Aesculetin methyltransferase protein in the traditional Chinese medicine Artemisia annua and its encoding gene and application

Abstract

The present invention discloses a esculetin methyltransferase protein from the traditional Chinese medicine Artemisia annua, as well as its encoding gene and application. The protein is the protein described in a) or b) below: a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing; b) a protein derived from a) in which the amino acid sequence shown in Sequence 2 in the sequence listing is substituted and / or deleted and / or added with one or more amino acid residues and has esculetin methyltransferase activity. The present invention is based on enriching scopoletin from the traditional Chinese medicine Artemisia annua, screening AaOMT1 using transcriptome and proteome, and identifying its ability to catalyze the production of scopoletin through enzymatic activity. Identifying a methyltransferase that directly and specifically produces scopoletin can provide a new pathway for the biosynthesis of scopoletin and a new reference for drug preparation, purification, and synthetic design.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to aesculetin methyltransferase protein in the traditional Chinese medicine Artemisia annua and its encoding gene and application. Background Art

[0002] Scopoletin (7-hydroxy-6-methoxycoumarin, Scopoletin) is a typical representative of the coumarin family. Figure 1 Scopoletin is derived from a carbon skeleton C6-C3, containing a quasi-cubic skeleton core in a 1,2-benzopyrone structure with methoxylated and hydroxylated modifications on the benzene ring (C6). Scopoletin was isolated from phenolic resins found in Arabidopsis thaliana and other plants with phenylpropanoid pathways, and its medicinal value has attracted great attention around the world. The in vitro pharmacological activities of scopoletin include antibacterial, antifungal, antituberculosis and antihypertensive properties. Proven in vivo pharmacoecological activities include anti-inflammatory, neurological, anti-diabetic and anti-hyperuricemic properties.

[0003] Scopoletin has been biosynthesized in Escherichia coli. The key enzymes involved include 4-coumarate:CoA ligase (4CL), caffeic acid oxygen methyltransferase (COMT), caffeoyl-CoA oxygen methyltransferase (CCoAOMT), feruloyl-CoA 6'-hydroxylase (F6'H), and coumarin synthase (COSY). From the molecular structure, it is clear that the direct product of methylation of the 7-hydroxyl group of aesculetin is scopoletin. However, to date, only POMT9 and PaOMT2 have been identified in poplar (Populus deltoids Marsh) and Plagiochasma appendiculatum that can methylate aesculetin to produce multiple products including scopoletin. In summary, identifying methyltransferases that directly and specifically produce scopoletin could provide a new pathway for the biosynthesis of scopoletin and a new reference for drug preparation, purification, and synthetic design. Summary of the Invention

[0004] In light of this, the present invention enriched scopoletin from the traditional Chinese medicinal herb Artemisia annua, identified AaOMT1 through transcriptomic and proteomic screening, and identified its enzyme activity as catalyzing the production of scopoletin. Identifying a methyltransferase that directly and specifically produces scopoletin could provide a new pathway for the biosynthesis of scopoletin and offer a new reference for drug preparation, purification, and synthetic design.

[0005] The technical solutions of the present invention are as follows:

[0006] A protein is a protein as described in a) or b) below:

[0007] a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

[0008] b) A protein derived from a) having aesculetin methyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is subjected to substitution and / or deletion and / or addition of one or more amino acid residues. That is, one or more of the three types of substitution and / or deletion and / or addition are selected for treatment.

[0009] The aesculetin methyltransferase activity is an activity that catalyzes the methylation of aesculetin to generate scopoletin.

[0010] The gene encoding the protein also falls within the protection scope of the present invention.

[0011] The coding gene is as shown in 1) or 2) or 3) below:

[0012] 1) The nucleotide sequence is the DNA molecule shown in Sequence 1 in the sequence listing;

[0013] 2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in 1);

[0014] 3) A DNA molecule having 90% or more homology with the DNA molecule defined in 1) or 2).

[0015] The coding gene contains 759 nucleotides, as shown in sequence 1 in the sequence table; it encodes a protein containing 252 amino acids, as shown in sequence 2 in the sequence table. The gene is named AaOMT1 and the protein it encodes is named AaOMT1.

