Coumarin o-methyltransferase, its encoding gene and application
By identifying and expressing PcOMT1, PcOMT2, PcOMT7, and PcOMT10 coumarin O-methyltransferases from Psoralea corylifolia, the problem of lack of key enzyme genes in the coumarin biosynthesis pathway was solved, specific O-methylation reaction was achieved, and the yield of target components was increased.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEIFANG MEDICAL UNIV
- Filing Date
- 2026-05-21
- Publication Date
- 2026-06-19
AI Technical Summary
The lack of systematic identification of key enzyme genes for the O-methylation reaction of coumarin in psoralea corylifolia in the current technology has affected the elucidation of the coumarin biosynthesis pathway and the improvement of the yield of the target component.
Four coumarin O-methyltransferases, namely PcOMT1, PcOMT2, PcOMT7 and PcOMT10, and their encoding genes were cloned and identified from Psoralea corylifolia. The catalytic reaction was carried out by recombinant expression vector and host cells to generate methoxycoumarin compounds.
Specific O-methylation of coumarin compounds was achieved, providing key functional genes for coumarin biosynthesis, which can be used to construct engineered bacteria or cell factories for the production of methoxycoumarins, thereby increasing the yield of the target components.
Smart Images
Figure CN122235103A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of genetic engineering, enzyme engineering, and natural product biosynthesis, and specifically relates to a coumarin O-methyltransferase, its encoding gene, and its applications. Background Technology
[0002] Psoralea corylifolia L. is an annual herb belonging to the genus Psoralea in the legume family. Its dried, mature fruit is a commonly used traditional Chinese medicine. Psoralea contains various chemical components, including coumarins, flavonoids, monoterpenoids, lipids, and volatile oils. Coumarin compounds are among its important active ingredients. Furanocoumarins such as psoralen, isopsoralen, bergamot lactone, and xanthotoxin have high medicinal and application value. The structural diversity of coumarins is closely related to their post-modification reactions. O-methylation is an important modification in the biosynthesis of natural products. Typically, S-adenosylmethionine is used as a methyl donor, and under the catalysis of O-methyltransferases, the methyl group is transferred to the phenolic hydroxyl oxygen atom in the substrate molecule. This reaction can alter the polarity, stability, lipophilicity, and biological activity of the substrate and is a crucial step in the formation of methoxycoumarins.
[0003] Currently, research on plant O-methyltransferases mainly focuses on the methylation modification of phenylpropane compounds, lignin monomers, flavonoids, and alkaloids. Previous studies have reported coumarin O-methyltransferases from plants or microorganisms such as *Peucedanum praeruptorum*, *Aconitum carmichaelii*, *Angelica dahurica*, and *Cnidium monnieri*, but the key enzyme gene involved in the O-methylation reaction of coumarin in *Psoralea corylifolia* lacks systematic identification. Since *Psoralea corylifolia* is rich in various coumarin active components, identifying and characterizing its coumarin O-methyltransferase is of great significance for elucidating the coumarin biosynthetic pathway, increasing the yield of target components, and constructing synthetic biology preparation systems. This invention discloses a coumarin O-methyltransferase derived from *Psoralea corylifolia*, its encoding gene, recombinant expression vector, recombinant host cell, and its application in catalyzing the O-methylation reaction of coumarin compounds and preparing methoxycoumarin compounds. Summary of the Invention
[0004] This invention provides a coumarin O-methyltransferase, characterized in that the coumarin O-methyltransferase is selected from one or more of PcOMT1, PcOMT2, PcOMT7 and PcOMT10, and its amino acid sequence is SEQ ID NO:1~4.
