Microorganism in-vivo continuous evolution system and application thereof

Abstract

The invention relates to a microorganism in-vivo continuous evolution system and application thereof, and belongs to the technical field of biology. A continuous evolution system for realizing directed evolution of microorganisms is constructed based on a CRISPR (clustered regularly interspaced short palindromic repeats) technology and a T5DNA polymerase mutant, and system elements comprise nCas9 protein, the T5DNA polymerase mutant and sgRNA (single guide ribonucleic acid) of a targeted target sequence. In order to further reduce the off-target rate, an MS2 hairpin structure is introduced into sgRNA, and the T5DNA polymerase mutant and MCP protein are subjected to fusion expression, so that the continuous evolution system guides nCas9 protein to generate a single-chain incision in target DNA through the sgRNA, and under the action of a protein recruitment system MS2-MCP, the T5DNA polymerase mutant is introduced into mutation. The system is introduced into a host strain, and is combined with application of screening pressure, so that a target mutant can be obtained through continuous subculture screening, and efficient and rapid evolution of a microbial cell factory is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a continuous evolution system in microorganism and application thereof. BACKGROUND

[0002] Directed evolution technology simulates the process of natural evolution, and through random mutation and recombination, and artificially exerting selection pressure, it screens out biological molecules with desired functions. It provides an effective strategy for improving protein functions (such as thermal stability, catalytic activity and substrate selectivity, etc.) and enhancing the performance of microbial cell factories. Traditional directed evolution technology needs to generate mutation library in vitro, which has the problems of complicated operation, time-consuming and low efficiency. In vivo continuous evolution technology can effectively overcome the above limitations. This technology can realize in situ diversification of DNA sequence in living cells, and automatically accumulate mutation library diversity with cell passage, which greatly reduces the need for artificial intervention. This feature not only facilitates the in-depth exploration of the evolutionary trajectory of microorganisms or proteins, but also provides an efficient tool for analyzing key molecular mechanisms.

[0003] At present, in vivo continuous evolution technologies mainly including error-prone replication and CRISPR-based technologies have been developed for the evolution of specific genes. For example, the error-prone replication-based in vivo evolution technology represented by OrthoRep system usually needs to construct the target sequence to a linear plasmid, which limits the in situ evolution of specific sites in the genome. Base editors and EvolvR technology rely on the specific recognition of target sites by CRISPR system and the introduction of mutations by mutagenic factors to achieve in situ evolution of the genome, but there are limitations in base preference and editing window length. In addition, in vivo continuous evolution systems usually rely on specific host genome environment, which limits the application of in situ evolution in different microbial hosts. Therefore, it is of great significance to develop an efficient in vivo targeted continuous evolution method with host universality to accelerate the evolution efficiency of specific genes in the genome. SUMMARY

[0004] To solve the above technical problems, the present application provides a continuous evolution system for realizing directed evolution of microorganisms. The system elements include nCas9 protein, T5 DNA polymerase mutant and sgRNA targeting target sequence, and further introducing MS2 hairpin structure in sgRNA, and fusing and expressing T5 DNA polymerase mutant with MCP protein, so that the continuous evolution system generates single-strand cut in target DNA by guiding nCas9 protein by sgRNA, and under the action of protein recruitment system MS2-MCP, T5 DNA polymerase mutant introduces mutations. The continuous evolution system is introduced into host strain, combined with screening pressure and continuous passage, and target positive mutant can be obtained.

[0005] The first object of the present application is to provide a continuous evolution system in a microorganism, which comprises the following elements:

[0006] (1) an nCas9 protein;

[0007] (2) a T5 DNA polymerase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 1;

[0008] (3) an sgRNA targeting a target sequence.

[0009] Further, the nCas9 protein is obtained by mutating the 10th aspartic acid of a Cas9 protein to alanine.

[0010] Further, an RBS sequence is present upstream of the nCas9 protein.

[0011] In an embodiment of the present application, the RBS sequence is shown in SEQ ID NO. 5.

[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. 4.

[0015] Further, the T5 DNA polymerase mutant is expressed in fusion with the MCP protein.

[0016] Further, a Linker is present between the T5 DNA polymerase mutant and the MCP protein.

[0017] In an embodiment of the present application, the amino acid sequence of the Linker is shown in SEQ ID NO. 3.

[0018] The second object of the present application is to provide the use of the above continuous evolution system in improving the transport efficiency of the di-arginine transport pathway of a host.

