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A strain of Escherichia coli engineered to synthesize muconic acid using glucose as a substrate

A technology of Escherichia coli and glucose, applied in the biological field, can solve problems such as increasing difficulty, expensive shikimic acid, and increasing production cost of muconic acid, and achieves the effect of reducing burden

Inactive Publication Date: 2018-12-04
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 muconic acid
However, the synthesis of anthranilic acid from chorismic acid is a rate-limiting reaction, which requires the regeneration of glutamic acid; while the conversion of salicylic acid to catechol requires the participation of NADH, which increases the construction of muconic acid biosynthetic recombinant bacteria. Difficulty, limiting further increases in output

Method used

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  • A strain of Escherichia coli engineered to synthesize muconic acid using glucose as a substrate
  • A strain of Escherichia coli engineered to synthesize muconic acid using glucose as a substrate
  • A strain of Escherichia coli engineered to synthesize muconic acid using glucose as a substrate

Examples

Experimental program
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Effect test

Embodiment 1

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

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

[0062] The entX (SEQ ID NO.1 encoding 2,3-dihydroxybenzoic acid decarboxylase) was cloned from the Klebsiella pneumoniae CICIM B7001 genome by PCR with primers A1 and A2; from the F1 genome of Pseudomonas putida by PCR with primers B1 and B2 The cloned catA (SEQ ID NO. 2 encodes catechol 1,2-dioxygenase). After the PCR products and expression vectors of the two genes were digested with corresponding restriction enzymes respectively, the DNA fragments were inserted into the corresponding digestion sites of pACYCDuet-1 and pETDuet-1 at the same time to obtain recombinant plasmids pACYC-XA and pET- XA.

Embodiment 2

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

[0064] PRSFDuet-1 was chosen as the expression vector of entC and entBA genes. EntC (encoding chorismate isomerase) was cloned from the genome of Escherichia coli JM109 by PCR with primers C1 and C2; entBA (encoding isochorismate lyase and 2,3 were cloned from the genome of Escherichia coli JM109 by PCR with primers D1 and D2). -Dihydro-2,3-dihydroxybenzoate dehydrogenase). After the PCR product and the expression vector were digested with the corresponding restriction enzymes, 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] Choose pCDFDuet-1 as aroG fbr And aroL gene expression vector. This vector has double T7 promoters, and the presence or absence of foreign genes can be expressed through the addition of IPTG. 3-deoxy-arabinoheptulose-7-phosphate synthetase is abbreviated as DAHP synthase, composed of AroG, AroF, and AroH. AroG encodes AroG. Among them, AroG is feedback-inhibited by phenylalanine, and the method of site-directed mutagenesis is used (primers E1, E2, E3 and E4), the aspartic acid (GAT) at position 146 of AroG was mutated to asparagine (AAT) to obtain the mutated gene aroG fbr , Lifting the feedback inhibition of phenylalanine. aroL was cloned from E. coli JM109 genome by PCR with primers F1 and F2. After the PCR product and the expression vector were digested with corresponding restriction enzymes, the DNA fragment was inserted into the corresponding digestion site of pCDFDuet-1 to obtain the recombinant plasmid pCDF-GL.

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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 engineered bacterium that uses glucose as a substrate to synthesize muconic acid, and belongs to the field of biotechnology. Background technique [0002] Muconic acid, also known as adiponic acid, has been widely studied because it can be used 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, which are used to make nylon 66, polyester synthetic fiber, dimethyl terephthalate, industrial plastics, pharmaceuticals, and plasticizing. 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 production enterprises is increasing by more than 10% every year. The traditional chemical production method of muconic acid mainly relies on non-renewable pet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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