Cytochrome p450 enzymes from tetradium cumingii and their use in the biosynthesis of aporphine alkaloids

Abstract

The application discloses a cytochrome P450 enzyme derived from Stephania sinica Diels and application thereof in biosynthesis of aporphine alkaloid compounds. The application first characterizes a key P450 involved in formation of a methylenedioxy bridge structure of aporphine alkaloids in Stephania sinica Diels, provides an important reference for analysis of an aporphine alkaloid biosynthesis pathway in other species, and lays a foundation for heterologous production of various aporphine alkaloids. The application screens a P450 enzyme CYP719C3 annotated as CYP719 in a genome of Stephania sinica Diels, performs enzymatic reactions with different aporphine compounds as substrates, and detects reaction results by using a liquid chromatograph-mass spectrometer. It is found that CYP719C3 can catalyze an enzyme forming a methylenedioxy bridge on an A ring of an aporphine alkaloid, and can be applied to further analysis and heterologous production of an aporphine alkaloid pathway.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to cytochrome P450 enzymes derived from Stephania tetrandra and their application in the biosynthesis of apophyte alkaloids. Background Technology

[0002] The CYP719 family is a family of P450 enzymes found only in the Ranunculales and Aristolochiales orders, and is involved in the BIA biosynthesis pathway. Apophene alkaloids belong to the isoquinoline alkaloid class and are a class of naturally occurring products widely found in nature. Modern pharmacological studies have found that apophene alkaloids possess various pharmacological activities, including antioxidant, antitumor, and antirheumatic activities.

[0003] The methylene dioxin bridge structure is an important pharmacodynamic group in apophene alkaloids, and most apophene compounds identified from Stephania tetrandra contain this structure. However, no reports have been published regarding P450, a highly efficient catalyzer for the formation of methylene dioxin bridges in apophene alkaloids. Summary of the Invention

[0004] The technical problems to be solved by the present invention are how to catalyze the formation of a methylene dioxin bridge of the A ring of apophene alkaloids and / or how to efficiently catalyze the formation of a methylene dioxin bridge of the A ring of apophene alkaloids and / or how to prepare and obtain rootless vine cinnamon, neo-limonene and / or cryptoodorine.

[0005] To address the aforementioned technical problems, the present invention first provides a protein, which may be one of the following:

[0006] A1) The amino acid sequence is that of sequence 2 in the sequence listing;

[0007] A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in A1);

[0008] A3) Proteins derived from or having more than 80% identity with the proteins shown in A1) or A2) that have the same function and are obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in Sequence 2 of the sequence listing.

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0012] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0013] To address the aforementioned technical problems, the present invention also provides biomaterials related to the proteins described above, wherein the biomaterials are any of the following:

[0014] D1) Nucleic acid molecules that encode the proteins described above;

[0015] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0016] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0017] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0018] D5) Nucleic acid molecules that promote or enhance the expression of the proteins mentioned above;

[0019] D6) Expression cassettes, recombinant vectors, or recombinant microorganisms containing the nucleic acid molecules described in D5).

[0020] In the above-mentioned biological materials, the nucleic acid molecule described in D1) may be the gene encoding the protein as shown below:

[0021] d1) The coding sequence is the DNA molecule of sequence 1 in the sequence listing;

[0022] cDNA or DNA molecules that hybridize with cDNA or DNA molecules defined by d2) and encode proteins with the same function.

[0023] To address the aforementioned technical problems, the present invention also provides any of the following applications of the proteins and / or the biomaterials described above:

[0024] Application of M1 in the catalytic formation of a methylene dioxin bridge on the A ring of apophene alkaloids;

[0025] Application of M2 in the production, preparation or development of apophyte alkaloid products containing methylene dioxin bridges;

[0026] Application of M3 in the breeding of Stephania tetrandra;

[0027] Application of M4 in the production, preparation or development of apophyte alkaloid-related products.

[0028] In the above applications, the apophytic alkaloids may be bordinine, isona davidin, Nordomesticine, and / or Liriodendron chinense alkaloid.

[0029] The bordinine is a compound with the chemical structural formula of Formula 2 below, the isordomesticine is a compound with the chemical structural formula of Formula 7 below, and the Nordomesticine is a compound with the chemical structural formula of Formula 9 below.

[0030]

[0031] The apophene alkaloid containing a methylene dioxin bridge may be root-root cinnamon, neolizardine and / or cryptoodorine;

[0032] The rootless vine cinnamon is a compound with the chemical structural formula of Formula 6 below, the neo-limonene has the chemical structural formula of Formula 8 below, and the cryptodorine has the chemical structural formula of Formula 10 below.

