Glycosyltransferase mutants and uses thereof

Abstract

The present application relates to glycosyltransferase MS1 mutant; meanwhile the present application provides a method for converting mogrol to mogroside IV and mogroside V by in vitro multi-enzyme catalysis, using mogrol and UDP-glucose (or UDP and sucrose) as raw materials in a multi-enzyme reaction system. In the whole mogroside synthesis pathway, only sucrose and mogrol or intermediate products need to be supplemented, so as to realize the efficient synthesis of mogroside IV and V. The method has low cost, simple process and high conversion efficiency, can improve the sweetness of immature momordica grosvenori fruit extract, and can be used as a substitute method for traditional planting to obtain mogroside.

Description

Technical Field

[0001] This invention relates to a method for the biological synthesis of mogrosides, belonging to the field of biosynthesis technology of sweeteners. Background Technology

[0002] Mogrosides are tetracyclic triterpenoid compounds isolated from monk fruit (Siraitia grosvenorii), and are its main sweetener, characterized by zero calories and high sweetness. In addition to its antitussive and cough-relieving effects, mogrosides have also been shown to possess anticancer, antitumor, and antibacterial bioactivities. However, due to the complex structure and low abundance of mogrosides in nature, coupled with the demanding environmental requirements of monk fruit cultivation, traditional cultivation methods limit their further application. Therefore, there is an urgent need to develop an alternative preparation method for mogrosides.

[0003] In the synthesis of monk fruit, the multi-stage glycosylation reaction catalyzed by glycosyltransferases is a key factor in the high sweetness of these substances. Currently, the number of glycosyltransferases reported in the literature capable of glycosylating mogrosides is limited, and those that exist suffer from low catalytic activity. Therefore, identifying glycosyltransferases capable of catalyzing the glycosylation of mogrosides and simultaneously improving the catalytic activity of these enzymes is of great significance for the development of sweeteners and for expanding the market for mogrosides.

[0004] We experimentally verified the application of glycosyltransferases MG1 and MS1 in the synthesis of mogrosides. Simultaneously, we designed, constructed, and experimentally confirmed the synthetic route for mogrosides. The process involved is as follows: a mutant glycosyltransferase MG1 catalyzes the sequential formation of mogrosides IE and IIE from mogrosides, followed by the formation of mogrosides IIIA, IIIE, IV, and V under the catalysis of MS1. In other words, the in vitro conversion synthesis from mogrosides to mogrosides IV and V was successfully achieved using these enzymes. Furthermore, through site-directed and combinatorial mutagenesis, the catalytic activity of MS1 was improved, thereby increasing the conversion rate of mogrosides. Summary of the Invention

[0005] This invention enhances the catalytic activity of glycosyltransferase MS1 for mogroside through site-directed mutagenesis and, through combination with different glycosyltransferases, successfully achieves the synthesis of mogroside from mogroside by converting mogroside from mogroside. It provides a method for preparing mogroside using mogroside and UDP-glucose as substrates, catalyzing the production of mogroside through a multi-enzyme reaction system. This verifies and fills the gap in the current understanding of the unrealized synthetic pathways from mogroside to mogrosides IV and V.

[0006] The present invention adopts the following technical solution:

[0007] This invention provides a glycosyltransferase MS1 mutant, characterized in that the amino acid sequence of the mutant contains a mutation in at least one of the following sites corresponding to SEQ ID NO. 2: 11, 13, 19, 25, 34, 37, 38, 40, 46, 62, 77, 82, 102, 107, 108, 116, 139, 142, 146, 16, 17, 18, 20, 21, 23, 24, 26, 354, 189, 195, 202, 225, 236, 243, 265, 267, 270, 292, 298, 344, 391, 313, 349, 359, 360, 366, 395, 417, 427, 438, 439, 222, 327.

[0008] According to the present invention, the amino acid sequence of the mutant contains a mutation of amino acid residues at at least one of the following sites corresponding to SEQ ID NO.2: 34, 77, 122, 144, 146, 225, 265, 298, 344, 391.

[0009] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any two sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at the aforementioned two sites, and at least one of the mutation sites is site 146. Preferably, one of the mutation sites in the amino acid sequence of the mutant is site 146A, and the other mutation site is any one of 34, 77, 122, 144, 225, 265, 298, 344, and 391.

[0010] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any three sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at sites 146 / 77 / 34.

[0011] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any four sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at sites 146 / 77 / 34 / 344.

[0012] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any five sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at sites 146 / 77 / 34 / 344 / 391.

[0013] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any six sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations in sites 146 / 77 / 34 / 344 / 391 / 313.

[0014] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any seven sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations in sites 146 / 77 / 34 / 344 / 391 / 313 / 360.

[0015] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any eight sites from SEQ ID NO. 2, namely 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at sites 146 / 77 / 34 / 344 / 391 / 313 / 360 / 122.

[0016] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any nine sites from SEQ ID NO. 2, namely 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations at sites 146 / 77 / 34 / 344 / 391 / 313 / 360 / 122 / 144.

[0017] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any ten sites from SEQ ID NO. 2, namely 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations in sites 146 / 77 / 34 / 344 / 391 / 313 / 360 / 122 / 144 / 225.

[0018] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any eleven sites among SEQ ID NO. 2: 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations in sites 146 / 77 / 34 / 344 / 391 / 313 / 360 / 122 / 144 / 225 / 265.

[0019] According to the present invention, the amino acid sequence of the mutant contains mutations in amino acid residues corresponding to any twelve sites from SEQ ID NO. 2, namely 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391. In one embodiment, the amino acid sequence of the mutant contains mutations in sites 146 / 77 / 34 / 344 / 391 / 313 / 360 / 122 / 144 / 225 / 265 / 298.

[0020] According to the present invention, the mutant has more than 70% homology with the amino acid sequence shown in SEQ ID NO.2, for example, more than 80% homology, and even more than 90%, more than 95%, or more than 98% homology.

[0021] The present invention also provides a nucleic acid encoding the above-mentioned glycosyltransferase MS1 mutant.

[0022] The present invention also provides a recombinant vector comprising nucleic acid encoding the above-mentioned glycosyltransferase MS1 mutant.

[0023] The present invention also provides a genetically engineered bacterium expressing the above-mentioned glycosyltransferase MS1 mutant, which contains nucleic acid encoding the glycosyltransferase MS1 mutant.

[0024] According to the present invention, the genetically engineered bacteria are recombinant strains obtained by ligating the nucleic acid vector to obtain a recombinant vector and then introducing it into a host bacterium.

[0025] According to the present invention, the host bacteria is any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, lactic acid bacteria, yeast, or Chinese hamster ovary cells, such as E. coli BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), EndoToxin-Free BL21(DE3), BL21 trxB(DE3), JM109, DH5α, top10, etc.

[0026] According to the present invention, the vector may be a prokaryotic expression vector or a eukaryotic expression vector, such as any one of the following: Escherichia coli expression vector, Bacillus subtilis expression vector, and Streptomyces expression vector. Preferably, the vector is selected from any one of pET15b, pET28a, pET32, pGEX4T1, and pGEX-6p-1.

[0027] According to the present invention, the nucleic acid and the vector are ligated by ligase or PCR recombination to form a recombinant vector.

[0028] According to the present invention, the genetically engineered bacteria express a glycosyltransferase MS1 mutant. Preferably, expression is induced, for example, by IPTG induction.

[0029] The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria, including the steps of obtaining a recombinant vector by linking the nucleic acid to the vector, and then introducing the recombinant strain into a host bacterium.

[0030] This invention provides the application of this genetically engineered bacterium in the preparation of glycosyltransferase MS1 mutants.

[0031] The present invention further provides a method for preparing a glycosyltransferase MS1 mutant, comprising the step of culturing the genetically engineered bacteria to express the nucleic acid encoding the glycosyltransferase MS1 mutant.

[0032] According to the present invention, the culture temperature is 35-40℃, preferably 37℃; the culture time is 1-3h, preferably 2h.

[0033] According to the present invention, the culture further includes an induction expression step, wherein after the inducer is added, the temperature is 10-20℃, preferably 16℃, and the culture time is 16-24h.

[0034] According to the present invention, the culture is carried out under stirring or oscillation conditions, for example, the stirring speed is 100-1000 rpm, preferably 200 rpm.

