Glycosyltransferase mutants, nucleic acids, recombinant expression vectors and uses thereof

Abstract

The present application relates to glycosyltransferase mutants, nucleic acids, recombinant expression vectors and their use. Specifically disclosed are mutants with improved activity and thermostability of glycosyltransferase YojK, nucleic acids encoding the glycosyltransferase mutants, recombinant expression vectors containing the nucleic acids, recombinant expression transformants containing the recombinant expression vectors, recombinant glycosyltransferase mutant catalysts, and the use of the glycosyltransferase mutants or the recombinant glycosyltransferase catalysts in the preparation of rebaudioside D. Compared with other glycosyltransferases, the glycosyltransferase mutants obtained by the present application can efficiently catalyze the synthesis of rebaudioside D from rebaudioside A as a substrate, or with the sucrose synthase from Arabidopsis thaliana At SuS The present application constructs a coupling reaction to prepare rebaudioside D. The method of the present application has the advantages of high substrate loading, high yield, low production cost and environmental friendliness, and has good industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to glycosyltransferase mutants, nucleic acids, recombinant expression vectors, and their applications. Specifically, this invention relates to glycosyltransferase mutants, nucleic acids encoding said glycosyltransferase mutants, recombinant expression vectors containing said nucleic acids, recombinant expression transformants containing said recombinant expression vectors, recombinant glycosyltransferase catalysts, and the application of glycosyltransferase mutants or glycosyltransferase catalysts in the preparation of rebaudioside D. Background Technology

[0002] Rebaudioside D (Reb D for short) is a tetracyclic diterpenoid glycoside extracted from the leaves of Stevia rebaudiana Bertoni, and is an important member of the steviol glycoside family. Its chemical structure uses steviol as the aglycone, with a trisaccharide chain (Glc-β(1→2)-Glc-β(1→3)-Glc) consisting of a glucose and a rhamnose group linked at C19, and a disaccharide chain (Glc-β(1→2)-Glc) consisting of two glucose molecules linked at C13. Rebaudioside D is approximately 200-300 times sweeter than sucrose, and retains a pure sweetness even at high concentrations. Its bitter aftertaste and metallic finish are significantly lower than those of earlier steviol glycosides such as Rebaudioside A.

[0003] As a new generation of high-sweetness, low-calorie natural sweetener, rebaudioside D has shown great application potential in the food, beverage, pharmaceutical, and health product industries. Compared with rebaudioside A, rebaudioside D has superior sensory characteristics: its sweetness initiates faster, lasts longer, and is more compatible with acidulants and flavorings. Clinical studies show that rebaudioside D does not participate in human metabolism and has no effect on blood sugar levels, making it suitable for diabetic patients and those managing their weight. In recent years, with the advancement of the global "sugar reduction movement," the market demand for natural, high-efficiency sweeteners has grown rapidly. However, the natural content of rebaudioside D in stevia is extremely low (approximately 0.1%-0.3% in dried leaves), and direct extraction from plants is costly, has limited yield, and is constrained by planting conditions, making it difficult to meet industrial needs. Therefore, developing efficient rebaudioside D synthesis technology has become an urgent need for the industry.

[0004] Previous studies have explored the chemical synthesis of rebaudioside D, using steviol or rebaudioside A as starting materials and introducing specific glycosyl groups through multiple chemical reactions. However, this method requires the use of large amounts of toxic reagents (such as heavy metal catalysts and halogenated sugar donors), and the regioselectivity and stereoselectivity of the glycosylation reaction are difficult to control, often resulting in isomer byproducts. Biosynthesis, on the other hand, offers advantages such as mild reaction conditions, environmental friendliness, high optical purity of the product, and the elimination of the need for toxic chemical reagents, making it a promising candidate for industrial applications.

[0005] Using glycosyltransferases as catalysts to catalyze the synthesis of rebaudioside D from rebaudioside A, which is abundant in stevia, is considered a feasible technical route to increase the production of rebaudioside D. Currently, wild-type glycosyltransferases exhibit low catalytic activity for rebaudioside A and poor thermal stability during the reaction.

[0006] Chinese patent CN114164191A discloses a mutant of Bacillus subtilis-derived glycosyltransferase YojK-I241T / G327N, which can catalyze the conversion of rebaudioside A to rebaudioside D. However, this scheme has only been verified in a shake-flask small-scale system and lacks fermentation scale-up data at high substrate concentrations.

[0007] Chinese patent CN116064454A discloses a YojK-derived mutant of glycosyltransferase with improved thermostability and activity, which can catalyze the conversion of rebaudioside A to rebaudioside D. However, the conversion rate is only 87.7% under the substrate condition of 24 g / L rebaudioside A, indicating substrate load limitation.

[0008] Therefore, it is necessary to develop enzyme catalysts with better catalytic performance to meet the technical requirements of industrial production. Summary of the Invention

[0009] The purpose of this invention is to overcome the problems of low catalytic activity of existing glycosyltransferases for rebaudioside A and poor thermal stability in the reaction, and to provide glycosyltransferase mutants, nucleic acids, recombinant expression vectors and their applications.

[0010] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a glycosyltransferase YojK mutant, which is a mutant formed by mutation at the following sites in the amino acid sequence shown in SEQ ID NO.2: a combination of one or more of the following: serine at position 18, valine at position 37, glycine at position 63, glutamic acid at position 70, threonine at position 75, phenylalanine at position 81, alanine at position 84, threonine at position 129, methionine at position 142, threonine at position 240, asparagine at position 244, proline at position 324, valine at position 332, and lysine at position 347, and the glycosyltransferase mutant has the function of the glycosyltransferase with the amino acid sequence shown in SEQ ID NO.2.

[0011] The glycosyltransferase described in this invention encodes glycosyltransferase YojK, which is derived from Bacillus subtilis ( ). Bacillus subtilis The amino acid sequence of the glycosyltransferase YojK is shown in SEQ ID No. 2. Methods for preparing the glycosyltransferase of this invention include, but are not limited to: 1) from Bacillus subtilis (…). Bacillus subtilis 1) Obtained by direct extraction and isolation from cells; 2) Obtained by heterologous recombination expression of the glycosyltransferase-encoded gene in accordance with conventional techniques in the art.

