Method for producing itaconic acid in escherichia coli

A technology of Escherichia coli and itaconic acid, which is applied in the field of genetic engineering, can solve problems such as the inability to meet high-yield industrial production, and achieve the effect of easy mutual recognition and assembly

Inactive Publication Date: 2018-02-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of method of producing itaconic acid in escherichia coli, th...

Method used

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  • Method for producing itaconic acid in escherichia coli
  • Method for producing itaconic acid in escherichia coli
  • Method for producing itaconic acid in escherichia coli

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Experimental program
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Embodiment 1 3

[0030] Construction and expression of embodiment 1 three-enzyme self-assembly plasmid

[0031] gltA (predicted as dimer enzyme) gene from Corynebacterium glutamicum (GenBank ID: 1018824), acnA (predicted as monomeric enzyme) from Corynebacterium glutamicum (GenBank ID: 1019512), cadA (predicted as Dimerase) was derived from Aspergillus terreus (GenBank ID: BAG49047.1). The genes gltA, acnA, and cadA are obtained through the whole gene synthesis of the biological company, and have undergone codon optimization. In addition, the genes PDZ and SH3 have been constructed in our laboratory and stored at -20°C.

[0032] Plasmid construction adopts a one-step cloning method. In this experiment, all plasmid backbones are inserted into the strong promoter BBa_J23100 for transformation, and the target gene is gene fused with the vector. The gltA and cadA genes were constructed in the plasmid pQE-30 to obtain the plasmid pQE30-cadA-gltA, and the ligand SH3lig of the SHM protein pair and ...

Embodiment 2

[0034] Embodiment 2 gltA, acnA and cadA enzymatic property characterization

[0035] Plasmids expressing gltA, acnA and cadA proteins alone were constructed and used as controls after separation and purification. The catalytic activities of the three fusion enzymes and their original enzymes were measured respectively, and their enzymatic properties were studied. The enzyme activity detection method is as follows:

[0036] 1) The enzyme activity of aconitic acid decarboxylase cadA was detected by the concentration of itaconic acid, and the cis-aconitic acid was reacted with the enzyme, and the amount of itaconic acid produced was detected by HPLC. The detection conditions are: Inertsil ODS-3column, 0.1M ammonium dihydrogen phosphate (pH 2.6) as the mobile phase, and the column temperature is 40°C. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 micromole of itaconic acid per minute at 37°C.

[0037] 2) The detection method o...

Embodiment 3

[0040] Example 3 Multi-enzyme in vitro assembly experiment

[0041] Multi-enzyme assembly, verification and characterization are carried out outside the cell, that is, non-denaturing electrophoresis, molecular exclusion chromatography, laser particle size analysis, dynamic light scattering and other technologies and activity detection are used to investigate the concentration, ratio and assembly time of each enzyme assembly on the assembly process. and cascade catalytic activity, and by means of atomic force microscopy (AFM) and scanning electron microscopy (SEM) to measure the morphology and surface morphology of the assembled multienzyme, and characterize the structure and compartmentalized geometric characteristics of the multienzyme. The intracellular assembly activity was verified by the amount of itaconic acid (HPLC detection), the product synthesized by final fermentation.

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Abstract

The invention provides a method for producing itaconic acid in escherichia coli, belonging to a method for efficiently producing itaconic acid in escherichia coli by simultaneously utilizing a multi-enzyme assembly technology and a metabolic pathway transformation technology. The method for producing itaconic acid in escherichia coli comprises the following steps: (1) carrying out self-assembly onthree key enzymes for synthesizing itaconic acid in cells, thus forming a multi-enzyme complex, wherein the three key enzymes comprise citrate synthase gltA, cis-aconitase acnA and aconitate decarboxylase cadA; (2) carrying out metabolic pathway transformation on escherichia coli by adopting the Crispr/cas9 technology; and (3) transferring the multi-enzyme complex into escherichia coli after themetabolic pathway transformation, thus constructing an escherichia coli engineering bacterium, and producing itaconic acid through fermentation. According to the method, the three key enzymes for synthesizing itaconic acid are taken as assembly objects, meanwhile, the Crispr/cas9 technology is adopted for transforming the metabolic pathways, thus the efficient production of itaconic acid in escherichia coli is realized, and a new path is provided for the industrial production of itaconic acid.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for producing itaconic acid in Escherichia coli. Background technique [0002] Itaconic acid (methylene succinic acid, methylene succinic acid) is an unsaturated dicarboxylic acid, which is an important industrial raw material and additive in the chemical industry. It is widely used as a building block in the production of plastics, chemical fibers, superabsorbents Agents, latex, anti-fouling agents, etc. Itaconic acid has received increasing attention as a renewable material. In 2004, itaconic acid was selected as one of the 12 bio-based platform compounds with the most development potential by the US Department of Energy. At present, the world's itaconic acid production capacity is 50,000 tons per year, and there is still a gap of at least 30,000 tons, and itaconic acid can replace acrylic acid and methacrylic acid for the production of degradable plastics, and its...

Claims

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Application Information

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IPC IPC(8): C12P7/44C12N15/70C12R1/19
CPCC12N15/70C12P7/44
Inventor 任宇红魏东芝杨中伟王红玲
Owner EAST CHINA UNIV OF SCI & TECH
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