In-vivo multi-site in-situ continuous evolution system and application thereof

Abstract

The invention relates to an in-vivo multi-site in-situ continuous evolution system and application thereof, and belongs to the technical field of biology. The invention provides a universal in-vivo multi-site continuous evolution system for microorganisms, which is composed of a plurality of sgRNAs, T7DNA polymerase mutants and nCas9 proteins, the sgRNAs respectively target different target sequences, the sgRNAs contain MS2 hairpin structures, and the T7DNA polymerase mutants and MCP proteins are subjected to fusion expression. After the continuous evolution system is introduced into a host cell, nCas9 protein generates a single-chain incision in a target sequence under the guidance of sgRNA, T7DNA polymerase error-prone mutants are recruited to the single incision through an MS2 hairpin structure to generate mutation, a mutation library is generated in the target sequence along with growth passage of a host, and the target sequence is obtained by combining application of selective pressure. The forward mutants of a plurality of sites can be rapidly obtained at one time, and the evolution requirements of a cell factory are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an in vivo multi-site in situ continuous evolution system and application thereof. BACKGROUND

[0002] Directed evolution technology is an effective strategy to modify important genetic elements, proteins and microbial cell factories. In vivo continuous evolution technology does not rely on multi-step molecular biology operations such as mutation-transformation-selection in vitro, and can perform mutation and selection in living microbial cells, reducing human intervention, having the advantages of not being limited by transformation efficiency, saving time and labor, etc., accelerating the evolution of microbial cell factories in laboratory or industrial environment, and thus having important application value in the fields of biological medicine, industrial biotechnology, synthetic biology, etc.

[0003] Although in vivo continuous evolution technology that introduces mutations in the genome can increase genetic diversity, it may interfere with the stability of the host genome, and thus affect the normal growth and function of the cells. Researchers have also developed various in vivo continuous evolution systems targeting specific genes in the genome in microorganisms to minimize non-specific effects on other parts of the genome, such as in vivo continuous evolution systems based on CRISPR technology, including base editors and EvolvR technology, but the base substitution type has preference and the mutation window is usually narrow, and when targeting multiple distant large genes, a large number of sgRNAs need to be designed to cover the target DNA region, making the continuous evolution process time-consuming and costly, limiting the efficiency of multi-site continuous evolution. Therefore, developing an efficient in vivo continuous evolution system targeting long fragments of the genome is of great significance to the development of synthetic biology and biotechnology. SUMMARY

[0004] To solve the above technical problems, the present application provides a microbial in vivo multi-site continuous evolution system based on CRISPR technology and T7 DNA polymerase, which is composed of multiple sgRNAs targeting different target sequences, a T7 DNA polymerase mutant and an nCas9 protein, wherein the sgRNA contains an MS2 hairpin structure, and the T7 DNA polymerase mutant is expressed in fusion with an MCP protein.

[0005] The first object of the present application is to provide a microbial in vivo multi-site in situ continuous evolution system, which comprises:

[0006] (1) multiple sgRNAs targeting different target sequences, respectively.

[0007] (2) a T7 DNA polymerase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 3.

[0008] (3) nCas9 protein.

[0009] Further, the nCas9 protein is obtained by mutating the 10th aspartic acid of the Cas protein to alanine.

[0010] Further, the nCas9 protein gene further has an RBS sequence downstream.

[0011] In an embodiment of the present application, the RBS sequence is shown in SEQ ID NO. 11.

[0012] Further, the sgRNA contains an MS2 hairpin structure.

[0013] Further, the continuous evolution system further comprises a MCP protein.

[0014] Further, the nucleotide sequence of the MCP protein is shown in SEQ ID NO. 2.

[0015] Further, the T7 DNA polymerase mutant is connected to the MCP protein through a Linker, and the amino acid sequence of the Linker is shown in SEQ ID NO. 10.

[0016] The second object of the present application is to provide the use of the above-mentioned multi-site in-situ continuous evolution system in improving the tolerance of a host to multiple antibiotics.

[0017] The third object of the present application is to provide a multi-site continuous evolution method for improving the tolerance of a host to multiple antibiotics, comprising the steps of introducing the continuous evolution system into a starting strain to obtain a recombinant strain, applying a screening pressure to the obtained recombinant strain, and obtaining a positive mutant strain after continuous passage.

[0018] Further, the antibiotics include one or more of rifampicin, streptomycin and spectinomycin.

[0019] The fourth object of the present application is to provide a 30S ribosomal protein S5 mutant, which comprises any one of the following mutations:

[0020] (1) taking the amino acid sequence shown in SEQ ID NO. 5 as the starting sequence, mutating the 25th valine to phenylalanine;

[0021] (2) taking the amino acid sequence shown in SEQ ID NO. 5 as the starting sequence, mutating the 26th lysine to asparagine.

[0022] The fifth object of the present application is to provide the use of the above-mentioned mutant in improving the spectinomycin resistance of E. coli.

