Recombinant escherichia coli and application thereof in preparation of Lipid II

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to recombinant Escherichia coli, the recombinant Escherichia coli is obtained by introducing 29th and 49th cysteine mutated murJ genes into an Escherichia coli BW25113 strain, and the nucleotide sequence of the murJ genes is shown as SEQ ID NO.1. The defects in the prior art are overcome, and the recombinant Escherichia coli is a recombinant Escherichia coli strain. Extra cysteine mutation is introduced to MurJ protein with cysteine mutation, so that the sensitivity of the MurJ protein to MTSES is increased, and the purpose of reducing the consumption of MTSES is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and specifically relates to a recombinant Escherichia coli and an application thereof in the preparation of Lipid II. Background Art

[0002] Bacterial infection has always been an important factor endangering human health, and in recent years, due to the abuse of antibiotics and the evolution of bacterial resistance mechanisms, new antibiotics are needed. Currently, many known antibiotics inhibit bacteria mainly by destroying cell wall synthesis, among which cell wall synthase is the key site of action.

[0003] Cell wall synthesizing enzymes all have a common substrate: Lipid II, which is a specific substrate for synthesizing cell walls. After being synthesized in the cytoplasmic membrane, it is flipped into the periplasmic space by the flip enzyme MurJ, which switches between the inward and outward conformations, to maintain the synthesis of the new cell wall.

[0004] There are antibiotics that specifically target Lipid II to inhibit cell wall synthesis, such as vancomycin, which is known as the last line of defense, and ramoplanin. Therefore, we speculate that there are other molecules targeting Lipid II to help solve the problem of bacterial resistance.

[0005] The preparation of Lipid II will help to efficiently screen new antibiotics. At the same time, it can be used to study the mechanism of cell wall synthesis. However, due to the complex structure of Lipid II, chemical synthesis is difficult, and there are few Lipid II monomers in the cell, so the yield of Lipid II directly extracted from bacteria is low. Qiao et al. used vancomycin to treat Staphylococcus aureus to obtain more Gram-positive bacteria Lipid II. However, due to the differences between Gram-negative bacteria Lipid II and Gram-positive bacteria Lipid II, and the protective effect of the Gram-negative bacteria outer membrane on it, vancomycin cannot be used to extract Gram-negative bacteria Lipid II, so the large-scale preparation of Gram-negative bacteria Lipid II still faces great difficulties. Qiao et al. used MTSES to react with Lipid II flippase MurJ with a specific site cysteine ​​mutation, and site-specifically changed the properties of MurJ, making it unable to change its conformation alternately, resulting in Lipid II accumulation in the plasma membrane to achieve the purpose of enriching Gram-negative bacteria Escherichia coli Lipid II, but MTSES is expensive and is not economical for large-scale preparation of Lipid II. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant Escherichia coli and its use in the preparation of Lipid II, which overcomes the shortcomings of the prior art and obtains a large amount of Lipid II with a lower MTSES consumption to help screen new antibiotics and study the mechanism of cell wall synthesis.

[0007] To solve the above problems, the technical solutions adopted by the present invention are as follows: A recombinant Escherichia coli, wherein the recombinant Escherichia coli is an Escherichia coli BW25113 strain into which the murJ gene with cysteine ​​mutations at positions 29 and 49 is introduced, and the nucleotide sequence of the murJ gene is shown in SEQ ID NO.1.

[0008] Further, the preparation method thereof comprises the following steps: A total of 200 bp of nucleotide sequences were selected upstream and downstream of the 49th amino acid site encoded by the murJ gene to find the original spacer sequence that can be recognized and cut by the CRISPR-Cas9 system; Construct pACYC-sgRNA plasmid expressing sgRNA; The pKD46 plasmid and the pET-Cas9 plasmid were transferred into an Escherichia coli strain encoding the murJ gene with a cysteine ​​mutation at position 29 in the genome; The constructed pACYC-sgRNA plasmid and single-stranded primer were transferred into the E. coli strain carrying pKD46 and pET-Cas9 plasmids; (5) The single clones grown on the plate are sequenced and identified, and the successfully edited strains are saved to obtain the Escherichia coli strain.

