Sucrose synthase mutant, method of preparation and use

Abstract

The application discloses a sucrose synthase mutant, a preparation method and application, and the amino acid sequence of the mutant comprises the mutation of the amino acid residues of at least one site corresponding to T22A, V47L, M48I, T49A, S60K, Y68G, C73S, Q80L, L90P, V104E, T150S, S212E, D219P, A221D, Q226E, K243T, M248L, I387L, V398I, E415D, G529S, Y532F, S533P, D549E, N581R, D596A, Q623E, V658Y, I667V, T719A, T729C, Q731E, G734S, A738K, D758E in SEQ ID NO:1. The application is based on sucrose synthase SrSUS1, single-point and combined mutations are carried out on the predicted amino acid, a sucrose synthase mutant with high enzyme activity is obtained, the mutant is coupled with glycosyltransferase, high-efficiency preparation of the glycosylation product of steviol glycoside RebM is realized, and the application is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a sucrose synthase mutant, a preparation method and application. BACKGROUND

[0002] Sucrose synthase (SuSy, EC 2.4.1.13) is a special glycosyltransferase existing in most plants. It can catalyze the decomposition and synthesis of sucrose, and plays a key role in sucrose metabolism. In recent years, the research on the construction of in situ UDP-Glc recycling system to recycle UDP-Glc by combining glycosyltransferase with sucrose synthase (SuSy) to promote biocatalytic glycosylation has attracted widespread attention. In the SuSy-GT cascade reaction, SuSy forms a recycling system with sucrose, thereby eliminating the inhibition of UDP accumulation on UGT during the catalytic reaction, and regenerating UDP as a glycosyl donor UDPG to promote the progress of glycosylation reaction.

[0003] Plant-derived sucrose synthase (SuSy) mainly uses UDP as its main nucleotide substrate. In contrast, bacterial sucrose synthase tends to use ADP or other nucleoside diphosphates (NDPs) as substrates, which limits their coupling application with uridine diphosphate glucosyltransferase (UGTs). After determining the types and functions of UGTs, the UGT-SuSy system is mainly subject to the enzyme activity and stability of SuSy. In the synthesis reaction of UDP-Glc, the temperature is usually kept between 37-45℃ to prevent SuSy from inactivating, thereby prolonging the reaction time. The thermal stability of bacterial-derived SuSy is relatively high, and the SuSy from Caldus Acidithiobacillus has extremely high thermal stability, with an optimum temperature of 60℃. Even if it is cultured at 60℃ for 15 minutes, its enzyme activity can still remain 96%. The optimum temperature of plant sucrose synthase is 40-55℃, and the thermal stability significantly decreases when the temperature exceeds 30℃. This limited thermal stability hinders the enzyme to maintain sufficient activity to complete the substrate conversion. Therefore, improving the optimum temperature and thermal stability of SuSy helps to improve the reaction temperature and shorten the reaction time, and ultimately improve the space-time yield (STY) and productivity.

[0004] Since plant SUS has higher specificity for UDP-sugar, and UDP-glucose is the main sugar donor for UGT to catalyze the glycosylation of plant natural products, it may indicate that plant SUS is more suitable for coupling with UGT compared with bacterial SUS. However, the low stability of plant SUS limits the further application of these enzymes. An ideal SUS should have the stability of bacterial enzymes and the high UDP affinity of plant enzymes.

[0005] In summary, it is of great significance to study the high-enzyme-activity SUS or to engineer the structure of SUS to improve its stability. SUMMARY

[0006] One of the technical problems to be solved by the present application is to provide a sucrose synthase mutant and a preparation method, based on sucrose synthase SrSUS1, to obtain a sucrose synthase mutant with high enzyme activity by single-point and combined mutation of predicted amino acids, in view of the deficiencies of the prior art.

[0007] Another technical problem to be solved by the present application is to provide an application of a sucrose synthase mutant, to realize efficient preparation of glycosylation products of steviol glycoside RebM by coupling SrSUS1 mutant with glycosyltransferase, in view of the deficiencies of the prior art.

[0008] To solve the first technical problem, the technical solution of the present application is as follows:

[0009] A sucrose synthase mutant, wherein the amino acid sequence of the mutant is a mutation of at least one amino acid residue at positions T22A, V47L, M48I, T49A, S60K, Y68G, C73S, Q80L, L90P, V104E, T150S, S212E, D219P, A221D, Q226E, K243T, M248L, I387L, V398I, E415D, G529S, Y532F, S533P, D549E, N581R, D596A, Q623E, V658Y, I667V, T719A, T729C, Q731E, G734S, A738K, D758E in SEQ ID NO: 1.

[0010] Preferably, the amino acid sequence of the sucrose synthase mutant is a mutation of amino acid residues at any one or more of positions T22A, T49A, L90P, and V104E in SEQ ID NO: 1.

[0011] Preferably, the mutant T22A is obtained by mutating the 22nd amino acid from threonine (T) to alanine (A) in the sucrose synthase mutant SrSUS1 sequence shown in SEQ ID NO: 1, and the amino acid sequence of the mutant T22A is shown in SEQ ID NO: 2; and the nucleotide sequence is shown in SEQ ID NO: 3.

[0012] The mutant V47L is obtained by mutating the 47th amino acid from valine (V) to leucine (L) in the sucrose synthase SrSUS1 sequence shown in SEQ ID NO: 1.

