Enzyme related to biosynthesis of effective component of traditional Chinese medicine radix stephaniae tetrandrae and biological material and application thereof

Abstract

The invention discloses an enzyme related to biosynthesis of an effective component of a traditional Chinese medicine radix stephaniae tetrandrae as well as a biological material and application thereof. According to the technical scheme, the cytochrome P450 enzyme from stephania tetrandra is applied to catalysis of a C-O coupling reaction of bisbenzylisoquinoline alkaloid to generate cyclized bisbenzylisoquinoline alkaloid, the name of the protein is CYP82BC3, and the amino acid sequence of the protein is a sequence 2 in a sequence table. According to the invention, microsome extracting solutions of CYP82BC3 and CYP80Q4 proteins are extracted to carry out three groups of substrate enzymatic reactions, and compared with a CYP80Q4 enzyme independent catalytic reaction, the CYP82BC3 can catalyze a catalytic product of the CYP80Q4 enzyme to continuously carry out cyclization so as to generate the cyclized bisbenzylisoquinoline alkaloid. The method can be applied to biosynthesis of cyclized bisbenzylisoquinoline alkaloid and / or an effective component tetrandrine of stephania tetrandra and breeding of stephania tetrandra.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and mainly relates to enzymes related to the biosynthesis of effective components of Stephania tetrandra, as well as biological materials and applications thereof. Background Art

[0002] Stephania tetrandra is a perennial vine of the genus Stephania in the family Menispermaceae. Its roots are the plant source of Stephania tetrandra in traditional Chinese medicine, and have the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling, 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.

[0003] Existing research believes that the upstream pathway of the tetrandrine biosynthesis pathway is the same as that of other bisbenzylisoquinoline 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 bisbenzylisoquinoline alkaloid (S)-N-methylcoclaurine. Currently, there is no report on CYP450 that catalyzes the cyclization reaction of bisbenzylisoquinoline alkaloids. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to synthesize cyclized bisbenzylisoquinoline alkaloids by biosynthesis method and / or how to synthesize cyclized bisbenzylisoquinoline alkaloids by cytochrome P450 and / or how to synthesize the effective components of Stephania tetrandra by biosynthesis method and / or how to breed Stephania tetrandra.

[0005] To solve the above technical problems, the present invention first provides any one of the following applications of a protein:

[0006] M1) The application of the protein in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to generate cyclized bisbenzylisoquinoline alkaloids;

[0007] 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 generate cyclized bisbenzylisoquinoline alkaloids

[0008] 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 generate cyclized bisbenzylisoquinoline alkaloids;

[0009] Use of the protein in catalyzing the C-O coupling of the O on C7' of Berbamunine with the C on C8 to generate a cyclized bisbenzylisoquinoline alkaloid;

[0010] M5) Use of the protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products;

[0011] M6) Use of the protein in the production, preparation or development of products related to Tetrandrine;

[0012] M7) Use of the protein in products for breeding Stephania tetrandra;

[0013] The protein may be the protein of any of the following A1), A2), A3) or A4):

[0014] A1) A protein with an amino acid sequence of Sequence 2 in the sequence listing;

[0015] A2) A fusion protein obtained by fusing a protein tag at the carboxyl terminus and / or amino terminus of the protein shown in A1);

[0016] 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 and having the same function, which is derived from A1) or A2) or has an identity of more than 80% with the protein shown in A1) or A2).

[0017] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically.

[0018] In the above-mentioned protein, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology for the purpose of facilitating 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 and / or SUMO tag, etc.

[0019] In the above-mentioned protein, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page on 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.

[0020] In the above-mentioned protein, 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.

[0021] In the above application, the bisbenzylisoquinoline alkaloid can be (R,S)-Lindoldhamine, 2’-Norberbamunine, or Berbamunine.

[0022] In the above application, the C-O coupling can be the C-O coupling of the O on C7’ and the C on C8 of the bisbenzylisoquinoline alkaloid.

[0023] The cyclized bisbenzylisoquinoline alkaloid in M2) can be Bisnorobamegine (m / z 567) and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloid in M3) can be 2’-Norobamegine (m / z 581) and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloid in M4) can be Obamegine (m / z 597, obamegine).

[0024] In the above application, the cyclized bisbenzylisoquinoline alkaloid can also be a compound with m / z of 569 or 567 or 583 or 581 or 597 detected by an ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer UPLC-QTOF-MS.

[0025] 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:

[0026] N1) Use of the said biological material in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to generate cyclized bisbenzylisoquinoline alkaloids;

[0027] N2) Use of the said biological material in catalyzing the C-O coupling of the O on C7’ of (R,S)-Lindoldhamine with the C on C8 to generate cyclized bisbenzylisoquinoline alkaloids;

[0028] N3) Use of the said biological material in catalyzing the C-O coupling of the O on C7’ of 2’-Norberbamunine with the C on C8 to generate cyclized bisbenzylisoquinoline alkaloids;

[0029] N4) Use of the said protein in catalyzing the C-O coupling of the O on C7’ of Berbamunine with the C on C8 to generate cyclized bisbenzylisoquinoline alkaloids;

[0030] N5) Use of the said protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products;

[0031] N6) Use of the said protein in the production, preparation or development of products related to tetrandrine;

[0032] N7) Use of the said protein in the breeding products of Stephania tetrandra;

[0033] In the above applications, the said biological material can be any one of the following D1) to D6):

[0034] D1) A nucleic acid molecule encoding the said protein described above;

[0035] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0036] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0037] 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);

[0038] D5) A nucleic acid molecule that promotes or enhances the expression of the said protein described above;

[0039] D6) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in D5).