[0019] The third object of the present application is to provide a method for improving the efficiency of the di-arginine transport pathway of E. coli by continuous evolution, the steps of which comprise introducing the continuous evolution system into a starting strain to obtain a recombinant strain, applying a selection pressure to the obtained recombinant strain, and obtaining a positive mutant strain after continuous passage.

[0020] Further, the method further comprises the step of introducing an auxiliary plasmid into the starting strain, the auxiliary plasmid expressing a TorA signal peptide and a fusion protein, the fusion protein consisting of a fluorescent protein and a beta-lactamase.

[0021] The TorA signal peptide can be recognized by the transmembrane protein Tat system of E. coli and guide the downstream fusion protein to be transported to the periplasmic space. The fusion protein realizes periplasmic space expression and has a biological activity function, allowing double screening by using periplasmic fluorescence detection and ampicillin resistance selection methods.

[0022] Further, the nucleotide sequence of the TorA signal peptide is shown as SEQ ID NO. 7.

[0023] Further, the fluorescent protein is green fluorescent protein EGFP.

[0024] Further, the nucleotide sequence of the EGFP-beta-lactamase fusion protein is shown as SEQ ID NO. 8.

[0025] A fourth object of the present application is to provide a TatC protein mutant, which comprises any one of the following mutations:

[0026] (1) taking the amino acid sequence shown in SEQ ID NO. 11 as the starting sequence, mutating the asparagine at position 242 to threonine;

[0027] (2) taking the amino acid sequence shown in SEQ ID NO. 11 as the starting sequence, mutating the amino acid coding sequence at position 244 from GAA to GGAA.

[0028] A fifth object of the present application is to provide a gene encoding the above-mentioned TatC protein mutant.

[0029] A sixth object of the present application is to provide the use of the above-mentioned TatC protein mutant or the above-mentioned gene in improving the transport efficiency of the double-arginine transport pathway of E. coli.

[0030] The present application has the following beneficial effects:

[0031] The present application provides a microbial continuous evolution system based on CRISPR technology and T5 DNA polymerase mutants. By continuous passage, the target sequence of the host microorganism accumulates mutations to produce a mutation library. The addition of an auxiliary plasmid enables the system to have two screening strategies: growth coupling and fluorescence detection. This allows the screening of strains with strong survival ability and high fluorescence expression, and efficient acquisition of target positive mutants. According to the Tat system evolution method established based on the continuous evolution system, the ampicillin resistance of E. coli is increased from 75 μg / mL to 400 μg / mL. Further application of the system to K. lactis increases the mutation rate of the strain by 6.6 x 10 4 Therefore, the microbial continuous evolution system has important application value for the evolution of the chassis strain. Attached Figure Description

[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0033] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pET-LT5 in Example 1 of the present invention;

[0034] Figure 2 This is a schematic diagram of the structure of the recombinant plasmid pET-MT5 in Example 1 of the present invention;

[0035] Figure 3 This is a schematic diagram of the structure of the report plasmid pReporter in Embodiment 1 of the present invention;

[0036] Figure 4 This is a schematic diagram of the structure of the auxiliary plasmid pTorA in Embodiment 1 of the present invention;

[0037] Figure 5 The mutation rate at different sites of the strain in Example 2 of this invention with the recombinant plasmid pET-LT5 introduced;

[0038] Figure 6 The mutation rate and off-target rate of the strains in Example 2 of this invention that have been introduced with recombinant plasmids pET-LT5 and pET-MT5;

[0039] Figure 7 The fluorescence intensity of the strain introduced with the helper plasmid pTorA in Example 3 of this invention after different inductions;

[0040] Figure 8 This demonstrates the survival ability of the strain with the helper plasmid pTorA introduced in Example 3 of the present invention under different concentrations of ampicillin.

[0041] Figure 9 This is a schematic diagram of the continuous in vivo evolution of the Tat protein in Example 4 of the present invention;

[0042] Figure 10 The survival ability of the shake-flask fermentation mutant strains CM1 and CM2 in Example 4 of this invention;

[0043] Figure 11 The fluorescence expression intensities of the shake-flask fermentation mutant strains CM1 and CM2 in Example 4 of this invention;

[0044] Figure 12 This is a map of the Kluyveromyces lactis expression plasmid pUDP-MT5 in Example 5 of the present invention;

[0045] Figure 13 The mutation probability of Kluyveromyces lactis in Example 5 of this invention. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0047] The primer sequences involved in the following examples are shown in Table 1.

