Application of PgUGT29 in synthesis of platycodin D3

Abstract

The invention discloses an application of PgUGT29 in synthesis of platycodin D3. The method specifically comprises cloning of the gene PgUGT29, codon optimization, expression and purification of the protein and enzyme activity application of the protein. The nucleotide sequence of the platycodon grandiflorum PgUGT gene is as shown in SEQ ID NO.1, the optimized nucleotide sequence is as shown in SEQ ID NO.2, and the amino acid sequence coded by the platycodon grandiflorum PgUGT gene is as shown in SEQ ID NO.3. The platycodon grandiflorum PgUGT29 gene has the function of specifically catalyzing platycodin D to generate platycodin D3, can be used for generating the platycodin D3, and is used for improving the quality of platycodon grandiflorum and improving the pharmacological activity of the platycodon grandiflorum.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to the application of PgUGT29 in the synthesis of platycodin D3. Background Art

[0002] Platycodon grandiflorum Platycodon grandiflorus (Jacq.) A. DC.) is a perennial herbaceous plant belonging to the genus Platycodon of the Campanulaceae family, and has various pharmacological activities such as anti-inflammatory, anti-tumor, anti-obesity and enhancing cognitive ability. The main active ingredients of Platycodon grandiflorum are platycodin D, platycodin D3 and platycodin E, and they all belong to oleanane-type pentacyclic triterpenoid saponins. The oleanane-type pentacyclic triterpenoid saponins in Platycodon grandiflorum are usually disaccharide-based saponins, and are mainly glycosylated at the C-3 and C-28 positions. The types of glycosyl groups linked mainly include D-glucose, L-arabinose, L-rhamnose, D-xylose, D-xylose and corresponding derivatives. The triterpenoid saponins in Platycodon grandiflorum can be divided into three types according to their parent nuclei: polygalacic acid type, platycodic acid type and platycogenic acid type, and among them, the most active platycodin D belongs to the platycodic acid type. At present, the platycodic acid type saponins isolated from Platycodon grandiflorum mainly include platycodigenin, platycodin F, platycodin D, platycodin D without apiose, platycodin E, platycodin E without apiose, 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 requires 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 the glycosyl chain is called glycosylation, and vice versa is called deglycosylation. Glycosyltransferases (GTs) are a class of enzymes that catalyze glycosylation reactions. They transfer the glycosyl group in the activated donor molecule to the receptor molecule, thereby generating various glycosides. Among them, glycosyltransferase family 1 (GT1) uses uridine diphosphate sugar as the glycosyl donor, so it is often called uridine diphosphate glycosyltransferase (UGT). At present, many reports have been found in plants that UGT can catalyze the combination of different types of glycosyl groups and triterpenoid saponin skeletons to generate new triterpenoid saponins. Because the structures of different types of Platycodon grandiflorum triterpenoid saponins are similar, it is currently difficult to extract different types of Platycodon grandiflorum saponins singly, and the process flow is complex and the cost is high. Moreover, due to its complex structure, it is very difficult to achieve total chemical synthesis. Therefore, obtaining and cloning the enzyme that can catalyze the synthesis of Platycodon grandiflorum triterpenoid saponins by biological means is of extremely important significance for obtaining more triterpenoid saponins and increasing the content of triterpenoid saponins by biological means. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of PgUGT29 in the synthesis of platycodin D3. The protease encoded by PgUGT29 can effectively catalyze the formation of platycodin D3 from platycodin D, providing a reference for further analyzing the synthesis pathway of platycodin, increasing the content of platycodin, and optimizing the germplasm of Platycodon grandiflorum.

[0005] The present invention is achieved by the following technical solutions: <First aspect> The present invention provides the application of Platycodon grandiflorum PgUGT29 in the synthesis of platycodin D3, and the amino acid sequence of the Platycodon grandiflorum PgUGT29 is as shown in SEQ ID NO. 3.

[0006] The nucleotide sequence of the Platycodon grandiflorum PgUGT29 is one of the following sequences: A. The nucleotide sequence as shown in SEQ ID NO.1; B. The nucleotide sequence as shown in SEQ ID NO.2 obtained by codon optimization of the sequence as shown in SEQ ID NO.1.

[0007] The synthesis of platycodin D3 is achieved by catalyzing the formation of platycodin D3 from platycodin D.

