Targeted evolution system based on orthogonal bacteriophage RNA polymerase and application

Abstract

The invention provides a targeted mutation system based on orthogonal bacteriophage RNA polymerase and an orthogonal transcription mutant. According to the orthogonal transcription mutants, phage RNA polymerase and deaminase are fused, and a large number of mutations can be rapidly and efficiently generated on a target gene. The bacteriophage RNA polymerase part of the orthogonal transcription mutants can specifically recognize corresponding promoters and transcribe downstream genes, and the deaminase part of the bacteriophage RNA polymerase part of the orthogonal transcription mutants can generate mutation in the transcription process. The optimized orthogonal transcription mutants can simultaneously generate mutation from C: G to T: A and mutation from A: T to G: C on a target gene. According to the method, mutation and screening can be carried out on target genes on plasmids or genomes or genes on metabolic pathways, the evolution efficiency of enzymes or other proteins can be greatly improved, and time and cost are saved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene and protein mutation, and more specifically, to a targeted evolution system and application based on orthogonal phage RNA polymerase. Background Art

[0002] Directed evolution is a protein engineering method that effectively simulates the mechanism of natural evolution. By applying selection pressure in the laboratory, researchers can force proteins to evolve new functions or properties under specific conditions. This method can produce a large number of genetic variations in a short period of time, improve the activity and specificity of proteins, and accelerate the evolution process (Molina et al., 2022.'In vivo hypermutation and continuous evolution', Nature Reviews Methods Primers, 2:36). The success of directed evolution lies in its ability to combine design and empirical methods, using the technology of synthetic biology to systematically explore protein sequence space. These methods can better understand the mechanism of evolution, and use this knowledge to develop biomacromolecules with new functions. The operation steps of traditional directed evolution methods (such as error-prone PCR) include constructing a mutant library of the target gene in vitro and then screening it in host cells. These methods usually produce a large number of relatively inefficient mutant libraries, and the lack of high-throughput screening methods makes searching these mutant libraries time-consuming, laborious and costly (Cirino, Patrick C, Kimberly M Mayer, and Daisuke Umeno. 2003. 'Generating mutant libraries using error-prone PCR', Directed evolution library creation: Methods and protocols: 3-9).

[0003] To address these issues, some in vivo targeted mutagenesis tools have been developed to enable the mutation, expression, and screening processes to proceed continuously, thereby improving the efficiency of mutagenesis and expanding the diversity of the variant library. For example, in vivo directed mutagenesis methods based on CRISPR-Cas technology use the sequence recognition specificity of sgRNA to generate mutations in specific regions, such as EvolR, CRISPR-X, base editors, and guide editors. However, the editing windows of these methods are relatively narrow, and many gRNAs usually need to be designed to cover the target gene. Methods based on positively transfected DNA polymerases, such as Orthorep in Saccharomyces cerevisiae, BacORep in Bacillus thuringiensis, and EcoRep in Escherichia coli, and methods based on viral replication, such as PACE and VEGAS, have been successfully established and applied to the evolution of many useful proteins (Zimmermann, Anna, Julian E. Prieto-Vivas, Karin Voordeckers, Changhao Bi, and Kevin J. Verstrepen. 2024. 'Mutagenesis techniques for evolutionary engineering of microbes–exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases', Trends in Microbiology). However, these methods will cause mutations in the entire linear plasmid or viral genome, and cannot achieve targeted mutations in specific genes or regions.

[0004] Therefore, it is urgent to develop a method that can quickly and efficiently produce mutations in specific target genes, thereby accelerating the speed of protein engineering and saving time and cost. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a targeted evolution system based on orthogonal phage RNA polymerase, which is characterized in that the phage RNA polymerase and deaminase are fused to form an orthogonal transcription mutant.

[0006] Therefore, in one aspect, the present invention provides a fusion protein comprising: (i) a bacteriophage RNA polymerase; and (ii) a base editing enzyme.

[0007] In a specific embodiment, the bacteriophage RNA polymerase is selected from MmP1 (SEQ ID NO: 1) or a variant thereof, K1F (SEQ ID NO: 2) or a variant thereof, and VP4 (SEQ ID NO: 3) or a variant thereof.

[0008] In a specific embodiment, the base editing enzyme comprises a deaminase, preferably adenosine deaminase, cytidine deaminase, or a combination thereof.

[0009] In a specific embodiment, the base editing enzyme is fused to the N-terminus of the bacteriophage RNA polymerase via an optional linker.

[0010] In one embodiment, the linker is selected from:

[0011] SGSETPGTSESATPES(XTEN)(SEQ ID NO:4)

[0012] SGGS (SEQ ID NO:5)

[0013] SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:6)

[0014] SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:7)

[0015] GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGG SGGS(SEQ ID NO:8)

[0016] SGGSGGSGGS (SEQ ID NO:9)

[0017] (GGGS)n

[0018] (GGGGS)n

[0019] (G)n

[0020] (EAAAK)n

[0021] (GGS)n,

[0022] Wherein n=1, 2, 3, 4, 5 or 6.

[0023] In one embodiment, the deaminase is selected from:

[0024] a deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases, preferably an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase or a variant thereof, preferably from rat or human,

[0025] Activation-induced cytidine deaminase (AID) or a variant thereof, preferably of human origin,

[0026] Sea lamprey (Petromyzon marinus) cytidine deaminase 1 (PmCDA1) (SEQ ID NO: 10) or a variant thereof (eg, evoPmCDA1) (SEQ ID NO: 11),

[0027] TadA adenosine deaminase or a variant thereof, preferably TadA7.10, TadA8.1, TadA8.2, TadA8.3, TadA8.4, TadA8.5, TadA8.6, TadA8.7, TadA8.8, TadA8.9, TadA8.10, TadA8.11, TadA8.12, TadA8.13, TadA8.14, TadA8.15, TadA8.16, TadA8.17, TadA8.18, TadA8.19, TadA8.20, TadA8.21, TadA8.22, TadA8.23 or TadA8.24, more preferably TadA7.10 (SEQ ID NO: 12), TadA8e (SEQ ID NO: 13), TadA9 (SEQ ID NO: 14), TadA9e (SEQ ID NO: 15), TadA9e (SEQ ID NO: 16), TadA9e (SEQ ID NO: 17), TadA9e (SEQ ID NO: 18), TadA9e (SEQ ID NO: 19), TadA9e (SEQ ID NO: 20), TadA9e (SEQ ID NO: 219), TadA9e (SEQ ID NO: 22), TadA9e (SEQ ID NO: 23), TadA9e (SEQ ID NO: 24), TadA9e (SEQ ID NO: 25), TadA9e (SEQ ID NO: 26), TadA9e (SEQ ID NO: 27), TadA9e (SEQ ID NO: 28), TadA9e (SEQ ID NO: 29), TadA9e (SEQ ID NO: 30), TadA9e (SEQ ID NO: 31), TadA9e (SEQ ID NO: 32), TadA9e NO: 14), CABET3.1, CABET3.155 and TadDE, preferably from Escherichia coli, and combinations thereof.

[0028] In a specific embodiment, the fusion protein further comprises an auxiliary mutation element, preferably an inhibitor of base excision repair, more preferably a uracil glycosylase inhibitor (UGI), and even more preferably a glycosylase inhibitor UGI (SEQ ID NO: 15) derived from Bacillus subtilis phage PBS1.

