Peucedanum saccharosyltransferase PpUGT22 and its application

Abstract

The present invention discloses the use of an oxyglycosyltransferase encoded by the PpUGT22 gene of Peucedanum peucedanum in the biosynthesis of scopoletin, belonging to the field of biotechnology. The nucleotide sequence of the PpUGT22 gene encodes a nucleotide sequence having an amino acid sequence as shown in SEQ ID NO. 2. Scopoletin is obtained by an in vitro enzymatic biosynthesis method using the PpUGT22 protein, providing a new method for synthesizing scopoletin.

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 praeruptorum Dunn, also known as Peucedanum praeruptorum Dunn, wild angelica or Duhuo in traditional Chinese medicine, is a perennial herb mainly distributed in Northeast China, North China and East China. Its roots are often used in traditional Chinese medicine and have certain pharmacological effects and clinical application value. The medicinal part of Peucedanum praeruptorum Dunn is mainly its dried roots, which contain a variety of active ingredients, including but not limited to: ·Coumarins: such as imperatorin and isoimperatorin, which are one of the most important active ingredients in Peucedanum praeruptorum Dunn and have multiple biological activities such as anti-inflammatory, antioxidant and anti-tumor. ·Volatile oil: Contains some ingredients that have therapeutic effects on respiratory diseases. ·Polysaccharides: May have the effect of enhancing immunity. ·Flavones: Have antioxidant, anti-inflammatory and other effects.

[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. As one of the most common modifications, glycosylation is often associated with altered physiological activity. 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 to date, few studies have examined O-glycosyltransferases in Peucedanum peucedanum. Cloning and validating novel O-glycosyltransferases could provide novel 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) The enzyme provided by the present invention is not ion-dependent.

[0028] 3) The enzyme provided by the present invention can quickly reach optimal catalytic efficiency.

[0029] 4) 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

[0030] The method of the present invention and its beneficial effects are described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Figure 1 This is the electrophoresis result of amplifying the target gene fragment using Peucedanum praeruptorum cDNA as a template, with the DNA Marker (2k bp) on the left and the target gene fragment on the right.

[0032] Figure 2 This is the SDS-PAGE gel electrophoresis diagram of the purified protein, with the Marker on the left and the target protein on the right.

[0033] Figure 3 This is the liquid phase detection result of the catalytic reaction of PpUGT22.

[0034] Figure 4 Mass spectrometry results of the catalytic reaction liquid of PpUGT22

[0035] Figure 5 This is the result diagram of enzyme kinetic parameters, where A is the divalent metal ion, B is the reaction pH, C is the reaction temperature, D is the reaction time, and E is the Michaelis constant. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] Example 1

[0039] Cloning of target oxygen-glycosyltransferase gene

[0040] 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 PpUGT22 gene. Specific primer information is shown in Table 1:

[0041] Table 1 Primers sequence

[0042]

[0043] 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 A clear band appeared at about 1 500 bp, which was basically consistent with the expected gene size.

[0044] 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.

[0045] The nucleotide sequence and encoded amino acid sequence obtained by sequencing are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively. The gene sequence contains 1,365 bases and encodes 454 amino acids.