[0033]

[0034] In the aforementioned biological materials, the expression cassette containing nucleic acid molecules described in D2) refers to DNA capable of expressing the proteins described in the above applications in host cells. This DNA may include not only promoters that initiate transcription of protein-coding genes but also terminators that terminate transcription of protein-coding genes. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0035] Recombinant expression vectors containing the protein-coding gene expression cassettes can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the carmine synthase gene Nos) and plant genes (such as the soybean storage protein gene). When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0036] In the aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0037] To address the aforementioned technical problems, the present invention also provides a product comprising any one of the following: the protein described above and / or the biological material described above:

[0038] P1, a product that catalyzes the formation of a methylene dioxin bridge on the A ring of apophyll alkaloids;

[0039] P2. Production, preparation or development of apophyll alkaloids containing methylene dioxin bridges;

[0040] P3. Products that produce, prepare or develop apophyte alkaloids;

[0041] The apophytic alkaloids may be bordinine, isona natansine, Nordomesticine and / or Liriodendron chinense alkaloid;

[0042] The bortine is a compound with the chemical structural formula of Formula 2 below, the isoomesticine is a compound with the chemical structural formula of Formula 7 below, and the Nordomesticine is a compound with the chemical structural formula of Formula 9 below.

[0043]

[0044] The apophene alkaloid containing a methylene dioxin bridge may be root-root cinnamon, neo-limonene, and / or crytodorine.

[0045] The rootless vine cinnamon is a compound with the chemical structural formula of Formula 6 below, the neo-limonene has the chemical structural formula of Formula 8 below, and the cryptodorine has the chemical structural formula of Formula 10 below.

[0046]

[0047] To address the aforementioned technical problems, the present invention also provides a method for preparing cytochrome P450 enzyme CYP719C3, which may include the following steps: expressing the gene encoding the protein described above in eukaryotic microorganisms to obtain the cytochrome P450 enzyme CYP719C3.

[0048] To address the aforementioned technical problems, the present invention also provides a method for preparing apophene alkaloids containing methylene dioxin bridges. The method may include the step of using the proteins and / or biological materials described above as described above to carry out a catalytic reaction with apophene alkaloids as substrates to obtain apophene alkaloids containing methylene dioxin bridges.

[0049] In the above method, the apophytic alkaloids may be bordinine, isonamidinine, Nordomesticine and / or Liriodendron chinense alkaloid;

[0050] The bordinine is a compound with the chemical structural formula of Formula 2 below, the isordomesticine is a compound with the chemical structural formula of Formula 7 below, and the Nordomesticine is a compound with the chemical structural formula of Formula 9 below.

[0051]

[0052] The apophene alkaloid containing a methylene dioxin bridge may be root-root cinnamon, neolizardine and / or cryptoodorine;

[0053] The rootless vine cinnamon is a compound with the chemical structural formula of Formula 6 below, the neo-limonene has the chemical structural formula of Formula 8 below, and the cryptodorine has the chemical structural formula of Formula 10 below.

[0054]

[0055] To address the aforementioned technical problems, the present invention also provides a protein composition for preparing neo-limonene and / or cryptoodorine, comprising the protein described above and protein B; wherein protein B is the following protein:

[0056] B1) The amino acid sequence is that of the protein in sequence 4 of the sequence listing;

[0057] B2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in B1);

[0058] B3) Proteins derived from or having more than 80% identity with the proteins shown in B1) or B2) that have the same function and are obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in sequence 4 of the sequence listing.

[0059] The chemical structural formula of the neo-limonene is as shown in Formula 8, and the chemical structural formula of the cryptodorine is as shown in Formula 10.

[0060]

[0061] Based on the previously screened P450 annotated as "CYP719" in the genome of Stephania tetrandra, this invention uses different apophene compounds as substrates to carry out enzymatic reactions. The reaction results were detected by liquid chromatography-mass spectrometry, and the enzymes that catalyze the formation of methylene dioxin bridges on the A and D rings of apophene alkaloids were discovered. This lays the foundation for further analysis of the apophene alkaloid pathway and heterogeneous production.

