A polypeptide with methylation function at n-position of a genus amaryllidaceae alkaloid compound, and a coding gene and application thereof
By identifying and expressing N-methyltransferases LrNMT1 and LrNMT2, which are alkaloids from the Amaryllidaceae family, the problem of unclear catalytic function was solved, and N-methylation reactions of various substrates were realized, enriching the understanding and application potential of biosynthetic pathways.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NORTHWESTERN POLYTECHNICAL UNIV
- Filing Date
- 2026-03-09
- Publication Date
- 2026-05-29
AI Technical Summary
In the existing technology, the gene source, substrate specificity and catalytic function of the N-methylation reaction catalytic enzyme of Amaryllidaceae alkaloids have not been clearly identified, which increases the difficulty of elucidating the biosynthetic pathway and its engineering application.
The study provides polypeptides and their encoding genes that enable N-methylation of Amaryllidaceae alkaloids, including LrNMT1 and LrNMT2 polypeptides and their encoding nucleotides, which are expressed in host cells via recombinant expression vectors to achieve N-methylation of various related substrates.
The catalytic function of key enzymes in the synthesis of Amaryllidaceae alkaloids has been clarified, providing a reliable biocatalytic tool applicable to the synthesis of Amaryllidaceae alkaloids and their structurally modified derivatives, and possessing both scientific research value and industrial application prospects.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a polypeptide with N-position methylation function of an Amaryllidaceae alkaloid compound, its encoding gene, and its applications. Background Technology
[0002] Amaryllidaceae plants are rich in various alkaloids with important medicinal value, among which alkaloids such as galantamine have attracted widespread attention due to their significant efficacy in treating nervous system diseases. Current research indicates that the biosynthesis of Amaryllidaceae alkaloids such as galantamine involves multiple enzymatic reactions, in which various structural modification reactions have a crucial impact on the formation of the final product.
[0003] In the synthesis of alkaloids in the Amaryllidaceae family, N-methylation is considered one of the important steps affecting the structural diversity and biological activity of alkaloids. This reaction is usually catalyzed by N-methyltransferases (NMTs), which use S-adenosylmethionine as a methyl donor to methylate nitrogen-containing intermediates. However, the specific gene origin, substrate specificity, and catalytic function of the N-methyltransferases involved in the biosynthesis of galantamine in Amaryllidaceae plants have not been clearly identified or systematically reported.
[0004] Current research largely focuses on methyltransferases from other plants or microorganisms, whose catalytic targets and reaction characteristics differ significantly from those of Amaryllidaceae alkaloid intermediates. Furthermore, the differences in reaction site selectivity and reaction sequence among different methyltransferases further complicate the elucidation and engineering application of Amaryllidaceae alkaloid biosynthetic pathways.
[0005] Therefore, there is an urgent need to discover and identify N-methyltransferases derived from Lycoris genus plants that can specifically catalyze the N-methylation reaction of key intermediates in the biosynthesis of galantamine, in order to solve the aforementioned problems in the existing technology. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of the existing technology. This invention targets N-methyltransferases in *Amaryllis* plants involved in the biosynthesis of galantamine, whose specific gene origin, substrate specificity, and catalytic function have not yet been clearly identified and systematically reported. This invention provides a polypeptide with N-position methylation function in *Amaryllis* alkaloids, its encoding gene, and its applications. This polypeptide exhibits clear enzymatic activity and good substrate adaptability, and can catalyze N-methylation reactions of various related substrates in vitro or in vivo, providing a reliable biocatalytic tool for the synthesis of *Amaryllis* alkaloids and their structurally modified derivatives.
[0007] The first objective of this invention is to provide a polypeptide that performs N-position methylation on an Amaryllidaceae alkaloid compound, wherein the polypeptide is selected from any one of (a) to (f): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2; (c) A polypeptide formed by substitution, deletion or addition of one or more amino acids from the amino acid sequence in (a) and having the function of methylation at the N-position of Amaryllidaceae alkaloids; (d) A polypeptide derived from the amino acid sequence in (b) by substitution, deletion or addition of one or more amino acids and possessing the ability to methylate at the N-position of Amaryllidaceae alkaloids; (e) has an amino acid sequence that is more than 95% identical to that in (a), and is a polypeptide that has the function of methylating at the N position of Amaryllidaceae alkaloids. (f) and (b) have amino acid sequences with more than 95% identity, and are polypeptides that can methylate at the N-position of Amaryllidaceae alkaloids.
[0008] Preferably, the polypeptide is obtained by artificial synthesis, or by synthesizing its encoding gene and then biologically expressing it.
[0009] A second objective of this invention is to provide a polynucleotide selected from the following (g) to (j): (g) Polynucleotides encoding the polypeptides described in (a) to (f); (h) Conserved variants of polypeptides, active fragments, derivatives or analogs of the polypeptides described in (a) to (f); (i) A polynucleotide that is hybridizable to the polynucleotide sequence defined in (g) or (h) under strict hybridization conditions and encodes a polypeptide having N-position methylation function of an Amaryllidaceae alkaloid, wherein the polynucleotide sequences have at least 70% identity with each other. (j) has at least 70% identity with the polynucleotide sequence defined in (g) or (h) and encodes a polypeptide with the same or similar N-methyltransferase biological activity and function as the polypeptides described in (a) to (f).
