CN-122012447-A - Application of phosphoenolpyruvate synthase T419D mutation site

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a construction method and application of a phosphoenolpyruvate synthase T419D mutation site. The mutation site is formed by mutating threonine at 419 th site of wild type phosphoenolpyruvate synthase of escherichia coli into aspartic acid, the sensitivity of an escherichia coli mutant strain with the mutation site to nalidixic acid, mitomycin C and polymyxin B sulfate is obviously improved, and the mutation site can be used for improving the sensitivity of bacteria to medicines.

Inventors

• LU TAO
• HAN XIULIN
• LI LIFANG
• ZHU WEILING

Assignees

• 云南大学

Dates

Publication Date

20260512

Application Date

20251114

Claims (4)

1. 1. A phosphoenolpyruvate synthase PpsA T D mutation site, wherein the mutation site is from threonine at position 419 of escherichia coli wild-type phosphoenolpyruvate synthase PpsA to aspartic acid.
2. 2. The coding gene sequence of the phosphoenolpyruvate synthase T419D mutation site according to claim 1, wherein the coding gene sequence is obtained by mutating the ACC coding for threonine at position 419 of a wild-type phosphoenolpyruvate synthase gene of Escherichia coli into GAC, and the nucleotide sequence of the PpsA T D mutant is shown as SEQ ID No. 1.
3. 3. The phosphoenolpyruvate synthase T419D mutation site according to claim 1-2, wherein the sensitivity of the E.coli mutant strain having the mutation site to nalidixic acid, mitomycin C and polymyxin B sulfate is significantly increased.
4. 4. The use of a phosphoenolpyruvate synthase T419D mutation site according to claims 1-3, wherein the phosphoenolpyruvate synthase T419D mutation site is used for increasing the sensitivity of bacteria to drugs.

Description

Application of phosphoenolpyruvate synthase T419D mutation site Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for constructing a T419D mutation site of escherichia coli (ESCHERICHIA COLI) phosphoenolpyruvate synthase (phosphoenolpyruvate synthase, ppsA) by using a molecular biological method and improving the sensitivity of bacteria to medicines. Background The development of drug resistance by an increasing number of pathogenic bacteria has become a global problem in the 21 st century that severely threatens human health. Studies have shown that tolerance of bacteria to drugs has an inseparable relationship with the metabolic activity of bacteria in specific environments. With the development of genomic sequencing and various histology techniques, thousands of Ser/Thr phosphorylation sites have been found in bacteria, a significant portion of which belong to the carbon metabolic pathway. Having so many phosphorylation sites in enzymes involved in carbon metabolism and their related factors suggests that they may play a very important role in bacterial carbon metabolism regulation. Meanwhile, there are more proteins phosphorylated by bacteria at the time of drug treatment, so that bacteria are likely to regulate carbon metabolism by phosphorylation to cope with external drug pressure. The inventors found in earlier studies that a serine/threonine protein kinase (Ser/THR KINASES, STKS) YihE in E.coli was able to protect bacteria by maintaining normal levels of intracellular reactive oxygen species (Reactive Oxygen Species, ROS) in bacteria when treated with multiple drugs. Through phosphoproteomic analysis, yihE kinase potential targets were found to be involved in bacterial carbon metabolism, including central carbon metabolism key enzyme PpsA. Thus, the phosphorylation modification of PpsA may be related to the susceptibility of bacteria to drugs. The inventors performed site-directed mutagenesis on the Ser/Thr phosphorylation site of E.coli PpsA, and found that the mutant was significantly altered in both carbon metabolism and drug sensitivity. The invention improves the sensitivity of bacteria to medicines by mutating the phosphorylation site of key enzyme PpsA for carbon metabolism of escherichia coli, and provides a new thought and method for coping with the problem of bacterial drug resistance. Disclosure of Invention The invention aims to provide an application of PpsA T D mutation site, and no report on the mutation site related to bacterial drug sensitivity exists at present. In particular to a method for constructing PpsA T D mutation site and application thereof, which are used for improving the sensitivity of bacteria to medicines. The sensitivity of the bacteria to the drugs is that of the colibacillus to nalidixic acid, mitomycin C and polymyxin B sulfate. In order to achieve the above purpose, the present invention adopts the following technical scheme: a) E.coli BW25113 is used as an initial strain, and a CRISPR/Cas9 gene editing method is adopted to construct PpsA phosphorylation site mutant strains. B) The point mutation primer is designed and synthesized, and PCR amplification is carried out by taking plasmid pCA24N-PpsA as a template. And (3) carrying out homologous recombination on the amplified product after DpnI digestion under Exnase II catalysis to complete cyclization of linear DNA, then converting and introducing the recombinant product into E.coli DH5 alpha competent cells by adopting a heat shock method, extracting plasmids in the transformant, and sequencing to verify phosphorylation site mutation constructed in the ppsA gene. C) Designing and synthesizing homologous arm primers, taking the constructed plasmid containing ppsA gene phosphorylation site mutation as a template, amplifying homologous arm fragments by PCR, and recovering amplified products by gel for later use. D) SgRNA is designed and synthesized, sgRNA is phosphorylated, annealed and then connected with the original pEcgRNA cut by Bsa I, the product of the connection reaction is transformed into competent cells of escherichia coli DH5 alpha by using a heat shock method, and pEcgRNA plasmid for the constructed mutation is extracted and subjected to sequencing verification. E) The pEcCas plasmid was extracted, the pEcCas plasmid was transformed into competent cells of E.coli BW25113 by heat shock, and sequencing was performed after the plasmid was extracted from the transformant. Then, E.coli BW25113 electrotransformation competent cells containing pEcCas plasmid were prepared for use. F) The pEcgRNA plasmid and the homology arm fragment described above were electrotransduced into E.coli BW25113 containing pEcCas plasmid, and then the mutant strain was selected on LB plates containing antibiotics. Primers were designed and synthesized, ppsA full-length gene was amplified using mutant genomic DNA as a template, and the constructed phosphorylation site mu