[0062] This invention discovers that two CYP719C subfamily genes, CYP719C3 and CYP719C4, derived from *Stephania tetrandra*, play important roles in the formation of methylene dioxin bridges on the A and D rings of apophene alkaloids, respectively. Previous literature has reported that CYP719A participates in the formation of methylene dioxin bridges on the A and D rings of protoberberine-type alkaloids. Existing CYP719A22 from lotus can catalyze the formation of methylene dioxin bridges on the A rings of lirinidine and caaverine, forming roemerine and anonaine, respectively. However, this invention, using commercially available lirinidine standards as substrates, found that CYP719A22 exhibits low catalytic activity against lirinidine standards, while the CYP719C3 identified in this invention from *Stephania tetrandra* can efficiently catalyze the formation of methylene dioxin bridges on the A ring of apophene alkaloids.

[0063] This invention is the first to characterize the key P450 involved in the formation of the methylene dioxin bridge structure of apophene alkaloids in Stephania tetrandra, providing an important reference for the analysis of the biosynthetic pathways of apophene alkaloids in other species, and laying the foundation for heterologous production of a variety of apophene alkaloids. Attached Figure Description

[0064] Figure 1 To illustrate the formation of methylene dioxin bridges from CYP719C3 and CYP719C4 catalyzing the catalytic reaction of boldine: A is the EIC mass spectrum of the catalytic reaction products; B is a schematic diagram of the catalytic reaction.

[0065] Figure 2 The formation of methylene dioxin bridges from norisoboldine catalyzed by CYP719C3 and CYP719C4: A is the EIC mass spectrum of the catalytic reaction products; B is a schematic diagram of the catalytic reaction.

[0066] Figure 3 The formation of methylene dioxin bridges from lirinidine catalyzed by CYP719A22: A is the EIC mass spectrum of the catalytic reaction product; B is a comparison of the secondary mass spectra of the reaction product and the standard; C is a schematic diagram of the catalytic reaction.

[0067] Figure 4 The following are secondary mass spectra of the substrates and products. The horizontal axis represents the mass-to-charge ratio, and the vertical axis represents the relative abundance. A is the secondary mass spectrum of boldine; B is the secondary mass spectrum of norisoboldine; C is the secondary mass spectrum of norisoboldine, a product of CYP719C3 catalyzing the formation of bordinine; D is the secondary mass spectrum of nandinarine, a product of CYP719C4 catalyzing the formation of bordinine; E is the secondary mass spectrum of norisoboldine, a product of CYP719C4 catalyzing the formation of norisoboldine; F is the secondary mass spectrum of neolidinone, a product of CYP719C3 catalyzing the formation of norisoboldine; G is the secondary mass spectrum of cryptoodorine, a product of CYP719C3 catalyzing the formation of norisoboldine.

[0068] Figure 5 The two-dimensional nuclear magnetic resonance (2D-NMR) correlation structure of the product nordomesticine is shown.

[0069] Figure 6The proton NMR spectrum (H NMR) of nordomesticine detected by one-dimensional nuclear magnetic resonance (600 MHz, methanol-d4) is as follows: 7.87 (1H, s, H-11), 6.84 (1H, s, H-8), 6.68 (1H, s, H-3), 5.98 (2H, d, J = 4.8 Hz, -OCH2O-), 4.16 (1H, dd, J = 14.4, 4.2 Hz, H-6a), 3.65 (1H, dd, J = 12.6, ... 6.0Hz,H-5eq),3.58(3H,s,OCH3),3.33(1H,overlapped,H-5ax),3.16(1H,td,J=17.4, 5.4Hz, H-4eq), 2.93 (1H, m, H-7eq), 2.91 (1H, m, H-4ax), 2.82 (1H, t, J = 13.8Hz, H-7ax).

[0070] Figure 7 The DEPT spectrum (Distortionless Enhancement by Polarization Transfer) of nordomesticine detected by nuclear magnetic resonance.

[0071] Figure 8 Carbon-13 NMR of nordomesticine for NMR detection 13 C NMR) spectrum (150MHz, Methanol-d4) δ150.9(C-1),147.4(C-9),147.1(C-10),144.1(C-2),127.4(C-7a),126.4(C-1a,3a),124.9(C-11a),1 20.2(C-3b),114.5(C-3),107.9(C-11),107.9(C-8),101.2(-OCH2O-),59.0(-OCH3),53.0(C-6a),41.3(C-5),33.6(C-7),24.9(C-4).

[0072] Figure 9 The gCOSY spectrum of nordomesticine detected by nuclear magnetic resonance.

[0073] Figure 10 The gHMBCAD spectrum of nordomesticine detected by nuclear magnetic resonance.