[0010] Preferably, the polynucleotide further includes variants of the polynucleotide, including substitution variants, deletion variants, or insertion variants.
[0011] Preferably, the polynucleotide is in the form of DNA or RNA, wherein the DNA includes cDNA, genomic DNA or artificially synthesized DNA, and the DNA is in the form of single-stranded or double-stranded strands, and is a coding strand or a non-coding strand.
[0012] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned polynucleotides.
[0013] The fourth objective of this invention is to provide a recombinant host cell into which the above-mentioned recombinant expression vector is inserted.
[0014] The fifth objective of this invention is to provide a method for preparing a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids, comprising the following steps: (1) Culture the above-mentioned recombinant host cells; (2) Obtaining a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide can be obtained as a crude cell extract or obtained after separation and purification.
[0015] The sixth objective of this invention is to provide the application of the aforementioned polypeptides, polynucleotides, recombinant expression vectors, or recombinant host cells in the N-position methylation of Amaryllidaceae alkaloids.
[0016] A seventh objective of this invention is to provide a method for N-position methylation of Amaryllidaceae alkaloids, the method comprising: In the presence of a methyl donor, a polypeptide is used to carry out an in vitro enzymatic reaction to induce N-methylation of the substrate, thereby generating the corresponding product. Alternatively, in the presence of a methyl donor, recombinant host cells can be used to N-methylate the substrate through in vivo or in vitro reactions, thereby generating the corresponding product. The methyl donor is S-adenosylmethionine; The substrates are N-demethylgalantamine, N-demethylnalvidin, and their structural analogs.
[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a polypeptide with N-position methylation function of an alkaloid compound from the Amaryllidaceae family, its encoding gene, and its applications. This invention provides a polypeptide derived from the Amaryllidaceae plant *Lycoris radiata* (…). Lycoris radiata N-methyltransferase (N-methyltransferase) Lr NMT1 and Lr The study elucidated for the first time the catalytic function of NMT2 and its encoding nucleotide sequence in the N-methylation reaction of related substrates, enriching our understanding of the types and functions of key enzymes in the biosynthesis of Amaryllidaceae alkaloids. Lr NMT1 and LrThe NMT2 protein possesses well-defined enzymatic activity and good substrate adaptability, enabling it to catalyze N-methylation reactions of various related substrates in vitro and in vivo. This provides a reliable biocatalytic tool for the synthesis of Amaryllidaceae alkaloids and their structurally modified derivatives. The present invention... Lr NMT1 and Lr The nucleotide encoding NMT2 can be obtained through artificial synthesis or conventional molecular biology methods, and can be stably expressed in various heterologous hosts such as Escherichia coli and yeast. The preparation method is simple, reproducible, and suitable for further scale-up applications. Furthermore, this invention provides a new key enzyme element for the elucidation of the biosynthetic pathway of Amaryllidaceae alkaloids, metabolic engineering modification, and synthetic biology research, possessing significant scientific research value and potential industrial application prospects. Simultaneously, its technical solution is complete and widely applicable, which helps improve the stability and resistance to patent circumvention. Attached Figure Description
[0018] Figure 1 for Lr NMT1 and Lr NMT2 purified SDS-PAGE electrophoresis image.
[0019] Figure 2 Recombinant plasmid Lr A schematic diagram of the spectrum of NMT1-pET-28a.
[0020] Figure 3 Recombinant plasmid Lr A schematic diagram of the spectrum of NMT2-pET-28a.
[0021] Figure 4 for Lr NMT1 and Lr HPLC detection of NMT2-catalyzed N-demethylgalanthamine to Galanthamine conversion. 1 is Lr NMT1 catalyst. 2 is Lr NMT2 catalysis. 3 is a Galanthamine standard. 4 is an N-demethylgalanthamine standard.
[0022] Figure 5 for Lr NMT1 and Lr HPLC detection of NMT2-catalyzed N-demethylnarwedine to Narwedine. 1 is Lr NMT1 catalyst. 2 is Lr NMT2 catalyst. 3 is Narwedine standard. 4 is N-demethylnarwedine standard. Detailed Implementation
[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Test methods in the following embodiments that do not specify specific conditions are generally operated under conventional conditions. Since they do not involve the inventive point, their steps are not described in detail.
[0024] This invention targets N-methyltransferases involved in the biosynthesis of galantamine in *Amaryllis* plants. The specific gene origin, substrate specificity, and catalytic function of these N-methyltransferases have not yet been clearly identified or systematically reported. The purpose of this invention is to provide a polypeptide with N-position methylation function in *Amaryllis* alkaloids, its encoding gene, and its applications.