[0005] Another embodiment of the present invention provides the application of the above-mentioned coumarin O-methyltransferases (one or more of PcOMT1, PcOMT2, PcOMT7, and PcOMT10) in catalyzing the O-methylation reaction of coumarin compounds. The coumarin compounds are selected from one or more of umbelliferone, bergamot, and xanthotoxin. Specifically, PcOMT1 can catalyze the formation of 7-methoxycoumarin from umbelliferone, the formation of bergamot lactone from bergamot lactone, and the formation of xanthotoxin from xanthotoxin; any of PcOMT2, PcOMT7, and PcOMT10 can catalyze the formation of bergamot lactone from bergamot lactone and the formation of xanthotoxin from xanthotoxin.
[0006] Another embodiment of the present invention provides a gene encoding the above-mentioned coumarin O-methyltransferase, characterized in that the nucleotide sequence of the gene is one or more of SEQ ID NO:5~8.
[0007] Another embodiment of the present invention provides the use of the above-mentioned gene encoding coumarin O-methyltransferase (one or more of SEQ ID NO: 5-8) in the preparation of recombinant expression vectors containing the gene and / or recombinant expression host cells.
[0008] Another embodiment of the present invention provides a recombinant expression vector or recombinant host cell containing the above-mentioned gene encoding coumarin O-methyltransferase (SEQ ID NO: 5-8 or more).
[0009] The recombinant expression vector described in this invention is preferably a pET series expression vector, more preferably a pET-32a expression vector. The host cell is preferably Escherichia coli, more preferably Escherichia coli BL21(DE3).
[0010] This invention also provides a method for preparing methoxycoumarin compounds, characterized by the following steps: adding a coumarin compound, S-adenosylmethionine, and the coumarin O-methyltransferases (one or more of PcOMT1, PcOMT2, PcOMT7, and PcOMT10) described in this invention to a buffer system for reaction, thereby obtaining the corresponding methoxycoumarin compound. The coumarin compound is selected from one or more of umbelliferone, bergamot, and xanthocyanin. The buffer system is a commonly used buffer system in biosynthesis, such as Tris-HCl buffer.
[0011] The amino acid sequence of PcOMT1 in this invention is shown in SEQ ID NO:1; the amino acid sequence of PcOMT2 is shown in SEQ ID NO:2; the amino acid sequence of PcOMT7 is shown in SEQ ID NO:3; the amino acid sequence of PcOMT10 is shown in SEQ ID NO:4; and the coding gene sequences of PcOMT1, PcOMT2, PcOMT7, and PcOMT10 are shown in SEQ ID NO:5–8, respectively. In this invention, "PcOMT" represents psoralen O-methyltransferase.
[0012] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention is the first to clone and successfully identify the O-methyltransferase involved in the O-methylation modification of coumarin from Psoralea corylifolia, providing a key functional gene for elucidating the biosynthetic pathway of coumarin from Psoralea corylifolia. (2) Through substrate spectrum analysis, the present invention found that among the eight candidate substrates tested, PcOMT1, PcOMT2, PcOMT7 and PcOMT10 showed catalytic activity for coumarin substrates, while no obvious methylation products were detected for the tested flavonoid and phenylpropane substrates, indicating that they have a coumarin substrate preference. (3) The enzymes and their encoding genes described in the present invention can be used as synthetic biology elements to construct engineered bacteria or cell factories for the production of methoxycoumarin compounds. Attached Figure Description
[0013] Figure 1 This is a phylogenetic analysis diagram of the PcOMT candidate genes.
[0014] Figure 2 This is an agarose gel electrophoresis image of the PCR amplification products of the target genes PcOMT1, PcOMT2, PcOMT7, and PcOMT10.
[0015] Figure 3 This is an SDS-PAGE electrophoresis image of the recombinant proteins PcOMT1, PcOMT2, PcOMT3, PcOMT7, and PcOMT10.
[0016] Figure 4 This is an LC-MS detection result of the recombinant proteins PcOMT1, PcOMT2, PcOMT7, and PcOMT10 catalyzing the formation of bergamot lactone from bergamotol.