[0008] <Second aspect> A recombinant plasmid comprising Platycodon grandiflorum PgUGT29, and the amino acid sequence of the Platycodon grandiflorum PgUGT29 is as shown in SEQ ID NO.3.

[0009] The nucleotide sequence of the Platycodon grandiflorum PgUGT29 is one of the following sequences: A. The nucleotide sequence as shown in SEQ ID NO.1; B. The nucleotide sequence as shown in SEQ ID NO.2 obtained by codon optimization of the sequence as shown in SEQ ID NO.1.

[0010] <Third aspect> An engineered bacterium comprising Platycodon grandiflorum PgUGT29, and the amino acid sequence of the Platycodon grandiflorum PgUGT29 is as shown in SEQ ID NO. 3.

[0011] The nucleotide sequence of the Platycodon grandiflorum PgUGT29 is one of the following sequences: A. The nucleotide sequence as shown in SEQ ID NO.1; B. The nucleotide sequence as shown in SEQ ID NO.2 obtained by codon optimization of the sequence as shown in SEQ ID NO.1.

[0012] <Fourth aspect> A method for synthesizing platycodin D3, comprising the following steps: (1) Introduce the plasmid containing PgUGT29 of Platycodon grandiflorum into a host bacterium to obtain a genetically engineered bacterium; the amino acid sequence of the PgUGT29 of Platycodon grandiflorum is as shown in SEQ ID NO. 3.

[0013] (2) Cultivate the genetically engineered bacterium, induce protein expression, collect the bacterial liquid after induced expression, collect the bacterial cells and perform fragmentation treatment to obtain a cell lysate containing the PgUGT29 protein of Platycodon grandiflorum. (3) Add platycodin D as a substrate to the cell lysate to generate platycodin D3.

[0014] The nucleotide sequence of the PgUGT29 of Platycodon grandiflorum is one of the following sequences: A. The nucleotide sequence as shown in SEQ ID NO.1; B. The nucleotide sequence as shown in SEQ ID NO.2 obtained by codon optimization of the sequence as shown in SEQ ID NO.1.

[0015] <Fifth aspect> A glycosyltransferase protein, the amino acid sequence of which is as shown in SEQ ID NO.3.

[0016] The nucleotide sequence of the PgUGT29 of Platycodon grandiflorum is one of the following sequences: A. The nucleotide sequence as shown in SEQ ID NO.1; B. The nucleotide sequence as shown in SEQ ID NO.2 obtained by codon optimization of the sequence as shown in SEQ ID NO.1.

[0017] The present invention also provides a method for rapidly detecting the enzymatic activity function of the enzyme protein encoded by PgUGT29 Step 1: Construct an enzyme reaction system: The reaction is carried out in a 100 μl system, which includes 50 mM Tris-HCl (pH 7.0) buffer, 50 μl of the protein encoded by PgUGT29, 14 mM β-mercaptoethanol, 5 mM UDP-Glc, and 1 mM receptor substrate. The reaction is carried out overnight at 30 °C and terminated with cold methanol the next day.

[0018] Step 2: HPLC detection: The column temperature is 35 °C. The gradient elution system consists of 0.1% aqueous phosphoric acid solution (A) and acetonitrile (B).

[0019] The gradient elution program is as follows: 0 minutes (12% B), 0 - 1 minute (15% B), 1 - 4 minutes (20% B), 4 - 8 minutes (28% B), 8 - 17 minutes (31% B), 17 - 25 minutes (33% B), 25 - 36 minutes (38% B), 36 - 50 minutes (44% B). The flow rate is maintained at 1 mL / min.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1), At present, it is difficult to extract different kinds of platycodin from Platycodon grandiflorum. In the prior art field, although there are reports that glycosyltransferases can gradually convert platycodin D into D3 and E, however, these enzymes cannot specifically catalyze the conversion step of platycodin D to platycodin D3 during the catalytic process. The present invention has achieved a major breakthrough and for the first time clearly reveals the unique application of PgUGT29 in Platycodon grandiflorum. Specifically, the protease encoded by PgUGT29 exhibits high substrate specificity and can accurately and specifically catalyze the conversion of platycodin D to platycodin D3. This discovery has opened up a new biological path for the synthesis of platycodin D3. PgUGT29 can more precisely recognize and bind to platycodin D, reducing non-specific interactions with other substrates, thereby improving the selectivity of the reaction.

