Glucosyltransferase mutant and application thereof

Abstract

The invention discloses a glucosyltransferase mutant and application thereof, and the glucosyltransferase mutant comprises six single mutants and six double mutants; the six single mutants are A146S, T286R, T286W, L344V, F378G and F378S, the six double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G and L344V / F378G, and the gene sequences of the double mutants are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 12. According to the method, mutation sites are predicted through computer-aided semi-rational design; the predicted mutant is expressed in an escherichia coli chassis and used for preparing the anthocyanin, the glycosylation efficiency is remarkably improved, efficient production of the anthocyanin is achieved, a new technical route is provided for industrial production of the anthocyanin, and the method is safe and environmentally friendly.

Description

Technical Field

[0001] The present invention relates to a class of glucosyltransferase mutants and their applications in the biosynthesis of anthocyanins, belonging to the technical field of bioengineering (enzyme engineering). Background Art

[0002] Anthocyanin is a water-soluble natural plant pigment with functions such as antioxidant property, free radical scavenging, and prevention of cardiovascular diseases, and is widely used in the food, cosmetic, and health product industries. In nature, anthocyanins rarely exist in a free state and often combine with glycosidic bonds to form anthocyanins, which have better stability and water solubility. Cyanidin-3-O-glucoside (C3G) is the most abundant and widely studied anthocyanin in nature and has various biological activities such as anti-inflammatory, antioxidant, and anti-aging effects. Traditional plant extraction requires a large amount of harmful chemicals, is prone to environmental pollution, and has high production costs, which limits the further application of C3G. Therefore, the green and sustainable solution of microbial cell factories is worthy of being developed and applied to the production of C3G.

[0003] The glycosylation of compounds can increase their structural diversity, effectively improve water solubility, stability, pharmacological activity, and bioavailability, and is crucial for the drug development of plant natural products. In the synthesis process of C3G with catechin as the substrate, the glycosylation reaction catalyzed by glucosyltransferase is the key to ensuring the stability of such substances. Currently, the catalytic activity, stability, and substrate specificity of natural glucosyltransferases capable of glycosylating cyanidin are relatively low, which greatly limits the development of C3G biosynthesis. Therefore, improving the biological activity of glucosyltransferase is of great significance for the industrial production of C3G.

[0004] With the rapid development of biotechnology and computational science, molecular modification technology has become an important means to optimize enzyme functions. Traditional molecular modification technologies mainly include rational design and directed evolution. Rational design relies on in-depth understanding of protein structure and function, and optimizes performance through precise amino acid substitution; while directed evolution simulates the natural evolution process through random mutation and screening. However, both methods have their limitations: rational design requires high-precision structural information and has a limited design scope; directed evolution is time-consuming and laborious, and it is difficult to control the mutation direction. Computer-aided semi-rational design combines the advantages of rational design and computational simulation, predicts potential mutation sites through computer tools, and combines experimental verification to achieve efficient and precise enzyme modification. This method shows great application potential in the fields of enzyme engineering, metabolic engineering, etc. Summary of the Invention

[0005] The present invention provides a class of glucosyltransferase mutants and their applications to overcome the problem of insufficient catalytic ability of existing glucosyltransferases.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] Glucosyltransferase mutants, including 6 single mutants and 6 double mutants;

[0008] The 6 single mutants are A146S, T286R, T286W, L344V, F378G and F378S. Among them, the gene sequence of A146S is shown in SEQ ID NO.1, the gene sequence of T286R is shown in SEQ ID NO.2, the gene sequence of T286W is shown in SEQ ID NO.3, the gene sequence of L344V is shown in SEQ ID NO.4, the gene sequence of F378G is shown in SEQ ID NO.5, and the gene sequence of F378S is shown in SEQ ID NO.6;

[0009] The 6 double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G, L344V / F378G. Among them, the gene sequence of A146S / T286R is shown in SEQ ID NO.7, the gene sequence of A146S / L344V is shown in SEQ ID NO.8, the gene sequence of A146S / F378G is shown in SEQ ID NO.9, the gene sequence of T286R / L344V is shown in SEQ ID NO.10, the gene sequence of T286R / F378G is shown in SEQ ID NO.11, and the gene sequence of L344V / F378G is shown in SEQ ID NO.12.

[0010] The above glucosyltransferase mutants can be used for the preparation of anthocyanins.

[0011] The above anthocyanins can be pelargonidin anthocyanins, cyanidin anthocyanins, peonidin anthocyanins, delphinidin anthocyanins, petunidin anthocyanins, malvidin anthocyanins, preferably cyanidin anthocyanins, and more preferably cyanidin-3-O-glucoside.

