Engineering bacterium for synthesizing dencichine and application

A technology of Panax notoginseng and engineering bacteria, which is applied to bacteria, enzymes, microorganism-based methods, etc., can solve the problems of tedious steps, low yield, and large environmental pollution problems of Panax notoginseng, and achieves simple method and high conversion rate. , the effect of low cost

Pending Publication Date: 2022-05-20
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the existing chemical methods for synthesizing Panax notoginsin usually have

Method used

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  • Engineering bacterium for synthesizing dencichine and application
  • Engineering bacterium for synthesizing dencichine and application
  • Engineering bacterium for synthesizing dencichine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Expression, purification and enzyme activity assay of embodiment 1panE

[0054] According to the acylated glyoxylate dehydrogenase gene published by NCBI sequence ID: WP_015822665.1, the gene fragment was obtained by chemical synthesis, and the recombinant plasmid pET-panE was constructed with BamHI and XhoI restriction sites; positive transformants were selected and transformed into BL21 (DE3) Competent cells, spread the transformation mixture on a solid LB plate, incubate at 37°C for 12 hours, pick a single colony and inoculate it into an LB ampicillin-resistant medium test tube, and incubate in a 200rpm constant temperature shaker at 37°C for 8 hours, According to the volume fraction of 1% to 10%, the inoculum was transferred to a Erlenmeyer flask containing 100mL LB ampicillin resistance medium, cultured at 37°C in a constant temperature shaker at 200rpm for 2h, and the inducer isopropyl-β-D-sulfur was added Substitute galactoside (IPTG) to a final concentration of ...

Embodiment 2

[0056] Expression, purification and enzyme activity assay of embodiment 2BAHD

[0057] The genome and transcriptome were extracted from selected bacteria, molds or plants, and compared with the known N-acylase sequence, it was preliminarily determined that it can catalyze the formation of 3-D from 2,3-diaminopropionic acid and oxalyl-CoA. The enzyme of Qisu is named as 2,3-diaminopropionic acid N-oxalyltransferase BAHD, and its sequence is shown in SEQ ID NO.1. The codon-optimized gene fragment was obtained by chemical synthesis, and the recombinant plasmid pET-BAHD was constructed with BamHI and XhoI restriction sites; positive transformants were selected and transformed into BL21 (DE3) competent cells, and the transformation mixture was spread on solid LB On the plate, cultivate at 37°C for 12 hours, pick a single colony and inoculate it into a test tube of LB ampicillin-resistant medium, culture it at 37°C in a 200rpm constant temperature shaker for 8 hours, and transfer it...

Embodiment 3

[0059] Expression, purification and enzyme activity assay of embodiment 3 Gloxdh

[0060] According to the glyoxylate dehydrogenase gene published by NCBI sequence ID: BAH29964.1, the gene fragment was obtained by chemical synthesis, and the recombinant plasmid pET-Fpgloxdh was constructed with BamHI and XhoI restriction sites; according to the published by NCBI sequence ID: BAH29964.1 The glyoxylate dehydrogenase gene sequence was compared with other sources, and the comparison results showed that the Cyb2p gene derived from Saccharomyces cerevisiae, NCBI sequence ID: AJS66626.1, had 50.30% homology with Fpgloxdh, and was named Sagloxdh. Using the Saccharomyces cerevisiae genome as a template for PCR, the recombinant plasmid pET-Sagloxdh was constructed with EcoRI and XhoI restriction sites. Select positive transformants and transform them into BL21(DE3) competent cells, spread the transformation mixture on a solid LB plate, incubate at 37°C for 12 hours, pick a single colony...

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Abstract

The invention provides an engineering bacterium for synthesizing dencichine, the engineering bacterium comprises a host bacterium and a plasmid vector transferred into the host bacterium, and an enzyme for coding and synthesizing oxalyl coenzyme A and a gene of 2, 3-diamino propionic acid N-oxalyltransferase BAHD are introduced into the plasmid vector at the same time. Three ways for synthesizing the oxalyl-coenzyme A enzyme are as follows: acylation glyoxylate dehydrogenase panE, glyoxylate dehydrogenase Gloxdh and oxalyl-coenzyme A ligase AAE, and oxaloacetic acid hydrolase Oah and oxalyl-coenzyme A ligase AAE. And a gene for coding and synthesizing the 2, 3-diaminopropionic acid biosynthase is also introduced into the plasmid vector. The engineering bacteria are optimized in manners of enhancing an upstream path, inhibiting competition and the like so as to improve the production capacity of dencichine. Therefore, the dencichine produced by using the engineering bacteria has the advantages of simple method, low cost, high conversion rate and the like, and is beneficial to industrial production and cost reduction.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, more precisely, the invention relates to an engineering bacterium for synthesizing notoginseng and its application. Background technique [0002] Panax notoginseng, also known as Tianqi, is a recognized strategic resource in the domestic pharmaceutical industry. It has the effects of promoting blood circulation and stopping bleeding, removing blood stasis and relieving pain. It is the key raw material of more than 360 kinds of Chinese patent medicine preparations including Yunnan Baiyao and Pien Tze Huang. Notoginseng (β-N-oxalyl-L-α, β-diaminopropionic acid, β-ODAP) is the main component of notoginseng exerting hemostatic effect, and its structural formula is [0003] Panax notoginseng is a special kind of amino acid, which is a white solid, which is toxic when eaten directly. External use promotes platelet aggregation to play a role similar to thrombin to shorten bleeding time ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/04C12R1/19
CPCC12N15/52C12N9/1029C12N9/0008C12N9/93C12N9/14C12P13/04C12Y203/01058C12Y102/01017C12Y602/01008C12Y307/01001Y02A50/30
Inventor 袁其朋孙新晓李文娜马琳申晓林王佳
Owner BEIJING UNIV OF CHEM TECH
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