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Method for producing succinic acid by means of fermentation

A technology of succinic acid and anaerobic fermentation, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve the problems of difficult cultivation of bacterial strains and low acid production capacity, and achieve simple and convenient methods and high acid production capacity Strong, simple and feasible fermentation method

Active Publication Date: 2016-05-11
态创生物科技(广州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the technology of the present invention is to provide a method for fermenting and producing succinic acid for the existing succinic acid-producing strains that are not easy to cultivate and have low acid-producing ability, and to use the method to produce succinic acid to obtain the method for acid-producing bacterial strains Simple and convenient, the fermentation method is simple and feasible, and the ability to produce acid is strong, which can greatly reduce production costs and improve economic benefits

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  • Method for producing succinic acid by means of fermentation
  • Method for producing succinic acid by means of fermentation
  • Method for producing succinic acid by means of fermentation

Examples

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Embodiment 1

[0030] This example illustrates the method of constructing an expression plasmid co-expressing isocitrate dehydrogenase to obtain a recombinant strain producing succinic acid.

[0031] Construct pLGZ920-icdh plasmid (such as figure 2 ), its process comprises: (1) synthesis has the primer of SacI and BamHI restriction site:

[0032] Upstream primer: 5'-CGGGATCCCAAACCAGTAGCGCTCGAAGGAGAG-3';

[0033] Downstream primer: 5'-CGAGCTCCGCCCGTTGCAATATTAAACACATG-3';

[0034] (2) Using E.coliBL21 genomic DNA as a template, PCR amplifies the target gene fragment. The reaction conditions are: 94°C, 10min; (94°C for 45s, 62°C for 45s, 72°C for 80s, 35 cycles); 72°C, 10min .

[0035] After purifying the amplified icdh gene (such as Figure 4 ), the expression plasmid pLGZ920 was digested with SacI and BamHI, and connected to obtain the recombinant plasmid pLGZ920-icdh (such as Figure 6 ).

Embodiment 2

[0037] This example illustrates the construction of an expression plasmid that co-expresses isocitrate dehydrogenase and citrate synthase, restores the ability of wild-type Actinobacillus succinogenes to synthesize glutamate in a synthetic medium, and obtains a recombinant strain that produces butyrate Methods.

[0038] 1. Construct the expression plasmid (such as image 3 ), the process includes:

[0039] (1) Synthesize primers for homologous recombination of one-step cloning with BamHI restriction sites both upstream and downstream,

[0040] Upstream primers:

[0041] 5'-ATGAGGTGATCTAGAGGATCCCAGGTTGATGTGCGAAGGC-3';

[0042] Downstream primers:

[0043] 5'-GCGCTACTGGTTTGGGATCCTCTATTAAAGGCGGGTCCGGAAAG-3';

[0044] (2) Using Escherichia coli E.coliBL21 genomic DNA as a template, PCR amplifies the target fragment, and the reaction conditions are: 94°C, 10min; (94°C for 45s, 55°C for 45s, 72°C for 100s, 35 cycles); 72°C, 10min. Purify the amplified cs (eg Figure 5 ) gene, ...

Embodiment 3

[0047] This example illustrates the comparison of sugar consumption and acid production ability of the overexpressed newly constructed recombinant Actinobacillus succinogenes BD106 and the starting strain NJ113 without adding glutamic acid in the synthetic medium.

[0048] When Actinobacillus succinogenes NJ113 was introduced into the plasmid pLGZ920-icdh-cs, the co-expression of isocitrate dehydrogenase and citrate synthase restored its ability to synthesize glutamate in synthetic medium.

[0049] Seed culture: activate Actinobacillus succinogenes BD106 on a plate, and cultivate overnight in an anaerobic box at 37°C. The activated strains on the plate were inserted into serum bottles, and cultured at 37°C and 200r / min for 12 hours as the primary seeds. Put the primary seeds into the serum bottle with 6% inoculum amount, and cultivate them at 37° C. and 200 r / min for 12 hours as the secondary seeds. The seed medium in the serum bottle was fed with CO 2 2min, culture temperat...

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Abstract

The invention discloses a method for producing succinic acid by means of fermentation. The method includes steps of constructing acid-producing bacterial strains; producing the succinic acid from the recombinant bacterial strains by means of fermentation. Original bacterial strains for constructing the recombinant bacterial strains are actinobacillus succinogenes NJ113 (with a preservation number of CGMCC No.1716). The method for producing the succinic acid has the advantages that processes for acquiring the acid-producing bacterial strains are simple and convenient, fermentation processes are simple and feasible, the method is high in acid-producing capacity, the production cost can be greatly reduced, and economic benefits can be increased; the recombinant bacterial strains can grow in synthetic culture media without culture media with added exogenous glutamic acid and are high in succinic acid yield; the 19.53 g / L succinic acid can be produced after 27 g / L glucose which is used as a carbon source is fermented in an anaerobic serum bottle for 72 h, and the succinic acid yield can reach 72.33%.

Description

technical field [0001] The invention relates to a method for producing succinic acid by fermentation, which belongs to the technical field of industrial microbes and fermentation. Background technique [0002] Succinic acid, also known as succinic acid, is a common natural organic acid widely found in the human body, animals, plants and microorganisms. As an intermediate product of the TCA cycle and one of the terminal reduction products of anaerobic metabolism, succinic acid plays a very important role in the process of biological metabolism. Research results in recent years have shown that succinic acid can also be used as a C4 platform compound to synthesize bulk chemicals such as 1,4-butanediol, tetrahydrofuran, and γ-butyrolactone, as well as polybutylene succinate (PBS) biodegradable compounds. Degrades polyester. In recent years, with the increasing depletion of fossil resources and the increasingly serious environmental problems, the use of biological methods to pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/46C12N1/21C12N15/76C12R1/01
Inventor 姜岷陈美丽马江锋吴明科韦萍欧阳平凯
Owner 态创生物科技(广州)有限公司
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