[0017] Figure 5 This is an LC-MS detection result of the recombinant proteins PcOMT1, PcOMT2, PcOMT7, and PcOMT10 catalyzing the formation of xanthotoxin from xanthotoxin.
[0018] Figure 6 This is an LC-MS detection result of the PcOMT1 recombinant protein catalyzing the production of 7-methoxycoumarin from umbelliferone.
[0019] Figure 7 This is a diagram showing the molecular docking results of PcOMT1 with xanthotoxin (A, C) and S-adenosylmethionine (B, D). Detailed Implementation
[0020] The following examples are used to illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make appropriate adjustments to the experimental conditions without departing from the essence of the present invention. Unless otherwise specified in the following examples, conventional conditions or conditions recommended by the manufacturer should be followed.
[0021] Example 1: Screening and phylogenetic analysis of candidate genes for psoralen-coumarin O-methyltransferase
[0022] Based on the transcriptomes reported in the NCBI database (ACS Synth Biol. 2024, 13(11):3600-3608, doi:10.1021 / acssynbio.4c00416) and the reported plant O-methyltransferase sequences (Plant Physiol Biochem. 2023, 204:108142, doi:10.1016 / j.plaphy.2023.108142 and Front Plant Sci.2016;7:722, doi:10.3389 / fpls.2016.00722) as references, potential O-methyltransferase genes in Psoralea corylifolia were screened. Multiple candidate PcOMT genes were obtained by combining sequence homology, gene expression characteristics, and phylogenetic relationships.
[0023] The candidate PcOMT protein sequence was subjected to multiple sequence alignment with the reported plant O-methyltransferase protein sequence (Plant Physiol Biochem. 2023, 204:108142), and a phylogenetic tree was constructed using phylogenetic analysis software. Figure 1 The analysis results showed that the candidate PcOMT protein of Psoralea corylifolia is evolutionarily associated with reported plant O-methyltransferases, and some members cluster with branches related to coumarin or phenolic O-methyltransferases, suggesting that they may be involved in the O-methylation modification of coumarin or phenolic compounds.
[0024] Further analysis of the cloning status of candidate PcOMT genes, recombinant protein expression, and in vitro enzyme activity results confirmed that PcOMT1, PcOMT2, PcOMT7, and PcOMT10 are psoralen-derived O-methyltransferases with coumarin O-methylation catalytic function. The amino acid sequences of PcOMT1, PcOMT2, PcOMT7, and PcOMT10 are shown in SEQ ID NO:1–4, and their encoding gene sequences are shown in SEQ ID NO:5–8, respectively.
[0025] Example 2 Cloning of the PcOMT gene
[0026] Fresh Psoralea corylifolia plant material was ground into powder using liquid nitrogen, and total RNA was extracted using a plant total RNA extraction kit (Jianshi Biosciences). Reverse transcription was performed using the total RNA as a template to obtain Psoralea corylifolia cDNA. Using the Psoralea corylifolia cDNA as a template, open reading frame sequences of PcOMT1, PcOMT2, PcOMT7, and PcOMT10 were amplified using specific primers.
[0027] The primers used for cloning the target gene and linearizing the pET-32a vector are shown in the table below.
[0028]
[0029] The PCR amplification system was as follows: 25 μL of 2× high-fidelity PCR Master Mix, 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of cDNA template, and ddH2O to a final volume of 50 μL. The PCR reaction program was: 95℃ pre-denaturation for 3–5 min; 95℃ denaturation for 15–30 s, 55–58℃ annealing for 15–30 s, 72℃ extension for 45 s–5 min, repeated 30–35 times; and 72℃ extension for 5 min. The PCR products were detected by agarose gel electrophoresis. Figure 2 The target strip is recovered and used for subsequent vector construction.
[0030] Example 3: Construction of recombinant expression vector and expression of recombinant protein
[0031] The recovered target fragments PcOMT1, PcOMT2, PcOMT7, and PcOMT10 were ligated into linearized pET-32a expression vectors to obtain pET32a-PcOMT1, pET32a-PcOMT2, pET32a-PcOMT7, and pET32a-PcOMT10 recombinant expression vectors.
