Methyltransferase, biological material related to methyltransferase and application of methyltransferase in preparation of bisbenzylisoquinoline alkaloid

Abstract

The invention discloses methyltransferase, a biological material related to the methyltransferase and application of the methyltransferase in preparation of bisbenzylisoquinoline alkaloid. The methyltransferase disclosed by the invention is StB2 'NMT1, StB7OMT, StB2' NMT2 and StB2NMT, and the sequences of the StB2 'NMT1, the StB7OMT, the StB2' NMT2 and the StB2NMT are respectively shown as SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 and SEQ ID No.10 in a sequence table. The methyltransferase disclosed by the invention can be used for producing isotetrandrine with StBOMT as shown in SEQ ID No.2 and CYP82BC4 as shown in SEQ ID No.13 by taking (R, S)-lindoldhamine as a substrate, and the isotetrandrine can be produced by the methyltransferase, the StBOMT as shown in SEQ ID No.2 and the CYP82BC4 as shown in SEQ ID No.3.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, methyltransferase and related biological materials and applications thereof in preparing bisbenzylisoquinoline alkaloids. Background Art

[0002] Secondary metabolites are the primary source of bioactive components in medicinal plants. The rapid development of omics technologies in recent years has laid a foundation for the discovery and identification of genes involved in the biosynthesis of secondary metabolites in medicinal plants. Cloning and functional validation of genes involved in secondary metabolite synthesis provide essential biological components for metabolic engineering and synthetic biology research in natural products, and also offer insights into the selection, breeding, and improvement of medicinal plants.

[0003] Stephania tetrandra (Stephania tetrandra S. Moore) is a perennial vine of the genus Stephania in the family Menispermaceae. Its roots are the botanical source of the traditional Chinese medicine Stephania tetrandra, which has the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling. Currently, the main types of compounds isolated and identified from Stephania tetrandra include alkaloids, steroids, and flavonoids. Alkaloids, with their abundant content, diverse skeletons, and significant pharmacological activity, have long been an important focus of research on the active substance basis of Stephania tetrandra in traditional Chinese medicine. A review of the alkaloids isolated from Stephania tetrandra revealed that they are primarily bisbenzylisoquinoline alkaloids (BBIQs), such as tetrandrine, isotetrandrine, fangchinoline, and berbamine.

[0004] Research has shown that the upstream pathway for the biosynthesis of BBIQ in Stephania tetrandra is consistent with that of other benzylisoquinoline alkaloids (BIAs) such as morphine. It begins with tyrosine and proceeds through decarboxylase, hydroxylase, transaminase, norcoclaurine synthase, norcoclaurine-6-O-methyltransferase, and norcoclaurine-N-methyltransferase to form the intermediate compound N-methylcoclaurine. Based on the structure, it is speculated that two molecules of N-methylcoclaurine undergo head-to-head and tail-to-tail CO coupling to form the basic skeleton of BBIQ, which is then modified by methylation to form various compounds. Therefore, methyltransferase (MT) is a key enzyme in the biosynthesis of BBIQ in Stephania tetrandra.

[0005] Methyltransferases are a relatively common class of enzymes in the BIA biosynthetic pathway, primarily including oxygen-methyltransferases (OMTs) and nitrogen-methyltransferases (NMTs). Some of these enzymes exhibit high substrate specificity, while others can simultaneously catalyze the methylation of multiple positions. All MTs in the BIA biosynthetic pathway do not require metal ions as cofactors; instead, they rely on S-adenosyl-L-methionine (AdoMet) as a methyl donor. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a methyltransferase that can be used to catalyze the production of isotetrandrine.

[0007] To solve the above technical problems, the present invention first provides a protein, which is StB2'NMT1, StB7OMT, StB2'NMT2 or StB2NMT;

[0008] The StB2'NMT1 is derived from Stephania tetrandra S. Moore and is the following A1), A2) or A3):

[0009] A1) the amino acid sequence is the protein of SEQ ID No. 4 in the sequence listing;

[0010] A2) a protein derived from A1) or having 70% or more identity with the protein of A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of the protein of A1) and having the same function;

[0011] A3) A fusion protein obtained by fusing a protein tag at the carboxyl or / and amino terminus of the protein shown in A1) or A2);

[0012] The StB7OMT is derived from Stephania tetrandra S. Moore and is as follows B1), B2) or B3):

[0013] B1) the amino acid sequence is the protein of SEQ ID No. 6 in the sequence listing;

[0014] B2) a protein derived from B1) or having 70% or more identity with the protein of B1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of B1) and having the same function;

[0015] B3) A fusion protein obtained by fusing a protein tag at the carboxyl or / and amino terminus of the protein shown in B1) or B2);

[0016] The StB2'NMT2 is derived from Stephania tetrandra S. Moore and is as follows C1), C2) or C3):

[0017] C1) the amino acid sequence is the protein of SEQ ID No. 8 in the sequence listing;

[0018] C2) a protein derived from C1) or having 70% or more identity with the protein of C1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of C1) and having the same function;

[0019] C3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in C1) or C2);

[0020] The StB2NMT is derived from Stephania tetrandra S. Moore and is as follows D1), D2) or D3):

[0021] D1) the amino acid sequence is the protein of SEQ ID No. 10 in the sequence listing;

[0022] D2) a protein derived from D1) or having 70% or more identity with the protein shown in D1) and having the same function as the protein shown in D1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein shown in D1);

[0023] D3) A fusion protein obtained by fusing a protein tag to the carboxyl terminus or / and amino terminus of the protein shown in D1) or D2).

[0024] The term "70% or more identity" refers to 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.

[0025] The proteins in A2), B2), C2) and D2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0026] The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence set forth in SEQ ID No. 3, and / or performing one or more base pair missense mutations, and / or attaching a tag coding sequence to its 5' and / or 3' ends. The DNA molecule set forth in SEQ ID No. 3 encodes the StB2'NMT1 protein set forth in SEQ ID No. 4.

