Mutation device system and application thereof

Abstract

The invention provides a mutator system and application thereof. The present disclosure provides a muter system comprising: a DNA polymerase IV, a nucleic acid molecule encoding the DNA polymerase IV or an accelerator thereof, and a DNA polymerase III alpha subunit mutant, a nucleic acid molecule encoding the DNA polymerase III alpha subunit mutant or an accelerator thereof; the kit further comprises an endonuclease NucS mutant, a nucleic acid molecule for coding the endonuclease NucS mutant or an accelerant of the nucleic acid molecule. According to the mutant system provided by the invention, the mutation efficiency can be improved, the mutation speed and the mutation diversity are remarkably improved, and the mutant system can be used for adaptive evolution of corynebacterium glutamicum to create a more efficient cell factory.

Description

Technical Field

[0001] The present disclosure belongs to the fields of molecular biology and bioengineering and relates to a mutator system and its applications. In particular, the present disclosure relates to a mutator system capable of increasing the mutation frequency, mutation rate, and mutation types of strains. Specifically, the present disclosure relates to a system that combines mutants of the DNA polymerase IV encoding gene DinP and the DNA polymerase III α subunit DnaE1 and / or the mismatch repair mechanism key protein NucS of Corynebacterium strains, particularly Corynebacterium glutamicum, to construct a series of mutator systems capable of increasing the mutation frequency, mutation rate, and mutation diversity of Corynebacterium. Background Art

[0002] Corynebacterium glutamicum is a non-pathogenic Gram-positive bacterium isolated from soil. It is also an important industrial strain for the production of bulk chemicals such as amino acids and organic acids, and is widely used in food and pharmaceuticals. Due to its biosafety (generally regarded as safe by the US FDA), rapid growth rate, low nutritional requirements, and broad substrate spectrum, Corynebacterium glutamicum is considered an ideal microbial platform for biomanufacturing. To continuously improve the physiological performance and product synthesis capacity of industrial strains, non-rational evolutionary engineering is an effective technical approach. High-fidelity DNA replication is crucial for the stable transmission of genetic information in organisms, and low-frequency random mutations during DNA replication play a key role in the adaptive evolution of bacteria to the external environment. Therefore, developing mutagens that can increase the frequency of random mutations in Corynebacterium glutamicum is crucial for the rapid evolution of bacterial strains.

[0003] During the natural evolution of bacteria, cellular metabolism and environmental stress can cause DNA damage. Through translesion DNA synthesis (TLS), bacteria replace high-fidelity DNA polymerases with error-prone DNA polymerases, allowing DNA replication to span mismatch sites, continuing DNA replication and contributing to genomic diversity. DNA polymerase IV is an error-prone DNA polymerase that performs translesion synthesis and is widely present in bacteria. Existing studies have shown that inducing DNA polymerase IV encoded by the dinB gene in Escherichia coli can increase the mutation frequency of the strain by 4.5 times (Biotechnology for Biofuels, 2015, 8:93.), overexpressing DNA polymerase IV encoded by the dinB1 gene in Mycobacterium smegmatis can increase the mutation frequency to about 6 times (Nature Communications, 2022, 13(1):4493.), and similarly, overexpressing DNA polymerase IV encoded by the dinP gene in Corynebacterium glutamicum can also increase the mutation frequency by 10.2 times (Metabolic Engineering, 2023, 79:182-191.). This indicates that increasing the expression of error-prone DNA polymerases can effectively increase the mutation frequency of strains, but the increase is limited.

[0004] The inventor started from the proofreading mechanism in the DNA replication process and the mismatch repair mechanism after replication in the early stage, and found that by overexpressing the mutant of DNA polymerase III α subunit DnaE1 or mismatch repair mechanism key protein NucS in Corynebacterium glutamicum, the mutation frequency of Corynebacterium glutamicum can be greatly improved, and based on this, a variety of genome mutator elements with different mutation frequencies have been developed, which has accelerated the adaptive evolution of strains (Nucleic Acids Research, 2023, 51 (16): 8623-8642.). In view of the difference in mutation mechanism, whether the above-mentioned mutator can be combined with the cross-lesion synthesis mechanism to continue to improve the strain mutation frequency is not clear. In addition, the mutators currently reported can mostly only improve the mutation frequency and mutation rate of strains, but the type of mutation is limited, which also makes the adaptive evolution of strains limited. Therefore, this area still needs to develop more types of genome efficient mutators, to further improve the artificial evolution speed of Corynebacterium glutamicum, enrich the diversity of evolution strain mutations, and create more efficient cell factories. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] The present invention discloses a series of mutators that can increase the mutation frequency, mutation rate, and mutation diversity of strains by mutating and combining the enzymes of the proofreading mechanism during DNA replication and the mismatch repair mechanism after replication from Corynebacterium glutamicum. The mutator system is composed of these mutators, which can increase the random mutation frequency and mutation diversity, reduce the DNA replication fidelity, and be used for the adaptive evolution of strains of the genus Corynebacterium, especially Corynebacterium glutamicum.

[0007] Solutions for solving problems

[0008] [1] A mutator system, comprising:

[0009] (i) DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV, or a promoter thereof; and

[0010] (ii) a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant, or a promoter thereof.

[0011] [2] According to the mutator system described in [1], the DNA polymerase IV is selected from any one of the following groups consisting of (a1) to (a3):

[0012] (a1) comprising the amino acid sequence shown in SEQ ID NO: 1;

[0013] (a2) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in (a1), and retaining DNA polymerase IV activity;

[0014] (a3) A polypeptide having an amino acid sequence as shown in (a1) or (a2) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus, and retaining DNA polymerase IV activity.

[0015] [3]. The mutator system according to [1] or [2], wherein the DNA polymerase IIIα subunit mutant corresponds to the amino acid sequence shown in SEQ ID NO: 2, and has a mutated amino acid at one or more of the following positions: position 20 is mutated to cysteine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, valine or tyrosine, and / or position 223 is mutated to alanine, glutamine, serine or asparagine.

[0016] [4] The mutator system according to any one of claims [1] to [3], characterized in that the mutator system also includes a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant, or a promoter thereof.

[0017] [5] The mutator system according to any one of [1] to [4], wherein the endonuclease NucS mutant corresponds to the amino acid sequence shown in SEQ ID NO: 3 and has the mutation E111L.

