Magnolia denudata caffeic acid methyltransferase YbOMT1 and application thereof in biosynthesis of ferulic acid
By providing the methyltransferase gene that catalyzes ferulic acid synthesis and its applications, the problem of difficult to achieve large-scale production of ferulic acid biosynthesis in the prior art is solved, and efficient and clean ferulic acid biosynthesis is achieved, with the advantages of simple steps, small pollution and single product.
Patent Information
- Application Number
- CN202510315703.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-07-01
AI Technical Summary
It is difficult to achieve large-scale production of ferulic acid biosynthesis in the prior art, and the existing preparation methods have problems such as complex steps, large pollution, and impure products.
It provides a methyltransferase gene that catalyzes the production of ferulic acid and its application. It obtains a methyltransferase through prokaryotic expression, and uses the enzyme to catalyze the synthesis of ferulic acid in an enzyme-active reaction system.
The efficient and clean production of ferulic acid by biosynthesis is achieved, with the advantages of simple steps, small contamination and single product, and the methyltransferase has a wide suitable reaction temperature range and a higher reaction rate.
Smart Images
Figure CN120230732A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to Magnolia sieboldii methyltransferase and its applications. Background Technology
[0002] Ferulic acid, chemically known as 3-methoxy-4-hydroxycinnamic acid, is found in high concentrations in traditional Chinese medicinal herbs such as asafoetida, angelica sinensis, chuanxiong rhizome, cimicifuga rhizome, and jujube seed, and is one of the active ingredients in these herbs. Ferulic acid possesses a wide range of pharmacological activities and is clinically used as an adjunct treatment for cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, cerebrovascular disease, glomerular diseases, pulmonary hypertension, diabetic vascular complications, and vasculitis. It can also be used to treat migraines and vascular headaches.
[0003] The main methods for preparing ferulic acid include the following: Direct extraction from plants: This method can obtain ferulic acid from plants through three pathways. The first is from the combination of ferulic acid with some small molecules; the second is from plant cell walls; and the third is through tissue culture. Ferulic acid in plants usually cross-links with polysaccharides and lignin through ester bonds, or undergoes self-esterification or etherification to form diferulic acid. Generally, ester bonds are broken using alkaline and enzymatic methods to release ferulic acid, followed by extraction with a suitable solvent. Chemical synthesis: The chemical synthesis of ferulic acid uses vanillin as the basic raw material, and the main organic reactions used are the Wittig-Horner reaction and the Kneoevenagel reaction. These reactions can synthesize ferulic acid in the laboratory through specific chemical steps. Biosynthesis: This method uses several microorganisms to convert ferulic acid precursors into ferulic acid. For example, eugenol cinnamate extracted from clove oil can be converted into ferulic acid. Biosynthesis is considered a clean and efficient synthetic method, but large-scale production has not yet been achieved. Therefore, it is of great significance to identify and develop enzymes with ferulic acid synthesis function and to develop corresponding biosynthetic methods. Summary of the Invention
[0004] This invention addresses the aforementioned technical problems by providing a methyltransferase gene for catalyzing the production of ferulic acid, as well as a method for biosynthesizing ferulic acid, thereby overcoming the shortcomings of existing methods for preparing ferulic acid.
[0005] Specifically, the present invention provides the following technical solution: On the one hand, the present invention provides a methyltransferase whose amino acid sequence includes the sequence shown in SEQ ID NO.2.
[0006] On the other hand, 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. Alternatively, based on the principle of complementary pairing, the methyltransferase gene provided by the present invention can be a sequence encoding a fully complementary pair of nucleotide sequences containing the amino acid sequence shown in SEQ ID NO. 2.
[0007] In a preferred embodiment, the nucleotide sequence of the methyltransferase gene described above 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.
[0008] As is known to those skilled in the art, gene sequences can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequence of the methyltransferase gene described above can also contain introns, promoters, and various regulatory elements.
[0009] On the other hand, the present invention provides an application of a methyltransferase gene in the synthesis of ferulic acid, 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 completely complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.
[0010] 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.
[0011] 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.
[0012] On the other hand, the present invention provides the application of the aforementioned methyltransferase in the synthesis of ferulic acid.
[0013] On the other hand, the present invention provides a method for synthesizing ferulic acid, the method comprising the following steps: 1) Obtain the aforementioned methyltransferase; 2) The methyltransferase from step 1) is used to catalyze the synthesis of ferulic acid in an enzyme-activated reaction system.
[0014] In a preferred embodiment, the above-mentioned methyltransferase was obtained by prokaryotic expression.
[0015] In a preferred embodiment, the above-mentioned methyltransferase is obtained by chemical synthesis.
[0016] In a preferred embodiment, the enzyme activation reaction system contains the above-mentioned methyltransferase, SAM, substrate for the synthesis of ferulic acid, and buffer solution.
