Escherichia coli engineering bacteria with improved acid stress resistance and application of escherichia coli engineering bacteria

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, osmotic stress, etc., and achieve the effect of simple operation, improved acid stress resistance, and improved acid stress resistance

Active Publication Date: 2021-02-23
JIANGNAN UNIV
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  • 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 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 bacteria with improved acid stress resistance and application of escherichia coli engineering bacteria
  • Escherichia coli engineering bacteria with improved acid stress resistance and application of escherichia coli engineering bacteria
  • Escherichia coli engineering bacteria with improved acid stress resistance and application of escherichia coli engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of recombinant strain E.coli K12 MG1655 / pTrc99a-PyrB

[0060] Specific steps are as follows:

[0061] (1) Based on the pyrB gene sequence in the NCBI database (encoding the catalytic subunit of aspartate carbamoyltransferase, the PyrB gene participates in the pyrimidine metabolism pathway and regulates aspartate carbamoyl transfer) designed as SEQ ID NO. 4 and the primer PTrc99a-PyrB-F shown in SEQ ID NO.5, PTrc99a-PyrB-R;

[0062] (2) Design the primers loop p-pTrc99a-F and loop p-pTrc99a-R shown in SEQ ID NO.10 and SEQ ID NO.11 respectively;

[0063] (3) Using the genome of E.coli K12 MG1655 as a template, using PTrc99a-PyrB-F and PTrc99a-PyrB-R as primers to amplify by PCR to obtain the gene fragment shown in SEQ ID NO.1;

[0064] (4) Using the vector pTrc99a as a template, using loop p-pTrc99a-F and loop p-pTrc99a-R as primers to amplify by PCR to obtain a linearized long fragment of the vector;

[0065] (5) Ligate the PCR products obtaine...

Embodiment 2

[0067] Example 2: Construction of recombinant strain E.coli K12 MG1655 / PTrc99a-PyrD

[0068] Specific steps are as follows:

[0069] (1) Based on the pyrD gene sequence in the NCBI database (the nucleotide sequence gene encoding dihydroorotate dehydrogenase PyrD, which participates in the pyrimidine metabolic pathway and regulates the dehydrogenation process of dihydroorotate), the design is as follows: SEQ ID Primers PTrc99a-PyrD-F and PTrc99a-PyrD-R shown in NO.6 and SEQID NO.7;

[0070] (2) Design the primers loop p-pTrc99a-F and loop p-pTrc99a-R shown in SEQ ID NO.10 and SEQ ID NO.11 respectively;

[0071] (3) Using the genome of E.coli K12 MG1655 as a template, using PTrc99a-PyrD-F and PTrc99a-PyrD-R as primers to amplify by PCR to obtain the gene fragment shown in SEQ ID NO.2;

[0072] (4) Using the vector pTrc99a as a template, using loop p-pTrc99a-F and loop p-pTrc99a-R as primers to amplify by PCR to obtain a linearized long fragment of the vector;

[0073](5) Liga...

Embodiment 3

[0075] Example 3: Construction of recombinant strain E.coli K12 MG1655 / PTrc99a-PyrF

[0076] Specific steps are as follows:

[0077] (1) Based on the pyrF gene sequence in the NCBI database (encoding Orotidine-5'-phosphate decarboxylase protein PyrF gene, participates in pyrimidine metabolism pathway, regulates the decarboxylation of Orotidine-5'-phosphate) design such as SEQ ID NO.8 and Primers PTrc99a-PyrF-F and PTrc99a-PyrF-R shown in SEQ ID NO.9;

[0078] (2) Design the primers loop p-pTrc99a-F and loop p-pTrc99a-R shown in SEQ ID NO.10 and SEQ ID NO.11 respectively;

[0079] (3) Using the genome of E.coli K12 MG1655 as a template and using PTrc99a-PyrF-F and PTrc99a-PyrF-R as primers to obtain the gene fragment shown in SEQ ID NO.3 by PCR amplification;

[0080] (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;

[0081] (5) Ligate the PCR products o...

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Abstract

The invention discloses escherichia coli engineering bacteria with improved acid stress resistance and application of the escherichia coli engineering bacteria, and belongs to the technical field of microbial engineering. According to the invention, aspartate carbamoyl transferase is overexpressed in escherichia coli to catalyze subunits PyrB, dihydroorotate dehydrogenase PyrD and Orotidine-5'-phosphate decarboxylase protein PyrF, so that the escherichia coli engineering bacteria with improved acid stress resistance, which can be widely applied to preparation of food, medicines, feeds and chemicals, is constructed successfully; and compared with a control strain, the stress resistance of the recombinant strain to itaconic acid, D-lactic acid and succinic acid is remarkably improved. The escherichia coli engineering bacteria are simple to operate and can be widely applied to industrial production.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium with improved acid stress resistance and an 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] High optical purity D-lactic acid (ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N15/53C12N15/60C12P7/56C12P7/54C12P7/40C12R1/19
CPCC12N15/52C12N9/1018C12N9/001C12N9/88C12N15/70C12P7/56C12P7/54C12P7/40C12Y201/03002C12Y103/05002C12Y401/01023Y02A50/30
Inventor 张娟杨谨华堵国成陈坚
Owner JIANGNAN UNIV
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