A kind of glycosyltransferase in Peucedanum peucedanum and its application in the synthesis of scopoletin

Abstract

The invention discloses the application of an oxygen-glycosyltransferase encoded by a PpUGT5 gene of Peucedanum peucedanum in the biosynthesis of scopolamine, and belongs to the field of biotechnology. The nucleotide sequence of the PpUGT5 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO.2. Scopolamine is obtained by an enzyme-catalyzed biosynthesis method using the PpUGT5 protein in vitro, providing a new method for synthesizing scopolamine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to peucedanum oxyglycosyltransferase and application thereof. Background Art

[0002] Peucedanum praeruptorumDunn. is a perennial upright herb, also known as chicken-foot peucedanum, official peucedanum, mountain angelica, etc. Peucedanum praeruptorumDunn. is adapted to warm and humid climatic conditions. It is distributed on sunny slopes at an altitude of 100-2000m, on the edges of sparse forests, in hillside grasses and roadside shrubs. It is mainly produced in Gansu, Henan, Guizhou, Guangxi, Sichuan, Hubei, Hunan and other places in my country. The "Pharmacopoeia of the People's Republic of China (2020 Edition)" (hereinafter referred to as "Chinese Pharmacopoeia") includes the dried roots of the Apiaceae plant Peucedanum praeruptorumDunn. It is dug up from winter to the following spring when the stems and leaves wither or before the flower stems emerge, remove the fibrous roots, wash, and dry in the sun or at a low temperature. Peucedanum praeruptorumDunn. has the effects of dispersing wind and heat, descending qi and resolving phlegm. It is clinically used for wind-heat cough with excessive phlegm, phlegm-heat wheezing, and coughing up phlegm. Studies have shown that Peucedanum peucedanum mainly contains coumarin chemical components, including simple coumarins, furanocoumarins and pyranocoumarins, and its main physiologically active ingredients are two coumarin compounds, peucedanum peucedanum A and peucedanum peucedanum B.

[0003] Peucedanum peucedanum contains numerous glycosylated coumarins. Glycosylation is the process by which glycosyl groups are transferred to small molecule acceptors under the action of proteases. Glycosylation is one of the most common and important reactions in biological systems. Glycosylated products have diverse functions, including information storage and transmission, energy storage, maintenance of cellular structural integrity, molecular recognition, signaling, virulence, and chemical defense. As one of the most common modifications, glycosylation is often associated with altered physiological activities. Some glycosylated metabolites exhibit improved biological activity, stability, and solubility. Glycosyltransferases (GTs) are primarily responsible for glycosylation reactions, transferring glycosyl donors to glycosyl acceptors to form glycosidic bonds. Glycosyltransferases are classified according to the glycosylation linkage pattern as O-, N-, C-, and S-glycosyltransferases. O-glycosyltransferases are key enzymes, but few studies have yet to investigate their role in Peucedanum peucedanum. Cloning and validating novel O-glycosyltransferases could provide new approaches for the enzymatic biosynthesis of glycosylated coumarins. Summary of the Invention

[0004] The present invention mainly addresses the above technical problems and provides an oxygen-glycosyltransferase gene that catalyzes the production of glycosylated coumarin compounds, as well as a method for biosynthesizing glycosylated coumarin compounds, to address the shortcomings of existing chemical synthesis of scopoletin.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides an oxygen-glycosyltransferase, the amino acid sequence of which comprises the sequence shown in SEQ ID NO.2.

[0007] In another aspect, the present invention provides an oxyglycosyltransferase gene, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0008] Alternatively, based on the principle of complementary pairing, the oxyglycosyltransferase gene provided by the present invention may be a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

[0009] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene comprises a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0010] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements. Therefore, the nucleotide sequence of the glycosyltransferase gene may also contain introns, promoters and various regulatory elements.

[0011] In another aspect, the present invention provides a recombinant expression vector encoding the aforementioned oxyglycosyltransferase.

[0012] In another aspect, the present invention provides a transgenic recombinant bacterium encoding the aforementioned oxyglycosyltransferase.

[0013] On the other hand, the present invention provides an application of an oxyglycosyltransferase gene in the synthesis of scopoletin, wherein the nucleic acid sequence of the oxyglycosyltransferase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0014] In a preferred embodiment, the above-mentioned glycosyltransferase gene sequence is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2, or a sequence that is completely complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

[0015] In a preferred embodiment, the nucleotide sequence of the above-mentioned glycosyltransferase gene is as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0016] In another aspect, the present invention provides use of the aforementioned oxyglycosyltransferase in the synthesis of scopoletin.

