O-methyltransferase catalyzing catalyzing catalyzing catalyzing catalyzing catalyzing catalyzing catalysis of apiole to osthol and its encoding gene and application

Abstract

The present application provides an O-methyltransferase derived from Cnidium monnieri, and an encoding gene and application thereof. Specifically, the O-methyltransferase of the present application can catalyze the methylation reaction of pulegol to generate ostruthin. The O-methyltransferase of the present application brings a broad space for improving the content of target components or directly producing effective components by using biotechnology, and has important application value in the fields of agriculture and medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an O-methyltransferase that catalyzes the formation of osthol from coumarin compounds such as apigenin, its encoding gene, and its applications. Background Technology

[0002] Cnidium monnieri is a traditional medicinal plant. Cnidium monnieri The dried, ripe fruit of Cnidium monnieri. Traditional medicine believes that Cnidium monnieri has the effects of drying dampness and dispelling wind, killing parasites and relieving itching, and warming the kidneys and strengthening yang. Chemical research shows that osthol is the main active ingredient of Cnidium monnieri. As a natural coumarin compound, osthol has biological activities such as anticancer, antiprotozoan, anti-osteoporosis, and anti-asthma, and is widely used in agriculture, medicine, cosmetics, and other fields. Osthol production mainly uses plant extraction methods, while microbial synthesis of osthol is not affected by seasons, has a short fermentation cycle, low production cost, and mild reaction conditions, and is expected to replace traditional production methods. However, the biosynthetic pathway of osthol is not yet fully elucidated, and the O-methyltransferase catalyzing the conversion of parsleyol to osthol has not been reported. Therefore, there is an urgent need in this field to develop a clone-related enzyme that can increase the content of the target ingredient or directly produce the active ingredient. Summary of the Invention

[0003] The purpose of this invention is to provide an O-methyltransferase CmOMT1 that catalyzes the production of osthol from parsleyol, its encoding gene, and its applications.

[0004] A first aspect of the present invention provides an isolated O-methyltransferase, namely the CmOMT1 polypeptide, wherein the polypeptide is selected from the group consisting of:

[0005] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0006] (b) A derivative protein having catalytic activity of parsleyol formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1.

[0007] (c) A protein derived from the sequence of (a) or (b);

[0008] (d) A derivative protein having ≥65% homology (preferably ≥80%, more preferably ≥90%, e.g. ≥95%, ≥99%) of the amino acid sequence shown in SEQ ID NO:1 and having catalytic activity against parsleyol.

[0009] In another preferred embodiment, the sequence (c) is a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (a) or (b).

[0010] In another preferred embodiment, the CmOMT1 polypeptide is derived from Cnidium monnieri.

[0011] In a preferred embodiment, the amino acid sequence of the CmOMT1 polypeptide is shown in SEQ ID NO:1.

[0012] A second aspect of the present invention provides an isolated polynucleotide selected from the group consisting of:

[0013] (a) The nucleotide sequence encoding the CmOMT1 polypeptide as shown in SEQ ID NO:1;

[0014] (b) The nucleotide sequence shown in SEQ ID NO:2;

[0015] (c) A nucleotide sequence that has ≥75% homology (preferably ≥80%, more preferably ≥90%, e.g. ≥95%, ≥99%) to the sequence shown in SEQ ID NO:2;

[0016] (d) A nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-12, more preferably 1-9) nucleotides to the 5' end and / or 3' end of the nucleotide sequence shown in SEQ ID NO:2;

[0017] (e) A nucleotide sequence that is complementary (preferably perfectly complementary) to any of the nucleotide sequences described in (a)-(d).

[0018] In a preferred embodiment, the sequence of the nucleotide is shown in SEQ ID NO:2.

[0019] In another preferred embodiment, the polynucleotide sequence as shown in SEQ ID NO:2 encodes a polypeptide with an amino acid sequence as shown in SEQ ID NO:1.

[0020] A third aspect of the present invention provides a recombinant vector containing the polynucleotides described in the second aspect of the present invention.

[0021] In some embodiments, the carrier is selected from the group consisting of: expression carriers, shuttle carriers, integration carriers, or combinations thereof.

[0022] In other embodiments, the vector is selected from the group consisting of bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or combinations thereof.

[0023] In a preferred embodiment, the vector includes a vector expressed in Escherichia coli, such as the pET series vectors.

