Genomic hypermutation system of halomonas sp. and application thereof in strain modification

Abstract

The application provides a Halomonas genome hypermutation system HaloMut and application thereof in rapid adaptive evolution of microorganisms. By combining expression of an engineered endogenous DNA polymerase, a mismatch repair protein and an exogenous deaminase, and by using a weak ribosome binding site and a multi-promoter modular expression strategy, the genome mutation rate of Halomonas is significantly improved, and the highest mutation rate reaches 1.0 x 10 ‑4 base per generation, which is more than 1 million times higher than that of the wild type, and is the highest record of genome mutation efficiency at present, and the mutation rate can be dynamically regulated by the concentration of an inducer. By using the system, an evolved strain with high tolerance to low salt, high-concentration amino acids (arginine, lysine) and furfural and the like can be rapidly obtained in several days to several weeks. The application provides an efficient platform for industrial chassis modification of Halomonas and other extremophiles, and has a wide application prospect in the fields of green biological manufacturing and biological remediation.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and synthetic biology, specifically relating to a method for the synthesis of halophilic monoclonal bacteria (Halophilus spp.). Halomonas The efficient and tunable genome hypermutation system HaloMut, constructed in [the study], and the method and application of using this system to rapidly evolve and obtain evolutionary strains with a variety of excellent industrial traits (such as low salt tolerance, high product tolerance, etc.). Background Technology

[0002] Extremophiles, due to their ability to survive in harsh environments such as high temperature, high salinity, high pH, ​​or high toxicity, are highly promising chassis cells in industrial biotechnology. Among them, halophilic monoclonal bacteria (Halophilomonas) Halomonas With its robust growth under high salinity and alkalinity conditions, availability of inexpensive substrates, and ability to perform open-loop, non-sterile fermentation, *S. spp.* has become an ideal host for next-generation industrial biotechnology (NGIB) and has been successfully used to produce high-value compounds such as polyhydroxyalkanoates (PHAs) and tetrahydropyrimidines (Shao M, Li M, Zhang Z, et al. Toward a circular bioeconomy: bioproduction based on Halomonas grown on non-food feedstocks[J]. Current Opinion inBiotechnology, 2026, 99: 103497.).

[0003] However, most extremophiles are still primarily obtained through isolation from the natural environment, and the engineering modification of their systems is limited by the lack of efficient genomic evolution tools. Even Halomonas bluephagenesis Such industrial chassis strains also experience growth inhibition when faced with stresses in industrial environments, such as high temperatures, toxic substrates, or high concentrations of metabolites (e.g., amino acids). Traditional adaptive laboratory evolution (ALE) methods are limited by the inherently low spontaneous mutation rate of organisms (approximately 10^6). -9 Up to 10 -10The use of bases ( / generation) makes strain optimization a time-consuming process, which is difficult to meet the needs of rapid industrial iteration. Although a variety of in vivo mutagenesis systems have been developed in model organisms such as Escherichia coli, these systems often show incompatibility or inefficiency in non-model extremophiles with distant phylogenetic relationships (He T, Zhuo B, Zhao X, et al. Beyondnature's clock: Accelerating genomic diversity through hypermutation[J].Biotechnology Advances, 2025, 83: 108638.).

[0004] Therefore, there is an urgent need to develop an efficient genome evolution platform suitable for halophilic monoclonal bacteria in order to overcome the bottleneck in strain development. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly efficient, tunable, and programmable genomic hypermutation system, HaloMut, for halophilic monoclonal bacteria. Another purpose of this invention is to provide a method for rapidly evolving halophilic monoclonal mutants with superior industrial traits (such as low salt tolerance, high amino acid tolerance, and tolerance to highly toxic substances) using the HaloMut system.

[0006] Therefore, the present invention provides a genomic hypermutation system for halophilic monoclonal bacteria, wherein the system comprises a combination of the following proteins (i.e., DNA polymerase subunits or variants thereof, DNA mismatch repair (MMR) pathway proteins or variants thereof, and exogenous deaminases) or a combination of expression elements of the following proteins: (1) A DNA polymerase subunit or variant thereof derived from Halophilic mononuclei, the variant having a reduced DNA replication fidelity function, wherein the DNA polymerase subunit or variant thereof is selected from: the ε subunit DnaQ, α subunit DnaE, ​​β subunit DnaN, τ subunit DnaX or δ subunit HolA of DNA polymerase III or variants thereof, or the PolA variant of DNA polymerase I; (2) DNA mismatch repair (MMR) pathway proteins or variants thereof derived from Haloxylon ammodendron; and (3) Exogenous deaminase.

[0007] In one embodiment, the genomic hypermutation system comprises variants of DNA polymerase subunits and / or variants of DNA mismatch repair (MMR) pathway proteins.

[0008] In one specific embodiment, the variant of the ε subunit of DNA polymerase III is DnaQ carrying the D7A and E9A double mutation. D7A,E9A .

[0009] In one specific embodiment, the variant of the DNA polymerase III α subunit DnaE is a DnaE carrying the E618K mutation. E618K .

[0010] In one specific embodiment, the PolA variant of the DNA polymerase I is PolA 3M carrying D420A, I705N, and A755R mutations, or PolA 5M carrying D420A, I705N, F738Y, A755R, and P792H mutations.

[0011] In one specific embodiment, the DNA mismatch repair (MMR) pathway protein or a variant thereof includes a MutL variant, preferably a MutL variant carrying the G101D mutation. G101D Variants.

[0012] In one specific embodiment, the deaminase is selected from one or more of cytosine deaminase, adenine deaminase, or bifunctional deaminases possessing both cytosine and adenine deaminase activities, preferably the cytosine deaminase is selected from sea lamprey (Gymnocypris spp.). Petromyzon marinus The adenine deaminase is selected from TadA adenine deaminase or its variant, and / or the bifunctional deaminase with both cytosine and adenine deaminase activities is selected from CABE T3.155, TadDE and CABE T3.1.

[0013] 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.

[0014] Preferably, the activation-induced cytidine deaminase (AID) or a variant thereof is of human origin.

[0015] 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.

[0016] In one specific embodiment, the expression element further includes an auxiliary mutagenesis 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 for expression.

[0017] In one specific embodiment, the expression elements of each protein are driven by independent promoters, including constitutive and inducible promoters, with inducible promoters being preferred. For example, the promoter includes P... MmP1 Promoter.

[0018] In one specific embodiment, the expression elements of each protein are regulated by ribosome binding sites of varying strengths, preferably wherein the expression elements with higher toxicity (e.g., TadA8e) are driven by weaker ribosome binding sites.

