A continuous evolution system based on T7 RNA polymerase mutants and application thereof

Abstract

The present application relates to a kind of continuous evolution system based on T7 RNA polymerase mutant and its application, belong to the technical field of evolutionary engineering.The present application is based on the T7 RNA polymerase mutant with the ability of synthesizing single-stranded DNA, constructs the continuous evolution system comprising single-stranded DNA synthesis template and T7 RNA polymerase mutant, the continuous evolution system is introduced into host, T7 RNA polymerase mutant is recombined with target gene by synthesizing single-stranded DNA, so that host is constructed to obtain mutant library in the process of continuous passage, host is subjected to screening pressure, and host realizes continuous evolution.The system is applied to complete the evolution of tetracycline efflux pump protein in 7 days, and the tryptophan deficiency of saccharomyces cerevisiae is repaired in 24 hours.The tetracycline efflux pump protein mutant screened can tolerate tetracycline and tigecycline, and the tolerance to tigecycline is increased by 8 times, and the tolerance to tetracycline is increased by 2 times, which is of great significance to improve the performance of cell factory.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of evolutionary engineering, and particularly relates to a continuous evolution system based on a T7 RNA polymerase mutant and application thereof. BACKGROUND

[0002] Microorganisms are widely distributed in nature and are subjected to various environmental stresses such as temperature, pH, nutrient limitation, and toxic substances. These stresses can affect the growth and reproduction of microorganisms, and only those microorganisms that can adapt to these stresses can survive and continue to reproduce. Through continuous evolution, microorganisms can gradually accumulate beneficial genetic variations and form strains that are more adapted to the environment, thereby improving their stress resistance and survival ability. Continuous evolution of microorganisms can also endow microorganisms with new functions, such as degrading specific pollutants and synthesizing new metabolites. These new functions are of great significance to environmental protection and biological manufacturing. Studying the genetic variations and adaptive changes of microorganisms during continuous evolution can provide a deeper understanding of the basic principles and dynamics of biological evolution.

[0003] Continuous evolution is a laboratory evolution technique that simulates the natural evolution process by generating targeted or non-targeted mutations in living cells and accumulating these mutations during passaging to form a mutant library. This process can improve the environmental tolerance, substrate utilization efficiency, and product synthesis efficiency of microbial cell factories. However, current strategies for continuous evolution of microorganisms often have various defects, such as the need for manual design of mutation libraries in the MAGE method, discontinuity, dependence on DNA Q polymerase in the ribosome switch method, inability to evolve specific genes, limited gene editing length in the EvolvR method, and the need for multiple elements to work together to induce mutations, which limits its universality. In addition, genome in situ large fragment targeted evolution techniques rely excessively on deaminases, which inevitably cause cytotoxicity and off-target risks. Therefore, a new continuous evolution strategy is urgently needed to achieve in situ evolution of large fragments of the genome. SUMMARY

[0004] To solve the above technical problems, the present application provides a continuous evolution system based on a T7 RNA polymerase mutant and application thereof. The present application is based on a T7 RNA polymerase mutant with the ability to synthesize single-stranded DNA, and constructs a continuous evolution system containing a single-stranded DNA synthesis template and a T7 RNA polymerase mutant. The continuous evolution system is introduced into a host, and the T7 RNA polymerase mutant recombines with the target gene by synthesizing single-stranded DNA, so that the host constructs a mutant library during continuous passaging. The host is subjected to a selection pressure, and the host realizes continuous evolution.

[0005] The first object of the present application is to provide a continuous evolution system comprising a single-stranded DNA synthesis template and a T7 RNA polymerase mutant, wherein the T7 RNA polymerase mutant has the following mutations: the 10th aspartic acid of the starting sequence shown in SEQ ID NO. 4 is mutated to glutamic acid, the 581st isoleucine is mutated to phenylalanine, the 586th alanine is mutated to valine, and the 615th alanine is mutated to threonine.

[0006] Further, the nucleotide sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO. 1.

[0007] The T7 RNA polymerase mutant has the ability to synthesize single-stranded DNA, and can recombine with the target gene and introduce mutations according to the single-stranded DNA synthesis template. The mutant library can be obtained by continuous passage of the host strain containing the above-mentioned first expression vector and the second expression vector.

[0008] Further, the single-stranded DNA synthesis template uses three T7 terminators in series to terminate expression.

[0009] Further, the continuous evolution system further comprises a single-stranded annealing protein coding gene.

[0010] Further, the coding gene of the T7 RNA polymerase mutant and the single-stranded annealing protein coding gene are expressed in the form of multiple cistrons.

[0011] Further, the continuous evolution system uses a medium copy plasmid as an expression vector. T7 RNA polymerase has a large metabolic burden on cells, and high copy plasmids cause cytotoxicity, resulting in slow cell growth, while the expression level of low copy plasmids is too low to affect the efficiency of the continuous evolution strategy.

