Oxy glycosyl transferase in peucedanum praeruptorum dunn and application of oxy glycosyl transferase in synthesis of scopolamine

Abstract

The invention discloses an application of oxygen glycosyl transferase coded by a peucedanum praeruptorum PpUGT5 gene in biosynthesis of scopolin, and belongs to the technical field of biology. The nucleotide sequence of the PpUGT5 gene encodes a nucleotide sequence of an amino acid sequence as shown in SEQ ID NO. 2. The scopolamine is obtained by a biosynthesis method for carrying out enzyme catalysis by utilizing the PpUGT5 protein in vitro, and a new method is provided for synthesizing the scopolamine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically 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 edge of sparse forests, on hillside grasses and on 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 the "Chinese Pharmacopoeia") includes the dried roots of the white flower peucedanum of the Umbelliferae plant. It is dug when the stems and leaves wither or when the flower stems are not drawn from winter to the following spring, remove the fibrous roots, wash, and dry in the sun or at 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 sputum, phlegm-heat wheezing, and sputum. Studies have shown that Peucedanum peucedanum mainly contains coumarin chemical components, including simple coumarins, furanocoumarins and pyranocoumarins. Its main physiologically active ingredients are two coumarin compounds, peucedanum peucedanum A and peucedanum peucedanum B.

[0003] Peucedanum peucedanum contains a large number of glycosylated coumarin compounds. The process of transferring glycosyl groups to small molecule receptors under the action of proteases is called glycosylation. Glycosylation is one of the most common and important reactions in biological systems. Glycosylated products have multiple functions, including information storage and transmission, energy storage, maintaining cell structural integrity, molecular recognition, signal transduction, toxicity and chemical defense. As one of the most common modifications, glycosylation is usually associated with changes in physiological activity. Some glycosylated metabolites show improved biological activity, stability and solubility. Glycosyltransferases (GTs) are mainly responsible for glycosylation reactions, transferring glycosyl donors to glycosyl acceptors to form glycosidic bonds. According to the connection pattern of glycosylation, they are divided into O-, N-, C- and S-glycosyltransferases. O-glycosyltransferase is the key enzyme, but so far, there are few reports on the study of O-glycosyltransferase in Peucedanum peucedanum. If new enzyme O-glycosyltransferases can be cloned and verified, it can provide a new method for the enzyme-catalyzed biosynthesis of glycosylated coumarin compounds. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides an oxygen-glycosyltransferase gene for catalyzing the production of glycosylated coumarin compounds, and a method for biosynthesizing glycosylated coumarin compounds, so as to solve the shortcomings of the existing chemical synthesis of scopolamine.

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

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

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

[0008] Or based on the principle of complementary pairing, the oxyglycosyltransferase gene provided by the present invention can 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, and thus the nucleotide sequence of the oxygen-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 oxygen-glycosyltransferase gene in the synthesis of scopoletin, wherein the nucleic acid sequence of the oxygen-glycosyltransferase 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 oxyglycosyltransferase 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, the method 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 scopolamine in an enzyme activity reaction system.

[0020] In a preferred embodiment, the above 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, sugar donor and buffer.

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

[0024] In the embodiment, the reaction system of step 3) also 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 uses an enzyme-catalyzed biosynthesis method to obtain scopoletin, which has the advantages of simpler steps, less pollution, and a more uniform product compared to chemical synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0029] Figure 1 This is the electrophoresis result 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 This is the SDS-PAGE gel electrophoresis diagram of the purified protein, where M: Maker 1: PpUGT5 pure enzyme.

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

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 creative work 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 ordinary technicians in the technical field to which this application belongs.

[0034] Example 1

[0035] Cloning of target oxygen-glycosyltransferase gene

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

[0037] Table 1 Primers sequence

[0038]

[0039] The PCR reaction system is: cDNA 1μL, 10μmol·L-1 upstream and downstream primers 1μL each, high-fidelity enzyme 2×PhantaMax Master Mix 12.5μL, ddH2O to 25μL. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 1.15min, 30 cycles; 72℃ extension for 5min; 4℃ storage. 1.0% agarose gel electrophoresis was used to detect PCR products, and DNA gel recovery kit was used to recover PCR products. Electrophoresis diagram is shown in 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 DH 5α chemically competent cells and sequenced after resistance screening.

[0041] The nucleotide sequence and the encoded amino acid sequence obtained by sequencing are shown as 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 BL21-Psj8-MBP bacterial solution obtained by expansion culture, and culture in a shaker at 37°C and 200 rpm for 2-3 h.

[0073] (2) Cultivate until OD 600 When the concentration reaches about 0.6-0.8, take it out from the shaker, add 100mM of inducer IPTG to make the final concentration 0.5mM, and continue shaking and culturing in a shaker at 16°C and 200rpm for 16h.

[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 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 of the centrifuge tubes in (4), dilute its liquid until it turns milky white, and then divide it into 2 mL centrifuge tubes.

[0080] (6) Take the 2 mL centrifuge tubes obtained in step (5) and place them in an ice bath for 8 min of ultrasonic cell wall disruption. Centrifuge at 4000 rpm for 10 min at 4°C. 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 carried out in a 200 μL mixture of 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 at pH 7.5. The protein inactivated by heating at 100 °C for 10 min was used as a negative control. The mixture was incubated at 37 °C for 12 h, terminated by adding 200 μL methanol, centrifuged at 12,000 × g at 4 °C for 5 min, and then removed by injection through a microporous filter membrane and then detected by UHPLC. The solvent gradient elution conditions were as follows:

[0084] Table 2 HPLC gradient elution conditions

[0085]

[0086] Test results: HPLC test results show ( Figure 3 ), the peak time of the standard control is: scopoletin (S1) 8.541min; scopoletin S2) 14.711min. Compared with the control group (B, C), the PpUGT5 enzymatic reaction group (A) showed chromatographic peaks at 8.541min and 14.711min, which indicated that the reaction solution contained scopoletin and scopoletin, that is, scopoletin was generated under the PpUGT5 enzymatic reaction.

[0087] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope 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 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.

3. A recombinant expression vector encoding the oxyglycosyltransferase 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 the synthesis of scopoletin.

6. Use of the vector according to claim 3 or the bacterium 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 according to claim 7, characterized in that: In step 1), the oxyglycosyltransferase is obtained by prokaryotic expression or by chemical synthesis.

9. A method for synthesizing scopolamine as claimed in claim 5, 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 5, characterized in that: The enzyme activity reaction system in step 2) also contains a buffer.

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