[0012] The amino acid sequence of the glycosyltransferase YojK is as follows: MANVLMIGFPGEGHINPSIGVMKELKSRGENITYYAVKEYKEKITALDIEFREYHDFRGDYFGKNATGDEERDFTEMLCAFLKACKDIATHIYEEVKHESYDYVIYDHHLLAGKVIANMLKLPRFSLCTTFAMNEEFAKEMMGAYMKGSLEDSPHYESYQQLAETLNADFQAEIKKPFDVFLADGDLTIVFTSRGFQPLAEQFG ERYVFVGPSITERAGNNDFPFDQIDNENVLFISMGTIFNNQKQFFNQCLEVCKDFDGKVVLSIGKHIKTSELNDIPENFIVRPYVPQLEILKRASLFVTHGG MNSTSEGLYFETPLVVIPMGGDQFVVADQVEKVGAGKVIKKEELSESLLKETIQEVMNNRSYAEKAKEIGQSLKAAGGSKKAADSILEAVKQKTQSANA(SEQ ID No. 2) From Bacillus subtilis ( Bacillus subtilis A new glycosyltransferase gene was cloned from *Bacillus subtilis*, and its nucleotide sequence is shown in SEQ ID No. 1. For example, *Bacillus subtilis* (*Bacillus* spp.) Bacillus subtilis Using the genomic DNA of the sample as a template, the complete nucleic acid molecule encoding the glycosyltransferase is obtained by employing conventional techniques in the art (such as polymerase chain reaction PCR).

[0013] The nucleotide sequence of the gene encoding the glycosyltransferase YojK is as follows: Furthermore, the amino acid sequence of the glycosyltransferase mutant is selected from one of the following: a) Replace serine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with valine; b) Replace the serine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with valine, and replace the valine at position 37 with proline; c) Replace the 18th serine in the amino acid sequence shown in SEQ ID No. 2 with valine, the 63rd glycine with leucine, and the 81st phenylalanine with methionine. d) Replace the 18th serine of the amino acid sequence shown in SEQ ID No. 2 with valine, the 70th glutamic acid with valine, the 75th threonine with valine, the 84th alanine with leucine, the 240th threonine with aspartic acid, the 324th proline with leucine, the 332nd valine with cysteine, and the 347th lysine with phenylalanine; e) Replace glycine at position 63 with leucine, threonine at position 75 with valine, and alanine at position 84 with leucine in the amino acid sequence shown in SEQ ID No. 2; f) Replace the glutamic acid at position 70 of the amino acid sequence shown in SEQ ID No. 2 with valine; g) Replace the 70th position of glutamic acid in the amino acid sequence shown in SEQ ID No.2 with valine, the 75th position of threonine with valine, the 129th position of threonine with alanine, the 142nd position of methionine with glycine, and the 324th position of proline with leucine. h) Replace the threonine at position 75 of the amino acid sequence shown in SEQ ID No. 2 with valine; i) Replace phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with methionine; j) Replace phenylalanine at position 81 with methionine, alanine at position 84 with leucine, and threonine at position 129 with alanine in the amino acid sequence shown in SEQ ID No.2. k) Replace phenylalanine at position 81 with methionine, alanine at position 84 with leucine, threonine at position 129 with alanine, proline at position 324 with leucine, and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No.2. l) Replace phenylalanine at position 81 with methionine, threonine at position 129 with alanine, and lysine at position 347 with phenylalanine in the amino acid sequence shown in SEQ ID No. 2. m) Replace the threonine at position 129 of the amino acid sequence shown in SEQ ID No. 2 with alanine; n) Replace threonine at position 129 with alanine, methionine at position 142 with glycine, valine at position 332 with cysteine, and lysine at position 347 with phenylalanine in the amino acid sequence shown in SEQ ID No.2. o) Replace the methionine at position 142 of the amino acid sequence shown in SEQ ID No. 2 with glycine; p) Replace the threonine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid; q) Replace threonine at position 240 with aspartic acid, proline at position 324 with leucine, and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No.2; r) Replace proline at position 324 of the amino acid sequence shown in SEQ ID No. 2 with leucine; s) Replace proline at position 324 with leucine and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No. 2; t) Replace the lysine at position 347 of the amino acid sequence shown in SEQ ID No.2 with phenylalanine.

[0014] Furthermore, the present invention is derived from the genus Bacillus subtilis ( Bacillus subtilis Using the glycosyltransferase as the parent material, molecular modification was performed to obtain mutants of the glycosyltransferase YojK. Compared with the parent material, all mutants showed increased activity, and some mutants also showed improved stability.

[0015] The method for obtaining the glycosyltransferase mutant in this invention is random mutation.

[0016] The method for preparing the glycosyltransferase YojK mutant described in this invention is a conventional method in the art. Preferably, the method involves: cloning a nucleic acid molecule encoding the glycosyltransferase YojK and carrying a point mutation into an expression vector; transforming the resulting recombinant expression vector into an expression host to obtain a recombinant expression transformant; culturing the obtained recombinant expression transformant; and isolating and purifying the glycosyltransferase mutant to obtain the mutant. Alternatively, the glycosyltransferase mutant can be obtained by artificially synthesizing amino acids from proteins.

[0017] The second technical solution of the present invention is: providing nucleic acid encoding the YojK mutant of the glycosyltransferase. The source of the encoding nucleic acid of the glycosyltransferase mutant includes: obtaining the encoding gene of the glycosyltransferase mutant through gene cloning technology; or obtaining the encoding gene of the glycosyltransferase mutant through artificial full-sequence synthesis.