[0025] To achieve the above objectives, the first aspect of the present invention provides a polypeptide with N-position methylation function of an Amaryllidaceae alkaloid compound, said polypeptide being selected from any one of (a) to (f): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2; (c) A polypeptide formed by substitution, deletion or addition of one or more amino acids from the amino acid sequence in (a) and having the function of methylation at the N-position of Amaryllidaceae alkaloids; (d) A polypeptide derived from the amino acid sequence in (b) by substitution, deletion or addition of one or more amino acids and possessing the ability to methylate at the N-position of Amaryllidaceae alkaloids; (e) has an amino acid sequence that is more than 95% identical to that in (a), and is a polypeptide that has the function of methylating at the N position of Amaryllidaceae alkaloids. (f) and (b) have amino acid sequences with more than 95% identity, and are polypeptides that can methylate at the N-position of Amaryllidaceae alkaloids.
[0026] In this invention, a polypeptide capable of methylating at the N-position of Amaryllidaceae alkaloids is named... Lr NMT1 peptide and Lr The NMT2 polypeptide, corresponding to the polypeptide composed of the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, also includes conserved variant polypeptides based on the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or their active fragments or active derivatives or analogs.
[0027] The term "conservative variant polypeptides, active fragments, active derivatives, and analogs" refers to polypeptides whose amino acid sequences have been altered relative to those shown in SEQ ID NO.1 or SEQ ID NO.2, but which are substantially similar to those shown in SEQ ID NO.2. Lr NMT1 peptide or Lr NMT2 peptides have the same or similar biological functions, namely, they still possess the activity of catalyzing the N-position methylation of Amaryllidaceae alkaloids.
[0028] The conserved variant polypeptide, active fragment, active derivative, or analogue may be any one or more of the following forms: (1) Based on the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, one or more amino acid residues are replaced by conserved or non-conserved amino acid residues, preferably by conserved amino acid residues; (2) In the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2, one or more amino acid residues are replaced by other groups without affecting their N-methyltransferase activity; (3) A fusion polypeptide formed by fusing the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 with other compounds or polypeptide sequences, preferably a compound or polypeptide used to improve protein stability, solubility or in vivo half-life, such as polyethylene glycol (PEG) or a tagged polypeptide; (4) A polypeptide formed by fusing an additional amino acid sequence to the N-terminus or C-terminus of the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2, wherein the additional amino acid sequence includes, but is not limited to, a signal peptide, a secretory peptide, an affinity purification tag, or a reporter protein sequence.
[0029] The present invention Lr NMT1 peptide or Lr The NMT2 polypeptide can be a natural polypeptide, a recombinant polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide can be obtained through natural purification, chemical synthesis, or expression in prokaryotic or eukaryotic host cells using recombinant DNA technology. The host cells include, but are not limited to, Escherichia coli, yeast, insect cells, or plant cells.
[0030] Depending on the expression system used, the present invention describes Lr NMT1 peptide or Lr The NMT2 polypeptide may or may not contain a starting methionine residue.
[0031] A second aspect of the present invention provides a polynucleotide selected from the following (g) to (j): (g) A polynucleotide encoding the polypeptides described in claims 1(a) to (f); (h) A conserved variant of the polypeptide, active fragment, derivative or analogue of the polypeptide described in claims (a) to (f) of claim 1; (i) A polynucleotide that is hybridizable to the polynucleotide sequence defined in (g) or (h) under strict hybridization conditions and encodes a polypeptide having N-position methylation function of an Amaryllidaceae alkaloid, wherein the polynucleotide sequences have at least 70% identity with each other. (j) has at least 70% identity with the polynucleotide sequence defined in (g) or (h), and the polypeptide encoded therefrom has the same or similar N-methyltransferase biological activity and function as the polypeptides described in (a) to (f) of claims 1.
[0032] For example, a polynucleotide, said polynucleotide is selected from any of the following: (g) Encoding Lr NMT1 peptide or Lr The NMT2 polypeptide is preferably a polynucleotide that encodes the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, respectively, forming the polypeptide. (h) encoding Lr NMT1 peptide or Lr Conserved variants of NMT2 peptides, active fragments, derivatives or analogs of polynucleotides; (i) A polynucleotide that is hybridizable to the polynucleotide sequence defined in (g) or (h) under strict hybridization conditions and encodes a polypeptide having N-position methylation function of an Amaryllidaceae alkaloid, wherein the polynucleotide sequences have at least 70% identity, preferably at least 80%, more preferably at least 90%; (j) has at least 70% identity with the polynucleotide sequence defined by (g) or (h), and the polypeptide it encodes has similarity to... Lr NMT1 or Lr Polynucleotides with N-methyltransferase biological activities and functions that are identical or similar to NMT2.
[0033] The polynucleotides described in this invention also include variants of the aforementioned polynucleotides, including substitution variants, deletion variants, and insertion variants. As long as the encoded polypeptide still has N-methyltransferase activity, they fall within the scope of protection of this invention.
