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 obtained by mutating threonine at the 419th site of phosphoenolpyruvate synthase of an escherichia coli wild type into aspartic acid, the sensitivity of an escherichia coli mutant strain with the mutation site to nalidixic acid, mitomycin C and polymyxin sulfate B is remarkably improved, and the mutation site can be used for improving the sensitivity of bacteria to drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to the construction of an Escherichia coli strain using molecular biology methods. Escherichia coli The T419D mutation site of phosphoenolpyruvate synthase (PpsA) is used to improve the sensitivity of bacteria to drugs. Background Technology

[0002] The increasing drug resistance of pathogenic bacteria has become a serious global problem threatening human health in the 21st century. Studies have shown that bacterial drug resistance is inextricably linked to their metabolic activities under specific environmental conditions. With the development of genome sequencing and various omics technologies, thousands of Ser / Thr phosphorylation sites have been discovered in bacteria, a significant portion of which belong to carbon metabolism pathways. The abundance of phosphorylation sites in enzymes and related factors involved in carbon metabolism suggests they may play a crucial role in the regulation of bacterial carbon metabolism. Furthermore, bacteria phosphorylate more proteins during drug treatment, suggesting that bacteria may regulate carbon metabolism through phosphorylation to cope with external drug stress.

[0003] In previous studies, the inventors discovered that YihE, a serine / threonine protein kinase (STK) in *E. coli*, can protect bacteria by maintaining normal levels of intracellular reactive oxygen species (ROS) under various drug treatments. Phosphoproteomic analysis revealed that the potential targets of YihE kinase are all related to bacterial carbon metabolism, including the central carbon metabolism key enzyme PpsA. Therefore, phosphorylation modification of PpsA may be related to bacterial sensitivity to drugs.

[0004] The inventors of this invention performed site-directed mutagenesis on the Ser / Thr phosphorylation site of PpsA in Escherichia coli, and found that the mutant strain exhibited significant changes in carbon metabolism and drug sensitivity. This invention improves bacterial drug sensitivity by mutating the phosphorylation site of PpsA, a key enzyme in E. coli carbon metabolism, providing a new approach and method for addressing bacterial drug resistance. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the PpsA T419D mutation site, which currently has no reported association with bacterial drug sensitivity.

[0006] Specifically, a method for constructing the PpsA T419D mutation site and its application are disclosed to improve bacterial sensitivity to drugs.

[0007] The bacterial drug sensitivity refers to the sensitivity of Escherichia coli to nalidixic acid, mitomycin C, and polymyxin B sulfate.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] a) Using Escherichia coli BW25113 as the starting strain, a PpsA phosphorylation site mutant strain was constructed using the CRISPR / Cas9 gene editing method.

[0010] b) Design and synthesize point mutation primers, and perform PCR amplification using plasmid pCA24N-PpsA as a template. The amplification product is then subjected to... Dpn Following digestion with enzyme I, homologous recombination was performed under the catalysis of Exnase II to complete the circularization of linear DNA. The recombinant product was then transformed into E. coli DH5α competent cells using a heat shock method. Plasmids were extracted from the transformants and sequenced for verification. ppsA Mutations at phosphorylation sites constructed in the gene.

[0011] c) Design and synthesize homologous arm primers to construct primers containing the above-mentioned homologous arms. ppsA The plasmid with the gene phosphorylation site mutation was used as a template to amplify the homologous arm fragment by PCR. The amplification product was then recovered by gel and used for later use.

[0012] d) Design and synthesize SgRNA, phosphorylate SgRNA, and anneal it with... Bsa The original pEcgRNA digested with enzyme I was ligated, and the ligation product was transformed into E. coli DH5α competent cells using a heat shock method. The pEcgRNA plasmid used for the constructed mutation was extracted and sequenced for verification.

[0013] e) Extract the pEcCas plasmid and transform it into E. coli BW25113 competent cells using a heat shock method. Extract the plasmid from the transformants and perform sequencing verification. Then, prepare E. coli BW25113 electroporation competent cells containing the pEcCas plasmid for later use.