[0018] [6] The mutator system according to any one of [1] to [5], wherein the mutator system is selected from any one of the following (d1) to (d7):

[0019] (d1) Mutator system DnaE1 D20R -NucS E111L -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof;

[0020] Among them, the DNA polymerase IIIα subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2; the endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO: 3;

[0021] (d2) Mutator system DnaE1 D20R -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0022] Wherein, the DNA polymerase III α subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0023] (d3) Mutator system DnaE1 D20M -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0024] Wherein, the DNA polymerase III α subunit mutant has a mutation D20M corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0025] (d4) Mutator system DnaE1 D20N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0026] Wherein, the DNA polymerase III α subunit mutant has a mutation D20N corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0027] (d5) Mutator system DnaE1 D223N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0028] Wherein, the DNA polymerase IIIα subunit mutant has a mutation D223N corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0029] (d6) Mutator system DnaE1 D20I -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0030] Wherein, the DNA polymerase III α subunit mutant has a mutation D20I corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0031] (d7) Mutator system NucS E111L -DinP, which comprises: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof;

[0032] The endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO: 3.

[0033] [7]. An isolated polynucleotide, wherein the polynucleotide encodes the mutator system as described in any one of [1] to [6].

[0034] [8]. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide described in [7].

[0035] [9] A recombinant host cell, wherein the recombinant host cell comprises the mutator system described in any one of [1] to [6], the polynucleotide described in [7], or the recombinant expression vector described in [8];

[0036] Optionally, the host cell is derived from the genus Corynebacterium;

[0037] Preferably, the host cell is derived from Corynebacterium glutamicum.

[0038]

[10] Use of the mutator system according to any one of [1] to [6], the polynucleotide according to [7], the recombinant expression vector according to [8], or the recombinant host cell according to [9] in at least one of the following (a) to (d):

[0039] (a) preparing a mutant strain having an increased random mutation frequency;

[0040] (b) preparing mutant strains with reduced DNA replication fidelity;

[0041] (c) preparing mutant strains with increased mutational diversity;

[0042] (d) used for adaptive evolution of strains;

[0043] Optionally, the mutant strain is derived from the genus Corynebacterium;

[0044] Preferably, the mutant strain is derived from Corynebacterium glutamicum.

[0045]

[11] . A method for adaptive evolution of a strain, wherein the method comprises introducing into the strain a mutator system as described in any one of [1] to [6], a polynucleotide as described in [7], and a recombinant expression vector as described in [8]; optionally, the method further comprises the step of isolating and purifying the adaptively evolved strain.

[0046] Effects of the Invention

[0047] In a specific embodiment, the mutator system provided by the present disclosure can improve the mutation efficiency, and significantly improve the mutation speed and mutation diversity, and can be used for the adaptive evolution of Corynebacterium glutamicum to create a more efficient cell factory.

[0048] In a specific embodiment, the present disclosure provides isolated polynucleotides, vectors, and recombinant host cells containing mutants in a mutant library, as well as uses of the aforementioned isolated polynucleotides, vectors, and recombinant host cells. Each of these can be used to increase the random mutation frequency of a strain or reduce the DNA replication fidelity of a strain, and can be used for the adaptive evolution of Corynebacterium glutamicum.

[0049] In a specific embodiment, the aforementioned source is a strain of the genus Corynebacterium, preferably a strain of Corynebacterium glutamicum, having an increased random mutation frequency or a decreased DNA replication fidelity or an increased mutation diversity. DETAILED DESCRIPTION

[0050] definition

[0051] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0052] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0053] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0054] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0055] When used in the claims or description, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0056] As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0057] As used in this disclosure, the term "amino acid" may include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. Amino acids and their abbreviations and English abbreviations in this disclosure are as follows:

[0058] Histidine (His, H); Serine (Ser, S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0059] In the present disclosure, "DNA polymerase IV (Pol IV)" is an important member of the γ family of polymerases, which is widely distributed in prokaryotes, eukaryotes and archaea. In Corynebacterium glutamicum, DNA polymerase IV is encoded by the dinP (Cgl2144) gene. Pol IV and its homologs have the ability of translesion DNA synthesis (TLS), can bypass various lesions for DNA replication, and are also involved in multiple biological processes of bacteria. In a specific embodiment of the present disclosure, the amino acid sequence of the wild-type DNA polymerase IV of Corynebacterium glutamicum involved in the present disclosure is shown in SEQ ID NO: 1.

[0060] In the present disclosure, "DNA polymerase III" is a multi-enzyme complex that plays a leading role in the elongation of DNA replication chains and is the main enzyme that catalyzes DNA replication synthesis.

[0061] In the present disclosure, the "α subunit of DNA polymerase III" is encoded by the dnaE1 gene.

[0062] In a specific embodiment of the present disclosure, the amino acid sequence of the α subunit of DNA polymerase III of Corynebacterium glutamicum involved in the present disclosure comprises the amino acid sequence encoded by the dnaE1 gene, and the amino acid sequence encoded by the dnaE1 gene is the sequence shown in SEQ ID NO: 2.

[0063] In the present disclosure, "endonuclease NucS" refers to a non-classical mismatch repair system present in Corynebacterium glutamicum, encoded by the nucS (Cgl1215) gene, and is an endonuclease that specifically recognizes and cuts mismatched bases.

[0064] In a specific embodiment of the present disclosure, the amino acid sequence of the wild-type endonuclease NucS of Corynebacterium glutamicum involved in the present disclosure is shown in SEQ ID NO: 3.

[0065] As used herein, the term "endogenous" refers to a polynucleotide, polypeptide, or other compound that is naturally expressed or produced within an organism or cell. That is, an endogenous polynucleotide, polypeptide, or other compound is not of exogenous origin. For example, an "endogenous" polynucleotide or polypeptide is present in a cell when the cell is initially isolated from nature.

[0066] As used in this disclosure, the term "exogenous" refers to any polynucleotide or polypeptide that is naturally found or expressed in the particular cell or organism in which expression is desired. An exogenous polynucleotide, polypeptide, or other compound is not endogenous.

[0067] As used herein, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used herein, "naturally occurring" and "wild-type" are synonyms.

[0068] As used in this disclosure, the term "mutant" refers to a polynucleotide or polypeptide that comprises an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild-type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing the nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0069] As used herein, the term "synonymous mutation" or "synonymous substitution" refers to the evolutionary replacement of one base pair with another in the exons of a gene encoding a protein, so that the resulting amino acid sequence is not modified. In other words, synonymous mutations are point mutations, which means that they are simply incorrectly copied DNA nucleotides that only change one base pair in the RNA copy of the DNA. In some embodiments, synonymous mutations are changes in the DNA sequence that encode an amino acid in a protein sequence but do not change the encoded amino acid. Due to the redundancy of the genetic code (the same amino acid is encoded by multiple codons), these changes usually occur at the third position of the codon. For example, GGT, GGA, GGC, and GGG all encode for glycine. Any change in the third position of the codon (e.g., A->G) will result in the same amino acid being incorporated into the protein sequence at that position.

