Three fad-dependent enzymes in mulberry and their application in synthesis of diels-alder adducts

Abstract

The application belongs to the field of genetic engineering, and relates to three FAD-dependent enzymes in Morus alba and application of the three FAD-dependent enzymes in synthesis of Diels-Alder adducts. Specifically, the three FAD-dependent enzymes MaFAD1, MaFAD2 and MaFAD3 in Morus alba and nucleic acid molecules of the coding proteins of the three FAD-dependent enzymes, expression vectors of the nucleic acid molecules and host cells containing the vectors, and a production method of the enzymes coded by the nucleic acid molecules are disclosed. The application also relates to application of the three FAD-dependent enzymes in catalyzing synthesis of Diels-Alder adducts such as chalcomoracin and kuwanon J in Morus alba, wherein MaFAD1 is an oxidase, MaFAD2 and MaFAD3 are both cyclases, and MaFAD1 catalyzes oxidation reaction to synthesize diene, and MaFAD2 and MaFAD3 catalyze [4+2] cyclization reaction to synthesize Diels-Alder adducts.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically involving novel nucleic acid molecules of three FAD-dependent enzymes—MaFAD1, MaFAD2, and MaFAD3—derived from mulberry and their encoded proteins, including expression vectors containing the nucleic acid molecules and host cells containing the vectors, as well as methods for producing enzymes encoded by the nucleic acid molecules; it also involves the application of these two types of enzymes in the catalytic synthesis of Diels-Alder adducts from mulberry, wherein MaFAD1 is an oxidase catalyzing the synthesis of dienes, and MaFAD2 and MaFAD3 are both cyclases catalyzing [4+2] cyclization reactions. Background Technology

[0002] Diels-Alder reactions, typically [4+2] cyclization reactions, are a class of highly efficient reactions capable of constructing complex polycyclic compounds. They are widely used in organic synthetic chemistry, particularly in the synthesis of medicinal natural products and pharmaceuticals, and are considered an important class of chemical reactions (Takao K., Munakata R., Tadano K. Recent advances in natural product synthesis by using intramolecular Diels-Alder reactions, Chemical Review, 2005, 105, 4779; Nicolaou, KC, Snyder, SA, Montagnon, T. & Vassilikogiannakis, G. The Diels-Alder reaction in total synthesis, Angewandte Chemie International Edition, 2002, 41, 1668). In recent years, with the development of molecular biology techniques, some intramolecular [4+2] cyclization-related enzymes have been obtained from microorganisms by knocking out genes on gene clusters related to the biosynthesis of secondary metabolites and combining them with heterologous expression. (Zanda M, et al. Enzyme-catalyzed [4+2]cycloaddition is a key step in the biosynthesis of spinosyn A, Synthesis, 2011, 19, 1134; Hashimoto T, Kuzuyama T. Mechanistic insights into Diels-Alder reactions in natural product biosynthesis, Current Opinion in Chemical Biology, 2016, 35, 123; Jeon BS, Wang SA, Ruszczycky MW, et al. Natural [4+2]-cyclases, Chemical Review, 2017, 117, 5388; Li L, Yu P, Tang MC, et al. Biochemical characterization of a eukaryotic decalin-forming) Diels-Alderase, Journal of the American Chemical Society, 2016, 138, 15840.).Most of these enzymes are intramolecular [4+2] cyclases and are derived from microorganisms, while intermolecular [4+2] cyclases derived from plants have not yet been reported.

[0003] Mulberry (Morus alba L.) is a plant belonging to the genus Morus in the family Moraceae. Its various parts are widely used not only as a traditional Chinese medicine in clinical practice, but also as an important economic crop in multiple fields. In the 1980s, Japanese scholar Taro Manuro conducted a systematic study on the chemical composition of mulberry root bark, discovering a series of Diels-Alder type adducts such as chalcomoracin and kuwanon J. From a biosynthetic perspective, the isopentenyl group in isopentenyl-substituted compounds may be oxidized by an oxidase to form a diene. This dienophile, along with chalcone or chalcone derivatives, may then form Diels-Alder adducts under the catalysis of an intermolecular [4+2] cyclase. However, the enzyme catalyzing this reaction and related genes have not yet been discovered, and the reaction mechanism remains unclear (Hirakura K., Hano Y., Fukai T., et al. Structures of three new natural Diels-Alder type adducts, kuwanons P and X, and mulberrofuran J, from the cultivated mulberry tree, Chemical & Pharmaceutical Bulletin, 1985, 33, 1096.). Related pharmacological studies have shown that these Diels-Alder adducts possess various activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, and cytotoxic activities. Due to the low content of these components in mulberry and limited resources, [further research is needed].While successful chemical synthesis has been reported, it lacks stereoselectivity. Therefore, efficient and stereoselective synthesis of this class of Diels-Alder adducts remains a challenging problem (Nomura, T.; Fukai, T. Prenylflavonoids from the root bark of the cultivated mulberry tree, Heterocycles, 1981, 15, 1531; Dai, SJ; Mi, ZM; Ma, ZB; Li, S.; Chen, RY; Yu, DQ, Bioactive Diels-Alder type adducts from the stem bark of Morus macroura, Planta Medica, 2004, 70, 758; Dai, S.-J.; Ma, Z.-B.; Wu, Y.; Chen, R.-Y.; Yu, D.-Q. Guangsangons FJ, Antioxidant and anti-inflammatory Diels-Alder type adducts, from Morus macroura). Miq., Phytochemistry, 2004, 65, 3135.).

[0004] Discovering enzymes and genes related to the biosynthesis of Diels-Alder adducts in mulberry plants and using genetic engineering technology to produce recombinant engineered enzymes for efficient catalytic synthesis is an effective technical method to solve the above problems.

[0005] In summary, current research on the synthesis of Diels-Alder adducts in mulberry plants faces the following limitations:

[0006] 1. The content of active Diels-Alder adducts in mulberry is low, chemical synthesis lacks selectivity, and the source is limited;

[0007] 2. The oxidases involved in the key diene formation and the cyclases involved in the intermolecular [4+2] cycloaddition reaction of the Diels-Alder adduct biosynthesis in mulberry have not been reported, and their related genes cannot be realized. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the technical problem solved by the present invention is to provide an oxidase for diene formation and a cyclase for intermolecular [4+2] cycloaddition reaction, which are key components in the biosynthesis of Diels-Alder adducts in mulberry, as well as their nucleic acid molecules, an expression vector containing the nucleic acid molecules and a host cell containing the vector, and a method for producing the oxidase that catalyzes diene formation and the cyclase that catalyzes the [4+2] cyclization reaction. The invention also relates to their application in the catalytic synthesis of Diels-Alder adducts in mulberry.

[0009] To solve the technical problem of this invention, the following technical solution is provided:

[0010] The first aspect of the present invention is to provide three FAD-dependent enzymes, MaFAD1, MaFAD2, and MaFAD3, wherein MaFAD1 is an oxidase catalyzing the formation of dienes, and MaFAD2 and MaFAD3 are both cyclases catalyzing intermolecular [4+2] cyclization reactions, and their amino acid sequences are as follows:

[0011] (1) The amino acid sequences of MaFAD1, MaFAD2 and MaFAD3 are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively;

[0012] (2) The amino acid sequences obtained by conserved mutation of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, that is, the amino acid sequences with equivalent functions formed by replacing, deleting or adding amino acids.

