A genome hypermutation system based on transcription-replication coupling and its application in rapid evolution of halomonas

Abstract

The present application constructs a hypermutator capable of efficiently introducing mutations in the process of transcription and replication by fusing cytosine or adenine deaminase to RNA polymerase subunit and DNA polymerase III subunit respectively or simultaneously. Further, by fusing bifunctional deaminase, introducing DNA polymerase subunit mutant with proofreading function defect, increasing the copy number of genome replication initiation site and optimizing the culture medium conditions, the mutation efficiency and mutation spectrum are significantly improved. The hypermutation system constructed by the present application can make the genome mutation frequency of halomonas sp. increase more than 330,000 times compared with the wild type, and can produce multiple mutation types such as C:G to T:A, A:T to G:C and A:T to T:A. Using the system, evolution strains tolerant to high-concentration lysine and furfural stress conditions can be quickly obtained in a few days to weeks. The present application provides a powerful tool for efficient modification of halomonas sp. and other industrial microbial platforms, and has important application value in the field of green biological manufacturing.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and synthetic biology, specifically relating to a genome hypermutation system based on transcription and replication processes, and the application of this system in halophilic monoclonal bacteria (Halophilus spp.). Halomonas Methods and applications for rapidly evolving mutant strains with excellent industrial characteristics (such as high product tolerance, high toxicity tolerance, etc.) in the field of chemistry. Background Technology

[0002] Genomic mutation is the fundamental driving force of biological evolution and adaptation to the environment. In synthetic biology, adaptive laboratory evolution (ALE) is widely used to optimize traits such as stress tolerance or product synthesis ability in microorganisms. However, the high fidelity of the in vivo DNA replication and repair system limits the frequency of natural mutations to an extremely low level, which greatly restricts the rapid evolution of microbial phenotypes (Dragosits M, Mattanovich D. Adaptive laboratory evolution–principles and applications for biotechnology[J]. Microbial cell factories, 2013, 12(1): 64.). Therefore, developing efficient and controllable in vivo mutation systems is crucial for accelerating strain evolution.

[0003] Halophilic bacteria ( Halomonas Halomonas spp. are ideal chassis cells for Next Generation Industrial Biotechnology (NGIB) because they can perform pollution-resistant open continuous fermentation under high salinity and alkalinity conditions, and have been used to produce high-value compounds such as polyhydroxyalkali esters (PHA) and tetrahydropyrimidine (Yan X, Wang J, Wen R, et al. The halo of future bio-industry based on engineering Halomonas[J]. Metabolic Engineering, 2025, 90: 16-32.). However, unlike model organisms such as Escherichia coli, Halomonas lacks an efficient genome mutation system, which has become a major bottleneck in the engineering modification of its strains.

[0004] Therefore, there is an urgent need to develop a high-efficiency genomic hypermutation platform suitable for Halophilic monoclonal bacteria with diverse mutation types. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a highly efficient genome hypermutation system based on transcription and replication coupling in halophilic monoclonal bacteria, which has a broad spectrum of mutation types, and to provide a method for rapidly evolving halophilic monoclonal mutants with excellent industrial traits using this system.

[0006] Therefore, in one aspect, the present invention provides a genome hypermutation system based on transcription and replication coupling, wherein the system comprises at least one fusion protein expression element, the fusion protein being formed by fusing a deaminase or a variant thereof with a protein related to the transcription or replication process via an optional linker peptide.

[0007] In one specific embodiment, the deaminase is selected from one or more of cytosine deaminase, adenine deaminase, or bifunctional deaminases with both cytosine and adenine deaminase activities; and / or the transcription or replication-related protein is selected from one or more of RNA polymerase subunit, DNA polymerase III subunit, or DNA helicase.

[0008] In one specific embodiment, the cytosine deaminase is selected from sea lamprey (Gymnocypris spp.) Petromyzon marinus The adenine deaminase is selected from TadA adenine deaminase or a variant thereof, and / or the bifunctional deaminase with both cytosine and adenine deaminase activities is selected from CABE T3.155, TadDE and CABE T3.1.

[0009] Deaminases derived from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases include APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or variants thereof, preferably derived from rats or humans.

[0010] Preferably, the activation-induced cytidine deaminase (AID) or a variant thereof is of human origin.

[0011] TadA adenosine deaminase or its variants include 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, CABE T3.1, or CABE. T3.155 and TadDE, preferably derived from Escherichia coli, and combinations thereof.

[0012] In one specific embodiment, the transcription-related protein is the α subunit RpoA, β subunit rpoB, or β' subunit RpoC of RNA polymerase, preferably RpoC; the replication-related protein is the ε subunit DnaQ, α subunit DnaE, ​​β subunit DnaN, γ / τ subunit DnaX, δ subunit HolA of DNA polymerase III, or DNA helicase DnaB, preferably the ε subunit DnaQ.

[0013] In one specific embodiment, the deaminase or a variant thereof is fused to the N-terminus or C-terminus of a protein associated with the transcription or replication process via an optional linker peptide.

[0014] In one specific embodiment, the linker peptide is selected from: SGSETPGTSESATPES (XTEN) (SEQ ID NO: 1) SGGS (SEQ ID NO: 2) SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 3) SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 4) GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 5) SGGSGGSGGS (SEQ ID NO: 6) (GGGS) n (GGGGS) n (G) n (EAAAK) n (GGS) n , Where n = 1, 2, 3, 4, 5 or 6.

[0015] In one specific embodiment, the fusion protein further includes an auxiliary mutant element, 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. Preferably, the UGI is fused to the C-terminus of a deaminase.

[0016] In one embodiment, the system simultaneously includes expression elements of a first fusion protein and a second fusion protein, wherein the first fusion protein is a fusion protein formed by fusing (i) a deaminase or a variant thereof with (ii) an RNA polymerase subunit via an optional linker peptide, and the second fusion protein is a fusion protein formed by fusing (i) a deaminase or a variant thereof with (ii) a DNA polymerase III subunit or a DNA helicase via an optional linker peptide.

[0017] In one specific embodiment, the expression elements of the first fusion protein and the second fusion protein may be contained in the same or different expression vectors, or the first fusion protein and the second fusion protein may be expressed under the control of the same or different promoters.

[0018] In one specific embodiment, the first fusion protein is formed by fusing (i) cytosine deaminase or a variant thereof with (ii) an RNA polymerase subunit via an optional linker peptide, and the second fusion protein is formed by fusing (i) adenine deaminase or a variant thereof with (ii) a DNA polymerase III subunit via an optional linker peptide.

[0019] In one specific embodiment, the ε subunit DnaQ of DNA polymerase III contains a mutation that causes it to lose its 3'-5' exonuclease proofreading function, the mutation being a D7A and E9A mutation.

[0020] In one specific embodiment, the first fusion protein is formed by fusing a bifunctional deaminase, a uracil glycosylation inhibitor (UGI), and an RNA polymerase β' subunit RpoC sequentially (from N to C-terminus) via optional linker peptides, and the second fusion protein is formed by fusing a DNA polymerase III ε subunit DnaQ carrying D7A and E9A mutations, a bifunctional deaminase, and a uracil glycosylation inhibitor (UGI) sequentially (from N to C-terminus) via optional linker peptides.