[0018] The third technical solution of this invention is to provide a recombinant expression vector containing the aforementioned glycosyltransferase mutant nucleic acid. The recombinant expression vector can be constructed by connecting the nucleotide sequence encoding the glycosyltransferase mutant of this invention to various commercially available empty vectors using conventional methods in the art. The commercially available empty vectors can be various plasmid vectors conventional in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the corresponding glycosyltransferase. Preferred plasmid vectors differ for different expression hosts. Those skilled in the art are generally aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0019] Preferably, for the *E. coli* host, the plasmid vector is preferably the pET-28a(+) plasmid. More preferably, the *E. coli* recombinant expression plasmid of the present invention can be prepared by the following method: the gene sequence DNA fragment of the glycosyltransferase mutant obtained by PCR amplification is processed using a restriction endonuclease... Nde I and Xho Double digestion with restriction enzymes, and simultaneously digestion of the empty vector plasmid pET-28a(+) with the same restriction endonuclease. Nde I and Xho The DNA fragments of the glycosyltransferase mutant and the empty vector plasmid were recovered by double digestion with enzyme I. The DNA fragments were then ligated using T4 DNA ligase to construct a recombinant expression vector containing the glycosyltransferase mutant gene described in this invention for expression in Escherichia coli.

[0020] The fourth technical solution of the present invention is to provide a recombinant expression transformant containing the glycosyltransferase mutant gene or its recombinant expression vector described in the present invention.

[0021] The recombinant expression transformant can be prepared by transforming the recombinant expression vector into the corresponding host cell using conventional techniques in the art. The host cell can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate spontaneously and effectively express the target protein after induction with an inducer. This invention preferentially uses *Escherichia coli* as the host cell, and more preferably *Escherichia coli*. E.coli BL21(DE3) is used for efficient expression of the glycosyltransferase mutant described in this invention.

[0022] The fifth technical solution of the present invention is: to provide a recombinant glycosyltransferase mutant catalyst, wherein the recombinant glycosyltransferase mutant catalyst is any one of the following forms: (1) Cultivate the recombinant expression transformant of the present invention, and isolate the recombinant expression transformant cells containing the glycosyltransferase mutant; (2) Cultivate the recombinant expression transformant of the present invention and separate the crude enzyme solution containing the glycosyltransferase mutant; (3) The lyophilized enzyme powder is obtained by freeze-drying the crude enzyme solution of the glycosyltransferase mutant.

[0023] The cultivation method and conditions of the recombinant expression transformant are conventional methods and conditions in the art, which include the following steps: culturing the recombinant expression transformant of the present invention to obtain recombinant glycosyltransferase.

[0024] For recombinant Escherichia coli, the preferred culture medium is LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 6.5–7.0.

[0025] The preferred culture method is as follows: The recombinant *E. coli* strain described above is inoculated into LB medium containing kanamycin and cultured overnight at 37°C and 180 rpm with shaking. Then, 1-2% (v / v) of the inoculum is transferred to a 2L Erlenmeyer flask containing 400 mL of LB medium (containing kanamycin) and cultured at 37°C and 180 rpm with shaking. When the OD600 of the culture medium reaches 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1-0.5 mmol / L is added as an inducer. Induction is performed at 16-25°C for 16-24 hours. The culture medium is then centrifuged, the precipitate is collected, and the cells are washed twice with physiological saline to obtain the recombinant expression-transformed cells. The harvested recombinant expression-transformed cells are freeze-dried to obtain frozen stem cells containing the glycosyltransferase mutant. The harvested recombinant expression transformants were resuspended in 5-10 volumes (v / w) of buffer solution, sonicated, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant glycosyltransferase mutant. The collected crude enzyme solution was frozen at -80°C and then dried at low temperature using a vacuum freeze dryer to obtain the lyophilized enzyme powder of the recombinant glycosyltransferase mutant. The obtained frozen stem cells or lyophilized enzyme powder can be conveniently used by storing them in a 4°C freezer.

[0026] The method for determining the activity of glycosyltransferase described in this invention is as follows: In a 1 mL system (100 mmol / L potassium phosphate buffer, pH 8.0) containing 10 mM substrate (rebaudioside A) and 1 mM UDPG, an appropriate amount of glycosyltransferase is added for measurement. The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of rebaudioside D per minute under the above reaction conditions; one enzyme activity unit, i.e., 1 U, is defined as 1 U.

[0027] The sixth technical solution of the present invention is: the application of the glycosyltransferase mutant or recombinant glycosyltransferase mutant catalyst in the preparation of rebaudine D, wherein the application is to add the glycosyltransferase of the present invention to a solution containing rebaudine A to catalyze the reaction of rebaudine A to generate rebaudine D.

[0028] In the aforementioned applications, the concentration of rebaudioside A can be 10–110 mM, the amount of sucrose used is 4–10 times the equivalent of rebaudioside A, the amount of recombinant glycosyltransferase can be selected as 2–30 g / L or 0.5–100 U / ml, the amount of sucrose synthase AtSuS can be 4–20 g / L, the amount of added UDP is 0–2 mM, and the buffer solution is phosphate buffer with pH 7.0–9.0.

[0029] When the above-mentioned glycosyltransferase mutant or recombinant glycosyltransferase mutant catalyst is recombinant transformed cells or lyophilized enzyme powder, the dosage is 2-30 g / L; when the glycosyltransferase mutant or recombinant glycosyltransferase mutant catalyst is crude enzyme solution, the dosage is 0.5-100 U / ml.

[0030] The sucrose synthase from Arabidopsis Arabidopsis thaliana (Genbank ID: NM_122090.4, Efficient enzymatic production of rebaudioside a from stevioside. Journal of the Agricultural Chemical Society of Japan , 2016, 80(1), 67-73.).

[0031] The reaction temperature is 20–55°C, preferably 30°C. The reaction time is defined as the time when the conversion rate stops increasing, preferably less than 24 hours. The conversion rate is analyzed by liquid chromatography under the following conditions: C18 column (250 mm × 4.6 mm), mobile phase acetonitrile:water = 28:72 (v / v), column temperature 30°C, flow rate 1.5 ml / min, and detection wavelength 210 nm.

[0032] The structure of rebaudioside A is shown in Formula I: The structure of lebodiin D is shown in Formula II: The structure of UDP is shown in Equation III: .

