Angelica cyclase genes AdDC and AdOD and their application in the preparation of furanocoumarin compounds

Abstract

This invention discloses an Angelica dahurica cyclase gene AdDC, AdOD, its encoded protein, and its applications. The Angelica dahurica cyclase gene includes a gene... AdDC and genes AdOD The gene AdDC The nucleotide sequence of the gene is SEQ ID No. 1; AdOD The nucleotide sequence is SEQ ID No. 2. This application marks the first successful cloning of two novel cyclase genes from Angelica dahurica. AdDC and AdOD The specific nucleotide sequence and encoded amino acid sequence were disclosed, eukaryotic expression vectors and plant overexpression vectors were constructed, and a method for gene preparation of furanocoumarin compounds was established, providing a new method for the industrial production of furanocoumarin medicinal components.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the angelica cyclase genes AdDC and AdOD and their application in the preparation of furanocoumarin compounds. Background Technology

[0002] The Chinese herbal medicine Angelica dahurica is a plant belonging to the genus Angelica in the family Apiaceae. Angelica dahurica (Fisch. ex Hoffm.)Benth. et Hook. f. or Angelica dahurica (Hangzhou Angelica) Angelica dahurica The dried root of *Angelica dahurica* (Fisch. ex Hoffm.) Benth. etHook. f. var. *formosana* (Bois s.) Shan et Yuan has a long history of medicinal use. As a plant used for both food and medicine, Angelica dahurica has wide applications in food flavoring and preservation, agricultural antibacterial agents, and cosmetic whitening. For example, Angelica dahurica burn tincture can be used for anti-infection and hemostasis, and Angelica dahurica extract can be used as a natural preservative for pickled vegetables or as a whitening ingredient in skin care products. Its coumarin compounds can inhibit plant pathogens, and its application prospects are broad.

[0003] The core pharmacologically active components of Angelica dahurica are furanocoumarins, whose biosynthetic pathway can be divided into three stages: nucleus formation, isopentenylation, and cyclization and structural modification. Cycloyltransferases catalyze the formation of the furan ring, marking the formal entry of the metabolic pathway into the furanocoumarin synthesis stage. Currently, two isopentenyltransferases (C-PTs) have been identified in Angelica dahurica, but the encoding genes, protein sequences, and enzymatic functions of the cyclyltransferases catalyzing the key cyclization stage have not yet been elucidated. This remains a key bottleneck in understanding the molecular mechanism of furanocoumarin biosynthesis in Angelica dahurica.

[0004] Cycloylases are widely distributed in plants, and those from different families and genera belong to different gene subfamilies. For example, the cyclase in fig (Moraceae) belongs to the CYP76 subfamily, while the cyclases reported in Peucedanum praeruptorum, Angelica sinensis, and Notopterygium incisum (Apiaceae) all belong to the CYP736 subfamily, providing a reference for the study of cyclases in the Apiaceae family. However, research on cyclases from the CYP736 subfamily of Angelica dahurica, an important medicinal plant in the Apiaceae family, remains lacking. To date, there are no reports on the extraction and functional verification of this type of cyclase gene, resulting in an unclear molecular regulatory mechanism for the biosynthesis of furanocoumarins in Angelica dahurica. This makes it difficult to regulate the expression of cyclase genes through genetic engineering, thus hindering the accumulation of this core active ingredient.

[0005] Currently, the acquisition of furanocoumarin components from Angelica dahurica mainly relies on traditional plant extraction and chemical synthesis methods, which suffer from problems such as low extraction rate, high resource consumption, complex processes, high costs, and environmental pollution. Due to the lack of key cyclase gene resources, researchers are unable to construct microbial heterologous synthesis systems, making it difficult to achieve green, efficient, and large-scale production of furanocoumarin products. This seriously restricts the in-depth development and industrial upgrading of Angelica dahurica medicinal resources, and also limits its wider application in the fields of medicine, food, agriculture, and cosmetics.

[0006] In summary, there is an urgent need for a method to efficiently extract furanocoumarin components from Angelica dahurica, so as to achieve green, efficient and large-scale production of furanocoumarin products. Summary of the Invention

[0007] The purpose of this application is to overcome the shortcomings of the prior art and provide two cyclase genes cloned for the first time from Angelica dahurica and their encoded amino acid sequences, as well as methods and results for functional verification of the above genes and proteins, clarifying their in vitro and in vivo catalytic activities. It also provides recombinant vectors containing the aforementioned genes and their application in the biosynthesis of furanocoumarin compounds, and UPLC-MS / MS and HPLC detection techniques for verifying and quantitatively analyzing related compounds.