[0013] The mutant T49A is obtained by mutating the 49th amino acid from threonine (T) to alanine (A) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0014] The mutant S60K is obtained by mutating the 60th amino acid from serine (S) to lysine (K) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0015] The mutant L90P is obtained by mutating the 90th amino acid from leucine (L) to proline (P) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0016] The mutant V104E is obtained by mutating the 104th amino acid from valine (V) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0017] The mutant T150S is obtained by mutating the 150th amino acid from threonine (T) to serine (S) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0018] The mutant A221D is obtained by mutating the 221st amino acid from alanine (A) to aspartic acid (D) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0019] The mutant Q226E is obtained by mutating the 226th amino acid from glutamine (Q) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0020] The mutant G529S is obtained by mutating the 529th amino acid from glycine (G) to serine (S) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0021] The mutant V658Y is obtained by mutating the 658th amino acid from valine (V) to tyrosine (Y) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0022] The mutant T719A is obtained by mutating the 719th amino acid from threonine (T) to alanine (A) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0023] The mutant T729C is obtained by mutating the amino acid at position 729 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to cysteine (C);

[0024] The mutant D758E is obtained by mutating the amino acid at position 758 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from aspartic acid (D) to glutamic acid (E);

[0025] The mutant T22A / T49A is obtained by mutating the amino acid at position 22 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to alanine (A), and mutating the amino acid at position 49 from threonine (T) to alanine (A);

[0026] The mutant T22A / L90P is obtained by mutating the amino acid at position 22 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to alanine (A), and mutating the amino acid at position 90 from leucine (L) to proline (P);

[0027] The T22A / V104E is obtained by mutating the amino acid at position 22 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to alanine (A), and mutating the amino acid at position 104 from valine (V) to glutamic acid (E);

[0028] The T49A / L90P is obtained by mutating the amino acid at position 49 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to alanine (A), and mutating the amino acid at position 90 from leucine (L) to proline (P);

[0029] The T49A / V104E is obtained by mutating the amino acid at position 49 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from threonine (T) to alanine (A), and mutating the amino acid at position 104 from valine (V) to glutamic acid (E);

[0030] The L90P / V104E is obtained by mutating the amino acid at position 90 in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1 from leucine (L) to proline (P), and mutating the amino acid at position 104 from valine (V) to glutamic acid (E);

[0031] T22A / T49A / L90P is that the 22th amino acid is mutated from threonine (T) to alanine (A), the 49th amino acid is mutated from threonine (T) to alanine (A), and the 90th amino acid is mutated from leucine (L) to proline (P) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0032] T22A / T49A / V104E is that the 22th amino acid is mutated from threonine (T) to alanine (A), the 49th amino acid is mutated from threonine (T) to alanine (A), and the 104th amino acid is mutated from valine (V) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0033] T22A / L90P / V104E is that the 22th amino acid is mutated from threonine (T) to alanine (A), the 90th amino acid is mutated from leucine (L) to proline (P), and the 104th amino acid is mutated from valine (V) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0034] T49A / L90P / V104E is that the 49th amino acid is mutated from threonine (T) to alanine (A), the 90th amino acid is mutated from leucine (L) to proline (P), and the 104th amino acid is mutated from valine (V) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1;

[0035] The mutant T22A / T49A / L90P / V104E is that the 22th amino acid is mutated from threonine (T) to alanine (A), the 49th amino acid is mutated from threonine (T) to alanine (A), the 90th amino acid is mutated from leucine (L) to proline (P), and the 104th amino acid is mutated from valine (V) to glutamic acid (E) in the sucrose synthase SrSUS1 sequence shown in the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the mutant T22A / T49A / L90P / V104E is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 5.

[0036] An expression gene encoding any of the above-mentioned sucrose synthase mutants.

[0037] A recombinant plasmid connected with the nucleotide sequence of the above-mentioned sucrose synthase mutant and the glycosyltransferase UGT76G4 gene.

[0038] A recombinant bacterium comprising the recombinant plasmid or the expression gene.

[0039] Further, the host bacterium of the recombinant bacterium is a recombinant Escherichia Coli BL21 (DE3) strain.

[0040] To solve the second technical problem, the present application provides the following technical solutions:

[0041] The application of a sucrose synthase mutant for catalyzing the synthesis of steviol glycoside RebM.

[0042] A method for catalyzing the preparation of steviol glycoside RebM by a sucrose synthase mutant, comprising the following steps:

[0043] S1: Constructing a recombinant strain containing double-enzyme co-expression:

[0044] A codon-optimized gene encoding glycosyltransferase UGT76G4 is cloned into an expression vector to obtain a first plasmid, and a gene fragment of a sucrose synthase mutant is cloned into the first plasmid to obtain a recombinant plasmid; the recombinant plasmid is transformed into a host bacterium to obtain a recombinant strain containing double-enzyme co-expression;

[0045] S2: Inducing enzyme production of the recombinant strain:

[0046] After the recombinant strain is activated, it is transferred to an LB liquid medium, an inducer IPTG is added for induction culture, and then low-temperature centrifugation is performed to collect the bacterial bodies, the bacterial bodies are resuspended in an appropriate amount of buffer and broken, and the supernatant collected by centrifugation is the crude enzyme solution;

[0047] S3: Biocatalytic synthesis of steviol glycoside RebM:

[0048] The crude enzyme solution, RebD, sucrose and UDP are added to the catalytic reaction system to perform glycosylation reaction, the reaction is terminated after being warmed up, centrifugation is performed, and the supernatant collected is the steviol glycoside RebM.

[0049] Further, in step S1, the expression vector is pRSFDuet-1.

[0050] Further, in step S2, the final concentration of the inducer is 0.02-1 mM, the temperature for induction culture is 10-30°C, the rotation speed is 150-300 r / min, and the induction time is 24 h.

[0051] Further, in step S3, in the catalytic reaction system, the initial concentration of RebD is 20 g / L, the concentration of sucrose is 1-500 mM, the concentration of the crude enzyme is 0.1-1000 g / L, and the concentration of UDP is 1 mM.

[0052] Further, the glycosylation reaction is biotransformed in a buffer solution with pH 7.2.

[0053] Further, the reaction temperature of the glycosylation reaction is 20-40 DEG C, and the reaction time is 1-24 h.