[0012] The second object of the present application is to provide the use of the above-mentioned continuous evolution system in restoring tryptophan deficiency in Saccharomyces cerevisiae, comprising introducing the continuous evolution system into a starting strain to obtain a recombinant strain, wherein the tryptophan synthesis gene in the genome of the starting strain has a nonsense mutation, and inoculating the recombinant strain on a culture medium without tryptophan to screen for colonies that can grow on the culture medium.

[0013] The third object of the present application is to provide the use of the above-mentioned continuous evolution system in screening for tetracycline efflux pump protein mutants.

[0014] Further, the steps include introducing the continuous evolution system into a starting strain to obtain a recombinant strain, inoculating the recombinant strain on a culture medium coated with tetracycline and / or tigecycline, and screening for colonies that can grow on the culture medium for continuous passage.

[0015] A fourth object of the present application is to provide a tetracycline efflux pump protein mutant, wherein the tetracycline efflux pump protein is obtained by modifying the starting sequence as shown in SEQ ID NO. 3 at any one of the following positions:

[0016] (1) the leucine at position 83 is mutated to proline, and the glycine at position 154 is mutated to serine;

[0017] (2) the leucine at position 83 is mutated to proline;

[0018] (3) the leucine at position 12 is mutated to valine;

[0019] (4) the tyrosine at position 58 is mutated to histidine.

[0020] Preferably, the tetracycline efflux pump protein is obtained by mutating the leucine at position 83 to proline in the starting sequence as shown in SEQ ID NO. 3.

[0021] Preferably, the tetracycline efflux pump protein is obtained by mutating the leucine at position 83 to proline and the glycine at position 154 to serine in the starting sequence as shown in SEQ ID NO. 3.

[0022] A fifth object of the present application is to provide a gene encoding the above-mentioned tetracycline efflux pump protein mutant.

[0023] A sixth object of the present application is to provide an expression vector comprising the above-mentioned gene.

[0024] A seventh object of the present application is to provide the use of the above-mentioned tetracycline efflux pump protein mutant, the above-mentioned gene, or the above-mentioned expression vector in improving the tolerance of a host to tetracycline and / or tobramycin.

[0025] The beneficial effects of the present application are as follows:

[0026] The present application provides a continuous evolution system based on T7 RNA polymerase mutants, which can effectively promote the continuous evolution of hosts, and is particularly suitable for screening host mutants with specific functions (such as tetracycline and / or tobramycin tolerance). In the system, a single-stranded DNA synthesis template uses three T7 terminators in series to terminate expression, ensuring precise regulation and high efficiency of gene expression. The T7 RNA polymerase mutant encoding gene and the single-strand annealing protein encoding gene are expressed in the form of multiple cistrons, improving the expression efficiency and system stability. The system completes the evolution of tetracycline efflux pump protein within 7 days and repairs the tryptophan deficiency of Saccharomyces cerevisiae within 24 hours. The tetracycline efflux pump protein mutant screened can tolerate tetracycline and tobramycin, and the tolerance to tobramycin is increased by 8 times, and the tolerance to tetracycline is increased by 2 times, which is of great significance for improving the performance of cell factories. BRIEF DESCRIPTION OF DRAWINGS

[0027] 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 accompanying drawings, in which

[0028] Figure 1 Schematic diagram of the continuous evolution system principle based on T7 RNA polymerase mutant of the present application;

[0029] Figure 2 Map of the recombinant plasmid pTM-EVO in Example 1 of the present application;

[0030] Figure 3 Map of the recombinant plasmid pCE-GOI in Example 2 of the present application;

[0031] Figure 4 Mutation frequency of different sites of tetracycline efflux pump protein in Example 4 of the present application;

[0032] Figure 5 Results of tetracycline and tigecycline tolerance experiments of wild-type and mutant strains in Example 4 of the present application;

[0033] Figure 6 Map of the evolution plasmid pY15_v6 for Saccharomyces cerevisiae in Example 5 of the present application;

[0034] Figure 7 Mutation rate of T7 RNA polymerase mutant in Saccharomyces cerevisiae in Example 5 of the present application. DETAILED DESCRIPTION

[0035] The present application will be further described below in conjunction with the accompanying 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.

[0036] Seed culture and fermentation of recombinant strains:

[0037] LB medium (g / L): tryptone 10, yeast powder 5, NaCl 10.

[0038] YPD medium (g / L): yeast powder 10, tryptone 20, glucose 20.

[0039] YNB plate: mother liquor purchased from Shenguo Biotech (Order NO. A610507), diluted according to the instructions and then added with agar with a final concentration of 2.5%.

[0040] Antibiotic concentration (mg / L): kanamycin 50, spectinomycin 50, tetracycline 50.