[0008] The objective of this invention is achieved through the following technical solution: Firstly, an angelica cyclase gene, including the gene... AdDC and genes AdOD The gene AdDC The nucleotide sequence of the gene is SEQ ID No. 1; AdOD The nucleotide sequence is SEQ ID No. 2; Among them, SEQ ID No. 1 is: SEQ ID No. 2 is: Specifically, the gene AdDC The encoded amino acid sequence is SEQ ID No. 3; the gene AdOD The encoded amino acid sequence is SEQ ID No. 4.

[0009] Among them, SEQ ID No. 3 is: MSPTQFATLLVLVGVLWWFIQLCRTILSNMPRQLHKPPPGPRGLPLIGHLHLLGKQPHRTLYNLSKKYGPIMSLRLGSIPTIVVSTSAASELFLKTHDTVFASRPKSQAAEYLWYGTKGMIFREYGT YWRNVRKFCTLELLSSTKIASMASMRREELVLLVDSLKIAARNREVVDVSQKLALLMEDMTCRMLFGKSRDERFDLNAIIHELTIVVGAFNVADFIPFLGALDLQGLTRRLKETSKAVDKVLETMIDE HEQDASDGCKKLERDFVDVLLSLKNSTHSSTHEQLGQKVDRSTIKAIVLDMIFGTIDTSQIAIAWIMSELVRHPRVMKQLQKEVENVVGDCDFVKESQLSKLDYLDMIVKESMRLHPVATLLPPHES MEDIVIDGYHIKKKSRIIVNNWGIGRDPRNWSENVDEFLPERFIGSSIDFAGNDFQLIPFGSGRRSCPGIHLGITMVKLVVTQLVHTFDWELPLGMSPDELDMEELFGATVPRATHLLAVPSLRNLP; SEQ ID No. 4 is: MSLIEFALFLLVAVAMWLFIHLRRTLTSHGGHKPPPGPIGLPLIGSLHQLGKHPHRSLYKMSQKYGPIMSLRLGLIPTIVVSSPAAVELFLKTHDSIFANRPKVQATEDLYGPKTIAFSEFGGYWR SLRKFCNIELLSPTKIDSMAGQRREELGFLVESLKNAAATGQVVNVTEKVAGLIEDMTCRMLLGKSRDDRFNLSKLIDEMTKATGSINIADFLPFLAPLDLQGLGRRIKVIVRELDKILDTIIDDH EQEASEGYKKLDKDFVDVLLSLKSNPPSIHEQLAKNIDRSNIRAIIVDIIFGSVETSITAIEWTMAELIRHKRVMKRVQEEIRNVIADCEYVEESHLSKLDYLHMVVKEIMRLHPIIPLLIPHEST EDIVVDGFYIQKKSRIIINGWGLAHDPKIWSENVEEFSPERFIDKDIDLRGKNFQLIPFGSGRRSCPGMHLGLTNVKLVIANLVHSFDWELPFGMSPDALNMDETFGLSLPRAKNLEAIPKIRQF. Secondly, the method for constructing a eukaryotic expression vector for the Angelica dahurica cyclase gene includes the following specific steps: The gene AdDC and / or genes AdOD Codon optimization and whole-genome synthesis were performed to obtain the optimized gene. AdDC and / or AdOD ; The optimized gene was recombinantly ligated with a yeast expression vector to obtain the eukaryotic recombinant plasmid. The recombinant plasmid pYES2 / CT- AdDC and / or pYES2 / CT- AdOD The yeast was transformed into Saccharomyces cerevisiae WAT11 competent cells and screened on SD-Ura-deficient medium to obtain a positive recombinant yeast strain pYES2 / CT- AdDC -WAT11 and pYES2 / CT- AdOD -WAT11; Preferably, the eukaryotic expression vector comprises pYES2 / CT-Amp; Preferably, the eukaryotic recombinant plasmid includes pYES2 / CT- AdDC or pYES2 / CT- AdOD ; Preferably, the brewing yeast includes brewing yeast WAT11; the positive recombinant yeast strain includes pYES2 / CT- AdDC -WAT11 or pYES2 / CT- AdOD -WAT11.