[0054] Due to the adoption of the above technical solutions, the present application has the following beneficial effects:

[0055] 1. The present application provides a sucrose synthase mutant, which improves the enzyme activity of sucrose synthase SrSUS1 through site-directed mutation, realizes efficient catalytic synthesis of natural glycosides by using the mutant, has mild synthesis method conditions, simple operation, short time, high catalytic efficiency, high yield, and good application prospect.

[0056] 2. The sucrose synthase multi-point mutant T22A / T49A / L90P / V104E constructed in the present application is the mutant with the most significant enzyme activity, and the specific enzyme activity is 14.74 U / mg, the specific enzyme activity of the original sucrose synthase as a control is 5.45 U / mg, and the specific enzyme activity of the mutant is 3.7 times that of the original specific enzyme activity.

[0057] 3. The sucrose synthase mutants constructed in the present application are coupled with glycosyltransferase UGT76G4 as a biological catalyst, and when RebD is catalyzed to synthesize RebM, the yield of the target product is improved.

[0058] 4. The mutant provided in the present application is simple to prepare, and under the same conditions, the enzyme activity of the mutant is improved by 41%-370% compared with the wild type. The reaction system constructed by coupling the mutant T22A / T49A / L90P / V104E with glycosyltransferase UGT76G4 has a RebM concentration of 93.35 g / L after 24 h of reaction, and the yield reaches 93.35%. BRIEF DESCRIPTION OF DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0060] Figure 1 is a standard curve graph of fructose;

[0061] Figure 2 is a concentration curve of steviol glycoside RebM in the system with reaction time and a supplemental curve of RebD with reaction time in the reaction process. DETAILED DESCRIPTION

[0062] In order to enable one skilled in the art to more clearly understand the above-mentioned objects, features and advantages of the present application, the solutions of the present application will be further described below. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0063] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein; obviously, the embodiments described in the specification are only a part of the embodiments of the present application, and not all the embodiments.

[0064] Consensus sequence design provides a promising strategy for designing high-stability proteins while retaining biological activity, as it draws on evolutionary history, in which residues important for both stability and function are likely to be conserved. In the context of enzyme evolution, amino acid residues that are more prevalent in homologous proteins play a more important role in improving the catalytic activity and stability of enzymes compared to non-conserved amino acid residues. These frequently occurring residues are referred to as consensus residues. Consensus residues located on the surface of a protein are associated with structural stability, while consensus residues located near the active site significantly affect the catalytic efficiency and regioselectivity of enzymes.

[0065] The present application is based on sucrose synthase SrSUS1, and the predicted residues are subjected to single-point and combined mutation. The catalytic ability of the sucrose synthase mutant is investigated by comparing the relative activity of the mutant strain. The sucrose synthase mutant is coupled with glycosyltransferase to catalyze the synthesis of steviol glycoside RebM from steviol glycoside RebD and an appropriate amount of sucrose.

[0066] A method for biocatalytic synthesis of RebM, comprising the following steps:

[0067] (1) Constructing a recombinant strain containing double-enzyme co-expression:

[0068] A codon-optimized gene encoding glycosyltransferase UGT76G4 is cloned into an expression vector to obtain a first plasmid, and a gene fragment of the sucrose synthase mutant is cloned into the first plasmid to obtain a recombinant plasmid. The above-mentioned recombinant plasmid is transformed into a host bacterium to obtain a recombinant strain containing double-enzyme co-expression. The amino acid sequence of the glycosyltransferase UGT76G4 gene is shown in SEQ ID NO: 6, and the nucleotide sequence of the codon-optimized glycosyltransferase UGT76G4 gene is shown in SEQ ID NO: 7;

[0069] (2) Inducing enzyme production of the recombinant strain:

[0070] After the above-mentioned recombinant strain is activated, it is transferred to LB liquid medium, and an inducer IPTG is added for induction culture. Then, low-temperature centrifugation is performed to collect the bacterial cells, the bacterial cells are resuspended in an appropriate amount of buffer and broken, and the supernatant is collected by centrifugation to obtain a crude enzyme solution.

[0071] (3) Bio-catalytic synthesis of steviol glycoside RebM:

[0072] The above crude enzyme solution, RebD, sucrose and UDP are added into the catalytic reaction system to perform glycosylation reaction, and after the reaction is completed, the reaction is terminated by heating, centrifugation, and collection of the supernatant, which is the steviol glycoside RebM.

[0073] With regard to step (1):

[0074] In some embodiments of the present application, the expression vector is pRSFDuet-1.

[0075] In some embodiments of the present application, the UGT76G4 gene fragment of the glycosyltransferase is cloned into the Nco I and Eco R I enzyme cutting sites of pRSFDuet-1 using the primers in Table 1, and the generated plasmid is named pRSF-UGT76G4. The target gene fragment of the sucrose synthase mutant is cloned into the Nde I and Xho I enzyme cutting sites of pRSF-UGT76G4 to obtain the recombinant plasmid.

[0076] Specifically, the synthesis of the sucrose synthase SrSUS1 gene fragment and the glycosyltransferase UGT76G4 gene fragment is completed by Nanjing Kingsriver Biotechnology Co., Ltd. The nucleotide sequence of the codon-optimized SrSUS1 is shown in SEQ ID NO: 8.

[0077] PCR amplification is performed using the primers shown in Table 1 and PrimeSTAR DNA polymerase (TaKaRa) with pRSFDuet-1 as the template. The PCR target plasmid amplification reaction system is as follows: 10 μmol / L of forward and reverse primers, 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterilized water dd H2O to 50 μL; PCR target plasmid amplification reaction conditions: 95°C pre-denaturation for 30 s; 30 cycles of 95°C denaturation for 15 s, 65°C annealing for 15 s, and 72°C extension for 7 min; 72°C thorough extension for 5 min; finally 16°C incubation, and agarose gel nucleic acid electrophoresis detection of the PCR amplification product, which is the pRSFDuet-1 linearized vector fragment.

