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Platform bacterium for producing L-aspartate, recombinant bacterium constructed based on platform bacterium for producing beta-alanine and construction method of recombinant bacterium

A technology of aspartate aminotransferase and aspartic acid, which is applied in the field of microorganisms, can solve problems such as unsuitable separation and purification, harsh reaction conditions of chemical synthesis methods, and environmental pollution.

Active Publication Date: 2020-10-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0018] To sum up, the chemical synthesis method generally faces problems such as harsh reaction conditions, unsuitable separation and purification, and easy to cause environmental pollution; while the biotransformation method needs to establish a cheap raw material route and increase the conversion rate of the product in order to reduce production costs and form a promising method for popularization. production mode

Method used

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  • Platform bacterium for producing L-aspartate, recombinant bacterium constructed based on platform bacterium for producing beta-alanine and construction method of recombinant bacterium
  • Platform bacterium for producing L-aspartate, recombinant bacterium constructed based on platform bacterium for producing beta-alanine and construction method of recombinant bacterium
  • Platform bacterium for producing L-aspartate, recombinant bacterium constructed based on platform bacterium for producing beta-alanine and construction method of recombinant bacterium

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Embodiment 1

[0231] Embodiment 1, the construction of genetic engineering strain

[0232] 1. Construction of a recombinant plasmid expressing Escherichia coli aspartate aminotransferase (i.e. recombinant plasmid pLB1a-E)

[0233] 1. The bacterial genome extraction kit was used to extract the genomic DNA of Escherichia coli BW25113.

[0234] 2. Using the Escherichia coli genomic DNA extracted in step 1 as a template, using aspC-GF and aspC-GR as primers, perform PCR amplification with high-fidelity TransStart FastPfu DNA polymerase to obtain PCR amplification products.

[0235] 3. Perform agarose gel electrophoresis on the PCR amplification product obtained in step 2, and then recover a DNA fragment of about 1240 bp. The DNA fragment contains the aspC gene, and the nucleotide sequence of the aspC gene is shown as sequence 2 in the sequence list. The aspC gene encodes the aspartate aminotransferase of E. coli.

[0236] 4. Digest the vector pLB1a with restriction endonucleases NcoI and Xho...

Embodiment 2

[0425] Embodiment 2, utilize the genetic engineering bacterial strain that embodiment 1 constructs to prepare L-aspartic acid or β-alanine

[0426] Autoinduction medium ZYM consisted of 100 mL of solution A, 2 mL of solution B, 2 mL of solution C, 200 μL of solution D, and 100 μL of solution E.

[0427] Solution A: an aqueous solution containing 1% (m / m) tryptone and 0.5% (m / m) yeast powder.

[0428] Solution B: Contains 1.25M Na 2 HPO 4 、1.25M KH 2 PO 4 , 2.5M NH 4 Cl and 0.25M Na 2 SO 4of aqueous solution.

[0429] Solution C: an aqueous solution containing 25% (m / m) glycerol, 2.5% (m / m) glucose and 10% (m / m) L-arabinose.

[0430] Solution D: MgSO at a concentration of 1M 4 aqueous solution.

[0431] Solution E: Contains 50mM FeCl 3 , 20mM CaCl 2 , 10mM MnCl 2 , 10mM ZnSO 4 , 2mM CoCl 2 , 2mMNiCl 2 , 2mM Na 2 Mo 4 , 2mM Na 2 SeO 3 and 2mM H 3 BO 3 of aqueous solution.

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Abstract

The invention discloses a platform bacterium for producing L-aspartate, a recombinant bacterium constructed based on the platform bacterium for producing beta-alanine and a construction method of therecombinant bacterium. By changing a glucose intake pathway and a glycolysis pathway of escherichia coli, phosphoenolpyruvic acid is accumulated in a large amount, so that a carbon fixation pathway isenhanced to obtain a large amount of precursor substance oxaloacetic acid, and related gene targets of an L-aspartate synthesis pathway are enhanced; by introducing a cofactor circulating system, theproblem of insufficient co-substrates is solved, and a central metabolic pathway tricarboxylic acid cycle is transformed and a byproduct competitive bypass is knocked out, so that the platform bacterium is obtained; and based on the platform bacterium, expression of aspartate decarboxylase is enhanced to obtain the recombinant bacterium. The platform bacterium can synthesize 50.10+ / -2.10 mM L-asp, and the conversion rate reaches 1.00 M / M glucose; and the recombinant bacterium can synthesize 76.01+ / -2.80 mM beta-alanine, and the conversion rate is 1.52 M / M glucose. The platform bacterium for producing the L-aspartate, the recombinant bacterium constructed based on the platform bacterium for producing the beta-alanine and the construction method of the recombinant bacterium have important application value.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a platform bacterium for producing L-aspartic acid, a recombinant bacterium for producing β-alanine constructed based on the platform bacterium and a construction method thereof. Background technique [0002] L-aspartate (L-asp), also known as α-aminobutyric acid, is a commonly used organic chemical raw material, in the form of colorless flaky crystals or white crystalline powder, odorless, slightly sour . L-aspartic acid is mainly used as food additives, intermediates of chemical products and pharmaceutical raw materials: in the food industry, L-aspartic acid is a good nutritional supplement, and it is a substitute for sugar aspartame. The main production raw materials; in the chemical industry, it can be used as a raw material for the manufacture of synthetic resins, and is widely used in the synthesis of polyaspartic acid, an environmentally friendly material. Polyas...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/20C12P13/06C12R1/19
CPCC12N9/88C12N9/104C12N9/0016C12N9/1205C12N9/0006C12N9/12C12N9/0008C12N9/001C07K14/245C12P13/20C12P13/06C12Y401/01031C12Y401/01C12Y203/02007C12Y104/01002C12Y207/0104C12Y101/01037C12Y102/03003C12Y402/01001C12Y401/01032C12Y103/01006
Inventor 刘伟丰朴晓宇林白雪陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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