[0010] Thirdly, the method for constructing a plant overexpression vector for the Angelica dahurica cyclase gene includes the following specific steps: The gene AdDC and / or genes AdOD The plasmid pCAMBIA2300- was recombined with the linearized plant overexpression vector pCAMBIA2300-eGFP-Kana to construct the recombinant plasmid. AdDC And / or pCAMBIA2300- AdOD ; The recombinant plasmid pCAMBIA2300- AdDC And / or pCAMBIA2300- AdOD The recombinant plasmids were introduced into GV3101 Agrobacterium competent cells using a heat shock method. After resistance selection and PCR identification, strain pCAMBIA2300- was obtained for plant transformation. AdDC -GV3101; Preferably, the plant overexpression vector comprises pCAMBIA2300-eGFP-Kana; Preferably, the plant recombinant plasmid includes pCAMBIA2300- AdDC or pCAMBIA2300- AdOD ; Preferably, the Agrobacterium includes GV3101 Agrobacterium; the plant transformation strain includes pCAMBIA2300- AdDC -GV3101 or pCAMBIA2300- AdOD -GV3101.

[0011] Fourthly, the gene AdDC Application of protein in catalyzing the cyclization of 7-demethylcorpin to isopyramidal lactone; Fifthly, the gene AdOD Application of protein in catalyzing the cyclization of 7-demethylcorpin to isopyrambutanol and the cyclization of sennaol to dihydroaperone. Among them, Angelica dahurica cyclase gene AdDC and genes AdOD It can catalyze the formation of an epoxy intermediate from the substrate. The hydroxyl group at the C5 position of the substrate is deprotonated to form an oxygen anion. This nucleophilic center launches an SN2 nucleophilic attack on the C2 carbon atom of the epoxy ring, driving the epoxy ring to open and cyclize through a 5-exterior-tetrahedral structure, thereby specifically constructing a five-membered furan ring structure.

[0012] Sixthly, a method for preparing furanocoumarin compounds using the angelica cyclase gene includes the following steps: S1. Construct the gene AdDC and / or genes AdOD The eukaryotic expression vector or the plant overexpression vector; S2. Transform the eukaryotic expression vector or the plant overexpression vector constructed in S1 into host cells to obtain recombinant engineered bacteria or transgenic plants; S3. Induce the recombinant engineered bacteria or transgenic plant to express the [specific expression]. AdDC and / or AdOD Proteins, through the use of substrates, generate the furanocoumarin compounds via a catalytic reaction; Specifically, the furanocoumarin compounds include at least one of 7-demethylcorpin, sennaol, dihydroaperitol and isopyramidalin; Specifically, the host cells include eukaryotic Saccharomyces cerevisiae and Agrobacterium tumefaciens; Preferably, the host cells include eukaryotic Saccharomyces cerevisiae WAT11 and Agrobacterium tumefaciens GV3101; Specifically, the furanocoumarin compounds were detected using UPLC-MS / MS and UPLC methods.

[0013] The beneficial effects of this application are: (1) This application is the first to extract two key cyclase genes for furanocoumarin biosynthesis from Angelica dahurica. AdDC and AdOD The specific nucleotide sequence and encoded amino acid sequence were disclosed, providing a molecular basis for elucidating the biosynthetic pathway of furanocoumarins in Angelica dahurica. (2) This application successfully verified the function of these two genes in Saccharomyces cerevisiae and plant systems by constructing eukaryotic expression vectors and plant overexpression vectors, and clarified their catalytic activities; (3) This application provides a complete method for preparing furanocoumarin compounds using the gene and successfully constructs a chassis-based heterologous synthesis system. This method overcomes the shortcomings of traditional plant extraction methods, such as long cycle, large resource consumption, and difficult separation. It realizes the directional and efficient production of target compounds under controllable conditions and has good industrialization prospects. (4) This application establishes UPLC-MS / MS and UPLC methods for simultaneous detection of multiple furanocoumarin compounds, providing accurate and efficient analytical means for gene function verification, fermentation process monitoring, and final product quality control. Attached Figure Description

[0014] Figure 1In Example 1 AdDC and AdOD Gene gel electrophoresis image (lane M is a 100~2000 bp DNA marker; lane 1 is: AdDC Lane 2 is: AdOC ); Figure 2 This is a sequencing diagram of the AdDC gene clone in Example 1; Figure 3 This is a sequencing diagram of the AdOD gene clone in Example 1; Figure 4 In Example 4 AdDC and AdOD Protein Western blot image (lane M is for 17~180 kDa protein markers; lane 1 is for: AdDC Lane 2 is: AdOC ); Figure 5 In Example 4 AdDC and AdOD In vitro enzyme activity analysis (Figure A) AdDC Extraction ion chromatogram of enzyme reactants; Figure B is AdOD Extraction ion chromatogram of enzyme reactants); Figure 6 In Example 4 AdDC Mass spectrum of isopyram lactone in enzyme reaction product; Figure 7 In Example 4 AdOD Mass spectrum of isopyram lactone in enzyme reaction product; Figure 8 In Example 4 AdOD Mass spectrum of dihydroapigenin in enzyme mixture; Figure 9 The transient overexpression material in Example 5 AdDC and AdOD Relative gene expression levels and product content (Figure A) AdDC Relative expression levels; Figure B shows AdOD Relative expression levels; Figure C shows the content of isopyrambutanol; Figure D shows the content of dihydroaperitol. Detailed Implementation