[0024] A fourth aspect of the present invention provides a genetically engineered host cell containing the recombinant vector described in the third aspect of the present invention, or having the polynucleotides described in the second aspect of the present invention integrated into its genome.

[0025] In another preferred embodiment, the host cell is a prokaryotic or eukaryotic cell. More preferably, the host cell is selected from the group consisting of bacteria, yeast, higher plants, insects, or mammalian cells. In some embodiments, the host cell is a lower eukaryotic cell, such as a yeast cell. In other embodiments, the host cell is a higher eukaryotic cell, such as a mammalian cell. In still other embodiments, the host cell is a prokaryotic cell, such as a bacterial cell, preferably *Escherichia coli*.

[0026] The fifth aspect of this invention provides a method for preparing a CmOMT1 polypeptide, the method comprising:

[0027] (a) Culturing the host cells described in the fourth aspect of the present invention under suitable expression conditions;

[0028] (b) The CmOMT1 polypeptide was isolated from the culture.

[0029] The sixth aspect of the present invention provides the use of the CmOMT1 polypeptide or its derivative polypeptide as described in the first aspect of the present invention, the carrier as described in the third aspect of the present invention, or the host cell as described in the fourth aspect of the present invention, for catalyzing the following reaction, or for preparing a catalytic agent for catalyzing the following reaction: catalyzing the methylation of the hydroxyl group of a coumarin compound, for example catalyzing the methylation of the 7-hydroxyl group of parsleyol to generate osthol.

[0030] The seventh aspect of the present invention provides a method for methylating the hydroxyl groups of coumarin compounds, comprising the following steps:

[0031] In the presence of the polypeptide or its derivatives described in the first aspect of this invention, a methylation reaction of the hydroxyl groups of a coumarin compound is catalyzed, for example, by catalyzing parsleyol, to obtain osthol (therefore, this is a method for preparing osthol); the reaction principle is as follows:

[0032]

[0033] In a further preferred embodiment, the method further includes providing an additive to the reaction system for regulating enzyme activity.

[0034] In other embodiments, the additive for regulating enzyme activity is an additive that enhances or inhibits enzyme activity. In another preferred embodiment, the additive for regulating enzyme activity is selected from the group consisting of: Mg 2+ Ca 2+ Co 2+ Mn 2+ Ba 2+ Al 3+ Ni 2+ Zn 2+ , or Fe 2+ .

[0035] In a preferred embodiment, the pH of the reaction system is 6.5-8.5, preferably 7.4-7.6.

[0036] In some other preferred embodiments, the temperature of the reaction system is 25 ℃-35 ℃, preferably 28 ℃-30 ℃.

[0037] In some other preferred embodiments, the reaction time is 0.5 h-24 h, more preferably 1 h-10 h, and even more preferably 2 h-3 h.

[0038] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new technical solutions, all of which fall within the scope of this invention. Due to space limitations, they will not be described in detail here.

[0039] Through extensive and in-depth research, the inventors have, for the first time, derived from Cnidium monnieri ( Cnidium monnieri The parsnipin O-methyltransferase CmOMT1 was discovered in the transcriptome and is a key enzyme in the biosynthesis of osthol. CmOMT1 can efficiently catalyze the conversion of coumarin compounds such as parsnipin into osthol, which has profound scientific significance and application value for the biosynthesis of osthol. Attached Figure Description

[0040] Figure 1 SDS-PAGE protein electrophoresis image of CmOMT1. M: Standard; 1: Lysis buffer; 2: Supernatant; 3: Purified protein. CmOMT1 size is 42.9 kDa (including a 3.5 kDa His tag).

[0041] Figure 2 HPLC detection results of the conversion products of CmOMT1 in vitro enzyme-catalyzed reaction. A: using parsleyol as substrate; B: using bergamotol as substrate.

[0042] Figure 3MS detection results of the in vitro enzyme-catalyzed conversion products of CmOMT1. A: using parsleyol as substrate; B: using bergamotol as substrate.

[0043] Figure 4 Kinetic curves of the reaction of CmOMT1 with osthenol. Detailed Implementation

[0044] As used herein, the terms “active polypeptide”, “polypeptide of the present invention and its derivative polypeptides”, “enzyme of the present invention”, and “CmOMT1 of the present invention” all refer to the CmOMT1 (SEQ ID NO:1) polypeptide and its derivative polypeptides.

