A glycosyltransferase CaUGT that catalyzes rebaudioside A to produce various steviol glycoside derivatives

Abstract

The present invention discloses a glycosyltransferase (CaUGT) capable of catalyzing rebaudioside A to produce multiple steviol glycoside derivatives. Recombinant Escherichia coli strains are constructed to construct the enzyme. The resulting crude recombinant enzyme solution and purified enzyme are capable of converting low-value rebaudioside A into multiple high-value steviol glycoside derivatives. The nucleic acid sequence is: a) the nucleotide sequence shown in SEQ ID NO. 1; or a nucleotide sequence different from the nucleotide sequence in a) that encodes the amino acid sequence shown in SEQ ID NO. 2. The enzyme gene is cloned into an Escherichia coli expression vector, achieving heterologous expression of the enzyme in Escherichia coli. The resulting recombinant protein, CaUGT, uses UDPG as a glycosyl donor to catalyze the substrate rebaudioside A to directly produce rebaudioside D, rebaudioside I, and rebaudioside M2. This recombinant CaUGT can convert low-value steviol glycosides into high-value steviol glycosides in a single step, and has significant industrial significance for the green conversion of high-value steviol glycosides by biochemical methods.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and molecular biology, and in particular to the application of glycosyltransferase CaUGT in the production of glycoside compounds. Background Art

[0002] Stevioside is a natural sweetener derived from the South American stevia plant. Due to its high sweetness (approximately 250-400 times that of sucrose) and zero calorie content, it is widely used in South America. In recent years, a variety of steviol glycoside derivatives (including steviol glycoside, rebaudioside A, rebaudioside D, and rebaudioside M) have been discovered in natural stevia. Subsequently, biocatalytic reactions have revealed that rebaudioside D and rebaudioside M exist as isomers, rebaudioside I and rebaudioside M2. Its structural formula is as follows:

[0003]

[0004] Rebaudioside A, rebaudioside D, and rebaudioside M received Generally Recognized as Safe (GRAS) certification from the United States Food and Drug Administration (USFDA) in 2008, 2013, and 2014, respectively.

[0005] Steviosides are linked to different numbers of glucose groups via β-1, β-(1-2), β-(1-3), and β-(1-6) glycosidic bonds, forming a variety of steviol glycoside derivatives. However, all steviol glycoside derivatives can ultimately be metabolized into steviol. Therefore, the safety data for a single steviol glycoside can be used to support the safety of other steviol glycoside derivatives.

[0006] In natural stevia, rebaudioside A is converted to rebaudioside D under the catalysis of UGT91D2. It is also converted to rebaudioside I under the catalysis of UGT76G1. Rebaudioside D and rebaudioside I are further glycosylated to produce rebaudioside M and rebaudioside M2. As the number of glycosyl groups increases, sweetness gradually increases and aftertaste decreases, but the natural content also decreases, resulting in low economic benefits for directly extracting high-value steviol glycosides from natural plants. Rebaudioside I and rebaudioside M2 ​​are isomers of rebaudioside D and rebaudioside M, differing only in the position of the glycosyl groups and possessing potential economic value. This invention can use cheap rebaudioside A as a substrate to directly catalyze the production of various steviol glycoside derivatives (including rebaudioside D, rebaudioside I and rebaudioside M2) in a one-step process. In particular, in the synthesis of rebaudioside M2, there is no need to add exogenous direct substrates rebaudioside D or rebaudioside I, which is of great significance to the economical production of steviol glycosides. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a glycosyltransferase CaUGT that can catalyze rebaudioside A to produce a variety of steviol glycoside derivatives, and the use of recombinant bacteria in catalyzing rebaudioside A to produce a variety of steviol glycoside derivatives (including rebaudioside D, rebaudioside I and rebaudioside M2), thereby solving the problem of low economic benefits of directly extracting high-value steviol glycosides from natural plants in the prior art.