[0021] 2) It provides an important reference for further analyzing the synthesis pathway of platycodin. By studying the role of PgUGT29 in the conversion of platycodin D to platycodin D3, it helps to deeply understand the entire platycodin synthesis network and lays a foundation for comprehensively revealing the biosynthesis process of platycodin.

[0022] 3) By using PgUGT29 to catalytically synthesize more platycodin D3, through further research and regulation of this enzyme, the content of other platycodins can be increased, thereby improving the quality and medicinal value of Platycodon grandiflorum medicinal materials.

[0023] 4) Through genetic improvement of Platycodon grandiflorum by biotechnological means, excellent Platycodon grandiflorum varieties that can efficiently synthesize platycodin are screened or cultivated, promoting the development of the Platycodon grandiflorum planting industry and related industries.

[0024] 5) Compared with the traditional method of simply extracting different kinds of platycodin from Platycodon grandiflorum, the present invention uses biological means to obtain more triterpenoid saponins by cloning the enzymes that can catalyze the synthesis of Platycodon grandiflorum triterpenoid saponins, reducing the difficulty of obtaining specific platycodin, simplifying the process flow, and reducing cost input. At the same time, it avoids the problem that it is difficult to achieve total chemical synthesis due to the complex structure of platycodin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other features, objects, and advantages of the present invention will become more apparent by reading the following detailed description of non - limiting embodiments with reference to the accompanying drawings: Figure 1 Heat map drawing for similarity scores; Figure 2 Expression patterns of PgUGT88 in Platycodon grandiflorum roots, stems, and leaves; Figure 3 Expression patterns of PgUGT91 in Platycodon grandiflorum roots, stems, and leaves; Figure 4 Expression patterns of PgUGT96 in Platycodon grandiflorum roots, stems, and leaves; Figure 5 Expression patterns of PgUGT29 in Platycodon grandiflorum roots, stems, and leaves; Figure 6 Expression patterns of PgUGT27 in Platycodon grandiflorum roots, stems, and leaves; Figure 7 Expression patterns of PgUGT72 in Platycodon grandiflorum roots, stems, and leaves; Figure 8 Expression patterns of PgUGT84 in Platycodon grandiflorum roots, stems, and leaves; Figure 9 SDS - PAGE analysis diagram, where A is the SDS - PAGE analysis result of NPE and DPE of PgUGT29; Ø is the uninduced strain (negative control), 1 is NPE, 2 is DPE; B is the SDS - PAGE analysis result after purification of the inclusion body protein of PgUGT29; Figure 10 Result analysis diagram of PgUGT29 catalyzing the binding of platycodin D and glucose glycosyl to form PD3 with platycodin D as the substrate and UDP - Glc as the donor; Figure 11 Catalysis diagram of PgUGT29 with platycodin D3 as the substrate and UDP - Glc as the donor; Figure 12 Catalysis diagram of PgUGT29 with platycodigenin as the substrate and UDP - Glc as the donor; Figure 13 Schematic diagram of PgUGT29 catalyzing the glycosylation of platycodin D to form platycodin D3. Detailed implementation manners

[0026] The present invention will be described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can still be made. These all belong to the protection scope of the present invention.

[0027] Example 1 Screening and Expression Pattern Analysis of Platycodon grandiflorus PgUGT Gene 1. Screening Process (1) The PgUGT genes in Platycodon grandiflorus were screened using the Hidden Markov Model (PF00201), and a total of 107 PgUGT genes were obtained, named PgUGT 1 - 107.

[0028] (2) To study the catalytic effects of these PgUGTs on grandiflorasaponins, 24 glycosyltransferases (GTs) with catalytic effects at the C3 position of the triterpenoid saponin skeleton were selected as references and compared with 107 PgUGTs in NCBI by blast. Heat maps were drawn through similarity scores (as Figure 1 ) to screen out 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) that were highly similar to the reference GTs as candidate enzyme genes.

[0029] Table 1

[0030] 2. Expression Pattern Analysis RT-qPCR analysis was performed on the 7 screened PgUGTs (PgUGT27, PgUGT29, PgUGT84, PgUGT72, PgUGT88, PgUGT96, PgUGT91) to study their expression patterns in the roots, stems, and leaves of Platycodon grandiflorus.