[0045] As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using its amino acid sequence.

[0046] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology.

[0047] The present invention also includes fragments, derivatives, and analogs of the said polypeptide. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the said polypeptide.

[0048] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0049] The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1.

[0050] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0051] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0052] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent (or stringent) conditions.

[0053] This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides long, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding the CmOMT1 protein.

[0054] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and are more preferably purified to homogenization.

[0055] The full-length nucleotide sequences or fragments thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0056] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0057] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0058] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0059] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0060] The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0061] Methods well known to those skilled in the art can be used to construct expression vectors containing the CmOMT1 polypeptide, as well as encoding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0062] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0063] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0064] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0065] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0066] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0067] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0068] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0069] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0070] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0071] Equipment, materials and reagents

[0072] PCR was performed using Mastercycler Pro (Eppendorf).

[0073] The constant temperature incubation was performed using an SGSP-03 300*35 constant temperature incubator (Huangshi Hengfeng Medical Equipment Co., Ltd.) and a ZQZY-75AN full temperature shaking incubator (Tianjin Boxin Biotechnology Co., Ltd.).

[0074] Centrifugation was performed using the 5418R high-speed refrigerated centrifuge and the 5418 mini centrifuge (Eppendorf).

[0075] OD600 was measured using a UV-1800 UV-Vis spectrophotometer (Shimadzu).

[0076] High performance liquid chromatography was performed using the LC-20AD liquid chromatography system (Shimadzu).

[0077] The results were obtained by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1200 HPLC system coupled with a Bruker-MicroTOF-II mass spectrometer.

[0078] Ultrasonic cell disruption was performed using the JY92-IIN cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0079] Oligonucleotide primers were purchased from Genewiz Biotechnology Co., Ltd.

[0080] Escherichia coli DH5α, BL21(DE3) strains and pET-28a(+) vector were used for gene cloning and protein expression.

[0081] The standard compounds, parsleyol and osthol, were purchased from Chengdu Zhibiao Chemical Pure Biotechnology Co., Ltd.

[0082] The standard compound bergamot was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0083] S-Adenosine-L-methionine (SAM) was purchased from Beijing Wokai Biotechnology Co., Ltd.

[0084] The gel recovery kit and the polysaccharide and polyphenol plant total RNA extraction kit were purchased from Tiangen Biotech Co., Ltd.

[0085] The TransScript One-step gDNA Removal and cDNA Synthesis Supermix reverse transcription kit was purchased from Beijing TransGen Co., Ltd.

[0086] Phanta Max high-fidelity DNA polymerase and seamless cloning kit were purchased from Novizan Biotechnology Co., Ltd.

[0087] Ni NTA Beads gravity columns and SDS-PAGE pre-adhesive were purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.

[0088] The 1M Tris-HCl (pH=7.5) buffer solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0089] Amicon® Ultra-15 centrifugal filter purchased from Meck Millipore

[0090] The BCA protein concentration assay kit was purchased from Biosharp.

[0091] Example 1: Recombinant preparation of the CmOMT1 gene and its encoded protein

[0092] 1. Discovery of the CmOMT1 gene

[0093] Transcriptome data of *Cnidium monnieri* (published in the literature "De novo transcriptome analysis of...) Cnidium monnieri(L.) Cuss and detection of genes related to coumarin biosynthesis) sequence analysis was performed. Based on annotation results, differential expression analysis, conserved sequence analysis, and amino acid sequence size, CmOMT1 was obtained as a candidate gene. Its DNA-encoded nucleotide sequence and protein amino acid sequence are shown in SEQ ID NO:2 and SEQ ID NO:1, respectively. Therefore, based on the above research, the inventors obtained CmOMT1 from Cnidium monnieri ( Cnidium monnieri A novel CmOMT1 protein catalyzing the conversion of parsleyol to osthol was screened from this species.

[0094] 2. Preparation of cDNA from Cnidium monnieri fruit

[0095] Fresh fruits of the plant *Cnidium monnieri* were flash-frozen in liquid nitrogen. RNA was extracted using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, following the kit's operating steps. cDNA was prepared by reverse transcription of RNA, referring to the TransScript One-step gDNA Removal and cDNA Synthesis Supermix Reverse Transcription Kit from TransGen.