[0034] The polynucleotide can be in the form of DNA or RNA, wherein the DNA includes, but is not limited to, cDNA, genomic DNA or artificially synthesized DNA, and the DNA can be in the form of single-stranded or double-stranded, and can be a coding strand or a non-coding strand.
[0035] The polynucleotide sequence encoding the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 may be identical to the corresponding coding sequence or a degenerate variant. As described herein, a "degenerate variant" refers to a different nucleotide sequence resulting from the degeneracy of the genetic codon, but whose encoded amino acid sequence is identical to that of the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2.
[0036] A third aspect of the present invention provides a recombinant expression vector containing the aforementioned polynucleotides.
[0037] Exemplary, a recombinant expression vector comprises a nucleic acid molecule encoding the N-methyltransferase. The aforementioned recombinant vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or other vectors well-known in the art. Vectors applicable in this invention include, but are not limited to: T7 promoter-based expression vectors for expression in bacteria, such as pET-28a; vectors for expression in yeast, such as the YEp series vectors; and MSXND expression vectors for expression in mammalian cells. In short, any vector that can stably replicate and exist within a host cell can be used to construct a recombinant expression vector.
[0038] A fourth aspect of the present invention provides a recombinant host cell into which the above-mentioned recombinant expression vector is inserted.
[0039] Exemplary, a recombinant host cell for expressing the N-methyltransferase, wherein the host cell of the recombinant expression system can be a prokaryotic or eukaryotic microorganism, including but not limited to *Escherichia coli*, yeast, or other microbial hosts suitable for heterologous expression. The recombinant expression system comprises: a nucleic acid molecule encoding the N-methyltransferase, an expression vector functionally linked to the nucleic acid molecule, and a recombinant host cell containing the expression vector. In a preferred embodiment, the N-methyltransferase obtained by expression in the recombinant host cell can be purified by using an affinity tag, the affinity tag including but not limited to a His tag.
[0040] The fifth aspect of this invention provides a method for preparing a polypeptide having N-position methylation activity of Amaryllidaceae alkaloids, comprising the following steps: (1) Culture the above-mentioned recombinant host cells; (2) Obtaining a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide can be obtained as a crude cell extract or obtained after separation and purification.
[0041] An exemplary method for preparing a polypeptide having N-position methylation activity of an Amaryllidaceae alkaloid compound, the method comprising the following steps: (1) The culture was transferred to a medium containing the coding. Lr NMT1 peptide or Lr Recombinant host cells of recombinant expression vectors of NMT2 polypeptide polynucleotides; (2) Obtaining a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids from the recombinant host cell, wherein the polypeptide can be obtained as a crude cell extract or after separation and purification.
[0042] This invention utilizes gene recombination technology to enable recombinant host cells to carry codes containing... Lr NMT1 or Lr An expression unit for the NMT2 peptide, enabling the host cell to spontaneously or inducedly express the peptide under suitable conditions. As described herein, an "expression unit" is a nucleic acid sequence capable of initiating transcription and / or translation in a host cell to produce a specific peptide, typically including a promoter, a coding sequence, and necessary regulatory elements. The recombinant host cell expresses... Lr NMT1 peptide or Lr The NMT2 peptide can be obtained by cell disruption to obtain a crude cell extract containing the peptide, or by using conventional protein separation and purification methods in the art to obtain a peptide with high purity. The preparation method of the peptide can be adjusted according to the selected expression vector and host cell type. Furthermore, it should be understood that the synthesis of the peptide described in this invention using a cell-free protein expression system (such as a cell-free system) is similar in principle to intracellular expression and also falls within the protection scope of the peptide preparation method of this invention.
[0043] The sixth aspect of this invention provides the application of the above-mentioned polypeptides, polynucleotides, recombinant expression vectors, or recombinant host cells in the N-position methylation of Amaryllidaceae alkaloids.
[0044] A seventh aspect of the present invention provides a method for N-position methylation of Amaryllidaceae alkaloids, the method comprising: In the presence of a methyl donor, the above-mentioned polypeptide is used to carry out an in vitro enzymatic reaction to N-methylate the substrate, thereby generating the corresponding product. Alternatively, in the presence of a methyl donor, the aforementioned recombinant host cells can be used to N-methylate the substrate through in vivo or in vitro reactions, thereby generating the corresponding product. The methyl donor is S-adenosylmethionine; The substrates are N-demethylgalantamine, N-demethylnalvidin, and their structural analogs.
[0045] Exemplary, one method of utilizing Lr NMT1 peptide or LrA method for N-position methylation of Amaryllidaceae alkaloids using NMT2 peptide, which can be performed intracellularly or extracellularly using N-demethylgalanthamine, N-demethylnarwedine, or their structural analogs as substrates, via... Lr NMT1 peptide or Lr A method for catalyzing the NMT2 peptide to generate the corresponding N-methylated product, the method comprising one of the following steps: (a) Providing N-methylation function Lr NMT1 peptide or Lr The NMT2 polypeptide undergoes an in vitro enzymatic reaction in the presence of a methyl donor to N-methylate the substrate, thereby generating the corresponding product. (b) Provided a transfer containing encoding Lr NMT1 peptide or Lr Recombinant host cells of NMT2 polypeptide polynucleotides, in the presence of methyl donors, can induce N-methylation of the substrate through in vivo or in vitro reactions, thereby generating the corresponding product.