[0031] The results are as Figures 2 - 8As shown, the expression levels of PgUGT27, PgUGT29, PgUGT72, and PgUGT88 were the highest in leaves, followed by stems, and finally roots, showing a consistent expression pattern. In previous studies, we found that the content of platycodin D in platycodon leaves was slightly lower than that in roots and stems, but the difference was not significant, while the content of platycodin E in platycodon leaves was significantly higher than that in roots and stems. The gene expression patterns of candidate PgUGTs were highly similar to the content distributions of platycodin D and platycodin E, that is, the parts with high expression levels of candidate PgUGTs also had relatively high PE content.

[0032] Therefore, it is speculated that these genes can transfer the glucose in UDP-glc to the C3 position of platycodin D, resulting in a decrease in the content of platycodin D and an increase in the content of platycodin E. In subsequent experiments, we selected PgUGT29 among them as an example for protein expression and purification, and verified their functions.

[0033] Example 2 Cloning of Platycodon grandiflorum PgUGT29 Gene and Construction of Recombinant Plasmid Platycodon grandiflorum variety: ( Platycodon grandiflorum (Jacq.) A.DC.) (1) Seedling cultivation: Growing in the medicinal herb garden of Anhui University of Chinese Medicine in Anhui Province, China, with a daytime temperature of 25 ± 2˚C and a nighttime temperature of 23 ± 2˚C.

[0034] (2) Total RNA extraction: Take 100 mg of young platycodon leaf tissue and place it in liquid nitrogen for thorough grinding until it becomes powdery. Extract the total RNA of the leaves according to the method in the plant total RNA extraction kit TransZol Kit instruction manual. Take 3 μL of the obtained plant total RNA for agarose gel electrophoresis to identify the quality, and then measure the concentration on a NanoDrop spectrophotometer.

[0035] (3) Gene cloning: Using the extracted total RNA as a template (500 ng), reverse transcribe to produce the first-strand cDNA according to the method in the reverse transcription kit FastKing RTKit instruction manual. The specific primer sequences are as follows: Forward primer P1: 5’-TTTCAAACTGTGGTGGAATGGC-3’, SEQ ID NO.14; Reverse primer P2: 5’-CAATAAGAGGCTTAGGAGAGGG-3’, SEQ ID NO.15.

[0036] PCR reaction system: Includes 5 μL of 10×KOD buffer, 5 μL of dNTPs, 4 μL of MgSO4, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of cDNA template, 1 μL of KOD enzyme, and ddH2O is added to make up to 50 μL.

[0037] PCR amplification conditions: pre-denaturation at 95°C for 3 min, 35 cycles (95°C for 30 sec; 54°C for 30 sec; 68°C for 100 sec), and finally extension at 68°C for 5 min.

[0038] After recovering and purifying the PCR products, they were ligated to the blunt-ended vector pLB (product of Tiangen Biochemical Co., Ltd.) and sequenced to obtain the recombinant plasmid pLB-PgUGT29.

[0039] Example 3. Expression and purification of the protein encoded by Platycodon grandiflorum PgUGT29 1. Codon optimization and subcloning The codons of PgUGT29 nucleotides were optimized for Escherichia coli preference (excluding BamHI / HindIII restriction enzyme sites. The amino acid sequence encoded by the Platycodon grandiflorum 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 optimized nucleotide sequence is shown in SEQ ID NO.2 (denoted as fragment 2).

[0040] Fragment 2 was digested with BamHI / HindIII, and the target fragment was ligated to the pET - 28β - 3C (Wuhan PuJian Biotechnology Co., Ltd.) vector that had been digested with the same enzymes to form the recombinant expression plasmid pET - 28β - 3C-PgUGT29.

[0041] BamHI: The recognized sequence is 5'-GGATCC - 3' HindIII: The recognized sequence is 5'-AAGCTT - 3'.

