A methyltransferase from Dendrobium huoshanense and its application in synthesizing flavonoid compounds with anti-tumor activity

Abstract

The invention discloses the application of methyltransferase encoded by DhOMT8 gene of Dendrobium huoshanense in biosynthesis of anti-tumor active flavonoid compounds, belonging to the field of biotechnology. The nucleotide sequence of DhOMT8 gene encodes the nucleotide sequence of amino acid sequence shown in SEQ ID NO.2. Methylated flavonoid compounds are obtained by biosynthesis method catalyzed by enzyme using DhOMT8 protein in vitro, providing a new method for synthesizing anti-tumor active flavonoid compounds.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a methyltransferase from Dendrobium huoshanense and an application thereof in synthesizing flavonoid compounds with antioxidant activity. Background Art

[0002] Huoshan Dendrobium has rich pharmacologically active ingredients, and its effects include: Anti-tumor: Some studies have shown that polysaccharides, flavonoids and other ingredients in Huoshan Dendrobium have inhibitory effects on certain cancer cells and have potential anti-tumor effects. Enhance immunity: Polysaccharides, flavonoids and alkaloids in Huoshan Dendrobium can enhance the body's immune function and improve disease resistance. Antioxidant: Polysaccharides and flavonoids in Huoshan Dendrobium have strong antioxidant effects, which can remove free radicals in the body and slow down the aging process. Protect the liver: Huoshan Dendrobium has a liver-protecting effect, which can promote liver cell regeneration and protect the liver from harmful substances. Nourishing yin and moistening the lungs: Traditional Chinese medicine believes that Huoshan Dendrobium has the effects of nourishing yin and moistening the lungs, clearing heat and promoting fluid production, and is often used to treat symptoms such as dry cough and pharyngitis. Promote digestion: Huoshan Dendrobium can promote gastric juice secretion, improve digestive function, and help treat problems such as loss of appetite and indigestion. The methoxytransferase of flavonoids is involved in the biosynthesis and metabolism of flavonoids in plants. These enzymes can catalyze the reaction in which the hydroxyl group (-OH) on the flavonoid skeleton is replaced by a methoxy group (-OCH3), thereby generating methoxylated flavonoid derivatives. Methoxytransferases introduce methyl groups at the hydroxyl position of flavonoids, changing their chemical structure and thus affecting their biological activity. Methylated flavonoids are generally more lipid-soluble and more easily pass through cell membranes, increasing their bioavailability and stability. Methylation modification can enhance the anti-tumor activity of flavonoids. For example, some methylated flavonoids (such as gentianin, isorhamnetin, isorhamnetin, etc.) show stronger anti-cancer activity in vivo, and can more effectively inhibit the proliferation of cancer cells and induce their apoptosis. In addition, methylated flavonoids also show enhanced antioxidant and anti-inflammatory effects, which are also of great significance for the prevention and treatment of cancer. Therefore, the study of flavonoid methoxytransferases helps to gain a deeper understanding of the biosynthetic mechanism of such compounds and provide a theoretical basis and potential biotechnological means for the development of new drugs.

[0003] Studies have shown that the chemical composition of Dendrobium huoshanense contains a rich class of methylated flavonoids with anti-tumor activity, and its methyltransferase can catalyze the methylation of the oxygen atoms of the hydroxyl groups in flavonoid compounds. Methylated flavonoid compounds mainly require chemical synthesis and enzyme synthesis. The chemical synthesis method has harsh reaction conditions, involves organic reagents, has a low yield and is prone to produce by-products that are harmful to the environment and human body. It is not the best choice for synthesizing methylated flavonoid compounds. In comparison, the enzyme synthesis method is more suitable. So far, no reports on the methyltransferase of Dendrobium huoshanense have been found. Therefore, it is of great significance to identify and develop enzymes with the function of synthesizing methylated flavonoid compounds and apply them in the synthesis of flavonoid compounds with anti-tumor activity. Summary of the invention

[0004] The present invention mainly aims at the above technical problems and provides a methyltransferase gene for catalyzing the production of anti-tumor active flavonoid compounds, as well as a method for biosynthesizing anti-tumor active flavonoid compounds, so as to solve the shortcomings of existing chemical synthesis of tumor flavonoid compounds.