[0046] The method described in this invention can utilize enzymes expressed in vivo by recombinant host cells, or synthesize N-methylated products using extracellular or cell-free systems. Its technical principle is similar to that of in vivo expression.
[0047] The invention is further illustrated in the following examples, but these do not limit the scope of the invention. Some molecular cloning methods and operational details may be adjusted according to the instructions of the reagent, enzyme, or kit provider used, and will not be repeated in the examples.
[0048] Example 1 Lr NMT1 and Lr Obtaining NMT2-encoded nucleotides According to the nucleotide sequence information disclosed in this invention, the following methods can be used to obtain... Lr NMT1 and Lr The nucleotide encoding NMT2.
[0049] Method 1: Through Lycoris radiata ( Lycoris radiata Obtained by amplification in tissues.
[0050] Fresh Lycoris radiata leaves were used as experimental material, quickly frozen in liquid nitrogen, and thoroughly ground. Total RNA was extracted using an RNA extraction kit (Tiangen), following the kit's instructions. After the extracted RNA passed quality testing, first-strand cDNA was synthesized using a reverse transcription kit (Tiangen) to serve as a template for subsequent amplification reactions.
[0051] Using the synthesized cDNA as a template, specific primers were designed, and the cDNA was amplified by PCR. Lr NMT1 and Lr NMT2.
[0052] Primer design: Lr NMT1, F terminus: CCGCGCGGCAGCCATATGGCGACGAAGACG (SEQ ID NO.5), Lr NMT1, R-terminus: CAGTGGTGGTGGTGGTGGTGTTATTCTGGTTTACGACATGC (SEQ ID NO. 6); Lr NMT2, F terminus: CCGCGCGGCAGCCATATGACGACGATGACGG (SEQ ID NO.7), Lr NMT2, R terminus: CAGTGGTGGTGGTGGTGGTGTTACTTTGGCTTACGACATGC (SEQ ID NO.8); The PCR system is shown in Table 1 below: Table 1 shows the parameters of the PCR reaction system.
[0053] The PCR reaction procedure is shown in Table 2 below: Table 2 shows the PCR reaction procedure parameters.
[0054] Method 2: Based on SEQ ID NO.1 ( Lr NMT1) and SEQ ID NO.2 Lr The nucleotide sequence information of NMT2 is used to obtain the target nucleic acid molecule through artificial synthesis. In order to adapt to the target host cell (such as E. coli, yeast or other suitable microorganisms for expression), the nucleotide sequence can be codon-optimized before artificial synthesis.
[0055] Example 2 Construction of Recombinant Vector Will Lr NMT1 and Lr The NMT2 CDS sequence was cloned into a suitable expression vector to construct a recombinant expression vector. Lr The sequence name of NMT1 CDS is SEQ ID NO.3; Lr The sequence name of NMT2 CDS is SEQ ID NO.4.
[0056] Will Lr NMT1 and Lr The NMT2-encoded nucleotides are amplified using templates synthesized artificially or obtained from plant tissues, and then cloned into vectors suitable for expression in *E. coli* or other suitable hosts (e.g., pET-28a) to obtain recombinant expression vectors. Details are as follows: The vector pET-28a(+) was digested using the restriction endonucleases NdeI and XhoI.
[0057] Inactivate the endonuclease according to the instructions for use.
[0058] The digested vector and PCR fragment were mixed, and the vector fragment and gene fragment were ligated using T4 DNA ligase.
[0059] The ligation product was transformed into E. coli competent cells DH5α and screened on LB + kanamycin solid plates (incubated overnight at 37°C).
[0060] Transformants were inoculated into 5 mL of liquid LB + kanamycin medium and cultured with shaking (37°C, 220 rpm for 9 hours). Plasmids were extracted using a plasmid extraction kit.
[0061] The extracted plasmids were subjected to DNA sequencing to screen for those carrying the correct DNA. Lr NMT1 and Lr The NMT2 sequence plasmid was sequenced using universal primers T7 and T7-ter.
[0062] See Figure 1 and Figure 2 As shown, recombinant plasmid Lr NMT1-pET-28a and Lr A schematic diagram of the spectrum of NMT2-pET-28a.
