Application of FsE4MT01 protein and / or FsE4MT02 protein in regulating content of forsythiaside in forsythia suspensa

Abstract

The application belongs to the technical field of biology and particularly relates to application of FsE4MT01 protein and / or FsE4MT02 protein in regulating content of forsythiaside in Forsythia suspensa. The application finds that Class I type oxygen methyltransferase (FsE4MT01 and FsE4MT02) with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 can catalyze 4-position oxygen methylation reaction of (+)-pinoresinol to generate forsythiaside. Through establishment of a Forsythia suspensa leaf transient expression system, the application finds that overexpression of FsE4MT01 and FsE4MT02 can significantly improve the yield of forsythiaside. On the contrary, inhibition of expression of the genes through a virus-induced gene silencing (VIGS) technology can reduce accumulation of the corresponding product.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of FsE4MT01 protein and / or FsE4MT02 protein in regulating the content of forsythosides in Forsythia suspensa. Background Technology

[0002] Phillygenin is a natural lignan compound extracted from the traditional Chinese medicine Forsythia suspensa. Belonging to the phenylpropanoid class of compounds, it possesses the core structural basis for the various pharmacological activities of Forsythia suspensa. Studies have shown that the phenolic hydroxyl and methoxy groups in its structure are closely related to its antioxidant activity. Phillygenin exhibits various pharmacological activities, such as participating in anti-inflammatory and immunomodulatory processes by inhibiting the TLR4 / MyD88 / NF-κB and PI3K / AKT / GSK3β signaling pathways, significantly reducing inflammatory responses. In a diabetic nephropathy model, it can reduce the expression of inflammatory factors such as IL-6 and TNF-α, improving renal function. Furthermore, it can combat liver fibrosis by regulating macrophage exosomes, inhibiting M1 macrophage polarization, and alleviating hepatic stellate cell activation. Other studies have shown that Phillygenin has lipid-lowering effects and inhibits low-density lipoprotein oxidation, demonstrating its potential in lipid metabolism-related diseases. Recent research has also found that it can affect glucose metabolism parameters by inhibiting Akt activity, providing new insights for the treatment of diabetes and its complications. In addition, as a natural antioxidant, Phillygenin has a significant scavenging ability against DPPH and ABTS free radicals, and its activity is superior to some synthetic antioxidants. This property makes it a potential candidate for applications in food preservation and anti-aging cosmetics.

[0003] Phillygenin can be prepared in several ways: (1) Plant extraction: The traditional method is to extract it from the fruit of Forsythia suspensa, but the yield is low (the content in Forsythia suspensa is about 0.1%-0.5%), and it is limited by the season and place of origin. (2) Chemical synthesis: Due to the complex structure of Phillygenin and the presence of multiple chiral centers, the total synthesis route is long and is not suitable for large-scale production at present.

[0004] Phillygenin, a key active ingredient in Forsythia suspensa, shows broad application prospects in anti-inflammatory, metabolic regulation, and antioxidant effects.

[0005] Phillygenin belongs to the class of bicyclic lignans, characterized by a bicyclic system consisting of two cis-configured tetrahydrofuran rings linked by an 8-O-4′ bond (Wang et al., 2018). Based on the chemical composition of Forsythia suspensa (Li et al., 2024; Wu et al., 2020) and the well-established lignan biosynthesis pathways in other plants (Chen et al., 2024; Fan et al., 2024), we hypothesize that the biosynthesis of phillygenin mainly involves three stages (…). Figure 1 This pathway begins with the shikimic acid pathway (Shende et al., 2024), which provides the phenylpropanoid precursor L-phenylalanine (Dong and Lin, 2021). Subsequently, L-phenylalanine is converted to coniferyl alcohol via the phenylpropane metabolic pathway (Dong and Lin, 2021; Fan et al., 2024; Pan et al., 2014). Directional proteins (DIRs) mediate the stereospecific coupling of coniferyl alcohol to form (+)-epiderminesin (Halls and Lewis, 2002; Pickel et al., 2010; Pickel and Schaller, 2013), which is ultimately converted to forsythosides via enzymatic modification through a lignan-specific synthesis pathway. Figure 1 ).

[0006] Although these upstream steps are relatively well understood, the unique terminal enzymatic step of forsythoside A remains incompletely elucidated, primarily involving a key reaction: oxygen methylation at position 4 of (+)-epiporesinin to generate forsythoside A. This reaction is presumably catalyzed by O-methyltransferases (OMTs). OMTs mainly use S-adenosylmethionine (SAM) as a methyl donor to catalyze the methylation of the oxygen atom on the hydroxyl group. However, the specific OMT responsible for this reaction in Forsythia suspensa has not yet been identified, hindering the reconstruction of this metabolic pathway. The large number of members in the OMT gene family further complicates identification. Another key obstacle is the lack of an efficient genetic transformation system for Forsythia suspensa, which further restricts gene function verification and related research.

[0007] The references are as follows:

[0008] Chen, R., Yu, J., Yu, L., Xiao, L., Xiao, Y., Chen, J., Gao, S.,Chen, X., Li, Q., Zhang, H., Chen, W., and Zhang, L. (2024). The ERFtranscription factor LTF1 activates DIR1 to control stereoselective synthesisof antiviral lignans and stress defense in Isatis indigotica roots. ActaPharm. Sin. B 14: 405-420.

[0009] Dong, N.-Q., and Lin, H.-X. (2021). Contribution of phenylpropanoidmetabolism to plant development and plant–environment interactions. J.Integr. Plant Biol. 63: 180-209.

[0010] Fan, H., Shen, X., Ding, Y., Li, Y., Liu, S., Yang, Y., Ding, Y., andGuan, C. (2024). DkWRKY transcription factors enhance persimmon resistance toColletotrichum horii by promoting lignin accumulation through DkCAD1 promotorinteraction. Stress Biology 4: 17.

[0011] Halls, S.C., and Lewis, N.G. (2002). Secondary and quaternarystructures of the (+)-pinoresinol-forming dirigent protein. Biochemistry 41:9455-9461.

[0012] Li, J.J., Chen, Z.H., Liu, C.J., Kang, Y.S., Tu, X.P., Liang, H.,Shi, W., and Zhang, F.X. (2024). The phytochemistry, pharmacology,pharmacokinetics, quality control, and toxicity of Forsythiae Fructus: Anupdated systematic review. Phytochemistry 222: 114096.

