CN116790535A - Glycosyltransferase mutant and application thereof - Google Patents

Abstract

The present invention relates to glycosyltransferase MS1 mutants; meanwhile, the invention provides a method for converting the mogrol into a plurality of mogrosides IV and mogroside V in a multienzyme reaction system by using the mogrol and UDP-glucose (or UDP and sucrose) as raw materials and catalyzing the mogrol to be converted into the mogroside IV and the mogroside V by the multienzyme in vitro. In the whole mogroside synthesis path, high-efficiency synthesis of mogrosides IV and V can be realized by only supplementing sucrose and mogrol or intermediate products. The method has low cost, simple process and high conversion efficiency, can improve the sweetness of the immature fructus momordicae fruit extract, and can be used as an alternative method for obtaining mogrosides by traditional planting.

Description

Glycosyltransferase mutant and application thereof

Technical Field

The invention relates to a method for synthesizing mogroside by a biological method, and belongs to the technical field of biosynthesis of sweeteners.

Background

The mogrosides are tetracyclic triterpene compounds separated from fructus Siraitiae Grosvenorii, are main sweet substances, and have zero calorie and high sweetness. In addition to the effects of relieving cough and treating dry cough, the mogrosides have also been shown to have anticancer, antitumor and antibacterial biological activities. Because the mogroside has a complex structure and low content in the nature, and meanwhile, the planting of the momordica grosvenori has severe environmental requirements, the further application of the mogroside is limited by the traditional planting method. Therefore, development of an alternative preparation method of mogrosides is needed.

In the synthesis process of momordica grosvenori, a multi-stage glycosylation reaction catalyzed by glycosyltransferase is a key factor of high sweetness of the substance. The glycosyltransferase capable of carrying out glycosylation modification on mogrosides has the problems of small quantity, low catalytic activity and the like reported in the prior literature. Therefore, the development of glycosyltransferases capable of catalyzing the glycosylation modification of mogrosides, while improving the catalytic activity of such enzymes, is of great importance in the development of sweeteners and in the development of the market for mogrosides.

We have experimentally verified the use of glycosyltransferases MG1 and MS1 in mogroside synthesis. Meanwhile, the synthetic route of mogrosides is designed, constructed and experimentally verified. The related process is as follows: the glycosyltransferase MG1 mutant catalyzes mogrols to sequentially generate mogroside IE and mogroside IIE, and then sequentially forms the mogrosides IIIA, IIIE, IV and V under the catalysis of MS 1. The in vitro conversion synthesis from mogrol to mogroside IV and mogroside V is successfully realized by using the enzyme. In addition, through site-directed mutagenesis and combined mutagenesis, the catalytic activity of MS1 is improved, and the conversion rate of mogrosides is improved.

Disclosure of Invention

The invention improves the catalytic activity of glycosyltransferase MS1 on mogrosides through a site-directed mutagenesis technology, and combines the glycosyltransferase MS1 with different glycosyltransferases to perform catalytic action, so that the synthesis of mogrosides by converting mogrol is successfully realized. The current situation that the synthesis path from the mogrol to the mogrosides IV and V is not realized at present is verified and made up.

The invention adopts the following technical scheme:

the present invention provides a glycosyltransferase MS1 mutant, characterized in that the amino acid sequence of the mutant comprises a mutation of an amino acid residue at least one of positions 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 corresponding to SEQ ID No. 2.

According to the invention, the amino acid sequence of the mutant comprises a mutation of an amino acid residue corresponding to at least one of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any two positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at both of the above sites, and at least one of the mutation sites is site 146. Preferably, one of the mutation sites of the amino acid sequence of the mutant is a 146A site, and the other mutation site is any one of 34, 77, 122, 144, 225, 265, 298, 344, 391.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any three of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any four of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344.

According to the invention, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any five of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any six of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any seven positions of 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any eight positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any nine of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any ten of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225.

According to the invention, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any eleven positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225/265.

According to the invention, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any twelve positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID NO. 2. In one embodiment, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225/265/298.

According to the invention, the mutant has a homology of 70% or more, for example 80% or more, for example 90% or more, 95% or more, 98% or more, with the amino acid sequence shown in SEQ ID NO. 2.

The invention also provides nucleic acids encoding the above glycosyltransferase MS1 mutants.

The invention also provides a recombinant vector comprising nucleic acid encoding the glycosyltransferase MS1 mutant.

The invention also provides a genetically engineered bacterium expressing the glycosyltransferase MS1 mutant, which comprises nucleic acid for encoding the glycosyltransferase MS1 mutant.

According to the invention, the genetically engineered bacterium is a recombinant strain obtained by introducing the recombinant vector obtained by connecting the nucleic acid with a vector and then introducing the recombinant vector into a host bacterium.

According to the present invention, the host bacteria may be any of E.coli, bacillus subtilis, corynebacterium glutamicum, lactobacillus, yeast or Chinese hamster ovary cells, and E.coli BL21 (DE 3), BL21 (DE 3) pLysS, rosetta (DE 3), endoToxin-Free BL21 (DE 3), BL21 trxB (DE 3), JM109, DH 5. Alpha., top10, etc. may be used.