[0096] 3. Cloning of the CmOMT1 gene and construction of expression plasmids

[0097] Design and synthesize primers (upstream primer: AGCAAATGGGTCGCGGA ATGGAAGGAATGACAAAAAAC; Downstream primer: CTCGAGTGCGGCCGCA CTAAGGATAAGCCTCGATAAC; the underlined part represents the homologous arm sequence. PCR amplification was performed using cDNA as a template, introducing homologous arm sequences complementary to pET-28a(+) upstream and downstream of the gene, respectively. PCR amplification system (50 μL): 10 μL 5X SF buffer, 1 μL dNTP Mix, Primer 1 / Primer 2 final concentration 0.2 μM; cDNA <200 ng; remaining volume made up with sterile distilled water. PCR reaction conditions: 95℃ pre-denaturation for 5 min, then 95℃ denaturation for 15 s, 45℃ annealing for 15 s, 72℃ extension for 90 s, 32 cycles. The target fragment was recovered using a Tiangen gel extraction kit.

[0098] Using a seamless cloning kit (Novozymes Biotechnology Co., Ltd.), the recovered fragment was ligated into the pET-28a(+) vector digested with BamHI and HindIII. The seamless cloning system (10 μL) consisted of 5 μL 2X ClonExpress Mix, 2 μL linearized vector, and 3 μL recovered fragment. Seamless cloning reaction conditions: incubation at 50°C for 5 min, followed by storage at 4°C. The seamless cloning product was transformed into *E. coli* DH5α competent cells. The plasmid that tested positive by colony PCR was sent to Genewiz Biotechnology Co., Ltd. for sequencing. The obtained DNA coding sequence and protein are shown in SEQ ID NO: 2 and 1, respectively. This recombinant plasmid is pET-28a(+)-CmOMT1.

[0099] 4. Expression of CmOMT1

[0100] The recombinant plasmid pET-28a(+)-CmOMT1 was transformed into competent Escherichia coli BL21(DE3) cells and plated on LB solid medium (kanamycin 50 μg / mL). The cells were incubated overnight at 37°C to obtain the recombinant strain BL21(DE3) / pET-28a(+)-CmOMT1. Single colonies were picked and cultured in 5 mL of LB liquid medium (kanamycin 50 μg / mL) overnight. After incubation, the colonies were transferred to 50 mL of fresh LB liquid medium and cultured at 200 rpm until OD500. 600 =0.6-0.8, add IPTG inducer to a final concentration of 0.1mM, and continue culturing at 200rpm and 16℃ for 20h. Centrifuge the bacterial culture (4000rpm, 10min, 4℃), discard the supernatant, resuspend the bacterial cells in 5mL of lysis buffer (25mM Tris-HCl, pH7.5), and sonicate on ice to obtain bacterial lysate.

[0101] Example 2: In vitro functional identification of CmOMT1

[0102] 1. Crude enzyme-catalyzed reaction

[0103] The total volume of the crude enzyme reaction system was 500 μL, including: 0.5 mM parsleyol, 2 mM S-adenosyl-L-methionine, 100 μL CmOMT1 crude enzyme solution, 1 mM DTT, 1 mM MgCl2, and 200 mM Tris-HCl (pH=7.5). After mixing, the reaction mixture was reacted at 30 °C for 12 h, and then 500 μL of methanol was added to terminate the reaction. After centrifugation (12000 rpm, 10 min) of the reaction mixture, the products were analyzed by HPLC and LC-MS.

[0104] 2. Product testing

[0105] The HPLC detection parameters are as follows: SilGreen C18 column (5μm, 4.6×250mm); column temperature 40℃; mobile phase consisting of solution A (0.1% formic acid aqueous solution) and solution B (methanol), with parsleyol as the substrate. The gradient elution method for the reaction system is as follows: maintain 30% mobile phase B for 0-5 min, increase mobile phase B from 30% to 100% for 5-25 min, maintain mobile phase B at 100% for 25-35 min, and decrease mobile phase B from 100% to 30% for 35-36 min. The cutoff wavelength is 326nm, and the flow rate is 1mL / min.

[0106] For LC-MS analysis, the chromatographic conditions remained unchanged. The mass spectrometry conditions were as follows: electrospray ionization (ESI) source in positive ion mode, capillary voltage 4500 V, nebulizer pressure 1 bar, nitrogen as the desolvent gas, flow rate 6.0 L / min, desolvent temperature and ion source temperature both 180 °C, and the scan range... m / z The range was 50-1000. The LC-MS data collection software was MassLynx 4.0 (Waters, USA). Sodium trifluoroacetate was used as the calibration solution for accurate molecular weight.