[0029] In a specific embodiment, the fusion protein comprises the following structure from the N-terminus to the C-terminus:

[0030] Deaminase-XTEN-bacteriophage RNA polymerase,

[0031] Preferably, the following structure is included:

[0032] Deaminase-UGI-XTEN-bacteriophage RNA polymerase,

[0033] Preferably, the deaminase is PmCDA1 or TadA adenosine deaminase or a variant thereof,

[0034] More preferably, the fusion protein is selected from:

[0035] PmCDA1-XTEN-MmP1,

[0036] PmCDA1-UGI-XTEN-MmP1,

[0037] evoPmCDA1-UGI-XTEN-MmP1,

[0038] PmCDA1-UGI-XTEN-K1F,

[0039] PmCDA1-UGI-XTEN-VP4,

[0040] TadA8e-XTEN-MmP1,

[0041] TadA8e-XTEN-K1F,

[0042] TadA8e-XTEN-VP4,

[0043] TadA7.10-XTEN-MmP1,

[0044] TadA9-XTEN-MmP1,

[0045] PmCDA1-UGI-TadA8e-XTEN-MmP1,

[0046] PmCDA1-UGI-TadA8e-XTEN-K1F,

[0047] PmCDA1-UGI-TadA8e-XTEN-VP4,

[0048] TadA8e-PmCDA1-UGI-XTEN-MmP1,

[0049] TadA8e-PmCDA1-UGI-XTEN-K1F,

[0050] TadA8e-PmCDA1-UGI-XTEN-VP4,

[0051] TadA8e-XTEN-MmP1(opt),

[0052] TadA8e-XTEN-K1F(opt), and

[0053] TadA8e-XTEN-VP4 (opt).

[0054] Another aspect of the present invention also provides a nucleic acid molecule encoding the fusion protein according to the present invention.

[0055] Another aspect of the present invention also provides a targeted evolution / mutation system, which comprises:

[0056] (i) a fusion protein or a nucleic acid molecule according to the present invention; and

[0057] (ii) a polynucleotide sequence comprising a promoter and a cloning site, wherein the cloning site is used to insert a DNA molecule of a target gene to be evolved / mutated,

[0058] The bacteriophage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter.

[0059] In a specific embodiment, the polynucleotide sequence is located on a plasmid or on the genome.

[0060] In a specific embodiment, one or more of the promoters are included upstream, downstream, or both upstream and downstream of the cloning site.

[0061] In a specific embodiment, the upstream, downstream, or both upstream and downstream of the cloning site include one or more bacteriophage RNA polymerase transcription terminators.

[0062] In a specific embodiment, the bacteriophage RNA polymerase is selected from MmP1 or its variants, K1F or its variants and VP4 or its variants, and their corresponding promoters are selected from pMmP1 (SEQ ID NO: 16), pK1F (SEQ ID NO: 17) and pVP4 (SEQ ID NO: 18).

[0063] In one embodiment, the targeted evolution / mutation system is based on an inducible expression system selected from the following or the fusion protein or nucleic acid molecule or polynucleotide sequence is expressed in the following expression system: Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acylhomoserine lactone AHL-inducible system), P Tet and its mutants (anhydrotetracycline aTc-inducible system), P Van and its mutants (vanillic acid inducible system).

[0064] Another aspect of the present invention also provides a targeted evolution / mutation system, which comprises:

[0065] (i) a fusion protein or a nucleic acid molecule according to the present invention; and

[0066] (ii) a target gene DNA molecule to be evolved / mutated connected to a promoter,

[0067] The bacteriophage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter, thereby transcribing the target gene DNA molecule.

[0068] In a specific embodiment, the target gene DNA molecule is located on a plasmid or on a genome.

[0069] In a specific embodiment, one or more promoters are included upstream, downstream, or both upstream and downstream of the target gene DNA molecule.

[0070] In a specific embodiment, the upstream, downstream or both upstream and downstream of the target gene DNA molecule include one or more bacteriophage RNA polymerase transcription terminators.

[0071] In a specific embodiment, the bacteriophage RNA polymerase is selected from MmP1 or a variant thereof, K1F or a variant thereof and VP4 or a variant thereof, and their corresponding promoters are selected from pMmP1, pK1F and pVP4.

[0072] In one embodiment, the targeted evolution / mutation system is based on an inducible expression system selected from the following or the fusion protein or nucleic acid molecule or polynucleotide sequence is expressed in the following expression system: Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acylhomoserine lactone AHL-inducible system), P Tet and its mutants (anhydrotetracycline aTc-inducible system), P Van and its mutants (vanillic acid inducible system).

[0073] Another aspect of the present invention also provides a method for targeted evolution / mutation of a target gene DNA molecule, the method comprising: using the targeted evolution / mutation system according to the present invention, contacting the target gene DNA molecule with the fusion protein, so that the phage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter, thereby transcribing the target gene DNA molecule, wherein the base editing enzyme introduces mutations during the transcription process.

[0074] In a specific embodiment, the contacting occurs in vivo / intracellularly or in vitro / extracellularly.

[0075] In a specific embodiment, the target gene DNA molecule is located on a plasmid or on a genome.

[0076] In a specific embodiment, the base editing enzyme comprises a deaminase, preferably an adenosine deaminase, a cytidine deaminase, or a combination thereof, and the mutation is C:G to T:A and / or A:T to G:C.

[0077] In a specific embodiment, the method further comprises the step of screening mutants by antibiotic resistance (eg erythromycin resistance), cytotoxicity (eg sacB gene) or fluorescence or color or morphology.

[0078] In a specific embodiment, the method is carried out in a prokaryote or a eukaryote, preferably a microorganism, more preferably the prokaryote is selected from Escherichia coli, Pseudomonas, Eutropha rosea, Aeromonas hydrophila, Corynebacterium glutamicum or halophilic bacteria, and even more preferably the halophilic bacteria is selected from Halomonas, including Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, Halomonas aydingkolgenesis or its derivatives, preferably Halomonas bluephagenesis TD01 (culture collection number CGMCC No.4353), Halomonasaydingkolgenesis M1 (culture collection number CGMCC No.19880), Halomonas campaniensis LS21 (culture collection number CGMCC No.6593), and / or the eukaryote is selected from yeast, fungi or algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0080] Figure 1 .Principles of the targeted evolution system based on orthogonal phage RNA polymerases;

[0081] Figure 2 . Test the expression of GFP driven by N-terminal fusion and C-terminal fusion;

[0082] Figure 3 .pSEVA341 plasmid map;

[0083] Figure 4 .Construct a C:GT:A mutation detection method;

[0084] Figure 5.C: GT:A type mutation detection plasmid map pMT64-MmP1;

[0085] Figure 6 .Construct A:TG:C mutation detection method;

[0086] Figure 7 .A:TG:C type mutation detection plasmid map pMT58-MmP1;

[0087] Figure 8 .A:TG:C type mutation sequencing results;

[0088] Fig. 9 .C: GT: A type mutation sequencing results;

[0089] Fig.10 .Design of orthogonality detection of three orthogonal transcriptional mutants;

[0090] Fig.11 . The orthogonality test results of three orthogonal transcription mutants showed high specificity;

[0091] Fig.12 . Fluorescent protein gene plasmid map;

[0092] Fig.13 .Pigment protein gene plasmid map;

[0093] Fig.14 . Mutation of three fluorescent protein genes produces multiple colors;

[0094] Fig.15 . Mutation of three pigment protein genes produces a variety of colors;

[0095] Fig.16 . Mutation of cell division-related gene ftsQAZ;

[0096] Fig.17 . Mutation of the cytoskeleton-related gene mreBCD; and

[0097] Fig.18 . Mutations in the cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ.

