CYP450 enzyme related to biosynthesis of cyclized bisbenzylisoquinoline alkaloid as well as biological material and application of CYP450 enzyme

Abstract

The invention discloses a CYP450 enzyme related to biosynthesis of cyclized bisbenzylisoquinoline alkaloids as well as a biological material and application of the CYP450 enzyme. The name of the CYP450 enzyme disclosed by the invention is CYP82BC2. Experiments prove that when the CYP82BC2 and the CYP80Q4 are used for catalyzing a substrate together, the bisbenzylisoquinoline alkaloid (R, S)-Lindeldhamine or 2 '-Norberbamunine generated by catalysis of the CYP80Q4 can be further subjected to a C-O coupling reaction to be converted into cyclized bisbenzylisoquinoline alkaloids, namely, Bisnorobamesine or 2'-Norobamesine and racemosinine A. The CYP82BC2 and the CYP80Q4 are used for catalyzing the substrate together, and the CYP82BC2 and the CYP80Q4 are used for catalyzing the substrate together, so that the CYP82BC2 and the CYP80Q4 are used for catalyzing the substrate together. The CYP82BC2 protein and related biological materials of the CYP82BC2 protein can be applied to synthetic biological production of cyclized bisbenzylisoquinoline alkaloids and breeding of stephania tetrandra.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and mainly relates to CYP450 enzymes related to the biosynthesis of cyclic bisbenzylisoquinoline alkaloids, their biological materials and applications. Background Art

[0002] Cytochrome P450 is a heme-containing membrane protein that widely exists in various organisms. Cytochrome P450 can catalyze various types of reactions, such as hydroxylation, epoxidation, isomerization, dealkylation, etc., but the C-O or C-C phenol coupling catalyzed by P450 is a relatively rare reaction. In recent years, with the popularization of the third-generation sequencing technology and the enrichment of high-throughput screening methods, more and more P450s in the biosynthesis pathways of plant secondary metabolites have been reported.

[0003] The secondary metabolites of plants are the effective components of many plant-derived traditional Chinese medicines. However, with the rapid growth of demand, the limited plant resources and relatively low production efficiency will not be able to meet the needs of the people.

[0004] Stephania tetrandra is a perennial vine of the genus Stephania in the Menispermaceae family. Its roots are the plant source of the traditional Chinese medicine Stephaniae Tetrandrae Radix, which has the effects of dispelling wind and relieving pain, promoting diuresis and reducing edema, etc. Tetrandrine is a bisbenzylisoquinoline alkaloid and the main active ingredient of Stephania tetrandra. Tetrandrine is a calcium ion antagonist that affects the transmembrane transport of calcium ions and has good therapeutic effects in the treatment of silicosis, pulmonary fibrosis, etc., and has pharmacological effects such as protecting liver cells, anti-hepatic fibrosis, and anti-tumor.

[0005] The main active ingredient of Stephania tetrandra, tetrandrine, is a bisbenzylisoquinoline alkaloid. Existing studies believe that the upstream pathway of the tetrandrine biosynthesis pathway is the same as that of other benzylisoquinoline alkaloids such as morphine, starting from tyrosine and catalyzed by decarboxylase, hydroxylase, transaminase, norcoclaurine synthase, norcoclaurine-6-O-methyltransferase, and coclaurine-N-methyltransferase to form the intermediate compound (S)-N-methylcoclaurine. Bisbenzylisoquinoline alkaloids also require the catalysis of CYP450 to undergo a second C-O coupling to form cyclic bisbenzylisoquinoline alkaloids, and finally complete the post-modification through methyltransferase to form different bisbenzylisoquinoline alkaloids. However, the CYP450 catalyzing the cyclization reaction has not been reported. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to synthesize cyclic bisbenzylisoquinoline alkaloids by biosynthesis method and / or how to catalyze the synthesis of cyclic bisbenzylisoquinoline alkaloids by cytochrome P450 enzyme.

[0007] To solve the above technical problem, the present invention first provides any one of the following applications of the protein:

[0008] M1) Use of the protein in catalyzing C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids;

[0009] M2) Use of the protein in catalyzing C-O coupling of the O on C7’ of (R,S)-Lindoldhamine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids;

[0010] M3) Use of the biomaterial in catalyzing C-O coupling of the O on C7’ of 2’-Norberbamunine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids

[0011] M4) Use of the protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products;

[0012] M5) Use of the protein in fangji breeding products;

[0013] M6) Use of the protein in the production, preparation or development of products related to tetrandrine;

[0014] The protein can be the protein of any of the following A1), A2), A3) or A4):

[0015] A1) A protein with an amino acid sequence of Sequence 2 in the sequence listing;

[0016] A2) A fusion protein obtained by fusing a protein tag to the carboxyl terminus and / or amino terminus of the protein shown in A1);

[0017] A3) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 of the sequence listing, having the same function, derived from A1) or A2), or having more than 80% identity with the protein shown in A1) or A2);

[0018] The structural formula of the (R,S)-Lindoldhamine is as follows Formula 1; the structural formula of the 2’-Norberbamunine is as follows

[0019] Formula 2:

[0020]

[0021] The above-mentioned protein can be derived from Stephania tetrandra.

[0022] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then biologically expressed.

[0023] In the above application, the protein tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag, etc.

[0024] In the above application, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by 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 a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0025] In the above application, the identity of more than 80% can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0026] In the above application, the bisbenzylisoquinoline alkaloid can be (R,S)-Lindoldhamine or 2’-norberbamunine. The cyclized bisbenzylisoquinoline alkaloid in M2) is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid in M3) is 2’-Norobamegine and / or racemosinine A.

