Use of pgugt29 in synthesis of platycodin d3

Abstract

The application discloses application of PgUGT29 in synthesis of kudou saponin D3. Specifically, the application includes cloning, codon optimization, protein expression and purification of the gene PgUGT29 and application of the enzyme activity of the protein. The nucleotide sequence of the kudou PgUGT gene is shown as SEQ ID NO. 1, the optimized nucleotide sequence is shown as SEQ ID NO. 2, and the encoded amino acid sequence is shown as SEQ ID NO. 3. The kudou PgUGT29 gene in the application has the function of specifically catalyzing kudou saponin D to generate kudou saponin D3, and can be used for generating kudou saponin D3, improving the quality of kudou and improving the pharmacological activity of kudou.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of PgUGT29 in the synthesis of platycodon saponin D3. Background Technology

[0002] Platycodon grandiflorus ( Platycodon grandiflorus (Jacq.) A. DC. is a perennial herb belonging to the genus Platycodon in the family Campanulaceae. It possesses various pharmacological activities, including anti-inflammatory, anti-tumor, anti-obesity, and cognitive enhancement. The main active components of Platycodon are platycodin D, platycodin D3, and platycodin E, all of which belong to the oleanane-type pentacyclic triterpenoid saponins. Oleanane-type pentacyclic triterpenoid saponins in Platycodon are typically disaccharidated saponins, primarily linked to glycosyl groups at the C-3 and C-28 positions. The types of glycosyl groups mainly include D-glucose, L-arabinose, L-rhamnose, D-xylose, D-xylose, and their corresponding derivatives. Based on their parent nucleus, the triterpenoid saponins in Platycodon can be divided into three types: polygalic acid, platycodic acid, and platycodic dicarboxylic acid, with the most active, platycodin D, belonging to the platycodic acid class. Currently, the saponins isolated from Platycodon grandiflorus mainly include platycodin aglycone, platycodin F, platycodin D, deapigenin platycodin D, platycodin E, deapigenin platycodin E, platycodin D2, and platycodin D3. The pharmacological and biological activities of saponins are mainly related to the number and type of glycosyl side chains.

[0003] The formation of different triterpenoid saponins involves the synthesis and modification of the triterpenoid saponin skeleton. The interconversion between triterpenoid saponins is a process of glycosylation modification; adding a glycosyl group to a glycosyl chain is called glycosylation, and vice versa, it is called deglycosylation. Glycosyltransferases (GTs) are a class of enzymes that catalyze glycosylation reactions, transferring a glycosyl group from an activated donor molecule to an acceptor molecule, thereby producing various glycosides. Among them, glycosyltransferase family 1 (GT1) uses uridine diphosphate glycosyl group as a glycosyl donor, and is therefore often called uridine diphosphate glycosyltransferase (UGT). Currently, many reports in plants have found that UGT can catalyze the combination of different types of glycosyl groups with the triterpenoid saponin skeleton to generate new triterpenoid saponins. Because different types of bellflower triterpenoid saponins have similar structures, the extraction of different types of bellflower saponins from bellflower alone is currently difficult, with complex processes, high costs, and, due to their complex structures, it is difficult to achieve complete chemical synthesis. Therefore, obtaining and cloning enzymes that can catalyze the synthesis of triterpenoid saponins from platycodon through biological means is of great significance for obtaining more triterpenoid saponins and increasing their content through biological methods. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the application of PgUGT29 in the synthesis of platycodon saponin D3. The protease encoded by PgUGT29 can effectively catalyze the conversion of platycodon saponin D to platycodon saponin D3, providing a reference for further elucidating the synthetic pathway of platycodon saponins, increasing platycodon saponin content, and optimizing platycodon germplasm.

[0005] This invention is achieved through the following technical solution:

[0006] <First Aspect>

[0007] This invention provides the application of Platycodon grandiflorus PgUGT29 in the synthesis of Platycodon grandiflorus saponin D3, wherein the amino acid sequence of Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO. 3.

[0008] The nucleotide sequence of the Platycodon grandiflorus PgUGT29 is one of the following sequences:

[0009] A. A nucleotide sequence as shown in SEQ ID NO.1;

[0010] B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

[0011] The synthesis of platycodon saponin D3 is achieved by catalytically converting platycodon saponin D into platycodon saponin D3.