[0078] The conditions for cloning the UGT76G4 gene fragment into the pRSFDuet-1 vector are as follows:

[0079] The UGT76G4 gene fragment and the linearized pRSFDuet-1 carrier are purified by a FastPure Gel DNA Extraction Mini Kit kit, and then recombined, and the recombination system is: linearized carrier 2 μL; UGT76G4 gene fragment 2 μL; 5×CE II buffer 4 μL; Exnase II 2 μL, and the recombination reaction is carried out at 37 °C for 30 min, and the plasmid pRSF-UGT76G4 is obtained.

[0080] Table 1

[0081]

[0082] The SrSUS1 mutant gene fragment is constructed into the plasmid pRSF-UGT76G4 between the Nde I and Xho I enzyme cutting sites, and the conditions are as follows:

[0083] The pRSF-UGT76G4 is used as a template, and the primers shown in Table 2 and PrimeSTAR DNA polymerase (TaKaRa) are used for PCR amplification, and the PCR target plasmid amplification reaction system is: 10 μmol / L forward primer and reverse primer 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, and sterilized water dd H2O is added to 50 μL; the PCR target plasmid amplification reaction condition is: 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 thorough extension for 5 min; finally 16 °C incubation, and the PCR amplification product is detected by agarose gel nucleic acid electrophoresis, and the PCR product is obtained, that is, the pRSF-UGT76G4 linearized carrier fragment.

[0084] The SrSUS1 mutant gene fragment and the pRSF-UGT76G4 linearized carrier are purified by a FastPure Gel DNA Extraction Mini Kit kit, and then recombined, and the recombination system is: linearized carrier 2 μL; SrSUS1 mutant gene fragment 2 μL; 5×CE II buffer 4 μL; Exnase II 2 μL, and the recombination reaction is carried out at 37 °C for 30 min.

[0085] Table 2

[0086]

[0087] In some embodiments of the application, the sucrose synthase SrSUS1 plasmid is obtained by the following steps:

[0088] A: The gene fragment of SrSUS1 is cloned into the Nde I and Eco RI enzyme cutting sites of pETDuet-1, and the generated plasmid is named as pET-SrSUS1, and the optimized SrSUS1 nucleotide sequence is shown as SEQ ID NO: 1;

[0089] Specifically, the sucrose synthase SrSUS1 gene fragment synthesis is completed by Nanjing Kingsrui Biotechnology Co., Ltd.

[0090] PCR amplification is performed by using the primers shown in Table 3 and PrimeSTAR DNA polymerase (TaKaRa) with pETDuet-1 as a template, and the PCR target plasmid amplification reaction system is as follows: 10 μmol / L of forward primer and reverse primer, 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterilized water dd H2O to 50 μL; the PCR target plasmid amplification reaction conditions are 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 thorough extension for 5 min; finally 16 °C incubation, and the PCR amplification product is detected by agarose gel nucleic acid electrophoresis to obtain the PCR product, i.e., the pETDuet-1 linearized vector fragment.

[0091] The gene fragment of SrSUS1 and the pETDuet-1 linearized vector are subjected to gel recovery and purification by using a FastPure Gel DNA Extraction Mini Kit kit, and then recombined, and the recombination system is as follows: linearized vector 2 μL; gene fragment of SrSUS1 2 μL; 5×CE II buffer 4 μL; Exnase II 2 μL, and the recombination reaction is performed at 37 °C for 30 min.

[0092] Table 3

[0093]

[0094] In some embodiments of the present application, the sucrose synthase SrSUS1 mutant is obtained by the following steps:

[0095] PCR amplification is performed by using the primers shown in Table 4 and PrimeSTAR DNA polymerase (TaKaRa) with pET-SrSUS1 as a template, and the PCR target plasmid amplification reaction system is as follows: 10 μmol / L of forward primer and reverse primer, 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterilized water dd H2O to 50 μL.

[0096] PCR target plasmid amplification reaction conditions: 95°C pre-denaturation 30s; 30 cycles (95°C denaturation 15s; 65°C annealing 15s; 72°C extension 7min); 72°C thorough extension 5min; finally 16°C incubation, and the PCR amplification product is detected by agarose gel nucleic acid electrophoresis. The primers of each mutant are shown in Table 4:

[0097] Table 4

[0098]

[0099] With regard to step (2):

[0100] The specific process of transforming the above-mentioned recombinant plasmid into a host bacterium is as follows: the recombinant plasmid constructed in step (1) is added to E. coli BL21 (DE3) competent cells (TsingKe), the recombinant plasmid and the competent cells are mixed by flicking, treated in an ice bath for 20-30min, then placed in a 40-42°C water bath for heat shock for 80-90s, then continue to be treated in an ice bath for 2-3min, then 400µL of LB liquid medium without resistance is added, mixed by blowing and sucking, and placed in a 37°C shaking bed for activation for 30-35min to obtain a bacterial solution, 70µL of the above-mentioned bacterial solution is spread on an LB plate containing 50mg / L of kanamycin resistance, and cultured in a 37°C incubator for 12h to obtain a recombinant strain.

[0101] With regard to step (3):

[0102] In some embodiments of the present application, the process of activating the recombinant strain is as follows: the recombinant strain of step (2) is spread on an LB solid plate containing corresponding resistance (kanamycin: 20-50µg / mL), and placed in a 37°C incubator for culture for 12h.

[0103] In some embodiments of the present application, the process of transferring the activated recombinant strain to LB liquid medium is as follows: the recombinant strain is inoculated in LB liquid medium at 1-3% v / v, cultured at 37°C until OD600 reaches 0.6-0.8, an inducer is added for induction culture, after the culture is completed, the bacterial slurry is frozen and centrifuged, resuspended in a buffer and broken by ultrasonic crushing method, the supernatant is collected by freezing and centrifugation to obtain a crude enzyme solution.

[0104] In some embodiments of the present application, the inducer is IPTG, and the final concentration is 0.02-1mM.

[0105] In some embodiments of the present application, the induction culture conditions are: temperature 10-30℃, rotation speed 150-300r / min, and induction time 24h.