[0008] The specific scheme of the present invention is:

[0009] A pepper glycosyltransferase CaUGT that catalyzes rebaudioside A to produce a variety of steviol glycoside derivatives. The pepper-derived glycosyltransferase CaUGT nucleic acid sequence is:

[0010] a) the nucleotide sequence shown in SEQ ID NO.1;

[0011] b) or a nucleotide sequence that is different from the nucleotide sequence in a) and can encode the amino acid sequence shown in SEQ ID NO.2.

[0012] A recombinant vector is constructed by cloning the pepper-derived glycosyltransferase CaUGT gene described in claim 1 into any one of pPICZα-A / B / C, pPIC9K, pPIC9, pPinkα-HC, pYES2, YCplac33, YEplac195, pHT01, pHT08, pHT43, pET series vectors, pMAL, pCOLD series vectors and pBAD series vectors.

[0013] A recombinant bacterium is obtained by transforming the recombinant vector into a host cell; the host cell includes any one of Escherichia, Pichia pastoris, Saccharomyces cerevisiae, and Bacillus subtilis.

[0014] The Escherichia species includes any one of E. coli BL21(DE3), BL21star(DE3), Tuner(DE3), T7Express and BL21-A1.

[0015] The application of glycosyltransferase CaUGT in catalyzing rebaudioside A to produce various steviol glycoside derivatives includes the following steps:

[0016] (1) inoculating a recombinant bacterium capable of expressing pepper glycosyltransferase CaUGT in a culture medium containing an inducer and culturing the culture medium;

[0017] (2) collecting and purifying the recombinant protein obtained in step (1) to obtain the purified enzyme CaUGT;

[0018] (3) adding the purified enzyme obtained in step (2) to a reaction system containing rebaudioside A, UDPG, metal cations and a buffer solution to perform a glycosylation reaction to catalyze the production of a variety of stevioside derivatives.

[0019] The induction expression method of the recombinant bacteria in step (1) is as follows: the recombinant bacteria are inoculated into LB culture medium and cultured until OD600 = 0.5-0.9, and then 0.1-1.2M isopropyl-β-D-thiogalactoside is added to induce expression.

[0020] The recombinant bacteria are induced to express at a temperature of 18-30° C. for 5-17 hours. The induced bacterial solution is centrifuged to collect the bacterial cells, which are then broken and centrifuged to obtain the recombinant enzyme.

[0021] The multiple steviol glycoside products include any one of rebaudioside D, rebaudioside I and rebaudioside M2.

[0022] The temperature of the glycosylation reaction in step (3) is 18° C.-60° C., the glycosylation reaction time is 6-96 hours, and the pH of the glycosylation reaction system is 5.0-10.5.

[0023] Step (1) is preferably: inoculating a recombinant bacterium capable of expressing glycosyltransferase CaUGT in a medium containing IPTG and culturing for 5-18 hours;

[0024] Step (2) is preferably: collecting the recombinant protein obtained in step (1), collecting the crude enzyme solution after ultrasonic disruption, collecting the supernatant by centrifugation to obtain the recombinant enzyme, and purifying the recombinant enzyme using a nickel resin column of GE to obtain the purified enzyme CaUGT;

[0025] Step (3) is preferably: constructing a reaction system containing 1-20 g / L rebaudioside A, 1-6 mM UDPG, 1-6 mM metal cation, a buffer solution at pH 6.0-10.5, and a CaUGT purified enzyme, placing the reaction system at 25-50° C. for 1-48 hours to obtain a variety of steviol glycoside derivatives (including rebaudioside D, rebaudioside I, and rebaudioside M2). In order to save the cost of UDPG, UDPG can also be added by adding sucrose synthase + sucrose substrate + UDP, using sucrose synthase to decompose sucrose into glucose and fructose, and glucose combines with UDP to form UDPG.

[0026] The method for constructing the recombinant strain is as follows: after connecting the pepper glycosyltransferase CaUGT nucleic acid sequence to an expression vector, constructing it into a host cell to obtain the recombinant bacteria.

[0027] In some embodiments, the present invention provides a recombinase comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO. 2.

