Construction and application of an engineered strain of Escherichia coli producing n-butyric acid

A genetically engineered bacteria and genetic engineering technology, applied in the direction of bacteria, microorganism-based methods, lytic enzymes, etc., to achieve the effects of low production cost, short fermentation cycle, and easy control

Active Publication Date: 2021-05-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Construction and application of an engineered strain of Escherichia coli producing n-butyric acid
  • Construction and application of an engineered strain of Escherichia coli producing n-butyric acid
  • Construction and application of an engineered strain of Escherichia coli producing n-butyric acid

Examples

Experimental program
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Effect test

Embodiment 1E

[0056] The knockout of embodiment 1E.coli ATCC8739 acetate kinase gene ackA

[0057] According to the 45 bp sequence of the 5' and 3' ends of the ackA (acetate kinase gene) gene in Escherichia coli ATCC8739 in the NCBI database, the upper and lower homology arm amplification primers ackA-1 and ackA-2 were designed and amplified using pKD4 as a template A knockout frame containing a homology arm was produced and named ackAK.

[0058] The ackAK was electrotransformed into E.coli ATCC8739 competent cells containing the pKD46 plasmid (electric transformation voltage and time were 2500V and 5mS, respectively). Quickly recover in 1 mL LB medium at 37°C and 150 rpm for 1 hour, and spread on LB solid medium plate containing kanamycin (30 g / mL). After 24 hours of inverted culture, positive transformants were identified by colony PCR using the identification primers ackA-U and ackA-D. The amplified fragment of the colony with the kanamycin resistance gene knockout frame successfully in...

Embodiment 2E

[0064] Example 2 Knockout of E.coli ATCC8739A Alcohol Dehydrogenase Gene adhE

[0065] According to the 45 bp sequence of the 5' and 3' ends of the adhE (alcohol dehydrogenase gene) gene in Escherichia coli ATCC8739 in the NCBI database, the upper and lower homology arm amplification primers adhE-1 and adhE-2 were designed, and pKD4 was used as a template A knockout box containing homology arms was amplified and named adhEK.

[0066] The adhEK was electrotransformed into E.coli ATCC8739A competent cells containing the pKD46 plasmid (electric transformation voltage and time were 2500V and 5ms, respectively). Quickly recover in 1 mL LB medium at 37°C and 150 rpm for 1 hour, and spread on LB solid medium plate containing kanamycin (30 g / mL). After 24 hours of inverted culture, positive transformants were identified by colony PCR using the identification primers adhE-U and adhE-D, and the amplified fragment of the colony where the kanamycin resistance gene knockout frame was succ...

Embodiment 3E

[0069] Example 3 Knockout of E.coli ATCC8739AA lactate dehydrogenase gene ldhA

[0070] According to the 45 bp sequence at the 5' and 3' ends of the ldhA (lactate dehydrogenase gene) gene in Escherichia coli ATCC8739 in the NCBI database, the upper and lower homology arm amplification primers ldhA-1 and ldhA-2 were designed, and pKD4 was used as a template A knockout cassette containing homology arms was amplified and named ldhAK.

[0071] The adhEK was electrotransformed into E.coli ATCC 8739AA competent cells containing the pKD46 plasmid (the electroporation voltage and time were 2500V and 5ms, respectively). Quickly recover in 1 mL LB medium at 37°C and 150 rpm for 1 hour, and spread on LB solid medium plate containing kanamycin (30 g / mL). After upside-down culture for 24 hours, positive transformants were identified by colony PCR using identification primers ldhA-U and ldhA-D. The amplified fragment of the colony with the kanamycin resistance gene knockout frame successfu...

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Abstract

The invention discloses the construction and application of an Escherichia coli engineering bacterium producing n-butyric acid, and belongs to the technical field of fermentation engineering. The present invention takes Escherichia coli ATCC 8739 as the starting strain, utilizes metabolic engineering means, constructs butyric acid-producing Escherichia coli genetically engineered bacteria by constructing a butyric acid synthesis path and cutting off redundant metabolic branches. After 72 hours of fermentation, the production of butyric acid reached 15g/L. The fermentation process is an aerobic fermentation, the bacteria grow fast, the fermentation period is short, and the acid production rate is high. At present, there is no report of directly using glycerol to ferment and produce butyric acid. The method adopts a simple fermentation process, is easy to control, has low production cost, and is beneficial to popularization and application of industrial production.

Description

technical field [0001] The invention relates to the construction and application of a butyric acid-producing Escherichia coli engineering bacterium, and belongs to the technical field of fermentation engineering. Background technique [0002] Butyric acid is a short-chain fatty acid, which is an important raw material for the synthesis of other fragrance substances and fine chemical products. Butyric acid and its derivatives are widely used in industries such as chemical industry, food, medicine, animal feed and cosmetics. Butyric acid derivatives, such as butyrate, are used in beverages, food and cosmetics as aroma and flavor enhancers. Polymers prepared from butyric acid and cellulose acetate are used to make plastics and textile fibers. Derivatives of butyrate, as highly active compounds, have been shown to have anticancer effects. Furthermore, the commercial value of butyric acid is highly regarded as a precursor for biofuels such as n-butanol. [0003] At present, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/52C12R1/19
CPCC12N9/0006C12N9/001C12N9/1029C12N9/16C12N9/88C12P7/52C12Y101/01157C12Y402/01055
Inventor 陈修来郭亮刘立明刁文文刘佳罗秋玲
Owner JIANGNAN UNIV
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