[0019] In one specific embodiment, the system comprises a combination of expression elements of the following proteins (e.g., all six proteins): DnaQ D7A, E9A PolA 3M, MutL G101D DnaE E618K ,TadA8e and PmCDA1-UGI.

[0020] In a preferred embodiment, the system comprises a combination of expression elements of the following proteins: 1) DnaQ D7A, E9A ; and 2) one or more of the following: PolA 3M, MutL G101D DnaE E618K ,TadA8e and PmCDA1-UGI.

[0021] In a preferred embodiment, DnaQ D7A, E9A Using relatively strong RBS expression (e.g., SEQ ID NO: 21), other mutants (such as PolA 3M, DnaE) E618K The expression uses a relatively weak RBS (e.g., SEQ ID NO: 22).

[0022] In a preferred embodiment, DnaQ D7A, E9A Use P MmP1 The promoter, expressed using a relatively strong RBS (e.g., SEQ ID NO: 21), uses another independent P MmP1 The promoter co-expresses PolA 3M and DnaE E618K 、TadA8e、MutL G101D And / or PmCDA1-UGI.

[0023] In a preferred embodiment, four independent Ps are used. MmP1 Promoter, co-expressing six mutant elements (DnaQ) D7A, E9A PolA 3M, MutL G101D DnaE E618K 、TadA8e、PmCDA1-UGI).

[0024] In a preferred embodiment, a P is used. MmP1 Promoter co-expression of DnaQ D7A, E9A And PolA 3M, preferably, wherein DnaQ D7A, E9A PolA 3M is expressed using a relatively strong RBS (e.g., SEQ ID NO: 21), while PolA 3M is expressed using a relatively weak RBS (e.g., SEQ ID NO: 22).

[0025] In a preferred embodiment, a P is used. MmP1 Promoter co-expression of DnaQ D7A, E9A and DnaE E618K Preferably, DnaQ D7A, E9A Using relatively strong RBS (e.g., SEQ ID NO: 21) expression, DnaE E618K Use relatively weak RBS (e.g., SEQ ID NO: 22) expression.

[0026] In a preferred embodiment, a P is used. MmP1 Promoter co-expression of DnaQ D7A, E9A And PolA 3M, preferably, wherein DnaQ D7A, E9AUsing a relatively strong RBS (e.g., SEQ ID NO: 21) expression, PolA 3M uses a relatively weak RBS (e.g., SEQ ID NO: 22) expression; meanwhile, using another independent P MmP1 Promoter expression of DnaE E618K Preferably, a relatively weak RBS (e.g., SEQ ID NO: 22) is used to express DnaE. E618K .

[0027] In a preferred embodiment, a P is used. MmP1 Promoter co-expression of DnaQ D7A, E9A And PolA 3M, preferably, wherein DnaQ D7A, E9A Using a relatively strong RBS (e.g., SEQ ID NO: 21) expression, PolA 3M uses a relatively weak RBS (e.g., SEQ ID NO: 22) expression; meanwhile, using another independent P MmP1 The promoter expresses TadA8e, preferably using a relatively weak RBS (e.g., SEQ ID NO: 22) to express TadA8e.

[0028] In a preferred embodiment, a P is used. MmP1 Promoter co-expression of DnaQ D7A, E9A And PolA 3M, preferably, wherein DnaQ D7A, E9A Using a relatively strong RBS (e.g., SEQ ID NO: 21) expression, PolA 3M uses a relatively weak RBS (e.g., SEQ ID NO: 22) expression; meanwhile, using another independent P MmP1 MutL promoter expression G101D Preferably, MutL is expressed using natural RBS (i.e., wild-type RBS of MutL). G101D .

[0029] In a preferred embodiment, the system comprises a combination of the following expressive elements: 1) Use a P MmP1 Promoter co-expression of DnaQ D7A, E9A And PolA 3M, preferably, wherein DnaQ D7A, E9A PolA 3M is expressed using a relatively strong RBS (e.g., SEQ ID NO: 21), while PolA 3M is expressed using a relatively weak RBS (e.g., SEQ ID NO: 22). 2) Use another independent P MmP1 MutL expressed by promoter G101D Preferably, MutL is expressed using natural RBS. G101D ; 3) Use another independent P MmP1 DnaE expressed by promoter E618K Preferably, a relatively weak RBS (e.g., SEQ ID NO: 22) is used to express DnaE. E618K ; 4) Use another independent P MmP1 The promoter co-expresses TadA8e and PmCDA1-UGI, preferably using a relatively weak RBS (e.g., SEQ ID NO: 22) to co-express TadA8e and PmCDA1-UGI.

[0030] Another aspect of the present invention provides a method for constructing the genome hypermutation system described herein, comprising the following steps: (1) Select at least one gene encoding a DNA polymerase subunit variant from Halophilus and at least one gene encoding a DNA mismatch repair pathway protein variant or exogenous deaminase from Halophilus. (2) Based on the cytotoxicity of each expression element, its expression level was regulated by using ribosome binding sites of different strengths; (3) Multiple independent inducible promoters were used to drive the expression of different expression elements to construct expression vectors; (4) The expression vector is introduced into the host cell of Halophilic monocytogenes.

[0031] In one specific embodiment, the DNA polymerase subunit variant in step (1) includes DnaQ carrying double mutations of D7A and E9A. D7A，E9A Variant, DnaE carrying the E618K mutation E618K The variant, the PolA3M variant carrying the D420A, I705N, or A755R mutations, or the PolA5M variant carrying the D420A, I705N, F738Y, A755R, or P792H mutations, is at least one of the following:

[0032] In one specific embodiment, the DNA mismatch repair pathway protein variant in step (1) is MutL carrying the G101D mutation. G101D The variant is that the exogenous deaminase is one or both of PmCDA1 and TadA8e.

[0033] In one specific implementation, the weak ribosome binding site in step (2) is used to drive TadA8e expression.

[0034] In one specific implementation, the inducible promoter in step (3) is P MmP1 The number of the plurality of independent inducible promoters is 2-6 (e.g., 2, 3, 4, 5 or 6).

[0035] In one specific embodiment, the halophilic monoclonal host cell in step (4) includes 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).

[0036] Another aspect of the present invention provides a method for obtaining a halophilic monoclonal mutant strain with a target phenotype using the systematic rapid evolution described in the present invention, comprising the following steps: (1) The supermutation system was introduced into the starting halophilic monoclonal strain; (2) The strain obtained in step (1) is cultured in a culture medium to induce the expression of the expression element in order to initiate a genome hypermutation process; (3) The culture from step (2) is continuously passaged or screened under selection pressure (such as low salt, high concentration of amino acids, furfural, etc.); and (4) Evolutionary strains with the target phenotype are isolated from cultures that can grow under selective pressure.