[0028] The recombinant enzyme is prepared by inducing any of the above-mentioned recombinant bacteria.

[0029] In some embodiments, the method further comprises the following steps: centrifuging the induced bacterial solution, collecting the bacterial cells, and lysing the cells and then centrifuging to obtain the recombinant enzyme.

[0030] In some embodiments, the crude enzyme solution is purified using GE's Ni-NTA His.Bind resin.

[0031] Beneficial effects:

[0032] The present invention discloses a glycosyltransferase CaUGT from pepper, which can catalyze rebaudioside A to produce a variety of steviol glycoside derivatives (including rebaudioside D, rebaudioside I, and rebaudioside M2). The enzyme gene is cloned into an Escherichia coli expression vector, achieving heterologous expression of the enzyme in Escherichia coli. The resulting recombinant protein CaUGT achieves a breakthrough in catalyzing the substrate rebaudioside A to directly produce rebaudioside D, rebaudioside I, and rebaudioside M2 ​​using UDPG as a glycosyl donor. The reaction does not require the addition of rebaudioside D, the direct substrate of rebaudioside M2, thereby saving economic costs, simplifying the process, and achieving a high yield. This is of great significance to the industrial and economical production of steviol glycosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The plasmid map containing the nucleic acid sequence of pepper glycosyltransferase CaUGT obtained in Example 1;

[0034] Figure 2 This is the SDS-PAGE protein electrophoresis pattern of the crude enzyme solution of the recombinant protein CaUGT in Example 2;

[0035] Figure 3 This is a graph showing the HPLC results of the recombinant protein CaUGT catalytic reaction in Example 3;

[0036] Figure 4 The MS and NMR spectra of the three products in Example 3 are as follows: A: MS and NMR spectra of the product with an HPLC retention time of 3.8 min; B: MS and NMR spectra of the product with an HPLC retention time of 6.4 min; C: MS and NMR spectra of the product with an HPLC retention time of 8.4 min;

[0037] Figure 5 is the relative catalytic activity of the CaUGT enzymatic reaction at various UDPG concentrations in Example 4;

[0038] Figure 6 is the relative catalytic activity of the CaUGT enzymatic reaction under different reaction temperature conditions in Example 5;

[0039] Figure 7 is the relative catalytic activity of the CaUGT enzymatic reaction under different reaction pH conditions in Example 6;

[0040] Figure 8 The effects of different metal ions on the CaUGT enzymatic reaction in Example 7; relative activities were calculated using the control without the addition of metal ions; A: relative activity was calculated based on RA conversion; B: relative activity was calculated based on RM2 generation; C: relative activity was calculated based on RD generation; D: relative activity was calculated based on RI generation. DETAILED DESCRIPTION

[0041] The abbreviations used in the present invention are as follows:

[0042] Rebaudioside A, rebaudioside D, rebaudioside I, and rebaudioside M2 ​​are abbreviated as RebA, Reb D, Reb I, and Reb M2, respectively.

[0043] Uridine diphosphate glucose is abbreviated as UDPG;

[0044] Uridine diphosphate is abbreviated as UDP;

[0045] Isopropyl-β-D-thiogalactopyranoside is referred to as IPTG;

[0046] The present invention will be further described below with reference to specific embodiments.

[0047] Example 1

[0048] Obtaining the glycosyltransferase CaUGT gene and constructing the recombinant strain

[0049] The amino acid sequence (SEQ ID NO: 2) and nucleic acid sequence (SEQ ID NO: 1) of pepper (Capsicum annuum L.) glycosyltransferase were downloaded from GenBank. The nucleic acid sequence was codon-optimized by GenScript, and the optimized synthetic gene was ligated into the vector pET28a(+) through the restriction sites Nco1 and Xho1 to obtain the plasmid pET28a(+)-CaUGT. The plasmid map is shown in Figure 1 .