[0012] <Second aspect>

[0013] A recombinant plasmid comprising Platycodon grandiflorus PgUGT29, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0014] The nucleotide sequence of the Platycodon grandiflorus PgUGT29 is one of the following sequences:

[0015] A. A nucleotide sequence as shown in SEQ ID NO.1;

[0016] B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

[0017] <Third aspect>

[0018] An engineered bacterium comprising Platycodon grandiflorus PgUGT29, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0019] The nucleotide sequence of the Platycodon grandiflorus PgUGT29 is one of the following sequences:

[0020] A. A nucleotide sequence as shown in SEQ ID NO.1;

[0021] B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

[0022] <Fourth Aspect>

[0023] A method for synthesizing platycodon saponin D3 includes the following steps:

[0024] (1) The plasmid containing Platycodon grandiflorus PgUGT29 was introduced into the host bacteria to obtain the genetically engineered bacteria; the amino acid sequence of Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO. 3.

[0025] (2) Culture the genetically engineered bacteria, induce protein expression, collect the induced bacterial culture, collect the bacterial cells for lysis, and obtain cell lysate containing Platycodon grandiflorus PgUGT29 protein.

[0026] (3) Platycodon saponin D was added to the cell lysate as a substrate to generate platycodon saponin D3.

[0027] The nucleotide sequence of the Platycodon grandiflorus PgUGT29 is one of the following sequences:

[0028] A. A nucleotide sequence as shown in SEQ ID NO.1;

[0029] B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

[0030] <Fifth Aspect>

[0031] A glycosyltransferase protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0032] The nucleotide sequence of the Platycodon grandiflorus PgUGT29 is one of the following sequences:

[0033] A. A nucleotide sequence as shown in SEQ ID NO.1;

[0034] B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

[0035] This invention also provides a method for rapidly detecting the enzymatic activity of an enzyme protein encoded by PgUGT29.

[0036] Step 1: Constructing the enzyme reaction system: The reaction was carried out in a 100 μl system, which included 50 mM Tris-HCl (pH 7.0) buffer, 50 μl of PgUGT29 encoded protein, 14 mM β-mercaptoethanol, 5 mM UDP-Glc, and 1 mM receptor substrate. The reaction was incubated overnight at 30°C and terminated the next day with cold methanol.

[0037] Step 2: HPLC detection: Column temperature was 35℃. The gradient elution system consisted of 0.1% phosphoric acid aqueous solution (A) and acetonitrile (B).

[0038] The gradient elution program is as follows: 0 min (12% B), 0–1 min (15% B), 1–4 min (20% B), 4–8 min (28% B), 8–17 min (31% B), 17–25 min (33% B), 25–36 min (38% B), 36–50 min (44% B). The flow rate is maintained at 1 mL / min.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1) Currently, extracting different types of platycodon saponins from Platycodon grandiflorus presents significant challenges. While existing technologies report the stepwise conversion of platycodon saponin D to D3 and E via glycoside transferases, these enzymes cannot specifically catalyze the conversion of platycodon saponin D to platycodon saponin D3. This invention represents a major breakthrough, clearly revealing for the first time the unique application of PgUGT29 in Platycodon grandiflorus. Specifically, the protease encoded by PgUGT29 exhibits high substrate specificity, precisely and specifically catalyzing the conversion of platycodon saponin D to platycodon saponin D3. This discovery opens a novel biological pathway for the synthesis of platycodon saponin D3, allowing PgUGT29 to more accurately recognize and bind to platycodon saponin D, reducing non-specific interactions with other substrates and thus improving reaction selectivity.

[0041] 2) This study provides an important reference for further elucidating the synthetic pathway of platycodon saponins. Investigating the role of PgUGT29 in the conversion of platycodon saponin D to platycodon saponin D3 helps to gain a deeper understanding of the entire platycodon saponin synthetic network, laying the foundation for a comprehensive reveal of the biosynthetic process of platycodon saponins.