[0035] According to the present invention, the preparation method further includes the step of isolating and / or purifying the glycosyltransferase MS1 mutant from the culture.

[0036] The present invention also provides the application of the above-mentioned glycosyltransferase MS1 mutant in the preparation of mogroside.

[0037] Furthermore, the present invention also provides a method for producing mogroside.

[0038] According to the present invention, the mogroside is one or more of mogroside IE, IIE, IIIE, IIIA, IVA, Sia I or V.

[0039] According to the present invention, the mutant uses mogroside IE, IIE, IIIE, IIIA, IVE, or Sia I as a substrate for catalytic reaction. Preferably, the mutant uses mogroside IIE, IIIE, IIIA, IVE, or Sia I as a substrate for catalytic reaction.

[0040] According to the present invention, the method further includes the use of glycosyltransferase MG1. The glycosyltransferase MG1 catalyzes the reaction using mogroside or mogroside IE as a substrate.

[0041] This invention also provides a method for synthesizing a monk fruit sweetener, wherein the monk fruit sweetener is one or more of mogrosides IE, IIE, IIIE, IIIA, IVA, Sia I, or V. The method is characterized by comprising contacting the aforementioned glycosyltransferase MS1 mutant with mogrosides, mogrosides IE, IIE, IIIE, IIIA, IVE, or Sia I to carry out a catalytic reaction. Preferably, the contact is with mogrosides IIE, IIIE, IIIA, IVE, or Sia I.

[0042] According to the present invention, the above method further includes primary glycosylation modification catalyzed by glycosyltransferase MG1.

[0043] According to the present invention, the glycosyltransferase MG1 catalyzes a reaction using mogroside iodine or mogroside IE as a substrate. Preferably, the amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO.4.

[0044] This invention also provides a method and application for the enzymatic biosynthesis of mogrosides using the above-mentioned glycosyltransferase and its mutants.

[0045] According to the present invention, the method for synthesizing mogroside and its application include the following steps:

[0046] 1) Cultivate the genetically engineered bacteria of the present invention to express the nucleic acid encoding the MS1 mutant glycosyltransferase;

[0047] 2) Isolate and / or purify the MS1 glycosyltransferase mutant from the culture;

[0048] 3) Add mogroside IE, IIE, IIIE, IIIA, IVE or Sia I, and react with the MS1 mutant glycosyltransferase obtained in step 2) to obtain mogroside.

[0049] According to the present invention, the method or application further includes UDP-glucose or UDP and sucrose. Preferably, the sucrose is synthesized by sucrose synthase. The amino acid sequence of the sucrose synthase is shown in SEQ ID NO. 6.

[0050] This invention also provides a method and application for the whole-cell biosynthesis of mogrosides from the above-mentioned glycosyltransferase and its mutants.

[0051] According to the present invention, the method for synthesizing mogroside and its application include the following steps:

[0052] 1) Cultivate the genetically engineered bacteria of the present invention to express the nucleic acid encoding the MS1 mutant glycosyltransferase;

[0053] 2) Collect cells;

[0054] 3) Add mogroside IE, IIE, IIIE, IIIA, IVE or Sia I to the cells collected in step 2) and react them.

[0055] According to the method or application of the present invention, the glycosyltransferase MS1 mutant preferably corresponds to the S34A / F77L / V146A / A313V / T344V / M360L / A391V or S34A / F77L / V146A mutant of SEQ ID NO.2.

[0056] The method or application according to the present invention further includes the use of glycosyltransferase MG1. The amino acid sequence of said glycosyltransferase MG1 is shown in SEQ ID NO.4.

[0057] Beneficial effects

[0058] This invention provides a mutation site affecting the activity of glycosyltransferase MS1. Simultaneously, this invention provides a multi-enzyme reaction system using mogroside IV and mogroside V as raw materials, through in vitro multi-enzyme catalysis of mogroside IV to mogroside V. In the entire mogroside synthesis pathway, only sucrose and mogroside IV or intermediate products need to be added to achieve efficient synthesis of mogroside IV and V. The method of this invention is low-cost, simple, and highly efficient, and can improve the sweetness of extracts from immature mogroside fruits, serving as an alternative to traditional methods for obtaining mogrosides through cultivation. Attached Figure Description

[0059] Figure 1 . SDS-PAGE diagram of glycosyltransferase (1: MG1; 2: MS1). Specific Implementation

[0060] To facilitate understanding of the present invention, it will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to make the disclosure of the present invention more thorough and complete.

[0061] The following description is for illustrative purposes only, and only a small portion of the examples are presented; however, it should not be construed as limiting the invention. Unless otherwise specified, all reagents used in this invention are commercially available.

[0062] Example 1. Mutant Design

[0063] First, the glycosyltransferase MS1 (nucleotide sequence as SEQ ID NO:1, amino acid sequence as SEQ ID NO:2) derived from monk fruit was subjected to amino acid multiple sequence alignment analysis with other glycosyltransferases with triterpenoid secondary glycosylation modifications to identify important amino acid residue sites in the MS1 sequence that are related to catalytic and substrate recognition functions.

[0064] Simultaneously, the structure of the glycosyltransferase MS1 was predicted. Homology modeling of the MS1 protein was performed using software such as Swiss-Model, Phyre2, and Discovery Studio. Molecular docking was then conducted using substrates such as mogroside or shikimic acid to predict the enzyme's catalytic and substrate-binding sites, and the roles of amino acid residues near these sites were analyzed.

[0065] Finally, the key sites identified by the above homology comparison analysis and protein structure simulation were subjected to site-directed mutagenesis, and these mutant enzymes were expressed in E. coli for activity assay.

[0066] Example 2. Mutant activity assay

[0067] Enzyme activity reaction system: 0.2 mM mogroside IIe, 1 mM UDP-glucose, 5 mM MgCl2, and approximately 100 μg of purified enzyme solution of MS1 or its mutant were added. The reaction solution was then brought to 300 μL with 50 mM Tris-HCl (pH 8). The reaction was carried out in a 35°C water bath for 30-120 min (depending on the activity of different mutants). After the reaction was complete, an equal volume of chromatographic grade methanol was added to terminate the reaction. The mixture was centrifuged at 14000 rpm for 5 min, filtered through a 0.22 μm filter, and detected using Aglilent 1260 high-performance liquid chromatography. Analytical methods: C18 analytical column (4.6×250mm, 5μm); injection volume 20μL; mobile phase A (acetonitrile + 0.1% formic acid) and B (water + 0.1% formic acid), gradient elution conditions: 0-25min, 25%-85% gradient elution with pump A; mobile phase flow rate 1mL / min; UV detection wavelength 203nm.

[0068] Screening revealed that mutations at sites 11, 13, 19, 25, 34, 37, 38, 40, 46, 62, 77, 82, 102, 107, 108, 116, 139, 142, 146, 16, 17, 18, 20, 21, 23, 24, 26, 354, 189, 195, 202, 225, 236, 243, 265, 267, 270, 292, 298, 344, 391, 313, 349, 359, 360, 366, 395, 417, 427, 438, 439, 222, and 327 significantly affected the enzyme's catalytic activity.

[0069] The relative enzyme activities of T11R, L13A, N37A, S46W, S108D, H116E, V225T, Q243G, and F267A were 1.1, 1.3, 1.3, 1.5, 1.6, 3.1, 1.7, 1.4, and 1.5 times that of the wild type, respectively. Furthermore, ten sites closely related to activity were obtained, numbered 34, 77, 122, 144, 146, 225, 265, 298, 344, and 391 in the overall sequence. Single-point saturation mutagenesis was performed on these ten activity-related sites as follows: using the recombinant plasmid pET32-MS1 as a template, and a pair of primers containing the mutation sites, high-fidelity PCR amplification of the whole plasmid was performed to obtain the recombinant plasmid with the specified mutation sites. The amplification products were digested with DpnI enzyme at 37°C for 2 hours to degrade the initial template. The digestion products were transformed into E. coli BL21, plated onto LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. 200 positive clones were screened from each site.

[0070] A high-throughput screening method for mutants was established to analyze and determine the catalytic activity of the enzymes. Glycosyltransferase mutants with enhanced catalytic activity were sequenced to identify the amino acid mutations at corresponding positions. The mutation sites and relative activities of the mutants with enhanced activity are shown in Table 1, with the wild-type relative activity being 1.