[0037] In one specific embodiment, the induced expression of the expression element can be achieved by adding an inducer (e.g., IPTG) to the culture medium, via an inducible promoter (e.g., P) in the expression element. MmP1 This is achieved through a promoter.

[0038] In one specific implementation, the target phenotype is the ability to withstand the selection pressure.

[0039] 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 TD1.0Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (culture preservation number CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593).

[0040] In one specific embodiment, the target phenotype includes: tolerance to low salt concentrations of 2-5 g / L NaCl, tolerance to 5-20 g / L L-arginine, tolerance to 20-50 g / L L-lysine, and tolerance to 10-20 mM furfural.

[0041] In one specific embodiment, the selection pressure is 2-5 g / L (e.g., 2 g / L) NaCl, 5-20 g / L (e.g., 10 g / L) L-arginine, 20-50 g / L (e.g., 50 g / L) L-lysine, or 10-20 mM (e.g., 20 mM) furfural.

[0042] In one specific embodiment, the culture medium is a mineral salt (MM) medium.

[0043] Another aspect of the present invention provides a halophilic monoclonal mutant strain obtained by the method described in the present invention.

[0044] Another aspect of the present invention provides the application of the system or method described in the invention in, for example, microbial breeding of halophilic monoclonal bacteria, chassis cell modification of extremophiles, or green biomanufacturing.

[0045] This invention significantly improves the genome mutation rate of *Halophyta* by combining the expression of engineered endogenous DNA polymerase, mismatch repair protein, and exogenous deaminase, and employing a modular expression strategy using weak ribosome binding sites and multiple promoters. The mutation rate of *Halophyta* can reach up to 1.0 × 10⁻⁶. -4 The mutation rate is increased by more than 1 million times compared to the wild type, and the mutation rate can be dynamically controlled by the concentration of the inducer. Using this system, highly tolerant evolved strains to various stress conditions, including low salt, high concentrations of amino acids (arginine, lysine), and furfural, can be rapidly obtained within days to weeks. This invention provides an efficient platform for the industrial chassis modification of extremophiles such as halophilic monoclonal bacteria, and has broad application prospects in the fields of green biomanufacturing and bioremediation. Attached Figure Description

[0046] 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 1Overview of the design, optimization, and industrial applications of the HaloMut genome mutation system.