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

[0006] In one aspect, the present invention provides a methyltransferase, whose amino acid sequence comprises the sequence shown in SEQ ID NO.2.

[0007] In another aspect, the present invention provides a methyltransferase 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 methyltransferase 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 methyltransferase 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 above-mentioned methyltransferase gene may also contain introns, promoters and various regulatory elements.

[0011] On the other hand, the present invention provides an application of a methyltransferase gene in the synthesis of flavonoid compounds with anti-tumor activity, wherein the nucleic acid sequence of the methyltransferase 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.

[0012] In a preferred embodiment, the methyltransferase 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.

[0013] In a preferred embodiment, the nucleotide sequence of the methyltransferase 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.

[0014] On the other hand, the present invention provides the use of the aforementioned methyltransferase in synthesizing flavonoid compounds with anti-tumor activity.

[0015] In another aspect, the present invention provides a method for synthesizing flavonoid compounds with anti-tumor activity, the method comprising the following steps:

[0016] 1) obtaining the aforementioned methyltransferase;

[0017] 2) Using the methyltransferase in step 1) to catalyze the synthesis of anti-tumor active flavonoid compounds in an enzyme activity reaction system.

[0018] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0019] In a preferred embodiment, the methyltransferase is obtained by chemical synthesis.

[0020] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing anti-tumor active flavonoid compounds and a buffer.

[0021] In another aspect, the present invention provides a method for synthesizing flavonoid compounds with anti-tumor activity, characterized in that the method comprises the following steps:

[0022] 1) obtaining a methyltransferase comprising the amino acid sequence shown in SEQ ID NO.2;

[0023] 2) Using the above methyltransferase to catalyze the synthesis of anti-tumor active flavonoid compounds in an enzyme activity reaction system.

[0024] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0025] In a preferred embodiment, the methyltransferase is obtained by chemical synthesis.

[0026] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing anti-tumor active flavonoid compounds and a buffer.

[0027] In a preferred embodiment, the enzyme activity reaction system also contains divalent cations.

[0028] In a preferred embodiment, the reaction temperature of the enzyme activity reaction system is 4-70°C.

[0029] In a preferred embodiment, the pH of the enzyme activity reaction system is 7.0-10.0.

[0030] In a preferred embodiment, the substrate used to synthesize the anti-tumor flavonoid compounds is luteolin.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1) Compared with the prior art, the present invention utilizes an enzyme-catalyzed biosynthesis method to obtain anti-tumor active flavonoid compounds, which has the advantages of simpler steps, less pollution, and more uniform products compared with chemical synthesis methods.

[0033] 2) The present invention provides a new methyltransferase having a wider suitable reaction temperature range (4-70°C).

[0034] 3) The methyltransferase in the present invention is not metal ion dependent.

[0035] 4) The methyltransferase of the present invention achieves a higher reaction rate in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 The results of cloned gene fragments. A: Gene cloning, M is a 2000 bp DNA marker, and lane 1 is DhOMT8.

[0038] Figure 2 The SDS-PAGE gel electrophoresis diagram of pET-28a(+)-DhOMT8 fusion protein. The left side is the protein marker (15-180 kDa), and the right side is the pure DhOMT8 enzyme (1, 2, and 3 are empty load, crude enzyme, and purified protein, respectively).

[0039] Figure 3 The results of the reaction between DhOMT8 and the substrate and the schematic diagram of the catalytic activity are shown in Figure 1. A: liquid phase diagram of the reaction between DhOMT8 and luteolin; B: structural diagram of the substrate and product; C: MS diagram of the catalytic product.