[0033] The seventh technical solution of the present invention is: a method for preparing rebaudioside D by enzymatic cascade conversion, using rebaudioside A as a substrate, and using sucrose synthase and the glycosyltransferase mutant or recombinant glycosyltransferase mutant catalyst of the present invention to carry out enzymatic cascade, sequentially catalyzing the glycosylation of UDP and the glycosylation of rebaudioside A to obtain rebaudioside D.

[0034] Furthermore, it includes the following steps: S1: In the presence of UDP, sucrase synthase catalyzes the conversion of sucrose into UDP-glucose; S2: Using the UDP-glucose obtained in step S1 as a glycosyl donor, the glycosyltransferase mutant or recombinant glycosyltransferase mutant catalyst of the present invention is used to catalyze the glycosylation of rebaudioside A to obtain rebaudioside D, while releasing UDP to achieve UDP recycling.

[0035] The structure of rebaudioside A is shown in Formula I: The structure of lebodiin D is shown in Formula II: The structure of UDP is shown in Equation III: .

[0036] Furthermore, the sucrose synthase from Arabidopsis thaliana Arabidopsis thaliana (Genbank ID: NM_122090.4, Efficient enzymatic production of rebaudioside a from stevioside. Journal of the Agricultural Chemical Society of Japan, 2016, 80(1), 67-73.) Compared with the prior art, the present invention has the following beneficial effects: This invention provides a glycosyltransferase mutant with improved catalytic performance and thermal stability, capable of efficiently catalyzing the preparation of rebaudioside D from rebaudioside A. The glycosyltransferase mutant can catalyze the reaction of 10³ mM rebaudioside A, achieving a conversion rate of over 90%. Compared to other glycosyltransferases, the glycosyltransferase mutant obtained in this invention exhibits advantages such as high catalytic activity, better thermal stability, large substrate loading capacity, high yield, low production cost, and environmental friendliness, demonstrating excellent prospects for industrial application. Detailed Implementation

[0037] The reaction or detection conditions described in this invention can be combined or modified based on common knowledge in the art, and can be verified experimentally. The technical solutions and effects of this invention will be clearly and completely described below with reference to specific embodiments. However, the scope of protection of this invention is not limited to these embodiments; all changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0038] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Based on common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0039] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual," 3rd edition (J. Sambrook and DW Russell, translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions could be determined through simple experiments if necessary.

[0040] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0041] The materials used in the following embodiments are sourced from: The plasmid vector pET28a was purchased from Novagen, model number 69864-3.

[0042] E.coli DH5α was purchased from Beijing Tiangen Biotech Co., Ltd., model number CB101; E.coli BL21(DE3) competent cells were purchased from Beijing Tiangen Biotech Co., Ltd., model number CB105; The 2×Taq PCR Master Mix was purchased from Beijing Tiangen Biotech Co., Ltd., model number KT201; The agarose gel DNA recovery kit was purchased from Beijing Tiangen Biotech Co., Ltd., model DP209.

[0043] Restriction endonucleases Nde I was purchased from New England Biolabs (NEB), model R0111. Restriction endonucleases Xho I was purchased from New England Biolabs (NEB), model number R0146; T4 DNA ligase was purchased from New England Biolabs (NEB), model number M0202.

[0044] Method for determining glycosyltransferase activity: Add an appropriate amount of glycosyltransferase to a 1 mL system (100 mmol / L potassium phosphate buffer, pH 8.0) containing 10 mM substrate (rebaudioside A) and 1 mM UDPG. The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of rebaudioside D per minute under the above reaction conditions; one enzyme activity unit is defined as 1 U.

[0045] Example 1: Gene Cloning of Glycosyltransferase This invention uses bioinformatics analysis to predict glycosyltransferase genes that may have high catalytic activity for rebaudioside A, and then sorts, clones, and expresses them to verify their function. Using this method, glycosyltransferase genes from the genus *Bacillus subtilis* (…) were identified. Bacillus subtilis A new glycosyltransferase, YojK (amino acid sequence shown in SEQ ID NO.2), was cloned from Bacillus subtilis, and its encoding gene nucleotide sequence is shown in SEQ ID NO.1. Bacillus subtilis Using the genomic DNA of the sample as a template, the complete nucleic acid molecule encoding the glycosyltransferase is obtained using conventional techniques in the art (such as polymerase chain reaction PCR). The preferred synthetic primers involved are shown in SEQ ID No. 3 and SEQ ID No. 4. Upstream primer SEQ ID No. 3: CCG CATATG GCTAATGTATTAATGATCGG Downstream primer SEQ ID No. 4: CCG CTCGAG CTATGCATTTGCTGATTGAG The sequence shown underlined in the upstream primer is... Nde The restriction site for enzyme I, and the underlined sequence of the downstream primer are: Xho I restriction enzyme site.

[0046] Bacillus subtilis ( Bacillus subtilis Using the genomic DNA (NCBI No.: GCF_000009045.1) as a template, the gene was amplified by polymerase chain reaction PCR.

[0047] PCR system (50 μL): 25 μL of 2×Taq PCR MasterMix, 1 μL of genomic DNA (100 ng / μL), 2.5 μL each of forward and reverse primers (10 μM), and sterile distilled water to make up to 50 μL.

[0048] PCR reaction procedure: (1) 95°C pre-denaturation for 5 min; (2) 94°C denaturation for 30 s; (3) 55°C annealing for 30 s; (4) 72°C extension for 2 min; 30 cycles of steps (2) to (4) were performed; finally, 72°C extension for 10 min was performed, and the product was stored at 4°C.

[0049] The PCR products were analyzed and verified by agarose gel electrophoresis, then purified by gel excision, and the target fragment was recovered using a DNA recovery kit. Bacillus subtilis (…) could be cloned. Bacillus subtilis ) glycosyltransferase gene.

