Biphenylcyclooctene-type lignan-oxymethyltransferases and their applications

Abstract

This invention relates to a polypeptide molecule whose amino acid sequence is selected from: (A) SEQ ID NO.2; (B) an amino acid sequence obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncated version of (A) or (B); wherein the polypeptide molecule has catalytic activity for lignan methylation modification. The biphenylcyclooctene-type lignan oxygen methyltransferase of this application can methylate biphenylcyclooctene-type lignans, which can be used to explore lignans with novel functions, laying the foundation for synthetic biology research on the diversification of lignans in Schisandra chinensis, and has significant application value. Compounds obtained using the biphenylcyclooctene-type lignan oxygen methyltransferase of this application, or compounds obtained using it as an intermediate, can become effective pharmaceutical ingredients, functional food materials, etc., making the biphenylcyclooctene-type lignan oxygen methyltransferase of this application have positive application prospects in the pharmaceutical and food industries.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and particularly to oxymethyltransferases involved in the biosynthesis of biphenylcyclooctene lignans from Schisandra chinensis and their applications. Background Technology

[0002] Biphenylcyclooctene lignans are a class of lignans containing a unique, highly oxidized eight-membered ring structure, polymerized from two C6-C3 units. They are concentrated in the Schisandraceae family. Since the 1970s, they have received widespread attention due to their significant activity, exhibiting hepatoprotective, lipid-lowering, anti-inflammatory, anti-tumor, and central nervous system sedative activities.

[0003] The Chinese medicinal herb Schisandra chinensis is the plant Schisandra chinensis of the Schisandraceae family. Schisandra chinensis) The dried, ripe fruit of Schisandra chinensis, commonly known as "Northern Schisandra," is included in the Chinese Pharmacopoeia and possesses astringent, qi-tonifying, and fluid-generating effects. Biphenylcyclooctene lignans, characteristic components of Schisandra chinensis, with schisandrin A, schisandrin B, and schisandrol A as representative compounds, are the pharmacological basis for its hepatoprotective, central nervous system-improving, lipid-lowering, anti-inflammatory, and anti-tumor activities. The activity of biphenylcyclooctene lignans is closely related to their diverse methylation modifications.

[0004] Methyltransferases are common modifying enzymes in plants, and a large number of methyltransferases have been reported to date, including those catalyzing flavonoids, coumarins, and alkaloids. However, methyltransferases catalyzing biphenylcyclooctene lignans have not yet been identified. Identifying methyltransferases that specifically catalyze biphenylcyclooctene lignans plays a crucial role in the structural exploration and discovery of active compounds.

[0005] Chemical structural studies of lignans have revealed that structural modifications can improve the physicochemical properties of drugs, facilitating interactions between drugs and receptors or enzymes and inducing corresponding biochemical and biophysical reactions. However, research on enzyme-induced chemical structural modifications of biphenylcyclooctene-type lignans remains lacking.

[0006] The findings are of great significance for elucidating the biosynthetic pathway of biphenylcyclooctene-type lignans characteristic of Schisandra chinensis and for the development of active drugs. Summary of the Invention

[0007] To address the technical problems existing in the prior art, the present invention proposes a polypeptide molecule whose amino acid sequence is selected from: (A) SEQ ID NO.2; (B) an amino acid sequence generated by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncated version of (A) or (B); wherein the polypeptide molecule has the activity of catalyzing lignan methylation modification, and further, the polypeptide molecule has the activity of catalyzing lignan phenolic hydroxymethylation.

[0008] The polypeptide molecule as described above, wherein the lignan is a biphenylcyclooctene type lignan.

[0009] The polypeptide molecule described above, wherein the lignan is Gomisin J.

[0010] A polynucleotide molecule encoding a polypeptide molecule as described above, said polynucleotide molecule being selected from: (a) SEQ ID NO.1; (b) a nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncated version of (a) or (b).

[0011] A recombinant vector includes: a polynucleotide molecule as described above; and an expression vector.

[0012] A fusion cell includes: a recombinant vector as described above; and an expression cell.

[0013] A method for preparing a protein for catalyzing the methylation modification of biphenylcyclooctene lignans includes: converting a nucleic acid molecule as described in 4 above into an expression cell; expressing the protein of the polypeptide molecule in the expression cell; and purifying the protein of the polypeptide molecule.

