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Escherichia coli gene engineering strain for synthesizing Dammar enediol and construction method

A genetically engineered strain and the technology of dammarene diol, applied in the biological field, can solve the problems of no biosynthesis of dammarene diol, etc., and achieve the effect of easy metabolic engineering transformation and simple pathway regulation

Active Publication Date: 2015-12-23
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 gene engineering strain for synthesizing Dammar enediol and construction method
  • Escherichia coli gene engineering strain for synthesizing Dammar enediol and construction method
  • Escherichia coli gene engineering strain for synthesizing Dammar enediol and construction method

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 (USA, ATCC) genome as a template, and using the sequences SEQIDNO.8 and SEQIDNO.9 as primers, the squalene synthase gene ScERG9 (SEQIDNO.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.

[0074] Th...

Embodiment 2

[0098] The construction of embodiment 2 dammarenediol Escherichia coli production strain

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

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

Embodiment 3

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

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

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

[0104] In order to carry out the control of the recombinant strains and facilitate the comparative analysis of metabolites, E. coli containing the 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 gene engineering strain for synthesizing Dammar enediol and a construction method. The escherichia coli gene engineering strain for synthesizing Dammar enediol contains a squalene synthase gene with 26 amino acid residues at the truncated C end, a 2,3-oxidized squalene synthase gene, a cytochrome-NADPH-reductase 1 gene in arabidopsis with 45 amino acid residues at the truncated C end, and a Dammar enediol synthase gene. The construction method disclosed by the invention is used for successfully constructing the escherichia coli gene engineering strain for synthesizing the Dammar enediol. Compared with other host bacteria, the escherichia coli gene engineering strain disclosed by the invention has the advantages that the way of synthesizing the Dammar enediol by escherichia coli is easy to regulate and control, and metabolic engineering transformation is facilitated, so that a foundation is laid for fermentation and production of the escherichia coli of Dammar cucuribitane saponin.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an Escherichia coli engineering bacteria for synthesizing dammarenediol and a construction method. Background technique [0002] As a precious Chinese medicinal material, ginseng has always been a research and application hotspot in disease treatment and health care. Ginsenosides are a class of triterpenoids and are the active ingredients in ginseng. According to the difference of 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 sapogenin protopanaxadiol have the pharmacological effects of protecting cardiovascular, anti-fatigue, anti-aging, anti-cancer, liver protection and enhancing immunity. The main raw material for the traditional production of ginsenosides is ginseng. So far, my country's wild ginseng resources have been on the verge of extinction, ginseng ...

Claims

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Application Information

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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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