[0071] Table 1

[0072]

[0073] Example 3. Combinatorial Mutation

[0074] Due to the relative activity of V146A, the above single mutation sites were combined to obtain double mutants with different combinations. The mutants with higher relative activity were then combined with other sites in round-by-round mutations, and the relative activities were measured. Table 2-3 lists the relative activities of the combined mutants with increased relative activity. The relative activity of wild type is 1.

[0075] Table 2

[0076]

[0077] Table 3

[0078]

[0079]

[0080] Example 4. Induced expression of glycosyltransferase

[0081] To obtain glycosyltransferases, *E. coli* containing pET32-MG1 (nucleotide sequence as shown in SEQ ID NO:3, amino acid sequence as shown in SEQ ID NO:4) and MS1 plasmids were inoculated into LB liquid medium containing 100 mg / mL ampicillin and cultured overnight at 37°C and 200 rpm to obtain seed culture. A 1% inoculum was then inoculated into 50 mL of fresh LB medium and cultured at 37°C and 200 rpm for 2 h. When OD... 600 When the pH reached approximately 0.7, isopropyl β-D-1-thiogalactopyranose (IPTG) was added to the culture medium to a final concentration of 0.4 mM, and induction was performed at 16°C and 200 rpm for 16 to 24 h. The bacteria were harvested by centrifugation at 6000 rpm, resuspended in 1 mL of Tris·HCl (pH = 8.0), sonicated, and centrifuged at 14000 rpm for 1 h. The supernatant was used as the crude enzyme solution. Purification was performed using Ni column affinity chromatography, followed by ultrafiltration using a 50 kDa ultrafiltration tube to obtain concentrated and purified wild-type and mutant proteins (e.g., ...). Figure 1 (As shown).

[0082] Example 5. Evaluation of Glycosyltransferase Activity

[0083] Tris-HCl (pH = 8.0), 0.2 mM mogroside or mogroside, 1 mM UDP-glucose, 10 mM MgCl2, 100 μg MS1 or its mutant pure enzyme solution, reacted at 40℃ for 12 h. After the glycosylation reaction was completed, an equal volume of methanol was added to terminate the reaction, and the determination was performed using liquid chromatography and mass spectrometry. High-performance liquid chromatography (HPLC) analysis was performed under the following conditions: Agilent HPLC 1200 instrument, C18 column, mobile phase: water + 0.1% formic acid, acetonitrile + 0.1% formic acid, flow rate: 1 mL / min, sample loading volume: 20 μL. Gradient elution conditions: 0-30 min, 25%-80% acetonitrile (0.1% formic acid) flow rate: 1 mL / min, UV detection wavelength: 203 nm. Mass spectrometry conditions: positive ion mode, ESI ion source.

[0084] Example 6. Enzymatic synthesis of mogroside IIIE

[0085] The 10 mL reaction system included 10 mg of mogroside ethanol, 50 mg of UDP-glucose, 25 mg of MG1 glycosyltransferase enzyme solution, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 2-5 hours, 300 μL of sample was taken and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged, filtered, and analyzed by HPLC (Example 5). The results showed that mogroside ethanol first generated mogroside IE, followed by mogroside IIE, and finally, mogroside ethanol was completely converted into the single product mogroside IIE.

[0086] Example 7. Whole-cell biosynthesis of mogroside IIIE

[0087] After induction of expression, *E. coli* cells (containing pET32-MG1) were collected, resuspended in PB buffer (50 mM, pH 8.0), and 10 mg of mogroside and 5% glucose were added. The mixture was reacted at 37°C for 12-24 hours, followed by HPLC analysis. The results showed that mogroside was completely converted to the single product mogroside IIE.

[0088] Example 8. Synthesis of mogroside IVA using MS1 mutant glycosyltransferase via enzymatic conversion.

[0089] The 10 mL reaction system included 10 mg of the substrate mogroside IIE or IIIA, 50 mg of UDP-glucose, 50 mg of glycosyltransferase MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant enzyme solution, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 24 hours, 300 μL of the sample was taken and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged, filtered, and analyzed by HPLC. The results showed that the mutants could completely convert the substrate to mogroside IVA.

[0090] Example 9. Synthesis of mogroside IVA using whole-cell conversion of MS1 mutant glycosyltransferase.

[0091] After induction of expression, E. coli cells (containing pET32-MS1) were collected. S34A / F77L / V146A / A313V / T344V / M360L / A391V The substrate was resuspended in PB buffer (50 mM, pH = 8.0), 5% glucose was added, and 10 mg of mogroside IIE or IIIA was added. After reacting at 37°C for 36 hours, HPLC analysis was performed. The results showed that the mutants could completely convert the substrate to mogroside IVA.

[0092] Example 10. Synthesis of mogroside Sia I using MS1 mutant glycosyltransferase via enzymatic conversion.

[0093] The 10 mL reaction system included 10 mg of the substrate mogroside IIIE, 50 mg of UDP-glucose, 50 mg of glycosyltransferase MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant enzyme solution, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 24 hours, 300 μL of the sample was taken and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged, filtered, and analyzed by HPLC. The results showed that the mutants could completely convert the substrate to generate mogroside Sia I (95%) and IVA (5%).

[0094] Example 11. Synthesis of mogroside Sia I using whole-cell method with MS1 mutant glycosyltransferase

[0095] After induction of expression, E. coli cells (containing pET32-MS1) were collected. S34A / F77L / V146A / A313V / T344V / M360L / A391V The mixture was resuspended in PB buffer (50 mM, pH 8.0), and 10 mg of mogroside IIIE and 5% glucose were added. After reacting at 37°C for 36 hours, HPLC analysis was performed. The results showed that the mutants could completely convert the substrate to mogroside Sia I.

[0096] Example 12. Synthesis of mogroside V using MS1 mutant glycosyltransferase

[0097] The 10 mL reaction system included 10 mg of the substrate mogroside Iv or Sia I, 50 mg of UDP-glucose, 50 mg of glycosyltransferase MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant enzyme solution, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 5–24 hours, 300 μL of sample was taken and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged, filtered, and analyzed by HPLC. The results showed that the mutants could completely transform the substrate and specifically generate mogroside V.

[0098] Example 13. Synthesis of mogroside V using whole-cell conversion of MS1 mutant glycosyltransferase.

[0099] After induction of expression, E. coli cells (containing pET32-MS1) were collected. S34A / F77L / V146A / A313V / T344V / M360L / A391V The mixture was resuspended in PB buffer (50 mM, pH 8.0), and 10 mg of mogroside IV or SiaI and 5% glucose were added. After reacting at 37°C for 36 hours, HPLC analysis was performed. The results showed that the mutants could specifically convert the substrate to mogroside V.

[0100] Example 14. Dual-enzyme tandem catalysis of mogroside V from mogroside V

[0101] The 10 mL reaction system included 10 mg of mogroside V, 130 mg of UDP-glucose, 125 mg of glycosyltransferase MG, 50 mg of MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant enzyme solution, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 24 hours, 300 μL of the sample was taken and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged, filtered, and analyzed by HPLC. The results showed complete substrate conversion, and the mogroside V content in the product was over 99%.

[0102] Example 15. Multi-enzyme tandem catalysis of mogroside V from mogroside V

[0103] The 10 mL reaction system included 10 mg of mogroside V, 130 mg of UDP-glucose, 125 mg of glycosyltransferase MG, 50 mg of MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant enzyme solution, 50 mg of MS2 (nucleotide sequence as shown in SEQ ID NO:9, amino acid sequence as shown in SEQ ID NO:10), 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 8.0). After reacting for 12, 16, and 24 hours, 300 μL of sample was added to an equal volume of methanol to terminate the reaction, followed by centrifugation, filtration, and HPLC analysis. The results showed complete substrate conversion after 16 hours, with mogroside V accounting for over 99% of the product. Compared to Example 14, the reaction time to reach the final reaction state was shortened.