[0042] 3) By utilizing PgUGT29 as a catalyst to synthesize more platycodon saponins D3, and through further research and regulation of this enzyme, the content of other platycodon saponins can be increased, thereby improving the quality and medicinal value of platycodon.

[0043] 4) Genetically improve Platycodon grandiflorus through biotechnology, screen or cultivate superior Platycodon grandiflorus varieties that can efficiently synthesize Platycodon saponins, and promote the development of Platycodon grandiflorus planting industry and related industries.

[0044] 5) Compared to traditional methods of extracting different types of platycodon saponins from platycodon, this invention utilizes biological methods to obtain more triterpenoid saponins by cloning enzymes that catalyze the synthesis of platycodon triterpenoid saponins. This reduces the difficulty of obtaining specific platycodon saponins, simplifies the process, and reduces costs. Simultaneously, it avoids the problem of achieving complete chemical synthesis due to the complex structure of platycodon saponins. Attached Figure Description

[0045] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0046] Figure 1 Heatmaps of similarity scores were generated.

[0047] Figure 2 The expression pattern of PgUGT88 in the roots, stems and leaves of Platycodon grandiflorus;

[0048] Figure 3 The expression pattern of PgUGT91 in the roots, stems and leaves of Platycodon grandiflorus;

[0049] Figure 4 The expression patterns of PgUGT96 in the roots, stems and leaves of Platycodon grandiflorus;

[0050] Figure 5 The expression pattern of PgUGT29 in the roots, stems and leaves of Platycodon grandiflorus;

[0051] Figure 6 The expression pattern of PgUGT27 in the roots, stems and leaves of Platycodon grandiflorus;

[0052] Figure 7 The expression patterns of PgUGT72 in the roots, stems and leaves of Platycodon grandiflorus;

[0053] Figure 8 The expression pattern of PgUGT84 in the roots, stems and leaves of Platycodon grandiflorus;

[0054] Figure 9 The images show SDS-PAGE analysis results, where A represents the SDS-PAGE analysis results of NPE and DPE of PgUGT29; Ø represents the uninduced strain (negative control), 1 represents NPE, and 2 represents DPE; B represents the SDS-PAGE analysis results of inclusion body proteins of PgUGT29 after purification.

[0055] Figure 10The result analysis diagram of the PgUGT29 catalyzing the binding of platycodon saponin D and glucose glycosyl group to generate PD3 when platycodon saponin D is used as substrate and UDP-Glc is used as donor;

[0056] Figure 11 The diagram shows the catalytic behavior of PgUGT29 when platycodon saponin D3 is used as the substrate and UDP-Glc is used as the donor.

[0057] Figure 12 The diagram shows the catalytic behavior of PgUGT29 when platycodon saponin aglycone is used as a substrate and UDP-Glc is used as a donor.

[0058] Figure 13 This is a schematic diagram of the PgUGT29-catalyzed glycosylation of platycodin D to platycodin D3. Detailed Implementation

[0059] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0060] Example 1: Screening and Expression Pattern Analysis of Platycodon grandiflorus PgUGT Gene

[0061] 1. Screening process

[0062] (1) The PgUGT gene in Platycodon grandiflorus was screened using the Hidden Markov Model (PF00201), and a total of 107 PgUGT genes were obtained, which were named PgUGT 1-107.

[0063] (2) To investigate the catalytic activity of these PgUGTs on triterpenoid saponins, 24 glycosyltransferases (GTs) with catalytic activity at the C3 position of the triterpenoid saponin backbone were selected as a reference and compared with 107 PgUGTs using BLAST in NCBI. A heatmap was generated based on the similarity scores (e.g., Figure 1 PgUGT27 (SEQ ID NO.8), PgUGT29 (SEQ ID NO.3), PgUGT84 (SEQ ID NO.9), PgUGT72 (SEQ ID NO.10), PgUGT88 (SEQ ID NO.11), PgUGT96 (SEQ ID NO.12), and PgUGT91 (SEQ ID NO.13), which are highly similar to reference GTs, were selected as candidate enzyme genes.