[0048] Table 1 Primers and Sequences

[0049]

[0050]

[0051] Example 1: Construction of an in vivo evolutionary system based on CRISPR technology and T5 DNA polymerase

[0052] (1) Construction of pET-LT5 plasmid

[0053] Conventional PCR amplification of pET28a vector and sgRNA expression cassette gene fragment: The vector was amplified using primers pET28a-F / pET28a-R, and the sgRNA-1 expression cassette gene fragment synthesized by Sangon Biotech (Shanghai) Co., Ltd. was amplified using primers sgRNA-F / sgRNA-R.

[0054] The pET28a vector and sgRNA-1 expression cassette gene fragment obtained by Gibson assembly were homologously recombined to obtain the pET-sgRNA1 plasmid.

[0055] Conventional PCR amplification of the pET-sgRNA1 vector, tetracycline-inducible promoter gene fragment, and T5 DNA polymerase error-prone mutant (T5 DNAP) D164A / E166A / A593R / I308V ) gene fragment (nucleotide sequence as shown in SEQ ID NO.1) and nCas9 (Cas9 D10A Gene fragments (nucleotide sequences as shown in SEQ ID NO.2): The vector was amplified using primers pET-sgRNA1-F / pET-sgRNA1-R, and the tetracycline-inducible promoter, T5 DNA polymerase error-prone mutant, and nCas9 gene fragment synthesized by Sangon Biotech (Shanghai) Co., Ltd. were amplified using primers pTetR-F / pTetR-R, T5-F / T5-R, and nCas9-F1 / nCas9-R, respectively.

[0056] The amplified pET-sgRNA1 vector, tetracycline-induced promoter, T5 DNA polymerase error-prone mutant, and nCas9 gene fragment were homologously recombined using Gibson assembly. The T5 DNA polymerase error-prone mutant and nCas9 protein were linked together via a linker (amino acid sequence shown in SEQ ID NO. 3) to obtain the pET-LT5 plasmid. The plasmid structure is shown below. Figure 1 As shown.

[0057] (2) Construction of pET-MT5 plasmid

[0058] The sgRNA-2 expression cassette gene fragment containing two MS2 hairpin structure sequences, synthesized by Sangon Biotech (Shanghai) Co., Ltd., was amplified by conventional PCR using primers sgRNA-F / sgRNA-R. The vector obtained in step 1 was then assembled with the gene fragment using Gibson assembly to obtain pET-sgRNA2.

[0059] Conventional PCR amplification of the pET-sgRNA2 vector, tetracycline-induced promoter gene fragment, and T5 DNA polymerase error-prone mutant (T5 DNAP) D164A / E166A / A593R / I308V Gene fragments, MCP protein gene fragments (amino acid sequences shown in SEQ ID NO.4), and nCas9 (Cas9) D10A Gene fragments: The vector was amplified using primers pET-sgRNA2-F / pET-sgRNA2-R. The tetracycline-inducible promoter, T5 DNA polymerase error-prone mutant, MCP and nCas9 gene fragments synthesized by Sangon Biotech (Shanghai) Co., Ltd. were amplified using primers pTetR-F / pTetR-R, T5-F / T5-R, MCP-F / MCP-R and nCas9-F2 / nCas9-R, respectively.

[0060] The pET-sgRNA2 vector, tetracycline-induced promoter, T5 DNA polymerase error-prone mutant, MCP, and nCas9 gene fragments obtained from the amplification of sample 6 were homologously recombined using Gibson assembly. The T5 DNA polymerase error-prone mutant and MCP protein were linked by a linker. An RBS sequence (nucleotide sequence shown in SEQ ID NO. 5) exists between the MCP and nCas9 proteins to obtain the pET-MT5 plasmid. The plasmid structure is shown below. Figure 2 As shown.

[0061] (3) Construction of pReporter plasmid

[0062] Conventional PCR amplification of the pACYCDuet-1 vector and the spectinomycin resistance gene aadA*(aadA) containing a nonsense mutation. L33StopThe pACYCDuet vector was amplified using primers pACYC-F / pACYC-R, and the laboratory-preserved aadA* gene fragment was amplified using primers aadA*-F / aadA*-R.

[0063] The amplified pACYCDuet vector and aadA* gene fragment were homologously recombined using Gibson assembly to obtain the pReporter plasmid. The plasmid structure is shown below. Figure 3 As shown.

