Continuous evolution system based on T7 RNA polymerase mutant and application thereof

Abstract

The invention relates to a continuous evolution system based on a T7RNA polymerase mutant and application of the continuous evolution system, and belongs to the technical field of evolution engineering. The method comprises the following steps: on the basis of a T7RNA polymerase mutant with a single-stranded DNA synthesis capability, constructing a continuous evolution system containing a single-stranded DNA synthesis template and the T7RNA polymerase mutant, introducing the continuous evolution system into a host, and recombining the T7RNA polymerase mutant with a target gene by synthesizing single-stranded DNA, so that the host constructs a mutant library in a continuous passage process, screening pressure is applied to the host, and the host realizes continuous evolution. By applying the system, evolution of tetracycline efflux pump protein is completed within 7 days, the tryptophan defect of saccharomyces cerevisiae is repaired within 24 hours, the tetracycline efflux pump protein mutant obtained through screening can tolerate tetracycline and tigecycline, the tolerance to the tigecycline is increased by 8 times, the tolerance to the tetracycline is increased by 2 times, and the tetracycline efflux pump protein mutant has the advantages that the tetracycline efflux pump protein mutant can tolerate the tetracycline and the tigecycline. The important significance is realized on improving the cell factory performance.

Description

Technical Field

[0001] The present invention relates to the technical field of evolutionary engineering, and in particular to a continuous evolution system based on T7 RNA polymerase mutants and application thereof. Background Art

[0002] Microorganisms are widely present in nature and face a variety of environmental pressures, such as temperature, pH value, nutrient limitation, toxic substances, etc. These pressures will affect the growth and reproduction of microorganisms, and only those that can adapt to these pressures can survive and continue to reproduce. Through continuous evolution, microorganisms can gradually accumulate favorable 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 give microorganisms new functions, such as degrading specific pollutants and synthesizing new metabolites. These new functions are of great significance to environmental protection, biomanufacturing and other fields. Studying the genetic variation and adaptive changes of microorganisms during continuous evolution can provide a deep understanding of the basic principles and driving forces 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 the passage process 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 continuous evolution strategies for microorganisms often have various defects. For example, the MAGE method requires artificially designed mutation libraries and is not continuous. The ribosome switch method relies on DNA Q polymerase and cannot evolve specific genes. The EvolvR method is limited in the length of editable genes, and these methods usually require multiple elements to cooperate to induce mutations, which limits their universality. In addition, the targeted evolution technology of large fragments of the genome in situ is overly dependent on deaminases, which brings inevitable 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 of the invention

[0004] In order to solve the above technical problems, the present invention provides a continuous evolution system based on T7 RNA polymerase mutants and its application. Based on the T7 RNA polymerase mutants with the ability to synthesize single-stranded DNA, the present invention constructs a continuous evolution system comprising a single-stranded DNA synthesis template and a T7 RNA polymerase mutant, introduces the continuous evolution system 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 passage, exerts screening pressure on the host, and the host achieves continuous evolution.

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

[0006] Furthermore, 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 recombinantly introduce mutations with the target gene according to the single-stranded DNA synthesis template. The mutant library can be obtained by continuously passage the host strain containing the first expression vector and the second expression vector.

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

[0009] Furthermore, the continuous evolution system also includes a single-stranded annealing protein encoding gene.

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

[0011] Furthermore, the continuous evolution system uses medium-copy plasmids as expression vectors. T7 RNA polymerase has a large metabolic burden on cells, high-copy plasmids bring cytotoxicity and slow cell growth, and the expression level of low-copy plasmids is too low, affecting the efficiency of the continuous evolution strategy.

[0012] The second object of the present invention is to provide an application of the above-mentioned continuous evolution system in restoring tryptophan deficiency in cerevisiae, the steps comprising introducing the continuous evolution system into a starting strain to obtain a recombinant strain, wherein a nonsense mutation occurs in the tryptophan synthesis gene in the genome of the starting strain, the recombinant strain is spread on a culture medium without tryptophan, and colonies that can grow on the culture medium are screened.

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

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

[0015] The fourth object of the present invention is to provide a tetracycline efflux pump protein mutant, wherein the tetracycline efflux pump protein is obtained by subjecting the starting sequence of the amino acid sequence as shown in SEQ ID NO.3 to any of the following modifications:

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

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

[0018] (3) mutation of leucine at position 12 to valine;

[0019] (4) The tyrosine at position 58 was mutated to histidine.