[0027] The C-O coupling can be the C-O coupling of the O on C7’ of the bisbenzylisoquinoline alkaloid with the C on C8.

[0028] The cyclized bisbenzylisoquinoline alkaloid can also be a compound detected by an ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer UPLC-QTOF-MS with m / z 569 or 567 or 583 or 581 or 597.

[0029] To solve the above technical problems, the present invention also provides any one of the following applications of biological materials related to the protein described above:

[0030] N1) Use of the biological material in catalyzing C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids;

[0031] N2) Use of the biological material in catalyzing C-O coupling of the O on C7’ and the C on C8 of (R,S)-Lindoldhamine to produce cyclized bisbenzylisoquinoline alkaloids;

[0032] N3) Use of the biological material in catalyzing C-O coupling of the O on C7’ and the C on C8 of 2’-Norberbamunine to produce cyclized bisbenzylisoquinoline alkaloids

[0033] N4) Use of the biological material in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products;

[0034] N5) Use of the biological material in fangji (Stephania tetrandra) breeding products;

[0035] N6) Use of the biological material in the production, preparation or development of products related to tetrandrine.

[0036] The biological material may be any one of the following D1) to D6):

[0037] D1) A nucleic acid molecule encoding the protein described above;

[0038] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0039] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0040] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0041] D5) A nucleic acid molecule that promotes or enhances the expression of the protein described above;

[0042] D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).

[0043] In the above applications, the nucleic acid molecule described in D1) may be the coding gene of the protein shown in the following d1), d2) or d3):

[0044] d1) A cDNA molecule or DNA molecule whose coding sequence is the cDNA molecule or DNA molecule of Sequence 1 in the sequence listing;

[0045] d2) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

[0046] In the above application, the expression cassette containing a nucleic acid molecule described in D2) refers to DNA that can express the protein described in the above application in a host cell. This DNA may not only include a promoter that initiates the transcription of the protein-coding gene, but also include a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may also include enhancer sequences. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters.

[0047] An existing plant expression vector can be used to construct a recombinant expression vector containing the expression cassette of the protein-coding gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the non-translated regions transcribed at the 3' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos) and plant genes (such as the soybean storage protein gene) have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence.

[0048] In the above application, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi.

[0049] In the above application, the bisbenzylisoquinoline alkaloid can be (R,S)-Lindoldhamine or 2'-norberbamunine. The cyclized bisbenzylisoquinoline alkaloid described in N2) is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid described in N3) is 2'-Norobamegine and / or racemosinine A.

[0050] The C-O coupling can be the C-O coupling of the O on C7' of the bisbenzylisoquinoline alkaloid with the C on C8.

[0051] The cyclized bisbenzylisoquinoline alkaloid may also be a compound detected by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) with m / z 569 or 567 or 583 or 581 or 597.

[0052] To solve the above technical problems, the present invention also provides any one of the following products containing the protein and / or biological material described above:

[0053] P1. A product for producing a cyclized bisbenzylisoquinoline alkaloid;

[0054] P2. A product for catalyzing the C-O coupling of the O on C7' of (R,S)-Lindoldhamine with the C on C8 to form a cyclized bisbenzylisoquinoline alkaloid;

[0055] P3. A product for catalyzing the C-O coupling of the O on C7' of 2'-Norberbamunine with the C on C8 to form a cyclized bisbenzylisoquinoline alkaloid.

[0056] P4. A product for preparing a catalyst for the C-O coupling of bisbenzylisoquinoline alkaloid to form a cyclized bisbenzylisoquinoline alkaloid;

[0057] P5. A product for producing, preparing or developing products related to tetrandrine.

[0058] In the above products, the bisbenzylisoquinoline alkaloid may be (R,S)-Lindoldhamine or 2'-norberbamunine.

[0059] The cyclized bisbenzylisoquinoline alkaloid in P2 is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid in P3 is 2'-Norobamegine and / or racemosinine A.

[0060] The above-mentioned Bisnorobamegine is a compound with the structural formula of Formula 3, the above-mentioned 2'-Norobamegine is a compound with the structural formula of Formula 4, and the above-mentioned racemosinine A is a compound with the structural formula of Formula 5:

[0061] <�

[0062] The C-O coupling may be the C-O coupling of the O on C7' of the bisbenzylisoquinoline alkaloid with the C on C8, or the C-O coupling of the O on C7 with the C on C8'.

[0063] The cyclized bisbenzylisoquinoline alkaloid may also be a compound detected by ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) with m / z 569 or 567 or 583 or 581 or 597.

[0064] To solve the above technical problems, the present invention also provides a method for preparing cytochrome P450 enzyme CYP82BC2, which may include the following steps: expressing the coding gene of the above-mentioned protein in a eukaryotic microorganism to obtain the cytochrome P450 enzyme CYP82BC2.

[0065] In the above method, the expression may include introducing the coding gene of the above-mentioned protein into a recipient microorganism to obtain a recombinant microorganism expressing the cytochrome P450 enzyme CYP82BC2, culturing the recombinant microorganism, and expressing to obtain the cytochrome P450 enzyme CYP82BC2.

[0066] The eukaryotic microorganism may be yeast.