[0013] Li, L.-F., Cushman, S.A., He, Y.-X., and Li, Y. (2020). Genomesequencing and population genomics modeling provide insights into the localadaptation of weeping forsythia. Horticulture Research 7.

[0014] Li, Y., Wang, F., Pei, N., Li, Q., Liu, H., Yuan, W., and Zhang, H.(2023). The updated weeping Forsythia genome reveals the genomic basis forthe evolution and the forsythin and forsythoside A biosynthesis. Hortic.Plant Journal 9: 1149-1161.

[0015] Pan, H., Zhou, R., Louie, G.V., Mühlemann, J.K., Bomati, E.K.,Bowman, M.E., Dudareva, N., Dixon, R.A., Noel, J.P., and Wang, X. (2014).Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcoholdehydrogenase, key enzymes of monolignol biosynthesis. Plant Cell 26: 3709-3727.

[0016] Pickel, B., Constantin, M.-A., Pfannstiel, J., Conrad, J., Beifuss,U., and Schaller, A. (2010). An enantiocomplementary dirigent protein for theenantioselective laccase-catalyzed oxidative coupling of phenols. Angew.Chem. Int. Ed. 49: 202-204.

[0017] Pickel, B., and Schaller, A. (2013). Dirigent proteins: molecularcharacteristics and potential biotechnological applications. Appl. Microbiol.Biotechnol. 97: 8427-8438.

[0018] Shende, V.V., Bauman, K.D., and Moore, B.S. (2024). The shikimatepathway: gateway to metabolic diversity. Nat. Prod. Rep. 41: 604-648.

[0019] Wang, Z., Xia, Q., Liu, X., Liu, W., Huang, W., Mei, X., Luo, J., Shan, M., Lin, R., Zou, D., and Ma, Z. (2018). Phytochemistry, pharmacology, quality control and future research of Forsythia suspensa (Thunb.) Vahl: Areview. J. Ethnopharmacol. 210: 318-339.

[0020] Wu, L., Chen, YL, Ma, YF, Yang, ZF, Yang, N., Deng, WZ, Chen,YB, Sun, YY, Li, YM, and Lin, L. (2020). Clinical practice guideline ontreating influenza in adult patients with Chinese patent medicines. Pharmacol. Res. 160: 105101. Summary of the Invention

[0021] This invention identifies and functionally characterizes two Class I oxygen methyltransferases, FsE4MT01 and FsE4MT02, which are involved in the biosynthesis of forsythosides in Forsythia suspensa.

[0022] The present invention specifically adopts the following technical solution:

[0023] This invention employs a combination of metabolomics and transcriptomics to investigate the tissue-specific biosynthesis of forsythosides in Forsythia suspensa. We identified two Class I oxygen methyltransferases (FsE4MT01 and FsE4MT02) that catalyze the methylation of (+)-epiporesinin to forsythosides. Through in vivo and in vitro experiments, we confirmed that FsE4MT01 and FsE4MT02 specifically catalyze the 4-O-methylation of (+)-epiporesinol to generate forsythosides. By establishing a transient expression system in Forsythia suspensa leaves, we found that overexpression of FsE4MT01 and FsE4MT02 significantly increased the yield of forsythosides. Conversely, inhibiting the expression of these genes using virus-induced gene silencing (VIGS) technology reduced the accumulation of the corresponding products.

[0024] Based on the above, in a first aspect, the present invention provides a Forsythia Class I oxygen methyltransferase, namely FsE4MTs protein, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0025] Secondly, the present invention provides a nucleic acid encoding the Forsythia Class I type O-methyltransferase described above. In a further embodiment, the sequence of the nucleic acid is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0026] Thirdly, the present invention provides biological materials containing the nucleic acid, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector or host cell.

[0027] Fourthly, the present invention provides the application of Forsythia Class I type O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, nucleic acid encoding Forsythia Class I type O-methyltransferase, and biological material containing said nucleic acid in regulating the content of forsythosides in Forsythia.

[0028] Fifthly, the present invention provides the application of Forsythia Class I OMT enzyme with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, a nucleic acid encoding Forsythia Class I OMT enzyme, and a biological material containing said nucleic acid in increasing the content of forsythosides in Forsythia. The biological material is Forsythia.

[0029] Based on the above, Class I oxygen methyltransferase can be used to increase the content of forsythosides in Forsythia suspensa. Therefore, this invention provides a method for increasing the content of forsythosides in Forsythia suspensa, comprising: increasing the expression level of Class I oxygen methyltransferase in Forsythia suspensa. Specifically, overexpressing Class I oxygen methyltransferase in Forsythia suspensa.

[0030] Furthermore, exogenous DNA encoding Class I oxymethyltransferase was transferred into Forsythia suspensa.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention discovers that in Forsythia suspensa, FsE4MT01 and FsE4MT02 can catalyze the 4-oxymethylation of (+)-epifolinin to generate forsythoside. Transient overexpression of FsE4MT01 and FsE4MT02 in Forsythia suspensa increased forsythoside accumulation by 2.5-fold and 2.2-fold, respectively, compared to the control, while silencing FsE4MT01 and FsE4MT02 in Forsythia suspensa significantly reduced forsythoside accumulation. Attached Figure Description

[0033] Figure 1 The proposed biosynthetic pathway of forsythoside in Forsythia suspensa. The proposed biosynthesis of forsythoside involves three main stages: (1) the shikimate pathway; (2) the phenylpropanoid pathway; and (3) the lingan-specific pathway. The key steps in the biosynthetic pathway of forsythoside are highlighted in orange, while the dashed arrows indicate proposed reactions that are yet to be verified. Abbreviations: E4P (erythrose-4-phosphate), PEP (phosphoenolpyruvate), FsDIR (dirigent protein).

[0034] Figure 2 Overview of metabolomics and transcriptomics data. (A) Phenotypic characteristics of Forsythia fruit at two harvest stages: green immature fruit stage (GFS) and red mature fruit stage (RFS). (B) Classification of metabolites detected in Forsythia. Pie charts are colored by metabolite category, with the area of ​​each sector representing its relative proportion in the overall metabolomics profile. Sample abbreviations: GFS-Leaf (young leaf, GFS); GFS-Branch (young branch, GFS); GFS-Shell (fruit shell, GFS); GFS-Seed (seed, GFS); GFS-Base (fruit stalk, GFS); RFS-Leaf (young leaf, RFS); RFS-Branch (young branch, RFS); RFS-Shell (fruit shell, RFS); RFS-Seed (seed, RFS); RFS-Base (fruit stalk, RFS). (C) Principal component analysis (PCA) of the metabolomics profile. (D) Principal component analysis (PCA) of the transcriptomics profile. (E) Phylogenetic tree and expression heatmap of 76 FsOMT genes. The circular plot consists of three layers: phylogenetic tree (innermost layer), gene expression level heatmap (middle layer), and gene identifiers (outermost layer). Different gene clusters (I-VII) are marked with different colored backgrounds. Differentially expressed OMT gene names are marked in black, and non-differentially expressed ones are marked in blue.

