Directed evolution system

Abstract

The invention relates to the technical field of biology, in particular to a directed evolution system and method. The system comprises an orthogonal transcription mutant, the orthogonal transcription mutant comprises bacteriophage RNA polymerase, deaminase and a first connecting sequence, and the deaminase is connected with the N end of the bacteriophage RNA polymerase through the first connecting sequence. Leakage expression can be reduced by increasing the number of regulatory elements in the inducible promoter, and controllable mutation is achieved; in addition, the negative screening gene sacB is added in the plasmid containing the orthogonal transcription mutants, the mutation plasmid can be rapidly removed after mutation is realized by adding cane sugar in a culture medium, and continuous mutation of the target gene is avoided. According to the directed evolution system and method, mutation can be rapidly and efficiently generated for a specific target gene, meanwhile, the mutation rate can be accurately regulated and controlled, leakage mutation is reduced, plasmid loss is rapidly achieved, and therefore the protein engineering modification speed is increased, and time and cost are saved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a directed evolution system, method, and vector. Background Art

[0002] Directed evolution is a protein engineering method that effectively mimics the mechanisms of natural evolution. By generating a large number of genetic variants in a short period of time and applying selective pressure in the laboratory, researchers can force proteins to evolve new functions or properties under specific circumstances, accelerating the evolutionary process. By combining design with experience and leveraging techniques from synthetic biology, directed evolution can systematically explore protein sequence space, better understand the mechanisms of evolution, and develop biomacromolecules with novel functions.

[0003] Directed evolution methods include in vitro construction of mutant libraries and in vivo targeted mutation construction of mutant libraries. The traditional directed evolution method (such as error-prone PCR) operation steps include constructing a mutant library of the target gene in vitro and then screening it in the host cell, which will produce a large number of relatively inefficient mutant libraries, and due to the lack of high-throughput screening means, mutant screening is also time-consuming, labor-intensive and costly. The method of constructing a mutant library using in vivo targeted mutation tools (such as CRISPR-Cas technology, positive translocation DNA polymerase method or virus replication-based method) improves the mutagenesis efficiency by making the mutation, expression and screening processes continuous, but the editing window of the in vivo directed mutagenesis method based on CRISPR-Cas technology is relatively narrow, and it is usually necessary to design many gRNAs to cover the target gene; while the method based on positive translocation DNA polymerase or the method based on virus replication will cause the entire linear plasmid or viral genome to mutate, and it is impossible to achieve directed mutation of specific genes or regions.

[0004] Therefore, there is an urgent need to develop a directed evolution system and method that can quickly and efficiently generate mutations in specific target genes, with reduced and controllable expression leakage, which will help to accelerate protein engineering and save time and costs, while accurately regulating the mutation rate, reducing leakage mutations and quickly achieving plasmid loss, and better screening for beneficial mutations. Summary of the Invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0006] To this end, embodiments of the first aspect of the present invention provide a directed evolution system comprising an orthogonal transcriptional mutagen, wherein the orthogonal transcriptional mutagen comprises a bacteriophage RNA polymerase, a deaminase, and a first linker sequence, wherein the deaminase is linked to the N-terminus of the bacteriophage RNA polymerase via the first linker sequence, and further comprising a mutation control element. This system can rapidly and efficiently generate mutations in specific target genes, thereby accelerating protein engineering and saving time and cost.

[0007] In some embodiments, the directed evolution system further comprises an orthogonal promoter, wherein the bacteriophage RNA polymerase specifically recognizes the orthogonal promoter.

[0008] In some embodiments, the orthogonal promoters include pMmP1, pK1F, and pVP4.

[0009] In some embodiments, the orthogonal transcription mutant further comprises an auxiliary mutation element, and the deaminase is connected to the auxiliary mutation element via a second linker sequence.

[0010] In some embodiments, the helper mutational element comprises UGI.

[0011] In some embodiments, the orthogonal transcription mutants are one or more orthogonal transcription mutants, wherein the phage RNA polymerase of the one or more orthogonal transcription mutants is independently selected from one or more of MmP1 RNA polymerase and variants thereof, K1F RNA polymerase and variants thereof, and VP4 RNA polymerase and variants thereof, wherein the deaminase of the one or more orthogonal transcription mutants is independently selected from one or more of deaminase APOBEC1 and variants thereof, deaminase AID and variants thereof, deaminase PmCDA1 and variants thereof, and deaminase TadA variants TadA7.10, TadA8e, TadA9, CABE T3.1, CABET3.155, and TadDE, wherein the first linker sequence comprises XTEN, (GGGGS) n .

[0012] In some embodiments, the directed evolution system further comprises an inducible expression system for controlling the orthogonal transcriptional mutants.

[0013] In some embodiments, the inducible expression system comprises one or more of an isopropylthiogalactoside (IPTG) induction system, an arabinose induction system, an acylhomoserine lactone (AHL) induction system, an anhydrotetracycline (ATC) induction system, and a vanillic acid induction system.

[0014] In some embodiments, the mutational regulatory element represses the directed evolution of the orthogonal transcriptional mutant under a first condition.

[0015] In some embodiments, based on the inducible expression system being an isopropylthiogalactoside (IPTG) inducible system, the mutation regulatory element is a lacO element, and the mutation regulatory element is exogenous IPTG; based on the inducible expression system being an anhydrotetracycline aTc inducible system, the mutation regulatory element is TetO, and the mutation regulatory element is exogenous aTc.

[0016] In some embodiments, the number of the lacO elements is 1-4; more preferably, the number of the lacO elements is 4.

[0017] In some embodiments, the directed evolution system further comprises a negative screening gene, wherein the negative screening gene removes the mutated plasmid subjected to the directed evolution by the orthogonal transcription mutant under a second condition, wherein the second condition is different from the first condition.

[0018] In some embodiments, the negative selection genes are sacB and pheS.

