Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof

A genetically engineered bacteria, a technology for producing succinic acid, applied in the field of bioengineering to achieve the effect of enhancing the range of adaptation

Inactive Publication Date: 2012-07-04
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The strain can efficiently use xylose to ferment succin

Method used

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  • Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof
  • Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof
  • Genetic engineering strain for producing succinic acid by utilizing glucose and acidogenic fermentation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example illustrates the use of homologous recombination technology to knock out phosphoenolpyruvate carboxylase in the starting strain NZN111 ppc gene, resulting in the elimination of apramycin-resistant strains.

[0039] 1. Using LB medium, cultivate Escherichia coli NZN111 to OD at 37°C under aerobic conditions 600 =0.4~0.6, prepared to be electrotransfer competent.

[0040] 2. Electrotransform plasmid pKD46 into competent Escherichia coli NZN111. The electric shock conditions were: 200 Ω, 25 μF, electric shock voltage 2.3 kV, electric shock time 4-5 ms. Immediately after the electric shock, the cells were added to pre-cooled 1 mL SOC medium, cultured at 150 r / min, 30°C for 1 h, and then spread on the LB medium plate with ampicillin (amp) to screen the positive transformant Escherichia coli NZN111 ( pKD46).

[0041] 3. Add 10 mM L-arabinose to LB medium, induce plasmid pKD46 to express λ recombinase at 30°C, and make electroporation competent.

[0042] 4. U...

Embodiment 2

[0053] This example illustrates the construction of an expression plasmid that overexpresses phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase, restores the ability of the recombinant strain to metabolize glucose under anaerobic conditions, and obtains a strain Escherichia coli Methods of BA205.

[0054] 1. Construct pTrc99a- pck Plasmids, the process of which involves:

[0055] (1) synthetic with Sac I and Xba Primers for I restriction sites,

[0056] Upstream primer: 5'-CGAGCTCATGAACTCAGTTGATTTGACCG-3'.

[0057] Downstream primer: 5'-GCTCTAGAGCATTCCGTCAATTAAAACAAG -3'.

[0058] (2) with Bacillus subtilis Genomic DNA was used as a template, and the target gene fragment was amplified by PCR. The reaction conditions were: 94°C for 5 min; (94°C for 45 s, 53°C for 45 s, 72°C for 100 s, 35 cycles); 72°C for 10 min. purified amplified pck After the gene, the expression plasmid was used respectively with pTrc99a Sac I and Xba I double...

Embodiment 3

[0066] This example illustrates the co-expression of a newly constructed recombinant E. coli strain Escherichia coli BA205 and the total amount of NAD (H) and NADH / NAD of the apramycin-resistant strain obtained in BA205 and Example 1 + The comparison of the ratio, and the comparison of the sugar consumption and acid production capacity during the fermentation process of the two.

[0067]When the plasmid pTrc99a- pck - pncB Finally, eliminating the overexpression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase in the apramycin-resistant strain restored the redox balance of the recombinant bacteria under anaerobic conditions, and the total amount of NAD(H) was Significantly improved, and also restored the ability to metabolize glucose under anaerobic conditions, while the main product is succinic acid, without the accumulation of formic acid and lactic acid.

[0068] Escherichia coli BA205 was inoculated into the aerobic stage fermentatio...

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Abstract

The invention belongs to the field of biology engineering technology, and relates to a genetic engineering strain for producing succinic acid by utilizing glucose and an acidogenic fermentation method of the genetic engineering strain. The genetic engineering strain for producing succinic acid by utilizing glucose is named as Escherichia coli BA205 and the preservation number is registered as CCTCC No.M2011447. In the construction process, Escherichia coli which is short of lactic dehydrogenase (LDH) gene and Pyruvate formate-lyase (PFL) gene activity is mainly used as an original strain; phosphoenolpyruvate carboxylase (PPC) gene is removed by utilizing a homologous recombination technology; and phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyl transferase are excessively co-expressed; therefore the synthesis efficiency of succinic acid is greatly increased. In the fermentation method, a two-stage fermentation manner is adopted, the biomass is improved in an aerobic stage and the acidogenic fermentation is carried out in an anaerobic stage.

Description

Technical field [0001] The present invention belongs to the field of biological engineering technology. It involves the use of glucose -produced dicedic acid strains and its fermented acidic acid method. Specifically, it is a efficient use of glucose to grow and produce dicenic acid reorganization strains and its use of the strain to produce Ding two.Sour. Background technique [0002] SUCCINIC Acid, also known as amber acid, is widely used in industries such as medicine, pesticides, dyes, spices, paint, food and plastic.Organic chemicals such as γ-butyl, as well as polycoticotoxol (PBS) biodegradable materials, are considered by the US Department of Energy to be one of the 12 most valuable biological products in the future. [0003] The production method of ditic acid mainly includes chemical synthesis and microbial fermentation methods. The use of microbial fermentation method to transform renewable resources (glucose, xylose, etc.). Due to the extensive sources of raw material...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/09C12P7/46C12R1/19
CPCC12Y101/01027C12N9/1077C12Y204/02011C12N9/0006C12Y401/01049C12N9/1029C12R1/19C12Y401/01031C12N9/88C12N15/09C12P7/46C12Y203/01054C12N1/205C12R2001/19
Inventor 姜岷梁丽亚刘嵘明苟冬梅张常青马江锋陈可泉韦萍欧阳平凯
Owner NANJING UNIV OF TECH
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