[0013] The three FAD-dependent enzymes MaFAD1, MaFAD2, and MaFAD3 mentioned above can be routinely modified; or the enzymes can be attached with tags for detection or purification.

[0014] The three FAD-dependent enzymes MaFAD1, MaFAD2, and MaFAD3 mentioned above are modified in the following ways: conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, and immobilization modification; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and ProfinityeXact.

[0015] The second aspect of the present invention is to provide nucleic acid molecules encoding three FAD-dependent enzymes, MaFAD1, MaFAD2 and MaFAD3, preferably the nucleic acid sequence shown in SEQ ID NO.4, named geneMaFAD1; the nucleic acid sequence shown in SEQ ID NO.5, named geneMaFAD2; and the nucleic acid sequence shown in SEQ ID NO.6, named geneMaFAD3.

[0016] A third aspect of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule described in the second aspect of the present invention. The nucleic acid molecule described in the second aspect of the present invention is ligated into the expression vector by enzyme digestion, preferably using the pPIC3.5K expression vector.

[0017] A fourth aspect of the present invention is to provide a host cell containing the recombinant expression vector described in the third aspect of the present invention. The host cell is selected from bacteria, yeast, plant cells, and animal cells, with Pichia pastoris being a preferred host cell.

[0018] The fifth aspect of the present invention is to provide a method for producing the three FAD-dependent enzymes MaFAD1, MaFAD2 and MaFAD3 described in the first aspect of the present invention, characterized in that host cells containing their gene sequences are cultured under suitable culture conditions to obtain cells containing MaFAD1, MaFAD2 and MaFAD3, crude enzyme solution or pure enzyme obtained by further extraction and purification.

[0019] The sixth aspect of this invention provides the application of the oxidase MaFAD1 described in the first aspect of this invention, combined with cyclases MaFAD2 and MaFAD3, in the preparation of structurally diverse and biologically active Diels-Alder compounds such as chalcomoracin and kuwanon J. The dienophilic precursor substrates include isopentenyl dibenzofuran compounds, isopentenyl chalcones, etc., with the dienophilic substrate being isopentenyl chalcones, etc.

[0020] The experiment involved the oxidation of moracin C by the oxidase MaFAD1 to form a diene, followed by a Diels-Alder reaction with the diephile moracalcone A under the catalysis of MaFAD2 or MaFAD3 cyclases. HPLC-MS analysis showed that MaFAD2 or MaFAD3 efficiently catalyzed the intermolecular [4+2] cyclization reaction to generate chalcomoracin. The oxidation of moracalcone A by MaFAD1 to form a diene, followed by a Diels-Alder reaction with the diephile moracalcone A under the enzymatic catalysis of MaFAD2 or MaFAD3, also showed that MaFAD2 or MaFAD3 efficiently catalyzed the intermolecular [4+2] cyclization reaction to generate kuwanon J. Currently, the oxidases that oxidize moracin C to form dienes and the cyclases that catalyze the intermolecular Diels-Alder reaction are not previously reported, making this novel approach. Furthermore, the products chalcomoracin and kuwanon J generated under enzymatic catalysis exhibit only the endo configuration, demonstrating strict stereoselectivity.

[0021] Beneficial technical effects:

[0022] 1. The three FAD-dependent enzymes MaFAD1, MaFAD2 and MaFAD3 described in this invention, including diene oxidase MaFAD1 and [4+2] cyclases MaFAD2 and MaFAD3, are previously unreported and can catalyze diene formation and [4+2] cyclization reactions, respectively.

[0023] 2. By using the diene oxidase MaFAD1 and [4+2] cyclases MaFAD2 and MaFAD3 described in this invention for combined catalysis, Diels-Alder adducts chalcomoracin and kuwanon J can be synthesized efficiently.

[0024] 3. The [4+2] cyclases MaFAD2 and MaFAD3 described in this invention have high stereoselectivity and can be developed into tool enzymes for catalyzing [4+2] cyclization reactions. They can be used to prepare optically and biologically active Diels-Alder compounds.

[0025] Supplementary Explanation

[0026] Unless otherwise stated, the technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. This invention may also be practiced using any methods and materials similar to or equivalent to those described herein. While specific embodiments and preferred methods and materials are described herein, they do not impose any limitation on the invention. Attached Figure Description

[0027] Figure 1 HPLC-MS analysis of the reaction of recombinant MaFAD1 and MaFAD2 or MaFAD3 proteins catalyzing the reaction of moracin C with morachalcone A[4+2].

[0028] Figure 2 HPLC-MS analysis of the reaction of recombinant MaFAD1 and MaFAD2 or MaFAD3 proteins catalyzing the moracalcone A[4+2] reaction. Detailed Implementation

[0029] The following embodiments are provided to further illustrate various aspects of the present invention. These embodiments are non-limiting and should not be construed as limiting any aspect of the invention. The scope of protection of the present invention is limited only by the claims. Various modifications and improvements can be made to various aspects of the present invention without departing from the scope of the claims, and these modifications and improvements also fall within the scope of protection of the present invention. For example, replacing the promoters and expression vectors used in the embodiments with other promoters and expression vectors commonly used in the art is something that those skilled in the art can understand and implement.

[0030] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0031] Example 1: Cloning of the geneMaFAD1, geneMaFAD2, and geneMaFAD3 genes

[0032] 1. Extraction of total RNA and synthesis of cDNA first strand

[0033] Select an appropriate amount of mulberry cell suspension as material, quickly transfer it to a mortar pre-cooled with liquid nitrogen, and grind it thoroughly into powder; transfer the powdered sample to a centrifuge tube, add 500 μL RCL buffer / 100 mg tissue (EZNA) TMPlant RNA kit: Homogenize using a vortex mixer and repeatedly pipette until well mixed; incubate at 55℃ for 3 min; centrifuge at 15,000×g for 5 min, transfer the supernatant to a filter column, and centrifuge at 14,000×g for 2 min. Add an equal volume of RCB buffer to the filtered liquid and invert 5-10 times. Transfer the liquid to an RNA adsorption column and centrifuge at 10,000×g for 1 min; discard the filtrate, add 400 μL of RWC elution buffer to the adsorption column, and centrifuge at 10,000×g for 1 min; allow the adsorption column to air dry at room temperature, and then elute the total RNA with 50 μL of ultrapure water. Detect the integrity of the total RNA using 1.0% non-denaturing agarose gel electrophoresis and measure the OD using a UV spectrophotometer. 260 / OD 280 Ratio and RNA concentration. Using SMARTer. TM RACE cDNA amplification kit (Clontech, USA) synthesizes cDNA.