[0019] In some embodiments, the second condition is exogenous sucrose and p-chlorophenylalanine. The directed evolution system of the present invention, based on the aforementioned elements, can reduce leaky expression and achieve controlled mutation by increasing the number of regulatory elements in the inducible promoter. Furthermore, by adding the negative selection gene sacB to the plasmid containing the orthogonal transcriptional mutagen, the mutant plasmid can be quickly removed after mutation by adding sucrose to the culture medium, thus preventing continued mutation of the target gene.

[0020] An embodiment of the second aspect of the present invention provides a vector, characterized in that the vector includes a first vector, which contains at least one polynucleotide encoding an orthogonal transcription mutant, phage RNA polymerase or deaminase as described in any embodiment of the first aspect of the present invention.

[0021] In some embodiments, the first vector further comprises a first linker sequence.

[0022] In some embodiments, the first vector further comprises an inducible expression system for use with the orthogonal transcriptional mutant.

[0023] In some embodiments, the vector includes a second vector for inserting an orthogonal promoter upstream of the target gene or inserting it in reverse direction downstream of the target gene.

[0024] In some embodiments, the system further comprises a mutation regulatory element, a negative selection gene, and / or other regulatory elements or proteins.

[0025] The embodiments of the fourth aspect of the present invention propose a directed evolution method, characterized in that it includes: using the directed evolution system as described in any embodiment of the first aspect of the present invention, or the vector as described in any embodiment of the second aspect of the present invention to perform targeted mutation to achieve directed evolution.

[0026] In some embodiments, the method reduces leaky expression by increasing the number of mutated regulatory elements; and by adding a negative selection gene sacB to the plasmid containing the orthogonal transcriptional mutant to quickly remove the mutant plasmid after mutation.

[0027] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0028] The orthogonal phage RNAP polymerase-based directed mutagenesis system provided by the present invention can rapidly and efficiently generate a large number of mutations specifically and efficiently in target genes on plasmids or genomes. The optimized orthogonal transcription mutagens can generate two types of mutations, C:G to T:A and A:T to G:C, separately or simultaneously. This greatly improves the efficiency of mutation and evolution of enzymes or other proteins, accelerates protein engineering, saves time and costs, and has broad application prospects. Furthermore, this directed mutagenesis system has reduced and controllable expression leakage, which facilitates better screening of beneficial mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the directed mutation principle of the directed evolution system according to an embodiment of the present invention.

[0030] Figure 2 Schematic diagram of orthogonality detection design for three orthogonal transcription mutants in the directed evolution system of an embodiment of the present invention.

[0031] Figure 3 Schematic diagram of orthogonality detection results of three orthogonal transcription mutants in the directed evolution system of an embodiment of the present invention.

[0032] Figure 4 This is a fluorescence microscopy result of a microorganism after the target fluorescent protein gene is mutated using the directed evolution system of an embodiment of the present invention.

[0033] Figure 5 This is a diagram of the microbial culture results after the target pigment protein gene is mutated using the directed evolution system of an embodiment of the present invention.

[0034] Figure 6 This is a cell scanning electron microscopy analysis result after the target cytoskeleton-related gene mreBCD is mutated using the directed evolution system of an embodiment of the present invention.

[0035] Figure 7This is a cell scanning electron microscopy analysis result after the target cell division-related gene ftsQAZ was mutated using the directed evolution system of an embodiment of the present invention.

[0036] Figure 8 This is a cell scanning electron microscopy analysis result diagram after the target cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ were mutated using the directed evolution system of an embodiment of the present invention.

[0037] Figure 9 This is a result diagram showing the reduction of leaky expression by increasing the number of LacO elements in the inducible promoter according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0039] Unless otherwise indicated, all scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures in the following cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are well known in the art and commonly used. Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are carried out according to the conventional methods described in this area and various general references (generally referring to Sambrook et al., "Molecular Cloning: A Laboratory Manual" (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (Cold Spring Harbor, NY), which are incorporated herein by reference), and are incorporated herein in their entirety. The nomenclature used herein and the laboratory procedures in the following analytical chemistry and organic synthesis are well known in the art and commonly used. Chemical synthesis or chemical analysis can also be carried out using standard techniques or variations thereof.

[0040] In the disclosed embodiments, "naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that has not been intentionally modified by human manipulation. A "mutant" means a sequence that has at least one amino acid alteration relative to a native or wild-type amino acid sequence. In some embodiments, the alteration (mutation) comprises at least one of a substitution, a deletion, and an insertion.

[0041] In the disclosed embodiments, amino acids may be represented by the common three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their recognized single-letter codes.

[0042] In the embodiments of the present disclosure, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., a carbon atom is bound to a hydrogen atom, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain a chemical structure that is essentially the same as that of naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of amino acids but act in a manner similar to that of naturally occurring amino acids. The amino acid sequences proposed in the embodiments of the present disclosure may contain the above-mentioned amino acid analogs and mimetics or related modifications, as long as they do not affect the basic properties of the corresponding amino acids and the activity of the entire enzyme or its active fragments.

[0043] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0044] Directed evolution is a protein engineering method that effectively mimics the mechanisms of natural evolution. By generating a large number of genetic variants in a short period of time and applying selective pressure in the laboratory, researchers can force proteins to evolve new functions or properties under specific circumstances, accelerating the evolutionary process. By combining design with experience and leveraging techniques from synthetic biology, directed evolution can systematically explore protein sequence space, better understand the mechanisms of evolution, and develop biomacromolecules with novel functions.

[0045] Traditional directed evolution methods (such as error-prone PCR) involve constructing a mutant library of the target gene in vitro and then screening it in host cells. This produces a large number of relatively inefficient mutant libraries, and due to the lack of high-throughput screening methods, mutant screening is time-consuming, labor-intensive and costly.

