Gly Thr Leu Ser Asp Arg Ser Lys Pro Ile Leu Phe

565 570 575

Ser Met Ala Arg Leu Asp Lys Val Lys Asn Ile Ser Gly Leu Val Glu

580 585 590

Met Tyr Ser Lys Asn Thr Lys Leu Arg Glu Leu Val Asn Leu Val Val

595 600 605

Ile Ala Gly Asn Ile Asp Val Asn Lys Ser Lys Asp Arg Glu Glu Ile

610 615 620

Val Glu Ile Glu Lys Met His Asn Leu Met Lys Asn Tyr Lys Leu Asp

625 630 635 640

Gly Gln Phe Arg Trp Ile Thr Ala Gln Thr Asn Arg Ala Arg Asn Gly

645 650 655

Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Ala Gln Pro

660 665 670

Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys

675 680 685

Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly Pro Ala Glu Ile Ile

690 695 700

Glu His Gly Leu Ser Gly Phe His Ile Asp Pro Tyr His Pro Glu Gln

705 710 715 720

Ala Gly Asn Ile Met Ala Asp Phe Phe Glu Arg Cys Lys Glu Asp Pro

725 730 735

Asn His Trp Lys Lys Val Ser Asp Ala Gly Leu Gln Arg Ile Tyr Glu

740 745 750

Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Met Thr Leu Ala Gly

755 760 765

Val Tyr Gly Phe Trp Lys Tyr Val Ser Lys Leu Glu Arg Arg Glu Thr

770 775 780

Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg Asp Leu Val

785 790 795 800

Lys Thr Val Pro Ser Thr Ala Asp Asp

805

<210> 4

<211> 2904

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 4

acgtcacttg tagcgaaaac agtatcaaga aaaagagaag atcaaacacg tcttcttttc 60

tctctctctc tttgtcgcct aaaattccag aatcactctg ctttttaccc ttttaatcaa 120

tgatttttcc ttttagtagc aatcgttggt gattcgaaaa accaaacttt tctcggacta 180

ggattctagg gttttagtga tcatctgaat attcatggca aaccctaagc tcactagggt 240

tctaagcaca agggatcgcg tgcaagacac gctttccgct caccgcaacg aactcgttgc 300

tcttctctcc aggtatgtgg atcaggggaa agggattctt caaccacata acttaattga 360

cgaactcgaa tctgttatcg gagacgatga aacaaagaag agtctctctg atggtccttt 420

tggagagatc cttaaatcag caatggaagc tatagttgta ccaccttttg ttgcgttagc 480

cgttagacca agacctggtg tttgggaata tgttcgtgtt aatgtcttcg agctaagtgt 540

tgaacaatta acagtctctg agtatcttcg tttcaaagaa gaactcgttg atggacctaa 600

tagtgaccct ttttgtcttg agcttgattt cgagcccttt aacgcaaacg tgccacgtcc 660

ttctcgttcg tcttcgattg gtaatggagt tcagtttctg aatcgtcact tgtcttctgt 720

tatgttccgt aacaaagatt gcttggagcc tctgcttgat ttccttagag ttcataagta 780

caaaggtcat ccgttgatgt tgaatgatcg gattcaaagc atatctaggc ttcaaatcca 840

gcttagtaaa gcagaagatc atatctctaa gctttcacaa gaaactccgt tctcggaatt 900

cgaatacgcg ttgcaaggaa tgggttttga gaaaggatgg ggagataccg cagggagagt 960

tcttgaaatg atgcatcttc tctctgatat tcttcaagct cctgatcctt cgtccttgga 1020

gaagtttctt gggatggtac caatggtttt caacgttgtg atcttatctc cacatggata 1080

tttcgggcaa gccaatgttt taggcttacc tgacactggt ggacaagttg tctatattct 1140

tgaccaagtc cgtgcccttg agactgaaat gctgttgaga ataaagagac aggggttgga 1200

tatatcacct agtattctta ttgtaactag gttgataccg gatgctaaag gaactacgtg 1260

taaccagcgg ttagagagag tcagcggaac agagcatact catattctcc gggttccttt 1320

taggtctgag aaaggaatcc tccgtaagtg gatttcaaga ttcgacgtat ggccttatct 1380

agagaactat gctcaggatg cagcaagcga gattgtcggt gaattgcaag gcgtaccgga 1440

ctttatcatc ggtaactata gtgacggaaa ccttgttgca tcgttaatgg cacatagaat 1500

gggtgttaca caatgtacta ttgcacatgc tttggagaaa accaagtatc cagattcaga 1560

catttactgg aaagacttcg acaacaagta tcatttctct tgtcaattca cagctgatct 1620