[0041] Inducer concentration (mM): Isopropyl-beta-D-thiogalactopyranoside (IPTG) 0.2, arabinose 0.5.

[0042] Culture condition: Seed cultured for 12 h at 37℃, 220 rpm was transferred into fermentation medium at 0.1% inoculation amount, and cultured at 37℃, 220 rpm for 12 h. Antibiotics and inducers were added according to the culture target.

[0043] Continuous passage: 1000 μL of fermentation medium and corresponding antibiotics and inducers were added to each well of a 24-well plate. After inoculation at 0.1%, it was transferred at 0.1% every 12 h, and it was considered to be passed for 10 generations.

[0044] Continuous evolution: Under appropriate culture conditions, a mutant library was generated in the fermentation broth, and a target mutant was obtained by combining a suitable screening strategy.

[0045] Example 1: Construction of T7 RNA polymerase mutant expression vector

[0046] A T7 RNA polymerase mutant expression vector was constructed using pTM plasmid as a template. The T7 RNA polymerase mutant (nucleotide sequence as shown in SEQ ID NO. 1) capable of synthesizing single-stranded DNA and the single-stranded annealing auxiliary protein CspRecT (GenBank: OP072625.1) were encoded on a medium copy plasmid containing a spectinomycin resistance gene, and were induced to express by arabinose. The single-stranded annealing auxiliary protein and the T7 RNA polymerase mutant were expressed in a multi-cistronic form, and the obtained recombinant plasmid was named pTM-EVO, as shown in Figure 2 .

[0047] Example 2: Construction of single-stranded DNA synthesis template

[0048] The single-stranded DNA synthesis template of the T7 RNA polymerase mutant (nucleotide sequence as shown in SEQ ID NO. 2) was encoded on a medium copy plasmid containing a kanamycin resistance gene, and was expressed using an IPTG inducible T7 promoter. Three T7 terminators in series were used to terminate expression, and the obtained recombinant plasmid was named pCE-GOI, as shown in Figure 3 .

[0049] Example 3: Construction of recombinant Escherichia coli

[0050] The constructed recombinant plasmid pTM-EVO was transformed into Escherichia coli TOP10, and the transformants were selected for colony PCR to verify that the construction of recombinant Escherichia coli was successful. The recombinant strain was named TOP11.

[0051] The constructed recombinant plasmid pTM-GOI was transformed into Escherichia coli TOP11, and the transformants were selected for colony PCR to verify that the recombinant E. coli was successfully constructed, and the recombinant strain was named TOP12.

[0052] The tetracycline efflux pump protein (TetK) (GenBank: CP033733.1, amino acid sequence as shown in SEQ ID NO. 3) was knocked into the genome of the strain TOP12 using the CRISPR / Cas9 tool to make it resistant to tetracycline. After colony PCR verification and sequencing, it was named TOP12-tetK. The primers used for gene knockout are as follows:

[0053] Tet-zb-f: 5'-cgacgaacaccaaaaaatgaccagc-3';

[0054] Tet-zb-r: 5'-gttaatcaacgtacaagcagctgccgtctaaactgggtcaccgttaaaagg-3';

[0055] Tet-F: 5'-gcagctgcttgtacgttgattaac-3';

[0056] Tet-R: 5'-ccatgagttgctagtaacatctgaccgag-3';

[0057] Tet-yb-f: 5'-gtcagatgttactagcaactcatggcgaacgtcatttgaaggtgatggtcg-3';

[0058] Tet-yb-r: 5'-ggtcctgaatgcgattatttaccgg-3'.

[0059] Example 4: Continuous passage of recombinant E. coli

[0060] The constructed pTM-GOI plasmid was transformed into Escherichia coli TOP11. The transformants were selected for colony PCR to verify that the recombinant E. coli was successfully constructed, and was named TOP12-CK. TOP12-CK was subjected to continuous evolution according to the description of the above examples.

[0061] TOP12-tetK was continuously evolved. The constructed recombinant E. coli TOP12 was streaked on a plate containing kanamycin and spectinomycin, and incubated at 37°C for about 14 hours. A single colony was inoculated in a culture medium containing kanamycin and spectinomycin, and incubated at 37°C, 220 rpm for 12 hours. Then, 0.1% of the culture was inoculated in a culture medium containing kanamycin, spectinomycin, tigecycline, IPTG and arabinose, and the library was started to be produced. Thereafter, every 12 hours, 0.1% of the culture was inoculated in a culture medium containing kanamycin, spectinomycin, tigecycline, IPTG and arabinose, and was regarded as being passed for 10 generations. During the continuous evolution, the tigecycline was increased by 8 μg / mL every 10 generations, and a total of 10 times, 100 generations of passage were performed. The mutant finally obtained which could tolerate high concentration of tigecycline was subjected to colony PCR, and the PCR product was subjected to Sanger sequencing, and the tetracycline efflux pump protein mutant was obtained.

