Colibacillus engineering bacterium taking glucose as substrate for synthesizing muconic acid

A technology of Escherichia coli and glucose, applied in the biological field, can solve problems such as increasing difficulty, limiting yield improvement, expensive shikimic acid, and achieving the effect of reducing burden

Inactive Publication Date: 2014-10-15
SHANDONG XINGQIANG CHEM IND TECH RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After 84 hours of fermentation in a 7.5L fermenter, the highest content of muconic acid reached 52g/L, which has application prospects, but a certain amount of shikimic acid was added to the medium to make up for the auxotrophic type of aromatic amino acids in Escherichia coli, and the price of shikimic acid Very expensive and increases the production cost of mucon

Method used

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  • Colibacillus engineering bacterium taking glucose as substrate for synthesizing muconic acid
  • Colibacillus engineering bacterium taking glucose as substrate for synthesizing muconic acid
  • Colibacillus engineering bacterium taking glucose as substrate for synthesizing muconic acid

Examples

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Effect test

Embodiment 1

[0060] Example 1 Construction of plasmids pACYC-XA and pET-XA.

[0061] Respectively, pACYCDuet-1 and pETDuet-1 were used as co-expression vectors for the synthesis of exogenous muconic acid genes.

[0062] Use primers A1 and A2 by PCR to obtain entX (SEQ ID NO.1 encoding 2,3-dihydroxybenzoic acid decarboxylase) from Klebsiella pneumoniae CICIM B7001 genome clone; The clone obtained catA (SEQ ID NO.2 encoding catechol 1,2-dioxygenase). After the PCR products and expression vectors of the two genes were digested with corresponding restriction enzymes, the DNA fragments were simultaneously inserted into the corresponding restriction sites of pACYCDuet-1 and pETDuet-1 to obtain recombinant plasmids pACYC-XA and pET- XA.

Embodiment 2

[0063] Example 2 Construction of plasmid pRSF-CBA.

[0064] Select pRSFDuet-1 as the expression vector of entC and entBA genes. entC (encoding chorismate isomerase) was cloned from the Escherichia coli JM109 genome by PCR using primers C1 and C2; entBA (encoding isochorisate lyase and 2,3 - dihydro-2,3-dihydroxybenzoate dehydrogenase). After the PCR product and the expression vector were digested with corresponding restriction endonucleases, the DNA fragment was inserted into the corresponding restriction site of pRSFDuet-1 to obtain the recombinant plasmid pRSF-CBA.

Embodiment 3

[0065] Example 3 Construction of plasmid pCDF-GL.

[0066] Select pCDFDuet-1 as aroG fbr and the expression vector of aroL gene. This vector has double T7 promoters, and the expression of foreign genes can be realized with or without addition of IPTG. 3-deoxy-arabinoheptulose-7-phosphate synthase is referred to as DAHP synthetase for short, consists of AroG, AroF, AroH, aroG encodes AroG, wherein AroG is subject to feedback inhibition by phenylalanine, using the site-directed mutagenesis method (primers E1, E2, E3 and E4), the aspartic acid (GAT) at position 46 of AroG is mutated into asparagine (AAT), and the gene aroG after mutation is obtained fbr , which relieves the feedback inhibition of phenylalanine. aroL, cloned from the genome of E. coli JM109 by PCR using primers F1 and F2. After the PCR product and the expression vector were digested with corresponding restriction endonucleases, the DNA fragment was inserted into the corresponding restriction site of pCDFDuet-1...

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Abstract

The invention discloses a colibacillus engineering bacterium taking glucose as a substrate for synthesizing muconic acid, and belongs to the field of biotechnology. 2,3-dihydroxy benzoic acid (2,3-DHB) is led to colibacillus during the metabolic pathway of muconic acid (MA); the metabolic pathway of colibacillus from glucose to 2,3-DHB is reinforced. The colibacillus engineering bacterium has the advantages that less foreign genes are required; the foreign gene expression load of a host bacterium is reduced; the incompatibility problem of the foreign genes is solved. Moreover, the host bacteria are not auxotroph; the foreign addition of expensive shikimic acid or other aromatic amino acid is avoided; the flask shaking fermentation yield of muconic acid is 1.15 g/L.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium which uses glucose as a substrate to synthesize muconic acid, and belongs to the field of biotechnology. Background technique [0002] Muconic acid, also known as hexadienedioic acid, has been extensively studied as a precursor and platform compound for the production of bioplastics. These products are mainly commercialized bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid used in the manufacture of nylon 66, polyester synthetic fibers, dimethyl terephthalate, industrial plastics, pharmaceuticals, plasticizers Agents and cosmetics, etc. The consumption of adipic acid in the world has reached 3 million tons. The demand for adipic acid in my country is increasing every year, and the demand for polyurethane manufacturers is increasing by more than 10% every year. Traditional muconic acid chemical production methods mainly rely on non-renewable petroleum raw mater...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/44C12R1/19
Inventor 郑璞王杰
Owner SHANDONG XINGQIANG CHEM IND TECH RES INST CO LTD
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