tatcgcaatg aacaacgcag atttcatcat cacaagcact taccaagaaa tcgcaggaac 1680

gaagaacacc gtcggtcaat atgaaagcca cggggctttt acgctcccgg gactatatag 1740

agtagtacac ggcatcgatg tgtttgatcc gaagttcaac atagtctcgc ccggtgcaga 1800

catgaccata tatttcccgt attccgaaga aactaggaga cttacagctt tacatggttc 1860

aatagaggaa atgctctata gccctgacca gactgatgag catgtcggta cactgagtga 1920

tcgatcaaag ccaatactct tctctatggc gaggctcgac aaagtgaaga acatctccgg 1980

tttagttgag atgtatagta agaacacaaa gttgagggag ctggttaatc tggttgtaat 2040

agctggtaac attgatgtga acaagtccaa agatagagaa gaaatcgtag agattgagaa 2100

aatgcataac cttatgaaga attacaagct tgatggacag tttcgttgga taactgctca 2160

gactaaccga gctcgaaatg gtgagcttta ccgctacatc gcggatacaa gaggtgcttt 2220

tgctcagcct gcgttctacg aggcttttgg acttacggta gtggaagcga tgacttgcgg 2280

gctcccgact tttgccactt gtcacggtgg tccagcagag atcatcgagc acgggctctc 2340

gggtttccac atcgatccat accatcctga gcaagcgggt aacataatgg ctgatttctt 2400

tgaacgttgt aaggaagatc caaaccattg gaagaaagta tcagacgctg gtctccaaag 2460

gatatacgaa aggtacacat ggaagatata ctcggagaga ttgatgacac tagctggtgt 2520

gtatggtttc tggaaatacg tatcgaaatt ggagcgtcgt gagactcggc gatatcttga 2580

aatgttctac attctcaaat tccgcgactt ggtgaaaact gttccttcaa ccgccgatga 2640

ctgacccggt tcagcgttgg atgaagtgtt ggactgtaaa aaagaataaa tgatgagtaa 2700

ctttggctat gtaccctatg ttgtgtgtat gtatgaaagg aagaatttgt tccatcttcc 2760

atggagtaat tacgaatttg gtggtacttt tgtgttgtct catgtgtgtc tgactgacat 2820

gttgaagatt attcacgtga agtttgtagc ttataataaa aagtcttcaa tggaacattt 2880

catttcacaa aaaaaaaaaa aaaa 2904

<210> 5

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 5

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccggaagatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcgaactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctacgtgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 6

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 6

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccgcaggatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcggactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctacgtgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 7

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 7

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccgcaggatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcgaactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctgaatgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 8

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 8

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccggaagatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcggactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctacgtgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 9

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 9

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccgcaggatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcggactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctgaatgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 10

<211> 1437

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 10

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggaaaata aaaccgaaac caccgtccgc cgtcgtcgcc gtatcattct gttcccggtc 120