According to the present invention, the vector may be selected from a prokaryotic expression vector or a eukaryotic expression vector, for example, any one of an E.coli expression vector, a Bacillus subtilis expression vector and a Streptomyces expression vector. Preferably, the vector is selected from any one of pET15b, pET28a, pET32, pGEX4T1, pGEX-6 p-1.

According to the invention, the nucleic acid is linked to the vector by means of ligase or PCR recombination to form a recombinant vector.

According to the invention, the genetically engineered bacterium expresses a glycosyltransferase MS1 mutant. Preferably, expression is induced by induction, e.g., IPTG.

The invention also provides a construction method of the genetically engineered bacterium, which comprises the steps of connecting the nucleic acid with a vector to obtain a recombinant vector, and then introducing the recombinant vector into a host bacterium to obtain a recombinant strain.

The invention provides application of the genetically engineered bacterium in preparation of glycosyltransferase MS1 mutants.

The invention further provides a preparation method of the glycosyltransferase MS1 mutant, which comprises the step of culturing the genetically engineered bacterium to express nucleic acid encoding the glycosyltransferase MS1 mutant.

According to the invention, the temperature of the culture is 35-40 ℃, preferably 37 ℃; the cultivation time is 1-3 hours, preferably 2 hours.

According to the invention, the culture further comprises an induction expression step, the temperature being 10-20 ℃, preferably 16 ℃, after the addition of the inducer; the culture time is 16-24h.

According to the invention, the cultivation is carried out under stirring or shaking conditions, for example at a stirring speed of 100-1000rpm, preferably 200rpm.

According to the invention, the preparation method further comprises the step of isolating and/or purifying the glycosyltransferase MS1 mutant from the culture.

The invention also provides application of the glycosyltransferase MS1 mutant in preparation of mogrosides.

Furthermore, the invention also provides a production method of the mogroside.

According to the invention, the mogroside is one or more of mogroside IE, IIE, IIIE, IIIA, IVA, sia I or V.

According to the invention, the mutant takes mogrol, mogroside IE, IIE, IIIE, IIIA, IVE or Sia I as a substrate to carry out catalytic reaction. Preferably, the mutant takes mogroside IIE, IIIE, IIIA, IVE or Sia I as a substrate to carry out catalytic reaction.

According to the invention, the method further comprises the use of glycosyltransferase MG1. Wherein, the glycosyltransferase MG1 takes mogrol or mogroside IE as a substrate to carry out catalytic reaction.

The invention also provides a method for synthesizing the momordica grosvenori sweetener, wherein the momordica grosvenori sweetener is one or more of mogroside IE, IIE, IIIE, IIIA, IVA, sia I or V, and the method is characterized by comprising the step of contacting the glycosyltransferase MS1 mutant with momordica grosvenori alcohol, mogroside IE, IIE, IIIE, IIIA, IVE or Sia I for catalytic reaction. Preferably, it is contacted with mogroside IIE, IIIE, IIIA, IVE or Sia I.

According to the invention, the above method further comprises a primary glycosylation modification catalyzed by glycosyltransferase MG1.

According to the invention, the glycosyltransferase MG1 takes mogrol or mogroside IE as a substrate to carry out catalytic reaction. Preferably, the amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO. 4.

The invention also provides a method for synthesizing mogroside by the glycosyltransferase and the mutant enzymatic biosynthesis thereof and application thereof.

According to the invention, the method for synthesizing mogrosides and the application thereof comprise the following steps:

1) Culturing the genetically engineered bacterium of the invention to express nucleic acid encoding the glycosyltransferase MS1 mutant;

2) Isolating and/or purifying the glycosyltransferase MS1 mutant in culture;

3) Adding mogrol, mogroside IE, IIE, IIIE, IIIA, IVE or Sia I, and carrying out catalytic reaction with the glycosyltransferase MS1 mutant obtained in the step 2) to obtain mogroside.

According to the invention, UDP-glucose or UDP and sucrose are also included in the method or application. Preferably, the sucrose is synthesized by a sucrose synthase. Wherein the amino acid sequence of the sucrose synthase is shown as SEQ ID NO. 6.

The invention also provides a method for synthesizing mogroside by using the glycosyltransferase and the mutant whole-cell biosynthesis thereof and application of the glycosyltransferase and the mutant whole-cell biosynthesis thereof.

According to the invention, the method for synthesizing mogrosides and the application thereof comprise the following steps:

1) Culturing the genetically engineered bacterium of the invention to express nucleic acid encoding the glycosyltransferase MS1 mutant;

2) Collecting cells;

3) Adding momordica alcohol, momordica glycoside IE, IIE, IIIE, IIIA, IVE or Sia I, and reacting with the cells collected in the step 2).

According to the method or use 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.

The method or use according to the invention further comprises the use of glycosyltransferase MG1. Wherein the amino acid sequence of the glycosyltransferase MG1 is shown as SEQ ID NO. 4.

Advantageous effects

The present invention provides a mutation site affecting glycosyltransferase MS1 activity; meanwhile, the invention provides a method for converting the mogrol into a plurality of mogrosides IV and mogroside V in a multienzyme reaction system by using the mogrol and UDP-glucose (or UDP and sucrose) as raw materials and catalyzing the mogrol to be converted into the mogroside IV and the mogroside V by the multienzyme in vitro. In the whole mogroside synthesis path, high-efficiency synthesis of mogrosides IV and V can be realized by only supplementing sucrose and mogrol or intermediate products. The method has low cost, simple process and high conversion efficiency, can improve the sweetness of the immature fructus momordicae fruit extract, and can be used as an alternative method for obtaining mogrosides by traditional planting.