[0067] In the above method, the coding gene of the protein may be first modified as follows and then introduced into the target plant to achieve a better expression effect:

[0068] 1) Connecting with promoters expressed in various plants to facilitate its expression in plants; the promoters may include constitutive, inducible, temporal regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters; the selection of promoters will vary with the time and space requirements of expression and also depends on the target species; for example, tissue- or organ-specific expression promoters depend on the developmental stage of the receptor as needed; although many promoters derived from dicotyledonous plants have been proven to be functional in monocotyledonous plants and vice versa, ideally, dicotyledonous plant promoters are selected for expression in dicotyledonous plants and monocotyledonous plant promoters are selected for expression in monocotyledonous plants;

[0069] 2) Connecting with suitable transcription terminators can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV and E9 derived from rbcS; any available terminator known to be functional in plants can be connected to the gene of the present invention;

[0070] 3) Introducing enhancer sequences, such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0071] The above-mentioned protein and / or the above-mentioned biological material also fall within the protection scope of the present invention. The present invention has carried out functional identification on a CYP450 in Stephania tetrandra through methods such as gene screening, heterologous expression, in vitro enzymatic reaction and product detection. This enzyme can catalyze the second-step C-O coupling reaction of bisbenzylisoquinoline alkaloids to form cyclized bisbenzylisoquinoline alkaloids. When this CYP450 and CYP80Q4 are used together to catalyze (S)-coclaurine, the (R,S)-Lindoldhamine produced by CYP80Q4 catalyzing (S)-coclaurine + (R)-coclaurine can further undergo a C-O coupling reaction to be transformed into cyclized bisbenzylisoquinoline alkaloid Bisnorobamegine. In addition, when this enzyme and CYP80Q4 are used together to catalyze the reaction of one molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine, the 2’-norberbamunine formed by CYP80Q4 catalysis can further undergo a C-O coupling reaction to be transformed into cyclized bisbenzylisoquinoline alkaloids 2’-norobamegine and racemosinine A. The Cytochrome P450 Nomenclature Committee named this gene CYP82BC2, and this gene can be applied to the synthetic biological production of cyclized bisbenzylisoquinoline alkaloids and the breeding research of Stephania tetrandra. Description of the Drawings

[0072] Figure 1 Schematic diagrams of two groups of substrate reactions catalyzed by CYP80Q4.

[0073] Figure 2 Chromatogram of the product (R,S)-Lindoldhamine of the enzymatic reaction catalyzed by CYP80Q4 for the substrate group ① (one molecule of (S)-coclaurine and one molecule of (R)-coclaurine). The position framed by the black box is the position where the C-O coupling reaction occurs. The ordinate is the relative abundance, and the abscissa is the time.

[0074] Figure 3 Mass spectrum of the product (R,S)-Lindoldhamine of the enzymatic reaction catalyzed by CYP80Q4 for the substrate group ① (one molecule of (S)-coclaurine and one molecule of (R)-coclaurine). The abscissa is the mass-to-charge ratio, and the ordinate is the relative abundance.

[0075] Figure 4Chromatogram of 2’-norberbamunine, the enzymatic reaction product of CYP80Q4 catalyzing the group ② substrates (one molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine). The position framed by the black box is the position where the C-O coupling reaction occurs. The vertical axis represents relative abundance, and the horizontal axis represents time.

[0076] Figure 5 Mass spectrum of 2’-norberbamunine, the enzymatic reaction product of CYP80Q4 catalyzing the group ② substrates (one molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine). The horizontal axis represents the mass-to-charge ratio, and the vertical axis represents relative abundance.

[0077] Figure 6 Schematic diagram of two groups of reactions jointly catalyzed by CYP80Q4 and CYP82BC2.

[0078] Figure 7 Chromatogram of the product of the reaction of CYP80Q4 and CYP82BC2 jointly catalyzing the group ① substrates. The position circled by the black round frame is the position where the C-O coupling reaction occurs. The vertical axis represents relative abundance, and the horizontal axis represents time.

[0079] Figure 8 Mass spectrum of the product of the reaction of CYP80Q4 and CYP82BC2 jointly catalyzing the group ① substrates. The horizontal axis represents the mass-to-charge ratio, and the vertical axis represents relative abundance.

[0080] Figure 9 Chromatogram of the product of the reaction of CYP80Q4 and CYP82BC2 jointly catalyzing the group ② substrates. The position circled by the black round frame is the position where the C-O coupling reaction occurs. The vertical axis represents relative abundance, and the horizontal axis represents time.

[0081] Figure 10 Mass spectrum of the product of the reaction of CYP80Q4 and CYP82BC2 jointly catalyzing the group ② substrates. The horizontal axis represents the mass-to-charge ratio, and the vertical axis represents relative abundance.

[0082] Figure 11 Chemical structure diagram of Bisnorobamegine.

[0083] Figure 12 For Bisnorobamegine in DMSO-d6 1 1H NMR.

[0084] Figure 13 For Bisnorobamegine in DMSO-d6 13 13C NMR. Detailed implementation mode

[0085] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0086] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0087] The expression vector pESC-URA used in the embodiments of the present invention is a product of Agilent Technologies, with the catalog number 217454.