[0027] The gene encoding the protein in B2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence shown in SEQ ID No. 5, and / or performing one or more base pair missense mutations, and / or attaching a tag to the coding sequence at its 5' and / or 3' end. The DNA molecule shown in SEQ ID No. 5 encodes the StB7OMT protein shown in SEQ ID No. 6.

[0028] The gene encoding the protein in C2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence set forth in SEQ ID No. 7, and / or performing one or more base pair missense mutations, and / or attaching a tag to the coding sequence at its 5' and / or 3' end. The DNA molecule set forth in SEQ ID No. 7 encodes the StB2'NMT2 protein set forth in SEQ ID No. 8.

[0029] The gene encoding the protein in D2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence set forth in SEQ ID No. 9, and / or performing one or more base pair missense mutations, and / or attaching a tag to the coding sequence at its 5′ and / or 3′ ends. The DNA molecule set forth in SEQ ID No. 9 encodes the StB2NMT protein set forth in SEQ ID No. 10.

[0030] The proteins described in A2), B2), C2) and D2) can all be derived from Stephania tetrandra S. Moore.

[0031] The tag described in A3), B3), C3), and D3) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be a Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0032] To solve the above technical problems, the present invention first provides a biomaterial related to the protein, wherein the biomaterial is any one of the following H1) to H9):

[0033] H1) a nucleic acid molecule encoding the protein;

[0034] H2) an expression cassette containing the nucleic acid molecule described in H1);

[0035] H3) a recombinant vector containing the nucleic acid molecule described in H1), or a recombinant vector containing the expression cassette described in H2);

[0036] H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3);

[0037] H5) a transgenic plant cell line containing the nucleic acid molecule described in H1) or a transgenic plant cell line containing the expression cassette described in H2);

[0038] H6) transgenic plant tissue containing the nucleic acid molecule described in H1) or transgenic plant tissue containing the expression cassette described in H2);

[0039] H7) a transgenic plant organ containing the nucleic acid molecule described in H1) or a transgenic plant organ containing the expression cassette described in H2);

[0040] H8) a nucleic acid molecule that promotes or increases the expression of the protein;

[0041] H9) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in H8).

[0042] In the above-mentioned biological material, the nucleic acid molecule in H1) may be any one of the following b1) to b10):

[0043] b1) a DNA molecule whose coding sequence is SEQ ID No.3 in the sequence listing; b2) a DNA molecule shown as SEQ ID No.3 in the sequence listing; b3) a DNA molecule whose coding sequence is SEQ ID No.5 in the sequence listing; b4) a DNA molecule shown as SEQ ID No.5 in the sequence listing; b5) a DNA molecule whose coding sequence is SEQ ID No.7 in the sequence listing; b6) a DNA molecule shown as SEQ ID No.7 in the sequence listing; b7) a DNA molecule whose coding sequence is SEQ ID No.9 in the sequence listing; b8) a DNA molecule shown as SEQ ID No.9 in the sequence listing; b9) a DNA molecule that has 75% or more identity with the nucleotide sequence specified in any one of b1) to b8) and encodes the protein; b10) a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence specified in any one of b1) to b9) and encodes the protein.

[0044] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0045] Those skilled in the art can readily mutate the nucleotide sequences encoding the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT proteins of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequences of the isolated StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT proteins of the present invention are derived from and are equivalent to the nucleotide sequences of the present invention, as long as they encode the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT proteins and have the function of the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT proteins.

[0046] As used herein, the term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, or SEQ ID No. 10 of the present invention. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences.

[0047] In the above biological materials, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 1×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC, 0.1% The membrane can be rinsed in SDS; alternatively, hybridization can be performed at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and then rinsed in 0.1×SSC, 0.1% SDS at 65°C; alternatively, hybridization can be performed at 65°C in a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS; alternatively, hybridization and washing the membrane twice at 68°C in a solution of 2×SSC, 0.1% SDS, each for 5 minutes, and then hybridization and washing the membrane twice at 68°C in a solution of 0.5×SSC, 0.1% SDS, each for 15 minutes; alternatively, hybridization and washing the membrane in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, at 65°C.

[0048] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0049] In the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule encoding the protein described in B2) (i.e., the StB2'NMT1 gene expression cassette, the StB7OMT gene expression cassette, the StB2'NMT2 gene expression cassette, or the StB2NMT gene expression cassette) refers to DNA capable of expressing the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT protein in a host cell. This DNA may include not only a promoter that initiates transcription of the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT gene, but also a terminator that terminates transcription of the StB2'NMT1, StB7OMT, StB2'NMT2, or StB2NMT gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0050] Existing expression vectors can be used to construct recombinant vectors containing the above-mentioned gene expression cassettes.

[0051] In the above-mentioned biological material, the vector can be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be a pET-32a(+) vector.

[0052] B3) The recombinant vector may specifically be StB2'NMT1-pET-32a, StB7OMT-pET-32a, StB2'NMT2-pET-32a, or StB2NMT-pET-32a. Among them, StB2'NMT1-pET-32a is a recombinant vector obtained by inserting the StB2'NMT1 gene shown in SEQ ID No. 3 between the multiple cloning sites of the pET-32a(+) vector, and the recombinant vector can express a recombinant protein formed by the StB2'NMT1 protein shown in SEQ ID No. 4 and the polypeptide shown in SEQ ID No. 11; StB7OMT-pET-32a is a recombinant vector obtained by inserting the StB7OMT gene shown in SEQ ID No. 5 between the multiple cloning sites of the pET-32a(+) vector, and the recombinant vector can express a recombinant protein formed by the StB7OMT protein shown in SEQ ID No. 6 and the polypeptide shown in SEQ ID No. 11; StB2'NMT2-pET-32a is a recombinant vector obtained by inserting the StB2'NMT2 gene shown in SEQ ID No. 7 between the multiple cloning sites of the pET-32a(+) vector, and the recombinant vector can express the StB2'NMT2 protein shown in SEQ ID No. 8 and the polypeptide shown in SEQ ID The recombinant protein formed by the polypeptide shown in No.11; StB2NMT-pET-32a is a recombinant vector obtained by inserting the StB2NMT gene shown in SEQ ID No.9 between the multiple cloning sites of the pET-32a(+) vector, and the recombinant vector can express the recombinant protein formed by the StB2NMT protein shown in SEQ ID No.10 and the polypeptide shown in SEQ ID No.11.