[0032] The recombinant expression vector was transformed into E. coli DH5α, and positive clones were screened and sequenced for verification. After confirming correct sequencing, the recombinant plasmid was extracted and transformed into E. coli BL21(DE3) competent cells to obtain the recombinant expression strain.
[0033] Positive single clones were selected and inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37°C and 200 rpm. The overnight bacterial culture was then inoculated into fresh LB medium at a ratio of 1:200, and cultured until the OD600 was approximately 0.5. IPTG was then added to a final concentration of 0.5 mM, and expression was induced for 16–18 h at 16°C and 180 rpm.
[0034] After induction, bacterial cells were collected by centrifugation, resuspended in binding buffer, and then sonicated before centrifugation to collect the supernatant. The supernatant was purified by Ni-NTA affinity chromatography, and the target protein was eluted with elution buffer containing 250 mM imidazole. The purified protein was concentrated by ultrafiltration and used for SDS-PAGE detection and in vitro enzyme activity assays. SDS-PAGE results showed ( Figure 3 PcOMT1, PcOMT2, PcOMT7 and PcOMT10 can all be solublely expressed in Escherichia coli BL21(DE3).
[0035] Example 4: In vitro enzyme activity detection of PcOMT recombinant protein
[0036] Using S-adenosylmethionine as a methyl donor, and umbelliferone, bergamot, xanthocyanin, p-coumaric acid, daidzein, quercetin, luteolin, and apigenin as candidate substrates, the in vitro catalytic activity of the PcOMT recombinant protein was detected. The expression product of *E. coli* transformed into the empty pET-32a vector served as a negative control.
[0037] The in vitro reaction system is shown in the table below.
[0038]
[0039] After mixing the above components, the reaction was carried out at 37°C for 2 h. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction, and the mixture was extracted twice with ethyl acetate. The combined organic phases were evaporated to dryness, redissolved in methanol, and the reaction products were detected by LC-MS.
[0040] LC-MS analysis was performed using a C18 column at a flow rate of 0.3 mL / min. Detection wavelengths of 254 nm, 280 nm, 320 nm, and 346 nm were selectable, with an injection volume of 4 μL. The chromatographic conditions are shown in the table below.
[0041]
[0042] The reaction products were identified by comparing the reaction system with the negative control for any difference peaks, and by combining the retention time of the standard with the mass spectrometry signal. The results showed that PcOMT1 can catalyze the formation of 7-methoxycoumarin from umbelliferone. Figure 6 PcOMT1, PcOMT2, PcOMT7, and PcOMT10 can catalyze the formation of bergamot lactone from bergamotol (); Figure 4 PcOMT1, PcOMT2, PcOMT7, and PcOMT10 can catalyze the formation of xanthotoxin from xanthotoxin ( ); Figure 5 ).
[0043] Example 5: Substrate profiling of PcOMT recombinant protein
[0044] To further clarify the substrate adaptability and substrate preference of PcOMT recombinant proteins, this embodiment selected coumarin, flavonoids and phenylpropane compounds as candidate substrates, and performed in vitro enzyme activity detection on successfully expressed and purified PcOMT1, PcOMT2, PcOMT7 and PcOMT10.
[0045] Candidate substrates include: coumarin substrates such as umbelliferone, bergamotol, and xanthotoxin; flavonoid substrates such as daidzein, quercetin, luteolin, and apigenin; and phenylpropane substrates such as p-coumaric acid.
[0046] The substrate spectrum results are shown in the table below.
[0047]
[0048] Note: "+" indicates that a product peak consistent with or corresponding to the corresponding methoxylated standard was detected under the detection conditions; "-" indicates that no significantly different product peak was detected under the detection conditions.