[0014] Application of any of the above-described polypeptide molecules in catalytic modification of biphenylcyclooctene lignans by methylation.

[0015] A method for preparing Gomisin K2, the method comprising: catalyzing Gomisin J using one or more of the following groups: a polypeptide molecule as described above; a polypeptide molecule encoded by a nucleic acid molecule as described above; a polypeptide molecule expressed by a recombinant vector as described above; a polypeptide molecule obtained from fusion cells as described above; and a polypeptide molecule prepared by the method as described above.

[0016] Gomisin K2 was prepared by the above method.

[0017] The lignan-oxymethyltransferase of this application can methylate biphenylcyclooctene-type lignans, which can be used to explore lignans with novel functions, laying the foundation for synthetic biology research on the diversification of lignans in Schisandra chinensis and has significant application value. Compounds obtained using the biphenylcyclooctene-type lignan-oxymethyltransferase of this application, or compounds obtained using it as an intermediate, can become effective pharmaceutical ingredients, functional food materials, etc., making the lignan-oxymethyltransferase of this application a promising candidate for applications in both the pharmaceutical and food industries. Attached Figure Description

[0018] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0019] Figure 1 This is according to an embodiment of the present invention. Sch SDS-PAGE electrophoresis image of OMT6 protein, where: M: protein molecular weight standard; lane 1: Sch OMT6 supernatant; Lane 2: Sch OMT6 precipitated bacterial cells; Lane 3: Sch OMT6: Unattached proteins; Lane 4: Miscellaneous proteins; Lane 5: Sch Purified protein of OMT6;

[0020] Figure 2 This is according to an embodiment of the present invention. Sc HPLC chromatogram of the enzyme activation catalysis reaction of hOMT6 with Gomicin J as substrate; wherein the enzyme activation catalysis reaction was compared with the catalysis reaction of empty support as a control.

[0021] Figure 3 This is according to an embodiment of the present invention. Sch LC-MS spectra and reaction formulas of the enzyme-catalyzed reaction of OMT6 with gomisin J as a substrate; among which... Figure 3 A is an embodiment of the present invention. Sch LC-MS spectrum of the enzyme-catalyzed reaction of OMT6 with Gomicin J as substrate; Figure 3 B is an embodiment of the present invention. Sch The enzyme-catalyzed reaction formula of OMT6 using Gomisin J as a substrate. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments may also be utilized, or structural, logical, or electrical changes may be made to the embodiments of the present application.

[0024] The technical terms used in this article have the following meanings:

[0025] The term "lignans" as used in this article refers to a class of natural organic compounds formed by the polymerization of two or three molecules of phenylpropanoid derivatives in various forms, mostly distributed in angiosperms and gymnosperms. They can be divided into two main categories: compounds formed by the polymerization of two phenylpropanoid derivatives through the β-position of their side chains are called lignans; compounds formed by the side chain of one phenylpropanoid molecule linked to the benzene ring of another molecule, or by the two parts being linked by oxygen atoms, are called neolignans. More than 200 lignans and more than 100 neolignans have been isolated from the plant kingdom. Traditional Chinese medicines such as Schisandra chinensis, Acanthopanax senticosus, Trachelospermum jasminoides, Arctium lappa, Polygala tenuifolia, Forsythia suspensa, Carthamus tinctorius, and Asarum heterotropoides all contain lignans. Many lignan components may have stereoisomers due to the saturated cyclic structure. Various lignans with anticancer, antifungal, insecticidal, white blood cell-raising, alanine transaminase-lowering, hepatoprotective, antitussive, and laxative activities have been discovered, but few are used medicinally. Derivatives of podophyllotoxin lignans are used as anticancer drugs, while lignans in Schisandra chinensis have the effect of lowering alanine aminotransferase (ALT) and are used to treat hepatitis.

[0026] The "Schisandra" mentioned in this article refers to Schisandra chinensis Schisandra chinensis is a woody plant belonging to the genus Schisandra in the family Schisandraceae. Studies have shown that Schisandra chinensis has extensive medicinal value, including: anti-hepatic effects, broad-spectrum central nervous system depressant effects, cardiotonic effects, enhancement of the body's defense against non-specific stimuli, and inhibitory effects against Bacillus anthracis, Staphylococcus aureus, Staphylococcus albus, Salmonella typhi, and Vibrio cholerae. Lignans are the main active components of Schisandra chinensis, with the fruit containing 18.1% total lignans and the stem containing 10.25%. These lignans exhibit significant antioxidant activity, protecting the heart from lipid peroxidation damage.