[0040] Figure 4 These are the results of enzymatic kinetic parameter detection of DhOMT8 catalyzing the conversion of luteolin to luteolin. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] Example 1

[0044] Cloning of target methyltransferase gene

[0045] 1. Total RNA extraction kit was used to extract total RNA from Dendrobium huoshanense. The experimental operation was carried out according to the instruction manual of IIQ RT SuperMix reverse transcription kit to obtain the ideal cDNA. The full-length fragment of DhOMT8 gene was amplified by polymerase chain reaction (PCR) using this as a template. The specific primers were designed according to the transcriptome data of this experiment, see Table 1:

[0046] Table 1 Primers sequence

[0047]

[0048] The cDNA of Dendrobium huoshanense extracted in the above experiment was used as a PCR template, and the target fragment was amplified using primers DhOMT8-F and DhOMT8-R. The polymerase used in PCR was Nanjing Novozyme 2×Phanta Max Master Mix. According to Table 10, 25 μL of PCR reaction system was prepared.

[0049] Table 2 25μL PCR reaction system for amplifying target gene (25μL)

[0050]

[0051]

[0052] Place the prepared PCR reaction system in a PCR instrument and amplify the target gene according to the PCR reaction program in 11.

[0053] Table 3 PCR reaction procedures for amplified genes

[0054]

[0055] After the reaction was completed, the product size was detected by 1% agarose gel electrophoresis. The electrophoresis diagram is shown in Figure A.

[0056] 2. Connect the amplified product to the T vector (pET-28a, Nanjing Novozymes Biotech Co., Ltd.) and transform it into E. coli DH5α. Select a single colony and culture it in LB medium and perform bacterial liquid PCR verification. The bacterial liquid PCR reaction system is: 12.5μL Green Taq Mix (P131, Nanjing Novozymes Biotech Co., Ltd.), 1μL of upstream and downstream primers, 2μL template (bacterial liquid), and add dd H2O to 25μL. Samples with correct bacterial liquid PCR results were sent for sequencing.

[0057] The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1. The gene sequence contains 1077 nucleotides and encodes 358 amino acids (shown in SEQ ID NO.2).

[0058]

[0059]

[0060] Example 2 Expression and detection of target protein

[0061] Expand the target bacterial solution with correct sequencing and extract the recombinant plasmid pET-28a-DhOMT8. Transform the recombinant plasmid into E. coli BL21 (DE3) competent cells, plate, and select clones to LB medium, culture at 37°C, 200rpm, and obtain the mother solution for 16 hours. Inoculate the mother solution into LB medium containing 50μg / mL KaN at a ratio of 1:100 and culture at 37°C until OD 600 =0.6-0.8 (with sterile LB medium as control), add IPTG with a final concentration of 0.5 mM, and finally culture at 16°C, 200 rpm for 18-20 h.

[0062] Collect the induced expression bacterial solution by centrifugation, resuspend the bacteria in pre-cooled Tris-HCl (50mM) buffer at pH = 9.0, centrifuge, collect the bacteria again, and repeat once to remove the residual culture medium. Finally, add 15mL of the above Tris-HCl buffer per gram of wet bacteria. The obtained bacterial suspension can be frozen in a -20°C refrigerator overnight, and then low-temperature crushed on an ultrasonic cell crusher with a power of 150W, working for 3s, and resting for 3s. When the bacterial suspension is clear, the crushing can be stopped. Finally, the crushed liquid is centrifuged at 4°C and 14000rpm for 4min. The supernatant is the crude enzyme solution, which is stored in a -20°C refrigerator.

[0063] According to the His-Tag tag on the fusion protein, the target protein was purified by nickel affinity chromatography, and the target protein was eluted with imidazole solutions of different concentrations. Finally, the molecular weight and purity of the target protein were detected by 10% SDS-PAGE electrophoresis. Figure 2 As shown, the target protein appeared around 43 kDa (1, 2, and 3 were used for empty load, crude enzyme, and purified protein, respectively), indicating that the protein was successfully expressed in the supernatant and the purified protein could be used for subsequent experiments.

