Application of a terpene synthase in the preparation of borneol
By cloning the AarTPS89 gene from the migrant plants and expressing the protein in E. coli, the problems of low efficiency of head synthesis and by-products in the prior art were solved, and efficient synthesis and yield of head synthesis were achieved.
Patent Information
- Application Number
- CN202211216081.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-30
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2042-09-30
AI Technical Summary
The prior art is difficult to efficiently synthesize the important monoterpene compound of the medicinal plant wormwood, and the catalytic efficiency is not specific, which has the problem of by-products.
By cloning the AarTPS89 gene from the migrant plants, the expression vector was constructed and the AarTPS89 protein was expressed in E. coli, which was used to catalyze the production of geranium pyrophosphate into the longan brain.
It has achieved efficient synthesis of longanopsis, with substrate specificity and product specificity, and improved the yield and medicinal quality of longanopsis.
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Figure CN116103274B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biology and genetic engineering of medicinal plants, and more specifically to a borneol-based diphosphate synthase AarTPS89 protein for efficiently synthesizing borneol, a coding gene and an application thereof. Background Art
[0002] Artemisia argyi is a medicinal plant of the genus Artemisia in the Asteraceae family. It is mainly concentrated in East Asia and is also found in the Far East, such as Russia. The whole plant is used as medicine, which has the effects of warming the meridians and dispelling cold, stopping bleeding and reducing inflammation. It has a history of thousands of years in folklore, health care, diet and medicine. Artemisia argyi is dried and made into moxa sticks for moxibustion, which can treat colitis, diarrhea, urinary incontinence, dysmenorrhea, knee osteoarthritis, asthma and other diseases. In addition, Artemisia argyi fumigation for antibacterial and Artemisia argyi sachets for insect repellent are also common applications.
[0003] Terpenoids are a very important class of compounds in mugwort, mainly monoterpenoids and their derivatives, such as 1,8-cineole, borneol, thujone, camphor, etc.; and sesquiterpenoids and their derivatives, such as caryophyllene, caryophyllene oxide, and germaene D. In the 2020 edition of the Chinese Pharmacopoeia, eucalyptol and borneol are specified as indicator ingredients. Among them, borneol has the effects of expelling intestinal worms, relieving fever, relieving smooth muscle spasms, and expectoration, and is also an important industrial raw material.
[0004] Bornyl diphosphate synthase (BPPS) is a special monoterpene synthase that catalyzes a special reaction, that is, during the cyclization of GPP, the phosphate group undergoes a process of separation and re-binding with a carbon cation to generate a phosphate-containing, water-soluble bornyl diphosphate (BPP). When alkaline phosphatase is used to depyrophosphate bornyl diphosphate, the final product borneol can be generated. The earliest BPPS was cloned in the aromatic plant Salvia officinalis. SoBPPS is a multi-product enzyme. Since then, it has been cloned in many plants such as lavender, sweet tongue grass, and Amomum villosum. However, similar to SoBPPS, the catalytic efficiency of the enzyme is not specific and by-products are produced.
[0005] AarTPS89 is a member of the terpenoid synthase gene family and belongs to the TPS-b subfamily. Studies have found that AarTPS89 catalyzes the production of borneol diphosphate in vitro, and further produces a single product, borneol, under the action of dephosphorylase. It has substrate specificity and product specificity and is identified as borneol diphosphate synthase. Therefore, how to use the AarTPS89 gene, construct an expression vector with it, and synthesize borneol in an organism is a problem that those skilled in the art urgently need to solve. Summary of the invention
[0006] The purpose of the present invention is to provide an AarTPS89 protein, a coding gene and an application.
[0007] The present invention first provides a protein, including the following 1)-3):
[0008] 1) the amino acid sequence shown in SEQ ID NO.1;
[0009] 2) A sequence having the same function as the sequence shown in SEQ ID NO.1, and obtained by substituting, deleting or adding one or more amino acids;
[0010] 3) A fusion protein having the same function as the protein shown in SEQ ID NO. 1 and having a tag connected to the N-terminus or C-terminus.
[0011] The above-mentioned protein, protein tag refers to a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and / or SUMO tag, etc.