[0035] Figure 3 Cluster analysis of Forsythia suspensa metabolite abundance profiles. Hierarchical clustering was performed based on abundance data of 1,013 metabolites in 30 Forsythia suspensa samples (five tissues at two developmental stages, with three biological replicates each). Data were Z-score normalized for each metabolite. The heatmap color gradient (0 to 1) corresponds to the Pearson correlation coefficient (PCC), reflecting the similarity of metabolite accumulation patterns.

[0036] Figure 4 Cluster analysis of Forsythia gene expression profiles. Hierarchical cluster analysis based on 37,475 expressed genes from 30 Forsythia samples. Rows represent samples, and columns represent genes. Color gradient (0 to 1) represents the Pearson correlation coefficient (PCC) calculated based on the log-transformed expression level (TPM).

[0037] Figure 5 Candidate FsOMT genes were screened using multi-omics analysis. (A) A heatmap showing the relative abundance of metabolites in the forsythoside biosynthesis pathway. Data are presented as Z-values ​​(scale: -2 to 2). (B) Gene expression patterns in the phenylpropane pathway. Expression levels are shown as Z-values ​​normalized per gene (scale: -2 to 4). (C) Cluster dendrogram of co-expression modules identified by WGCNA. 23 principal modules are distinguished by different colors. (D) Heatmap of the correlation between gene co-expression modules (columns, colored by module) and pathway metabolites (rows). Red indicates a positive correlation, and blue indicates a negative correlation. (EH) A scatter plot of superimposed density curves showing the correlation between forsythoside content (x-axis) and candidate FsOMT gene expression levels (y-axis). Yellow shading represents the density distribution of forsythoside content, and blue shading represents the density distribution of gene expression. R and P represent the Pearson correlation coefficient and its significance level, respectively.

[0038] Figure 6 Heatmap of lignan accumulation patterns in various tissues of Forsythia suspensa. Data are presented as Z-scores (scale: -2 to 3) based on 30 samples (five tissues at two developmental stages, three biological replicates each).

[0039] Figure 7 Correlation analysis of (+)-epidermopinen accumulation and FsDIR gene expression. A scatter plot of superimposed density curves shows the relationship between the relative content of (+)-epidermopinen (x-axis, yellow density curve) and the relative expression levels of FsDIR1 (A) and FsDIR2 (B) genes (y-axis, blue density curve). R and P represent the Pearson correlation coefficient and its statistical significance, respectively.

[0040] Figure 8 Co-expression analysis of FsDIR1 and FsE4MT. The scatter plot of superimposed density curves shows the pairwise correlations between the relative expression level of the FsDIR1 gene (x-axis, yellow density curve) and the relative expression levels of FsE4MT01 (A), FsE4MT02 (B), FsE4MT03 (C), and FsE4MT04 (D) genes (y-axis, blue density curve). R and P represent the Pearson correlation coefficient and its statistical significance, respectively.

[0041] Figure 9In vivo functional verification of candidate genes in *Nicotiana benthamiana*. (A) Workflow diagram for screening candidate gene function in *Nicotiana benthamiana* leaves. (B) Conversion of exogenously added (+)-epistine to forsythoside in *Nicotiana benthamiana* leaves expressing four FsE4MT candidate genes. Data are expressed as mean ± standard deviation (n=3). One-way ANOVA was used to determine statistical significance relative to the empty vector control (*P < 0.05, **P < 0.01). (C) Subcellular localization of FsE4MT01 and FsE4MT02 fused with GFP in the epidermal cells of *Nicotiana benthamiana* leaves. Fluorescent signals co-localized with the endoplasmic reticulum (ER) marker protein ER WAK2 RFP. Scale bar = 20 micrometers.

[0042] Figure 10 Characterization of the in vitro enzymatic properties of FsE4MT01 and FsE4MT02. (A) Phylogenetic analysis and classification of FsE4MT01 and FsE4MT02 proteases in identified plant OMTs. (B) Schematic diagram of the 4-O-methylation reaction catalyzed by FsE4MTs, using (+)-epiporesinin as substrate and S-adenosyl-L-methionine (SAM) as methyl donor to generate forsythoside. (C) Extraction ion chromatogram (XIC) analyzed by LC-MS / MS, showing the conversion of (+)-epiporesinin to forsythoside by recombinant FsE4MT01 and FsE4MT02. (D–F) High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis of the enzymatic reaction products and forsythoside standards. Steady-state kinetic parameters of (G–H)FsE4MT01(G) and FsE4MT02(H) for the substrate (+)-epiraminin. Data are expressed as mean ± standard deviation of three independent experiments.

[0043] Figure 11 (A, B) Purification of recombinant proteins FsE4MT01 (A) and FsE4MT02 (B). The N-terminal HIS–SUMO fusion protein, heterologously expressed and purified in *E. coli* BL21, was analyzed by SDS-PAGE.

[0044] Figure 12 Molecular docking analysis of candidate enzymes with substrates. (A, B) FsE4MT01 (A) and FsE4MT02 (B) with substrates (+)-epifenaffinin, methyl donor SAM, and Mg. 2+ Predicted binding conformation and interaction energy of the complex formed by the cofactor. The calculated binding free energies (ΔG) are -10.385 kcal / mol and -10.162 kcal / mol, respectively.

[0045] Figure 13 FsE4MT01 and FsE4MT02 in the absence of Mg 2+Molecular docking analysis under the specified conditions. (A) Without Mg 2+ Under cofactor conditions, the predicted binding conformation and interaction energy of the complex of FsE4MT01 with the substrate (+)-epifenain and the methyl donor SAM were calculated, and the binding free energy (ΔG) was -9.655 kcal / mol; (B) without Mg 2+ Under cofactor conditions, the predicted binding conformation and interaction energy of the complex of FsE4MT02 with (+)-epiporesinin and SAM were obtained, and the binding free energy (ΔG) was calculated to be -9.218 kcal / mol.

