A method for directed evolution of escherichia coli antibiotic resistant strains based on cytidine deaminase

Abstract

This invention discloses a method for directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, belonging to the field of microbial directed evolution and genetic engineering technology. This method uses E. coli as the host, introducing a recombinant plasmid expressing an optimized double-stranded cytidine deaminase mutant. Utilizing the low toxicity and high mutagenicity of this mutant, continuous passage evolution is carried out under gradient concentrations of aminoglycoside antibiotics (kanamycin and streptomycin). Combined with whole-genome sequencing, molecular docking, and reverse genetics verification, the A145T missense mutation in the wcaE gene is identified as the core functional site. This invention overcomes the shortcomings of traditional spontaneous and chemical mutagenesis, which suffer from low efficiency and significant strain damage. The mutation type is controllable, the evolutionary cycle is short, and the obtained engineered strains can tolerate up to 300 mg / L kanamycin while exhibiting streptomycin cross-resistance, and the genetic stability of the tolerance trait is strong. This method is simple, highly reproducible, and suitable for industrial breeding of stress-resistant E. coli, and can be widely applied in antibiotic fermentation, industrial microbial culture, and other scenarios.

Description

Technical Field

[0001] This invention relates to the fields of directed evolution of microorganisms, genetic engineering, and industrial microbial stress-resistant breeding, specifically to a method for directed evolution of antibiotic-resistant strains of Escherichia coli based on cytidine deaminase. Background Technology

[0002] Escherichia coli is the most widely used model microorganism in industrial fermentation and molecular biology research, and it has important applications in antibiotic synthesis, enzyme production, and fermentation of chemical intermediates. In culture systems containing aminoglycoside antibiotics, common E. coli is easily inhibited in growth or even dies. Therefore, selecting engineered strains with high antibiotic resistance is a key technology for improving the adaptability of industrial strains and lowering the production threshold.

[0003] Current strain evolution technology has many shortcomings: 1. Traditional adaptive laboratory evolution (ALE) relies on spontaneous mutation of microorganisms, which has extremely low mutation efficiency, a continuous passage cycle of up to tens of days, and low overall breeding efficiency. 2. Chemical mutagenesis and physical mutagenesis (ultraviolet light, radiation) are random mutagenesis, and the mutation sites are uncontrollable. They can easily cause large-scale damage to the genome, leading to a decline in the growth capacity of the strain and high subsequent repair costs. 3. Although the natural double-stranded cytidine deaminase DddA can directly act on double-stranded DNA to achieve base mutations, it is extremely cytotoxic and will severely inhibit the proliferation of host bacteria, making it unsuitable for long-term continuous passage evolution. 4. Most existing evolutionary schemes cannot distinguish between "environmental stress-induced resistance" and "genome-driven mutation resistance", resulting in poor genetic stability of the selected strains and easy loss of resistance after scale-up culture; 5. Most studies only focus on the strain phenotype, making it difficult to locate core functional mutation sites, resulting in weak technical scalability.

[0004] To address the aforementioned technical deficiencies, this invention employs a rationally designed and site-directedly mutated low-toxicity double-stranded cytidine deaminase mutant to construct a controllable whole-genome hypermutation system. Combined with gradient antibiotic stress passage and stress-free stability verification processes, it efficiently selects highly tolerant Escherichia coli while simultaneously elucidating the core mutation mechanism, thus overcoming the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, which solves the problems of low efficiency, large strain damage, uncontrollable mutations, and unstable traits in traditional mutagenesis. This method can rapidly obtain engineered Escherichia coli resistant to high concentrations of kanamycin and streptomycin, while identifying core functional mutation sites and improving the versatility and scalability of the technology.

[0006] As described in claims 1 to 6, the present invention uses an optimized low-toxicity double-stranded cytidine deaminase mutant as a mutagenic element to construct a recombinant plasmid and transform it into Escherichia coli; genomic directional CG→TA base mutation is achieved through IPTG-induced expression, and high-tolerance strains are screened by continuous passage under gradient kanamycin selection pressure; then, the stability of the trait is verified by passage without antibiotics, and cross-tolerance and mutation site analysis experiments are carried out simultaneously.