[0027] The term "methylation" as used in this article refers to the catalytic transfer of methyl groups from an active methyl compound to other compounds, which can form various methyl compounds or chemically modify certain proteins or nucleic acids to form methylated products. Within biological systems, methylation is enzymatically catalyzed and involves the regulation of gene expression, the regulation of protein function, and ribonucleic acid processing.

[0028] The "OMT6" mentioned in this article belongs to the OMTs family and is an O-Methyltransferase. OMTs are important enzymes that depend on S-adenosylmethionine to catalyze the production of various secondary metabolites such as flavonoids, alkaloids, and phytoalexins. They play a crucial role in various stages of plant growth and development, as well as in resisting the invasion of external pathogens. OMTs typically exist in plants and animals as a gene family.

[0029] The protein of the present invention, for example, the amino acid sequence shown in SEQ ID No. 2, comprises (A) a protein having the amino acid sequence shown in SEQ ID No. 2, or (B) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the above amino acid sequence, and having the function of catalyzing the phenolic hydroxymethylation of gomisin J, or (C) a truncated form of the above two, such as truncating 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids at the N-terminus or C-terminus, or extending 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids at the N-terminus or C-terminus, and having the function of catalyzing the phenolic hydroxymethylation of gomisin J.

[0030] The terms "replacement, deletion, or addition of one or more bases" or "replacement, deletion, or addition of one or more amino acids" as used herein refer to the use of known mutagenesis methods such as localized mutagenesis [Hashimoto-Gotoh, Gene 152, 271-275 (1995) others] to mutate a nucleotide or amino acid sequence into a sequence having at least 80% homology to the original sequence. For example, the mutated sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology to the original sequence. In some embodiments, in the above-described proteins and genes, the term "more than 95% homology" may mean at least 96%, 97%, or 98% identity. The term "more than 90% homology" may mean at least 91%, 92%, 93%, or 94% identity. The term "more than 85% homology" can mean at least 86%, 87%, 88%, or 89% identity. The term "more than 80% homology" can mean at least 81%, 82%, 83%, or 84% identity.

[0031] The terms "coding gene" and "nucleic acid molecule" refer to ribonucleotide (RNA) or deoxyribonucleotide (DNA) sequences that encode a specific amino acid peptide chain. In some embodiments, the coding gene or nucleic acid molecule can be obtained through whole-gene synthesis, PCR amplification, chemical synthesis, etc. This application does not limit the method of obtaining the coding gene and nucleic acid molecule.

[0032] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. Vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. Vectors also include vectors having multiple of the above-described functions. A vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0033] In some embodiments of the present invention, the term "recombinant vector" specifically refers to cloning the coding gene of lignan-oxymethyltransferase into an expression vector, such as the pET28a vector (which contains restriction enzyme sites, such as restriction enzyme sites as...). Bam HⅠ and XhoThe recombinant vector obtained between (I) and (II) is used. In other embodiments, the expression vector is selected from one or more of the following vectors: pPIC9, pPIC9K, pPICZαB, pPICZαB vector, pET series vectors, pGEX series vectors, pMAL series vectors, pQE series vectors, pBADmycHis series vectors, pTrcHis series vectors, pTXB series, T series vectors, and other vectors, as well as modified vectors of the above vectors. In some embodiments, the recombinant expression vector may be selected from prokaryotic expression vectors such as the pET series, pGEX series, and pCold series, yeast expression vectors such as pPIC9, pHIL-D2, and pPIC3.5, and plant expression vectors such as the PBI series and pCAMBIA series. This application does not limit the method of obtaining the recombinant vector; recombinant vectors obtained by other means are also within the scope of protection of this application.

[0034] The terms "recombinant expression cell," "recombinant cell," and "expression cell" refer to cells whose genomes contain foreign genes, or host cells containing expression vectors. In some embodiments, "recombinant expression cell," "recombinant cell," and "expression cell" can be bacteria, fungi, higher plant cells, etc. In some embodiments, it can be obtained by introducing the encoding gene of lignan-oxymethyltransferase into *Escherichia coli*, mammalian cells, yeast cells, or *Pichia pastoris* genetically modified via a glycosylation pathway.