[0046] Figure 14 High-resolution mass spectrometry characterization of (+)-epidermopine. High-resolution time-of-flight mass spectrometry (HR-TOF-MS) image acquired in positive ion mode. [M+H] at m / z 359.0664. + The ion corresponds to (+)-epifenaffinin (C 20 H 22 Protonated molecular ions of O6.

[0047] Figure 15 Biochemical properties of recombinant proteins FsE4MT01 and FsE4MT02 were characterized. The effects of pH (A–B) and temperature (C–D) on the in vitro activity of purified recombinant proteins FsE4MT01 (A, C) and FsE4MT02 (B, D) were determined, and bell curve fitting was performed using GraphPadPrism 8 software to determine their optimal reaction conditions.

[0048] Figure 16Functional validation of candidate genes FsE4MT01 and FsE4MT02 in Forsythia suspensa and Forsythia australis using transient overexpression and virus-induced gene silencing (VIGS) technology. (A) Schematic diagram of the workflow of transient expression and VIGS experiment in Forsythia suspensa leaves. (B, C) Relative mRNA levels of the genes detected by RT-qPCR after transient overexpression of FsE4MT01 (B) and FsE4MT02 (C) in Forsythia suspensa leaves. (D) Accumulation of forsythosides and forsythosides in Forsythia suspensa leaves with transient overexpression of FsE4MT01 or FsE4MT02. (E, F) Relative mRNA levels of FsE4MT01 (E) and FsE4MT02 (F) in Forsythia suspensa leaves after VIGS-mediated gene knockdown. (G) Accumulation of forsythosides in Forsythia suspensa leaves after VIGS-mediated knockdown of FsE4MT01 or FsE4MT02. Data are expressed as mean ± standard deviation. Student's t-test (B, C, E, F), one-way ANOVA (G), and two-way ANOVA (D) were used to determine statistical significance relative to the empty vector control (* P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001). Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0050] The Forsythia suspensa materials used in this embodiment were collected from its natural growing area—Fanli Town, Lushi County, Henan Province, China (34°10′26.99″N, 111°10′49.78″E, altitude 650 meters), a typical production area for Forsythia suspensa. Tissue samples were collected from six-year-old healthy plants in the fruiting stage.

[0051] In the examples, the vectors pEAQ-HT-DEST2 / pCambia1300-CFP / pTRV1, pTRV2, pET28a, and Agrobacterium tumefaciens GV3101 were all commercially available.

[0052] In the examples, S-adenosyl-L-methionine (SAM; Cat. No. s832588, Lot No. C16929940) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; forsythoside (Cat. No. CFN90511, Lot No. CFS202302, purity ≥98%), forsythoside (Cat. No. CFN99998, Lot No. CFS202302, purity ≥98%) and (+)-epidermosiderin (Cat. No. CFN92288, Lot No. CFS202401, purity ≥98%) were all purchased from Wuhan Tianzhi Huajing Chemical Reagent Co., Ltd. (ChemFaces).

[0053] The methods for determining the content of forsythosides and forsythosides in Forsythia suspensa and tobacco leaves in the examples are as follows:

[0054] Collected Forsythia or tobacco leaf samples were freeze-dried under vacuum in a Scientz-100F freeze dryer. After drying, 50 mg of powder sample was accurately weighed and extracted with 1,200 μL of pre-cooled (-20°C) 70% methanol-water extraction buffer. The mixture was vortexed for 30 seconds every 30 minutes, repeated 6 times, followed by centrifugation at 12,000 rpm for 3 minutes. The supernatant was collected, filtered through a 0.22 μm microporous membrane, and the filtrate was stored in a sample vial for HPLC-UV analysis.

[0055] Forsythoside and forsythoside content analysis was performed using a Waters HPLC system equipped with an Atlantis™ dC18 column (4.6 × 250 mm, 5 µm). The mobile phase was an aqueous solution (A) containing 0.3% formic acid and methanol (B). The gradient elution program was as follows: 0–15 min, A:B changed from 90:10 to 75:25; 15–23 min, A:B changed from 75:25 to 67:33; 23–33 min, A:B changed from 67:33 to 62:38; 33–40 min, A:B changed from 62:38 to 60:40; 40–50 min, A:B changed from 60:40 to 40:60; 50–55 min, A:B changed from 40:60 to 90:10; 55–60 min, A:B remained at 90:10. The detection wavelength was 235 nm, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was maintained at 35 °C. The metabolite content was calculated by comparing the peak areas with the standard curve obtained from the standards.

[0056] In this embodiment, forsythoside A and forsythoside B were analyzed by high-resolution electrospray ionization mass spectrometry using a Vanquish Flex ultra-high performance liquid chromatography system coupled with a Q Exactive Plus mass spectrometer, as detailed below:

[0057] For freeze-dried Forsythia suspensa or tobacco leaf samples, forsythosides and forsythosides were extracted with 1,200 μL of pre-cooled (-20°C) 70% methanol, intermittently vortexed (30 seconds every 30 minutes, for a total of 6 times), followed by centrifugation (12,000 rpm, 3 min). The supernatant was filtered through a 0.22 μm nylon needle filter into amber sample vials and stored at -20°C until UPLC-MS analysis. Chromatographic separation was performed using a Hypersil GOLD column (2.1 × 100 mm, 1.9 μm) at 30°C with an injection volume of 5 μL. The mobile phase consisted of an aqueous solution (A) containing 0.1% formic acid and acetonitrile (B), with gradient elution (0–18 min, phase B increasing from 10% to 95%) at a flow rate of 0.3 mL / min. Mass spectrometry detection was performed using an electrospray ionization source in both positive and negative ion modes (spray voltage: 3.5 kV for positive ion mode, 3.2 kV for negative ion mode) at a capillary temperature of 320℃. Data acquisition was performed using full-scan MS / ddMS2 mode (resolution: 70,000 / 17,500; AGC target: 1e6; TopN: 8) with multi-level collision energies (20%, 40%, 60%).