[0012] Also provided is a gene encoding the protein, wherein the gene encoding the protein is at least one of the following:
[0013] 1) the nucleotide sequence shown in SEQ ID NO.2;
[0014] 2) a DNA molecule that hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the protein;
[0015] 3) a nucleotide sequence that has 90% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the protein;
[0016] 4) A sequence obtained by substituting, deleting or adding nucleotides from the sequence shown in SEQ ID NO.2;
[0017] 5) Producing different transcripts or homologous gene sequences from the nucleotide sequence shown in SEQ ID NO.2.
[0018] Also provided are a recombinant vector, an expression box, a recombinant bacterium, a recombinant cell and a transgenic plant containing the encoding gene.
[0019] Based on the differential expression analysis of the whole genome of Artemisia argyi and the transcriptomes of different organs / tissues and the phylogenetic evolutionary tree analysis constructed with the BPPS protein of known function, the nucleotide sequence shown in SEQ ID NO.2 of the BDH gene that may be involved in camphor synthesis was screened out, or the nucleotide sequence was recombined with an expression vector and introduced into a recipient microorganism, the expression vector was specifically pET-28a, and the recipient microorganism was specifically Escherichia coli BL21 (DE3), to obtain a recombinant microorganism expressing the AarTPS89 protein or a protein with the same function as the AarTPS89 protein, and the recombinant microorganism was cultured to express the protein.
[0020] Also provided is the use of the encoding gene in catalyzing geranyl pyrophosphate to produce borneol.
[0021] Also provided is the use of the recombinant vector, expression box, recombinant bacteria, recombinant cells and transgenic plants in catalyzing geranyl pyrophosphate to produce borneol.
[0022] A method for preparing the proteins 1)-3) is also provided, comprising: recombining the nucleotide sequence of the coding gene with an expression vector and introducing the vector into a recipient microorganism to obtain a recombinant strain, culturing the recombinant strain, and expressing the protein.
[0023] A method for preparing borneol is also provided, the method comprising using the proteins 1) to 3) to catalyze geranyl pyrophosphate to produce borneol. AarTPS89 protein was purified by NTA resin, and in vitro enzymatic reaction and dephosphorylation reaction of AarTPS89 protein were carried out in buffer. The catalytic products of AarTPS89 gene in prokaryotic expression system in vitro were detected by solid phase microextraction and gas chromatography-mass spectrometry (GC-MS) technology to obtain the catalytic product borneol.
[0024] The present invention clones AarTPS89, i.e., borneol diphosphate synthase, from mugwort. This gene is a key enzyme gene for the synthesis of borneol, an important indicator component monoterpenoid compound, obtained for the first time from mugwort. Experiments have shown that AarTPS89 of the present invention can catalyze GPP to form borneol, which plays an important role in further analyzing the synthesis of terpenoid compounds in mugwort, and is of great significance for further improving the yield of borneol and enhancing the quality of medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0026] Figure 1 The attached figure is a phylogenetic evolutionary tree of candidate BPPS genes of Ai and other identified BPPS genes;
[0027] Figure 2 The attached figure is a heat map of the expression of candidate BPPS genes in different tissue parts;
[0028] Figure 3 The attached figure is a multiple sequence alignment of AarTPS89 and other identified BPPS genes;
[0029] Figure 4 The attached figure shows the catalytic product detected by GC-MS in the in vitro expression system;
[0030] Figure 5 The attached figure is a mass spectrum of the catalytic product of AarTPS89, wherein the upper end of the horizontal axis is the standard ion peak, and the lower end of the horizontal axis is the product ion peak. DETAILED DESCRIPTION
[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0032] Example 1 AarTPS screening
[0033] 1) Based on the Ai genome information and the known BPPS gene sequence, AarTPS with potential borneol biosynthesis function were screened. A total of 8 genes may have important functions in borneol biosynthesis, and their amino acid sequence lengths ranged from 580 to 610.
[0034] 2) Select the identified functional CbTPS1 (MW196671), LaBPPS (AJW68082), Ld BPPS (ATY48638), AvBPPS (AWW87313) SoBPPS (AF051900), and use DNAMAN for multiple sequence alignment, such as Figure 3 As shown in Figure 1, MEGA was used to build a phylogenetic tree. Figure 1 shown.
[0035] 3) RNA-seq technology was used to analyze the expression profiles of candidate TPS genes in different organs such as roots, stems, leaves, and flowers. Figure 2 shown.