[0088] The sources of the reagents in the embodiments of the present invention are as follows:

[0089] Tris-HCl: Sangon Biotech (Shanghai) Co., Ltd., catalog number B548124-0500;

[0090] EDTA: Beijing Solarbio Science & Technology Co., Ltd., catalog number E8040;

[0091] KCl: From Beijing Solarbio Science & Technology Co., Ltd., catalog number P9921;

[0092] Glycerol: Beijing Solarbio Science & Technology Co., Ltd., catalog number G8190;

[0093] PEG4000: From Beijing Solarbio Science & Technology Co., Ltd., catalog number P8240;

[0094] D-Sorbitol: Beijing Solarbio Science & Technology Co., Ltd., catalog number S8090;

[0095] Reduced nicotinamide adenine dinucleotide phosphate (NADPH): Beijing Solarbio Science & Technology Co., Ltd., catalog number N8100;

[0096] Flavin adenine dinucleotide (FAD): Catalog number F8384 from Sigma-Aldrich;

[0097] Flavin mononucleotide (FMN): Catalog number F6750 from Sigma-Aldrich;

[0098] Glucose-6-phosphate (G6P): Beijing Solarbio Science & Technology Co., Ltd., catalog number G8870;

[0099] Glucose-6-phosphate dehydrogenase (G6PDH): Sangon Biotech (Shanghai) Co., Ltd., catalog number A003997-0002;

[0100] Dithiothreitol (DTT): Solarbio Science & Technology Co., Ltd., Beijing, Catalog No. D8220;

[0101] Sources of compound standards used in the examples of the present invention:

[0102] (S)-coclaurine, (R)-coclaurine and (S)-reticuline (Catalog No. B21083) are from Yuanye Bio-Technology Co., Ltd., Shanghai. Among them, coclaurine (Catalog No. B30141) is a racemate and is resolved into (S)-coclaurine and (R)-coclaurine by a chiral column. (R)-N-methylcoclaurine is from WuXi Labex (Wuhan) Chemical Technology Co., Ltd. (R,S)-Lindoldhamine is from BioDuro (Jiangsu) Co., Ltd.

[0103] The chiral resolution method of (S)-coclaurine and (R)-coclaurine is as follows:

[0104] Preparative liquid chromatography: YMC K-Prep LAB 100G

[0105] Chromatographic column: ChiralPAK IBN 2.5 cm I.D.×25 cm L, 5 μm

[0106] Mobile phase: n-hexane / isopropanol 0.1% diethanolamine = 50 / 50 (V / V)

[0107] Flow rate: 50 mL / min

[0108] UV detection wavelength: 210 nm

[0109] Column temperature: 40 °C.

[0110] The expression strain WAT11 yeast competent cells used in the examples of the present invention are products of Coolaber Technology Co., Ltd., Beijing, Catalog No. CC311.

[0111] Example 1. Cloning of CYP82BC2 gene and construction of expression vector

[0112] 1. Cloning of CYP82BC2 gene:

[0113] Through the sequenced Stephania tetrandra genomic data, by methods such as splicing, annotation, and screening, the cDNA sequence of the candidate cytochrome P450 in the alkaloid biosynthesis pathway of Stephania tetrandra was obtained. The primers for the candidate cytochrome P450 were designed using the bioinformatics software DNAMAN. The primers were synthesized by Beijing Ribobio Co., Ltd., and the primer sequences were:

[0114] Forward primer: 5’-ATGATGATCGAGATCCAAAATT-3’

[0115] Reverse primer: 5’-TCAGGAAGAAGGAAGCCG-3’

[0116] Take the rhizomes of the Stephania tetrandra plants with vigorous growth, extract the total RNA using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen, DP441), perform reverse transcription using the reverse transcription kit to obtain cDNA, and use the cDNA as a template to amplify the gene sequence of CYP82BC2 to obtain the amplification product.

[0117] Perform agarose gel electrophoresis on the amplification product, and a specific band appears near about 1500 bp. Recover the band from the gel, ligate the recovered product to the Blunt Zero Cloning Vector (TransGen Biotech, CB501-01) and transform the Escherichia coli competent cells Trans1-T1 (TransGen Biotech, CD501-02). Pick positive clones for sequencing (Beijing Novogene Bioinformatics Technology Co., Ltd.), and select and preserve the monoclonal colonies containing the CDS sequence of the CYP82BC2 gene (shown as Sequence 1 in the Sequence Listing) for the construction of the subsequent expression vector. The amino acid sequence of the cytochrome P450 enzyme CYP82BC2 is Sequence 2 in the Sequence Listing.

[0118] 2. Construction of the CYP82BC2 recombinant vector

[0119] Design primers with BamHⅠ restriction sites as follows:

[0120] Primer F: 5’-AGGAGAAAAAACCCCGGATCCATGATGATCGAGATCCAAAATT-3’

[0121] Primer R: 5’-AGTGAGTCGTATTACGGATCCTCAGGAAGAAGGAAGCCG-3’

[0122] Using the primer pair, the ORF of CYP82BC2 was amplified with the cDNA of the rhizome of Stephania tetrandra plants as a template to obtain an amplification product. The amplification product was ligated to the pESC-URA expression vector using seamless splicing technology (seamless splicing enzyme: TransGen Company, Basic Seamless Cloning and Assembly Kit CU201) to obtain the recombinant expression vector pESC-ura-CYP82BC2. The recombinant vector pESC-ura-CYP82BC2 was transformed into Escherichia coli competent cells Trans1-T1. Positive clone strains were screened through solid LB medium containing 50 mg / mL sodium ampicillin (formula: 1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% Agar, the rest is sterile water, add filter-sterilized sodium ampicillin before autoclaving and cooling). Positive monoclonal strains were picked into liquid LB medium containing 50 mg / mL sodium ampicillin (formula: 1.0% Tryptone, 0.5% Yeast Extract, 0.5% NaCl, the rest is sterile water, add sterilized sodium ampicillin after autoclaving and cooling) for shaking culture. The bacterial solution was sent to a sequencing company for sequencing. After the monoclonal bacterial solution with correct sequencing was amplified and cultured, the plasmid was extracted to obtain the recombinant expression vector plasmid pESC-ura-CYP82BC2. The recombinant expression vector pESC-ura-CYP82BC2 contains the CDS sequence of the CYP82BC2 gene shown in Sequence 1 in the Sequence Listing and can express the CYP82BC2 protein with the amino acid sequence shown in Sequence 2 in the Sequence Listing.

