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Recombinant eukaryotic strain for generating amorphadiene and method for preparing amorphadiene with recombinant eukaryotic strain

A technology of artemisinene and artemisinene synthase, applied in the field of bioengineering, can solve the problems of lack of glycosylation and post-translational modification, unfavorable artemisinene modification and the like

Active Publication Date: 2016-02-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of glycosylation and post-translational modification functions in the Escherichia coli system, it is not conducive to the modification in the subsequent production of artemisinin, which limits its application.

Method used

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  • Recombinant eukaryotic strain for generating amorphadiene and method for preparing amorphadiene with recombinant eukaryotic strain
  • Recombinant eukaryotic strain for generating amorphadiene and method for preparing amorphadiene with recombinant eukaryotic strain
  • Recombinant eukaryotic strain for generating amorphadiene and method for preparing amorphadiene with recombinant eukaryotic strain

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Experimental program
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Effect test

Embodiment 1

[0090] The construction of embodiment 1 recombinant eukaryotic strain

[0091] (1) Construction of gene expression module

[0092] Step 1: Construction of pRS425K-promoter-terminator module:

[0093] Using the Saccharomyces cerevisiae BY4741 genomic DNA as a primer, the promoter and terminator unit and the Delta homology arm region were cloned with specific primers; the Amp resistance gene sequence in the original pRS425 plasmid was replaced with the Kan resistance gene sequence, and the resulting plasmid became pRS425K, linearized with restriction endonuclease NotI, was used as a vector for subsequent construction.

[0094] Construction of the homology arm unit module LDL: Delta1 upstream sequence, URA3 gene sequence and different terminator unit sequences were amplified by overlapPCR, digested with restriction endonuclease NotI, ligated with the linearized vector pRS425K by T4 ligase, Obtain recombinant vector pRS425K:Delta1-URA3-T according to this method CYC1 . Gene expr...

Embodiment 2

[0112] Example 2 Fermentative production of artemisinin by recombinant strains

[0113] Experimental Materials:

[0114] Recombinant strain: the recombinant strain obtained by constructing the method provided in Example 1, SyBE_000111001 recombinant strain.

[0115] Medium: SC-ura liquid medium, the concentration of glucose in the medium is 40g / L, the concentration of YNB is 6.7g / L, the concentration of amino acid mixture is 2.0g / L, and the concentration of leucine is 0.1g / L L, the concentration of histidine is 0.02g / L, and the concentration of tryptophan is 0.02g / L.

[0116] (1) Shake flask fermentation

[0117] The correct positive clone SyBE_000111001 recombinant strain obtained by screening was inoculated into 5mL SC-ura liquid medium, and cultivated to OD at 30°C and 200rpm 600 The value is about 5.0; take the obtained bacterial liquid, and take the initial OD 600 Transfer to SC-ura liquid medium with a value of 0.1, and culture at 30°C and 200rpm for 12 hours, accord...

Embodiment 3

[0137] Example 3 Preparation of artemisinin by using the recombinant eukaryotic strain provided by the present invention

[0138] Fermentation medium: SC-ura liquid medium, the concentration of glucose in the medium is 25g / L, the concentration of YNB is 6.7g / L, the concentration of amino acid mixture is 2.0g / L, and the concentration of leucine is 0.8g / L, the concentration of histidine is 0.16g / L, and the concentration of tryptophan is 0.16g / L.

[0139] Fermentation method:

[0140] The correct positive clone SyBE_000111001 recombinant strain obtained by screening was inoculated into 5mL SC-ura liquid medium, and cultivated to OD at 30°C and 200rpm 600 The value is about 5.0; take the obtained bacterial liquid, and take the initial OD 600 Values ​​of 0.05 were respectively transferred to the fermentation medium. The initial medium volume was 2L, the temperature was 28°C, the pH was maintained at 6.0, the rotation speed was maintained at 500rpm, and the air flow rate was 4LPM...

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Abstract

The invention belongs to the technical field of bioengineering and discloses a recombinant eukaryotic strain for generating amorphadiene and a method for preparing the amorphadiene with the recombinant eukaryotic strain. Expression genes of 3-hydroxyl-3-methylglutaryl coenzyme A, FPP (farnesylpyrophosphate) synthase and amorphadiene synthase are integrated in a genome of the eukaryotic strain and integrated at a multi-copy locus of the genome of the eukaryotic strain. The recombinant eukaryotic strain for generating the amorphadiene is an integrated eukaryotic strain, greatly increases yield of the amorphadiene and is beneficial to amorphadiene production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant eukaryotic strain producing artemisinin and a method for preparing artemisinic acid by using the recombinant eukaryotic strain. Background technique [0002] Natural compounds are mainly obtained from the following ways: (1) extraction from plants; (2) synthesis by chemical synthesis; (3) synthesis by means of synthetic biology. Synthetic biology is a new discipline for the production of large-scale and stable natural compounds. It is an important alternative to direct extraction from plants and chemical total synthesis. Genes are introduced into some commonly used model chassis cells such as yeast, Escherichia coli or plants, and the pathways that can produce natural compounds are rebuilt in the chassis cells, and then the natural compounds are expressed. [0003] Artemisinin and its related derivatives have been recognized by the World Health O...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P17/18C12R1/865
Inventor 元英进郭睿丁明珠
Owner TIANJIN UNIV
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