[0023] The beneficial effects of the present application are as follows:

[0024] The in-vivo multi-site in-situ continuous evolution system provided by the present application has universality, and can construct a mutation library in a variety of microorganisms, so that the host bacteria can be continuously evolved. The continuous evolution system is introduced into E. coli, and three antibiotics, spectinomycin, streptomycin and rifampicin, are used as screening pressure. After continuous passage, multiple genomic sites are rapidly and effectively evolved, and the tolerance concentration of the obtained mutant strain to rifampicin, spectinomycin and streptomycin is increased from 0 to 200 μg / mL, which is beneficial to the efficient construction of target chassis strains, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0026] Figure 1 The figure is the map of E. coli expression plasmid pET-MT7-3sg in Example 1 of the present application;

[0027] Figure 2 The table shows the rifampicin resistance mutation frequencies of single gene sites and multiple gene sites of E. coli in Example 2 of the present application;

[0028] Figure 3 The table shows the streptomycin resistance mutation frequencies of single gene sites and multiple gene sites of E. coli in Example 2 of the present application;

[0029] Figure 4 The table shows the spectinomycin resistance mutation frequencies of single gene sites and multiple gene sites of E. coli in Example 2 of the present application;

[0030] Figure 5 The table shows the survival ability verification of mutant strains in Example 2 of the present application. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

[0032] The primer sequences involved in the embodiments are shown in Table 1.

[0033] Table 1: Primers and sequences

[0034]

[0035]

[0036] Example 1: Construction of CRISPR-T7 DNA polymerase in-vivo continuous evolution system

[0037] (1) Construction of pET-MT7 plasmid

[0038] The pET28a vector was amplified by conventional PCR using primers pET28a-F1 / pET28a-R1, and the sgRNA expression cassette gene fragment synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. was amplified by primers sgRNA-F / sgRNA-R, wherein the stem loop structure sequence of sgRNA was inserted into 2 MS2 hairpin short sequences.

[0039] The pET28a vector and the sgRNA expression cassette gene fragment obtained by amplification were subjected to homologous recombination by Gibson assembly to obtain the pET-sgRNA plasmid.

[0040] The pET-sgRNA vector, tetracycline inducible promoter gene, nCas9 (Cas9 D10A ) gene (nucleotide sequence as shown in SEQ ID NO. 1), MCP gene (nucleotide sequence as shown in SEQ ID NO. 2) and T7 DNA polymerase error-prone mutant (T7 DNAP D5A / E7A / Y64C / F120L / S399T ) gene fragment (nucleotide sequence as shown in SEQ ID NO. 3) were amplified by conventional PCR: the vector was amplified using primers pET28a-F2 / pET28a-R2, and the tetracycline inducible promoter, nCas9, MCP and T7 DNA polymerase error-prone mutant gene fragments synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. were amplified using primers pTetR-F1 / pTetR-R1, nCas9-F1 / nCas9-R1, MCP-F1 / MCP-R1 and T7-F / T7-R, respectively.

[0041] The vector and the gene fragment obtained by amplification were subjected to homologous recombination by Gibson assembly to obtain the pET-MT7 plasmid.

[0042] (2) Construction of pET-MT7-3sg plasmid

[0043] The pET-MT7 plasmid was amplified by reverse PCR using primers sg1-F / sg1-R, sg2-F / sg2-R and sg3-F / sg3-R, respectively, to obtain sgRNA1 (targeting rpoB, nucleotide sequence as shown in SEQ ID NO. 7) targeting the rpoB (rifampicin resistance related gene, amino acid sequence as shown in SEQ ID NO. 4), sgRNA2 (sgRNA targeting rpsE, nucleotide sequence as shown in SEQ ID NO. 8) targeting the rpsE (spectinomycin resistance related gene, amino acid sequence as shown in SEQ ID NO. 5) and sgRNA3 (sgRNA targeting rpsL, nucleotide sequence as shown in SEQ ID NO. 9) targeting the rpsL gene (streptomycin resistance related gene, amino acid sequence as shown in SEQ ID NO. 6) in the E. coli TG1 genome, to obtain the pET-MT7-1sg (rpoB / rpsE / rpsL) plasmid; then the pET-MT7-1sg (rpoB), pET-MT7-1sg (rpsE) and pET-MT7-1sg (rpsL) plasmids were amplified by conventional PCR using primers pET28a-F3 / pET28a-R3, sg2-F / sg2-R and sg3-F / sg3-R, respectively, and the obtained vector and gene fragments were assembled by Gibson to obtain the plasmid pET-MT7-3sg containing simultaneous targeting of the above three genes, and the plasmid structure is as shown in Figure 1 The T7 DNA polymerase mutant is connected by a Linker (amino acid sequence as shown in SEQ ID NO. 10) and an MCP protein, and an RBS sequence (nucleotide sequence as shown in SEQ ID NO. 11) is present downstream of the nCas9 protein.