[0047] Sequence List Description The preferred embodiments of the present invention and the specific sequences used in the embodiments are as follows: The amino acid sequence of PmCDA1 cytosine deaminase (SEQ ID NO: 1): MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV The gene sequence of PmCDA1 cytosine deaminase (SEQ ID NO: 2) atgaccgacgctgagtacgtgagaatccatgagaagttggacatctacacgtttaagaaacagtttttcaacaacaaaaaatccgtgtcgcatagatgctacgttctctttgaattaaaacgacggggtgaacgtagagcgtgtttttggggctatgctgtgaataaaccacagagcgggacagaacgtggcattcacgccgaaatctttagcattagaaaagtcgaagaatacctgcgcgacaaccccggacaattcacgataaattggtactcatcctggagtccttgtgcagattgcgctgaaaagatcttagaatggtataaccaggagctgcgggggaacggccacactttgaaaatctgggcttgcaaactctattacgagaaaaatgcgaggaatcaaattgggctgtggaatctcagagataacggggttgggttgaatgtaatggtaagtgaacactaccaatgttgcaggaaaatattcatccaatcgtcgcacaatcaattgaatgagaatagatggcttgagaagactttgaagcgagctgaaaaacgacggagcgagttgtccattatgattcaggtaaaaatactccacaccactaagagtcctgctgtt Amino acid sequence of UGI glycosylase inhibitor (SEQ ID NO: 3): MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML Gene sequence of UGI glycosylase inhibitor (SEQ ID NO: 4): atgaccaacctttccgacatcatagagaaggaaacaggcaaacagttggtcatccaagagtcgatactcatgcttcctgaagaagttgaggaggtcattgggaataagccggaaagtgacattctc gtacacactgcgtatgatgagagcaccgatgagaacgtgatgctgctcacgtcagatgccccagagtacaaaccctgggctctggtgattcaggactctaatggagagaacaagatcaagatgcta The amino acid sequence of TadA8e adenine deaminase (SEQ ID NO: 5): MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN The gene sequence of TadA8e adenine deaminase (SEQ ID NO: 6): atgtctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccactctaggatcggccgcgtggtgtttggatggagaaattctaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaactga DnaQ D7A, E9A Amino acid sequence of (SEQ ID NO: 7): MRQVILATATTGIDPKDGHRLVEIGAVEMINRRFTGRSYHQYINPERHIDAEVVAVHGIDDAKVANEPVFAEIADDFWAFIEGAELVIHNAPFDVGFIDHELTMLNQRRRSPALGPVSDHCRILDTLVMARQMHPGQRNSLDALCKRYDIDNGHRVLHGALLDAEILADVYLAMTGGQTALTLDSEASSGEQQDNQASEGLSVQRLSLTPGQLRVVQPSDEERAAHQAKCQAHQLRWFDGGWSEGNSADA DnaQ D7A, E9A Gene sequence of (SEQ ID NO: 8): atgcgccaagtgatcttggctacggcaacgaccggcatcgaccctaaagatggccaccgattagttgaaattggtgccgtcgaaatgattaaccggcgttttacagggcgctcctatcatcaatacatcaaccccgaacggcatattgatgctgaagtcgtggcggttcacggtattgatgatgcaaaggttgctaacgaaccggtgtttgccgaaatagcggacgacttctgggcatttatagaaggtgccgagctggtgattcacaacgccccctttgatgtggggtttattgatcatgagctaacaatgttaaaccaacggcgacggtcacccgcattaggccctgttagtgatcattgccgtattctggatacgctggtgatggcccgtcagatgcacccaggccagcgtaatagtctcgatgcgttatgtaagcgctacgatattgacaacggtcatcgcgtactgcacggcgcattgcttgatgctgagatcttagccgacgtctatttggcgatgacgggcgggcaaacggcgctgacgcttgattcagaagcttcgtcaggcgaacagcaagataatcaagccagtgaaggactgtcggtacagcgtttatcgctaacgccaggacagctgagggtagtccagcctagtgatgaagagcgcgccgcccaccaagctaagtgccaggcgcatcagttacgttggtttgatggtggttggtctgaaggtaatagtgccgatgcttag DnaE E618K Amino acid sequence (SEQ ID NO: 9) of DnaE E618K The gene sequence (SEQ ID NO: 10): Amino acid sequence of PolA2M (SEQ ID NO: 11): MARAPIVLVDGSSYLYRAFHALPPLTTSNGQPTGAVKGVLNMLKRLIKDYPESPMAVVFDAPGKTFRDEMYSEYKAHRPPMPDDLRSQIKPLHACVKALGLPLLCIEGVEADDVIGTLAHHATQAGRDAVISTGDKDMAQLVNDHITLVNTMKEETLDEAGVKEKFGLPPSLIIDFLALMGDKVDNIPGVPGVGEKTAIGLLQGMEGGLETIYGDLERVKTLSFRGAKTLPKKLEEHREQAFLSYQLATIKTDCELPVGLDDLDIAHPDREALVELYKEMEFKQWLAELLAGNDEGVDDVKGGEPAPDNITTDAAESAVPSSQRNDHVIVEQAEFDAWLERLKQAERFCFALATTSLNYMDADIVGVGLALEAGEAAYIPLAHDYLDAPAQLDRQQVLQALKPLLEDPEKTKIGQNLKYDISVLANYDIHVVGPLADTMLASYVLNSTATRHDMDSLALKYLGEKTISFEEIAGKGAKQLTFNQIALEQAVPYACEDVDITLRLQETLRPQVEREGRLADVLDHLELPLINVLSRIERNGVALDAERLHEQSQQLERRIRELESEAFELAGREFNLGSPKQLGQILFEEQKIPVIKKTPKGAPSTAEAVLEELALDYPLPKVIMQHRGLAKLKSTYTDKLPRLLNKTTGRVHTSYHQAVTATGRLSSSDPNLQNIPIRTEEGRKIRQAFVARPGYRIVAADYSQIELRIMAHLSEDKGLLNAFAEGRDIHTATAAEVFGTALEKVSADQRRSAKAINFGLIYGMSAWGLSRQLHIDRNQAQTYIDRYFDRYPGVARYMDRIRTQAAEDGFVETVLGRRLYLPEIHSQNRNRRQGAERTAINAPMQGTAADIIKQAMIDVDAWLAEEEFDALMVMQVHDELVFEVAEVQVEAFIEQVRARMQAAATLSVPLIVEAESGANWDEAH The gene sequence of PolA2M (SEQ ID NO: 12): Amino acid sequence of PolA3M (SEQ ID NO: 13): MARAPIVLVDGSSYLYRAFHALPPLTTSNGQPTGAVKGVLNMLKRLIKDYPESPMAVVFDAPGKTFRDEMYSEYKAHRPPMPDDLRSQIKPLHACVKALGLPLLCIEGVEADDVIGTLAHHATQAGRDAVISTGDKDMAQLVNDHITLVNTMKEETLDEAGVKEKFGLPPSLIIDFLALMGDKVDNIPGVPGVGEKTAIGLLQGMEGGLETIYGDLERVKTLSFRGAKTLPKKLEEHREQAFLSYQLATIKTDCELPVGLDDLDIAHPDREALVELYKEMEFKQWLAELLAGNDEGVDDVKGGEPAPDNITTDAAESAVPSSQRNDHVIVEQAEFDAWLERLKQAERFCFDLETTSLNYMDADIVGVGLALEAGEAAYIPLAHDYLDAPAQLDRQQVLQALKPLLEDPEKTKIGQNLKYAISVLANYDIHVVGPLADTMLASYVLNSTATRHDMDSLALKYLGEKTISFEEIAGKGAKQLTFNQIALEQAVPYACEDVDITLRLQETLRPQVEREGRLADVLDHLELPLINVLSRIERNGVALDAERLHEQSQQLERRIRELESEAFELAGREFNLGSPKQLGQILFEEQKIPVIKKTPKGAPSTAEAVLEELALDYPLPKVIMQHRGLAKLKSTYTDKLPRLLNKTTGRVHTSYHQAVTATGRLSSSDPNLQNIPIRTEEGRKIRQAFVARPGYRIVAADYSQNELRIMAHLSEDKGLLNAFAEGRDIHTATAAEVFGTALEKVSADQRRSAKRINFGLIYGMSAWGLSRQLHIDRNQAQTYIDRYFDRYPGVARYMDRIRTQAAEDGFVETVLGRRLYLPEIHSQNRNRRQGAERTAINAPMQGTAADIIKQAMIDVDAWLAEEEFDALMVMQVHDELVFEVAEVQVEAFIEQVRARMQAAATLSVPLIVEAESGANWDEAH The gene sequence of PolA3M (SEQ ID NO: 14): Amino acid sequence of PolA5M (SEQ ID NO: 15): MARAPIVLVDGSSYLYRAFHALPPLTTSNGQPTGAVKGVLNMLKRLIKDYPESPMAVVFDAPGKTFRDEMYSEYKAHRPPMPDDLRSQIKPLHACVKALGLPLLCIEGVEADDVIGTLAHHATQAGRDAVISTGDKDMAQLVNDHITLVNTMKEETLDEAGVKEKFGLPPSLIIDFLALMGDKVDNIPGVPGVGEKTAIGLLQGMEGGLETIYGDLERVKTLSFRGAKTLPKKLEEHREQAFLSYQLATIKTDCELPVGLDDLDIAHPDREALVELYKEMEFKQWLAELLAGNDEGVDDVKGGEPAPDNITTDAAESAVPSSQRNDHVIVEQAEFDAWLERLKQAERFCFDLETTSLNYMDADIVGVGLALEAGEAAYIPLAHDYLDAPAQLDRQQVLQALKPLLEDPEKTKIGQNLKYAISVLANYDIHVVGPLADTMLASYVLNSTATRHDMDSLALKYLGEKTISFEEIAGKGAKQLTFNQIALEQAVPYACEDVDITLRLQETLRPQVEREGRLADVLDHLELPLINVLSRIERNGVALDAERLHEQSQQLERRIRELESEAFELAGREFNLGSPKQLGQILFEEQKIPVIKKTPKGAPSTAEAVLEELALDYPLPKVIMQHRGLAKLKSTYTDKLPRLLNKTTGRVHTSYHQAVTATGRLSSSDPNLQNIPIRTEEGRKIRQAFVARPGYRIVAADYSQNELRIMAHLSEDKGLLNAFAEGRDIHTATAAEVYGTALEKVSADQRRSAKRINFGLIYGMSAWGLSRQLHIDRNQAQTYIDRYFDRYHGVARYMDRIRTQAAEDGFVETVLGRRLYLPEIHSQNRNRRQGAERTAINAPMQGTAADIIKQAMIDVDAWLAEEEFDALMVMQVHDELVFEVAEVQVEAFIEQVRARMQAAATLSVPLIVEAESGANWDEAH The gene sequence of PolA5M (SEQ ID NO: 16): MutL G101D Amino acid sequence of (SEQ ID NO: 17): MSELISSPTRIHVLDPRLANQIAAGEVVERPSSVTKELIENAIDAGSQRIEVEIEQGGARLIKVRDDGIGIGEQDLPLALARHATSKINSLEDLEGVSSLGFRDEALASISSVSRLELISNAEEDPRQGWRVVAEGRGMEARVTPAPHPRGTSVAVRDLFFNTPARRKFLRTEKTEFAHVEEAFRRQALSRYDVAWVLRHNQKVVHQLPPGVTPAARERRIASLLGKNFIEHARYIEREAGGLRISGWVGLPTHSRSQADQQYFFVNGRVVRDRLVAHAVRQAYRDVLYNGRHPVFVLYLELDPDVVDVNVHPTKHEVRFRDGRMVHDFLYSSLHHCLASSKPAEEEQNMSHGHEDDVTDAPGSAGEVDVQTPAPRWQQQGMALSDSPDRHPGAERVRRFMQGYQALHPDHEESLLTPQPSSPSISTSKATEVREAPLAMPEHDATAAPPLGFALGQLHGVYILAQNAQGLVLVDMHAAHERIVYERMKNQLAAANGIDTQPLLVPVSLAASRAEVATAESERDAIAQLGVELDVAGPETLLVRQLPALLAQADPEALIRQMLEELARFGRTHQVEARIHELLSTMACHGSVRANRRLTIDEMNALLRDMERTERSDQCNHGRPTWTQMSMKALDRLFLRGQ MutL G101D Gene sequence of (SEQ ID NO: 18): The amino acid sequence of RpoB (SEQ ID NO: 19): The gene sequence of RpoB (SEQ ID NO: 20): Gene sequence of strong RBS (SEQ ID NO: 21): tactagagaaagaggagaaatactag Gene sequence of weak RBS (SEQ ID NO: 22): tctagagtcacacaggaaacctactag P MmP1 Promoter sequence (SEQ ID NO: 23): atatttgtggcattatagggaattgtgagcgctcacaattagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctctacaaataattttgtttaa Detailed Implementation