[0046] SEQ ID NO.1

[0047] ATGGAGAAGAGCAAAAAATGTCCTCATGCTCTGGTTCTTCCTTTCCCTACACAAGGCCACATAAACCCCATGCTCCAGTT

[0048] TTGCAAACGCTTGGTCTCGAAAGGCATCAAAGTCACACTTGCAAACACTATTCTCATCTCCAAATCCATGCATGCTGACC

[0049] CTCAAAGCCCCGTCTCTCTCGAAACTTACTCAGATGGTTTTGATCAAGTTGCAGGCTATGCTCAAGCAGGAAGCACTGAT

[0050] ATCTACCTCTCCAGTCTACGCAATGTCGGCTCTCAAACACTGGGCAATCTTGTCAAGAAACTTGATGAGGCTGGCAACCC

[0051] TGTGACTGTGCTCATTTACGATAGTTTGTTGCCTTGGGCTTTTGATGTTGCTAACAACTTTGGACTAAAGAAAGCTGTGT

[0052] TTTTTACTCAGTCATGTGCTGTGAACACTATATATTATCATGTTTATAAGGGTCTTATCTCTCTCCCACCGCGAGATGAC

[0053] GAATCAACAGTTATTTCGATTCCTGGATTGCCCCTGTTTAAATCATGGGAAGCACCAGATCCTCGTGACTACAAAGATAT

[0054] TCTAATAAATCAGTTCTCTAATGTTGATGATGTAGATTGGGTCTTTGTTAACACATTCCACAAATTGGAGGAAGAGGAGC

[0055] TTAAGTATGTGCCAAAATGTGGAAACTGAGGACAATAGGACCAACACTTCCATCTATGTACCTAGACCAGAGACTTGAA

[0056] GATGATAAAGGTTATGGTATTAATATTCAAGCCAGATAATGGGGTTTGTATGAAGTGGCTAAGTGAAAAACCTAGTGG

[0057] CTCAGTCATATATGTTTCGTTTGGAAGCTTCGTACAAGTTGAAGCAGAACAATTGGAAGAAGTAGCATGGGGGCTCAAGG

[0058] ACAGCAACTTCAACTTTTTGTGGGTGGTAAGGGAATCGGAAAAGCCAAAGCTTCCAAAATAACGTCATTAATGAGATATCT

[0059] GATAAGGGTCTGGTTGTGACATGGAGTCCACAGTTGGAGATATTAATACATGAGTCAATAGTTGTTTCGTGACACACTG

[0060] TGGTTTCAAATTCTGTTCTTGAAGCCATAAGTTTGGGAGTGCCTATGGTGGGGATACCGCAGTGGACTGATCAACCTACCA

[0061] ATGGCAAGTATATGGAGGATGTTTGGGGCGTGGGGGTCAGAGCTAGACCAGACGAAGGGAATTTGTGAAACGAAATGTT

[0062] ATAAAATCATCGATAAAAGAGGTAATGGAGGGAGAAAGGGGAAACAATCAAGAAAAATGCTGCCAAGTGGAAGAATTT

[0063] GGCTACAGAAGCTCTGGCTGAAGGTGGAACTTCTGATAAGAATATTAACGAGTTTGCAGCTGAATTGTTGCACTCTGTTG

[0064] NO

[0065] SEQ ID NO.2

[0066] MEKSKKCPHALVLPFPTQGHINPMLQFCKRLVSKGIKVTLANTIFISKSMHADPQSPVSLETYSDGFDQVAGYAQAGSTD

[0067] IYLSSLRNVGSQTLGNLVKKLDEAGNPVTVLIYDSLLPWAFDVANNFGLKKAVFFTQSCAVNTIYYHVYKGLISLPPRDD

[0068] ESTVISIPGLPLFKSWEAPDPRDYKDILINQFSNVDDVDWVFVNTFHKLEEEELKYVPKLWKLRTIGPTLPSMYLDQRLE

[0069] DDKGYGINIFKPDNGVCMKWLSEKPSGSVIYVSFGSFVQVEAEQLEEVAWGLKDSNFNFLWVVRESEKPKLPNNVINEIS

[0070] DKGLVVTWSPQLEILIHESIGCFVTHCGFNSVLEAISLGVPMVGIPQWTDQPTNGKYMEDVWGVGVRARPDEKGIVKRNV

[0071] IKSSIKEVMEGEKGETIKKNAAKWKNLATEALAEGGTSDKNINEFAAELLHSVE

[0072] Example 2 Induced expression and purification of PpUGT22 protein

[0073] Induction of PpUGT22 protein

[0074] (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.

[0075] (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.

[0076] Protein extraction

[0077] (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.

[0078] (2) Discard the supernatant, add 15 mL of Tris-HCl buffer to wash the bacterial pellet, and mix thoroughly by pipetting.

[0079] (3) Repeat steps (1) and (2) once.

[0080] (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.

[0081] (5) Take one centrifuge tube from (4), dilute its liquid until it turns milky white, and then divide it into 2 mL centrifuge tubes.

[0082] (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.

[0083] The protein expression was detected by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, and the results showed that ( Figure 2 ).

[0084] Example 3 In vitro enzyme activity detection

[0085] 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 sodium dihydrogen phosphate / sodium dihydrogen phosphate buffer, pH 8. 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:

[0086] Table 2 HPLC gradient elution conditions

[0087]

[0088] LC-MS conditions:

[0089] Column model: Waters ACQUITY UPLC BEH C18 column (2.1×100mm, 1.7μm)

[0090] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0091] Elution gradient: 0-5 min: 5-20% B; 5-8 min: 20-22% B; 8-17 min: 22-25% B; 17-23 min: 25-35% B; 23-25 ​​min: 35-50% B; 25-32 min: 50-95% B; 32-38 min: 100% B; 38-40 min: 100-5% B.

[0092] Mass spectrometry conditions: electrospray ion source; acquisition mode: AutoMS2; positive ion mode; capillary voltage: 3500 V; sheath gas temperature: 280°C, flow rate: 11 L / min; drying gas temperature: 320°C, flow rate: 10 L / min; mass-to-charge ratio scanning range: 100-1700 m / z; collision voltage: 175 V; collision energy: 15-50 eV.

[0093] Test results: HPLC test results showed ( Figure 3 ), by comparing the peak diagram with the standard, it was shown that the reaction solution contained scopoletin and scopoletin, which means that scopoletin was converted into scopoletin under the enzymatic reaction of PpUGT22. The conversion efficiency reached 74%. The mass spectrometry results also showed that the product was scopoletin ( Figure 4 ).

[0094] Example 4

[0095] Enzyme kinetic parameter detection

[0096] Scopoletin was used as substrate and UDP-Glc was used as glycosyl donor to investigate the effects of pH, temperature, metal ions and reaction time on the catalytic activity.