[0107] HPLC results of CmOMT1 catalyzing parsley phenol are as follows: Figure 2 As shown in Figure A, a new absorption peak appeared in the reaction system of the crude CmOMT1 enzyme, indicating that CmOMT1 can effectively convert the substrate parsleyol to generate a new product. The retention time of the new absorption peak is consistent with that of osthol. LC-MS detection was performed... Figure 3 (A), the exact molecular weight of this compound ([M+H)). + =245.1125) represents the exact molecular weight of parsleyol plus a methyl group. Therefore, the product is identified as osthol.

[0108] Example 3: In vitro reaction of CmOMT1

[0109] The crude enzyme solution was centrifuged (12000 rpm, 10 min, 4℃), and the supernatant was collected. CmOMT1 with the HIS tag was purified by affinity chromatography. Three column volumes of Ni NTA Beads gravity column were equilibrated with equilibration buffer (25 mM Tris-HCl (pH 7.5), 20 mM imidazole, 150 mM NaCl). The supernatant was added, and the column was held for 3 min to allow the target protein to fully bind to the nickel column. Ten column volumes were eluted with the equilibration buffer. Three column volumes were eluted with buffer A (25 mM Tris-HCl (pH 7.5), 100 mM imidazole, 150 mM NaCl) to ensure complete elution of contaminating proteins. Three column volumes were eluted with buffer B (25 mM Tris-HCl (pH 7.5), 250 mM imidazole, 150 mM NaCl). The target protein was collected and analyzed by SDS-PAGE. Figure 1 The collected solution was added to a 30 kDa ultrafiltration tube and centrifuged (4000 rpm, 20 min, 4 °C). The filtrate in the collection tube was discarded. 25 mM Tris-HCl (pH 7.5) was added to the ultrafiltration tube and centrifuged again (4000 rpm, 20 min, 4 °C) to achieve desalting. The solution in the ultrafiltration tube was aspirated with a pipette and added to buffer C (25 mM Tris-HCl (pH 7.5), 10% glycerol). The protein concentration was determined using the BCA method. The solution was aliquoted and stored at -20 °C.

[0110] The Km value of CmOMT1 to parsleyol was further determined. The reaction system was 100 μL, containing 1 mM S-adenosyl-L-methionine (SAM), parsleyol concentrations ranging from 10 μM to 320 μM, and 2 μg of purified protein CmOMT1. The reaction was carried out at 37℃ for 5 min, and the reaction was terminated by adding 100 μL of methanol. HPLC analysis was performed. The kinetic curve is shown below. Figure 4 CmOMT1 on parsleyol K m , K cat 、K cat / K m The values ​​were 20.285±3.445 μM and 0.137±0.006 s, respectively. -1 6.987±1.484s -1 ·mM -1 .

