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Construction method for producing succinic acid Escherichia coli gene engineering bacteria by means of xylose-metabolism

A genetically engineered bacteria and succinic acid-producing technology, applied in the field of bioengineering, can solve problems such as polluting the environment and wasting resources, and achieve the effect of promoting progress and development and increasing ATP supply.

Inactive Publication Date: 2012-08-01
NANJING TECH UNIV
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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.

Method used

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  • Construction method for producing succinic acid Escherichia coli gene engineering bacteria by means of xylose-metabolism
  • Construction method for producing succinic acid Escherichia coli gene engineering bacteria by means of xylose-metabolism
  • Construction method for producing succinic acid Escherichia coli gene engineering bacteria by means of xylose-metabolism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] This example illustrates the construction of an expression plasmid that overexpresses phosphoenolpyruvate carboxykinase, restores the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and obtains the strain Escherichia coli BA204.

[0062] 1. Lack of lactate dehydrogenase gene ( wxya ), pyruvate formate lyase gene ( PPML ) Active E. coli The NZN111 strain was the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out to obtain simultaneous deficiency wxya , PPML and PPC competent strains.

[0063] Knockout of the phosphoenolpyruvate carboxylase (PPC) gene using homologous recombination technology: using the apramycin resistance gene with FRT sites on both sides as a template, using a high-fidelity PCR amplification system, and designing Amplification primers with PPC homologous fragments at both ends successfully amplified linear DNA homologous fragments; a plasmid capable of inducing the expressi...

Embodiment 2

[0085] This example illustrates the construction of an expression plasmid co-expressing phosphoenolpyruvate carboxykinase and malic enzyme, restoring the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and obtaining the strain Escherichia coli BA205.

[0086] 1. Lack of lactate dehydrogenase gene ( wxya ), pyruvate formate lyase gene ( PPML ) Active E. coli The NZN111 strain was the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out to obtain simultaneous deficiency wxya , PPML And the competent bacterial strain of PPC (same as embodiment 1).

[0087] 2. Construction of an expression plasmid co-expressing phosphoenolpyruvate carboxykinase and malic enzyme, the process comprising:

[0088] (1) Both the upstream and downstream of the synthesis have Hin primers for the dIII restriction site,

[0089] Upstream primer: 5'- CCCAAGCTTATGAACTCAGTTGATTTGACCG -3';

[0090] Downstream primer: 5'-CCCA...

Embodiment 3

[0094] This example illustrates the construction of an expression plasmid co-expressing phosphoenolpyruvate carboxykinase and malate dehydrogenase, restoring the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and obtaining the strain Escherichia coli BA206.

[0095] 1. Lack of lactate dehydrogenase gene ( wxya ), pyruvate formate lyase gene ( PPML ) Active E. coli The NZN111 strain was the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out to obtain simultaneous deficiency wxya , PPML And the competent bacterial strain of PPC (same as embodiment 1).

[0096] 2. Construction of an expression plasmid co-expressing phosphoenolpyruvate carboxykinase and malate dehydrogenase, the process comprising:

[0097] (1) Both the upstream and downstream of the synthesis have Hin primers for the dIII restriction site,

[0098] Upstream primer: 5'- CCCAAGCTTATGAACTCAGTTGATTTGACCG -3';

[0099] Downstream...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a construction method for producing succinic acid Escherichia coli gene engineering bacteria by means of xylose-metabolism and a method for fermenting succinic acid. The ATP (adenosine triphosphate) biosynthesis pathway of Escherichia coli is modified by molecular biology means, over-expression of activity related to the pathway is realized, the total quantity of ATP in an Escherichia coli cell is effectively increased, recombined Escherichia coli can grow by means of xylose-metabolism, and the synthetic efficiency of the succinic acid is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for constructing a genetically engineered bacterium of Escherichia coli producing succinate by utilizing xylose metabolism. 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 US Department of Energy to be among the 12 most valuable biorefinery products in the future. [0003] The production methods of succinic acid mainly include chemical synthesis and microbial fermentation, using microbial fermentation to convert renewable resources (glucose, xylose, etc.). can absorb and fix CO 2 , can effectively alleviate the greenhouse effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12R1/19
CPCC12N15/52C12N15/70C12N9/93C12Y401/01031C12N9/88C12P7/46C12R1/19C12N15/63C12N9/0006C12N15/67
Inventor 姜岷刘嵘明梁丽亚马江锋陈可泉韦萍
Owner NANJING TECH UNIV
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