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Escherichia coli engineering bacterium with high acid stress resistance and application

A technology of Escherichia coli and engineering bacteria, applied in the field of microbial engineering, can solve the problems of easy degradation of bacterial species, osmotic stress, weakening existing metabolic pathways, etc., and achieves the improvement of acid stress resistance and D-lactic acid resistance. Effect

Active Publication Date: 2021-03-05
JIANGNAN UNIV
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  • 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
[0005] At present, the methods for improving the acid stress resistance such as lactic acid and acetic acid of Escherichia coli mainly contain: (1) mutagenesis breeding, which has the characteristics of simplicity and various types, but its main disadvantages are heavy workload and low efficiency, and The strain after mutagenesis is easy to degenerate; (2) metabolic engineering strategy, the method that utilizes metabolic engineering strategy to improve E. This method has the problems of high cost and low success rate

Method used

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  • Escherichia coli engineering bacterium with high acid stress resistance and application
  • Escherichia coli engineering bacterium with high acid stress resistance and application
  • Escherichia coli engineering bacterium with high acid stress resistance and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the construction of recombinant escherichia coli Eschericha coli K12 MG1655 / pTrc99a-RffH

[0036] Specific steps are as follows:

[0037] (1) Obtain the rffH gene sequence (encoding dTDP-glucose pyrophosphorylase 2 as shown in SEQ ID NO.1) based on the NCBI database, participate in the biosynthesis of polyketose units, regulate glucose-1-phosphate thymidine transfer), design primers pTrc99a-rffH-F, pTrc99a-rffH-R shown in SEQ ID NO.3 and SEQ ID NO.4 respectively;

[0038] (2) According to the gene sequence to be recombined, design primers loop p-pTrc99a-F and loop p-pTrc99a-R shown in SEQ ID NO.7 and SEQ ID NO.8 respectively;

[0039] (3) using the genome of E.coli K12 MG1655 as a template, pTrc99a-rffH-F, and pTrc99a-rffH-R as primers to obtain the gene fragment shown in SEQ ID NO.1 through PCR amplification;

[0040] (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 o...

Embodiment 2

[0043] Embodiment 2: Construction of recombinant Escherichia coli Eschericha coli K12 MG1655 / pTrc99a-RffG

[0044] Specific steps are as follows:

[0045] (1) Obtain the rffG gene sequence shown in SEQ ID NO.2 based on the NCBI database (encoding dTDP-glucose 4,6-dehydratase, participate in the biosynthesis of polyketose units, and regulate the dehydration of dTDP-glucose), Design primers pTrc99a-rffG-F, pTrc99a-rffG-R shown in SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0046] (2) According to the gene sequence to be recombined, design primers loop p-pTrc99a-F and loop p-pTrc99a-R shown in SEQ ID NO.7 and SEQ ID NO.8 respectively;

[0047] (3) using the genome of E.coli K12 MG1655 as a template, pTrc99a-rffG-F, and pTrc99a-rffG-R as primers to obtain the gene fragment shown in SEQ ID NO.2 through PCR amplification;

[0048] (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...

Embodiment 3

[0050] Embodiment 3: the growth situation of recombinant bacterial strain and control bacterial strain under normal conditions

[0051] Specific steps are as follows:

[0052] (1) The bacterial strain E.coli K12 MG1655 / pTrc99a-RffH, E.coliK12MG1655 / pTrc99a-RffG obtained in Example 1 and 2 and the control bacterial strain E.coli K12 MG1655 / pTrc99a were inoculated in LB liquid medium for activation , placed in a shaker at 37°C at 220rpm and cultured overnight;

[0053] (2) Transfer the seed solution obtained in step (1) to LB liquid medium with an inoculum amount of 2% (v / v), and place it in a shaker at 37°C at 220rpm for cultivation;

[0054] (3) During the cultivation of bacterial strains in step (2), sampling is taken every 2 hours, and the OD value under the measured 600nm wavelength is drawn, and the growth curve is drawn (the growth curve obtained by drawing is as follows: figure 1 shown).

[0055] The result is as figure 1 As shown, through growth performance test ana...

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Abstract

The invention discloses an escherichia coli engineering bacterium with high acid stress resistance and an application, and belongs to the technical field of microbial engineering. According to the invention, a gene for encoding dTDP-glucose pyrophosphorylase 2RffH and / or a gene for encoding dTDP-glucose 4, 6-dehydratase RffG is used as a target gene, and escherichia coli is used as an expression host, so that an escherichia coli engineering bacterium which can be widely applied to preparation of foods, medicines, feeds and chemicals is successfully constructed. The D-lactic acid stress resistance of the escherichia coli engineering bacterium is remarkably improved and is respectively improved by 28.9 times and 4509.6 times to the maximum compared with that of a wild strain.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium with strong resistance to acid stress and its application, 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 lactic acid is α-hyd...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12P7/56C12P7/54C12P7/40C12R1/19
CPCC12N9/1241C12N9/88C12N15/70C12P7/56C12P7/54C12P7/40C12Y402/01046C12Y207/07024
Inventor 张娟杨谨华堵国成陈坚
Owner JIANGNAN UNIV
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