[0048] 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.

[0049] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0050] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0051] 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).

[0052] 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.

[0053] 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".

[0054] 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 marinus The 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 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.

[0055] As used herein, the term "expression element" refers to an element used to express a 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.

[0056] The expression elements described in the system of this invention can be constitutively expressed in the host. Preferably, they are used for *Halomonas* species (…). Halomonas The promoter for constitutive expression of ) can be selected from the wild-type porin gene P. porin The promoter or a mutant thereof, wherein the mutant is selected from P porin1 P porin3 P porin42 P porin51 P porin58 P porin59 P porin68 P porin140 P porin141 P porin183 P porin192 P porin194 P porin203 P porin221 P porin226 P porin259 and P porin278 More preferably, the porin59 promoter or the porin140 promoter. These P porin The promoter and its mutants are disclosed, for example, in CN117143793B.

[0057] The expression elements described in the system of this invention can also be induced to express in the host. Preferably, the induction expression system used to control the expression of expression elements 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 Tet and its mutant (ahydrotetracycline aTc-induced system), P Van One or a combination of the promoter and its mutants (vanillic acid-induced system). Preferably, the promoter may be P MmP1 The promoter can be induced to express by IPTG.

[0058] 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.

[0059] 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 , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Even better Halomonas bluephagenesis TD1.0 ( Halomonas bluephagenesis TD1.0 is in Halomonas bluephagenesis Inserted on the basis of TD01 lacI For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. et al. (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) Halomonas campaniensis 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).

[0060] 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...). et al. , SEVA 2.0: anupdate of the Standard European Vector Architecture for de- / re-construction of bacterial functionalities. Nucleic Acids Res 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). ACS Synth. Biol 2023, 13 (1): 61-67.

[0061] 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, community competition, plasmid maintenance stress, etc. In a preferred embodiment, the microorganism is a halophilic monoclonal bacterium, and the selection pressure may be a low salt (NaCl) concentration, a high concentration of arginine or lysine, furfural, etc., in the culture medium.

[0062] As used herein, the term "mineral salt (MM) medium" is a synthetic basic culture medium prepared from a variety of inorganic mineral salts to provide the mineral nutrition required for microbial growth and to maintain osmotic pressure and physiological environment.

[0063] First, this invention provides a HaloMut genome hypermutation system for Halophilic monoclonal bacteria. This system significantly improves the genome mutation rate by combining the expression of multiple types of mutant elements through a synergistic engineering strategy. The mutant elements include: 1. Endogenous DNA polymerase variants: These reduce the replication fidelity of DNA polymerase by introducing specific mutations, including but not limited to variants of the ε subunit DnaQ of DNA polymerase III (such as DnaQ). D7A, E9A ), α-subunit DnaE variants (such as DnaE) E618K ) and PolA variants of DNA polymerase I (such as PolA 3M, PolA 5M).

[0064] 2. DNA mismatch repair (MMR) pathway protein variants: expressing dominant-negative MMR proteins to interfere with mismatch repair function, including but not limited to MutL variants (such as MutL...). G101D ).

[0065] 3. Exogenous deaminases: Introducing enzymes that catalyze base deamination reactions to directly generate mutations, including but not limited to cytosine deaminase PmCDA1 (expressed in fusion with the glycosylation inhibitor UGI) and adenine deaminase TadA variants (such as TadA8e). Secondly, this invention optimizes system performance through precise expression regulation. Research has found that the optimal expression level of the mutant element is the balance between maximizing mutation efficiency and minimizing cytotoxicity. This invention achieves optimization through the following strategies: 1. Ribosome binding site (RBS) engineering: Different strengths of RBS are used for different mutant elements. In particular, for highly toxic elements (such as TadA8e), weak RBS is used to drive their low-level expression in order to maintain normal cell growth while obtaining a high mutation rate.