[0034] 2. RT-PCR amplification of the target gene fragment

[0035] Based on the transcriptome information of mulberry suspension cells, specific primers (SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12) were designed for candidate genes geneMaFAD1, geneMaFAD2, and geneMaFAD3. PCR amplification was performed using KOD DNA polymerase to obtain the full-length genes geneMaFAD1, geneMaFAD2, and geneMaFAD3. The PCR products (target gene fragments) were recovered from the gel, ligated into the pEASY-blunt vector, transformed into Trans1-T1 competent cells, and screened using blue-white and colony PCR. Positive clones were cultured and sequenced to confirm the sequence information of the candidate genes.

[0036] 2.1 PCR reaction system:

[0037]

[0038] 2.2 PCR Cycle:

[0039] 1) 94℃ 2min; 2) 98℃ 10sec; 3) 58℃ 30sec; 4) 68℃ 1min;

[0040] 5) 98℃ for 10 seconds, 35 cycles; 6) 68℃ for 10 minutes; 7) Hold at 10℃.

[0041] 3. Comparison and analysis of the similarity between the cloned gene and the published FAD-dependent monooxygenase sequences.

[0042] The cloned gene was compared with the published FAD-dependent monooxygenase gene using NCBI (http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0043] 4. Bioinformatics analysis of proteins encoded by the geneMaFAD1, geneMaFAD2, and geneMaFAD3 genes.

[0044] The physicochemical properties of the target gene-encoded protein obtained from previous cloning were predicted using expasy (http: / / web.expasy.org / cgi-bin / protparam / protparam); the transmembrane region of the target gene-encoded protein was predicted using TMHMM Server v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ); and the amino acid sequence was compared and analyzed using NCBI (http: / / www.ncbi.nlm.nih.gov / BLAST / ) and Vector NTI.

[0045] Example 2: Expression of geneMaFAD1, geneMaFAD2, and geneMaFAD3 genes and isolation and purification of recombinant proteins

[0046] 1. Construction of the SMD1168-pPIC3.5K recombinant expression strain and induction of recombinant protein expression

[0047] Based on the gene sequences flanking the multiple cloning site of the pPIC3.5K vector, primers containing homologous arms of the vector were designed. Homologous arms were introduced into the N-terminus and C-terminus of the target gene coding sequence using PCR, and the PCR products were recovered via gel electrophoresis. The target gene was ligated to the linearized pPIC3.5K vector using homologous recombinase to construct a recombinant plasmid. This plasmid was transformed into *E. coli* (Top10) cloning hosts, and positive transformants were screened by PCR. Sequencing was then used to verify the correctness of the reading frame. Transformants with correct sequencing were amplified, cultured, and the plasmid was extracted, linearized, and transformed into *Pichia pastoris* competent cells. Positive clones were screened by PCR. Positive transformants were stored in 15% glycerol at –80°C.

[0048] The steps for inducing recombinant protein expression, crude enzyme extraction, and detection are as follows: 1) Strand the positive transformants with correct sequences as identified by sequencing onto fresh MD solid plates and activate them at 30℃ for 1–2 days; 2) Inoculate the activated recombinant Pichia pastoris onto BMGY liquid medium (1% yeast extract, 2% protein powder, 100mM phosphate buffer pH 6.0, 1.34% YNB, 4×10⁻⁶ oz.) from the surface of the MD plate.-5 1% biotin, 1% glycerol), incubate overnight at 30°C and 250 rpm until OD reaches zero. 600 ≈0.6; collect cells by centrifugation at 3000 rpm for 5 min; 3) transfer to mBMMY liquid medium (modified BMMY, 1% yeast extract, 2% protein powder, 100 mM citrate buffer pH 5.5, 1.34% YNB, 4 × 10 -5 The culture medium was induced at 20°C and 90 rpm with 1% biotin, 0.5% casein extract, 1% methanol, and 10 mg / L riboflavin. Methanol was added to the medium every 24 hours to bring the final concentration to 1%, for a total of 6 days. After induction, the medium and cells were separated by centrifugation at 10,000 × g for 5 min. The supernatant was the crude enzyme solution. The supernatant was directly added to a 15 mL ultrafiltration tube. Ultra-15 (30K) 4000×g centrifugation concentration 10 times.

[0049] 2. Isolation and purification of recombinant proteins MaFAD1, MaFAD2, and MaFAD3

[0050] Protein isolation and purification were performed using recombinant expression strains. The target gene was expressed by fusion with 6×His·tag, therefore Ni Sepharose was used. TM The following steps are used to separate and purify His-tagged fusion proteins using 6-Fast Flow resin (GE Healthcare) affinity chromatography columns and PD-10 desalting columns (GE Healthcare):

[0051] 2.1 Preparation of buffer solution:

[0052] 1) Loading buffer: 20mM phosphate buffer, 500mM NaCl, 20mM imidazole, pH 7.2;

[0053] 2) Elution buffer: 20mM phosphate buffer, 500mM NaCl, 500mM imidazole, pH 7.2;

[0054] 3) Desalting buffer: 20mM Tris-HCl.

[0055] 2.2, Ni Sepharose TM Pretreatment of 6-Fast Flow resin affinity chromatography column:

[0056] According to 1 mL Ni Sepharose TMFor 6-Fast Flow resin column bed, select an appropriate size affinity chromatography column based on the proportion of protein expressed by 2L recombinant strain culture medium. Elute sequentially with 10 column volumes of ultrapure water, loading buffer, and elution buffer. Finally, equilibrate the chromatography column with loading buffer.

[0057] 2.3 Preparation and loading of crude extracts of recombinant proteins MaFAD1, MaFAD2, and MaFAD3

[0058] The recombinant strain culture and protein induction expression steps were the same as in Example 1. The recombinant bacteria were centrifuged at 4°C and 6,000×g to separate the supernatant and bacterial cells. The supernatant was directly added to a 15mL ultrafiltration tube. Concentrate the sample 10-fold by centrifugation at 4000×g (Ultra-15, 30K), and mix it with loading buffer at a volume ratio of 1:10. Filter the mixture through a 0.45 μm filter membrane and load it into an affinity chromatography column at a flow rate of 1 mL / min.

[0059] 2.4 Elution of recombinant proteins MaFAD1, MaFAD2, and MaFAD3

[0060] Elute contaminating proteins with 10 column volumes of loading buffer at a flow rate of 1 mL / min. Elute proteins not tightly bound to the column bed. Once the absorbance of the eluent at 280 nm approaches 0, elute the target protein. Prepare gradient elution buffers with different imidazole concentrations for elution of the target proteins MaFAD1, MaFAD2, and MaFAD3 at concentrations of 20 mM, 50 mM, 100 mM, and 200 mM. Elute with elution buffer for each concentration gradient, using 5 column volumes. Analyze the fractions using SDS-PAGE. Concentrate the high-purity fraction containing the target protein to 1 / 10 of its original volume using ultrafiltration centrifuge tubes (30 kDa, Millipore).