[0050] The resulting plasmid pET28a-CaUGT was transformed into E. coli BL21 competent cells and screened using LB (1% peptone, 0.5% yeast extract, 1% NaCl, 1.6% agar powder) solid plates containing 50 μg / ml (or 50-100 μg / ml) kanamycin. The screened monoclonal transformants were identified by colony PCR to obtain the recombinant strain E. coli BL21 (pET28a-CaUGT).

[0051] The above recombinant plasmids can also be transformed into competent cells such as E. coli Tuner (DE3), BL21star (DE3), T7Express and BL21-A1 to obtain corresponding recombinant strains.

[0052] Example 2

[0053] Induced expression of recombinant strains and preparation of crude enzyme solution

[0054] Taking the recombinant strain BL21(DE3)(pET28a-CaUGT) as an example, the expression of the pepper glycosyltransferase CaUGT gene in Escherichia coli was described.

[0055] The strain BL21 (DE3) (pET28a-CaUGT) was cultured in TB liquid medium (1.2% peptone, 2.4% yeast extract, 0.4% glycerol) containing 50 μg / ml (or any value between 50 and 100 μg / ml) of kanamycin at 37°C and 220 rpm until the OD 600 The pH value was 0.6-0.9, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1-1 mM, and the expression was induced at 18°C-30°C for 5-17 h.

[0056] The induced expression culture was centrifuged (12,000 rpm, 4°C, 10 min), the supernatant discarded, and the bacterial pellet collected. The collected cells were then washed once with 10 mM PBS (pH 7.2) to remove any residual culture medium. The cells were resuspended in 10 mM PBS (pH 7.2) at a ratio of 1 / 20 of the original bacterial volume and sonicated in an ice bath at 150 W for 5 seconds on, 8 seconds off, and a total of 8 minutes. The supernatant was collected after centrifugation (12,000 rpm, 4°C, 10 min). This was the crude enzyme solution of the glycosyltransferase CaUGT.

[0057] Take 5-20 μL of crude enzyme solution, add 5× protein loading buffer, mix well, denature and inactivate at 100°C for 10 min, centrifuge (12000 rpm, 4°C, 2-10 min), and use the supernatant for 10% SDS-PAGE gel protein electrophoresis. Figure 2 It can be seen that there is an obvious band at 52 kDa, which is basically consistent with the estimated size of the target protein, indicating that the crude enzyme solution of the recombinant protein CaUGT has been successfully prepared.

[0058] Example 3

[0059] CaUGT catalyzes the glycosylation of rebaudioside A to various steviol glycoside derivatives

[0060] The crude enzyme solution prepared in Example 2 was added to 0.5 mL of an enzymatic reaction system (1 g / L RebA, 1 mM UDPG, 3 mM MgCl2, 50 mM PBS buffer, 361 μL of the crude enzyme solution). After incubation at 30°C for 24 h, the reaction was terminated by the addition of 0.2 mL of 100% acetonitrile. After mixing, the mixture was allowed to stand for 10 min, centrifuged at 12,000 rpm for 10 min, and filtered through a 0.22 μm organic membrane. The substrate and product concentrations were analyzed by HPLC.

[0061] HPLC was performed using Shimadzu TECAN INFINITE 200M PLEX high performance liquid chromatography with a mobile phase of 25% acetonitrile, a flow rate of 1 ml / min, a column temperature of 40°C, a detector wavelength of 210 nm, and an injection volume of 50 μL.

[0062] Liquid phase analysis revealed that glycosyltransferase CaUGT could use UDPG as a glycosyl donor to catalyze RebA to produce three steviol glycoside derivatives with retention times of 3.8 min, 6.4 min, and 8.4 min, respectively. Figure 3 ), the three products were separated by high performance liquid chromatography, and the structures of the generated compounds were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy ( Figure 4 ).