[0021] In one specific embodiment, the fusion protein comprises the following structure from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: Deaminases – optional XTEN – proteins involved in the transcription process. Preferably, it includes the following structure: Deaminase-UGI-optional XTEN-transcriptional protein, More preferably, the fusion protein includes at least one of the following (wherein the N-terminus or C-terminus of the deaminase or deaminase-UGI fusion protein may optionally include the linker peptide XTEN): RpoA-PmCDA1-UGI ​​(MTR1) RpoB-PmCDA1-UGI ​​(MTR3) PmCDA1-UGI-RpoB (MTR4) RpoC-PmCDA1-UGI ​​(MTR5) PmCDA1-UGI-RpoC (MTR6) DnaB-PmCDA1-UGI ​​(MTD1) PmCDA1-UGI-DnaB (MTD2) DnaN-PmCDA1-UGI ​​(MTD3) PmCDA1-UGI-DnaN (MTD4) DnaQ-PmCDA1-UGI ​​(MTD5) PmCDA1-UGI-DnaQ (MTD6) HolA-PmCDA1-UGI ​​(MTD8) PmCDA1-UGI-HolA (MTD9) RpoA-TadA8e (MTR1.2) TadA8e-RpoC (MTR6.2) DnaQ-TadA8e (MTD5.2) CABE T3.155-RpoC (MTR6-T3.155) TadDE-RpoC (MTR6-TadDE) CABE T3.155-UGI-RpoC (MTR6-dual1) TadDE-UGI- RpoC (MTR6-dual2) DnaQ-CABE T3.155 (MTD5-T3.155) DnaQ-TadDE (MTD5-TadDE) DnaQ-CABE T3.155-UGI (MTD5-dual1) DnaQ-TadDE-UGI (MTD5-dual2) DnaQ D7A,E9A -TadDE-UGI (MTD5-d2-926).

[0022] In a preferred embodiment, the genomic hypermutation system comprises a combination of transcription-coupled mutant / fusion protein expression element (PmCDA1-UGI-RpoC) and replication-coupled mutant / fusion protein expression element (DnaQ-TadA8e) (i.e., MTDR1).

[0023] In a preferred embodiment, the genomic hypermutation system comprises a combination of transcription-coupled mutant / fusion protein expression element (TadA8e-RpoC) and replication-coupled mutant / fusion protein expression element (DnaQ-PmCDA1-UGI) (i.e., MTDR2).

[0024] In a preferred embodiment, the genome hypermutation system includes a transcription-coupled mutant / fusion protein expression element (TadDE-UGI-RpoC) and a replication-coupled mutant / fusion protein expression element (DnaQ). D7A,E9A The combination of -TadDE-UGI (i.e. MTDR3 or MTDR4).

[0025] Another aspect of the present invention provides a nucleic acid molecule encoding a fusion protein in a system according to the present invention.

[0026] Another aspect of the present invention provides a method for rapidly evolving microbial (e.g., halophilic monoclonal bacteria) mutants with a target phenotype using the system described in the present invention, comprising the following steps: (1) The supermutation system is introduced into the starting microorganism (e.g., halophilic monoclonal bacteria) strain; (2) The strain obtained in step (1) is cultured in a culture medium to induce the expression of the fusion protein in order to initiate a genomic hypermutation process; (3) The bacterial culture from step (2) is continuously passaged or screened under selective pressure (such as high concentrations of lysine, sodium furfural, etc.); and (4) Evolutionary strains with the target phenotype are isolated from cultures that can grow under selective pressure.

[0027] In one specific embodiment, the induced expression of the fusion protein can be achieved by adding an inducer (e.g., IPTG) to the culture medium, via an inducible promoter (e.g., P) included in the expression element. MmP1 This is achieved through a promoter.

[0028] In one specific implementation, the target phenotype is the ability to withstand the selection pressure.

[0029] In one specific embodiment, the halophilic monoclonal bacteria include Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (culture preservation number CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593).

[0030] In one specific embodiment, the transcription or replication-related proteins in the hypermutation system are derived from microorganisms (e.g., Halophilic bacteria) or from the same genus or species of microorganisms (e.g., Halophilic bacteria), or are endogenous to the microorganisms (e.g., Halophilic bacteria).

[0031] In one specific embodiment, the target phenotype includes tolerance to 20-50 g / L L-lysine or tolerance to 10-20 mM furfural.

[0032] In one specific embodiment, the selection pressure is 20-50 g / L (e.g., 50 g / L) of L-lysine or 10-20 mM (e.g., 20 mM) of furfural.

[0033] In one specific embodiment, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) replication origin sites are inserted into the genome of the microbial strain (e.g., Halophilus). oriC copy.

[0034] In one specific embodiment, the culture medium is a mineral salt (MM) medium.

[0035] Another aspect of the present invention provides a mutant strain of a microorganism (e.g., halophilic monoclonal bacteria) evolved by the method described in the present invention.

[0036] Another aspect of the present invention provides the application of the systems, nucleic acid molecules or methods described in the present invention in, for example, microbial breeding of halophilic monoclonal bacteria, chassis cell modification of extremophiles or green biomanufacturing.

[0037] This invention utilizes the editing window created during the unwinding of the DNA double helix during transcriptional bubbling and replication fork formation by fusing a deaminase to an RNA polymerase subunit or DNA polymerase III subunit. This allows for targeted deamination of transiently exposed single-stranded DNA, introducing C:G to T:A or A:T to G:C mutations. Furthermore, by employing a bifunctional deaminase, introducing a proofreading-deficient DnaQ mutant, increasing the copy number at the genome replication initiation site, and optimizing culture medium conditions, the mutation frequency in mineral salt medium reaches 3.9 × 10⁻⁶. -3 Compared to the wild type, the efficiency is over 330,000 times higher, and it can simultaneously generate three mutation types: C:G to T:A, A:T to G:C, and A:T to T:A. Using this system, evolved strains of Halophilic bacteria tolerant to industrial stress conditions such as 50 g / L lysine and 20 mM furfural can be rapidly obtained within weeks, providing a powerful tool platform for the efficient modification of non-model industrial microorganisms such as Halophilic bacteria. Attached Figure Description

[0038] 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, wherein: Figure 1 Design principles and applications of genome hypermutation systems based on transcription and replication coupling; Figure 2 Construct mutants based on the PmCDA1-UGI ​​fusion RNA polymerase subunit; Figure 3 Construct mutants based on PmCDA1-UGI ​​fusion helicase or DNA polymerase subunits; Figure 4 Mutation frequency of transcription- and replication-coupled C:G to T:A supermutants; Figure 5 Mutation types in transcription- and replication-coupled C:G to T:A supermutants; Figure 6 Construct mutants based on TadA8e fusion RNA or DNA polymerase subunits; Figure 7Mutation frequency of TadA8e fusion RNA or DNA polymerase subunit mutants; Figure 8 Mutation types of TadA8e fusion RNA or DNA polymerase subunit mutants; Figure 9 Construct dual mutants based on PmCDA1-UGI ​​or TadA8e fusion RNA or DNA polymerase subunits; Figure 10 Mutation rate and mutation type of double mutants; Figure 11 Constructing supermutants with multiple mutation types; Figure 12 Mutation frequency of supermutants with multiple mutation types; Figure 13 Mutation types of supermutants with multiple mutation types; Figure 14 . H. bluephagenesis Different strains oriC Mutation frequency of copies; Figure 15 Construct combinatorially optimized supermutation systems; Figure 16 Mutation frequency of the supermutation system after combinatorial optimization; Figure 17 Mutation frequency of the hypermutation system after culture medium optimization.