Drawings

FIG. 1 shows the SDS-PAGE pattern of glycosyltransferases (1: MG1;2: MS1).

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention will be described in more detail below in order to facilitate understanding of the present invention. It should be understood, however, that this invention may be embodied in many different forms and should not be limited to the implementations or embodiments described herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.

Hereinafter, only a few of them are described in the examples for illustration only, which should not be construed as limiting the present invention. Unless otherwise specified, the reagents used in the present invention are commercially available.

Example 1 mutant design

Firstly, glycosyltransferase MS1 (nucleotide sequence is shown as SEQ ID NO:1, amino acid sequence is shown as SEQ ID NO: 2) from fructus momordicae is subjected to amino acid multiple sequence comparison analysis with other glycosyltransferases with triterpene secondary glycosylation modification, and important amino acid residue sites with catalytic function and substrate recognition function in the sequence of the glycosyltransferase MS1 are identified.

Meanwhile, the structure prediction was performed for glycosyltransferase MS 1. The MS1 protein is subjected to homologous modeling by software such as Swiss-Model, phyre2, discovery Studio and the like, and molecular docking is performed by substrates such as mogrol or mogroside and the like, so that the catalytic site and the substrate binding site of the enzyme are predicted, and the effect of amino acid residues near the sites is analyzed.

Finally, site-directed mutagenesis is performed on the key sites found by the homology alignment analysis and the protein structure simulation, and the mutant enzymes are expressed in escherichia coli to perform activity measurement.

EXAMPLE 2 mutant Activity assay

Enzymatic activity reaction system: 0.2mM mogroside IIE, 1mM UDP-glucose, mgCl ₂ 5. 5 mM and adding about 100. Mu.g of MS1 or its mutant in pure enzyme solution, 50mM Tris-HCl (pH 8) to make up the reaction solution to 300. Mu.L, and reacting in a 35℃water bath for 30-120min (depending on the activity of the different mutants). After the reaction is finished, adding methanol with the same volume of chromatographic grade to terminate the reaction. Centrifugal force of 14000rpm for 5min, filtering with 0.22 μm filter membrane, and detecting with high performance liquid chromatography Agilent 1260. The analysis method comprises the following steps: c18 analytical chromatographic column (4.6X250 mm,5 μm); the sample injection amount is 20 mu L; mobile phases a (acetonitrile+0.1% formic acid) and B (water+0.1% formic acid), gradient elution conditions were: gradient elution is carried out for 0-25min by a pump A with the concentration of 25% -85%; the flow rate of the mobile phase is 1mL/min, and the ultraviolet detection wavelength is 203nm.

Screening found that 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, 327 were mutated to have a greater effect on the catalytic activity of the enzyme.

Wherein the relative enzyme activity of T11R, L13A, N37A, S W, S108D, H116E, V T, Q243G, F A is 1.1, 1.3, 1.5, 1.6, 3.1, 1.7, 1.4, 1.5 times that of wild type. In addition, 10 sites closely related to activity were obtained, numbered 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 at the total positions of the sequence. The 10 sites closely related to the activity are subjected to single-point saturation mutation, and the specific operation is as follows: the recombinant plasmid pET32-MS1 is used as a template, a pair of primers with mutation sites are used for carrying out full plasmid PCR amplification by high-fidelity enzyme, and the recombinant plasmid with the designated mutation sites is obtained. The amplified product was digested with DpnI enzyme at 37℃for 2 hours to degrade the original template. The digested product was transformed into E.coli BL21, plated onto LB agar plates containing 100. Mu.g/mL ampicillin, and incubated overnight at 37℃to screen 200 positive clones at each site.

And establishing a mutant flux screening method, analyzing and measuring the catalytic activity of the enzyme, and sequencing and analyzing amino acid mutation at the corresponding position of the glycosyltransferase mutant with improved catalytic activity. The mutation sites and the relative activities of the mutants with improved activities are shown in Table 1, and the wild type relative activity is 1.

TABLE 1

Example 3 combinatorial mutation

Since the relative activities of V146A were combined at the single mutation sites described above, double mutants of different combinations were obtained, and the mutants having higher relative activities were further subjected to round-by-round combination mutation with other sites, and the relative activities were measured, and tables 2 to 3 list the relative activities of the combination mutants having improved relative activities, and the wild type relative activity was 1.