[0035] In this article, "including" generally means to contain, summarize, include, or encompass. In some cases, it also means "to be" or "composed of".

[0036] O-methyltransferases, as an important class of structural modifying enzymes, are key enzymes in the biosynthesis of pharmacodynamic compounds in Schisandra chinensis. Studies on the biosynthetic pathway of biphenylcyclooctene lignans in Schisandra chinensis suggest that the pathway involves the formation of a biphenylcyclooctene lignan structure from a dibenzylbutane-type lignan via a benzene ring linkage. Methylation modification may occur before or after coupling and is a crucial step in the synthetic pathway of compounds containing oxymethyl groups.

[0037] Therefore, this invention focuses on screening and identifying structural modification genes (OMTs) of schisandra lignans. The results have significant theoretical and practical implications for elucidating the lignan synthesis pathways and the mechanisms underlying the structural diversity of schisandra compounds.

[0038] The purpose of this invention is to provide an oxygen methyltransferase gene involved in the methylation modification of biphenylcyclooctene lignans in Schisandra chinensis and its encoded protein. Studies have shown that the Schisandra chinensis oxygen methyltransferase SchOMT6 obtained in this invention can catalyze the methylation of gomisin J.

[0039] The present invention provides Sch The OMT6 gene, whose nucleotide sequence is shown in SEQ ID No. 1, or its mutated sequence.

[0040] The present invention provides Sch The protein encoded by the OMT6 gene has the amino acid sequence shown in SEQ ID No. 2, or a mutated sequence thereof.

[0041] The objective of this invention can be achieved through the following technical solutions:

[0042] Technical Solution 1: Screening of key enzyme genes for methylation modification of Schisandra chinensis biphenylcyclooctene lignans based on transcriptome analysis, with the following steps:

[0043] 1) UPLC was used to detect the differences in the content of oxymethyl biphenylcyclooctene lignans in Schisandra chinensis fruit, mature stems, old leaves and roots.

[0044] 2) Based on the Schisandra chinensis genome, identify members of the Schisandra chinensis OMTs gene family, and use the HMM model of the OMT gene family to preliminarily screen the key enzyme protein sequences of OMTs in Schisandra chinensis.

[0045] 3) Based on transcriptome data from different tissues of Schisandra chinensis, differential gene expression was analyzed to further screen key enzyme genes that modify hydroxymethylation in the biosynthetic pathway of biphenylcyclooctene lignans in Schisandra chinensis.

[0046] Technical Solution 2: Key Enzyme Gene Sch Functional validation of OMT6. A prokaryotic expression system was used, with gomisin J as a substrate, to identify OMT6 transoxidases in vitro. Sch The functions of OMT6.

[0047] This application relates to a polypeptide molecule whose amino acid sequence is selected from: (A) SEQ ID NO.2; (B) an amino acid sequence obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncated version of (A) or (B); wherein the polypeptide molecule has catalytic activity for lignan methylation modification, and further, the polypeptide molecule has catalytic activity for lignan phenolic hydroxyl methylation. Further, the above-mentioned polypeptide molecule is encoded by a coding gene selected from the following polynucleotide molecules: (a) SEQ ID NO.1; (b) a nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncated version of (a) or (b).

[0048] In some embodiments, the lignan is a biphenylcyclooctene-type lignan. Further, in some embodiments, the biphenylcyclooctene-type lignan is Gomisin J.

[0049] In some embodiments, this application relates to a recombinant vector comprising: the aforementioned polynucleotide molecule, wherein the nucleic acid molecule is capable of expressing the aforementioned polypeptide molecule. In some embodiments, the polypeptide molecule has catalytic activity for lignan methylation modification; and an expression vector. In some embodiments, the polypeptide molecule has catalytic activity for lignan phenolic hydroxyl methylation. In some embodiments, the expression vector may be selected from prokaryotic expression vectors such as the pET series, pGEX series, and pCold series; yeast expression vectors such as pPIC9, pHIL-D2, and pPIC3.5; and plant expression vectors such as the PBI series and pCAMBIA series.