[0016] Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the encoding gene also fall within the scope of protection of the present invention.

[0017] The application of the protein as aesculetin methyltransferase also falls within the protection scope of the present invention.

[0018] The aesculetin methyltransferase activity is an activity that catalyzes the methylation of aesculetin to generate scopoletin.

[0019] The application of the protein in catalyzing the conversion of aesculetin to scopoletin also falls within the protection scope of the present invention.

[0020] The use of the encoding gene in catalyzing the conversion of aesculetin to scopoletin also falls within the scope of protection of the present invention.

[0021] The present invention is based on the inventors' discovery of a specific enrichment of scopoletin (up to 2.24 mg / g dry weight) in the traditional Chinese medicinal herb Artemisia annua. Combined with other literature reports of the presence of trace amounts of aesculetin, the inventors hypothesized the presence of a methyltransferase that directly catalyzes the conversion of aesculetin to scopoletin. Through transcriptome and proteome screening of two ecotypes of the traditional Chinese medicinal herb Artemisia annua, the inventors discovered and identified the methyltransferase AaOMT1 in Artemisia annua that catalyzes the methylation of aesculetin to scopoletin. This provided the aesculetin methyltransferase protein and its coding sequence for the biosynthesis of scopoletin. Unlike the two identified OMTs (POMT9 and PaOMT2 of poplar and blunt-scale purple-backed moss both catalyze the conversion of scopoletin to scopoletin and isoscopoletin), AaOMT1 can specifically produce a single product, scopoletin, avoiding the difficulty of separating and purifying analogues in the later stage, providing a methyltransferase sequence for the biosynthesis of scopoletin and offering new ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For purposes of illustration and not limitation, the present invention will now be described with reference to preferred embodiments thereof, particularly with reference to the accompanying drawings, in which:

[0023] Figure 1 This is the biosynthetic pathway of scopoletin; among them, COMT: caffeic acid oxygen methyltransferase; CCoAOMT: caffeoyl-CoA oxygen methyltransferase; OMTs: methyltransferase; 4CL: 4-coumaric acid:CoA ligase; F6'H: feruloyl-CoA 6'-hydroxylase; COSY: coumarin synthase.

[0024] Figure 2 It is to clone the gene into the pEASY blunt cloning vector; where M stands for maker.

[0025] Figure 3 AaOMT1 is subcloned into an expression vector and expression bacteria; wherein M stands for maker.

[0026] Figure 4 This is an SDS-Page gel image of the AaOMT1 recombinant protein; M stands for maker; the AaOMT1 fusion protein contains an MBP-tagged protein (approximately 42.5 kDa).

[0027] Figure 5 This is the UPLC chart for the identification of the catalytic activity of AaOMT1 towards aesculetin.

[0028] Figure 6 This is the MS identification diagram of the products catalyzed by AaOMT1 on aesculetin; wherein, peak PI represents the methylated product. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0031] Example 1. Esculetin methyltransferase protein in the traditional Chinese medicine Artemisia annua and its encoding gene and application

[0032] 1. Methods and steps of gene cloning

[0033] Based on the transcriptome and proteome of the traditional Chinese medicine Artemisia annua, and with AaCOMT and AaCCoAOMTs found in Arabidopsis as references, 6 AaOMTs sequences were obtained, and AaOMT1 was finally locked.