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

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

[0022] The fifth object of the present invention is to provide a gene encoding the above tetracycline efflux pump protein mutant.

[0023] The sixth object of the present invention is to provide an expression vector comprising the above gene.

[0024] The seventh objective of the present invention is to provide the use of the above tetracycline efflux pump protein mutant, the above gene or the above expression vector in improving the host's tolerance to tetracycline and / or tigecycline.

[0025] Beneficial effects of the present invention:

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

[0027] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0028] Figure 1 This is a schematic diagram of the principle of the continuous evolution system based on T7 RNA polymerase mutants of the present invention;

[0029] Figure 2 is a map of the recombinant plasmid pTM-EVO in Example 1 of the present invention;

[0030] Figure 3 is a map of the recombinant plasmid pCE-GOI in Example 2 of the present invention;

[0031] Figure 4 is the mutation frequency of different sites of the tetracycline efflux pump protein in Example 4 of the present invention;

[0032] Figure 5 The results of the tetracycline and tigecycline tolerance experiments of the wild strain and the mutant strain in Example 4 of the present invention are shown;

[0033] Figure 6 This is a map of the evolutionary plasmid pY15_v6 used for Saccharomyces cerevisiae in Example 5 of the present invention;

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

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0036] Recombinant strain seed culture and fermentation:

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

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

[0039] YNB plate: The stock solution was purchased from Sangon Biotechnology (Order NO. A610507), diluted according to the instructions and added with agar to a final concentration of 2.5%.

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

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

[0042] Culture conditions: The seeds cultured at 37°C and 220 rpm for 12 h were transferred into the fermentation medium at an inoculum rate of 0.1%, and cultured at 37°C and 220 rpm for 12 h. Antibiotics and inducers were added according to the culture objectives.

[0043] Continuous subculture: Add 1000 μL of fermentation medium and corresponding antibiotics and inducers to each well of a 24-well plate. After inoculation at a 0.1% inoculum, transfer at 0.1% every 12 h, which is considered as 10 generations.

[0044] Continuous evolution: Continuous subculture under appropriate culture conditions will produce a mutant library in the fermentation broth, and the target mutants can be obtained by combining appropriate screening strategies.

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

[0046] The pTM plasmid was used as a template to construct a T7 RNA polymerase mutant expression vector, and the T7 RNA polymerase mutant (nucleotide sequence as shown in SEQ ID NO.1) that can synthesize 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 the expression was induced by arabinose, wherein the single-stranded annealing auxiliary protein and the T7 RNA polymerase mutant were expressed in the form of polycistronic. The obtained recombinant plasmid was named pTM-EVO, as shown in FIG. Figure 2 shown.

[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, expressed using an IPTG-inducible T7 promoter, and terminated using three tandem T7 terminators. The resulting recombinant plasmid was named pCE-GOI, as shown in Figure 3 shown.

[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 the successful construction of the recombinant Escherichia coli. 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 the successful construction of the recombinant Escherichia coli. 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 strain TOP12 using CRISPR / Cas9 to make it resistant to tetracycline. After colony PCR verified the correct sequencing, it was named TOP12-tetK. The primers used for gene knock-in 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 Escherichia coli

[0060] The constructed pTM-GOI plasmid was transformed into Escherichia coli TOP11. The transformants were selected for colony PCR to verify the successful construction of the recombinant Escherichia coli, which was named TOP12-CK. TOP12-CK was continuously evolved according to the description of the above embodiment.

[0061] TOP12-tetK was continuously evolved. The constructed recombinant Escherichia coli TOP12 was streaked on a plate containing kanamycin and spectinomycin, cultured at 37°C for about 14 hours, and a single colony was picked and inoculated in a medium containing kanamycin and spectinomycin. After culturing at 37°C and 220rpm for 12 hours, 0.1% was inoculated in a medium containing kanamycin, spectinomycin, tigecycline, IPTG and arabinose to start generating the library. Thereafter, 0.1% was transferred to a medium containing kanamycin, spectinomycin, tigecycline, IPTG and arabinose every 12 hours, which was considered to be 10 generations. During the continuous evolution process, 8 μg / mL of tigecycline was increased every 10 generations, and a total of 10 times and 100 generations of passage were performed. Colony PCR was performed on the mutants that could tolerate high concentrations of tigecycline, and the PCR products were Sanger sequenced to obtain tetracycline efflux pump protein mutants.