[0064] Table 1

[0065]

[0066] 2. Expression Pattern Analysis

[0067] Seven PgUGTs (PgUGT27, PgUGT29, PgUGT84, PgUGT72, PgUGT88, PgUGT96, and PgUGT91) were screened and analyzed by RT-qPCR to investigate their expression patterns in the roots, stems, and leaves of Platycodon grandiflorus.

[0068] The results are as follows Figure 2-8 As shown, PgUGT27, PgUGT29, PgUGT72, and PgUGT88 were expressed at the highest levels in leaves, followed by stems, and finally roots, exhibiting a consistent expression pattern. In previous studies, we found that the content of platycodon D in Platycodon grandiflorus leaves was slightly lower than that in roots and stems, but the difference was not significant, while the content of platycodon E in Platycodon grandiflorus leaves was significantly higher than that in roots and stems. The gene expression pattern of candidate PgUGTs is highly similar to the content distribution of platycodon D and platycodon E, that is, the PE content is correspondingly higher in the parts with high expression of candidate PgUGTs.

[0069] Therefore, it is hypothesized that these genes can transfer glucose from UDP-glc to the C3 position of platycodin D, leading to a decrease in platycodin D content and an increase in platycodin E content. In subsequent experiments, we selected PgUGT29 as an example for protein expression and purification, and verified its function.

[0070] Example 2 Cloning and Recombinant Plasmid Construction of Platycodon grandiflorus PgUGT29 Gene

[0071] Platycodon varieties: ( Platycodon grandiflorum (Jacq.)A.DC.)

[0072] (1) Seedling cultivation: The seedlings were grown in the medicinal herb garden of Anhui University of Traditional Chinese Medicine in Anhui Province, China, with a daytime temperature of 25±2˚C and a nighttime temperature of 23±2˚C.

[0073] (2) Total RNA extraction: 100 mg of young Platycodon grandiflorus leaf tissue was taken and ground into powder in liquid nitrogen. Total RNA was extracted from the leaves according to the instructions of the TransZol Kit. 3 μL of the obtained total RNA was subjected to agarose gel electrophoresis to identify its quality, and then the concentration was determined on a NanoDrop spectrophotometer.

[0074] (3) Gene cloning: Using the extracted total RNA as a template (500 ng), first-strand cDNA was produced by reverse transcription according to the instructions of the FastKing RT Kit. The specific primer sequences are as follows:

[0075] Forward primer P1: 5'-TTTCAAACTGTGGTGGAATGGC-3', SEQ ID NO.14;

[0076] Reverse primer P2: 5'-CAATAAGAGGCTTAGGAGAGGG-3', SEQ ID NO.15.

[0077] PCR reaction system: including 5 μL 10×KOD buffer, 5 μL dNTPs, 4 μL MgSO4, 1 μL forward primer, 1 μL reverse primer, 1 μL cDNA template, 1 μL KOD enzyme, and ddH2O to bring the total to 50 μL.

[0078] PCR amplification conditions: pre-denaturation at 95℃ for 3 min, 35 cycles (95℃ for 30 sec; 54℃ for 30 sec; 68℃ for 100 sec), and final extension at 68℃ for 5 min.

[0079] After the PCR product was recovered and purified, it was ligated into the blunt-ended vector pLB (a product of Tiangen Biotech Co., Ltd.) and sequenced to obtain the pLB-PgUGT29 recombinant plasmid.

[0080] Example 3: Expression and purification of the protein encoded by Platycodon grandiflorus PgUGT29

[0081] 1. Codon optimization and subcloning

[0082] The PgUGT29 nucleotide sequence was optimized using E. coli preferred codons (excluding BamHI / HindIII restriction endonuclease sites). The amino acid sequence encoded by the Platycodon grandiflorus PgUGT29 gene is shown in SEQ ID NO.3; the nucleotide sequence before optimization is shown in SEQ ID NO.1 (denoted as fragment 1), and the nucleotide sequence after optimization is shown in SEQ ID NO.2 (denoted as fragment 2).

[0083] Fragment 2 was digested with BamHI / HindIII, and the target fragment was ligated with the pET-28β-3C (Wuhan Pujian Biotechnology Co., Ltd.) vector that had been digested with the same enzymes using DNA ligase to form the recombinant expression plasmid pET-28β-3C-PgUGT29.