[0066] 2. Modular Architecture with Multiple Promoters: It was found that single-promoter-driven polycistronic operons suffer from transcription unit length limitations, hindering further improvements in mutation efficiency. Therefore, this invention employs multiple independent inducible promoters (such as P...). MmP1 By driving the expression modules of different mutation elements respectively, this bottleneck was successfully overcome, and synergistic effects of multiple mutation mechanisms were achieved.

[0067] Third, using the optimized HaloMut system described above, this invention provides a method for the rapid evolution of halophilic monoclonal bacteria. The method includes: introducing a HaloMut system plasmid into a starting strain, performing short-term culture under induction conditions to generate a highly diverse mutant library, followed by screening or continuous passage under specific selection pressures (such as low salt concentration, high amino acid concentration, or high furfural concentration), ultimately isolating evolved strains with significantly enhanced target tolerance traits.

[0068] The beneficial effects of this invention are as follows: 1. Extremely high mutation rate: The HaloMut system (such as the HaloM70 plasmid) constructed in this invention exhibits extremely high mutation rates. H. bluephagenesis It achieved a resolution of up to 1.0 × 10 -4 The mutation rate of the / base / generation is more than 1,000,000 times higher than the wild-type background, which is the highest mutation rate reported in all organisms to date.

[0069] 2. Adjustable mutation rate: By changing the concentration of the inducer (such as IPTG), the mutation rate can be precisely controlled within a dynamic range of more than 10,000 times to meet the needs of different evolutionary scenarios.

[0070] 3. Programmable mutation spectrum: By selecting different combinations of mutation elements, mutation systems with a preference for specific mutation types (such as A:TT:A transversion or A:GT:C / C:TG:A transition) can be constructed to achieve customized evolutionary paths.

[0071] 4. Rapid Evolution: This system can rapidly obtain evolved strains with high tolerance to various industrial stresses within days to weeks, greatly shortening the strain development cycle. For example, it has successfully obtained low-salt mutants tolerant to 2 g / L NaCl, high-amino acid mutants tolerant to 10 g / L arginine and 50 g / L lysine, and mutants tolerant to 20 mM furfural.

[0072] 5. Traceless and portable: The system is entirely plasmid-based, requiring no modification to the host genome, avoiding the use of chemical mutagens or special instruments, and is easy to transfer and apply between different strains.

[0073] 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.

[0074] 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.

[0075] Example 1: Design Principles of the HaloMut Genome Hypermutation System for Halophilic Monotrophs See Figure 1 In this embodiment, the HaloMut system of the present invention systematically combines and finely modulates multiple mutant elements through a synergistic engineering strategy, including halophilic mononuclear endogenous DNA polymerase fidelity-reducing variants (such as DnaQ). D7A, E9A DnaE E618K PolA 3M / 5M), dominant-negative mismatch repair protein variants (such as MutL) G101D The HaloMut system, along with exogenous deaminases (such as PmCDA1-UGI ​​and TadA8e), works simultaneously at three levels—introduction of replication errors, inhibition of repair pathways, and direct chemical modification of bases—to maximize the genome mutation rate. Building upon this, ribosome binding site (RBS) engineering is used to balance the expression levels of various elements to mitigate cytotoxicity, and a multi-promoter modular architecture is employed to overcome the limitations of single-promoter transcription unit length, achieving synergistic effects from multiple mutation mechanisms. After optimization, the HaloMut system (such as HaloM70) has achieved a mutation rate as high as 1.0 × 10⁻⁶ in Haloxylon ammodendron. -4 The mutation rate of the / base / generation is increased by more than 1 million times compared to the wild type, and the mutation rate can be dynamically controlled within a range of more than 10,000 times by changing the concentration of the inducer. Furthermore, the mutation spectrum can be programmed by combining different mutant elements. Using this system, within days to weeks, low-salt mutants tolerant to 2 g / L NaCl, high-amino acid mutants tolerant to 10 g / L arginine and 50 g / L lysine, and mutants tolerant to 20 mM furfural capable of synthesizing selenium nanoparticles were successfully obtained, fully demonstrating its high efficiency and practicality in the rapid modification of industrial chassis for extremophiles.

[0076] Example 2: Construction and Screening of Various Mutant Elements in the HaloMut System This invention firstly H. bluephagegenesis In TD1.0, mutant elements of different origins and types were systematically screened and identified (Table 1). All mutant genes were cloned into the low-copy plasmid pSEVA321 (plasmid reference: 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), P induced by IPTG MmP1 Promoter control of expression. Utilizing a rifampicin resistance gene (… rpoB The mutation accumulation assay of (SEQ ID NO: 20) was used to detect the genomic mutation rate of various mutants in the HaloMut system. The specific principle is as follows: rifampin binds to the β subunit of RNA polymerase (composed of...). rpoB Gene encoding) represses transcription, when rpoB Bacteria can acquire rifampicin resistance after a specific point mutation occurs in a gene. Therefore, the frequency of rifampicin-resistant colonies can directly reflect the level of genome mutation rate.

[0077] The specific operating steps are as follows: The starting strain is... Halomonas bluephagenesis TD1.0, which is in Halomonas bluephagenesis Based on TD01 (CGMCC No. 4353), the following was inserted: drawer For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. et al. (2017) Novel T7-1ike 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. Plasmids carrying blank control plasmids or different HaloMut mutants (sequences of each mutant are listed in the sequence listing) will be used. H. bluephagegenesis 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 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...-7 100 μL of the dilution was spread onto 60 LB agar plates (without rifampin, for counting total viable bacteria N0) and 60 LB agar plates (containing 100 mg / L rifampin, for counting mutant colonies N1), with three replicates for each dilution. The plates were incubated at 37°C for 48 hours before colony counting.

[0078] 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. To obtain the mutation rate per base per generation (spb), the Ma-Sandri-Sarkar (MSS) maximum likelihood method was used. The phenotypic mutation frequency was converted to the number of mutation events m using the FluCalc online tool, and then divided by the genome copy number (1). rpoB The number of possible mutation sites in the gene that could produce rifampicin resistance (26 types identified in Halophilic bacteria) was determined, and the mutation rate per base per generation was obtained. Three independent biological replicates were set up for each experiment, and the mean mutation rate and 95% confidence interval were calculated.

[0079]

[0080] 1. Construction of DNA polymerase-based mutant elements: Through the H. bluephagegenesis Sequence and structural conservation analyses were performed on endogenous DNA polymerases, and a series of mutants with reduced fidelity were designed and constructed.