[0074] Figure 11 The gHSQCAD spectrum of nordomesticine detected by nuclear magnetic resonance. Detailed Implementation

[0075] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0077] The reagent information in this embodiment of the invention is as follows:

[0078] Norisoboldine is an apophene alkaloid with the following chemical structural formula (Formula 1) and CAS number 23599-69-1.

[0079] Boldine is an apophyte alkaloid with the following chemical structural formula (Formula 2) and CAS number: 476-70-0.

[0080] Lirinidine, an apophene alkaloid, has the following chemical structural formula (Formula 3) and CAS number: 54383-28-7.

[0081] Caaverine is an apophene alkaloid with the following chemical structural formula (4) and CAS number: 6899-64-5.

[0082]

[0083] Roemerine is an apophene alkaloid with the following chemical structural formula (Formula 5) and CAS number: 548-08-3.

[0084]

[0085] The expression vector pESC-URA used in the embodiments of the present invention is a product of Agilent Technologies, catalog number 217454; the deuterated methanol used in the embodiments of the present invention was purchased from Cambridge Isotope Laboratories.

[0086] Lirinidine and roemerine standards were purchased from Baoji Chenguang Biotechnology Co., Ltd.

[0087] The mobile phase method for purifying the products of the large-scale norisoboldine reaction catalyzed by CYP719C4 is shown in Table 1.

[0088] Table 1. Liquid mobile phase methods for preparing enzyme-catalyzed reaction products

[0089] 0 14％ 86％ 1 14％ 86％ 20 16％ 84％ 21 95％ 5％ 30 95％ 5％ 31 14％ 86％ 40 14％ 86％

[0090] The prepared product was concentrated to dryness by rotary evaporation, weighed to 1 mg, redissolved in a small amount of methanol, transferred to a vial, concentrated to dryness again, dissolved in deuterated methanol, transferred to an NMR tube, and detected using a 600M NMR spectrometer.

[0091] Example 1: Cloning and vector construction of CYP719C3 and CYP719C4 genes

[0092] 1. Cloning and expression vector construction of candidate CYP719 family genes

[0093] Using cDNA obtained by reverse transcription of RNA extracted from the roots of Stephania tetrandra as a template, high-fidelity PCR was performed using specific primer pairs with homologous arms containing restriction enzyme sites (primer pair CYP719C3F / CYP719C3R and primer pair CYP719C4F / CYP719C4R). The PCR products obtained were the sequences of the full-length CYP719C3 gene and the CYP719C4 gene with homologous arms.

[0094] The specific primer sequences are as follows:

[0095] CYP719C3F: 5'-AGGAGAAAAAACCCCGGATCCatggagttggcagcgat-3';

[0096] CYP719C3R: 5'-AGTGAGTCGTATTACGGATCCttaagtccgtctgggtactat-3';

[0097] CYP719C4F: 5'-AGGAGAAAAAACCCCGGATCCatggaacagagcactgtgt-3';

[0098] CYP719C4R: 5'-AGTGAGTCGTATTACGGATCCttaagaccaacgacgcact-3';

[0099] The CYP719C3 and CYP719C4 gene sequence fragments were ligated with the pESC-URA linear vector using a seamless splicing kit to obtain ligation products. The ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened using solid LB medium containing 50 mg / mL ampicillin sodium (formula: 1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% Agar, the remainder being sterile water; sterile ampicillin sodium was added before cooling). Positive single clones were picked and cultured in liquid LB medium containing 50 mg / mL ampicillin sodium (formula: 1.0% Tryptone, 0.5% Yeast Extract, 0.5% NaCl, the remainder being sterile water; sterile ampicillin sodium was added after cooling). The bacterial culture was sent to a sequencing company for sequencing. After expanding the culture of the correctly sequenced single clones, plasmids were extracted to obtain two recombinant expression vector plasmids, pESC-Ura-CYP719C3 and pESC-Ura-CYP719C4.

[0100] pESC-Ura-CYP719C3 contains the coding sequence of the CYP719C3 protein shown in Sequence 1 of the sequence listing and can express the CYP719C3 protein shown in Sequence 2 of the sequence listing; pESC-Ura-CYP719C4 contains the coding sequence of the CYP719C4 protein shown in Sequence 3 of the sequence listing and can express the CYP719C4 protein shown in Sequence 4 of the sequence listing.