[0040] In the above applications, the nucleic acid molecule described in D1) can be the coding gene of the said protein shown as the following d1), d2) or d3):

[0041] d1) The coding sequence is a cDNA molecule or a DNA molecule of the nucleotides of Sequence 1 in the Sequence Listing;

[0042] d2) A cDNA molecule or a DNA molecule that hybridizes with the cDNA or DNA molecule defined in d1) and encodes a protein with the same function.

[0043] In the above application, the bisbenzylisoquinoline alkaloid may be (R,S)-Lindoldhamine, 2’-Norberbamunine or Berbamunine.

[0044] In the above application, the C-O coupling may be the C-O coupling between the O on C7’ and the C on C8 of the bisbenzylisoquinoline alkaloid.

[0045] In the above application, the cyclized bisbenzylisoquinoline alkaloid in N2) may be Bisnorobamegine (m / z 567) and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloid in N3) may be 2’-Norobamegine (m / z 581) and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloid in N4) may be Obamegine (m / z 597, obamegine).

[0046] In the above application, the cyclized bisbenzylisoquinoline alkaloid may also be a compound with m / z of 569 or 567 or 583 or 581 or 597 detected by an ultra performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer (UPLC-QTOF-MS).

[0047] In the above application, the expression cassette containing the 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 not only includes a promoter that initiates the transcription of the protein-coding gene, but also includes a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may also include an enhancer sequence. 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.

[0048] The recombinant expression vector containing the protein coding gene expression cassette can be constructed using existing plant expression vectors. 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 further 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 nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3' untranslated region transcribed by plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, 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.

[0049] Among the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae, and fungi.

[0050] To solve the above technical problems, the present invention also provides any of the following products containing the protein described above and / or the biological material described above:

[0051] P1. A product for producing cyclized bisbenzylisoquinoline alkaloids;

[0052] P2. A product for preparing a cyclized bisbenzylisoquinoline alkaloid by catalyzing the C-O coupling of the O on C7' of (R,S)-Lindoldhamine with the C on C8;

[0053] P3. A product for preparing a cyclized bisbenzylisoquinoline alkaloid by catalyzing the C-O coupling of the O on C7' of 2'-Norberbamunine with the C on C8;

[0054] P4. A product for preparing a cyclized bisbenzylisoquinoline alkaloid by catalyzing the C-O coupling of the O on C7' of Berbamunine with the C on C8;

[0055] P5. A product for producing, preparing, or developing products related to tetrandrine.

[0056] Among the above products, the bisbenzylisoquinoline alkaloid may be (R,S)-Lindoldhamine, 2’-Norberbamunine or Berbamunine.

[0057] Among the above products, the C-O coupling may be the C-O coupling between the O on C7’ and the C on C8 of the bisbenzylisoquinoline alkaloid.

[0058] In the above application, the cyclized bisbenzylisoquinoline alkaloid in P2) may be Bisnorobamegine (m / z 567) and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloid in P3) may be 2’-Norobamegine (m / z 581) and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloid in P4) may be Obamegine (m / z 597, obamegine).

[0059] The above-mentioned (R,S)-Lindoldhamine may be a compound with the structural formula of Formula 1 or Formula 1-1, the above-mentioned 2’-Norberbamunine is a compound with the structural formula of Formula 2 or Formula 2-1, and the above-mentioned Berbamunine is a compound with the structural formula of Formula 3 or Formula 3-1:

[0060]

[0061] The above-mentioned Bisnorobamegine may be a compound with the structural formula of Formula 4, the above-mentioned 2’-Norobamegine may be a compound with the structural formula of Formula 5, and the above-mentioned Obamegine may be a compound with the structural formula of Formula 6:

[0062]

[0063] The chemical structural formula of the said Bisnoraromoline is as follows in Formula 7; the chemical structural formula of the said racemosinine A is as follows in Formula 8:

[0064]

[0065] Among the above products, the cyclized bisbenzylisoquinoline alkaloid may also be a compound with m / z of 569 or 567 or 583 or 581 or 597 detected by an ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer UPLC-QTOF-MS.

[0066] As described above, R can represent the configuration of the chiral C atom as the R configuration, and S as described above can represent the configuration of the chiral C atom as the S configuration.

[0067] To solve the above technical problems, the present invention also provides a method for preparing cytochrome P450 enzyme CYP82BC3, and the method may include the following steps: expressing the coding gene of the above-mentioned protein in eukaryotic microorganisms to obtain the cytochrome P450 enzyme CYP82BC3.