[0017] On the other hand, the present invention provides a method for synthesizing ferulic acid, characterized in that the method comprises the following steps: 1) Obtain a methyltransferase containing the amino acid sequence shown in SEQ ID NO.2; 2) Ferulic acid was synthesized by catalyzing the above-mentioned methyltransferase in an enzyme-activated reaction system.
[0018] In a preferred embodiment, the above-mentioned methyltransferase was obtained by prokaryotic expression.
[0019] In a preferred embodiment, the above-mentioned methyltransferase is obtained by chemical synthesis.
[0020] In a preferred embodiment, the enzyme activation reaction system contains the above-mentioned methyltransferase, SAM, substrate for the synthesis of ferulic acid, and buffer solution.
[0021] In a preferred embodiment, the enzyme activation reaction system also contains divalent cations.
[0022] In a preferred embodiment, the reaction temperature of the enzyme activation reaction system is 4~70℃.
[0023] In a preferred embodiment, the pH of the enzyme activation reaction system is 6.0-10.0.
[0024] In a preferred embodiment, the substrate used to synthesize ferulic acid is caffeic acid.
[0025] Compared with the prior art, the present invention has the following advantages: 1) Compared with the prior art, the present invention uses enzyme-catalyzed biosynthesis to obtain ferulic acid, which has the advantages of simpler steps, less pollution and more singular products than chemical synthesis.
[0026] 2) The methyltransferase in this invention has a wide suitable reaction temperature range (4~70℃).
[0027] 3) The methyltransferase of the present invention achieves a high reaction rate in a short time.
[0028] 4) The methyltransferase of the present invention can almost completely catalyze caffeic acid to ferulic acid. Attached Figure Description
[0029] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.
[0030] Figure 1 This is the cloned gene fragment. M is a 2000bp DNA marker, and lane 1 is YbOMT1.
[0031] Figure 2 This is an SDS-PAGE gel electrophoresis image of the pET-28a(+)-YbOMT1 fusion protein. M is the protein marker (15-100kDa), and lane 1 is the crude YbOMT1 enzyme.
[0032] Figure 3 The results of the reaction between YbOMT1 and the substrate are shown in Figure A: Liquid phase diagram of the reaction between YbOMT1 and caffeic acid; and Figure B: Mass spectrum of the catalytic product.
[0033] Figure 4 The results show the enzyme kinetic parameters for the conversion of caffeic acid to ferulic acid catalyzed by YbOMT1. Detailed Implementation
[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] 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 pertains.
[0036] Example 1 Cloning of the target methyltransferase gene 1. Total RNA extraction kit was used to extract total RNA from *Yulania biondii*. The extracted total RNA was then processed according to the instructions of the Nanjing Novizan HiScript® II Q RT SuperMix reverse transcription kit to obtain the desired cDNA. This cDNA was then used as a template for polymerase chain reaction (PCR) amplification of the full-length YbOMT1 gene fragment. Specific primers were designed based on the transcriptome data of this experiment, as shown in Table 1. Table 1 Primer Sequences Using the cDNA extracted from Magnolia biondii in the above experiments as PCR templates, the target fragment was amplified using primers YbOMT1-F and YbOMT1-R. The polymerase used for PCR was Nanjing Novizan 2×Phanta Max Master Mix. A 25 μL PCR reaction system was prepared according to Table 2.
[0037] Table 2. PCR reaction system for amplifying the target gene (25 μL) Place the prepared PCR reaction system in a PCR instrument and amplify the target gene according to the PCR reaction procedure in Table 3.
[0038] Table 3 PCR reaction procedure for gene amplification After the reaction, the product size was determined by 1% agarose gel electrophoresis. The electrophoresis pattern is shown below. Figure 1 .
[0039] 2. The amplified product was ligated to the T vector (pET-28a, Nanjing Novizan Biotechnology Co., Ltd.) and transformed into *E. coli* DH5α. Single colonies were selected and cultured in LB medium for bacterial culture PCR verification. The bacterial culture PCR reaction system was: 12.5 μL Green Taq Mix (P131, Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of forward and reverse primers, 2 μL template (bacterial culture), and dd H2O was added to a final volume of 25 μL. Samples with correct bacterial culture PCR results were sent for sequencing.
[0040] The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1. This gene sequence contains 1083 nucleotides and encodes 360 amino acids (shown in SEQ ID NO.2).
