A kind of Escherichia coli engineering bacterium with improved acid stress ability and its application

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

Active Publication Date: 2022-03-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A kind of Escherichia coli engineering bacterium with improved acid stress ability and its application
  • A kind of Escherichia coli engineering bacterium with improved acid stress ability and its application
  • A kind of Escherichia coli engineering bacterium with improved acid stress ability and its application

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 (the NikA gene encoding the periplasmic binding protein of nickel ion ABC transporter, involved in the ABC transport metabolic pathway of nickel ions, and regulating the binding of substrate proteins in the nickel transport system), the designs are 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 loops 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, using p-pTrc99a / NikA-F, p-pTrc99a / NikA-R as primers, the gene fragment shown in SEQ ID NO.1 was obtained by PCR amplification, and PCR was obtained product;

[0045] (4) using the vector pTrc99a as the template, using the loop p-pTrc99a-F and the loop p-pT...

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 control strain E.coli K12MG1655 / pTrc99a obtained in Example 1 were inoculated respectively Activated in LB liquid medium, placed in a shaker at 37°C and cultured at 220 rpm for 12 hours to obtain seed liquid;

[0053] (2) transfer the seed liquid obtained in the above-mentioned step (1) to LB liquid medium with an inoculation amount of 2% (v / v) respectively, and place it in a 37° C. shaker for cultivation at 220 rpm; sampling every 2 hours , measure the OD value under the wavelength of 600nm, draw the growth curve (drawing the obtained growth curve such as figure 1 ).

[0054] The result is as figure 1 As shown in the growth performance test analysis, after culturing for 12 hours, the growth of the recombinant strain overe...

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.coliK12 MG1655 / pTrc99a-NikA obtained in Example 1 were respectively inoculated into LB liquid medium for activation, and cultured at 220 rpm in a shaker at 37°C 12h to obtain seed liquid;

[0059] (2) The seed liquid obtained in the above (1) was transferred to fresh LB liquid medium with an inoculum of 2% (v / v) respectively, and cultured at 220 rpm in a shaker at 37°C for 4.5 hours, and cultured to logarithmic In the middle growth stage, the OD600 at this time is 1.4-1.5, and the culture medium is obtained;

[0060] (3) Centrifuge the culture solution obtained in step (2) at 6000 rpm for 5 min, collect the cells, wash the obtained cells twice with 0.85% PBS buffer, and resuspend them in an equal volume of fresh coat Conic acid L...

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Abstract

The invention discloses an Escherichia coli engineering bacterium with improved acid stress ability and its application, belonging to the technical field of microbial engineering. The present invention uses the gene of the nickel ion ABC transporter periplasmic binding protein NikA as the target gene and Escherichia coli as the expression host to successfully construct a class of Escherichia coli engineering bacteria that can be widely used in the preparation of food, medicine, feed and chemicals; The acid stress ability of the strain has been significantly improved, and its resistance to itaconic acid is 6.3 times that of the control strain; the resistance to D-lactic acid is 3.3 times that of the control strain; the resistance to succinic acid is 3.3 times that of the control strain 1.6 times.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium with improved acid stress capability and its application, and belongs to the technical field of microbial engineering. Background technique [0002] Escherichia coli is an important host bacteria in prokaryotes. Such 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, 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, animal husbandry, food and medicine. Very high. At present, a variety of high-value organic acid bio-fermentation methods have been successfully applied, and some people have also tried to express them using Escherichia coli as the host, but there are often problems of acid stress. [0003] Itaconic acid is scientifically calle...

Claims

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Application Information

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Patent Type & Authority Patents(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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