Terpene synthase for producing germacrene A and application thereof

Abstract

A terpenoid synthase for producing germacrene A and application thereof.The present application relates to a germacrene A synthase AarTPS34 gene and its coding product and application.The AarTPS34 gene is cloned from folium artemisiae argyi, and the AarTPS34 gene is a key enzyme gene for synthesizing germacrene A, a sesquiterpenoid compound, which is obtained from artemisia for the first time.The experiment proves that the AarTPS34 protein of the present application can catalyze the precursor material farnesyl pyrophosphate (FPP) to generate the sesquiterpenoid compound germacrene A, and germacrene A is a synthetic precursor of anticancer active ingredient beta-elemene, and the research on germacrene A has important theoretical and practical significance for improving the quality of folium artemisiae argyi and producing plant monoterpene compounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicinal plant molecular biology and genetic engineering, and more particularly to a terpene synthase for producing germacrene A and application thereof. BACKGROUND

[0002] Artemisia argyi is a medicinal plant of the genus Artemisia in the family Asteraceae. The whole plant is used as medicine, which has the effects of warming meridians and dispelling cold, stopping bleeding and inflammation, relieving cough and asthma, preventing miscarriage, and resisting allergy. The dried and crushed leaves of Artemisia argyi can be made into moxa sticks for moxibustion. In addition, the whole plant of Artemisia argyi can drive parasites, and the fumigation smoke can be used for room disinfection. Artemisia argyi has very high economic and medicinal value. Studies have found that volatile oil is the main material basis of Artemisia argyi, which has broad-spectrum antibacterial, antioxidant, anti-inflammatory, antitumor, analgesic, antiasthmatic, and immunoregulatory abilities. The main components are monoterpenes, sesquiterpenes and their derivatives. Terpenes are synthesized by the relatively conservative terpene synthase (TPS) in plants. By using biological engineering technology to express the terpene synthase gene heterologously, a large amount of target terpenes can be obtained by directional catalysis, thereby obtaining terpene products with development value or practical use.

[0003] Terpene synthase germacrene A synthase (GAS) is a key enzyme that catalyzes the precursor farnesyl pyrophosphate (FPP) to generate sesquiterpenes germacrene A. The product germacrene A can directly obtain β-elemene in vitro through simple Cope rearrangement. β-Elemene is a natural terpene compound with high efficiency, low toxicity, and broad-spectrum anticancer activity. Elemene oral emulsion and injection drugs have achieved remarkable clinical efficacy as a national class II new drug. At present, the production of β-elemene mainly relies on plant extraction and chemical synthesis, which is complicated and easy to pollute the environment. Therefore, it has great application prospects to use biological engineering technology to express germacrene A synthase heterologously and construct a microbial factory.

[0004] AarTPS34 is a member of the terpene synthase gene family of Artemisia argyi and belongs to the TPS-b subfamily. Studies have found that AarTPS34 catalyzes the synthesis of sesquiterpenes germacrene A in a heterologous biosynthesis system, has substrate specificity and product specificity, and is identified as germacrene A synthase.

[0005] Therefore, how to use the AarTPS34 gene to construct an expression vector and synthesize the sesquiterpenes germacrene A in an organism is a problem that those skilled in the art need to solve. SUMMARY

[0006] The present application aims to provide a gene of a germacrene A synthase gene AarTPS34 for synthesizing germacrene A, a protein encoded by the gene and a coding product and application thereof.

[0007] The present application first provides a nucleotide derived from a plant of Artemisia of the family Asteraceae, i.e., a nucleotide of any one of the following 1), 2) or 3):

[0008] A1) the nucleotide is a nucleotide sequence of the AarTPS34 gene, as shown in SEQ ID NO. 1;

[0009] A2) or a sequence having 85-99% identity with the nucleotide sequence shown in SEQ ID NO. 1, wherein the encoded protein has a function of catalyzing the generation of camphor;

[0010] A3) or a sequence obtained by substitution and / or deletion, or addition / deletion of one or more nucleotides from the sequence shown in SEQ ID NO. 1, wherein the encoded protein has a function of catalyzing the generation of germacrene A;

[0011] A4) or a different transcript or homologous gene sequence derived from the nucleotide sequence shown in SEQ ID NO. 1.

[0012] Secondly, the present application provides a protein derived from a plant of Artemisia of the family Asteraceae, i.e., a protein of germacrene A synthase AarTPS34 of Artemisia, as any one of the following 1), 2) or 3):

[0013] B1) the protein is AarTPS34, and the amino acid sequence is shown in SEQ ID NO. 2;

[0014] B2) or a sequence having 85-99% identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the protein has a function of catalyzing the generation of germacrene A;

[0015] B3) or a sequence obtained by substitution and / or deletion, or addition / deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO. 2, wherein the protein has a function of catalyzing the generation of germacrene A;

[0016] B4) or a fusion protein obtained by connecting a tag to the N-terminus or C-terminus of the protein shown in SEQ ID NO. 2.