[0034] Seeds of the traditional Chinese medicinal herb Artemisia annua were collected in Hainan and Gansu (non-patent literature describing Artemisia annua includes: B. Liao, X. Shen, L. Xiang, S. Guo, S. Chen, Y. Meng, Y. Liang, D. Ding, J. Bai, D. Zhang, et al., Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield. Mol Plant. 15(2022)1310-1328. https: / / doi.org / 10.1016 / j.molp.2022.05.013.) and planted in a greenhouse for 3 months. RNA was extracted and reverse transcribed into cDNA. Primer sequences were designed (as shown in Table 1). Using mixed cDNA from roots and leaves as a template, an AaOMT gene fragment was cloned using KOD high-fidelity enzyme (the total volume of the KOD high-fidelity enzyme PCR system was 50 μL: 5 μL). 10X Buffer, 3μL MgSO4, 5μL dNTPs (2mM), 3μL primers (10mM), 1μL template, and 32μL water. The reaction procedure is shown in Table 2. Using the pEASY-Blunt vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CB111-01 (20rxns)), the AaOMT gene fragment was successfully ligated into the vector (total ligation volume: 2.5μL: 0.5μL MixBuffer with enzyme and 2μL template, reaction at 25°C for 2h). The ligation system was directly transformed into TransT1 competent cells, and positive clones were selected for sequencing (total colony PCR volume: 20μL: 13μL MixBuffer, 1μL template, 1μL primers, and 5μL water, procedure is shown in Table 3). Figure 2 ), compared with the transcript sequence, the nucleotide sequence has 100% similarity with the original data, and the actual sequencing results shall prevail.

[0035] Table 1 Primer sequences used for cloning genes

[0036]

[0037] Note: Only the primer sequence of one gene that successfully expressed the recombinant protein is listed.

[0038] Table 2 KOD high-fidelity enzyme PCR reaction program

[0039]

[0040]

[0041] Table 3 Colony PCR reaction procedure

[0042]

[0043] 2. Obtaining the gene sequence and its encoded protein sequence

[0044] The sequencing results showed that the gene amplified using the primers in Table 1 contained 759 nucleotides, as shown in Sequence 1 in the sequence listing; it encoded a protein containing 252 amino acids, as shown in Sequence 2 in the sequence listing. The gene was named AaOMT1, and the protein it encoded was named AaOMT1.

[0045] 3. Verification of gene function

[0046] The gene function was verified with the help of a prokaryotic system, and the pMAL-c2X-AaOMT1 vector was constructed. After sequencing confirmed that the sequence was correct, the vector was successfully transferred into the prokaryotic expression strain Novablue for in vitro verification.

[0047] First, the AaOMT1 fragment was ligated with a linker containing a restriction site (the system and procedure were consistent with the gene cloning method described above; primer information is shown in Table 4). After the AaOMT1 fragment (with the restriction site) was digested, it was constructed into the expression vector pMAL-c2X (purchased from New England Biolabs, catalog number E8200S) using T4-DNA ligase (the total ligation system was 7 μL: 3.5 μL Mix Buffer, 2.8 μL AaOMT1 fragment, and 0.7 μL pMAL-c2X; the reaction was carried out at 4°C overnight). Second, the ligation system was transformed into TransT1 (provided as a gift with the purchase of the pEASY-Blunt vector), and positive clones were selected. After plasmid extraction, the plasmid was transformed into the expression strain Novablue (purchased from Merck Millipore, a life science business of Merck KGaA, Darmstadt, Germany, catalog number 69284-3). Positive clones were identified using the colony PCR procedure (Table 3). Figure 3 ).

[0048] Table 4 Primer sequences used to construct prokaryotic expression

[0049]

[0050] Table Notes: Lowercase letters in the sequence are protected bases, and underlined bases are enzyme cleavage site bases.

[0051] The induction, purification, enzyme activity analysis and product identification of the recombinant protein are as follows:

[0052] 1) Induction of recombinant protein

[0053] Single clones of pMAL-c2X-AaOMT1 and pMAL-c2X were picked and cultured in 3 mL LB (containing 100 mg / L Amp) liquid culture medium at 37°C with shaking (200 rpm) overnight.

[0054] Take 1 mL of overnight cultured bacterial solution and add it to 100 mL of fresh LB culture medium (containing 100 mg / L Amp and 0.2% sterilized glucose). Cultivate at 37°C in a shaking incubator until the OD value at 600 nm reaches 0.5-0.8. Then take 1 mL of bacterial solution and collect the bacteria as a control.

[0055] 30 μL IPTG (stock concentration 1 M, isopropyl-β-D-thiogalactoside) was added to 100 mL of bacterial solution to a final concentration of 0.3 mM, and cultured at 16° C. for 48 h.