[0017] In another aspect, the present invention provides a method for synthesizing scopoletin, comprising the following steps:

[0018] 1) obtaining the aforementioned oxygen-glycosyltransferase;

[0019] 2) Using the oxygen-glycosyltransferase in step 1) to catalyze the synthesis of scopoletin in an enzyme activity reaction system.

[0020] In a preferred embodiment, the above-mentioned oxygen-glycosyltransferase is obtained by prokaryotic expression.

[0021] In a preferred embodiment, the above-mentioned oxyglycosyltransferase is obtained by chemical synthesis.

[0022] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned oxygen-glycosyltransferase, aglycone, a sugar donor and a buffer.

[0023] In a preferred embodiment, the aforementioned aglycone is scopoletin.

[0024] In the embodiment, the reaction system in step 3) further contains other necessary components known in the art for synthesizing coumarin compounds.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1) The present invention provides a novel oxygen-glycosyltransferase.

[0027] 2) Compared with the prior art, the present invention utilizes an enzyme-catalyzed biosynthesis method to obtain scopoletin, which has the advantages of simpler steps, less pollution, and a simpler product compared to chemical synthesis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The method of the present invention and its beneficial effects are described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Figure 1 The figure shows the electrophoresis results of amplifying the target gene fragment using Peucedanum peucedanum cDNA as a template, where M is DNA Marker (5k bp) and 1 is the target gene fragment.

[0030] Figure 2 The figure is the SDS-PAGE gel electrophoresis diagram of the purified protein, where M: Maker 1: PpUGT5 pure enzyme.

[0031] Figure 3 The following are the detection results of the catalytic reaction of PpUGT5, including: A. mass spectrometry detection result of the reaction product of the recombinant protein and the substrate; B. liquid chromatography detection result of the standard substance of scopoletin; C. liquid chromatography detection result of the standard substance of scopoletin. DETAILED DESCRIPTION

[0032] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] Example 1

[0035] Cloning of target oxygen-glycosyltransferase gene

[0036] 1. Total RNA was extracted from Peucedanum praeruptorum using a total RNA extraction kit, and single-stranded cDNA was generated using a reverse transcription kit with a gDNA wiper. This was used as a template for polymerase chain reaction (PCR) amplification of the full-length fragment of the PpUGT5 gene. Specific primer information is shown in Table 1:

[0037] Table 1 Primers sequence

[0038]

[0039] The PCR reaction system was as follows: 1 μL cDNA, 1 μL each of 10 μmol L-1 upstream and downstream primers, 12.5 μL of high-fidelity enzyme 2× PhantaMax Master Mix, and ddH2O to 25 μL. The reaction procedure was as follows: 95°C initial denaturation for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 1.15 min; extension at 72°C for 5 min; and storage at 4°C. PCR products were detected by 1.0% agarose gel electrophoresis and recovered with a DNA gel recovery kit. See the electropherogram for details. Figure 1 (M: DL 5000 marker; Lane 1: PpUGT5 gene), a clear band appeared at about 1 000 bp, which was basically consistent with the expected gene size.

[0040] 2. Use homologous recombinase to connect the PCR product with the pET-28a linear vector double-digested with Nde I and BamH I to obtain a recombinant plasmid, which is then transformed into DH5α chemically competent cells and sequenced after resistance screening.

[0041] The nucleotide sequence and encoded amino acid sequence obtained by sequencing are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The gene sequence contains 1473 nucleotides and encodes 490 amino acids.