[0123] The sequence of Sequence 1 in the Sequence Listing is as follows (5'-3'):

[0124]

[0125] The sequence 2 in the sequence listing is as follows:

[0126] MMIEIQNFFPSHIVCGVLLAVISAYYLLTKYKNKPARAAAPEPAGAWPVIGHILQFGPLDLWHRKLGDMADEIGPAFVIRLGMLRVLIINNWELAKECFTLNDKIFASRPGSITSKYMGYDGAMLGFAPDGPFMLELRKVAMQELFSNHRLRLLRHIRTSEIEIMIKGLNELYHRNFNNATGGASMLVHLDPWLDALTINVMLRSIAGKRYYDGGAAVEGEEAKRWKEAFGKVMLVFKMLLVSEMFPRLEMVDVMLGTTRAMKKAYEEMDLLLSGWLEEHRSKKDRGDHEQDFVDVLLNVGNNNPKLFSQYDVDSVVKATCLDILIAASDSTTLTLSWALSLLLNNRRVLKKVQEELDNQIGKDRHAEEEDIKNLPYLQAVVKETWRLYPPSPLSLPHEASKDCVVGGFHIPKGTSLMTNVWKIHRDPKVWQEDPLEFKPERFLSASHAHVDFRGLHFEFIPFGSGRRMCLGTNLAANIAHLTLARLLHEFELGAPDDAPLDMTESPEVSLKRASPLNVLIAPRLPSS。

[0127] 3. Cloning of the full-length CYP80Q4 gene and construction of recombinant vectors

[0128] Using the sequenced genome data of Stephania tetrandra, the cDNA sequence of the candidate cytochrome P450 in the alkaloid biosynthesis pathway of Stephania tetrandra was obtained by methods such as splicing, annotation, and screening. The primers for the candidate cytochrome P450 were designed using the bioinformatics software DNAMAN, and the primer sequences are:

[0129] Forward primer F1: 5’-ATGGATCCAATCACACTGT-3’;

[0130] Reverse primer R1: 5’-TTAAACCGCAGTTGCTGAT-3’.

[0131] Using the same gene cloning method as in Step 1, the gene sequence of CYP80Q4 was amplified with Stephania tetrandra cDNA as a template to obtain an amplification product, and the amplification product was transformed into Escherichia coli competent cells Trans1-T1 to obtain positive monoclonal colonies containing the CDS sequence of the CYP80Q4 gene.

[0132] According to the gene coding sequence and restriction enzyme sites, primers with BamHⅠ restriction enzyme sites were designed for CYP80Q4 as follows:

[0133] Primer F2: 5’-AGGAGAAAAAACCCCGGATCCATGGATCCAATCACACTGT-3’;

[0134] Primer R2: 5’-AGTGAGTCGTATTACGGATCCTTAAACCGCAGTTGCTGAT-3’;

[0135] Using the same recombinant vector construction method as in Step 2, the CDS sequence containing the CYP80Q4 gene was ligated to the pESC-URA expression vector by seamless splicing technology to obtain the recombinant expression vector pESC-ura-CYP80Q4. The recombinant expression vector pESC-ura-CYP80Q4 contains the CDS sequence of the CYP80Q4 gene shown in Sequence 3 of the Sequence Listing and can express the CYP80Q4 protein with the amino acid sequence shown in Sequence 4 of the Sequence Listing.

[0136] Sequence 3 in the Sequence Listing is as follows (5’-3’):

[0137]

[0138] SEQ ID NO: 4 in the Sequence Listing is as follows:

[0139] MDPITLLLILFTLLSSLFFSFEFKRYISPKKLPPGPFAWPLIGNTVLSVSEGQLHVALADLAKKFGPLMMLKFGLVPPLMVASNHVAAAEILKTQDRACSGRNVAHSVQVPGYIQYSMVWADCTEHWKMVRRIWRTELFSTKMVDLQTSIREEKVRELLGFLRRKEGQVVQLSDAIFGCIINVLGSIIFNQNVYDYEGKVDNEKGMKGMIRQLMVLAAKPNLPDFYPVFGPLDLQGIRRTTAECVKQMNEYWGGIVKERRASKDHSRNDFLDVLIQANFTDAQIDALLLEIFGPGSDTSSCTIEWAMSELIKNPNKLLKLQEELNRVIGQSREVRESDLENLPYLHACIKETLRLHPPVTFLLPHRATETCEVMNCTIPKDSQIFVNAYSIQRDPEVWEDPLIFKPERFLNSTVDYQGNDFHYIPFGAGRRICPALNMASRATRFMLGSLIHNFEWSLPNGMKPSELDMRDAFVLVLAKTVPLSVIPKARSSATAV。

[0140] 4. Obtaining of recombinant yeast

[0141] 4.1 Yeast transformation and culture:

[0142] Using Frozen-EZ Yeast TransformationⅡ TMThe kit (ZYMO RESEARCH, T2001) was used to transfer the recombinant vectors pESC-ura-CYP82BC2 and pESC-ura-CYP80Q4 into the competent cells of Saccharomyces cerevisiae WAT11 respectively. Positive monoclonal colonies were picked and inoculated into 10 mL of uracil-deficient liquid medium (Phygene, 8 g / L, containing 2% glucose), and cultured at 30 °C and 200 rpm for 48 hours. Then, they were inoculated into 100 mL of uracil-deficient liquid medium according to an inoculation ratio of 1:20, and cultured at 30 °C and 200 rpm for 24 hours. The supernatant was removed by centrifugation at 6000 g, and the cells were resuspended with 100 mL of YPL medium (1% Yeast extract, 2% peptone, 2% galactose, and the rest was sterile water, sterilized by high temperature), and then continued to be cultured at 30 °C and 200 rpm for 12 hours to obtain the culture solutions of recombinant yeast (WAT11 / pESC-ura-CYP82BC2) and recombinant yeast (WAT11 / pESC-ura-CYP80Q4) respectively. At the same time, the pESC-URA empty plasmid was transformed into the competent cells of Saccharomyces cerevisiae WAT11 to obtain the culture solution of recombinant empty yeast (WAT11 / pESC-URA).

[0143] 4.2 Extraction of yeast microsomes:

[0144] 1) The culture solutions of recombinant yeast (WAT11 / pESC-ura-CYP82BC2), recombinant yeast (WAT11 / pESC-ura-CYP80Q4), and recombinant empty yeast (WAT11 / pESC-URA) were centrifuged at 6500 g for 10 min to collect the cells. The supernatant was discarded, and the cells were inverted on absorbent paper for several seconds. Then, 10 mL of TEK (50 mM Tris-HCl, 1 mM EDTA, 100 mM KCl, pH 7.5, and the rest was water) (1 / 10 of the volume of YPL) was added to resuspend the cells, and then the cells were placed at room temperature for 5 min;

[0145] 2) Centrifuge at 6500 g for 10 min, and resuspend the cells with 100 mL of TESB (50 mM Tris-HCl, 1 mM EDTA, 600 mM D-sorbitol, and the rest was water, pH 7.5) (equal volume to YPL), and then place them on ice;

[0146] 3) The cell suspension was broken on a homogenizer (the condensed water was pre-cooled to 2 °C in advance) at a pressure of more than 1000 bar for 15 - 20 times until the cell suspension became milky and turbid; after each break, wait for the temperature to drop back to 2 °C before breaking the next bottle;

[0147] 4) Centrifuge the bacterial solution at 12,000 rpm for 20 min at 4°C, pour the supernatant into a bottle containing 10 g of PEG4000 and 0.88 g of NaCl, and incubate on ice for 15 - 30 min, shaking 3 - 5 times during this period until all the solids are dissolved;

[0148] 5) Centrifuge at 12,000 rpm for 20 min at 4°C, discard the supernatant, invert it on absorbent paper for a few seconds and then place it on ice;

[0149] 6) Add 1000 μL of TEG (50 mM Tris - HCl, 1 mM EDTA, 20% (v / v) glycerol, pH 7.5), pipette evenly to obtain the microsome extracts of recombinant yeast (WAT11 / pESC - ura - CYP82BC2), recombinant yeast (WAT11 / pESC - ura - CYP80Q4) and recombinant empty - vector yeast (WAT11 / pESC - URA) respectively, and store at - 80°C or directly use for enzymatic reactions. The microsome extract of recombinant yeast (WAT11 / pESC - ura - CYP82BC2) contains CYP82BC2 protein (the amino acid sequence is sequence 2 in the sequence listing), and the microsome extract of recombinant yeast (WAT11 / pESC - ura - CYP80Q4) contains CYP80Q4 protein (the amino acid sequence is sequence 4 in the sequence listing).

[0150] 5. Enzymatic reaction of CYP80Q4 alone and product extraction

[0151] The total enzymatic reaction system is 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) of microsomal CYP80Q4 enzyme, and 20 μM of substrate.

[0152] Among them, the enzyme in the experimental group's enzymatic reaction system is the microsome extract of recombinant yeast WAT11 / pESC - ura - CYP80Q4 obtained in step 4 (containing CYP80Q4 protein); the enzyme in the control group's enzymatic reaction system is the microsome extract of recombinant empty - vector yeast WAT11 / pESC - URA obtained in step 4.

[0153] The substrates were divided into two groups, with two compounds in each group, and 20 μM of each was added. After the reaction system was prepared, it was placed in a shaker at 30 °C and oscillated at 150 rpm for 2 h. Then, 500 μL of ethyl acetate (Beijing Chemical Works) was added, and ultrasonic extraction was carried out for 30 min. It was placed in a centrifuge and centrifuged at 12,000 g for 20 min. The upper organic phase solution was taken, dried in a nitrogen blower, redissolved in 150 μL of methanol (Merck, USA), filtered through a 0.22 μm filter membrane, and then injected into an ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer UPLC-QTOF-MS for detection.

[0154] The two groups of substrates are as follows:

[0155] ①: One molecule of (S)-coclaurine and one molecule of (R)-coclaurine;

[0156] ②: One molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine;

[0157] Among them, the Chinese name of (S)-coclaurine is (S)-liriodendrine; the Chinese name of (R)-coclaurine is (R)-liriodendrine; the Chinese name of (R)-N-methylcoclaurine is (R)-N-methyl-liriodendrine.