[0017] The protein tag refers to a polypeptide or protein fused with the target protein for expression, detection, tracing and / or purification of the target protein by using DNA in vitro recombination technology. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0018] The present application provides the above-mentioned AarTPS34-related biological material as any one of the following:

[0019] C1) an expression cassette containing the isolated nucleic acid molecule;

[0020] C2) or, a recombinant vector containing the nucleic acid molecule according to any one of A1), A2) or A3);

[0021] C3) or, a host cell containing the nucleic acid molecule according to any one of A1), A2) or A3);

[0022] C4) or, a host cell containing the isolated protein according to any one of B1), B2) or B3);

[0023] C5) or, a host cell containing any one of the recombinant vectors according to C2);

[0024] C6) a transgenic plant containing the nucleic acid molecule according to any one of A1), A2) or A3);

[0025] C7) a transgenic plant containing the isolated protein according to any one of B1), B2) or B3);

[0026] C8) a transgenic plant containing any one of the recombinant vectors according to C2);

[0027] C9) a transgenic plant containing any one of the host cells according to C3), C4) or C5).

[0028] The present application further provides the application of the above-mentioned nucleotide, protein or related biological material. The specific application is any one of the following:

[0029] D1) the application of the above-mentioned protein as a germacrene A synthase;

[0030] D2) the application of the above-mentioned related biological material in the preparation of a germacrene A synthase;

[0031] D3) the application of the above-mentioned protein or related biological material in the preparation or synthesis of germacrene A;

[0032] D4) the application of the above-mentioned protein or related biological material in the formation of FPP into germacrene A.

[0033] The object of the present application can be achieved by the following technical solutions:

[0034] Based on the genome of Artemisia argyi and the differential expression analysis of the transcriptome of different organs / tissues, and the phylogenetic tree analysis of the known function of germacrene synthase GS protein, the coding gene of AarTPS34 which may be involved in the synthesis of germacrene A is screened.

[0035] The nucleotide sequence described in the above A1)-A4) is introduced into a receptor microorganism after recombination with an expression vector, the expression vector is specifically pET-28a, and the receptor microorganism is specifically Escherichia coli BL21 (DE3), so as to obtain a recombinant microorganism expressing AarTPS34 protein, and the recombinant microorganism is cultured to obtain AarTPS34 protein.

[0036] By The AarTPS34 protein is purified by Ni NTA resin, the in vitro enzymatic reaction of the AarTPS34 protein is carried out in a buffer, the catalytic product of the AarTPS34 gene in the prokaryotic expression system in vitro is detected by solid-phase microextraction technology and gas chromatography-mass spectrometry (GC-MS) technology, and camphor is obtained as the catalytic product.

[0037] The AarTPS34, i.e., germacrene A synthase, is cloned from Artemisia argyi, and the gene is a key enzyme gene of germacrene A biosynthesis which is obtained from Artemisia argyi for the first time. It is proved by experiments that the AarTPS34 of the present application can catalyze FPP to form germacrene A, and the research on germacrene A has important theoretical and practical significance for improving the quality of Artemisia argyi medicinal materials and producing plant monoterpene compounds. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0039] Figure 1 The drawing is the catalytic product of AarTPS34 in the prokaryotic in vivo expression system detected by GC-MS;

[0040] Figure 2 The drawing is the catalytic product of AarTPS34 in the prokaryotic in vitro expression system detected by GC-MS;

[0041] Figure 3 The drawing is the mass spectrum of the catalytic product of AarTPS34 in the prokaryotic expression system. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0043] Example 1 Gene cloning of AarTPS34 and sequence of protein coded by the gene

[0044] Primers were designed according to the sequence of AarTPS34 in the genome of A. arguta, and the base sequences of the primers are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The A. arguta cDNA was used as a template for amplification, and a nucleotide sequence with a length of 1683 bp was obtained, as shown in SEQ ID NO. 1. The amino acid sequence coded by AarTPS34 was obtained after translation according to the full-length cDNA sequence, as shown in SEQ ID NO. 2.

[0045] Example 2 Construction of a prokaryotic expression system of AarTPS34 gene and detection of catalytic products

[0046] 1) BamH I / EcoR I was selected as the enzyme cutting site, and the pET-28a vector was cut, and the gene fragment AarTPS34 was connected to the cut vector using a seamless cloning kit to construct a pET-28a-AarTPS34 gene expression vector;

[0047] 2) The pET-28a empty vector (control) and the pET-28a-AarTPS34 were transformed into BL21 competent cells, and the transformed cells were plated on plates containing 50 mg / L Kana (kanamycin) to select positive clones. Single colonies were inoculated in LB liquid medium containing the corresponding antibiotic, and after overnight culture, 1:100 transfer was performed for expansion culture. When the OD 600 of the bacterial liquid reached 0.6, 0.5 mM IPTG was added, and the culture was induced at 16°C in the dark for 16 h at a shaking speed of 110 r / min;

[0048] 3) 5 mL of the induced bacterial liquid was taken and placed in a 20 mL headspace bottle, the extraction temperature was 60°C, and the extraction time was about 30 min. Solid-phase microextraction column (PDMS / DVB, 100 μm) was used for extraction;