[0101] 2. Obtaining recombinant yeast and extracting yeast microsomes

[0102] 2.1 Obtaining recombinant yeast

[0103] The two recombinant expression vector plasmids pESC-Ura-CYP719C3 and pESC-Ura-CYP719C4 constructed in step 1 were transformed into yeast strains, respectively. Specifically, Frozen-EZ Yeast Transformation II was used. TMThe kit (ZYMO RESEARCH, T2001) was used to transform four recombinant vectors into competent Saccharomyces cerevisiae WAT11 cells. Positive single clones were picked and transferred to 10 mL of ura-deficient liquid medium (Pankino, 8 g / L, containing 2% glucose), and cultured at 30°C and 200 rpm for 48 hours. The cells were then inoculated into 100 mL of ura-deficient liquid medium at a 1:20 ratio and cultured at 30°C and 200 rpm for 24 hours. After centrifugation at 6000 g to remove the supernatant, the cells were resuspended in 100 mL of YPL medium (1% Yeast extract, 2% peptone, 2% galactose, with the remainder being sterile water, sterilized at high temperature). The cells were then cultured at 30°C and 200 rpm for 12 hours to obtain two recombinant yeast cultures (WAT11 / pESC-Ura-CYP719C3 and WAT11 / pESC-Ura-CYP719C4).

[0104] 2.2 Yeast microsome extraction:

[0105] The two recombinant yeast culture media obtained in step 2.1 were used to extract yeast microsomes using the TESB method to obtain two recombinant yeast microsome extracts (WAT11 / pESC-Ura-CYP719C3 and WAT11 / pESC-Ura-CYP719C4).

[0106] 2.3 Enzymatic reaction and product extraction

[0107] Two recombinant yeast microsomal extracts were subjected to enzymatic reactions using boldine as a substrate. The total enzymatic reaction system was 500 μL, containing 100 mM Tris-HCl, 1 mM NADPH (reduced nicotinamide adenine dinucleotide phosphate), 5 μM FAD (flavin adenine dinucleotide), 5 μM FMN (flavin mononucleotide), 4 mM G6P (glucose-6-phosphate), 1 μg UG6PDH (glucose-6-phosphate dehydrogenase), 2 μM DTT (dithiothreitol), 500 μg (250 μL) of yeast microsomal extract, and 20 μM boldine substrate.

[0108] After the reaction, the reaction solution was alkalized with ammonia, and the reaction product was extracted with ethyl acetate and detected by liquid chromatography-mass spectrometry (LC-MS). The results showed that new chromatographic peaks were generated in both the CYP719C3 and CYP719C4 reaction systems. CYP719C3 and CYP719C4 are P450 enzymes. New chromatographic peaks with a mass-to-charge ratio of 326, reduced by 2 compared to the substrate boldine, were detected in both reaction systems. The compounds represented by these two new peaks are more polar than those of the substrate boldine. Figure 1The concentration of ethylene glycol (boldine + EV) in A was significantly reduced, and the retention times varied but were similar. Figure 1 The compounds represented by Boldine + CYP719C3 and Boldine + CYP719C4 (representing ring A) can both catalyze the enzymatic reaction of Boldine, forming methylene dioxin bridges on the A and D rings of the substrate Boldine, respectively. Based on secondary mass spectrometry peak comparison analysis, it was ultimately determined that CYP719C3 catalyzes the production of cassythicine, a product with methylene dioxin bridges on the A ring, while CYP719C4 catalyzes the production of isodomesticine, a product with methylene dioxin bridges on the D ring. Figure 1 (Middle right figure). In the secondary mass spectrometry experiment, significant substrate residue was still detected in the CYP719C3 reaction system. Figure 1 The purple peak in CYP719C3 (boldine + CYP719C3) is visible in the reaction system, while the substrate boldine is almost undetectable in the CYP719C4 reaction system. Therefore, CYP719C4 has a higher catalytic efficiency for bodline than CYP719C3. Figure 1 ).

[0109] The chemical structural formula of Cassythicine (methyl cassythicine) is shown in Formula 6 below, CAS No.: 5890-28-8; the chemical structural formula of Isodomesticine is shown in Formula 7 below, PubChem CID: 69523059;

[0110]

[0111] 3. Validation of catalytic function

[0112] To further explore whether reaction product 2 (containing cassythicine) obtained by CYP719C3 catalyzing boomine and reaction product 3 (isodomesticine) obtained by CYP719C4 catalyzing boomine in step 2.3 can be used as substrates, a methylene dioxin bridge was formed again on another ring under the catalysis of the two P450 enzymes. Reaction product 2 (containing cassythicine) and reaction product 3 (isodomesticine) were recovered and concentrated completely. Then, another P450 recombinant yeast microsomal extract was added: CYP719C4 recombinant yeast microsomal extract was added to reaction product 2 catalyzed by CYP719C3 catalyzing boomine; CYP719C3 recombinant yeast microsomal extract was added to reaction product 3 catalyzed by CYP719C4 catalyzing boomine. After the reaction was completed, the extracted products were analyzed by mass spectrometry.