TABLE 2

TABLE 3 Table 3

EXAMPLE 4 induced expression of glycosyltransferases

To obtain glycosyltransferase, E.coli containing pET32-MG1 (nucleotide sequence shown as SEQ ID NO:3, amino acid sequence shown as SEQ ID NO: 4) and MS1 plasmid are inoculated into LB liquid medium containing 100MG/mL ampicillin, and cultured overnight at 37deg.C and 200rpm to obtain seed solution. The cells were inoculated into 50mL of fresh LB medium at an inoculum size of 1%, and cultured at 37℃for 2 hours at 200rpm. When OD is ₆₀₀ When the value reached about 0.7, isopropyl β -D-1-thiogalactopyranose (IPTG) was added to the medium at a final concentration of 0.4mM, and induction was carried out at 16℃and 200rpm for 16 to 24 hours. The bacteria were collected by centrifugation at 6000rpm, the bacteria suspension was sonicated with 1mL of Tris-HCl (pH=8.0), and the supernatant was centrifuged at 14000rpm for 1 hour to obtain a crude enzyme solution. Purification was performed by Ni column affinity chromatography and further ultrafiltration was performed using a 50kDa ultrafiltration tube to obtain concentrated purified wild-type and mutant proteins (shown in FIG. 1).

EXAMPLE 5 evaluation of glycosyltransferase Activity

Tris-HCl (pH=8.0), 0.2mM mogrol or mogroside, 1mM UDP-glucose, 10mM MgCl ₂ 100. Mu.g of MS1 or its mutant in pure enzyme solution was reacted at 40℃for 12 hours. After the glycosylation reaction was completed, the reaction was terminated by adding an equal volume of methanol, and the measurement was performed by using liquid phase and mass spectrometry. The high performance liquid chromatography analysis was performed as follows: the instrument is Agilent high performance liquid chromatograph 1200, the column is a C18 column, mobile phase: water +0.1% formic acid, acetonitrile +0.1% formic acid, flow rate: 1mL/min, the loading was 20. Mu.L. The gradient elution condition is 0-30min, the flow rate of acetonitrile (0.1% formic acid) is 1mL/min, and the ultraviolet detection wavelength is 203nm. The mass spectrometry conditions were positive ion mode, ESI ion source.

EXAMPLE 6 enzymatic Synthesis of mogroside IIE

In a 10mL reaction system, the substrate mogrol 10MG,50MG UDP-glucose, glycosyltransferase MG1 enzyme solution 25MG and 10mM MgCl are included ₂ 50mM Tris-HCl buffer (pH=8.0). After 2-5 hours of reaction, 300. Mu.L of the sample was taken and the reaction was stopped by adding an equal volume of methanol, and the reaction was centrifuged and filtered for HPLC detection (example 5). The results show that the mogrol firstly generates mogroside IE and then generates mogroside IIE, and finally the mogrol is completely converted into a single product of mogroside IIE.

EXAMPLE 7 Whole cell biosynthesis of mogroside IIE

After the induction of expression was completed, E.coli cells (containing pET32-MG 1) were collected, resuspended in PB buffer (50 mM, pH=8.0), and then 10MG of mogrol, 5% glucose, was added and after 12-24 hours of reaction at 37℃HPLC detection was performed. The results showed complete conversion of the final mogrol to the single product mogroside IIE.

Example 8 enzymatic conversion of mogroside IVA Synthesis Using glycosyltransferase MS1 mutant

In a 10mL reaction system, the reaction system comprises 50mg of substrate mogroside IIE or IIIA 10mg,50mg of UDP-glucose, 50mg of glycosyltransferase MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant enzyme solution and 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=8.0). After 24 hours of reaction, 300. Mu.L of the sample was taken and the reaction was stopped by adding an equal volume of methanol, and the reaction was centrifuged and filtered for HPLC detection. Results show a processThe variants all can completely convert the substrate into mogroside IVA.

EXAMPLE 9 Synthesis of mogroside IVA by Whole-cell transformation Using glycosyltransferase MS1 mutant

After the induction of expression was completed, 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), 5% glucose, 10mg of mogroside IIE or IIIA was added and reacted at 37℃for 36 hours, after which the HPLC detection was performed. The results show that the mutants can completely convert the substrate into mogroside IVA.

Example 10 enzymatic conversion of mogroside Sia I Using glycosyltransferase MS1 mutant

In a 10mL reaction system, the reaction system comprises substrate mogroside IIIE 10mg,50mg UDP-glucose, glycosyltransferase MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant enzyme solution 50mg and 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=8.0). After 24 hours of reaction, 300. Mu.L of the sample was taken and the reaction was stopped by adding an equal volume of methanol, and the reaction was centrifuged and filtered for HPLC detection. The results showed that the mutants were able to fully convert the substrate and produce mogrosides Sia I (95%) and IVA (5%).

EXAMPLE 11 Synthesis of mogroside Sia I by Whole-cell transformation Using glycosyltransferase MS1 mutant

After the induction of expression was completed, 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 then subjected to HPLC detection after adding mogroside IIIE 10mg, glucose 5%, and reacting at 37℃for 36 hours. The results show that the mutants can completely convert the substrate into mogroside Sia I.

EXAMPLE 12 enzymatic Synthesis of mogroside V Using glycosyltransferase MS1 mutant

In a 10mL reaction system, the reaction system comprises 50mg of substrate mogroside IVE or Sia I10 mg,50mg UDP-glucose, glycosyltransferase MS 1-S34A/F77L/V146A/A146V/T344V/M360L/A391V mutant enzyme solution and 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=8.0). After 5-24 hours of reaction, 300. Mu.L of sample was taken and added with equal volume of methanol to terminate the reactionThe HPLC detection should be performed by centrifugation. The results show that the mutants can completely convert the substrate and specifically generate the mogroside V.