[0064] (4) Construction of pTorA plasmid

[0065] Conventional PCR amplification of the pACYCDuet vector backbone, arabinose-inducible promoter, TorA signal peptide, and EGFP-β-lactamase gene fragment: pACYCDuet was amplified using primers pACYCDuet-F / pACYCDuet-R as the vector, and the arabinose-inducible promoter (nucleotide sequence as shown in SEQ ID NO. 6), TorA signal peptide (nucleotide sequence as shown in SEQ ID NO. 7), and EGFP-β-lactamase gene fragment (nucleotide sequence as shown in SEQ ID NO. 8) were amplified using primers Para-F / Para-R, TorA-F / TorA-R, EGFP-F / EGFP-R, and Bla-F / Bla-R.

[0066] The amplified pACYCDuet vector, arabinose-inducible promoter, TorA signal peptide, and EGFP and β-lactamase gene fragments were homologously recombined using Gibson assembly to obtain the pTorA plasmid. The plasmid structure is shown below. Figure 4 As shown.

[0067] (5) Construction of pUDP-MT5 plasmid

[0068] The yeast codon-optimized nCas9 gene fragment synthesized by Sangon Biotech (Shanghai) Co., Ltd. was amplified by conventional PCR using primers ynCas9-F1 / ynCas9-R1, and the pUDP002 vector was amplified by conventional PCR using primers pUDP-F1 / pUDP-R1.

[0069] The pUDP002 vector and the nCas9 expression cassette gene fragment obtained by Gibson assembly were homologously recombinated to obtain the pUDP-nCas9 plasmid.

[0070] Conventional PCR amplification of the pUDP002 vector backbone, the fused T5 DNA polymerase mutant-MCP, nCas9, and sgRNA gene fragments: Conventional PCR amplification of the pUDP002 vector using primers pUDP-F2 / pUDP-R2 and pUDP-F3 / pUDP-R3 yielded pUDP002-1 and pUDP002-2 vector fragments. Conventional PCR amplification of the yeast codon-optimized T5 DNA polymerase mutant-MCP gene fusion fragment synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the pUDP-nCas9 plasmid yielded the T5-MCP fusion gene, nCas9 gene expression cassette, and sgRNA gene fragment.

[0071] The pUDP002-1 and pUDP002-2 vector fragments obtained by Gibson assembly were homologously recombined with the T5-MCP fusion gene, the nCas9 gene expression cassette, and the sgRNA gene fragment to obtain the pUDP-MT5 plasmid.

[0072] Example 2: Mutation rate in Escherichia coli using an in vivo evolutionary system based on CRISPR technology and T5 DNA polymerase.

[0073] The pET-LT5 and pReporter plasmids were co-transformed into *E. coli* TG1 strain. After recovery at 37°C with shaking for 1.5 h, tetracycline-inducing agent was added, and the culture was transferred to 25 mL of LB medium and incubated at 37°C with shaking for approximately 12 h. The culture was then appropriately diluted and plated onto plates containing and without spectinomycin. The mutation rate per base per generation was calculated based on the number of spectinomycin resistance reversion mutant colonies and the total number of colonies. The results are as follows: Figure 5 As shown, compared with the wild-type strain, the mutation rate at the target site of the pET-LT5-expressing strain was increased by approximately 7.1 × 10⁻⁶ at a distance of 1 bp from the nCas9 nick. 5 The mutation rate was 1000 times higher than that of the wild-type strain at a position 2044 bp away from the nCas9 incision site, indicating that the system can effectively mutate the target gene with an action length of up to 2-kb.

[0074] The pET-MT5 recombinant plasmid was constructed using the MS2-MCP system, replacing the T5 DNA polymerase mutant with a flexible linker for nCas9 fusion expression. In this system, the T5 DNA polymerase mutant fused with MCP protein is recruited to the vicinity of the nCas9 protein due to the interaction between MCP and the MS2 hairpin structure in the sgRNA stem-loop, thereby increasing the mutation rate in the single-strand nick region. The pET-MT5 plasmid and pReporter plasmid were co-transformed into *E. coli* TG1 strain. After recovery at 37°C with shaking for 1.5 h, tetracycline inducer was added, and the culture was transferred to 25 mL of LB medium and incubated at 37°C with shaking for approximately 12 h. After appropriate dilution, the culture was plated on plates containing and without spectinomycin. The mutation rate per base per generation was calculated based on the number of spectinomycin resistance reversion mutant colonies and the total number of colonies. The results are as follows: Figure 6 As shown, compared with the pET-LT5 strain, the mutation rate of pET-MT5 was not significantly affected, but the off-target rate was reduced by 96.8%. The off-target mutation rate of pET-MT5 was about 43 times higher than that of the wild-type strain, indicating that the in vivo evolution system based on CRISPR technology and T5 DNA polymerase in this invention is an efficient in vivo continuous evolution method.