[0097] 1. Temperature: Investigate the changes in enzyme activity at different reaction temperatures (20°C, 30°C, 37°C, 40°C, 45°C, 50°C, 60°C, and 70°C). The reaction system contained 100 μL of crude enzyme, 0.5 mM substrate, 5 mM UDP-GLC, and 14 mM β-mercaptoethanol, and was diluted to 200 μL with 50 mM, pH 8.0 phosphate buffer. The reaction solution was reacted at each temperature for 12 hours. After the reaction, two volumes of pre-cooled methanol were immediately added to the reaction system to terminate the reaction. After centrifugation, the supernatant was filtered and tested by HPLC. Three parallel experiments were set up for each reaction. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0098] 2. pH: The effects of different 50 mM pH buffers on enzyme activity were investigated: 5.0-6.0 citric acid-sodium citrate buffer; 7.0-8.0 Na₂HPO₄-NaH₂PO₄ buffer; 8.0-9.0 Tris-HCl buffer; and 9.0-10.0 Na₂CO₃-NaHCO₃ buffer. The reaction system consisted of a 200 μL reaction system containing 14 mM β-mercaptoethanol, 5 mM UDP-GlyCl₂, 100 μL crude enzyme, and 0.5 mM scopoletin. The volume was then made up with the appropriate buffer. The reaction solution was incubated at 37°C for 12 h. Immediately after completion, two volumes of pre-chilled methanol were added. After centrifugation, the supernatant was filtered and analyzed by HPLC. To ensure data reliability, three replicates were performed for each reaction. Conversion was calculated based on peak area in the chromatogram.

[0099] 3. Metal ions: The receptor molecule of the reaction solution is selected as the compound scopoletin, and different divalent metal ions are investigated: Fe 2+ 、Mn 2+ 、Co 2+ 、Zn 2+ 、Ba 2+ , Ca 2+ Mg 2+ The effect of EDTA on enzyme activity. A 200 μL reaction system contained 14 mM β-mercaptoethanol, 5 mM UDP-GLC donor, 100 μL crude enzyme, 0.5 mM substrate, 5 mM divalent metal ions, and 50 mM phosphate buffer, pH = 8.0, to the specified volume. The reaction solution was placed at 37°C for 6 hours. Immediately after the reaction, two volumes of pre-cooled methanol were added and centrifuged at 14,000 rpm and 4°C for 4 minutes. The supernatant was filtered and analyzed by HPLC. Three replicates were set up for each reaction. The final conversion rate was calculated by the peak area of ​​the chromatogram.

[0100] 4. Reaction time: A 200 μL phosphate buffered saline reaction system containing 14 mM β-mercaptoethanol, 5 mM UDP-GLC, 100 μL crude enzyme, and 0.5 mM substrate was incubated in a 37°C water bath for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, and 28 hours, respectively. After completion of the reaction, each reaction solution was quickly terminated by adding two volumes of pre-chilled methanol. The mixture was then centrifuged at 14,000 rpm and 4°C for 4 minutes. The supernatant was filtered and analyzed on an HPLC system, and the substrate conversion was calculated. To ensure data accuracy, three replicates were performed for each group. The substrate conversion was calculated as the ratio of the peak areas in the HPLC spectrum.

[0101] 5. Michaelis constant (Km): The total volume of the enzyme activity reaction system was 200 μL, containing: 5 μL crude enzyme and 5 mM UDP-Glc. The substrate concentration ranged from 50 to 900 μM (50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, and 900 μM, respectively). The volume was made up to 200 μL with 50 mM phosphate buffer, pH 8.0. The reaction was incubated at 37°C for 10 min, then immediately terminated with an equal volume of pre-cooled methanol. After high-speed centrifugation at 14,000 rpm for 5 min, 20 μL was sampled and analyzed by HPLC. The liquid chromatography conditions were as above. The Michaelis constant was calculated using the Michaelis equation and the Lineweaver-Burk plot method.

[0102] Test results:

[0103] Enzyme kinetic studies have shown that ( Figure 4 ), PpUGT22 has the highest enzyme activity at 37°C and has a good conversion rate in the range of 30-40°C. The optimal pH of the reaction solution is 8.0, and the reaction is suitable for the reaction in the pH range of 7.0-8.0, and the catalytic efficiency is highest in phosphate buffer. The enzyme is not dependent on metal ions, Mg 2+ It can significantly increase the catalytic efficiency. The relative catalytic efficiency can reach more than 70 in 12 hours, and the relative conversion rate can reach the maximum catalytic efficiency in 22 hours. Finally, through calculation, the Km value of the above recombinant protein for kaempferol is 356.6mmol / L. The above enzyme kinetics related results are shown in Figure 5 A-5E.

[0104] 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.

3. A recombinant expression vector encoding the oxyglycosyltransferase according to claim 1.

4. A transgenic recombinant bacterium expressing the oxyglycosyltransferase 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 bacterium according to claim 4 in synthesizing scopoletin.

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

8. A method for synthesizing scopoletin according to claim 7, characterized in that: In step 1), the oxyglycosyltransferase is obtained by prokaryotic expression or chemical synthesis.

9. The method for synthesizing scopoletin according to claim 7, wherein: In step 2), the enzyme activity reaction system contains scopoletin and UDP-Glc.

10. The method for synthesizing scopoletin according to claim 7, wherein: The enzyme activity reaction system in step 2) also contains a buffer solution.

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