[0111] In addition, bergamot was used as a substrate for the reaction. The enzymatic reaction system was 100 μL, containing 100 μM bergamot, 1 mM S-adenosyl-L-methionine (SAM), 2 μg purified protein CmOMT1, and 25 mM Tris-HCl (pH 7.5). The reaction was carried out at 37℃ for 30 min, and terminated with 100 μL of methanol. No CmOMT1 was added in the blank control experiment. The product formation was identified by HPLC after the enzymatic reaction. The gradient elution method for the reaction system was as follows: 50% mobile phase B was maintained for 0-5 min; from 5-13 min, mobile phase B was increased from 50% to 100%; from 13-18 min, mobile phase B was maintained at 100%; from 18-20 min, mobile phase B was decreased from 100% to 50%; the cutoff wavelength was 308 nm; the flow rate was 1 mL / min. The HPLC results of CmOMT1 catalyzing bergamot are shown below. Figure 2 As shown in Figure B, a new absorption peak appears in the reaction system, indicating that CmOMT1 can effectively convert the substrate bergamotol to generate a new product. This was confirmed by LC-MS detection. Figure 3 (B), the exact molecular weight of this compound ([M+H)). +=217.0510) is the exact molecular weight of bergamotol plus a methyl group. Therefore, the product is identified as bergamot lactone. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> An O-methyltransferase that catalyzes the conversion of parsleyol to osthol, its encoding gene, and its applications. <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 358 <212> PRT <213> Cnidium monnieri <400> 1 MEGMTKNEATHENEDTAQAQVDIWRFVYGFAETAAVKCAVELGIPDILENHTEPMTLSQLSSALACRSTPLFRIMRFLMNIGLFKEKRTNQGSMGYVQTPMSRLLVKDGKNSLASMFLFVNGSVMTAPWNFLSAHVLDDRASPFTYAHGMGSWKLAAGNPDFRKLIDDAMACDTKMTVP AVLDGCPELFNGLSSVVNVGGGNGAAVRILVERCPWIHGINFDLPQVVSVAPVCEGIEHVGGDMFMSVPKANAAFLKWVLHNWSDDECIKILKNCREAIAEYGTSGKVIIVDAVIEGNGGDKPKNMGVLLDLIMLAQTDKGKERTAREWTFIFREAGFTRHTIKNIPFLTEITVIEAYP 358 <210> 2 <211> 1077 <212> DNA <213> Cnidium monnieri <400> 2 <210> 3 <211> 38 <212> DNA <213> Artificial sequence <400> 3 AGCAAATGGGTCGCGGAATGGAAGGAATGACAAAAAAC 38 <210> 4 <211> 37 <212> DNA <213> Artificial sequence <400> 4 CTCGAGTGCGGCCGCACTAAGGATAAGCCTCGATAAC 37

Claims

1. An isolated O-methyltransferase, characterized in that, It is a polypeptide with the amino acid sequence shown in SEQ ID NO:1, or a fusion protein formed by adding a tag sequence, signal sequence or secretion signal sequence to the amino acid sequence shown in SEQ ID NO:

1.

2. The O-methyltransferase as described in claim 1, characterized in that... From Cnidium monnieri.

3. A nucleic acid encoding an isolated O-methyltransferase as described in claim 1.

4. A recombinant vector, characterized in that, It contains the nucleic acid as described in claim 3.

5. The recombinant vector as described in claim 4, characterized in that, The originating vector can be a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, animal cell virus, or retrovirus.

6. A recombinant host cell, characterized in that, The host cell contains the vector of claim 4 or 5, or the nucleic acid of claim 3 is integrated into its genome.

7. The recombinant host cell as described in claim 6, characterized in that, These are bacteria, yeast, higher plant cells, insect cells, or mammalian cells.

8. The recombinant host cell as described in claim 7, characterized in that, It is either brewer's yeast or Escherichia coli.

9. A method for preparing an O-methyltransferase, characterized in that, include: (a) Culturing the recombinant host cells according to any one of claims 6 to 8 under suitable expression conditions; (b) Isolate the O-methyltransferase from the culture.

10. Use of an O-methyltransferase as described in claim 1 or 2, a recombinant vector as described in claim 4 or 5, or a recombinant host cell as described in any one of claims 6 to 8, characterized in that, Used to catalyze the following reaction, or used to prepare a catalyst for catalyzing the following reaction: methylating the hydroxyl groups of a coumarin compound to obtain the corresponding product; said coumarin compound is parsleyol or bergamotol.

11. A method for methylating the hydroxyl groups of coumarin compounds, characterized in that, The method includes the following steps: catalyzing the methylation of the hydroxyl groups of a coumarin compound in a reaction system in the presence of the O-methyltransferase as described in claim 1 or 2; wherein the coumarin compound is parsleyol or bergamotol.

12. The method as described in claim 11, characterized in that, The reaction system also contains an enzyme-activating additive, which is selected from the following: Mg 2+ Ca 2+ Co 2+ Mn 2+ Ba 2+ Al 3+ Ni 2+ Zn 2+ , or Fe 2+ Mg may be generated 2+ Ca 2 + Co 2+ Mn 2+ Ba 2+ Al 3+ Ni 2+ Zn 2+ , or Fe 2+ The substance.

13. The method as described in claim 12, characterized in that, The pH of the reaction system is 6.5-8.5; the temperature of the reaction system is 25℃-40℃; and the reaction time is 0.5 h-24 h.

14. The method as described in claim 13, characterized in that, The pH of the reaction system was 7.4-7.6; the temperature of the reaction system was 28℃-37℃; and the reaction time was 1 h-10 h.

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