Escherichia coli genetic engineering strain and construction method for synthesizing dammarenediol

A technology of genetically engineered strains and dammarediol, applied in the biological field, can solve the problem of no biosynthesis of dammarediol, etc., and achieve the effects of easy metabolic engineering transformation and simple pathway regulation.

Active Publication Date: 2018-05-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Escherichia coli has been successfully used for the biosynthesis of prodrugs such as carotenoids, paclitaxel, and artemisinic acid, but there is no report on the biosynthesis of dammarenediol.

Method used

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  • Escherichia coli genetic engineering strain and construction method for synthesizing dammarenediol
  • Escherichia coli genetic engineering strain and construction method for synthesizing dammarenediol
  • Escherichia coli genetic engineering strain and construction method for synthesizing dammarenediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 Construction of dammarenediol synthesis expression vector

[0070] (1) Acquisition of related genes

[0071] Using the Saccharomyces cerevisiae W303-1a (U.S., ATCC) genome as a template, and using the sequences SEQ ID NO.8 and SEQ ID NO.9 as primers, the squalene synthase gene ScERG9 ( SEQ ID NO. 1).

[0072] According to the amino acid sequence of 2,3-oxosqualene synthase in Methylococcus capsula, codon optimization for Escherichia coli was carried out, and the 2,3-oxosqualene synthase gene was synthesized by GenScript Biotechnology Co., Ltd. McSE, (SEQ ID NO. 2).

[0073] According to the amino acid sequence of cytochrome-NADPH-reductase 1 in Arabidopsis thaliana truncated by 45 amino acid residues at the C-terminus, the codon optimization for Escherichia coli was carried out, and the truncated C-terminus was synthesized by Jinweizhi Biotechnology Co., Ltd. Arabidopsis cytochrome-NADPH-reductase 1 gene ATR1 (SEQ ID NO.3) of 45 amino acid residues.

[...

Embodiment 2

[0098] Embodiment 2 Construction of dammarenediol Escherichia coli production strain

[0099] The plasmid 5 was transformed into Escherichia coli (Escherichia coli) BL21 (DE3) to obtain a genetically engineered Escherichia coli strain for synthesizing dammarenediol, which was named strain 1.

[0100] The Escherichia coli transformation method of plasmid 5 is as follows: add plasmid 5 into 50ul Escherichia coli BL21(DE3) competent cells, bathe in ice for 30min, and heat shock at 42°C for 90s for transformation. Add 1ml of LB medium, rejuvenate at 37°C for 1 hour, collect the cells and spread them on LB plates for kanamycin resistance screening, and culture overnight at 37°C. After overnight culture, colonies were removed for colony PCR verification, positive transformants were selected for sequencing verification, and the positive transformants with correct sequencing were named strain 1.

Embodiment 3

[0101] Embodiment 3 Construction of Escherichia coli genetically engineered strains for synthesizing dammarenediol

[0102] The plasmid 6 was transformed into Escherichia coli (Escherichia coli) BL21 (DE3) to obtain a genetically engineered Escherichia coli strain for synthesizing dammarenediol, which was named strain 2.

[0103] The Escherichia coli transformation method of plasmid 6 is the same as that in Example 2.

[0104] In order to control the recombinant strains and facilitate the comparative analysis of metabolites, Escherichia coli containing pET28a(+) empty plasmid was constructed according to the above transformation method, and named as strain 5.

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Abstract

The invention discloses an Escherichia coli genetic engineering strain for synthesizing dammarenediol and a construction method. The Escherichia coli genetic engineering strain for synthesizing dammarenediol contains a squalene synthase gene with 26 amino acid residues truncated at the C-terminal, 2 , 3‑oxidized squalene synthase gene, cytochrome‑NADPH‑reductase 1 gene and dammarenediol synthase gene in Arabidopsis thaliana truncated by 45 amino acid residues at the C-terminus. The invention successfully constructs the Escherichia coli genetic engineering strain for synthesizing dammarenediol. Compared with other host bacteria, Escherichia coli synthesizes dammarenediol pathway with simple regulation and easy metabolic engineering, which lays the foundation for Escherichia coli fermentation production of dammarane-type saponins.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an Escherichia coli engineering bacterium for synthesizing dammarenediol and a construction method thereof. Background technique [0002] Ginseng, as a precious Chinese medicinal material, has always been a research and application hotspot in disease treatment and health care. Ginsenosides are a class of triterpenoids, which are active ingredients in ginseng. According to the different triterpenoid saponins, they can be divided into dammarane-type saponins and oleanane-type saponins. Among them, dammarane-type ginsenosides such as Rb1, Rg3, Rh2, CK and saponin protopanaxadiol have the pharmacological effects of protecting cardiovascular, anti-fatigue, anti-aging, anti-cancer, protecting liver and enhancing immunity. The main raw material for the traditional production of ginsenosides is ginseng. So far, wild ginseng resources in our country are on the verge of extinction...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
Inventor 卢文玉李大帅张强赵方龙
Owner TIANJIN UNIV
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