[0070] As used herein, the term "non-synonymous mutation" or "non-synonymous substitution" refers to a nucleotide mutation that changes the amino acid sequence of a protein. Non-synonymous substitutions are different from synonymous substitutions, which do not change the amino acid sequence and are (sometimes) silent mutations. Because non-synonymous substitutions can cause biological changes in organisms. Compared with synonymous mutations, non-synonymous mutations have a much greater impact on individuals. The insertion or deletion of a single nucleotide in a sequence during transcription is only one possible source of non-synonymous mutations. However, most non-synonymous mutations are caused by the substitution of a single nucleotide. Non-synonymous mutations with single nucleotide substitutions will change the amino acid sequence by replacing a different amino acid called a missense mutation or replacing the original amino acid with a stop codon called a nonsense mutation. Nonsense mutations can cause RNA transcription to terminate prematurely.

[0071] As used in this disclosure, the term "mutated amino acid" includes "one or more amino acids that are substituted, repeated, deleted, or added." In this disclosure, the term "mutation" refers to a change in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution."

[0072] In one embodiment, the "mutation" of the present disclosure can be selected from "conservative mutations". In the present disclosure, the term "conservative mutation" refers to a mutation that can maintain the normal function of the protein. A representative example of a conservative mutation is a conservative substitution.

[0073] As used in the present disclosure, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). As used in the present disclosure, a "conservative substitution" typically exchanges an amino acid at one or more sites in a protein. Such substitutions can be conservative. In addition, conservative mutations also include naturally occurring mutations that arise from individual differences, strains, and species differences in the genes from which they are derived.

[0074] As used in the present disclosure, the term "mutant strain" refers to a strain different from the "wild-type strain" or "starting strain" obtained by random mutation of the "wild-type strain" or "starting strain", that is, the polynucleotide or polypeptide of the "mutant strain" contains changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to the polynucleotide or polypeptide of the "wild-type strain" or "starting strain". In a specific embodiment, the "mutant strain" described in the present disclosure refers to a strain different from the "wild-type strain" or "starting strain" obtained by random mutation of the "wild-type strain" or "starting strain" using the mutator system of the present disclosure.

[0075] As used herein, the term "mutator" or "mutator system" has a meaning well known to those skilled in the art and refers to an element or combination of elements that can increase the frequency of random mutations in a strain. In a specific embodiment, the mutator (system) includes a polynucleotide encoding a DNA polymerase IV, DNA polymerase III α subunit mutant, and / or endonuclease NucS mutant as described herein, a polynucleotide having transcription initiation activity operably linked to a gene encoding a DNA polymerase IV, DNA polymerase III α subunit mutant, and / or endonuclease NucS mutant as described herein, and an expression vector capable of replicating in a host cell.

[0076] As used in this disclosure, the term "adaptive evolution", also known as directed evolution or domestication, is a bacterial strain improvement technology that can effectively change certain phenotypes and physiological characteristics of the strain (such as bacterial growth rate, substrate consumption rate, tolerance to high / low temperatures, high / low pH and different organic solvents, etc.) in a relatively short period of time without affecting other excellent traits except the target phenotype.

[0077] As used herein, the term "operably linked" refers to the functional linkage of a polynucleotide having transcription initiation activity to a DNA polymerase IV, DNA polymerase III α subunit mutant, and / or endonuclease NucS mutant encoding gene sequence of the present disclosure to initiate and mediate transcription of the DNA polymerase IV, DNA polymerase III α subunit mutant, and / or endonuclease NucS mutant. Operable linkage can be achieved using genetic recombination techniques known in the art, and restriction enzymes and ligases known in the art can be used for site-specific DNA cleavage and ligation, but are not limited thereto.

[0078] As used in the present disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biology methods (e.g., using a cloning vector). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T." In other words, a "polynucleotide" refers to a nucleotide polymer that has been removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "Recombinant polynucleotide" is a type of "polynucleotide."

[0079] As used in the present disclosure, the terms "sequence identity" and "percentage identity" refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). A polypeptide can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotides or polypeptides. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0080] In some embodiments, two or more sequences or subsequences have a "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both compared biopolymers (e.g., polynucleotides).

[0081] As used herein, the term "corresponding to" has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in the other sequence after alignment for homology or sequence identity. Thus, for example, with respect to "the amino acid residue corresponding to position 150 of the amino acid sequence set forth in SEQ ID NO: 1," if a 6×His tag is added to one end of the amino acid sequence set forth in SEQ ID NO: 1, then the position corresponding to position 150 in the resulting mutant may be position 156.

[0082] As used in this disclosure, the term "expression vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences to express a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. A recombinant expression vector may include, for example, a transcriptional subunit comprising i) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation start and stop sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, as a plasmid vector, pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used, specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pXMJ19 vectors, etc. can be used, but are not limited to these, as long as they can be replicated and expressed in Corynebacterium glutamicum. As a phage vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used.

[0083] The term "host cell" in the present disclosure refers to any cell type that is susceptible to the use of a mutagen (system) or a polynucleotide encoding a mutagen, or a recombinant expression vector comprising the mutagen of the present disclosure.

[0084] The term "recombinant host cell" in the present disclosure encompasses host cells that are different from parent cells after the introduction of exogenous polynucleotides, nucleic acid constructs or recombinant expression vectors, or mutations. Recombinant host cells are specifically achieved by transformation.

[0085] The term "transformation" as used herein has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0086] The host cells of the present invention can be cultured according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0087] <Mutator System>

[0088] In some aspects of the present disclosure, a mutator system is provided, comprising:

[0089] (i) DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV, or a promoter thereof;

[0090] (ii) a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant, or a promoter thereof;

[0091] and, optionally, (iii) an endonuclease NucS mutant, a nucleic acid molecule encoding an endonuclease NucS mutant, or a promoter thereof.

[0092] In some specific embodiments, the mutator system comprises:

[0093] (i) DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV, or a promoter thereof;

[0094] (ii) a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant, or a promoter thereof;

[0095] and (iii) a mutant of endonuclease NucS, a nucleic acid molecule encoding a mutant of endonuclease NucS, or a promoter thereof.