[0012] When used for the preparation of anthocyanins, the glucosyltransferase mutant is any one of the 6 single mutants and 6 double mutants; the 6 single mutants are A146S, T286R, T286W, L344V, F378G and F378S, and the 6 double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G, L344V / F378G.

[0013] The above 6 single mutants and 6 double mutants all have higher activities than the wild type.

[0014] To achieve a more significant increase in the production of anthocyanins, the glucosyltransferase mutant is any one of the 6 double mutants. Further preferably, the glucosyltransferase mutant is 146S / F378G, T286R / L344V, A146S / T286R, T286R / F378G or L344V / F378G. More preferably, the glucosyltransferase mutant is 146S / T286R, T286R / F378G or L344V / F378G. Most preferably, the glucosyltransferase mutant is L344V / F378G. The mutant L344V / F378G has the highest activity.

[0015] Construction of the Escherichia coli chassis for synthesizing cyanidin-3-O-glucoside: Using a plasmid system, the anthocyanin synthase PhANS from hybrid petunia and the glucosyltransferase At3GT from Arabidopsis thaliana carrying the mutant were inserted into Escherichia coli. After induction culture, the Escherichia coli chassis for synthesizing cyanidin-3-O-glucoside was obtained, which was used for the conversion of catechin to cyanidin-3-O-glucoside, with UDP-glucose as the glycosyl donor.

[0016] Preferably, the mutant L344V / F378G with the highest activity is expressed in the Escherichia coli chassis.

[0017] The medium used for the above induction culture can be LB, YT, TB, or TSB medium, preferably YT medium (1% yeast powder, 1.6% peptone, 0.5% NaCl); the optimal induction time is OD 600 = 1; the optimal addition amount of IPTG inducer is a final concentration of 0.5 mM; the optimal culture temperature is 25°C.

[0018] In the construction of the Escherichia coli chassis for synthesizing cyanidin-3-O-glucoside described above, the Escherichia coli used was BL21(DE3) carrying the plasmid PACYCDuet-cscB-cep-UgpA (the acquisition method can be referred to: Yang Bao Wu, Huan Wang, Yang Liu, Linguo Zhao, and Jianjun Pei. An efficient preparation and biocatalytic synthesis of novel C-glycosylflavonols kaempferol 8-C-glucoside and quercetin 8-C-glucoside through using resting cells and macroporous resins. Biotechnology for Biofuels and Bioproducts, vol. 15, 2022, p. 129. DOI: 10.1186 / s13068-022-02228-5); the plasmid used was pETDuet-1.

[0019] In the construction of the Escherichia coli chassis for synthesizing cyanidin-3-O-glucoside described above, the two genes were co-expressed under the regulation of a single Tac promoter in the form of a dicistronic expression cassette.

[0020] The method for obtaining the glucosyltransferase mutant of this application includes the following steps:

[0021] Obtain the three-dimensional structure of the wild-type glucosyltransferase;

[0022] Simulate the binding of the substrate and the enzyme through molecular docking;

[0023] Based on strategies such as thermal stability prediction, sequence alignment, and substrate pocket expansion, predict potential mutation sites;

[0024] Experimentally verify the predicted mutation sites and screen out mutants with significantly improved catalytic efficiency.

[0025] Unless otherwise specified in this application, all are mass percentages.

[0026] Technologies not mentioned in the present invention all refer to the prior art.

[0027] The present invention predicts mutation sites through computer-aided semi-rational design; expresses the predicted mutants in an Escherichia coli chassis for the preparation of anthocyanins, significantly improves the glycosylation efficiency, realizes the efficient production of anthocyanins, provides a new technical route for the industrial production of anthocyanins, and is safe and environmentally friendly. Description of the Drawings

[0028] Figure 1 It is a docking result diagram of wild-type At3GT protein with ligands cyanidin and UDP-glucose;

[0029] Figure 2 It is a diagram of the mutant energy of At3GT mutants constructed by the thermal stability simulation method and the yield of C3G produced in Escherichia coli;

[0030] Figure 3 It is a diagram of the sequence alignment result of the key active sites of wild-type At3GT;

[0031] Figure 4 It is a diagram of the yield of C3G produced by At3GT mutants in Escherichia coli obtained by the sequence alignment method;

[0032] Figure 5 It is a visualization diagram of the interaction between threonine at position 286 in wild At3GT and the substrate UDP-glucose;

[0033] Figure 6 It is a visualization diagram of the substrate channel of wild-type At3GT;

[0034] Figure 7 It is a diagram of the yield of C3G produced by At3GT mutants in Escherichia coli obtained by the method of expanding the substrate pocket;

[0035] Figure 8 It is a diagram of the yield of C3G produced by double mutants of At3GT in Escherichia coli;

[0036] Figure 9 It is a diagram of the C3G yield of the double mutants of At3GT with optimized expression conditions in Escherichia coli. Specific implementation mode

[0037] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the content of the present invention is not limited to the following embodiments only.

