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Gene engineering bacterium for producing succinic acid, and method for producing succinic acid by fermentation by using same

A genetically engineered bacteria and a succinic acid-producing technology, which are applied in the field of succinic acid-producing genetically engineered bacteria and their fermentation to produce succinic acid, can solve the problems of wasting resources, pollute the environment and the like, and achieve the effect of improving biosynthesis capacity

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 20g / L NaOH at 121°C for 1 hour. The total mass concentration of glucose and xylose is about 50g / L
[0007] 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 to obtain a total sugar mass of 50g / L , of which xylose accounts for more than 80%

Method used

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  • Gene engineering bacterium for producing succinic acid, and method for producing succinic acid by fermentation by using same
  • Gene engineering bacterium for producing succinic acid, and method for producing succinic acid by fermentation by using same
  • Gene engineering bacterium for producing succinic acid, and method for producing succinic acid by fermentation by using same

Examples

Experimental program
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Effect test

Embodiment 1

[0046] This example illustrates the process of using the homologous recombination technology to knock out the phosphoenolpyruvate carboxylase ppc gene in the starting strain NZN111 to obtain the apramycin-resistant strain.

[0047] 1. Use LB medium to cultivate Escherichia coli NZN111 to OD at 37°C under aerobic conditions 600 =0.4~0.6, prepared into electrotransformation competent.

[0048] 2. The plasmid pKD46 was electroporated into competent E. coli NZN111. The electric shock conditions are: 200Ω, 25μF, electric shock voltage 2.3kV, electric shock time 4-5ms. Immediately after electric shock, the cells were added to pre-cooled 1 mL SOC medium, cultured at 150 r / min at 30°C for 1 h, and then spread on LB medium plate with ampicillin (amp) to select positive transformants Escherichia coli NZN111 (pKD46).

[0049] 3. Add 10 mM L-arabinose to the LB medium, induce the plasmid pKD46 to express λ recombinase at 30°C, and make the electrotransformation competent.

[0050] 4. U...

Embodiment 2

[0063] This example illustrates the process of using the ppc gene-knocked out strain obtained in Example 1, and again using homologous recombination technology to knock out the ptsG gene in the PTS transport system to obtain an apramycin-resistant strain.

[0064] 1. Use LB medium to cultivate the ppc gene knockout strain obtained in Case 1 at 37°C under aerobic conditions to OD 600 =0.4~0.6, prepared into electrotransformation competent.

[0065] 2. The plasmid pKD46 was electroporated into the competent cells. The electric shock conditions are: 200Ω, 25μF, electric shock voltage 2.3kV, electric shock time 4-5ms. Immediately after the electric shock, the bacterial cells were added to the pre-cooled SOC medium of 1 mL, cultured at 150 r / min at 30 °C for 1 h, and then spread on the LB medium plate with ampicillin (amp) to select the positive transformants Escherichia coli NZN111 / △ppc ( pKD46).

[0066] 3. Add 10 mM L-arabinose to the LB medium, induce the plasmid pKD46 to ex...

Embodiment 3

[0080] This example illustrates the construction of an expression plasmid overexpressing phosphoenolpyruvate carboxykinase, which can efficiently utilize monosaccharides such as glucose, xylose, arabinose and fructose, and efficiently utilize various proportions of mixed sugars and cellulose hydrolyzate fermentation , and a large amount of succinic acid is accumulated to obtain the method of strain Escherichia coli BA305.

[0081] 1. Construction of an expression plasmid for overexpressing phosphoenolpyruvate carboxykinase, the process of which includes:

[0082] (1) Synthesize primers with SacI and XbaI restriction sites,

[0083] Upstream primer: 5'-CGAGCTCATGAACTCAGTTGATTTGACCG-3';

[0084] Downstream primer: 5'-GCTCTAGAGCATTCCGTCAATTAAAACAAG-3'.

[0085] (2) Using the genome of Bacillus subtilis as the template, PCR amplifies the target gene fragment. The reaction conditions are: 94°C, 5min; (94°C, 45s, 53°C, 45s, 72°C, 100s, 35 cycles); 72°C, 10min. After purifying the...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a gene engineering bacterium strain for producing succinic acid, and a method for producing succinic acid by fermentation by using the same. The classification designation of the gene engineering bacterium strain for producing succinic acid is Escherichia coli BA305, and the preservation number is CCTCC NO:M2012102. The construction process mainly comprises the following steps: inactivating or knocking out phosphoenolpyruvic acid carboxylase gene and ptsG gene in a phosphate translocation system; and overexpressing the phosphoenolpyruvic acid carboxylase so that the recombinant colibacillus can efficiently utilize glucose, xylose, arabinose, levulose and other monosaccharides and efficiently utilize mixed saccharides in various proportions and cellulose hydrolysate, thereby greatly enhancing the synthesis efficiency of the succinic acid. The fermentation method adopts a two-stage fermentation mode: the aerobic stage can enhance the biomass, and the anaerobic stage is used for fermentation to produce the acid.

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 monosaccharides such as glucose, xylose, arabinose, and fructose. The invention discloses a recombinant strain that grows and produces succinic acid by mixing sugar and cellulose hydrolyzate in various proportions and a method for fermenting and producing succinic acid by using the strain. Background technique [0002] Succinic acid, also known as succinic acid, is widely used in pharmaceuticals, pesticides, dyes, fragrances, paints, food and plastics and other industries. As a C4 platform compound, it can be used to synthesize 1,4-butanediol, tetrahydrofuran, Organic chemicals such as gamma-butyrolactone and biodegradabl...

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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