[0098] Description of Sequence Listing

[0099] The preferred embodiments of the present invention and the specific sequences used in the examples are as follows:

[0100] Amino acid sequence of bacteriophage RNA polymerase MmP1 (SEQ ID NO: 1)

[0101] MSIAAAVNKNDFSDVELAAIPFNTLADHYGADLAREQLQLEHESYVMGEERFRKM

[0102] LERQEKAEEFGDSSVAKPLIITLLPKVTQRITDWLNEWADPNKKGRKPIAYTHLKDIK

[0103] PETLAFITIKVVLNKLAGKDDAFMQPLAYAIGSSIEDEARFGRIRELEMAHFKKCAEE

[0104] NLNKRRGTAYRKAFLSVVEADMLDKGLLGGESWGTWNKTDVMNIGISMLEKLIEA

[0105] TGLVELREKRNFEEMDRIVIAEEYVKAMATRAQSLAGISPMYQPCVVPPKPWVSITG

[0106] GGYWANGRKPTALIRTHTRKALYRYEDVYMPEVYKAINYAQETPWRINRKVLAVV

[0107] NELVKWKNNPVKDMPSIDKLELPQRPDDIDTNEEALRSWKREAAAAVYRKDEQRKS

[0108] RYLSMSFALEQANKFSNKKAIYFPYNMDWRGRVYALPMFNPQGNDMVKGLLTLAK

[0109] GKPIGKDGFYWLKIHGANTAGVDKVTFPERIKFIEDNHDNIMQCAESPLDNLWWTE

[0110] QDSFPFCFLAFCFEYAQVTKKGLGWVCSLPIALDGSCSGIQHFSAMLRDDIGGRAVNL

[0111] LPSETVQDIYGIVADKVNEALKELVINGTDNYTDTVTDKSTGEIIERYRLGEKELARQ

[0112] WLEFGVTRSVTKRSVMTLAYGSKEYGFRDQVLEDTIRPAIDSGKGAMFTNPSQAAS

[0113] FMAKRIWEAVSVTVVAAVGAMKWLQSSAKLMAAEVKDKKTKEVLRKRCAVHWV

[0114] TPDGFPVWQEYRKPKQKRVHLMFLGSYYDARMKETSSDCSIDAHKQESGISPNFVH

[0115] SQDGNHLRMTVVYAREKYNVESFALIHDSFGTIPADVPNLFKAVRETMVNMYENND

[0116] VLADFYEQFADQLHESQLDKMPALPPKGKLNLQDILKSDFAFA*

[0117] Amino acid sequence of bacteriophage RNA polymerase K1F (SEQ ID NO:2)

[0118] MSVISIDKHDFSDVSNAIEPFNLLADHYGQDLAVKQLQLEHEAYTEGERRFIKNLER

[0119] QTERGELADNQVAKPLMQTLVPKIAQAVKEWHEGPDGKLSTSRPSVAFTMLSTEER

[0120] AVKDRSLRISCESAAVIILKVILSKLVKPEGIPITPMASAIGRTLEDEIRFGRIRDKEKEH

[0121] FKKAIADNLNKRAGASYKKAYMQAVEASMLEQGQLEDAWGTWSPTEAVHVGIKM

[0122] LEIVIQSTQLVELKRYGAGNAAADVEMVHLSDFWVKKMAQRGFSLAGIAPVYQPC

[0123] VVPPKPWTGVVGGGYWAKGRRPLPLIRLGSKSAVARYEDVYMPEVYEAVNIIQNTP

[0124] WKVNKKVLDVVNMVEKLNNTPIDDIPQMEPLKPEAYAGETEEELKAWKKAAAGIY

[0125] RREKARQSRRLSLSFIVNQANKFSQFKAIWFPYNMDWRGRVYAVPMFNPQGNDMQ

[0126] KGLLTLAVGKPIGADGFKWLKVHGANCAGVDKVTFEERIKWVEDNHDNIMAAAK

[0127] APMDSIEWWGKLDSPFCFLAFCFEYAGVMHHGLSYSCSLPIAFDGSCSGIQHFSAML

[0128] RDHIGGHAVNLTPSGKVQDIYRIVSDRIEEELKVLLVNGTDNEMVTHEDKKTGEITE

[0129] RLKLGTRELARQWLTYGMSRKVTKRSVMTLAYGSKEYGFADQVYEDIVMPAIDSG

[0130] SGAMFTEPSQASRFMAKMIWEAVSVTVVAAVDAMKWLQGAAKLLAAEVKDKKTG

[0131] EILKPCLPVHWVTPDGFPVWQEYRKKDTTRLNLMFLGSFNLQPTVNKGTKKELDK

[0132] HKQESGISPNFVHSQDGSHLRKTVVHTHRKYGVMSFAVIHDSFGTIPADAEYLFRGVRETMVETYRDNDVLLDFYEQFEYQLHESQRDKLPELPKKGKLNIEDILSSDFAFA*

[0133] Amino acid sequence of phage RNA polymerase VP4 (SEQ ID NO:3)

[0134] MANVIKPQSHNFSDISAAILPFNVLADSYGEALAAEQLMLEHESYQLGEARFIKAME

[0135] RQVERGEVSDNAVAKPLLDTLIPALAARITEFVEMKQRGKPHVSKGYFAMIKPESAA

[0136] FIIVKTTLNILAKEESVPVQRVAMAIGGNIEDEIRFGRIRDEEIKHFKERVKPNLDKRN

[0137] GFIYKKAYMEAVEAGMQDKGELNSTHEAWEKDVKFHVGIRAIEMLIEATGMVQLE

[0138] RKFKGIPDKDHEALHLAPEYVEKLTNRAHALAGISPMYQPMIVKPKRWTGVQGGG

[0139] YWAKGRRPLNLIRVGSKRALDRYRQVDMPEVYDAINTIQETAWRINKDVLAVVNN

[0140] VVTWTNCCPVEDVPSIDKLALPEKPEDIDNNEESLKKWKKAAAAIYRKEKARQSRRI

[0141] SLEFALSQANKFSKYNEIYFPYNMDWRGRVYAIPMFNPQGNDMVKGLLTFAKKVPV

[0142] GIDGGYWLAVHGANCAGVDKVSLEDRVKWVNDNEANIIASAEAPLDFTWWAEQD

[0143] SPFCFLAFCFEWAAYVKAGKKPSFESSLPLAFDGTCSGLQHFSAMLRDEIGGAAVNL

[0144] LPADKPQDIYGIVAVKVNEVLRDLVISGTEDEMQTLEDKKTGEITERLVGTRTLAAQ

[0145] WLEYGVTRSVTKRSVMTLAYGSKEYGFADQVFEDTVMPAIDNGKGTMFTEPSQAC

[0146] RFMAKLIWDAVSKTVVAAVEAMQWLQSAAKLVSSEVKDKKSGEILKHAMPVHWT

[0147] TPNGFPVWSEYCKQEQKVIDCVIlgSMRLQLKLNMRDKKEIDTAKQASGIAPNFVH

[0148] SMDASHLQMTVNKCFKVYGIHSFAMIHDSFGCHAGFASKMFRAVRETMVETYEEHDVIQEFYNQFEKQLHESQIEKMPALPRKGNLELREILKSLYTFS*

[0149] Linker

[0150] SGSETPGTSESATPES(XTEN)(SEQ ID NO:4)

[0151] SGGS (SEQ ID NO:5)

[0152] SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:6)

[0153] SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:7)

[0154] GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS(SEQ ID NO:8)SGGSGGSGGS(SEQ ID NO:9)

[0155] (GGGS)n

[0156] (GGGGS)n

[0157] (G)n

[0158] (EAAAK)n

[0159] (GGS)n,

[0160] Where n = 1, 2, 3, 4, 5 or 6

[0161] PMmP1 promoter sequence (SEQ ID NO: 10):

[0162] cccatgagttaattatatttgtggcattataggg

[0163] PK1F promoter sequence (SEQ ID NO: 11):

[0164] gacatggctcaagcctaaactatcactatagg

[0165] PVP4 promoter sequence (SEQ ID NO: 12):