[0063] Example 3: Expression of Recombinant Escherichia coli Lr NMT1 and Lr NMT2 and its separation and purification The plasmids selected in Example 2 were introduced into suitable host cells, such as Escherichia coli BL21(DE3), and positive clones were screened using kanamycin-resistant plates (Kan+, 100 mg / mL) and cultured overnight at 37°C. Positive clones were obtained through selective culture. Single colonies were picked and added to 5 mL of LB liquid medium (Kan+, 100 mg / mL) and cultured at 37°C and 220 rpm until the OD600 was 0.7. The bacterial culture in 5 mL of LB medium was transferred to 800 mL of 2YT medium (Kan+, 100 mg / mL), and cultured at 37℃ and 220 rpm until the OD600 reached 0.7. Then, the temperature was lowered to 16℃, and 0.5 mMIPTG was added to induce expression for 16 h. Collect the above-mentioned bacterial culture into a collection bottle, centrifuge at 5500 rpm for 10 min, and collect the bacterial cells; The bacterial cells were fully resuspended in buffer A (50 mM Tris-HCl, 200 mM NaCl, pH 8.0); The resuspended bacterial cells were disrupted using a high-pressure cell disruptor. The resulting lysate was centrifuged at 4°C and 10,000 rpm for 40 min, and the supernatant was collected. The supernatant was loaded onto a Ni Sepharose 6 Fast Flow nickel column equilibrated with buffer A to adsorb the target protein. Elution was performed stepwise using buffers containing 50 mM, 100 mM, and 300 mM imidazole to remove contaminating proteins and obtain the target protein. The eluted protein solution was concentrated to 500 µL using a Millipore ultrafiltration tube, and the protein concentration was determined using a BCA protein assay kit (Pierce, USA), with bovine serum albumin (2 mg / mL) as the standard. The finally purified protein was flash-frozen in liquid nitrogen and then placed in... The protein was stored at 80℃ for later use, and its purity and subunit molecular weight were analyzed by SDS-PAGE. Results are as follows: Figure 3 As shown, Lr NMT1 and Lr NMT2 purified SDS-PAGE electrophoresis image.
[0064] The method described in this embodiment can be performed in prokaryotic or eukaryotic hosts, and is not limited to *E. coli*; the expression and purification conditions of the peptide can be optimized according to the host characteristics. The obtained peptide can be used for downstream N-methylation reactions or other applications.
[0065] Example 4 utilizes Lr NMT1 or Lr NMT2 in vitro catalytic N-methylation reaction of Amaryllidaceae alkaloid intermediates Lr NMT1 peptide or LrThe NMT2 peptide catalyzes the N-position methylation of nitrogen-containing intermediates in the biosynthesis of Amaryllidaceae alkaloids, using S-adenosylmethionine (SAM) as a methyl donor to convert N-demethylated substrates into the corresponding N-methylated products. In this embodiment, N-demethylgalanthamine and N-demethylnarwedine were selected as substrates and verified under in vitro conditions. Lr NMT1 or Lr Catalytic activity of NMT2.
[0066] 1) The standard in vitro enzymatic reaction system is as follows: The total reaction volume is 200 μL, and it includes the following components: The buffer solution is Tris-HCl, pH=7; N-Demethylated Amaryllidaceae alkaloid substrates, such as N-demethylgalantamine or N-demethylnalvidin; Methyl donor S-adenosylmethionine (SAM); Lr NMT1 or Lr NMT2 protein (preparation method as in Example 3); 2) Reaction conditions and termination The reaction system was incubated at 30°C for 2 hours to allow N-methylation to occur. Then, 200 μL of methanol was added to terminate the reaction. Finally, the reaction mixture was centrifuged and filtered to remove protein components, and the reaction supernatant was obtained for subsequent analysis.
[0067] 3) Product Analysis The reaction products could be detected and identified using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), or other analytical methods. Product analysis was performed using an HPLC-MS system (Agilent 1200, Agilent Technologies, USA), and chromatographic separation was performed using a Waters C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), with a gradient elution program as follows: 0–9 min, fraction B 2%; 9–19 min, fraction B increased to 25%; 19–23 min, fraction B increased to 95%; 23–25 min, fraction B maintained at 95%; 25–29 min, fraction B decreased to 2%. The flow rate was set to 1 mL / min, and the injection volume was 50 μL. Mass spectrometry was performed using an ESI ion source in negative ion mode, with a scan range of m / z 50–500.
[0068] The results showed that, Lr NMT1 or Lr Under the catalytic action of NMT2: N-Demethylgalanthamine can be converted to galanthamine via the following reaction route:
[0069] See Figure 4 As shown, Lr NMT1 and Lr HPLC detection of NMT2-catalyzed N-demethylgalanthamine to Galanthamine conversion. 1 is Lr NMT1 catalyst. 2 is Lr NMT2 catalysis. 3 is a Galanthamine standard. 4 is an N-demethylgalanthamine standard.
[0070] N-Demethylnalidin can be converted to narwedine via the following reaction route:
[0071] See Figure 5 As shown, Lr NMT1 and Lr HPLC detection of NMT2-catalyzed N-demethylnarwedine to Narwedine. 1 is Lr NMT1 catalyst. 2 is Lr NMT2 catalyst. 3 is Narwedine standard. 4 is N-demethylnarwedine standard.