[0063] The mass spectrum of the product with a retention time of 3.8 min ( Figure 4 A left) shows that the mass-to-charge ratio of the ion peak is 1313.5199 [M + Na] +, compared with its theoretical value (1314.2928, C 56 H 90 NaO 33 ), indicating that the sample molecular formula is C 56 H 90 O 33 , which is consistent with the structure of Reb M2. Figure 4 (A, right) Data analysis reveals that chemical shifts around 0.5-2.5 ppm are primarily due to methylene hydrogen signals from the aglycone portion of the structure; around 3.0-4.0 ppm, these are primarily due to hydrogen signals from the glucose terminal group; and at 5.5-4.8 ppm, these signals correspond to hydrogen signals from the glucose terminal groups, corresponding to H-20, 25, 32, 44, 60, 67, and 79 in the structure. The integrated area here is approximately 10H. Comparison of this H NMR spectrum with the RD H NMR spectrum reveals additional hydrogen signals near 3.8-4.3 ppm, highlighted in red, further confirming that the product is Reb M2.

[0064] The mass spectrum of the product with a retention time of 6.4 min ( Figure 4 B left) shows that the mass-to-charge ratio of the ion peak is 1151.4665[M+Na]+, compared with its theoretical value (1151.4734, C50 H 80 NaO 28 ), indicating that the sample molecular formula is C 50 H 80 O 28 , which is consistent with the structure of RD. Figure 4 (B, right) Data analysis revealed that chemical shifts around 0.5-2.5 ppm are primarily due to the methylene hydrogen signals of the aglycone portion of the structure. Signals from 3.0-4.0 ppm are primarily due to the hydrogen signals of the glucose terminal groups. The hydrogen signals at 5.5-4.8 ppm correspond to the terminal hydrogen signals of glucose, corresponding to H-20, 25, 32, 44, 60, and 67. The integrated area here is approximately 12H. Combined with the consistent liquid phase retention time of RD, which is consistent with that of the standard, further confirms the structure of RD.

[0065] The mass spectrum of the product with a retention time of 6.4 min ( Figure 4 C (left) The mass spectrum shows the mass-to-charge ratio of the ion peak is 1151.4668 [M+Na] + , compared with its theoretical value (1151.4734,C 50 H 80 NaO 28 ), indicating that the sample molecular formula is C 50 H 80 O 28 , which is consistent with the structure of RI.

[0066] H NMR spectroscopy ( Figure 4 (C right) Data analysis reveals that chemical shifts around 0.5-2.5 ppm are primarily due to methylene hydrogen signals from the aglycone portion of the structure. Those around 3.0-4.0 ppm are primarily due to hydrogen signals from the glucose terminal groups. The hydrogen signals at 5.5-4.8 ppm correspond to the terminal hydrogen signals of glucose, corresponding to H-20, 25, 32, 44, 60, and 67 in the structure. The integrated area here is approximately 12H. Comparison of the RD H NMR spectrum with the RI H NMR spectrum reveals substantial agreement, but a slight difference in the hydrogen signals around 4.0-4.5 ppm is attributed to the different glucose attachment methods in RD and RI, resulting from the different hydrogen chemical environments at this location. Combined with the consistent liquid phase retention time of RD and the RD standard, this further confirms the RI structure.

[0067] Example 4

[0068] CaUGT catalytic activity in reaction systems containing various UDPG concentrations

[0069] The purified enzyme CaUGT was added to a 50 mM PBS (pH = 7.2) containing 1 g / L RebA and 3 mM Mg 2+(MgCl2) was added to a 0.5 mL reaction mixture, and then 1 mM, 2 mM, 3 mM, and 4 mM uridine diphosphate glucose (UDPG) were added respectively. The reaction was allowed to stand at 30°C for 24 h. The reaction samples were subjected to liquid phase analysis to detect their catalytic activity in order to observe the effect of different UDPG concentrations on the enzymatic reaction. Figure 2 As shown, when the final UDPG concentration ranged from 1mM to 4mM, the catalytic activity increased significantly with increasing UDPG concentration and gradually approached equilibrium. At 4mM UDPG, the catalytic activity reached its maximum, with an RA conversion rate of 85.53%. At 3mM UDPG, the catalytic activity reached 98.71% of the maximum catalytic activity, approaching the maximum value and representing a 48.23% increase over the catalytic activity at 1mM UDPG. Considering economic factors, a 3mM UDPG concentration can be selected as the optimal enzymatic reaction condition.