[0039] Sequence List Description The preferred embodiments of the present invention and the specific sequences used in the embodiments are as follows: Linking peptides SGSETPGTSESATPES (XTEN) (SEQ ID NO: 1) SGGS (SEQ ID NO: 2) SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 3) SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 4) GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 5) SGGSGGSGGS (SEQ ID NO: 6) Amino acid sequence of PmCDA1 cytosine deaminase (SEQ ID NO: 7): MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV Gene sequence of PmCDA1 cytosine deaminase (SEQ ID NO: 8): atgaccgacgctgagtacgtgagaatccatgagaagttggacatctacacgtttaagaaacagtttttcaacaacaaaaaatccgtgtcgcatagatgctacgttctctttgaattaaaacgacggggtgaacgtagagcgtgtttttggggctatgctgtgaataaaccacagagcgggacagaacgtggcattcacgccgaaatctttagcattagaaaagtcgaagaatacctgcgcgacaaccccggacaattcacgataaattggtactcatcctggagtccttgtgcagattgcgctgaaaagatcttagaatggtataaccaggagctgcgggggaacggccacactttgaaaatctgggcttgcaaactctattacgagaaaaatgcgaggaatcaaattgggctgtggaatctcagagataacggggttgggttgaatgtaatggtaagtgaacactaccaatgttgcaggaaaatattcatccaatcgtcgcacaatcaattgaatgagaatagatggcttgagaagactttgaagcgagctgaaaaacgacggagcgagttgtccattatgattcaggtaaaaatactccacaccactaagagtcctgctgtt Amino acid sequence of UGI glycosylase inhibitor (SEQ ID NO: 9): MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML Gene sequence of UGI glycosylation inhibitor (SEQ ID NO: 10): atgaccaacctttccgacatcatagagaaggaaacaggcaaacagttggtcatccaagagtcgatactcatgcttcctgaagaagttgaggaggtcattgggaataagccggaaagtgacattctc gtacacactgcgtatgatgagagcaccgatgagaacgtgatgctgctcacgtcagatgccccagagtacaaaccctgggctctggtgattcaggactctaatggagagaacaagatcaagatgcta The amino acid sequence of TadA8e adenine deaminase (SEQ ID NO: 11): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN The gene sequence of TadA8e adenine deaminase (SEQ ID NO: 12): atgtctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccactctaggatcggccgcgtggtgtttggatggagaaattctaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaactga Amino acid sequence of CABE T3.155 (SEQ ID NO: 13): MSEVEFSHEYWMRHALTLAKRARDERSVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD Gene sequence of CABE T3.155 (SEQ ID NO: 14): atgtccgaagtcgagttttcccatgagtactggatgagacacgcattgactctcgcaaagagggctcgagatgaacgcagcgtgcccgtgggggcagtactcgtgctcaacaatcgcgtaatcggcgaaggttggaatagggcaatcggactccacgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtgatgcagaattatcgactttatgatgcgacgctgtacaccacgtttgaaccttgcgtaatgtgcgcgggagctatgattcactcccgcattggacgagttgtattcggtgttcgcaacgccaagacgggtgccgcaggttcactgatggacgtgctgcatcatccaggcatgaaccaccgggtagaaatcacagaaggcatattggcggacgaatgtgcggcgctgttgtgtcgtttttttcgcatgcccaggcgggtctttaacgcccagaaaaaagcacaatcctctactgactaa Amino acid sequence of TadDE (SEQ ID NO: 15): MSEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGWNRRIGLHDPTAHAEIMALRQGGLVMQNSRLIDATLYVTFEPCVMCAGAMINSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN Gene sequence of TadDE (SEQ ID NO: 16): atgtctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagggagaggcgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagacgtatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactccagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatcaactctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaac Amino acid sequence of RpoA (SEQ ID NO: 17): MQRSVTEFLRPRDIKVEEISAHHAKIVLEPFERGFGHTLGNALRRILLSSMPGAAVVEAEIAGVEHEYSALEGVQEDVIEILLNLKDVAIKMHSRDEAVLSLNKQGPAVVTAGDIALDHSVEIVNPDHVIAHVNEGAELKIQLKVALGRGYEPADARGSDEETRAIGRLQLDATFSPVRRVSYSVEAARVEQRTDLDKLIIDLETDGTLDPEEAIRRSATILQEQLAAFVDLEADKEQEVEEEEDLVDPILLRPVDDLELTVRSANCLKAENIYYIGDLIQRTEVELLKTPNLGKKSLNEIKDVLAARGLSLGMRLENWPPASLKDDKASA Gene sequence of RpoA (SEQ ID NO: 18): atgcagcgttcagtgacagagtttcttcgccctcgcgacatcaaggtcgaagaaatcagcgcacatcatgccaaaatcgttctcgaaccgttcgagcgcggatttggccacaccctggggaatgcacttcgtcgcattctgctttcgtccatgcccggcgctgccgtggtagaggctgaaattgccggtgtagagcacgaatacagtgcactcgaaggggtgcaggaagatgtcatcgaaatcctcctgaacttgaaagatgttgcgatcaagatgcacagccgcgatgaggcggtgctctcgctgaacaagcagggcccagctgtcgtcaccgctggcgacattgcgcttgatcatagcgtcgaaatcgtcaacccggatcacgtcattgctcatgtcaatgaaggtgccgagctgaaaattcagcttaaggtagcgctgggtcgtggttacgaaccggctgacgctcgtggctcagatgaagagacacgtgctattggccgcctgcagctggatgccaccttcagccctgttcgccgtgtttcctactcggttgaggccgctcgtgttgagcagcgcaccgacctcgataagttaattatcgatctggaaaccgacggtacactggatccggaagaggctatccgtcgcagtgcgaccattctgcaagagcagctggccgccttcgtcgacctggaagctgataaagaacaggaagtcgaagaggaagaggatcttgttgatcctatcctattgcgccccgtagacgatcttgagttgacagttcgcagcgctaactgcctaaaagccgagaatatttactacattggtgatcttattcagcgcactgaagttgagctgctgaagacaccgaatcttggtaaaaagtctttgaatgaaatcaaagacgtattggcagcgcgtggcttgtccctcggcatgcggttggaaaattggccacccgctagcctgaaggacgacaaggcctccgcgtga The amino acid sequence of RpoB (SEQ ID NO: 19): The gene sequence of RpoB (SEQ ID NO: 20): The amino acid sequence of RpoC (SEQ ID NO: 21): The gene sequence of RpoC (SEQ ID NO: 22): Amino acid sequence of DnaB (SEQ ID NO: 23): MQDQPSADQETAAIKLPPHSLEAEQSVLGGLMLDNQAWDNVSERLVADDFYRYEHRLVFNVMIHLAESGQPLDVITLSEALEARDQLDTVGGLAFLAELARNTPSASNIRAYADIVRERATLRKLIRAANQIADGAFSPQGRPADELLNEAERLVFQIAEERPKTGGPIGMSELLTKAVDRIDELFNLKGEMTGLSSGFRDLDEMTSGLQPSDLVIIAGRPSMGKTTFAMNLVEHAVIASDKPVMVFSMEMPAESLMLRMLSSLGRIDQTRVRSGQLEDEDWPRLTSAVNLLKDKQLFIDDTAALSPNEMRSRLRRVVREHGNMALIMIDYLQLMQIPGFSENRTGEISEISRSLKGLAKEFQCPVVALSQLNRSLEQRPNKRPVMSDLRESGAIEQDADVIAFVYRDEVYNPDNPDNQGIAELIIGKQRNGPIGTVHMAFIGKYTRFEDLAPDSYGEAFGD Gene sequence of DnaB (SEQ ID NO: 24): Amino acid sequence of DnaN (SEQ ID NO: 25): MKFTISREALLRPLTLVAGVVERRQTLPVLSNVLIQVEGDQVALTGTDLEVELVGRTVASQVDQEGAATVPARKLMDICKSLPDQSEIQLAVEEGRAVLRSGRSRFTLSTLPVAEFPNIEDADGSQELSVPRGTLKHLIESTSFAMAQQDVRYYLNGMLLEIQSNLLRTVATDGHRLAMCSRPIDIVVDQAQKLIVPRKGILELSRLLDDSDEPVSLTLGSTHVRAHTGDFTFTSKLIDGKFPDYERVVPRNGDKVLIAERAELRQVLSRTAILSNEKYRGVRLYLEENNLKVMANNPEQEEAEENVAVEYNGGAMEVGFNVGYLVDVLSVLDEDRVQMTLADPNSSALLEEPGGGDALYVVMPMRL