[0096] DNA polymerase IV

[0097] In some embodiments of the present disclosure, the DNA polymerase IV (DinP) is selected from any one of the following groups consisting of (a1)-(a3):

[0098] (a1) comprising the amino acid sequence shown in SEQ ID NO: 1;

[0099] (a2) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in (a1), and retaining DNA polymerase IV activity;

[0100] (a3) A polypeptide having an amino acid sequence as shown in (a1) or (a2) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus, and retaining DNA polymerase IV activity.

[0101] Corynebacterium glutamicum DNA polymerase IV (DinP) amino acid sequence (SEQ ID NO: 1):

[0102] MQRWVLHIDMDAFFASCEQLTRPTLRGRPVLVGGVSGRGVVAGASYEARKFGARSAMPMHQAKARVGFGAVVVTPRHIVYSAASRRVFQIVEKRAGIVERLSIDEGFMEPEALVGA TPEEVKQWAEELRAEIKEVTGLPSSVGAGSGKQIAKIGSGEAKPDGVFVVPVDKQHDLLDPLPVGALWGVGPVTGSKLASMGVETIGDLAALTQKEVEISLGATIGISLWNLARGID DRPVEPRAEAKQISQEHTYEKDLLTRQQVDAAIIRSAEGAHRRLLKDGRGARTVSVKLRMADFRIESRSYTLSYATDDYATLEATAFRLARYPGEVGPIRLVGVSFSGLEESRQDIL FPELDQQIIVPPAPDTDYEVGVQSSSSSESTQVEAPQDVALSMWCATQDVYHPEYGHGWVQGAGHGVVSVRFETRSTTKGRTKSFSMDDPDLTPADPLDSLDWADWFAENGETGDDE

[0103] DNA polymerase III α subunit mutants

[0104] In the present disclosure, the DNA polymerase III α subunit mutant is as disclosed in CN116606833A, which is incorporated herein by reference.

[0105] In some embodiments of the present disclosure, the DNA polymerase III α subunit mutant is selected from any one of the following groups consisting of (b1)-(b3):

[0106] (b1) the amino acid sequence of the mutant corresponds to the sequence shown in SEQ ID NO: 2, having a mutated amino acid at one or more of the following positions: position 20 is mutated to cysteine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, valine or tyrosine, and / or position 223 is mutated to alanine, glutamine, serine or asparagine;

[0107] (b2) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in (b1); and a mutant strain containing the mutant has an increased random mutation frequency or decreased DNA replication fidelity relative to the wild-type strain;

[0108] (b3) A polypeptide having an amino acid sequence as shown in (b1) or (b2) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus; and a mutant strain containing the polypeptide has an increased random mutation frequency or decreased DNA replication fidelity relative to the wild-type strain.

[0109] Corynebacterium glutamicum DnaE1 amino acid sequence (SEQ ID NO: 2):

[0110]

[0111] Endonuclease NucS mutant

[0112] In the present disclosure, the endonuclease NucS mutant is as disclosed in Chinese patent application 202310311874.8, which is incorporated herein by reference.

[0113] In some embodiments of the present disclosure, the endonuclease NucS mutant is selected from any one of the following groups consisting of (c1)-(c3):

[0114] (c1) The amino acid sequence of the endonuclease NucS mutant corresponds to the amino acid sequence shown in SEQ ID NO: 3, and the amino acid at position 111 is mutated to leucine (E111L).

[0115] (c2) a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (c1);

[0116] (c3) A polypeptide having an amino acid sequence as shown in (c1) or (c2) wherein one or more amino acids are added to or deleted from at least one of the N-terminus and the C-terminus.

[0117] Corynebacterium glutamicum NucS amino acid sequence (SEQ ID NO: 3):

[0118] MRLVIARCSVDYVGRLEAHLPSADRLLMVKADGSVSIHADDRAYKPLNWMTPPCSLVETPITDEDGEATGESLWVVENKKGEQLRITVEEIHSEQNFDLGQDPGLVKDGVEDHLQ ELLAEHITTLGDGYTLIRREYPTAIGPVDILCRNSDGETVAVEIKRRGGIDGVEQLTRYLELLNRDELLKPVHGVFAAQEIKPQAKTLAEDRGIKCVTLDYQALRGIESNELTLF

[0119] Encoding protein (DNA polymerase IV, DNA polymerase III α subunit mutant or endonuclease NucS mutant) Nucleic acid molecules

[0120] In this specification, the terms "DNA polymerase IV / DNA polymerase IIIα subunit mutant / nuclease NucS mutant gene", "DNA polymerase IV / DNA polymerase IIIα subunit mutant / nuclease NucS mutant encoding gene", "DNA polymerase IV / DNA polymerase IIIα subunit mutant / nuclease NucS mutant encoding sequence" or "nucleic acid molecule encoding DNA polymerase IV / DNA polymerase IIIα subunit mutant / nuclease NucS mutant" are used interchangeably, all referring to a nucleotide sequence encoding a protein or polypeptide of the DNA polymerase IV, DNA polymerase IIIα subunit mutant, or nuclease NucS mutant described in the present disclosure, or a molecule that hybridizes with these sequences under stringent conditions, or a family gene molecule that is highly homologous to the above molecules.

[0121] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85% or greater than 90%, and more preferably greater than 95%.

[0122] The nucleic acid molecules encoding DNA polymerase IV / DNA polymerase IIIα subunit mutants / endonuclease NucS mutants disclosed herein (e.g., full-length nucleotide sequences or fragments thereof) can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0123] Promoter of protein or nucleic acid molecule encoding protein

[0124] The present disclosure also relates to a "promoter" of a protein (DNA polymerase IV, DNA polymerase IIIα subunit mutant, or endonuclease NucS mutant) or a nucleic acid molecule encoding a protein (DNA polymerase IV, DNA polymerase IIIα subunit mutant, or endonuclease NucS mutant). The term "promoter" refers to a substance that can increase the level or activity of a protein (DNA polymerase IV, DNA polymerase IIIα subunit mutant, or endonuclease NucS mutant) or a nucleic acid molecule encoding a protein (DNA polymerase IV, DNA polymerase IIIα subunit mutant, or endonuclease NucS mutant). Promotor agents that can be used in the present disclosure include, but are not limited to, protein expression vectors, exogenous proteins, naked DNA of protein coding sequences, liposome-encapsulated DNA of proteins or protein coding sequences, proteins (DNA polymerase IV, DNA polymerase IIIα subunit mutant, or endonuclease NucS mutant).