[0053] In the above applications, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacteria may be Escherichia coli, such as Escherichia coli BL21 (DE3).

[0054] In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0055] The use of StB2'NMT1, StB7OMT, StB2'NMT2 or StB2NMT as a methyltransferase also falls within the scope of protection of the present invention.

[0056] In the above application, the StB2NMT, the StB2'NMT1 and the StB2'NMT2 are all nitrogen methyltransferases;

[0057] The StB7OMT is an oxygen methyltransferase.

[0058] In one embodiment of the present invention, the substrate is (R,S)-lindoldhamine, whose structural formula is shown in Formula I. StB2'NMT1 and StB2'NMT2 can both catalyze the methylation of the substrate at the N2' position, StB2NMT can catalyze the methylation of the substrate at the N2 position, and StB7OMT can catalyze the methylation of the hydroxyl group at the 7 position.

[0059]

[0060] The present invention also provides the use of StB2'NMT1, StB7OMT, StB2'NMT2 or StB2NMT or the biological materials in producing bisbenzylisoquinoline alkaloids, or in preparing products for producing bisbenzylisoquinoline alkaloids, or in catalyzing the methylation of the parent nucleus of bisbenzylisoquinoline alkaloids, or in preparing products for catalyzing the methylation of the parent nucleus of bisbenzylisoquinoline alkaloids.

[0061] In the above application, the bisbenzylisoquinoline alkaloid may be isotetrandrine.

[0062] The present invention also provides a product, which contains M1), M2) and / or M3):

[0063] M1) the StB2NMT or the biological material related to the StB2NMT;

[0064] M2) the StB7OMT or the biological material related to the StB7OMT;

[0065] M3) the StB2'NMT1, the biological material related to the StB2'NMT1, the StB2'NMT2, or the biological material related to the StB2'NMT2.

[0066] The above-mentioned product may further contain StBOMT or biological materials related to said StBOMT, and / or, CYP82BC4 or biological materials related to said CYP82BC4;

[0067] The CYP82BC4 is the following E1), E2) or E3):

[0068] E1) the amino acid sequence is the protein of SEQ ID No. 13 in the sequence listing;

[0069] E2) a protein derived from E1) or having 70% or more identity with the protein of E1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of E1) and having the same function;

[0070] E3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in E1) or E2);

[0071] The biological material related to the CYP82BC4 is any one of the following I1) to I9):

[0072] I1) a nucleic acid molecule encoding the CYP82BC4;

[0073] I2) an expression cassette containing the nucleic acid molecule described in I1);

[0074] I3) a recombinant vector containing the nucleic acid molecule described in I1), or a recombinant vector containing the expression cassette described in I2);

[0075] I4) a recombinant microorganism containing the nucleic acid molecule described in I1), or a recombinant microorganism containing the expression cassette described in I2), or a recombinant microorganism containing the recombinant vector described in I3);

[0076] I5) a transgenic plant cell line containing the nucleic acid molecule described in I1), or a transgenic plant cell line containing the expression cassette described in I2);

[0077] I6) transgenic plant tissue containing the nucleic acid molecule described in I1), or transgenic plant tissue containing the expression cassette described in I2);

[0078] I7) a transgenic plant organ containing the nucleic acid molecule described in I1), or a transgenic plant organ containing the expression cassette described in I2);

[0079] I8) a nucleic acid molecule that promotes or increases the expression of CYP82BC4;

[0080] I9) an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in I8);

[0081] The StBOMT is as follows G1), G2) or G3):

[0082] G1) the amino acid sequence is the protein of SEQ ID No. 2 in the sequence listing;

[0083] G2) a protein derived from G1) or having 70% or more identity with the protein of G1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of G1) and having the same function;

[0084] G3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in G1) or G2);

[0085] The biological material related to the StBOMT is any one of the following K1) to K9):

[0086] K1) a nucleic acid molecule encoding the StBOMT;

[0087] K2) an expression cassette containing the nucleic acid molecule described in K1);

[0088] K3) a recombinant vector containing the nucleic acid molecule described in K1), or a recombinant vector containing the expression cassette described in K2);

[0089] K4) a recombinant microorganism containing the nucleic acid molecule described in K1), or a recombinant microorganism containing the expression cassette described in K2), or a recombinant microorganism containing the recombinant vector described in K3);

[0090] K5) a transgenic plant cell line containing the nucleic acid molecule described in K1) or a transgenic plant cell line containing the expression cassette described in K2);

[0091] K6) transgenic plant tissue containing the nucleic acid molecule described in K1) or transgenic plant tissue containing the expression cassette described in K2);

[0092] K7) A transgenic plant organ containing the nucleic acid molecule described in K1) or a transgenic plant organ containing the expression cassette described in K2);

[0093] K8) a nucleic acid molecule that promotes or increases the expression of StBOMT;

[0094] K9) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in K8).

[0095] The term "70% or more identity" refers to 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Perresidue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.