Gene sequence of DnaN (SEQ ID NO: 26): The amino acid sequence of DnaQ (SEQ ID NO: 27): MRQVILDTETTGIDPKDGHRLVEIGAVEMINRRFTGRSYHQYINPERHIDAEVVAVHGIDDAKVANEPVFAEIADDFWAFIEGAELVIHNAPFDVGFIDHELTMLNQRRRSPALGPVSDHCRILD TLVMARQMHPGQRNSLDALCKRYDIDNGHRVLHGALLDAEILADVYLAMTGGQTALTLDSEASSGEQQDNQASEGLSVQRLSLTPGQLRVVQPSDEERAAHQAKCQAHQLRWFDGGWSEGNSADA The gene sequence of DnaQ (SEQ ID NO: 28): atgcgccaagtgatcttggatacggaaacgaccggcatcgaccctaaagatggccaccgattagttgaaattggtgccgtcgaaatgattaaccggcgttttacagggcgctcctatcatcaatacatcaaccccgaacggcatattgatgctgaagtcgtggcggttcacggtattgatgatgcaaaggttgctaacgaaccggtgtttgccgaaatagcggacgacttctgggcatttatagaaggtgccgagctggtgattcacaacgccccctttgatgtggggtttattgatcatgagctaacaatgttaaaccaacggcgacggtcacccgcattaggccctgttagtgatcattgccgtattctggatacgctggtgatggcccgtcagatgcacccaggccagcgtaatagtctcgatgcgttatgtaagcgctacgatattgacaacggtcatcgcgtactgcacggcgcattgcttgatgctgagatcttagccgacgtctatttggcgatgacgggcgggcaaacggcgctgacgcttgattcagaagcttcgtcaggcgaacagcaagataatcaagccagtgaaggactgtcggtacagcgtttatcgctaacgccaggacagctgagggtagtccagcctagtgatgaagagcgcgccgcccaccaagctaagtgccaggcgcatcagttacgttggtttgatggtggttggtctgaaggtaatagtgccgatgcttag DnaQ D7A, E9A Amino acid sequence of (SEQ ID NO: 29): MRQVILATATTGIDPKDGHRLVEIGAVEMINRRFTGRSYHQYINPERHIDAEVVAVHGIDDAKVANEPVFAEIADDFWAFIEGAELVIHNAPFDVGFIDHELTMLNQRRRSPALGPVSDHCRILDTLVMARQMHPGQRNSLDALCKRYDIDNGHRVLHGALLDAEILADVYLAMTGGQTALTLDSEASSGEQQDNQASEGLSVQRLSLTPGQLRVVQPSDEERAAHQAKCQAHQLRWFDGGWSEGNSADA DnaQ D7A, E9A Gene sequence of (SEQ ID NO: 30): atgcgccaagtgatcttggctacggcaacgaccggcatcgaccctaaagatggccaccgattagttgaaattggtgccgtcgaaatgattaaccggcgttttacagggcgctcctatcatcaatacatcaaccccgaacggcatattgatgctgaagtcgtggcggttcacggtattgatgatgcaaaggttgctaacgaaccggtgtttgccgaaatagcggacgacttctgggcatttatagaaggtgccgagctggtgattcacaacgccccctttgatgtggggtttattgatcatgagctaacaatgttaaaccaacggcgacggtcacccgcattaggccctgttagtgatcattgccgtattctggatacgctggtgatggcccgtcagatgcacccaggccagcgtaatagtctcgatgcgttatgtaagcgctacgatattgacaacggtcatcgcgtactgcacggcgcattgcttgatgctgagatcttagccgacgtctatttggcgatgacgggcgggcaaacggcgctgacgcttgattcagaagcttcgtcaggcgaacagcaagataatcaagccagtgaaggactgtcggtacagcgtttatcgctaacgccaggacagctgagggtagtccagcctagtgatgaagagcgcgccgcccaccaagctaagtgccaggcgcatcagttacgttggtttgatggtggttggtctgaaggtaatagtgccgatgcttag Amino acid sequence of DnaX (SEQ ID NO: 31): MSYQVLARKWRPRTFHELVGQAHVQRALVNALDQGRLHHAYLFTGTRGVGKTTLARILAKCLNCTANGRGDEGITSTPCGQCDSCRAIDEGRFVDLIEVDAASRTKVEDTRELLDNVQYAPTQGRYKVYLIDEVHMLSTSSFNALLKTLEEPPPHVKFLLATTDPQKLPATVLSRCLQFTLKHMPPERVVEHLTYVLGEEGVAYDESALWLLGKAAEGSMRDAMSLTDQAIAFGQGAIRHADVAAMLGTLDHRHVLALVEALADVDVQRLLAEVAQLSEQGPDFAAVLDELSAVLHRLAVAQMVPDAVDNSHGDRALIQQLASRFTAEDIQLYYQIGIQGRGDMVHAPDLRSALEMTLLRMLAFRPQGVPKPAATPLPLRRESPSNAASQEAVPHEPNSHEPVSHEPVSHESISQVSASDNSSVSESASAHEPTTKKPEPALPSNQVEAPDSAAVQASVEPVTNEASSALDASLEQQAPPWSLDEVESAAMLSPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPVSADVPVNVASRQGASGDRLDHAGWLACFSALGLGGLTRNLAAHCQLESDDGHTVVLRLDPGQSAMQADVHNGRIERALSSYGLPRRIEFTVADLASDIETPRQQEERQQQERHSLAVDLLHRDPNIQKLQQAFGATLIESTVKPTSDKRS Gene sequence of DnaX (SEQ ID NO: 32): Amino acid sequence of HolA (SEQ ID NO: 33): MKVFSDQLPAALAKKLPKVVIVAGDEPLQHRDACDAVRLAARQAGVEEREVLDVEPNFAWGRLLETASNLSLFASNKLLELRLGTHKLGQDGSKALAQYAEMMSGSDDLLLISMGKLDAKQQKSAWFKALDKQGLFVPVWPVDASRLGYWLRDRASLHGLQIDLEAARLLGERTEGNLLAADQELQKLALIHPPNTRLNVESIAQGVEDSTRFDVFNLADACLKGEPTRTSRIVNGLRSEGVEAPIVLWALSRELRTLLSLHQHLDQGQSFEHACKTQKPMIFDKRRPAYQKAISRLSMKRLHKLLLMAQRLDLAVKGASVVPLWPGLHDLALTMAGGKGLLAETPWTYRISANN Gene sequence of HolA (SEQ ID NO: 34): Codon-optimized TadDE-UGI gene sequence (SEQ ID NO: 35): atgagcgaagtggaattcagccacgaatactggatgcgccacgccctgactctggccaaacgtgccagagatgaaggcgaagccccggttggtgctgttctggttctgaacaatcgcgttatcggtgaaggctggaaccgccgcattggcctgcacgatcctactgcccatgccgaaattatggccctgcgtcagggtggtctggtgatgcaaaatagccgcctgattgacgccaccctgtacgtgacttttgaaccgtgcgtgatgtgcgccggcgccatgattaatagccgtattggccgtgtggtgtttggtgtgcgcaacagcaaacgcggcgccgctggttctctgatgaatgttctgaattacccgggcatgaaccaccgcgtggaaatcaccgaaggcatcctggccgatgaatgcgccgcccttctttgtgatttttatcgcatgccgcgccaggtgttcaacgcccaaaaaaaagcccagagcagcatcaacatgaccaacctgagcgacatcatcgaaaaagaaaccggcaaacagctggtgatccaggaaagcatcctgatgctgccggaagaagtggaagaagtgatcggcaacaaaccggaaagcgacatcctggtgcacaccgcctatgacgaaagcaccgacgaaaacgtgatgctgctgaccagcgacgccccggaatataaaccgtgggccctggttattcaggacagcaatggcgaaaacaaaatcaaaatgctgtga P MmP1 Promoter sequence (SEQ ID NO: 36): atatttgtggcattatagggaattgtgagcgctcacaattagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctctacaaataattttgtttaa OriC Gene sequence (SEQ ID NO: 37): tcatctcaccatcggttgaaaacggccgaccattgtagcgcccaacgaagcggttatccacacgcactcaagcctctttttgacctgtggacaactattcataagagataaggaaaaactggtaatggatcaaggacaaaaactgatctgaggtgttttataacct gttgaacacatcaaattaacagcttatttaaagcttatccgaaggttatgcacagattttttatgattttaagatgttgataaaaatcagctaaaatggcttatccaaataagtatccccactattaataacagtaatctttaaataacttcattagtaatagta XTEN gene sequence (SEQ ID NO: 38): agcggcagcgagactcccgggacctcagagtccgccacacccgaaagt Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0042] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0043] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0044] The term "comprising" or "including" as used in this invention is an open-ended description, encompassing all specified components or steps described, as well as other specified components or steps that do not substantially affect the meaning; when used to describe the sequence of a protein or nucleic acid, 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 possess the activity described in this invention.