[0125] In some preferred embodiments, the mutator system is selected from any one of the following (d1) to (d7):

[0126] (d1) Mutator system DnaE1 D20R -NucS E111L -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof;

[0127] Among them, the DNA polymerase IIIα subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2; the endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO: 3;

[0128] (d2) Mutator system DnaE1 D20R -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0129] Wherein, the DNA polymerase III α subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0130] (d3) Mutator system DnaE1 D20M-DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0131] Wherein, the DNA polymerase III α subunit mutant has a mutation D20M corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0132] (d4) Mutator system DnaE1 D20N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0133] Wherein, the DNA polymerase III α subunit mutant has a mutation D20N corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0134] (d5) Mutator system DnaE1 D223N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0135] Wherein, the DNA polymerase IIIα subunit mutant has a mutation D223N corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0136] (d6) Mutator system DnaE1 D20I -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof;

[0137] Wherein, the DNA polymerase III α subunit mutant has a mutation D20I corresponding to the amino acid sequence shown in SEQ ID NO: 2;

[0138] (d7) Mutator system NucS E111L -DinP, which comprises: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof;

[0139] The endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO: 3.

[0140] The present invention obtains a series of mutants that can improve the mutation frequency, mutation speed and mutation diversity of strains by mutating and combining the enzymes of the proofreading mechanism during DNA replication and the mismatch repair mechanism after replication from Corynebacterium glutamicum, especially DnaE1. D20R -NucS E111L -DinP mutator system can double the mutation efficiency and significantly improve the mutation speed and mutation diversity. It can be used for the adaptive evolution of Corynebacterium glutamicum and create more efficient cell factories.

[0141] <Polynucleotide>

[0142] In some aspects of the present disclosure, an isolated polynucleotide is provided, wherein the polynucleotide encodes the above-mentioned mutator system.

[0143] In some specific embodiments, the polynucleotide comprises one or more of the following:

[0144] i) a polynucleotide encoding the DNA polymerase IV in the above-mentioned mutant system;

[0145] ii) a polynucleotide encoding a DNA polymerase III α subunit mutant in the above-mentioned mutant system;

[0146] iii) A polynucleotide encoding the endonuclease NucS mutant in the above-mentioned mutator system.

[0147] The polynucleotides disclosed herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0148] Polynucleotides encoding the mutator system of the present disclosure include: coding sequences encoding only the mutator system; coding sequences for the mutator system and various additional coding sequences; coding sequences for the mutator system (and optional additional coding sequences) and non-coding sequences.

[0149] <Vectors and recombinant host cells>

[0150] In some aspects of the present disclosure, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the above-mentioned polynucleotide.

[0151] In some specific embodiments, the recombinant expression vector comprises one or more of the following:

[0152] i) a polynucleotide encoding the DNA polymerase IV in the above-mentioned mutant system;

[0153] ii) a polynucleotide encoding a DNA polymerase III α subunit mutant in the above-mentioned mutant system;

[0154] iii) A polynucleotide encoding the endonuclease NucS mutant in the above-mentioned mutator system.

[0155] In some embodiments, the above i) to iii) can be in the same recombinant expression vector. In other embodiments, the above i) to iii) can be in different recombinant expression vectors.

[0156] In some aspects of the present disclosure, a recombinant host cell is provided, wherein the recombinant host cell comprises the mutator system described above, the polynucleotide described above, or the recombinant expression vector described above.

[0157] In some specific embodiments, the host cell is derived from the genus Corynebacterium;

[0158] In some preferred embodiments, the host cell is derived from Corynebacterium glutamicum.

[0159] <Use and Method>

[0160] In some aspects of the present disclosure, there is provided use of the mutator system as described above, the polynucleotide as described above, the recombinant expression vector as described above, or the recombinant host cell as described above in at least one of the following (a)-(d):

[0161] (a) preparing a mutant strain having an increased random mutation frequency;

[0162] (b) preparing mutant strains with reduced DNA replication fidelity;

[0163] (c) preparing mutant strains with increased mutational diversity;

[0164] (d) Used for adaptive evolution of strains.

[0165] Optionally, the mutant strain is derived from the genus Corynebacterium.

[0166] Preferably, the mutant strain is derived from Corynebacterium glutamicum.

[0167] In some aspects of the present disclosure, there is also provided the use of DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV, or a promoter thereof for increasing the random mutation frequency of a mutant strain and / or reducing the fidelity of DNA replication, wherein the mutant strain contains: a DNA polymerase IIIα subunit mutant, a nucleic acid molecule encoding a DNA polymerase IIIα subunit mutant, or a promoter thereof; and / or a nuclease NucS mutant, a nucleic acid molecule encoding a nuclease NucS mutant, or a promoter thereof.

[0168] Optionally, the mutant strain is derived from the genus Corynebacterium.

[0169] Preferably, the mutant strain is derived from Corynebacterium glutamicum.

[0170] In some aspects of the present disclosure, a method for adaptive evolution of a strain is also provided, wherein the method comprises introducing into the strain a mutator system as described above, a polynucleotide as described above, and a recombinant expression vector as described above. In some optional embodiments, the method further comprises the step of isolating and purifying the adaptively evolved strain.

[0171] Example

[0172] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0173] Experimental Materials:

[0174] The components of TSB medium are: 5 g / L glucose, 5 g / L yeast powder, 9 g / L soy peptone, 3 g / L urea, 0.5 g / L succinic acid, 1 g / L K2HPO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.01 mg / L biotin, 0.1 mg / L vitamin B1, and 20 g / L MOPS.

[0175] Example 1 Construction of DinP overexpression vector

[0176] Primers DinP-F and DinP-R were designed based on the genomic sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI (NCBI accession number: 2830649). The wild-type dinP gene (Cgl2144; NCBI accession number: NP_601346.1) was obtained by PCR amplification using the genome of Corynebacterium glutamicum ATCC 13032 as a template. Primers P19-F / R were designed based on the sequence of plasmid pXMJ19 (Biotechnology Techniques, 1999, 13(6), 437-441). The pXMJ19 linearized vector fragment was obtained by PCR amplification using plasmid pXMJ19 as a template. The two fragments were recovered and recombined. The ligation product was transformed into Trans T1 competent cells (TransGen Biotech) and plated on LB plates containing 34 μg / mL chloramphenicol. The cells were cultured overnight and positive clones were selected for colony PCR and sequencing verification. The verified recombinant vector was named pXMJ19-DinP.

[0177] The specific sequences of the above primers are shown in Table 1.