[0061] 2.5 Desalting of recombinant proteins MaFAD1, MaFAD2, and MaFAD3

[0062] The PD-10 Desalting Column was equilibrated with 10 column volumes of pre-cooled ultrapure water and desalting buffer. The concentrated protein solution obtained from ultrafiltration was slowly loaded onto the upper layer of the desalting column. After the sample had completely entered the column, it was eluted with desalting buffer and sample collection began. Each 1 mL fraction was collected for denaturing gel electrophoresis (SDS-PAGE). According to the elution principle of the PD-10 desalting column, the fractions were combined, diluted to an appropriate volume with desalting buffer, and the protein content was determined.

[0063] Example 3: Synthesis of chalcomoracin and kuwanon J adducts catalyzed by recombinant protein combination

[0064] The induced expression and purification of the recombinant protein were as described in Example 2. moracin C was oxidized by the dienophile oxidase MaFAD1 to generate a diene, which then reacted with the dienophile morachalcone A in a Diels-Alder reaction catalyzed by the cyclases MaFAD2 or MaFAD3. HPLC-MS analysis revealed that MaFAD2 or MaFAD3 efficiently catalyzed the intermolecular [4+2] cyclization reaction to generate chalcomoracin. morachalcone A could also be oxidized by the oxidase MaFAD1 to generate a diene, which then reacted with the dienophile morachalcone A in a Diels-Alder reaction catalyzed by the cyclases MaFAD2 or MaFAD3. HPLC-MS analysis revealed that… Figure 1-2 MaFAD2 or MaFAD3 can efficiently catalyze intermolecular [4+2] cyclization reactions to generate kuwanon J. The standard enzymatic catalytic reaction system for the synthesis of Diels-Alder compounds catalyzed by recombinant proteins is as follows: 10 mM moracin C or moracalcone A (dienophile precursor), 10 mM moracalcone A (dienophile), 0.02 μg each of recombinant MaFAD1 and MaFAD2 (or MaFAD3) proteins, total reaction volume 100 μL, reaction buffer (20 mM Tris-HCl, pH 8.0), reaction in a 30°C water bath for 10 min, 200 μL of methanol to terminate the reaction, centrifuge at 15,000 × g for 30 min, and the supernatant is collected for HPLC-MS analysis. The results showed that the combined recombinant protein MaFAD1 could catalyze the formation of dienophiles from the aforementioned diene precursors, and MaFAD2 and MaFAD3 could further catalyze the formation of Diels-Alder adducts chalcomoracin and kuwanon J from the diene and dienophiles.

[0065] Example 4: Preparation of Diels-Alder adduct chalcomoracin using recombinant MaFAD1 with MaFAD2 or MaFAD3

[0066]

[0067] 1. Recombinant protein amplification enzymatic reaction

[0068] Recombinant protein amplification enzymatic reaction conditions: 20 mL reaction system, 10 mM moracin C (diene precursor), 10 mM morachalcone A (dieneophile), containing 0.02 mg each of recombinant MaFAD1 and MaFAD2 (or MaFAD3) proteins, reaction buffer (20 mM Tris-HCl, pH 8.0), reaction at 30℃ for 30 min, the conversion rate can reach 100%. The reaction solution was directly evaporated to dryness under reduced pressure, resuspended in methanol, filtered through a 0.45 μm membrane, and separated by semi-preparative HPLC.

[0069] 2. Isolation, purification, and structural identification of Chalcomoracin, a Diels-Alder adduct product

[0070] The reaction products were separated and purified by semi-preparative HPLC. The HPLC system used a Shiseido MGIII C18 reversed-phase column (250 mm × 10.0 mm ID, 5 μm). The separated reaction products were further purified by... 1 H NMR, 13 The structure was identified as chalcomoracin by C10 NMR and MS. The product, chalcomoracin, showed only the endo configuration, indicating strict stereoselectivity of the enzymatic reaction. MS and NMR data of the product are as follows:

[0071] Chalcomoracin: ESI-MS m / z 647.54 [MH] - ; 1H NMR(Acetone-d6,600MHz)δ:8.44(1H,d,J=9.0Hz,H-14″),7.34(1H,d,J=8.4Hz,H-4),6.98(1H,d,J=8.4Hz,H-20″),6.93(1H,s,H-7),6.92(1H,s,H-3),6.76(2H,s,H-2′,H-6′),6.75(1H,dd,J=8.4,2.4Hz,H-5),6.52(1H,d,J=2.4Hz,H-17″),6.46(1H,d,J=9.0Hz,H-13″),6.31(1H,dd,J=8.4,2.0Hz,H-19″),5.77(1H,br s,H-2″),5.16(1H,t,J=7.2Hz,H-22″),4.65(1H,t,J=4.8Hz,H-4″),4.10(1H,br s,H-3″),3.75(1H,br s,H-5″),3.25(2H,d,J=7.2Hz,H-21″),2.48(1H,m,H-6″),2.11(1H,m,H-6″),1.94(3H,s,H-7″),1.71(3H,s,H-24″),1.57(3H,s,H-25″); 13 C NMR(Acetone-d6,150MHz)δ:155.4(C-2),101.9(C-3),121.9(C-4),113.2(C-5),156.6(C-6),98.4(C-7),122.7(C-3a),156.7(C-7a),131.6(C-1′),103.5(C-2′),157.9(C-3′,C-5′),116.6(C-4′),104.9(C-6′),133.8(C-1″),124.4(C-2″),33.2(C-3″),47.8(C-4″),36.6(C-5″),32.2(C-6″),23.9(C-7″),209.9(C-8″),113.2(C-9″),164.7(C-10″),116.0(C-11″),163.3(C-12″),108.2(C-13″),132.2(C-14″),122.7(C-15″),157.9(C-16″),103.6(C-17″),157.8(C-18″),107.6(C-19″),128.8(C-20″),22.2(C-21″),123.2(C-22″),131.8(C-23″),17.9(C-24″),25.9(C-25″).

[0072] Example 5: Preparation of Diels-Alder adduct Kuwanon J using MaFAD1 and MaFAD2 or MaFAD3

[0073]

[0074] Recombinant protein amplification enzymatic reaction conditions: 20 mL reaction system, 20 mM moracalcone A (as dienophile precursor and dienophile substrate respectively), containing 0.02 mg each of recombinant MaFAD1 and MaFAD2 (or MaFAD3) proteins, reaction buffer (20 mM Tris-HCl, pH 8.0), reaction at 30℃ for 30 min, the conversion rate can reach 70%. The separation, purification and structural identification steps of the Diels-Alder adduct product Kuwanon J are the same as those described in Example 4. The reaction product was separated and purified by semi-preparative HPLC, and the separated reaction product was further processed... 1 HNMR, 13 The structure was identified as kuwanon J by C NMR and MS. The product, kuwanon J, has only the endo configuration, indicating strict stereoselectivity in the enzymatic reaction. The MS and NMR data of the product are as follows:

[0075] Kuwanon J: (c 0.03,MeOH); ESI-MS m / z 677.54[MH] - ; 1H NMR(Acetone-d6,600 MHz)δ:14.37(1H,s),12.88(1H,s),8.39(1H,d,J=9.0 Hz,H-14″),8.15(1H,d,J=15.6 Hz,H-β),7.85(1H,d,J=9.0 Hz,H-6′),7.72(1H,d,J=15.6 Hz,H-α),7.66(1H,d,J=9.0 Hz,H-6),6.98(1H,d,J=8.0 Hz,H-20″),6.53(1H,d,J=2.4Hz,H-17″),6.48(1H,d,J=2.4 Hz,H-3),6.43(1H,d,J=9.0 Hz,H-5′),6.43(1H,dd,J=2.4,9.0Hz,H-5),6.36(1H,d,J=9.0 Hz,H-13″),6.32(1H,dd,J=2.4,8.4 Hz,H-19″),5.68(1H,s,H-2″),5.16(1H,t,J=7.2 Hz,H-22″),4.67(1H,t,J=4.0 Hz,H-4″),4.14(1H br s,H-3″),3.78(1H br s,H-5″),3.26(2H,d,J=7.2 Hz,H-21″),2.53(1H,br d,J=18.0 Hz,H-6″),2.25(1H,br d,J=18.0 Hz,H-6″),1.92(3H,s,H-7″),1.71(3H,s,H-24″),1.58(3H,s,H-25″); 13C NMR(Acetone-d6,150 MHz)δ:114.4(C-1),160.7(C-2),108.0(C-3),162.7(C-4),103.7(C-5),132.3(C-6),117.8(C-α),141.9(C-β),115.6(C-1′),163.9(C-2′) ,116.7(C-3′),165.8(C-4′),110.0(C-5′),131.0(C-6′),134.7(C-1 ″),123.9(C-2″),33.3(C-3″,C-6″),47.9(C-4″),36.7(C-5″),23.8(C -7″),209.8(C-8″),113.9(C-9″),164.5(C-10″),116.1(C-11″),163 .9(C-12″),109.0(C-13″),132.3(C-14″),122.9(C-15″),157.0(C-16 ″),103.5(C-17″),157.8(C-18″),107.3(C-19″),129.0(C-20″),22. 4(C-21″), 123.6(C-22″), 131.8(C-23″), 17.9(C-24″), 25.9(C-25″). sequence list <110> Institute of Materia Medica, Chinese Academy of Medical Sciences <120> Three FAD-dependent enzymes from mulberry and their application in the synthesis of Diels-Alder adducts <160> 12 <170> SIPOSequenceListing 1.0 <210> 2 <211> 547 <212> PRT <213> Morus alba <400> 2 Met Lys Tyr Phe Ala Leu Ser Ser Leu Phe Ala Lys Ile Leu Ile Leu 1 5 10 15 Leu Phe Ser Thr Ser Leu Ala Asp Leu Glu His Thr Arg Glu Asp Phe 20 25 30 Leu Gln Cys Leu Val Thr Arg Leu Ser Pro Ser Thr Tyr Asn Pro Gln 35 40 45 Pro Ile Ile Tyr Thr Pro Asn Asn Ser Ser Tyr Tyr Ser Val Leu Asn 50 55 60 Ser Ser Ile Gln Asn Arg Arg Phe Ser Ser Pro Phe Thr Pro Lys Pro 65 70 75 80 Tyr Val Ile Val Thr Pro Phe Thr Pro Thr His Val Gln Asn Thr Val 85 90 95 Tyr Cys Ser Glu Lys His Gly Met Gln Ile Arg Thr Arg Ser Gly Gly 100 105 110 His Asp Tyr Glu Gly Leu Ser Tyr Val Ser Asn Val Pro Phe Val Val 115 120 125 Ile Asp Leu Arg Asn Leu Ser Ser Ile Ser Val Asp Val Glu Ser Lys 130 135 140 Tyr Ala Trp Val Gln Ala Gly Ala Thr Leu Gly Glu Leu Tyr Tyr Arg 145 150 155 160 Ile Gly Glu Lys Thr Gly Asn Leu Gly Phe Pro Ala Gly Asp Cys His 165 170 175 Thr Val Gly Val Gly Gly Gln Ile Gly Gly Gly Gly Tyr Gly Tyr Leu 180 185 190 Thr Arg Lys Tyr Gly Leu Ala Ala Asp Asn Ile Leu Asp Ala Lys Leu 195 200 205 Ile Asp Ala Lys Gly Arg Val Leu Asp Arg Lys Ser Met Gly Val Asp 210 215 220 Leu Phe Trp Ala Ile Arg Gly Gly Gly Pro Ser Ser Phe Gly Val Val 225 230 235 240 Leu Ala Trp Lys Leu Arg Leu Val Pro Val Pro Pro Thr Val Thr Val 245 250 255 Phe Asp Val Arg Arg Asn Met Glu Asp Asn Ala Thr Arg Lys Phe Val 260 265 270 His Gln Trp Gln Leu Arg Ala Asn Lys Val Asp Asp Asp Leu Thr Ile 275 280 285 Tyr Ile Thr Phe Leu Ser Ser Ser Ser Ile Asp Lys Glu Gly Asn Lys 290 295 300 Lys Ile Ile Ile Glu Ala Ser Val Arg Ala Thr Tyr His Gly Gly Ile 305 310 315 320 Asp Lys Leu Leu Gln Leu Met Gln Glu Glu Phe Pro Glu Leu Gly Leu 325 330 335 Leu Arg Gln Glu Cys Thr Glu Met Arg Trp Ala Glu Ser Phe Leu Tyr 340 345 350 Phe Asn Leu Phe Arg Asn Gly Glu Ser Leu Asp Ala Leu Leu Ser Arg 355 360 365 Asn Ser Tyr Phe Asn Leu Ser Ser Phe Lys Ala Lys Ser Asp Tyr Val 370 375 380 Thr Lys Pro Ile Pro Asp Asp Val Leu Glu Glu Met Leu Gly Arg Leu 385 390 395 400 Leu Glu Glu Glu Val Gly Gln Ala Arg Ile Asp Val Phe Pro Tyr Gly 405 410 415 Gly Lys Met Asp Glu Phe Ser Glu Ser Thr Ile Pro Phe Pro His Arg 420 425 430 Ile Gly Asn Leu Tyr Met Ile His Tyr Leu Val Asn Trp Gln Glu Glu 435 440 445 Gly Asn Leu Thr Ala Ser Glu Lys His Ile Thr Trp Val Arg Arg Leu 450 455 460 Tyr Asn Tyr Met Thr Pro Tyr Val Ser Lys Asn Pro Arg Val Thr Tyr 465 470 475 480 Leu Asn Phe Arg Asp Leu Asp Ile Gly Met Asn Glu Asn Glu Asp Gly 485 490 495 Ala Thr Ser Thr Leu Asp Asn Ile Ala Arg Ala Arg Ile Trp Gly Arg 500 505 510 Lys Tyr Phe Lys Asn Asn Phe Asn Lys Leu Val Ser Val Lys Thr Ile 515 520 525 Val Asp Pro Thr Asn Phe Phe Thr Asp Glu Gln Ser Ile Pro Pro Ile 530 535 540 Val Met His 545 <210> 2 <211> 550 <212> PRT <213> Mulberry (Morus alba) <400> 2 Met Lys Tyr Phe Ser Leu Ser Leu Ser Phe Ala Lys Ile Ser Ile Phe 1 5 10 15 Leu Phe Ser Phe Val Leu Val Ala Ser Ala Asp Gln Ile Gly His Glu 20 25 30 Gly Phe Leu Lys Cys Leu Ile Thr Arg Ile Ser Lys Ser Asn Ser Thr 35 40 45 Ser Thr Ser Glu Ser Ile Ile Tyr Thr Gln Asn Asn Pro Ser Tyr Ser 50 55 60 Thr Ile Leu Thr Ser Thr Met Gln Asn Pro Arg Phe Leu Ser Leu Pro 65 70 75 80 Ile Pro Lys Pro Phe Val Ile Val Thr Pro Leu His Val Ser His Val 85 90 95 Gln Ala Thr Leu Tyr Cys Ala Lys Lys His Asp Ile Gln Ile Arg Ile 100 105 110 Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser Tyr Met Ser Asn Val 115 120 125 Thr Phe Val Ile Leu Asp Leu Arg Asn Leu Ser Ser Ile Asn Ile Asp 130 135 140 Val Lys Arg Lys Ser Ala Trp Val Gln Ser Gly Ala Thr Ile Gly Glu 145 150 155 160 Leu Tyr Tyr Arg Ile Ala Glu Lys Ser Leu Ser Leu Ala Phe Pro Gly 165 170 175 Gly Leu Gly His Thr Ile Gly Val Gly Gly Gln Leu Gly Gly Gly Gly 180 185 190 Tyr Gly Tyr Ser Thr Arg Lys Tyr Gly Leu Ala Ser Asp Asn Ile Ile 195 200 205 Asp Ala Gln Phe Met Asp Val Gln Gly Arg Ile Leu Asn Arg Lys Ser 210 215 220 Met Gly Glu Asp Leu Phe Trp Ala Ile Arg Gly Gly Gly Ala Gly Ser 225 230 235 240 Phe Gly Ile Val