[0104] Example 16. Biosynthesis of UDP-glucose and mogroside V supplied by sucrose synthase

[0105] Using the pET21 plasmid and with the aid of BamHI and XhoI restriction sites, an expression vector for sucrose synthase (Arabidopsisthaliana sucrose synthase, abbreviated as AtSUS1, nucleotide sequence as shown in SEQ ID NO:5, amino acid sequence as shown in SEQ ID NO:6) was constructed and introduced into *E. coli* BL21. Following the method in Example 4, strains containing glycosyltransferases MG1 and MS1 and sucrose synthase AtSUS1 were induced to express the enzymes. After completion, the bacterial cells were collected, lysed, and purified to obtain the aforementioned enzyme proteins. A biosynthetic system (10 mL) of mogroside based on the UDP-glucose cycle was constructed: 10 mg of mogroside substrate, 10 mg of UDP, 25 mg of glycosyltransferase MG1, 50 mg of MS1-S34A / F77L / V146A mutant enzyme solution, 10 mg of sucrose synthase AtSUS1, 10 mM MgCl2, and 50 mM Tris-HCl buffer (pH = 7.0). The reaction was carried out for 6-24 hours, and samples were taken for liquid chromatography analysis. The results showed that the conversion efficiency was good and the substrate conversion efficiency was faster than that in Example 14. This indicates that the combined use with sucrose synthase is more conducive to glycoside synthesis.

[0106] Example 17. Whole-cell biosynthesis of mogroside V in yeast

[0107] First, the Exg hydrolase gene in *Saccharomyces cerevisiae* was replaced and knocked out using a resistance gene to obtain the engineered yeast strain Sg-01. Then, a yeast expression plasmid containing the glycosyltransferase MG1 and the MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant was transformed into strain Sg-01. Through defect screening, strain Sg-02 was obtained. Recombinant yeast cells were picked and inoculated into 5 mL of SD medium and cultured overnight at 30°C. Then, at a 1% inoculation rate, the cells were inoculated into 50 mL of fresh SD medium (500 mL Erlenmeyer flasks) and cultured at 30°C and 220 rpm for 96 h. The cells were then collected by centrifugation and washed with PBS buffer (pH 8.0). The bacterial cells were resuspended in 1 mL of PBS buffer (pH 8.0, containing 2% glucose), and 0.2 mM mogroside was added. The reaction was carried out at 40℃ and 700 rpm for 48 h to generate mogroside V.

[0108] Example 18. Optimization of the UDP-glucose biosynthesis pathway and transformation of MS1 yeast strain with mogrosides

[0109] First, the synthetic UDP-glucose genes GPD1 (nucleotide sequence as shown in SEQ ID NO:7) and PGM2 (nucleotide sequence as shown in SEQ ID NO:8), along with their respective promoters and terminators, were inserted into the Exg hydrolase gene to obtain a yeast strain Sg-03 with increased UDP-glucose content and a knockout of the glycoside hydrolase gene. Then, yeast expression plasmids containing glycosyltransferase MG1 and MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutants were transformed into strain Sg-03. After defect screening, strain Sg-04 was obtained. Recombinant yeast cells were inoculated into 5 mL of SD medium and cultured overnight at 30°C. Then, at a 1% inoculation rate, the cells were inoculated into 50 mL of fresh SD medium (500 mL Erlenmeyer flasks) and cultured at 30°C and 220 rpm for 96 h. The cells were then collected by centrifugation and washed with PBS buffer (pH 8.0). The bacterial cells were resuspended in 1 mL of PBS buffer (pH 8.0, containing 2% glucose), and 0.2 mM mogroside was added. The reaction was carried out at 40℃ and 700 rpm for 48 h to generate mogroside V. The product generation rate was faster than that of strain Sg-02.

[0110] Example 19. Optimization of UDP-glucose biosynthesis pathway and transformation of MS1 yeast strain with mogroside

[0111] First, the synthetic UDP-glucose genes GPD1 (nucleotide sequence as shown in SEQ ID NO:7) and PGM2 (nucleotide sequence as shown in SEQ ID NO:8), along with their respective promoters and terminators, were inserted into the Exg hydrolase gene to obtain a yeast strain Sg-03 with increased UDP-glucose content and a knockout of the glycoside hydrolase gene. Then, yeast expression plasmids containing glycosyltransferases MG1, the MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V mutant, and MS2 were transformed into strain Sg-03. After defect screening, strain Sg-05 was obtained. Recombinant yeast cells were inoculated into 5 mL of SD medium and cultured overnight at 30°C. Then, at a 1% inoculation rate, the cells were inoculated into 50 mL of fresh SD medium (500 mL Erlenmeyer flasks) and cultured at 30°C and 220 rpm for 96 h. The cells were then collected by centrifugation and washed with PBS buffer (pH 8.0). The bacterial cells were resuspended in 1 mL of PBS buffer (pH 8.0, containing 2% glucose), and 0.2 mM mogroside was added. The reaction was carried out at 40℃ and 700 rpm for 48 h to generate mogroside V.

[0112] Example 19. Construction of a yeast strain with increased MS1 copy number of glycosyltransferase and conversion with mogroside

[0113] Yeast expression plasmids containing 2-4 copies of the glycosyltransferase mutant MS1-S34A / F77L / V146A / A313V / T344V / M360L / A391V were transformed into strain Sg-03. After defect screening, strains Sg-06 to Sg-08 were obtained. Recombinant yeast cells were inoculated into 5 mL of SD medium and cultured overnight at 30°C. Then, 1% of the cells were inoculated into 50 mL of fresh SD medium (500 mL Erlenmeyer flasks) and cultured at 30°C and 220 rpm for 96 h. The cells were collected by centrifugation and washed with PBS buffer (pH 8.0). The cells were resuspended in 1 mL of PBS buffer (pH 8.0, containing 2% glucose), and 0.2 mM mogroside IIE was added. The transformation was carried out at 40°C and 700 rpm for 24-48 h to generate mogroside V, with the product formation rate gradually increasing with increasing copy number.