[0178] Table 1 DinP amplification primers

[0179] Primer name Sequence information SEQ ID NO DinP-F agcttaaaggagttgagaatgcaacgctgggtgcttca SEQ ID NO: 4 DinP-R tctagagtcgacctgcagctattcgtcatcccccgtttcac SEQ ID NO: 5 P19-F CTGCAGGTCGACTCTAGAGGATC SEQ ID NO: 6 P19-R TCTCAACTCCTTTAAGCTTAATTAATTCTGTTT SEQ ID NO: 7

[0180] Example 2 Construction of DinP overexpression strain and mutation frequency test

[0181] Corynebacterium glutamicum ATCC 13032 competent cells were prepared by the method reported in the literature (Biotechnology Letters, 2015, 37: 2445-52; which is incorporated herein by reference), and 1 μg of pXMJ19 and pXMJ19-DinP plasmids were electroporated into the competent cells prepared above, and 1 mL of 46° C. preheated TSB medium was added, incubated at 46° C. for 6 min and 30° C. for 2 h, and then spread on TSB solid medium containing 5 μg / mL chloramphenicol. The cells were cultured at 30° C. for 1 day to obtain transformants and obtain recombinant strains ATCC 13032 / pXMJ19 and ATCC 13032 / pXMJ19-DinP.

[0182] The effect of overexpression of DinP on the mutation frequency of Corynebacterium glutamicum was tested by the method reported in the literature for rifampicin resistance (Nucleic Acids Research, 2023, 51(16):8623-8642; which is incorporated into the present disclosure by reference). The above recombinant strains were inoculated into TSB liquid medium containing 5 μg / mL chloramphenicol and 0.1 mM IPTG and cultured overnight until OD 600Dilute to 5×10 with TSB liquid medium. 6 The cells were plated on TSB solid medium without chloramphenicol and containing 5 μg / mL chloramphenicol, respectively. Another 100 μL bacterial liquid was plated on TSB solid medium containing 5 μg / mL chloramphenicol and 2 μg / mL rifampicin. The cells were cultured at 30°C overnight. After single colonies grew, they were counted and the mutation frequency was calculated using the following formula.

[0183]

[0184] The mutation frequency of the DinP overexpressing strain is shown in Table 2. Compared with the strain overexpressing the empty plasmid pXMJ19, the overexpression of DinP can slightly increase the mutation frequency of the strain, and the mutation frequency of the recombinant strain is increased by about 2.6 times.

[0185] Table 2 Mutation frequency of DinP overexpressing strains

[0186] strain Mutation frequency ATCC 13032 / pXMJ19 <![CDATA[0.14±0.01×10 -7 ]]> ATCC 13032 / pXMJ19-DinP <![CDATA[0.51±0.12×10 -7 ]]>

[0187] Example 3 Construction of a double mutant combining DinP overexpression with NucS or DnaE1 mutation

[0188] The inventors' previous studies have shown that overexpression of mutant DNA polymerase IIIα subunit DnaE1 and mutation of the key protein NucS of the mismatch repair mechanism in Corynebacterium glutamicum can lead to a higher mutation frequency of the strain (Nucleic Acids Research, 2023, 51(16):8623-8642.). Therefore, the present invention discloses that DnaE1 D20R Mutants and NucS E111L The mutant was combined with DinP to construct a new double mutant.

[0189] According to plasmid pXMJ19-DnaE1 D20R Primers E1-F and E1-R were designed based on the sequence of plasmid pXMJ19-DnaE1 D20R (Nucleic Acids Research, 2023, 51(16): 8623-8642.) was used as a template to obtain a linearized vector fragment by reverse PCR amplification. Using the pXMJ19-DinP recombinant vector as a template, primers PEF and PER were designed to amplify the dinP gene fragment. After the two fragments were recovered, they were recombined and connected. The connection product was transformed into Trans T1 competent cells, spread on LB resistance plates containing 34μg / mL chloramphenicol, cultured overnight, and positive clones were selected for colony PCR and sequencing verification. The verified correct recombinant vector was named pXMJ19-DnaE1 D20R -DinP (in this vector, DnaE1D20R and DinP were expressed in their respective expression frames, and the same was true in the subsequent examples).

[0190] According to the plasmid pXMJ19-NucS E111L Primers SF and SR were designed based on the sequence of plasmid pXMJ19-NucS E111L (Nucleic Acids Research, 2023, 51(16): 8623-8642.) was used as a template to obtain a linearized vector fragment by reverse PCR amplification. Using the pXMJ19-DinP recombinant vector as a template, primers PSF and PSR were designed to amplify the dinP gene fragment. After the two fragments were recovered, they were recombined and connected. The connection product was transformed into Trans T1 competent cells, spread on LB resistance plates containing 34μg / mL chloramphenicol, cultured overnight, and positive clones were selected for colony PCR and sequencing verification. The verified correct recombinant vector was named pXMJ19-NucS E111L -DinP.

[0191] The specific sequences of the above primers are shown in Table 3.

[0192] Table 3 Primers for amplification of DnaE1 or NucS double mutants

[0193] Primer name Sequence information SEQ ID NO E1-F attcggggtcgttcactggt SEQ ID NO: 8 E1-R ttaaccgaggatgcctggcc SEQ ID NO: 9 PEF ccaggcatcctcggttaaaaaggagttgagaatgcaacgct SEQ ID NO: 10 PER accagtgaacgaccccgaat SEQ ID NO: 11 SF ctgcaggtcgactctagagg SEQ ID NO: 12 SR TTAGAACAATGTCAGCTCATTGG SEQ ID NO: 13 PSF GAGCTGACATTGTTCTAAaaaggagttgagaatgcaacgct SEQ ID NO: 14 PSR cctctagagtcgacctgcag SEQ ID NO: 15

[0194] Example 4 Detection of mutation frequency of double mutants with DinP overexpression combined with NucS or DnaE1 mutants

[0195] The strain construction method described in Example 2 was used to construct the above recombinant vector and pXMJ19-DnaE1 D20R 、pXMJ19-NucS E111L The plasmids were transformed into competent cells of Corynebacterium glutamicum ATCC 13032 to obtain the recombinant strain ATCC 13032 / pXMJ19-DnaE1 D20R and ATCC 13032 / pXMJ19-DnaE1 D20R -DinP, ATCC 13032 / pXMJ19-NucS E111L and ATCC 13032 / pXMJ19-NucS E111L -DinP. At the same time, the mutation frequency detection method described in Example 2 was used to test the simultaneous overexpression of DnaE1 D20R and DinP, NucS E111L The mutation frequencies of the strains with the double mutants of β-DinP and β-DinP are shown in Table 4.