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0016] Example 1 Angelica dahurica cyclase gene AdDC and AdOD Cloning Using 'Chuanzhi No. 2' Angelica dahurica as material, samples were flash-frozen in liquid nitrogen and then ground. Total RNA was extracted using the Trizol Total RNA Extraction Kit and reverse transcribed using the Novizan HiScript II 1st Strand cDNA Synthesis Kit. Primer 5 software was used to design... AdDC and AdOD Gene-specific primers (Table 1) were used for PCR amplification using Phanta® Max Super-FideLity DNA PoLymerase. The products were recovered by 1% agarose gel electrophoresis, followed by the recovery of the target band (e.g., [missing information]) using a Biomed agarose gel purification and recovery kit. Figure 1 (As shown). E. coli were transformed according to the instructions of the pClone007 Blunt Vector Kit, plated, cultured, and sequenced. Plasmids were extracted from the correct strain (OMEGA kit) and stored at -20°C. The cloned genes were then analyzed. AdDC and AdOD Gene sequencing and gene alignment were performed, and the gene sequencing maps are shown below. Figure 2 and Figure 3 As shown in the sequencing results, the cloned gene sequence is consistent with the target gene reference sequence, indicating that the gene was successfully cloned from Angelica dahurica. AdDC and AdOD Gene.

[0017] Table 1 AdDC and AdOD Gene-specific primers Example 2 Angelica dahurica cyclase gene AdDC and AdOD eukaryotic expression vectors and bacterial cultures The results obtained in Example 1 AdDC and AdOD The gene sequence was optimized for codons and synthesized by Shanghai Sangon Biotech. Using the Novizan ClonExpress II One Step Cloning Kit, the optimized gene was recombinated and ligated with the linearized yeast expression vector pYES2 / CT-Amp to construct the recombinant plasmid pYES2 / CT- AdDC and pYES2 / CT- AdOD Subsequently, the recombinant plasmid was transformed into *Saccharomyces cerevisiae* WAT11 competent cells (Shanghai Weidi Biotechnology Co., Ltd.), and screened on SD-Ura deficient medium to obtain positive recombinant yeast strains pYES2 / CT- AdDC -WAT11 and pYES2 / CT- AdOD-WAT11 is used for subsequent protein induction expression.

[0018] Example 3 Angelica dahurica cyclase gene AdDC and AdOD Plant overexpression vectors and bacterial cultures Using the Novizan ClonExpress II One Step Cloning Kit, AdDC and AdOD The gene was recombined with the linearized plant overexpression vector pCAMBIA2300-eGFP-Kana to construct the recombinant plasmid pCAMBIA2300- AdDC and pCAMBIA2300- AdOD The recombinant plasmids were introduced into GV3101 Agrobacterium competent cells (Shanghai Weidi Biotechnology Co., Ltd.) using a heat shock method. After resistance screening and PCR identification, strain pCAMBIA2300- was obtained, which can be used for plant transformation. AdDC -GV3101 and pCAMBIA2300- AdOD -GV3101.

[0019] Example 4 Angelica dahurica cyclase gene AdDC and AdOD In vitro functional verification (1) Protein induction expression and detection The recombinant yeast strain obtained in Example 2 was cultured in galactose-containing SC-URA induction medium to induce protein expression. Cells were collected, and microsomal proteins were extracted after cell lysis. Western blot analysis showed that the recombinant… AdDC and AdOD The proteins (molecular weights of 58.07 kDa and 55.60 kDa, respectively) were successfully expressed in yeast, while the empty vector control showed no band. Figure 4 ).

[0020] (2) In vitro catalytic activity analysis Using 7-demethylcorkol and crocin as substrates, respectively, with substances containing AdDC or AdOD The protein reaction mixture was subjected to an in vitro catalytic reaction, using empty vector microsomal protein as a negative control. The reaction products were analyzed by UPLC-MS / MS, and the results are shown below. Figure 5 .

