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A kind of microorganism and method for biosynthesizing arachidine

A technology of arachicarpine and microorganisms, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effects of fast growth, mature genetic manipulation technology, and cost reduction

Active Publication Date: 2022-06-14
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long growth cycle of plants and the low content of benzylisoquinoline alkaloids, the cost of extracting benzylisoquinoline alkaloids from plants is relatively high

Method used

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  • A kind of microorganism and method for biosynthesizing arachidine
  • A kind of microorganism and method for biosynthesizing arachidine
  • A kind of microorganism and method for biosynthesizing arachidine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 constructs arachiline-producing Escherichia coli engineering bacteria

[0040] 1. Construction of gene knockout strain DY

[0041] Using the pKD4 plasmid as a template, PCR amplifies the targeting fragment, which contains (kan r Sequence and homology arms complementary to both ends of the target gene to be knocked out); electroporate the amplified targeting fragment into E. coli carrying the pKD46 plasmid. Using the Red homologous recombination system on the pKD46 plasmid, the target gene to be knocked out is recombined with the targeting fragment, and the target gene is replaced by the resistance gene. Then, the temperature-sensitive plasmid pCP20 was introduced into the strain, and the FLP flippase on the plasmid was used to remove the resistance gene on the chromosome of the target strain. The dkgB, yeaE, yqhC, yqhD, dkgA, yahK and yjgB genes in Escherichia coli MG1655 strain were sequentially knocked out by the above method to obtain Escherichia coli ...

Embodiment 2

[0064] Embodiment 2 Escherichia coli engineering bacterium DY1 produces the fermentation experiment of argocarpine

[0065] 1. Inoculate the Escherichia coli engineering bacteria DY1 cultivated overnight in 37-degree LB into 50 ml of M9 fermentation medium (including 2% glucose), and carry out 30-degree shake flask fermentation until the OD of the bacteria is 600 is about 0.8, add 0.1mM IPTG to induce the expression of the target gene.

[0066] 2. After 24 hours of fermentation and cultivation, the bacteria were collected, the cells were broken by ultrasonic waves, and the product was extracted with chloroform.

[0067] 3. Use a rotary evaporator to spin dry the solvent chloroform, and then dissolve the product with a certain amount of chloroform.

[0068] 4. GC-MS (gas chromatography-mass spectrometry) detects the sample.

[0069] 5. Experimental results: The GC-MS detection results of E. figure 2 As shown in A, the identification of arachidine by GC-MS is as follows im...

Embodiment 3

[0070] Embodiment 3 Escherichia coli engineering bacteria DY2 produces the fermentation experiment of argocarpine

[0071] 1. Inoculate the Escherichia coli engineering bacteria DY2 cultivated overnight in 37-degree LB into 50 ml of M9 fermentation medium (including 2% glucose), and carry out 30-degree shake flask fermentation until the OD of the bacteria is 600 is about 0.8, add 0.1mM IPTG to induce the expression of the target gene.

[0072] 2. After 24 hours of fermentation and cultivation, the bacteria were collected, the cells were broken by ultrasonic waves, and the product was extracted with chloroform.

[0073] 3. Use a rotary evaporator to spin dry the solvent chloroform, and then dissolve the product with a certain amount of chloroform.

[0074] 4. GC-MS (gas chromatography-mass spectrometry) detects the sample.

[0075] 5. Experimental results: The GC-MS detection results of Escherichia coli DY2 using glucose to synthesize arhatine are as follows: figure 2 B, sh...

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Abstract

The invention discloses a microorganism for biosynthesizing argocarpine and a method thereof. The microorganism does not contain or knock out dkgB, yeaE, yqhC, yqhD, dkgA, yahK and yjgB genes, and highly expresses or introduces aroG fbr , tyrA fb , KDC, HpaB, HpaC, DODC, CAR, sfp, aldH, CNS, 4OMT, 6OMT and CNMT genes. The transformed microorganism of the present invention can realize the microbial conversion from monosaccharide or glycerol to arachidine, and provides a feasible way for large-scale biosynthesis of arachidine.

Description

technical field [0001] The invention belongs to the field of microbial metabolism engineering, and in particular relates to a method for directly synthesizing arachidine from monosaccharide or glycerol by transforming microbial metabolic pathways. Background technique [0002] Benzylisoquinoline alkaloids include more than 2500 species, many of which, such as morphine, berberine and noscapine, have broad application prospects and economic status in the medical field. Aragoline is a common intermediate of benzylisoquinoline alkaloids. [0003] Currently commercialized benzylisoquinoline alkaloids are mainly extracted from plants. Due to the long growth cycle of plants and the low content of benzylisoquinoline alkaloids, the cost of extracting benzylisoquinoline alkaloids from plants is relatively high. By means of biotechnology, microbial fermentation of monosaccharides or glycerol to synthesize benzylisoquinoline alkaloids common intermediate aracine can effectively reduce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P17/12C12R1/19
CPCC12N9/0006C12Y101/01021C12P17/12
Inventor 郭道义潘虹孔思佳
Owner GANNAN NORMAL UNIV
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