[0113] The results showed that chromatographic peaks with a mass-to-charge ratio of 324 and consistent retention times were detected in both the reactions involving CYP719C3 and CYP719C4. Figure 1 The light blue peaks in (boldine+CYP719C3)+CYP719C4 and (boldine+CYP719C4)+CYP719C3 in group A were analyzed by secondary mass spectrometry and identified as neolitine (neolidinine), where both rings A and D are bridged by methylene dioxide. However, in this experiment, the abundance of the product peak detected in the reaction system involving CYP719C3 was significantly higher than that detected in the reaction system involving CYP719C4. Based on the results of the first round of enzymatic reactions, it can be inferred that, from the perspective of favoring the downstream metabolic flow, the catalytic effect of CYP719C4 should occur before that of CYP719C3. Figure 1 (B)

[0114] The chemical structural formula of neolitine is shown in Formula 8 below, CAS No.: 2466-42-4;

[0115]

[0116] The above results indicate that CYP719C3 and CYP719C4 catalyze the formation of methylene dioxin bridges on rings A and D of the apophene alkaloid boldine, respectively, regardless of whether a methylene dioxin bridge has already formed on the other ring, ultimately resulting in neolitine, a compound with methylene dioxin bridges on both rings A and D. However, the catalytic efficiency of CYP719C3 and CYP719C4 varies depending on the catalytic order; CYP719C4 should catalyze the reaction before CYP719C3.

[0117] The coding sequence for CYP719C3 is sequence 1 in the sequence listing, and the amino acid sequence of the corresponding CYP719C3 protein is sequence 2 in the sequence listing. The coding sequence for CYP719C4 is sequence 3 in the sequence listing, and the amino acid sequence of the corresponding CYP719C4 protein is sequence 4 in the sequence listing.

[0118] Sequence 1 in the sequence list is as follows (5'-3'):

[0119]

[0120] Sequence 2 in the sequence listing is as follows (5'-3'):

[0121] MELAAMSDAWMIVGTVLLLGLLMILLDQTFLWSASKQQWPPGPKRLPLIGNLHQLNRGGKLVHVTLAKMAKEHGGIMTVWFGGQQPSVVVSDHDLVWEVMVTKAADFGARALPKITKIFTADWGTIATCDLGTYWQTLRKGLQSSAINPLTISSQTQLQEKDVADFVSSFEQEASLNNGIVDPLPKLRKIAIRLLARFCFGREFPIEEHFVEQMDRAMLDENRLMGHTRLQDVFAFTRYIPGLWRPFKEAENLRRRIKELIRPYIRSTTPNCFLSFLVSQGFPEEVVIFNLFELFALAVDSTSNTLAWALAFLIHNEEVQERMYKEMNDKLGRRRMVSAEEVMSGMEYVHAVVKETMRMRPIVSLAPLRAGADSELKGFKIREGTAVMLNLYAVHHDDKVWKEENKFMPERFMEDGGKARERSFVPFGGGRRICGGMELAKLHMALLLANLVNRFQWRSVEEGQPPDLTENLTFLLMMKTPLVARIVPRRT*.

[0122] Sequence 3 in the sequence listing is as follows (5'-3'):

[0123]

[0124] Sequence 4 in the sequence list is as follows (5'-3'):

[0125] MEQSTVYIAVATLASLALLSKLLAQSLFCPNTKQLWPPGPIKLPLIGNLHHLGGDLIHWTLANLAQKHGKLMTVWFGSQQPFIVVTDLDLAWELLVTKGVDFWSRKMPYLSRITSAGYRTLATC DGGPYWETLRRGLQSTALNPQTISSQTKLQEQDIADMISSMQREADLNNGVVKPLNHLRKLAIRLLSRLCFGTDFPNEGRFVKRMDELIEDEMRLSTEARLVDVYEFTRHIPGLKPPLREIEGHT ERIKELIRPCLAAARKYSSSGNSHMSFLLSQGFSEDVILLNLFEVFAFGVDSTSATIAWALGFLILERETQEKLFKEVETKLGGTRRMVRVEDVSGMEYVHAVVKETLRMRPVAPLAVPHRAAR DSELKGLKVKEGTALLVNLYALLHDEKVWKEPNRFVPERFVKRDGVEDLKKMERYYVPFGAGRRACPGMELAKVEVAVALANLVNSFKWRNAVEGQPPDLSEALSPLLGMKTPLEARIVRRWS*.