[0041] >SEQ ID NO.1 >SEQ ID NO.2 MASPLSKPKTSDEDEQCLFAMQLASASVLPMALKAALELEVLEIIAKAGPGAHLSPSEIAAHLPTENPEAPVMLDRILRLLASYCILACSVVTLDDGQVERRYGLAPVCNFLVRNREGVSIAPLVLMNQDKVLMESWYHLKEAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNRGMSDH STLTMKRILDTYKGFEGLKSVVDVGGGVGATLNMIISKYPQIKGINFDLPHVIADAPDYSGVEHLGGDMFVSVPSGEAIFMKWILHDWSDDHCLKLLKNCYKALPDSGKVIIVESILPVVPETNLAANCVIQQDLIMLAHNPGGKERVENEFEALANGAGFTSFKVLCCAYNSWVMEFHK Example 2: Expression and detection of the target protein Expand the culture of the target bacterial culture that has been correctly sequenced and extract the recombinant plasmid pET-28a-YbOMT1. Transform the recombinant plasmid into *E. coli* BL21(DE3) competent cells, plate the cells, and select clones for incubation in LB medium at 37°C and 200 rpm for 16 h to obtain the stock solution. Inoculate the stock solution at a 1:100 ratio into LB medium containing 50 μg / mL KaN and incubate at 37°C until OD500. 600 When the concentration of the medium is 0.6~0.8 (using sterile LB medium as a control), add IPTG to a final concentration of 0.5 mM, and then incubate at 16℃ and 200 rpm for 18-20 h.
[0042] The induced bacterial culture was collected by centrifugation, and the cells were resuspended in pre-chilled Tris-HCl (50 mM) buffer (pH 9.0). Centrifugation was repeated, and the cells were collected again to remove residual culture medium. Finally, 15 mL of the above Tris-HCl buffer was added to each gram of wet cells. The resulting cell suspension was frozen overnight at -20°C, and then disrupted at low temperature using an ultrasonic cell disruptor at 300 W for 3 seconds followed by a 3-second interval. Disruption was stopped when the cell suspension became clear. Finally, the disrupted solution was centrifuged at 14000 rpm for 4 minutes at 4°C. The supernatant was the crude enzyme solution, which was stored at -20°C.
[0043] Based on the His-Tag tag on the fusion protein, the target protein was purified using a nickel affinity chromatography column. The target protein was eluted with imidazole solutions of different concentrations, and finally, the molecular weight and purity of the target protein were determined by 10% SDS-PAGE electrophoresis. Figure 2 As shown, the target protein appeared in the range of 40-50 kDa (lanes 1, 2, and 3 represent the empty vector, crude enzyme, and purified protein, respectively), indicating that the protein was successfully expressed in the supernatant and that the purified protein could be used for subsequent experiments.
[0044] Example 3 In vitro enzyme activity detection 1. Enzyme activation reaction system: 10 μg purified enzyme, 0.5 mM caffeic acid, 5 mM SAM methyl donor, and 14 mM β-mercaptoethanol were added and diluted to 200 μL with 50 mM Tris-HCl buffer (pH 8.0). After incubating the reaction solution in a water bath at 45°C for 45 min, double the volume of pre-cooled methanol was added, the mixture was vortexed, and centrifuged at 14000 rpm for 4 min at 4°C. The supernatant was filtered and analyzed by high-performance liquid chromatography (HPLC).
[0045] 2. HPLC conditions 3. LC-MS conditions: The detection mode was negative ion mode, the capillary voltage was 3500 V, the sheath gas temperature was 280℃, and the flow rate was 11 L·min. -1 The drying gas temperature was 320 °C, and the flow rate was 10 L·min. -1 Nozzle voltage 155 V, flow rate 0.250 mL·min -1 .
[0046] 4. Test Results: See results Figure 3 When YbOMT1 is used as a substrate, a single compound peak can be observed in the reaction. By comparing the retention time and mass-to-charge ratio (m / z) of the standard, the product of this peak is found to be ferulic acid.
[0047] Example 4 Detection of Enzyme Kinetic Parameters Using caffeic acid as the substrate and SAM as the methyl donor, the effects of pH, temperature, metal ions, and reaction time on catalytic activity were investigated.
[0048] 1. Temperature: Using caffeic acid as the methyl acceptor and SAM as the methyl donor, the changes in enzyme activity at different reaction temperatures (4℃, 25℃, 30℃, 40℃, 45℃, 50℃, 60℃, and 70℃) were investigated. The reaction system contained 10 μg of purified protein, 0.5 mM of substrate, 5 mM SAM, and 14 mM β-mercaptoethanol, and was brought to a final volume of 200 μL with 50 mM Tris-HCl buffer (pH 8.0). The reaction solution was reacted at each temperature for 45 min. Immediately after the reaction, twice the volume of pre-cooled methanol was added to terminate the reaction. After centrifugation, the supernatant was filtered and analyzed by HPLC. Each reaction was performed in triplicate. The substrate conversion rate was estimated based on the peak area ratio in the chromatogram.