[0009] Furthermore, the single-stranded primer comprises a base sequence encoding cysteine ​​at position 49, and the single-stranded primer has 45 nt homologous sequences upstream and downstream of the murJ gene editing site.

[0010] The invention also protects the use of the recombinant Escherichia coli in preparing Lipid II.

[0011] Further, the following steps are included: The obtained recombinant E. coli strain was transferred to 500 ml of culture medium for cultivation; At OD600 of 0.1-0.2, 100 nM anhydrotetracycline was used to induce the expression of UppS and MraY; After 45 minutes of induction, MTSES was added for 20 minutes; The cultured E. coli was collected by centrifugation, PBS was added to re-select the culture, chloroform and methanol were added in a ratio of 1:2, and stirred for 1 hour to fully disrupt the cells; After centrifugation of the broken cells, the supernatant was transferred to a new tube containing PBS and chloroform, stirred at room temperature for 1 hour, and after centrifugation, the middle layer was transferred and dried by rotary evaporation; Use a solution of pyridine acetate: n-butanol = 1:2 to dissolve the product after rotary evaporation, add n-butanol saturated water to extract, repeat three times, and take the upper phase; The upper phase was dried by rotary evaporation at 40 °C; (8) Dissolve the mixture in a solution of chloroform:methanol = 1:1, blow dry under nitrogen, and collect the LipidII.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The invention introduces an additional cysteine ​​mutation into the MurJ protein which already has the cysteine ​​mutation, so as to increase the sensitivity of the MurJ protein to MTSES, thereby achieving the purpose of reducing the consumption of MTSES.

[0013] Based on the strain, the present invention upregulates the expression of key synthases in the Lipid II synthesis pathway, and further improves the yield of Lipid II on the basis of reducing the MTSES consumption; a large amount of Lipid II is obtained with a lower MTSES consumption to help screen new antibiotics and study the mechanism of cell wall synthesis. DETAILED DESCRIPTION

[0014] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Example

[0015] This embodiment discloses a recombinant Escherichia coli, into which the murJ gene with cysteine ​​mutations at positions 29 and 49 is introduced into the Escherichia coli BW25113 strain, and the nucleotide sequence of the murJ gene is shown in SEQ ID NO.1.

[0016] The method for preparing recombinant Escherichia coli comprises the following steps: A total of 200 bp of nucleotide sequences were selected upstream and downstream of the 49th amino acid site encoded by the murJ gene to find the original spacer sequence that can be recognized and cut by the CRISPR-Cas9 system; Construct pACYC-sgRNA plasmid expressing sgRNA; The pKD46 plasmid and the pET-Cas9 plasmid were transferred into an Escherichia coli strain encoding the murJ gene with a cysteine ​​mutation at position 29 in the genome; The constructed pACYC-sgRNA plasmid and a single-stranded primer were transferred into the E. coli strain with pKD46 and pET-Cas9 plasmids. The single-stranded primer contained a base sequence encoding cysteine ​​at position 49, and the single-stranded primer had 45 nt homologous sequences upstream and downstream of the murJ gene editing site. (5) The single clones grown on the plate are sequenced and identified, and the successfully edited strains are saved to obtain the Escherichia coli strain. Example