[0045] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As 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".

[0046] As used herein, the term "fusion protein," also known as a chimeric protein, refers to an artificial protein constructed by intentionally linking and expressing two or more genes encoding functional proteins using genetic engineering techniques. As those skilled in the art will understand, the functions of the individual functional proteins in a fusion protein should be substantially independent of each other.

[0047] As used herein, the term "deaminase" refers to an enzyme that catalyzes the removal of an amino group from a molecule or, for example, deamination via hydrolysis. In some embodiments, the deaminase is a cytidine deaminase that catalyzes the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methylcytidine to thymidine (T, 5-methyl-U), respectively. In some embodiments, the deaminase is an adenosine deaminase that 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, and those from sea lampreys (Gymnocypris spp.). Petromyzon marinusThe deaminases include cytidine deaminase 1 (PmCDA1) or variants thereof, and TadA adenosine deaminases or variants thereof, such as TadA7.10, TadA8e, and TadA9. These deaminases and their variants are known in the art and are described in detail, for example, in CN114929287A and CN118284692A. Preferably, the deaminases include, but are not limited to, rat cytosine nucleoside deaminase APOBEC1 and its variants, human activation-induced cytidine deaminase AID and its variants, lamprey cytidine deaminase PmCDA1 and its variants, adenosine deaminase TadA variants TadA7.10, TadA8e, and TadA9 derived 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.

[0048] As used herein, the term "replication-related protein" refers to all enzymes and accessory proteins involved in DNA replication initiation, unwinding, primer synthesis, strand polymerization, mismatch correction, primer excision, Okazaki fragment ligation, and replication fork maintenance. In some embodiments, the replication-related protein is selected from one or more of RNA polymerase subunits, DNA polymerase III subunits, or DNA helicase. Preferably, the transcription-related protein is the α subunit RpoA, β subunit rpoB, or β' subunit RpoC of RNA polymerase, preferably RpoC; the replication-related protein is the ε subunit DnaQ, α subunit DnaE, ​​β subunit DnaN, γ / τ subunit DnaX, δ subunit HolA, or DNA helicase DnaB of DNA polymerase III, preferably the ε subunit DnaQ. In a preferred embodiment, the transcription-related protein is the ε subunit DnaQ with D7A and E9A mutations introduced. In a Chinese patent application filed on the same day by the applicant entitled "A genomic hypermutation system of halophilic monoclonal bacteria and its application in strain modification," the 7th aspartic acid (D) and 9th glutamic acid (E) responsible for metal ion coordination in the Exo I domain of the DnaQ protein (ε subunit) were mutated to alanine (A), respectively, to obtain DnaQ. D7A,E9A The mutant was experimentally demonstrated to disrupt the 3'-5' exonuclease proofreading function, thereby enabling an increased mutation rate when included in the genomic hypermutation system of the present invention.

[0049] As used herein, the term "linker peptide" refers to a peptide fragment that can be used to link two protein or domains of a fusion protein. In some embodiments, the linker peptide can be about 5 to 100 amino acids in length. 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 peptide comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as the XTEN linker peptide. In some implementations, the linker peptide can be SGGS, SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS, SGGSGGSGGS, (GGGS) n (GGGGS) n (G) n (EAAAK) n (GGS) n etc., where n = 1, 2, 3, 4, 5 or 6.

[0050] Preferably, the linker peptides used include, but are not limited to, XTEN (GGGGS). n wait.

[0051] As used herein, the term "expression element" refers to an element used to express a protein (e.g., a fusion protein), including promoters, ribosome binding sites, target protein coding sequences, stop codons, etc., and optionally may also include purification tags or label tags, signal peptides, etc. These elements are well known to those skilled in the art. Expression elements may be included, for example, on plasmid expression vectors, viral expression vectors, or integrated into the host genome.

[0052] The fusion protein described in the system of this invention can be induced to express in a host. Preferably, the induction expression system used to control the expression of the fusion protein in the genome hypermutation system of this invention includes, but is not limited to, P... Tac and its mutant (isopropyl galactothioglycoside IPTG induction system), P BAD and its mutant (arabinose-induced system), P Lux and its mutant (acylhomoserine lactone AHL-induced system), P Tetand its mutant (ahydrotetracycline aTc-induced system), P Van One or a combination of its mutants (vanillic acid-induced system).

[0053] Preferably, the microorganisms suitable for the genome hypermutation system of the present invention are prokaryotic and eukaryotic microorganisms. The prokaryotic microorganisms are selected from, but are not limited to, halophilic bacteria, *Escherichia coli*, *Pseudomonas*, *E. rovovaginii*, *Aeromonas hydrophila*, and *Corynebacterium glutamicum*. Preferably, the halophilic bacteria are *Halomonas* genus (…). Halomonas (bacteria). Applicable eukaryotic microorganisms include any one of yeast, fungi, or algae.

[0054] In this invention, the term "halophilic genus (Halophilus)" is used. Halomonas ")" includes but is not limited to Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , sp. KM1、 Halomonas and Halomonas elongata Even better Halomonas smyrnensis TD1.0 ( Halomonas bluephagenesis Halomonas TD1.0 is in bluephagenesis Inserted on the basis of TD01 Halomonas bluephagenesis For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. lacI (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140), et al. Halomonas TD01 (CGMCC No. 4353) bluephagenesis M1 (CGMCC No. 19880) Halomonas aydingkolgenesis LS21 (CGMCC No. 6593) (All of the above strains have been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the Budapest Treaty and have been disclosed in previous patent applications. For example, CGMCC No. 4353 has been disclosed in CN102120973A, CGMCC No. 19880 has been disclosed in CN111593006A, and CGMCC No. 6593 has been disclosed in CN102925382A).

[0055] In this invention, the term "expression" can refer to "overexpression," which is defined as a gene expression level higher than the natural state, possibly achieved through increased transcription levels (producing more messenger mRNA) or improved translation efficiency (generating more functional proteins). In one specific embodiment, preferably, the expression or overexpression of the exogenous gene (fusion protein) can be achieved by inserting the target gene into a non-translation site of the genome, or by plasmid overexpression. Preferably, gene insertion is performed using the CRISPR / Cas9 method. Preferably, plasmid overexpression is introduced into the chassis strain via electroporation or conjugation transformation. When the host is *Halomonas*, the plasmid vector can be a pSEVA series vector (Martinez-Garcia E...). Halomonas campaniensis , SEVA 2.0: anupdate of the Standard European Vector Architecture for de- / re-construction of bacterial functionalities. et al. 2015, 43 (Database issue): D1183-1189.) or toxin-antitoxin plasmid pHbPBC vector (Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpCToxin-Antitoxin System of Halomonas bluephagenesis). Nucleic Acids Res 2023, 13 (1): 61-67.