[0166] gaagtaacttgattaattaaccctgactataggga

[0167] Amino acid sequence of PmCDA1 cytosine deaminase (SEQ ID NO: 13): MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV*

[0168] Amino acid sequence of evoPmCDA1 cytosine deaminase (SEQ ID NO: 14): MTDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV*

[0169] Amino acid sequence of UGI glycosylase inhibitor (SEQ ID NO: 15): MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML*

[0170] Amino acid sequence of TadA7.10 adenine deaminase (SEQ ID NO: 16): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD*

[0171] Amino acid sequence of TadA8e adenine deaminase (SEQ ID NO: 17): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN*

[0172] Amino acid sequence of TadA9 adenine deaminase (SEQ ID NO: 18): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRRVFNAQKKAQSSIN*

[0173] MmP1 codon optimized sequence (SEQ ID NO: 19)

[0174] K1F codon optimized sequence (SEQ ID NO: 20)

[0175] VP4 codon optimized sequence (SEQ ID NO: 21)

[0176] T7 RNAP (RNase) amino acid sequence (SEQ ID NO: 22)

[0177] T7 RNAP encoding nucleotide sequence (SEQ ID NO: 23)

[0178] sacB gene sequence (SEQ ID NO:24)

[0179] ftsQAZ gene cluster sequence (SEQ ID NO:25)

[0180] mreBCD gene cluster sequence (SEQ ID NO: 26)

[0181] ermC gene nucleotide sequence (SEQ ID NO: 27)

[0182] ATGAACGAGAAAAATATAAAACACAGTCAAAACTTTATTACTTCAAAACATAATA

[0183] TAGATAAAATAATGACAAATATAAGATTAAATGAACATGATAATATCTTTGAAATCG

[0184] GCTCAGGAAAAGGCCATTTTACCCTTGAATTAGTAAAGAGGTGTAATTTCGTAAC

[0185] TGCCATTGAAATAGACCATAAATTATGCAAAACTACAGAAAAATAACTTGTTGATC

[0186] ACGATAATTTCCAAGTTTTAAACAAGGATATATTGCAGTTTAAATTTCCTAAAAAC

[0187] CAATCCTATAAAATATGGTAATATACCTTATAACATAAGTACGGATATAATACGCA

[0188] AAATTGTTTTTGATAGTATAGCTAATGAGATTTATTTAATCGTGGAATACGGGTTTG

[0189] CTAAAAGATTATTAAATACAAACGCTCATTGGCATTACTTTTAATGGCAGAAGTT

[0190] GATATTTCTATATTAAGTATGGTTCCAAGAGAATATTTTCATCCTAAACCTAAAGTG

[0191] AATAGCTCACTTATCAGATTAAGTAGAAAAAAATCAAGAATATCACACAAAGATA

[0192] AAAAAAGTATAATTATTTCGTTATGAAATGGGTTAACAAAGAATACAAGAAAAT

[0193] ATTTACAAAAAAATCAATTTAACAATTCCTTAAAACATGCAGGAATTGACGATTTAA

[0194] ACAATATTAGCTTTGAACAATTCTTATCTCTTTTCAATAGCTATAAATTATTTAATAA

[0195] GTAA

[0196] Amino acid sequence encoded by ermC (SEQ ID NO: 28)

[0197] MNEKNIKHSQNFITSKHNIDKIMTNIRLNEHDNIFEIGSGKGHFTLELVKRCNFVTAIE

[0198] IDHKLCKTTENKLVDHDNFQVLNKDILQFKFPKNQSYKIYGNIPYNISTDIIRKIVFDS

[0199] IANEIYLIVEYGFAKRLLNTKRSLALLLMAEVDISILSMVPREYFHPKPKVNSSLIRLS

[0200] RKKSRISHKDKQKYNYFVMKWVNKEYKKIFTKNQFNNSLKHAGIDDLNNISFEQFLSLFNSYKLFNK*

[0201] mCherry gene nucleotide sequence (SEQ ID NO: 29)

[0202] sfGFP gene nucleotide sequence (SEQ ID NO:30)

[0203] TagBFP gene nucleotide sequence (SEQ ID NO:31)

[0204] spisPink gene nucleotide sequence (SEQ ID NO:32)

[0205] fwYellow gene nucleotide sequence (SEQ ID NO: 33)

[0206] amajLime gene nucleotide sequence (SEQ ID NO: 34)

[0207] Amino acid sequence of PmCDA1-XTEN-MmP1 (SEQ ID NO: 35)

[0208] Amino acid sequence of PmCDA1-UGI-XTEN-MmP1 (SEQ ID NO: 36)

[0209] Amino acid sequence of evoPmCDA1-UGI-XTEN-MmP1 (SEQ ID NO: 37)

[0210] Amino acid sequence of PmCDA1-UGI-XTEN-K1F (SEQ ID NO: 38)

[0211] Amino acid sequence of PmCDA1-UGI-XTEN-VP4 (SEQ ID NO: 39)

[0212] Amino acid sequence of TadA8e-XTEN-MmP1 (SEQ ID NO: 40)

[0213] Amino acid sequence of TadA7.10-XTEN-MmP1 (SEQ ID NO:41)

[0214] Amino acid sequence of TadA9-XTEN-MmP1 (SEQ ID NO:42)

[0215] Amino acid sequence of TadA8e-XTEN-K1F (SEQ ID NO:43)

[0216] Amino acid sequence of TadA8e-XTEN-VP4 (SEQ ID NO: 44)

[0217] Amino acid sequence of PmCDA1-UGI-XTEN-TadA8e-XTEN-MmP1 (SEQ ID NO:45) Amino acid sequence of TadA8e-XTEN-PmCDA1-UGI-XTEN-MmP1 (SEQ ID NO:46) Amino acid sequence of PmCDA1-UGI-XTEN-TadA8e-XTEN-K1F (SEQ ID NO:47) Amino acid sequence of TadA8e-XTEN-PmCDA1-UGI-XTEN-K1F (SEQ ID NO:48) Amino acid sequence of PmCDA1-UGI-XTEN-TadA8e-XTEN-VP4 (SEQ ID NO:49) Amino acid sequence of TadA8e-XTEN-PmCDA1-UGI-XTEN-VP4 (SEQ ID NO:50) DETAILED DESCRIPTION

[0218] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0219] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0220] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0221] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the technician refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0222] The words “include” or “comprising” described in the present invention are open-ended descriptions, including all the specified components or steps described, as well as other specified components or steps that will not be substantially affected; when used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0223] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0224] As used herein, the term "fusion protein" is also called chimeric protein, which refers to an artificial protein constructed by purposefully connecting two or more genes encoding functional proteins together and expressing them using genetic engineering technology. As those skilled in the art understand, the functions of the functional proteins in the fusion protein should not substantially affect each other.

[0225] As used herein, the term "bacteriophage RNA polymerase" refers to an RNA polymerase derived from a bacteriophage. The present invention selects bacteriophage RNA polymerase because it has high specificity and transcriptional activity, that is, it can specifically bind / recognize the corresponding phage promoter, but will not recognize other promoters, thereby avoiding interference with the host endogenous promoter, etc., and when used in the targeted mutation system according to the present invention, with the aid of base editing enzymes such as deaminases, it is possible to generate mutations only on the targeted gene downstream of the phage promoter, and will not generate mutations at other non-targeted positions. In the present invention, the preferred phage RNA polymerase is selected from MmP1 (SEQ ID NO: 1) or its variants, K1F (SEQ ID NO: 2) or its variants and VP4 (SEQ ID NO: 3) or its variants, and their corresponding specific promoters are pMmP1 (SEQ ID NO: 10), pK1F (SEQ ID NO: 11) and pVP4 (SEQ ID NO: 12), respectively.

