A Platycodin Glycosyltransferase PgGT1, Its Encoding Gene and Applications

Abstract

The present invention discloses a platycodin glycosyltransferase PgGT1, its encoding gene and applications, and claims a platycodin PgGT1 protein, which is a protein as follows (a1) or (a2): (a1) a protein encoded by the amino acid sequence shown in SEQ ID NO.2; (a2) a protein derived from SEQ ID NO.2 with a homology of ≧95% to the amino acid sequence shown in SEQ ID NO.2 and related to the regulation of the biosynthesis of platycodin D3 and / or platycodin E. The platycodin PgGT1 protein provided by the present invention can catalyze the addition of one molecule of glucose to the substrate platycodin D to generate platycodin D3 within ten minutes, and further rapidly and completely convert it into platycodin E within 1 h, having high economic value and broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the biological field, and in particular relates to a platycodon saponin glycosyltransferase PgGT1 and an encoding gene and application thereof. Background Art

[0002] Platycodon grandiflorum is a perennial plant of the Campanulaceae family and genus Platycodon. It has a strong ability to adapt to its ecological environment and is not only a highly valuable traditional Chinese medicine but also a common food in Southeast Asia, serving as both a medicinal and edible herb. The pentacyclic triterpenoid saponins platycoside D3 and platycoside E are active anti-inflammatory and expectorant components of Platycodon grandiflorum. They share the same sugar side chain attached to C-28 of the glycoside, differing only in the number of sugar groups attached to C-3. PD3 exerts anti-hepatitis C virus activity by regulating the production and secretion of airway mucins and is used as an expectorant for inflammatory lung diseases. PE has excellent pharmacological activity for the prevention or treatment of atopic dermatitis and myocardial infarction.

[0003] However, the preparation of these compounds is difficult due to the complex extraction and separation processes. Chemical synthesis methods also suffer from disadvantages such as side reactions, low regioselectivity, and low yields. In recent years, significant progress has been made in the enzymatic synthesis of glycosylation. However, relatively little research has been conducted on glycosyltransferases derived from Platycodon grandiflorum. Therefore, studying the enzymes and their encoding genes involved in the synthesis of PE from the medicinal and edible plant Platycodon grandiflorum has high economic value and broad application prospects. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a platycodon saponin glycosyltransferase PgGT1 and its encoding gene and application.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A Platycodon grandiflorum PgGT1 protein is the following protein (a1) or (a2):

[0007] (a1) a protein encoded by the amino acid sequence shown in SEQ ID NO. 2;

[0008] (a2) A protein derived from SEQ ID NO. 2 that has a homology of ≥95% with the amino acid sequence shown in SEQ ID NO. 2 and is involved in regulating the biosynthesis of platycoside D3 and / or platycoside E.

[0009] A gene encoding the above-mentioned Platycodon grandiflorum PgGT1 protein.

[0010] Preferably, the above-mentioned encoding gene is as follows (b1) or (b2):

[0011] (b1) the nucleotide sequence as shown in positions 1 to 1392 of SEQ ID NO. 1;

[0012] (b2) A nucleotide sequence having a homology of ≥95% with the nucleotide sequence shown at positions 1 to 1392 in SEQ ID NO. 1.

[0013] An expression vector, expression cassette or recombinant bacterium containing the above encoding gene.

[0014] The Platycodon grandiflorum PgGT1 protein is used as a glycosyltransferase.

[0015] The above coding gene is used for preparing glycosyltransferase.

[0016] The above-mentioned expression vector, expression cassette or recombinant bacteria is used to prepare glycosyltransferase.

[0017] The catalytic application of the above-mentioned Platycodon PgGT1 protein refers to catalyzing the substrate Platycodon saponin D to produce Platycodon saponin D3, or catalyzing the substrate Platycodon saponin D3 to produce Platycodon saponin E, or catalyzing the substrate Platycodon saponin D to produce Platycodon saponin D3 and further catalyzing the production of Platycodon saponin E.

[0018] Beneficial effects:

[0019] The Platycodon grandiflorum PgGT1 protein provided by the present invention can catalyze the addition of a substrate platycoside D to a molecule of glucose to produce platycoside D3 within ten minutes, and further rapidly and completely convert it into platycoside E within 1 hour, thus having high economic value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Agarose gel electrophoresis diagram of the amplified target gene PgGT1;

[0021] Figure 2 The figure is the SDS-PAGE electrophoresis of PgGT1 protein; M is the protein molecular weight standard; lane 1 is pET-32a (+) empty protein; lane 2 is the supernatant of PgGT1 crude enzyme; lane 3 is the purified protein of PgGT1;

[0022] Figure 3 This is the LC-MS detection result of the in vitro enzymatic activity reaction between PgGT1 and the substrate platycoside D. DETAILED DESCRIPTION

[0023] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] The amino acid sequence of glycosyltransferase PgGT1 is shown in Sequence No. 1, and the nucleotide sequence of the gene encoding PgGT1 is shown in Sequence No. 2.