[0058] The recombinant protein expression and purification methods in the examples are as follows:

[0059] To perform gene cloning and vector construction, total RNA was first extracted from Forsythia suspensa tissue using a plant RNA extraction kit (Tiangen, China), and cDNA was synthesized using a reverse transcription kit (Vazyme, China). Subsequently, the coding sequences of the target genes (FsE4MT01, FsE4MT02, and FsP4'GT) were amplified by PCR using Phanta Max high-fidelity DNA polymerase (Vazyme). The PCR products were cloned into the pET28a expression vector with an N-terminal 6×His tag using the ClonExpress II one-step cloning kit (Vazyme). The recombinant vector was then transformed into E. coli for induced expression of the recombinant proteins. The primer pair for amplifying FsE4MT01 is shown below:

[0060] FsE4MT01-His-F:5'- CAGATTGGTGGATCC ATGAAAATGAAAACTAGTGCA-3' (SEQ ID NO:5),

[0061] FsE4MT01-His-R:5'- CTCGAATTCGGATCC TCATTCAGGAGAAAGAAGAAGAATT-3' (SEQ IDNO:6);

[0062] The primer pair for amplifying FsE4MT02 is shown below:

[0063] FsE4MT02-His-F:5'- CAGATTGGTGGATCC ATGAGAGCAAAAACACAAGC-3' (SEQ ID NO:7),

[0064] FsE4MT02-His-R:5'- CTCGAATTCGGATCC CTAAGGACAAAATGGGTTTGTTCAAG-3' (SEQ IDNO:8);

[0065] During protein expression induction, 0.5 mM IPTG was added to the bacterial culture, and the culture was incubated at 22°C and 120 rpm for 24 hours with shaking to increase the yield of soluble protein. After induction, the bacterial cells were collected by centrifugation (10,000 × g, 1 min, 4°C), washed and resuspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), and then lysed using a high-pressure homogenizer (4°C). After centrifugation (12,000 × g, 10 min, 4°C), the supernatant was loaded onto a nickel-NTA gravity column (HyPur T 6FF, Cytiva) pre-equilibrated with binding buffer. Impurities were washed away with washing buffer (20 mM imidazole), and the target protein with the His tag was eluted with elution buffer (250 mM imidazole). Protein purity was verified using 12% SDS-PAGE, and protein quantification was performed using the BCA protein concentration assay kit (Sangon Biotech, China).

[0066] Example 1

[0067] 1. Spatiotemporal dynamics analysis of Forsythia metabolism and transcriptome

[0068] To elucidate the spatiotemporal dynamics of secondary metabolites related to forsythoside biosynthesis in Forsythia suspensa, we collected tissue samples from five organs (seeds (fully developed), pericarp (pericarp), pedicel (pedicel), young branches (current year's shoots), and young leaves (fully expanded)) at two different fruit ripening stages: green immature fruit stage (GFS, harvested in August 2022) and red mature fruit stage (RFS, harvested in October 2022). Figure 2 A) All samples were immediately flash-frozen in liquid nitrogen after collection and stored at -80°C until subsequent processing. Three biological replicates were set up for each sample type (each replicate represents an independent plant sample), for a total of 30 samples for secondary metabolomics analysis.

[0069] The above 30 samples were freeze-dried at -100℃ under vacuum for 72 hours using a Scientz-100F freeze dryer. The freeze-dried tissues were then ground into a fine powder using a Retsch MM 400 ball mill (frequency 30 Hz, duration 1.5 min). 50 mg of the powder sample was accurately weighed and added to 1.2 mL of 70% methanol solution pre-cooled to -20℃. Complete extraction was ensured by pulsed vortexing (30 seconds per cycle, 30 min intervals, for a total of 6 cycles). After centrifugation (12,000 rpm, 3 min, 4℃), the supernatant was filtered through a 0.22 μm nylon membrane (Millipore), and the filtrate was stored at -80℃ until ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) multiple reaction monitoring (MRM) metabolomics analysis.

[0070] Ultra-high performance liquid chromatography (UHPLC) was performed using an Agilent SB-C18 reversed-phase column (1.8 μm particle size, 2.1 × 100 mm) in ExionLC. TM Separation was performed using an AD ultra-high performance liquid chromatography system. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B), with the following gradient elution program: initial ratio 5% B (0 min), linear ramp to 95% B (0–9 min), hold (9–10 min), regression to 5% B (10–11.1 min), column equilibration (11.1–14 min). The flow rate was 0.35 mL / min, the column temperature was 40 °C, and the injection volume was 2 μL. Mass spectrometry detection was performed using an electrospray ionization source, switching between positive ion mode (+5,500 V) and negative ion mode (-4,500 V), with an ion source temperature of 500 °C. Gas source parameters: ion source gas I / II were 50 / 60 psi, curtain gas was 25 psi, and collision gas was nitrogen (medium pressure mode). Multiple reaction monitoring (MRM) mode was used, and the declustering voltage and collision energy were optimized through collision-induced dissociation. The raw data was analyzed using Analyst® 1.6.3 software for peak identification, alignment, and comparison with the standard database.

[0071] The identification of all compounds (especially lignans) was based on retention time, precise molecular weight, and tandem mass spectrometry fragmentation characteristics. For compounds with available standards, confirmation was achieved by comparing the retention time and mass spectrometry data of the analyte with those of the standards; for compounds without standards, preliminary identification was achieved by matching the obtained high-resolution tandem mass spectra with spectra in the Maiwei database.

[0072] Using the aforementioned ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) multiple reaction monitoring (MRM) mode, a total of 1,013 metabolites were detected. The main categories were: flavonoids (17.67%), lignans and coumarins (11.45%), terpenes (19.25%), alkaloids (8.29%), quinones (1.58%), tannins (0.3%), phenolic acids (30.5%), and other unclassified compounds. Figure 2 B).

[0073] Principal component analysis (PCA) of metabolites was performed using R software (version 4.2.0). Unit variance scaling was applied to the data to eliminate dimensional differences and avoid excessive influence of variables with large absolute values ​​on the model. The results showed a clear separation of metabolome profiles between different tissues from the two harvest periods, indicating high tissue specificity of the metabolome. Figure 2 C). This result was further validated by hierarchical cluster analysis. Figure 3 Notably, among the detected lignans, several important compounds were identified in each sample, including forsythoside (C). 27 H 34 O 11 The negative ion mode detection m / z was 533.20→371.15), and forsythoside C (C 21 H 24 O6, detected in positive ion mode (m / z 373.16 > 337.14) and its derivatives, such as syringaresinol (C 22 H 26 O8, negative ion mode detection m / z 417.16→402.10), (+)-epipinoresinol (C 20 H 22 O6, detected in negative ion mode m / z 357.13→151.04), 8-hydroxypinoresinol (C 26 H 22 O7, negative ion mode detection m / z 373.13→343.12), 1-hydroxypinoresinol-4'-O-glucoside (C 26 H 32 O 12 The negative ion mode detection m / z 535.18→373.13) and pinoresinol-4-O-glucoside (C 26 H 32 O 11(Detected in negative ion mode, m / z 519.19→357.14). The molecular formulas and retention times of these compounds are consistent with those of known diepoxylignans. The prevalence of these compounds in the metabolomic profile highlights their importance and suggests that they may play an important role in the forsythoside biosynthetic network.