[0226] As used herein, the term "base editing enzyme" refers to an enzyme that can modify / edit DNA bases. Typically, base editing enzymes are divided into cytosine base editing enzymes and adenine base editing enzymes, which can ultimately achieve base replacement of CT (GA) or AG (TC), respectively. Various base editing enzymes are disclosed, for example, in the following documents: CN111801345A, CN118284692A, CN118272370A, CN119053697A, CN113348245A, CN118202041A. In the present invention, preferred base editing enzymes include deaminases, preferably adenosine deaminase, cytidine deaminase or a combination thereof. When used in a targeted mutation system according to the present invention, the above-mentioned base editing enzymes such as deaminases can produce mutations from C:G to T:A and A:T to G:C in the target gene.

[0227] As used herein, the term "linker" refers to a covalent linker, a non-covalent linker, a chemical group or a molecule that can be used to connect two proteins or domains of a fusion protein (e.g., phage RNA polymerase and base editing enzymes such as deaminases). In some embodiments, the linker can be an amino acid or multiple amino acids, for example, a linker can be a length of about 5 to 100 amino acids. For example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker can be SGGS (SEQ ID NO: 5), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 6), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 7),

[0228] GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS(SEQ ID NO:8),SGGSGGSGGS(SEQ ID NO: 9), (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, etc., where n=1, 2, 3, 4, 5 or 6.

[0229] As used herein, the term "deaminase" refers to an enzyme that catalyzes the removal of an amine group from a molecule or deaminates, for example, by hydrolysis. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the deamination of cytidine (C) to uridine (U), the deamination of deoxycytidine (dC) to deoxyuridine (dU), or the deamination of 5-methyl-cytidine to thymidine (T, 5-methyl-U), respectively. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes and promotes the conversion of adenine to guanine. Non-limiting examples of deaminases include deaminases from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases, activation-induced cytidine deaminase (AID) or variants thereof, cytidine deaminase 1 (PmCDA1) (SEQ ID NO: 13) or variants thereof (SEQ ID NO: 14) of sea lamprey (Petromyzon marinus), and TadA adenosine deaminase or variants thereof, such as TadA7.10 (SEQ ID NO: 16), TadA8e (SEQ ID NO: 17) and TadA9 (SEQ ID NO: 18). These deaminases and variants thereof are known in the art and are described in detail in, for example, CN114929287A and CN118284692A.

[0230] The present invention provides an orthogonal transcription mutant, characterized in that the deaminase is connected to the N-terminus of the bacteriophage RNA polymerase via a suitable linker.

[0231] Preferably, the bacteriophage RNA polymerase includes but is not limited to one or more combinations of MmP1 RNA polymerase and its variants, K1F RNA polymerase and its variants and VP4 RNA polymerase and its variants, and can specifically recognize corresponding phage promoters and their variants, pMmP1, pK1F and pVP4.

[0232] Preferably, the deaminase includes but is not limited to 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 having both cytosine deaminase and adenine deaminase activities, such as one or more combinations of CABE T3.1, CABE T3.155 and TadDE.

[0233] Preferably, the linker used includes but is not limited to XTEN, (GGGGS)n, etc.

[0234] Preferably, the auxiliary mutation element used includes the glycosylase inhibitor UGI (SEQ ID NO: 15) derived from Bacillus subtilis phage PBS1.

[0235] Preferably, the inducible expression system used to control the orthogonal transcriptional mutants includes but is not limited to P Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acylhomoserine 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).

[0236] Preferably, the microorganism applicable to the above-mentioned orthogonal transcription mutant is a prokaryotic microorganism selected from but not limited to halophilic bacteria, Escherichia coli, Pseudomonas, Eutropha rhodobacterium, Aeromonas hydrophila and Corynebacterium glutamicum.

[0237] Preferably, the halophilic microorganism is a bacterium of the genus Halomonas, including but not limited to Halomonas bluephagenesis TD01 (CGMCC No.4353), Halomonas campaniensis LS21 (CGMCC No.6593), Halomonas aydingkolgenesis M1 (CGMCC No.19880), Halomonas bluephagenesis WZY254, Halomonas bluephagenesis WZY278 and their derivative strains or combinations thereof. Applicable eukaryotic microorganisms include any one of yeast, fungi or algae.

[0238] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0239] All patents and publications mentioned in this application are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention in detail and should not be considered to limit the scope of the present invention or the specific method described in the present invention.

[0240] Example 1 Design of a targeted evolution system based on orthogonal phage RNA polymerases

[0241] In this example, we designed a targeted evolution system based on orthogonal phage RNA polymerase (schematic diagram as shown in Figure 1The principle is that the orthogonal transcription mutant can generate a large number of mutations on the target gene quickly and efficiently by fusing the phage RNA polymerase with the deaminase. The phage RNA polymerase part of the orthogonal transcription mutant can specifically recognize the corresponding promoter and transcribe the downstream gene. During the transcription process, the double-stranded DNA will open to expose some single-stranded regions. Subsequently, the deaminase part of the orthogonal transcription mutant can undergo a deamination reaction on the nucleotides of the single-stranded DNA and generate mutations in the subsequent replication and repair process.

[0242] The specific process includes the following aspects:

[0243] (1) Select a suitable phage RNA polymerase. The selected phage RNA polymerase has high specificity and transcription activity. The specificity is reflected in the fact that the phage RNA polymerase can only specifically recognize the corresponding phage promoter, but not other promoters, thereby avoiding interference with the host endogenous promoter or other phage promoters, and thus ensuring that mutations will only occur in the downstream targeted gene of the phage promoter, and will not occur in other non-targeted positions; the characteristic of high transcription activity is reflected in the fact that the phage RNA polymerase can efficiently initiate the transcription of downstream genes, open the DNA double helix to expose part of the single-stranded DNA region, and create conditions for subsequent deamination reactions.

[0244] (2) Select appropriate deaminases and auxiliary mutation elements. There are many types of deaminases that can recognize pyrimidine or purine bases to produce deamination reactions and generate mutations during the subsequent replication process. Deaminases need to have strong mutation activity to ensure that they can produce enough mutations and increase the mutation rate. Expressing appropriate auxiliary mutation elements such as the glycosylase inhibitor UGI can prevent mutations caused by cytosine deaminase deamination from being repaired by the endogenous repair system, thereby further increasing the mutation rate.

[0245] (3) Connecting the deaminase and the auxiliary mutation element to the N-terminus of the phage RNA polymerase via a suitable linker. The inventors have found through experimental studies that only by connecting the deaminase and the auxiliary mutation element to the N-terminus of the phage RNA polymerase can their influence on the transcription activity of the phage RNA polymerase be avoided (see Figure 2: The N-terminal and C-terminal combination drives the expression of GFP. 1 to 3 are lamprey cytidine deaminase PmCDA1 or adenosine deaminase TadA8e connected to the N-terminus of phage RNA polymerase MmP1, which can normally drive the expression of GFP, while 4 and 5 are deaminase connected to the C-terminus of phage RNA polymerase, and the resulting fusion protein cannot normally drive the expression of GFP. The construction method of the plasmid expressing the above fusion protein (the plasmid backbone is pSEVA341) is shown in Example 2 below, and the construction method of the targeting plasmid expressing the target gene GFP (the plasmid backbone is pSEVA321) is shown in Example 4 below).

[0246] (4) Insert the promoter of bacteriophage RNA polymerase upstream or downstream of the target gene on the plasmid or genome. In order to generate mutations on the target gene, it is necessary to insert the corresponding promoter of bacteriophage RNA polymerase upstream or downstream. This promoter can recruit orthogonal transcriptional mutagen, thereby turning on transcriptional mutations.

