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Recombinant escherichia coli with high osmotic pressure resistance and application thereof

A technology for recombining Escherichia coli and Escherichia coli, applied in the field of genetic engineering of Escherichia coli, can solve the problems of cell growth and metabolism inhibition, affecting strain growth and metabolism, high osmotic pressure, etc.

Active Publication Date: 2015-10-14
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the glucose concentration is too high, the growth and metabolism of the strain will be affected due to the excessive osmotic pressure.
In addition, when organic acid fermentation is carried out at neutral pH, the organic acid salt of the fermentation product will cause high osmotic pressure, which will inhibit cell growth and metabolism

Method used

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  • Recombinant escherichia coli with high osmotic pressure resistance and application thereof
  • Recombinant escherichia coli with high osmotic pressure resistance and application thereof
  • Recombinant escherichia coli with high osmotic pressure resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1: Construction of strain Suc-T110 and HX-024

[0120] Using wild-type Escherichia coli ATCC8739 as the starting strain, the lactate dehydrogenase gene ldhA was knocked out, the pyruvate formate lyase encoding gene pflB was knocked out, the phosphoenolpyruvate-sugar phosphotransferase I gene ptsI was knocked out, and half The lactose MFS transporter GalP activates phosphoenolpyruvate carboxykinase PCK to obtain the strain Suc-T110 (for the specific construction process, please refer to Chinese patent application 201310198953.9, and Tan et al., Appl Environ Microbiol.2013,79:4838- 4844).

[0121] Starting from Suc-T110, continue to knock out the phosphoacetyltransferase gene pta and the acetate kinase gene ackA, activate the malate synthase AceA and isocitrate lyase AceB, and activate the dicarboxylic acid Dcu transporter DcuC to obtain the strain NZ-037.

[0122] Then NZ-037 was evolved for 1080 generations to obtain strain HX021.

[0123] Starting from r...

Embodiment 2

[0129] Embodiment 2: Construction of recombinant Escherichia coli NZ-502

[0130] (1) Construction of plasmid pXZ-CS for gene knockout, regulation of gene expression and integration of exogenous genes.

[0131] There are four steps in the plasmid construction procedure:

[0132] The first step, using the pACYC184 plasmid (Mok et al., 1991, Nucleic Acids Res19:2321-2323) DNA as a template, using primers 184-cat-up / 184-cat-down (SEQ ID No.: 16 / SEQ ID No.: 17), the amplified chloramphenicol resistance gene, the gene fragment size is 994bp, contains the chloramphenicol gene promoter sequence, called fragment I.

[0133] Amplification system: 10 μl of NewEngland Biolabs Phusion5X buffer, 1 μl of dNTP (10 mM for each dNTP), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), distilled water 33.5 μl for a total volume of 50 μl.

[0134] Amplification conditions were pre-denaturation at 98°C for 2 minutes (1 cycle); denat...

Embodiment 3

[0155] Embodiment 3: Construction of recombinant Escherichia coli NZ-504

[0156] Starting from the recombinant Escherichia coli Suc-T110, the cusS* mutant gene was integrated to replace the wild-type cusS gene according to the method of two-step homologous recombination in part (2) of Example 2 to obtain recombinant Escherichia coli NZ-504. The primer sequences used are shown in Table 3, where the names of the primers correspond to the names of the primers used in the process of rpoB*mutating the gene, and only rpoB is replaced with cusS.

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Abstract

The invention relates to the field of transforming escherichia coli by virtue of genetic engineering. Specifically, the invention provides recombinant escherichia coli containing mutated rpoB, cusS and / or mreC genes. The invention also relates to an application of the recombinant escherichia coli in production of chemical raw materials including butanedioic acid and the like. The invention also provides a method for producing the chemical raw materials including butanedioic acid and the like by using the recombinant escherichia coli, and a method for improving the osmotic pressure resistance of escherichia coli by introducing the mutated rpoB, cusS and / or mreC genes.

Description

field of invention [0001] The present invention relates to the field of transforming Escherichia coli by genetic engineering. Specifically, the present invention provides a recombinant Escherichia coli containing mutated rpoB, cusS and / or mreC genes. The present invention also relates to the use of the Escherichia coli for producing chemical raw materials such as succinic acid and the like. The invention also provides a method for producing chemical raw materials such as succinic acid by using the Escherichia coli, and a method for improving the osmotic pressure resistance of the Escherichia coli by introducing mutated rpoB, cusS and / or mreC genes. Background of the invention [0002] The physiological performance of microbial cells is one of the core issues in the fermentation of microbial engineering bacteria. Good microbial engineering bacteria need to have the following physiological characteristics: tolerance to high concentration of glucose, tolerance to high concent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/46C12R1/19
Inventor 张学礼朱欣娜肖孟雍陈晶马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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