[0070] Example 5

[0071] Catalytic activity of CaUGT at various reaction temperatures

[0072] The purified enzyme CaUGT was added to a 50 mM PBS (pH = 7.2) containing 1 g / L RebA, 1 mM uridine diphosphate glucose (UDPG) and 3 mM Mg 2+( The reaction mixture was added to 0.5 mL of the reaction mixture and allowed to react at 25–50 °C for 24 h. The reaction samples were analyzed by liquid phase to detect their catalytic activity to determine the optimal enzymatic reaction temperature ( Figure 3 The optimal temperature for the enzymatic reaction is 40°C, with relative activity exceeding 75% at 30–40°C. Therefore, CaUGT can effectively catalyze the conversion of RA within the 30–40°C temperature range.

[0073] Example 6

[0074] Catalytic activity of CaUGT at various reaction pH

[0075] Purified enzyme CaUGT was added to a culture medium containing 1 g / L RebA, 1 mM uridine diphosphate glucose (UDPG) and 3 mM Mg 2+ (MgCl2) was added to a 0.5 mL reaction mixture, and then buffers of different pH values ​​were added: potassium phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 8.0–9.0) and glycine buffer (pH 9.0–10.5). The reaction was allowed to stand at 30°C for 24 h. The reaction samples were subjected to liquid phase analysis to detect their catalytic activity. The activity of CaUGT in the pH range of 5.0–10.5 was determined to determine the optimal enzymatic reaction pH ( Figure 4The optimal reaction buffer for CaUGT is Tris-HCl pH 8.5, and it exhibits over 87% activity at pH 8.0-9.5. Therefore, CaUGT can effectively catalyze the conversion of RA within the pH range of 8.0-9.5.

[0076] Example 7

[0077] Catalytic tendency of CaUGT in reaction systems with various metal ions

[0078] The purified enzyme CaUGT was added to a 0.5 mL reaction mixture containing 1 g / LRebA, 1 mM uridine diphosphate glucose (UDPG), and 50 mM PBS (pH = 7.2). Six divalent metal ions of three different concentrations (1 mM, 3 mM, and 6 mM) were then added, including Ca 2+ (Calcium nitrate), Pb 2+ (lead acetate), Ba 2+ (barium chloride), Zn 2+ (zinc sulfate), Mn2+ (manganese chloride), Mg 2+ (magnesium chloride), and allowed to react at 30°C for 24 hours. The reaction samples were subjected to liquid phase analysis to detect their catalytic activity. The reaction without metal ions was used as the control group. The relative activity under various metal ion reaction conditions was calculated to determine the most suitable metal ion and concentration ( Figure 5 ).