[0074] To investigate the genetic basis of secondary metabolite biosynthesis, we performed transcriptome analysis on multiple tissues and maturation stages. Specifically, total RNA was sequenced from frozen Forsythia suspensa tissue samples using the Illumina HiSeq 2000 platform (2 × 150 bp paired-end sequencing). After filtering low-quality reads using Trimmomatic (v0.39), the quality-controlled valid sequences were aligned to the Forsythia suspensa reference genome using HISAT2 (v2.2.1). Transcript assembly was performed using StringTie (v2.2.1), and differential expression analysis was conducted using DESeq2 (v1.38.3) (screening criteria: |log2FC| > 1 and FDR < 0.05). Gene expression levels were quantified using FPKM (number of transcript fragments per kilobase length per million mapped reads) for functional annotation and enrichment analysis to screen key genes involved in forsythoside biosynthesis. Results showed a total of 250.74 Gb of high-quality clean data, with each sample producing over 6 Gb of clean reads, and Q30 values ​​exceeding 92%, indicating that the sequencing quality met the requirements for subsequent analysis. A total of 37,474 genes were predicted, of which 7,436 were compared to previous studies. This is a newly identified finding. Consistent with the metabolomics findings, transcriptomic analysis also revealed distinct tissue-specific expression patterns. Figure 2 D; Figure 4 ).

[0075] Of the 23,724 differentially expressed genes identified (between groups |log2Fold Change| ≥ 1, FDR < 0.05), over 90% were predicted to be associated with metabolic processes, suggesting their potential involvement in mediating tissue-specific biosynthesis of metabolites. KEGG annotation analysis revealed 76 putative OMTs (divided into 7 clades; Figure 2 E) participates in the methylation step of forsythoside biosynthesis. Among them, 51 OMTs showed differential expression ( Figure 2 (E), indicating that they may play a role in the biosynthesis of forsythosides. Therefore, these genes were prioritized as candidate genes for further functional studies.

[0076] 2. Identification of candidate genes FsOMTs for the biosynthesis of forsythosides

[0077] Metabolites and related genes in the forsythoside biosynthesis pathway all exhibit significant spatiotemporal expression patterns. Figure 5 A and 5B). Specifically, the final product forsythoside mainly accumulates in leaves during the GFS stage and branches during the RFS stage, while its direct precursors (+)-epistine and forsythoside are mainly found in branches during the RFS stage. Figure 5 A). Consistent with these metabolic patterns, most genes involved in forsythoside biosynthesis (including multiple Fs4CLs (4-coumarate-CoA ligases), FsCCRs (cinnamoyl-CoA reductases), FsCADs (cinnamyl alcohol dehydrogenases), FsDIRs (dirigent proteins), and FsCOMTs (Catechol-O-methyltransferases) were highly expressed in branches at both the GFS and RFS stages. Figure 5 B). These results indicate that the biosynthesis of forsythosides mainly occurs in the branches and may subsequently be transported to other tissues. Furthermore, LC-MS-based metabolomics analysis identified several other bioactive lignan compounds, such as arctigenin, arctiin, matairesinol, secoisolariciresinol diglucoside, and lariciresinol (4-coumarate-CoA ligases). These compounds also exhibited complex spatiotemporal accumulation patterns. Figure 6 The pattern is similar to the distribution characteristics of forsythoside and its metabolites in the synthetic pathway.

[0078] To identify key genes contributing to metabolic differences, we calculated the Pearson correlation coefficient (PCC) between the log-transformed expression levels of all 37,474 expressed genes (TPM ≥ 1 in at least one sample) and the relative abundance of metabolites. A total of 10,375 genes showed a significant correlation with at least one metabolite (|PCC| ≥ 0.80, P < 0.05). To further elucidate the dynamic regulatory network of forsythoside biosynthesis, we performed a weighted gene co-expression network analysis (WGCNA) on differentially expressed genes associated with forsythoside and its precursors. Figure 5C). Specifically, the weighted gene co-expression network was constructed using the blockwiseModules function in the WGCNA R package. Key parameters were optimized as follows: soft threshold power was set to 14, TOM type was set to 'signed', module merge sensitivity was mergeCutHeight = 0.25, and minimum module size was 50 genes. All other parameters were left at their default settings to conform to the standard WGCNA workflow. Among the obtained co-expression modules, the blue module showed the strongest positive correlation with forsythoside and its direct precursors (forsythoside A and (+)-epiporesin) (PCC > 0.90; Figure 5 D). In this blue module, we screened four FsOMT genes and two FsDIR genes as key candidate genes that may be involved in this metabolic pathway.

[0079] Correlation analysis showed that the accumulation of (+)-epifenain was significantly positively correlated with the transcriptional expression of the FsDIR1 gene (R = 0.51). Figure 7 A), but there was no significant correlation with the transcriptional expression of the FsDIR2 gene (R = 0.106). Figure 7 B), which is consistent with the known catalytic function of DIR proteins in mediating the conversion of coniferyl alcohol to (+)-pineneol. Furthermore, the expression of the four FsOMTs genes showed a strong positive correlation with the accumulation of forsythosides (all R > 0.70). Figure 5 E–5H). Therefore, these four genes were identified as candidate OMTs and named FsE4MT01–FsE4MT04. Notably, intergene correlation analysis showed that the expression of FsDIR1 was highly correlated with the expression of FsE4MT01 (R = 0.70) and FsE4MT02 (R = 0.53). Figure 8 A-8B), but no significant correlation was found with the expression of FsE4MT03 (R = 0.024) and FsE4MT02 (R = 0.428). Figure 8 (C-8D). This tight transcriptional co-regulation further supports the functional correlation of these enzymes in the sequential steps of the forsythoside biosynthesis pathway.