[0126] Example 2: CYP719C3 and CYP719C4 catalyze the enzymatic reaction of norisoboldine.

[0127] Similar to Example 1, this example uses norisoboldine as a substrate, and the enzymatic reaction is still carried out using two recombinant yeast microsomal extracts (WAT11 / pESC-Ura-CYP719C3 and WAT11 / pESC-Ura-CYP719C4) with norisoboldine as the substrate.

[0128] The total enzymatic reaction system was 500 μL, containing 100 mM Tris-HCl, 1 mM NADPH (reduced nicotinamide adenine dinucleotide phosphate), 5 μM FAD (flavin adenine dinucleotide), 5 μM FMN (flavin mononucleotide), 4 mM G6P (glucose-6-phosphate), 1 U G6PDH (glucose-6-phosphate dehydrogenase), 2 μM DTT (dithiothreitol), 500 μg (250 μL) yeast microsomal extract, and 20 μM norisoboldine substrate.

[0129] Mass spectrometry results showed that a new chromatographic peak was generated in the reaction system catalyzed only by CYP719C4, with a mass-to-charge ratio of 312, which was 2 lower than that of the substrate (the mass-to-charge ratio of the substrate norisoboldine was 314). This suggests that the compound may be a product formed after the formation of a methylene dioxanone bridge on the D ring of the substrate norisoboldine. Figure 2 The reaction is represented by norisoboldine + CYP719C4 (A). This step has high reaction efficiency, and the substrate norisoboldine is almost undetectable in the reaction system. Expanding the reaction system and preparing the reaction product, analysis using a 600M NMR spectrometer (DRX Avance-600, Bruker, Germany) confirmed that the product is indeed nordomesticine, the product formed after the formation of a methylene dioxan bridge on the D ring. Figures 5-11 ).

[0130] The chemical structural formula of Nordomesticine ((S)-(+)-nordomesticine) is as follows: Formula 9, CAS No.: 14787-35-0;

[0131]

[0132] Further, using the reaction product of CYP719C4 catalyzing norisoboldine (containing nordomesticine) as a substrate, a second round of catalytic reaction was performed using the CYP719C3 enzyme. The results of the second round of enzymatic reaction showed that a new chromatographic peak with a mass-to-charge ratio of 310 was detected in the CYP719C3 catalytic product. Figure 2 The yellow chromatographic peak in (norisoboldine+CYP719C4)+CYP719C3 in group A represents the compound. Comparative analysis of secondary mass spectrometry peaks indicates that this compound is cryptoodorine, which is obtained by further catalyzing the formation of a methylene dioxin bridge on the A ring of nordomesticine.

[0133] The chemical structural formula of cryptodorine (norneolitsine) is shown in Formula 10 below, CAS number: 41787-55-7.

[0134]

[0135] The above results indicate that CYP719C4 can efficiently catalyze the formation of a methylene dioxin bridge on the D ring of norisoboldine, regardless of whether the N atom is methylated or not, or the different sequences of hydroxyl and methoxy groups on the A ring. Unlike boldine, norisoboldine lacks a methyl group on its N atom, and the sequences of hydroxyl and methoxy groups on the A ring are different, causing CYP719C3 to lose its catalytic activity. However, when norisoboldine is catalyzed by CYP719C4 to form nordomesticine, CYP719C3 exhibits catalytic activity towards nordomesticine, generating the crystalrine product, which is a methylene dioxin bridge on the A ring.

[0136] Example 3: Comparison of the catalytic activity of CYP719Cs screened in this invention with that of existing CYP719A22

[0137] CYP719A22, derived from lotus, has previously been reported to catalyze the formation of a methylene dioxin bridge from the A ring of apophene alkaloids in lotus. Previous experiments, using only a mixed extract of lotus alkaloids as substrates, observed a decrease in the corresponding substrate and an increase in the product, thus confirming its function. To further expand its substrate range and clarify its function and reaction efficiency using standards, this invention cloned the CYP719A22 gene and obtained a recombinant yeast microsomal extract containing the CYP719A22 protein using *Saccharomyces cerevisiae* (the method of obtaining the extract is the same as in Example 1).