[0046] In addition, methods for constructing mutant libraries using in vivo targeted mutagenesis tools (such as CRISPR-Cas technology, positive translocation DNA polymerase methods, or viral replication-based methods) improve mutagenesis efficiency and expand the diversity of variant libraries by enabling continuous mutation, expression, and screening. In vivo targeted mutagenesis methods based on CRISPR-Cas technology (such as EvolR, CRISPR-X, base editors, and guide editors) exploit the sequence recognition specificity of sgRNAs to generate mutations in specific regions. However, these methods have relatively narrow editing windows, typically requiring the design of multiple gRNAs to cover the target gene. Methods based on positive translocation DNA polymerases (such as the Orthorep system in Saccharomyces cerevisiae, the BacORep system in Bacillus thuringiensis, and the EcoRep system in Escherichia coli) and viral replication-based methods (such as PACE and VEGAS) have been successfully established and applied to the evolution of many useful proteins. However, these methods mutate the entire linear plasmid or viral genome, making them incapable of achieving targeted mutagenesis of specific genes or regions.

[0047] To this end, an embodiment of the first aspect of the present invention proposes a directed evolution system, comprising an orthogonal transcription mutant, wherein the orthogonal transcription mutant comprises a phage RNA polymerase, a deaminase, and a first linker sequence, wherein the deaminase is connected to the N-terminus of the phage RNA polymerase via the first linker sequence, and the directed evolution system further comprises a mutation control element. The system can quickly and efficiently generate mutations in specific target genes, thereby accelerating the speed of protein engineering and saving time and cost. The schematic diagram of the directed mutation principle of the directed evolution system is shown in FIG. Figure 1 As shown, specifically, an orthogonal transcription mutant obtained by fusing a bacteriophage RNA polymerase with a deaminase is provided to the host microorganism. The bacteriophage RNA polymerase portion of the orthogonal transcription mutant can specifically recognize the corresponding promoter (i.e., an orthogonal promoter) located upstream of the target gene and transcribe the downstream gene. During the transcription process, the double-stranded DNA opens to expose a portion of the single-stranded region. Subsequently, the deaminase portion of the orthogonal transcription mutant can undergo a deamination reaction on the nucleotides of the single-stranded DNA and generate mutations during the subsequent replication and repair process, thereby achieving rapid and efficient generation of a large number of mutations on the target gene.

[0048] In some embodiments, the specific process of applying the directed evolution system of the present invention includes selecting a suitable phage RNA polymerase; selecting a suitable deaminase and auxiliary mutation element; connecting the deaminase to the N-terminus of the phage RNA polymerase via a suitable linker sequence; and inserting a phage RNA polymerase-specific promoter upstream or downstream of the target gene on a plasmid or genome. This promoter can then recruit orthogonal transcriptional mutagens, thereby initiating transcriptional mutagenesis.

[0049] It should be noted that the selected phage RNA polymerase has high specificity and transcriptional activity. Specificity is reflected in the phage RNA polymerase's ability to specifically recognize the corresponding phage promoter and not other promoters, thereby avoiding interference with the host's endogenous promoter or other phage promoters, and thus ensuring that mutations only occur in the targeted gene downstream of the phage promoter and not in other non-targeted locations. High transcriptional activity is reflected in the phage RNA polymerase's ability to efficiently initiate transcription of downstream genes, opening the DNA double helix to expose some single-stranded DNA regions, creating conditions for subsequent deamination reactions.

[0050] It should be noted that deaminases can recognize pyrimidine or purine bases and produce deamination reactions, which can then generate mutations during the subsequent replication process. Deaminases must have strong mutagenic activity to ensure they can generate sufficient mutations and increase the mutation rate. Expressing appropriate auxiliary mutagenic elements, such as the glycosylase inhibitor UGI, can prevent mutations generated by cytosine deaminase from being repaired by the endogenous repair system, further increasing the mutation rate.

[0051] It should be noted that because the C-terminus of the phage RNA polymerase plays a crucial role in its transcriptional activity, only by linking the deaminase and auxiliary mutation element to the N-terminus of the phage RNA polymerase can this effect be avoided. Furthermore, it is important to select an appropriate linker sequence to avoid interfering with the functions of the various components of the fusion protein.

[0052] In some embodiments, the directed evolution system further comprises an orthogonal promoter, wherein the bacteriophage RNA polymerase specifically recognizes the orthogonal promoter.

[0053] In some embodiments, the orthogonal transcription mutant further comprises an auxiliary mutation element, and the deaminase is connected to the auxiliary mutation element via a second linker sequence.

[0054] In some embodiments, the auxiliary mutation element comprises UGI, which is a glycosylase inhibitor derived from Bacillus subtilis phage PBS1, and the amino acid sequence of UGI is shown in SEQ ID NO: 4: MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWAL VIQDSNGENKIKML* (SEQ ID NO: 4).

[0055] In some embodiments, the orthogonal transcriptional mutant is one or more orthogonal transcriptional mutants. Figure 2 or Figure 3In the directed evolution system of the embodiment of the present invention, multiple orthogonal transcription mutants can be provided simultaneously to complete multiple mutations, and the orthogonality test results of the multiple orthogonal transcription mutants show that each of them has high specificity.

[0056] In some embodiments, the phage RNA polymerase of the one or more orthogonal transcription mutants is independently selected from one or more of MmP1 RNA polymerase and variants thereof, K1F RNA polymerase and variants thereof, and VP4 RNA polymerase and variants thereof.

