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Succinic acid genetic engineering bacterium and method for fermenting and producing succinic acid

A genetically engineered bacterium producing succinic acid technology, applied in the field of a succinic acid-producing genetically engineered bacterium and its fermentation to produce succinic acid, can solve the problems of wasting resources, polluting the environment, etc., and achieve improved production capacity and increased output And the effect of huge changes in production capacity and acid production characteristics

Inactive Publication Date: 2012-08-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, except for the use in the paper industry, most of them are discarded, which seriously wastes resources and pollutes the environment.
Its main components are cellulose, hemicellulose and lignin, so its hydrolyzate is a good sustainable green carbon source for microbial fermentation, but its hydrolyzate contains high concentration of xylose, so it is now In the prior art, most succinate-producing Escherichia coli cannot use rice straw hydrolyzate to ferment and produce succinic acid. Tao Wenyi et al. treated rice straw with dilute sulfuric acid at 121°C for 1 hour, and then treated the straw with 20 g / L NaOH at 121°C 1h, the total mass concentration of glucose and xylose reaches about 50 g / L
[0009] Bagasse is the main component left after sugar cane sugar is squeezed, so its hydrolyzate is a good sustainable green carbon source for microbial fermentation, but its hydrolyzate contains high concentration of xylose, Therefore, most succinate-producing Escherichia coli in the prior art cannot use rice straw hydrolyzate to ferment and produce succinic acid, and the cellulose containing about 50% can be pretreated by crushing and alkali / oxidation method to obtain a total sugar mass of 50 g / L, of which xylose accounts for more than 80%

Method used

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  • Succinic acid genetic engineering bacterium and method for fermenting and producing succinic acid
  • Succinic acid genetic engineering bacterium and method for fermenting and producing succinic acid
  • Succinic acid genetic engineering bacterium and method for fermenting and producing succinic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0050] This example illustrates the process of knocking out the phosphoenolpyruvate carboxylase ppc gene in the starting strain NZN111 by using homologous recombination technology to obtain a strain that eliminates apramycin resistance.

[0051] 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.

[0052] 2. The plasmid pKD46 was electrotransformed 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 out the positive transformant Escherichia coli NZN1 11 (pKD46).

[0053] 3. Add 10 mM L-arabinose to LB medium, induce plasmid pKD46 to express λ recombinase at 30°C, and make electroporation c...

Embodiment 2

[0067] This example illustrates the process of using the ppc gene-knockout strain obtained in Example 1 to knock out the ptsG gene in the PTS transport system by using homologous recombination technology again to obtain an apramycin-resistant strain.

[0068] 1. Using LB medium, cultivate the ppc gene-knockout strain obtained in Case 1 at 37°C under aerobic conditions to OD 600 =0.4~0.6, prepared to be electrotransfer competent.

[0069] 2. Electrotransfer the plasmid pKD46 into the competent cells. 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 out the positive transformant Escherichia coli NZN1 11 / Δppc(pKD46).

[0070] 3. Add 10 mM L-arabinose to LB medium, induce plasmid pKD46 to express λ recombinase at 30°C, and ...

Embodiment 3

[0084] This example illustrates the construction of an expression plasmid for overexpressing phosphoenolpyruvate carboxykinase.

[0085] 1. Construction of an expression plasmid for overexpressing phosphoenolpyruvate carboxykinase, the process comprising:

[0086] (1) Synthesize primers with SacI and XbaI restriction sites,

[0087] Upstream primer: 5'-CGAGCTCATGAACTCAGTTGATTTGACCG-3';

[0088] Downstream primer: 5'-GCTCTAGAGCATTCCGTCAATTAAAACAAG-3'.

[0089] (2) Use the Bacillus subtilis genome as a template to amplify the target gene fragment by PCR. The reaction conditions are: 94°C, 5 min; (94°C for 45 s, 53°C for 45 s, 72°C for 100 s, 35 cycles); 72°C , 10 min. After purifying the amplified pck gene, the expression plasmid was digested with pTrc99a with SacI and XbaI respectively, and ligated to obtain the recombinant plasmid pTrc99a-pck. Double restriction electrophoresis identification of plasmid pTrc99a-pck as Image 6 shown.

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Abstract

The invention belongs to the technical field of bioengineering, and relates to a succinic acid genetic engineering bacterium and a method for fermenting and producing succinic acid. Bacterial strains of the succinic acid genetic engineering bacterium are classified and named as Escherichia coli (Escherichia coli) BA306 with the preservation number of CCTCC NO:M2012103. A construction process mainly comprises inactivating or knocking phosphoric acid enol pyruvic carboxylase genes and ptsG genes in a phosphoric acid movement system, conducting excessive coexpression on phosphoric acid enol pyruvic carboxylase and niacin ribose phosphate transferring enzyme, enabling recombination of colon bacillus to be capable of efficiently using monosaccharide of glucose, xylose, arabinose, fructose and the like, simultaneously efficiently using mixed sugar and cellulose hydrolysate in various proportions for growth, and substantially improving combined efficiency of the succinic acid. A fermentation process adopts a two-stage fermentation mode, biomass liveweight is improved in an aerobic stage, and fermentation and acid production are carried out in an anaerobic stage.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a succinic acid-producing genetically engineered bacterium and a method for fermenting and producing succinic acid, in particular to a strain that efficiently utilizes monosaccharides such as glucose, xylose, arabinose and fructose, and efficiently utilizes A recombinant bacterial strain that grows by mixing sugar and cellulose hydrolyzate in various proportions to produce succinic acid and a method for fermenting and producing succinic acid using the bacterial strain. Background technique [0002] Succinic acid, also known as succinic acid, is widely used in industries such as medicine, pesticides, dyes, spices, paints, food and plastics. As a C4 platform compound, it can be used to synthesize 1,4-butanediol, tetrahydrofuran, Organic chemicals such as γ-butyrolactone and biodegradable materials such as polybutylene succinate (PBS) are considered by the U.S. Department of En...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/46C12R1/19
Inventor 姜岷刘嵘明梁丽亚吴明科曹伟佳马江锋陈可泉韦萍
Owner NANJING UNIV OF TECH
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