[0072] The above results indicate that Lr NMT1 or Lr NMT2 has the ability to catalyze the key N-methylation step in the synthesis of Amaryllidaceae alkaloids.
[0073] 4) Explanation It should be understood that this embodiment is merely an illustrative example. Parameters such as substrate type, reaction system composition, buffer conditions, reaction temperature, and time can all be adjusted according to actual needs and should not be construed as limiting the scope of protection of this invention. Lr NMT1 or Lr NMT2 can also catalyze the N-methylation of other nitrogen-containing Amaryllidaceae alkaloid intermediates with similar structures.
[0074] Example 5 Same as Example 4, except that, The buffer is HEPES, pH = 8.0; The reaction can be carried out at 25°C.
[0075] SEQ ID NO.1 Lr Amino acid sequence of NMT1: > Lr NMT1 MATKTEELTKGMVAWYDETATQDAVWGEYLHHGYYGLDEPAHVSTNHAAERRMVDEVLKFAGVSDDPSKRLKNIIDVGCGFGAAAIYLAKKYGANCCGINLSPAQIQKAKELAAASGLGDRASFIVGDALNQPFPDGQFDLVWSMEVIEHIPDKLKFVSELERIAAPGATIIFTSCCHRDLSPDETSLKPDEKNLIDKIRKTFHQYSYISPSDTVKVVQSLALHDVKMANFSENVIPYFRAIVQSQWTFQGLTSMLLNGWTNFKVSQNWPLMLKAYEKGVTKYAVIACRKPE SEQ ID NO.2 Lr Amino acid sequence of NMT2: > Lr NMT2 MTTMTDELTKGMVAWYDETAKQDAVWGEYLHHGYYGPGEQAHVSTNQAAERRMVDEVLKFAGVSDDPMKKPKNIIDVGCGFGGAAIYLAKKYGAYCSGINLSPAQIQKAKELAAANGLGDKASFVVGDALNQPFPDGQFDLVWSMEVIEHTPDKLKFISELARIAAPGATIIFTSCCHRDLAPDEKSLKPDEKNLIDKIRKTFHQSSYISPSDTVKVVQSLELQDIKMADFSENVLPYFRAIVQSQRTWQGLASMLLNGWTNFKVSQNWPLMLKGYEKNVTKYIVIACRKPK SEQ ID NO.3 Lr CDS sequence of NMT1: > Lr NMT1-cds ATGGCGACGAAGACGGAAGAACTGACCAAAGGCATGGTCGCTTGGTACGACGAGACGGCGACGCAGGATGCCGTGTGGGGGGAGTACTTGCACCACGGTTACTACGGACTCGACGAGCCGGCCCACGTTTCGACTAACCACGCCGCCGAGCGACGCATGGTCGATGAGGTTCTCAAATTTGCTGGCGTCTCCGATGATCCTTCGAAAAGGCTTAAAAATATAATCGATGTTGGTTGTGGATTCGGGGCAGCTGCAATATATCTGGCAAAGAAATATGGGGCCAATTGTTGTGGCATAAACTTGAGCCCTGCCCAAATTCAAAAAGCTAAAGAACTTGCCGCTGCTAGTGGATTAGGAGACAGAGCTTCCTTTATAGTTGGTGATGCGTTGAACCAGCCATTTCCTGATGGACAATTTGACCTAGTTTGGTCCATGGAAGTCATCGAGCATATACCTGACAAGTTAAAGTTCGTTAGCGAATTGGAACGTATAGCGGCACCAGGAGCTACCATCATATTTACATCATGCTGTCATAGAGATCTCTCTCCTGACGAAACGTCTCTAAAGCCTGATGAGAAGAACCTTATAGATAAAATACGCAAAACATTTCATCAATATTCTTATATTTCACCCTCGGACACTGTCAAAGTTGTTCAATCCTTAGCACTCCATGATGTCAAAATGGCTAATTTTTCAGAGAACGTAATACCATACTTTAGAGCTATCGTCCAATCGCAGTGGACATTTCAAGGCTTAACCTCAATGTTGCTAAATGGATGGACTAACTTTAAAGTGTCACAAAATTGGCCGTTGATGCTCAAGGCATACGAAAAGGGGGTGACTAAGTACGCTGTCATTGCATGTCGTAAACCAGAATGA SEQ ID NO.4 Lr NMT2 cds sequence: > Lr NMT2-cds ATGACGACGATGACGGATGAACTGACCAAAGGGATGGTCGCCTGGTATGACGAGACGGCAAAGCAGGATGCGGTATGGGGGGAGTACTTGCACCACGGTTACTACGGGCCTGGCGAGCAGGCCCATGTTTCGACTAACCAGGCTGCTGAGCGGCGCATGGTCGATGAGGTTCTCAAATTCGCCGGCGTCTCTGATGATCCTATGAAAAAGCCTAAGAAT ATAATTGACGTTGGTTGTGGGTTCGGTGGAGCTGCTATATATCTAGCAAAGAAATATGGGGCCTATTGTAGTGGCATAAACTTGAGCCCGGCCCAAATTCAAAAAGCTAAAGAACTTGCTGCGGCTAATGGACTCGGAGACAAAGCTTCCTTCGTAGTTGGTGATGCATTGAACCAACCTTTTCCTGATGGACAATTTGATTTAGTTTGGTCCATGGAAG TTATCGAGCATACACCTGACAAGTTAAAGTTCATTAGCGAATTGGCACGTATAGCGGCACCAGGGGCTACTATTATATTTACATCATGCTGTCATAGGGATCTCGCTCCTGACGAAAAATCTCTAAAACCTGATGAGAAGAACCTTATAGACAAAATACGCAAAACATTTCATCAATCTTCTTATATTTCACCCTCTGATACTGTCAAAGTTGTTCAATC CTTGGAACTCCAAGATATCAAAATGGCGGATTTTTCAGAATGTATTACCGTACTTCAGAGCTATCGTCCAATCGCAACGGACATGGCAAGGTTTAGCCTCTATGTTGCTAAATGGATGGACTAACTTTAAAGTGTCACAAAATTGGCCGTTGATGCTCAAGGGATACGAAAAGAATGTGACTAAGTATATTGTCATTGCATGTCGTAAGCCAAAGTAA While preferred embodiments of the present invention have been described above, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.