[0057] The amino acid sequence of MmP1 RNA polymerase is shown in SEQ ID NO: 1:

[0058] MSIAAAVNKNDFSDVELAAIPFNTLADHYGADLAREQLQLEHESYVMGEERFRKMLERQEKAEEFGDSSVAKPLIITLLPKVTQRITDWLNEWADPNKKGRKPIAYTHLKDIKPETLAFITIKVVLNKLAGKDDAFMQPLAYAIGSSIEDEARFGRIRELEMAHFKKCAEENLNKRRGTAYRKAFLSVVEADMLDKGLLGGESWGTWNKTDVMNIGISMLEKLIEATGLVELREKRNFEEMDRIVIAEEYVKAMATRAQSLAGISPMYQPCVVPPKPWVSITGGGYWANGRKPTALIRTHTRKALYRYEDVYMPEVYKAINYAQETPWRINRKVLAVVNELVKWKNNPVKDMPSIDKLELPQRPDDIDTNEEALRSWKREAAAVYRKDEQRKSRYLSMSFALEQANKFSNKKAIYFPYNMDWRGRVYALPMFNPQGNDMVKGLLTLAKGKPIGKDGFYWLKIHGANTAGVDKVTFPERIKFIEDNHDNIMQCAESPLDNLWWTEQDSPFCFLAFCFEYAQVTKKGLGWVCSLPIALDGSCSGIQHFSAMLRDDIGGRAVNLLPSETVQDIYGIVADKVNEALKELVINGTDNYTDTVTDKSTGEIIERYRLGEKELARQWLEFGVTRSVTKRSVMTLAYGSKEYGFRDQVLEDTIRPAIDSGKGAMFTNPSQAASFMAKRIWEAVSVTVVAAVGAMKWLQSSAKLMAAEVKDKKTKEVLRKRCAVHWVTPDGFPVWQEYRKPKQKRVHLMFLGSYYDARMKETSSDCSIDAHKQESGISPNFVHSQDGNHLRMTVVYAREKYNVESFALIHDSFGTIPADVPNLFKAVRETMVNMYENNDVLADFYEQFADQLHESQLDKMPALPPKGKLNLQDILKSDFAFA*(SEQ ID NO:1).

[0059] The amino acid sequence of K1F RNA polymerase is shown as SEQ ID NO:2 below:

[0060] MSVISIDKHDFSDVSNAIEPFNLLADHYGQDLAVKQLQLEHEAYTEGERRFIKNLERQTERGELADNQVAKPLMQTLVPKIAQAVKEWHEGPDGKLSTSRPSVAFTMLSTEERAVKDRSLRISCESAAVIILKVILSKLVKPEGIPITPMASAIGRTLEDEIRFGRIRDKEKEHFKKAIADNLNKRAGASYKKAYMQAVEASMLEQGQLEDAWGTWSPTEAVHVGIKMLEIVIQSTQLVELKRYGAGNAAADVEMVHLSDFWVKKMAQRGFSLAGIAPVYQPCVVPPKPWTGVVGGGYWAKGRRPLPLIRLGSKSAVARYEDVYMPEVYEAVNIIQNTPWKVNKKVLDVVNMVEKLNNTPIDDIPQMEPLKPEAYAGETEEELKAWKKAAAGIYRREKARQSRRLSLSFIVNQANKFSQFKAIWFPYNMDWRGRVYAVPMFNPQGNDMQKGLLTLAVGKPIGADGFKWLKVHGANCAGVDKVTFEERIKWVEDNHDNIMAAAKAPMDSIEWWGKLDSPFCFLAFCFEYAGVMHHGLSYSCSLPIAFDGSCSGIQHFSAMLRDHIGGHAVNLTPSGKVQDIYRIVSDRIEEELKVLLVNGTDNEMVTHEDKKTGEITERLKLGTRELARQWLTYGMSRKVTKRSVMTLAYGSKEYGFADQVYEDIVMPAIDSGSGAMFTEPSQASRFMAKMIWEAVSVTVVAAVDAMKWLQGAAKLLAAEVKDKKTGEILKPCLPVHWVTPDGFPVWQEYRKKDTTRLNLMFLGSFNLQPTVNKGTKKELDKHKQESGISPNFVHSQDGSHLRKTVVHTHRKYGVMSFAVIHDSFGTIPADAEYLFRGVRETMVETYRDNDVLLDFYEQFEYQLHESQRDKLPELPKKGKLNIEDILSSDFAFA*(SEQ ID NO:2).

[0061] The amino acid sequence of VP4 RNA polymerase is as shown in SEQ ID NO:3:

[0062] *(SEQ ID NO:3).

[0063] In some embodiments, the deaminase of the one or more orthogonal transcription mutants is independently selected from one or more of deaminase APOBEC1 and variants thereof, deaminase AID and variants thereof, deaminase PmCDA1 and variants thereof, and deaminase TadA variants TadA7.10, TadA8e, TadA9, CABE T3.1, CABE T3.155, and TadDE. Specifically, one or more of rat cytidine deaminase APOBEC1 and variants thereof, human activation-induced cytidine deaminase AID and variants thereof, lamprey cytidine deaminase PmCDA1 and variants thereof, adenosine deaminase TadA variants TadA7.10, TadA8e, and TadA9 from Escherichia coli tRNA, and TadA variants CABE T3.1, CABE T3.155, and TadDE having both cytosine deaminase and adenine deaminase activity.

[0064] The amino acid sequence of PmCDA1 cytosine deaminase is shown in SEQ ID NO: 5:

[0065] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILE WYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV*(SEQ ID NO:5).

[0066] The amino acid sequence of the variant of deaminase PmCDA1, evoPmCDA1 cytosine deaminase, is shown in SEQ ID NO: 6:

[0067] MTDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILE WYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV*(SEQ ID NO:6).

[0068] The amino acid sequence of TadA7.10 adenine deaminase is shown in SEQ ID NO: 7:

[0069] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRV EITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD* (SEQ ID NO: 7).

[0070] The amino acid sequence of TadA8e adenine deaminase is shown in SEQ ID NO: 8:

[0071] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRV EITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN* (SEQ ID NO: 8).

[0072] The amino acid sequence of TadA9 adenine deaminase is shown in SEQ ID NO: 9:

[0073] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRV EITEGILADECAALLCDFYRMPRRVFNAQKKAQSSIN* (SEQ ID NO: 9).

[0074] In some embodiments, the first linker sequence comprises XTEN and (GGGGS) n .

[0075] The nucleotide sequence of XTEN is as follows: agcggcagcgagactcccgggacctcagagtccgccacacccgaaagt (SEQ ID NO: 10).

