Method for synthesizing 3-geranyl-4-hydroxybenzoic acid and xiamenmycin by utilizing escherichia coli

A technology of hydroxybenzoic acid and Escherichia coli, applied in the field of synthetic biology, can solve problems such as long production cycle, and achieve the effect of increasing yield

Active Publication Date: 2018-11-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The yield of Xiamenmycin in wild-type Streptomyces

Method used

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  • Method for synthesizing 3-geranyl-4-hydroxybenzoic acid and xiamenmycin by utilizing escherichia coli
  • Method for synthesizing 3-geranyl-4-hydroxybenzoic acid and xiamenmycin by utilizing escherichia coli
  • Method for synthesizing 3-geranyl-4-hydroxybenzoic acid and xiamenmycin by utilizing escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Plasmid construction of 3-geranyl-4-hydroxybenzoic acid and its final product Xiamenmycin synthesis pathway

[0050] Plasmid pXM was transformed from pET24b(+) (Novagen, Darmstadt, Germany) and contained isologase XbaI / SpeI restriction sites. Plasmid pXM3 contains three genes in the xiamenensis gene cluster: the ximB gene (4-hydroxybenzoate prenyltransferase), the ximD gene (monooxygenase) and the ximE gene (cyclization enzyme). All genes were amplified by PCR, among which the ximB gene and ximD gene were amplified from Streptomyces xiamenensis 318 genome, and the ximEsyn gene was codon-optimized for Escherichia coli.

[0051] Xiamenmycin synthetic schematic diagram of the present invention is as figure 1 As shown, the schematic diagram of the heterologous synthesis of the 3-geranyl-4-hydroxybenzoic acid and Xiamenmycin is as follows figure 2 shown.

[0052] The primers used in the examples are shown in Table 1:

[0053] Table 1 Primer list

[0054] ...

Embodiment 2

[0069] Example 2 Construction of 3-geranyl-4-hydroxybenzoic acid production strain

[0070]The plasmid pXM-IDS2-ximB was directly transformed into Escherichia coli Rosetta (DE3) to obtain the strain XM01, and then pMVA was transformed into the strain XM01 to obtain the XM02 strain.

Embodiment 3

[0071] The construction of embodiment 3 Xiamenmycin production strains

[0072] Plasmids pXM4 and pMVA were transformed into Escherichia coli Rosetta (DE3) to obtain strain XM03.

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Abstract

The invention discloses a method for synthesizing 3-geranyl-4-hydroxybenzoic acid and xiamenmycin by utilizing escherichia coli. According to the method, an adopted escherichia coli strain contains xiamenmycin synthesis related genes and mevalonic acid pathway related genes which are partially or entirely subjected to codon optimization; three genes required by synthesis of the xiamenmycin are imported into the escherichia coli; the three genes are subjected to inducible expression, and GBA and the xiamenmycin are synthesized in the escherichia coli; the feed of intracellular geranyl pyrophosphate of the escherichia coli is further improved by introducing a mevalonic acid synthesis pathway, and the yield of the GBA is further increased by adding 4-hydroxybenzoic acid into a fermentation medium; the yield of the synthesized 3-geranyl-4-hydroxybenzoic acid reaches 94 mg/L, and the yield of the xiamenmycin reaches 11 mg/L; and the biosynthesis of the 3-geranyl-4-hydroxybenzoic acid and the xiamenmycin in the escherichia coli is realized for the first time.

Description

technical field [0001] The invention belongs to the field of synthetic biology and relates to a bacterial strain for producing 3-geranyl-4-hydroxybenzoic acid and its final product Xiamenmycin. Background technique [0002] Xiamenmycin is a benzopyran compound produced by Streptomyces xiamen. Animal experiments show that it can inhibit the contraction ability of fibroblasts, and can significantly weaken hypertrophic scars. At present, fibrotic diseases and cardiovascular fibrosis have seriously endangered human health. We have demonstrated that Xiamenmycin is a novel small molecule drug candidate compound with anti-fibrotic activity. [0003] The yield of Xiamenmycin in wild-type Streptomyces xiamenis is about 10mg / L, and the production cycle is long. The construction of genetically engineered bacteria capable of efficiently synthesizing the precursor and final product of Xiamenmycin has potential application value. [0004] 3-geranyl-4-hydroxybenzoic acid is an importan...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/06C12P7/42C12R1/19
CPCC12N9/0065C12N9/0069C12N9/1085C12N15/70C12P7/42C12P17/06
Inventor 徐岷涓徐俊贺贝贝步绪亮周婷
Owner SHANGHAI JIAO TONG UNIV
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