[0114] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. SEQUENCE LISTING <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A glycosyltransferase mutant and its application <130> CPCN22410276 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 1365 <212> DNA <213> Artificial synthesis <400> 1 atggatgctg cccaacaagg tgacaccaca accattttga tgcttccatg gctcggctat 60 ggccatcttt cagcttttct cgagctggcc aaaagcctct caaggaggaa cttccatatc 120 tacttctgtt caacctctgt taatcttgac gccattaaac caaagcttcc ttcttctttc 180 tctgattcca ttcaatttgt ggagctccat ctcccttctt ctcctgagct ccctcctcat 240 cttcacacaa ccaacggcct tccccctacc ctcatgcccg ctctccacca agccttctcc 300 atggctgccc agcactttga gtccatttta caaacacttg ccccgcacct tctcatttat 360 gactctcttc aaccttgggc tcctcgggta gcttcatccc tcaaaattcc ggccatcaac 420 ttcaatacta cgggagtttt cgtcatttct caagggcttc accctattca ctacccacat 480 tctaaattcc cattctcaga gttcgttctt cacaatcatt ggaaagccat gtactccact 540 gccgatggag cttctaccga aagaacccgc aaacgtggag aagcgtttct gtattgcttg 600 catgcttctt gtagtgtgat tctaatcaat agtttcagag agctcgaggg gaaatatatg 660 gattatctct ctgttctctt gaacaagaaa gttgttccgg ttggtccttt ggtttacgaa 720 ccgaatcaag acggggaaga tgaaggttat tcaagcatca aaaattggct tgacaaaaag 780 gaaccgtcct ccaccgtctt cgtgtcattt ggaagcgaat acttcccgtc aaaggaagaa 840 atggaagaga tagcccatgg gttagaggcg agcgaggtta atttcatctg ggtcgttagg 900 tttcctcaag gagacaacac cagcggcatt gaagatgcct tgccgaaggg ttttctggag 960 agggcgggag agagagggat ggtggtgaag ggttgggctc ctcaggcgaa gatactgaag 1020 cattggagca cagggggatt cgtgagccac tgtggatgga actcggtgat ggagagcatg 1080 atgtttggcg ttcccataat aggggttccg atgcatgtgg accagccctt taacgccgga 1140 ctcgtggaag aagctggcgt cggcgtggag gccaagcgag atccagacgg aaaaattcaa 1200 agagacgaag ttgcaaagtt gatcaaagaa gtggtggttg agaaaaccag agaagatgtg 1260 cggaagaaag caagagaaat gagtgagatt ttgaggagca agggagagga gaagtttgat 1320 gagatggtcg ctgaaatttc tctcttgctt aaaatactcg agtaa 1365 <210> 2 <211> 452 <212> PRT <213> Artificial sequence <400> 2 Met Asp Ala Ala Gln Gln Gly Asp Thr Thr Thr Ile Leu Met Leu Pro 1 5 10 15 Trp Leu Gly Tyr Gly His Leu Ser Ala Phe Leu Glu Leu Ala Lys Ser 20 25 30 Leu Ser Arg Arg Asn Phe His Ile Tyr Phe Cys Ser Thr Ser Val Asn 35 40 45 Leu Asp Ala Ile Lys Pro Lys Leu Pro Ser Ser Phe Ser Asp Ser Ile 50 55 60 Gln Phe Val Glu Leu His Leu Pro Ser Ser Pro Glu Phe Pro Pro His 65 70 75 80 Leu His Thr Thr Asn Gly Leu Pro Pro Thr Leu Met Pro Ala Leu His 85 90 95 Gln Ala Phe Ser Met Ala Ala Gln His Phe Glu Ser Ile Leu Gln Thr 100 105 110 Leu Ala Pro His Leu Leu Ile Tyr Asp Ser Leu Gln Pro Trp Ala Pro 115 120 125 Arg Val Ala Ser Ser Leu Lys Ile Pro Ala Ile Asn Phe Asn Thr Thr 130 135 140 Gly Val Phe Val Ile Ser Gln Gly Leu His Pro Ile His Tyr Pro His 145 150 155 160 Ser Lys Phe Pro Phe Ser Glu Phe Val Leu His Asn His Trp Lys Ala 165 170 175 Met Tyr Ser Thr Ala Asp Gly Ala Ser Thr Glu Arg Thr Arg Lys Arg 180 185 190 Gly Glu Ala Phe Leu Tyr Cys Leu His Ala Ser Cys Ser Val Ile Leu 195 200 205 Ile Asn Ser Phe Arg Glu Leu Glu Gly Lys Tyr Met Asp Tyr Leu Ser 210 215 220 Val Leu Leu Asn Lys Lys Val Val Pro Val Gly Pro Leu Val Tyr Glu 225 230 235 240 Pro Asn Gln Asp Gly Glu Asp Glu Gly Tyr Ser Ser Ile Lys Asn Trp 245 250 255 Leu Asp Lys Lys Glu Pro Ser Ser Thr Val Phe Val Ser Phe Gly Ser 260 265 270 Glu Tyr Phe Pro Ser Lys Glu Glu Met Glu Glu Ile Ala His Gly Leu 275 280 285 Glu Ala Ser Glu Val Asn Phe Ile Trp Val Val Arg Phe Pro Gln Gly 290 295 300 Asp Asn Thr Ser Gly Ile Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu 305 310 315 320 Arg Ala Gly Glu Arg Gly Met Val Val Lys Gly Trp Ala Pro Gln Ala 325 330 335 Lys Ile Leu Lys His Trp Ser Thr Gly Gly Phe Val Ser His Cys Gly 340 345 350 Trp Asn Ser Val Met Glu Ser Met Met Phe Gly Val Pro Ile Ile Gly 355 360 365 Val Pro Met His Val Asp Gln Pro Phe Asn Ala Gly Leu Val Glu Glu 370 375 380 Ala Gly Val Gly Val Glu Ala Lys Arg Asp Pro Asp Gly Lys Ile Gln 385 390 395 400 Arg Asp Glu Val Ala Lys Leu Ile Lys Glu Val Val Val Glu Lys Thr 405 410 415 Arg Glu Asp Val Arg Lys Lys Ala Arg Glu Met Ser Glu Ile Leu Arg 420 425 430 Ser Lys Gly Glu Glu Lys Phe Asp Glu Met Val Ala Glu Ile Ser Leu 435 440 445 Leu Leu Lys Ile 450 <210> 3 <211> 1362 <212> DNA <213> Artificial sequence <400> 3 atggaaaagg gtgacactca catcttggtt ttcccattcc cagctcaagg tcacattaac 60 ccattgttgc aattgtctaa gcacttgatc gctaagggta ttaaggtttc tttggtcact 120 accttgcacg tctctaacag aatgcaattg caaggtgctt actctaactc tgttaagatc 180 gaagtcattt ctgacggttc tgaagacaga ttggaaactg acaccttgag acaatacttg 240 gacagattca gacaaaagat gaccaagaac ttggaagact tcttgcaaaa ggctatggtt 300 tcttctaacc caccaaagtt catcatctac gactctacta tgccatgggt tttggaagtc 360 gctaaggaat tcggtttgga cagagctcca ttctacaccc aatcttgtgc tttgaactct 420 atcaactacc acgttttgca cggtcaattg aagttgccac cagaaactcc aaccatttct 480 ttgccatcta tgccattgtt aagaccatct gacttgccag cttacgactt cgacccagct 540 tctactgaca ccatcattga cttgttgact tctcaatact ctaacatcca agacgctaac 600 ttgttgttct gtaacacttt cgacaagttg gaaggtgaaa tcattcaatg gatggaaacc 660 ttgggtagac cagttaagac tgtcggtcca accgttccat ctgcttactt ggacaagaga 720 gtcgaaaacg acaagcacta cggtttgtct ttgttcaagc caaacgaaga cgtttgtttg 780 aagtggttgg actctaagcc atctggttct gttttgtacg tctcttacgg ttctttggtc 840 gaaatgggtg aagaacaatt gaaggaattg gctttgggta ttaaggaaac tggtaaattc 900 ttcttgtggg ttgtcagaga caccgaagct gaaaagttgc caccaaactt cgttgaatct 960 gtcgctgaaa agggtttggt tgtctcttgg tgttctcaat tggaagtttt ggctcaccca 1020 tctgtcggtt gtttcttcac tcactgtggt tggaactcta ccttggaagc tttgtgtttg 1080 ggtgttccag ttgtcgcttt cccacaatgg gctgaccaag tcactaacgc taagttcttg 1140 gaagacgttt ggaaggtcgg taaaagagtt aagagaaacg aacaaagatt ggcttctaag 1200 gaagaagtta gatcttgtat ctgggaagtc atggaaggtg aaagagcttc tgaattcaag 1260 tctaactcta tggaatggaa gaagtgggct aaggaagctg ttgacgaagg tggttcttct 1320 gacaagaaca ttgaagaatt cgtcgctatg ttgaagcaaa cc 1362 <210> 4 <211> 454 <212> PRT <213> Artificial sequence <400> 4 Met Glu Lys Gly Asp Thr His Ile Leu Val Phe Pro Phe Pro Ala Gln 1 5 10 15 Gly His Ile Asn Pro Leu Leu Gln Leu Ser Lys His Leu Ile Ala Lys 20 25 30 Gly Ile Lys Val Ser Leu Val Thr Thr Leu His Val Ser Asn Arg Met 35 40 45 Gln Leu Gln Gly Ala Tyr Ser Asn Ser Val Lys Ile Glu Val Ile Ser 50 55 60 Asp Gly Ser Glu Asp Arg Leu Glu Thr Asp Thr Leu Arg Gln Tyr Leu 65 70 75 80 Asp Arg Phe Arg Gln Lys Met Thr Lys Asn Leu Glu Asp Phe Leu Gln 85 90 95 Lys Ala Met Val Ser Ser Asn Pro Pro Lys Phe Ile Ile Tyr Asp Ser 100 105 110 Thr Met Pro Trp Val Leu Glu Val Ala Lys Glu Phe Gly Leu Asp Arg 115 120 125 Ala Pro Phe Tyr Thr Gln Ser Cys Ala Leu Asn Ser Ile Asn Tyr His 130 135 140 Val Leu His Gly Gln Leu Lys Leu Pro Pro Glu Thr Pro Thr Ile Ser 145 150 155 160 Leu Pro Ser Met Pro Leu Leu Arg Pro Ser Asp Leu Pro Ala Tyr Asp 165 170 175 Phe Asp Pro Ala Ser Thr Asp Thr Ile Ile Asp Leu Leu Thr Ser Gln 180 185 190 Tyr Ser Asn Ile Gln Asp Ala Asn Leu Leu Phe Cys Asn Thr Phe Asp 195 200 205 Lys Leu Glu Gly Glu Ile Ile Gln Trp Met Glu Thr Leu Gly Arg Pro 210 215 220 Val Lys Thr Val Gly Pro Thr Val Pro Ser Ala Tyr Leu Asp Lys Arg 225 230 235 240 Val Glu Asn Asp Lys His Tyr Gly Leu Ser Leu Phe Lys Pro Asn Glu 245 250 255 Asp Val Cys Leu Lys Trp Leu Asp Ser Lys Pro Ser Gly Ser Val Leu 260 265 270 Tyr Val Ser Tyr Gly Ser Leu Val Glu Met Gly Glu Glu Gln Leu Lys 275 280 285 Glu Leu Ala Leu Gly Ile Lys Glu Thr Gly Lys Phe Phe Leu Trp Val 290 295 300 Val Arg Asp Thr Glu Ala Glu Lys Leu Pro Pro Asn Phe Val Glu Ser 305 310 315 320 Val Ala Glu Lys Gly Leu Val Val Ser Trp Cys Ser Gln Leu Glu Val 325 330 335 Leu Ala His Pro Ser Val Gly Cys Phe Phe Thr His Cys Gly Trp