[0068] In the above method, the expression includes introducing the coding gene of the above-mentioned protein into a recipient microorganism to obtain a recombinant microorganism expressing the cytochrome P450 enzyme CYP82BC3, culturing the recombinant microorganism, and expressing to obtain the cytochrome P450 enzyme CYP82BC3.

[0069] In the above method, the recipient microorganism can be a eukaryotic microorganism, and the eukaryotic microorganism can be yeast.

[0070] The above-mentioned protein and / or the above-mentioned biological material also belong to the protection scope of the present invention.

[0071] The present invention has carried out functional identification on 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 catalyze (S)-coclaurine and (R)-coclaurine together, it can cause the (R,S)-Lindoldhamine produced by CYP80Q4 catalyzing (S)-coclaurine + (R)-coclaurine to further undergo a C-O coupling reaction and be transformed into cyclized bisbenzylisoquinoline alkaloid Bisnorobamegine.

[0072] In addition, when this enzyme and CYP80Q4 catalyze the reaction of one molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine together, it can cause the 2’-Norberbamunine formed by CYP80Q4 catalysis to further undergo a C-O coupling reaction and be transformed into the second cyclized bisbenzylisoquinoline alkaloid 2’-Norobamegine. This P450 can also catalyze the C-O coupling reaction of Berbamunine to generate the cyclized bisbenzylisoquinoline alkaloid Obamegine. The Cytochrome P450 Nomenclature Committee named this gene CYP82BC3, and this gene can be applied to the synthetic biology production of cyclized bisbenzylisoquinoline alkaloids and the breeding research of Stephania tetrandra. Description of the Drawings

[0073] Figure 1 Schematic diagram of three groups of substrate reactions catalyzed by CYP80Q4.

[0074] Figure 2 Chromatogram of the product (R,S)-Lindoldhamine of the enzymatic reaction catalyzed by CYP80Q4 for the group ① substrates (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 vertical axis is the relative abundance, and the horizontal axis is the time.

[0075] Figure 3 Mass spectrum of the product (R,S)-Lindoldhamine of the enzymatic reaction catalyzed by CYP80Q4 for the group ① substrates (one molecule of (S)-coclaurine and one molecule of (R)-coclaurine). The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0076] Figure 4 Chromatogram of the product 2’-norberbamunine of the enzymatic reaction catalyzed by CYP80Q4 for 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 is the relative abundance, and the horizontal axis is the time.

[0077] Figure 5 Mass spectrum of the product 2’-norberbamunine of the enzymatic reaction catalyzed by CYP80Q4 for the group ② substrates (one molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine). The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0078] Figure 6 Chromatogram of the product Berbamunine of the enzymatic reaction catalyzed by CYP80Q4 for the group ③ substrates (one molecule of (S)-N-methylcoclaurine 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 is the relative abundance, and the horizontal axis is the time.

[0079] Figure 7 Mass spectrum of the product Berbamunine of the enzymatic reaction catalyzed by CYP80Q4 for the group ③ substrates (one molecule of (S)-N-methylcoclaurine and one molecule of (R)-N-methylcoclaurine). The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0080] Figure 8 Chemical structure diagrams of three catalytic products of CYP80Q4, bisbenzylisoquinoline alkaloids, and schematic diagrams of three groups of reactions catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. A is the chemical structure diagram of the bisbenzylisoquinoline alkaloid (R,S)-Lindoldhamine; B is the chemical structure diagram of the bisbenzylisoquinoline alkaloid 2’-Norberbamunine; C is the chemical structure diagram of the bisbenzylisoquinoline alkaloid Berbamunine; D is the schematic diagram of three groups of reactions catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The positions circled by the black round frames are the positions where C-O coupling reactions occur.

[0081] Figure 9 Product chromatogram of the reaction of group ① substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The positions circled by the black round frames are the positions where C-O coupling reactions occur. The vertical axis is the relative abundance, and the horizontal axis is the time.

[0082] Figure 10 Product mass spectrum of the reaction of group ① substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0083] Figure 11 Product chromatogram of the reaction of group ② substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The positions circled by the black round frames are the positions where C-O coupling reactions occur. The vertical axis is the relative abundance, and the horizontal axis is the time.

[0084] Figure 12 Product mass spectrum of the reaction of group ② substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0085] Figure 13 Product chromatogram of the reaction of group ③ substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The positions circled by the black round frames are the positions where C-O coupling reactions occur. The vertical axis is the relative abundance, and the horizontal axis is the time.

[0086] Figure 14 Product mass spectrum of the reaction of group ③ substrates catalyzed by CYP80Q4 and co-catalyzed by CYP80Q4 and CYP82BC3. The horizontal axis is the mass-to-charge ratio, and the vertical axis is the relative abundance.

[0087] Figure 15 Chemical structure diagram of Bisnorobamegine.

[0088] Figure 16 1H NMR of Bisnorobamegine in DMSO-d6 1 H NMR.

[0089] Figure 17 13C NMR of Bisnorobamegine in DMSO-d6 13 13C NMR

[0090] Figure 18 Chemical structure of bisnoraromoline

[0091] Figure 19 1H NMR of bisnoraromoline in DMSO-d6 1 1H NMR

[0092] Figure 20 13C NMR of bisnoraromoline in DMSO-d6 13 13C NMR Detailed implementation manners

[0093] The present invention will be further described in detail below in combination with the detailed implementation manners. The provided embodiments are only for clarifying the present invention, rather than 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.