[0096] The proteins in E2) and G2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0097] The gene encoding the protein in E2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence set forth in SEQ ID No. 12, and / or performing one or more base pair missense mutations, and / or attaching a tag to the coding sequence at its 5′ and / or 3′ end. The DNA molecule set forth in SEQ ID No. 12 encodes the CYP82BC4 protein set forth in SEQ ID No. 13.

[0098] The gene encoding the protein in G2) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence shown in SEQ ID No. 1, and / or performing one or more base pair missense mutations, and / or attaching a tag to the coding sequence at its 5′ and / or 3′ end. The DNA molecule shown in SEQ ID No. 1 encodes the StBOMT protein shown in SEQ ID No. 2.

[0099] The proteins described in E2) and G2) can both be derived from Stephania tetrandra S. Moore.

[0100] The tag in E2) and G2) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0101] In the above product, the nucleic acid molecule described in I1) may be any one of the following c1) to c4):

[0102] c1) the coding sequence is the DNA molecule of SEQ ID No. 12 in the sequence listing;

[0103] c2) the DNA molecule shown in SEQ ID No. 12 in the sequence listing;

[0104] c3) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in c1) or c2) and encodes CYP82BC4;

[0105] c4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence specified in any one of c1) to c3) and encodes CYP82BC4.

[0106] In the above product, the nucleic acid molecule in K1) may be any one of the following d1) to d4):

[0107] d1) the coding sequence is the DNA molecule of SEQ ID No. 1 in the sequence listing;

[0108] d2) the DNA molecule shown in SEQ ID No. 1 in the sequence listing;

[0109] d3) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) or d2) and encodes StBOMT;

[0110] d4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence specified in any one of d1) to d3) and encodes StBOMT.

[0111] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0112] In the above-mentioned products, the expression cassette containing a nucleic acid molecule encoding CYP82BC4 (i.e., a CYP82BC4 gene expression cassette) described in I2) refers to DNA capable of expressing the CYP82BC4 protein in a host cell. This DNA may include not only a promoter that initiates transcription of the CYP82BC4 gene, but also a terminator that terminates transcription of the CYP82BC4 gene. The expression cassette containing a nucleic acid molecule encoding StBOMT (i.e., a StBOMT gene expression cassette) described in K2) refers to DNA capable of expressing the StBOMT protein in a host cell. This DNA may include not only a promoter that initiates transcription of the StBOMT gene, but also a terminator that terminates transcription of the StBOMT gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0113] Existing expression vectors can be used to construct recombinant vectors containing the above-mentioned gene expression cassettes.

[0114] The vector may be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid may be a pET-32a(+) vector or a pESC-URA vector.

[0115] I3) The recombinant vector may specifically be CYP82BC4-pESC-URA, wherein CYP82BC4-pESC-URA is a recombinant vector obtained by inserting the CYP82BC4 gene shown in SEQ ID No. 12 in the sequence list into the BamH1 site of the pESC-URA vector using BamH1. The recombinant vector can express a recombinant protein formed by the CYP82BC4 protein shown in SEQ ID No. 13 and the corresponding polypeptide encoded on the pESC-URA vector.

[0116] K3) The recombinant vector may specifically be StBOMT-pET-32a. StBOMT-pET-32a is a recombinant vector obtained by inserting the StBOMT gene shown in SEQ ID No. 1 between the BamH1 sites of the pET-32a(+) vector using BamH1. This recombinant vector is capable of expressing a recombinant protein formed by the StBOMT protein shown in SEQ ID No. 2 and the polypeptide shown in SEQ ID No. 11.

[0117] In the above product, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Escherichia coli, such as Escherichia coli BL21 (DE3). The yeast can be Saccharomyces cerevisiae, such as Saccharomyces cerevisiae WAT11U.

[0118] In the above products, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include reproductive materials.

[0119] The above products may also contain (R,S)-lindoldhamine.

[0120] The above-mentioned product may be the above-mentioned M1), M2) and / or M3), and may also be composed of at least one of the above-mentioned M1), M2) and M3) and StBOMT or a biomaterial related to the StBOMT, and may also be composed of at least one of the above-mentioned M1), M2) and M3) and CYP82BC4 or a biomaterial related to the CYP82BC4, and may also be composed of at least one of the above-mentioned M1), M2) and M3) and StBOMT or a biomaterial related to the StBOMT, as well as CYP82BC4 or a biomaterial related to the CYP82BC4.

[0121] The product can be used for producing bisbenzylisoquinoline alkaloids and can also be used for preparing reagents for producing bisbenzylisoquinoline alkaloids.

[0122] In the above product, the bisbenzylisoquinoline alkaloid may be isotetrandrine.

[0123] The present invention also provides a method for producing isotetrandrine, comprising: using (R, S)-lindoldhamine as a substrate, utilizing the CYP82BC4 and the StBOMT, the StB2'NMT1 or the StB2'NMT2, the StB7OMT and the StB2NMT to carry out a catalytic reaction to produce isotetrandrine.

[0124] The reaction system of the above method may further contain a methyl donor, NADPH, FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), G6P (glucose-6-phosphate), G6PDH (glucose-6-phosphate dehydrogenase), DTT (dithiothreitol), and / or potassium phosphate. In one embodiment of the present invention, the methyl donor is S-adenosylmethionine.

[0125] The reaction of the above method can be carried out at 30°C.

[0126] This study systematically investigates the methyltransferases involved in the biosynthesis of bisbenzylisoquinoline alkaloids (BBIQs). The study characterizes one oxygen methyltransferase (StB7OMT) and three nitrogen methyltransferases (StB2'NMT1, StB2'NMT2, and StB2NMT) that specifically catalyze the formation of BBIQ compounds. These enzymes, using AdoMet as a methyl donor, catalyze the methylation of (R,S)-lindoldhamine to produce isotetrandrine. The methyltransferase proteins and their encoding genes can be used to construct engineered bacteria for the production of BIAs.