[0247] Example 2 Construction and testing of orthogonal transcriptional mutagens that produce a single type of mutation

[0248] In this example, the transformation host was Escherichia coli S17-1, with the genotype of RP4-2 (Km::Tn7, Tc::Mu-1), pro-82, LAMpir, recA1, endA1, thiE1, hsdR17, creC510, which was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0249] In this example, we used the lamprey cytidine deaminase PmCDA1, the fusion of PmCDA1 and the glycosylase inhibitor UGI, the fusion of evoPmCDA1 (PmCDA1 variant) and the glycosylase inhibitor UGI, and the adenosine deaminase TadA variants TadA7.10, TadA8e and TadA9 from Escherichia coli tRNA, and used XTEN linkers to fuse with the N-terminus of one of three phage RNA polymerases (i.e., MmP1, K1F and VP4) to construct various types of orthogonal transcription mutants that produce single-type mutations. The plasmid expressing only phage RNA polymerase was set as the control group. The orthogonal transcription mutants of single-type mutations and the control group are shown in Table 1. These orthogonal transcription mutants are composed of P TacThe expression is driven by an inducible promoter, and the plasmid vector is the high-copy number pSEVA341 (plasmid pSEVA341 see Martínez-García E, Fraile S, Algar E, et al. SEVA4.0: an update of the Standard European Vector Architecture databasefor advanced analysis and programming of bacterial phenotypes[J]. Nucleic Acids Research, 2023, 51(D1): D1558-D1567). Figure 3 This is the plasmid map of pSEVA341.

[0250] Table 1. Orthogonal transcriptional mutagen for generating single-type mutations

[0251]

[0252] The mutation rate of these single-type mutation orthogonal transcription mutants was detected by using a functional restoration experiment based on the erythromycin resistance gene. In order to detect the C:G to T:A type mutation, a mutation was introduced at the Y104 site of the erythromycin resistance gene ermC. The Y104S mutation completely inactivates the erythromycin resistance gene, while the Y104F mutation can maintain erythromycin resistance. The mutation of C:G to T:A at the 104 site will cause serine (S104, TCT) to mutate to phenylalanine (F104, TTT), thereby restoring resistance ( Figure 4 ), and a targeting plasmid was constructed based on this ErmC Y104S mutant, and the plasmid name was pMT64-MmP1 (see the plasmid map for details). Figure 5), the plasmid backbone is pSEVA321 (for plasmid, see the literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41.D1 (2012): D666-D675), and its promoters include two: the constitutive promoter Pporin42 and the inducible promoter pMmP1. The constitutive promoter Pporin42 is used to continuously express the erythromycin resistance gene, and pMmP1 is used to recruit mutation elements to produce mutations. In order to detect mutations of the A:T to G:C type, an early termination mutation is introduced at the Q10 site of the erythromycin resistance gene ermC. The Q10* early termination mutation will inactivate erythromycin resistance, while the A:T to G:C mutation can mutate the TAA premature termination codon at site 10 to CAA (encoding glutamine Q), thereby restoring activity ( Figure 6 ). Based on this mutant, the targeting plasmid pMT58-MmP1 was constructed (see the plasmid map for details). Figure 7 ), the plasmid backbone is pSEVA321, and its promoters include constitutive promoter Pporin42 and inducible promoter PMmP1. The specific steps are to culture the targeted plasmid expressing the inactivated erythromycin resistance gene and different orthogonal transcription mutants in a medium containing chloramphenicol and spectinomycin, and culture them at 37°C for 10 hours. Then, transfer the secondary seed solution at a concentration of 1:100 and add 200 mg / L ITPG inducer, and culture them at 37°C for 20 hours. Dilute the culture solution to 10 in a 10-fold gradient to 10 -7 , and were spread on plates containing or not containing 200 mg / L erythromycin, and cultured at 37°C for 48 hours. The mutation rate was calculated as N1 / N0, where N1 represents the number of colonies grown on erythromycin plates and N0 represents the number of colonies grown on plates without erythromycin. Each group had three independent repeated experiments, and the mean and standard error of the mutation rate were calculated. The results are shown in Table 2 and Table 3. The use of different combinations of deaminases and phage RNA polymerases can increase the targeted mutation rate, which is 564 to 799118 times higher than that of the control group expressing only phage RNA polymerase. Among them, the mutation effect of PmCDA1-UGI ​​or TadA8e fusion phage RNA polymerase is the best and the mutation efficiency is the highest. The above results show that single-type mutations from C:G to T:A or A:T to G:C can be efficiently generated using orthogonal transcription mutagen.

[0253] Table 2. Mutation rates of orthogonal transcription mutants based on MmP1 phage RNA polymerase

[0254]

[0255] Table 3. Mutation rates of orthogonal transcription mutants based on K1F and VP4 phage RNA polymerases

[0256]

[0257] Example 3 Construction and testing of double mutant orthogonal transcription mutants

[0258] In this example, the transformation host was Escherichia coli S17-1, with the genotype of RP4-2 (Km::Tn7, Tc::Mu-1), pro-82, LAMpir, recA1, endA1, thiE1, hsdR17, creC510, which was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0259] In this example, we rationally combined cytosine deaminase, adenine deaminase and phage RNA polymerase to obtain mutations that can simultaneously produce C:G to T:A and A:T to G:C. We fused the two deaminases, PmCDA1-UGI ​​and TadA8e, to the N-terminus of phage RNA polymerase in different orders (PmCDA1-UGI-TadA8e-phage RNAP and TadA8e-PmCDA1-UGI-phage RNAP), or simultaneously expressed PmCDA1-UGI-phage RNAP and TadA8e-phage RNAP on one plasmid (opt). In order to avoid homologous recombination, the DNA sequence of the phage RNA polymerase fused to TadA8e was codon optimized (the codon-optimized sequences of MmP1, K1F and VP4 are shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively), as shown in Table 4 (opt represents the inclusion of optimized codons).

[0260] Table 4. Orthogonal transcriptional mutants that generate double mutations

[0261]

[0262] The mutation rates of these double mutation orthogonal transcription mutants were detected using the functional restoration experiment based on the erythromycin resistance gene described above, and the results are shown in Table 5. These orthogonal transcription mutants can simultaneously produce C:G to T:A type mutations and A:T to G:C type mutations (A:TG:C type mutation sequencing results and C:GT:A type mutation sequencing results, respectively, see Figure 8 and Fig. 9 ), thereby expanding the mutation range, which is beneficial to improving the diversity of the target gene mutation library and improving the mutation efficiency.

[0263] Table 5. Mutation rates of orthogonal transcriptional mutants producing double mutations

[0264]

[0265] We then designed and tested the orthogonality of orthogonal transcription mutants based on three bacteriophage RNA polymerases. We mutated the three orthogonal transcription mutants pMT23opt-MmP1, pMT23opt-K1F and pMT23opt-VP4 and the corresponding control groups pMT0-MmP1, pMT0-K1F and pMT0-VP4 respectively. The sacB gene encoding secreted sucrose fructanase, which can convert sucrose into toxic fructan, was inserted into the corresponding phage promoters pMmP1, pK1F and pVP4. The principle is as follows Fig.10 The specific steps are as follows: the targeting plasmid with the sacB gene and different orthogonal transcription mutants are cultured in the culture medium at 37°C for 10 hours. Then, the secondary seed solution is transferred at a concentration of 1:100 and 200 mg / L of ITPG inducer is added, and the culture is cultured at 37°C for 20 hours. The culture solution is diluted 10 times to 10 -7 , and were spread on plates containing or not containing 100g / L sucrose, respectively, and cultured at 37°C for 48h. The mutation rate multiple was calculated as M1 / M0, where M1 represents the mutation rate using the orthogonal transcription mutant, and M0 represents the background mutation rate of the corresponding phage RNA polymerase control group. The calculation results are shown in Fig.11 As shown, when the corresponding promoters pMmP1, pK1F and pVP4 were recognized, the mutation rates of pMT23opt-MmP1, pMT23opt-K1F and pMT23opt-VP4 orthogonal transcription mutants increased by 46608, 46470 and 8388 times, respectively. However, when the other two promoters were recognized, the mutation rate was only 6 to 27 times higher than the control. The above results show that orthogonal transcription mutants based on different RNA polymerases (RNAPs) have high specificity and almost no mutual interference.