[0094] 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.

[0095] The expression vector pESC-URA used in the embodiments of the present invention is a product of Agilent Technologies, with the product number 217454.

[0096] The sources of the reagents in the embodiments of the present invention are as follows:

[0097] Tris-HCl: Sangon Biotech (Shanghai) Co., Ltd., product number B548124-0500;

[0098] EDTA: Beijing Solarbio Science & Technology Co., Ltd., product number E8040;

[0099] KCl: from Beijing Solarbio Science & Technology Co., Ltd., product number P9921;

[0100] Glycerol: Beijing Solarbio Science & Technology Co., Ltd., product number G8190;

[0101] PEG4000: from Beijing Solarbio Science & Technology Co., Ltd., product number P8240;

[0102] D-Sorbitol: Beijing Solarbio Science & Technology Co., Ltd., product number S8090;

[0103] Reduced nicotinamide adenine dinucleotide phosphate (NADPH): Beijing Solarbio Science & Technology Co., Ltd., product number N8100;

[0104] Flavin adenine dinucleotide (FAD): Sigma-Aldrich, product number F8384;

[0105] Flavin mononucleotide (FMN): Sigma-Aldrich, product number F6750;

[0106] Glucose-6-phosphate (G6P): Beijing Solarbio Science & Technology Co., Ltd., product number G8870;

[0107] Glucose-6-phosphate dehydrogenase (G6PDH): Shanghai Sangon Biotech Co., Ltd., product number A003997-0002;

[0108] Dithiothreitol (DTT): Beijing Solarbio Science & Technology Co., Ltd., product number D8220;

[0109] Sources of compound standards used in the examples of the present invention:

[0110] (S)-coclaurine, (R)-coclaurine and (S)-reticuline (product number B21083) are from Shanghai Yuanye Bio-Technology Co., Ltd., where coclaurine (product number B30141) is a racemate and is resolved into (S)-coclaurine and (R)-coclaurine by a chiral column. (S)-N-methylcoclaurine and (R)-N-methylcoclaurine are from WuXi Labex (Wuhan) Chemical Technology Co., Ltd. (R,S)-Lindoldhamine is from BioDuro (Jiangsu) Co., Ltd.

[0111] The chiral resolution method of (S)-coclaurine and (R)-coclaurine is as follows:

[0112] Preparative liquid chromatography: YMC K-Prep LAB 100G

[0113] Chromatographic column: ChiralPAK IBN 2.5cm I.D.×25cm L, 5μm

[0114] Mobile phase: n-hexane / isopropanol 0.1% diethanolamine = 50 / 50 (V / V)

[0115] Flow rate: 50 mL / min

[0116] Ultraviolet detection wavelength: 210 nm

[0117] Column temperature: 40 °C.

[0118] In the examples of the present invention, the expression strain WAT11 yeast competent cells used are products of Beijing Coolaber Technology Co., Ltd., with the product number CC311.

[0119] Example 1, Cloning of the full-length CYP82BC3 gene and vector construction

[0120] 1. Cloning of the full-length CYP80Q4 gene and construction of recombinant vector

[0121] 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, and the primer sequences are as follows:

[0122] Forward primer F1: 5’-ATGGATCCAATCACACTGT-3’;

[0123] Reverse primer R1: 5’-TTAAACCGCAGTTGCTGAT-3’.

[0124] Take the rhizomes of the vigorously growing Stephania tetrandra plants, extract the total RNA using the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen, DP441), perform reverse transcription using the reverse transcription kit to obtain cDNA, and amplify the gene sequence of CYP80Q4 with the cDNA as a template to obtain the amplification product. 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.

[0125] According to the gene coding sequence and restriction enzyme sites, primers with BamHⅠ restriction enzyme sites were designed for CYP80Q4 as follows:

[0126] Primer F2: 5’-AGGAGAAAAAACCCCGGATCCATGGATCCAATCACACTGT-3’;

[0127] Primer R2: 5’-AGTGAGTCGTATTACGGATCCTTAAACCGCAGTTGCTGAT-3’;

[0128] Using the primer pair, the ORF of CYP80Q4 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 Biotech, Basic Seamless Cloning and Assembly Kit CU201) to obtain the recombinant expression vector pESC-ura-CYP80Q4. The recombinant vector pESC-ura-CYP80Q4 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, and sodium ampicillin sterilized by filter is added before high-temperature sterilization 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, and sterilized sodium ampicillin is added after high-temperature sterilization and cooling) for shaking culture. The bacterial liquid was sent to a sequencing company for sequencing. After the correctly sequenced monoclonal bacterial liquid was amplified and cultured, the plasmid was extracted 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 in the Sequence Listing and can express the CYP80Q4 protein with the amino acid sequence shown in Sequence 4 in the Sequence Listing.