Leu Ala Trp Lys Ile Arg Leu Val Asp Val Pro Thr 245 250 255 Thr Val Thr Val Phe Glu Ala Val Arg Lys Trp Glu Asn Asn Ala Thr 260 265 270 Lys Lys Phe Val His Arg Tyr Gln Arg Arg Ile Ala Asp Ile Asp Lys 275 280 285 Asp Leu Thr Ile Phe Leu Gly Phe Gln Thr Ala Asn Thr Gly Asp Glu 290 295 300 Gln Gly Asn Thr Lys Ile Glu Val Leu Ala Val Ile Ser Ala Thr Phe 305 310 315 320 His Gly Ser Gln Asp Lys Val Leu Pro Leu Met Gln Lys Glu Phe Pro 325 330 335 Glu Leu Gly Leu Leu Lys Glu Glu Cys Ile Glu Met Pro Trp Val Arg 340 345 350 Ser Ile Met His Tyr Asn Phe Phe Arg Asn Gly Glu Pro Leu Glu Val 355 360 365 Leu Leu Asn Arg Thr Leu Asn Phe Glu Met Lys Ala Phe Lys Leu Lys 370 375 380 Ser Asp Tyr Val Lys Glu Pro Ile Pro Asp Asp Val Leu Glu Lys Leu 385 390 395 400 Leu Gly Lys Leu Tyr Glu Glu Glu Ile Gly Glu Gly Tyr Ile Glu Leu 405 410 415 Phe Pro Tyr Gly Gly Lys Met Asn Glu Ile Ser Glu Ser Glu Ile Pro 420 425 430 Phe Pro His Arg Ala Gly Asn Leu Tyr Asn Leu Arg Tyr Leu Val Ser 435 440 445 Trp Ile Asp Asp Gly Asn Ile Thr Arg Thr Asn Glu His Ile Arg Trp 450 455 460 Val Arg Ser Ala Tyr Asp Tyr Met Thr Pro Phe Val Ser Lys Asn Pro 465 470 475 480 Arg Gly Ala Tyr Leu Asn Phe Arg Asp Leu Asp Ile Gly Ile Asn Ser 485 490 495 Asp Glu Asp Asp Tyr Asn Tyr Val Ala Gln Ala Ser Ile Trp Gly Thr 500 505 510 Lys Tyr Phe Lys Ser Asn Phe Tyr Arg Leu Val Tyr Val Lys Thr Leu 515 520 525 Val Asp Pro Thr Asn Phe Phe Thr Tyr Glu Gln Ser Ile Pro Pro Leu 530 535 540 Ser Pro His Tyr Lys Arg 545 550 <210> 3 <211> 539 <212> PRT <213> Mulberry (Morus alba) <400> 3 Met Lys Ser Ser Phe Val Phe Ala Lys Ile Ala Ile Leu Leu Phe Ser 1 5 10 15 Leu Val Leu Leu Ala Ser Ala Asn His Thr His Glu Glu Phe Leu Gln 20 25 30 Cys Leu Ser Ser Arg Ile Pro Lys Ser Ile Ile Tyr Ala Ser Asn Asn 35 40 45 Pro Ser Tyr Ser Asn Val Leu Asp Ser Thr Thr Gln Asn Pro Arg Phe 50 55 60 Leu Ser Ser Ser Thr Arg Asn Pro Ser Val Ile Val Thr Pro Phe Lys 65 70 75 80 Ile Ser His Ile Gln Pro Thr Ile Tyr Cys Ser Lys Lys His Gly Val 85 90 95 Gln Ile Arg Ile Arg Ser Gly Gly His Asp Tyr Glu Gly Leu Ser Tyr 100 105 110 Gln Ser Ser Val Pro Phe Phe Ile Leu Asp Leu Arg Asn Ile Asn Ser 115 120 125 Ile Gln Val Asp Val Glu Lys Lys Ser Ala Trp Val Glu Ala Gly Ala 130 135 140 Thr Leu Gly Glu Leu Tyr Tyr Ser Ile Ala Lys Lys Ser Lys Thr Leu 145 150 155 160 Gly Phe Pro Gly Gly Leu Cys Ser Thr Val Gly Val Gly Gly Gln Leu 165 170 175 Gly Gly Gly Gly Tyr Gly Tyr Gln Ser Arg Thr Tyr Gly Leu Ala Ser 180 185 190 Asp Asn Ile Ile Asp Ala Gln Leu Ile Asp Ala Arg Gly Arg Ile Leu 195 200 205 Asn Arg Lys Ser Met Gly Glu Asp Leu Phe Trp Ala Ile Arg Gly Gly 210 215 220 Gly Ala Gly Ser Phe Gly Ile Val Ile Ala Trp Lys Val Arg Leu Ile 225 230 235 240 Asp Val Pro Ser Thr Val Thr Val Phe Glu Thr Val Arg Met Trp Glu 245 250 255 Asp Asn Val Thr Lys Lys Phe Val His Arg Tyr Gln Arg Arg Ala Ser 260 265 270 Asn Ile Asp Lys Asp Leu Thr Ile Phe Leu Gly Phe Arg Thr Thr Asn 275 280 285 Thr Ser Asp Glu Gln Gly Asn Ser Lys Ile Gln Ile Ile Thr Ile Ile 290 295 300 Ser Ala Thr Phe His Gly Ser Arg Asp Arg Leu Leu Pro Leu Met Gln 305 310 315 320 Glu Glu Phe Pro Glu Leu Gly Leu Gly Lys Glu Asp Phe Lys Glu Met 325 330 335 Ser Trp Val Gln Ser Ile Val His Tyr Asn Asn Tyr Lys Asp Asp Asp 340 345 350 Pro Leu Glu Val Leu Leu Asn Lys Thr Val Asn Phe Glu Pro Asn Pro 355 360 365 Phe Lys Leu Lys Ser Asp Tyr Val Lys Lys Pro Ile Pro Asp Asp Val 370 375 380 Leu Glu Lys Leu Leu Ala Arg Leu Tyr Glu Glu Asp Ile Gly Tyr Asp 385 390 395 400 Phe Val Glu Phe Phe Pro Tyr Gly Gly Lys Leu Ser Glu Ile Ser Glu 405 410 415 Ser Glu Ile Pro Phe Pro His Arg Ala Gly Asn Leu Tyr Asn Leu Arg 420 425 430 Tyr Met Ala Ser Trp Lys Gln Gly Glu Asn Thr Thr Arg Ile Asn Asn 435 440 445 His Leu Ser Trp Val Arg Ser Val Tyr Asp Ser Met Thr Pro Tyr Val 450 455 460 Ser Lys Asn Pro Arg Gly Ala Tyr Leu Asn Phe Arg Asp Leu Asp Ile 465 470 475 480 Gly Val Asn Pro Asn Glu Ser Asp Thr Thr Ser Ala Tyr Asn Tyr Val 485 490 495 Lys Gln Ala Ser Val Trp Gly Thr Lys Tyr Phe Lys Asn Asn Phe Tyr 500 505 510 Lys Met Val Phe Ile Lys Thr Leu Val Asp Pro Thr Asn Phe Phe Thr 515 520 525 Tyr Glu Gln Ser Ile Pro Pro Ile Leu His His 530 535 <210> 4 <211> 1644 <212> DNA <213> Mulberry (Morus alba) <400> 4 atgaagtact ttgcattgtc ttcattattt gccaaaattt tgatccttct tttttcaact 60 tcgttagcag atttggaaca cactcgtgaa gactttcttc agtgtcttgt gacacgtttg 120 tctccttcta cctacaatcc ccaacccata atctacactc caaataattc atcgtattac 180 agtgtactaa attcatccat acaaaaccgt cgcttttctt ctcctttcac cccaaaacca 240 tatgttatcg ttacaccatt tactcccact catgttcaaa atactgttta ctgctccgag 300 aaacatggca tgcaaatcag aacacggagc ggtggccatg attatgaggg cctttcatat 360 gtgtccaatg ttccgtttgt tgtaattgac ttgagaaacc tagctcgat tagcgtagat 420 gtggagagca aatatgcatg ggttcaagct ggagctacgc ttggtgaact ttattataga 480 attggagaga aaactggaaa tcttggcttc ccagctggcg attgccacac tgtgggtgtt 540 ggtggacaga tcggtggagg aggctatggc tatttgacac gaaaatatg cctcgcagct 600 gataatattc ttgatgcgaa gttgatagat gctaaaggaa gagttcttga taggaaatcc 660 atgggggtag atttgttttg ggctatacgt ggtggtggac cgtcaagctt tggagttgtt 720 cttgcatgga aacttcgatt agttccggtg ccaccgacag tgactgtgtt tgatgttagg 780 aggaacatgg aagacaatgc aacaagaaag ttcgttcatc aatggcaact tcgtgctaac 840 aaagtcgatg acgatctaac aatctacatc acattcctga gtagcagttc tattgataaa 900 gaagggaata agaaaattat cattgaagct tctgttcgag ccacttatca tggtggtatt 960 