Shewanella piezotolerans genetic engineering bacteria for producing eicosapentaenoic acid (EPA) in high yield

A technology of eicosapentaenoic acid and genetically engineered bacteria, which can be used in genetic engineering, plant genetic improvement, bacteria, etc., and can solve problems such as ignorance of EPA regulation

Inactive Publication Date: 2011-01-12
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Claims
  • Application Information

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Problems solved by technology

[0004] Many deep-sea bacteria have been found to synthesize E

Method used

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  • Shewanella piezotolerans genetic engineering bacteria for producing eicosapentaenoic acid (EPA) in high yield
  • Shewanella piezotolerans genetic engineering bacteria for producing eicosapentaenoic acid (EPA) in high yield
  • Shewanella piezotolerans genetic engineering bacteria for producing eicosapentaenoic acid (EPA) in high yield

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Experimental program
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Embodiment 1

[0017] Embodiment 1: Deletion of the Fis gene (ORF4) in the gene cluster and obtaining genetically engineered bacteria

[0018] First, the DNA containing the EPA gene was extracted from the wild Shewanella piezotolerans WP3 strain, and then the DNA fragment (SEQ ID NO 1) with the deletion of the target gene was obtained by two rounds of PCR amplification, which was cloned into the suicide vector pRE112 to obtain the Fis deletion mutant vector pRE112-Fis. The pRE112-Fis was introduced into the diaminopimelic acid (DAP) auxotrophic Escherichia coli WM3064 by chemical transformation method, and then the plasmid pRE112-Fis was introduced from the donor strain WM3064 into WP3 by the method of combined transfer, and then passed two rounds Orfswp3548-deleted genetically engineered strain WP3 was obtained by DNA homologous recombination screening ΔFis .

[0019] details as follows:

[0020] 1. Materials:

[0021] 1. Suicide vector: pRE112, see its structure figure 1 , figure 1 m...

Embodiment 3

[0042] The method that embodiment 3WP3 cultivates and EPA detects:

[0043] 1. Bacterial culture under different conditions:

[0044] 2216E Medium (1000ml)

[0045] Yeast extract 1g

[0046] Tryptone 2g

[0047] NaCl 34g

[0048] Fe 3 PO 4 0.1g

[0049] (1) Cultivation of WP3 under different temperature growth conditions: after overnight culture in normal 2216E medium (salinity 3.4%), the bacterial liquid was diluted 50 times into fresh medium, and placed in low temperature (4°C) and Cultivate at room temperature (20°C).

[0050] (2) Cultivation of WP3 under high-pressure conditions: carried out with a simple pressure simulation system. After the bacteria liquid cultured overnight was grown to the mid-logarithmic growth phase in normal medium (salinity 3.4%), the bacteria liquid was diluted 100 times and then filled into a Pasteur tube to drive away the air bubbles and seal it, put it into a pressure valve, adjust to 20MP for 2 days. Another part of the...

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Abstract

The invention provides shewanella piezotolerans WP3 genetic engineering bacteria for producing eicosapentaenoic acid (EPA) in high yield. When the strain is compared with wild WPS, the Fis type transcription factor swp3548 which is closely positioned on the upstream of a pfa gene for EPA synthesis is deleted, so the transcription of genes required by EPA synthesis is increased by 3 to 12 times respectively, and the yield of EPA under each condition is improved by about 50 to 60 percent.

Description

technical field [0001] The invention relates to a Shewanella genetically engineered bacterium, in particular to a Shewanella piezotolerans WP3 genetically engineered bacterium with high yield of eicosapentaenoic acid (EPA). Background technique [0002] The fatty acid system plays a pivotal role in the adaptation of microorganisms to the extreme living environment of the deep sea. The two biggest characteristics of the deep-sea environment are low temperature and high pressure. It has been found that low temperature and high pressure can reduce the fluidity of the cell membrane and make the cell membrane appear in an orderly crystal state, so that the cell membrane cannot perform normal physiological functions and eventually hinder the normal growth of cells. . In order to cope with the reduction of cell membrane fluidity, the most common strategy used by organisms is to increase the cell membrane fluidity by adjusting the composition and content of fatty acids in the cell ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/55C12N15/54C12N15/52C12N15/31C12N15/74C12P7/64C12R1/01
Inventor 王风平肖湘姜丽晶王峰彭华煜
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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