[0106] In some embodiments of the present application, the low-temperature centrifugation conditions are: temperature 4-5℃, centrifugation speed 8000rpm, and time 3-5min.

[0107] In some embodiments of the present application, the buffer is potassium phosphate buffer with pH 7.2, and the mass ratio of the bacterial slurry to the buffer is 1:2-5 during resuspension.

[0108] In some embodiments of the present application, the freeze centrifugation conditions are: temperature 3-5℃, centrifugation speed 8000rpm, and centrifugation time 3-5min.

[0109] In some embodiments of the present application, the ultrasonic disruption conditions are: ultrasonic disruption of the bacterial cells in an ice-water mixture using an ultrasonic disruptor with parameter settings of Ф6, 300-350W, and 15min.

[0110] With regard to step (4):

[0111] In some embodiments of the present application, in the catalytic reaction system, the initial concentration of RebD is 20g / L; and / or the concentration of sucrose is 1-500mM, and / or the concentration of the crude enzyme is 0.1-1000g / L, and / or the concentration of UDP is 1mM.

[0112] Further, the reaction temperature of the glycosylation reaction is 20-40℃, and the reaction time is 1-24h.

[0113] Further, 20g / L of RebD is supplemented every 3h during the glycosylation reaction.

[0114] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations to the claims of the present application.

[0115] In the following examples, the raw materials are commercially available unless otherwise specified, and the conditions are conventional conditions in the art unless otherwise specified. Example 1

[0116] Construction of the recombinant strain of sucrose synthase SrSUS1:

[0117] S1: Preparation of the plasmid of sucrose synthase SrSUS1:

[0118] S1-1: The gene fragment of SrSUS1 was cloned into the Nde I and Eco RI enzyme sites of pETDuet-1 to generate a plasmid named pET-SrSUS1, and the optimized SrSUS1 nucleotide sequence is shown as SEQ ID NO: 8; wherein the synthesis of the sucrose synthase SrSUS1 gene fragment was completed by Nanjing Kings River Biotechnology Co., Ltd.

[0119] S1-2: PCR amplification was performed using the primers shown in Table 3 and PrimeSTAR DNA polymerase (TaKaRa) with pETDuet-1 as the template, and the PCR target plasmid amplification reaction system was as follows: 10 μM of each of the forward primer and the reverse primer, 2 μL; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterilized water dd H2O to 50 μL; the PCR target plasmid amplification reaction conditions were as follows: 95 °C pre-denaturation for 30 s; 30 cycles of 95 °C denaturation for 15 s; 65 °C annealing for 15 s; 72 °C extension for 7 min; 72 °C thorough extension for 5 min; finally 16 °C incubation; the PCR amplification product was detected by agarose gel nucleic acid electrophoresis, and the PCR product, i.e., the pETDuet-1 linearized vector fragment, was obtained.

[0120] S1-3: The gene fragment of SrSUS1 and the pETDuet-1 linearized vector were purified by gel recovery after using the FastPure Gel DNA Extraction Mini Kit kit for recombination, and the recombination system was as follows: linearized vector 2 μL; SrSUS1 gene fragment 2 μL; 5×CE II buffer 4 μL; Exnase II 2 μL, recombination reaction at 37 °C for 30 min. The optimized SrSUS1 nucleotide sequence is shown as SEQ ID NO: 8.

[0121] S2: Construction of recombinant strain pET-SrSUS1:

[0122] The recombinant plasmid pET-SrSUS1 was added to the E. coli BL21 (DE3) competent cells (TransGen Biotech), and the recombinant plasmid and the competent cells were mixed and homogenized by flicking, ice-bathed for 30 min, then placed in a 42 °C water bath for heat shock for 90 s, ice-bathed for 2 min, and then 400 µL of LB liquid medium without resistance was added, mixed by blowing and sucking, and placed in a 37 °C shaking bed for activation for 30 min to obtain a bacterial solution. 70 µL of the bacterial solution was spread on an LB plate containing 50 mg / ml kanamycin resistance, and cultured in a 37 °C incubator for 12 h to obtain the recombinant strain pET-SrSUS1. Example 2

[0123] Construction of sucrose synthase mutant enzyme expression system:

[0124] (1) Construction of mutant recombinant strain:

[0125] Construction of single mutant recombinant strain:

[0126] Using the recombinant plasmid pET-SrSUS1 of Example 1 as a template, the primers of the single mutants in Table 1 and PrimeSTAR DNA polymerase (TaKaRa) were used for PCR amplification, and after verification by agarose gel electrophoresis, 1 μL of Dpn I endonuclease was added to the PCR reaction product. The reaction system was then incubated at 37°C for 2h to digest the template, and then the digested PCR reaction product was transformed into E. coli BL21 (DE3) according to the method of Example 1. Finally, a single colony was picked from the plate and inoculated into LB liquid medium (10 g / L peptone, yeast extract 5 g / L, 10 g / L NaCl) containing 50 mg / L kanamycin, and incubated at 37°C, 200 rpm for 12 h. The plasmid was extracted and sent for identification.

[0127] The PCR target plasmid amplification reaction system was as follows: 10 μmol / L forward primer and reverse primer, 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterile water dd H2O to 50 μL.

[0128] The PCR target plasmid amplification reaction conditions were as follows: 95°C pre-denaturation for 30s; 30 cycles of 95°C denaturation for 15s, 65°C annealing for 15s, and 72°C extension for 7min; 72°C thorough extension for 5min; and finally 16°C incubation. The PCR amplification product was detected by agarose gel nucleic acid electrophoresis.

[0129] Construction of double mutant recombinant strain:

[0130] The forward single-point mutation was combined with mutation, and the mutation effect of the double-point combination mutation was evaluated. The combination mutation method was as follows:

[0131] T22A / T49A mutant:

[0132] Using the recombinant plasmid pET-SrSUS1-T22A as a template, the above mutation method was used for combination mutation of T49A, and finally sent for identification. The mutation primers of T22A / T49A are shown in Table 4.