[0021] Mass spectrometry EIC spectra show that, AdDC and AdOD In all reaction systems, a target ion peak with a mass-to-charge ratio (m / z) of 246.26 was detected, an increase of 16 Da compared to the substrate (m / z 230.26), consistent with the characteristics of an oxygenation cyclization reaction. Further identification confirmed... AdDC The protein can catalyze the cyclization of 7-demethylcorpin to generate isopyramidal lactone. AdOD The protein is a bifunctional enzyme that can catalyze the cyclization of 7-demethylcorpin and sennaol to produce isopyramidal lactone and dihydroaperitol.

[0022] (3) Detection was performed using UPLC-MS / MS. like Figure 6-8 As shown, a Waters ACQUITY UPLC HSS T3 2.1×100 mm×1.8 μm (LC-208) was used with acetonitrile (A) and 0.1% formic acid aqueous solution (B) as the mobile phase. The elution conditions were: 0 min: 0:100 (v / v), 6 min: 35:65 (v / v), 9 min: 60:40 (v / v), 13 min: 90:10 (v / v), 13.5 min: 90:10 (v / v), and 18 min: 0:100 (v / v). The ESI source conditions were: dry gas (N2) flow rate 8.0 L·min -1 Drying gas temperature 350℃, sheath gas temperature 350℃, sheath gas flow rate 11 L·min -1 Collision energy gradient: 10, 20, 40, 60 Ev; nozzle voltage: 4.0 kV; cone voltage: 100V; scanning mode: full scan; scanning range: m / z 50~1700; detection wavelength: 254 nm.

[0023] Example 5 Angelica dahurica cyclase gene AdDC and AdOD In vivo functional verification (1) Transient transformation and expression analysis of Angelica dahurica Angelica dahurica seedlings treated with empty vector infection solution were used as a control, and overexpressing bacterial solution pCAMBIA2300- was used as a control. AdDC -GV3101 and pCAMBIA2300- AdOD Agrobacterium GV3101 was used to treat Angelica dahurica leaves using a vacuum infection method for transient transformation. After successful transformation was confirmed by fluorescence microscopy, plant RNA was extracted for qRT-PCR analysis. The results are as follows: Figure 9 As shown in A and B, in overexpressing plants AdDC and AdOD Gene expression levels were significantly higher than those in the control group, at 39-fold and 23-fold respectively. P < 0.05).

[0024] (2) Determination of coumarin content by HPLC HPLC analysis showed that the contents of isopyram and dihydroaporphyrin in the control plants were 0.1420 mg / g.-1 and 0.3260 mg·g -1 In overexpression AdDC In the plants, the content of isopyram lactone increased to 0.3836 mg / g. -1 Overexpression AdOD In the plants, the content of dihydroaporphyrin increased to 0.7550 mg·g⁻¹. -1 The content of isopyramidal lactone was 0.3236 mg·g. -1 ( Figure 9 C, D).

[0025] The HPLC detection method is as follows: a Shimadzu LC-16 system (Shimadzu, Japan), 4.6 × 250 mm, 5 μM, Inertsil ODS-3 C18 column (Agilent, USA), column temperature 35℃, mobile phase flow rate 1.0 mL·min -1 The injection volume was 10 μL. The mobile phase was methanol (A)-acetonitrile (B)-0.2% phosphoric acid solution (C), and the elution gradient was as follows: 0 min: 0% A, 20% B, 80% C; 20 min: 2% A, 20% B, 78% C; 25 min: 5% A, 25% B, 70% C; 30 min: 5% A, 35% B, 60% C; 35 min: 5% A, 38% B, 57% C; 40 min: 5% A, 40% B, 55% C; 50 min: 5% A, 45% B, 50% C; 55 min: 5% A, 90% B, 5% C; 65 min: 5% A, 90% B, 5% C; 66 min: 0% A, 20% B, 80% C; 78 min: 0% A, 20% B, 80% C. The detection wavelength was 254 nm. The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. An Angelica dahurica cyclase gene, characterized in that, Including genes AdDC and genes AdOD The gene AdDC The nucleotide sequence of the gene is SEQ ID No. 1; AdOD The nucleotide sequence is SEQ ID No.

2.

2. The Angelica dahurica cyclase gene according to claim 1, characterized in that, The gene AdDC The encoded amino acid sequence is SEQ ID No. 3; the gene AdOD The encoded amino acid sequence is SEQ ID No.

4.