[0014] f) The pEcgRNA plasmid and homologous arm fragment were electroporated into *E. coli* BW25113 containing the pEcCas plasmid, and mutant strains were then screened on LB agar plates containing antibiotics. Primers were designed and synthesized to amplify the mutant strain's genomic DNA as a template. ppsA The full-length gene was sequenced and the constructed phosphorylation site mutations were verified.

[0015] g) Screening was performed using LB medium containing rhamnose and the corresponding antibiotic, as well as glucose / sucrose and the corresponding antibiotic, to eliminate [the contaminants]. Mutants of pEcCas and pEcgRNA plasmids.

[0016] h) Detection ppsA Sensitivity of wild-type strains and phosphorylation site mutants to drugs. Attached Figure Description

[0017] Figure 1. Construction of the gene encoding phosphoenolpyruvate synthase containing the T419D phosphorylation mutation site. (A) pCA24N-PpsA (Thr 419 -Asp 419 (B) Design of point mutation primers for pCA24N-PpsA (Thr 419 -Asp 419 Sequencing verification.

[0018] Figure 2. Construction of the Escherichia coli phosphoenolpyruvate synthase T419D mutant strain. (A) Design of SgRNA and homologous arms of the point mutation encoding the phosphoenolpyruvate synthase gene. (B) Sequencing verification of the mutation site of the phosphoenolpyruvate synthase gene in the mutant strain.

[0019] Figure 3. Drug sensitivity of Escherichia coli phosphoenolpyruvate synthase T419D mutant strain. (A) Nalidixic acid; (B) Mitomycin C; (C) Polymyxin B sulfate. Detailed Implementation

[0020] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the processes mentioned in the embodiments, such as genomic DNA extraction, plasmid extraction, electrophoresis, gel recovery, competent cell preparation, transformation, and plate screening, can all be implemented using conventional methods in the art, and therefore will not be described in detail further.

[0021] Example 1: Construction of the PpsA-encoding gene containing the T419D phosphorylation mutation site: Using pCA24N-PpsA (from the ASKA library) as a template, a plasmid containing the PpsA-encoding gene containing the T419D phosphorylation mutation site was constructed. Figure 1 In this method, point mutation primers are first designed and retrieved from the NCBI database. E. coli pps The base sequence of gene A and its corresponding amino acid sequence. Based on the pairing rules of genetic codons, it was located to... pps The 419th threonine (Thr) encoded by gene A 419 The ACC sequence of the bases is used to determine the positions of the bases to be mutated. To improve the success rate of point mutations, the base sequence of the point mutation is further analyzed. ppsA Both the sense and antisense strands of the gene underwent point mutations. The original base ACC (encoding threonine) in the pCA24N-PpsA template strand was mutated to GAC, encoding aspartic acid (…). Figure 1 -A). Then, for those containing wild typeppsA Site-directed mutagenesis was performed on the gene plasmid. First, PCR amplification was performed using point mutation primers with pCA24N-PpsA (from the ASKA library) as a template. The PCR primers were: 5′-GTCGTgacTGTCACGCAGCGATCATCGCTCGT-3′ and 5′-TGCGTGACAgtcACGACCGCCACGGTTGGTGA-3′. The PCR amplification conditions were: 95℃ preheating for 3 minutes, 1 cycle; 95℃ denaturation for 15 seconds, 56-70℃ annealing for 20 seconds, 72℃ amplification for 30-60 seconds / kb DNA, 35 cycles; 72℃ amplification for 10 minutes; 4℃ for 1 hour. Afterwards, the PCR products were... Dpn Digestion was used to remove the original plasmid. The reaction conditions were: 37℃, 1-2 hours. Since the designed point mutation primers have an approximately 20 bp inverse complementary fragment at the 5' end, Exnase II can be used to catalyze homologous recombination of the target fragment, cyclizing the linear fragment. The reaction conditions were: 37℃, 30 min. After the reaction was complete, the temperature was immediately lowered to 4℃. The cyclized target fragment was then transformed into [a specific organism] using a heat shock method. E. coli DH5α was plated on chloramphenicol plates (final concentration 30 μg / mL) and incubated overnight at 37°C. Several transformants were randomly selected and incubated overnight. Plasmids were extracted and sequenced for verification. Sequencing results showed that the pCA24N-PpsA point mutant plasmid was successfully constructed. Figure 1 -B).

