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Recombinant bacterium with improved acid tolerance and construction method and application thereof

A technology of recombinant bacteria and acid tolerance, applied in the field of genetic engineering, can solve problems such as poor acid tolerance and environmental pollution, achieve the effects of improving economic rationality and environmental friendliness, reducing production costs, and avoiding additional consumption

Pending Publication Date: 2020-12-29
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of poor acid tolerance of Escherichia coli, organic acids in the biosynthesis of 3-hydroxypropionic acid inhibiting the growth of strains and environmental pollution, the invention provides a recombinant bacterium with improved acid tolerance, the recombinant bacterium overexpresses Hydroxyacyl-ACP dehydroisomerase gene fabA, host bacteria Escherichia coli

Method used

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  • Recombinant bacterium with improved acid tolerance and construction method and application thereof
  • Recombinant bacterium with improved acid tolerance and construction method and application thereof
  • Recombinant bacterium with improved acid tolerance and construction method and application thereof

Examples

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Embodiment 1

[0044]Example 1: Construction of recombinant bacteria with improved acid tolerance.

[0045]1) Using the genomic DNA of E. coli BL21 (DE3) as a template, the hydroxyacyl-ACP dehydroisomerase gene fabA was obtained by PCR cloning with primers SEQ ID NO. 1 and SEQ ID NO. 2;

[0046]2) After the hydroxyacyl-ACP dehydroisomerase gene fabA and the vector pACYCDuet1 obtained in step 1) were digested with SalI and NotI respectively, the target fragment fabA and the vector pACYCDuet-1 after digestion were recovered using the recovery kit, and then Use T4DNA ligase was used for ligation, the ligation product was transformed into E. coli DH5α, and the positive clones were screened using primers SEQ ID NO. 3 and SEQ ID NO. 2 to obtain the recombinant plasmid pACYCDuet1-fabA.

[0047]3) Prepare the recipient strain E.coli BL21(DE3) competent according to the operating steps of the TAKARA Competent Preparation Kit, and transform the recombinant plasmid pACYCDuet1-fabA into the host cell E.coli BL21(DE3) ...

Embodiment 2

[0057]Example 2. Application of the recombinant bacteria prepared in Example 1 in the production of 3-hydroxypropionic acid and its derivatives.

[0058]In this example, the acetyl-CoA carboxylase gene acc and the malonyl-CoA reductase gene mcr were overexpressed in the recombinant bacteria obtained in Example 1. The synthetic gene of hydroxypropionic acid, acetyl-Coenzyme A carboxylase gene acc, comes from Escherichia coli, where the GeneBank ID of subunit accA is 6062185, the GeneBank ID of subunit accB is 6058890, and the GeneBank ID of subunit accC is The GeneBank ID of subunit accD is 60588863, and the GeneBank ID of the subunit accD is 6059083; a genetically engineered strain of 3-hydroxypropionic acid with improved acid tolerance is obtained, and the engineered strain is inoculated into a fermentation medium and cultured to obtain 3-hydroxypropionic acid Its derivatives; the above-mentioned malonyl-CoA reductase gene mcr consists of an N-terminal modified fragment mcrN and a C-t...

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Abstract

The invention relates to a recombinant bacterium with improved acid tolerance and a construction method and application thereof, belongs to the field of genetic engineering technology. In order to improve the acid tolerance of escherichia coli, the escherichia coli is used as an original strain to overexpress a key gene in the unsaturated fatty acid synthesis pathway , a hydroxyacyl ACP dehydration isomerase gene fabA. The recombinant bacterium with improved acid tolerance is prepared. Furthermore, a synthetic gene acetyl-CoA carboxylase gene acc and a malonyl-CoA reductase gene mcr for overexpressing 3HP in the obtained recombinant bacterium can be used for producing 3HP and derivatives thereof, which solves the problem of adding alkali to adjust pH during the fermentation process, and has important application value.

Description

Technical field[0001]The invention relates to a recombinant bacteria with improved acid tolerance and a construction method and application thereof, belonging to the technical field of genetic engineering.Background technique[0002]Escherichia coli has a sophisticated system to maintain a stable pH in the cell under an acidic environment. Five types of acid resistance (AR) pathways are known. At present, little is known about the acid-resistant system, but the AR1 system requires at least one of the signal factor σs and cAMP receptor protein (CRP) to function. The AR2-AR5 pathway is dependent on extracellular glutamate, arginine, lysine and ornithine, respectively. In practical applications, the organic acid fermentation often uses glucose as the carbon source, which causes the AR1 system to fail to function, while AR2-AR5 requires additional amino acids to be added to the culture medium, resulting in increased fermentation costs. Therefore, there are currently five known E. coli ARs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/88C12N15/70C12P7/42C12Y402/01
Inventor 赵广刘敏许莹
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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