[0042] SEQ ID NO.1

[0043] ATGAGAGCAGAGCTAATTTTCATCCCTTCGCCTGGAGTTGGTCATCTGGTATCTGCCGTTGAGTTTGCGAAGCTCCTTGC

[0044] TTGTCGAGATGAACGGATTTCCATCACCATTCTGGTAATGAAGTATCCTTTTGACTCCAACATAGGAGCATTGGCTCAGA

[0045] ACCTTAAGAAAGAGGCTCCTGAACGCATAGCCTTTGTAGACATCCCTGATTTAGATGAGACCACTCGGACAGAGCTCATG

[0046] TCTTTACCACAGATGTCGTTCTTCACTTTCTTCATTGAGAGCCAGCGGCCACTGGTGAGAGATGTAGTTGCAAAATTGTT

[0047] GAAACAGTCCGAGTCTGGTAAGCTTGGTGGATTCGTTATTGATATGTTCTGCGGCTCGATGATAGACGTGGCTAATGAAT

[0048] TCAATGTCCCAACATATGTATTCTTCACTTCAGGTGCTGCTTTTCTTTCCCTCACGTTTTATATCCAAAATCTCACAGGC

[0049] AACGAAAGCAAGGAAATTTCTGAGTACAAGGATTTAGAGGCTGACCTCTCCGTTCCTGGTTTCATCAACCCGGTACCTCT

[0050] TAAGGTTTTGCCTTCAGTGATGCTTACTAAGGAAGGGTCTGCCTTGGTCGTAGGTGTAGCTCGAAGGTTACGAGAGACCA

[0051] GAGCTATCTTGGTGAACACAGTGTGGGAACTGGAAGCCCATGCTATCAAGTCCCTTGCTGACGATGAAAACACCCCTCTG

[0052] ATTTACCATGTAGGCCCTTTAATTAATTTTACGACAGGTGGGGCAACAGCTGATAAGAAAAGGTCAGAAGAGGACATTGT

[0053] TTGCTGGTTGGACTGTCAACCGCCTCTTTCTGTAGTATTTCTGTGCTTTGGCAGTATGGGAAGCTTTGATACGGAGCAAG

[0054] TAACTGAGATAGCACAAGCACTGGAACTCAGCGGACAACGTTTCTTGTGGTCTCTACGACGCCCATCTCAAGAGAAAGAA

[0055] AATATGAAGCCGCCAACAGACTATGAGGATTACAGTGAGGTGTTGCCTGAGGGATTCTTGGAGCGGACATCAGAAATAGG

[0056] AAAGGTTATTGGATGGGCACCGCAGGTGACCATCCTTTCCCATCCATCTGTTGGAGGATTTGTATCCCATTGTGGATGGA

[0057] ACTCAACATTAGAGAGTATCTGGTGTGGCGTTCCAATGGCTACTTGGCCTCTGCATTCTGAGCAACAGATCAATGCGTTC

[0058] CAGTTAGTGAAGGAATTAGGAATTGCAGTGGAGATTAAAATGGACTACAGAAACGACCATTTTACTAACCTTGTATCAAC

[0059] AGAAGTTGTGACAGCAGATGTGATAGAAAGGGGTATAAGATATCTGATGGATGGAGAAAGTGAAGTCAGAAGTAAGATGA

[0060] AAGAAATGAAAAACAAATGCAGAGAAGCCACTGTAGAGGGTGGCTCATCTTATACTTCTCTTGGACAGTTTATCGACACT

[0061] TTCATGGATAACATCCGGGAAGGAGCATTTAATTGA

[0062] SEQ ID NO.2

[0063] MRAELIFIPSPGVGHLVSAVEFAKLLACRDERISITILVMKYPFDSNIGALAQNLKKEAPERIAFVDIPDLDETTRTELM

[0064] SLPQMSFFTFFIESQRPLVRDVVAKLLKQSESGKLGGFVIDMFCGSMIDVANEFNVPTYVFFTSGAAFLSLTFYIQNLTG

[0065] NESKEISEYKDLEADLSVPGFINPVPLKVLPSVMLTKEGSALVVGVARRLRETRAILVNTVWELEAHAIKSLADDENTPL

[0066] IYHVGPLINFTTGGATADKKRSEEDIVCWLDCQPPLSVVFLCFGSMGSFDTEQVTEIAQALELSGQRFLWSLRRPSQEKE

[0067] NMKPPTDYEDYSEVLPEGFLERTSEIGKVIGWAPQVTILSHPSVGGFVSHCGWNSTLESIWCGVPMATWPLHSEQQINAF

[0068] QLVKELGIAVEIKMDYRNDHFTNLVSTEVVTADVIERGIRYLMDGESEVRSKMKEMKNKCREATVEGGSSYTSLGQFIDT

[0069] FMDNIREGAFN

[0070] Example 2 Induced Expression and Purification of PpUGT5 Protein

[0071] Induction of PpUGT5 Protein

[0072] (1) Add 10 μL of Amp to 100 mL of liquid LB medium, and then add 1 mL of the BL21-Psj8-MBP bacterial solution obtained by the expanded culture. Incubate the culture in a shaker at 37°C and 200 rpm for 2-3 hours.