[0133] T22A / L90P mutant:

[0134] The recombinant plasmid pET-SrSUS1-T22A is used as a template, and the above mutation method is used to combine mutation of L90P. Finally, it is sent for test and identification. The mutation primer of T22A / L90P is shown in Table 4.

[0135] T22A / V104E mutant:

[0136] The recombinant plasmid pET-SrSUS1-T22A is used as a template, and the above mutation method is used to combine mutation of V104E. Finally, it is sent for test and identification. The mutation primer of T22A / V104E is shown in Table 4.

[0137] T49A / L90P mutant:

[0138] The recombinant plasmid pET-SrSUS1-T49A is used as a template, and the above mutation method is used to combine mutation of L90P. Finally, it is sent for test and identification. The mutation primer of T49A / L90P is shown in Table 4.

[0139] T49A / V104E mutant:

[0140] The recombinant plasmid pET-SrSUS1-T22A is used as a template, and the above mutation method is used to combine mutation of T49A. Finally, it is sent for test and identification. The mutation primer of T22A / T49A is shown in Table 4.

[0141] L90P / V104E mutant:

[0142] The recombinant plasmid pET-SrSUS1-L90P is used as a template, and the above mutation method is used to combine mutation of V104E. Finally, it is sent for test and identification. The mutation primer of L90P / V104E is shown in Table 4.

[0143] T22A / T49A / L90P mutant:

[0144] The recombinant plasmid pET-SrSUS1-T22A / T49A is used as a template, and the above mutation method is used to combine mutation of L90P. Finally, it is sent for test and identification. The mutation primer of T22A / T49A / L90P is shown in Table 4.

[0145] T22A / T49A / V104E mutant:

[0146] The recombinant plasmid pET-SrSUS1-T22A / T49A is used as a template, and the above mutation method is used to combine mutation of V104E. Finally, it is sent for test and identification. The mutation primer of T22A / T49A / V104E is shown in Table 4.

[0147] T22A / L90P / V104E mutant:

[0148] The recombinant plasmid pET-SrSUS1_T22A / L90P was used as a template, and the combination mutation of V104E was carried out by the above mutation method, and finally sent for test identification. The mutation primer of T22A / L90P / V104E is shown in Table 4.

[0149] T49A / L90P / V104E mutant:

[0150] The recombinant plasmid pET-SrSUS1_T49A / L90P was used as a template, and the combination mutation of V104E was carried out by the above mutation method, and finally sent for test identification. The mutation primer of T49A / L90P / V104E is shown in Table 4.

[0151] T22A / T49A / L90P / V104E mutant:

[0152] The recombinant plasmid pET-SrSUS1_T22A / T49A / L90P was used as a template, and the combination mutation of V104E was carried out by the above mutation method, and finally sent for test identification. The mutation primer of T22A / T49A / L90P / V104E is shown in Table 4.

[0153] (3) Construction of sucrose synthase mutant enzyme system:

[0154] The plasmids verified successfully in step (2) were transformed into E. coli BL21 (DE3) by the method in Example 1, and the recombinant strains were obtained. The recombinant strains were coated on LB solid plates containing corresponding resistance (kanamycin: 20-50 μg / mL) and incubated in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates and inoculated into LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) and incubated at 37°C, 200 rpm. When the absorbance (OD600) reached 0.6, IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 16°C for 24 h. The fermentation broth was collected and frozen and centrifuged (4°C, 8000 rpm, 5 min), and the supernatant was discarded to obtain the bacterial slurry. The bacterial slurry was washed twice with potassium phosphate buffer, and then potassium phosphate buffer was added (the mass ratio of bacterial slurry to potassium phosphate buffer was 1:5). The mixture was placed in an ice-water mixture and ultrasonically broken by an ultrasonic disrupter with the following parameters: Ф6, 300 W, 15 min. The bacterial solution was centrifuged using a refrigerated centrifuge with the following parameters: 4°C, 8000 rpm, 20 min. The supernatant was the crude enzyme solution, which was stored in a 4°C refrigerator for later use. Example 3

[0155] Construction of double-enzyme expression system:

[0156] The synthesis of the sucrose synthase SrSUS1 gene fragment and the glycosyltransferase UGT76G4 gene fragment was completed by Nanjing Kingsriver Biotechnology Co., Ltd.

[0157] PCR amplification was performed using the primers shown in Table 1 and PrimeSTAR DNA polymerase (TaKaRa) with pRSFDuet-1 as a template. The PCR target plasmid amplification reaction system was as follows: 10 μmol / L of forward primer and reverse primer, 2 μL each; template plasmid, 1 μL; PrimeSTAR DNA polymerase, 25 μL; and dd H2O, supplemented to 50 μL. The PCR target plasmid amplification reaction conditions were as follows: 95 °C pre-denaturation for 30 s; 30 cycles of 95 °C denaturation for 15 s, 65 °C annealing for 15 s, and 72 °C extension for 7 min; 72 °C thorough extension for 5 min; and finally 16 °C incubation. The PCR amplification product was detected by agarose gel nucleic acid electrophoresis, and the PCR product was obtained, which was the pRSFDuet-1 linearized vector fragment;

[0158] The UGT76G4 gene fragment was cloned into the pRSFDuet-1 vector under the following conditions:

[0159] After the UGT76G4 gene fragment and the pRSFDuet-1 linearized vector were purified by gel recovery using the FastPure Gel DNA Extraction Mini Kit kit, recombination was performed. The recombination system was as follows: linearized vector, 2 μL; UGT76G4 gene fragment, 2 μL; 5×CE II buffer, 4 μL; and Exnase II, 2 μL. The recombination reaction was performed at 37 °C for 30 min, and the plasmid pRSF-UGT76G4 was obtained.