[0056] As used herein, the term "selection pressure" refers to the restrictive conditions, whether artificially imposed or naturally present, such as physicochemical, nutritional, antibacterial, and survival competition conditions, during the artificial breeding or natural selection of microorganisms. These conditions allow superior mutant / recombinant strains in the microbial community to survive and proliferate, while inferior wild-type or degenerate individuals are suppressed or eliminated, thereby selectively screening and enriching target superior strains. Preferably, selection pressure may include antibiotic resistance, nutritional auxotrophic factors, high salt / high / low temperature / pH, osmotic pressure, organic acids, heavy metals, substrate analogs, microbial community competition, plasmid maintenance stress, etc. In a preferred embodiment, the microorganism is a halophilic monoclonal bacterium, and the selection pressure may be a high concentration of lysine, furfural, etc., in the culture medium.

[0057] As used in this article, the term "mineral salt culture medium" is a synthetic basic culture medium prepared from a variety of inorganic mineral salts to provide the mineral nutrients required for microbial growth and to maintain osmotic pressure and physiological environment.

[0058] This invention provides a genome hypermutation system based on transcription and replication coupling, the core of which lies in fusing deaminases to key proteins of the transcription or replication machinery via flexible linker peptides.

[0059] 1. Transcription-coupled mutagenic elements: fusion of deaminases into subunits of RNA polymerases (such as RpoA, ...). ACS Synth. Biol RpoC is preferred. During transcription, RNA polymerase opens the DNA double helix to form a transcription bubble, and the fused deaminase can deaminate the bases on the exposed single-stranded DNA (ssDNA), thereby introducing mutations into the transcription region.

[0060] 2. Replication-coupled mutagenic element: A deaminase is fused to a subunit of DNA polymerase III (such as DnaQ, DnaN, DnaX, HolA) or DNA helicase (DnaB), preferably the ε subunit DnaQ. During DNA replication, the helicase unwinds the double strand, and the DNA polymerase synthesizes the fused deaminase, which deaminates the ssDNA at the replication fork, achieving genome-wide mutation.

[0061] This invention also significantly improves mutation efficiency and the breadth of the mutation spectrum through the synergistic effect of multiple strategies.

[0062] 1. Applications of polyaminases and bifunctional deaminases: By fusing cytosine deaminase (PmCDA1-UGI) and adenine deaminase (TadA8e) to different target proteins (such as RpoC and DnaQ), or by using bifunctional deaminases with both activities (such as CABE T3.155 and TadDE), mutations from C:G to T:A and from A:T to G:C can be generated simultaneously, greatly broadening the mutation spectrum.

[0063] 2. Introducing DnaQ mutants with proofreading defects: By introducing D7A and E9A mutations into DnaQ, it loses the proofreading function of the 3'-5' exonuclease, thereby introducing an additional A:T to T:A transversion mutation during replication, further enriching the mutation types and improving the mutation efficiency.

[0064] 3. Increase the number of genome replication initiation sites ( rpoB Copy number: This is determined by inserting additional copies into the host bacterial genome. oriC Copying (e.g., increasing from a single copy to three copies) increases the frequency of replication initiation and the number of replication forks, thereby increasing the probability of replication-coupled mutations and overall mutation efficiency.

[0065] 4. Optimize culture conditions: Replacing the culture environment from nutrient-rich LB medium with a mineral salt (MM) medium of defined composition can further increase the mutation frequency of the hypermutation system.

[0066] Using the optimized hypermutation system described above, this invention provides a method for the rapid evolution of halophilic monoclonal bacteria. This method involves introducing the hypermutation system into a starting strain, generating a highly diverse mutant library under induction conditions, and then subjecting it to continuous passage or screening under specific selection pressures to rapidly obtain evolved strains with the target tolerance trait.

[0067] The technical advantages of this invention include: 1. Extremely high mutation efficiency: The optimized hypermutation system (such as MTDR3) constructed in this invention achieves extremely high mutation efficiency. H. oriC It can achieve a maximum of 3.9 × 10⁻⁶. -3 The mutation frequency was more than 330,000 times higher than the spontaneous mutation level of wild type.

[0068] 2. Broad mutation spectrum: By combining different strategies, this system can simultaneously generate multiple types of mutations, such as C:G to T:A transition, A:T to G:C transition, and A:T to T:A transversion, providing a rich genetic basis for screening diverse phenotypes.

[0069] 3. Flexible design and high portability: The design principle of this invention is based on targeting transient single-stranded regions of DNA, which has low dependence on species-specific pathways. Therefore, it has good portability and can be easily extended to other biological or non-model industrial strains.

[0070] 4. Rapid evolution speed: Using this system, halophilic monoclonal bacteria evolution strains with high tolerance to industrial stress conditions such as high concentrations of lysine (50 g / L) and furfural (20 mM) can be rapidly obtained within a few weeks, greatly shortening the development cycle of industrial strains.

[0071] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0072] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications can be made to this invention without departing from its spirit or scope. The following embodiments further illustrate the invention in detail and should not be considered as limiting the scope of the invention or the specific methods described herein.

[0073] Example 1: Design principles and applications of genome hypermutation systems based on transcription and replication coupling See bluephagenesisThe transcription and replication coupled hypermutation system of the present invention directly fuses cytosine deaminase (PmCDA1-UGI), adenine deaminase (TadA8e), or bifunctional deaminases (such as CABE T3.155, TadDE) to a flexible linker peptide. Figure 1 On the core subunit of endogenous RNA polymerase (preferably RpoC) or the core subunit of DNA polymerase III (preferably DnaQ), the deaminase can utilize the editing window of DNA double-strand unwinding during transcription vesicle and replication fork formation to efficiently target transiently exposed single-stranded DNA regions for base deamination, thereby introducing C:G to T:A or A:T to G:C mutations; further, by introducing proofreading defective DnaQ... D7A, E9A Mutants, combinations of multiple deaminase fusion proteins, and increased genome replication initiation sites ( Halomonas The synergistic effect of multiple strategies, including copy number reduction and optimized culture medium conditions, resulted in a mutation frequency in systems (such as MTDR3) in mineral salt media that was more than 330,000 times higher than that of wild-type, and the ability to simultaneously generate a broad spectrum of mutation types, including C:G to T:A, A:T to G:C transitions, and A:T to T:A transversions. Using this system, a mutant tolerant to 50 g / L lysine and 20 mM furfural was successfully evolved within weeks. oriC Superior mutant strains provide an efficient, broad-spectrum, and versatile genomic mutation platform for the rapid adaptive evolution of non-model industrial microorganisms such as Halophilic monoclonal bacteria.

[0074] Example 2: Construction and screening of transcription- and replication-coupled C:G to T:A supermutants This embodiment constructs a supermutant that targets the transcriptional and replication machinery with a fusion protein (PmCDA1-UGI) of the cytosine deaminase PmCDA1 and the uracil glycosylation inhibitor UGI. This is achieved using a rifampicin resistance gene (…). Halomonas bluephagenesis The mutation accumulation assay was used to detect the frequency of genomic mutations in the transcription-replication coupled hypermutation system. The specific principle is that rifampin inhibits transcription by binding to the β subunit of RNA polymerase. rpoB A specific mutation in the gene (SEQ ID NO: 20) can confer rifampicin resistance to the strain. Therefore, the frequency of rifampicin-resistant colonies can directly measure the mutation efficiency of the system.

[0075] The specific operating steps are as follows: Starting strain rpoB TD1.0 is in Halomonas bluephagenesis Halomonas Based on TD01 (CGMCC No. 4353), the following was inserted: bluephagenesis For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. lacI(2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140, and disclosed in granted Chinese patent applications Nos. 2021108133976, 2022113288365, and 2021100113917; the bacterium can be obtained from Tsinghua University. It carries blank control plasmids or various supermutant plasmids (such as the MTR series, MTD series, and MTDR series). et al. TD1.0 strains were inoculated into 60 LB liquid medium (i.e., LB medium containing 60 g / L NaCl) containing 25 mg / L chloramphenicol and cultured at 37°C and 200 rpm for 10 hours to obtain primary seed culture. Subsequently, the primary seed culture was transferred at a 1:100 volume ratio to fresh 60 LB liquid medium (or 50 mm mineral salt medium for condition optimization experiments) containing 25 mg / L chloramphenicol, and IPTG inducer was added to a final concentration of 200 mg / L to initiate mutant expression. Cultured for another 20 hours at 37°C and 200 rpm. After culture, an appropriate amount of bacterial culture was serially diluted 10-fold to 10⁻⁶. -7 100 μL of the dilution was spread onto corresponding solid plates without rifampicin (for counting total viable bacteria N0) and corresponding solid plates containing 100 mg / L rifampicin (for counting mutant colonies N1), with three replicates for each dilution. The plates were incubated at 37°C for 48 hours before colony counting.