[0049] 4) GC-MS instrument is Agilent 7890b-5977a (HP-5MS: 30m x 0.25mm x 0.25μm), direct injection from solid phase microextraction column without split. Chromatographic conditions: 40℃ for 1min, then ramp to 240℃ at 5℃ / min and hold for 5min, helium flow rate 1ml / min, injection port temperature 250℃. Mass spectrometric conditions: ion source temperature 230℃, scan mode acquisition 40-550m / z, electron impact ionization source energy 70eV. GC-MS detection results: relative to the control strain (pET-28a empty vector transformed strain), the strain containing pET-28a-AarTPS34 appeared a single product β-elemene at 20.3min, as shown in Figure 2. It indicates that AarTPS34 in prokaryotic expression system, only with E. coli endogenous FPP as substrate, specifically synthesizes the direct precursor of β-elemene, germacrene A (germacrene A undergoes coper rearrangement to generate β-elemene at high temperature of GC-MS injection port), indicating that AarTPS34 is a germacrene A synthase. Figure 1

[0050] Example 3 Construction of prokaryotic in vitro expression system of AarTPS34 gene and detection of catalytic products

[0051] 1) Collect the induced bacterial liquid in Example 2 and pET-28a empty vector bacterial liquid, centrifuge at 7000rpm / min for 10min to obtain bacterial precipitate, resuspend the bacterial precipitate with 5ml cell lysis solution and 2μL 50mg / ml protease inhibitor, ultrasonically break the bacterial precipitate at low temperature, then centrifuge at 7000rpm / min for 3min to obtain sample supernatant protein and control supernatant protein.

[0052] 2) Dilute the sample supernatant protein with binding buffer (20mM Tis-HCl pH=8.0, 10mM Imidazole, 0.5M NaCl), and hang it on ​Ni NTA, after eluting the impurity proteins with binding buffer, eluting the target protein with elution buffer containing 50, 150, 200, 300, 500 mM imidazole, and detecting the purified protein AarTPS34 under the optimal imidazole elution concentration by SDS-PAGE electrophoresis. The purified protein concentration was determined using a BCA detection kit. The determination of the in vitro enzyme activity of AarTPS34 was performed in 1 ml Tis-HCl (100 mM, pH = 8.0) buffer containing 10 μg of purified protein, 10 μg of farnesyl pyrophosphate (FPP) / geranyl pyrophosphate (GPP) / geranylgeranyl pyrophosphate (GGPP), 30 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 5 mM dithiothreitol (DTT), 25 mM magnesium chloride (MgCl2). The mixture was incubated at 30°C for 1 hour, and then the volatile product was detected by the solid-phase microextraction GC-MS method described in Example 2.

[0053] 3) GC-MS detection results: compared with the control protein (pET-28a empty vector supernatant protein), AarTPS34 only appeared a single product β-elemene at 20.3 min in the in vitro enzymatic reaction with FPP as the substrate, as shown in Figure 2 . This indicates that AarTPS34 has substrate specificity and product specificity in the prokaryotic expression system, indicating that AarTPS34 is expected to be developed as a functional protein for the direct precursor of β-elemene, germacrene A.

[0054] Based on the genome information of A. arborescens, the present application performs gene cloning, function verification and product detection on the TPS family member AarTPS34, and finds that AarTPS34 specifically catalyzes the synthesis of germacrene A with specific substrate FPP in the prokaryotic expression system. The present application provides an alternative and effective method for producing the target compound catalyzed by AarTPS34 by using biological engineering technology, and also provides a research basis for constructing an efficient heterologous synthesis platform for the direct precursor of β-elemene, germacrene A.

[0055] The embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other.

[0056] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

1.

2. A protein, characterized in that, The protein is AarTPS34 protein, and the amino acid sequence is shown as SEQ ID NO.

2.

3. Biomaterials associated with the nucleic acid molecule of claim 1 or the protein of claim 2, characterized in that, The related biological material is shown as any one of the following: A1) an expression cassette containing the nucleic acid molecule of claim 1; A2) or, a recombinant vector containing the nucleic acid molecule of claim 1; A3) or, A host cell containing the nucleic acid molecule of claim 1 or the protein of claim 2 or containing the expression cassette of A1) or containing the recombinant vector of A2).

4. Use of the nucleic acid molecule of claim 1, the protein of claim 2 or the related biological material of claim 3 in catalyzing the generation of germacrene A from farnesyl pyrophosphate (FPP) as a substrate.

5. A method of producing the protein of claim 2, characterized by, The method comprises the following steps: recombining the nucleic acid molecule of claim 1 with an expression vector and introducing into a recipient microorganism to obtain a recombinant microorganism expressing the protein of claim 2, culturing the recombinant microorganism to obtain the protein of claim 2.

6. A method of preparing gymnenic acid, characterized by: The method comprises catalyzing farnesyl pyrophosphate (FPP) with the protein of claim 2.

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