3. A method for constructing a eukaryotic expression vector of the Angelica dahurica cyclase gene as described in any one of claims 1 to 2, characterized in that, The specific steps include: The gene AdDC and / or genes AdOD Codon optimization and whole-genome synthesis were performed to obtain the optimized gene. AdDC and / or AdOD ; The optimized gene was recombinantly ligated with a eukaryotic expression vector to obtain the eukaryotic recombinant plasmid. The eukaryotic recombinant plasmid was transformed into competent cells of Saccharomyces cerevisiae, and positive recombinant yeast strains were obtained by screening on SD-Ura deficient medium.

4. The construction method according to claim 3, characterized in that, The eukaryotic expression vector includes pYES2 / CT-Amp; The eukaryotic recombinant plasmid includes pYES2 / CT- AdDC or pYES2 / CT- AdOD ; The brewing yeast includes brewing yeast WAT11; The positive recombinant yeast strains include pYES2 / CT- AdDC -WAT11 or pYES2 / CT- AdOD -WAT11.

5. A method for constructing a plant overexpression vector of the Angelica dahurica cyclase gene as described in any one of claims 1 to 2, characterized in that, The specific steps include: The gene AdDC and / or genes AdOD The plant overexpression vector was recombined with the linearized plant overexpression vector to construct a plant recombinant plasmid; The plant recombinant plasmids were introduced into Agrobacterium competent cells by heat shock, and strains for plant transformation were obtained after resistance screening and PCR identification.

6. The construction method according to claim 5, characterized in that, The plant overexpression vector includes pCAMBIA2300-eGFP-Kana; The plant recombinant plasmid includes pCAMBIA2300- AdDC or pCAMBIA2300- AdOD ; The Agrobacterium species includes Agrobacterium GV3101; The plant transformation strain includes pCAMBIA2300- AdDC -GV3101 or pCAMBIA2300- AdOD -GV3101.

7. The Angelica dahurica cyclase gene as described in claims 1-2 AdDC Application in the catalytic cyclization of 7-demethylcorpin to isopyramidal lactone.

8. The Angelica dahurica cyclase gene as described in claims 1-2 AdOD Applications in catalytic cyclization of 7-demethylcorpin to isopyramidal lactone and catalytic cyclization of sennaol to dihydroaperitol.

9. A method for preparing furanocoumarin compounds using the angelica cyclase gene, characterized in that, Includes the following steps: S1. Construct the gene AdDC and / or genes AdOD The eukaryotic expression vector or the plant overexpression vector; S2. Transform the eukaryotic expression vector or the plant overexpression vector constructed in S1 into host cells to obtain recombinant engineered bacteria or transgenic plants; S3. Induce the recombinant engineered bacteria or transgenic plant to express the [specific expression]. AdDC and / or AdOD Proteins, through a catalytic reaction using substrates, generate the furanocoumarin compounds described above; The furanocoumarin compounds include at least one of 7-demethylcorpin, sennaol, dihydroaperitol and isopyramidalin. The host cells include eukaryotic Saccharomyces cerevisiae and Agrobacterium.

10. The method according to claim 9, characterized in that, The detection of furanocoumarin compounds in step S4 was performed using UPLC-MS / MS. The UPLC-MS / MS method included: using a Waters ACQUITY UPLC HSS T3 column as the separation column, acetonitrile-0.1% formic acid aqueous solution as the mobile phase, and a gradient elution program as follows: Elution time was 0 min, and the volume ratio of the acetonitrile to the 0.1% formic acid solution was 0:

100. Elution was performed for 6 min, with the volume ratio of acetonitrile to 0.1% formic acid solution being 35:

65. Elution was performed for 9 min, with the volume ratio of acetonitrile to 0.1% formic acid solution being 60:

40. Elution was performed for 13 min, with the volume ratio of acetonitrile to 0.1% formic acid solution being 90:

10. Elution was performed for 13.5 min, with the volume ratio of acetonitrile to 0.1% formic acid solution being 90:

10. Elution was performed for 18 min, with the volume ratio of the acetonitrile to the 0.1% formic acid solution being 0:

100. Mass spectrometry conditions: ESI source, drying gas flow rate 8.0 L·min -1 Drying gas temperature 350℃, sheath gas temperature 350℃, sheath gas flow rate 11 L·min -1 The collision energy gradient is 10-60 eV, the nozzle voltage is 4.0 kV, the cone voltage is 100 V, the scanning mode is full scan, the full scan range is 50~1700 m / z, and the detection wavelength is 254 nm.

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