[0138] Then, using the same enzymatic reaction system as in Example 2, enzymatic reactions were carried out with boldine, norisoboldine, and lirinidine as substrates. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that only when lirinidine was used as a substrate could a new chromatographic peak with a mass-to-charge ratio reduced by 2 compared to the substrate and a lower abundance be detected. Figure 3 The lirinidine+CYP719A22 in the middle A spectrum represents the standard roemerine, and its retention time and secondary mass spectrometry peak are consistent with those of the standard roemerine. Figure 3 (B) When other compounds (boldine, nordomesticine, or caaverine) were used as substrates, no obvious products were detected. This demonstrates a significant difference between CYP719A22 and CYP719C3 of this invention, with no overlapping catalytic substrates between them.

[0139] The above results indicate that the formation mechanisms of methylene dioxin bridges may differ among different types of apophene alkaloids. Given the relatively weak catalytic activity of CYP719A22, it may not be the main gene responsible for this type of catalytic activity in plants.

[0140] In summary, this invention identifies two CYP719C subfamily genes from *Stephania tetrandra*: CYP719C3 and CYP719C4. They play important roles in the formation of methylene dioxin bridges on the A and D rings of apophene alkaloids, respectively. Recent studies have also reported a CYP719A22 from lotus, which can catalyze the formation of methylene dioxin bridges on the A rings of lirinidine and caaverine, forming roemerine and anonaine, respectively. However, this embodiment uses a commercially available lirinidine standard as a substrate to further investigate the catalytic activity of CYP719A22. The results showed that its catalytic activity against the standard was low. In contrast, the two CYP719Cs identified from *Stephania tetrandra* in this chapter can efficiently catalyze the formation of methylene dioxin bridges on the A and D rings of apophene alkaloids.

[0141] The CYP719C subfamily also exhibits catalytic sequentiality in its catalytic reactions. Two consecutive rounds of enzymatic reactions show that CYP719C4 and CYP719C3 can sequentially catalyze reactions on rings A and D, ultimately forming a product with both rings A and D bridging with methylene dioxins. Comparing the abundance of products and intermediates detected in the two rounds of reactions suggests that when CYP719C4 acts before CYP719C3, the overall system has a higher efficiency in generating the final product.

[0142] In summary, this invention is the first to characterize the key P450 involved in the formation of two methylene dioxin bridge structures in apophene alkaloids in Stephania tetrandra, providing an important reference for elucidating the biosynthetic pathways of apophene alkaloids in other species, and laying the foundation for heterologous production of a variety of apophene alkaloids.

[0143] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, characterized by: The protein in question is the following: A1) The amino acid sequence is that of the protein in sequence 2 of the sequence listing; A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in A1).

2. A biomaterial relating to the protein of claim 1, wherein the biomaterial is any of the following: D1) A nucleic acid molecule encoding the protein described in claim 1; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule, and the coding sequence of the DNA molecule is shown in Sequence 1 of the sequence listing.

4. The application of the protein of claim 1 and / or the biomaterial of claim 2 or 3 in the preparation of apophene alkaloids containing methylene dioxin bridges; the application includes the step of using the protein of claim 1 and / or the biomaterial of claim 2 or 3 to carry out a catalytic reaction with apophene alkaloids as substrates to obtain apophene alkaloids containing methylene dioxin bridges; wherein the apophene alkaloids are bordinine, isonadonine, and / or Nordomesticine; The bordinine is a compound with the chemical structural formula of Formula 2 below, the isordomesticine is a compound with the chemical structural formula of Formula 7 below, and the Nordomesticine is a compound with the chemical structural formula of Formula 9 below. ； ； ； The apophene alkaloids containing methylene dioxin bridges are root-root cinnamic acid, neolizardine and / or cryptoodorine; The rootless vine cinnamon is a compound with the chemical structural formula of Formula 6 below, the neo-limonene has the chemical structural formula of Formula 8 below, and the cryptodorine has the chemical structural formula of Formula 10 below. ； ； 。 5. A method for preparing cytochrome P450 enzyme CYP719C3, comprising the following steps: expressing a nucleic acid molecule encoding the protein of claim 1 in a eukaryotic microorganism to obtain the cytochrome P450 enzyme CYP719C3.

6. A protein composition comprising the protein of claim 1 and protein B; wherein protein B is a protein of the following type: B1) The amino acid sequence is that of sequence 4 in the sequence listing; B2) A fusion protein obtained by fusion protein tagging the carboxyl terminus and / or amino terminus of the protein shown in B1).

7. The use of the protein composition of claim 6 in the preparation of neo-Litsea cubebaine and / or cryptoodorine; The chemical structural formula of the neo-limonene is as shown in Formula 8, and the chemical structural formula of the cryptodorine is as shown in Formula 10. ； 。

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