[0079] Adding Zn to the enzymatic reaction catalytic system 2+ It will significantly inhibit the catalytic activity of CaUGT enzyme, reducing the relative activity of CaUGT to 12%-62%; Mg 2+ The use of Mg will enhance the catalytic activity of the enzyme. 2+ When the final concentration was 6mM, the relative activity of CaUGT was the highest, and the relative activity of RA conversion (121.36%) and the formation of three products (RM2: 137.70%; RD: 124.63%; RI: 119.55%) were all the highest. It is speculated that the main reason is that different metal ions can produce strong affinity with substrates, enzyme active products and the enzyme itself in different ways, resulting in changes in enzyme activity. Therefore, 6mM Mg can be selected. 2+ The next step of exploration is to use metal ions as the optimal reaction system. SEQUENCE LISTING <110> Sinochem Health Industry Development Co., Ltd. Tianjin University <120> A glycosyltransferase CaUGT that catalyzes rebaudioside A to produce various steviol glycoside derivatives <160> 2 <h2 style=";text-align:left;direction:ltr"><170> PatentIn version 3.5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 1362<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Capsicum annuum<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgggtaccc tgcgtgttct gatgttcccg tttctggcgt acggccacat cagcccgtat 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgaacgtgg cgaagaaact ggcggatcgt ggtttcctga tctacttttg cagcaccgcg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcaacctga agtacaccat caagaaaatt ccggagaagt atagcgacag catccagctg 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gttgaactgc acctgccgga gctgccggaa ctgccgccgc actatcatac caccaacggc 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgccgccgc acctgaacca caccctgcaa aaggcgctga aaatgagcaa gccgaacttc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcgaaaatcc tgcgtagcct gaagccggat ctggtgattt acgacgttct gcaacaatgg 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcgcagagcg tggcgaacga tcaaaacatc ccggcggtta aaatgctgac cagcggtgcg 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcggtgatta gctatttctt taacctgcgt aagaaaccgg gcgtggagtt cccgtacccg 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcgatctatc tgaccaaaat tgagcaggtg aaggttgcgg aactgctggc gaaggcggat 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaggagaaag aaccggacga tgttgatccg tttgcggacg gtaacatgca aatcatgctg 600<h2 style=";text-align:left;direction:ltr"> atgagcacca gccgtgtgct ggaggcgaaa tacattgact tctttaccga actgggtcac 660 tggaaggttg ttccggtggg tccgccggtt caggacccga tcattgacga ggtggacgat 720 gttgaactga tcgattggct gggtatgaaa gacgagagaca gcaccgtgtt cgttagcttt 780 ggcagcgagt atttcctgag caaggaac atggaggaaa tcgcgtttgg tctggaaaac 840 900 gatgttctgc cgaagggctt cctggaacgt atcggtgacc gtggccgtgt gctgaacaaa 960 tttgcgccgc aaccgcgtat tctgaaccac ccgagcaccg gtggcttcat cagccactgc 1020 ggttggaaca gcattatgga aagcatggat tttggcgttc cgatcattgc gatcccgatg 1080 cacctggacc agccgatgaa cgcgcgtctg atggtggagc tgggtgttgc gatcgaaatt 1140 gttcgtgacg atgacggcaa gttccaccgt ggcgagattg cggaaaccct gaaagatgtg 1200 atcaccccgta agcgtggcga gattctgcgt gcgaaagttc gtgacatcag caaaaaacctg 1260 aagagcgtgc gtgatgagga agcggacgtg gttgcgaagg aactgattca actgtgcaaa 1320 splash-scratch agtg-scratch-scratch-scratch ac 1362 <210> 2 <211> 454 <212> PRT <213> Capsicum annuum <400> 2 Met Gly Thr Leu Arg Val Leu Met Phe Pro Phe Leu Ala Tyr Gly His 1 5 10 15 Ile Ser Tyr Leu Asn Val Ala Lys Lys Leu Ala Asp Arg Gly Phe 20 25 30 Leu Ile Tyr Phe Cys Ser Thr Ala Ile Asn Leu Lys Tyr Thr Ile Lys 35 40 45 Lys Ile Pro Glu Lys Tyr Ser Asp Ser Ile Gln Leu Val Glu Leu His 50 55 60 Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn Gly 65 70 75 80 Your Pro Pro His Your Asn His Thr Your Gln Lys Only Your Lys Met Ser 85 90 95 Lys Pro Asn Phe Ala Lys Ile Leu Arg Ser Leu Lys Pro Asp Leu Val 100 105 110 Ile Tyr Asp Will Leu Gln Gln Trp Only Gln Ser Only Only Asn Asp Gln 115 120 125 Asn Ile Pro Ala Val Lys Met Leu Thr Ser Gly Ala Ala Val Ile Ser 130 135 140 Tyr Phe Phe Asn Leu Arg Lys Lys Pro Gly Val Glu Phe Pro Tyr Pro 145 150 155 160 Ala Ile Tyr Leu Thr Lys Ile Glu Gln Val Lys Val Ala Glu Leu Leu 165 170 175 Ala Lys Ala Asp Lys Glu Lys Glu Pro Asp Asp Val Asp Pro Phe Ala 180 185 190 Asp Gly Asn Met Gln Ile Met Leu Met Ser Thr Ser Arg Val Leu Glu 195 200 205 Ala Lys Tyr Ile Asp Phe Phe Thr Glu Leu Gly His Trp Lys Val Val 210 215 220 Pro Val Gly Pro Pro Val Gln Asp Pro Ile Ile Asp Glu Val Asp Asp 225 230 235 240 Val Glu Leu Ile Asp Trp Leu Gly Met Lys Asp Glu Ser Ser Thr Val 245 250 255 Phe Val Ser Phe Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu 260 265 270 Glu Ile Ala Phe Gly Leu Glu Asn Ser Asn Val Asn Phe Ile Trp Val 275 280 285 Ala Arg Phe Pro Lys Gly Glu Glu Gln Asn Leu Glu Asp Val Leu Pro 290 295 300 Lys Gly Phe Leu Glu Arg Ile Gly Asp Arg Gly Arg Val Leu Asn Lys 305 310 315 320 Phe Ala Pro Gln Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe 325 330 335 Ile Ser His Cys Gly Trp Asn Ser Ile Met Glu Ser Met Asp Phe Gly 340 345 350 Val Pro Ile Ile Ala Ile Pro Met His Leu Asp Gln Pro Met Asn Ala 355 360 365 Arg Leu Met Val Glu Leu Gly Val Ala Ile Glu Ile Val Arg Asp Asp 370 375 380 Asp Gly Lys Phe His Arg Gly Glu Ile Ala Glu Thr Leu Lys Asp Val 385 390 395 400 Ile Thr Arg Lys Arg Gly Glu Ile Leu Arg Ala Lys Val Arg Asp Ile 405 410 415 Ser Lys Asn Leu Lys Ser Val Arg Asp Glu Glu Ala Asp Val Val Ala 420 425 430 Lys Glu Leu Ile Gln Leu Cys Lys Asp Ser Asn Lys Cys Asn Leu Glu 435 440 445 His His His His His His 450