Asn 340 345 350 Ser Thr Leu Glu Ala Leu Cys Leu Gly Val Pro Val Val Ala Phe Pro 355 360 365 Gln Trp Ala Asp Gln Val Thr Asn Ala Lys Phe Leu Glu Asp Val Trp 370 375 380 Lys Val Gly Lys Arg Val Lys Arg Asn Glu Gln Arg Leu Ala Ser Lys 385 390 395 400 Glu Glu Val Arg Ser Cys Ile Trp Glu Val Met Glu Gly Glu Arg Ala 405 410 415 Ser Glu Phe Lys Ser Asn Ser Met Glu Trp Lys Lys Trp Ala Lys Glu 420 425 430 Ala Val Asp Glu Gly Gly Ser Ser Asp Lys Asn Ile Glu Glu Phe Val 435 440 445 Ala Met Leu Lys Gln Thr 450 <210> 5 <211> 3082 <212> DNA <213> Artificial Sequence <400> 5 gaactttcct attttcctaa aacacccttt tccttggact tttaaatcca ttatatttag 60 tcttctgggt ttggagccca agaaataaaa cgtagaagga aacagaggga agtaaatgat 120 tcatatcagc ttaccaaaat ttgtatcaag cttaaaaat tcgtctctct ataaatctga 180 aacatattga acattaacct ccacatctca ctcaaaacca cttctctata ctccagtttt 240 caaaaacttt ctcactgtta tctgcgtctc ttttttggat caatggcaaa cgctgaacgt 300 atgataacgc gcgtccacag ccaacgtgag cgtttgaacg aaacgcttgt ttctgagaga 360 aacgaagtcc ttgccttgct ttccagggtt gaagccaaag gtaaaggtat tttacaacaa 420 aaccagatca ttgctgaatt cgaagctttg cctgaacaaa cccggaagaa acttgaaggt 480 ggtccttct ttgaccttct caaatccact caggaagcaa ttgtgttgcc accatgggtt 540 gctctagctg tgaggccaag gcctggtgtt tgggaatact tacgagtcaa tctccatgct 600 cttgtcgttg aagaactcca acctgctgag tttcttcatt tcaaggaaga actcgttgat 660 ggagttaaga atggtaattt cactcttgag cttgatttcg agccattcaa tgcgtctatc 720 cctcgtccaa cactccacaa atacattgga aatggtgttg acttccttaa ccgtcattta 780 tcggctaagc tcttccatga caaggagagt ttgcttccat tgcttaagtt ccttcgtctt 840 cacagccacc agggcaagaa cctgatgttg agcgagaaga ttcagaacct caacactctg 900 caacacacct tgaggaaagc agaagagtat ctagcagagc ttaagtccga aacactgtat 960 gaagagtttg aggccaagtt tgaggagatt ggtcttgaga ggggatgggg agacaatgca 1020 gagcgtgtcc ttgacatgat acgtcttctt ttggaccttc ttgaggcgcc tgatccttgc 1080 actcttgaga cttttcttgg aagagtacca atggtgttca acgttgtgat cctctctcca 1140 catggttact ttgctcagga caatgttctt ggttaccctg acactggtgg acaggttgtt 1200 tacattcttg atcaagttcg tgctctggag atagagatgc ttcaacgtat taagcaacaa 1260 ggactcaaca ttaaaccaag gattctcatt ctaactcgac ttctacctga tgcggtagga 1320 actacatgcg gtgaacgtct cgagagagtt tatgattctg agtactgtga tattcttcgt 1380 gtgcccttca gaacagagaa gggtattgtt cgcaaatgga tctcaaggtt cgaagtctgg 1440 ccatatctag agacttacac cgaggatgct gcggttgagc tatcgaaaga attgaatggc 1500 aagcctgacc ttatcattgg taactacagt gatggaaatc ttgttgcttc tttattggct 1560 cacaacttg gtgtcactca gtgtaccatt gctcatgctc ttgagaaac aaagtacccg 1620 gattctgata tctactggaa gaagcttgac gaagtacc atttctcatg ccagttcact 1680 gcggatattt tcgcaatgaa ccacactgat ttcatcatca ctagtacttt ccaagaaatt 1740 gctggaagca aagaactgt tgggcagtat gaaagccaca cagcctttac tctcccgga 1800 ttgtatcgag ttgttcacgg gattgatgtg ttgatccca agttcacat tgtctctcct 1860 ggtgctgata tgagcatcta cttcccttac acgaggaga agcgtagatt gactaagttc 1920 cactctgaga tcgaggagct cctctacagc gatgttgaga aaagagca cttatgtgtg 1980 ctcaggaca agagaagcc gattctctc acaatggcta ggctgatcg tgtcaagac 2040 ttgtcaggtc ttgttgagtg gtacgggaag aacacccgct tgcgtgagct agctacttg 2100 gttgttgttg gaggle gagggag tchaaaaaaagggaagaaaaaaggg 2160 aagaaaatgt atgatctcat tgaggaatac aagctaaacg gtcagttcag gtggatctcc 2220 tctcagatgg accggggtaag gaacggtgag ctgtaccggt acatctgtga caccaagggt 2280 gcttttgtcc aacctgcatt atatgaagcc tttgggttaa ctgttgtgga ggctatgact 2340 tgtggtttac cgactttcgc cacttgcaaa ggtggtccag ctgagatcat tgtgcacggt aaatcgggtt tccacattga cccttaccat ggtgatcagg ctgctgatac tcttgctgat ttcttcacca agtgtaagga ggatccatct cactgggatg agatctcaaa aggagggctt cagaggattg aggagaaata cacttggcaa atctattcac aggctctt gacattgact ggtgtgtatg gattctgga gcatgtctcg aaccttgacc gtcttgaggc tcgccgttac 2640 cttgaaatgt tctatgcatt gaagtatcgc ccattggctc aggctgttcc tcttgcacaa gatgattga gagagaga aagactcgga accagtaag agtttgaga gagtggttcc ggtgtttgaa gaataaaca agatctcctt ttgattctta ttatcattcg gattgggaag cgcattttgt gttcctctgc ttctttgtta tttcaatcat ttgtctttgt tttccttgc 2880. ctctgtggca ttgctttgaa ttctggctgt tgggagattc cccttctaat tgcagtaaag tgtcttttgc aaaatttc tcaatattta catcttccag taaccaaatg caccttatta gcaaagaagt gtgaccatta tcagatcaca aaacaatatc aatatttgtt tgttaaagtt 3060 gggcttattt ctatggtgga ct 3082 <210> 6 <211> 808 <212> PRT <213> Artificial synthesis <400> 6 Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu 1 5 10 15 Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Path Leu 20 25 30 Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln 35 40 45 Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu 50 55 60 Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile 65 70 75 80 Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu 100 105 110 Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val 115 120 125 Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155 160 Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser 165 170 175 Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys 180 185 190 Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His 195 200 205 Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr 210 215 220 Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg 225 230 235 240 Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu 245 250 255 Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu 260 265 270 Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280 285 Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln 290 295 300 Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu 305 310 315 320 Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile 325 330 335 Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg 340 345 350 Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro 355 360 365 Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu 370 375 380 Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu 385 390 395 400 Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser 405 410 415 Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu 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 Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln 450 455 460 Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr 465 470 475 480 Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr 485 490 495 Glu Ser His Thr 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 Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr 530 535 540 Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn 545 550 555 560 Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe 565 570 575 Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu 580 585 590 Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val 595 600 605 Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala 610 615 620 Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly 625 630 635 640 Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu 645 650 655 Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala 660 665 670 Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly 675 680 685 Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val 690 695 700 His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala 705 710 715 720 Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser 725 730 735 His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys 740 745 750 Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val 755 760 765 Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg 770 775 780 Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln 785 790 795 800 Ala Val Pro Leu Ala Gln Asp Asp 805 <210> 7 <211> 1500 <212> DNA <213> Artificial synthesis <400> 7 atgtccacta agaagcacac caaaacacat tccacttatg