[0025] Example 1: Cloning of Platycodon grandiflorum saponin glycosyltransferase PgGT1

[0026] Extraction of total RNA from Platycodon grandiflorum and reverse transcription to cDNA: Total RNA was extracted according to the instructions of the trace sample total RNA extraction kit from Beijing Biotech Biotechnology Co., Ltd., and after determination of concentration and purity, it was stored at -80°C until use. Total RNA was reverse transcribed into cDNA using the following protocol. Reaction conditions: 37°C for 15 minutes; denaturation at 85°C for 15 seconds; incubation at 4°C, and storage at -20°C until use.

[0027]

[0028] PCR amplification of the target gene: Using the above cDNA as a template, primers PgGT1-F (the restriction endonuclease recognition sequence is underlined) and PgGT1-R (the restriction endonuclease recognition sequence is underlined) were used to amplify the ORF. The amplification program was 95°C pre-denaturation for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 68°C for 1.5 min; and extension at 68°C for 5 min. The amplification primers were as follows:

[0029] PgGT1-F:5'-CC GGATCC ATGGATAGGGAAAATGAGGGGAGTT-3';

[0030] PgGT1-R:5'-CC CTCGAG TCATCTCAAAGTGGACATTTTCCCA-3'.

[0031] The PCR reaction system is as follows:

[0032]

[0033] The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the target band was recovered using an Axygen gel recovery kit. The recovered product was double-digested with BamHI and XhoI. At the same time, the pET-32a(+) vector was also double-digested with the following enzyme digestion system:

[0034]

[0035] Ligate the target gene with sticky ends to the plasmid using the following ligation system: incubate in a metal bath at 22°C for 1 hour. The ligation product can be temporarily stored at 4°C and promptly transformed into competent E. coli DH5ɑ.

[0036]

[0037] Spread the transformed product onto LB solid medium containing ampicillin. Incubate in a 37°C incubator for approximately 16 hours. Pick a single colony and transfer it to 800 μL of LB liquid medium containing ampicillin. Incubate at 37°C with shaking for 5-6 hours. After the bacterial solution becomes turbid, perform PCR verification. The reaction system is as follows:

[0038]

[0039] The amplification procedure is the same as the above gene cloning procedure. After the PCR is completed, agarose gel electrophoresis is performed and the positive bacterial solution is sent to Genewise for sequencing. The sequencing results show that the ORF electrophoresis detection results obtained by PCR amplification are as follows: Figure 1 The nucleotide sequence is shown in the sequence listing as SEQ ID NO. 1, and the gene is named PgGT1 gene. The amino acid sequence is shown in the sequence listing as SEQ ID NO. 2, and the protein is named PgGT1 protein. A single clone stock with correct sequencing was obtained and the PgGT1-pET32a(+) plasmid was extracted. The constructed prokaryotic expression vector was transformed into Escherichia coli BL21(DE3) competent cells using the plasmid heat shock method. The transformation, screening, and identification methods were the same as above.

[0040] Example 2: Prokaryotic expression of recombinant protein of Platycodon saponin glycosyltransferase PgGT1

[0041] Protein induction: Pick a single colony grown after streaking with pET-32a(+)-PgGT1 and culture it in LB liquid medium containing ampicillin antibiotics at 37°C overnight with shaking until the bacterial solution OD 600 When the value is within the range of 0.6-0.8, IPTG (final concentration reaches 0.5 mM) is added for induction (16°C, 160 rpm). After culturing for 20 h, the bacteria are collected by centrifugation and stored at -80°C.

[0042] Protein purification: ① Enzymatic hydrolysis and ultrasonic cell disruption: Place the bacterial solution in an ice-water mixture, ultrasonically lyse the bacteria, centrifuge at 4°C, 12000rpm, and collect the supernatant for purification. ② Add the collected supernatant to the equilibrated Ni-NTA column, add an equal column volume of eluent (containing 20mM imidazole) to remove impurities, and then add Elution buffer (containing imidazole at a concentration of 250mM) to collect the target recombinant protein. After determining the protein concentration using the Bradford protein concentration assay kit (BCA method), take a small amount of purified protein and detect it using SDS-PAGE electrophoresis gel. The detection figure is as shown below. Figure 2As shown, M represents a protein molecular weight standard; lane 1 represents pET-32a(+) empty protein; lane 2 represents crude PgGT1 enzyme supernatant; and lane 3 represents purified PgGT1 protein. The molecular weight of PgGT1 protein is 52.3 kDa. Combined with the tag protein contained in the pET-32a(+) vector, the molecular weight of the recombinant protein is approximately 70 kDa, consistent with the molecular weight of the purified protein, indicating that the recombinant protein was successfully induced and purified.

