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Construction and application of Escherichia coli engineering bacterium for immobilizing CO2 and producing malic acid

A technology of Escherichia coli and malic acid, applied in bacteria, microorganism-based methods, DNA/RNA fragments, etc., can solve the problems of inability to fix and the third level is not fully realized, and achieve the effect of alleviating the greenhouse effect

Active Publication Date: 2020-04-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The obvious defect of the first level is that only a few special compounds can be produced, and CO cannot be fixed for products without carboxylation reactions in the synthetic pathway. 2 , and the third level has not yet been fully realized, the best research progress is to obtain a semi-autotrophic Escherichia coli

Method used

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  • Construction and application of Escherichia coli engineering bacterium for immobilizing CO2 and producing malic acid
  • Construction and application of Escherichia coli engineering bacterium for immobilizing CO2 and producing malic acid
  • Construction and application of Escherichia coli engineering bacterium for immobilizing CO2 and producing malic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Constructing a method capable of fixing CO 2 Escherichia coli engineered strain producing malic acid

[0042] (1) Knockout of fumarate reductase gene frdBC in E.coli MG1655

[0043] Knockout primers QCfrdBC-S and QCfrdBC-A were designed according to the frdBC gene sequence of Escherichia coli MG1655 in the NCBI database (Table 1), and the frdBC knockout frame was amplified using the pKD4 plasmid as a template and gel recovery was performed.

[0044] Remarks: The pKD4 plasmid contains two FRT sites (which can be folded under the action of flippase to eliminate the DNA sequence between the FRT sites), and between the two FRT sites is kanamycin (kan, used as a gene knockout screening pressure), the gene encoding FRT-kan-FRT. When the gene is knocked out, the primers are designed to amplify the DNA fragment of FRT-kan-FRT. It should be pointed out that the designed two amplification primers respectively contain the upstream and downstream 39-49bp of the target...

Embodiment 2

[0068] Example 2: Fermentative production of malic acid by engineering Escherichia coli GH0402 and GH0407

[0069] Plate activation medium and activation culture conditions: plate activation medium is LB medium, activation conditions: 37 ° C incubator inverted for 12 hours.

[0070] The fermentation medium used for fermentation contains: glucose 50g / L, Na 2 HPO 3 ·5H 2 O 20mM, KHCO 3 50mM, Na 2 HPO 4 12H 2 O 15.11g / L, KH 2 PO 4 3g / L NH 4 Cl 1g / L, NaCl 0.5g / L, trace element solution 1mL / L; trace element solution: FeCl 3 ·6H 2 O 2.4g / L, CoCl 2 ·6H 2 O 0.3g / L, CuCl 2 0.15g / L, ZnCl 2 4H 2 O 0.3g / L, NaMnO 4 0.3g / L, H 3 BO 3 0.075g / L, MnCl 2 4H 2 O 0.495g / L, with 0.1M HCl as solvent.

[0071] After activating the engineered Escherichia coli GH0402 and GH0407 on the plate, pick a single colony in the liquid LB seed medium, and cultivate it at 37°C and 200rpm for 12h (OD 600 =3-4). After the seed culture is completed, inoculate it into the fermentation medi...

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Abstract

The invention discloses construction and application of Escherichia coli engineering bacterium for immobilizing CO2 and producing malic acid, and belongs to the field of fermentation. The engineeringbacterium is a strain obtained by carrying out gene engineering modification on Escherichia coli MG1655. The genetic engineering transformation is as follows: 1, knocking out a fumarate reductase gene, a fumarase gene, a lactic dehydrogenase gene and an ethanol dehydrogenase gene, and carrying out free over-expression on formate dehydrogenase, acetyl coenzyme A synthetase, acylated acetaldehyde dehydrogenase, formaldehyde lyase, dihydroxyacetone kinase, malic enzyme and phosphorous acid oxidordeuctase so as to obtain a strain GH0407. The strain is used for fermentation production of malic acid, CO2 and glucose are used as co-substrates for anaerobic fermentation for 72 h, the malic acid yield reaches 39 g / L, the yield is 1.53 mol / mol, and malic acid is not accumulated in an original starting strain.

Description

technical field [0001] The present invention relates to a plant that fixes CO 2 The construction and application of malic acid-producing Escherichia coli engineering bacteria belong to the field of fermentation engineering. Background technique [0002] Atmospheric CO 2 The increasing concentration affects global climate change, so it is urgent to develop effective CO 2 storage technologies to enhance CO 2 capture or reduce CO 2 release. Conventional CO2 sequestration technologies based on physical and chemical methods, including CO2 capture (such as post-combustion and oxy-fuel combustion), CO2 separation (such as adsorption and membrane separation), and CO2 storage (such as saline-alkaline aquifers, offshore geological formations), are of great importance to Mitigation of carbon dioxide is of great significance. However, these methods have some obvious disadvantages, such as high energy consumption, high operating costs, or generation of degradation products that are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/46C12R1/19
CPCC12N9/0006C12N9/0004C12N9/0008C12N9/1205C12N9/1029C12N9/88C12Y402/01002C12Y101/01027C12Y101/01028C12Y101/01001C12Y102/01002C12Y102/0101C12Y207/01029C12Y101/0104C12N15/70C12P7/46C12N1/20C12N9/001C12N9/0093C12N9/93C12Y103/05004C12Y117/01C12Y602/01001C12N15/52C12Y120/01001C12R2001/19
Inventor 刘立明胡贵鹏陈修来马丹蕾刘佳罗秋玲高聪宋伟
Owner JIANGNAN UNIV
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