[0127] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1 Figure 2 is the SDS-PAGE electrophoresis of the purified StB2'NMT1, StB2'NMT2, StB2NMT, and St7OMT fusion proteins. M is a protein marker.

[0129] Figure 2 This is the reaction process of StB2'NMT1, StB2'NMT2, StB2NMT, St7OMT and the synthesis process of isotetrandrine.

[0130] Figure 3 The mass spectra of each methylation product, the final product and the isotetrandrine standard are shown in FIG. DETAILED DESCRIPTION

[0131] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0132] BL21(DE3) Chemically Competent Cell: Product of Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD601-02.

[0133] pET-32a(+): Invitrogen, catalog number 69-015-3.

[0134] The Saccharomyces cerevisiae WAT11U used in the following examples is described in "Juan Guoa et al., CYP76AH1 catalyzes turnover of miltiradiene intanshinones biosynthesis and enables heterologous production of ferruginol in yeasts, PNAS, July 16, 2013, vol. 110, no. 29." The public can obtain this biological material from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0135] Example 1. StB2'NMT1, StB2'NMT2, StB2NMT and StB7OMT gene cloning and vector construction

[0136] Table 1. Specific primers with homology arms of BamHI site in pET-32a(+) vector

[0137]

[0138] Total RNA was extracted from the roots of Stephania tetrandra, then reverse-transcribed into cDNA. Primers were designed to amplify the complete open reading frame based on the coding sequence of the StMTs gene, and restriction endonuclease sites (BamHI) were added to the upstream and downstream primers (Table 1). PCR was performed using the cDNA as a template using each primer. After PCR amplification, the PCR product was ligated into the expression vector pET-32a(+) using the Gibson Assembly seamless splicing method to obtain the recombinant vector StMTs-pET-32a (a total of four recombinant vectors for the four genes). The four recombinant StMTs-pET-32a vectors were transformed into E. coli Trans T1 competent cells. Positive clones were screened using solid LB medium containing 50 μg / mL ampicillin sodium. Positive clones were then cultured in liquid LB medium containing 50 μg / mL ampicillin sodium, and the plasmids were extracted to obtain the positive recombinant vectors.

[0139] After sequencing, the four recombinant vectors with correct sequences were StB2'NMT1-pET-32a, StB7OMT-pET-32a, StB2'NMT2-pET-32a, and StB2NMT-pET-32a. StB2'NMT1-pET-32a contains the StB2'NMT1 gene shown in SEQ ID No.3, and can express a recombinant protein formed by the StB2'NMT1 protein shown in SEQ ID No.4 and the polypeptide shown in SEQ ID No.11 (i.e., recombinant StB2'NMT1 protein); StB7OMT-pET-32a contains the StB7OMT gene shown in SEQ ID No.5, and can express a recombinant protein formed by the StB7OMT protein shown in SEQ ID No.6 and the polypeptide shown in SEQ ID No.11 (i.e., recombinant StB7OMT); StB2'NMT2-pET-32a contains the StB2'NMT2 gene shown in SEQ ID No.7, and can express a recombinant protein formed by the StB2'NMT2 protein shown in SEQ ID No.8 and the polypeptide shown in SEQ ID No.11 (i.e., recombinant StB2'NMT2); StB2NMT-pET-32a contains the StB2NMT gene shown in SEQ ID No.9, and can express a recombinant protein formed by the StB2NMT protein shown in SEQ ID No.10 and the polypeptide shown in SEQ ID No.11. The recombinant protein formed by the polypeptide shown in ID No.11 (i.e., recombinant StB2NMT protein).

[0140] Example 2. Acquisition of recombinant protein and analysis of enzymatic reaction

[0141] 1. Protein Induction and Extraction

[0142] The four recombinant vectors StMTs-pET-32a were transformed into BL21(DE3) Chemically Competent Cells to obtain four recombinant E. coli BL21(DE3) / StMTs-pET-32a. The pET-32a(+) empty vector plasmid was transformed into BL21(DE3) Chemically Competent Cells to obtain an empty control recombinant E. coli BL21(DE3) / pET-32a.

[0143] The four recombinant Escherichia coli BL21 (DE3) / StMTs-pET-32a and the empty control recombinant Escherichia coli BL21 (DE3) / pET-32a were cultured according to the following steps: the strains were cultured in 1 mL of liquid LB medium containing 50 μg / mL ampicillin sodium at 37°C and 200 rpm for 7 h, 1 mL of the bacterial solution was transferred to 100 mL of liquid LB medium containing 50 μg / mL ampicillin sodium, and cultured at 37°C and 200 rpm for 3 h until the bacterial solution OD reached 0. 600nm After the value reaches between 0.6-0.8, the bacterial solution is taken out, cooled to 16°C, IPTG is added to a final concentration of 0.5 mM, and the culture is shaken at 16°C and 200 rpm in a shaker. The fermentation liquid is collected after 16 hours of induced expression. The four fermentation liquids obtained by induced expression of recombinant Escherichia coli BL21 (DE3) / StMTs-pET-32a are named induced StMTs-pET-32a whole bacterial liquid, and the fermentation liquid obtained by induced expression of recombinant Escherichia coli BL21 (DE3) / pET-32a is named induced empty whole bacterial liquid.