Claims

1. An application of pepper glycosyltransferase CaUGT in catalyzing rebaudioside A to produce a variety of steviol glycoside derivatives, characterized in that: The nucleic acid sequence of the pepper-derived glycosyltransferase CaUGT is: a) a nucleotide sequence shown in SEQ ID NO.1; or b) a nucleotide sequence that is different from the nucleotide sequence in a) and can encode the amino acid sequence shown in SEQ ID NO.

2.

2. The use of the glycosyltransferase CaUGT according to claim 1 in catalyzing rebaudioside A to produce a variety of steviol glycoside derivatives, characterized in that: The method comprises the following steps: (1) inoculating a recombinant bacterium capable of expressing a capsicum glycosyltransferase CaUGT into a culture medium containing an inducer for cultivation; (2) collecting and purifying the recombinant protein obtained in step (1) to obtain a purified enzyme CaUGT; and (3) adding the purified enzyme obtained in step (2) into a reaction system containing rebaudioside A, UDPG, a metal cation and a buffer solution to carry out a glycosylation reaction to catalyze the generation of a plurality of stevioside derivatives.

3. The use according to claim 2, characterized in that The induction expression method of the recombinant bacteria in step (1) is as follows: the recombinant bacteria are inoculated into LB culture medium and cultured until OD600 = 0.5-0.9, and then 0.1-1.2M isopropyl-β-D-thiogalactoside is added to induce expression.

4. The use according to claim 2, characterized in that The recombinant bacteria are induced to express at a temperature of 18-30° C. for 5-17 hours. The induced bacterial solution is centrifuged to collect the bacterial cells, which are then broken and centrifuged to obtain the recombinant enzyme.

5. The use according to claim 2, characterized in that The multiple steviol glycoside products include any one of rebaudioside D, rebaudioside I and rebaudioside M2.

6. The use according to claim 2, characterized in that The temperature of the glycosylation reaction in step (3) is 18° C.-60° C., the glycosylation reaction time is 6-96 hours, and the pH of the glycosylation reaction system is 5.0-10.5.

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