[0196] Table 4 Overexpression of DnaE1 D20Ror NucS E111L Mutation frequency of strains with DinP double mutants

[0197] strain Mutation frequency ATCC 13032 / pXMJ19 <![CDATA[0.14±0.01×10 -7 ]]> <![CDATA[ATCC 13032 / pXMJ19-DnaE1 D20R ]]> <![CDATA[20.25±2.05×10 -7 ]]> <![CDATA[ATCC 13032 / pXMJ19-DnaE1 D20R -DinP]]> <![CDATA[109.07±7.49×10 -7 ]]> <![CDATA[ATCC 13032 / pXMJ19-NucS E111L ]]> <![CDATA[11.63±1.50×10 -7 ]]> <![CDATA[ATCC 13032 / pXMJ19-NucS E111L -DinP]]> <![CDATA[15.39±1.55×10 -7 ]]>

[0198] The results showed that overexpression of NucS E111L After the mutant was treated with DinP, the expression of the mutant was increased by 109 times compared with the strain overexpressing the empty plasmid pXMJ19, but was significantly higher than that of the strain overexpressing NucS. E111L The mutant strain only increased by 32%, indicating that the introduction of DinP after weakening the mismatch repair system will not increase the mutation frequency of the strain by the same proportion. D20R After the mutant and DinP, the expression of DinP increased by 778 times compared with the strain overexpressing the empty plasmid pXMJ19 and by 778 times compared with the strain overexpressing DnaE1. D20R The mutant strain increased its mutation rate by 4.4 times, which is higher than the rate achieved by overexpressing DinP alone. These results indicate that enhancing the translesion repair capacity of C. glutamicum (DinP) can be combined with strategies that interfere with the proofreading function of DNA replication (DnaE1 mutants) to increase the mutation frequency of the strain. While combining it with a mutant that interferes with mismatch repair (NucS mutants) can also produce a certain additive effect, the effect is far less significant than the previous strategy.

[0199] Example 5: Construction of a new high-performance mutant by overexpressing DinP in combination with different mutants

[0200] The applicant's previous research showed that overexpression of DNA polymerase IIIα subunit DnaE1 mutations in Corynebacterium glutamicum can make the strain have a higher mutation frequency, and the mutation frequencies of different mutants are different (Nucleic Acids Research, 2023, 51(16):8623-8642.). Therefore, the present disclosure selected some different DnaE1 mutants and combined them with DinP to construct corresponding double mutants. At the same time, DnaE1 D20R and NucS E111L The mutants were combined with DinP to construct a triple mutant based on DnaE1, NucS mutant, and DinP.

[0201] Referring to the construction method of Example 3, primers E1-F and E1-R were used to construct plasmid pXMJ19-DnaE1 D20I 、pXMJ19-DnaE1 D20M 、pXMJ19-DnaE1 D223N 、pXMJ19-DnaE1 D20N(Nucleic Acids Research, 2023, 51(16): 8623-8642.) was used as a template to obtain a linearized vector fragment by reverse PCR amplification. Using the pXMJ19-DinP recombinant vector as a template, primers PEF and PER were designed to amplify the dinP gene fragment. After the two fragments were recovered, they were recombined and connected. The connection product was transformed into Trans T1 competent cells, spread on LB resistance plates containing 34μg / mL chloramphenicol, cultured overnight, and positive clones were selected for colony PCR and sequencing verification. The verified correct recombinant vector was named pXMJ19-DnaE1 D20I -DinP, pXMJ19-DnaE1 D20M -DinP, pXMJ19-DnaE1 D223N -DinP, pXMJ19-DnaE1 D20N -DinP.

[0202] According to plasmid pXMJ19-DnaE1 D20R -NucS E111L Primers were designed based on the sequence of plasmid pXMJ19-DnaE1 D20R (Nucleic Acids Research, 2023, 51(16): 8623-8642.) was used as a template, and primers SF and Sr, as well as Sf and SR, were used to obtain a linearized vector fragment by reverse PCR amplification. Using the pXMJ19-DinP recombinant vector as a template, primers PSF and PSR were designed to amplify the dinP gene fragment. After the three fragments were recovered, they were recombined and connected, and the connection products were transformed into Trans T1 competent cells, spread on LB resistance plates containing 34μg / mL chloramphenicol, cultured overnight, and positive clones were selected for colony PCR and sequencing verification. The verified correct recombinant vector was named pXMJ19-DnaE1 D20R -NucS E111L -DinP.

[0203] The specific sequences of the above primers are shown in Table 5.

[0204] Table 5 Triple mutant amplification primers

[0205] Primer name Sequence information SEQ ID NO S-r agccccggatgctttggata SEQ ID NO: 16 Sf tatccaaagcatccggggct SEQ ID NO: 17

[0206] The recombinant vector was transformed into competent cells of Corynebacterium glutamicum ATCC13032 using the strain construction method described in Example 2 to obtain the recombinant strain ATCC 13032 / pXMJ19-DnaE1. D20I 、ATCC 13032 / pXMJ19-DnaE1 D20I-DinP, ATCC 13032 / pXMJ19-DnaE1 D20M 、ATCC 13032 / pXMJ19-DnaE1 D20M -DinP, ATCC13032 / pXMJ19-DnaE1 D223N 、ATCC 13032 / pXMJ19-DnaE1 D223N -DinP, ATCC 13032 / pXMJ19-DnaE1 D20N 、ATCC 13032 / pXMJ19-DnaE1 D20N -DinP, ATCC 13032 / pXMJ19-DnaE1 D20R -NucS E111L At the same time, the mutation frequency of the strains simultaneously overexpressing the DnaE1 mutant and the DinP double mutant was tested using the mutation frequency detection method described in Example 2, as well as the mutation frequency of the strains overexpressing the DnaE1 mutant and the DinP double mutant. D20R 、NucS E111L The mutation frequencies of the mutants and DinP strains are shown in Table 6 .

[0207] Table 6 Mutation frequencies of strains overexpressing DnaE1 mutants, DinP, and triple mutants

[0208]

[0209] The mutation frequency of strains overexpressing DinP and different DnaE1 mutants was significantly increased, 4.1-8.1 times compared to strains overexpressing only the corresponding DnaE1 mutants, and 383-778 times compared to control strains carrying empty plasmid pXMJ19. D20R 、NucS E111L The mutation frequency of the mutant and DinP strains reached 252.36±14.69×10 -7 , is the overexpression of DnaE1 only D20R and NucS E111L The mutant double mutant strain had a mutation rate of about 2 times that of the double mutant strain and an increase of 1802 times compared to the control strain carrying the empty plasmid pXMJ19. These results further indicate that overexpression of the error-prone DNA polymerase DinP can be combined with the strategy of interfering with the proofreading function in DNA replication to further increase the mutation frequency of the strain.