[0022] Example 2: Construction of Escherichia coli PpsA T419D mutant: Using Escherichia coli BW25113 as the starting strain, a PpsA phosphorylation site mutant was constructed using the CRISPR / Cas9 gene editing method. Figure 2 In this method, SgRNA and homologous arm designs were employed. ppsA SgRNAs with site-directed gene mutations ( Figure 2 -A), the PAM site is CGG, and its upstream 20 bp is SgRNA. Its homologous arm is approximately 500 bp long. A mutation of ACC to GAC at the 419th Thr site encoding PpsA results in PpsAThr. 419 Mutate to Asp 419 Simultaneously, a synonymous mutation was introduced at the PAM site, mutating CGG to CAG.

[0023] Genome editing of *E. coli* was performed using a modified pCas / pTargetF system. First, the mutants were prepared... ppsA Gene homologous arm fragment, with pCA24N - PpsA (Thr) 419 -Asp419 Using 5′-GCCACACCGGTAAACTGTTC-3′ and 5′-CAGGCGAAGTCGAAAGCAC-3′ as templates, amplification was performed under the aforementioned conditions. ppsA Homologous arm fragments of the gene were amplified, and the amplified products were stored at -20°C after gel re-extraction.

[0024] The designed sgRNA sequence was synthesized by Qingke Company, and then phosphorylated using T4 PNK (37℃, 3 hours). 2.5 μL of 1 M NaCl was added to the phosphorylated sgRNA, annealed at 95℃ for 5 min, and then slowly cooled to room temperature (30 minutes–1 hour). The annealed sgRNA was then diluted 10-fold with ddH2O for later use. Bsa pEcgRNA was digested with NEB (37°C, 5 hours). The digested pEcgRNA was subjected to agarose gel electrophoresis, followed by gel recovery using a SanPrep column DNA gel recovery kit. The DNA was then ligated using T4 ligase. Bsa The gel-recovered pEcgRNA digested with enzyme I was ligated to the above-mentioned SgRNA (16°C, 2 hours). The ligation product was then transformed into [a specific enzyme] via heat shock. E. coli DH5α competent cells were plated on streptomycin plates with a final concentration of 100 μg / ml and incubated overnight at 37°C. Transformants were picked and plasmids were extracted for sequencing verification.

[0025] Wild-type *Escherichia coli* BW25113 competent cells were prepared using the CaCl2 method. pEcCas was then transformed into BW25113 competent cells via heat shock. The transformed cells were plated on kanamycin plates with a final concentration of 50 μg / mL and cultured overnight at 37°C. Transformants were picked, plasmids were extracted, and sequencing was performed for verification. Subsequently, BW25113 / pEcCas competent cells for electroporation were prepared, and 10 mM arabinose was added during culture to induce λ-Red system expression.

[0026] pEcgRNA and homologous arm fragments were electroporated at a ratio of 2:8 into BW25113 / pEcCas competent cells. The cells were plated on streptomycin (final concentration 100 μg / mL) plates and incubated overnight at 37°C. Transformants were picked, and their genomic DNA was extracted. Using this DNA as a template, primers 5′-GCAGGATGTCTGTGAAGAGATTG-3′ and 5′-GTTCGATGTCCAACAATGGCTC-3′ were used for amplification under the aforementioned conditions. ppsA The full-length gene was amplified, and the amplified products were recovered from the gel and then sequenced for verification. Sequencing results are shown below. Figure 2 -B, PpsA's Thr 419Mutate to Asp 419 The PAM site showed a synonymous mutation, proving that the mutant strain was successfully constructed.