[0266] Example 4 Comparison of mutation rates between orthogonal transcriptional mutagens based on MmP1, K1F and VP4 and a mutagenesis system based on T7 RNA polymerase

[0267] In this example, we compared the mutation rates of the orthogonal transcription mutants based on MmP1, K1F and VP4 according to the present invention with the mutation system based on the known T7 RNA polymerase in Halomonas and Escherichia coli, respectively.

[0268] In Halomonas bluephagenesis TD01, a targeting plasmid expressing the sacB gene, three orthogonal transcription mutants pMT23-MmP1, pMT23-K1F and pMT23-VP4, and a T7 mutant plasmid obtained by replacing MmP1 / K1F / VP4 in the above mutants with T7 polymerase were respectively transferred into Halomonas. The culture was cultured at 37°C for 10 hours. Then, the secondary seed solution was transferred at a concentration of 1:100 and 200 mg / L of ITPG inducer was added, and the culture was cultured at 37°C for 20 hours. The culture solution was diluted to 10 in a 10-fold gradient. -7 , and were spread on plates containing or not containing 100g / L sucrose, and cultured at 37°C for 48h. The mutation rate multiple was calculated as M1 / M0, where M1 represents the mutation rate using the orthogonal transcription mutant, and M0 represents the background mutation rate of the corresponding phage RNA polymerase control group. The mutation rates are shown in Table 6:

[0269] Table 6. Comparison of mutation rates in halomonas

[0270]

[0271] It can be seen from the results that the mutation system using the existing T7 RNA polymerase is not applicable in Halobacterium, and the mutation rate is comparable to that of the control group with an empty plasmid, while the orthogonal transcription mutants based on the MmP1, K1F and VP4 polymerases of the present invention show a mutation rate of more than 10 -1 The mutation rate is much better than that of the T7 mutation system.

[0272] In E. coli MG1655 (purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.), the targeting plasmid expressing the sacB gene, three orthogonal transcription mutants pMT23-MmP1, pMT23-K1F and pMT23-VP4 and the above-mentioned T7 mutant plasmid were respectively transferred into E. coli. The cells were cultured at 37°C for 10 hours. Then, the secondary seed solution was transferred at a concentration of 1:100 and 200 mg / L of ITPG inducer was added, and the cells were cultured at 37°C for 20 hours. The culture solution was diluted to 10 by a 10-fold gradient. -7 , and were spread on plates containing or not containing 100 g / L sucrose, and cultured at 37°C for 48 h. The mutation rates are shown in Table 7:

[0273] Table 7. Comparison of mutation rates in E. coli

[0274]

[0275] From the results, we can see that in E. coli MG1655, the mutation rate of the T7 mutation system is only 10 -4 , while the mutation rate of orthogonal transcriptional mutants based on MmP1, K1F and VP4 exceeded 10 -1 This indicates that in E. coli, the orthogonal transcription mutants based on MmP1, K1F and VP4 according to the present invention are also significantly superior to the T7 RNA polymerase mutation system.

[0276] Example 5 Using orthogonal transcriptional mutagen to mutate the target gene on the plasmid

[0277] In this example, we tested the application of orthogonal transcriptional mutagenesis in mutating target genes on plasmids. We used three fluorescent protein genes and three pigment protein genes as the genes to be mutated, and produced cells with different colors by mutating these genes in Escherichia coli MG1655 (commercially available from Beijing Zhuangmeng International Biogene Technology Co., Ltd.). We used pSEVA321 as the backbone plasmid to construct two targeting plasmids, which contained three fluorescent protein genes mCheery, sfGFP and TagBFP, and three pigment protein genes amajLime, fwYellow and spisPink (see plasmid maps for details). Fig.12 and Fig.13 ). The pMmP1 promoter was inserted into the upstream and downstream of the two gene clusters. Then, the pMT23opt-MmP1 double mutant plasmid and the pMT0-MmP1 control plasmid were transferred to perform induction experiments. The bacterial solution was taken to observe the color change under a fluorescent confocal microscope, and the bacterial solution was spread on a plate. The colonies with color changes were selected for colony PCR and sequencing analysis.

[0278] The results are as follows Fig.14 As shown, compared with the control group, the use of pMT23opt-MmP1 orthogonal transcription mutants can produce a variety of colors, such as purple, cyan, etc. The sequencing results show (see Table 8) that different types of mutations exist in the three fluorescent protein genes of the mutants.

[0279] Table 8. Sequencing results of fluorescent protein mutants

[0280]

[0281] The results of the mutation of pigment protein show (see Fig.15 ), compared with the control group, the colonies showed a variety of colors after using the orthogonal transcription mutant. At the same time, the sequencing results of colonies with obvious color differences were selected (see Table 9), indicating that different types of mutations existed in the three pigment protein genes of the mutants.

[0282] These results indicate 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.

[0283] Table 9. Sequencing results of pigment protein mutants

[0284]

[0285] Example 6 Using orthogonal transcriptional mutants to mutate target genes on the genome

[0286] In this embodiment, we tested the application of orthogonal transcription mutants in target genes on the mutant genome. We selected the cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ in the Halomonas genome as target genes. The pMmP1 promoter sequence was inserted into the back of 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 transcription mutants. Then, the pMT23opt-MmP1 double mutation plasmid and the pMT0-MmP1 control plasmid were transferred into the Halomonas bluephagenesis TD01 strain after the pMmP1 promoter was inserted, and an induction experiment was performed, followed by scanning electron microscopy analysis. The results showed that compared with the control group, the pMT23opt-MmP1 double mutant plasmid had significantly elongated cell morphology after mutation of the cell division-related gene ftsQAZ (see Table 10 for sequencing results) (see Table 10 for sequencing results). Fig.16 ); after mutation of the cytoskeleton-related gene mreBCD (sequencing results see Table 11), the cell morphology changed from rod-shaped to spherical (see Fig.17 ); after the cytoskeleton-related gene mreBCD and the cell division-related gene ftsQAZ were mutated at the same time (see Table 12 for sequencing results), the cells showed ellipsoidal, long rod-shaped and other irregular shapes (see Fig.18 ). The above results show that the use of orthogonal transcriptional mutagen to mutate the cytoskeleton and cell division-related genes on the genome can successfully make cells present a variety of morphologies, proving the effectiveness of this system in mutating target genes on the genome.

[0287] Table 10. Sequencing results of mutant FtsQAZ

[0288]

[0289] Table 11. Sequencing results of mutant MreBCD

[0290]

[0291] Table 12. Sequencing results of simultaneous mutations of FtsQAZ and MreBCD

[0292]

[0293]

[0294] The above results show that the targeted mutagenesis system based on orthogonal phage RNA polymerase can quickly and efficiently generate a large number of mutations on the target gene. The optimized orthogonal transcription mutagen can simultaneously generate 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, greatly improve the mutation and evolution efficiency of enzymes or other proteins, and has a very broad application prospect.