[0075] Example 3: Construction of a screening method for Tat protein mutants

[0076] The TorA signal peptide can be recognized by the Tat transporter system, which guides the transport of downstream proteins to the periplasmic space. The helper plasmid PtorA was transformed into *E. coli* TG1, and single colonies were picked and placed into 14 mL tubes containing 2 mL of liquid LB. The cells were cultured overnight at 37°C. A 1% inoculum was then added to 250 mL Erlenmeyer flasks containing 20 mL of liquid LB, and 50 mmol / L arabinose inducer was added. Fermentation was carried out at 37°C for 10 h, and the fluorescence expression intensity in the fermentation broth and periplasmic space, as well as the cell viability on LB agar plates containing different concentrations of ampicillin, were measured.

[0077] Periplasmic space proteins were obtained by treating cells with arginine solution: Cells were first collected by centrifugation, washed once with PBS buffer at 4°C, and then resuspended in arginine solution (0.4 mol / L, pH 8.0) at a 1:10 dilution. After incubation at 4°C for 45 minutes, the supernatant was collected by centrifugation, and the fluorescence intensity was detected using a microplate reader; this was the periplasmic space fluorescence expression intensity. Cell growth capacity was characterized by serially diluting the fermentation culture 10-fold with phosphate buffer and spotting 1 μL of each dilution onto LB agar plates containing different concentrations of ampicillin.

[0078] The results are as follows Figure 7As shown, the periplasmic fluorescence expression intensity of the strain containing the PtorA plasmid was basically the same as that of the blank strain without the addition of an inducer; after induction with 50 mmol / L arabinose, the periplasmic spatial fluorescence intensity of the strain increased by about 1-fold. Meanwhile, the strain containing the PtorA plasmid showed growth inhibition on plates containing more than 100 μg / mL ampicillin. Figure 8 This indicates that the EGFP-β-lactamase fusion protein achieves periplasmic spatial expression and possesses biological activity, allowing for dual screening of the Tat transporter mutant library using periplasmic fluorescence detection and ampicillin resistance selection.

[0079] Example 4: Continuous evolution and screening validation of Tat protein

[0080] The *E. coli* transmembrane transport system (Tat) consists of TatA, TatB, and TatC proteins, with their corresponding coding genes arranged sequentially in the genome to form the tatABC gene cluster. Plasmid pET-MT5 is targeted at the C-terminus of the tatC protein gene in the Tat system (the original spacer sequence of the sgRNA is shown in SEQ ID NO. 9) to cover the tatABC gene cluster and generate mutations, thereby constructing a Tat protein mutation library (the principle is illustrated in the diagram). Figure 9 (As shown).

[0081] Since plasmid pET-MT5 and helper plasmid PtorA were co-transformed into *E. coli* TG1, after resuscitation culture at 37°C for 1.5 h, the culture was inoculated at a 1:500 dilution into fresh LB liquid medium containing chloramphenicol and kanamycin, with 50 ng / mL of adipic tetracycline added. The culture was then incubated at 37°C with shaking for approximately 14 h. The mutagenic culture was then transferred at a 1:100 inoculum to fresh LB liquid medium containing 75 μg / mL ampicillin. When the culture grew to OD... 600 When the concentration of the induced mutant culture reaches approximately 1, it is transferred to the next round of subculture, and the ampicillin concentration gradient is gradually increased to increase the selection pressure and automatically enrich the positive mutant strain. During the continuous evolution process, the culture was transferred 8 times, and the ampicillin concentration was increased from 75 μg / mL to 400 μg / mL.