[0044] Example 2: Continuous evolution of multiple genomic sites of E. coli

[0045] To evaluate the efficiency of the in vivo evolution system in simultaneously mutating multiple sites in the genome, the recombinant plasmid pET-MT7-1sg or pET-MT7-3sg was transformed into E. coli TG1, and after 1.5 hours of recovery culture at 37°C, the recovery culture was inoculated into fresh LB medium containing kanamycin and 75 ng / mL dehydrated tetracycline at a dilution of 1:500, and after about 14 hours of culture at 37°C, the culture was plated on LB medium with / without the corresponding antibiotic (100 mg / mL rifampicin, 50 mg / mL spectinomycin or 50 mg / mL streptomycin), and the corresponding drug resistance mutation frequency was calculated by calculating the number of drug-resistant colonies and the total number of colonies.

[0046] In E. coli, mutations in rpoB, rpsL and rpsE genes result in resistance to rifampicin, streptomycin and spectinomycin, respectively. As shown in Table 1, the pET-MT7-1 sg strain containing a single sgRNA targeting a single gene resulted in about 100-1000 fold increase in the mutation frequency of rifampicin / streptomycin / spectinomycin compared to the wild type. The pET-MT7-3 sg strain containing three sgRNA expression cassettes in tandem resulted in similar mutation frequency at the single gene sites as the pET-MT7-1 sg strain containing a single sgRNA targeting a single gene. This indicates that the CRISPR-T7 DNA polymerase-based in vivo evolution system has the potential to evolve multiple gene sites in situ. Figures 2-4

[0047] To obtain mutant strains resistant to all three antibiotics, the initial mutagenized culture of pET-MT7-3 sg (OD 600 about 1) was transferred to fresh liquid LB medium at a ratio of 1:10 3 and the final concentrations of the three antibiotics in the LB medium were gradually increased (from 0 to 200 μg / mL) during the continuous transfer culture. The obtained mutagenized culture was plated on LB solid plates containing the above three antibiotics to select for drug-resistant strains. The rpoB, rpsL and rpsE genes were amplified by colony PCR, and the PCR products were subjected to Sanger sequencing.

[0048] After 12 transfers in 8 days, two mutant strains were obtained, both carrying the rpsL K88R mutation, and different mutations in the rpoB and rpsL genes, i.e. M1 (rpoB S508P / L511R , rpsE V25F ) and M2 (rpoB H526L , rpsE K26N ), with a significant increase in the resistance concentration of rifampicin-spectinomycin-streptomycin from 0 to 200 μg / mL. Compared with the starting strain before continuous transfer, strains M1 and M2 showed significantly enhanced growth on plates containing a single antibiotic Figure 5 . This indicates that the in vivo continuous evolution system established in the present application can rapidly and effectively evolve multiple genomic sites.

[0049] Obviously, the above examples are merely illustrative and not limiting. Based on the above description, those skilled in the art can make other different forms of changes or variations. Here, it is not necessary or possible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.​

Claims

1. A multi-site in situ continuous evolution system in a microorganism, characterized by, The multi-site in-situ continuous evolution system comprises: (1) a plurality of sgRNAs respectively targeting different target sequences. (2) a T7 DNA polymerase mutant, wherein the nucleotide sequence of the T7 DNA polymerase mutant is shown as SEQ ID NO.

3. (3) an nCas9 protein.

2. The multi-site in situ sequential evolution system of claim 1, wherein: The nCas9 protein is obtained by mutating the 10th aspartic acid of a Cas9 protein to alanine.

3. The multi-site in situ continuous evolution system of claim 1, wherein: The sgRNA contains an MS2 hairpin structure.

4. The multi-site in situ continuous evolution system of claim 3, wherein: The continuous evolution system further comprises a MCP protein.

5. The multi-site in situ sequential evolution system continuous evolution system of claim 4, wherein: The T7 DNA polymerase mutant is connected to the MCP protein through a Linker, and the amino acid sequence of the Linker is shown as SEQ ID NO.

10.

6. Use of the multi-site in-situ continuous evolution system according to any one of claims 1-4 in improving the antibiotic resistance of a host.

7. A method of multilocus successive evolution for increasing the tolerance of a host to a plurality of antibiotics, characterized in that: The steps comprise introducing the continuous evolution system into a starting strain to obtain a recombinant strain, applying a screening pressure to the obtained recombinant strain, and obtaining a positive mutant strain after continuous passage.

8. The method of multisite sequential evolution of claim 7, wherein: The antibiotics comprise one or more of rifampicin, streptomycin and spectinomycin.

9. A 30S ribosomal protein S5 mutant, characterized in that, The mutant comprises any one of the following mutations: (1) taking the amino acid sequence shown as SEQ ID NO. 5 as a starting sequence, and mutating the 25th valine to phenylalanine; (2) taking the amino acid sequence shown as SEQ ID NO. 5 as a starting sequence, and mutating the 26th lysine to asparagine.

10. Use of the mutant according to claim 9 in improving the spectinomycin resistance of E. coli.

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