gataagctcc ttcaattaat gcaagaggaa tttcctgagt taggtttgct aagacaagag 1020 tgcactgaaa tgagatgggc tgaatccttt ctatattca atttattcag aaatggagaa 1080 tccttagatg ctttacttag taggaattct tatttcaatt tgtcatcgtt caaagcaaag 1140 tctgattatg tgacaaagcc gattccagat gacgtgttag aggaaatgtt aggaaggttg 1200 cttgaagaag aagtaggaca agctcggatt gatgtatttc cttacggtgg aaaaatggac 1260 gagttttcag aatccacaat cccatttccc caccgaattg gaaacctcta tatgatacat 1320 tacctcgtga attggcaaga agaaggaaat cttacggcgt ccgaaaaaca tataacttgg 1380 gtaagaaggc tttacaatta catgactcct tatgtgtcaa aaaatccgag ggttacatat 1440 ctcaacttta gagatcttga cattgggatg aatgaaaatg aggatggcgc aacaagtacc 1500 cttgataata ttgcaagagc aagaatctgg ggtaggaagt atttcaagaa taatttcaac 1560 aagttggtta gtgtaaagac tatagtggat ccaactaact tctttacaga tgaacaaagc 1620 atcccaccga tcgttatgca ttaa 1644 <210> 5 <211> 1653 <212> DNA <213> Morus alba <400> 5 atgaagtact tttccttatc tttatcgttt gccaaaattt ccatctttct tttttcattt 60 gtattggtag cttcagctga tcaaattggt catgaaggct ttcttaagtg cctgatcact 120 cgtatatcca aatccaactc tacctccact tctgaatcca ttatctacac tcaaataat 180 ccctcttatt caactatatt gacttcaacg atgcagaatc ctcgttttct ttctcttcca 240 atcccaaaac cattcgttat cgtaacacca ttacatgtct cccacgtcca agccactctt 300 tactgcgcca agaaacatga catacaaatc agaatccgaa gtggtggcca tgattacgag 360 ggcctttctt atatgtctaa tgtcactttt gtcatacttg acttgagaaa cttaagttct 420 attaacattg acgtgaagag gaagtctgca tgggttcagt ccggagcaac cattggcgaa 480 ctttattata ggattgctga gaaaagccta agtcttgcct tccctggagg gcttggccac 540 actattggtg ttggagaca gttaggtgga ggaggctatg gctattcgac gcgaaagtac 600 gggctcgcat ctgataatat tattgacgcc caatttatgg acgtgcaagg aagaattctc 660 aatcggaaat ctatggggga agatttgttt tgggccatac gcggtggtgg agctgggaagc 720 ttcggaattg ttctcgcctg gaaaatccga ctggtggacg tgcctacgac agtgaccgta 780 tttgaagccg taaggaagtg ggaaaacaat gcaacaaaga agtttgttca tcggtatcaa 840 cgccgtattg ccgacatcga taaggatcta actatctttc ttggattcca aactgcgaat 900 actggcgatg aacaagggaa cacgaaaatt gaagtattag ctgtcatctc agcaacattt 960 cacggcagtc aagataaggt ccttccattg atgcagaagg agtttcccga gttgggtttg 1020 cttaaagaag aatgcataga aatgccgtgg gtccgatcca ttatgcatta caactttttc 1080 cgaaacggag agcccttaga agttctactc aatagaacac ttaatttcga gatgaaggct 1140 ttcaaattga aatctgacta cgtgaaagag cctattccag atgacgtgtt ggaaaaattg 1200 ttgggcaagt tgtatgagga agaaatagga gaaggttaca ttgaactttt tccttatgga 1260 gggaagatga atgagatttc agaatctgaa attccgttcc cacatcgagc tgggaacctc 1320 tacaaccttc ggtacttggt gtcatggata gacgatggaa atattacgag aaccaacgag 1380 catattcgct gggtaagaag tgcttacgat tacatgactc cttttgtttc aaagaatcct 1440 aggggtgcgt atctcaactt cagagacctt gacatcggga ttaattccga tgaggatgat 1500 tacaactatg ttgcacaagc aagcatttgg ggcactaagt attttaaaag caatttctat 1560 aggttggttt atgtaaagac tttagttgat ccgactaatt tctttacata cgaacaaagc 1620 atcccacctc tttcaccaca ttacaaaagg tga 1653 <210> 6 <211> 1620 <212> DNA <213> Morus alba <400> 6 atgaagtcct ctttcgtgtt tgctaaaatt gccatccttc ttttctcgct tgttctgcta 60 gcttcggcta atcacactca tgaagagttt cttcagtgcc tgagctctcg tatacccaag 120 tccattatct atgcttcaaa taacccctcg tattcaaatg tattagattc gacgactcaa 180 aatcctcgtt tcctttcttc ttcgaccaga aatccatctg ttatcgtcac accgtttaaa 240 atctcccaca tacaacccac catttactgc tccaagaaac atggcgtgca gataagaatt 300 cgaagcggtg ggcatgatta tgaaggcctt tcttatcagt ccagtgtccc atttttcata 360 ctcgacttga gaaacataaa ttccattcaa gttgatgtgg agaagaagag tgcatgggtt 420 gaggcaggtg cgacgctcgg cgaactttac tacagtatcg ctaaaaaaag caaaacgctt 480 ggcttccctg gcggtctttg cagcaccgtt ggtgtcggtg agamgttagg tggaggc 540 tatggctatc aatcgcgaac atatgggctc gcatctgata atattattga tgcgcaatta 600 atcgacgctc gaggagaat tctcaatcgg aaatccatgg gggagattt gttctggcc 660 attcgcggtg gtggagcagg aagcttcgga attgtattg cctggaaggt tcgactcatt 720 gacgtgcctt cgacagtgac tgtctttgaa actgtacgca tgtgggaaga taatgtaacg 780 aagaagtttg ttcatcgata tcaacgtcgt gcttccaaca tcgatagga tctaactatc 840 ttctttgggat tccgaaccac aaatactagt gatgacaag ggaattcaa gattcaata 900 ataaccatca tctcagccac attccatggc agcaggata ggctccttcc attgatgcaa 960 gaggagtttc ccgagttggg tttggcaaa gaagattca aagaatgtc atgggtccaa 1020 tctattgtcc attacataa ttacaagac gatgatccct tggagttct actcacaa 1080 acagtcatt tcgaacccaa ccctttcaa ttgaatctg actatgtgaa aaagcctatt 1140 ccagatgacg tgttggaaa attgctggct cggttgtacg agagacat aggatatgat 1200 tttgtggaat tttttccata tggaggaaaa ttgagcgaga tttcagaatc tgaaatccca 1260 ttcccacatc gagctggaaa cctctacaac cttcggtaca tggcttcatg gaaacaaggc 1320 gaaaatacta caagaatcaa caaccatctt agctgggtaa gaagtgttta tgattccatg 1380 actccttatg tgtcaaagaa tccaaggggt gcatatctca actttagaga ccttgacatc 1440 ggggttaatc ctaatgagag tgacaccaca agtgcttata actatgttaa acaagcaagc 1500 gtttggggta ctaagtattt taagaacaat ttctacaaaa tggtgtttat aaagacttta 1560 gttgatccaa ctaatttctt tacatacgaa caaagcatcc cacctattct tcaccattaa 1620 <210> 7 <211> 21 <212> DNA <213> Mulberry (Morus alba) <400> 7 atgaagtact ttgcattgtc t 21 <210> 8 <211> 23 <212> DNA <213> Mulberry (Morus alba) <400> 8 ttaatgcata acgatcggtg gga 23 <210> 9 <211> 18 <212> DNA <213> Mulberry (Morus alba) <400> 9 atgaagtact tttcctta 18 <210> 10 <211> 19 <212> DNA <213> Mulberry (Morus alba) <400> 10 tcaccttttg taatgtggt 19 <210> 11 <211> 21 <212> DNA <213> Mulberry (Morus alba) <400> 11 atgaagtcct ctttcgtgtt t 21 <210> 12 <211> 26 <212> DNA <213> Mulberry (Morus alba) <400> 12 ttaatggtga agaataggtg ggatgc 26