[0076] The frequency of genomic mutations was calculated as f = N1 / N0, where N1 represents the number of resistant colonies grown on rifampicin plates and N0 represents the total number of viable colonies grown on rifampicin-free plates. Three independent biological replicates were set up for each experiment, and the mean and standard error of the mutation frequency were calculated, characterized by the fold increase relative to the wild-type control.

[0077] pSEVA321 (see reference: Silva-Rocha, Rafael, pSEVA321 plasmid) H. bluephagenesis"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) was used as an expression plasmid, and the PmCDA1-UGI ​​coding sequence (the coding gene sequences of PmCDA1 and UGI are shown in SEQ ID NO: 8 and SEQ ID NO: 10, respectively) was fused to the XTEN linker peptide (SEQ ID NO: 1) via the XTEN linker peptide. et al. Plasmid MTR1-MTR6 was constructed by using the N-terminus or C-terminus of endogenous RNA polymerase subunits RpoA, RpoB, and RpoC (amino acid and gene sequences are shown in the sequence listing). P... MmP1 The promoter (SEQ ID NO: 36) induces expression of ( Halomonas bluephagenesis PmCDA1-UGI ​​was fused to the N-terminus or C-terminus of the endogenous DNA helicase DnaB and the endogenous DNA polymerase III subunits DnaN, DnaQ, DnaX, and HolA (amino acid and gene sequences are shown in the sequence listing) via an XTEN linker peptide (SEQ ID NO: 1), thus constructing plasmid MTD1-MTD9. Figure 2 The results showed that among the mutants of RNA polymerase subunit fusion deaminase, MTR1 (RpoA-PmCDA1-UGI) and MTR6 (PmCDA1-UGI-RpoC) exhibited high activity, with mutation frequencies reaching 3.7 × 10⁻⁶. -7 and 9.5 × 10 -7 The mutation rates were 145-fold and 377-fold higher than those of the wild type. Among the mutants of DNA polymerase subunit fusion deaminase, MTD5 (DnaQ-PmCDA1-UGI) showed the highest mutation frequency, reaching 6.6 × 10⁻⁶. -7 263 times higher than the wild type ( Figure 3 Sequencing analysis confirmed that these mutants only produce C:G to T:A transition mutations. Figure 4 ).

[0078] Example 3: Construction and screening of transcription- and replication-coupled A:T to G:C and double mutants In this embodiment, adenine deaminase TadA8e (SEQ ID NO: 11) was fused to the selected optimal targets RpoC and DnaQ via an XTEN linker peptide, and a dual mutant was further constructed by combining them. The experimental detection method for genomic mutation frequency was the same as in Example 2.

[0079] A:T to G:C mutants: MTR1.2 (RpoA-TadA8e), MTR6.2 (TadA8e-RpoC), and MTD5.2 (DnaQ-TadA8e) plasmids were constructed. Figure 5 Among them, the mutation frequencies of MTR6.2 (TadA8e-RpoC) and MTD5.2 (DnaQ-TadA8e) reached 4.4 × 10⁻⁶. -6 and 6.9 × 10 -6 It is 726 times and 1147 times higher than the wild type. Figure 6 Sequencing confirmed that it only produces A:T to G:C conversion mutations. Figure 7 ).

[0080] PmCDA1-UGI ​​and TadA8e were fused to RpoC and DnaQ respectively via XTEN linker peptides to construct MTDR1 and MTDR2 dual mutant plasmids. Figure 8 Among them, the mutation frequency of MTDR1 reached 2.0 × 10⁻⁶. -5 It is more than 2900 times higher than the wild type. Figure 9 Sequencing showed that it can simultaneously generate C:G to T:A and A:T to G:C mutations. Figure 10 ).

[0081] Example 4: Constructing a multimutation-type supermutant using a bifunctional deaminase and a proofreading-deficient DnaQ To obtain a more balanced mutation spectrum and higher mutation efficiency, this embodiment introduces a bifunctional deaminase and a DnaQ proofreading defect mutant. The experimental detection method for genomic mutation frequency is the same as in Example 2.

[0082] Bifunctional deaminase fusion: Bifunctional deaminases CABE T3.155 (SEQ ID NO: 13) and TadDE (SEQ ID NO: 15) were fused to RpoC (SEQ ID NO: 21) and DnaQ (SEQ ID NO: 27) respectively via XTEN linker peptides. To enhance the mutation efficiency from C:G to T:A, UGI (SEQ ID NO: 9) was further fused to its C-terminus.

[0083] Proofreading defective DnaQ fusion: This involves fusing TadDE-UGI with proofreading defective DnaQ carrying D7A and E9A mutations (DnaQ).D7A, E9A The MTD5-d2-926 plasmid (SEQ ID NO: 29) was constructed by XTEN-linked peptide fusion. Figure 10 ).

[0084] The rifampicin assay results showed that MTR6-dual2 had the highest mutation efficiency, with a mutation frequency of 9.2 × 10⁻⁶. -5 This represents an increase of over 12,000 times compared to the wild type. Furthermore, the mutation frequency of MTD5-d2-926 reached 2.1 × 10⁻⁶. -5 Compared to the wild type, it is more than 2700 times higher ( Figure 11 This plasmid can not only generate C:G to T:A and A:T to G:C conversion mutations, but also additionally generate A:T to T:A transversion mutations. Figure 12 ).

[0085] Figure 13 Copy number optimization: using CRISPR-Cas9 technology (Qin, Q. et al. CRISPR / Cas9 editing genome of extremophile) oriC spp. Halomonas 47, 219-229 (2018)), [This refers to the use of endogenous halophilic monoclonal antibodies in *Halophilus*.] Metab. Eng oriC Insert wild type H. bluephagenesis In the TD1.0 strain, two and three were constructed. oriC Copy H. bluephagenesis Strains. The MTD5-dual2 plasmid was introduced into the genome containing one, two, and three... oriC Copy H. bluephagenesis In the strain. The results showed that in three copies oriC strain ( oriC In 3), the mutation frequency reached 6.3 × 10⁻⁶. -5 It is 21 times that of the wild-type strain (single copy). Figure 14 ).

[0086] Example 5: Combinatorial and Conditional Optimization of Hypermutation Systems This embodiment will compare the best-performing transcription-coupled mutant (TadDE-UGI-RpoC) and replication-coupled mutant (DnaQ). D7A,E9A -TadDE-UGI) were co-expressed in combination (where DnaQ) D7A,E9A -TadDE-UGI in TadDE-UGI according to H. bluephagenesisThe genomic codon frequency table was optimized (see SEQ ID NO: 35), and the effects of different culture media were tested. The experimental method for detecting genomic mutation frequencies was the same as in Example 2.

[0087] Combinatorial expression: Constructed using two independent P MmP1 The promoter (SEQ ID NO: 36) drives the plasmid MTDR3 of the two expression cassettes mentioned above, and uses a single P... MmP1 Promoter-driven dual expression cassette plasmid MTDR4 ( Figure 15 Rifampicin assay results showed that the mutation frequency of MTDR3 reached 5.0 × 10⁻⁶. -4 Compared to the wild type, it is more than 130,000 times better.