[0158] Detection of reaction products:

[0159] The enzymatic reaction products were detected using UPLC-QTOF-MS (Waters Xevo G2-S QTOF instrument), and it was found that in the experimental group catalyzed by CYP80Q4 enzyme, the above two groups of substrates underwent C-O coupling reactions to form bisbenzylisoquinoline alkaloids ( Figure 1 in ①-②). The specific reactions and products are as follows:

[0160] ①: One molecule of (S)-coclaurine ((S)-liriodendrine) and one molecule of (R)-coclaurine were catalyzed by CYP80Q4 enzyme to undergo intermolecular C-O coupling to form the bisbenzylisoquinoline alkaloid (R,S)-Lindoldhamine ((R,S)-thalictrine, m / z is 569), Figure 2 is the chromatogram of the reaction product, Figure 3 is the mass spectrum of the reaction product:

[0161] (S)-coclaurine + (R)-coclaurine → (R,S)-Lindoldhamine;

[0162] ②: One molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine undergo a C-O coupling reaction to form 2’-norberbamunine (2’-demethylberberine, CID: 441063). Figure 4 is the chromatogram of the reaction product. Figure 5 is the mass spectrum of the reaction product:

[0163] (S)-coclaurine + (R)-N-methylcoclaurine → 2’-Norberbamunine.

[0164] Among them, the structural formula of (R,S)-Lindoldhamine is as Formula 1 or Formula 1-1 below; the structural formula of 2’-Norberbamunine is as Formula 2 or Formula 2-1 below:

[0165]

[0166] 6. Combined enzymatic reaction of CYP80Q4 and CYP82BC2 proteins and product extraction:

[0167] 6.1 Combined enzymatic reaction

[0168] The total enzymatic reaction system is 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), 250 μg CYP80Q4 microsomal protein (microsomal extract of recombinant yeast WAT11 / pESC-ura-CYP80Q4 obtained in Step 4), 250 μg CYP82BC2 microsomal protein (microsomal extract of recombinant yeast WAT11 / pESC-ura-CYP82BC2 obtained in Step 2), and 30 μM substrate.

[0169] Among them, the substrates were also divided into 2 groups: ① (S)-coclaurine, ② (S)-coclaurine + (R)-N-methylcoclaurine. The substrates were two compounds, and 30 μM of each was added. After the reaction system was prepared, it was placed in a shaker at 30 °C and oscillated at 150 rpm for 2 h. Then, 500 μL of ethyl acetate (Beijing Chemical Works) was added, and ultrasonic extraction was carried out for 30 min. It was placed in a centrifuge and centrifuged at 12,000 g for 20 min. The upper organic phase solution was taken, dried in a nitrogen blower, redissolved in 150 μL of methanol (Merck, USA), filtered through a 0.22 μm filter membrane, and then injected into UPLC-QTOF-MS for detection.

[0170] 6.2 Detection of enzymatic reaction products:

[0171] The enzymatic reaction products were detected using an ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer UPLC-QTOF-MS. The results showed that compared with the reaction catalyzed by CYP80Q4 alone in step 5, new compounds were generated in the system co-catalyzed by CYP82BC2 and CYP80Q4. According to the mass spectrometry fragmentation rules, nuclear magnetic resonance data, and literature reports (related literature: Chen J, Zhao Q, Si D, Nie A, Wang Y, Deng Z, Wen Y, Chen F, Zhang L, Dong B, Yang J. Comprehensive profiling of Stephania tetrandra (Fangji) by stepwise DFI and NL-dependent structure annotation algorithm-based UHPLC-Q-TOF-MS and direct authentication by LMJ-HRMS. J Pharm Biomed Anal. 2020 Jun 5;185:113225.), it was determined that they were bisbenzylisoquinoline alkaloids formed by the C-O coupling reaction of bisbenzylisoquinoline alkaloids catalyzed by CYP80Q4. The results indicated that CYP82BC2 had the function of catalyzing the formation of cyclized bisbenzylisoquinoline alkaloids. The specific processes of reactions ① and ② are as Figure 6 shown.

[0172] In the enzymatic reaction of Group ① substrates, CYP80Q4 alone catalyzes one molecule of (S)-coclaurine and one molecule of (R)-coclaurine to generate (R,S)-Lindoldhamine ((R,S)-lindoldhamine); CYP82BC2 then catalyzes the C-O coupling of the O on C7’ of (R,S)-lindoldhamine with the C on C8 to generate the cyclized bisbenzylisoquinoline alkaloid Bisnorobamegine (m / z is 567, the chromatogram and mass spectrum are as shown in Figure 7 and Figure 8 shown, the chemical structure diagram is as shown in Figure 11 shown, and the nuclear magnetic resonance results are as shown in Figures 12 - 13 shown);

[0173] In the enzymatic reaction of Group ② substrates, CYP80Q4 catalyzes the C-O coupling of the O atom at the C4’ position of (S)-coclaurine with the C3’ of (R)-N-methylcoclaurine to generate 2’-Norberbamunine; CYP82BC2 then catalyzes the C-O coupling of the O on C7’ of 2’-Norberbamunine with the C on C8 to generate the cyclized bisbenzylisoquinoline alkaloid 2’-Norobamegine (m / z is 581, the chromatogram and mass spectrum are as shown in Figure 9 and Figure 10 ), and at the same time, the product racemosinine A (m / z is 581, the chromatogram and mass spectrum are also as shown in Figure 9 and Figure 10 shown) is also generated by the C-O coupling of the O on C7 with the C on C8’.