[0076] In some embodiments, the orthogonal promoters include pMmP1, pK1F, and pVP4. The nucleotide sequence of the pMmP1 promoter is as follows: cccatgagttaattatatttgtggcattataggg (SEQ ID NO: 11). The nucleotide sequence of the pK1F promoter is as follows: gacatggctcaagcctaaactatcactatagg (SEQ ID NO: 12). The nucleotide sequence of the pVP4 promoter is as follows: gaagtaacttgattaattaaccctgactataggga (SEQ ID NO: 13).

[0077] In some embodiments, the directed evolution system further comprises an inducible expression system for controlling the orthogonal transcriptional mutants.

[0078] In some embodiments, the inducible expression system comprises one or more of an isopropylthiogalactoside (IPTG) induction system, an arabinose induction system, an acylhomoserine lactone (AHL) induction system, an anhydrotetracycline (ATC) induction system, and a vanillic acid induction system.

[0079] In some embodiments, the inducible expression system comprises P Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acyl homoserine lactone AHL inducible system), P Tet and its mutants (anhydrotetracycline aTc inducible system), P Van and one or a combination of its mutants (vanillic acid inducible system). A person skilled in the art will understand that a mutant refers to a base mutation at a core site in a promoter to have different induction strengths.

[0080] In some embodiments, the mutational regulatory element represses the directed evolution of the orthogonal transcriptional mutant under a first condition.

[0081] In some embodiments, based on the inducible expression system being an isopropylthiogalactoside (IPTG) inducible system, the mutation regulatory element is a lacO element, and the mutation regulatory element is exogenous IPTG; based on the inducible expression system being an anhydrotetracycline aTc inducible system, the mutation regulatory element is TetO, and the mutation regulatory element is exogenous aTc.

[0082] In some embodiments, the number of the lacO elements is 1-4; more preferably, the number of the lacO elements is 4.

[0083] In some embodiments, the directed evolution system further comprises a negative screening gene, wherein the negative screening gene removes the mutated plasmid subjected to the directed evolution by the orthogonal transcription mutant under a second condition, wherein the second condition is different from the first condition.

[0084] In some embodiments, the negative selection genes are sacB and pheS.

[0085] In some embodiments, the second condition is exogenous sucrose and p-chlorophenylalanine.

[0086] It should be noted that the directed evolution system of the present invention suffers from problems such as leaky expression and difficulty in losing the plasmid, which leads to continuous mutations, potentially rendering beneficial mutations ineffective or deleterious. Therefore, leaky expression is reduced by increasing the number of mutation regulatory elements and by adding the negative selection gene sacB to the plasmid containing the orthogonal transcriptional mutagen to rapidly remove the mutant plasmid after mutation. This results in a more controllable targeted system.

[0087] In some embodiments, the microorganisms to which the orthogonal transcriptional mutants are applicable include prokaryotic microorganisms and eukaryotic microorganisms, wherein the eukaryotic microorganisms include yeast, fungi or algae, and the prokaryotic microorganisms are selected from at least one of Escherichia coli, Pseudomonas, Eutropha rhodesi, Aeromonas hydrophila, Corynebacterium glutamicum or halophilic bacteria.

[0088] In some embodiments, the halophilic microorganism includes Halomonas bacteria and its derivative strains, and the halophilic microorganism includes Halomonas bluephagenesis TD01 (CGMCC No. 4353), Halomonas campaniensis LS21 (CGMCC No. 6593), and Halomonas aydingkolgenesis M1 (CGMCC No. 19880).

[0089] An embodiment of the second aspect of the present invention provides a polynucleotide, wherein the polynucleotide comprises at least one polynucleotide encoding an orthogonal transcription mutant, a phage RNA polymerase, or a deaminase as described in any embodiment of the first aspect of the present invention.

[0090] In some embodiments, the polynucleotide further comprises a first linker sequence.

[0091] An embodiment of the third aspect of the present invention provides a vector, characterized in that the vector includes a first vector, which contains at least one polynucleotide encoding an orthogonal transcription mutant, phage RNA polymerase or deaminase as described in any embodiment of the first aspect of the present invention.

[0092] In some embodiments, the first vector further comprises a first linker sequence.

[0093] In some embodiments, the first vector further comprises an inducible expression system for use with the orthogonal transcriptional mutant.

[0094] In some embodiments, the vector includes a second vector for inserting an orthogonal promoter upstream of the target gene or inserting it in reverse direction downstream of the target gene.

[0095] In some embodiments, the system further comprises a mutation regulatory element, a negative selection gene, and / or other regulatory elements or proteins.

[0096] The embodiments of the fourth aspect of the present invention propose a directed evolution method, characterized in that it includes: using the directed evolution system as described in any embodiment of the first aspect of the present invention, or the polynucleotide as described in any embodiment of the second aspect of the present invention, or the vector as described in any embodiment of the third aspect of the present invention to perform targeted mutation to achieve directed evolution.

[0097] In some embodiments, the method reduces leaky expression by increasing the number of mutated regulatory elements; and by adding a negative selection gene sacB to the plasmid containing the orthogonal transcriptional mutant to quickly remove the mutant plasmid after mutation.

[0098] It should be noted that the above explanations of the embodiments proposed in the first aspect of the present disclosure are also applicable to the polynucleotides proposed in the second aspect of the present disclosure, the vectors proposed in the third aspect of the present disclosure, and the methods proposed in the fourth aspect of the present disclosure, and will not be repeated here.

[0099] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0100] Example

[0101] Example 1 Construction and testing of orthogonal transcriptional mutagens that produce single-type mutations

[0102] In this example, multiple orthogonal transcriptional mutants that produce single-type mutations from C:G to T:A or A:T to G:C were constructed, and the mutation rates of the orthogonal transcriptional mutants were tested.