cattcgagag caacacaaac 60 agcgttgctg cctcacaaat gagaaacgcc ttaaacaagt tggcggactc tagtaaactt 120 gacgatgctg ctcgcgctaa gtttgagaac gaactggatt cgtttttcac gcttttcagg 180 agatatttgg tagagaagtc ttctagaacc accttggaat gggacaagat caagtctccc 240 aacccggatg aagtggttaa gtatgaaatt atttctcagc agcccgagaa tgtctcaaac 300 ctttccaaat tggctgtttt gaagttgaac ggtgggctgg gtacctccat gggctgcgtt 360 ggccctaaat ctgttattga agtgagagag ggaaacacct ttttggattt gtctgttcgt 420 caaattgaat acttgaacag acagtacgat agcgacgtgc cattgttatt gatgaattct 480 ttcaacactg acaaggatac ggaacacttg attaagaagt attccgctaa cagaatcaga 540 atcagatctt tcaatcaatc caggttccca agagtctaca aggattcttt attgcctgtc 600 ccccaccgaat acgattctcc actggatgct tggtatccac caggtcacgg tgatttgttt 660 gaatctttac acgtatctgg tgaactggat gccttaattg cccaaggaag agaaatatta 720 tttgtttcta acggtgacaa cttgggtgct accgtcgact taaaaatttt aaaccacatg 780 atcgagactg gtgccgaata tataatggaa ttgactgata agaccagagc cgatgttaaa 840 ggtggtactt tgatttctta cgatggtcaa gtccgtttat tggaagtcgc ccaagttcca 900 aaagaacaca ttgacgaatt caaaaatatc agaaagttta ccaacttcaa cacgaataac 960 ttatggatca atctgaaagc agtaaagagg ttgatcgaat cgagcaattt ggagatggaa 1020 atcattccaa accaaaaaac tataacaaga gacggtcatg aaattaatgt cttacaatta 1080 gaaaccgctt gtggtgctgc tatcaggcat tttgatggtg ctcacggtgt tgtcgttcca 1140 agatcaagat tcttgcctgt caagacctgt tccgatttgt tgctggttaa atcagatcta 1200 ttccgtctgg aacacggttc tttgaagtta gacccatccc gttttggtcc aaacccatta 1260 atcaagttgg gctcgcattt caaaaaggtt tctggtttta acgcaagaat ccctcacatc 1320 ccaaaaatcg tcgagctaga tcatttgacc atcactggta acgtcttttt aggtaaagat 1380 gtcactttga ggggtactgt catcatcgtt tgctccgacg gtcataaaat cgatattcca 1440 aacggctcca tattggaaaa tgttgtcgtt actggtaatt tgcaaatctt ggaacattga 1500 <210> 8 <211> 1710 <212> DNA <213> Artificial sequence <400> 8 atgtcatttc aaattgaaac ggttcccacc aaaccatatg aagaccaaaa gcctggtacc 60 tctggtttgc gtaagaagac aaaggtgttt aaagacgaac ctaactacac agaaaatttc 120 attcaatcga tcatggaagc tattccagag ggttctaaag gtgccactct tgttgtcggt 180 ggtgatgggc gttactacaa tgatgtcatt cttcataaga ttgccgctat cggtgctgcc 240 aacggtatta aaaagttagt tattggccag catggtcttc tgtctacgcc agccgcttct 300 cacatcatga gaacctacga ggaaaaatgt actggtggta ttatcttaac cgcctcacat 360 aatccaggtg gtccagaaaa tgacatgggt attaagtata acttatccaa tgggggtcct 420 gctcctgaat ccgtcacaaa tgctatttgg gagatttcca aaaagcttac cagctataag 480 attatcaaag acttcccaga actagacttg ggtacgatag gcaagaacaa gaaatacggt 540 ccattactcg ttgacattat cgatattaca aaagattatg tcaacttctt gaaggaaatc 600 ttcgatttcg acttaatcaa gaaattcatc gataatcaac gttctactaa gaattggaag 660 ttactgtttg acagtatgaa cggtgtaact ggaccatacg gtaaggctat tttcgttgat 720 gaatttggtt taccggcgga tgaggtttta caaaactggc atccttctcc ggattttggt 780 ggtatgcatc cagatccaaa cttaacttat gccagttcgt tagtgaaaag agtagatcgt 840 gaaaagattg agtttggtgc tgcatccgat ggtgatggtg atagaaatat gatttacggt 900 tacggcccat ctttcgtttc tccaggtgac tccgtcgcaa ttattgccga atatgcagct 960 gaaatcccat atttcgccaa gcaagtata tatggtctgg cccgttcatt ccctacctca 1020 ggagccatag accgtgttgc CAggccat ggtctaact gttatgaggt cccaactggc 1080 tggaaatttttgtgcttt gttcgacgct aaaaattatttttgtgg tgagaatcg 1140 ttggtactg gttccaacca cgtaagggaa aaggacggtg tttggccat tatggcgtgg 1200 ttgaacatct tggccattta cacaaccat catccggaga acgaagctc tattagacg 1260 atacagaatg aattctgggc aaagtacggc cgtactttct tcactcgtta tgattttgaa 1320 aaagttgaaa cagaaaaagc taacaagatt gtcgatcaat tgagagcata tgttaccaaa 1380 tcggtgttg ttaattccgc cttcccagcc gatgagtctc ttaggtcac cgattgtggt 1440 gatttttcat acacagattt ggacggttct gttttctgacc atcaggtttt atatgtcag 1500 ctttccaatg gtgcaagatt cgttctaaga ttgtcaggta caggttctc aggtgctacc 1560 attagattgt acattgaaaa atactgcgat gataaatcac ataccaaaa gagagctgaa 1620 gaatacttga agccaattat taactcggtc atcaagttct tgaactttaa acaagtttta 1680 ggaactgaag aaccaacggt tcgtacttaa 1710 <210> 9 <211> 1389 <212> DNA <213> Artificial Synthesis <400> 9 atggatagtg gctactcctc atcttatgct gctgccgctg gtatgcacgt tgtgatctgc 60 ccttggttgg cctttggtca cctgttacca tgtctggatt tagcccaaag actggcctca 120 agaggccata gagtatcatt tgtgtctact cctagaaata tctctcgttt accaccagtc 180 agacctgctc tagctcctct agttgcattc gttgctcttc cacttccaag agtagaagga 240 ttgccagacg gcgctgaatc tactaatgac gtaccacatg atagacctga catggtcgaa 300 ttgcatagaa gagcctttga tggattggca gctccatttt ctgagttcct gggcacagca 360 tgtgcagact gggttatagt cgatgtattt catcactggg ctgctgcagc cgcattggaa 420 cataaggtgc cttgtgctat gatgttgtta gggtcagcac acatgatcgc atccatagct 480 gatagaagat tggaaagagc tgaaacagaa tccccagccg cagcaggaca aggtaggcca 540 gctgccgccc caacctttga agtggctaga atgaaattga ttcgtactaa aggtagttca 600 gggatgagtc ttgctgaaag gttttctctg acattatcta gatcatcatt agttgtaggt 660 agatcctgcg tcgagttcga acctgaaaca gtacctttac tatctacttt gagaggcaaa 720 cctattactt tccttggtct aatgcctcca ttacatgaag gaaggagaga agatggtgaa 780 gatgctactg ttaggtggtt agatgcccaa cctgctaagt ctgttgtta cgttgcattg 840 ggttctgagg taccactagg gttggaaaag gtgcatgaat tagcattagg acttgagctg 900 gccggaacaa gattcctttg ggctttgaga aaaccaaccg gtgtttctga cgccgacttg 960 ctaccagctg ggttcgaaga gagaacaaga ggccgtggtg tcgttgctac tagatgggtc 1020 ccacaaatga gtattctagc tcatgcagct gtaggggcct ttctaaccca ttgcggttgg 1080 aactcaacaa tagaaggact gatgtttggt catccactta ttatgttacc aatcgcgggc 1140 gatcagggac ctaacgcaag attgattgag gcaaagaacg caggtctgca ggttgcacgt 1200 aatgatggtg atggttcctt tgatagagaa ggcgttgcag ctgccatcag agcagtcgcc 1260 gttgaggaag agtcatctaa agttttccaa gctaaggcca aaaaattaca agagattgtg 1320 gctgacatgg cttgtcacga aagatacatc gatggtttca tccaacaatt gagaagttat 1380 aaagactaa 1389 <210> 10 <211> 462 <212> PRT <213> Artificial Synthesis <400> 10 Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His 1 5 10 15 Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu 20 25 30 Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val 35 40 45 Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu 50 55 60 Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly 65 70 75 80 Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro 85 90 95 Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro 100 105 110 Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp 115 120 125 Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro 130 135 140 Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala 145 150 155 160 Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly 165 170 175 Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys 180 185 190 Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Phe 195 200 205 Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val 210 215 220 Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys 225 230 235 240 Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg 245 250 255 Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala 260 265 270 Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val 275 280 285 Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg 290 295 300 Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu 305 310 315 320 Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala 325 330 335 Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly 340 345 350 Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met 355 360 365 Phe Gly His Pro Leu Ile Met Leu Pro Ile Ala Gly Asp Gln Gly Pro 370 375 380 Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg 385 390 395 400 Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile 405 410 415 Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys 420 425 430 Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg 435 440 445 Tyr with Asp Gly Phe with Gln Gln Leu Arg Ser Tyr Lys Asp 450 455 460

