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Escherichia coli engineering bacteria with improved acid stress capability and application thereof

A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of microbial engineering, can solve the problems of easy degradation of strains, accumulation of by-products, osmotic stress, etc., and achieve the effect of improving acid stress resistance and simple operation

Active Publication Date: 2021-02-12
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
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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  • Escherichia coli engineering bacteria with improved acid stress capability and application thereof
  • Escherichia coli engineering bacteria with improved acid stress capability and application thereof
  • Escherichia coli engineering bacteria with improved acid stress capability and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040]Example 1: Construction of recombinant strain E.coli K12 MG1655 / pTrc99a-NikA

[0041]Specific steps are as follows:

[0042](1) Based on the nikA gene sequence in the NCBI database (NikA gene encoding nickel ion ABC transporter periplasmic binding protein, involved in the ABC transport metabolic pathway of nickel ions, regulating the binding of substrate proteins in the nickel transport system) design respectively as SEQ The primers shown in ID NO. 2 and SEQ ID NO. 3 are pTrc99a / NikA-F, pTrc99a / NikA-R;

[0043](2) Design the primer loop p-pTrc99a-F and loop p-pTrc99a-R as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively;

[0044](3) Using the genome of E.coli K12 MG1655 as a template, and p-pTrc99a / NikA-F, p-pTrc99a / NikA-R as primers, the gene fragment shown in SEQ ID NO.1 is obtained by PCR amplification, and PCR is obtained product;

[0045](4) Using the vector pTrc99a as a template, using the loop p-pTrc99a-F and loop p-pTrc99a-R as primers to obtain a long linearized fragment of the vec...

Embodiment 2

[0050]Example 2: Growth of recombinant strains and control strains under normal conditions

[0051]Specific steps are as follows:

[0052](1) The strains E.coli K12 MG1655 / pTrc99a-NikA, E.coli K12MG1655 / pTrc99a-NikB, E.coli K12 MG1655 / pTrc99a-NikC and the control strain E.coli K12MG1655 / pTrc99a obtained in Example 1 were respectively inoculated Activate it in LB liquid medium and place it in a 37°C shaker at 220 rpm for 12 hours to obtain seed liquid;

[0053](2) Transfer the seed solution obtained in the above step (1) to LB liquid medium with an inoculum of 2% (v / v), and place it in a 37°C shaker at 220 rpm for incubation; take samples every 2 hours , Measure the OD value at the wavelength of 600nm, and draw the growth curve (the resulting growth curve is asfigure 1 ).

[0054]The result isfigure 1 As shown, after growth performance test analysis, after 12 hours of culture, the growth of the recombinant strain overexpressing the gene nikA lags slightly behind the growth cycle of the control s...

Embodiment 3

[0056]Example 3: Tolerance test of recombinant strain E.coli K12 MG1655 / pTrc99a-NikA under itaconic acid stress (pH 4.2)

[0057]Specific steps are as follows:

[0058](1) The control strain E.coli K12 MG1655 / pTrc99a and the recombinant strain E.coli K12 MG1655 / pTrc99a-NikA obtained in Example 1 were respectively inoculated into LB liquid medium for activation, and cultured in a shaker at 37°C at 220 rpm 12h, get the seed liquid;

[0059](2) Transfer the seed solution obtained in (1) above to fresh LB liquid medium at an inoculum of 2% (v / v), and incubate in a shaker at 37°C at 220 rpm for 4.5 hours, and cultivate to logarithm In the middle of growth, when the OD600 is 1.4-1.5, the culture solution is obtained;

[0060](3) Centrifuge the culture solution obtained in step (2) for 5 minutes at 6000 rpm, collect the bacteria, wash the obtained bacteria twice with 0.85% PBS buffer, and resuspend in an equal volume of fresh clothes In aconic acid LB liquid medium (pH4.2), stress for different time;

[...

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Abstract

The invention discloses escherichia coli engineering bacteria with improved acid stress capability and application, and belongs to the technical field of microbial engineering. According to the invention, a gene of nickel ion ABC transporter periplasmic binding protein NikA is taken as a target gene, and escherichia coli is taken as an expression host, so escherichia coli engineering bacteria which can be widely applied to preparation of foods, medicines, feeds and chemicals are successfully constructed; the acid stress capacity of the strain is remarkably improved, and the resistance of the strain to itaconic acid is 6.3 times that of a control strain; the resistance to D-lactic acid is 3.3 times that of a control strain; the succinic acid resistance of the strain is 1.6 times that of a control strain.

Description

Technical field[0001]The invention relates to an Escherichia coli engineered bacterium with improved acid stress ability and an application, and belongs to the technical field of microbial engineering.Background technique[0002]Escherichia coli is an important host bacteria in prokaryotes. This type of bacteria is extremely widespread in nature and has a rich diversity of species. They are not only ideal materials for postgraduates, genetics, molecular biology and genetic engineering, but also have important academic value in theory, but also have application value in important fields closely related to human life such as industry, agriculture and animal husbandry, food and medicine. Extremely high. At present, a variety of high-value organic acid biological fermentation methods have been successfully applied, and some people have tried to use E. coli as a host for expression, but there is often a problem of acid stress.[0003]The scientific name of itaconic acid is methylene succinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/70C12P7/40C12P7/54C12P7/56C07K14/245C12R1/19
CPCC07K14/245C12N15/70C12P7/40C12P7/54C12P7/56
Inventor 张娟杨谨华堵国成陈坚
Owner JIANGNAN UNIV
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