[0064] Example 3 In vitro enzyme activity detection

[0065] 1. Enzyme activity reaction system: 10 μg pure enzyme, 0.5 mM luteolin, 5 mM SAM methyl donor, 14 mM β-mercaptoethanol, dilute to 200 μL with 50 mM, pH = 9.0 Tris-HCl buffer. After the reaction solution was incubated at 45°C for 45 minutes, an equal volume of pre-cooled methanol was added, vortexed and mixed, and centrifuged at 4°C, 14000 rpm for 4 minutes. The supernatant was filtered and detected by high performance liquid chromatography.

[0066] 2. HPLC conditions

[0067] Table 4 HPLC gradient elution conditions

[0068]

[0069] 3. LC-MS conditions:

[0070] The detection mode was negative ion mode, the capillary voltage was 3500 V, the sheath gas temperature was 280 °C, and the flow rate was 11 L min -1 , drying gas temperature 320℃, flow rate 10L·min -1 , nozzle voltage 155V, flow rate 0.250mL·min -1 .

[0071] 4. Test results:

[0072] Results Figure 3When DhOMT8 used luteolin as substrate, two product peaks were observed in the reaction. By comparing the retention time and mass-to-charge ratio m / z of the standard, it was found that peak a was the substrate luteolin and peak b was the product chrysanthemum yellow.

[0073] Example 4

[0074] Enzyme kinetic parameters detection

[0075] Using luteolin as substrate and SAM as methyl donor, the effects of pH, temperature, metal ions and reaction time on the catalytic activity were investigated.

[0076] 1. Temperature: With luteolin as the methyl acceptor and SAM as the methyl donor, the changes in enzyme activity at different reaction temperatures (4℃, 25℃, 30℃, 40℃, 50℃, 60℃ and 70℃) were investigated. The reaction system contained 10μg of pure protein, 0.5mM substrate, 5mM SAM, 14mM β-mercaptoethanol, and the volume was fixed to 200μL with 50mM, pH=9.0 Tris-HCl buffer. The reaction solution was reacted at each temperature for 45min. 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 tests were set for each group of reactions. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0077] 2. pH: Investigate the effects of different pH buffers of 50mM: 5.0-6.0, Citric acid-sodium citrate Buffer; 6.0-8.0, Na2HPO4-NaH2PO4 Buffer; 7.0-9.0, Tris-HCl Buffer; 9.0-10.0, Na2CO3-NaHCO3 Buffer on enzyme activity. The reaction system is: 14mMβ-mercaptoethanol, 5mMSAM, 10μg protein, 0.5mM compound luteolin in a 200μL reaction system, and the corresponding buffer is added to the specified volume. The reaction solution is placed at 45℃ for 45min, and immediately after the reaction, two volumes of pre-cooled methanol are added. After centrifugation, the supernatant is filtered and tested by HPLC. To ensure the reliability of the data, three replicate samples are set up for each reaction, and the final conversion rate is calculated by the peak area of ​​the chromatogram.

[0078] 3. Metal ions: The acceptor molecule of the reaction solution is selected as the compound luteolin, and the donor is selected as SAM. Different divalent metal ions are investigated: Mn 2+ 、Co 2+ 、Zn 2+ , Ba 2+ , Ca 2+ Mg 2+The effect of EDTA on enzyme activity. A 200μL reaction system contains 14mMβ-mercaptoethanol, 5mM SAM donor, 10μg protein, 0.5mM substrate, 10mM divalent metal ions, and 50mM Tris-HCl buffer at pH=9.0 is added to the specified volume. The reaction solution is placed at 45℃ for 10min, and immediately after the reaction, two volumes of pre-cooled methanol are added, and centrifuged at 14000rpm, 4℃ for 4min. The supernatant is filtered and tested by HPLC. Three replicates are set up for each reaction, and the final conversion rate is calculated by the peak area of ​​the chromatogram.