Claims

1. A glycosyltransferase MS1 mutant, characterized in that, The amino acid sequence of the mutant is a mutation of amino acid residues corresponding to the following sites in SEQ ID NO.

2. (1) S34A, F77L, V146A, A313V, T344V, M360L and A391V; or (2) S34A, F77L and V146A.

2. The nucleic acid encoding the MS1 mutant of the glycosyltransferase of claim 1.

3. A recombinant vector containing the glycosyltransferase MS1 mutant of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The vector can be a prokaryotic expression vector or a eukaryotic expression vector.

5. The recombinant vector according to claim 4, characterized in that, The vector is selected from any one of the following: Escherichia coli expression vector, Bacillus subtilis expression vector, and Streptomyces expression vector.

6. The recombinant vector according to claim 5, characterized in that, The carrier is selected from any one of pET15b, pET28a, pET32, pGEX4T1, and pGEX-6p-1.

7. The recombinant vector according to claim 3, characterized in that, The nucleic acid and the vector are ligated together by ligase or PCR recombination to form a recombinant vector.

8. Genetically engineered bacteria comprising the nucleic acid of claim 2 or the recombinant vector of claim 3.

9. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacteria are recombinant strains obtained by ligating the nucleic acid vector to obtain a recombinant vector and then introducing it into a host bacterium.

10. The genetically engineered bacterium according to claim 9, characterized in that, The host bacteria are any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, lactic acid bacteria, yeast, or Chinese hamster ovary cells.

11. The genetically engineered bacterium according to claim 10, characterized in that, The host bacteria is E. coli BL21(DE3), BL21(DE3) pLysS, Rosetta(DE3), EndoToxin-Free BL21(DE3), BL21 trxB (DE3), JM109, DH5α, top10.

12. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacteria express a glycosyltransferase MS1 mutant.

13. The genetically engineered bacterium according to claim 12, characterized in that, The expression described is an induced expression.

14. The genetically engineered bacterium according to claim 13, characterized in that, The induced expression was IPTG-induced expression.

15. The method for constructing the genetically engineered bacteria according to claim 8, comprising the steps of obtaining a recombinant vector by ligating the nucleic acid into the vector, and then introducing the recombinant strain into a host bacterium.

16. The use of the genetically engineered bacteria of claim 8 in the preparation of the MS1 mutant glycosyltransferase.

17. A method for preparing a glycosyltransferase MS1 mutant, comprising the step of culturing the genetically engineered bacteria of claim 8 to express nucleic acid encoding the glycosyltransferase MS1 mutant.

18. The method for preparing a glycosyltransferase MS1 mutant according to claim 17, characterized in that, The culture temperature is 35-40℃; the culture time is 1-3 h.

19. The method for preparing a glycosyltransferase MS1 mutant according to claim 18, characterized in that, The culture temperature was 37℃; the culture time was 2 h.

20. The method for preparing a glycosyltransferase MS1 mutant according to claim 17, characterized in that, The culture also includes an induction expression step, where the temperature is 10-20℃ after the inducer is added, and the culture time is 16-24h.

21. The method for preparing a glycosyltransferase MS1 mutant according to claim 20, characterized in that, The temperature is 16℃.

22. The method for preparing a glycosyltransferase MS1 mutant according to claim 17, characterized in that, The culture was carried out under stirring or shaking conditions.

23. The method for preparing a glycosyltransferase MS1 mutant according to claim 22, characterized in that, The stirring speed is 100-1000 rpm.

24. The method for preparing a glycosyltransferase MS1 mutant according to claim 23, characterized in that, The stirring speed is 200 rpm.

25. The method for preparing a glycosyltransferase MS1 mutant according to claim 17, characterized in that, The preparation method further includes the step of isolating and purifying the glycosyltransferase MS1 mutant from the culture.

26. The application of the MS1 mutant glycosyltransferase according to claim 1 in the preparation of mogroside; in, When the mogroside is mogroside IVA or Sia I, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1); When the mogroside is mogroside V, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1) or (2).

27. The application according to claim 26, characterized in that, It also includes using glycosyltransferase MG1 to catalyze reactions with mogroside iodine or mogroside IE as substrates.

28. The application according to claim 27, characterized in that, The amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO.

4.

29. A method for synthesizing monk fruit sweetener, wherein the monk fruit sweetener is one or more of mogroside IVA, Sia I or V, characterized in that, The method includes contacting the MS1 mutant of glycosyltransferase (1) according to claim 1 with mogroside IIE, IIIE, IIIA, IVE or Sia I to carry out a catalytic reaction; or contacting the MS1 mutant of glycosyltransferase (1) or (2) according to claim 1 with mogroside to carry out a catalytic reaction.

30. The method for synthesizing monk fruit sweetener according to claim 29, characterized in that, The method also includes primary glycosylation modification catalyzed by glycosyltransferase MG1, wherein MG1 catalyzes the reaction using mogroside or mogroside IE as substrates.

31. The method for synthesizing monk fruit sweetener according to claim 30, characterized in that, The amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO.

4.

32. The application of the glycosyltransferase mutant as described in claim 1 for the enzymatic biosynthesis of mogroside; in, When the mogroside is mogroside IVA or Sia I, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1); When the mogroside is mogroside V, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1) or (2).

33. The application according to claim 32, characterized in that, The application of the synthesized mogroside includes the following steps: 1) Cultivate the genetically engineered bacteria of claim 8 to express the nucleic acid encoding the MS1 mutant glycosyltransferase; 2) Isolate and purify the MS1 mutant glycosyltransferase from the culture; 3) Add mogroside IIE, IIIE, IIIA, IVE or Sia I, and react with the MS1 mutant glycosyltransferase obtained in step 2) to obtain the target mogroside.

34. The application according to claim 32, characterized in that, The application also includes UDP-glucose or UDP and sucrose.

35. The application according to claim 34, characterized in that, The sucrose is synthesized by sucrose synthase.

36. The application according to claim 35, characterized in that, The amino acid sequence of the sucrose synthase is shown in SEQ ID NO.

6.

37. The application according to claim 32, characterized in that, It also includes the use of glycosyltransferase MG1.

38. The application according to claim 37, characterized in that, The amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO.

4.

39. The application of the glycosyltransferase mutant of claim 1 in the whole-cell biosynthesis of mogroside; in, When the mogroside is mogroside IVA or Sia I, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1); When the mogroside is mogroside V, the glycosyltransferase MS1 mutant is the mutant shown in claim 1 (1) or (2).

40. The application according to claim 39, characterized in that, The application of the synthesized mogroside includes the following steps: 1) Cultivate the genetically engineered bacteria of claim 8 to express the nucleic acid encoding the MS1 mutant glycosyltransferase; 2) Collect cells; 3) Add mogroside IE, IIE, IIIE, IIIA, IVE or Sia I to the cells collected in step 2) and react them.

41. The application according to claim 39, characterized in that, The application also includes the use of glycosyltransferase MG1.

42. The application according to claim 41, characterized in that, The amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO.4.

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