EXAMPLE 13 Synthesis of mogroside V by Whole cell transformation Using glycosyltransferase MS1 mutant

After the induction of expression was completed, 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 then assayed by HPLC after adding mogroside IVE or Sia I10 mg, glucose 5%, and reacting at 37℃for 36 hours. The results show that the mutants can specifically generate the mogroside V from the substrate.

Example 14 double enzyme tandem catalysis of mogrol to mogroside V

In a 10mL reaction system, the reaction system comprises substrate mogrol 10MG, 130MG UDP-glucose, glycosyltransferase MG1 25MG, MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant enzyme solution 50MG and 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=8.0). After 24 hours of reaction, 300. Mu.L of the sample was taken and the reaction was stopped by adding an equal volume of methanol, and the reaction was centrifuged and filtered for HPLC detection. The result shows that the substrate is completely converted, and the ratio of mogroside V in the product is more than 99%.

Example 15 Multi-enzyme tandem catalysis of mogrol to mogroside V

In a 10mL reaction system, the substrate mogrol 10MG, 130MG UDP-glucose, glycosyltransferase MG1 25MG, MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant enzyme solution 50MG, MS2 (nucleotide sequence is shown as SEQ ID NO:9, amino acid sequence is shown as SEQ ID NO: 10) 50MG, 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=8.0). After 12, 16 and 24 hours of reaction, 300. Mu.L of sample was taken and added with an equal volume of methanol to terminate the reaction, and the reaction was centrifuged and filtered for HPLC detection. The result shows that the substrate is completely converted within 16 hours, and the mogroside V in the product accounts for more than 99 percent. The time taken for the reaction to reach the final reaction state was shortened as compared with example 14.

EXAMPLE 16 biosynthesis of sucrose synthase supplied with UDP-glucose and mogroside V

pET21 plasmid is adopted, and enzyme cutting sites such as BamHI and XhoI are utilizedAn expression vector of sucrose synthase (Arabidopsis thaliana sucrose synthase is abbreviated as AtSUS1, the nucleotide sequence of which is shown as SEQ ID NO:5, and the amino acid sequence of which is shown as SEQ ID NO: 6) is constructed and introduced into escherichia coli BL21. Strains containing glycosyltransferases MG1, MS1 and sucrose synthase AtSUS1 were respectively induced and expressed according to the method in example 4, and after completion, bacterial cells were collected and crushed and purified to obtain the above enzyme protein. Construction of a mogroside biosynthesis system based on UDP-glucose cycle (10 mL): the substrate mogrol is 10MG, UDP 10MG, glycosyltransferase MG1 25MG, MS1-S34A/F77L/V146A mutant enzyme solution 50MG, sucrose synthase AtSUS 110 MG, 10mM MgCl ₂ 50mM Tris-HCl buffer (pH=7.0). The reaction was carried out for 6 to 24 hours, and sampling was carried out for liquid phase detection, and the results showed that the conversion efficiency was good and the substrate conversion efficiency was faster than that in example 14. Indicating that the use of the enzyme in combination with sucrose synthase is more advantageous for the synthesis of glycosides.

EXAMPLE 17 Yeast Whole cell biosynthesis of mogroside V

Firstly, a Exg hydrolase gene in saccharomyces cerevisiae is replaced and knocked out by utilizing a resistance gene to obtain a yeast engineering strain S.g-01, then a yeast expression plasmid containing glycosyltransferase MG1 and MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant is transformed into the strain S.g-01, and the strain S.g-02 is obtained through defect screening. Recombinant yeasts were picked up and inoculated into 5mL of SD medium, cultured overnight at 30℃and then inoculated into 50mL of fresh SD medium (500 mL Erlenmeyer flask) at 1% of the inoculum size, cultured at 220rpm for 96 hours at 30℃and harvested by centrifugation, and the cells were washed with PBS buffer (pH 8.0). The cells were resuspended in 1mL PBS buffer (pH 8.0, containing 2% glucose), 0.2mM mogrol was added and reacted at a conversion temperature of 40℃and 700rpm for 48h to yield mogroside V.

Example 18 optimization of UDP-glucose biosynthetic pathway and conversion of mogrosides by MS1 Yeast Strain

Firstly, a yeast engineering strain S.g-03 with increased UDP-glucose content and knocked out glycoside hydrolase genes is obtained by inserting synthetic UDP-glucose genes GPD1 (nucleotide sequence shown as SEQ ID NO: 7) and PGM2 (nucleotide sequence shown as SEQ ID NO: 8) together with respective promoters and terminators into Exg hydrolase genes, and then a yeast expression plasmid containing glycosyltransferase MG1 and MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutants is transformed into the strain S.g-03, and strain S.g-04 is obtained by defect screening. Recombinant yeasts were picked up and inoculated into 5mL of SD medium, cultured overnight at 30℃and then inoculated into 50mL of fresh SD medium (500 mL Erlenmeyer flask) at 1% of the inoculum size, cultured at 220rpm for 96 hours at 30℃and harvested by centrifugation, and the cells were washed with PBS buffer (pH 8.0). The thallus is resuspended by using 1mL PBS buffer solution (pH 8.0, containing 2% glucose), 0.2mM mogrol is added, the conversion temperature is 40 ℃, the reaction is carried out at 700rpm for 48 hours, the mogroside V is produced, and the production speed of the product is faster than that of the strain S.g-02.