[0050] In some embodiments, this application relates to a fusion cell comprising the aforementioned recombinant vector and an expression cell. In some embodiments, the expression cell may be bacteria, fungi, higher plant cells, etc. In some embodiments, it may be obtained by introducing the encoding gene of lignan-oxymethyltransferase into the *Escherichia coli*, mammalian cells, yeast cells, or *Pichia pastoris* genetically modified via a glycosylation pathway.

[0051] In some embodiments, the aforementioned polypeptide molecule, or the polynucleotide molecule encoding the aforementioned polypeptide molecule, can be used to catalyze methylation modification of lignans. In some embodiments, the lignan is a biphenylcyclooctene-type lignan. Further, the biphenylcyclooctene-type lignan is Gomisin J. In some embodiments, Gomisin J can be converted to Gomisin K2 by the catalytic activity of the aforementioned polypeptide molecule under the influence of its oxygen methyltransferase activity. In some embodiments, Gomisin K2 has the following structural formula:

[0052] .

[0053] This invention discloses the gene encoding the oxymethyltransferase that catalyzes hydroxymethylation in the lignan biosynthesis pathway of Schisandra chinensis. Sch OMT6 screening and functional identification methods, verification Sch OMT6 has the function of catalyzing the formation of gomisin J from gomisin K2, and can catalyze the hydroxymethylation of progomisin to generate Schisandrathera D, laying the foundation for the synthetic biology research on the diversity of lignans in Schisandrathera and having significant application value.

[0054] The technical solution of this application will be explained through the following embodiments.

[0055] Example 1: Gene Expression Sch OMT6 Cloning

[0056] 1.1 Extraction of total RNA from Schisandra chinensis using the CTAB-PVP method

[0057] (1) Take fresh Schisandra chinensis plant material (leaves or fruit) and grind it into powder quickly in liquid nitrogen.

[0058] (2) Use a centrifuge tube to estimate 50-100 mg of powder and put it into a pre-cooled 2 mL imported centrifuge tube. Add 600-800 μL of CTAB-PVP extract solution preheated at 65℃ and place it in a vortex shaker for 30 seconds to allow it to fully lyse.

[0059] The preparation method for the above CTAB-PVP extraction buffer is as follows:

[0060] 100 mM Tris·HCl (pH 8.0), 2% CTAB (w / v), 2% PVP (w / v), 25 mM EDTA, 2 M NaCl, and β-mercaptoethanol were added to 0.2% after autoclaving; the solution was prepared using DEPC-treated double-distilled water (ddH2O), which was then autoclaved and ready for use.

[0061] (3) Bathe in a 65℃ water bath for 30 minutes, inverting and mixing once every 10 minutes.

[0062] (4) After cooling to room temperature, add 600-800 μL of chloroform, mix by inversion, and centrifuge at 13,000 rpm for 10 min at 4℃.

[0063] (5) Transfer the supernatant to a new 2 mL imported centrifuge tube, add 600-800 μL of chloroform, shake to mix evenly, and centrifuge at 13,000 rpm for 10 min at 4℃.

[0064] (6) Repeat the previous step (i.e., extract with chloroform three times).

[0065] (7) Carefully aspirate the supernatant into a new 1.5 mL imported centrifuge tube, add 1 / 3 volume of 8 M LiCl, and let stand overnight at -20℃.

[0066] (8) Centrifuge at 13,000 rpm for 10 min at 4℃ and discard the supernatant.

[0067] (9) Add 700 μL of 75% ethanol (prepared with DEPC water) and wash the precipitate 2-3 times. Centrifuge, discard the supernatant, and evaporate the remaining ethanol.

[0068] (10) Add 30 μL of sterile water treated with Proteinase K to dissolve the RNA and obtain total RNA. The concentration and quality of the extracted RNA were determined using a BioPhotometer plus nucleic acid and protein analyzer.

[0069] 1.2 Sch OMT6 gene full-length amplification

[0070] 1.2.1 Primer Design

[0071] Full-length primers were designed on both sides of the open reading frame (ORF) using the software SnapGene. Sch OMT6-F / R amplifies the gene.

[0072] 1.2.2 cDNA Synthesis

[0073] Using total RNA extracted from Schisandra chinensis as a template, cDNA template strands were obtained by PCR using the PrimerScript RT Master Mix reverse transcription system.

[0074] The reverse transcription system and reverse transcription procedure are as follows:

[0075] Reverse transcription PCR system:

[0076]

[0077] Reverse transcription program: 37℃, 15 min;

[0078] 85℃, 15 s.