[0084] BamHI: Identified sequence 5'-GGATCC - 3'

[0085] HindIII: The recognized sequence is 5'-AAGCTT-3'.

[0086] 2. Plasmid transformation and expression strains

[0087] (1) Take 2 μL of the recombinant plasmid pET-28β-3C-PgUGT29 from step 1 and add it to Escherichia coli BL21(DE3) competent cells;

[0088] (2) After being placed on ice for 30 min, it was subjected to heat shock in a 42℃ water bath for 90 s;

[0089] (3) Add antibiotic-free LB liquid medium (Shanghai Sangon Biotech Co., Ltd.), incubate at 37℃ and 150 r / min for 40 min;

[0090] (4) Spread the mixture onto a culture dish prepared with kanamycin-resistant LB medium (Shanghai Sangon Biotech Co., Ltd.) and incubate overnight in a constant temperature incubator at 37°C.

[0091] 3. Protein-induced expression

[0092] Pick single clones from the culture dish in step 2 and transfer them to a culture container filled with kanamycin-resistant LB liquid medium. Incubate at 37°C and 200 r / min for 3-4 h. Transfer 1.5 mL of bacterial culture to a new 15 mL centrifuge tube and add 1.5 u LIPTG (final concentration 1 mM). Induce culture at 37°C and 200 r / min for 4 h. After induction, transfer the bacterial culture to a 1.5 mL centrifuge tube, centrifuge at 10000 g for 1 min, discard the supernatant, and retain the precipitate.

[0093] 4. Inclusion body protein purification

[0094] (1) Add an appropriate amount of lysis buffer (PBS pH 7.5, 10% Glyerol, 1 mM PMSF) to the precipitate from step 3 to lyse the cells. After mixing thoroughly, lyse the cells using an ultrasonic homogenizer for 20 min. Centrifuge at 12000 g for 10 min to separate the supernatant (the supernatant protein mixture to be purified, NPE) and retain the precipitate.

[0095] (2) The precipitate retained in step (1) was resuspended in 1×PBS (pH 7.5) containing 8 M urea and lysed by ultrasonic disruption for 20 min; after lysis, it was centrifuged at 12000 g for 10 min, and the supernatant (the mixture of inclusion body proteins to be purified, DPE) was separated and the precipitate was discarded.

[0096] (3) Add 25uL of 5× reducing loading buffer to NPE and DPE respectively, boil for 10 min and perform SDS-PAGE detection, with the uninduced strain as the negative control.

[0097] (4) Add a certain amount of Ni resin to the inclusion body protein (DPE) mixture to be purified, incubate in a shaker at 4°C for 30 min, collect the resin, temporarily retain the permeate (FT), and then purify according to the following steps: elute non-specifically bound proteins with purification buffer (1×PBS pH 7.5, 8M urea), and then elute the target protein with elution buffer (PBS pH 7.5, 300mM imidazole, 8M urea). Add the collected protein solution to a dialysis bag and dialyze overnight with 50mM Tris-HCl (pH 7.0) buffer. Collect the purified protein solution and perform SDS-PAGE detection using the method in step (3), and determine the concentration of purified protein using the Bradford method.

[0098] result:

[0099] The SDS-PAGE analysis results of NPE and DPE of PgUGT29 are as follows: Figure 9 As shown in Figure A, Ø represents the uninduced strain (negative control), 1 represents NPE, and 2 represents DPE. The expected PgUGT29 protein is 56.57 kDa. SDS-PAGE results showed that after 4 hours of induction culture at 37℃ and 1 mMIPTG, the target inclusion body protein DPE was well expressed.

[0100] After purification of inclusion body proteins, SDS-PAGE was performed again. Figure 9 (B). The final results showed that PgUGT29 protein was successfully expressed, with a final concentration of 0.21 mg / mL.