[0082] Mutagenic cultures containing 400 μg / mL ampicillin were streaked, and single colonies were picked for PCR amplification of the TatABC gene in the genome. Sanger sequencing was then used to obtain Tat transporter mutant strains. All mutations occurred in the TatC gene (nucleotide sequence as shown in SEQ ID NO. 10, amino acid sequence as shown in SEQ ID NO. 11). The mutant strains were CM1 (TatC...). N242TCM1 and CM2 (the amino acid coding sequence at position 244 of TatC is changed from GAA to GGAA). Mutant strains CM1 and CM2 were shake-flask fermented, with the starting strain as the control. The fluorescence expression intensity per unit pericosteal space and the survival ability on LB agar plates containing high concentrations of ampicillin were measured. Results are as follows: Figure 10 As shown, in the presence of 400 μg / mL ampicillin, the survival abilities of strains CM1 and CM2 were significantly improved compared to the original strain, and strain CM2 showed slightly higher tolerance to ampicillin than strain CM1. Further analysis of fluorescence expression intensity in the periplasmic space revealed that the fluorescence intensity of strains CM1 and CM2 was 3.4 and 5.1 times higher than that of the wild-type strain, respectively. Figure 11 This demonstrates that the present invention can efficiently achieve rapid evolution of microbial cell factories using an in vivo continuous evolution system based on CRISPR technology and T5 DNA polymerase.

[0083] Example 5: Evaluation of Kluyveromyces lactis mutation efficiency

[0084] The T5 DNA polymerase error-prone mutant (T5 DNAP) D164A / E166A / A593R / I308V ), a flexible linker composed of 23 amino acids, the MCP gene, and nCas9 (Cas9 D10A After codon optimization, the gene was constructed into the pUDP002 vector to obtain the pUDP-MT5 plasmid. Figure 12 The Ura3 gene in the genome of *Kluyveromyces lactis* GG799 was integrated and modified into the complete Ura gene Ura*(UraA131STOP) containing the premature stop codon TAA (nucleotide sequence shown in SEQ ID NO. 12). The recombinant plasmid was electroporated into GG799 competent cells and incubated at 30°C for 1 hour. Then, 1 mL of the resuscitation culture was transferred to 25 mL of fresh YPD medium containing hygromycin B, and the cells were grown at 30°C until saturation. Cells were collected and washed twice with sterile water, and an appropriate amount of cells were plated on SD plates with or without uracil.

[0085] The results showed that targeting the Ura* gene in the Kluyveromyces lactis genome with sgRNA, causing the premature stop codon TAA to be 11 bp away from the single-strand cut in nCas9, increased the mutation rate of the strain by 6.6 × 10⁻⁶ compared to the wild-type strain. 4 times ( Figure 13 This demonstrates the universality of the in vivo continuous evolution system based on CRISPR-T5 DNA polymerase in various industrial microorganisms.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A system for continuous evolution in a microorganism, comprising, The continuous evolution system comprises the following elements: (1) nCas9 protein; (2) T5 DNA polymerase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 1; (3) sgRNA targeting target sequence.

2. The continuous evolution system of claim 1, wherein: The nCas9 protein is obtained by mutating the 10th aspartic acid of Cas9 protein to alanine.

3. The continuous evolution system of claim 1, wherein: The sgRNA contains MS2 hairpin structure.

4. The continuous evolution system of claim 3, wherein: The continuous evolution system further comprises MCP protein.

5. Use of the continuous evolution system according to any one of claims 1-4 in improving the efficiency of double-arginine transport pathway of host.

6. A method for improving the transport efficiency of the di-arginine transport pathway in E. coli by directed evolution, characterized by: The steps comprise introducing the continuous evolution system into the starting strain to obtain a recombinant strain, applying screening pressure to the obtained recombinant strain, and obtaining a positive mutant strain after continuous passage.

7. The method of claim 6, wherein: The steps further comprise introducing an auxiliary plasmid into the starting strain, the auxiliary plasmid expressing TorA signal peptide and fusion protein, the fusion protein consisting of fluorescent protein and beta-lactamase.

8. A mutant of a TatC protein, characterized in that, The TatC protein mutant comprises any one of the following mutations: (1) taking the amino acid sequence shown in SEQ ID NO. 11 as the starting sequence, mutating the 242nd asparagine to threonine; (2) taking the amino acid sequence shown in SEQ ID NO. 11 as the starting sequence, mutating the 244th amino acid coding sequence from GAA to GGAA.

9. Gene encoding the TatC protein mutant of claim 8.

10. Use of the TatC protein mutant of claim 8 or the gene of claim 9 in improving the transport efficiency of double-arginine transport pathway of Escherichia coli.

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