[0079] The reverse transcription product should be stored at -20°C and diluted before use.

[0080] 1.2.3 Target gene amplification

[0081] Using diluted reverse-transcribed Schisandra chinensis cDNA as a template, SchOMT6-F / R primers were used for amplification.

[0082] The amplification system and amplification procedure are as follows:

[0083]

[0084] Add the above components to a 200 μL PCR tube, mix thoroughly, centrifuge at low speed, and then place in a PCR instrument for amplification according to the following procedure:

[0085] 95℃, 3 min;

[0086] 95℃, 15 s; 52℃, 15 s; 72℃, 45 s, 30 cycles;

[0087] 72℃, 5 min.

[0088] The PCR reaction products were detected by agarose gel electrophoresis, and the target size bands were cut and recovered from the gel.

[0089] Example 2 Gene Sch Construction of OMT6 expression vector

[0090] 2.1 Amplification of cells with homologous arms Sch OMT6 segment

[0091] Primers with homologous arms Sch OMT6-pET28a-F / R Sch The OMT6 fragment was amplified. The amplification system and procedure are shown in Example 1.

[0092] 2.2 Homologous recombination to construct expression vectors

[0093] 2.2.1 Vector double enzyme digestion

[0094] pET28a vector Bam HI and Xho I was subjected to enzyme digestion, and the enzyme digestion system is as follows:

[0095]

[0096] Enzyme digestion was performed in a 37°C water bath for 30 min. After stopping the reaction by adding 10× loading buffer to the digestion product, agarose gel electrophoresis was performed, and appropriate bands were selected for gel recovery. The gel recovery method is the same as above and will not be repeated.

[0097] 2.2.2 Homologous recombination, transformation and positive verification

[0098] The target fragment with homologous arms was ligated to the enzyme-digested vector pET28a using a homologous recombination kit, as follows:

[0099]

[0100] After thoroughly mixing the above components, react at 37°C for 30 min, and immediately cool on ice. Transform E. coli DH5α competent cells with the ligation product. After thawing E. coli DH5α competent cells stored at -80°C, add all the ligation product, gently pipette to mix, and place on ice for 30 min; heat shock in a 42°C water bath for 45 s, then quickly place on ice for 2 min, add 500 μL of antibiotic-free LB medium, and incubate at 37°C with shaking for 1 h. Spread 200 μL of the transformation solution onto LB solid medium (containing 100 μg / mL kanamycin resistance) and incubate statically at 37°C for 12-16 h.

[0101] Select single clones for colony PCR verification to confirm a positive result. A single clone that amplifies a bright and single target band is considered a positive clone. Send the positive clones for sequencing, and store and extract the bacteria from the clones that correctly sequence. Sch The OMT6-pET28a plasmid was used to transform E. coli BL21 (DE3) competent cells using a heat shock method. The transformation, screening, and identification methods were the same as those described above and will not be repeated here.

[0102] Example 3: Analysis of gene and protein expression and enzyme activity

[0103] 3.1 Sch OMT6 recombinant protein prokaryotic expression

[0104] (1) Pick strains Sch OMT6-pET28a-BL21 positive clones were inoculated into 4 mL of LB medium containing kanamycin (Kana) resistance and cultured overnight at 37°C and 110 rpm on a shaker.

[0105] (2) The cultured bacterial solution was inoculated into 200 mL of Kana-resistant medium at a ratio of 1:100 and cultured under the same conditions until OD. 600 ≈ 0.5. Add 0.3 mM IPTG to the bacterial culture and incubate at 16℃ and 110 rpm for 16-18 h to induce the expression of the target protein.

[0106] (3) Collecting bacteria: Centrifuge the bacterial solution at 5000 rpm for 5 min and then discard the supernatant.

[0107] (4) Washing: Add an appropriate amount of binding buffer to the centrifuge tube according to the bacterial count to resuspend the bacterial cells. Centrifuge at 5,000 rpm for 5 min, collect the bacterial cells, wash twice, and then add 15-20 mL of binding buffer to resuspend the bacterial cells.

[0108] (5) Lysis: Place the bacterial culture in an ice-water mixture, sonicate to lyse the bacterial cells, centrifuge at 4℃ and 12,000 rpm for 20 min, and collect the supernatant to obtain the crude enzyme.