[0144] The induced StMTs-pET-32a whole bacterial solution was centrifuged at 8000rpm for 20min, and the resulting precipitate was the bacterial cell containing the recombinant StMTs protein. The bacterial precipitate was resuspended in pre-cooled 5mL Tris-HCl (100mM, pH 7.5) and ultrasonically disrupted (power 60%, ultrasonic 5s, stop 5s, total time 20min), and then centrifuged at 4°C, 10000rpm for 20min. The supernatant was the recombinant StMTs crude protein. The supernatant was transferred to a 1ml nickel column (Kangwei Century, Beijing) for affinity chromatography, washed with 10mM imidazole buffer until Coomassie Brilliant Blue did not turn blue, the eluate was discarded, and then washed with 75mM imidazole buffer until Coomassie Brilliant Blue did not turn blue, and the eluate was collected, which was the purified recombinant StMTs protein. The SDS-PAGE electrophoresis results are shown in FIG. Figure 1 As shown, the target recombinant StOMT protein has a size between 55-70 kDa, which is in line with expectations.

[0145] The empty whole bacterial suspension was treated in the same way to obtain the purified protein of the empty control.

[0146] A recombinant vector, StBOMT-pET-32a, containing the StBOMT gene of SEQ ID No. 1 was constructed according to the method of Example 1. StBOMT-pET-32a contains the StBOMT gene of SEQ ID No. 1 and is capable of expressing a recombinant protein formed by the StBOMT protein of SEQ ID No. 2 and the polypeptide of SEQ ID No. 11. The recombinant vector was then replaced with StBOMT-pET-32a according to the above-described protein expression and purification methods to obtain purified recombinant StBOMT protein.

[0147] 2. Preparation of CYP82BC4 Microsomes

[0148] 2.1 Preparation of recombinant vector

[0149] The CYP82BC4 gene shown in SEQ ID No. 12 in the sequence list was inserted into the BamH1 site of the pESC-URA vector using BamH1 to obtain the recombinant vector CYP82BC4-pESC-URA, which can express a recombinant protein (denoted as CYP82BC4 recombinant protein) formed by the CYP82BC4 protein shown in SEQ ID No. 13 and the corresponding polypeptide encoded on the pESC-URA vector.

[0150] 2.2 Yeast transformation and culture

[0151] The recombinant vector CYP82BC4-pESC-URA was transformed into Saccharomyces cerevisiae WAT11U competent cells using the Frozen-EZ Yeast Transformation II™ Kit (ZYMO RESEARCH, T2001). Positive single colonies were picked and transferred to 10 mL of ura-deficient liquid medium (8 g / L pancreatin, containing 2% glucose). After incubation at 30°C, 200 rpm for 48 h, the cells were inoculated into 100 mL of ura-deficient liquid medium at a ratio of 1:20 and incubated at 30°C, 200 rpm for 24 h. The supernatant was removed by centrifugation at 6000 g and the cells were resuspended in 100 mL of YPL medium (1% yeast extract, 2% peptone, 2% galactose, and the remainder was sterile water, sterilized at high temperature). The cells were then incubated at 30°C, 200 rpm for 12 h to obtain the recombinant yeast (WAT11U / CYP82BC4-pESC-URA) culture medium. At the same time, the pESC-URA empty vector plasmid was transformed into Saccharomyces cerevisiae WAT11U competent cells to obtain the recombinant empty vector yeast (WAT11U / pESC-URA) culture fluid.

[0152] 2.3 Yeast microsome extraction:

[0153] The culture medium of recombinant yeast (WAT11U / CYP82BC4-pESC-URA) and the culture medium of recombinant empty yeast (WAT11U / pESC-URA) were operated according to the following steps: the cells were collected by centrifugation at 6500g for 10 minutes, the supernatant was discarded, and the cells were inverted on absorbent paper for a few seconds; then 10 mL of TEK (50 mM Tris-HCl, 1 mM EDTA, 100 mM KCl, pH 7.5, the rest is water) was added to resuspend the cells, and then incubated at room temperature for 5 minutes; centrifuged at 6500g for 10 minutes, and washed with 100 mL of TESB (50 mM Tris-HCl, 1 mM EDTA, 600 mM D-sorbitol, the rest is water, pH 7.5). 7.5) Resuspend the cells and place on ice; crush the cells 15-20 times in a homogenizer (pre-cooled to 2°C with condensed water) at a pressure of 1000 bar or higher until the cells are milky white and turbid; after each crushing, wait until the temperature returns to 2°C before crushing the next vial; centrifuge the crushed cells at 12000 rpm at 4°C for 20 minutes, pour the supernatant into a bottle containing 10g PEG4000 and 0.88g NaCl, and incubate on ice for 15-30 minutes, shaking 3-5 times during the process, until all solids are dissolved; centrifuge at 12000 rpm at 4°C for 20 minutes, discard the supernatant, invert onto absorbent paper for a few seconds, and then place on ice; add 1000μL TEG (50mM Tris-HCl, 1mM EDTA, 20% (v / v) glycerol, pH 7.5), pipette evenly to obtain recombinant yeast (WAT11U / CYP82BC4-pESC-URA) microsomal extracts and recombinant empty yeast (WAT11U / pESC-URA) microsomal extracts, respectively. Store at -80°C or use directly in enzymatic reactions. Recombinant yeast (WAT11U / CYP82BC4-pESC-URA) microsomal extracts contain CYP82BC4 recombinant protein.

[0154] 3. Enzymatic reaction and product extraction

[0155] Take an appropriate amount of the purified recombinant StMTs protein obtained in step 1 and add (R, S)-lindoldhamine (obtained by chemical synthesis, its structural formula is shown in Figure 2 ) as substrate, and S-adenosylmethionine (AdoMet) (Sigma, USA) as methyl donor to carry out enzymatic reaction, catalyzing the methylation of small (R, S)-lindoldhamine to generate isotetrandrine. Figure 2 shown.

[0156] The enzymatic reaction system is as follows: (R,S)-lindoldhamine (10 mM) 1 μL, AdoMet (10 mM) 10 μL, 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), recombinant StMTs protein, recombinant yeast (WAT11U / CYP82BC4-pESC-URA) microsomal extract (containing 250 μg of CYP82BC4 microsomes), and potassium phosphate (100 mM, pH 7.0) added to 500 μL.