[0210] Example 6 Testing of mutation types of different mutators

[0211] To test the mutation type preference of different mutagenizers, ATCC 13032 / pXMJ19-DnaE1 was used. D20R、ATCC 13032 / pXMJ19-DnaE1 D20R -DinP, ATCC 13032 / pXMJ19-DnaE1 D20R -NucS E111L 、ATCC13032 / pXMJ19-DnaE1 D20R -NucS E111L -DinP strains were subjected to mutation accumulation (MA) test (Nucleic Acids Research, 2018, 46(12): 6152-6165.). The above strains were respectively streaked on TSB plates containing 5 μg / mL chloramphenicol and 0.1 mM IPTG, with 2 parallels for each strain, and cultured overnight at 30 ° C. After growing a single clone of the same size, the single clone was picked and continued to be passaged on a new plate. After 20 consecutive passages, the single clones were picked and inoculated into TSB liquid medium containing 5 μg / mL chloramphenicol for overnight culture. After centrifugation to collect the bacteria, the genome was extracted, the whole genome second generation sequencing was performed, and the genome sequence was compared with the genome sequence of the starting strain to calculate the ratio of the mutated base types.

[0212] The results are shown in Table 7. There are obvious differences in the mutation preferences of different mutators. E111L Combined DnaE1 D20R It only reduced the frameshift mutation and further increased the base conversion ratio, while overexpression of DinP combined with DnaE1 D20R Then the ratio of base transversion can be greatly increased, base transition and frameshift mutations can be reduced, and a larger difference in mutation types can be shown, which can produce more diverse genotypes in the process of evolution. After the above three mutator elements are combined, the base mutation types are further concentrated on base transitions, which can avoid the large-scale genotype and phenotypic disturbances caused by random mutations and maintain the stability of the evolving bacterial colony. Therefore, the two novel mutator combination systems disclosed in the present invention can give bacterial strains a wider range of base mutation types relative to the prior art, which will contribute to the evolution of different adaptive phenotypes.

[0213] Table 7 Statistics of mutation type ratios of different mutators

[0214]

[0215] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0216] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A mutant system, characterized in that: The mutant system comprises: (i) DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV, or a promoter thereof; and (ii) a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant, or a promoter thereof.

2. The mutator system according to claim 1, characterized in that The DNA polymerase IV is selected from any one of the following groups consisting of (a1)-(a3): (a1) comprising the amino acid sequence shown in SEQ ID NO: 1; (a2) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in (a1), and retaining DNA polymerase IV activity; (a3) A polypeptide having an amino acid sequence as shown in (a1) or (a2) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus, and retaining DNA polymerase IV activity.

3. The mutator system according to claim 1 or 2, characterized in that: The DNA polymerase IIIα subunit mutant corresponds to the amino acid sequence shown in SEQ ID NO: 2, and has a mutated amino acid at one or more of the following positions: position 20 is mutated to cysteine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, valine or tyrosine, and / or position 223 is mutated to alanine, glutamine, serine or asparagine.

4. The mutant system according to any one of claims 1 to 3, characterized in that: The mutator system further includes a mutant of the endonuclease NucS, a nucleic acid molecule encoding the mutant of the endonuclease NucS, or a promoter thereof.

5. The mutant system according to any one of claim 4, characterized in that: The endonuclease NucS mutant corresponds to the amino acid sequence shown in SEQ ID NO: 3, and has the mutation E111L.

6. The mutant system according to any one of claims 1 to 5, characterized in that: The mutant system is selected from any one of the following (d1) to (d7): (d1) Mutator system DnaE1 D20R -NucS E111L -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof; Among them, the DNA polymerase IIIα subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2; the endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO: 3; (d2) Mutator system DnaE1 D20R -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof; Wherein, the DNA polymerase III α subunit mutant has a mutation D20R corresponding to the amino acid sequence shown in SEQ ID NO: 2; (d3) Mutator system DnaE1 D20M -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof; Wherein, the DNA polymerase III α subunit mutant has a mutation D20M corresponding to the amino acid sequence shown in SEQ ID NO: 2; (d4) Mutator system DnaE1 D20N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof; Wherein, the DNA polymerase III α subunit mutant has a mutation D20N corresponding to the amino acid sequence shown in SEQ ID NO: 2; (d5) Mutator system DnaE1 D223N -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof; Wherein, the DNA polymerase IIIα subunit mutant has a mutation D223N corresponding to the amino acid sequence shown in SEQ ID NO: 2; (d6) Mutator system DnaE1 D20I -DinP, which includes: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a DNA polymerase III α subunit mutant, a nucleic acid molecule encoding a DNA polymerase III α subunit mutant or a promoter thereof; Wherein, the DNA polymerase III α subunit mutant has a mutation D20I corresponding to the amino acid sequence shown in SEQ ID NO: 2; (d7) Mutator system NucS E111L -DinP, which comprises: DNA polymerase IV, a nucleic acid molecule encoding DNA polymerase IV or a promoter thereof, and a nuclease endonuclease NucS mutant, a nucleic acid molecule encoding a nuclease endonuclease NucS mutant or a promoter thereof; The endonuclease NucS mutant has a mutation E111L corresponding to the amino acid sequence shown in SEQ ID NO:

3.

7. An isolated polynucleotide, wherein The polynucleotide encodes the mutator system according to any one of claims 1 to 6.

8. A recombinant expression vector, wherein: The recombinant expression vector comprises the polynucleotide according to claim 7.

9. A recombinant host cell, wherein The recombinant host cell comprises the mutator system according to any one of claims 1 to 6, the polynucleotide according to claim 7, or the recombinant expression vector according to claim 8; Optionally, the host cell is derived from the genus Corynebacterium; Preferably, the host cell is derived from Corynebacterium glutamicum.

10. Use of the mutator system according to any one of claims 1 to 6, the polynucleotide according to claim 7, the recombinant expression vector according to claim 8, or the recombinant host cell according to claim 9 in at least one of the following (a) to (d): (a) preparing a mutant strain having an increased random mutation frequency; (b) preparing mutant strains with reduced DNA replication fidelity; (c) preparing mutant strains with increased mutational diversity; (d) used for adaptive evolution of strains; Optionally, the mutant strain is derived from the genus Corynebacterium; Preferably, the mutant strain is derived from Corynebacterium glutamicum.

11. A method for adaptive evolution of a strain, wherein: The method comprises introducing the mutator system according to any one of claims 1 to 6, the polynucleotide according to claim 7, and the recombinant expression vector according to claim 8 into the strain; optionally, the method further comprises the step of isolating and purifying the adaptively evolved strain.

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