[0049] The results showed that PcOMT1 catalyzed the O-methylation of umbelliferone, bergamot, and xanthotoxin to generate 7-methoxycoumarin, bergamot lactone, and xanthotoxin, respectively; PcOMT2, PcOMT7, and PcOMT10 catalyzed the O-methylation of bergamot and xanthotoxin to generate bergamot lactone and xanthotoxin, respectively. Figure 4-6 Meanwhile, when daidzein, p-coumaric acid, quercetin, luteolin, and apigenin were used as substrates, no obvious O-methylation products were detected in PcOMT1, PcOMT2, PcOMT7, and PcOMT10. No obvious O-methylation products were detected under the above detection conditions. These results indicate that PcOMT1, PcOMT2, PcOMT7, and PcOMT10 exhibit catalytic activity towards coumarin substrates within the detected substrate range and demonstrate coumarin substrate preference.
[0050] Example 6 Molecular docking analysis of PcOMT1 with substrates and SAM
[0051] The three-dimensional structure model of the PcOMT1 protein was predicted using AlphaFold2, and molecular docking analysis was performed using AutoDock Vina. A docking grid was set centered on the active pocket region of the predicted PcOMT1 model, and xanthocyanin and S-adenosylmethionine were docked into the PcOMT1 structural model, respectively. The results were visualized using PyMOL.
[0052] The results showed that xanthotoxin could bind to the active pocket of PcOMT1, surrounded by multiple amino acid residues related to substrate binding, including ASN177, MET180, ALA181, SER184, ASP206, GLY208, GLY209, GLY210, THR211, GLY212, ALA213, VAL214, ILE215, PHE263, and LYS265.
[0053] S-adenosylmethionine can form a stable binding with PcOMT1, and residues such as TRP266, PHE163, ASP231, HIS166, ASN173, LYS265, ILE267, and HIS269 may be involved in methyl donor recognition and binding. These results further demonstrate that PcOMT1 has a structural basis for binding coumarin substrates and S-adenosylmethionine.
[0054] The PcOMT1, PcOMT2, PcOMT7, and PcOMT10 provided by this invention can catalyze the O-methylation reaction of coumarin compounds to generate methoxycoumarins such as 7-methoxycoumarin, bergamot lactone, and xanthotoxin. This invention also provides the encoding genes, expression vectors, and recombinant host cells for the above enzymes, enabling the acquisition of recombinant proteins through genetic engineering. The enzymes can be used for in vitro enzyme-catalyzed reactions or introduced as synthetic biology elements into engineered microorganisms or plant cells to construct biosynthetic systems for methoxycoumarins. Therefore, this invention has promising industrial application prospects.
Claims
1. A coumarin O-methyltransferase, characterized in that... The coumarin O-methyltransferase is selected from one or more of PcOMT1, PcOMT2, PcOMT7 and PcOMT10, and its amino acid sequences are SEQ ID NO:1~4 in sequence.
2. The application of the coumarin O-methyltransferase according to claim 1 in catalyzing the O-methylation reaction of coumarin compounds, characterized in that... The coumarin compounds are selected from one or more of umbelliferone, bergamotol, and xanthotoxin.
3. A gene encoding the coumarin O-methyltransferase of claim 1, characterized in that... The nucleotide sequence of the gene is one or more of SEQ ID NO:5~8.
4. The use of the gene of claim 3 in the preparation of a recombinant expression vector containing the gene and / or a recombinant expression host cell.
5. A recombinant expression vector or recombinant host cell containing the gene of claim 3.
6. A method for preparing methoxycoumarin compounds, characterized in that... The method includes the following steps: adding coumarin compounds, S-adenosylmethionine, and the coumarin O-methyltransferase described in claim 1 to a buffer system for reaction to obtain the corresponding methoxycoumarin compounds; wherein the coumarin compounds are selected from one or more of umbelliferone, bergamotol, and xanthotoxin.