[0007] (1) Mutagenic element optimization: The tDddA series mutants were selected. Compared with natural DddA, the cytotoxicity was greatly reduced. The high mutation activity and normal growth of the strain were balanced, and long-term continuous passage could be supported. (2) Controllable mutation: Specific catalysis of CG→TA base conversion, single and predictable mutation spectrum, which facilitates subsequent mechanism analysis; (3) High evolution efficiency: Compared with traditional ALE technology, the evolution cycle is shortened by more than 70%, and strains resistant to 300 mg / L kanamycin can be obtained quickly; (4) Stable traits: Antibiotic-free passage was set up to verify that the resistance was driven by genomic mutations and not induced by the environment, and the trait was not lost after industrial scale-up culture; (5) Broad-spectrum tolerance: The obtained strains are resistant to both kanamycin and streptomycin, two types of aminoglycoside antibiotics, and have a wider range of applications; (6) The process is simple, the whole process can be realized in a conventional microbiology laboratory, it is highly reproducible and suitable for large-scale breeding. Attached Figure Description

[0008] Figure 1: Overall process flow diagram of the present invention Figure 2: Schematic diagram of recombinant plasmid structure Figure 3: Growth curves of strains at different kanamycin concentrations Figure 4: Bar chart of streptomycin cross-tolerance growth of target strains Figure 5: Comparison of docking structures between WcaE protein and kanamycin molecules Detailed Implementation

[0009] 1. Recombinant plasmid construction: The tDddA-S1416P gene sequence was synthesized and inserted into the pACYC vector to construct the recombinant expression plasmid pACYC-tDddA-S1416P, which carries a chloramphenicol resistance tag.

[0010] 2. Transformation of the strain: The recombinant plasmid was introduced into Escherichia coli BL21 (DE3) competent cells, plated on LB agar plates containing 34 mg / L chloramphenicol, and incubated at 37 ℃ for 12 h. Single colonies were picked to obtain the initial evolutionary engineered strain.

[0011] 3. Induction of expression and primary culture: Single colonies were inoculated into LB liquid medium (containing 34 mg / L chloramphenicol and 10 mg / L kanamycin), and IPTG was added to a final concentration of 0.1 mM. The culture was carried out at 30 ℃ and 200 r / min for 48 h to induce deaminase expression and initiate preliminary mutagenesis.

[0012] 4. Gradient pressure subcultures were performed, with the cells sequentially transferred to LB medium at kanamycin concentrations of 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L. Each generation was cultured for 48 h, with IPTG and chloramphenicol concentrations maintained throughout the process, and multiple subcultures were completed continuously.

[0013] 5. Target strain screening: OD values ​​were selected from 300 mg / L kanamycin medium. 600 Strains with normal values ​​were selected as candidate highly tolerant strains.

[0014] 6. Stability verification: The candidate strain was transferred to antibiotic-free LB medium and passaged 18 times (total 204 h). It was then inoculated again into 300 mg / L kanamycin medium. The strain could still grow normally, proving that the tolerance was stable.

[0015] 7. Cross-tolerance verification: The strain was inoculated into streptomycin medium at concentrations of 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, respectively. The results showed that the strain could grow stably in the 200 mg / L streptomycin environment and had cross-tolerance.

[0016] 8. Mutation site analysis: Whole genome sequencing of the target strain was performed. Combined with reverse genetics and molecular docking experiments, it was verified that the A145T missense mutation in the wcaE gene is the core functional site. This mutation reconstructs the interaction network between the protein and kanamycin, increases the binding affinity, and thus enhances the strain's tolerance.

Claims

1. A method for directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, characterized in that, Includes the following steps: S1. Construct a recombinant expression plasmid carrying a double-stranded cytidine deaminase mutant gene, wherein the double-stranded cytidine deaminase mutant is any one of tDddA-1416, tDddA-S1416P, or tDddA-S1416L; S2. Transform the recombinant expression plasmid into an *E. coli* host strain to obtain an initial evolutionary engineered bacterium; S3. Add IPTG inducer to the culture system to induce expression of the double-stranded cytidine deaminase mutant, causing a CG→TA directional base mutation in the engineered bacterium genome; S4. Use a culture medium with a gradient increasing kanamycin concentration to perform multiple rounds of continuous subculturing of the engineered bacterium, gradually increasing the antibiotic selection pressure; S5. Screen for target mutant strains that can stably grow under 200–300 mg / L kanamycin conditions; S6. Place the target mutant strain in an antibiotic-free culture medium for continuous subculturing to verify the genetic stability of antibiotic resistance traits.

2. The method according to claim 1, characterized in that: The host strain of Escherichia coli mentioned in step S2 is Escherichia coli BL21 (DE3).

3. The method according to claim 1, characterized in that: In step S4, the kanamycin concentration gradient was set sequentially to 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L, with each round of subculture lasting 48 h.

4. The method according to claim 1, characterized in that: In step S3, the final IPTG concentration is 0.1 mM, the culture temperature is 30 ℃, and the shaking speed is 200 r / min.

5. The method according to claim 1, characterized in that: It also includes a cross-tolerance verification step: the selected target mutant strains are inoculated into culture media containing different concentrations of streptomycin, the growth status of the strains is detected, and the tolerance of the strains to streptomycin is verified.

6. The method according to claim 1, characterized in that: It also includes a mutation site verification step: whole genome sequencing of the target mutant strain, combined with reverse genetics experiments and molecular docking analysis, to determine that the A145T missense mutation of the wcaE gene is the core functional mutation that enhances antibiotic resistance.

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