[0103] 1.1 Construction of orthogonal transcriptional mutants

[0104] The orthogonal transcription mutants constructed are specifically shown in Table 1. Table 1 shows that PmCDA1, a fusion of PmCDA1 and the glycosylase inhibitor UGI, a fusion of evoPmCDA1 (PmCDA1 variant) and the glycosylase inhibitor UGI, and TadA7.10, TadA8e, and TadA9, adenosine deaminase variants from Escherichia coli tRNA, were fused to the N-termini of three phage RNA polymerases, MmP1, K1F, and VP4, respectively, using XTEN linker sequences to construct various types of orthogonal transcription mutants that produce single-type mutations. Plasmids expressing only the three phage RNA polymerases, MmP1, K1F, or VP4, were set as control groups. These orthogonal transcription mutants were generated by PmCDA1. Tac The expression is driven by an inducible promoter and the plasmid vector is the high-copy number pSEVA341.

[0105] Table 1

[0106]

[0107]

[0108] 1.2 Testing the mutation rate of orthogonal transcriptional mutants

[0109] Functional restoration experiments based on the erythromycin resistance gene were used to detect the mutation rates of orthogonal transcriptional mutants that produced single-type mutations.

[0110] (1) The targeting plasmid expressing the inactivated erythromycin resistance gene and the plasmids of different orthogonal transcription mutants as shown in Table 1 were co-cultured in a medium containing chloramphenicol and spectinomycin at 37°C for 10 hours.

[0111] (2) Then, the secondary seed solution was transferred at a concentration of 1:100 and 200 mg / L ITPG inducer was added, and cultured at 37°C for 20 hours.

[0112] (3) Dilute the culture medium in (2) to 10 by 10-fold gradient. -7 , and spread them on plates containing or not containing 200 mg / L erythromycin, and cultured at 37℃ for 48h.

[0113] (4) Count the number of colonies growing on the erythromycin plate (N1) and the number of colonies growing on the plate without erythromycin (N0), and calculate the mutation rate based on N1 / N0. Each group had three independent replicates, and the mean and standard error of the mutation rate were calculated.

[0114] The results are shown in Tables 2 and 3. Table 2 shows the mutation rates of orthogonally transcribed mutants using MmP1 phage RNA polymerase. Table 3 shows the mutation rates of orthogonally transcribed mutants using K1F and VP4 phage RNA polymerases.

[0115] Table 2

[0116]

[0117] Table 3

[0118]

[0119] Results showed that all orthogonal mutagens constructed using different combinations of deaminases and phage RNA polymerases increased the targeted mutation rate by 564-fold to 799,118-fold compared to controls expressing only phage RNA polymerase. Mutagens using either PmCDA1-UGI ​​or TadA8e fusions of phage RNA polymerase showed the best results, achieving the highest mutation efficiency. Therefore, orthogonal mutagens can efficiently generate single-type mutations, such as C:G to T:A or A:T to G:C.

[0120] Example 2 Construction and testing of orthogonal transcriptional mutants producing double mutations

[0121] In this example, multiple orthogonal transcriptional mutants that simultaneously produce double mutations from C:G to T:A and A:T to G:C were constructed, and the mutation rates of the orthogonal transcriptional mutants that produce double mutations were tested.

[0122] 2.1 Construction of orthogonal transcriptional mutants

[0123] The orthogonal transcriptional mutagen constructed to generate double mutations is shown in Table 4. This orthogonal transcriptional mutagen was implemented by fusing the two deaminases, PmCDA1-UGI ​​and TadA8e, to the N-terminus of a bacteriophage RNA polymerase in different sequences (PmCDA1-UGI-TadA8e-phage RNAP and TadA8e-PmCDA1-UGI-phage RNAP), or by co-expressing PmCDA1-UGI-phage RNAP and TadA8e-phage RNAP on a single plasmid (opt). To prevent homologous recombination, the DNA sequence of the phage RNA polymerase fused to TadA8e was codon-optimized.

[0124] Table 4

[0125]

[0126]

[0127] 2.2 Testing the mutation rate of orthogonal transcriptional mutants

[0128] Referring to the steps in Example 1.2, a functional restoration experiment based on the erythromycin resistance gene was used to detect the mutation rate of the orthogonal transcription mutants that produced the double mutation.

[0129] The results are shown in Table 5. These orthogonal transcriptional mutagens can simultaneously produce C:G to T:A and A:T to G:C mutations, thereby expanding the mutation range, which is beneficial to improving the diversity of the target gene mutation library and enhancing the mutation efficiency.

[0130] Table 5: Mutation rates of orthogonal transcriptional mutants producing double mutations

[0131]

[0132] Example 3 Orthogonality Detection of Three Orthogonal Transcription Mutants

[0133] In this example, the orthogonality of orthogonal transcription mutants based on three phage RNA polymerases was designed and tested.

[0134] (1) Three orthogonal transcription mutants and their control groups were constructed, and the corresponding phage promoters were inserted upstream and downstream of the target gene sacB gene (encoding secreted sucrose fructanase, which can convert sucrose into toxic fructan) to obtain the targeting plasmid. Figure 2 The three orthogonal transcription mutants include pMT23opt-MmP1, pMT23opt-K1F and pMT23opt-VP4, and the corresponding control groups are pMT0-MmP1, pMT0-K1F and pMT0-VP4. The principle is as follows Figure 2 shown.

[0135] (2) The targeting plasmid expressing the sacB gene and different orthogonal transcription mutants were cultured in culture medium at 37°C for 10 hours.

[0136] (3) Then transfer the secondary seed solution at a concentration of 1:100 and add 200 mg / L ITPG inducer, and culture at 37°C for 20 hours.

[0137] (4) Dilute the culture medium in a 10-fold gradient to 10 -7 , and spread them on plates containing or not containing 100 g / L sucrose, and cultured at 37°C for 48 h.

[0138] (5) The mutation rate multiplier was calculated as M1 / M0, where M1 represents the mutation rate using the orthogonal transcriptional mutagen and M0 represents the background mutation rate of the corresponding phage RNA polymerase control group.

