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Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use

A technology of Escherichia coli and succinic acid, which is applied in the field of bioengineering, can solve the problems of limited flux and achieve the effects of accelerating the growth rate of bacteria, increasing the production rate, and increasing the density of bacteria

Active Publication Date: 2013-12-11
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the glyoxylate cycle is regulated by many factors, the effect of regulating its flux by means of genetic engineering is relatively limited

Method used

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  • Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use
  • Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use
  • Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Construction of departure strains for directed pathway evolution

[0029] (1) Knockout the phosphoenolpyruvate carboxylase gene of Escherichia coli BL21(DE3) (purchased from Novagen), and overexpress the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, to obtain The starting strain for evolution;

[0030] The specific operation of knocking out the Escherichia coli phosphoenolpyruvate carboxylase gene ppc is as follows:

[0031] The chloramphenicol resistance gene was amplified using amplification primers Kan1 / Kan2 and plasmid pKD3 (purchased from CGSC) as a template, and cloned into plasmid pUC18 (purchased from Invitrogen) at the Hind III / Sal I site to obtain plasmid pCM01.

[0032] The upstream homology arm of the ppc gene was amplified using the amplification primers Pout1 / Pout2 and the genome of Escherichia coli BL21 (DE3) as a template, and cloned into the plasmid pCM01 at the HindⅢ site to obtain the plasmid pCMppcH.

[0033] Using ampl...

Embodiment 2

[0040] Enhancing glyoxylate cycle flux through directed pathway evolution

[0041] In order to improve the flux of glyoxylate cycle and reduce the accumulation of α-ketoglutarate, a byproduct of succinic acid production, the strain BPB was used as a starting point to enhance the flux of glyoxylate cycle by directed pathway evolution.

[0042] The specific operation is as follows: the bacterial strain BPB obtained in Example 1 is cultivated in the M9 basic salt medium with glycerol as the only carbon source, the culture conditions are 37 ° C, 220 rpm, and continuous transfer is carried out in the logarithmic phase of bacterial growth Culture, the time interval of transfer is 12 hours, and when cultured to 100 generations, isolate to the specific growth rate of 0.4h in the M9 basic salt medium with glycerol as the only carbon source -1 One of the strains grown was named BPB18. A single gene knockout was performed on the strain BPB18, and it was found that the glyoxylate cycle b...

Embodiment 3

[0046] Construction of high-yielding strains of succinic acid by genetic engineering

[0047] Taking the evolved strain BPB18 as the starting strain, through genetic engineering methods including knocking out the succinate dehydrogenase gene, overexpressing phosphoenolpyruvate carboxylase and α-ketoglutarate dehydrogenase to construct a system that can use glycerol as Substrate strains that efficiently produce succinic acid. The overexpression of phosphoenolpyruvate carboxylase and α-ketoglutarate dehydrogenase is achieved by replacing their respective promoters with strong trc promoters.

[0048] (1) The specific operation of knocking out the succinate dehydrogenase gene sdhCDAB is as follows: transfer the plasmid pKD46 into the evolved strain BPB18 obtained in Example 2 and make it competent for electroporation; use the primer Sdh1 / Sdh2, using pKD4 as a template, amplified the fragment containing the kanamycin resistance gene and the upstream and downstream homology arms, ...

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Abstract

The invention discloses an Escherichia coli strain for producing succinic acid with glycerol as well as a construction method and use. The Escherichia coli strain for producing succinic acid with glycerol has the preservation number of CGMCC No. 7999. The construction method comprises the following steps: knocking out the phosphoenolpyruvate carboxylase gene of Escherichia coli, and performing over-expression of key enzymes (isocitrate lyase and malate synthetase) of the glyoxylate cycle to obtain an evolved starting strain; by virtue of evolution, obtaining an evolved strain which can grow in an M9 basic salt medium with glycerol as unique carbon source and has enhanced glyoxylate cycle flux; knocking out the succinate dehydrogenase gene, and performing over-expression of phosphoenolpyruvate carboxylase and alpha-ketoglutaric dehydrogenase systems to obtain an Escherichia coli strain for producing succinic acid by using glycerol. The strain grows rapidly and has a high cell density, so that the rate of producing succinic acid with glycerol is increased.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a bacterial strain for producing succinic acid by using glycerol, a construction method and application thereof. Background technique [0002] Succinic acid, also known as succinic acid, is widely used as an important raw material in industries such as medicine, food, plastics, paints, pesticides, dyes and surfactants. It is also an important platform compound, which can be used in the synthesis of tetrahydrofuran, n-methylpyrrolidone, 2-pyrrolidone and butanediol. [0003] The synthesis methods of succinic acid include chemical synthesis and microbial fermentation. Among them, the microbial fermentation method has attracted more and more attention from scientific research workers in various countries because of its low environmental pollution and low cost. The strains used for the production of succinic acid by microbial fermentation mainly include Actinobacillus succinat...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/46C12R1/19
Inventor 陈涛李宁王智文赵学明
Owner TIANJIN UNIV
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