[0056] Centrifuge at 8,000 × g for 3 min at 4°C, discard the supernatant, and collect the bacteria.

[0057] 2) Purification of recombinant protein

[0058] Recombinant AaOMT1 protein was purified according to the pMAL Fusion Protein and Purification System (New England BioLab Inc.) manual. Briefly, the collected bacterial pellet was resuspended in column buffer and stored at -20°C overnight. The next day, the sample was thawed and disrupted using an ultrasonic disruptor to release the protein. The sample was centrifuged at 9,000 × g for 30 min before loading. The affinity column was activated with 8 column volumes of column buffer (flow rate 1 mL / min). The sample was diluted 5-fold before loading. After the sample had completely flowed through the affinity column, contaminants were washed with 12 column volumes of column buffer. Finally, the target protein was eluted with 5 column volumes of column buffer (freshly prepared, 10 mM maltose). The sample was concentrated using a Millipore (30 kDa) low-speed centrifugation (3,000 × g, 4°C) and replaced with enzyme activity reaction buffer. After SDS-PAGE electrophoresis and Coomassie brilliant blue staining, the size of the recombinant protein was found to be approximately 70 kDa (the tag protein - MBP or maltose binding protein is 42.5 kDa), which is basically consistent with the predicted size of AaOMT1 of 27.7 kDa (e.g. Figure 4 ).

[0059] 3) Determination of enzyme activity

[0060] The enzyme activity reaction system was 100 μL, as shown in Table 5. After incubation at 30°C for 30 min, the reaction was terminated with an equal volume of methanol and centrifuged at 13,000 rpm for 10 min. 2 μL of the sample was passed through a 0.22 μm membrane and loaded.

[0061] Table 5 Recombinant protease activity reaction system

[0062]

[0063] 4) Analysis and identification of enzyme activity products

[0064] UPLC spectrum of enzyme activity products ( Figure 5 ), a new product peak was found in the reaction system of AaOMT1 and aesculetin.

[0065] UPLC conditions:

[0066] UPLC model: Nexera UHPLC LC-30A system (SHIMADZU, Japan).

[0067] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile.

[0068] Elution gradient: 0-7 mins, 5%-100% B; 7-9 mins, 100% B; 9-10.5 mins, 100%-5% B; 10.5-11.5 mins, 5% B.

[0069] DAD detection wavelength: 335 nm.

[0070] Further identification by mass spectrometry revealed that the mass-to-charge ratio of the enzyme activity product peak P1 of AaOMT1 to aesculetin was 14 (the molecular weight of a methyl group) higher than that of the substrate, indicating that the product was a methylated product of aesculetin. We found that the retention time of the enzyme activity product peak in UPLC and its mass spectrometric fragmentation information were consistent with those of the scopoletin standard ( Figure 6 In summary, the in vitro enzymatic activity evidence showed that AaOMT1 encodes a methyltransferase that catalyzes the methylation of aesculetin to scopoletin.

[0071] Mass spectrometry conditions:

[0072] The samples were separated by UPLC-MS / MS using an Eclipse Plus C18 RRHD column (1.8 μm, 2.1×50 mm id; Agilent). The mobile phase was the same as that for UPLC, with an elution gradient of 95% A in 0 min, 5% A in 7 min, and a final equilibrium at 95% A (1 min). The flow rate was 0.30 mL / min, and the detection wavelength was the same as above.

[0073] UPLC MS / MS conditions: electrospray ionization, full ion scan, negative-ion (EI) mass spectrometry analysis. Heat gas temperature: 300°C; gas flow: 5.0 L / min; capillary voltage: 3500 V; nozzle voltage: 500 V; delta EMV: 200 V. MS TOF (Expt): fragmentor voltage: 120 V; skimmer: 65 V; mass spectrum acquisition range: m / z 100-1000.

[0074] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. The protein having the amino acid sequence as shown in SEQ ID NO: 2 is used in catalyzing the conversion of aesculetin to scopoletin.

2. The coding gene with the nucleotide sequence shown in SEQ ID NO: 1 is used to catalyze the conversion of aesculetin to scopoletin.

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