[0073] (2) Cultivate to OD 600 When the C value reaches about 0.6-0.8, the cells are taken out from the shaker, and an inducer IPTG with a concentration of 100 mM is added to make the final concentration 0.5 mM. The cells are placed in a shaker at 16°C and 200 rpm and continue to be shaken and cultured for 16 h.

[0074] Protein extraction

[0075] (1) The induced bacterial solution was divided into two 50 mL centrifuge tubes, balanced to a weight difference of less than 0.2 g, and centrifuged at 4500 rpm for 10 min at 4°C.

[0076] (2) Discard the supernatant, add 15 mL of Tris-HCl buffer to wash the bacterial pellet, and mix thoroughly by pipetting.

[0077] (3) Repeat steps (1) and (2) once.

[0078] (4) Centrifuge again at 4°C, 4500 rpm for 10 min, discard the supernatant, add 7 mL of Tris-HCl buffer, and transfer to two 5 mL centrifuge tubes.

[0079] (5) Take one centrifuge tube from (4), dilute its liquid to milky white, and then divide it into 2 mL centrifuge tubes.

[0080] (6) Take the 2 mL centrifuge tubes from step (5) and place them in an ice bath for 8 min of ultrasonic cell wall disruption. Centrifuge them at 4000 rpm at 4°C for 10 min. Dispense the supernatant and precipitate to obtain the crude enzyme.

[0081] The protein expression was detected by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and the protein was purified by Ni-NTA affinity chromatography column. The results showed that ( Figure 2 ), compared with the empty load, the pure enzyme showed a single, clear band around 95 kDa, which was basically consistent with the predicted protein size, indicating that it was the induced expressed PpUGT5 recombinant protein.

[0082] Example 3 In vitro enzyme activity detection

[0083] The initial reaction was performed in a 200 μL mixture containing 0.5 mM scopoletin, 5 mM UDP-Glc, 14 mM reducing agent, and 100 μL crude enzyme, which was then filled with Tris-HCl buffer, pH 7.5. A negative control was used, in which the protein was inactivated by heating at 100°C for 10 minutes. The mixture was incubated at 37°C for 12 hours, terminated by the addition of 200 μL of methanol, and centrifuged at 12,000 × g for 5 minutes at 4°C. The solution was then removed by injection and filtered through a microporous filter membrane before being analyzed by UHPLC. The solvent gradient elution conditions were as follows:

[0084] Table 2 HPLC gradient elution conditions

[0085]

[0086] Test results: HPLC test results showed ( Figure 3 The peak times of the standard control were: 8.541 min for scopoletin (S1); 14.711 min for scopoletin (S2). Compared with the control groups (B and C), the PpUGT5 enzymatic reaction group (A) showed chromatographic peaks at both 8.541 and 14.711 min, indicating that the reaction solution contained scopoletin and scopoletin, indicating that scopoletin was converted to scopoletin through the PpUGT5 enzymatic reaction.

[0087] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

Claims

1. An oxygen-glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. An oxygen-glycosyltransferase gene, the sequence of which is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector encoding the oxygen-glycosyltransferase according to claim 1.

4. A transgenic recombinant bacterium expressing the oxygen-glycosyltransferase according to claim 1.

5. Use of the enzyme according to claim 1 or the gene according to claim 2 in synthesizing scopoletin.

6. Use of the vector according to claim 3 or the bacteria according to claim 4 in synthesizing scopoletin.

7. A method for synthesizing scopoletin, the method comprising the following steps: 1) obtaining the oxygen-glycosyltransferase as claimed in claim 1; 2) Using the oxygen-glycosyltransferase in step 1) to catalyze the synthesis of scopolamine in an enzyme activity reaction system.

8. A method for synthesizing scopolamine as claimed in claim 7, characterized in that: In step 1), the oxygen-glycosyltransferase is obtained by prokaryotic expression or by chemical synthesis.

9. A method for synthesizing scopolamine according to claim 7, characterized in that: In step 2), the enzyme activity reaction system contains scopoletin and UDP-Glc.

10. A method for synthesizing scopolamine according to claim 7, characterized in that: The enzyme activity reaction system in step 2) also contains a buffer.

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