[0088] Performance testing: In LB medium, the mutation frequency of MTDR3 reached 5.0 × 10⁻⁶. -4 It is more than 130,000 times higher than the wild type. Figure 16 When MTDR3 was further cultured in mineral salt (MM) medium, the mutation frequency reached as high as 3.9 × 10⁻⁶. -3 It is more than 330,000 times higher than the wild type. Figure 17 MM medium consists of a basal salt solution, a carbon source, and trace elements. Its 50 mL standard system includes: 41.3 mL of basal solution (containing 1 g / L yeast extract, 50 g / L NaCl, and 1 g / L urea), 4 mL of glucose stock solution (final concentration 40 g / L), 1 mL of trace element solution I (50x, containing 10 g / L MgSO4 and 25 g / L urea), 1 mL of trace element solution II (50x, containing 482.5 g / L Na2HPO4·12H2O and 75 g / L KH2PO4), 1 mL of final trace element mixture (50x, composed of solution III containing 2 g / L CaCl2 and 5 g / L ferric ammonium citrate, solution IV containing various metal salts such as Mn, Cu, Co, Mo, Ni, Zn, and B, and deionized water in a volume ratio of 10:1:9), and 0.2 mL of 5 M NaOH to adjust the pH to 8.5.

[0089] Example 6: Rapid evolution of high lysine tolerance using a hypermutation system H. bluephagenesis The MTDR1 mutant (i.e., a plasmid vector expressing the combination of the fusion proteins PmCDA1-UGI-RpoC and DnaQ-TadA8e) was introduced into... H. bluephagenesisTD1.0 was first inoculated with a primary seed culture in 60 LB medium, and then transferred at a volume ratio of 1:100 to 60 LB medium containing 200 mg / L IPTG. Continuous subculturing was performed in MM medium with progressively increasing lysine concentrations (20-50 g / L). After 16 days (8 subcultures), the mutant group showed significant growth advantage, while the control group showed growth stagnation. Four mutant strains (50RD-1 to 50RD-4) that exhibited vigorous growth at 50 g / L lysine were successfully isolated.

[0090]

[0091] Example 7: Rapid evolution of highly furfural tolerant strains using a hypermutation system H. bluephagenesis Similar to Example 6, the MTDR1 mutant was used and evolved in MM medium with gradually increasing furfural concentrations (10-20 mM). After 32 days (11 passages), the mutant group resumed growth and gained dominance, and three mutants (20RD-1 to 20RD-3) tolerant to 20 mM furfural were successfully isolated.

[0092]

[0093] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0094] 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 describe the various possible combinations separately.

[0095] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A transcription- and replication-coupled genomic hypermutation system, wherein the system comprises at least one fusion protein expression element, the fusion protein being formed by fusing a deaminase or a variant thereof with a protein associated with the transcription or replication process via an optional linker peptide, wherein: The deaminase is selected from one or more of cytosine deaminase, adenine deaminase, or bifunctional deaminases with both cytosine and adenine deaminase activities; and / or the transcription or replication-related protein is selected from one or more of RNA polymerase subunit, DNA polymerase III subunit, or DNA helicase.

2. The system according to claim 1, wherein: 1) The cytosine deaminase is selected from sea lamprey (Gymnocypris chinensis). Petromyzon marinus Cytidine deaminase 1 (PmCDA1) or a variant thereof, cytosine deaminase APOBEC or a variant thereof, and activation-induced cytosine deaminase AID or a variant thereof, wherein the adenine deaminase is selected from TadA adenine deaminase or a variant thereof, and / or the bifunctional deaminase possessing both cytosine and adenine deaminase activities is selected from CABE T3.155, TadDE, and CABE T3.1; and / or 2) The transcription-related protein is the α subunit RpoA, β subunit rpoB, or β' subunit RpoC of RNA polymerase, preferably RpoC; the replication-related protein is the ε subunit DnaQ, α subunit DnaE, ​​β subunit DnaN, γ / τ subunit DnaX, δ subunit HolA of DNA polymerase III, or DNA helicase DnaB, preferably the ε subunit DnaQ.

3. The system according to claim 1 or 2, wherein: 1) The deaminase or a variant thereof is fused to the N-terminus or C-terminus of a protein associated with the transcription or replication process via an optional linker peptide; 2) The linker peptide is selected from: SGSETPGTSESATPES (XTEN) (SEQ ID NO: 1) SGGS (SEQ ID NO: 2) SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 3) SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 4) GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 5) SGGSGGSGGS (SEQ ID NO: 6) (GGGS) n (GGGGS) n (G) n (EAAAAK) n (GGS) n , Where n = 1, 2, 3, 4, 5, or 6; and / or 3) The fusion protein further includes 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 derived from Bacillus subtilis phage PBS1.

4. The system according to any one of claims 1 to 3, comprising expression elements of a first fusion protein and a second fusion protein, wherein the first fusion protein is a fusion protein formed by fusing (i) a deaminase or a variant thereof with (ii) an RNA polymerase subunit via an optional linker peptide, and the second fusion protein is a fusion protein formed by fusing (i) a deaminase or a variant thereof with (ii) a DNA polymerase III subunit or a DNA helicase via an optional linker peptide, preferably wherein the first fusion protein is formed by fusing (i) a cytosine deaminase or a variant thereof with (ii) an RNA polymerase subunit via an optional linker peptide, and the second fusion protein is formed by fusing (i) adenine deaminase or a variant thereof with (ii) a DNA polymerase III subunit via an optional linker peptide.

5. The system according to any one of claims 2 to 4, wherein the ε subunit DnaQ of said DNA polymerase III contains a mutation that causes it to lose its 3'-5' exonuclease proofreading function, said mutation being a D7A and E9A mutation.

6. The system according to claim 4 or 5, wherein the first fusion protein is formed by sequentially fusing a bifunctional deaminase, a uracil glycosylase inhibitor (UGI), and an RNA polymerase β' subunit RpoC via an optional linker, and the second fusion protein is formed by sequentially fusing the ε subunit DnaQ of DNA polymerase III carrying D7A and E9A mutations, a bifunctional deaminase, and a uracil glycosylase inhibitor (UGI) via an optional linker.

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

8. A method for obtaining a mutant strain of a microorganism (e.g., halophilic monoclonal bacteria) with a target phenotype through rapid evolution of systems according to any one of claims 1 to 6, comprising the following steps: (1) The supermutation system described above is introduced into the starting strain; (2) The strain obtained in step (1) is cultured in a culture medium to induce the expression of the fusion protein in order to initiate a genomic hypermutation process; (3) The bacterial culture from step (2) is continuously passaged or screened under selection pressure (such as high concentrations of lysine and furfural); and (4) Evolutionary strains with the target phenotype are isolated from cultures that can grow under selective pressure.

9. The method according to claim 8, wherein: 1) The halophilic monoclonal bacteria include Halomonas bluephagenesis , Halomonas aydingkolgenesis Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593); 2) The transcription or replication-related proteins in the hypermutant system are derived from microorganisms (e.g., Halophilic bacteria) or from the same genus or species of microorganisms (e.g., Halophilic bacteria), or are endogenous to the microorganisms (e.g., Halophilic bacteria). 3) The target phenotype includes: tolerance to 20-50 g / L L-lysine or tolerance to 10-20 mM furfural; and / or 4) The selected pressure is 20-50 g / L of L-lysine or 10-20 mM of furfural.

10. The method according to claim 8 or 9, wherein: 1) The genome of the microorganism (e.g., Halophilic monoclonal bacteria) strain contains two or more replication origin sites. oriC Copy; and / or 2) The culture medium is a mineral salt culture medium.

11. A mutant strain of a microorganism (e.g., halophilic monoclonal bacteria) evolved by the method according to any one of claims 8 to 10, preferably the mutant strain having significantly enhanced growth capacity under high concentrations of lysine or furfural.

12. The application of the system according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the method according to any one of claims 8 to 10 in microbial breeding, extreme microbial chassis cell modification, or green biomanufacturing.

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