[0038] Example 1

[0039] Molecular docking was performed by Autodock 4.0 software to find key active sites:

[0040] The three-dimensional model of glucosyltransferase (At3GT) (PDB ID: 2C1X) was obtained from the Protein Data Bank (PDB). The chemical molecular structures of the substrates cyanidin and UDP-glucose were obtained from the Chemical Structure Database (Pubchem). The protein was docked with the ligand using Autodock 4.0 software, and the docking results are as Figure 1 shown.

[0041] Example 2

[0042] Based on the prediction of thermal stability, single-point mutation simulations were performed on the residues within the range of the substrate to mutate into 20 common amino acids and calculate the mutation energy. According to the analysis of the prediction results, 15 mutants were finally obtained, namely G21Y, T22W, T145R, A146S, G285W, T286W, S312F, A339V, H356D, N360K, S361K, Q381H, V342Y, Q341E, W359L.

[0043] The construction method of the mutant At3GT* (the general name of the mutants) is as follows:

[0044] (1) According to the codon preference of Escherichia coli, the At3GT and PhANS genes were codon-optimized. The optimized genes were synthesized by Jiutian Gene Biology Company, and the At3GT and PhANS genes were cloned into the pETDuet-1 plasmid. The At3GT gene was upstream of the PhANS gene and encoded in the downstream coding region of two tac promoters respectively, obtaining the plasmid pETDuet-tac-At3GT-tac-PhANS.

[0045] (2) Using the pETDuet-tac-At3GT-tac-PhANS plasmid as a template, primers were designed at the mutation sites, and reverse PCR was performed using Takara's PrimeSTAR MAX high-fidelity enzyme to obtain the mutated linear plasmid. Then, the original plasmid was further digested with DNA methyltransferase (DMT) at 37 °C for 1 h. The linear vector after DMT was phosphorylated, and finally circularized using T4 ligase at 4 °C for 8 h. The primers, related reaction systems, and reaction procedures are shown in the following table.

[0046] Mutant primer design

[0047]

[0048]

[0049]

[0050]

[0051] PrimeSTAR MAX high-fidelity enzyme reaction system

[0052]

[0053]

[0054] PrimeSTAR MAX High-Fidelity Enzyme Reaction Program

[0055]

[0056] Phosphorylation Reaction System

[0057]

[0058] Phosphorylation Reaction Program

[0059]

[0060] Ligation Reaction System

[0061]

[0062] The method for verifying the catalytic efficiency of the obtained mutant in Escherichia coli includes the following steps in sequence:

[0063] (1) By heat shock method, the pETDuet-tac-At3GT*-PhANS expression vector was transformed into the BL21(DE3) competent cells carrying the PACYCDuet-cscB-cep-UgpA plasmid (the acquisition method can be found in: Yang Bao Wu, Huan Wang, Yang Liu, Linguo Zhao, and Jianjun Pei. An efficient preparation and biocatalytic synthesis of novel C-glycosylflavonols kaempferol 8-C-glucoside and quercetin 8-C-glucoside through using resting cells and macroporous resins. Biotechnology for Biofuels and Bioproducts, vol. 15, 2022, p. 129. DOI: 10.1186 / s13068-022-02228-5. Preserved in the laboratory, the original strain was purchased from Shanghai Weidi Biotechnology Co., Ltd.). Take 100 μl of the pre-prepared competent cells and thaw them on ice. Then, add 1 ng of the plasmid to the thawed competent cells, gently mix well, let it stand on ice for 10 min. Place the centrifuge tube containing the competent cells in a 42 °C water bath for 60 s, take it out and immediately let it stand on ice for 3 min. Then, add the SOC liquid medium preheated to 40 °C to the centrifuge tube, mix it by inverting up and down. Finally, place the centrifuge tube in a shaker at 37 °C and 250 rpm for 50 min. After taking it out, evenly spread it on a plate containing 100 mg / L ampicillin and 33 mg / L streptomycin;