[0042] 2. Plasmid transformation and expression strain (1) Take the recombinant plasmid pET - 28β - 3C-PgUGT29 (2 μl) from step 1 and add it to Escherichia coli BL21(DE3) competent cells; (2) After placing on ice for 30 min, heat shock at 42°C in a water bath for 90 s; (3) Add antibiotic-free LB liquid medium (Shanghai Sangon Biotech Co., Ltd.), incubate at 37°C and 150 r / min for 40 min; (4) Spread the mixture onto a culture dish prepared with kanamycin-resistant LB medium (Shanghai Sangon Biotech Co., Ltd.) and incubate overnight in a 37°C constant temperature incubator.

[0043] 3. Protein induction expression Pick monoclonal colonies from the petri dish in step 2 into a culture container filled with kanamycin-resistant LB liquid medium, and culture at 37°C and 200 r / min for 3 - 4 h; take 1.5 mL of the bacterial solution into a new 15 mL centrifuge tube, add 1.5 μL IPTG (final concentration 1 mM), and induce culture at 37°C and 200 r / min for 4 h; collect the induced bacterial solution into a 1.5 mL centrifuge tube, centrifuge at 10000 g for 1 min, discard the supernatant, and retain the precipitate.

[0044] 4. Inclusion body protein purification (1) Add an appropriate amount of lysis buffer (PBS pH 7.5, 10% Glyerol, 1 mM PMSF) to the precipitate in step 3 to lyse the cells. After thorough mixing, lyse with an ultrasonic disruptor 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; (2) Resuspend the precipitate retained in step (1) with 1×PBS (pH 7.5) containing 8 M urea, and lyse with an ultrasonic disruptor for 20 min; after lysis, centrifuge at 12000 g for 10 min to separate the supernatant (the inclusion body protein mixture to be purified, DPE), and discard the precipitate; (3) Add 25 μL of 5× reducing Loading buffer to NPE and DPE respectively, boil for 10 min for SDS-PAGE detection, using the uninduced strain as a negative control.

[0045] (4) Add a certain amount of Ni resin to the inclusion body protein (DPE) mixture to be purified, incubate at 4°C in a shaker for 30 min, then collect the resin, temporarily retain the flow-through (FT), and then perform purification according to the following steps: elute the non-specifically bound proteins with purification buffer (1×PBS pH 7.5, 8 M urea), and then elute the target protein with elution buffer (PBS pH 7.5, 300 mM imidazole, 8 M urea). Add the collected protein solution into a dialysis bag and dialyze overnight with 50 mM 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 the purified protein by the Bradford method.

[0046] Results: The SDS-PAGE analysis results of NPE and DPE of PgUGT29 are as Figure 9As shown in A, Ø represents the uninduced strain (negative control), 1 represents NPE, and 2 represents DPE. The expected PgUGT29 protein is 56.57 kDa. The SDS-PAGE results showed that after induction culture at 37 °C for 4 hours under the condition of 1 mM IPTG, the target inclusion body protein DPE was well expressed.

[0047] After purification of the inclusion body protein, SDS-PAGE was performed again ( Figure 9 as shown in B). The final results showed that the PgUGT29 protein was successfully expressed, and the measured final concentrations were 0.21 mg / mL respectively.

[0048] Example 3 Verification of in vitro enzyme activity of Platycodon grandiflorum PgUGT29 (1)Construct an enzyme reaction system: The reaction was carried out in a 100 μl system, which included 50 mM Tris-HCl (pH7.0) buffer, 50 μl PgUGT-encoded protein (PgUGT29), 14 mM β-mercaptoethanol, 5 mM UDP-Glc, and 1 mM receptor substrate (platycodin D, platycodin D3, platycodigenin). The reaction was carried out at 30 °C for 12 h and terminated with cold methanol (4 °C) the next day.

[0049] (2)HPLC detection: High performance liquid chromatography analysis was performed using an Agilent 1260 Infinity II LC system (Agilent, USA). The chromatographic column was a Topsil C18 chromatographic column (4.6 mm × 250 mm; Agilent, USA), and the column temperature was 35 °C. The gradient elution system consisted of 0.1% phosphoric acid water (A) and acetonitrile (B). The gradient elution program was as follows: 0 minutes (12% B), 0 - 1 minute (15% B), 1 - 4 minutes (20% B), 4 - 8 minutes (28% B), 8 - 17 minutes (31% B), 17 - 25 minutes (33% B), 25 - 36 minutes (38% B), 36 - 50 minutes (44% B). The flow rate was maintained at 1 mL / min.