[0160] The SrSUS1 mutant gene fragment was constructed into the pRSF-UGT76G4 plasmid between the Nde I and Xho I enzyme digestion sites under the following conditions:

[0161] The pRSF-UGT76G4 was used as a template, and the primers shown in Table 2 and PrimeSTAR DNA polymerase (TaKaRa) were used for PCR amplification. The PCR target plasmid amplification reaction system was as follows: 10 μmol / L of forward primer and reverse primer, 2 μL each; template plasmid 1 μL; PrimeSTAR DNA polymerase 25 μL, supplemented with sterile water dd H2O to 50 μL; the PCR target plasmid amplification reaction conditions were as follows: 95 °C pre-denaturation for 30 s; 30 cycles of 95 °C denaturation for 15 s, 65 °C annealing for 15 s, and 72 °C extension for 7 min; 72 °C thorough extension for 5 min; finally 16 °C incubation; the PCR amplification product was detected by agarose gel nucleic acid electrophoresis, and the PCR product was obtained, which was the pRSF-UGT76G4 linearized vector fragment.

[0162] The SrSUS1 mutant gene fragment and the pRSF-UGT76G4 linearized vector were purified by gel recovery using the FastPure Gel DNA Extraction Mini Kit kit, and then recombined. The recombination system was as follows: linearized vector 2 μL; SrSUS1 mutant gene fragment 2 μL; 5×CE II buffer 4 μL; Exnase II 2 μL, recombination reaction at 37 °C for 30 min. The optimized nucleotide sequence of SrSUS1_T22A / T49A / L90P / V104E is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 4;

[0163] (2) Preparation of recombinant strain:

[0164] The above recombinant plasmid pRSF-SrSUS1_T22A / T49A / L90P / V104E-76G4 was added to the E. coli E. coli BL21 (DE3) competent cells (TransGen Biotech), and the recombinant plasmid and competent cells were mixed and shaken on ice for 30 min, then placed in a 42 °C water bath for heat shock for 90 s, and then placed in an ice bath for 2 min. 400 μL of LB liquid medium without resistance was added, mixed by blowing and sucking, and placed in a 37 °C shaking bed for activation for 30 min to obtain a bacterial solution. 70 μL of the bacterial solution was spread on an LB plate containing 50 mg / L of kanamycin resistance, and cultured in a 37 °C incubator for 12 h to obtain the recombinant strain SrSUS1_T22A / T49A / L90P / V104E-76G4.

[0165] (3) Construction of double-enzyme expression system:

[0166] The above recombinant strain SrSUS1_T22A / T49A / L90P / V104E-76G4 was coated on LB solid plates (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl, agar 15 g / L) containing corresponding resistance (kanamycin: 50 μg / mL), and incubated in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates into LB liquid medium (10 g / L peptone, yeast powder 5 g / L, 10 g / L NaCl) and cultured at 37°C, 200 rpm. When the absorbance (OD600) reached 0.6, the inducer IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 16°C for 24 h. After the induction culture was completed, the bacterial solution was collected and frozen and centrifuged (4°C, 8000 rpm, 5 min) to obtain bacterial slurry, which was washed twice with potassium phosphate buffer, and then potassium phosphate buffer (mass ratio of bacterial slurry to potassium phosphate buffer 1:5) was added and placed in an ice-water mixture for ultrasonic disruption treatment with an ultrasonic disruptor, with parameters set to Ф6, 300 W, 15 min. Then, the supernatant was collected by centrifugation with a frozen centrifuge at 4°C, 8000 rpm, 20 min, which was the crude enzyme solution, and was stored in a 4°C refrigerator for later use. Example 4

[0167] Compared with Example 3, the difference is that in step (1), SrSUS1_T22A is used instead of SrSUS1_T22A / T49A / L90P / V104E, and other conditions are the same as in Example 3. Example 5

[0168] Compared with Example 3, the difference is that in step (1), SrSUS1_L90P / V104E is used instead of SrSUS1_T22A / T49A / L90P / V104E, and other conditions are the same as in Example 3.

[0169] The enzyme activity and thermal stability of the mutant enzymes prepared in the above examples were tested, and the testing methods and testing results are as follows:

[0170] 1. Comparison of enzyme activities of sucrose synthase SrSUS1 and single-point mutants thereof:

[0171] The single-point mutants obtained by screening in the above examples were subjected to enzyme activity determination, and the effect of single-point mutation was evaluated. Among them, the sucrose synthase enzyme activity is defined (U) as 1 enzyme unit for the amount of enzyme required to convert 1 μmol of fructose in 1 min.

[0172] DNS method was used to determine the enzyme activity of the crude enzyme, and the fructose content generated by the crude enzyme cleaving sucrose was determined. 0.02 mg / mL crude enzyme was added to 100 μL reaction solution containing 200 mM sucrose, 5 mM UDP and HEPES buffer (50 mM, pH 7.0), and reacted at 37°C for 20 min, and then heated at 95°C for 5 min to terminate the reaction. At the same time, the reaction without adding the crude enzyme was used as a control experiment to exclude the influence of heat treatment on sucrose decomposition. 200 μL DNS reagent was added to the inactivated reaction solution, heated in a boiling water bath for 2 min, then cooled to room temperature, 700 μL distilled water was added, and the absorbance of the sample at 540 nm was determined. The fructose content was calculated by comparing with the DNS standard curve as shown in Figure 1 Table 5.

[0173] Table 5

[0174]

[0175] As can be seen from the test results in Table 5, the enzyme activity of some single-point mutants is better than that of the wild type, wherein the enzyme activity of M48I, C73S, Y529S, N581R is significantly lower than that of the wild type, and the enzyme activity of T22A, T49A, L90P, V104E is significantly higher than that of the wild type.