[0027] The mutant strain containing pEcCas and pEcgRNA plasmids was inoculated into LB liquid medium containing rhamnose (final concentration 10 mM) and kanamycin (final concentration 50 μg / mL) and incubated at 37°C and 180 rpm for 12 hours. The bacterial culture was then diluted, and 10 μL was plated onto a kanamycin (final concentration 50 μg / mL) plate and incubated upside down at 37°C for 12 hours. The next day, several colonies growing on the kanamycin plate were randomly selected and spotted onto a streptomycin (final concentration 100 μg / mL) plate using a sterile toothpick and incubated overnight at 37°C. Colonies that did not grow on the streptomycin plate but grew on the kanamycin plate were then selected and inoculated into LB liquid medium containing glucose (5 g / L) and incubated overnight at 37°C and 180 rpm. Spread 10 μL of bacterial culture onto LB agar plates containing glucose (5 g / L) and sucrose (10 g / L), and incubate overnight at 37°C. Randomly select single colonies and use sterile toothpicks to spot them sequentially onto ordinary LB agar plates, kanamycin (final concentration 50 μg / mL) plates, and streptomycin (final concentration 100 μg / mL) plates for screening. Single colonies that can grow on ordinary LB agar plates but cannot grow on kanamycin and streptomycin plates are PpsA phosphorylation site mutant strains with successfully eliminated pEcCas and pEcgRNA plasmids.

[0028] Example 3: Drug susceptibility testing of Escherichia coli PpsA T419D phosphorylation site mutant strain: Both the PpsA T419D phosphorylation site mutant strain and the wild-type strain were inoculated at a 1% inoculum volume in LB broth and cultured until mid-log (OD2). 600 ≈0.3-0.5). Take 100 μL of bacterial culture that has grown to mid-log phase and add it to 900 μL of LB liquid medium for serial dilution (10). -1 ~10 -6 For each dilution, 10 μL of sample was plated as a control group without drug treatment. Simultaneously, 900 μL of bacterial culture grown to mid-log phase was mixed with 100 μL of the corresponding drug concentration and cultured for another 2 hours. After drug treatment, serial dilutions and plate spotting were performed as described above, serving as the drug-treated experimental group. The plates were then incubated overnight at 37°C with the plates inverted. After colonies appeared, the number of colonies on the plates was recorded. The survival rate was calculated using the following formula: Survival rate = Number of drug-treated colonies / Number of untreated colonies × 100%. Drug sensitivity testing showed that when threonine at position 419 of PpsA was mutated to aspartic acid, the mutant strain was more sensitive to the drug than the wild-type strain. Figure 3This indicates that the PpsA T419D phosphorylation site mutation increases the bacteria's sensitivity to drugs.

Claims

1. A phosphoenolpyruvate synthase PpsA T419D mutation site, characterized in that, The mutation site is derived from the mutation of threonine at position 419 of wild-type Escherichia coli phosphoenolpyruvate synthase PpsA to aspartic acid.

2. The gene sequence encoding the T419D mutation site of phosphoenolpyruvate synthase according to claim 1, characterized in that, The sequence is derived from the mutation of ACC to GAC at threonine 419 in the wild-type Escherichia coli phosphoenolpyruvate synthase gene; the nucleotide sequence of the PpsA T419D mutant is shown in SEQ ID No.

1.

3. The T419D mutation site of phosphoenolpyruvate synthase according to claims 1-2, characterized in that, Escherichia coli mutants with this mutation site showed significantly increased susceptibility to nalidixic acid, mitomycin C, and polymyxin B sulfate.

4. The application of the phosphoenolpyruvate synthase T419D mutation site according to claims 1-3, characterized in that, Used to improve the sensitivity of bacteria to drugs.

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