[0036] Example 2 Gene cloning of AarTPS89 and its encoded protein sequence
[0037] Primers were designed based on the AarTPS89 sequence in the mugwort genome, and amplification was performed using mugwort leaf cDNA as a template to obtain a nucleotide sequence of 1812 bp in length, such as SEQ ID NO. 2. The amino acid sequence encoded by AarTPS89 was obtained after translation based on the full-length cDNA sequence, such as SEQ ID NO. 1.
[0038] Example 3 In vitro catalytic reaction of AarTPS89 and product detection
[0039] 1) Select BamH I / EcoR I as restriction sites, digest the pET-28a vector, and use a seamless cloning kit to connect the target gene fragment AarTPS89 to the restriction vector to construct the pET-28a-Aar TPS89 gene expression vector;
[0040] 2) Transform the pET-28a empty vector (control) and pET-28a-AarTPS89 into BL21 competent cells respectively, and spread the transformed cells on a plate containing 50 mg / L Kana (kanamycin) to screen positive clones. Select a single colony and inoculate it in LB liquid medium containing the corresponding antibiotics. After culturing overnight, transfer it at a ratio of 1:100 for expansion culture. When the OD value of the bacterial solution reaches 1.0, the bacterial strain will be 600 When it reached 0.6, 0.5 mM IPTG was added and induced for 16 h in a shaking incubator at 16 °C in the dark at a speed of 110 r / min;
[0041] 3) The induced bacterial solution and the pET-28a empty vector bacterial solution were centrifuged at 7000 rpm / min for 10 min to obtain bacterial pellets, and the bacteria were resuspended in 5 ml of cell lysis buffer and 2 μL of 50 mg / ml protease inhibitor. The bacteria were ultrasonically disrupted at low temperature and centrifuged at 7000 rpm / min for 3 min to obtain sample supernatant protein and control supernatant protein.
[0042] 4) Dilute the sample supernatant protein with binding buffer (20mM Tis-HCl pH=8.0, 10mM Imidazole, 0.5MNaCl) and hang it on On NTA; after eluting the impurities with binding buffer, the target protein was eluted with a gradient elution buffer containing 50, 150, 200, 300, and 500 mM Imidazole, and the purified protein AarTPS89 was obtained at the optimal Imidazole elution concentration by SDS-PAGE electrophoresis. The concentration of the purified protein was determined using a BCA detection kit.
[0043] 5) The in vitro enzyme activity of AarTPS89 was determined in a total of 200 μL buffer system containing 10 μg of purified protein, 10 μL geranyl pyrophosphate (GPP), 25 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 5 mM dithiothreitol (DTT), 5 mM magnesium chloride (MgCl2), and covered with 200 μL of n-hexane on the liquid surface. The mixture was incubated at 30°C for 2 hours, and after mixing, 1.5 μL of dephosphorylase was added and incubated at 37°C for 1 hour. The n-hexane phase was then collected and the volatile products were detected by GC-MS method.
[0044] 6) GC-MS test results: Compared with the control protein (pET-28a empty vector supernatant protein), Aa rTPS89 produced a single product, borneol, in the in vitro enzymatic reaction with GPP as a substrate, such as Figure 4 As shown in the mass spectrum Figure 5 This indicates that AarTPS89 has substrate specificity and product specificity in the prokaryotic expression system, indicating that AarTPS89 is expected to be developed into a functional protein for the efficient and specific synthesis of borneol.
[0045] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0046] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.
1.
2. A gene encoding the protein according to claim 1.
3. The coding gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.
2.
4. A recombinant vector, expression cassette, recombinant bacterium or recombinant cell containing the coding gene according to claim 2 or 3.
5. Use of the coding gene according to claim 2 or 3 in catalyzing geranyl pyrophosphate to produce borneol.
6. Use of the recombinant vector, expression cassette, recombinant bacteria or recombinant cell according to claim 4 in catalyzing geranyl pyrophosphate to produce borneol.
7. The method for preparing the protein according to claim 1, characterized in that: The method comprises: recombining the nucleotide sequence of the coding gene of claim 2 or 3 with an expression vector and then introducing the vector into a recipient microorganism to obtain a recombinant strain, culturing the recombinant strain, and expressing the protein of claim 1.
8. A method for preparing borneol, characterized in that: The method comprises using the protein of claim 1 to catalyze geranyl pyrophosphate to produce borneol.