[0139] The orthogonality test results are as follows Figure 3 As shown, when targeting the corresponding promoters pMmP1, pK1F, and pVP4, the mutation rates of the pMT23opt-MmP1, pMT23opt-K1F, and pMT23opt-VP4 orthogonal transcription mutants increased by 46,608-, 46,470-, and 8,388-fold, respectively. However, when targeting the other two promoters, the mutation rates increased only 6-27-fold compared to the control. These results demonstrate that orthogonal transcription mutants based on different RNAPs are highly specific and exhibit little cross-talk.

[0140] Example 4: Using orthogonal transcriptional mutagen to mutate target genes on plasmids

[0141] In this example, orthogonal transcriptional mutagens were used to mutate the target genes on the plasmid, namely, three fluorescent protein genes and three pigment protein genes, and the mutations were detected by cell color and sequencing.

[0142] (1) Using pSEVA321 as the backbone plasmid, targeting plasmids containing three fluorescent protein genes, mCheery, sfGFP, and TagBFP, as well as three pigment protein genes, amajLime, fwYellow, and spisPink, were constructed.

[0143] (2) The pMmP1 promoter was inserted upstream and downstream of the two gene clusters mentioned above.

[0144] (3) Then, the pMT23opt-MmP1 double mutant plasmid and the pMT0-MmP1 control plasmid were transferred to induce expression.

[0145] (4) Take the bacterial solution and observe the color change using a fluorescence confocal microscope, and then spread the bacterial solution on a plate. Select the colonies with color changes for colony PCR and sequencing analysis.

[0146] Figure 4 This image shows fluorescence microscopy results from microorganisms mutated with the targeted fluorescent protein genes using the directed evolution system of an embodiment of the present invention. Compared to the control group, the pMT23opt-MmP1 orthogonal transcription mutant produced a variety of colors, such as purple and cyan. Sequencing results, shown in Table 6, indicate the presence of different types of mutations in the three fluorescent protein genes of these mutants.

[0147] Figure 5Figure 7 shows the results of microbial culture after mutation of the target pigment protein gene using the directed evolution system of an embodiment of the present invention. Compared to the control group, colonies using orthogonal transcription mutants exhibited a variety of colors. Sequencing results for selected colonies with significant color differences are shown in Table 7. These results indicate that the mutants harbor different types of mutations in the three pigment protein genes.

[0148] These results demonstrate that orthogonal transcriptional mutagens can rapidly mutate fluorescent protein and pigment protein genes to produce a variety of colors, demonstrating the effectiveness of this system in mutating target genes in plasmids.

[0149] Table 6

[0150]

[0151] Table 7

[0152]

[0153] Example 5 Using orthogonal transcriptional mutagen to mutate target genes on the genome

[0154] In this example, orthogonal transcriptional mutagens were used to mutate target genes on the genome, namely, the cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ, separately or simultaneously, and the mutations were detected by scanning electron microscopy and sequencing.

[0155] (1) The cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ in the Halomonas genome were used as target genes. The pMmP1 promoter sequence was inserted behind these two gene clusters using the CIRSPR / Cas9 system (for insertion methods, see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp[J]. Metabolic engineering, 2018, 47: 219-229) to recruit orthogonal transcriptional mutants.

[0156] (2) The pMT23opt-MmP1 double mutant plasmid and the pMT0-MmP1 control plasmid were transferred into Halomonas for induced expression.

[0157] (3) Scanning electron microscopy analysis.

[0158] The results are as follows Figure 6-8As shown in the figure, compared to the control group, the pMT23opt-MmP1 double mutant plasmid mutated the cell division-related gene ftsQAZ, resulting in significantly elongated cell morphology. Mutating the cytoskeleton-related gene mreBCD changed the cell morphology from rod-shaped to spherical. Furthermore, mutating both the cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ simultaneously resulted in cells exhibiting ellipsoidal, elongated, and other irregular shapes. These results demonstrate that using orthogonal transcriptional mutagens to mutate cytoskeleton and cell division-related genes within a genome can successfully induce cells to exhibit a variety of morphologies, demonstrating the effectiveness of this system in mutating target genes within a genome.

[0159] Example 6: Increasing the number of LacO elements to reduce the leaky mutation rate

[0160] In this embodiment, by increasing the number of LacO elements in the inducible promoter, leaky expression can be reduced and controllable mutation can be achieved.

[0161] (1) Construction of inducible promoters with different numbers of LacO elements. One, two, three, and four LacO elements were constructed in the inducible promoter pTac that controls the orthogonal transcription mutant. The principle is as follows Figure 9 shown.

[0162] (2) The targeting plasmid expressing the sacB gene and orthogonal transcription mutants containing different numbers of LacO elements were cultured in culture medium at 37°C for 10 hours.

[0163] (3) The secondary seed solution was transferred at a concentration of 1:100 without adding inducer and cultured at 37°C for 20 hours.

[0164] (4) Dilute the culture medium in a 10-fold gradient to 10 -7 , and spread them on plates containing or not containing 100 g / L sucrose, and cultured at 37°C for 48 h.

[0165] (5) The mutation rate multiplier was calculated as N2 / N0, where N2 represents the number of sacB gene mutations caused by leaky mutations of orthogonal transcriptional mutants and N0 represents the total number of cells.

[0166] The results, shown in Table 8, show that increasing the number of LacO elements significantly reduced the leaky mutation rate in the absence of inducers. Compared to a single LacO element, the leaky mutation rate decreased over 100-fold after adding four LacO elements. These results demonstrate that increasing the number of LacO elements in pTac can reduce leaky expression and achieve more controlled mutagenesis.

[0167] Table 8: Leaky mutation rates at different numbers of LacO elements

[0168]

[0169] Example 7: Rapid Removal of Mutant Plasmids Using Negative Screening Genes

[0170] In this embodiment, by adding the negative selection gene sacB to the mutant plasmid, sucrose can be added to the culture medium after the mutation is completed to achieve rapid removal of the mutant plasmid and avoid the generation of continuous mutations.

[0171] (1) A negative screening module was constructed in the plasmid of the orthogonal transcription mutant to control the expression of sacB by the constitutive promoter Porin42.