[0129] The sequence of Sequence 3 in the Sequence Listing is as follows (5’-3’):

[0130]

[0131] The sequence 4 in the sequence listing is as follows:

[0132] MDPITLLLILFTLLSSLFFSFEFKRYISPKKLPPGPFAWPLIGNTVLSVSEGQLHVALADLAKKFGPLMMLKFGLVPPLMVASNHVAAAEILKTQDRACSGRNVAHSVQVPGYIQYSMVWADCTEHWKMVRRIWRTELFSTKMVDLQTSIREEKVRELLGFLRRKEGQVVQLSDAIFGCIINVLGSIIFNQNVYDYEGKVDNEKGMKGMIRQLMVLAAKPNLPDFYPVFGPLDLQGIRRTTAECVKQMNEYWGGIVKERRASKDHSRNDFLDVLIQANFTDAQIDALLLEIFGPGSDTSSCTIEWAMSELIKNPNKLLKLQEELNRVIGQSREVRESDLENLPYLHACIKETLRLHPPVTFLLPHRATETCEVMNCTIPKDSQIFVNAYSIQRDPEVWEDPLIFKPERFLNSTVDYQGNDFHYIPFGAGRRICPALNMASRATRFMLGSLIHNFEWSLPNGMKPSELDMRDAFVLVLAKTVPLSVIPKARSSATAV.

[0133] 2. Cloning of CYP82BC3 gene and construction of recombinant vector

[0134] Through the sequenced genome data of Stephania tetrandra, by methods such as splicing, annotation, and screening, the cDNA sequence (sequence 1 in the sequence listing) 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, and the primer sequences were:

[0135] Forward primer F1: 5’-ATGATCGAGATCCAAAACTT-3’

[0136] Reverse primer R1: 5’-TCAAGGAGAAGGAAGTCTCG-3’

[0137] Using the same gene cloning method as in step 1, the gene sequence of CYP82BC3 was amplified with Stephania tetrandra cDNA as the template to obtain an amplification product, and the amplification product was transformed into the Escherichia coli competent cell Trans1-T1 to obtain a positive monoclonal colony containing the CDS sequence of the CYP82BC3 gene.

[0138] According to the gene coding sequence and restriction enzyme cleavage sites, the primers with SpeⅠ restriction enzyme cleavage sites were designed for CYP82BC3 as follows:

[0139] Primer F2: 5’-TAAAGGGCGGCCGCACTAGTATGATCGAGATCCAAAACTT-3’;

[0140] Primer R2: 5’-TGTAATCCATCGATACTAGTTCAAGGAGAAGGAAGTCTCG-3’.

[0141] Using the same recombinant vector construction method as in step 1, the CDS sequence containing the CYP82BC3 gene was ligated to the pESC-URA expression vector by seamless splicing technology to obtain the recombinant expression vector pESC-ura-CYP82BC3. The recombinant expression vector pESC-ura-CYP82BC3 contains the CDS sequence of the CYP82BC3 gene shown in Sequence 1 in the sequence listing and can express the CYP82BC3 protein with the amino acid sequence shown in Sequence 2 in the sequence listing.

[0142] Sequence 1 in the sequence listing is as follows (5’-3’):

[0143]

[0144] The sequence 2 in the sequence listing is as follows:

[0145] MIEIQNFFRSDIVCGVLLAFISAYYILIKWKKHYHERSDETKNAAWPRPAPEAAGAWPIIGHVLQFGPLDLWHRKLGDMADEVGPAFMIRLGMRRVLIINNWELAKECFTSHDKTFAGRPGAISSKYMGYGGAMLGFAPDGPYMVELRKVAMQELFSNHRLRALKHIRMSEIEIMIKSLNELYHRNFNNARGGASMLVHLDPWLDALTLNVMLRSIAGKRYYNGDPCAAQDEEAKRWKEGFGKLIHVFRTFVVSEMFPGLEFVDVMLGATRAMEKASKDVDMLLSRWVEEHRSKMNSKDYQGGLGEGDFVDVLLNVDKNAKLFSHYDVDSVVKATCLDILIAASDSTTLTLSWALSLLLNNRHVLKKIQEELDNQIGKGRHAEEEDIKNLPYLQAVVKETWRLYPPSPLSLPHEASKDCIIGGFHVPKGTSLMTNIWKIHRDPNVWKDPLEFKPERFLSSSHAHVDFRGLHFEFIPFGSGRRMCLGTNLAANIVHLMLARLLHEFELGTPNDAPVDMTEGPDVSLNRASPLNVLIAPRLPSP。

[0146] 3. Obtaining of recombinant yeast

[0147] 3.1 Yeast transformation and culture

[0148] Using Frozen-EZ Yeast TransformationⅡ TMThe kit (ZYMO RESEARCH, T2001) was used to transfer the recombinant vectors pESC-ura-CYP82BC3 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). After culturing at 30 °C and 200 rpm for 48 hours, they were inoculated into 100 mL of uracil-deficient liquid medium at an inoculation ratio of 1:20, and cultured at 30 °C and 200 rpm for 24 hours. Then, the supernatant was removed by centrifugation at 6000 g. After resuspending the cells with 100 mL of YPL medium (1% yeast extract, 2% peptone, 2% galactose, and the rest was sterile water, sterilized by high temperature), the cells were continued to be cultured at 30 °C and 200 rpm for 12 hours to obtain the culture solutions of recombinant yeast (WAT11 / pESC-ura-CYP82BC3) 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).