[0043] Example 3: In vitro enzymatic activity and functional characterization of Platycodon grandiflorum saponin glycosyltransferase PgGT1

[0044] In vitro enzyme activity analysis: After the concentration of the purified pET-32a(+)-PgGT1 protein was determined by BCA, an in vitro enzyme activity assay was performed. The reaction system was as follows:

[0045]

[0046] Platycoside D was used as the substrate and UDP-Glc was used as the sugar donor. The recombinant protein pET-32a(+)-PgGT1 was added and reacted at 42°C with low-speed shaking for 10, 20, 30, 40, 50, and 60 minutes. A control was performed under parallel conditions, with the addition of induced pET-32a(+) empty protein. The reaction was terminated with 100 μL of methanol and centrifuged at 12,000 rpm for 15 minutes. The supernatant was collected and analyzed by LC-MS.

[0047] Enzyme Activity Product Analysis: The product components of the enzyme activity reaction were detected using LC-MS coupled to an Agilent 1290 Ultra-High Performance Liquid Chromatography system. A ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm; Agilent, USA) was used. The column oven temperature was 35°C. The mobile phase consisted of pure water (A) and pure acetonitrile (B). The flow rate was 1 mL / min, and the injection volume was 10 μL. Mass spectrometry analysis was performed using an Agilent 1290LC-6530Q-TOF MS with quadrupole-time-of-flight mass spectrometer. The run parameters were set to negative ion mode for sample detection, and the gradient elution conditions were set to:

[0048]

[0049] The product was analyzed by LC-MS. PgGT1 converted platycoside D into compound 2 in ten minutes and completely converted compound 2 into compound 3 within 1 hour. Comparative analysis with the standard showed that the retention times of compound 2 and the standard platycoside D3, and compound 3 and the standard platycoside E were consistent. Further mass spectrometry analysis of the product showed that the molecular ion peak m / z 1385.6398 and the characteristic fragment ion peak m / z 843.4482 produced by compound 2 were consistent with the results of the standard platycoside D3, and the main molecular ion peak m / z 1547.6980 and the characteristic fragment ion peaks m / z 1415.6458, 1385.6285, 1283.6000, and 1005.5058 produced by compound 3 were consistent with the results of the standard platycoside E (such as Figure 3 Finally, it was confirmed that PgGT1 has obvious catalytic activity and can catalyze platycoside D to produce platycoside D3, and further catalyze platycoside D3 to produce platycoside E with 100% catalytic efficiency, which provides a research basis for subsequent synthetic biology research related to platycosides. The LC-MS detection results of the in vitro enzymatic reaction of PgGT1 with the substrate platycoside D are shown in Figure 2. Figure 3 shown.

[0050] SEQ ID NO.1: DNA, 1392, Platycodon grandiflorus

[0051]

[0052] SEQ ID NO.2: PRT, 463, Platycodon grandiflorus

[0053]

[0054]

[0055]

[0056] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A Platycodon grandiflorus PgGT1 protein, which is a protein encoded by the amino acid sequence shown in SEQ ID NO.

2.

2. A coding gene for the Platycodon grandiflorus PgGT1 protein according to claim 1.

3. The coding gene according to claim 2, wherein The gene is a nucleotide sequence shown in positions 1 to 1392 of SEQ ID NO.

1.

4. An expression vector, expression cassette or recombinant bacterium containing the coding gene according to claim 2 or 3.

5. Use of the Platycodon grandiflorus PgGT1 protein according to claim 1 as a glycosyltransferase.

6. Use of the coding gene according to claim 2 or 3 for preparing a glycosyltransferase.

7. Use of the expression vector, expression cassette or recombinant bacterium according to claim 4 for preparing a glycosyltransferase.

8. The catalytic application of the Platycodon grandiflorus PgGT1 protein according to claim 1, wherein the catalysis refers to catalyzing the substrate platycodin D to produce platycodin D3, or catalyzing the substrate platycodin D3 to produce platycodin E, or catalyzing the substrate platycodin D to produce platycodin D3 and further catalyzing to produce platycodin E.

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