[0109] 3.2 Sch Functional verification of OMT6 crude enzyme

[0110] Sch OMT6 was used for in vitro enzyme activity assays, with the reaction system containing pET28a protein serving as the control group. The substrates included progomisin, gomisin J, and gomisin K1. The enzyme activity reaction system is as follows:

[0111]

[0112] After mixing the above components and reacting overnight at 30°C, the reaction was terminated by adding 50 μL of methanol. After centrifugation at 12,000 rpm for 30 min, the supernatant was collected and analyzed by HPLC for enzyme activity.

[0113] The results are as follows Figure 2 As shown, compared with the blank control, Sch OMT6 can catalyze the methylation of biphenylcyclooctene lignan gomisin J. The retention time is consistent with that of the standard gomisin K1, and it is initially judged to be a product of monohydroxy methylation. However, due to the stereoconfiguration of the substrate, it is speculated that the product is gomisin K2, which has the same structural formula as gomisin K1 but a different stereoconfiguration.

[0114] 3.3 Sch Purification and functional validation of OMT6 recombinant protein

[0115] As described in 3.1, the supernatant of the lysed bacterial culture was purified by column chromatography, and a portion of the supernatant was reserved for SDS-PAGE to observe protein expression.

[0116] (1) Separation: Add the collected supernatant to an equilibrated Ni-NTA column. After the supernatant has flowed out, add one column volume of elution buffer (containing 20 mM imidazole) to wash away the impurities. Then, use 5 mL of elution buffer (containing 250 mM imidazole) to collect the target recombinant protein.

[0117] (2) Ultrafiltration: Place the eluted protein solution into an ultrafiltration tube with a protein molecular weight of 30,000 Da, centrifuge at 4,000 rcf for 10 min, and add binding buffer to change the medium 2-3 times, and then concentrate the target protein.

[0118] (3) Pipette the concentrate into a 2 mL collection tube, measure the protein concentration, and retain the sample.

[0119] (4) After adding 10% glycerol to the protein, quick-freeze it with liquid nitrogen and store it in a -80°C freezer for later use.

[0120] Binding buffer: Weigh 2.42 g Tris-HCl and 29.22 g NaCl, dissolve in water, adjust pH to 8.0, bring volume to 1000 mL, sterilize, add 70 μL β-mercaptoethanol, and store at 4 °C.

[0121] Elution buffer: Weigh 2.42 g Tris-HCl, 29.22 g NaCl, and 34 g gimidazole, dissolve them in water, adjust the pH to 8.0, bring the volume to 1000 mL, sterilize, add 70 μL β-mercaptoethanol, and store at 4 °C.

[0122] The activity of the purified protein was verified as described in section 3.2, and enzyme activity was analyzed using LC-MS. The results are as follows: Figure 3 As shown. Among them, Figure 3 The displayed Sch The product generated by OMT6 catalysis of Gomicin J has an added methyl group, making it a monohydroxymethylated product.

[0123] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A polypeptide molecule having the amino acid sequence shown in SEQ ID NO.2; in, The polypeptide molecule has the activity of catalyzing the hydroxymethylation of lignans.

2. A polynucleotide molecule encoding the polypeptide molecule as described in claim 1, said polynucleotide molecule being shown in SEQ ID NO.

1.

3. A recombinant vector, comprising: The polynucleotide molecule as described in claim 2; as well as Carrier of expression.

4. A fused cell, comprising: The recombinant vector as described in claim 3; as well as Expression cells.

5. A method for preparing a protein for catalyzing the methylation modification of Gomisin J, comprising: Transform the polynucleotide molecule as described in claim 2 into expression cells; Proteins that express the polypeptide molecule within the expressing cell; as well as Purify the protein of the polypeptide molecule; The polypeptide molecule catalyzes the methylation modification of gomisin J to produce gomisin K2.

6. The application of the polypeptide molecule as described in claim 1 in catalyzing the methylation modification of Gomisin J; wherein, The polypeptide molecule catalyzes the methylation modification of gomisin J to produce gomisin K2.

7. A method for preparing Gomisin K2, the method comprising: The polypeptide molecule as described in claim 1 is used to catalyze Gomisin J.

8. Use of the polypeptide molecule as described in claim 1 in the preparation of Gomisin K2.

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