[0064] (2) Pick a single colony growing normally on the plate and inoculate it into 5 ml of LB liquid medium containing 100 mg / L ampicillin and 33 mg / L streptomycin, and culture it at 37 °C and 250 rpm for 12 h;

[0065] (3) Take 500 μl of the bacterial solution and inoculate it into 50 ml of LB liquid medium containing 100 mg / L ampicillin and 33 mg / L streptomycin, and culture it at 37 °C and 250 rpm for 2 h;

[0066] (4) When the OD600 is about 0.8, add 25 μL of the inducer IPTG (1 M), and the final concentration is 0.5 mM. Place the flask in a shaker at 25 °C and 200 rpm for 3 h.

[0067] (5) Transfer the bacterial solution into a 50 ml centrifuge tube, centrifuge at 4 °C and 4000 rpm for 10 min to collect the bacterial cell precipitate, and resuspend it in acidified M9 medium (pH = 5). Add 0.4% glucose, 1 mM MgSO4, 0.1 mM CaCl2, 2.5 mM substrate catechin, 5 g / L cellobiose, 100 mg / L ampicillin, 33 mg / L streptomycin, 1 mM IPTG, cofactor 0.1 mM α-ketoglutaric acid, and 2.5 mM sodium ascorbate.

[0068] (6) Ferment the resuspended bacterial solution at 25 °C, 200 rpm, and in the dark. After culturing for 48 hours, take the bacterial solution sample for detection.

[0069] The detection method of C3G is as follows:

[0070] Dissolve 5 mg of C3G standard in 500 μL of DMSO and store it in a -20 °C refrigerator. Prepare C3G solutions with concentrations of 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 800 mg / L using a methanol solution with 1% HCl concentration and 50% concentration (that is, in the solution, the mass content of HCl is 1%, the mass content of methanol is 50%, and the balance is water) as the solvent to draw a standard curve for the quantification of C3G. In the C3G culture experiment, add an equal volume of 1% HCl methanol to the taken bacterial solution sample, centrifuge the sample at 12000 g for 10 min for pretreatment, take the supernatant and filter it through a 22 μm filter membrane, and detect the sample using a high-performance liquid chromatograph (Agilen 1260, Agilent Technologies) equipped with a UV detector and an Eclipse XDB-C18 column (4.6 mm × 250 mm × 5 μm). An aqueous solution containing one-thousandth (mass content) formic acid (solvent A) and acetonitrile (solvent B) are used as the mobile phase, with a flow rate of 1 mL / min and the following gradient: 10 - 40% A (0 - 10 min) and 10 - 40% A (10 - 15 min). The column temperature is 35 °C.

[0071] Among them, the expression of A146S and T286W mutants in the E. coli chassis significantly increased the C3G production by 24.31% and 5.41% respectively ( Figure 2 ). The mutation energy of T286W is -2 kcal / mol, indicating enhanced thermal stability, while the mutation energy of A146S is only -0.11 kcal / mol ( Figure 2 )). We speculate that the increased production of A146S is due to the improved solubility - the replacement of hydrophobic Ala with hydrophilic Ser enhances the protein solubility.

[0072] Example 3

[0073] Screening for beneficial mutations by sequence alignment:

[0074] Based on sequence alignment, the contribution of conserved amino acids to protein fitness is greater than that of non-conserved amino acids on average, as they are retained during evolution. We selected the key active sites reported by NCBI and identified by molecular docking results, and aligned them with At3GT homologous proteins with a sequence similarity > 80% ( Figure 3 ). Ten mutants were obtained, namely A25S, P192Q, V196S, T286R, L344V, T350V, V354L, V362T, V362L, K414Q. Among them, the expression of T286R and L344V in the Escherichia coli chassis increased the production of C3G by 11.49% and 25.05% respectively ( Figure 4 )(see Example 2 for the specific method). Molecular docking showed that T286 formed two hydrogen bonds with UDPG (uridine diphosphate glucose) ( Figure 5 ). Mutating to the positively charged Arg can produce electrostatic interactions with the negatively charged phosphate group of UDPG, thereby shortening the distance to the substrate and increasing the substrate binding affinity. The change of Leu to Val at position 344 reduced the steric hindrance and facilitated the entry of the substrate into the active center.