[0080] 3. Functional verification of FsE4MTs protease in forsythoside biosynthesis

[0081] Subcellular localization analysis showed that both FsE4MT01 and FsE4MT02 co-localized with the endoplasmic reticulum marker protein ER-WAK2. Figure 9 C). To verify the catalytic function of the candidate genes, FsE4MT01–FsE4MT04 were cloned into the pEAQ expression vector and heterologously expressed in tobacco leaves. Figure 9A) Specifically, the coding sequences of the FsE4MT01–FsE4MT04 genes shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:10 were cloned into the pEAQ-HT-DEST2 vector and transformed into Agrobacterium tumefaciens strain GV3101 (empty vector as a control). The bacterial culture was resuspended in a solution containing 10 mM MES, 10 mM MgCl2, and 150 μM acetylsyleugenone to OD200. 600 = 0.6-0.8) The needleless syringe was used to soak 4-week-old tobacco leaves, followed by 1 day of dark culture and 3 days of light culture. When amplifying the coding sequences of FsE4MT01~FsE4MT04 genes, the template used was cDNA reverse transcribed from RNA extracted from tender leaves of Forsythia suspensa. The primers used are detailed in Table 1.

[0082] For the substrate feeding experiment, on day 4 after Agrobacterium infiltration, the substrate ((+)-epidermopinesin) aqueous solution was infiltrated to the same sites. Forty-eight hours after substrate infiltration, the labeled leaf regions were collected, flash-frozen in liquid nitrogen, and freeze-dried for extraction of the metabolite forsythosides and HPLC-UV analysis. The results of the substrate feeding experiment showed that after the addition of (+)-epidermopinesin, the accumulation of forsythosides in leaves expressing FsE4MT01 or FsE4MT02 (but not FsE4MT03 or FsE4MT04) was significantly increased (up to 44%) compared to the empty vector control. Figure 9 (B) This result indicates that only FsE4MT01 and FsE4MT02 can specifically catalyze the 4-position oxygen methylation of (+)-epidermines. Notably, even the empty vector control leaves showed some basic conversion activity of (+)-epidermines to forsythosides, which may originate from the activity of endogenous OMTs in tobacco. These in vivo experimental results collectively confirm that FsE4MT01 and FsE4MT02 are responsible for catalyzing the methylation of epiidermines to forsythosides.

[0083] Table 1. Primers used for cloning FsE4MTs

[0084]

[0085] Note: The underlined parts in the primers in Table 1 are homologous arm sequences.

[0086] Phylogenetic analysis of terrestrial plant lineages indicates that O-methyltransferases (OMTs) appeared early in the evolution of terrestrial plants and can be divided into two main categories. The catalytic activity of class I enzymes requires Mg... 2+Ions are involved, while the function of class II enzymes is independent of metal ions. Therefore, we performed a phylogenetic analysis of OMTs proteases from different terrestrial plant lineages. Specifically, gene sequences were extracted according to the KEGG pathway annotation system, and the corresponding protein sequences were obtained for phylogenetic analysis. Multiple sequence alignment was performed using the ClustalW program in MEGA12 software with default parameters. After alignment, the terminal regions were manually proofread, and inconsistent parts were pruned to improve alignment quality. The phylogenetic tree was reconstructed using the neighbor-joining method, the pairwise evolutionary distance was calculated using the p-distance model, and the node support rate was evaluated by 1500 bootstrap replicates. The final generated Newick format tree file (including bootstrap support rate) was imported into the R language (version 4.3.3) environment, and advanced visualization analysis was performed using the "ggtree" and "ggtreeExtra" packages. The results showed that FsE4MT01~FsE4MT04 were all predicted to belong to class I OMTs ( Figure 10 A, Table 2), whose molecular weight is similar to that of other members in this category ( Figure 11 This suggests that its catalytic activity depends on Mg. 2+ This hypothesis is further supported by molecular docking analysis. The analysis showed that in Mg... 2+ Under the presence of these conditions, both FsE4MT01 and FsE4MT02 exhibit stronger substrate binding capabilities. Figure 12 A, 12B and Figure 13 FsE4MT01 ( Figure 12 A) and FsE4MT02 ( Figure 12 B) The active site is located within a semi-closed pocket formed by the surrounding secondary structures. This structural feature facilitates interaction with the substrate (+)-epifenaffinin, the methyl donor S-adenosine-L-methionine (SAM), and the essential cofactor Mg. 2+ Specific interactions occur. Hydrogen bonds and hydrophobic interactions stabilize these bonds, ensuring precise localization of reactants to facilitate catalysis. In vitro enzyme activity experiments confirmed the presence of this Mg... 2+ Dependence: When Mg is removed from the reaction system 2+ At that time, the synthesis of forsythoside A was completely inhibited. Figure 10 C).

[0087] Table 2. O-methyltransferases used for phylogenetic analysis

[0088]

[0089] To assess in vitro enzyme activity, we expressed FsE4MT01~FsE4MT02 with an N-terminal His-SUMO fusion tag in E. coli and purified the recombinant protein ( Figure 11Using (+)-epifenaffinin and S-adenosyl-L-methionine (SAM) as substrates, and with the addition of the essential cofactor Mg... 2+ Enzyme activity assay ( Figure 10 B). The reaction products were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). By comparing the retention times and mass spectra with those of the standards, it was confirmed that only FsE4MT01 and FsE4MT02 could catalyze the 4-oxymethylation of (+)-epifolinin to generate forsythoside (). Figure 10 C). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis confirmed the enzymatic conversion of (+)-epidermopinesinin to forsythopinesinin. In positive ion mode, the substrate epiidermopinesinin was detected with a [M+H]⁺ ion (C⁻) at m / z 359.1464. 20 H 23 O6⁺ theoretical value: 359.1495), accompanied by the characteristic fragment ion [M−CH3+H]⁺ with m / z 344.0413. Figure 14 The product, forsythoside, shows a [M+H]⁺ ion (C) at m / z 373.1630. 21 H 25 Theoretical value of O6: 373.1651), and dehydration-induced fragment ions [M−H2O+H]⁺ (C) were detected at m / z 355.1538. 21 H 24 O5 theoretical value: 355.1545 Figure 10 D–10F).

[0090] To characterize the enzyme kinetics of FsE4MT01 and FsE4MT02 in the catalytic (+)-epidermopinesin methylation reaction, we determined their steady-state kinetic parameters using purified substrates. Specifically, the methyltransferase activity assay for FsE4MT01 and FsE4MT02 was performed in a 100 μL reaction volume containing 100 mM Tris-HCl buffer (pH 7.0), 100 μM MgCl2 (cofactor), 0.5 mM SAM (methyl donor), and 50 μM epipinesin (substrate). The reaction was initiated by adding 20 μg of purified enzyme solution and incubated at 40°C for 3 hours. Both enzymes were measured at pH 7.5 (… Figure 15 A, 15B) and 40℃ ( Figure 15 The best activity was observed under conditions C and 15D. The apparent catalytic efficiencies (Kcat / Km) for FsE4MT01 were 2.18 × 10⁻⁶. 3 M -1 s -1 FsE4MT02 is 2.98 × 10 3 M -1 s -1( Figure 10 G, 10H).