[0081] (1) DnaQ variant (HaloM5): The 7th aspartic acid (D) and the 9th glutamic acid (E) responsible for metal ion coordination in the Exo I domain of the DnaQ protein (ε subunit) are mutated to alanine (A), respectively, to obtain DnaQ. D7A,E9A A mutant was found that disrupts the 3'-5' exonuclease reading function (Table 1). Rifampicin resistance experiments showed that HaloM5 increased the mutation rate to 1.1 × 10⁻⁶ under 200 mg / L IPTG induction. -8 / base / generation, 693 times higher than wild type.

[0082] (2) DnaE variant (HaloM7): The glutamic acid (E) at position 618 of the Pol3 domain of the DnaE protein (α subunit) is mutated to lysine (K) to obtain DnaE. E618K The mutant (Table 1) affects the selective incorporation of nucleotides. HaloM7 exhibits a high mutation rate (1.6 × 10⁻⁶). -9 The efficacy was 219 times higher than that of the control group.

[0083] (3) PolA variants (HaloM11, HaloM12, HaloM13): Multisite mutants of the PolA protein (DNA polymerase I) were constructed (Table 1), including the double mutant D351A / E353A, the triple mutant PolA 3M (D420A / I705N / A755R), and the quintuplet mutant PolA 5M (D420A / I705N / F738Y / A755R / P792H). The mutation rates of the three variants were 1.1 × 10⁻⁶. -10 2.1×10 -9 and 2.7×10 -9 Among the spb strains, PolA 5M exhibited the highest mutational activity, 365 times higher than the wild type.

[0084] 2. Construction of mutational elements based on mismatch repair pathways: MutL, a key protein in the endogenous MMR pathway of Halophilic monoclonal bacteria, was modified to construct a fault-prone mutant, MutL. G101D (HaloM19). This mutation is located in the ATPase domain of the MutL protein and interferes with ATP binding or hydrolysis, thereby weakening the function of the entire MMR complex. Its mutation rate is 1.7 × 10⁻⁶. -9 spb was 41 times higher than that of the wild type.

[0085] 3. Construction of mutant elements based on exogenous deaminases: Introduced exogenous deaminase system: Cytosine deaminase (HaloM24): Expresses lamprey-derived PmCDA1 and fused with a uracil DNA glycosylase inhibitor (UGI) to inhibit the repair of deamination products via the base excision repair pathway, thereby increasing the C:G→T:A mutation efficiency. The mutation rate of HaloM24 is 1.1 × 10⁻⁶. -9 SPb was 105 times higher than that of the wild type.

[0086] Adenine deaminase (HaloM34): Expresses the highly active adenine deaminase TadA8e, derived through phage-assisted evolution, catalyzing the A:T→G:C mutation. By employing weak RBS (SEQ ID NO: 22) (HaloM34) to control its low-level expression, the mutation rate was increased to 7.5 × 10⁻⁶ while maintaining cell viability. -9 / base / substitution.

[0087] Example 3: Combination and expression optimization of mutant elements to improve mutation efficiency To obtain a higher mutation rate, this invention optimized the combined expression and expression intensity of different mutant elements (Table 2, the sequences of strong RBS and weak RBS are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively). The experimental detection method for genomic mutation frequency is the same as in Example 2.

[0088]

[0089] 1. RBS optimization for dual / multiple mutant elements under a single promoter: With the highly efficient mutant DnaQ D7A, E9A Based on this, it was compared with other mutants (such as PolA 3M, DnaE) E618K ) Constructed in the same P MmP1 Under the promoter. Through screening, it was found that using weak RBS to drive the expression of a second mutant (such as HaloM42 and HaloM47) can minimize the background mutation and significantly improve the mutation efficiency after induction, reaching 3.7 × 10⁻⁶. -8 and 4.5 × 10 -8 / base / substitution.

[0090] 2. Design and application of a modular expression architecture with multiple starters: To address the problem of limited mutation efficiency caused by excessively long single-promoter transcription units, this invention designs a multi-promoter modular expression system. Based on the double-mutant plasmid HaloM42 (P MmP1 -DnaQ D7A, E9A -PolA 3M), introducing a second independent P MmP1 The promoters drive DnaE respectively E618K ,TadA8e or MutL G101D The expression of [the gene] was used to construct the HaloM63-65 plasmid. Rifampicin resistance experiments showed that the mutation rate was significantly increased after introducing multiple promoters, with the mutation rate of HaloM63-65 reaching 9.7 × 10⁻⁶. -7 5.1 × 10 -8 and 8.9 × 10 -7 / base / substitution.

[0091] Based on this, by iteratively adding expression modules, a final structure containing four independent P was constructed. MmP1 Promoter, co-expressing six mutant elements (DnaQ) D7A, E9A PolA 3M, MutL G101D DnaE E618K The ultimate version of HaloM70 (P, TadA8e, PmCDA1-UGI) MmP1 “DnaQ D7A, E9A(Strong RBS), PolA 3M (Weak RBS), P MmP1 MutL G101D (nativeRBS) and P MmP1 “DnaE E618K (weak RBS) and P MmP1 "TadA8e (weak RBS), PmCDA1-UGI ​​(weak RBS)"). The mutation rate of HaloM70 reached 1.0 × 10⁻⁶. -4 The mutation rate of HaloM70 increased by more than 1 million times compared to the wild type. The mutation rate of HaloM70 could be dynamically regulated within a range exceeding 10,000 times by changing the IPTG concentration (0-200 mg / L). Analysis of its mutation spectrum showed that HaloM70 mainly produced A:GT:C and C:TG:A conversion mutations.

[0092] Example 4: Rapid evolution of low-salt-tolerant organisms using the HaloMut system H. bluephagegenesis This invention utilizes mutant plasmids to accelerate the evolution of organisms capable of tolerating low salt. H. bluephagegenesis First, the product containing HaloM42... H. bluephagegenesisTD1.0 was induced and passaged 18 times in 60 LB medium to construct a mutant library. 200 mg / L IPTG was used, and the mutants were transferred to a new 60 LB medium at a volume ratio of 1:100 every 24 h. Subsequently, the mutant library was inoculated into mineral salt (MM) medium containing 10 g / L NaCl. This medium consists of a basal salt solution, carbon source, and trace elements. A 50 mL standard system includes: 41.3 mL of basal solution (containing 1 g / L yeast extract, 2–50 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 (50×, containing 10 g / L MgSO4 and 25 g / L urea), 1 mL of trace element solution II (50×, containing 482.5 g / L Na2HPO4·12H2O and 75 g / L KH2PO4), 1 mL of final trace element mixture (50×, 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 10:1:9 volume ratio), and 0.2 mL of 5 M... NaOH was used to adjust the pH to 8.5 (10MM) (10MM means 10 g / L NaCl, 2MM means 2 g / L NaCl), and the salt concentration of the culture medium was gradually reduced to 2 g / L (2MM). The entire evolution process took only 44 days. Four mutant strains (LS1-LS4) were finally isolated that grew vigorously in 2MM and 5MM media, while the wild type could not grow. Shake-flask experiments showed that in 2MM medium, the true cell mass (TCM) of the LS1-LS4 mutant strains reached 2.8-3.2 g / L, far exceeding the 0.2 g / L of the wild type.