Example 19 optimization of UDP-glucose biosynthetic pathway and conversion of mogrosides by MS1 Yeast Strain

Firstly, a yeast engineering strain S.g-03 with increased UDP-glucose content and knocked out glycoside hydrolase genes is obtained by inserting synthetic UDP-glucose genes GPD1 (nucleotide sequence shown as SEQ ID NO: 7) and PGM2 (nucleotide sequence shown as SEQ ID NO: 8) together with respective promoters and terminators into Exg hydrolase genes, and then yeast expression plasmids containing glycosyltransferase MG1, MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutants and MS2 are transformed into the strain S.g-03, and strain S.g-05 is obtained through defect screening. Recombinant yeasts were picked up and inoculated into 5mL of SD medium, cultured overnight at 30℃and then inoculated into 50mL of fresh SD medium (500 mL Erlenmeyer flask) at 1% of the inoculum size, cultured at 220rpm for 96 hours at 30℃and harvested by centrifugation, and the cells were washed with PBS buffer (pH 8.0). The cells were resuspended in 1mL PBS buffer (pH 8.0, containing 2% glucose), 0.2mM mogrol was added and reacted at a conversion temperature of 40℃and 700rpm for 48h to yield mogroside V.

Example 19 construction of Yeast Strain to increase the copy number of glycosyltransferase MS1 and mogroside transformation

Yeast expression plasmids containing 2-4 copies of the glycosyltransferase MS1-S34A/F77L/V146A/A313V/T344V/M360L/A391V mutant were transformed into strain S.g-03 and strain S.g-06 to S.g-08 were obtained by defective screening. Recombinant yeast was selected and inoculated in 5mL of SD medium, cultured overnight at 30℃and then inoculated in 50mL of fresh SD medium (500 mL Erlenmeyer flask) at 1% of the inoculum size, cultured at 30℃and 220rpm for 96 hours, and the cells were collected by centrifugation and washed with PBS buffer (pH 8.0). The cells were resuspended in 1ml of buffer (pH 8.0, containing 2% glucose) and 0.2mM mogroside IIE was added to react at 40℃and 700rpm for 24-48h to give mogroside V, with the rate of product formation increasing progressively with increasing copy number.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> institute of Tianjin Industrial biotechnology, national academy of sciences

<120> glycosyltransferase mutant and application thereof

<130> CPCN22410276

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 1365

<212> DNA

<213> 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> Synthesis

<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> Synthesis

<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> Synthesis

<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> Synthesis

<400> 5

gaactttcct attttcctaa aacacccttt tccttggact tttaaatcca ttatatttag 60

tcttctgggt ttggagccca agaaataaaa cgtagaagga aacagaggga agtaaatgat 120

tcatatcagc ttaccaaaat ttgtatcaag ctttaaaaat 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

ggtcctttct 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

cacaaacttg gtgtcactca gtgtaccatt gctcatgctc ttgagaaaac aaagtacccg 1620

gattctgata tctactggaa gaagcttgac gacaagtacc atttctcatg ccagttcact 1680

gcggatattt tcgcaatgaa ccacactgat ttcatcatca ctagtacttt ccaagaaatt 1740

gctggaagca aagaaactgt tgggcagtat gaaagccaca cagcctttac tcttcccgga 1800

ttgtatcgag ttgttcacgg gattgatgtg tttgatccca agttcaacat tgtctctcct 1860

ggtgctgata tgagcatcta cttcccttac acagaggaga agcgtagatt gactaagttc 1920

cactctgaga tcgaggagct cctctacagc gatgttgaga acaaagagca cttatgtgtg 1980

ctcaaggaca agaagaagcc gattctcttc acaatggcta ggcttgatcg tgtcaagaac 2040

ttgtcaggtc ttgttgagtg gtacgggaag aacacccgct tgcgtgagct agctaacttg 2100

gttgttgttg gaggagacag gaggaaagag tcaaaggaca atgaagagaa agcagagatg 2160

aagaaaatgt atgatctcat tgaggaatac aagctaaacg gtcagttcag gtggatctcc 2220

tctcagatgg accgggtaag gaacggtgag ctgtaccggt acatctgtga caccaagggt 2280

gcttttgtcc aacctgcatt atatgaagcc tttgggttaa ctgttgtgga ggctatgact 2340

tgtggtttac cgactttcgc cacttgcaaa ggtggtccag ctgagatcat tgtgcacggt 2400

aaatcgggtt tccacattga cccttaccat ggtgatcagg ctgctgatac tcttgctgat 2460

ttcttcacca agtgtaagga ggatccatct cactgggatg agatctcaaa aggagggctt 2520

cagaggattg aggagaaata cacttggcaa atctattcac agaggctctt gacattgact 2580

ggtgtgtatg gattctggaa gcatgtctcg aaccttgacc gtcttgaggc tcgccgttac 2640

cttgaaatgt tctatgcatt gaagtatcgc ccattggctc aggctgttcc tcttgcacaa 2700

gatgattgaa gagaagagaa aagactcgga accagtaaag agtttgaaga gagtggttcc 2760

ggtgtttgaa gaataaaaca agatctcctt ttgattctta ttatcattcg gattgggaag 2820

cgcattttgt gttcctctgc ttctttgtta tttcaatcat ttgtctttgt tttcctttgc 2880

ctctgtggca ttgctttgaa ttctggctgt tgggagattc cccttctaat tgcagtaaag 2940

tgtcttttgc aaaatatttc tcaatattta catcttccag taaccaaatg caccttatta 3000

gcaaagaagt gtgaccatta tcagatcaca aaacaatatc aatatttgtt tgttaaagtt 3060

gggcttattt ctatggtgga ct 3082

<210> 6

<211> 808

<212> PRT

<213> 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 Ala 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> 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