[0050] Example 2 Preparation of recombinant expression vector and recombinant expression transformant for glycosyltransferase The glycosyltransferase target gene obtained by PCR amplification in Example 1 and the empty vector plasmid pET28a were respectively processed with restriction endonucleases. Nde I and Xho I was double-digested at 37°C for 12 h. The digestion products were analyzed by agarose gel electrophoresis and then purified by gel extraction to obtain the linearized pET28a plasmid and the purified target gene fragment. The linearized pET28a plasmid and the purified target gene fragment were ligated using T4 DNA ligase at 16°C overnight to obtain the ligation product. The ligation product was transformed into *E. coli*. E.coli In BL21(DE3) competent cells, the colonies were evenly spread onto LB agar plates containing 50 μg / mL kanamycin and incubated statically at 37°C for approximately 12 hours. Colony growth was verified by colony PCR, and clones showing positive colony PCR results were selected for sequencing verification. After successful sequencing verification, the corresponding plasmid was extracted to obtain the recombinant expression vector pET28a-YojK for glycosyltransferase.

[0051] The obtained recombinant expression vector was further transformed into... E.coli Positive clones were selected from BL21(DE3) competent cells to obtain recombinant expression transformants of glycosyltransferase. E.coli BL21(DE3) / pET28a-YojK.

[0052] Example 3 Construction and high-throughput screening of a glycosyltransferase mutant library The nucleotide sequence encoding the glycosyltransferase YojK, as shown in SEQ ID NO.1, was randomly mutated using error-prone PCR technology.

[0053] Using pET28a-YojK plasmid as a template and the gene sequences shown in SEQ ID No. 3 and SEQ ID No. 4 as primers, error-prone PCR was performed with rTaq DNA polymerase to construct a random mutant library of glycosyltransferase YojK.

[0054] PCR system (50 μL): rTaq DNA polymerase 0.5 μL, 10× PCR buffer (Mg) 2+ Add 5.0 μL of Plus, 4.0 μL of dNTPMixture (2.0 mM each), MnCl2 to a final concentration of 100 μmol / L, 0.5 ng of pET28a-YojK plasmid, 2 μL each of forward and reverse primers (10 μM), and sterile distilled water to a final volume of 50 μL.

[0055] PCR reaction procedure: (1) 95°C pre-denaturation for 5 min; (2) 94°C denaturation for 30 s; (3) 55°C annealing for 30 s; (4) 72°C extension for 2 min; 30 cycles of steps (2) to (4) were performed; finally, 72°C extension for 10 min was performed, and the product was stored at 4°C.

[0056] The PCR products were verified by agarose gel electrophoresis, and then purified and recovered by gel excision. The recovered target gene and empty vector plasmid pET28a were then processed with restriction endonucleases. Nde I and Xho I was double-digested at 37°C for 12 h. The digestion products were analyzed by agarose gel electrophoresis and then purified by gel extraction to obtain the linearized pET28a plasmid and the purified target gene fragment. The linearized pET28a plasmid and the purified target gene fragment were ligated using T4 DNA ligase at 16°C overnight to obtain the ligation product. The ligation product was transformed into *E. coli*. E. coli BL21(DE3) competent cells were evenly spread on LB agar plates containing 50 μg / ml kanamycin and incubated statically at 37°C for about 12 h to produce transformants (recombinant transformants containing glycosyltransferase mutants).

[0057] Transformants grown on the above transformation plates were picked and inoculated into deep-well plates containing 300 μL of LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C with shaking. Then, 50 μL was transferred to a secondary deep-well plate containing 600 μL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking for 3 h. IPTG was then added to a final concentration of 0.2 mmol / L, and induction was performed at 16°C for 24 h. Finally, the plates were incubated at 4°C and 3500 × 10⁻⁶ mcg / mL. g Centrifuge for 10 min, discard the supernatant, add 200 μL of lysozyme solution (750 mg lysozyme and 10 mg DNase dissolved in 1 L deionized water) to each well, vortex to mix, and then incubate at 37 °C on a shaker for 2 h. Subsequently, centrifuge at 3500 × g for 10 min at 4 °C, and transfer 10 μL of appropriately diluted cell lysate supernatant to a 96-well microplate containing 90 μL of reaction solution (reaction solution formulation: 10 mM rebaudioside A, 100 mM sucrose, pH 8.0 potassium phosphate buffer, 20 g / L sucrose synthase). AtSuS The supernatant was broken up, and the mixture was reacted with 1 mM UDP at 30°C for 1 h with shaking. The reaction was quenched with 10 times the volume of methanol, and the mixture was analyzed by liquid chromatography. Mutants with improved performance were screened in this way, and the corresponding genes were sequenced to determine the amino acid mutation sites.

[0058] The enzyme activities of each strain in the mutant library were determined, and mutants with significantly enhanced activity were screened. The thermostability of these mutants at 45°C was then characterized, and a series of mutants with increased activity and thermostability were selected. The sequences of these mutants, along with their activities and stability, are listed in Table 1. In Table 1, the sequence numbers correspond to a series of sequences listed later. In the activity column, compared to the parent strain YojK, one plus sign "+" indicates a 1-5 fold increase in mutant protein activity; two plus signs "++" indicate a 6-25 fold increase; and three plus signs "+++" indicate a 26-50 fold increase. In the thermostability column, one plus sign “+” corresponds to a residual activity of 5.0-30.0% of the mutant protein after incubation at 45°C for 30 min; two plus signs “++” correspond to a residual activity of 30.1-50.0% of the mutant protein after incubation at 45°C for 30 min; and three plus signs “+++” correspond to a residual activity of 50.1-90.0% of the mutant protein after incubation at 45°C for 30 min.