[0091] 4. Effects of regulating the expression of FsE4MT01 and FsE4MT02 on the synthesis of forsythosides in Forsythia leaves

[0092] To address the lack of a stable genetic transformation system for Forsythia suspensa, we have developed a transient leaf expression method based on minimally invasive pretreatment combined with Agrobacterium-mediated vacuum infiltration. Figure 16 A) Specifically, the coding sequences of the FsE4MT01 and FsE4MT02 genes were cloned into the pCambia1300-eCFP vector (primers used are detailed in Table 1) to obtain recombinant plasmids pCambia1300-FsE4MT01 and pCambia1300-FsE4MT02. These recombinant plasmids were then transformed into *Agrobacterium tumefaciens* strain GV3101 (empty vector served as a control) to obtain the 35S::FsE4MT01 and 35S::FsE4MT02 constructs. *Agrobacterium tumefaciens* was cultured in LB medium containing 50 μg / mL kanamycin, 25 μg / mL rifampin, 10 mM MES, and 0.2 mM acetylsyleugenol until OD200. 600 The OD value was 0.8-1.0 (28℃). The cells were resuspended in 10 mM MgCl2 solution until the OD value was reached. 600 The concentration of the culture medium was 0.6-0.8, and 200 μM acetylsyringone was added for induction for 3 hours. Forsythia suspensa young leaves treated with micro-trauma were subjected to vacuum permeation of the bacterial solution (-100 kPa, 30 min), followed by co-culturing at 22℃ for 48 hours. Finally, the leaves were freeze-dried and metabolites were analyzed. The results showed that the 35S::FsE4MT01 and 35S::FsE4MT02 constructs were successfully introduced into Forsythia suspensa young leaves using this technique. RT-qPCR analysis confirmed that, compared with the control group, the expression levels of FsE4MT01 and FsE4MT02 in the transformed leaves were significantly increased ( ). Figure 16 B, 16C); primers used for RT-qPCR analysis are shown in Table 3. HPLC-UV analysis showed that transient overexpression of FsE4MT01 and FsE4MT02 increased the accumulation of forsythoside lipoprotein by 2.5 times and 2.2 times, respectively, compared with the empty vector control. Figure 16 D). These results further confirm that FsE4MT01 / FsE4MT02 catalyzes the conversion of (+)-epidermosinin to forsythosinin.

[0093] Table 3. qRT-PCR primers

[0094]

[0095] In addition, virus-induced gene silencing (VIGS) technology was used to knock down the FsE4MT01 and FsE4MT02 genes. Specifically, using the recombinant plasmids pCambia1300-FsE4MT01 and pCambia1300-FsE4MT02 constructed above as templates, a recombinant pTRV2 vector containing FsE4MT01 and FsE4MT02 gene fragments was constructed using the primers listed in Table 1. VIGS experiments were then performed on Forsythia suspensa using Agrobacterium-mediated transformation. Forsythia suspensa infected with Agrobacterium GV3101 carrying the empty pTRV2 vector was used as a control. Silent leaves were collected 2 days after infection and analyzed by HPLC. The results showed that the mRNA levels of FsE4MT01 and FsE4MT02 were significantly lower than those of the pTRV2 empty vector control. Figure 16 E, 16F). Furthermore, silencing FsE4MT01 and FsE4MT02 leads to a significant reduction in the accumulation of forsythoside lipoproteins (E, 16F). Figure 16 G). These gain-of-function and loss-of-function experimental results together confirm that FsE4MT01 and FsE4MT02 are essential key enzymes for the biosynthesis of forsythosides in Forsythia suspensa.

[0096] Given the crucial roles of FsE4MT01 and FsE4MT02 proteases in the biosynthesis of forsythosides, we employed computational molecular docking to elucidate their catalytic mechanism. Specifically, the three-dimensional structural models of the target proteins were generated using AlphaFold3 (https: / / alphafoldserver.com / ), while the structures of the ligands (including (+)-epiporesinin) were obtained from the PubChem database. Molecular docking was performed using AutoDock Vina v1.2.0 software, and binding sites were explicitly defined for each enzyme: FsE4MT01 and FsE4MT02 docked with (+)-epiporesinin and Mg... 2+ And SAM. During the docking process, the initial placement of ligands was first performed using the triangle matching method, followed by flexible docking optimization through binding-induced conformation optimization. The resulting docking conformations were compared and verified with the resolved enzyme-ligand crystal complex structures, and the optimal conformation was selected based on structural rationality and catalytic feasibility. The PLIP tool was used to analyze the protein-ligand interaction, with particular attention paid to the hydrogen bond network involving metal ions. All structural visualizations and renderings were generated using PyMOL (Schrödinger) software for detailed examination of the binding interface. Precise substrate selectivity ensures that the synthesis of forsythoside is highly dependent on the activity and abundance of the enzyme, which also explains the significant increase in forsythoside content after overexpression of the enzyme.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Forsythia Class I type O-methyltransferase, characterized in that, The amino acid sequence of the Forsythia Class I type O-methyltransferase is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The nucleic acid encoding the Forsythia Class I type O-methyltransferase as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. A biomaterial containing the nucleic acid of claim 2 or 3, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

5. The application of Forsythia Class I OMT enzyme with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, the nucleic acid encoding Forsythia Class I OMT enzyme, and biological materials containing said nucleic acid in regulating the content of forsythosides in Forsythia, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

6. The application of Forsythia Class I OMT enzyme with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, the nucleic acid encoding Forsythia Class I OMT enzyme, and biomaterials containing said nucleic acid in increasing the content of forsythosides in Forsythia, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

7. A method for increasing the content of forsythosides in Forsythia suspensa, characterized in that, include: To increase the expression level of Class I oxygen methyltransferase in Forsythia suspensa, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:

2.

8. A method for increasing the content of forsythosides in Forsythia suspensa according to claim 7, characterized in that, Exogenous DNA encoding Class I oxymethyltransferase was transferred into Forsythia suspensa.

9. A method for increasing the content of forsythosides in Forsythia suspensa according to claim 8, characterized in that, The exogenous DNA sequence encoding Class I oxygen methyltransferase is shown in SEQ ID NO:3 or SEQ ID NO:4.

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