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Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli

A technology for Escherichia coli and succinic acid production, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of difficult screening of transporters and few reports

Inactive Publication Date: 2017-05-17
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, although there are related reports indicating that the production of succinic acid can be increased by enhancing the transport capacity of strains, it is still difficult to obtain a suitable transporter and there are few reports

Method used

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  • Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli
  • Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli
  • Method for constructing succinic-acid-producing Escherichia coli and application of Escherichia coli

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Embodiment 1

[0039] In the present embodiment, the specific method of recombinant escherichia coli construction is as follows:

[0040] Using the genome of Corynebacterium glutamicum ATCC13032 as a template, the gene fragment of Corynebacterium glutamicum NCgl2130 was amplified.

[0041] Among them, the upstream primer sequence:

[0042]5-GCTCTAGAGAACGTGCCCAGTTCCACATCAAATAACGC-3

[0043] Downstream primer sequence:

[0044] 5-CCCAAGCTTTGCCGAGAAGTTGAACAGCAATCTTGCAG-3

[0045] The PCR amplification system is: 1 μL of Corynebacterium glutamicum genome template (not less than 200ng), 1 μL of primers upstream Primer1 and downstream Primer 2, 4 μL of dNTP, 10 μL of 5×Primer star buffer, 0.5 μL of Primer star polymerase, ddH 2 O 32.5 μL.

[0046] The PCR reaction program was as follows: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 45 s; then annealing at 57°C for 45 s, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 10 min.

[0047] PCR products were subjected ...

Embodiment 2

[0053] In this embodiment, the specific method for extracting recombinant Escherichia coli membrane protein is as follows:

[0054] Plate culture: the recombinant bacteria obtained in Example 1 were inoculated on the plate medium for anaerobic culture, the culture temperature was 37°C, and the culture time was 12 hours;

[0055] Seed culture: Inoculate the recombinant bacteria cultivated on the plate into the test tube seed medium, the liquid volume is 5mL, the culture temperature is 37°C, the rotation speed is 200r / min, and the culture time is 14h;

[0056] Aerobic induction: the recombinant bacteria cultured in test tubes were inoculated into 500mL shake flasks, the liquid volume was 100mL, the inoculation volume was 1% (v / v), and the inducer IPTG (isopropyl-β-D-thiogalactoside ) concentration is 0.5mmol / L, the induction temperature is 37°C, the rotation speed is 200r / min, and the incubation time is 8h;

[0057] Membrane protein extraction: according to the method provided ...

Embodiment 3

[0061] In the present embodiment, the specific method for optimizing the anaerobic fermentation temperature condition of recombinant bacteria is as follows:

[0062] Plate culture: the recombinant bacteria obtained in Example 1 were inoculated on the plate medium for anaerobic culture, the culture temperature was 37°C, and the culture time was 12 hours;

[0063] Seed culture: inoculate the recombinant bacteria cultured on the plate into the test tube seed medium, the liquid volume is 5mL, the culture temperature is 37°C, the rotation speed is 200r / min, and the culture time is 14h; then the recombinant bacteria cultured in the test tube are inoculated into 500mL shake In the bottle, the liquid volume is 100mL, the inoculum volume is 1% (v / v), the culture temperature is 37°C, the rotation speed is 200r / min, and the culture time is 8h;

[0064] Anaerobic fermentation: Inoculate the seed culture solution into 100mL anaerobic shake flask fermentation medium, the filling volume is 3...

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Abstract

The invention relates to a method for constructing succinic-acid-producing Escherichia coli and the application of Escherichia coli. The method comprises the following steps: taking Escherichia coli which lacks of activities of lactate dehydrogenase genes and pyruvate formate-lyase genes and in which a ptsG chromosome of a phosphotransferase system is subjected to spontaneous mutation as an original strain, and constructing recombinant Escherichia coli containing corynebacterium glutamicum NCg12130. According to the recombinant Escherichia coli constructed by the method disclosed by the invention, the secretion ability of succinic acid is obviously improved, and the bacterial growth, sugar consumption and succinic acid production capacity in the whole process are obviously enhanced. The method disclosed by the invention provides a novel excellent transport protein for enhancing the capacity of transporting the succinic acid by the Escherichia coli and further provides a new way for enhancing the succinic acid secretion performance.

Description

technical field [0001] The present invention relates to the field of biochemical engineering, and more specifically, the present invention relates to a construction method and application of succinate-producing Escherichia coli capable of enhancing succinic acid secretion. Background technique [0002] Succinic acid, also known as succinic acid, is an important C4 platform compound. It is widely used in medicine, spices, paints, dyes, food and other industries. It can be used as a precursor to synthesize tetrahydrofuran, γ-butyrolactone, 1,4- Organic chemicals such as butanediol and biodegradable materials such as polybutylene succinate. In addition, succinic acid is also used to produce intermediates that are beneficial to human health, such as antibiotics, amino acids, and vitamins. It is considered by the US Department of Energy to be one of the 12 most valuable biorefinery products in the future, and the market demand remains above 10%. annual growth rate. [0003] At ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/46C12R1/19
CPCC12P7/46C07K14/34
Inventor 姜岷李晓展章文明信丰学马江锋吴明科陆家声
Owner NANJING UNIV OF TECH
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