[0049] 2. pH: The effects of different 50 mM pH buffers on enzyme activity were investigated: 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. The reaction system consisted of 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg protein, and 0.5 mM caffeic acid in 200 μL of the reaction system, with the corresponding buffer added to the specified volume. The reaction solution was incubated at 45℃ for 45 min. Immediately after the reaction, twice the volume of pre-cooled methanol was added. After centrifugation, the supernatant was filtered and analyzed by HPLC. To ensure data reliability, three replicates were set up for each reaction. The final conversion rate was calculated from the peak area of the chromatogram.
[0050] 3. Metal ions: The acceptor molecule in the reaction solution is caffeic acid, and the donor is 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 was investigated. A 200 μL reaction system contained 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg protein, 0.5 mM substrate, and 5 mM divalent metal ions, and was supplemented to the specified volume with 50 mM Tris-HCl buffer (pH 8.0). The reaction solution was incubated at 45 °C for 10 min. Immediately after incubation, an equal volume of pre-cooled methanol was added, and the mixture was centrifuged at 14000 rpm at 4 °C for 4 min. Three replicates were established for each reaction group. The supernatant was collected for HPLC analysis, and the final conversion rate was calculated from the peak area of the chromatogram.
[0051] 4. Reaction Time: A 200 μL Tris-HCl (50 mM, pH=8.0) reaction system containing 14 mM β-mercaptoethanol, 5 mM SAM, 10 μg purified protein, and 0.5 mM caffeic acid substrate was reacted at 30℃ for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. After the reaction, twice the volume of pre-cooled methanol was quickly added to each reaction solution to terminate the reaction, and the mixture was centrifuged at 14000 rpm and 4℃ for 4 min. The supernatant was filtered and analyzed by HPLC, and the substrate conversion rate was calculated. To ensure the accuracy of the data, three parallel experiments were conducted for each group, and the substrate conversion rate was calculated based on the ratio of peak areas in the HPLC chromatogram.
[0052] 5. Michaelis constant Km: The reaction system for YbOMT1 was 200 μL Na2HPO4-NaH2PO4 (50 mM, pH=8.0) containing 14 mM β-mercaptoethanol, 5 mM SAM, and 10 μg purified protein. Caffeic acid was selected as the substrate, with concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM. The reaction was carried out at 60℃ for 1 min. Immediately after the reaction, two volumes of pre-cooled methanol were added to each reaction mixture. After centrifugation, the supernatant was collected and analyzed by HPLC at the maximum absorption wavelength of the substrate. The conversion rate of the substrate was calculated based on the peak area. To ensure data accuracy, three parallel experiments were conducted for each group. The Michaelis constant was obtained by fitting the Michaelis equation.
[0053] 6. Test Results: Enzyme kinetic studies have shown that ( Figure 4 YbOMT1 exhibits the highest enzyme activity at 45℃, and maintains a certain conversion rate within the temperature range of 4–70℃. The optimal pH for the reaction solution is 8.0, and it is suitable for reactions within the pH range of 6.0–10.0. Furthermore, its catalytic activity is higher in Na2HPO3-NaHPO3 or Tric-CH1 buffer than in other buffers. This enzyme is metal ion dependent; among divalent metal ions, Mg... 2+ , Ca 2+ Both can increase enzyme activity, among which Mg 2+ The promoting effect was optimal. Furthermore, the maximum reaction rate was reached after 30 minutes. Using SAM as the methyl donor at a constant concentration of 5 mM, enzyme kinetics were investigated with different substrate concentrations of caffeic acid. The product caffeic acid was measured, and calculations were performed using the Michaelis-Menten equation. The results showed that YbOMT1 had a Km of 11.2 μM at pH 8 and a reaction temperature of 45℃.
[0054] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded 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 an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is completely complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.
2.
3. Use of the methyltransferase according to claim 1 in the synthesis of ferulic acid.
4. Use of the methyltransferase gene according to claim 2 in synthesizing ferulic acid.
5. A method for synthesizing ferulic acid, the method comprising the steps of: 1) obtaining the methyltransferase according to claim 1; 2) Using the methyltransferase in step 1) to catalyze the synthesis of ferulic acid in an enzyme activity reaction system.
6. A method for synthesizing ferulic acid as claimed in claim 5, characterized in that, In step 1), the methyltransferase is obtained by prokaryotic expression or by chemical synthesis.
7. A method for synthesizing ferulic acid as claimed in claim 5, characterized in that, The enzyme activity reaction system in step 2) contains the methyltransferase described in step 1), S-adenosylmethionine and a substrate for synthesizing ferulic acid.
8. A method for synthesizing ferulic acid as claimed in claim 5, characterized in that, In step 2), the enzyme activity reaction system also contains divalent cations.
9. A method for synthesizing ferulic acid as claimed in claim 5, characterized in that, The reaction temperature of the enzyme activity reaction system in step 2) is 5-70°C.
10. A method for synthesizing ferulic acid as claimed in claim 5, characterized in that: The pH of the enzyme activity reaction system in step 2) is 7.0-10.0.