[0157] In the enzymatic reaction system, the addition of recombinant StMTs protein is set as follows:

[0158] ① 100 μL of recombinant StB2'NMT1 protein; ② 100 μL of recombinant StB2'NMT2 protein; ③ 100 μL of recombinant StB2NMT protein; ④ 100 μL of recombinant St7OMT protein; ⑤ 100 μL of recombinant StBOMT protein; ⑥ 100 μL each of recombinant StB2'NMT1, StB2NMT, St7OMT, and StBOMT proteins; ⑦ 100 μL each of recombinant StB2'NMT2, StB2NMT, St7OMT, and StBOMT proteins.

[0159] After the reaction system was prepared, it was shaken at 200 rpm and 30°C for 3 h. Then, 500 μL of ethyl acetate (Beijing Chemical Plant) was added, and ultrasonic extraction was performed for 30 min. The mixture was centrifuged at 12,000 g for 20 min. The upper organic phase was removed and dried in a nitrogen blower. 150 μL of methanol (Merck, USA) was added for reconstitution, and the mixture was centrifuged at 12,000 g for 20 min. 100 μL of the supernatant was injected into a UPLC-QTOF-MS (Waters Technologies, Milford, MA, USA) for detection.

[0160] 3. Reaction Product Detection

[0161] UPLC-QTOF-MS was used to detect the products of the enzymatic reaction. The results showed that with AdoMet as the methyl donor, the substrate (R,S)-lindoldhamine can be catalyzed by recombinant CYP82BC4, StB2'NMT1 / StB2'NMT2, StB2NMT, St7OMT, and StBOMT proteins to produce isotetrandrine. StB2'NMT1 and StB2'NMT2 can both catalyze N2' methylation, StB2NMT catalyzes N2 methylation, StB7OMT catalyzes 7-hydroxymethylation, and StBOMT catalyzes 12-hydroxymethylation. The chromatogram of the detection product of the recombinant StMTs enzyme catalyzed reaction is shown in Figure 2. Figure 2 The mass spectra of the target reaction product Isotetrandrine and Isotetrandrine standard (Chengdu Ruifensi Biotechnology Co., Ltd., DFZY-5mg) are shown in Figure 3 shown.

[0162] The chromatographic conditions were as follows: the chromatographic column was a T3 column (2.1 mm × 100 mm, 2.7 μm); the mobile phase was acetonitrile (A, Merck, USA) and 0.1% formic acid-water (B, Thermo Fisher, USA), and the gradient elution conditions were as follows: 0-6.0 min: 5%-30% A, 6.0-12.0 min: 30%-60% A, 12.0-13.5 min: 60%-90% A, 13.5-15.0 min: 90%-5% A, 15.0-18.0 min: 5%-5% A; the injection volume was 4 μL, the column temperature was 40°C, and the mobile phase flow rate was 0.4 mL / min.

[0163] The mass spectrometry conditions were as follows: electrospray ion source, positive ion mode, scanning detection range of m / z 50-1500; capillary voltage of 0.5 kV; sample cone voltage of 40 V; ion source temperature of 100°C; desolvation temperature of 300°C; desolvation gas flow rate of 800 L / h; trap collision energy of low-energy function was set to 6 eV; ramp trap collision energy of high-energy function was set to 30-50 eV. MassLynx software was used for data acquisition and processing.

[0164] In summary, the recombinant StMTs protein is a methyltransferase that can catalyze the methylation of (R, S)-lindoldhamine using AdoMet as a methyl donor to generate isotetrandrine. The recombinant StMTs protein and its encoding gene of the present invention can be used to construct BIA engineering bacteria.

[0165] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A protein, which is StB2'NMT1, StB7OMT, StB2'NMT2 or StB2NMT; the StB2'NMT1 is as follows A1), A2) or A3): A1) the amino acid sequence is the protein of SEQ ID No. 4 in the sequence listing; A2) a protein derived from A1) or having 70% or more identity with the protein of A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of the protein of A1) and having the same function; A3) A fusion protein obtained by fusing a protein tag at the carboxyl or / and amino terminus of the protein shown in A1) or A2); The StB7OMT is as follows B1), B2) or B3): B1) the amino acid sequence is the protein of SEQ ID No. 6 in the sequence listing; B2) a protein derived from B1) or having 70% or more identity with the protein of B1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of the protein of B1) and having the same function; B3) A fusion protein obtained by fusing a protein tag at the carboxyl or / and amino terminus of the protein shown in B1) or B2); The StB2'NMT2 is as follows C1), C2) or C3): C1) the amino acid sequence is the protein of SEQ ID No. 8 in the sequence listing; C2) a protein derived from C1) or having 70% or more identity with the protein of C1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of C1) and having the same function; C3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in C1) or C2); The StB2NMT is as follows D1), D2) or D3): D1) the amino acid sequence is the protein of SEQ ID No. 10 in the sequence listing; D2) a protein derived from D1) or having 70% or more identity with the protein of D1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of D1) and having the same function; D3) A fusion protein obtained by fusing a protein tag to the carboxyl terminus or / and amino terminus of the protein shown in D1) or D2).

2. The biomaterial related to the protein according to claim 1, which is any one of the following H1) to H9): H1) a nucleic acid molecule encoding the protein according to claim 1; H2) an expression cassette containing the nucleic acid molecule described in H1); H3) a recombinant vector containing the nucleic acid molecule described in H1), or a recombinant vector containing the expression cassette described in H2); H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3); H5) a transgenic plant cell line containing the nucleic acid molecule described in H1) or a transgenic plant cell line containing the expression cassette described in H2); H6) transgenic plant tissue containing the nucleic acid molecule described in H1) or transgenic plant tissue containing the expression cassette described in H2); H7) a transgenic plant organ containing the nucleic acid molecule described in H1) or a transgenic plant organ containing the expression cassette described in H2); H8) a nucleic acid molecule that promotes or increases the expression of the protein according to claim 1; H9) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in H8).

