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Recombinant escherichia coli for overexpression of GatA gene and application thereof

An Escherichia coli and gene technology, applied in the field of microbial engineering, can solve the problems of easy degradation of bacteria, accumulation of by-products, osmotic stress, etc., and achieve the effects of improved acid stress resistance and simple operation.

Active Publication Date: 2021-02-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the addition of alkaline substances often leads to the accumulation of by-products, and the salts formed in the by-products will once again lead to a hypertonic environment for cells, resulting in osmotic stress, which will affect the growth and metabolism of bacteria again
[0008] At present, the methods for improving the acid stress resistance of Escherichia coli mainly include: (1) mutation breeding. Strains are easy to degrade; (2) Metabolic engineering strategy, the current method of using metabolic engineering strategy to improve the environmental stress of E. coli mainly includes constructing new metabolic pathways, expanding existing metabolic pathways and weakening existing metabolic pathways. However, this method has a cost Problems with high and low success rates

Method used

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  • Recombinant escherichia coli for overexpression of GatA gene and application thereof
  • Recombinant escherichia coli for overexpression of GatA gene and application thereof
  • Recombinant escherichia coli for overexpression of GatA gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of recombinant strain E.coli K12 MG1655 / pTrc99a-GatA

[0042] Specific steps are as follows:

[0043] (1) Based on the gatA gene sequence in the NCBI database (the gene encoding the unique IIA component GatA of galactitol, which participates in the galactose metabolism pathway and regulates the metabolism of galactitol), the designs are shown as SEQ ID NO.2 and SEQ ID NO. Primers pTrc99a / GatA-F, pTrc99a / GatA-R shown in 3;

[0044] (2) designing the primer loop p-pTrc99a-F and the loop p-pTrc99a-R respectively shown in SEQ ID NO.4 and SEQ ID NO.5;

[0045] (3) Using the genome of E.coli K12 MG1655 as a template, using pTrc99a / GatA-F, pTrc99a / GatA-R as primers to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification;

[0046] (4) Using the vector pTrc99a as a template, using loop p-pTrc99a-F and loop p-pTrc99a-R as primers to obtain a linearized long fragment of the vector by PCR amplification;

[0047] (5) Ligate the PCR products o...

Embodiment 2

[0049] Embodiment 2: the growth situation of recombinant bacterial strain and control bacterial strain under normal conditions

[0050] (1) The recombinant strain E.coli K12 MG1655 / pTrc99a-GatA obtained in Example 1 and the control strain E.coli K12 MG1655 / pTrc99a were respectively inoculated in LB liquid medium for activation, and placed in a shaker at 37°C at 220rpm Culture overnight;

[0051] (2) Transfer the seed solution obtained in the above step (1) to LB liquid medium with an inoculation amount of 2% (v / v), and place it in a shaker at 37°C at 220rpm for cultivation; sampling every 2 hours , measure the OD value under the 600nm wavelength, and draw the growth curve (the growth curve obtained by drawing is as follows: figure 1 ).

[0052] The result is as figure 1 As shown, through the growth performance test analysis, after culturing for 10 hours, the growth of the recombinant strain E.coli K12 MG1655 / PTrc99a-GatA was not significantly different from that of the cont...

Embodiment 3

[0053] Example 3: The specific steps of the itaconic acid stress (pH 4.2) tolerance test of the recombinant strain E.coli K12 MG1655 / pTrc99a-GatA are as follows:

[0054] (1) The control strain E.coli K12 MG1655 / pTrc99a and the recombinant strain E.coliK12 MG1655 / pTrc99a-GatA obtained in Example 1 were respectively inoculated in LB liquid medium for activation, and cultured in a shaker at 37°C at 220rpm 12h, obtain seed liquor;

[0055] (2) Transfer the seed solution obtained in the above (1) to fresh LB liquid medium with an inoculum amount of 2% (v / v), culture at 220rpm in a shaker at 37°C for 4.5h, and cultivate to logarithm In the mid-growth period, the OD600 at this time is 1.4-1.5, and the culture medium is obtained;

[0056] (3) Centrifuge the culture solution obtained in step (2) for 5 min at 6000 rpm, collect the thalline, wash the thalline obtained twice with 0.85% PBS buffer solution, and resuspend in an equal volume of fresh clothing In conic acid LB liquid mediu...

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Abstract

The invention discloses recombinant escherichia coli for overexpression of a GatA gene and application thereof, and belongs to the technical field of genetic engineering and microbial engineering. According to the recombinant escherichia coli, the gatA gene of the specific IIA component of galactitol is used as a target gene, and the escherichia coli is used as an expression host, so that escherichia coli engineering bacteria which can be widely applied to preparation of medicines, feeds and chemicals are successfully constructed. The itaconic acid stress resistance of the escherichia coli engineering bacteria is remarkably improved and is increased by 29.4 times to the maximum compared with that of a wild strain, and the Dlactic acid stress resistance of the escherichia coli engineering bacteria is remarkably improved and is increased by 101 times to the maximum compared with that of the wild strain. The succinic acid stress resistance of the escherichia coli engineering bacteria is remarkably improved and is improved by 1.6 times to the maximum compared with that of the wild strain.

Description

technical field [0001] The invention relates to a recombinant escherichia coli overexpressing GatA gene and application thereof, belonging to the technical field of microbial engineering. Background technique [0002] Escherichia coli is an important host bacteria in prokaryotes. These bacteria are widely distributed in nature and have rich species diversity. They are not only ideal materials for studying chemistry, genetics, molecular biology and genetic engineering, and have important academic value in theory, but also have application value in important fields closely related to human life, such as industry, agriculture, animal husbandry, food and medicine. extremely high. At present, a variety of high-value organic acid bio-fermentation methods have been successfully applied, and some of them have tried to use Escherichia coli as the host to express, but there is often the problem of acid stress. [0003] The scientific name of itaconic acid is methylene succinic acid...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/31C12P7/56C12P7/54C12P7/40C12R1/19
CPCC07K14/245C12N15/70C12P7/56C12P7/54C12P7/40
Inventor 张娟杨谨华堵国成陈坚
Owner JIANGNAN UNIV
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