Claims

1. The FAD-dependent enzyme MaFAD1 from mulberry, characterized in that, The amino acid sequence of MaFAD1 is the amino acid sequence shown in SEQ ID NO.1; or the amino acid sequence obtained by linking the amino acid sequence shown in SEQ ID NO.1 with a tag for detection or purification.

2. The FAD-dependent enzyme MaFAD1 according to claim 1, characterized in that, The tags are selected from His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or Profinity eXact.

3. A nucleic acid molecule encoding the FAD-dependent enzyme MaFAD1 of claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A recombinant expression vector comprising the nucleic acid molecule as described in any one of claims 3-4.

6. A host cell containing the nucleic acid molecule of any one of claims 3-4 or the expression vector of claim 5, wherein the host cell is selected from bacteria or yeast.

7. The host cell according to claim 6, characterized in that, The yeast mentioned is Pichia pastoris.

8. A method for producing the FAD-dependent enzyme MaFAD1 according to claim 1, characterized in that, Culture the host cells of any one of claims 6-7 under the culture conditions to obtain cells containing MaFAD1, crude enzyme solution, or pure enzyme obtained through further extraction and purification.

9. The application of the FAD-dependent enzyme MaFAD1 in claim 1 in the catalytic synthesis of dienes, wherein MaFAD1 is an oxidase that catalyzes the synthesis of dienes.

10. The application according to claim 9, characterized in that, The reaction substrates of the oxidase MaFAD1 include isopentenyl dibenzofuran compounds or isopentenyl chalcone compounds. In the above substrates, the isopentenyl group is oxidized by oxidase MaFAD1 to a diene, which provides diene substrates for [4+2] cyclases MaFAD2 and MaFAD3 to carry out [4+2] cyclization reactions to synthesize Diels-Alder adducts. The amino acid sequences encoding MaFAD2 and MaFAD3 are shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

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