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Engineering strain for producing beta-elemene and application of engineering strain

A technology for engineering strains and elemene, applied in the application, genetic engineering, bacteria and other directions, can solve the problems of unfavorable environmental protection, low purity, difficult to achieve chemical synthesis reaction conditions and the like

Inactive Publication Date: 2020-02-21
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous increase of market demand for elemene, the use of traditional extraction and separation methods to obtain elemene from natural plant turmeric has been unable to meet market demand, and the products obtained through extraction and separation have problems such as low yield and low purity. High cost for secondary separation
However, the synthesis of elemene by chemical synthesis requires the use of highly toxic chemical reagents such as dichloromethane, sodium cyanide, toluene, which is not conducive to environmental protection; and the reaction conditions of chemical synthesis are difficult to achieve, and the product yield is also low. very low

Method used

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  • Engineering strain for producing beta-elemene and application of engineering strain
  • Engineering strain for producing beta-elemene and application of engineering strain
  • Engineering strain for producing beta-elemene and application of engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of engineering bacteria E.Coil BL21codon pius / pACYCDuet-1 / ERG20

[0035] 1.1 Full-length cloning of XsGAS gene

[0036] Xanthium seedling RNA was extracted and reverse transcribed into cDNA as a template. Primers were designed with the GAS sequence of Xanthium annulus published on NCBI (upstream primer (SEQ ID NO.9): 5'-ATGGCAGCAGTTGGAGCCAATG-3'; downstream primer (SEQ ID NO.10): 5'-TTACATGGGTAAAGAATCGACAAACAAGAG-3'), and cDNA Carry out high-fidelity PCR reaction for the template, and the PCR reaction parameters are: first, denaturation at 98°C for 3 minutes; second, denaturation at 98°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 1 min and 30 sec, and 35 cycles; finally, extension at 72°C for 10 min; The product was recovered to obtain the sesquiterpene synthase coding gene XsGAS derived from Xanthium annulus, its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0037] 1.2 Cons...

Embodiment 2

[0058] Example 2 Fermentation and detection of β-elemene engineering bacteria

[0059] 2.1 Activation of engineering bacteria

[0060] The engineering bacteria E.Coil BL21codon pius / pRSFDuet-XsGAS, E.CoilBL21codon pius / pACYCDuet-ERG20 and E.Coil BL21codon pius / pRSFDuet-XsGAS / pACYCDuet-ERG20 prepared in Example 1 were respectively added to 50 μg / mL kanamycin , 34μg / mL chloramphenicol and 20mL LB liquid medium containing 50μg / mL kanamycin and 34μg / mL chloramphenicol at the same time, culture and activate overnight at 37℃ on a shaker.

[0061] 2.2 Detection of fermentation and post-fermentation products and detection of protein expression

[0062] Take 200 μL of the activated bacterial solution in step 2.1 and transfer it to 20 mL of TB medium containing the antibiotic corresponding to the activation, and culture at 37°C until OD 600 When it reaches 0.6-0.8, add IPTG to a final concentration of 0.5mM, and add 10% n-dodecane at the same time to collect the product. After inducti...

Embodiment 3

[0068] Example 3 Optimization of Induced Expression of Xanthii Sesquiterpene Synthase XsGAS

[0069] 3.1 Types of carbon sources

[0070] The engineered bacteria E.Coil BL21codon pius / pRSFDuet-XsGAS / pACYCDuet-ERG20 were activated and then transferred to 20mL TB medium containing 5g / L glucose, sucrose, glycerol, maltose, and lactose respectively and cultured at 37°C until OD 600 0.6-0.8, add IPTG final concentration 0.5mM to induce, ferment at 20°C for 20h, and detect with the detection method of β-elemene in method 2.

[0071] The recombinant bacteria could detect β-elemene after fermentation in five mediums containing different carbon sources, and glycerol was the carbon source for the highest fermentation yield, so glycerol was selected as the carbon source for subsequent fermentation. ( Figure 4 )

[0072] 3.2 Carbon source concentration

[0073] Activated engineering bacteria E.Coil BL21codon pius / pRSFDuet-XsGAS / pACYCDuet-ERG20 were transferred to 20mL TB containing 5...

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Abstract

The invention discloses an engineering strain for producing beta-elemene and an application of the engineering strain. The engineering strain for producing beta-elemene is obtained by simultaneously converting a recombinant plasmid containing a sesquiterpene synthase coding gene XsGAS derived from xanthium sibiricum and a recombinant plasmid containing a phosphate synthase coding gene ERG20 derived from saccharomyces cerevisiae into an E.coli BL21 competent cell for construction. According to the invention, the constructed recombinant engineering bacteria is utilized for fermentation in a terrific broth (TB) culture medium to synthesize beta-elemene, and after carbon source, carbon source concentration, inducer concentration, induction time and induction OD600 are optimized, the yield of beta-elemene reaches 1.17mg / L. Compared with a chemical synthesis method, the application has the advantages that the engineering bacteria provided by invention can efficiently produce beta-elemene without pollution, and is suitable for industrial production.

Description

technical field [0001] The invention relates to the field of traditional Chinese medicine biotechnology, in particular to the construction and expression of a β-elemene-producing strain. Background technique [0002] β-elemene is one of the active ingredients of traditional Chinese medicine Wenyujin, which has anti-inflammatory, antibacterial, anti-tumor and other pharmacological activities. At present, elemene injection is widely used clinically to treat various malignant tumors such as lung cancer, liver cancer, esophageal cancer, and nasopharyngeal cancer. Radiation therapy drugs enter the cancer tissue to work. With the continuous increase of market demand for elemene, the use of traditional extraction and separation methods to obtain elemene from natural plant turmeric has been unable to meet market demand, and the products obtained through extraction and separation have problems such as low yield and low purity. The cost of secondary separation is high. However, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12N15/60C12N1/21C12P5/00C12R1/19
CPCC12N9/1085C12N9/88C12N15/70C12P5/002C12Y205/0101
Inventor 开国银黄超冯岳尤大伟张通范三鹏
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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