[0149] 3.2 Yeast microsome extraction:

[0150] Centrifuge the culture broths of recombinant yeast (WAT11 / pESC-ura-CYP82BC3), recombinant yeast (WAT11 / pESC-ura-CYP80Q4), and recombinant empty vector yeast (WAT11 / pESC-URA) at 6500 g for 10 min to collect the cells, discard the supernatant, and invert the centrifuge tubes on absorbent paper for several seconds; then add 10 mL of TEK (1 / 10 of the volume of YPL medium) (50 mM Tris-HCl, 1 mM EDTA, 100 mM KCl, pH 7.5, the rest is water) to resuspend the cells, and then place them at room temperature for 5 min; centrifuge at 6500 g for 10 min, and resuspend the cell pellet with 100 mL of TESB (equal volume to YPL medium) (50 mM Tris-HCl, 1 mM EDTA, 600 mM D-sorbitol, the rest is water, pH 7.5), and then place it on ice; break the cell suspension on a homogenizer (the cooling water is pre-cooled to 2 °C) at a pressure above 1000 bar for 15 - 20 times until the cell suspension becomes milky and turbid; after each break, wait for the temperature to drop back to 2 °C before breaking the next tube; centrifuge the broken cell suspension at 12000 rpm at 4 °C for 20 min, pour the supernatant into a bottle containing 10 g of PEG4000 and 0.88 g of NaCl, and ice-bath for 15 - 30 min, shaking 3 - 5 times during this period until all the solids are dissolved; centrifuge at 12000 rpm at 4 °C for 20 min, discard the supernatant, invert the centrifuge tubes on absorbent paper for several seconds and then place them on ice; add 1000 μL of TEG (50 mM Tris-HCl, 1 mM EDTA, 20% (v / v) glycerol, pH 7.5), and after pipetting evenly, obtain the microsomal extracts of recombinant yeast (WAT11 / pESC-ura-CYP82BC3), recombinant yeast (WAT11 / pESC-ura-CYP80Q4), and recombinant empty vector yeast (WAT11 / pESC-URA) respectively, and store them at -80 °C or use them directly for enzymatic reactions. The microsomal extract of recombinant yeast (WAT11 / pESC-ura-CYP82BC3) contains the recombinant enzyme CYP82BC3 with the amino acid sequence as sequence 2 in the sequence listing, and the microsomal extract of recombinant yeast (WAT11 / pESC-ura-CYP80Q4) contains the CYP80Q4 protein (the amino acid sequence is sequence 4 in the sequence listing).

[0151] 4. Enzymatic reaction of CYP80Q4 enzyme alone and product extraction

[0152] 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) of microsomal CYP80Q4 enzyme, and 20 μM of substrate.

[0153] Among them, the enzyme in the experimental group's enzymatic reaction system was the microsomal extract of the recombinant yeast WAT11 / pESC-ura-CYP80Q4 obtained in step 1 (containing CYP80Q4 protein); the enzyme in the control group's enzymatic reaction system was the microsomal extract of the recombinant empty vector yeast WAT11 / pESC-URA obtained in step 4.

[0154] The substrate was divided into three groups, with each group consisting of two compounds, 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 Factory) was added, and ultrasonic extraction was performed 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 evaporator, 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.

[0155] The three groups of substrates were as follows:

[0156] ①: One molecule of (S)-coclaurine and one molecule of (R)-coclaurine;

[0157] ②: One molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine;

[0158] ③: One molecule of (S)-N-methylcoclaurine and one molecule of (R)-N-methylcoclaurine;

[0159] Among them, the Chinese name of (S)-coclaurine is (S)-linderalactam; the Chinese name of (R)-coclaurine is (R)-linderalactam; the Chinese name of (R)-N-methylcoclaurine is (R)-N-methyl-linderalactam; the Chinese name of (S)-N-methylcoclaurine is (S)-N-methyl-linderalactam.

[0160] Detection of reaction products:

[0161] 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 three groups of substrates underwent C-O coupling reactions to generate bisbenzylisoquinoline alkaloids ( Figure 1 ① - ③ below). The specific reactions and products are as follows:

[0162] ①: One molecule of (S)-coclaurine 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)-lindoldhamine, m / z is 569). Figure 2 The chromatogram of the reaction product is shown in Figure 3 The mass spectrum of the reaction product is shown in

[0163] (S)-coclaurine + (R)-coclaurine → (R,S)-Lindoldhamine;

[0164] ②: One molecule of (S)-coclaurine and one molecule of (R)-N-methylcoclaurine underwent a C-O coupling reaction to form 2’-Norberbamunine (2’-norberbamine, CID: 441063). Figure 4 The chromatogram of the reaction product is shown in Figure 5 The mass spectrum of the reaction product is shown in

[0165] (S)-coclaurine + (R)-N-methylcoclaurine → 2’-Norberbamunine;

[0166] ③: One molecule of (S)-N-methylcoclaurine and one molecule of (R)-N-methylcoclaurine underwent C-O coupling to form the bisbenzylisoquinoline alkaloid Berbamunine (berbamine, m / z is 597). Figure 6 The chromatogram of the reaction product is shown in Figure 7 The mass spectrum of the reaction product is shown in

[0167] (S)-N-methylcoclaurine + (R)-N-methylcoclaurine → Berbamunine.