[0062] Escherichia coli TOP11 without the pTM-GOI plasmid was named as TOP12-CK. While TOP12-tetK was continuously evolved, TOP12-CK was continuously passed, and the passing process of TOP12-CK did not contain antibiotics and inducers, and was only for proving that the mutant produced by TOP12-tetK was from the continuous evolution system, rather than natural mutation. TOP12-CK participated in the test of antibiotic tolerance after the passing was ended.

[0063] The mutants were analyzed and visualized using pymol software, and the mutation frequency of different sites was as shown in Figure 4 .

[0064] Table 1 TetK mutant obtained by continuous evolution

[0065] Mutant Mutation Evol-T1 G154S, L83P Evol-T2 L83P Evol-T3 L12V Evol-T4 Y58H

[0066] The tetracycline efflux pump protein (TetK) was knocked into the genome of the strain TOP10 using the CRISPR / Cas9 tool to obtain tetracycline resistance. After colony PCR verification and sequencing were correct, it was named as TOP10-tetK-CK. According to the above method, TOP10-tetK-CK was continuously evolved, and served as a WT group control.

[0067] WT, Evol-T1 and Evol-T2 were subjected to tigecycline and tetracycline tolerance experiments, respectively, and the experimental results were as shown in Figure 5As shown, strains Evol-T1 and Evol-T2 showed significantly improved tolerance to tetracycline and tigecycline, with tolerance to tigecycline increasing by 8-fold and tolerance to tetracycline increasing by 2-fold. TetK itself does not induce tigecycline resistance in the host, demonstrating the ability of the T7 RNA polymerase-based continuous evolution system provided by this invention to mediate large-fragment in situ evolution of the genome as a single element.

[0068] Example 5: Continuous evolution to repair tryptophan deficiency in Saccharomyces cerevisiae

[0069] The evolutionary plasmid pY15_v6 (image shown) will be used. Figure 6 As shown in SEQ ID NO.5, the nucleotide sequence was transformed into *Saccharomyces cerevisiae* CEN.PK2-1Ca (GenBank: JRIV00000000), resulting in a nonsense mutation in the tryptophan synthesis gene of the *Saccharomyces cerevisiae* genome. After verifying positive clones by colony PCR, single colonies were picked and incubated overnight on YPD medium. Then, a 1% inoculum was transferred to fresh YNB medium and cultured at 30°C and 220 rpm for 24 hours. 100 μL of the culture was then plated onto YNB plates that did not contain tryptophan.

[0070] Saccharomyces cerevisiae containing an evolution plasmid targets an evolutionary sequence (SEQ ID NO. 6), specifically a 150 bp region near a nonsense mutation. Mutations accumulate rapidly within 24 hours; for example, in the recovery of tryptophan deficiency, the evaporated group accumulates at a rate of 1.5 × 10⁻⁶ bp. -8 The mutation rate (bases / replication) of the target gene evolves, while the control group does not undergo mutations (e.g., Figure 7 As shown in the figure, this demonstrates the species universality of the strategy.

[0071] 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 continuously evolving system, characterized in that: The continuous evolution system includes a single-stranded DNA synthesis template and a T7 RNA polymerase mutant, which is obtained by mutating aspartic acid at position 10 to glutamic acid, isoleucine at position 581 to phenylalanine, alanine at position 586 to valine, and alanine at position 615 to threonine in the starting sequence shown in SEQ ID NO.

4.

2. The continuous evolution system according to claim 1, characterized in that: The single-stranded DNA synthesis template is used to terminate expression using three tandem T7 terminators.

3. The continuous evolution system according to claim 1, characterized in that: The continuous evolution system also includes genes encoding single-chain annealed proteins.

4. The continuous evolution system according to claim 3, characterized in that: The coding gene for the T7 RNA polymerase mutant and the coding gene for the single-stranded annealing protein are expressed in a polycistronic form.

5. The application of the continuous evolution system according to any one of claims 1-4 in the recovery of tryptophan deficiency in Saccharomyces cerevisiae, characterized in that: The steps include introducing the sequential evolution system into the starting strain to obtain a recombinant strain, wherein the tryptophan synthesis gene in the genome of the starting strain undergoes a nonsense mutation, plating the recombinant strain on a tryptophan-free medium, and screening for colonies that can grow on the medium.

6. The application of the continuous evolution system according to any one of claims 1-4 in screening tetracycline efflux pump protein mutants, characterized in that: The steps include introducing the continuous evolution system into the starting strain to obtain a recombinant strain, inoculating the recombinant strain onto a medium coated with tetracycline and / or tigecycline, and screening for colonies that can grow on the medium for continuous passage.

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