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Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid

A technology of genetically engineered bacteria and Escherichia coli is applied in the field of constructing fumaric acid-producing Escherichia coli genetically engineered bacteria to achieve better and ideal results

Inactive Publication Date: 2012-08-01
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fumaric acid gene involved in the above research is either introduced from an external source, or is a related gene in the mixed acid pathway, and there is no report on promoting fumaric acid production by strengthening related genes in TCA

Method used

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  • Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid
  • Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid
  • Method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid

Examples

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Embodiment 1

[0032] This example illustrates the use of RED recombination technology to knock out the starting strain parent E. coli Methods of encoding the TCA cycle fumarase (FUM) gene in MG1655.

[0033] 1. Using LB medium, culture at 37°C under aerobic conditions E. coli MG1655 to OD600=0.6, prepared to be electroporation competent.

[0034] 2. Introduce plasmid pKD46 into competent Escherichia coli, cultivate overnight at 30°C, inoculate into LB medium (containing ampicillin, 100 μg / L) the next day, cultivate at 30°C until OD=0.25, add 10 mM L-arabinose, induced plasmid pKD46 to express three proteins EXo, Bet and Gam at 37°C, and prepared competent cells again E. coli MG1655 1 .

[0035] 3. Using pKD3 with FRT sites on both sides and chloramphenicol resistance as a template, use a high-fidelity PCR amplification system, and design fumA Amplification primers for homologous fragments, amplified to obtain linear DNA homologous fragments. Similarly, combining various subunits...

Embodiment 2

[0051] This example describes the E. coli HH1 is the starting strain, using RED recombinant technology to knock out the protein gene (arcA) that inhibits the TCA cycle, so as to realize the normal opening of the TCA cycle under anaerobic conditions.

[0052] 1. Using LB medium, culture at 37°C under aerobic conditions E. coli From HH1 to OD600=0.6, it was prepared as a competent state for electroconversion.

[0053] 2. Introduce plasmid pKD46 into competent Escherichia coli, cultivate overnight at 30°C, inoculate into LB medium (containing ampicillin, 100 μg / L) the next day, cultivate at 30°C until OD=0.25, add 10 mM L-arabinose, induced plasmid pKD46 to express three proteins EXo, Bet and Gam at 37°C, and prepared competent cells again E. coli HH1 1 .

[0054] 2. Using pKD3 with FRT sites on both sides and chloramphenicol resistance as a template, use a high-fidelity PCR amplification system, and design amplification primers with FRD homologous fragments at both ends...

Embodiment 3

[0062] This embodiment is an optimized implementation scheme on the basis of embodiment 2, illustrating the following E. coli HH2 is the starting strain, using RED recombinant technology to knock out a subunit of fumarate reductase FRD wxya , to achieve the destruction of FRD and block the conversion of fumaric acid to succinic acid.

[0063] 1. Using LB medium, culture at 37°C under aerobic conditions E. coli HH2 to OD600=0.6, prepared to be electroporation competent.

[0064] 2. Introduce plasmid pKD46 into competent Escherichia coli, cultivate overnight at 30°C, inoculate into LB medium (containing ampicillin, 100 μg / L) the next day, cultivate at 30°C until OD=0.25, add 10 mM L-arabinose, induced plasmid pKD46 to express three proteins EXo, Bet and Gam at 37°C, and prepared competent cells again E. coli HH2 1 .

[0065] 2. Using pKD3 with FRT sites on both sides and chloramphenicol resistance as a template, use a high-fidelity PCR amplification system, and design...

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Abstract

The invention belongs to the field of biochemical engineering, and particularly relates to a method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid. The method for constructing the escherichia coli genetic engineering bacteria mainly comprises the following steps of: inactivating or knocking fumarase serving as key enzyme for converting fumaric acid into malic acid in a tricarboxylic acid (TCA) cycle out, and knocking a key gene arcA for inhibiting the TCA cycle out. Further, key enzyme genes in paths of succinic acid, lactic acid and formic acid can also be knocked out, so the fumaric acid can be accumulated under the anaerobic condition.

Description

technical field [0001] The invention belongs to the field of biochemical industry, in particular to a method for constructing fumaric acid-producing Escherichia coli genetically engineered bacteria. Background technique [0002] Fumaric acid is an important four-carbon organic acid with an acidity 1.5 times that of citric acid. Because of the special molecular structure containing a double bond and two carboxyl groups, fumaric acid can be further processed through ammoniation, hydration, hydrogenation, iso L-aspartic acid, malic acid, succinic acid, polymers, etc. are produced by processes such as structure and polymerization, making it an important platform chemical raw material and widely used in resins, coatings, plasticizers, food, feed and other fields. At present, the fumaric acid sold in the market is mainly obtained through petrochemical methods. With the continuous consumption of petroleum resources, the continuous fluctuation of petroleum prices, and the environmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/46C12R1/19
Inventor 黄和徐晴李霜江凌
Owner NANJING UNIV OF TECH
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