[0075] Example 4

[0076] Screening for beneficial mutations by expanding the substrate pocket:

[0077] The UDP-glucose molecule has a relatively large volume. In the molecular docking results, we observed that the substrate channel of AT3GT is relatively small. Therefore, we replaced the larger amino acids in the substrate channel with smaller amino acids to expand the substrate channel and improve the catalytic rate. The substrate channel was predicted using the caver 3.0 plugin on the pymol software ( Figure 6 ). Six selected large amino acids were mutated to the smallest glycine and the relatively small and hydrophilic serine respectively, and 13 mutants were obtained, namely F18G, F20S, F20G, Y281S, Y281G, F284S, F284G, W338S, W338G, W259S, W259G, F378S, F378G. Among them, mutating the phenylalanine at position 378 to either glycine or serine could increase the yield, by 32.75% and 31.52% respectively ( Figure 7 )(see Example 2 for the specific method). Mutating it to the smaller glycine increased the yield more, confirming that the phenylalanine at this site caused steric hindrance due to its large volume, and mutating to glycine eliminated this hindrance.

[0078] Example 5

[0079] Constructing combinatorial mutants:

[0080] Finally, six mutants, namely A146S, T286W, T286R, L344V, F378G, and F378S, were obtained at the four sites of 146, 286, 344, and 378. The best mutation results at each site were combined pairwise to construct six double mutants, namely A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G, and L344V / F378G. Among them, the double mutant L344V / F378G increased the production of C3G by 92.226% when expressed in the Escherichia coli chassis ( Figure 8 )(For the specific method, refer to Example 2, and the mutant primers are the superposition of the corresponding single mutant primers).

[0081] Example 6

[0082] Using glucosyltransferase mutants to increase the yield of C3G and optimize the culture conditions:

[0083] The plasmid containing the best mutant L344V / F378G of glucosyltransferase At3GT and anthocyanin synthase PhANS (for the specific method, refer to Example 2) was transformed into the BL21(DE3) competent cells carrying the PACYCDuet-cscB-cep-UgpA plasmid, inoculated on the LB plate medium, and after overnight culture at 37°C, single colonies were picked and inoculated into 5 mL of LB medium, and cultured overnight at 37°C and 250 rpm to obtain the seed solution.

[0084] 1. Medium optimization:

[0085] The 1% seed solution was inoculated into 50 mL of LB, YT, TB, and TSB liquid media containing 100 mg / L ampicillin and 33 mg / L streptomycin respectively. Wait for OD 600When it reached 1.0, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction, and the culture was incubated at 30 °C and 200 rpm for 3 h. Subsequently, the culture broth was transferred to a 50 mL centrifuge tube and centrifuged at 4 °C and 4000 rpm for 10 min. The cell pellet was collected and resuspended in acidified M9 medium (pH = 5), and 0.4% glucose, 1 mM MgSO4, 0.1 mM CaCl2, 2.5 mM substrate catechin, 5 g / L cellobiose, 100 mg / L ampicillin, 33 mg / L streptomycin, cofactor 0.1 mM α-ketoglutaric acid, and 2.5 mM sodium ascorbate were added. 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction. After 48 h, 500 μL of the bacterial liquid was taken and an equal volume of 1% HCl methanol (prepared by mixing methanol with 37% hydrochloric acid, and the mass content of HCl in the final solution was 1%, and the same expression meaning was the same elsewhere) was added. The sample was pretreated by centrifuging at 12000 g for 10 min, and the supernatant was detected using a high performance liquid chromatograph. Finally, it was determined that the highest C3G yield was obtained using YT medium (as Figure 9 A) (see Example 2 for specific method).

[0086] 2. Optimization of induction time:

[0087] 1% of the seed solution was inoculated into 50 mL of YT medium containing 100 mg / L ampicillin and 33 mg / L streptomycin respectively. When OD 600 reached 0.2, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 respectively, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction, and the culture was incubated at 30 °C and 200 rpm for 3 h. Subsequently, the culture broth was transferred to a 50 mL centrifuge tube and centrifuged at 4 °C and 4000 rpm for 10 min. The cell pellet was collected and resuspended in acidified M9 medium, and 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction. After 48 h, the sample was taken and an equal volume of 1% HCl methanol was added. The sample was pretreated by centrifuging at 12000 g for 10 min, and the supernatant was detected using a high performance liquid chromatograph. Finally, it was determined that the highest C3G yield was obtained when the induction time was at OD 600 of 1.0 (as Figure 9 B).