[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A polypeptide that undergoes N-position methylation of an Amaryllidaceae alkaloid compound, characterized in that, The polypeptide is selected from any one of the following (a) to (f): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2; (c) A polypeptide formed by substitution, deletion or addition of one or more amino acids from the amino acid sequence in (a) and having the function of methylation at the N-position of Amaryllidaceae alkaloids; (d) A polypeptide derived from the amino acid sequence in (b) by substitution, deletion or addition of one or more amino acids and possessing the ability to methylate at the N-position of Amaryllidaceae alkaloids; (e) has an amino acid sequence that is more than 95% identical to that in (a), and is a polypeptide that has the function of methylating at the N position of Amaryllidaceae alkaloids. (f) and (b) have amino acid sequences with more than 95% identity, and are polypeptides that can methylate at the N-position of Amaryllidaceae alkaloids.
2. The polypeptide with N-position methylation function of the Amaryllidaceae alkaloid compound according to claim 1, characterized in that, The polypeptide is obtained through artificial synthesis, or by synthesizing its encoding gene and then expressing it biologically.
3. A polynucleotide, characterized in that, Selected from the following (g) to (j): (g) A polynucleotide encoding the polypeptides described in claims 1(a) to (f); (h) A conserved variant of the polypeptide, active fragment, derivative or analogue of the polypeptide described in claims (a) to (f) of claim 1; (i) A polynucleotide that is hybridizable to the polynucleotide sequence defined in (g) or (h) under strict hybridization conditions and encodes a polypeptide having N-position methylation function of an Amaryllidaceae alkaloid, wherein the polynucleotide sequences have at least 70% identity with each other. (j) has at least 70% identity with the polynucleotide sequence defined in (g) or (h), and the polypeptide encoded therefrom has the same or similar N-methyltransferase biological activity and function as the polypeptides described in (a) to (f) of claims 1.
4. The polynucleotide according to claim 3, characterized in that, The polynucleotide also includes polynucleotide variants, including substitution variants, deletion variants, or insertion variants.
5. The polynucleotide according to claim 3, characterized in that, The polynucleotide is in the form of DNA or RNA, wherein the DNA includes cDNA, genomic DNA or artificially synthesized DNA, and the DNA is in the form of single-stranded or double-stranded strands, and is a coding strand or a non-coding strand.
6. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in any one of claims 2 to 5.
7. A recombinant host cell, characterized in that, The recombinant expression vector of claim 5 was transferred.
8. A method for preparing a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids, characterized in that, Includes the following steps: (1) Culturing the recombinant host cells as described in claim 7; (2) Obtaining a polypeptide with N-position methylation activity of Amaryllidaceae alkaloids from recombinant host cells, wherein the polypeptide can be obtained as a crude cell extract or obtained after separation and purification.
9. The use of the polypeptide of claim 1, the polynucleotide of any one of claims 2 to 5, the recombinant expression vector of claim 6, or the recombinant host cell of claim 7 in the N-position methylation of Amaryllidaceae alkaloids.
10. A method for N-position methylation of Amaryllidaceae alkaloids, characterized in that, The method includes: In the presence of a methyl donor, the polypeptide of claim 1 is used to carry out an in vitro enzymatic reaction to N-methylate the substrate, thereby generating the corresponding product. Alternatively, in the presence of a methyl donor, the recombinant host cell described in claim 7 can be used to N-methylate the substrate through in vivo or in vitro reactions, thereby generating the corresponding product. The methyl donor is S-adenosylmethionine; The substrates are N-demethylgalantamine, N-demethylnalvidin, and their structural analogs.