[0174] Among them, the chemical structure formula of the cyclized bisbenzylisoquinoline alkaloid Bisnorobamegine is as Formula No. 3 below; the chemical structure formula of the cyclized bisbenzylisoquinoline alkaloid 2’-Norobamegine is as Formula No. 4 below; the chemical structure formula of the cyclized bisbenzylisoquinoline alkaloid racemosinine A is as Formula No. 5 below:

[0175]

[0176] Therefore, this CYP82BC2 can be applied to the synthesis, biological production and fangji breeding of cyclized bisbenzylisoquinoline alkaloids.

[0177] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. Any of the following applications of the protein: M1) The application of the protein in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids; M2) The application of the protein in catalyzing the C-O coupling of the O on C7' of (R,S)-Lindoldhamine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids; M3) The application of the protein in catalyzing the C-O coupling of the O on C7' of 2'-Norberbamunine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids; M4) The application of the protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products; M5) The application of the protein in the preparation of Stephania tetrandra breeding products; M6) The application of the protein in the production, preparation or development of products related to tetrandrine; The protein is the following protein: A1) A protein with the amino acid sequence of sequence 2 in the sequence listing; A2) A fusion protein obtained by fusing a protein tag at the carboxyl terminus and / or amino terminus of the protein shown in A1); A3) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in sequence 2 of the sequence listing and having the same function, which is derived from A1) or A2) or has more than 80% identity with the protein shown in A1) or A2); The (R,S)-Lindoldhamine is a compound with the structural formula of formula 1: The 2'-Norberbamunine is a compound with the structural formula of formula 2:

2. The application according to claim 1, wherein: The bisbenzylisoquinoline alkaloid is (R,S)-Lindoldhamine or 2'-Norberbamunine; the cyclized bisbenzylisoquinoline alkaloid in M2) is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid in M3) is 2'-Norobamegine and / or racemosinine A; The Bisnorobamegine is a compound with the structural formula of formula 3: The 2'-Norobamegine is a compound with the structural formula of formula 4: The racemosinine A is a compound with the structural formula of formula 5:

3. Any of the following applications of the biological material related to the protein in claim 1: N1) The application of the biological material in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids; N2) The application of the biological material in catalyzing the C-O coupling of the O on C7' of (R,S)-Lindoldhamine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids; N3) The application of the biological material in catalyzing the C-O coupling of the O on C7' of 2'-Norberbamunine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids; N4) The application of the biological material in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products; N5) Use of the biological material in fangji (Stephania tetrandra) breeding products; N6) Use of the biological material in the production, preparation or development of products related to tetrandrine; The biological material 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 molecule 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) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A nucleic acid molecule that promotes or enhances the expression of the protein described in claim 1; D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5); The (R,S)-Lindoldhamine is a compound with the structural formula of Formula 1: The 2’-Norberbamunine is a compound with the structural formula of Formula 2:

4. The application according to claim 3, wherein: The nucleic acid molecule described in D1) is the coding gene of the protein as shown below: d1) A cDNA molecule or DNA molecule whose coding sequence is the cDNA molecule or DNA molecule of Sequence 1 in the sequence listing; d2) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

5. The application according to claim 3 or 4, characterized in that: The bisbenzylisoquinoline alkaloid is (R,S)-Lindoldhamine or 2’-norberbamunine; the cyclized bisbenzylisoquinoline alkaloid described in N2) is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid described in N3) is 2’-Norobamegine and / or racemosinine A; The Bisnorobamegine is a compound with the structural formula of Formula 3: The 2’-Norobamegine is a compound with the structural formula of Formula 4: The racemosinine A is a compound with the structural formula of Formula 5:

6. Any of the following products containing the protein described in claim 1 or 2 and / or the biological material described in any one of claims 3-5: P1. A product for producing cyclized bisbenzylisoquinoline alkaloids; P2. A product for catalyzing the C-O coupling of the O on C7’ of (R,S)-Lindoldhamine with the C on C8 to form a cyclized bisbenzylisoquinoline alkaloid; P3. A product for catalyzing the C-O coupling of the O on C7’ of 2’-Norberbamunine with the C on C8 to form a cyclized bisbenzylisoquinoline alkaloid P4. A product for preparing a product for catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to form cyclized bisbenzylisoquinoline alkaloids; P5. A product for the production, preparation or development of products related to tetrandrine; The (R,S)-Lindoldhamine is a compound with the structural formula of Formula 1: The 2’-Norberbamunine is a compound with the structural formula of Formula 2:

7. The product according to claim 6, characterized in that: The bisbenzylisoquinoline alkaloid is (R,S)-Lindoldhamine or 2’-norberbamunine; the cyclized bisbenzylisoquinoline alkaloid described in P2 is Bisnorobamegine; the cyclized bisbenzylisoquinoline alkaloid described in P3 is 2’-Norobamegine and / or racemosinine A; The Bisnorobamegine is a compound with the structural formula of Formula 3: The 2’-Norobamegine is a compound with the structural formula of Formula 4: The racemosinine A is a compound with the structural formula of Formula 5:

8. A method for preparing cytochrome P450 enzyme CYP82BC2, comprising the following steps: expressing the coding gene of the protein described in claim 1 in a eukaryotic microorganism to obtain the cytochrome P450 enzyme CYP82BC2.

9. The protein described in claim 1 and / or the biological material described in claim 3 or 4.

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