[0101] Example 3: In vitro enzyme activity verification of Platycodon grandiflorus PgUGT29

[0102] (1) Construction of the enzyme reaction system: The reaction was carried out in a 100 μl system, which included 50 mM Tris-HCl (pH 7.0) buffer, 50 μl of PgUGT-encoded protein (PgUGT29), 14 mM β-mercaptoethanol, 5 mM UDP-Glc and 1 mM receptor substrate (platycoside D, platycoside D3, platycoside aglycone). The reaction was carried out at 30 °C for 12 h and terminated with cold methanol (4 °C) the next day.

[0103] (2) HPLC detection: High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 Infinity II LC system (Agilent Technologies, USA). The chromatographic column was a Topsil C18 column (4.6 mm × 250 mm; Agilent Technologies, USA), and the column temperature was 35 °C. The gradient elution system consisted of 0.1% phosphoric acid (A) and acetonitrile (B). The gradient elution program was as follows: 0 min (12% B), 0–1 min (15% B), 1–4 min (20% B), 4–8 min (28% B), 8–17 min (31% B), 17–25 min (33% B), 25–36 min (38% B), 36–50 min (44% B). The flow rate was maintained at 1 mL / min.

[0104] The results show:

[0105] When platycodon saponin D is used as a substrate and UDP-Glc is used as a donor, PgUGT29 can catalyze the binding of platycodon saponin D and glucose glycosyl group to generate PD3. Figure 10 ).

[0106] When platycodon D3 is used as a substrate and UDP-Glc is used as a donor, PgUGT29 cannot catalyze the formation of platycodon E from platycodon D3. Figure 11 ).

[0107] When platycodon aglycone is used as a substrate and UDP-Glc is used as a donor, PgUGT29 cannot catalyze the formation of platycodon aglycone from platycodon aglycone to platycodon-3-O-β-D-glucopyranoside. Figure 12 ).

[0108] Figure 13 This is a schematic diagram of the PgUGT29-catalyzed glycosylation of platycodin D to produce platycodin D3.

[0109] The results showed that PgUGT29 could catalyze the binding of platycodin D and glucose glycosyl groups to form PD3, but it could not catalyze the formation of platycodin D3 from platycodin E, nor could it catalyze the formation of platycodin-3-O-β-D-glucopyranoside from platycodin aglycone. This indicates that platycodin PgUGT29 possesses specific substrate specificity and catalytic activity.

[0110] This invention discloses the application of PgUGT29 in the synthesis of platycodon saponin D3. Specifically, it includes the cloning of the PgUGT29 gene, codon optimization, protein expression and purification, and enzymatic activation of the protein. The nucleotide sequence of the above-mentioned platycodon PgUGT gene is shown in SEQ ID NO.1, the optimized nucleotide sequence is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3. The platycodon PgUGT29 gene of this invention has the function of specifically catalyzing the conversion of platycodon saponin D to platycodon saponin D3, and can be used to generate platycodon saponin D3 for the quality improvement of platycodon and to enhance the pharmacological activity of platycodon.

[0111] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The application of Platycodon grandiflorus PgUGT29 in the synthesis of platycodon saponin D3, characterized in that, The amino acid sequence of the Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The synthesis of platycodon saponin D3 is achieved by catalytically converting platycodon saponin D into platycodon saponin D3.

3. A recombinant plasmid comprising the nucleotide sequence encoding Platycodon grandiflorus PgUGT29, characterized in that, The amino acid sequence of the Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO.

3.

4. An engineered bacterium comprising the nucleotide sequence encoding Platycodon grandiflorus PgUGT29, characterized in that, The amino acid sequence of the Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO.

3.

5. A method for synthesizing platycodon saponin D3, characterized in that, The synthesis method includes the following steps: (1) A recombinant plasmid containing the nucleotide sequence encoding Platycodon grandiflorus PgUGT29 was introduced into a host bacterium to obtain a genetically engineered bacterium; the amino acid sequence of Platycodon grandiflorus PgUGT29 is shown in SEQ ID NO. 3; (2) Cultivate the genetically engineered bacteria, induce protein expression, collect the bacterial culture after induction, collect the bacterial cells and break them up to obtain cell lysate containing Platycodon grandiflorus PgUGT29 protein; (3) Platycodon saponin D was added to the cell lysate as a substrate to generate platycodon saponin D3.

6. A glycosyltransferase protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO. 3.

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