[0059] Table 1. List of YojK mutant sequences of glycosyltransferase and corresponding activity improvements The amino acid sequences of the glycosyltransferase mutants corresponding to the sequence numbers are as follows: (1) Replace the serine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with valine; (2) Replace the 18th serine in the amino acid sequence shown in SEQ ID No.2 with valine, and replace the 37th valine with proline; (3) Replace the 18th serine in the amino acid sequence shown in SEQ ID No.2 with valine, the 63rd glycine with leucine, and the 81st phenylalanine with methionine. (4) Replace the 18th serine in the amino acid sequence shown in SEQ ID No.2 with valine, the 70th glutamic acid with valine, the 75th threonine with valine, the 84th alanine with leucine, the 240th threonine with aspartic acid, the 324th proline with leucine, the 332nd valine with cysteine, and the 347th lysine with phenylalanine; (5) Replace glycine at position 63 of the amino acid sequence shown in SEQ ID No.2 with leucine, threonine at position 75 with valine, and alanine at position 84 with leucine; (6) Replace the 70th glutamic acid in the amino acid sequence shown in SEQ ID No. 2 with valine; (7) Replace the 70th position of glutamic acid in the amino acid sequence shown in SEQ ID No.2 with valine, the 75th position of threonine with valine, the 129th position of threonine with alanine, the 142nd position of methionine with glycine, and the 324th position of proline with leucine. (8) Replace the threonine at position 75 of the amino acid sequence shown in SEQ ID No. 2 with valine; (9) Replace the phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with methionine; (10) Replace the phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No.2 with methionine, the alanine at position 84 with leucine, and the threonine at position 129 with alanine; (11) Replace the phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No.2 with methionine, the alanine at position 84 with leucine, the threonine at position 129 with alanine, the proline at position 324 with leucine, and the valine at position 332 with cysteine. (12) Replace the phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No.2 with methionine, the threonine at position 129 with alanine, and the lysine at position 347 with phenylalanine. (13) Replace the threonine at position 129 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (14) Replace threonine at position 129 of the amino acid sequence shown in SEQ ID No.2 with alanine, methionine at position 142 with glycine, valine at position 332 with cysteine, and lysine at position 347 with phenylalanine. (15) Replace the methionine at position 142 of the amino acid sequence shown in SEQ ID No. 2 with glycine; (16) Replace the threonine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid; (17) Replace the threonine at position 240 of the amino acid sequence shown in SEQ ID No.2 with aspartic acid, the proline at position 324 with leucine, and the valine at position 332 with cysteine; (18) Replace the proline at position 324 of the amino acid sequence shown in SEQ ID No. 2 with leucine; (19) Replace proline at position 324 of the amino acid sequence shown in SEQ ID No.2 with leucine and valine at position 332 with cysteine; (20) Replace the lysine at position 347 of the amino acid sequence shown in SEQ ID No.2 with phenylalanine.

[0060] Statistical analysis revealed that the mutation sites of some mutants with increased enzyme activity relative to glycosyltransferase YojK were mainly concentrated at serine position 18, glycine position 63, glutamic acid position 70, threonine position 75, phenylalanine position 81, alanine position 84, threonine position 129, methionine position 142, threonine position 240, proline position 324, valine position 332, and lysine position 347. Some of these mutations exhibited partial repetition, i.e., regularity, suggesting that these amino acid residues may be key or important sites in the enzyme's active pocket / channel, warranting further investigation.

[0061] In addition, mutant M4 exhibits the best overall performance in terms of enzyme activity and thermostability. Compared with glycosyltransferase YojK, it has undergone mutations in which serine at position 18 is replaced with valine, glutamate at position 70 is replaced with valine, threonine at position 75 is replaced with valine, alanine at position 84 is replaced with leucine, threonine at position 240 is replaced with aspartic acid, proline at position 324 is replaced with leucine, valine at position 332 is replaced with cysteine, and lysine at position 347 is replaced with phenylalanine.

[0062] Example 4: Induced expression and fermentation activity determination of glycosyltransferase The recombinant expression transformants obtained in Examples 2 and 3 were inoculated into LB medium containing 50 μg / ml kanamycin and cultured at 37°C and 180 rpm for 12 h as seed culture. Then, at an inoculation rate of 1% (v / v), the seed culture was inoculated into 2L Erlenmeyer flasks containing 400 ml of LB medium (containing 50 μg / ml kanamycin) and cultured at 37°C and 180 rpm. When the OD of the culture medium... 600 When the concentration reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L as an inducer, and the mixture was induced at 16°C for 24 h to obtain the induced culture medium. The induced culture medium was then subjected to a flow rate of 8000 mL / L. g Centrifuge for 10 minutes, collect the precipitate, and wash twice with physiological saline to obtain resting cells.

[0063] Centrifuge 100 ml of the induced culture medium, collect the precipitate, and wash to obtain resting cells (approximately 1 × 10⁻⁶). 13 (Number of cells) were suspended in 10 ml of potassium phosphate buffer (100 mM, pH 7.0) and subjected to sonication in an ice-water bath as follows: 300 W power, 2 s operation, 2 s interval, 12 min sonication, followed by 12000 Hz sonication at 4°C. g Centrifuge for 40 minutes, collect the supernatant to obtain the crude enzyme solution; freeze the collected crude enzyme solution at -80℃, and then dry it at low temperature using a vacuum freeze dryer to obtain the lyophilized enzyme powder. The obtained frozen stem cells or lyophilized enzyme powder can be conveniently used by storing them in a 4℃ freezer. Viability determination is performed according to the method detailed above.

[0064] The glycosyltransferase mutant YojK of this invention was determined to be... M4 The activity of the crude enzyme solution was 100 U / mL, and the activity of the lyophilized enzyme powder was 500 U / mg.

[0065] Example 5 Synthesis of Rebaddi Glycosyltransferase Catalyzed by Recombinant Glycosyltransferase for Rebaddi Glycoside D The reaction was carried out in a 20 mL jacketed reactor. 10 mL of potassium phosphate buffer (100 mM, pH 8.0) was supplemented with 10 mM rebaudioside A, 100 mM sucrose, and 20 g / L sucrose synthase. AtSuS The lysed supernatant was infused with 1 mM UDP and 0.5 U / mL recombinant glycosyltransferase YojK. M4 The crude enzyme solution was prepared. The reaction was carried out under magnetic stirring at 30°C. NaOH solution (1M) was added dropwise using an automatic potentiometric titrator to maintain the pH of the reaction solution at 8.0. After 24 hours of reaction, the substrate conversion rate was determined according to the method detailed in the invention above, and the results are shown in Table 2.

