Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof

A kind of amorphadiene and mutant technology, which is applied in the direction of application, lyase, and introduction of foreign genetic material by using vectors, which can solve the problems of high cost and complicated artemisinin process

Inactive Publication Date: 2014-07-16
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Artificial total synthesis of artemisinin is complex and costly

Method used

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  • Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof
  • Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof
  • Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1, Extraction of Artemisia annua total RNA, PCR amplification of target gene ADS and site-directed mutagenesis

[0082] a. Extraction of Artemisia annua Total RNA

[0083] Artemisia annua material (about 100 mg) was thoroughly ground in liquid nitrogen. Transfer to a 1.5ml centrifuge tube, add 1mL Trizol (Invitrogen, Cat. 15596-018), mix well, and stand at room temperature for 5min. Centrifuge at 12,000rpm for 10min and discard the precipitate. Add 200 μL of chloroform to the supernatant, mix well, and centrifuge at 12,000 rpm for 10 min. Take the supernatant and add 500 μL of isopropanol to precipitate the RNA. Centrifuge at 12,000rpm for 10min, wash the pellet with 70% ethanol, dry in vacuum, dissolve in 20-50μL H 2 O (RNase free). Properly dilute the RNA with 10mM Tris-HCl (pH7.5), and measure the UV absorption value at a wavelength between 200-300nm. RNA concentration = 40 μg / mL × A 260 ×Dilution factor. A 260 / A 280 Should be between 1.9 and 2.1. ...

Embodiment 2

[0110] Embodiment 2, vector construction and Escherichia coli transformation

[0111] a. Vector construction

[0112] ADS coding region fragment (ADS) and mutant fragments (ADS-T399S, ADS-T399A, ADS-T399N, ADS-T399D, ADS-T399R) were amplified by KOD-plus DNA polymerase, and ligated into pET by NcoI / BamHI digestion -32a carrier (Amp r , Novagen, America), to obtain the recombinant vector.

[0113] b. Competent cell preparation

[0114] Escherichia coli DH5α or BL-21 stored at -70°C were streaked on a solid LB plate and cultured overnight at 37°C; a single colony was picked and cultured in 5 mL of liquid LB medium at 250 rpm overnight. The next day, scale up and inoculate into 500mL liquid LB medium at a ratio of 1 / 50, culture at 18-22°C until OD 600 ≈0.5 (about 5-6h), cooling on ice for 10min. Centrifuge at 2,500 g for 10 min at 4°C, resuspend the cells with 160 mL of transformation buffer, discard the supernatant, and finally resuspend the cells with 40 mL of transformati...

Embodiment 3

[0120] Embodiment 3, RT-PCR

[0121] 1 μg total RNA, Oligo(dt) 20 As primers, 10 μL reaction system was used for reverse transcription according to the requirements of the reverse transcription kit (TOYOBO, Osaka, Japan). React at 42°C for 30 minutes. After boiling water for 5 minutes, place on ice. The reverse transcription product (or diluted 10 times) can be directly used for PCR detection.

[0122] According to the known ADS sequence, PCR primers were synthesized. When analyzing the expression of ADS, Artemisia annua Actin1 (GenBank: EU531837) was used as an internal control to correct the amount of template in the RT-PCR reaction. The PCR conditions are as follows: the annealing temperature and extension time of the reaction are determined by the primers and the length of the amplified fragment. The general reaction conditions are: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 72°C for 30 s, amplification fo...

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Abstract

The invention relates to an Artemisia annua amorpha-4,11-diene synthase mutant with improved enzyme activity and application thereof. The amorpha-4,11-diene synthase mutant has enzyme activity substantially higher than that of wild type amorpha-4,11-diene synthase under the condition that catalysate maintains unchanged. Compared to a traditional method, a method of applying the Artemisia annua amorpha-4,11-diene synthase mutant provided by the invention in enzymatic production of artemisinin has the advantages of high efficiency and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a mutant of Artemisia amorphadiene synthase with improved enzyme activity and application thereof. Background technique [0002] Artemisinin is a peroxybridge-containing sesquiterpene lactone isolated and identified for the first time by Chinese scientists from Artemisia annua. It is currently the most effective drug for the treatment of cerebral malaria and chloroquine-resistant falciparum malaria. Artemisinin-based combined therapy has been confirmed and recommended by the World Health Organization and Western countries. The content of artemisinin in Artemisia annua is low (0.01-1% dry weight), and it is greatly affected by species and regional planting, and resources are in short supply. [0003] In the biosynthesis of artemisinin, the sesquiterpene component amorphadiene (Amorphadiene, Amorpha-4, 11-diene) is synthesized by the amorphadiene synthase (a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P5/00C12P17/18
Inventor 陈晓亚李建戌王凌健洪高洁胡文利阮菊新杨长青毛颖波
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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