[0168] Among them, the structural formula of (R,S)-Lindoldhamine is as shown in Formula 1 or Formula 1-1 below; the structural formula of 2’-Norberbamunine is as shown in Formula 2 or Formula 2-1 below; the structural formula of Berbamunine is as shown in Formula 3 or Formula 3-1 below:

[0169]

[0170]

[0171] 5. Combined enzymatic reaction of CYP80Q4 and CYP82BC3 proteins and product extraction:

[0172] 5.1 Combined enzymatic reaction

[0173] 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, 250 μg CYP82BC3 microsomal protein, and 30 μM substrate (30 μM for each substrate).

[0174] Among them, the CYP80Q4 microsomal protein is the microsomal extract of the recombinant yeast WAT11 / pESC-ura-CYP80Q4 obtained in step 3; the CYP82BC3 microsomal protein is the microsomal extract of the recombinant yeast WAT11 / pESC-ura-CYP82BC3 obtained in step 3 (containing the recombinant enzyme CYP82BC3); the enzyme in the control group enzymatic reaction system is the microsomal extract of the recombinant empty yeast WAT11 / pESC-URA obtained in step 3.

[0175] Among them, the substrates are divided into 3 groups:

[0176] ① (S)-coclaurine and (R)-coclaurine;

[0177] ② (S)-coclaurine and (R)-N-methylcoclaurine;

[0178] ③ (S)-N-methylcoclaurine and (R)-N-methylcoclaurine.

[0179] 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 performed 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 evaporator, redissolved with 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 (Waters Xevo G2-S QTOF instrument) for detection.

[0180] 5.2 Detection of reaction products:

[0181] The enzymatic reaction products were detected by UPLC-QTOF-MS. The results showed that compared with the reaction products of CYP80Q4 alone catalyzing the substrate in step 4 ( Figure 2 , Figure 4 and Figure 6 ), new compounds were generated in the enzymatic reaction system of CYP82BC3 and CYP80Q4 mixed catalyzing the substrate. 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 it was the bisbenzylisoquinoline alkaloid after the C-O coupling reaction and cyclization of the bisbenzylisoquinoline alkaloid catalyzed by CYP80Q4 ( Figure 8 ). Therefore, the enzymatic reaction results showed that CYP82BC3 had the function of catalyzing the formation of cyclic bisbenzylisoquinoline alkaloids from bisbenzylisoquinoline alkaloids.

[0182] In the enzymatic reaction of Group ① substrates, CYP80Q4 catalyzes the formation of (R,S)-Lindoldhamine ((R,S)-lindoldhamine) from (S)-coclaurine and (R)-coclaurine. CYP82BC3 catalyzes the C-O coupling of the O on C7’ of (R,S)-Lindoldhamine with the C on C8 to form Bisnorobamegine (m / z 567, chromatogram and mass spectrum as shown in Figure 9 and Figure 10 shown, chemical structure diagram as shown in Figure 15 shown, NMR results as shown in Figures 16 - 17 shown), and at the same time, a compound Bisnoraromoline with a relatively low yield is also produced (m / z 567, chromatogram and mass spectrum as shown in Figure 9 and Figure 10 shown, chemical structure diagram as shown in Figure 18 shown, NMR results as shown in Figures 19 - 20 shown).

[0183] 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 form 2’-Norberbamunine (2’-demethylberberine, CID: 441063). CYP82BC3 then catalyzes the C-O coupling of the O on C7’ of 2’-Norberbamunine with the C on C8 to form 2’-Norobamegine (m / z 581, chromatogram and mass spectrum as shown in Figure 11 and Figure 12 ), and at the same time, a compound racemosinine A with a relatively low yield is also produced (m / z 581, chromatogram and mass spectrum as shown in Figure 11 and Figure 12 ).

[0184] In Reaction ③, CYP80Q4 catalyzes the C-O coupling of (S)-N-methylcoclaurine and (R)-N-methylcoclaurine to form Berbamunine, and CYP82BC3 then catalyzes the C-O coupling of the O on C7’ of Berbamunine with the C on C8 to form Obamegine (chromatogram and mass spectrum as shown in Figure 13 and Figure 14 ). The specific process is as shown in Figure 8 shown.

[0185] The chemical structural formula of the cyclized bisbenzylisoquinoline alkaloid Bisnorobamegine is as follows in Formula 4; the chemical structural formula of the cyclized bisbenzylisoquinoline alkaloid 2’-Norobamegine is as follows in Formula 5; the chemical structural formula of the cyclized bisbenzylisoquinoline alkaloid Obamegine is as follows in Formula 6:

[0186]

[0187] The chemical structural formula of Bisnoraromoline is as follows in Formula 7; the chemical structural formula of racemosinine A is as follows in Formula 8:

[0188]

[0189] From the catalytic results, it can be seen that the products generated by CYP82BC3 in this application are more specific, that is, more products with C-O coupling between the O on C7’ and the C on C8 are generated, and very few unknown compounds are produced. When used for heterologous production of bisbenzylisoquinoline compounds, it can more specifically produce cyclized bisbenzylisoquinoline alkaloids with C7’-C8 coupling. In addition to catalyzing reaction ③, CYP82BC3 can also convert the substrate of reaction ② into products to a greater extent, and can be better used for the production of bisbenzylisoquinoline alkaloids with both S- and R-types present.