[0062] The Escherichia coli TOP11 without the pTM-GOI plasmid was named TOP12-CK. While TOP12-tetK was continuously evolving, TOP12-CK was continuously passaged. The passage process of TOP12-CK did not contain antibiotics and inducers, only to prove that the mutants produced by TOP12-tetK came from the continuous evolution system, not natural mutations. After the passage, TOP12-CK participated in the antibiotic tolerance test.

[0063] The mutants were analyzed and visualized using pymol software. TOP12-CK was used as a control. The mutation frequencies at different sites were shown in Figure 4 shown.

[0064] Table 1 TetK mutants obtained by continuous evolution

[0065] Mutants 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 strain TOP10 using the CRISPR / Cas9 tool to make it tetracycline-resistant. After colony PCR verified the correct sequencing, it was named TOP10-tetK-CK. TOP10-tetK-CK was continuously evolved according to the above method and used as the WT group control.

[0067] The tolerance test of tigecycline and tetracycline was performed on WT, Evol-T1 and Evol-T2, and the experimental results are shown in Figure 5As shown, the tolerance of strains Evol-T1 and Evol-T2 to tetracycline and tigecycline was significantly improved, with tolerance to tigecycline increased by 8 times and tolerance to tetracycline increased by 2 times, while TetK itself would not cause the host to produce tigecycline resistance, demonstrating the ability of the T7 RNA polymerase-based continuous evolution system provided by the present invention to mediate the in situ evolution of large genome fragments as a single element.

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

[0069] The evolution plasmid pY15_v6 (map as Figure 6 The nucleotide sequence is shown in SEQ ID NO.5) was transformed into Saccharomyces cerevisiae CEN.PK2-1Ca (GenBank: JRIV00000000), in which a nonsense mutation occurred in the tryptophan synthesis gene of the Saccharomyces cerevisiae genome. After the positive clones were verified by colony PCR, a single colony was picked and inoculated in YPD medium overnight, and then transferred to fresh YNB medium at a 1% inoculation amount, and cultured at 30°C, 220rpm for 24 hours, and 100 μL was spread on a YNB plate without tryptophan.

[0070] The Saccharomyces cerevisiae containing the evolution plasmid targeted the evolution target sequence (shown in SEQ ID NO.6), which was 150 bp near the nonsense mutation. Mutations were rapidly accumulated within 24 hours. Taking the recovery of tryptophan deficiency as an example, the evolution group (Evolved) was 1.5×10 -8 The target gene is evolved with a mutation rate of (bases / each replication), while the control group (Control) does not mutate (e.g. Figure 7 ), demonstrating the species universality of this strategy.

[0071] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A continuous evolution system, characterized in that: The continuous evolution system includes a single-stranded DNA synthesis template and a T7 RNA polymerase mutant, wherein the T7 RNA polymerase mutant mutates the 10th aspartic acid of the starting sequence shown in SEQ ID NO.4 to glutamic acid, the 581st isoleucine to phenylalanine, the 586th alanine to valine, and the 615th alanine to threonine.

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

3. The continuous evolution system according to claim 1, characterized in that: The continuous evolution system also includes a gene encoding a single-stranded annealing protein.

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

5. Use of the continuous evolution system according to any one of claims 1 to 4 in restoring tryptophan deficiency in Saccharomyces cerevisiae, characterized in that: The steps include introducing the continuous evolution system into the starting strain to obtain a recombinant strain, causing a nonsense mutation in the tryptophan synthesis gene of the starting strain genome, coating the recombinant strain on a culture medium without tryptophan, and screening colonies that can grow on the culture medium.

6. Use of the continuous evolution system according to any one of claims 1 to 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 on a culture medium coated with tetracycline and / or tigecycline, and screening colonies that can grow on the culture medium for continuous subculture.

7. A tetracycline efflux pump protein mutant, characterized in that: The tetracycline efflux pump protein is prepared by subjecting the starting sequence of the amino acid sequence as shown in SEQ ID NO.3 to any of the following modifications: (1) Leucine at position 83 mutated to proline, and glycine at position 154 mutated to serine; (2) leucine at position 83 mutated to proline; (3) mutation of leucine at position 12 to valine; (4) The tyrosine at position 58 was mutated to histidine.

8. A gene encoding the tetracycline efflux pump protein mutant according to claim 7.

9. An expression vector comprising the gene according to claim 8.

10. Use of the tetracycline efflux pump protein mutant according to claim 7, the gene according to claim 8 or the expression vector according to claim 9 in improving the host's tolerance to tetracycline and / or tigecycline.

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