ccgttccagg gccacatcaa cccgattctg caactggcga acgtgctgta ttcgaaaggt 180

ttcagcatca ccatcttcca tacgaacttc aacaagccga agaccagcaa ttacccgcac 240

tttacgttcc gttttattct ggataacgac ccggaagatg aacgcatctc taatctgccg 300

acccacggcc cgctggcggg tatgcgtatt ccgattatca acgaacacgg cgcagatgaa 360

ctgcgtcgcg aactggaact gctgatgctg gccagcgaag aagatgaaga agtttcttgc 420

ctgatcaccg acgcactgtg gtattttgcc cagtctgttg cagatagtct gaacctgcgt 480

cgcctggtcc tgatgaccag cagcctgttc aattttcatg cccacgttag tctgccgcag 540

ttcgatgaac tgggttatct ggacccggat gacaaaaccc gcctggaaga acaggcgagc 600

ggctttccga tgctgaaagt caaggatatt aagtcagcgt actcggactg gcagattctg 660

aaagaaatcc tgggtaaaat gattaagcaa accaaagcaa gttccggcgt catctggaat 720

agtttcaaag aactggaaga atccgaactg gaaacggtga ttcgtgaaat cccggctccg 780

agttttctga ttccgctgcc gaagcatctg accgcgagca gcagcagcct gctggatcac 840

gaccgcacgg tgtttcagtg gctggatcag caaccgccga gttccgtgct gtatgttagc 900

ttcggtagta cctcggaagt ggatgaaaag gactttctgg aaatcgctcg tggcctggtt 960

gatagcaaac aatctttcct gtgggtggtt cgcccgggtt ttgtgaaggg ctctgaatgg 1020

gttgaaccgc tgccggacgg cttcctgggt gaacgtggcc gcattgtcaa atgggtgccg 1080

cagcaagaag tgctggcgca tggcgcgatt ggcgcgtttt ggacccactc cggttggaac 1140

tcaacgctgg aatcggtttg tgaaggtgtc ccgatgattt tctcagattt tggcctggac 1200

cagccgctga atgcacgtta tatgtcggat gttctgaaag tcggtgtgta cctggaaaac 1260

ggttgggaac gcggcgaaat tgcgaatgcc atccgtcgcg ttatggtcga tgaagaaggc 1320

gaatacattc gtcagaatgc tcgcgtcctg aaacaaaagg cggacgtgag cctgatgaaa 1380

ggcggttcat cgtatgaaag tctggaatcc ctggtttcat acatcagctc tctgtaa 1437

<210> 11

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 11

aacgacccgg aagatgaacg catctctaat ctgccg 36

<210> 12

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 12

gcgttcatct tccgggtcgt tatccagaat aaaacg 36

<210> 13

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 13

gcgtactcgg actggcagat tctgaaagaa atcctg 36

<210> 14

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 14

aatctgccag tccgagtacg ctgacttaat atcctt 36

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 15

aagggctctg aatgggttga accgctgccg gacggc 36

<210> 16

<211> 36

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 16

ttcaacccat tcagagccct tcacaaaacc cgggcg 36

Claims

1. A glycosyltransferase mutant characterized in that the mutant is an amino acid sequence mutated on the basis of the amino acid sequence of the glycosyltransferase shown as SEQ ID NO. 1, and the mutation is any single-point or multi-point mutation of Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160, Thr319 or Thr 81.

2. The glycosyltransferase mutant of claim 1, wherein the mutation is a Q72E-N196D-T319E multiple point stacking mutation.

3. An expressed gene encoding the glycosyltransferase mutant of claim 1.

4. A recombinant plasmid to which the expressible gene of claim 3 is linked.

5. A recombinant cell comprising an expression gene of the recombinant expression plasmid or the glycosyltransferase mutant.

6. A method for catalytically synthesizing rebaudioside A by utilizing a glycosyltransferase mutant is characterized by comprising the following steps:

1) constructing a recombinant strain containing double-enzyme coexpression: constructing genes of the mutant of glycosyltransferase UGT76G1 and sucrose synthase genes into plasmids to obtain recombinant plasmids;

2) transforming the recombinant plasmid into a competent cell of escherichia coli BL21(DE3) to obtain a recombinant strain containing a double-enzyme co-expression system;

3) activating the recombinant strain, transferring the recombinant strain to a TB induction culture medium, adding an inducer for induction culture, centrifuging at low temperature and collecting thalli, suspending the thalli in a proper amount of buffer solution and crushing, centrifuging and collecting supernatant, namely crude enzyme solution;

4) catalytic synthesis of rebaudioside a: adding stevioside, cane sugar and crude enzyme liquid into a catalytic reaction system, reacting for 5-30h, inactivating the enzyme at high temperature, and centrifuging to obtain supernatant, namely rebaudioside-A.

7. The method of claim 6, wherein the final concentration of the inducer is 0.02g/L and the induction time is 20 h.

8. The method of claim 6, wherein the concentration of stevioside is 30 g/L; the concentration of the sucrose is 90 g/L; the amount of the crude enzyme added was 5 g/L.