cccaccgaat 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> Synthesis

<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 gcaaggtata tatggtctgg cccgttcatt ccctacctca 1020

ggagccatag accgtgttgc caaggcccat ggtctaaact gttatgaggt cccaactggc 1080

tggaaatttt tctgtgcttt gttcgacgct aaaaaattat ctatttgtgg tgaagaatcg 1140

tttggtactg gttccaacca cgtaagggaa aaggacggtg tttgggccat tatggcgtgg 1200

ttgaacatct tggccattta caacaagcat catccggaga acgaagcttc tattaagacg 1260

atacagaatg aattctgggc aaagtacggc cgtactttct tcactcgtta tgattttgaa 1320

aaagttgaaa cagaaaaagc taacaagatt gtcgatcaat tgagagcata tgttaccaaa 1380

tcgggtgttg ttaattccgc cttcccagcc gatgagtctc ttaaggtcac cgattgtggt 1440

gatttttcat acacagattt ggacggttct gtttctgacc atcaaggttt atatgtcaag 1500

ctttccaatg gtgcaagatt cgttctaaga ttgtcaggta caggttcttc aggtgctacc 1560

attagattgt acattgaaaa atactgcgat gataaatcac aataccaaaa gacagctgaa 1620

gaatacttga agccaattat taactcggtc atcaagttct tgaactttaa acaagtttta 1680

ggaactgaag aaccaacggt tcgtacttaa 1710

<210> 9

<211> 1389

<212> DNA

<213> 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 ctgttgttta cgttgcattg 840

ggttctgagg taccactagg ggtggaaaag 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> 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 Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

Claims (10)

1. A glycosyltransferase MS1 mutant, characterized in that the amino acid sequence of the mutant comprises a mutation of an amino acid residue at least one of positions 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 corresponding to SEQ ID No. 2.

Preferably, the amino acid sequence of the mutant comprises a mutation of an amino acid residue corresponding to at least one of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2.

Preferably, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any two of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at both of the above sites, and at least one of the mutation sites is site 146. Preferably, one of the mutation sites of the amino acid sequence of the mutant is a 146A site, and the other mutation site is any one of 34, 77, 122, 144, 225, 265, 298, 344, 391.

Preferably, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any three of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34.

Preferably, the amino acid sequence of the mutant comprises a mutation of an amino acid residue corresponding to any four of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344.

Preferably, the amino acid sequence of the mutant comprises a mutation of an amino acid residue corresponding to any five of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391.

Preferably, the amino acid sequence of the mutant comprises mutations corresponding to amino acid residues at any six of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313.

Preferably, the amino acid sequence of the mutant comprises mutations of amino acid residues corresponding to any seven positions of 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360.

Preferably, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any eight of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122.

Preferably, the amino acid sequence of the mutant comprises a mutation of the amino acid residues corresponding to any nine of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144.

Preferably, the amino acid sequence of the mutant comprises mutations corresponding to amino acid residues at any ten of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225.

Preferably, the amino acid sequence of the mutant comprises a mutation of an amino acid residue corresponding to any eleven positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225/265.

Preferably, the amino acid sequence of the mutant comprises mutations corresponding to amino acid residues at any twelve of positions 34, 77, 122, 144, 146, 225, 265, 298, 344, 391 of SEQ ID No. 2. More preferably, the amino acid sequence of the mutant comprises a mutation at position 146/77/34/344/391/313/360/122/144/225/265/298.

Preferably, the mutant has more than 70% homology, for example more than 80% homology, further for example more than 90%, more than 95% homology, more than 98% homology with the amino acid sequence shown in SEQ ID NO. 2.

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

3. Recombinant vectors and genetically engineered bacteria comprising the nucleic acid of the glycosyltransferase MS1 mutant of claim 2. Preferably, the genetically engineered bacterium is a recombinant strain obtained by introducing a recombinant vector obtained by ligating the nucleic acid into a vector and then introducing the recombinant vector into a host bacterium.

Preferably, the host bacteria are any of E.coli, bacillus subtilis, corynebacterium glutamicum, lactobacillus, yeast or Chinese hamster ovary cells, for example E.coli BL21 (DE 3), BL21 (DE 3) pLysS, rosetta (DE 3), endoToxin-Free BL21 (DE 3), BL21 trxB (DE 3), JM109, DH 5. Alpha., top10, etc. can be used.

Preferably, the vector may be selected from a prokaryotic or eukaryotic expression vector, such as any one of E.coli, B.subtilis and Streptomyces. Preferably, the vector is selected from any one of pET15b, pET28a, pET32, pGEX4T1, pGEX-6 p-1.