[0079] 4. Reaction time: 200 μL Tris-HCl (50 mM, pH = 9.0) reaction system contains 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg purified protein, 0.5 mM substrate luteolin, and reacts in a 45 ° C water bath for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. After the reaction is completed, two volumes of pre-cooled methanol are quickly added to each reaction solution to terminate the reaction, and centrifuged at 14000 rpm and 4 ° C for 4 min. The supernatant is filtered and tested on HPLC, and the conversion rate of the substrate is calculated. To ensure the accuracy of the data, three parallel experiments are set up for each group, and the substrate conversion rate is calculated according to the ratio of the peak area in the HPLC spectrum.

[0080] 5. Michaelis constant Km: The reaction system of DhOMT8 is: 200 μL Tris-HCl (50 mM, pH = 9.0) contains 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg purified protein, and luteolin is selected as the substrate, with concentrations of 100, 200, 500, 800, 1100, 1600, 2000 μM, respectively, and the reaction is carried out at 50 ° C for 1.5 min. Immediately after the reaction, two volumes of pre-cooled methanol were added to each reaction, and the supernatant was taken after centrifugation and detected on HPLC. The detection wavelength is the maximum absorption wavelength of the substrate, and the conversion rate of the substrate is calculated based on the peak area. To ensure the accuracy of the data, three parallel experiments were set up for each group. The Michaelis constant was obtained by fitting the Michaelis equation.

[0081] 6. Test results:

[0082] Enzyme kinetic studies have shown that ( Figure 4 ), DhOMT8 has the highest enzyme activity at 50℃, and has a good conversion rate in the range of 4~70℃. The optimum pH of the reaction solution is 9.0, and it is suitable for reactions in the pH range of 8.0~9.0, and its catalytic activity in Tris-HCl buffer is higher than that in Na2CO3-NaHCO3. The enzyme is not dependent on metal ions. Among divalent metal ions, Ba 2+ Mg 2+, Ca 2+ Both can increase enzyme activity, among which Mg 2+ The promotion effect was the best. SAM was selected as the methyl donor with a constant concentration of 5mM. The enzyme kinetics of luteolin with different substrate concentrations was carried out, and the product luteolin was determined. The results showed that DhOMT8 had a Km of 98.42μM at pH = 9 and a reaction temperature of 50℃.

[0083] 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. A methyltransferase, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A methyltransferase gene, the sequence of which is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

3. Use of the methyltransferase according to claim 1 in the synthesis of flavonoid compounds with anti-tumor activity, wherein the flavonoid compound is luteolin prepared using luteolin as a substrate and S-adenosylmethionine as a methyl donor.

4. Use of the methyltransferase gene of claim 2 in the synthesis of flavonoid compounds with anti-tumor activity, wherein the flavonoid compound is luteolin prepared using luteolin as a substrate and S-adenosylmethionine as a methyl donor.

5. A method for synthesizing flavonoid compounds with anti-tumor activity, the method comprising the following steps: 1) obtaining the methyltransferase according to claim 1; 2) Using the methyltransferase in step 1) to catalyze the synthesis of anti-tumor active flavonoid compounds in an enzyme activity reaction system, wherein the enzyme activity system comprises the methyltransferase in step 1), S-adenosylmethionine and luteolin.

6. The method according to claim 5, characterized in that In step 1), the methyltransferase is obtained by prokaryotic expression or by chemical synthesis.

7. The method according to claim 5, characterized in that: In step 2), the enzyme activity reaction system also contains divalent cations, wherein the divalent cations are Mn 2+ 、Co 2+ 、Zn 2+ , Ba 2+ , Ca 2+ Mg 2+ Any cation.

8. The method according to claim 5, characterized in that The reaction temperature of the enzyme activity reaction system in step 2) is 4-70°C.

9. The method for synthesizing flavonoid compounds with anti-tumor activity as claimed in claim 5, characterized in that: The pH of the enzyme activity reaction system in step 2 is 7.0-10.0.

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