[0295] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0296] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0297] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A fusion protein comprising: (i) a bacteriophage RNA polymerase; and (ii) a base editing enzyme. 2 . The fusion protein according to claim 1 , wherein the bacteriophage RNA polymerase is selected from MmP1 (SEQ ID NO: 1) or a variant thereof, K1F (SEQ ID NO: 2) or a variant thereof, and VP4 (SEQ ID NO: 3) or a variant thereof.

3. The fusion protein according to claim 1 or 2, wherein the base editing enzyme comprises a deaminase, preferably adenosine deaminase, cytidine deaminase or a combination thereof.

4. The fusion protein according to any one of claims 1 to 3, wherein: 1) The base editing enzyme is fused to the N-terminus of the bacteriophage RNA polymerase via an optional linker; 2) The linker is selected from: SGSETPGTSESATPES(XTEN)(SEQ ID NO:4) SGGS (SEQ ID NO:5) SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:6) SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:7) GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGG SGGS(SEQ ID NO:8) SGGSGGSGGS (SEQ ID NO:9) (GGGS)n (GGGGS)n (G)n (EAAAK)n (GGS)n, Where n = 1, 2, 3, 4, 5 or 6; 3) The deaminase is selected from: a deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases, preferably an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase or a variant thereof, preferably from rat or human, Activation-induced cytidine deaminase (AID) or a variant thereof, preferably of human origin, Sea lamprey (Petromyzon marinus) cytidine deaminase 1 (PmCDA1) or a variant thereof (e.g., evoPmCDA1), TadA adenosine deaminase or a variant thereof, preferably TadA7.10, TadA8.1, TadA8.2, TadA8.3, TadA8.4, TadA8.5, TadA8.6, TadA8.7, TadA8.8, TadA8.9, TadA8.10, TadA8.11, TadA8.12, TadA8.13, TadA8.14, TadA8. .15, TadA8.16, TadA8.17, TadA8.18, TadA8.19, TadA8.20, TadA8.21, TadA8.22, TadA8.23 or TadA8.24, more preferably TadA7.10, TadA8e, TadA9, CABET3.1, CABET3.155 and TadDE, preferably from Escherichia coli, and A combination of these; and / or 4) The fusion protein further comprises an auxiliary mutation element, which is preferably an inhibitor of base excision repair, more preferably a uracil glycosylase inhibitor (UGI), and even more preferably a glycosylase inhibitor UGI derived from Bacillus subtilis phage PBS1.

5. The fusion protein according to any one of claims 1 to 4, comprising the following structure from N-terminus to C-terminus: Deaminase-XTEN-bacteriophage RNA polymerase, Preferably, the following structure is included: Deaminase-UGI-XTEN-bacteriophage RNA polymerase, Preferably, the deaminase is PmCDA1 or TadA adenosine deaminase or a variant thereof, More preferably, the fusion protein is selected from: PmCDA1-XTEN-MmP1, PmCDA1-UGI-XTEN-MmP1, evoPmCDA1-UGI-XTEN-MmP1, PmCDA1-UGI-XTEN-K1F, PmCDA1-UGI-XTEN-VP4, TadA8e-XTEN-MmP1, TadA8e-XTEN-K1F, TadA8e-XTEN-VP4, TadA7.10-XTEN-MmP1, TadA9-XTEN-MmP1, PmCDA1-UGI-TadA8e-XTEN-MmP1, PmCDA1-UGI-TadA8e-XTEN-K1F, PmCDA1-UGI-TadA8e-XTEN-VP4, TadA8e-PmCDA1-UGI-XTEN-MmP1, TadA8e-PmCDA1-UGI-XTEN-K1F, TadA8e-PmCDA1-UGI-XTEN-VP4, TadA8e-XTEN-MmP1(opt), TadA8e-XTEN-K1F(opt), and TadA8e-XTEN-VP4 (opt).

6. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5.

7. A targeted evolution / mutation system, comprising: (i) the fusion protein according to any one of claims 1 to 5 or the nucleic acid molecule according to claim 6; and (ii) a polynucleotide sequence comprising a promoter and a cloning site, wherein the cloning site is used to insert a DNA molecule of a target gene to be evolved / mutated, The bacteriophage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter.

8. The targeted evolution / mutation system according to claim 7, wherein: 1) The polynucleotide sequence is located on a plasmid or on a genome; 2) including one or more of the promoters upstream, downstream, or upstream and downstream of the cloning site; 3) the upstream, downstream or both upstream and downstream of the cloning site include one or more bacteriophage RNA polymerase transcription terminators; 4) the bacteriophage RNA polymerase is selected from MmP1 or its variants, K1F or its variants and VP4 or its variants, and their corresponding promoters are selected from pMmP1, pK1F and pVP4; and / or 5) The targeted evolution / mutation system is based on an inducible expression system selected from the following: Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acylhomoserine lactone AHL-inducible system), P Tet and its mutants (anhydrotetracycline aTc-inducible system), P Van and its mutants (vanillic acid inducible system).

9. A targeted evolution / mutation system, comprising: (i) the fusion protein according to any one of claims 1 to 5 or the nucleic acid molecule according to claim 6; and (ii) a target gene DNA molecule to be evolved / mutated connected to a promoter, The bacteriophage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter, thereby transcribing the target gene DNA molecule, preferably wherein: 1) The target gene DNA molecule is located on a plasmid or genome; 2) including one or more of the promoters upstream, downstream, or upstream and downstream of the target gene DNA molecule; 3) The upstream, downstream, or upstream and downstream of the target gene DNA molecule respectively include one or more bacteriophage RNA polymerase transcription terminators; 4) the bacteriophage RNA polymerase is selected from MmP1 or its variants, K1F or its variants and VP4 or its variants, and their corresponding promoters are selected from pMmP1, pK1F and pVP4; and / or 5) The targeted evolution / mutation system is based on an inducible expression system selected from the following: Tac and its mutants (isopropylthiogalactoside IPTG induction system), P BAD and its mutants (arabinose inducible system), P Lux and its mutants (acylhomoserine lactone AHL-inducible system), P Tet and its mutants (anhydrotetracycline aTc-inducible system), P Van and its mutants (vanillic acid inducible system).

10. A method for targeted evolution / mutation of a target gene DNA molecule, the method comprising: Using the targeted evolution / mutation system according to any one of claims 7 to 9, the target gene DNA molecule is contacted with the fusion protein, so that the phage RNA polymerase in the fusion protein specifically recognizes / binds to the promoter, thereby transcribing the target gene DNA molecule, wherein the base editing enzyme introduces mutations during the transcription process, preferably wherein: 1) The contact occurs in vivo / intracellular or in vitro / extracellular; 2) The target gene DNA molecule is located on a plasmid or genome; 3) the base editing enzyme comprises a deaminase, preferably adenosine deaminase, cytidine deaminase or a combination thereof, and the mutation is C:G to T:A and / or A:T to G:C; 4) The method further comprises the step of screening the mutants by antibiotic resistance, cytotoxicity or fluorescence or color or morphology; and / or 5) The method is carried out in a prokaryote or a eukaryote, preferably a microorganism, more preferably the prokaryote is selected from Escherichia coli, Pseudomonas, Eutropha rosea, Aeromonas hydrophila, Corynebacterium glutamicum or halophilic bacteria, and even more preferably the halophilic bacteria is selected from Halomonas, including Halomonas bluephagenesis or its derivatives, Halomonascampaniensis or its derivatives, Halomonas aydingkolgenesis or its derivatives, preferably Halomonasbluephagenesis TD01 (Cultivation Number CGMCC No.4353), Halomonas aydingkolgenesis M1 (Cultivation Number CGMCC No.19880), Halomonas campaniensis LS21 (Cultivation Number CGMCC No.6593), and / or the eukaryote is selected from yeast, fungi or algae.

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