Preferably, the nucleic acid is linked to the vector by means of ligase or PCR recombination to form a recombinant vector.

Preferably, the genetically engineered bacterium expresses a glycosyltransferase MS1 mutant. Preferably, expression is induced, e.g., IPTG-induced expression.

4. The method for constructing a genetically engineered bacterium according to claim 3, comprising the step of obtaining a recombinant vector by ligating the nucleic acid into the vector, and introducing the recombinant strain into a host bacterium.

5. The use of the genetically engineered bacterium of claim 3 in the preparation of glycosyltransferase MS1 mutants.

6. A method for preparing a glycosyltransferase MS1 mutant, comprising the step of culturing the genetically engineered bacterium so that it expresses a nucleic acid encoding the glycosyltransferase MS1 mutant.

Preferably, the temperature of the culture is 35-40 ℃, preferably 37 ℃; the cultivation time is 1-3 hours, preferably 2 hours.

Preferably, the culturing further comprises an induction expression step, the temperature being 10-20 ℃, preferably 16 ℃, after the addition of the inducer; the culture time is 16-24h.

Preferably, the cultivation is carried out under stirring or shaking conditions, for example at a stirring speed of 100-1000rpm, preferably 200rpm.

Preferably, the preparation method further comprises the step of isolating and purifying the glycosyltransferase MS1 mutant from the culture.

7. Use of the glycosyltransferase MS1 mutant according to claim 1 for the preparation of mogrosides.

Preferably, the mogroside is one or more of mogroside IE, IIE, IIIE, IIIA, IVA, sia I or V.

Preferably, the mutant takes mogrol, mogroside IE, IIE, IIIE, IIIA, IVE or Sia I as a substrate to carry out catalytic reaction. Preferably, the mutant takes mogroside IIE, IIIE, IIIA, IVE or Sia I as a substrate to carry out catalytic reaction.

Preferably, the method further comprises using glycosyltransferase MG1 to catalyze a reaction with mogrol or mogroside IE as a substrate. Preferably, the amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO. 4.

8. A method of synthesizing a luo han guo sweetener, wherein the luo han guo sweetener is one or more of mogroside IE, IIE, IIIE, IIIA, IVA, sia I or V, the method comprising contacting the glycosyltransferase MS1 mutant of claim 1 with luo han guo alcohol, mogroside IE, IIE, IIIE, IIIA, IVE or Sia I to perform a catalytic reaction.

Preferably, the glycosyltransferase MS1 mutant according to claim 1 is contacted with mogroside IIE, IIIE, IIIA, IVE or Sia I for catalytic reaction.

Preferably, the method further comprises primary glycosylation modification catalyzed by glycosyltransferase MG1, and is characterized in that the glycosyltransferase MG1 carries out catalytic reaction by taking mogrol or mogroside IE as a substrate. Preferably, the amino acid sequence of the glycosyltransferase MG1 is shown in SEQ ID NO. 4.

9. The method or use of glycosyltransferase mutant enzyme biosynthesis of mogrosides according to claim 1.

Preferably, the method or application for synthesizing mogrosides comprises the following steps:

1) Culturing the genetically engineered bacterium of claim 3 so that it expresses a nucleic acid encoding said glycosyltransferase MS1 mutant;

2) Isolating and purifying the glycosyltransferase MS1 mutant from the culture;

3) Adding momordica grosvenori alcohol, momordica grosvenori glycoside IE, IIE, IIIE, IIIA, IVE or Sia I, and carrying out catalytic reaction on the obtained glycosyltransferase MS1 mutant obtained in the step 2) to obtain target momordica grosvenori glycoside.

Preferably, the method or application further comprises UDP-glucose or UDP and sucrose. Preferably, the sucrose is synthesized by a sucrose synthase. Wherein the amino acid sequence of the sucrose synthase is shown as SEQ ID NO. 6.

Preferably, the method or use further comprises the use of glycosyltransferase MG1. Wherein the amino acid sequence of the glycosyltransferase MG1 is shown as SEQ ID NO.4

Preferably, the glycosyltransferase MS1 mutant corresponds to the S34A/F77L/V146A/A313V/T344V/M360L/A391V or S34A/F77L/V146A mutant of SEQ ID NO. 2.

10. The method or use of glycosyltransferase mutant whole cell biosynthesis of mogrosides according to claim 1.

Preferably, the method or application for synthesizing mogrosides comprises the following steps:

1) Culturing the genetically engineered bacterium of claim 3 so that it expresses a nucleic acid encoding said glycosyltransferase MS1 mutant;

2) Collecting cells;

3) Adding momordica alcohol, momordica glycoside IE, IIE, IIIE, IIIA, IVE or Sia I, and reacting with the cells collected in the step 2).

Preferably, the method or use further comprises the use of glycosyltransferase MG1. Wherein the amino acid sequence of the glycosyltransferase MG1 is shown as SEQ ID NO. 4.

Preferably, the glycosyltransferase MS1 mutant corresponds to the S34A/F77L/V146A/A313V/T344V/M360L/A391V or S34A/F77L/V146A mutant of SEQ ID NO. 2.