[0066] Table 2. Experimental results of recombinant glycosyltransferase-catalyzed synthesis of rebaudioside D. Examples 6-26: Synthesis of Rebaudioside D by Glycosyltransferases and Their Mutants The reaction was carried out in a 20 mL jacketed reactor. 10 mL of potassium phosphate buffer (100 mM, pH 8.0) was supplemented with 10 mM rebaudioside A, 100 mM sucrose, and 20 g / L sucrose synthase. AtSuS The supernatant was broken up, and 1 mM UDP and 10 g / L resting cell catalyst as described in Example 4 were added. The reaction was carried out with magnetic stirring at 30°C. NaOH solution (1 M) was added dropwise using an automatic potentiometric titrator to maintain the pH of the reaction solution at 8.0. After 12 hours of reaction, the substrate conversion rate was determined according to the method detailed in the invention above, and the results are shown in Table 3.

[0067] Table 3. Glycosyltransferases and their mutants catalyze the preparation of rebodiin D from rebodiin A. As shown in Table 3, the mutant strain M4 used in Example 10 exhibited the best enzymatic catalytic reaction performance, similar to the results in Table 1. The following sections will focus on: The mutant strain M4 was named YojK. M4 .

[0068] Example 27 Recombinant YojK M4 Catalytic synthesis of rebaudioside A into rebaudioside D The reaction was carried out in a 20 mL jacketed reactor, with 100 g / L (103 mM) rebaudioside A, 1000 mM sucrose, and 50 g / L sucrose synthase added to 10 mL of potassium phosphate buffer (100 mM, pH 8.0). AtSuS The lysate supernatant contained 1 mM UDP and 100 U / mL recombinant glycosyltransferase YojK. M4 The crude enzyme solution was prepared and reacted under magnetic stirring at 30°C. NaOH solution (1M) was added dropwise using an automatic potentiometric titrator to maintain the pH of the reaction solution at 8.0. The reaction conversion rate was monitored intermittently. After 12 hours of reaction, the concentration of rebaudioside D in the aqueous phase reached 105 g / L, indicating a conversion rate of approximately 90%.

[0069] Example 28 Recombinant YojK M4 Catalytic synthesis of rebaudioside A into rebaudioside D The reaction was carried out in a 20 mL jacketed reactor, with 100 g / L (103 mM) rebaudioside A, 1000 mM sucrose, and 50 g / L sucrose synthase added to 10 mL of potassium phosphate buffer (100 mM, pH 8.0). AtSuS Dissociated supernatant, 1 mM UDP, 5 g / L recombinant glycosyltransferase YojK M4 The lyophilized enzyme powder was reacted with magnetic stirring at 30°C. NaOH solution (1M) was added dropwise using an automatic potentiometric titrator to maintain the pH of the reaction solution at 8.0. The reaction conversion rate was measured intermittently. After 8 hours of reaction, the concentration of rebaudioside D in the aqueous phase reached 107 g / L, with a conversion rate of approximately 91.6%.

[0070] Example 29 1-L Scale Recombination of YojK M4 Catalytic synthesis of rebaudioside D The reaction was carried out in a 2L jacketed reactor, with 100 g / L (103 mM) rebaudioside A, 1000 mM sucrose, and 50 g / L sucrose synthase added to 1L potassium phosphate buffer (100 mM, pH 8.0). AtSuS Dissociated supernatant, 1 mM UDP, 5 g / L recombinant glycosyltransferase YojK M4 The lyophilized enzyme powder was reacted under mechanical stirring at 30°C and a stirring speed of 250 rpm. NaOH solution (1M) was added dropwise using an automatic potentiometric titrator to maintain the pH of the reaction solution at 8.0. The reaction conversion rate was measured intermittently. After 8 hours of reaction, the concentration of rebaudioside D in the aqueous phase reached 106 g / L, with a conversion rate of approximately 90.7%.

[0071] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention. The gene encoding glycosyltransferase (SEQ ID No. 1) is derived from *Bacillus subtilis*. Bacillus subtilis . The amino acid sequence of the glycosyltransferase YojK (SEQ ID No. 2) is derived from Bacillus subtilis. Bacillus subtilis . MANVLMIGFPGEGHINPSIGVMKELKSRGENITYYAVKEYKEKITALDIEFREYHDFRGDYFGKNATGDEERDFTEMLCAFLKACKDIATHIYEEVKHESYDYVIYDHHLLAGKVIANMLKLPRFSLCTTFAMNEEFAKEMMGAYMKGSLEDSPHYESYQQLAETLNADFQAEIKKPFDVFLADGDLTIVFTSRGFQPLAEQ FGERYVFVGPSITERAGNNDFPFDQIDNENVLFISMGTIFNNQKQFFNQCLEVCKDFDGKVVLSIGKHIKTSELNDIPENFIVRPYVPQLEILKRASLFVT HGGMNSTSEGLYFETPLVVIPMGGDQFVVADQVEKVGAGKVIKKEELSESLLKETIQEVMNNRSYAEKAKEIGQSLKAAGGSKKAADSILEAVKQKTQSANA Upstream primer (SEQ ID No. 3): CCGCATATGGCTAATGTATTAATGATCGG Downstream primer (SEQ ID No. 4): CCGCTCGAGCTATGCATTTGCTGATTGAG.

Claims

1. A glycosyltransferase mutant, characterized in that, It is a mutant formed by mutation at one or more of the following sites in the amino acid sequence shown in SEQ ID NO.2: serine at position 18, valine at position 37, glycine at position 63, glutamic acid at position 70, threonine at position 75, phenylalanine at position 81, alanine at position 84, threonine at position 129, methionine at position 142, threonine at position 240, asparagine at position 244, proline at position 324, valine at position 332, and lysine at position 347, and the glycosyltransferase mutant has higher activity than the glycosyltransferase shown in the amino acid sequence of SEQ ID NO.

2.