[0017] This example discloses the preparation of Lipid II using the recombinant Escherichia coli prepared in Example 1. The specific preparation method includes the following steps: The obtained recombinant E. coli strain was transferred to 500 ml of culture medium for cultivation; At OD600 of 0.1-0.2, 100 nM anhydrotetracycline was used to induce the expression of UppS and MraY; After 45 minutes of induction, MTSES was added for 20 minutes; The cultured E. coli was collected by centrifugation, PBS was added to re-select the culture, chloroform and methanol were added in a ratio of 1:2, and stirred for 1 hour to fully disrupt the cells; After centrifugation of the broken cells, the supernatant was transferred to a new tube containing PBS and chloroform, stirred at room temperature for 1 hour, and after centrifugation, the middle layer was transferred and dried by rotary evaporation; Use a solution of pyridine acetate: n-butanol = 1:2 to dissolve the product after rotary evaporation, add n-butanol saturated water to extract, repeat three times, and take the upper phase; The upper phase was dried by rotary evaporation at 40 °C; Use a solution of chloroform:methanol=1:1 to dissolve, blow dry under a nitrogen environment, and collect the LipidII.

[0018] The present invention introduces a cysteine ​​mutation at position 49 into a sequence encoding a MurJ protein having a cysteine ​​mutation at position 29 on the genome, thereby obtaining a strain with double cysteine ​​mutations on the MurJ protein, thereby reducing the consumption of MTSES to a certain extent. By transferring a pZH509 plasmid containing Lipid II synthetase into the MurJ double cysteine ​​mutant strain, the yield of Lipid II is significantly improved after induction. By culturing and inducing the strain in a nutrient-rich medium and treating it with MTSES, Lipid II is further improved.

[0019] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A recombinant Escherichia coli, characterized in that: The recombinant Escherichia coli is Escherichia coli BW25113 strain into which the murJ gene with cysteine ​​mutations at positions 29 and 49 is introduced. The nucleotide sequence of the murJ gene is shown in SEQ ID NO.

1.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The preparation method comprises the following steps: (1) A total of 200 bp of nucleotide sequences were selected upstream and downstream of the 49th amino acid site encoded by the murJ gene to search for the original spacer sequence that can be recognized and cut by the CRISPR-Cas9 system; (2) Construction of pACYC-sgRNA plasmid expressing sgRNA; (3) Introducing the pKD46 plasmid and the pET-Cas9 plasmid into an Escherichia coli strain encoding the murJ gene with a cysteine ​​mutation at position 29 in the genome; (4) Introducing the constructed pACYC-sgRNA plasmid and single-stranded primer into the E. coli strain harboring pKD46 and pET-Cas9 plasmids; (5) The single clones grown on the plate are sequenced and identified, and the successfully edited strains are saved to obtain the Escherichia coli strain.

3. The recombinant Escherichia coli according to claim 2, characterized in that: The single-stranded primer comprises a base sequence encoding cysteine ​​at position 49, and the single-stranded primer has 45 nt homologous sequences upstream and downstream of the murJ gene editing site.

4. A use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the preparation of Lipid II.

5. The use of a recombinant Escherichia coli in the preparation of Lipid II according to claim 4, characterized in that: The following steps are involved: (1) Transfer the obtained recombinant E. coli strain into 500 ml of culture medium for cultivation; (2) When OD600 was 0.1-0.2, 100 nM dehydrotetracycline was used to induce the expression of UppS and MraY; (3) 45 minutes after induction, MTSES was added for 20 minutes; (4) Collect the cultured E. coli by centrifugation, add PBS to re-select the culture, add chloroform and methanol in a ratio of 1:2, and stir for 1 hour to fully disrupt the cells; (5) After the cells are broken and centrifuged, the supernatant is transferred to a new test tube containing PBS and chloroform, stirred at room temperature for 1 hour, and after centrifugation, the middle layer is transferred and dried by rotary evaporation; (6) Dissolve the product after rotary evaporation in a solution of pyridine acetate and n-butanol in a ratio of 1:2, add n-butanol saturated water for extraction, repeat three times, and take the upper phase; (7) Drying the upper phase by rotary evaporation at 40 °C; (8) Dissolve the mixture in a solution of chloroform:methanol = 1:1, blow dry under nitrogen, and collect the Lipid II.