[0093] Example 5: Rapid evolution of high amino acid tolerance using the HaloMut system H. bluephagegenesis Feedback inhibition from high concentrations of amino acid products is a common bottleneck in microbial fermentation. Take L-arginine and L-lysine as examples: Arginine tolerance: [This refers to products containing HaloM70.] H. bluephagegenesis After 24 hours of induction in arginine-free conditions, TD1.0 was directly inoculated into 50 mm medium containing 10 g / L arginine for three rounds of enrichment screening. The HaloM70 group showed significant growth recovery and dominance, successfully isolating mutant strains (10Arg-1 to 10Arg-3) that grew normally under 10 g / L arginine conditions, while the control group showed no growth. OD values ​​of strains 10Arg-1 to 10Arg-3 at 10 g / L arginine concentration were also measured. 600 The values ​​were 15.2, 8.2, and 5.4, respectively, while the wild-type strain had a value of only 0.1.

[0094] Lysine tolerance: Using a sequential subculturing strategy, strains containing HaloM70 were passaged in 50MM medium with lysine concentrations gradually increased from 20 g / L to 50 g / L. After 19 subcultures, mutant strains (50Lys-1 to 50Lys-3) stably growing at 50 g / L lysine were successfully obtained. OD values ​​of strains 50Lys-1 to 50Lys-3 at 50 g / L lysine concentration are shown in the figures. 600 The values ​​were 2.0, 1.6, and 1.8 respectively, which are much higher than the wild type's 0.2.

[0095] Example 6: Rapid Evolution of Tolerance to Highly Toxic Substances Using the HaloMut System H. bluephagegenesis Furfural tolerance: Furfural is a major inhibitor in lignocellulose hydrolysates. [The following appears to be a separate, unrelated sentence:] [The product contains HaloM70.] H. bluephagegenesis Strains of strain TD1.0 were passaged 11 times in media with progressively increasing furfural concentrations (10-20 mM) to obtain mutant strains (20F-1 to 20F-3) that were stably grown at 20 mM furfural. The OD values ​​of strains 20F-1 to 20F-3 at 20 mM furfural concentration are shown in the figures. 600 The values ​​were 15.9, 11.5, and 9.2 respectively, while the wild type had only 0.4.

[0096] 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.

[0097] 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.

[0098] 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 genome hypermutation system, wherein the system comprises a combination of the following proteins or a combination of expression elements of the following proteins: (1) A DNA polymerase subunit or variant thereof derived from Halophilic mononuclei, the variant having a reduced DNA replication fidelity function, wherein the DNA polymerase subunit or variant thereof is selected from: the ε subunit DnaQ, α subunit DnaE, ​​β subunit DnaN, τ subunit DnaX or δ subunit HolA of DNA polymerase III or variants thereof, or the PolA variant of DNA polymerase I; (2) DNA mismatch repair (MMR) pathway proteins or variants thereof derived from Haloxylon ammodendron; and (3) Exogenous deaminase.

2. The system of claim 1, comprising a variant of the ε subunit of DNA polymerase III, a variant of the α subunit DnaE of DNA polymerase III, and / or a variant of the PolA subunit of DNA polymerase I, wherein: 1) The variant of the ε subunit of DNA polymerase III is DnaQ carrying the double mutations of D7A and E9A. D7A,E9A ; 2) The variant of the α subunit DnaE of DNA polymerase III is DnaE carrying the E618K mutation. E618K ; and / or 3) The PolA variant of DNA polymerase I is PolA 3M carrying D420A, I705N and A755R mutations, or PolA 5M carrying D420A, I705N, F738Y, A755R and P792H mutations.

3. The system according to claim 1 or 2, wherein the DNA mismatch repair (MMR) pathway protein or a variant thereof comprises a MutL variant, preferably a MutL carrying the G101D mutation. G101D Variants.

4. The system according to any one of claims 1 to 3, wherein the deaminase is selected from one or more of cytosine deaminase, adenine deaminase, or a bifunctional deaminase possessing both cytosine and adenine deaminase activities, preferably the cytosine deaminase is selected from sea lamprey (Gymnocypris spp.) Petromyzon marinus The adenine deaminase is selected from TadA adenine deaminase or its variant, and / or the bifunctional deaminase with both cytosine and adenine deaminase activities is selected from CABE T3.155, TadDE and CABE T3.

1.

5. The system according to any one of claims 1 to 4, wherein: 1) The expression elements of each protein are each driven by an independent promoter, the promoters including constitutive and inducible promoters, preferably inducible promoters; and / or 2) The expression elements of each protein are regulated by ribosome binding sites of different strengths, and preferably, the expression elements with stronger toxicity (e.g., TadA8e) are driven by weak ribosome binding sites.

6. The system according to any one of claims 1 to 5, wherein the system comprises a combination of expression elements of the following proteins: DnaQ D7A, E9A PolA 3M, MutL G101D DnaE E618K ,TadA8e and PmCDA1-UGI.

7. A method for obtaining a halophilic monotypic mutant strain 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 was introduced into the starting halophilic monoclonal strain; (2) The strain obtained in step (1) is cultured in a culture medium to induce the expression of the expression element in order to initiate a genome hypermutation process; (3) The culture from step (2) is continuously passaged or screened under selection pressure (such as low salt, high concentration of amino acids, furfural, etc.); and (4) Evolutionary strains with the target phenotype are isolated from cultures that can grow under selective pressure.

8. The method according to claim 7, 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 target phenotype includes: tolerance to low salt concentrations of 2-5 g / L NaCl, tolerance to 5-20 g / L L-arginine, tolerance to 20-50 g / L L-lysine, or tolerance to 10-20 mM furfural; and / or 3) The selected pressure is 2-5 g / L NaCl, 5-20 g / L L-arginine or 20-50 g / L L-lysine, and 10-20 mM furfural.

9. A halophilic monoclonal mutant strain evolved by the method according to claim 7 or 8, preferably the mutant strain exhibiting significantly enhanced growth capacity under low salt, high amino acid, furfural, or sodium selenite stress.

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

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