[0088] 3. Optimization of inducer addition amount:

[0089] 1% of the seed solution was inoculated into 50 mL of YT medium containing 100 mg / L ampicillin and 33 mg / L streptomycin respectively. At OD 600When they reached 1.0 respectively, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction, and the culture was carried out at 30 °C and 200 rpm for 3 h. Subsequently, the culture solution was transferred to a 50 mL centrifuge tube, centrifuged at 4 °C and 4000 rpm for 10 min, the cell precipitate was collected and resuspended in acidified M9 medium, and 0, 0.1, 0.5, 1, 1.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) were added for induction respectively. After sampling 48 h later, an equal volume of acidified methanol was added, and the sample was pretreated by centrifugation at 12000 g for 10 min. The supernatant was taken and detected by high performance liquid chromatography. Finally, it was determined that the highest yield of C3G was obtained when the final concentration of IPTG was 0.5 mM (as Figure 9 C).

[0090] 4. Optimization of culture temperature:

[0091] 1% of the seed solution was inoculated into 50 mL YT medium containing 100 mg / L ampicillin and 33 mg / L streptomycin respectively. When the OD 600 reached 1.0, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction respectively, and the culture was carried out at a rotation speed of 200 rpm and temperatures of 16 °C, 20 °C, 25 °C, 30 °C for 3 h. Subsequently, the culture solution was transferred to a 50 mL centrifuge tube, centrifuged at 4 °C and 4000 rpm for 10 min, the cell precipitate was collected and resuspended in acidified M9 medium, and 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added for induction. After sampling 48 h later, an equal volume of 1% HCl methanol was added, and the sample was pretreated by centrifugation at 12000 g for 10 min. The supernatant was taken and detected by high performance liquid chromatography. Finally, it was determined that the highest yield of C3G was obtained at 25 °C (as Figure 9 D).

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Claims

1. A class of glucosyltransferase mutants, characterized in that: It includes 6 single mutants and 6 double mutants; The 6 single mutants are A146S, T286R, T286W, L344V, F378G, and F378S. Among them, the gene sequence of A146S is as shown in SEQ ID NO.1, the gene sequence of T286R is as shown in SEQ ID NO.2, the gene sequence of T286W is as shown in SEQ ID NO.3, the gene sequence of L344V is as shown in SEQ ID NO.4, the gene sequence of F378G is as shown in SEQ ID NO.5, and the gene sequence of F378S is as shown in SEQ ID NO.6; The 6 double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G, L344V / F378G. Among them, the gene sequence of A146S / T286R is as shown in SEQ ID NO.7, the gene sequence of A146S / L344V is as shown in SEQ ID NO.8, the gene sequence of A146S / F378G is as shown in SEQ ID NO.9, the gene sequence of T286R / L344V is as shown in SEQ ID NO.10, the gene sequence of T286R / F378G is as shown in SEQ ID NO.11, and the gene sequence of L344V / F378G is as shown in SEQ ID NO.

12.

2. Use of the glucosyltransferase mutant according to claim 1, characterized in that: It is used for the preparation of anthocyanins.

3. The application according to claim 2, characterized in that: The anthocyanin is a cyanidin - type anthocyanin.

4. The application according to claim 2 or 3, characterized in that: The glucosyltransferase mutant is any one of the 6 single mutants and 6 double mutants; the 6 single mutants are A146S, T286R, T286W, L344V, F378G, and F378S, and the 6 double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G, L344V / F378G.

5. The application according to claim 4, characterized in that: The glucosyltransferase mutant is any one of the 6 double mutants.

6. The application according to claim 5, wherein: The glucosyltransferase mutant is 146S / F378G, T286R / L344V, A146S / T286R, T286R / F378G, or L344V / F378G.

7. The application according to claim 6, characterized in that: The glucosyltransferase mutant is 146S / T286R, T286R / F378G, or L344V / F378G.

8. The application according to claim 6, wherein: The glucosyltransferase mutant is L344V / F378G.

9. The application according to claim 2 or 3, characterized in that: Using the plasmid system, the anthocyanidin synthase PhANS from hybrid petunia and the glucosyltransferase At3GT from Arabidopsis thaliana carrying the mutant are inserted into Escherichia coli. After induction culture, an Escherichia coli chassis for synthesizing cyanidin - 3 - O - glucoside is obtained, which is used for the conversion of catechin to cyanidin - 3 - O - glucoside, where UDP - glucose is the glycosyl donor.

10. The application according to claim 9, characterized in that: Induction culture was carried out using YT medium, and the induction time was OD 600 = 1; The addition amount of IPTG inducer was a final concentration of 0.5 mM, and the culture temperature was 25 ± 2 °C.

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