3. The biomaterial according to claim 2, characterized in that: H1) The nucleic acid molecule is any one of the following b1) to b10): b1) the coding sequence is the DNA molecule of SEQ ID No. 3 in the sequence listing; b2) the DNA molecule shown in SEQ ID No. 3 in the sequence listing; b3) the coding sequence is a DNA molecule of SEQ ID No. 5 in the sequence listing; b4) the DNA molecule shown in SEQ ID No. 5 in the sequence listing; b5) the coding sequence is a DNA molecule of SEQ ID No. 7 in the sequence listing; b6) the DNA molecule shown in SEQ ID No. 7 in the sequence listing; b7) the coding sequence is a DNA molecule of SEQ ID No. 9 in the sequence listing; b8) the DNA molecule shown in SEQ ID No. 9 in the sequence listing; b9) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in any one of b1) to b8) and encodes the protein; b10) A DNA molecule that hybridizes under stringent conditions to a nucleotide sequence specified in any one of b1) to b9) and encodes the protein.

4. Use of the protein according to claim 1 as a methyltransferase.

5. The use according to claim 4, characterized in that: The StB2NMT, the B2'NMT1 and the StB2'NMT2 are all nitrogen methyltransferases; The StB7OMT is an oxygen methyltransferase.

6. Use of the protein according to claim 1 or the biomaterial according to claim 2 or 3 in the production of bisbenzylisoquinoline alkaloids, or in the preparation of a product for the production of bisbenzylisoquinoline alkaloids, or in the catalysis of the methylation of the parent nucleus of bisbenzylisoquinoline alkaloids, or in the preparation of a product for the catalysis of the methylation of the parent nucleus of bisbenzylisoquinoline alkaloids.

7. The use according to claim 6, characterized in that: The bisbenzylisoquinoline alkaloid is isotetrandrine.

8. A product containing M1), M2) and / or M3): M1) the StB2NMT or a biological material related to the StB2NMT according to claim 1; M2) the StB7OMT or a biological material related to the StB7OMT according to claim 1; M3) The StB2'NMT1 of claim 1, a biological material related to the StB2'NMT1, the StB2'NMT2 of claim 1, or a biological material related to the StB2'NMT2.

9. The product according to claim 8, characterized in that: The product further contains StBOMT or biological materials related to StBOMT, and / or CYP82BC4 or biological materials related to CYP82BC4; The CYP82BC4 is the following E1), E2) or E3): E1) the amino acid sequence is the protein of SEQ ID No. 13 in the sequence listing; E2) a protein derived from E1) or having 70% or more identity with the protein of E1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of E1) and having the same function; E3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in E1) or E2); The biological material related to the CYP82BC4 is any one of the following I1) to I9): I1) a nucleic acid molecule encoding the CYP82BC4; I2) an expression cassette containing the nucleic acid molecule described in I1); I3) a recombinant vector containing the nucleic acid molecule described in I1), or a recombinant vector containing the expression cassette described in I2); I4) a recombinant microorganism containing the nucleic acid molecule described in I1), or a recombinant microorganism containing the expression cassette described in I2), or a recombinant microorganism containing the recombinant vector described in I3); I5) a transgenic plant cell line containing the nucleic acid molecule described in I1), or a transgenic plant cell line containing the expression cassette described in I2); I6) transgenic plant tissue containing the nucleic acid molecule described in I1), or transgenic plant tissue containing the expression cassette described in I2); I7) a transgenic plant organ containing the nucleic acid molecule described in I1), or a transgenic plant organ containing the expression cassette described in I2); I8) a nucleic acid molecule that promotes or increases the expression of CYP82BC4; I9) an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in I8); The StBOMT is as follows G1), G2) or G3): G1) the amino acid sequence is the protein of SEQ ID No. 2 in the sequence listing; G2) a protein derived from G1) or having 70% or more identity with the protein of G1) by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of G1) and having the same function; G3) A fusion protein obtained by fusing a protein tag at the carboxyl terminus or / and amino terminus of the protein shown in G1) or G2); The biological material related to the StBOMT is any one of the following K1) to K9): K1) a nucleic acid molecule encoding the StBOMT; K2) an expression cassette containing the nucleic acid molecule described in K1); K3) a recombinant vector containing the nucleic acid molecule described in K1), or a recombinant vector containing the expression cassette described in K2); K4) a recombinant microorganism containing the nucleic acid molecule described in K1), or a recombinant microorganism containing the expression cassette described in K2), or a recombinant microorganism containing the recombinant vector described in K3); K5) a transgenic plant cell line containing the nucleic acid molecule described in K1) or a transgenic plant cell line containing the expression cassette described in K2); K6) transgenic plant tissue containing the nucleic acid molecule described in K1) or transgenic plant tissue containing the expression cassette described in K2); K7) A transgenic plant organ containing the nucleic acid molecule described in K1) or a transgenic plant organ containing the expression cassette described in K2); K8) a nucleic acid molecule that promotes or increases the expression of StBOMT; K9) An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule described in K8).

10. The product according to claim 8 or 9, characterized in that: The product also contains (R,S)-lindoldhamine.

11. The product according to any one of claims 8 to 10, characterized in that: The bisbenzylisoquinoline alkaloid is isotetrandrine.

12. A method for producing isotetrandrine, comprising: Using (R, S)-lindoldhamine as a substrate, the CYP82BC4 of claim 9 and the StBOMT, the StB2'NMT1 or the StB2'NMT2 of claim 1, the StB7OMT and the StB2NMT are used to carry out a catalytic reaction to generate isotetrandrine.

Images