[0176] 2. Comparison of enzyme activity of sucrose synthase SrSUS1 and its multi-point combination mutants:

[0177] DNS method was used to determine the enzyme activity of the crude enzyme, and the fructose content generated by the crude enzyme cleaving sucrose was determined. 0.02 mg / mL crude enzyme was added to 100 μL reaction solution containing 200 mM sucrose, 5 mM UDP and HEPES buffer (50 mM, pH 7.0), and reacted at 37°C for 20 min, and then heated at 95°C for 5 min to terminate the reaction. At the same time, the reaction without adding the crude enzyme was used as a control experiment to exclude the influence of heat treatment on sucrose decomposition. 200 μL DNS reagent was added to the inactivated reaction solution, heated in a boiling water bath for 2 min, then cooled to room temperature, 700 μL distilled water was added, and the absorbance of the sample at 540 nm was determined. The fructose content was calculated by comparing with the DNS standard curve as shown in Figure 1 Table 6.

[0178] Table 6

[0179]

[0180] As can be seen from the test results in Table 6, the enzyme activity of the multi-point combination mutants is higher than that of the wild type, and the specific data are shown in Table 6. As can be seen from the results, the multi-point combination mutation effectively maintains higher enzyme activity than the wild type. Among them, the T49A / L90P / V104E mutant has the highest enzyme activity.

[0181] Application Example One

[0182] Biocatalytic synthesis of RebM:

[0183] In the catalytic reaction system (10 mL), 20 g / L RebD, 120 g / L sucrose, 1 mM UDP, 10 mg / mL SrSUS1_T22A / T49A / L90P / V104E-76G4 of Example 3 and 100 mM potassium phosphate buffer solution (pH 7.2) were added, and the reaction was carried out at 40℃ for 24 h. During the reaction, 20 g / L RebD was added at 3h, 6h, 9h and 12h, and 120 g / L sucrose was added at 9h. After 24h of high temperature inactivation, the supernatant was obtained by centrifugation, which was RebM. The RebD supplement curve is shown in Figure 2 As can be seen from Figure 2 , the concentration of RebM at 24h of reaction is 93.35 g / L, and the yield reaches 93.35%.

[0184] Application Example Two

[0185] Compared with application example one, an equal amount of SrSUS1_T22A-76G4 was used to replace SrSUS1_T22A / T49A / L90P / V104E-76G4, and other conditions were the same as application example one. The concentration of RebM in the supernatant at 24h of reaction was 80.26 g / L, and the yield reached 80.26%.

[0186] Application Example Three

[0187] Compared with application example one, an equal amount of SrSUS1_L90P / V104E-76G4 was used to replace SrSUS1_T22A / T49A / L90P / V104E-76G4, and other conditions were the same as application example one. The concentration of RebM in the supernatant at 24h of reaction was 85.48 g / L, and the yield reached 85.48%.

[0188] The principles and implementations of the present application are described herein with specific examples, and the above descriptions of the examples are only used to help understand the method of the present application and its core ideas, including the best mode, and also enable any person skilled in the art to practice the present application, including manufacturing and using any device or system, and implementing any combined method. It should be noted that, for those skilled in the art, some improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application. The scope of patent protection of the present application is defined by the claims, and can include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to the literal expressions of the claims, or if they include equivalent structural elements that are not substantially different from the literal expressions of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A sucrose synthase mutant, characterized in that: The amino acid sequence of the mutant corresponds to any of the mutations in SEQ ID NO:1: T22A, V47L, T49A, S60K, L90P, V104E, T150S, A221D, Q226E, G529S, V658Y, T719A, T729C, D758E, T22A / T49A, T22A / L90P, T22A / V104E, T49A / L90P, T49A / V104E, L90P / V104E, T22A / T49A / L90P, T22A / T49A / V104E, T22A / L90P / V104E, T49A / L90P / V104E, T22A / T49A / L90P / V104E.

2. An expressed gene, characterized in that: The nucleotide sequence encoding the sucrose synthase mutant of claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is linked with the nucleotide sequence of the sucrose synthase mutant of claim 2 and the glycosyltransferase UGT76G4 gene.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant plasmid of claim 3 or the expression gene of claim 2.

5. The application of a sucrose synthase mutant, characterized in that: Used to catalyze the synthesis of steviol glycoside RebM.

6. The application of the sucrose synthase mutant according to claim 1, characterized in that, Includes the following steps: S1: Constructing a recombinant strain containing dual enzyme co-expression: The codon-optimized gene encoding the glycosyltransferase UGT76G4 was cloned into an expression vector to obtain the first plasmid. The gene fragment of the sucrose synthase mutant was cloned into the first plasmid to obtain the recombinant plasmid. The above recombinant plasmid was transformed into the host bacteria to obtain a recombinant strain containing co-expression of the two enzymes. S2: Induced enzyme production by recombinant strains: After activation, the above recombinant strains were transferred to LB liquid medium, and IPTG was added to induce culture. After low-temperature centrifugation, the bacterial cells were collected, resuspended in an appropriate amount of buffer, and lysed. The supernatant was collected by centrifugation and was the crude enzyme solution. S3: Biocatalytic synthesis of steviol glycoside RebM: The above crude enzyme solution, RebD, sucrose, and UDP were added to the catalytic reaction system to carry out the glycosylation reaction. After the reaction was completed, the temperature was raised to terminate the reaction, centrifuged, and the supernatant was collected, which is the stevioside RebM.

7. The application of the sucrose synthase mutant according to claim 6, characterized in that: In step S1, the expression vector is pRSFDuet-1; Alternatively, in step S2, the final concentration of the inducer is 0.02-1 mM, the induction culture temperature is 10-30℃, the rotation speed is 150-300 r / min, and the induction time is 24 h.

8. The application of the sucrose synthase mutant according to claim 6, characterized in that: In step S3, the initial concentration of RebD in the catalytic reaction system is 20 g / L, the concentration of sucrose is 1-500 mM, the concentration of crude enzyme is 0.1-1000 g / L, and the concentration of UDP is 1 mM. Alternatively, the reaction temperature for glycosylation is 20-40℃, and the reaction time is 1-24h.

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