[0172] (2) After the mutation is completed, the culture medium is replaced with LB medium without antibiotics containing 100 g / L sucrose and cultured at 37°C for 20 hours for rapid plasmid removal.

[0173] (3) Dilute the culture medium in a 10-fold gradient to 10 -7 , and spread them on plates with or without antibiotics, and cultured at 37℃ for 48h.

[0174] (4) The plasmid removal rate was calculated as (N4-N3) / N4*100%, where N3 represents the number of cells grown on the antibiotic plate (representing cells that did not drop the plasmid) and N4 represents the total number of cells.

[0175] The results, shown in Table 9, significantly improved the plasmid removal rate by adding a negative selection gene to the mutant plasmid. After adding the sacB gene, the plasmid removal rate reached 91%. These results demonstrate that by adding the negative selection gene sacB to a plasmid containing an orthogonal transcriptional mutant, the mutant plasmid can be rapidly removed after mutation by adding sucrose to the culture medium.

[0176] Table 9: Plasmid removal efficiency after adding negative selection genes

[0177]

[0178] The above results show that the targeted mutagenesis system based on orthogonal phage RNAP polymerase can quickly and efficiently generate a large number of mutations on the target gene. The optimized orthogonal transcription mutagen can simultaneously produce two types of mutations: C:G to T:A and A:T to G:C. The present invention can rapidly mutate the target gene on the plasmid or genome, which can greatly improve the mutation and evolution efficiency of enzymes or other proteins, and has a very broad application prospect. In addition, by increasing the number of regulatory elements in the inducible promoter, leaky expression can be reduced and controllable mutation can be achieved; in addition, by adding the negative screening gene sacB to the plasmid containing the orthogonal transcription mutagen, the mutant plasmid can be quickly removed after the mutation is achieved by adding sucrose to the culture medium, thereby avoiding continuous mutations in the target gene. In this way, the mutation rate can be precisely controlled, leaky mutations can be reduced, and plasmid loss can be quickly achieved, which helps to better screen beneficial mutations.

[0179] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0180] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0181] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A directed evolution system, characterized in that: The orthogonal transcription mutant comprises a phage RNA polymerase, a deaminase, and a first connecting sequence, wherein the deaminase is connected to the N-terminus of the phage RNA polymerase through the first connecting sequence, and the directed evolution system further comprises a mutation control element.

2. The directed evolution system according to claim 1, characterized in that The directed evolution system further comprises an orthogonal promoter, wherein the bacteriophage RNA polymerase specifically recognizes the orthogonal promoter. Optionally, the orthogonal promoter includes pMmP1, pK1F and pVP4.

3. The directed evolution system according to claim 1, characterized in that wherein the orthogonal transcription mutant further comprises an auxiliary mutation element, and the deaminase is connected to the auxiliary mutation element via a second linker sequence, Optionally, the helper mutation element comprises UGI.

4. The directed evolution system according to claim 1, characterized in that The orthogonal transcription mutant is one or more orthogonal transcription mutants, wherein the phage RNA polymerase of the one or more orthogonal transcription mutants is independently selected from one or more of MmP1 RNA polymerase and variants thereof, K1F RNA polymerase and variants thereof, and VP4 RNA polymerase and variants thereof, wherein the deaminase of the one or more orthogonal transcription mutants is independently selected from one or more of deaminase APOBEC1 and its variants, deaminase AID and its variants, deaminase PmCDA1 and its variants, and deaminase TadA variants TadA7.10, TadA8e, TadA9, CABE T3.1, CABE T3.155, and TadDE, Wherein, the first linker sequence comprises XTEN and (GGGGS) n .

5. The directed evolution system according to claim 1, characterized in that The directed evolution system also includes an inducible expression system for controlling the orthogonal transcription mutants. Optionally, the inducible expression system comprises one or more of an isopropylthiogalactoside IPTG induction system, an arabinose induction system, an acylhomoserine lactone AHL induction system, an anhydrotetracycline aTc induction system, and a vanillic acid induction system.

6. The directed evolution system according to claim 5, characterized in that The mutation control element, under the first condition, inhibits the orthogonal transcription mutant from undergoing the directed evolution. Optionally, Based on the fact that the inducible expression system is an isopropylthiogalactoside IPTG inducible system, the mutation regulatory element is a lacO element, and the mutation regulatory element is exogenous IPTG; Based on the fact that the inducible expression system is a dehydrotetracycline aTc inducible system, the mutation regulatory element is TetO, and the mutation regulatory element is exogenous aTc; Preferably, the number of the lacO elements is 1-4; more preferably, the number of the lacO elements is 4.

7. The directed evolution system according to claim 6, characterized in that The directed evolution system further comprises a negative screening gene, wherein the negative screening gene removes the mutated plasmid subjected to the directed evolution by the orthogonal transcription mutant under the second condition, wherein the second condition is different from the first condition, Optionally, the negative selection genes are sacB and pheS; Optionally, the second condition is exogenous sucrose and p-chlorophenylalanine.

8. A carrier, characterized in that The vector comprises a first vector comprising at least one polynucleotide encoding an orthogonal transcription mutant, a bacteriophage RNA polymerase, or a deaminase according to any one of claims 1 to 7, Optionally, the first vector further comprises a first linker sequence, Optionally, the first vector further comprises an inducible expression system used in conjunction with the orthogonal transcription mutant, Optionally, the vector includes a second vector for inserting an orthogonal promoter upstream of the target gene or inversely into the downstream of the target gene. Optionally, it further comprises a mutation regulatory element, a negative selection gene and / or other regulatory elements or proteins.

9. A directed evolution method, characterized in that include: Directed evolution is achieved by performing targeted mutagenesis using the directed evolution system according to any one of claims 1 to 7 or the vector according to claim 8.

10. The directed evolution method according to claim 9, characterized in that Reducing leaky expression by increasing the number of mutated regulatory elements; and By adding the negative selection gene sacB to the plasmid containing the orthogonal transcription mutant, the mutant plasmid can be quickly removed after mutagenesis.

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