[0190] The above details the present invention. For those skilled in the art, without departing from the gist and scope of the present invention and without 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 changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of any one of the following proteins: M1) Use of the protein in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids; M2) Use 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) Use 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) Use of the protein in catalyzing the C-O coupling of the O on C7’ of Berbamunine with the C on C8 to produce cyclized bisbenzylisoquinoline alkaloids; M5) Use of the protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products; M6) Use of the protein in the production, preparation or development of products related to tetrandrine; M7) Use of the protein in the breeding of Stephania tetrandra; The protein is a protein of any of the following A1), A2) or A3): 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 one or several 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: The Berbamunine is a compound with the structural formula of formula 3:

2. The application according to claim 1, characterized in that: The bisbenzylisoquinoline alkaloids are (R,S)-Lindoldhamine, 2’-Norberbamunine or Berbamunine; the cyclized bisbenzylisoquinoline alkaloids in M2) are Bisnorobamegine and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloids in M3) are 2’-Norobamegine and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloids in M4) are Obamegine; The Bisnorobamegine is a compound with the structural formula of formula 4: The 2’-Norobamegine is a compound with the structural formula of formula 5: The Obamegine is a compound with the structural formula of formula 6:

3. Use of any one of the following biomaterials related to the protein described in claim 1: N1) Use of the biomaterial in catalyzing the C-O coupling of bisbenzylisoquinoline alkaloids to produce cyclized bisbenzylisoquinoline alkaloids; N2) Use of the biological material in catalyzing the C-O coupling of the O on C7' and the C on C8 of (R,S)-Lindoldhamine to form a cyclized bisbenzylisoquinoline alkaloid; N3) Use of the biological material in catalyzing the C-O coupling of the O on C7' and the C on C8 of 2'-Norberbamunine to form a cyclized bisbenzylisoquinoline alkaloid; N4) Use of the protein in catalyzing the C-O coupling of the O on C7' and the C on C8 of Berbamunine to form a cyclized bisbenzylisoquinoline alkaloid; N5) Use of the protein in the production, preparation or development of cyclized bisbenzylisoquinoline alkaloid products; N6) Use of the protein in the production, preparation or development of products related to tetrandrine; N7) Use of the protein in fangji breeding products; The biological material is any one of the following D1) to D6): 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: The Berbamunine is a compound with the structural formula of Formula 3:

4. The application according to claim 3, wherein: D1) The nucleic acid molecule is the coding gene of the protein shown in the following d1) or d2): d1) A cDNA molecule or DNA molecule whose coding sequence is the nucleotide 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, 2'-Norberbamunine or Berbamunine; the cyclized bisbenzylisoquinoline alkaloid described in N2) is Bisnorobamegine and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloid described in N3) is 2'-Norobamegine and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloid described in N4) is Obamegine; The Bisnorobamegine is a compound with the structural formula of Formula 4: The 2'-Norobamegine 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 preparing a product that catalyzes the C-O coupling of the O on C7' and the C on C8 of (R,S)-Lindoldhamine to form cyclized bisbenzylisoquinoline alkaloids; P3, a product for preparing a product that catalyzes the C-O coupling of the O on C7' and the C on C8 of 2'-Norberbamunine to form cyclized bisbenzylisoquinoline alkaloids; P4, a product for preparing a product that catalyzes the C-O coupling of the O on C7' and the C on C8 of Berbamunine to form cyclized bisbenzylisoquinoline alkaloids; P5, a product for producing, preparing or developing 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: The Berbamunine is a compound with the structural formula of Formula 3:

7. The product according to claim 6, wherein: The bisbenzylisoquinoline alkaloids are (R,S)-Lindoldhamine, 2'-Norberbamunine or Berbamunine; the cyclized bisbenzylisoquinoline alkaloids in P2) are Bisnorobamegine and / or Bisnoraromoline; the cyclized bisbenzylisoquinoline alkaloids in P3) are 2'-Norobamegine and / or racemosinine A; the cyclized bisbenzylisoquinoline alkaloids in P4) are Obamegine; the Bisnorobamegine is a compound with the structural formula of Formula 4: The 2'-Norobamegine is a compound with the structural formula of Formula 5: The Obamegine is a compound with the structural formula of Formula 6:

8. A method for preparing cytochrome P450 enzyme CYP82BC3, 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 CYP82BC3.

9. The method according to claim 8, characterized in that: The expression includes introducing the coding gene of the protein described in claim 1 into a recipient microorganism to obtain a recombinant microorganism expressing the cytochrome P450 enzyme CYP82BC3, culturing the recombinant microorganism, and expressing to obtain the cytochrome P450 enzyme CYP82BC3.

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

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