[0050] The results showed that: When platycodin D was used as the substrate and UDP-Glc was used as the donor, PgUGT29 could catalyze the binding of platycodin D and glucose glycosyl to generate PD3 ( Figure 10 ).

[0051] When platycodin D3 was used as the substrate and UDP-Glc was used as the donor, PgUGT29 could not catalyze the formation of platycodin E from platycodin D3 ( Figure 11 ).

[0052] When using platycodin aglycone as the substrate and UDP-Glc as the donor, PgUGT29 cannot catalyze the formation of platycodin aglycone-3-O-β-D-glucopyranoside from platycodin aglycone ( Figure 12 ).

[0053] Figure 13 Schematic diagram of PgUGT29 catalyzing the glycosylation of platycodin D to produce platycodin D3.

[0054] The results showed that PgUGT29 could catalyze the glycosylation of platycodin D with glucose to produce PD3, but could not catalyze the formation of platycodin E from platycodin D3, nor could it catalyze the formation of platycodin aglycone-3-O-β-D-glucopyranoside from platycodin aglycone. This indicates that PgUGT29 from Platycodon grandiflorum has specific substrate specificity and catalytic activity.

[0055] The present invention discloses an application of PgUGT29 in the synthesis of platycodin D3. Specifically, it includes the cloning of the gene PgUGT29, codon optimization, expression and purification of the protein, and the enzymatic activity application of the protein. The nucleotide sequence of the above-mentioned PgUGT gene from Platycodon grandiflorum is shown in SEQ ID NO.1, the optimized nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence encoded by it is shown in SEQ ID NO.3. The PgUGT29 gene from Platycodon grandiflorum in the present invention has the function of specifically catalyzing the formation of platycodin D3 from platycodin D, and can be used to produce platycodin D3 for improving the quality of Platycodon grandiflorum and enhancing its pharmacological activity.

[0056] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Application of Platycodon grandiflorum PgUGT29 in the synthesis of Platycodon grandiflorum saponin D3, characterized in that: The amino acid sequence of the Platycodon grandiflorum PgUGT29 is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The nucleotide sequence of Platycodon grandiflorum PgUGT29 is one of the following sequences: A, the nucleotide sequence shown in SEQ ID NO.1; B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The synthesis of platycodon saponin D3 is through catalyzing platycodon saponin D to generate platycodon saponin D3.

4. A recombinant plasmid comprising Platycodon grandiflorum PgUGT29, characterized in that: The amino acid sequence of Platycodon grandiflorum PgUGT29 is shown in SEQ ID NO.

3.

5. The recombinant plasmid according to claim 4, characterized in that The nucleotide sequence of Platycodon grandiflorum PgUGT29 is one of the following sequences: A, the nucleotide sequence shown in SEQ ID NO.1; B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.

1.

6. An engineered bacterium comprising Platycodon grandiflorum PgUGT29, characterized in that: The amino acid sequence of the Platycodon grandiflorum PgUGT29 is shown in SEQ ID NO.

3.

7. A method for synthesizing platycodon saponin D3, characterized in that: The synthesis method comprises the following steps: (1) introducing a plasmid containing Platycodon grandiflorum PgUGT29 into a host bacterium to obtain a genetically engineered bacterium; the amino acid sequence of Platycodon grandiflorum PgUGT29 is shown in SEQ ID NO. 3; (2) culturing the genetically engineered bacteria, inducing protein expression, collecting the bacterial solution after induced expression, and crushing the collected bacteria to obtain a cell lysate containing the Platycodon grandiflorum PgUGT29 protein; (3) Add platycodon saponin D as a substrate to the cell lysate to generate platycodon saponin D3.

8. The method for synthesizing platycodon saponin D3 according to claim 7, characterized in that: The nucleotide sequence of Platycodon grandiflorum PgUGT29 is one of the following sequences: A, the nucleotide sequence shown in SEQ ID NO.1; B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.

1.

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

3.

10. The glycosyltransferase protein according to claim 9, characterized in that The nucleotide sequence encoding the glycosyltransferase protein is one of the following sequences: A, the nucleotide sequence shown in SEQ ID NO.1; B. The nucleotide sequence shown in SEQ ID NO.2 obtained after codon optimization of the sequence shown in SEQ ID NO.1.

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