2. The glycosyltransferase mutant as described in claim 1, characterized in that, The amino acid sequence of the glycosyltransferase mutant is selected from one of the following: a) Replace serine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with valine; b) Replace the serine at position 18 of the amino acid sequence shown in SEQ ID No. 2 with valine, and replace the valine at position 37 with proline; c) Replace the 18th serine in the amino acid sequence shown in SEQ ID No. 2 with valine, the 63rd glycine with leucine, and the 81st phenylalanine with methionine. d) Replace the 18th serine of the amino acid sequence shown in SEQ ID No. 2 with valine, the 70th glutamic acid with valine, the 75th threonine with valine, the 84th alanine with leucine, the 240th threonine with aspartic acid, the 324th proline with leucine, the 332nd valine with cysteine, and the 347th lysine with phenylalanine; e) Replace glycine at position 63 with leucine, threonine at position 75 with valine, and alanine at position 84 with leucine in the amino acid sequence shown in SEQ ID No. 2; f) Replace the glutamic acid at position 70 of the amino acid sequence shown in SEQ ID No. 2 with valine; g) Replace the 70th position of glutamic acid in the amino acid sequence shown in SEQ ID No.2 with valine, the 75th position of threonine with valine, the 129th position of threonine with alanine, the 142nd position of methionine with glycine, and the 324th position of proline with leucine. h) Replace the threonine at position 75 of the amino acid sequence shown in SEQ ID No. 2 with valine; i) Replace phenylalanine at position 81 of the amino acid sequence shown in SEQ ID No. 2 with methionine; j) Replace phenylalanine at position 81 with methionine, alanine at position 84 with leucine, and threonine at position 129 with alanine in the amino acid sequence shown in SEQ ID No.

2. k) Replace phenylalanine at position 81 with methionine, alanine at position 84 with leucine, threonine at position 129 with alanine, proline at position 324 with leucine, and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No.

2. l) Replace phenylalanine at position 81 with methionine, threonine at position 129 with alanine, and lysine at position 347 with phenylalanine in the amino acid sequence shown in SEQ ID No.

2. m) Replace the threonine at position 129 of the amino acid sequence shown in SEQ ID No. 2 with alanine; n) Replace threonine at position 129 with alanine, methionine at position 142 with glycine, valine at position 332 with cysteine, and lysine at position 347 with phenylalanine in the amino acid sequence shown in SEQ ID No.

2. o) Replace the methionine at position 142 of the amino acid sequence shown in SEQ ID No. 2 with glycine; p) Replace the threonine at position 240 of the amino acid sequence shown in SEQ ID No. 2 with aspartic acid; q) Replace threonine at position 240 with aspartic acid, proline at position 324 with leucine, and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No.2; r) Replace proline at position 324 of the amino acid sequence shown in SEQ ID No. 2 with leucine; s) Replace proline at position 324 with leucine and valine at position 332 with cysteine ​​in the amino acid sequence shown in SEQ ID No. 2; t) Replace the lysine at position 347 of the amino acid sequence shown in SEQ ID No.2 with phenylalanine.

3. An isolated nucleic acid, characterized in that, The nucleic acid is a nucleic acid molecule encoding a glycosyltransferase mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 3.

5. A recombinant expression transformant, characterized in that, The recombinant expression transformant comprises the nucleic acid as described in claim 3 or the recombinant expression vector as described in claim 4.

6. A recombinant glycosyltransferase mutant catalyst, characterized in that, It is any of the following forms: a) Cultivate the recombinant expression transformant as described in claim 5, and isolate recombinant transformant cells containing the glycosyltransferase mutant as described in claim 1 or 2; b) Cultivate the recombinant expression transformant as described in claim 5, and separate the crude enzyme solution containing the glycosyltransferase mutant as described in claim 1 or 2; c) Freeze-dried enzyme powder obtained by freeze-drying the crude enzyme solution of the recombinant glycosyltransferase mutant.

7. The use of the glycosyltransferase mutant as described in claim 1 or 2, or the recombinant glycosyltransferase mutant catalyst as described in claim 6, in the preparation of rebaudioside D, characterized in that, Rebadini glycoside D was prepared by catalysis of rebadini glycoside A in a buffer solution in the presence of the cosubstrate UDP and sucrose using the glycosyltransferase mutant as described in claim 1 or 2 or the recombinant glycosyltransferase mutant as described in claim 6. The structure of rebaudioside A is shown in Formula I: The structure of lebodiin D is shown in Formula II: The structure of UDP is shown in Equation III: 。 8. The application as described in claim 7, characterized in that, The concentration of rebaudioside A is 10–110 mM; the amount of the glycosyltransferase mutant as described in claim 1 or 2 or the recombinant glycosyltransferase mutant catalyst as described in claim 6 is 2–30 g / L or 0.5–100 U / ml; the amount of sucrose is 4–10 times the equivalent of rebaudioside A; the amount of added UDP is 0–2 mM; the buffer is a phosphate buffer with pH 6.0–9.0; and the reaction temperature is 20–55 °C. When the glycosyltransferase mutant as described in claim 1 or 2 or the recombinant glycosyltransferase mutant catalyst as described in claim 6 is recombinant transformed cells or lyophilized enzyme powder, the amount is 2–30 g / L; when the glycosyltransferase mutant or the recombinant glycosyltransferase mutant catalyst is crude enzyme solution, the amount is 0.5–100 U / ml.

9. A method for preparing rebaudioside D via enzymatic cascade transformation, characterized in that, Using rebaudioside A as a substrate, sucrase is used in an enzymatic cascade with a glycosyltransferase mutant as described in claim 1 or 2 or a recombinant glycosyltransferase mutant as described in claim 6 to sequentially catalyze the glycosylation of UDP and the glycosylation of rebaudioside A, thereby obtaining rebaudioside D. The structure of rebaudioside A is shown in Formula I: The structure of lebodiin D is shown in Formula II: The structure of UDP is shown in Equation III: 。 10. The method according to claim 9, characterized in that, Includes the following steps: S1: In the presence of UDP, sucrase synthase catalyzes the conversion of sucrose into UDP-glucose; S2: Using the UDP-glucose obtained in step S1 as a glycosyl donor, glycosylation of rebaudioside A is catalyzed by the glycosyltransferase mutant as described in claim 1 or 2 or the recombinant glycosyltransferase mutant as described in claim 6 to obtain rebaudioside D, while simultaneously generating UDP, thereby achieving UDP recycling.

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